^^^^I'fga. HX64141764 QP519 -Si5 I H&?Urmt? ©brarg &m §§3r^ m m ik ,v- -->• C\. Digitized by the Internet Archive in 2010 with funding from Open Knowledge Commons (for the Medical Heritage Library project) http://www.archive.org/details/textbookofphysio1901simo A TEXT-BOOK Physiological Chemistry FOR STUDENTS OF MEDICINE AND PHYSICIANS. BY CHARLES E. SIMON, M.D., Of Baltimore, Md. LEA BROTHERS & CO., 1MI I I. \ LI. I.I'll I A A N I) N EW YORK. I 901 . Entered according to Act of Congress, in the year 1901, by LEA BROTHERS & CO., In the Office of the Librarian of Congress, at Washington. All rights reserved. WESTOOTT & THOMSON, ELECTROTYPERS, PHILADA. PRESS OF WILLIAM J. DORNAN. PHILADA. TO MY UNCLE, MR. HENRY T. SIMON, THIS VOLUME IS AFFECTIONATELY DEDICATED BY THE AUTHOR. PREFACE. In preparing the present volume on Physiological Chemistry I have endeavored to adapt the book as much as possible to the wants of the medical student, and the physician who in the past has been unable to devote the attention to the subject which it merits. The work is intended as a text-book for the lecture-room and as a guide in the physiological-chemical laboratory. Theoretical discussions have been avoided as far as possible, and it has been my aim to present ascertained facts as concisely as appeared consistent with the importance of the problems under consideration. The various chemical methods have been described with all due regard to necessary detail, but with the supposition that the student's course in physiological chemistry has been preceded by a course in general chemistry, such as is offered now in the majority of our medical colleges. The subject-matter has been arranged in such a manner that in the first section of the work a general survey is given of the origin and the chemical nature of the three great classes of food-stuffs, and also of the most important products of their decomposition; the second section deals essentially with the processes of digestion, re- sorption, and excretion ; while the third portion of the work is devoted to the chemical study of the elementary tissues and the various organs of the animal body, the specific products of their activity and their relation to physiological function. This arrange- ment has suggested itself to me as the most satisfactory for purposes of teaching. References to literature have been omitted, as they did not appear t<» Ik- necessary in a work which is intended primarily for the student. The names of tin- grand-masters of physiological chem- vi PREFACE. istry and physiology, however, have been introduced into the text as a matter of historical interest. To my friend, Mr. Charles Glaser, of Baltimore, I wish to express my sincere thanks for many valuable suggestions and aid in the revision of the manuscript. To Messrs. Lea Bros. & Co. I am indebted for many acts of courtesy. 1302 Madison Avenue, Baltimore, Md., 1901. CONTENTS. CHAPTER I. INTRODUCTION. PAGE General chemical composition of living matter 17 Forces at work in the living world 17 Character of chemical changes 18 Synthetic processes in plants 18 Oxidations and hydrations in the animal body 19 Chlorophyl 21 Chemical nature of chlorophyl 21 The food-stuffs of the plant 23 Synthesis of the carbohydrates 24 Glucosides 25 Mannides 26 Synthesis of the fats 26 Synthesis of the albumins 26 CHAPTER II. THE ALBUMINS. Elementary composition 30 Crystallization 30 Solubility 31 Behavior toward neutral salts and alcohol 31 Diffusion 31 Coagulation 32 Denaturization 33 Behavior toward polarized light 33 • lolor-reactiooB 34 The xanthoproteic reaction 34 Millon's reaction 34 The reaction of Adamkiewicz 34 The biuret reaction 34 Boiling witli hydrochloric acid^furfurol reaction 35 Sulphur t() The spermatozoa . 421 The ovaries 4>22 The ovum 4^2 The yolk . . |.j(; Incubation aoq CHAPTER XXII. THE DUCTLE8S GLANDS. The Thyroid Gland 439 Thyreoglobulin 433 Thyreo-niicleo-albumin 434 Extractives ... 435 Tin. Adrenal Glands i.;, PHYSIOLOGICAL CHEMISTRY. CHAPTER I. INTRODUCTION. The science of physiological chemistry has for its object the study of the various chemical processes which take place in the bodies of animals and plants, and which are more or less intimately associated with the phenomena of life. As the phenomena of life, moreover, are essentially dependent upon the transformation of living matter into non-living matter, and vice versa, physiological chemistry deals primarily with the chemical processes of nutrition in the widest sense of the term. Its study therefore comprises a consideration of the various substances which are generally desig- nated as food-stuffs, their origin, their transformation into living tissue, and their ultimate fate. General Composition of Living Matter. — Chemical examina- tion shows that plants and animals consist essentially of carbon, hydrogen, nitrogen, oxygen, sulphur, phosphorus, chlorine, potas- sium, sodium, calcium, magnesium, and iron — that is, of elements which occur also widely distributed in the non-organized world. In the bodies of animals and plants these elements are built up to form bodies of highly complex chemical constitution, which belong to the class of albumins, carbohydrates, and fats. Upon their pres- ence both animals and plants are dependent for their existence, and as these bodies are constantly being broken down and transformed into Bimpler chemical compounds, as the result of the various mani- festations of lite, it follows, from the law of the indestructibility of matter, that for their replacement the living body is forced to depend upon such simpler matter as is pre-e.\istent. I nis matter it is capable of transforming into the complex substances of which it - tissues are composed. Forces at Work in the Living- World. — The forces which are at work in effecting these various changes are the same as those which we meet with in the non-organized world. They represent essentially n transformation of energy, of which, as we shall presently Bee, the -nnlight is the ultimate source. Ii would thus appear that life iii itself i- solely a physical phenomenon, and that its various 2 17 1 8 INTR OD UCTION. manifestations can be reduced to simple physical laws. This state- ment, however, is true only in part, for although the forces of which we have cognizance as being at work in living bodies are the same as those with which we are familiar in the non-organized world, we are nevertheless unable to explain the phenomena of life upon this basis, and are as yet forced to accept the existence of a vital principle, of the nature of which we know nothing. Character of Chemical Changes. — The chemical processes which are involved in the transformation of non-living matter into living tissue are qualitatively the same in plants and animals. Quantitative differences, however, exist, which are sufficiently pro- nounced to serve as marks of distinction between animals and plants. Thus plants are capable of evolving from relatively simple compounds those complex chemical substances which go to form their structure, while animals apparently do not possess this power. They are hence dependent for their existence upon food-stuffs which are preformed ; and the potential energy which animals require for the functioning of their various organs, and which they trans- form into kinetic energy, is, as a matter of fact, derived in every instance, either directly or indirectly, from plant-life. Plants, in turn, obtain the potential energy which is stored in their tissues from the kinetic energy of sunlight, and in virtue of this energy can elaborate those simple chemical substances which are at their dis- posal as food-stuffs into the complex bodies which constitute their tissues. We thus observe that while in plant-life synthetic chemical processes prevail, analytical processes are foremost in animal life. These analytical processes, moreover, are largely of the character of oxidations, while the syntheses which are effected in the bodies of plants are essentially of the nature of reductions. But just as syn- thetic processes are not absolutely characteristic of plant-life, so also do oxidation-processes occur in plants, and synthetic reductions in animals. This becomes especially noticeable as we descend in the scale of both animal and vegetable life. Primitive vegetable organisms are thus met with which, like the highly organized mam- mal, are almost entirely dependent for their existence upon already elaborated food-stuffs, and low forms of animal life similarly occur in which the processes of nutrition are essentially the same as those which occur in the higher plants. The differences which thus exist between animal life and plant-life are therefore, as has been stated, more of a quantitative than a qualitative kind. Synthetic Processes in Plants. — I have said that plants are capable of elaborating from simpler compounds the complex chem- ical substances of which they are composed, and that the chemical processes here involved are essentially of the nature of synthetic reductions. Formerly, it was believed that the various organic sub- stances which occur in animals and plants could be produced only through the agency of a special vital force ; but we now know that OXIDATIONS AND HYDRATIONS IN THE ANIMAL BODY. 19 this is not necessarily the case, and that as a matter of fact a large number of such bodies can be produced artificially in the chemical laboratory. Wohler, in 1829, was the first to demonstrate this possibility by preparing urea from ammonium cyanate. This he accomplished by heating the substance to a temperature of 100° C, when a transposition of atoms apparently takes place, and urea results. The force which is necessary to effect such a change is here, as in many syntheses which can artificially be brought about, a relatively high temperature. In the bodies of animals and plants a like temperature, of course, would destroy life, and there must hence be a different mechanism at the disposal of living beings to effect such a change. Of the nature of this mechanism, however, we know but little, and we are forced to admit that while it is pos- sible to produce chemical substances, such as those which are found in the living world, by artificial means, plants and animals have manifestly other forces at their command which are more or less intimately associated with that peculiar phenomenon we term life. We know that under the influence of sunlight certain plants are capable of effecting the synthesis of carbohydrates, fats, and albu- mins from the carbon dioxide of the air, and the water and certain mineral salts of the soil, and that the ability to bring about these changes is in a large measure dependent upon the presence of a chemical substance which is found in the green parts of plants, and which is termed chlorophyl. We know further that chlorophyl requires exposure to sunlight to effect these changes, but of the mechanism through which these changes are brought about we know nothing. Oxidations and Hydrations in the Animal Body. — The oxida- tion-processes which prevail in animals, and in consequence of which the more complex substances which go to form the various tissues and organs of the body are retransformed into those simple com- pounds which plant- require for their existence, we are also un- able to explain on the basis of simple physical laws. We know that the oxygen of the air, as also that of the blood, exists in a neutral molecular form, and is as such incapable of effecting the oxidation of such complex substances as the albumins and fats. The older view that oxygen exists in the body as ozone, and that the various oxidation-processes take place in the animal fluids, has been abandoned, and it is now generally accepted that these changes occur iii the individual cells. Here, then, a splitting up of the neutral oxygen must take place, but of the forces which effect this decomposition we know next to nothing. Whether we believe with Pfluger that the organized living albumin, in contradistinction to the non-organized circulating albumin, i< characterized by a greater motility of it- atom-, iii consequence of which the neutral oxygen i- decomposed, or whether we accept the view that reducing- -uli-ianee- are formed during the decomposition of the albuminous molecule in consequence of the activity of a third factor, we are 20 IX TB OD UCTION. as far removed from an adequate explanation of these phenomena as in the beginning. Within recent years repeated observations have shown that from various organs of the body certain substances can be extracted which are apparently identical with or closely related to the so-called en- zymes. Certain representatives of this class, such as pepsin, trypsin, ptyalin, and others, are, as we shall see, formed in the cells of the digestive glands of the body, and serve the purpose of transforming the various food-stuffs which are furnished the animal by the plant into forms which can be absorbed and built up into its tissues. The chemical processes which are here involved are essentially of the character of hydrations. Other bodies, however, of this order which can be obtained from living tissues, and which are also capable of manifesting their special activity after the death of their parent-cells, apparently possess the power of oxidation, and it is hence supposed by some that these processes in the living tissues may also be referable to such enzymotic activity. Whether this is actually the case is not definitely known. But if so, we are apparently approaching a time when what we have heretofore been forced to ascribe to the activity of a special vital force may be explained upon the basis of physical laws which are seen also at work in the non-organized Avorld. For we know that properties which are supposedly characteristic of the enzymes are possessed also by cer- tain elements which are found only in the inorganic world. The most notable properties of the enzymes are their ability to effect an amount of chemical change which is out of all proportion to the quantity of the enzyme present, and the fact that the enzyme itself apparently does not enter into the reaction. These same properties, however, are common to certain metals and their oxides.. Bredig and von Berneck showed that a gram-atomic weight (193 grams) of colloidal platinum diffused through 70,000,000 liters of water shows a perceptible action on more than 1,000,000 times the quantity of hydrogen peroxide ; and H. C. Jones demonstrated that the reac- tion which here takes place is a mono-molecular reaction, which indicates that the platinum itself does not enter into the reac- tion. Curiously enough, the analogy between the action of such metallic solutions and that of the enzymes goes still further. Finely divided platinum, palladium, iridium, osmium, etc., thus have the power of inverting cane-sugar, like one of the enzymes, invertin ; and certain poisons, such as hydrocyanic acid, sulphuretted hy- drogen, carbon disulphide, and mercuric chloride, which inhibit or even suspend the action of the enzymes entirely, exert a similar influence upon a solution of colloidal platinum. Without entering upon this very interesting subject further, it is clear that a path has been opened upon which it may be possible to penetrate into the mysteries of the so-called vital forces, and to show ultimately that such forces are essentially the same as those met with in the non- living world. CHLOROPHYL. 21 Chlorophyl. — In the light of more reeent investigation, it seems probable that some of the synthetic processes which occur in plant- life may also be referable to the action of enzymes. As a matter of fact, such bodies are abundantly present in the vegetable world, and we know that some of these at least, and probably all, are characterized by a reversible activity. Maltase, a ferment, which, as we shall see later, causes inversion of the disaccharide maltose to glucose, is thus similarly able to bring about the synthetic formation of maltose from two molecules of glucose. On the other hand, it appears that the primary formation of food-stuffs in plants does not occur in this manner, but is referable to the activity of a special body which, as has been stated, is present in the exposed green parts of most plants, and which is termed chlorophyl. This sub- stance occurs widely distributed in the vegetable world, but is also found in those low forms of animal life in which the processes of nutrition are essentially the same as those met with in the higher plants. In itself, however, chlorophyl is incapable of bringing about those syntheses which are characteristic of vegetable life, and in the cells of the foliage of plants it occurs in combination with certain albuminous bodies, in the form of the so-called chlorophvlic granules. These are apparently special elementary organisms, and endowed with a power of locomotion analogous to that of amcebse and leucocytes, so that they can approach the surface of the leaf to seek the sunlight, or retreat when this becomes too intense. In a germinating plant which has not been exposed to sunlight the green color is wanting, but in the cotyledons is found a differentiation of the cellular protoplasm into small yellowish bodies, which have been termed leucltes. When these bodies are exposed to light, even for a relatively short time, they assume a green color, and then constitute the chlorophvlic grannies. I have said that chlorophyl — that is, the green coloring-matter of plants — is unable to effect synthetic changes, and the same is true of the colorless leucites. Chlo- rophyl thus apparently forms an integral constituent of the function- ing leucites, and all those chemical and physical influences which bring about the destruction of the protoplasm similarly destroy the power of chlorophyl to manifest its special activity. In the living plant, however, it becomes active at once upon exposure to sunlight, and i- then capable of effecting those complicated syntheses of which mention has been made. In the dark it again becomes inactive, and the plant i~ then obliged to live like an animal — by consuming its stored energy. Chemical Nature of Chlorophyl. — Of the chemical composi- tion and Structure of chlorophyl we know little that is definite. Numerous attempts have been made to isolate it from the living plant, but it i- doubtful whether any of these attempts has yielded the actual substance. Only its decomposition-prodilcts, or at best very impure form-, have apparently been obtained, (iautier, it is true, claims to bave isolated the substance in crystalline'fbrm by 22 INTRODUCTION. methods which are- calculated to avoid its chemical alteration. Others, however, have not been successful in repeating his work. The substance which Gautier obtained from spinach-leaves oc- curred in the form of small crystals of a dark-green color, which on exposure to light turned brown, then yellow, and finally became colorless. Its composition corresponded to the formula C^H^NgC^. The mineral ash consisted of about 1.75 per cent, of magnesium phosphate, traces of calcium and sulphates, while iron was absent. Treated with hydrochloric acid, it was decomposed into phylloxanthin and phyllocyanic acid, C19H22N2303 or C,8H20N2O3. This latter is thus a homologue of bilirubin, [C16H18N203]2, which in turn is derived from haematin, and is isomeric with hcematoporphyrin. A most interesting relationship between the blood coloring-matter haemoglobin and the vegetable coloring-matter chlorophyl thus becomes apparent, and constitutes a further link connecting the animal with the vegetable world. Recent investigations have shown that a substance can be obtained from chlorophyl, termed phylloporphyrin, which differs only from hsematoporphyrin anhy- dride in containing three atoms less of oxygen, viz., C32H34N4(32. Both bodies are thus clearly different oxidation-products of one and the same substance. Moderately concentrated solutions of chlorophyl in alcohol or petroleum-ether show seven bands of absorption. The first of these, I, is situated in the red portion of the spectrum between B and C, and is well pronounced and sharply defined on both sides. The bands II, III, and IV are rather indistinct and scattered through the orange-yellow, the yellow and the yellowish-green portion be- tween C and E. From F off, the greater portion of the spectrum is absorbed by the remaining bands, V, VI, and VII, of which V is seen to the right of F, VI most marked about C, while VII occu- pies the extreme violet end. Very concentrated solutions allow the red rays to pass only as far as B, while in greater dilution the green rays likewise appear. Such solutions, therefore, appear green when viewed with transmitted light, while with reflected light they are red and fluorescent. When a fresh leaf is similarly examined, a spectrum is obtained which is essentially the same as that just described. There is lack- ing, however, the band that corresponds to the red fluorescent rays of chlorophyl solutions. This is explained by the assumption that the red rays are absorbed by living chlorophyl and transformed into chemical energy. In accordance with this view, we find that when living plants are successively exposed to the various rays constitut- ing sunlight, decomposition of carbon dioxide with liberation of oxygen — which, as we shall presently see, takes place in the green portions of every plant whenever it is exposed to sunlight — occurs with special intensity when the plant is exposed to the rays corre- sponding to the bands I, II, and III. In this manner, then, chloro- phyl-bearing plants derive their kinetic energy from sunlight, and THE FOOD-STUFFS OF PLANTS. 23 thus become enabled to elaborate the simple food-stuffs which are at their disposal into the complex substances which constitute their tissues. The Food-stuffs of Plants.— The most essential elements which enter into the composition of the tissues of plants are, as has been pointed out, carbon, hydrogen, oxygen, and nitrogen. These sub- stances are available to the plant as carbon dioxide, water, and cer- tain nitrates. The origin of the first mentioned is, of course, obvious, while that of the last is at first sight somewhat obscure. The nitrates are present in any soil which contains organic matter, and are now known to result from this through the special activity of certain bacteria. Decomposing animal and vegetable matter is, however, not the only source of the nitrates, for it can be demonstrated that arable soil, apparently devoid of vegetable life is capable, unless sterilized, of fixing a very considerable amount of nitrogen, which must of necessity be derived from the atmosphere. It is not in its elementary form, however, that the nitrogen which we thus find stored reaches the soil, but in all probability as an ammo- niacal compound. This the bacteria then transform into nitrates, which the plant requires for its existence. I do not wish to convey the impression, however, that all plants require their nitrogen in this form, for we know that the Saccharomyces cerevisia?, for example, can elaborate its nitrogen not only from ammoniacal salts directly but is even incapable of utilizing that which is furnished in the form of nitrates. Under certain conditions, moreover, probably all plants can, for a while at least, grow in the presence of ammoniacal nitrogen only. From what has been said, it is clear that a certain symbiosis exists between the bacteria of the soil and plants, in virtue of which the former transform the ammoniacal nitrogen that is present in the soil into nitrates, which can be utilized by plants, while they in turn probably aid the nutrition of the bacteria by furnishing them with humus and the ternary matter which is necessary for their develop- ment. The necessary mineral salts the plant likewise obtains from the .-oil. The question now arises: In what manner do plants effect the synthesis of those complex chemical substances which go to form their tissues from the simple bodies which serve as their food-stuffs? The kinetic energy which is necessary to effeel these changes is, as has been stated, derived from the sunlight and transformed into potential energy by the chloropbyl. We should thus expect to find |" those part- in which this is present the origin of those final products which we meet with in the tissues of the plant. These products nmy be divided into three groups, and in the following pages ;m attempt will he made to describe the manner in which representatives of each are formed. I -hall accordingly consider the origin of the carbohydrates, the fats, albumins, and certain non- 24 • INTRODUCTION. albuminous, nitrogenous bodies, all of which are also found in the animal body, and which represent the essential food-stuifs of the animal world. In doing so, I am aware that I am trespassing to a certain extent upon what will follow in subsequent chapters ; but as I shall deal with the chemistry of animal life more exclusively in the present work, it has been deemed best to consider briefly the principal syntheses which are effected by plants before proceeding to a more detailed study of the subject proper. Synthesis of the Carbohydrates. — It has been pointed out that during exposure of chlorophyl-bearing plants to sunlight the carbon dioxide of the air is decomposed, with liberation of oxygen. The volume of gas thus set free is equivalent to the volume of carbon dioxide that is decomposed. At the same time a reduction of water takes place, as is apparent from the observation that a larger amount of hydrogen is found in the plant than is necessary to form water with all of the oxygen that is present at the same time. It thus follows that one-half of the oxygen per volume must be derived from carbon dioxide, and the other from water, according to the equation : C02 + H20 = r + 20, 2 volumes. 2 volumes. in which r represents one atom of carbon, one atom of oxygen, and two atoms of hydrogen, which have been retained by the plant. A combination of these atoms in one molecule, however, would repre- sent one molecule of formic aldehyde, CH20. Should this be actually formed in the plant, we would at once have a probable explanation of the manner in which the carbohydrates are con- structed, as these are polymeric compounds of formic aldehyde, or their anhydrides. In fact, it is possible artificially to effect the synthesis of many of the carbohydrates which are found in the living world by starting with this simple aldehyde. Formic alde- hyde, it is true, has not as yet been isolated as such from the leaves of plants, as its existence here is probably only momentary, but its oxidation-product, formic acid, has frequently been obtained. It is thus extremely probable that this is actually the starting-point in the elaboration of the carbohydrates by the plant. As to the exact manner in which the aldehyde is derived from carbon dioxide and water, we are as yet uncertain ; but it appears from the interesting researches of Gautier and TimiriazefF that the chlorophyl first decomposes water, under the influence of sunlight, and forms a hydride of chlorophyl, which is colorless, and that this product subsequently reduces carbon dioxide, with liberation of oxygen and restitution of the green chlorophyl. These changes may be rep- resented by the equations : (1) x +H20 = xH2 + 0. (2) *H2 + C02 =z + H2CO + 0. SYNTHESIS OF THE CARBOHYDRATES. 25 where x represents the chlorophyl. As a matter of fact, Gautier and Timiriazeff succeeded in obtaining sneh a hydride — -protophyllin — which turned green on exposure to air, or in an atmosphere of carbon dioxide under the influence of sunlight ; while in the dark, or on exposure to sunlight in an atmosphere of hydrogen, no change occurred. The existence of this body in etiolated plants, more- over, has likewise been established. By a process of polymerization and subsequent hydrolysis, then, formic aldehyde gives rise to the large number of carbohydrates which are found in the vegetable world. Of the manner in which these polymerizations and subsequent changes are brought about, however, we know little ; but there is reason to believe that they are largely effected through the activity of special ferments, as has been indicated. That some of these changes take place in the chlorophyl- bearing parts of the plant can readily be demonstrated. If a spiro- gvra. for example, is exposed to sunlight after having been kept in darkness for some time, so as to remove any sugar that may have been present in the cell, it will be observed that after a very few minutes starch granules appear, which can readily be detected by the addition of a little iodine solution. On subsequent removal from the light the starch soon disappears from the green parts of the plant, and is carried to the storage-cells proper, where it is trans- formed into dextrin, glucose, and various soluble gums, which may be further transformed into celluloses, certain mucilages, etc. Glucosides. — Closely related to the carbohydrates proper, the origin of which has just been considered, is a group of substances which likewise occur widely distributed in the vegetable kingdom. These are the so-called glucosides. They are so termed from the fact that glucose is invariably formed during their hydrolytic decomposition, which, as an anhydride, thus constitutes an integral part of their molecule. This observation at once suggests their origin also from formic aldehyde. Such substances are salicin, which on hydrolytic decomposition yields glucose and saligenin ; arbutin, which yields glucose and hydroquinon ; phloridzin, which gives rise to glucose and phlorc- tin, etc. (i) c13h19ot + n./> = c,h8o2 + cyi,2o6. Salicin. Saligenin. oinuose. (2) f„irlfio7 + h2o c6ii6o, i c6n„v the observation that hydrocyanic acid, as Buch, or in the form of cyanides, occurs widely distributed in the vegetable world, and is characterized by the readiness with which it combines with a large Dumber of* organic substances to form highly complex chemical coin- pounds. A study of the decomposition-products of the various albumins further shows that their molecule can be reduced to urea, /MI CO— NIL ( ( ) vnJ, -'"id oxamide, | , the hydrogen atoms of which \.mi, CO— NH2 have either entirely or partly been replaced by the chains ro—cn,— fii, — en— Nil— rir,— CH— Nil — CH2— < <><>n. On studying this chai ore closelv.il will be observed thai the 28 INTB OD UCTION. formic aldehyde radicle CH2 is here repeatedly encountered, and that these radicles are united by the group CH — NH, which in turn represents hydrocyanic acid plus one atom of hydrogen. This, as we have just seen, results from the action of formic alde- hyde on nitric acid. Through a union of such aldehyde groups with hydrocyanic acid chains could then result of the composition : H H H H III II I = C-NH-C-C-C-NH-C-C-NH-C-C = M, III II I OHOH OHOH in which through a process of hydration the first and last C — NH groups can be transformed into CO and COOH, according to the equations : HON + H20 = CO + NH3 and HCN + 2H20 = H.COOH + NH3. In the presence of nascent hydrogen, moreover, the aldehyde and hydrocyanic acid groups would be transformed into the groups H H II C and C — N — , thus giving rise to the chains : I I H H — CO — CH2 — CH2 — CH.NH — CH2 — CH2 — CH.NH — CH2 — COOH. That hydrogen is actually available in the leaves for this purpose we know, as during the formation of formic acid from its aldehyde hydrogen is constantly being liberated. The formation of urea and oxamide, finally, the radicles of which, as we shall see, are possibly present as such in the albuminous mole- cule, could further be explained upon the basis just outlined, as we know that both can be formed by hydration from hydrocyanic acid, as is shown by the equation : 2HCN + 2H20 = CO (NH2)2 + CH2 O. According to Gautier, then, these chains are further united to tyrosin, so that the structural formula of albumins could be repre- sented by the general formula : /COOH C,Hq NOH H— C ^c c 1/ h/ 1 1 \h E v I |E )c c( Hx \ / XH H— C I OH in which R represents the chains in question. SYNTHESIS OF THE ALBUMINS. 29 Should this theory as to the origin of the albumins in plants prove correct, it would thus be clear that all three of the great classes of food-stuffs which animals require for their existence are formed synthetically under the influence of sunlight, and through the special activity of chlorophyl. Subsequent changes, of course, take place, whereby the albumins, like the carbohydrates and fats, arc transformed into those peculiar modifications of the original compounds which are required by the various organs of the plant. These changes, however, are in a manner of only secondary impor- tance, and not to be compared in complexity to the elaborate syn- thetic processes which have previously occurred. They are brought about through the specific activity of the various cells of the organism, and in part, at least, through the agency of ferments. The food-stuffs which are thus elaborated by the plants cannot all be utilized by the animal as such, however, and previous to their assimilation they are further modified. The albumins are thus trans- formed into albumoses and peptones, starch is inverted to maltose, and the fats are decomposed and saponified. These processes of what may be termed primary assimilation, or digestion, render the food-stuffs capable of passing through the mucous membrane of the gastro-intestinal canal. During this passage the albumoses and peptones are retransformed into albumins proper, maltose is changed into glucose, and the fats are reconstructed from their two com- ponents. Subsequently all these bodies are further modified accord- ing to the character of the tissues in which they are to be utilized. Ultimately, however, they give rise to the formation of those simple substances which plants require for their existence — that is, into carbon dioxide, water, and certain nitrogenous bodies which readily give rise to the formation of amnion iacal salts. The passage through the body of the various elements which go to form the tissues and organs of both plants and animals, and the various chemical and physical changes which are here involved, con- stitute the phenomena of metabolism ; and we may thus state that physiological chemistry deals primarily with the various metabolic processes which occur in the living world. lief'ore proceeding to a study of these various changes in the animal body, however, it will be well to review the chemical proper- tic- and the composition of the various foot 1-st nil's which enter into tin- construction of its tissues. We shall accordingly consider the chemistry of the albumins, the carbohydrates, and fats, and then attempt to follow the course of these bodies through the living organism so far as this is possible with the present state of our knowledge. CHAPTEK II. THE ALBUMINS. The albumins, or proteins, are the most important food-stuffs ■which animals require for their existence. Albumins enter into the construction of all the tissues and organs of the body, and form the groundwork of every living cell. The phenomena of life, indeed, are dependent upon and centre in their presence. While many different forms of albumin exist, they all present certain general chemical and physical characteristics which serve to distinguish them as a class, and show that a close genetic relation- ship exists between them. Elementary Composition. — All albumins contain carbon, hydro- gen, nitrogen, oxygen, and sulphur in certain definite proportions, which vary but little in the different members of the group. The variations which occur are shown in the following table : Carbon 50.0-55.0 per cent. Hydrogen 6.5- 7.3 " " Nitrogen 15.0-17.6 " " Oxygen 19.0-24.0 " " Sulphur 0.3- 2.4 " " Other elements are not found in the albumins proper, but may occur in certain compound albumins, which are formed, through the union of an albuminous group with other more or less complex radicles. The coloring-matter of the blood thus contains iron ; the most important constituents of the nuclei of cells are more or less rich in phosphorus ; other bodies belonging to this order contain iodine, etc. All albumins, moreover, contain variable amounts of mineral salts, which are closely bound to the albuminous molecule. The most important and constant of these are the chlorides and phos- phates of the alkalies and the alkaline earths. Crystallization. — In the eggs of certain fish and amphibia so-called yolk-platelets may be observed, which apparently possess a crystalline structure. Chemical examination, however, has shown that these bodies do not consist of pure albumins, but also contain a large percentage of lecithins and mineral salts. The so-called aleuron crystals, which have been found in the seeds of certain plants, are thus likewise not composed of a pure albuminous sub- stance, and the same probably holds good of the little eosinophilic crystalloids which may be seen in the blood of birds. Artificially 30 DIFFUSION. 