:i!|il!:yilii:iy'lf!lillll!il lii!li-iiil llillii.iiiili^ i::!:; l.\ AMERUA TO XuOvici Ibelitoen THIS BOOK IS RESPECTFULLY DEDICATED. AS A SLIGHT TOKEN OF THE GRATITUDE AND ESTEEM OF HIS PUPIL nSOBOl PREFACE TO THE THIRD EDITION Despite the war, active investigations in the chemical problems of disease have continued, even in those countries most deeply involved in the conflict. Although some of the later publications of foreign countries have not been directly accessible, but few have not been avail- able at least through abstracts, and it is believed that most of the literature of importance, within the scope of this book, has been con- sidered in its revision, although the rule previously followed of quot- ing only from the original articles has of necessity been violated in several instances. The new additions to our knowledge in the three years since the second edition was issued have been so numerous that it has again been necessary to reprint the entire work. Several sub- jects have been largely rewritten, especially Gout, Specificity of Im- munological Reactions, Anaphylaxis, Icterus, Acidosis, Diabetes and Uremia. New sections have been added on the Abderhalden Reaction, Specificity, Chemical Basis of Growth, Atrophy, and the Pressor Bases, as well as many briefer additions. As previously, every effort has been made to make the discussion of each topic as brief as con- sistent with reasonable clearness and completeness. Where new articles including older references have been quoted, the latter have, in most instances, been omitted from the book. Again I must express my indebtedness to the several colleagues who have been so kind as to read over various sections of the book and to help me with their suggestions; and also to the members of my Department who have helped me so generously with the proof reading, especially Dr. Lydia ]\I. De Witt and Miss Harriet F. Holmes. Dr. R. T. Woodyatt has kindly revised the chapter on Diabetes, which he contributed to the second edition. Since that edition was printed much valuable information on the subject of carbohydrate metabolism has been contributed from Dr. Woodyatt 's own labora- tory, and through his new method for accurately timed and measured intravenous injections the way has been opened for many advances in the study of metabolism under both normal and pathological condi- tions. H. G. W. Chicago, III., November, 1917. PREFACE TO THE FIRST EDITION DxjKiNG the past score of years the subject of biological chemistry has attracted the attention and labors of a constantly increasing num- ber of investigators, many of whom have, for one reason or another, been interested in ])atlu)logical conditions. Sometimes the physiologist has souglit for light on his problems in the evidence afforded by related pathological conditions. Frequently clinicians have studied the meta- bolic clianges and the composition of the products of disease processes. Relatively seldom, unfortunately, has the pathologist attacked his problems by chemical methods. From the above and other sources have come scattered fragments of information concerning the chemi- cal changes that occur in pathological phenomena. Only when bearing upon conditions such as gout and diabetes, which concern alike the physiologist, the clinician, and the pathologist, have the fragments been moulded together into a homogeneous whole. For the most part they still remain isolated, uncorrelated, frequently unconfirmed items of information, scattered through medical, chemical, physiological, and physical literature. It has been the aim of the writer to collect these scattered fragments as completely as possible, and to use them as a basis for a consideration of General Pathology from the standpoint of the chemical processes which occur in pathological conditions. Owing to the diffuselj^ scat- tered conditions of the literature on which this work is based, it cannot be claimed that all of the many contributions from which useful in- formation might be obtained have been noticed ; but it is hoped that a sufficiently thorough collection of material has been made to afford a fair basis for a consideration of "Chemical Pathology." The time seems ripe for an effort of this nature. Within the past few years great and encouraging advances have been made in biological chem- istry, which in many instances seem to throw light upon pathological processes. In medicine, the use of chemical methods in the study of clinical manifestations has become more general, and has yielded valuable information. Pathologists have come to feel that the op- portunities for the acquirement of knowledge by means of morphologi- cal studies have become reduced to a mininmm, while the fields of pathological phj^siology and chemistry lie still almost unexplored. The development of research upon the subject of natural and acquired immunity has presented innumerable problems, all of which are essentialiv chemical. And perhaps most important of all is the general 8 P KEF ACE TO THE FIRST EDITION av:akonIn^ of an appreciation of the importance of physiological chem- it.tr> to medical science, wtiich has led to the introduction of laboratory courses on this subject in every medical school worthy of the name. A book on Chemical Pathology should, therefore, seek to supply information to a varied group of readers. It sliould furnish collateral reading to the student who for the first time goes over the subject of General Pathology, which his text-books usually consider chiefly from the morphological standpoint. It should exploit to the graduate in medicine tlie advances that are being made along lines that are of fundamental importance to clinical medicine. It should serve for the investigator in biological chemistry or in pathology as a source of information concerning the ground upon which the two subjects over- lap— the "Grenzgebiete" of Pathology and Physiological Chemistry. And, above all, it should afford a guide to the sources of our knowledge of these subjects, since nothing but direct familiarity with the original reports of the investigators themselves can give the student an im- personal view of the actual status of the questions under consideration. On account of this multiplicity of the objects in view, it has often been necessary to consider certain topics from more than one standpoint ; which explains, perhaps, certain apparent irregularities in the style and manner of treatment. It has been assumed that the reader has at least an elementary knowledge of organic and physiological chemistry. For the benefit of those whose studies in these subjects date back some years, it has seemed advisable to include in an introductory chapter an epitome of the more modern views concerning the chemistry of the protein mole- cule, the composition of the animal cell, and the principles of physical chemistry, in as far as they apply to biological problems. The general consideration of ''Enzymes" in Chapter II is written with a similar object. In discussing these fundamental topics it has seemed advis- able to omit detailed references to the numerous original sources, — these may be found quoted in the special text-books cited in the foot- notes; but in presenting the more distinctly pathological topics the attempt has been made to render all the important literature available to the reader and investigator. To economize space, a complete bibli- ography has not been inserted when this exists already eollected in some readily accessible review or original article ; hence the references cited in the foot-notes will generally be found to include only the more recent publications. These references have been so selected, however, that they will be found to furnish bibliogra]ihical matter sufficient to lead the investigator to all the important litei-ature on the topics covered in this book. As to those subjects (such as gout, diabetes, and gastro-intestinal putrefaction) which, because of their great practical clinical interest, have already been discussed in available monographs at greater length than the scope of this work would })ermit, it has seemed appropriate merely to summarize the most recent views and PREFACE TO THE FIRST EOmON 9 advances, referring the reader to tlie ,s])eeial treatises for the general and historical discussions. It is with the greatest pleasure that I acknowledge my indebtedness to many colleagues in the University of Chicago, who have kindly read the sections of my manuscript tliat touch upon tlieir own special fields, and whose criticism and advice have been of the greatest assistance ; their number alone prevents my thanking them by name. Most par- ticularly, however, must I express my debt to my former instructor, Professor Lafayette B. jMendel, of Yale University, whose kindly criticism and suggestions have been of inestimable value. For con- stant assistance in the preparation of the manuscript, and for the revision of the bibliography, I am indebted to my wife. H. G. W. CONTENTS CHAPTEU i I'AGK iMKODUCnoN 17 The ClIEMlSTKY and rilYSlCS OF THE CELL 17 Chemistry of the Essential Cell Constituents 18 Proteins It) Fats and Lipoids (Lipins) 23 Carboliydiates 25 Inorganic iSubstances 26 The Physical Chemistry of the Cell and Its Constituents 26 Crystalloids and iheir Properties 27 Colloids 34 The (Structure of the Cell in Relation to Its Chemistry and Physics . . 43 The iS'ucleus 44 The Cytoplasm 46 The Cell-wall 49 CHAPTER II Enzymes 53 The Nature of Enzymes and Their Actions 54 The Principles of Enzyme Action 56 The Toxicity of Enzymes 61 Anti-enzymes 63 The Intracellular Enzymes 68 Oxidizing Enzymes 68 Lipase 77 Amylase SO CHAPTER III Enzymes (Coniinued) 81 Intracellular Proteases (Proteolytic Enzymes), Including a Considera- tion of Autolysis 81 Autolysis 82 Relation of Autolysis to Metabolism 87 Defense of the Cells Against Their Autolytic Enzymes 88 Autolysis in Pathological Processes 90 CHAPTER IV The Chemistry of Bacteria and Their Products 106 Structure and Physical Properties lOti Chemical Composition 107 Bacterial Enzymes 113 Poisonous Bacterial Products 120 Ptomains 120 Toxins 125 Endotoxins 129 Poisonous Bacterial Proteins 13] Bacterial Pigments 132 11 12 CONtENTS CHAPTER V PAGE Chemistry of the Animal Parasites 134 Protozoa 135 Cestodes 137 Nematodes 140 CHAPTER VI Phytotoxins and Zootoxins 144 Phytotoxins 144 Tlie Toxin Causing Hay-fever 147 Zootoxins 148 Snake Venoms 148 Scorpion Poison 157 Spider Poison 158 Centipedes 159 Bee Poison 160 Poisons of Dermal Glands of Toads and Salamanders 161 Poisonous Fish 162 Eel Serum 164 CHAPTER VII Chemistry of the Immunity Reactions — Antigens, Specificity, Anti- toxins, Agglutinins, Precipitins, Anaphylaxis or Allergy, Ab- derhalden Reaction, Opsonins, and Related Subjects .... 165 Antigens 166 Non-Protein Antigens 167 Specificity of Immune Reactions 171 Toxins and Antitoxins 177 Chemical Nature of Antitoxins 180 Agglutinins and Agglutination 183 Precipitins 189 Anaphylaxis or Allergy 193 The Abderhalden Reaction 204 Opsonins 207 Tlie Meiostagtiiin Reaction 208 The Epiphanin Reaction 209 CHAPTER VIII Chemistry of the Immunity Reactions (Continued) — Bacteriolysis, Hemolysis, Complement Fixation, and Serum Cytotoxins . . 210 Scrum Bacteriolysis 210 Cytotoxins 214 Hemolysis or Erythrocytolysis 215 Hemolysis liy Known Chemical and Physical Agencies 215 Hemolysis by Serum 218 Hemolysis by ]5acteria 224 Hemolysis by Vegetable Poisons ', 225 Hemolysis 1)y Venoms 228 Hemolysis in Disease 229 Complement Fixation and Wassermann Reactions 234 Cytolysis in General 238 CHAPTER IX Chemical Means of Defense Against Non-Antigenic Poisons .... 243 Inorganic Poisons 246 Organic Poisons 248 CONTENTS 13 CHAPTER X PAGE Inflammation, Rkcknkkation, GIrowth 253 Ameboid Motion and Phagocytosis 254 Chemotaxis 254 Chemotaxis of Leucocytes 256 Phagocytosis 262 Theories of Chemotaxis and Phagocytosis 266 Artificial Imitations of Ameboid Movement . . .• 267 Relation of the Above Experiments to tlie Plienomena Exhibited by Leu- cocytes in Inflammation 271 Suppuration 276 Composition of Pus 277 Sputum 280 Proliferation and Regeneration 283 Growth and Repair. "'Vitamines." 285 CHAPTER XI Disturbances of Circut>ation and Diseases of the Blood 289 The Composition of the Blood 289 Hemorrhage 293 Hemophilia 297 Anemia and the Specific Anemias 300 Secondary Anemias 300 Chlorosis 302 Pernicious Anemia 305 Leukemia 307 Hyperemia 312 Active Hyperemia 312 Passive Hyperemia 313 Thrombosis 315 Fibrin Formation 315 The Formation of Thrombi 322 Embolism 325 Infarction 327 CHAPTER XII Edema 330 Formation of Lymph 331 Absorption of Lymph 338 The Causes of Edema 339 Special Causes of Edema 348 Composition of Edematous Fluids 352 Varieties of Edematous Fluids 359 Chemistry of Pneumothorax 365 CHAPTER XIII Retrogressive Changes (Necrosis, Gangrene, Rigor Mortis, Parenchy- matous Degeneration) 367 Necrosis 36/ Causes of Necrosis 3/1 Varieties of Necrosis 381 Fat Necrosis 384 Gangrene 388 Rigor Mortis 390 Atrophy 393 Cloudv Swelling 394 14 CONTENTS CHAPTER XIV PAGE Retrogressive Processes (Continned), Fatty, Amyloid, Hyaline, Colloid. AND GLYCOGf:XIC INFILTRATION AND DEGENERATION 397 Fatty Metamorpliosis 397 Physiological Formation of Fat 397 Pathological Fat Accunuilation 399 Causes of Fatty .Metamorphosis 406 Processes Related to Fatty Metamorphosis 410 Adipocere 410 Lipemia 412 Pathological Occurrence of Fatty Acids 414 Pathological Occurrence of Cholesterol 415 Amyloid Degeneration 417 The Origin of Amyloid 421 Hyaline Degeneration 423 Colloid Degeneration 425 Mucoid Degeneration 427 Glycogen in Pathological Processes 428 Physiological Occurrence 429 Pathological Occurrence 431 CHAPTER XV Calcification, Concretions, and Incrustations 435 Calcification 435 Occurrence of Pathological Calcification 438 Chemistry of the Process of Calcification 439 Osteomalacia 443 Rickets 445 Concretions 447 Biliary Calculi 448 Urinary Calculi 454 Corpora Amylacea 400 Other Less Common Concretions 461 Pncumonokoniosis 465 CHAPTER XVI I Pathological Pigmentation 467 Melanin 467 Lipochrome 474 Blood Pigments 476 Icterus 484 Congenital Hemolytic Icterus 489 CIL\PTER X^^I The Chemistry of Tumors 492 A. Chemistry of Tumors in General 404 B. (licniistry of Certain Specific Tumors 50!) (1 I Benign Tumors 50!) (2) Malignant Tumors 515 Multiple Myelomas and ^lyclopatliic "Alliuniosuria" 51S CHAPTER XVIII Pathological Conditions Due to, or Associated with. Abnormalities IN Metabolism, Including Autointoxication 523 I'remia 525 Toxemias of Pregnancy 533 COyTEMS 15 PAGE Eclampsia 533 Acute Yellow Atrophy of the Liver 53: cell division, when they seem to lose part of the combined protein and approach more nearly pure nucleic acid — just as inorganic salts occur with the acids and bases saturating each other more or less incompletely, e. g., mono-, di-, and tribasie ])li()sphates. in this we have a chemical explanation of the intensity of the staining of dividing nuclei by basic dyes.'' Phosphoproteins. Of these, by an luifortunate similarity of name, the so-called "nucleo-albumins" are often confused with nueleo-pro- teins by non-chemical writers, a difficulty increased by an actual re- semblance to the extent that they also yield phosphoric acid, and are somewhat similar in solubility and digestibility. They are essentially different, however, in that they do not yield nucleic acid or purine bases on cleavage. Probably members of this group are also constant components of cells. Glycoproteins (or gluco-proteins) and pliospho-glycoproteins are also believed to occur frequently or constantly in protoplasm. They are compounds of proteins with a sugar or sugar-like group, which probably usually contains nitrogen, thus differing from the ordinaiy hexoses and pentoses. Insoluble proteins, or bodies resembling the coagulated proteins in their lack of solubility in various fluids, are left behind after the other proteins have been extracted from the cells. Their signitieance is not known : whether to a large extent artificially produced or whether a normal structural element of the cell. FATS AND LIPOIDS (LIPINS) Lipoids is a term in common use but of indefinite significance ; most usually it comprehends the intracellular substances which are soluble in ordinaiy fat solvents, but which are not simple fats or fatty acids, lecithin and cholesterol being the most important of the lipoids. For the entire group of fats and lipoids the term lipins has been proposed by Gies and Rosenbloom. Lipoids and ordinary fats, that is lipins, occur in all cells, but their demonstration is not always readily pos- sible. The microscopic appearance of a cell, even when special stains for fat are used, gives no correct idea of the amount of lipins actually present. Thus normal kidneys contain 15 to 18 per cent, in their dry substance, but none of this can be detected readily with the micro- scope. A kidney which seems microscopically the site of marked fatty degeneration may show no more fat when examined chemically than a normal kidney, which in section appears to be quite free from fat. This is because some of the intracellular fat is bound chemically with 8 The oheniistrv of the niicleo-proteins is discussed in the chapter on Frio Acid Metabolism and Gout. Chap. xxi. 24 77//; CHEMISTRY AND PHYSICS OF THE CELL the proteins,) and when so bound it cannot be seen, nor can it be stained by the dyes ordinarily used for that purpose ; onl}- when de- generative changes of certain khids have liberated it from combina- tion does it become visible and stainable by ordinary methods (Rosen- feld). By the special fixation method of Ciaccio the fatty compounds of even normal cells may be made stainable (Bell),^ showing that the so-called masked fat is really in a not altogether invisible form. Whether the intracellular fat has any function other than that of serving as a food-stuff is not known, but there can be no question of the importance of the phosphorized fats, or phospholipins. Lecithin is a primarj^ cell-constituent, and is probably important both in metabolism and physically. liammarsten regards it as con- cerned in the building up of the nucleus. As will be shown later, many of the most essential physical properties of the living cell de- pend upon the presence in it of lipoids, of which lecithin is appar- ently the chief. Of the ether-soluble substances in the heart, for ex- ample, 60 to 70 per cent, is lecithin, which constitutes about 8 per cent, of the dry weight of the myocardium, There are several varieties of lecithin, depending upon the fatty acid radical they contain. The structural formula of one lecithin, stearyloleyl lecithin, is as follows: CH,— 0— C— H3,0 CH— 0— Cis— H33O I CH„— 0— PO— OH I 0— CH„— CH2— N =E (CHala. OH It differs from ordinary fats, therefore, in having two special groups, one the phosphoric acid, the other the choline radical, which last may be of some importance in pathological processes. In its ]ihysical properties it is quite similar to tlie ordinary fats, although it forms even finer emulsions in water, which are .practically colloidal solutions (W. Koch). Cephalin, a closely related body differing in having but one methyl grouj), is also pi-obably as M^dely spread in the tissues as lecithin, according to Koch and Woods. ^° Cholesterol, which is another lipoiil, is nearly as universally present as lecithin. ^^ It exists both free and in combination with fatty acids, for chf)lesterol is an alcohol and not at all similar to the fats chemically, aitliough very similar physically. The empirical formula is CoJT.,,011 or C,-H.t,blT, and it is ivJal.'d I0 llic terpenes. It n.Tour. ^^\od. Eos., mil (10), 539. if>.Tour. Biol. C'hoin., 100.5 (1), 20.1. 11 Recent literature piven liv Clikin, llioclicm. Cciilr.. lOOS (7). 2Sn. CARBOHYDRATES 25 seems to be relatively inert chemically, and therefore is probably im- portant only because of its effect on the physical properties of the cells. By some it is considered to be a decomposition or cleavage product of the proteins, which is in accordance witli its abundance in masses of old necrotic tissue, e. I'lIYHICH OF THE CELL Since glj^eogen is formed from dextrose and is constantly breaking down into dextrose, it is probable that the latter is also constantly present in the cells. INORGANIC SUBSTANCES Up to this point the substances of the cytoplasm that have been dis- cussed have all been organic compounds which do not naturally exist independent from living or once living cells, yet the inorganic sub- stances of the protoplasm are also of vital importance. As Mann says, "so-called pure ash-free proteins are chemically inert, and, in the true sense of the word, dead bodies. AVhat puts life into them is the presence of electrolytes." The various salts of i)otassium, so- dium, calcium, magnesium, and iron which all cells contain do not exist merely dissolved in the water of the cell, but in part they are com- bined with tlie organic constituents of the protoplasm. They are not combined as simple additions of the salts to the proteins ; but ions, both anions and cations, are united in chemical combination to the large pro- tein molecule (ion-proteins). Possibly the proteins participate in vi- tal chemical processes only as ion compounds with inorganic elements. It is extremely difficult, indeed almost impossible, to secure proteins entirelj^ free from inorganic substances (ash-free proteins). The fact that inorganic substances are held in the cells chemically rather than by simple diffusion into them from the surrounding fluids is sho^^m by the great difference in the proportions of various salts in the cells and in the extra-cellular fluids. Thus potassium is nearly always much more abundant in the cells than in the tissue fluids, while so- dium is more abundant in the fluids. Phosphoric acid is also more abundant in the cells, and chlorin in the plasma. In cells iron seems to exist chiefly in combination with the nucleo-proteins. These mat- ters will be taken up in greater detail in considering the physical chemistry of the cell." THE PHYSICAL CHEMISTRY OF THE CELL AND ITS CONSTIT- UENTS "^ From the standpoint of physical chemistry the cell consists of a collection of colloids and crystalloids, electrolytes and non-electro- lytes, dissolved in water, in lipoids, and in each other, surrounded by a semipermeable membrane, and perhaps subdivided by similar mem- branes or surfaces. Physical chemical processes, as we shall see later, play an all-important part in the life phenomena of the cell, and therefore some s])ace may l)e occupied ])rofitably in explaining tlie nature of these changes and of the substances that particii)ate in them. 14 Soe ^raf'.alluni on ^tici'oclioinistry, l^rfxobnissc Pliysiol., lOOS (7), 5;">2. i-t'i S(>e I'.ayliss, ■"I'liiiciph's of (icMicral Pliysiok)gy,"' London, 1015, for a more extensive diseiissinii of lliese (opii-s. vinsTM.t.ofDs A\n Tin:/!,- /'i!ori:irrfi:s 27 CRYSTALLOIDS AND THEIR PROPERTIES Crystalloids, or substances tliat tend under favorable conditions to form crystals, aiul whicli diffuse readily tlu'()u<;h most diffusion mem- branes, form a I'elatively small part of tlie total mass of tbe cell, but they are fully as essential as the colloids. The chief representatives of this group that are found usually or constantly in the cell are the inorganic salts, sugar, and the innumerable decomposition products of the proteins, including particularly urea, creatine, purine bases, amino-acids, etc. ^Most of these are by no means so characteristic of living things as are the colloids, sometimes occurring quite inde- pendently of a cellular origin, which the proteins never do. The inor- ganic salts in particular seem quite foreign to living processes, and as they enter and leave the body practically unchanged they are evidently not a source of energy through chemical change. Their importance to the cell lies almost entirely in their physical or physico- chemical properties. The organic crystalloids, although of initritional value, also have physical properties in some respects similar to those of the inorganic crystalloids, and therefore to this extent they exert similar influences, but the essential difference between the organic and the inorganic crystalloids is that all the latter are electrolytes, while many of the organic crystalloids that occur in cells are non- electrolytes. The importance of this distinction lies not in the utility or non-utility of these substances as conductors of electrical cur- rents in the ordinaiy sense, but rather on the existence of those properties which determine their conductive ability. Electrical con- ductivity is an index of ionization, and upon ionization depends the chief influence of the electrohi:es upon vital activities. The impor- tance of this process of dissociation or ionization lies in the fact that with most substances no chemical reaction can occur while the sub- stance is in the non-ionized state. The chemical properties of ioniza- ble substances are produced largely by the ions they liberate on dissociation. As a consecjuence, the physiological effects of electro- lytes are due to their ionic condition, and through the ions that are present in the cell many of its various chemical processes are brought about. Not all substances ionize with the same readiness, which causes a great difference in their properties. The reason that acetic acid is a weaker acid than hydrochloric acid is that it does not ionize to such an extent, and so a corresponding quantity does not introduce as large a number of hydrogen ions into a solution. Larger molecules, as a rule, ionize less than smaller ones of similar nature, e. g., stearic acid ionizes less than acetic acid and therefore is a weaker acid. Likewise the properties of a substance which depend upon its ions will be less marked when it is in a solvent that produces little ionization. For example, bichloride of mercury owes its antiseptic properties to the Hg ions that it sets free when in solu- 28 77//; ciiKMisTny wn riivs/cs of the cell tion. It is woll known tliat solutions of mercury, and for tliat matter most other antisp]:)tics, are much less actively germicidal in alcohol than when in water, because their ionization is less in alcohol; and the germicidal properties decrease as the proportion of alcohol increases, until the germicidal effect of the mixture is no greater than that of alcohol alone in the same strength. If we had no electrolytes in the cell, electric charges could not be carried about in it, and hence chemical reactions could not occur. It is this fact that makes the inorganic salts of such vital importance to the cell life. To repeat Mann's words, it is the electrolytes that put life into the proteins. AYater itself is almost absolutely non-dissoci- ated, and proteins so little that for some time it w^as doubted if they I'eally did ionize. Probably all soluble substances do dissociate to a certain minimal degree, but it is so slight for most of the constitu- ents of the cell except the inorganic salts (the organic acids and alka- lies, and a few dissociable organic products of protein metabolism, occur in such insignificant amounts as to be almost negligible) that without them there would be little chemical activity possible, and hence life would be absent or at a very low ebb indeed. As before mentioned, the inorganic salts probably exist in the cell not only as salts, but also, and perhaps chiefly, as ions and ionic compounds w^ith the cell proteins. For the most part it seems to be the cations that play the chief role in forming ion-protein compounds, althoiigh un- doubtedly the anions do combine wdth the proteins also, and in some instances they exert very characteristic and important effects; e. g., the differences between the effects of chlorides, bromides, and iodides, or of CNH as compared with HCl, both of wdiich liberate the same cation and differ only in their anions. IMany applications of the facts and theories of ionization have been made in physiology, and a few applications have also been made in pathology, especially the relation of ions to edema, to diuresis and glycosuria, and also to problems of immunity. No attempt will be made here to go further in the observations and theories concerning ionization or its role in physiology, but for more extensive informa- tion as well as for the complete bibliography the works mentioned below may be referred to.^^ The applications in pathology will be brought out as the subject under discussion in subsequent chapters necessitates, and it is largely to facilitate the understanding of such references that this brief summary of the subject of ionization has 15 "Physical Chemistry in tlio Service of Medicine." Wolfsanir Paul!, transla- tion by M. TT. Fischer, New York. IflO". "Physikalisohe Chemie der Zelle und der Gewebe." ITiiber, Leipzig, 191;"). "Osniotische Driick iind Tonenlehre in den medicinischen Wissensehaften," Hamburjrer, Wiesbaden. "Studies in General Physiolopy." Loeb, University of Chicairo Press, 1005. "Dynamics of Livinjj ]\ratter," Loeb, Columbia University Press. New York, 1000. Bottaz/i, Er^eb- nisse d. Pliysiol., 1008 (7), IGl. Spiro and J. Loeb, Oppenheimer's "Handbuch der Biochemie," 1008 (2), 1-141. DIFFUSION AM) OSMOSIS 29 been introduced. In the same spirit we take up tlie subjects of dif- fusion and osmosis. Diffusion and Osmosis. — Although the non-electrolytes do not ion- ize to any considerable extent, and therefore are relatively inactive chemically, the crystalloidal non-electrolytes, of which sugar and urea are the two chief examples among the cell constituents, possess in common with the electrolytes the important property of diffusion. By this process the exchange of chemical substances between the blood and the cell is brought about, by it the chemical composition of the different parts of the cell and between different cells is equalized, and without it chemical change would be practically impossible. Dif- fusion occurs most simply between two solutions of mil ike nature, or between a solution of a substance and the solvent alone, when placed directly in contact with one another. If we place in tlie bot- tom of a cylindrical vessel a solution of copper sulphate and above it some water, carefully avoiding mixing, it will be found after some time tliat the fluid has become equally blue throughout. This is brought about by the movement of the dissolved particles which gradualh' carries them through the entire mass of fluid, and as their migration is against the force of gravity, they evidenth' accomplish work. This process is not dependent upon ionization, for a solution of cane-sugar or of urea will show the same diffusion. A solution of protein or other colloid does so much more slowly, however, indeed quite imperceptibly. If we were to introduce a piece of filter-paper between the water and the copper sulphate solution, the diffusion would go on the same, the pores of the paper permitting the passage of the molecules with- out hindrance. If, instead of filter-paper, there were introduced a sheet of some substance free from pores, the diffusion would be much more affected. If the septum was of such a nature that the sub- stances in solution were insoluble in it (e. g., glass), diffusion would of necessity stop ; but if it were something in which the solvent or the solute was soluble, such as a gelatin plate, then these substances would dissolve in it, and diffusing through its substance escape into the fluid on the other side. The last example indicates the conditions afforded in the animal cell, and also in the usual laboratory diffusion experiments when the membrane is generally either an animal mem- brane or a parchment paper, both of which are composed of colloids. Crystalloids are generally soluble in colloids and hence pass through such diffusion membranes; colloids dissolve but slightly in colloids, and hence they do not pass through a diffusion membrane readily, and are, therefore, but very slightly diffusible. The process of diffusion, if uninterrujited, always continues until the solution is of exactly the same composition throughout. If on one side of the diffusion membrane there is a substance that passes through the membrane rapidly, and on the other a substance that passes 30 Tin: (iiEMisruY wn rinsicH of the cell through slowly or not at all, tliere will soon be an unequal condition on the two sides of the membrane, for tlie diffusible substance would ac- cumulate in equal amounts on each side, while the non-diffusible would remain where it was. On one side there would then be more material exerting osmotic pressure than on the other, and if the membrane were flexible, it would bulge toward the opposite side. The pressure is supposed to be due to the bombardment of the containing walls by molecules or ions of the substances in solution, and hence the more molecules and ions in solution, the more pressure. When equal num- bers of particles are on each side of the partition, the pressure is equalized. It is quite possible to have membranes readily permeable to one substance and almost entirely impermeable to another; such membranes are called semipermeable. To produce osmotic pressure it is not necessary that the membrane be absokitely impermeable to any of the substances — it may only be relatively less permeable for the solute than for the solvent. If, for example, we fill a parchment bag with concentrated sugar solution, tie up the top tightly and throw into water, it will swell up rapidly and eventually burst. But if the parchment is in the form of a tube, open at the top, and the lower end is placed in water, the amount of fluid inside the tube will in- crease at first, but eventually the sugar will diffuse out to such an extent that the solution is of the same concentration inside and out- side of the tube, and the column of fluid will again become of equal height on both sides. These results indicate that the water passes through the membrane more rapidly than does the sugar, but that eventually the sugar can all pass through. Exacth' similar conditions exist in cells, particularly plant cells. The typical cell of plant tissue consists of a distinct wall, usually cellulose or ehitin, lined internally by a layer of protoplasm which incloses a mass of aqueous solution, the cell sap, containing sugar and various other solutes. The outer wall is readily permeable by water and by most solutes, whereas the protoplasmic layer inside it behaves like a semipermeable membrane, which permits water to pass through readily but hinders greatly the passage of most solutes; that it is somewhat permeable is attested by the fact that the cell sap contains solutes derived fnmi the external fluids. As a result of this arrange- ment there is a constant tendency for the cavity of the cell to be distended by water and for the solutes within it to exert their con- siderable pressure upon tlie cell wall. Because of the strengtli of the cellulose layer the cell can withstand great pressures that would tear apart tlie tender protopla.smic layer that really determines the osmotic conditions, just as in the exjierimental membi-ane the clay cylinder supports the delicate i)recipitation membrane. It is the osmotic pressure that causes the rigidity oi- turgor of plant cells, and explains the ability of a tender gi-ecii shoot to hold itself up- j'iglit oi- horizontal in the air; and it is the foi-ce that enables growing OSMOTIC iMji:ssri;K 31 roots to lift <>reat stoiu's or tear apart rocks in whose clefts they grow. If certain plant cells are placed in distilled water, the pressure may rise to such an extent that the cells burst, and it was throuf^h studyin<>: this ])lu'n()nieiion that Pfett'er worked out the basis of our present knowledge of osmotic pressure. If the cell is placed in a so- lution of greater concentration than its cell sap, the pressure outside will be greater than that inside and the protoplasmic membrane will be forced away from the cellulose wall, while its central cavity shrinks and perhaps disappears entirely, the protoplasm forming a ball in the center. This is practically what occurs when a plant stem is cut and it "wilts" — the water is removed by evaporation, the osmotic pressure outside the cells becomes greater than that inside, and the water passes out. Likewise when a plant cell dies the turgor is lost because the membrane becomes permeal)U\ and so pressure soon be- comes the same on both sides of the cell wall. I21 animal cells the wall is not so highly developed as in plants, nor is it backed up by a rigid material like cellulose ; indeed, for many animal cells there is no well-defined wall and the protoplasm appears to be naked. Nevertheless the behavior of the animal cells indicates that they do possess what resembles a cell wall, in that they behave when in solutions as if the^^ were surrounded by a dif- fusion membrane. The degree to which phenomena of this nature are shown varies with different cells; with red corpuscles, for example, the osmotic pressure influences are very marked, as shown by the wrinkling or crenation of the corpuscles when they are placed in fluids of higher concentration than the blood plasma, and by their swelling and disintegration with escape of the hemogiobin (hemoly- sis) when they are put into distilled water or solutions of less con- centration than the plasma. Other tissue cells seem to undergo more or less alteration from changes in the osmotic pressure in the fluids surrounding them. The diffusion membrane that surrounds the cell is generally not well defined, and for most cells seems to be but a surface condensation of the protoplasm, perhaps formed through the effects of surface tension. It seems probable that this surface dif- fusion membrane contains a large proportion of cell lipoids, i. e., cholesterol and lecithin (for the red corpuscles this is practically certain) ; hence substances soluble in lipoids penetrate the cell read- ily, while to many substances insoluble in lipoids the cell is nearly or quite impermeable (Overton). Probably the wall of the aninuil cell is not so nearly semipermeable as is that of the plant cell, for nowhere in the animal body do we get such turgor in the cells as we see in plant tissues. Lacking a cellulose wall, animal cells could not develop such an internal pressure without rupturing, and such a process of rupturing {plasinorrhexis, plasmoptysis) does not seem to be a normal occurrence in animal tissues. AVe shall be most nearly correct, probably, if we look upon the animal cell as possess- 32 THE CHEMIHTRY AM) I'llY^WS OF THE CELL ing a delicate diffusion membrane at its surface, through which water passes more readily than do most crystalloids, and through which colloids pass almost not at all, but the exclusion of each of these t^'pes of substances is merely relative and not absolute. AVithin the cell, also, the colloids probably exist as a more or less well-developed emulsion, so that we have here a practically limitless amount of surface formation all through the protoplasm; such a structure could permit the endless number of reactions of a living cell to go on side by side in the same cell. Recent studies of G. L. Kite seem to show that all the protoplasm has much the same relation to solu- tions as does the external layer or cell membrane, for he found that if drops of solutions which can penetrate a cell from outside be injected directly into a cell they diffuse through it, but sub- stances which camiot penetrate from outside are also unable to diffuse through the cell after they have been injected into it. Since osmotic pressure, exactly like gas pressure, is presumably produced by the bombarding of the walls of the container by parti- cles in the solution, the amount of pressure will vary in proportion to the number of particles present. With such substances as sugar and urea, the non-electrolytes, the moving particles seem to be mole- cules, and so a solution of sugar or urea will produce an osmotic pressure directly proportional to the number of molecules it con- tains. In the case of the electrolytes, however, the ions produce pressure as well as the molecules, and hence an electrolyte in solution will produce a relativelj^ high osmotic pressure as compared with an equivalent solution of a non-electrolyte, since each molecule yields two or more ions. Colloids, however, exert so slight an osmotic pressure that it is difficult of detection; this probably depends on the great size and slight motility of their molecules. In the many and important osmotic processes of the animal organism, therefore, the colloids take no part except in helping to form the diffusion membrane, and in preventing the diffusion of one another.^® It is interesting to consider also that colloids under ordinary conditions do not greatly modify the diffusion of crystalloids through a solution containing both classes of matter. The fact that a cell is full of dis- solved colloids does not seriously affect the osmotic properties of the intracellular crystalloids, provided the colloids are not condensed in such a way as to form diffusion nuMiibranes. But as all the eleav- age products of proteins after tliey liave passed the peptone stage are crystalloids, by decomposition of the intracelhdar proteins the os- motic pressure may be greatly raised. As long as the cell is living there can be no constancy in composition, for metabolic processes, 10 Undor cxpciiinontal conditions it is found that tlie nature of the membrane preatly modifies tlic osmotic ])reasure; for if a jjfiven colloid is soluble in a cer- tain membrane and a certain crystalloid is not, the colloid will ditVuse tln-oujrh tlie membrane wliile the crystalloid is held back. (Kahlenberff, Jour. Physical Chem., 1 !)()() (10), 141.) OFMOTIC I'fx'ESSrUE 33 ])}' ])rodnc'ino- from proteins that liave no osmotic pressure erystal- !t)i(lal substances tluit do luive osmotie pressure, cause intracellular osmotic conditions to be continually varjdng. As a result, streams of diffusin<>- particles arc m()vin<«- a])out in every direction, settinjr up new chemical reactions and consequent new osmotic currents. The {2:reater the ditlt'erence in osmotic pressure between a cell and its environs, and between the ditferent parts of the same cell, the more powerful the osmotic efiPects, and as a result the g^reater the capacity for accomplish inji' work. The storing- up of insoluble and inditfusiblc forms of substance, such as glycogen, fat, and proteins, is an im- portant factor in maintaining inequalities in osmotic pressure, and in this way of increasing work Capacity. Indeed, we may look upon cell life as a constant attempt at the esta])lishment of equilibrium, both chemical and osmotic, which is never achieved because the move towards one sort of equilibrium is always against the other. All the food-stut?s — fats, carbohydrates and proteins— are characterized by being colloids when intact and crystalloids when disintegrated, thus: colloidal proteins ?^ crystalloid amino acids colloidal o'lycQcren 5=^ crystalloid sugar nondiffusihlo fats ^ diffusible soaps and glycerol. In consequence of this, if the crystalloids dififuse from the blood into a cell there is at once an excess of this end of the equation, and, hastened by the intracellular enzjones, partial synthesis to the colloid soon occurs to establish chemical equilibrium. Chemical changes in the crystalloids, by oxidation, reduction or hydrolysis, upset this chem- ical equilibrium, and hence further diffusion, synthesis and hydrolysis continue, one upsetting the other continuously. If equilibrium were established we should have no further reactions, and the cells would be inactive. The constant upsetting of the equilibrium is what con- stitutes cell life. The relation of osmotic pressure and osmosis to physiological prob- lems is only beginning to be studied. It is apparent that they must be of essential importance in absorption from the alimentary canal, in absorption and excretion between the cells and the blood stream, and in secretion by glandular organs; but it is also certain that they are no less important in all the less obvious chemical and phys- ical processes of the cell.^'^ In pathological processes osmotic pressure may play an equally important role, and the facts discussed in the preceding paragraphs will be alluded to frequently in subsequent chapters. 1' For further consideration of the subject of osmotic pressure in these rela- tions see: Livingston, "The Role of Diffusion and Osmotic Pressure in Plants." University of Chicaeo Press, Cliicago. 100.3; Czapek. "Biocheniie der Pflanzen," Jena, lfl03. Also, Spiro, Pauli and Ilobcr, all previously cited. 3 34 TUB CHEMISTRY AND PHYSICS OF THE CELL COLLOIDS 18 Since Graham in 1861 studied the differences between the sub- stances that did or did not diffuse readily through animal or parch- ment membranes, soluble substances have been classified in the two main groups of colloids and crystalloids, which distinction Graham believed separated two entirely different classes of matter. Although at the present time the differences between the two classes do not seem so great, yet the same division is found useful in classification. By colloids Graham indicated those substances which were dissolved to the extent of showing no visible particles in suspension, but which either did not pass through diffusion membranes at all, or did so very si owl}' indeed, as compared to the crystalloid substances. Under cer- tain conditions they tended to assume a sticky, glue-like nature, hence the name. (Many substances are now known which have the chief properties of the colloids and are therefore classified among them, but never are glue-like, e. g., the colloidal metals, so that the name has lost some of its original significance.) The physical prop- erty which Graham particularly noted in the colloids, besides their non-diffusibility, was the tendency to assume various states of solidity. Not only can they be in solution, when he called them "sols" (when the solvent Avas water, "hydrosols"), but they can become quite firm although containing much water (then called "gels" or "hydrogels"). The gels may assume a firm, coagulated condition, the so-called "pec- tous" state, which state is permanent in that the gel form cannot be reobtained from the pectous modification. Finally the colloid can be in a dry, solid state, quite free from water, and then not a sol at all. Included in the great class of colloids are all forms of proteins, and also giims, starch, dextrin, glycogen, tannin, chondrin, probably the enzymes, and also the greater number of organic dyes; also there are inorganic colloids, such as silicic acid, arsenic sulphide, hydrated oxide of iron, and many other similar compounds, besides the ele- ments themselves, especially the noble metals, which may exist in col- loidal form. It will be seen at once that the chief constituents of the cells, in fact nearly all the primaiy constituents except the inorganic salts, are organic colloids, and therefore the properties of the cells are largely dependent upon the properties of the colloids. In considering the characteristics of the colloids we at once meet the question — "What distinguishes the colloids from the crystalloids, on the one side, and from susjx'nsions or emulsions on the other? The sum and substance of our present conception of the nature of colloidal solution may be briefly summarized as follows: 1^ For full disnissinns of tho nature of colloids soo: TTolior. "Physikalisclie Clicinio (Icr Zcllc." Loip/,i), 10;"); r.ocliliold, "Die Kolloidc in Biolo^io und :\1(Hlizin," Drosdon. 1!)12: Wo. Ost- wald, "Crundriss dor Kolloidcliomio,"' Drosdon, 1000; franslafod by 'M. IT. Fischer, 101.'). A good brief discussion of colloids is f!;iven by Young in Zinsser's "Infection and Itesistance," 1014. COLLOIDS 35 It is possible for solid substances to be so divided among the par- ticles of a solvent that they remain permanently in this condition, neither aggregating into masses nor separating out through the action of gravity. Witli some substances, as sugar, for example, the solid seems to divide up into its molecular form, eacli molecule being free from all others of its kind except during occasional contacts. Some other substances, as salt, go still further, and the molecule divides into two or more parts, which have different electric charges (ionization) . The first of these classes of substances forms a solution which con- tains no particles visible by any known means, does not contain particles large enough to reflect light impinging upon them, exerts a large osmotic pressure, but does not conduct electricity. The other, in which ionization has occurred, differs solely in its capacity to conduct electricity readily. Both are true solutions of crj-stal- loids; the one which does not ionize is a non-electrolijte ; the other, by virtue of its ionization, is an electrolyte, the ions carrying electric charges through the solution. At the other end of the scale we have substances which are quite insoluble when in masses, but which, when very finely divided by me- chanical means, can be suspended and uniformly distributed through a fluid without having any marked tendency to aggregate or settle out. Such suspensions or emulsions contain particles visible under the microscope, usually appear turbid, refract light, are non-diftusible, exert no osmotic pressure, and do not transmit electricity. Such mix- tures are obviously very different from the true solutions above de- scribed. Between these two extremes stand the colloids, which vary in their properties so that they approach sometimes the suspensions (e. g., lecitliin, or' coagulated egg-albumin in colloidal suspension), and sometimes more nearly the true solutions (e. g., dextrin). No sharp boundaries can be drawn between any of tlie members of the series. Indeed, one substance may present all the different stages under different conditions, some agreeing with the properties of the typical suspensions, and some with the properties of the true solutions. The colloids stand in an intermediary position, differing quanti- tatively in one way or another from the true solutions, but yet ap- proaching them closely and sometimes almost indistinguishably re- sembling them. For the most part, however, they show character- istics decided enough to entitle them to separate classification, and to make any confusion with the crystalloids impossible. The Characteristics of Colloids. — The chief properties of the colloids are, then, as follows : Amorphous Fcrai. — This, like almost all other "colloidal properties," is not absolute, for in egg-albumin, hemoglobin, and various globulins w^e have proteins which in every respect are typical colloids, yet they form crystals readily and abundantly. Oxyhemoglobin, the mo- lecular weight of which is calculated at about 14,000, exhibits Tyn- 36 THE CHEMISTRY AHiD PHYSICS OF THE CELL dall's phenomenon and will not pass through a very fine porcelain filter, and therefore resembles the colloids decidedly, yet it forms beautiful crystals. The very fact that crystals are formed, Spiro points out, is proof that when in soluti(jn tlie individual molecules must have been free and separate, for otherwise they could scarcely unite in the definite spatial relations necessary to produce crystalline foi-ms. With these few exceptions, however, the colloids do not pre- sent any typical structure, and are not crystalline under any visible condition. But when they are made insoluble by chemical means they may, under certain conditions, produce rather characteristic non-crystalline structures, a matter that will be discussed in a sub- sequent ])aragTaph. Solubility. — Although we speak of "colloidal solutions," this term does not commit us to the theory of the identity of the solution of colloids with that of crystalloids. We have above stated w^hat seems to be a fair view of the matter as shown by many methods of experi- mentation. Most colloids seem to be, in fact, suspensions of masses of molecules, or perhaps of very large sing:le molecules, and a true solution is likewise a suspension of single molecules or of ions. When the aggregations of molecules are sufficiently large, w^e have an ordi- nary suspension ; but a single protein molecule is as large as a very great number of molecules of such substances as sugar (crystalloid) ; or tannin, C14H10O9 (colloid) ; or calcium carbonate (insoluble, sus- pension) ; and it would be strange if a true solution of a protein did not behave in many particulars like a suspension of molecular aggregates of dimensions similar to the dimensions of protein mole- cules. Nearly all colloidal solutions show Tyndall's phenomenon, which demonstrates the existence of particles in suspension large enough to reflect light from their surfaces. Most of the colloids are held back by very fine filters to a greater or less degree ; some are al- most entirely retained by a hardened paper filter, while others pass through the finest-pored clay filters. Furthermore, the metallic col- loids, such as those of platinum, gold, and silver, are unquestionably suspensions of finely divided particles of metal, yet they exhibit all the typical phenomena of colloids, passing through many sorts of filtei-s, and even accomplishing the same hydrolytic changes as many en- zymes. It must also be mentioned that the solvent is probably an im- portant factor in determining tlie colloidal or noii-colloidal nature of a substance ; e. g., soaps form true solutions in alcoliol and colloidal solutions in water; gelatin forms colloidal solutions in water but not in ether, whereas rubber forms colloidal solutions in ether but not in water. Closely I'elati'd to solubility is tiie ])li('ii(itiieii()n ol' iuihihittnn (the "Quellung" of German writers), which inny he deliuiMl ;is the tak- COLLOIDS 37 inp: up of a fluid hy a solid body witliout chemical change. Not all colloids possess this i)roperty, but it is shown by most of the organic colloids, particularly the proteins. Fick distinguishes cap- illary, osmotic, and molecular imbibition, the latter of which is the form exhibited by colloids, and it occurs independent of the existence of pores or other preformed spaces in the imbibing body. The imbibi- tion of water by colloids is more than a simple mechanical process, for it is accompanied by a contraction in the total volume of solid iiiid water, and by the evolution of lieat. The forces developed are far greater than those of osmotic pressure ; e. g., to prevent imbibi- tion of water by starch requires a pressure of over 2500 atmospheres. On the other hand, the ]^hysical properties of an aqueous colloidal so- lution show that the colloid is not chemically combined in the form of a hydrate. To describe this peculiar relation Hofmeister and Os- wald recommend the term "mechanical affinity." Hardy has shown that water held in a gelatin jelly cannot be removed by g-reat pres- sures (400 pounds to the square inch), but after the nature of the jelly is so changed by formalin that it is no longer liquefiable by heat, the water can be easily expressed from the loose meshwork that is formed. It would seem from this that the imbibition and retention of water by colloids may be closely related to surface phenomena. Hofmeister has shown that organized animal tissues obey the same laws of imbibition as do simple gelatin plates, and probably this phe- nomenon of colloids is very important in physiological and patho- logical processes. Non-diffusibility. — The lack of power to pass through animal and parchment membranes, wiiich was Graham's starting-point in the study of colloids, is also only a relative condition. This is shown by the following figures, giving the relative time required by the same amount of different substances to pass through a certain diffusion membrane : Sodium chloride 2.3.3 Sugar 7.00 Magnesium sulphate 7.00 Protein 49.00 Caramel 98.00 This difference of time is so great, however, as to pennit of separation of salts from proteins, etc., by dialyzation, a process in constant u.se. Primarily the ability to diffuse through a given membrane requires that the diffusing substance be soluble in the membrane. Diffusion membranes are always composed of colloids, e. g., animal bladders, or parchment, which is a colloidal cellulose. Crystalloids are generally soluble in colloids, while colloids are little or not at all soluble in other colloids, and hence do not diffuse through one another readily and permeate diffusion membranes very slowly. For example, if 38 THE CHEMISTRY AND PHYSICS OF THE CELL a stick of agar jelly be placed in a solution of aminoniated cop- per sulphate (a crystalloid), and another be placed in a solution of Prussian blue (a colloid), it will be found that the copper solution penetrates the agar completely before the colloidal solution of Prus- sian blue has penetrated it at all. This property is of great im- portance, undoubtedly, in keeping different colloidal constituents of the cell in given localities within its protoplasm, e. g., the oxidizing ferments seem to be chiefly localized within the nucleus; the colloidal glj'cogen remains where it is formed in the cytoplasm, unable to escape from the cell, whereas the crystalloidal sugar from which it is formed and into which it is converted, dififuses rapidlj' into or out of the cell. The osmotic pressure of the colloids is extremely small. The closely related phenomena of diffusion, depression of freezing -point, and ele- vation of hoiUng-point, are also exhibited by colloids to but an ex- tremely slight degree. For example, in one experiment, the dissolv- ing of from 14 per cent, to 44 per cent, of egg-albumin in water low- ered the freezing-point but 0.02° to 0.06° ; and some other colloids have even less effect. The results of the latest and best experiments seem to indicate that the trifling effects of colloids upon osmotic pres- sure and upon freezing- and boiling-points observed in colloidal solu- tions are due to the colloids themselves, rather than to included im- purities, although it may possibly be that some of these effects are due to the high surface tension and cohesion affinity of the colloids. In all cellular processes accompanied by manifestations of osmotic pressure or dififusion, however, the crystalloids may be considered as almost entirely responsible. Electrical Phenomena. — As colloids do not separate freely into ions when dissolved, they do not conduct electricity appreciably. How- ever, when an electric current is passed through water containing ■colloids in solution, the colloidal particles tend to pass to one pole or the other. Most colloids move toward the anode. This phenomenon. eataphoresis, is also generally exhibited by suspensions, and hence in this particular the colloids resemble suspensions rather than solutions. Helmholtz has explained the movement of the suspended particles as due to the accunmlation of electrical charges u])on the surfaces of two heterogeneous media when in contact. The nature of the charge de- pends upon both the suspended substance and the fluid ; e. g., sulphur or graphite particles suspended in water assume a negative charge and move toward the anode, but when suspended in oil of turpentine they become positively charged and move toward the cathode. AYater has such a high dielectric constant that most substances suspended in water become negatively charged as eoni])ared with the water, and move toward the positive pole or anode. Hardy has observed that colloidal solutions of coagulated proteins move toward the anode when in alkaline solnlion, and toward the COLLOIDS 39 cathode when in acid solution.^" Tliis peculiar property of proteins sugorests that perhaps simple surface phenomena do not snfifice to ac- count for the electrification of all colloid particles. Knowing the pe- culiar amphoteric character of ])r()tcins, which is probably due to the presence of both NII2 and COOII groups in the molecule, we can readily understand that in an acid solution the NH2 radicles are com- bined with the acid, leaving the COOH iradicles free. The molecule would then have acid properties, and could dissociate into an acid II ion and a basic or electrically positive colloid ion. The colloid ion would then go toward the negative pole slowly, because of its great size. When a suitable concentration of both ions is produced the pro- teins will move towards both poles, this concentration being, in the case of serum albumin. H^IO " (^lichaelis). Living protoplasm be- haves in most instances, as if the proteins were acids bound to inor- ganic cations (Robertson), and is usually stimulated at the cathode on the '"make" of the current. It is permeable to ions, and the vitality of a tissue is so dependent on the maintenance of normal permeability that the permeability may be employed as a sensitive and reliable indicator of its vitality (Osterhout ^°^). This may be done by determining the electrical resistance of the cells, which is lowered by anything that lowers their vitality. Surface tension,-" which may be described as the force icith tchich a fluid is strii'i)ig to reduce its free surface to a minimum, is highly exhibited by colloids as compared with crystalloids. The formation of emulsions and the spreading out of oil upon the surface of water de- pend upon surface tension. Ameboid movement may be attributed to changes in surface tension, as also may phagocytosis. (The relation of surface tension to these processes will be considered under the subject of Inflammation.) The effect of colloids upon chemical processes going on within their solutions or gels is surprisingly small. Salts in solution in a thick gel of agar or gelatin will diffuse almost as rapidly as in water ; they will also ionize as rapidly as in watery solutions, and chemical reactions occur with nearly the same speed and completeness as if the colloids were absent. Furthermore it makes little difference whether these processes are measured in a colloid solution that is liquid, or after it has set in the gel form. These facts merely indicate that the colloids do not greatly impede the movements of molecules or ions in solutions. On the other hand, as before mentioned, colloids diffuse very slowly into each other. Hence, in the cell the colloids are quite fixed in their positions, whereas the crystalloids may wander about freely, and this arrangement is certainly of great importance in bio- 19 According to Field and Teague (Jour. Exper. Med., 1007 (9), 222). vative proteins in serum move towards the cathode, no matter what the reaction. ina Science. 1904 (40), 4SS. 20 See article on "Surface Tension and Vital Phenomena." hv ^Macallum. Erirel)- nisse d. Phvsiol., 1011 (11). (\()2. 40 Tin: CHEMISTRY Ayo riivfiics of the cell logic processes. Pauli suggests the probability that the fixation of the colloid causes the cell to have different properties in different parts, and so various reactions may occur independently in different areas of the cytoplasm. The possibility of the correctness of this view is increased when we consider that the enzymes are colloids, for there is inuch evidence to show that they are distributed in just such an un- even manner within the cells. Although colloids permit the passage of dissolved crystalloids, through them, they greatly interfere with the movement of larger particles. This property accounts for the ability of colloids to hold many insoluble substances in such extremely fine suspensions that they seem superficially to be in true solution. If, for example, sodium phosphate is added to a solution of casein in lime-water, tlie calcium phosphate formed does not precipitate. It is not in solution, how- ever, but rather exists as a suspension of very finely divided particles of the salt which the colloid keeps from aggregating into particles, large enough to be visible or to overcome the viscosity of the fluid and sink to the bottom. Probably in this way many substances, in- cluding calcium salts, are carried in the blood, held in permanent suspension b3' the proteins. Substances thus finely divided will have extremely large surface area for reactions, and, therefore, will un- doubtedly undergo changes with considerable rapidity and facility, although not in solution. Precipitation and Coagulation of Colloids. — Because of the rather slender marghi by which the colloids are separated from the suspen- sions, their persistence in solution is generally in a rather precarious condition. Kelatively slight changes suffice to throw the colloids out of solution, and when once precipitated, they are often incapable of again dissolving in the same solvent. Solutions of albumin may un- dergo spontaneous coagulation on standing for some time, and agita- tion rapidly produces the same effect in many protein solutions. Some inorganic colloids are as readily coagulated as the proteins. A comparatively small rise in temperature, less than to 50° C. with some proteins, renders the protein perfectly insoluble. Further- more, we have coagulation of protein solutions by enzyme action. The inorganic "colloidal suspensions" may be precipitated by the addition of very small quantities of electrolytes. Colloidal solutions of the type of the proteins are not so readily i)recipitated by most electrolytes, but if to the solution large quantities of crystalloids are added, the protein molecules are practically crowded out of solution, as in the "salting-out" process used in separating proteins by am- monium sul])liate and other salts. Tlie effect of heat U])on different folloids is ])ecu]iar, in that some vai'ieties, a.s silicic acid, aluminium liydrate, and many proteins are rendcrcM] so insolnble tliat they can- not again be dissolved in any fluid williont lii-st l)eing modified in some way; whereas colloids of the type of gelatin and agar are made- COLLOIDS 41 more soluble by heat. The change of colloids into insoluble forms, the 'Specious'' condition of (irahani, re(|uir('.s the presence of water, for the dry colloids ni;i\ lie iieated to relatively hiprh temperatures witliout losino- their s()lul)ilit y. On the other hand, deliydration of colloids while in solution will result in their precipitation and coagu- lation, as occurs, in protein solutions when alcohol is added. If solutions of two oppositely chard. 20. Erfriinzunpslipft. 44 nil-: vhemistry a\j> I'livsics of the cell THE NUCLEUS 2* Although the luieleus i)reseiits morphologically a sharp isolation from the cytoplasm, and displays eciually sharp tinctorial ditt'erences, it is probable that chemically the ditt'erences between nucleus and cy- toplasm are quantitative rather than qualitative. The characteristic affinit}' of certain elements of the nucleus for basic stains depends upon the presence in the nucleus of nucleoproteins in large proportion, and to a limited degree nucleoproteins are characteristic of nuclei. Their affinity for basic dyes depends upon the nucleic acid radical.-*'* For exami)le, the heads of spermatozoa contain nucleic acid bound to simple proteins in such a way that it readily forms a salt or salt-like combination with basic dyes, and so the sperm heads appear intensely stained by alum-hematoxylin, etc. Ordinary chromatin threads of nuclei appear to contain somewhat more firmly bound protein in their nucleoprotein molecules, and hence stain less intensely than do the spermatozoa heads, except when in karyokinesis, when the chromatin nucleoprotein seems to approach that of the spermatozoa in avidity for basic dyes. We also have nucleoproteins with the nucleic acid so thor- oughly saturated by protein that they do not stain at all by basic dyes, and these seem to exist principally in the cytoplasm, and also to form the ground-substance of the nuclei, occupying the spaces between the chromatin particles (this achromatic substance of the nuclei is called linin or plastin by some cytologists). Besides the chromatin and the nucleoli, there is a peculiar chromatophile substance, suspended in the liner part of the nuclear structure in the same manner as the chromatin itself is in the coarser portions ; this was called lanthanin by Heiden- hain,-^ and is probably similar to the substances also described as para- ehromatin and paraUnin. Undoubtedly the other forais of proteins found in the cell, such as globulin, albumin, and nucleoalbumin, exist both in the nucleoplasm and in the cytoplasm, the essential difference being that the proportion of nucleoprotein is much greater in the nu- cleus. As nucleoproteins are little affected by peptic digestion, it is possible to isolate nuclear elements, especially the chromatin, for analy- tic purposes, and it has been demonstrated by this means also that nuclein is the chief constituent of the staining elements. The distribu- tion in the nucleus, of the other primary constituents of the cyto- plasm, such as lecithin, cholesterol, and inorganic salts has not yet been worked out, except that IMacallum -*' has found that nuclei contain no chloi-ide, as indicated by their not staining with silver nitrate, and 2^ Karlicr litcraturo by Alhroclit, "Pathologio der Zcllc." Luliarscli-Ostortafr. Erjrcl). view that they are mi(deic acid com pounds. ■■^■- Fcstsdir. f. K.illikcr. 1802, p. 128. -'•i I'rocecdinL's of Ihc K'oval Societv. IHO.-) (Tfi). 217. COMl'OSHT/OX OF MCLEI 45 •ioi()>'V," ]!)05. THE CELL WALL 49 on tlic influt'iice of different ions on emnlsion formation and mem- brane l)ermeabilit3^•'''" The influence of inorganic salts on the swell- ing of tissue colloids is also tliscussed from another standpoint by M. H. Fischer in his work on ''Edema." In any event it is important ior the cell that the proportion of the inorganic constituents be main- tained in rather constant conditions of quality and ([uantity. The various secretory granules, fat-droplets, pigment-granules, gh'cogen granules, keratin, etc., that may lie in the cytopla.sm, are inconstant constituents, varying with different cells, and under varj^- iug conditions in the same cells, and lie beyond the scope of our dis- cussion of the general coin])osition of the cell. According to Ruzicka ^"^ there is contained in all cells, both in nucleus and cytoplasm, an in- soluble substance which corresponds structurally to the "plastin" of the cytologists, and chemically is related to the reticulins and other albuminoids; this he looks upon as the ground substance of the cells, corresponding to the albuminoid ground substance of stroma of or- ganized tissues. Certain of the granulations observed in the cj'toplasm of cells seem to be definite, constant structures of the living protoplasm, and these are now called mitochondria, which term includes many forms of granules described under various names.^"" Their solubility and staining reactions suggest that they contain phospholipins, perhaps associated with proteins. Their functional importance is indicated by the fact that usually their number varies directly with the meta- bolic activity of the cells, and they may be related to histogenesis. Other histological cellular structures also permit of more or less satisfactory identification by microchemical methods, and Unna^^*" especially has contributed to this field. By staining sections with dyes of varying reaction, after extracting the sections with various solvents, he has obtained evidence of the chemical nature of some of the cell structures, although it is by no means certain that the con- clusions drawn will all be verified. In the nucleolus he finds a sub- stance resembling globulin, the granuloplasm of the cell body he re- gards as an albumose, the spongioplasm as histone, mast cell granules as mucin or mucoid substances. Nissl bodies he holds to be albumose, altho others have believed them to be nucleins.^^'^ THE CELL-WALL 37 The cell membrane in most animal cells is inconspicuous struc- turally, but in discussing osmosis it was sho^^^l that it is of the greatest 35a Jour. Physical Chem., 1916 (20), 407. scArch. f. Zellforsch., 1008 (1), 587. 36a See review bv Cowdrv. Amer. Jour. Anat., 1916 (19), 42.3. sfibSee review bv Gans, "Dent. med. Wooh., 191.3 (39), 1944. 36c See Unna, Berl. klin. Woeh.. 1914 (51), 444: :Miihliiiann. Arch. mikr. Anat.. 1914 (85). 361. 3" See Zangger, '"Ueber ^lembranen und IMcjnljrancnfunktionen," Krgcbiiisse d. 4 50 THE CHEMISTRY AND PHYSICS OF THE CELL biological importance. There is no direct chemical or microscopical evidence at hand showing the composition of the animal cell mem- brane, but b}^ observations on its behavior when the cells are in solu- tions of different sorts, facts have been collected indicating that lecitliin and cholesterol, and probably the allied fat-like bodies, "protagon" and cerebrin, are prominent constituents. The sub- stances that diffuse through most cell walls are just the substances that are soluble in or dissolve these lipoids, e. g., alcohol, chloroform, ether^ etc., and it is prol)able that the anesthetic effects of many of these substances depend in some w'ay on their fat-dissolving power and the large proportion of lipoids in nerve-cells. These observations were made first by Overton ^^ and Meyer,^^ and led to the now prominent but disputed hypothesis that the permeability of cells is determined by the lipoids. Of particular interest for our purpose are Over- ton's observations on the effects of dyes on living cells. The best known vital stains {i. e., stains that will enter the living cell without requiring or causing injury to it) are neutral red, methylene blue,, toluidin blue, thionin, and safranin. If uninjured cells, e. g., frog eggs, are placed in watery solutions of these dyes they soon become filled with the coloring-matter, which seems to penetrate the cell mem- brane quite uniformly at all points ; if the dyed eggs are then placed in clear water, the stain diffuses out again, show'ing it to be simply absorbed, rather than chemically combined. In contrast to these stains the sulphonic acid dyes, such as indigo carmine and water- soluble indulin, nigrosin, and anilin blue, do not penetrate the living cell at all. Overton tested the solubility of dyes which are not vital stains and found them all insoluble in oils, fats, and fatty acids ; but the dyes staining living cells were readily soluble in lecithin, choles- terol, "protagon," and cerebrin, the so-called cell lipoids. Further- more, if crumbs of lecithin, "protagon," or cerebrin were placed in very dilute watery solutions of these dyes, they were found to absorb from the water the vital stains, but not the others, whieli indicates- that stains that penetrate living cells are more soluble in lipoids than they are in water. Many exceptions to this rule of the fat solubility of dyes which can penetrate living cells have been found, especially by Ruhland,^" and the universal applicability of the Overton-IMeyer hypothesis has been questioned. It is at once evident that the common foodstuffs which enter the cell, such as water, sugar, amino-acids, and salts are not li})()id-soluble, hence it has been suggested that the cell membranes must have a "mosaic" striiclui-e, some of the blocks being lipoids or Physiol.. 1008 (7), 99; also T{. S. l.illic. "TIh' TJule of Moiiibraiios in Coll Proc- esses," Popular Science Montlily, Feb., I'M:!. 38.Tahrb. f. wissontsohafll. I'joianik, 1!H)0 (34), 609. 30 .Arch. f. exp. Patli. u. PliarTii., 1S99 (42), 109. 4n.Talirb. f. Wis.seiischaft. Botanik, 1912 (f)!), .-{TO. THE CELL WALL 51 lipoid conipoiinds, and others proteins without lipoids, (Robertson *^ suggests that there is a supei-fieial film of concentrated protein about the cells, underlaid by a discontinuous lipoid layer.) There is, fur- thermore, evidence that the entire cell substance has a profound effect upon diffusion within the cell, so that it is at present impossible to say whether the osmotic phenomena of cells depend upon a cell mem- brane or upon the entire cell substance. ^^-^ It may be that there are membranes or surfaces within the cell, as postulated in the foam structure hypothesis of protoplasm, or that a homogeneous protoplasm develops surfaces where in contact with substances entering from the outside. Many facts indicate that either the delicate external membrane of animal cells or the entire cytoplasm has the features of a semi- permeable membrane, to the extent of permitting certain substances to diffuse through and not others. Had they the property of some of the artificial semipermeable membranes, of letting water pass through but holding back almost absolutely all crystalloids, the re- sult would be the development of an enormous disproportion in the pressure between the inside and the outside of the cell. Furthermore, the exchange of nutritive material and excretion products between the blood and the cells would be impossible. But permitting some sub- stances to pass into the cell results in their accumulation within the cell, until they are in sufficient concentration to neutralize the osmotic pressure exerted on the outside of the cell. As evidence of this elec- tive permeability we have the fact that the proportion of certain salts within the cell is quite different from what it is in the fluids bathing them; e. g., animal cells generally contain more potassium and less sodium than the fluids surrounding them. The inorganic constituents of red cells are different from those of the plasma, the corpuscles not containing any calcium at all, while the magnesium seems to enter them freely; in other words, the red coi'puscle seems to be impermeable to calcium and permeable to magnesium. If the salts in a corpuscle are in smaller proportion than in the surrounding fluid, it indicates that the cell membrane is not freely penneable for them ; if in greater proportion, that some constituent of the cell is holding them in combination, possibly as ion-protein compounds. Probably inorganic salts are present in the cell by virtue of both physical and chemical influences, some simply diffusing in and out, others com- bining with the proteins and being held chemically. The intercellular substance varies greatly in different tissues. In the case of the supportive tissues it is the important element, and the cells seem to exist chiefly for the purpose of forming and keeping it in repair as it is worn out. In the epithelial and secreting tissues, however, the intercellular substance is reduced to a minimum, except "Jour. Biol. Chem., 1908 (4), 1. 4ia See Kite, Amer. Jour. Pliysiol., 1915 (37), 282. 52 THE CHEMISTRY A.AD PHYSICS OF THE CELL in SO far as a cement substance is required, and the cells generally lie in almost immediate apposition. It is probable that there is a greater or less amount of cement substance, even between the most closely applied cells, and this substance seems to be related to mucin. It (;an generally be brought out by staining with silver nitrate, and ]\racallum ^" points out that this reaction is merely a micro-chemical test for chlorides, and indicates that the cement substance contains them in larger proportion than does the cytoplasm. 42 Proceedings of the Royal Society, 1905 (76), 217. CHAPTER II ENZYMES Every cell is eoiistaiitly aeeoinplishin<>: an enormous luiinber of chemical reactions of varied natures, at one and the same time ; how many we do not know, but the score or more that we do know to be constantly going on in the liver cell, for example, are probably but a part of the whole. Furthermore, reactions take place between sub- stances that show no inclination to affect each other outside the body, and proceed in directions that we find it difficult to make them take in the laboratory. Proteins are being continually broken down into urea, carbon dioxide, and water ; yet to split proteins even as far as the amino-acid stage requires prolonged action of concentrated acids or alkalies, or super-heated steam under great pressure. But all the time in the cell a multitude of equally difficult changes is going on at once, within its tiny mass, always keeping the resulting heat within a frac- tion of a degree of constant, and the resulting products within narrow limits of concentration. We have already indicated the means used to keep the concentration of the cell products withm safe limits; namely, the processes of diffusion and osmosis and their modification by the cell structure. The forces that bring about the chemical reac- tions reside, we say, in enzymes, although in so doing we only shift the attribute formerly conceded to the cell, to certain constituents of the cell whose nature and manner of action are equally unkno\\ai. AVlien the only enzymes that were known were limited to those se- creted from the cell, and found free in fluids, such as pepsin and tryp- sin, the chemical changes that went on in the cell were ascribed to its "vital activity." Buchner, by devising a method to crush j^east cells, and finding the expressed cell contents able to produce the same changes in carbohydrates that the cells themselves did, ]n'oved the ex- istence within living cells of enzymes similar to those excreted by cer- tain cells, and substantiated the belief of their existence that had become general before it was thus finally corroborated. Growing out from this and subsequent experiments has come a larger and larger amount of evidence that many of the chemical activities of the cells are due to the enzymes they contain, until now the point is reached where one may rightfully ask if cell life is not entirely a matter of enzyme activity. There are certain facts, however, which seem to in- dicate that there are some essential differences between cells and enzymes. One of the most importaiit of these is the difference in the 5.3 54 ENZYMES susceptibility to poisons of enzymes and cells.^ Strengths of certain antiseptics that will either destroj^ or inhibit the action of living cells, such as alcohol, ether, salicylic acid, thymol, chloroform, toluene and sodium fluoride, will harm free enzymes in solution little or not at all. This fact has been of great assistance in distinguishing between the action of enzymes and of possible contaminating bacteria in experi- mental work. Although this difference between enzymes and cells is characteristic, it does not finally decide that tlie cell actions are not enzyme actions, for it may well be that the poisons act chiefly by altering the physical conditions of the cell so that diffusion is inter- fered with, thus seriously interfering with the exchange of cleavage products between different parts of the cell, and cheeking intracellu- lar enzyme action, which we shall see later requires free diffusion of the products for its continuance. At the very least, however, we may look upon the intracellular enzymes as the most important known agents of cell metabolism, and consequently of all life manifestations, and the changes they undergo or produce in pathological conditions must be fully as fundamentally important as is their relation to physiological processes. It therefore becomes necessary for us to consider carefully — THE NATURE OF ENZYMES AND THEIR ACTIONS - Since up to the present time no ferment has been isolated in an absolutely pure condition we are entirely unfamiliar with their chemi- cal characters, and consequently are obliged to recognize them solely by their action. As far as we know, true enzymes never occur except as the result of cell life — they are produced within the cell, and in- creased in aniount by each new cell that is formed, and, furthermore, they are present in every living cell without exception. As the same facts are equally true of the proteins, and apjiarently true of nothing else, it is natural to associate the enzymes with proteins, and so ex- plain the importance of the proteins for cell life.^ If enzymes are obtained in any of the usual ways from animal cells or secretions they are always found to give the reactions for proteins, even if re- purified many times. But it is well known that whenever proteins are precipitated the other substances in the solution tend to bo dragged 1 See discussion by Vernon, Erayliss, "The Nature of l^'n/.ynie AcUon." I\l(Uioij;ra|)lis on Bio- chemistry, London: Stern, "Pliysico-cliemical Pasis of I'erinent Action," in Oppen- heimer's "Ilandbuch d. P.iochemie," Vol. 4, pt. 2: Samuelcy, "Animal Ferments." ibid. Vol. I; A. E. Taylor, "On Fermentation," T^niv. of California Publications; Euler, "General Chemistrv of tlie Enzvmes," translated bv T. IT. Pope, New York, 1912. 3 Another impfirtaiit point is that the closest imitation of cn/yin(>s, Bredig's "inorganic ferments," seem to owe their action to th(>ir colloidal nature. PROPERTIES OF EX/A.UES 55 down by the colloids, and it is possible that the enzymes are merely associated with the proteins in this way. Furthermore, enzymes are known to become so closely attached to stringy protein masses, such as fibrin and silk, that they cannot be removed by washing. Some have claimed that they have secured active preparations of pepsin and invertase that did not give protein reactions and contained very little or no ash or carbohydrate ; but it has so far been impossible to secure trypsin free from protein, and diastase seems to be certainly of protein nature. Analyses of enzymes purified as completely as pos- sible do not have great worth, for the ''purified" enzymes are prob- ably far from pure; however, it is of some importance that they vary greatly in the proportions of carbon, hydrogen, and nitrogen which they contain, indicating that possibly different enzymes may be of very different nature. The enzymes have been found to possess defi- nite electrical charges, in neutral solutions trypsin is negative or amphoteric, pepsin and invertase negative (INIichaelis).* jNIacallum has shown microchemieally that phosphorus is closely associated with the formation of zymogen granules in cells, which seem to be started in the nucleus; and there are many other observations suggesting that certain ferments are closely related to the nucleo-proteins. This is particularly true of the oxidases, which seem also to contain iron and manganese. A final point of importance in support of the pro- tein nature of enzymes is that pepsin destroys tr\'psin and diastase, while trypsin destroys pepsin. So uncertain, however, is our information concerning the chemical nature of the enzymes, that it has become possible for an hypothesis to be developed urging that enzymes are immaterial, that the actions Ave consider as characterizing enzj^mes are the result of physical forces which may reside in many substances, and perhaps even free from visible matter, but the weight of evidence at present available is en- lirel}' in favor of the view that enzjones are colloidal substances, al- though perhaps of widely differing chemical nature. A valuable piece of evidence of the material existence of enzymes is their specific na- ture, lipase affecting only fats, and trypsin only proteins, indicating chemical individuality. They are true secretions, formed A\ithin the cell by recognizable steps; and, furthemiore, when injected into the body of an animal, they give rise to the formation of specific innnune bodies that antagonize their action. Emil Fischer's work with the sugar-splitting enzymes, moreover, indicates that they owe their action to their stereochemical configiiration. He prepared two .sets of sugar derivatives which differed from each other solelj' in the arrangement of their atoms in space (i. e., isomers) and found that one specific enzyme w^ould split members of only one of the varieties, while nn- other enzyme would act only on the variety with the opposite isomeric form. These experiments make it very probable that there must be a ^Biochem. Zeit., 1909 (16), 81 and 486; (17), 231. 56 EXZYMEfi certain relation of jreometrieal stnicture between an enzyme and the substances it acts upon, and leaves little question of its material na- ture. Bredif? has found that colloidal sohifio)is of metals have many of the properties of true enzymes, aeeomi)lishino- many of the decom- positions produced by enzymes, being affected by temperatures of nearly the same degree, and even being ''poisoned" by substances that destroy or check enzymes. The only possible explanation of these observations seems to ])e that the enzyme effects are brought about by surface phenomena. A colloidal solution of platinum, so far as is known, differs from a piece of metallic platinum solely in the enor- mously great amount of surface it offers in proportion to its weiglit, and it is well known that surfaces may affect chemical action. Hence we have the possibility that some enzyme actions, at least, may depend upon the existence of a very large surface, and since by no means all colloids are enzymes, that this surface must bear a certain relation in form to the surface of the body that is to be acted upon. THE PRINCIPLES OF ENZYME ACTION The effects produced by enzymes, which at one time were con- sidered ciuite unique and remarkable, have now been made compara- tively plain, chiefly through the observations of Ostwald on related chemical reactions ; and by the investigations of Croft Hill, Kastle and Loevenhart, and others, on enzymes, which show that enzyme action is in no way different from chemical action observed independ- ent of enzymes. The fundamental consideration is that chemical re- actions are reversible, that is, that their tendency is to establish an equilibrium, and that the change may be from either side of the equa- tion.^ The action of enzymes is similar to that of all catalytic agents, that is, they increase the speed of reaction. In the case of such a reaction as that of NaOH and HCl, the reaction is so ra])id that the effect of catalyzers could hardly be noticed ; but with many other substances the reaction is very slow, and without the ])resence of catalyzers it would go on almost or quite imperceptibly. For ex- ample, ethyl butyrate saponifies on the addition of water according to the following equation : CJI— 0— OC— CJI: -f- R,O^CJI,OH + IIOOC— C,H;. On the other hand, if ethyl alcohol aiid butyric acid, the products of this reaction, are placed together, they will combine to form ethyl butyrate; in otlier words, the reaction is rexci-sible, as indicated by the arrows in the e(|uation. In any event, however, the reaction is not complete, but continues only until a certain deHnite pro})ortion of 5 See Taylor, Arcli. Jut. ^\vd., IJIOS (2), 14S THE Ph'i\rfpLi:s OF Kx/.VMi: AcTioy 57 ethyl alcohol, butyric acid, etliyl l)utyrati', and water exists. -wIk'h the chaiijro will stop, i. e., eqiiUibriKin is cstubiishcd. The time that would be required for this reaction to occur at room temperature would be extremely long, the change being hardly noticeable, but in the presence of a catalytic agent the reaction goes on much more rapidly. Catalytic agents, therefore, merely hasten reactions which would go ou without them, and they do not initiate or change the na- ture of chemical reactions at all. AVhen equilibrium is established, the reaction stops and the en/ymc has notliing more to do. Furthermore, and this is a recently a])preciated fact, enzymes will hasten synthesis just as well as they hasten catalysis. Croft Hill first showed that maltase would synthesize glucose into maltose ; Kastle and Loevenhart soon after established the synthesis of ethyl butyrate under the in- fluence of lipase. Taylor '^ first synthesized one of the normal body fats, triolein, by the action of lipase (from the ca.stor-oil bean) iipon oleic acid and glycerol. Successful synthesis of fats by pan- creatic lipase is described by Lombroso.'^ It may seem improbable at first sight that the synthesis of proteins can be accomplished by enzymes, as is the relatively very simple synthesis of carbohydrates and fats, but the improbability disappears when we recall that the jjroducts of protein cleavage are reconverted into body proteins after absorption from the intestines. Proteins manifestly are synthesized and we have not a little reason to believe that this is accomplished by enzymes, presumably by a reversal of their action in the establishment of equilibrium. Taylor ^ was able to synthesize protamin, one of the simplest proteins, by the action of trypsin upon its cleavage products, and it has been found that the addition of proteolytic enzymes to solu- tions of pure albumose leads to the formation of a jelly-like, insoluble protein substance, ' ' plastein, ' ' which seems to be the effect of a reversed action on the part of the enzymes." Another well known synthetic ac- tion that seems to be due to reversible ferment action is the formation of hippuric acid from benzoic acid and glycocoU in the kidney; the formation of glucose into glycogen and its reformation are also prob- ably both accomplished by one and the same enzyme acting reversibly. Other reversible reactions less closely related to animal cells liave also been described. The reversible nature of enzyme action explains many problems of metabolism, and makes the whole field much clearer. The following consideration of the newer understanding of fat metabolism on this 6 Univ. of California Publications (Pathology), 1004 (1), 33. 7 Arch, di farmacol., 1012 (14), 420. 8. Tour. Biol. Cliem.. 1000 (r,), 381. 0 See Micheli, Arch. ital. biol., 1000 (46). 185: Lcvcnc aiul Van Slykc. r.iocliom. Zeit, 1008 (13), 4.58; Tavlor, .Tour. Biol. Cheni.. 1000 (5), .300; Gay and Robert- son, ibid. 1912 (12), 233; Abderhalden. Fermentforsch., 1014 (1), 47; v. Knaffl- Lenz and Pick, Arch. exp. Path., 1013 (71), 206, 407. 58 ENZYMES beisis may explain the manner in which chemical changes are believed to occur in the cells and fluids of the body : " In the intestines fat is split by lipase into a mixture of fat, fatty acid, and glj'cerol; but as the fatty acid and glycerol are diffusible, while the fat is not, they are separated from the fat by absorption into the wall of the intestine. Hence an equilibrium is not reached in the intestine, so the splitting continues until practically all tiie fat has been decomposed and the products absorbed. When this mixture of fatty acid and glycerol first enters the epithelial cells lining the intestines there is no equilibrium, for there is no fat absorbed with them as such. Therefore the lipase, which Kastle and Loevenhart showed was present in these cells, sets about to establish equilibrium bj' combining them. As a result we have in the cell a mixture of fat, fatty acid, and glycerol, which will attain equilibrium only when new additions of the two last substances cease to enter the cell. Now another factor also appears, for on the other side of the cell is the tissue fluid, containing relatively little fatty acid and. glycerol. Into this the diffusible contents of the cell v.'ill tend to pass to establish an osmotic e(iuilibrium, which is quite independent of the chemical equilibrium. This abstraction of part of the cell contents tends to again overthrow chemical equilibrium, there now being an excess of fat in the cell. Of course, the lipase will, under this condition, reverse its action and split the fat it has just built into fatty acid and glycerol. It is evident that these processes are all going on together, and that, as the composi- tion of the contents of the intestines and of tlie blood-vessels varies, the direction of the enzyme action will also vary. In the blood-serum, and also in the lym- phatic fluid, there is more lipase, which will unite part of the fatty acid and glycerol, and by removing them from the fluid abotit the cells favor osmotic diffu- sion from the intestinal epithelitmi, thus facilitating absorption. Quite similar must be the process tliat takes place in the tissue cells through- out the body. In the blood-serum batliing the cells is a mixture of fat and its constituents, probably nearly in equilibrium, since lipase accompanies them. If the diffusible substances enter a cell containing lipase, e. g., a liver cell, the process of building and splitting will be quite the same as in the intestinal epitlielium. The only difference is that here the fatty acid may be removed from the cell by being utilized by oxidation or some other chemical transformation.! i To summarize, it may be stated that throughout the body there is constantly taking- place both splitting and building of fat. Fat enters the cells, leaves them, and is utilized only in the form of its acid and alcohol, never as the fat itself. Fat constitutes a resting stage in its own metabolism. If proteolytic enzymes also act reversibly, then the phenomena of protein metabolism are similarly explained, for there is no doubt that every cell and body fluid contains proteolytic enzymes. All metabolism, then, may be considered as a continuous attempt at establishment of equilibrium bij enzymes, perpetuated by prevention of attainment of actual equilibrium through destruction of some of the participating substances by oxidation or other chemical processes, 10 See Loevenhart, Amer. Jour, of Physiol., 1002 (G), 331; Wells, Journal Amer. Med. Assoc, 1902 (38), 220. The discrepancies between tiie action of lipase in the tissties and in vitro are well explained liy Taylor, Jour. Biol. Chem., 1906 (2), 103. 11 Bradley (.Tour. Biol. Chem., 1910 (8), 2r)l; 1913 (13). 407-439) calls atten- tion to tlic great conc(>ntration necessary for fat synthesis by lipase in vitro, and tlie lack of correspondence between Die amount of fat and of lipase in various tissues, questioning tlie importance of lipase for fat synthesis in the living tissues as well as the significance of reversed enzyme reaction for biological processes in general. GENERM. i'if<)i'i:irrii:s or i:\/.y\ii:h 59 or by removal from the cell or entrance into it of materials which over- halance one side of the equation. In just Avhat manner the enzymes accomplish tlicir catalytic effect is yet unknown.^- A favorite idea is that they form loose compounds Avith the substance to be split and with water ; the resulting compound being unstable and breaking down, the water remains attached to the components of the substance. Enzymes do not act eatalytically on all substances by any means, but show a decidedly specific nature. The}' affect only organic sub- stances, and the actions are limited to two processes — hydrolysis and oxidation, or the reverse processes of dehydration and reduction.^^ The most essential difference between the enzymes and the chemicals that can accomplish hydrolysis or oxidation is this : the ordinary -chemical reagents produce their effects on many sorts of substances, whereas the enzymes are specific ; thus hydrochloric acid will hydrolyze starch or protein with equal facility, but pepsin will not affect starch at all. The very specific nature of the enzymes, their activation by other l^ody products, the fact that they seem to be bound to the substance upon which they act, that they are susceptible to heat, and that they produce immune bodies when injected into experimental animals, all suggest the probability of a relationship hettveen enzymes and toxins. This matter will be discussed more fully in considering the chemistry of immunity against enzymes. General Properties of Enzymes. — Other properties of enzymes may be briefly mentioned. The speed of reaction they produce in- creases with the amount of enzymes present, but not in direct propor- tion (except with rennin). Very dilute acids favor the action of nearly all fennents, and alkalies are unfavorable for all but trypsin, ptyalin, and a few others. Weak salt solutions also are more favor- able than distilled water. (These facts suggest strongly the possi- l)ility that ions play an important role in the process.) Water and dilute glycerol dissolve enzymes, which form always colloidal solutions that are very slightly dialyzable ; and they may be precipitated from solution by alcohol, and redissolved again with but slight impairment of strength. Filtration through porcelain filters is not complete, from 10 to 25 per cent, of most enzymes being lost in each filtration and enzj'mes are subject to great absorption by surfaces, e. g., charcoal, kaolin." As before mentioned, many chemicals poisonous to bacteria have little influence on most enzymes, but nearly all substances when concentrated are injurious or destructive, and some enzymes are 12 See Euler, "Chemical Dynamics of Enzyme Reactions." Ergebnisse d. Pliysiol., 1910 (9), 241. * " . _ 13 Alcoholic fermentation may be an exception, the change being C„II,;Oc — 2CoH,, -f 2C0.,, but it is very possibly an intramolecular oxidation. 14 See Hedin, Ergebnisse d. Phvsiol'., 1910 (9), 433. 60 ENZYMES known that are more susceptible to antiseptics than are the cells that contain tliein. Formaldehyde is very destructive to enzymes, even when dilute. The efifect of protein-coag-ulatiu^ antiseptics upon en- zymes is, of course, greatly modified by tlie amount of protein sub- stances mingled with the enzymes; and the effects of heat and other injurious influences are greatly decreased by the presence of proteins and other impurities. All enzymes are most active between 35° and 45° C, and it is inter- esting to note that Kobert found this equally true for enzymes derived from cold-blooded.animals. Although enzymes can stand temperatures of 100°' C. or more when dry, in water they are generally destroyed somewhat below 70° C. Low temperature, even — 190° C, (liquid air), does not destroy them. The loss of power through heating occurs gradually, and there is no sharp line at which their action disappears. Sunlight is harmful to enzymes in solution, but only in the presence of oxygen ; this effect is augmented by the presence of fluorescent substances. Nascent oxygen is destructive to enzymes.^** Radium and a:;-rays seem to have a deleterious effect upon most en- zymes, and retard their rate of action; but apparently, autolytic en- zymes (Neuberg^") and tyrosinase (Willcock^") are not injured by these agencies.^*"* Ultra violet rays are also injurious to enzymes,^^ and they can be destroyed by violent shaking (Shaklee and INIeltzer ^^). Labile as enzymes are, their persistence when diy is remarkable ; Kobert found active trypsin in the bodies of spiders that had been in the Nuremberg ]\Iuseum for 150 years, and Sehrt ^^ found that the muscle tissue of mummies contained active glycolytic ferment. All enzymes as ordinarily prepared have the property of decom- posing hydrogen peroxide, a property possessed by substances of varied nature ; this effect is prevented by CNIT, wOiich does not pre- vent other enzyme manifestations, indicating that this property is due to an associated enzyme, catalase. The retardation of enzyme action by aecuinuhitioii oi" the products of their action is simply explained as being due to establislnnent of o((uilibrium ; in some instancies, however, tlie substances jiroduced are of themselves harmful to tlie enzymes, c. r/., alcohol and acetic acid. Activation of Enzymes. — AVithin the cell, the enzymes — at least those 1hat are excreted, sncli as trypsin and pejisin — exist with few excep- tions ill ;iii active form, the z]iu\()()cu. Tlieir activation a|)pears to take i)lace normally only after they have been discharged from the 34a See Buifje. Anicr. Jour. I'livsiol, 1014 (34). 140. i--Ecr]. klin. Wocli., in()4 (41), lOSl. in Jour, of Pliysiol., lOOG (.34), 207. i'!ar;ii(lz('ii1 (Zcil. Siriililoiitlicr.. 1914 (4). (KKI) denies lliiil rndium nets on ciizyiiies. 1" Ajriillidii. Ann. Inst, rusteur, lill-J (1^(1). .'iS ; Hiir^'e i / <(/., Anier. .lour. PliVBiol.. l!)lt) (40), 42(i. i"s Ainer. .T analogy of zymogen-kinase combinations to coniplement-amboceptor eomliination. Walker, however, finds evidence that many enzymes consist of a specitic ambo- ceptor and a non-specific complement or kinase (Jour, of Physiol.. IKiKl (.3.3), p. xxi.). 2iVircho\v's Archiv, 1800 (121), 1. 62 ENZYMES bacteria is shown by Kionka and by Achalme -- who obtained similar results witli enzymes made sterile by filtration through porcelain. Achalme found that such sterile preparations of pancreatic juice in- jected subcutaneously into guinea-pigs produce a marked local pink gelatinous edema, followed by gangrene; if the animal dies, the blood is non-coagulable. Apparently cells of nearly all types can be de- stroyed b}^ trypsin, which may cause necrosis in one-fourth hour ; how- ever, spermatozoa and surface epithelium resist strong trypsin solu- tions. Intravenous injections cause death with lesions in the heart muscle and severe hemorrhages. After recover}^ from one injection of trypsin the animal is temporarily somewhat more resistant to another injection, and there are other resemblances to anaphylactic intoxication (Kirchheim -^). Fiquet -* also observed that trypsin and pepsin rendered the blood incoagulable, but after some time the coagulability of the blood is increased and thrombosis is frequent. Wells -^ found that pancreatic extracts containing veiy active trypsin and lipase, injected intraperitoneally, produced an acute inflammatory reaction, but no fat necrosis. Extracts containing active lipase and inactive trypsin were less toxic, but produced fat necrosis. Extracts of liver and blood serum, rich in lipase, were almost without effect on dogs and cats. Papain was found to be much more toxic than any animal enzyme, causing violent local hemorrhagic inflammation. Schepilewsky -^ also found papain much more toxic than rennin and pancreatin; repeated injection of the two latter caused amyloidosis in rabbits. Active immunity does not follow repeated injections of papain.-^ Lombroso -* found that inactive pancreatic juice was much less toxic than the activated, showing that it is the trypsin that is the important toxic agent. He also found that succus entericus in doses of 1 to 5 c.c. is toxic, but not lethal for dogs. Pancreatic lipase is hemolytic (Noguchi-^) if activated by fats, which suggests that when this enzyme gets into the blood it may cause hemolysis. Hildebrandt ^^ observed that enzymes Avere positively chemotactic, but it is probable that the products of their action on the tissues are the chief chemo- tactic agents. The enzymes that are secreted into the gastro-intestinal tract seem to be chiefly destroyed, but part is eliminated in the feces, and part that is absorbed apparentl}^ reappears in the urine in ver}^ small quantities.^^ Pepsin, diastase, and rennin all have been found in nor- 22 Ann. d. I'Inst. Pasteur, 1001 (15), 737. 23 Arch. exp. Patli. u. Pliarni., mil (GO), 352; 1914 (78), 99; 1913 (74), 374. 24 Arch. d. M^'d. Kxpor., 1899 (11), 145. 25 Jour. Med. Hesearcli, 1903 (9), 92. 26 Cent. f. Pakt.. 1899 (25), 849. 2TStenitzer, Piocliom. Zeit.. 1908 (9), 382. 2R Al)8tract in Piuchoni. Contralblatt, 1903 (1), 712. 20 Pioclicni. Zeit., 1907 ((i), 185. so Virchow's Arch., 1893 (131), 5. 31 Falk and Kolicb, Zeit. klin. 'Mod., 1909 (08), 15G. A\Ti-E\zy.][i:s 63 mal urine; but trypsin is present chiefly as trypsinogen, especially abundant after a meat diet.''- Pepsin and rennin enter the urine as the zymogens, in quantities in proportion to the amount in the stomach, and are absent in gastric carcinoma (Puld and Hirayama^^). During resolution of pneumonia, leucocytic protease may appear in the urine (Bittorf ^*). Ferments injected subcutaneously seem sel- dom to be eliminated in any considerable amounts in the urine, but Opie ^^ has demonstrated the presence of lipase in the urine in pan- creatitis with fat necrosis. Hildebrandt was able to prove that emulsin remained active for at least six houi's after it was injected into animals subcutaneous!}', by its splitting amygdalin which was then injected, the CNH liberated by the cleavage of the amygdalin causing death. ANTI-ENZYMES Injection of enzymes into animals leads to the appearance of sub- stances in the senim of the animals that antagonize the action of the enzymes.^^ The principles involved are quite the same as in the immunization of animals against bacterial toxins or against foreign proteins. This seems to have been first observed by Hildebrandt, and it has been taken up extensively in recent years in the stud}' of the problems of immunity. An interesting observation that was made rather early in these studies was that normal blood-serum possesses a marked resistance against the action of proteolytic enzymes, not being at all digested by dilutions of enzymes that will rapidly digest a serum that has been heated. This property seems to be shared by egg-white ^^'^ and by the tissues and organs of the body (Levene and Stookey^^). The anti-enzyme action is easily destroyed, by heat of about 70°, by the action of dilute acids, and even by prolonged stand- ing. It is exerted not only against the secreted ^proteolytic enzjones, pepsin and trypsin, but also against the intracellular enzymes of various organs. It seems highly probable that the resistajice of the body tissues to digestion by their own enzymes and by the enzymes of one another depends in some way upon the presence of anti-enzymes in the cells ;ind tissue fluids, for self -digestion of tissues is greatly impeded by serum.^^ Weiland^*' has demonstrated that certain intestinal worms 32 V. Schoenborn. Zeit. f. Biol.. 1010 (53), 386. 33Berl. klin. Woch., 1010 (47), 1062. 3* Dent. Arch. klin. :\led.. 1007 (91), 212. 30 Johns Hopkins Hosp. Bull., 1902 (13), 117. 37 According to Porter (Quart. .Joxir. Exper. Physiol., 1910 (3), 37o) enzymes in contact with various membranes are inactivated, and substances appear which are strongly inhibitivo to the enzymes: it is possible that this effect depends largely on zvmoids, which unite with the substrate and deviate the enzymes. 37aSugimoto. Arch. exp. Path.. 1013 (74), 14. 38 Jour. Medical Pesearch. 1003 (10), 217. 39 Wells, Jour. Med. Pesearch, lOOG (10). 149. •to Zeit. f. Biol.. 1003 (44), 45: see also Dastre and Stassano. Compt. Pend. Soc. Biol., 1003 (55), 130 and 254: and TTamill, Jour, of Physiol., 1906 (33), 470. 64 ENZYMES contain a strong- antitrypsin, to which lie attributes their ability to live bathed in pancreatic juice without being digested.*"'^ Similar properties have been ascribed by other observers to the cells of the mucosa of the stomach*^ and intestine, and to the mucus itself (de Klug)/- but the work of Bensley and Harvey ^^ indicates that the ab- sence of free acid in the gland cells and lumen is perhaps the chief protection of the stomach from pepsin. Kirchheim ** holds that the intestines are protected less by anti-enzymes than by rapid absorption and removal of the enzymes, which are really not present in any con- siderable excess in the intestinal contents. The anti-enzymes seem only to inhibit enzyme action, and not to destroy the enzyme itself.*"' Normal anti-enzj-mes do not seem to be at all specific, according to V. Eisler,*" that is, human serum is no more resistant to human tryp- sin than is pig serum — indeed, it is less so.*' Cathcart ** found that antitrypsin is connected ^\^th the ' ' albumin fraction" of the serum, i. e., the fraction precipitated between half and full saturation with ammonium sulphate. Globulins do not pos- sess this action, but they are not easily digested. Antitrypsin is found in all varieties of serum, and is little or not at all specific. It is destroyed by 65-70° C.*" for one-half hour, but retains its anti-enzy- matic activity after drying, and is equally effective against all sorts of proteins. The normal anti-tryptic activity decreases during fast- ing and increases during digestion (Rosenthal ^") ; it is increased during pregnancy ^"^ and the blood of the fetus shows less than that of the mother. Normal antitrj^psin unites with trypsin according to the law of multiple proportions (^leyer) and the reaction is not re- versible (Rondoni). It is found in the urine, and in inflammator}' exudates, but not in normal serous fluids, and it resists putrefaction. Normal serum does not seem to inhibit the enzymes which act upon purines. Fuld and Spiro '^^ found that the natural antirennin of 4oa Burge (Jour. Parasitol., 1915 (1), 179) suggests that the protection of parasites, and perhaps of the alimentary epithelium, depends on tlie active oxidative pro])erties of tliese tissues destroyinsr tlie en/.vmes. 41 See Blum and Fuld, Zeit. Iclin. Med., 'lOOG (fiS), 505; LangensldoUl. Skand. Arch. Physiol., 1914 (31), 1. •»2 Arch, internat. d. phvsiol., 1007 (5), 297. ■»3 Biological Bulletin, 1912 (2.3), 225. 44Arch. exp. Bath. u. Pharm., 1912 (71), 1 45 l^ayliss and Starling (Jour, of Physiol.. 1905 (.32). 129; and Meyer. Biochem. Zeit., 1909 (23), OS. o])pose the view of Dele/enne that the antitryptic action of the hlood is due to an antikinase. and believe the antibody acts upon trypsin. ■»« Ber. d. Wien. Akad., 1905 (104), 119. 4" This is contradicted by Claessncr; llofmeister's Beitriige, 1903 (4), 79. 48 .Tour, of Phvsiol., 1904 (31), 497; also see Kiimmercr and Aubrv . l?i(iclicni. Zeit., 1913 (48),' 247. 40 Unless otherwise specified, all tem])craturcs are given according to the Centi- grade scale. ->o Folia Serologica. 1910 (0), 2S5 ; also Fran/, and Jarisdi, Wien. klin. Woch.. 1912 f25). 1441. noaSee Franz, Arch. f. CJvn., 1914 (102). 579. 51 Zeit. f. phvsiol. Chem." 1900 (31). 132. A\Tf-K\Z)]n:,<< 65 normal horse serum is in the pseu(l()ines ajipear one hy one in the d(>v('lo])nHMit of the ovum. Tlieir activity is modified considerahly by infections (Sieher. Piocliem. Zeit.. 1911 (32). 108) and other diseases (Grossinaiin. (7)(V7.. 1912 (41). 181). 74 f"om})lete 1)il)iio" BattclH and Stern do not include aldeliydai^o anion<,' tin- oxidizinj; cn/ynips. on the ground tliat its action is not oxidative but hydrolytic. oxii)i7A\<; j:\zy ]n:s 73 abundant in the liver'''' and si)l(M'n, and is )»rcsriii in |)i'. .Jacoby luis ol)tained a body with the properties of aldehydase which diil not <^ive protein re- actions. It is a true enzyme, since it oxidizes aldeliydes without itself being used up. Its range of action is limited, for Jacoby found it without effect upon acetic acid and stearic acid. Tyrosinase. — This enzyme, which is found both in animal and plant tissues, is particularly interesting in relation to the formation of pigments. Bertrand found that the transformation of the juice of lac-yielding plants into the black lacquer was brought about by the action of an oxidizing ferment, laccase, upon an easily oxidized sub- stance, laccol, which is a member of the aromatic series. He later found, in a number of plants an enzyme acting on tyrosine, distinct from the laccase, which he named tyrosinase. Biederman later found tyrosinase in the intestinal fluid of meal worms, v. Fiirth and Schneider found a similar enzyme in the hemolymph of insects and arthropods, which explains its darkening when exposed to air. This enzyme, as obtained from different sources, is not always specific for tyrosine, frequently oxidizing other substances. As yet the chemical processes and end results of the oxidation of tyrosine by tyrosinase are unknown. Bach ^"^'^ obtained evidence that tyrosinase is not a specific oxidizing enzyme, but consists of an aminoacidase, which dis- integrates the tyrosine and makes it susceptible to the action of pheno- lase which is the oxidizing agent, v. Fiirth and Schneider found the product of oxidation of tyrosine by animal tyrosinase related to cer- tain of the melanins of animal tissues, and believe that tyrosinase is responsible for the production of many normal pigments. In the ink-sacs of the squid, which eject an inky fluid containing melanin- like pig-ment, tyrosinase was also found, corroborating this hypothesis, and it is probable that tyrosinase in the skins of aninmls is responsible for their pigmentation.*" Bacteria also contain tyrosinase,"" and this or similar enzymes seem to be present in melano-sarcomas."^ Gonnermann "- found that tyrosinase from beet-root produced homogentisic acid by acting on tyrosine, which is of interest in con- nection with the congenital hereditary disease, alkaptonuria {q. v.)y in which the urine becomes dark upon exposure because of the pres- ence of homogentisic acid. The action of tyrosinase upon the aromatic radicals of proteins is of great importance in the study of both physiological and pathological pigment formation, and hence has re- ssBattelli and Stern. Biocliem. Zeit., 1910 (29), 130. ssaBiochem. Zeit., 1914 (60). 221. 89 Meirovvsky, Cent. f. Path., 1909 (20), 301. 90 Lehmann "and Sano, Arch. f. Hyg., 1908 (67), 99. 91 Alsberg, Jour. Med. Res., 1907' (16). 117: Neuberfj:, Virchow's Arcliiv., 190S (192), 514; Gessard, Compt. Rend. Soc. Biol., 1902 (54), 1305. 92 Pfluger's Arch., 1900 (82), 289. 74 ENZYMES ceived extensive study, which will he found fully described in the monograph b}' Kastle {loc. cit.) '^* and under the appropriate subjects in subsequent chapters. Other Oxidizing Enzymes. — Of the great numlier of other less studied oxidizing enzj-mes little can be definitely stated. Some con- sider that they are largely different manifestations of the action of one oxidizing ferment, but against this view Jacoby mentions that they occur distributed unequally in different organs, can be separated from each other, and they cause different reactions. For the catalase and for laccase (which produces the Japanese lacquer by an oxidizing process) and perhaps for other oxidizing ferments, iron and man- ganese may be essential constituents. Of particular significance for pathology are the enzymes which accomplish the oxidation of purines to uric acid and the subsequent destruction of uric acid. These are discussed in Chapter xxi. Also the enzymatic oxidation and reduc- tion of ^-oxj^butyric acid and aceto-acetic acid in the liver, as studied by Dakin and Wakeman,"^ are of great importance in acidosis {q. v.). Reducing enzymes have not yet been satisfactorily demonstrated."* It is possil)le that they do not exist, and that the intracellular re- ductions that are carried on within the cells are brought about by simple chemical reactions independent of catalysis. The best known intracellular reduction is that of methylene blue, which can be readily studied experimentally because the blue color disappears on reduction of the dye. It is open to question if this particular reduction is due to a reducing enzyme. According to Ricketts ^^ the reduction depends upon two bodies, one thermostabile, the other thermolabile, recalling the reaction of complement and amboceptor. Strassner ^"^ found evi- dence that the SH groups of the tissues are responsible for the reduc- tion of methylene blue ; their activity is impaired by heating, but a thermostable element of tissues augments the reducing activity of SH compounds, thus corroborating and explaining the observations of llicketts. Harris,"^ however, believes that the evidence for the exist- ence of a true reducing enzyme is as good as for most other cellular enzymes. An enzyme has been found in the liver, muscle and kidney which transforms aceto-acetic acid into l-/3-oxybutyric acid, and called ketoroductase (Friedmann and Maase)."'^" Oxidizing Enzymes in Pathological Processes. — Although the oxidizing enzymes undoubtedly play an important part in pathologi- cal conditions, they have been but little investigated from this stand- point. Jacoby found that they did not disappear from the degen- erated liver in phosphorus poisoning or in diabetes, or when the liver 03 Jour. .Amor. IVfod. Assoc, 1910 (54), 14-11. o4Soo TIofTtPr. Arc-li. exp. Path. u. Pharm.. lOOS, Suj)])!.. p. 25.3. "•"•Jour of Infoft.ions Disoaaos, 1004 (1), 500. (•« Biocliem. Zcit., 1010 (20), 205. "7 Biofiioni. .loiir., 1010 (5), 14:?. 07a iJiocliom. Zeit., 1012 (27), 474; 101.3 (55), 4,58. (}\inr/.i\(! i:\'/.y\ii:H 75 undergoes self-digestion, which speaks against Sjjitzer's contention that oxidase is a nucleoprotein.'-''* JSelilesinger '-'''' found that it is less in amount in livers of children dead from gastro-iutestinal diseases than in normal livers, as also did Briining.^ I am inclined to believe that fatty metamorphosis, wlien brought about by poisons, is often due to inhibition of the oxidizing enzymes (v. fatty metamorphosis), although I found that livers the seat of the most profound fatty de- generation showed no evident impairment of their power to oxidize xanthine and uric acid.- Buxton^ failed to find in tumors any en- zyme giving the guaiac test alone, but found enzymes that did so in the presence of HgOg (peroxidases). Catalase was present, but no very positive reactions for oxidizing enzymes were obtained by the indo-phenol reaction, the hydrochiuon reaction, or with tyrosine for tyrosianase. v. Fiirth and Jerusalem * have found evidence tliat the melanin of melanotic tumors of horses is produced by tyrosinase. Peroxidase has been demonstrated in the granules of pus cells iFisclieP). Meyer ® found that leucocytes, whether from pus or leukemic or pneumonic blood, contained a substance oxidizing guaiac directly, without the presence of H^O,, which is not liberated until the cells are destroyed. By microchemical reactions oxidases have been found present in the myelocytes and nucleated erythrocj'tes in leukemia, be- ing absent from the polynuclear cells.' The observation of Natalie Sieber * that oxidases of the blood and of vegetable origin destroy diphtheria toxin rapidly, and also tetanus toxin and ricin, has been confirmed by Loewenstein as far as destruction by peroxide, with or without the presence of catalase, is concerned. Oxidation is un- doubtedly an important process in defending the bod}' against other forms of poisons, including the so-called ' ' fatigue toxins, ' ' and Battelli and Stern consider that all the oxidizing enzymes so far definitely identified are concerned only in protective processes. (See Chapter vii). Schmidt" has found that by oxidation certain poisonous mor- phin derivatives are rendered non-poisonous by liver extracts. Oxalic acid and poisonous fatty acids are also oxidized into harmless sub- stances; phosphorus and sulphur are oxidized into their acids, which 9s Duccheschi and Almagia (Arch. ital. Biol., 1903 (39), 29) also found tlie aldeliydase in livers of phosphorus poisoning usually no less abundant tlian in normal livers. 9'J Hofmeister's Beitr., 1903 (4), 87. iMonat. f. Kinderheilk., 1903 (2), 129. 2. Tour. Exper. Med., 1910 (12), 607. 3. Jour. Med. Research, 1903 (9), 356. 4 Hofmeister's Beitr., 1907 (10), 131. 5\Vien. klin. Woch., 1910 (23), 1557. sMiinch, med. Woch., 1903 (50), 1489. 7 Fiessinger and Roudowska, Arch, de med. exper., 1912 (24), 585. sZeit. physiol. Chem., 1901 (32), 573. 9 Dissertation, Heidelberg, 1901. 76 ENZYMES are then neutralized. Indole and skatole are oxidized into less harm- ful substances. The Indophenol Reaction. i" — Alplui-naplitliol and dimethyl-para-plicnylendia- min. wlieii lirouglit louctln'r in alkaline solntioii, become oxidized in the pres^ence of air and form an insoluble blue dye, indophenol. Tliis reaction is jireatiy accelerated by oxidizing' agents, and it has liien found that certain tissues pos- sess this property, hence the indophenol synthesis has been used for microchemical study of the presence and distribution of oxidizing enzymes in cells. As the in- tracellular agent which causes this reaction is, ho\.ever, so resistant to heat and chemicals that it can be demonstrated in sections fixed in formalin and prejiared by the ordinary paralbn imbedding method (Dunn), there is room for much doubt as to whetlier' it represents a true enzyme of the polyphenol oxidase class. It may be that it is identical with phenolase.n In tiie presence of small amounts of peroxide tiie granules of leucocytes and myelocytes are stained with alpha- naphthol alone, which Graham na interprets as oxidation by an enzyme of the peroxidase type. The indophenol reaction is observed best in the granules of neutroi>hile leucocytes of blood and in myeloid cells of bone marrow, leukemic blood and fetal organs; eosinophiles and basophile leucocytes also give reactions, but not lymphocytes, mature erythrocytes, or most fixt tissue cells. ( See Dunn, Quart. Jour. Med., 1913 (6), 293.) By using alkali-free, unfixt tissues Gierke found granules present in tissue ceils generally, and Griiff states that they occur in proportion to the metabolic activity of the cells; they are abundant in carcinomas, scanty in sarcoma and connective tissue growths generally, arc not destroyed in cloudy swelling or fatty changes, but disappear in infarcts and autolyzing tissues, and in tissues asphyxiated with illuminating gas.i- Lung tissue is especially poor in this form of oxidative activity,i2a but giant cells of tubercles contain oxidase granules. 12b During experimental pneumococcus sep- ticemia the indophenol oxidase reaction is decreased in the tissues. 12c Glycolytic Enzymes.^^ — The oxidation of sugar by the tissues, which is one of the chief sources of energy in the animal body, presum- ably takes place through several steps. Of these, it is believed by some that the first is the formation of glycuronic acid — CH,OH— (CHOH),C— H + 0, = COOH— ( CHOH ) ,C— H + H,0, (glucose) (glycuronic acid) but the subsequent changes which involve decomposition of the straight chain are not at present understood. Attempts to isolate from various organs an enzyme oxidizing glucose, particularly from the pancreas, muscle, and liver, have led to varying results and nnich dissension, but it is probable, because of these failures, that no such enzyme exists in quantities sufficient to account for the amount of 10 Literature given bv Schultze, Ziegler's Beitr., 1909 (45), 127: Dunn. Jour. Path, and Bact., 1910 ('l5), 20; Griiff, Frankfurter Zeit. f. Path., 1912.(12), 358. 11 Bach and Marvanovitsch, Biochem. Zeit.. 1912 (42), 417. 1 la Jour. Med. Ues., .1916 (35), 231. 12 See Klojifcr, Zeit. exp. Pharm., 1912 (11), 407. 12a Weiss, Wien. klin. Woch., 1912 (25), t)97. i2bMakino, Verh. Jai)an. Patli. Gesell., 1915 (5), 71. i2.Medigrcceanu. Jour. Exp. .Med., 1914 (19), ,303. 13 Also discussed under "Diabetes," chap. xxil. As glycolysis by blood and tissues can <)(- mono- and triaeetin, lecithin and cephalin.^^^ Little is known about the part played by lipase in pathological con- ditions. According to Achard and Clerc/** the amount of splitting of ethyl butyrate by the blood-serum is lessened in most diseases, and in- creases and decreases Avith the health of the patient; according to Pribram ^* and Sagal ^''^ it is increased in the blood during fevers. Clerc ^'•' found that acute arsenic, phosphorus and diphtheria-toxin poisoning increased this property of the serum, while chronic poison- ing and staphylococcus intoxication lowered it. Somewhat similar results were obtained by Grossmann,-" but Saxl found no increased activity in phosphorus poisoning. Using the ethyl butyrate test, AYintemitz and ]Meloy -^ found that the more nearly normal an organ is the more cleavage of the ester; lipolytic activity is low at birth, increases rapidly during the first few days of life, and does not de- crease in old age. There is a decline in activity of tissues in diabetes, tuberculosis, and the toxemia of pregnancy, in the livers of passive congestion and fatty degeneration, in the pneumonic lung and the cirrhotic liver. After taking food there is a slight increase in esterase, reaching a maximum in three hours. -'''^ Wliipple -'^°- finds the blood lipase (butyrase) increased whenever there is injury to the liver, such as in chloroform anesthesia and puerperal eclampsia ; it is lowered in cirrhosis. Poulain -- found that the butyric-splitting power of lym])h-g]ands draining infected areas Avas decreased. Fisclier ^^ observed, in a case of extreme lipemia in diabetes, that the lipolytic power of the blood was absent. The lipase of lipomas presents no demonstrable difference from that of ordinary fatty areolar tissues.-* Lipase has also been demonstrated in pus by a number of ob- servers,-' Avho agree that there is more in exudates than in transu- dates. Zeri -" found lipase in the urine only when pus or blood was also present, but Pribram and Loewy -'^ found it in nephritis, con- isoEnplisli and IVIacArtlnir (Jour. Aiiicr. Chom. Soc. 1915 (.37). Or).'?). who have also found in sheop l)rain, erepsin, amylase, catalase. enzymes deeomposinfj arluitin and salol, probably pepsin and trypsin, but not peroxidase, oxidase, reductase, jjuanase, urease or rennin. i« Compt. Rend. Soc. Biol.. 1002 (54), 1144. IS Cent. inn. Med., 1908 (29), 81. loCompt. Rend. Soc. Biol., 1901 (53), 1131. zoBiochem, Zeit., 1912 (41), 181. 21 Jour. Med. Res., 1910 (22), 107. 20a.Toblinfr et al.. Jour. Exp. Med., 1915 (22), 129. 2ia Whipple et al., Bull. Jolms Hopkins Hosp., 1913 (24), 207 and 357. 22 Com p. Rend. Soc. Biol., 1901 (53), 7Sfl. 23Virchow's Arch., 1903 (172), 218. 24 Wells, Arch. Int. ]\led., 1912 (10), 297. 2'-. Aclialme. C'ompt. Rend. Soc. Biol., 1899 (51), 5(iS -. Zeri. 11 Bolicliiiieo. 1903 (10), 43.-]; Meiiniii, Clin. ]\led. Ital., 1905 (44), 129. 2" 11 Policlinico. 1905 (12), 733. 27 Zeit. j)l)ysiol. f'liein., 1912 (70), 489. LIPASE 79 gestion, polyuria and other conditions. Lorenzini,-'''^ however, re- ports that in albuniinnria tlie lipase content of the urine is reduced, in common with other enzj-mes, there being a simultaneous accumula- tion of enzymes in the blood. Fiessinger and ]\Iarie -* contend that the lymphocytes of exudates are the chief source of lipase, and sug:gest that this may be of effect in defense against the fatty tubercle bacilli. Toxines were found by Pesci -^ to increase the butyrase but not the other lipases of liver tissue. In syphilis the lipolytic activity of the serum is increased,^" which may be related to Bergell's^^ observation on the origin of lipase in lymphocytes (corroborating Fiessinger and ^Marie). Jobling and Bull ^- state that a specific serum lipase increase occurs in animals immunized to red corpuscles, and that this lipase has to do with hemolysis ; but ^Mendel ^^ found no evidence that hemoh^sis by ricin is related to lipase. Abderhalden and Rona ^* found that excess feeding of fats leads to an increase in the lipase of the blood. The part played by lipase in fatty degeneration must be of great importance, but as yet it has been little considered, except that Loeven- hart. and Duccheschi and Almagia '"' found no appreciable difference in the lipase content of nonnal and phosphorus-poisoned livers, but in chloroform poisoning Quinan ^^'^ found a decrease in the butyrase of the liver, although it was increased in the kidneys and muscles. This question will be considered more fully in discussing fatty meta- morphosis. An improved method of testing for lipase action has been devised by Rona and Michaelis,^® by measuring the change in surface tension caused by hydrolysis of a soluble ester, usually tributyrin. Using this, Bauer found that every human serum contains fat-splitting enzymes, which are greatly decreased in carcinoma and advanced phthisis, some- what decreased in syphilis and exophthalmic goitre, and increased in early pulmonary tuberculosis. Caro ^'^^ found a decrease in all cases of cachexia, but there was no relation between the lipolytic enzyme and the blood picture. The blood contains no thermostable antilipase analogous to the antitrypsin. Red corpuscles are said to contain an enz\'me splitting cholesterol esters, " cholesterase." ^'^ 27a Policlinico, 1015 (22), 35S. 2sCompt. Rend. Soc. Biol., 1909 (67), 177. See also Eesch. Dent. Arch. klin. Med., 1915 (118), 179. 29 Pathologrica. 1912 (.3), 207. 30 Citron and Reieher, Berl. klin. Woch., 1908 (45), 1.39S. 31 Miinch. med. Woch., 1909 (56), 64. 32 .Jour. Exp. Med.. 1912 (16), 48.3. 33 Arch. Fisiol., 1909 (7). 168. 34Zeit. phvsiol. Chem.. 1911 (75). 30. 35 Arch. Ital. Biol., 1903 (39). 29. 35a .Jour. :\Ied. Res., 1915 (32), 73. 36 See Bauer, Wien. klin. Woch.. 1912 (25), 1376 (bibliography). 3Ga Zeit. klin. Med., 1913 (781,286. 37 See Cytronberg, Biochem. Zeit., 1912 (45), 281. 80 i:\'/.)MEH Fat necrosis, resulting from the escape of pancreatic juice into the peripanereatic tissues and abdominal cavity, undoubtedly is largely the result of lipase action. (See "Fat Necrosis," Chapter xiii, for complete consideration.) AMYLASE 3H Although undt'i* ordinary conditions starch is not supposed to enter the blood stream and tissues, yet all tissues and body fluids are capable of hydrolyzing starch. Apparently the amylase is derived from the pancreas and salivary glands, and i)ossibly from many or all other tissues (King), but it is not (piantitatively related to the amount of carbohydrate in the diet of a species or an individual (Carlson and Luckhardt). In llic bloiMJ it occurs in the albumin fraction.'"' There is disagreement in the literature as to the variations in amount of amylase in the blood during disease, and little information concerning its distribution in the tissues. Normally the kidneys and liver seem to be most active. During acute infections the blood amylase is in- creased, presumably coming from the leucocytes (King). It is greatly increased when the pancreas is acutely inflamed or injured (Stocks). Intravenous or subcutaneous injection of starch is said to increase the blood amylase, presumably as a defensive reaction (Abderhalden), but the amylase ordinarily in the blood seems to be a waste substance on its way to excretion, rather than a functionating enz^one of the blood. There appears to be no normal antiamylase in the blood. Because of possible diagnostic signiticance, the amylolytic activity of the urine has been particularly studied, and found normally to be approximately constant for 24 hour specimens of the same individual.*" Anything impairing the excretory capacity of the kidney decreases the urinary amylase, although sometimes when the urine contains blood, pus, or much all)umen there may be an increased amylase excretion in spite of diminished functional activity. There may be an increase in the amylase in the blood when the urinary amylase is decreased, but with normal kidneys increase of the blood amylase causes an increase in the urine; hence, acute pancreatic diseases cause an increased urinary amylase TStocks), but this is not constant (McClure and Pratt). In diabetic urine it is said to l)e usually decreased, but this is mostly accounted for by the dilution of the urine. Parenteral in- jection of stai-ch causes a marked increase in the amount of diastase in the urine (King) .'^^ 3« Litcraluro {.nvcn bv Kinff, .Amor. Jour. Phvsiol.. 1914 (S.'i), 301; Govelin, Arc-h. Int. Med., 1014 (13), 06; Storks. Quart. .Toiir. ^rcd., 1010 (0), 210; :^[c'Clure and Pratt. An-h. Int. Med., 1017 HO). .'>GS. 3»Satta, Arcli. Sci. "MM., lOlo (.10), 4(i. ■"> In infants llio urino anivlaso is low f^[c("'Iuro and riiaiiccUdr, Zoit. Kindor- hcilk.. 1014 (11), 4S:{. 'I rrnr. So.-. Kvii, lii.il,. I'.HT ( 1.-)). 1(11. CHAPTER III ENZYMES (Continued) INTRACELLULAR PROTEASES' (PROTEOLYTIC ENZYMES), IN- CLUDING A CONSIDERATION OF AUTOLYSIS To what extent synthesis of proteins goes on in the body is still a problem ; still more uncertain is the part played by reversible action of proteases. There is evidence enough that somewhere in the body the amino-acids can be rebuilt into protein, for several investigators have succeeded in keeping animals in nitrogenous equilibrium by feed- ing them products of proteolysis that contained no protei-us whatever, and as the proteins of the animal body are being broken down in- cessantly, it must be that they were replaced by synthesis of the non- protein material fed to the animals. In addition, it has long been questioned whether amino-acids absorbed from the intestines are not resynthesized into proteins while passing through the intestinal wall. Cohnheim found that in the intestinal epithelium there is an enzyme, erepsin, capable of splitting albumoses and peptones into the amino- acids, which enzyme presumably exists for the purpose of securing complete cleavage of all ingested proteins into their ultimate "build- ing stones." This may be looked upon as a provision to reduce all varieties of proteins to their common elements, so that the body by quantitative selection can resynthesize them into its own types of protein, for, as is well known, foreign proteins (e. g., egg-albumin) introduced directly into the blood stream cannot be utilized, but are excreted unaltered in the urine. ^ As was shown for lipase, the as- sumption that such synthesis occurs as a normal physiological process by reverse enzyme action, requires that the proper enzymes be present in the cells throughout the body, and within the past few years it has been abundantly demonstrated that such is the case. For over half a century it has been known that amebre digest solid proteins within their bodies, but it is only within a few years that proteolytic enzymes have been definitely isolated from them. It has been nuieh the same w'ith the intracellular proteases of the higher 1 As the possibility exists tliat ferments T\-hiart by trausforming pro/ynics into en/.ymes and by destroying in- hibiting substances, hence the value of the results obtained in autolysis with these acids is questionable. However, sodium salicylate and benzoate are said to favor autolysis (Laqueur^i). Toluene seems to interfere much less with auto- lysis than chloroform or thymol (Benson and Wells -2), and bromides are less harmful tiian toluene (Laqueur). Toluene vapor, acting on solid aseptic tis- sues, seems to cause more depression of autolysis than is usually observed in autolysis in solution. 23 Dorothy Court -* found the only satisfactory antiseptics to be chloroform, formaldehyde, benzoic and salicylic acids, and IIXC; she em- phasizes the fact that for dillVrcnt sorts of niat(>rials tiie diti'erent antiseptics give variable results, so that the antiseptic used should be selected with reference to the material. Autolysis proceeds rapidly in weak ethyl alcohol, 5 per cent, being the minimum strength that will prevent putrefaction: for complete sup- pression t)f autolysis by alcohol the strength must be at least 00 per cent, net, after allowing for the water content of the tissues (Wells and Caldwell) .2-i;i Certain inorganic substances in proper concentrations may increase the rate of autolysis [mercury 2s and silver, 20 (colloidal or salts)], yellow phosphorus, 2" iodides, 2s arsenic, 2!> CaClo,30 salts of Fe, Mg, and cobalt,''i as well as salts of selenium, tellurium, 32 and manganese, 32a colloidal sulfur "^i) but not colloidal carbon. 3'.;i- The favorable concentrations of these metals are very low: thus the o])tim\un proportion of arsenic is 0.007 milligrams |)er 1 gm. tissue, while 0.04 mg. inhibits autolysis. ('()._. increases and oxygen decreases autolysis 3-ia jn litro is'cSee Schul/. Miiii.l:, nied. Wodi. ]!)l:i ((iO), 2.)12; Mandelbaum, ibid.. 1014 (61), 401. ii'«l See Sloan, Anier. .b>ur. I'liysiol., litl.-) (30), 0. 2oZeit. physiol. Chem., 1908 (58), 341. 21 Zeit. phVsiol. Chem., 1012 (70), 38 and tif). 22 .Jour. Hiol. Chem., 1010 (8), 01. 23 Cruick.shank, Jour. Path, and Bact., 1011 (l(i), 107. 24 Proc. Rov. Soc, Edinburgh, 1012 (32), 251. 2-Ja,Tour. Biol. Chem.. 1014 (10). 57. 2-. Trufli, Biochem. Zeit., 1010 (23), 270. 2«Izar, ihiiL, 1000 (20). 240. 2i Sa\l, llofmeister's Beitr.. 1007 (10), 447; Vin-liow's Arch.. 1010 i202), 140. 2*' Kepiiiow. Hiocheni. Zeit., 1011 (37*. 23S : Kaschiwali;ii-a, Zeit. ]ili\si<)l. Cliein., 1012 (H2), -42.'). 2» Izar, Miochem. Zeit., 1000 (21), 4(); Laqueur and I'.ttiuLier. Zeit. phxsiol Chem., 1012 (70). 1. aoBriill, Biochem. Zeit., 1010 (20), 408. 31 Pn-ti. Zeit. phvsiol. Clieiii., IMOil (OOi, 317: I'niliiii. I!ineheiii. Zeit.. 1012 (47). 30(i. 32 Kasiani, .\rcli. sci. med., 1012 (30), 430. •■"2.1 I'.iadlev, .lour. I5i(.l. (hem.. 1015 (21), 200: 1015 (221, 11.3. ;>2i> Kagiiioli. JtiiM-heMi. Zeit.. 101.3 (50). 20 1. •«2<- i/ar and I'atane. ihiil.. \>. .307. •''•''II .M. .Moise found iiwgen uithnut eMeet mi aMtul\si>. Hiocheni. Uullef I0l5 (5). 143. RELATION OF AUTOrAHfS TO M F.TMiOIJSM 87 (I.aqm'ur). Thoro is disagrooiiient as to vvlietlier radium rays auf,'ment autolysis. •■'•■* Injection of iodids into animals is said to increase the jjostinortem autolysis of their tissues (Stookey, Kepinow), as also do iron salts,3:ib while lar>re doses of salicylates decrease it (Laciucur). ]Morse ''■*f attributes the acceleratinj; action of iodin and Itromin to increased acidity from formation of halogen acids, and Bradley " finds evidence that most inorganic salts that stimulate autolysis act by increasing Il-ion concentration. Addition of tuberculin to tissues at first delays and tiien increases the autolysis ( Pe.sci ;^» ) . and dii)htheria toxin in snuiU amounts increases autolysis ( Barlocco,-*'' Bertolini 3n. ■••''.Tonr. Biol, riicin., lOl.'i (22), 12r). <" Opifi (loc cit.) found, liovvovor, that autolysis of loueocytoa was more rai)id in an alkalino incditun. Docho/, (Proc. Sop. Exp. Biol, and ^I'^d.. 1010 (7). 07) stat-oH that llvfT alno contains an cn/ynip active in an alkaliiii' iiu'diiim. Ii\it which exists as an inactive zyniofrcn until activated by acids. ••7 Festschrift f. liainniarston, Upsala, 190G. DEFENSE OF CELLS AGAIXST THEIR AUTOLYTIC EX/AMES 89 to a minimum, since during life the tissue fluids, and presumably the cell contents, are preponderatingly alkaline. Perhaps a better ex- planation of the attack of the cells by their own enzymes after death is to be sought in the conditions of chemical equilibrium. During life constant new supplies of protein are being brought to the cell, and at the same time the products of proteolysis are presumably being car- ried away by the circulation or being changed by oxidative processes. When circulation stops, the processes of splitting go on without the introduction of new supplies of material, and hence the tissues are not replaced as fast as they are destroj'ed, and the products of their decomposition accumulate, for lack of any means of destroying or re- moving them. The control of autolysis by maintenance of a low H-ion concentration is, however, undoubtedly an important factor, for Bradley ^ found that a reaction equal to that of blood almost com- pletely inhibits autolysis, while the degree of increased H-ion con- centration that may develop in local asphyxia, or after death, produces optimum conditions for autolj^sis. Still another possible defense of the living cells may be found in the existence of specific antienzymes. Just as the serum contains anti- trypsin, so it seems to contain substances antagonistic to the autolytic enzjones. Levene and Stookey found that tissue juices show a resist- ance to digestion, and Opie found that the serum of inflammatory exudates retarded the action of the autolytic enzymes that are con- tained within the leucocytes. Serum also inhibits autolysis of the tissues, so it is probable that continuance of the circulation may pro- vide antibodies to the tissues to hold the intracellular enzymes in check, possibly without interfering with their action on other pro- teins than those of the cell structure.*^* (See Antienzymes.) Lack of oxygen cannot be held solely responsible, according to the studies of Morse,*^'' wdio found that autolysis occurs in muscles with divided nerves but intact blood supply. Nevertheless, reduced blood supply results in increased H-ion concentration which greatly facilitates auto- lysis, and it cannot he denied that autolysis is observed chiefly if not solely in asphyxiated tissues. There can be no question that the supply of food-stuff is of essential importance in determining autolytic changes, for it has been found by Conradi,-*^ Rettger.^" and Effront ^'*' that bacteria and yeasts begin to undergo autolysis when they are placed in distilled water or salt solution, which they do not do, to an}- such extent at least, when in 47a According to Gusrgenheimer (Deut. Arch. klin. Med., 1013 {112). 248: Dent, med. Woch., 1914 (40), 63), the serum in various diseases has a characteristic stimulating or inhibiting effect on in vitro tissue autolysis, hut the conditions of such experiments are so complex as to make their significance doubtful. 47b Amer. Jour. Phvsiol.. 101.5 (3fi). 147. 48 Deut. med. Woch., 1003 (20). 26. 49 , Jour. Med. Research. 1004 (13). 70. 50 Bull. Soc. Chim., 1005 (33), 847. 90 f;.v/y.i//;.s' iiutriciil iiiciliii. ( 111 tliis way it has liccii iniiiid ])()ssil)le to obtain the intracH'llular toxins of sueli hactt'ria as typlioid and cholera.) Autolysis is not marked so lonj; as tlif bacteria are supplied with nourishment. l)ut wlicii nutrient iiialfiial is laekinjz:, autolytic decom- position is no l()ii«ier repaired aiul tiie bacteria disintegrate. Pre- sumably the (•lian.^). r'-' Scionro. 1004 MO). .'{.^O. '•"■Many nulliors snirp'st flint flic jciicocvf cs iiicrclv ciinv oh/mik's from ono orpin, parliciilarly tlic pancreas. f \Tlli)l.<)(;i(\L I'mx'EfSFfEH 91 iilbuinin to catgut ligatures. 'I'lic lictci-olysis iiuiy be iutracellular ■\vliou the material to be digested luis tirst beeu taken up by the cells (phagocytosis) ; or extra-cellular, either by enzymes normally con- tained in the blood ])lasma and tissue fluids, or by enzymes liberated by the leucocytes and tixed tissue cells. On death and dissolution of a cell the intracellular enzj'mes are released,'*"^ but it is not known to what extent the enzymes may be secreted from intact living cells. As far as pathological processes show, the amount of liberation of en- zymes from normal cells is very slight, if any, and the digestive en- zymes of the blood plasma seem to be very feeble, but this is perhaps because they are largely held in check by the anti-enzymatic substances of the serum. I'athological autolysis and heterolysis, therefore, are brought about chiefly by enzymes liberated from dead or injured cells. Bacteria, however, can multiply upon a medium of coagulated protein, M'hich suggests that they also secrete proteolytic substances, for other- wise it would be difficult to explain how they secure their nourish- ment. In pathological conditions, digestion of degenerated tissues seems usually to be the result of both autolysis and heterolysis. An infarct softens because the intracellular enzymes digest the dead cells, exactly as they do when the tissue is removed from the body, ground up, and put in the incubator under toluene. In addition leucocytes wander in, disintegrate, and their liberated enzymes help in the proc- ess, as also do, to a less degree, the enzymes of the blood plasma. It is because of the heterolysis by leucocytic enzymes that a septic infarct becomes softened so much more rapidly than does a sterile infarct, and by comparing the rate of softening in septic and aseptic infarcts we see that the cellular autolysis is a very slow process as compared to the heterolysis accomplished by the leucocytes. The explanation of this may lie in the fact that most intracellular proteases act best in an acid medium (Wiener), while leucocytic proteases act best in an alkaline medium (Opie), and the infarcts of small size are seeped through by alkaline blood fluids. When an infarct is large, we find it undergoing central softening while the periphery remains firm ; this corroborates our hypothesis, for acids are developed during autolysis (Magnus-Levy), which at the periphery are neutralized by the blood plasma, so that only at the center is autolysis active. The inhibiting formed by the leuooevte itself. Opie (Jour. Exp. ^led., 10(1.7 (7). T.IO ) Juis shown, liowever. that the bone-marrow contains proteolytic enzymes which are like those of the leucocytes in that tliey act best in an alkaline medium, whereas the autolytic enzymes of the lymphatic tflands and most otliei- tissues act best in an acid medium. This leaves little room for doubt that the leucocytes are equipped with their characteristic enzymes when they leave the bone-marrow, and that they are not obtained later in the pancreas or elsewhere. ^Fore re- cently, however, van Calcar (Pfliijier's Archiv., 1012 (148), 2rt~) has revived the idea of the origin of leucocytic enzymes in the digestive ^rlands. ooa Peptolytic enzymes appear in the urine after severe superficial burnincr, pre- sumably comins: from the disintegrated cells. (Pfeiffer, INIiinch. med. Wocli , 1914 (61), 1329.) 92 ENZYMES action of the serum also has a similar effect, limiting autolysis at the periphery. In the case of septic softeniny; the action of the bacteria needs also to be taken into consideration, since they also produce proteolytic ferments, but their effect seems to be relatively small as compared with leucocytic digestion. Intracellular digestion of necrotic tissue by leucocytes seems also to be relativelj^ unimportant. Suppuration, therefore, must be considered as the result of digestion of dead tissue by enzymes derived from the leucocytes, the plasma, the bacteria, and the destroyed cells themselves. A tubercle does not ordinarily sup- purate, because the tul)er('le bacillus and the substances it produces are not strongly chemotactic, and hence not enough leucocytes enter the necrotic area to produce a digestive softening. The enzymes of stai)liylo('Occus are much more strongly proteolytic than those of streptococcus (Knapp^'), which may be one reason why the latter so much more frequently produces lesions without suppuration than does the former. Necrotic areas of any kind are absorbed by similar processes. Autolysis of tumors is quite active in specimens removed from the body, and the areas of necrosis that occur commonly in tumors are absorbed in this way. Apparently all varieties of cells are subject to autolj'sis or heterolj^sis whenever they are killed or sufficiently injured. Involution of the uterus probably depends upon autolj'sis, which is much more active in the puerperal uteinis (Ferroni^^), and creatine is found in the urine when such autolysis occurs,^^ although A. INIorse -'^ considers this to be independent of the uterine autolysis. Atrophy may be looked upon as an autolysis in the normal course of catabolism, not met by a corresponding build- ing up of the proteins, l)ut ]\I. ^Morse ^''''^ could tind no evidence that the atrophy and involution of the tadpole tail is accompanied by an accelerated autolysis. The solution of fibrin by tissues, fihrinolysis, is considered to be distinct from tissue autolysis by Fleisher and Loeb.'"''"' In atrophic cirrhosis the fibrinolytic activity of the blood is increased, wliidi may explain the lieniorrliagic tendency of this dis- ease.^®° The products of autolysis may of themselves be toxic; albumoses and pe])tones certainly are, and the other cleavage products are prob- abh^ not altogether innocuous. (See " Autoinloxication.") Some of the symptoms of suppuration, ])articularly llic fever and chills, have been ascribed to the autolytic products i-atlni- tlian to the bacterial "Zcit. f. ITeilk. (riiir.). 1902 (2.3). 2.30. f'SAnn. (li Ostctrica e. Ginocol., IflOfi (2), .'i5.3: set' also Slciuons, Bull. Johns Hopkins Ifosp., 1014 (2.'5), 195; Arthur Morso, Jour. Amer. Med., Assoc, 1915 (0-)), 1(11.3. so SliiifTor. Amor. .lour. I'hvsiol., 190S (23). 1. snnMiix Morso, Am. .lour. I'hvsiol., lOl.'i (3G), 14r). si'b.Tour. IJiol. Chom., IfM.'} (21). 477. '•!'■• Coodpnslurc, I'.uli. .lolins Hopkins ITosp., 1914 (25), 330. AUTOLYSIS IX I'ATUOLOGICAL PROCESSES 93 poisons, particularly as aseptic suppuration is accompanied by fever, Joclimanu '^'^ lias found evidence that the protease of leucoytes can cause fever and also reduce the coagulability of the blood. The work of Vaughan and other recent students of the reaction to foreign pro- teins, shows that typical fevers can be produced by the enzymatic dis- integration of proteins in the body."'^'' Degenerative changes in nerv- ous tissue are associated with autolytic decomposition of the lecithin (NoU"^) and the liberated choline, or its more toxic derivatives, may be a source of intoxication.''- In all conditions associated with auto- lysis, such as resolving pneumonic exudates, large abscesses, softening tumors, etc., albumoses (and peptones?) may appear in the urine. Autolytic products may also be hemolytic (Levaditi "^), and thej^ may prevent clotting of the blood (Conradi "*). It is probable that among tlie products of autolysis are bactericidal substances,"^'^ although it is doubtful if the concentration is often sufficient for them to be of influence except in well walled areas. Work has been reported upon autolytic processes in a number of pathological conditions, which may be discussed briefly as follows: Exudates. — The presence of leucine, tyrosine, proteoses, and pep- tones in pus has been known for many years, and the reason for their appearance is now clear. Miiller,^^ many years ago, observed that purulent sputum digested fibrin, but that non-purulent sputum did not have this property. Achalme "*' found that pus would dissolve gelatin, fibrin, and egg-albumen. Ascoli and jMareschi ®" detected autol,ysis in sterile exudates obtained experimentally. Umber ^^ found that ascitic fluid exhibited autolytic changes, which observa- tion could not be confirmed by Schiitz ^° in pleural exudates and as- citic fluids. Zak ^° found that autolysis was inconstant in various exudates. The ditferences in these results are explained by Opie's"^ observation that in experimental inflammatory exudates the leuco- cytes are capable of marked autolysis, whereas the serum contains an antibody which holds this autolysis in check; if the antibody is de- stroyed by heat, then tlie serum proteins are also digested by the leu- eocytic enzymes. This antibody seems to be contained normally in GoVirchow's Arch.. 1908 (104). 342. 60a See Vaughan, "Protein Split Products." Philadelpliia. 101.3. Gi Zeit. physiol. Chemie, 1809 (27), 380. G2 See Haliibvirton, Erpebnisse der Plivsiol., 1904 (4), 24. G3 Ann. d. I'Inst. Pasteur, 1903 (17), 187; also Fukuhara. Zeit. f. exp. Path. u. Pharm.. 1907 (4), 658. 64 Hofmeister's Beitr., 1901 (1), 130. 64a See Bilancioni, Arch, farmacol., 1911 (11), 491. GsKossel, Zeit. f. klin. ]\Ied.. 1S8S (13), 149. «« Compt. Rend. Soc. Biol., 1899 (.51), 568 G7 See Malv's Jahresbericht. 1902 (32), 568. osMiinch. ined. Woch., 1902 (49). 1169. 69 Cent. f. inn. Med., 1902 (23), 1161. 70Wien. klin. Woch., 1905 (18), 376. 71 Jour, of Exper. Med., 1905 (7), 316 and 759; 1906 (8), 410 and 536; 1907 (9), 207, 391 and 414; also a full review in Arch. Int. Med., 1910 (5), 541. 94 EXZYMES the albumin of the blood-serum. In old exudates the antibodies are decreased, and autolysis then occurs, explaining the variable results of Umber, Schiitz and Zak. The intracellular proteases of the poly- nuelear leucocytes act best in an alkaline medium; those of the mononuclears in an acid medium. If the proi)()rti()n of serum to leucocytes is high, then there is no autolysis, as in serous exudates; but if the leucocytes are abundant, then the antibody is overcome and we get autolysis, as in ordinary suppurative exudates. Animals with but little protease in their leucocytes (e. g., rabbits), do not ordinarily produce a liquid pus (Opie). Exudates produced by bac- terial infection also seem to possess the properties above described. Galdi '- found autolysis greater in exudates than in transudates, but observed no constant relation between the number of leucocytes, or the amount of chlorides, and the rate of autolysis. All exudates, ac- cording to Lenk and Follak,'' contain enzymes splitting glycyl-gly- cine (peptolytic enzymes) ; the most active exudates are those of cancer and tuberculosis, the least active are passive congestion fluids; pleural exuchites contain more active enzymes tlian peritoneal exudates of similar character. Knapp '* holds that in pus the cocci and tlie enzymes they produce are responsible for much of the digestion. Pus cells alone do not undergo digestion so rapidly as wlien bacteria are present, and di- gestion is more rapid if the bacteria are alive than when inhibited or . killed by antiseptics. Streptococcus is almost inactive, staphylococcus is quite active, and B. coli still more so. However, pus corpuscles free from bacteria are highly proteolytic, causing digestion in serum plates in dilutions of 1-700 (Jochmann). Knapp could find no rela- tion between the autolytic power of the pus and the severity of the in- fection from which it resulted. A constant constituent of pus is d-lactic aeid,^^^ and it increases during autolysis ; this may well modify the rate of autolysis of pus. (See also tlie discussion of the "Chem- istry of Tiis, " Cha]). X.) Proteolytic Enzymes of the Leucocytes.'-' — By tlie introduction of the plate method of testing the proteolytic activity of leucocytes, Miiller and Jochmann brought the study of this particular vital activity into the range of clinical laboratories, and aroused nuu-h general interest in what had previously concerned only a few pathol- ogists, especially E. L. Opic The jn-inciple is that of permitting the leucocytes or other cells to act ui)oii a blood .serum plate at a tem- pei'ature of 55'^', which prevents bacterial action but permits the pro- 7-' See Folia llcmat., 1!)().") (2), .")2n. 73 Dout. Arcli. kliii. Med.. 1013 (100), ViTiO: see iilso Wiener. Biocliem. Zeit., 1012 (41), 140; Miuidclliainii. :Miiiieli. ined. Woeli., l!tl4 (CI). 4(il. 7* Zeitsclir. f. lleilk., I!l(i2 (2:5. Cliir. .Mil.), 2:i(i. 7-ialto, .Tour. ]?i<.l. Ciieiii.. 101(1 (2(1). 17.S. "■'• i"'ull liililio^'riipliv liv \\ieTis, Kr), 1: ■locliinaiin, Koiie and Wasserniaiin's Ilandliueli. 1012 (2). 1:301. AUTOLYSIS I\ I'ATUOLOaiCAL I'h'OCESSES 95 toolytic on/ymes of the cells to (IirEsii:ic action of the lecocytes has been found limited to the neutro- phile granules. In neutral media evidence is obtained of the presence of protease in the lymphoc}i:es of chronic lymphatic leukemia and the leucocytes of acute and chronic myeloid leukemia ; maltase, lipase and amylase are found in both types of cells, and oxidase in the granular cells derived from the marrow (^Morris and Boggs).-^ v. Jaksch." Erben.-^ and others have noted the occurrence of peptones and albu- moses in leukemic blood, particularly if removed postmortem. The improvement in leukemia that follows j:--ray treatment is associated with an increased nitrogen elimination, probably due to autolysis of disintegrating cells,-* although j--rays have no appreciable effect upon the leueocytic proteases in vitro (^Miiller and Jochmann). (See also "Leukemia," Chap, xi.) IT Jour. Med. Research, 1001 (6), 120. isBiochem. Zeit., 1913 (48), 448. 19 Hofmeisters Beitr., 1003 (.3), 576: 1905 (7). 175. 20 See discussion of leueocytic enzymes, p. 94. Longcope and Donhauser (.Jour. Exper. !Med., 1908 (10), 618) found proteases in the large lymphocytes in acute leukemia, which were most active in an alkaline medium. 21 Arch. Int. :\red., 1911 (8), 806. 22 Zeit. f. phvsiol. Chem.. 1892 (16). 243. 23 Zeit. f. klin. Med., 1900 (40), 282; Zeit. f. Heilkunde. 1903 (24), 70: Hof- meister's Beitr.. 1904 (5). 461. 24Mus5er and Edsall. Univ. Penn. Med. Bull., 1905 (18), 174. 104 i:\/.yMiJs Tumors. — l^i-obably because of tlie jiTeat amount of necrosis that is constantly ino\v. Zeit. f. K rebsforscli.. I'.Ki'.i (7i. r)i7. 31 Zeit. plixsiol. CIm'iii., 1910 ((>(i), 277. s2Bio< licin'. /cii., 1 1) 1(1 (2(i), 344. T! Moh'X 105 tioii of these enz.ymes.'''' Tumors also contain nuclease^* to disinte- {^rate their nucleic acid, and the same outfit of purine-splitting en- zymes as normal tissues,-'"' so that in rep:ard to the nucleoproteins of tumors autolysis follows the same course as in normal tissues. The non-cancerous livers of cancerous patients were found by Yous- souf •'" to jiroduce more lactic acid duringr antiseptic autolysis than did livers in other conditions. Autolysis of orfjans of cancer patients is about as rapid as normal ( Cohvell "' ). Several observations have sugrp-ested that tumor tissues mig-ht contain ])r()teolytic enzymes dif- fering from those of normal tissues especially in their ability to di- gest heterologous normal tissues, ])ut at present this work needs con- firmation and amplification before it can carry the weight of specula- tion which has been heaped upon it.''- ^licheli and Donati ■■' attribute the hemolytic properties possessed by extracts of malignant tumors to the products of autolysis that are present, which Petry has also demonstrated to produce hemolysis. Emerson *° attributes the disappearance of HCl from the gastric juice in carcinoma of the stomach to neutralization by basic products of autolysis, a hypothesis that nmy well be questioned. (See also "Tumors," Chap, xvii.) Varioiis other intracellular enzymes have been described, -wliich for the most part iiave as yet no significanee in pathology. An exception is fiJn-ln fcnnent, which will be considered fully in discussing thrombosis. Ferments coagulating milk seem to be widely spread in the tissues. The precipitation of plastein from proteose solution by organ extracts (Xiirnberg) may be either the effect of a coagulating ferment or due to reverse action of the proteases. Ferments s[)lit- ting specifically maltose, lactose, sucrose, glucosides. and nucleoproteins have been described, and the glycogenolytic ferment is probably nearly universallv pres- ent. Other enzymes decomposing amino-acids into ammonium compounds may also exist. The enzymes acting specifically upon the nucleic acids and the ])urine bodies are discussed in Chapter xxi. 33 See Jacques and ^Yoodyatt, Arch. Int. :\Ied., 1012 (10), .lOO: Ilamlmrger, Jour. Amer. Med. Assoc. 1912 (59), 847. 34 Goodman, Jour. Exp. ^^led.. 1912 (15), 477. 33 Wells and Long, Zeit. Krebforsch., 1913 (12), .598. 36Virchow's Arch., 1912 (207), .374. 37 Arch. Middlesex ITosp.. 1910 (19). 55. 38 See, for example, Rulf, Zeit. Krebsforsch., 1906 (4). 417: Miillcr, Cent. inn. Med., 1909 (30), 89. 3oRiforma med.. 1903 (19). 1037. 40 Deut. Arcli. klin. Med., 1902 (72), 415. CHAPTER IV THE CHEMISTRY OF BACTERIA AND THEIR PRODUCTS STRUCTURE AND PHYSICAL PROPERTIES ' In structure, as in uearly all other respects, bacterial cells stand inteiinediate between the cells of ordinary plant and animal tissues. Their cell wall seems to be generally more hig-hl}- developed than that of animal cells, and less so than the wall of most plant cells. In composition, however, the wall is more closely related to animal than to vegetable tissues. The much vexed question as to the existence or non-existence of a nucleus seems to be best answered by Zettnow, who considers that the portion of the bacterial cell usually made evident by ordinary staining methods consists of a mixture of nuclear substance {chroiiiati)i) with non-chromatic substance {endoplasm) ; the outer membrane, which requires special methods for its satisfac- tory demonstration, consists of a modified cytoplasm {ectoplasm). Some bacteria consist chiefly of chromatin (e. g., vibrios), but the proportion of the ditit'erent elements varies greatly, not onh^ in dif- ferent varieties, but also in the same variety under ditferent con- ditions. The fact that the chromatin is not aggregated into the usual nuclear form may be ascribed to the low stage of development reached by bacteria in the scale of evolution ; or, as Vejdovosky^ has suggested, to the extremely rapid rate of cell division in the bacteria which pre- vents the chromatin from appearing in the resting stage whicli a nucleus constitutes. Finer structures witliiu tlie bacterial cell have as 3'et been onl}^ imperfectly discerned. The thickness of the ectoplasm varies greatly even in the same species, being generally greatest in older cultures. In some forms the ectoplasm may constitute one-half of the total mass of the cells. The capsule seems to arise through a swelling of the ectoplasm, and is probably present in at least a rudiniontary stage in all bacteria LMiguhi).' F^lasmolysis and Plasmoptysis. — I'mler conditions of altered osmotic pressure the bacterial cell behaves quite similarly to the plant 1 In this chapter reforcnees will not ponorally he iinann's TIandliucli. A jxcncral consideration of the Bio]o<,'y of the Bacteria, incliidinjj refercn it ranks tluMU with tlie plant 4Rtip('ll, Cent. f. I?akt., 1007 (4.3), 4S7. •'"' Jinx ton, Zcit. ]»liysil. .ri/liuuDi ; he therefore considers it probable that bacterial cell walls do not always consist of the same substance. Cramer could find no glucose in any variety, although there are some bacteria that contain material reacting like starch with iodin. Levene,^*' how- ever, found in B. tuhcixulosis a substance with the properties of glyco<:'('ii. Bacterial Fats. — By staining methods, fats have been recognized in many species, and by extraction with fat solvents lecithin, cholesterol, simple fats, and specific bacterial fats have been isolated ; this is par- ticularly true of B. tuberculosis, which owes its characteristic staining ])roperties to the specific fat-like bodies which make up a large pro- portion of its entire mass.^^ Numerous studies of these fats of B. tuherculosis have been made ^^ and by using different extractives, from 20 to 40 per cent, of the entire weight of the bacilli has been found s()lul)le in fat solvents. Kresling found that the substance soluble in chloroform had the following composition: Free fatty acid 14. ."^R per cent. Neutral fats and fatty acid osters 77.2.5 " " Alcohols obtained from fatty acid esters .... 30.10 " " Lecithin " O.lfi " Substances soluble in water 0.73 " " Bulloch and ^Maclcod found that ethereal extracts did not contain the acid-fast substance which they consider to be a wax-like alcohol, soluble in hot, but insoluble in cold absolute alcohol or in ether. The simple fats seem to be formed by oleic, isocetinic, and myristinic wall of veasts contains a hemicellulose and a nianno-dextran. See also Kozniewski, Zeit. phvsiol. Chem.. 1914 (00). 208. mSeoViehofer. Ber. Dent, riiem. fies.. 1012 (301. 443. i-'i Mor;rulis states that chitin consists of two j)arts. one containincr all the glucose and amino <:roups. tlie other bcin? a stable nitrogenous compound yielding no plucoHC. (Science. l!»lf. (44). SCO.) isbZeit. physiol. Cliem.. 1014 (.SO). 304. I'-cPharm. Wcekbhid.. lOlC. (.".3). 11S3. 11 Jour. Med. Research. 1001 (0), 13r>. "Sec Tamils and Pai.miez. Compt. Rend. Soc. Riol.. lOO.") i,-)!)), 701. J 8 For literature see Bulloch iiiid MachMKl. .Tour, of lly-^i.-ne. 1004 (4). 1. CHEMICAL COMPOSITIOX OF BACTERIA 111 acids, and there is some laiiric acid in the form of a soap. Cholesterol could not be found in tubercle, diphtheria and other bacteria examined by Tamura, although there probably are lipochroines giving the cul- tures their color. There is still, however, much disagreement as to whether the acid fastness of tubercle bacilli depends upon waxes, alcohols, fatty acids, or lipoid-protein compounds.^" It must be ad- mitted that a high content of fatty materials is regularly present in acid-fast bacilli; thus, in an acid-fast bacillus isolated from leprous lesions, 34.7 per cent, of fats, fatty acids and cholesterol, and 1.7 per cent, of lecithin were found by Gurd and Denis.-" Tamura-"'' states that the phosphatids of B. tuberculosis and a saphrophyte examined by him were not lecithin but a diaminophos- phatid, although diphtheria bacilli seemed to contain lecithin.-"*^ He found in both a high molecular alcohol, "mykol," to which he ascribes acid- and Gram-fastness. In a Gram-negative bacillus -°*^ he found lecithin, but no cholesterol or mykol. By growing tubercle bacilli on suitable media they can be made to lose their acid-fast property, although still Gram-positive (Wherry ^'"^). The observation of ]\Iiss Sherman,-^ that tubercle bacilli are almost absolutely impenneable to fat-soluble dyes which stain their isolated fats well, and her cor- roboration of Benians' demonstration that acid-fastness depends on the integrity of the bacillary envelope, make the role of the fatty sub- stances uncertain. Their high content in unsaturated fatty acids gives them a high antitryptic power which may be concerned in the defense of the bacteria, and also in the persistence of caseous ma- terial in tubercles (Jobling and Petersen).-^-'' By staining with Sudan III, Sata -- demonstrated fats, not only in the acid-fast bacilli, but also in anthrax. Staphylococcus aureus, B. mucosus, and actinomyces; but not in diphtheria, pseudo-diphtheria, plague, cholera, and chicken cholera bacilli, or in members of the colon group. -^ Only a few bacteria form fat on agar free from glycerine, but potato is a favorable medium. Ritchie -* obtained positive fat staining in B. diphtheria' and anthracis, but not in S. pyogenes aureus or M. tetragenus, although these last forms contain 19 See Camus and Pagniez, Presse MM., 1007 (15), 65; Devkc, ^liinfh. mod. Woch., 1910 (57), 633. 20 .Jour. Exper. :\[ed., 1911 (14), 606. 20aZeit. phvsiol. Chem.. 1913 (87), 85. 20hlhid., 1914 (89), 289. 20c Ihid., 1914 (90), 286. 20d Jour. Infect. Dis., 1913 (13), 144. 21 Jour. Infect. Dis., 1913 (12), 249. 2ia.Joiir. Exp. Med., 1914 (19), 239. 22 Cent. f. allg. Path.. 1900 (11). 97. 23 Auclair (Arch. Med. Exper.. 1903 (15), 725) contends that the ether and chloroform extracts of many patho>renic bacteria contain important toxic sub- stances. Holmes (CJuy's TIosp. Reports, 1905 (59), 155) states that injection of fattv acids from tuljercle bacilli into rabbits causes a lymphocytosis. 24 Jour. Pathol, and Bact., 1905 (10), 334. 112 CHEMlSTh'Y or BAVTKltl \ AM) THEIR I'UODLCTH cliomically demonstrable lipiiis. Analyses of dififereiit bacteria show a relatively low content of lipins as i-oiajjared with tubercle bacilli, vary- ing from 1.7 per cent in B. suhtilis to 8.5 per cent, in staphylococci (Jobling and Petersen).'-'-' However, the degree of unsaturation of the fatty acids is less with tuhei-clc liacilli than with other bacteria examined by these authoi's. Extensive studies of bacterial fat stains are rej)()rted by Eiseiilxn'g,-'' but ])ractically nothing is known of the character of the fally or lipoid cDnslituonts of l)a('teria outside the acid-fast grou]). Spores diil'er from llicir jiareiil l)acteria in containing a nuich greater proportion of the solid constituents and less water. In molds Drymont found that the spores contained over 60 per cent, of dry substance, and almost all the water was so held as to resist drying by temperatures below boiling; the dry substance is very rich in protein and poor in salts. As the spores may lose their chromatin content without loss of capacity to propagate, it would seem that this is not a nuclear chromatin but merely a reserve food supply.-""'^ The wall of the spore consists of a "cellulose-like" substance and a very hygroscopic extractive matter. The great resistance of spores to dry- ing and to heat can be readily understood in view of these facts. They contain, and perhaps secrete, active enzymes (Eifront).-'^ Flagella also soom to be composed of a relatively condensed protein. Staining Reactions. — The staining reactions of bacterial cells are much as if the bacteria consisted entirely of chronuitin, so that at one time the theory prevailed that bacteria consisted merely of a nucleus and a cell wall, without any true cytoplasm. The demon- stration of abundant nucleoprotein in the contents of bacterial cells explains their staining affinity for basic anilin dyes. Owing to some unknown differences in composition, not all bacteria are stained equally well by the same basic dyes. Although the staining of bac- teria depends upon a chemical reaction between the nucleoproteins and the basic dye, yet the combination is not usually a firm one, being readily broken by weak acids in most cases. That the decolorization of bacteria depends u})oii dissociation of the dye-])roteiii couiponnd is shown by the fact that absolutely water-free alcohol will not de- colorize dry bacteria, nor do water-free alcoholic solutions of dyes stain dehydrated bacteria. Gram's ^NFetiiod of staining has been ascribed to llie format ion of an iodiii-])ararosanilin-protein compound which is not easily disso- ciated b\' water' in the case of bacteria that stain b\" tins nu^thod, and which is readily dissociated and dissolved out in the case of bacteria 24.1 .Tour. Ivs]). Mfd.. 1!II4 CJO). 4.")(t. 2-' Vircliow's .Arcliiv., 1!>1(I (1!»!M. ">n2. 2-.a Ruzifka, ("cnt. f. l?ali!s of W. T. Sod^^wick. r.KMI. p. IJ). 37 (Vnl. f. Hakl.. 1 !»(»:! (:(.-,). 'l . 3« Aiiicricaii .Med., l!l(i:{ ( (l ) . i:i7. BACTERIAL ES'/A MKS 115 ENZYMES IIYDKATIXG CARBOHYDRATES 39 Amylase. Maltase. Invertase. Lactase. 1. Antlirax + 2. Cliolera + '^. Coli foiniiiunis — 4. 'ryi)lu)i(l — 5. l)i|)litlioria — (i. Slapli. ])yo<;eiios aureus — 7. J.ac-tis aerogenes .... — 8. Pyocyaiieus — 0. Violacoii.s — 1(K Mycoidcs + 11. Prodigiosus — 12. Saecharomyces niger ... — 13. Saecharomyces neoformaus — 14. Aspergillus niger . . . . + 15. Aspergillus oryzoe . . . . + PROTEOLYTIC ENZYMES, DIGESTING Inulase. + + + + + + + + + + + + + + + + — + Milk. Gela- tin. Serum Egg- albu- men. Fibrin. Red blood- Coagul. Diges- tion. corpus- cles. + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + -4- 2. Cholera + 3. Coli communis 4. Staph, pyogenes aureus. 5. Streptococcus pyogenes. 6. Pyocyaneus + + + 8 Mvcoides + + 10. Aspergillus niger 11. Aspergillus oryzoe .... + Rennin is produced by many bacteria, as is shown by their coagu- lating milk, independent of any acid reaction," and protease from pyocyaneus causes "pilastein" formation in albumose solutions (Zak).*^ Bacteria which give negative results by the plate method may con- tain active lipase demonstrable in killed bacteria by direct action upon fats and esters, these lipases behaving exactly like the lipase of animal tissues (Wells and Corper) ; ■*- staphylococcus and pyocyaneus are more actively lipolytic than B. coli, B. dysenteri. 40 Contradicted bv DeWaele, Cent. f. Bakt., 1905 (39), 353. 41 Hofmeister's Beitr., 1907 (10), 287. 42 Jour. Infect. Dis., 1912 (11), 388; literature on bacterial lipases. See also Kendall. 43 Wratschebnaja Gazetta, 1902. p. 52. 44 Jour. Med. Research, 1903 (10), 42. ]16 CHEMISTRY OF BACTERIA AND THEIR PRODUCTS on bouillon. There does not seem to be any important relation be- tween enzj'me production and pathogenicity.*^" In general, bacterial proteolytic enzymes resemble trypsin more closely than they do pepsin, acting best in an alkaline medium; but the enzymes extracted from bacterial cultures are very feeble as com- pared with pancreatic trypsin. It is probable that there are several distinct proteolytic enzymes in bacterial cells, gelatinase being a dis- tinct protease (Jordan)."'' Abbott and Gildersleeve found that the gelatin-dissolving enzyme of bacteria resists a temperature of 100° C. for as long as fifteen to thirty minutes, but Jordan found that the reaction of the medium modified greatly this heat resistance. Schmailowitsch '^^ states that some bacteria produce an enzyme acting in acid medium upon gelatin but not upon albumin, and this enzyme carries the digestion only as far as the gelatin-peptone stage, whereas the enzymes acting in an alkaline medium carry the splitting through to leucine, tyrosine, etc. Kendall and Walker ^^-'^ state that the pro- teolytic enzymes of B. pruteus are not formed when the bacteria have enough carbohydrate supplied so that they need not depend on pro- teins for their energy requirements; deaminization is independent of proteolysis and represents intracellular enzj^me action. Plenge *" suggests that there is a special enzyme digesting nucleoproteins. Bacteria are able to split nucleic acids and to convert amino-purines into oxypurines, but they do not carry the oxidation to uric acid; putrefactive bacteria can slowly destroy uric acid (Schittenhelm),*^ and B. coli destroys purines.*'^^ Cacace *^ investigated the splitting products of gelatin and coagu- lated blood when digested by B. anthracis, Staph, pyogenes aureus, and Sarcina aurantiaca, and found that proteoses and peptone are produced, which disappear in the later stages of digestion. Rettger *^ found leucine, tyrosine, tryptophane, as well as phenols, skatole, in- dole, aromatic oxy-acids, and mercaptan, among the products of bac- terial decomposition of egg-albumen and meat; proteoses and pep- tones appear in the early stages, but later disappear, as also eventu- ally do the leucine, tyrosine, etc. Choline has also been found in the l)roducts of autolysis.^" The digestive power of the filtrates of cultures and of killed bac- teria is far less than that of the living bacteria (Knapp).^^ Strepto- 4«a Rosenthal and Patai. Cent. f. Bakt., 1014 (7.'?), 400; (74), 3011. 4-Ji) Corroborated bv Bertiau, Cent. f. Bakt.. 1914 (74), 374. 4.-. Abst. in Biocliein. Centr., 1903 (1), 230; see also DeWaele. Cent. f. Bakt., 1905 (39), .353. 45a.l,Hir. Infect. Dis.. 1915 (17), 442. 40Zeit. f. i.livsiol. C'liem., 1903 (39), 190. 47 Zeit. physiol. Ciiem., 190S (57). 21. 4Ta Siven, Zeit. phvsiol. Chem., 1914 (91), 33(). 48 Cent. f. Bakt.. 1901 (30), 244. 40Amer. Jour, of Physiol., 1903 (8), 284. r.o KiitHcher and Loliiiiann, Zeit. plivsiol. Ch(>ni., 1903 (39), 313. M Zeit. f. lleilk. (Cliir. Abt.) 1902 (23), 230. IMMU^aiTY AGAiysr BACTEIilAh ES/AMEfi 117 cocci digest proteins of exudates feebly, staphylococci more rapidly, and colon bacilli are still more active. He could find no relation between the proteolytic power of the bacteria and the severity of the infection from which they came. Staphylococci can cause coagula- tion of pla.sma and then dissolve the coagulum, showing the presence of two enzymes, staphylokiimse and fihrinolysin (Kleinschmidt).^^ Sperry and Rettger,"* however, found that even the most actively putrefactive bacteria are unable to attack or grow upon carefully purified proteins, although the presence of small amounts of peptone or other available nutrient makes the proteins available to the bac- teria; apparently they must have some nutrient more available than intact protein molecules to enable them to grow sufficiently to produce enough free enzymes to attack the proteins. By virtue of their pro- teolytic enzymes, filtrates of bacteria that liquefy gelatin also can digest hardened liver, kidney and other tissue elements in vitro, the changes resembling those of necrobiosis.^-^ Catalase is demonstrable in bacteria, the anaerobic forms showing the least activity (Rywosch),^^ but practically no species is entirely inactive ( Jorns) ; ^* it may exist as either endo- or ecto-enzyme. Cer- tain bacteria and actinomyces exliibit oxidative effects, resembling tyrosinase, but such an enzyme could not be extracted by Lehmann and Sano.^^ Immunity against bacterial enzymes may be secured as it is against other enzymes. Abbott and Gildersleeve ^* found that by injections into animals of proteolytic bacterial filtrates which were only slightly toxic, the serum of the animals acquired a slight but specific in- crease in resistance to the proteolytic enzymes of the filtrates.^^ Normal serum contains a certain amount of enzjTne-resisting sub- stance. Other observers have found that immunization against living or dead bacteria leads to the production of substances antagonistic to their enzj-mes, but the degree of resistance acquired is never great, v. Dungern ^' found that the serum of animals infected with various bacteria prevented digestion of gelatin by the enzymes ob- tained from cultures of the same species of bacteria. He applied this fact to the diagnosis of infectious conditions, finding that the serum of a patient with osteomyelitis was over twenty times as strongly inhibitory to staphylococcus enzymes as was serum of nor- mal persons. The reaction is specific, cholera vibrio enzjTiies not be- ing inhibited to any corresponding degree. 52Zeit. Immunitat., 1900 (3). 516. 52a .Jour. Biol. Chem., 1915 (20), 445. 52bBittrolff. Ziegler's Bcitr., 1915 (60), 3.37. 53 Cent. f. Bakt., 1907 (44), 295. 54 Arch. f. Hvcr., 1908 (67), 134. 55 Arch. f. Hyg., 1908 (67), 99. 56 Antigelatinase has also been obtained bv Bertiau. Cent. f. Bakt., 1914 (74), 374. 57 Munch, med. Woch., 1898 (45), 1040. 118 CHEMISTRY OF BACTERIA A\D THEIR PRODUCTS Kaiitorowicz ""'' and de AVaele ''•' state tliat bacteria contain an in- tracellular anti-protease wiiicli, with most bacteria, holds in check the proteolytic action; only with tlie liquefying bacteria are the proteases in excess. Bacteria grow well in strong solutions of en- zymes, and without destroying the enzymes (Fermi).®" After Gram- negative bacteria have been heatetl to 80° they are readily digested by trypsin, pepsin or leucocytic proteases; but Gram-positive bacteria are resistant even after heating. This is ascribed by Jobling and Petersen""'' to the unsaturated fatty acids, wliicli are present in greater amounts in Gram-positive bacteria. Autolysis of Bacteria. — Autolysis occurs also in bacteria, their proteolytic enzymes digesting the cell substance whenever the organ- isms are killed by agents (chloroform, toluene, etc.) that do not de- stroy these enzymes, and which, being fat solvents, may facilitate di- gestion by removing the inhibitory lipoids. Even the absence of food leads to autolysis, presumably because the normally existing auto- lytic processes are not counteracted by synthesis of new protein ma- terial ; hence, autolysis occurs M^hen bacteria are placed in salt solution or distilled water. Although it had been known for many years that yeast cells digest one another when there is nothing else for them to live upon, the first definite study of bacterial autolysis seems to have been made by Levy and PfersdorfP "^ and Conradi.^- The former digested anthrax bacilli (in whose bodies are contained rennin, lipase and protease) under toluene for several weeks and obtained a slightly toxic product. Conradi permitted dysentery bacilli and typhoid bacilli to digest themselves in normal salt solution for twenty-four to forty-eight hours at 37° C, and obtained in this way the soluble, highly poisonous endotoxins of the bacteria, which are liberated by the destruction of the bacterial structure by the autolytic enzymes. Longer autolysis results in the destruction by the enzymes of the en- dotoxins themselves. Rettger '''^ found among the autolytic products of bacteria, leucine, tyrosine, basic substances, and phosphoric acid. Under favorable conditions complete autolysis can occur in two to ten days. Brieger and IMayer "* found that at room temperature (15° C.) practically no autolysis occurs with typhoid bacilli in distilled water, and the soluble products thus obtained are quite non-toxic, although if injected into animals they give rise to the production of agglu- tinins and bacteriolysins. Bertarelli '''■' has used tlie products of ssMiinfh. mod. Woch., 1909 (.'56), 897. •'•oCont. f. Bakt., 1909 (50), 40. 00 Arch. Farmac'ol., 1909 (S), 481. «oa,Tour. Kxy). Med., 1914 (20), 321. «i Dc'ut. nicd. Wocli.. 1902 (2S), 879. 12 Ibid., ]'M):\ (29), 2(). 03 Jour. :M('d. Kcsoaroli, 1904 (13), 79. «4Dcut. iiK'd. WOcli., 1904 (.30), !)S0 05 Cent. f. Bakt., 1905 (38), 5S4. .\( Toi.Ysis OF ii.\("n:ni.\ 110 autolysis of cliolcra vibrios sncccssi'uily in the j)i'0(hicti()i) of iiniiiu- iiity, and states tliat the i)ro(lu('ts of autolysis consist larfi;('ly of nu- c'leins. It is ])i'ol)al)l(' tliat in cvciy culture jjactcria arc couslantly Ijcinfj destroyed, eitlier by their own enzymes or by tlie proteolytic enzymes of the other bacteria. Some bacteria are much more rapidly auto- lyzed tliau others, cholera vibrios, colon, typhoid, and dysentery bacilli being rapidly di CH— CH,— CH,— NHj ^„ >CH— CH,— CH— NH, CHa^^ CJI3/ X (iso-amylamine) (leiicinp) ^('OOH. 73 All tlipse matters are discussed at Icny^th hy ^ an^'liaii and Xovy. to wliose hook llie reader is referred. -•Zeit. physiol. riiem., 1910 (69), 205. CIIOLIM-: a ROUP 123 Putrescine, C^HjoN^,, structural formula, NH,— CH„— CH:;— CH„— CH,— XI r,, and cadaverine, C-II14N.,, structural formula, NHo— CHo— CH,— CH,— CH,— CH,— NH„ are of interest because they have been found in the intestinal con- tents, arising" from putrefaction of proteins, and also are sometimes present in the urine in cystinuria.''' They are closely related to the diamino-acids, lysine and ornithine. They are but slightly toxic, although capable of causing local necrosis when injected subcutane- ously. (See further discussion on these and the Pressor Bases in Chap, xix.) The Choline Group. — Another group of ptomai'ns, including choline and closely related substances, is also of interest. These ptomai'ns are: Choi ine, CH^OH— CH„— X ( CH3 ) 3— OH Xeurine, CH„=CH— N ( CH, ) 3— OH IMuscarine, CH ( OH ) ,— CH„— X ( CH, ) 3— OH Betaine, COOH— CH,— X^ ( CH3 ) 3— OH The first point of importance is that choline is present in every cell normally, forming the nitrogenous portion of the lecithin mole- cule. Its source in putrefaction of tissues is, therefore, plain. It is possible' that choline is liberated from nerve tissues when they break down in the body during life,'^® and there is a considerable literature on the supposed finding of choline in the blood and cerebrospinal fluid in diseases of the central nervous system and experimental lesions in nervous tissues. At present it seems probable that these observations depend upon faulty methods of analysis, and it is ex- tremely doubtful if enough choline is ever set free at one time from even severe acute nervous lesions to be detected in the body fluids by chemical means.'^" Hunt ''-'' has devised a physiological test that per- mits of the detection of as little as 0.00001 mg., but he was unable to obtain evidence that choline is of any significance in either physiologi- i^al or pathological processes. Normally the largest amounts by far are obtained from the adrenals, which also seem to contain choline deriva- tives of much greater physiological activity. Choline itself is some- 73Udranszkv and Baumann, Zeit. phvsiol. Chem., ISSn (1.3), 562: 1880 (15), 77. 70 Coriat (Amor. Jour, of Physiol., 1904 (12). .35.3) has studied the conditions \inder wliich choline niav lie produced from lecitliin. Putrefaction of lecitliin or lecithin-rich tissues liberates choline, as also does autolysis of brain tissue; neither pepsin nor trypsin, however, splits it from the lecithin. Tn brain tissue, therefore, there seems to be an enzyme different from trypsin, whicli splits choline out of the lecitliin molecule. 77 See Webster, Biocliem. .Tour., inon (4), 123; Kajiura, Ouart. Jour. Exper. Physiol., inOS (1), 201; Handelsmann, Dent. Zeit. Xervenheilk., 1908 (35), 428; Doree and Golla, Biochem. .Jour., 1910 (5), 306. 77a Jour. Pharmacol., 1915 (7), 301. 124 CHEMISTRY OF BACTERIA AND THEIR PRODUCTS what toxic, but the closely related body, neurine, into which it may be transformed, is highl}' poisonous, which makes choline an important indirect source of intoxication. It is possible, for example, that lecithin taken in the food splits off choline in the gastro-intestinal tract, and this being converted into neurine gives rise to intoxication which may be ascribed to food intoxication. Likewise it has been suggested that the intoxication of fatigue may be due, at least in part, to choline and neurine produced from lecithin decomposed during the period of cellular activit}^ The close structural relation to choline and neurine, of the mushroom poison, muscarine, which produces physiological effects very similar to those of neurine, indicates the close relationship of the putrefactive ptomains and the vegetable alkaloids. Indeed a muscarine apparently- identical with that of the mushroom has been found in decomposing flesh, and neurine, presum- ably derived from lecithin, may be found in human urine. '^^ Betaine, the fourth member of the group, which has but slight tox- icity, is particularly well known as a constituent of plant tissues; possibly betaine or other basic bodies may occur substituted for choline in certain varieties of lecithin (Lippmann). Both neurine and muscarine are extremely poisonous and quite similar in their effects. Subcutaneous injection of but 1 to 3 mg. of muscarine in man produces salivation, rapid pulse, reddening of the face, weakness, depression, profuse sweating, vomiting, and di- arrhoea. Neurine, likewise, causes salivation, lachrymation, vomiting, and diarrhoea. In fatal poisoning respiration ceases before the heart stops. Both poisons resemble phj'sostigmine in their stimulation of secretion and are equally well counteracted by atropine. The toxicity of these substances is so great that not a large amount would need to be formed by oxidation of choline to produce severe symptoms, altliough it is not kno\\ai that this actually occurs in the body. When introduced by mouth, the lethal dose of neurine is ten times as great as when injected subcutaneously, indicating that chemical changes in the gastro-intestiiial tract or liver offer some protection against in- toxication by these substances when taken in tainted food. Choline, although by no means so poisonous as neurine, has a similar action when administered in sufficiently large doses. According to Brieger, it is about one-tenth to one-twentieth as toxic as neurine.'''' Choline seems to be rapidly destroyed in the body, not aj^pearing in the urine **^ but forming formic acid and perhaps glyoxylic acid. Donath ''^ 78Kutscher and Loliiiiaiiii. /oil. pliysiol. C1u>mi.. 1000 (48). 1. 70 Halliburtxm, "Cliciii. of Musclo and Xorvo," 1004, p. 110. states that elioline produces a fall in blood |)ressure by dilatinj; tlie porii)heral vessels, whereas neurine constricts tlie periplicral vessels; he uses tliis dillerence in pliysiolopical elTeet as a means of distinjiuisliinLr the two substances. Injected into animals, choline causes a consideralile liut transient decrease in tlie number of leucocytes in tlie blood, followed later 1)V an increase (Werner and Liehtenberg, Deut. med. Woch., 1000 (32), 22). 80 V. Iloesslin, Ilofmoister's Beitr., lOOG (8), 271. TOXfxs 125 fouud that choline injected directly into the cortex or under tlie dura is extremely toxic, causing severe tonic and clonic convulsions, and believes that choline may be responsible for epileptic convulsions. This view has been opposed, and properly so, by Handelsmann "^ and others. The attempt to ascribe importance to choline as a cause of cither toxic or therapeutic effect of j"-rays seems also to be entitled to but slight consideration.*- It is probably a factor in the lowering of blood pressure which results from injection of extracts of various tis- sues, in which it is commonly present in minute amounts,-' for very minute amounts of choline will produce a decided fall in blood pres- sures^ The Pressor Bases. — By decarboxylation of amino acids, amines are obtained, and some of them, notably those derived from leucine, tyrosine, phenylalanine and liistidine, have a marked effect on non- striated muscle. These are discussed in Chapter xix. TOXINS Certain bacteria produce soluble poisons by sj'nthetic processes, which poisons are secreted into the surrounding medium and repre- sent the chief poisonous products of the bacteria, being capable of causing most or all of the symptoms attributed to infection by the specific bacteria that have manufactured them. To this class of solu- ble poisons the term toxin has now become limited (for reasons that will be mentioned below), including not only toxins of bacterial ori- gin, but also poisons of similar nature produced by animals (snake venoms, eel serum, etc.) and by plants (ricin, abrin, crotin). The chief bacteria secreting true toxins are B. diphtherke, B. tetani, B. ■pyocyaneus, and B. hotulimis (not including bacteria producing hem- olytic substances resembling toxins). It will be seen that the tenn toxin has been greatly narrowed since the time when all ptomai'ns and other poisonous bacterial products were called toxins, until now it ha.s come to include the specific poisons of but four of the great group of pathogenic bacteria.*^ Chemical Properties of Toxins. — The chemical nature of the toxins is entirely unknown. Hy various precipitation methods they may be carried down, but included with them are masses of impuri- ties, chiefly proteins. They behave like electro-positive colloids,®^ but siZeit. f. physiol. Chem., 1003 (39), 526; also see Mcd« Xews, 100.5 (86), 107, for literature and methods of analysis. 82 See Schenk. Deut. med. Woch.', 1910 (36), 1130. 83 Schwarz and Lederer, Pfliiger's Arch., 1008 (124), 3.)3 ; Kinosliita, ihi. 05. ENDOTOXINS 129 the cellular constituents (receptors) -which bind the haptophure o-roups to the cells, these excessive receptors being secreted into the blood, where they combine with the toxin or enzyme so that it cannot enter into combination with the cells. This "side chain theory" of Ehrlich has been a useful working hypothesis, although it is becoming highly probable that it does not picture the exact method of toxin and antitoxin action."^'* Immune substances cannot 1)C produced against ptomdins, or for that matter against the vegetable alkaloids, or against any chemical bodies of knoicn constitution. Another difference between the action of toxins and simpler chemical poisons is, that while with the latter the effects are produced in a very short time after injection, there is a latent period of several hours before symptoms appear after inject- ing toxins. What occurs during this latent period is not fully known, but that there is a latent period suggests a resemblance to enzyme action. An alkaloidal or other chemical poison enters the cell, and its harm is done at once. A toxin combines with the cell, and then, if it produces its effects by an enzymatic alteration of the cellular struc- ture, some time must elapse before the changes are great enough to cause the appearance of symptoms. ENDOTOXINS og By far the greater number of pathogenic bacteria do not secrete their poisons as toxins into the surrounding medium, although they manifestly cause disease by poisoning their host. Among them are such organisms as the typhoid bacillus, pneumococcus, the pus cocci, cholera vibrios, and many others. If cultures of these organisms are iiltered, the tiltrate will be found to be but slightly toxic (except for the hemolytic poisons), although the bodies of the bacteria after they have been killed by chloroform or other antiseptics are highly poison- ous if injected into an animal. These bacteria, then, produce poisons which do not escape from the cells into the culture-medium, but are firmly held within them. By using various means these intracellular toxins, or endotoxins, can be obtained independent of the bacterial cells. One of these is to grind up the cells, which can be particularly well done if they are first made brittle by freezing at the temperature of liquid air (MacFadyen's method). By very great pressure in the Buchner press the cellular contents can be expressed. They may also be obtained by letting the bacteria autolyze themselves for a short time in non-nutrient fluids (Conradi," et al.) Endotoxins obtained in this way are soluble and highly poisonous, and it is undoubtedly through their action that the characteristic diseases are produced by the bacteria that contain them. Presumably the endotoxins are liber- 95a See Coca; Jour. Infect. Dis., 1915 (17), 3.51. 96 See general review by Pfeiffer, Jahresber. d. Innimnitatsforsch.. 1910 (6). 13. 97Deut. med. Woch., 1903 (29), 26. 9 130 CHEMISTRY OF BACTERIA AND THEIR PRODUCTS ated in the body either by autolysis, or by heterolysis by the enzymes of the body cells and fluids, and there is some question as to whether they are preformed specific constituents of the bacteria, or merely the poisonous product of enzymatic disintegration of the bacterial proteins, similar to the " anaphylatoxins. " "^'^ Endotoxins differ from the true toxins, however, in one important respect: namely, it is difficult or impossible to obtain cm antitoxin for endotoxins by immunization of animals°^ Animals immunized against endotoxins develop in their serum substances that are bacteri- cidal and agglutinative to the bacteria from Avhich the poisons are derived, but the serum will not neutralize the endotoxins. As a re- sult, we are unable to perform experiments indicating whether endo- toxins have the same structure as the true toxins, i. e., a haptophore and a toxophore group, but presumably their nature is different in some essential particular. The chemical nature of the endotoxins is also unknown, for they are always obtained mixed with the other con- stituents of the bacteria.'*'' Tuberculin, once supposed to be an albumose, is produced even when the bacilli are grown on a protein-free medium, and in the active solu- tion no albumose or other protein is then found. Hence it seems probable that tuberculin is of the nature of a polypeptid, which gives no biuret reaction but is destroyed by pepsin and trypsin, according to Loevenstein and Pick,^ but not by erepsin (Pfeiffer).- Whether tuberculin should be considered an endotoxin liberated by the disin- tegration of the bacilli in the cultures is unknown ; Pick looks upon it as a secretion of the bacilli, and closely related to the true toxins. Since far more bacterial diseases are brought about by endotoxins than by true toxins, the failure to secure antitoxins for these sub- stances has been a great check in the progress of serum therapy, and the problem of the endotoxins is one of the most important in the en- tire field of immunity. 97a See Dold and Hanau, Zeit. Immunitiit., 1913 (10), 31; Zinsser. "Infection and Resistance," N. Y., 1914, Chap. xvii. 98 Positive results are claimed by Besredka (Ann. Inst. Pasteur, 1906 (20), 304). and some others; see Kraiis, Wien. klin. Wocli.. 1900 (19). 6,5,') : Zeit. Immunitiit., 1909 (3), 646. It is suggested by VVassermann (Kolle and \Yasser- maim's Ilandbuch, 1912 (2), 246) that this dilTiculty in obtaining antiondoloxins depends on the large size of the molecule, — the small diffusible toxin molecule is so altered in its physical condition tlirough union with the antibody that its properties are much altered, whereas the large endotoxin molecule must be di- gested by com])lement before its toxicity is destroyed. 99 'J'lie Afi(/7-eNsivs of Pail, to which lie ascrilies the pathogenicity of bacteria, are too little esta])lished to permit of a discussion from the chemical standpoint. By many they are Ixdieved to be iiolhing more tluin endotoxins. (Literature given by Miilier, Oppenhcimer'a Ilandb. d. Piochem., 1909 (II (1) ), 6S1 : Dud- geon, Lancet, 1912 (1S2), 1673). According to Ingravelle (Ann. d' ig. sperim., 1910 (20), 483), typhoid aggressins arc found in the albumins. 1 Piwhem. Zeit.," 1911 (31), 142. 2 Wien. klin. Woch., PMl (24), 11 If); see also Lockiiiaiiii. Zeit. pbvsiol. C'liem.. 1911 (73), 389. POISONOUS BACTERIAL PROTEINS 131 POISONOUS BACTERIAL PROTEINS If we filter a bouillon culture of diphtheria bacilli through porce- lain, wash thoroughly with salt solution the bacteria remaining, and collect them thus freed from their secretion products, it will be found that extracts of the bacterial substance or the bodies of the killed bac- teria themselves are quite free from the typical toxin. This indicates that the toxin is eliminated from the bacteria as fast as it is formed, and no considerable quantity is retained within the cell. The bac- terial substance, however, or proteins isolated from it, is found to pro- duce severe local changes when injected into the bodies of animals, necrosis and a strong inflammatory reaction with pus-formation being the chief features. This local effect is not a specific property of the diphtheria bacillus, for other bacterial proteins, including proteins from non-pathogenic bacteria, will produce the same changes; in- deed, many proteins that are derived from vegetable and animal sources have equally marked pyogenic properties. All foreign pro- teins when introduced into the circulation of animals are more or less toxic, and the toxic effects of the bacterial proteins are, for the most part, neither specific nor particularly striking. There are a few pathogenic organisms, however, which seem to produce neither true toxins nor endotoxins, notably the tubercle bacillus and the anthrax bacillus, and with these there may be a relation between their protein constituents and their specific effects. Numerous protein substances have been extracted from bacterial cells, particularly nucleoproteins, but also proteins resembling al- bumins, nucleo-albumin, and globulins. In all probability the chief proteins of the bacterial cell are nuelein compounds, wliicli is indi- cated both by their nuclear staining and by the analyses of Iwanoff ; * and many of the nucleoproteins, both of bacterial and non-bacterial origin, cause considerable local inflammatoiy reaction when injected into animals. Tiberti * claims that vaccination with non-lethal doses of the nucleoproteins of anthrax bacilli will protect animals against inoculations of virulent anthrax bacilli. Some of the earlier observa- tions on the toxicity of bacterial proteins were erroneous because im- pure proteins, containing toxins, endotoxins, and ptomai'ns were used. Schittenhelm and Weichardt ^ have found, however, that bacterial proteins are much more toxic than any ordinary proteins, as indi- cated by loss of nitrogen, temperature changes and alterations in the leucocytes of injected animals. Vaughan and his students have been able to split off from the bodies of various pathogenic bacteria toxic materials which are stated to resemble in some respects the protamins,^^ although they do not all 3 Hofmeister's Beitr., 1002 (1), 524. 4 Cent. f. Bakt., 1006 (40), 742. sMiinch. med. Woch., 1011 (58), 841. 5a A full review of this work is given in Vaucrhan's "Protein Split Products," Philadelphia, 1013; and in Jour. Lab. Clin. Med., 1916, Vols. 1 and 2. 132 CHEMISTRY OF BACTERIA ASD THEIR PRODUCTS give a satisfactory biuret test. These toxic materials are evidently quite different from either the true soluble toxins or the endotoxins, since they resist heatinp: for ten minutes, at 110° in the autoclave with 1 per cent, sulphuric acid, this being- a method used for securing the substance. Since the sarcinas and B. prudigiosus also yield similar toxic products, they cannot be considered as the specific toxic substances of the pathogenic bacteria, but apparently are com- mon to all proteins of whatever origin. With some bacteria the split- ting process with sulphuric acid separates completely the toxic from the non-toxic insoluble bacterial substance,'' e. g., B. coli communis; with others a toxic portion remains insoluble. The colon bacillus pro- tein gives all the protein reactions, is synthesized on Uschinsky's medium, and does not yield a reducing carbohydrate. From B. fifphosiis about 10 per cent, by weight of protein can be split off by dilute acid, of which at least a part seems to be a phospliorized glyco- protein.'^ Poisonous substances have also been obtained from B. diph- therice, B. anthracis, B. tuherculosis ^ and B. pyocyaneus. They pro- duce death without the usual latent period obsen'ed with toxins, and are very toxic, a few (10-20) milligrams of colon bacillus poison kill- ing guinea-pigs in less than ten minutes.'' A certain degree of immu- nity can be obtained against them.^° Their relation to endotoxins has yet to be determined. BACTERIAL PIGMENTS i^ The formation of pigment by bacteria seems to be, for the most part, an adventitious, unessential property. There are a few bacteria wliicli possess pigments of the nature of chlorophyll, or allied to it, and this pigment is undoubtedly of great importance in the life processes of these particular forms. Other varieties of pigment- forming bacteria, of which but very few are pathogenic {Bacillus pyocyaneus, B. proteus fluorescens, S. pyogenes aureus and citreus, M. cereus flavus), seem to produce pigment as a waste product which is excreted from the cell as fast as formed. Generally the pigments are produced in a colorless form (leuco-hase) which is oxidized by the air into the pigment, e. g., in pyocj/aneus infections the soiled dress- ings are most colored about the portions most exposed to air. Since pigment-forming bacteria produce pigments only under certain condi- tions, and can grow abundantly without producing any pigment, it is evident that the pigment formation is no very essential part of llicif iiK'tjiholisiii. It is ])()ssi])](' to modify pigment ])r()ducti()ii al- G Wlioelcr, Jour. Amor. ^Tcd. Assoc, 1905 (44), 1271. ■! Ihid., in04 (42). 1000. «S(>o White and Avery, Jour. IVfed. Pxes., 1912 (20), 317. 0 Jour. Amcr. ]\Ied. Assoe.. lOOfi (44), 1.340; American Medic iiie. inO.l (10). 14"!. lOVaufrhan (Jr.), Jour, of INIed. riesearch, 1905 (14), ()7. 13 For complete bibliography and r^surn^ see Sullivan. Jour. ^led. Research, 1905 (14), 109. BACTERIAL PIGMENTS 133 most at will, and even to develop races of bacteria that do not produce pigment at all, from races that ordinarily are pigment-producers. Of nuniorous classifications of pigment-forming bacteria, all faulty because of our slight knowledge of the chemistry of the process, that of Migula seems the best; it is based on the solubility of the pigments formed, as follows: (1) Pig-ments Soluble in Water. — This includes the pigments of all fluorescent bacteria, as well as those giving a red or brown color to gelatin media. Most important among these is Bacillus pyo- cyaneus, whose pigments have been considerably studied. There seem to be two pigments, one, pyocyanin, characteristic for this organism; and a fluorescent pigment which numerous other organisms also pro- duce. Pyocyanin has been analyzed by Ledderhose, who found it to be a ptoma'in-like body, a derivative of the aromatic series, probably re- lated to the anthracenes. It can be reduced to a colorless leuco-base, in which form it is probably produced by the bacteria, and then is oxidized in the air into the pigment. Its composition is Cj^Hj^NoO (the sulphur-containing pyocyanin which has been described is proba- bly impure). The fluorescent pigment is insoluble in alcohol and chloroform, and can thus be separated from pyocyanin, which is solu- ble in chloroform. Although related to the ptomains, pyocyanin seems to be altogether non-poisonous to animals. Jordan ^* and Sullivan ^^ have studied the conditions under which pigments are formed, and found that pyocyanin can be produced in protein-free media, and without the presence of either phosphates or sulphates; but both sulphur and phosphorus must be present to pro- duce the fluorescent pigment. As pigments can be produced on media containing only ammonium salts of succinic, lactic, or aspartic acid, or asparagin, they are evidently formed synthetically, and not by cleavage of the media. (2) Pigments Soluble in Alcohol and Insoluble in Water. — The most important bacteria of this group are the Staphylococcus pyogenes aureus and citreus. Their pigment is of a fatty nature, a lipochrome, which lies among the bacteria in the form of dendritic crystals. Be- ing a fat, it can be saponified, and when decomposed it gives the acrolein reactions and odor, from the breaking down of the glycerol of the fat molecule. Acted upon by strong sulphuric acid, the yel- low pigment changes into blue granules and crystals {lipocyanin re- action). The lipochromes are soluble in the usual fat solvents, and fonn fat spots on paper. (3) Pigments Insoluble in Water and in Alcohol. — The pigment of Micrococcus cereus flavus belongs to this class; its nature is quite un- known. i*Jour. Exper. Med., 1899 (4), 627. CHAPTER V CHEMISTRY OF THE ANIMAL PARASITES ' This subject has received muck less consideration than its import- ance deserves, and we are quite in the dark as to how much of the effects produced by animal parasites are not merely mechanical, but are due to soluble poisons that they may secrete or excrete. Some of the parasites probabl.y cause harm mechanically and in no other way, but with most of them there is more or less evidence of the forma- tion of poisonous substances. The composition of the bodies of the animal parasites is an almost unexplored field, but we have no reason to believe that the composition of the cells of invertebrates differs essentially from that of the cells of higher organisms. Perhaps the most characteristic constituent observed in many forms is chit in, which forms a large part of the outer covering of the encysted forms, and probably of many of the worms. Glycogen is usually abundant in the invertebrates, and the animal parasites form no exception,- this carbohydrate having been found in their bodies by many observers. Eosinophilia. — One of the most characteristic features of the animal parasites is that they exert a positive chemotaxis, particularly for eosinophile leucocytes.^ An increase in the number of these cells in the blood, as well as a local accumulation in the tissues nearest the parasite, has been observed in infection with practically all the animal parasites.* Of these, infection with Trichinella spiralis causes the most pronounced eosinophilia, presumably because of the great number of parasites present in the tissues at once. That the eosinophilia is due to the ac- tion of the soluble products or constituents of the parasites has been shown by experimental injection into animals of extracts from the bodies of the parasites. Calamida lias found that extracts of dog tapeworms also, when placed in the tissues in a capillary tube, cause an accumulation of eosinophile cells in the tube.^' Experimental in- fection with excessive numbers of trichinella causes a rapid diminu- 1 General references to this subject will be found in v. Fiirtli's "Verirli'ichende chemische Physiolofjie dor niederen Tiere." .Tena, 190.3; Faust's "Tierische Gifte."' Braunscliweip, 100(»; Kocb. Er<;ebnisse Pathol.. 1010 (xiv (1) ), 41. 2 See Pniifrer, Pniiper's Arch.. 190:? (OH), l.l.'^ 3 Mtorature bv Oi)ie. Amer. .Tour. Med. Sci., 1904 (127). 477: Rtiiubli. Deut. Arch. klin. Med." 1906 (S.5), 280; Hubner, ihid., 1911 (104), 2HG; Schwarz. Frgeb. allfr. Pathol.. 1914 (17,). 1.38. 4 Litcratino by Pruns. Liefmann and Miickel, Miinch. nied. Wocli.. 190,") (ri2), 253: Vallillo, Arch. wiss. u. prakt. Tierhk., 1908 (34), 50.'). 0 Nefrative results were obtained with extracts of flclrrostoiiia cipniiiiu) by Grosso (Folia llcniatol., 1912 (14), 18). 134 PROTOZOA 135 tiou iu tlie number of eosinophile leucocytes, Avhicli also show evi- dences of disintegration in the bone-marrow and lymph-glands. Such large injections are fatal, which suggests that the eosinophilia has a protective influence. In favor of this view is the observation of Milian,*^ who found that sarcosporidia in beef are destroyed by a violent leucocytic reaction, the prevailing cell being the eosinophile. As the eosinophile increase does not occur until several days after the infected flesh is eaten, the chemotactic substance is not liberated from the encapsulated trichinella? when tlieir capsules are digested ofit" in the gastric juice, but comes either from the free larvae, or from the degenerated muscles in which they burrow. Coincident bacterial infection may reduce the number of eosinophiles. Herrick " finds that extracts of Ascaris himhricoides cause a notable eosinophilia, but only when the animal has been sensitized previously with the same extract, the active agent of which is a protein ; this suggests a rela- tionship between parasitic and anaphylactic eosinophilia.'^^ That the eosin()j)hiles play a part in the immunity reactions obsei"\'ed in the hosts of animal parasites is indicated by the fact that hydatid fluid loses its antigenic properties when in contact with eosinophiles.'*^ PROTOZOA These unicellular forms possess all the chemical characters of the (^ells of higher forms, even to the more specialized constituents. Thus it has been demonstrated that protozoa contain proteolj^tic enzymes,^ and that they secrete an acid into their digestive vacuoles.^ On the other hand, Amcfha coll does not seem to digest the red corpuscles and the bacteria that it takes up.^° Whether the Anmha coli produces any toxic materials, specific or non-specific, has not yet been deter- mined, but the necrosis that it produces in liver abscesses, when bac- terial cooperation can often be excluded by culture, strongly indi- cates the production of necrogenic substances. Apparently these sub- stances are not chemotactic, in view of the absence of leucocytic ac- cumulation which is characteristic of the lesions of amebic dj^sentery. There is also no evidence, clinical or experimental, that amebic in- fection causes the formation of anti-substances of any kind in the body of the host. The spontaneous recovery from amebic and other protozoan infections, however, may be considered as indicating the development of an immunity against these organisms.^^ Numerous 6 Bull, et Mem. Soc. Anat.. 1901 (Ser. G, T. 3i, 32.3. 7 Arch. Int. Med., 1913 (11), 165. Ta Supported liv Paulian, Tresse IMed.. 191.1 (23), 403. ■b Weinberg' and Sepuin. Ann. Inst. Pasteur. 1916 (30), 323. sMouton. Conipt. Rend. Soc. Biol., 1901 (.13), 801. 9 Le Dantec, Ann. Inst. Pasteur, 1890 (4), 776; Greenwood and Saunders, .Tour, of Phvsiol., 1894 (16), 441. Jo^fusgrave and Clegg, Bureau of Gov't. Laboratories, ^lanila, 1904, Xo. 18, p. 38. 11 Concerninfr immunitv to protozoan infections see Schilling-, Kolle and Wasser- mann's Handbuch, 1913 '(7), 566. 136 CnEMISTBY OF THE AXIMAL PARASITES observers have suggestecl the possil)ility of obtaining artificial im- munity against protozoa, and Rossle ^- has obtained immune sera against infusoria. The serum of rabbits immunized against amoebae was found by Sellards ^^ to be cytolytic for the same amoebae, but no antibodies could be found in the blood of patients with amebic dysentery. Novy ^* has obtained immunity against trypanosomes, but the serum of immune animals will not confer passive immunity. Braun and Teichmann,^^ however, claim positive results with immune serum from rabbits; they found no poisonous agent in trypanosome sub- stance.^^" The fact that trypanosomes themselves readily become im- mune to various trypanocidal chemicals has been demonstrated and extensively studied in Ehrlich's laboratory. Gonder ^"^ has made the niteresting observation that trypanosomes which can be stained by certain vital stains, become unstainable while alive if immune to arsenic compounds, suggesting that this immunity is associated with considerable structural or chemical changes. Plasmodium malarise undoubtedly produces toxic substances, which seem to be of such a nature that they do not diffuse from the red corpuscle, but are only liberated when the corpuscle breaks up on the maturation of the parasite. In this way the characteristic par- oxysmal manifestations of the disease are produced. The nature of the poison or poisons is unknown, but we have evidence that it is hemolytic, since malarial serum may hemolyze normal corpuscles,^" and extracts of the parasites are strongly hemolytic (Brem^®) ; prob- ably the malarial hemoglobinuria is caused by this hemolysis. Pre- sumably malarial poisons are not extremely toxic for parenchymatous cells, since the parenchymatous lesions in malaria seem to be relatively slight as compared with the intensity and duration of the intoxication. Some authors state that the toxicity of the urine is increased after the paroxysm,^** which, however, does not necessarily indicate that a poison formed by the parasites is excreted in the urine. Immunity seems to be seldom developed against the malarial poison or against the parasite itself, although some persons seem to be naturally im- 12 Arch. f. Hvfj., 1905 (54), 1; full review of this topic. 13 Philippine" Jour. Sci., 1911 (6). 281. 14 Jour. Infec. Dis., 1912 (11). 411. isZcit. Immunitiit., Ref., 1912 (6), 4G5. • isallintze (Zeit. f. Hyp:., 1915 (80), .377) obtained little immunity with T. hrucei. hut Schillinfj and Rondoni (Zeit. Immunitiit., 1913 (18), 651) obtained a yxiison from Napana trypanosomes which produced active immunity in mice. When trypanosomes are killed by weak electric currents they may liberate an active poison (Uhlenhuth and Sevderhelm, Zeit. Tmiminitiit., 1914 (21), 30()). 10 Zeit. Immunitiit., 1913 (15)," 257. 17 See Repnault, Revue de IVIed.. 1903 (23), 729. 18 Arch. Int. Med., 1912 (9), 129. 19 Quoted from Blanchard, Arch. d. Parasitol., 1905 (10), 83; this article pives a resume of the subject of the toxic substances produced by the animal parasites. CKHTOliEK l^*^ mune while some acquire innnunity through previous nifection.- The blood of persons with malaria seems to contain no antibodies tor the parasite (Ferrannini)r^ although it seems to have some antihemo- lytic power (Brem). (Concerning the pigment present m the ma- larial parasites see "Pigmentation," Chap, xvi.) , , ,, ^ Sarcosporidia of sheep yield aqueous and glycerol extracts that are hiohlv toxic for rabbits (Pfeiffer), the poisonous constituent of which %as called sarcocusiin by Laveran and ^lesnil.- Tins is so highlv toxic that 0.0001 gm. is fatal to rabbits (per kilo), other ani- mals "being less susceptible. It loses its toxicity on heating at 80 for twentv minutes, and is impaired at 55-57° for two hours It produces pruritis and other anaphylactic symptoms, and although the serum of sheep with this parasite does not confer passive anaphylaxis to sarcosporidia, vet it does give positive complement fixation.-- Ihat it is a true toxin is shou^n by Teichmann and Braun,-^ who produced an effective antitoxin by immunizing rabbits; only rabbits seem to be susceptible to the toxin. The sarcosporidia contain also a distinct theinnostable agglutinin. The lethal dose of dried substance of sar- cosporidia is. for rabbits, but 0.0002 gm., and the poison seems to unite with the lipoids of the nervous system (Teichmann).- it is probable that the pathogenic protozoa, at least in some instances have a semipermeable membrane about them, for Goebel -^ found that trypanosomes are very susceptible to changes in osmotic conditions. CESTODES T^nia echinococcus has been by far the most studied, its abundant fluid content furnishing suitable material for investigation. That this fluid is toxic has been repeatedly observed when, through rup- ture or puncture, the fluid has escaped into the body cavities; such accidents are aften followed by violent intoxication, sometimes by death =" As long as the cvst is unopened no toxic manifestations are observed The most constant symptoms are local irritation and in- flammation, accompanied by urticaria, which may also be produced experimentally in man if the cyst contents are injected subcutane- """"The svmptoms are so strikingly similar to those of anaphylactic intoxication, that it is now generally believed that they are the result of such a reaction in a pei^son sensitized by absorption of antigenic substances from the cyst." Carriers of echinococcus cysts have been 20 See Celli. Cent. f. Bakt.. 1900 (27). 107. 2iRiformaMed.. 1911 (27), 177. 22Compt. Rend. Soc. Biol.. 1800 (51). .311. 22a McGowan. .Tour. Path, and Bact., 1913 (18), \lo. 24 7b7rf ' l01o7''0)*'."96: see also Knebel. Cent. f. Bakt., 1912 (66), 52.3. 25Ann.*Soe. Med. d. le Gand. 1900 (86). 11. 26Rpp 4ehard \rch "en. de Med.. 1887 (22), 410 and 5(2. 27!:: BoS^in and Lar'-oche. Presse Med., 1910 (18), 329; Ghedini and Zamorani, Cent. f. Bakt., 1910 (55), 49. 138 CHEMISTRY OF THE ANIMAL PARASITES found to have in their blood antibodies giving precipitin ^^ and com- plement fixation -" reactions with extracts of echiuococcus, and some- times with other taenia.^" The antigen of the echinococcus is be- lieved by some to be a lipoid ;^^ in the case of Taenia sagitiata, at least, it seems to be associated with the lecithin (Meyer ^"). Graetz,^^ however, states that the protein of the hydatid cyst is derived from the host, and that it is therefore incapable of causing anaphylaxis in that host, but it may undergo alterations in the cyst so that it is toxic after the order of anaphylatoxins {q. v.). The complement fixa- tion reaction with echiuococcus fluid has been found quite reliable in the clinic, 93 per cent, of positive reactions having been obtained in 500 cases collected by Zapelloni,^-'' while controls were always negative. The fluid of the echinococcus cysts has generally a specific gravity of 1005-1015, and contains 1.4-2 per cent, of solids. Most abundant are sodium chloride, about 0.8 per cent., and sugar, 0.25 per cent., the latter presumably coming from the glycogen contained in the wall. Cholesterol is often abundant, while inosite, creatin, and suc- cinic acid are often found. Clerc has found traces of lipase, but other enzymes seem to be absent or in very small amounts. Proteins are present only in traces, unless inflammation has occurred. Schil- ling ^^ found the molecular concentration of the cyst fluid to be quite the same as that of the patient's blood. The fluid is said not to be toxic to laboratory animals.^* The cyst wall consists of a hyaline substance which seems to stand between the chitin and the proteins, and probably consists of a mix- ture of both. Because of the chitin it yields about 50 per cent, of a reducing, sugar-like body when boiled with acid. Glycogen is also usually present, but it is limited to the germinating membrane.^^ Other cestodes, when in the cystic form, contain fluids which are more or less toxic. Thus Moursou and Schlagdenhauffen ^^ found a "leucoma'in'' in the Cysticercus tenuicollis, the larva of Taenia mar- ginata, which causes urticaria and other toxic symptoms when in- jected into animals. The fluids of Cysticercus pisiformis (the com- mon cestode of rabbits) have been found toxic for frogs, and Vaulle- geard ^^ has determined the presence of an "alkaloid" and a ''fer- ment toxin" in this fluid. The fluids of the cysts of Caenurus cere- hralis, Coenurus serialis, and Echinococcus polymorphiis have all been 28 Welch, et al., Lancet, 1009, Apr. 17. 29Kreuter, Miinoh. mcd. Woch., 1900 (50), 1828; Weinberg, Ann. Inst. Pasteur 1909 (2.3), 472. 3o\Ii.v(.r, Berl. klin. Woch., 1910 (47). 1310; Zeit. Inmiunitjit., 1910 (7). 732. 31 Israel, Zeit. ITyg., 1910 (00), 487; Meyer, Zeit. Immunitiit.. 1911 (9), 530. 32 Zeit. Iiniiumiliit., 19)2 (15), GO: general review. 32aPoliclinico, Surg., 1915 (22), Nos. 0-11. 33 Cent. inn. Med., 1904 (25), 833. 340raet/„ Cent. f. Hakt., 1910 (55), 234; ZiMt. IniimiMitiit .. 1912 (15), 00. 85 Brault and Loeper, Jour. Phys. et Patli. g^n., 1904 (0), 295. soCompt. Rend. Soc. Riol., 1882 (95), 791. 37 Bull. Soc. linneenne de Normandie, 1901 (4), 84. CESTODES 139 found toxic, and it is probable that this is a general rule with the cestodes,^^ but human forms other than the echinoeoccus seem not to have been investigated ; ^^ according to Jammes and Mandoul, extracts of taniia are bactericidal.^" Dibothriocephalus latus frequently causes anemia, which has been attributed to a poison liberated by the parasite when it undergoes disintegration, and possibly as a secretion of the living worm.*^ All the intestinal cestodes are equipped with a well-developed excretoiy apparatus, and it is easy to imagine that their excretory products may be toxic to the animal into whose intestine they are excreted. Tallqvist ^- has made extensive studies of bothriocephalus, which show that the active hemolytic agent is contained in the lipoids of the parasites, presumably as a cholesterol ester of oleic acid.^^ The proglottides contain a proteolytic enzyme, which apparently digests the substance of dead segments, liberating the hemolytic lipoid, which constitutes about ten per cent, of the solids of the parasite. There is also a hemagglutinin, which, unlike the hemolytic substance, is thermolabile, and causes the appearance of an antibody in immunized animals. In common with other parasites, antitrj^jtic and antipeptic effects are exhibited by extracts. Rosenqvist ** has studied the metabolism of twenty-one cases of bothriocephalus anemia, and found evidence in nearly all of a toxo- genic destruction of protein, which ceases promptly when the worms are removed. He has found that these worms produce a poison which is globulicidal, and probably also generally cytotoxic, since in the anemias that they produce, the elimination of purine bodies of tissue origin (endogenous purine) is increased. The nitrogenous metabolism is quite the same in pernicious anemia and in bothrio- cephalus anemia. Isaac and v. d. Velden ^^ state that the blood of patients infected with this parasite gives a precipitin reaction with autolytic fluid obtained from bothriocephalus, and that rabbits im- munized with such autolytic fluids developed a precipitin. Other Taenia. — There is much less evidence that other forms of taenia produce toxic substances which injure their host, although the clinical manifestations observed in persons harboring ta?nia are often of such a nature as to indicate strongly an intoxication. Jammes and IMandoul *® found no toxic manifestations produced by extracts of Taenia saginata, which negative finding is supported by Cao,"*" Tall- 38 Blanchard., loc cit. •^n vSemaine med., 190.5 (25), 55. •10 See also Joyeux, Arch. d. Parasitol., 1007 (11), 400. 41 Literature by Blanchard, Joe rif. 42Zeit. klin. iVied., 1007 (61), 427. 43 Faust and Tallqvist, Arch. exp. Path. u. Pharm., 1007 (57), .307 44Zeit. klin. Med., 100,3 (40), 103. ■*-> Dout. med. Woeh., 1904 (30), 082. 4fi Compt. Rend. Acad. Sci., 1904 (138), 1734. 47Riforma med., 1901 (3), 795. 140 CIIEMTFiTRY OF THE AMMAL PARASITES qvist and Boycott/** using various sorts of tienia. These results con- tradict the earlier positive finding's of Messineo and Calamida/'' who found extracts of taenia from dog's to be hemolytic, chemotactic (especially for eosinophilos), and to cause local fatty degeneration in the liver. Extracts of 2\ perfoliata and plicata (of the horse) were found highly toxic for guinea-pigs by Pomella,'^" the hema- topoietic organs being greatly stimulated. Bedson ^"^ found that ex- tracts of all sorts of helminths produced similar effects on guinea-pigs, the chief lesions being in the adrenals and thyroid. Possibly these dif- ferences in results are due to the fact that different parasites were studied by different investigators; furthermore, tests of toxicity of human parasites upon rabbits and guinea-pigs can hardly be consid- ered conclusive. Le Dantec did not find a precipitin for Taenia saginata extracts in the blood of persons harboring this parasite, and negative results with several other taenia were obtained by Langer,^^ but complement fixation reactions may be given. ^^ Picou and Ramond °^ state that tienia extracts undergo putrefaction very slowly, and attribute this to a bactericidal property, which was observed with several forms of t^nia by Allesandrini. AVeinland '"* has found that many intestinal parasites exhibit antitryptic proper- ties,^^' but in a study of the histological changes of autolysis I observed a taenia in the intestine of a dog undergo more rapid karyolytic changes than did the intestinal epithelium. Dastre and Stessano ^"^ state that extracts of Taenia serrata act upon enterokinase rather than on tryp- sinogen. NEMATODES Ascaris. — The toxicity of members of this group has been a matter of dispute, although, as with the Taenia, there have been observed in patients symptoms that were more easily explained as due to chemical substances than as due to mechanical irritation. INIiram, while study- ing Ascaris megalocephala, suffered from attacks of sneezing, lachry- mation, itching, and swelling of the fingers, v. Linstow suffered from a severe attack of conjunctivitis with chemosis after touching his eye with a finger that had been in contact with one of these worms. Others have had similar experiences, and it has been found that the fluid from these worms is toxic to rabbits. In man it seems to affect 48 Jour. Pathol, ajid Bacteriol., 1905 (10), 383. 49 Cent. f. Bakt., 1001 (;iO), 346 and 374. •"'OCompt. Rend. Soc. Biol., 1912 (73), 445. 50a Ann. Inst. Pasteur, 1913 (27), 682. •"•i Munch, med. Wocli., 1905 (52), 1665. •'••2Mever, Zeit. Imniuuitiit., 1910 (7), 732. saCompt. Rend. Soe. Biol., 1899 (51), 176. MZeit. f. Biol.. 1902 (44), 1 and 45. 5fj ('orrol)orated for Taenia sa")), ISTfl; Schwartz. Kri;i'b. allp. Pat.liol., lit 14 (17), l.^ti. BSbMiincli. med. Wocli., 1!)14 (01), 045. 58r.Tour. Amer. Mod. Assoc, 1010 (67), .')79. 58dStroebel, Miinch. mod. Woch., 1911 (58), G72. NEMATODES 143 of tliis loss of blood is explained by L. Loeb ''''' as due to the presence, in the anterior portion of the parasite (they studied Ankylostonia caniniim), of a substance that inhibits the coa^lation of the blood. However, Preti "^ would ascribe importance to a lipoidal or lipoid- like hemolytic constituent of the parasitic tissues of the European ankylostoma, but Whipijle,*"'^ who lias observed a weak hemolysin in the American hook worm, considers it too ineffective to be of practical importance. In Sclerostoma equinmn, however, Bondonoy "- found active hemolytic agents, ascribed by him to lipase ; also a ptomain, an alkaloid and other substances. Correspondinfj to Flury's analyses of ascaris, he found that the cuticle is albuminoid and not chitinous, and that the parasite produces much volatile fatty acids, especially butyric; both lecithin and cholesterol were absent. The dermatitis produced by uncinaria larva? is ascribed by C. A. Smith ^^ to an alco- hol-soluble substance. Wateiy extracts of Sclerostoma were found by Grosso "* to cause but slight chemotaxis without eosinophilia. Filaria seem not to produce any appreciable amount of toxic ma- terial, if we may judge by the slight evidence of intoxication shown by infected individuals. An exception may be made in the case of the guinea-worm {Dracunculus or F. medinensis) . This parasite causes chiefly mechanical injury unless its body is ruptured, which may happen in attempting to remove it forcibly; this accident is fol- lowed by violent local inflammation or gangrene, which indicates that some powerfully irritant substance is liberated from the torn body of the worm.^^ 59 Cent. f. Bakt., 1004 (37), 93; 1906 (40), 740; Loeb and Fleischer, Jour. Infec. Dis., 1910 (7), 625. eoMiinch. med. Woch., 1908 (5.5). 436. 61 Jour. Exp. Med., 1909 (11), 331. 62 Arch. Parasitol., 1910 (14), 5: see also Ashcroft, Compt. Rend. Soc. Biol., 1914 (77), 442. 63 Jour. Amer. Med. Assoc, 1906 (47), 1693. 64 Folia Hematol., 1912 (14), 18. 65 Earthworms are said by Yagi (Arch, internat. phaimacodyn.. 1911 (21), 105) to contain a hemolytic substance, "lumbricin," the properties of -which he describes. Nukada and Tenaka (Mitt. med. Fakult., Tokio. 1915 (14), 1), found an antipyretic agent which seems to be derived from tyrosine. CHAPTER VI PHYTOTOXINS AND ZOOTOXINS The production of substances possessing the essential features of true toxins is by no means limited to the bacterial cell. In the plant kingdom such substances are formed, and called phytotoxins. Of these, the best known are ricin, abrin, crotin, and robin. Among the toxins of animal origin, zootoxins, are the venoms of poisonous snakes, lizards, spiders and scorpions, and the serum of eels and snakes. These may be briefly considered as follows : PHYTOTOXINS The chief phytotoxins ^ are as follows : Ricin, from the castor-oil bean {Bicinus communis) . Abrin, from the seeds of Ahrus prccatorius. Crotin, from the seeds of C rot on tigliiim. Robin, from the leaves and bark of the locust, Bolinia pseudo- acacia. Curcin, from the seeds of Jatropha curcus. In their general properties all these substances are very similar and may be considered together. They resemble proteins in many re- spects, for they can be salted out of sohitions in definite fractions of the precipitate, are, precipitated by alcohol, and are slowly de- stroyed by proteolytic enzymes. For some time they were referred to in the literature as toxalbumins, until Jacoby stated that, by com- bining the salting-out method with trypsin digestion, he was able to secure preparations of ricin and abrin that did not give the pro- tein reactions. This seemed to pUice tliem in the same category with bacterial toxins and enz.ymes, /. r., large molecular colloids, closely resembling the proteins with which they are associated, but still not giving the usual protein reactions. Because of their great similarity to bacterial toxins tliis seemed a very probable description, and it has been generally accepted. More recent work by Osborne, Mendel, and Harris,- however, does not support Jacoby 's contention. They found the toxic properties of ricin associated inseparably with the coagulable albumin of the castor beans, and were able to isohite it in such purity 1 Rr-suni(5 of litpi-atuiv hv Ford, Cent. f. Bakt., 101.3 (aS). 12!); .lacohv. Kollo and Wasscrmann's Iland))uch. 1!)1,3 (2), 14;-).'?. 2 Amcr. Jour, of Plivsiol., 100.') (14). 2.'i0. 144 PHYTOTOXIN IMMIXITY 145 that one one-thousandth of a milligram (0.000001 gram) was fatal per lilo of rabbit, and solutions of 0.001 per eent. would agglutinate red corpuscles. The toxicity was also impaired or destroyed by tryptic digestion. They consider that probably, because of its extremely great toxicity, Jacoby was able to get active preparations that con- tained too little active substance to give the protein reactions. As they remark: "If one-thousandth of a milligram of a compound giving on analysis every indication of being a relatively pure protein, is phj^siologically active in the degree characterized by our experiments, the toxicity of any impurity must be infinitely greater than tliat of any known toxins." Against the claim that the toxic princii)le is simply carried down with the protein is the fact that it does not come down in the first fraction that is precipitated, the globulin, which usu- ally carries down all impurities. All the ricin comes down between the limits of one-fifth and one-third saturation with ammonium sul- ]ihate, exactly as does the albumin. Field ^ has found evidence that the agglutinin and toxin of pure ricin are separable, but Reid believes them identical. Of 21 varieties of ricinus seeds examined by Agul- hon,^^ all yielded hemagglutinins. During germination of the castor bean the ricin disappears with the albumin.^'' Ricin agglutinates not only corpuscles, but tissue cells of all sorts, and causes precipitates in normal serum.'* Curcin alone seems to have no hemagglutinative action.*^ Immunity. — The phytotoxins have been very serviceable in the study of immunity, since they obey the same laws as bacterial toxins and can be handled in more definite quantities. By their use Ehrlich first determined that toxin and antitoxin act quantitatively. They seem to possess haptophore and toxophore groups, and immunity is readily obtained against them, not only by subcutaneous injection, but by dropping into the conjunctival sac, and also by feeding, show- ing their direct absorbability and their resistance to digestion. The antitoxin is present in the milk of the immunized mother and im- munizes the suckling; but little is carried through the placenta into the fetal blood. The immunity is specific, ricin antitoxin, for exam- ple, not protecting against abrin (although it is said to protect against robin). Roemer found that in animals immunized by conjunctival application the eye so used became immune to the local action of the poison before the other eye did, indicating a local development of immune substance. In general immunization the immune substance appears first in the spleen and bone-marrow. Normal serum gives a precipitate with ricin, but immune serum gives a much heavier one. 3 Jour. Exper. Med., 1910 (12), o.il ; Keid, Landwirtsch. Versuchst.. 101.3 (F2), 393. 3a Ann. Inst. Pasteur. 1014 (28). SIO. 3bAar paralysis. The pulse is of moderate strength until a few minutes after the cessation of respiration ; the latter becomes slower, labored, and more and more superficial until it dies out almost imperceptibly. Death occurs at the latest within fifteen hours; in 32 per cent, of all cases in three hours. There are very few local changes." Cushny sna finds that cobra venom produces paralysis of the motor nerve terminations of muscle, resembling the action of curare ; the central nervous system is not directly involved. Death re- sults from failure of tlie motor nerve ends in the respiratory muscles to transmit impulses to the muscles. Alkaloids that are antagonistic to curare (physostig- mine, guanidine) are not eff"ective in cobra poisoning, but are themselves rendered inactive. Viper Poisoning. — "After the bite of a viper the local changes are most pro- nounced; there are violent pains in the bleeding wound, hemorrhagic discolora- tion of its surroundings, bloody exudations on all the mucous memln-anes, and hemoglobinuria. Usually somewliat later than in cobra poisoninir constitutional symptoms develop; viz., great prostration with nansea and vomiting, blood pres- sure falls continuously, and respiration grows slow and stertorous. After a tem- porary increase in reflexes, paresis supervenes, with paraplegia of tlie lower ex- tremities, (>xtending in an upward direction and ending in a comiilete ))aralysis. It therefore rescmliles an acute ascending spinal paralysis. If tlio patient re- covers from tlie paralysis, a septic fccr may develop; not rarely there remain suppurating gangrenous wounds, which heal poorly." It will be noticed that there is lacking tlu' usual ]ioriod of incuba- tion that follows injection of bacterial toxins, and if it happens that the venom has been injected directly into one of the veins, death may 20a Trans. Koy. Soc, London (B), 1016 (208), 1. NATURE OF VENOMS 153 occur within a few minutes. AVhen recovery occurs, the disappear- ance of symptoms is remarkably abrupt, within a few hours a des- perately sick person becoming almost entirely free from all evidences of the intoxication. Pathological Anatomy. — Postmortem examination shows changes varying with the nature of the poisonous shake that has caused death. In the case of a cohra bite, according to JMartin. the areolar tissue aVmut tlie wound is infiltrated with pinkish fluid; the blood is often lluid; the \eins of the jiia are congested, and the ventricles often contain turbid lluid; the kidneys inay show much congestion. When death occurs in a few minutes, enormous general intravascular clotting is found, which seems to be the cause of death. After death from a viper bite the site of the wound is the seat of intense edema and extravasation of blood; if in the muscles, these are much softened and disorganized. Hemorrliages are fovind in all organs and in the intestinal tract. If death occurs after several days it is generally because of sepsis, and shows the usual changes of this condition ; in addition, as a rule, to marked gangrenous, ulcerative, and sloughing processes at the site of the bite. Histologically there are found, in addition to innumerable hemorrhages in nearly all the organs, many vessels plugged with thrombi composed of more or less hemoh'zed, agglutinated erythrocytes. The changes produced in tlie nervous tissue by the Australian tiger snake are described by Kilvington.so ^vho foimd marked chromatolysis, the Nissl bodies breaking into dust-like particles, and eventually all stainable substance disappearing from the cytoplasm; the nucleus retains its central position, but often loses its outline and may disappear. The cells aroimd the central canal of the cord are most affected. There are no inflammatory changes in the nervous system, and if death occurs very quickly there may be no microscopic alterations. Hunter si found similar changes in the Nissl bodies in both krait and cobra poisoning: in the medullated fibers he found the myelin sheath converted into ordinary fat. The venom of sea snakes (Enhijdrina valaka- dien) has a severe action on the nervous tissues, while Daboia has none (Lamb and Hunter 32). Nowak 33 studied experimental animals, and found mucli fatty change in the livers, even if death occurred one-half hour after poisoning; also focal necrosis in the liver, acute parenchymatous alterations in the kidney, and pneumonic patches in the lungs. Effects on the Blood. — Tlicre has been much discussion concerning the part played by tlie abvnidant and prominent intravascular clotting in causing death after snake-bite. Lamb s* states that when venoms are slowly absorbed the coagulability of the blood is decreased and it is found fluid after death, but when a fatal dose of venom (viper) is rapidly absorbed, clotting is increased and throm- bosis is the chief cause of death. ]\Iartin has demonstrated very active fibrin ferments in snake venom (loc. cit.). It is highly probable, however, that many of the thrombi of venom poisoning are not produced by coagulation of fibrin, but by agglutination of the red corpuscles, which Flexner 35 has shown can cause large clots in the heart and great vessels, as well as "hyalin" thrombi in the small vessels. Nature of Venoms. — The varied effects produced by venoms have been found to' be due to a number of poisonous elements which they contain, and which have been distinguished and separated from one another by Flexner and Nognchi.^'' These are hemotoxins (hemoly- sins and hemagglutinins) , leucocytolysins, neurotoxins, and endothel- 30 Jour, of Physiol., 1902 (28), 420. 31 Glasgow Med. -Jour., 1003 (59), 98. 32 Lancet, 1907 (ii), 1017. 33 Ann. d. I'Inst. Pasteur, 1898 (12), 369. 34 Indian Medical Gazette, Dec, 1901. 35 Univ. of Penn. Med. Bull.. 1902 (15). 324 36 Jour. Exp. Med., 1903 (9), 257; Univ. Penn. Med. Bull., 1902 (15), 345. 154 pnyTOToxi\i< axd zootoxixs iotoxins (hemorrhagin) , but it must be taken into consideration that Faust ^' believes tliat the single o-lucosidal poison which he has found in rattlesnake venom is responsible for all the effects of the venom, except the hemao-glutination. [In another place (see "Hemolysis") the nature of the hemolytic agent is discussed.] Venom agglutinin is quite independent of the hemolysin, for it is destroyed by heating to 75°-80°, whereas the hemolysin is destroyed only partly at 100°. Agglutinin acts in the absence of serum complement, and therefore is not an amboceptor; it is apparently more like the toxins in its na- ture. The agglutination of the corpuscles does not interfere with their subsequent hemolysis. Michel states that the agglutinin of cobra venom can be separated from the hemolysin and the toxin by means of ultrafiltration through collodion membranes, as the agglutinin exists in larger molecular aggregates.^*'-'' The leucocytotoxins were found by Flexner and Noguchi to be quite distinct from the hemolysins, for after saturating all the hemoly- sin with red corpuscles, the venom still shows its efit'eets on the leu- cocytes, which effects consist in cessation of motility and disintegra- tion, affecting particularly the granular cells. The leucocytotoxin, however, resembles the homolysin in that it appears to be an ambo- ceptor. Leucocytes are also agglutinated by venom, possibly by the same agglutinin that acts on the red corpuscles. Serum complement is inactivated in vitro by cobra venom through changes in the globu- lins brought about by the venoms.^^'' By saturating venom with either red corpuscles or nerve-cells it was found by these authors that the toxic principle for each is dis- tinct and separate.^*^ Other sorts of cells, however, are able to com- bine, or at least remove some parts of the toxic elements, but to a much less degree. The neurotoxin, like the hemolysin, resembles an amboceptor, and since venom contains no complement, the neurotoxin has first to be supplied with complement by the victim's blood or tis- sues before it can harm the cells. The venoms are not only toxic for mammalian cells, but also for simple unicellular organisms, including bacteria; tadpoles are paralyzed in solutions containing one part of cobra venom per million.^" The pronounced hemorrhage-producing projjcrty of serums, partic- ularly that of the rattlesnake, was also found to be due to a specific toxin acting on the endothelium of the capillaries and small veins, and not to the changes in the blood itself, as had formerly been thought. This endotheliotoxin, which Plexiu^r and Noguchi call "hemorrhagin," is quite distinct from the other toxic substances, being destroyed at 37 Arch. Expor. Patli. ii. I'lunni., 1!)11 (f>4), 244. 3«a Conipt. Itoiul. Soc. ]?ioI., l!)lf) (77). ISO. 37anirsclif<'Id and Klinj^or. Ilioohem. Zcit., 101;i (70). .SOS. 38 r5an liite of CENTIPEDES 159 A number of common spiders investigated by Kobert ^^ were ap- parently not poisonous for mammals, except the ''cross spider" {Epe- ira diadema), which has since been thoroughly studied by him and by Sachs.'^*' Walbum *'°* states that the chief poison of these spiders is found in the ovaries, the salivary poison being much weaker, and the hemolysin is found chiefly in the albumin fraction. Epeiratoxin re- sembles the snake venoms strikingly, according to Sachs, for it eon- tains a powerful hemolysin which he calls " arachnolysin, " acting very differently with different sorts of blood, and destroj'ed by heating at 70^-72° for forty minutes, and it behaves with lecithin and cholesterol like cobra venom. "^ The agglutinin is quite distinct from the hemo- lysin."- Only such blood is hemolyzed as is able to bind the poison in the stroma of the red corpuscles. By immunizing a guinea-pig Sachs succeeded in securing an antitoxin of some strength. The dis- covery of this hemolysin explains Robert's observation of hemoglobin, methemoglobin, etc., in the urine of persons bitten by spiders. Von Fiirtli considers that the bite of the historically famous Italian tarantula is able to cause no more than local inflammation, and Ko- bert found that the entire extract of six Russian tarantulas (w^liich are supposed to be more poisonous than the Italian) caused no symp- toms when injected into a cat. In all probability the other poisonous spiders possess toxic sub- stances allied to those of the venoms, with hemolytic, agglutinative, and neurotoxic products, Sachs' studies indicating the general sim- ilarity of all the zootoxins. An antitoxin is said to have been secured against the Russian tarantula."^ CENTIPEDES Undoubtedly the severity of centipede poisoning has been greatly exaggerated, the results being usually limited to local inflammation, frequently spreading some distance in an erysipelas-like manner. An authentic case of fatal poisoning of a child four years old by a centipede (Scolopendra heros) has been reported from Texas by G. Linceicum,*'^ death resulting five to six hours after the bite was re- ceived. Besides the local pain and inflammation, vomiting was marked, occurring also in five other non-fatal cases. Centipedes secrete their poison in relatively large glands, which which is capable of causing severe spasm of Uie abdominal muscles, according to Atwood (Southern Californ. Pract.. Vols. 10. 12 and 16). Kellogg and Coleman (.Jour, of Parasitol., 1015 (1). IflJ), found extracts of the poison glands of this spider to be highly toxic. 59 "Beitriige ziir Kentnisse der Ciftspinnen,"' Stuttgart. 1001. eoHofmeister's Beitr.. 1902 (2), 12.5. GoaZeit. Tmmunitiit., 1015 (2.3), 623. «iPini. II Policlinico (Sez. Med.), 1009 (16), 208. 62 V. Szily, Zeit. Tmmunitiit., 1910 (.5). 2S0. <5» Konstanzoff, Russkv Wratsch., 1007, Xo. 17. 64Amer. Jour. Med. Sci., 1866 (.52), 575. 160 PHYTOTOXIXS AND ZOOTOXINS discliarge at the apices of a pair of specialized claws that take the place of the first pair of legs. The nature of this poison seems not to have been investigated. Numerous chemical substances are de- scribed as secreted by other glands of these animals, including prus- sic acid and a camphor-like matter (see v. Fiirth). BEE POISON Bee poison has been better studied than most insect poisons, begin- ning with the work of Paul Bert (1865). It is secreted by the glands into a small poison sac, and stored up until ejected. Cloez found that bee poison was precipitated by ammonia, tannin, and platinic chloride, and Langer proved it to be a non-volatile organic base. As excreted, it is acid, contains 30 per cent, of solids, and one honey-bee secretes 0.0003-0.0004 gm. It contains formic acid and much pro- tein, but it has been stated that the poison is protein-free, and is not destroyed by heat (100°), weak acids, or alkalies. On the other hand, it is said to be destroyed by proteolytic enzymes, which would indicate that it is of protein nature. Hemolysis is produced both in v-itro and in vivo with all sorts of blood, but to very different degrees, thus resembling spider toxin. The hemolytic action is greatly in- creased by the presence of lecithin, forming a toxolecithid like "cobra lecithid. " ''^ Locally bee poison causes necrosis, with marked hyper- emia and edema. A 4500 gm. dog was killed by intravenous injection of 6 c.c. of a 1.5 per cent, solution of pure poison (Langer).*'*' Immunity is undoubtedly possible, for bee-keepers frequently show a great decrease in susceptibility. On the other hand, abnormally great susceptibility is frequently seen, some cases of fatal poisoning having been obsei'ved.*''^ Wasps and Hornets presumably produce poisons similar to those of the bees. A study by Bertarelli and Tedeschi '^'"^ establishes this for a species of wasp (Vespa crahro L) . Ants also produce formic acid, a fact so well known that it has come to be considered that this is the source of their toxicit.y. Von Fiirth, however, suggests the probability that ant poison, like that of the bees, owes its chief effects to other more complex, unkno\vn poi- sons.*'^ «5 Mor SALAMAXDERS 161 POISONS OF DERMAL GLANDS OF TOADS AND SALAMANDERS It has been known for centuries that toads produce poisonous sub- stances. Pare in 1575 havin), 1.531; Jour, of Pharm., 1912 (3), 319. 11 162 PHYTOTOXINS AXD ZOOTOXiys and it is extremely active. The toad is relative!}^ immune to bufagiu, but not at all to the epinephrine. A Chinese drug derived from toad skins has been found to contain similar ing-redients (Shimizu"^), as well as a substance resembling picrotoxin in action. Salamanders also produce poisonous secretions in their dermal glands, which have been studied especially b}^ Faust,'* and earlier by Zalesk}^,'^^ who isolated an organic base which he named saman- darin. Faust describes samandarin as first stimulating and then paralyzing the automatic centers in the medulla. The poison resem- bles the alkaloids, having the formula C2,;Il4„N.O, and produces death in doses of 0.7-0.9 mg. per kilo (dogs) with respirator}^ failure. Im- munization of rabbits was practically impossible. A second alkaloid, samandaridin (CooH-jiNO) is also present in even greater quantities than the samandarin, and differs only in being weaker. Fro^s also have similar poisons in their skins, extracts of Rena es- cidenta skin being highly toxic."'' The dermal secretions of most of the amphibians are poisonous, not only for mammals, but also for rep- tiles, and in large doses for the animals producing them (Phisalix)." Bert '^^ and also Dutartre '^^ have described a digitalis-like poison in the secretion of the dermal glands of frogs. It is evident that all these poisons are quite distinct from the venoms, and from the tn^e toxins, apparently being simple chemical compounds not related to the proteins and not capable of causing im- munization. POISONOUS FISH SI There are numerous fish, especially in tropical waters, which de- fend themselves by injecting poisons into their enemies. This is ac- complished by spines, to which are attached poison glands.^- Dun- bar-Brunton ^^ has described two such fish (Trachinis draco and Scor- pcena scorpha) of Mediterranean waters. Wounds by these spines cause in animals intense local irritation and edema and paralysis of the part, followed by gangrene about the site of the wound; in fatal poisoning death occurs in from one to sixteen hours, with general par- alysis. The sufferings of persons so poisoned are said to be extreme, and death may occur either directly from the poison or later from sep- sis following the local gangrene. Presumably this poison is not dissim- ilar to that of the snakes; it probably is not an alkaloid, as Dunbar- 73a .Tour. Pharmacol., 1910 (S). .347. 74Arfh. pxpor. Patli. ii. l^liarm., 1S9S (41). 229 (litoraturo) : 1900 (43), 84. 75 Hoppe-Seyier's Med. Chein. T'litrrsucli., 1801!, p. 8."i. 70 Caspar! and Loowy, Med. Klinik. 1911 (7), 1204. 77 .Jour. Phvs. et Path, pen., 1910 (12), 325. 78Compt. Pend. Soc. Biol., 188,5, p. 524. T^Ihid., 1890, p. 199. 81 Full discussion and literature given by Faust. "Tierische Gifte." p. 134. 82 For a list of fish with poison {glands see Pawlowskv, Zool. .Tahrb., 1912 (.31), 529. 83 Lancet, 189G (ii), 000. P0I80N0US FISH 1^^ +o Tf nffppts chiefly the heart, according to Pohl,«* and r:r :ifer,v.i"pSe w,Lu l.e,.ave. UUe the ve„..,n hemo- lysins in that it is acnvated by se™^ EvanV ^^^^^^ ^^ ^^^^^ ^^.^^^ to have caused fatal intoxication m s::^^;^r„ra::." rz^i^^^^^- -- - iJ kept but ^-/;-^°|^;^_^;^'^:;;:ttle cannot be safely marketed. Tr+n he developed and contained in the ovaries and eggs, and :; *., £r ...1 1.™. ~. aw,, ..a —"■•J":"; ;';; •„ +i.a «viim and leo-s- this terminates m collapse, coma, ana Sr^re^resp-t'oryor eardiae paralysis. Jhe^entire eo„. nf tl,e process may be but ten to twenty minutes, or it may be as many lirs '^On aeeount of the localization of the poisoii in the eggs and ovaries not all persons who eat the fish are poisoned, and not all who are poisoneclreeeive a fatal dose. In the gastro-intestinal form the sWom appear later, consist chiefly of gastro-intestmal disturbances ?eZlZ. more elosely ptomain poisoning, and the prognosis is not "The paJhofogilf ailmy of this foi-m of poisoning has not been carefully studied, but no characteristic or striking anatomical changes have been noted in the bodies examined. Tahara>' has described a 84Prager med. Wocli., 1S93 (18), 31. 85 British Med. Jour.. 1007 (i).73. ssaKonstanzoff and Manoiloff. ^^ re.^'^i"; ? J-'; iSfifi Hi ;rature) 86 "Atlas des Poissons Vencneux," St. Petersburg, 1886 (hteratine). STBiochem. Zeit., 1010 (30), 2.56. 164 PnYTOTOXIXS ASD ZOOTOXINS toxic body, tetrodo-toxin, isolated from the ovaries of Tetrodon.^'''^ The purest preparations had a minimum lethal dose of 0.0025 to 0.004 grm. per kilo, and a provisional formula of CieHgiNOig was given to it. Tetrodotoxin is neither protein nor alkaloid, nor yet a protamin. In tliis connection may be mentioned the peculiar erysipelas-like lesions caused by bites of crabs, which indicates the formation of some toxic product by tliese crustaceans. Gilchrist ** obtained a history of bites or injuries by crabs in 323 of 329 cases of " er^-sipeloid. " Crabs, in turn, may be poisoned by cephalopods which secrete an active poison from their salivary glands.*'' ^lany coelenterates produce active poisons (most famous of these being the Portuguese-man-o'-war), which have especially a paralyzing and a local irritant effect.*"'^ EEL SERUM In 1888 IMosso ^° studied the toxic properties of eel serum, which he found was extremely poisonous for experimental animals, 0.1 to 0.3 c.c. per kilo being fatal for rabbits and dogs in a few minutes if in- travenously injected; introduced into the stomach it is not toxic, but it produces a violent conjunctivitis when it enters the eye, the poison- ous agent being contained in the albumin fraction.^^ The poisonous principle Mosso called ichtJiyoto.rin. Death results from respiratory failure with large doses ; small doses lead to cachexia and death after a few days. The coagulability of the blood is greatly reduced. Kos- sel ®^ found histological changes in the central nervous system in such animals, that resembled the lesions of tetanus. He succeeded in se- curing an active antitoxin which neutralized the strongly hemolytic action of eel serum in vitro, and also prevented fatal effects in ani- mals. Camus and Gley ^^ have studied the physiological action of eel serum and found it strongly hemolytic, and also apparently neuro- toxic. The toxicity is destroyed by heating" to 58° for fifteen minutes. B}^ immunization an antitoxic serum can be obtained which neutralizes the eel toxin completely. Tchistovitch ^* secured antitoxic serum, which acted also as a precipitin for eel serum. De Lisle "^ found that eel serum does not act like an amboceptor, since after heating it can- not be reactivated with fresh mammalian serum, and it seems, there- fore, to be different from snake serum in its. structure. Lamprey serum is likewise toxic, ^^ as is also that of the Rays. 87aAroli. exp. Path. u. Pharm., 1890 (26). 401 and 4r).3. 88 Jour. Cutaneous Diseases, Novemher, 1904. soBafrlioni, Zeit. f. T5iol., 1908 (f)2), 130. soa Roe von Fiirth. Verjrl. chem. Phvsiol.; also Lojacono, .Tour. d. plivsiol., 1908 (10), 1001. ooAreli. Ital. de Biol., ISSS (10), 141; 1SS9 (12), 229. 01 Prdlot and Palilson, Graefe's Areli., 1911 (72), 1S:1. 02Borl. klin. Woeh., 1898 (.35), ]52. 03 Arcli. inU-rnat. d. Piiarm.. 1899 (.''>), 247. 04 Ann. Inst. Pasteur, 1899 (13), 40fi. 05 Jour, of Mod. Researoli, 1902 (8). 39fi. ooCIev, ('omi)t. Rend. Soe. Biol., 1915 (78), 110: Camus and Olev. ibid., p. 203. CHAPTER VII CHEMISTRY OF THE IMMUNITY REACTIONS- ANTIGENS, SPECIFICITY, ANTITOXINS, AGGLU- TININS, PRECIPITINS, ANAPHYLAXIS OR AL- LERGY, ABDERHALDEN REACTION, OPSONINS, AND RELATED SUBJECTS Although immunity was first iuvestigated in relation to bacterial infection, it was soon learned that the reactions by which the animal body defends itself against bacteria have not been^.-developed as speeitic means of defense against bacteria alone, but are reactions against foreign substances of similar chemical nature, whether bac- terial, animal, vegetable or artificially synthetic in origin. Further- more, these reactions are chemical reactions, and the problems of immunity are chemical problems, although as yet most of the react- ing substances are not accessible to chemical investigation. In this place, where our concern is with the chemical aspects of pathological processes, the subject of immunity will be discussed only from the standpoint of the chemistry of the processes and substances involved, leaving to other works the clinical and bacteriological aspects of the subject.^ The reactions of immunity are, we find, reactions to chemical sub- stances entering the body from without, or abnormally developed within the body by invading organisms or by changes in the chemi- cal processes of the body. Furthermore, there seems to be an essen- tial difference between the reactions incited by simple chemical com- pounds to which the animal body can develop a certain degree of resistance (such as morphine, alcohol, and arsenic), and the reactions against more complex substances such as bacterial toxins, foreign pro- teins, venoms, etc. The complex substances of the latter group incite reactions wliicli are to a greater or less degree specific, and usually very highly augment the defense of the body against the foreign sub- stances ; with the simple poisons the reactions are largely or altogether non-specific, and the resulting resistance is usually relatively slight. Substances of the first class we usually refer to as antigens. 1 Especially to be recommended for a discussion of the scientific problems of immunology is Zinsser's "Infection and Resistance," Macmillan, New York, 1914; and for methods and applications see Kolmer's "'Infection, Immunity and Specific Therapy," W. B. Saunders, Philadelphia, 1915. Also see Kolle and* Wassermann, "Handbuch der path. Mikroorganismen"; Weichardt "Jahresbericht der Inimuni- tUtsf orschung" ; and for more recent literature consult the Zeit. f. Immuni- tiitsfrsch., Referate. 165 166 CHEMISTRY OF THE IMMUXITY REACTIOXS ANTIGENS ■' This term includes those substances which, Avhen introduced into the blood or tissues' of an animal, in proper amounts and under suitable conditions, cause the generation and appearance in the blood of specific antibodies capable of reacting with the antigen. Con- cerning the chemistry of antigens we can say that all antigens, so far as now known, are colloids. Furthermore, with one exception, every known soluble, complete protein may act at least to some degree as an antigen, and, as yet, it has not been finally established that any colloids other than proteins can act as antigens. The exception is the racemized protein of Dakin, which Ten Broeck -"^ found to be en- tirely non-antigenic although soluble and possessed of all the amino- acids present in the egg albumin used in preparing it. Solubility is an essential character for antigenic action, for proteins that have been coagulated by heat lose their antigenic capacity, while proteins that are not coagulated (e. g., casein, ovomucoid) retain their antigenic properties after boiling.-'' Of the cleavage products of proteins it is certain that none of the amino-acids and simple polypeptids can act as antigens, and it is not yet fully established that even such large complexes as the pro- teoses are antigenic, although there is some evidence in favor of this view. AYhether the entire protein molecule, or only groups thereof, determine the characteristics of the antigen, is not known, there be- ing evidence w^hich can be interpreted in favor of either view, but TVells and Osborne ^ have submitted evidence which indicates that a single protein molecule can act with and engender more than one antibody; this is supported by Kliein's demonstration of the produc- tion of two distinct antibodies by immunizing with casein.^'' It has been shown by Gay and Robertson,* moreover, that if the non- antigenic cleavage products of casein are resynthesized by the re- A^erse action of pepsin, into a protein resembling paranuclein, this synthetic protein is capable of acting as an antigen. Protamins and globin, they found, were not antigenic,^ although globin when com- bined with casein forms a compound wliicli engenders an antibody that gives complement fixation reactions witli globin. Schmidt also found that protomain edestinate is antigenic for edestin and for itself, but not for protamins, whereas a compound protein, both elements of which were non-antigenic (globin-albumose), was not antigenic. °''- 2 See the Tlevicw on Antifrens by E. P. Pick, Kolle ami Wassorinaiin's ITaiullnicli d. path. Mikroorpanismcn, 1912 (1), G85. -2aJour. Biol. Chem., 1914 (17), 369. 2b See Wells, Jour. Infect. Dis., 1908 (5), 449; Jour. Biol. Clicm.. 1910 (28), 11. 3 Jour. Infec. Dis., 1913 (12), 341. -3a Folia Microbiol., 1912 (1), 101. , 4 Jour. Biol, f'lioni., 1912 (12), 233. ■ 5 Jour. Exp. Mod.. 1912 (IG), 479: 1913 (17). 535. 5a Univ. of Calif. Puld., Pathol.. 1910 (2). l.-i7. Koviow and biblioirrajiliy on specificity. NON-PROTEIN ANTIGENS 167 NON-PROTEIN ANTIGENS Amono- the many aeeouiits of what the autliors interpret as the successful production of specific antibodies as a reaction to non-pro- tein antigens, are the following: Ford ** found that rabbits can be immunized to extracts of Aman- ita phalloides, and that the serum of such rabbits will neutralize five to ei^-lit times the lethal dose for guinea-pi«>'s. and is anti-hemolytic for tlie hemolysin of amanita when diluted to 1-1000. As he and Abel " had found this hemolytic poison of Amanita to be a glucoside, this observation is to be interpreted as a successful production of an antibody for a non-protein poison, a glucoside. This work was fur- ther 'supported by successfully immunizing rabbits to extracts of Rhiis toxicodendron, and finding that their serum in doses of 1 cc. will protect guinea-pigs from 5-6 lethal doses of the poison, which was found by Acree and Syme ^ to be a glucoside. Subsequent work by the same author confirms the main point, showing that an active hemolysin can be obtained free from demonstrable protein, and that immunization with this protein-free hemolj'sin will result in stronglj' active (1-1000) antihemolytic serum.^ Ajiother. non-hemolytic poison from Amanita, which Ford desig- nates as Amanita toxin, was found to contain neither protein nor glucoside, and no antitoxic serum or definite artificial immunity can be obtained for it. The antihemolysin unites with the hemoly- sin in simple multiple proportions. ^° Jaeoby believed that he had obtained the phytotoxin ricin free from protein, in which case the well-known and active antiricin must rep- resent an antibody for a non-protein antigen. However, the work of Osborne, iMendel and Harris ^^ has shown that ricin is, in all probability, an albumin, and this, for the present at least, places ricin with the protein antigens. The work of Ford is, in our estimation, the strongest evidence yet presented as to the possibility of non-protein antigens. The newer developments in immunological research, moreover, make it seem entirely plausible that a complex glucoside, which can be hydroh'zed by enzymes, can act as an antigen. If we consider the evidence that immunity consists in the development of a special power to hydro- lyze foreign substances, when these substances are of such a nature as to stimulate the cells to activity, and that Abderhalden and others have found evidence that specific enzymatic properties appear in the blood of animals injected with carbohydrates and fats, it seems c.Tour. Tnfec. Dis., 1907 (4), 541. T Jour. Biol. Chem., 1907 (2), 273. 8. Jour. Biol. Chem.. 1007 (2), 547. 9 Jour. Pharmacol.. 1910 (2), 145. 10 Jour. Pharmacol., 191.3 (4), 235. 11 Amer. Jour. Physiol., 1905 (14), 259. 168 CHEMISTRY OF THE IMMUNITY REACTIONS entirely reasonable that a toxic glucoside can have antigenic proper- ties. A similar line of reasoning will apply to the question of lipoid antigens. The evident participation of lipoids ^- in innnunity reactions, es- pecially the complement-fixation and allied reactions, has naturally led to investigation of the possibility that lipoids may act as true antigens, a possibility^ made conspicuous by the fact that lipoids can be substituted for true antigens in the "Wassermann reaction (q. v.). Bang and Forssmann immunized with ethereal extracts of red cor- puscles and obtained hemolysins, so that they concluded that the antigenic constituent of the corpuscles is a lipoid, probably a phos- phatid. This Avork has caused much controversy and many workers have failed to confirm their results.^^ It is a striking fact that when purified phosphatids, from sources favorable for obtaining pure ma- terials, are used, the results are always negative, while the positive results are generally reported with lipoids of more or less dubious purity. IMuch and others have worked with lipoids from a streptothrix, which is called "nastin," and they state that sera are obtained which give complement fixation reactions with nastin used as the antigen.^* Similar results are described for the fatty materials from tubercle bacilli ( ' ' tuberculonastin " ) . Meyers ^^ has reported the production of specific complement fix- ation antibodies by immunizing rabbits with acetone-insoluble lipoidal material obtained from tape worms and echinococcus. He has found the acetone-insoluble fraction of tubercle bacilli, presum- ably phosphatids, to serve as antigen in complement fixation reac- tions with antibodies for tubercle bacilli,^*' and much more effectively than the protein residue of the bacilli, wherefore he concludes that the reactions obtained with the lipoids certainly cannot be ascribed to adherent traces of protein. Bergel " observed after lecithin injections in rabbits, not only an increase in the lipase con- tent of the blood and tissues, but also the presence of complement- binding antibodies, and -Tobling and Bull ^^ have found an increase in -:erum li])ase after imiimnizing with red corpuscles. Bogomolez ^" suggests that the lipoids themselves may be produced in excess for defense against various poisons, which they serve to inhibit, especially the toxin of B. hotulinus. 12 Bibliography on Lipoids and Immunity pivon by Landsteincr. KoUo and Was- sormann's TTandbnoh, 101.3 (2), 1240; .Toblino;, Jour. Tmmnnol., 1010 (1). 401. 13 Review of literature bv T.andsteiner. Jaliresb. Imnninitiitsfrscli.. 1010 (fi),. 200. 14 Literature in Beitr. Klinik d. Tuberlc, 1011 f20). 341. isZeit. Immunitiit., 1010 (7), 7.32; 1011 (9), 530; 1012 (14), 355. if> Ihid., 1012 (14), 350; 1012 (15). 245. i7Deut. Areh. klin. Med.. 1012 (106), 47. 15 Jour. Kxp. Med., 1012 (10), 4S3. i«Zeit. Immunitiit., 1010 (S), 35. yoS-rilOTEIS ASTKIESH 169 The number of reputed positive results witli lipoids makes it im- possible at this time to state dogmatically that lipoids may not pos- sess antio-enic propcM-ties, but it must be taken into account that the successful use of lipoids as antigens in complement fixation reactions is not proof of their true antigenic nature, in view of our present lack (,f knowledge of the actual nature of this reaction itself, i^ urther- more we have the testimony of Fitzgerald and Leathes ^« that a lipoidal material from liver, which was itself capable of serving as antio-en in the Wassermann reaction, did not engender complement- fixiiro- antibodies in rabbits immunized with this lipoid. Kitchie and Mille"r==^ could find no antigenic activity in the lipoids of serum or corpuscles. Also Kleinschmidt,^^ who accepts the antigenic nature of nastin was unable to secure antibodies by immunizing rabbits with nastin. Thiele =^^ calls attention to the fact that lipoids possess no specificity, and therefore cannot give rise to antibodies. Neufeld found that "rabbits immunized with lecithin developed no opsonins for lecitliin emulsions. A suggestive observation is that of Pick and Schwarz -* who found that the presence of lecithin increases the anti- genic power of bacteria, which may help to explain the activity of possible traces of proteins in lipoid preparations used as antigens. Many drugs cause a hypersensitization, and in this respect seem to behave as antigens produciirg anaphylactic antibodies. It happens that most of these chemicals are of such a nature as to permit of their nnion with proteins, and it seems probable that such protein com- pounds behave as foreign proteins to the animal in which they are formed, for it has been found that guinea-pig seram treated with iodin can render guinea-pigs sensitive to the same iodized serum.- Hence, hypersensitiveness to iodin compounds would be a reaction to iodized proteins,-" and not to the non-protein iodin compound: the same applies to anaphylactic reactions observed with salvarsan, atoxyl," and perhaps aspirin and antipyrin.=« Zieler, however, has questioned the validity of many of the experiments on which these views are based.^« It is possible that certain chemicals may react m such a way with the tissue or blood proteins as to make them sensi- tive to the animal's own complement, which then forms anaphyla- 20 Univ. of Calif. Publ., 1912 (2) , 39. 2iJoiir. Path, and Bact., 1013 (17), 429. 22Berl. klin. Woch., 1910 (47), 57. 23Zeit. Imnmnitat.. 1913 (IG), 160. 24Biochem. Zeit.. 1909 (15). 4.53. 25 Friedborger and Ito, Zeit. Immunitiit., 1912 (12), 241. 2GAccordincr to Block (Zeit. exp. Path., 1911 (9). 509) iodoform uhosyncrasv depends upon the CH. rather than on the iodin, and is a local cellular reaction rather than a humoral reaction, the protoplasm havm- an moroased affinity for methyl radicals. (See Weil. Zeit. Chemotherapie 1913 (1-412.) _^ 27Moro and Stheeman, Miinch. med. Woch., 1909 (56), 1414. , „. , - 28Bruck, Berl. klin. Woch., 1910 (47), 1928; Klausner, Miinch. med. ^^ och., 1911 (5S), 138. 29 Munch, med. Woch., 1912 (59), 401. 170 CHEMISTRY OF THE IMMUXITY REACTIOXS toxin,^° and thus causes reactions, but the whole anaphylatoxin ques- tion is in so uncertain a state at the time of writing that further speculation in this direction is not justifiable. The attempts to produce antitoxin against cantharidin have not yielded convincing results,-'^ nor against epinephrine.^- De Angelis ^^ claimed that he had produced specific precipitins for various natural and syntlietic dyes, but this woi'k has, as was to be expected, failed of confirmation.^^ Elschnig and Salus "" state that melanin from the eye is antigenic, producing complement-fijiing antibodies specific for melanin but not for the species. We know too little concerning the composition of melanin to interpret this observation; furthermore, their preparation was not tested for proteins. In general terms, therefore, antigens are protein molecules, and the reactions of immunity are reactions against proteins foreign to the body of the host, and manifested by the presence in the blood of the reacting animal of substances which combine with and cause recognizable changes in the foreign protein.'" These changes are recognized in many ways, such as precipitation, agglutination, com- plement-fixation, etc., and the question at once arises as to whether these diiferent manifestations depend each upon a separate antibody, or if several or all of them are not caused by a single antibod}^ the action of which is indicated by the different reactions which are made manifest by different procedures in each case. This question will be discussed further in later paragraplis. Knowing that the antigens are merely foreign proteins which have been introduced into the body of an animal, there naturally occurs the thought that the animal body is continually receiving in its food foreign proteins, and against which it defends itself in the alimentary canal by enzymatic action, which disintegrates these proteins until they have lost their colloidal character. Logically following this comes the idea that perhaps the reactions of immunity are simply the same or similar disintegrative enzymatic actions, carried on within the blood and tissues to protect the body in the same way against 30 See Manoilov, Wien. klin. Woch., 1912 (25), 1701. 31 Champy, Compt. Rend. Soc. Biol., Ifl07 (62), 1128. 32Pollak, Zeit. pliysiol. Clieni., 1010 (68), GO. 33 Ann. di Ig. Sperim., 1009 (19), 33. 34Takemura. Zoit. Imnuinitiit., 1010 (5), 607. __ 35Graefe's Arch., 1011 (70), 428. 3C Drew lias found no evidence of antibody formation Ity imnuinii'inti molluscs and ecliinoderms (Jour, of Hyp., 1011 (11), 188), from which ho conchules that the reaction to foreign pioteins is not a imiversal ]iroperty of pmloplasm ; a sweeping generalization which requires more extensive investigation for its es- tablisliiiiciil. ("antacuzenp (('omi)t. Rend. Soc. Biol., 1013 (74). Ill) obtained precipitins l)y iiniiiuni/.ing J'linlliisia itiiimiJlatd witii mammalian blood, but no licmolysins with this or A plirodilr aciilcala and Elcdonc vioschatn. Carrel and Ingebrigtsen (.Tour. Kxp. ]\Ied., 1012 (l.'i), 287) have found that tissues growing iti vitro with foreign blood jjroduce hemolytic antibodies for that blood, indicat- ing that isolated cells can react to antigens by antiliody ]>niduction. SPECIFICITY OF IMMVyF h'FACTIOXS 171 foreign proteins whicli tiic alimentary digestive apparatus has not had the opportunity to destroy. This conception of the nature of im- mune reactions to antigens has been especially advanced and in- vestigated by Victor C. Vaughan ^^"^ and his co-workers, and l)y Kmil Abderhalden, who has demonstrated in various ways an increased proteolytic power in the blood of animals which have received pa- renteral injections of foreign proteins.'"'' Thus, if the antiserum re- acts on the specific protein within a dialyzing sac, the products of proteolysis diffuse into the surrounding medium, where they can be detected by simple chemical reactions. Also, changes in the spe- cific rotation of the protein or peptid solution can be observed by the polariscopic reading before and after the action of the antiserum. A particularly important corroboration of Vaughan 's theory is fur- nished by tlie behavior of the racemized i)rotein of Dakin. Although soluble, this protein cannot be attacked by the digestive proteolytic enzymes, presumably because of its altered configuration ; and it" is non-antigenic, presumably because it cannot be attacked by the pro- teases of the blood and tissues. Likewise it cannot be nietabolized, w^hether fed or injected subcutaneously.^"^ Here we have good evi- dence of the fundamental identity of the three processes, digestion, metabolism, antigenic activity. As immunity reactions manifest themselves, however, there are many steps in the process besides simple hydrolysis of proteins, even if This be the ultimate goal of them all. SPECIFICITY OF IMMUNE REACTIONS The many attempts to explain the various reactions of immunity solely on the basis of known physico-chemical properties of colloids all flatten out when the striking, characteristic, and often extreme specificity of these reactions is considered. Chemical explanations are but little more satisfactory. In enzyme action we find many com- parable examples of specificity, — but this does not help, as the enzymes are as mysterious as the antibodies. But no proposed explanation of any of the reactions incited by antigens can be of value if it fails to take into account the specificity of the reactions. We lack the space here to consider the many ideas and the items of evidence which have been advanced concerning this all-important chemical problem, but refer the reader to the excellent discussion by E. P. Pick-^^"* The main facts at present available are the following: Specificity was at first supposed to depend solely upon biological relationships, for it was found easy to distinguish the serum of animals of unlike nature by n cans of the precipitin and other reactions, but the more closely re- -6a See Vaiiglmn, "Protein Split Products," Pliiladelpliia, 1913. 3" Abderhalden, "Abwehrferniento dos tierisclicn Oriranismus," Perlin. 10] 3. "a See Ten Broeck, Jour. Biol. Cliem., 1014 (17). 3(1!). 3S Kolle and Wassermann's Ilandhuch d. path. ^Mikroor^ranismen, 1012 (1), 685 ; full bibliography. 172 CHEMISTRY OF THE IMMVSITY REACTIONS lated the animals the less sharply these reactions distinguish them, until, with such closely related animals as dog and fox, or man and apes, antisera for the blood of one react nearly as well with the blood of the other, the existing differences being only quantitative. The opinion therefore gained ground that the specificity depended upon some peculiar biological relationship of the antigens, and, as serum proteins which seemed to be quite similar chemically but which were from unrelated species, were sharply differentiated by the bio- logical reactions, that the specificity must depend upon something quite distinct from ordinary chemical differences. But even with closely related species, differences can often be brought out by means of the process of saturation (which consists in treating the antiserum with sufficient quantities of an antigen until it no longer reacts with additional quantities of this antigen, and then trying its reactive power with the other related antigen which one wishes to test). As use began to be made of other materials than serum, and especially when more or less purified proteins were employed, it was found that within the tissues of a single animal or plant there might exist antigens which were quite distinct from one another — more so, indeed, than some of the chemically similar substances of different biological origins. Thus, in the hen's egg, by means of the anaph}'- laxis reaction, I have been able to distinguish five distinct antigens, and these correspond to as many different proteins which have been distinguished by chemical means. ^'' Also, for another example, in the crystalline lens are found proteins which are specific for lens proteins, in that they produce antibodies reacting with lens proteins from varied species of animals, but not with the serum proteins of the species from which the antigenic lens substance was derived.*" Here the chemical character of the protein is undoubtedly more ^significant than its biological relations. These and other observa- tions leave little room for doubt that specificity does depend upon chemical composition, and that the differences in species as exhibited hy their biological reactions depend iipon distinct differences in the chemistry of their proteins.*°'^ Chemically distinct proteins {e. g. lens and serum proteins) of one animal may be immunologically distinct, and chemically related proteins of dissimilar species (c. g. casein from goat and cow milk) may shoAV immunological relationship. Crystalline albumin from hen's eggs shows no immunological distinc- tion fi-om that of ducks' eggs, whereas eacli of the three ju'oteins sep- arable fi'om horse serum — euglobulin, pseudoglobulin and albumin — can l)e distinguished from the other two l)y the anaphylaxis reac- tion.''"'' Furthermore, it h.as been shown by AVells and Osborne *^ 30 Jour. Infeo. Dis., 1911 (9), 147. 40 Spc Knisiua, Zcit. Immunitiit., 1910 (5), tlOi). 40ii8ee WoUs and Oaboriic, Jour. Infect. Dis.. 1910 (19), 183. •if'hDalc and Ilardov, Hiocliem. Jour., 191G (10), 408. ■ii.ldwr. Infect. Dis.", 191.3 (12), 341. si'Kcu'JciT) or ni]ii \K /.'/v.kwo.v.s' 173 that a single pure protein may exliibit jiiultiple antigenic properties, and react or fail to react with other pure proteins according to whether chemical differences can be demonstrated by recognized analytical methods. A striking example of the existence of identical antigenic prop- erties in materials of biologically unrelated origins, is furnished by the sheep corpuscle hemolysin discovered by Forssner,*^-'' who found that many different materials, when injected into rabbits, engender in the rabbits' serum a(!tive hemolytic amboceptors for sheep corpuscles. This antigenic property has been demonstrated in the organs of the guinea-i)ig, horse, cat, dog, mouse, cliicken, turtle, and several species of fish,*^^ although not exhibited by organs of many closely related species (>. g. pig, ox, rabbit, goose, frog, eel, man, pigeon, rat). It is not present in the red corpuscles of these animals, but is present in the corpuscles of the sheep, whose organs do not have this property. It has also been found in paratyphoid and Gartner bacilli, mouse tumors and sheep spermatozoa. Not only does the serum of rabbits thus inmiunized show active hemolysis for sheep corpuscles, but if injected into the vein of an animal whose organs contain this antigen there results a prompt, severe anaphylactic intoxication, presumably Through reaction between the antigen present in their tissues and the antibodies of the rabbit serum. Furthermore, the antibody can be specitically removed from the immune rabbit serum by contact with any of the antigen-containing tissues, but not by tissues that do not exhibit this antigenic property. The antigen seems to remain in the tissues when the fluids are forced out by pressure, and Doerr and Pick believe it to be associated with the nucleoproteins. This series of observations, which seem to have been quite gen- erally corroborated, indicates conclusively that the immunological specificity of an antigen is not necessarily related to the biological specificity of the living organism from which it is derived. The logi- cal explanation is that there ma.v exist proteins in different species which have chemical resemblances or identity, and this is scarcely to be doubted. We find identical lipoids, fats, nucleic acids, and carbo- hydrates in different species ; many peculiar types of proteins show apparent chemical identity in different species (e. g. gelatin, keratin) ; some chemically similar, derived proteins also seem immunologically identical or closely related (e. g. lens protein, casein). Therefore, it is highly probable that many tissue proteins may be identical in dif- ferent forms of animal cells, and even in animal and plant cells. Another sort of manifestation of apparently non-specific immunity reactions has been observed especially in therapeutic immunizations.*^'' Beginning with the classical observation of Matthes that the tuber- 4ia Review by Doerr and Pick, Rioeliem. Zeit.. 1014 (00), 2r)T. 4ibTsunecka* Zeit. Inimiinitat., 1014 (22), ,567. 4ic See review by Jobling, .Tour. Amer. Med. Assoc., 1916 (66), 1753. 174 CHEMISTRY OF THE IMMUNITY REACTIOXS ciiliii reaction could be produced with deutero-albumose, many sim- ilar non-specific reactions have been observed. Particularly the sharp reaction that follows intravenous injections of killed typhoid bacilli into typhoid patients has been found to result equally well if colon bacilli are used, or deutero-albumose. One possible explana- tion of this type of reaction is that the injected substance acts as a common antigen, which causes the production of common antibodies that react also with the antigens of the cause of the disease. Another possibility is that the foreign protein stimulates the tissues that form antibodies, presumabl.y the red marrow, so that they produce not only antibodies for this antigen, but also for the antigens of the specific etiologic factor of the disease that have been stimulating the bone marrow previously. INIoreover, the febrile reaction, the leu- cocytosis, and other phenomena, such as the antiferment index of the serum ( Jobling), that injection of nonspecific protein produces, may be responsible for favorably affecting the disease, rather than actual antibody formation. An interesting illustration of the fact that whatever stimulates the bone marrow may cause it to form, among other blood elements, spe- cific antibodies, is furnished by the behavior of antitoxin-producing horses. If a horse that has been imnuinized to diphtheria toxin is bled as much as possible, it will be found to have regenerated the lost antitoxin within 48 hours,*^*^ even although the last immunizing dose of toxin was received long before. Also, it is stated that persons who have once had typhoid, but whose blood no longer contains much agglutinin, may show a high typhoid agglutinin content when infected by some other organism, or after any sharp febrile attack. It is highly possible that many therapeutic agents may similarly act by stimulating the marrow to increased formation of specific antibodies, e. g. arsenic, mercury and other metals, lieliotlierapy, hemorrhage or phlel)otom.y, hot baths. The other aspect of specificity, i. e., the presence of several antigens in a single organism, entirely distinct from other antigeus in the same organism, has been repeatedly demonstrated. Besides the identifica- tion of five distinct antigens in tlie hen's ogfc. mentioned previously, we have the repeatedly demonstrated individuality of serum pro- teins and milk casein of the same animal, and even the differentiation of casein from lactalbumin in the same milk, as contrasted witli the common inter-reactions of caseins from different sources,^^® e. g., cow and goat. A certain but slight distinguishable specificity may be ob- served between proteins from different organs of the same animal, which differentiation is still sharper between the tissue proteins and serum proteins of tlic }iniiii;i].^"' Sex cells esjiecially seiMu to be iidO'Rripn. .Jour. Palli. iiiul Had., ini.3 (18), SO. 4ic.Sce VorscU. Zeit. liiiiimnitiit., 1915 (24), 2()7. 4ifSee Salus, liiocliein. Zeit., ION (GO), 1. SPECIFICITY OF IMMUNE REACTIONS 175 rather distinct iminnnol()ver, state that alteration of proteins by simply treatin\ 183 tissues, part may be destroyed, since only traces appear in the urine. It resists autolysis. ^^ AGGLUTININS AND AGGLUTINATION ''^ This wi'll-kiiown plienonienon. the clumping or agglutination of bacteria when acted upon by the serum of immunized or infected animals, can hardly be considered as a means of defense, since we have no evidence that it in any way protects the aniraal.^*^ Agglu- tinated bacteria seem not to be severely injured by the process, and can grow vigorously in agglutinative serum. Possibly agglutina- tion favors phagocytosis and lessens dissemination of the infecting organisms, but it is improbable that the influence on the course of infection is great. Agglutination, therefore, may be looked upon as an incident in the infection, rather than as a definite method of re- sistance, and it is equally well produced hj immunizing with foreign cells or any foreign protein masses of suitable size which contain soluble antigens. For the production of agglutination it is necessary that the cell contain an antigen {agglutinogen) which has an ai^nity for the specific constituent of the serum, agglutinin. Normal serum may contain agglutinin ; e. g., typhoid bacilli are sometimes agglutinated by normal serum, even when it is diluted thirty times, but by immunization this property can be greatly increased until agglutination may be obtained with dilutions as high as one to a million. Whether normal agglutinins are essentially different from immune agglutinins is not known.^® Many protein solutions, especially extracts of plant tissues and legum- inous seeds, cause marked non-specific hemagglutination. "'' Likewise, bacterial extracts may agglutinate red corpuscles.''^ In immunization the agglutinogen, which is probably an intracellular protein, acts as a stimulator to the formation of the specific agglutinin. Hence, when we inject either extracts of cells or entire cells, we secure agglutinins, for the agglutinogens are liberated from the cells upon their disinte- gration. In erythrocytes the agglutinogen seems to be in the stroma.®^ We can obtain agglutinins against nearly all bacteria, including non-pathogenic forms, but in varying strengths. Agglutinins are found in the blood stream in the highest concentrations, but they are — 57 Wolff-Eisner and Eosenbaum. Boii. klin. Wndi.. lOOfi (43), 94.5. 58 Bibliocrraphy piven by Miiller, Oppenheimer's Handbuch der Bioohemie. 1900 (II (1) ). .592: Landsteiner, ihid., p. 428: Paltanf, Kolle and Wasserniann's Hand- buch., 1913 (II), 483. 58a Bull, however, would ascribe much importance to aff^lutination of bacteria for their removal from the circulation (.Tour. Exp. Med., 191,5 (22), 484). 59 See Andrejew, Arb. kaiserl. Gesundhtsamt., 1910 (33), 84. 60 Mendel, Arch. Fisiol., 1909 (7), 168. Gi Fukuhara. Zeit. Immunitat., 1909 (2), 313. •■'^ Chyosa, Arch. f. Hyg., 1910 (72), 191 184 CHEMLSTRY OF THE IMMUMTY REACTIONS also present in the various org-ans, and to greater or less extent in the other body fluids, excepting usually the spinal fluid (Greer and Beeht).®^ The place of their formation is unknown. Since bacteria contained within a collodion sac implanted in an animal give rise to the production of agglutinins, it is evident that the agglutinogens are diffusible to some extent, at least, through collodion. Old cul- tures of bacteria contain free agglutinogens, probably liberated from disintegrated cells, and filtrates of such cultures will neutralize ag- glutinins, showing both that the agglutinogens are filterable, and that the reaction of agglutination is a eliemical one and not depend- ent upon the presence of cells. Agglutinogens are said to pass through dialyzing membranes, while agglutinins do not. So it is evi- dent that the agglutinogen is of smaller molecular dimensions than tlie agglutinin, just as toxin molecules are smaller than antitoxin molecules. Agglutinogens are not destroyed by formalin, heat, or ultraviolet rays in concentrations sufficient to kill the bacteria con- taining them.*'^ Stuber holds that bacterial agglutinogens are lip- ine.^*^' Properties of Agg'lutinins. — Like most of the other substances produced in immunity, agglutinins are precipitated out of the serum in the globulin fraction. All attempts to separate them from pro- teins have been unsuccessful. Stark ^" found that trypsin does not attack the agglutinins readily, corresponding to the resistance of the serum globulins to this enzyme ; alkaline papayotin solution destroys them slowly, while pepsin acts much more rapidly. Alkalies are de- structive even when quite dilute, while acids are much less harmful. The temperature resistance of agglutinins seems to be variable, plague agglutinin being destroyed at 56°, while purified typhoid ag- glutinin may resist 80°-90° ; most agglutinin serums lose their activ- ity at 60°-65°. The rate of reaction of agglutinins increases with the temperature, as long as this is not high enough to injure the reacting substances."*' They are not precipitated by specific precip- itins, but are readily absorbed by charcoal. They can be formed by spleen tissue grown in artificial tissue cultures."*''^ The structure of the agglutinins (in the Elirlieh theory) is sim- ilar to that of the toxin; /. e., there is a haptophore group by which they combine with the agglutinogen, and a toxophore group by which they produce the changes that cause agglutination. The agglutino- gen is probably related to the antitoxins in structure, having a sin- gle haptophore to unite w\\h tlie agglutinin. By degeneration of the loxo])lior()us gi'oup of tlie agglutinin, agglntinolds nxay be formed. «3,Toiir. Infoct. Dia., 1910 (7), 127. 04Stiissano and L<'niailc, Coiii))!. Kcnd. Acail. Sei.. 1011 {\'r2) . (S2?,. 04a HioclKMii. Zoit.., li)16 (77), 388. 05 Jnaufr. Dissert., Wiirzhurj,', lOOo. oclMadscn, ct nl.. Jonr. V.\\wv. Mod., 1900 (8), 337. con I'ly/gode, Wien. klin. Wocli., 1913 (20), 841. THE MECHANISM OF AOGLUTINATIOy 185 It is believed that ag'glutiMins are eell receptors, wliich have a group n'itli a chemical affinity for the agglutinogen of the bacterial proto- plasm, and also another group which brings about the agglutination. They are, therefore, more complex than the simple receptors that unite with toxins, and are called receptors of the second order. Ac- cording to Ohno **' the reaction of agglutinin and antigen is in con- stant proportions, and seems to be a chemical rather than a physical reaction. Coplans '^^ finds this reaction associated with an increase in conductivity in the solutions, but whether this depends upon the agglutinin reaction itself, or upon associated processes, is question- able. Just what constituent of the bacteria acts as the stimulus to the production of the agglutinin is unknown. Apparently, there are at least two bacterial substances with this property, one of which seems not to be a protein, since it is soluble in alcohol and gives no biuret reaction, and resists temperatures up to 165°. The other gives all protein reactions, and is destroyed by heating to 62°. We con- sider, therefore, that there are two agglutinogens in the bacterial cell, one, thermostable, the other, thermolabile. The difference in the function of these two agglutinogens is still a matter of dispute. Likewise, the question as to whether they occur in the membrane or within the bacterial cell is still open, but Craw found that the insolu- ble residue of crushed typhoid bacilli, after being washed free of all soluble constituents, was but slightly agglutinated by active serum; therefore, the agglutinogens are probably soluble intracellular sub- stances. Agglutinated bacteria can be again separated from one another by the action of organic and inorganic acids, alkalies, acid salts, and by heating to 70° or 75°, and after once being separated they can- not be reagglutinated by fresh semm.®^ The Mechanism of Agglutination. — Tliis has been a fruitful field of research, in which the application of physical chemistry has been very profitable. At first it was believed that the clumping was brought about by loss of motility, until it was found that non-motile bacilli were equally affected. Similarly, the hypothesis of adhesion of the flagellfe was disposed of. Gruber'" and others supposed that a sticky substance, " glahrificm,'" was absorbed from the senim by the bacilli, which caused them to adhere on contact with one another; but this does not explain the flocking together of non-motile bacilli. Paltauf considered that the specific precipitin (see next section) pro- duced by immunization carried the bacilli down in the precipitate formed, and there is reason to believe that this reaction is of im- 67 Philippine Jour. Sci., 1008 (3). 47. fiSJour. Path, and Bact., 1912 (17). 130. eoEisenberg and Volk, Zeit. f. Infektionskr., 1002 (40). 102. "0 For complete bibliojrraphy, see Craw, Jour, of Hygiene. 1005 (.">). 113. 186 CHEMISTRY OF THE IMMUMTY REACTIOX^ portance, but it does not explain all the facts of agglutination, nor is the relation between agglutinating and precipitating power of im- mune serums a constant one. In support of this hypothesis is the observation of Scheller ' ^ that mixtures of typhoid bacilli and agglu- tinating serum lose their agglutinability by vigorous shaking, which may be interpreted as the result of disintegration of the agglutinating precipitate. Shaking of either 'bacteria or serum alone is without effect. Neisser and Frieduiann '- found that if the bacterial cells were saturated with lead acetate, washed in water until all soluble lead was removed, and then treated with IToS, they were promptly agglutin- ated and precipitated, supporting other observations that indicate that precipitation within the bacterial cells can lead to agglutina- tion. This sort of agglutination is related to the process of formation of" coarse flocculi in solutions, and probably depends upon alterations in surface tension. Bordet and Gay described, under the term conglutination, the observation that in ox serum there is a substance which combines with corpuscles (or bacteria) that have been acted upon by aggluti- nating sera, and augments the agglutination.'^^ Dean finds that, in general, agglutination requires two agents, one being the specific antibody, and the other a precipitable substance, probably a globu- lin. When cells have combined with the antibody the precipitable substance is aggregated on their surfaces and, presumably, determines the agglutination. Co-agglutination, described by Bordet and Gen- gou as the agglutination by an antigen and the homologous antibody, of the corpuscles of another animal, is probably closely related to these phenomena (Dean). Bordet ^* made the important observation that agglutination would not occur if both the bacterial suspension and the agglutinating serum were dialyzed free from salts before mixing; but if, to such mixtures, a small amount of NaCl was added, agglutination and pre- cipitation of the bacteria occurred at once. This observation brought the phenomenon of bacterial agglutination into close relation with the precipitation of colloids by electrolytes, Bordet comparing it to the precipitation of particles of inorganic matter suspended in the fresh water of rivers that occurs when the fresh water meets the salt water of the ocean. He found that the agglutinin combined with the bac- teria in the absence of the salts, and the resulting compound was pre- cii)itated by the addition of minute amounts of electrolytes,"''' which alone did not ])recipitate or agglutinate the bacteria or the serum. 71 Cent. f. Bakt., 1910 (.'54), l.'iO. "Miinch. med. Woeli., lOO-l (51), 4G5 and 827. T3 Litoraturo jrivon bv Dean, Proc. IJoval Soc. (V.) . 1011 (S4), 41(i: ITall. Univ. Calif. Publ., Pathol., 1013 (2), 111." 74 Ann. d. I'lnst. Pasteur. 1800 (1.3), 22.'). 74a Corroborated for sensitized red corpuscles bv Eisner and Kriedeniann. Zcit. Imniniiiiiit., 1014 (21), ,''>20. THE MECII.WISM OF ACI(lLrTI\\TfO\ 187 Tlii.s indicates that the ap'g'hitinins cause a eliaii^e in tht- bacteria which brings them under the same physical laws as the inorganic colloidal suspensions, which are characterized by being precipitated by the addition of traces of electrolytes.'^^ This precipitation is imdoubtedly due to changes in solution tension and surface tension (see ''Precipi- tation of Colloids," introductory chapter). Before the agglutinin combines with the bacteria they behave like the colloidal solutions of organic colloids, lieing precipitated only by the salts of heavy inetals, alcohol, formalin, etc., or by great concentrations of neutral salts. Field and Teague '^® have found that agglutinins carry positive charges while bacteria are negative, and that by the electric current agglutinins can be separated from bacteria with which they have com- bined; this shows that the agglutinin is not destroyed in the reaction. Teague and Buxton ^^ consider that neutralization of the electric charge of the bacteria is not, however, the only important factor in agglutination. According to Bechhold ~^ normal bacteria behave like inorganic suspensions that have each particle protected by an albumin-like membrane, which prevents them from being thrown out of suspen- sion by solutions of alkali salts, etc. After being acted on by agglu- tinin they are so altered that they behave like the unprotected inor- ganic suspensions, and are precipitated by salts and other electro- lytes. This suggests the possibility that the agglutinin makes the bacteria permeable for these electrolytes. Buxton and ShaflPer "^ also found that bacteria which have been acted upon by agglutinin be- have as if their proteins had been so changed that they are more capable of absorbing or combining with salts than when in their nor- mal condition. Strong salt solutions inhibit agglutination by prevent- ing the binding of the agglutinin.