31 also the crystallization of albumins is apparently possible only in the presence of certain mineral salts. Numerous attempts to bring this about in the absence of salts have so far at least yielded only nega- tive results. Of vegetable albumins, the phytovitellin of para-nuts, the castor-oil bean, etc., yields well-defined crystals when the sub- stance is dissolved in solutions of neutral salts at a temperature of about 40° C, and is subsequently allowed to cool or evaporate. Egg-albumin, the serum-albumin of the horse, and pure casein may similarly be made to crystallize. The material thus obtained does not represent pure albumins, however, but is apparently a compound with the salts employed. The tendency to crystallization, moreover, increases with repeated exposure to the various salt solutions in which crystallization is to take place. The globulins have thus far not been obtained in crystalline form by artificial means, but Paton has shown that after their passage through the kidneys they may at times separate out in crystalline form spontaneously. In the dry state the albumins usually occur in the form of a white powder, or as yellowish, brittle, more or less opaque lamellse, which are both odorless and tasteless. Solubility. — Some of the albumins, such as serum-albumin and egg-albumin, are soluble in water. Others are insoluble in water, but dissolve in dilute saline solution, wThile still others are insoluble in both water and dilute saline solution, but dissolve in dilute alka- line or acid solutions. All albumins are soluble in concentrated acetic acid and in strong solutions of the caustic alkalies, but in undergoing solution they are more or less modified and transformed into syntonins or alkaline albuminates, as the case may be (see below). In cold absolute alcohol and ether albumins are insoluble, but in dilute alcohol some of them dissolve with comparative ease. Behavior toward Neutral Salts and Alcohol. — All albumins, with the exception of certain deutero-albumoses and peptones, may be precipitated from their neutral or feebly acid solutions by satura- tion with ammonium sulphate. Other neutral salts of the alkalies and alkaline earths, such as sodium chloride and magnesium siil- phate, behave in a different manner toward the individual representa- tives of the group, and it is thus possible nut only to separate the albumins from a large number of other substances, but also from each other. In being thus precipitated their structure and proper- tie- are not altered iii the Least. Strong alcohol act- in very much the same manner, but it is to be noted that after prolonged exposure, and especially in the presence of -.1I1-, the albumins pass over into the coagulated state, and then remain refractory to all neutral solvents. Diffusion. — Like the colloids of the inorganic world, so also are the albumin- practically incapable of diffusing through animal mem- branes. This peculiarity Graham explained by the assumption that 32 THE ALBUMINS. such bodies do not occur in a state of actual solution. This, how- ever, is not necessarily the case, and it is more likely that their inability to pass through animal membranes is to be explained by the great size of their molecule. This property of the albumins is very important, as it enables us to separate these bodies from a large number of other substances which may be present in the same solu- tion, and to some extent also from each other. Coagulation. — It has just been stated that according to Graham's view the albumins do not occur in a state of actual solution. While this may be questionable, we know nevertheless that solutions of these substances are quite unstable and possess a marked tendency to revert to the solid state. In this respect also they behave very much like the inorganic colloids. Thus, when a solution of sodium silicate is added to a large excess of dilute hydrochloric acid the silicic acid which is thus formed is apparently held in solution. If then the excess of hydrochloric acid, together with the sodium chloride which was formed during the reaction, are removed by dialysis, an apparently clear solution of silicic acid remains in the dialyzer. This, however, is at once transformed into a thick, gelat- inous mass when a small amount of carbonic acid is passed through the solution. Some of the albumins, such as the globu- lins, behave in much the same manner. In undergoing such changes the albumins may retain their original properties and structure, or they may be altered in such a manner that they are no longer soluble in the original neutral media. Then they are said to be coagulated. The phenomenon of coagulation is common to all true albumins, and upon this property the ability of certain forms to occur in a more or less solid state in the tissues of the body is no doubt dependent. With this statement, however, I do not wish to convey the idea that the albumins which go to form the groundwork of such structures as connective tissue, cartilage, and the like, occur in a state of actual coagulation, analogous to that which can be brought about through the influence of heat. The phenomenon simply indi- cates the direction which we shall have to follow in seeking for an explanation of the occurrence in the tissues of living animals of cer- tain albumins, in the solid or semisolid state. In certain groups of albumins, such as the albumins proper and the globulins, coagulation is brought about in the most characteristic manner through the influence of heat, providing that the solution presents a neutral, or, better, a feebly acid reaction. If the reaction is alkaline, coagulation is not complete, and in the presence of a certain amount of free alkali or an alkaline carbonate it may not occur. . An excess of organic acids similarly prevents coagulation, and care is therefore necessary to insure only a feebly acid reac- tion when it is desired to free a solution from all its coagulable albumins As the temperature at which coagulation of the various albumins BEHAVIOR TOWARD POLARIZED LIGHT. 33 takes place varies in the different representatives of the group, it is possible thus to separate them from each other, and to identify the individual forms. A certain care, however, is necessary in this process, as the temperature of coagulation for any one albumin varies within fairly wide limits according to the concentration and reaction of the solution, and the kind and amount of salts that may at the same time be present. Denaturization. — It has been pointed out that coagulation alters the character of all albumins in a profound manner, as is evidenced by the fact that they are then no longer soluble in the usual media. Of the changes which take place in the albuminous molecule during this process we know nothing. When once coagulated, however, it is only possible to effect their solution by chemical processes, which are calculated to bring about definite changes in structure. Aside from their digestion with ferments, the coagulated albumins may be dissolved by treating with dilute solutions of the alkalies or mineral acids, or with concentrated organic acids under the influence of heat. They are thus transformed into alkaline albuminates and acid albu- mins, or syntonins. The changes which the albumins thus undergo, Neumeister has termed the denaturization of albumins. As a con- sequence, the products which are thus formed differ not only from the native albumins in their general chemical composition, but also in their properties. They are thus insoluble in neutral solutions, but dissolve with ease in solutions of the alkaline hydrates, of sodium carbonate, and in hydrochloric acid. From their acid solutions they are precipitated by saturation with sodium chloride or ammonium sulphate. To undergo this change, it is not necessary, however, that the native albumins have been previously coagulated, as solu- tions of the albumins, which have been boiled after the addition of alkalies, or large amounts of organic acids, and have thus been prevented from undergoing coagulation, behave in the same manner. Behavior toward Polarized Light. — All albumins are lawo- rotatory — /'. r., they turn the plane of polarized light to the left. A- the degree of rotation varies with the different members of the group and the amount of albumin present, it is thus possible, not only to identify the individual bodies, but also to determine the amount present. The specific rotatory power of some of the more important representatives of the group, for the yellow line 1 >, is as follow- : albumin («) D 33°-38° 3 Mm albumin " 56° Lactalbumin " :«;°-T7° im-globulin . " .V.)°-75° Fibrinogen " 43° Syntonin (from mvosin) " 72° hi (dissolved in magnesium sulphate solution) . " 80° Alkaline nil. urn i. i:iic " 62.2° Various albumoseti " 70°-80° 3-4 THE ALBUMINS. Color-reactions. — The color-reactions to be described are not exclusively characteristic of the albumins, and in examinations in this direction it is always necessary to employ a number of these tests before drawing conclusions as to the presence or absence of an albuminous substance. 1. The Xanthoproteic Reaction. — This reaction depends upon the formation of certain nitro-derivatives when albumins are treated with concentrated nitric acid. The test is conducted as follows : A few c.c. of the solution in question are treated with a few drops of concentrated nitric acid, when in the presence of certain albumins a white flaky precipitate develops, which turns yellow on boiling. With other forms the solution remains clear, but also turns yellow on boiling. If in either case ammonia is then added in excess, a deep-orange color results which is very characteristic. The reaction is supposedly dependent upon the existence in the albuminous molecule of a certain aromatic radicle or radicles belong- ing to the phenol or phenyl group. It is therefore also obtained with tyrosin, phenol, cresol, phenyl-acetic and phenyl-propionic acid, as also with leucin. 2. Millon's Reaction. — This is apparently referable to the pres- ence of the oxybenzoic acid radicle in the albuminous molecule, and is accordingly obtained with all those albumins which on tryptic digestion yield tyrosin. The reagent is prepared as follows : A few grammes of mercuric nitrate are treated with an amount of water that is just sufficient for their solution. Any basic salt that may have been formed is dis- solved with fuming nitric acid, when a solution of sodium acetate is added, drop by drop, until the mixture reacts with a dilute solution of phenol, as described below. The test is conducted by adding a few drops of the solution to be examined to a few c.c. of the reagent, when in the presence of albu- mins a white precipitate is formed, which turns a brick red on the application of heat. If undissolved albumins are examined in this manner, they are transformed into brownish-red flakes. 3. The Reaction of Adamkiewicz. — This reaction is now thought to be referable to the simultaneous presence in the albuminous mole- cule of a carbohydrate radicle, together with the aromatic groups which give Millon's reaction. The test is conducted as follows : A particle of the dry albumi- nous substance is dissolved in a small amount of glacial acetic acid by the aid of heat, and then treated with one-half its volume of concentrated sulphuric acid. Immediately, or on boiling, a violet color develops, and the fluid at the same time becomes slightly fluo- rescent. The test, however, is not altogether reliable, and with albumoses and peptones gives a positive reaction only when these are present in concentrated form. 4. The Biuret Reaction. — It is thought that this reaction is depen- dent upon the presence of a urea-forming radicle in the albuminous COLOR-REACTIONS. 35 molecule, a> a very similar reaction is obtained with urea, and is in that ease referable to the formation of biuret. It is possible, how- ever, that its occurrence may be due to a cyanic radicle, which is likewise contained in biuret, and, as we have seen in the preceding chapter, hydrocyanic acid is in all probability intimately concerned in the synthesis of albuminous substances. Both hydrocyanic acid and hydrocyanuric acid, moreover, give this reaction, and it is to be noted that the former yields the purplish color of the albumoses, while the latter gives rise to the violet color of the native albumins. The test is conducted as follows : A few c.c. of the solution to be examined are treated with an excess of a strong solution of sodium hydrate, and then drop by drop with a 2 per cent, solution of copper sulphate. In the presence of albumins, with the exception of phy- tovitellin, a pure violet color is obtained, while with albumoses and peptones a rose color develops. If larger amounts of albumin are present, the reaction is obtained without difficulty. Should traces only be present, great care must be taken not to add too much of the copper solution, as otherwise the violet color is obscured by the blue of the copper solution. Where larger amounts are present, it is necessary to add more of the reagent. An excess of neutral salts, which is often present when this test is employed, does not interfere with the reaction. If ammonium sulphate is present, however, it is advisable to use a large quantity of the sodium hydrate solution, in order to bring out the color. Should magnesium sulphate be con- tained in the solution, a precipitate of magnesium hydroxide is formed, and is allowed to settle. Sodium chloride does not interfere with the reaction. 5. Boiling with Hydrochloric Acid. — The reaction apparently depends upon the formation of furfurol, which yields a violet color when brought in contact with some other substance which is formed from the albuminous molecule at the same time. What thia other substance is, however, we do not know. The albuminous material, best after extraction with hot alcohol and subsequently with ether, is boiled for several minutes with concentrated hydrochloric acid, to which a drop of concentrated sulphuric acid has been added. The albumin passes into solution and a deep-violet color results. 6. The Sulphur-test. — The albuminous solution is heated with an excess of sodium hydrate in the presence of a small amount of a<'*'< )_, and CnE^„_lN02. After decomposition the nitrogen would accordingly be found as amido-nitrogen, while in the albu- minoid molecule itself if is supposedly present as imido-nitrogen. This theory is based essentially upon the observation that during the decomposition of the albumins with superheated baryta-water, carbon dioxide, oxalic acid, and ammonia are formed in the same relative proportions as during the decomposition of urea and oxamid. With fibroin Schiitzenberger thus obtained the following complex result : - II \ ,«>-. 24H20 5QHA ll<<> 5C2H4Oa Fibroin. Oxalic acid. lei I Ic add. ami the mixture of amido-acids yielded , N'W, C,HnNO, 7< ,||.,\o, 7' II, \<>, 2C4H„NOa IC4H7N0 Ty rosin. Glycocoll. Alanin. Amido Amido-acid butyric acid, of i he acrylic plea. 40 THE ALBUMINS. The theory is ingenious, but open to many objections, upon which it is not necessary, however, to enter at this place. Latham regards the living albumins as consisting of a chain of cyanic alcohols which are united to a benzol radicle. Such alcohols are formed through union of an aldehyde with hydrocyanic acid, and we thus see that his idea of the composition of the albumins is essentially the same as that originally suggested by Gautier. The formation of the various decomposition-products of the albumins Latham explains on the basis of the extreme instability of these compound alcohols. Kossel, on the other hand, divides the albumins into four classes, and assumes that in each a protamin radicle is the essential nucleus. Those bodies in which this is present by itself he assigns to the first group, and it is accordingly represented by the protamins them- selves. The second group is formed by albumins in which the primary protamin nucleus is variously combined with mono-amiclo- acids of the aliphatic series, viz., with leucin, amiclo-valerianic acid, asparaginic acid, glutaminic acid, and glycocoll. Most of these contain in addition sulphur in more or less intimate combination, and in some iodine and other elements also may be found. Other albumins contain an aromatic radicle in addition to the pro- tamin group and the acid radicles of the fatty series, and, ac- cordingto the absence or presence of a sulphur group, he fur- ther divides these into two classes, his third and fourth group, respectively. Through a union of any two or more of these groups with each other, or with new prosthetic groups, still more complicated albumins result, such as the histons and the common proteids. Classification of the Albumins. — The various albumins may be divided into four classes, viz., the albumins proper, the pro- teids, the albuminoids, and what may be termed the derived albumins. They are further subdivided, as is shown in the follow- ing table : Albumins The native albumins 1 ~, , , * (jrlobul ins ^ Vitellins f Serum-albumin. J Egg-albumin. J Lactalbumin. ^ Vegetable albumin. Fibrinogen. Serum-globulin. Fibrinoglobulin. Vegetable globulins. Myosin. f Phytovitellin. \ Crystallins. i THE NATIVE ALBUMINS. 41 f Xucleins. The proteids J Nueleoproteids. j (jlucoproteids. ^ Haemoglobins. f Keratins. The albuminoids < Albumoids. ^ Amyloids. The derived albumins r Albumoses. Peptones. Albuminates, j Coagulated albumins. ^ Fibrin. THE NATIVE ALBUMINS. These have been described in a general way in the foregoing pages. They are subdivided as above indicated. The Albumins. — The albumins, in the narrower sense of the term, comprise serum-albumin, egg-albumin, lactalbumin, and the so-called vegetable albumin. They are all soluble in water, but may be precipitated from their neutral aqueous solutions by salting with ammonium sulphate. Sodium chloride and magnesium sulphate cause their precipitation only if the solution has been acidified with acetic acid. The addition of small amounts of acids or alkalies to their aqueous solutions is without effect. Larger amounts of mineral arid-, as also the salts of the heavy metals, cause their precipitation. Coagulation occurs on boiling, and in the presence of a certain amount of neutral salts, while this does not occur if the solution contains only a trace of salts. The albumins of this order are very rich in sulphur, containing from 1.6 to 2.2 per cent. The nitrogen is held in part as so-called amido-nitrogen, partly as diamino-nitrogen, and partly as tnono- amino-nitrogen. The Globulins. — These comprise serum-globulin, fibrinogen, fibrinoglobulin, myosin, and various vegetable globulins. They are all soluble in dilute solutions of* the neutral salts, and may be pre- cipitated from these solutions by saturation with magnesium sul- phate or by 50 per cent, saturation with ammonium sulphate. Sodium chloride precipitates them only in part. Some of them are insoluble in water, while others are soluble without difficulty. II" a dilute saline solution of the common serum-globulin of* the blood- plasma, for example, i- subjected to dialysis, a certain portion of the globulin i- precipitated. Another portion, however, remains in solution, and may lie demonstrated By saturating with magnesium sulphate or by saturation with 60 per cent, ammonium sulphate. It 42 THE ALBUMINS. is to be noted, moreover, that the portion which remains in solution represents from-three-fifths to four-fifths of the entire amount that was originally present, but it appears that, barring their different solubility in water, both portions are identical. Some of the globulins may also, in part at least, be precipitated from their neutral solutions by copious dilution with water, by pass- ing a current of carbon dioxide through the solution, or by acidifying with acetic acid or some other organic acid. If an excess of the acid, however, is added, they again dissolve. All globulins are coagulated by heat, and it is to be noted that the greater number also pass into the coagulated state when kept long under water. Like the true albumins, the globulins contain nitrogen in at least three forms, viz., as amido-nitrogen, as diamino-nitrogen, and as mono-amino-nitrogen. They contain less sulphur than the albu- mins, but not less than 1 per cent. The Vitellins. — The vitellins are apparently closely related to the globulins and the albumoses. Some of them, such as the aleurons of seeds and the so-called yolk-platelets, which are found in the eggs of certain fish and amphibia, occur in crystalline form, and still others may also be made to crystallize artificially. As has been mentioned, these crystalline bodies do not represent the pure albumins, however, but are probably compounds of albu- mins with various salts and lecithins. Both animal and vegetable vitellins are soluble in dilute saline and alkaline solutions ; they are precipitated from these by acidify- ing with dilute acetic acid, by passing a current of carbon dioxide through the solutions, and by salting with magnesium sulphate or sodium sulphate to saturation. Unlike the globulins, they cannot be precipitated from their solutions by saturation with sodium chloride. The vitellins proper do not contain phosphorus, as is frequently stated, but in the eggs of birds and fish they are commonly found in combination with lecithins and nucleins, both of which are rich in phosphorus. Like the common albumins and globulins, they contain also sulphur, but in variable amounts. THE PROTEIDS. The proteids differ from the albumins in being more complex bodies, and consist essentially of an albuminous radicle, which is variously combined with a non-albuminous group. This may be of the nature of a phosphoric acid radicle, or a carbohydrate group, or a pigment. In this manner the nucleins, the glucoproteids, and the haemoglobins result. The nucleo-albumins, further, which also belong to this group, are formed through the union of an albu- minous radicle with a nuclein. The Nucleins. — The nucleins differ from the true albumins in containing, in addition to carbon, hydrogen, nitrogen, oxygen, THE PROTEIDS. 43 and sulphur, a variable amount of phosphorus, and in some instances also iron Their quantitative composition, moreover, is different, as is ap- parent from the following table : Yolk-nuclein. Veast-nuclein. Carbon 42.11 40.81 Hvdrogen 6.08 5.38 Nitrogen 14.73 15.98 Oxygen 31.05 31.26 Sulphur . . . . 0,-,:, 0.38 Phosphorus 5.19 6.19 Iron 0.29 . . The nucleins occur widely distributed both in the animal and the vegetable world, and are of special importance as food-stuffs, in so far as the iron which some contain is only accessible to animals in this form. They are essentially albumins which are closely com- bined with a phosphoric acid radicle. In certain forms, however, this group is not only united to an albuminous radicle, but also with certain basic substances, such as adenin, hypoxanthin, guanin, and xanthin. These bodies belong to the class of the so-called xanthin, alloxuric, or purin bases, and in combination with a phosphoric acid radicle constitute the so-called nucleinic acids. Individually these various bodies will be considered in another section of this work, but it may here be mentioned that the nucleinic acids and the nucleinic bases not only occur in the animal body in combination with albumins, but also as such. According to the combination of the albuminous group with phosphoric acid only, or through this with the nucleinic bases, the nucleins are now divided into two groups, viz., the so-called para- nucleiri8, or pseudonueleins, and the nuclear nucleins proper. All nucleins possess the character of strong acids. They are soluble in solutions of the hydrates of the alkalies, less readily so in dilute solutions of the alkaline carbonates and in concentrated hydrochloric acid. In water and alcohol they are for the most part insoluble. They are coagulated by heat, as also by alcohol, and are then insoluble in solutions of the alkaline hydrates. In dilute acids and in artificial gastric juice they are practically insoluble, and it is thus possible to separate them from any albumins that may be present at the same time. Like the albumins proper, they give the various color-reactions which are characteristic of the albumins as a class. The Nucleo-albumins. — The nucleo-albumins are compounds of the aucleine and paranucleins with a special albuminous radicle. Like the nucleins, they hence contain phosphorus but their quan- titative composition varies but little from that of the albumins proper. This is no doubt owing to the fad thai the nucleinic or paranucleinic radicle, which enters into their construction, repre- sents only a -mall portion of the entire molecule. Like the nucleins, they occur widely distributed in the animal and vegetable world, 44 THE ALBUMINS. and are of special importance as food-stuffs. Some of them also contain iron. They possess markedly acid properties, and can hence combine with bases to form salt-like products. Most of the nucleo-albumins are insoluble in distilled water, in neutral salt solutions, and in weak acids, while they dissolve with ease in the presence of a small amount of an alkaline hydrate or lime-water. The most important member of this group, casein, occurs in solu- tion in the milk as a calcium compound. In combination with the alkalies or the alkaline earths, the nucleo-albumins dissolve in water upon the application of heat, and it is to be noted that such solu- tions do not coagulate on boiling. Coagulation occurs, however, as in the case of the albumins proper, as soon as the basic component is removed by means of an acid. Other nucleo-albumins, such as those which can be obtained from the yolk of birds' eggs, and leucocytes, are soluble in dilute acids and in a 10 per cent, saline solution, but are also insoluble in water. From their solutions they are partly coagulated by heat. Pepsin in the presence of 0.2 per cent, of hydrochloric acid decomposes the nucleo-albumins with the liberation of the nucleins. The Glucoproteids. — In the glucoproteids an albuminous radicle is combined with a carbohydrate group, or a carbohydrate deriva- tive which may or may not be nitrogenous. They all contain carbon, hydrogen, nitrogen, oxygen, and sulphur. In addition, phosphorus has been found in certain representatives of this group, and these have accordingly been termed phosphoglucoproteids. The gluco- proteids proper comprise the mucins, the mucoids or mucinoids, and the hyalogens, all of which are peculiar to the animal world. Of the phosphoglucoproteids, on the other hand, only two repre- sentatives are known at the present time, viz., the ichthulin of carp eggs, and the helicoproteid which may be obtained from the albu- minous gland of Helix pomata. The carbohydrate radicle, which may be separated from the albu- minous group on boiling with dilute mineral acids, is apparently not the same in all glucoproteids, and in most cases its true chemical nature has not as yet been ascertained. From certain mucins, Landwehr claims to have obtained the so-called animal gum, which is a dextrin-like carbohydrate, when the substance was ex- posed to the action of superheated steam. On carrying the decom- position further, as on boiling with strong mineral acids, lsevulinic acid was found, besides leucin, tyrosin, and other bodies of this order. From the helicoproteid Hammarsten succeeded in splitting off a gum-like dextrorotatory substance, which he regards as animal sinistrin. Beyond these few data, however, practically nothing is known of the character of the reducing substance. While all glucoproteids show a structural composition which warrants their classification as a separate group of proteids, the THE PROTEIDS. 45 subgroups differ from each other in many respects, and the differ- ent representatives of each group, moreover, possess certain features which serve to distinguish them from each other. The mucins proper, which include the mucin that is furnished by the large mucinous glands, the mucin that is found in tendons and the umbilical cord, that which is secreted by snails, and that found in the capsule of frogs' eggs, are insoluble in water. They possess acid properties, and dissolve in water after neutralization with an alkali. Such solutions do not coagulate on heating, but are precip- itated on acidifying with acetic acid. This precipitate is insoluble in an excess of the acid. In the presence of from 5 to 10 per cent, of sodium chloride, however, they are not precipitated in this manner. From such acid solutions they are not thrown down by potassium ferrocyanide, while tannic acid causes the mucin to sepa- rate out. Neutral solutions of the mucins are precipitated by alcohol in the presence of neutral salts. Similar results are ob- tained with some of the salts of the heavy metals. When heated on a water-bath with dilute hydrochloric acid (2 per cent.) the mucins are decomposed, with liberation of the carbohydrate group, which can be demonstrated with Fehling's test (see Urine). Ac- cording to more modern investigations, however, it appears that the radicle which is thus split off is not a true carbohydrate. Miiller and Seemann were thus able to isolate a crystalline substance from mucins which was apparently identical with glucosamin, and Leathes obtained a body which he regards as a reduced chondrosin. Levene concludes from his recent investigations that the mucins contain the complex of chondroitin-sulphuric acid. This would account in a satisfactory manner for the acid properties of the mucins, and the fact that they yield a reducing-substance on decomposition. This, however, is not a carbohydrate proper, but glucosamin. All mucinous solutions are more or less viscid, and are therefore extremely difficult to filter. In the dry state they area white or yellowish-gray powder. The mucoids, or mminoids, are found in the cornea and the vitreous humor of the eye, in the white portion of birds' eggs, in cartilage, and arc very abundant in certain ovarian cysts, where two distinct varieties have been encountered, viz., the so-called metalbu- min or paralbumin, or pseudomucin, and colloid. They will be con- sidered in detail later. The hyalogem comprise ;i class of substances which, according to Krukenberg, are essentially characterized by the fact that on treat- ment with alkalies they are decomposed into an albuminous sub- stance and into nitrogenous carbohydrate-like bodies, the so-called hyaliri8, which in turn are gaid to yield a carbohydrate proper on further decomposition. Among the hyalogens may be mentioned the neossin which is found in edible Chinese swallow nests; membranin, obtained from I' emet'fi membrane and the capsule of the crystalline lens; 46 THE ALBUMINS. spirograph in, from the spirographic membrane ; the holothurian mucin • the chondrosin of certain mushrooms ; and others. The hyalin which is found in echinococcus cysts, and the onuphin of the tubes of Onuphis tubicola, which have both been regarded as hyalogens, are apparently not proteids. Hyalins can, however, also occur as such, or as closely related bodies which are not combined with an albuminous group. They are principally found in the extra-skeletal or intra-skeletal parts of various animals. Among these may be mentioned the so-called chondroitin, which occurs in the matrix of the cartilage of the higher animals, as chondroitin- sulphuric acid ; and chitin, which forms the greater portion of the carapace of the arthropods and the inner skeletal structures of cer- tain cephalopods and brachiopods. From both these substances glucosamin can be obtained on hydrolytic decomposition. The hyalogens are for the most part insoluble in water, and, as we have seen, are decomposed by treating with dilute alkaline solutions. They give the general color-reactions of the albumins, and, like the mucins and mucoids, consist of carbon, hydrogen, nitrogen, oxygen, and sulphur. Their albuminous radicles, however, are unknown. Of the jyhosphoglucoproteids little is known. They are appar- ently related to the nucleo-albumins, and yield paranuclein on diges- tion with artificial gastric juice. The haemoglobins are essentially compounds which contain an albuminous radicle that is variously combined with an organic pigment. They will be considered in detail in the chapter on the Blood. THE ALBUMINOIDS. The albuminoids, as they are commonly termed, are closely related to the albumins, but differ from these in many important par- ticulars. For the most part, they contain less carbon and more oxygen than the albumins proper, and can hence be regarded as early products of decomposition and oxidation. They are not found in the vegetable world, and must therefore be produced in the animal body through a certain rearrangement of atoms from the vegetable albumins." During the reconstruction of the molecule in the animal body, however, a certain amount of carbon is mani- festly lost. We find, as a matter of fact, that in certain representa- tives of this group the aromatic radicle is lacking, and among the decomposition-products of such substances we accordingly find neither indol nor tyrosin. Their nutritive value is therefore also less than that of the albumins, and Yoit actually demonstrated that gelatin, for example, is in itself insufficient to maintain life. Certain members of this group, moreover, cannot be regarded as food-stuffs at all, owing to the extreme resistance which they offer to most solvents, including the digestive fluids. As a class the albuminoids occur widely distributed in the animal THE ALBUMINOIDS. 