^" Tulloch ^°^ observed that in the presence of salts of mono- and di-valent cations, unsensitized bacteria do not readily precipitate or agglutinate, but sensitized bacteria, as Bordet show^ed, agglutinate with small quantities of salts. In this respect unsensitized bacteria behave like "non-rigid colloids," such as fresh egg white, while sensitized bacteria resemble "rigid colloids." such as denatured egg white. Hence he advances the hypothesis that the process of sensitization is akin to that of denaturation of proteins, the specificity perhaps depending on difiPerent degrees of denatura- 75 Arrheiiius (Zeit. physikal. Chem., in03 (46), 415) lias attempted to show that the gas laws are applicable to tlie partition of afrslutinin between the bac- teria and the medium, which he compares to tlie partition of iodin between water and carbon disuli)hid. This idea is not accepted by Craw (loc. rit.). nor by Dreyer and Douglas, Proc. TJoval Soc, 1910 (S2), 185. 76, Jour. Exper. INFed., 1007 (0), SO. 77 Zeit. physikal. Chem., 1007 (57). 70. 78 Zeit. f. physikal. Chem., 1004 (48), .385. 79 Zeit. physikal. Chem., 1007 (57), 47. 80 Landsteiner and St. Welecki, Zeit. Immunitat., 1910 (8), 397. 80a Biochem. Jour.. 1014 (8), 203. 188 CHEMISTRY OF Till; IMMUyiTY REACTIONS lion. Agglutination obeys the same laws as other similar physical phenomena ; the rate of agglutination depends upon the concentration of the suspension and of the electrolytes, and varies with the valence of the cations. Although bacteria in an electric stream move toward the anode like all suspensions, after being acted on by agglutinin they are agglutinated by the current between the poles ; ^^ this indicates the importance of the electrical charges of the bacterial surfaces in tlieir agglutination reactions. In all respects the behavior of bacteria and agglutinin resembles the behavior of colloidal mixtures in suspension (Neisser and Friede- mann) ^~ which form an electrically amphoteric colloidal suspension, so that the ions of electrolytes or the electric currents, by discharging them unequally, cause precipitation. Pliysico-chemical researches, however, have yet failed to explain the specific character of the ag- glutinins for specific bacteria, but IMiehaelis *^ has developed an inter- esting analogy in the specific agglutination of bacteria by acids. This is based on the fact that the optimum concentration of H ions which precipitates proteins from solution is characteristic and constant for each protein, and the same is true for the agglutination of bacteria by acids, the agglutination by acids being even more sharply specific in some cases than the agglutination by immune sera ; e. g., typhoid and paratyphoid bacilli are readily distinguished because the former are agglutinated by a concentration of H ions from -4 to 8 X 10"'', while paratyphoids require 16 to 32 X 10'^, and colon bacilli are not agglu- tinated at all by acids. The acid agglutination, however, does not al- ways affect all strains in the same way, some strains which are not readily agglutinable by antisera also resisting acid agglutination.®^* According to Arkwright,®^" typhoid bacilli contain two extractable proteins that are agglutinated by acids, one at 3.6 X 10"^ and the other at 1.1 X 10"'' ; the former seems to be related to, if not identical with, the substance that is precipitated by immune serum. Apparently acid agglutination of bacteria belongs to the same class of reactions as the coagulation by H ions of amphoteric colloids of preponderatingly acid character. Bacteria which have been sensitized by serum are more sensitive to acid agglutination than are normal bacteria.^* Alterations in the agglutinability of bacteria are marked, e. g., strains of typhoid bacilli freshly cultivated from human infections SI Ilcclihold: liowcver, Buxton and Teag-ue (Kolloid Zoitsolir., 1908, II, Suppl. 2) state that agfjlutinin bacteria do move towards tlie anode, but slower tlian normal bacteria. 82 Miincli. med. \A'ocli., 1!)04 (51), M\'-> and S'27 ; sec also (iiiard-MaiiL:iii and Henri, C'ompt. Rend. Soe. IJiol., ]i)04, vol. 5fl ; and Zanggcr, Cent. f. Hakt! ( ref. ) , ino.5 (.'^(i), 225. 83 Folia Serologica, 1911 (7). 1010; also Benias.li, Zeil. InninuiitiU., 1!U2 (12), 268. 83a See Kemper, .Tour. Tnf. Di.s., ]!)1G (18), 200. 831. Z,Mt. Imnuniitiit., 1014 (22), .SOO; Jour. Ilyg., 1014 (H). -'(W. 84 Krumwiede and Pratt, Zeit. Immunitiit., 1013 (10). 7^\'i . PRECIPITINS 189 may be practically inagglutinable even by active scrum, but after pro- longed cultivation on media they may or may not develop agglutina- bility. This phenomenon has not yet been satisfactorily explained, but it may depend on an active immunity of the bacteria against the agglutinins. Such bacteria injected into rabbits produce antisera that will agglutiiuitc ordinary agglutinablc strains, but not themselves; hence they do not lack agglutinogens. They give normal complement fixation reactions, and hence do not lack receptors, and they agglu- tinate with acids and chemicals much tlie same as ordinary agglutinable strains. ^^'"^ PRECIPITINS *■' If to a solution containing proteins we add in proper proportions the serum of an animal inmiunized against the same protein, a pre- cipitate will soon form. AVliile not absolutely specific, the quantitative specificity of the precipitin reaction is sufficiently characteristic to be of great value in biological, bacteriological, and medicolegal work, and it is of importance to the physiological chemist, since it furnishes a means of distinguishing between closely related forms of proteins, more delicate by far than any known chemical reagent. The serum reactions also prove that there are sometimes essential differences be- tween the proteins of different species of animals, even when by all other methods these proteins seem to be practically identical ; e. g., lactalbumin of cow's milk is in some respect different from lactal- bumin of goat's milk since it produces a different precipitin. Medi- colegally they offer an accvirate method of determining the origin of blood and serum stains, no matter how old the stain may be ; thus Hansemann *** found that material obtained from a mummy 5000 years old gave the precipitin reaction.-" Production of Precipitins. — For the production of the precipita- tion reaction it is necessary to have in the substance used for immu- nization a certain group, the precipitinogen, which when injected gives rise to production of precipitin by the animal. Apparently almost any protein may act as a precipitinogen if injected into the proper ani- mal, but it must he a foreign protein; rabbit serum will not produce precipitins if injected into a rabbit,^^ probably because it is normally present in the blood of the rabbit and therefore does not stimulate any reaction; but certain chemical alterations in the proteins of an animal, such as heating, iodizing, or partial digestion, may render them 84a Mcintosh and McQueen, Jour. Hyg., 1914 (1.3), 409. 85 For complete bibliocraphy of the subject of "Precipitins" see the resume by Michaelis, Oppenheimor's Handb. d. Biochemie. 1009. II (1), ,5.52; Kraus. Kolje and Wassermann's Handb.. 1913, II; Uhlenhuili and Stefl'enhairen. ihid.. III. 2.i7: Zinsser. "Infection and Eesi stance."' sGMiinch. med. Woch.. 1904 (.30), 572. 87 Not corroborated by Scliniidt. Zeit. allor. Pliysiol., 1907 (7), 369. 88 Rarely a slight reaction against homologous proteins has been obtained {iso- precipitins ) . 190 CHEMISTRY OF THE IMMUNITY REACTIONS SO different from the normal proteins of the same animal that they will act as an antigen when present in the blood of that animal, or an- other of the same species, from which they were derived. Of the natural proteins of serum the giobulins are much more active precipi- tinogens than the albumins. In general the more foreign the protein, the greater the amount of precipitin; closel}^ related animals, e. g., rab- bit and guinea-pig, produce little precipitin for one another's pro- teins. This indicates distinctly that difference in species depends upon or is associated with difference in chemical composition of the proteins. DiffereJit sioecies of animals have very different capacity for producing precipitins, rabbits producing active sera, while guinea- pigs can produce but feebly precipitating sera. Cantacuzene ^'^ be- lieves that precipitins are formed chiefly in the lymphoid tissues and bone marrow, and that the mononuclear macrophages are most active in their formation.*"'' Only proteins can produce precipitins; when split to the peptone stage they lose this property, but the proteins of serum resist tryptic digestion a long time before losing their precip- itinogenic property,"*^ which is destroyed much more quickly by pep- sin-HCl mixtures. The precipitate itself is very resistant to disin- tegrative agencies, including putrefaction (Friedberger),"^ but is soluble in dilute acids and alkalies. It has the power of binding complement (Gay^-) and if the complement causes solution of the precipitate, poisonous substances are formed (Friedberger). Ex- cess of antigen prevents the formation of precipitate, or redissolves it, but excess of antiserum has no effect. Since both reacting sub- stances are colloids tliey follow the laws governing other mutually precipitating colloids, and precipitation occurs only when they are brought together in concentrations that lie within definite zones of relative proportions. It is, of course,' perfectly possible to have a union of precipitin and antigen without anj^ visible precipitate occurring, since the product of the reaction is not necessarily insoluble under all conditions; in this case the occurrence of a reaction must be demon- strated by some other method, e. g., the complement fixation reaction. No precipitins can be secured against lipoids or other non-protein sub- stances. Possibly precipitins can be produced for closely related sub- stances with molecules approximating in size the protein molecule, e. g., certain substances present in supposedly protein-free filtrates of bac- terial cultures. As with the agglutinin reaction, electrolytes must be present or precipitation will not occur. Neither the precipitin nor the so Ann. Inst. Pasteur. 11)08 (22), 04. 8»a Spleen tissue eiiltivated artificially in tlie presence of liorse serum jiroduces specific precipitins for liorse serum, and tissue from the spleon of a guinea pisr that has received injections of horse serum also develops precipitins for horse serum wlien grown in cultures (Przygode, Wien. klin. WoSoc. Biol., 1906, Vol. 61, and subsequent volumes. 94a .Tour. Infect. Dis., 1916 (19), 452. 94b Precipitinogens are relatively resistant to moderate heating, and heat«d extracts of bacteria are used for precipitin tests imder the name thermoprecipi- tins. See review by A. Ascoli, Virchow's Arch., 1913 (213), 182. 95 Moll, Zeit., exp. Path. u. Ther., 1906 (3), 325; Welsh and Chapman, Proc. Royal Soc., B., 1908 (80), 161; Zeit. Immunitat., 1911 (9), 517. 95a Jour. Immunol., 1916 (1), 35. 192 CIIEMLSTIx'V OF THE JMMUMTY KEACTI0N8 precipitate consists of more than the precipitin alone ; it ma}'' be added that the precipitate is always less in amount than the total giobuliu of the antiserum."'^ It is always greater when the reaction is between homologous antiserum and antigen, than with even closely related but heterologous antigens,^' so that the quantitative measurement of the amount of i)recipitate is of value in applying this reaction to deter- mine the imture of protein solutions. The dilution of the reacting solutions is of influence, however, for if in too dilute solutions weak precipitins may fail to give reactions; with strong precipitins the influence of dilution is nuich less (Michaelis). According to the source of the protein used we recognize bacterial precipitins, phyto-precipitins (for plant proteins),'*^ and zooprecipi- tins (for animal proteins). Although tissue extracts, body fluids, and exudates are generally used in immunizing, i)urified constitu- ents of these protein mixtures will also excite precipitin formation, e. (J., we may immunize with caseinogen as well as with milk. Com- plete pepsin digestion of proteins deprives them both of their pre- cipitability and their power to produce precipitins, the former prop- erty being lost first. Trypsin seems to produce the same effect more slowly. Heating to coagulation — indeed, heating in the autoclave — does not destroy the precipitinogenous property of proteins, but modi- fies somewhat the reactions of the precipitin obtained,^ and precipi- tinogen is destroyed by alkalies. The specificity of precipitinogens is so modified by heating that the precipitins engendered by a boiled antigen react with the boiled antigen and with similarly heated anti- gens from other species, but not with unheated antigens even from the homologous species. - As proteins introduced into the stomach are normally destroyed before being absorbed, they do not enter the blood and cause pre- cipitin formation. However, as is well known, eating of excessive amounts of egg-albumen or other easily absorbed proteins may re- sult in their passing the barriers and entering the blood stream, and in this way precipitins have been experimentally produced. Pre- sumably the precipitin reaction is a means of throwing such foreign proteins out of solution and rendering them harmless. According to Zinsser ^ and others, the function of the precipitin is to sensitize 06 Francescliclli, Arch. f. llyg., ]!)07 (00). 207. 07 Welsh and Cliapman, .Toiir. Hygieno, 1910 (10), 177. 98 Literatures on precipitins for vegetable proteins given bv Wells and Osborne, Jour. Infect. Dis., 1!)11 (8), 66. 1 See Oberniayer and Pick, who consider in detail the efVects of various modilica- tions of proteins upon their ))o\ver to incite precipitin formalion (Wien. kiln. Woch., 1006 (19), 327). The precipitability of the serum, or its power to pro- duce precipitins, is not affected by disease "(Pribram, Zeit. exp. Path. u. Ther.. 1006 (3). 28). -'Schmidt, Bioclieiu. Zeit., 1908 (14), 294; 1910 (24). 4.".; Zeif. lininunitiil., I!tl2 (i:i), 173; also Zinsser, "Infection and Resistance," l!»14. p. 2t;(). ••'.Jour. Kxper. Med., 1912 (15), 529; 1913 (18), 219. ,l.\.l/'//17,.l.\7N O/.' M.LKItCY 193 tlie uufoniHHl forcijiii pmrtcins to the difjestive complement, a view in harmony with tlie prevailing tendeiiey to correlate tlie inunimity reaction witli defense through enzj'niatic hydrol^'sis. Precii)itiii appeals in the blood generally about six days after in- jection of the ])rotein, but disappears after injection of eacli subse- quent dose of protein, to reappear again after a somewhat sliorter lapse of time. After injections are stopped, the precipitin disap- pears rather rapidly, but never appears in the urine, although it may enter the fetal blood from the blood of pregnant female animals. The presence of precipitins in the blood does not seem to prevent the excretion of the foreign protein in the urine, nor are the animals less susceptible to the toxic action of the foreign protein; indeed, the reaction is even stronger in the immunized animals, and sometimes the ordinary dose becomes fatal. Certain antibodies are carried down with the precipitates formed when the serum containing them reacts under proper conditions with an antiserum ; e. g., diphtheria antitoxin is precipitated when added to the serum of a rabbit immunized to horse serum. This is not true of all antibodies, however.^^ As the pre- cipitates formed in the precipitin reaction, when injected into a guinea- pig make it passively hypersensitive to the protein used as antigen in the precipitin reaction, it would seem that the precipitin and the anaphylactin are identical (Weil),^^ or at least closely associated. Chemical Properties. — In its chemical nature precipitin resembles the "antibodies'' generally, being precipitated in the euglobulin fraction of the serum/ and slowly destroj'ed by trypsin, rapidly by pepsin. It cannot be separated from the serum proteins. The pre- cipitation by precipitins is not an enzyme action, for the precipitins are used up in the process. It apparently does not differ from pre- cipitations of colloids by other colloids of opposite electrical charges, except in that the reaction is specific. ANAPHYLAXIS OR ALLERGY In many instances the injection of a foreign protein into an ani- mal produces severe, perhaps fatal, intoxication. With some pro- teins this natural toxicity is very marked, — thus eel serum is fatal to rabbits and dog-s in doses of 0.1 to 0.3 c.e. per kilo, and foreign sera are commonly toxic to other animals; e. g., fresh bovine and human serum are quite toxic to" guinea-pigs. This so-called "pri- mary" toxicity is reduced or destroyed in most cases by heating to 56° for 30 minutes.*^ Almost any non-toxic soluble protein, however, may be made toxic for animals by giving the animal a small dose of 3a See Gay and Stone. Jour. Immunol., 1916 (1), 83. 3b Jour. Immunol., 191 6 (1), 1. *Fiuick (Cent. f. Bakt. ( Ref. ) . 100.5 (36). 744) states tliat if tlie precii)itin serum is very strono:, part of the precipitin comes down in the pseudoiriobulin. 4a The nature of the toxic agent is unknown, hut there is reason to helieve that it is formed, at least in part, during- the coagulation of the drawn blood. 13 194 CflEMlsTh'Y OF THE JilMiMrV REACTIOXS this same protein at least eight days previous!}'. This preliminary dose, which may be extremely minute, renders the animal hypersen- sitive to the same protein, so that a relatively small quantity (a few milligrams in the case of the guinea-pig) of an ordinary entirely harmless protein, such as egg white or milk, produces violent, often fatal, symptpms when introduced into the blood of the animal. We have not the space to discuss the general features of the reaction, its history and its relation to biology and pathology, which are fully covered in many easily accessible reviews,-' but shall limit our consid- eration to the more definitely chemical aspects of the reaction.^ The Substances Involved (Anaphylactogens) . — So far as now kno^vn. these are always proteins, and with the exception of gelatin ^^ and a few others, practically any soluble protein will produce sen- sitization and intoxication of susceptible animals, i. e., almost any soluble protein may be an anaphylactogen. As with the other immun- ity reactions, observations have been made which are interpreted as indicating that non-protein substances can produce this reaction, but these interpretations are not generally accepted.'"' It is possible, how- ever, for non-protein substances to combine with or alter the pro- teins of an animal so that they become as foreign proteins to that animal, and thus cause sensitization; in this way can be explained apparent anaphylactic reactions to iodin and arsenic compounds, and other non-protein substances. So far as my own experiments show, nothing less than an entire protein molecule will suffice," the products of protein cleavage all being inactive.^ Presumably the inefficiency of gelatin as an anaphylactogen depends upon its de- ficiency in aromatic radicals, since these radicals have been shown (Vaughan, Obermeyer and Pick) to be particularly r important in immunological reactions. It is not necessary for a protein to con- tain all the knowni amino-acids of proteins to be active, however, for certain vegetable proteins (zein, hordein, gliadin) which lack one or 5 Doerr, Kolle and Wassermann's Handbuch, 1913, Vol. II: and Zeit. f. Tm- miinitat., 1010: (2, ref.), 49; also v. Pirquet, Aroh. Int. Med.. 1911 I 7), 2.'>9; Friodmann, Jaliresbcr. Erpeb. Immunitiitfrsch., 1911 (6). 31; Schittenliehn. ibid., p. 115; Hcktoen, Jour. Amer. Med. Assoc, 1912 (fiS), 1081; Zinsser, Areh. Int. Med., 1915 (16), 223. Concerning anaphylaxis in man see Lonpcope. Ainer. .lour. Med. Sci., 1916 (152), 625. 6 Many of the chemical features of anaphylaxis I have covered in the foUowinj: series of articles: .Tour. Inf. Dis., 190S (5). 449; 1909 (6). 506; 1911 (S), 66; 1911 (9), 147; 1913 (12), 341; 1914 (14), 364 and 377; 1915 (17), 259; 1916 (19), 183. 6a Wells, Jour. Amer. Med. Assoc. 1908 (50), 527; Jour. Infect. Dis., 1908 (5), 459. <5b Concerning lipoids as antigens see Meyer, Zeit. Inimunitiit., 1914 (21), 654. 7 Zunz (Zeit. Inimunitiit., 1913 (60), 580), however, claims to get typical re- actions with the higher proteoses from fibrin, a tiling T have never succeeded in doing with proteoses from egg albumen. sAbdcrhalden (Zeit. physiol. Chem., 1912 (81), 314) states that he has ob- tained a positive reaction with a synthetic polypeptid containing 14 amino-acid molecules, including only leucine and glycocoll. ^.Y.l/'//r/,.lA7.S' OR ALLERGY 195 more of such amino-acids as glycocoll, tryptophane, or lysine, pro- duce typical reactions. Some compound proteins are efficient ana- phylactoprens (mucin,*"" casein) but with alpha-nucleoproteins which have been thoroughly purified I have obtained only negative results ; **• as also with histon and nucleic acid, the isolated components of nu- eleins. Bacterial substances, extracts of plant tissues, purified plant proteins, and proteins obtained from invertebrates and cold-blooded vertebrates, have all been found to be anaphylactogens, if they can be introduced by any means into the blood or tissues in a soluble unaltered condition. If the proteins are rendered insoluble by coagulation they become inert, but proteins which cannot be made insoluble by heating (e. g., casein, ovomucoid) withstand boiling temperatures. Trypsin destroys anaphylactogens in just the same proportion as it splits the protein molecules ; thus, globulins resist trypsin longer than albumins, both as regards coagulability and anaphylactic activity. Acids, alkalies and other chemical agents may modify the reactivity of proteins in propor- tion to the changes in solubility or constitution which they produce.^ The amounts of protein necessary to produce reactions in guinea- pigs are very small. With crystallized egg albumin sensitivity has been produced with one twenty-millionth of a gram (0.000,000,05 gm.) and fatal reactions are obtained after sensitization with one- millionth of a gram. No other animal seems to be so sensitive to this reaction as the guinea-pig, however, and rabbits and dogs require larger, and in many instances, repeated doses to render them ana- ]5hylactie. Within certain limits large doses are less effective in sen- sitizing guinea-pigs than small, e. g., one milligram of most proteins will usually be much more effective than one hundred milligrams. Wliite and Avery ^^ found that there is a certain relation between the minimum sensitizing and the minimum intoxicating dose ; with ex- tremely minute sensitizing doses a larger intoxicating dose is required to produce fatal reaction than when the sensitizing dose is larger. It is now generally accepted that both the sensitizing and intox- icating agent are (or are derived from) one and the same protein, but the minimum intoxicating dose is always larger than the mini- mum sensitizing dose: thus, with pure egg albumin the minimum lethal dose for sensitized pigs was one-twentieth to one-tenth milli- gram by intravascular injection, or about one hundred times more than the minimum fatal sensitizing dose. With less soluble proteins the disparity is even greater, for with such the sensitizing dose is not ?■! Elliott, Jour. Infect. Dis., 1914 (15), 501. sb See review in Zeit. Immunitat., 101.3 (10), 500. conrernino- alplia-nucleopro- teins. Avhicli is the type nsiially desip:natofl as "niirlooproteins." T have found beta-nnoleoproteins to be more effective antigens (Jour. Biol. Cliem., 1016 (28), 11). 9 See Dold and Aoki. Cent. f. Bakt., Ref. Bellage, 1912 (54), 246. 9a Jour. Infect. Dis., 1013 (13), 103. 196 niEMlSTRY OF Tin: niMI MTV }{EACTIO\S much changed, but the mininumi iutoxieatiiig' close is relatively much increased. Apparently an animal may be killed by much less antigen than is required to saturate the antibodies present in its body (Weil). The proteins concerned must be foreign to the circulating blood of the injected animal, but they ma}^ be tissue proteins of the same ani- mal (e. g., placenta elements, organ extracts, lens proteins) which are not normally present in its blood. Indeed it has been claimed that by injecting a guinea-pig with the dissolved lens of one eye it will become sensitized so that it will react to a subsequent injection of the lens from the other e^-e.^" It is also possible that vai'ious chemicals may so alter the blood proteins that they, too, behave as foreign proteins to the same animal, rendering it sensitive to the same altered proteins if they are formed subsequently by another injection of the chemical (e. g., iodin, salvarsan). In general, tis- sue proteins are less active antigens than the proteins of the blood, lymph, and secretions, but even keratins may produce anaphylaxis when dissolved ^^ and positive results have been obtained with pro- teins from mummies. ^- The Poisonous Agent ( Anaphylatoxin) . — The symptomatology of the intoxication which follows injection of the protein into an animal sensitized with the same protein, is such as to leave no question that a poison is responsible, and this is established as a fact in several ways, although as yet the poison has not been isolated. As the symp- tom complex is practically the same no matter what sort of protein is being used, it would seem that the poison must always be the same or similar — a striking and surprising fact in view of the extremely varied nature of the proteins capable of inciting anaphylactic in- toxication. Probably the poison is a product of cleavage of the pro- tein by tissue or blood enzymes, which act only in the presence of the specific antibodies which unite the protein to the enzjnue (or complemejit). A^aughau and his collaborators showed that i)roteins boiled with an alcoholic NaOlI solution might be split into two frac- tions, one toxic and alcohol-soluble, the other non-toxic and insoluble in alcohol. The toxic fraction gives all the protein reactions (except that of jVlolisch for carbohydrates) and in doses of 8 to 100 mg. kills guinea-pigs witli symptoms practically identical with those of ana})hy- lactic intoxication. The uniformity of the toxic effects with prepara- tions from different sorts of proteins suggests the existence in every protein molecule of some fundamental toxic group, common to all y proteins, the specificity residing in other non-toxic attached groups. This and other observations led him to the hypothesis that specific enzymes are develoix'd in response to the presence of foreign pro- 10 IJlilcnlmtli and Haendcl, Zoit. f. Tniiminiliit., 1910 (4), 761. JiKriisiiis, Arch. f. Aufrcnlioilk., Sujipl., 1010 (47), 47; Clougli. Aib. kuis. GesuiKDitsiuntc, lt)ll (;n), 431. 12 Ulilcnliuth, Zeit. f. Iminuiiitiit., 1!)10 (4), 774. 4A'.l/'//17..1A7.s' on ALLKIiOY 197 teius in the blood stream, and that upon injection of a second dose of the same protein these enzymes at once disintegrate it, and some of the cleavage products being toxic the anaphylactic in- toxication results. ]\Iany of the later developments in this field, especially Abderhalden's studies on "protective ferments," have added support to this hypothesis, so that in its fundamental concep- tions it is now the most generally accepted explanation of the processes involved in anaphylaxis.^^ Friedberger caiTied the matter a step farther by showing that if serum from a sensitized animal is incubated for a short time with the same protein, and in the presence of enough complement, a poison is developed which produces the typical symptoms of anaphylactic intoxication when injected into guinea-pigs. Tliis poison resists heat- ing at 56°, but not at 65°, and is not a true toxin, for it will not produce an antitoxin immunity. In the absence of complement, or when the complement fixation is prevented by strong salt solution,^* the poison (anaphylatoxin) does not develop, so that the anaphylactic reaction falls into the same class as the lytic reactions, in which the non-speeiMc serum complement is united to a cell by the specific am- boceptor, and then causes lysis of the cell ; in anaphylaxis not an organized cell but a complex protein molecule is disintegrated by the complement, but in either case a poisonous substance may be liberated. This agrees with Vaughan's hypothesis in ascribing the poisoning to products of protein disintegration formed by enzyme action, but differs in that specific intermediary substances or amboceptors are sup- posed to be developed by sensitization, rather than specific enz;ymes. Friedberger is of the opinion that many or all the different immunity reactions depend upon a single antibody, the different reactions merely being different methods of demonstrating the presence of the anti- body in tiie serum. The precipitin reaction differs from the anaphy- lactic reaction, he contends, only in that in the latter the specific pre- cipitate is (L'ssolved by complement, yielding the anaphylatoxin. There are many objections ^^ to accepting this idea in its entirety, which we shall discuss later, but the formation of a poison resembling that of anaphylaxis, by a digestive action of complement fixed to the antigen bj' the antibody, seems to be well established, both as regards in vitro and in riro reactions. It would seem probable that proteins may yield a similar poison in whatever way their hydrolysis is brought about, provided the cleav- age is not too deep-seated. For example, Rosenow ^® has found that 13 See Vaiighan, Amer. Jour. Med. Sci., 1913 (145), 161; Zeit. Immunitiit., 1011 (9), 458. Also a full review in his "Protein Split Products," Philadelpiiia, 191.3. 14 Fricdherger's explanation of the inhibiting efieot of salt as interference witli complement action, has been questioned. (See Zinsser, Arch. Int. Med 1915 (16), 238.) 13 See Besredka et a/., Zeit. Immunitiit., 1912 (16), 249. 16 Jour. Infec. Dis., 1912 (11), 94 and 235. 198 CHEMISTRY OF THE niMiyiTY REACTIOXS pneumoeocci and other bacteria, permitted to autolyze for a proper length of time, produce poisonous substances with all the toxicologic characters of the anaphylatoxin. Too extensive autolysis again de- stroys the poison, which is also produced by digestion of pneumo- eocci with serum from normal guinea-pigs, and more rapidly with serum from sensitized animals, which likewise causes a demonstrably more rapid proteolysis. The pneumococcus anaphylatoxic poison is soluble in ether and seems to be a base, containing amino-acids, but Friedberger did not find anaphylatoxin made from serum proteins to be soluble in ether or alcohol, nor was it precipitated with the globu- lins. The so-called "Abderhalden method" of sero-diagnosis of preg- nancy, which depends on the presence of specific proteolytic properties in the blood, is an especially studied instance of these principles, and is discussed later. Presumahly anaphylactic intoxication is hut an exaggeration of the normal process of defense of the body against foreign proteins (in- cluding bacteria) through digestion. Normally this is accomplished in the alimentary tract, and complete disintegration past the toxic stage is made certain by the presence of erepsin in the intestinal wall ; but if intact foreign protein molecules reach the blood in any way, this same digestive destruction is performed by the enzymes of the blood or tissues. So abnormal is the ''parenteral" introduction of foreign proteins that, once it has happened, the protective mechanism is stimulated to the production of large amounts of proteolytic sub- stances, and on this account if another quantity of the same protein is again parenterally introduced the breaking down of the protein is ■extremely rapid. Certain of the disintegration products are toxic, but with the normal rate of disintegration the amount present at any one time is inadequate to cause poisoning ; when the proteolysis is ac- celerated, as in the sensitized animal, a poisonous dose may be pro- duced, with the resulting anaphylactic intoxication.^" Whether this proteolysis takes place both in the blood and tissues is not known. It has been found that the specific proteolytic power of the blood is in- creased in sensitized animals, but on the other liand, there is evidence that without the intervention of the liver (at least in dogs) anaphy- lactic intoxication cannot take place (INlanwaring and others'). During the reaction, in any event, ])i'()du('ts of jirottMii liydi-olysis a])pear in the blood (Abderhalden).'^ Among possible cleavage products of proteins which may be the tf)xic agent in anajiliylaxis, is /3-imidazolylethylamine ("histamine"), which is derivable from liistidine, as indicated by the structural for- mulas, and which produces effects resembling acute anaphylactic in- 17 Heilner (Zeit, Biol., 1012 (58), .S.3.3) bolievos t.liat tlio anaphyhu'tio poisons arc substances wliich normally arc dostroyod by jirotoolysia, but tliat in the sensitized animals there is a depressed catiibolism \\hic]i prcvtMits tlieir destruc- tion. 18 Zeit. physiol. Chem., 1912 (82), 100. ANAPHYLAXIS OR ALLERGY 199 toxicatioii/'^ Methylguanidine is said to produce somewhat similar symptoms,-" and other amines possibly may be involved. (See Chap- ter iv, Ptomains; Chapter xix, Pressor liases.) CTT ] I X X HC=C— CH,— C'T[ ( Nil.,) —coon Histiclinc fir /^ HX X HO=(_'— CH,— CH,— NH, /3 — imidazolyletliylaniine ( ITistainiiu' ) . The relation of the normal toxicity of certain foreign sera to ana- phylactic intoxication has not been determined, but there seem to be both definite similarities and differences,-^ which have been discussed by Loewit; '-"^ chief of these diflferences is the absence of the bronchial spasm with pulmonary emphysema which is characteristic of anaphy- laxis. The anaphylactic poison would seem to be after the order of the alkaloidal poisons, at least from the pharmacological standpoint, since it produces its effects quickly, and these effects, no matter how se- vere, are strictly transitory, passing off completely in a few hours, which indicates that (like morphine, strychnine, etc.) they do not produce any deep-seated structural alterations in the tissues. Ac- cording to Schultz -- the chief effects are directly on the smooth muscles. Such anatomical alterations as are produced, of which hemorrhages and waxy degeneration of the voluntary muscles of respiration -^ are most noticeable, are ascribable to the effect on respiration, which in the guinea-pig often amounts to total asphyxia- tion through spasm of the musculature of the bronchioles (Auer and Lewis) with profound permanent emphysematous distension of the lungs. This effect is peripheral, and is inhibited by atropine.-* Cal- cium salts also reduce anaphylactic reactions.-' The poisonous frac- tion obtained from proteins by Vaughan's method resembles anaphyla- toxin, in that it causes a fall in blood pressure by paralyzing the vaso- 19 See Barper, "The Simpler Natural Bases," London, 1914, p. 30. 20Heyde. Cent. f. Physiol., 1911 (2.5), 441; 1912 (26), 401. 21 It lias been found that extracts of various orsjans are especially toxic to animals, but that this toxicity may be suppressed by a minute dose, for a few minutes later larsre doses can be injected with impunity, alihoui/h the l)lood of the animal is highly toxic durinjj the immune period, which is of brief duration. This condition is called slcpto-phi/la.ris. (See Lambert, Ancel and Bouin. Compt. Rend. Acad. Sci., 1911 (154). 21.) Vauffhan reports the findinjjr in normal tissues of substances resembling his "protein poisons," which perhaps come from autolysis or tissue metabolism and may be related to the "primary toxicity" of organ ex- tracts. 2iaArch. exp. Path. u. Pharm., 191:? (73). 1. 22 Bull. llvfT. Lab., U. S. P. H. and ]\f. TL Service, 1912 (80), 1. 23 See Cent. f. Pathol., 1912 (23), 945. 24 Jour. Exp. Med., 1910 (12), 151; Schultz, .Jour. Pharm. and Fxi). Tlier.. 1913 (3), 299. 25 Kastle, Healy and Buckner, Jour. Infec. Dis., 1913 (12), 127. 200 CIIKMISTJ,')- OF THE niMCMTY h'EACTJOXS motor endings in the blood vessels (Edmunds -"'''). It also produces local urticaria when rubbed into the skin and behaves much like histamine, with which, however, it is not identical. One gram of casein yields enough of Vaughan's poison to kill 800 guinea-pigs, and the poison seems to contain most of the aromatic radicals of the pro- teins. There is also much other evidence of the importance of the aromatic radicals' in anaphylaxis.-^'' Other effects of the anaphylactic toxin are leucopenia, eosinophilia,-'' reduced coagulability of the blood, and a severe fall of temperature unless the dose of antigen is very small when the temperature may rise.-^ The antitrypsin content of the blood is not increased in the anaphylactic animal (Ando -'^). Poisonous substances similar to ana- phylatoxin appear in the urine during the anaphylactic intoxication (PfeifiPer).-® Anaphylactic reactions are commonly associated with jnarked eosinophilia, both local and general.-" As with other poisons, anaphylatoxin produces different symptoms in different animals. In. dogs the chief effects are a great fall in blood pressure,^" loss of coagula- l)ility of the blood, hemorrhagic enteritis, but no bronchial spasm. In rabbits the heart is severely affected, while in guinea-pigs there is a remarkable lack of interference with the heart, so that it beats long after respiration ceases. A pressor substance has been found in the serum of intoxicated guinea-pigs, which is not present in the artificial anaphylatoxin and therefore presumably is produced in the body of the animal.^^ In man the symptoms are most like those in the guinea- pig. If the protein is injected into the skin of a sensitized animal there follows a severe local reaction, — hyperemia, edema, even necrosis, — indicating that in this specific proteolysis, poisons are formed which have a profound local effect, especially on the blood vessels. Repeated anaphylactic intoxication may result in structural changes in the kidneys, heart muscle and liver (Longcope ^^''). Metabolism studies may show an increased toxicogenic destruction of protein, ''"" but the increase in amino-acids presumably resulting from proteolysis in the sensitized individual, is not large enough, if it does occur, to be demon- strated by chemical methods.^^'^ 25a Zeit. ImmimitJit., 1013 (17), 105. Soe also Underliill and llciulrix. .Tour. Biol. Chcm., 1915 (22), 4G5. 25b Seo Baohr and Pick, Arch. Exp. Path., 1913 (74), 73. 26 Schlecht and Sclnvenkor, Dent. Arch. klin. Med., 1912 (108), 405. 27 See Vauglian, et al., Zeit. Immunitiit., 1911 (9), 458. 27a Zeit. Iiiinuinitiit, 191:5 (18), 1. 28 Zeit. f. Immunitiit., 1911 (10), 550. 21' I^iiteraturc hy Moscliowitz, New York INIed. .Tour., -Ian. 7, 1911; SdiU'cht and Scliwenker, Arch. exp. Patli. u. Pharm., 1912 (68). Ki.i. 30 Probahlv from influence U])()n the nerve ondinfrs of the vessels (Pearce and Eisenhrev, .Tour. Infec. Dis., 1910 (7), 5115). •ii Hir.sclifeld, Zeit. Immunitiit., 1912 (14). 4G(i. 3iaJour. Exp. Med., 1913 (18), G78; 1915 (22). 7!t3 : also lUniuhlon. .lour. Immunol., 1910 (1), 105. •'ill. See Major. Deut. Ardi. klin. Me.l.. 1!I14 (llti), 248. •■•■"•See .\uer a!id Van Slvke, Jour. I'lxj). .Med.. 1913 (18), 210; P.ar^icr and Dale. I'.iochcni. .lour. 1914 (8), G70. .1 \ t/'//r/..i \/N ni; M.i.r.iu.y 201 There is, liowcvcr, imu-li (lou])t as to the identity of tlie process of anaphylatoxin formation (as it occurs when antigren, antibody and complement are incubated in vitro) and the process of anaphylactic intoxication. In the first place, a poisono^^s character, apparently identical with this "anapliylatoxin,'' may be griven to serum without the use of any specific antibody whatever; merely agitating fresh serum with any finely divided foreign material that offers large total surfaces, such as kaolin, agar, or starch, is sufficient, as also is treat- ment with li])oid solvents, such as chloroform (Jobling). In fact, merely removing the fibrin from the plasma may make the resultant serum highly toxic, even for the very animal from which it came. Furthermore, if anaphylactic shock were the result of anaphylatoxin formation in the sensitized animal through the reaction of antigen with antibody and complement, the intoxication should occur if antibod}'" and antigen are injected simultaneously into an animal ; but as a mat- ter of fact the animal receiving antibody in passive sensitization will not react unless the antigen is injected at least three hours after the sensitizing serum is injected. ^^'^ This incubation period is sup- posed to be required for the anaphylactic antibody to be fixed in the cells where the reaction takes place (Otto), and perhaps in modifica- tion of the antibody so that it has a greater afifinity for the antigen than it has while free in the serum (Weil) ^^*'; also in acquiring the capacity to affect the cells after union with the specific antigen. Fi- nally, the isolated noustriated muscle tissue (uterus) of a sensitized animal gives specific reactions when brought in contact with the specific antigen, no matter how thoroughly the animal's blood has been removed from the tissues ; whereas, the uterine muscle of an ani- mal injected with sensitizing immune serum only one hour before kill- ing does not react when in contact with specific antigen. Weil dis- putes the toxic nature of anaphylaxis, even in the intracellular reac- tion, which he calls a "cellular discharge." Nevertheless, the formation of anaphylatoxin is an interesting phe- nomenon which may well be of importance in human intoxications, even if it is not the essential phenomenon of the anaphylactic intoxica- tion. So readily is blood serum made toxic in vitro that it seems most highly probable that a similar development of toxicity may take place in the body. Jobling ^^^ has found that intoxication from anaphyla- toxin formation seems to occur when kaolin is injected intravenously into animals, and hence it is quite possible that the presence in the blood of abnonnal, finely divided bodies, such as precipitated proteins, cellular fragments, even bacteria, may cause anaphylatoxin formation in vivo just as they do in vitro. The mechanism of anaphylatoxin formation is not yet understood^ sidSee Weil, Jour. Med. Ees.. 1014 (30), 87: Jour. Tnimuiiol., lOlf, (1), 100. sieJour. Med. Res., 1015 (32), 107. 3if Jobling, Petersen and Eggstein, Jonr. Exp. Med., 1015 (22), 500. 202 CHEMISTRY OF THE IMMUXITY REACTIOXS but there is no lack of theories. The original explanation was that anaphylatoxin formation by specific antisera is the result of digestion of antigen in vitro by the action of complement united to the antigen by the immune antibody. For the formation of anaphylatoxin by inert finely divided particles, the explanation advanced was that the highly developed surfaces of these particles either activated comple- ment, or united it to the serum proteins so that it digested them. Jobling^^^ has advanced the hypothesis that normal serum antifer- inents, which are believed by him to be lii)oidal in nature, are bound by the particles or by specific precipitates, so that the complement is free to attack the serum proteins. In any case, it is now generally agreed that the poisonous substance is derived chiefly, if not entirely, from the serum and not from the antigen, even in the case of ana- phylatoxin formation by specific antigen-antibody-complement reac- tions.^^"^ Furthermore it seems to be the same, as far as we can analyze it by pharmacological methods, no matter what protein it is derived from, or whether manufactured by immune or by nonspecific reactions, or by chemical means, such as that of Vaughan. Jobling, who holds to the importance of anaphylatoxin formation as the cause of anaphylactic intoxication, presents the following con- ception of anaphylaxis : During the course of sensitization there oc- curs the mobilization of a nonspecific protease, which is greatly in- creased during acute anaphylactic shock; at this time there is also a decrease in antiferment which permits proteolysis of the animal's own proteins. As a result, there is to be found an increase in noncoagula- ble nitrogen and amino-acids of the blood, and a decrease in serum proteases. "The acute intoxication is brought about by the cleavage of serum proteins through the peptone stage by a non-specific protease. The specific elements lie in the rapid mobilization of this ferment and the colloidal serum changes which bring about the change in anti- ferment titer." The Anaphylactic Antibody (Anaphylactin). — That anaphylaxis, like other immunity reactions, depends upon the presence of sjiecific anti- bodies in the blood of the sensitized animal, is sho^\^l by the produc- tion of passive anaphylaxis in normal animals, by injecting into them a few cubic centimeters of blood or serum from a sensitized animal. Such animals become sensitive in a few hours to the specific antigen, no matter what species of animal furnishes the serum, showing that various anaphylactins can unite with the same complement, altliough strongly specific as to the antigen. In active sensitization the ana- phylactin appears in the blood in ap])re('iable (luantities about eight siKZeit. Tnimunilii).. 1014 (2:n, 71: Jour. Kxp. Med.. 101.1 (22). 401. 3iii 1'liat tlip antigen nuist be dijiostihlo, liowin-or, ia siijri;osto(l l)y the observa- tion of Ten. Broeok (Jour. Biol. Chem., 1014 (17), IM'tO) that proteins raeemi/ed by Dakin's method, wliicli cannot be dijjested by proteolylie en/yines. a)'e unable to cause anapliylaxis. 77//; .1 \1 /'//!/,. I C"/7C' .WTIIiOhV i A \ A I'll YLACTIX) 203 clays after the sensitizing injection, increases to a maximum between the 15th. and 30th days, and then very slowly decreases. The reaction of antibody and antigen is strictly quantitative, as with all ambo- ceptor reactions. Tlie amount of antibody developed seems to be limited, for after a sensitized animal is given a sub-lethal intoxicating dose of protein it may be no longer sensitive to this protein, and this refractory or anti-anaphylactic condition persists for three weeks or more. It has been demonstrated, especially conclusively by Weil and Coca,^^ that this refractory condition is, as Friedberger suggested, de- pendent upon saturation or exhaustion of all the anaphylactic anti- bodies, and hence the amount of these antibodies present free in the blood of a sensitized animal must be relatively small, for a few milli- grams of the specific protein is sufficient to saturate them, e. g., the amount of antibody present in 3 c.c. of serum from a guinea-pig sen- sitized with horse serum could be neutralized with from 0.0005 to 0.01 c.c. of horse serum. ^^ They are, however, very persistent, remain- nig in guinea-pigs through the entire life of an animal sensitized when young. They also pass from the mother to the fetus, conferring a passive sensitization which, like passive sensitization from injec- tion of serum from a sensitized animal, is of relatively brief duration, in contrast to the persistence of active sensitization. ^^'^ Anaphylactin, like amboceptor, resists heating at 56° for one hour, and is salted out from serum in the globulin fraction.^^'^ ■ Friedberger contends that it is identical with the precipitin, a view yet under discussion,'* but strongly supported by Weil 's observations.^*^ Weil ^■' has observed certain phenomena which led him to conclude that in anaphylaxis the specific antibody must be largely fixed in the cells, and that it is in the cells that the reaction occurs; the anti- bodies present in the blood of the sensitized animal are insufficient to protect its cells from the foreign protein, hence the cellular intoxica- tion. In support of this idea is the observation of Dale ^*' that the isolated smooth muscle of sensitized guinea-pigs is specifically sensitive to the foreign protein. Weil states that ' ' all the evidence proves con- 32Zeit. Immiinit-it, 1013 (17), 141. 33 Anderson and Frost, Jour. Med. Res., 1010 (23), 31. 33a The brief duration of passive sensitization presumably dejiends on the forma- tion of antibodies for the foreign sensitizing serum, constitutintr tlie condition of "antiscnsitization" as contrasted with the refractory period wliich results from the exliaustion of antil)odies by antigen. (See Weil, Zeit. Immunitiit., 1013 (20), 199; 1014 (23), 1.) 3_3b However, Scliiff and ]Moore state that in immune sera the allKunin fraction contains both the agent that confers passive sensitization and llic constituent that causes the "primary toxicity" of foreign sera. (Zeit. Immunitiit.. 1014 (22), CIO.) 34 See Zinsser, Jour. Exp. Med.. 1012 (15), 529. '34a Jour. Immunol.. 1916 (1), 1. 35Jour. Med. Research, 1913 (27), 407: 1014 (30). 200-364: 1015 (321, 107. 3G Jour. Pharm.. 1013 (4), 167. 204 CHEMISTRY o/' THE fMMCX/TY REACTIONf? elusively that anaphylactic shock is induced by reaction between an- chored antibody and antigen, and tliat circulating antibody plays abso- lutely no role in its production." The anaphylactin shows quite the same characteristics of specificity as the other immune antibodies,"' in that proteins of closely related species tend to interact, while proteins of \ery distinct biological or chemical nature are easily distinguished. Thus, guinea-pigs sensitized with ape serum will react with human serum, but not with serum from dog or ox or fowl. However, in the final analysis, the speci- ficity depends upon the chemical composition of the antigenic protein, rather than its biological origin, for I have found it possible to dis- tinguish in the hen's egg five distinctly different antigens, and these correspond to five proteins which have been distinguished by chemical measures. Together with Dr. T. B. Osborne, working with purified vegetable proteins, I have found evidence that a single isolated protein (hordein or gliadin) may contain more than one antigenic radical. ^^ As Osborne ^^ has said, "chemically identical proteins apparently do not occur in animals and plants of different species, unless thej' are biologically very closely related." Whether the chemical differences that determine specificity are of quantitative nature, which can be disclosed by analytic means, or whether they are sometimes dependent upon spatial relationships of the amino-acid radicals, as Pick sug- gests, remains to be determined. ]\Iy own experience indicates that usually, at least, proteins distinguishable by anaphylactic reactions also show readily distinguishable chemical differences. THE ABDERHALDEN REACTION This reaction is based upon ,the hypothesis that the animal body reacts to the presence of foreign proteins by providing specific means of destroying them through proteolysis, and hence is fundamentally the same as the anaphylaxis reaction as conceived by Vaughan, Friede- mann, Friedberger and others. It differs from the other reactions of this class merely in that the metliods used for determining the proteo- lysis are chemical rather than biological. The occurrence of a reac- tion is indicated by the production of diffusible products of protein hydrolysis, which may be detected by any one of several methods, altliough most used is "ninhydrin" (triketohydrindene hydrate) wiiich reacts with any alpha-amino acid, the resulting condensation compound being a blue or violet color, or by observing the change in optical rotation that occurs in a solution of i)eptone under the hydro- lytic action of the serum. It has undergone nnich llie same scries of shifting explanations as the other reactions of tliis ehiss. At first, like the other proteolytic ;" See review in Jour. Tnfoet. Dis.. 191 1 (S). 7:?. a«.T()ur. In fee. Dis.. 1013 (12), 341. 3'JHarvev Leetures, 1010 11. THE MiltF.UIIM.DRX RKM'TIOS 205 roat'tioiis, it was assumed that the antigen was digested; but, as with the preeipitiii and anaphylaxis reactions, evidence was found by iiuuierous observers tiiat not the antigen but the proteins of the im- mune serum are the chief or sole source of the cleavage products. For some reason, liard to explain, it has always been referred to as if it were the result of the formation of specific enzymes which attacked the antigen, in spite of the repeated demonstration that sera giving posi- tive reactions can be inactivated by heat and reactivated by normal jserum,^®^ thus throwing it into the class of amboceptor-complement reactions, with which it agrees in principle. Having been introduced first as a method for diagnosing pregnancy, on the principle that in pregnancy the chorionic cells of the placenta enter the maternal circulation and as foreign proteins cause the forma- tion of specific "defensive ferments," it was at once taken up as a clinical procedure, and as a result an enormous literature on this reaction was rapidly produced. Much of this represents highlj- un- critical work, largely from workers not trained or experienced in immunological principles, and hence it is not profitable to review it in extenso here. Abderhalden's own views are given in full in his monographs """ and there exist numerous critical reviews.^*"^ The status of the reaction at this writing seems to be as follows : Animals, or man, after having foreign proteins of any sort enter the blood stream, may, and commonly do show an altered condition f)f their serum, whereby when their serum is incubated with the anti- g:en under suitable conditions very minute quantities of the products of protein cleavage may be set free, and recognized when dialyzed away from the digesting mixture ; or, a measurable change in optical rotation of the digestion mixture occurs. However, perfectly normal sera may at times cause a similar proteoh'sis, usually but not always less than with the immune serum. The digestion seems to involve chiefly the serum proteins rather than the antigen, although under certain conditions there may be some digestion of the antigen. Bronfenbrenner holds that the en- zymes exhibit no selectivity, digesting both the antigen and the serum impartially.^*"* Apparently the digestion is accomplished by serum complement, or at least normal serum enzymes, rather than by any new-formed specific enzyme, although enzymes set free from the tissues have been held responsible by some. 39a See Stephan, Miinch. med. Woch.. 1914 (61), SOI: Ilrtuptmann, ibid., p. 1167; Betteneourt and !Menezes, Conipt. Rend. Soc. Biol., 1!)1G (77), 162. 39b Emil Abderhalden, "Scluitzfermente des tierisclien Orpanismus." 39c See Wallis, Quart. Jour. Med., 1016 (9), 138; Bronfenbrenner, Jour. Lab. Clin. Med., 1915 (1), 79; 1916 (1), 573. Hulton, .Jour. Biol. Chcm., 1916 (25), 163. 39d Supported by Smitb and Cook, .Jour. Infect. Dis., 1916 (18), 14. De Waele states tbat it is the serum globulin that is digested (Conipt. Rend. Soc. Biol., 1914 (76), 627). 206 CHEMISTRY OF THE nniUXITY KEACTIOXS The mechanism of the reaction is not understood. Jobling and Petersen have suggested that the antigen-antibody combination may adsorb or bind the antipro teases of the serum, so that the normal pro- tease digests the serum proteins. Or it may be that union of antigen and antibody activates the complement, or binds it to the antibody so that it digests either the antibody or other proteins of the seinim. It also is suggested that enzymes are set free from the tissues injured by the specific protein, or by disease, which digest the foreign protein or the cellular joroteins that maj' have escaped from the tissues into the blood stream. The reaction possesses a certain specificity, but just the degree of this specificity has not been agreed upon. The claim of Abder- halden ^^^ and his followers, that it is by far the most specific of im- munity reactions, whereby disintegrati'^n of small amounts of any given organ of an individual can be determined by specific reactions- between his serum and that organ, with such refinement that even cerebral localization is possible, is scarcely credible. There are so many possible sources of error in the original technic that even with great care the charge of incorrect results fi-om incorrect technic cannot be escaped, and therefore, those who do not accept the doctrine of its specificity are always on the defensive. Nevertheless, so many careful and experienced investigators have found the original Abderhalden reaction to give at times absolutely non-specific and hopelessly para- doxical results, that its diagnostic value for either clinical or scientific purposes must be considered at present as unproved,''^^ whatever the final decision as to its standing as a specific reaction may be. Serum treated with various inert, finely divided particles, such as kaolin, starch, silicates, etc., may acquire the property of giving posi- tive reactions. This is another point of resemblance to anaphylatoxin foraiation, and against the specificity of the reaction, indicating that the antigen merely acts as a' non-specific adsorbent. By far the most satisfactory results have been recorded in the diagnosis of pregnancy by means of placental antigen. This may be explained by the fact that the protease activity of the serum seems to be 39e A reply to numerous criticisms is given by Abderhalden, Fermentforscliung, 1916 (1), 351; this and other numbers of this journal also consist lavjroly of articles on the Abderlialden reaction. 39tO. J. Elsesser (Jour. Infect. Dis.. mifi (19), 055), wirking in my labora- tory with the purified vegetable proteins of Osborne, foimd tlnit at the best tlie specificity of the reaction was less than that of tlie anaplivlaxis reactioji, and there were many absolut<']y non-specific and irrational reactions. As tliese j)ure proteins furnish a much more appropriate material for studying specificity tlian the tissues or sera commonly used, it would seem (liat the results thus obtaiiied are excellent prof)f of tlie uncertainty and unreliability of the reaction. Careful quantitative studies of tlie setting free of amino-acids by serum incul)ated with placenta, liy Van Slyke and his a^-^sociates, also showed a c()m))letc lack of spe- cific proteolysis by pregnancy scrum (Arcli. Int. ^led., 1017 (10), 5fl; Jour. Biol. Chem.,"l915 (23), 377: see also Hulton. ibid., 1010 (25), 103). OPSOMNki 207 increased in pregnancy,^"*^ and hence the reaction with placenta is more marked tlian witli the serum of non-jiregnant individuals. But simply sliaking nornud serum with kaolin or other foreign substances may cause it to give strong reactions with placenta antigen (AVallis) . OPSONINS 40 The correlation of phagocytic and serum immunitj' was accom- plished when A. E. AV right showed that, before any considerable phagoej'tosis of bacteria can take place, the bacteria must first be acted upon by serum, which in some way prepares them to be in- gested by the leucocytes. The hypothetical substances accomplish- ing this sensitization of the bacteria were called opsonins by Wright, and the}' exist to a certain extent in normal serum, being increased by immunization. Not only bacteria, but cellular elements in general, including especially red corpuscles, and even unorganized particles (such as melanin),*^ are sensitized for phagocytosis by opsonins. Probably phagocytosis by endothelial *- and other cells also requires sensitization of the bacteria by opsonins. Although there have been many expressions of the opinion that the opsonins are not distinct antibodies, but are identical with agglutinins, bacteriolytic ambo- ceptors, or other antibodies, there is much evidence to the contrary.*^ There are two opsonizing elements in serum, one thermostable and one thermolabile, it being the former which is increased during immuniza- tion ; the thermostable element unites firmly with the object whieli is to be opsonized, while the thermolabile element seems to remain free in the fluid (Hektoen)." It would seem that opsonization and phagocytosis constitute but one of a series of similar processes by which foreign proteins are removed from the blood and tissues; i. e., by lysis by extracellular enzymes when this is possible, as it is vsdth simple protein aggregates (albu- minolysis) and with some of the more labile cells (hemolysis, bacterio- lysis) ; but in the case of more resistant structures, notable Gram- positive cocci and acid-fast bacilli, extracellular lysis being unsuc- cessful, these protein structures are taken within the cells where a 30g See Sloan, Amer. Jour. Physiol.. 1915 (39), 9. 40 Bibliography given bv Xeufeld, Kolle and \Yassermann's Handbuch, 191.3 (2), 440. 41 Shattock and Dudgeon. Proc. Eoyal Soo. (B), 1908 (SO). 165. 42 Briscoe, Jour. Path, and Bact., 1907 (12), 66. See also ]\Ian\varing and Coe, who found that the Kupffer cells can take up onlv opsonized pneuinococci (Proc. Soc. Exp. Biol., 1916 (13), 171). 43 See Hektoen. .Jour. Tnfec. Dis., 1909 (6). 7S: 1913 (12), 1. 44Sawtchenko (Arch. Sci. Biol., 1910 (15), 145: 1911 (16), 161) holds that there are two steps in phagocytosis: (1) Fixation of the bacteria to the leuco- cyte because of modification of surface tension by the fixative substance (opsonin or aniboceptor-coniplement complex); (2) Ameboid motion of the phagocyte; an entirely independent phenomenon. Neither phase of phagocytosis can occur in the absence of electrolvtes. 208 (lIF.MlSTh'Y OF Tin: IMMIMTY NEACTIOXS greater concentration oi" enzymes may destroy them. Fundamentally serum bacteriolysis and phagocytosis seem to he the same — in each case specific antibody sensitization prepares the hacterium for lysis by enzymes, either inside or outside the cells that fur)iish the lytic enzyme. As yet nothing- is known concerning the cliange brought about in the bacteria by the opsonin, although it has been established that it is the bacteria that are modified and not the leucocytes. The chemical nature of the opsonins is likewise unknown, except that they may combine with certain inorganic ions and are then inert (Hektoen and Ruediger) ,-*^ since addition of CaCL, BaCl., SrCL MgCU, K.SO^, NaHCO.j, sodium oxalate and potassium ferroc^-anide, inhibit the opsonic effect of serum. On the contrary, calcium salts stimulate the phagocytic effect of leucocytes, salts of barium and strontium being inactive.*® In common with other immune bodies, opsonins are thrown down in the soluble serum globulins.^" They are very sensitive to acids and alkalies, being destroyed by a concentration of n/^ and their maximum effect is at the neutral point.** However, treatment of either the bacteria or the leucocytes with very weak acids or alkalies, increases the rate and amount of phagocytosis (Oker-Blum ).•*'■* Op- sonins may be developed by immunizing against substances practically free from protein, e. g., melanin granules. ""^ Injection of nuclein preparations may increase the amount of opsonin present in the blood. °°^ Cholesterol in excess diminishes phagocytosis, but appar- ently through its action on the leucocytes.^'"' Both the sensitization of bacteria and their ingestion by leucocytes, either with or without sensitization, take place in accordance with the laws regulating an adsorption process (Ledingham,"'"'' Schiitze*"). THE MEIOSTAGMIN REACTION Reaction of antigens with their specific antibodies results in lower- ing the surface tension of the solution in which the reaction occurs, which may be demonstrated by counting the number of drops of the fluid per minute, under constant conditions. Ascoli and Izar ^^ worked out methods for practical application of this phenomenon, giving it the name of "meiostagmin reaction," from the Greek, mean- ing ''small drop." The numl)er of drops from a stalagmometer is counted, and an increase of two or more per minute is considered a 45 .Tour. Infect. Dis., 190.'') (2), 120. 4" llamburfjer, Biochem. Zeit., 1910 (24), 470; 1910 (2()K M. 47 See Simon et al., Jour. Exp. ]\Ied., 1906 (8), G51; I li'iiiciiiaiin ami (lato- wood, .lour. Infcc-. Dis., 1912 (10), 410. 48 No-ruclii, .lour. Kxp. Mod., 1907 (9). 4.)4. 49 Zeit. linnnuiitiit., 1912 (14), 48.5; Sdiiitzo. Jour. llv>:., 1!>14 (14). 201. ■'-n Lcdiii^haiii. Zeit. liinnunitiit., 1909 (.3). ll'.i. ■"'Oa lU'dson, .lour. Patli. and I5act., 1914 (19). I'.H. cob Dewev and Nu/.um, .Tour. Infect. Dis., 1914 (hi). 472. •"•oo.Tour.'lIyff., 1912 (12). 320. 51 :\Iiincli. nied. Woch., 1910 (57), (12. 182 and 403. THE KI'irilAMS h'KACTlOX 209 positive reaction, after two hours' incubation of the reacting mixture; the increase is seldom above eight drops. This reaction is said to be sharply specific and extremely delicate, detecting antigens diluted \\\) to 1 in 100,000,000 or more. The antigens used are soluble in alcohol, but their nature is unknown ; the antibody involved in the reaction is referred to as the meiostagmin, but its relation to other antibodies is likewise unknown. THE EPIPHANIN REACTION Besides reduction in surface tension, other physico-chemical changes result from antigen-antibody reactions, including the rate of diffusion, the osmotic pressure, and, in consequence, according to Weichardt, the neutral point to phenol-phthalein of a mixture of barium hydrox- ide and sulphuric acid, is also changed towards the acid side by anti- gen-antibody reactions taking place in the mixture.'^- This phenom- enon has been utilized by Weichardt, under the name of "epiphanin reaction," to determine the occurrence of such interaction of antigen and antibody. The reaction probably depends upon absorption phe- nomena, but the exact nature of the change is not yet understood. According to Rosenthal," the epiphanin reaction is especially suitable for demonstrating cancer antibodies and antigens, but Burmeister ^* and others have not been successful with this procedure. 52 See Weichardt. Berl. klin. Woch., 1911 (48), 1935; Rosenthal, Zeit. Im- nuinitat., 1912 (13), 383; Angerer and Stotter, Miinch. med. Woch., 1912 (59), 2035. 53 Zeit. Chemotherapie, 1912 (1), 156. 54 Jour. Infec. Dis., 1913 (12), 459. 14 CHAPTER VIII CHEMISTRY OF THE IMMUNITY REACTIONS (Con- tinued)—BACTERIOLYSIS, HEMOLYSIS, COMPLE- MENT FIXATION, AND SERUM CYTOTOXINS SERUM BACTERIOLYSIS ' The bactericidal property of serum may be shown by its destruc- tion of the life manifestations of bacteria without marked alteration in their structure, or it may be accompanied by dissolution of the bacterial cell {'bacteriolysis). How much of the bacteriolytic process is performed by the serum itself, or how much by the autolytic enzymes of the bacterial cell, is unknown, but the latter is probably a factor. The bactericidal property of immune seriTm has been sho^^^l to be quite independent of the antitoxic properties and also to have quite a different mechanism. This last is shown in the following- manner : If w^e heat bactericidal serum made by immunizing an animal against bacteria, say the cholera vibrio, at 55° for fifteen minutes, it will be found to have lost its power of destroying these organisms. Normal serum of non-immunized animals is equally without effect upon the vibrios. If however, we add to the inactivated heated serum an equal quantity of inactive normal serum, the mixture will be found to be as actively bactericidal as the original unheated immune serum. This phenomenon is interpreted to mean that, by immuniza- tion, some new substance has been developed which, although by itself incapable of destroying bacteria, is able, when united with some sub- stance present in normal serum, to destroy bacteria readily. The substance present in normal serum is also incapable of affecting bac- teria by itself, but needs the presence of the substance developed by immunizing to render it bactericidal. Hence the hactericidal prop- erty in this case depends on two suhstancrs acting together: one, de- veloped during immunization and therefore caHed the inintioie hody, is specific for the variety of bacteria used in immunization, and is not destroyed by heating at 55°. The other, present in normal serum, is not increased during immunization, is not (altogether) specific in character, and is destroyed by heating at 55° ; as its action is eom- plementar}' to that of tlie specific innnune body, it is called the com- plement.^ 1 Review and liiljlio^rrapliv l)v ^Vfiiller, Oppeiilieimer's TTaiull). d. Bioelicm., 1009 (II (1) ), 629. - 'J'lie i)olyiiuc]ear Icucocyies also coniain l)actoriolytic agents, "endolysins," of 210 AMBOCEPTOR A^D COMPLEMENT 211 It is believed tliat the action of these substances is as follows : The immune body is, like antitoxin, a cell receptor which unites the bac- teria to the cell. It differs from the antitoxin, however, in that it has two affinities, one for the complement and the other for the bac- terial substance. On account of the existence of the two affinities it is called an amboceptor. Some serums contain such amboceptors for certain bacteria without previous immunization, hence the term im- mune amboceptor is reserved for amboceptors developed by immuniza- tion. Amboceptor and Complement. — The function of the amboceptor is to unite the bacterial protoplasm, to which it is attached by one affinity, to the complement which it holds by its other affinity, or, to put it in a more strictly chemical way, the addition of the ambocep- tors to the bacteria gives them a chemical affinity for complement. It is, therefore, an intermediary body, uniting the complement to the bacterial protoplasm. The complement ^ is the substance that actually destroys the bacteria, in which respect, as well as in its susceptibility to heat, it resembles the enzymes. Complement is present in normal serums, and, as it is not increased in amount during immunization, it may not be sufficient to satisfy all the amboceptors, hence it may be impossible to secure marked bactericidal effects even when many amboceptors have been formed. If the complement in an immune serum has been destroyed by heating, it may be replaced by adding normal serum from another animal, even of some other species; indi- cating either that the complement is not absolutely specific in its nature, or that quite the same complement may be present in the blood of many different animals. The origin of the complement is unknown, but it has been urged that the leucocytes are an important source of this substance, if not its chief one ; ^^ there is evidence, how- ever, that various organs and cells may also produce complement.^*^ Its most important characteristics are its extreme susceptibility to heat, and the resemblance of its action to the action of enzymes.'* Hektoen ^ found that it could be made to unite vsdth Mg, Ca, Ba, Sr, and SO^ ions, which rendered the complement (for typhoid bacilli and red corpuscles) inactive. ]\Ianwaring '^ found that these ions could be separated again from the complement by simple chemical precipita- a similar complex structure, but quite distinct from the serum bacti.Tiolvsins. (See Kling, Zeit. Immunitat., 1910 (7), 1). 3 Review and bibliography by Noguchi, Biochem. Zeit., 1907 (6), 327. 3a Cholera antiserum will produce the Pfeiffer phenomenon of lysis of cholera vibrios in animals made leucocyte-free with thorium, showing that the presence of the leucocytes themselves is not essential. (Lippmann, Zeit. Immunitiit., 1915 (24), 107.) 3b See Dick, Jour. Infect. Dis., 1913 (12), 111; and Lippmann and Plesch, Zeit. Immunitiit.. 1913 (17), 54S. 4 See Walker, Jour, of Phvsiol.. 1906 (33), p. xxi. 5 Trans. Chicago Path. Soe., 1903 (5), 303. 6 Jour. Infectious Diseases, 1904 (1), 112. 212 CHEMIfiTRY OF THE IMMUNITY REACTIONS tion. Acids stronger than COo and of the higher saturated or un- saturated fatty acid series, inactivate complement in strengths greater than n/^f,, and alkalies are equally inhibitiveJ Ultraviolet rays destroy complement.''^ Sherwood "'' has made a study of various sub- stances that maj" be present in the blood in excessive amounts during pathological conditions, such as CO,, lactic acid, acetone, etc., and finds that they interfere seriously with the action of complement, wliich suggests that they may favor infection or interfere with recovery from infection. Presumably the complement is a protein, for it has antigenic prop- erties, so that immunization with sera containing either complement or complementoid causes anticomplement activity in the blood of the immune animal. Also, it is destroyed b^^ tiypsin free from lipase,^ and, like other colloids, is readily adsorbed by surfaces; like enzymes, complement is destroyed by shaking,'' and gradually disappears on standing. There are some striking resemblances between the be- havior of complement and of certain compounds of protein with soaps and lipoids, as pointed out especially by Noguehi, but that these are identical with true complement is doubtful. (See Hemolysis.) Its colloid nature is attested by the large loss when complement is filtered through Berkefeld filters.^" A careful review of the evidence has led Liefmami " to the conclusion that the reaction of complement to sen- sitized corpuscles is more like that of ferment to substrate than of antigen to antibody. According to the Ehrlich theory, complement, like toxins and en- zymes, possesses at least two groups : one, the haptophore, with which it unites with the amboceptor; the other, the toxophore (or zymo- phore, because of its enzyme-like action), which attacks the bacterial protoplasm. It may degenerate and lose its toxophore group while retaining the power to combine by means of its haptophore group, thus forming a complementoid. Complement and amboceptor exist side by side in the serum, not uniting with one another until the amboceptor has become attached to the bacterial protoplasm. It is generally stated that if serum containing complement be so treated as to separate the globulins from the albumin, it is found that the complement has been divided into two parts, one present in each of the protein fractions. The globulin fraction of the com- plement will unite to amboceptor which is fixed to cells, and hence is called the mid-piece of the complement, for it will unite also with TNofruchi, Biochem. Zeit., 1907 (6), 172. TaCourmont et nl.. C. R. Soc. Biol., 1913 (74), 1152. 7b Jour. Infect, llis., 1917 (20), 18,5. 8 Michaelis and Skwirsky, Zeit. Immunitiit., 1910 (7), 497. 1) Nu<,riichi and Bronfonlircniior, Jour. K\-i>. Med., 19H) (1.'}), 229; Rilz, Zeit. Immunitiit., 1912 (15), 145. 10 See Sclimidt, Arch. f. Hy}j., 1912 (76), 284; Jour. llviJ:., 1914 (14), 4.37. 11 Zeit. Immunitiit., 1913 (10), 503. AMBOCEPTOR AND COMPLEMENT 213 the end-piece of tlie complement contained in the albumin fraction, and then cytolysis can take place. Without the intervention of the globulin mid-piece the albumin end-piece cannot unite with the am- boceptor, while in the absence of end-piece the amboceptor mid-piece complex can cause no cytolysis. Both fractions of the complement are destroyed by heat, but if the mid-piece is bound to the ambo- ceptor it resists heating. The mid-piece corresponds to Ehrlich's haptophore, the end-piece to the toxophore group, and this complex structure is common to both bacteriolytic and hemolytic complement. Bronfenbrenner and Noguehi,"-'' however, contend that the supposed cleavage of complement is merely an inactivation by the agencies em- ployed, all the complement being in the albumin fraction in a condi- tion capable of reactivation, not only by globulin but by simple amphoteric substances, a view which has not been generally accepted. In its effect of dissolving bacteria (and also other cells against which animals may have been immunized) complement resemJjles the enzymes, and by many it is looked upon as related to. them, but the changes it produces do not resemble those produced by proteolytic en- zj-mes in all details."'' In particular, complement seems to participate in reactions according to the law of definite proportions, unlike the enzymes.^- In certain immune reactions, colloids (lecithin, silicic acid) ^^ can play the role of complement and immune body, but these reactions are probably quite different from those of bacteriolysis by immune serum. Amboceptors are formed, according to Wassermann, and Pfeift'er and ]\Iarx, in the spleen and hemopoietic organs, since in immuniza- tion i\\ey can be demonstrated in these organs before they appear in the circulating blood. The stability of the amboceptors is very con- siderable : serum prepared in 1895 by Pfeiffer against cholera vib- rios was found to have lost almost none of its activity after eight years in an ice-box (Friedberger). Heating twenty hours at 60° scarcely injures them, but 70'^ for one hour destroys them almost completely, and heating the serum to 100° destroj^s all the immune bodies. They are quite resistant to putrefaction, and, like the anti- toxins, do not dialyze. Strong salt solutions will prevent the union of complement and amboceptor in vitro, and probably to greater or less degree in the animal body, but the union of antigen and ambo- ceptor is not prevented by salt.^* Alkalies may prevent the union of amboceptor with the cells, or extract it from the cell to w^hich it has united; and they may also inhibit the union of amboceptor and iia Jour. Exp. Med., 1912 (5), 598; good review of literature, lib The curve of coiupleraent action resembles that of enzvnic action. (Tliiele and Embleton. -Jour. Patli. and Bact... 191,5 (19), .372.) 12 See Liebermann, Dent, nietl. \Yoch., 1906 (32), 249. 13 Landsteiner and Jagic, Wien. klin. Woch., 1904 (17). 03: Miincli. med. Woch., 1904 (.51), 1185. i4Angerer, Zeit. Immunitat., 1909 (4), 243. 214 CHEMISTRY OF THE IMMUNITY REACTIONS complement. Amboceptors are not inactivated by shaking, as is com- plement, but they are destroyed alike by nltraviolet rays, and both resist a;-rays."^ According to Pfeiffer and Proskauer,^^ digestion of the globulin precipitate, in which amboceptors are carried down, does not destroy their activity completely even when all the proteins are thus re- moved. Removal of the nucleo-albumin or nuclein does not remove the amboceptors from the serum. Immune serum kept three months in alcohol yielded an extract with distilled water that was rich in immune bodies, but almost free from protein. Pick, Rhodain, and Fuhnnann found that immune bodies are precipitated entirely in the euglobulin fraction of the serum protein. From these experiments it has been thought by some that the bacteriolytic amboceptor is not itself a protein, although closely associated with the serum globu- lins.'® CYTOTOXINS Just as precipitins can be obtained for proteins derived from other sources than bacterial cells, so also upon immunizing an animal against various types of cells other than bacteria, substances appear in its serum that exercise a destructive effect upon the type of cells injected. In other words, the reactions of animals to infection are not specially devised for combating bacteria and their products, but can be equally exerted against non-bacterial cells and their products. In the case of soluble proteins, as before mentioned, the antibodies show their effects by precipitating them, with agglutination of the par- ticles into flocculi and perhaps a subsequent digestion ; in the case of cells, whether bacterial or tissue cells, the antibodies cause agglutina- tion and loss or impairment of vitality. This injury may be mani- fested by loss of motion in the motile cells (bacteria, spermatozoa, ciliated epithelium) or by solution of their contents (bacteriolysis, erythrocytolysis, leucocytolysis, etc.), or by cell death without marked morphological alterations (B. typhosus, spermatozoa). If we inject red corpuscles, leucocytes, spermatozoa, renal epithelium, or any other foreign cell, the reaction is as specific as it is if we inject bacteria, and of exactly the same nature. Therefore, all that has been said pre- viously concerning bactericidal substances and agglutinins can be transposed to apply to immunity against tissue cells. As a matter of fact, however, the transposition is generally made in the other direc- tion, for red corpuscles are much easier cells to study than bacteria, because their hiking gives ])ronipt and readily recognized evidence i*aScamcli, Uiochoni. Zeit., 1015 (60). 102. in Cent. f. Bakt., 189G (19), 101. 10 Ascoli found that tlie active substance of antliracidal seruni, which is not an amhoceptor, is contained in the pseu(h>-j,'h'hulin fraction of asses' serum, but in poat's serum part is in tlie euf,'h)bulin fraction. (IJiochem. Centr., 1006 (5), 458.) HEMOLYSIS 215 tliat the toxic serum has brought about changes. Much of our knowl- edge of bactericidal serum has been obtained through studies of the mechanism of erythrocytolysis, tlie results of which have then been applied to the subject of bacteriolysis. Both on this account, there- fore, and because solution of red corpuscles is of itself an important process in manj- intoxications and diseases, the subject is of great theoretical and practical importance. HEMOLYSIS 1' OR ERYTHROCYTOLYSIS In hemolysis the essential phenouienon consists in tlie escape of the hemoglobin from the stroma of the corpuscles into the surround- ing fluid. As it is not exactly known in what way the stroma holds the liemoglobin normally, whether purely physically or in part chem- ically, or whether the stroma consists of a spongioplasm or sac-like membranes, or both, the ultimate processes that permit the escape of the liemoglobin are not finally solved. However, the agents by which the escape is brought about are well known and extensively studied, and they are found to be of extremely various natures. They may be roughly classified as: (1) known physical and chemical agents; (2) unkno^^Ti constituents of blood-serum; (3) bacterial products; (4) certain vegetable poisons ; (5) snake venoms. HEMOLYSIS BY KNOWN CHEMICAL AND PHYSICAL AGENCIES The Mechanism of Hemolysis. — If distilled water is added to corpuscles of any kind, osmotic changes are bound to occur, since within the cells are abundant salts, soluble in water, which will begin to diffuse outward in an attempt to establish osmotic equilibrium be- tween the corpuscles and the surrounding fluid., Converselj^, water enters the corpuscles at the same time, and accumulating there leads to swelling until such injurv^ has been produced as permits the hemo- globin to escape and enter the surrounding fluid. Before this oc- curs the fluid is opaque because of the obstruction to light offered by the red cells, but on the completion of hemolysis the fluid becomes transparent. The stroma now settles to the bottom, while the hemo- globin diffuses into the fluid, making- it red, but perfectly transparent. This process has long been known as the "laking" of blood, and is essentially the condition present in all forms of hemolysis. That the hemoglobin escapes only through injure* of the stroma and not through simple osmotic diffusion, is shown l)y the fact that if salt .solution of the same concentration as normal serum is used instead of distilled water, no such escape of hemoglobin occurs. As hemo- globin is perfectly soluble in salt solution, it should pass out if it dif- 17 Through usage this term has been limited to the solution of the red cor- puscles, which is more accurately described by the term erf/throriitolf/is. For bibliography see Sachs, Ererebniss'e der Pathol.,' 1002 (7), 714: 1900 (11), 515; Kolle and Wassermann's Handbuch. 191.3 (II), 793; Landsteiner, Handbuch d. Biochem., 1909 (II (1)), 395. 216 CHEMISTRX OF THE IMMVSITY REACTIOXS fused as do the salts. Since there is no escape of hemoglobin in such a salt solution, it is evident either that the stroma is not permeable to hemoglobin, or else the hemoglobin is in some way attached to or combined with the stroma. Again, if the corpuscles are placed in a solution of salt more concentrated than their own fluids, water es- capes and the corpuscles shrink; as no hemoglobin escapes with the Avater, it is evident that the stroma is not permeable to hemoglobin when intact. Because of the resemblance of the process of hemolysis to the rupture of plant cells with escape of their contents when they are placed in distilled water, it might be assumed that hemolysis is largely a physical matter, but if a red corpuscle in an isotonic solu- tion is cut into pieces, the hemoglobin does not escape, indicating that its structure is quite dissimilar to that of the simple vegetable cell, and that there is some union of stroma and of hemoglobin, whether a physical or a chemical union. ^^ ]\I. H. Fischer ^° interprets hemolysis as a separation of lipoid-protein stroma and adsorbed hemo- globin, which process can be duplicated experimentally with a com- bination consisting of a corresponding solid hydrophilic colloid, fibrin, and a hydrophobic colloid dye, carmine; this artificial combi- nation behaves exactly like a corpuscle to simple hemolytic agents.^'"' Repeated alternate freezing and thawing is another physical means of bringing on hemolysis. Heating to 62°-64° C. causes hemolysis of mammalian corpuscles; in cold-blooded animals this seems to occur at a slightly lower temperature. Some chemical agents are capable of liberating hemoglobin, even when the corpuscles are in isotonic solutions. The ordinary salts of serum, of course, do not have this property, but annnonium salts are strongly hemolytic. The chemical agents that dissolve red cor- puscles seem to be those that have the power of penetrating the stroma. Ammonium salts and urea penetrate the coii^useles freelj' and cause hemolysis. Sugar and NaCl seem not to penetrate the corpuscles, and therefore do not produce hemolysis. Of the perme- ating substances, there seem to be two types : one, like urea, does not produce hemolysis when in a solution of NaCl isotonic with the serum ; the other, like ammonium chloride, is not prevented from producing hemolysis by the presence of NaCl.-° 18 Stewart (Jour, of Physiol., 1890 (24), 211) found that in hemolysis by pliysioal means or under the influence of servuns, tliere is no marked increase in the electrical cfmductivity, but luMnclysis liy sapoJiin and by water causes an increase of conductivity, presumahl.v liccaiisc of llie escajjc of electrolytes; cor- roborated by A. ^^'oelfei, Biocliem. ,Jour., 1908 (3), 140; see also IMoorc and Roaf, ibid., p. 55. loKolloid Zeit., 1909 (5), 14G. ifa Conccrnin from the substance in blood whicli unites with these antibodies; tlierefore. they conclude, tlie "recejitors" of cells are iiot identical with tlH> antibodies. (See con- troversy with l*]lirlich in Miinch. med. Woch., Vols. oG and 57.) 3!> Corpuscles treated with f)smic acid will unite with hemolysins of diverse oripin, but when used for immunizinfi they engender no hemolysins (Coca: also V. Szily, Zeit. Imminiitiit., 1909 (3). 451). ITeatinfr corpuscle stroma alters frreatlv the reactivity (Landsteiner and Praselv, ihid., 1912 (13), 403). •to Miinch. med. Woch., 1902 (49), 8. 41 Jour. Med. Research, 1904 (12), 191. THE COMPLEMENT 221 ferent constituents of eorpuselcs, usiii<^ — (1) pure crystalline hemo- globin; (2) proteins of the stroma soluble in salt solutions; (3) an extract with alcohol-ether; and (4) an extract in 1.5 per cent, sodium bicarbonate. Only the last gave positive results, and the serum was almost devoid of agglutinative ])roperties. Injection with corpuscles that liad been digested witli trypsin gave about the same results as alkaline extracts; corpuscles digested by pepsin gave a much weaker serum ; in neither was agglutination obtained. According to Bang and Forssmann '- ethereal extracts of red corpuscles give rise to pro- duction of hemolysins on imnumization, and this "lysinogen" sub- stance can be precipitated with acetone, is insoluble in alcohol, is not destroyed by boiling, and gives rise to no agglutinin. Ford and Hal- sey *^ obtained serum with both lytic and agglutinative powers by in- jecting either the stroma or the laked blood free of stroma ; results with pure hemoglobin were indefinite. Stewart *^ obtained similar re- sults by immunizing with corpuscles laked by physical means, by serums, or by saponin. According to Guerrini,*^ nucleoprotein ob- tained from dog's blood will give rise to specific hemolysins, and Beebe states that nucleoproteins from visceral organs do not have this effect. Levene's alkaline extracts probably also contained nucleoproteins. Immunization with extracts of tissues and cells of various sorts, even when entirely free from blood (e. g., spermatozoa), may produce hemolytic sera. The fact that various tissues from many different species of animals, when used as antigen, may give rise to hemolysin for sheep corpuscles, is an interesting but so far unexplained phe- nomenon, which is discussed under "Specificity" in the preceding chapter. The Complement. — Hemolj'tic complement possesses the same properties as bacteriolytic complement, resembling enzymes to the ex- tent that it is susceptible to heat and causes a disintegration of cells, and is largely retained by Berkefeld filters.*" The joint action of amboceptor and complement is strikingly like the activation of tryp- sinogen by kinase. On the other hand, hemolysis by serum is quite different from the effect of trypsin on corpuscles, as trypsin completely disorganizes the hemoglobin and destroys the stroma, while in hemo- lysis the stroma and hemoglobin seem to be merely separated from one another but not chemieallj' altered. Again, hemolysin acts quanti- tatively, although that may be due to a difference in the way the bind- ing to the cell occurs, rather than in the method of action of the com- plement. Landsteiner and others have suggested that a lipoidal complement dissolves the corpuscle lipoids, liberating the hemoglobin, while Neuberg and others haA^e supported the hypothesis that comple- 42 Hofmeister's Beitr., 1906 (8), 238. — 43 Jour. Med. Research. 1004 (11), 40.3. - 44Amer. Jour, of Phvsiol., 1004 (11). 250. 45Puv. crit. di din. liied.. 1003 (4), 561. 4GMuir and Browning, Jour. Path, and Bact., 1009 (13), 232. 222 VUKMfsTh'Y OF THE JMMi.MTY L'EACTIOSS ineut is virtually a lipase which splits the lipoids out of the corpuscles. Bordet believes that the hemolysin causes a lesion of the stroma which changes the resistance to osmotic influences. Dick'*^ has found evi- dence that the complement is a ferment formed in the liver, and that it causes actual proteolytic changes. Jobliug ^^ associates the serum lipase with the hemolytic complement.^*"' Ohta'"' observed no increase in non-coagulable nitrogen during hemolj'sis, but Dick found an increase in the free amino acids ; therefore, as yet agreement has not been reached as to whether hemolysis depends in any way upon proteolysis or lipolysis in the corpuscle stroma. Although the serum of one animal may complement the immune bodies in serum of several other varieties, and also produce lysis of many sort of cells, it may be that not one complement does all the complementing ; Elirlich and others have asserted tliat one serum may contain several complements of slightly differing natures. Noguchi,°° Liebermann and Fenyvessy, and others have pointed out the striking resemblance between hemolytic complement and certain compounds of soaps or lipoids with serum proteins, and it is possible that such compounds are of importance in serum hemolysis; but there seems also to be evidence of the existence of distinct protein complements, entirely different from these,^^ and it is possible that the protein com- plements are the important agents in specific hemolysis by immune sera." Antibodies can be obtained for both complement and hemolytic amboceptor by immunizing against serum containing them, and in many serums antihcmolysins exist normally. Against certain vegeta- ble hemolysins this antihemolytic action is \evy strong (Kobert). Antihcmolysins are generally anticomplements, but in a number of instances anti-amboceptors have been obtained. The existence of im- mune bodies specific for hemolytic amboceptor and complement, sup- ports the view that both of these agents are proteins. Hemagglutinin. — Agglutination of red corpuscles occurs under the influence of immune serum as well as under the influence of some normal serums. In all respects the principles seem to be the same as 47 .Tour. Infect. Dis.. 1913 (12), 111. •ts.Toblinfr and Bull, .Tour. Exper. Med., 101.3 (17), (51: also Bergel. Deut. Arch, klin. Med., 1012 (100), 47. 48a Thiele and Embleton, however, state that hemolysin is not a lipase, and that tlie hemohtic power of serum has no relation to its lipolytic power (Jour. Path, and Bact., 1914 ( 10) , 349) . "49Biochem. Zeit., 1012 (40), 247. soBiochem. Zeit., 1907 (6), 172 and 327; Jour. Exper. Med., 1907 (0), 430. •'■1 See Liefmann. et al., Zeit. Imnuuiitiit., 1012 (13), 150. 52 Liebermann and Fenyvessy (loc. cit.) si ludieve that serum hemolysis takes place as follows: First, tlie aml)0('eptor acts on the corpust'Ic, injurinL; it so that it becomes h-ss resistant; second, tliis combination acts upon the comple- ment (a soap compound) and frees the soap so tliat it can unite with tlie ambo- ceptor-corpuscle system; third, tlie soaj) causes liemolysis; fourth (aa a separate step), the escape of the hcnio^'h)])iii from tlie corpuscles. llEMAdCIJ TIMN 223 those described for bacterial aygiutiiiatioji. Tlie hemagglutinating antibody behaves like the other antibodies and proteins under the in- fluence of chemical and physical agencies, but Landsteiner and Jagic have obtained strong agglutinating solutions containing very little protein. Bergel '^ contends that hemagglutination is produced by lipase from the lymphocytes, which alters the lipoid membranes of the erythrocytes. Agglutination occurs at much lower temperatures than hemolysis, and also is not checked h\ heating the serum to 55° ; hence it is possible to observe hemagglutination independent of hemo- lysis. Serums may contain hemagglutinins and not "be hemolytic; the reverse is also true. The conglutinin effect of beef serum (Bordet and Gay) is also observed with corpuscles as with bacteria. As agglu- tination occurs in corpuscles that have been fixed in formalin or sub- linuite, it is probably not the proteins that are affected, but some other of the ingredients of the stroma, of which lecithin and cholesterol seem to be the chief. Certain vegetable poisons also produce agglutination of red cor- puscles, especially ricin, abrin, and crotin, and the fact that ricin has little or no hemolytic action shows the independence of the proc- esses. Antisera for these vegetable poisons are also antiaggiutina- tive, acting, as Ehrlich showed, on the poison and not on the corpus- cles. The seeds of many non-poisonous leguminous plants, and also of Solanacece, jdeld extracts that are strongly agglutinative for red corpuscles; in Phaseolus multiflorus the active substance is found in the proteose of the seed, and seems to be a part of the stored food (Schneider).^* It is not present in other parts of the plant. Snake venoms contain agglutinins, destroyed by heating to 75° ; their ag- g-lutinating power being in inverse ratio to their hemolytic power. Corpuscles agglutinated by venoms may be again separated by po- tassium permanganate solutions.^^ Silicic acid and certain other colloids may act as agglutinins, their effects bearing a relation to the effects of electrical charges upon agglutination of bacteria or of col- loids {q. v.).-'^ Corpuscles that have been sensitized by hemolytic am- boceptors are much more readily agglutinated by salts of heavy metals, especiall}'^ copper and zinc, presumably because of quantitative altera- tions in the electrical charge of the corpuscles induced by the anti- body.""'"^ Agglutination of the corpuscles during life va&y be of great patho- logical importance, for such masses of agglutinated corpuscles may readily produce capillary thrombi and emboli, which, if wide-spread, may create much disturbance. Sometimes the serum of one indi- vidual of a species agglutinates the corpuscles of another individual 53Zeit. Immunitat., 1912 (14), 255; 1913 (17), 169. 54 Jour. Biol. Chem., 1912 (11), 47; bibliographv. 55 See Flexner, Univ. of Penn. Med. Bull., 1902 (1.5). .*324 and 301. 56 See Landsteiner and Jagic, Miinoh. med. Woch.. 1904 (51), 1185. 56a Eisner and Friedemann, Zeit. Immunitiit., 1914 (21), 520. 224 CHEMISTRY OF TIIK IMMUMTY REACTIONS of the same species {isoayglutination) , a fact which must be taken into account in performing" transfusion of blood, lest dangerous ag- glutination take place. Agglutination of an individual's corpuscles by his own seinim (autuaggliitination) , nuiy also be observed under experimental, and perhaps under pathological conditions (Land- steiuer), this pathological autoagglutination probably occurring espe- cially at temperatures below 37°. (See Parox.ysmal Hemoglobin- uria.) ]\Iany bacteria produce substances that are agglutinative for human red corpuscles, among them being B. typhosus, pyocyaneus, and staphylococcus. Flexner ^^~ has found in typhoid fever thrombi that seemed to be composed of agglutinated red corpuscles, almost free from fibrin and leucocytes. Probably many of the so-called "hy- aline thrombi" found freciuently in infectious diseases are really com- posed of agglutinated, partly hemolyzed red corpuscles (see "Throm- bosis," Chap. xi). HEMOLYSIS BY BACTERIA -s Both pathogenic and non-pathogenic bacteria produce hemolytic substances that are excreted into the fluids in which they grow. Dur- ing many infectious diseases marked hemolysis occurs, especially in those diseases accompanied by septicemia. After death the hemo- globin of the blood goes into solution, and the resulting staining of the walls of the blood-vessels, and later of the tissues everywhere, is generally familiar. In the post-mortem hemolysis probably the pu- trefactive organisms are chiefly concerned, although it is marked a very short time after death in many cases of septicemia, particularly when the infecting organism is the streptococcus, and here probably the pathogenic organism is the chief cause of the hemolysis. The hemolytic action of bacteria can be studied both {}i vitro and in vii'o. Among the best known hemolytic bacterial toxins are tetaiwlysin, pyocyanolysin, typholysin, staphylolysin,^^ and streptocolysin, as they have been termed. Of these, the case of pyocyanolysin is question- able, because it has been described as resisting heat above the boiling- point, and Jordan *"' seems to have proved that the hemolysis is a.scriba- ble to the alkalinity that this organism produces in culture-media. Other bacterial hemolysins are, however, destroyed by heat at 70° or less for two hours ; but they are altogether different from ordinary cellular hemolysins. Apparently streptocolysin is simply a toxin for 57 Univ. of R-nn. IMod. Bull., 1002 (15), 324'; Aini-r. Jour. Mod. Sci., I'M);! ( 12(i), 202. 58 See Pribram, Kolle and Wassermann's Handliuch.. lOi;^ (II). l:?2S. 59 Analysis of staphylolysin ])v Burkhardt (Arcli. cxp. I'itth. \uul IMiarni.. I!tl0 (63), 107), showed it to 1)p diiily/.ahlc, protein- and biiiiot-ficc. tlicrniolaliilo and soluble in other. From 1i. piitidum lie isolated a luMnolytic substance which seems to be a derivative by oxidation of erucacic acid ( oxydiiut'lliylthiolerucacic acid ) . '^I'.lour. Medical Research, 100:] (10), 31. HEMOLYSIS BY YEGETABIJ-: POISONS 225 red cells,"' and unites directly to the cell receptors without the inter- vention of any intermediary body. As a similar structure has been shown for stai)hylolysin and tetanolysin, it is probable that the hac- ierial hemolysins are all merely toxins tcith a particular affinity for red cells, and ajrainst some of these bacterial hemotoxins antitoxic sera are obtainable, althouo-h there is usually some question as to how much of tlu:" antagonistic effect depends on true antitoxins and how much upon the cholesterol in the scrum. Of course bacteria may also fonii many non-specific hemolytic sid)stances as products of their metab- olism, such as acids and bases. Secondary anemia occurring in the infectious diseases is probably to be explained largely by this hemolj'tic property of bacterial toxins. Hemoglobinuria may also be produced in the same way in some in- stances. Intravenous injections of filtrates of the saprophyte, B. megatherinm, will produce hemoglobinuria in guinea-pigs, hence hemolysis is not an exclusive property of pathogenic bacteria, and with streptococci Lyall '^^^ found that the hemolysin titer did not afford a criterion of virulence. No immunity is produced in animals immun- ized with streptococcus hemolysin."'^ Pneumoeocci produce an intra- cellular hemolytic toxin which is very labile and antigenic ; living pneumoeocci convert hemoglobin into methemogiobin, but this the hemolytic extracts of pneumoeocci cannot do (Cole).^^'^ Streptococcus viridans has the same property,*^'* which may play a part in the effects of infections with these organisms. HEMOLYSIS BY VEGETABLE POISONS A nuudjer of plant poisons are strongly hemolytic, and some of them owe much of their toxicity to their effect on the erythroc^'tes. One group consists of the bodies often called "vegetable toxalbu- mins, " because they seem to be proteins, and includes ricin, abrin, crotin, curcin and robin. Of these, crotin and curcin are particularly actively hemolytic, while ricin, abrin, and robin are more marked by their agglutinating action, hemolysis being produced only by relatively large doses. Their effects varv^ greatly, however, according to the species of animals whose blood is used. They resemble the bacterial toxins, in that immunity can be secured against them, and the imnume serum will prevent their hemolytic action. Heating the toxalbumins to 65° or 70° does not destroy the hemolytic or agglu- tinating action except with phallin, but 100° does. The action of these substances is not like that of the enzymes, in that it is quanti- tative,, a given amount acting on a given amount of corpuscles to 61 Jour. Amer. Med. Assoc, 1003 (41), 0G2; Jour. Infect. Dis., 1007 (4), 277. cia.Jour. Med. Res., 1914 (30), 51.5. sibMcLeod and McXeo. .Tour. Path, and Bact., 1013 (17), 524. -> sicJour. Exper. ^led.. 1914 (20), 347. 3(!3. - eid Blake, Jour. Exper. Med., 1916 (24), 315. 15 226 CHE^[IsTl,•y or rni: immimtv reactioxs which it is bound. ^Madsen and Walbuni '^- observed that red corpiis- t'les had the power of dissociatino- neutral mixtures of ricin and anti- riein, the ricin entering the corpuscles from which it could be recov- ered.®^ Ford and Abel believe the hemolj^tic agent of amanita to be a glucoside. (The general nature and other properties of these sub- stances are considered under the heading of "Phytotoxins, " in Chap, vi.) Saponin Group. — Another quite distinct group of vegetable hemolyzing agents consists of the ^'saponin substances." ^^ These are a closely related group of glucosides, found in at lea.st 46 differ- ent families of plants, and they are strong protoplasmic as well as hemolytic poisons. They differ altogether from the true toxins, be- ing heat resistant, having no resemblance to proteins, and not giving rise to antibodies on immunization of animals. "^^ The degree of their toxicity is not directly proportional to their hemolytic activity ; they seem to injure chiefly the nerve-cells. Apparently hemolysis is brought about by action upon the lipoids of the red corpuscles, for addition of cholesterol to saponin prevents its hemolytic effect ; "*' leci- thin does not have the same property.®^ Both cholesterol and leci- thin combine M'ith saponin, the cholesterol compound being quite inert, whereas the lecithin compound is both hemolytic and toxic. The compound formed between a typical saponin, digitonin, and cholesterol, is so insoluble that it has been found useful in the quan- titative analysis of cholesterol."^ Normal serum seems to contain an antihemolysin for saponin, and therefore hemoglobinuria is not produced by all saponins on intravenous injection. Careful immu- nization leads to a slight increase in this antihemolytic action of the serum, possibly due to an increased formation of cholesterol (Ro- bert). The resistance of corpuscles to saponin hemolysis varies in disease, being especially low in jaundice (M'Neil).*"' A study of the toxicity of the members of this group by Kobert '° shows that in general they have similar properties, but that minor c2Cent. f. Bakt., in04 (36). 242. IS According to Pascueoi (Hofmeister's Bcitr.. 1905 (7). 4.5"). riein combines directly with lecitliin, the compound beinjr strongly liemolytic. f* Completo litoraturc on saponin pivon by Kobert. "Die Raponinsnbstanzen." Stuttgart, 1004: also Kuiikel. "Handi)iicli der 'I'oxokolotrie," .Tena. 6''' Saponins are cliaracterized by tlieir ready solubility in water and the foamino:, soapy character possessed by tlie solution; hence their teclinical appli- cations as soap bark, etc. Heated with dilute acids they split ofT sugar; also when acted on by frlucoside-spliUin"r enzymes (from spiders), accordinsr to Kobert. Saponin from QiiiUnja (soajj-bark) has the formula f",„lT;,„0,o (Stiitz). Most are colloids, but some crystallize. 66 Ransom, Dent. mcd. Woch., 1001 (27). 104: INbidsen and Xounulii. Cent. f. Bakt.. 100.5 (.'^7). .367; Pascucci, Hofmeister's Beitr.. 1005 (6). .543. OTXopuchi, Univ. of Benn. Med. Bull., 1002 (15), .327: Meyer. Hofmeister's Beitr.. 1008 (11), 357. «8Windaus, C'hem. Berichte. 1000 (42), 238. co.Tour. Path, and Bact.. 1010 (15), 56. 70 Arch. exp. Path. u. Pharm., 1887 (23), 233. THE ^AI'OM.y a ROUP 227 differences exist between tlieni. All cause hemolysis, some in .Illa- tion as great as 1 :100,000. Some produce hemoglobinuria when in- iected intravenously, others do not. All paralyze the heart, but the injuries to the central nervous system are the chief cause of death, ^larked local changes are produced at the site of injection, but the leucocytes are apparently not injured, although sterile suppuration is produced. There is a period of latency after intravenous injection of small doses— twenty-four hours or more— before the appearance of symptoms. Sapotoxin is one of the most actively toxic and hemolytic products of quiUaja. . -, ^ ^ ^ \ Cyclamix is also a member of this group (derived from Cyclamen), and is said to be the most active of all as a hemolytic agent (Tufa- now). , ^ 1 • J SoLANix '^ is obtained from all parts of the potato plant, combined with malic acid; it is found particularly in young sprouts, but not in any considerable amounts in normal potatoes.^^ Its formula is unknown but as it splits up into an alkaloid (solanidin) and sugar it is called a glyco-alkaloid. In its action it resembles the saponins, being a powerful protoplasmic poison, killing bacteria, and hemolyz- ing blood in very great dilutions. A great number of hemolytic poisons are obtained from poisonous mushrooms. Best known of these is : Helvellic Acid, from HelveUa esculenta, which has the empiric formula Ci,H„oO,." Intravenously injected it produces hemoglobin- uria and icterus, with hemoglobin infarcts in the kidneys (Bos- troem).'^* , Phallix, or Amanita hemolysin, described by Robert as a toxal- bumin has been found by Abel and Ford to be a glucoside, and thus belongs to the saponin group. (See Chap. vi. for further discussion.) In the leaves of the ivy, Hcdera helix, a hemolytic glucoside has been found by Moore.'' It" is of interest that Faust believes the poisonous agent of cobra venom to be a glucoside, closely resembling sapo- toxin. As will be seen, all these last-mentioned vegetable hemolytic agents are essentially different from either the bacterial or serum hemolysins, or from the abrin, ricin, crotin, or robin group, in that they are of relatively simple chemical composition, and quite unlike proteins, en- zymes, or toxins. The manner in wdiich they cause hemolysis is unknown, but from their relation to saponin it is probable that, like it, they cause injury- by combining with or dissolving the lipoids of Ti Literature, see Mever and Schmiedeberg, Arch. f. exp. Path. u. rharm., 1895 (36). 361: Perles, ibid., 1890 (26), 88. 72 See Kunkel, "Handbueh der Toxokoloie," p. 873. 73Boehm and Kiilz, Arch. exp. Path. u. Pharm., 1885 (19), 403. 74Deut. Arch. klin. Med., 1883 (32), 209. T5 Jour. Pharmacol., 1913 (4). 263. 228 CHEMLSTRY OF rilE IMMl MTV REACTIONS the stroma of the corpuscles. Extracts of Morchella esculcnta do not hemolyze corpuscles in vitro, although powerfull}^ hemolytic when injected into animals, and causing severe hemoglobinuria; so that it is probable that they cause their hemolytic effects indirectly through the changes which they produce in the tissues of the poisoned animal.'^'' HEMOLYSIS BY VENOMS" The laking of blood-corpuscles by venoms is of peculiar interest from the standpoint of immunity phenomena, since it was demon- strated by Flexner and Noguchi that the hemolytic principle of the venoms resembles an amboceptor, in that some substance behaving like complement has to be furnished by the blood. Kyes demon- strated that this complementing agent is lecithin,^^'' and was able to produce what he considers to be compounds of the hemolysin with lecithin, called "leeithids." The hemolytic activity of these lecithids is very great, and they seem to be free from the neurotoxic princi- ple of the venoms. Whether thej^ represent true compounds of a hemolytic amboceptor with lecithin, or are simpl}^ actively hemolytic products of the cleavage of lecithin by an enzymatic activity of the venom, is at present unsettled ; ^^ it seems probable, however, that the hemolysin of cobra venom is a lipase that splits lecithin into two hemolytic components, oleic acid and "desoleolecithin" (Coca)." Noguchi suggests that not only lecithin, l)ut also soaps, especially of unsaturated fatty acids, and probably protein compounds of soaps jind lecithin, may act as the hemolytic "complement" which activates venoms. The hemolytic agents of venom seem to be secreted by the salivary glands of the reptiles from their blood, wliieh contains almost identical amboceptors, differing chiefly in that they can be activated only by agents contained in snake blood, while the amboceptors of venom can be activated by nearly all sorts of blood. Venoms from cobra, rattlesnake, moccasin, and copperhead possess in each a variety of intermediary bodies (amboceptors) that seem to be at least partly identical in nature, although they may vary in quantity. In order of decreasing hemolytic power for mammalian corpuscles come venoms from cobra, water moccasin, copperhead, and rattlesnake. These venoms are also agglutinative for all corpuscles tried, and agglutina- tion will occur at 0° C. Exposure for thirty minutes at 75°-80° C. destroys the agglutinating property. In general, the hemolytic power of the venoms for different sorts of corpuscles varies in inverse ]iro]ior- 70 Freidborcrer and Brossa, Zoit. Immunitiit., 1912 (15), .506. 77 Geneial review of literature on the lieniolytie jirojjerties of animal poisons pivcn by Saelis, r.ioelieiii. Centrallilatt, IHOG (.ij. 257; No-ruclii, Jour. Kxp. Med., 1907 (!)). 4:m. 77a Cruicksliank also found tliat. oilier ]i])oids tlian lecitliin niav activate eobra venom (Jour. Patli. and IJaet., 1013 (17), 019). 78 See Kyea, Jour. Infeet. Dis., 1910 (7), 181: v. Dun,u:erii and Coea. ihiiJ.. 1912 (10), 57 ; "Manwarinj:. Zeit. Immunitiit., 1910 ((5), r)i:{; I?aii;r, ihid.. I'.Hi) (S), 202; Coca, Jour. Infect. Dis., 1915 (17), 351. HEMOLYSIS IX DISEASE 229 tion to their agglutinative power. The hemolytic iutermediary bodies are resistant to heat, suffering but slight loss of power at 100° C. •Red corpuscles of the frog are not hemolyzed by venom, and those of ■necturus (mud pui)py ) but slightly, agreeing with the known resist- ance of cold-blooded animals to snake-bites. The erythrocytes of different individuals show considerable varia- tions in their resistance to hemolytic agents, perhaps depending upon the amount or upon the manner of fixation of the lipoids in the cor- puscles; thus, the corpuscles of syphilitics show a heightened resist- ance to hemolysis by cobra venom (Weil) " except in the earliest stages, when they are hypersensitive. Also, the serum of persons suf- fering from various diseases, especially mental diseases, inhibits the hemolysis of human corpuscles by cobra venom.'*" After splenectomy there is an increased resistance to venom hemolysis.^"'^ Eel serum is remarkably hemolytic, so much so that a quantity of 0.1 c.c. per kilogram of body weight will kill a rabbit or guinea-pig in three minutes when injected intravenously. Heating at 5-4° C. for fifteen minutes destroys the hemolytic action, and, unlike ordinary serum hemolysins the addition of complement does not restore its ac- tivity. Animals can be immunized against this serum. Introduced into the stomach in ordinary quantities eel serum is not toxic. It can be dried and redissolved without losing its activity, but acids and alkalies readily destroy it. Mosso, who first discovered the toxicity of eel serum, called the unknown active principle ichthyotoxin. ]\Iany other animals produce hemolytic poisons (e. g., spiders, bees) which are diseussetl under Zootoxins, Chapter vi. HEMOLYSIS IN DISEASE During health there is always going on a certain amount of de- struction of red corpuscles that have outlived their usefulness; hence in disease we may have to deal with either an alteration in the nor- mal processes of blood destruction or the introduction of entirely new processes. Although the place and manner of normal red corpuscle destruction is not completely known, yet it seems probable that there is relatively little hemolysis wathin the circulating blood. When a red corpuscle becomes damaged, it seems to become more susceptible to phagocytosis, and it is then picked out of the blood, chiefly by the endothelial cells of the sinuses of the liver, spleen, hemolymph glands, and bone-marrow. Within these cells it apparently undergoes hemo- lysis. Eventually, the resulting pigment is split up by the liver, the non-ferruginous portion forming the bile-pigments, while the iron seems to be mostly withheld to be worked over into new hemoglobin.*"'' 79 Jour. Infect. Dis., 1909 (6), 688; Stone and Schottstaedt, Arch. Int. :Med., 1912 (10), 8. 80 See articles on this siibject in the !Miinch. med. Woch., 1909, Vol. .56. soaKolmer, .Tonr. Exp. Med., 1917 (25), 195. sobMuir and Dunn (Jour. Path, and Bact, 1915 (20), 41), find that after 230 CHEMISTRY OF THE IMMUMTY h'EACTIOXS (See ' ' Pigineutation, " Chap, xvi.) Wlioiievor during disease red cor- puscles are more rapidly injured than they are under normal condi- tions, these processes of normal hemolysis are exaggerated and we not only find the phagoc.vtic cells of tlie spleen and glands packed with corpuscles, but endothelial cells elsewhere, and also leucocytes, take on the hemolytic function. At the same time there results an exces- sive production of bile-pigment from the destroyed red corpuscles, which has an etiological relation to the so-called " hemato-hepatogen- ous" jaundice. If hemolysis is very excessive, the blood pigment ac- cumulates in other organs than the liver and spleen. According to Pearce *^ and his associates, when the blood contains at one time more than 0.06 gm. of free hemoglobin per kilo of body weight, it begins to be excreted by the kidneys ; smaller amounts are cared for chiefly by the liver, and even when much larger amounts of hemoglobin are present in the blood the liver takes care of most of it, only a rela- tively small proportion, 17 to 36%, being excreted in the urine. Hence it is possible to have hemolytic jaundice without hemoglobin- uria. Part of the pigment is converted into urobilin, and the amount of this pigment in the stool is an index of the amount of hemolysis.^^* In persons with hemolytic hemoglobinemia, intravenous injection of hemoglobin will produce hemoglobinuria with snuiller dosage than in normal persons, who require at least 17 c.c. of laked corpuscles to pro- duce hemoglobinuria.^^'' It is possible that the globin, which is quite toxic when f ree,^- may play a part in the symptomatology of hemolytic poisons. The stroma of the erythrocytes also seems to be toxic. ^-^ The resistance of erythrocytes to hemolytic agents varies greatly in disease conditions ^^ and often specifically, — i. e., resistance may be increased to one agent, decreased for another, and normal with a third. Attempts have been made to use this resistance as a diagnostic or prognostic index, but not with great success in most cases. A])- parently changes in the plasma lead to alterations in the permea- bility of the corpuscles, which determines their behavior with hemo- lytic agents ; also changes in the proportion of lipoids and hemo- globin may modify hemolysis. As an example of this condition may be cited observations on hemolysis by cobra venom, the corpuscles hav- ing been found less resistant in dementia precox, more resistant in carcinoma and sy])hilis. Hutler®* states that fragility of the cor- acute li(»ni()l\iic niicTiiia in raliliils llio oxooss iron stored in tlic oryans has liccii nearly all alisorbod by tlie time re: No^iiclii. "Sciiiin Diagnosis of Svpliilis and l.uclin Head ion." I'liila- dclpliia, 1912. <'()Mri.i:\n:\T fixation and wassermann ke action 235 reiuuved by licatiiiy, no liemolysis will occur, i'or we have added no free complement. But if our original mixture had contained dysen- tery hacilli instead of tyi)lioid bacilli the complement would not have been fixed, and the addition of this mixture, containing free comple- ment, to the sensitized sheej) eoi'puseles would cause prompt hemol- ysis. This reaction was at first used for the detection of antibodies in sera,''* and for the identification of bacteria, and was found to be ex- (iuisitely delicate, detecting most minute amounts of antigens with the sharpest specificity limits of any of the immunity reactions. On account of the delicacy of this reaction it can be used to determine the presence in tissues of specific organisms which cannot be culti- vated ; thus, it has been possible to demonstrate the existence of a specific scarlatinal virus "" in the tissues during this disease, although the actual organism cannot be isolated. This fact led Wasser-inann to use extracts of the livers of congenital syphilitic fetuses, which contain great quantities of spirochetes, as an antigen for complement fixation reactions, whereby it should be possible to determine in a given serum the presence of specific amboceptors for the virus of syphilis, such amboceptors being present in persons infected with syphilis as a result of the reaction to the infection. As originally in- troduced, then, the Wassermann reaction was supposed to be simply a specific reaction between syphilitic antigen, specific syphilitic am- boceptors, and non-specific complement. It was soon learned, how- ever, that the reaction as it occurred in syphilis was decidedly dif- ferent from the original complement fixation reaction of Bordet and Gengou, for it was found possible to substitute in the reaction for extracts of tissues containing syphilitic virus (spirochetes), the most varied sorts of tissue extracts, coming from tissues certainly free from spirochetes (e. g., ox heart). Noguchi and Bronfenbrenner ^ summarize the i)resent state of the matter in these words: "We know merely this : that complement in the presence of syphilitic anti- gen may be rendered inactive by one or more substances in the body fluids of a syphilitic or parasyphilitic patient." Extended investigation of these non-specific antigens whicli give specific complement fixation with syphilitic sera, has shown them to be related to the lipoids, especiallj" the lecithins, as indicated by the fact that the most efficient antigens contain the aceton-insoluble frac- tion of the tissue lipoids. The antigenic value of this fraction of 3S Accordinfi to Gay (I^niv. of Calif. Publ., rathol., mil (2), ], full dispus- s-on) complement fixation is jirodueod by an antioen-antihody complex' distinct from precipitino<;en-j)reci[)itin, l)ut Dean (Zeit. f. Tmnuinitiit., 1012 (1.31, S4) be- lieves that they represent two phases or stages of tlie same reaction. Thiele and Embleton (Zeit. Tmmnnitiit., I!tl3 (10), 4.10) consider tliat in sypliilis it is not a specific antibody, but an anti-complementary sul)stance wliicli arises from the disintefjrating tissues. on Koessler and Koessler, .Tour. Tnfec. Dis., 1012 (!)). .30(1. iJour. E.\-p. Med., 1911 (13), 43. 236 CHEMISTRY OF THE IMMUNITY REACTIONS different liver extracts varies nearly directly with its power to com- bine with iodin - (Noguchi and Bronfenbrenner) , which indicates that the unsaturated fatty acids are important in the reaction.^ Lecitliins from different sources vary in efficiency, heart lecithin being jnore active than liver lecithin, brain and egg yolk lecithin following. Addition of cholesterol to the lecithin solutions increases greatly their activity.* An acetone-precipitated "antigen" of this class is not a true antigen, however, for fixation antibodies are not developed in aninuils injected witli such a lipoid which has been shown to be en- tirely efficient in the Wassermann reaction.'^ As for the substance in the syphilitic serum which participates in the Wassermann reaction, it w-ould seem to be related to the globulins, which are decidedly increased in the blood and spinal fluid '^ of sypliilitics,'''^ especially the euglobulin.' P. Schmidt ^ ascribes the reaction to the physico-chemical properties of the globulins of the syphilitic serum, which, he believes, possess a greater affinity for the colloids of the antigen than normal globulins; this affinity is held in check in normal serum b}' the albumins of the serum, which are rela- tively or absolutely decreased. That physico-chemical factors do play a part is evidenced by the common observation that the turbidity of the antigen suspension is closely related to its efficiency, clear solu- tions being less active. Slight changes in H-ion concentration will change a reaction from negative to positive, or reverse; and neutral salts can change a negative to a positive reaction, but not the reverse (Gumming).'*'^ The lipoids in syphilitic sera are said by Peritz ^ to be increased, but the lipoid content and the antibody titer do not show any constant relation (Bauer and Skutezky ).■'-'' The cholesterol con- tent of syphilitic blood shows no evidence of a quantitative relation to the Wassermann reaction.'*'' Friedemann ^° believes that a globulin- 2 Not corroborated by Browning, Cniioksliank and Gilmour. 3 An interesting obsorvation made by Xognclii and Bronfenbrenner. is that ex- tracts frona fatty livers arc almost devoid of antigenic properties; but So (Cent. f. Bakt., 1912 (63), 438) found that the extract from fatty hearts of guinea-pigs was more active than from normal hearts. 4 Browning et al., Zeit. Immunitiit., 1012 (14). 284; Jour. Pathol, and Bact., 1911 (16), 135 and 225. Klein and Fraenkel believe the "antigen" of ox heart extracts to be a comltination of lecithin with cliolosterol and small amounts of a soap-like substance similar to jecorin (IMiincli. mcd. \Voch., 1914 (61), 651). 5 Fitzgerald and LeaOics. Tniv. of Calif. I'ul)!., Path., 1912 (2), 39. ePfein'er, Kol)cr aiid Field, Broc. Soc. Exp. Biol., 1915 (12), 153. oaSee Kowe, Arch. Int. ^Nled., 1916 (18), 455. TMiiller and Hough, Wien. klin. Woch., 1911 (24), 167. 8 Zeit. f. Hvg., 1911 (69), 513. See also Hirschfeld and Klinger, Zeit. Immuni- tUt., 1914 (2i), 40. 8a Jour. Infect. Dis., 1916 (18), 151. 0 Zeit. exp. Path., 1910 (8), 255. 'JaWicn. klin. Wodi., 1913 (26), 830. «b Weston, Jour. Med. Pes., 1914 (30), 377; Stein, Zeit. exp. Med., 1914 (3), 309. 10 Zeit. f. Ilvg., 1910 (67), 279. COMPLEMEXT FIXATIOS AM) W ASSEltM WX REACT/OX 237 soap conipouiid is the active substance in sypliilitic sera. ^Mcintosh " says that the active component differs from typical antibodies in not passing through collodion or porcelain filters, and there are many who hold that the reacting substance is a product of tissue disintegration. AVassermann ^^'"^ has found evidence that the antibody is derived from the lymphocytes, at least in the spinal fluid of syphilitics. Whether true antibodies are concerned in the Wassennann reaction is a question. In favor of this view is the fact that the serum of rabbits immunized with congenital syphilis livers contains an anti- body giving the Wassennann reaction, exactly like the serum of syphilitics.'- On the other hand, the actual substance of pure cultures •of spirochetes does not ordinarily act as antigen with syphilitic sera in the Wassennann reaction (Noguchi). It is highly probable that when syphilitic liver extracts are used as antigen in the Wassermann reaction, we have a true Bordet-Gengou reaction of complement fixa- tion with the syphilitic substance present in this extract, in addition to the reaction which is accomplished by the lipoids. Whether the complement is destroyed by enzymes,^^ or is inhibited by anti-comple- ment present in syphilitic serum, or is destroyed by some toxic sub- stance in the serum " are matters still under discussion. A favorite interpretation of the Wassermann reaction, which seems to harmonize with the known facts, is that there is a precipitation of serum globu- lin by the lipoidal colloids of the antigen, and adsorption of the com- plement by this precipitate. Klausner's Serum Reaction. — When distilled water is added in cer- tain proportions to fresh serum, a distinct flocculent precipitate separates out in a few hours, and this property- is much more marked in syphilitic than in normal sera. While not specific for syphilis, this reaction is almost invariably present in certain stages of syphilis. This property is not due to the excess of globulin present in sj^phi- litic sera, according to the later studies of Klausner,^^ who be- lieves that the high lipoid content of syphilitic serum is responsi- ble.'-^ Porges-Hermann-Perutz Reaction. — If equal parts of a 2% solution of sodium glycocholate and an alcoholic cholesterol suspension (0.4%) oisons have been particularly considered by E. Fromm,^^ whose out- line is here partially followed, and to which the reader is referred for bibliography. INORGANIC POISONS Metallic poisons, such as lead, silver, mercury, and arsenic, are made insoluble, particularly by forming compounds with proteins in sSantesson, Skand. Arch. Physiol.. 1011 (25), 2S. 9 Hofmeister's Reitr., 1001 (1), 281; 1002 (2), 307. 10 Denied by ITeffter (Arch, inteinat. de PharmaPodyn.. 1005 (15), 300). who considers it more a physico-chemical ])rocess. 11 Stessano, Conipt. kend. Acad. Sci., 1900 (131), 72. 12 See Graham, .Tour. Inf. Dis., 1011 (8), 147. 13 V. Lhota, Arch, internal. ])harniacodyn.. 1012 (22), QA . 14 "Die chemischen R<'hiit/niittel des TierkJirpers hei Vcr<.Mf(iiii eliminated in 'the breath as methyl telluride, and also in the sweat and fecesjT^ Selenium, pyridine, and some other substances also combine with methane. The source of the methane is possibly in the xanthine molecule. Summary. — There are, therefore, three chief reactions used against 15 Arch. exp. Path. u. Pharm.,'1906 (54), 196: Corresponbl. Schweizer Aerzte, 1911 (41), 737. 16 Not accepted by Hausmann. Ergebnisse Phvsiol., 1907 (6), .58: or Joachi- moglu, Arch. exp. Path., 1916 (79), 419. IT Arch. exp. Path. u. Pharm., 1911 (64), 352. 17a See Osterhout, Proc. Phil. See, 1916 (55), 533. 18 Increased tolerance to phosphorus may be obtained by repeated small doses, but it lasts only while the poison is beinor given continuously (Oppol, Ziegler's Beitr., 1910 (49), 543). Accompanying the tolerance are structural changes in the liver cells to which are ascribed some significance bv Oppel. loAmer. Jour. Physiol., 1902 (7), 412. 20 See Mead and Gies, Amer. Jour. Physiol., 1901 (5), 105. Caffein niav be demethylated in the liver, Kotake, Zeit., 'physiol. Chem., 1908 (57), 378. 248 DfJFEXSE AGAINST NON-ANTIOENW POISONS inorganic i)ois()ns in the body, oxidation, reduction, and splitting off of tvater; neutralization of acids or alkalies and the formation of al- buminates and sulphides being included under the last heading, since in these reactions the splitting off of water is an essential step. ORGANIC POISONS In the case of organic poisons an equally small number of primary reactions is em])loyed in their detoxication, but in more complicated manners and condjinations corresponding with the complexity of organic compounds. Oxidation, which has already been mentioned as a means of de- struction of bacterial toxins, is naturally one of the most effective agents in the destruction of simpler organic substances, since the ordinary decomposition of all organic food-stuifs is through oxidation. There are numbers of specific examples of the conversion of a poisonous into a less poisonous or non-poisonous substance by oxidation. All acids of the fatty acid series are oxidized vigorouslj^ in the body, eventually into COg and H^O ; and occasionally pathologically pro- duced oxalic, acetic and lactic acids are destroyed in this way. The liver contains an oxidase destroying alcohol, which is not increased in the livers of animals made tolerant to alcohol (J. Hirsch).-^ Uric acid is oxidized vigorously by many organs, as are other members of the purine series, such as caffeine and theobromine. Presumably oxi- dation of organic poisons as well as of food-stuffs is brought about by the oxidizing enz,ymes of the cells, as shown by Ehrlich's indophenol reaction, which consists of the oxidation of paraphenylene diamine and a-naphthol, with a resulting synthesis. This reaction is said by Lillie " to occur principally in and about the cell nuclei or cell mem- branes. Combination, with or without Preliminary Oxidation, — Oxida- tion is also an essential preliminar.y step to many of the protecting com- binations, in which a cell constituent is united to an organic poison. The most important of these combining substances are : 1. Sulphuric Acid. — One of the earliest and most impoi'tant observa- tions on tlie jjrotective action of sulphuric acid was made by Baumann and Herter,-- who showed that phenol is eliminate*! as a potassium salt of the sulphuric acid derivative, as follows: ('„II,()Tr + TTO-S(\t\ = r„Tl,p-SO,l\ + Il,(). a reaction that has been put to practical use in treating phenol jioison- ing. As phenol and cresols are produced constantly in intestinal de- composition, this reaction is uiuloubtedly of great service, since the salt foi-med is relatively hariidcss. Indole and skatolc are similarly de- 21 Bioclicin. /('it., line. (771. 129. 22 Zi-it. i)liysi()l. Cliciii.. 1S77 (1). 247. ORGANIC POISONS 249 toxicatetl by being converted into corresponding salts, but only after a preliminary oxidation into indoxyl and skatoxyl, according to the following reaction: CM C(on) /-^ /\. Coll, CII + 0 = C'„H, CH. \ / \ / NH Nil (indole) (indoxyl) C(OH) ' C— 0— SO.OK /^ /\v CJI, ( 11 + HO— SOjOK = CH, CH + H..0. \ / \ / KII NH (indoxyl) (indican) A host of other aromatic organic substances are similarly combined with sulphuric acid,-^ with or without preliminary oxidation, includ- ing all substances resembling phenol or which through oxidation are changed into phenols, such as cresol, thymol, anilin, naphthalin, pyro- gallol, and tannin. By this means a poisonous substance is converted into a relatively harmless one, which is readily soluble and rapidlj^ eliminated. 2. Glycuronic acid occupies the same position as sulphuric acid, com- bining particularly with naphthol, thymol, camphor, chloral hydrate, and butyl chloral. Sometimes a substance may appear in the urine combined in part with sulphuric, in part with glycuronic acid, show- ing the similarity of their function. Apparently when there is not sufficient sulphuric acid in the body to combine with all the poison, the excess unites with glycuronic acid,-* although combination between glycuronic acid and the aromatic substance begins to occur before all the sulphuric acid is exhausted.-'' Glycuronic acid represents merely a first step in the oxidation of glucose, as follows : OHC-(CHOH),-CH,OH + 0, = OHC- (CHOH),-COOH + H.O. (glucose) (glycuronic acid) This oxidation occurs after the aldehyde group of the glucose has been combined by some other substance ; hence the aldehyde group escapes oxidation, although ordinarily more easily oxidized than the alcohol group. Just as with the addition of sulphuric acid, oxidation may be a preliminary step to the addition of glycuronic acid : e. g., naphthalin is oxidized into a-naphthol, before uniting to glycuronic acid, as fol- lows : 23 See Hammarsten's Text-book (fourth American ed. ), p. 542. 24 See Austin and Barron, Boston ]Med. and Surg. Jour.,. 190,5 (152), 260. Wohlgemuth has observed a case in which all the sulphuric acid of the urine was in organic combination (Berl. klin. Woch.. lOOG (43). 508). 25 See Salkowski, Zeit. physiol. Chem.. 1004 (42), 230. 250 DEFENSE AGAINST NON-ANTIGENIC POISONS U 11 A'=C\ H H H HCC J>C—C^ /C=C\ OH V — Cf >CH + 0 = HCC \C — Ca H V = r^ V — cf )CH H H H \C=C^ H H (iiaphtlialiii) (a-iiaplitliol) The same is the case with mam- camphors and terpenes. Reduction may be the preliminary step, as with chloral hydrate, which is first reduced to trichlor-ethyl-alcohol. In still other cases splitting off of w^ater is the chief preliminary step. 3. Glycocoll is one of the longest known combining substances, the observation of the combination of glycocoll with benzoic acid to form hippuric acid being the first proof of synthesis in the animal body dis- covered by Wohler (1824). The reaction is as follows: C„H,,COOH + H,N-CHo-COOH = aH-,CO — HN-CH„-CnOH. (benzoic acid) (glycocoll) (hippuric acid) A special enzyme has been found in kidney substance which can bring about this reaction outside the body. Normally this enzyme occurs chiefly in the kidney but may also occur in other organs. ]\Iany other aromatic compounds also combine with glycocoll before elimination, e. g., salicylic acid. Some are first altered to a suitable form by oxidation ; e. g., toluene is oxidized to benzoic acid, xylene to toluic acid, nitro-benzaldehyde to nitro-benzoic acid. INIany of the sub- stances that can be made to combine with glycocoll in the body are of such a foreign nature that they never could need neutralization under any other than experimental conditions, but here, as with the sul- phuric and glyeuronic acid reactions, combination occui*s whenever a suitable substance is present in the blood, glycocoll always being abun- dant as a cleavage product of the proteins. 4. Urea may also be a means of defense, forming salts with organic acids which are rapidly eliminated ; e. g., amido-benzoic acid and nitro- hippuric acid. 5. Methane. — IMothylation, which occurs also with tellurium, is observed on administration of pyridine, as shown by the following equation : IT H H H C— C C— 0 CIT, // W // ^ y TIC K + CH, + 0 - = BC X \ / \ / \ c=c C=C OH H H H n (pyridine) This reaction is of special importance, because many alkaloids contain a pyridine group ; and the resulting methyl compound may be less toxic than the original alkaloid — e. g., methyl mori)hine. OROANIC POISONS 251 6. Sulphur si)lit off from proteins may coml)ine with CNII and CNK. oonvertinp: them into the much less toxic sulphocyanides.-"' 7. Bile Acids. — All tlie above-mentioned reactions are protective largely beeaiise the substances formed are solultie and rapidly elim- inated, as well as being less toxic than the original poison. Com- pounds of many poisons are formed with bile acids which are insoluble, and therefore only slowly dissolve or decompose, thus protecting the body from overwlielming doses of the poison. Sncli eoiu])ounds are fonned, not only with inorganic ])oisons, but also with alkaloids, espe- cially strychnin, brucin, and (|uiiiin. They are then deposited in the liver, to be slowly dissolved and eliminated. (Occasionally acetic acid and cijsteine have been observed to act as <'ombining substances. Calcium may be considered a defensive agent against certain poisons [oxalic and citric acids} with which it forms insoluble compounds, although it is probable that the toxicity of oxa- lates dejiends largely upon their robbing the cells of calcium.-') Neutralization of organic acids entering the body or formed in metabolism is accomplished by the sodium carbonate of the blood W'hen in small amounts; if excessive in quantity (e. g., diabetic coma), a portion is combined with ammonia and appears as an ammonium salt in the urine. Magnesium and calcium salts may also help in the neutralization, probably at the expense of the bone tissue.-^ (See ^'Acid Intoxication," Chap, xviii.) Dehydration, wdiich plays a prominent part in a number of the above-mentioned syntheses, is particularly important in the change of ammonium carbonate into urea : XH,— 0 NH„ "^('0 = ^CO + 2H2O. XH,— 0 XH„ As ammonium salts of all sorts are very toxic, especially hemolytic, w^hile urea is not, this process is probably one of the most important detoxicating reactions of the body because of the great amount of ammonium compounds thaf^s constantly being formed in nitrogenous metabolism. Summary. — As Fromm points out, the variety of reactions and the variety of defensive substances are both remarkably small in num- ber. The reactions are : oxidation and reduction, hydration and de- hydration, and perhaps simple addition (methylation). The chief known protective substances are the alkalies of the blood, proteins, hydrogen sulphide, sulphuric acid, glycocoll, urea, cysteine, bile acids, 2G See ;Nreurice, Arch. int. Pliarmaeodyn., IflOO (7), 11. 27 See Robertson and Burnett, Jour. Pharmacol., 1012 (.3), 6.3.5. 28 In this connection it may l)e mentionerl that the bactericidal power of the blood is increased if the blood is more alkaline, decreased if it is less alkaline, than usual. 252 DEFEXSE AGAINST NOX-AyTIGEXIC POISONS glj'curoiiic acid, and acetic acid. .1/^ these substances are normally present in the body, and none of them is specific against any one poison, but each combines with several poisons. This last fact is interesting in comparison with the highly specific nature of the im- mune substances against bacteria and their products. As far as we know, no particular increase in the neutralizing sub- stances results from the administration of inorganic or organic poisons. The body does not appear to produce any excessive amounts of sulphuric acid in carbolic-acid poisoning, or of glycocoll when benzoic acid is administered. Both substances are present in the body normally, and as much as is available combines with the poison ; if there is not enough, the remaining poison combines with something else, or goes uncombined. In other words, the neutralizing substances described above do not appear to be the result of any special adapta- tion to meet a pathological condition. They are present in the body as a result of normal metabolism ; they have an affinity for various chemical substances, some of w^hicli happen to be poisons ; if these poisons happen to enter the body, they may be combined and neutral- ized to some extent, but, as a rule, very incompletely. There appears to be no elaborate process of defense against the chemically simple poisons, such as seems to be called into action by bacterial infection, and hence a degree of resistance or immunity similar to that present after an attack of scarlet fever or smallpox does not exist for strychnin or phosphorus. It is also of interest to consider that unicellular organisms may show a marked capacity to increase their resistance to poisons, as shown especially by Ehrlich's studies on trypanosomes, which readily become immune to various trypanocidal drugs, including arsenic compounds, and which transmit this acquired immunity through suc- ceeding generations. Yeasts and bacteria can also exhibit increased tolerance to antiseptics, and Effront found that yeasts owe their aug- mented tolerance to fluorides to an increased content of calcium, which precipitates the fluoride which enters the cells ; this tolerance is also transmitted to new generations of yeasts. The acquired tolerance is specific in all these cases, and may, indeed, be accompanied by a decreased resistance to other poisons ; thus, protozoa acclimated to alcohol may be more susceptible to other chemicals.-" Paramecia made immune to antimony are not immune to arsenic, and this specific immunity is transmitted to succeeding generations (Neuhaus).^" 20 Daniel, .Jour. Exper. Zool., 1000 (fi), .571. 30 Arch. Internat. Pliarmacoydn., 1910 (20), .303. CHAPTER X INFLAMMATION,' REGENERATION, GROWTH Although morphological alterations are prominent features of the reaction of the tissues to local injury and infection, yet at the bottom the processes of intlamniation are brought about by and result in chemical alterations. The causes of inflammation are in nearly all cases chemically active substances, but for the most part their nature is too little known to permit of speculation as to what chemical char- acteristic or characteristics a substance must possess to exhibit the power of causing- an inflammatory reaction. Even in the case of in- flammation due to mechanical, thermal, and electrical injuries, it seems probable that most of the features of the inflammatory reaction are brought about by the action of chemical substances produced by alterations in the tissue constituents at the point of injury,- for tissue proteins that have been altered in necrosis are chemotactic,-'^ as also are extracts of tissues. The essential features of inflammation, namely, local hyperemia and related vascular disturbances, exudation of plasma, migration of leucocytes and their phagocytic action, all may be caused by the action of chemical substances upon the vessels and leucocytes. Active hy- peremia in the case of inflammation is due to stimulation of the vasodilator nerves or paralysis of the vaso-eonstrictors, or direct par- alysis of the muscular fibers of the arterioles ; these may result from mechanical, thermal, or electrical stimuli, but in local infection the cause is usually chemical products of bacterial growth or of tissue disintegration. The escape of hlood plasma (inflammatory edema) appears to depend upon a number of factors (discussed more fully 1 For extensive reviews and bibliography see Adanii, in Allbutt's Rvstem of Medicine; reprinted also as a monograph. "TnflamTnation," 1000; also Opie. Arch. Int. ^ted.. 1910 (.5), 541. Some interesting ideas are advanced by Klemensiewicz, "Die Entziindung," G. Fischer, .Tena. li'OS. 2Schlaepfer (Zeit. exp. Path.. 1010 fS). ISl) finds tliat tlic reduction of methylene blue is decreased in inflammatory areas, and advances the hypothesis that inflammatory stimulants are oxidation stimulants, inflammation occurring only when the amount of oxidation aroused l)y the stimulant is insufficient. In accord with this is the observation of Amberg (Zeit. exp. ^Ted., 101."} (2). 10) that substances facilitating oxidation reduce inflammatory reactions. (See also Woolley, Jour. Amer. ^Med., Assoc, 1014 (6.3), 2270.) Another observation of similar significance is that phagocytosis is stimulated by H2O2, and that phago- cytes react to HXC in the same wav as the respiratorv center (Hamburger; In- ternat. Zeit. plivs.-chem. Biol.. 101.3 "(2), 24.5-2(54). 2a Burger and Dold, Zeit. Immunitiit.. 1014 (21), .378. 2,-S.3 254 INFLAMMATION, REGENERATION, GROWTH under "Edema," Chap, xii) of which the most important seem to be: (1) injury to the capiHary M'alls, produced largely by the chemical causes or products of the inflammation-. (2) increased osmotic pres- sure in the tissues, due to increased or abnormal formation of crystal- ioidal substances with high osmotic pressure from large molecular compounds, many of which are colloids (proteins) without apprecia- ble osmotic pressure; (3) alterations in the hydration capacity of the colloids, whereby, tlirough decrease in salts or increase in acidity, thej^ come to possess a greater affinity for w^ater (^I. H. Fischer). By far the most characteristic feature of inflammation, however, is the hehavior of the leucocytes — their increase in number in the blood, their migration from the vessels and accumulation about the point of injury, and their engulfing and destroying various solid particles, such as bacteria and degenerating tissue elements. These processes, which seem to indicate something approaching independent volition on the part of the leucocytes, may, however, be well explained by ap- plication of known laws of chemistrv' and physics, without passing into the realms of the metaphysical. This will be attempted under the heading of : AMEBOID MOTION AND PHAGOCYTOSIS The accumulation of leucocytes at a given point in the body indi- cates that some means of communication must exist between this point and the leucocytes in the circulating blood. No direct com- munication by the nervous system or other stnictural method existing, the only possible explanation is that the connnunication is through the fluids of the body, and depends upon changes in their chemical composition or physical condition. As the latter generally depends upon the former, the communication is considered to be accomplished by chemical agencies, and called chemotaxis. CHEMOTAXIS Changes in the chemical composition of a fluid have been shown frequently to aflPect the motion of living organisms suspended in it. One of the earliest observations was that of Engelmann,^ who no- ticed that Bacterium termo suspended in water tended to accumulate about a bubble of oxj^gen in the water. Pfeffer * discovered that the spermatozoids of certain ferns were attracted powerfully by veiy dilute solutions of malic acid, which is contained in the female sperm cell, indicating that the migration of the sperm cells in the proper direc- tion depends on a chemical communication, and he proposed the term chemotaxis for this phenomenon. Strong solutions of malic acid, on the other hand, repelled spermatozoids. Cane-sugar was found to at- 3 Botanisoho Zoitiinp, ISSl (.30). 441. ♦Untersuoh. aiis dem Bot. Institut in Tiiliiii;:(>n, 1SS1-1SSS. Bd. 1 und 2. CHEMOTAXIS 255 tract tlic spermato/oids of a certain I'oliacoous moss. In the case of the malic acid, it seems to be the anion that produces tlie effect, since salts of malic acid have exactly the same property. Stahl's •"' ex])eriment with a large jelly-like Plasmodium {Aethal- twn scpticu)))) p'rowing on hark in wet places, has become classical. He found that if the plasmodium was placed on a moist surface, and nearby was placed a drop of an infusion of oak bark, the organism moved by the process of ])rotoplasmic streaming toward and into the infusion. If a piece of oak bark was placed in the water, plasmodial arms were sti'ctched out to it and the piece of bark was soon com- pletely surrounded by the organism. These movements were found to occur in any direction, even exactly against the force of gravity. Other substances, as acids or strong solutions of salt or sugar, were found to cause the phismodium to move away from them, although when sufficiently dilute they exerted an attraction. A plasmodium might, however, move into a strong sugar solution if kept with a scanty supply of moisture for some time, and after it had lived in such a strong solution (2 per cent.) for some time, a weaker solution or pure water was as injurious as the concentrated sugar solution previously had been. Temperature was also found to exert a marked thermotactic effect. If a Plasmodium was placed on a filter-paper, on^ end of which was in water at 7°, and the other in water at 30°, it would move toward the warmer end. The Theory of Tropisms. — Ciliated protozoa, which can move about freely in water, show very distinct reactions to stimuli of all sorts. The first step in their change of direction of movement is considered by many observers to be an orientation of the organism until it is headed in the axis along which it is to move. This is ascribed by J. Loeb ** to the existence of a certain degree of equality of irritability of sjrmmetrical parts of the body. The stimulant, Avhether it be rays of light, or diffusing chemicals, or heat-waves, moves along definite lines, and the organism receives at first unequal stimuli on symmetrical parts of the body, unless the axis of the organ- ism is parallel to the lines of motion of the stimulant. As long as the stimulant acts on symmetrical parts of the body unequally, these parts will react unequally until at length the body is swung into a position where the stimulation is equal, when it will stay in this position and move along a line parallel to the line taken by the stimulant. Not only protozoa, but much higher forms, including some vertebrates are believed to react in this way to stimuli — e. g., the maintenance bj fish of a position heading up stream. The above constitutes the so- called 'theory of tropism," and we have such reactions to stimuli of all sorts, not only chemotropism and thermotropism, but also helio- sBotanisehe Zeitunp, 1884 (42), 145 and 161. 6 Comparative Physiology of the Brain, New York, 1900, p. 7. 256 IXFLAMMATION, NEGENEKATIOX, GROWTH tropism (reaction to light) ; geotropism (to gravity), electropism (to electricity), thigmotropism (reaction to contact), etc. The work done upon tropisms applies particularly to ciliated, freely motile organisms, and interests us less in connection with leucocytes than do the observations on such forms as AmcehaJ In passing maj' be mentioned the fhiynwtaxis or thigmotropism (reac- tion to mechanical stimuli) shown by spermatozoa, which explains their apparently difficult feat of advancing in opposition to the cilia of the epithelial lining of the female generative tract. It may also be noted that the nature of reactions of organisms to various stimuli is not constant for even the same organism. Copepods (minute crus- taceae) may be negatively heliotropic in the day and go away from the bright surface of the water, whereas at night the same animals are positively heliotropic and swarm to the surface illuminated brightly by a lantern. Variations in heliotropism may, in some cases, be explained as due to chemical changes that occur in the organism, which explanation is made more probable by J. Loeb's experiments, Avhich show that change in composition in the fluid in which animals are suspended may cause a complete reversal in their reaction to a constant stimulus. IMotile bacteria seem to behave much like ciliated protozoa in their reaction to stimuli. CHEMOTAXIS OF LEUCOCYTES « That leucocytes come to the site of an infection because of chemical substances produced by bacteria at this point, that is to say, through chemotaxis, was first clearly pointed out by Leber ^ in 1879, who likened the attraction of such substances for leucocytes to the effect of malic acid upon spermatozoids as shown by Pfeffer. He found that in keratitis, leucocytes invaded the avascular cornea from the dis- tant vessels, not in an irregular manner, but all moved directly toward the point of infection, where they collected. As dead cultures of staphylococci produced a similar, although less marked, accumlilation of leucocytes, he sought the chemotactic substance in their bodies, and isolated a crystalline, heat-resisting substance, phlogosin, which at- tracted leucocytes in animal tissues. He also observed that capillary tubes filled with phlogosin or with staphylococci were soon invaded by masses of leucocytes. Since Leber's experiments, many other investigations have been made showing that chemical substances of many different origins other than Ijacterial exert a chemotactic influence on leucocytes. Some sub- stances are indifferent in effect, most are positive, while some are be- lieved to repel leucocytes; /. e., are negatively chemotactic. 7 For full details see .Toiiniii'xs (Publication Xo. 10. Caniotjie tustituto. Wash- ington, l!tn4: also J. Loch, "Studies in (Icncral Pliysiolojiv." 8 Keview of literature on leucocytes bv TTellv, Krjieb. allix. Patliol., I!tl4 (17,,,), 1. 0 Fortschritte der :\Ied., 1888 (6), 4Gf>. CHEMOTAXIS OF LEUCOCYTES 257 Negative Chemotaxis. — Probably the substances that repel leuco- cytes are iV-w in number; Kantliack, indeed, doubted the existence of really negative cliemotactic action upon leucocytes. Verigo ^° also considoi's that as yet no actual negative chemotaxis has been satisfac- torily demonstrated; but, by analogy with the effects of chemicals on amebae, ciliata, and plasmodial forms, which all siiow a decided nega- tive chemotaxis under certain influences, it would seem most probable that leucocytes also should be repelled as well as attracted by chem- icals.^^ Non=bacterial Chemotactic Substances. — One of the earliest sig- nificant studies of the effects of non-bacterial substances upon chem- otaxis was made by ]\Iassart and Bordet,^- who showed that products of the disintegration of leucocytes and other cells had a strong posi- tive chemotactic influence. They also corroborated the statement of Yaillard and Vincent that lactic acid is an actively- repellant sub- stance, for they found that tubes containing a pyocyaneus culture, which ordinarily became filled with leucocytes rapidly, did not become invaded at all if lactic acid was also added in a strength of 1 : 500, although leucocytes did enter when the dilution was 1 : 1000. Gabritchevsky ^^ studied the chemical influence of a large number of substances on leucocytes and divided them into three groups : I. Substances exerting "negative chemotaxis," including those that at- tracted only a few leucocytes.^* II. Substances with "indifferent chemotaxis," which attracted moderate numbers of leucocytes. III. Substances with positive chemotaxis. If we correct the groupings made by Gabritchevsky we have the following classification: I. Substances negatively chemotactic or indifferent : (a) Concentrated solutions of sodium and potassium salts; (&) Lactic acid in all concentrations; (c) quinine (0.5 per cent.) ; (d) alcohol (10 per cent.) ; (e) chloroform in wa- tery solution; (/) jequirity (2 per cent., passed through Chamberland filter) ; (g) glycerol (10 per cent, to 1 per cent.); (h) bile; (i) B. cliolerae gaUinarium. II. Substances with feeble chemotaxis : (a) Distilled water; (&) dilute solutions of sodium and 10 Arch. d. MM. exper., 1001 (13), .585. 11 Salomonsen's observation (Festskrift ved indvielsen af Statens Sorum In- stitut, Kopenhagen. 1902, Art. XII), that ciliated infusoria when killed show a strong negative effect on other ciliates, is of much interest, particularly as it seems to be the opposite of tlie positively chemotactic effect of dead upon living leucocytes. The negative reaction of different ciliata was specific for their own kind rjuantitatively. but not qualitativelv. 12 Ann. d. I'Inst! Pasteur, 1891 (5), 417. 13 Ann. d. Tlnst. Pasteur, 1890 (4), 346. 1* Evidently these substances were not all negatively chemotactic, but were relatively sliQ;htly chemotactic or indifferent: yet in the literature generally these experiments Jiave been cited as indicating a negative chemotactic influence of the substances .studied. 17 258 IXFLAMMATIOX, REGENERATION, GROWTH potassium salts (1-0.1 per cent.); (c) phenol; (d) anti- pyrin; (e) phloridzin ; (/) papayotin (in frog) ; ( g) glyco- gen; (h) peptone; (i) bouillon; (j) blood and aqueous humor; (k) carmine. III. Substances with strong positive chemotaxis : (a) Papayotin (in rabbits) ; (b) sterilized living cultures of bacteria, whether pathogenic or non-pathogenic. These results can only be considered as suggestive and not as accu- rate findings, in view of other contradictory- results. Buchner ^^ obtained from the piwumohacillus of Friedlander, a protein which ex- erted a strong chemotactic influence, thus showing the chemical na- ture of the attraction of leucocytes by bacteria, and he isolated other similar proteins from other bacteria. He also obtained a "glutin- casein" from grain which was related chemically to the bacterial pro- teins, and which was equally chemotactic. The metabolic products of bacteria, however, he -found to be negatively chemotactic. Alkali albuminate and hemi-albumose were strongly positive, but peptone was not. GlycocoU and leucine were found to be chemotactic, but urea, ammonium urate, skatole, tyrosine, and trimethylamine were not. It was also observed that if the positively chemotactic sub- stances were injected subcutaneously, they produced general as well as local leucocytosis. The products of the action of serum on bacteria, " anaphylatoxin, " produce inflammatory reactions, and probably are important factors in pathology ; the products of tissue disintegration have similar effects.^^'^ Certain drugs (notably quinine, morphine and chloral) when injected subcutaneously seem to reduce the amount of leucocytic emigration at a point of local injury (Ikeda).^^'' V. Sicherer ^® found that chemotaxis of leucocytes may be observed outside the body. If a tube containing positively chemotactic sub- stances (dead beer-yeast cells and dead staphylococci were the strong- est) is placed with one end in a leucocyte-containing exudate, the leu- cocytes pass up into it against gravity. Bloch ^■^ demonstrated that carbol-glycerol extracts made from each of the different viscera and tissues exerted a positive chemotaxis, dis- crediting the statements of Goldscheider and Jacob that onlj- extracts of hematogenetic tissues showed positive chemotaxis. Egg-albumen, gelatine, albumen-peptone, and alkali albuminate were also positive, carboliydrates feebly so, and fat not at all. ^Metallic copper, iron, mercury, and their salts have also been found to be chemotactic sub- stances, but it is very probable that they act in part through destroy- ing the tissues in their vicinity, which give rise to decomposition-prod- 15 Berl. klin. Wochensehr., 1890 (27), 1084. 15a See Bold, Arh. Patli. Inst. Tiibingen. 1J)14 (i1). :J(). 15b .Jour. Pharniacol., litHi (8), 137. 10 Cent. f. Bakt., lS!)i) (2(i), 300. 17 Cent. f. allf,'. I'atli., IBJIO (7), 785. yON-BACTERIAL CHEMOTACTIC SUBSTANCES 259 nets having a positive effect. Adler,^* however, found that bichloride of mercury as dilute as 1 : 3000 caused more leucocytic invasion of a piece of saturated older pith than did even cultures of pyogenic bac- teria.^" ^Tetehnikoft' obsemcd tliat leucocytes might, after a time, be at- tracted toward substances that at first seemed to repel them. If the blood is full of toxins, the subcutaneous introduction of bacteria does not lead to a local accumulation of leucocytes, presumably because the difference in chemotaxis between the blood and the tissue fluids is not great enough to cause emigration ; in this connection should be men- tioned Pfeffer's observation that B. termo in a peptone solution will not migrate toward another stronger peptone solution, unless the lat- ter is at least five times as strong as the former. Leucocytes will mi- grate freely toward substances that kill them; of the bacterial prod- ucts the toxins of pyocyaneus and diphtheria bacilli being especially destructive and causing typical karyorrhexjs.-° Substances soluble in lipoids are said by Hamburger ^^ to increase phagocytic activity when in extreme dilutions, although stronger concentrations are highly toxic for leucocytes. If an electric current is passed through two fin- gers there will be found more leucocytes in the tissues of the cathode' finger than in the anode finger, presumably because the OH-ions in- crease ameboid movement. "^^ ]\rany substances have been used to increase the number of leuco- cytes in the circulating blood in the hope of increasing resistance to infections, a result that does not seem to follow artificial leucocytosis with any recognizable uniformity. A compilation of the literature on this subject by Gehrig -'^'^ shows such contradictor}' findings as to indicate that most of the recorded M'ork is of little value. He was unable to corroborate the current statement that antipyretic drugs increase the number of leucocytes in the blood. Nucleinic acid and tissue extracts seem to increase circulating leucocytes with considerable regularity, while witli thorium-A' and benzol they can be reduced to almost complete extinction. The behavior of inflammatory processes in animals thus deprived of available leucocytes has considerable ex- perimental interest.-^'^ If less than 1000 leucocytes per cubic mm. are present in the blood, no leucocytic exudate can be produced, ^^"* although the other features of inflammation occur as usual. Relation of Cell Types to Migration. — Of the leucocytes, the most actively affected by chemotaxis is the polymorphonuclear vari- is Festschr. for A. Jacobi, 1000, Xew York. 10 Concernincr th^ effects of iodin and iodides Tij>on tlio leueocvtes, see Heinz, Virohow's Arch., 1809 (1.55). 44. 20 Schiirmaiin, Cent. f. Pathol.. 1010 (21). .3.37. 21 Arch. N^erland.. 1912 (III, B), 1.34: Brit. Med. .Jour., 1016 (1), 37. 2iaSchwvzer, Bio<'heni. Zeit.. 1914 (60), 454. 2ibZeit. exp. Path.. 1015 (17), 161. 2ieSee G. Rosenow, Zeit. exp. Med.. 1914 (3). 42. 2irodu('e supjiuration unless they are hroken down so that their pi/ogenic proteins are released: e. (j.. anthrax bacilli cause suppuration when actin^r locally, as in malipnant pustule, hut not when they are causing septicemia, because only in the former case are their pvopenic proteins liberated. '"•""Zcit. klin. Med., 1904 (.'">-)). 508. "" .\j)parently su)iy)iirat ion mav occur in herpes 70ster vesicles in the absence of bacteria, accordin 685.8.5 673.69 Insoluble bodies 205.66 J Lecithin ~l i^qqq 75.64 Fat J" '^^•^■^'^ 75.00 Cholesterol 74.00 . . 72.83 Cerebrin 51.99 ) iaqr-i Extractive bodies 44.33 J ' ' i"-o-* Mineral Substances in 1000 Parts of the Dried y'^ubstance. NaCI 4.35 Ca,(P04)o 2.05 Mg3(PO,)2 1.13 FePO^ 1.06 PO4 9.16 Na 0.68 K trace As abnormal constituents of the leucocytes contained in abscesses may be mentioned glycogen, fat (from phagocytosis and from ''fatty degeneration" of the leucocytes), and ''peptone" (Hofmeister).^° Pus serum differs from blood-serum chiefly in the substances added to it through the proteolytic changes that occur in the pus, and also in that it has lost its antiproteolytic property, containing instead free leueoprotease. The fibrinogen that escapes from the vessels into sup- purating areas becomes so altered that pus will not coagulate, even upon addition of fibrin ferment (defibrinated blood). The reaction of the serum is usually slightly alkaline, becoming strongly alkaline if much ammonia is produced, which occurs especially if there is sec- ondary infection with the organisms of putrefaction. Sometimes, liowever, lipase derived either from bacteria or from the cells causes the splitting of sufficient amounts of fatty acids from the fats to make the reaction acid ; lactic and other fatty acids are also sometimes formed. Presumably the nature of the infecting organism will mod- ify the reaction, for some (e. g., sfnphjflococcus) cause an acid forma- tion in media, while otliers (e. g., pj/ocj/ancus) cause an alkaline reac- tion. Hoppe-Seyler 's analysis of pus serinn gave the following re- 68 Mod.-r'hom. ITntersnoluinficn. TOZeit. physiol Cheni., 1880 (4), 268. COMPOSITION OF PUS 279 suits, wliicli show no considerable deviation from the composition of blood plasma, except in an increased proportion of fatty matter and extractive substances. Tarle II Quantitative composition Plasma of pus serum [normal ) . I II III Water 913.7 905.65 90S.4 Solids 86.3 94.35 91.6 Proteins 63.23 77.21 77.6 Lecithin 1.50 0.56 "1 Fat 0.26 0.29 } 1.2 Cholesterol 0.53 0.87 J Alcohol extractives . . . 1.52 0.73 ) .^ Water extractives . . . 11.53 6.92 ( ^^ Inorganic salts .... 7.73 7.77 8.1 Quantitatively the chief abnormal constituent of pus serum is the so-called " pyin" of the older writers, which is nucleoprotein de- rived from the decomposing leucocytes, and hence increasing in amount progressively with the age of the pus; '^ it is characterized by its insolubility in acetic acid. The same substance is found more abundantly in the entire pus, on account of the presence of the cells, and when treated with 10 per cent. NaCl solution it forms a stringy mass which was formerly called "Rovida's hyalin substance." Glu- cothionic acid, derived from the leucocytes, is also present in pus.'^- In the pus serum are found all the other constituents of the leuco- cytes, including particularly lecithin, cholesterol, fats (and soaps), cerebrin, "jecorin, " and glycogen; and also the usual components of the blood-serum as well as some small quantities of pigment derived from decomposed red corpuscles. The products of autolysis are of particular interest, and they are found in varying amount, but usually less abundantly than might be expected, probablj'- because of their solubility and consequent rapid absorption. Albumoses and peptones seem to be constantly present (Shattock).'^ The common occurrence of albumosuria during sup- puration presumably depends on the absorption of digestion products from the pus,'^* but true peptone has not been satisfactorily identified in the urine. Leucine and tyrosine have also frequently been found in pus,^^ but Taylor ''^ could find no workable traces of either monoam- 71 Strada. Biochem. Zeit.. 1909 (16), 193. "2 Mandel and Levene, Biochem. Zeit.. 1907 (4), 78. -3 Trans. London Path. Soc, 1892 (43), 225. "4 Literature on albumosuria, see Yarrow. Amer. 'Mod.. 1903 (5). 452: Elmer, ihirl.. 1906 (11), 169; Senator, International Clinics, 1905 (IV), series 14. p. 85. See also "Albumosuria," Chap. xix. "5 Mfiller (Cent. inn. Mod., 1907 (28), 297) recommends the tyrosine reaction with Millon's reagent as a means of difTerentiatiTineumonia and infarct a high protein content speaks for the former. Rogers ^^ stated that the sputum in every case of tuberculosis shows albumin,^*'-'' but this has been questioned, especially as to chronic or quiescent cases. ^^ Albumin, or better, coagulable protein is also pres- ent in the sputum of patients with pulmonary edema and pleurisy. According to Works ^^ in active tuberculosis there is usually 0.2 per cent, or more of coagulable protein in the sputum. The mucin of sputum yields 33.6 per cent, of glucosamin when split with HCl, which gives an index of the quantity of mucin ; this is highest in chronic bronchitis and lowest in pneumonia and phthisis. Kossel found 0.1- 0.33 gm. of nucleins in the sputum daily. The following table by Bokay (taken from Ott) gives the pro- portion of the organic constituents of sputum in parts per thou- sand : On account of the digestion of the exudates by the leucocytes, spu- tum contains proteoses, peptones, and amino-acids, generally in pro- portion to the richness of the exudate in leucoej-tes; they are, there- ssZeit. f. Biol., 187.5 (11). 102. 84Plesch (Zeit. exp. Path. u. Ther.. 1906, Bd. iii. .Tuly) found that 4.8 per cent, of all the absorbed calories were lost in the sputum in an advanced case of phthisis. Under similar conditions the amount of salts excreted by tlie sputum mav equal or exceed that in the urine ( Falk, loc. cit. ) . ssDeut. Arch. klin. Med.. 1003 (75). .347. ssPresse Mfd., 1910 (18), 289; 1911 (19). 409; also Ganz and Hertz, ibid., 1911 (19), 41; Kaufmann, Beitr. Klin. d. Tuberk, 1913 (26), 269; Hempel- Jfirgensen. ibid., p. 392. 86a RevieAv by Cocke. Amer. Jour. Med. Sci.. 1914 (148). 724. 8" Fischberfr and Felberbaum, Medical Record, Oct. 21, 1911; Acs-Xagv, A^'ien. klin. Woch., 1912 (25), 1904. 88 Jour. Amer. Med. Assoc, 1912 (59), 1537. 282 IXFLAMMATIOX, liEGEXEh'ATIOX, GROWTH Table III. Bronchitis Phthisis, Phthisis, in Fibroid early in Phthisis, Phthisis, ad- typhoid phthisis apex cavities advanced vanced Fatty acids as fat 0.224 0.845 0.462 2.468 3.468 9.725 Free fatty acids . trace 0.184 0.521 0.370 0.307 0.902 Soaps .... traces 0.380 0.430 0.537 0.516 3.973 Cholesterol traces 0.4 1.617 0.172 1.160 0.141 Lecithin traces traces 1.543 1.165 1.245 Xuclein traces 0.102 0.260 0.489 Protein 0.898 2.040 4.430 3.455 5.115 fore, most abundant in pneumonia. Simon '^^ found considerable al- bumose in phthisical sputum, but no nucleohiston or free histon. In febrile cases of tuberculosis the amount of albumose may exceed the coagulable albumen, which rarely exceeds one per cent, of the moist weight."" Staffregen, however, could find no true peptone in phthisical sputum, but Stadelmann -'^ found that such sputum con- tained enzymes hydrolyzing fibrin, and attributed this largely to bacteria. Probably most of the enzjones present in sputum come from the leucocytes. In the early stage of pneumonia the sputum has no proteolytic action, presumably because inhibited by the large amount of serum present ; but with resolution active proteolji:ic prop- erties appear (Rittorf).^- In tuberculosis sputum the tryptic and antitryptic properties fluctuate, and lipase is absent (Eiselt).®^ Pneumonic sputum before the crisis has but slight action on peptids, but acquires marked peptolytic activity thereafter.^* Most sputa contain enzymes splitting casein and polj'peptids.''*'' Sputum may contain indole, derived either from the putrefying proteins or ex- creted from the blood."*'' The amount of fats seems to depend directly upon the number of pus-corpuscles and the age of the pus (i. e., the amount of fatty de- generation). Jacobson found from 0.08 to 1.6 grams of fatty matter per day, containing on an average 14.76 per cent, of soaps, 15.79 per cent, of higher fatty acids, 0-10 per cent, of water-soluble fatty acids, 13.58 per cent, lecithin, and 10.49 per cent, cholesterol. As to the inorganic substances. Bamberger found two types of spu- tum, catarrlial and inflammatory. In tlie inflammatory there is a deficiency in alkali phosphate, SO;,- constitutes more than 8 per cent. Na O . 15 of the salts, and the ratio, KO equals 41 the alkali phosphates constitute 10-14 per cent., -rr^ In catarrhal sputum Na O 31 ^, and the KO 89 Arch. exp. Path. u. Pharm., 1903 (49), 449. ooProrok. Miinoh. mod. Wodi.. 1909 (56). 2053. fi Zeit. klin. Med.. 1SS9 (16). 128. 02Deut. Arch. klin. Mod., 1907 (91). 212. 93Zoit. klin. Mod., 1912 (75), 91. 04 Ahdorlialdon. Zoit. phvsiol. C'liom.. 1912 (78), 344. n4a:Maliwa, Dout. Aroh. klin. Med., 1914 (115), 407. 94b Binda and Cassarini. Oaz. Med. Ital., 1913 (64), 461. J'h'<)IJI/:U.\T/(>\ AM) h'KdENERATION 283 SO;, is from 0.6-1.2 per cent. Chlorine is about the same in botli forms. These differences are, however, not as constant as Bamberger believes, aecordino- to several later inv('sti 4.(i7 0.78 35.78 0.70 13.05 24.07 27.1)0 1.G3 0.09 1.20 1.74 0.0 Acute phthisis 93.38 6.88 0.74 33.40 0.80 14.15 19.99 31.09 4.32'- 0.14 0.22 0.3 PROLIFERATION AND REGENERATION The factors that incite cells to proliferate, as well as those that cause the cessation of proliferation after it has once started, are too entirely unknown to permit of speculation as to their exact nature. It seems probable, however, that they are, as Ziegler says, "identical with the stimuli which excite or increase functional and nutritive activity," and these are certainly in many instances of chemical na- ture. Thus the application of various irritating substances in not too concentrated a form (e. g., painting the skin with iodin) may lead to proliferation without causing discernible degeneration of the cells. Mallory's^"' observations on the phenomena of proliferation and phHgocvlT)susjjhowJii^rit the same baj-terial products whicli destroy the cells when concentrated, when sufficiently dilute cause prolifera- tion of similar cells. Carnot and Lalievre "" have obtained evidence that actively growing kidney tissue, whether fetal or adult regener- ating kidney, contains something which is capable of stimulating growth of renal epithelium when injected into other animals. (The importance of this observation calls for its corroboration, but no repe- tition of the work is known to us.) Many other instances of prolifera- ^•5 Including magnesium. 98 Jour. Exp. Med., 1900 (5), 15. 97 Arch. M6d. Exper., 1907 (19), 388. 284 JXFLAMMATIOX, REGENERATION, GROWTH tiou in response to chemical stimuli might be cited, but in nearly all cases it is extremely difficult to determine that the proliferation is not, after all, reparative in compensation for degenerative changes, and, therefore, possibly obeying some other biological law than that of a simple reaction to a chemical stimulus. Although proper nutrition is necessary for cell proliferation, yet it does not seem that excessive nourishment can lead to excessive cell mul- tiplication, or by itself cause cell proliferation to take place. Oxygen and certain inorganic salts are essential for cell division even in the lowest forms, and among such simple organisms as sea-urchins and certain other marine forms segmentation of the unfertilized ova may be incited by changes in osmotic concentration, leading eventualh* to formation of perfect larvje (J. Loeb, et. al.).^^ In lower animals verj' dilute solutions of alkalies stimulate the rate of cell growth, and somewhat higher concentrations cause extremely irregular cell division ; in mammals the feeding of alkalies causes great wasting as if through cell stimulation.^ The products of nuclein hydrolysis are said to stimulate cell growth.- Potassium salts seem to be particularly im- portant for proliferating cells, and Beebe and also Clowes and Fris- bie ^ have found that actively growing malignant tumors are rich in potassium and poor in calcium, whereas in slow-growing tumors the reverse is the case. Dennstedt and Rumpf * also found that in hypertrophy of the heart the amount of potassium is increased, while in chronic degeneration of the myocardium the calcium and mag- nesium are usually increased. The proportion of nitrogen in the dif- ferent parts of the heart is not changed during hypertrophy (Benee),^ but the amount of NaCl is much increased in hypertrophy." Chemical studies of proliferation are lacking, except in regard to the development of the embryo, etc."'"^ New tissues difiPer from adult tissues in having a large proportion of water, and in having a larger proportion of the "primary" cell constituents and a smaller propor- tion of the various secondary constituents, since these last are largely products of the activity of the adult cell. Of the primaiy constitu- ents, the proportion of the nucleoproteins is particularly high, and a number of interesting facts concerning the nucleoproteins in cell di- vision have been determined. IMost important, perhaps, are the clas- sical observation of INIiescher, who found that during the migration of salmon up stream to the spawning grounds, during which time no food is taken, the proteins of the muscular tissue become largely 08 Sep J. Loob, Studios in Conoral Plivsiolofrv, Chioapo. 1005. 1 Moore et al., Rioolicm. Jour.. 1900 h), 2\H: 1012 (6), 162. 2 Calkins et al.. Jour. Infect. Di.s., 1912 (10), 421. 8 See "Tumors," Cliap. xvii. 4Zeit. klin. Med., 190.5 (58). 84. BZeit. klin. Med.. 1908 (06). 441. « Rzentkowski, ihid., 1910 (70), 3.37. "a. l.iterature on the ehemistry of firowtli piven liy Aron, llandliueli d. Bioeliem., Ergiinzungsband, 1913. CHEMICAL /?.hS7i<1.. 1012 (34). 444. oAmer. .Tour. Phvsiol.. 1001 ffi). .54. 11 See IMcClendon', Carnegie Inst. Publ.. 1014, Xo. 1S3. 12 See Mendel, "Nutrition and Growth," Harvev Society Lectures. 1014- I.t: Amer. Jour. Med. Sci., 1917 (153), 1. 286 JXFLAMMATJOX. RECEy ERATIOy . aROWTH not only provide a sufficient amount of nitrogen, but they must also provide certain specific amino-acids, as has been especially demon- strated by the investigations of Osborne and JMendel.'^ Apparentlj' the presence of some of the simj)le straight-chain amino-acids can be dispensed with (c. g., g-lycine), and the animal will grow and thrive if other nutritive supplies are adequate, but certain, at least, of the more complex cyclic amino-acids must be provided. Furthermore, the requirements for growth (quantitatively speaking at least), seem to be sometliing more than the requirements for mere preservation of health and equilibrium, for it was found that animals could live and preserve nitrogen equilibrium when the protein of the diet furnished at most small quantities of lysine, but young animals were unable to grow with such a restricted supply of this amino-acid. If lysine was added to the defective protein (gliadin from wheat) the animal would then be able to grow at a normal rate. Of particular importance is the fact that animals can be kept in a stunted condition on such a deficient diet until they have reached an age at which normally all growth would have long since ceased, and then when supplied with sufficient lysine they will begin to grow and continue until full size is reached.^* This last observation proves that growth is not condi- tioned by age, and that we do not stop growing because a certain age is reached : the capacity for growth may remain latent and capable of exhibiting itself when proper conditions are furnished. But no amount of any amino-acid will cause a fully grown animal to grow any more, so it would seem that the capacity for growth becomes ex- tinguished when it has been utilized to a certain fixed extent, and re- mains potent until it has been completely utilized. If the only protein furnished contains no tryptophane the animal cannot maintain itself and slowly loses weight until it dies, unless tryptophane is supplied. If zein from corn, which yields neither h'- sine nor tryptophane, is the sole protein, then the animal cannot grow unless both lysine and tryptophane are added to the diet. That the pure isolated amino-acids can meet the deficiencies when added to the imperfect protein ration, demonstrates that proteins ser^'e for food as amino-acids, and not as larger complexes. Not only must the proteins present certain essential chemical com- pounds to the living and growing organism, but also an adequate su])])ly of the essential inorganic salts and certain other less well de- fined substances are also necessary to permit of maintenance, growth and repair. It has long been recognized clinically that certain dis- eases, notably scurvy, may result from tlu> absence of some essential food supply. More recently other diseases have been proved or sus- pected of having a similar cause, and the study of one of these, beri- 13 Series of papers in Jour. Biol. Clioiii.. 1!)12, ct scf/. ^•».Tnnr. P.inl. Diem., 1915 (23), 4:W. \nA.\u\Ei^ 287 beri, lias led to a closer approximation to the nature of the food essen- tials concerned. This disease seems to result from the use of pol- ished rice as the chief constituent of the diet, and can be checked by feeding unpolished rice, or rice polishings, or even extracts of rice polishiugs. A somewhat similar condition may be produced readily in birds by feeding- polished rice, the chief feature being a severe neuritis, which is relieved with remarkable rapidity by supplying the food deficiency. This experimental neuritis of fowls {polyneuritis gallinarum) has served as a valuable means of study of diseases of this class, and led to the demonstration that not only extracts of rice polishings, but also many other food materials, contain the essential materials without which health cannot be maintained. One of the earl}' investigators of this subject, Casimir Funk,^"' gave to "the hitherto unrecognized essential dietary factors" the name "vitamines, " which, in spite of certain logical objections, has been generally adopted. Although so essential for life the amount required is very small, for whole rice is said to contain not over 0.1 gm. per kilo., and perhaps much less, of the active substance. Besides beriberi, the following may be considered as of the nature of ' ' deficiency diseases ' ' : Scurvy and infantile scurvy (Barlow's disease) and possibly pellagra, rick- ets and tetany. Furthermore, the "vitamines" are necessary for growth, no matter how much and what proteins, carbohydrates and fats are supplied. McCollum '■^ has summarized the evidence that two classes of sub- stances are necessary for maintenance, the more important in pre- venting neuritis' being water-soluble, although for growth to occur an unidentified lipoid-soluble substance is essential. As yet the exact identity of the active agents in water or lipoid solutions has not been determined, but the pursuit is being gradually brought closer to the goal. Funk believed them to be pyrimidine derivatives. AVilliams and Seidell ^^ have found that hydroxypurines have marked anti-neu- ritic effects, and they suggested that an isomer of adenine is responsible for the antineuritic action of yeast extracts. Later Williams ^''"^ found an active hydroxypyridene, and suggested that the curative properties of yeast and rice polishings may be due to an isomeric form of nicotinic acid. The lipoid-soluble "vitamines" seem to be especially abundant in butter, egg yolk, and cod liver oil, which presumably ac- counts for the commonly accepted values of these particular fats. Why the vitamines are essential and how they act is unknown. Ved- der ^^ has suggested that the antineuritic vitamine is essential for growth and repair of the nervous tissue, and in its absence normal 15 See Ergeb. Phvsiol., 1913 (13), 12.i, for review of liis work. ifiJour. Biol. Chem., 1916 (24), 491. 17 Jour. Biol. Chem., 1916 (26), 431. 17a Jour. Biol. Chein., 1917 (29). 49"). 18 Jour. Amer. Med. Assoc, 1916 (67), 1494. 288 INFLAMMATIOy, REaENERATlOX, GROWTH Avear cannot be made good. It is probable that more than one vita- mine is neeessaiy for maintaining normal conditions, and deficiency of one causes beriberi, of another scurvy, for some dietaries lead to one disease and some to the other. CHAPTER XI DISTURBANCES OF CIRCULATION AND DISEASES OF THE BLOOD THE COMPOSITION OF THE BLOOD The function of the blood being- to maintain an equilibrium in the temperature, chemical composition and osmotic pressure between all parts of the body, it follows that it is never of exactly the same composition in any two places or at any two times. To the extent that every tissue is continually giving off something to the blood, we may consider that every organ is a factor in its formation, .and as a result of this multiplex origin of the blood, the substances it may con- tain are beyond enumeration. There are probably but few chemical substances occurring in. the tissue-cells that do not also occur iii greater or less amount in the blood. In addition to these there are also the substances characteristic of the blood itself, besides a host of substances of unknown nature, apparently manufactured in re- sponse to the stimulation of substances entering the body from out- side ; for we find that the blood of every adult individual contains substances that make him immune to a multitude of diseases that he has had in childhood, as well as substances that in later life protect him to a greater or less degree from infection by such organisms as the colon bacilli of his intestine, the pneumococci and streptococci in his throat, etc. We have learned of these defensive substances within very recent times, and also of the " antienzymes " that possi- bly protect the blood from the digestive enzymes of the body cells. Wliat other substances of importance we may yet find in the blood is an open question. There are no apparent limits to the possibilities of the study of the blood, for it represents a little of every organ, and much that is characteristic of itself. In discussing briefly the substances that have been isolated from the normal blood, before considering the changes that occur in it during pathological conditions, we may roughly divide the blood into the formed elements and the plasma in which they are suspended. THE FORMED ELEMENTS. — By weijjht, tlie red corpiisclos constitute from 40 to 50 per cent, of tlie l)lo(id, the percentage varying under difl'erent conditions, while the total weight of tlie leucocytes and platelets is insignificant. The hemo- globin constitutes from Sii to 04 per cent, liy weight of tlie solids of the red cor- puscles, but the physical and chemical relations that it bears to the stroma of the corpuscles are as yet undetermined (see ''Hemolysis"). Of tlie remainin£r constit- uents of the corpuscles, from .5 to 12 per cent, consist of proteins, probably chiefly globulins and nucleoproteins; 0.3 to 0.7 per cent, of lecithin; and about 0.2 to 0.3 19 289 290 DISTURBANCES OF CIRCULATION per cent, of cholesterol (Hoppe-Seyler) . The outer coat of tlie red corpuscles does not seem to be equally permeable for all substances, and tlierefore we find the composition of the fluid portion of the cell quite different from tliat of the plasma about it. The salts of the corpuscles consist largely of potassixun ])hos- phate, a little sodium chloride, some magnesium, but no calcium.i which is quite different from their proportion in tlie plasma. Probably many of tlie other con- stituents of the plasma, especially urea, penetrate the red corpuscles to a greater or less degree, but most of them, particularly the sugar, remain chiefly in the plasma. Hemoglobin, the most characteristic constituent of all the heterogeneous com- ponents of the blood, is a compound protein, and probably exists combined with some other constituent of the corpuscle, most probably the lecitliin. It splits up readily into a protein, glohin, and an iron-cont^iining substance, he^nochromo- gen, which readily takes up oxygen to form hematin. Only about 4 to 5 per cent, of the hemoglobin is heniochromogen, and iron constitutes but about 0.4 per cent. Hematin may be further split up into other substances, which will be considered in the discussion of "Hemorrhage." The leucocytes consist chiefly of nucleoproteins, with probaldy some globulin, and they also contain glycogen, lecithin, and cholesterol. The hiood-pUitclrts are believed to Ik^ largely nucleoprotein, but little is known of tlicir actual composi- tion; miiTocliciuical examination shows no evidence of either fat or glycogen. 2 BLOOD PLASMA differs from blood-serum in that the latter is formed from the former through the conversion of the fibrinogen into fibrin. Serum, therefore, contains no fibrinogen, but more fibrin ferment: otherwise it is practically the same as the plasma. It is well for us to appreciate that the blood is funda- mentally a tissue, with its more solid structural elements lying in a protoplasm, the plasma, somewhat more dilute than the protoplasm of other tissues but in other respects much the same. Proteins. — Fibrinogen, has the general properties of a globulin, with also a peculiar tendency to go into the insoluble form, fibrin. (Tliis process will be discussed imder "Thrombosis.") In the plasma are also other globulins.-'a one soluble in water (pseudo-glolmlin) , the other insoluble in water (euglobulin) . Serum-albtimin, another protein of the plasma, probably consists of two or more varieties of albumin. There are also nucleoproteins (prothrombin) and non- coagulable proteins, which being poorly understood have been variously considered as glycoproteins, or mucoids, or albumoses. The serum proteins seem to be closely related to, or compovmded with, the lipins of the plasma. Other Constituents. — The fnt of the plasma varies much according to tlie time which has elapsed after tlie taking of f^od ; in fasting animals it amounts to from 0.1 to 0.7 per cent. The sugar fluctuates' less, being normally about 0.1 pei- cent., while the urea has been estimated at 0.05 per cent. ]\Tost of the sugar is dex- trose; but probably there is some levulose, possibly some pentose and otlier forms, and possibly also sugar combined with lecithin (jecnrin) or other substances. Soaps, cholesterol, and lecithin also exist free in the plasma. Plasma difTers strikingly from the corpuscles in that its inorganic substances are chiefly sodium and chlorine, while potassium and phosphoric acid are almost entirely absent. Another imjiortant fact is that wIumi the plasma is combusted, the acid radicals remaining do not suffice to balance the bases, indicating that much of tlie inorganic bases is joined with organic sulistances. probably as ion- protein compounds. The alkali joined to tlie protein is non-diffusible, and con- stitutes aliout five-sixths of tlie total alkali. The concentration of the electrolytes of the blood has been determined ^ly ascertainiiiir iht> lowering of the f-ce/jim-iioint, which in human Idood averages about 0..52(i° ; this corresponds closcb- to the efi'ect of a salt solution of 0.0 per cent, strength. About three-fcnirtlis of the dissolved molecules of the blood-serum are electrolytes, and about three-fourths of these an* molecules of NaCl. most of which are in the dissociated states 1 The current statement that corpuscles are impermeable for calcium is refuted by Ilamlnirgcr (Zeit. physikal. ("hem.. 1000 (CO), C0.3). " 2 Ayiiaud. Ann. Inst. Pasteur, 1011 (25), 56. 2a Literature given by Powe. Arch. Int. j\Ied.. lOlfi (IS), 455. 3 Concerning relation of conductivil v to free/.ing-poiiif see Wilson. .Xmer. .Tour, of Physiol., 1006 (16), 438. THE COMI'OHITIOX OF THE BLOOD 291 Enzymes. — A large number of enzymes exist in tlie blood, the followiufi; being among those tliat have been detected: diastase, glucase, lipase, thrombin, rennin, and proteases. The proteases and [lerhaps the other enzymes are held in elieck to a large extent by ''antifer7HCtits'' that are also present (see "Hnzyines'") . In relation to the antiferments are the innumerable antibodies tiiat exist normally in the serum for foreign proteins, foreign cells, and for bacteria and their toxins, as well as those resulting from reaction to infection, etc. The proportions in which the constituents of the plasma normally occur have been determined by Hoppe-Seyler and by Ilammarsten as follows: * Table I No. 1 Xo. 2 Water 908.4 1)17.6 Solids 91.6 S2.4 Total proteins 77.6 69.5 Fibrin 10.1 6.5 Globulin 38.4 Seralbumin 24.6 Fat 1.2 Extractive substances 4.0 ,„„ ^ ^ Soluble salts 6.4 ^'^•^ "^ Insoluble salts 1.7 No. 1 is an analysis by Hoppe-Seyler. No. 2 is the average of three analyses made by Hammarsten. Reaction. — It i.s very difficult to determine the exact reaction of tlie blood plasma. If we titrate with an acid, we liberate much of the alkali from the proteins, dissociate all the NagCOg present, as well as the NaHCO.j and the sodium phosphate, and find in this way that the entire fresh blood contains neutralizahle alkali corresponding to a solution of NajCOg of about 0.443 per cent, strength (Strauss). In other words, the blood has a quantity of alkali in combination that can be drawn upon to neutralize acids to the extent indicated by the above figures. The real alkalinity of a fluid, however, is dependent upon the number of free OH ions in the solution ; and Hober has de- termined by physico-chemical methods that the concentration of OH ions in blood is but little greater than in distilled water." Michaelis ^* has found the H+ concentration of the blood to be 0.45 X 10"^, as contrasted with neutrality at 38° which is H+ = 1.5 X 10"^ The in- terchange between COo, phosphates and carbonates in the blood is such that it is impossible for any considerable quantities of free H or OH ions to exist, and the protoplasm is thus protected from an excess of either. The capacity of the blood to neutralize acids and alkalies is sometimes referred to as its "buffer value." ^^ According to Hen- derson ^ not more than five parts of excess free H or OH ions can be present in ten billion parts of protoplasm. An alkalinity is impos- sible because this would cause an increased osmotic pressure which 4 For complete analyses of the blood see Abderhalden, Zeit. physiol. Chem., 1898 (25), 106. ^> For bibliograpbv on Alkalinity of Blood, see Henderson, Ergebnisse Physiol., 1909 (8), 254. saDeut. med. Woch.. 1914 (40). 1170. 5b See 'Levx and Rowntree. Arch. Int. Med., 1916 (17), 525. 6 Amer. Jour. Phvsiol., 1907 (18), 250; 1908 (21), 427. 292 DISTURBANCES OF CIRCULATIOX the kidneys would reflate ; acidity is impossible because death would result from the inability of the blood to carry COj. The blood and tissue proteins also can bind much of either H or OH ions/ so that the preservation of neutrality is elaborately guarded. In the tissues, be- cause of the production of acids during metabolism, the H-ion con- centration is slightly higher than that of the blood, being estimated by JVIichaelis at exact neutrality, 1.5 X 10"'^. Presumably one important purpose of the exact regulation of reaction is to provide proper con- ditions for enzyme action. The alkali of the blood exists in part as alkaline salts, carbonate and phosphate (the diffusible alkali), and partly combined with pro- tein {non-diffusible alkali). As the corpuscles are richer in diffusible alkali than the plasma or serum, the number of corpuscles modifies the alkalinity of the blood decidedly. j\Iuch importance is attached to the question of the alkalinity of the blood for two reasons : first, in certain conditions of disease the blood contains so much of organic acids that the alkali is partly saturated and the power of the blood to carry CO, is lessened, with serious results (see "Acid Intoxica- tion," Chap, xviii) ; and, second, the bactericidal power of the blood is found to vary according to its alkalinity. In fact, metabolic activ- ity seems generally to be favored by certain degrees of alkalinity ; for example, J. Loeb ^ found that sea-urchin eggs develop with much greater rapidity if a small amount of OH ions is free in the sea- water. Brandenburg ^ states that the non-diffusible alkali varies ac- cording to the amount of protein in the blood ; in pneumonia and acute nephritis he found it low. In cancer the titrable alkalinity is distinctly increased, and IMoore and Walker ^° find that this is due to an increased alkalinity of the proteins of the blood. Orlowsky ^- could find no decrease in the alkalinity of the blood in leucoeytosis, or when virulent, bacteria were introduced into the blood. By gas chain measurements of H and OH concentration, Roily '^^ found prac- tically no change in reaction during starvation or after bilateral nephrectomy. Abnormally low alkalescence was rarely found except in diabetic acidosis, while increased alkalescence was obtained chiefly in liver diseases. Awerbach ^^ claims that in severe high fevers the bac- tericidal effect of the blood alkalinity is increased (see also "Passive Congestion" for further discussion concerning the relation of alka- linity to bactericidal power). Viscosity of the Blood.^" — Xonnal l)lood is about five times (4.5 7 See Robertson. Jour. Uiol. Cliem., 1909 (fi), 313; 1910 (7). 3.">1. 8 Arch. f. Ent.\vicklun<:sinoolianik, 189S (7), 031. oDout. mod. Woch., 1902 (28), 78; Zeit. f. klin. ^fed.. 1902 (45), l.>7. If* Biocliem. Jour., 1900 (1), 297; pood discussion of blood reaction. 12 Dent. mod. Wocli., 1903 (29). (iOl. i3Miincb. nied. Wocli.. 1912 (r)9), 1201 and 1274. 14 Med. Obosrenijo, 1903, p. .')90. 15 Review of literature by Determann. Zeit. klin. Med., 1910 (70), 185; also HEMORRHAGE 293 times, Austrian) more viscous than water, chiefly because of the corpuscles and the dissolved proteins. This viscosity does not vary directly Avith the specific gravity or the hemoglobin, but is closely related to the number of red corpuscles (Burton-Opitz) ; laking the corpuscles increases the viscosity considerably. ^lost salts increase the viseosit}', but some, especially iodides, are said to reduce it. Car- bon dioxide increases viscosity greatly, even when in amounts possible in the circulating blood. Anemia decreases the viscosity, approxi- mateh' in proportion to the number of corpuscles ; polycythemia is ac- companied by a corresponding increase ; leukemia, because of anemia, shows a decrease ; in nephritis there may either be an increase or a de- crease in the viscosity, not corresponding in any way to the blood pres- sure. Cardiac disease with edema shows low viscosity" because of the anemia and hydremia, but if there is polycythemia and no edema the viscosity may be high. Jaundice, causes an increase, diabetes gives variable results. Typhoid causes no characteristic change beyond that resulting from anemia, and in pneumonia the cyanosis and salt reten- tion usually cause an increase (Austrian). Gullbriug ^^'^ found the viscosity to vary directl}' with the per cent, of neutrophiles. HEMORRHAGE Hemorrhages result from an altered condition in the vessel-walls, which may be due either to trauma or to chemical injuries. Of the chemical agencies causing hemorrhages, bacterial products are the most important practically, but manj' poisons, such as phosphoinis, formaldehyde, phytotoxins (ricin, abrin, and crotin), and zootoxins (snake venoms) cause numerous and abundant hemorrhages. For- merh', the tendency was to ascribe hemorrhages from the above causes to mechanical injury of the vessels by thrombi, or by emboli of ag- glutinated corpuscles, but the work of Flexner " has shown that venoms cause hemorrhages by injuring the capillary walls, so that actual rents are produced by the intravascular pressure, and it seems highly probable that hemorrhages are produced by other chemical substances in a similar way. We may, therefore, refer such hemor- rhages to an endotheliotoxic action of the poison, or to a solvent effect upon the intercellular cement substance. In the case of ordinary chemical poisons the endotheliotoxic action is not specific, but with some of the toxins it seems to be quite so; for example, rattlesnake venom contains an endotheliotoxic substance (hemorrhagin) , which seems to be a specific poison for endothelium, and which is the most dangerous constituent of the venom. If we immunize animals against tissues containing iiiucli endotlielium ( c. g., lymph-glands), their serum Austrian, Johns Hopkins Hosp. Bull., 1911 (22), 9. See also Traube, Internat Zeit. phvsik.-chem. Biol., 1014 (1), 380. isaBeitr. klin. Tuberk., 1014 (30), 1. 16 Univ. of Penn. Med. Bull., 1902 (15), 355. 294 DISTURBANCES OF CIRCULATION will be found to contain endotlieliotoxins, so that when this serum is injected subcutaneously into a susceptible animal, large local hemor- rhages result; if injected into the peritoneal cavity, there results marked desquamation of the endotlielial cells, which soon undergo de- generative changes (Ricketts).^^ It is quite probable that the bac- terial poisons that cause marked hemorrhagic manifestations likewise contain endotlieliotoxins, although tliis matter does not seem to have been investigated. Even hemorrhage by diapcdesis seems to be due to, or at least associated with, chemical changes in the capillary walls, for Arnold ^* found that when capillaries from M^hich diapedesis had occurred were stained by silver nitrate, dark areas were found between the endothelial cells. As silver nitrate is a stain for chlorides, and dark- ens intercellular substance because it is rich in sodium chloride (Macallum), it is probable that there is an increase in the amount or a difference in the method of combination of the chlorides of the cement substance between the endothelial cells at the places where red corpuscles escape. ]\I. H. Fischer ^^ suggests that diapedesis re- sults from a change in the endothelial cells, which under the influence of acids or other agents of metabolic origin become excessively hydro- philic, swell up, and become so softened that corpuscles may pass di- rectly through the cell, just as a drop of mercury can pass through a sufficiently soft jelly without leaving a hole in the jelly. Hemorrhage in cachetic conditions is often ascribed to changes in the vessel-walls due to malnutrition, but it is difficult to imagine capillary walls suffering from lack of nourishment, even with the poorest of blood, and it seems more probable that the hemorrhages are due, even in cachexia, to chemical constituents of the blood that in- jure the endothelium. Hemorrhages that follow re-establishment of the circulation after complete occlusion, however, may be the result of asphyxial changes in the capillary walls, presumably colloidal swell- ing of the cells. After severe hemorrhages the blood shows a decrease in specific gravity and viscosity, an increase in surface tension and electrical resistance, and either increase or decrease of the freezing-point de- pression, all these changes being transient if the individual is other- wise normal.-" (See also Secondary Anemia.) There is a rapid absorption of fluid from the tissues and tissue spaces, resulting in a dilution of protein and formed elements, but not of salts. There is ?aid to be a decreased permeability of vessels, resulting in reduced exudative processes.^ "^ The proportion of the several blood proteins 17 Trans. Chicago Path. Soc, 1902 (5), 181. 18 Virehow's Arch., 1S75 (62), 157. m "Nophriiis," New ^'ork, 1012, p. 78. 2"01iva, Folia cliiiicii, 1!»12 C?), 21:^ »«ii.Liiitlil('ii, M.'d. Klin.. 1913 (9), 1713. iiKMoiiiiiiAdi: 295 is variably altci't'd aftrr repeated lieiuorrliag'es; the sugar is little affected l>ut the non-jjroteiu nitrogen and urea are increased.'''" Rapid lieniorrhagos cause a decrease in the coagulation time l)ecause of a decrease in antitlirond)in and a sligiit increase in prothrombin, in spite of a decrease in fibrinogen.^-" If the blood is withdrawn repeatedly in large amounts, centrifuged, and the washed corpuscles reinjected suspended in isotonic salt solution (plasmaphaeresis), life can be maintained even after 4 to 5 times the total volume of blood has l)een removed and washed. This is possible because of rapid reformation of the plasma, and the ])lood sliows the changes characteristic of sec- ondary anemias. ^"'^ Changes in the Extravasated Blood. — These begin soon after its escape. In most situations sufficient fibrin ferment is formed to cause prompt clotting, but in the pleura and other serous cavities the blood may remain fluid for some time, possibly because of lack of cellular injury that miglit cause liberation of fibrin ferment.-''^ If the blood does not become infected, the rapidity, of subsequent changes depends chiefly upon the location and amount of blood. Small ex- travasations of blood into the tissues are subjected to the action of the tissue cells and of leucocytes emigrating freely from the capillaries; large masses of blood are but little affected by these agencies, the leucocytes within the mass soon die, and secondary changes go on very slowly. In small subcutaneous hemorrhages (e. (/., a bruise) enzymes from the invading leucocytes and tissue-cells soon dissolve the small quantities of fibrin present ; even earlier the stroma of the red cor- puscles is so altered that hemolysis occurs and the hemoglobin escapes and diffuses into the tissues. This hemolysis may be brought about by the action of proteolytic enzjTnes on the corpuscles, or by the hemo- lytic action of the products of protein splitting. Soon the hemoglo- bin disintegrates, forming the masses of pigment so characteristic of old hemorrhagic areas, and also giving rise to the discoloration observed beneath the skin in the later stages of resolution of hemor- rhagic extravasations. The first products of the splitting of hemo- globin are: (1) The protein, glohin, which constitutes 94 per cent, of the hemoglobin; and (2) the iron-containing coloring-matter, hem- atin (in the absence of oxygen the pigment is reduced hematin or hemochromogen). As hematin may be experimentally obtained by the action of proteases upon hemoglobin, the decomposition of the hemoglobin in the tissues is probably accomplished in a similar way lOb Taylor and Lewis, Jour. 'Riol. Chom., 19X5 ^22), 71. i9i- Drinker, Amer. .Jour. Plivsiol., 1915 '.36). :^0o. iMAbel et al. .Tour, rharmacol., Ifll4 (5). (i2.5 ; 101,5 (7), 120. 20a Denny and Minot (Amor. Jour. Physiol., 1016 (.30). 4-55) believe that the blood really does clot, and that it remains lluid when withdrawn because the fibrinogen has been removed by olottino;. Zahn and Walker (Biochem. Zeit.. 101.3 (58). 130). however, consider that the fibrinogen is altered by the pleural endo- thelium, so that it cannot clot. 296 DISTURBAXCES OF CIRCULATION b}^ the proteases of the leueoeytes, tissue-cells and blood plasma ; the g'lobin is thus digested away and the soluble products carried off, while the insoluble liematin remains.-^ The hematin gradually un- dergoes further changes, forming an iron-free i)igment (hcinatoulin) and an iron-containing pigment (hemosiderin) . Hematoidin is nearly or quite identical with the bile-pigment, hili- rubin, and is absorbed from the hemorrhagic extravasation and elimi- nated as bilirubin in the bile. Possibly some of the hematoidin un- dergoes transformation into nrobiUn, and is then eliminated in the urine. Hemosiderin seems to be relatively insoluble and, therefore, is more slowly removed, so that it may be found at the site of a hem- orrhage after the other evidences of blood extravasation have been removed. It may be easily demonstrated by staining with potassium ferrocyanide, the Prussian blue that is formed being readily dis- tinguished. Unstained hemosiderin generally appears in the form of brown or yellowish-brown granules, never as crystals. After a time the hemosiderin is taken away, and probably is to a greater or less extent deposited in the liver and spleen, either as hemosiderin or as some other iron compound. Eventually it is probably utilized to make new hemoglobin ; at any rate, the iron liberated by the breaking- up of hematin within the body does not appear to be eliminated. "- The changes in the red corpuscles described above are not at all peculiar to extravasated blood, but are quite the same as the changes that are going on continuously and normally in the blood. Red cor- puscles are short-lived, being but non-nucleated fragments of cells, and they are continually disintegrating with the production of iron- free pigments that are excreted as the coloring-matters of the bile and urine, while the iron is worked over again into new hemoglobin after a varying period of storage in the tissues, particularly in the spleen and liver. The destruction of red corpuscles under normal conditions seems to take place chiefly in the spleen, bone-marrow, and hemolymph glands, where injured or decrepit c()ri)uscles are taken out of the blood by the phagocytic endothelial cells, and de- composed by intracellular enzymes. In hemorrhagic extravasations the changes are essentially the same; some corpuscles are destroyed by phagocytes, but more by extracellular enzymes. The products of decomposition also seem to be no different from those formed dur- ing normal katabolism of hemoglobin, and they meet the same fate in the end. If the hemorrhages are very abundant, some hemoglobin may be absorbed as such and appear in the urine, but this ])rol)nbly seldom happens unless red corpus('le^'. are also being destroyed in tlie circu- lating blood.--'' An increased amount of iron accumulates in the 21 More fullv disciisscHl in tlie consideration of "Pitrnu'ntaiion." Clia|). xvi. 22 See Morishima, Arch. f. cxp. Tath., 1898 (41), 2!)]. 22a In cerebral lieniorrliase the blood serum niav be frreenisli and somewhat IIEMOI'IIILIA 297 liver, but if iniicli blood has been lost by lieiiioiThage on free surfaces, the iron conteiit of the liver is decreased, as it is taken away to form new hemoglobin (Quincke).-^ Excretion of bile-pigments is in- creased by destruction of blood (Stadelmann), but not greatly in the case of hemorrhages, for the blood is decomposed and absorbed too slowly. Schurig -* found that hemoglobin injected into the tis- sues is partly decomi)osed in situ with formation of iron compounds, but the greater part enters the circulation as hemoglobin, and is partly converted into bile-pigment by the liver-cells, the rest being converted into simpler iron compounds by the spleen, bone-marrow, and renal cortex. If the hemorrhagic extravasation has been large in amount, the deeper ])ortioiis of the mass are not soon, if ever, invaded by leuco- cytes or tissue-cells. Consequently the blood is acted upon very slowly by the enzymes liberated by the leucocytes it contains itself, and by the small amounts of proteases in the serum. Furthermore, the products of decomposition are not soon absorbed, but accumulate in considerable amounts, so that we often find crystalline deposits of hematoidin, sometimes even of hematin, hemoglobin, or parahcmoglo- hin- (Nencki) -"' or methemogJohin. The least soluble constituent of the red corpuscle stroma, choles- terol, also accumulates in such extravasations as large, thin plates ; after most of- the other products of distintegration have been absorbed from such accumulations of blood, the most conspicuous part of the residue may be a mass of cholesterol crystals imbedded in prolifer- ating connective tissue. HEMOPHILIA 2G There are several pathological conditions associated with increased tendency to bleeding, notably scurvy and the various forms of pur- puras, but especially the remarkable hereditary condition, hemophilia. In the purpuric diseases various of the factors concerned in coagula- tion of the blood have been found altered,-"''' notably the blood plate- lets,-^'' but Howell found no change in either prothrombin or anti- thrombin in purpura hemorrhagica and other related conditions. Similar negative results were obtained in scurvy by Hess.-*"= Melena fluorescent from absorbed pigment, according to !Marie and Lcri, Union Pharm., Aug. 15, 1914. 23Deut. Arch. klin. Med., 1880 (25), 567: 1880 (27), 103. , 24 Arch. exp. Path. u. Pharm., 1808 (41), 29. 23 Arch. exp. Path. u. Pharm., 1886 (20), .3.S2. 26 Literature and resume given bv Stonipcl, Cent. f. Grenzgel). ^Ted. ni. Chir.. 1900 (3). 753; Sahli, Zeit. f. klin. :\ied.. lOO.i (56), 294: :Marchand, in Krehl and Marchand's Handb. allg. Pathol., 1912. II (1), 307. Also later references in this text. 26a See Hurwitz and Lucas, Arch. Int. Med.. 1916 (17), 543: Minot ei ah, ibid.^ 1916 (17), 101. r">. 26b See Lee and Robertson, Jour. Med. Res., 1916 (33>, 32*r 26cAmer. Jour. Dis. Children, 1914 (8), 386. 298 iJlSTURBAyvE.S OF CIRCULATION neonatorum exhibits decreased prothrombiu in tlie blood, while in leukemias and anemias there may be an excess of antithrombin,-"'' leading to severe hemorrhage (see also Thrombosis). Since hemophilia seems, superficially at least, to depend upon some alteration in a chemical property of the blood, namely, coagulability, it is frequently i-egarded as an example of hereditary transmission of a chemical abnormality. The exact cause of this peculiar tendency to prolonged bleeding from insignificant or perhaps imperceptible wounds has been sought vigorously by both histological and chemical means, but as yet without avail. Various observers have described abnormal thinness, or increased cellularity or fatty degeneration of the vessel-walls, but the findings have been far too inconstant to afford a satisfactory anatomical explanation of all the features of hemophilia. Likewise increased blood pressure can be ruled out, for although the left heart is frequently enlarged, there is usually no in- creased blood pressure demonstrable; furthermore, conditions of high blood pressure, such as nephritis, do not cause hemophilia. The theory of "hydremic plethora" is also without good foundation. The most natural place to look for the fundamental fault is in the blood, but speaking strongly against this is the occasional occurrence of "local" hemophilia; e. g., in this type of hemophilia wounds of the skin may behave as in normal individuals, whereas any injury of the mucous surfaces is followed by pronounced hemophilic bleeding ; - ' in other cases the hemophilic bleeding is limited to regions above the shoulders; in still another class the bleeding is always from one or- gan, e. g., the kidney's. Nevertheless, a great deal of investigation of the blood has been done, at first chiefly with negative results. There are no characteristic changes in the cellular elements of the blood, beyond the changes common to all secondary anemias, excepting pos- sibly a decrease in the number of white corpuscles with a relative increase in the number of lymphocytes as observed hy Sahli; the platelet count is normal. No constant alterations in the salts of the blood have been found, calcium usually being normal ; and the propor- tion of water, fibrinogen and the several other proteins, the alkalinity, and the osmotic pressure of the serum all seem to be normal. IMetab- olism is unchanged, except possibly for calcium loss in some cases.-* Since bleeding is normally stopped principally by coagulation, a de- ficiency in fibrin oi' its antecedents might be expected, but most studies on this point have shown a noi-mal amount of fibrinogen in the blood of hemophilics, the fre(pient formation of large tumors of clotted blood at the bleeding points supporting the experimental evidence that the blood contains an abundance of fibrinogen. The 2od Whipple, Arcli. Int. Aled., 1913 (12), 637. 27 Ahdi'ilialdcii, Zicfrl,.,'s I'.oiir., 1!)04 ( .T) ) . '213. 2« Kulin, Amer. Jour. Dis. Cliildroii, lOKJ ( 11 ), 103: Laws and Cowie, ibid., 1917 (13), 236; Hess, Bull. Joiins Hopkins Hosp., 1916 (26), 372. iiKMoi'iiii.ix 299 ■'blectliii^' time"" t'ullowing puiiclurcs in tliu skin is not excessive. As to the rate of clotting, Sahli,-° who avoided a number of errors made in earlier investio-ations, found that in the intervals between the at- tacks of hemorrhafi-c the rate of the coa^'ulation of the blood is con- stantly Huicli slower than noi-iiial. Dui'iii;^- an attack of bleeding the coagulation time approaches the normal ; indeed, it may be faster than normal ; apparently this is due to a reaction on the part of the organism to the loss of blood. If blood is collected directly from the site of bleeding the coagulation time is very rapid, because of the ac- cumulation of fibrin ferment from the clot over which the escaping blood flows. Yet in spite of the normal coagidability of the blood and the rapid clotting after the blood escapes from the vessel, bleeding continues for long periods before it can be stopped. As he found no general change in the properties of the blood to account for the bleeding, and as local influences seem to be important in hemophilia, Sahli advanced the plausible hypothesis that the cause of hemophilia lies in hereditary deficiency of the fibrin-forming substances, throm- bokinase or zymoplastic substance (see "Thrombosis"), in the vessel- walls, so that when the vessels are injured there is no local produc- tion of fibrin such as occurs normally. Local hemo])hilia may be ex- plained readily as a local deficiency in fibrinoplastic material. In general hemophilia even the leucocytes may exhibit the same defect, in which case clotting of the blood is diminished even outside the tis- sues. This hypothesis seems to be in excellent agreement with many of the facts now known, but there yet remains to be demonstrated such a lack of fibrin-forming elements in the vessel-walls and other tissues of a hemophilic subject, and a single autopsy of a hemolytic subject gave, on the contrarj^ a very active thromboplastic extract from the vessels fGressot).-^ "With the improved methods of study of the factors in coagulation of blood introduced by Howell, it has been found by him and cor- roborated by others ^° that in hemophilia there is constantly a defi- ciency in prothrombin, the other factors being practically normal in amount, and as in other hemorrhagic conditions there is no equal alteration in the prothrombin, they look upon this change as an essential characteristic of hemophilia. Fonio, and ]\Iinot and Lee, however, find that the blond platelets of hemophilics are remarkably ineffective in causing coagulation of either normal or hemophilic plasma, although normal platelets cause normal coagulation of hemo- philic plasma, and therefore conclude that there is some deficient ac- tivity on the part of the platelets in spite of their occurrence in normal luimbers in hemophilia. The significance of the platelets is shown 20 Zeit. klin. Med., 1012 (70), 104. Since corroborated by Minot and Lee. 30 Howell, Arch. Int. Med., 1014 (1.3), 76; Hurwitz and Lucas. ihicL. 1016 (17), 543: ^linot and Lee, ihid., 1010 (18), 474; these papers review re<'ent work on hemophilia. 300 DLSTCRliAXCIJS OF CIRCCLATION especiallj" clearly by the observation of Ledingham and Bedson ^^ that anti])lat('let seruiu will ])rotliice a ]Mir]nirie condition when injected into animals of the species furnishing the platelets, al- though no similar effect is produced by antileucocyte or antiery- throcyte serum. Hess^- states that there may be an hereditary jiurpura, sometimes occurring in the females of hemophilic families, differing from hemophilia in a deficiency in the number of platelets, hemorrhages following local congestions or puncture wounds and exhibiting an increase in the bleeding time. ANEMIA AND THE SPECIFIC ANEMIAS ^^ The customar}^ division of the anemias is into — (a) priinary, i. e., those in which the anemia seems to depend upon some abnormality in the blood-forming organs or in the blood itself; and (&) secondary, embracing anemias the result of some obvious cause, such as hemor- rhage, poisoning with blood-destroying poisons, cachexia, etc. In these various forms of anemia certain chemical differences prevail, but they are by no means so striking as are the histological differences in the formed elements of the blood. ^* SECONDARY ANEMIAS As the simplest variety, anemia following a single large hemorrhage may be considered first. If loss of blood by hemorrhage is rapid, the effects are naturally much more serious than when the loss is slow. The total quantity' of blood in the average adult is estimated at about ]{r, to ^i.j the total body weight (therefore about 10 to 12 pounds), although this pro- portion does not hold for extremely obese or extremely thin indi- viduals; ^^ in infants the proportion is lower — about ^20- When one- third of the total amount of blood is lost rapidly, a marked fall of blood pressure occurs; loss of one-half of the total amount may be fatal, and loss of more than that at one time usually is fatal. The chief cause of death following large hemorrhages is the low blood pressure rather than the loss of any of the constituents of the blood ; hence the successful results of the use of physiological salt solution after severe hemorrhage. The number of corpuscles may be greatl.v reduced after several small hemorrhages, even to as low as 11 per cent, of the normal number (Hayem), without fatal results, because in the intervals between the hemorrhages enougli fluid lias been taken up b}' the blood to maintain the blood pressuiv within safe limits. 31 Lancet, Fcl). l."?. 101.5. 32 Arch. Int. Med., 1!)1() (17), 203. 33 M(.(abolism in anemia reviewed I)v ^lolir, nandbucli d. Bioclioni., liUO ( I\' (2) ), 372. 34 Concerning local anemia, see "Infarcts." 35TIaldane and Smith (Jour, of Physiol., 1000 (25), 331) estimated tiie blood of adults at but J^q "f ^'"' 1>o(l.v weiglit. ANEMIA AND THE HPEVIFIC ANEMIAS 301 After a severe heiuorrliag-e tlie composition of the blood changes rapidly, for the fluids contained within the tissues and lymph-spaces nass into the blood in large amounts. This helps to maintain blood pressure, but results in. the blood containing a large proportion of water and salts and a smaller amount of protein and red corpuscles ; the "total alkalinity" also falls, largely because of the scarcity of "fixed alkali," on account of the poverty in corpuscles and blood proteins. The proportion of water increases at first more rapidly than the proportion of salts, and as a consequence the size of the red corpuscles is increased because of imbibition of water ; indeed, it is possible that this may even be sufficient to cause hemolysis, which wall happen if the isotonic streng-th of the blood becomes less than that of a 0.46 per cent. NaCl solution (Limbeck), while swelling may occur whenever the strength is below 0.8 per cent. The specific gravity of the erythrocytes is decreased ; ^^ the depression of the freez- ing point increases," while the viscosity falls. The number of plate- lets is high. Regeneration of the blood begins veiy soon, and for some tniie the number of corpuscles exceeds the proportion of hemoglobin. During this time the amount of iron in the liver and spleen is decreased, it being taken up to be used in the formation of new hemoglobin. If the hemorrhages are numerous and the condition of anemia prolonged, secondary changes in the viscera may occur, fatty metamorphosis be- ing most' marked, supposedly because of decreased oxidation. Indeed, many observers state that repeated bleedings greatly increase body weight by causing increased fat deposition. Metabolic Changes. — Gies ^'^ studied the metabolism of dogs after Avithdrawing a total amount of blood equal to 11.5 per cent, of the body weight during four bleedings, and found that a slight and tem- porary increase in nitrogenous elimination followed the bleedings, owing to an increased protein katabolism. Sugar increases in the blood, while albumin and laetic acid appear in the urine. After each successive hemorrhage the proportion of fibrin and the coagulability of the blood increase, while the proportion of the ash obtained from both blood and serum remains practically unchanged (:\Ieyer and Gies). Baumann ''« states that in regeneration after hemorrhage the serum albumins increase more rapidly than the globulins, while others have observed the opposite relation. The urine in secondary anemia shows the effects of increased protein katabolism. its specific gravity, total solids, and total nitrogen being raised ; the total amount of urine is at first diminished because of lowered blood pressure, but it soon rises above normal and later falls back to normal. The view soBonninger, Zeit. exp. Path.. 1012 (11), 1. 37 Hoesslin. Hofnieister's Boitr.. 1906 (8). 431. 38 American IMed., 1004 (8), 155 (resume of literature). 39 Jour. Phvsiol., 1903 (29), 18. 302 DISTURBANCES OF CIRCULATION formerly held tliat oxidation is decreased in anemia has been con- siderably modified by more recent investig'aticms;^" in fact, respira- tion stndies indicate heightened gas exchange in secondary anemia.*"* Secondary anemia due to cachexia, or to malnutrition, is accom- panied ])y a general decrease in all the elements of the blood, both cellular and chemical. The proteins of the plasma, particularly, show a decrease in starvation, being drawn on by the cells for food, and the total quantity of blood as well as of each of its constituents is de- creased (Panum),*^ but the proportion of blood to body weight re- mains about normal. With protracted starvation there is only a slight loss of hemoglobin and an increased coagulability, but practi- cally no other changes.'*^'' In aplastic anemias the prothrombin and platelet content are likely to be low, with normal fibrinogen. ^^'^ Anemia due to hemolytic agencies presents quite different fea- tures, in that red corpuscles are almost solely attacked and the prod- ucts of their disintegration are present in the plasma. As a result, the plasma or serum may contain free hemoglobin, and if the hemo- globin is in large amounts, it may escape into the urine. Thus par- oxysmal hemoglohinuria. is probably due to the presence in the blood of hemolytic substances, which can be demonstrated in the blood of the patients during the attack. (See Chapter viii.) The products of the decomposition of the hemoglobin set free by hemolysis are present not only in the blood, but also in the organs, particularly the liver and spleen, which become rich in iron. In acute anemia pro- duced by hemolytic sera, with destruction of more than half the blood in three days, nearly all the iron from the destroyed hemoglobin can be found in the liver, spleen and kidneys, there being but little lost through the urine even in so severe an anemia as this (INIuir and Dunn).*^" Excretion of bile-pigments increases, and ^^hematogenous jaundice" may result, the bile-pigments that are present in the blood being derived from the hematoidin of the hemoglobin molecule. Changes in metabolism occur which are quite similar to those ob- served in other forms of anemia, with fatty changes in all the paren- chymatous organs, increased protein katabolism, and an excessive quantity of pigmentary substances, particularly urobilin, in the urine. CHLOROSIS The characteristic feature of the blood in chlorosis is the rela- tively small amount of licmoglobin in pro])()rtion to the number of corpuscles. Apparently, therefore, the fault lies rather in the manu- 40 See Mohr, Zeit. exp. Path.. lOOfi (2), 4.35. 40aGrafe, Deiit. Arch. klin. Med.. 11)1,'> (US), 14S. 41 Virchow's Arch., 1864 (20), 241. 4iaA8h. Arch. Int. Med., 1014 (14), 8. 41b Drinker and Ilurwitz, Arch. Int. Med, I'M.') (If)), 73:]; Jour. Exp. yied., 1915 (21), 401. 4ic Jour. Patli. and I'-act.. IDlf) (10), 417. cuiJtuosL^ 303 facture of hemoglobin than in cither a destruction or a deficient formation of red corpuscles. Er])cn's '- analyses of chlorotic blood showed that the total amount of protein is decreased, chiefly because of the deficiency of hemoglobin; the relation of serum globulins and serum albumins is unchanged, wliile tlie proportion of fi])rinogen is increased. There is much more fatty substance than normal in both the serum and the erythrocytes, but the lecithin is decreased both in the serum and in the total blood, although somewhat increased in the red cells. Cholesterol is decreased in both serum and corpuscles. In the asli, pliosphoric acid, potassium, and iron are decreased, w-hile calcium and magnesium are both increased. An apparent increase in sodium chloride exists, but it is only apparent, being the result of the increase in the proportion of plasma in the blood. The total amount of plasma is greatly increased (polyplasmia). The decrease in hemoglobin is demonstrable chemically as well as microscopicall}', Becquerel and Rodier *^ having found the amount of iron in the total blood decreased in direct proportion to the apparent decrease in hemoglobin, which frequently falls to 30^0 per cent., and may drop to 20 per cent, or possibly less. Alkalinity, as determined by titration, is diminished in some cases, but generally remains nearly rionnal. The corpuscles are said to contain a larger proportion of water than normal, independent of the proportion of water present in the serum. Limbeck found their isotanicity {i. e., the strength of NaCl necessary to prevent hemolysis) veiy low — about 0.38-0.4 per cent. NaCl. Very few changes seem to occur in the organs of the body; the usual tendency to lay on fat, and the occurrence of fatty degenera- tion observed commonly in anemias, may be exhibited, and are cor- related with Erben's observation of an increased fat content in the blood; but these changes are often absent. The hypoplasia of the aorta, upon which Virchow laid so much stress, is now considered to be of little or no significance. Thrombosis is a not infrequent com- plication of chlorosis,** and is probably favored by the increased platelet and fibrin-content of the blood and the tendency to fatty changes in the vessel-walls. Studies of nitrogenous mctaholism by Yannini *° showed practically no alterations except a slight retention of nitrogen. Etiology. — As to the etiology of chlorosis, chemical findings indi- cate some possibilities and negative others, but decide nothing. That chlorosis does not depend upon a hemolytic poison is well established *2Zeit. klin. ^led., 1002 (47), 302. See also Frohmaier. Folia Hematol., 1015 (20), 115: Boumer and Burger, Zeit. exp. Path.. 1013 (13), 351. *3 For literature see Krehl, "Basis of Symptoms," lOlfl, p. 106; Ewing, "Clinical Pathology- of the Blood," 1001. p. 167: Kossler, Cent. f. inn. Med., 1807 (18), 657. ** See Schweitzer. Vircliow's Arch., 1898 (152), 337, and Lcichtenstern, Miinch. med. Woch., 1809 (46), 1603. *5Virchow's Arch., 1004 (176), 375. 304 DISTURBANCES OF CIIiCULATIOX hy the following facts : there is no free hemoglobin in the blood plasma, and even less iron in the serum ash than normal ; lecithin and cholesterol, important products of disintegration of erythrocytes, are both decreased in the serum ; hematogenous icterus does not occur, and the amount of pigments in the urine and feces is decreased. Apparently, therefore, hematogenesis is at fault, particularly the formation of hemoglobin, since this is more deficient than is the total number of red corpuscles. The rapid improvement in the condition that follows the administration of iron would seem to indicate that a deficient supply of iron is the cause of chlorosis, but numerous ob- jections exist to this hypothesis. Bunge advanced the idea that under normal conditions the only form of iron that can be absorbed is that which is combined with proteins, particularly nucleoproteins ; iron ad- ministered in inorganic form, or as compounds with organic acids, he believed, can all be recovered from the feces, and, therefore, is not absorbed. He suggested that in chlorosis the iron taken with the ordinary food is precipitated in the intestines by sulphides or other products of intestinal putrefaction, and hence there results a de- ficiency in the amount of iron absorbed and available for the manu- facture of hemoglobin. The inorganic iron given in chlorosis, Bunge believes, owes its efficiency to its saturating all of these sulphides so that the nucleoprotein-iron is not precipitated, and can, therefore, be absorbed. ]\Iany objections have been raised to Bunge 's hypothesis, however, for competent observers have failed to find any abnormal putrefaction in chlorosis, and others have found that sulphide of iron itself gives good results in the treatment of chlorosis, while bismuth and other sulphur-binding substances are without effect. Further- more, Bunge 's contention that iron administered in medicinal form is not absorbed seems to have been completely disproved by several ex- perimenters.^** As a consequence of all these conflicting data we are at present completely in the dark as to the reason for that failure properly to manufacture hemoglobin which seems to be at the bottom of chlorosis. The hypothesis that iron and arsenic favor recoveiy by stimulating the hemogenetic tissues, which is urged by v. Noorden and others, is unsatisfactory in the extreme, and explains nothing. There is abso- lutely no question tluit administration of iron restores the composi- tion of the blood to normal, usually quite rapidly, and this seems to leave as most probable the explanation that in some way an iron starvation is the fundamental cause of cldorosis. However, as Ewing says, any theory must be inadequate that fails to take into account the age of puberty, tlie female sex, and tlio function of menstruation. 40 Full review witli biljliofjiapliv bv E. Clever, Krpebiiisse Phvsiol.. 1005 (.5), 698; Meinertz, Cent, riiysiol. u. Path". StolTwedi., lOOT (2), 652." PERNICIOUS ANEMIA 305 PERNICIOUS ANEMIA 111 contrast to chlorosis many evidences of hematolysis may be found in pernicious anemia, particularly the increased amounts of iron in the liver, spleen, and kidneys; hemoglobinemia and hemoglo- binuria; increase in ur()l)ilin, and not infreqneiitly icterus. Chemical Changes. — l-'rlicn's i" analyses of tlie blood in ])ornici()us anemia prave the f(>ilo\\inu- losiilts: Tlic proteins are decreased, both in the sernm *'a and in the blood as a whole: part ieularly in the latter, because of the <.'reat decrease in the number of corpuscles. The <|uantity of proteins in the individual corpuscles is increased, correspondintj to their increased size. Fibrin is decreased in total amount, but is relatively normal as compared with the total proteins: alliuniin is normal ; serum olobulin much decreased. The proportion of water is much in- creased, both in the serum and in tlie corjiuscles. Fat is present in normal amounts; cholesterol is decreased, althoujjh in relatively normal quantities in the corpuscles. Lecithin is decreased in the total blood, but increased propor- tionately in the corpuscles. The total ash is increased, owin^ have foiuid some decrease in total alkalinity, which probably depends on the loss of proteins and their fixed alkali. ■'''•'' The red corpuscles are very susceptible to hemolysis by lowering of osmotic pressure ("high isotonicity," equal to 0.;')4 per cent. XaCl — v. Limbeck). The specific gravity of the whole blood is, of course, decreased, but the corpuscles themselves *" Zeit. klin. :Med., 1900 (40), 266. Reumer and Riirger. Zeit. exp. Path.. 1013 (13), 343. *"a See also Heudorfer, Zeit. klin. IMed., 1!)1.3 (70), 103. ■t^Rerl. klin. ^Yoch.. 1001 (38), 477. 40 Abst. in Folia Ilematnl.. 1004 (1), 2S3 and 580. •'i'l Quart. Jour. :Med.. 1013 (6). 170. ■"■1 Ilofmeister's Reitr., 1003 (4). 4r)3. siaZeit. physiol. Chem., 1016 (07), 32. ■"■ih Drinker and Hurwitz, Arch. Int. Med., 1915 (15), 733. 52 "Klin. Pathol, des Blutes," Jena. 1896, p. 311. 53 See Brandenburg, Zeit. klin. Med.. 1902 (45), 157. 20 306 DISTURBANCES OF CIRCULATIOX have practically iioinial specilic gravity, while tlie decrease is chielly in the serum. 54 111 six cases of pernicious anemia Stiihlen ^r. found ahundant iron in the liver and spleen microscopically, and less constantly in the kidneys and bone-marrow. Hunter '"J gives the following results of analysis of the liver, kidney, and spleen for iron: Liver and kidney. Spleen. Pernicious anemia, seven cases average . . . 0.360 per cent. 0.125 per cent. Other conditions (with anemia), average . ". 0.079 " 0.3(52 " Healthy organs 0.084 " 0.090 Iron is also fouiul in the hemolymph glands, sometimes more abundantly than in the spleon (\\'arthin) .57 Extensive studies on the protein mctaholism of pernicious anemia by Rosen- quist 58 showed tliat there is a considerable destruction of tissue proteins, as indicated by nitrogen loss, but that at times nitrogen may be stored up for brief periods. At times there may also be an excessive elimination of purine nitrogen, indicating destruction of nuclear elements. Calorimetric studies show the metab- olism to be slightly above normal. 5' XH, thus being similar to, although not identical with, piperazin. The entire question of the composition of spermin is still im- settled,9o however; and it is probable, furthermore, that the crystals found in leukemia are not identical with the crystals observed in semen. Crystals that appear similar are also found in asthmatic sputum, empyema, and ascites fluid, bone-marrow, and tumors, and it has been suggested that they are derived from or related to the oxyphile granules of the eosinophiles.9" This view implies an agi'eement with Gumprecht's opinion that the crystals seen in bone-marrow, asthmatic sputum, etc., are not spermin, but of protein nature. As can be seen, the nature and significance of Charcot's crystals are, at the pres- ent time, quite undetermined. Summary. — The chemical changes observed in leukemia depend ss Radiiim also causes marked metabolic changes in leukemia, with enormously increased excretion of urea, ammonia and total X. and especially of P^0_ ; never- theless tlie increase in uric acid excretion is slight (Ordwav, Knudson and Erdos, Boston :Nred. and Surg. .Jour.. 1917 (176), 490). 89Miinch. med. Woch., 1906 (53), 1063. 90 Hofmeister's Beitr.. 1905 (7), 527. 91 Literature on effects of x-ravs in leukemia, see Arneth. Berl. klin. Wocli., 1905 (42), 1204: Musser and Eds'all, I'niv. of Penn. Med. Bull.. 1905 '18), 174: Rosenberger. Miinch. med. Woch.. 1906 (53). 209: Williams, Biocheia. Jour.. 1906 (1). 249; Lossen and IMorawitz, Deut. Arch. klin. Med., 1905 (S3), 288; Koniger, Deut. Arch. klin. Med., 1906 (87). 31. 92Munch. med. Woch., 1905 (52), 2409. 93 Jour. Exp. Med., 1907 (9), 51; see also Klieneberger u. Zoeppritz. ^liinch. med. Woch., 190G (53), Xo. 18: Milchner u. Wolfi". Berl. klin. Woch., 1906 (43). No. 23. 94 Literature given Ijv v. Levden, Festschrift fiir Salkowski. Berlin, 1904, p. 1. 95Deiit. med. Woch.. "1895 (21). 475. 90 Literature, see Hammarsten, Amer. Transl., 1904, p. 420. 97 Literature, see Floderer, Wien. klin. Woch., 1903 (16), 276; Predtetschcnskv, Zeit. klin. Med., 1906 (59), 29. 312 DISTURBANCES OF CIRCULATION upon the excessive quantity of leucocytes and lymplioid tissue, which undergo processes of disintegration at irregular intervals, with the result that the products of nucleoprotein destruction (uric acid, purine bases, and phosphoric acid) appear in the urine in increased quantities. As the large neutrophiles contain abundant autolytic enzymes, the products of cell autolysis (proteoses, amino-acids, and products of nucleoprotein destruction) may appear at times in the urine and in the blood; hecause of the small amount of such enzymes in the lymphocytes, these changes are all much less marked in lymph- atic leukemia. Charcot's crystals, which are perhaps derived from leucocytic nucleoproteins, may be found in the blood and tissues. The changes in the red cells are chiefly those of a secondary anemia, with occasionally some chlorotic features. The chemical findings of leukemia throw no light whatever upon the cause of the disease. Pseudoleukemia and Hodgkin's disease show only the evidences of a secondary anemia, without the chemical changes of either leukemia or pernicious anemia. There seems to have been little study of the chemical processes of these diseases. Moraczewski ^* reports a study of metabolism in one case, designated by him as pseudoleukemia and so quoted in subsequent literature, although the only leucocyte count mentioned in the original article was 171,000. This case showed some retention of nitrogen and calcium, with little change in the phosphorus and purine bases in the urine. HYPEREMIA ACTIVE HYPEREMIA This condition is associated with but few chemical changes. Cer- tain chemicals may cause active hyperemia ; some locally, as in the case of irritants, such as alcohol, ether, ammonia, mustard, etc., which act either by producing a local vasodilator stimulus or by paralyzing the vasoconstrictors. Other substances may produce active hyperemia in special vascular areas, e. g., cantharides causes active hyperemia in the kidneys, probably because of its elimination through these organs ; pilocarpin causes active hyperemia in the salivarj^ glands and skin, which is associated with increased function. In general, functional activit.y is associated with active hyperemia, and Gaskell ^ has suggested that this is due to atonicity of the vascular muscle, the result of de- creased alkalinity of the lymph flowing away from the active organ along the vessel-walls, it having been found that alkalies cause a tonic contraction and acids an atonic dilation of arterial muscle.^'' osVirdiow's Arch., ISOS (151), 22. 1 Quoted by Lazanis-Barlow, "]\Tamial of General Patliolo Vircliow's Arch., 1899 (156), 329; also, "OsmotisHuT Dnick \iiid loiu'iilehre." 7 Roo Bier, "Hvpersemie als Heilmittel," Leipsic, 190;]. 7ii Glasewald, Cent. Grenz. Med. Cliir., 1915 (18), 50". 8 Cent. f. 15akt., 1890 (7), 753. 0 Literature, see Hanilmrfjer (loc. citfi), p. 281. 10 Vircliow's Arch., 1899 (156). 329. 11 Zoit. exp. Patli. u. Therap., 1905 (1), 670. uaBeitr. klin. Cliir.. 1913 (84), IT. 1. i-^CIrawitz, Dent. Arch. f. klin. Med., 1895 (54), 588. 13 See Krehl, "Pathologische Pliysiologie," 1904, p. 201. Tiii{(> ]fiiosis 315 trated blood of passive congestion the corpuscles may number six to eight millions per .cubic millimeter, while the concentration of the solids of the serum may be at tlie same time reduced CKrehl). The viscosity of such blood is higher than that of normal l)lood.^* In acute stasis the proi)orti()n of serum proteins, especially the albumin, increases with the duration of the stasis ; no changes occur in the non- protein constituents of the blood (Rowe).^^^ THROMBOSIS The cliemistry of thrombosis in most respects resolves itself into the chemistry of fibrin formation, a subject which is so extensively con- sidered in most treatises on physiological chemistry and physiology that it does not seem desirable to give here anything more than the essential principles involved in the clotting of the blood, as now under- stood, as an introduction to the consideration of the same process as it occurs under pathological conditions. In spite of innumerable in- vestigations, our knowledge of the actual participants and processes involved in the formation of fibrin is in a veiy unsatisfactory and fragmentary state. Some facts seem well established, however, and we have a general idea of the subject that may be applied with ad- vantage to the consideration of thrombosis. FIBRIN FORMATION.i- Several difFeront substances seem to l)e eoneerned in the forniation of fibrin, of -which tlie first of importance is its antecedent, fibrinogen. Fibrinogen is a simple protein, related to the globulins, and diflfering chiefly in its icady coagula- bility, not only by fibrin ferment, but also by heat, salts, and other coagulating agencies. By itself, however, it shows no tendency to coasrulate spontaneously. According to Goodpasture.ifi fibrinosen is formed through tlie combined activity of the liver and intestines, although earlier writers have, variously described its formation in the bone marrow, leucocytes, liver or intestines. The amount of fibrinogen present in the blood is actually quite small, the fibrin formed in nor- mal clotting being but 0.1 to 0.4 per cent, of tlie weiffht of the blood. Acted upon by the fibrin-ferment, it yields the characteristic insoluble protein fibrin, in crystalline form under favorable conditions,!" but we do not know definitely what changes the fibrinogen undercroes in this process. Fibrin resembles in its insolu- bility the proteins coagulated by heat, alcohol, etc., but when kept aseptically for some time, it becomes asain dissolved; this process of fibrinoh/sis probably de- pends upon proteolytic enzymes, which fibrin, in common with other sulistances of similar physical nature, has the property of dragging out of solution and holding firmly. Undonljtedly entangled leucocytes are also an important factor in the fibrinolysis. lo which is greatly increased in phosphorus poisoning and when the liver is excluded from the circulation, a fact suggesting that tlie liver may form inhibiting substances. i4Determann. Zeit. klin. :Med.. inOO (.5n), U. 2-4. 14a .Tour. Lab. flin. :\fed., 1016 (1), 48.5. 15 For literature and full discussion see Hammarsten's or ^lathews' Physiolog- ical Chemistry; ;Morawitz, Ergebnisse der Phvsiol., Abt. 1, 1004 (4), 307. and the andbuch "d. Biochem., 1008 II (2), 40; 'Leo Loeb, Biochem, Centr.. 1907 (6), 829. ifi Amer. Jour. Physiol., 1914 (3.3), 70. 1' See Howell. Amer. Jour. Phvsiol., 1014 (3.5), 143; Hekma, Internat. Zeit. physik. chem. Biol., 191.5 (2), 279. 19 See Morawitz, loc. cit.; also Pvulot, Arch, internat. d. Physiol.. 1904 (1), 152. 316 DISTURBAyCEl^ OF CIRCULATION Theories of Fibrin Formation. — The great problem is the nature and the place and manner of uriuin of the librin-forming enzyme, generally called fihrin-ferment (also ])lasmasc. thrombin and coaulin was not decreased in or<;ans altered by phospliorus jioisoning, althou^di duriufj experimental autolysis the coajrulins disajjpear. Wiien tissue coagulins and blood coaiiulins act together, the elTect is greater than the sum of their independent actions, indicating the probability that they combine in some way to [jroduce a particularly active coagulin. The blood eoagulins are (piite dili'erent from the tissue eoagulins in many important respects, and the eoagulins cannot be l(M)ked ujion as a single substance of dillVrcnt origins. Blood-platelets. — It is still undetennined just what jiart the platelets ])lay in coagulation. The well-known observation that in thrombosis the fibrin is often first formed about masses of platelets clinging to the wall of the ve.ssel indicates that they participate in the process, and Bizzozero and others have maintained that the platelets and not the leucocytes are the source of the prothrombin. Numerous studies on the relation of the platelets to disease conditions have in- dicated a certain parallelism l)et\veen their number and the tendency to coagulation observed in the various diseases (Welch). Howell I)elieves the platelets to be the chief source of thromboplastin, which neutralizes the antithrombin of the blood and tims causes clotting. Eordet and Delange consider the ])latelets of more importance than the leucocytes in producing participant* of the coagulat- ing mechanism. The histological evidence of the importance of the ])latelets in thrombus formation is conclusive (see Zurhelle, Derewenko), and Cramer and Pringle -« state that coagulation cannot occur without platelets. Kemp 29 con- cludes, from a thorough review of the subject, that the blood-platelets are visually normal or subnormal in number during acute infectious diseases, but increase rapidly if the disease terminates by crisis: in pernicious anemia the number is always greatly diminished, although in secondary anemias they may sometimes be increased; in purpura lurmorhagica the number of plates is enormously di- minished, which is perhaps related to the slowness of the clotting of the i)lood in this condition. Duke 3'^ states that when the i)latelet count falls below 10,(100 per cubic mm. there is delayed coagulation and a tendency to purpura : with counts above 40,000 tliere is usually no hemorrhagic tendency. If the platelet count is reduced artificially (by benzene, diphtheria toxin) a similar purpuric tendency is observed. Poisons that in large doses reduce the platelet count, will increase it if in small doses. Calcium Salts. — The exact significance of calcium in fibrin formation is still imsettled. Blood from which the calcium has been precipitated will not coagu- late, and the addition of calcium salts v'\\\ promptly cause it to do so. The vari- ous hypotheses advanced to explain the way in which calcium influences the clotting process are not in agreement. One hypothesis is tliat the calcium ions are necessary for the transformation of prothrombin into thrombin (Pekelharing, Hammarsten, IMorawitz), the thrombin consisting of a compoimd of prothrombin, calcium salts, and thrombokinase. Howell considers that no kinase is necessary, the calcium activating the prothrombin whenever it is not inhibited by anti- thrombin. Modification of Coagulability. — Another important matter for consideration is the etit'eet of various substanees in moclifyin<>- the rate or completeness of the coagulation of the blood. In the first place, we have the well-known fact that if blood is drawn into a glass vessel well coated with oil or vaseline, through a cannula similarly ]n-otected, no coagulation will take place ; but if any unoiled foreign stibstance enters, even particles of dust, coagulation begins at once. The ex- 20 Leo Loeb, Univ. of Penn. Med. Bull.. 1004 (16), .3S2 ; Muraschew. Deut \rch klin. Med., 1904 (SO). 187. 28 Quart. Jour. Exper. Physiol., 1913 (6), 1 29 Jour. Amer. ^Med. Assoc., in06 (46). 1022. 30 Jour. Exp. Med., 1011 (14). 265; Arch. Int. :\red., 1012 (10). 44.1: Jour Amer. Med. Assoc, 1915 (65), 1600. 318 DISTVRBAyCES OF CIRCULATION planation is that the leucocytes do not liberate their coagulating sub- stances until they have been injured by contact with some foreign body, and the experiment proves the importance of this action of the leucocytes, as well as explaining wliy the blood does not coagulate dur- ing life. The classical experiment of the ligation of a vein without injury' to the endothelium, which permits the blood to remain stag- nant for a long period without clotting, depends upon the same fact, namely, that normal endothelium neither liberates coagulin itself nor injures the leucocytes so that they disintegrate. Loeb recalls the observation of Overton that lipoids are important constituents of the cell membranes, and suggests a similarity between the vessel lining and the oiled cannula, but analyses of aortic endothelium have shown a rather low lipin content (8.41-9.25 per cent.), although peritoneal endothelium has much more (13 to 15 per cent.).^^ The suggestion that the vessel walls contain an anti-coagulin could not be confirmed by Loeb. Since leucocytes are constantly undergoing disintegration in the blood and tissues under normal conditions, it might be asked why they do not cause clotting then and there. In explanation Loeb ad- vances his observation that the coagulins are destroyed during cell autolysis, and suggests that when leucocytes normally disintegrate, the coagulins are first destroyed by autolysis. It has also been shown that the cells and serum contain substances which inhibit or prevent coagu- lation, and it is possible that these play an important part under nor- mal conditions in preventing coagulation by products of cell disintegra- tion, much as other antienzymes are supposed to act in preventing autodigestion of living cells. Coagulation of drawn blood may be retarded experimentally by re- moval of the calcium by precipitation as oxalate, fluoride, etc. ; also by diminishing the oxygen and increasing the COo, by addition of solu- tions of neutral salts in large amounts, l)y diluting greatly with water, or by keeping the blood cold. Coagulation may be hastened by moder- ate heat, by whipping, exposure to air, by contact with much foreign matter, and by the addition of watery extracts from many different tissues and organs. Poisons that destroy the ])latplets reduce the coagulation (Duke). Of particular interest i)ath<)l()gically is the re- tardation of coagulation that follows injections of proteoses (the so- called "peptone" solutions) and also various other protein-containing solutions, such as organ extracts, bacterial toxins, snake venoms, eel serum, extract of leeches or of Vneinarm, impure nucleo-protein solu- tions, or solutions of various colloids. ^lost of tlies(^ substances (>. g., peptone, eel serum) cause reduction of coagulability when injected into animals, and are without effect on blood removed from the body. A few, however, prevent coagulation of di-awn blood (snake venom, extract of leeches). When substances of the first class are injected in sufficient f(uantities, there occurs first a period of accelerated co- st Tait. Quart. .Iniir. Exj.. Physiol., ini.T (8), 301. COAGULATIOS OF THE ULOOD 319 agulation whieli may, particularly in the ease of org'au extracts, cause prompt death from intravascular clotting; if the animal sur- vives, there follows a period of decrease or total iiihil)ition of co- agulability of the blood, both within the vessels and after removal from the body. The first period of increased coagulability undoubt- edly depends upon the formation of a large amount of fibrin-ferment, but it has not yet been satisfactorily explained how the inhibition of coagulation is i)r()duced. Apparently the fibrin-ferment formed at first is rai)idly destroyed, but it is thought by some that it is con- verted into a substance that neutralizes the fibrin-ferment that may be formed later, or that a true anticoagulin is formed. It is also among the possibilities that all the available prothrombin or throm- bokinase is used up during the first stage of acceleration. As before ]nentioned, the blood and tissues contain substances that inhibit coagulation, and it may be that these are secreted in excessive amounts, a view which is receiving much support from recent observa- tions. According to Davis "'- injection of tlirombin is followed quickly by an increase in the amount of antithrombin in the blood. It has been found that in animals deprived of the liver no coagulation- inhibiting substances are formed in the blood after injection of pro- teoses, hence Delezenne believes that the substances of this class act by causing a destruction of leucoc3'tes, thus liberating a substance which increases coagulation and also another substance retarding co- agulation ; the first of these is destroyed by the liver, leaving the re- tarding substance to act unopposed."' Leech extract {hirudin) pre- vents clotting by means of an antiferment action, combining with the thrombin.''* Snake venom, however, acts upon the thrombokinase (Morawitz). Coagulability of the Blood in Disease. — In disease the alterations in the coagulability of the blood depend upon much the same factors. In all conditions associated with suppuration and leucocytosis the amount of fibrinogen is increased. This is especially true of pneu- monia.^^ The fluidity of the blood in septicemia is probably dependent upon the appearance of the coagulation-inhibiting pha.se that follows the action of the products of cell destruction, including among them proteoses. In this connection should be mentioned the observation of Conradi,^*^ who found that among the products of autolysis is a coagulation-inhibiting substance which is not destroyed by heat, dif- fuses readily, and in general behaves unlike the proteins. This or 32Amer. Jour. Physiol., 1011 (29), 160. 33 The manner in whieh gelatin injections affect tlie blood coaffulability is not yet understood (see Boggs, Deut. Arcli. klin. :\Ied., 1004 (70), 530) ; Moll (Wien. klin. Woch., 1003 (16), 1215) found an increase in fibrinogen. 3* Hirudin mav contain antikinase (Mellanby, Jour, of Phvsiol.. 1000 (38), 441). 35Dochez, Jour. Exp. Med., 1012 (16), 603. 36 Hofmeister's Beitr., 1901 (1), 137. 320 DISTURBANCES OF CIRCULATION similar substances may well play a part in affecting coagulation in infectious diseases, and Whipple ^' has found a decreased coagula- bility in sei)ticemia because of the })resence of an excess of anti- thrombin. It may also be mentioned that animals soon acquire an immunity against proteoses, so that their inhibiting influence is no longer shown. This corresponds to the observation of Kanthack ^^ that immune serum against venom, neutralizes very effectively the anticoagulating principle of venom ; an amount of antiserum alto- gether insufficient to neutralize the toxic properties of venom will neutralize its property of preventing clotting. The bacterial prod- ucts may also modify coagulation, and L. Loeb '" has found that different organisms are unequally effective in this respect, Staphylo- coccus aureus being much more powerful in causing coagulation than any others tested ; *° typhoid, diphtheria, tubercle, and xerosis bacilli and streptococci being without any apparent effect, while pyocyan- eus, prodigiosus. and colon bacilli occupy an intermediate position. Furthermore, after the organisms are killed by boiling, this effect is greatly reduced, showing that it does not depend merely upon the mechanical action of the bacteria, but probably upon bacterial prod- ucts contained in the culture-media. After phosphorus-poisoning the blood may become non-coagulable, which Jacoby *^ ascribed to an absence of fibrinogen in the blood, be- cause of a fibrinogen-destroying ferment in the liver. Doyon *- has made a similar finding in chloroform necrosis of the liver, but he at- tributes especial importance to an excess of antitlirombin liberated from the liver in these conditions. Whipple has also found a de- crease in fibrinogen with chloroform necrosis and cirrhosis of the liver.*-'' In other instances of decreased coagulability the fibrinogen is present, generally in normal amounts. After death the blood be- comes incoagulable because the fibrinogen is destroyed through a process similar to that of fibrinolysis; *^ this fibrinolysis may be com- plete as early as ten hours after death. The other proteins of the blood do not seem to be corresi)on(lingly attacked. Thrombokinase is also scanty in cadaver blood, but there seem to be no coagulation- inhibiting substances present. In anaphylactic shock the coagula- bility is reduced or abolished, associated wath which is a leucopenia.** 37 Arch. Int. Med.. 1012 (9), 305. -« Cited by La/.anis-Barlow, p. 141. 30 .Tour. Med. Hesearcli, \'M):\ (10), 407. 41 ]\Tueli (Biocliem. Zeit., 1!»0S (14), 14:5) states that stai>hyloeoeeus oontains tlirf)nit)()kinase. •»i Zeit. phvsiol. Chem., 1000 (30), IT;")-, also Doyoii rt a]., Compt. Bend. Soc. Biol., 1005 (58), 403. 42 Compt. Bend. Soc. Biol., 1005 (58), 704; .lour, piiys. et path., 101-2 (14), 229. 42a P.ull. .lohna Hopkins Ilosp., 1013 (24). 207. 43 Morawitz, Hofmeister's Beitr., lOOf, (8). 1. 44 The ineoagulaliility of menstrual blood is ascribed to a lack of lilirin ferment COAGULATION OF THE BLOOf) 321 Whipple ^ ' states that the antithrombin-prothrombin balance in the ■ blood is in delicate equilibrium, but preserved by strong factors of ^safety. The prothrombin factor is rarely involved, most notably in melena neonatorum and aplastic anemia, and such conditions may be relieved by injecting normal blood, through the added prothrombin. The antithroinhiu factor is often excessive in hemorrhagic conditions, especially with hepatic injury, or it may be lowered and lead to throm- bosis from relatively slight injuries. Obviously the injection of nor- mal blood will harm rather than help patients with hemorrhage due to excessive antithrnmbin. Antithrombin is often found increased in diseases of the blood-forming organs, e. g., leukemia, possibly as a reaction to the products of disintegration of corpuscles; and hence hemorrhagic tendencies are noted in these diseases. In icterus the notable tendency to hemorrhage seems to depend upon the binding of the calcium of the blood by the bile pigments,**' and administration of calcium may bring the coagulation time back to normal with a cor- responding decrease in the hemorrhagic tendency. PfeifPer ^' estimated the fibrin content of the blood in disease, and found it increased in diseases with leucocytosis (pneumonia, rheuma- tism, erysipelas, scarlet fever), except leukemia, where it was normal; in diseases without leucocytosis (typhoid, malaria, nephritis), the fibrin was normal in amount. Stassano and Billon *^ have, further- more, shown that the amount of fibrin-ferment varies directly with the number of leucocytes in the blood. Kollmann *'-^ found an increase in the fibrin of eclampsia, wdiich Lewinski ^° could not substantiate. In experimental infections of animals Langstein and ^Mayer ^^ found a specific increase in pneumococcus sepsis, which undoubtedly bears an important relation both to the characteristic fibrinous nature of the alveolar exudate in pneumonia, and the striking amount of fibrin found in pneumococcus pleuritis, peritonitis, etc. ^Mathews ^'- found an increase in the fibrin with all experimental suppurations. The coagulation time of the drawn blood has been the subject of much study by various methods,^^ but as yet very little agreement has been obtained. By different methods, in which different conditions for coagulation are presented, the normal coagulation time varies from 2 to 30 minutes ; with most methods it is 5 to 8 minutes. In general, by Bell (Jour. Path, and Bact., 1914 (18), 462) and to an excess of antitlirombin bv Dienst (Miinch. med. Woch., 1912 (51), 2709). 45 Arch. Int. Med.. 1913 (12), 037. 46 Lee and Vincent, Arch. Int. Med., 1915 (16), 59. .47Zeit. klin. Med.. 1897 (33), 214: Cent. f. inn. Med., 1S9S (19). 1. 48Compt. Rend; Soc. Riol., 1903 (55), 511. 49 Cent. f. Gvniik., 1897 (21). .341. sopfliitrer's Arch., 1903 (100), 611. 51 Hofmeister's Beitr.. 1903 (5), 69. 52 Amer. Jour. Physiol.. 1899 (3), 53. 53 Full review and bibliography by Cohen, Arch. Int. Med., 1911 (8), 684 and 820. 21 322 DISTUh'BAXCES OF CIRCULATION coagulability is not constantly if at all alteral by fever, cancer, dia- betes, slight secondary anemias, or many other diseases, and in nor- mal conditions it remains fairly constant. In infants the coagulation time is slightly shorter than in adults. The coagulation is hastened after considei-able hemorrhages, in endocarditis, and perhaps in aneu- rism and thrombosis ; and is commonl}- delaj'ed in the acute exan- themata, in hemophilia, in purpura neonatorum, and occasionally in some other diseases.'* There is entire lack of agreement concerning the reputed acceleration of coag'ulation by oral administration of cal- cium salts, and retardation by citrates; and the supposed thrombo- plastic influence of gelatin cannot be shown consistently by direct ob- servations. In jaundice, calcium salts probably have an effect, since here the cause of the deficient coagulation seems to be the fixation or precipitation of the blood calcium by the bile pigments. It seems probable that the measurement of the time required for coagulation to take place in vitro does not exactly represent the tendency of the same blood to coagulate in the body of the person from whom it is obtained ; for example, the injection of foreign serum has a notable effect in stop- ping hemorrhages, but the coagulation time of the recipient's blood is not correspondingly altered. Whipple's observations that with a low fibrinogen content the blood may coagulate in normal time, and yet the clots be too delicate to stop hemorrhage, explains at least part of the discrepancy; and of similar significance is the fact that with a very low platelet count the blood may coagulate a.s rapidly as normal, but the clots do not shrink and become firm (Duke). Hence with a se- vere purpura hemorrhagica we may have a normal clotting time. In other conditions with normal coagulability, hemorrhages may re- sult from excessive fibrinolysis which causes solution of the clot, espe- cially in hepatic diseases.'^'*^ THE FORMATION OF THROMBI If we apply the facts brought out in the precetling discussion rela- tive to the factors in the coagulation of blood, to the manner and conditions under which thrombi are formed in the circulating blood, we find explanations for many of the features of thrombosis. Welch '^ describes the steps in the formation of a thrombus after injury to the vessel-wall, as follows: First, there is an accumulation of blood- platelets adhering to the wall at the point of injury. Leucocytes, 54 See Dochez, (.Jour. Exp. MckI., 1912 (16), 69:?), wlio found s.mic delay in coagulation in pneumonia. Corroborated by Minot and l.cc, .lour. Aiiur. ^led. Assof.. 1917 (6S), 545. 5411 See (ioodpasture. Bull. .Johns Hopkins Hosj)., 1914 (25). :?30. •>^> Albutt's System, vol. (!, complete (liscusaion of the {general featuri's of throm- bosis; also see Kiister, P^rpeb. inn. IMed., 1913 (12), 667; Zurhelle, Ziejrler's Beitriifie, 1910 (47), 5.39; Sdiwalbe, Krfjebnisse Pathol. . 1907 (XI (2) ). 901; I..ubar8ch, All-2<>. ns Tliis subject is fully discussed by Leonard Hill in ■rvccciit Advances in Physiolof^v and Biocliemistrv," T»ndon, 190G. eoAmer. Jour. Med. Sci., 'lOlO (130), 373; Erdman. ibid., V^\^^ (145). friO. jyFAncTiox 327 (luring- dec()uii)ivssion.'" At body temperature fats can dissolve five times as much nitrogen as serum or plasma/^ which probably accounts for the severity of the changes in the nervous system with its rich lipoid content and delicate structure. Air embolism following obstet- rical operations oi- surgical operations about the neck and chest pre- sents chiefly mechanical features,'- and large (luantities of air nuist be present to cause dangerous obstruction to circuhition.'^ Gas-bubbles may be produced in the blood soon after death by B. aerogenes cap- sulatus, but it is not probable that they are formed before death and cause air embolism. INFARCTION The changes that occur in infarcted. areas are of much interest in connection with the study of autolysis, for the absoi^ption of the ne- crotic tissue of aseptic infarcts is purely a matter of autolysis. Ja- coby '* found by ligating off a portion of a dog's liver, and keeping the dog alive for some time afterward, that in the infarcted tissues so produced leucine and tyrosine could be detected, just as they are found in liver tissue undergoing autolysis outside of the bodj'. So, too, proteoses may appear in the urine when any considerable amount of tissue is cut off from its blood-supply. The processes of autolysis which occur in ordinary sterile infarcts are, however, extremely slow, and it is doubtful if enough of the products are ever in the blood or urine at any one time to be detected or to cause noticeable effects. For example, in an infarct of the kidney which was known to be al- most exactly fourteen weeks old, there still remained a la.yer of ne- crotic cortex one millimeter thick, quite unabsorbed during this time. If we examine such aseptic infarcts in various stages, we get the im- pression that the digestion is accomplished by leucocytes acting on the j;eri])hery of the infarct, and not entering the dead area deeply, pre- sumably because of a lack of cheraotactic substances in the dead cells. On the other hand, it seems probable that the tissue enzymes them- selves play an important part in the autolysis, for if we implant into inimals pieces of tissue in which the enzymes have been destroyed by heating to boiling, it will be found that the cells and their nuclei re- main unaffected for man}' weeks; whereas if sterile unheated pieces of tissue in which the enzymes are still active are implanted, they lose their nuclear stain and begin to disintegrate relatively soon, without apparent participation by the leucocytes."'' Ribbert '*' found that in experimentally produced anemic infarcts in the kidneys of rabbits the nuclei retain their staining property well for nearly twenty-four hours^ -oHill and Greenwood, Proc. Roval Soc. (B). 1907 (79), 284. 71 Vernon, i1>id., p. Sfifi: Quincke", Arcli exp. Path. u. Pharni.. 1910 (02). 404. "2 Review of literature by Wolff, Virchow'.s Archiv., 190,3 (174). 454. T3 See Hare, Amer. Jour.' :\rod. Sciences, 1902 (124), 84:5. ■^Zeit. phvsiol. Chem., 1900 (.30). 149. T3 Wells, Jour. :Med. Research. 1906 (15), 149. ToVirchow's Arch., 1899 (155), 201. 328 DISTURBAXCES OF CIRCULATIOy becoming tlieii small and deeply stained, undergoing karyorrhexis^ and in large part disappearing from the convoluted tubules inside of forty-eight hours, although some nuclei may persist in the glomerules- for three or more days. In human infarcts, Rihbert believes, the process goes on faster, for he has observed here a loss of nuclei within twenty-four hours. These nuclear changes undoubtedly depend upon, autolysis, but it is probable that the enzymes concerned reside in the cytoplasm rather than in tlie nucleus, for I have observed that cells of lymphoid type, with practically no cytoplasm, generally retain their nuclear stain much longer than cells with more cytoplasm; this- is particularly noticeable in splenic infarcts, where the ]\Ialpighian corpuscles retain their staining affinities much longer than the pulp elements. AVhether the destruction of the nuclei is accomplished by the ordinary intracellular proteases, or by special nucleoprotein- splitting enzymes (nuclease, ^^ etc.), remains to be determined. It is quite possible, however, that the first changes consist of a splitting of the nucleoproteins of the nucleus by the autolytic enzymes, liber- ating the nucleic acid, which gives the nuclei the characteristic intense- staining with basic dyes (pycnosis) observed in areas of early anemic necrosis. The nucleic acid may then be further decomposed by the nuclease or similar enzymes. Taken all together, then, it would seem that the nuclear and cellular alterations that make up the character- istic picture of anemic necrosis are brought about by the intracellular enzj^mes — an autolytic process. The removal of the dead substance, however, seems to be accomplished rather by the invading leucocytes, through heterolysis. The relatively small part taken by the intracel- lular enzymes may possibly be due to the seeping through them of alka- line blood-plasma, for autolytic enzymes are not active in an alkaline medium; the leucocytic enzymes, however, act best in an alkaline medium.'^^ About the periphery of infarcts is usually observed more or less fat deposition (Fischler),'^'' particularly in the endothelial cells (Ribbert). This is not peculiar to infarcts, however, for Sata ^" found a similar peripheral fatty metamorphosis common to all necrotic areas. The basis of this is possibly the persistence of the cell lipase, which syn- thesizes fatty acid and glycerol dififusing into the necrotic area with the plasma, unchecked by the normal oxidative destruction of these substances. (See "Fatty Degeneration," Chap, xiv.) Hemorrhagic infarcts offer, in addition to the changes common to anemic infarcts, the alterations ocQurring in the blood-corpuscles. Panski ^^ found that after ligation of the splenic vein of dogs the red T7 Sachs, Zoit. physiol. Cliem., lOUu (4C), 337; Schiltonliclm. ibid.. 3.")4. 78 More fullv discusapd bv WoUs.ts Joe. cit., and under necrosis. Cluip. xiii. -"Cent. f. Patlt., 1002 (13), 417. «oZiepler'8 Beitr., 1000 (2R), 4()1. SI "Untcrsucliuii^^-n iilicr den I'i^niieiil j^clialt der Stamingsmilz," Dor|)at. 1800. INFAUCTIOX 329 corpuscles begin to give up their heuioglobiu in about tliree hours. After twelve hours fibrin formation begins in the tissues, the corpus- cles continue to give up hemoglobin and become cloudy in appearance. Later, iron-containing pigment is formed in the cells beneatli the cap- sule, but in the deeper tissue even the iron normally present in the spleen tissue seems to disappear;®- this possibly depends upon the fact that pigment reacting for iron, hemosiderin, is formed only in living colls under the influence of oxygen, or it may be that acids formed during autolysis dissolve it. During autolysis iji vitro, how- ever, Corper "*•' found no evidence of removal of iron from insoluble or coagulable compounds. The hemolysis is probably produced either by the action of autolytic products, which are notoriously hemolytic, or perhaps also by direct attack of tissue and blood proteases upon the corpuscles. Other retrogressive changes that may occur in infarcts, such as sep- tic softening and calcification, are not greatly different from the same processes occurring in other conditions, and will be considered with the discussion of these processes. s2See also M. B. Schmidt, Cent. f. Path., 1908 (19), 416. S3 Jour. Exper. Med., 1912 (15), 429. CHAPTER XII EDEMA 1 As the term edema indicates the excessive accumuhitioii of lymph (which may be either normal or modified in composition ) in the cells, intercellular spaces, or serous cavities of the body, the problems of edema are inseparably connected Avith the consideration of the proc- esses of physiological formation and removal of lymph. For many, years the study of these processes has been a favorite field of investi- gation by physiologists, and the great battle-place of the "vitalistic" and "mechanistic" schools; and to this day the forces that determine the formation of lymph and its subsequent absorption have not been completely understood. By the application of the principles of phys- ical chemistry to the problem, however, great advances have recently ])een made, which seem to render our understanding of both lymph- formation and its pathological accumulation in the tissues much clearer and more nearly accurate than they were before. AVe shall first consider, therefore, the physiological formation of lymph, before taking up the subject of edema. Composition of Lymph. — Lymph consists of material derived from two chief sources. The greater part consists of fluid passing out of tlie capiUaries into the tissue spaces: here it is modified by the addition of products of metabolism derived from the tissue-cells, and by the sul)traction of materials tliat the cells utilize in their metabolism. It is, therefore, essentially a modified blood plasma, and the modifications the plasma undergoes are so slight tliat. under ordinary conditions, lymph shows on analysis no considerable differences from blood plasma, except a relative poverty in proteins, due chiefly to the impermeability of the capillary walls for colloids. Its quantitative composition varies greatly, depending upon the conditions under which it is collected, whether during activity or rest, etc. Tiie following tables of analyses have been collected by Ilammarsten: 1 2 .*? 4 Water 0.30.0 0.34.S 057.6 0.55.4 Solids 00.1 05.2 42.4 44.6 0.4 2.2 Fibrin 0.5 0.0 Albumin 42.7 42.8 Fat, Cholesterol, Lecithin . .S.S 0.2 Extractive bodies . 5.7 4.4 Salts 7.3 S.2 .34.7 35.0 1 and 2 are analyses of lymph from tlie tliigli of a woman. .'! is from tlic contents of sac-like dilated vessels of the spermatic cord, 4 is lymph from the neck of a colt. lA complete bibliograpliy is given by Afeltzer. .\merican l\redicine, 1004 (S), 10 et seq. ; also bv Klemeiisiewicz, in Krehl and Marchand's Ihuullmeli d. allg. Path., 1012, II (1), 341: ]\Iagnus. TTandlmeh d. Hiochem.. 1!)(1S, 11 |2), 90; Gorhartz, ihid., ]>. 11(5. 330 Foinf.iTJox or L) Mi'if 331 Cliyle difl'ers from lym]ili chit'lly in tlie prosem-t' of larjic (juantities of fat; (hiring starvatiim the lymj)li and tiie t-liyle arc of jiractically tlie same composi- tion. Normal lymph contains much less fibrinogen tlian docs tlic hlocvd plasma, and hence coagulates slowly. Lipase and oilier enzymes ha\-e lieen found in the lymph, as in the plasma. 'I'lie products of tissue metabolism added to the lymph by the cells may render it toxic (Asher and Barbera^). I'nder patii- ological conditions llie lym])h may l)e greatly altered, liecoming jxwrer in solids under some conditions of edema, and bee nning rich in proteins and l)loo(l-cor- puscles under intlammatory conditions, luitil it ])artakes of tlie characteristics of an inflammator}- exudate (see analyses of transudates and exudates). An important fact to consider is, that of the entire water of the body hut about one-tenth is in the blood. About two-thirds of the entire weight of the body is water, which is mostly in the cells and tissues, firmly bound by the colloids, only an unknown but smaller portion being as free movable fluid, and even here always associated with more or less colloid. A body weighing 60 kilos will, therefore, have 40 kilos of water, of which but about 4 kilos is blood. FORMATION OF LYMPHS Filtration Theory. — The simplest possible conception of lymph foruuition is that it is merely the result of filtration of the liquid con- stituents of the blood through the capillary walls under the influence of the blood pressure. This "filtration theory" was supported origi- nally by Ludwig, and it w^as a prominent factor in the early appli- cations of mechanical principles to biological processes. In support of this theory were advanced the results of numerous experiments in which it was shown that increasing the blood pressure by means of ligating the veins, or by causing arterial dilatation, resulted in an in- crease of the lymph flowing out of the lymph-vessels of the part. Also, when the blood pressure is raised by epinephrine or by other means, a large proportion of the fluid leaves the blood vessels; con- versely, M^hen the blood pressure is suddenly lowered by hemorrhage there is a rapid passage of fluid from the tissues into the blood. The experimental results were not always favorable to the theory, how- ever, particularly in the experiments in which blood pressure was raised by arterial dilatation ; often the flow of lymph was little in- creased, even w^hen the arterial flow and pressure were greatly in- creased. Nevertheless, the filtration theory held for many years, not only as an explanation of lymph formation, but also as an explanation of urinary secretion and of the secretion by other organs. It w^as only within a comparatively short time that it became clear that filtra- t^'on alone ccmld not account for all the phenomena of secretion. For example, in many lower forms with undeveloped circulatory systems, and almost no blood pressure, secretion goes on vigorously ; the pres- sure of glandular secretions may be much higher than the blood 2Zeit. f. Biol.. 1808 (36), 1.54. 3 See review by Asher, Biochem. Centralblalt. 1005 (4), 1. 332 EDEMA pressure within the capillaries ; the activity of secretion is by no means in proportion to blood pressure, etc. If in *^landular' secretion, there- fore, fluids are removed from the blood and transferred into an ex- cretory duct through the action of some force other than that of the blood pressure, it is probable that lym])h formation is eciually independent of blood pressure. On this basis Ileidenliain advanced his — Secretory theory of lymph formation, in which he suggested that lymph is the product of an active secretion by the endothelial cells of the capillaries, just as saliva is the product of the activity of the glandular cells. He showed that certain chemical substances may stimulate lymph flow, independent of blood pressure, just as pilocar- pine and other drugs may stimulate the secretion of saliva. These hmiph-stimulating substances, which he named lyinphagogues, fall into two distinct classes. One which includes such substances as peptone, leech extract, strawberry juice, extracts of crayfish, mussel or oysters, and numerous other tissue extracts, are characterized by causing the secretion of a lymph which is rich in proteins, even richer in proteins than the blood plasma; and, furthermore, there is no simultaneous increase in urinary secretion. Heidenhain considered that these sub- stances caused lymph secretion by stimulating the capillary endothe- lium in a specific manner ; as they caused no appreciable rise in blood pressure the increased lymph secretion certainly could not be attrib- uted to filtration. This independence of the lymph flow of blood pressure is most conclusively shown by postmortem lymph secretion; for example, Mendel and Hooker * observed lymph flow for four hours after death, in a dog that had received an injection of peptone eight minutes before being killed.^ The second class of lymphag-ogues includes crystalloidal substances, such as sugar, urea, and salts. ^'^ Lymph secreted under the influence of these substances is poorer in protein than ordinary lymph, and at the same time an increased urinary secretion is produced. With these crystalloidal lymphagogues the amount of effect is in inverse proportion to their molecular M^eight, which means that their effects depend upon the number of molecules in solution rather than upon their nature ; in other words, the stimulation of lymph by crystalloids is dependent upon the osmotic pressure of the crystalloids. Heiden- hain explained their action as follows: The crystalloids are secreted into the lymph-spaces by the action of the capillary endothelium, and there, owing to their raising osmotic pressure, cause a flowing of water out of the vessels. The difificulfy here is to explain why the 4Anier. Jour, of Physiol., 1902 (7), 380. OA fact not sufTiciently takon into account is that blisters filled witli scrum, i. e., an inflanimatorv odoma, mav be ])rodticcd in dead bodies bv burns or scalds. (See Leers and Kaysky. Virclio\v''s Arch., IDOO (197), .324). 5a The action of many other substances lias In-en invest ipatcnl by Vanaj^awa, Jour. Pharmacol., 1916 "(9), 75. FORMATION OF JA Wff'H 333 crystalloids while still in the vessels do not attract the fluids from the lymph-spaces into the blood, and so canse rather a lessened lymph secretion. "Wliile admitting- that in pathological conditions (e. g., passive con- j^estion) pressure and filtration may play an important part, Heiden- haiu considered that an active secretion by the endothelial cells is the chief factor in the normal formation of lymph. The means by which the cells perform this function was unknown ; it was considered as an example of "vital activity," Ileidenhain meaning by this term such chemical and physical forces of living cells as are unliJiown or not understood at the present time, rather than any metaphysical concep- tion of living matter, such as many vitalists assume. Other observers, corroborating Heidenhain's results for the most part, liave modified, or amplified his theory. Asher and his collabo- rators, for example, ascribe the work done in causing lymph forma- tion to the cells of the various tissues and organs, rather than to those of the capillary wall. The increased flow of lymph from the salivary gland that occurs during its activity they consider due to the work of the gland cells, and its function the removal of products of metab- olism. The action of such a lymphagogue as peptone they ascribe to its stimulation of cellular activity, particularly in the liver, where it causes an increased formation of bile. Gies *' and Asher also ob- served that after an injection of crystalloidal lymphagogues, such as sugar, a prolonged flow of lymph occurred after the death of the animal, proving completely that such lymphagogic action is inde- pendent of blood pressure. Potocytosis. — In explanation of the process l)y which the cells, whether en- dothelial or tissue-cells, pass fluids throuph themselves from one place to another, Meltzer 1 has made an interesting suggestion, as follows: Considering the prop- erty of endothelial cells to act as phagocytes, MacCallum " has shown tliat solid granules (e. f/., coal pigment, carmin) are taken throujrh the walls of the lymphat- ics by the phagocytic activity of their endothelial cells. ]\Ieltzer suggests tliat in a similar way the endothelial cells may transport through the vessel-walls not only solid particles, ])ut also, by the same mechanism, substances in solution; and for this hypothetical process he suggests tlie name "potoci/tosis." There can be little question that cells do take up substances in solution, and sometimes this is done in an apparently selective manner; e. g., the taking up of bacterial toxins and vegetable poisons in the peritoneal cavity by the leucocytes. Presumably the mechanism of "potocytosis" is not different from that of phagocytosis, chemo- tactic forces determining the occurrence of tlie process. Xo experimental evi- dence has been advanced as yet for this very plausible hypothesis. Permeability of Capillaries. — In explanation of the variability in the amount and composition of the lymph. Starling ^ has introduced the factor of altered permeability of the capillary walls, which pre- sumably depends upon the number and size of the pores. He found that normally the lymph coming from the lower extremities contains sAmer. Jour. Physiol.. IflOO (.3), p. xix: Zeit. f. Biol.. 1000 UO), 207. 7 Johns Hopkins Hosp. Bull., 1903 (14), 1 s Lancet, 1896 (i), Jlay 9, et seq.; Sch:i,fer's Text-book of Physiology, vol. 1. 334 EDEMA only 2 per cent, to 3 per eent. of proteins, while lymph from the intes- tines contains 4 per eent. to 6 per eent., and lymph from the liver eon- tains 6 per cent, to 8 per eent. of proteins ; hence he considers that the liver capillaries are the most permeable, i. e., have the largest pores, so that more of the laroe colloid molecules can escape from them. The effect of lymphafi'oo'ues of the first class (peptones, etc.) he attributes to their poisonous properties, and the consetiuent injury to, and alter- ations in, the capillary wall. The crystalloidal lymphagogues, he believes, act by first attracting fluids from the tissues into the blood with a resulting ''hydremic plethora," which in turn leads to in- creased blood pressure and consequent filtration of a watery fluid out of the vessels. He considers, therefore, that the amount and qii_ality of the IxiBph produced in any part are determined solely by two factors, the intracapillary;J)lood_:pjceasm:e and the permeability ^f_ the capillary wjiJjs. In connection with this question of the permeability of the capil- lary walls, Meltzer suggests that the contractility and irritability of the endothelium may be a potent factor in determining the size of the pores in the capillary walls. When in a tonic condition, the endothe- lium is firmly contracted about the pores, keeping their size small ; when the endothelial cells become relaxed by any cause, such as poi- sons, high blood pressure, poor nourishment, etc., the pores are en- larged, and increased escape of fluids results. It must be borne in mind, however, that most histologists do not now admit that capillary walls contain pores. M. H. Fischer holds that the endothelial cells undergo changes in consistency through changes in the affinity of the cell colloids for wa- ter; especially under the influence of acids the endothelium may be- come much more fluid and of greater permeability. Adolf Oswald " says that the normal capillary wall is somewhat permeable for the less viscous blood proteins (albumin and pseudoglobulin), and in in- flammation this permeability becomes increased so that the more vis- cous euglobulin and fibrinogen can pass through. Osmotic Pressure. — Still another possible factor in causing fluid to leave the vessels is osmotic pressure. Heidenhain refers to this cause the transudation produced by crystalloid lymphagogues, al- though in a rather unsatisfactory manner. As a result of the more recent studies of physical chemistry, and its application to biological processes, vce have learned to appreciate the importance of osmotic pressure in cell activities (see Introductory Chapter), and in the question of lymph formation it occupies a ])articularly important ])lace. AVe may consider it as follows: In the blood we have certain proportions of readily diffusible crystalloids and of non-diffusible colloids. If no metabolic processes were going on in the tissues, we should have the diffusible substances leaving the vessel-walls (leaving » Zcit. f. oxp. Patli.. 1010 (S), 22(). FORM AT/OX OF JAM I'll 335 out, for tile j)rc.sent, any question of activity on the part of the endo- Tlielium) until an osmotic equilibrium is established in the tissues and in the blood. As a matter of fact, however, the blood proteins are not absolutely non-diifusible, but small quantities do pass through the cap- illary walls, and so lymph under such a hypothetical condition would consist of a mixture of the same osmotic concentration as the blood plasma, with about the same proportion of crystalloids, but a smaller proportion of proteins ; this, it will be noticed, is just about the com- position of normal lymph. During life, however, the cells of the tis- sues are causing metabolic changes in these lym])hatic constituents, and these changes consist chiefly in breaking down large molecules of proteins, carbohydrates, and fats into much smaller molecules. Now the osmotic pressure of a solution is dependent upon the numher of molecules and ions it contains, hence by breaking down these few large molecules with verj^ little osmotic pressure into many small mol- ecules, the osmotic pressure in these cells and tissues becomes raised above that of the blood-vessels, and consequently water flows out of the vessels because of the increased pressure. We see here the prob- able explanation of the stimulating influence of metabolic products upon the formation of lymph, noted by Hamburger, Heidenhain, and others. For suggesting and urging the importance of osmotic pres- sure in the formation of lymph we are indebted particularly to Hei- denhain, V. Koranyi,^" J. Loeb,^^ and Roth.^- Loeb show^s very clearly the relative greatness of the water-driving force of osmotic pressure as compared to that of blood-pressure, by his statement that the os- motic pressure of a physiological salt solution is about 4.9 atmospheres, which is twenty ti)nes as great as the hJood jjressure with which we have to do in ordinary physiological experiments. In varying osmotic conditions we may readily see an explanation for the increased lymph flow that occurs during tissue activity ; namely, it is due to the in- creased formation of metabolic products. ]Many of the lymphagogues may act similarly by stimulating metabolic activity, with resulting in- crease in the formation of osmotic pressure-raising products of metab- olism in the organs ; e. g., the increased lymph flow from the thoracic duct that follows stimulation of hepatic activity by injection of pep- tone (Heidenhain) or ammonium tartrate (Asher and Busch).^^ As we shall see later in considering edema, osmotic pressure may play an important part in the pathological formation of lymph. It must be admitted, however, that there are many difficulties in the way of accepting unqualifiedly the original views as to the importance of osmotic pressure in lymph formation. For example, the lymph con- tains more chlorides and may have a much higher osmotic pressure loZeit. f. klin. Med.. 1807 (33), 1: 1898 (34), 1. iiPfliicrer's Arch., 1898 (71), 457. 12 Englemann's Arch., 1899, p. 416. i3Zeit. f. Biol., 190O (40), 333. 336 EDEMA than the serum of the same animal (Hamburger, Carlson, et al.) }^^ Variable Capacity of Colloids for Water. — Colloids of the type of the tissue proteins, i. c, liydrophil colloids, imbibe water with great avid- ity, until a certain proportion of water is present, the proportion varying under different conditions. The importance of tliis force in the production of edema and related processes was first pointed out by Martin H. Fischer, and has been developed extensively by him." The amount of water which a given hydrophil colloid, such, for exam- ple, as gelatin, or fibrin, will take up, is greatly modified by the reac- tion of the solution and by its content of electrolytes. Very small concentrations of acids or alkalies will greatly increase the amount of water absorbed, while salts reduce it, and the different acids and salts vary in their effects; thus hydrochloric acid causes a greater swelling of colloids than a corresponding strength of sulphuric acid, and calcium chloride depresses the swelling more than potassium chloride. The effect of the salts is made up of their constituent ions. Non-electrolytes have relatively little effect. The forces developed by this affinity of colloids for water are enormous ; thus, to prevent the taking up of water by starch requires a pressure of over 2500 at- mospheres, and dried gelatin will take up 25 times its weight of water, and fibrin as much as forty times. Different colloids differ greatly in their affinity for water, and in the way in which this affinity is mod- ified by electrolytes, and change in a colloid may greatly alter its ca- pacity for swelling; thus, ;8-gelatin, which can be formed from ordi- nary gelatin by the action of proteolytic enzymes, has greater capacity for swelling than the original gelatin. Gies especially lays stress on this factor, that is, the alterations of the hydrophilic tendencies of the tissue colk)ids by enzymes. ^^ On the basis of the facts briefly summarized above, the proportion of water present in any cell or in any fluid of the body which contains colloids, is assumed to be determined by certain factors, namely (1) the cliaracter of the colloids themselves; (2) the proportion and na- ture of acids or alkalies present in the fluids in and about the colloids ; (3) the proportion and nature of the salts. All these factors are changeable, and tlierefore the amount of water present in the cell or fluid varies accordingly. Thus, if a cell through its metabolism de- velops from such a non-electrolyte as sugar (which has no consider- able effect on the water content of the protoplasm), an organic acid, i3aAmer. Jour. Physiol., in07 (10), .-^eO: 1008 (22), 01. 1* See Fischer's INlonopraph, "Oedema and Nepliritis."' New York. 1015; also numerous articles in tlie Zeit. f. Chem. u. Ind. d. Kolloide. An especially thor- ou^'h discussion of this theory is contained in the biochemical l^villctin. Vol. T., pivinj^ a bihliofijraphy of Fischer's work, together with articles on Gies' observa- tions on the modification of the hydrophilic tendency of proteins by enzyme action. 1' A definite and clear-cut example of tlu^ swelling' of a tissue under the in- fluence of acid of metabolic orifjin is shown in the muscle cell in Zenker's waxy degeneration (Wells, Jour. Exper. Med., 1000 (11), 1). FORMATION OF JAMI'JI 337 such as lactic acid, which has a large effect in increasing the affinity of tile colloids for water, the cell will, presumably, take on more water, perhaps to a degree to cause intracellular edema. The acids diffusing from the cell into the intercellular spaces or into the lymph will cause equally well an increased affinity for water in the colloids here present, leatling to intercellular edema. Conversely, neutralization of acids present in a colloidal solution, by alkaline salts brought by the blood, will decrease the affinity of the colloids for water which will escape from the colloids as they shrink. This theory, which introduces a hitherto unappreciated factor into the considerations of lymph formation and edema, is of the utmost importance. It practically eliminates osmotic pressure, also the cell membranes so essential for the efficiency of this force, and in view of the difficulties that have arisen in trying to fit the cell membrane hypothesis and osmotic pressure to many facts of normal and patho- logical biology, an alternative hypothesis is welcome. As pointed out above, the forces involved in the swelling of colloids are so large as to be of great significance, and the amounts of electrolytes necessary to cause considerable variations in colloidal swelling are not more than can be present under normal and pathological conditions; conse- quently the possible influence of colloidal swelling must be taken into account in all consideration of pathological processes. Whether or not it is capable of as universal application as Fischer maintains, remains to be demonstrated, and there are, indeed, some facts that do not seem to be in harmony with this theory. Summary. — We see from the above discussion that numerous the- ories have been advanced to explain the normal formation of lymph, and as their basis exist several different possible factors. Filtration, active secretion by the capillary endothelium, attraction by the tissue- cells, osmosis in response to formation of crystalloids outside the ves- sels, and changes in the affinity of colloids for water; all have been shown to be possible causes of lymph formation. It is highly prob- able that in a certain way all are involved, particularly if we accept the view of the physical school that "secretion" and "attraction" by the cells are merely the outcome of physical forces ; the causes of lymph formation then reduce themselves to absorption, filtration and diffusion. There has been, until recenth-, no question but that lymph does escape from the vessels through simple filtration, for the pressure inside the capillaries is presumably greater than outside, the capil- lary walls are not water-tight, and the}^ are not impermeable to the substances dissolved in the plasma.^' Likewise osmotic exchanges 17 Hill ("Recent Advances in Physiolo^iy and Biochemistry," 1000, p. GIS) dis- putes tlie possibility of such a thing as filtration pressure, on the n#ler altered capillary permeability as an essential factor in edema^^ characterized by protein-rich fluids (exudates), and iglc'ffe^that'.tlie influence of al- tered permeability in the producti^* of protein-poor fluids (trans- udates) is not proved, and is perhaps not of importance, although the evidence of recent studies on- experimental nephritis seems to 344 EDEMA point more and more to the importance of vascular chang:es in acute nephritis, at least.-" 5. Increased Filterability of the Blood Plasma. — This takes us back to Richard Bright 's conception of renal dropsy. He im- agined that through the great loss of albumin in the urine the blood became so thinned and watery that it could filter through the vessel- walls, while normal plasma, he thought, was too thick and viscid to do so. The same idea was applied to the edemas of cachexia in cancer, etc., chlorosis, and all forms of edema associated with a decrease in the corpuscular or protein elements of the blood. With our present knowledge of diffusion of crystalloids and colloids we can readily ap- preciate that a decrease in the blood colloids, such as might occur in these diseases, could not modify the passage of fluids through the capillary walls to any considerable degree. Stewart and Bartels con- sidered that in renal dropsy the increased filterability of the plasma was not due so much to the loss in albumin as to retention of water, which caused an hydremic plethora. But this factor was soon elimi- nated, for it was found that complete anuria, produced by ligating both ureters, does not cause edema ; and also that to produce an edema by increasing the water of the blood it was necessary to increase it many times as much as it can ever be increased by disease. Simpl}^ increasing the proportion of water by removing part of the blood and injecting a corresponding amount of salt solution did not cause edema iCohnheim and Lichtheim). We may, therefore, look upon the hy- pothesis of increased filterability of the blood as chiefly of historic interest, and not an important factor in the causation of edema. In the presence of other factors for the production of edema, however, the amount of fluid in the vessels is important; thus Pearce -' found that in experimental uranium nephritis hydremia exerted a marked in- fluence on the production of edema. 6. Disparity of Osmotic Pressure in Favor of the Tissues and Lymph over the Blood. — On a preceding page we have already considered the means by which changes in osmotic pressure in the tis- sues are brought about, and how they may lead to an accumulation of fluid. The importance of osmotic pressure in causing pathological edema was suggested by J. Loeb -^ in his studies on the physiological action of ions. He stated that edema occurred when the osmotic pressure was higher in the tissues than it was in the blood and lympli, and the cause was to be sought in conditions that lowered the osmotic pressure of the blood and lympli or raised that of the tissues. This condition he found in the accumulation of metabolic products : — in the case of muscle, totanization of a fi-og's muscle for ten minutes raised the osmotic pressure over one atmosphere ; separating a muscle 26 See Schmid and Sdilavcr, Dent. Arch. klin. Med., 1011 (104). 44. 27 Arch. Int. Med., 1908 "(3), 422. 28Pfluger's Arch., 1898 (71), 457. THE CAUSES OF EDEMA 345 from its l)l()()(l-supi)ly led to sueli an increase in osmotic pressure tliat it took uj) water from a 4.!) per cent. NaCl solution, wliich lias a j)res- sure of over thirty atmospheres. AVhen we consider that in his studies on lung edema Welch was able by ligation of the aorta to raise the blood pressure less than ^^o atmosphere, we begin to appreciate how much more powerful are the physico-chemical forces that are at work in the body than is the blood pressure, even of the aorta itself. ^ Loeb found that whenever oxidation is impaired in a tissue its osmotic pressure rises, which he ascribed to the accumulation of in- completely oxidized metabolic products, particularly acids, and as a result the muscle takes up water and becomes edematous. On this basis we might explain the edema of venous stagnation as due to ac- cumulation of products of metabolism, partly because of impaired oxidation, partly, perhaps, because of their slow removal in the blood on account of the circulatory disturbance. The so-called ''neurotic" edemas may possibly be explained by local increase in metabolic ac- tivity brought about by nervous stimuli, which causes increased forma- tion of substances raising osmotic pressure in the stimulated tissues. In renal edema the retention of water also seems to depend rather on osmotic pressure than on circulatory disturbances or alterations in the vessel- walls, for it has been shown that retention of chlorides, which the diseased kidneys do not eliminate normally, is an important cause of the dropsy in some cases. The chlorides accumulating in the tissues lead to an increased osmotic pressure, which causes the abstraction of water from the blood and its retention in the tissues. (The details of this subject will be considered later.) Convereely, Meltzer and Salant found that salt solution is absorbed from the peritoneal cavity more rapidly in nephrectomized rabbits than in normal rabbits, because metabolic products accumulate in the blood and raise its osmotic pres- sure above normal ; and it was observed by Fleisher and L. Loeb ^^ that the rate of absorption of fluid from the peritoneal cavity is in- creased when the osmotic pressure of the blood is raised. There are some difficulties, however, in applying the influence of osmotic pressure as an explanation of all edemas. For example, in edema of the lungs, as Meltzer points out, what is the force that drives the fluid into the empty air-cells? Equally difficult to explain as the result of osmotic disturbance is the distribution of fluid that is seen in cardiac dropsy. The fluid does not accumulate in the tissues where metabolism is greatest, or where the most oxygen is used ; but rather in the inactive subcutaneous tissues and in the serous cavities. Possi- bly the original transudation does occur in the muscles and solid viscera, and the fluid is then mechanically forced out of them into the surrounding tissue-spaces, later settling according to the laws of gravity or according to the distensibility of the tissues. It is im- portant to take into consideration the fact that demonstrable edema 29 Jour. Exper. Med., 1910 (12), 510. 