47 world, and form the greater portion of the internal as well as the external skeleton. The most important members of the group are the keratins, the principal constituents of the epidermal structures of the animal body; elastin, which is found in the connective tissue of the higher animals ; collagen, which is present also in connective tissue and in the organic portions of the bones; gelatin, or glutin, which is soluble collagen, viz., collagen plus water ; the skeletins, spongin, conchiolin, kornein, fibroin, sericin, and elastoidin, which have been mentioned as occurring among the invertebrate animals ; and, finally, the so-called amyloid substance, which is encountered under various pathologic conditions. Of these various substances, the keratins and elastin are more closely related to the albumins proper than the remainder. They both give rise to the same decomposition-products as the albumins, though elastin yields but very little tyrosin and no glutaminic or asparaginic acid. They give the various color-reactions of the albumins, but it appears that elastin only contains in its molecule that form of sulphur which is easily split off on boiling with dilute alkalies. Keratin, on the other hand, contains much sulphur — 3 to 5 per cent. ; and it is inter- esting to note that during its decomposition a fairly large proportion may be obtained in the form of cystin. Both the keratins and elastin can be brought into solution only by means of superheated -team or by boiling with strong alkalies, but the substance is at the same time decomposed. Concentrated mineral acids also dissolve elastin with varying ease, and with or without the application of heat, according to the origin of the material. Collagen and its hydrate glutin, or gelatin, on the other hand, are structurally further removed from the true albumins. They appar- ently contain no aromatic radicle, and hence on decomposition yield neither tyrosin nor indol. Leucin, glycocoll, glutaminic acid, and asparaginic acid are, however, always obtained. Pure solutions of gelatin give the biuret reaction and the xanthoproteic reaction, while the reactions of Millon and Adamkiewicz are negative. The sulphur is apparently present only as closely combined sulphur, as hydrogen Bulphide doe- not develop on boiling with dilute alkalies. Solution- of collagen gelatinize on cooling and redissolve on the application of heat. The behavior of the substance is in this respect exactly the contrary of what we see in the albumins proper. The mineral acids, potassium ferrocyanide in the presence of acetic acid, and -i mineral salts do not precipitate the gelatin from its solutions. Solutions of cartilaginous glutin, which was formerly termed c/io, uliin, possess characteristics which are different from those of glutin thai is obtained from connective tissue or decalcified bones. These differences, however,are not owing to the glutins as such, but to the presence of certain soluble compounds of chondroitin-sul- (ilinric acid, the diondroit in radicle 01 which, a- we have seen, belongs to the so-called hyalins. 48 THE ALBUMINS. The skeletins are in part related to elastin and partly to collagen. Spongin aud conchiolin thus do not give Millon's reaction, and accordingly yield no tyrosin on decomposition, while both are obtained from fibroin, kornein, and elastoidin. It appears, on the other hand, that, with the possible exception of kornein, all the skeletins contain no sulphur. The amyloid substance, finally, occupies a unique position among the albuminoids. It is apparently met with only under pathologic conditions, and is then found in the connective tissues. Like the true albumins, it consists of carbon, hydrogen, nitrogen, oxygen, and sulphur, and on decomposition yields both leucin and tyrosin. It gives Millon's reaction, that of Adamkiewicz, and the xantho- proteic reaction. It is insoluble in water, alcohol, ether, dilute hydrochloric acid, and acetic acid. Concentrated hydrochloric acid and solutions of the alkaline hydrates cause its solution, but at the same time transform it into acid albumin or alkaline albuminate. The gastric juice, contrary to what has been claimed, likewise causes the substance to dissolve. Most characteristic is its behavior toward iodine and aniline green. The latter is colored red. Dilute aqueous solutions of iodine color the substance a brownish red or a bluish violet, which passes into blue on treating with sulphuric acid. Iodo- methyl aniline stains the substance red, especially after previous treatment with acetic acid. The more important members of the various groups which have been briefly considered in the preceding pages, their specific proper- ties, and methods of isolation, will be dealt with in greater detail in connection with the tissues in which they are principally encountered. The derived albumins also, which are now to occupy our attention, are likewise considered only in a general way at this place, as we shall have opportunity to study them in greater detail in the chapters on the Blood and Digestion. THE DERIVED ALBUMINS. Fibrin. — Fibrin occupies a unique position among the albumins. So far as its general chemical composition goes, it is unquestionably closely related to the albumins proper. It contains carbon, hydro- gen, nitrogen, oxygen, and sulphur in very much the same propor- tion as the true albumins, and, like these, yields leucin, tyrosin, glutaminic acid, asparaginic acid, and glycocoll on decomposition. On the other hand, fibrin is insoluble in the common solvents of the true albumins, viz., in water and neutral saline solutions. Acids and alkalies cause its dissolution, but during this process the substance itself is transformed into acid albumin or alkaline albuminate, as the case may be. In this respect fibrin is closely related to the coagulated albumins. It further merits a place among the derived albumins, however, by reason of its being itself a derivative of a true albumin, namely, fibrinogen, which is transformed into fibrin through THE DERIVED ALBUMINS. 49 the agency of a specific ferment. Of the manner in which this transformation is effected we know but little. According; to some observers, the process is essentially an oxidation-process. Others maintain that the fibrin is present in the fibrinogenous molecule in combination with another albuminous group, and that it results from the fibrinogen through decomposition of its molecule under the influence of the fibrin-ferment. However this may be, it is certain that the fibrinogen is not changed into fibrin through any rearrange- ment of its atoms, and that a certain amount of another — but soluble — proteid, fibrinoglobulin, is obtained whenever fibrin itself is formed. It is hence a derivative of a true albumin, but not a native albumin itself. The Coagulated Albumins. — The coagnlated albumins result from the albumins proper through the influence of heat, prolonged exposure to strong alcohol, especially in the presence of a neutral -alt, and in the case of fibrin at least, which, as we have just seen, is closely related to this group, through the activity of a specific ferment. They differ from the true albumins in their extreme resistance to ail neutral solvents, and also to dilute acids and alkalies. Stronger acids and alkalies cause their dissolu- tion, with the simultaneous formation of acid albumins or alkaline albuminates. The Albuminates. — The albuminates, as has been pointed out, result from the native albumins through a process of denaturization, as Neunieister terms it, in consequence of which their original char- acteristics are entirely lost. Aside from their quantitative composi- tion, they differ from each other only in so far as they have resulted through the action of an acid or an alkali. The alkaline albu- minates thus contain less sulphur and less nitrogen than the acid albumins, as a portion of the sulphur and the so-called amido- oitrogen have been split off. Both the acid albumins and the albu- minates are insoluble in neutral solvents, and are therefore precipi- tated from their solutions on neutralization. They are soluble, on the other hand, in solutions of the alkaline hydrates, in dilute solu- tion- of sodium carbonate, in hydrochloric acid, and with a little more difficulty in strong acetic acid. From their acid solutions they are precipitated by salting with ammonium sulphate or sodium chloride. Through the action of an alkali acid albumin can be transformed into alkaline albuminate, but it is, of course, manifest that tin- reverse cannot occur. In the living body the denaturiza- tion of all albumins is effected during the process of digestion, and invariably precedes the formation of albumoses and peptones. The Albumoses. — The albumoses result from the albumins proper, and also from the albuminoids and the albuminous radicles of the proteids, through the action of the so-called proteolytic fci-- ments, or during their hydrolytic decomposition l>v means of acids or alkalies. In every case their formation is preceded by the denaturization of the original molecule. i 50 THE ALBUMINS. Collectively the albumoses which are derived from the true albu- mins, in contradistinction to those which are obtained from the albuminoids, are also termed proteoses. According to their origin, we further distinguish between globulinoses, vitelloses, caseoses, myosinoses, keratinoses, elastoses, gelatoses, etc. One and the same albumin, mqreover, can give rise to the formation of different albu- moses. During the decomposition of fibrin, for example, primary albumoses first result, which are then transformed into secondary albumoses, and these into the so-called peptones. Formerly a dis- tinction was made between hemi-albumoses and anti-albumoses, according to the varying degree of resistance which the individual substances offered to the action of trypsin. While we still recognize the existence of hemi- and anti-groups in the original albumins, it appears from recent researches that a complete separation of these groups does not occur at the stage of digestion at which the albu- moses only are found. These terms have hence been abandoned. The primary, albumoses were formerly also divided into two groups, viz., the proto-albumoses and the hetero-albunioses. We now know, however, that still others are primarily formed, as will be shown later. The term dysalbumose has been applied to a variety of albu- mose which apparently results from the hetero-albumoses when these are dried or kept under water for some time; dysalbumose is then insoluble in dilute saline solution. In their quantitative composition the albumoses very curiously do not differ materially from the original albumins, and it is hence dif- ficult to explain the relationship which exists between the two groups. According to most authorities, the albumoses represent hydrolytic decomposition-products of the albumins, and it has been shown as a matter of fact that through the influence of acetic anhy- dride upon the so-called peptones, which, as we shall presently see, represent the final products of albuminous digestion, albuminate-like substances can be obtained. Others, however, regard the trans- formation of albumins into albumoses as a depolymerization of the original substance, while still others look upon both as isomeric bodies. The albumoses give the same color-reactions as their mother- substances. With the biuret test, however, the original violet is absent, and instead a beautiful rose color is obtained. Their final decomposition-products are the same as those of their antecedents. Unlike the albumins, the albumoses are not entirely indiffusible, and it appears that the power to pass through animal membranes increases as they become structurally further and further removed from their mother-substances. As a class the albumoses are much more readily soluble than the albumins. Most of them are soluble in water or in dilute saline solutions, and also in dilute acids and alkalies. From their solu- tions they are readily precipitated by certain neutral salts, notably ammonium sulphate, which precipitates all albumoses when added to THE DERIVED ALB V MISS. 51 saturation, the reaction being slightly acid. Most of them, indeed, are thrown down when the salt is added to the extent of 75 per cent. Each albumose, in fact, appears to possess certain special limits of precipitation with ammonium sulphate which enables ns to separate the individual substances from each other, and also from other albumins which may be present at the same time. Zinc sul- phate behaves in a very similar manner. Sodium chloride when added in substance to saturation causes a partial precipitation of the albumoses from their neutral solutions, while a fairly complete sepa- ration is obtained if to the saturated fluid is added a small amount of acetic acid that has been saturated with the salt. Neutral or acid solutions of albumoses are not coagulated by heat nor on treating with alcohol, although they are precipitated when this is present in considerable concentration. After precipitation, however, they are as soluble as before, and in this respect they differ very markedly from the albumins proper. Like the native albumins, the albumoses are precipitated by nitric acid, potassium ferrocyanide and acetic acid, metaphosphoric acid, phosphotungstic acid in the presence of hydrochloric acid, tannic acid, picric acid, trichloracetic acid, etc. ; but it must be noted that on the subsequent application of heat the precipitate redissolves, but reappears on cooling. The same result is obtained by treating a solution of albumoses with an equal volume of a saturated solution of sodium chloride and acidifying with acetic acid. The Peptones. — The term peptone is generally used to designate those final products of albuminous decomposition which result from the albumoses on further digestion with the proteolytic ferments, in -'i far as they still possess an albuminous character. According to Kuhne and his school, they differ from the albumoses and all other albumins in not being precipitated from their solutions on saturation with ammonium sulphate. Such substances are obtained in abun- dance during the process of tryptic digestion, in vitro at least, while during peptic digestion they are formed only in small amounts and on prolonged exposure to the ferment. I have pointed out above, that Kuhne distinguishes between a heini- and an anti-group in the albuminous molecule, and he accord- ingly divide- the peptones into hemipeptone and antipeptone. Both are supposedly formed during peptic as well as pancreatic digestion, in the Former instance the mixture of the two substances is spoken <>f ,-i- amphopeptone. It is to be noted, however, that hemipeptone has thus far not been Isolated as such, and it appears indeed that the substance ifl only theoretically existent, and on artificial digestion with trypsin i- further decomposed into amido-acids, while in the digestive trad it is supposedly taken up at once by the epithelial cells ami transformed into albumin proper. Antipeptone, on the other hand, to judge from recent investigations, represents a mixture of acid and basic substances, among which Latter the so-called hexon DflSdfl are found. It is thus dear that with the possible exception 52 THE ALBUMINS. of the theoretical hem i peptone, we are not warranted in speaking of peptones as a well-defined chemical unity, and it is questionable, indeed, whether the final products of proteolytic digestion can actually be classed as albumins. We shall accordingly not give an account of the various properties of the peptones which would be based upon the conception that we are dealing with a well-defined chemical substance. We shall have occasion, however, at another place to deal in detail with some of the better known constituents of antipeptone. CHAPTER III. THE CARBOHYDRATES. It has been pointed out in the preceding chapter that while plants are capable of effecting from relatively simple compounds the svnthesis of those complex albumins which are found in their various tissues and organs, animals do not possess this power, and are therefore dependent for their supply of nitrogen upon the albuminous food-stuff's that have been elaborated by plants. The carbohydrate supply of animals is also derived from plants, but for the maintenance of life it is not necessary that the carbohydrates should be furnished as such, as animals are not only capable of splitting off the carbohydrate radicle of the albuminous molecule as occasion demands, but, as we shall see later, they can also form carbo- hydrates directly from the fats which are stored in their tissues. The carbohydrates cannot therefore be regarded as essential food-stuffs, and we see, as a matter of fact, that carnivorous animals, at least, are capable of existing on albuminous food exclusively. Carbo- hydrates are important, however, as the stored energy which is thus supplied to animals represents a considerable caloric value, and they can hence protect the albumins from undue destruction. The im- portance of the carbohydrates as food-stuffs is thus only secondary, and they are totally unable to take the place of the albumins. All living matter requires a definite amount of nitrogen so that life may be maintained, and if this is withdrawn death inevitably results. It is to be noted, however, that whereas animals can exist without carbohydrate food, and whereas the albumins largely predominate in ii- (issues, the reverse holds good for plants. Here the carbo- hydrates prevail, while the albumins are much less abundant. Con- sequently we may expect to find a far greater diversity of carbo- hydrate-, in the vegetable than in the animal world. This is actually the case. Ajb it would lead too far, in a work of this -'•ope, however, to consider all those carbohydrates which occur in the vegetable world, we -hall confine our attention in the subsequent pagi - 10 those form- which may be regarded as common food-stuffs, OT those which are more or Less peculiar to the animal body. All carbohydrates consisl of carbon, hydrogen, and oxygen, and in mo-t members of the group tin' elements hydrogen and oxygen are presenf in such proportion a- to form water, [n others, how- ever, this i- not the case; and there are substances, such as lactic acid .Mid acetic acid which likewise contain hydrogen and oxygen in this proportion, but which are manifestly not carbohydrates. As 54 THE CARBOHYDRATES. there are no specific properties peculiar to these substances as a class, it is impossible to give an adequate definition of what is meant by the term carbohydrate. Chemically speaking, they are deriva- tives of polyatomic alcohols, and of the nature of aldehydes or ketones. They are conveniently divided into monosaccharides, disaccharides, and polysaccharides. The disaccharides and poly- saccharides differ from the monosaccharides in being more complex substances and apparently built up from the monosaccharides through a condensation of monosaccharine anhydrides to form a double or a multiple group. Accordingly, on hydrolytic decom- position they yield two or more monosaccharine molecules for every original molecule, as is shown below : (1) C6H1206 — glucose, viz., leevulose. (2) C12H22On + H20 = CfiH1206 + C6rl]206. Cane-sugar Glucose. Lsevulose. (Disaccharide.) (Monosaccharides.) THE MONOSACCHARIDES. According to the number of carbon atoms which are present in the molecule, the monosaccharides can be divided into trioses, tet- roses, pentoses, hexoses, heptoses, octoses, etc. Of these, the hex- oses only will be considered, as the remaining groups are of practi- cally no significance as animal food-stuffs, and are in man, at least, mostly eliminated through the kidneys as foreign matter. The most important representatives of the hexoses are glucose,, which is also termed dextrose ; lsevulose or fructose ; mannose and galactose. Some of these, such as glucose and lsevulose, are found free in nature, or they result as hydrolytic decomposition-products from the more complex carbohydrates and related nitrogenous sub- stances, the so-called glucosides. They are all derivatives of the stereo-isomeric hexatomic alcohols of the composition CH2.OH — (CH.OH)4 — CH2.OH. Of these, three are known to occur in the natural state, viz., sorbite, or glucite, mannite, and dulcite. As has been pointed out above, the monosaccharides are either aldehydes or ketones, and we accordingly find that glucose, mannose, and galac- tose represent the aldehydes (aldoses) of sorbite, mannite, and dulcite, respectively, while lsevulose is the ketone (ketose) of mannite. They can therefore be represented by the structural formulse : (1) CH2(OH).CH(OH).CH(OH).CH(OH).CH(OH).CHO (glucose, mannose, and galactose). (2) CH2(OH).CH(OH).CH(OH).CH(OH).CO.CH2(OH) (kevulose). As a matter of fact it is possible to transform these hexoses into their corresponding alcohols by careful reduction, and vice versa. In accordance with their character as aldehydes or ketones, the aldoses on oxidation yield oxyacids, which have the same number THE MONOSACCHARIDES. 55 of carbon atoms as the original substances, while the ketoses give rise to acids which have a smaller number of carbon atoms. The oxyacids which arc derived from the aldoses, moreover, are either monobasic or dibasic, according to the extent to which the oxida- tion has been carried. These changes are represented by the equations : (1) CH2(OH).CH(OH).CH(OH).CH(OH).CH(OH).CHO + O = Glucose. CHa(OH).(CH.OH )4.COOH. Gluconic acid. (2) CH2(OH).CH(OH).CH(OH).CH(OH).CH(OH).CHO + 30 = Glucose. COOH.(CH.OH)4.COOH + H,0. Saccharinic acid. The acids which can thus be obtained from the aldoses glucose, mannose, and galactose, are the monobasic acids — gluconic, man- nonie, and galactonic acid ; and the dibasic acids — saccharinic, man- nosaccharinic, and mucinic acid. Of these, saccharinic acid is of special interest, as it can readily be transformed into saccharolactonic acid, which in turn yields glucuronic acid. This latter, as we shall see later, is found also in the animal body. It is an aldehydic acid, and stands midway between gluconic acid and saccharinic acid. It is represented bv the formula COOH.CH(OH).CH(OH).CH(OH). CH(OH).COH. The hexoses are colorless and odorless substances of a sweetish taste ; they present a neutral reaction, and are readily soluble in water, with difficulty in absolute alcohol, and insoluble in ether. They can be obtained in crystalline form, and diffuse through animal membranes. Some of them are dextrorotatory, others laevorotatory, while still others are optically inactive. They are strong reducing- substances, and for the most part fermentable with yeast. Espe- cially interesting further is the behavior of the hexoses toward the hydrazins in the presence of acetic acid, with which they form hydrazo7t8. These can be further transformed into osazons, which arc very characteristic substances, and may serve to distinguish the various sugars from each other. On decomposition with fuming hydrochloric acid the osazons then give rise to the formation of ogon8 — /'.'•., keto-aldehydes, which can be further reduced to ketoses. By starting with an aldose, it is thus possible to obtain an isomeric ketose. These changes may be represented by the equations: I CH,(OH).CH(OH).CH(OH).CH(OH).CH(OH).CHO • < •;H,i.xir.\IT,= Glucose Phenylhydrazin. CH,(OH).CH(OH).CH(OH |.CH(0H).CH(0H).CH:N.NH.C6H6 H,0 Phony lglucohydrazon. ■ II OH M II "II .< II "II I II on ,CH(OH).CH:N.NH.CaH6 | <;n ..\n.Nir!l = CH,(OH CH(OH).CH(OH).CH(OH |.C.CH.N.NH.CeH, |- H2<> + 211. Phenylglucosazon. ssmru6. 56 THE CARBOHYDRATES. (3) CH2(0H).CH(0H).CH.(0H).CH(0H).C.CHN.NH.C6H5+2H30+2HC1 = N.NHC6H5. 2NH3.NH.C6H5.HCl + CH2(OH).CH(OH).CH(OH).CH(OH).CO.COH. Oson. (4) CH2(OH).CH(OH).CH(OH).CH.(OH).CO.CHO + 2H= CH2(OH). [CH(OH)]3.COCH2(OH) Lsevulose. The same result may be reached when the corresponding osazon of the aldose is directly reduced, and the resulting osamin is treated with nitrous acid. The glucosamin thus obtained as an intermediary product is of special interest in so far as it also results from the de- composition of the hyalins chitin and chondroitin. By oxidation with bromine glucosamin then yields chitonic acid, from which the cor- responding sugar, chitose, can be obtained on reduction. The changes which are here involved may be represented by the equa- tions : (1) CH2(OH).CH(OH).CH(OH).CH(OH).C.CH.N.NH.C6H6 + H20 + 4H = Phenylglucosazon N.NH.C6H5 CH2(OH).CH(OH).CH(OH).CH(OH).CO.CH2(NH,) + Glucosamin C6H5.NH2.NH + C6HB.NH2 Phenylhydrazin anilin (2) CH2(OH).CH(OH).CH(OH.)CH(OH)CO.CH2(NH2) + HN02 = Glucosamin H20 + 2N + CH2(OH).CH(OH).CH(OH).CH(OH).CO.CH2(OH) Leevulose. (1) C,sH,0N2O12 + 4H20==2CH2(OH).CH(OH).CH(OH):CH(OH). Chitin. ' Glucosamin. CO.CH2(NH2) + 3CH3.COOH (2) CH.,(OH).CH(OH).CH(OH.)CH(OH).CO.CH2(NH2) + 40 = CH2(OH).CH(OH).CH(OH).CH.(OH).CH(OH).COOH + HN02 Chitonic acid (3) CH2(OH).CH(OH).CH(OH).CH(OH).CH(OH).COOH + 2H = CH2(OH).[CH(OH)]4.COH + H20 Chitose On boiling with dilute mineral acids the hexoses are decomposed into formic acid, lsevulinic acid, and certain humin substances. With the alkalies, on the other hand, they yield, besides other products, also Isevulinic acid and a ketonic acid of the composition CH3.CO. CH2.CH2.COOH. On the application of dry heat they form so- called caramel, and are finally carbonized. As stated above, most of "the hexoses are capable of undergoing fermentation — i. e., a decomposition which is effected through the activity of certain minute organisms. According to the character of the specific organism present, we distinguish between alcoholic and acid fermentation, such as lactic acid, butyric acid, and acetic acid fermentation. The former is brought about through the influ- ence of various varieties of yeast, while the latter are referable to the activity of certain bacteria, such as the Bacterium lactis aerogenes, the Bacillus acidi butyrici, the Mycoderma aceti, etc. The decom- THE DISACOHABIDES. 57 positions which are thus effected may be represented by the equa- tions : (1) CeH^Og = 2C,II5(OH) + C02. Ethyl alcohol. (2) C6H1206 = 2CH3— CH.OH-COOH. Lactic acid. (3) 2C3H603 = C3HT.COOH 2COa + 4H. Lactic acid. Butyric acid. Of the hexoses, glucose only is found in the animal body ; while laevulose, mannose, and galactose do not occur as such, and on reach- ing the liver are apparently immediately transformed, together with glucose, into the polysaccharide glycogen. Whether or not a trans- formation into glucose first takes place, and whether this can occur in the intestinal mucosa, is unknown. The amount of glucose which may be found in the blood and lymph, and in the various tissues of the body, is always small. Laevulose occurs in nature together with glucose, most abundantly in various fruits, the roots and seeds of many vegetables, and also in honey. It further results during the hydrolytic decomposition of cane-sugar, inulin, and other carbohydrates. It is readily soluble in water, and its aqueous solutions, in contradistinction to common glucose, are hevorotatory. It may be obtained in crystalline form, but with difficulty. It is fermentable, and gives the same reduc- tion-tests as glucose (which see). With phenylhydrazin laevulose yields the same osazon. Galactose is formed during the hydrolytic decomposition of lactose and many other carbohydrates. It is also obtained from eerebrin on heating with dilute mineral acids. It is not so readily soluble in water as glucose, but like it is dextrorotatory. Galactose crystallizes in needles and platelets which melt at 168° C. It is fermentable, and yield- an osazon which melts at 193° C. It re- duce- an alkaline solution of cupric oxide, but to a less marked degree than glucose. On oxidation it yields first galactonic add and later mucinic arid. Glucose will be considered in a subsequent chapter, where the methods of testing for the simple sugars in general, and also their quantitative estimation, will be described. THE DISACCHARIDES. The disaccharidea result from the monosaccharides through a con- densation of the anhydrides of two monosaccharine molecules, analo- gous to the formation of* ethers from alcohol-. On hydrolytic de- composition they accordingly yield two monosaccharine molecules, which represent either one ana the same substance or two isomeric bodies. Some of the disaccharidea occur in nature as such, while other- result from the decomposition of still more complex carbo- hydrates. 58 THE CARBOHYDRATES. The most important members of the group are cane-sugar or saccharose, lactose, maltose, and isomaltose. They are all hexo- . bioses — i. e., they represent the union of the anhydrides of two hexoses, and can therefore be represented by the general formula C12H22Ou. Of these, cane-sugar is formed through the union of one molecule of glucose and one molecule of lsevulose ; lactose from glucose and galactose; while maltose contains two molecules of glucose. In their general properties the disaccharides closely resemble the monosaccharides. Like these, they have a sweet taste. They are crystallizable, capable of passing through animal membranes, and are optically active. In certain particulars, however, differences exist. Lactose, maltose, and isomaltose are thus capable of reducing metallic oxides in alkaline solution, and yield osazons with phenyl- hydrazin, while saccharose does not react in this manner. The disaccharides as such are not fermentable, but only after inver- sion to monosaccharides. It is true that a solution of cane-sugar or of lactose will undergo alcoholic fermentation when exposed to the action of yeast; but we now know that the yeast-cell is capable of furnishing certain ferments which belong to the class of the so-called non-organized ferments, and which themselves are capable of bring- ing about the inversion of these more complex carbohydrates.. Emil Fischer, moreover, has shown that for the inversion of a special disaccharide a specific ferment is necessary. As the various fermentative agents, however, possess a varying number of inverting ferments, it is clear that a certain disaccharide may be inverted by one form of yeast, but not by another ; while, on the other hand, one special form may be capable of inverting all forms of the disaccharides. This is actually the case, and we thus find that the so-called kefir granules, as also the Bacterium lactis, can invert cane- sugar as well as maltose and lactose. Common yeast, on the other hand, inverts only cane sugar and maltose, while lactose is not attacked. According to their specific power of inversion, these ferments are spoken of as invertin, maltase, and lactase. The derivation of the two latter names is, of course, apparent. The significance of the term invertin, however, is not so clear. It has reference to the mixture of glucose and lsevulose which is obtained from cane-sugar by inversion, and which was originally spoken of as invert-sugar. Invertin is thus a ferment which inverts cane-sugar. After inversion the disaccharides undergo fermentation, like the monosaccharides, and here, as there, we may distinguish between alcoholic, lactic acid, butyric acid, and acetic acid fermentation. Cane-sugar is found in nature most abundantly in sugar-cane, in the roots of the sugar beet, and in the stems of certain plants. The pure substance is crystalline, and melts at 160° C. On further heating it turns brown and forms so-called caramel. It is easily soluble in water, and turns the plane of polarization to the right. THE POLYSACCHARIDES. 59 As stated, it does not yield an osazon and does not reduce metal- lic oxides. After inversion with invertin it undergoes the same fermentations as the resulting monosaccharides. On oxidation it vields, in addition to other substances, saccharinic and oxalic acids. Maltose does not occur in nature as such, but results during the digestion of starch and glycogen in the alimentary canal. It is a crystalline substance, which is easily soluble in water and turns the plane of polarization to the right. With phenylhydrazin it yields an osazon — maltosazon — which melts at 206° C. It readily under- goes fermentation, and like glucose reduces metallic oxides in alka- line solution, but to a less degree. Isomaltose results from starch through the action of a diastatic ferment. In the intestinal canal it is thus found together with maltose. It is easily soluble in water and turns the plane of polari- zation to the right. Its osazon melts at 150° to 153° C. It under- goes fermentation, but much more slowly than maltose. Lactose, which is almost exclusively found in the animal world, will be considered in a subsequent chapter (see Milk). THE POLYSACCHARIDES. The polysaccharides result from the monosaccharides in the same wav as the disaccharides. In other words, they represent the anhydrides of the monosaccharides, of which many molecules, how- ever, are condensed to form the resulting polysaccharine molecule. Their general formula therefore is (C6H1(JOr))x, in which x is a vari- able factor, but exceeds two. In many cases the value of x is unknown, but it is probably always large. From a determination of the -izc of the starch molecule, for example, we may conclude that x in this case is equivalent to 108. In others, such as glycogen and the dextrins, however, it is certainly much smaller. In con- formity with their structure, the polysaccharides all yield mono- saccharides on hydrolytic decomposition. During this process, however, a variable number of intermediary products are formed, which may themselves be polysaccharides, though of a lower order, and which in turn yield disaccharides and finally monosaccharides. Starch is thus first transformed into erythrodextrin, which in turn yield- achroodextrin ; this is further changed to isomaltose, and then to maltose, which finally yields glucose. In other eases, ;is with glycogen, the disaccharides isomaltose and maltose are formed directly. Cellulose likewise yields glucose as a, final product, while Uevulose results from inulin, and mannose from the so-called reserve celluloses, which are found in the cell-walls of many seeds. Galac- tose i- similarly obtained from many gums, and from a variety of cellulose, which Schultze hae termed galactose-cellulose, in con- tradistinction to the mannose-cellulose and the true dextrose-cellu- loses. J 1 1 many instances, however, the exaci mode of decomposi- 60 THE CARBOHYDRATES. tion, as also the character and number of the intermediary products, is but imperfectly understood. In their physical and chemical properties considerable differences exist between the polysaccharides and the other carbohydrates which have so far been described. They are thus non-crystal lizable substances and devoid of a sweet taste. In alcohol and ether they are insoluble. In water most of them are more or less soluble, but as a class they are incapable of diffusing through animal membranes. From their solutions they can be precipitated by saturation with neutral salts, and notably with ammonium sulphate. Like the monosaccharides and disaccharides, they are optically active, but with the exception of the dextrins they do not reduce metallic oxides in alkaline solution, and none of them combine with phenyl- hydrazin to form osazons. As such, they are incapable of under- going fermentation ; but, like the disaccharides, they may be inverted to monosaccharides through various ferments or acids, and can then be further decomposed. Especially important is their behavior toward iodine, with which most of the polysaccharides combine to form colored compounds that are quite characteristic. Starch is thus colored blue, glycogen a mahogany brown, and erythrodextrin red. The polysaccharides which are used as food-stuffs are conveni- ently divided into starches, vegetable gums and dextrins, and cellu- loses. Of these, the starches are by far the most important, as they include not only vegetable starches and glycogen, but also give rise to formation of the dextrins. Like the disaccharides, all these substances finally give rise to the formation of glycogen, but it appears that they are previously trans- formed into glucose, and that this transformation takes place in the epithelial lining of the intestinal canal. Starch occurs widely distributed in the vegetable world, and con- stitutes the most important reserve food of most of the higher plants. It is found in the form of distinct granules, which, on microscopic examination exhibit a marked concentric striation, and which differ in size and form in different plants. The individual granules are enclosed in a capsule of so-called starch cellulose, which is insoluble in water, but which can be made to open by heating in the presence of much water. The contained starch -prarm fo.se can thus be obtained, and constitutes the so-called soluble starch, amylum or amylodextrin. During this process no doubt a still more complex molecular group of monosaccharine anhydrides is decomposed, but of the intermediary products which are thereby formed, if any, nothing is known. In the alimentary canal this change is effected through the activity of certain ferments, which then further give rise to the formation of dextrin, maltose, isomaltose, and a certain amount of glucose. On boiling with dilute acids glucose is formed, with various dextrins as intermediary products. Among these, as has been shown, erythrodextrin is apparently the first to develop, THE POLYSACCHARIDES. 