346 EDEMA does not manifest itself until a very large quantity of fluid has been retained by the body — as much as six kilos, according to AVidal. Increased Hydration Capacity of the Tissue Colloids. — According to Fischer's tlieory this factor is of greater importance than any of the preceding, and of chief importance in increasing the amount of water present in the tissues are organic acids formed during metabolism. For example, the great power of asphyxiated muscle to take up water from a strong salt solution, which J. Loeb ascribed to the osmotic, pressure of the acids formed in asphyxia, is attributed by Fischer to the influence of tliese acids upon the capacity of the colloids for water, and this explanation seems to be in better agreement with the facts, especially since Overton has shown that even if all tlie proteins, earboliydrates and fats in a muscle were split into the greatest possi- ble number of simple molecules and ions, the resulting osmotic pres- sure would not be sufficient to account for the amount of water taken up. Furthermore, when cells with demonstrable semi-permeability die, they at once lose their semi-permeability, and in consecjuence their osmotic pressure falls — but dead cells and tissues often exhibit great power of taking up water and becoming edematous.''" It is an in- disputable fact that edema is especially associated Math conditions of asphyxiation, and the attempt to explain this by the increased osmotic pressure of the products of incomplete oxidation seem to harmonize with the facts far less successfully than the application of the prin- ciple of colloidal swelling. A common error of the critics of this theory is that of assuming that free acid must be present to cause swelling. This is not at all true. An amount of acid far less than enough to saturate the acid-binding property of a protein or to be detected by indicators will greatly increase the amount of water which this protein will combine. Presumably the colloidal carbohydrates and lipoids may also play a part in the water absorption of tissues. Fischer's theory of edema, in his own words, is this: "A state of edema is induced whenever, in the presence of an adequate supply of water, the affinity of the colloids of the tissues for water is in- creased above that which we are pleased to call normal. The ac- cumulation of acids within the tissues brought about either through their abnormal production, or through the inadequate removal of such as some consider normally produced in the tissues, is chiefly responsi- ble for this increase in the affinity of the colloids for water, though the possibility of explaining at least some of the increased affinity for water tlirough the production or accunnilation of substances which af- ^0 The secreted fluid of postmortem tlioraeie lymiili How dilVers from normal thoracic lymph in beinp more cloudy, often l)looh flow dejiends upcm ehanpes in the cells, caused liy as))hyxia and not dissimilar E EDEMA 347 feet the eolloitl-s in a way similar to acids, or tlirougli the conversion of colloids M^hieh have but little affinity for water into such as have a fjfreater affinity, must also be bonie in mind." In support of this theory he advances evidence which lie interprets as indicatin*^ that: (1) "xVn abnormal production or accumulation of acids, or condi- tions predisposing thereto, exist in all states in which we encounter the development of an edema. (2) The development of an edema in tissues is antagonized by the same substances which decrease the affinity of the (hydrophilic) enuilsion colloids for water (salts) and is unaffected by the presence of substances which do not do this (non- ■eleetrol5i;es). (3) Any chemical means by which we render possible the abnormal production or accumulation of acids in the tissues is accompanied by an edema." There are many features of lymph formation and edema with which this theory seems to harmonize well, and others with w'hich it •does not seem to agree so well, if at all, so that at this time it is a fair statement that the theory is under consideration, but the limita- tions of its applicability have not yet been agreed upon. It has met with much adverse criticism, some of w^hich was poorly founded, but the fact cannot be disputed that the amount of water that colloids will hold varies greatly with changes in the colloids. "We may not T\now absolutely, at present, whether the changes that take place in the colloids during life are great enough to alter tlieir water content appreciably, but it is highly probable that they are. In many in- stances the principles of colloidal hydration offer the best explanation ■of observed conditions, and their application often elucidates matters more satisfactorily than any other working hypothesis. Certainly they cannot be disregarded in considering the factors that may come into iilay in producing edema. Summary. — We find that a number of factors may be considered as responsible for edema, some of them being prominent in one in- stance, some in another, but in feiv cases can we consider one factor alone as the sole cause. In most of the forms of edema, such as those due to renal disease and cardiac disease, it now seems probable that either osmotic pressure changes or changes in the affinity of the tissue <3olloids for water, play the most important part ; whereas in inflamma-\ tory edema there can be no question that alteration in the capillary ■walls is the most essential factor. But the mechanical factor of blood pressure cannot be disregarded, although by itself seldom sufficient to cause edema ; associated with other factors it is undoubtedly an im- portant agency, for there are few edemas that are not associated with increased blood pressure. Hydremia and hydremic ])lethora may al- most he disregarded, except in so far as they may cause altered metab- olism in the tissues, injuiy to vessel-walls, over-saturation of the blood ■colloids, and decreased osmotic pressure within the vessels. Lymph- 348 EDEMA atic obstruction is possibly a factor of some secondary importance if we consider that distended vessels and tense tissues may occlude the lymph capillaries. SPECIAL CAUSES OF EDEMA We may now consider which of the above factors are at work in bringing about edema under the conditions in which it is usuallj^ observed clinically. Before taking up the detailed consideration of edematous conditions, however, it may be well to call attention to the fact that our knowledge of edema, and especially its clinical recog- nition and study, has been handicapped by the lack of a suitable ob- jective method of detecting and measuring edema. We are in the same position in respect to edema that we were to blood pressure when the only clinical measure was the clinician's forefinger. An attempt to remedy this defect has been made by Schade,^"'' whose "elastometer" reveals and measures degrees of edema not discernible by the palpating finger. A study of edema with this instrument in the hands of Schwartz ^°^ has revealed many interesting facts, but as yet the apparatus is too complicated for general clinical use. "Cardiac" Edema. — Passive congestion introduces nearly all these factors, for in addition to the increased blood pressure there is also an opportunity for changes in the capillary wall, either from stretch- ing and thinning of the cells and cement substances, or from "loss of tone" in the endothelium surrounding the stomata (Meltzer), or from toxic injury by accumulated products of tissue metabolism. AVhen the stasis is nearly complete, or if it is complete for a time and then relieved, the endothelium may be injured through lack of nourishment. As the edematous fluid in chronic passive congestion is usually of a watery type, poor in proteins, the edema is prob- ably less dependent upon capillary permeability than upon other factors, except in the case of acute stasis, when the fluid partakes of the character of the exudates. Presumably the accumulation of crystalloids within the tissues also plays a part in this form of edema, as the osmotic pressure is raised in tissues having deficient oxygen supply. But Fischer holds that the reduction in oxidation acts chiefly by increased production of acids, which greatly increase the affinity of the tissue colloids for water and at the same time alter the colloidal state of the capillary endothelium so that the capillaries become more permeable. Finally, there is probably more or less obstruction to lymphatic outflow because of the increased pressure on the lymphatic channels, and perhaps, also, in the case of cardiac incompetence, ob- struction to the discharge of lymph from the thoracic duet into the sul)clavian vein against the higli intravenous ])i-essnre. Renal Edema. — AVe must recognize under tliis heading two dififer- soaZeit. cxp. Path. u. Thor., 1!)]2 (11), .309. 30b Arch. Int. Med., 1910 (17), 396 and 459. SPECIAL CAUSES OF EDEMA 349 ent types of edema. In acute nephritis {e. g., in scarlatina) toxic materials appear to be the chief cause, and, as Senator contends, in- jure alike the capillaries of the renal glomerules and of the sub- cutaneous tissues; in each case there results an increased permeability which is manifested by albuminuria as a result of the injury to the renal capillaries, and by edema as a result of the injury to the tissue capillaries. This sort of edema is allied to that produced by peptone and similar lymphag-o. 41 .Tour. Pharm. et Chim., 1910 (102), 209. ••- Many data are jriven l)v Cerliartz, llandhuch der Hiocheinie, 190S, 11 43 Adarni, Allbutfs System, 1896 (1), 97. 44 Quoted by Hammarsten, "Physiological Chemistry." Hope 2), 137, COMI'OS/'noX OF EDKMATOl s j'l.iins Table II 353 Ascitic fluid in Parts of protein to 1000 c. v'. fluid Max. Min. Mean Cirrhosis of the liver Bright's disease 34.5 Ki.ll 5.6 10.10 0.69-21.06 15.6 -10.36 Tuborculosis and idiopatliic peritonitis Carpinomatous peritonitis 55.8 54.20 1S.72 27.00 30.7-37.95 35.1-58.96 Tlie specific g-ravity varies nearly in direct proportion to the amount of proteins, that of traiisndates nsnally being below 1.015, and exu- dates above 1.018, although there are many exceptions. Indeed, it is often very difficult to decide whether a given fluid is an exudate or a transudate.^'' According to Rzentkowski,*" the transudates at the moment they pass out of the vessels are simply solutions of crystal- loids in water and quite free from protein ; the small amount of protein found in transudates he ascribes to protein pre-existing in the tissue- spaces. This idea is hardly acceptable in view of the known per- meability' of the vessel-walls for proteins in normal conditions ; more probably in cardiac and renal dropsies the quantity of protein escap- ing from the vessels is not greatly different from normal, but the excessive fluid escaping in these conditions carries with it no addi- tional proteins, and to this extent transudates in statu nascendi are protein-free. Transudates, even when produced by the same cause, vary in com- position in different parts of the body, presumably because of varia- tions in the permeability of the vessels in different vascular areas; just as pleural, pericardial, peritoneal, and meningeal fluids normally differ from one another. Thus C. S. Schmidt *' found the composition 4s Rivalta (Eif. Med., 1903: Bioehem. Centr., 1904 (2), 529) has siiirtrested the following test to distinguish exudates and transudates: Into a beaker con- taining 200 o.c. of water with 4 drops of glacial acetic acid, let fall a few drojjs of the fluid to be tested. If an exudate, a bluish-white line is left transiently behind the sinking drops, due to precipitation of the euglobulin and filjrinoyen. Tliis test, and also certain modifications (see Rivalta. Policlinico, 1910 (17). (i7fl), seem to give quite reliable results. (See I'jihard, Berl. klin. Woch., 1914 (51), 1112. With tuberculous effusions Rivalta's test is positive, but not Mo- relli's test, which consists in dropping tlie fluid into saturated HgClo solution, a yellowisii ring of all)uminate forming with non-tuber:'ulous exudates, and a gran- ular precipitate with transudates. (See Zannini, Gaz. degli Osped., 1914 (4), 461). ]\Iemnii (Clin. Med. Ital., 1905, No. 3) suggests the larfrer content of lipase as a means of distinction of exudates. Tedeschi (Caz. degli Osped.. 1905 (26), SS) states that e]fl'<)S/T/(>\ or KI>K\I.\T()V8 FLUIDS 355 centration, while lioldiii*-- back the organic substances. Transiidatfis contain an excess of NaCl over other electrolytes, while in exudates the proportion of electrolytes other than chlorides is increased over the finding:s in transudates.^^ The surface tension of exudates is lower than that of transudates,'^ depending chiefly upon the glol)ulin con- tent. Rzentkowski "'"' found some slight differences in molecular con- centration as indicated by the freezing-point : in tuberculous pleurisy the average lowering was 0.523°, that of the serum being — 0.56°; in cardiac dropsy the subcutaneous fluid gave — 0.548°, and in renal dropsy — 0.583° ; tuberculous peritonitis, — 0.523° ; cirrhosis — 0.536° ; carcinomatous edema — 0.547°. Of these figures, the most significant is the comparatively high molecular concentration of the fluid in nephritis, supporting the contention that the cause of renal edema is retention of crystalloids.'" Tieken '"' has found the results in transudates, exudates, and other body fluids shown in Table IV. Table IV Freezing- Freezing- Nature of Fluid Sp. gr. point of effusion, — ° C. point of blood, — ° C. Disease Pleuritic effusion . 1.016 — 0..55 —0.56 Pneumonia, lobar. (( a 1.018 —0.55 —0.55 it it " " . . 1.018 —0.54 —0.56 Tuberculosis. a li 1.020 —0.55 —0.56 It " " . . 1,016 —0.55 —0.56 it a It 1.018 —0.64 —0.56 Valvular heart disease. It It 1.0.30 —0.60 —0.58 Empyema ; cyanosis. Pericardial " . 1.018 —0.55 —0.56 Pericarditis. It it 1.016 —0.56 —0.56 " It it 1.012 —0.56 —0.56 Hydropericardium. Ascitic fluid 1.024 —0.60 —0.56 Cirrhosis of liver. te it 1.020 —0.57 —0.56 a a (.' j;er's Arch., l!)(l.3 (!>:]), .irjS. f'3 See Epstein. .Jour. Kxp. Med., 1!)14 (20), .3:U. <>•! Zeit. klin. Med., 1!»0:$ (48), 3(54: also llolst. Tpsalalakar. Forhand.. 1!)04, p. 304. COMJ'OSIT/OX OF EDFAIATOU^ FLUIDS 357 similar Id tiic synovial iiniciii isolated in arthritis by Salkowski, and calls it scrosaimicin. Non=Protein Organic Contents. — Proteoses,'^ leucine, and tyro- sine may be present in small quantities in exudates, being produced by autolysis'''"' (Umber) ; and also mucoid substances (TTammarsten). Nucleoproteins nuiy be ])resent from leucocytic disintegration in exu- dates, as well as the products of their further splitting, such as purine's and phosphates, (ilaldi and Appiani "' found uric acid con- stantly' in amounts between 0.0055 g. and 0.0714 i:., in all exudates, of which seven were tuberculous and two neoplastic. In three trans- udates amounts from 0.006 g. to 0.011 g. were found. Allantoin is said to have been found in exudates (Moscatelli),"* but this is doubtful. All the other innumerable components of plasma may be found in edematous fluids; thus sugar'"'' and urea (Carriere) ^* are often present, as well as other extractives. The amount of urea varies quite as it does in the blood of the same individual, ^^ and it seems probable that all the crystalloid substances present in the blood pass freely into and from inflammatory exudates, so that an equilibrium between ]»lood and exudates is approximated.'- Sugar is said sometimes to be greater in amount in transudates than in the blood, but in exudates it is usually, if not always, lower than 0.1 per eent.^** Lecithin is al- ways present, partly bound to globulin and partly free (Christen)."^ Cholesterol is present particularly in fluids that have been standing for a long time in the body, appearing often as visible crystals shining in the fluid ; it probably originates from degenerating cells. Glycogen is not present (Carriere)."* Toxicity. — Contrary to earlier ideas, transudates are not toxic, even in nephritis (Baylac,"' Boy-Teissier,"^ Laiforcade '"), and there- fore the toxic manifestations frequently observed after reduction of edema in nephritis, and ascribed to absorption of poisons in the trans- udates, are probably due to some other cause. In inflammatory exu- osOpie, Jour. Exp. Med., 1007 (0), 391. 66 Histidine and arginine were found in a carcinomatous exudate hv Wiener (Biochem. Zeit., 1912 (41), 149). 67 Riforma Med., 1904, p. 1.373: also Carriere, Compt. Rend. Soc. Biol., 1899 (51), 467. 68 Zeit. physiol. Cliem., 1899 (13), 202. 69 Sugar was found in only 8 of 23 fluids by Sittig (Biochem. Zeit., 1909 (21). 14) ; but is present in pulmonary edema fluid in proportion equal to or even greater than the blood (Kleiner and !Meltzer). 71 Javal and Adler, Compt. Rend. Soc. Biol., 190G (61), 235; Roseiil)ero:, Berl. klin. Woch., 1916 (53), 1314. 72 Wells and Hedenburg, Jour. Infect. Dis., 1912 (11), 349; Scheol, Xord. :\red. Laeg.. 1916 (77), 610. 70Hegler and Schumm. :\red. Klinik. 1913 (9), 1810. 73 Cent. f. inn. :\Ipd., 1905 (26), .329. 74 Compt. Rend. Soc. Biol., 1899 (51), 467. 75 Compt. Rend. Soc. Biol., 1901 (53), 519. -G Ihid., 1904 (56), 1119. 77 Gaz. heb. Med. et Chir., Jan. 28, 1900. 358 EDEMA dates, of course, the causative agents as well as the products of cell desti'uction render tlie fluids ])()is()nous. Enzymes and Immune Bodies. — All the enzj'mes of the plasma nuiy appear in edematous fluids, being- in all cases probably more abundant in exudates than in transudates. According to Carriere,'^ oxidases are inconstant, even in exudates. Lipase is said to be much more abundant in exudates than in transudates."'' (Concerning pro- teolytic enzymes see "Autolysis of Exudates," Chap, iii.) The various immune bodies, cytotoxins, hemolysins, bacteriolysins, ag- glutinins, etc., seem to pass freely into both transudates and exudates, and their presence is not characteristic of either,-" but as a rule the proportion is much higher in exudates. ^^ Peptid-splitting enzymes are usually found in such fluids,'*- especially tuberculous exudates,^-'^ and these enzymes seem to be difi:'erent from both erepsin and trypsin. Probably this type of enzyme is more often present than trypsin. Antitryptic activity is usually high, unless exhausted by the presence of much, trypsin from cell-rich exudates. Purulent fluids are usually poor in ojosonins ; ®^ in non-purulent fluids the opsonin content varies with the amount of proteins."*^ Turpentine exudates may sometimes be more strongly bactericidal than the serum of the same animal.*^ Exudates usually contain about as much complement as the serum, but in suppuration the complement disappears ; transudates contain little of either complement or hemolysins. ^^''^ Precipitin Reactions, etc. — Edematous fluids have been often used as a source of material in immunizing animals against human proteins. The precipitins thus formed are specific for human serum or for the proteins of the effusion, but cannot be used to differentiate a transudate from an exudate, or a hydrothorax fluid from an ascites fluid (Quadrone ).'*'' Immune bodies, complement, agglutinins, and antitoxins are present in effusions ; ^° e. g., the common use of blister fluid for the Widal test. Furthermore, according to Hamburger,*® edema fluid is distinctly more bactericidal than normal lymph. 78Compt. Kend. Soo. Biol., ISflO (51), oOl. TO Zeri, 11 roliclinico, 1J10.3 (10), No. 11; :\lemmi, ("lin. :\lt'd. Ital., ino.'>. No. 3: Galletta, Clin, iiiwl. Ital., 1011 (50), 14.3. 80 Granstriiiii, Tnaug. Dissort., St. Petersburg, lOO.i. 81 Not corroborated by Liidke, Cent. f. Bakt., 1907 (44). 2()S. 82 Hall and Willianiscm. .lour. Path, and Bact., inil (15), .'551. 82a See H. Koeh, Zeit. Kinderhoilk.. 1014 (10), 1. 83 0pie. .lour. E.xper. Med.. 1!M)7 (0), 515. 84 Brill me, Deut. Areli. klin. Med.. 1000 (96), 105. ssRastaedt, Zeit. Tnimunitiit., 1012 (13), 421. 85a Aronstanim. Cent. f. Bakt.. 1014 (74), 32(1. 80 Cent. f. Bakt. (Ref.), 1905 (36), 270. 88 Virohow's Arch., 1S09 (15(1). 320. VAh'lIJTIKH OF KDEMATOrx FfJ IDH 359 VARIETIES OF EDEMATOUS FLUIDS -<:' On tilt' ])i-('c(Mliiiji- ])ag<'s liavc been mentioned the chief differences in the characters of the effusions in the usual sites,"" with their varia- tions in protfMii contents, which variation agrees with Starling's state- ment tliat the |)('rmeal)ility of the capillary wall for proteins ditlt'ers normally in different localities. Some of the other ett'usion fluids not mentioned jjreviously have particular properties of some interest. Subcutaneous Effusions.'"^'' — When of non-inflammatory origin these are very watery, having ordinarily a protein content of from 0.1 to 0.2 gm. per 100 c.c. tliere being more globulin in nei)hritic than in cardiac dropsy. The non-coagulable nitrogen and chloride content are not so high as in the blood of the same patients, but the ash is the same as that of the serum. The specific gravity may be as low as 1.00."), but the solids increase with the duration of the edema. Hydrocele and Spermatocele Fluids. — These have been studied particularly by Hammarsten, who found the average results of analyses of seventeen hydrocele fluids and four spermatocele fluids as follows: Table V Hydrocele Spermatocele Water 9,38.85 986.8.3 Solids 61.15 13.17 Fibrin 0.59 Globulin 13.25 0.59 Seralbumin 35.94 1.82 Ether-extractive bodies . . 4.02 1 Soluble salts 8.60 \- 10.76 Insoluble salts 0.66 J Marchetti ^^ found in ten specimens of hydrocele fluid rather higher results for the solids than did Hammarsten. He found 57.8 to 104.2 p. m. of solids, contain- ing organic substances 48.8 to 95.02, and inorganic substances 8.10 to 9.56; pro- teins, 33.5 to 90.19; ratio of globulin to albumin as 2.56 to 9.11. Among the proteins is found 1 to 4 p. m. that is not precipitated by heat. Corresponding with the analytic results, tlie specific gravity of hydrocele fluid is higher. l.Olti to 1.026 as against 1.006 to 1.010 for spermatocele fluid. Cholesterol is often abun- dant in hydrocele fluids, appearing to the naked eye as glistening scales. Patein ^^ found sugar in most specimens of hydrocele. Apparently hydrocele fluid stands intermediate in properties between transudates and exudates. -'3 Meningeal Effusions.-'* — Normal meningeal fluid differs from all other serons fluids in being clear and Avatery, in its low specific gravity (1.004 to 1.007), in containing but a trace of protein which is chiefly 80 Chemistry of Pus and Sputum are discussed under Inflammation, Chapter x. 90 Literature and resume on pleuritic exudates, see Ott, Chem. Pathol, der Tuberc, 1903, p. 392. noaSee Epstein, Jour. Exper. Med., 1914 (20). 334. 91 Lo Sperimentale, 1902 (56). 297. 92 Jour, pharm. et chim., 190(i (23), 239; also Conipt. Rend. Soc. Piol., 1906 (60), 303. 93 Vecchi, Gaz. Med. Ital., 1912 (63 1, 211; Epstein, Jour. Exp. Med., 1914 (20), 344. 94 Resume by Blumenthal, Ergeb. der Physiol., 1902 (1), 285; Blatters and Lederer, Jour.Amer. Med. Assoc, 1913 (60)j 811, 360 EDEMA globulin (with a trace of proteose ( ?)), and 0.05-0.13 per cent, of a reducing- substance that is proba])ly <«-lucose,''^' which is decreased in acute suppurative meningeal intlannnation (Jacob).'"' Halliburton gives the following analyses of pathological accumulations of such fluids : Table VI. (Spina bifida.) Case 1 Case 2 Case 3 Water 989.75 989.877 991.6.5S Solids 10.25 10.123 8.342 Proteins 0.S42 1.(502 0.199 Salts ) . . . . f.f..^f. ( 0.631 3.02S Extractives i . . . . '^- " * 7.890 5.115 The percentage of solids in spina bifida is thus a little higher than in normal meningeal fluids. In hydrocephalus the percentage of solids is rather greater, as seen in Table VII. T.\.DLE VII. (Hydrocephalus.) Case 1 Case 2 Case 3 Water 986.78 984.59 980.77 Solids 13.22 15.41 19.23 Proteins and extractives . . 3.74 6.49 11.35 Salts 9.48 8.92 7.88 Normal cerebrospinal fluid seems to be hypertonic to the serum of the same animal,**' and slightly more alkaline than the blood.**^ In meningitis the alkalinity is often lowered.''*'' According to Fuchs and Kosenthal,-*" the average freezing-point of the cerebrospinal fluid is loW'Cred about the same in all diseases (A = — 0.52° to — 0.54°) ex- cept in tuberculous meningitis, wdiere it is much less (average — 0.43°). The amount of potassium is about the same as in the blood/ and not increased in degenerative diseases of the central nervous system ; ^°- after death the amount is much increased by post-mortem changes. Calcium is almost constant at 5 mg. per 100 c.c, or about one-half as much as in the plasma.^" In diseases associated with destruction of brain tissue, such as general paralysis and epilepsy, choline or some other base - may be found in the spinal fluid. (See "Choline," Chap. iv.) Under pathological conditions the amount of protein varies greatly and to some extent characteristically. Thus, in syphilis the euglobulin is so greatly- increased that it is readily identified by 95 Schloss and Schroeder, Amer. Jour. Dis. Child.. 191(1 (11), 1; Hopkins, Amer. .Jour. Med. Sci., 1915 (150), 847. no Brit. :Mcd. .lour., 1912, Oct. 26. 07 Ravaut, Presse nied., 1900 (8), 128; Zanier, Cent. f. Phvsiol., 1896 (10), 353. 98Hur\vitz and Tranter, Arch. Int. Med., 1916 (17), 828. ' n«aLevinson, Arch. Pediatri'-^. 1916 (33), 241. onWien. med. Presse, 1904 (4.H. 2081 and 2135. 1 Mvers, Jour. Biol. Cheni.. PHi'.l (6), 115. literiilure. la Pvosenhloom and Andrews. Arch. Int. ^Med., 1914 (14), 536. ih llalvcrson and IJerfrciin, .lour. Biol. Cli.'in., 1917 (29), 337. 2 Kaufmann, Zeit. piiyniol. ("hem., 1910 IM)), 343; Lai,i:nel-Liivastiiu> and Lasusse, Compt. Rend. Soc. Biol.. 1910 (OS), 803. \.\ini:Tii:s or j:i>i:\i \t<>( s fij ids :^61 various |)i'('('i|)ilat ioii met liods, ■ while in more acute iuHaiumatious libriuoficii appears.' Acc()rdiiintiiig pi-eeipitation of colloidal suspensions by electrolytes (the "Goldzahl" of Zsigmondy)." The surface tension is higher than that of the serum and is not characteristically altered in disease.'"" The increased organic matter of pathological fluids raises the permanganate reduction index.'"' Cholesterol can be found in all cases of mental disease, the amount not bearing any relation to the type of psychosis (Weston) ; ^ ordinar- ily 0.2 to 0.7 mg. per 100 c.c. is found. The changes in P2O-, content in disease are doubtful,** while the amount of reducing substances is said to be increased in disease.-' In general the inflammatoiy fluids in the spinal canal resemble exudates elsewhere, but usually the con- centration of the different components is relatively low, except the chlorides.^" Normal cerebrospinal fluid contains no antiprotease (for leucoprotease), as does the fluid in many cases of chronic inflamma- tions; in acute inflammation proteases may appear (Dochez ^^). Pep- tid-splitting enzymes are especially abundant in meningitis. ^^'^ Anti- bodies pass from the serum into the cerebrospinal fluid only in minimal amounts or not at all, except when inflannnatory exudation occurs, and even then the antibody concefitration is usually low,^- and even simple chemicals enter the normal spinal fluid but very little, ^^ ex- cept perhaps alcohol.^* According to Rosenbloom "-''^ there is no crea- tin or creatinine. It contains normally from 2 to 4 mg. of amino-N. per 100 c.c, or about half that in the blood, without definite changes in syphilis.^'"' There is almost the same amount of urea as in the serum of the same person, i. e., 20 to 42 mg. per 100 c.c.^*'= Substances giving the ninhydrin test appear in meningitis,'^'' but Rosenberg states 3 See Xogiiclii, Jour. Exp. Med., inon (11), 604. 4 See :\Iestrczat., Rev. d. Med., ]!)10, )). ISO; KafYka. Dciit. nied. \Voch., 1013 (39), 1874. 5 Lancet. July 0, 1910. 6Lange, Zeit." Cliemother., 1012 (1), 44; Spiit, Zeit. Tmnniiiitat., I'll.i (23), 426. eaKisch and Kemcrtz. IMiinch Med. Woch., 1914 (20), 1097. 6b See Hoffmann and Schwartz, Arch. Int. Med., 1916 (17), 293. 7 Jour. Med. Res., 1915 (33), 119. 8 Apelt and Schumm. Arch. Psvchiat u. Xervenkr., 190S (44), 84.5. 9,]»acob, Brit. Med. Jour., Oct. '26, 1912. 10 Java!, Jour. pbys. ct ])atli. gen.. 1911 (15), 508. 11 Jour. Exp. Med., 1909 (11), 718. iia Major and Nobel, Arch. Int. Med., 1914 (14), 383. isLeniaire and Debre, Jour, physiol. et patli. gen., 1911 (13), 233. 13 See Eotkv, Zeit. klin. :Med., "l912 (75), 494. 14 Schottmiiller and Scliumm, Neurol. Zbl., 1912 (31), 1020. i4aRiochem. Bull., 1916 (5), 22. 14b Ellis, et al.. Jour. Amer. Med. Assoc, 1915 (04). 126. 14c Ellis and Cullen, Jour. Biol. Chem., 1915 (20), 511. i4dNol}el, jMiinch. med. Woch., 1915 (62), 1355, 1786. 362 EDEMA that even with the hig'liest indieanemia '^"^ no indicaii is found in the spinal fluid. Su- once been present ; the amount of fat is small, usuall\' about 1 per cent., and the fluid is rich in solids. If due to a ruptured thoracic duct, it may ])e ])()ssible to detect special fats taken in the food, e. g., butter-fats (Straus).-'' The reaction is usually alkaline or neuti-al. and some specimens coagulate spontane- ously. Specific gravity varies from 1.007 to 1.040, the average being about 1.017. Perhaps the most important characteristic is the varia- tion produced by changes in diet.-' Zdarek -* found in a chyle-cyst 2.7 per cent, of fats, 7.2 per cent, of proteins, and 0.05 per cent, of sugar; feeding of fats increased their amount in the cyst and star- vation decreased it. Schumm -^ found in the solids of such a cyst, 35.76 per cent, of fat, some of which was in the form of calcium soap. Chijlothorax fluid is, of course, quite similar to that of chylous ascites. Thus. Buchtala ^"^ found 91.34 per cent, of water, 8.66 per cent, solid, 4.86 per cent, protein, 2.5 per cent, fat, 0.26 per cent, cholesterol, and 0.94 per cent. ash. Similar figiires were obtained by Salkowski "^ and others. Chyluria,^^'^ which seems to depend upon an abnormal connnunica- tion between the lymphatics of the receptaculum chyli and the kid- ney,^- shows no particular chemical features beyond those of an ad- mixture of a considerable amount (100 to 1000 c.c. per day) of chyle with the urine. Carter ^^ found the amount of fat in the urine to rise with increase of fat in the food. In some cases chyle escapes directly into the bladder or ureter from the lymphatics, in others the fat may 23 ;Medicine. 1S95 (1), 257; also see "Chem. ii. morph. Eigensehaften fett- haltiiie Exsiulaten," St. ^Mutermilch, \Varscliau. 1003; Comev and IMeKibhen, Boston Med. and Surg. Jour., 1003 (14S), 100. 24 .Jour. Amer. Med. Assoc, 1905 (44), 513. 25 For example, v. Tabora ( Deut. nied. Wocli., 1004 I 30), 1505) found as high as 0.864 per cent, of sugar in a tvpical case. 26 Arch. Physiol, et Pathol.. 1886' (Ser. 3, vol. 8), 367. 2T A sample of the composition of 1 liter of chylous ascitic fluid is shown by the analysis in the case studied by Comev and McKibben Hoc. cit.) : Specific gravity, 1.010; solids, 21 gm. ; protein, 0.75 gm.; urea, 1.28 gm.: fat, 1.45 gm. : inortranic matter. 8 gm.; peptone (?) and sugar, present; fibrinogen, mucin, nucleo-albumin. and uric acid absent. 2SZeit. f. Heilk., 1006 (27), 1. 2n Zeit. phvsiol. Chem., 1006 (40), 266. soZeit. phvsiol. Chem., 1010 (67), 42. 3iVirchow"s Arch.. 1000 (108), 180; also Tulev and Graves, Jour. Amer. :\Ied. Assoc. 1016 (66), 1844; Patein, Jour, pharm. Chim., 1015 (11), 265. 3ia Review of literature bv Sanes and Kahn, Arch. Int. iled., 1016 (17), 181. 32 See Magnus-Lex-v. Zeit. klin. Med., 1008 (66), 482. 33 Arch. Int. Med.," 1916 (18), 541. 364 EDEMA be excreted directly from the blood, independent of lymphatic abnor- mality ; in some cases the fluid entering' the urine is true chyle and in others it is lymph. Ascites adiposus is characterized by the absence of sugar and by a liigher percentage of fat, the maximum observed being 6.4 per cent. It is ascribed to fatty metamorphosis of cells, particularly in carcino- matous and tuberculous exudates; Edwards was able to show experi- mentally that a transudate may change from serous to cellular, and later come to contain fat. Pseudochylous effusions are also observed, not onl}^ in the abdom- inal and thoracic cavities, but even in the fluid of the edematous legs and scrotum ; these resemble chylous fluids in being turbid or milky, but are said to contain little or no fat. The turbidity is ascribed chiefly to lecithin, which is largely combined with the pseudoglobulin of the fluid ( Joachim). ^^ Possibly in some cases the turbidity is j)artly or largely (Poljakotf) ^'' due to poorly dissolved proteins. Strauss ^"^ has noted the occurrence of this form of ascites particu- larly in chronic parenchymatous nephritis, but believes the turbidity has a local origin. Hammarsten has observed a similar turbidit}^ due to mucoid substances, as also have Gouraud and Corset.^' The pseudo-chylous effusions have a lower freezing point, a lower specific gravity, lower fat and greater lecithin content than typical chylous ascites. Gandin,^'' however, questions the possibility of always differ- entiating the three types of turbid fluids as above indicated. Collect- ing all the recorded analyses in the literature he finds wide discrep- ancies, as indicated in the following table: (The maximum and mini- mum percentage figures are given for each component determined quantitatively, with the average in parentheses.) Chylous Adipose (Chyliform) Pseudochylous Ether extract O.O60-9.2 (1.65) 0.1-4.3 (1.15) 0.007-1.86 (0.25) Cholesterol + in 7, — in 2 + in 4 + in 3, — in 2 Lecithin + in 4, — in 1 + in 3 + in 20, — in 2 Sugar + in 46, — in 28 + in 1, — in 4 -f- in 15, — in 14 Dry residue ;?. 1-10.6 (6.2) 1.6-11.7 (5.1) 1.2-7.6 (2.0) Protein (».!»-7.7 (;5.5) 0.6-6.8 (3.0) 0.1-4.2 (1.4) "Pepton."' + in (!, — in 4 4- in 1, — in 2 + in 1, — in 5 Asii 0.1-1.0 (0.5!)) 0.45-1.03 (O.6.- i") 0.40-0.00 (0.73) It is quite evident that although the pseudochylous fluids usually contain little fat, they often contain more than the minimal content found in the other forms. P^ach type of fluid overlaps the others in one respect or anotiier. Gandin states that to produce a turbid fluid but 0.01-0.1 per cent, of finely emiilsionizcd fat is necessary, and lie 34Munch nied. W'odi., 1003 (50), 1015; also thristen. Cent., f. inn. Med.. 1005 (26), 320; Wallis and ScJiolherj,', Quart, .lour. Med., 1010 (3), 301; 1011 (4). 153. 35 Fortsclir. d. Med., 1003 (21), lOSl ; also iiauslialter, Coinj)!. Heiid. Soc l!i(d., 1910 (6H), 550. 30 Note to Poljakoll's article; also Hiochein. Ceiitr., l!(i):{ (1). 437. 37Conipt. Rend. Soc. Biol., 1006 (60), 23. VllEMISTh'Y or l'.\i:i MOTHnUAX 365 believes tliat milky fluids always incaii admixture of cliyle, rejecting the terms pseudochylous and chyliform as unwarranted. He admits that fluids may contain droplets of fats not emulsionized, and hence not millrs', which may be properly called adipose fluids. There are no characteristic chemical differences in tlie fats extracted from the dif- ferent types of fluids. CHEMISTRY OF PNEUMOTHORAX Til conneetioii witli the subject of exudates the above topic iiuw apjn'opriately be considered. The composition of the gases found in the pleural cavity in pneumothorax will necessarily vary greatly according to the cause. If the pleural cavity is in free communica- tion with the exterior, the gas will be simply slightly modified air: for example, Ewald ^^ found the following proportions in the gases in such a pneumothorax : CO., 1.76 per cent. ; 0, 18.93 per cent. ; and 79.31 per cent. X. Here the proportion of CO, is even a little less than in ordinary expired air, which contains 3.3-3.5 per cent. When air enters a closed pleural cavity and no effusion follows, it is slowly absorbed until a mixture of about 90 per cent. N, 4 per cent. 0 and 6 per cent. CO^ results ; but if there is a serous effusion the oxygen disappears nearly or quite completely (Tobiesen).^^ In a seropneu- mothorax Ewald found. 8.13 per cent, of CO2, 1.26 per cent, of 0, and 90.61 per cent, of X, which is quite similar to the proportions of the gases in dry pneumothorax. Purulent pneumothorax generally shows more COo than the serous form, the average in the former being 15-20 per cent., in the latter 7.5-11.5 per cent. The average of the analyses in six cases of pyopneumothorax is given bj' Ew^ald as 18.13 per cent. CO., 2.6 per cent. 0, and 79.81 per cent. X'. In open pyopneumothorax the gas approaches more closely the com- position of air, but usually shows a slight excess of CO,; it is thus possible by a determination of the carbon dioxide to determine quite accurately whether a given pneumothorax is in communication with the outside air. The transformation of a purulent into a putrid pneumothorax is accompanied by an increase of COo, even as high as 40 per cent, having been found. The products of decomposition by the putrefactive saprophytes also are present, one analysis having shown 4.3 per cent, of hydrogen. 6.25 per cent, of methane, and traces cf hydrogen sulphide. Infection of a pleural effusion by gas-producing organisms may also convert it into a pneumothorax, although this is not a common occurrence. The gases then present are the same as the organisms produce in similar culture-media, modified somewhat by absorption. The anaerobic gas-producing organisms have been found as the cause 38 Complete literature and resume given bv Clemens, in Ott's '"Cliem. Patli. der Tubereulose,"' Berlin, 1008. p. 4f)ti. 30 Beitr., z. Klin. d. Tuberk., 1011 (1!)). 451; 1011 (21), 100; Peut. Arcb. klin. Med., 1914 (115), 399. 366 EDEMA of suuli gaseous accumulations; it is questionable if the ordinary path- ogenic organisms can cause a pneumothorax, since they are for the most part not capable of producing gas. The colon bacillus produces gas in sugar-containing media, but the amount of sugar in the patho- logical exudates is too small to yield any considerable amount of gas; an exception is the pleural eflfusion in diabetes, and pneumothorax from infection of the pleural effusion in a diabetic by B. coli has been reported. Complete quantitative analyses of the gas in this form of pneumothorax seem not to have been made, but ^lay found about 20 per cent, of CO,. The combustibility of the gas has frequently been noted, and is probably due to hydrogen and methane. CHAPTER XIII RETROGRESSIVE CHANGES (NECROSIS, GANGRENE, RIGOR MORTIS, PARENCHYMATOUS DEGENERA- TION) NECROSIS We recognize that a cell is alive through its reproducing, func- tioning, and its taking on and utilizing nutritive substances; yet at tlie same time we appreciate that a cell may do none of these things and still be alive. For example, a bacterial spore is quite inert physically, and exhibits no chemical activity, yet it is by no means dead, since it still possesses the latent power to assume again an active existence under suitable conditions. In pathological condi- tions we are accustomed to recognize the fact that a cell is dead by certain alterations in its structural appearance, particularly disin- tegrative changes in the nucleus ; but this is exactly equivalent to recognizing that an animal is dead by the appearance of postmortem decomposition, for most of the characteristic histological changes of necrosis are merely postmortem changes in the cell. A cell may be dead and show absolutely none of these microscopic disintegrative changes, either because it has not been dead long enough for them to have taken place, or because the changes have been prevented by some means, just as we can prevent the appearance of postmor- tem decomposition by embalming. For example, if we examine mi- croscopically the mucous membrane of the stomach of a person who has died immediately after taking a large ([uantity of carbolic acid, although to the naked eye this mucous membrane is hard, white, and definitely necrotic, yet we find the histological picture presented by the cells almost absolutely unchanged from the normal. The cells are dead, but they have been so "fixed" that postmortem changes could not affect their structure. All cells examined by ordi- nary histological methods are, of course, dead — killed by the fixing agents outside of the body, in the same way that the carbolic acid fixes them within the body. It is evident, therefore, that it ma.v be very difficult to determine always whether a cell is dead or not. Part of the difficulty, perhaps, lies in our failure to appreciate that not all parts of a cell die at the same time; i. e., the causes of different chem- ical processes of the cell reside in its different intracellular enzjones, and these are not necessarily destroyed alike by the same agents. AVe recognize that after an animal is dead as a whole the various 367 368 RETROGRESSIVE CHANGES cells of its body do not die for some time, as shown by the following examples: (1) We can cause the heart to beat for a considerable period after its removal from the body; (2) if we perfuse a mixture of g'lycocoll and benzoic acid throug-h the kidney of a recently killed animal, synthesis of these substances into hippuric acid will occur; and (3) the epithelium of the skin can be removed from the body of an animal lono: after death and transplanted successfully on another animal. So, too, in ordinary cell death (necrobiosis) not all the enzymes are destroyed together. When all are destroyed at once, as by strong chemicals or by heat, the customary disintegrative changes do not take place. If, however, not all the enzymes are thrown out of function, then the others may be able to act, producing the disintegrative changes by which histologists ordinarily recognize cell death. These tlisintegrative changes are, for the most part, ap- parently brought about by the intracellular proteases, that is, through autolysis. This may be shown as follows:^ If we take two pieces of fresh normal tissues from an animal, and in one kill the enzymes by heating to 100° C, then implant both aseptically into the abdom- inal cavity of an animal of the same species, it will be found that the changes that follow in the two will be very unlike. In the un- heated tissue nuclear changes soon occur, so that they lose their ca- pacity for taking up basic stains, the cytoplasm becomes granular and fragmented, the tissue becomes friable so that it is difficult to secure good sections, and the changes are in general similar to those seen in areas of necrosis. The boiled tissue, on the other hand, retains its capacity for nuclear staining for months, except at the periphery, where it is slowly attacked by leucocytes and the enzymes of the blood plasma. Therefore it would seem that the characteristic changes of necrosis depend ehietly upon the intracellular enzymes, rather than upon the infiltrating plasma as Weigert - and other early writers imagined. In areas of anemic necrosis (see "Infarcts") we have another case, in which the oxidizing enzymes are thrown out of function through lack of oxygen, while the otlier enzymes are, presumably, at first luuiffected. From studies of infarcts it would seem that the intracellular proteases bring about the subsequent nuclear and cytoplasmic alterations, but that the eventual digestion of the area is accomplished by the invading leucocytes working slowly inward from the periphery. Apparently when the sui^i)]y of materials from outside ceases, and when the oxidation ]>ro('esses of the cells no longer accomplish necessary steps of synthetic reai-tions or destroy products of protein catabolism, the proteases continue to split proteins without the balancing by the above-mentioned factors, with a resulting disintegration of the cells. Karyoljjsis and Ldri/orrhf.rls are, Ihcu, Ihe i-esult of an autolytic 1 Wells, .Tour. Med. Kcsoarcli, liMXI (15 1. lift. 2 Cent. f. Path., 1801 (2), 78,5. NECROSIS 369 process, whicli is perhaps due to intracellular proteases that act spe- cifically on nucleoproteins, and which may be designated as nucleases.^ Nuclear staining by the usual methods depends upon an affinity of tlie acid nucleoproteins (in which the nucleic acid is not completely saturated by j)r()teins) for basic dyes. Presumably in karyolysis the first step consists in a splitting of the nucleoprotein of the chromatin into nucleic acid and protein; this can be accomplished, according to Sachs, by the ordinary trj'psin, and presumably, therefore, by the trypsin-like enzymes of the cell. Corresponding with this cliange we should expect the free nucleic acid to give an intense staining with basic stains, and this has frequently been described by those who have studied the cytological changes in anemic necrosis,* and called pijoiosis. As supporting this view still further may be quoted Arnheim 's "' observation that in alkaline solutions the nucleus soon stains diffusely and weakly, and not at all after twelve to eighteen hours; this is to be explained by the fact that nucleic acid is both dissolved and neutralized by alkaline solutions. Acids developed in injured cells may, by combining with the basic elements of the n\\- cleoproteins, render them still more acid and highly basophilic ; thus, in muscles showing waxy degeneration from accumulation of lactic acid the muscle nuclei will be found pycnotic (see waxy degenera- tion). After the nucleic acid has been freed from the protein by the autolytic enzymes, it is still further decomposed by the "nu- clease" or similar intracellular enzymes that have the property of splitting nucleic acid into the purine bases that compose it — cor- responding with this change the hyperchromatic nucleus loses its affinity for stains, and karyolysis is complete. "When extensive ne- crosis occurs there will result, therefore, an increased elimination of purines, as was found by Jackson and Pearce ^ in animals with severe hepatic necrosis from hemotoxic serum. A careful analytical study of the chanses taking place in the autolyzinor spleen, for the purpose of correlatino^ the chemical and microscopical chancres, has been made by Corper," whicli corroborates tlie interpretation of necrosis advaiu-e l)econie moi-e turbid, associated with which are alterations in tlieir per- meability to crystalloids. A gelatin mass possesses its maximum elasticity tiiree or four hours after it is first formed; and crystalloids penetrate fresh, quickly- formed gels at first more rapidly than later. As Bechhold says, we can inuigine (1) a relation of such facts to the greater elasticity of young tissues; (2) to a ))resumably greater permeability for crystalloids and hence more rapid metab- olism: (3) to the decreasing water of tlie tissue with age (94 per cent, of water in the fetus of three months. 00-60 per cent, at birth, and 58 per cent, in adults) : (4) to the demonstrated greater permeability of yoiuig nerve tissues for vital stains, etc. ''In general we can say that the tissue colloids decrease in their water attinity (Qiiellharl-eit) both in animal organisms, which become poorer in water with age, and in plants, as shown by the hardening of older j^lant tissues.'' The bearing of these principles on the problem of senility and degeneration of elastic tissue, regeneration and many other subjects is obvious. CAUSES OF NECROSIS Anemia. — After the cutting off of blood-supply, cells soon undergo morphological changes that we recognize as indicating tlieir death, and after a time they also become incapable of returning to their nor- mal condition when the blood-supply is re-established, probably be- cause of these structural changes. In just what way lack of nourish- ment causes death has not been determined, but, as has been before suggested, it seems probable that it is because catabolic processes are no longer balanced by anabolic processes, and with these latter oxi- dizing enzymes seem to be inseparably associated, as far as our pres- ent knowledge shows us. That the loss of oxygen alone, with other materials presumably supplied to the cells in adequate amount, may cause necrosis, is shown by the presence of marked hepatic necrosis in animals kept a week in atmospheres extremely low in oxygen (5-9 per cent.).^-^ The nature of the chemical changes taking place in a cell when oxygen is deficient must be very different from the normal changes, and hence abnormal toxic substances maj accumulate, e. g., excessive amounts of organic acids. Were it not that the proteolytic 11 Oaleotti's earlier observations with animal tissues (Zeit. f. Biol., 100.3 (45). 65) do not harmonize with Osterhout's results, and Galeotti's idea that there is a special degree of ionization cliaracteristic of living cells is not established. loe Botan. Gaz., 1015 (50), 242. 12 See H. Bechhold, "Die Kolloide in Biologie und Medizin." Dresden, 1012, p. 65 12a Martin, Bunting and Loevenhart, Jour. Pharmacol., Proc, 1916 (8), 112. 372 RETROGRESSIVE CHANGES enzymes continue in action after nutrition is shut off, the cells might remain in a completely unaltered condition for an indefinite period, and capable of resuming- their function when nourishment is again supplied, which is decidedly contrary to the facts. (The general features of anemic necrosis have been already discussed in the pre- ceding paragraphs, and also under the subject of infarction.) Thermic Alterations. — These have been studied particularly in conneetion with the cells of the lower organisms.^^ While some uni- cellular organisms can survive a temperature of 69°, most of them are killed at from 40°-45°. For the great majority of metazoa the nicfximum temi)erature lies below 45°, and in the case of marine species below 40°." The heating is accompanied by the appearance of granules in the cytoplasm, which become larger until the condi- tion of "heat rigor" sets in. Kiihue, in 1864, showed that in muscle cells, at least, there is contained a protein which becomes turbid through partial coagulation at 40°, and Halliburton ^^ has found that in nearly all tissues are globulins coagulating at from 45°-50° ; it is probable, therefore, that the granules formed in heated cells are produced through coagulation of these proteins. The importance of this coagulation in determining death is not yet fully established, but it would seem to be very great. Halliburton has observed that in both muscles and nerves to which heat is applied, contractions occur at various temperatures, corresponding exactlj^ with the tem- peratures at which the several varieties of the proteins of the cells coagulate. Furthermore, Mott ^"^ has found that the temperature that is immediately fatal to mammals (47°) is exactly the same as the coagulating temperature of the lowest coagulating protein of nerve-cells. This fact is undoubtedly of great practical importance in causing death from fever, for although 47° C. (117° F.) is prob- ably never reached in man, yet application of much lower tempera- tures, even 42° (108° F.), for a few hours will cause coagulation of these proteins (all proteins coagulate at less than their ordinary coagulation point if the heating is continued for a long time). It would seem from the above observation that heat may cause cell death through coagulation of the proteins. AVhethei- the cell death is in any way dependent upon destruction of the enzymes by heat has not been ascertained ; but as most enzymes are not destroyed much be- low 60°-70°, it seems improbable that they are greatly injured at the temperatures at which cells are killed. It is possible, however, that under the conditions in which enzymes exist in the cell they i- Literature, see Davenport, "Experimental Aforphology," New 'Sork, 1S!)7; Schmaus and Albrecht, Erpebnisse der Pathol.. 1896 (,1, Abt. 1), 470. 14 The adaptation of animal cells to hipli temperatures is an interosliiii,^ topic, especially in view of sueli results as tliose of Daliinfjer, wiio, by raisiui: tlie teni- peraturo gradually durines in temperature, and such .alterations miglit well have disas- trous results. Different tissues show unequal susceptibility to heat. Werhov- sky ^^ found the blood most affected by raising the temperature of living animals, next the liver, kidneys, and myocardium in order, the other tissues being little or not at all structurally injured. Cold is well withstood by unicellular forms, and relatively poorly by more complex organisms, particularly by those with a highly de- veloped circulatory system ; this is because individual cells are not greatly affected by freezing, whereas the circulatory channels are readily blocked by this cause. Bacterial cells are not killed by ex- posure for long periods to the temperature of liquid air ^^ ( — 190°). Reduction of the temperature of plant cells to — 13° may result in a granular transformation of the cytoplasm, often with rather seri- ous structural alterations. Cytoplasm seems to be more affected than the nucleus, for mitosis may occur slowly in plant cells at — 8°, and Uschinsky ^^ noted that in animal tissues the nuclei were less af- fected by cold than the cytoplasm. Blood seems little affected by freezing temperature, for du Cornu found that dog's blood kept on ice for five to ten days could be employed for transfusion without causing hemoglobinuria. Grawitz saw motion persist in human cili- ated epithelium kept for seven to nine days on ice. Ciliated epi- thelium from the mouth of the frog may survive cooling to — 90°, and frog eggs are not killed by — 60°. In many cells, however, the physical changes produced by freezing, and also bj^ the subsequent thawing, are sufficient to render them incapable of further exist- ence.-'' Cells devoid of or poor in water cannot be killed by freez- ing, hence it is probable that the currents set up about the crystals of ice in thawing, as well as the rapid contraction and expansion under the influence of the cold and the ice formation, are the cause of the effects of freezing, which, therefore, are not dependent upon chemical, but upon physical, alterations. In the case of warm-blooded animals, the gangrene following freez- i7Ziegler's Beitr., 1895 (18), 72. isMacFadven, Lancet. 1900 (i), 849. i9Ziegler's Beitr., 1893 (12), 115. 20 In plant cells it is the freezing and not the thawing that causes the harm (Maximow, Berichte Deut. Bot. Gesell., 1912 (30), 504). 374 h'i:Tii'(Kih'i:ssni-: ciiAyaEH ing depends not so mueli u])()n the freezing of the cells themselves as upon the formation of hyalin thrombi in the injured vessels (v. Recklinghausen, Hodara).-^ Kriege -- found that if the freezing is transitory, the thrombi may again disappear; if over two hours in duration, tliey are persistent. Rischpler,-' however, considers that cell death is due primaril}- to the eft'ect of the cold upon the cells. On tlie other hand, Stechelmacher -^^ found that freezing of liver tissue produced the same changes as ligation of the hepatic artery, i. e., in- creased permeability of the cell wall followed by similar changes in the nucleus, suggesting that the changes produced by freezing depend on the vascular changes. Light.-"' — Light may atfect tissues seriously, apart from the efifects of accompanying heat, although the experiments of xVron -^ indicate that insolation does not depend on the light rays, but solely on the heat. Tn the treatment of lupus by the Finsen method with concen- trated light rays, the action is largely a stimulating one, but associ- ated with or subsequent to a certain degree of cell injury. Ogneff -'' found that moderate action of electric light, rich in violet and ultra- violet rays, causes mitotic cell division : if the action is stronger, the cells undergo amitotic division and then become necrotic. Blue rays have but slight cytotoxic action, and rays further towards the red end of the spectrum are without demonstrable effect. Light l)aths are said by Oerum -•' to increase greatly the quantity of corpuscles and hemo- globin, while residence in the dark reduces these elements. The de- struction of bacteria by light is a well-known phenomenon,-" but it has been suggested that their destruction depends rather upon the action of substances produced in the culture-medium under the influ- ence of light than upon the effect of the light ujion the bacterial cells themselves. In view of the fact that enzymes and antibodies in solu- tion are quite readily weakened or destroyed by the action of light, it is possible that intracellular enzymes may be similarly destroyed by light, with resulting cell death. However, in the case of bacteria, at least, the effects of light seem to depend upon oxidation processes, for in the absence of oxygen, bacteria are not seriously injured by light, and D'Arcy and TIardy -* fouiul that "active oxygen" is formed by the same ])()rtion of tlie sjiectrum that is most active in destroying 21 Miinch. med. Woch., 1896 (43), 341. 22Virc'how's Arch., 1880 (11(5), f>4. 23Ziefrl('r's Beitr., 1900 (2S), .541. 23aBoitr. path. Anat., 1913 (57), 314. 23bT{ovi(nv l)y Bering', Er (17). 390. CAUSKH OF SECR08L-! 375 Lacteria.-" Liill., 1907 (IS), 349) reports a case of multiple subcutaneous fat necrosis witliout pancreatic lesions, in a 14 days' old l)aby, and j^ivcs a re- view of other similar cases. 8« Virchow's Arch., 1H90 (122), 2r)2. 8T Dissertation, Gottingen, 1895. FAT NECROSIS 385 pancreatic juice was the active agent. Flexner ^^ supported this con- tention by demonstratinfj: tlie presence of a fat-splitting enzyme in foci of fat necrosis, wliich was corroborated by Opie.-^ The latter'*" was also able to (lenioiistrate the presence of lipase in the urine of a patient with fat necrosis,'" and the highest values for amylase in the blood and urine are found in pancreatitis (Stocks).'*^'' In a study of the pathogenesis of fat necrosis, particularly with reference to the question whether the lipase or the trypsin of the pancreatic juice was responsible. Wells ^- found that typical fat necrosis could be produced by injecting extracts of fresh pancreas into animals, either of the same species as that from which the pan- creas was obtained, or into a foreign species. Commercial "pan- ereatins" were also quite effective, whether in weak acetic acid or weak alkaline solutions. The power of these materials to cause fat necrosis was reduced by heating to or above 60° for five minutes, and completely destroyed at 71°, indicating that the active agent is an enzyme. But, as in the same material trypsin was injured by temperatures above 60°, and destro.yed at between 70° and 72°, and lipase was weakened above 50°, and destroyed above 70°, it was im- possible to determine, by heating pancreatic preparations, whether the lipase or the trypsin was the essential factor. By permitting pancreatic extracts to digest themselves it was found that the power to produce fat necrosis decreased, pari passu, with the decrease in lipolytic strength. Preparations strongly tryptic, but very weak in lipase, produced no fat necrosis, and, on the other hand, extracts of pig's liver or of cat's serum, both rich in lipase but devoid of tryp- sin, were equally ineflPective. Furthermore, mixtures of liver or serum lipase and trypsin were incapable of causing fat necrosis. Fresh pancreatic extracts from fasting dogs, containing lipase but almost no trypsin (which in fresh extracts is still in the form of inactive trypsinogen), produced abundant fat necrosis, whereas after the trypsinogen in such extracts was activated by enterokinase, no fat necrosis could be produced. It therefore seems certain that trypsin alone cannot produce fat necrosis, and that the decrease in strength of lipase in a pancreatic extract is associated with a cor- responding decrease in power to produce fat necrosis. But, on the other hand, lipase of liver or blood-serum alone, or when mixed with 88 Jour. Exper. Med., 1807 (2), 413. 89Contrib. of pupils of W. IT. Welch, Baltimore. 1000, p. 850; .Tolnis Hopkins Hosp. Rep., 1000 (0), 8.50. 00 Opie, "Diseases of the Pancreas," Lippincott, 1003, p. ].")6: .Johns Ildiikins Hosp. Bull., 1002 (13), 117. 91 It yet remains to be seen if this is a constant occurrence: and also if tlie lipase so excreted comes from the pancreas, for Zeri (II Policlinico. 100.5 (12), "3.3) has found lipase in the urine in hcmorrhajric nepliritis and inihimiiiation of the urinary tract: also Pribram and Loewv, Zeit. phvsiol. Chem., 1012 (76), 480 9ia Quart. .lour. Med., 1016 (0), 216. 92 Jour. Med. Research, 1003 (9), 70. 25 386 RETROGRESSIVE CHANGES trypsin, will not produce fat necrosis. The possibility remains that pancreatic lipase is different from liver or serum lipase, and can by itself cause fat necrosis; more probably, however, the production of fat necrosis depends upon a double action, tr\'psin causing the death of the cells, and lipase splitting the fats."^ The fatty acids alone will not cause necrosis of fat-cells, and it was shown that the first steps in the process consist of a necrosis of the surface endothelium extending into the connective and fat tissue; this may occur in a few minutes, while evidence of fat-splitting can be ob- tained onlj^ after about three hours, and the splitting occurs only in cells that have already become necrotic ; hence the fat-splitting is not the cause of the necrosis, but occurs subsequent to the necrosis. After about four hours a substance appears in the decomposed fat that stains with hematoxylin, which is probably calcium. Fat necrosis may be produced by any means that will cause the escape of pancreatic juice from the natural channels within the gland. In human pathology it has followed trauma and acute in- fection of the gland, and the blocking of the ampulla of Vater b^^ gall- stones which permits the bile to back up into the pancreatic duct, where it produces an acute inflammation of the pancreas (Opie)."* Flexner °^ has shown that it is the bile salts that cause the inflamma- tion, and also that this effect is decreased or prevented by the presence of large amounts of colloids. ]\Iuch emphasis is laid by some au- thors ^** upon the necessity of enterokinase passing up the ducts to activate the trypsinogen (an idea first advanced by Starling and Ba}^- Hss in 1902), but it should be remembered that there are kinases pres- ent in leucocytes, and that kinases can develop in the pancreas itself during autolysis, which can activate the trypsinogen ; hence the pres- ence of entero-kmase is not essential for sufficient activation of tryp- sinogen to account for pancreatitis and fat necrosis. Lattes °'^ believes that fresh pancreatic juice, which digests tissues very' slowly, can pro- duce typical fat necrosis but not the characteristic intoxication; this results from the action of juice which has been activated by entero- kinase, or by products of pancreatic autolysis M'hicli have a similar effect. The kinases of leucocytes he found unable to activate pan- !i3 Wlion fat tissue dies in the liody from other causes, tlie lipase normally eon- tain<'d within the fat tissue does not cause the changes seen in fat necrosis. It is possible, therefore, that the coml)ininrenhuyze, Zeit. phvsiol. Chem." 1014 (91),' 151). 7 See Wliipple, et al, Johns Hopkins TIosp. ■Bull.. 1010 (-21). XV.) -. Karas. Zeit. klin. Med., 1013 (77). 125. 7a Arch. klin. C'hir., 1012 (OS). TT. 2. 7b Whipple and Coodpastiire, Siir";.. Gvn. and OI)st.. lOi;? (17), 541. sChiari, Zeit. f. Ileilk., 180(1 (17), 00 ;" Pforrinjrer, Virehow's Arch.. ISOO (15S), 126; Liepmann, ibid., 1002 (100), 532; WulfT, Bcrl. klin. Woch., 1002 (30), 734. GANGRENE 389 (lead tissue, and as a result such tissue contains all the innumerable products of the decomposition of proteins and fats. Thus Ziegler mentions as morphological elements that may be present in gan- grenous tissue: Fat needles, the so-called "margarin" crystals (a mixture of stearic and palmitic acids), fine acicular crystals of tyrosine, globules of leucine, rhombic plates of triple phosphate, black and brown nuisses of pigment, and crystals of hematoidin. In solu- tion we also have, beyond a doubt, all the substances formed in the decomposition of proteins, from proteoses and peptones down through the different amino-acids to such final products as ammonia and its salts, while CO.^ and 1L,S are abundantly given off. In addition occur, undoubtedly, many of the ptomains which are formed by the action of the bacteria upon the amino-acids derived from the pro- teins." In the sputum from pulmonary gangrene there is but little soluble protein, most of the nitrogen, of which there is much, is in the formed elements. ^'^ The fetid plugs which occur in the bron- chioles in gangrene, the "Dittrich's plugs," were found by Traube to be composed chiefly of fatty acid crystals, and Schwartz and Kayser ^^ ascribe their formation to the action of lipolytic staphy- lococci. If the necrotic tissue is in contact with living tissue over a con- siderable area, enough of these products of autolysis and putrefaction may be absorbed to cause intoxication (sapremia) . At the same time, the formation of such large quantities of crystalloids from the pro- teins of the dead tissue leads to a dififusion of water into this area, with consequent swelling, and often a lifting up of the skin in the form of blisters. Emphysematous gangrene,^- usually produced by gas-forming anaerobic bacteria, particularly by B. aerogenes capsulatus, may also possibly be produced by B. coli communis in diabetic patients in whose blood and tissues there may occur sufficient sugar to permit of gas-formation. Ilitsehmann and Lindenthal ^^ found that the gas produced in cultures by an anaerobic organism which they isolated from a case of emphysematous gangrene, consisted of 67.55 per cent, hydrogen, 30.62 per cent, carbon dioxide, and traces of ammonia and nitrogen ; this corresponds to the statement of Welch and Nut- tall that the gas in the tissues of infected animals is inflammable. Dunham ^* found that the gas produced by B. aerogenes capsulatus in cultures has the following composition : Hydrogen, 64.3 per cent. ; 9 An interestinor observation concerning: gangrene of the lung has been made by Eijkman (Cent. f. Bakt., Abt. 1, 1003 (35), 1), who found in this condition bacteria that secrete an enzyme dissolving elastic tissue. loOrszag. Zeit. klin. Med.,' 1909 (67), 204. 11 Zeit. klin. Med., 1905 (5G), 111. 12 Complete literature bv Fraenkel, Ergebnisse der Pathol., 1902 (8), 403; and by Welch, Johns Hopkins Hosp. Bull., 1900 (11), 185. 13 Quoted by Fraenkel. 14 Johns Hopkins Hosp. Bull., 1897 (8), 68. 390 RETROGRESSIVE CHANGES carbon dioxide, 27.6 per cent. ; other gases, probably chiefly nitrogen, 8.1 per cent. RIGOR MORTIS 15 This topic may be appropriately considered in connection with cell death, since it is a characteristic change occurring after general death. All forms of muscle, striped, smooth, and cardiac, undergo this change, which is shown by a shortening and thickening of the muscle, which also becomes opaque and hard. Rigor mortis begins first in the heart muscle, according to Fuchs,^'' but it is generally observed first in the eyelids, then in the muscles of the jaw, from which point it proceeds downward, although the ujjper extremities may not become rigid before the lower. The time of onset is ex- iremely variable, but the following general rules may be stated : All conditions that lead to excessive muscular metabolism, with its re- sulting increase in the acidity of the muscle fluids, will hasten the onset of rigor mortis ; thus, people killed suddenly during violent activity may remain almost in the position in which they met death. Acute fevers, strychnine poisoning, tetanus, etc., cause likewise a rapid onset of rigor, which may, indeed, appear almost simultane- ously with death or even before the heart has stopped beating. When a liealthy individual meets death without previous exertion, rigor does not usually appear for four or six hours, but will be hastened by heat and retarded by cold. Death from hemorrhage or asphyxia is followed by a slow development of the rigor. Under ordinary conditions rigor usually begins between the first and second hour after death and is complete in one or two more hours. ^' The duration of rigor mortis also is influenced by many factors. In general, it may be said that the duration is in inverse relation to the rapidity of onset, and directly to the musculature of the in- dividual. Therefore, in an emaciated individual dying with fever, rigor may appear and disappear again within two or three hours, or, indeed, escape observation altogether. The body of a muscular man dying from accident or hemorrhage may, on the other hand, show rigor for two or three weeks if kept in a cold place. Once the rigor has been broken by force, it does not again return. Rigor mortis may be produced even before death, through poisons (monobromacetic acid, quinine), and its occurrence, even postmor- tem, does not necessarily mean that the nniscle is dead, for if the part is transfused with a salt solution the rigor may be removed, and the 15 Literature, see v. Fiirlli, TTini(ll)ucli d. Tlioclipm., 100!) (II (2), 252; also Molt/cr and Aiicr, Jour. Kxp. Med., IltOS (10), 45). inZeit. f. Ileilk., IHOO (21, Patli. Abt.), 1. 17 Kifror mortis may develop in tlie dead fetus wliile in the womb, but it cen- erallv disajipears witliin live or six lioiirs. Literature hv WolfT, Areli. f. Gvn., ]!)0:}' (G8), 549; Das, Brit. Jour, of Obstet., 1903 (4), 545. RIGOR MORTIS 391 muscle will thou be found to react to stimuli. This indicates that the chemical chano:es of rigor mortis are not very profound.^** The chemistry of the changes involved in rigor mortis has been a much-contested problem. Two chief doctrines have been sup- ported : one that rigor was not essentially different from ordinary muscular contraction except in degree, and perhaps due to a loss of inhibition to contraction. The other looks upon it as a coagula- tion similar to the coagulation of the blood ; and this idea, it may be said, has had the most general acceptance. Brlicke in 1842 supported this view, and in 1859 Kiihne extracted from muscle a plasjua which coagulated like ordinary blood plasma. The protein which formed the clot is called myosin, and its coagulable antecedent, myosinogen. This experiment ha.s been since repeatedly verified and amplified, especiall}' by v. Fiirth and by Halliburton," who have separated more definitely the proteins concerned in coagulation, and found them to be globulins. There seem to be two : one, coagulating at 47°, called paramyosinogen (Halliburton), constitutes but about one-fifth of the total clotting globulin, and passes readily into the insoluble clot, myosin: the other, which coagulates at 56°, constitutes the re- maining four-fifths, is called myosinogen (Halliburton), or myogen (v. Fiirth), and before becoming changed into myosin it passes through a soluble stage called soluble myogen-fibrin, which is coagu- lated at the remarkably low temperature of 40°. By analogy with fibrin-formation we should expect this clotting also to be brought about by an enzyme, but this has not been proved. Calcium is of influence, favoring coagulation greatly, but its presence is not absolutely essential (v. Fiirth). Of particular importance is the acid reaction of the dead muscle. Normal muscle is amphoteric when at rest, but when active the reaction becomes more and more acid, as it also does when the circulation is shut off, and hence acidity increases greatly after death. The acidity is due chiefly to lactic acid (although the neutral phosphates may become converted into acid phosphates in the presence of the lactic acid, and thus seem to contribute to the acidity), and may increase in twenty-four hours after death by from 6.7 to 12.8 c.c. of "/^o acid for each 100 grams of muscle (v. Fiirth -°). The same author found that although the amount of acid might become in time sufScient to cause coagula- tion of the muscle proteins by itself, yet actually rigor mortis appears before the acidity has reached any such degree. Meigs -^ advanced the hypothesis that the rigor is due to the swelling of the muscle col- loids under the influence of acids, a view which is accepted by von isSoe Mangold. Pfliiper':, Arch., 1003 (Ofi), 4nS. 19 "Chemistry of ^fuscle and Xerve." 1004. 20 Hofnieister's Beitr., 1003 (3), r)43 : see also Fletcher and Hopkins, Jour, of Phvsiol.. 1007 (3.5), 247; Wacker. Biochem. Zeit., 1016 (75), 101. 2iAmer. Jour. Physiol., 1010 (26), 101. 392 RETROGRESSIVE CHANGES Fiirtli and Lenk.-- When sufficient acid is formed in the muscle the swelling may be so great that the structure of the muscle cell is destroyed entirely, and it goes into the condition of "waxj- degenera- tion."-'^ This readih' explains why the time of appearance of rigor is so modified by the amount of muscle metabolism before death. It is, indeed, possible to produce rigor in living animals by transfusing a limb with slightly acid salt solution,-* and in strychnine-poisoning the muscular spasm may pass imperceptibly into rigor mortis. It has been suggested that the disappearance of rigor mortis depends upon beginning autolj'sis of the clot by the intracellular proteases of the muscle, which act best in an acid medium, but proteoses and pep- tones cannot be found in such muscle. It is improbable that the de- gree of acidity ever becomes so high that the myosin is redissolved through a conversion into acid albumin (syntonin), as was formerly supposed. V. Fiirth holds that the re-solution of the rigor is caused by coagulation of the proteins, thus reducing this hydrophilic tend- ency, a view in harmony with recent developments in colloid chemis- try.-^ "Waxy" degeneration of muscles, although usually resulting from the action of toxic substances, is entirely different from cloudj- swell- ing, in that the cytoplasm has become homogeneous and not granular. This is undoubtedly due to the increased accumulation of acid which takes place in muscles when they suffer from a defective oxygen sup- ply, for I have found it possible to produce the tj'pical appearance of Zenker's waxy degeneration by letting weak solutions of lactic or other acids act on muscle fibers.-^^ Even excessive stimulation of muscles was found to be sufficient to cause waxy degeneration, the acid being formed faster than it can be removed.-*^ INIuscles showing the ' ' reaction of degeneration ' ' have been analyzed by Rumpf and Schumm,-^ who found a great increase in the fatty matter, which was about fifteen times the normal amount. The muscle^ deducting the fat, showed a loss of solid matter and an increase of water; sodium and calcium were increased, potassium decreased. There is also a great relative increase in the proportion of phosphorus. 22Biochcm. Zeit., 1011 (33), 341; Wien. klin. Woch., 1!U1 (24), 1070. 23 Wells, Jour. Exper. Med., 1000 (11), 1. 24 The hardness of a linili from which the blood-supply has been shut ofl' by tlirombosis or einbolisin, and also much of the cramp-like i)ain. is probably due to rijror mortis in the muscles caused by acid formation under conditions of sub-oxidation. 25 Corroborated bv Lentz, Zeit. anpew. Chem., 1012 (25), 1513: and Schwarz, Biochem. Zeit., 1012 (37), 35. 2'ia Jour. Exper. ]\Icd., 1000 (11), 1. Corroborated bv Steinmler, \irc]io\v"s Arch., 1014 (21fi), 57. 2" As this work antedates much of the recent work on the influence of acids of metabolic origin upon the swelling of cell structures, attention nuiy be called to the fact that a preliminary report of these experiments was made in the first, edition of this book, written in 100(i. 27 Deut. Zeit. f. Nervenheilk., 1001 (20), 445. ATROPHY 393 bound to })r()teiii in imisrlcs wiiicli have atrophied after nerve section, because of the jx'i-sistenee of iiuelear and loss of non-nueh'ar elements (.Grund -"''), but there is little ehanjie in the i^roportion of mono- and di-amino nitrogen.^^ ATROPHY The chemical chanpfes of simple atroi)liy have not, so far as I can find, been definitely studied. It is to be presumed, in view of the structural changes, that analysis of atrophied tissues would show a relatively high nucleic acid and collagen content. It is known that in atrophy the cell lipoids are not much altered, while the simi)ler fats may be increased in parenchymatous organs. In fatty tissues, of course, the fat is greatly reduced, its place being partly taken by serum (serous atrophy of fat). In the heart muscle, especially, but also to a less extent in the liver and kidney, during atrophy there is an increased pigmentation (brown atrophy) apparently consisting of lipochromes or lipofuscins ; but it is to be doubted that this represents so much an actual increase in pigment as a relative increase through loss of other cellular elements. Atrophied tissues also tend to undergo a marked compensatory invasion by fatty areolar tissue if located in contact with such tissue ; e. g., atrophy of muscles after nerve section, specific muscular dystrophies, and atrophy of the pancreas. Starvation, of course, produces typical atrophic changes in the tissues, and the general effects on metabolism have been especially fully worked out by Benedict.-'*-'' The structural changes in parenchy- matous cells are described ~^^ as of two types ; first, granular changes and vacuolization of the cytoplasm, resembling the effects of osmotic pressure alterations; second and later, lysis of cytoplasm with also some involvement of the nuclei, after the order of autolytic changes. The cell walls may also become indistinct, so that the cells resemble a syncytium. -*'° In the atrophied muscle after nerve section Wake- man ^^^ found a decrease in solids, and a lowered proportion of diamino acids. Morse has considered, by experimental methods, the question of the mechanism involved in atrophy, using especially the involuting tail of the tadpole as his test object.-^® He could find no evidence that autolysis is accelerated during this involution, nor in the atrophying muscle after nerve section. The involution of the puerperal uterus, whether it can properly be called atrophy or not, seems to be the result of heightened autolysis, the products of which are excreted 2TaArch. exp. Path., 1912 (67), .39.3. 28Wakeman, .Tour. Biol. Chem., 190S (4), r37. 28aCarnefrie Inst. Piihl., 1915, No. 203. 28b Cesa-Biandii. Frankf. Zeit. Path., 1909 (3). 723. 28cMorgulis, Howe and Hawk, Biol. Bull., 1915 (28), 397, 28dJour. Biol. Chem., 1908 (4), 137. 28eAmer. Jour. Physiol., 1915 (36), 145. 394 ri:tro(:ri:s>ns, Bull. .Toliiis Hopkins llosp., 1914 (25). l!)."). 2RKjour. Biol. Chcni.. lltlfi (25), 2(51. 20 Review of ^enoral features bv Landstcinev, Zic-^lcr's T^cilr.. 1!)0:? iX]). 2'M . 30 Verb. Deut. Patb. Ccsoll., 100.3 (6), (>:{. CLOUDY fiWEIJAM} 395 of cell i)r()t(Miis, and luMice heat j^reeipitation may hv partly responsi- ble for the tui'hidity of eells in cloudy swi'lling, l)ut it is d()ul)tful if the o-raimles thus formed would be soluble in acetic acid. A careful discussion of the character and characteristics of this process is given by Hell,^"" who concludes that the term cloudy swelling is sound only as a gross description, since microscopically the cells may be found to show albuminous »>i-anules, or fatty metamorpliosis or simple edema. "When present, the granules are of unknown nature — tiiey are not identical with Altmann's granules, although Aschoff and Ernst ^"^ both consider that many of them are derived from the mitochondria. An enormous number of granules may be present in the renal cells without demonstrable impairment of function.'"'' They may disap- pear during acute infections, and they bear no constant relation to fatty changes. We may si)eak with more assurance concerning the swelling of the cell, and attribute it to an edema of the cell contents, it having been shown that in cloudy swelling the water content of the organs is in- creased.^^ This might be produced by a rise in osmotic pressure due to abnormally rapid splitting of proteins with incomplete oxidation of the substances formed, which results in formation of many crys- talloid molecules with high total osmotic pressure, from a smaller num- ber of colloid molecules with almost no osmotic pressure. It has fre- quently been shown that the cell-walls do not lose their semipermea- ble character until the death of the cell occurs ; hence in cloudy swell- ing water diffuses in much more rapidly than the crystalloids can ditfuse out,^- causing a hydropic swelling. This hypothesis is sup- ported by the observations of Cesaris Demel,^^ who found that by modifying the osmotic conditions of the cells, particularly epithelial cells, he could closely reproduce many of the characteristic features of parenchymatous degeneration. It is possible, also, that too high concentration of crystalloids within the cells may be a factor in the precipitation of the cell colloids. In view of the fact that in the earliest stages of autolysis histologic and microscopic changes closely resembling those of cloudy swelling are pronounced, and that organs the seat of cloudy swelling notoriously undergo autolysis with extreme rapidity after death, ^^"^ we may also consider that this process is possibly in part responsible for the change of ordinary in- tra vitani cloudy sw^elling. The appearance of fine granules of lipoid substance ^^ (myelin or "protagon" (?)) in cells during autolysis 30a .Tour. Amer. Med. Assoc, 1013 (61), 4.5.5. 30b Verb. Dent. Path. Gesellsch., 1914 (17), 43 and 103. 'oc Shannon, Jour. Lab. Clin. :\red., 1916 (1), 541. 31 Sc'hwenkenbecber and Tnpaki, Arch. exp. Patli. u. Pbarm.. 1906 (55). 203. 32 See introdiu'torv oliapter conoerninu osmosis: also discussion of edema. 33 Lo Sperimentale. 1905: Cent. f. Path., 1905 (16), 613. 33a See MedisTeceanu. Jour. Exp. :\Ied.. 1914 (19). 309. 34 0rgler, Virchow's Arch., 1904 (176), 413; Hess and Saxl, ibid.. 1910 (202), 149. 396 RETROGRESSIVE CTIANGES and durinj; cloudy swelling is in support of this idea, and chemical analysis of organs showing cloudy swelling gives definite evidence of autolytic decomposition of the proteins and an increase in the water content.^'' Presumably this increase in water is the cause of the low- ered specific gravity of organs exhibiting parenchymatous degener- ation.^° Landsteiner, through his studies of cloudy swelling in human material, also came to the conclusion that autolysis is an important element in its production. Martin H. Fischer ^^ applies the principles of colloidal chemistry to the problem, and concludes that the changes of cloudy swelling may be ascribed to acids developed in the cell. Electro-negative proteins in the cell are precipitated by weak concentrations of acids, fonning the granules in the cells, which can be dissolved again by a stronger concentration of acid as in the characteristic clearing of granular cells by acetic acid. The swelling is explainable by the increased affinity for water of other cell proteins under the influence of acids. This theory is supported by good experimental evidence and has much in its favor, the chief question being whether the blood cannot, under ordinary conditions of circulation, furnish suf- ficient neutralizing salts to prevent adequate acidification in the cells to cause cloudy swelling. 35 Verb. Deut. Path. Gesell.. 1903 (6). 76. 36 See Olsho, Arch. Int. Med., 1908 (2), 171. 37 "Oedema and Nephritis," New York, 1915, p. 455; also Zeit. Chem. u. Indust. Colloide, 1911 (8), 159. CHAPTER XIV RETROGRESSIVE CHANGES (Continued) Fatty, Amyloid, Hyaline, Colloid, and Glycogenic Infiltration and Degeneration FATTY METAMORPHOSIS In 1847, ill the tirst number of his Archiv, Virchow divided the forms of fatty changes that may occur in pathological conditions into two groups — ''infiltration" and "degeneration" — a division tliat has since become classical. By infiltration he indicated the ex- cessive accumulation of fat in the cells in the form of large drop- lets, without destruction of the nucleus or irreparable damage to the cells, and by the use of the term infiltration he implied his belief that the fat entered the cell from without. When the fat remained in the form of fine droplets and the cell became much disintegrated, Virchow considered that the fat was derived from the breaking dow^^ of the cell proteins, and hence the process was considered to be a fatty degeneration of the protoplasm. Since that time scarcely any other subject in pathology has been more warmly discussed than that of the origin of the fat in fatty degeneration, and an appalling amount of literature has accumulated concerning the questions in- volved. It will be impossible to give more than the essential facts that have been developed, referring the reader for the full details of the discussion and evidence to the numerous compilations of litera- ture, particularly those of Rosenfeld,^ and to the original articles cited in the text. PHYSIOLOGICAL FORMATION OF FAT Concerning the normal formation of fat we may summarize the evidence as follows : 1 "Fat Formation," Erfrelmisse der Physiol.. Al)t.. 1, 1002 (1), ti.")l: ibid.. 190.3 (2), 50. Also see discussion in the Verh. Dent. Path. Oosell.. 1004 (Ti). .37-108, and the review bv Leathes in his ''Prolilems in Animal ^Metabolism." IflOO, pp. 71—121, and "Tlie Fats," ^ionoGrraphs on Biochemistry, London, 1010; von Fiirth, "Chemistry of Metabolism," Amer. Transl.. New York. 1916. Concerning theories of role of li])ase in fat metabolism see Chap. iii. Other reviews of literature on patholo£rical fat formation bv Christian. .Johns Hopkins ITosp. tiull.. IflOo (16), 1: Lohlein, Virchow's Arch.,' 190.5 (180), 1; Pratt. Johns TTopkins IJosp. Bull., 1904 (15), 301 (particular reference to heart) ; Wohlpemuth. JTandbucli d. Bio-- chem., 1909, TIT (1), 150; :Ma?nus-Levv and Mever, ihid.. 1910, JV (1). 445; Dietrich, Erpebnisse der Pathol., 1909. XIII (2), 283. Concerning Obesity see v. Bercrmann, Handbuch d. Biochera., 1910. IV (2), 208. Later references of im- portance cited in the text. 397 398 RETROGRESSIVE CHANGES (1) A large proportion of the fat of the body comes from the fat taken in the food, as also does the fat of the milk. This can be shown, as Rosenfeld particularly demonstrated, by starving- an ani- mal until it is as free from fat as possible, then feeding with a large amount of some fat that is of a type different from that normally found in the animal ; the new fat that is then laid up in the fat de- pots of the animal will partake of the characters of the fat given in the food. In case the animal is lactating, the milk-fat will also resem- ble the fat of the food. As a matter of fact, the bod}- fat is not of constant composition, even in the same individual ; it varies greatly with age, having much less olein in infancy than in later years, vary- ing somewhat in composition in the different fat depots in the same body, and apj^areiitly being more or less modified hy diet. (2) Fat may also be formed from carbohydrates. According to Rosenfeld, this fat differs from the fat formed on mixed diet in ha'v- ing less olein in proportion to the palmitin and stearin, and it is de- posited particularly in the subcutaneous and mesenteric tissues rather than in the liver. ]\Ian does not seem to form fat readily from car- bohydrates, but rather burns them to protect his proteins: on the other hand, swine and geese readily form fat from carbohydrates. As the fatty acid radicals of ordinary fat (CigH„|,,Oo, Cif.,H^2025 CigHg^Oo), are much larger than the carbohydrate radicals, a process of synthesis must be involved in the formation of, fat from carbo- hydrates.- (3) Proteins are a possible source of fat, but it has not been estab- lished that they are either a common or an important source of fat in either physiological or pathological conditions, or, indeed, that they really ever do form fat. Upon this statement rests our present tendency to refute the long-cherished conception of fatty degeneration as a true degeneration of cell proteins into fat, as suggested by A^ir- chow. This view was supported by the earlier work of Voit and his school, who believed that they had demonstrated that animals could form fat from protein food, and their work was for a long time ac- cepted as correct. Later Pfliiger and his pupils pointed out what seem to have been essential errors in these investigations, and, after much discussion and experimentation, the majority of physiologists now support the view advanced in the sentence opening this para- graph. Since proteins contain carbohydrate groups, and since fats can be formed from carbohydrates, the ]iossibility of the foi-mation of fats from the proteins in this indirect way cannot be denied. It is also possible that the nitrogen-containing groups may be split out of the amino-acids of the protein molecule, and that the non-nitrogenous residues can then be built up into fatty acid molecules as large as the 2'1'liis. Ma^'inis Levy siifjposts, may 1k' iicc()iii|plislicil (lirduiih lactic acid wliicli is forniod from siifjar. and tlicn, after reduction to an aldciiydo. several of tliese molecules are eombined into the liijrlier fatty aeid. See Loathes, loc. cit., p. S2. I'Al II()I.(K1/<'M. J-'AT ACCVMILATION 399 molecules of stearic, palmitic, and oleic acids; l)ut wc have no proof that either of tliese i)roces.ses occurs in the normal cell or in the cell that is undergoing degeneration. PATHOLOGICAL FAT ACCUMULATION For a long time fatty degeneration was looked upon as one of the chief evidences that fat was formed directly from protein, for the cell protoplasm seemed, morphologically, to be changed directly into fat in this process. Additional support was also claimed from the sujijiosed increase in fat in the ripening of cheese ; ^ from the forma- tion of abundant fat by maggots living in fat-poor blood or fibrin ; and by the apparent conversion of proteins into fatty acids and soaps in the postmortem change, aclipocere. But it has now been well established that there is no true conversion of protein into fat in the fatty degeneration produced experimentally by poisoning with pho-sphonis, etc.,* and the other supposed instances of fat-formation above cited have been discredited by various methods which it will not serve our purpose to discuss here, beyond mentioning that one of the chief sources of error lies in the fact that many fungi and bac- teria ^ can form fat from protein. It having been rendered probable that fat was not formed by dis- integration of the protein of the degenerating cells, it remained to de- termine what the source of the fat observed in the cells under patho- logical conditions might be. and this part of the problem has been largely cleared up by Rosenfeld. This investigator proceeded as fol- lows: Animals were starved until they were extremely poor in fat, then fed upon easily identified foreign fats, such as mutton tallow (which has a high melting-point and can combine with little iodin) or linseed oil (which has a low melting-point and can combine with. much iodin). The animals under these conditions laid up in their fat depots, including the liver as well as the subcutaneous tissues, large quantities of these foreign fats. By starving again for a few days the foreign fat was removed from the liver, leaving still a large amount in the other storehouses, and the animals were then poisoned with phosphorus or other poisons that cause a typical fatty degener- ation of the liver and other viscera. When the fat was extracted from the fatty liver of these animals, it was found that the new fat that had appeared in the liver during the process was not normal dog fat C which it should have been if formed by degeneration of the cell pro- teins), but was, in part, of the same type as the foreign fat which the animals had deposited in their subcutaneous tissues and other fat 3 Even tlie increase of fat in ripcninsr cheese is doiilitful (Xierenstein. Proc. Royal Soc. B., 1911 (S.3). .301: Kondo. P.iochem. Zeit.. 1014 (50), 113). 4 See Tavlor, Jour. Exp. :Med., 1800 (4), 300; Shihata, Biochem. Zeit., 1011 (37), 345. 5 See Beebe and Buxton, Amer. Jour, of Plivsiol., 1005 (12), 466; Slosse, Arch. Internat. Physiol., 1004 (1), 348. 400 RETROGRESSIVE CHANGES storehouses. Furthermore, it was found that animals stai*ved to an extremely low fat content do not develop the typical fatty liver of phosphorus-poisoning:, a fact which Lebedeff had already noted in a case of phosphorus-poisoning in an emaciated patient. Of similar sigiiiticance is the fact that in fatty human livers the iodin number, normally high, falls as the amount of fat increases until it is ap- proximately that of adipose connective tissue.® Therefore, it seems evident that the fat accumulating in the liver during fatty degenera- tion is not derived, as Virchow thought, through a transformation of cell proteins into fat, hut rather is an infiltrated fat brought in the blood from the fat deposits of the body to the disintegrating organ. This work has since been corroborated and extended by many ob- servers, and its correctness can now hardly be questioned.' "Fatty degeneration," therefore, differs from "fatty infiltration" chietiy in the fact that in the former the process is associated with serious in- jury to the cell, caused by the action of toxins or loss of nutrition, while in the latter the cell is not seriously injured and is capable of returning to its nonnal condition whenever the fat is removed.* Fatty "Degeneration" without Infiltration. — By showing that new fat in fatty livers is infiltrated fat, Rosenfeld did not entirely clear up the subject, for, in the course of his analyses of organs that were macro- or micro-scopically the seat of fatty degeneration, he found that there is not always any correspondence between the amount of fat that seems to be present, as determined by microscopic methods, and the amount that chemical analj^sis shows to be present. This is particularly true of the kidney. Thus, the amount of fat and lipoids, or lipins (to adopt the more comprehensive term recom- mended by Rosenbloom and Gies ^ to include neutral fats, fatty acids, soaps, lipoids, and their compounds), present in normal kidneys (dog) was found to vary between 18.5 per cent, and 29.12 per cent, of the dry weight, the average being 21.8 per cent. ; whereas, after producing a typical "fatty degeneration" by means of phosphorus and other poisons, the lipin content was still found to be between 16.9 per cent, and 22.6 per cent.^" In all instances the amount of lipins in kidneys fi Leathos, Lanect, Feb. 27, 1000; Hartley and IMavrojiordato, Jour. Patli. and Bact., 1008 (12), 371; Jaekson and Peare'e, .Tour. Kxp! ^led.. 1007 (0), 57S. 7 Sehwalbe (Verli. der Deut. Path. Cesell., 1003 (0). 71) claims tliat in a sim- ilar way iodin eomjiounds of fat can lie demonstrated to lie Iransjiorted into tlie fatt}" organs. Tlis analyses were merely qualitative, and hy (|uantitative deter- minations I was unable to corroborate his conclusions (Zeit. f. plivsiol. Chem., 1905 (45), 412). 8 A strikinfT proof of the lack of injury associated Avitli fatty in(iltra(ion is shown by the fatty infiltration frequently seen in the liver, especially of alcoholics, in which it may be diOicult to find, microscopically, any cell cytojdasm Ixn-ause of the fat, the tissue looking like fatty areolar tissue; and yet there may be no clinical evidence wliatevcr that tlie lix'cr function has be(Mi im])aire(I i)y tlie jirocess. !> Bioclicm. I'.iillctiii, 1011 (1), -A. 10 Concerning the normal intracellular fats see introductory chapter. I'ATHOlJXilC.lL FAT ACri'Ml I.ATIOS 401 sliowiiiy typiral Tatty degoueratiuu under the microscope was found equal to or less than the normal amount — it was never increased. The same conditions were found to obtain in human kidneys that showed fatty nietaniorpliosis. .Microscoj)ic examination of s])ecimeiis stained with tlie specific fat stains," therefore, uives no indication of tlie amount of fat contained in a degenerated kidney. A pathok)i])on analysis a decreased fat content (Dietrieh, Verh. Deut. Path." CJesellsch., IDOf) (n),'212). -s Literature by I.ieathea, "The Fata," London, 1!M0; l^anp, l\rd as judical injr decreased oxi- dative power. Wells (.Tour. Exper. ^Med., 1010 (12), 007) found that the power of liver tissue to oxidize i)urines was Tiot decreased bv the maximum dearee of fatty defjeneration, but Waldvo .Anthony, .lour. Mod. Itos., 1911 (20), .S5!). ci Faust, Suppl. V,d.. ScliniicdclxTfi's Arch., lOOS, p. 171. •••2 Z. Inimunitiit., Kef., 1!)11 (4), (i.")6. "sa.Joblinp and Petersen, Jour. K.\p. Mod., 1014 (10), 251. 03 Fischler, Cent. f. Path., 1004 (15), 013. I'ATIlOLOalVAL OVCUh'KEXCE OF CHOLKSTEROL 415 salt, while the neutral fats are stained red by the oxazone base (J. L. Smith). Fischler and Gross"'' state that fatty acids are present in atheromatous areas and about the margin of anemic infarcts, but are not recognizable by this method in such fatty degenerations as ])neuin()nic exudates, caseation, etc. Klotz "^ considers that calcium soaps are formed as the first step in pathological calcitication, accord- ing to microchemical evidence ; but a chemical investigation of the same question did not give tlie writer positive results.^' In fatty cells, especially in the liver, crystals are often found and interpreted as fatt\- ac'ds, whirli hre really crystals of neutral fats."' PATHOLOGICAL OCCURRENCE OF CHOLESTEROL 's Cholesterol in crystals is found under somewhat the same conditions as the fatty acids, and although cholesterol is not a fat, but an alco- hol, its phj'sical properties are so similar that it may be considered in this place. (See "Gall-stones," Chap, xv, for further discvission.) The characteristic large flat plates of cholesterol may be found in any tissue in which cells are undergoing slow destniction, and where absorp- tion is poor. Therefore, they are found frequently in atheromatous patches in the blood-vessels, encapsulated caseous areas, old infarcts and hematomas, inspissated pus-collections, dermoid cysts, hydrocele fluids, etc. ; especially large amounts occur in the cholesteatomatous tumors of the ear and cranial cavity. *'*' In degenerative conditions of the central nervous system ^^^ choles- terol may be present in the spinal fluid (Pighini'°), and in an old pleural effusion as much as 1.25 per cent, of cholesterol has been found (Ruppert "^). Windaus ''- found that normal aortas contain about 0.15 per cent, cholesterol, while in two atheromatous aortas he found 1.8 per cent, and 1.4 per cent., the increase being more in the cholesterol esters than in the free cholesterol. Amyloid kidne,ys, however, show an increase only in the cholesterol esters, and not at all in the free choles- terol. (See Relation of Lipoids to Fatty Metamorphosis, p. 404.) Ameseder ^^ found that 28.56 per cent, of the ether extract of athero- matous aortas was cholesterol. The claim of Chauffard that arcus senilis, xanthelasma, and other ocular conditions depend on choles- terol deposition is not substantiated by Mawas."* In liquids the crys- GiZiegler's Beitr., lOO.l (7th suppl.), 343. G5 Jour. Exp. :\rocl., 100.5 (7), 033. 66 Wells, Jour. Med. Research, 190G (14). 491. 67 Smith and White, Jour. Path, and Bact., 1907 (12), 126. 68 Concerning the chemistry of cholesterol see introductory chapter. 69 See Bostrocm, Cent. f. Path., 1897 (8), 1. 69a Southard has described cholesterol concretions up to 2 cm. diameter in the brain and cord. (Joiir. Amer. Med. Assoc, 1905 (45), 1731.) ToRiforma Med.. 1909 (25), 67. 7iMiinch. med. Woch., 1908 (55), 510. T2Zeit. physiol. Chem., 1910 (67), 174. 73Zeit. physiol. Chem., 1911 (70), 458. T4Monatsbl. f. Augenheilk., 1912 (13), 604. 416 RETROGRESSIVE CHANGES tals form <"" Lit.'rature -riven 1)V Hosenl)looni, Arcli. Int. :\Ied., 1013 (12). 30,-). ><'>b Pil,li,,trnipliv hv licwcv. Arch. Tut. .Med., 101(5 (17), 7r>7. 8o<- l)<'iit iiicil. 'W.irh.. l!il';! ( :!".»), r>44. AMYLOID 417 some observations, in nepliritis the amount of cholesterol beare no re- lation to tlie albuminuria, and in uremia it may be low; acute febrile diseases usually show a lowered cholesterol, which is unchanfjed in tuberculosis. The blood content has been reported as low in febrile cutaneous diseases, but high in afebrile cutaneous diseases associated with eosinoi)hilia/"'' However, Denis -'-"^ states, after examination of a large number of cases, that hypercholesterolemia was found only in diabetes, and that low cholesterol values are found in cachexia or pros- tration, but are not characteristic of any particular disease. Experimental hypercholesterolemia in animals leads to a deposition of cholesterol in various organs, especially the aorta, kidneys and liver, accompanied by degeneration in the parenchymatous structures, and excretion of cholesterol in the urine and bile ; gall stones may be formed (Dewey). Sometimes lipoid-filled endothelial cells become so abundant in the spleen as to resemble Gaucher 's disease (Anichkov, ]\Ic^Ieans ~""). Cholesterol in the blood reduces phagocytic activity and antibody formation in experimental animals.^°^ The ratio of free cholesterol to cholesterol esters in normal human blood is nearly constant, the esters being about 33.5 per cent, in the blood and 58 per cent, in the plasma ; in pregnancy the proportion of cholesterol esters is high, in cancer and nephritis it is low.®°^ AMYLOID «i Virchow, in 1853, made the first study of the nature of the substance characteristic of "lardaceous" degeneration, and considered it to be a sort of animal cellulose, because it often became blue if treated with iodin followed by sulphuric acid. To this resemblance in staining reaction we owe the unfortunate, misleading, but generally used, name amyloid.^- It was but a few years (1859) before Friedreich and Kekule showed that the substance in question was of protein nature ; their methods were very crude, but the main fact was soon better substantiated by KiOine and Rudneff (1865). Krawkow,*^ however, sodFischl. Wien. klin. Woch., 1914 (27), 982. soeJour. Biol. Chem., 1917 (29), 9.3. 80f Jour. Med. Res.. 1916 (.33), 481. 80g Dewey and Nnzuni, Jour. Infect. Dis., 1914 (In). 472. 80h Bloor and Knudson, Jour. Biol. Chem., 1917 (29). 7. 81 General literature to 1893, see Wichmann, Ziepler's Beitr.. 1893 (13). 487: also Lubarsch, Ergeb. allg. Path., 1897 (4), 449; discussion in the Verb. Deut. Path. Gesellsch.. 1904 (7), 2-,51: Davidsohn. Virchow's Arch., 1908 (192), 226, and Ergebnisse allg. Path.. 1908 (12), 424. 82 In view of the fact that this substance is cheniically related to chondrin, and that it also closely resembles this substance physically, it has seemed to the writer that the name "chondroid" would be inuch more appropriate than any of the many more or less misleading and inappropriate titles tliat are at present in use. The very multiplicity of these terms, however, prohibits any attempt to introduce still another. A particularly unfortunate source of confusion exists in the use of the name amyloid for a vegetable substance, formed by the action of acids upon cellulose. 83 Arch. exp. Path. u. Pharm., 1897 (40), 196. 27 418 RETROGRESSIVE CHANGES in 1897 g-ave us the first ^ood idea of the composition of amyloid sub- stance through his amplification of Oddi's®^ observation that amyloid organs contain chondroitin-sulphuric acid, finding that amyloid is a compound of protein with this acid, similar to nucleoprotein, which is a compound of nucleic acid and protein. This work has received general acceptance, although a later paper by Hanssen ®^ reports a study of amyloid material isolated in pure condition from sago spleens by mechanical means, which contained no chondroitin-sulphuric acid, although the amyloid organs taken i)i toto do contain an excess of sul- phur as sulphate. This important contradiction to prevailing ideas has not, so far as I can find, been subjected to investigation by others, with the exception of a casual remark by Mayeda ^^ that a prepara- tion of amyloid which he had made did not yield sulphuric acid. Chondroitin-sulphuric acid, which has been studied especially hy jMorner and hy Sclimicdeborg,8" has tlie formula C\sH^^NS0,7, accordinr^). 4S.1 ; Davidsohn, VirchowV Arcli.. 1008 (192), 226. THE ORIGIN OF AMYLOID 421 observations on the difference in composition of amyloid of different origins. Krawkow studied these reactions witli pure, isolated amyloid, and found evidence that the iodin reaction depends upon the physical properties of the amyloid, while tlie methyl-violet stain is a chemical reaction, and hence the iodin reaction is much the more readily altered or lost. As Dickinson ''^ says, amyloid stains with iodin simply as if it absorbed the iodin more than does the surrounding tissue. The methyl-violet reaction is due to the dye forming a compound with the ehondn)itin-sali)huric acid, for Krawkow found that these substances unite witli one another to form a rose-red precipitate. Hanssen, how- ever, holds tliat the dyes react with the protein, the iodin with some other, unknown labile substance. Schmidt found that implanted pieces of amyloid lost their iodin reaction as they underwent auto- lysis, while the methyl-violet reaction was still very distinct.'*'^ It is evident, therefore, that iodin is not by itself a specific stain for amyloid, especially as glycogen gives a similar reaction,''" while true amyloid may not react. THE ORIGIN OF AMYLOID This question has not been at all cleared up as j^et by the advances made in our knowledge of the chemistry of amyloid substance. The fact that chondroitin-sulphuric acid is a characteristic constituent suggests that this body may be liberated in considerable amount dur- ing the destructive processes to which amyloidosis is usually sec- ondary: this idea is further supported by the fact that amyloidosis occurs particularly after chronic suppuration in bone and lungs, both of which tissues, according to Krawkow, contain chondroitin-sulphuric acid. This idea was not substantiated, however, by the experiments made by Oddi and by Kettner,^^ who fed and injected into animals large quantities of the sodium salt of chondroitin-sulphuric acid with- out producing amyloid changes. Unpublished experiments of the writer wdth the same material, as well as with ground-up cartilage and with mucin, were equally unsuccessful. Likewise mice injected by Strada "" with the nucleoprotein of pus, the so-called pyin, or with chrondroitin-sulphuric acid, did not develop amyloidosis. Oestreich ^ injected cancer patients with chondroitin-sulphuric acid for thera- peutic purposes, but no amyloidosis resulted. As it is possible to 95 Allbutt's System, vol. 3, p. 22.^. 96Litten (Verb. Dent. Path. Gesell.. 1904 (7), 47) states that thionin and kresyl-violet are the most specific stains for amyloid, which they color blue; whereas methyl-violet stains red not only amyloid but also mucin, mast cell granules, and tlie groinul substance of cartilage. V. Gieson's stain usually colors amvloid pale vellow, and hvalin red. 97 See Wichmann, Ziegler's Reitr., 1803 (13), 487. 98 Arch. exp. Path. u. Pharm., 1902 (47), 178. 99Biochem. Zeit., 1909 (16), 195. 1 Zeit. Krebsforsch.. 1911 (11), 44. 422 RETROGRnSSIVE CnAXGES cause amj'loidosis experimentalh' in animals, especially chickens and rabbits, by causing protracted suppuration or chronic intoxication with bacterial filtrates, these negative results speak strongly against the idea of a transportation of chondroitin-sulplmric acid, but do not detenuine it finally. They may also, with propriety', be used in sup- port of the statement of Hanssen that amyloid does not contain chon- droitin-sulphuric acid. There is usually much difficulty in producing amyloid experimentally, for in only a certain proportion of cases are the experiments positive (in but about one-third of Davidsohn's- 100 trials, and many other experimenters have been much less successful ) .^ Davidsohn, failing always to get amyloid experimentally after the spleen had been removed, suggests that this organ (in which amyloid is usually earliest and most abundantly observed) produces an enzyme, which causes a precipitation of amyloid in the tissues from a soluble precursor brought in the blood from the site of cell destruction. Schmidt * considers it probable that some enzymatic action causes a precipitation or coagulation of the substance in the tissue-spaces or lymph-vessels. Amyloid is never deposited in the cells themselves,*^ and it seems to be now generally considered that the amyloid material is infiltrated in the form of a soluble modification or precursor and that it is not manufactured in the organ where it is found. It is an interesting fact that a practically identical substance is formed in all tissues and in all species of animals, even when the cause is quite dif- ferent. Whether the precursors are brought to the organ in solution, or in leucocytes, is unknown — probably the former. Pollitzer ^ states that in various infections, especially coccus infections, chondroitin- sulphuric acid is excreted in the urine ; if this is correct it has an undoubted bearing on the genesis of amyloidosis. The presence of glycothionic acid in pus ^ is of similar significance. The hypothesis that amyloid is formed from disintegrating red corpuscles is probably incorrect. Ann'loidosis is produced by the most varied species of bacteria and by their toxins, although the staphylococcus is usually most effective in experimental work.'^ Neither is suppuration abso- lutely essential, for injection of toxins alone {e. g., in preparing diph- theria antitoxin^), without suppuration, may produce amyloidosis, as iilso frequently does syphilis without suppuration and, less often, many other non-suppurative conditions {r. rj., tinnors). 2 Verb. Deut. Path. Gesell., 1904 (7), 39. 3 See Tarchetti, Deut. Arch. klin. iled.. 1903 (75), 526. 4 Verb. Deut. Path. Cesell., 1904 (7), 2. 4a See Ebert, A'ircliow's Arch., 1914 (216), 77. •-'Deut. med. Wocb., 1912 (3S), 1538. bits bv Witte's "peptom\" which consists chiellv of proteoses (Trans. Chicafjo Path. Soc., 1903 (5). 240). 8 See Lewis, Jour. Med. Research, 1906 (15), 449. UYAfJXK DEGENERATIOX 423 Local amyloid accumulations are of some interest in considering the genesis of the usual generalized form. They occur particularly as small tumors in the larynx, bronchi, nasal septum, and eyelids; as all these tissues are normally rich in chondroitin-sulphuric acid, it seems probable that the amyloid arises from a local overproduction of cliondroitin-sulphuric acid, which becomes bound with proteins in .liitu. This makes it seem more probable that, in spite of the lack of positive experimental evidence, general amyloidosis is due to liberation of excessive quantities of chondroitin-sulphuric acid in the sites of tissue destruction. Another form of local amyloid is seen particularly in the regional lymph-glands of suppurating areas; e. g., the lumbar glands in verte- bral caries, the axillary glands in shoulder- joint suppuration. This local amyloidosis is undoubtedly due simply to the fact that these glands receive first, and in largest amounts, the cause, whatever it may be, of the amyloid production. ° Less readily explained are cases of extensive amyloidosis limited to the heart.^° Corpora amylacea will be found discussed under "Concretions" (Chap. xv). HYALINE DEGENEHATION '^ ]\Iuch confusion concerning this condition may be avoided if we ap- preciate that the term hyaline indicates a certain physical condition, which may be exhibited by many substances of widely different na- ture and origin. There is no one chemical compound, "hijnlin," which, accumulating in cells or tissues, produces a hyaline appear- rince. The limits of the application of the term "hyaline degenera- tion," even to histological findings, is not agreed upon, but in gen- eral it is used to apply to clear, homogeneous, pathological substances that possess a decided affinity for acid stains, such as eosin. Somewhat similar substances, usually of epithelial origin, which do not take either acid or basic stains strongly, are usually called "col- loid." We may properly consider that pathological hyalin can be divided into two chief classes according to its origin: (1) connective- tissue hyalin ; (2) epithelial hyalin. Connective=tissue hyalin is characterized, like amyloid, by being deposited in or among the fibrillar substance of connective tissues, and not within the cells themselves, but there are undoubtedly several dif- ferent sorts of chemical substances responsible for various forms of 9 Quite unexplained is the eause of the rarelv observed localization of amyloid in the wall of the urinary bladder. See Luckseli (Verb. Deut. path. Gesell., 1904 (7), 34). Concretions ofivinp the amvloid reactions have been found in the pelvis of the kidnev. (Schmidt, Cent. f. Pathol., 1912 (23), 86.5. Mivauchi. ihid., 1915 (26). 289.) ■ 10 See Hecht. Virchow's Arch., 1910 (202), 168. 11 General literature, see Lubarsch, Ergeb. allg. Path., 1897 (4), 449. 424 JiETJiOGRESSJVE CHAA^GES connective-tissue hyalin. One form is closely associated with amyloid, being- found in organs showing amyloid degeneration, or in other tis- sues in the same body. In experimentally produced amyloidosis in animals it has been shown that such a hyaline substance may appear before the amyloid, wliicii eventually replaces it; hence, it has been suggested that hyalin is a precursor of amyloid.^- Such hyalin differs from true amyloid only in its failure to give the characteristic stain- ing reaction of amyloid; in all other respects, e. g., cause, location, termination, it is the same. As it has been shown (see preceding sec- tion) that the staining properties of amyloid are vers^ inconstant, it is probable that the above-described variety of hyalin is merely an in- completely developed, or occasionally a reirogressively altered amy- loid. However, it is probably not necessary, as some authors have thought, that amyloid should always pass through this hyaline stage in its formation. Quite different, without doubt, is the form of hyalin observed in .scar tissue. This variety develops almost constantly in any scar-tissue after the blood-supply has been reduced to a minimum through con- traction, and is seen characteristically in the corjDora fibrosa of the ovary, fibroid glomerules in chronic nephritis, thickened pleural, peri- cardial, and episplenitis scars, etc. Such hyaline substance occurs independent of the usual causes of amyloid, affects only abnormal fibrous tissue, never changes into amyloid, and is prone to undergo calcification — it surely has no close chemical relation to the form of hyalin that does become amyloid. Presumably, it is similar in na- ture to the collagen of normal fibrous tissue intercellular substance, Avhich has undergone physical rather than chemical changes into a homogeneous hyaline substance. For its physiological prototype it has the thick "collagenous" fibers of the subcutaneous connective tis- sue. Probably of quite different origin is the hyalin that develops from elastic tissue, as seen best in the thick-walled, partly obliterated arteries of the senile spleen; and less characteristically in the early stages of arteriosclerosis, since here the preceding form of connective- tissue hyalin may also occur. Although arterial elastic tissue is related chemically to amyloid, these hyaline vessels do not develop the usual amyloid reaction, but retain more or less of the specific, elastic tissue stains. Presumably this form of hyalin is an increased and physically altered elastin.^^ Epithelial hyalin occurs within the cells, and includes substances of ])r('siima])ly widely diverse chemical nature, from the keratin of s(|uamous epithelium to the snudl intracellular hyaline granules of carcinoma and other degenerating cells (Russell's fuchsin bodies).^* 12 Roe Lubarsch, Cent. f. Pafliol.. 1910 (21). 97. laSco Schmidt, Verh. Dout. path. Cosoll.. 1904 (7). 2. 1* Literature, see llektoen, Progressive ^led., 1899 (ii), 241. coLuun J )i:( n:\JuUATioN 425 Fiiclisin l)0(li(\s are foniid also in plasma cells and, less often, in other cells, inelu(lin<>- granulation tissue; the fuchsin ])odies of this class are believed by Brown ^'' to be derived from red corpuscles, a view also held by Saltykow, but not accepted by all pathologists.'" Extracellu- lar substances of hyaline character, but of unknown composition, may also be i)i"()(luc('(l by epitliclium, c. g., hyaline casts in the renal tu- bules. The composition of none of these forms of hyalin is known, except that by using microchemical methods Unna^^' has found evidence that keratohyalin consists of two elements, one of acid character, ap- parently derived from the chromatin, and a basic substance resem- bling the globulins. Many other pathological materials of widely differing nature may, under certain conditions, assume a hyaline appearance; e. g., fibrinous exudates and thrombi, degenerated muscle-fibers (Zenker's or *'waxy" degeneration), tumor-cells (cylindroma), etc. In all of these the chemical nature of the parent substance or substances is probably much less altered than its physical appearance, but whether the change is related to the process of protein coagulation or not is unknown. Occasionally hyalin, both in epithelium and connective-tissue, takes on a crystalline structure (Freifeld).^^ COLLOID DEGENERATION This term, also, has a very indefinite meaning, and is applied to many different conditions by various authors. Thus, v. Reckling- hausen includes under this name amyloid, epithelial hyaline, and mu- coid degeneration. ^Nlarchand includes hyaline connective-tissue de- generation, and, also, as do most other writers, the mucoid degeneration of carcinoma. Ziegler rightly protests against the inclusion of mucin under this heading, but includes the corpora amylacea. On account of the discovery by Baumann of the specific chemical nature of thyroid colloid it becomes particularly unfortunate that the term "colloid" has such a wide and uncertain application. It would seem that the safest view to take is that the word coUold is merely morpJiologically and macroscopicaUii descriptive of certain products of cell activity or disintegration, which have nothing in common except the fact that they form a thick, glue-like or gelatinous, often yellowish or brownish substance. There is no one definite suhstance colloid, according to the usual usage of the word in pathological literature, but many dif- ferent protein substances may assume the appearance to which the 15 Jour. Exp. MecL, 1010 (12), 5,33. 16 See discussion, Verh. Deut. path. Gesell., lOOS (12), 26.'S: :\liintcr, Virrhow's Arch.. 1909 (198), 105. iGaBerl. klin. Woch., 1914 (51), 598. iTZiesler's Beitr., 1912 (55), 168; also Goodpasture, Jour. Med. Res., 1917 (35), 259. 426 ji'ETh'odh'iussn j: vii amies name "colloid"' is given. Looking at the matter in this waj^ we must recognize as the usual "colloid" substances, the following chemical bodies : Thyroid colloid, the physiological prototype of the group. This consists of a compound of globulin with an iodin-eontaining substance, thyroiodin, the compound protein being called bj^ Oswald iodothyreo- globulin. It occurs pathologically only in cystic and similar chauges in the thyroid or accessory thyroids. Being a specific product of the thyroid (and perhaps of the hypophysis) with definite physiological properties, it manifestly has only a morphological relation to the other forms of colloid found in degenerating tumors, etc. In cysts of the thyroid, and less often in tumors, there is occasionally found a more dense "colloid" material of deeper color, the "caoutchouc colloid" of the Germans; this seems to result largely from transformation of red corpuscles in hemorrhagic cysts (Wiget).^* (The nature of thy- roid colloid is discussed more fully under "Diseases of the Thyroid," Chap. XX.) Mucin, when secreted in closed cavities, as in tumore, where it be- comes thickened by partial absorption of the water, may take on a "colloid" appearance while retaining its chemical and tinctorial char- acteristics. This is particularly observed in the "colloid" carcinomas which arise especially from the mucous membrane of the alimentary tract. This substance is, of course, quite specific both in its chemical luiture and its origin from specialized epithelial cells, and the process should properly be considered as a "mucoid degeneration." Pseudomucin, which difi'ers from mucin in not being precipitated by acetic acid, is a common component of ovarian cysts, and when somewhat concentrated by absorption of water, forms a "typical colloid." Because it is alkaline, this form of colloid tends to stain rather with the acid dyes (eosin, acid fuchsin, etc.), while true mucin stains with basic dyes. Several varieties of pseudomucin have been described by Pfannenstiel, and their properties will be considered more fully in the section on "Ovarian Tumors" (Chap. xvii). The clear, glassy, yellowish substance contained in small cavities of ovarian tu- mors, which is usually called ' ' colloid, ' ' consists of nearly pure pseudo- mucin. All these substances yield a reducing substance on boiling with acids, which is a nitrogen-containing body, f/Jucosamin^^ Simple proteins {e. g., serum-globulin, serum-albumin, nucleo- albumin, etc.) may, when in solution in closed cavities, become con- centrated through absoi*ption of water until they produce the physical appearance of "colloid." Probably the colloid contents of dilated renal tubules, cavities in various mesoblastic tumors, etc., are pro- duced in this way. isVirchow's Arcli., 1000 (IS;")), 410: von Simior, ibid., lOl.j (210), 2711. 10 Ziinpcrle. Miindi. mod. Wodi., 10(10 (47). 414. MUCOID DEaEXEh'ATWy ^27 MUCOID DEGENERATION ATnoin in its typical form, is a compound protein, consisting of a :\Iucm, in Its IH ica ' ^ ^^^^i^ carbohydrate, rjlucosamm. S:;:r:t;r1>oerwir:crds mucin ;Ma. . substanee reducing Sno-'s so ution. Mucin is acid in reaction, probably because of : thZc'dvL* It r;rdily dissolved in very weak alkaline solu^ n s is prec Stated br acetic acid, and its physical properties when n inrirarfc ui.e characteristic. The t;""™-". >;---; ^tl Wv covers a number of related but distinct bodies. Some, sucli as tne "fc. r^c^rare readily distinguished by not b^ing preeipi a - b^ acetic acid and bv being alkaline in reaction: others jield ieducin„ suWances without previous decomposition with acids (paramucm ; while even Tmong tile "true" mucins certain differences in solubility 'tnThe mammalian body we find mucin <"=.<;"■■>;!"= '■;,f™2f'ta tt calitics: (1) as a product of secretion of ePf ^^^ Is (2^ m the interstices of connective tissue, especially of t ndo. . (^^^ J^^J Hanee of svnovial fluid to mucin is more physica than ™emical.) Thpre is a so evidence that mucin or a related body constitutes the cement suSance between all the bodycells. Corresponding to these Zc^iief sources of mucin we And mucoid degeneration occnrring as ;^stincrprocesses in mucous membranes (or tissues derived therefrom) '^Eprthl'ilLT M^cin!-! epithelial mucin represents a distinct prod- uct ospcialized cells, it is questionable if the ordinary app. cation o th tenndegeneration in the sense of the conversion of eell-proto- nlasr^ nto mucin, is correct. Certainly the mucin formation of ca_ t'arhal nflaliation is merely an excess of a -"-> -- '^j^^;^ tbP deo-enerative changes that may be present m the epithelial cells are nroducedbv the cause of the inflammation, and are not dependent upon mucirfornn. ion. Even in the extreme example of mucoid degenera- tion" en i^ carcinomas derived from mucous membranes (the so-c Tn mouse tvimora TTaaland found fflycofren only in squamous coll caroinoma, and in the connective tissue aurroundinfr other tumors (Jour. Path, and Tiact., H»OH (12). 4.3!)). 40Virchow's Arch., 1007 flSn), 1:58. 41 Virchow's Arch., 1900 (183). 188. •*- Elaborate treatise on occurrence of usch. Arch, internat. phvsiol.. 1005 (3), 49; Brault and Loeper, Jour. Phys. et Path. G(n\., 1004 (6), 205 and 720. dLYVOUKS l\ rATII()L()(;l('AL PltOCKHtiES 433 perhaps contained in blood-platelets) in all blood, wliether normal or patliolo<^ieal. '■'■ nirscliberut a trace of calcium and much sodium and potassium. Structure of Calcified Areas. — As before mentioned, in calcifi- cation there is not the same uniform infiltration of the ground sub- stance with lime salts that occurs in bone, yet the calcified area is pos- sessed of a ground substance of organic material which does not dis- solve in acids that remove the salts. There is no definite ratio be- tween the lime salts and this albuminoid matrix, however. At first the salts occur in granules, which may become fused to a greater or less degree. It has been thought b}' some that the deposition occurs in the form of ' ^ calcospherites." These are small calcareous bodies, iisiially of concentric structure, which were first described by Harting. Tliey appear to occur widely distributed in normal tissues, both animal and plant, and seem to be the result of the formation of insoluble calcium salts in the presence of some organic substances, just as urinary and other concretions are formed about an organic nucleus. If calcium chloride and soluble carbonates are allowed to combine very slowly to form calcium car- bonate in a solution of e£rg-albumen, these or indistinguishable bodies are formed, which on being dissolved are found to possess an organic stroma that exhibits a marked affinity for any pigmentary substance that may be present. Apparently, Avhen the proper concentration exists, the salts in crystallizing hold between the crystals tlie albuminous substances by which they are surrounded. Dastre and Morat believe that the substratum is lecithin, which others have found occupying a similar place in prostatic concretions. Calcospherites have been found in tumors, in cystic cavities, and in bodies with beginning decomposition. It may be men- tioned in passing that Littlejohn » observed the abundant formation of calcium l)hosphate crystals in bodies that had been immersed for some time in sea water. Oliver has found calcospherites in the tissues of a cancer of tlie breast. Pettit lo found calcospherites in a. sarcoma of the maxilla, presenting insensible transi- tions into the substance of the osseous tissue, and he suggests the possibility that the calcospherite formation may be related to the formation of bone. It seems, however, that tliey are probably more closely related to the formation of the shells of invertebrates, which are largely composed of carbonates in crystalline structure with an organic ground substance between them, and very little phos- phate indeed. sSee Gigon, Ziegler's Beitr., 1912 (55), 4G; Sprunt, Jour. Exp. Med., 1011 (14), 50. « Cent. f. Pathol.. 1000 (17), 177. ■Virchow's Arch., 1010 (200). 220. sZiegler's Beitr., 1011 (50), 14.3. 9 Edinburgh ^Nled. Jour., 100.3 (13), 127. 10 Arch. d. Anat. Micros., 1897 (1), 107. 438 dALCIFICATION, CONCRETIONS, AND INCRUSTATIONS OCCURRENCE OF PATHOLOGICAL CALCIFICATION As far as we know, calcification seldom occurs in normal tissue, except in the formation of bone. Often the infiltrated tissue is com- pletely dead, as in infarcts, organic foreign bodies, caseous areas, and particularly in old inspissated collections of pus. It may be said that any area of dead tissue that is not infected, and that is so large or so situated that it cannot be absorbed, will probably become infil- trated with lime salts. i\Iost frequently calcified, next to totally ne- crotic tissues, are masses of scar-tissue that have become hyaline sub- sequent to the shutting off of circulation in the scar by contraction of the tissue about the vessels. Elastic tissue also seems prone to an early calcification, and it is not uncommon to see the elastic laminaB of small arteries calcified in an apparently selective manner. A pe- culiar form of calcification is that frequently found in ganglion-cells of the brain which have become degenerated or necrotic, particularly in the vicinity of old hemorrhages; the cells become infiltrated with lime salts until a complete cast of the cell, with dendrites and axis- cylinder well impregnated, is formed. The calcification of renal epi- thelium obtained experimentally by temporary ligation of the renal vessels or by the administration of certain poisons, is more closely related to the formation of ordinary urinary eoneretions than to tissue calcification, the calcium being present as the phosphate only.^^ Cal- cification of epithelial cells does occur, however, and seems to be pre- ceded by hyaline changes, in which hyaline substance the calcium is later deposited, as in epithelial pearls, for example. Metastatic Calcification. — What is perhaps the only exception to the rule that some form of tissue degeneration is required before cal- cification occurs, is the ''metastatic calcification" of Virchow.^- In conditions with much destruction of bone, as osteomalacia, caries, osteosarcoma, etc., deposits of lime salts have been found distributed diffuseh^ in various organs, particularly in the lungs and stomach. As much as 13.38 per cent, of the dry weight of the lung and 12.15 per cent, of the kidney have been found as CaO in such a case.^--'' As there is no evidence that these organs have been the site of any dif- fuse tissue necrobiosis before the calcification occurred, it seems prob- able that the deposits have been made in practically or quite normal organs, because of oversaturation of the tissue fluids by calcium salts. The fact that the lung and stomach, and also to a less degree the kid- ney, are picked out, suggests that the calcification is related to the fact that in these same organs we have the excretion of acids into their cavities, which leaves the fluids in the substance of the organs 11 Jour. Med. Researeli. 1011 (2.")), :17.1. 12 Virohow'a Arrli.. IS.'i.'S (S), 10.3: review by Koekel. Dent. .\reh. klin. IMed., 1899 (64), 332. Biblioj,r,aphv and review bv Wells, Arch. Int. Med., 1015 (15), 574. i-'a Virchow's .\rcli., 1000 (107), 112. ,,,;.; U/NVA'V O/' 77//; /'A'OCESS OF CALCIFIC.iTIOy 439 T 1 .,lk.,lino and an increase in the alkalinity of the oorresponchngly ^^^'^.^"\'' 'Vt'^ecidedlv less soluble. In the stomach fluids makes the calcumi ^f ^^J^^^^'^ ^^^ter-landular tissue about the ealeium deposits ^^ ^J^i::^:':..^ corresponding the upper portion of the ?U>^^*^ .^.^^osed to secrete the acid. Pre- to the parietal cells which '^V^ "^^^j ^''' J,,,,,t of calcium in the blood IS too slignt to ue uiiu skeleton, precipita- attemrted to include 'l;^"^!"'*',!^^'^.^^ ^cX , h oVi.in of the in old age in the metastatic =«1"«°'^*'°°'' *'";"" bably dependent salts to the senile f -'T'*-" .^^ ^''™ :;„tltfon oft^^^^^ tis- :^:JZ:^ rtrs:r iSe\ot:"Tchan,e .... .een. to he rCo'lra^tpoSnffaeto't^tt: solTtion of ealeinn. salt. ,n 111 rt the Uo d%s tcreS hviniectin, or feedin. calcium : t deposi«ons ot calcium salts may take place m injured tissues, or even in normal tissues, as in Tanaka's experiments. • CHEMISTRY OF THE PROCESS OF CALCIFICATION Tn analv^iuK the etiological factors in the production of pathologi- bo at an phosphate themselves, or as ealcium-ion-protem com- ^o nds or perhaps both. This suspension or solution is an unstable eondt;., poss ble only because of the extremely small proportion o calctm"n ^e blood (about 1 :in,O0O), and. therefore, capable of be n"mcrtl rown bv increased alkalinity of the blood, changes m the prtSiiis or CO, content, or changes in the quantity or composition r u 1 • „„+ " -NT T5 «5p>innAt has described a case with ,eS^/^;ST„„°l,':;,'fcZ no. ^L »^^ „™..K- allele, in physiol. Chem'., 1913 (S5). 324. iqnTn) 1 "^:STzSi^^r\^^r^rnif:^': i« a.Vo Kata., Be... pa.„. Anat., 1914 (57), 516. 440 CALCIFICATION, CONCRETIOXS, AND IXCRUSTATI0X8 of tlie ealciiim salts. It is probable, from the work of Barille, that the calcium of the blood exists as a soluble complex double salt, tri- basic calcium-earbon-phosphate (PoOgCa,!!, : 2COo(C03H)2Ca), this compound being possible because of an excess of CO,. (2) Retrog-ressive changes in the tissues are a sine qua non. Hya- line degeneration, the chemical nature of which is not understood, is a veiy favorable condition, as also is necrosis when absorption is deficient. (3) In the areas that are to become calcified the circulation is very feeble, the blood plasma seeping through the tissue as through any dead foreign substance of similar structure, without the presence of red corpuscles to permit of oxidative changes. We may, therefore, imagine that the dei)osition of calcium salts in such areas of tissue degeneration depends upon one or more of the following conditions : (1) Increased alkalinity or decreased CO, in the degenerating tissues, causing precipitation of the inorganic salts in the fluids seep- ing slowly through them. (2) Utilization of the protein of the fluids by the starved tissues so completely, because of its slow passage through them, that the calcium cannot be held longer in solution. (3) The formation within tlie degenerated area of a substance or substances having a special affinity for calcium. (4) Production of a physical condition favoring the local absorp- tion of salts, the least soluble salts accumulating in excess. The first of these conditions seems to come into play especially in metastatic calcification, already discussed. We have no evidence that in degenerating tissues, much less in normal ossification, there is an alkaline reaction developed ; but rather the contrary^ an acid reaction is more usual. But, as explained below, decrease in the CO, content in calcifying tissues, especially when combined with other changes, may be of importance. Lichtwitz ^^ especially has laid emphasis on the possible part played by changes in the proteins in inducing calcification. He advances the idea that precipitation of the colloids in the degenerated area, as in caseation, decreases the amount of crystalloids which can be held in solution, wherefore the least soluble salts, tliose of calcium, are precipitated; by laws of osmotic pressure more calcium in solu- tion will then enter to establisli ('()uilibrium, be precipitated, and make way for more calcium, until the amount of deposit prevents further osmotic diffusion. Altliough suggestive in regard to patho- logical calcification, and probably of importance in the fonnation of concretions, this conception is difficult to aiii)ly to normal ossifi- cation ; also in pathological calcification one would exjiect precipi- tation of calcium to occur in tlie outermost surface of the degener- 11 Doiit. mcd. Wocli., 1010 (aO), 704. l'()l!M\n<)\ OF CM.illU SOM'S 441 ated area, soon loading to a sliell of inorganic material which would limit the deposition. The possibility of the formation of calcium-binding substances within the degenerated area has always seemed the most attractive, and has received the most attention by investigators. Of the special substances that might be present in such areas that would have a high afifinity for calcium, phosphoric acid usually receives first con- sideration, since it is as phos])hate tliat most of the calcium is bound, and also since the possible sources of phosphoric acid in decomposed nucleoproteins and lecithin are so obvious. Less considered in the past, fatty acids offer another possibility, especially in view of the fatty degeneration that so frequently precedes calcification. Proteins might also be formed that Avould combine calcium, especially deutero- albumose, which Croftan ^^ states has a high degree of affinity for calcium, and which would be present in areas undergoing autoly- sis. Formation of Calcium Soaps. — In favor of the possibility that the calcium is first bound as soaps are the following facts : Calcifica- tion occurs chiefly in places where fatty degeneration has occurred, such as tubercles, atheromatous vessels, etc. In fat necrosis fatty acids are formed, which soon combine with calcium to form calcium soaps. Virchow observed calcification in the form of soaps in a lipoma, and Jaeckle ^* found that a calcifying lipoma contained 29.5 per cent, of its calcium in the form of calcium soaps. Klotz ^^ ob- tained staining reactions in calcifying tissues that suggested the pres- ence of soaps, which he also extracted by solvents, and he strongly urges, as the first step in the formation of pathological calcified masses, that the calcium is first laid down as soaps, afterward under- going a transformation into the less soluble phosphate and carbonate. Fischler and Gross -" also obtained microchemical reactions for soaps in the margins of infarcts and in atheromatous areas, but not in caseous areas; they therefore consider that calcium-soap formation is an important step in the process of pathological calcification, but that it is not essential. The value and the interpretation of the his- tological evidence of the participation of calcium soaps is, however, open to question. On the other hand, Wells,-^ studying large quantities of material chemically, found at most doubtful traces of calcium soaps in calci- fying matter, even in the earliest stages, and also very small amounts of other soaps or fatty acids, and, therefore, questions the occurrence of calcium soaps as an essential step in calcification, although not doubting that under certain conditions (e. g., calcifying lipomas, fat I'.Tour. of Tuberculosis. in03 (5), 22. isZeit. physiol. Chem., inn2 (36). .53. 19 Jour. Exper. :Nred., ion.5 (7), 633; 1906 (S), 322. 20 Ziegler's Beitr., 1005 (7th suppl.). 339. 21 See review in Arch. Int. Med., 1911 (7), 721. 442 CALCIFIC ATlOy, COyCREllOyfi, A.VZ) lyCRU STATIONS necrosis) this may occur. In calcification at all stages the propor- tion of calcium carbonate and phosphate was found quite constant, and exactly the same as in normal bone; namely, in the proportion expressed by the formula 3(Ca.5(POJo) : CaCO,, which Hoppe-JSeyler advanced to express the composition of the salts of bone. Hence it seems probable that there are no essential differences between the processes of ossification and pathological calcification,-^'' and there seems to be as yet no reason for assuming that in the former calcium soaps constitute an essential step in the process. Phosphoric Acid in Calcification. — It has generally been assumed that in normal ossification the calcium is combined by phosphoric acid, which probably is derived from the cartilage cells, possibly through autolysis of the nucleoproteins or some similar process.-- Grandis and Mainini,-^ by using mierochemical methods, thought that thej^ found evidence that the phosphorus of ossifying cartilage is converted from an organic combination into an inorganic form (PoOJ, which then takes up calcium from the blood. The methods used have been questioned, and Pacchioni,-* from his studies, was in- clined to the opinion that the calcium entered the cartilage already combined as phosphate. Wells implanted into the abdominal cavity of rabbits various tissues that had been killed and sterilized by boil- ing, and found that tissues rich in nucleoproteins showed no tendency to take up calcium in greater amounts than did tissues poor in nucleo- proteins, which result speaks against the idea that phosphoric acid derived from nucleic acid combines the calcium. On the other hand, implanted dead cartilage soon became thoroughly impregimted with calcium salts, which seemed to be deposited in the same proportion as to carbonate and phosphate as in bone. Physical Absorption of Calcium Salts. — As there could be no question of "vital activity" on the part of this boiled cartilage, it seems most probable that there exists in cartilage a specific absoi"p- tion affinity for calcium salts, similar to the absorption affinity that ITof meister -^ observed exhibited by other organic colloids (gelatin disks) toward various crystalline substances in solution. It is of sig- nificance that the substances in which calcium is deposited are, in most instances, of similar character, being homogeneous and often hyaline, although of the most varied chemical composition; in other words, they agree much more in physical tlian in chonical stnicture. Also we find tliat liyalino tissues witli an affinity for calcium often exhibit a similar affinity for othei- substances, such as pigment and '.iiii Dyps tliat stain the bones \\\wn fed to livinji: animals (madder) also stain patholof^ical calcific deposits (Macklin, Anal, rvccord. 1!)17 (11). 387). 22 TTanes, who observed that the phosphatids disappear from tlie liver of the developing chick, supfjests this as a source of the iijiosphoric acid rcijuired for ossification (Jour. Exper. ]\led., 1012 (10), 512). 23 Arch, per la sci. ]\Ted. Torino, 1000 (24), 07. 2'» Jahrb. f. Kinderheilk., 1002 (56). 327. 2.'. Arch, exper. Path. n. Pharm., 1801 (28), 210 OSTEOMALACIA 443 iron.-" Ilofmeister advances the hypothesis that when the cartilage or other matrix becomes saturated with calcium salts, any decrease in COo content of the solution will lead to a precipitation of calcium salts, thus restoriiijj; to the cartilage its power of absorbing more calcium salts whenever the fluid comes to it with a higher degree of saturation with calcium salts and COg. This hypothesis is in har- mony with Barille's observation that when the CO2 is reduced the complex car])oii-pliosphate of calcium precipitates a mixture of car- bonate and phosphate in the same proportions as found in bones and calcific deposits generality. The fact that this ratio (10 to 15 per cent. CaCO.j and 85 to 90 per cent. Ca;j(P04)o), is found in all stages of calcification, is entirely in favor of the above hypothesis, and opposed to the idea that any special chemical precipitant formed in the calcifying area is responsible for the deposition of calcium. Taken all in all, the evidence seems in favor of the view that normal ossification and pathological calcification (except metastatic calcifica- tion and the calcification of fat necrosis and other areas of necrotic fat tissue) depend more upon physico-chemical factors and variations in CO2 concentration than upon the presence of chemical precipitants in the tissues. OSTEOMALACIA 27 In this condition the quantity of inorganic salts in the bone is greatly decreased, while, at the same time, their place is taken in part by new-formed osteoid tissue ; as a result, the proportion of the weight of the bone formed by inorganic salts is reduced to as low as 20 to 40 per cent., instead of being from 56 to 60 per cent., as in nor- mal bone. This has suggested that the cause of the disease may be a solution of the lime salts by some acid, but Levy -^ found that in osteomalacia the proportion of calcium carbonate and phosphate in the bones remains constant, as also does the proportion of cal- cium and phosphoric acid ; if the decalcification occurred through solution by lactic or other acids, the carbonate should be decora- posed first, whereas the lime salts seem to be taken out as mole- cules of calcium carbonate-phosphate ; i. e., in the same propor- tion as they exist in the bone. On the other hand, it has been found in Pawlow's laboratory that dogs kept for long periods after a pan- creatic fistula has been established, develop a condition resembling osteomalacia, "° which would seem most reasonably explained as due to the constant loss of alkali in the pancreatic juice. Furthermore, investigation of Levy's objection to the acid solution theory has led 26 See Sprunt, Joiir. Exp. Med., 1011 (14), i^O. 27 See also review in Albn and Neuberp's "^rineralstofTwechsel," Berlin, lfl06, pp. 124-127; biblioirraphy by Zesas, Cent. Crenz. :\led. u. C'hir., 1007 (10), 801; full discussion bv ]\IcCru'dde'n, Arch. Int. INIed., 1010 (•>), riW: 1012 (0), 27.3. 28Zeit.. physiol. Chem., 1804 (10), 2.30. 29 Babkin, Zeit. Stoffwechsel, 1010 (11), .561; Looser, Verh. Dent. Path. Gesell., 1007 (11), 201. 444 CALCIFICATION, COXCRETIOXS, AXD INCRUSTATIONS to the observation that when mixtures of calcium carbonate and phos- phate are in colloids they are dissolved at equal rates.-''^ McCrudden found tliat in the bones in human osteomalacia, together with the de- crease in calcium there is an increase in the magnesium ^° and sul- phur, because of newly deposited tissue poor in calcium. Histologic- ally, absorption seems to depend largely upon a direct eating out of bone tissue, both organic and inorganic substance, by osteoclasts (Cohnheim), followed by a formation of an uncalcified osteoid tis- sue. (Senile osteoporosis differs chiefly in that no new osteoid tissue is formed.) According to Dibbelt ^^ when osteomalacia is experimen- tally induced in pregnant dogs and then recovery is allowed to take place, the decalcified bone substance present in the active stage does not become calcified, but is absorbed and replaced by new bone. Studies of metabolism in osteomalacia have shown a loss of calcium by the body, especially in the urine, as shown by the following table given by Goldthw^ait et al. : ^' Limbeck Neumann Goldthwait CaO in urine (gm.) CaO in feces 1.773 3.S34 3.859 1.800 Total excreted Total in food 5.607 2.965 2.965 11.65 11.26 5.66 4.56 Loss of CaO 0.39 1.10 McCrudden also found a considerable retention of nitrogen and sulphur, which may be retained in the new-formed osteoid tissue ; magnesium is also retained, probably being substituted for calcium in the bones. It is known that when magnesium and strontium are given to growing animals they will partially replace the calcium in the bones,^-'' while it is said by Etienne ^^ that excessive feeding of cal- cium itself leads in time to decalcification of the bones. Zuntz ^* found the respiratoiy metabolism in osteomalacia, within normal lim- its, but tending to be low; protein metabolism shows nothing strik- ing, but there is a high excretion of phosphoric acid througli the feces. Castration of women with osteomalacia has been frequently, but not always, followed by improvement or recovery,^^"^ and Neumann, and also Coldthwait, have found that in these cases the calcium loss is rejjlaced by a marked calcium retention after the 0])eration. AVhat tlie relation of the ovaries to calcium metabolism or to osteomalacia 2naKran/. and Lioso<,'anfr, Dent. Monat. Zalniheilk., 1914, p. 62S. 30 Corrolmratod by C'ai)pez/.U(>li, Biocliem. Zeit., 1909 (16). 355. 31 Arbeit. Patli. Tnst. Tuhinf,'en, 1911 (7), 559. 32Goldlli\vait, Painter, Osfjood and I\IcCr\idden, Amcr. .lour. I'livsiol.. 1905 (14), 389. 32a See Lelinerdt, Zeit. exp. Med., 1<)1;5 (1). 175. 33 Jour. I'livsiol. et Path., 1912 (14). lOS. 34Areli. f. fiyn., 1913 (99), 145. 34a Bibliography by Schnell, Zeit. Oeb. u. (Jyn., 1913 (75), 178. h'lChlJT.S 445 may be has not yet been ascertained, ^charf e ^■' and Bucura ^" both state that tliere are no cliaracteristic or constant structural altera- tions in the ovaries in osteonuUacia. McCrudden "' found that the improvement in calcium metabolism observed after castration may be but temporary, and therefore believes that the primary cause of the disease does not lie in the ovaries. He is of the opinion that re- peated drains on the calcium of the bones, incited most often by pregnancy, occasionally by tumors, sometimes by unknown causes, result in an excessive reaction to the stinuili, so that eventually the losses become too great to be made up ; that is, osteomalacia is an exaggei-ation of a normal process resulting either from excessive stimulation of that process, or a failure to recover when the stimulus ceases. The beneficial effects of castration are probalily ascribable chiefly or solely to the prevention of pregnancy. Osteitis deformans seemed to be a localized osteomalacia. The relation of the adrenals to osteomalacia advocated by Bossi,^* is of questionable significance, and there is no definite evidence as to any relation of exophthalmic goiter ^^ or the parathyroids,^" although hyperplasia of the parathyroids has been described." RICKETS i-^ As with osteomalacia, chemical studies of the bones in rickets have thrown little light upon the etiology or pathogenesis of this condition. As the following table (taken from Vierordt*^) shows, there is a marked deficiency in the proportion of inorganic salts in the bones in rickets. The proportion of the different salts seems to be quite the same as in normal bone. Normal bone of a two months old child Rachitic bones Tibia. Ulna. Femur. Tibia. Humerus. 18.88 81.12 Ribs. 37.19 62.91 Vertebras. Inorganic matter Organic substance .... 65.32 34.68 57.54 1.03 6.02 0.73 33.86 0.82 64.07 35.93 56.35 1.00 6.07 1.65 34.92 1.01 20.60 79.40 14.78 0.80 3.00 1.02 72.20 7.20 33.64 66.36 26.94 \ 0.81 / 4.88 1.08 60.14 \ 6.22 / 32.29 67.71 Calcium phosphate .... Magnesium phosphate. . Calcium carbonate .... Soluble salts Collagen (or ossein) . . . 15.60 2.66 0.62 81.22 445. 35 Cent. f. Gyn., 1900 (24), 1216. 36Zeit. f. Heilk., 1907 (28), 209. 37Amer. Jour, of Physiol., 1906 (17), 211. 38Zent. f. Gyn., 1907 (31), 69 and 172. 39Tolot and Sarvonat, Rev. d. Med., 1906 (26 4"Erdheim, Cent. nied. Wiss., 1908 (46), 163. ■41 Bauer. Frankfurter Zeit. Pathol., 1911 (7), 231. •12 Complete literature and full discussion bv Pfatmdler. -lalir. f. 1904 (60), 123: also see Albu and Xeuberg. ".Mineralstotl'weclisel," pp. 119-124; symposium in the Verhandl. Deut. Path. Gesellseh., Metabolism studies by Meyer, Jahrb. Kinderheilk., 1913 (77), 28. 43 Xothnagel's System, vol. 7, part ii, p. 21. Kin Ber 1909 dcrheilk.. n. 1906, (13), 1. 