61 achroodextrin appears later, and from this isomaltose, maltose, and glucose are finally obtained. It appears, however, that during the decomposition of achroodextrin at least still other dextrins of lower molecular weight are simultaneously formed, which in turn yield maltose and glucose. But finally one dextrin is obtained which undergoes no further change, and which is termed maltodextrin. Most characteristic is the behavior of starch toward iodine, with which it gives an intense blue color that disappears on heating, but reappears on cooling. In a solution of sodium or potassium hydrate starch swells up and forms a paste. Inulin and lichenin, which also belong to the starches, and which occur in the roots of various composites and in lichens, respectively, are insignificant as food-stuffs and need not be considered. Glycogen, which likewise belongs to this class, and is known also as animal starch, is largely formed in the animal body and represents one of its most important constituents. It will be considered in detail in a subsequent chapter. Dextrins. — The dextrins, as has been shown, are formed from starch during its hydrolytic decomposition by means of ferments or on boiling with dilute mineral acids. To a certain extent they result also when starch is heated to a temperature of from 200° to 210° C. Through continued decomposition they give rise to maltose and isomaltose, and finally to glucose. As a class the dextrins are easily soluble in water and turn the plane of polarization to the right. From the other polysaccharides they differ in their ability to dissolve cupric hydroxide in alkaline solution. With erythrodextrin iodine strikes a red color, while achroodextrin is unaffected. The so-called vegetable gums and vegetable mucins will not be considered, as they are of no importance as food-stuff's. To this class belong the so-called gum Arabic, wood gum, cherry gum, etc., as also the various pectins. Celluloses. — As food-stuffs the celluloses are likewise of second- arv importance. They arc considered, however, at this place owing to their wide distribution in the vegetable world, where they form the greater portion of all cell-envelopes. In the animal world they are Likewise encountered, and enter largely into the composi- tion of tlic external skeleton of the tunicates, (lie arthropods, and Bome of tic cephalopoda. They arc characterized by their extreme resistance to the mosl diver- solvents, and arc indeed soluble only in a solution of CUpric hydroxide in Strong ammonia — the so-called Schweitzer's reagent. From this solution the substance can !><■ obtained in amorphous form on precipitation with acids. Moder- ately concentrated sulphuric acid transforms cellulose into vegetable amyloid, which i- colored blue by iodine. With concentrated nitric acid, or with ;i mixture of nitric acid and concentrated sulphuric acid, it yields the highly explosive nitrocelluloses. Wood (lignin)and cork arc derivatives of cellulose. On hydro- 62 THE CARBOHYDRATES. lytic decomposition the common cellulose yields glucose, while the so-called hemicelluloses give rise to galactose or rnannose, as also to certain pentoses, such as arabinose and xylose. In the intestinal canal a certain portion of the ingested cellulose is unquestionably dissolved. The products, however, to which it gives rise are for the most part unknown. Certain micro-organisms which are present at the time bring about a fermentation of the substance, during which marsh gas, acetic acid, and butyric acid are formed, but the greater portion no doubt is eliminated in the feces as such. CHAPTER IV. THE FATS. The origin of fats in the animal body is threefold : one portion is derived from the fats which have been ingested as food ; another portion is formed from the carbohydrates ; while a third portion results from the decomposition of albumins. As food-stuffs the fats are of great importance, because their caloric value is quite high — higher in fact than that of the carbohydrates and albumins ; but, like the carbohydrates, they are unable to take the place of the albumins. Animals that are fed exclusively on fats die sooner or later, although they may become quite fat during the period of their special diet. In the animal body they represent a variable amount of reserve food, which is conveniently stored in the subcutaneous areolar tissue, in the omentum and mesentery, in the bone-marrow, etc. In case of inanition it is utilized long before the tissues of the body proper are attacked, and we accordingly find that in persons who have died from wasting diseases every vestige of fat may have disappeared, while the muscular nutrition may still be fair. That portion of the body fat which is derived from the fats ingested as such is really the smallest portion, and by far the greater amount results from the carbohydrates. The manner in which this transformation takes place is unknown, but it is probable that the carbohydrates which are utilized for this purpose are first decom- posed, and then reduced ; and that the fats finally result through a synthesis of Buch redaction-products. Such syntheses, however, cannot at once be compared to those which take place in plants, for here we have PO(OH)j Di Btearyl-glycerln-phoBphorlc acid. 66 THE FATS. (3) CH2.O.C18H350 f (CH3)3 CH2.O.C18H350 I II I CH.O.Cl8H350 + N \ CH2— CH2.OH = CH.O.C18H350 I II I CH2.0— PO(OH)2 L OH CH2.0— PO.C2H4 Cholin. OH (CH3)3 J- N OH Lecithin. On decomposition of the lecithins with acids or alkalies we accordingly obtain glycerin-phosphoric acid, fatty acids, and cholin. At the same time, however, another basic substance, ?ieurin, is usually found, and it is to be noted that, in contradistinction to cholin, neurin possesses extremely toxic properties. It results from cholin through the loss of two atoms of hydrogen and one atom of oxygen, and is also formed during bacterial decomposition of the lecithins in the presence of much oxygen. Chemically it is (CH3)3 / trimethyl-vinyl-ammonium hydroxide, N — CH=CH2, while cholin \ OH must be regarded as trimethyl-oxyethyl-ammonium hydroxide. Another derivative of cholin which may be obtained through the action of fuming nitric acid, is a basic substance that is apparently isomeric with muscarin, and, like this, extremely toxic. Chemically (CH3)3 / it may possibly be represented by the formula N — CH2.COH, and OH could accordingly be regarded as the aldehyde of oxyneurin (tri- methyl glycocoll). The lecithin which is most commonly found in the aninial body is the cholin compound of distearyl-glycerin-phosphoric acid ; other lecithins can, of course, also occur, in which the glycerin hydroxyl groups have been replaced by the radicles of oleic and palmitic acids, for example, but they are but little known. In its dry state the common lecithin occurs as a wax-like, plastic mass, which is soluble in alcohol (at 40°-50° C), ether (less readily), chloroform, benzol, carbon disulphide, and in the fatty oils, while in water it is insoluble. Placed in water it swells and becomes pasty, and on microscopical examination it will be noted that the substance occurs in the form of peculiar slimy droplets and threads, which are generally termed its myelin forms. From its alcoholic solution it crystallizes in wart-like masses, which consist of small platelets. Of special interest is the tendency of the lecithins to combine with albumins to form more or less stable compounds, which have been termed lecithalbumins. Such compounds have been found in the mucosa of the stomach, in the lungs, the liver, and the spleen. In THE CHOLESTERLXS. 67 the yolk of eggs it occurs in combination with vitellin, but is here apparently not closely bound. A certain similarity thus exists between the lecithins and the nucleins ; both contain phosphorus in their molecules, and both combine with albumins to form more complex substances. The lecithins occur widely distributed in both the animal and vegetable world. According to Hoppe-Seyler, they are found in all cells and bodily fluids. They are especially abun- dant in nerve-tissue and also in the eggs and semen of most animals. Their isolation and special tests will be considered in a subsequent chapter. THE CHOLESTERINS. The cholesterins are monatomic alcohols of the formula C2-H45. OH + H20, and occur widely distributed both in the vegetable and the animal world. They are especially abundant in nerve-tissue and in the bile. In the gall-bladder they are frequently found in the form of so-called gall-stones, and not uncommonly constitute the greater portion of their solids. Different varieties apparently exist, such as the common cholesterin of the concretions just men- tioned, isocholesterin (which has been obtained from lanolin), phy- tosterin, paracholesterin, and kaulosterin, which are found in plants. Their structural composition is unknown. Like the fats, they are insoluble in water, but soluble in ether, alcohol, and chloro- form, from which solutions they may be obtained either in the form of very characteristic platelets or as needle-like crystals. In solu- tions of the alkalies, in the absence of alcohol, they are entirely insoluble, even on boiling, in which respect they differ from the fats. Like glycerin, cholesterin combines with fatty acids to form com- pound ethers, and in this form it is frequently found in nature. In wool-fat, for example, it is thus present in large amounts, and from it such ethers can be readily obtained. In pure form they constitute the lanolin of the shops. These ethers show a remarkable difference, as compared with the fats, in their behavior toward water. Of this they apparently take up one-quarter of their own weight, and on stirring give rise to a pasty, frothy mass. From their ethereal compounds eholesterin can readily be sepa- rated by treating with diacetic-ethy] ether, which dissolves the cholesterin and leaves the ethers behind. Their special tests, and also their mode of isolation, will be taken up in a subsequent chapter. After having thus studied the. three great classes of food-stuffs which plants are capable of elaborating from water, carbon dioxide, and certain mineral -alt-, and which are also represented ill the animal body, we shall now proceed to a similar survey of the natural decomposition-products of these substances which are formed during their passage through the animal body, and which are of more or less interesl in indicating the manner in which these decompositions 68 THE FATS. are effected. Like the substances that have already been con- sidered, these products will be taken up only in a general way at first ; while their special study, as well as their methods of isolation and quantitative estimation, will be considered in succeeding chapters, in connection with the chemical study of those tissues in which they are principally encountered. At this place I wish only to impress general facts upon the mind of the student, so that he will be pre- pared to understand the composite chemical structure of the various tissues and organs of the body, as will be described later. CHAPTER V. THE NITROGENOUS DERIVATIVES OF THE ALBUMINS. THE PROTAMINS. The term protamin was first introduced by Miescher to designate a basic substance which he was able to isolate from the spermatozoa of the salmon, and which is there apparently united with a nuclemic acid radicle. Kossel then showed that a very similar substance can be obtained from the spermatozoa of the sturgeon ; and his pupil, Kuraieff isolated a third body of this order from the spermatozoa of the mackerel. It was thus shown that different protamins occur in nature, and the use of the term has since been extended to the entire class. Miescher's original protamin is now spoken of as sXLand is identical with Kossel's clupem, which was obtained from the spermatozoa of the herring. The two other protamms which have thus far been isolated are termed storm and scombnn, according to their origin from the sturgeon and the mackerel, re- SPA Jo^ding to Kossel, the protamins are essentially albumins of the lowest order, and he assumes that a radicle of this kind forms the nucleus of all the more complex albumins. This assumption is largely based upon the observation that all protamins yied certain dewmposition-products, which may also be obtained from the various albumin aiounini:-. , , • i • These products are the so-called hexon bases, and comprise his- tidin arginin, and lysin. But whereas sturin and all the complex albumins which have been examined in this direction give rise to the formation of all three of these bodies apparently, salmin (clupem) and scombrin only contain the arginin group. It consequently fol- lows that the protamin radicle of the complex' albuminous molecule mu.t be of the nature of sturin. Whether other complex albumins exisl which also contain the salmin or Bcombnn radicle is as yet unknown. , . , \- regards the quantitative relations which exist between the three hexon bases within the sturin molecule, our knowledge is incom- plete. Kossel suggested that six molecules here unite in such-a manner thai every two combining molecules lose two molecules ot water, and that the formation of sturin might bence be represented by the equation : M!,no; ::i,n„N40; I ^j!Mvo. r:i6n«,N,A | 5H20 Hifltldin. Arginin. Sturin. However this may he, it must not be Hippo.., I thai the quantita- .','.1 70 THE NITROGENOUS DERIVATIVES OF THE ALBUMINS. tive relations between the hexon bases are always the same, and as a matter of fact we have reason to assume that the nucleus of most of the complex albumins contains more lysin groups than are found in the sturin molecule. As regards the further development of the complex albumins from protamins, Kossel imagines that these are formed through the union of a protamin radicle with various other radicles, which partly belong to the class of the mo no-am ido-acids, and partly to the aro- matic series, to which a sulphur or an iodine radicle can further be added. These various bodies can then also combine with each other or with foreign radicles to form still more complex substances. A relation is thus established between the lowest forms of the albumins and the carbohydrates, and just as the polysaccharides can be decom- posed by hydrolysis into the hexoses, so also are the protamins trans- formed into hexons. This decomposition in both instances, more- over, can be effected through the agency of ferments. Diastase thus causes inversion of the polysaccharides to hexoses, and trypsin can similarly break down the protamins, with the formation of the hexon bases. Whether a reversion is here also possible has not as yet been ascertained. As the size of the protamin molecule has not been established, it is possible only to indicate the composition of salmin, sturin, and scombrin by the respective formula? (C30H57N17O6)a;, (C36H69N1907)k, and (C30H69N16O6)«. Like the complex albumins, the protamins give the bluish-violet biuret reaction, and from what has been said we have reason to assume that this reaction in the case of all albumins is referable to the presence of a protamin group. The xanthoproteic reaction, Millon's reaction, Molisch's reaction, and the sulphur-test, on the other hand, are negative. The aqueous solutions of the protamins are strongly alkaline ; from these solutions they are precipitated by picric acid, phospho- tungstic acid, iodopotassic iodide, potassium ferrocyanide in the presence of acetic acid, by salting with sodium chloride or ammo- nium sulphate, etc. They combine with acids to form salts, among which the sulphates are especially characteristic, as on cooling or upon the addition of ether they separate from their aqueous solution in the form of an oily material. With the salts of the heavy metals they further combine to form double salts. With the coagulable albumins and the so-called primary albumoses, in ammoniacal solu- tion, the protamins combine to form compounds which are apparently identical with the histons, and are precipitated as such. Neither the protamins nor their salts have thus far been obtained in the crystal- line state.1 1 Quite recently Kurajeff announced that he has been able to isolate pro- tamins also from the mature spermatozoa of the pike, of Silurus glanis and Acci- pensis stallatus. The products from the two latter he has termed silurin and accipenserin, respectively. For the accipenserin sulphate he gives the formula C35H72N1809.4H2S04. He regards it as closely related to sturin. THE HEX ON BASES. 71 Closely related to the protamins on the one hand, and to the histons on the other, are two bodies which have been obtained from the spermatozoa of an Arbacia and Cyclopterus lumpus, and which are termed arbacin and cyclopterin, respectively. Of these cyclop- terin is more closely related to the protamins proper, and, like these, yields a sulphate, which may be obtained as an oily material. Unlike the protamins, however, it gives Millon's reaction, but does not form a precipitate on heating; on cooling, it separates out, . and then presents a rose color. It contains much less oxygen than the protamins. From most of the histons it differs m containing no sulphur and in not being precipitated by ammonia. Its formula has not as vet been ascertained. The sulphate contains 42.03 per cent, of carbon, 6.90 per cent, of hydrogen, and 22.08 per cent, of nitrogen. Arbacin differs from the protamins and cyclopterin in containing much less nitrogen, but, like cyclopterin, it gives Millon's reaction. It is not completely precipitated from its solutions by ammonia, but resembles the histons in other respects. Kossel indeed seems now to regard it as such. It is precipitated from its solutions bythe neutral alkaloidal reagents, and itself precipitates albumins. It gives the biuret reaction.1 THE PROTONS. The protons are substances closely related to the protamins, and are formed as intermediary products during the hydrolytic decom- position of the latter into hexon bases. Individually they are but little known. Thev differ from the protamins in the greater solubility of their sulphates and in the fact that they are not thrown "down bv tin- protamin preeipitants, or, if so, are more readily soluble. ' With the coagulable albumins and the primary albumoses in ammoniacal solution, moreover, they do not give rise to a precipitate, or to a slight turbidity only, which may be due to tracer of andecomposed protamin. From clupein three protons have been obtained! One of these apparently has the same com- position aa clupein itself, while the others contain more hydrogen, but less carbon and nitrogen, and may hence be regarded as clupein hydrates. The formula of one of these is ( ':;jHfi,\17< >„ and n is interesting bo note that the analogous product of sturin has the same composition. THE HEXON BASES. The hexon bases comprise arginin, histidin, and lysin. As has been stated, they are formed during the hydrolytic decomposition of i From Lota vulgaris Klirstrom obtained a biBton-like body, which be terms lota hwton This is insoluble in water and solutions of the neutral sails, but dissolves in acids and alkalies. From its acid solutions il is precipitated by ammonia. It gives armlet biuret reaction. The xanthoproteic reaction a positive, that ol Millui. h-.-M. but distinct Molisch's reaction is quite intense, and thai of Adamkiewicz uigni. \ -i„,ihr body was found by kossel in the mature testicles of Gadus marrhuft, I by Bang in the immature organs of the mackerel. The twoarespoken of biston and »combron, respectively. 72 THE NITROGENOUS DERIVATIVES OF THE ALBUMINS. the protamins, as also of all complex albumins, both of animal and vegetable character, which have been examined in this direction. Among these may be mentioned fibrin, keratin, spongin, collagen, conglutin, elastin, egg-albumin, casein, etc. Kutscher, moreover, obtained these bodies among the final decomposition-products of tryptic digestion, and showed that that portion of Kiihne's anti- peptone which can be precipitated with phosphotungstic acid con- sists to the extent of from 30 to 31 per cent, of these hexon bases. The free bases, like the protamins, are lsevorotatory, while their salts, which are formed through union with acids and salts of the heavy metals, are dextrorotatory. These salts can be obtained in crystalline form, and it has thus been possible to determine the formulae of the free bases. Arginin. — On hydrolytic decomposition arginin yields urea and Ornithin. The substance is therefore regarded as a guanidin deriva- tive, similar to kreatin, in which one amido-group has been replaced by the ornithin radicle. The correctness of this supposition has since been established through the synthesis of arginin from cyana- mide and ornithin. Ornithin, moreover, is now known to be a, o-diamido-valerianic acid, and the structural formula of arginin may hence be represented as follows : NH2 NH, I I NH = C — NH.CH2.CH2.CH2.CHCOOH Its relation to guanidin and kreatin, as also its decomposition into urea and ornithin, is further shown : /NH2 .NH, Cf(NH) <(NH) \nH, \N(CH3).CH2.COOH Guanidin. Kreatin. /NH, CN.NH, + CH(CH,).NH2.COOH = C^(NH) Cyanamide. Methy'l-glycocoll. \N(CH3).CH2.COOH Kreatin. /NH, CN.NH, + CH2.CH2.CH2.CH.COOH = C^(NH) | | \NH.CH2.CH2.CH2.CH.COOH Cyanamide. NH2 NH2 I Ornithin. " NH2 Arginin. NH2 Cf(NH) \NH.CH2.CH2.CH,.CH(NH2).COOH + HO = Arginin. NH2 CO( + CH2(NH2).CH2.CH2.CH(NH2)COOH ^NH, Ornithin. Urea. On oxidizing arginin with barium permanganate Kutscher obtained guanidin, guaniclin-butyric acid, and ethylene-succinic acid. He concludes that the process occurs in two phases, as 'represented by the equations : THE HEXON BASES. 73 /NH, NH2 (1) (T.NH | +20 = \NH. CH2.CH2. CH,. CH. COOH Arginin. /NH2 C(- Nil + C02 + NH3 \NH. CH,.CH2.CH2.COOH Guanidin-butyric acid. /NH2 (2) (T.NH + 20 = NII.CH2.CH2.CH2.C(X)H Guanidin-butyric "acid. /NE, C(-NH + C00HCH2.CH2 COOH \\H, Succinic acid. Guanidin. Of great interest, further, is the fact that ornithin can give rise to putrescin, viz., to tetramethylene-diamine, a ptomain which is formed during the putrefaction of albuminous material, and which has also been found in the urine in association with cyst in. Thus far this transformation has been effected only through the agency of micro-organisms, but there is no reason to suppose that their presence is essential, and that in the tissues of the living body the same proc- ess cannot also occur. This transformation may be represented by the equation : CH2(NH2).CH,CH,,CH.(NH2).COOH = C02 + CH2(NH,).CH.,CH2.CH2.(NH2) Ornithin. Putrescin. Should the formula of ornithin, as above indicated, be correct — and it may be added that there is every reason to suppose that this is the case — we can readily understand how pyridin derivatives can develop from the albumins without being forced to assume the existence of a pyridin radicle in the albuminous molecule directly. The active principle of the suprarenal gland, which von Fiirth regards as tetrahydro-dioxypyridin, could thus result from ornithin by tli«' replacement of the a-amido-group by hydroxy] and the elim- ination of water. That oxypiperidin results from <5-amido-valerianic acid in an analogous manner lias indeed been demonstrated. These relations may he expressed by the formulae: •II Ml .< I[,.( ll,cil,.(0()ll ||(» ( 'H,i Ml i.UU'l !.,.<'] I2.CO. 8-amido-valerianic acid. oxypiperidin. I) CH Ml « II .1 Il..< ll.MU.COOH II,o Ornithin. < 1 1.,( M I . ..< I I < I I < I I Ol I i.< < ><>I I | Ml,. a-hydroxy, i amido valerianic acid. 2 'II Ml, .' HPCHa.CH(OH).OOOH II.o ■ .,./_' I ' ..<>, Thymonucleinic acid ' ,} l:n;\,< >.,„l's Guanylic acid < •..■.."31^ 'm( W^ [nosinic acid < 'i„l l| ,;N ' ,' > . I' On decomposition the primary nucleinic acids give rise to the 76 THE NITROGENOUS DERIVATIVES OF THE ALBUMINS. formation of what I have termed the secondary nucleinic acids. These contain more phosphorus than the primary acids, and may or may not give rise to xanthin bases on further decomposition. They may accordingly be divided into acids of the type of plasminic acid and of thyminic acid, respectively. The former result from the primary acids through a splitting off of atomic groups, which are free from phosphorus, but ultimately the characteristic decomposi- tion-products of the primary acids result, viz., the nucleinic bases, phosphoric acid, etc. Thyminic acid, on the other hand, is obtained from the primary acids, with the possible exception of inosinic acid and guanylic acid, after the separation of the nucleinic bases. For its barium salt Kossel obtained the formula C16H23N3P2012Ba. On decomposi- tion with strong sulphuric acid thyminic acid yields a crystalline product termed thymin. As this substance is obtained as a con- stant decomposition-product from all the primary nucleinic acids which have been examined in this direction, with the exception, as has been indicated, of inosinic acid and guanylic acid, Kossel now divides these primary acids into thymonucleinic acids, and a second group which is represented by the two exceptions just mentioned. Thymin (C5H6]N!"202), according to Steudel, may be regarded as a methyl-dioxy-pyrimidin, and is thus isomeric with methyl-uracil. It is closely related to the ureids (barbituric acid, etc.) and the purin bases (see below), and it is possible, as a matter of fact, to condense isodialuric acid, which is closely related to uracil, with urea to uric acid. The position of the hydrogen and oxygen atoms, as also of the methyl group, in their relation to the pyrimidin nucleus, is, how- ever, as yet uncertain. The relation existing between these bodies may be seen from the following formulae : N NH N /-\ /\ /\ CH CH CO C— CH3 CH CH I II I II II N CH NH CH N C— NHX \/ \/ W >CH CH CO C N^ - Pyrimidin. Methyl-uracil. . Purin. The primary nucleinic acids are amorphous and possess a strongly acid reaction. They are soluble in dilute solutions of the alkalies, and are precipitated from these solutions, especially in the presence of alcohol, by adding a slight excess of hydrochloric acid. In alcohol and ether they are insoluble. In acid solutions (acetic; acid) they give rise to precipitates with albumins, which are apparently identical with the nucleins. From the nucleins they are obtained by treating with a dilute solution of sodium hydrate, which is subse- quently neutralized with dilute hydrochloric acid. The separated albumin is then precipitated by adding an excess of acetic acid, when THE NVCLEINIO BASES. 77 the filtrate is treated with an equal volume of alcohol and hydro- chloric acid to the extent of from 3 to 5 pro mille. In this manner impure nucleinic acid is thrown down, which can be further purified by solution in ammoniacal water and further treatment with acetic acid, hydrochloric acid, and alcohol, as just described. Thyminic acid differs from the nucleinic acids proper in its ready solubility in cold water, and in the fact that it is not precipitated from its solutions by the mineral acids. Like the nucleinic acids, it gives a precipitate with albumins or primary albumoses (propep- tones) in acetic acid solution, but, in contradistinction to the nucleinic acids, this precipitate is soluble in hydrochloric acid and in solu- tions of many salts. Plasminic acid likewise precipitates albumins in acid solution, but, unlike the nucleinic acids, is easily soluble in water; on treating with ammonia a yellow color develops. Its phosphoric acid radicle is capable of binding iron in such form that it appears like a true organic iron compound. According to Ascoli, the substance contains 1 per cent, of iron. It does not give Millon's reaction nor the biuret reaction, and contains no sulphur. On decomposition with mineral acids by boiling it yields nucleinic bases and phosphoric acid. The substance may be obtained from yeast. The question whether the paranucleim contain an acid radicle which is analogous to the nucleinic acids is still undecided. If they occur, such paranucleinic acids, as they would be termed, could, of course, not contain any basic radicle of the character of the nucleinic bases. A few isolated observations seem to show that such acids exist. Altaian thus obtained an acid from the yolk of eggs which he re- garded as a nucleinic acid. As the nucleinic bases, however, cannot be obtained from the same source, it follows that the substance in question could not be a true nucleinic acid. Wildenow further speaks of a phosphorus-containing substance, which she was able to obtain from the paranuclein of casein, and which precipitated albumins. Neither of these bodies, however, has as yet been isolated in a form suitable for analysis. Whether KossePs SO- called paranucleinic acid, which was later shown to be the same as thymonucleinic acid, is identical with the prosthetic group of the paranuclein-, remains to be seen. Its properties certainly are such as would a •priori be expected from a true paranucleinic acid. But even if a group of this order were found in some of the paranucleus, it- presence in all would not necessarily follow, and it is quite con- ceivable that in others the albumin is directly combined with a phos- phoric acid. THE NUCLEINIC BASES. The nucleinic bases, which are also spoken of as the xanthin, aUoxuriCf or purin bases, are found widely distributed both in fix; animal and the vegetable world. They occur either in the lice state or a- OODStitUtente of tin; nucleinic acids and the nucleins. 78 THE NITROGENOUS DERIVATIVES OF THE ALBUMINS. They comprise xanthin, hypoxanthin (or sarcin), episarcin, hetero- xanthin, paraxanthin, theophyllin, theobromin, caffein, guanin, epi- guanin, adenin, and carnin. Of these, paraxanthin, heteroxanthin, epiguanin, and episarcin have thus far been found only in the urine. Theophyllin, theobromin, and caffein are confined to the vegetable world, while the remaining members of the group are common con- stituents of both animal and vegetable cells. All these bodies are closely related to each other and to uric acid, which, as we shall see later, constitutes one of the most important end-products of animal metabolism. According to Emil Fischer, they are derived from a hypothetical substance, which has been termed purin, and it has previously been pointed out that in this manner a further relation- ship is established between the nucleinic bases and thymin, as it is probable that this substance, or allied bodies, constitute the ante- cedents of the purin radicle. Behrend, in fact, has pointed out that through the condensation of urea and isodialuric acid, which is closely related to uracil, uric acid results. These relations are shown below : (6) (l) N^=CH I I (V) (2)HC (5)C NHs || >CH(8) Purin = C5H4N4. (3) n C N * (4) 0) By substituting the group NH2 for the hydrogen atom at 6, adenin results, and is hence spoken of as a 6-amino-purin : N C.NH2 I I HC C NHX I! || >CH Adenin = C5H5N5. N C N * ■ Hypoxanthin, according to this conception, would be _ 6-oxypurin, xanthin 2, 6-dioxypurin, and guanin 2-amino-6-oxypurin : HN CO HN CO II II HC C NJEL CO C NH. II I >CH | || >CH N C N " HN C N V Hypoxanthin = C5H4N4O. Xanthin = C5H4N4O2. HN CO I I HN=C C NHX I II >H HN C N * Guanin = C6H5N60. From these primary bodies the remaining ones then result through a substitution of methyl groups. Heteroxanthin is thus formed from xanthin by replacing the hydrogen atom at 7 with CH3, and is therefore 7-methyl-2,6-dioxypurin. Paraxanthin is accordingly THE NUCLEINIO BASES. 