446 CALCIFICAriOX, COyCKETIOXH, AXD IXCRL STATIONS INIore modern analyses** show a relative increase in water and magnesium, with a persistence of the normal ratio of calcium phos- phate and carbonate.**'' Cattaneo *"^ finds the increase in magnesium to var}- in different parts of the skeleton, being greatest in the ribs. The blood of children with rickets shows greater variations from the usual CaO content (8-10 mg. per 100 c.c.) than are found in normal children ( Asehenheim) .*'"' As an essential difference from osteomalacia is the fact that in rickets there is a failure on the part of the osteoid tissues to calcify, Avhereas in osteomalacia absorption of calcified tissue takes place with subsequent substitution by osteoid tissue. Furthermore, in rickets the deficiency in calcium is said to be present only in the bones/'^ whereas in osteomalacia the soft tissues are also poor in lime salts. According to Schmorl *'^ the first structural abnonnality in rickets is a failure to lay on calcium by small islands of cartilage in the zone of preparatory calcification. None of the various hypotheses as yet advanced to explain this defective ossification has satisfactorily explained all the observed facts. That a deficiency of calcium in the food is the cause of rickets is a most natural assumption, but it has not been proved that this is the case. Young animals fed on calcium-poor foods show, naturally enough, defective development of the bone,*'^ but this differs essentially from rickets in that the bone formed is defective chiefly in amount rather than in quality (Stoltzner). Furthermore, such "pseudo-rachitic bone" jDOSsesses' a marked affinity for calcium salts^ and takes them up as soon as they are supplied (Pfaundler). In view of the fact that rickets is not solely a disease of bone tissue, but that all the various important viscera, as well as the muscles and tendons, show pathological changes, it seems most reasonable that rickets should be looked upon as a constitutional disease, in which the bone changes are prominent chiefly because the disease occurs at a time when the bone tissue is most actively forming and when the other organs are relatively quite completely developed. Stoltzner,*^ finding evidence that rickets does not depend upon either lack of calcium in the food or deficient absorption of calcium, and that the blood in rickets is of normal alkalinity, looks upon the failure of calcification as depending upon an abnormality in the calcified bone tissue itself. He finds evidence of a preliminary alteration in normal 44 Gassniann. Zoit. pliysiol. Clicm., iniO (70), 101. •»414. 5-.a Bi),lio;,rrapliy liy Haciiicislcr, Kr;,^^!). inn. Mod.. l!)l:? (11), 1. r,ob Fischer and Kosc found about 0.1 ,'all stones from cattle. (Zeit. physiol., Cheni., li)13 (8S), 331.) JUIJAUY CALCULI 449 traces of copper/"' iron, and iiiang-anese." The quantity of bile salts, the chief constituent of the bile, is usually extremely minute, appar- ently only so much as may percolate into the crevices of the concre- tion. However many stones there may be in a gall-bladder, they usually are all of approximately the same composition and structure. In o-all-stones from the domestic animals the i)rop()rtion of inorganic salts is usually much higher than it is in man. Naunyn has classified gall-stones according to their composition, as follows : 1. "Pure" Cholesterol Stones. — The purity is only relative, since even the ])urost always contain some pigment as well as a stroma and a nucleus; but the amount of cholesterol may reach 98 per cent., and is usually over 90 per cent. Crystalline structure is usually well marked, while stratification is slight. The color varies from nearly ]iure white to yellow, or even brown on the surface. 2. Laminated Cholesterol Stones. — These consist of about 75-90 per cent, of cholesterol, and differ from the preceding form in con- taining more pigment, which is deposited in layers alternating with the white layers of cholesterol. The pigrment here, as in all other gall-stones, consists always of the calcium salts of the pigments — not of pure bilirubin and biliverdin themselves. Considerable calcium carbonate is also usually present, particularly in the green layers of biliverdin-calcium. 3. Common Gall-bladder Stones. — The composition of this form is but little different from the above, the chief difference being in the structure. They present externally a firmer crust, usually distinctly laminated ; in the center is a softer pigmented nucleus which fre- quently shows a central cavity containing fluid. Such calculi are not distinctly crystalline in structure, and are small, seldom larger than a cherry. 4. Mixed Bilirubin-calcium Calculi. — These generally occur singly, but sometimes in groups of three or four, and are of large size. Although the chief constituent is bilirubin-calcium, there is always much cholesterol, often over 25 per cent. Copper and traces of iron may also be present. Their structure is laminated, with sometimes a crystalline cholesterol nucleus. 5. "Pure" Bilirubin-calcium Calculi. — In addition to the chief con- stituent, 'biliverdiii-calcmm, hilifuscin and hilihumin ^^ are practically 56 See Mizokuclii, Cent. f. Pathol., 1912 (23), 3.37. 57 Gall-stones have been found enclosing droplets of merpury. (Xaunyn. Frerichs. ) 39 Biliverdin differs from hiliruhin in containing- one more atom of oxygen in the molecule, and it is easilv formed from Ijilirubin — even exposure to air will slowly bring about the oxidation. Bilifuscin is a still more oxidized deriva- tive— so much so that it does not give Gmelin's reaction (with PTXO. + HNOj) for bile-pigments. Bilihumin represents the most oxidized of these products, is brown in color, and is the chief constituent of the residue left after treating gall-stones with ether, alcohol, and chloroform to dissolve out the cholesterol. 29 450 CALCIFICATIOW ('<)\Cin:TI()\S, AM) IWhTSTATlOXS always present. liilihumiii is at times tlie chief iiiKi'eilient. and niay form over half of the substance; bilicijanin is rarely present. There is always some cholesterol, but sometimes onlj^ traces. These calculi are small, from the size of a grain of sand to that of a pea, and they occur in two distinct forms. One form is of wax-like consistence; the other is harder. steel-<>Tay or black in color, with a metallic luster. Pure bi]irul)iii and hiliverdin, not coinhiiicd with calcium, are prac- tically never ]i resent in concretions. 6. Rarer Forms. — (a) AniorpJwus (okI incomplete]]) cri/stdlJine cho- lesterol gravel. Cholesterol externally giving' them a pearly luster: pigment in the center. (6) Calcareous Stones. — Consist chiefly of a mixture of calcium carbonate and bilirubin-calcium. Calcium carbonate may occur either as a superficial crust, or as small masses within an ordinary calculus; calcium sulphate and phosphate occur rarely in traces. Stones consisting mainly of calcium carbonate are extremely rare in man, but more frecjuent in cattle and other herbivora, in which all forms of concretions contain much calcium, either combined with pigment or as carbonate and phosphate. A calcium oxalate gall-stone has also been described.*^" (c) Concretions ivith included bodies, and conglomerate stones. (d) Casts of Bile-ducts. — Occur particularly in cattle, and consist chiefly of bilirubin-calcium. Rarely and imperfectly formed in man. Formation of GalUstones. — We owe much of our present under- standing of the chemistry and pathology of the formation of gall- stones to Naunyn "^ and his pupils. Former observers, having learned that bile normally contains cholesterol (Hammarsten found from 0.06-0.16 per cent, in human bile), sought the cause of gall- stones in either an increased elimination of cholesterol by the liver, or a decrease in the power of the bile to hold the cholesterol in solution. Thus Frerichs, finding that the presence of large amounts of bile salts and an alkaline reaction favored the solution of cholestei-ol, im- agined that a diminution of either bile salts or alkalinity led to the precipitation of the cholesterol. Naunyn and his pupil's, however, observing that the amount of cholesterol present in the bile does not depend upon the amount taken in the food or tlie amount present in the blood, and that it did not vary in disease, exce]it when gall- stones were present, concluded lliat the cholesterol of the bile is neither a product of geiu'ral metabolism nor a specific secretion-prod- uct of the liver. Finding that ])us and the secretions from inflamed iiiiii'ous iiK'inbranes (bronchitis) contained as much cholesterol as did iioniial bile, and often more, they concluded that the chief source of '^'1 Mniit lain-. Hull. sci. |pliaiiiiaf(il.. \'ol. IS, p. 111. '•' All l-^iifrlisli translation of tliis classic work, by .\. K. (iarrod, lias Iuhmi |)u1)- lislicd by the Sydenham Society, ISiXi, vol. l.'iS. "Nfoio recently excejition has been biken to certain of Nanii.\ii's \ icws, cs|)ccia]ly by AsdiolV and nacnicister. "Cholelithiasis," Custav Fisclicr, .Iciia. l!l()!l. lill.lMH rM.ci /.I 451 eliolestorol in ^all-stoiu' roniiatioii was fi-oiii tin- (lc;:riK'ratiii^ and desquamated epithelial cells of the jiall-hladdcr and l)ilr tracts. This idea was supported by the larjie amount of cholesterol found in the coutents of tiall-l)ladders shut off from the conunon duct, and by the formation of (jall-stones in such isolated 3 Aschotr, Miinch. med.' Woch.. 1906 (53). 1847 and 1913 (00), 1753; Laroche and Flandin, Compt. Rend. Soc. P.iol.. 1912 (721. (i60. 04 See Cushing (Jt)hns Hopkins Hosp. Bull., 1899 (10). l()(i), who produced gall-stones experimentally by injecting typhoid bacilli into the circulation after injuring the gall-bladder. Literature (m the relation of bacteria to gall-stones given by Fnnke, Proc. Path. Soc, Philadelphia. 1908 (11), 17: also see Uosenow who finds that streptococci are often res!)onsible (Jour. Infect. Dis., 191fl (19), 527). «3Jour. of Physiol., 1903 (29), 341; see also Hansemann. Vircli. Arch., 1913 (212), 139. 452 CALCIFICATION, CONCRETIOXS, .l.VZ) IXCRl'STATIOyS masses, consisting chiefly of cholesterol and bilirubin-calcium. From this material calculi may eventually form, and by their irritation lead to further formation of cholesterol and increased o:rowth.''*'^ But bacterioloo-ical studies indicate that generally an infectious influence is present in cholelithiasis, and bacilli may be found alive in gall-stones for remarkably long periods. Recent applications of colloidal chemistry' add much to our under- standing of gall-stone formation. Thus, Lichtwitz points out that the colloids of normal bile, all of which are electro-negative, may be precipitated by positive serum colloids coming from the blood when the gall-bladder is inflamed ; hence we get a precipitate of cholesterol, bilirubin and proteins. Wlien the colloids are thus thrown down the solvent power of the bile for the alkali earths it contains is decreased, and so calcium or magnesium are added to the mixture. Cholesterol is in solution in the bile as an emulsion colloid, and when stagnation of the bile leads to absorption or disintegration of the cholates and fats which keep it in solution, the droplets become confluent, and then ciystallization takes place (Schade) with formation of sphero- liths, and eventually a crystalline cholesterol calculus. If cA'en the slightest pressure is brought to bear on the myelin-like masses before they crystallize, however, they will be pressed into scales, and the common laminated structure results ; hence crystalline calculi are single, while multiple gall-stones are laminated, with perhaps partial crystallization between the lamellae. Also when the gall-stones result from inflammation, and there is much serum colloid present, the stones are lamellated because these colloids deposit in that form (e. g., corpora amylaeea and other protein concretions). These considera- tions explain the formation of gall-stones in the gall-bladder from either inflammation, or stagnation without inflammation. AschofiP and Bacmeister,*'" however, hold that the usual series of events in the fonnation of gall-stones is first the formation of a pure cholesterol stone without inflammatory cause, because of actual in- creased excretion of cholesterol by the liver, because of cholester- olemia, or because of resorption of solvent substances from stagnating bile; these primary cholesterol stones then cause inflammation and occlusion, leading to the formation of the common mixed stones. Bacmeister ascribes more importance to calcium than do most other investigators, while Kuru ^^ states that fibrin is usually present. More recent studies of the cholesterol content of the blood and bile also have reacted against the concept that all the cliolesterol of gall- stones comes from the wall of the bile tract througli inflammatory changes. It has been found that patients with gall-stones often show "^a Concerninfj the stnicturo of frail stonos sec Kibhort. VirchowV Aroli.. IHlo (220), 20. nn Zioplor's Beitr., lOOR (44), .'i2S. «7Virc]iow's Arch., 1912 (210), 4.1.'^. BILIAKY CALCULI 453 a hypereliolesterolemia (Ilenes""'') ; that pregnancy, which seems to be a predisposing cause of cholelithiasis, is accompanied by hyperchol- esterolemia; that with hypercholesterolemia there is an increased out- put of cholesterol in the bile, and that experimental hypercholesterole- mia may lead to the formation of gall-stones without evident infection of the bile tracts (Dewey °'^). As far as the existing evidence permits one to draw conclusions, it would seeui probable that both local and systemic conditions are of importance in gall-stone formation. Ap- parently, gall-stones may form from cholesterol derived from the in- flamed bile tract walls, independent of the amount of cholesterol pres- ent in the bile ; but presumably they may derive part if not all the cholesterol from the bile in some cases. In either event, a hyper- cholesterolemia will favor their formation, and hence any given con- dition of injury to the gall bladder will more often give rise to con- cretions in persons with a high cholesterol content in the blood. Changes in the bile itself may be produced by disease of the liver that will alter its composition in such a way that its capacity to sustain cholesterol in solution or suspension will be lowered,"*^ and this fac- tor also cannot be dismissed as without importance ; transient thicken- ing of the bile, such as may occur in any febrile disease, may also very possibly initiate precipitation and stone formation.*^'*^ It w-as formerly supposed that the calcium-pigment concretions were produced by the presence of excessive calcium in the bile, de- rived particularly from lime-laden drinking-water, but it has been demonstrated that increase of calcium in the food does not cause an increase in the amount in the bile. Furthermore, on concentrating bile, which contains both bilirubin and calcium, the free bilirabiu separates out and not the calcium compound of bilirubin; and also, Naunyn found that the bile salts prevent precipitation of calcium- bilirubin, even when calcium salts are added in considerable amounts. Apparently it is the presence of positively charged protein substances that leads to the precipitation of this electro-negative substance from bile, and hence the formation of pigment calculi is also favored or initiated by inflammation of the bile tracts, particularly as most of the calcium salts seem to come from the mucous membrane ; ^^ later, as we have seen, these pigment concretions often become covered with cholesterol derived from the injured epithelium, and the common mixed calculi are then formed. In view of the fact that much of the pig- ment in these calculi is composed of the oxidation products of bili- 67a Surg., Gvn. and Obst.. lOlf, (2.3), 91. sTbArch. Int. Med., 1016 (17), 757; see also Aoyama, Deut. Zeit. Chir., 1914 (1.32), 234. cTcSee D'Amato, Biochem. Zeit., 1915 (69), .353. 67dSee Rovsing. Hospitalstidende, 1915 (58), 249. (■■8 This commonly-held view is denied by Lichtwitz and Bock (Deut. med. Woch., 1915 (41), 1215), who found the calcium content of bile from fistulas to be from 65-84 mg. per liter, and in l)hidder bile to vary from 85 to 325 mg., but not according to the presence or absence of inflammation. 454 CALCJFicAriox, coxcnirr/oxs. \\i> ixchTsTAriows rubin, especially billhunilii, it is p()ssil)l(' that oxidatiou processes in the stagnating bile are im])ortant causes of the precipitation; Naunyn suggests that bacteria may be the cause of the oxidation. Pigment calculi are particularly important as the starting-point of the larger mixed calculi. It is possible, Naunyn believes, for the pigment to be later gradually replaced hy cholesterol. URINARY CALCULI >■•■> These differ from the l)ile concretions in two imi)ortant respects: First, there is no evidence that any considerable part of tlieir con- stituents may come from the walls of the cavities that contain them; they are usually deposited on account of an over-saturation of the urine, or on account of a change in composition of the urine, which renders them insoluble. Second, the composition of urinary calculi is usually less mixed than that of biliary calculi, although seldom, if ever, is it pure. Thus, Finsterer found but six concretions composed of only one substance, in a collection of 114 calculi. As with the bile, the chief constituent of the urine (urea) is so soluble that it never forms concretions, but only the less soluble minor constituents are thrown down. For the formation of calculi, however, it is not sufficient to have merely an excess of a substance in the urine, for we may have deposition of urates, phosphates, or uric acid in simple crystalline form without the formation of calculi. A nucleus of some sort is present as well as a binding substance,''^ which is often mucus derived from the walls of the passages, although the center of the concretion most often consists of uric acid or urates. Although the amount of colloidal material in urine is relatively small, yet it undoubtedly plays an important part in maintaining in solution the less soluble crystalloids, which are especially the urates and calcium oxalate. Normal urine contains no colloids which form irreversible gels, and hence ordinary deposits can be readily dissolved, but in inflammatory conditions there appears fibrinogen which readily forms the irreversible fibrin, and conditions thus become favorable for the formation of concretions of any crystalloid with which the urine m'dy be saturated or over-saturated at the time (Schadeh Possibly other colloids may play a similar role. Aschoff' and Kleiu- schmidt "' hold that most urinary calculi begin as primai-y calculi, formed independent of inflammation from excess of the nmin con- stituent (uric acid, oxalates, xanthine, but chiefly ammonium urate) : this calculus foniis the ciystalline nucleus of tlie laminated second- ary deposits of other sul)stances, chiefly uric acid, oxahites and phos- •■'!• fipneral liihiiofrrapliy ;iivoii liy Fiiistoror. Dcut. Zcit. kliii. Ch'w.. I'.iOi; (SO). 414; and Liclitwitz.'^^'' 70 Hippocrates a])preciatpil tlie existence and iniijoiiaiicc S, AXD IXCRU^TATIONS as furnishing the primary nucleus of calculi of preponderatingly cal- careous or mixed composition. Apparently there are marked differ- ences in the prevailing composition of calculi in different countries ; in China, for example, Pfister "'^ found eleven of twelve calculi composed of uric acid. Uric acid is eliminated combined chiefly with sodium, potassium^ and ammonium ; according to some authors, as a biurate, according to others, as a quadriurate. If the urine is excessively acid, it con- tains much acid phosphates, which withdraw part of the bases from the uric acid, and this, when free, crystallizes out if in excess. Hence tlie foruuition of uric-acid concretions is favored by high acidity of the urine, by concentration of the urine, or by an increased elimina- tion of the uric acid. The last may result from excessive nuclein- rich food, or from excessive catabolism of the tissue nucleoproteins (e. g., leucocytosis from inflammatory diseases or leukemia), which conditions are also usually associated with an increased urinary acid- ity. (The chemistry of uric acid is discussed more fully in the chap- ter on ''Gout," Chap, xxi.) Uric-acid calculi are formed chiefly in the pelvis of the kidney, but many pass into the bladder. They are quite hard, and yellow or reddish-yellow in color, because of the presence of vrochrome and K'rohilin, the former of which seems to be chemically combined and the latter but physically, since it can be washed out with v/ater. Uraerythrin or uromelanin (a decomposition product of urochrome) may also be present. Not infrequently calcium oxalate is present, sometimes in considerable quantities. Other urinary constituents may be present in small amounts. In case the calculus enters the urinary bladder it may set up irritation leading to infection; the urine then becoming alkaline, calcium and ammonio-magnesium phosphate will be deposited upon the surface, and the uric acid will be more or less dissolved out and replaced by the phosphates (metamorpho- sis). Urate calculi occur chiefly in new-born or young infants, and rarely in adults. In the young thoy are related to, and may originate in, the deposits of urates in the pyramids of the kidney (tlie so-called urate or uric-acid "infarcts"), which have been supposed to result from the decomposition of the nucleoproteins of the nucleated fetal red corpuscles. (See "Uric Acid," Chap, xxi.) The concretions are composed chiefly of either ammonium or sodium urate, but potassium and even calcium and magnesium urate may be admixed. Their genesis in the young probably depends upon injury to epithelium by the excessive urates of tlie "infarcts," whicli affords a suitable nucleus for their start; their growth depends chiefly upon the con- centration of the infant's urine. In adults they may arise secondary to an ammoniacal decomposition of the urine. Urate concretions are 73aZeit. Urol., 1M13 (7), 945. LV.'/A l/.T VALVULI 457 not coiiimuu ; they are generally rather soft, and often mueh colored by pigments. Calcium oxalate calculi are, according to recent observers,'^ the most oonunon urinai-y concretions.'^ Often they show admixtures of urates or uric acid, which latter frc(iuently constitutes the nucleus, and when urinary infection occurs they may in turn serve as the nucleus to phosphatic deposits. On account of the hardness and roughness of these stones they frequently cause bleeding, which may result in their being very dark in color and containing blood-pigment. They are usually first formed in the pelvis of the kidney, and arise chiefly in persons excreting excessive quantities of oxalic acid. Normally but about 0.02-0.05 gram of oxalic acid is eliminated daily in the urine, apparently all as calcium oxalate, which is kept in solution by the acid phosphates. The amount may be increased by certain foods rich in oxalates, particularly rhubarb, grapes, spinach, etc. ; also probably by gastric fermentation."^ Oxalic acid may possibly be formed from uric acid, and perhaps also from the carbohydrate group of pro- teins,'*^ and it is possible that abnormally large amounts arise from these sources under pathological conditions. During bacterial de- composition of the urine oxalic acid miay be formed from uric acid (Austin).^' Phosphate calculi are formed as a result of decomposition of the urine, with formation of ammonia from the urea. In the ammoniacal solution thus formed the magnesium is precipitated as NH^MgPO^, the calcium as Ca3(P04)„, and calcium oxalate and ammonium urate are also thrown down, so that the concretions consist of a mixture of these substances, the magnesium salt being the most abundant. In none does one substance occur in a pure state. Pigments of various kinds, and more or less mucus or other organic constituents of the framework are also present. Phosphate calculi are the typical "sec- ondary" concretions, and they are formed usually in the bladder as a consequence of cystitis, but may be formed in the renal pelvis or in the urethra. In some cases the salts are precipitated in such large quantities that they form great masses of a sediment which does not aggregate into concretions. Occasionally stones consisting princi- pally of Ca.,(POJ. or CaHPO^ are formed, but these are rarities. As the calcium taken in the food is chiefly eliminated in the feces, the amount in the urine does not vary directly with the amount in the food, and the formation of phosphatic concretions is always a matter or urinaiy reaction and not of diet.'* As these stones fuse to a black, 7* Concerning their structure see Fowler, Johns Hopkins Hospital Reports, 1906 (13)..';07. "5 Baldwin, Jour. Exp. Med., 1900 (5), 27. -« See Austin, Boston Med. and Surg. Journal, 1901 (145), 181. Contradicted by Wegrzynowski. Zeit. phvsiol. Cliem.. 1913 (83), 112. TT.Tour. Med. Research. 1906 (15), 314. 78 Under the name "struvit stone," Poninier (Verh. deut. Path. Gesell.. 1905 (9), 28) describes a urinary calculus composed of very pure ammonio-magnesium 458 cALcii'icATiow c()\('i>'i:ti(>\s, am) /xrh'i statioxs enaiiu'l-like mass under the l)l()W-i)ipe, they have been called "fusible calculi." Calcium carbonate calculi are formed freciuently in herbivora, but tliey are xcry rare in the \irinary passages of man, althoufi-h oe- currinw elsewhere in the body not infrequently. Occasionally these are soft and chalky, but if well crystallized, they are the hardest of concretions. Cystine calculi '" are rare but very interesting formations. Cys- S-CII(NIL)-C'0011 tine I is important as the sulphur-containing por- S-CH(XIL)-COOH tion of the protein molecule. Under normal conditions all the cystine taken in food is completely oxidized and none (or uncertain traces) appears in the urine. In certain individuals the urine contains con- siderable quantities of cystine constantly {(jjstiuuria, see Chap, xix), and occasionallj" in these cases soft concretions of nearly pure cystine are formed in the urinary passages. Cystine calculi may reach the size of a hen's egg, are crystalline in structure, and in the urine of such patients the characteristic hexagonal crystals may usually be found. The cystine of calculi is identical with that from proteins.^" Xanthine Calculi. — Xanthine is the most abundant of the purine bases normally present in urine, but the total amount is extremely small. Like uric acid, it fluctuates in amount according to the amount of destruction of nucleoproteins, either of the food or of the tissues. Concretions consisting chiefly of xanthine, which is often mixed with uric acid, are extremely rare, but a few isolated si)eci- mens having been described. Rosenbloom could collect but six cases in the literature, adding one himself. ^^ Indigo calculi, derived from the indican of the urine through oxi- dation, have also been described a few times. Urostealith calculi, composed of fatty matter, have been occasion- ally ol)served. Although some of the concretions described under this head have really represented foreign bodies introduced through the urethra (e. g., Kruckenberg 's concretion of paraffin from a bou- gie), yet true fat concretions do occur. The origin of the fat in tliese stealiths is unknown; possibly it comes from degenerated epithelium. TTorbaczewski ^' analyzed such a specimen which had the following percentage composition : Water 2..'> Tnoriranio matt«M" .... OS Orfjaiiic matter (cliiclly ])riitciii ) 11.7 Fatty acids .')]..") Nculral fal :?:5..-, Cliolcstcnil traces ])li()S|)lia1e, foriiiiii^ tlic liard, rlioinliic crystals known to iiiiiicralouisis as "struvit." 'I'liis is an exaini)lc of a l)lios|)Iiate stone formed independent of am- inf)niaea] deeom]H>8iti()ii, a raic occurrence. '» T>iteratnre concerniiij,' cystine, see Kricdmann, ImltcIi. der IMi\siol., ]'J()2 (iK 1.'): Marriott and Wolf. Am." .lour. Med. Sci., 1!1()(> (i:?!). \'M. '^'1 .M.dcriialden. Zcit. iilivsioi. Clicm.. 1!»07 (51), :{91. «i X. V. Med. .loiir., .lati. Ill, lUlf). / A'/N I A') CM. CI I.I 459 The fatty acids coiisisti'd of stearic, j)aliiiitic, and pi'ohalily inyi-istic aeid. Cholesterol calculi have been found in the urinary bladder in a few instances, the cause being unknown. Horbaczewski ''- descril)es one weij2;hiiig 25.4 ••■rains, found in a patient who had i)i-eviously had cystine calculi; it contained 1)5.87 per cent, of cholesterol and but 0.55 per cent, of inorganic inatei'ial. (iall-stones have been known to enter the urinary bladder through a fistula between the gall-bladdei- and urinary bladder."" Fibrin "calculi," formed from blood-clots, often moi"e or less im- pregnated with urinary salts, have occasionally been observed. Other proteins may also form similar calculi.*^ General Properties of Urinary Concretions/"* — The hardness de- ])ends partly upon the chemical composition of the calculus, hut more upon the rate and condition of formation (Rowlands, Kahn). I'nder comparable conditions it is said that those composed of amorphous phosphates are the softest ; next come those with some admixture of crystalline phosphates. IVate concretions are harder than these, but are still softer than the uric acid and crystalline phosphate calculi. Oxalates are usually the hardest, except for the rare crystallized calcium carbonate stones. Cystine and amorphous concretions can be scratched with the finger-nail, while even the hardest varieties of calculi can be scratched with a wire nail. Genersich -" gives the following degrees of hardness for different calculi: Cholesterol, 1.5- 1.6; ammonium urate, 2.5; soft phosphate (^Ig), 2.6; hard phos- phate (Ca). 2.75: uric-acid stones (also salivary and prostatic calculi, atheromatous patches, and phleboliths), 2.9; calcium oxalate (also rhinoliths and lung stones), 3.3-3.5; calcium carbonate stones of herbivora, 4.5. But the hardness or gross appearance of a urinary calculus give little or no indication of its chemical composition. The rate of growth also varies according to composition, but is, of course, mucli modified by other factors. Oxalate and urate stones grow most slowly, phosphate stones most rapidly. A urate stone has been known to increase by about two ounces during seven and one half years, while a catheter fragment or other foreign body may be- come covered with a crust several millimeters thick in a few weeks."" Spontaneous disintegration of urinary concretions is limited almost solely to calculi composed entirely or largely of uric acid. Out of 121 cases collected by Englisch,*^ in all but 7 this was the case, these being 82 Zeit. physiol. Cliem., 1804 (IS), Xir^. 83 See Finsterer, Deut. Zeit. kliii. Chir., lOm; ISO), 4-i(). S4 See ]\Iorawitz and Adrian, Mitt. Orcnz. Med. u. Chir., l!li)7 i 17). .■)7!). f<"> Systems for i)rocediire in detenninin'/ tlie nature of luinarv ealeuli are given by Hammarsten (Text-l)ooi< of Phvsiol. C'liem.) and bv Smith ( Reference Hand- book of Med. Sci.). ■ sfi Virehow's Arch.. 180.3 (131), 185. s" Zuckerkandl. Xothnasjel's System, vol. 10, pt. 2, p. 220. ssAroh. klin. Chir., 100.> (76), Or,l (elaborate review). 460 CALCIFICATION, CONCRETIONS, AND INCRUSTATIONS composed of calcium and niagnesiuin phosphate (5), or calcium phos- phate or carbonate (1 each). The tlisintegration is brouglit about through solution of the binding substance and mechanical shattering of the stone into fragments. This occurs but rarely, Bastos ®*^ esti- mating that perhaps one calculus in ten thousand undergoes disinte- gration. CORPORA AMYLACEA ^^ In the case of these widely-spread concentric bodies we find the name misleading, for the bodies are not a form of animal starch, as was suggested by their laminated structure and iodin reaction, nor are they so closely related to amyloid material as the name implies. Different authors disagree decidedly concerning the staining reactions of these bodies, but it may be said that the reactions are extremely inconstant. Sometimes the corpora are stained bluish or green with iodin, sometimes brown, often little at all ; occasionally they react partly with methyl-violet, but more often they do not ; sometimes por- tions of one body react one way, while the remainder behaves differ- ently. Seldom if ever do the ordinary concretions of the prostate give all the amyloid reactions characteristically, but the corpora amylacea of the lungs are much more likely to do so (Stumpf).''° It seems improbable that these bodies, which occur in the prostate of every adult, can be the same as the amyloid, which is seldom observed except as the result of serious processes of tissue destruction. Accord- ing to their structure they obey the usual laws of the formation of concretions, having a central nucleus and a structural framework of different composition from the chief substance. It seems most prob- able that they should be interpreted as simple concretions of protein nature, Avhich form under certain conditions when a nucleus of some sort (usually pigment, degenerated cells, or inorganic crystals) exists in a stagnating, protein-rich fluid. At times the resulting concretion may be of such a physical nature that it absorbs iodin readily (just as they often show a marked absorption-affinity for pigments), and occasionally it may react metachromatically with methyl-violet, pos- sibly because of the presence of chondroitin-sulphuric acid derived from the mucin of the cavities where the concretions form, but per- haps for some other unknown reasons. Occasionally pure amyloid may form in the tissues typically concentric (or even crystalline) bodies, as in Ophlil's case, but this is the exception. It seems prob- able that corpora amylacea are usually protein concretions,"^ and 88a Folia Tirol., 101.'? (8), SI. 80 Gonoral literature, Posncr, Zcit. kliii. Mod., ISSO (Ifi), 144: Luharscli, Erpcl). allfr. Patliol.. 1894 (L). ISO; Opliiils, .Tour. Exp. :\lo(l.. 1900 (5). Ill; Nunf)I), 29,5. 03 Posner, Zeit. f. ITrologie, 1911 (,5), 722. ^i Tbid., 1912 (6), 30. 95 Literature by Scheunert and Berpliolz, Zeit. plivsiol. Chom.. 1907 (-52), 33S. 96 See Lazarus,' Zeit. klin. Med.. 1901 (,51), 530. Literatin-e. 9fia Rosenthal, Arch. f. Verdauunuskr., 1914 (20). G19. 97 Brit. Med. .Tour., 1896 (i), 1034. 98 Lancet, Dec. 18, 1909. 99 Schmidt's Jahrb., 1900 (268), 210. 462 cALciFKwnow coscinrnoxs. .\\i> i\Ch'rsT.[Ti<)\^< Cholesterol 7.69 Pifrnients and soaj) 40.01 Uiuletormiiied 6.01 rsually. liowc'ver, pancreas stones consist chiefly of inorganic sub- stances. .Johnson and Wollaston report analyses of two stones, one containing 72.30 per cent, calcium phosphate and but 8.80 per cent. organic matter; the other 91.65 per cent, calcium carbonate, 4.15 per cent, magnesium carbonate, and but 3 per cent, organic matter. Legrand ^ found only 0.7 per cent, organic matter i)i another concre- tion which contained 93.1 per cent, calcium carbonate. Pancreatic juice, being strongly alkaline, can hold but a small quantity of calcium salts in solution (normally but 0.22 part per thousand — C. Schmidt) ; presumably the little normally present is held in the form of a colloidal suspension by the proteins. Possibly when stasis occurs, digestion of the proteins leads to the precipitation of the calcium salts, or, more probably, the excessive calcium is largely derived from the exudate from the inflamed ducts, as seems to be the case with the calcium of biliary calculi. Salivary Calculi.- — These have a similar composition, in the main, to the concretions of the pancreatic duct, except that they generally contain more organic matter, resembling in this respect the "tar- tar" of the teeth. Bessanez found in one 81.3 per cent, of calcium carbonate and 4.1 per cent, of calcium phosphate, whereas in an- other the carbonate was but 2 per cent, and the phosphate 75 per cent. Potties has described a calculus with a central portion com- posed chiefly of uric acid and a peripheral portion containing 69 per cent, of calcium pliospliate and 20.1 per cent, of calcium carbonate. Harlay ^ found in one specimen 15.9 per cent, organic matter, 75.3 per cent, calcium phosphate, 6.1 per cent, calcium carbonate. Ro- berg believes that bacteria alone do not usually cause salivary calculi to form, but that a foreign body entering the duct is the chief factor. Increased alkalinity nuiy also favor ]irecipitation of calcium from the saliva. In Roberg's case of sialolitliiasis the saliva was of normal composition. Intestinal Concretions. — These always have a nuclens of some indigestible foreign su])stance. most often hair, but sometimes eellu- lose structures or solid indigestible j^articles, including gall-stones. fruit-stones, bone, etc. The bulk of the eoiu'retions is usually made U]i chiefly of ammonio-magnesium ))hosj)hate, with some calcium ])hos])hate, carbonate, and sulphate, |)rotein matter, and occasionally calcium and magnesium soa])s. Two iulestiual coiu'retions analyzed by Scliuberg ^ had the following percentage com{)osition when dried: 1 .Idiir. I'lianii. ct Cliiiii.. I'.IOI (14), 21. - I.iteratiire, see KoIht'.', Annals of Siiruerw l!>(»4 (."!!»), (Itl!). a.Ioiir. I'liarm. et Cliiin., l'.Mt:{ (IS), 11. ■• \'irclio\v's .Arcii., ISSii (!l(l|, ~:i. IXTEiiTl\AL COS VRET loss M).\ Ainnionio-iiiayiicsiuin pliospliate .... ■)7.1 (i;5.!t Calcium phospliatf '"i-T 23.S Calcium carbonate 4.(5 Calcium sulpiiate -J.*' ".7 Alcoiiol-ctlicr extract l.'.t n.s Other orgajiic substances -lo ti.n 111 L'uuntries wliere oatmeal is largely eaten, intestinal eoneretions are not infrel : also Kahn. .\rch. Int. Med., 1913 (11). 92. and M. B. Schmidt, Deut. med. Woch., 1913 (39), 59. 20 Cent. f. Path.. 1S98 (9), 296. 30 466 CALCIFICATIOX, COXCRETIOXS, AND INCRUSTATIOXS in the peribrone-liial glands than in the lungs, constituting from 6 to 36 per cent, of the ash, corresponding with Arnold's observation that in gold-beaters the glands contain more metal than the lungs. In stone-workers Schmidt found a higher proportion of SiO. in the lungs than in the glands. In normal adults the amount of coal-pigment is greater than the amount of silicates; in children the reverse is the case. Thorel -^ reports that the lungs of a worker in soapstone contained 3.25 per cent, of ash, including 2.43 per cent, of soapstone. In siderosis iron has been found in the lungs in proportions varying from 0.5 per cent, to 7.9 per cent, of the dry weight, the last amount having been found by Langguth " in the lungs of an iron miner, which contained also 11.92 per cent, of SiOo. An analysis of a lung from a knife-grinder is reported by Iloden- pyl,-^ Avhich gave the following results : Total weight of dried and powdered lung, 48.1009 grams ; total solids, 44.7986 ; ether-soluble sub- stance, 14.6017. Composition of the ether-soluble substance : free fatty acids, 7.498; neutral fats, 4.044; cholesterol, 3.037. Proteins, 15.4759; charcoal (total carbon less protein carbon), 7.198; ash, 4.2903. The composition of the ash. (in grams) was as follows: KoO, 0.2167; Xa..O, 0.3523; CaO, 0.0965; Fe,0;,„ 0.0879; AUO„ 1.4628; SO,, 0.0704; P,0-„ 0.9565; SiO„ 1.2043. The amount of emery, rep- resented by the oxides of aluminum and silicon made up more than one-half of the ash, and the iron constituted about one-fourth. The man had worked at the trade of knife-grinder for about fifteen years. ]\IcCrae -^ has analyzed the lungs of six gold mine workers, in South Africa, finding from 9 to 21.7 grams of ash per lung, of which 29 to 48 per cent, was silica; aluminum was also high, and an increased P2O.., content was ascribed to the accompanying fibrosis. Klotz -■' found from 1.2 to 5.3 grams of free carbon in each lung, of dwellers of Pittsburg, as contrasted with 0.145 and 0.405 grams found in the lungs of residents of Ann Arbor. Hirsch -•' analyzed four average Chicago lungs, finding in grams per lung: I Carbon 2.72 Silica O.IS Calcium O.xidc . . 0.45 21 Ziegler's Beitr., 1896 (20), 85. 22Dcut. Arch. klin. Med., 1895 (55), 255. 23 Medical Record. 1890 (56), 942. 24 "The Ash of Silicolic T.niifrs." John McCrae, Johannesburg, 1914. 2''' Anicr. .T(nir. Piibl. Tb'altli, 1914 (4), 887. General review on antliracosis 20 Jour. Amer Med Assoc, 191() (66), 9.50. TT Til IV 0.71 1.20 0.19 0.28 0.69 0.04 0.12 0.02 0.05 CHAPTER XVI PATHOLOGICAL PIGMENTATION ' MELANIN - Melanin occurs normally as the coloring-matter of hair, of the choroid of the eye, of the skin, in the pigment matter of many lower animals, and most strikingly as a defensive substance in the "ink'' ejected by squids to render themselves invisible in the water. Path- ologically melanin occurs chiefly as the result of an excessive pro- duction of this pigment by cells normally forming it, as in freckles, melanotic tumors, and Addison's disease (probably). Cells that do not normally form melanin probably do not acquire this power in pathological conditions.-" Pathological failure to form melanin is also observed, as in skin formed in the healing of wounds and after syphilitic lesions; or in albinism, in which the failure to form me- lanin may be attributed to hereditary influences.^ The function of melanin is evidently that of protection from light rays, and Young ^'' has found that isolated melanin from human skin absorbs violet and ultra-violet rays. Probably this protection is responsible, at least in part, for the relative infrequency of skin cancers in the colored races.^'' Melanin seems always to be produced through metabolic activity of specialized cells. The idea, which was formerly advanced, that it is derived from hemoglobin as a product of disintegration, seems to have failed entirely of substantiation. In malaria we frequently find a diffuse pigmentation of the skin of such a nature as to suggest strongly a melanin formation, and this has been cited as an example of the production of melanin from hemoglobin. Carbone has proved, however, that this malarial pigment is derived from hematin. The amount of iron contained in melanin has been much investigated, as 1 Literature bv Oberndorfer, Ergebnisse Pathol., 1908 (12), 460, and Hueck, Zieofler's Beitr., 1012 (54), (58. 2 Literature and resume given by v. Fiirth. Cent. f. Pathol., 1004 (15), 617; Handb. d. Biochem.. 1, 742. 2a The pigment of the so-called "melanosis" of the large intestine is neither true melanin nor ordinary "waste" pigment (Henschen and Bergstrand, Ziegler's Beitr., 1013 (56), 10.3). 3 Gortner holds that dominant whites are due to the presence of antioxidase. while regressive whites have neither the power to form pigments nor to inhibit their formation (Amer. Naturalist, 1010 (44), 497). 3a Biochem. Jour., 1014 (8), 460. 3b However. Hanawa found white areas in sl;in less aflfocted by cliemical irri- tants and infections than dark areas. (Dermatol. Zeit., 1013 (20), 761.) 467 468 PATHOLOGICAL PIGMENTATION bearing upon the question as to whether the melanin is derived from hemoglobin or not, and the results obtained by the best methods indi- cate that the amount of iron present is usually extremely small, and often it is entirely absent; furthermore, the presence of iron is no proof that the pigment is derived from hemoglobin, since other iron- protein compounds undoubtedly exist, — especially nucleoproteins, and chemical examination shows that melanin does not contain hemopyr- role groups.* Composition of Melanin. — The elementary composition of differ- ent specimens of melanin examined by various observers has been found to vary greatly. This probably depends on three factors: First, it is extremely difficult to obtain melanin in a pure condition; second, the process of purification requires the action of strong acids and alkalies, which undoubtedly modify the composition of the mel- anin; thirdly, melanin is probably not a single substance of definite composition, but includes several related but different bodies. The values found vary for carbon from 48.95 to 60.02 per cent. ; for hy- drogen from 3.05 to 7.57 per cent. ; for nitrogen, 8.1 to 13.77 per cent. Hofmeister gives, as a characteristic of melanins, that their elementary molecular composition is always nearlv in the proportions N : H : C = 1 : 5 : 5. Gortner's ^ studies have led him to accept the general principle that melanin is formed through the action of an oxidase on an oxidizable chromogen, but that in keratinous structures there exist at least two types of melanins, one, a ''melano-protein," soluble in dilute acids and existing dissolved in the keratins; the other, insoluble in dilute acids, exists as pigment granules and is of unknown nature. Piettre '^ believes that melanin from sarcoma of the horse consists of a protein united to a pigment. Those whose studies of melanin formation have been made with the microscope, state that the nucleus is active in the process," and some find the melanin so closely related to the lipoids that they consider it a lipochrome.^ A particularly prominent constituent of some melanins is sulphur, which has been found in as high proportions as 10 per cent, in mel- anin from sarcomas, and even 12 per cent, in sepia from the squid ; in melanin from hair the sulphur is usually about 2-4 per cent.; but in choroid melanin, and in some other forms, sulphur seems to be ab- sent. The proportions of sulphur obtained from the same specimen purified by different methods show wide variations, and hence v. 4 Spioprlcr, TTofmoistor's Beitr., 1007 (10), 253. •'". Biofhom. Bulletin. liHl (1), 207: rr'suni^. oConipt. Bend. Acad. Sci., 1011 (I'l:^), 782; also sec Ri'priiil frnm 1st Intor- nat. ronfr. Compar. Pathol., Paris, 1912. T StanVi, Vcrli. Df'iit. Path. Ges., 1007 (11), 130; Schultz, Jour. Mod. Rca.. 1912 (20), 05. sDvaon, Jour. Path, and Bact., 1011 (15), 298; KriMbieii. Wien. klin. Woi'h., ion "(24), 117. COMPOSITIOX OF MELAMX 469 Fiirth consiilers that iioitlier the .suli)hur nor the iron are indispensa- ble constituents of the melanin. Probably the melanin molecule con- tains at(mi-coinploxes that have a tendency to bind certain sulphur and iron comi)()Uiids (e. g., cystine or hematin derivatives). There is much reason to believe that the melanin is derived from certain p:roups of the protein molecule that seem readily to form col- ored compounds. The aromatic compounds of the protein inolecule, such as tyrosine, ]i]ienylalaiiine, and tryptophane, readily condense with elimination of water and absorption of oxygen, to produce dark- colored substances. When proteins are heated in strong hydrochloric acid, we obtain a dark-brown material, which closely resembles the melanins both in elementary composition and in general properties, so that it is referred to as "artificial melanin" or "melanoid sub- stance." These substances, like the natural melanins, when decom- posed by fusing with caustic potash, yield skatole, indole, and pyrrole derivatives, which are undoubtedly derived from the tyrosine and tryptopliane of the protein molecule. Therefore, it seems probable that both the melanoid substances and the true melanins are formed from the chromogen groups of the protein molecule through processes of condensation, elimination of water, and the taking up of oxygen. ^^ Tyrosinase. — In the sepia sacs of the cuttle-fish, in meal-worms which form a melanin-like pigment, and in plants that produce the black Japanese lacquer, have been found oxidizing enzymes that have the property of producing black pigment by their action upon tjTO- sine and other aromatic compounds. Neuberg ° found that extracts of a melanosarcoma of the adrenal could produce pigment from epinephrin and ^-oxyphenylethylamine, but not from tyrosine. The ink sacs of the squid contain an enzyme forming a pigment from epinephrin, apparently through oxidation and condensation. These enz^'mes may, therefore, possibly be responsible for the production of melanin in animal tissues, by causing oxidative changes in the chromogen groups of the protein molecule that are liberated by auto- lysis (see "Tyrosinase" p. 73). v. Fiirth urges strongly the view that both normal and pathological melanin formation depend upon the action of the tyrosinase or allied enzymes in conjunction with autolytic enzjones; the latter split free the chromogen groups of the protein molecule, which are then oxidized by the tyrosinase, undergo condensation, and take up sulphur- and iron-holding groups and also other organic compounds, the entire complex forming the melanin. Properties of Melanin. — Wlien isolated in a pure condition, melanin is a dark-brown substance of amorphous structure, no mat- ter how black the material from which it is derived may be.^" It is SiiSee Herzmark and von Fiirtli, Biochcm. Zeit.. lill."? (40), 130. 9 Zoit. f. Krebsforsch.. IflO!) (S), 195. 10 Spiegler, (Hofmeister's Beitr., 1903 (4). 40) claims to have isolated from white wool a white chromogen, closely related to melanin chemically, but Gortner 470 PATHOLOGICAL PIGMENTATION quite insoluble in all ordinary reagents except alkalies, in which some melanins dissolve easily, and some with difficulty. Strong boiling hydrochloric acid scarcely affects non-i)rotein melanins. By the action of sunlight or oxidizing agents on melanin-containing sections the pigment can be bleached out. The chief decomposition-products formed on fusing with alkalies are indole, skatole, and "melanic acid"; no cystine, leucine, tyrosine, or other amino-acids can be iso- lated. ]\rost authors, therefore, consider the melanins as heterocyclic compounds standing in some relation to the indole nucleus. If melanin is injected subcutaneously into animals (rabbits and guinea-pigs), there appears in the urine a substance which turns dark brown after the urine has stood for some time (Kobert, Helman). The pigment is apparently reduced, particularly by the liver, to a colorless melanogen, which is eliminated in the urine. The same process occurs when melanin is produced in excess and enters the blood, as in the case of melanosarcoma, a colorless melanogen being formed which is excreted in the urine, constituting "melanuria." Occasionally the urine is dark when first passed, because of the pres- ence of melanin, but usually it must be subjected to oxidizing agen- cies (bromine water, nitric acid, hypochlorites, etc.), or exposed to air to bring out the brown color. Helman ^^ says that true melano- gen may be considered to' be present in urine : ( 1 ) If the careful addition of ferric chloride causes the development of a black precipi- tate. (2) If this precipitate dissolves in sodium carbonate, forming a black solution. (3) If from this solution mineral acids precipitate a black or brownish-black powder. All three reactions must be obtained, for substances other than melanin may give the first two. The coloring power of melanin is very great, for urine containing but 0.1 per cent, of melanin has the color of dark beer (Hensen and Nfilke), and the entire skin of a negro contains only about 1 gram of melanin (Abel and Davis). ^- Excessive quantities of melanin ma.y be in part deposited in the lymph-glands and skin, causing diffuse pigmentation ; it may be deposited in the endothelium lining the blood-vessels, hi a pigmented colon Al)derhalden ^-'' found melanin- like substances which seemed to be derived from tryptophane. Nik- las,'-'' however, Ijelieves t3a'osinase activity to be responsible for this type of intestinal melanosis. Kobert injected melanin into albino ra])l)its, but did not succeed in getting any dejiosition in the choroid (Amor. Xatiiralist, 1010 (44), 407) boliovos this to bo a docDmiKisif imi ])ro(liiot of koratin, iinrolalod to incdanin. iiCViit. f. inn. Mod., 1002 (2:5), 1017: Aroh. iiiloniat. riianiial to r.tr the Dvrrole rin- is reduced, and instead it undergoes reduc- tTLZ^^n, ^ion with sulphuric ^id to W ^^^al s„lnhate of methvlpvrrolidlne-hydroxy-earbonic acid (CH,-b,li.lN..U4 ) . XbdJhalto-' also found a relation to tryptophane, for m the urme ^f atianuric was present a substance rich in tr,-p.opl.ane ; and 13 \roh internat. Plmnnakodynam., 1003 (1-). 14 Jour. Med. Res., V.m (16), 117. isVirchow's Arch.. 1000 (108). 62. 16-Biochem. Zeit., 1010 (28). ISl iTZeit. physiol. Chem., 1912 (/8), 159. 472 PATHOLOGICAL PIGMEXTATION Priinavera '^ found the iiriiie in a case of melanosarcoma contain- ing free tyrosine, fluctuating in amount with the pigment. Addison's disease is associated with the deposition of a pigment in the skin that is generally considered to be a melanin, differing from that produced normally in the skin only in quantity' and not in origin or composition.^'' No satisfactory explanation of the relation of the adrenal to this pigmentation seems yet to have been made, al- though it is natural to assume that when the function of the adrenal is destroyed, substances accumulate in the blood that have a stimu- lating effect on the pigment-forming cells. Abnormal protein catab- olism, with excessive accumulation of the chromogenic constituents of the protein molecule, has been suggested, as also have alterations in the influence of the sympathetic nervous system upon the chromo- phore cells, for nerve lesions (e. g., neurofibroma) often are accom- panied by pathological pigmentation of the skin.-" It is significant that the active constituent of the adrenal medulla, the epinephrin, is an aromatic derivative closely related to tyrosine, since the production of pigment by the action of oxidizing enzymes upon such substances is well known. Furthermore, Neuberg has de- scribed a melanotic adrenal tumor which produced pigment by oxi- dizing epinephrin. On this basis the pigmentation of Addison's dis- ease would seem to be the result of an abnormal accumulation or dis- tribution of aromatic compounds, because of their failure to be con- verted into epinephrin. In support of this hypothesis is the obser- vation of ^leirowsky that the human skin contains an enzyme capable of oxidizing e])inephrin to a pigment, and that pieces of skin kept warm will develop a postmortem pigmentation, and this is supported by Konegstein -^ who found that the pigmentation was greater in animals deprived of their adrenals or given injections of epineph- rin. As exact chemical studies of the pigment in Addison's disease have not been made, however, we have no positive proof that it is a mela- nin, hence any speculation as to the cause of its formation is prema- ture. Carbone -- claims to have isolated from the urine in Addison's disease a pi^iment that contains much suljiliur, and which he considers similar to or identical with the melanogen of melanuria. A similar observation is reported by Eiselt.--' v. Kahlden,-^ however, has ob- served crystals resembling hematoidiii in the pigmented tissues. isfiiorn. Int. Scicnze Med., 1908 (29), 978. 19 Concerning histogenesis of the pigment see Pfuiringcr, Cent. f. Path., 1900 (11), 1. 20 See r68iim^ hv Schmidt, Ergeb. der Pathol., 1896 (Bd. ,3. Abt. 1), 551. -■1 Wicn. ll). 100. 34 Frankfurtor Zoit. Patliol., lOl.S (12), 21S; also Toutsclilaoiuler. Virdiow's Arch., 1014 (217). .-^O.^. 35 .Tour. Exp. l\To(l.. 1002 (fi), 110. 30 Jour. Exppr. Med., 1011 (1.3). 200. 37 Cent. f. Pathol., 1002 (13). SSI. 3SVirchow's Ardi., 1004 (177), 24S. S(>o also Afavor et ah. Jmir. plnsiol. ct path. ji<-"n., 1014 (16), 581. L/I'OCHROME 475 by osiiiic aeitl ; they are dissolved by the usual fat solvents. It is questionable if all pigments that stain for fat should be considered as true lipochromos, however, for their other reactions are variable; and Rorst would distin<;-uisli these path()loo('V(>nliart, Jour. Exp. ^Nlcd., 1913 (IS), 107. 52 Cent. f. Path.. 1909 (20). 900. 5.3SPO Xeun.ann, Vircliow's Arch., 1888 (111), 25; 1900 (101), 422; 1904 (177), 401; also Arnold, ihid., 1900 (ICl), 284; Leupold, Beitr. path. Anat., 1914 (59), 501. jii.oon I'KiM i:\TS 479 deposited in the liver, spleen and kidneys within 2-1 hours.''^'' In in- farcts hemosiderin soon disappears (Schmidt),'^* presumably because dissolved by the acids formed during- autolysis. According- to Neu- mann, hemosiderin is produced only under the influence of living cells and in the presence of oxygen, while hematoidin arises independent of cellular activity ; '"'' but ]^rown ■''' has found that hemosiderin can be formed during autolysis of the liver, especially when air is present, and thei-efore pi'obably by an oxidizing enzyme. He suggests that in hemosiderin the pigment is still hematoidin, and that the formation of hemosiderin takes place in the nuclei, the hemosiderin being made directly from hemoglobin without the intervention of hematin. It may also be formed from the iron-containing protein of the cells dur- ing autolysis, independent of hemoglobin."'" jNIilner ^* considers that, under similar conditions, an iron-containing pigment is also formed, which differs from hemosiderin in having the iron so combined that it cannot react with the usual reagents ; this pigment may later change into hemosiderin. Up to the present time we do not know the chem- ical nature of hemosiderin, nor its exact fate in the body, but it is probabl}' utilized in the manufacture of new hemoglobin, for it is known that the iron liberated when hematin is broken up in the body under experimental conditions is deposited and not eliminated (Mor- ishima).'"'^ Unstained hemosiderin generally appears in the form of brown or yellowish-brown granules, and not as crystals. After a time it is taken up and deposited to a large extent in the liver, spleen, bone- marrow, and kidney, either as hemosiderin or possibly as some other iron compound of similar nature. From these sites it seems to be later taken up to be utilized in the manufacture of new red cor- puscles. All told the average human body contains about 3.2 grams of iron, of which 2.4 to 2.7 grams is in the blood. According to Meyer ""^ iron is present in the body in three forms : 1. Not demonstrable by reagents because so firmly bound (hemoglobin). 2. Loosely bound iron, colored by (NIT4)oS acting for a long time (ferratin). 3. Salt- like compounds with proteins, and inorganic compounds, reacting at once with reagents. Ferratin is the iron compound in the liver, con- taining 6 per cent. iron. He believes that probably hemosiderin is not a definite substance, but merely indicates compounds of the third 33aMiiir and Dunn. Jour. Path, and Baot., 1015 (10), 417. 54 Verb. Deut. Path. Cesell., lOOS (12), 271. 55 The aocuninlation of iron in tho liver wliich follows poisoninijf with hemolytic agents, is not prevented or diminished bv preliminary removal of the spleen (Meinertz, Zeit. exp. Path. u. Ther., 1906 (2), 602). 56 Jour. Exper. Med., 1910 (12), 623. 5T Sprunt et al., Jour. Exp. Med., 1912 (16), 607. ssVirchow's Arch., 1903 (174), 475. 59 Arch. exp. Path. u. Pharm., 1898 (41), 291. eoErgeb. der Physiol, 1905 (5), 698; literature. 480 PATIIOLOaiCAL PIGMEXTATIOX class. Iron pigments may be transformed from one class to another, e. g., in corpus lutenm scars, whose age can be estimated, class three may be replaced by class two. We may have in the sputum and lungs " Herzf ehlerzellen " that either do or do not stain with ferrocyanide. In morbus macidosus, Kunkel found the pigment of the internal or- gans to be pure iron oxide. Hueck also hohls that hemosiderin is an inorganic iron compound, loosely bound to proteins and fats, and that it never forms an iron-free pigment, as has been stated. He be- lieves that there is very little iron in the tissues in a firm union like hemoglobin, and that by proper technic some iron can be stained in every organ which contains iron chemicallj' demonstrable. Ischida '^^ believes that an iron-containing pigment may be formed in striated muscles from the iron normally there, w^ithout requiring a hematoge- nous origin. Hematoporphyrin."- — There are several closely related pigments de- rived from hematin that are appropriately grouped under the desig- nation of porphyrins, for they are not all identical with the pigments prepared artificially from hematin b}^ Nencki and called by him heniatoporphyrin and mesoporphyrin, the former apparently repre- senting a reduction, the latter an oxidation product. ''^ The porphy- rins found in the urine and feces are different from each other and from those prepared by Nencki.*'* Physiologically, these pigments are of great interest, because of the close chemical relation they have been found to bear to chlorophyll,^'^ with which hemoglobin is so closely re- lated functionally. It is also interesting to consider that whereas car- nivora obtain much hemoglobin in their food, herbivora obtain much chlorophyll. Pathologically, porphyrin is of interest as a urinary pigment, being found normally in the urine in traces, but present in considerable quantities in many diseases,'^*' such as rheumatism, tuber- culosis, various liver diseases, and, most strikingly, after the admin- istration of sulphonal, veronal or trional. When in abundance it may color the urine a rich Burgundy red, and it is sometimes accom- panied by a precursor, vro-fuscin. It is present in the bones of animals showing hemochromatosis and in the bones of persons *""' exhibiting a congenital form of "hematoporphyria, " described by Giinther, which is accompanied by severe skin lesions that are ascribed to the action of light upon the skin sensitized by the hematoporphyrin. Hausmann •'^ and others have studied extensively the photosensitizing action ex- «i Virehow's Arch., 1012 (210), 67. "s Literature and full review bv Giintlier, Deut. Arcli. kiiu. :\[ed., 1012 (105), 80; and by Jesionek, Erjreb. inn. Med., 1013 (11), .52"). «■■! Fischer and Meyer-Bet/, Zeit. physiol. Chem., 1912 (82). 06. 04 11. Fischer, Miiiich. nied. Woch.. 1016 (63). 377; Zeit. phvsiol. Chem., 1016 (97), 100 and 148; Schumni, ibid., 1015 (06). 183. «5 For literature see Aliderhalfh'ii, "Lehrbucli der plivsiol. C'lieniie." 1000. «<■■ See Carnid, .lour, of Phvsiol., 1802 (1:5), .'"jOS. O'iallegler ct a/., J)eut. iiied. Wodi., 1013 (30), 842. 07 Biochcm. Zeit., 1010 (3(1), 27(;; 1014 (67), 300. PSEUDOMELANOSIS 481 Libited by liematoporpbyrin and other porphyrins, and find evidence suggesting a rehitionship between hematoporpliyria and "hydroa aestiva," and other conditions in which the skin is abnormally sensi- tive to light. After injection of 0.2 gm. hematoporphyi-in into his own veins, Mej'^er-Betz '^'^^ found himself so sensitized to light that exposure to the sun caused severe skin reactions during a period of weeks, and exposure to the Finsen light produced severe ulceration ; but little hematojiorphyrin escaped in the urine. Many other products of blood destruction tested on animals were without sensitizing effects. Meth^-lation of the p^'rrol groups only partially removes the activity of hematoporphyrin. Porphyrin obtained from urine and feces by Fischer also sensitized mice to light. Sufficient doses of hematopor- phyrin may sensitize mice so that they become narcotized and die in a few minutes after exposure to intense light, a true "light stroke." Pseudomelanosis. — When loosely bound iron is present in the tis- sues, and in the same tissues sulphides are produced through bacterial action, a discoloration with sulphide of iron will result, which is 'Called pseudomelanosis, because the pigment resembles true melanin in its blackness. This is most frequently observed as a postmortem phenomenon in and about the abdominal cavity, and in the ordinary postmortem discoloration both the liberation of the iron from its firm organic combination, and the production of hydrogen sulphide, are the ■work of bacteria. Pseudomelanosis may also occur intra vitam, par- ticularly in the margins of infected areas, and it may also be observed in the intestines, liver and spleen, and about the peritoneum, in bodies examined immediately after death, before any evident postmortem decomposition has set in. This seems to depend upon the previous intra vitam formation of hemosiderin, which is then combined by sul- phur liberated from tissue proteins through bacterial action.''^ If hj'drogen sulphide acts upon hemoglobin that has not been decom- posed, a greenish compound of sulphur-mctheniogloTjin is formed (Harnack^^), which is the cause of the greenish color seen in the abdominal walls and along the vessels of cadavers. This union of hemoglobin and hydrogen sulphide occurs only when oxygen is pres- ent (oxyhemoglobin). The sulphur-hemoglobin compound is readily decomposed by weak acids, even by CO., with the formation of methemoglohin, which in turn readily becomes decomposed to form hematin. During life sulphemoglohin may form, the sulphur presumably coming from ijitestinal putrefaction, and hence called "enterogenous cyanosis," which term also covers metliemoglohinemia produced by nitrites formed in the intestines.^" The latter condition is also pres- 67a Dent. Arch. klin. Med., 191.3 (112). 476. G8 Ernst, Virchow's Arch., 189S (152), 418. Literature. 69Zeit. physiol. Chem., 1899 (20), .558. 70 West and CMarke. Lancet. Feb. 2, 1907: Davis, ihid., Oct. 26, 1912; Gibson Quart. Jour. Med., 1907 (1), 29; Wallis, Hid., Oct., 1913. 31 482 I'ATIIOLOaiCAL PldMEXTATION ent in poisoning by plienacetin,'"'^ aniline and acetanilid, and related pigments appear in the blood in poisoning with chlorates and uitro- benzol. Pneumocoeei and ^Streptococcus viriduns, as well as some other bacteria, may produce methemoglobin.^'"' In infections with B. empliysematosus, Scluimm found this pigment free in the blood, and probably it could be found in other conditions if sought. Hemofuscin is the name given by von Recklinghausen to the brownish pigment found in involuntary muscle-tibers, particularly in the wall of the intestine. It does not react for iron, and is insoluble in alcohol, ether, chloroform, or acids; therefore it is not a iipochrome. It is bleached by HoOo, and is often found associated with hemosiderin which is not bleached. Von Recklinghausen, and also Goebel,^^ ascribe this pigment to an alteration of hemoglobin which enters the cells in dissolved form, but Rosenfeld,'- who has submitted the material to analysis after isolation, found 3.70 per cent, of sulphur, from which he considers that it is related to the melanins or melanoid substances. The substance is readily dissolved by alkalies, and contains no iron. According to Taranoukhine,^^ the pigment in the myocardium in hroioi atrophy of the heart is also derived from proteins, and is neither a Iipochrome nor a hemoglobin derivative. Other observers, however, consider this pigment a Iipochrome or a lipofusein. It is probable that the name hemofuscin has been given to several different pigments, which resemble one another only in that they do not contain iron. Strater "^'^ says that the name hemofuscin cannot be used for the pigment of the involuntary muscles, as he finds evidence that it does not arise from hemoglobin and is probably a waste pigment : but hemofuscin is found in e}>ithelial and connective tissue cells. Hemochromatosis.'* — This name was given by von Recklinghausen to a condition in which the organs and tissues throughout the body are abundantly infiltrated with two pigments : one, iron-containing, iden- tical with hemosiderin ; the other seems to be the same as the hemo- fuscin described above. It is to be distinguished from general hemo- siderosis in which only the iron pigment is deposited.'*'' In hemo- chromatosis the hemosiderin is found chiefly in the parenchyma cells of the glandular organs, especiall.y the liver and pancreas, which or- gans usually show marked interstitial proliferation. Hess and Zur- TOaRee PTciibner. Arch. exp. Path., 1913 (72), 241. 70b CoU'. Jour. Exp. ;Med.. 1914 (20). .303; Blake ihid.. 1910 (24). 315; Sohumni, Zoit. phvsiol. Ciicm., 1913 (87), 171. Ti Virchow's Arch., 1894 (130), 4S2. "Arch. exp. Path. ii. Pharm.. 1900 (45), 40. "Rousskv Arch. Patol., 1900 (10), 441. -3a Vircliow's Arch., 1914 (218), 1. 74 Literature piven by Sprunt, Arch. Int. Med., 1911 (8). 75; Potter and Milne, Aincr. Jour. Med! Sci., 1911 (143), 46; Roth, Deut. Arcli. kiln. Mod.. 1915 (117), 224. 7+a In lower animals occurs a form of liemochromatosis afTectinir especially the bones, and sometimes mistaken for ochronosis. (See Teutschlaender X'irchow's Arch., 1914 (217), 393.) iii:M<)('/ih'<)\iAT0Sis 483 lu'lle i'oniul ;)S.7 ^in. of iron in llic livci- in one case (the normal amount is 0.3 gm.), and Bemouille " found 18.3 gm. or 2,95 per cent, of the dry weifrht in the liver, 2.65 per cent, in the pancreas, and the same in tlie spleen. Anschiitz found 14.69 per cent, in the lymph glands, 7.62 per cent, in the liver, and 5 per cent, in the pancreas of a case, ^hi'ir and Dunn "'' obtained the following percenttige figures : Liver, 6.43 ; pancreas, 2.49 ; spleen, 0.825 ; retroperitoneal glands, 11.64; kidneys, 0.406: adrenals, 0.121; heart, 0.714; skin, 0.188; small intestine, 0.14. The hemofuscin is found in the smooth muscle- fibers of the gastro-intestinal tract, blood-vessels, and genito-urinary tract. Under the heading of local hemochromatosis, von Reckling- hausen grouped such conditions as brown atrophy of the heart, and pig-mentation of the intestinal wall, which probably are quite dis- tinct from the generalized hemochromatosis, since the local form oc- curs as a physiological process in old age. In a considerable proportion (50 of 63 collected by Sprunt) of the cases of generalized hemochromatosis there occurs diabetes, called by Hanot, "bronzed diabetes," because of the coloration of the skin. It has been suggested that the pigmentation is due to decomposition of the blood-corpuscles in the diabetic blood, but the pigmentation and sclerotic changes precede the diabetes, which is secondary to the atrophic and sclerotic changes in the pancreas. There can be little question that both the pigment formation and the tissue changes de- pend upon some intoxication, the origin and nature of the toxic agent being entirely unknown. In many cases it has seemed probable that alcohol might have been the inciting cause. There is no evidence of any abnormal blood destruction which might account for the pigmen- tation, and Parker suggests that the difficulty lies in the inability of the tissues to get rid of the iron set free in normal catabolism.'^^'' Roessle believes that the primarv^ change is in the capillaries, whereby hemorrhagic extravasations take place, and phagocytosis of red cor- puscles by gland cells results in pigmentation. Opie's conclusions concerning this subject are as follows: (1) There is a distinct morbid entity, hemochromatosis, characterized by widespread deposition of an iron-containing pigment in certain cells, and an associated formation of iron-free pigments in a variety of localities in which pigment is found in moderate amount under physi- ological conditions. (2) With the pigment accumulation there occur degeneration and death of the containing cells and consequent inter- stitial inflammation, notably of the liver and pancreas, which become the seat of inflammatory changes accompanied by hypertrophy of the organ. (3) When chronic interstitial pancreatitis has reached a cer- tain grade of intensity, diabetes ensues, and is the terminal event in T5Corr.-Bl. Scliweiz. Aertze, 1911 (40). 010. Tsa.Jour. Path, and Ract., 1914 (19), 226. -EbSee Quart. Jour. Med., 1914 (7), 129. 484 PATHOLOGICAL PIGMEXTATION the disease. Spmnt suggests that the diabetes may be referable to diminished oxidative power because of disturbances in the iron-con- taining constituents of the tissues, assuming these iron compounds to be catalytic agents in oxidizing processes. ICTERUS ■« Pigmentation of the tissues of the hody in jaundice depends upon the presence in them of bile-pigments, which usually have been formed in the liver and reabsorbed either into the lymph or blood (or both). However, a pigment that seems to be chemically identical with bili- rubin {hematoidin) may be formed from hemoglobin liberated on the breaking up of red corpuscles, and possibly this may be produced in sufficient amounts outside of the liver to give rise to general icterus. Certainly the local greenish-yellow pigmentation occurring in the vicinity of extravasations of blood, due to hematoidin formation, may be looked upon as a "local jaundice. "^^ and in icterus hematoidin crystals may be found in the tissues." Bile-pigments. — Biliruhin is of a reddish-yellow color, and it is the chief pig- ment of human hile. Its formula is Cg^H^sNiOu or C.,3H3oN40,;, and its relation to hematin, from wliich it is formed, is sliown by the following formula, which ex- presses the manner in which blood pigment may Ijc converted into bilirubin by the liver under normal conditions, and into hematoidin (its isomer) in the tissues and fluids of the body in pathological conditions: Ca^H^^N.O.Fe + 2HoO = C^JI.s'^fi, f FeO. (hematin) (hematoidin or bilirubin) Bilirubin is not soluble in water, but dissolves in the alkaline body fluids as a soluble compound, "bilirubin alkali." It is very slightly soluble in ether, ben- zene, carbon disulphide, amyl-alcohol, fatty oils, and glycerol, Init is more soluble in alcohol and in chloroform. Biliverdin, C;,4H38N40s, as its formula indicates, is an oxidation product of bilirubin. Bilirubin in alkaline solutions will oxidize into biliverdin merely on exposure to tlie air, and the change from yellow to green of icteric specimens when placed in oxidizing solutions (e. g., dicliromate hardening fluids) is due to the formation of the green biliverdin. Biliverdin is the chief pigment of tlie bile of carnivora, but it is also present in varying amounts in human bile. The various other biliary pigments, namely, hilifuscin, hiliprdsin. rliolrprnniii.'^ hilihininn, and hilicyanin, are probably not normal C(mstituents of bile, but are oxidation products of bilirubin, and are found chiefly- in gall stones ( (/. c. ) . A pigment similar to urobilin may be present in normal bilo. The total amount of pigment's present in liile is probably not far from one gram per liter; ratlier under than above this amount. Etiology of Icterus. — Although hematoidin, wliich is isomeric if not identical with bilirubin, may be formed outside of the liver when red corijuscles are broken u]) in hemoiThagic extravasations, and . 76 Literature by Stadelmann, "Der Icterus," Sluttgart, 1S91; Minkowski, Ergebnisse der Pathol., 1895 (2), 079. 77 See Ouillain and Troisier, Semaine MM., 1909 (29). l;?:5: Widul and .Jolt rain, Arch. mr-d. expC'r., 1909 (21). (541. 78l)iinzelt, Cent. f. Patli., 1909 (20). 900. 70 See Kiister, Zeit. physiol. Chem., 1906 (47), 294. ICTERUS 485 possibly also when they are broken up within the vessels by hemolytic agents, yet it has generally been considered that a true general icterus does not occur without the liver being implicated. This view rested on evidence of various sorts. First, the classical experiments of ^Minkowski and Nannyn,"^' which demonstrated that in geese the pro- duction of hemolj-sis by means of arseniuretted hydrogen leads to icterus, but if the livers of the geese have been previously removed, no icterus follows the poisoning. Second, the repeated demonstration that in icterus produced by septic conditions, poisoning, etc., which was formerly looked upon as a ''hematogenous" icterus, the urine contains bile salts as Avell as pigment, indicating an absorption of bile from the liver. Third, the finding of histological evidence that in so-called hematogenous icterus there occur occlusions or lesions of some sort in the bile capillaries, which can account for the reabsorp- tion of the bile into the general circulation.®^ Therefore, it was be- lieved that the pigments that produce the general discoloration of icterus are, at least for the most part, manufactured by the liver, whatever the cause of the reabsorption of the bile from the liver into the blood may be. That hemolytic agents cause icterus was explained by the fact that on account of the large amounts of free hemoglobin brought to the liver, excessive amounts of bile-pigments are formed, which render the bile so viscid that it blocks up the fine bile capil- laries; on account of the low pressure at which bile is secreted, a slight obstruction of this kind is sufficient to stop entirely the outflow of bile, which then enters the capillaries of the liver and also, to a less extent, the lymphatics.^^ It is also possible that the hemolytic poisons injure the liver-cells so much that the minute intra- and intercellular bile capillaries become disorganized, and permit of escape of bile into the lymph-spaces and its absorption into the blood-vessels.®^ Swelling of the degenerated liver-cells may also be an important factor in the occlusion of the bile capillaries; swelling of the lining cells of the bile capillaries may also coexist, and fibrin may occlude them in toxic or infectious icterus. However, Whipple and Hooper,®* have obtained experimental evi- dence that after intravenous injection of hemoglobin into dogs with the liver excluded from the circulation, bile pigments appear in the urine and icterus is manifested in the fat tissues, from which observa- tions it is concluded that the liver may not be the only place in which 80 Arch. f. exp. Pathol, u. Pharm., 1886 (21), 1. 81 See Eppin^er, Ziegler's Beitr.. 1903 (33), 123; Gerhardt, Miindi. med. Wooh., 1905 (52), 889. Lang (Zeit. exp. Path. u. Ther., July, 1906 (3), 473) has demonstrated the presence of fibrinogen in the bile in phosphorus-poisoning, which perhaps accounts for the "l)ile thrombi" observed by Eppinger in toxic icterus. 82 See :Mendel and Underbill, Amer. Jour. Phvsiol., 1905 (14), 252; Whipple and King, Jour. Exp. ]\red., 1911 (13), 115. s'? Sterling, Arch. exp. Path., 1911 (64), 468; Fiessinger, Jour. Phvsiol, et Pathol., 1910 (12), 958. 84 Jour. Exper. Med., 1913 (17), 593 and 612. 486 PATHOLOGICAL PIGMEXTATION bile pigment can be formed from hemoglobin. Several authors have found bilirubin produced in hemorrhagic elfusions located where the liver could have had no influence.®^'' We also recognize types of hemo- lytic icterus in which the liver does not seem to be concerned, and with bile pigments present in the blood and urine unaccompanied by bile salts (dissociated icterus), so that the old dictum of the essential" implication of the liver in icterus seems to be incorrect.^*'' Joanno- vics " gives, as a result of a comparative study of icterus from bile obstruction and icterus from hemolysis, the following chief differ- ences: Icterus due to hemolysis appears sooner than icterus from bile-duct occlusion, and reaches a much higher degree ; the obstruction in hemolytic icterus, Avhen present, is intra-acinous ; in stasis it is cliiefly inter-acinous ; in hemolytic icterus there is a large splenic tumor due to accumulation of degenerated red cells in the spleen, where they become disintegrated preliminary to the formation of bile- pigment. If the spleen is removed, hemolytic agents may not cause icterus, because the corpuscles are not then prepared for pigment formation. ^^-'^ Toxicity of Bile. — In any event, we must appreciate that in icterus not only are abnormally large quantities of bile-pigment present in the blood, but also the other less conspicuous constituents of the bile. Whole bile of rabbits is fatal to rabbits in doses of 0.25 to 0.5 cc. per kilo, by intraperitoneal injection, and about half as much intraven- ously (Bunting and Brown ^'^). Death is the result of changes in the myocardium, where necrosis is produced ; and severe degenerative changes are also found in the kidneys and liver; when the bile is in- jected into the peritoneum, pancreatitis and fat necrosis result. The relative toxicity of the bile-pigments and the bile salts is not as yet uni- formly agreed upon. Bile-pigments. — Bouchard *^ and others have claimed that the bile- pigments are far more toxic than the bile salts, which is contradicted by Rywosch and others. A series of analyses by Gilbert '^'"^ and others gave the following results : Normal blood-serum contains 0.027-0.08 gram bilirubin per liter; in obstructive ictenis they found 0.7 to 1.0 gram of bilirubin per liter, in biliary cirrhosis 0.33 gram per liter, in icterus neonatorum 0.2 to 0.5 gram ; in pneumonia 0.068 gram was found. King and Stewart -^ state that the amount of pigment in a s<4aTIoop(M- and ^^■llippl(>, Jour. Exp. Mod., inifi (2:1), 137. S4b Att('iiij)ts to produce hile pifjments from liemojiloliin by bacterial action have been un.successful. (Quadri, Fol. Clin. C'liim.. 1914, Xo. 10). ssZeit. f. Hellk., Path. Al)t.. l')()4 (25), 25. S5a 'I'lic etiology of icterus neonatorum (when not obstructive) has not been ascertained, but a nalurai tendency towards icterus is said to exist in tiie new- l)orn, llieir l)b>od containin5 (2), 709. 90 Jour. Exp. Med., 1906 (8), 128; review and literature concerning toxicity of bile. 91 See King, Bigelow and Pearce, Jour. Exper. Med., 1912 (14), 159. 92Neufeld and Hiindel, Arb. kaiserl. Ges.-Amte, 1908 (28), 572. 488 PATHOLOGICAL PIGMENTATION mentary jaundice of congenital hemolytic icterus. There is also an increase in the cholesterol in the blood, which may be related to the "xanthomas" that form in chronic icterus."^ A remarkable tendency to spontaneous hemorrhages, frequently ob- served in icterus, probably depends upon injury to the capillary endothelium by the bile salts,"^ while the protracted, often uncontrol- lable, hemorrhage that may occur from operation wounds in icteric pa- tients, is related to the slowed coagulation of the blood observed in icterus. The cytotoxic effect of the bile salts is also shown by the albuminuria of icteric persons, which frequently results from the renal lesions the bile produces. Although bile itself is toxic to many bacteria, especially the pneumococcus,^^ yet in icterus the bactericidal power of the blood is lowered, and infections are prone to develop and to be severe; moreover, the growth of several species of bacteria is favored by bile."*^ Croftan ^^ summarizes the physiological effects of bile acids as fol- lows: (1) A powerful cytolytic action, affecting both blood-cor- puscles and tissue-cells. (2) A distinct cholagogue action. (3) In small doses (1-500) they aid coagulation. (4) In large doses (1-250 and over) they retard coagulation. (5) Slow the heart action.*'^'^ (6) In small doses they act as vasodilators; in large doses, as vasocon- strictors. (7) Reduce motor and sensory irrita-bility. (8) Act on the higher cerebral centers, causing coma, stupor, and death. Sel- lards °* found that injection of bile salts into guinea pigs causes ulcer- ation and hemorrhage in the stomach. It is difficult to decide how much of the profound intoxication that is sometimes present in icterus (''cholemia" and "icterus gravis") to ascribe to the reabsorbed bile, for frequently there is an accompany- ing infection, and even if there is no infection the impairment of liver function by the obstruction of bile outflow must also be reckoned with. The liver is not only the great destroyer of toxic substances absorbed from the alimentary canal, but it is also an important seat of nitrogenous metabolism, interference with which may lead to ac- cumulation of many toxic nitrogenous substances in the blood. ^ The long duration of severe icterus in some cases of occlusion of the bile- ducts, with relatively slight evidences of intoxication, would seem to indicate, however, that on the whole the bile is not so nuich respon- ds ChaufTard, Prosso Mr>d., 1913 (21), SI; Chvostok. Zcit. klin. Med., 1!)11 (73), 470; Pinkiis and Pick, Dout. med. Woch., 1008 (34), 1427. n-tSpo Morawitz, Areh. cxp. Path.. 1907 (56), 115. "•'> Roe Noiifpld and TTaendpl, Inr. cit. n« See Mcyorstoin, fVnt. f. Pnkt.. 1907 ^44), 434. f'7New York Mod. .Tour., inn; (;.3). SIO; see also Faust, "Die tiorisohe Cifto," Braunschwoifr, 1906, p. 29. oTaSoo Porti, Oaz. depli Ospod.. 1910 (37), 1233. !>«Aroli. Tnt. Mod., 1909 (4), .'>02. 1 See Bickel, Exper. Untersuch. liber der Pathol, der Cholaemie, Wiesbaden, 1900. CONGENITAL HEMOLYTIC ICTERUS 489 sible for the iiitoxieation observed in icterus as are the associated conditions. On the other hand, in not a few instances it has been observed that escape of large quantities of bile into the peritoneal cavity may be followed by symptoms similar to those of icterus gravis; in these cases only the bile can be held responsible for the intoxica- tion.- Dissociated Jaundice -i^ is the oxistcneo of cillicr l)ilc salts or l)ilo piil)lio Anipr. Jour. Phvsiol., 190r) (1.3), 341. loZeit. physiol. Chem., 1899 (27), 398. CHE Ml ST in or TUMONS J\ (IKSEK.XL 495 lated to Bergell's iiiuliii6r., 1011 (23), 370. viii:mistIx'Y of tumohs j\ aESEitAi, 497 squamous epitlieliuni, whiuli tissues iioniuilly cuntaiu inucli g-lyeogen, are likewise provided with an abundance of this material. Second, the occurrence of areas of impaired cell-iuitrition favors the accumu- lation of lilycogen in the degenerating tumor-cells, just as it leads to a similar accumulation in all other tissues (Gierke).-^ The most ex- tensive consideration of this topic is reported by Lubarsch,-" who found glycogen microscopically in 447 (or 29 per cent.) of 1544 tumors examined. Jt was i)resent in but 3 out of 184 fibromas, osteomas, glionuis, hemangiomas, lipomas, and lymphangiomas, and in but 2 out of 260 adenomas from various parts of the body. It occurred in all teratomas, rhabdomyomas, hypernephromas, and syncytiomas. In 138 sarcouuis glycogen was present in 70 (50.7 per cent.) ; of 415 carcino- mas it was found in 181 (43.6 per cent.). In the squamous epithelial cancers 70 per cent, contained glycogen, while the mucoid or colloid cancers were always free from glycogen. The glycogen undoubtedly enters the cells from without, probably entering as sugar, and being converted into glj'cogen by intracellular enzymes. "We have no re- liable studies of the actual quantity of glj'cogen in various tumors, al- though Meillere ^° states that the microscopic and chemical examina- tion of tumors give corresponding comparative results, which Gierke states is generally true with glycogen estimations. Pentoses. — Neuberg ^^ reports finding, as a product of autolysis of a carcinoma of the liver, a pentose which was not produced by autolysis of either normal liver tissue or the primary growth in the stomach. Beebe ^- found that in carcinoma of the mammary gland the per- centage of pentose {xylose) is somewhat higher than the amount in normal mammary glands (about 0.23 per cent.). Carcinoma in the liver did not show any constant excess of pentose above that of normal liver tissue (about 0.38 per cent.). A primary carcinoma of the liver showed quite the same pentose and phosphorus content as normal liver tissue. In general, no constant relation of pentose to origin, malignancy, or degeneration of tumors was observed. Purines and Purine Enzymes. — The purines of both benign and malignant tumors have been studied by Wells and Long,^" who found them the same as those in nonnal tissues, and in much the same rela- tive proportions. The proportion of the total nitrogen of tumors which is constituted by the purine nitrogen is less than would be expected from the histological evidence of the amount of nuclear material con- tained in the tumors. Tumors also seem to contain much the same purine enzymes as the normal tissues. Thus, guanase seems uni- versally present in tumors derived from human tissues, and adenase 28 Ziegler's Beitr., 1005 (37), 502. 29Virchow's Arch., 1006 (183), 18S. 3oCompt. Pvend. Soc. Biol., 1000 (52), 324. 31 Berl. klin. Woch.. 1004 (41). lOSl ; 1005 (42), 118. 32Amer. Jour. Physiol., 1005 (14), 231. 33 Zeit. f. Krebsforsch., 1013 (12), 508. 32 498 THE CHEMIi^TIiV OF TUMORS is missing, although autolyzing tumors can disintegrate their nucleic acid (nuclease) and change the adenine radicals of the nucleic acid into hypoxanthine, presumably by way of adenosine and inosine (Amberg and Jones). Secondary tumors growing in the human liver do not accjuire the enzyme, xanthine-oxidase, which is a characteristic enzyme of tliis organ. The liver tissue between the cancer nodules seems to oxidize purines less activelj^ than normal liver tissue. Long ^* has also found similar conditions in tumors from sheep, pigs and cattle, observing that primary carcinoma of the liver does not con- tain xanthine oxidase, a point of interest in view of the fact that in the development of mammals the xanthine oxidase does not appear until late. Lipins. — Tumor cells seem to contain much the same fats and lipoids as normal cells, and, so far as known, in much the same pro])ortions as characterize the cells from which the tumors arose. Thus Wells ^^ found that hypernephromas show the same high proportions of leeitlii]i and cholesterol as he found in normal adrenal, and as are found in the renal cortex. Other malignant tumors have much less lipoids and fats (see Hypernephromas). A secondaiy carcinoma of liver cells^ metastatic in the skull, was found by Prym ^^ to show the same sort of fatty infiltration that is characteristic of fatty liver cells. On account of the poor blood supply of many tumors, fatty changes are usual, occurring under the same conditions and showing the same microscopic features as fatty degeneration in other tissues,^^ being more common in malignant than in benign tumors ; especially abun- dant in squamous cell carcinomas, and scanty in sarcomas. Crystals of cholesterol or cholesterol compounds are described in tumors by White.^* Even lipoma fat shows no difference from normal fat,^** and the depot fat of tumor patients is quite the same as in patients with other diseases associated with equal wasting,^'^ in whom some increase in unsaponifiable material (cholesterol) is usual. INIurray '*^ saj's that the lipoids of degenerating uterine fibroids are strongly hemolytic, which may account for the so-called ''red degeneration" of these tumors. Freuncl and Kaminer *^ suggest that the fatty acids of tissues are of importance in determining whether a tissue is a suitable soil for secondary growth, these substances being deficient in tissues where growths develop. There has been some effort to correlate the cholesterol and lecithin contents of blood and tissues with the rate of 34, Tour. Exper. Med., 1013 (18), rA2. 35 .Tour. Med. Res., 1908 (17), 461. 3«l'>ankf. Zeit. Palli., 1012 (10), 170. 37 See lliifra, IJorl. klin. Wocli., ]:;]2 (40), ,342; Joamiovics, Wien. klin. Woeli., 1912 (25). 37. 38 .Tour. Patli. and Haci.. 1!)0S (I.']). .3. 39 See WellH, Arcli. Int. .Med., 1912 (10), 2!l7. ■»" \\'acker, Zeit. ])hyfiiol. Cheni., 1!)12 (7S), :54!>; l!)12 (SO), ;5S.3. 41 Jour. Obst. Gvn.'lJrit. Knip., 1010 (17), 534. 4-:Wien. klin. Woch., 1012 (25), KiOS. JXOh'dAMC COX^TITLEXTS OF TUMOUH 499 cancer o;rowth ; apparently lecithin inhibits gi-o\vth and cholesterol stimulates/'' However, Bullock and Cramer *^'' found much more cholesterol in a slowly jifrowing- mouse carcinoma than in a rapidly growing one. somewhat more phosphatid in the latter, much more phosphatid in a sarcoma than in the carcinoma, and* cerebrosides only in the latter; in necrotic portions of tumors they found an increase in simple fats. These tigiires are based on too few observations to be in- terpreted as yet. (3) Inorganic Constituents. — These have been studied under ex- ceptionally favorable conditions, in that the age of the tumor could be accurately estimated, in the inoculable carcinoma of mice (Jensen), by Clowes and Frisbie.^^ They found that rapidly growing tumors contain a high percentage of potassium and little or no calcium, whereas in old, slowly growing, relativel}' necrobiotic tumors the rela- tion is reversed, the potassium decreasing greatly while the calcium in- creases. Magnesium is present only in traces, while the proportion of sodium fluctuates much less, but is usually greater than either the potassium or calcium, although in very old tumors the latter may be- come excessive. The most rapid growth, however, seems to occur in tumors in which both calcium and potassium arfe present in the ratio of K 2 3 — = - or - Ca 1 2 Beebe ^'' analyzed a number of human tumors with the following results: PhosphoTOS was found in proportion to the amount of nu- clear material, varying from 0.139 per cent, (uterine fibroid) to 1.06 per cent, (sarcoma). Iron varied from 0.013 per cent, to 0.064 per cent., probably depending on the amount of blood and nucleoproteins. Calcium is most abundant in old degenerated tumors, and potassium in rapidly growing tumors. These results, supported by Clowes and Frisbie's findings, indicate the importance of potassium for cell growth. Injection of potassium salts into mice increases their suscep- tibilitj' to inoculation (Clowes),""^ while calcium decreases cancer growth (Goldzieher).'*" A greater proportion of potassium was found in primary than in secondary growths by Mottram ; '*^ sodium was the same in each ; there is more potassium in squamous cell carcinoma than in round cell sarcoma. Robin ^^ states that in cancerous livers the cancer tissue contains more inorganic matter than the normal liver 43 See Robertson and Burnett. Jour. Exp. Med., 1013 (17), 344; 1916 (23), 631; Sweet et al.. Jour. Biol. Cliem., 1915 (21), 309. 43aProc, Roval Soc, London (B), 1914 (87), 236. 4*Amer. Joiir. Physiol., 190.5 (14), 173. 45 Amer. Jour. Plivsiol., 1904 (12), 167. 48 British Med. Joiir., Deo. 1, 1906. 4TVerhandI. Deiit. Path. Gesellsch., 1912 (15), 283. 48 Arch. Middlesex Hospital, 1910 (19), 40. 49Conipt. Rend. Acad. Sci., 1913 (156), 334. 500 THE CHEMISTRY OF TUMORS tissue about it. Cattley •""' found tiie inierochemic distribution of po- tassium the same in cancer as in normal cells, and the same seems to be true of maup:anese.'''^ Schwalbe ^^ found that cancer-cells contain iron in a condition demonstrable b}^ the Berlin-blue reaction, and occurring independent of hemorrhages. Tracy ■'* found that tumors reacted microscopically for iron, either free or in the form of an albuminate, only in areas where hemorrhages had occurred. Nuclear or organic iron could be detected in the nuclei, occurring in a network arrangement. In other words, iron occurs in tumors, both quantitatively and qualitatively, exactly as in normal cells of the same type. The same writer ^^ found in tumors, by microchemical reactions, that phosphorus in the form of nucleo])roteins likewise shows no essential dift'erences from its dis- tribution in normal tissues. In this connection may be mentioned the observations of Hem- meter,"" who found that the cells of carcinoma of the mammarv gland will shrink when placed in physiological salt solution or in the serum of the patient, whereas normal cells swell when placed in cancer-juice. This suggests that the osmotic pressure, and, by infer- ence, the amount of inorganic constituents, is lower than in normal tissues. Crj'stalloids, such as KI, diffuse readily into cancer tissue. ^'•'^ (4) Enzymes. — The rapid and extensive autolysis that occurs in tumors, as showm both morphologically and by the presence of the products of protein cleavage in them, indicates that tumor cells resemble all other cells in possessing intracellular proteolytic enzymes. Because of autolysis, puncture fluids in cancer of serous surfaces show an increased amount of incoagulable nitrogen (Morris), ''' and they may show free amino-acids (Wiener) j"^^ while there is a slight increase in the incoagulable nitrogen of the blood (Takemura)."'** There is considerable but not undisputed evidence that cancer tis- sue autolyzes somewhat more rapidly than corresponding normal tis- sues,**" and, according to Neuberg, Blumenthal and others,'^^ that cancer extracts digest other tissues than themselves (heterolysis), a property not exhibited by extracts of normal tissues. ]\Iiiller and others would ascribe this heterolysis to the leucocytes present in the soLancot, 1907 (172), 13. ci Modifrrofoaini, Proc. Roval Soc, B, 1912 (86), 174. 53 ('(.111. f. I'atli., inoi (12), 874. f'^ Jour. IMcd. Kpscarch, 1905 (14), 1. 55 Martha Tracy, Jour. Mod. rjescaroli, 1906 (14). 447. 50 Auier. Jour. Med. Soi., 190.3 (125), 080. .Ida Van den Volden, P.ioohem Zeit., 1908 (9), 54; aeo also Wells ami Tledon- hiiTfr, Jour. Infect. Di.s., 1912 (11), 349. 57 Arch. Int. Med.. 1911 (8), 457. 58 Hiochem. Zeit., 1912 (41), 149. 50 I hid., 1<)10 (25), 505. «oSc,. ^(oliirnolo, P»iochem. Zeit., 1909 (22), 299; Daels and Dclenz^, Bull. Acad. .Med. J'.clj;., 1913 (26), 833. 01 Bibliography by Hamburger, Jour. Amer. Med. Assoc, 1912 (59), 847. ENZYMES OF TUMORS 501 tumors. Nucleases have been found in tumors as in otlier tissues,"^ and in general the enzymes which deamidize adenine and guanine (ad- enase and guanase) are usually present if the original tissue possessed these enzymes, but no instance of tlie presence of xanthine oxidase or uricolytie enzyme has been obtained (Wells and Long, loc. cit.^^). Hamburger finds that the enzymes of cancer tissue upon which the glycyl-tryptophane and other enzyme tests for cancer are based, are ereptases, resembling in all thinr properties the ereptases of -normal tissues, and not })resent in particularly large amount. However, Ab- derhalden ^^ has found evidence that certain peptids may be split in a different way by cancer than by normal tissues, supporting those who hold that cancer enzymes are different from normal tissue enzymes. Autolysis of tumors is said to be augmented by x-ray, and especially by radium (Neuberg), and tumor tissue is readilj^ digested by tryp- sin. The presence of ereptases in carcinomatous gastric juice has been especially studied because of its diagnostic possibilities, and the care- ful investigation of Jacques and Woodyatt "^ seems to show conclu- sively that such an enzyme is rarely present in gastric juice except when derived from a cancer present in the wall of the stomach, pro- vided peptolytic bacteria are excluded by filtration. Deaminizing enzymes may also be found in gastric cancer secretions.*'^" In the blood of cancer patients there is usually an increased antitrj^ptic activity, ascribable to the reaction against enzymes absorbed from the cancer; it is less pronounced with sarcoma.*'^ The body tissues of patients dying with cancer show a low ereptic activity, but the same is true of persons d.ying from other wasting diseases ( Colwell ) ."'' The same seems to be true of other tissue enzymes ; — at least purine oxidizing enzymes are deficient in the liver tissue between secondary cancers (Wells and Long^^) and the eatalase is also reduced in liver tumors (Blumenthal and Brahn) ^'' and in the blood of tumor mice (Rosen- thal)''^ ; in human blood the eatalase may vary either side of normal.®*^ Brahn °^^ found that liver metastases of gastric cancer contained no lipase or lecithinase, which enzymes were also reduced in the liver tis- sue between cancer nodules. However, choline has been found in necrotic sarcomas of rats,"*'' which would seem to indicate the presence of enzymes disintegrating lecithin. As mentioned elsewhere (Mela- 62 Goodman. Jour. Exp. Med., 1912 (15), 477. 63 Zeit. Krebsforscli., 1010 (9), 2fi6. 6-tArch. Int. INled., 1912 (10), 560. 64aHalporn, Mitt. Grenz. Med. Chir., 1915 (28), 709. 65 Citronblatt, Med. Klin., 1912 (8), 1.38. 66 Arch. Middlesex Hosp., 1909 (15), 96. 6T Zeit. f. Krebsforsoh., 1910 (8), 436. csDeut. med. Wocli., 1912 (.38), 2270. 68a Rohdonburo:, X. Y. ]\Ied. .Tour., 1913 (97), 824. esbSitzber. kpl. preuss. Akad. Wiss.. 1916 (20), 47-8. 68c Euinger, Miinch. mod. Woch., 1914 (61), 2336. 502 THE CHEMISTRY OF TUMORS nin), melanotic tumors may contain enzymes oxidizing tyrosine, epine- phrin, pj^rocatechin, or other related aromatic substances, with the formation of pigmentary substances. Catalase is low in tumor tissues (Blumenthal and Brahn). (See also, Autolysis in Tumors, chap, iii.) Other enzymes are also present in tumor cells. Buxton '^^ exam- ined a large number of tumors for their enzymes by the plate {aiixan- ographic) method, and found considerable variations in different growths. All contained amj'lase (splitting starch) and lipase (split- ting butyrin). IMost, but not all, tumors coagulated milk and liquefied casein, and also liquefied gelatin (rennin, proteases). Peroxidase was nearly ahvays, and catalase always, present. Digestion of fibrin, co- agulated seinim, and coagulated egg albumen could not be observed. Practically all tumors split glycogen. Tj-^rosinase could not be demon- strated. The fact that early embryonic tissues were found poor in enzymes ""^ speaks against the common assumption that tumors repre- sent strictly an embryonic formation, but Long "^ found that xanthine- oxidase, which in normal development does not appear until late in fetal life, was absent from primary- carcinomas of sheep livers, al- though normal adiilt sheep liver tissue is rich in this enzyme. ]\lacFadyen and Harden ^- studied the juices obtained by grinding up tumor cells made brittle by liquid air, and found by direct meth- ods (chiefly in breast cancers) invertase, maltase, amylase, proteases acting in both acid and alkaline solutions, catalase, oxidase, with per- haps traces of lipase and peroxidase, but no lactase. Tumors arising from the gastric mucosa, according to AYaring,'^^ contain both pepsin and rennin ; those from the pancreas, both pri- mary and secondary growths, contain trypsin, steapsin, amylase, and rennin. (5) Internal Secretion. — If tumors are derived from an organ with an important internal secretion, the tumor cells in many cases produce the same internal secretion, which seems to have the same functional properties as the normally produced' secretion. Thus a metastatic growth from a thyroid tumor has been said to functionate in place of the resected gland ; Gierke ^* found in about 20 grams of material from metastatic thyroid tissue in the vertebral column about 5 mg. of iodin, which was a trifle larger proportion than was present in the thyroid itself. Carlson and Woelfel "'' found much iodin in the metastases of a thyroid carcinoma of a dog, while in another dog whose cancerous thyroid contained no iodin the secondary tumors were also devoid of this element. ^Marine and Johnson '^ found that fin.Tour. Med. Research, inn.3 (0), :]r-,(\. ■!0 Ihid., mOo (l.*?), .54.S. Ti.Toiir. Expor. Mod., 101.3 (18), 512. -2 Lancet. 100.3 (ii), 224. 73 .Tour. .Anat. and Phvsiol., 1804 (28). 142. 74TTofrneisior's ■Reitr.."l0n2 (3), 280. -". Amer. Jour. Plivsiol.. 1010 (20), 32. 70 Arch. Int. Med." 1013 (11), 288. INTERNAL SECRJJTIOX OF TUMORS 503 in two cases of caucer of the thyroid in man, and one in the dog, tlie cancer tissne sliowed no ability to retain iodin given by month, in con- trast to nonnal thyroid and simple adenomas. Meyer-Hiirlimann and Oswald '"'* have described a remarkable ease of cystic carcinoma of the thyroid, from which in six weeks 2840 c.c. of secretion was obtained by pnncture. It contained 0.077 mg. iodin per 10 c.c. (the patient having previously been given KI) as compared with normal thyroid which contains 0.4 to 4 mg. per 10 gm. It contained both globulin and albumin, the former corresponding to true thyroglobulin, even to in- creasing vagus irritability experimentally. The "adenomatous" nodules of the thyroid often show evidence of active secretion, Goetsch'*''' having found their cells rich in mitochondria, while Gra- ham ^"'^ found that they take up iodin and metabolize it so that the adenomatous tissue produces the typical thyroid effect on the develop- ment of tadpoles. Adrenal cancers do not usually cause Addison's disease, because they functionate in place of the destroj^ed gland (Lubarsch). In the peculiar and characteristic production of cachexia, often ap- parently out of all proportion to the amount of tumor tissue, there would seem to be evidence that a peculiar and abnormal product of metabolism is formed by cancer-cells, and extracts from cancers have been found toxic for protozoa.^^ As yet, however, it has been im- possible to demonstrate any characteristic toxic substance in cancers.'"'' Girard-^Iangin '^ claims that malignant tumors contain colloidal poi- sonous substances in proportion to their softness, extracts causing paralysis and fall of blood pressure ; but others have failed to substan- tiate this.'-' Because of the constant disintegration of the tumor tis- sues, products of autolysis are formed, and undoubtedly enter the cir- culation in small quantities ; possibly they are a factor in the systemic manifestations of malignant growths, analogous to the action of cleav- age products of foreign proteins which may produce "protein fever" and other toxic effects. Since all normal tissue-cells produce substances through their me- tabolism that enter the circulation, it is quite certain that tumor-cells do likewise, and it is highly probable that the presence of abnormal quantities of such products, even if they are of quite normal compo- sition, may cause disturbances in the body. As yet, however, no such substances, either normal or abnormal, have been isolated, nor has their presence been demonstrated. Numerous isolated observations of -6aKorr.-Bl. Sohweizer Aerzte, 1013 (43), 1468. 76b Bull. Johns Hopkins Hosp., 1916 (27), 120. 76c .Tour. Exp. :\rc(l., 1916 (24), 34.>. T7 Woodruff and Underbill, Jour. Biol. Cheni., 1913 (15). 401; Calkins, .Tour. Cancer Res.. 1916 (1), 205 and 399. 7 7a See Blunientlial, Festschr. f. Salkowski, Berlin, 1904; HansenuTun, Zeit. Krebsforscli., 1906 (4), 565. TsPresse Med.. 1906, p. 1709; Compt. Rend. Soc. Biol., 1909 (67), 117. 79 See Brusehettini and Barloeco, Cent. f. Bakt., 1907 (43), 064. 504 THE CHEMISTRY OF TUMORS ptoinains or similar substances in tlie urine of cancer patients may be found in the literature,®" but their importance is extremely question- able. Hemolytic Substances. — A number of observers have described the finding of hemolytic substcinces in cancer extracts. Bard ®^ observed that in hemorrhagic carcinomatous exudates in serous cavities the blood is rapidly hemolyzed, which is not the case in exudates from other causes, but this was not corroborated by Weil.®' Kullmann ®*^ found that extracts of carcinomas contain hemolytic substances acting energetically both in the body and in vitro; these are soluble in alcohol and in water, are not complex in composition, are not specific for hu- man corpuscles, but are toxic for all varieties of corpuscles. IMicheli and Donati ®^ likewise found hemolytic substances in 8 of 15 tumors, of which 5 acted on all varieties of corjDuscles, and 3 acted on only certain varieties; they regard the hemolj'tic substances as the products of autolysis in the tumors. Weil ®* also found the hemolytic property of tumor extracts to vary with the amount of necrosis, from which are derived dialysable hemolytic substances distinct from the hemolysins of normal tissues. It is well known tliat among the products of autolysis of normal tissues are hemolytic substances. Whether the severe anemia frequently present in carcinoma is due, either largely or in part, to these products of autolysis is unknown, but it is very probable that they have some effect. Hemolysis in Cancer. — The blood serum of cancer patients has often a hemolytic action on the corpuscles of normal persons (Crile), but this property is quite inconstant, being present in 67 per cent, of a series of 472 cancer cases collected by Krida, while 15 per cent, of cases of other diseases and 2.6 per cent, of normal persons showed hemolytic activity of the serum. ®^ Elsberg found that normal corpus- cles injected subcutaneously into cancer patients are hemolyzed, but Gorham and Lisser found this reaction positive in but 60 per cent, of their eases, the subcutaneous hemolysis not corresponding at all to the hemolytic activity of the patient's serum in the test tube. The stomach contents in cancer of the stomach, when ulcerated, are hemo- lytic (Grafe and Rohmer).®*^ The red corpuscles of cancer patients are said to have usually a greater resistance to hemolysis by cobra- venom than normal corpuscles, but this is not eluiracteristic, tliere being simi- lar alterations in other diseases.®' The reputed power of the serum 80 See Kullmann, Zcit. klin. IMcd.. in04 (.')3), 2fl-'?. 81 La Scmaine ^tecL, 1001 (21), 201. 82 Jour. ]\Iecl. EcH.. 1010 (23), 80. 83 Rift.rma Mod., 100.1 (19), 10.37. 84 Jour. Mod. Pu's., 1007 (K!), 287. sn Liioraturo l»v (iorliain and Liasor, Amor. Jour. ^lod. Si'i., li*]2 (144), 10.'?. 80 Dcut. Arcli. klin. ]\lod., 1008 (04), 230. 87 Kraua, Kanzi and II. Klirlidi, Sit/.. Bor. Akad. Wion., 1010 (110). 3: spo also Grunbaum, Jour. rath, and lUiot., 1012 (17), 82. METMiOLlSM i\ qAycEii 505 in eaiK'or to i)r()toc't I'orjju.st-lc.s fi-om liemolysis b}- oleic and lactic acid could not be demonstrated by Sweek aijd Fleisher.'*^ An extensive review of the literature and methods led Cohnreich **" to the conclusion that resistance of erythrocytes to hypotonic solutions and to poisons vary independently of one another. He has devised an improved method for testing resistance to hypotonic solutions, which seems to vary directly with the' amount of stroma and PO4 content, and finds that determinations of maximum and minimum resistance is of little value, as these concern only a small part of the corpuscles; he therefore determines the "plurinnnn" resistance, involving most of the corpuscles. The most significant results were obtained in cancer of the alimentary tract, in which an increased resistance was always demonstrable. Farmachidis ^^^ finds the cobra venom resistance more specific for cancer than most other investigators. (6) Metabolism in Cancer. — Speaking against any specific na- ture in the cause of cancer cachexia are numerous observations, indi- cating that the cachexia is in no way different from the cachexia of other conditions. The behavior of the nitrogen metabolism seems to be quite the same as in tuberculosis and other wasting diseases. There is the same excessive elimination of aromatic substances (phenol, indi- can) and oxyacids (Lewin,^® Blumenthal ^°), which Lewin considers to arise from the abnormal metabolism of proteins, and not from putre- factive decomposition in the tumor or in the intestines. In rats with sarcoma, increased excretion of uric acid and creatin has been ob- served.°°^ There is also the same excessive elimination of mineral salts that is observed in pulmonarv^ tuberculosis, and termed "demin- eralization" by Robin, ^^ but no alteration in the excretion of chlo- rides."^'^ As in other cachexias, the creatin content of the muscles is decreased.''- Fraenkel ^^ finds evidence that there maj' be some diffi- culty in tryptophane metabolism in tumors and in tumor patients. Extensive respiratory studies by "Wallersteiner ^^'^ showed enormous variations in the amount of heat production in different cases, in about 10 per cent, of which figures as high as those of severe fevers or exophthalmic goiter were obtained repeatedly; most of the cases showed high normal figures. Nitrogen loss did not ordinarilj^ occur if ssJour. Med. Res., 1013 (27). 3S.3. 88a Folia HematoL, 1913 (16). 307. full bibliography. 88b Gaz. degli Osped., 1915 (30), fiSO. s9Deut. med. Woch., 190,5 (31), 218. 90 Festschr. f. Salkowski, Berlin. 1904. 90aOrdway, Jour. Med. Res., 1913 (23), 301. 91 Quoted by Lewin, loe. cit. Clowes ct al. (5th Ann. Rep., X. Y. State Dept. of Health. 190.3-4) report observing a slight chloride retention in cancer pa- tients, and review tlie literature of metabolism in cancer. 9ia Robin. Compt. Rend. Acad. Sci.. 1913 (156), 1262. 92 Chisholm, Riocliem. Jour., 1912 (6), 243. 93 Wien. klin. Woch., 1912 (25), 1041. 93aDeut. Arch. klin. Med., 1914 (116), 145. 506 THE en KM I ST If y of timors the ealorimetric finding's were considered in the calculations; nitrogen equilibrium was maintained if sufficient nourishment was obtained and utilized. In general, metabolism iu cancer resembles that of fever, and warrants the assumption of a toxic stimulation of tissue destruction. It is entirely possible that the products of cancer protein destruction are responsible for this toxicogenic metabolic abnormality, since Vaughan has demonstrated that the effects of bacteria and foreign proteins are quite the same in their pyretic and toxic action. Salkowski demonstrated that the amount of colloidal nitrogenous material, precipitated from the urine by strong alcohol, is increased in cancer. Numerous observers have corroborated this, but find that a similar condition obtains in other cachectic diseases, although in cancer the amount of colloidal nitrogen seldom is as low as normal unless the tmnor is removed.''* ]\Iuch of this colloidal nitrogen seems to be in the form of "oxy-proteic acid" (Salomen and Saxl),^^ which is a mixture of incompletely oxidized polypeptids, containing much unoxidized sul- phur. The proportion of neutral sulphur in the total sulphur in the urine seems to be increased in cancer (Weiss), but not so constantly or characteristically as to be of diagnostic value.^^ Much clinical in- vestigation has been made of these urinary changes, which has gen- erally substantiated the fact that there usually is more increase in colloidal nitrogen and ethereal sulphate in the urine of cancer than in other diseases, but that in no sense are these changes specific for cancer, and the fundamental metabolic disturbances responsible have not been ascertained.^®^ They seem more indicative of the excessive catabolism of cachexia than of cancer tissue itself. Saxl ^^^ has ascribed part of the increased sulphur elimination to abnormal excre- tion of sulphoeyanid, and as small doses of sulphocyanides lead to increased oxyproteic acid in the urine he suggests that in cancer there is a specific disturbance in sulphoeyanid metabolism, an hypothesis that awaits confirmation. Of similar status is the excessive excretion of glycuronic acid described by Roger. ""- Israel, and also Engelmann, have reported the occurrence of a marked increase in the lowering of the freezing-point of the blood in carcinoma (as low as — 0.60° to — 0.63°, the normal being — 0.56°), which they attributed to the presence of excessive products of protein decomposition in the blood. Engel,^" however, found no such increased lowering of the freezing-point in his cases, and questions the signifi- !>4See Mancini. Dent. Arch. klin. l\Tod,. I'Ml (103), 288-. Seniciuiw. Folia T'rol., 1912 (7), 21.5; de Bloemo et nl., Bioolioin. Zcit.. 1014 (0.5). .34;"). »•'•• WicTi. klin. Woch., 1!)11 (24), 440. !"! RliuUmiillor and Koscnblooni. .\rcli. Inf. Alrd.. 101:i (12), 27{>; Intorstato Med. Jour., 1916 (23), Xo. 2: bildiojrrapliy. f'Oa See Goodridfie and Kaliii. Hioclicni. l^iill.. 10].") (4). US: Damask. ^Vion. klin. Woch., lOl.') (28). 499; Rassa, Biodicni. Zoit.. 1914 ( f)4 ) , 19."). n«!b Biodiom. Zoit., 1913 (.'5")). 224. oi' P.ull. Soo. :Mpd. Hop.. Paris, 191") (31). 499. t'TBcrl. klin. Woch., 1904 (41). 82S. IMMUNITY RfJACTlOXS IX CANCER 507 cance of the results of Israel aiul Eiigelniann. According to Moore and Wilson "** the acid-neutralizing- power of the blood ("alkalinity") is increased in cancer; this is probably related to if not the cause of the decreased HCl content of the gastric juice, which occurs whether the cancer is in the stomach or not. As this alkalinity is not associated with an increase in the inorganic bases of the blood, it may be that the proteins have an increased basicity. However, numerous other ob- servers describe a decreased alkalinity as in other cachetic conditions.®" The blood in cancer contains less calcium than normal, which results in a tendency to osteoporosis ^ and to deposition of calcium in the kid- ney ei)itlielium ; ^'^ there is an increase in the i)otassium of both the blood and tissues. - (7) Immunity Reactions in Cancer. — The fact that a certain degree of specific innnunity can be developed against normal tissue cells (see Cytotoxins, Chap, ix), has encouraged study of the possibility of se- <;uring immune antibodies which might be specific for cancer, and has led to much research on this subject,^ with results as yet of more sci- entific than practical value. There is no doubt that the body has distinct powers to inhibit to a greater or less degree the growth of tu- mors, and to destroy many of the cells which escape from cancers into the lymph and blood,* while in experimental animals inoculated tumors are in most instances unable to grow, and they may, after growth has 'once begun, recede or even disappear. Furthermore, ani- mals may be made immune to tumors to which they would otherwise be susceptible. Many schemes of immunization of patients by injec- tion of extracts or autolysates made from their own tumors, or similar tumors of others, have been tried ; but in the hands of competent and critical observers the results seem to have been practically negative.'"' There is no lack of evidence that cancers do produce, in greater or less amounts, various antibodies of some degree of specificity for cancer, which must be inteii^reted as evidence that cancer proteins are in some respects different from the normal proteins of the host ; however, the amount and specificity of these antibodies seems to be low,**^ and, in many observations, they have failed to be demonstrated. Indeed, Coca in his review states unqualifiedly, "The usual biological tests of complement deviation and specific precipitation fail to show the hypo- as Biocliem. Jour., 1906 (1), 207: Watson, Jour. Path, and Baot.. 1000 (1,3), 429: Sturrock, Brit. Med. Jour., 1913 (2), 780. Tlie OH content ot the blood is constantly increased in cancer (^Tenten, Jour. Cancer Res., 1917 (2), 170). 99 See Traube. Int. Zeit. Phvsik.-Chem. Biol., 1014 (1), 380. iGoldzieher, Verb. Deut. Path. Ges., 1012 (15), 283. laM. B. Schmidt, Verb. Deut. Path. Ges., 1913 (16), 329. 2Mottram, x\rch. :\Iiddlesex Hosp., 1910 (19), 40. 3 Literature by Coca, Zeit. InimunitJit., 1912 (13), 525; Kraus et al.. Wien. klin. Woch., 1911 (24), 1003. 4 Reviewed by Wells, Jour. Amer. Med. Assoc, 1009 (52), 1731. 4a See Blumenthal, Zeit. Krebsforsch., 1914 (14), 491: Bauer, Latzel and Wessely, Zeit. klin. Med., 1915 (81), 420. 4b See Morgenroth and Bieling, Biochem. Zeit., 1915 (68), 85. 508 THE CHEMISTRY OF TUMORS thetical antibodies, tliongh a distinct cytotoxic influence can be demon- strated in the plasma of animals of foreign species that have been actively immunized against a tumor." His own experiments failed to demonstrate specific complement-fixation antibodies in patients in- jected with extracts of their own tumors. Lewin ^ also fails to find conclusive evidence of the demonstration of specific antibodies in cancer, yet accepts the immunity which is produced by injections of a virulent cancer material as an active immunity dependent upon cancer antibodies. It may, however, depend on a stimulation of the local cellular reactions that inhibit cancer growth.^" Pfeiffer ^ claims to find specific anaphylactic antibodies in the blood of cancer patients, but this has not been confirmed by several other observers.^'^ V. Dungern ^ has claimed to secure positive complement fixation reactions, partially specific for cancer and benign tumors, by using alcoholic extracts of the tumors or acetone extracts of human ery- throcytes as antigen, but he interprets these reactions as not due to specific antibodies, but to abnormal products of metabolism. The complement content of the blood is said to be slightly increased in cancer ( Engel ) ,* but there is nothing characteristic about this. Ascoli and Izar ** have applied the meiostagmin test (g, v.) and state that this gives very positive results in determining the existence of cancer, their work having been corroborated by many but not by all of those who have repeated it.-'^ Burmeister ^'^ could obtain no reliable results with the epiphanin reaction. Freund and Kaminer ^^ have found that the serum of cancer pa- tients is unable to dissolve cancer cells, as normal serum does, and even protects them against the lytic power of normal serum. The lysis is ascribed to a non-nitrogenous fatty acid, while the protective agent of cancer serum is said to be a "nucleo-globulin" which is in- creased in the serum in cancer. They also find that cancer extracts give a specific turbidity or precipitation with cancer serum, which is attributed to a carbohydrate content of the extract. According to Kraus and v. Graff ^- the serum of full term, pregnant women, and normal umbilical cord serum, behave like serum from cancer 5 Folia Serologica, 1911 (7), 1013: literature. saTvzzor, Jour. Cancer. Res., 1916 (1), 12.5. cWien klin. Wodi., 1909 (22), 989; Zeit. Imiminitiit., 1910 (4), 45.5. f.aSoe Weil, .Jour. Exp. Med., Oct., 1913. TMiincli. ined. Wocli., 1912 (59), 65, 1093 and 2854; also Rosenberg, Dent, iiied. Woch., 1912 (38), 1225. sDeut. nied. Woch., 1910 (36), 986. Not corroborated I)V Ordwav and Kcllert, Jour. :Med. Research, 1913 (28), 287. oMiinch. med. Woch., 1910 (57), 2129; Biocheni. Zeit., 1910 (29), 13. oaSee Rosenberg, Deut. med. Wocli., 1913 (39), 926; Wissung, Rerl. klin. Woch.. 1915 (52), 998.' 10 See Burmeister, Jour. Inf. Dis., 1913 (12), 4.59; Bruiriremann, ^Slitt. (irenz. Med. u. C'hir., 1913 (25), 877. 11 Biochem. Zeit., 1912 (46), 470; Wicn. klin. Wo.Ii., 1911 (24), 1759; 1913 (26), 2108. i^Wien. klin. Wodi., 1911 (24), 191. CHEMISTRY or ('i:irr\i\ si-rr/ric ri uons 509 patients, lu ibUpport of Freuiul and Kaniiner's observation is the experiment of Neuberg ^^ who found that cancer cells plus noi'mal serum underwent digestion more rapidly than cancer cells plus cancer serum, as measured by the incoagulable nitrogen. A critical test of many reconunended methods of serum diagnosis of cancer by Hal- pern ^^ gave disappointing results. With the von Uungern technic he obtained 80 per cent, of positive results, with, the meiostagmin reaction 85 per cent., but with the Abdei'halden method but 30 per cent. The other methods he finds of little value. The testimony concerning the specificity of the Abderhalden reaction in cancer is so conflicting that it seems unprofitable to discuss it, the results varying from such as those cited by Ilalpern above, to 100 per cent, correct reactions de- scribed by others. INfuch weight, however, must be given to the en- tirely unsuccessful attempts to establish the principle of this reac- tion with refined chemical methods by Van Slyke.^*'' Coca '■^^ obtained entirely unsatisfactory results with both the von Dungern comple- ment fixation test and the Freund-Kaminer reaction. ]Many observations have been made on the antitryptic activity of the blood in cancer (see Chap, iii) which has usually shown an in- crease (in all but about 10 per cent, of the cases) ; but many other conditions, especially cachexia, may cause positive reactions. Cancer serum is said to have a heightened power to activate pancreatic lipase.^^ B. CHEMISTRY OF CERTAIN SPECIFIC TUMORS In the literature are to be found a few studies of chemical features of some forms of tumors, which may be briefly discussed to advantage. (1) BENIGN TUMORS (a) Fibromas and Myomas. — The few specimens studied show but a small amount of nucleoprotein, as might be expected from the small amount of their nuclear material. Because of the tendency of fibromas to undergo retrogressive changes, the amount of calcium is likely to be large. No studies as to the special features of their col- lagen, as compared with normal connective-tissue collagen, seem to have been made. Lubarsch ^^ found no glycogen (microscopically) in any of 66 fibromas he examined. Wells and Long ^" found that in uterine fibro-myomas but one per cent, of the total nitrogen is purine nitrogen, distributed as guanine, 44 per cent, ; adenine, 31 per cent. ; i3Biocliem. Zeit., 1910 (26), 344. i4Mitt. Grenz. Med. Cliir., 1013 (27), 370. See also Mioni, Tiimrri. 1014 (3), 697. i4aArdi. Int. :\Ied.. 1917 (10), .56. i*t( Jovir. Cancer Research, 1917 (2), 01. 15 Shaw-Mackenzie, Proc. Rov. Soc. Med. (Pharmacol), 1012 (5), 152. leVirchow's .Arch., 1906 (183), 1S8. IT Zeit. Krebsforsch., 1913 (12), 598. 510 Till-: Clll^MlSTh') OF Tl MORS liypoxanthine, 25 per cent. The relatively large proportion of pre- formed hypoxantliine corresponds with the abundance of this purine free in unstriated muscle. Fibromyomas are able to deamidize their guanine and adenine to xanthine and hypoxantliine, and contain guanase but not adenase. Extracts from uterine fibromyomas show practically^ the same composition as extracts of normal uterus. ^''^ A uterine fibroid analyzed by Becbe ^* contained 14.56 per cent, of nitrogen, 0.981 per cent, of sulphur, 0.139 per cent, of phosphorus, 0.013 per cent, of iron, 0.12 per cent, of calcium oxide, 0.44 per cent, of potassium, and 1.115 per cent, of sodium. The proportions of ni- trogen and sulphur are high as compared with most tumors; the phos- pliorus, iron, and potassium are low, corresponding to the small amount of nucleoprotein and the slow rate of growth. If degenera- tion is marked, the amount of calcium is greatly increased. Kraw- kow ^" found a trace of chondroitin-sulphuric acid in a uterine fibroid. Lubarsch found glycogen occasionally in richly cellular uterine leio- myomas, and in the vicinity of degenerating areas; however, 76 out of 85 showed no glycogen. Pfannenstiel -° analyzed the alkaline fluid of a cystic fibromyoma, which coagulated spontaneously ; it contained sugar, but no mucin or pseudomucin. The cysts were dilated lymph- spaces, and the fluid corresponded to lymph in composition. A simi- lar result was obtained by Oerum,-^ who found in the fluid serum- albumin, serum-globulin, and 0.358 per cent, of fibrin; the total pro- teins constituted 6.3056 per cent. Sollmann — found in the "colloid" of a cystic degenerated fibromyoma both pseudomucin and paramucin (see "Ovarian Cysts"), which differed somewhat from the same sub- stances found in ovarian tumors. From a myxoma of the back Os- wald --'■' obtained a mucin with the following elementally composition : C, 50.82 ; H, 7.27 ; N, 12.24 ; S, 1.19 ; P, 0.25 per cent. This differs from other mammalian mucins in the presence of phosphorus, but Oswald does not consider this a contamination. It also contained 12 per cent, of carbohydrate, apparently glucosamine. The common occurrence of marked cardiac weakness in patients with uterine fibroids has led to the suggestion that in the fibroids some toxic product is formed which acts on the heart, or that both the fibroid and the heart defect might result from a common cause. The experi- mental evidence concerning the relationship is not convincing, and there is much ground for the belief that the heart suffers from the anemia common in these eases.^^ There is said to be a hemolytic poi- i7aWini\vaifArch. cxp. Path! u. Pharm., 1898 (40), 195. 20 Areh. f. Ovn., 1890 (38), 4(58. 2iMalv's Jahrosbcr., 1884 (14), 462. 22 Amcr. Oynccol., 1903 (2), 232. 22a Zcit. i)li.VKi(.!. ("hcni., 1914 (92). Ml. 23 Sw Jasciiko, Mitt. Crenz. Mod. u, Cliir., 1912 (lo). 249; IMcCliini, Suifr., Gvn., Olist., 1914 (18), 180. (JllEMlaTRy OF BESIUS TLMOJi.S 511 son, a lipoid according to ]\Iurray,-* formed in the degenerating fibroids whicli causes local liemolysis and "red degeneration," and there are cases of acute aseptic degeneration of til)roinyonias wiiieli seem to have caused systemic intoxication. (h) Chondromas, like normal cartilage, always contain much glycogen (Luharsch). ^Alorner -"' found cliondroitin-sulphuric acid in several chondromas that he examined, althougli Schiuiedeberg had failed to do so. (c) Lipomas have been studied by Schulz -^ and by Jaeckle.-^ The former found in a retroperitoneal lipoma 75.75 per cent, of fat, 2.25 per cent, of connective tissue, and 22 per cent, of water. Of the fat, 7.81 per cent, was in the form of the free fatty acids and 92.7 per cent, as neutral fats. The fatty acids of the fat consisted of 65.57 per cent, oleic acid ; 29.84 per cent, stearic acid ; 4.59 per cent, pal- mitic acid. Cholesterol was only qualitatively demonstrable. In the (.•onnective tissue was found chondroitin-sulphuric acid. Lubarsch found glj'cogen in lipomas only when they were degenerated. Jaeckle observed the formation of calcium soaps in a calcifj-ing li- poma, the calcium being distributed as follows : calcium soaps, 29.5 per cent. ; calcium carbonate, 28.61 per cent. ; calcium phosphate, 41.89 per cent. The fats of lipomas he found practically^ identical with those of the subcutaneous tissues, except sometimes for a deficiency in lecithin, as shown by the following figures : Composition of Fats in — Subcutane- Lipoma Lipoma Lipoma ous tissue. I. II. III. Eefraction. at 40° 50.6 50.1 50.9 50.5 Saponitication number . 197.3 197.7 197.7 195.9 Eeichert-Meisser number 0.25 0.33 0.35 0.35 lodin number 63.7 59.0 64.0 64.1 Olein 74.1 68.6 74.4 74.5 Oleic acid .... 70.9 65.7 71.2 71.3 Acid number .... 0.39 0.31 0.48 0.67 Free acid 0.196 0.1.55 0.24 0.34 Palmitic acid 18.5 24.9 18.5 Stearic acid .... 6.2 5.1 5.9 Lecithin 0.084 0.015 Cholesterol .... 0.32 0.34 Lipomas are able to hydrolyze fats and esters, their lipase behaving in all respects like the lipase of normal areolar tissue.-'* Lipoma fat is hydrolyzed by lipase as readily as is nonnal human fat. No rea- 24 Jour. Obs. and Gvn., 1910 (17), 534. 25 Zeit. phvsiol. Chem.. 1895 (20), 357. sePfluger's Arch., 1S93 (55), 231. 2T Zeit. phvsiol. Chem., 1902 (36), 53. 28 Wells, Arch. Int. Med., 1912 (10), 297. 512 TllL {JllEiUSTRY OF TLMORH son for the reputed unavailability of lipoma fat for the metabolism of the host could be fouud.--' {d) Ovarian cyst contents have been studied more than almost any other tumor products, because in their gelatinous or slimy sub- stance are contained numerous interesting forms of proteins, many of which are combined with carbohydrates and related to the true miucins. These substances are frequently referred to under the names of pseudomucin, paralbumin, metalbiimin, and ovarian ''colloid," and belong to the class of '^ mucoids." ^^ In view of the fact that the flu- ids in the Graafian follicles of the ovary do not contain these particu- lar forms of protein, their presence in cj^sts derived from adventitious structures (Pfliiger's epithelial tubes) suggests a specific form of metabolism on the part of the epithelium of these structures. Serous cysts, formed by dilation of Graafian follicles, usually are small in size, and the contents resemble those of the normal follicles (Oerum),^^ consisting of a serous fluid with a specific gravity usually from 1.005 to 1.014 (occasionally 1.020 or more), and containing 1.0-4.0 per cent, of solids. Occasionally in these cysts the contents become solidified through absorption of the water, and a gelatinous or glue-like "colloid" content results. Mucoids are never present (Pfan- nenstiel)."- Proliferating cystomas contain the peculiar characteristic mucoid proteins mentioned above. Usually the contents are fluid, but of a peculiar slimy, stringy character, due to the mucoid substance, and often opalescent or slightly turbid. The specific gravity is generally high — 1.015-1.030. The reaction is usually slightly alkaline to lit- mus, and neutral or slightly acid to phenolphthalein. If hemorrhage has occurred into them, the fluid is discolored, and may contain blood- pigments in ciystalline and amorphous forms. Small cysts often show a condensation of the proteins into a semisolid "colloid" ma- terial, but sometimes their contents resemble those of a serous cyst. Often masses of proteins fall out of solution, forming yellowish floc- culi or large deposits half filling the cysts. As with all stagnant flu- ids of this type, cholesterol crystals are frequently found. The char- acteristic proteins are members of the class of pseudomucins, which are constantly present (Oerum). 2» III xanthoma tuhvrosum multiplex, wliicli shows local deposits composed largely of cholesterol esters and contains also ]>igment with tlie properties of a lipochrome, the jjresence of hvper-choiesterolemia is dis])uted. ( lvosenl)looni. Arch. Int. Med., IDl.S (12), .Siif) ; Scliniidt, Dermatol. Zeit.. 1!)14 (21), 1:57). Edsall fonnd the comijosition of tlie fat in the fatty txmiors of adipoNis dolorosa but little different from tliat of normal fat. ( (,)uo{ed hv Dercum and ?ilc('arthy, Amer. Jour. Med. 8ci., 1!)02 (124), 994.) 30 Concernin{^ mucoids see Mann's "Chemistry oi the Proteins," llHttl. iiji. 541- 551. 31 See Malv's Jahresbericht. 1884 (14), 4.59. 32 Arch. f. Viyna-k., 1890 (.•?S),407 (literature). CHEMISTRY OF BEXKIX TUMORS 513 Chemistry of the Mucoids of Ovarian Cysts. — Pseudormuin lias tlie following ■elfiucntaiy compositiuii : C, 4!).75; II, (i.ilS; X, 10.2S; S, 1.25; 0, 31.74 per cent. ( Hammarsten) . In eoimnon with the true mucins it yields a sugar-like reducing body, which has been investigated by numerous chemists (Miiller, Panzer, Zangerle, Ix^vthes, Neubcrg, and ileymann^s). Panzer considers that this re- ducing substance is in the form of a sulphuric-acid compound, similar to, but not identical with, chondroitin-sulphuric acid. Hammarsten, however, did not find this substance constantly present. Leathes determined for the carbohydrate group the composition C,;lL:,NOi„, named it ''paramucosin," and considers it a reduced <^houdrosin (which is the carbohydrate group of chondroitin-sulphuric acid). Neuberg and lleymann established, however, that tlie reducing body must come from chitosainin (CjHi-iNO.-,) , and do not consider paranuicosin a constant con- stituent of ovarian mucoids. The amount of reducing substance varies greatly in the mucoids found in different cysts; in some the mucoid yields but about 3 to 5 per cent., in others as much as 30 or 35 per cent., of reducing substance. Psendomucin dissolves readily in weak alkalies, and differs from true mucin in that it is not precipitated by acetic acid, and from the simple proteins in that its solutions are not coagulated by boiling. With water a slimy, stringy semi- solution is formed, resembling in appearance the material found in ovarian cysts. Leathes distinguishes two forms of ovarian mucoids: (3ne, paramucin, occurs as a firm, jellj'-like substance, which is converted by peptic digestion into the easily soluble pseudomucin. Ovarian "coUoid" probably consists of a thickened pseudo- mucin. often mixed with other proteins. Pfannenstiel 32 considers the "colloid" material as representing a modified pseudomucin, strongly alkaline and relatively insoluble, which he calls "pseudo-mucin /3." He also describes a very soluble mucoid found only in certain ovarian cysts, naming it "pseudo-mucin 7." The reasons why these variations in the pseiidomucins exist is not understood ; they cannot be explained as due to variations in the cell type in the cyst wall, althoug-h pseudomucin is probably the result of true secretion. The smallest cavities of ovarian cystadenomas con- tain nearly pure pseudomucin, which presents a clear, glassy struc- ture ; the larger the cysts become, and the more turbid and thinner the fluid is, the more simple are the proteins it contains. True mucin is never present in ovarian cysts. Pseudomucin occurs only in the glandular proliferating cystomas and the papillary proliferating cystadenomas, in the former appearing constantly and abundantly, in the latter not constantly and never abundantly (Pfannenstiel) . Paral- humin (Scherer) is a mixture of pseudomucin with variable amounts of simple proteins. Metalhumin (Scherer) is the same body that is called pseudomucin by Hammarsten. Paramucin (Mitjukoff) ^^ is a mucoid differing from mucin and pseudomucin in reducing Fehling's solution directly, without having the carbohydrate group first split off by boiling with an acid. Hj^drolysis of paranuicin by Pregl ^'' showed an absence of glycocoll, but traces of diamino-acids, and the presence of leucine, alanine, proline, aspartic and glutamic acids, tryptophane and tyrosine. Substances similar to pseudomucin have been occasionally found in cancerous ascitic fluid and in cystic fibromyoraas (Sollmann) ; and they are abundant as constituents of the contents of the peritoneum 33Hofmeister's Beitr., 1902 (2), 201 (literature). 35 Arch. f. Gynaek.. 1805 (40), 278. seZeit. physiol. Chem., 1908 (58), 229. 33 514 THE CHEMISTRY OF TLMOIiS in the condition known as "pseudomyxoma peritoniei," ^'' when the material is in reality the product of cells implanted on the peritoneal surface through the bursting- of an ovarian cyst (or a cyst of the verm- iform appendix (Friinkel) ).^* The phj-sically similar substance found in pathological sj^novial membranes by Hammarsten differs in yield- ing no reducing substance. Parovarian cysts arising from the Wolff- ian bod}' present an entirely different content, which is a clear, wa- tery fluid, with specific gravity- usually under 1.010; the solids amount to but 1 or 2 per cent., and consist chiefly of salts (the ash being often over 80 per cent.), mostly sulphates and chlorides. They are usually (or always) free from pseudomucin, mucin, or other sugar-containing substances, and other proteins occur only in small amounts, unless the cyst is inflamed. Apparently mucoids do not form in cysts lined b}' ciliated epithelium (Pfannenstiel). Santi ^^ has studied the physical chemistry of ovarian cysts, and finds the freezing point very near that of blood, having no relation to den- sity, viscosity or nitrogen content; the specific electrical conductivity is higher than that of blood serum. The physicochemical properties are less dependent upon chlorides, and more on other substances (Gnmer)." Intraligamentary papillary cysts contain a yellow, yellowish-green, or brownish-green liquid, which contains little or no pseudomucin ; the specific gravity is usually high (1.032-1.036) and the fluid con- tains 9 to 10 per cent, of solids. The principal constituents are the simple proteins of blood serum (Hammarsten). According to the same author, the rare iuho-ovarian cysts contain a watery serous fluid with no pseudomucin. (e) Dermoid cysts of the ovary contain, as their chief and most characteristic constituent, a yellow fat, which melts at 3-4°-39° and solidifies at 20°-25°. Ludwig and Zeynek *^ have examined over sixty such tumors, and found that the fatty material constantly contains two chief constituents : one, crystallizing out readily, they believed to be cctyl alcohol, (CH3— (CIL),, — CILOII) ; the other, remaining as an oily fluid, seems to be closely related to cholesterol, although not consisting of one substance alone. Small quantities of arachidic acid (C^nH^oOo), as well as stearic, palmitic, and myristie acid (Ci4H.,sOo), existing as glycerides, are also pres- ent. Ameseder,"*^ however, found evidence that the supposed cetyl al- cohol is reall.y eikosyl alcohol (CooH^oO). Tliese substances are 37 Litoraturo l)v Potcrs. :\r()iiatsclir. f. Cch. u. C,\u.. ISOO (10), 74!)-. Wobor, St. Pctorsb. inod.' Woeh., 1001 (26), XU. ssMiincli. mod. Woc-li., 1901 (4S), OfJf). 3!'l''()]iii dill, cliiniica ot inicrosco])., 1010 (2), 73. 40 7{i()cli('in. Jour.. 1007 (2). 3S;{. 41 Zoit. jilivsiol. f'licni.. 1S07 (2.3), 40. *^ Jhid., 1007 (.'")2), 121. CHEMISTRY OF MALIONAyT TUMOh'S 515 secreted by tlie glands of the cutaneous structures of the cyst, and resemble in composition sebaceous material, which is characterized by containing a large proportion of cholesterol partly combined with fatty acids. Dermoids sometimes contain masses of fatty concretions which seem not to depend on chemical changes but on the presence of forma- tive nuclei and framework of desquamated epithelium ; they consist of a mixture of neutral fats and cholesterol esters, with some free cho- lesterol."-'^ Cholesteatomas, in addition to their abundant cholesterol content, contain keratin.'^ (/) "Butter" Cysts. — In the mammary gland retention cysts form filled with products of alteration of the milk, including butyric acid 'and lactose (Klotz),"'" and these are called "butter cysts" or milk cysts. Analysis of the contents of such a cyst by Smita *■' gave the following results, as compared with human milk : Fat . . Casein . Albvunin Milk-sugar Ash Watei- . Cyst contents. Human milk 72 07 3.nO . 4.37 0.63 . 1.91 1.31 0.88 6.04 0.36 0.49 . 20.81 87.09 Fats consisted of — , Cyst. Cows' milk. Stearin and palmitin 37.0 50.0 Olein ^''•" Butyrin 9.0 7.8 Occurring independent of lactation usually, but not always, are the ''soap cystl" which contain chiefly calcium and magnesium soaps, but also neutral fats, free fatty acids, and traces of cholesterol (Freund*''). (2) MALIGNANT TUMORS The chief general features of the composition of these growths have been considered in the discussion of the chemistry of tumors in general (pases 494-509). A malignant tumor differs from a similar benign tumor chiefly in having usually a larger proportion of the primary cell constituents, and a smaller proportion of the sec- ondary constituents and intercellular substances, since these are largelv the product of the functional activity of the cells, which, in malignant tumors, do not often develop sufficiently to functionate extensively. Hence malignant tumors usually show a rather high proportion of the characteristic constituents of nucleoproteins ; i. e., phosphonis and iron. If rapidly growing, they contain much potas- 42aLippert, Frankf. Zeit. Path., ini3 (14), 477. 43Risel. Verh. Deut. Path. Gesell., 1900 (13), 322. 44Arch. klin. Chir.. 1880 (25), 40. v, • i ri >, 45Wien. klin. Woch., 1800 (3), 551; see also Zdarek. Zeit. physiol. Chem., 1008 (57), 461. 46 Virchow's Arch., 1809 (156), 151. 516 THE CUEMIKTRV OF TUMORS sium; if iinderfjoin states that normal human hone marrow mav contain true albumoses. 62a Taylor et aJ., Jour. Biol. Chem., 1917 (29), 425. 63 Biochem. Zeit., 190S (10), 167. 64 Arch. Int. Med., 1912 (9), 236. 65 Folia Hematol., 1909 (8), 14. 66 Zeit. physiol. Chem., 1905 (46), 125. 522 THE CHL'MlsTh') OF TLMORS found that the serum of rabbits immunized with Bence-Jones protein gives the precipitin reaction witli human serum, is evidence that the protein is a human tissue protein and not merely an absorbed and excreted food protein. This has been corroborated by Hopkins and Savorj-,*'^ who also found that the amount of protein in the urine, which contained about one-third the total nitrogen excreted, varied with the general metabolism and was not controlled by the diet. i\Iassini "^^ reports securing positive complement fixation tests with immune sera, differentiating the Bence-Jones protein from normal serum proteins; positive sensitization tests were not obtained by cu- taneous injections of the protein by Boggs and Guthrie. Injected into the blood it is non-toxic and does not lower coagulability as a proteose would. It is capable of acting as an antigen in anaphylaxis reactions, wliich also indicates that it is a complete protein and not a cleavage product.®" When injected into dogs it is partly utilized, although nephritic animals excrete it partly hydrolyzed into proteose.*'-^ 67 Corroborated also bv Bosprs and Guthrie, Amer. Jour. Med. Sci., 1012 (144), 803; Folin and Denis, Jour. Biol. Chem.. 1014 (18), 277. esDeut. Arch. klin. Med., 1911 (104). 20. 69Tavlor and Miller, Jour. Biol. Chem.. lOlG (25). 281. C PI A P T K R X AM 1 I PATHOLOGICAL CONDITIONS DUE TO, OR ASSOCI- ATED WITH, ABNORMALITIES IN METABOLISM, INCLUDING AUTOINTOXICATION During the course of metabolism innumerable organic compounds are formed, some of which are of a more or less poisonous nature. As long as the body is in a nonual condition, these injurious sub- stances are kept from accumulating in sufificient quantities to do hann; this is accomplished, in one of the following ways: (1) elimi- nation from the body in the urine, feces, etc.; (2) combination with other substances into harmless, or relatively harmless, compounds; (3) chemical alteration into compounds that are non-toxic or relatively innocuous. Therefore a harmful accumulation of metabolic products may be the result of any one of the following conditions : (1) Failure of elimination because of abnormal conditions in the eliminating organs ; e. g., uremia. (2) Failure of neutralization by chemical combination, presumably due to abnormalities in the organs or tissues through whose activities the neutralization is normally accomplished; e. g., diseases of the liver. (3) Failure in the chemical transformation of the metabolic prod- ucts; this may result either from abnormalities in the functionating tissues, or through a checking of the normal steps of metabolism by the failure of elimination of the end-products. (4) Excessive formation of certain normal products of metabolism; e. g., hyperactivity of the thyroid. (5) Production of abnormal toxic chemical substances; e. g., the intoxication following superficial burns. Numerous classifications of autointoxication have been proposed by various authors, some excluding from the causes of autointoxication all but the products of metabolism within the blood and tissues of the body, as has been done in the preceding consideration ; many in- cluding intoxications caused by the products of gastro-intestinal fer- mentation and putrefaction; and still others (v. Jaksch) including even the intoxications produced by bacterial invasion of the body.^ It is extremely difficult to draw the line as to just what should be 1 See resume by Weintraud, Ergeb. der Path., 1897 (4), 1. 523 524 ABWORMALITli:S /X METABOLISM included under the temi autointoxication, and particularly difficult to decide the proper placing of the intoxication resulting from fecal retention and from processes of decomposition in the alimentary canal. For example, the poisoning followinu,' the eating of partially decomposed canned food could not be looked upon as an autointoxi- cation, and yet there is no fundamental difference whether the decom- position occurs, as in this case, before the food enters the body, or whether it occurs in the intestinal tract because of abnormal bacteri- ological or anatomical conditions. On the other hand, since many of the obnoxious products of metabolism are eliminated through the bowels, failure of elimination through this channel may lead to a true autointoxication as much as may deficient renal elimination. On the M'hole. it seems best to restrict the term autointoxication, as far as possible, to the disturbances produced by products of metabolism that have been formed within the tissues of the body {intermediary metaholism), considering as a distinct but related subject gastro-in- testinal autointoxication. In the discussion of autointoxication from the standpoint of chem- ical pathology, we are interested particularly in the chemical nature of the substances that cause the intoxication, and in the chemical processes by which their action is kept at a minimum, rather than in the clinical features or anatomical results that may be produced. Unfortunately, in but a few instances have the exact chemical sub- stances causing these intoxications been accurately determined, prob- ably because in most cases not one but a number of poisonous sub- stances are present; and, furthermore, we do not always know ex- actly when a certain disease is to be ascribed to autointoxication, nor can we always determine that the cause of a certain intoxication lies in an abnormality in metabolism and not in an infection of hidden nature. It is, therefore, quite impossible, with the uncertain infor- mation available at this time, to consider autointoxication in a sys- tematic way, and we must limit ourselves to a consideration of cer- tain pathological conditions in which there appears to be an element of abnormal metabolism with resulting intoxication. In some cases this intoxication is a prominent feature of the disorder, in others it is subordinate to other manifestations of the disease ; and, finally, we may have marked alterations in metabolism without evidences of dis- turbance of health {e. g., cystinuria, alkaptonuria). Of the autointoxications due to the retention of poisonous products of metabolism that should be excreted from the body, first in order of importance stand uremia and cholemia (the latter has already been considered in connection with the discussion of Icterus, Chap. xvi). Of apparently less significance are autointoxications due to failure of elimination of gaseous metabolic products by tlie lungs, and failure of the excretorv functions of the skin. UREMIA 525 UREMIA - The cause or causes of the severe, often fatal, intoxication that may occur when the outflow of urine is completely checked, or when it is qualitatively and quantitatively altered for long periods of time, have not j^et been definitely determined. As the kidney seems to be the chief organ for the removal of the products of nitrogenous metab- olism, it is naturally assumed that uremia is the result of a retention of these products, but as yet it has not been ascertained which of the many products is responsible, and, indeed, there are very good rea- sons for questioning if the substances present in normal urine do or can cause uremia when their elimination by the kidney is defective. There is no question but that the urine contains toxic substances. Among them are the salts of potassium, which, however, cannot alone explain all the urinary toxicity, for the symptoms produced by the injection of urine are different from those produced by potassium salts, and it has been found that the inorganic constituents (ash) of urine are less poisonous than the entire urine. Furthermore, toxic mixtures of organic, ash-free substances have been obtained from nor- mal urine.^ Of the known normal constituents of the urine there -are few, however, that are toxic to any considerable degree, and these •occur in but very small quantities. Urea is generally considered as almost absolutely non-toxic, the animal body withstanding injection of large quantities without appreciable injury. Uric acid, the purine bases, hippuric acid, creatinine, and the urinary' pigments are all possessed of very slight toxicity, and their effects do not explain uremia. Injections of urine into animals may cause more or less disturbance, but it is different, on the whole, from the manifestations of uremia. (The experiments of Bouchard and his school present such serious errors of technique and interpretation that they are now largely disregarded.) For these and other reasons, it is generally considered that the intoxication of uremia is not due solely or chiefly to the substances that are normally eliminated in the urine, but rather to more toxic antecedents of the nitrogenous constituents of the urine. Urea repre- sents but the final product of a long series of reactions by which the huge protein molecule is broken up into its "building-stones," the various amino-acids, and these in turn are decomposed in such a way that their NHo groups are combined with carbonic acid * and eliminated as the diamido-compound of carbonic acid, namely urea, 2 General r^snin^ with literature bv: Honigmann. Ergeb. der Pathol., 1894 (Bd. 1, Abt. 2), 639; 1902 (S), .'549 • Ascoli, Vorlesimgen iibcr Uriimie. Jena, 1903. 