79 l,-7-dimethyl-2,6-dioxypurin, and caffein l,3,7-trimethyl-2,6-dioxy- purin. Theophyllin and theobromin are isomeric with paraxanthin, and structurally l,3-dimethyl-2,6-dioxypurin and 3,7-dimethyl-2,6- dioxypurin, respectively. Epiguanin is similarly derived from guanin by the replacement of the hydrogen atom at 7 with methyl, and is hence 7-methyl-guanin. HX CO CO C N.CH3X HX C X^^ Hetcroxanthin = C6H6N4Oo. Caffein CH3.X CO Co C XHX CO C X.CH3 CH,.X C X * CH3.X C N^^ Theophyllin = C7H8N402. Theobromin = C7H8N402. HX CO HX = C C X.CH3X I II ^CH HX C X^^ Epiguanin = C6H7N50. Carnin is apparently closely related to hypoxanthin, as on treatment with bromine it yields methyl bromide, carbon dioxide, and the brom- hydrate of hypoxanthin, according to the equation : C7HsX4Os + 2Br = C5H4X4O.HRr + CH3Br + C02. Its structural formula is thus possibly the following : (II .X CO I I CHOH ( XH v I XCH 1 1 X-CO = CO X ^ Episarcin is but little known. Its formula is given as C4H6NsO, but it is possible that this is not correct. The substance has un- doubtedly many properties in common with epiguanin, and future researches inCO + C02 Guanin. \NH2 CO— NEK Guanidin. Parabanic acid. C5H5N50 + HN02 = C3H4N402 + 2N + H20. Guanin. Xanthin. C5H4N402 + 2H = C5H4N40 + H20. Xanthin. Hypoxanthin. Xanthin, however, on oxidation and hydration yields urea and alloxan, which latter is further oxidized to parabanic acid and carbon dioxide, as shown in the equations : CO-NHx I \ /NH, C5H4N402 i- 20 + H20 = CO >CO + CO< Xanthin. | / XNH2 CO— NH/ Urea. Alloxan. CO— NHX | \ CO— NH, CO >CO + 0=| >co + co2 I / CO— nh/ CO— NET Alloxan. Parabanic acid. The close relationship which exists between uric acid and the nucle- inic bases thus becomes apparent, as uric acid on oxidation and hydration gives rise to the same products as xanthin : C5H4N403 + O + H20 = C4H2N204 + CON2H4. Uric acid. Alloxan. Urea. This relationship is further shown by decomposing the primary xanthin bases and uric acid Avith fuming hydrochloric acid or hydriodic acid under high pressure. Qualitatively the same prod- ucts are thus obtained, viz., ammonia, carbon dioxide, glycocoll, and formic acid, while the quantitative relations, of course, vary with the nature of the individual substance. C5H5N5 + 8H20 = 4NH3 + C02 4- CH2.NH2.COOH + 2H.C00H. Adenin. Glycocoll. Formic acid. C5H4N40 -I- 7H20 = 3NH3 + C02 + CH2.NH2.COOH + 2H.C00H. Hypoxanthin. THE UREIDS. 81 C5H5X50 + 7H20 = 4NH, + 2C02 + CH2.NH2.COOH + H.COOH. Guanin. C;H(XA - 6H20 = 3NH3 + 2C02 + CH2.NH2.COOH + H.COOH. Xanthin. ( ,H4X A + 5H20 = 3NH, + 3C02 + CH2.NH2.COOH. Uric acid. According to Emil Fischer, the structural formula of uric acid can be represented as follows : HX — CO CO C— NHX I I >co HN— C— XHX Uric acid. and it is thus seen that, like the nucleinic bases, it contains the purin radicle. All the nucleinic bases combine with acids and alkalies to torm salts, many of which are readily crystallizable. On boiling with acetate of copper most of them are thrown down as insoluble com- pounds. From their neutral solutions, or in the presence of a little ammonia, they are precipitated by ammoniacal silver nitrate solu- tion. Tin's precipitate dissolves in nitric acid on the application of heat, but reappears on cooling, Most of the nucleinic bases, more- over, when evaporated to dryness in the presence of nitric acid, leave a yellowish residue, which changes to orange and often to a temporary purple on the addition of an alkali. In this respect also these substances resemble uric acid, which gives a very similar reac- tion (see Murexid test). When exposed to the action of putrefactive organisms adenin is transformed into hypoxanthin, and guanin into xanthin, so that these two only are found in decomposed material. For a description of the individual members of this group which are found in the animal body, as well for the method of their isola- tion and quantitative estimation, the reader is referred to subsequent chapters (see especially pages 241 and 363). THE UREIDS. The ureids comprise a number of nitrogenous crystallizable bodies characterized by the fact that on hydrolytie decomposition alone, or on simultaneous oxidation, they yield" urea. They are hence derivatives of urea, and may contain one or more molecules of this^ substance, in which one ormore hydrogen atom- are replaced by radicles of mono- basicor polybasic acid-. On decomposition they yield either urea and a oon-nitrogenous acid directly, or they give rise to urea and a less complex iireid, which is then further decomposed, as in the li,M instance. They are accordingly divided into mono-ureids and di-ureids. The former generall) contain two atoms of nitrogen in their molecule, while the latter possess four atoms of nitrogen. All ,!„._,. bodies are closely related to each other and to the nuclemio bases, from which they are, in part at least, derived. 82 THE NITROGENOUS DERIVATIVES OF THE ALBUMINS. The mono-ureids comprise alloxan, alloxanic acid, dialuric acid, barbituric acid or rnalonyl-iirea and its derivatives, all of which can be decomposed into urea and non-nitrogenous acids containing three atoms of carbon. From these another series of mono-ureids is de- rived, which also yield urea and non-nitrogenous acids ; but the latter, in contradistinction to the first group, contain only two atoms of carbon. These include parabanic acid, oxaluric acid, allanturic acid, and hydantoin and its derivatives, viz., hydantoic acid or glyco- luric acid, and methyl-hydantoin. The di-ureids are similarly divided into two groups, the first of which comprises uric acid, alloxantin, murexid or ammonium purp urate, and hydurilic acid ; while the second is represented by allantoin, allantoic acid, and allituric acid. * The best known representative of the ureids is uric acid. From it all others can be derived, and a description of its general proper- ties and reactions may serve as an illustration of the entire class. Uric acid is a crystallizable dibasic acid of the formula C5H4N403. Structurally it may be regarded as a purin derivative, and may be represented by the formula : HN— CO I I CO C— NHS ! Ii HN — O-NH' )CO. It was first obtained synthetically by treating a mixture of glycocoll and urea at a temperature of 230° C. until the melted mass assumed a yellowish color and became turbid. The reaction which took place may be represented by the equation : CH2.NH2.COOH + 3CO(NH2)2 = C5H4N403 + 2H20 + 3NH3 Glycocoll. Urea. Uric acid. The same result may be reached by heating a mixture of urea and trichlorolactic amide : C3H4C13N02 + 2C0(NH2)2 = C5H4N403 + H20 + HC1 + NH4C1 Trichlorolactic amide. As indicated in the previous section, moreover, uric acid also results through the condensation of isodialuric acid and urea, according to the equation : HN— CO HN-CO II II CO C + CO(NH2)2 = CO C— NH. || | || >CO + 2H20 HN— C(OH) HN— C— NH^ When treated in the dry state uric acid is decomposed into hydro- cyanic acid, cyanuric acid, ammonium carbonate, ammonium cyanate, biuret, etc. Fused with an excess of potassium hydrate, it similarly yields ammonia, potassium carbonate, oxalate, cyanate, and cyanide. On reduction with nascent hydrogen, in the presence of water and THE UREIDS. 83 sodium amalgam uric acid is first transformed into xanthin, and sub- sequently into hypoxanthin. The close relationship which exists between the nucleinic bases and uric acid is thus further shown (see page 80). Analogous to the synthetic formation of uric acid from urea and glvcocoll, we find that on decomposition with hvdriodic acid the substance yields carbon dioxide, ammonia, and glycocoll, viz., the same products which are obtained from the nucleinic bases. From the reactions which have thus far been described the nature of uric acid as a ureid is not apparent. If the hydrolytic decom- position of the substance is effected less energetically, this becomes manifest at once. On prolonged boiling with water it is decom- posed into urea and dialuric acid, which -latter further yields urea and tartronic acid. In this manner the character of uric acid as a diureid of the first order is demonstrated. The reactions which take place are represented by the equations : (1 ) C5H4NA + 2H20 = 04H4NA + CO(NH^a Uric acid. Dialuric acid. Urea. (2) C,HtX,04 + 2H20 = C3HA + CO(NH2)2 Dialuric acid. Tartronic acid. Urea. On treating with an oxidizing agent, in the presence of water, uric acid is similarly decomposed into urea and the mono-ureid alloxan, which can be further decomposed into urea and mesoxalic acid : (1) C5H4N,03 + H20 + O = C4H2N204 + CO(NH2)2 Uric acid. Alloxan. (2) €4H2X.A + H20 = C3HA + COfNH,), Alloxan. Mesoxalic acid. Its relation to the di-ureids of the second order is shown by oxidiz- ing the substance with peroxide of manganese in neutral solution at a moderate temperature. In this manner allantoin is formed, from which, on further oxidation, urea and oxalic acid result: (1) C5H4X403 + H20 + O = qftNA + C02 Allantoin. (2) C4H6X403 + 2H20 + O = C2HA + 2CO(NH2)2 Oxalic acid. Of special interest, further, is the formation of murexid, or ammonium pur put-ate, which results when uric acid, even in minimal amount--, is evaporated together with nitric acid, and the reddish r< sidue i- brought in contact with ammonia. A beautiful purplish- red color then develops, which is characteristic of uric acid and its -alt- (murexid test). The reactions which take place maybe rep- resented by the equations : I) c.H.NA +2H20 =(',ir,NA +CO(NH2)2 Dialuric acid. (2) r.H.N'A -f-NH4OH =C4H8(NH4)NA + HSQ \m 111'. niiim (liillur.it' (?,, 2CIII./\H ,)N20( + 0 =('.]|/\|[,).\<)1; I 311,0 Ammonium dialurate. Murexid. 84 THE NITROGENOUS DERIVATIVES OF THE ALBUMINS. The relation of uric acid to the mono-ureids of the second order, finally, is shown by treating one part of uric acid with three parts of nitric acid (50 per cent, solution), and heating to 70° C. On sub- sequent evaporation to a syrup and cooling, parabanic acid crystal- lizes out, and on decomposition yields urea and oxalic acid : (1) C5H4N403 + H20 + O = C4H2N204 + CO (NH,), Alloxan. (2) C4H2N204 + O = C3H2N203 + C02 Parabanic acid. (3) C3H2N203 + 2H20 = C2H204 + CO(NH2)2 Oxalic acid. Guanin, as has been shown, under the same conditions yields guanidin and parabanic acid, which further illustrates the close relationship between the two classes. For a consideration of the methods employed in the isolation of uric acid and its quantitative estimation, the reader is referred to the chapter on the Urine. THE KREATINS. The kreatins, or kreatinic leucomains, as they are also termed by Gautier, are basic substances which are closely related to the nucleinic bases and to the ureids, which have just been considered. They comprise kreatin, kreatinin, crusokreatinin, xanthokreatinin, amphikreatinin, and two similar substances of doubtful composition. Kreatin, moreover, is related to arginin, and can be produced syntheti- cally through the union of cyanamide and methyl-glycocoll, as arginin results from cyanamide and ornithin. While arginin, however, can be obtained artificially from albuminous material, kreatin has thus far not been isolated in this manner. Its formation from cyanamide and methyl-glycocoll is represented by the equation : /NH2 N = C — NH2 + NH(CH3) — CH2— COOH = NH = C< XN(CH3) — CH2— COOH Cyanamide. Methyl-glycocoll. Kreatin. It is thus a homologue of glycocyamin, which results through the union of cyanamide and glycocoll : /NH2 N = C — NH2 + CH2 — NH2 — COOH = NH2 = C< \NH — CH2-COOH Cyanamide. Glycocoll. Glucocyamin. On dehydration kreatin loses one molecule of water and is trans- formed into kreatinin, which may thus be regarded as the anhydride of kreatin : XNH2 .NH CO NH = C< = NH = C( + H20 XN(CH3) — CH2 — COOH ' NN(CHS) — CH, THE AMIDO- ACIDS. 85 On oxidation with mercuric oxide kreatin gives rise to the forma- tion of the oxalate of methyl-guanidin or methyl-urarnin, which in turn results from guanidin, and this from guanin. -ClII9X302 + 50 = (C,H7X3)2.C,H204 + 2C02 + H20 Kreatin.' Methyl-guanidin oxalate. On decomposition with baryta-water kreatin yields urea, methyl- glycocollj and a small amount of hydantoic acid. Kreatinin is similarly transformed into methyl-hydantoin, which is then likewise decomposed into urea and methyl-glycocoll, thus demonstrating the close relationship which exists between the kreatins and the ureid-. .NH2 CH2 — NH.CH3 NH = C{ + H20 = I + CO(NH2), NN(CH3) — CH2 — COOH COOH Urea. Kreatin. Methyl-glycocoll. /XH CO /NH CO NH = C I +H20 = NH3 + CO< I X(CH3) — CH2 XN(CH3) — CH2 Kreatinin. Methyl-hydantoin. Kreatin and kreatinin are homologous with lysatin and lysatinin, which, as has been seen, result from albuminous material when this is decomposed with boiling mineral acids. While kreatin, however, contains but four atoms of carbon, lysatin contains six, and is represented by the formula C6H13N302. Like kreatin and kreatinin, lysatin and lysatinin yield urea on hydrolytic decomposition. The individual representatives of the group will be considered later. THE AMIDO-ACIDS. The amido-acids represent one of the most important groups of chemical substances which are found in the animal and the vegetable world. They are intimately concerned in the construction of the albuminous molecule, and are accordingly always found among the decomposition-products of all albumins. Some of them belong to the BO-called diamido-acids, while others are mono-amido-acids. With the latter we shall deal more exclusively at this place. They are generally of the character of a fatty acid or of an aromatic acid, in which one hydrogen atom of the group CH8 or C,.H5 has been replaced by the amido-radicle NHj. Acetic acid thus gives rise to tnono-amido-acetic acid, and benzoic acid to mono-amido-benzoic acid, etc, a- shown by the formulae: CHg.COOH, acetic acid. CHj MI, 1.' noil, mono-amido-acetic acid. I ,11 ' « 1' ill. benzoic add. I ,,l I, ' \ 1 1 1 < K )OH, mono-amidobenzoic acid. 86 THE NITROGENOUS DERIVATIVES OF THE ALBUMINS. The most important members of the group which will here be considered are glycocoll, leucin, asparaginic acid, glutaminic acid, and tyrosin. They are represented by the formulae : CH2.(NH2).COOH, glycocoll. (CH3)2.CH2.CH(NH2).COOH, leucin. CH2.CH(NH2).(COOH)2, asparaginic acid. (CH2)2.CH(NH2).(COOH)2, glutaminic acid. C6H4(OHJ.CH2.CH(NH2).COOH, tyrosin. It will thus be seen that glycocoll and leucin are amido-deriva- tives of the mono-basic acids of the formic series, viz., amido-acetic acid and a-amido-capronic acid, while asparaginic acid and glutam- inic acid are dibasic acids of the oxalic series, viz., amido-succinic and amido-glutaric acids. Tyrosin, on the other hand, is an amido- derivative of the aromatic series, viz., para-oxy-a-amido-propionic acid. They are all derivatives of the albumins, and, as has been indi- cated, integral constituents of the albuminous molecule. Their quantitative relations, however, are subject to considerable variation, and from some albumins indeed not all can be obtained. Tyrosin, for example, is lacking in glutin and collagen, and glycocoll is similarly not found in casein. In some leucin predominates, while in others tyrosin stands in the foreground. These variations will be considered in greater detail when we shall deal with the various digestive products of the albumins. The amido-acids of the fatty series are of special interest to the physiological chemist, owing to the fact that they are apparently intimately concerned in the production of urea. Von Schroder, Nencki, and others have thus shown that in the liver the ammonium salt of carbamic acid, viz., amido-formic acid, is transformed into urea, and we also know that in the mammalian organism leucin, glycocoll, and asparaginic acid are likewise transformed into urea and eliminated as such. That a portion of the urea, indeed, origin- ates in this manner can scarcely be doubted. As regards the nature of the chemical changes which take place during the trans- formation of the amido-acids into urea, our knowledge is not complete. It was formerly supposed that uric acid represented the immediate antecedent of urea and was transformed into this by oxidation. We find, as a matter of fact, that in birds and reptiles uric acid constitutes the final decomposition-product of the nitro- genous metabolism, and is thus analogous to the urea of mammals. I have also pointed out that as a ureid, uric acid on oxidation can yield urea. But as far as is known, the uric acid of mammals is exclusively derived from the nucleinic bases, and is thus scarcely found in sufficient quantity to give rise to the large amount of urea which is daily eliminated in the urine. That a small fraction of THE AMIDO-ACIDS. 87 the urea may result from uric acid by simple oxidation is possible, and indeed probable, but the greater portion must of necessity originate in a different manner. In birds, on the other hand, some of the uric acid apparently results from glycocoll, and we thus see that in both classes of animals the amido-acids may be the antecedents of the final products of nitrogenous metabolism. It is conceivable that in mammals cyanic acid may be produced as an intermediary product, and that urea then results through a condensation of two molecules of this substance in statu nascendi, according to the equation : /NH2 COXH + CONH = CO< + C02. XNH2 Then again we may imagine that a transformation of the amido- acids occurs into the ammonium salts of the fatty acids standing next in order in the downward scale, and that by further oxidation these are transformed into ammonium carbonate, and this into urea. It has been shown as a matter of fact that a fair amount of urea is thus produced when blood containing ammonium carbonate or ammo- nium formate is allowed to flow through the isolated livers of dogs. According to Drechsel, finally, the amido-acids are transformed into carbamic acid, from which urea may then result, as indicated by the equation : /NH2 XNH2 /NH, CO< + CO< = CO< + C02 + H20. OH xOH XH2 In a subsequent chapter this subject will be treated at greater detail. In addition to the important rdle which the amido-acids thus play in the formation of urea, these bodies are of further interest from the part which they take in some of the syntheses that occur in the animal organism. In this manner they give rise to a Dumber 'if complex substances which can hence be regarded as amido-derivatives. With benzoic acid glycocoll thus combines to form hippuric acid, as shown by the equation: < II MI i.OOOB C6H5.COOB MI ,.MI< < JU'Oi.COOII II,0 Glycocoll. Benzoic acid. Hippuric acid. With phenyl-acetic acid glycocoll similarly combines to form phenaceturic acid : rii,.(XH,> .moil mi < ii .COOH CeHj.CHj.CO — NH.CHj.COOH II.o. That uric acid on bydrolytic decomposition will yield ammonia, carbon dioxide, and glycocol] hag been shown. There is evidence, moreover, to show thai in the organism of birds and reptiles, at least, it- synthesis can similarly occur. Ornithuric acid results through the union of benzoic acid with the diamide ornithin : 88 THE NITROGENOUS DERIVATIVES OF THE ALBUMINS. C4H7(NH2)2.COOH + 2C6H5.COOH = C4H7(NH.C6H5.CO).,.COOH + 2H20 Ornithin. Benzoic acid. Hippuric acid. The amido-acids also are closely related to the biliary acids, as on decomposition with baryta-water, glycocholic acid is decomposed into glycocoll and cholalic acid, as shown in the equation : C26H43N06 + H20 = CH2(NH2).COOH + C24H40O5 Glycocholic Glycocoll. Cholalic acid. acid. Taurocholic acid similarly gives rise to cholalic acid and taurin, which latter can be regarded as amido-isethionic acid — that is, as isethionic acid, H(C2H4.OH)S03, in which the hydroxyl group has been replaced by the amido-radicle : C26H45N07S + H20 = C24H40O5 + C2H7N03S Taurocholic Cholalic Taurin. acid. acid. After the ingestion of amido-acids, moreover, the corresponding compounds of carbamide, — CONH, appear in the urine. These are spoken of as the uramic acids, and comprise methyl-hydantoinic acid, taurocarbamic acid, uramido-benzoic acid, and tyrosin-hydantoinic acid, or hydantoin-hydroparacumaric acid. They are found after the ingestion of sarcosin or methyl-glycocoll, of taurin, amido- benzoic acid, and tyrosin, respectively. The syntheses which are thus effected may be represented by the equations : C3H702 + CONH = C4H8N203 Sarcosin. Methyl-hydantoinic acid. C2H7NS03 + CONH = C,H8N2S04 Taurin. Taurocarbamic acid. C7H7N02 + CONH = C8H8N203 Amidobenzoic Uramido-benzoic acid. acid. C9HHN02 + CONH = CipH10N2O3 + H20 Tyrosin. Tyrosin hydantoinic acid. Among the mono-amido acids, finally, which may be formed in the animal body, we must mention cystein, or a-amido-thiolactic acid. This is of special interest, as its disulphide — cystin — is occa- sionally found in human urine, and is then commonly associated with the diamins putrescin and cadaverin. The relation which exists between cystin and cystein is thus similar to that of a mer- captan to its disulphide, and may be represented by the equation : H3C v /SH H3C \ /S S\ /CH3 2 >C< +0= ^ >C( >C< +H20 h2n/ xcooh h2n/ xCOOH COOH' xnh2 Cystein. Cystin. Analogous to the synthetic formation of hippuric acid after the ingestion of benzoic acid, we find in dogs and rabbits that following the administration of mono-bromobenzol bromophenyl-mercapturic THE AMIDO-ACIDS. 89 acid appears in the urine. The reaction which here takes place may be represented by the equation : HoC \ /oil \c< - CJi5Br-CH2(XH2).COOH = H2N XCOOH GlycocoU. Cvbteiu. H,C\ /S.CGH4.Br >C< + NH3 + 2H20 CH^.CO.NH/ \COOH Bromophenyl-mereapturic acid. On reduction theamido-acids are transformed into the correspond- ing acids from which they are derived. Glycocoll is thus transformed into acetic acid, leucin into capronic acid, asparaginic acid into suc- cinic acid, glntaminic acid into glutaric acid, tyrosin into para-oxy- phenyl-propionic acid (hydroparacumaric acid), etc., as shown by the equations : CH2(XH,).COOH + 2H = CH3.COOH + NH3 Glycocoll. Acetic acid. /COOH /COOH CH,.CH(NH,)/ + 2H = CH2.CH2.< + NH3 " COOH XCOOH Asparaginic acid. Succinic acid. XCH /OH C6H4 +2H = C6H4/ ' +NH, \CH2.CH(NH2).COOH xCH2.CH2.COOH Tyrosin. Para-oxy-phenyl-propionic acid. On oxidation these are further changed into the acids standing next in order in the downward scale. Acetic acid thus gives rise to the formation of formic acid, succinic acid to malonic acid, para-oxy- phenyl-propionic acid to para-oxy-phenyl-acetic acid, etc., as shown by the equations : CH3.COOH + 30 = H.COOH + H20 + C02 Acetic acid. Formic acid. /COOH /COOH CH./H,. + 30 = CH, + H20 + C02 COOH xCOOH Succinic acid. Malonic acid. OH /OH C6ir, +30 = CBH1 + H20 + CO2 CHj.CHj.COOH CHj.COOH Para-oxy-phenyl-propionic Para-oxy-plieiiyl-acetic acid. acid. Through a splitting off of carbon dioxide para-oxy-phenyl-acetic acid theu further gives rise to paracresol, from which phenol is finally obtained on oxidation : OB OH (,!l, =C6H4< +C02 I II ..ex )H OH, ParacresoL oil I ,11, +30 = (',,11, OH -f CO, + H,0 < |[ PhenoL 90 THE NITROGENOUS DERIVATIVES OF THE ALBUMINS. In the animal body paracresol and phenol, which are formed from tyrosin during the process of intestinal putrefaction, then combine with sulphuric acid, and are eliminated through the urine in the form of their potassium salts : /OH .OH /O.HSO3 C6HZ + S02< =C6H4< +H20. XCH3 \OH \CH3 /OH C6H5.OH + S02< = QH.O.HSO, + H20. XOH During the process of albuminous putrefaction other aromatic compounds besides tyrosin and its derivatives also result, but these, in contradistinction to tyrosin, belong to the ortho-group. They comprise indol, skatol, and skatol-carbonic acid. According to some observers, these substances are derived from a special aromatic radicle, which is contained in the albuminous molecule, and which differs from the tyrosin radicle, while others believe that they result syn- thetically through the influence of the living bacteria from certain aromatic groups, which on hydrolytic decomposition with acids give rise to tyrosin. Indol, skatol, and skatol-carbonic acid are closely related to each other and to indigo. Skatol thus results from indol through a sub- stitution of the methyl-group for a hydrogen atom of one of the CH groups, as shown by the formula : /CH ^ /C(CH3)CH +0 = C6H / J^CH \NH/ \NH^^ Indol. Indoxyl. /C(OH) . ^C(O.SOsH)< (2) C6H4< ^CH + H2SO, = C6H4< Indoxyl. THE PTO MAINS. 91 On decomposition with strong hydrochloric acid indican is accord- ingly decomposed into sulphuric acid and indoxyl, which latter can then be oxidized to indigo-blue : /C(OH) ^ /CO x /CO x 2C6H/ >CH + 20 = C6H4< >C = C< >C6H4 + 2H20 Indoxyl. Indigo-blue. On reduction indigo-blue is transformed into indigo-white, C8H6NO, which, when boiled with zinc and water, then further yields indol : C8H5NO + H = C8H6NO. C8H6NO + 3H r- C8H7N + H20. Animal indican, however, must not be confused with vegetable indican, which is a glucoside, and yields indigo-blue and indiglucin on hydrolytic decomposition : C26HS1N017 + 2H20 = C8H5NO + 3C6H10O6 Vegetable indican. Indigo-blue. Indiglucin. A small amount of skatoxyl, indoxyl, and phenol is also eliminated in the urine, in combination with glucuronic acid, as skatoxyl, indoxyl, and phenol glucuronates, respectively. This acid may be derived from glucose by the substitution of one atom of oxygen for two atoms of hydrogen, and is accordingly represented by the formula COOH.(CH.OH)4.COH. It is possible, however, that glu- curonic acid may also be derived from chondroitin-sulphuric acid, which is normally found in cartilage, and, as a matter of fact, we have seen that through a series of simple reactions glucuronic acid can be obtained from this source. On oxidation it is transformed into saccharinic acid, the relation of which to glucose has already been considered. THE PTOMAINS. The term ptomain was originally applied by Selmi to certain alkaloidal bodies which are formed during the process of albu- minous putrefaction. Gautier then extended its use to include all those alkaloidal substances which result from anaerobic fermenta- tion, as also those which are formed in the tissues of the higher animals in the absence of air, or in the presence at least of an insuf- ficient Bupplyof oxygen. In contradistinction to these substances, Gautier terms those alkaloidal bodies which are formed during the normal and aerobic life of the tissues leucomains. Under this latter heading, as has been -ecu, he comprises the nucleinic bases and the kreatins. Both classes of substances are of special interest to the physician, as their formation or undue accumulation in the body may give rise to serious disturbances. This is true more particularly of the ptomains, some of which are extremely toxic. Gautier divides the ptomains into acyclic and cyclic ptomains, 92 THE NITROGENOUS DERIVATIVES OF THE ALBUMINS. some of which are free from oxygen, while in others this is present. They are all derived from ammonia by the substitution of various radicles for one or all of the nitrogen atoms of ammonia, and are hence analogous to the amins. The acyclic ptomains which are free from oxygen comprise the following substances : Methylamin NH2.CH3 = CH5N Dimethylamin NH(CH3)2 = C2H7N Trimethylamin N(CH3)3 = CgHgN Butylainin ]STH2(C4H9) = C4HnN Amylamin NH2(C5Hn) = C5H13]Sr Hexylamin NH2(G6HI3) = C6H15N JSTeuridin C5HUN2 Saprin C5HUN2 Pentamethylene-diaruin or cadaverin NH2(CH2)5.NH2 Ethylene-diamin NH2(C2H4)4NH2 Tetraniethylene-diamin or putrescin NH2(CH2)4.NH2 Dimethylene-imide or spermin (CH2)2NH Mydalein Metliyl-guanidin CH4(CH3)N3 The oxygen-containing acyclic ptomains are the following : Cholin or trimethyl-oxyethylene-am- 1 N(CH3)3.(C2H4.OH).OH = C5HI5N02 monium hydrate J v i,A * ° ° i NhUvdrate trimethy1-viny1"ammonium } N(CH3)3.(C2H3) .OH = C5H13NO Muscarin '. '. '. '. '. '. '. . . '. '. '. . N(CH3)3.(CH2.COH).OH = C5H15N03 Betain or oxycholin N(CH3)3.(CH2.COOH).OH = C5H,;N02 Mvdatoxin C6H13N02 Mydin C8HnNO Gadinin C7HI6N02 Methyl-gadinin C8H18N02 Mytilotoxin C6Hl5N02 Propyl-glucocyamin C6H13N203 The remaining ptornains are partly cyclic and in part not classified : Collidin (iso-phenyl-ethyiamin) . . . . C6H5.CH(CH3).NH2 = C8HnN Hydrocollidin C8H13N Parvolin C9H,3N Corindin C10H15N Hydrolutidin C7HHN Hydrocornidin C10H17N Scombrin Cj7H38N4 Morrhuin C19H27N3 Asellin C25H32N4 Morrhuicacid C5H3(OH)(C3H6.COOH).NH = CqH13N03 Typhotoxin C7H17N02 Tetanin Ci3H30N2O4 Tetanotoxin C5H,,N Spasmotoxin Tyrotoxin Pyocyanin Pyoxanthin Some of these substances and their origin from the albumins have already been considered, and we shall have further occasion to study them in greater detail. Others are scarcely known, and require no further description at this place. They are all intimately related to THE ORGANIC SOX-NITROGENOUS ACIDS. 93 the vegetable alkaloids, with which they have many reactions in common. They have strongly basic properties, and are capable of combining with acids to form salts. Like the albumins from which they are derived, they are precipitated with the chlorides of platinum, mercury, and gold, as also with tannic acid, picric acid, phospho- molybdic acid, phosphotungstic acid, etc. With these they form well-defined crystalline salts, which serve for their differentiation from each other and as a basis for the determination of their ele- mentary composition. The methods which are employed for the separation of ptomains will be considered in a subsequent chapter. THE ORGANIC NON-NITROGENOUS ACIDS. The organic non-nitrogenous acids which are formed in the animal body are largely members of the tatty acid series. Others belong to the glycolic series, still others to the acrylic series, some are repre- sentatives of the oxalic series, and still others belong to the aromatic oxy-acids. The first group comprises the following acids, which as a class may be represented by the formula C„H2n02. Formic acid H.COOII = CH.,02 Acetic acid CH3.COOH = C2H402 Propionic acid CH,.CH3.COOH = C3H60„ Butvricacid (CH2)2.CH3.COOH = C4H802" Valerianic acid (CH2)3.CH3.COOH = C5H10O2 Capmnicacid (CH2)4.CH3.COOH = C6H„02 Palmitic acid (CH2)14.CH3:COOH = C16H320, Stearic acid (CH2)16.CH3.COOH = C18H36(J2 The two last, as has been seen, are integral constituents of the fats, in which they are present in combination with glycerin as tri- glycerides. From these the others may in part be derived, but to the greatest extent no doubt they result from the amido-acids through a process of oxidation or reduction, as illustrated by the equations : CH2(XII,).COOH + 20 = H.COOH + NH3 + C02 Glycocoll. Formic acid. ny nh2).cooh + 2H = ch8.cooh + nh3 Glycocoll. Acetic acid. Through further oxidation these acids are then transformed into those standing nexl in order in the downward scale, and so on, until finally carbon dioxide and water result, as seen in the equations : fh < II, ( Il.moH + 30 = CH,.COOH | C02 + II,<> Propionic acid. Acetic acid. (2) CHg.COOH +30 = H.C00H -(- C02 + H20 e acid. Formic acid. B.COOH <) OOj | U,p Formic add. To some extent, however, the fatty acids are derived also from lactic acid and related acids, which, as will !><■ seen later, arc constantly 94 THE NITROGENOUS DERIVATIVES OF THE ALBUMINS. being formed in the metabolism of the albumins. Their transforma- tion into the fatty acids is then probably analogous to the production of butyric acid during the process of butyric acid fermentation. 2C3H603 = C4H802 + 2C02 + 4H Lactic Butyric acid. acid. The glycolic acids which are found in the animal body may be represented by the general formula CnH2„03. They comprise the following bodies : Glycolic acid CH2.OH.COOH = C.2H403 Ethylidene-lactic acid (paralactic acid) . CH3.CH(OH).COOH =C3H603 Ethylidene- lactic acid(optically inactive) CH3.CH(OH).COOH =C3H603 Ethvlidene-lsevo-lacticacid CH3.CH(OH).COOH =C,H603 /3-oxybutvric acid CH.OH.CH2.CH3.COOH = C,H803 Leucinicacid (CH3)2.CH.CH2.CH.OH.COOH = C6H1203 Of these acids, glycolic acid does not occur in the animal body, so far as is known, but it is of interest owing to the fact that it is closely related to glycocoll, and is derived from this in the same manner in which leucinic acid is obtained from leucin, viz., by the substitution of a hydroxyl group for the amido-group, as shown by the equations : CH2.(NH2).COOH + H20 = CH2(OH).COOH +NH3 Glycocoll. Glycolic acid. (CH3)2.CH.CH2.CH.(NH2).COOH + H20 = Leucin. (CH3)2.CH.CH2.CH(OH).COOH + NH3 Leucinic acid. /3-oxybutyric acid is found only under pathologic conditions. Lactic acid and its isomeric compounds, as well as leucinic acid and /3-oxybutyric acid, are all albuminous decomposition-products, and in part, at least, derived from the amido-acids. The origin of lactic acid, however, is not so clear, but we shall consider this ques- tion in greater detail in a subsequent chapter. On reduction these acids can be transformed into fatty acids. Lactic acid, as has just been shown, thus gives rise to butyric acid, leucinic acid is similarly changed to capronic acid, etc. On oxidation /3-oxybutyric acid is transformed into diacetic acid, which in turn is decomposed, with the formation of acetone and carbon dioxide : CH,.CH.OH.CH2.COOH + O = (CH3.CO)CH2COOH + H20 J3-oxybutyric acid. Diacetic acid. (CH3.CO)CH2.COOH = CO(CH3)2 + C02 Acetone. The bodies which are thus formed are of special interest to the pathologist, as their accumulation in the animal body is apparently capable of causing very serious disturbances. Acetone, however, is also met with under normal conditions, and apparently represents a constant product of albuminous decomposition. On boiling with dilute mineral acids /3-oxybutyric acid is trans- THE ORGANIC NON-NITROGENOUS ACIDS. 95 formed into an acid of the acrylic series (see below), viz., a-crotonic acid : CH3.CH(OH).CH2.COOH = 2H20 + CH3 — CH = CH.COOH a-Capronie acid. The. acids of the acrylic series can be represented by the general formula CBH2„_202. Representatives of these are the a-capronic acid, just referred to, and oleic acid, which as a triglyceride rep- resents a must important constituent of many of the vegetable and animal fats. On heating with hydriodic acid and red phosphorus to a temperature of 210° C, oleic acid takes up two atoms of hydro- gen, and is thus reduced to stearic acid : CH3. ( 'II , |, ,CH = CH.CH2.COOH + 2H = CH3.(CH2)16.COOH Oleic acid, Stearic acid. On treating with nitrous acid, in statu nascendi, it is transformed into the solid elaidinic acid, which is isomeric with oleic acid and belongs to the same series. The dibasic acids of the oxalic series can be represented by the formula C„H2n_204. They include oxalic acid, succinic acid, and glutaric acid, of which the two latter are generally met with in the form of their amides, viz., as asparaginic acid and glutaminic acid, respectively. In this form they are invariably obtained, together with oxalic acid, as decomposition-products of most albumins. Oxalic acid and succinic acid, however, may also be observed as such. The relation of oxalic acid to the ureids has already been con- sidered. They are represented by the formulae : Oxalic acid (COOH)2 = C2H204 Succinic acid (CH2)2.(COOH)2 = C4H*< >< Glutaric acid (CH2)3.((X)OH)2 = C5H8G4 On oxidation they are decomposed into water and carbon dioxide, but it is probable that during this transformation fatty acids are formed as intermediary products : ( JI204 = C02 + H.COOH. C4HA = C02 + C3H602 Propionic acid. The principal aromatic oxy-acids which may be found in the animal body are hydroparacumaric acid or para-oxy-phenyl-pro- pionic acid, para-oxy-phenyl-acetic acid, para-oxy-plienyl-lactic acid or oxy-hydroparacumaric acid, and para-oxy-phenyl-glycolic acid. They are derivatives of phenol, in which one hydrogen atom has been replaced by tin- radicle of the corresponding non-aromatic acid, a- shown by the equation : on r If oil CH..CH.OH.COOH 0 CJ3L + II20 ooL actic acid. (II. (II CH.COOH oxy-phenyl- Lactic acid. 96 THE NITROGENOUS DERIVATIVES OF THE ALBUMINS. They are probably all derivatives of tyrosin, and it has already been shown (p. 89) how hydroparacu marie acid results from this on reduction, and is then transformed into para-oxy-phenyl-acetic acid by oxidation. The formulae of these acids and their relation to tyrosin are seen below : .OH Tyrosin (para-oxy-phenyl-amido-propionic acid) = C6H4<^ xCH2.CH(NH2).COOH X)H Para-oxy-phenyl-propionic acid = C6H4<( \CH2.CH2.COOH .OH Para-oxy-phenyl-acetic acid = C6H4f the cells, but also arrest or annihilate the action of the ferments. Such sub- stances are the bichloride of mercury, carbolic acid, the mineral acids, and to a less marked degree other metallic salts, as also picric acid, tannic acid, etc. It is to be noted, however, that one ferment, ;it Least, viz., pep-in, i- not destroyed by dilute acids. The 102 THE FERMENTS. activity of the ferments is further decreased with an increase of their specific products beyond a certain degree. Absence of water like- wise inhibits the action of the ferments, but this is at once re- established when the necessary degree of moisture is supplied, and it is possible therefore to preserve the ferments in the dry state. During the process of drying, however, care must be had that the temperature does not exceed a certain limit. This varies with the different ferments, but it may be stated as a general rule that all animal ferments are killed by a temperature of 75° C, while the vegetable ferments cannot survive a temperature of 80° C. In the absence of moisture, however, they can apparently withstand much greater heat, and it is said that dry trypsin, pepsin, and diastase may be heated to a temperature of from 150° to 160° C without losing their activity. Strong alcohol destroys the action of certain ferments, such as pepsin and diastase, while others, like the fibrin-ferment, are not affected. The most peculiar property of the ferments, and the one which is characteristic of all, is the power to bring about an amount of chemical change which is out of all proportion to the quantity of the ferment present, while the ferment itself undergoes no apparent change. The common pepsin preparations of the market are thus of a strength that 1 part by weight of the pepsin will digest 6000 parts by weight of coagulated egg-albumin, and Petit claims that a preparation from his laboratory was capable of dissolving even 500,000 times its weight of fibrin in seven hours. That the ferments themselves undergo no change while exerting their specific action can be readily shown, as it is possible to re- obtain them from the various digestive mixtures and to test their efficacy as before. The rapidity with which the action of ferments takes place is often most remarkable, and is especially well shown during the coagulation of milk under the influence of chymosin. In order that the ferments may exhibit their activity to best advantage a definite temperature is necessary, which varies some- what with the different ferments, but is generally about that of the body. Higher as well as lower temperatures gradually inhibit their action, and, as has been seen, destroy it entirely when 75°-80° C. is reached. Very low temperature has the same effect. The presence or absence of oxygen has no effect upon the action of ferments, and they thus show a distinct difference from the organized ferments, which are more or less dependent upon either its presence or its absence. The reaction of the medium in which the ferments are to display their activity is very important, and varies with the different fer- ments. Some of these, such as pepsin, can act to advantage only in an acid medium ; while others, such as ptyalin, require an alka- line reaction ; and still others can act in acid, alkaline, and neutral media, but exhibit certain preferences. CHEMICAL COMPOSITION AND GENERAL REACTIONS. 103 In conclusion, the reversible action of ferments, which has recently been established, must be briefly considered. It has long been known that the hydrolytic decomposition effected by ferments is never carried to an end, and it is usually stated that this is owing to the fact that a gradual increase in the production of decom- position-products inhibits the action of the ferments in question. In the light of more recent investigations, however, this explanation is not satisfactory. It has been demonstrated that maltase, when added to a solution of maltose, will cause inversion of the latter to glucose. An end-reaction is then not obtained ; but if this solution is now added to a solution of glucose in turn, at a time when further inversion does not occur, it will be noted that a retransformation of glucose to maltose takes place, which, however, is likewise not complete. It thus appears that the ferment is not only capable of causing the hydrolytic decomposition, but also the synthesis of mal- tose ; but that in so doing, its action ceases as soon as a certain equilibrium of reaction has been established. This reversible action cm the part of ferments is, of course, of the greatest interest to the physiological chemist, in showing that the complex syntheses which take place in plant-life may, to a certain extent at least, be referable to such action, and to forces which are probably also at work in the non-organized world. Further research will show whether this action is common to all ferments. Chemical Composition and General Reactions. — Of the chem- ical composition of the ferments but little is known that is definite. This is owing to the fact that isolation of any one of the ferments in a chemically pure form has thus far not been accomplished. They apparently contain nitrogen, and are usually regarded as albumi- nous substances ; but it is still a matter of doubt whether this is actually the case, and it is possible that the supposition of their albuminous nature is owing to their being contaminated with albumins. Like the albumins, they are as a class non-diffusible. They are soluble in water, and can be precipitated from their aqueous solu- tions by salting with ammonium sulphate or by the addition of strong alcohol. When kept under alcohol for any length of time some of the fer- ments, such as pepsin and diastase, are rendered inactive and are apparently coagulated, while the activity of others, such as the fibrin ferment, remains unaffected. Characteristic general reactions, which are common to all fer- ments, are unknown. Formerly it was supposed that they all pos- sessed the power of decomposing hydrogen peroxide, but it appears that this property does not belong to the ferments proper, but to adherent particles of protoplasm. Asa matter of fact, it is possible in a number of ferments to destroy this power of decomposing hydrogen peroxide without influencing their specific activity in the 104 THE FERMENTS. least. If pancreatic juice is thus heated to a temperature of 60° C, and then allowed to cool to 40° C, it will be observed that the fluid is still capable of digesting albumins and of inverting starch, while it has lost the power of decomposing hydrogen peroxide entirely. Similar results may be obtained on heating the dry ferments to a somewhat higher temperature, by treating with alcohol, or by satu- rating their solutions with neutral salts. Mode of Action. — The decompositions which the ferments are capable of effecting in suitable media are essentially of a hydrolytic character. This can be readily shown by comparing the decom- position-products to which the ferments give rise with the original substances, when it will be found that, practically without exception, the former contain more water. Nasse, moreover, could demon- strate a distinct increase in the electrical conductivity of watery solutions of starch, for example, when these were treated with dias- tase, showing that dissociated molecules of water must have been present. As to the manner, however, in which these hydrolytic phenomena are brought about Ave are very much in the dark. On the one hand, we may suppose that the molecular oscillations which take place in the molecules of the ferments are of such a nature as to bring about an increase in the molecular oscillations of the substances upon which the ferments exert their specific activity, and that in consequence of this increase in the oscillations the labile equilibrium of the large albuminous or polysaccharine molecules is disturbed, which in turn would lead to new combinations of atoms to form molecules that are more stable, and the oscillations of which would be more nearly like those of the ferments. According to this theory, then, the action of the ferments would be what has been termed a katalytic action, and analogous to the katalytic action of various metals, such as platinum, gold, silver, etc., which in fine suspension behave in very much the same manner 'as the ferments (see page 20). On the other hand, we may suppose that the action of the ferments is an action by- contact, such that one molecule of the ferment causes hydrolytic decomposition of one molecule of an albuminous substance, for example, and that this decomposition in turn causes decomposition of the adjacent albuminous molecules, and so on. The action of certain ferments, such as the fibrin ferment, and that which causes the coagulation of milk, might very well be explained upon such a basis. For here we see a rapidity of action which scarcely admits of any other explanation ; and direct contact of the ferments with all parts of the surrounding material, moreover, is excluded, as each, ferment-molecule must of necessity be at once surrounded by a layer of the coagulated albumin. Classification. — It has been pointed out that there are no general reactions which are characteristic of all ferments. The ferments can be separated into groups, however, which are fairly well char- acterized through their specific activity and the decomposition- CLASSIFICATION. 105 products to which they gave rise. They are accordingly divided into the following classes : 1. The Proteolytic Ferments. — These comprise the animal ferments pepsin and trypsin, various vegetable ferments such as papayotin, and others which may be obtained from germinating seeds of Lupinus angustifoleus, Lupinus luteus, Vicia Faba and Ricinus* major, and certain ferments which may be obtained from bacteria. They all digest the various albumins, with the formation of albu- naoses, and some of them also cause the further decomposition of these to amido-acids, hexon bases, etc. 2. The Amylolytic Ferments. — These include the ptyalin of the saliva and the diastatie ferment of the pancreatic juice, the so-called vegetable diastase, and related ferments, which may be obtained from bacteria. Some of these only render starch soluble, while others carry the hydrolysis further to the formation of monosac- charide. 3. The Inverting Ferments. — These are apparently closely related to the amylolytic ferments, and are to a certain extent identical with them. They invert the disaccharides to monosaccharides, and, according to their specific effect upon cane-sugar, maltose, and lactose, arc termed invertins, maltases, and lactases, respectively. Such ferments are found in the saliva, the pancreatic juice, and the intestinal juice, in many of the higher plants, and also in numerous organized ferments. 4. The Steatolytic Ferments. — Such ferments cause decomposition of fats into glycerin and fatty acids. Representatives of this order arc the so-called steapsin of the pancreatic juice, and analogous fer- ments that have been found in the vegetable world, notably in the ,„i5.C12H2A,] + 45H20 = o4C12H22Ou = 54Cl2H22On Isomaltose. Maltose. We shall return to these reactions in the next chapter, when the digestion of the food is considered in detail. To isolate the ptyalin from saliva, the following method, which has been suggested by Gautier, may be employed: To a large quantity of saliva (J8 per cent, alcohol is added so long as a floccu- lent precipitate is seen to form. This is collected on a small filter and dissolved with a small amount of distilled water. The solu- tion is treated with a few drops of a solution of bichloride of mercury, in order to remove any albuminous material that may be present. In the filtrate the excess of the bichloride is removed with hydrogen Bulphide, when the remaining liquid is evaporated to dryness at a temperature not exceeding 40° C, and the residue is treated with Strong alcohol. The insoluble portion is then dissolved with a small amount oi* distilled water, filtered, dialyzed in order to remove inor- ganic -alts, and finally precipitated with absolute alcohol, when the ptyalin will separate out in light Hakes. Obtained in this manner, ptyalin is a white amorphous substance, which is soluble in water, dilute alcohol, and glycerin. In neutral or slightly alkaline solu- tion, but not iii acid solution, it rapidly transforms boiled starch into sugar at a temperature of from 36° to 40° C. Heated to a temperature of 60° C., its solutions lose this power, and it is thus possible to distinguish between ptyalin and the diastatic ferment of vegetable origin, tor which the optimum temperature Lies between 60° and 65° C. Of special interesl i- the feci that the transformation of starch into sugar ceases as soon as the latter is presenl to the extent oi from 2 to 2.5 per cent. This phenomenon is common to all eniy- 112 THE DIGESTIVE FLUIDS. matic processes, and is probably referable to the establishment of a certain equilibrium of reaction. A complete transformation of the starch could occur only if the resulting sugar were removed as rapidly as it is formed. So long as it is present, the reversible action of the enzyme becomes manifest, and, analogous to the rever- sion of glucose to maltose, a similar retransformation of maltose to dextrin no doubt occurs. The amount of ptyalin which is secreted in the twenty-four hours has not been determined. Its activity, as would be ex- pected, is subject to considerable variations. It is greatest in the morning on rising, and then steadily diminishes during the day. Immediately before meals, however, a temporary increase is ob- served, which is then followed by a marked decrease. Of the chemical nature of ptyalin but little is known. Like all other ferments, it is generally regarded as an albuminous substance, and on the application of dry heat it develops the characteristic odor of burning albumins. It is nitrogenous, but does not give the xanthoproteic reaction. From its solutions it can be precipitated with acetate and subacetate of lead, while bichloride of mercury and the salts of platinum, as also tannic acid, are without effect. In the human being ptyalin is formed in the parotid and the submaxillary glands and, as will be seen later, also in the pancreas, while the sublingual glands apparently yield no ptyalin. In other animals its presence in the saliva is variable. In the typical carnivora it is said to be absent, while in the saliva of all herbivor- ous auimals it is uniformly found. Of other ferments, human saliva apparently also contains traces of maltase, and of an oxydase of unknown character ; invertin, however, has not been found. Consequently an inversion of maltose to glucose may also take place in the early stages of carbohydrate digestion, but is certainly insignificant in extent. The digestive importance of the saliva is, in man, at least, but slight, as ptyalin is rapidly destroyed by contact with the acid gastric juice. During the process of mastication and deglutition, moreover, it has scarcely time to effect much change, and in experi- ments in vitro we find that the amount of maltose which is formed by the saliva from starch is small. The importance of the salivary glands as digestive glands has thus been much overrated, and it has been conclusively demonstrated that their function has mostly to do with the preparation of the food for the act of deglutition. This is, of course, greatly facilitated by its thorough lubrication with the mucus that is furnished by the salivary glands, and which in reality represents the most important constituent of the saliva. Mucin. — The mucin of the saliva is derived from the submaxil- lary and sublingual glands, as also from the small mucous glands which are found imbedded in the mucous membrane of the mouth. Its formation in the salivary glands is apparently under the control of the sympathetic nervous system, as it is secreted in much larger THE SALIVA. 113 amounts on stimulation of these fibres than of the corresponding cerebral fibres. According to Levene, the submaxillary mucin eon- tains the chondroitin-sulphuric aeid complex, or a closely allied group. To the presence of the mucin the viscid, stringy character of the saliva is due. The substance can be obtained by precipitation with acetic acid, and it is to be noted that, in contradistinction to the mucin-like substances which belong to the class of the nucleo-albu- mins, the precipitated mucin is insoluble in an excess- of the acid. In dilute solutions of the alkalies it is soluble, and it is thus possible by repeated precipitation and solution to obtain the mucin in fairly pure form. In alcohol and water it is insoluble, although in the latter it swells to form a jelly-like material. Unlike the albumins, it is not coagulated by heat; but, like these, it gives the xanthoproteic reaction, the biuret reaction, and Millon's reaction. It contains also a small amount of sulphur. On boiling with dilute mineral acids mucin is decomposed into a substance which resembles mid albumin, and into a carbohydrate-like body which reduces Fehlingr's solution. This has been regarded as identical with Land- wehr's animal gum ; Hammarsten, however, states that from the mucin of the submaxillary gland a gum-like substance is obtained which contains nitrogen. On decomposition with strong mineral acids mucin yields leucin, tyrosin, and hevulinic acid (see also page 45). In the dry state it occurs as a white or yellowish-gray powder. Within the cells mucin exists as so-called mu&nog&ri, which prob- ably represents a compound of mucin with an additional albuminous substance. Sulphocyanides. — Traces of sodium sulphoeyanide are in man usually found in every specimen of normal saliva. It is secreted by all the salivary glands, but in largest amount by the parotids. In other animals its presence is not so constant, and in some indeed it i- not found. In man also it is at times absent. To demonstrate the presence of sulphocyanides, it usually suffices to treat a few cubic centimeters of saliva, which have been slightly acidified with hydrochloric acid, with a few drops of a very dilute solution of perchloride of iron, when a red color will be seen to develop. If no residt is obtained in this manner, a larger quantity, such as 10() c.C IS evaporated to a small volume and tested as described. Nitrites. — Small amounts of nitrites may also at times be observed, and are no doubt derived from the nitrates ingested. To tesf for tli<-<-, ;i|)()iit lo <•.<•. of saliva are treated with a few drops of [lasvay'e reagent, and beated t<» a temperature of 80° C, when in the presence of nitrite- ;i red color develops. [lasvay's reagent i- prepared as follows: 0.5 gramme of sulph- anilic acid in 150 c.c. of dilute acetic acid i- treated with 0.1 gramme of naphtylamin, and dissolved in 20 c.c. of boiling water. 114 THE DIGESTIVE FLUIDS. After standing for some time the supernatant fluid is poured off, and the blue sediment dissolved in 150 c.c. of dilute acetic acid. The solution is kept in a sealed bottle. Extractives. — Of extractives, normal saliva contains a small amount of urea, and traces of cholesterin, lecithin, and leucin. In gouty conditions uric acid has been found ; sugar, the biliary pig- ments, and biliary acids are not eliminated through the saliva. Mineral Constituents. — The mineral constituents of saliva con- sist to the extent of 90 to 92 per cent, of soluble salts, among which the chlorides greatly predominate, and of about 6 per cent, of salts, which are principally represented by the carbonates and phosphates of calcium and magnesium, which are held in solution by the free carbonic acid of the saliva. In addition, a trace of iron is found. Following the administration of bromides and iodides a notable elimination of these salts occurs through this channel. Gases. — Of gases, which are present in a state of solution, we find about 20 c.c. for every 100 grammes of saliva. Of these, 19 c.c. are represented by carbon dioxide, while oxygen and nitrogen together amount to only 1 c.c. THE GASTRIC JUICE. General Considerations. — The gastric juice is the secretory product of the glandular structures of the stomach, and the only digestive fluid which presents an acid reaction. In pure form it is best obtained from animals after ligating the ducts of the salivary glands and establishing a fistulous opening on the outer abdominal walls. If the mucous membrane is then appropriately stimulated, a clear or but slightly opalescent yellowish fluid is obtained, which has a very characteristic odor and a strongly acid reaction. Its density varies between 1.001 and 1.010. On microscopic examination are found epithelial cells from the lining of the glandular ducts, goblet-cells, mucous corpuscles, free nuclei, and a variable number of bacteria. In addition, we often observe small tapioca-like bodies, which under the microscope are seen to contain numerous formations resembling snail-shells, and which probably consist of altered mucin. Amount. — Of the total amount of gastric juice secreted in the twenty-four hours, but little is known. Its secretion is influenced by numerous factors, such as the appetite, the quality and quantity of the food ingested, the age and sex of the individual, the time of day (notably in relation to the taking of food), the various emotions, etc. According to Bidder and Schmidt, the amount corresponds to about one-tenth of the body-weight, so that a man weighing 70 kilo- grammes would secrete about 7000 grammes in the twenty-four hours. This figure, however, I regard as too high, and am inclined to place the amount at from 2000 to 3000 c.c. The non-digesting stomach of the dog and other animals is said THE GASTRIC JUICE. 115 to contain no fluid ; in man, however, a small amount of gastric juice can usually be obtained by means of the stomach-tube, vary- ing between 1 and 60 c.c. Larger amounts may be found under pathologic conditions, and in the so-called Magensaftfluss of the Germans it is not rare to find as much as 1000 c.c. in the early morning, before any food has been taken. The amount of fluid which can be normally obtained from the digesting organ is likewise variable. It depends upon the amount of liquid ingested, the period of digestion, the character of the food, the size and motor power of the stomach, etc. Exact figures, how- ever, are lacking to represent these relations, and it is manifest that such figures must always have reference to more or less diluted gastric juice. Chemical Composition. — A general idea of the chemical com- position of the gastric juice may be formed from the following analy- ses, which are taken from C. Schmidt ; but it is to be noted that the specimen of human gastric juice was contaminated with saliva and somewhat diluted with water (the figures have reference to 1000 parts) : Human gastric juice, Gastric juice of containing saliva dog, free with water. from saliva. Water 994.40 973.0 Solids 5.60 27.0 Organic material 3.10 17.1 Mineral salts 2.19 6.7 Sodium chloride 1.46 2.5 Calcium chloride 0.06 0.6 Potassium chloride 0.55 ....... 1.1 Ammonium chloride 0.5 Calcium phosphate 1.7 Magnesium phosphate 0.2 Iron 0.12 0.1 Free hydrochloric acid 0.20 0.1 Acidity of the Gastric Juice. — It has now been definitely established that the acidity of normal gastric juice is referable to the presence of free hydrochloric acid, and to this only. This can be shown by estimating the amount of chlorine and all basic sub- stances that are present, when it will bo found that after the acid affinities of tin- latter have been saturated, a certain amount of chlorine still remains, which can be referable only to hydrochloric acid, and corresponds in its degree of acidity to that observed in th<- gastric juice itself. During the process of digestion, however, other factors enter into consideration. In the beginning of digestion lactic acid is always {tre-eiit when carbohydrates form part of the meal. Its amount, lowever, i- then quite small, and after the ingestion of Ewald's test-breakfkst, for example, does not exceed 0.3 pro niille. The occurrence of larger quantities of lactic acid, as from 1 to ."> pro mille, is always abnormal, and in many cases indicative of the existence of carcinoma of the stomach. During the later stages 116 THE DIGESTIVE FLUIDS. of digestion, when hydrochloric acid is found in a free state, lactic acid disappears. Its origin, under normal conditions at least, is referable to the action of certain bacteria, such as the Bacterium lactis, the Bacillus lactis aerogenes, etc., upon starches and sugars, as represented by the equations : (1) 2C5HI0O5 + H20 = C12H22On Starch. Lactose. (2) C12H22CU + H20 = 4C,H603 Lactose. Lactic acid. Other organic acids, such as butyric acid and acetic acid, are usu- ally not found in the gastric contents unless large amounts of milk, carbohydrates, or alcohol have been ingested. In such event, how- ever, they may be present, and, like lactic acid, are then referable to the action of certain micro-organisms. They are essentially of patho- logical significance. It is thus seen that even during the process of digestion the acidity of the gastric contents is, under normal conditions, scarcely influenced by acids other than hydrochloric acid. It should be noted, however, that following the ingestion of food hydro- chloric acid does not appear in a free state at once, but only after the affinities of the albuminous constituents of the food have been saturated. We consequently find that while in the begin- ning of digestion the acidity of the stomach-contents is largely referable to such combined acid, in the later phases of digestion two factors enter into consideration, viz., free and combined hydrochloric acid. The period at which free acid appears as such varies, of course, with the character of the meal, and directly with the amount of proteids ingested. After the administration of Ewald's test- breakfast free acid is thus found only at the expiration of about thirty-five minutes ; while after the administration of Riegel's test- dinner, which contains much larger amounts of albumin, two hours must elapse before free acid can be demonstrated. Acid salts, finally, play only a small part in determining the total acidity of the gastric juice ; and it is thus clear that unless carbo- hydrates, much fat, and alcohol have been ingested, hydrochloric acid, either in a free state or in combination with albumin, or both, is the sole factor which enters into consideration. Under pathological conditions, on the other hand, lactic acid, butyric acid, and acetic acid may also play a part ; but then hydrochloric acid is usually not present, and the acidity of the gastric contents is hence largely referable to fermentative changes which have taken place in the stomach. Determination of the Total Acidity of the Gastric Contents. — Five or 10 c.c. of the filtered gastric contents are titrated with a decinormal solution of sodium hydrate, using phenolphthalein as an indicator, until the rose color, which appears on the addition of each drop of the sodium hydrate solution, no longer disappears on stirring or is intensified by the addition of a further drop. The number of THE GASTRIC JUICE. 117 cubic centimeters employed to bring about this reaction, multiplied by 0.00365, indicates the acidity of the 5 or 10 c.c. of gastric juice in terms of hydrochloric acid. Amount. — The degree of acidity of the gastric juice is usually fairly constant, and in man varies between 0.15 and 0.25 per cent. It is influenced to a certain extent by the character of the food ; for instance, following the administration of a meal rich in proteids, somewhat larger amounts are obtained than after the ingestion of carbohydrates or fats. The smallest amounts are found after the ingestion of water. After Ewald's test-breakfast, which consists of from 35 to 70 grammes of wheat bread and 300 to 400 c.c. of water, or weak tea without sugar, the maximum acidity is reached in about one hour, and corresponds to 1.5 to 1.75 pro mille. Follow- ing the ingestion of Riegel's test-meal, on the other hand, which eon-ists of a plate of soup (400 c.c), 200 grammes of beefsteak, 50 gramme-; of wheat bread, and 200 c.c. of water, the amount of hydrochloric acid increases to 2.7 pro mille, after from one hundred and eighty to two hundred and ten minutes. In disease still higher figures (5 p. m.) may be observed ; or its secretion may diminish below the normal, and may even cease altogether. Hydrochloric Acid. — Origin. — The hydrochloric acid of the gastric juice is furnished by the so-called parietal, adelomorphous, or oxyntic cells, which are principally found in the glands of the fundus. This can be demonstrated by resecting the fundus, then closing one end with a fine suture, and sewing the other into the abdominal wound, while the cardiac portion of the stomach is joined to the pyloric end. If food be now administered to the animal, a fluid will be secreted by the isolated fundus in which the presence of free hydrochloric acid can easily be shown. If, on the other hand, the pyloric- end of the stomach, in which no parietal cells arc found, is similarly isolated, no acid is obtained, but, instead, a Btrongly alkaline mucus. While it is thus clear that the hydrochloric acid is furnished by the parietal eel!-, we are as yet ignorant of the mechanism by which this is accomplished. A free acid is manifestly not present in these cells, as can be shown by testing with litmus-paper, or still better by. injecting potassium ferrocyanide and lactate of iron into the circula- tion of an animal, when it will be observed that JJerlin-blue is formed in the Stomach-cavity, while the cells themselves remain unstained, it thus follows that a substance must either be present in tin- cells which is capable of yielding hydrochloric acid when secreted to the outside, or n mechanism must exist by which the hydrochloric acid, though formed within the cells, is at once elim- inated. The hitter v;ew i< now generally held. That the hydro- chloric acid i- derived from the chlorides of the blood can be regarded ;i- an established feet. It may thus be secreted even though no food— tuff- have been ingested; and Kahn, moreover,has shown that animals in which the chlorides of the bodv have been 118 THE DIGESTIVE FLUIDS. artificially reduced to that minimum which is tenaciously retained are no longer capable of secreting hydrochloric acid. The mech- anism, however, by which the formation of hydrochloric acid takes place is, as has been stated, unknown. Ludwig formerly thought that it resulted through electrolytic influences within the cells, and that the acid then diffused out into the lumen of the glandular duct before the alkaline elements of the cell could bring about its neutral- ization. This, however, is improbable, and the view that is now held by most observers is that expressed by Maly, according to which the hydrochloric acid results from a mass-action on the part of the carbonic acid of the blood upon the chlorides in the body of cells, and is immediately eliminated to the outside, while the result- ing bicarbonate is returned to the blood. We know that within the cells carbon dioxide is present under great pressure. Schlierbeck thus found that water which is introduced into the stomach of living dogs after a variable length of time contains a certain amount of carbon dioxide, and that its tension rises from 30 to 40 Hgram. while fasting, to 130 to 140 Hgmm. during the process of active digestion. Of late, Liebermann has further suggested that lecithalbumin may be present in the parietal cells in combination with sodium chloride, and that hydrochloric acid may result from this through a mass- action on the part of carbonic acid, which, in turn, is formed within the cells as a result of an increase of their functional activity. Significance of the Hydrochloric Acid. — The assumption that the principal function of the hydrochloric acid consists in its power to render the pepsin of the gastric juice physiologically active, viz., capable of bringing about the transformation of albumins into albu- moses and peptones, has now been largely abandoned. We know that life can go on in the entire absence of the stomach, as has been proved not only by experiments on animals, but also by operations which have been performed on the human being. A dog whose stomach was almost entirely removed by Czerny in 1876, lived for more than six years after the operation, when it was killed in Lud- wig's laboratory ; and it is reported that the animal was normal in every respect, and had increased in weight from 5850 grammes to 7000 grammes. It is thus manifest that while the hydrochloric acid of the gastric juice no doubt aids in the process of albuminous di- gestion, its presence to this end is not imperative, and the question naturally suggests itself, whether the secretion of such large amounts of acid does not serve another and perhaps more important purpose. This purpose is now thought to be the prevention of putrefactive changes in the contents of the stomach, and we find, as a matter of fact, that albuminous material that has been removed from the stomach at the height of digestion can be preserved for a long time without undergoing decomposition. It has been noted, moreover, that the gastric juice is also capable of arresting putrefactive proc- esses when these have begun before the ingestion of such materiaL THE GASTRIC JUICE. 119 This problem has been carefully investigated, and we now know that the amount of hydrochloric acid which is present at the height of digestion is sufficient at least to arrest the activity of most bacteria which are usually ingested together with food. Some of these, however, are more resistant than others, and among them the lactic acid bacteria are especially stable. We can accordingly under- stand why in the beginning of the process of digestion traces of lactic acid are usually found. So soon, however, as the percen- tage of hydrochloric acid has increased to 0.7 pro mille the activity of these organisms also ceases. Whether or not the gastric jnice actually destroys all of the more common micro-organisms that are swallowed has not been determined, but there is evidence to show that in some instances at least the spores remain unaffected and can later develop in the alkaline contents of the small intes- tine. In this manner we can account for the presence of the innumerable bacteria which are found in the lower intestinal tract. It must be remembered, moreover, that in the beginning of digestion — i. r.