3 See Dresbach. Jour. Exp. Mod., 1900 (."i). 31;"). 4 Arginine alone of all the amino-acids splits off urea directly from its molecule. 526 AB.\OiiUALlTlE>i IS METABOLISM 0 = c/ . "We know that the liver is able to accomplish the con- ,NH, \nil • version of amino-aeids to urea, for it has been experimentally shown that if leucine and glycocoll are passed through the vessels of the iso- lated liver they disappear in part, while an increased amount of urea escapes from the hepatic veins. It is probable that the liver is the chief site of urea formation, but it is also probable that urea can be formed in other organs. We do not know, however, the in- termediate steps by which the amiuo-acids of the protein molecule are converted into urea. It has been repeatedly shown that urea can be formed from ammonium salts of organic acids (including ammonium carbonate), and ammonia is a constant product of autolysis, being characteristically more abundant as a product of autolytic proteolysis than as a product of tryptic proteolysis ; therefore, one of the ante- cedents of urea is probably ammonia, which is somewhat toxic and especially hemolytic." Another antecedent of urea is ammonium car- bamate, which stands in structure intermediate between urea and am- monium carbonate, as shown by the following graphic formulae : ,0H .0 — NH, .NH. .NH. 0=C< 0=C< 0=C< 0=C< (carbonic acid) (ammonium carbonate) (ammonium carbamate) . (urea) That ammonium carbamate is possibly an important precursor of urea has been shown particularly through the results of studies of dogs with Eck's fistula,*' which consists of a fistula between the portal vein and the inferior vena cava, the blood from the portal system then passing directly into the general circulation without first passing through the liver. In such animals the urine becomes poor in urea and relatively rich in ammonium carbamate. At the same time, the dogs show severe symptoms of intoxication from which they die, and which are similar to the symptoms that follow intravenous injection of ammonium earl)anmte. Ammonium carbamate, being a substance of considerable toxicity ^ when free in tlie bh)()d, it has, therefore, been quite widely considered that it may be an important factor in the production of uremic symptoms. On the other hand, it seems most pr()ba])]e that the condition of uremia does not depend upon one but upon many various and varying su])stances, especially as Hawk '^ found that sodium carbamate did not produce uremic symptoms in his Eck fistula dogs, while Liebig's extract did." Clinically the symptoms 5 Concerning the toxicity of iunmoniuiii sails sco IJacliford and Craiio. 'Medical News, 1902 (81), 778. 6 See Hahn, Masson, Xciicki, and rawlow, Arcli. f. cxp. l*alli, u. riiarm.. 1S0.3 (32), 161. 7 See Bickcl, "Exp. ITntersucli. iibor Cliolaciiiic," W'icsliadcii. 1000. 8 Amor. .Tour. Pliysi(d., 1008 (21), 2(10. 0 Fisclilcr l»(di('ves tlie iiiloxicatioii wliicli occurs after fcc(liii) . 207). 11 See Tieken, Amer. Med., 1905 (10). pp. .303. .507, and 822. 12 See table of freezinfj points of blood and clfusions on page 35.5. 13 Deut. med. Woch.. 1002 (28), 501. 1-4 Die chronischen Nierenentziindungen. etc., Berlin, 1002. 15 Stern (Med. Record, 1003 (63), 121) notes that the electrical eondtictivity is reduced by the presence of excessive quantities of non-electrolytes in uremia, and regards this lowered conductivity as a factor of some possiljle importance. i«Levene et al.. Jour. Exper. :Med.," 1000 (11), 825. I'Zeit. klin. Med.. 1003 (50), 441: 1905 (57), 30. 17a Arch. Int. Med., 1017 (19), 3.54. 928 ABX0R1IAIJTIL\S IX METABOLISM ered iu chronic nephritis with uremia, an increased proportion of globulin being present; with uremia the total protein content is nor- mal or slightly higher, with usually increased globulin, while nephritis without edema or uremia produces a marked increase in the globulin. The decrease in red corpuscles and hemoglobin in nephritis is a well- known feature. Orlowski ^* found that an accumulation of acids occurs in uremia, but not until just before death, and, therefore, the reduction of blood alkalinity is not the cause, but an accompaniment of the uremia. Fur- thermore, in other diseases a corresponding or greater reduction in alkalinity may occur without uremia. Measurements of the partial pressure of COo in the alveolar air in uremia indicate, however, a certain degree of acidosis.^'' This seems to occur to a sufficient degree to be responsible for definite clinical symptoms of acidosis only in advanced nephritis, but earlier in nephritis an acidosis may be demonstrable by the alkali tolerance test when it is not sufficient to affect the alveolar air.-° The development of this terminal acidity, together with the finding of albumose in the blood of a nephritic by Schumm,-^ suggests the probability of active autolytic processes occurring in uremia. Neuberg and Strauss -- have also found glyco- coll in considerable quantities (1.5 per mille) in the blood-serum of a uremic patient and in the blood of nephrectomized rabbits. The amount of colloidal material present in the urine is decreased in nephritis, according to Pribram,-^ who suggests that retention of this material, which is rich in aromatic radicals, may be of importance in the toxicity of uremia. Rumpf found that the organs of nephritics contain an excess of potassium, and Blumenfeldt -* attributes this to a defective elimination of potassium salts which he observed in ne- phritis. Numerous attempts have been made by both chemical and immu- nological methods to determine wdiether the proteins in the urine in nephritis come from the food, the blood, or from the renal cells them- selves. In alimentary albuminuria the urinary proteins seem not to be those of the food, but human proteins.-^ In nephritis, however, differentiation between serum proteins and kidney proteins has not yet been satisfactorily accomplished.-" The development of improved methods of analysis of small quan- tities of blood, and other fluids, especially by Folin and Denis, ]\Iar- isZontr. f. StofTwpchsol \i. Vcrdauunpskr., 1902 (3), 123. 19 Straub and Sclilavor, Miinch. med. Woch., 1912 (59), 5G9. 20Peabody, Arch. Iiit. INFod., 1915 (16), 955. 21 Hofmeister's Bcitr., 1903 (4), 4.53. 22 13erl. klin. Woch., 190G (43), 258. 23 Fortschr. d. Mod., 1911 (29), 951. 24Zeit. exper. PatlioL. 1913 (12), 523. 25 Wells, Jour. Aincr. ^Fcd. Assoc. 1909 (.■)3), 803. 20 Cameron and Wells, Arch. Int. Med., lOl.'j (l.")). 746. UREMIA 529 shall, and \'aii Slykc, has enabled us to obtain exact knowledge of many of the chemical changes of nephritis and uremia."^ It has been found that the normal blood contains from 20 to 30 mg. of nitrogen in noncoagulable form in each 100 c.c, there being usually about 5 mg. increase after meals, and ordinarily about one half, or a little more, of this nitrogen is in the form of urea. In all conditions that impair renal function, whether renal changes or circulatory deficiency, there is a rise in this noncoagulable nitrogen, and when there is ex- cessive tissue destruction there may also be a slight rise independent of renal injury. As a general rule, but with some exceptions, the amount increases with increased renal impairment, the highest figures being seen in uremia, in which figures as high as 350 mg. have been obtained. Tn 130 uremics, Foster found the average to be 84 mg. of nitrogen. There is no constant relationship between the blood pres- sure and the nitrogen figure, but functional tests usually show a cor- respondence between the excretory power of the kidney and the re- tention of metabolites in the blood. The symptoms of asthenic uremia are rarely well defined when the concentration of urea in the blood is less than 100 mg. per 100 c.c, and thej^ are rarely absent Avhen the concentration exceeds 200 mg.-* Along with the other nitrogenous constituents the uric acid is in- creased from a normal 2 to 3 mg. up to 7 to 10 mg., and even higher. Creatinine rises from 1 or 2 mg. up to 5 to 20 mg.-'' On the other hand the amino-acid nitrogen may be normal in the blood even with extremely high nonprotein nitrogen figures,^" although sometimes it is much increased, as high as 30 mg. amino acid N having been found by Bock ^"^ in uremia (the normal figure being 7 mg.). Ammonia nitro- gen may show a slight increase, rising in half of Foster's cases from the normal 0.5 mg. to from 0.7 mg. to 2.2 mg. per 100 c.c. Indicanemia may also be present, but it is not a toxic factor. (Dorner.)^^ The blood normally contains about 0.05 mg. per 100 c.c. ; in uremia it may rise to 0.2 mg., and as much as 2.2 mg. has been found in one case.^- The Etiology of Uremia. — The fact that the highest figures for non-protein nitrogen are usually found in uremia might be accepted as proving that uremia is caused by poisoning with these metabolites, were it not for certain contradictoiy observations. 27 Good I'eviews and l)ihlioo;raphips are given hv Tileston and Comfort, Arch. Int. Med., 1914 (14), 620; Schwartz and :\rcGill', ibid., 1910 (17). 42: Woods, ibid., 1915 (16), .577; Karsner, Jour. Lab. Clin. :Med., 1916 (1), 910. 28 Hewlett, Cxilbert and Wickett. Arcli. Int. Med., 191G (18). 6.36. 29 See :Mvers and Fine, Arch. Int. :Med , 1915 (16), 536; 1916 (17), 570. 30 Foster, Arch. Int. INled., 1915 (15). 356. •"■oa .Tour. Biol. Chem., 1917 (29), 191. 31 Deut. Arch. klin. :Med., 1914 (113), 342: Rosenberg Arch. exp. Path.. 1916 (79), 260: TscherkotT, Deut. nied. Woch., 1914 (40), 1713. 32Hass, Deut. Arch. klin. Med., 1916 (119), 177. 34 530 ABNORMALITIES IN METABOLISM (1) Occasionally quite typical attacks of uremia are observed with- out high nonprotein nitrogen figures for the blood, even as low as 28 mg. having been recorded in a fatal case.^^ (2) Extremely high nonprotein nitrogen content may be observed without uremia. Thus Tileston and Comfort found 169 and 150 mg. in two cases of acute intestinal obstruction without uremic symptoms, and similar results have been obtained in bicliloride of mercury pois- oning,^* and mechanical anuria. The occurrence of albuminuric re- tinitis also seems to bear no relation to the nitrogen retention (Woods). (3) None of the known nitrogenous constituents of the urine can be held responsible for all the manifestations of typical uremia pois- oning. The highest purine, uric acid and creatinine concentration in a given case may occur entirely independent of uremic couditions,^'^ the amino-nitrogen is not increased in uremia and urea is not sup- posed to be toxic in this degree. To be sure, an unknown toxic sub- stance may be responsible, but in some cases of uremia the total non- protein nitrogen can be accounted for by the known nitrogenous com- ponents found in the blood (Foster). We therefore are driven to one of the following alternatives : (1) The nerve cells may be made hypersensitive to some one of the known constituents by the excessive amounts of the other metabolites. This is a purely speculative hypothesis, without any actual evidence in its support. (2) The portion of unidentified nitrogen usually present in the blood may contain a specific, highly efficient poison. In support of this hypothesis is the finding in a series of cases that the pro- portion of noncoaguhible blood nitrogen that couhl not be accounted for by tlie known nitrogenous metabolites seemed to vary directly with the severity of the symptoms (Woods). 35a ' Ilartnian 3ob has suggested that the substance which causes the characteristic odor of the lu-ine may be responsible for at least some of the intoxication of uremia. This substance, which he has isolated and described under the name "urinod," he believes to be a cyclic ketone with the empirical formula CgHsO; it is highly toxic, and causes mental symptoms. This important observation awaits confirmation. Foster s^r has described the finding of a toxic base in the Ijlood of uremics, absent from the blood in other conditions, whicli causes death of guinea pigs with symptoms suggestive of tlie eclamptic type of uremia. Further development of this work is also awaited. 33 There arc few who would go to tlie extreme of Strauss ( IJerl. kliii. Woch., 1915 (.52), .S08) and limit tlie term lu-emia to cases showing a liigli non -protein nitrogen in the blood, no matter wliat the symptomatology and patliology may be. A totally difi"erent view])()int is expressed liy iJeiss, Zeit. klin. Med., 1014 (80), 97, 424, 4.')2. 34 See Foster, Arch. Int. IVfed., l!)ir) (],')), 754. 35Mvers and Fine, .Tour. IJiol. Cliein., 1915 (20), :V.n. 35a Arch. Int. Med., V.U', (Hi), .-)77, sab Ibid., 1915 (Ifi), 9H. 30c Trans. Assoc. Anier. i'livs., 1!)15 (.'JO), 305. UREMIA 531 (3) Uremia may not depend on intoxication of tlie nerve cells, but upon the nieclianical effects of edema involving these cells. One of the striking features of autopsies of uremics is often the ."wet brain" and the excessive amount of cerebrospinal iluid wiiicii, during life, may be found lo be under a heightened pressure. We l. 4iaAmer. .Tour. j\Ied. St-i., 1914 (147), or.fi ••lb Amor. Jour. ^Nled. Sci.. 1917 (15.3), 94. 4-.iAroh. f. Gyn., 1905 (7(5), 537. 43 Arch. f. Gyn., 1912 (96), 43. ECLAMPSIA 535 icity of tlie blood in eclampsia leave nothiiif^ proved concerning this point, but more recent studies by Graf and Landsteiner *^ affirm an increase of toxicity of the blood, not due to any special poison but to an increase in the amount of the toxic substances ordinarily present. The antitryptic titer of the blood may be much increased. ^^'^ Zang- meister ^^ ascribes importance to edema of the brain, liallerini *" found that the physico-chemical changes in tlie blood are quite tlie same as in corresponding conditions of nephritis. An increase in the sugar content of the blood has been observed by Benthin/^ but no other abnonnality of carboh^'drate metabolism is usually present. Blood lipase is much increased because of the hepatic injury (Whip- ple ).-''••' Theories as to Etiology. — The anatomical changes of eclampsia are such as to leave little or no room for doubt that there is a severe intoxication with poisons that have a markedly toxic effect upon all the organs of the body, thus differing from the toxic materials at work in uremia, which seem to affect chiefly the central nervous system. Repeated bacteriological and histological studies have failed to dem- onstrate that infection with either vegetable or animal parasites is the cause, and clinical observations do not support such an hypothe- sis. The association of the condition with pregnancy, and particularly the rapid improvement that often follows the removal of the con- tents of the uterus, almost compels us to admit that the causative agent is produced by the fetus or the placenta. Some investigators (Politi, Liepmann) believe that they have found a greater degree of toxicity in extracts from the placentas from eclamptic than from nor- mal women. We have no exact ideas as to the nature of the supposed toxic substances, except that recent developments in the study of immunity reactions point to their origin from proteolysis of tissue proteins, presumably from the placenta. The hypothesis of Zweifel that lactic acid is responsible seems untenable, and the degree of acidosis present is not sufficient to account for the intoxication (Losee and Van Sh-ke). The Placenta as a Source of Intoxication. — Histologists having fre- quently observed placental cells in the blood and vessels of eclamptic patients, it was once suggested that multiple capillary emholi of pla- cental cells, detached from chorionic villi and forced into the pla- cental circulation, cause the manifestations of the disease ; this theoiy is entirely inadequate, however, to explain all the features of eclamp- sia. Eelated to this hypothesis is the idea that the placental tissues, "Cent. f. Gyn., 1909 (3.3), 142. 44a Franz, Arch. f. Gvn., 1914 (102), 579. 45Deut. med. Woch.,"l911 (37), 1879. 40Annali Ostet. e Gin., 1910 (32), 273. 4TMonats. Geb. u. Gvn., 1913 (37), 305; Rvser, Deut. Arch. klin. Med., 1916 (118), 408. 47a Jour. Med. Res., 1913 (24), 357. 536 AIi.\URMALITIJ:,S 1\ inrrABOLLSM being- foreign to the maternal organism in so far as they are derived from the ovum, give rise to the production of antibodies {syncytioly- si)is) by the mother, which are toxic for pregnant animals (Ascoli), and which may have to do with eclampsia in some unknown way. Kosenau and Anderson found that guinea pigs could be made anaphy- lactic to guinea-pig placenta, showing conclusively that the placenta contains i)roteins foreign to the motlier. Attempts to establish the anaphylactic nature of eclampsia have, like so many other theories, foundered on the fact of the characteristic anatomy of this disease, wliich is never seen in anaphylaxis.^'* The studies of Abderhalden have shown that the blood of ever}' pregnant female animal contains enzj'mes which have a specific proteolytic action, and so the possibility exists that abnormal or excessive products of such proteolj^sis, or a lack of adequate defensive digestive action, may be responsible for the toxemias of pregnanc3^ Esch " and Franz ^^ have, indeed, found evidence of the presence in the serum and urine of eclamptics, of sub- stances resembling anaphylactic poisons in their action, and presum- ably derived from proteolysis somewhere in the body. Franz found That if the poison injures the kidneys seriously it is retained in the bod;,', the urine ceasing to be toxic, wliich has, presumably, a relation to the toxicosis of eclampsia.^^ Liepman " and others have reported the finding of a considerable degree of toxicity in eclamptic placentas, but this is probably related to the increased autolysis observed in eclamptic placentas by Dry- fuss.'*^ According to Mohr and Heimann,^''^ the eclamptic placenta shows a great decrease in lecithin, which they ascribe to the increased autolysis, and to the hydrolyzed lecithin they attribute the hemotoxic effects. On the other hand JMurray and Bienenfeld ''^ report the find- ing of an increased amount of lipoids in eclamptic placenta.''" The Fetus as a Source of Intoxication. — A reasonable view of the cause of eclampsia is that it is initiated by the excessive products 48 See Felliinflcr. Zoit. Gelj. u. Gvn., 1911 (68), 26; :\rosbaclicr. Dent. med. Woch., 1911 (37), ]()-21. However, Vertes (Monat. (ieb. u. Gyn., 1914 (40), 361, 4(il' ) states tliat animals dyiiij^ from aiiajihylaxis may sliow typieal eelainptic tissue clianges, whieli is not in accordance willi tlie observations of manv otliers. 49Miincli. med. Wocli., 1912 (59), 461. 50lhid., pajje 1702. 51 Hull and Ehodenlmrfj (Amer. Jour. Obst., 1914 (70), 919) ascribe impor- tance to leucine derived from proteolysis of the placental elements, while Kiutsi (Zeit. Gel), u. ^iyn., 1912 (72), 57(i) considei-s the nuclcins of the {)lacenta llie toxic ajjents; both statements beinfj unconlirn\ed and improbable. ■•sMiinch. med. Woch., 1905 (52), (;S7 and 24S4; ]?oos, P.oslon .Med. and Surj;. Jour., 1908 (158), 612. •"]?iochem. Zeit., 1908 (7), 493. 5iJbid., 1912 (4(i), 367. 55Jour. Obst. and Cvn. Urit. Empire, 1!»1() (18). 225; lii.nlicm. Zcil.. 1912 (43), 245. •''« The hypothesis of ^lohr and I'rcund llial oleic aejd fidiii llic cehun])tic placenta is a hcniohiic fa<'1nr, is not corroborated bv I'olano (Zeit. ly to tlie disease as a whole. "2 Babes, Ann. Inst. Path. Bucarest, vol. 6. '3 Frey, Zeit. klin. Med.. 1012 (75). 4.'}.5. '* Jour. Amer. Med. Assoc, 1904 (44), 685. 540 AHSOiniAlATIES 1\ .UI:TA HOLISM suits. In the countries wliere phosphorus poisoning- is common (es- pecialh' Austria) there has been found much difficulty in distin- guishing in many cases the results of phosphorus poisoning from acute yellow atrophy of the liver, and many have contended that there is no real difference ; i. e., that phosphorus, as well as unknown poisons, may cause acute yellow atrophy. The present trend of opin- ion, however, seems to favor the view that there is a primary liver atrophy which is different from that caused by phosphorus or other known poisons in several essential respects.''* Phosphorus Poisoning. — Between phosphorus poisoning and ^"pri- mary" hepatic atrophjj the following chief differences may he dis- cerned: Phosphonis produces a general injurious effect upon all the organs of the body, the liver merely showing the most marked anatomical changes, which at first consist of a fatty metamorphosis of the liver, due to migration of the body fat from the fat deposits into the injured cell (Rosenfeld, Taylor) ; subsequently the liver cells disintegrate, the cytoplasm being aft'ected before the nucleus, and the liver may become smaller than normal, although it is usu- ally enlarged because of the fat deposition. Typical acute yellow atrophy is characterized by an early necrosis of a large proportion of the liver-cells, the nucleus becoming unstainable while the cyto- plasm is still little altered in appearance, and fatty changes play a subordinate role or are absent. As Anchiitz says, the poison seems to strike at the life of the cell, its nucleus, while phosphorus attacks the cytoplasm. Furthermore, the poison of yellow atrophy seems to be very specific, for it attacks the other organs of the body almost not at all, and within the liver it affects only the hepatic cells proper, while the bile-duct epithelium and the stroma cells are so little in- jured that they are able to proliferate greatly, this proliferation being a prominent feature. There are also clinical and chemical dif- ferences that will be discussed later, but yet, on the whole, the re- semblances of yellow atrophy and phospliorus poisoning are so great that we have obtained much information concerning the former by means of experimental studies of phosphorus poisoning. Delayed Chloroform Poisoning. — After chloroform narcosis, and rarely after etlier, there occasionally develops a severe intoxication, with clinical and anatomical findings very similar to acute yelloAV atrophy and phosj)horus poisoning;"" in point of the fatty changes the cases usually stand intermediate between acute yellow atro]ili,\' wnd phosphorus poisoning. Tliis action of chloroform would seem, fioni 75 See Anschiitz, Arb. a. <1. I'atli. hist. Tiiljiiiucn. 1!M>2 ( :! ) , -I'-W: raltaiif. \vy\\. Deut. Path. Gesell., VMY.\ (5), !)1: liicss, ]?('rl. klin. WO.li., litOf) (42). No. 44a, p. 54. 7« Complete review and lileratvire bv Hevan ami Favill. .Tour. Amer. Med. Assoc., 100,5 (45), 091; :Muskeiis. Mitt, (irenz. :\led. u. (hir., IDll (22), .lliS. Full dineuKsion of clieniislry of clilorofonu necrosis liy Wells, .lour. Riol. Cheni.. 1!>()S (5), 12!>. Kxpciimcnial necrosis — see \\'liip))le and Sperrv, .lohns Hopkins Hosp. Bull., I'JU!) (20), 278; (Jraiuim, .lour. Ivxper Med., 1!)12 (15), '^Ol. ACITK YELLOW ATh'OI'll) <)l' Tin: Ll\ El! 541 the studies of Evarts Graham,'"-' to be produced by the hydrochloric acid formed from it in the liver. Some cases of puerperal eclampsia also i)resent such profound liver changes that they are distinguished as eclampsia chictiy on the basis of the convulsive manifestations, rather than on the ground of anatomical changes. So, too, the hepa- tic changes in certain septicemias and acute syphilis may resemble those of acute yellow atrophy to a greater or less degree. Summary of Views on Etiology. — From a review of the literature and the study of a few cases, the writer has reached the following understanding of the condition described as acute yellow atrophy of the liver: The "atrophy" is due entireh' to autolysis of necrotic liver-cells by their own enzymes. In the most typical cases of "pri- mary'' or "idiopathic"' yellow atrophy we have to do with a poison having a very specific effect on the liver-cells, which destroys their "life" (i. e., stops synthetic activities) without injuring their intra- cellular proteolytic enzymes," and consequently autolysis occurs; as the poison affects other organs but little, the necrosis and autolysis continue until there is so much loss of liver function that systemic poisoning results from the hepatic insufficiency and from the result- ing accumulation of poisonous products of incomplete metabolism. Tliat the intoxication comes in large measure from the changes in the liver, even in phosphorus poisoning, is shown by the greater re- sistance to phosphorus of dogs with Eck's fistulas."* The patient dies from this poisoning,'^ and the liver is found at autopsy to have decreased by from one-third to one-half or more in its volume. This great change would not be possible if the poisons affected the heart, kidneys, or brain as much as they do the liver structure, which is probabl}' the reason that phosphorus, bacterial poisons, snake poisons, and other poisons that destroy liver-cells do not ordinarily produce the typical picture of liver atrophy. When these poisons affect the liver more and the other tissues less, we approach the condition of acute yellow atrophy; e. g., if the dose of phosphorus is not so great as to kill the patient through injury- of other more vital organs, after a few days the necrosed liver-cells undergo autolysis, and if enough liver-cells have been destroj-ed, hepatic insufficiency may cause death, with the finding of an anatomical condition in the liver that can be properly designated as acute atrophy. Hence it is pos- sible for many poisons to cause this condition under certain circum- stances, and there seem to be certain unknown poisons (probably of 76a .Jour. Exp. Med., 1015 (22), 48. ~~ According to some investigators phosphorus augments autolysis even in vitro (see Krontowski, Zeit. f. Biol."; IfllO (54) , 479) . T8 Fischler and Bardach. Zeit. pliysiol. Chem.. 1912 (78). 4:^5. 79 The mortality of cases sutticiently typical to be diagnosed antemortem is estimated by Rondaky (Roussky Vratch.'Oct. 28. 1900) at 97 to 98 per cent. Concerning the regenerative changes in the cases which recover, see Yamasaki (Zeit. f. Heilk., Path. Abt., 1903 (24), 248). 542 ABXOiniMJTIES l.\ METABOlAfiM intestinal origin -") that are of suck a iiature that they cause spe- cifically acute hepatic atrophy. The above hypothesis seems to ex- plain all the known facts concerning this disease. That phosphorus, chloroform, and some other poisons lead particularly to fatty changes may, perhaps, be due to tlieir acting especially upon the oxidizing en- zymes,*^ leaving the autolytic enzymes and the lipase free to digest the cell and to form fat.^- That it is particularly the oxidizing en- zymes that are attacked is well shown by the chemical findings, and also by Loewy's^^ observation that in poisoning with CNH, which acts by impairing oxidation, the alterations in protein metabolism are ver}' similar to those of phosphonis poisoning.^* To be sure, Lusk ^^ found no deficiency in general oxidation in phosphonis poisoning, but this does not signify that tlie local changes do not depend upon local defective oxidative processes. Not only phosphorus but many metals, especially mercury, seem able to cause the anatomical changes of acute yellow atrophy, for the condition has been observed very frequently in persons receiving mercurial and arsenical treatment for syphilis.^*' Here the syphilis has been held responsible hj some, but the fact that in many of the eases the syphilis was quiescent or chronic at the time, and that mer- cury and arsenic are known to kill cells and stimulate autolj'sis, seems to incriminate the metals,^' at least in some cases. CHEMICAL CHANGES OF ACUTE YELLOW ATROPHY The Urine. — Most striking, and long regarded as pathognomonic, is the presence of leucine and tyrosine in the urine, first described by Frerichs. While we now know that these and other amino-acids may occur in the urine in any conditions in which there is a great breaking down of tissue within the body, yet it is true that in no other condition are they found so abundantly as in acute hepatic atrophy (as high as 1.5 gm. of tyrosine per diem has been found). ®^ 80 See Carbone, Riforma Med., 1902 (1), 687 and 608. 81 See Verworn, Dout. med. Woch., 1009 (35), 1593; Joannovics and Pick, Arch. fjes. Physiol., 1911 (140), 327. 82 Wells, Jour. Amer. Mod. Assoc, 1006 (46), 341. S3 Cent. f. Physiol., 1906 (19), 23. 8-t The liypothosis siiofffcstod by Quincke (Xothnatrel's Handbook, 1899. vol. 18, p. 307) that possibly rofjurfjitation of pancreatic juice up tlie bile ducts mipht lie responsible for the dopenerative chanfjes in the liver, is contradicted by the fact tliat the bile pressure is greater tlian tiie pancreatic juice pressure, and that the bile-ducts and periplieral portions of tlie h>bules are least afTected. Nor could Best "0 prove that trypsin injected int^ tlie liver by way of the bile-duets is able to cause such c]ian CH„ — CHOH — COOH which can be demonstrated in the urine of dogs poisoned with phosphorus, and which represents a simple deaminization of tyrosine without further oxidation. It is evident from the urinary findings, therefore, that oxidation is decreased, which is presumably because of the destruction of liver tissue with its important oxidizing func- tals in the urine of a healthy precmant woman, and cites other cases of tyrosin uria witliout hepatic atrophy (X. Y. ^led. Jour., Sept. 1!), 1914). 80 Rerl. klin. Woch., 1905 '(42), Xo. 44 a., p. 54. 90 Salkowski (Berl. klin. Woch., inO,5 (42), 15S1 ) found in the urine of a case of acute yellow atrophy a large quantity of nitrogen in a colloidal but non- protein form, apparently of carbohydrate nature. Mancini (Arch, di farm, sperim., 1906, Bd. v) also observed an increase in the colloidal nitrogen of the urine in liver diseases. oiVirchow's Arch., 1909 (198), 188. 92Takeda. Pfliigcr's Arch., 1910 (133), 365. 93 See Welsch, Arch. int. pharm. et th^r., 1905 (14), 211. 94 See Voegtlin, .Johns Hopkins Hosp. Bull., 1908 (19), 50: White, Boston Med. and Surg. .Jour., 1908 (158), 729. 95Zeit. physiol. Chem., 1910 (65), 397 and 402; also Fromherz, ibid.. 1911 (70), 351. 544 AB\OR.UALITJi:S IN METABOLISM tions. The reduction of oxidation eau also be shown experimentally by studying the respiratory exchange, Welseh having found the oxi- dation decreased by from % to Y:, in phosphorus poisoning. Carba- mates do not seem to be present in reeogni/.al)le amounts, and sugar is also absent. In phosphorus poisoning the urinary findings are similar, but with marked (pumtitative differences. Tyrosine cannot usually be de- tected, at least by ordinary methods, being found by Riess in but 7 of 36 cases of (human) phosphorus poisoning, and in but 4 of these was it abundant. Leucine is even less frequently found. With ex- perimental animals glycocoll and other amino-acids have been found ^ in the urine, and they could probably be found in acute hepatic atro- phy if the same delicate methods were employed. AVohlgemuth - has indeed found glycocoll, alanine, and arginine in human urine after phosphorus poisoning. The small quantity of amino-acids in phos- phorus poisoning is probably due to the relative slowness of the auto- lytic changes. On the other hand, the deficiency of oxidation in phos- phorus poisoning is shown by the abundant elimination of organic acids, Riess having obtained as high as 4 to 6 grams of the zinc salt of paralactic acid from the urine (per liter) in human cases, and its presence seems to be constant. The Liver.^ — In the liver may be found an abundance of the free amino-acids that have not yet escaped by diffusion, their presence having been first detected by Frerichs microscopically. Taylor * was able to isolate from a liver weighing 900 grams, 0.35 gm. of leucine and 0.612 gm. aspartic acid, which probably represent much less than the total amount present. Deuteroalbumose was also found, but no peptone, arginine, histidine, or lysine, and glj^cogen was also absent. In another case that appeared to be the result of chloroform intoxi- cation, Taylor ^ obtained 4 grams of leucine, 2.2 grams of tyrosine, and 2.3 grams of arginine nitrate. AVells found several amino-acids free in sufificient quantity to identify in the liver in cases of acute yel- low atrophy and chloroform necrosis, an increase in gelatigenous substance in tlie former, and of organic non-lipoidal phosphorus in both, sulphur being unchanged. The increase in tissue phosphorus is striking, and agrees with Slowtzoff's and Wohlgemuth 's " finding that the tissue phosphorus persists in experimental phosphorus poi- soning. Wakeman ' found that in phospliorus poisoning of dogs the 1 Abderhalclen and Barker. Zcit. plivsiol. Cliom.. l!)04 (42), 524; AlKlorliald.-n and Bcrgcll, ibid., li)()3 CM), 4G4. ^ Zeit. physiol. Cliem., 1!)(),5 (44), 74. 3 Full analyses and discussion of the clieinistry of the liver in acute yellow atrophv and clioloroforni necrosis <;iven hv Wells. Jour. Kxper. iled., 11I07 (H), 627; Arch. Jnt. .Med., litOS (1), .-)S!t: .lour." Biol. Cheni., l!tOS ( f) ) . 12!). 4 Zeit. pliysiol. Ciieni., I!t(t2 (;i4). r,A{); .lour. .Med. Research. 1!)02 (8). 424. 5 Tniv. of Calif. Pui)lications (Pathol.), l'.»04 (1), 4.S. '• Biodiem. Zeit.. 1011 (.S2), 172. -Jour. Kxper. Med., 1!)05 (7), 292; Jour. Jiiol. V\wm., lUUS (4), 11!). ACUTE YELLOW ATROPHY OF THE LI] Eli 545 liver shows a diniimitioii of the hexone bases as a whole, the arginiue being especially reduced; but no such change was found by him in acute yellow atrophy, nor by Wells in chloroform necrosis. Jack- son and Pearce ^ found an increase in the diaraino nitrogen with ex- tensive necrosis of the liver in dogs and liorses. Wohlgemuth '■* found arginine in the urine in phosphoiiis poisoning. The lecithin of the liver is also decreased (Heffter^° and Wells), and the increase in P0O3 observed in the urine presumably comes partly from this source ; cholesterol is unchanged. Beebe ^^ found the pentose of the liver not greatly altered from the normal relations. The typical idiopathic atrophied liver shows little or no inorease in fat, either chemically or microscopically, whereas there is considerable replacement of the lost liver substance by water, as shown in the following table: Fat-free Dried "Water Fat Substance Normal liver (Quincke) 76.1 3.0 20.9 Normal liver (Wells) 77.6 5.0 17.4 Acute atrophy (Perls) 81.6 8.7 9.7 Acute atrophy (Perls) 76.0 7.6 15.5 Acute atrophy (v. Starck) 80.5 4.2 15.5 Acute atrophy (Taylor) 85.8 2.0 12.2 Acute atrophv (Wakeman) 79.3 . . . . Acute atrophy (Wells) 83.8 2.5 13.7 Acute atrophy (Voegtlin) 78.0 6.6 15.4 Phosphorus poisoning (v. Starck) 60.0 29.8 10.0 Fatty degeneration (v. Starck) 64.0 25.0 11.0 Chloroform necrosis (Wells) 72.4 8.8 18.8 Similar results have been obtained frequently by other observers. Tay- lor estimating that in his ease about three-fourths of the liver paren- chyma had disappeared. The yellow color of the liver tissue charac- teristic of this condition seems to be due to bilirubin rather than to fat, because as soon as the tissues are put into oxidizing agents {e. g., dichromate hardening fluids) they turn grass-green from the oxida- tion of the bilirubin into biliverdin. There seems to be a marked in- crease in free fatty acids, probably the unsaturated higher fatty acids, which are strongly hemolytic.^" Jacoby ^^ found that the livers from phosphorus-poisoned dogs underwent autolysis with greater rapidity than normal livers, which was attributed to increased activity or amount of the autolytic en- zymes, although addition of phosphorus to a solution containing liver feraients was not found to increase their activity. The aldehydase was not found decreased, and tyrosinase could not be demonstrated, s Jour. Exper. Med., 1907 (9), 520. 9 Zeit. physiol. Chem., 1905 (44), 74. 10 Arch. exp. Path. u. Pharm., 1891 (28), 97. 11 Amer. Jour, of Physiol., 1905 (14), 237. i2Joannovics and Pick, Berl. klin. Woch., 1910 (47), 928. 13 Zeit. physiol. Chem.. 1900 (30), 174; see also Porges and Pribram, Arch, exp. Path. u. Pharm., 1908 (59), 20. 35 546 Aii\oiniMJTii:s /.v metauolism but SloAvtzoff ^* found both peroxidase and protease decreased, and iitti-ibutcd tlie increased autolysis to a g'reater acidity of the liver. The Blood. — In the blood marked changes are found, one of the most prominent, besides the icterus, being the decreased coagulability of the blood. This seems due to a loss of fibrinogen, ^^ wliich, with the giobuliu, is greatly decreased, the albumin remaining less al- tered.^® The fibrin-ferment also seems to be decreased. These changes may be due to direct autolysis of the blood constituents (Ja- coby having found that thrombi become rapidly dissolved in phos- pliorus-poisoning) or to the changes in the liver. The icterus de- pends apparently upon lesions of the finest bile capillaries,^^ although there is also some increase in hemolj-sis, and a decrease in the total blood and all its elements (Welsch) ; ^^ and both bile salts and pig- ments appear in the urine. In all these diseases with marked liver changes there is an increase in the lipase of the blood. ^^^ Neuberg and Richter ^^ have analyzed the blood drawn during life from a pa- tient with acute hepatic atrophy, and isolated from 355 c.c. of blood 0.787 gm. tyrosine, 1.102 gm. leucine, and 0.240 gm. of lysine ; they estimated the amount of free amino-acids in the entire blood to be about 30 grams. This amount is so large that they question the pos- sibility of it all arising from the degenerated liver tissue ; but more analyses are necessary before conclusions on this point can be drawn,-*' especially by the use of the newer methods. Certainly in dogs suf- fering from ehlorofomi necrosis of the liver or phosphorus poisoning the amount of free amino acids in the blood and urine is usually very small. -"•■' Origin of the Amino=acids. — The earliest conception of the source of the leucine and tyrosine found in the urine was that it came from the products of tryptic digestion absorbed from the intestinal tract, which the liver could not convert into urea because of its damaged condition. On the demonstration by Jacoby -^ that these same bodies i4Biocliom. Zcit., mil (31). 227. 1^' Whipple and ITurwitz (Jour. Exper. IVled., 1011 (l:^). i:}(i) find a <,noat (IccToase in fi1)rinof;on during experimental cliloroforin necrosis of the liver. i''.Jacobv, lor cit.; see also Doyon, C'ompt. Rend. Soc. Biol., 1!M(.") (.IS). 403; and 1900, Vol. 06. 1" Lang (Zoit. exp. Path., 1006 (3), 473) found fihrinogen in the liih" of a dog poisoned with phospliorus, which may account for the oeel\isien of the bile vessels and tlie resulting jaundice. 18 Arch. int. Pharm. et Ther.', lOO.") (14), 107. i«a Whipple et al.. Pull. Johns Hopkins ITosp., 1013 (24), 207 and 357. Quinan found the lipase content of liver tissue much reduced in chlorofium necrosis (.Tour. ^led. Res., 1015 (32), 73). A review of work- |)uhlished on blood dianges and liver fuTiction in phosphoiMis and cliloroforin ]ioisoninL; is <^iveii by ^^arshalI and liowntree, Jour. Exp. Med.. 1015 (22), 33;i. ii'Dcut.med. Woch., 1004 (30), 400. 20 V. Bergmann (llofnieister's l^eit., 1004 (6), 40) was able to isolate 2.3 grams of amino-acids combined with the chloride of naphthalene sulplioiiic arid, from 270 c.c. of blood in a case of acute vellow atrophv. ■iOaSee Van Slvke, Arch. Int. IMed., 1017 (10)," 77. 21 Zeit. physiol. Chcm., 1000 (30), 174. ACID JSTOXICATION 547 were present in the livers of phosphorus-poisoned animals because of autolysis, it became probable that the leucine and tyrosine found in the urine were formed from the degenerated liver-cells rather than in the intestine, which view has become generally accepted. It seems most probable, however, that the urinarj^ amino-acids are derived parti}' (and perhaps chiefly) from the autolysis of the liver, and partly from amino-acids produced both in the intestine and within the body during tissue metabolism, and which the liver cannot trans- form into urea as it normally does, for several observers have re- ported that even relatively slight disturbances in hepatic function are accompanied by a considerable rise in the amino-acids in the urine.^- ACID INTOXICATION -3 If a rabbit is given in repeated small doses by mouth considerable quantities of inorganic acids, such as hydrochloric or phosphoric acids, which it cannot destroy by oxidation, it soon becomes extremely ill. The manifestations are characteristic — unsteadiness of motion and stupor being followed by coma, in which the striking feature is the excessively active respiration, as if the animal were being asphyxi- ated (the so-called "air hunger"), while at the same time there is no cyanosis and the blood is bright red, containing much less COo than normal, while the amount of oxygen remains quite normal. The cur- rent explanation of this interesting condition is as follows : Normall}^ the blood carries the C0„ away from the tissues to the lungs in com- bination with the inorganic alkalies of the blood, of which sodium is by *far the most abundant. This combination is the bicarbonate of sodium for other base), which in the lungs is decomposed into the carbonate, the COo escaping into the alveolar air, according to this equation : 2X:iHCO,^Xa,C0, -f H,0 + CO, The carbonate thus formed goes back to the tissues to combine again with more COo and form bicarbonate. If acids are introduced into the blood they combine with the alkalies there, forming neutral salts which are eliminated in the urine, and in this way the amount of alkali in the blood is reduced, with a consequent reduction in the ca- pacity of the blood to carr\^ CO., away from the tissues; the amount of COo in the blood sinking to as low as 2.5 and 3 per cent. (Walter). Consequently, in acid poisoning the CO, produced in metabolism ac- cumulates in the tissues where it is formed, and blocks the processes 22 See. Masuda. Zeit. exp. Path., 1911 (S), r>20: Labhe and Bitli. Compt. Rend. Soc. Biol.. 1012 (73), 210. 23 General literature to lOOS, piven by Ewing, Arcli. Tnt. "Nfed.. inns (2), .3.30: also see Magnus-Levy, Ergebnisse inn. Med., 1008 (1), 374: Lusk, Arch. Int. Med., 1900 (3), 1. More recent literature given by PTurtley. Quart. ,Tour. ]\Ted., 1016 (9), 301, and an excellent review of recent work bv ^^^litnev, Bost. ^led. Surg. Jour., 1017 (176), 22-5. 548 ABNORMALITIES IX METABOLISM of oxidation, so that tlie animal suffers from asphyxia exactly as if it were deprived of air. In other Avords, the lack of alkalies in the blood in acid intoxication checks the "internal respiration," as in- tracellular gas exchange is called, by preventing the removal of COg from the cells. The acids stimulate the respiratory center, which is extremely sensitive to them, and the increased respiration tends to reduce the acidity by getting rid of the CO,,-* but under the condi- tions of the experiment this is not sufficient to prevent asphyxia. If the urine of such an animal is analyzed, it is found to contain increased quantities of the four chief inorganic bases, Na, K, Ca, and Mg (the last two apparently being derived from the bones), but in addition to these it is found that the amount of ammonia in the urine is decidedly increased. If instead of a rabbit a carnivorous animal, such as a dog, is given acids, it will be found relatively insusceptible, so that great quantities can be given without causing acid intoxica- tion. Examination of the urine of such a dog will show that the elimination of ammonia is increased much more than it is in the herbivora, while the inorganic alkalies are increased but little. From this it is deduced that in acid intoxication part of the nitrogen that normally goes to form urea becomes, while in the antecedent form of ammonia, combined with part of the acid that has entered the blood. In this way much of the neutralization of the acids is accomplished by ammonia, and the inorganic alkalies of the blood are spared. As in carnivora the amount of protein metabolism is much greater and more rapid than in herbivora, the ammonia available for neutral- ization of acids is much- greater than in the latter, and hence the rela- tive lack of susceptibility of carnivora to acid poisoning.-^ Accord- ing to Landau,-*' the proteins of the blood also combine much of the acid. Another factor, which is commonly overlooked, is the possible accumulation of acids within the cells, which must modify greatly any conclusions based upon studies of the blood and urine. It is witliin the cells that the effects of acids must be manifested, and it is per- fectly possible, and indeed almost certain, that we may have degrees of acidity and alkalinity in the cells which are quite different from those in the blood. As pointed out especially by Henderson,-"'^ the normal reaction of the body is kept practically constant chietiy by : 1. The salts of COo and H3PO4, existing in such proportions of carbonate, bicarbonate and carbonic acid, or disodium- and monosodinm-hydrogen-phos- 24 Sop Porpos, Wion. klin. Wooli.. 1011 (24). 1147. 2r. Tliis lias Ih'oti iiiooly sliown liy Kppin},'6r (\\ii>ii. klin. Wooh.. 1006 (10). 111). who found tliat administration of considorablo quantities of amino-acids (plyco- ooll, alanine, aspartic acid) to rabbits proatly inoroasod tlieir resistance to acid intoxication, presunialdy by yielding ammonia tlirougli normal steps of pro- tein metabolism. 2ur. P.iol. Cliem., lOlS fl4), .128. 45 Arch. c\p. i'atli. u. I'harni.. 1S90 (42), 149; Erjrch. inn. Aled.. 1908 (1). 374. omais OF THE acetone bodies 555 The studies of Knoop,'" and his associates have indicated that ill the catabolisni of fatty acids, the chains are bi'okeii (hjwn by oxidation of the cafboii atom lliii'd ffoiii tlic ciid, tliat is, the ^-position, and the two end carbon atoms are tlien split off. Tliere- fore, two carbon atoms are always split off at a time, and hence every fatty acid wliich contains an even numl)er of carl)ou atoms can l^e oxidized into oxybntyric acid, which includes the ordinary fatty acids (oleic, palmitic and stearic) of fat tissue, which have each an even number of carbon atoms (16 or 18), and also butyric, caproie and sim- ilar acids. Normal fatty acids which contain an odd number of car- bon atoms cannot yield oxybutyric acid. However, according to A. Loeb,*' aceto-acetic acid may be built up from acetic acid in the liver, and the urine in diabetes may contain acetic acid. "The for- mation of oxybutyric acid and of diaeetic acid in all these cases may be said to be due to the fact that the diabetic oroanism is not able quite to finish the attack on the beta-carbon atom of butyric acid" (Folin). From the results of tliese studies it seems that the acetone bodies can, theoretically be formed from any of the three classes of food- stuffs, but that ordinarily they come chiefly from the fats, and in severe diabetes also to considerable extent from fatty acids formed hy deaminization of amino-acids. Although it is probable that the acetone bodies are formed in many if not all tissues, yet there is abundant evidence that the liver plays an important part in ketogene- sis, as shown by the decrease in acetone bodies in Eck fistula dogs, and their great increase when the blood supply of the liver is augmented.*^ In addition to the sources of acidosis substances from metabolic proc- esses, as outlined above, it is also possible that they may be derived from organic acids formed by bacterial action in the alimentary canal, as emphasized by Palacios.^*'' Sarcolactic Acid often is found in the urine, but in origin and sig- nificance it is entirely different from the acetone bodies, and it prob- ably is never present in sufficient amounts to cause an acid intoxica- tion by abstraction of alkalies from the blood. //( vitro, we obtain sarcolactic acid whenever sugar is placed in an alkaline solution, provided the supply of oxygen to the solution is deficient: but if the oxygen supply is adequate, sugar will not yield lactic acid with alka- lies (Nef). Similarl}', an isolated surviving muscle, when asphyxi- ated by any means, shows a rapid accumulation of lactic acid, which fails to occur when sufficient oxygen is supplied. This lactic acid comes chiefly from sugar, but about 25 to ;30 per cent, of it can have it:; origin in protein (or fat?) (Woodyatt). If an organism as a 40 Full bibliofirraphy and discussion by Porges. Frsjcbnisse Physiol.. 1010 (10), G. See also Rinfrer, Jour. Piol. (hem., 1!)1.3, Vol. 14. 4V Biochem. Zeit., 1012 (47), US. 48 Fischer and Kossow. Dent. Arch. klin. INfed.. 101.3 (101). 470. 48aAmer. Jour. Med. Sci., 1015 (149), 267; Med. Eecord. starch 25, 1916. 556 ABXOliMALITIES IX METABOLIHU whole is iiisufifieiently supplied with oxygeu, lactic acid accumulates in the tissues and appears in the urine, disappearing- when the oxy- gen supply is restored. Lactic acid often appears after poisoning with a large number of drugs, which Loewy has classified as drugs whose action in the hody resembles that of lack of oxygen (arsenic, phosphorus, hydrazine, chloroform, etc.). These poisons are all char- acterized by causing" impoverishment of glycogen, fatty liver, and acute degenerative changes especially in the liver cells and the endo- thelium. Therefore the assumption seems justified that the poisons and conditions which lead to lactic acid excretion depend ultimately upon impairment of the interchange of oxygen in the cells. Wood- yatt states that, so far as known, lactic acid has never been demon- strated in any tissue in which deficient oxygenation can be excluded, and regards lactic acid as the metabolite of asphyxia or its equivalent. Over against this view is that of Embden and his associates, which is shared b^- others, that lactic acid is a normal intermediary in the breakdoA\Ti of the sugars in the body, its direct antecedent being a triose, but perusal of their work only emphasizes that in all the con- ditions in which their data were obtained asphyxial conditions were present; furthermore, this conception of lactic acid as a chief inter- mediate in normal sugar catabolism is not in harmony with the best ideas of carbohydrate chemistrj^ (Woodyatt). This author has fur- thermore found, by direct observation of the utilization of lactic acid wdien injected intravenously, that it cannot well be an important inter- mediate in carbohydrate catabolism. ^^"^ It is possible that the presence of lactic acid in the urine may also result from defective transformation of ammonia into urea by a dis- eased liver, the acid neutralizing, and being excreted with, the am- monia; in this case no defective oxidation need be assumed. How- ever, administration of phlorhizin to phosphorus poisoned dogs causes both ammonia and lactic acid to disappear from the urine, indicating that the ammonia is the protective substance which neutralizes the lactic acid, and not the reverse. Sarcolactic acid, which is dextrorotary, must be distinguished from its optical isomer, the inactive lactic acid that is produced by fer- mentation. AVhen this fermentation lactic acid is formed in the stom- ach and enters the blood, it ordinarily, like other ingested organic acids, is combined by the blood alkalies and oxidized to carbonates. It is doubtful if it ever enters the urine.*" As a general rule sarcolactic acid is not found abundant in the urine together with the acetone bodies, but is, indeed, antiketogenic. Its appearance in the ui-ine indicates tliat glycogen is not com]ilctoly ■•^th TTarvey Society Lectures, lOlG. <" Tlie theory of Boix that cirrhosis of (lie liver may he jirodiiced by butyric acid formed in fiastric ferment^Ttictn coiild im< lie corroborated bv Joaniiovics, Arch. int. Pharmacodyn., 190.') (l.'j), 241. ACID IXTOMCAT/OX I\ XOXDIAnHTKJ COXDITIOyH 557 burned, and this condition is usually aeconipanied witJL fatty changes in the liver, which also depend on lack of oxidation. Throughout the clinical forms of acidosis, lactic acid and fatty degeneration are always associated (Ewiug). To assume, as has been generally done, that the lactic acid appears in the urine when hepatic alterations are marked, because of a loss of the liver tissue which should destroy it, is probably not warranted. Rather, the liver conditions and the for- mation of lactic acid depend upon the same cause, which is a de- fective oxygen supply or interchange, either general or local. ''''"^ ACID INTOXICATION IN CONDITIONS OTHER THAN DIABETES «b Clinical Types of Acidosis. — Ewing has divided acidosis as it oc- curs in man, into two main types: 1. Acidosis resembling in its effects the acidosis produced by experimental injection of acids. This is characterized by the excretion of the acetone bodies in large amounts with a corresponding amount of ammonia in the urine, and by the absence of marked and characteristic anatomical changes. Diabetic acidosis and the acidosis of starvation are the clinical condi- tions showing this type. 2. Acidosis resembling that produced experimentally by extirpa- tion of the liver or by the Eck fistula. Here the urine contains much lactic acid and relatively little acetone bodies, the ammonia being in excess of any acetone compounds, and there is also much incom- pletely changed nitrogenous compounds. The clinical prototj^pes are phosphorus and chloroform poisoning, the toxemias of pregnancy and cyclic vomiting and acute yellow atrophy. Anatomically it is char- acterized by severe hepatic degeneration, usually fatty. This classification is purely one of convenience, for typical and ex- treme fatty liver may occur in phlorhizin diabetes with the typical coma, provided the animal was fat when the experiment was begun, and that the experiment was not carried on in such a way as to ex- haust the fat temporarily present in the liver. It is tnie that high- grade fatty livers are not usually observed in most cases of long- standing diabetes, but fatty changes are severe in the acute fulmi- nating types of diabetes of infancy and childhood, and here we have typical acidosis. The reason that lactic acid does not occur in the urine of a completely diabetic animal or man, is probably not be- cause of any assumed destructive power of the comparatively nor- mal liver, but because here the carbohydrate equilibrium is so dis- turbed that even if lactic acid were formed it would necessarily be converted into glucose and appear in the urine as such. ]\Iandel and Lusk have indeed shown that even dogs poisoned with phos- phorus excrete no lactic acid in the urine if, in addition, they are 49a See Macleod and Wedd (.Tour. Biol. Clicm., 1014 (IS), 446) wlio found that reducing the oxygen supply to the liver caused a marked rise in the lactic acid content of the hepatic blood. 49b See resuna^ by Frothingham, Arch. Int. Med., 1916 (IS), 717. 558 AIi\ORMALITli:s I\ METAliOIAHM made completely diabetic Avith phlorliizin, and that lactic acid in- jected subcutaneoiisly into a phlorliizinized animal is excreted gram for o-ram as sugar. Not infrequently acetone and diacetic acid, less often oxybutyric acid, are found in the urine of patients suffering from the most di- verse diseases. It is customary to refer to this condition as " aceto- tieniia" or "acetomiria," and to ascribe many of the observed symp- toms to "acid intoxication." The presence of these substances in the urine, however, is by no means evidence of acidosis, for excretion of considerable amounts of acetone bodies may occur Avithout reduced COg-carrying capacity' of the blood, and they may be absent with marked acidosis. In addition, it must be kept in mind that acidosis may result from other causes than over-production of organic acids; e. g., acid phosphate retention in nephritis, or loss of bases from biliary or pancreatic fistula. In no other condition do the amounts of organic acids in the urine approximate the amounts found in diabetic coma. Therefore, the intoxication in these cases is probably not due to the acids, but, on the contrary, the presence of the acetone bodies is due more often to the effects of toxic substances of diverse origins and na- tures. Anesthesia. — As shown first by Greven (1895), and especially by Brewer and by Helen Baldwin,^" acetone is nearly always present in the urine during the first twenty-four hours after administration of either chloroform or ether, and occasionally diacetic acid appears on the second or third day after; but ordinarily there is no increase in organic acids in the urine. The starvation preceding and following the operation is also a factor of considerable importance. It does not seem probable that the symptoms observed in typical cases of delayed chloroform-poisoning are due chiefly, if at all, to acid intoxication per se, but rather are the result of extensive injury to the parenchy- matous organs, particularly the liver, by the chloroform, which causes a condition resembling acute yellow atrophy or phosphorus-poison- ing.^^ Cachectic Acetonuria. — Acetone and diacetic acid, but less abun- dantly the oxybutyric acid, are found in the urine in many condi- tions associated with wasting, among Avhich may be especially men- tioned : Infantile marasmus,"- in which increased ammonia is found in the urine, and sometimes sym])toms resembling acid intoxications occur. Normally the urine of suckling infants contains 1-4 mg. per day of acetone bodies, which may be increased to 15-35 mg. by simple hun- ger. In fact, "acidosis" seems to occur particularly frequently in infants from relatively slight causes, such as gastro-enteritis and ■in .I.Hir. .,f T^iol. Choni., lOOG (1), 239. •"'i Wells, .lour. Amcr. ]\lcd. Assoc. l!)(l)i ()('.]. :M1. 52 See Meyer and Laii{i;stein. .Jalirb. f Isiiidrrhcilk.. 1!M)(1 (fi.T). .10. \()\-I)/\I!i:tic Ac/n nrox/r \'ni)\ 559 other infectious conditions. 'J'liis is perliaps due to a lower oxidizing power on the part of the infantile organism ( Pfaundler),''^ since the proportion of nitrogen in the urine of infants in forms other than urea, is higher than in adults. Even an unusually fatty diet may cause aeetonuria in infants. It has also been suggested that ex- cessive formation of acids in the intestine through bactei-ial decompo- sition may cause withdrawal of tlic bases from tlic IjIooiI. wliich are lost to tlie body through excretion in the feces. Starvation. — Acetone, which is normally excreted through the lungs for tlie most part (80-90 per cent, of that produced) appears in excess in the urine verj' soon after fasting begins, there being more produced than can be exhaled. After 24 to 36 hours of fasting, diacetic acid appears, and then oxybutyric acid, which may reach 10 to 20 grams per day in starvation, and even higher figures are re- corded. The urinary ammonia nitrogen runs parallel to the acidosis. The use of a carbohydrate-free diet is also accompanied by a marked acetonuria,^^'' no matter how much fat is supplied, which may reach a point where several grams of oxybutyric acid are being excreted per day without symptoms of serious intoxication. A relatively small amount of carbohydrate (80 grams) is sufficient to prevent this acidosis. If the meat-fat diet is continued for some time, however, there seems to be some sort of adaptation so that the aeetonuria di- minishes until practically normal figures may be reached. Acidosis of Pre^ancy. — During pregnancy the urine usually con- tains acetone in slight excess, and occasionally is in large excess in women who are suffering from the toxemias of pregnancy. Here there is a rise in ammonia far beyond the proportion of acetone bod- ies, partly because of the large amounfs of lactic acid which are ex- creted, and partly from abnormal protein metabolism and tissue de- struction, but the proportion of the urinarv^ nitrogen which is consti- tuted by ammonia is too inconstant to serve as a prognostic and operative guide. Ewing has observed a case of pernicious vomiting with 75 per cent, of the total nitrogen as ammonia, and no urea, — while there may occur fatal cases without large excess of ammonia. Higher ammonia figures are usually reached in pernicious vomiting of pregnancy than in eclampsia ; in neither is the acidosis present suffi- cient to account for the intoxication. (See discussion of "Eclamp- sia.") Even normal pregnant women seem to show a reduced ability to tolerate a deficiency in the carbohydrates of the diet.^* Cyclic Vomiting. — Here the urine usually shows acetone bodies, lac- tic acid, indican in excess, and a rise in the proportion of neutral to oxidized sulphur (Howland and Richards). As these findings may persist in spite of absorption of carbohydrates, they are not en- 33,Jahrb. f. Kinderheilk.. 1001 (.54), 247. 53a See Higgins. Peabodv and Fit/. Jour. ^Fed. T!os.. 1910 (.341, 203. 54Porges and Novak, Berl. klin. Woch., 1911 (4S), I7")7. 560 ABNORMALITIES IX METABOLISM tirely due to starvation, and there are severe fatty changes in the liver and kidneys, indicating a toxemic origin associated with de- fective oxidation. ^Iclhinl)y ■'"' fuuntl a considerable creatine elimina- tion in a typical case, together with the acidosis. Inanition and Cachexia^ — Under this heading may be grouped the acetonuria observed in intestinal disturbances in children,'^^'^ hys- terical vomiting, psychoses, and cancer. In each of these conditions coma of the type of diabetic coma has sometimes been observed, and in all of them acetonuria is common, the reasons being obvious after the above discussion. A relative acidosis may also result from de- ficiency of bases in the diet of growing infants. In many cases of acidosis of infants there is not sufficient increase in the acetone bodies of the blood to account for the acidosis ; "'^ on the other hand, most of the children excreting acetone bodies in the urine do not have acidosis. Retention of placenta or fetus, acetonuria being considered of di- agnostic value in determining the death of the fetus in utero,^'' but not in extrauterine pregnancy (Wechsberg).-'* In uremia, as previously mentioned, organic acids may appear in the urine, but apparently as a result, and not as the cause, of the uremia (Orlowski). There is usually some acidosis in advanced nephritis, but marked only in uremia. ^*^ Here, according to ]\Iarriott and Howland,^^'' acid phosphates which the kidney has failed to ex- crete, may be an important factor. Sometimes in advanced nephritis the acidosis may be of such a degree as to simulate diabetic coma, and the nocturnal hyperopnoea of nephritis probablj" is the result of acidosis (Whitney-^). Other Conditions. — Acetonuria is observed inconstantly in fever, especially in children; also after poisoning by many drugs, includ- ing, besides the heavy metals, morphine, atropine, antipyrine, and phlorhizin. Asiatic cholera shows a marked acidosis, in manj^ re- spects resembling diabetic acidosis,'**'^ and gastro-intestinal infections of similar sorts may cause severe acetonuria. "Whitney -^ finds acid- osis to be a common terminal event in many diseases, and often the immediate cause of death. Pneumonia is accompanied by acidosis,^*^ often of serious degree, subsiding rapidly after the crisis. At high altitudes there is always an acidosis, which stimulates the respiratory center to increased activity. In asphyxial conditions of all sorts S5 Lant'ot, Jiilv 1, UMl. 55a See Ilowland and .Marriott, Amer. Jour. Dis. Child.. lOlC, (11), ,?0!) : (12), 459. ■">'! Moore, Amcr. .Tour. Dis. Cliild., miC, (12), 244. 57 See Frojumer. Borl. Ulin. Woch., 100;! (42), 1008. ssWien. klin. Wocli., 1000 (10), 053. r.'^aSee Scllards, Hull. Jolins Hopkins Hosp., 1014 (2.")). 141: Toabodv, Arch. Int. :Med., 1015 (10), 455. 58i> Arcli. Int. :M('d., lOlG (IS), 70S. f'Sc Sellards and Sluikloo. Pliilipi)ine Jour. Sci., 1011 (G), 53. 50 Lewis and Barcroft, Quart. Jour. Med.. l!)15 (S), lOS. FATiaUE 5G1 aeiduisis is present, c. g., uncompensated cardiac defects, severe anemia, gas poisoning. FATIGUE The symptoms of fatigue, whether general or local, seem to be due to an intoxication with the products of the excessive metabolic activ- ity, and part of the symptoms, at least, seem to be due to acid intox- ication. Among the metabolic products of muscular activity are known to be creatin, creatinin, sarcolactic acid, and carbon dioxide. The amount of acid developed in an active muscle is quite consider- able, and when the activity is violent or prolonged the sarcolactic acid accumulates, being formed faster than it can be removed. Part of the acidity of the muscle is due, however, not to the sarcolactic acid itself, but to monopotassium phosphate (KHoPO^), which is formed by the action of the sarcolactic acid upon the dipotassium phosphate present in the blood and muscle. The effect of these various substances upon muscular fatigue has been studied experi- mentally, and while the creatin seems not to be a " fatigue substance, ' ' sarcolactic acid, monopotassium phosphate, potassium sarcolactate, and carbon dioxide all cause muscle tissue to react to stimuli in the same way that a fatigued muscle does (Lee).*"^ It is quite probable that the muscular weakness of diabetics, and the exhaustion associated with many conditions in which organic acids appear in the urine in abnormal quantities, depend, at least in part, upon the effect of these acids upon the muscle tissue, for Lee found that |8-oxybutyric acid causes the same fatigue reaction in mus- cles as does sarcolactic acid. Furthermore, sarcolactic acid itself often appears in the urine in these conditions. It may be added that in fatigued animals the alkalinity of the blood (by titration) has been found decreased (Geppert and Zuntz), and the proportion of the urinary nitrogen that appears in other combinations than urea is increased (Poehl).'^^ The "Toxins" of Fatigue. — In extreme exhaustion the evidences of a general intoxication often become severe, so that the condition may resemble an acute febrile disease and last for several days. It seems very probable that substances more toxic than the above-men- tioned acids are involved. Weichardt '- claims to have demonstrated as produced by muscular fatigue a toxic substance, which in structure resembles the bacterial toxins, called by him kenotoxin,'^-"' and against which an antitoxin may be obtained. This toxic material is, he be- lieves, formed from the protein molecule in the first stages of its de- 60 Jour. Amer. Med. Assoc, 1906 (46), 1401; where is given a complete review of the subject of fatigue, with tiie literature. eiDeut. med. Woch., 1001 (27), 706. «2 "Ueber Enniidungsstoffe," Enke, Stuttgart, 1012; Kolle and Wassermann's Handbuch, 1013 (2), 1400. 02a See Weichardt and Schwenk, Zeit. phvsiol. Chem.. 191.3 (83), 381. 36 562 ABXOKMALITIES IX METABOLISM composition, as a side product which is normally protected against by a formation of an antitoxin, rather than bj^ being split up further^ as is the case with the rest of the protein molecule. It is excreted not only in the urine, but also in the breath, and may be produced in vitro by disintegrating protein at temperatures under 40°. Various poisons, cause kenotoxin to appear in the urine, and it is found in the urine of many animals, as well as in plant tissues.**^ The study of anaphy- laxis has led to so many evidences of the remarkable toxicity of the products of protein cleavage, that the possibility that some of these may be responsible for fatigue, as Weichardt has so vigorously main- tained, is receiving much support."* Whether this work is coiitirmed or not, there remains the fact that the blood of fatigued animals contains toxic substances, which ^losso proved as follows: Tf blood is transfused from an exhausted dog to a normal dog, from which an equivalent amount of blood has been withdrawn, this second dog^ will show the usual manifestations of fatigue. Mental Fatigue. — The chemical changes of mental fatigue are not known, but it is known that the ganglion-cells show marked struc- tural alterations as a result of fatigue, chromatolysis often being very striking. Since lecithin forms so important a part of the nerv- ous system, it is tempting to imagine that in fatigue excessive quan- tities of its toxic decomposition-product, choline, and the still more toxic derivative of choline, neurine, are formed in considerable amounts and cause part, at least, of the intoxication. That choline or neurine actually are the cause of any of the symp- toms of fatigue, however, has not been established ; but Donath '^^ considers choline an important factor in the production of epileptic convulsions J^*^ Animals kept for a long time from sleeping show the presence in their blood, cerebro-spinal fluid and brain tissues, of a poisonous property causing somnolence in other animals (Legendre and Pieron).**' This cannot well be choline or any similar substance, for it does not filter, is insoluble in alcohol, and is destroyed by heating at 65°. THE POISONS PRODUCED IN SUPERFICIAL BURNS «* In a certain proportion of cases of extensive but superficial burns,, death follows after an interval of from six hours to a few- days, ap- 03 Cent. f. Bakt.. in07 (4.3), .312. •■'♦ The failure of various investigators to corroborate Woiiliardt is disciissod l)v Konricli. Zcit. f. TTvp., 1014 (7S). 1: Korflf-Petersen. ihUJ.. p. 37. ■ o^' Zeit. pliysiol. Chem.. 1003 (.''0), -,20. •"■• Confeiiiin the review of ^lasoin. Aroh. internat. de Pharniaeodvnainie, 1004 (13), 3S7. «7 Zeit. alljr. Physiol., 1012' (14). 235. «s Literature piven li\- P.ardeen. .Tolins Hopkins llosp. Peports. ISOS (7). 137; Eyff, Cent. Orenzf^'el). :\lc(l. u. Cliir.. 1001 i4), 42S: PfeillVr, Virdiow's Areh... I'uisoxs I'Roui ri:u IS jiLJtxs 563 parently because of a profound intoxication. As evidence of intoxica- tion we Imve not oidy clinical manifestations, such as delirium, iiemo- ^lobinuria, and albuminuria, vomitinj>^, bloody diarrhea, etc., but, more convincinoly, the anatomical lindings at autopsy, which are strikingly similar to those resulting from acute intoxication with bacterial prod- ucts. Uardeen found (juite constantly cloiuly swelling and focal and parenchymatous degeneration in the liver and kidneys: softening and enlargement of the spleen with focal degeneration in the ^Iali)ighian bodies ; and particularly degenerative changes in the lymph-glands and intestinal follicles resembling those observed in diphtheria, which ^McCrae '••' considers due to proliferation and phagocytosis by the endothelial cells of the lymphatic structures. IMarked changes are usually present in the blood, consisting of fragmentation and dis- tortion of the red corpuscles, hemoglobinemia, loss of water with a relative increase in the number of corpuscles by from one to four millions per cubic millimeter, an increase in the blood platelets, and a rise in the number of leucocytes as high as 30,000 to 50,000.'^° Hem- oglobinuria is also frequently present, and almost constantly gastro- intestinal irritation occurs, with anatomical evidences of acute enter- itis, acute gastritis, and occasionally gastric or duodenal ulcers. Ac- cording to Korolenko,"^ the sympathetic nervous system is seriously involved. It therefore seems probable that poisons are formed as a result of superficial burns, which have the effect of causing hemolysis, and which are also cytotoxic for parenchymatous cells and particularly for nervous tissues. These hypothetical poisons seem to be eliminated by the intestines and kidneys, which are injured by the poisons in their passage through these organs. The attempts to explain all the observed effects of burns as due to thrombosis or to embolism by al- tered red corpuscles seem to have failed, for the peculiar location of the lesions (e. g., duodenal ulcers, necrosis in the Malpighian bodies of the spleen, etc.) does not agree with this hypothesis, and there are too many evidences of the presence of some decidedly toxic substance in the blood. There can be no question that the poisonous substance or substances are formed in the burned area, and not in the internal organs as a result of hyperpyrexia, as shown by numerous observa- tions. Thus, if the burned area is removed immediately (in narco- tized experimental animals), death will be prevented, whereas if the burned tissue is permitted to remain for a few hours, death will occur. If the burned skin is transplanted to a normal animal, this animal will develop symptoms of intoxication, while the burned ani- mal may be saved by the transplantation (Vogt). The poison ap- 190.5 (180). 367. Full discussion of theories by Vopt, Zeit. exp. Path. u. Pliarm., 1912 (11), 191. 69Amer. :Med.. 1901 (2), 735. TO Locke, Boston :\led. and Surg. Jour., 1902 (147), 480. 71 Cent. f. Path., 1903 (10), 663. 564 ABNORMALITIES IX METABOLISM pears to be absorbed from the burned area into the blood, for if the circulation is shut off from the burned area, no intoxication results; this probably explains in part why deep destructive bums of small areas, which are associated with local thrombosis, are much less seri- ous than a superficial slight scalding over a large area. Apparently the poison is produced chiefly or solely in the skin, for burning of muscle is not followed by intoxication (Eijkman and Hoogenhuyze).'- AVhen one of a pair of animals united to another by operative pro- cedure (parabiosis) is burned, the other animal may become intoxi- cated, while the intoxication of the burned animal is less than it would be if it were alone (Vogt). Numerous investigators have reported finding poisonous sub- stances in the blood, tissues, or urine of burned men and animals, but the reports disagree widely in details.'^ Thus Dietrichs states that the blood of burned animals contains hemolysins and hemao:glutinins, which could not be corroborated by Burkhardt ^* or b}^ Pfeiffer/' The latter, however, finds that the urine, serum, and organs of burned animals contain substances poisonous for the same and for dififerent species, which is in accord with the results of numerous earlier inves- tigators. The poisons, according to Pfeiffer, are neurotoxic and necro- genic in their properties, and act without a period of incubation ; they are rapidly w^eakened on standing in solution and by the action of sunlight, are absorbed from the gastro-intestinal tract, are soluble in water, alcohol, and glycerol, but not in chloroform or ether, are precipitated by HgCL in acid solution, and by phosphotungstic acid, and they are not volatile. Apparently, according to Pfeiffer. they are not ptomains, nor yet pyridine derivatives, as many investigators have contended, but resemble more closely the labile poisons of snake venom, and have effects similar to the unknown poisons that are con- cerned in uremia. The neurotoxic substance is more thermostable than the necrogenic substance, which is very easily destroyed by heat. Pfeiffer believes it probable that the poisons are derived from the cleavage of proteins altered in composition by burning, and he finds an enzyme splitting glj'cyltryptophane in the blood and urine of burned animals."'"' The hemolysis he attributes to direct injury of the blood in its passage through the heated area, and not to the action of poisons ; this is very possible, since red corpuscles fragment after being heated to 52°, and may be seriously impaired functionally at 45°. There are many authors, indeed, who consider tlie blood changes T2Vircl)o\v's Arch., 1900 (183), 377. 7:! Kavcnna and ^Tiiiassian ( IJcf. in Tiioc-honi. C'(>Mtr.. 1003 (IK 34S ) stato tliat blood heated outside tlie body to .'ir)°-60° is toxic, and causes the same anatomical chan<,'es as does death from l)\irning, vhicli findinff is corroborated bv ireisted. Arch. klin. riiir., lOOfi (70), 414. 74 Arch. klin. f'hir., 1005 (7r)), S4.'i. 75 Virehow's Arch.. 1005 (ISO), 3(i7: Zeit. f. Ilyg., lOOG (54). 419. 75aZeit. Tmmuniliit., 1015 (23), 473. POISONS PRODUCED IN BURNS 565 the chief cause of death, but the weight of evidence is iu favor of the theory of the development of toxic substances in the burned skin. Kutscher and Ileyde '"^ believe methyl guanidine to be the toxic substance eliminated in the urine, stating that it produces effects sim- ilar to that caused by injections of the toxic urine from burn cases. These symptoms are quite similar to those characteristic of anaphy- laxis, and Heyde states that small burned areas sensitize an animal to later injections of extracts of burned tissue. He, as well as Vogt, are therefore inclined to believe that some cases, especially those dy- ing unexpectedly 12 or 13 days after the burning, may be the result of anaphylactic reaction to proteins made of foreign character by the heat.'^ The newer observations concerning the presence of toxic sub- stances in the urine during anaphylactic intoxication are in harmony with the findings in burn cases,"''' although the identity of methyl guanidine with the toxic agent is questionable. Burn Blisters. — The contents of burn blisters resemble the fluid of inflammatory edemas generally. K. Morner ^^ found 5.031 per cent, of proteins, which included 1.359 per cent, of globulin and 0.011 per cent, of fibrin ; there was also present a substance reducing cop- per oxide, but no pyrocateehin. 76 Cent. f. Physiol., lOll (25), 441. 77 Heyde, Med. Klinik, 1912 (8), 263. 77a See Pfeiflfer, Zeit. Immunitiit., 1913 (18), 75. 78Skand. Arch. Physiol., 1895 (5), 272. CHAPTER XIX GASTRO-INTESTINAL "AUTOINTOXICATION" AND RELATED METABOLIC DISTURBANCES Under this heading are commonly included all intoxications that can be ascribed to the absorption from the gastro-intestinal tract of toxic substances that have been formed within its contents, either by the action of the digestive ferments or of putrefactive bacteria. The propriety of considering such conditions as examples of auto- intoxication is properly questioned, since it is often difficult to deter- mine whether the putrefaction occurred within the body, or had already taken place in the food before it was eaten. But even those who would limit the use of the term autointoxication to intoxication with the products of cellular metabolism, must admit the possibility of products of metabolism reentering the blood from the contents of the bowels through the intestinal wall, since the bile, and perhaps also the intestinal juice, contain excrementitious substances which may, in case of defective fecal elimination, be reabsorbed into the blood. Therefore, in gastro-intestinal disturbances we have the pos- sibility of both true autointoxication and intoxication by putrefactive products occurring together in an inseparable way, and the common inclusion of gastro-intestinal intoxication in the discussion of auto- intoxication would seem to be justifiable as well as expedient. The sources of poisonous substances arising in the gastro-intestinal tract are numerous. They may be formed either from the food-stuffs, or from the secretions and excretions of the body that enter the ali- mentary canal ; and they may be formed either by the digestive fer- ments or by the bacteria of the intestinal contents. Hence the number of these products is enormous, and we are by no means sure that those that have yet been identified include the most important or most toxic. To classify the poisonous substances that are known to be formed in the alimentary canal, and which might, under certain conditions, cause an intoxication, is extremely difficult, because of the uncertainty of our information; but, using as a basis the sources of the substances, they may be classilied as follows: ^ I. The con.stituents of the digestive secretions, including the bile salts and pigments, pepsin, and trypsin. 1 ^r<)(lifie(l from Weintravul. Erl' l>lS '^^' 11. Products of >^"7^\^^V*'^'r^oses peptones, amino-acids. (ft) From proteins— proteoses pM"^ (b) From fats-fatty acids and ^dycerol. III. Products of putrefaction and fermentation: (a) From protenis: ,..,,-,:„„i„ (tyrosine, phenylalanine, M^ T^rnm the aromatic radicals vk^^"*^'" ' t .-^ . (1) J^rom tne a skatole-carbonic (or in- ,2^ ^^r'tlirfattv acid radieals-fatty acids (especially '^ butyric nd acetic), acetone, »>»™'"-- »'"'™:,t;'.'=:r 1«„ dioxide hvdrogen, ma.sh-gas. Also pto.muiis . ca ; rputresci„e%thylide„d,amine, >soamylamn. (3) Fvom the s«lpln,r-conta,nmg rad.cals--H,S, methyl ' ' mereaptan, ethyl mereaptan, ethyl sulphid. <^^ ^Xttncid- r Mlowin, havi,., --'-'«^™rc' acetic, propionic, butync, vale"*™- ^f- »>1'»'^"'=- and succinic; also acetone, CO,, t^ti,, n,. ''' '^ XheTfatty acids, as well as bntyric acid; also gly- cerol. From lecithin-choline, neurine, and muscarme- like bodies. I THE CONSTITUENTS OF THE DIGESTIVE FLUIDS These call for but brief -nsideration for althov^h many of tbem are known to be toxic, ff .*- - - -''^»- ''^^ j'l^,,, ,,ypsin, intoxication ctbe.. >» j'^™ ^«^tS when in^ect^d experimentally especially the latter, are ucemtrL y on^ear ever to pass :SiX»r::gen. t&;"n f:nbu;;;: a:tion „f .. .... "^Th/brsllts are also toxic. especia% hemolytic, ""* tj- «-^ -;. reabsorbed from the intestines are taken ^^^ "'*» '^^ , tTor a 1 ^ 1 Tiiw T^vntpctive arransrement seems to be sutncuuT lui ^^^^•^^^^^J^^Tirble^i'^ments become converted into urohihnogen emergencies. Ihe bile pi^mt ^ absorbed and eliminated as through reduction, and this is huge > ^^^^^' ^^ ^ .^^^^^^^^^ ^ urnhiJin Icterus and cholemia do not seem e^tI \" ^^ i WL of bile-pigments and bile salts from the nitestn.es. 568 G ASTRO-INTESTINAL ''AUTOINTOXICATION II. PRODUCTS OF NORMAL DIGESTION Proteoses and Peptones. — Under normal conditions, these are broken uj) in the intestinal wall into the amino-acids, through the agency of erepsin, and do not appear in the blood in appreciable quantities. To be sure, certain authors claim to have found albumose in nonnal blood, but if present the amounts are extremely minute. In conditions in which ulceration or other lesions are present in the gastro-intestinal tract it is possible to find small amounts of proteoses in the urine, probably absorbed through the abnormal areas, but not in quantities sufficient to account for any appreciable intoxication, although proteoses are distinctly toxic. This last statement has been much contested, because the difficulty of purifying proteoses ob- tained from ordinary sources has left open the possibility that such toxic effects as have been observed are due to contaminating sub- stances, and not to the proteoses themselves. More recent work, how- ever, particularly that of Underbill,- Gibson -^ and Zunz,^ seems to have established affirmatively the toxicity of proteoses, whether from animal or vegetable proteins. Besides the classical effect of inhibiting the coagulation of the blood, the proteoses have a lymphagogue effect (Ileidenhain),'* cause a marked febrile reaction,^' and in doses of some size are fatal to experimental animals (rabbits being much less sus- ceptible than dogs and many other animals). Locally they cause a mild inflammatory reaction, which is followed by the appearance of much connective-tissue formation.** Long continued injection of pro- teoses does not produce visceral lesions."'"^ The careful studies of Zunz show that intravenous injection of hetero-albumose, thio-albumose, deutero-albumose and proto-albumose cause a rise in blood pressure, but large doses may cause a fall in pressure ; the abiuret products of 2Amer. Jour. Physiol., 1903 (9), ?A5; Jour. Biol. Chcni., 1015 (22), 443, (literature) . 2a Philippine Jour. Sei., 1914 (9). 490. 3 Arch, intcrnat. plivsiol., 1911 (73), 110. 4 F5ee also Nolf, Arch, internat de Diysiol., 1906 (3). 343. 5 Gibson finds that carefully purified proteoses liave but a slight. ])vrofrenio ef- fect. (Philippine Jour. Sci./l913 (8), 475.) 6 In a paper appearing in tlie Transactions of the Chicago Pathological Society, 1903 (5), 240, I published the observation that repeated injections of Witte's "peptone" (which consists chiefly of proteoses) into rabbits led to tlie ]>roduction of marked cirrhosis of the liver, and sujrgested the possil)ility that jiroteoses escaping tlirougli a diseased gastr'c or intestinal wall into the blood might be a factor in tlie production of cirrhos's in man. Subse()uent. observations, how- ever, have shown tliat rei)eated injection of almost any foreign protein material (e. g., emulsions of organs, foreign })lood. etc., used in immunization exjieriments) will cause a similar cirrliosis in ral)bits, which animals, indeed, often spon- taneously sliow this condition when apparently otherwise normal. "Peptone" in- jections in dogs and guinea-pigs have failed to cause a similar cirrhosis, and lu'nce the value of tliese and all other rabbit. exi)eriments on cirrliosis of the liver is very questionable: however, the possibility of the correctness of the original con- clusions still remains open. flaWoolley et al., Jour. Exp. Med., 1915 (22), 114. PRODUCTS OF .Yr>AM/.l/> D/dESTION 569 tryptie digestion are iiiurli more actively depi'essor than tlie allnmioscs. As a general rule, however, it has been observed that the first products of protein hj-drolysis are the most toxic, and with further cleavage the toxicity lessens and finally disappears, as sliown especially in the studies on ana])hy]axis and anaphylatoxin formation."" Thus AVolf ^ found that the amino-acids do not cause a fall of blood pressure, nor do polypeptids.^ Most of the attempts to obtain antibodies for the products of pro- tein hydrolysis have failed, for it seems to require an intact protein molecule to act as antigen. The most authentic positive results so far reported are those of Zunz," who claims that with the higher cleavage products, the ''primary proteoses" from fibrin hydrolysis, he secured some degree of anaphylactic reaction. This work has failed of con- firmation and with the cleavage products of egg albumen Wells ^*' obtained no positive results. "Albumosuria." ""^ — If proteoses enter the blood stream they ap- pear in large part in the urine, indicating that the tissues do not read- ily utilize them in this form." Consequently, when proteoses are pro- duced in considerable amounts by autolysis of pathological tissues they appear in the urine, and their presence is considered to be of di- agnostic value. ^^ True peptone seems rarel.y, and according to manj' observers never, to appear in the urine. But in view of the observa- tions that polypeptids often appear in the urine,^-'' it is probable that true peptones also do. Albumoses, therefore, may be found in the urine whenever any considerable amount of tissue or exudate is being autolyzed and absorbed, and it has been found in the following con- ditions: Suppuration of all kinds; resolution of pneumonia; involu- tion of the puerperal uterus; carcinoma (two-thirds of all cases — Ury and Lilienthal ) , and other malignant growths ; febrile conditions vrith tissue destruction (37.5 per cent, of all eases, ]\[orawitz and Dietschy) : " acute yellow atrophy, phosphorus poisoning, and eclamp- sia; leukemia, especially under .r-ray treatment; absorption of simple and inflammatory exudates; ulcerating pulmonary tuberculosis,^* and 6b The statement of v. Knaffl-Lenz (Arch. exp. Path. u. Pharm., 1913 (73), 292) that the toxicity of the cleavage products varies directly with their trypto- phane content could not be corroborated bv Underbill and Hendrix, Jour. Biol. Cheni., 1915 (22). 443. 7. Tour, of Phvsiol., 100.5 (32), 171. 8 Halliburton, \biVZ., 190.5 (32), 174. 9 Bull. Acad. Roval de M^^d. Belgique, Mav 27, 1911. 10 Jour. Infect. Dis., 1900 (6), 506. lOaGood critical review given by Pollak. Zeit. exp. ^led.. 1014 (2). 314. 11 They may be partly liydrolyzed into smaller complexes, however, primary pro- teoses being partly changed to deutero-proteoses, and the latter partly to peptones (Chittenden, ^lendel, and Henderson, Amcr. Jour. Phvsiol., 1S99 (2), 142). 12 See Yarrow. Amer. Med., 1903 (5), 452; Try and Lilientlial, Arch. f. Ver- dauungskr., 1905 (11), 72; Senator, International Clinics, 1905 (4, series 14), 85. i2aChodat and Kummer, Biochem. Zeit.. 1914 (05), 392. 13 Arch. f. exp. Path. u. Pharm., 1905 (54), 88. 14 See Parkinson, Practitioner, 190G (76), 219. 570 GASTROIXTESTIXAL 'AUTOIXTOXICaTIOX" after tuberculin reactions ( Deist ).^^ Albumosuria is present in small- pox and may serve in differential diagnosis.' ■'^'' In ulcerative condi- tions of the alimentary' canal albumoses may be absorbed unchanged and cause alimentary albumosuria. The normal kidney seems to be impermeable to the small amounts of proteose that may be present nor- mally in the blood, or even after large oral ingestion of proteoses, but in parenchymatous nephritis it may escape in the urine (Henderson/** Pollak^"^).' It is possi])le that some of the symptoms of these conditions are due to intoxication with proteoses, for 0.07 to 0.1 gram deutero-albu- mose will cause a febrile reaction in a healthy man/' but probably their amount is usually too small to cause appreciable etfects.^^ It is well known, however, that the characteristic rise of temperature fol- lowing tlie injection of tuberculin into tuberculous individuals is also produced if minute quantities of proteose solutions are injected in place of tuberculin ; therefore, proteoses arising from autolysis in tu- berculosis may be of importance in causing fever and other symp- toms.^^ Tuberculous animals are said to succumb to a much smaller dose of deutero-albumose than normal auimals.^®^ The so-called "Bence- Jones albumose" that appears in the urine of patients with multiple bone-marrow tumors is not a true albumose, but is more closely related to the simple proteins, and is discussed under the head of "Chemistry of Tumors." III. PRODUCTS OF PUTREFACTION AND FERMENTATION -° AVe may perhaps gain some appreciation of the enormous amount of bacterial action that goes on in the normal intestinal digestive processes by considering the fact that as much as one-third of the total weight of the solids of normal feces may consist of bacteria (Stras- burger), their proportion being increased in diarrheal disorders and decreased in constipation. They attack all food-stuff's, and among the decomposition-products formed through their activity are un- doubtedly many of considerable toxicity. ]\Iost of the products of in- testinal putrefaction that have as yet been isolated are, however, not extremely poisonous ; but many of them are toxic to some degree, and their long-continued absorption may well lead to serious disturbances. 15 Boilr. z. kliii. 'I'lilH-rk.. I!tl2 (23), .147. isa Primavora, Cay.. Int. Med. o Cliir., IIU.3, No. 10. i«Laiu-pt, :Mar. 6, IflOO. 17 See :\rattlies. Arch, exper. Patli. u. Pliarni.. ISO.") CM)). 4:i7. 1** In a series of uni)ul»lislu'd experiments I was nnable to cause amylnid de- feneration in rabbits by ]>r()tracted intoxication with proteose solutions. i!» Simon, .\rch. ex]). :Med.. 190:? (4!t), 44!>. Concernini: relation of tuberculin to proteoses see review liy -lolles in Ott's "C'hemisi'he l\itliol. der Tubcrculose." i!ia Kirchlieim aiid Tuczek. Arcli. exj). Path. u. I'harm.. 1014 (77). ."^87. 2" Complete bibiiy^rajjliv jriven in the resumf' on "Intestinal Putrefaction" bv Gerhardt, Erpebnisse der Physiol., 1004 (III. Abt. 1). 107. Chemistry of Putre- faction is reviewed by Ellinjjer, ibid., 1007 ( ti ) , 20. I'h'ODl t-TS or rUTREFACTION AND FERMENTATWX 571 Considering- tlu'iu lirst according to their origin and chemical nature, we take up iirst the products of: A. PROTEIN PUTREFACTION (1) SUBSTANCES DERIVED FROM THE AROMATIC RADICALS OF THE PROTEIN MOLECULE In the protein molecule are contained the following amino-acids with an aromatic nucleus : Tyrosine, Ho/ y-CH^— CH — COOH riu'iiylalaniiu', <^ \ cH.- — CH — COOH NH2 Ti yptopliane, / N_ C — CH2 — CH — COOH \ CH V H In the intestinal contents have been found a number of substances that are undoubtedly derived from these aromatic radicals. They are (1) phenol, . — . which is formed in small quantities, presumabl}^ from tyrosine, as also is the closely related (2) paracresol, and also (3) para-oxyphenyl acetic acid, HO / \ CH, — COOH and (4) para-oxyphenyl-propionic acid, HO / N CH, — CH.. — COOH From the tryptophane are formed numerous important substances, as follows: NH2 _ C — CH2 — CH — COOH \/ '" XH (tryptophane) readily yields, through splitting off the NIT, group and addition of H, iiidole propionic acid (formerly incorrectly called sl-atole acetic acid), which is 572 GASTROINTESTINAL "AUTOINTOXICATION" CH= — CH2 — COOH / C \^ I CII I / NI[ and from which in turn may readily be formed indole acetic acid (erroneously called skatole carhoxylic acid), which is 0H= — COOH CH I / NH Both of these substances have been formed in the intestinal contents. From these substances are formed the better known skatole, cm / c , W CH I / . NH and indole, / — v < >CH CH NH In dogs, but not in man, kynurcnic acid. / — \ < >— C.OH ^-/ \^ C.COOH N = CH is also formed from tryptophane.-^ The greatest interest concerning these bodies arises from the fact that after they are absorbed from the intestine they become combined with sulphuric or glycuronic acid, and are excreted in the urine as salts of these acids ; consequently the amount of sulphuric acid ap- pearing in the urine in such organic combination ("ethereal sul- phuric acid") is considered as an index of the amount of intestinal putrefaction. In the case of indole and skatole, which have no hydroxyl group, a preliminary oxidation occure, whereby inHole is converted into indoxyl, >C — OH I CH I / NH and skatole into skatoxyl. \ C — ClJs roH / XH 21 See Ellingcr, Zcit. pliysiol. CIiciii., 1!)04 (43), 325. PRODUCTH OF PUTREFACTION A\D FEIIMENTATIOX 573 and they are thou combined with sulphuric or glycuronie acid, as follows : _ c — ; OH + H : 0 — SO, — OK = < > _ c — o — SO2 — ok N\ ■• ' V_y N\ \ CH \ pB. (indican) HN -\"H B}^ far the greater part of these aromatic substances, when ex- creted in the urine, is combined with sulphuric acid, and but a small part with glycuronie acid ; but in case the amount of sulphuric acid available is too small to combine with all the aromatic radicals enter- ing the blood, a large amount of the glycuronie acid compound ap- pears in the urine (e. g., after therapeutic administration of phenol, cresol, thymol, camphor, etc.)- Both the preliminary oxidation and the combining with acids seem to occur chiefly in the liver, this process constituting one of the most important of the many pro- tective offices of that organ, since the resulting compounds are much less toxic than are the original substances.-^'' Herter and Wakeman -- have shown that living cells have the power of acting upon indole and phenol (and presumably upon the rest of this group) in such a way that they cannot be recovered by distillation. Most active in this respect is the liver, then in order come kidney, muscle, blood, and brain. The change seems to be a loose chemical combination with the protoplasm of the cells, and the power of the tissues to bring about this combination is not greatly, decreased by serious pathological changes in the organs (e. g., ricin poisoning).-^ Indole. — This is probably the most important member of this group of substances, the striking color of its derivatives making its detec- tion in the urine easy, so that it is generally used as the most available index of the amount of putrefaction that is occurring in the intes- tines.^* The greatest quantities are found when intestinal putre- faction is marked, especially in intestinal obstruction involving the small intestine ; obstruction of the large intestine, as Jaife first dem- onstrated, does not cause marked indicanuria unless the stagnation involves the ileum, as it may in the latter stages of obstruction. With marked impairment of renal function indican may accumulate in the blood (see Uremia). There can be no question that the indican of the urine is derived, at least in part, from the indole formed in the intes- tine, for administration of indole by mouth to either animals or man causes a considerable increase in the indican present in the urine ; how- ever, but 40 to 60 per cent, can be recovered in this way, the rest ap- parently being oxidized ^0 other compounds, part of which may also 21a Metchnikoff insisted that these sulfo-compounds still retain considerable toxicity. (Ann. Inst. Pasteur, 1014 (27), 80."^). 22 .Tour. Exper. :^red.. ISOt) (4), 307. 23 For further discussion of this topic, see "Chemical Defences against Poisons of Known Composition," Chapter ix. 24 See Houghton, Amer. Jour. Med. Sci., 1908 (135), 567. 574 GASTRO-INTESTIXAL '■AVTOiyTOXICATlOX" aiipear in tlie urine. -■' Whether i)art of tlie urinary indiean is derived from tryptophane liberated during intracellular protein metabolism, and not from intestinal putrefaction, has long been a disputed point among physiological chemists.-" The demonstration hj Ellinger and Gentzen -' that try]ito])hane, when fed or injected su1)cutaneously, causes no increase in uriiuiry indican, whereas its injection into the cecum causes much indicanuria, would indicate that indole is formed from tryptophane only through putrefaction, and not in cellular nietabolisiu. Othei- expei'imeuts support the same view.-*^ However, it is possible that part of the indican present in the urine during conditions associated with gangrene, putrid cancers, putrid placentas, or puti'id purulent exudates, may be derived from these decomposing materials. The statement that indicanuria is of significance in in- sanity could not be substantiated by Borden,-" who used quantitative methods and careful controls. A large proportion of the data and conclusions in the literature concerning indicanuria are valueless be- cause of improper or inadequate methods. Probably the chief agent in the formation of indole in the intes- tines and in putrid tissues is the colon bacillus, which, as is well known, produces indole in ordinary culture-media. Toxicity of Indole. — Although the toxicity of indole seems to be relatively slight, and this toxicity is further reduced by the conver- sion of indole into indoxyl and indican, yet Herter ^^ found that ad- ministration to healthy men of indole in quantities of 0.025 to 2 grams per day caused frontal headache, irritability, insomnia, and confu- sion ; the continued absorption of enough indole to cause a constant strong reaction for indican in the urine is sutificient to cause neuras- thenic symptoms. Long-continued injection of indole leads/to hyper- trophy of the adrenal medulla and slight interstitial changes in the kidneys,^^ but the reputed responsibility of indole for arteriosclerosis is most doubtful. ^^^ Lee ^- has also demonstrated that iiulole. skatole, and methyl mercaptan cause muscles to react to stimuli like fatigued muscles. Normal urine contains but about 12 milligrams of indican per day, which amount is so insigiiificant in proportion to the above- mentioned doses that were found necessary to produce symptoms, that we may well doubt the occurrence of noticeable intoxication -^' If frt'latin is subaiitutod for proteins in tlic diotary, indican is not oxrrcted, because {gelatin does not contain tryptophane (Underliill, Amer. .lour. Piivsiol., 1!KM (VI), 17(i). 20 Literature by Gerliardt, Erfrel). der I'liysiol., 1!)(I4 ( 11 [. Abt. T), LSI. 27 TL)fineister's Beitr., 1903 (4), 17L 2sSee Scliolz. Zeit. physiol. Clieni.. 1!)0.3 (.'JS), fiL'?; ITnderhill. Jor. cit. Slierwin and Hawk found an absence of indican in the urine in tlie latter part of a long fast (ISiocliem. Bull., 1914 (3), 410). 2ft Jour. Uiol. Cheni., 1907 (2), r)75. 30 X,.\v York Med. dour., 1S9S (6S), 89. 31 W.xdlev and Xewburgb, .lour. Amer. IMed. Assoc. 1911 (oO), 1796. siaSee Steenliuis, Folia Mikroliiol.. 1915 (3). 7(>. 32 Jour. Amer. Med. .\ssoc., 190() (40), 149!t. I'noitt \ \\i) ji:inii:\T \'H()\ 575 from tliis siil)stiiii('t' iiiidcr ordinary coinlitious. Nesbitt "•' states that twenty tiuics as iiindi iiulok' or skatole as are excreted daily by an adult man may be injected into the ju CIL — CHXH„ — COOH. OH Uroleucic aci(i,->4 / \ CH„ — CHOH — COOH. HO OH Homogcntisic acid, /^ \ CK, — COOH. HO Apparently the condition depends upon an abnormality in the in- termediary metabolism, and not upon an abnormal formatiou of homo- urentisie acid through intestinal putrefaction, as was at first believed. Alkaptonuria is never observed in slight degrees; if there is any homogcntisic acid in the urine at all it is there in large amounts (4-5 grams per day), depending on the diet, for when the error in metabo- lism is present at all it is complete. On a mixed diet the ratio of homogcntisic acid to nitrogen in the uriue is 40^5 to 100. The pre- vailing idea has been that the abnormality consists not in the excessive formation of homogcntisic acid, but in a lack of ability on the part of the alkaptonurie individual to split open the benzene ring. It is generally stated that tyrosine and phenylalanine first suffer a split- ting out of the nitrogen radical from the alanine side-chain, and then are oxidized into homogcntisic acid, following which changes comes a disintegration of the benzene ring, with subsequent complete oxida- tion. On this basis the alkaptouuric accomplishes the conversion into the ox3^-acid, but the process stops there. Wakeman and Dakin,*^ hoAvever, have obtained evidence that in the normal oxidation of tyro- sine and phenylalanine, homogcntisic acid is not an intermediary product, and Dakin statop that the alkaptonurie can destroy simple derivatives of plienylalanine and tyrosine, provided their structure is such that the formation of substances of tlu^ type of homogentisic acid is precluded. He believes that in alkaptonuria there is abnoi'mal formation of homogentisic acid as well as a failure to destroy it when formed. On the other hand, Abderhalden '**' has been able to cause dence of syphilis, and in one case this reaction disappeared wlien the ]>atient was given large amounts of tyrosine (Siiderliergh, Nord. Med. Arkiv., IHI.") (48), 1). ; 4» The older writers stated that uroleucic acid commonly accompanied homo- gentisic acid in the urine of alkaptonuria, hut later observations do not confirm this. (Oswald, Zeit. phvsiol. Chem.. 1914 (0.3), n07 ) . ■•5 .Tour. Biol. Cliem., 1011 (f)), 130 and 151. <«Zeit. physiol. Chem., 1012 (77), 454. .\IJ\AI'TOMh'/A 579 the appearaiK'c in tlio urine of liomogentisic acid in a normal individ- ual by feedin<>: large amounts of tyrosine, which is in favor of the view that it is a normal intermediary in tj-rosine catabolism. In any case the administration of tyrosine or phenylalanine, or of tyrosine- rich foods — i. e., proteins — causes a marked increase in the amount of homogentisic acid eliminated in the urine; indeed, this increase is almost quantitative. Normal individuals when jjiven these substances in moderate amounts, or homogentisie acid itself, destroy them com- pletely, so that the latter does not appear at all in the urine.^"'' If alkaptonurics are kept Avithout protein food for some time, the elimi- nation of alkaptonuric acids goes on, although in diminished amounts, indicating that the aromatic amino-acids formed in tissue catabolism also fail to be destroyed and, therefore, appear in the urine as these derivatives. Since gentisic acid, OH COOH, HO >\'hen given by mouth, is also eliminated unchanged by alkaptonurics, although completely destroyed by normal individuals, it seems evident that the difficulty in metabolism affects the benzene ring itself and does not depend upon the character of the side-chain. Nonnal organ- isms seem to be capable of destroying such aromatic compounds as pass through a stage of homogentisic acid in being oxidized, which indicates that the benzene ring can be broken up only Avhen oxidized in this particular manner (the 2, 5 position) ; the alkaptonuric differs in being unable to break up even this form (Falta). According to Garrod *' the conversion of tyrosine and phenylalanine into homogen- tisic acid is so complete that the ratio of homogentisic acid to nitro- gen is constant and the same in all cases. Frommherz and Her- manns ^"'^ advance the suggestion that normal oxidation of the aromatic radicals may take place by two routes, one by way of homogentisic acid, the other by way of the 3-4 dioxy-derivatives (i. e., pyrocate- chin), since such derivatives can be readily oxidized in the metabolism of alkaptonurics who cannot destroy homogentesic acid. That is, their deficiency involves onl}^ one of two possible methods of oxidizing aromatic compounds, leaving them considerable capacity for this im- portant metabolic function. The tissues of the alkaptonuric are prob- ably not chemically affected in this condition, for Abderhalden *^ found that the hair and nails of an alkaptonuric contained normal propor- tions of tyrosine. 46a Gross states that normal scrum destroys liomogentisio acid, which property is lacking in the serum of alkaptonurics ( Biochem. Zeit., 1914 ( (il ) . l(i.i). 47 Garrod and Clarke, T!iocli«>ni. Zeit., 1!1()7 (2), 217. 4"aZeit. physiol. Chem.. 1914 (01), 194. 48 Zeit. physiol. Chem., 1907 (52), 435. 580 GASTRO-INTESTIXAL "AUTOIXTOXICATIOX" In some cases of alkaptonuria a pignuMitation of the cartilages also occurs, ochronosis, but the association is not constant; ochronosis may occur without alkaptonuria, and conversely. (See "Ochronosis.") (2) SUBSTANCES ARISING FROM THE FATTY ACID RADICALS (AMINO-ACIDS) OF PROTEINS As stated in the introductory chajitei', the protein molecule con- sists of a combination of a great number of organic acids, of various sorts, all of which have as a common characteristic the presence of an NHo group attached to the carbon atom nearest the acid radical, the a position; thus, R — CHNHo — COOH. A few of the amino-acids con- tain an aromatic group, and the relation of these to intestinal decom- position has been considered above. The greater number have a simple fatty acid radical (the simplest amino-acid being glycocoll,. CHoNHo — COOH), and from them are derived by intestinal putre- faction substances that are, for the most part, chemically simple and, as far as known, pathologically unimportant. From leucine alone is derived a substance of known considerable toxicity, the pressor base isoann/linitine. >CH — CH, — CH, — NH, CH3 which is less powerful than the cyclic pressor bases described prev- iously. Bain '^^^ found it the most abundant pressor base of the urine. Fattj^ acids may readily be formed from them by splitting out of the NH, group ; thus acetic acid may be formed from glycocoll, propionic acid from alanine, etc. Apparently butyric and acetic acid are the acids most commonly formed in this way. Gaseous deriva- tives, such as hydrogen, ammonia, carbon dioxide, and marsh-gas, are also produced. Acet&ne is perhaps formed from these fatty acids; it is often present in the intestinal contents, but may come from other sources. Certain conditions of cyanosis have been designated as enteroijenous cfjanosis, because of tlie belief that the methem()gh)bin responsible for the cyanosis is caused by nitrites derived from intestinal putrefaction, and demonstrable in the blood. ^" Presumably tlie nitrites come from llic Xll.j groui)s of the protein molecule, the ('(don bacillus being an active fornuM- of nitrites. Tender tlie same term are included the cases of sulph-hcinoglohinemia. This condition is ascribed by Wallis"'" to bacteria which produce from the proteins a hydroxylamine derivative, capable of reducing oxyhemoglobin, and whicli lie finds jiresent in the blood of patients with sul])li-hemogl()binemia. Diamines. — Of much interest are the substances that arc formed •"■"gwart. .)()iir. Kxp. Plivsiol., 1014 (S). 2-2!t. 4(1 Sec rjilison. Quart. Jour. :Mo(1., 100" (1). -i!) : Wost and Clarke. Laiieet, Feb. 2, in(»7; Davis. Lancet. Oct. 2ti. VM'l. •-■•o Quart. Jour. Med., Oct., ]!»!:{. i>i:in\ .\Ti\ i:s of siu'iiiu rosTAisisa I'ifOTEiN radicals 581 from tlio ainino-aeids by bacterial action, wliich still retain their nitro- gen radicals — the ptoindins. Two of these, the diamines putrescine, NII2 (CIIn)4 NIL, and cadaverine, Nil, (0X12)5 NIIj are of particular interest,^^ because they have been observed in the feces and urine of persons with cystinurw. The stools in cholera also seem to contain these ptoma'i'ns frequently. Their etiological relation to the cystinuria is no longer accepted, however, and their toxicity is slight. They are probably derived from the diamino-acids of the protein molecule, pu- trescine being closelj' related to ornithine,''^'' and is probably formed from it as follows : NHa NHj NHo XH, CH2 — CH, — CH, — CH — COOH p=, CH, — CTI, — CH, — CH, + CO, ( ornithine ) ( putrescine ) while cadaverine is probably formed from, lysine, NH„ NHo NH2 NH, CH,— (CH,), — CH — COOH .=. CH,— (CH,), — CH, + CO, (lysine) (cadaverine) .NH, Ethylidendiamine, CH^-CH < which is somewhat toxic, has also ^NH,, been detected in the contents of the gastro-intestinal tract. Apparently these substances are absent from normal feces, but this does not exclude the possibility of their normal formation, ab- sorption, and destnietion. There is no evidence that they ever cause symptoms or pathological alterations. (3) SUBSTANCES ARISING FROM THE SULPHUR-CONTAINING RADICAL OF PROTEINS Most if not all of the sulphur in the protein molecule seems to be contained in the amino-acid. cystine, which has the following compo- sition : S — CH, — CHXH, — COOH S — CH, — CHNH, — COOH. From this is formed the hydrogen sulphide of the intestinal gases, of which about 0.058-0.066 gram is present in each one hundred grams of normal colon contents. Although Senator has described a case in which an intoxication with HoS of intestinal origin occurred, this gas seems not to be a frequent cause of intoxication, and Senator's case stands almost alone. Under normal conditions H^S does not appear in the urine, any that may be absorbed probabh^ being oxidized 51 For discussion of formation and properties of these t\vo ptomains, see Vanghan and No\'\''s ''Cellular Toxins." 51a Ornithine forms part of tlie arginine molecule, which is the most universally present (in proteins) of all tlie amino-acids, ornithine being formed when urea is split from arginine. 582 GASTRO-INTESTIKAL 'AUTOIXTOXICATIOX" to SO4. If enough H2S should enter the blood so that it was not completely destroyed, it might well cause harm, for it is decidedly toxic, particularly att'ecting the nervous system; but we have no evi- dence that this often happens. Van der Bergh ^- has observed cases of intestinal obstruction in which the presence of sulphemoglo'bin in the patient's blood was demonstrated. Methyl mercaptan, CII3SII, has also been found in the feces, al- though it seems not to be abundantly or constantly present, according to Herter,^^ who found also that mixed bacteria from normal feces rarely produce mercaptan in cultures. However, bacteria from the feces of persons suffering with various diseases often produce mercap- tan. Ethyl mercaptan, CoH-SH, and ethyl sulphide, CoHj-S-CjHj, have also been described as fecal constituents. It is not known that the mercaptans are a cause of intoxication. CYSTINE AND CYSTINURIA '■* The presence of cystine in the urine has been observed in a num- ber of cases, and when present at all it is usually present in consider- able quantities. Because of its slight solubility it appears as a de- posit of hexagonal crj'stals, and frequently forms cystine concretions (q. V.) in the urinary bladder."'^ According to Garrod it is more common than alkaptonuria, and, like the rest of the "Inborn Errors of Metabolism," occurs much more often in males than in females. Hofmann ^^ was able to collect from the literature to 1907 a total of 175 cases, of which 85 were males and 45 females. Baumann and others observed that in cystinuria the urine often contains, besides the cystine, the diamines cadaverine and putrescine, which are formed from lysine and ornithine respectively in the intestines througli putre- faction, and they naturally suspected that cystine arose in the same way. Another view was that the diamines interfered with the oxida- tion of sulphur in the body, so that it was eliminated in the unoxidized form of cystine. But it has been demonstrated that neither of these hypotheses is correct, for (1) cystine could not be found in the feces; (2) if given by mouth, it is completely oxidized, and causes only the appearance of excessive amounts of sulphates in the urine; (3) cys- tinuria has been observed to occur independent of the presence of the diamines, and not to be modified or caused by their administration or pathological formation. The view now prevalent is that the cystine that escapes in the urine in c^-stinuria is not derived from intestinal 52Deut. Arch. klin. Med., 1905 (S3), SG. 53 .Tour. Biol. Cliem., 1906 (1), 421. 54 Literature coneerninfj cystine {,'ivoii l)y Fricdmanii, Kifiohiiissc der Physiol., 1902 (I. Aht. 1). If); and l)y Maiui. "Chemistry of the Proteins." jjp. r)()-(i4. Cys- tinuria reviewed l)y I5(')dtker. Zeit. ])hysiol. Chom., 1905 (45), 393; C.arrod, "Inborn Errors of Metaholism," and Lancet, duly, P.tOS. 5-'> Ahderhaldcn (Zeit. physiol. Chem., 1003 I3S). f).!?) has described a ease in a child in which tlie or^ians were inliJtrated witii masses of tlie cystine crystals. ooCent. Grenz. Med. u. Chir., 1907 (KD. 721. i'i,-(U)i <'rs or Fh'h'\ii:\TAT/<>.\ or <• AUiioiiyiu; M-rn" 583 putrefac'tioiu l»ut is formed in the tissues from the protein molecule, and fails to he further decomposed because of some anomaly of metab- olism. This view is supported by the fact that cj'stinuria often ap- pears to be an hereditary disease, occurrino: in families for several jivnerations; it is independent of the diet, cystine appearin<>- even if proteins are withheld, and also independent of intestinal putrefac- tion."'" It having been found that leucine and tyrosine may also occur in the urine in cystinuria, it seems probable that this condition de- pends upon a general abnormality of protein metabolism. The rela- tion of the diamines to the condition is, however, very uncertain. Cystine does not seem to exert any toxic effect, and patients with cystinuria do not usually appear to sutfer greatly from the abnormal metabolism, the chief trouble observed being due to the formation of the concretions in the bladder.""'' Sometimes in children, however, emaciation and early death, without other apparent cause, have been observed, and may be due to the metabolic anomaly. The metabolic error in cystinuria is not complete, for only a por- tion of the total cystine of the catabolized proteins is excreted as such (Garrod). This would amount to some five grams per day, whereas the average excretion is only about 0.3-0.5 gram, and sul- phates and other neutral sulphur compounds are always present in the urine. In no condition other than cystinuria have putrescine and cadaverine been found in quantities which could be detected by ordi- nary methods in 2-4-hour specimens ; they may also be found in the feces of cystinurics, where cystine is never found. In the urine their presence is inconstant, and the amounts are at best very small. Leu- cine and tyrosine are found much less often than the diamines ; lysine, has been found in one case,^* which supports the view that cadaverine and putrescine come from the diamino-acids of the protein molecule by metabolism rather than by putrefaction. B. PRODUCTS OF FERMENTATION OF CARBOHYDRATES These include practically all the members of the fatty acid series, from formic acid to valerianic ackl; and the oxy-acids, lactic, succinic, and o.ryhutijric; also, oxalic acid, acetone, ethyl alcohol, and the fol- lowing gases: CO^, CH^, H,. For the most part, the various organic acids are absorbed through the intestinal walls, and are oxidized completely in the tissues without causing any harm whatever. The possibility that acid intoxication may be produced in this way has been suggested, but it is generally believed that this does not occur, except possibly in infants. Lactic and butyric ''^^ acids are formed 57 An isolated case of transient cystinuria in a patient with Raynand's disease is described by Githens (Penn. ^led. Jour., 1910 (1), 507). 57a This may be avoidetl by decreasinrr the cystine by means of a low jirotein diet, and increasinof its solubility by keeping the reaction of tlie urine alkaline (Smillie. Arch. Int. Med.. 1915 (IG), 50.3). •''•'^ Ackermann and Kutscher, Zeit. f. Biol., 1011 (57), 355. 5sa Coleman (Ann. Inst. Pasteur, 1915 (29), 139) attempts to incriminate 584 GASTh'oi\ri:sTJ.\AL '-AUToiy toxic ATioy particularly in gastric fermentations in persons with deticient hydro- chloric acid, motor insufficiency, or or<>-anic obstruction. ]\Iost of the disturbances observed in these conditions seem to be due to distention of the stomach with gas, chiefly CO2, which is formed during the fer- mentation. It is possible, however, that the organic acids exercise some irritant effects on the mucous membrane; and they may also cause diarrhea, lactic and acetic acid often being present in diar- riieal discharges due to excessive feeding with carbohydrates (Herter). These acids or their salts do not appear in the urine, unless possibly as minute traces, except the oxalic acid. ^linute quantities (0.02 gm. per day) of this substance are present in normal urine, but larger quantities (oxaluria) seem to depend either upon the taking of food containing much oxalic acid (rhubarb, spinach, etc.) or upon excessive gastric fermentation of carbohydrates (Baldwin),"'' and perhaps upon excessive destruction of purines, from which oxalic acid may be de- rived. . Of the amino-acids it is presumably the diatomic acids, glu- tamic and aspartic, which yield oxalic acid (Jastrowitz).*'^* Others, however, do not admit that any appreciable amount of oxalic acid is derived from proteins.'^"-'' Probably the small quantities of oxalie acid thus formed do not cause toxic etfects, and are important chiefly as causing urinary concretions of calcium oxalate, although there is. evidence that long-continued excretion of oxalic acid may cause renal lesions. (See also consideration of oxalic calculi. Chap, xv.) C. PRODUCTS OF THE DECOMPOSITION OF FATS These differ but little in nature from the products of carbohydrate fennentation, the large fatty acid molecules being broken down to smaller ones. In infants these fatty acids have been believed to be a cause of acid intoxication and acetonuria,"^ but probably they are sel- dom, if ever, of pathological importance. It is possible, however, that a serious reduction in the bases of the blood may result from the formation of excessive amounts of fatty acids in the intestines, the bases being combined to unite with the fatty acids, and then excreted in the feces. It is quite otherwise with the products of decom])ositi()n of leci- thin.^^ From its molecule is split o1"f the ptonui'in, cJwlinc, {( 11:,) ,. = X — C'll. — CH,OH, I OH Imtyric acid in tlic production of iutorioscloiosis, while Oswald Loeh believed lax-tic at'id to be important, a " 'e-v wliicli could not be alto (8), 27. o-iZeit. physiol. Chem., 1906 (48), 1. 6-. Biochem. Jour.. 1910 (5), 306. 65a Jour. Pharmacol.. 191;) (7), nOl. 65b See Dale, Jour. Pharmaeol., 1914 (6), 147. 586 (V . 1 N Th'o-ix Ti:^ ri vi /, '•. i ltoix toxic. i tiox" and luilk products. Thus Clairinoiit and Raiizi "" found lieat-resistant toxic substances in the intestinal contents in ileus (experimental), and similar substances could also be obtained b}' growing cultures of the intestinal contents on bouillon. Another explanation is that many unidentified poisonous substances are produced in the alimentary canal which ordinarily are destroyed, but under certain conditions may be reabsorbed. That unrecognized toxic substances are formed in the intestines is almost certain, for it has been repeatedly shown that extracts of the contents of the alimentary canal are very poison- ous. Although the teehnic of many of these experiments has been questionable, the results have been obtained so often as to render it probable that the main contention is correct."^' Thus ^lagnus-Alsle- ben '^"'^ has found in the upper part of the small intestine of dogs (ex- cept when on milk diet) a very poisonous substance which killed rab- bits by respiratory paralysis, but which is inert when injected into the portal vein. Extracts of the wall of the large intestine are also toxic, and lose their toxicity at 60°, by passing through porcelain filters and by treatment with alcohol ; extracts of fetal intestines are not toxic (Distaso)."^ Whipple "" has demonstrated that closed duodenal loops in dogs come to contain a highly toxic substance of unknown nature, appar- ently formed in the epithelium of the gut rather than in its contents, which causes severe splanchnic congestion, vomiting and diarrhoea when injected into normal dogs. The agent is not destroyed hy autolysis, filtration or heating at 60°, yet dogs can be made somewhat immune. The origin and nature of this poison have not yet been de- termined, but it seems probable that it is an important factor in the intoxication of intestinal obstruction. Apparently the liver does not have much detoxicating ett'ect, for dogs with p]ck fistula behave much the same when the intestine is obstructed as normal dogs. A similar material cannot be obtained by hydrolysis or autolysis of normal duo- denal mucosa, the obstruction being an essential feature. Obstruction of lower portions of the intestine has much less effect,'^ and it has been suggested that the poiscm formed in the duodenum is neutralized or destroyed farther down in the intestine.^- In any case, correctly or incorrectly, a great num])er of disease con- ditions have been attributed to poisons of gastro-intestinal orig'in, including not only such minor conditions as headache, nuilaise, lassi- tude, etc., but also sciatica, tetany, epilepsy, eclampsia, many forms of dermatitis, various forms of nervous diseases, myxedema and cretin- "« Arch. klin. Chir., 1904 (73), 090. (17 For example, see Roijer and (ianiicr, Coiiipt. I\eiul. Soc. IJiol., 190.") {'i9) , 388 and f)74 ; 190(1 (60), CM). OS IlofmeiBter's Beitr., 190.5 (fi), i)0.'l. If Zcit. Inimunitiit., 191:5 (10), 400. ■'I \\liil)j)le. Sloiie and Bcrnlieini. .lour. K\]H'r. ^\^H\.. 19l;i (17), 280. 71 See liimtiii^'. .(..iir. K\]u-v. Mod., 1 9 1 ;i (17). 192. 72 Maury, .\iiici-. .Idiii-. Med. S.-i., 19i)-l\Ti:sT/\\l. I \T()\ l7), IT); Jour. Exp. Med., 1910 (23), 12:{: l!tl7 (2.")), 2:51 and 401. «:< See Murpliy and IJrooks. Areli. Int. Med.. ini.-> (l.")), .302; Curd.. .Tour. Infect. Dis.. 1014 (1.')). 124: Draper. .Tour. Anier. Med. Assoe.. 1010 (67), 1079; Dragstadt, Moorliead and Hurcky. .lour. K\p. ^led.. 1017 (2.1), 421. ACUTE INTESTINAL OBSTRUCTIOS 589 nitrog-en content uf tlu- l.lo.ul, W^nvv. similar to those of fatal uremic coma being common- which may be the result ot absor-ption of c eav- age products from the intestine, or toxogenic destruction ot tissue proteins, or both. S4 Cooke, Rodenbaugl. and Whipple, Jour. Exp. Med., 191G (23), 123. CHAPTER XX CHEMICAL PATHOLOGY OF THE DUCTLESS GLANDS 1 DISEASES OF THE THYROID - As we liave miieh evidence that the thyroid has a marked influence upon metabolism, and also that it may be of importance in preventing autointoxication, the chemistry of diseases of the thyroid may be ap- propriately considered in connection with the autointoxications. THE FUNCTIONS OF THE THYROID Metabolic Function. — That the thyroid has an important relation to metabolism, especially of proteins, is shown by the following- facts : (1) Administration of the gland substance, or active preparations made from it, to healthy men or animals, causes a greatly increased elimination of nitrogen in the form of urea. This nitrogen comes not only from the food, but also from increased tissue-destruction, as is shown by the loss of weight and strength, and by the increased ex- cretion of sulphur and phosphorus. An increased destruction of the body fat also occurs, so that thyroid therapy has been found efficient in the treatment of obesity, but often dangerous because of the rela- tively great amount of tissue-destruction. Basal metabolism is most markedly raised in hyperthyroidism, and is lower in cretinism and myxedema than in any other disease.-'' (2) Loss of thyroid tissue, either through operation or disease, greatly reduces both nitrogenous metabolism and oxidative processes. Administration of thyroid preparations under these conditions will bring the nitrogen elimination and the gas exchange back to normal. (3) Deficient thyroid secretion in young animals prevents their de- veloping normally, the amount of deficiency varying from nearly total lack of development in extreme cretinism to slight grades of defective development (infantilism) or delayed maturity. In adult animals, besides decreased metabolism there occur also various trophic changes in the skin and its appendages, an increased amount of mucin-like material in the tissues, and greatly decreased nervous and mental ac- 1 Thorough reviews of tin- ciitiic sulijcct (if llic diictlfss glands nro lmvcii hy Biedl, "Tnnero Sekretioii,"' rrhaii and Scliwar/ciilK'rjr. r>crlin. ini;i: and Vincent, Erffchnissc I'livsioi.. lltlo (!)), 451; 11)11 (10). 21S. - Coiicerninfr tlie thyroid see besides iiiedl and \'ineent, the review bv llirelier, Ergebnisse Pathol., 1011, XV (,), 82. iiaDu Bois, Arch. Int. Med., 1916 (17). i)15. 590 77//; Fr\(Tio\s or Tin-: Tiirnoih 591 tivity. All these conditions are relieved to jrreater or less degree by adiniiiistratioii of thyroid tissue or its preparations.'* Evidently, therefore, tiie thyroid exerts an influence upon growth and tissue changes ; whether this depends upon its influence upon metabolism, or is an independent and specific function, cannot be determined.* IIow the thyroid or its secretion modifies metabolism is not yet un- derstood. One is reminded of the effects of kinases upon enz^-mes and their antecedents, and it may be imagined that the thyroid secre- tion activates both proteolytic and oxidative enzymes vv^ithin the cells. Shryver,"^ indeed, did find that administration of thyroid to dogs for some time before killing them causes their liver tissue to undergo autolysis more rapidly than normal, although AVells " had been unable to observe any increased amount of autolysis when thyroid extracts acted upon liver tissue in vitro. Experimental observations show that carbohydrate metabolism is much influenced by the thyroid, so that thyroidectomized animals may fail to show glyeosviria from vari- ous procedures that usually produce it (King),^ and they are incapable of utilizing sugar injected parenterally as well as normal animals ; ^ they also exhibit an excessive creatine output, but otherwise show no striking changes.'*'' Detoxicatory Function. — The evidence that the thyroid has for its function the destruction or neutralization of poisonous substances formed in metabolism or through intestinal putrefaction is as follows : (1) After total removal of the thyroid from many species of ani- mals acute symptoms develop that suggest strongh' an intoxication. (2) After removal of the thyroid, marked changes occur in the blood, there being a severe anemia (as low as 2,000,000 red corpus- cles), Avith some leucocytosis, and there occur structural changes in the blood-vessel walls (Kishi).'* Cytoplasmic degeneration of the liver, kidneys, and myocardium may also result (Bensen).^'^ These effects suggest strongly the presence of poisonous substances in the blood of persons or animals lacking sufficient thyroid tissue. (3) All the effects of thyroidectomy are more marked in carnivor- ous animals than in herbivora ; indeed, the latter may be able to live 3 Concerning tlie influence of thyroid on skeletal growth see Holmgren. Xordiskt Med. Arkiv, 1910 (43), No. 2. Literature given bv Basingcr, Arch. Int. 31ed., 1916 (17), 260. 4 See the interesting experiments of Oudernatsch (Arch. Entwickl., 1J)12 (35), 457; Amer. Jour. Anat., 1014 (15), 431: Anat. Record, 1017 (11), 357), Avho found that feeding thyroid to tadpoles hastens their differentiation but checks growth. 5 Jour, of Physiol., 1005 (32), 150. 6 Amer. Jour. Phvsiol., 1004 (11), 351; corroborated by Morse, Jour. Biol. Chem., 1915 (22), 125. 7 Jour. Exper. IVled., 1000 (11), 665. sUnderliill and Saiki, Jour. Biol. Chem., 190S (5), 225. 8a Hunter, Quart. Jour. Phvsiol., 1014 (8), 23. 9Virchow's Arch., 1004 (176), 260. loVirchow's Arch., 1902 (170). 220. 592 CHEMICAL /'ATHOLOGY OF THE DUCTLESS GLAyDS in fair condition for several years without a thj-roid,^^ Administra- tion of meat to thyroidcetomized lierbivora or omnivora causes a great increase in the syinptoms, while tliyroideetomized carnivora do much better if kept without meat. Tims, Hluui ^- found that thj-roidecto- mized dogs, which were doing well on a milk diet, developed symptoms of athyreosis immediately they were given meat. This fact has been interpreted as indicating that toxic materials are formed from meat in tiie intestinal tract, which under -normal conditions are neutralized by the thyroid. On the other hand, one may well imagine that the so- called autointoxication in athyreosis is not from intestinal putrefac- tion, but is due to the products of incomplete metabolism of proteins within the tissues, which are destroyed when protein metabolism is normal, but not when the metabolism-favoring influence of the thyroid is wanting. It should also be added that the presence of specific poisonous substances in the blood or urine of thyroidcetomized animals has not been conclusively established.^"' (4) Reid Hunt ^* found that mice fed thyroid preparations have a greatly increased resistance to poisoning by aceto-nitrile ; however, this is not necessarily nor even probably a direct detoxication, but more likely it results from alterations in metabolism. Rats and guinea pigs behave just the opposite, showing a decreased resistance to aceto- nitrile after being fed thyroid, and according to some authors morphine is more toxic for such animals. ^"^^ "Whether the thyroid exercises its detoxicating effect, assuming that it has one, by a direct neutralizing action of its secretion upon the toxic substances in the blood or in diverse tissues, or indirectly by stimulation of the function of other tissues which perform the de- toxication. or in part locally within the gland itself, is an unsettled problem. In relation to the last-named hypothesis is the extreme vas- cularity of the thyroid, which, according to Burton-Opitz ^" has passed through it much more blood in proportion to its weight than any other gland. Against the idea of a local detoxication is the fact that after extir]iation of the thyroid all abnormal conditions may be pre- vented l)y i)r()pcr administration of thyroid substance. Biedl summarizes his views as to the function of the thyi'oid, in 11 I'arl of tlicse results may be due to tlie faet tliat in some lierbivora the parathyroids are so far separated from the thyroid that they are not ordinarily removed in thyroidectomy, whereas in many carnivora eom]ilete removal of paratiiyroids witli tlie thyroids is more likely to be aeeom])lished. 12 Virchow's Arch.. IHOO (1(12), .'575. i-i Heinedi ( Lo Sperinientale, 1!)()2; abst. in Cent. f. Path., litO.S (14), fi!)5) claims tliat tetanus toxin and other l)acterial |)oisons, when injected into the thyroid fjland, are harmless, which he attrii)utes to a neutralization by the colloid. This observation is discredited bv the work of Basinger, Jour. Infect. Dis., 1017 (20), 1.31. 14 Jour. Amer. ^led. Assoc, 1!H)7 (4!M, 240; llyuicnic Lul). IbiU., 1!>{)!I, No. 47; Jour. Pharmacol., 1910 (2), 15. ir-See Olds, Amer. Jour. Physiol., 1010 (2(1), .Sr)4. I'UJuarl. Jour. Phvsiol.. 101(> (.'{). 207. cii i:\iisTin OF Tin: riiynoiit 503 tilt" t'ollowiiiLj staltMiiciit : "Tlic tliyi'oid is a secretory orjraii which (lischaf thyroid, and no storaf^o of bromin ta'^o* T-'aoe. Administration of pilocarpine docs not. increase ilie amount of iodin iTi tlic thyroid. 20 Niirn1)cri:. llofincisicr's Bcitr.. 1007 (10), 125. •■;i Jour. l?iol. Cliem., 101:5 (14), 101. -'-•Jour. Pliarni. and F\p. Ther., 1010 (1). 123. 1!'^' Jour. l?iol. C'hcm., 1015 (10), 421. i'"'Arcli. ^'cs. Phvs.. 1015 (102), 00. CHEMISTRY OF Till-: THYUOin 595 dialyzcd, tlie active suhstaiiee diffused thnnimh the membrane, indi- cating that it is not a colh)idal complex, and that if derived from the proteins it must be an iodized amino-acid or some related compound. This observation is to be expected in view of the fact that thyroid pro- duces its effects when fed and is presumably hydrolyzed before ab- sorption. Lenhart ^'-"^ considers the effect of thyroid on tadpoles to be merely an expression of the (>oneral stinndatio]i of metabolism, rather than a specitic effect on differentiation.""^ liy decomposing thyreoglobulin by boiling with 30 per cent, sul- phuric acid, a body is obtained containing as high as 14.5 per cent, of iodin ; this is the tJnjroiodin of Baumann, which gives no biuret re- action, yet is physiologically active. The stability of this active con- stituent of the thyreoglobulin explains the successful administration of thyroid preparations by mouth. It appears to be absorbed un- changed and, unless enormous doses are given, none appears in the urine (Oswald -■''). Long-continued digestion with trypsin, or auto- lysis of thyroid glands, causes a complete splitting-out of the iodin. One part of the iodin seems to be more firmly bound than the rest. Kendall ~* has isolated from the thyroid, after alkaline hydrolysis, a crystalline compound containing 60 per cent, of iodin, which he sug- gests may be di-iodo-di-hydroxy-indole. This is highly active, causing rapid pulse, nervous irritability, and increased metabolism. It does not contain all the iodin of the thyroid, but the nature of the other iodin compounds is unknown beyond the observation that they have no appreciable effects on normal persons but greatly improve the con- dition of cretins. A small amount of the iodin may exist as inorganic and lipoid compounds.-*^ When fed to tadpoles, Kendall's active principle produces the characteristic thyroid effect."*'' The amount of iodin in the thyroid is greatest in middle age, greater in females than in males, and it is decreased in acute infectious dis- eases and in tuberculosis, alcoholism, and circulatory disturbances (Aeschbacher).'^ The thyroid is very rich in lipase, catalase and peroxidase; ex- tirpation is followed by a decrease in these enzymes in the blood, while thyroid feeding increases them as well as the antitrypsin ( Juschtschenko) .^® The physiological activity of thyroid preparations, according to nearly all investigators, is in direct proportion to the iodin content,-'' 19c Jour. Exp. Med., 1015 (22), 739. iMSee also Kahn, Arch. (jes. Physiol., 1016 (163), 384. 23 Arch. exp. Path. u. Pharm., 1910 (63). 263. 24 Jour. Amer. Med. Assoc, 1015 (64), 2042; Jour. Biol. Cheni., 1915 (20), 501. 2-ta Blum and Griitzner, Zeit. physio]. Cheni., 1914 (91), 400. 24b Rogoff and ^Marine, Jour. Pharmacol., 1910 (9), 57. 25 :Mit't. a. d. Grenzgeb. med. u. C'hir., 1905 (15), 209; Pellegrini, Arch. sci. med., 1915 (39), 276. 26Biochem. Zeit., 1910 (25), 49; Zeit. phvsiol. Chem., 1911 (75), 141. 27 Fonio, Mitt. Grenz. Med. u. Chir., 1911 (24), 123; Frey, ibid., 1914 (28), 596 CHEMICAL PATHOLOGY OF THE DUCTLESS GLAyOS which is the best of evidence that the formation of this compound is one of the chief functions of the gland, and that the iodin in the thy- roid is not merely stored there as an undesirable foreign substance like copper in the liver. The selective deposition of iodin in the thy- roid is remarkable, and when iodin is fed to animals it is stored very rapidlj' in the thyroid, bvit it seems to require several hours before the active growth-modifying hormone is formed.-^" INIarine and Lenhart -^ find that the normal human gland contains an average of 0.4 mg. of iodin per gram of fresh weight (2.17 mg. per gram of dry weight), be- ing less than that of domestic animals in the same part of the country. These figures agree closely with tliose obtained in thyroids from various parts of America by Wells--' (2.10 mg. per gram dry weight). They found, as Oswald and Kocher also have, that the amount of iodin varies directly with the amount of colloid, being decreased when cellular hy- perplasia is present, in direct proportion to the amount of hyper- plasia, and administration of iodin causes a reduction in the In'per- plasia and a return to the colloid type of gland, while the iodin is de- posited in the gland. Kocher, however, disputes the regularity of the variation of iodin and colloid content, stating that it is especially the concentrated follicle contents which hold the iodin. Seidell and Fen- ger ^° have found a marked seasonal variation in the thyroid iodin of animals, there being about three times as much between June and No- vember as between December and May.""'' In man it has been found that before birth the thyroid of the fetus contains little or no iodin, but in domestic animals the fetal glands contain not a little iodin (Fenger).^^ The cells of the gland contain very little iodin (A. Kocher). Extracts of the thyroid have little effect on the blood pres- sure, except for an alcohol-soluble fraction, poor in iodin, which is a depressor.^^^ Wasting diseases are associated with a considerable decrease in the size of the thyroid and the amount of colloid, and with this a decrease in the iodin ; especially is this true of tuberculosis.^- Patients or ani- mals to whom iodin compounds are administered deposit it in the thyroid in large amounts, especially if the gland is previously de- fective in iodin, and at times there results even an acute thyroiditis from the iodin administration.^^ Iodides are said to increase the 340; Hunt. Jour. Amer. Med. Assoc, 1907 (49), 1323; and Jour. Pharm. and oxp. Therap., 1910 (2), 15. 27a Marine and l^ogofi", Jour. Pliarm., 191G (9), 1. 28 Arch. Int. Med., 1909 (4), 440. 29 Jour. Amer. :Med. Assw., 1897 (29), 897. 30 .Tour. P.iol. Clieni., 1913 (13), r,]l. 30a \'alual)k' fifjures on tlic iodin content of foods are given bv Forhes ct at., Bullet. Oliio Agric. Exi)t. Stotion, June, 1910. No. 299. 31 Jour. Piol. Cliem., 1912 (11), 489; 1912 (12), .5;); 1913 (14), 397. 3ia Fawcett et al., Amer. .Four. Pliysiol., 1915 (.3(5), 113. 32 See Vitrev and Hiraud, Conijjt.'Rend. Soc. Biol., 1908 (65), 405. 33 See Mendel, .Med. Klinik, 19()(; (2), 833. 77//; l'Mr\TII )!,'() IDS 597 ainoiiiit of tliyi-('()rh., 190G. XI, Sujipl. s" An excellent review of tliis subject is given by Thom})son in "The Surgery and PathologT of the Thyroid and Parathyroid Giands." bv A. J. Ochsner and R. L. Thompson, St. Louis, 1910. See also MacCallum, Ergeb. inn. :\Ied., 1913 (11), 509. 37 Roussy and C'lunet, Compt. Rend. Soc. Biol. 38 See Edmunds, Jour. Path, and Pact., 1910 39 See ^MacCallum, Johns Hopkins Hosp. Pull., io fhid., 1907 (18), 331: also Cameron, J.mr. Biol. Clu-in.. 1914 (10), 465. 41 Amer. Jour. Med. Sci., 1908 (19), 135. 1910 (68), 818. 14). 288. 1907 (IS). 341. Biol. Cliein.. 1914 598 CHEMICAL PATHOLOGY OF THE DUCTLESS GLAXDS many investigators the acute manifestations of athyreosis, while the more chronic changes of myxedema are attributed to the loss of the thyroid. MacCallum's studies support this \dew, for he found the results of parathyroidectomy in dogs very different from the results of thyroidectomy. The most prominent symptoms are muscular twitchings, gradually passing into tetanic spasms, and due to nervous impulse rather than to muscular changes, since they did not appear in muscles from which the nerve-supply has been cut off. Trismus, protrusion of the eyes, and rapid respiration without cyanosis {i. e.. air hunger) were also observed, and death usually resulted from ex- haustion. Apparently these sj^mptoms are due to some toxic sub- stance which, accumulates on account of the absence of the parathy- roids, for it was found that simply diluting the dog's blood by with- drawing part of it, and injecting a corresponding amount of salt so- lution, caused a temporary cessation of the tetanic symptoms ; and injections of emulsions of parathyroid checked the sj'mptoms for some time, presumably through neutralizing the hypothetical poisons. Degenerative changes that were observed in the cerebral ganglion- cells also favor the view that some unneutralized toxin is responsible for the symptoms following parathyroidectomy, and W. F. Koch *^^ found that the urine of parathyroidectomized animals contains an abundance of toxic bases, especially methyl cyanamide. On the other hand, profound mental symptoms and insomnia have resulted from feeding parathyroid to man.*^'' The metabolism after parathyroid ectomii may show tlie following ohaiities : -12 There is a reduction in the assimilation limit for carbohydrates (Hirsch, Under- hill 42a and others). Concernino; inorganic metabolism there is disasjreement, for while ilacCalliim and Voegtlin *^ found an increased elimination of calciinn and a loss of tlie same element from the blood and brain (which they would make responsible for the increased nervous irritability), Cooke found no such loss of calciiun,^3a but she did find an increased urinary excretion of magnesiiun. Ac- cording to most observers, nitrogenous metabolism is altered as shown by the in- creased excretion of nitrogen, and especially of anunonia, which suggests tlie ex- istence of an acidosis. Greenwald ** found increased anunonia less conspicuous than increased undetermined nitrogen and sul])hur, and decreased ])hos])horus excretion. There may occur an increase in the bases of the blo;id (alkalosis) which disappears under the acidosis that results from tetany. ■♦^i> In view of the conflicting facts, the theory that the increased irritability and spasm of tetany result from hyi)ocalcification of tlie nerve tissue is at present unproved. Calcium does diminish nervous irrital)ility, as shown by J. Loeb, and hence when administered it may favorably intlueiice the symptoms of tetany para.tli\re()])riva., liut Ihis does not establish tlie tlieory. Tliat luunerous experi- menters have been able to stop tliese symptoms, both in man antl animals, by feeding of parathyroid,*5 or parathjroid nucleoprotein, establishes the relation- 4ia.rour. Uiol. Chem., 11)1.3 (1,5). 4.3; Jour. Lab. Clin. :\led., lOK] (1), 2!1!). 41b Morris, .lour. F.ab. Clin. Med., IDl.'i ( 1 ) , 2(i. ■J 2 Sec review Ijv Cooke, .\mer. dour. :Med. Sci.. 1010 (UOi, 404. 42a .Tour. Biol. Cliem., 1014 (IS), 87. 43 Jour. K\p. Med., 1009 (11), 118; 101.3 (IS). CIS. 43a See also IJergeim, Stewart and Hawk, Jour. Kxp. :Med., 1014 (20), 225. 44Amer. Jour. I'liysiol., 1011 (2S), 10.3; Jour. Biol. Chem., 1013 (14), 3()3. 44a Wilson, Stearns and 'i'hurlow, .lour. Biol. Chem.. I!)!') (23). SO, 123. 45 See Ilalsted, Amer. .lour. ISied Sei., 1007 n34), 1. cnHuisTJi'Y OF (!1. "2 Reported by Bassoe, Trans. Cliicufio I'atli. Soe., 1!)()3 (;1), 2'M. "•■''Metabolism in exopiitlialniic j^oiter, see Du ]?ois, Areli. Int. Mi'd., l!'l(i (17), *»l;i: llalverson, l?er-,reini and Hawk, ihid., 1010 (IS), 800. T.jii Sufrar niilizat ion is decreased, as sliown by study of tlie utiii/ation of suffar {liven intravenously (\^'ilder and Sansuni, Areh. Int. 'Nted., 1017 (10), 311). T4See Gliedini, Wien." klin. Woeli., 1011 (-24), 73lJ: Hunt and Seidell, .lour. Pharm. and Exp. Tlier., 1010 (2), 1.5. 7-«aCoet8eh, Bull. Johns ITojjkins Tlosp., 101(1 (27), 120. ■!^ Dent. Zeit. Chir.. 1008 (01), .302. j:\<)i'iiTii\i.\iic (:<)iTi:i{ OOf) ill ('.\()|)litlialiiii(' ^'•oitcr, Mai'ine contends that it has hccii pn-ccdcd by a hyi)('i'j)histi(' stajiO."'' Based on tlie tlieory that the iioi-mal function of tlie thyroid is the detoxieation of metabolic products, is the sernni treatment advocated first by Ballet and Kni-i(iu('/., and later by Lanz, and Burghart and ]-}lnnienthal;'' On tlic ])rinei})le that after thyroidectomy tiie l)lood should contain an acennudation of those substances, which the thyroid normally neutralizes, they injected the serum of thyroidectomized goats into patients with exophthalmic goiter, in the hope that these ac- cumulated substances might in turn neutralize any excessive thyroid secretion. Favorable results were obtained, and it was subsequently found that the milk of thyroidectomized goats possesses the same qual- ities, and may be administered by mouth ; this has led to ([uite exten- sive clinical use of this method of treatment, which has failed to show any regidar beneficial effects in the hands of most careful observers."-'' Of similar significance are the favorable effects obtained by Beebe''* and Rogers '" with a serum made by immunization of animals with the mu'leoproteins of the thyroid, which have not been corroborated by others. Oswald *'* found that the thyroid in exophthalmic goiter contains generally a smaller proportion of iodin than normal glands, but with the total amount approximately normal. However, the findings are very inconstant, corresponding with the fact that in some cases of exophthalmic goiter the amount of colloid is abundant (in which case the amount of iodin may be large), while usually the amount of colloid is small, and its highly vacuolated condition in hardened sections suggests that it is of unusually fluid consistency. A. Kocher *^ found that either the amount of iodin is small, which is usual, or else very high, but it is seldom the same as in normal thyroids ; the more dense the colloid in the follicles the higher iodin content he observed; the phosphorus content is both relatively and absolutely increased. ^la- rine has found that in exophthalmic goiter as well as in other conditions the amount of iodin is in direct proportion to the colloid and inverse to the hyperplasia. E. V. Smith ^^'' obtained in simple hyperplastic glands an average of 0.54 mg. of iodin per gram dry weight, as com- pared with 1.52 mg. in hyperi)lastic glands showing retrogressive changes with more densely staining colloid. P^onio found that, as with normal thyroids, the physiological effect of exophthalmic goiter glands 70 See also Wilson. Amer. Jour. Med. Sci., lilOS (136). .S.jI. TTDeut. med. Woch., 1899 (25), 627. Also Mobiua, Miiiicli. mo.l. Woi-li.. I'.tOl (48), 185.3; v. Levden, :\Ied. Klinik. 1904 (1), 1; Kuloiihor'r. I'.crl. kiiii. Woch.. 1905 (42), 3. "a See Sonne, Zeit. klin. Mod.. 1914 (?0), 229. 78 Jour. Amer. ^Med. Assoc, 1906 (4()), 484; 1900 (47). 6.)5. -^ Ihid., 1900 (40), 487; 1006 (47), 001. soVirchow's Areli., 1902 (109), 475. siVirchow's Ardi., 1912 (208). 80. 81a Jour. Amer. Med. Assoc., 1914 i02). 113. 606 CIIKMJCAJ, PATflOLOaV OF THE DVCTLEfi^ GLAyOS varies directly with the proijortioii of iodin, and such glands take up iodin administered tlierajjeutically just as a normal thyroid does (Koeher,**- i\Iarine and Leiiliart).^'^ These results, therefore, indicate n()thiii<>- either for or a-oiter is due to autointoxication with the secretion of the thyroid, but Wilson and Kendall ^^^ find that in the toxic type of goiters there is but Vio-Vi r. as much of the active iodin compound of Kendall as in normal glands, and hence they suggest that in thyroid intoxication this toxic material has been discharged from the thyroid into the circulation. On the other hand, it is impossible to produce a symptom-complex resembling exophthalmic goiter ®*^ in animals by excessive feeding of thyroid,^* either normal or from exophthalmic goiter; and after ex- tensive study of the subject Marine and Lenhart have come to the con- clusion that "the essential physiological disturbance of the thyroid in exophthalmic goiter is insufficiency, its reaction compensatory and its significance symptomatic." This view, however, certainly fails to agree with the excellent results which come from partial extirpation of the thyroid in exophthalmic goiter. Oswald,-^ also an experienced investigator in this field, invokes an abnormally irritable nervous system, which stimulates the thyroid and in turn is stimulated by the thyroid secretion, constituting a vicious circle. Other observers are of the opinion that not an excessive, but a perverted, secretion is at fault,^** a view not confirmed by tests of the effects of thyroid extracts on animals.^^ However, it is stated by Blackford and Sanford,*^ that extracts of the thyroid in this disease, as well as the blood of patients in the acute toxic stages, exhibit a marked depressor effect on blood pressure, which is distinct from that of choline, and which they believe to be specific for exophthalmic goiter. There can be no doubt that the thyroid secretion is capable of caus- ing serious intoxication, for patients who have overused thyroid prep- arations in the treatment of obesity, skin diseases, etc., have often suf- fered severely from the symptoms mentioned previously, and, in at least one such case, a diagnosis of exophthalmic goiter was made be- fore the cause of the disturbance was detected. Not infre(|uently evidences of acute intoxication have followed immediately after opera- tions upon the thyroid, and these have been considered as due to intoxication with the large quantities of thyroid secretion that have R2Arcli. klin. Cliir.. 1010 (1)2). 442; 1!)11 (90), 403. 83Arfli. Int. Mi'd., 1!»11 (S), 205. !^3a Anier. Jour. Mod. Sci., 1010 (151), 70. '<4aT]if> patlio<" See Sclir.nltorn, Arch. exp. I'atli. u. riiarni.. 1000 i(;o), 300. f'WJour. Anier. Med. Assoc., 1014 (02), 117. EXoi'iiriiM.Mic (;<)iii:i{ 607 escaped from llic <:l;iii(l (lui-iiiii- the njx'i-at i\r iii;iiii|Mil;it ion. Tlic fact tliat (i))ibli/(jpi(i, r('S('iiil)liii;oit('r ])aralh'ls tlic nitro^icn (Towles. Amer. Jour. Med. Sci., 1!)10 (140), 100). "•'■'Literature '■• See Kolmer, lMlfi->er"s .\rcli., ]!H2 (144), .'Itil. 97 See (JIvnii, Quart, .lour. Med., litl'i I.")), 157; .lump ct al.. Amer. .lour. Med. Sci., 1014 (147), 5(iS. f»7a It does not liave a marked ell'ect on tlie development of tadpoles, lience dif- ferinj,' from thyroid and tiiymus ( (Judernatseli ) . 'J"i> See also Crowe and \\"ish)cki. Bull, .lohns Hopkins llosp., 1!)14 ('25). 