} when free hydrochloric acid is as yet not pres- ent— large numbers of bacteria may also pass through the stomach unaffected, as combined hydrochloric acid possesses no anti-fermenta- tive properties. Some of the more important pathogenic bacteria are unfortunately more resistant than the common benign forms. This is true especially of the tubercle bacillus, and in many cases of the anthrax bacillus and some of the pus organisms, while the cholera bacillus, on the other hand, is readily destroyed. Tests for Free Hydrochloric Acid. — A large number of tests have been devised for the purpose of demonstrating the presence of free hydrochloric acid in the stomach-contents. At this place only the more important ones will be described, which are employed in the clinical laboratory. Topper's Test. — A small amount of the filtered gastric contents is treated with a few drops of a 0.5 per cent, alcoholic solution of dimethyl-amido-azobenzol, when in the presence of free hydro- chloric acid a beautiful cherry-red color develops at once, which varies in intensity with the amount of the free acid present. Com- bined hydrochloric acid, as well as acid salts and organic acids, in the concentration in which they may be met with in the stomach- conterits, does not produce this color. The delicacy of the reagent is such that the norma] yellow color of the indicator is changed to a reddish tinge upon the addition of hut one drop of a X normal solution of hydrochloric acid in 5 cc of distilled water, viz., 0.7 per cent. GtTNZBURo/s Test. — The reageni consists of 2 grammes of phloro- glucin and 1 gramme of vanillin, dissolved in 100 grammes of 80 per cent, alcohol. It should be kept in a dark-colored, glass- stoppered bottle. A few drops of the filtered gastric content- are carefully evapo- rated with an equal amount of the reageni on a plate of thin porce- 120 THE DIGESTIVE FLUIDS. lain or glass, when in the presence of free hydrochloric acid a rose-colored mirror is obtained, which varies in intensity with the amount of the acid. Organic acids do not produce the reaction. The delicacy of the test is such that the presence of 0.05 gramme of hydrochloric acid in 100 parts of water can be demonstrated. Boas' Test. — The reagent consists of 5 grammes of resublimed resorcin and 3 grammes of cane-sugar, dissolved in 100 grammes of 94 per cent, alcohol. The test is conducted like that of Giinz- burg, but it is necessary to heat a little more strongly, especially after the fluid has been evaporated. A similarly colored mirror is obtained, which gradually fades on cooling. The delicacy of the test is the same as that of Giinzburg. Examination for the Presence of Combined Hydrochloric Acid. — The presence of combined hydrochloric acid cannot be demonstrated by means of simple tests like those just described, but is inferred indirectly, as shown in the following method : Separate Estimation of the Free and Combined Hydrochloric Acid of the Gastric Contents. — Topfer's Method. — The total acidity of a given amount of the gastric contents is first determined, as already described, and termed A. This indicates the amount of the physiologically active hydrochloric acid, viz., the free and the combined hydrochloric acid, as well as that of any acid salts and organic acids that may be present. In a second specimen the total amount of free acids and acid salts is determined by titrating, as before, with a -^ normal solu- tion of sodium hydrate, but using a few drops of a 1 per cent, aqueous solution of alizarin (alizarin-monosulphonate of sodium) as an indicator. The titration is carried to a point Mrhere a pure violet color is obtained. The result is termed B. The difference between A and B is thus referable to the presence of the combined hydro- chloric acid, and termed C. In a third specimen the amount of free hydrochloric acid is determined by titrating with the decinormal solution of sodium hydrate, using a few drops of a 0.5 per cent, alcoholic solution of dimethyl-amido-azobenzol as indicator, until the red color which first appears has changed to yellow. The result is termed F. F plus C will then represent the amount of physiologically active hydrochloric acid P, viz., the combined and free acid, while the difference between A and P corresponds to the acid salts and organic acids that may also be present. Method of Morner and Sjoqvist. — By this method the amount of physiologically active hydrochloric acid can also be estimated. It is somewhat more complicated and time-consuming than the one just described, but more accurate. It is based upon the fact that on evaporating the gastric contents to dryness in the presence of barium carbonate, and subsequently incinerating the residue, the organic THE GASTRIC JUICE. 121 acids are destroyed, while the hydrochloric acid combines with the barium, and can thus be estimated as barium chloride. To this end, 10 c.c. of the filtered stomach-contents are treated with a pinch of chemically pure barium carbonate and evaporated to dryness. The residue is ignited at a moderate temperature until white, and the remaining ash extracted with hot water. After filtering the solution (about 50 c.c.) it is treated with an equal volume of alcohol (94 per cent.) and 0.75 c.c. of a 10 per cent, solution of sodium acetate in dilute acetic acid (10 per cent, solution). The barium is then estimated by titrating with a standardized solution of potassium bichromate, containing 8.5 grammes of the chemically pure substance in the liter, and using tetramethyl-paraphenyldiamin paper as an indicator, until a drop of the titrated fluid causes a distinct blue coloration of the paper within one minute. From the number of cubic centimeters employed to bring about the end-reaction the corresponding amount of hydrochloric acid can then be calculated by multiplying with 0.00405, if the bichromate solution was standardized with a ^ normal solution of barium chloride. Method of Leo. — This method is based upon the observation that calcium carbonate combines with free and loosely bound hydro- chloric acid at ordinary temperatures to form neutral calcium chlo- ride, while the acid phosphates are not affected. If then the total acidity of the stomach-contents is first determined, and the acidity referable to acid salts deducted from this figure, the amount of physiologically active hydrochloric acid is ascertained. Organic acids, of course, must first be removed by extracting with ether (50-100 c.c. for 10 c.c. of gastric juice). As the monophosphates of potassium and sodium, however, are changed to monocalcium phos- phate in the presence of calcium chloride, which requires double the quantity of sodium hydrate solution for its neutralization than the corresponding amount of the alkaline phosphates, it is either neces- sary to divide the number of cubic centimeters of the sodium hydrate solution which is used in the second titration by 2, or to make the firsl titration under the same conditions as the first, viz., after adding an excess of calcium chloride solution. To this end, then, we proceed as follows: 15 c.c. of the filtered gastric contents are treated with a pinch of dry and chemically pure calcium carbonate. The mixture is well stirred and passed at once through a dry filter, 'fen c.c, from which the carbon dioxide is expelled by a current of air, are then treated with 5 cv. of a con- centrated solution of calcium chloride and titrated as usual. The resulting value is termed I', and represents the acid phosphates. The total acidity is then determined in another specimen, after adding the same amounl of the calcium chloride solution, and the result termed T. T minus A will then represent the amount of the physiologically active hydrochloric acid. The combined hydrochloric acid may, of course, he readily deter- mined with either of the two method- which have jnst been 122 THE DIGESTIVE FLUIDS. described, by separately estimating the amount of free hydrochloric- acid by Topfer's method, and deducting the result from the total amount of the physiologically active acid. More accurate results are probably reached in this manner than with Topfer's method, unless some experience has been gained in the titration with alizarin. Should organic acids also be present, their amount may be esti- mated by deducting from the total acidity the result reached with the above method. If monophosphates are present at the same time, the resulting figures will be a little too low ; but the error which is thus incurred is trifling. It may be obviated, however, by making use of Leo's method (see above). Lactic Acid. — Tests for Lactic Acid. — In order to assure our- selves that any lactic acid that may be found in the gastric contents has not been introduced into the stomach from without, it is neces- sary to make such examinations after the administration of a test- meal, in which the acid in question does not occur preformed. The meal which is almost exclusively used for this purpose in clinical work is the so-called test-meal of Boas. It consists of a plateful of oatmeal soup, which is prepared by adding a tablespoonful of rolled oats and a little salt to a liter of water, and boiling down to about 500 c.c. The contents of the stomach are then drawn off after one hour, filtered, and treated as described below. Uffelmann's Test. — About 10 c.c. of the filtered gastric con- tents are extracted with ether (50-100 c.c.) by shakiug in a sepa- rating funnel for from twenty to thirty minutes. The ethereal ex- tract is then evaporated to dryness by distilling on a water-bath ; the residue is taken up with a few cubic centimeters of distilled water, and treated as follows : 3 drops of a saturated aqueous solu- tion of ferric chloride are mixed with an equal number of drops of a concentrated solution of pure carbolic acid, and diluted with water until a light-amethyst color is obtained. To this solution a portion of the ethereal extract is added, when in the presence of lactic acid a lemon or canary color develops. The delicacy of the test is such that the presence of 0.1 per cent, of lactic acid can be demonstrated. Kelling's Test. — Five or 10 c.c. of the filtered stomach-contents are diluted from ten to twenty times with water and treated with 1 or 2 drops of a 5 per cent, aqueous solution of ferric chloride. In the presence of lactic acid a distinct greenish-yellow color is obtained when the tube is held to the light. Boas' Test. — This test is more accurate than the two just de- scribed, but more time-consuming and complicated. It is based upon the decomposition of lactic acid into formic acid and acetic aldehyde, and the demonstration of the presence of the latter. To this end, from 10 to 20 c.c. of the filtered stomach-contents are- treated with a slight excess of barium carbonate, and evaporated on a water-bath. The resulting syrup is acidified with a few drops THE GASTRIC JUICE. 123 of phosphoric acid, and freed from carbon dioxide by momentary ebullition. On cooling, it is extracted with 100 c.c. of ether by shaking for about thirty minutes. The ethereal extract is poured off, the ether distilled, and the residue taken up with 45 c.c. of water. After filtering, the solution is then treated with 5 c.c. of concentrated sulphuric acid and a pinch of manganese dioxide, and carefully heated to boiling. Should lactic acid be present, this is now decomposed, and acetic aldehyde liberated, which ran be demon- strated by passing the vapor into a test-tube containing Nessler's reagent or an alkaline solution of iodopotassic iodide. In the first instance, yellowish-red aldehyde of mercury is formed, while iodoform results in the latter, and can be readily recognized from its odor, which becomes marked when the solution is heated. Tests for Acetic Acid and Butyric Acid. — These acids can usually be recognized by their odor. Chemically they can be demonstrated as follows : Test for Acetic Acid. — Ten c.c. of the filtered stomach-contents are extracted with ether as above. The ether is distilled off, the residue taken up with a few drops of water and accurately neutral- ized with sodium hydrate. To this solution a drop or two of a very dilute solution of ferric chloride is added, when in the presence of acetic acid a dark-red color develops. With nitrate of silver, on the other hand, a precipitate is obtained which is soluble in hot water. Test for Butyric Acid. — The ethereal extract of 10 c.c. of the stomach-contents is freed from ether by distillation, the residue is dissolved in a few cubic centimeters of water, and treated with a trace of calcium chloride in substance. In the presence of butyric acid small oil droplets separate out, the nature of which is readily recog- nized from the pungent odor. If, in the place of calcium chloride, a slight excess of baryta-water is used, highly refractive rhombic platelets or granular, wart-like masses are obtained on evaporation, which consist of barium butyrate. Butyric acid can also be recognized by the peculiar odor of pine- apple which develops when the dry residue of the ethereal solution is treatfd with a little sulphuric acid and alcohol. The reaction is due to the formation of butyl ethylate, C,H702.C2H5. Quantitative Estimation of Lactic Acid. — This is best accom- plished by means of Boat? method: The decomposition of the lactic acid is effected as described above. After the addition of the sul- phuric add and manganese dioxide the flask is closed with a doubly perforated stopper. Through one aperture a bent lube passes to the condenser, while a Btraighl tube passes through the other opening, and i- provided at its i'v<-<- end with a small piece of rubber tubing that is clamped ; thi~ tube should dip well into the liquid, and serves for passing a current of air through the solution when the distilla- tion i- completed. The mixture is then distilled until about four- lif'th- of the contents have passed over, excessive heat being carefully .avoided, -(, :i- to prevent decomposition of the aldehyde-. The dis- 124 THE DIGESTIVE FLUIDS. tillate, which is received in a high Erlenmeyer flask, is heated with 20 c.c. of a Jq- normal solution of iodine and the same amount of a 5.6 per cent, solution of potassium hydrate. The mixture is thor- oughly shaken and set aside for a few minutes. The excess of iodine is then estimated after adding 20 c.c. of hydrochloric acid (specific gravity 1.018), by titrating with a -^ normal solution of sodium thiosulphate. The titration is carried almost to the point of decolorization, when a little starch solution is added, and the titra- tion continued until the last trace of blue has disappeared. The dif- ference between the number of cubic centimeters of the thiosulphate solution employed to bring about this end and the amount of the iodine solution added, viz., 20, will indicate the number of cubic centimeters of the latter which were utilized in the formation of iodoform. By multiplying this number by 0.003388 the corresponding amount of lactic acid is ascertained. Quantitative Estimation of the Organic Acids. — The organic acids in toto may be estimated by one of the methods already described (pages 120 and 121), or according to the following procedure, as suggested by Hehner-Seemann. This method is based upon the transformation of the organic acids into their alkaline salts, and their subsequent combustion, with the formation of the correspond- ing carbonates, which are then estimated by titration. At the same time the amount of physiologically active hydrochloric acid can be obtained as follows : 10 c.c. of the filtered gastric contents are neu- tralized with a decinormal solution of sodium hydrate, and the total acidity thus ascertained. The neutralized solution is then evaporated to dryness and the residue incinerated. Care should be had, however, that the application of heat is discontinued as soon as the ash has ceased to burn with a luminous flame. The residue is then taken up with a small amount of water and titrated with a decinormal solution of hydrochloric acid. The number of cubic centimeters employed to bring about the end-reaction, multiplied by 0.00365, will indicate in terms of hydrochloric acid the amount of organic acids which were originally present. By deducting this value from the total acidity, as first ascertained, the amount of physiologically active hydro- chloric acid is found. The Ferments of the Gastric Juice and their Proenzymes. In the gastric juice of almost all vertebrate animals two fer- ments are constantly found. These are termed pepsin and chy- mosin, or rennin, and are supposedly furnished by the so-called adelomorphous or central cells of the gastric glands. This has been established by resecting the pyloric end of the stomach and convert- ing it into a blind pouch, with a fistulous opening on the anterior abdominal walls, while the fundus was united to the duodenum. It was then noted that this resected portion of the stomach, in which no delomorphous cells are found, furnished an alkaline and markedly THE GASTRIC JUICE. 125 viscid secretion, which contained pepsin and large amounts of mucin, but no hydrochloric acid. A reversal of the experiment, on the other hand, in which the fundus was thus isolated, showed that here both pepsin and hydrochloric acid are secreted. As this portion of the stomach contains both delomorphous and adelo- morphous cells, the conclusion naturally suggests itself that the hydrochloric acid is furnished only by the delomorphous cells, while the pepsin— and the same apparently holds good for chy- niosin — is secreted by the adelomorphous cells. The latter are hence also spoken of as pepsin calls, while the delomorphous cells are similarly termed the oxyntic cells of the stomach. As in the case of the ptyalin of the saliva, however, it appears that the ferments in question do not exist in the cells as such, but in the form of proenzymes or zymogens, which are termed propepsin or pepsinogen and prorennin or chymosinogen, respectively. It has thus been shown that an aqueous extract of the gastric mucosa when treated with 1 per cent, of soda, and- kept at a tem- perature of 40° C, even for several hours, does not lose its digestive power, and can be rendered physiologically active by subsequently acidifving with hydrochloric acid to from 0.2 to 0.3 per cent,, pro- viding that the animal has previously fasted. If then, however, such artificial gastric juice is neutralized, and then alkalinized with soda to the extent of only 0.5 per cent., the solution is rendered entirely inactive after a few seconds, when warmed to the tempera- ture of the body, and it is to be noted that the subsequent addition of hydrochloric acid is now no longer capable of restoring ' the activity of the enzyme. This demonstrates, of course, that while the proenzyme is more or less resistant to soda, the ferment is thereby rapidly destroyed. On the other hand, it appears that pepsin is more resistant to the influence of carbonic acid than propepsin. Between chymosin and its zymogen similar relations exist. Of the chemical nature of the proenzymes and the manner in which they arc produced by the cells, practically nothing is known. Nerve-influences, no doubt, are here at work, as in the case of the salivary glands. At the same time the blood-supply is of moment, and we' find that during the process of digestion the blood-vessels are dilated, and that the venous circulation is more rapid and the blood of a light-red color. But as in the salivary glands, it is certain that the height of the blood-pressure has only indirectly to do with the activitv of the glands. The proenzymes here, as there, are formed through a specific activity on the part of the cells, from food- material which is supplied by the lymph. Whether «.r not the transformation of the proenzymes into the corresponding ferments occurs in the bodiesof the cells has not been definitely decided. It appears, however, that in the majority of animal-' which have been examined in this direction the glands secrete only the proenzymes, and that these are then rendered physiologically active by the hydrochloric acid of the gastric juice. 126 THE DIGESTIVE FLUIDS. A solution of propepsin, which may be obtained by macerating in glycerin the mucous membrane of a fasting animal, is thus in itself inert, but is rendered active at once when hydrochloric acid is added to the extent of from 0.1 to 0.2 per cent. It is indeed supposed that pepsin, which in itself is inactive like its zymogen, combines with hydrochloric acid, which alone is similarly inert, as regards its digestive ability, to form a compound acid, the so-called pepsin- hydrochlorie acid. On coming in contact with albuminous mate- rial this is supposedly decomposed, with the formation of nascent hydrochloric acid, which then acts as the active digestive principle, while the liberated pepsin combines with a new portion of hydro- chloric acid, and thus serves as an acid-carrier. On this question, however, a uniformity of opinion does not exist ; still, the hypoth- esis is an attractive one, and has a good deal in its favor. If we thus regard the action of a ferment as essentially influencing the rapidity of reaction, the action of the weak hydrochloric acid of the gastric juice could be compared to the effect of stronger solutions upon albumins under the application of heat. Pepsin. — In pure form pepsin has thus far not been obtained. In the form we are able to isolate the substance, it occurs as an amor- phous white or yellowish-white powder which is not hygroscopic. It is soluble in water, dilute acids, and glycerin. When acidified with hydrochloric acid to the extent of from 0.1 to 0.3 per cent., it is capable of dissolving albumins, with the formation of albumoses and so-called amphopeptone (see below). This can readily be demonstrated as follows : an artificial gastric juice is prepared by dissolving a pinch of one of the commercial preparations of pepsin in dilute hydrochloric acid (0.1-0.2 per cent.), to which a flake of boiled beef-fibrin is then added. The mixture is kept at a temperature of about 40° C, when it will be noted that after a short time the fibrin begins to swell and is subsequently dissolved. In the solution which thus results albumoses and peptones can be demon- strated (see page 183). Other acids, such as sulphuric acid, nitric acid, phosphoric acid, lactic acid, and even acetic acid, are also cap- able of rendering pepsin physiologically active, but much larger amounts of these are necessary to bring about the same result. In the case of phosphoric acid, for example, an acidity of 10-12 pro mille is necessary. Carbonic acid and hydrocyanic acid, on the other hand, are without effect. Unlike chymosin, pepsin does not bring about coagulation of casein. In neutral and alkaline solutions pepsin is inactive, and, as has been seen, it is rapidly destroyed by sodium carbonate, even in very small amount. Its resistance to higher temperatures is to a great extent dependent upon the reaction of its solutions. In neutral solution it is destroyed at 55° C. ; in the presence of 0.2 per cent, of hydrochloric acid this result is reached only at 65° C, and in the presence of peptones and certain salts a temperature of 70° C is necessary to bring about the same end. In the dry state, on the THE GASTRIC JUICE. 127 other hand, the ferment may be heated to 100° C, and even higher, without being destroyed. At temperatures lower than 40° C. pepsin is still active, but less energetically so, and at 0° C. its action ceases altogether. Alcohol precipitates pepsin from its solutions without affecting its subsequent activity, unless the exposure has been prolonged. Some of the salts of the heavy metals, such as the acetates of lead and platinum chloride, as also tannic acid, magnesium carbonate, and ammonium sulphate, likewise cause the precipitation of impure forms, at least, but are without effect upon the ferment itself. Like the albumins, pepsin docs not diffuse through animal membranes. To a certain extent the rapidity of digestion is dependent upon the amount of pepsin that is available ; but, as in the case of all ferments, very small quantities are sufficient to effect an amount of chemical change that is apparently out of all proportion to the amount present. Thus, Petit claims that a pepsin preparation, which he prepared himself, was capable of dissolving 500,000 times its weight of fibrin in seven hours. The much more impure commericial forms are, of course, far less active, but many of them possess remarkable digestive power. The ability on the part of pepsin to digest albumins is, however, limited ; and with an increase in the amount of digestive products formed, its activity gradually diminishes and finally ceases. This can be obviated in a measure by removing these products as they are formed, and may be artificially accomplished by allowing the digestion to take place in a parchment tube which has been sus- pended in dilute hydrochloric acid. The peptones which are formed then pass from the tube by dialysis, and in this manner digestion can be carried much further than under other conditions. Com- plete digestion, however, may even then not be achieved, which is probably to be explained by the reversible action of the fer- ment, as already described (page 111). Little is known of the chemical nature of pepsin. At first sight, it is apparently related to the albumins ; but it is to be noted that the reactions of the purer forms become further and further re- moved from those of the albumins as the degree of purity increases. An analysis of a fairly pure specimen has given the following result: C, 47.75; H, 6.5; N, 14.24; (), 30.20j 8, 1.31 per cent. < Specific teste for the demonstration of the pepsin of the gastric juice, as compared with other proteolytic ferments which similarly act in acid solution-, are unknown. As a result, all such ferments have been designated as pepsin, although it is very likely that they are not identical. Such ferments have been observed in the secre- tion of the glands of Brunner, in the muscles, the kidneys, the brain, tin; saliva, ami the urine. Isolation of Pepsin. — If it i- merely desired to obtain an effective solution of pep-in without regard to the purity of the substance, the following procedure may be employed : 128 THE DIGESTIVE FLUIDS. v. Wittich's Method. — The mucous membrane of a pig's stomach is carefully dissected off, freed from mucus by washing with water, hashed, rubbed together with pure quartz sand, and finally treated with glycerin, containing 0.1 per cent, of hydrochloric acid, in the proportion of 10-20 grammes for 1 part of mucous membrane. The mixture is kept at a temperature of 40° C for from one to two weeks, when it is filtered. The extract which is thus obtained may then be used for experimental purposes by diluting with 0.1-0.4 per cent, of hydrochloric acid, in the propor- tion of 2-3 : 100. To obtain as pure a preparation as possible, Briicke's method, or one of its many modifications, is usually employed. But it is to be noted, as Gautier has pointed out, that while the substance is thus obtained in large amount, it is but little active. We find, as a matter of fact, that a small flake of fibrin, which has been previously caused to swell by placing it in dilute hydrochloric acid, dissolves in a solution of Briicke's pepsin only after five minutes, while other and more impure preparations are decidedly more effective. Briicke's method is as follows : Brucke's Method. — The mucous membrane, which has been carefully dissected off and washed with water, is placed in a solu- tion of dilute phosphoric acid (12 pro mille) and allowed to stand for about one week. By this time digestion has usually proceeded to a point where a precipitate is no longer obtained on render- ing the mixture nearly neutral with sodium hydrate. It is then neutralized with lime-water, which causes a precipitation of cal- cium phosphate, while the pepsin is at the same time carried down mechanically, and adheres so firmly to the phosphates that these can subsequently be washed with water without losing any of the pepsin. The precipitate is then dissolved in dilute hydro- chloric acid, and the solution dialyzed until the phosphates and the hydrochloric acid have diffused out. The remaining solution, which contains the pepsin, is now treated with large quantities of strong alcohol. In this manner the ferment is precipitated, and is readily collected on a filter. Other ferments, such as ptyalin, do not adhere to the phosphates so firmly as pepsin, and can hence be removed by suspending the precipitate in water and passing a current of air through, the solu- tion for some time. Instead of dialyzing the acid solution, as just described, and then precipitating with alcohol, it is also possible to obtain the ferment by treating such acid solutions with a concentrated alco- holic-ethereal solution of cholesterin. When this is added the cholesterin is immediately thrown down, and carries the pepsin with it. If this precipitate is then collected on a filter and washed with alcohol, the cholesterin is dissolved, while the ferment remains. Such preparations, however, are impure, and probably contain THE GASTRIC JUICE. 129 mucin-peptones, which have resulted during the digestion of the mucous membrane. Purer forms may possibly be obtained according to Kuhxe's method. To this end, pigs' stomachs are placed in large quantities of dilute hydrochloric acid, and are allowed to digest for several week-. As soon as albumoses are only present in comparatively small amounts, owing to their transformation into peptones, the solution is saturated with ammonium sulphate. In this manner the remaining albumoses, and with them the pepsin, are precipitated. This mass is then further treated with hydrochloric acid and allowed to stand, when a further portion of the albumoses is transformed into peptones. By repeating this process all the albumoses are finally peptonized, and ultimately a nearly pure pepsin is thrown down by saturating with the salt. This is dissolved in water, dialyzed, and finally precipitated with strong alcohol, and rapidly collected on a filter. Instead of using the stomachs of animals, some of the more active commercial products may be directly employed and purified, as just described. Such preparations give scarcely any of the characteristic color- reactions of the albumins, and are not precipitated by tannic acid. Quantitative Estimation of Pepsin. — Accurate methods for the quantitative estimation of pepsin are not available. Relative values, however, can be obtained by the following method, as suggested by Hammerschlag : three Esbach tubes (albuminimeters) are em- ployed. Tube 1 is filled to the mark U with a mixture of 10 c.c. of a 1 per cent, solution of serum-albumin in 0.4 per cent, of hydrochloric acid and 5 c.c. of filtered gastric juice. Tube 2, which is the standard, is filled to the same mark with the serum- albumin solution, but receives in addition 0.5 gramme of pepsin. Tube :>> contain- merely 10 c.c. of the albumin solution and 5 c.c. of di -til led water. Esbach' 8 reagent, which consists of 10 grammes of picric acid and 20 grammes of citric acid, dissolved in 1000 c.c. of distilled water, is then added to each tube to the mark K. A tier twenty-four hours the amount of precipitate is read off and the difference between tubes 1 and 3 compared witli that of tube 2. Pepsinogen. — The presence <»f pepsinogen in the gastric juice '•an be ascertained only when hydrochloric acid is absent, as it is otherwise transformed into the active enzyme. Its occurrence, as such, i- hence essentially a pathologic phenomenon, and indicates the absence of free hydrochloric acid. Hut while the latter may be absent in many diseases which are not associated with structural abnormalities of the gastric mucous membrane, pepsinogen, and con- sequently also pepsin, are found lacking only in disease of the stomach it-elf mid when complete atrophy of the glandular struct- ures ha- occurred. Test for Pepsinogen. — Specimens of gastric juice in which pepsin- 130 THE DIGESTIVE FLUIDS. ogen only is present are incapable of digesting albumins. In such cases, as I have just said, hydrochloric acid is absent. If then the solution is acidified to the extent of from 0.1 to 0.3 per cent., and the dissolution of a flake of boiled beef-fibrin now occurs, the presence of the zymogen may be inferred. Quantitative Estimation of Pepsinogen. — The determination of the absolute quantity of pepsinogen in the gastric juice, as that of pep- sin, is not possible. Relative values, however, may be obtained by the following method, as suggested by Boas : To this end, specimens of the gastric juice are variously diluted with distilled water in the proportion of 1 : 5, 1 : 10, 1 : 20, etc. A known quantity of coagu- lated egg-albumin or serum-albumin is then added to each tube, as also 1 or 2 drops of a 0.3 per cent, solution of hydrochloric acid, for every 10 c.c. The tubes are kept at a temperature of about 40° C, when the degree of dilution is noted at which the albumin is still dissolved. The greater the degree of dilution at which this occurs, of course the greater is the amount of pepsinogen — that is, of pepsin — present. Should it be desired to exclude definitely the presence of pepsin and pepsinogen in the stomach, 200 c.c. of a decinormal solution of hydrochloric acid are introduced into the organ through a tube, and the remaining liquid removed after one-half hour. If this fluid then contains no pepsin, the absence of the zymogen may also be inferred. Chymosin. — Chymosin, or rennin, like pepsin, is secreted by the central cells of the gastric glands in the form of a pro-enzyme, which, like the pepsinogen, is then transformed into the corresponding fer- ment by the hydrochloric acid of the gastric juice. It is to be noted, however, that calcium chloride or any other soluble calcium salt is likewise capable of bringing about this result. The specific action of chymosin is exerted upon milk or lime-containing solutions of casein, which are coagulated in neutral and even feebly alkaline solutions. Unlike pepsin, chymosin is not a proteolytic ferment, and its action ceases with the formation of paracasein. It is there- fore surprising to note that chymosin is found not only in the stomachs of mammals, but also in other vertebrate animals, and even in certain plants, where casein as a food-stuff certainly does not enter into consideration. Our knowledge of ferments in general, how- ever, is as yet very defective, and, as a matter of fact, we are acquainted only with the more manifest reactions of these bodies, while it is quite possible that they possess other important properties of which we are now in ignorance. It is conceivable, moreover, that differ- ent varieties of chymosin exist, which, as a class, are all capable of coagulating casein, but wrhich differ from each other in other respects and serve other purposes. While chymosin is also active in feebly acid solution, it is gradu- ally destroyed at a temperature of from 37° to 40° C. when exposed to the action of gastric juice containing 0.3 per cent, of hydrochloric THE GASTRIC JUICE. 