287. "7c See Fulk and MacLeod (Amer. .liMir. i'iiysiol., lilHl (40), 21) wlio found that the active j)rinci])le of otlier cliromallin tissues lias tlie same physiok)fricaI efTect as tliat of the adrenal medulla. THE ADh'i:\.\f.S \\l> \ltn/sn\-s DISEASE 609 Iji-iiiciplc. lilt' cpiiu'phi'in, and by containing a very large proportion of lil)()i(ls. Thus, in water-free human adrenals (cortex and medulla botli iiu'ludcd) there was found 36.;^ per cent, of ether-soluble material, of wliich 20. () per cent, was cholesterol and 33 per cent, was let-ithin.'-"* The proportion of fats and lipoids varies greatly during changes of age, disease, and perhaps of function, and there are those who believe the adrenal cortex to be a chief source of the lipoids of the blood, to which much imi)ortant function is ascribed in the reactions of immu- nity. (See Lipoids, under Fatty ^[ctamorpliosis.) When cholesterol is fed in large amounts some is deposited in the adrenal cortex,"'-' while in nuiny diseases, notably delirium tremens ( Ilirsch ) ,"'-"' the lipoid content of the adrenals is greatly decreased. In renal and arterial disease the adrenal lii)oids have been found increased.''"*^ The lipins of the adrenal cortex are said to contain little or no neutral fat,^"*" but free fatty acids which may be increased when the cholesterol decreases. Loss of body fats is not accompanied by a loss of adrenal lipoids ordinarily, although they decrease in acute infections, especially pneu- monia.'""^ A vaso-depressor effect is produced by extracts of adrenal cortex, probably caused by choline which has been found in such extracts. The medulla is characterized, besides, by its pigmentary content, by the remarkably active internal secretion, epinephrin,^ which it always contains in greater or less amount. Presumably epinephrin, of which the formula is HO /^"^CIIOH — CH, (XH) — CH3 HO is derived from the aromatic radical of the proteins, its close relation- ship to tyrosine being seen when the formula of the latter is com- pared ho/ \cH„ — CH (XH,) — COOH That epinephrin is formed from tyrosine directly, is, however, not yet demonstrated. There are also other amines and aromatic com- pounds which might be formed in the body, that have a pressor effect, and which perhaps are formed, although not yet identified.- It is to 98 Wells, Jour. INIed. Res., 1!)08 (17), 461. 99Krylov, Beitr. patli. Anat., Iftl4 (.icS), 434. »9a Jour. Amer. :\led. AssOc, ini4 (63), 2186. 99bChauffard, Compt. ?>end. Sof. Biol.. 1914 (76), 529. 99c FJorberg. Skand. Areli. Plivsiol., 191") (32), 2S7. 99d Elliott, Quart. Jour. Mcd.^ 1914 (S). 47. 1 Tins name, given by Abel and Crawford, is to be preferred to the others in common use, especially the niost-iised term "adrenalin," wliich has been copy- riplited bv a manufacturinif establishment so that this name means specifically tiieir product, and not the active principle of tlie adrenal from whatever source. 2 See Barjrcr and Dale, Jour. Plivsiol.. 1910 (41), 19. 39 610 CHEMICAL PATHOLOGY OF THE DUCTLESS GLAXDS be borne in mind that the formation of ei)inep]irin is not limited to tbe adrenals, but that otlier islands of chromaffin sympathetic tissue can do the same,^ which explains the observed discrepancies between the anatomic chantres in the adrenals and the clinical manifestations of a deficiency in epinephrin. Accordino- to Goldzieher ^ the normal human adrenals contain to- gether about 4 mg. epinephrin, which ma}' be increased in conditions with high blood pressure, such as arteriosclerosis and nephritis, in which he found an average of 5.8 mg. ; and in septic conditions with low pressure he found it reduced to an average of 1.5 mg.*^ The human adrenal contains no epinephrin before birth,"' but Fenger ^ found it present in the adrenal of unborn domestic animals. Autolysis of the adrenal decreases the amount,' but not all of the epinephrin is destroyed even several days after death, as shown by Ingier and Schmorl,^ who, using both morphological and chemical methods, also found a gradual increase in the epinephrin content of normal glands from birth to the ninth year, after which it remains practically con- stant at about 4.5 mg. (males 4.4, females 4.71 mg.). They also found a slight increase in arteriosclerosis, more in acute and chronic nephritis, and a decrease in diabetes and narcosis, there being practically no epinephrin in the adrenal of Addison's disease. In most of the infec- tious diseases they found no changes, and in amyloid infiltration the amount was about normal. The amount of chromaffin substance and epinephrin do not always run parallel, although Borberg " found a close parallelism ; this author also failed to observe any marked de- crease of chromaffin substance in narcosis. Elliott ""'' found a low epinephrin content in acute-infectious diseases, and especially low in acute cardiac failure associated with great mental distress: he did not find any increase in the epinephrin in nephritis or in any otlier dis- ease. The function of the epinephrin is manifestly to modify the tone of the non-striated muscle fibers which are under control of the sympa- thetic nervous system, acting upon some receptive substance present in the muscle, perhaps at the nerve endings. But it is a fact of much practical importance that administration of epinephrin will not com- pensate successfully for the loss of the adrenals, whether because the gland secretes other things, or because the intermittent artificial ad- ministration of the epinephrin will not compensate for the regulated 3 See Vincent, Proc. Ro.y. Soc., B, 1908 (82), 502. 4Wion. klin. Wooh., 1010 (23), SOn. •1" See also Keicli and Bcresne-j^owski, T?eitr. klin. Chir.. 1!)14 (91), 40.'^. Oliiui (Verb. .Japan. Palli. riescll., lOKJ (('.). l.'j) found tlie normal content lo ho ahout 5.0 mg., averai,nn\' Cushiiifr. "Tlio Piiiiii:u\ Podv and ils Disorders,"' IMiiladclpliia, 1012 :' also .\sclni.T. Ptliii;or"s .Vrcli.'. 1012 (]4(i), 1. 32 Wells, Jour. Biol, flicin.. 1010 (7). 2.-)0. 3-tT>(>\vis. :\lillor and IMatllunvs. Arch. Tnt. IMod.. 1011 (7), 7S.-) : llerriujr. Quart. Jour. Kxp. Phvsiol., 1014 (S). 24") and 207. 30 Sec also Pull. .lolms Hopkins' IIosp., 1013 (24). 40. Till-: in I'oi'in s/s .i\/> ACh'oMjyi.iiA' &15 inaclivity ami undL'rdevL'lopment, .subiKjnnal body temperature ami increased carbohj'drate tolerance.^'^ These manifestations correspond to those observed in certain Innnan conditions (Froehlicli's syndrome) associated witli defects in tlie hyi)opliysis. Removal of the posterior lobe does not produce any characteristic and constant effects, altliongh marked polyuria and erotism have resulted. The anterior lobe fed to 3^oun^ rats has a stimulating effect on growth, and especially on sexual development and activity, while posterior lobe feeding has a retarding influence (Goetsch^^). Robertson describes a modification of growtli in mice fed anterior lobe substance, which he attributes to a specific substance, tethelin, containing phosphorus and probably an iminazolyl group, and hence related to the active constituent of the posterior lobe, although it has no pressor effect. ^^ Puncture of the hypophysis produces the same effect as puncture of Bernard's diabetic center in the fourth ventricle,^^ and stimulation of the glalid has a similar effect, presumably because of the secretion of a glycogenolytic agent. A diminution of posterior lobe secretion occurring in certain conditions of hypopituitarism leads to an acquired high tolerance for sugars, with the resultant accumulation of fat. In hibernating animals, also, the adiposity and lowered temperature are associated with hypoplasia of the anterior lobe of the hypophysis, ac- cording to the same author. There also seems to be some relation be- tween the hypophj'sis and urinary secretion, for extracts of the posterior lobe cause marked polyuria, and in some instances of "dia- betes insipidus," lesions have been found in the hypophysis. Sim- monds ^^ holds that the pars intermedia is responsible. Like the thyroid, the hypophysis enlarges during pregnancy.^" Feeding of hypophysis is said to increase both gaseous and nitrogenous metab- olism, and in a case of hypopituitarism the urine has been found to contain a high proportion of undetermined nitrogen and of neutral sulphur."'^ Varying results have been obtained in studies on the basal metabolism of hypopituitarism.^^'' Acromegaly. — The accumulating evidence seems to have practically proved that acromegaly depends upon a hyperfunctionating of the anterior lobe tissue of the hypophysis, one of the most imjxn-tant facts being the improvement which has followed removal of the hyper- plastic tissues in several cases successfully operated. Although there are many cases of tumor of the hypophysis without acromegaly, this is of no significance since it is not to be expected that all tumors will 3T Conoernino: motaholism after hvpophvscctoniv see Benedict and Ilonians, Jour. Med. Pvcs., 1012 (-2.")). 40<). 33,Tohns Hopkins Hospital Bulletin, 1016 (27), 20. 35, Jour. Biol. ChenK. 1!»16 (24). 400. 38Amer. .Tour. PliYsioI., 1013 (.31), xiii. 39Mnneh. med. Woeli.. 1013 (60), 127. 40 See Krdheini and Stumnie. Ziegler's Beitr., 1000 (40). 1. 41 Stetten and Rosenbloom, Proc. Soe. exp. Biol, and ^Med.. 1013 (10), 100. 41a Means, Jour. Med. Res., 101.5 (32), 121. 616 CHEMICAL PATHOLOGY OF THE DLCTLE^^ GLAyOfi carry on tlie functions of the tissue in which they arise. Acromegaly without hypoi)hyseal changes is rare, especially if we consider the liner cytological evidence of cellular activity/- So far, little of chem- ical interest has been learned concerning this disease. The metabo- lism studies generally indicate a retention of nitrogen, phosphorus and calcium, because of the overgrowth of bone and soft tissues.^--' Ac- cording to some observers this retention is decreased, or changed to an excess elimination, by administration of hypophyseal substance. ^^ The elimination of endogenous uric acid is said to be greatlj^ increased in acromegaly, and decreased in eases with hypofunction of the gland. ^* A considerable excretion of creatine was 'observed by Ellis.-'-^ Glycosuria and actual diabetes is frequently present in acromegaly (40 per cent, of the cases collected by Borchardt),*" presumably from interference with the regulating function of the hypophysis, but this assumption has been questioned because of the fact that lesions in this location might also produce glycosuria by affecting the "diabetic center." However, since puncture of the hypophysis causes glyco- suria, while injection of posterior lobe extract produces glycosuria dependent upon hyperglycemia (Gushing), and in view of the fact brought out by Borchardt that in cases of tumor of the hy})ophysis without acromegaly, glycosuria has never been observed, there is much probability that in many if not all of the cases of glycosuria with acromegaly, it is the liypophysis itself that is concerned, and that both the acromegaly and the glycosuria are caused by hyperactivity of the gland. In later stages of acromegaly there may develop a hypoactivity because of pressure upon the posterior lobe or infundibular stalk, whereupon the sugar disappears and is replaced by an increased toler- ance for sugar.^^ THYMUS-': AND OTHER DUCTLESS GLANDS From the clicrnical stand|)()iiit little of iiiterost is known coneerning this orpan. Tt is frequently used as a souree of nucleic acids, in which it is rich, but there is no study of its clieniical clianges tliat is of interest in i>atholoufy. l-Alirpation of the thymus in youiii;' animals is followed by marked defects in tlie (le\('loi)inent of the skeleton, and changes in tlie development of the sex organs.-''" Some au- thors state that thymus extirpation causes a loss of calcium, and tlial a calciinn 42 See Lewis, 'Bull. Johns Hopkins' TTosp.. 1005 (16), InT. 42;i See Hergeim, Stewart and lii'wk. Jour. Exp. ^led., ini4 (20). 21S. 4^ See Kidiinraiit, Dissert., Zuricl', (Jebr. l.ceman, 1012: Mediureccanu and Kristeller, .Tour. Biol, ("hem., 101 1 ( !) ) , 100. ■"Falta and Nowaczynski, Berl. klin. Woch.. 1012 (40), ITSl. ■'•"'Jour. Amer. Med. Assoc. 1011 (5(>), 1S70. ••« Zeit. klin. Med., lOOS (fifi), X\2. ••7 Full discussion in .Johns Hopkins TIosp. Bull., 1011 (22). M\'->: 101:? (24), 40. 2" In addition to Biedl's "Innere Sekretion." see Wiesel, Krgebnisse Plnsiol., 1011 (XV (,) ), 41(>: Klose and Vogt, Beitr. z. klin. Chir., lOlO ((10), 1; Matti, .Mitt. (Jrenz. .Med. u. Chir.. 1012 (24), H. 4-5. 2"ia Not corroborated iiv Taiipcnlieimer (.Tour. Kx]). Med.. 1014 (10). :nO; (20), 477) or by Nordmann, (Ardi. klin. Ciiir.. 1014 (KH!), 172). TUYMVH AM) o'lHEIi DUCTLESS GLAyDS 617 rotentioii results fidiii feeding' tliviiius, Iml the ri'sulls (|iintc(l ari' not at all in liar- mony. It is certain, liowovcr, tiiat (h'lici(>n<-y in tlie tliynuis causes a defect in ossification. Also llierc occurs a period of adiposity, followed hy cacliexia witli liyperplasia of tiic lynijihatic tissues, thyroid, pancreas, ovaries and testicles ( Klose and \'o','t ) . These authors attrilnite the defects in ossification to aii acidosis, which liypothesis is, liowever. far from established. ( ludernatscli 27 found that the fecdinut Gomez 30 obtained only lowering of blood pressure fi-om extracts of this gland, and bilateral removal causes no characteristic effects.soa 2T Arch. Entwicklgs., ini2 (35), 457: Amer. Jour. Anat., 1914 (15), 431. 27a Arch. ges. Physiol., 1915 (162), 99. 27b Literature given by Basch, Zeit. exp. Path., 1913 (12), 180. 270 See review by Halsted, Bull. Johns Hopkins Hosp., 1914 (25), 223. 28 Jordan and Eyster, Amer. Jour. Physiol., 1911 (29), 115: Dixon and Halli- burton, Quart. Jour. Exper. Phvsiol., 1909 (2), 283. Dana and Berkelev. Med Record, 1913 (S3), Xo. 19. 29 See Pappenheimer, Virchow's Arch., 1910 (200), 122. 29a Jour. Exp. :Med., 1915 (22), 237. 29b Jour. Amer. Med. Assoc, 1914 (63), 232 and 517. 29c Concerning the composition of the pineal gland see Fenger, Jour. Amer. ^led Assoc. 1916 (67), 1836. 30 Amer. Jour. IVIed. Sci., Julv, 1908. 30a Massaglia, Frankf. Zeit. Path., 1916 (18), 333. CHAPTER XXI URIC-ACID METABOLISM AND GOUT ' These subjects have been the o])jeet of such a prodigious amount of research that it is far beyond the seope of tliis work to review the histor}' and the details of the investigations. Such a review is also particularly unnecessary, since it can be found in the works on phys- iological chemistry and various treatises on metabolism. Conse- quently the attempt will be made in this chapter merely to give, as brietiy as possible, the views now most generally accepted concerning the nature and metabolism of uric acid, and its relation to patho- logical processes. For the historical discussion, indicating by what devious steps we have reached our present understanding concerning this long-disputed subject, the reader is referred to the articles men- tioned below, upon which I have drawn freely. A particularly clear summary of the subject is given by Walter Jones in his monograph on nucleic acids.^ THE CHEMISTRY OF TTRIC ACID It is the very great service of Emil Fischer to have shown us the structure of the uric-acid molecule, the empirical formula of which, C3H4N4O3, had long been known. He demonstrated that it is a mem- ber of a group of substances, which are all characterized by being built up about a certain nucleus, C-N^. As the simplest member of the group is a synthetically formed body, purine, the nucleus is called the "purine nucleus." The structural relations of the better-known "purine todies" to this purine nucleus and to each other is clearly shown by their structural formula?, as given below : The atoms in the "purine nucleus" are arranged as follows: N(i)— C(6) I I C(2)— C(5)— N(t) N(3)-C(4)-N(9) To each atom has been given a inimber, as shown, for the purpose of facilitating reference to the location of various atoms and groups that are attached to this nucleus. The structure of i)uriiu' itself is as shown on the following page : - 1 CompU'te rpviows arp irivcn l)y 1'. TI. .McCniddcii, "rric At'id," Xcw ^Orlc, lOOn; Wionor, Krpol)nissf dor Plivsiol.," 1002 (1), fj;");") ; ibid., l!t().S (2), :?77; Burian and Sdmr, Pfliifrcr's Arcli., ino'o (SO), 241; 1001 ( S7 ) . 2:5!); Sc'liiltonludni. Ilaiidh. d. Tliocliciii.. 1010, IV (,), 4S0; Bru'-srh and Scliittcnholm, "Die NukleinstolTwoHisel und seine St.r)run<,'('n," .Jena, 1010; Waller -lones, "Niudeie Acids," Monograplis on JJioclieniistrv, 1014. An excellent sinnniai\- of i-eccnt woik is i;iven bv Benedict, Jour. l.ab. Clin. Med., lOlG (2), 1. -In these formuhe the symbols of llie atoms foiining tiie purine nucleus are in heavy type. 618 THE CHEMISTRY OF URIC ACID 619 N=CH I I HC C-NH II II X CH N-C-N Purine The derivatives of purine are described by stating to which atom of tlie purine nucleus the combining groups are attached. Thus, adenine is referred to as 6-aniino-purine, and therefore has the follow- ing formula : N=C-NH2 I I HC C— NH II II r^" N-C-N Adenine (6-amino-purine) Other important members of this group of "purine bodies,*' (also called xunthine bodies, alloxuric bodies, and nuclein bodies) are built up about the purine nucleus as shown below : HN-C=0 HN-C=0 II I I H2N.C C-NH 0=C C-NH II II >^» i II >^"- N-C-N HN-C-N Guanine Xanthine (2-aniinoUoxypurine) (2, 6-dioxypurine) HN-C=0 HN-C=0 II II HC C-NH 0=C C-NH II II ^^" i II /*^=^- N-C-N HN-C-i^H Hvpoxanthine Uric acid (6-oxypurine) (2-6-8trioxypurine) H3C-N-C=0 H>J=C=0 I I .CHa I I CH3 o=c c-n/ o=c C-N/ ! II ^cH ! II >^" H3C-N-C-N H3C-N-C-N Caffeine Theobromine (1-3-7 trimethyl-2-6 dioxypurine) (3-7-dimethyl, 2-ti di.ixypurine) As shown by their structural formula?, the purimidinrs present in the nucleic acids are also closely related to the ])urines, viz: N — CH X — C — NH= HN — C = 0 HX — Cc=.0 HO CH 0 = C CH 0 = C CH 0<=.C C — CH» N = CH HN^ — CH TIN- — CH HN — CH Pvrimidine Cvtosine Uracil Thymine (2-oxy. 6-animo- (2-6-dioxy- (5-methyl. 2-6-dioxy- pyrimidine) pyrimidine) pyrimidine). 620 CRIC.WID METABOLISM AXD GOUT Properties of Uric Acid. — Uric acid, when pure, is white, and crystallizes in rhombic tablets. Its solubility is very slifi'ht ; at room temperature (18°) it dissolves but about one part to 40,000 of water, so that a saturated solution contains but 0.0253 gram to the liter. It is much more soluble in blood-serum, dissolving in 1000 parts,^ ])robably held in some complex combination. His and Paul have shown that in a saturated solution oidy 9.5 per cent, of the molecules are dissociated, tlie dissociation occurring in two steps; the Urst and chief dissociation is into H and C.,H..5N^0.j, which then undergoes fur- ther dissociation into H and Cr.HoN^O.,, the latter dissociation being very slight. If any other acid is present in the solution, its dissocia- tion and liberation of free hydrogen ions interferes with the dissocia- tion of the uric acid, and as the undissociated uric acid is extremely in- soluble, the amount dissolved in an acid solution is much less than in a neutral solution.^'' Gudzent * found that saturated solutions of urates gradually precipitate out the salts because of a transformation of part of the uric acid into what he believes to be a I-actim form. (The lactim form is shown in the following formula, as compared with the isomeric lactam form shown above, in which uric acid is supposed to exist ordinarily.) N = C — OH HO — ( fi- ll ■NH \ r- // — OH ] kiiis, .lour, liiol. Clieiii., I'tlO (26), 205. «Z('it. pliysiol. Chcin., I'KI!) ((iO), .38. •'■' .Xs <'i. matter of fact, l)olli salts ^ive a sli- H^PO, + C,,H ,0, - C,H,N,0 guanylic acid phospho-nuclease guanosine and this enzyme is therefore designated as jjhospho-nuclcasc. Another enzyme, purine nuclease, splits off, instead, the purine radi- cal, thus: HjPO, - C,H,03 - r,H,N,0 + 11,0 y ILPO, - C,HA. + C.HiN^O guanylic acid i)uriiie-nuclease guanine Following either of these cleavages, the enzymes which deaminize l)ui-ijies ])egin to act, and we have formed as a result either the free oxypurines or the oxy purines still boimd in the glucoside-like combi- nation with sugar. If the purines are free the reaction will be: ■:> Jdiu's and ITcad (Jour, l^iol. Clicni., 1!)17 (2!>), 11]) liavo advanced evidence to indicate* that tlie liid.\ OF I inc Ar/n 023 C\.II,N,0 + ILO >-C',n,X,(),, + XII3 guanine ;;uaua!!K xantliiiiu or, hi case the guanine glueoside is present: C5HA-C,H4N„0 + H,0 >- CJIA - C,H3N A + ^ih suanosinc guanosinc-deaniinasc xanthosine lu the latter case a hydrolytic euzynie, xaiithosine-hydrolase, then splits off the xanthine, so that by either route the end result is the same. By a similar series of changes the adenine radical is converted into hypoxanthine, either directly by adenase: C5H3N, + ILO >-Cjr,N,0 + XH3 adenine aden;:se h\ poxanthine or by adenosine-deaminase the hypoxanthine-glucoside (inosine) is formed, and later the hypoxanthine is split off. We now have hypoxanthine and xanthine, which, in the presence of oxygen, are oxidized to form uric acid, thus : QH^N.O + O ^ >^C,H,N,Oo hypoxanthine hypoxanthine-oxidase xanthine CH^NA + O y C-,H,N,03 xanthine xanthine-oxidase uric acid Further oxidation of the uric acid causes its conversion into the much more soluble allantoin, thus : C5H,N,03 + 0 + H,0— >-C,HeN,03 + CO^ uric acid uricase allantoin It is thus evident that the steps of the disintegration of nucleic acid are numerous, but that each separate process is a simple one ; and also, that it has been possible to follow out and distinguish the several steps and to establish the fact that each step depends on a distinct and specific enzyme. Not every tissue possesses all the enzymes of purine destiniction, and in different species of animals the distribution of the enzymes is different. For example, the enzyme xanthine-oxidase, which oxidizes xanthine into uric acid, is found in man only in the liver, and also in other animals it is of limited distribution, being found usually only in the liver or in the liver and kidney, but in the dog it seems to be present in several tissues. The deaminizing en- zymes, adenase and guanase, are much more widely distributed, but by no means universally. Adenase, for example, is not present in the tissues of the rat, and not in the tissues of adult human beings.^ Guanase is absent from the spleen and liver of the pig and from human spleen, although present in most other tissues. Uricase, the enzyme which destroys uric acid, also has peculiarities of distribution, being seldom found in any other tissue than the liver or kidney, and being absent entireh- from the tissues of man, and from the birds and 8 There liave l)cen some rejiorts indicatiiisj tlie presence of adenase in fetal liuman tissues (Long, Jour. Biol. Chem., 101.3 (la), 440). 624 URIC-ACID METABOIJ^M A\D GOUT reptiles so far examined. The significance of this distribution of iiri- case will be discussed at greater length a little later. The following graphic expression of the series of steps leading to the formation of uric acid has been presented by Amberg and Jones:'' 0--P'-Q CsHjOj Cs HjN, (NHj) ^u.n.n Uric add /OH C,HN-OH oxidcie Xanthine ^ adenosine [anrhlne ypm, C.HjN^O H Another possible source of uric acid is through synthesis. In birds, which eliminate most of their nitrogen in the form of uric acid, syn- thesis of uric acid undoubtedly occurs. It must also be considered that young mammals can synthesize the purines necessary for their growth from foods which contain no purines.^*' It would seem pos- sible, therefore, for synthesis of uric acid to occur in adult mammals, but as yet satisfactory experimental evidence is lacking that such synthesis does occur, although an apparently reversed reaction, whereby uric acid destroyed by liver tissue can be resj^nthesized by the same tissue acting upon it in the absence of oxygen, has been de- scribed by Ascoli and Izar.^^ Their work has not been repeated suc- cessfully by others. I have failed in several attempts to secure re- synthesis of uric acid by dog livers, and Spiers/^'' who made a more extensive investigation, was unable to corroborate their findings. It should also be mentioned that not all of the purine bases of the body is bound in the form of nucleic acid. A considerable amount is present in a free condition, or at least not bound in luicleic acid, espe- cially in muscle tissue. Uric acid can be fonned as well from the free purine bases as from purine bases liberated from nucleic acid — indeed, evidence has been brought forward indicating that a large proportion of the uric acid arising during metabolism (endogenous) comes from the free hypoxanthine of the muscles. As to the place where uric acid is formed, it seems probable that in different animals different organs arc chiefly coiu'erned. for it has oZeit. plivsiol. Chvm., 1011 (7.S), 407. lOMK'olluin, .Amcr. .lour. Plivsiol.. 100!) (2r>). 120. 11 Sf'c Zeit. physiol. Clicin.. 1010 (05), 78. iiaBiochem. .lour., l!)l.'i (0), -.VM. BEST ix' I cm )\ (>r uuic acid 625 been found tliat tlie (listrihution of the en/ymes mentioned above varies jjreatly in tlie various organs and tissues of different species/'- In most animals tiie xanthine oxidase, wliieli forms uric acid from xan- thine, is localized chiefly or solely in the liver, and this is the case in man ; therefore it is presumable that uric acid is formed chiefly in the liver from jnirines hy the steps described above. That there may be otlier metliods of foniiiiiu' uric acid is possible. DESTRUCTION OF URIC ACID i '• With most mammals but little of the total amount of purine bases taken as food or set free in the tissues, appears in the urine as uric acid, most of it being converted into allantoin, which seems to be ex- ereted witli little or no loss. Thus, when dogs, pigs or rabbits are fed nucleic acid, about 93 to 95 per cent, can be recovered as allantoin, 3 to 6 per cent, as uric acid, and 1 to 2 per cent, as purine bases (Schittenhelm). It would seem that practically all the purines can be found in these three forms combined, the proportions varying in different species. In man alone, except for the chimpanzee ^* and orang-utan, does a considerable proportion escape as uric acid, a fact in complete harmony with repeated observation that the tissues of man have no power whatever to destroy uric acid in vitro; the earlier reports of positive uricolysis undoubtedly being erroneous. Even the monkey has active uricolytic enzymes in its liver, and therefore excretes its purines chiefly as allantoin. With mammals as a whole, therefore, uric acid is destroyed to the extent of being converted into allantoin,^*'' the close relationship of which to uric acid is shown by the structural formula: NH — PH — NH o = i^l— i = 0 I I I NH — CO — NH= (allantoin) With most mammals the oxidation of uric acid takes place chiefly in the liver, but in some of the herbivora the kidneys are more active, as far as experiments in vitro can show. Whether man can destroy uric acid at all has been a matter of dis- pute. It has been shown by Wiechowski and others that uric acid injected subcutaneouslj^ is excreted almost quantitatively and un- changed in the urine. To be sure, human urine does contain a very little allantoin. 7 to 14 mg. per day, but this amount is too small to be of much significance, for it is possibly all derived from the food, as 12 A compilation of this distribution is given hv Wells, Jour. PJiol. Clieni., 1010 (7), 171. 13 See discussion by Wells. Jour. Lab. Clin. "Med., inio (1). 104. 1* Wiechowski, Pras-er nied. Woch.. 1012 (37), 27o : Wells and Caldwell. .Tour. Biol. Chem., 1014 (18), 157. 14a See Hunter and Civens, .Jour. Biol. Chem., 1914 (IS), 403. 40 626 VBICACID METAHOLISM A\D aOT'T 1lie liuman organism cannot desti-ny allaiitoiii.^^'' On tlu' other hand, it lias been found repeatedly that nneleie aeid or purines given by mouth are by no moans (|uantitatively excreted in the urine, even when uric acid, allantoin and purine bases are added together. Ap- parently a considerable proportion of the purine nitrogen fed, about half in most experiments, is excreted as urea.^*'= As allantoin seems not to be at all disintegrated in the human body it would seem prob- able that if purines are destroyed, as these experiments indicate, they pass through some other route than allantoin, and possibly that part of the purines which is destroyed does not pass through the stage of uric acid. Experiments show that outside the body uric acid can be destroyed by other routes than through allantoin; thus, it can be dis- integrated into glycocoll, ammonia and CO2 ; or by another method of destruction it yields first alloxan (C4H2N2O4), then parabanic acid (CjHoNjO.t) which in turn yields oxalic acid and urea. There is no evidence, however, that any of these alternative routes is ever fol- lowed in the aiiimal body. It is possible that the failure to find all the purines of the food as uric acid in the urine depends on their par- tial destruction in the intestine by bacteria.""^ It is highly probable, in view of all available evidence, that in man most of the purine ab- sorbed from the food, and practically all the purine from cell metabo- ■ lism, is converted into uric acid and excreted as such. THE OCCTJRRENCE OF URIC ACID IN THE BLOOD, TISSUES. AND URINE As can be seen from the foregoing discussion, the amount of uric acid that appears in the urine depends upon a number of factors, which may be enumerated as follows: (1) The amount of purine bodies taken in the food, upon which, chiefly, depends the amount of exogenous uric acid. (2) The amount of destruction of tissue nucleo- proteins. (3) The amount of purine bases formed in the muscle tissue. (4) The amount of conversion of purine bases into the uric acid. (5) The amount of destruction of uric acid, if any, occurring in the body. (6) Possibly upon the capacity of the tissues to syn- thesize uric acid ; and in case such power to synthesize uric acid exists, uDon the presence of the precursors of uric acid in the body. (7) The retention of uric acid in the blood and tissues. fS'i The power of the kidneys to excrete uric acid. If we also take into account the fact that the solubility of uric acid in the urine depends chiefly upon the amount of neutral phosphates present in the urine, and also upon the temperature, reaction, and 14b See Ac-krovd. T.iodiciti. .Tour., 1011 (.T), 217, 400, 442. Hf" Soe Taylor. Jour, r.i.il. Clioni.. 101.3 (14), 410; Hivons and TTuiitor. ihld.. lOlf) (2.3), 200. A1)ont nno-toTilli as miieli uri(" acid is ovcrclcd in tlic swoat as in tlio urine, sweat containinfr 0.1 niur. Biol. Cliom., lOlfi (20), ."^lO. IT Zcit. physiol. Cliem., 1909 (63), 4.55. GOUT G29 deficiency in the cliniinatoi-y jjowers (jf the kidneys. There seems to be no particular relation between the amount of uric acid in the blood and the occurrence or severity of attacks.'"'' This uric acid is, ac- cording: to the best evidence, in a free state, and not combined, as was at one time urged by several students of gout. In the intervals be- tween tlie attacks of acute gout the elimination of uric acid remains within the normal limits; however, for a period of one to three days before each acute attack the amount of uric acid is usually decreased considerably. With the onset of the attack the amount of uric acid excreted becomes increased, and for a few days remains above the average, then subsides to about the normal. Of these two features, the increased output of uric acid during the attack seems to be more constant than the reduced output preceding it, but cases occur in which the uric acid excretion shows no variation from that of normal persons. In certain cases of rheumatoid arthritis the behavior of the purine metabolism resembles that of gout.^* As yet we have no definite information either as to the cause of this behavior of the uric acid during the paroxysms of acute gout, or as to its part in causing the paroxj^sm. However, in view of the fact that monosodium urate is found in the joints during the attacks, it seems most probable that for some as yet unknown reason there occurs a precipitation or anchoring of the urates in the tissues, which is associated with the attacks of pain and swelling. We do not know, however, that it is the deposition of urates that causes the attacks. Indeed, the fact that uric-acid retention precedes the attack, rather than accompanies it. seems to suggest that it is the absorption of the urate rather than its deposition in the joints that is responsible for the local disturbances. It is also possible that during the period of re- tention the uric acid is held in the blood in some form that cannot be eliminated by the kidney, and that its deposition in the joints in an absorbable form occurs simultaneously with the attack. The fail- ure of recent studies- on the enzymatic transformation of purines to locate anywhere in the human body an enzyme destroying uric acid, makes hazardous the attempt to explain gouty metabolism as a result of enzymatic abnormalities. However, there can be little doubt that the fundamental reason for the existence of uric acid gout in man lies in the inability of the human organism to destroy uric acid. Because man cannot destroy uric acid rapidly by oxidation, as can all other mammals, he is always a potential victim of uric acid retention and deposition. It should be mentioned in addition that it is not the uric-acid metabolism alone that is altered in gout. Irregular periods of nitro- iTa Pratt, Amer. Jour. Med. Sei., 1916 (151), 92; Bass and Herzherg. Deut. Arch. klin. :\red., 1916 (110). 482. 18 W. J. Mallory. .Tour. Path, and Bact.. 1910 (15), 207; Ljungdahl, Zeit. klin. Med., 1914 (49), 177. 630 URIC-ACID METABOLISM AyD (JOIT gen retention and nitrogen loss are quite eonstant features. The cause of this variability, and the form in which the nitrogen is re- tained, are quite unknown, although there is some evidence that the retained nitrogen is in the form of purine bodies (Vogt). Most of the excessive loss occurs during the acute attacks,^'' and the retention of nitrogen between attacks may be partly to repair the loss; against this, however, is the fact that there is not sufficient gain in weight to account for all of the nitrogen retention. Associated M'ith the de- layed excretion of ingested purines is also a delayed excretion of the other nitrogenous products of i)rotein food.-" The proportion of purine bases to uric acid is not altered in gouty urine. -^ The state- ments in regard to phosphoric acid elimination, which depends largely on decomposition of nucleins, are contradictory, but it seems probable that it shows no characteristic alterations in gout. Amino acids, espe- cially glycocoll, are said to be excreted in excess.-^^ It may be seen from the foregoing discussion that we neither under- stand fully the intricacies of metabolism in gout, nor know whether uric acid is responsible for either the acute painful attacks or for the anatomical alterations in the kidneys, heart, and bloodvessels. Indeed, Daniels and McCrudden -^^ have shown that it is possible for gouty patients to have a persistently low content of uric acid in the blood, below the average normal quantity, and to have typical acute attacks without change in either the uric acid content of the blood or its excretion ; attacks were even observed to occur when the blood uric acid was at a subnormal figure from administration of atophan, which increases its elimination. Furthermore, Bass and Herzberg -^"^ found that uric acid can be injected into the blood of gouty subjects until the blood contains as much as 10 mg. per 100 c.c. without causing any joint symptoms. It is very possible that some entirely difiPerent product of metabo- lism than uric acid is responsible for most of the changes and symp- toms of gout -- — indeed, this would seem to be the case were it not for the great frequency of the deposition of monosodium urate in the joints and cartilages, both during the acute attacks and in chronic gout. This indicates that there is surely something abnormal in the conditions of uric-acid solution and circulation. Why the urate is precipitated in these definite places is another of the many unsolved problems of gout. The local nature of the deposition indicates that it must depend upon local changes; but the hypothesis that there occur in r{ruf,'scli, Zcit. cxp. Patli. u. Tlicr., 1000 (2), filO. ^" I.ovcno and Kristeller, .Tour. Kxp. Mod., 1012 (Ki), 'MVA. ai llcfrtor, DiMit. Arcl). klin. Mod., 1!>1;{ (109), :522. 2i;i IJiirtrcr and Scliwcririor, Arcli. oxp. Tatli.. lOi:! (74). :?5;?. 21b Arcli. Int. Med., 1!)].') (l.'-i), 1040. •■:i\v's Arch., lOOo (179). 359. 2V Almajria (Hofnioistor's Rcitr.. 1905 (7), 466) has found that joint cartilage ]>laee Arncr. Jour. Phvsiol., 1904 (10), 4r)2 ; 1907 (20), 4:?9. ■' 2fiH .Tour. Anier. :\Ied. Assoc. lOlH ((JO), 20.')1. • 20b See also Denis, Jonr. Hiol. Chein.. 191.5 (2.'?). 147. LA'/f ACJD JMWh'VTS 633 URIC-ACID INFARCTS ■'■" Uric-aeitl ini'arL-ts, as the deposits of urates and urie acid observed in the kidneys of at least half of all children dying within the first two weeks of life are called, ^ive evidence of tlie slijihtiiess of the toxic effects of these substances upon the tissues. Usually little or no change occurs in the renal tubules as a result of these depositions, except such as can be attributed to their mechanical effect,^^ but they may serve as the starting point of calculi. The reason for the for- mation of these infarcts is not at all understood. Spiegelberg ^- found it possible to cause them experimentally in young dogs, in which they do not occur naturally, by injection of 0.25 gram of uric acid per kilo. He was unable to explain wh}^ this deposition should occur in young animals but not in old, for he could not find evidence of lessened oxidative power on the part of young animals, and the solvent power of infants' urine was found equal to or greater than that of adults. Other authors, however, have found a lower oxidative power in young animals, and ^Mendel and Mitchell ^^ have found that in the embryo pig uricolytic enzymes do not appear until just at or just after the time of birth. As human tissues have no demonstrable power to oxidize uric acid, however, these animal experiments cannot be applied to the uric acid infarcts in human infants. Possibly the uric-acid infarcts of infants are the result of the great destruction of nucleoproteins that results from the change of the nucleated fetal red corpuscles to the non-nucleated adult form. Flensburg believes that a hyaline substance is secreted in the urine of new-born infants which acts as a matrix for urate deposition. jMcCrudden considers the high concentration of infants' urine an important factor. ^Minkowski ^^ observed that administration of adenine to dogs led to a deposition of uric acid or some similar substance in the kidneys. Schittenhelra ^^ found the same deposits in the kidneys of rabbits fed adenine, but not w^hen they w^re fed guanine. According to Nicolaier.^" the crystals thus deposited are not uric acid or urates, but 6-amino-2-8-dioxy- purine, derived from the adenine (6-amino-purine) by direct but in- complete oxidation. He could not find this substance in either hu- man urine or in a uric-acid calculus. Eckert ^'""■* obtained urate de- 30 See discussion hy Wells and Corper, .Tour. Biol. Cliein., 1000 ((>). .')21. 31 1 have obsej'ved a case of fatal hematuria veon-atonnti. associated with most extensive hemorrha:en, etc. The fol- lowing may serve to suggest other factors. It mifiht 1)0 ooiu'oivcd tliat llu' cell coTilaiiicd nioleciUcs of a trhicolytic catalyst or enzyme similar in its effects to metallic hydroxides, that jjlucose molecules as fast as they entered the cells would come into collision with catalyst molecules, perhaps combining witli tliem, and that as a result of the encounter the glucose molecules would he dissociated into unsaturated frajjments or ions. From the mo- ment of imion or dissociation they would cease to behave as glucose molecules. Tlie unsaturated fragments might suhs('(|ueiitly sutler various fates, depending ujion tlie cliaracter and quantities of various substances in tlie cell. Thus, some might combine with oxygen to yield, finally, carbon dioxide and water. Others might combine with each other to form polymers like glycogen, others again undergo reduction to fat or molecular rearrangement to give lactic acid. The relative quantities imdergoing those several changes, would depend upon the relative quantities of H and OH ions, of available oxygen, salts, etc., foimd in the various phases of the cell. This conception is based on that used by Nef to ex- plain the behavior of sugars in alkaline solutions. For a concrete conception of the dvnamics of a reaction between an organic substrate and catalyst the reader is referred to Van Slyke's study of the enzyme urease.s The general principles outlined above may be illustrated by experi- ments with timed intravenous injections of glucose. It has long been known that if a comjiaratively large dose of glucose is injected rapidly into a peripheral vein a marked glycosuria usually results. Pavy, however, emphasized the fact that a material fraction of a dose so given fails to be excreted and appears to be utilized. Doyon and Du Fourt demonstrated that with a standard dose of gluco.se the per- centages excreted and utilized respectively are influenced by the time consumed in injection, the slower rates of injection causing lower per- centage excretions and vice versa. Blumenthal chose a standard in- jection time of about 10 seconds and varied the weight of sugar given in that time. He found that a certain dose of glucose might be injected into the ear vein of a rabbit without causing any glycosuria at all. However, the maximum dose which could be so injected once could not be repeated 15 minutes later without causing glycosuria. He assumed from this that the first dose "sattirated" the tissues and that fifteen minutes later the utilization of sugar had only resulted in a partial desaturation. He, therefore, determined the dose of glucose which might be injected repeatedly at 15 minute intervals for as long as 3 hours without ever causing glycosuria. His figures varied be- tween 0.6 and 1.3 gm. per kg. of body weight per hour. This he termed the "utilization limit," whereas the largest dose which could be given within 10 seconds once without causing glycosuria he called the "saturation limit." The latter he placed at 0.8 gm. per kg. but 7 Jour. Biol. Chem., 101 fi ^27), 490. 8 Jour. Biol. Chem., 1014 (10), 141. 640 DIABETES E. ]\r. Wilder has been unable to confirm this observation. Woodyatt, Sansum and Wilder ^ made continued intravenous injections of glu- cose at uniform rates by means of a motor driven pump for 2 to 17 hours with the following- findings : If chemically pure glucose in aqueous solution is injected con- tinuously into the peripheral venous blood of a normal resting man, dog or rabbit at the rate of 0.8 gm. per kg. of body weight per hour, or at any slower rate, the injection may be sustained in most cases, hour after hour for 7 hours and probably longer without producing any glycosuria in the usual sense of the word. If the rate is advanced to 0.9 gm. of glucose per kg. of body weight per hour, while all other conditions remain the same, the injection may be sustained for a short time without causing glycosuria, but in nearly all cases abnormal quantities of glucose beg^in to appear in the urine after 5 to 30 minutes of injection, the time depending upon the previous degree of satura- tion. Once established,- the glycosuria then tends to proceed at a uniform rate as long as the rate of injection and other conditions re- main fixed. However, if the injection rate is again reduced to 0.8 gm. per kg., glycosuria promptly ceases. Thus during the continuance of an injection at the latter (0.8 gm.) rate there can be no continued accumulation of unchanged glucose in the body, but the rate of injec- tion is equalled hy the rate of utilization. It is important to note that it makes no appreciable difference whether one uses an 18 or a 72 per cent, glucose solution for injection. The tolerance limit for glucose may be demonstrated at the same point regardless of wide variation in the quantity of water administered with the glucose, even 1 hough the blood volume and the blood sugar percentages may be influenced by variation of the water supply. Also, if glucose is in- jected continuously and uniformly at a rate productive of some glycosuria, the glucose excretion may proceed at a constant rate in spite of marked variation in the water supply during successive hours. A certain dog receiving by vein 20 gm. of glucose per 10 kg. per hour for 8 hours, excreted every hour close to 0.42 gm. of sugar per 10 kg. of body weight. Yet during the experiment water was injected at varying rates into the same vein with the glucose, so that the hourly volume of urine varied between 6 c.c. and 128 c.c. and the percentages of sugar in the ui'ine varied*betvveen 0.35 and 4.9. This em])hasizes the fundamental importance of the rate at which sugar is supplied to the organism in determining the occurrence or non-occurrence of glycosuria and in fixing the rate of excretion when the latter occurs. In view of the above generalities several specific mechanisms sug- gest themselves by which glycosuria might be produced : (1) An increased supply of preformed glucose to the whole organ- ism from without (alimentary glycosuria). f Jour. Amor. M(>d. Assoc, 101.') ((>")). 2007 (preliminary report); Woodyatt, ITarvev Sociptv Loftnros, IDIO; Wilder and Sansnm, Arch. Int. Med.. 1017 (10), 311; Woodyatt and Sansiun, Jour. Biol. Ciiem., 1017 (30), 155. 77//; iii.(K)h srcM! 641 (2) A (Iccrcascd utilization in tlie orgjaiiism as a whole (j)aiicreatic diabetes). (8) All iiit-reased supply to tlie kidneys resultinj^ from the libera- tion into the blood of sugar previously stored or combined in other orp-ans. Thus, the rapid hydrolysis of glycogen following ])un('ture of the floor of the fourth ventricle and analogous nerve stiimilations, and occurring in the acid, asphyxial, narcotic, thyroid, epinephrine, and hypophysis glycosurias. In an analogous manner lactose may enter the circulating blood from the mammary gland, and pentose from unknown sources. (4) An increased supply to the kidneys due to decrea,sed utilization in other organs. The breaking down of glycogen mentioned in (3) might be so interpreted. (5) Decreased utilization in the kidney itself. (6) Increased physical penetrability of the kidney membrane to glucose. Both (5) and (6) are hypothetical conditions, the latter having been proposed as the basis of so called kidney diabetes, a state in which glycosuria occurs with a normal or subnormal percent age of sugar in the blood and in which the rate of sugar excretion is, in comparison with other forms of glycosuria, little influenced by the diet.-"^ THE BLOOD SUGAR * The normal blood sugar concentration is found to average 0.10 per cent., but, as statistics show, it may vary at least between 0.06 and 0.11 per cent. The literature contains numerous references to that blood sugar concentration which if exceeded leads to glycosuria ("threshhold" value). In accox'dance with the general principles above discussed we should expect this value to vary. It has been placed at 0.147 to 0.164 per cent, by Foster, between 0.17 and 0.18 per cent, by Hainan and Hirschman, at about 0.20 per cent, by Pavy, and other writers have reported greater variations, due in part doubt- less to differences in the analytical methods used. How widely the threshhold blood sugar percentage may be varied by extreme variations of the blood volume and other factors has not been settled. Following the ingestion of free glucose the blood sugar percentage ordinarily rises, and in a similar way, but more slowly, after feedings of starch. Fisher and "Wishart gave 50 gm. of glucose in 150 c.c. of water by stomach to dogs weighing 8 to 0 kg. and found in the first hour blood sugar percentages of 0.16 and 0.13. In succeeding hours there was little variation from 0.11 per cent. In harmony Avith the previous work of Gilbert and Baudoin and the more recent studies of others on man. these experiments showed that the l)lood sugar percentage rises during the first hour, then falls and thereafter remains iKU-mal. There was no increase of the blood volume during the first hour, the hemo- 9a Cf. Epstein, A. A. ^rosenthal. 41 642 DIA BETES globiu pereeiitajio remaiiiinp' iiiiehaiigod, jn-obably because the large quantity of ghieose in the bowel held water there. But in the second hour the blood volume became large and the hemoglobin showed the effects of dilution. In this same hour the sugar percentage returned to normal. But the absorption of glucose was only completed in the fourth lumr and calorinietric observations by Lusk showed that the metabolism also ran at a uniform rate 20 per cent, above the basal level into the fourth hour. Accordingly the observed blood sugar per- centages first rose as tlie rate of sugar supply was increased, but fell again during the )i\ainte)iance of the increased siipphj and ichih the metabolism was constant, owing to the shifting of water. When concentrated (54 to 72 per cent.) glucose solutions are in- jected continuously into the blood at rates of 0.4 to 0.8 gm. per kg. per hour, there is at first a steep rise of the blood sugar percentage, followed by a fall coincident with an increased hydremia, after whicli a new equilibrium is established and the blood sugar percentage may become constant at a "normal" level exactly as in the above. By injecting glucose at the same rates in sufficiently dilute solutions this initial rise may be very much reduced and the ])lood sugar percentage established in later hours may even be lower than that observed before injection began. On the other hand, if glucose is injected at rates above 0.9 gm. per kg. per hour, glycosuria begins, and if the rate of injection is rapid enough may be made intense. As glucose passes through the kidney membrane, water tends to accumulate with the glucose on the urinary side of the membrane (increased diuresis, polyuria ). In the same way that glucose in the bowel lumen may tend to withhold water from the blood, so a sufficient quantity of glucose in the urinary tubules may manifest the same tendency in this local- ity. Whether the glucose in the urinary tubules will have the effect of concentrating the blood or vice versa will depend on the cpiantitative distribution of free sugar between tliese two fluids, and the ((uantity of water available for distribution between the blood sugar and the urinary sugar. During continuous intravenous injections of glucose at rates from 2.7 gm. per kg. per hour upward. 80 to 40 per cent, of the glucose injected may be excreted and there is a strong tendency toward dehydration of the whole body. This may be neutralized b\" su])plying water with the sugar as fast as it flows away in the urine, provided the rate of injection is not so great that tlie necessary traffic in water overtaxes the cardio-i-enal mechanism. By employing these high rates of injections and maintaining the water balance at as low levels as c(mipatil)le with life and recovery, it is possible to produce and maintain for houi's blood sugar concenti'ations as higli as 2.;?8 ]K'r cent. Joslin obsei-ved 1.40 ])er cent, of sugai' in llic blood of a fatal case of diabetes with nephi'itis. This is ])rol)ably the highest on record. Tlie blood sugai- of diabetics passing sugar in the ni'ine is as 77//; N7M77-; OF Till: srcM! i\ Tin: r.i.ooh 643 a rule hiiilicr tliaii iiDniial. l)iit not iicct'ssafily so, iiiucli (l('])eM(liii per cent. THE STATE OF THE SUGAR IN THE BLOOD It has k)n<>- been believed that tlie siif lactic acid which burned in the presence of oxyars of the formula CoH,„0„. The behavior of the hexoses when dissolved in very dilute alkali makes it convenient to consider tliem in four natural series of oifrht members each. Thus one series comprises tlic S hexoses whose structural formuUie ai)pear below. This may be called tlie d-filucose series. (1) (2) (3) (4) (5) (6) (7) (8) CHO CHO CHiOH CHO CHO CH.OH CH^OH CHoOH H-i-OH HO-C-H CO H-C-OH HO-C-H CO H-COH HO-C-H I I I I I I I I HO-C-H HO-C-H HO-C-H H-C-OH H-C-OH H-C-OH CO CO I I I I I I ! I H-6-0H H-C-OH H-C-OH H-C-OH H-C-OH H-C-OH H-C-OH H-C-OH II I I I I I I n-(^'-OH H-r-OH H-COH H-C-OH H-C-OH H-C-OH H-C-OH H-C-OH r r 1 I I I I CH,OH Cn.OH CH.OH CH,OH Cn,OH CH.OH CH.OH CHsOH d-i)seudo dglucase d-mannose d-fructose d-allose d-latose fructose ad-ghitose /3-d ghitose Tliere is also an l-<:lucose series in which the members are the mirror imajres of the above. There is a tliird series coniprisins/:s G,')! ciplos coininoii to all. l-Aiun'malion of tlic S toiiimhu' sIkiws tlial iiiiinhLTs 1 2, 4 and .> have aldfliydc fjroiijjs ( 11 — C^ O) at tlic end of the eliaiii and arc licncc aldolii'xoscs. Mcndici's :? and t> have kctom- (C^f)) >rroii,))s, at tlic second car- bon atom, and are therefore 2-keto-liexoses, while numbers 7 and S havinjj ketone jjroups at the third carbon atom are .'5-keto-hcxoses. Since each series of S sugars has a like nnmber of the diircrcnt types there are in all Iti aldoln-xoscs. ci^lit 2-keto- and ciiiht ;i-keto-liexoses. Jt had lonjr been known that if a solution of any optically active su^'ar. such as d-jihicose. was alkalini/.ed the solution i('lMHi(lcianco xcluding the bowel as a (M)iiti-il)iit('f to the d(M're;ised tolerance. It is unlikely also lliat 1li(' ki(hieys in pliosplioi'izcd animals wei'c iHMulci'ed abnoniially ))t'nneal)h' for galactose, since when the kidneys alone are ))liosi)liorized without affeeting the liver, the excretion of galactose after adminis- tration l)y mouth oi- into a xcin lias been retarded rather than ha.stened. These princii)les ha\c iK'couie ineorjioi-ated in a clinical test for dis- ease of the lie|»;itie | )a I'eneliv ma. When galactose is administered to a /. /; 17 /.0,S7v ( Fh'CCTOSE ) 655 fully (liahctic art in "assimilating" levulose as with other sugars. Spontaneons Alimeniarj) LcvuJosuria, i. c, the appearance of lev- ulose in the urine from such small quantities of levulose as occur naturally in the food, has been demonstrated in eight cases. In five of these levulose apjiears to have been the only sugar present. These persons showed a decreased tolerance for ingested levulose and ceased passing the sugar when the diet was cai-bohydrate-free. The tend- 31 Bauer, Dent. nied. Wocli.. IftdS (.3.)), 150."): Roiss u. .Tohn, Dcut. Arcli. f. klin. Med., 1012 (108), 1S7: Roiibitscheck, Dcut. Areli. f. klin. Mod., 1012 (108), 225; Naiuiyn, "Bcitriifio 7,ur Lchre von Ikterus, etc.", Reichcrt-Duboisschcs Archiv fiir Anatomie, 18G0, p. 570; Schupffer, Arch. f. exp. Path. u. Pharm, 1873 (1), 73. 656 DIABETES ency of tli()Uut if the exposure to cold is long and intense enough a time conies when it ceases to have this ett'ect, and if epinephrine is given subcutaneously in the dosage of about 0.4 mg. per kg. of body weight once ever}- three hours there is for a tinie a heavy increase of the glucose output, but this becomes less and less until after 6 or 8 doses the ratio becomes constant again, regardless of whether epincj)hrine is given or not. In such dogs neither cold nor narcosis nor other toxic effects will increase the output of glucose, and analyses of the liver and muscles reveal no glycogen. In a long series of dogs so treated Sansum and the writer have not encountered ratios above 3.2 to 1, and the 2.8 ratio recurs frequently. Since the glycogen is gone and the dog is fasting, the sugar which continues to appear in the urine must have its origin in body fat or protein, or both. Sugar from Fat. — If such a dog be given large cpiantities of fat in the diet no change occurs in the G : N ratio, nor any increase in the glycosuria, except such as may be ascribed to the glycerol of the fat (Lusk). On the other hand, propionic acid, according to Ringer, may cause a rise in the sugar excretion and a corresponding rise in the G : N ratio.*^ From this it is concluded that the fats of the food do not as a rule form sugar in the body, although sugar formation from at least one lower fatty acid is possible in view of Ringer's experiment. Von Noorden and Falta and their associates have regarded sugar formation from fat as a regular normal ]>henomenon, because in dia- betes melitus they believe that high ratios occur wliich make this view necessary. Sugar from Protein. — If instead of fat, ]irotein be given to the dog above mentioned, there occurs an absolute rise in the sugar of the uriiu^ and a corres])onding rise in the nitrogen, hut the G:X ratio remains constant. Following a meat feeding there may be fluc- tuations of the ratio during short periods, but this statement generally holds if the time of observation is 12 to 24 hours. These facts have led to the conclusion that when in a fasting, fully phlorhizinized ani- mal, or one fed on meat and fat alone, a constant (r : X ratio of 3.65 : 1 is seen ; this means that the glucose and the nitrogen are coming from one and the same source, viz., protein. A cram of uitrotren corresponds *^ The doo whose exhaustive monograph reviews the literature to 1910, concludes tliat the internal function of the pancreas is not monopolized by islet cells. Bensley si developed intra-vital staining methods which, for tlie first time, made possible the sure differentiation of islet cells from duct or acinus epithelium witliout reference to form or arrangement, and appears to have proved tliat these cells are regenerated from duct epithelivun. lie also showed the great normal variations in size and number of islets in different individuals (guinea pigs). His study explains certain of the discrepancies which occur in tlie litera- ture, es])ecially in tlie estimation of the quantity of islet tissue in pancreatic rests, grafts, etc. Recently Allen 52 has reported that when proper sized fragments of pancreas, in connecticm with the ducts, are left in situ, and the remainder of the gland is removed, the subsequent development of severe diabetes may be coincident with disappearance of islet tissue while acinus cells and ducts are unaffected. This operation, according to Allen, is eminently satisfactory for producing ex- perimental diabetes without infection and without loss of the external secretions. The Nature of the Internal Secretion of the Pancreas. — Direct evi- dence on this subject is lacking. Such a secretion has never been isolated. Even the experiments made with tlie feeding of fresh pan- creas and with extracts of the gland have led to no definite advance. R<'ports of iniiirovement following the administration of any sub- stance in dialx'tes are worthless unless accompanied by proof of the constancy of the diet, of th(> amount of work performed, and of other factors wliich are known to influence the course of diabetes. Some 48 "Diseases of the Pancreas,"' LipjiincoH >!c Co., 1910. 40 See llomans, .Tour. MvA. Kes.. 1014 (.30), 49. noErgeb. der Plivsiol., 1910 (10), 1. •'.lAm. Jour, of Anat., 1911 (12), 297. 52 Gl.vcosuria and Diabetes, Bostojj, 1913. /'.I \r/,'/;.iN hiMii:Ti:s .i\/> m \iu:ti:s \ii:i.iTUi^ 667 jrlinimcr of success ajjpcarcd to liavc altcndcd the iiilravcuous use of an extract made 1»\ Zurl/.ci-. ■ ■ although delclci'ioiis hy-cffects occurred, and the apparent ini])rovenient could have been due wliolly to reten- tion. According to lledon and Drennan, amelioration of tiio severity of pancreas diabetes as evidenced by a dimimition of glycosuria has followed the transfusion of blood from a healthy animal or the injec- tion of fresh detibrinated blood, and Forschbach. working with a ])arabiosis (or two animals so joined by oi)erative means that pei-ma- nent intermingling of their blood occurs) performed pancreatectomy ill one of the animals without producing diabetes in either; from which it might seem that the internal secretion was carried by the blood. Ill harmony with these results were the investigations of Knowlton and Starling,''* who found that an isolated beating heart taken from a depancreatized animal (cat) was capable of removing less sugar from the blood used as a perfusion medium than are hearts of normal animals, but these latter experiments have not been confirmed and are subject to criticism. Tn most of the transfusion experiments re- ported the standardization of the metabolism prior to giving the fresh blood has not been such as to make the results certain. Carlson and Drennan found that pancreatectomy in a pregnant animal near term might fail to cause diabetes, but that diabetes developed at once fol- lowing delivery. This could be explained on the basis that an in- ternal secretion passed from fetus to mother, or that sugar failing of utilization in the mother was utilized by the fetuses. Kramer and JNTurlin failed to note any increase of the respiratory quotient in de- pancreatized dogs following blood transfusion, and Sansum and Woodyatt saw no improvement following transfusion in a human case."" Symptoms. — Tn the absence of extracts which contain the active ]iriiici]ile in measurable fpiantity, the attention must be turned to a more detailed study of the effects which follow its lack. Now it is well known that in diabetes melitus there are all grades of severity. WTiai follows has reference onily to the severest cases — those ivhich maif hr called "complete diahetes." In the severest cases of dia- betes, glycosuria ])ersists even when the individual subsists on a fat- protein diet, and after the glycogen in the body has been reduced to a mere trace. When this stage has been reached, and provided no carbohydrate food is eaten, it is found that tlie total glucose in the urine bears from day to day a constant ratio to the total nitrogen in ihe urine as.already described for })lilorhizin dialietes. This "G :N" ratio" is not always the same. Tn depancreatized dogs nourished solely on fat and protein, it is often found, as Minkowski first recog- nized, at 2.8 :1, and in human diabetes the same value for G : N is •-••iZoit. f. oxp. Pali).. innS-n (5), .107. 54 .Tom-, of Physiol., 1913 (45), 140. 55 Jour. Amer. Med. Assoc, 1914 (62), 006 for lit. references. 668 DIABETES sometimes seen. But, as in phlorhizinized dogs, higher ratios may occur in the human disease. If to such a case of diabetes as this we give by mouth 40 grams of glucose there may appear in the urine close to 40 grams of extra sugar. Plainly such extra sugar has escaped utilization of any kind. It cannot have been oxidized or converted into fat, since these proc- esses are irreversible, although it might have existed momentarily in the body as glycogen or other isomer of glucose. What phase in the utilization of this glucose is primaril}' disturbed is another question. To say that 40 grams of ingested glucose causes the appearance of 40 grams of extra sugar in the urine does not prove that the diabetic body is inherently incapable of using any sugar or every carbohydrate. It might still be capable of using a two, three, or four carbon atom sugar, some other member of the group of 32 hexoses, or, as some have it (von Noorden), sugar which has first been built up into glycogen, etc., provided these substances could be kept from undergoing trans- formations into the non-utilizable glucose. As a rule, however, when other sugars are fed to complete diabetics, they are transformed into glucose and appear as such in the urine. This phenomenon has much of significance for the general theory of sugar metabolism and is an indication of the nature of the primary disturbance in diabetes, as will now be shown. Theory of Diabetes. — What sort of a chemical process is involved when levulose, for example, is converted in the body into glucose? As already stated in the chemical introduction, the reciprocal trans- formations of hexoses one into another in the alkaline solution in liiro depend upon a preliminary ionization of the sugars followed by salt formation, the salts then undergoing dissociation which, according to ^lathews and IMichaelis, is still purely electrolytic with rearrange- ments of the anion ; but which, according to Nef, is a non-electrolytic dissociation of the type which he calls methylene dissociation. Some form of dissociation must he a prelude also to these transformations in the hody. This view is logically just as necessary as it has been found to be for the organic chemist, and, it may be added, that for the oxida- tion of sugars as well as for their polymerization a preliminary dissoci- ation is essential. Now since the diabetic body can transpose other sugars into glucose, it must be able at least to dissociate the former sugars deeply enough for this process. These trans])ositions are ac- complished chiefly in the portal system and perhaj^s in other places too, but certainly- levulose and man}' other substances can be made in the liver into glycogen, whose hj'drolysis then yields glucose. The degree or character of the dissociation necessarj'' for reciprocal transfonnations differs from that which is a necessary prelude to destructive reactions such as oxidation. A very weak alkali suffices in vitro for the former, while for the latter it is necessary to use a THEORY OF DIABETES 669 soinewliat stronger alkali t'oiiceiitratioii. '"■ The diabetit' body tlK-re- fore behaves as tliouj^h it were weakened witli respect to the alkali eou- ceiitratioiis which it cau bring to bear on sugars. As far back as 1871, Sehultzen suggested that the error in diabetes might be found in the disability of the body to dissociate the glucose juolecule into two 3-carbon substances.-" i^aunigarten "'^ also supported the idea of a "fermentative splitting" which precedes oxidation, be- cause he found a greater percentage utilization of certain substances closely allied to glucose (such as gluconic acid, saccharic acid, mucic acid, etc.), than of glucose itself; whereas gluconic acid and glucose, for example, differ only in that the sugar has an aldehyde group where the acid has carboxyl. Similar general ideas have been expressed from time to time by others. The present writer has urged in place of the vaguer terms, the adoption of chemical "dissociation" in the sense which is rapidh' finding favor in the field of pure organic chem- istry, notably for the explanation of the behavior of aldehydes, ketones and alcohols.'^'-' There can be no doubt that the dissociation of glucose in the body is a normal occurrence. This is directly and conclusively shown whenever muscles make lactic acid (C.jHqO.J out of glucose (CgHijOo), since in this process no chemical phenomenon is involved save cleavage of the hexose and intramolecular rearrangement. The polymerization of sugar into glycogen might be similarly interpreted. Direct proof of a failure of glucose dissociation in diabetes has not yet been brought, although its absence would explain all the metabolic phenomena more directly and simply than any other single physi- ologic error which has been hypothecated. It is, moreover, a tangible chemical conception, whereas to say that the body loses its power to oxidize sugar or to "fix" it as glycogen is merely to name effects in phj^siologic terms. (Cf. Naunyn's diszoamylie). It might be assumed that all sugars upon entering certain phases of the cells (phases especially well represented in liver cells), meet con- ditions which are equivalent to those met in a weakly alkaline solution, favoring reciprocal transformations, and, as A. P. Mathews points out, polymerization; but not conditions conducive to the deeper de- structive reactions. That is, especially in the liver, there may be the equivalent of dilute alkali for all sugars. Glucose, being the least dissociable, represents the form into which all other sugars tend to accumulate. But in the normal body a special glucolytic enzyme Calkali carrier or intensifier?) destroys glucose selectively. All other sugars must become glucose before destruction. In diabetes the enzyme necessary for the deep dissociation of glucose is lacking or in- 50 See Woodyatt, Jour. Biol. Chom., 1015 (20), 129. 57 Zeit. f. oxp. Path. u. Pliarm.. 1005 (2) . 53. 58 Glyceric aldehyde and glycerol, according to Sehultzen. 59 Cf. Xef, loc. cit., and Stieglitz, Qualitative Chemical Analysis. New York, 1912, I, pp. 289-292. 670 DfAnKTES active. Tlie recent studies of Murliii, Kramer,' Sweet ami Karver, show that alkali administration (NaoCO;,) may increase glucose utili- zation, especially when introduced into the duodenum where it may neutralize acid entering the bowel from the stomach and thus spare the liver and ])anereas from the effects of absorbed acid. Underbill 's experiments"" with bicarbonate feeding in diabetes confirm these ob- servations. One difference between diabetes melitus and phlorhizin diabetes is that in the former the glycosuria is due to hyperglj-cemia, the sugar loss being an overflow like water escaping from an overfilled tank; whereas in phlorhizin ])oisoning there is ajiparently an hypoglycemia — the loss resulting in this case, to carry out the simile, from a leak in the bottom of the tank which keeps the water at a lower level. But the results are the same. ^Moreover, if in diabetes melitus we could meas- ure only the chemically active or dissociated blood sugar, it is possible we should again find for this kind of sugar an hypoglycemia compara- ble to that of phlorhizin diabetes. This conception coincides with the doctrine that in diabetes melitus there is a primari) underconsnmp- iion of sugar as opposed to the idea of a primary overproduction. Overproduction vs. Underconsumption. — At the present time the chief ex])onents of overproduction are the followers of Kraus, and of von Noorden in whose books ''Die Zuckerkrankheit" and ''New Aspects of Diabetes" will be found the arguments favoring this idea. A translation of Minkowski's criticism of the latter has been made by Lusk."^ In this place it may be briefly recalled that the chief argu- ments favoring underconsumption in addition to what has already been said are the following: (1) The respiratory quotient in diabetes is freciuently found to be low, and when carbohydrate food is admin- istered this quotient rises but little, wdiereas in health the administra- tion of carbohydrate food results in a greater rise.''- (2) The acetone bodies (acetone, aceto-acetic acid and beta-hydroxybutyric acid) ap- pear in the urine when for any reason the quantity of sugar burning in the body falls below a certain minimum, as in starvation, or when a })erson accustomed to a mixed diet is suddenly switched to a full calory diet composed exclusively of fat, or of fat and carbohydrates, with the carbohydrate calories representing less than 10 per cent, and the fat calories more than f)0 per cent, of the total (Zeller "■''). In these cases the restoration of sugar to the diet abruptly and ])ernuinently stops the output of actone bodies. But in severe diabetes the excretion of acetone bodies is less affected by the 60 Jour. Amer. Med. Assoc, 1017 (OS), 407. «i IMpflieal Rword. Feb. 1, 1013.- >'- I'^ir tlie literalnre of res])irati(>n sdidies in dialtotes see Josliji. Troalment of Diabetes Melitus, New York, lOlfi; Du Bois, Harvey Society Lectures, 1916; and "Studies from tlie Department of niysioloic doses there may indeed Ix- a t(Mii])orary fall in the acidosis, but this is never perinaiiently attain- able. One interpretation made of these facts is as follows. In dia- betes there is an acetone body output because sujiar, althouuh brought to the cells, fails to take part in. certain cheniical i-eactions which nor- mally occur between sugars and certain of the breakdown i)roducts of butyric acid and which normally prevent the diabetic acidosis. Hence the bringing of more sugar has little effect. A)id iiere again one might suggest that in diabetes glucose fails to interact with the pi-od- ucts mentioned because tlie glucose is not sufficiently dissociated. Another intei-pretation has been to the effect that the sugar simply causes a compensatory decrease of the fat metabolism, i.e., spare fat, thereby decreasing the formation of the acidosis bodies. The mech- anism of the process is in any case still a theme for research. There are numerous other theories of diabetes, for the presentation of which the reader is referred to the larger works. Lepine has long stood for the view that the pancreas secretes a glycolytic oxidizing ferment. Xaunyn's theory pays particular regard to the ability of the body to "fix"' glycogen, while glycogen formation is held to be a necessary preliminary step in the utilization of sugar. The fail- ure to fix glycogen he calls " diszoamylie ," and the other metabolic disturbances he regards as sequences. The complex develo])meiit of this same general idea by von Noorden, with the added element of primary sugar overproduction, has already been alluded to. Pavy saw in the diabetic a failure to assimilate sugar; that is, a failure of the body to incorporate sugar in a colloidal combination which would at once permit of its transportation to the points of utilization, and prevent its prenmture excretion. The assimilation he held occurred in the villi of the intestines, and the lymphocytes he regarded as the morphologic elements which carry the sugar. Cohnheim's theory that the muscles formed glycolytic enzymes, for which the pancreas supplies an essential activator, is without any substantial experimen- tal support at the present writing. Allen proposed that the ])ancreas supplies an "amboceptor" which is essential for the proper colloidal blood sugar combination. Bronzed diabetes, the name given to that form of hemochromatosis in which, along with the hepatic cirrhosis, there is an associated fibrosis of the pancreas, and, as a result of this, the sym]itoms of pancreatic diabetes, will be found discussed under the heading "hemochromatosis," chapter xvi. Diabetic coma is discussed under "acid intoxication,"" chapter xviii. Lipemia, which is observed frequently and most severely in diabetes, is discussed in chapter xiv. THE END INDEX XoTE. — Tho minibcrs printed in bold-face type refer to pai;es upon wliich the topic is speeitically diseiissed. AiiDKiuiAr.UKX roaotioii, 1!»S, 204-207 specilic-ity of, 20(j witli veji'etable proteins, 206 Abriii, 144, 177, 2-i;5, 225, 293 poisoning', liistologie changes, 146 Abscess, 4:5."] cold tuberculous, 280 liver, 135 Absorption, 337 impaired, 246 in dead bodies. 338 partly due to lymph cliannels, 338 physical, 240 Acetanilid, 217. 482 Acetic, 248, 251, 567 Aceto-acetic acid, 550 poisoning, 553 Acetone, 550, 567 bodies, origin of, 554 toxicity of. 553 Acetonemia. 558 Acetonitrile, 592 test. 604 Acetonuria, 558, 560, 584 cachectic, 558 in fever, 560 Acid, 71 dves, sul])honic. 50 fastness. 111. 207 intoxication. 292. 534, 547-550, 584 non-di:il)ctic. 557-561 lactic. 647, 648 phloretinic, (i59 phospliate retention, 558 within cells. 548 Acidity, abnormal, 347 high, 456 increased, 348 of gastric juice, 245 of n\iclei, 46 relation of risror to. 391 Acidosis, 74, 412, 549-550, 614 as cause of death, 560 at high altitudes. 560 diabetic. 551, 71. 292 estimation of. 549 general. 531 local. 409 of ]iregnancy. 559 relation to diabetic coma, 552 Acquired tolerance. 246 Acrolein test, 475 Acromegaly, 614-616 Actinosphaerium, 377 Acute yellow atrophy of liver. 379, 403, '538, 539 557, 569 blood in, 546 Addison's disease. 4(i7. 472, 60S 613-614 unstriated muscle in, 613 Adenase, 85, 497, 023 Adenine. 287, 619, 623 Adenoma, 432 Adenomatous goiter, 601 Adenosine-deaminase. 623 Adipocere, 399. 410-412 composition of. 410 formation, 411 Adipose connective tissue. 400 fluids, 302 Adiposis dolorosa, 512 Adiposity, 614 Adrenals, 445, 60S acute insufficiency. Oil cancers. 503 choline in. 123 cortex, 404 relation to generative system. hypertrophy, 532 li])oids in arterial disease, 009 in ])neumonia. 609 in renal disease. 609 medulla. 472 relation to carboliydriites. 611 resemblance of ln])erneplirom; 518 tumor, melanotic, 472 Adrenalin, 609 Aethalium septicum. 255 Agglutination. 183 189, 324 by acids. 188 mechanism of, 185 relation of salts to. 186 relation to resistance. 183 Agirlutinative thrombi. 325 Agglutinins. 118, 170, 183-189. cell receptors. 185 electric charges of, 187 plague. 184 properties of. 184 typhoid. 174. 184 venom. 154 Agglutinogen, 183 100. 550, 610, 608 I to. .358 673 43 67i INDEX Aggressins, 130 Air einbolisni, 326-327 Alanine, 495 AIl)inisui, 4(i7, 577 A lliiuiKMi- peptone, 258 Albuminuid matrix, 437 Albuminul\ sis, 207 Albnminous soaps, 268 AU.nniins. 21, 351. 350 Allnuninnria, 349. 417, 563 alimentary, 528 Albuminuric retinitis, 530 Albumose, 279, 508 Bence- Jones, 309, 518, 570 constitution of. 520 reaction of. 519 Albumosuria, 279, 569-570 myelopathic, 519 521 occurrence of, 520-521 Alcohol, 50, 105, 244, 257, 301, 483 cetyl, 514 efi'ect of, on germicides, 28 oxidase, 71 in liver, 248 Alcoholism. 412, 595 acute. 531 lipemia in. 413 Aldehydase, 72, 100, 545 Alimentary albuminuria, 528 levulosuria, 055 spontaneous, 055 tract, 247 Alkali, 71 albuminate. 258 dill'usible, 292 free, 247 non-difl"usible. 292 Alkaline salts, 337 Alkalinitv, increased. 440 of bile,' 245 of blood, 292. 303, 314, 534 relation to bactericidal power, 292 total, 301 real, 291 relation of calcification to, 439 Alkaloids, 379 Abalosis, 598 Alkaptonuria. 09, 73. 473, 524, 577, 580 Allantoin, 357, 023. 025 Allergy, 193-204 Alloxan. 020 Alloxuric bodies. 019 Alpha-inicleoproteins. 1 95 Altmann's granules. 98 Aluminium hydrate, 40 Amanita hemolysin, 227 niuscaria, 140. 147 jiliallobles. 140. 147. 107 Amanita toxin, 147 Amblyopia. 007 Ambocei)tor. 211 214. 218. 228 action of. 220 derivation of, 219 Amljoceptor. hemolytic, 219-221, 232 properties of, 219 immune, 211 lelation to proteins, 214 stability of, 213 union with cell, 220 where formed, 213 Amboceptor-complement bacteriolysins, 205 Ambrosia. 147 Ameb.T, 81, 250 artificial, 268-271 taking of food, 209 coli, 135 relation of leucocyte to, 200 Ameboid motion. 39, 254-256 artificial imitations of, 267- 271 by inorganic substances, 208 Amibodiastase, 202 Amino-acids, 20. 279, 281, 311, 546, 507, 508 cyclic, 280 derivatives, 580-581 nitrogen, 529 radicals, 176 Ammonia, 567, 620 Amnion iacal decomposition, 455 Ammonio-magnesium phosphate. 455, 450 Ammonium carbonate, 251, 520 compounds, 231 urate. 258, 454, 455 Amoeba. See Aniebce Amygdalin, 280 Amylase, 07, SO, 95. 103 in urine. SO Amyloid, 417-423, 4(>0 accumulations, local, 423 chemistry of, 418 420 en/.yme, 422 intiUration, 010 kidneys, 404, 415 concretions, 423 origin of, 421 423 relation of liyaiin to, 424 si)lenic, 120 staining properties, 420-421 Amyloidosis. 02, 421 Anabolic ))rocesses. 371 , Anaerobic gas-producing organisms. 305 A-iiapbthol. 248 Anaphylactic difl'erentiation of blood corpuscles. 175 of hemoglobins. 175 intoxication. 02. 198 poison, character of, 199 reactions with salvarsan, 109 shock, 320 Aiuiphylactin. 202 relation of ]>recipitin to. 193 Anaphylactogens, 194 INDEX 675 Anaplivhitoxin, 120, 130, 170, 197, by lUitolysis of bacteiia, 1!>8 formation. ")(!!• from kaolin in lilood, 201 relation to anaphylaxis, 201 Anapliylaxis, 172, 193 204. 509 dne to protein cleavaye \<\ protease, 202 pathologic changes in, 19!t relation of anaphylatoxin to. 201 Anatomical and ciiemieal fat changes, 402 Ancistrodon contortrix, 149 piscivorus, 149 Anemia, 70, 142, 231. 293, 29S. 371- 372, 407, 413, 602 acute, 302 iiemolytic. 478 aplastic. 321 bothriocephahis, 139, 300, 414 due to hemolytic agencies, 302 hemolytic, 230 Ijeruicious, 231, 305-307, 317, 477. 587 analysis of organs in, 305 calorimetric studies in, 3116 cavises of, 307 chemical changes in, 305 due to hemolytic poisons, 300 iron in corpuscles, 305 protein metabolism in, 300 secondary, 225. 294, 317. 322. 300- 302 severe. 561 transitory. 394 Anemic heart murmur, 431 infarct, 327. 381. 407, 415 necrosis, 328. 368, 372, 381 Anesthesia. 558 chloroform. 78 Anesthetics. 245 Aneurism, 322 Angioneurotic edema, 351 Anilin dve cancer, 493 Anilines," 218. 249, 482 Animal parasites, 260. 340, 432 chemistry of, 134-143 eosinophilia (chemotaxis) . 134 Anistropic bodies, 404 lipoids, 25 Antagonism of ions, 247 Antelope dorcas, 463 Antemortem rigor, 390 Anthracene fractions of tar, 493 Anthracosis, 265 Anthrax, 276 bacillus, 108, 131. 132 Anti-amboceptors, 222 Anti-anaphylactic condition, 203 Antibodies,' 126, 128, 417 echinococcus, 138 effect of light on, 374 Antibodies, hemolytic, in vitro tissue cultures, 170 relation of lipoids to, 169 specific, therapeutic stimulation of, 174 Anticatalase, 70 Anticoagulin, 318 Anticomplement, 239 Anti-endotoxins, 130 Anti-enzymes, 63 68, 89, 277, 289 specificity of, 66 Antiferments, 291 Antigelatinase, 117 Antigen-antibody-com|)lement, 202 Antigenic activity, 171 Antigens, 166-171 etl'ect on non-stiiated muscle, 201 excess of, 190 from echinococcus lijjoids, 168 from ta])e worm lipoids, 168 from tubercle bacillus, 168 immunological specificity of, 173 in vegetable proteins, 204 monovalent, 179 nature of, 166 non-specific. 235 several in single organism, 174 specific. 235 Antihemolysins, 222, 231 Antiketogenesis, 554 Antiketogenic agents, 554 Antikinase, 64, 319 Antilijiase, thermostable, 79 Antimony, 247 Antioxidase, 467 Antipepsin, 65 Antiplatelet serum, 239, 300 Antiiuioumin. 69 Antiprotease. 361 in bacteria, 118 serum. 277 Antiy>^■retic drugs. 259 Antipyrin. 160, 243, 258, 560 Antirennin. 64 Antisensiti/ation. 203 Antiseptic substances. 378 Antiserum, 171 cholera. 211 excess o^, 190 Antithrombin, 295. 308, 316 Antithrombin - prothrombin balance, 321 Antitoxin. 177-183 difl'usion of. 182 filterability of. 182 against cantharidin. 170 associated -with pseudoirlobulin. 181 chemical nature of, 180 diphtheria. 174 Ehrlich's theory of. 177 for endotoxin. 130 neutralization of toxins by. 179 putrefaction of, 182 676 INDEX Antitoxin, relation of, to enzymes, 180 to proteins, 181 specilicity of, ITS Antitrypsin, 63- 68 content of blood, 200 Antitryptic titer, 535 Antivenin, 155, 156-157 Ants, 160 Anuria, 344 Aorta, atheromatous degeneration of, 012 Aphrodite aeuleata, 170 Aplastic anemia, 321 Aporrhegma, 122 Appendicitis, 433 chronic, 414 Arabinose, 649 Arachidic acid, 514 Arachnolysin, 159 Arcns senilis, 415 Arginine, 20, 84, 90, 100, 285, 544, 545, 581 nitrate, 544 Aromatic compounds, chromogenic, 613 radicals, 176, 194 Arsenic, 165, 244, 246, 542 fixation to nucleus, 246 immunization against, 247 poisoning, 342, 539 sulphide, 34. 41 Arseniuretted hydrogen, 217, 232, 485 Arterial degeneration from epinephrin, 612 disease, adrenal lipoids in, 609 Arteries, large, 439 Arteriosclerosis, 416, 424, 428, 584, 587, 610, 612, 614 Arthritis, 357 gonorrheal, 433 rheumatoid, 629 Arthroi)athv. tabetic, 351 Artificial a'mel)a', 268-271 takinir of food. 269 Ascaris, 140-141 chemistry of, 141 luml)ricoides, 135 megalocepliala, 140 pioducts of metabolism, 141 toxicity of. 141 Ascites adii)osus, 364 chvhnis. 340 lluid. 311 liemorrhagic, 490 Aseptic li<|uefaction necrosis, 276 Asiatic cliolera. 560 Asparaginic acid. 495 Aspartic acid. 544. 584 Asphyxia, 263, 390. 538 Asphyxia! conditions, 560 Asphyxiation, 346 As|)irin, 169 Astlienia. gastro-int(>stinal. lil 1 muscular, 614 Asthenic uremia, 529, 532 Asthmatic sputum, 311 Atheroma. 414 Atheromatous areas, 441 degeneration of aorta, 612 masses, cholesterol in, 25 patches, 405, 415, 459 Athyreosis, 592, 598 Atophan, 630 -^toxvl, 169 Atrophy, 92. 393 394 acute yellow, 100. 379, 403, 538, 539- 550. 557, 569 brown, 393, 474, 482 of kidneys, 474 of liver, 474 of pancreas, 393 simple, 393 a^-ray, of ovaries, 376 of testicles, 376 Atrojjine, 560 Autocvtotoxins. 240 Autod'igestiun, 82, 318 Autohemolysin. 233 Autoimmunization. 311 Autointoxication, 523-565 gastro-intestinal. 566-589 Autolysis. 82-105. 317. 327, 368 bacterial, rate of, 119 defense of cells against, 88 histology and chemistry of. 99-100 inlluence of cliemicals in, 86 liver, efi'cct of thyroid on, 591 of ])acteria. 118-120 of cell proteins, 403 of leucocytes. 308 of tumors, 496 pancreatic. 386 products of. 279 relation of age to. 88 to asi)hvxiation. 89 to toxins. 102-103 witliin Ixnly. 370 Autolytic enzymes, 67 of tissue cells, 277 products, bactericidal ]>ower of, 119 ju'oteolysis. 526 Auto-o]isonin. 233 Auxanograi)hic nu^thod. 114. 502 Auxetics. 493 Bactt.t.is. acid-fast. 207 aerogones capsulatus. 307. 327, 389 anllirax, 108, 131, 132 botiilinns. 177. 585 toxin. 16S cholera' gallinariuni. 257 coli communis. 70. 94. 132. 264, 289, 320. 3S9. 451. 574. 633 diphtheria. 70, 177. 131, 132. 259, 320 lecithin in. Ill eTni)h\senn»1osns. 478. 482 enteritidis. 122 INDEX 677 Bacillus, iMicdlaiuU'r's, 258 (iiirtuiT, 17:5 liu^'-cliolora, .■524 niegatliorium, 22.') l>aratyiilioicl, 17:5 plajiue, 107 prodi-iiosus. 114, 320 ]iroti'Us. Ill) Uiiorosceiis. l."{2 l.vo.'vanevis. 11-1. 132, 177. 224. 259, 27S, 320 siihlilis, 114 tetanus, 70. 177 tubercle, 70, 70, 00, 107, 131, 132, 204. 320 antijien from, 108 foiiipdsitiou of. lOS fats of. 110 111 fatty acids in, 112 modification of acid fastness of, 111 no cholesterol in. 111 phosphatids in, 111 typhosus. 70. 120. 132, 183. 211, 214, 224, 2G4, 320, 451 xerosis, 320 Ba CL, 200 Bacteria. 73, 411 anti-protease in. 118 autolysis of. 89. 90. 118-120 chemical comjiosition. 107-113 chemistry of. 106-113 chemotaxis in. 107 cholesterol in. Ill chromatin of. 100 com])osition of cell wall, 110 decolorization of. 112 ectoplasm of, 106 endoplasm of. 106 inhibition of. 102 Gram's method of staininfr. 112- 113 motile. 250 nucleus of. 100 patlio.acnic. endotoxins in. 119 plasmolysis in, 107 plasmophysis in. 107 putrefactive. 506 reducinji power. 114 relation of nutriments to. lOS staining reactions. 112-113 synthetic activity of, 100 tolerance of. 252 typhoid colon. difTerentiated by acid agglutination. 188 Bacterial autolysis, rate of. 110 carbohydrates. 109-110 caseins. 108 catalase, 117 cell wall, animal or vegetable, 110 cultures, sterilized, 258 decomposition. 10 digestion, products of, 116 Bacterial endotoxins, 118 enzymes, 113 120, 27i;, 3S2 immunity again^l, 117 relation to pathngonicity, 110 role of, in infectious diseases, 110 fats, 110 112 and fatty acids, 111 112 ell'ect of media on, 1 1 1 formation, 390 nucleic acid, 109 nucleoprotcin, 108 pigments, 132-133, 280 alcohol s.>inl)lc, 133 insoluble, 133 Avater soluble, 133 ])oisons, 71, 502 ])recipitins, 192 proteins. 270, 280 poisonous. 131-132 toxic not specilic, 132 toxicity of, 131 proteolytic enzymes, resemblance to trypsin, 110 power, relation to virulence, 117 spore, 367 substances, 195 toxalbumins, 108 toxins, 125, 165, 265, 318, 407 Bacteriolysins, 118. 128. 358 amboceptor-complement, 265 Bacteriolysis. 207, 213. 214 serum," 208, 210-214 Bacteriolytic endolysins, 280 Bacterium ternio, 254 Barium, 240 Barlow's disease, 446 Basal metabolism, 590, 602 Bee poison. 160 toxolecithidin in. 100 Bence-Jones albumose. 300, 518, 570 constitution of 520 reaction of, 519 Benzene. 317 Benzoic acid, 250 Benzol, 259 Benzopurpurin, 493 Beriberi, 286 Beta-iminazolyl-ethylamine, 576, 014 Beta-oxvbutric acid, 550 Betaine." 123, 124 Bezoar stones. 463 Bibasic urate. 620 Bichloride of mercury, 43 poisoning, 532 Bile, 127. 232 acids. 218, 246. 251 alkalinity of. 245 colloids, "electro-negative, 452 fat content. 326. 403 pigments. 229. 484. 486-487. 560 calcium salts of. 448 salts, 487. 506 thickening of, 453 678 INDEX Bile thronil)i. 485 toxicity of. 486 tracts, inflammation of, 453 Bile-chicts. casts of, 450 Biliary calculi. 448-454 cirrhosis, 486 fistula. 558 Bilicyanin, 450, 484 Bilifiiscin, 449, 484 Bilihumin, 449, 450, 484 Biliprasin, 484 Bilirubin, 296, 375, 449, 474, 477, 484 Bilirubin-calcium calculi, mixed, 449 ])ure. 449 Biliverdin, 375, 449, 484 Biliverdincalcium, 449 Binding substance, 454 Biurate, 620 Black flies, 160 Blackwater fever, 232 Bladder. 457 urinary, 423 Blastomycetic dermatitis, 274 Blisters, burn. 565 fluid. 362 Blood. 247 alkalinity of, 292, 303. 314. 534 relation to bactericidal power, 292 total, 301 amount of water in. 331 antitrypsin content, 200 buffer value, 291 cadaver, 320 calcium in, 440 cells, red. See Erythrocj/tes white. See Lencori/tes changes after hemorrhage. 294 in passive hyperemia. 313 chemistrv of. in leukemia. 308 coagulability of. 200, 316. 321-322 in disease. 319 modification. 317 318 retarding of, 318 composition of, 289 corjuiscles. aiiaphviactic differentia- tion of. 175 red, 70, 289. See also Eryth- rnrj/tes white. See Levrofytefi effect of venoms on. 153 electrolyte concentration of. 290 enzvmos. 291 extravasatod, 29.-) 297 fat in, 326 fibrin content, in dispiisc. 321 formed, elements of. 289. 290 freezing-point. 301. 308 lowering of. 5f)6 imbibition of, 412 in acute Acliow atrophv. 546 hiking of.' 215 leukemic, coagulat inn of, 308 lipase, 535 Blood, menstrual. 320 nitrogen, non-jjroteiii, 534 pigments, 476 484 plasma. 290 increased filterabilitv of, 344 reaction of. 291-292 relation of hinjjli to. ."i.'JO platelets, 289, 290, 29!), ;517 poisons. 31S ))oisons, 406 pressure. 298, 331, 349 high. 612 increased. 341-342 intracapillary. 334 ]u-oteins, 290 serum. 200 normal. 486 state of sugar in. 643 sugar. 641 concentration, 641 content, 535 toad, ]>nison in. 161 viscosity of. 292-293 Bloody diarrhea, 563 Body temperature, subnormal. 615 Boiling-point, elevation of, of colloids, 38 Bone-marrow, 91, 296, 311 tumors. 570 Bones, congenital fragility, 447 Botliriocephalus anemia, 139, 306, 414 Botulism. 122, 567 Brain. 77, 83 edema of. 535 enzymes in. 78 softening, 342 tissue. 123 tumor of, 531 wet, 531 Breast, cancer of, 437 Brick-dust deposit, 455 Brilliant green, sensitization by, 220 Bronchi. 423 Bronchial catarrh, 434 secretions. 404 Bronchiectasis, 281 Bronchiectatic sputum. 414 Bronchitis. 281, 450-454 pufrid. 434 Bronzed diabetes, 483. 671 Brown atrophy. 393, 474, 482 Brucin. 251 Bruise. 295 Bufagin. 161 Buffer value of blood. 291 Bufo agua. 161 Bufonin, 161 Bufotalin. 161 Burn blisters. 565 Burns. S5 hemolysis in. 232 leucocytosis in, 261 INDEX 07!) Burns, siiiifiliiinl. imisoiis produced in, 562 565 Butter, 2S7 cysts, r.l.") Butyl chloral. -J 1!» Butyrase, 77. 7'.t Butyric acid, ^u, M7 Caciiixtic acetoiuiria. 008 conditions, 2!)4, .S;)4 diseases, oOG edema, ."$44 Cachexia, (Hi, 711, 2SI, 302, 417, .")(iO cancer. :5()!». 492 Cadaver blood, 320 Cadaverine. 123, 5*57, 582, 583 Caffein, 248, (il9. (327 demetliylated in liver, 247 Caisson disease. 32(5 Calcareous stones, 450 Calcification, 325, 329, 424, 435- 447 chemistry of. 439 441 deposits, coniixisitioii of, 436-437 metastatic, 438 439 of gan,u;lion-cells. 43S of renal epithelium. 438 patholo-rical. 415, 435, 438-439 pt'ricai'dial. 43(1 ]ihi's|)lioric acid in, 442 relation to alkalinity. 439 to ossification, 435-436 to retrogressive clianues, 440 Calcified areas, structure of, 437 ]ilaqiu's, 323 tidiercdes, 464 Calci'^ving lii)oma. 441 Calcium. 251. 285. 303, 308. 456, 487. 499 carhoiuite calculi, 458 chloride, 336 oout. 439 in blood. 440 in fibrin formation. 316 oxalate. 454. 45G. 457 calculi. 457 L'allstoiies. 450 salts. 48. 317 absorption of. 442-443 effect of. on phajjocytosis. 263 of bile pigments. 448 soaps. 387 formation of. 441-442 Calcium iron incrustations. 437 Calcospherites. 437 Calculus be/oar. 463 biliary, 448-454 cali'areous, 450 calcium carbonate. 458 oxalate. 457 cholesterol. 459 crystalline. 452 cystine. 458 fecal. 463 Calculus, librin, 459 fusible, 458 in Chiiui. 456 iiulifjo, 458 intestinal, 462 463 luni;-. 459, 4(i4 metaniorpliosed, 455 mixed bilirubin-calcium, 449 oxalic, 584 jiaiHTeatic, 461 462 l)hosplialc. 457 458 pure liilii'uhin-calciuin. \ 19 salivary. 462 str\ivi(. 457 urate, 456-457 uric acid. 455 456. 027 urinary, 454 460 disinteoration of. 459-460 general jfroperties, 459-460 urostealith. 458 xanthine, 458 Calories, 281 Calorimetric stiulies in ])eriii(i(ius aiu»- mia, 306 Calves' brains, 585 Camphor. 249 Cancer. 66. 71, 79, 94. 230. 231. 292, 322, 344. 356, 424, 425. 432, 497, 560. 569. 634 adrenal. 503 anilin dve. 493 cachexia. 309, 492 chemical stimuli causing. 493 colloid, 427, 516 extracts, 104 hemolytic, 492 glycyl-tryptoidiane test for. 501 hemolysis in, 504-505 increase of Oil in blood in. 507 induced cell reproduction and, 493 lysins, 241 metabolism in, 505-507 of breast. 437 o*^ stomach. 104. 504 of thyroid. 604 putrid. 574 skin, in colored races. 467 squamous cell, 516 a"-ray, 377 Cantharides. 312 blisters. 260 Cantharidin. 340 antiioxin a>jainst. 170 Caoutchouc colloid. 426 Capillaries, injury to. 254 obsl ruction. 326 permeability of, 333-334. 349 walls. increased permeability of, 342 344 Capra aecraurrus. 463 Carbohydrate uu-talxdism. 602. 635- 671 effect of tlnroid on. 591 680 INDEX Carboliydratos. :V.], 35, 25S, 335, 551 aiitiketouciiic act ion. 37'J bacttM-ial. 109 110 colloidal, 34(j fermentation of, products of, 583- 584 in food, fat from, 398 relation of adrenals to, 611 tolerance, increased, 615 Carbolic acid gangrene, 378 Carbon dioxide, 53, 293, 326, 501. 5Q7 Carcinoma. See Cancer Carcinomatous exudate, 304 peritonitis. ;553 Cardiac defects, 5(il disease, 71 with edema. 293 dropsy. 345, 349 edenui, 348 failure, acute, 610 incompetence. 324, 350 edema o*', 341 Cardiolvsin, 241 Caries. '438 Carotid gland, 617 Carotin. 448, 475 Cartilase. normal. 420 Caseation. 98. 99. 382-384, 415 Casein. 195, 282 bacterial. 108 Caseous areas. 438, 441 encapsulated, 415 material, fat in, 383 tubercles, 276 Castration, 444 Catabolic. See KatahoUc Catalase, 60, 67, 69-71, 280, 502, 595 bacterial, 117 in urine, 71 Catalytic agents, 56 Cataphoresis. 38 Cataract diabetic, 375 Catarrh, bronchial, 434 Catarrhal inflammation, 427 Cations, 28 Cell, acids within. 548 autolysis, inti'avitam. 408 chemistry and i)]iysics, 17-52 colloid eipiilibrium in, 373 dead. eflVct of dyes on. 370 death, 43 from d'CTuicals. 378 physical changes in. 370 recognition of, 307 divisifni. 23 imitation of. 271 endothelial, 219 death of. 343 lar stimulus. 284 causing cancers, 493 substances, abnormal toxic. 523 transformation. 523 Chemorecentors. 243 Chemotactic substances, non-hacterial, 257 259 Chemotnxis. 62. 254 261. 314 necatiye. 257 o*" bacteria. 107 of leucocytes, 250 nositive. 250 271, 270 theories of. 266-275 Chomot ronism 255 Chimi>anzee, 625 INDEX 681 Chitiii, ;i(), 110, i;Ji Chitosamia, 513 Chloial, 258 Chlorides, 335 retention of, 345. 350 Chluroforni, 5(i, 257 anesthesia. 78 narcosis, 540 541 necrosis, 4iis of liver, 320 poisoning, 37'J, 53!), 557 Chloroma, 47t) Chlorophyll. 4S(I Chlorosis, 302 304, 344, 587, G12 etiology of, 303 iron in, 304 starvation canse of, 304 Cliolagiigue action, 488 Cholalic acid, 463 Cholelithiasis. 453 Cholemia, 488, 490, 567 Clioleprasin, 484 Cholera, 11!) antiserum. 211 Asiatic, 560 vibrios, 70, 120 Cholesteatoma, 515 Cholesteatomatous tumors, 415 Cholesterase, 79 Cholesterol. 23-25, 85. 100, 138, 217 226, 236, 237, 244. 290, 297 303, 357, 361, 401, 404, 410 413. 448, 545, 609 calculi, 459 crystalline, 452 laminated. 449 pure, 449 esters, 25, 415 gravel, 450 in bacteria, 111 pathological occurrence, 415-417 relation of ]ihagocytosis to. 263 steatosis, 405 tests for. 415-416 Cholesteroleni ia . 4 1 (I — 1 1 7 Choline, 93. 123-125. 360, 501, 562, 567, 584, 609 in adrenals, 123 Choluria. 490 Chondrin. 34, 417 T'liondrodystropliia f(ptalis. 603 Cliondr()itin-suli)liuric acid. 418 Chondroma, 432, 511 Chondrosin, 513 Chorioepithelioma, 432, 516 Chromatin, 44 of bacteria, 106 threads, 47. 48 Chromatolysis, 562 of cortical ganglion cells, 531 Chromium ]>oisoning. 477 Hiromogen. white. 469 Chromogenic aromatic compounds, 613 Chyle. lonipo.Nilion ol', 362 lat from, 412 relation to lymph, 331 Chyliform lluids, 302 Chylothorax, 340 fluid, 363 Chylous ascites, 340 clVusions, 362-364 Chyluria. ."Uit, 36;i Ciaccoi's method, 402, 405 Cilia, 275 Ciliated ei)itlielium, 238 proto/oa, 255-256 Circulation, 338 feeble, 440 ( ircuhitory disturijances, 595 Cirrhosis. 78, 92, 356, 587 biliary, 4S6 of liver, 320, 355 Citric acid, 251 Cloudy swelling. 394-396 CO^ content, decrease in, 440 Co-agglutination, 186 Coagulated protein, 383 CoagT-ilating temperature of nerve cells, 372 Coag-ulation of blood. 200, 298, 302, 316 decreased, 546 in disease, 319 modification of, 317-318 retarding of, ."ilS necrosis. 381-382 of cell Liloliulins, ;i73 of coll.iids. 40 of leukemic blood, 308 reaction, 238 time of blood. 219. 321-322 Coagulins, 316, 317 tissue. 323 Coal-jngment. 465 Cobra poisoning. 152 venom, G>^, 102, 150. 151, 227, 228, 241 resistance, 505 Coccus. Cram-positive. 207 infections. 422 Cod liver oil. 287 Coelenteratts, poisons in. 164 Ccenurus cerehralis. 138 scrialis, 138 Co-enzymes. 61 Cohesion allinity, 267 ])ressure, 2(i7 Cold, effect on life, 373-374 tuberculous abscesses. 280 Cold-blooded vertebrates, 195 Collagen. 424 Colloid. 32. 34-43, 318. 334, 448 altered hydratioTi. 254 anu)rj)hous form. 35 bile, electro-negative. 452 cancer, 427, oK! caoutcliouc, 426 capacity for water, 336-337 682 INDEX Colloid dianges with time., 371 cliaii^es with time, 371 cliarafteristics of. 35 39 (U'^cncratioii, 425-428 depression of freeziugpoiiit, 38 diffusion of, 38 effect of, on chemical processes, 3!) electrical phciioineiia of, 38 elevation of lioiling-point, 38 emulsion, 452 goiter, iodin content, 600-(301 liydrophilic, 330, 409 tendency, 343 molecules, 395 non-dillusihility of. 37-38 osmotic pressure, 38 ovarian, 513 permeability to, 371 precipitation, 187, 301 and coaj^ulation, 40, 41 serum, electro-positive, 452 soluhilitv of, 36 structure <"ytic. 2!)(! sudaiiopiiilc clianjip. 272 Eiulotlu'liolytic scniiii, 23!) Eiulotlii'lioiiiii, 432 Endotheliotoxii' action, 293 Eiulotlu'liotoxin, 153, 240 Endotoxins, 103, 120. 129 130 antitoxin for, 130 hacterial. 118 in ]>atlioL;(Miic bacteria. 119 Enterojrcnous cyanosis, 580 Kntorokinase, 61, 385 Enteroliths, 463 Enzvnies. 34. 40. 53-105. 280. 358. 500 502 amyloid, 422 autolytic, 67 of tissue cells. 277 hartcrial. 113-120. 270. 3S2 ininmnity against, 117 ' proteolytic, resemblance to tryp- sin, 116 relation to pathooenicitv, 116 rrde o*, in infectious diseases, 110 blood, 201 dcaminiziiiir. 50] effect of light on. 374 electrical charges, 55 ethyl bntyrate. 77 glycolytic, 76 hydrolvzing fibrin, 282 in brain. 78 in kidney. 250 in venoms, 150 inorganic, 56 intracellular. 68-105. 264. 296, 368 lecithin, in tissues, 77 letu'ocvtic, 276 lipolytic. 77 in lymphoid cells. 77 oxidizing. 68. 60. 371 effect of liyhi on. 375 peptid-splitting, 361 jicptolytic. 01 protein nature. 54 protenlytic. 280. 315 enzymes of. 94-96 of leucocytes. 277 purine. 497-498 reducing. 74 relation of antitoxins to. 180 of complement to, 211 to toxins. 59, 126 resemblance of toxins to. 67. 68 specificity- of, 59 sugar-splitting. 55 synthesis by. 57 toxicity of. 61-63 uricolvtic, 501. 633 Eosin, 375, 493 K(jsiMophil('s, 76 Eosinoi)hilia, 200, 363, 417 in relation to glycogen, 432 parasitic, relation to anaphylactic, 135 5Jpeira diodcnia, 159 E|)idemic meningitis, 355 Epidermis, superficial, decomposition of. 412 Ei)ididyniis. 474 Epilepsy, 3(i0. 562, 586 Ejjileptic conxulsions, 562 Epinephrin. 472, 576. 609 arterial dcgcncratidii t'roni. 612 Epi]>hanin reaction. 209, 50S Kpisplenitis scars, 424 Kpithclial hyalin, 424-425 nuicin, 427 pearls. 438 ])roIifcration from dyes. 493 Epithelioid cells, 273 E])itheliolvsin. 241 E]iitheli(.nia, 432 cntaneous, 516 Epitheliinn, ciliated, 238 renal, calcil'cation of, 438 Equilibrium, 33, 56 nitrogen, 506 osmotic, 335 Erepsin. 81. 358, 568 Ereptase, 104 Ergot, 576 Erotism, 615 Erucacic acid, 224 Erysipelas, 276, 321 Erythema, 349 Erythrocytes. 70, 289, 301 infected, 474 resistance of, 230 Erythrocytohsis. 214, 215-234. See also Hemoli/sis Erythrolvsis. 218 Esterase,' 77, 408 Ether, 50 liyer necrosis, 325 Ethereal sul]>hates, 506, 575 sulphuric acid, 5. _ Etiiyl alcohol. 57 butyrate. 57 enzymes, 77 mercaptan. 567, 582 sulphid, 567. 582 Ethylidendiamine. 567 Eu^lobulin. 181. 236. 290. 351. 353. 356 Exanthemata, aoite. 322 Exophthalmic -joiter. 66. 79. 231. 241, 445, 604 608. 612 iddiii content. 605 parathyroids in. 607-608 jdiospliorus in. 605 serum treatment. 605 Exophthalmos, 606 Exosmosis. 350 Explosion of fulminate. 380 6S6 INDEX Extirpation of organs, 71 Extracellular lysis, 207 Extract of crayfish, 332 of leeches, 318. 332 of mussel, 332 of oysters, 332 Extravasated blood, 295-297 Extravasations, 47S Extravascular ])ressure. decreased. 342 E.xudates, 04, 93-94, 331, 433 carcinomatous, 364 cholesterol in, 25 inllanimatory, 331, 569 l)neumonic, 404, 415 putrid jiurulent, 574 tuliorculous. 358, 364 turpentine. 358 Exudation of plasma, 253 Eyelids, 423 Factors ■which influence supply of sugar to kidnevs, 637, 638 Fat, 23-25, 33, 84, 127, 278, 279, 335, 383 accumulation, pathological, 399-400 bacterial. 110-112 and fatty acids. 111 effect of media on. 111 changes, anatomical and chemical, 402-404 content, in organs, 403 of bile, 403 * decomposition of. products of, 584- 585 embolism, 325. 413 foreign, de])o;^ition of, 399 formation, bacterial, 399 by fungi, 399 from carbohydrates in food, 308 from chyle. 412 from fat in food. 398 from proteins possible, 398 in bile, 326 in blood. 326 in caseous material, 383 in chemical comliination. 401 in fattv organs, analysis of. 401 in urine, 326 increase of, in dc^cMicrating organ. 402 infiltration. 397 invisible, made visible. 403 iodin comiiounds. 400 masked. 401, 409 metabolism of, 58, 602 necrosis, 62. 80. 384-388. 414. 442. 4S6 nculrul, migralinn of. 412 of tulK'rcle bacillus. 110 111 solubility of dves. 50 staining in tubercle liacilhi.--. 1 1 I stains. 401 sugar from. 663 Fat tissue, 474 Fat droplets. 49 Fatigue, 561-562 mental, 562 toxins of. 75, 561-562 Fatty acids. 100, 278, 407, 441, 567, 580 crystallization of, 412 (Ives, 414 free, 280. 401 in tubercle bacilli, 112 pathological occurrence, 414 soaps of, 384 toxicity of, 414 volatile, 280 degeneration, 79, 397 due to poisons, 407 infiltration, 407 metamoi'phosis, 75, 301, 328, 397— 399 by absorj)tion, 408 causes of, 406-410 relation of lipoids to, 404-406 reaction, 508 substance, 303 Fecal elimination, defective, 506 retention. 524 stones. 403 Fermentation. 570-589 of carbohydrates, products of. 583- 584 Fermentoid, 120 Ferments, defensive, 205 digestive, 506 fibrin, 278 glycolytic, 60 protective, 197 Ferratin. 479 Fertilization, 285 Fetal tissue, 428 Fetus, intoxication from, 536 retention of. 560 Fever. 83. 322 acetonuria in, 500 acute, 390 Fibrin, 290 calculus, 459 content of blood in disease. 321 enz\ines hvdroiv/.ing, 282 ferment. 67, 10.5. 278, 316 thrombosis. 325 formation, 315-317 calcium in, 310 theories of. 316 ludrohsis. 509 Fil)rinogen. 181. 290. 295. 315, 351, 353, 356 formation of. 315 Fibrinolvsin. 117 Fil)rinolysis. 92. 315. 320. 325 {•"ibrinous thrombi. .323 {'"iliroid gloMHMules. 424 uterine. 510 degenerating, 498 INDEX 687 Fibroma, 432 edeiiiutous, 428 Filaria, 143, 340 niediiieiisis, 14;? Fillration, 337 theory of l\iiiiili I'm maliDii, 331- 332 Finson li<^lit. 4S1 Fisli, lK)i^^uIl(m.s. 162 164 plomaiiis, 1()3 Fistula biliary, 358 Eck's, 520 pancreatic, 443, 558 Fixation in organs, 245 Flagella, 112 Flies, black, KiO Fluid, distribution of, 345 migration of, 412 Fluids, normal, diil'erences in, 353 Fluorescent substances, 375 Fluorides fixed to bones, 246 Foam structure, 268 hypothesis of protoplasm, 51 Foamy phagocytic cells, 405 Food, fat from fat in, 398 intoxication, 124 poisoning, 122 supplies, 338 Foreign body giant-cell, 272 organic, 438 proteins, 165 serum, 322 Formaldehyde. 293 Formalin, 43 Formic acid, 567, 583 Fragility, congenital of bones, 447 Freckles, 467 Free acids, 247 alkalies, 247 fatty acids, 280. 401 Freezing, elVect of, 373 Freezing-point, depression of. of col- loids, 38 of blood. 301, 308 lowering of, 50(i of cerebrosiiinal fluid, 360 Friedlander's pneumobacillus, 258 Frog poi.sons, 150 in skin of, 162 red corpuscles of. 229 Fructose, 655, 656 Fuchsin, 493 bodies. 424 Fungus, fat formation by. 399 Fusible calculi, 458 Galactose, 654 Gall-stones, 448. 449 calcium oxalate. 450 common, 449 formation of, 450-454 relation to infection. 451 Ganglion-cells, 474 Ganglion-cells, calcification of, 438 cortical, chromatolysis of, 531 Ga.ngrene, 122, 373*, 388 390, 574 carbolic acid, 378 dry, 388 emphyseniat(jus, 3S'.l moist, 388 of lungs, 281, 389, 414 ic-ray, 376 CJjirtner l)acilli. 17.'! (las exchange. 302 poisoning. 5tJl Gas-])roducing organisms, anaerobic, 3(>3 Gastric carcinoma, 03 fermentation, 457 juice, acidity of, 245 Gastro-intcstinal asthenia. 614 autointoxication, 566-589 diseases, 75 infections, 560 Gaucher 's disease, 405. 417 Gelatin, 20, 36, 41, 194, 258, 319 Gelatinase, 115, 280 Gels, 34 Generative system, relation of adrenal coVtex to, 608 Geometrical structure, relation of, 56 Geotropism, 256 Germicides, effect of alcohol on, 28 Giant-cells, 273, 416. 631 foreign body, 272 formation of, 264, 273-274 of tubercles, 76 (Jlla monster. 155 Glabrificin, 185 (Hand decomposition, 412 Glandular secretions, 331 (iliadin. 194, 286 (Jlioma. 432 (Jlobin. 20, 290. 295. 476 (ilobulins. 22. 35, 64, 195. 289 cell, coagulation of. 373 onatorum. 033 Hemicellulose, 109 Hemochromatosis, 482-484 local, 483 Hemochromogen, 290, 295. 476 Hemofuscin, 482 Hemoglobin, 21, 35. 217. 229, 289, 290. 302. 476-477 anaphylactic dill'erentiation of, 175 excretion, 230 infarcts, 227 metal>i)lism of, 477 Hcmoglobineinia, 230, 305 Hemoglobinuria. 225, 227, 228, 230, 305, 503, 477 malarial. 130 paroxvsnial. 232-233, 302 Henndvinph glands. 232, 290 Hemolysin. 128, 153, 240, 358 (piantitative action. 221 Hemolvsis, 31, (i2, 207. 215-234, 487. 5(i3 action of, on stiiuiia. 217 acute toxic, 478 by chemical agents, 216 by phagocytes. 230 bv ]di\sical means. 210 bv serum. 218 219 INDEX 689 Hemolysis by tissue extracts, 218 by vegetable poisons, 225-226 by veuonis, 228 229 in burns, 2.52 in cancer, 504 .")(!,") in disease, 229 232 in poisoning, -'.i2 inliiliition ol, 217 ineclianisni of, 21.3 pathology of, 233-234 postniorieni, 224 serum, made of, 222 splenic, 232 variable resistance in disease, 217 Hemolytic amboceptors, 219-221, 232 ])roperties of, 211) anemia, 230 antibody, 218 in in vitro tissue cultures, 170 cancer extracts, 492 complement, 21'J, 221-222 jaundice, congenital, 231, 489 lipoid, 307 poisons, 177 substances in tiunors, 504-505 Hemophilia, 297-300, 322 coagulation time in, 2'J9 icterus, 487 local, 298 Hemopyrrole, 468 Hemorrhage, 293-297, 305 blood changes after, 294 by diapedesis, 294 death from, 390 from asphyxial changes, 294 metabolic changes alter, 301-302 Hemorrhagic ascites, 490 cysts, 42G infarcts, 328, 611, 633 nephritis, 385 Hemorrhagin, 154, 240, 293 Hemosiderin, 296, 329, 478-480 Hemotoxins, 153 Hepatic necrosis, 371 Hepatolysins, 238, 241 Herbivora, 459 Hernia, 433 strangulated, 341 Heroin tolerance, 244 Herpes zoster, 276 Heterocyclic compounds, 470 Heterolysis, 90, 328, 500 Heteronephrolvsin, 240 Hexoses, 22, 25, 650-654 H g Cl„, 451 H-ion concentration, 236 Hippuric acid, 250, 525 Hirudin. 319 Histamine, 198. 576 Histidine. 96, 100, 285, 544. 576 Histon, 282, 309 nucleinate, 494 sperm, 177 44 Histon thymus, 419 llisloretention, 350 llodgkins disease, 71, 312 llog-clioiera bacillus, 324 Homogentisic acid, 73, 473, 577, 578 Hordein, 194 Horniouo, disassimilatory, 593 Hornets, 100 Horniiication, 432 Hyalin casts, 425 connective-tissue, 423-424 degeneration, 3b2, 423-425 epithelial, 424-425 relation to amyloid, 424 sunstance of Itovida, 279 thromlii, 224, 324-325, 374 Hydatid cysts, 432 fluid, chemistry of, 138 wall, 138 Hydrated oxide of iron, 34 Hydration, colloidal, 347 Hydremia, 293 Hydremic pletliora, 298, 334, 342, 347 Hydroa aestiva, 481 Hydrocele fluid, 359, 415 Hydroce])halus, 360 Hydrochinon, 575, 577 Hydrochloric acid, 336 Hydrogels, 34 Hydrogen, 567 ' arseniuretted, 485 sulphide, 245, 567, 581 Hydrolysis, 19, 33, 429, 478, 569 fibrin, 569 Hydrophilic colloids, 336, 409 tendencies changed by enzymes, 336 of colloids, 343 Hydrophina?, 148, 149 Hydrothorax, 356 Hydroxy 1, 21 Hydroxypurines, 287 Hydro.xypyridene, 287 Hydroxy-stearic acids, 410 Hypercholesterolemia, 417, 453 Hyperemia, 312-315 'active, 312-313, 341 local, 253 passive, 313 blood changes in, 313 pulmonary, 341 Hyperemic pressure on lymph channels, 340 Hyperirritability, 531 Hypernephroma^ 432, 492, 497, 498, 516, 517-518 resemblance to adrenal, 518 Hyperopnoea, nocturnal, 500 Hyperplastic goiter, iodin content, 601 Hyperpyrexia, 563 Hypersensitiveness of nerve cells, 530 Hypersensitization, 169 Hyperthyreosis, 71 Hyperthyroidism, 604 690 lyoEX llyportrophy, 394 adrenal, 532 of heart, 284, 611 Ilypcealcification. 598 H\i)()i)ln'seet(>mv, ()]5 Ilypopliysis. 42'(i, 4!)3, 614-616 anterior lolie, 014-015 defects in, 015 posterior lobe, 615 ]nuu'tiire of, 615 Hypopituitarism, 015 Hypotliyreosis, 71 Hyijoxanthine, 4HS. (i]!i. 023 Hysterical vomit iiiii', .')(iO IcHTiiyoToxiN, 104, 229 Icterus. See Jaundice Idiojiathic peritonitis, 353 Idiosyncrasy, 243 llhiminatinc('ptor. 211 bodies, 210. 358 reaction inlluenced by electric charges, 175 specificity of, 171-177 Immunity, 277 against bacterial enzj'mes, 117 malaria, 130-137 non antigenic poisons, 243 phytotoxins, 145-146 ]>rotozoa, 135 toxins, 128 cellular, 244 natural, 178 reactions, chemistry of, 165-209 lipoids in, 16S relation of ions to, 28 Immunization, 07, 178 against arsenic, 247 therapeutic, 173 Imjiaired absorption, 246 local nourishment, 341 Inanition, 560 Indicanemia, .■»02, 529, 573, 574 Indi'^o calculi, 45S Indole, 282. 40!l, 471, 507, 572, 573- 575 acetic acid, 572 propionic acid, 571 toxicity of, 574 575 Indole-acetic acid, 575 liuloh'iie, 248 Indoplienol reaction, 7(i, 248 hidoplienoloxidase, 72 hwlowi, 249, 572 Infant ih' marasmus, 558 559 s.Mirvv, 287 Infantilism, 590, 603 intestinal, 587 Infants, coairulation time oi blood in, 322 Infarction, 327-329 hemorrli.igic. Oil Infarcts, 76, 91, 97, 276, 281, 327, 308, 431, 438, 441 anemic, 327, 381, 407, 415 choii'stcrol in, 25 hemoglobin, 227 Jiemorrhagic, 328, 033 iinman, 328 old, 415 splenic, 328 • uric-acid, 456, 627, 633-634 Infected areas, 481 infection, 455 Infectious diseases, 320, 610 acute, 317, 595 role of bacterial enzymes in, 119 Infiltration, fat, 397 Iniiamed areas, 431 Inllammation, 05, 253-288, 452 acute, 361 catarrhal, 427 clironic, 273, 405 of l>ile tracts, 453 pulmonary, 405 relation to chemical alterations, 253 Inllammatory edema, 253, 340, 343, 350-351 exudate, 331, 509 Infusoria, 130 Inhibition of bacteria, 102 Injection of glucose solutions, 642 Inorganic en/\ines. 50 poisons, 246-248 salts, 28, 48, 51 Inosine, 623 Insects, sting of, 351 Insoluble proteins, 23 Intercellular edema, 337 snl)stance. 51 solution of, 343 Intermediary metabolism. 524 Internal organs, 247 secretion of tumors. 502-504 Interstitial nephritis, 349. Oil. (i27 chronic, 527 Intestinal concretions, 462-463 infantilism, 587 mehinosis, 470 obstruction. 433. 532, 580 acut.-, 588 589 ])utrefact ion, 245, 588 sand, 403 \vornis, 03. 432 Intoxication, 050 acid, 292. 534, 547-550, 584 non-diabetic, 557-561 anaphylactic, (i2 from fetus, 5.30 uric-acid, (127 Intracai)illa)y blood ])rcssui-c, 334 Intracellular edema, ■3.")7 enzymes, 68 105, 204, 290, 308 lysis, 208 INDEX 691 Jiitraci'lluliir oxidasos, 4U7 parasite, 47-4 liilraliepatic necrosis, 387 liiliali^iameiitary papillary cysts, 514 Jntravitain cell autolysis, 408 liuilin. JSt) liivertase. lil, l&I Invertebrates, IDj shells of. 4;J7 Invertin, 07 Jnvdliitioii of uterus, !)2 Iodides. 71. 2!)3 lodin, (iOU compounds of fat. 4()(» content of colloid goiter, 600- 601 of exoiiiitiialniic goiter, OO.j ot hyperplastic goiter. GUI Iodoform, •24;5, 270 lodophilia, 4.5.S Ionization. 27-29 Ion-protein. 26 compounds, 28 Ions. 20 antagonism of, 247 relation of, to diuresis, 28 to edema, 28 to slycosuria, 28 to immunity. 28 ■ Iron, 20, 45, 258, 303 hydrated oxide of, 34 in tumors, 500 starvation cause of chlorosis. 304 Isoagglutination, 224, 231 Isoamylamine. 507 Isocetinic acid, 110 Isohemolysins, 220 Isoneplirotoxins. 240 Isotonicity, 303, 305 Japanese lacquer, 409 Jaundice, 71, 218. 220. 227. 203. .305, 321. 410. 478. 484 491. 507 congenital hemolytic. 231, 489 dissociated, 48!) etiology of, 484 486 gravis, 488 hematogenous, 302 heniato-hepatogenous, 230 hemophilia. 487 in newborn, 480 local, 484 necrosis. 487 obstructive. 231. 410. 480 pigmentation in. 489-490 relation to infections. 488 , Jecorin. 100. 270, 200 Jequirity. 257 Kauyokixests. 45, 285 Karyolysis. 368 369 luiclear chanires in. 360 Karvorrhexis. 218. 250. .328. 368-369. 380 Kalaljolic [iroccsses, 371 Katabolisni, normal, 200 nuelein, 310 protein, increased, .302 Kenotoxin. 501 Keratin, 40, 285, 424, 408, 510 Keratoiiyalin, 425, 402 Keratitis, 250 Ketone, cyclic, 530 Ketoreductase, 74 Kidneys, 480 amyU.id. 404, 415 atrophy of, 474 concretions, amyloid, 423 cn/.ynie in, 250 large white, 405 lipins in, 23 sugar sup])ly to, 637 Kinase, 01, 501 Klausner's serum reaction, 237 Krait venom, 155 Kupfl'er cells. 474 Kynurenic acid, 572 Laccase, 07, 73 Laccol, 73 Lactic acid, 85, 248, 257, 278, 047. 648 relation to drug jjoisoning, 556 Lactim, 020. 028 Lactose, 280 Lactosuria. 656 Laking of blood, 215 Lamprey serum, toxicity of, 104 Lanthanin, 44 Lardaceous degeneration. 417 Large white kidnej^s, 405 Larynx, 423 Lassitude, 585 j^athrodectes tredecim-guttatas, 158 Laurie acid, 111 Lead. 240 Lecithids, 228 Lecithin. 23, 24. 25, 85, 03, 100, 217, 270, 285, 280, 303. 357, 401, 437, 401. 502, 000 as antigens, 236 decomposition. 584 enzyme in tissues, 77 in diphtheria bacillus. Ill relation to opsonins. 160 stearvlolevl. 24 Leech extract. 318. 332 Leptothrix. 405 Leucine, 20. 87. 06. 258. 270. 309. 327. 357. 380. 542 1 eucocidins. 230 Leucocytes. 70. 75. 88, 01. 103, 238. 280. 290, 404 action of thorium-x on. 377 autolysis o^ 308 chemotaxis of, 250 dispersion of. 273 e'lect of electric current on. 250 glycogen in, 432 433 692 INDEX Lenoocytos, migration of, 253 due to c'liaiige of surface tension, 271 relation of cell types to, 259- 261 phagocytic. 631 action, 253 proteolytic enzymes of. 94-96, 277 relation to ameba. 2G() thermotaxis of. 261-262 Lencocytic enzymes, 270 protease, (i3, 6.1, 103 walls, 272 Leucocyto-agghitinin, 240 Leiicocytolysins, 1.53 Leiicocytolysis, 214 Leucoc'vtolvtic serum, 239 Leucocytosis, 239. 292, 319, 375, 450 general, 2 J '3 Leucocytosis in burns. 261 mastccU, 261 Leucocytotoxins, 154, 239, 240, 311 Leueolvsis, 218 Leucoiienia, 200, 239, 320 Leukemia, 71, 75, 70, 103, 293. 298, 307-312, 321, 376, 450, 031. 034 chemistry of blood in, 308 eliroiiic. 521 lymi)hatic. 260, 307 myeloid, 433 myelogenous, 95, 307 protein metabolism in. 309 a-ray, 377 Leukemic blood, coagulation of, 308 Leukoprotease, 95, 277, 278 Lerulose, 655-656 I^evulosuria. 031") alimentary, 055 idiopathic. 056 mixed. 656 spontaneous, 656 alimentary. 655 Light, bactericidal action of, 374 dillVrent rays of. 374 ^ effect on antibodies, 374 on enzymes. 374 on oxidation processes, 375 on oxidizing enzymes, 375 on ]iroteiti solubility, 375 on tissues, 374 377 on toxins, 375 stroke, 481 Lime laden drinking-water, 453 Linin. 44, 45 Lipase, 58, 62, 67, 77-80, 95, 103, 138, 262, 278. 280, 291, 358. 407, 535, 595 in 1\ inphocytes, 79 in ui'ine. 78 pancreatic. 3*^6 Linemia. 412 414 in iilc, holism. 413 ]>ipins. 23 25, 400, 498-499 Lipochrome, 393, 474-476 tests ft)r, 475 Lipoeyanin, 475 _L,ipofuscins, 393, 475 Liooidal degeneration, 410 Lipoidemia. 413 Lipoids. 23-25, 65, 127, 243, 461 adrenal, in arteiial disease, 009 in pneumonia, tiOO in renal disease, 6!)9 anisotropic, 25 doubly refractive. 25 echinococcus, antigen from. 108 hemolytic, 307 in immunity reactions. 108 relation t-,) antiliodies, 169 to fatty metamorphosis, 404-406 tape worm, antigen from, 168 Lipolysis, 413 Lipolytic enzymes, 77 in lymphoid cells, 77 Lipoma." 432, 511-512 calci'ying, 441 Lipo-peptids. 401 Liquefaction, 276 necrosis, 382 aseptic, 276 Lithemia. ()27 Lithofellic acid, 463 Liver, 83. 486 abscesses, 135 acute yellow atrophy, 379, 403, 538, 539-550. 557. 569 l)lood in. 546 alcohol oxidase in, 248 atroi)hy of. 474 autolysis of, 231 effect of thyroid on, 591 caffein demethylated in, 247 chhu'oform necrosis of. 320 cirrhosis of, 320. 355 degenerations, 100—102 diseases, 292. 480 edema of, 340 necrosis, 324 ether, 325 passive congestion. 431 site of urea formation, 526 Luml)ricin, 143 Liuigs, e08 -Melanemia, 471 Melanin, ,.i, 207, 467-472, 577 as antigen, 170 composiuon ol, 468 properties of, 469 loxicity of, 471 tumor, 496 Melanogeii, tej>ts for, 470 -Meuino-prolein, 4(»b AieUlno.•^arcoma, 469 -Melanosis, 467 intcBtmal, 470 Melanotic adrenal tumor, 472 tumors, 467, 471-472, 502, 577 Melanuria, 4(0, 473 -Melena neonatorum, 298, 321 Melituria, 636. fcsee also Diabetes Membrane, nuclear, 45, 47 semipermeable, 30 Meningeal ellusions, 359 -Ueningitis, 360, 361 epidemic, 355 tuberculous, 360 Menstrual blood, 320 Mental diseases, 229. 362 latigue, 562 Mercury, 246, 258, 276, 542 bichloride of, 43 hxation, 246 Metabolic activitj', 335 changes after hemorrhage, 301-302 disturbances, 566-589 Metabolism, 57, 58, 59, S7, 171 abnormalities in, 523-565 basal, 590. 602 carbohydrate, 602, 635-671 ell'ect of thyroid on, 591 fat, 602 gouty, 629 111 cancer, 505-507 intermediary, 524 of hemoglobin, 477 protein, 444 in leukemia, 309 in pernicious anemia, 306 j>urine. 629 relation of thyroid to, 590-591 respiratory, 444 uric-acid, 618-634 Metalbumin, 513 Metallic coi)per, 258 poisons, 246 sulphides. 247 Metal-protein compounds, 247 .Mctanu)r])liosed calculi. 455 -Metamorpliosis, fatty, 75, 301, 328, 397-399 by absorption. 408 causes of, 406^10 relation of lipoids to, 404-406 Metaplasia. 285 -Metastatic calcification, 438^39 694 INDEX Metazou, 372 :Motliiino. 2r)0 Methemoploljin, 297 ^Metlicnioglobineniia, 481 Methyl cyanainide, 598 guanidino, 505 mercaptan, 5(17, 582 Metliylation, 247, 250 Mctlivk'iu' blue, reduction of, 253 Metliylfiuauidine, 199 Micrococcus cereus tiavus, 132 Migration of fluid. 412 of leucocytes, 253 due to change of surface tension, 271 relation of cell types to, 259-261 of neutral fat, 412 Mitochondria, 47, 49, 395 Mocassin venom, 150, 228 ]\Ioist gangrene, 388 Molecule, colloid, 395 radicals in, arrangement of, 176 Monobasic urate, G20 Mononuclear macrophages, 190 ^l()n(>]iotassium phosphate, 561 Monosddium urate, 620, 629 Monovalent antigens, 179 ]\Iorbus maculosus, 480 Morchella esculenta, 228 Morner's body, 309 Morphine, 165, 244, 258, 379, 560, 592 tolerance, 244 Motile bacteria, 256 Mouse tumors, 173, 432 Mucin, 109, 195, 281, 356, 426 connective-tissue, 428 epithelial. 427 in myxedema, 603 Mucoid degeneration. 426, 427-428 Mucoids of ovarian cysts, 513 Multiple myeloma. 518-519 sclerosis, 414 Mummies. 60 Egyptian, 411 MurirnidiT?, 163 Muscarine. 123, 124, 260, 567. 585 ^Muscles, 22. 83 decomposition of. 412 waxy degeneration, 392-393 Muscular asllienia, 614 dystrophies. 393 Musli rooms. 227 poisons, 146, 177, 539 Mussel, extract of, 332 Myelins. 25, 404, 461 Myelol)last8, 476 Myelocytes, 476 Myelof/enous leukemia.. 95. 307 M'veloid leukemia, 4.33 ATveloma, multiide. 518 519 Myelopathic al1)umosuria. 519 521 occurrence of. 520 521 Myelotoxic seiiim, 240 Mykol, 111 Myocardium, 486 chronic degeneration of, 284 lecithin in, 24 Mvogen-fibrin, soluble, 391 JMvoma. 432, 509 :\Iyosin, 391 Myosinogen, 391 Myristic acid, 514 Myristinic acid, 110 Mvrosin, 61 Myxedema. 428, 586, 590, 601-604 mucin in, 603 Myxofibroma, 428 :\rVxoma. 428 Myxosarcoma, 428 Naphthalin, 249 Narcosis chloroform. 540-541 Narcotic poisons, 87 Narcotics, 246 Nasal septum, 423 Nascent oxygen, 60 Nastin as antigen. 168 Necrobiosis. 99, 117, 368, 438 Necrogenic substances, 135 Necrosis, 253, 276, 367-390, 631 anemic, 328, 368, 372, 381 aseptic liquefaction, 276 causes of, 371-381 chloroform. 408 of liver, 320 coagulation, 381-382 due to physical agents, 380-381 due to ic-ravs, 376 fat, 62, 80," 384-388, 414, 442, 486 hepatic, 371 icteric, 487 intrahepatic, 387 liquefaction, 382 liver, 324 ether. 325 radium, 376 Necrotic areas, central. 273 tissue, 97 Necturus, 229 Negative charg(\ increased. 285 chemotaxis, 257 Nematodes, 140 143, 432 Nephrectomy, 292 Nephritic transudates, coiu])ositi(m. 354 Nephritides. chronic, 240 Nephritis, 70. 78. 231, 293. 208, 321. 343. 416. 417, 558. 587. 610, 631 acute, 292. 349. 379 autolysis in. 98 chroTiic. 349. 424. 527 interstitial. 527 liemorrhagic, 385 interstitial. 349. 611. 627 parenehvmatous, 349, 350, 527. 570 clironic. 364 uranium, 344 iNimx 695 Xoi)liiolysins, 238 Ncplirolysis, a',i2 Ni'Kluohtic sc'ium, 240 Xephrotoxins. 240 Nerve cells, foajiulatiiij; teiiiiiorature. 372 hypoisensitiveness of, 530 neuralpie, 351 Nerves, vasoconstrictor, 253 vasodilator, 253 Nervous diseases, 58G system, sympathetic, 472, 503 Neuralgic nerve, 351 Neurine, 123, 124. 562. 507, 585 toxicity of. 124 Neuritis, 287 Neurotibroma. 472 Neurofrlia, 4(il Neurolytic scrum, 240-241 Neuropathic edema. 351 Neurotic edema, 345 Neurotoxins, 153 venom, 154 Neutral fat, migration of. 412 phos])hates. 021') Neutralization. ?4S by chemical comliination. 523 of organic acids. 251 Neutrophiles, 203 Newborn, jaundice in, 486 Nicoll prisn;s, 404 Nicotine, 012 Nidus, 451 Nile blue sulphate, 401 Ninhvdrin, 204 test, 301 Nissl bodies, 46, 49 Nitrobenzol. 217 Nitrogen, 326 amino-acid, 520 blood, Jion-protein, 534 colloidal, 500 dianiiiio, 545 e(|uilibrium, 506 non -protein, 520 Nitroglycerin, 217 Nitro-proteins. 176 Nocturnal hvperopnoea. 560 Non-electrolytes, 35, 330, 347 Nonoic acid. 414 Non-protein organic contents. 357 Non-specific hemagglutination, 183 reactions in typhoid fever, 174 Nuclear membrane. 45, 47 Niwlcase, 44, 85, 07, 105, 300, 501 purine, 622 Nucleic acid. 22. 23. 616 bacterial. 100 thymus, 621 Xiiclein. 22 bodies. 610 hydrolysis, 284 katabolism, 310 Nuclcinic acid, 250 Nucleo albumins, 23 Nucleohiston, 282 Nucleohis, IS, 45, 47, 48 Nucleo-profeins, 21, 22, 44. 105. 221, 238, 241. 270, 285, 280, 357, 45(;, 632 bacterial, 108 impure, 318 Nucleoside, 022 Nucleotids, 622 Nucleus. 18, 454 acidity of, 40 chemistry of, 44-46 of bacteria, 100 structure of, 48 Obesity, 416, 500 Obstructive jaundice, 231. 416 Occlusion of thoracic duct, 340 Ochronosis, 473, 482, 577 Oil, croton, 270 of turpentine, 276 rancid, in alkaline solution, 267 Oleic acid. 110, 22S Oleum pulgeii, 400 Ophiotoxin, 150 Opsonins. 207-208. 264, 358 action of chemicals on. 208 chemical nature nf, 208 relation of lecithin to, 100 Orang-utan, 025 Organ extracts, 318 Organic acids, 346 contents, non-protein, 357 dves, 34 poisons. 248 252 Organs of cancerous patients, 105 Ornithine, 581 Osazone, 387 Osmic acid, 43 Osmosis, 29 33. 337 Osmotic concentration. 284 equilibrium. 335 exchanges. 337 pressure. 30. 32. 210. 332. 334-336, 371. 305. .527. 643 changes in. 380 disparity of. 344-346 increase of, 254 of colloids, 38 Ossification, defect in, 617 relation of calcification to. 435-436 Osteitis deformans. 445 Osteogenesis imperfecta. 447 Osteohemachromatosis. 474 Osteoid change, 285 Osteoma. 432 Osteomalacia. 438, 443 445. 446, 520, 500 Osteomyelitis, 117 Osteoporosis, 500 senile, 444 Osteosarcoma. 438 Ova. 285 696 IXDEX Ovarian colloid, 513 cystadenoina, 513 cysts, contents of, 512 dermoid, 514 mucoids of, 513 tumors, -42(5 Ovaries, a;-ray atrophy, 37G Overproduction vs underconsumption in diabetes mellitus, 070 Overton theory, 31, 50 Ovomucoid, 1!)5 Oxalic acid, 248, 251, G20 calculi, 584 Oxaluria, 5S4 Oxazone base, 401 Oxidases, 55, 71, 05, 103, 280, 358 alcohol, 71 in tumors, 76 intracellular, 407 reaction, 309 xanthine, 71 Oxidation, 33, 68, 69, 248, 638, 639 impairment, 345 of cystine sulphur, 614 processes, 374 efl'ect of lights on, 375 reduction in, 348 uric acid, 248 Oxidative processes, 69 Oxidizing enzymes, 68, 60, 371 efl'ect of light on, 375 Oxy-acids, 101 Oxvbutyric acid, 567 Oxygen, 326 active, 374 nascent, 60 relation to sarcolactic acid, 555 Oxygenase, 72 Oxyhemogloliin, 35, 477 Oxyniandelic acid, 543 Oxypurines, 622 Oysters, extract of, 332 Palmitic acid, 514 Palsy. See Paralysis Pancreas, 80, 85 activating substance, 77 atropliy of, 393 cells, eml)oli of, 387 diabetes, 665 671 historical, (165 symptoms, 667 extiry)ation, 430 effects of, 6()5 islet theory, 666 nature of inlernal sccr(>tion of. 666 self -digest ion of, 388 Pancreatic aulolvsis, 386 calculi. 461 462 dial)etes, 412 fistula, 443, 558 juice, 127. 245, 542 lipase, 386 Pancreatitis, 385, 48(;, 6(t7 Papain, 62, 103 Papayotin, 258 Papillary cysts, intraligamentary, 514 Parabanic acid, 626 Parabiosis, 564 Paracentesis, 356 Parachroniatin, 44 Paracresol, 571, 576 Paraheinoglobin, 297, 477 Para-hydroxy-phenyl-ethylamine, 576 Paralactic acid, 544 Paralinin, 44 Paralysis, 487, 531 agitans, 599 diver's, 326 general, 360 progressive, 301 vasoconstrictor, 312 Paramecia, 252 Paramoecium, 266 Paramucin, 427, 513 Paramucosin, 513 Paramyosinogen, 391 Para-oxyphenyl acetic acid, 571, 576 Para-oxyphenyl-propionic acid, 571, 576 Paraphenylene diamine, 248 Parasites, animal, 260, 340, 432 chemistry of, 134-143 eosinophilia (chemotaxis) , 134 intracellular, 474 Parasitic eosinophilia, relation to ana- phylactic, 135 Parathyreopriva, tetany, 598 Parathyroid, 597-599 in exophthalmic goiter, 607-608 tetany, 549 tissue, 241 tumors, 432 Paratliyroidectomy, 598 Paratyphoid bacilli, 173 Parenchymatous degeneration, 395 goiter, 601 nephritis, 349, 350, 527, 570 chronic, 364 Parotid, (i03 Parovarian cysts, 514 Parowsmal hemoglnlunuiia, 232-233, 302 Pars intermedia, 015 Passive congestion, 314, 333 of liver, 431 hyperemia, 313 blood changes in, 313 lympliatic coiigestiou, .341 sensiti/ation. 191, 203 Pattiogenic bacteria, <'ii(loto\iiis in. 119 Pellagra, 287 Pt'lvis, renal, 457 Pentoses, 22, 25, ini. 497, 649. 650 Peiitx)suria, 577 chronic, 650 Pepsin, (il. 62. 04, 07, 103. 127. 5(i6 P(>ptid-splitf ing enzymes. .'iOl Peptolylie enzymes. 91 INDEX 697 r.'ptuncs, 27S, 27!l, 2Sl, .iOi), 544, 'iiil, .HiS l*eii'fiitad sufjar, (i41, (i4'2 Perilnoiieliial {jlamls, 4ti4 rericardial fuKillcatitin, 4:50 rericarditis, 355 ureiiiic-, ").!1 Peritonitis, 71, 355 carciiiuinatuiis, 353 general, 433 idiopathic, 353 IVniiangaiiate reduction index, 361 rerineability, 3i), 370 tor vital stains. 371 of capillaries, 333-334 lo colloids, 371 IVniicious anemia, 231, 305-307, 317, 477, 587 analysis of orjjans in, 305 calorinietric studies in, 306 causes of, 307 ciieniical changes in, 305 due to hemolytic poisons, 300 iron ill corpuscles, 305 l)rotein nn'tabolism in. 300 vomiting of pregnancy, 538 Peroxidase, 72, 595 Peroxides, GO Phagocytes, hemolysis by, 230 Phagocytic activity, 417 cells," 219 foamy, 405 cndotiu'lial cells, 296 leucocytes, 031 Phagocytosis, 39, 91, 207, 208, 253. 254 256, 261, 262-267 273, 314 by protozoa, 202 etfect of calcium salts on, 263 magnesium salts on, 203 inlluencc of serum on, 264 relation to cholesterol, 263 results of. 204 theories of, 266-275 Phallin, 147, 227 Phallusia mamilhita. 170 Phanerosis, 402 Phaseolus nuiltillorus, 223 PluMiacetin. 4S2 Plienol. 24S. 25S. 451, 507, 571, 575 ])ois()ning, 245 Phenolase, 73 PlK'iiylalanine. 469, 495, 507, 576, 578 Plienyl-ethyl amine, 576 Philo'catalase. 70 Phlclxdiths. 459 Plilogosin. 250 Phloretin. 059 Phloretiiiie acid. 059 Phlorhizin, 500, 659. (JCO diabetes, 493, 557, 659-664 Phlorose, 659 Phosphate anunonio-magnesium, 455, 456 Phospliate calculi, 457 458 triple, 3H9 Phosphates, 357 neutral, 626 Phospluitids in tubercle bacillus, 111 Phosi)ho-glycn proteins, 23 Phospholipin, 4!l protein, 310 I'liospiio-nuclease, 022 IMiospiioproteins, 23 Phosphorescence, 380 Phosphoric acid, 20, 303, 441, 444 excretion, 310 in calcitication, 442 Phosphorus, 247, 252. 293. 399, 407, 499 in exophthalmic goiter, 005 poisoning, 74, 100, 317, 320. 413, 539, 540, 557, 569 Photosensitizing action, 480 Phthisis, 79. 281, Physical absorption, 24() chemistry of cell, 26-43 solution, 246 Physico-chemical factors, 349 Phvto-preciptins. 192 Phytotoxins, 144-148. 107. 293 immunity against, 145-146 relation to proteins, 144-145 toxic action, 140 Pigment, 279. 295. 389 bacterial, 132-133, 2S0 alcohol soluble, 133 insoluble, 133 water solulile, 133 bile. 486 487, 566 blood, 476-484 fornuition, 73 malarial, 467 urinary, 525 Pigment-granules, 49 Pigmentation, 73 in jaundice, 489-490 malaiial, 474 patliological, 467-491 Pilocarpin, 260, 312 Pineal gland, 017 Piper az in, 311 Pituitrin, 014 Placenta, putrid, 574 retention of, 560 Placental cnd)oli. 535 Plague agglutinin, 1S4 bacilli. 107 Plant tissues. 195 Plasma, exudation of. 253 fat of, 290 sugar of. 290 Plasma phaer(>sis, 295 Plasmodia. 231 Plasmodium malari:c. 136-137 PlasuKdysis. 107 Plasmoptysis, 31, 107 698 INDEX Plasmorrhexis, 31 Plasmose, 316 Plasnioaomes, 430 Plastein. 57, 105, 115 Plastin, 44, 45, 49 Platypus venom, 157 Pletiiora, hydremic, 298, 334, 342, 347 Pleurisy. 281, 35G tubereulous, 350 Pleuritic fluid, 521 Pluriunim resistance, 305 Pnein. 09 Pneumoliacillus, Friodlandor's, 258 Pneuniocoecus. 70, 129, 198, 225, 289, 482. 488 anaijhvlatoxic poison. 198 Pneumonia. 03, 78, 281, 282. 292, 293, 319, 321, 350, 355, 413, 431. 433. 480. 500, 509, 034 adrenal lipoids in, 009 autolysis in, 96 Pneumonic exudates, 404, 415 sputum. 490 Pneumonoknniosis. 465-466 Pneumothorax, chemistry of, 365-366 dry. 305 purulent, 305 putrid, 305 Poison, ants, 160 bacterial. 592 bee, 160 toxolccitliidin in. 100 black flies, 100 blood, 400 causing somnolence. 502 centipede. 159 160 convulsive, 87 crab, 164 froof. 150 hemolytic. 177 hornets. 100 in coelenterates, 164 in eel serum, 104 in skin f)f frf)fr. 102 of salaniandcis. 162 inorr'anic. 246 248 I'letallic. 240 musliroom. 140, 177 naicotic, 87 non-antifrenic, defense apainst, 243- 252 immunity a<;ainsl. 243 orpanic, 248 252 prodiu'cd in su])erficial burns. 562 565 protoplasmic, 379 scorpion. 157 158 spider, 158 159 steato-renetic. 403. 409 toad, 150. 161-102 in blood, 101 vcfretable, hemolysis by, 225 226 wasps, 100 Poisoning, 485 abrin, histologic changes, 140 aceto-acetic acid, 553 arsenic. 342, 539 bichlorid, 532 chloroform, 379, 539, 557 chromium, 477 cobra, 152 drug, relation of lactic acid to. 550 food, 122 gas, 501 mushroom, 539 ])henol. 245 phosidiorus. 74. 100. 317, 320. 413, 539, 540, 557, 569 ptonuiin, sources of, 122 ricin, 245. 573 histologic changes, 146 shelUisli, 342 snake, pathological anatomy. 153 strawberry, 342 stiychnine. 390, 392 venom, loss of bacterial power, 155 thrombi from, 153 viper, 152 Poisonous bacterial proteins, 131-132 fish, 162-164 Polished rice. 287 Pollen, active toxic constituent an al- bumin, 147 Polyamino-acids, 285 Polycythemia, 293, 313 Polymerization, 429, 638 Polyneuritis gallinarum, 287 Polypeptid, 20. 282 synthetic, 194 Polvphenoloxidases, 71 Polyplasmia, 303 Polypoid tumors'. 428 Polysaccharides. 656 Polyuria. 015 Porges-Heriiiaun-Perutz reaction. 237- 238 Porphyrins. 480 Portal vein thrombosis, 354 Positive cheiiiotaxis. 250, 271. 270 Postmortem changes. 71, 101-102 decomposition. 477 hemolysis. 224 lymph' flow. 346 secretion. 332 Potassium, 26. 45. 303, 499 chlorate. 232 cliloride, 336 ])liosphate. 290 salts. 48. 257. 284. 525 Potocytosis. 333 P-oxyphenyl-lactic acid. 543 Pieci])itate, resistance of. 190 Precipitation of colloids. 40. ]S7. 301 S|iecific, 507 Precipitinogen. 189 Precipitins. 179, 189 193. 240 barterial. 192 INDEX 699 Precipitins, cluiiiical jjiopcrties, 193 for ilyt's, ITii iuiptopliori', I'.H reactions. l:i!t. 358 relation to anapliylact in, li);5 rrecipitoid, 1!»1 Precocity, sexual, OOS Piefrnuncv, 04, 8(), 410, 4:50, 453, (507, 012 acidosis of, 559 diagnosis of, 200 pernicious voinitinjr of, 538 toxemias of, 533 539. .").")7 Preputial concretions. 463 464 Pressor bases, 125, 507, 576 577 substance, 007, 014 Primary toxicity of foreign sera, 203 Proliferation, 283 285 chemistry of, 2S4 Propionic acid, 507 Prostatic concretions, 437, 404 Prota-ron. 25. 100 Prolamin. 20, 131, 285, 420 sperm, 177 Protease. 81-105. 277, 201, 301 leucocytic, 03, 05, 103 Protective ferments. 107 Protein, 19-23, 33. 335 bacterial. 270, 2.S0 poisonous, 131-132 toxic, not specific, 132 toxicity of, 131 Pence- Joiies, 30!), 518, 570 constitution of, 520 reaction of, 519 blood, 290 cell, autolysis of, 403 chanp:es in, 440 cleavajre products, 194 coagulated, 383 compound, 21 concentrated, 420 concretions, 452 contents of edema fluids. 356-357 etTects of skin injection, 200 electro-negative, 396 foreign, 165 hydrolysis, 569 insoluble, 23 intoxicating dose, 195 katabolism, increased, 302 metabolism, 58, 444 in l(ndrculosis, 79. 407, 505 ulcerating. 509 veins, 439 700 INDEX Purine, 04, 248, 357, 497-498, 584, 618 bases. 22, 310. 525 bodies, 280, (IIS enzymes, 497 498 excretion, (il4 nictaljolisiM. fi20 nitrogen in tumors, 4!t5 nuclease, (522 nucleus, 618 Purine-splitting enzvmes, 105 Purpura, 2n7 hemorrhafiica, 230, 2!)7, 317 neonatorum. 322 Purul(>nt tluicls. 358 pneumotliorax. 3ti5 Pus, 70, 78, 93-94 cocci, 129 composition of. 277-280 decomposed, 414 inspissated collections of, 438 serum. 278 279 Pus-collections. ins])issated, 415 Pus-cor])nselcs, 278 Pustule, malignant. 276 Putrefaction, 122, 388, 410, 570-589 intestinal, 245, 588 lack of, 362 of antitoxin, 182 protein. 571-583 Putrefactive bacteria. 566 Pntrescine. 123, 567. 582, 583 Putrid bronchitis. 434 cancers. 574 ]>lacenta, 574 pneiunothorax, 365 purulent exudates, 574 Pvcnosis, 07, 285, 328, 369 Pyin, 279, 421 Pvocvaneus toxins, 218 Pyocyanin, 133 Pyocyanolysin, 224 Pyogenetic proteins, 108 Pyogenic ]>roteins. 276 Pyonephrosis. 433 Pyopneumothorax, 365 open. 365 Pvosalpinx, 451 Pyridine. 247, 250. 521 Pyrimidine. 22. 287. 619 Pvrocatechin. 575. 612 Pvrogallol. 217. 249 Pyrrole. 469 ring. 471 QUADRU'RATE, 620 Quinin, 251, 257, 258 l!Ari:Mi/.i:i) ])r(itciii of Dakin, l(i6 Padiating structure. 448 Padicals, aminoacid, 176 aromatic, 176. 191 in molecule, arrangement of. 176 Radium. 60. 104, 501 necrosis. 376 Pancid oils in alkaline solution, 207 Hauid elimination, 245 Hattlesnake venom, 150, 228 Rays serum, toxicity of, 164 Reaction of degeneration, 392 reversibilitv of, 56 Real alkalinity- 291 Receptaculum chvli, 303 Receptors, 129 Red blood cells. 70 corpuscles. 289 degeneration. 498 Reducing enzymes. 74 Reduction. 247, 248 chemical changes in crystalloids bv, 33 of methylene blue, 253 Refractoriness, 2-14 Regeneration, 253-288. 371 Renal disease, lipoids in, 609 dropsy, 344 edema. 348-350 elimination, 524 e])ithelium, calcification of. 438 pelvis, 457 Rennin, 61, 67. 115. 291 Repair and growth, chemical basis, 285-288 Respiration, internal, 548 Respiratory metabolism, 444 Retinitis, albuminuric, 530 Reversibility of reactions, 56 Rhabdomyoma. 432. 497 Rheumatism, 321, 480 Rheumatoid arthritis, 629 Rhinoliths, 459. 464 Rhi/opods. 262 Rhubarb, 457, 584 Rims diversiloba, 147 toxicodendron, 147, 167 Rice, polished, 287 Ricin. 75. 144. 177. 223. 225. 293, 451 agglutinating poAver, 145 phytotoxin. 167 ]ioisonintr. 245, 573 histologic changes, 146 toxicity of, 145 Rickets, 287, 445-447 relation to osteomalacia, 446 Rigor, antemortcm. 390 mortis. 101. 390 393 relation to acidity. 391 Robertson film theory, 51 Robin. 144. 225 Roentgen rays. See also X-ratts Rovida's hvalin substance, 279 Rubber. 36' Russell's fm-hsin bodies, 424 SAcruAUosK. 280 Saccharosuria. 657 INDEX 701 Siifiiuiiii, I'M) JSiifjo spliH'ii, 4 IS Saliiinaiiders, poisons in skin of, 162 Salicyl-alcU'liyde, 72 Saliovlic atid, 250 Salivary calculi, 462 phyalin, 42!) Salmon, spawn inj,', 420 Salpin<,ntis. 4:!;? Salts, 71, 290, :332, -.Ul alkaline, ;3."37 bile, 487, 500 . calcium, 4S, 317 fractionation iiictliod. ISl inoi'^'aiiii'. 2S, 4S, .") 1 j)otassiuni. 4S, 32") relation of, to ag<:lut inal ion, ISO Salvarsaii, 243 anapiiylactic reactions with, Iti!) Samanilaridin, 102 Saniandarin, lti2 Sand, intestinal. 4(i;! Saponin. 217. 226 228. 244 etrect of, 227 Sajionin-cJiolesterol coinpound. 220 Sajjonin-lecitliin compound, 220 Sapotoxin, 227 .Sapremia, 380 Sareoeystin, 137 Sarcolaetic acid, 543, 555-557, 501 relation of oxygen to, 555 Sarcoma, 437, 468," 497, 634 spindle-cell. 494 Sarcosporidia, 135. 137 Saturation limit, ()39 Scarlatinal virus, specific. 235 Scarlet fever, 252, 321, 349 Sears, corpus luteum, 480 episplenitis. 424 tissue, 424 Sciatica, 586 Sclercnchymatous ^articles. 463 Sclerosis, multiple. 414 senile. 439 Selerostoma equinuni. 143 Scorpion poison, 157-158 toxin, 177 Scorpcena scor]ilia. 162 Scurvy, 286. 297 infantile, 287 Sea-snake venom, 155 Sea-urchins. 2S4 eggs, 47. 70 developing. 2S5 Sebaceous glands, 465 material. 515 Secondary anemia, 225. 294, 300-302, 317, 322 Secretory activity, increased, 343 grannies. 49 theory of Ivnipli formation. 332 Selenium. 247 Self-digestion of liver, 75 Seminal vesicles, 474 Semip('iiiicai)if mcniliraMes, 30 Senile osteoporosis, 444 sclerosis. 439 Senility, 371, 5S7 Sensitization, 187 active, 191, 202 by brilliant green, 220 by silicic acid, 220 passive, 191, 203 Sepia from squid, 468 Sepsis, 350 local, 433 Sej)tic conditions. 485, 610 infections. 7 1 softening, 329 Septicemia. 224. 319, 433. 5.39 Septum, nasal, 423 Serine, 495 Serosamucin, 357 Serozyme. 31(i Serum albumin. 21. 29() antiplatelet, 239. 300 anti|U()tense, 277 bacteriolysis, 208, 210-214 Idood. 29*0 colloids, electro-positive. 452 eel, 177, 229, 318 poisons in. 164 endotiieliolytic, 239 foreign. 322 globulin. 205 iieniolysis, mode of. 222 influence of. (m phagocytosis. 264 Lami>rev, toxicit\' of. 164 leucocvtolvtic. 239 lymph'atolytic, 240 myelotoxic, 240 neurolvtic, 240-241 l)us, 278 279 Rays, toxicity of. 1()4 reaction. Klausner's. 237 snake, 156 therapy. 130 thyrolytic. 241 treatnient of e\i>phthalmic goiter, 605 Sex cells, 174 Sexual inactivity. 614 precocity. 608 und(>velopment. 614 Sheep spermatozoa. 173 Shellfish jioisoning, 342 Shells of invertebrates. 437 Shock, ana])hylactic. 320 Sialolithiasis." 4ti2 Siderosis, 46(5 Silicates. 465 Silicic acid. 34. 40, 223 sensitization l»y, 220 Silver. 246 nitrate. 276. 294 Sistrurus. 149 Skatole. 248. 258, 469, 567. 572, 575 Skatole-carbonic acid, 567 702 INDEX Skatoxyl, 240. 572 Skeletal growth, 591 Skepto-phylaxis, 199 Skill cancels in colored races, 467 changes in, 614 Smallpox, 252, 570 Smegma, 463 Snake poisoning, patliological anatomv, 153 serum. 156 venom, 148-157, 177, 223, 293, 318, 319, 564 Snake-bites, mortality from. 151-152 Soaps, 25, 65, 102, 279, 4U7 calcium, 387 formation of, 441-442 cysts, 515 of fatty acids, 384 toxicity of, 414 Soda dropsy. 350 Sodium bicarbonate, 218 diphosphate, 455 glvcocholate, 237 salts, 257 Solanacese, 223 Solanidin, 227 Solanin, 227 Solidago, 147 Sols, 34 Solubility of colloids, 36 Solution of intercellular substance, 343 tension. 187 true, of crystalloids. 35 Spawning salmon, 420 Species specificity, 176 Specific antibodies, therapeutic stimu- lation of, 174 precipitation, 507 scarlatinal virus, 235 Specificity altered by cliemical charac- teristics. 17(i by physical measures, 176 its dependence of biological relations, 171 172 on chemical compusitioii, 172-173 of anti-cnzynies, M'> physico-chemical factors. 175 species. 176 Sperm histones, 177 prot.amines, 177 Spermatocele fiuid, 359 Spermatotoxin, 241 Spermatozoa. 62, 214. 23S. 256, 285 heads of. 44 shec]), 173 Spermatozoids, 254 Sperm in. 2S0 crystals. 311 Sphcroliths, 452 Spideis, 60 poison, 158-159 toxin, 177 Spina bifida. 360 Si)inach, 457, 584 Spindle-cell sarcoma, 494 Spleen, 296 autolysis, microscopical and chemi- cal changes, 369-370 sago, 418 Splenectomy, 229. 232 Splenic amyloid, 420 hemolysis, 232 infarcts, 328 tumor, 486 Splenolysin, 241 Splitting olT of water, 248 Spores, 112 bacterial. 367 Sputum, 93, 96, 280-283, 433 asthmatic. 311 bronchiectatic, 4 1 4 chemistry of, 280 283 pneumonic, 490 Squamous cell cancer, 516 Stagnation, 323, 452 Staining bacteria. Gram's method, 112-113 fat, in tubercle bacillus. 111 properties of amyloid, 420—421 reactions of bacteria, 112-113 Stains, fat, 401 vital, 50 permeability for, 371 Stalagmometer, 208 Staphvlococcus, 70, 78, 92, 94, 224, 256, 278 aureus, 320 pyogenes, 264, 276 aureus, 114, 132, 475 citreiis, 132 Staphylokimase, 117 Staphylolysin. 224 characteristics of, 224 Starch, 34 Starvation, 292, 302, 393, 394, 559 relation to autolysis, 87 Stasis, 314 general, 341 Stearic acid. 514 Stearyloleyl lecithin, 24 Steatogenetic poisons, 403, 409 Steatosis, cholesterol, 405 Sterilized bacterial cultures, 258 Stimulation, mechanical, 263 tactile, 262 Sting of insects, 351 Stomach, cancer of, 104. 504 effect of venom in, 152 Stone. See Cdlciiliis Strangulati'd hernia. 341 Strawberry juice, 332 ])oisoiiing, 342 Strci)t70 Testicular tumors. 432 Tetanization. 344 Tetanolvsin, 224 Tetanus, 390 bacillus. 70, 177 toxin. 70, 75, 103, 128, 260, 379, 592 digestion of, 127 Tetany, 287, 581!, 587 parathyreopriva, 598 parathyroid. 549 Tethelin, 015 Tetrodo-toxin. 104 Tetrodon, 164 poison in ovaries and eggs, 103 Tetroses, ()48 Theobromine. 248, 019 Theophyllin, 022 Therapeutic immunizations, 173 Thermal injuries. 253 Tliermic alterations, 372-373 Tliermoprecipitins, 191 Thermostable antilipase, 79 Thermotactic edect, 255 Thermotaxis. 2()9 of leucocytes, 261-262 Thermotro])ism. 255 Thigmotropism. 25(1 Thoracic duct, occlusion of. 340 lymph, osmotic pressure of. 33!> Thorium. 21 1 Thorium-J". 259 action on leiu'ocytes, 377 Thrombin, 291. 316 Thrombogen. 310 Thromhokinase, 299, 310, 320. 380 Thrond)oplastin. 310 Thrombosis, 105. 303, 315-325, 340 tibrin-ferment, 325 portal vein, 354 Throndius. 223. 293 agglutiiuitive. 325 bile. 485 librinous. 323 formation of. 322-325 hyalin. 224. 324 325. 374 secoiulary clianges in, 325 tvphoid."224 Thvmine. 109. 019 704 INDEX Thviiiol. 24!1 Tliyiims. 4!t4. 616 617 extirpation of, (iHi liiston, 4 lit mick'ic ai-i!*;! cystic, 42(1 detoxicatoi'X' fuiictidii of, .")!)! diseases of! 590 593 effect on carhnln drate iiietai)olisni, r)!)l on growth. .'Jlll on liver autolysis. .191 extract, therapeutic use, G02 functions of, 590-593 lipase. o05 peroxidase, 595 relation to metabolism. 590-591 secretion, deficient, 590-591 tissue loss of, 590 Thvroidectomv. 241. 002 effects of. 591 Tlivroidisnuis. 004 Tliyroiodin, 128. 420. 593 Tliyrolytic serum, 241 Tissue coagulins, 323 cultures, 40S disintegration, 258 effect of light on. 374-377 necrotic, 97 relative susceptibility to heat, 373 Tissue-cells, autolytic enzymes of, 277 beluivior of. 273 Toad poison, 150, 161-162 in blood, 101 Tolerance, acquired, 246 Tonsillar concretions, 4()5 Tonsillitis, 433 Tophi, gouty, 631 Toxalbumins, 144 bacterial, 108 vegetable, 225 Toxemia of pregnancy, 533-539, 557 Toxic bacterial proteins, not specific, 132 Toxicity of acetone, 553 of ascaris, 141 of en/viiies. 61 63 ..f indole. 574 575 of ui'ine. 534 '{"oxin-antitoxin. 180 Toxins. 120, 125 129. 177. 394 and antitoxin, dilfusion of, 1S2 niterabilily of. 182 adaorj)tion of, 127 agencies destroving or modifving. 127 bacterial, 125, 105, 265, 318, 407 chenucal ])ii)])ertie8 of, 125 Toxins. di]jhtheria, 75, 87, 102, 103, 127, 128, 260, 317 digestion of, 127 etrect of acids, bases, and salts on, 127 of light on, 375 of j'-rays on, 127 Elirlich's conception of nature of, 128 theory of, 177-178 fatigue, 75 immunity against, 128 neutialization of, bv antitoxins, 170 of fatigue, 561-562 relation to <'nzymes, 126 to proteins, 126 to ptomai'ns, 128 resemijlaiice to enzymes, 67, 68 scorpion, 177 specific, synthetic products, 128 spider, 177 susceptiliilitv to. 128 tetanus, 70,' 75, 103, 128, 260, 379, 592 digestion of, 127 Toxoid, 120 Toxolecithidin in bee poison, 100 Toxophore. 07, 128, 178, 212 grouj), 120 Trachinis draco, 162 Transudates, 94, 331 nephritic, composition of, 354 Trichinella, chemistry of, 142 infection, complement fixation of, 142 spiralis, 134 Trichinosis, intoxication of, 142 Trimetliylamine, 258 Trional. 480 Trioses. 645-649 Triton ta?niatus. 262 Tropisms, theory of, 255-256 True solutions of crystalloids, 35 Trypanosomes. 130 tolerance of, 252 Trypsin, 62, 05, 07, 103. 128, 358. 566 Trvpsinogen, activation by kinase, 221 Trvi)tic proteolvsis, 526 Tryptophane, 195. 286. 469-471, 571, 574 Tubercle bacillus. 70, 79, 99, 107, 131, 132, 204, 320 antigen from, 108 composilioii of, 108 fat stainiiiLT in. Ill fats of, 110 111 fatty acids in, 1 12 modification of acid fastness of, 111 no cholesterol in, 111 ])lios])hati(ls in. 1 1 1 Tubercles. 382, 431 calcified, 464 caseous, 27() Tiibercnlin, 87, 103, 130 injections, 260 INDEX 705 Tulh'iculin rcai't ioii, 570 witli (Icntt'io-alliuiiKisc, 17 t 'I'liliiTculonastiii, Ids 'rulicifuldMamin, .SS.'{ Tubi'rc\ tes of, 2()0 piiliiioiiaiy, 7!t. 407, 50,5 uleeratiiifj, 50!) 'riil)eicui<)iis aliseess, cold, 280 etl'usion, 353, 433 exudates, 358. 304 lueuiujritis. 300 pleurisy 350 ])roiesses, 340 'J'iil)i)-()varian cysts, 514 Tuu^^stcu. 240 ' 'luiiiors, 104-105, 311. 340, 405, 422 adrenal, melanotic, 472 autolysis of, 4!)() l)cnij:n, chemistry of. 509 515 hone-marrow, 570 cells. 404 chemistry of. 492-522 cholesteatomatous, 415 colloidal poisons in, 503 efVect of diet on growth, 493 embryonic orifjin, 4!)(i en/ymes in, 75 ferments in, 105 •rlycoiren in, 431-432 hemolytic sulistanccs in. 504 505 inorganic constituents of. 499- 500 internal secretio!i. 502-504 iron in. 500 malignant, 284, 5(;!» chemistry of. 515 518 melanotic. '4G7, 471 472, 496, 502, 577 mouse. 173. 432 of brain. 531 ovarian. 420 oxidase in. 70 paratlivi-oid, 432 polypoid, 428 proteins of. same as normal jtroteins, 494 purine nitrogen in, 495 splenic. 480 testicular, 432 Turgor of plant cells, 30 Tur])entine exudates. 358 oil of. 270 Tvi)lioid agtrlutinin, 174. 184 bacillus, 70. 129. 132. 183. 211. 214. 224. 204. 320, 451 colon bacteria, dilTerentiated by acid agglutination, 188 ■lyi'lKiid fever, 71, 119, 293, 321, .{50 niin-specirK- reactions in, 174 inlectidii, .'JOS tlirnnibi, 224 'lypholysin, -224 Tyramine, 570 Tyrosinase, 00, 07. 71, 73. 409, 545 Tyrosine, 20. 87, !)0. 258, 279, 309, .327, 357. 389. 4()!», 542, 507, 57S, 009 Tyrotoxicon, 585 Ui.CKRATixo pulmonarv tuberculosis, 509 I'lceration, 508 ntra-violet rays. 00. 182, 375 I'ncinaria, 318 duodenalis. 142-143 Unicellular organisms, 252 I'racil, 109, 019 Uranium nephritis, 344 Lrate. 454 bibasic. 020 calculi, 456-457 monobasic, ()20 nionosodium. 020, 029 Urea, 53, 250, 251, 258, 332, 357, 525, 626, 627 Urease, 67 Uremia, 70, 417. 525-533. 534, 560 acids in, 528 asthenic, 529, 532 chemical changes in, 527-529 etiolofry nf. 529-533 relation of eclampsia to, 533 Ureu'ic coma, 355 endocarditis, 531 pericarditis, 531 Urethra, 457 Uric acid 75, 103, 357, 525, 529, 619 calculi, 455 456. 027 chemistry of. 618 619 concretions, 310 deposition, 029 destruction of, 625-626 diathesis, 027 endogenous. 010, 021 exogenous. (i21 increased elimination of. 456 infarcts. 450. 027. 633-634 intoxication, 027 metabolism. 618 634 oxidation, 248 properties of, 620-621 i-eteiition, 029 sympathetic formation. 624-625 Uric-acidemia. 032 T'ricase. 71. 95, 623 Uricemia. 627, 631 Uricolvsis, 625 Uricolyfic en/ymes. 501, 633 T'rinarv bladder. 42.3 calculi. 454 460 706 INDEX Urinary calculi, disintegration of, 459- 460 general properties, 459-460 changes, HXi constituents, toxicity of, 52.5 dextrin, 3SS pigments, 525 secretion, .'i.Sl toxiiit\, 525 Urine, (i4, 542-544 amylase in. SO catalase in, 71 concentration of, 45(J fat in, 320 lipase in, 78 toxicity of, 53-4 Urinod, 530 Urobilin, 230, 2!)U, 302, 450, 474, 477, 4!)0 Urobilinogen, 477 Urobilinogenuria. 4'.I0 Urochronie, 450 Ura?rytlirin, 450 Uro-fuscin, 480 Uroleucie acid, 578 Uronielanin. 450 I'rorosein, 575 Urostealitli calculi. 458 Urticaria, 351 factitia. 380 local, 342 Uterine fibroid, 510 degenerating. 4!)8 Uterus, involution of, 02 puerperal, 303, 50!) Utilization of sugar, 038 Vacuomzao'ion, 378 A'alerianic acid, 507, 583 Valvular heart disease. 355 Vaquez-Osler disease. 313 Avascular disturbances, 253 Vasoconstrictor nerves, 253 paralysis, 312 Vasoconstrictors. 488 Vaso-depressor, GOO Vasodilator nerves, 253 stinuihis, 312 stimulation, 351 Vasodilatoi's, 488 Vegetalile ])ois(>iis. heniolvsis by, 225- 226 proteins, 270 toxall)umins, 225 Veins, pulmonary, 430 Venom. 105. 245 agglutinin, 154 as antigens, 150 chemicnl constitution of, 150 cobra. 150. 151, 227, 228, 241 resistance to, 505 <(il)pcrhead, 228 crotiiJMs, 151 Venom, ell'ect of, in stomach, 152 on bluod, 153 enzymes in, 150 gland, 140 hemolysis by, 228 229 immune serum, 320 Krait, 155 nu)cassin, 150, 228 nature of, 153-155 ni'urotoxin, 154 platypus, 157 poisoning, loss of bactericidal power, 155 thrombi from, 153 properties of, 149 rattlesnake, 150, 228 snake, 148-157, 177, 223, 203, 318, 310, 504 toxicity of, 151 variations in, 155 Veronal, 480 Vertebrates, cold-blooded, 105 Vesicants, 351 Vessel injury, 323 Vinegar eels, 370 Viper poisoning, 152 Viperida^ 148, 149 Viscosity. 203 of blood, 292-293. 301 Vital activity, 53, 333, 338 stains, 50 Vitalistic school, 330 Vitamines, 285-288 Volatile fatty acids. 280 ^'omiting, 245, 5()3 cyclic, 557, 559-560 hysterical, 500 pernicious, of pregnancy, 538 Wasps, 100 Wassermann reaction, 108, 234-238 Water aliinity, 371 amount in Ijlood, 331 caj)acity of colloids for. 336-337 dielectric constant, 38 distilled, 257 Waxy degeneration. 3(i0. 370 of muscles. 392 393 Wet brain, 531 White chromogen. 400 kidneys, large, 405 wool, 400 Wool, white, 400 Worms, intestinal. 03, 432 nu-al, 400 Woniid secretions, 302 Xantiiki.asma. 415 nuiltiplex, 47(i Xanthine, 75, 247, 454. Glfl, 023 bases. 101 iiodies, 010 calculi, 458 oxidase, 71, 408, 501 INDEX 707 -Xaiitlioiiiii, 4().>, 4SS tiilifitisiMii imilliplc.v, r)12 Xiiiit lioiiiatoiis iiiassi's, 41(i Xaii11i(>iiliyll, 47.') Xaiit liosiiu*. &2-i Xaiitliosiiu'-Iiydrolasc. i'fl'.i Xerosis bacillus, ."{'JO A'-rays, (iO, M\ atr()])liy of ovaries from. ."J"!! of testieles from, ."^7(1 cancer from, .■?77 eilect of. on toxins, 127 jjaiifirene from, .■?7ti leukemia from, .■>77 necrosis due to, 376 A'-rays treatiiicnl, lo:{, 5G9 Xylose, 4i)7, 04!) Yea.st extracts, 287 tolerance of, 252 Zeix, 194, 280 Zenker's waxv defeneration, 33G Zinc, 223 Zofiprecipitins. I'.lii Zootoxins. 148-164, 203 Zyiiioi^cn, ()() Zynioids, (i3, (17 Zymophore group, 120 Zymoplastic substance, 299, 31G THIS BOOK IS DUE ON THE LAST DATE STAMPED BELOW AN INITIAL FINE OF 25 CENTS WILL BE ASSESSED FOR FAILURE TO RETURN THIS BOOK ON THE DATE DUE. THE PENALTY WILL INCREASE TO SO CENTS ON THE FOURTH DAY AND TO $1.00 ON THE SEVENTH DAY OVERDUE. BIOLOGY LIBRARY APR "s 1941 NiftY 1 2 1941 AH< J 1942 1 V W3 "I'M '^AA »..■'(" ■ '4 '4 LD 21-10x1-7. '39 (402.« •^^0801 6'OLOG UBRAR 4> w UNIVERSITY OF CALIFORNIA LIBRARY i 1 i m .1