131 acid. The ferment is here apparently digested by the pepsin, and it is thus easily possible to obtain solutions of pepsin which are alto- gether free from chymosin. In neutral solution it is more resistant, and can be heated to a temperature of 50° C. ; at 70° C, however, it becomes permanently inactive. In its dry state, on the other hand, it can be heated to 110° C. without losing its activity. Alka- lies when present beyond traces destroy the substance, as they do pepsin. Like all other ferments, it is capable of effecting an exten- sive reaction, even when present in small amount. The quantity of ferment contained in 1 gramme of the dried and pulverized mucous membrane of the fourth stomach of the calf, when dissolved in water, is thus capable of coagulating 200 liters of milk in one minute at a temperature of 50° C Of the chemical nature of chymosin, nothing is known ; but, as in the case of pepsin, the purer preparations do not give the usual reactions of albumins. It is precipitated from its neutral solutions by subacetate of lead, while the acetate and tannic acid are without effect. Alcohol likewise precipitates the ferment and gradually renders it inactive. Like pepsin, it is not dialyzable. Under normal conditions chymosin is always present in the gastric juice of man. In certain diseases of the stomach, however, which are associated with the death of its glandular elements, the ferment, as also its zymogen, is lacking. Tests for Chymosin and Chymosinogen. — To test for the presence of chymosin, 5 or 10 c.c. of milk are treated with a few drops of the filtered gastric juice and kept at a temperature of from 37° to 40° C. If coagulation occurs within ten or fifteen minutes, the presence of chymosin may be assumed. Should the gastric juice, however, be markedly acid, it is necessary first to neutralize it with sodium hydrate. To test for chymosinogen, the milk is treated with 2-3 c.c. of a 1 per cent, solution of calcium chloride, and 10 c.c. of filtered gastric juice which has been rendered feebly alkaline with sodium hydrate. The mixture is kept at a temperature of from 37° to 40° (\, when in the presence of the zymogen a thick cake of casein is formed within a few minutes. Isolation of Chymosin. — To isolate chymosin in comparatively pure form, the following method, as suggested by Ilammarsten, may be employed : The mucous membrane of the fourth stomach of the calf is carefully dissected off, washed with water, and extracted with an o.l percent, solution "I' hydrochloric acid, as already described. The infusion is then neutralized mid repeatedly shaken with powdered magnesium carbonate until the pepsin has been removed. The filtrate is treated with subacetate of lead, the precipitate decomposed with very dilute sulphuric acid, and the acid filtrate further precipitated with an aqueous solution of stearin soap. The fermenl i- thus thrown down together with the fatty acids, from which it i- then separated by suspending the precipitate in w;it< r 132 THE DIGESTIVE FLUIDS. and extracting the fatty acids with ether. The chymosin remains in aqueous solution, and may now be precipitated with strong alcohol. It is then rapidly collected on a filter and dried. Quantitative Estimation of Chymosin and Chymosinogen. — As in the case of pepsin and pepsinogen, relative values only can be obtained. The gastric juice is neutralized with a very dilute solu- tion of sodium hydrate. Tubes are then prepared, containing 5 or 10 c.c. of the gastric juice, variously diluted in the proportion of 1 : 10, 1 : 20, 1 : 30, etc., to which an equal volume of neutral or amphoteric milk is further added. These tubes are kept at a temperature of from 37° to 40° C, when the degree of dilution is noted at which coagulation still occurs. Under normal conditions a positive reaction can thus be obtained in man with a degree of dilution varying between 1 : 30 and 1 : 40. In the case of the zymogen, the gastric juice is rendered feebly alkaline, when tubes are prepared as just described. Normally a positive reaction can thus still be obtained with a dilution varying between 1 : 100 and 1 : 150. Other Constituents of the Gastric Juice. — Of other con- stituents, the gastric juice normally contains traces of sulpho- cyanides, which are secreted by the stomach itself; a variable amount of mucin ; a small amount of coagulable albumin, or, if the fluid has stood for some time, a corresponding quantity of albumoses or peptones, and, as already shown, certain mineral salts. The gases which are found in the stomach have in part been swallowed with the food. A small portion is further referable to eructations from the duodenum, while a third portion is probably secreted by the stomach itself. This is true more especially of the carbon dioxide, and Schierbeck has shown that the tension of this gas gradually increases from 30 to 40 Hgmm. while fasting, to 130 to 140 Hgmm. during the process of digestion, and is apparently directly proportionate to the acidity of the gastric juice. An idea of the relative amounts of the gases which are normally found in the stomach may be formed from the accompanying table, which is taken from Planer : Man. Dog. Vegetable diet. Veg. diet. Meat diet, vol. per cent. vol. per cent. vol. per cent. Carbon dioxide 20.79-33.83 32.9 25.2 Oxygen 0.37 . . 0.8 6.1 Nitrogen 72.50-33.22 66.3 68.7 Hydrogen 6.71-27.58 Other gases, such as marsh gas, olefiant gas, ammonia, and hy- drogen sulphide, are found only under pathologic conditions, and are referable to certain fermentative and putrefactive changes which take place in the ingested food. THE PANCREATIC JUICE. 133 THE PANCREATIC JUICE. As has been pointed out, the digestive glands which have so far been considered are not essential to the maintenance of life. The salivary glands and the stomach, moreover, can in certain animals be eliminated without seriously interfering with the process of digestion, and the ferments which in man are secreted by these structures are in many animals absent, The pancreas, on the other hand, either as such or as a so-called hepatopancreas, is found in all vertebrate and invertebrate animals in which the process of diges- tion is carried on in a well-defined digestive tube. In many, indeed, it represents the only digestive gland of the body. Its removal, even in the higher animals, invariably leads to death. In dogs, in which this operation has been repeatedly performed, and in which life may go on for a few weeks thereafter, it has been observed that as a result of such interference the resorption of fats is seriously impeded, so that practically all that has been ingested reappears in the feces. In the case of the albumins, it is similarly found that but 44 per cent, is absorbed, and of the ingested starches from 20 to 40 per cent, is eliminated as such. Analogous results are obtained in the human being where atrophy of the pancreas is at times observed. As a consequence, rapid emaciation occurs, and, as has been stated, death ultimately results. It appears, however, that the fatal issue in these cases is not exclusively referable to impaired nutrition as a result of defective absorption. It is, indeed, possible to counteract this effect by administering a sufficient amount of raw pancreas together with the food, whereby the resorption of both fats and albumins is greatly improved. Death, however, takes place never- theless. It is thus apparent that besides its digestive function the pancreas must play an additional and important rdle in the metab- olism of the animal body. We find, as a matter of fact, that fol- lowing the extirpation of the pancreas in dogs a severe form of dia- betes rapidly develops, and is accompanied by the appearance of acetone, diacetic acid, and at times of ,9-oxvbutyric acid in the urine. That this is not due to suspension of the pancreatic digestion can be proved in various ways. If the animal thus receives an adequate amount of raw pancreas together with its food, the absorption of albumins and fats is, as just stated, greatly increased, while the diabetes persists. It has been further noted thai ligation of the secretory dud does not lead to the appearance of sugar in the urine, and that the diabetes continues after extirpation even when no food i- consumed for several days. The conclusion hence suggests itself ihat the pancreas, like the thyroid, the adrenal body, and other glands, probably furnishes an internal secretion also, which in some manner, as yet unknown, controls the metabolism of glucose within the animal body. Arthaud and Butte, it is true, claim that diabetes doe- n,,i follow ligation of the pancreatic veinsj bul it can readily 134 THE DIGESTIVE FLUIDS. be imagined that in such cases, and perhaps even under normal conditions the internal secretion of the gland is removed through the lymph-channels. It has been shown, moreover, that diabetes does not occur after extirpation of the pancreas if a piece of the gland has been previously transplanted under the skin. Of the nature of the substance or substances which are thus secreted by the pancreas, and in the presence of which the carbo- hydrate metabolism continues in a normal manner, Ave know nothing. According to Lepine and his school, the gland is supposed to furnish a glucolytic ferment, which brings about oxidation of the sugar in the tissues, and it will be seen that such a ferment can actually be demon- strated in the blood. The time has not come, however, when we can speak definitely on this subject. / The secretion of the pancreatic digestive fluid, like that of the saliva, is partly under the control of cerebrospinal nerve-fibres, which are derived from the vagus, and partly of sympathetic fibres. The material from which the secretion is elaborated through the specific activity of the glandular cells is obtained from the lymph, and ulti- mately, of course, from the blood. In carnivorous animals, in which the secretion of the pancreas is intermittent and dependent upon the ingestion of food, we accordingly find that in its stage of activity the gland assumes a bright rose-color, and is much increased in size, while in the resting stage it is pale and shrunken. General Properties. — Fresh pancreatic juice can be obtained only by establishing an artificial, fistula in the pancreatic duct. But as the least interference with the integrity of the gland leads at once to the secretion of an abnormal fluid, great care must be exercised to operate as gently and rapidly as possible. It is best to do so about three hours after the animal has received a large meal. For a short while at least, a normal secretion can then be obtained. This represents a clear, thick, colorless and odorless, very concentrated fluid, of a strongly alkaline reaction, which actively digests albumins, inverts starches and the more complex sugars, and emulsifies fats. After a variable length of time, however, the secretion becomes thinner, more deficient in solids, and otherwise altered, so that it can scarcely be regarded as normal. Specific Gravity. — The specific gravity of the pancreatic juice varies between 1.008 and 1.010, corresponding in man to the pres- ence of from 2.5 to 7 per cent, of solids. When kept for a few hours at ordinary temperatures it loses its viscosity and transparency, and rapidly undergoes putrefaction. Crystals are then deposited which consist of leucin and tyrosin ; they result from the digestion and subsequent decomposition of contained albumins. To prevent these changes, the secretion must be placed on ice at once, or treated with chloroform- water or a similar antiseptic solution. Owing to the presence of the large amounts of albumin Avhich the pancreatic juice contains, the liquid coagulates to a dense mass when heated to a temperature of 74° C. On cooling to 0° C, or when dropped into THE PANCREATIC JUICE. 135 water, a clot is formed, which redissolves on warming the solution or on adding an excess of sodium chloride. Amount. — The amount of pancreatic juice which is secreted in the twenty-four hours is variable, and largely dependent upon the quantity and quality of the food ingested. From permanent fistula? 45-100 grammes are supposedly secreted pro kilogramme of the animal. This figure, however, is no doubt too high, and Bidder and Schmidt have estimated that under normal conditions the secretion probably does not exceed 5 grammes pro kilo. In a recent case of pancreatic fistula, observed in man, Pfaff re- ports that ()00 c.c. of the secretion were obtained in twenty-four hours. This figure thus more nearly corresponds to the results reached by Bidder and Schmidt. Chemical Composition. — A general idea of the chemical composi- tion of the pancreatic juice of the dog may be formed from the accompanying analyses, which are taken from C. Schmidt and Kriiger. In these the essential points of difference which exist between the normal fluid, as compared with the secretion obtained from permanent fistuhe, are well shown. I have further added two analyses of human pancreatic juice which were made by Herter and Zawadsky. In Herter's case an accumulation of the secretion had taken place in the duct, owing to a pressure-stenosis, which was pro- duced by a carcinomatous growth ; while Zawadsky's material was obtained from a pancreatic fistula which had remained after removal of a pancreatic tumor. Of the two secretions, the first is manifestly abnormal (see below), while the analytical results in the second case conform closely with the figures which were obtained by Schmidt in what may be regarded as the normal pancreatic juice of the dog. I have finally appended an analysis of the contents of a pancreatic cyst, which in its general composition resembles the secretion ob- tained from permanent fistuke in animals. Trypsin, however, was absent. Secretion from a tem- Secretion from a per- porary fistula of the manent fistula of the dog. dog. (C Schmidt.) (Kruger.) Water 900.8 980.11 Solids 99.2 19. CO Albumins, peptones, and } ro ., q ,o ferments. ( Organic matter, comprising "J lenein, tyrosin, zantbins, [ 30.4 3.3 goaps, and fats. J Mineral constituents 8.8 3.57 Sodium chloride 7.35 0.93 Potassium chloride .... 0.02 0.07 Phosphate of calcium . . . 0.41 0.01 Phosphate of magnesium . . 0.12 0.02 Lime and magnesia .... 0.32 2.63 136 THE DIGESTIVE FLUIDS. Human. Human. (Herter's case.) (Zawadsky's case.) Water 975.9 864.05 Solids 24.2 135.95 Peptones and enzymes ) .. .. _ (no albumin.) / • • • ■ li-° 92.05 Alcoholic extract 6.4 43.90 Mineral ash 6.2 3.44 Analysis of the Contents of a Pancreatic Cyst (Lenarcic), Water 928.1 Solids 17.9 Organic matter 10.05 Mineral ash 7.85 The Ferments and their Zymogens. The most important constituents of the pancreatic juice are the ferments, of which five different forms are said to be present. These are trypsin, steapsin, an amylolytic ferment (which is said to be identical with the salivary ptyalin), maltase, and chymosin. Like the ferments which are furnished by the salivary glands and the central cells of the gastric glands, these enzymes occur also in the pancreatic cells in the form of zymogens, which are subsequently transformed into the active ferments. If a fresh pancreas is thus extracted with glycerin, it will be noted that the resulting extract has no proteolytic properties whatever, while an extract obtained after the gland has been hashed and exposed to the air for some time, or an aqueous extract of the fresh gland, digests albumins with ease. If the fresh gland, further, is hashed and briefly treated with a 1 per cent, solution of acetic acid, and then extracted with glycerin, an active preparation is obtained at once. Similar results are reached in the case of the pancreatic ptyalin. If a fresh pancreas is thus repeatedly extracted with glycerin until ptyalin no longer passes into solution, and the gland is then washed in water and left exposed to the air for a short time, an additional amount of the ferment can be obtained by further extraction with the same reagent. In what manner the transformation of trypsinogen, ptyalinogen, steapsinogen, and the other zymogens, into the corresponding fer- ments is normally effected, is unknown. But, as has been seen, this can be brought about artificially through the influence of water, dilute acetic acid, and probably also through the activity of acid- forming bacteria or the oxygen of the air. Of the chemical composition of the zymogens we know little, but it appears that they are of an albuminous nature and of a more complex composition than the ferments themselves. On decomposi- tion trypsinogen thus yields the corresponding ferment and an unknown albuminous substance. Of the origin of the zymogens, and of the manner in which they are produced in the cells, we know nothing. Trypsin. — Trypsin is the most important proteolytic ferment THE PANCREATIC JUICE. 137 which is found in the animal world, and in its action on albumins is fully capable of replacing pepsin when this is absent. Its digestive power, moreover, is much more extensive than that of pepsin, and it is further capable of decomposing albumins to amido-acids and hexon bases. Its hydrolytic effect may thus be compared to the action of strong mineral acids under the application of heat. ^This explains also the fact that leucin and tyrosin are so frequently found in the pancreatic juice. The extensive digestive activity of trypsin is well shown when the gland is finely hashed and treated with a large amount of chloroform- water, so as to guard against putrefac- tive changes. When kept at a temperature of 40° C. autodigestion rapidly takes place, and after several days it will be noted that while trypsin is -till present in its full activity, the other ferments have disappeared. Together with the various albumins of the gland they have apparently been digested by the more powerful ferment. While trypsin acts most energetically in feebly alkaline or neutral solutions (0.25-1.0 per cent, of sodium carbonate), it is also capable of digesting albumins in slightly acid media, providing that the acidity is not due to the presence of a free mineral acid; the diges- tive process is under such conditions, however, much less active. Free mineral acids rapidly destroy the ferment. Its optimum tem- perature lies between 37b and 40° C. In neutral solution it is destroyed at 45° C, while in feebly alkaline media, and especially in the" presence of albumoses and certain ammoniacal salts, it can be heated somewhat higher without impairment of its digestive power. A- in the case of pepsin, the digestive effect of trypsin is to a certain extent dependent upon the amount of the ferment present, and here as there the action of the enzyme ultimately ceases when the digestive products accumulate beyond a certain amount. Impure extracts can be prepared, as lias been pointed out, by extracting the gland with glycerin after it has been left exposed to the air. The purer forms, on the other hand, which can be obtained according to Iviilme's method (see below), are insoluble in glycerin and alcohol, but soluble in water. Of the chemical nature of trypsin little is known. In acid solu- tion it is coagulated by heat, and, according to Kiihne, decomposed into an albumin and peptone. An analysis of a fairly pure prepara- tion has given the following results: carbon, 02.75 percent.; hydro- gen, 7.51; nitrogen, 16.55 ; oxygen pins sulphur, 2:5.19 (Loew). Its composition is thus very similar to that of the peptones, and it is hence possible that, unlike the other digestive ferments which we have thus far considered, trypsin may be an albuminous substance. Test for Trypsin. — The tesl for trypsin resolves itself into the demonstration of the presence of a proteolytic ferment which is capable of digesting albumins in alkaline solution, with the ultimate formation of amido-acids. To this end, the solution in question is rendered alkaline with sodium carbonate to the extent of from 0.25 ,,, i.i, per cent. A -mall flake of fibrin is then added and an 138 THE DIGESTIVE FLUIDS. amount of thymol sufficient to saturate the solution, so as to guard against putrefactive processes. The mixture is kept at a tempera- ture of 40° C. If the fibrin is then dissolved and leucin or tyrosin can be demonstrated in the resulting solution, the presence of trypsin may be inferred. To this end, it is only necessary to evaporate the solution to a thick syrup and to examine this microscopically (see page 188). Should the solution to be tested contain a free mineral acid, or larger amounts of organic acids, no result will, of course, be obtained, as the trypsin has then been destroyed. Isolation of Trypsin. — Unless it is desired to obtain trypsin in as pure a form, as possible, alkaline solutions of the common pancreatin preparations which are sold in the shops can be used for experimental purposes. Otherwise the method of Kiihne, as modified by Gautier, should be employed : The fresh pancreas is finely hashed and washed with ice-cold water, to which 1 pro mille of salicylic acid has been added. After four hours the mass is treated with a large amount of a 5 pro mille solution of sodium carbonate, containing an excess of thymol, and is kept for twelve hours at a temperature of from 37° to 40° C. The acid and alkaline extracts are then mixed, treated with 0.5 per cent, of sodium carbonate, filtered, feebly acidulated with acetic acid, and saturated with ammonium sulphate in substance. The precipitate which then separates out contains all the trypsin. It is dissolved in water, filtered, and dialyzed, so as to remove the salt which is still present, as also traces of peptones and leucin. The resulting solution is concentrated at a very low temperature, and the trypsin finally pre- cipitated with strong alcohol. It is then rapidly filtered off and dried. The amount of material which can thus be obtained from one gland is always very small, but sufficient to digest a large quantity of albumin when dissolved in about 100 c.c. of water. The so-called dry pancreas of Kiihne, from which trypsin can likewise be obtained, and which is also used in digestive experiments as such, is prepared by extracting the fresh gland with alcohol and subsequently with ether until it is free from fats. The remaining material, which contains the active ferment, is then dried and pul- verized, and can be kept in this form indefinitely. The Amylolytic Ferment of the Pancreatic Juice (Amylop- sin). — The amylolytic ferment of the pancreatic juice is by many thought to be identical with the ptyalin of the saliva. It can be isolated, according to Gautier's method, from an aqueous infusion of the fresh gland that has remained exposed to the air for about twenty-four hours, as already described. To demonstrate its action, a few cubic centimeters of such an infusion, or a glycerin extract of the gland, are added to a small amount of starch paste and kept at a temperature of 40° C. The mixture is then tested for the presence of maltose, as already described. Steapsin. — It has long been known that the pancreatic juice pos- sesses the power of emulsifying fats, and of decomposing these into THE PANCREATIC JUICE. 139 glycerin and the corresponding fatty acids. While this phenomenon has by some been referred to the action of bacteria, others hold that it is dependent upon the presence of a specific ferment, which has been termed steapsin. That the latter view is probably the correct one, appears from the fact that the same result is obtained if the perfectly fresh gland is used and care is taken to prevent the action of micro-organisms by adding a small amount of hydro- cyanic acid or of mercuric chloride. Of the nature of the ferment nothing is known, but it is manifestly very unstable, as extracts prepared from glands that have been left exposed^ to the air for twenty-four hours are perfectly inert when brought in contact with neutral fats. Apparently it is digested during this time by the trvpsin. To demonstrate the action of steapsin on fats, a small amount of perfectly fresh pancreas is finely hashed and dehydrated with 90 per cent, alcohol. It is then dried between filter-paper and placed in an ethereal solution of neutral butyrin (Butterfett). On evaporation of the ether the remaining material is kept between two watch-crystals at a temperature of from 37° to 40° C, when after awhile a distinct odor of butyric acid becomes manifest (CI. Bernard). Or, a small amount of neutral fat is treated with a few cubic centim- eters of a feebly alkaline glycerin extract of the fresh gland (9 parte of glycerin and 1 part of al per cent, solution for each gramme of the gland), to which a few drops of tincture of litmus are added. If this mixture is then kept at a temperature of 37° C, it will be noted that the alkalinity gradually decreases, and the reaction finally becomes acid, owing to the liberation of free fatty acids (Hammar- sten). The emulsifying action of the pancreatic juice is owing to the decomposition of the neutral fats and the subsequent saponification of the resulting acids by the alkaline salts which are at the same time present. Maltase. — The presence of maltase in the pancreatic juice can only be inferred indirectly from the fact that small amounts of glucose are invariably formed during the action of the aqueous nr glycerin extract of the gland upon starch. Chymosin. — Chymosin can be demonstrated by adding a small amount of the pancreatic juice of the ox, pig, or sheep to milk, when coagulation results, as hi the case of the chymosin of the gastric juice. The ferment, however, is no! present in all mammals; in dogs, for example, it i- absent. The glucolytic ferment of Lepine has thus far not been isolated either from the pancreatic juice or from the gland itself. I,, conclusion, it is to !»<'■ noted that in some animals, Buch as the rabbit,:: secretion analogous to that of the pancreas is furnished also by the glands of Brunner, which are found in the upper portion <>f the duodenum. In other animals, however, such as the dog, the function of these glands i- to be compared to that of the pyloric glands of the stomach ; and according to Grutzner, extract- of this 140 THE DIGESTIVE FLUIDS. portion of the mucous membrane contain pepsin in considerable amount. Whether or not a diastatic ferment also is here secreted is as yet undecided, as the secretion can scarcely be obtained uncon- taminated by pancreatic juice, and it is hence difficult to make definite statements regarding its composition. THE ENTERIC JUICE. The enteric juice is essentially the secretory product of the glands of Lieberkiihu, which are found imbedded in the mucous membrane of the small intestine, and to some extent also in the large intestine. It may be procured by resecting a loop of the intestine, measuring about 0.15-0.20 meter in length, and closing the proximal end, while the distal end is sutured to the abdominal wall. The mes- entery, however, is allowed to remain, so that the nerve- and blood- supply of the portion which has thus been isolated is interfered with as little as possible. Instead of thus forming a blind pouch, as was first done by Thiry, both ends of the resected loop may also be sutured to the anterior abdominal wall. Such fistulse were first established by Vella, and they accordingly bear his name. The free ends of the divided gut are in either case then united with each other and the abdominal wound closed. In animals which have thus been operated it is noted that after five hours following the ingestion of food a copious secretion of fluid takes place in the small intestine, which continues for about six hours. During this period of activity the mucous membrane presents a rose-red color, while it is pale when at rest. As in the case of the pancreas, the secretion is intermittent. It can be mani- festly excited in a reflex manner, as a moderate secretion may be observed within an hour after the ingestion of food — that is, at a time when but little chyme has passed into the small intestine. Later, when the food has passed the stomach, its presence alone or that of the digestive products which have been formed already, apparently excites the increased secretion which is then observed. This may be further increased artificially by mechanical and espe- cially by electrical stimulation, and it is indeed possible to cause the secretion of enteric juice in this manner even at a time when diges- tion is not going on. In the upper portion of the duodenum of the dog the secretion is said to be small in amount, mucoid, and jelly-like, while further on it becomes more fluid. As obtained, the enteric juice always contains a not inconsiderable amount of mucus, which is derived from the goblet-cells that are found along the entire length of the intestinal canal. The small amorphous flakes which are always found in the secretion consist entirely of mucus. The juice itself, which can be separated from the greater portion of the mucus by filtration, is in the lower por- tion of the small intestine a thin light-yellowish fluid, of a strongly THE BILE. 1-11 alkaline reaction. This is largely due to the presence of consider- able amounts of sodium carbonate, and we accordingly find that on the addition of an acid effervescence of carbon dioxide occurs. Its specific gravity in the dog is fairly constant, and corresponds to about 1.01 0-1.011. The amount of solids is largely dependent upon the character and the quantity of the food ingested, and in the dog may vary between 12.2 and 24.1 pro mille. These variations are mainly referable to the presence of albumins, which are always found in the enteric juice, while the inorganic constituents are fairly constant. A general idea of the chemical composition of the secretion may be formed from the accompanying analyses : Dog Horse (Thiry). (Colin). "Water 97.59 per cent. 98.10 per cent. Solids 2.41 « « 1.90 " " Albumins 0.80 "1 o 45 " " Other organic matter (mucin) . 0.73 " " / Mineral asli 0.88 " " 1.45 " " Sodium carbonate .... 0.40 " Sodium chloride 0.48 " " Of the amount of enteric juice which is secreted under normal conditions in twenty-four hours we know nothing. In disease, how- ever, and notably in Asiatic cholera exceedingly large quantities may be observed ; but it is probable that in such cases we are deal- ing with a direct transudation from the blood, and not with an actual secretory product of the cells. On section of the correspond- ing nerves hypersecretion can be artificially brought about. This may be compared to the paralytic saliva which is obtained from the' sublingual gland on section of the chorda and of the sympathetic fibres that supply the gland. The question whether or not the enteric juice plays a part in the process of digestion is still undecided. This uncertainty is largely owing to the fact that innumerable bacteria are always present in the enteric secretion, and that some of these possess the power of inverting starch and of digesting albumins. Schiff, on the other hand, claims that these digestive phenomena are directly referable to the action of ferments, which are secreted by the glands of Lieberkuhn. However this may be, it appears certain that the enteric juice contains ferments which are capable of inverting saccharose and maltose, and that these ferments are furnished by the glands in question. Like the pancreatic juice, the enteric secretion is capable of emul- sifying fats, bul it is doubtful whether it can also bring about their saponification. THE BILE. Formerly it was generally supposed thai the bile played an im- portant part in the process of digestion, and was further capable of 142 THE DIGESTIVE FLUIDS. controlling the intensity of the putrefactive and fermentative proc- esses which even normally take place in the lower intestinal tract. It has now been definitely established, however, that, aside from its emulsifying action upon fats, the secretion possesses no digestive properties whatever, and is likewise without effect upon the bacteria which are normally found in the intestinal canal. We accordingly find that in animals and in man the processes of nutrition are in no way interfered with if the bile is prevented from entering the digestive tube, but is carried to the outside through the establish- ment of a fistulous opening in the common duct, provided that food is administered which contains but little fat. With a diet consist- ing of albumins and carbohydrates digestion thus continues unim- paired, and the animal is capable of maintaining its nitrogenous equilibrium practically as before. If fats, however, are given at the same time in large amounts, more or less serious digestive disturb- ances soon develop, and the animal loses weight. In such cases it has been ascertained that whereas normally from 2 to 10 per cent, of the ingested fat is eliminated in the feces, from 31 to 47 per cent, now escapes resorption. The offensive gases which are then passed by the animal are referable to an increase of the putrefactive proc- esses in the intestines, it is true. This is, however, not owing to the absence of bile per se, but to the fact that the unabsorbed fats envelop the albuminous material, and thus prevent its further diges- tion, so that in the lower portion of the digestive canal, where the putrefactive processes are most intense, the bacteria find an increased amount of pabulum at their disposal, and an increase of the putre- factive products accordingly results. In the presence of bile, on the other hand, this does not occur, as its emulsifying effect upon the fats soon leads to their absorption, and thus leaves the albumins exposed to the action of the digestive juices, and to their resorption in turn. Indirectly, it can thus control the process of putrefaction, but such action is not due to any germicidal or antiseptic properties of its own. On withdrawing fats from the food and giving an ade- quate supply of carbohydrates, normal relations are soon re-estab- lished, though the bile is absent as before. The bile in reality represents a most important excretory product of the animal body, and may in this sense be compared to the urine. It appears, moreover, that those waste-products which are markedly toxic in action, and could not be carried to the kidneys through the blood-current without seriously disturbing the general health, are formed in the liver directly, and are hence removed through separate channels in the bile. Substances are further eliminated in this manner which, like cholesterin, are insoluble in water, and could hence not be excreted by the kidneys. For convenience' sake, however, the bile is described at this place. Secretion. — As found in the gall-bladder, the bile represents the secretory product of the liver-cells which is eliminated into the THE BILE. 143 radicles of the biliary passages, together with so-called mucus which is derived from the epithelial lining of the greater trunks and the gall-bladder itself. Its secretion is continuous, but liable to exacer- bations which are essentially dependent upon the ingestion of food. According to Heidenhain, the curve of secretion, in reference to amount, shows two periods of greatest activity, which in the dog corre.-jxmd to the third to the fifth and the thirteenth to the fifteenth hour, respectively, after the administration of food. This curve, however, is further influenced by the character of the food ingested. With an albuminous' diet three stages of maximal secretion are thus noted : The first after two to three hours, a second stage after five to eight hours, and a third stage after twelve to fourteen hours. With a diet of albumins and fits, on the other hand, the period of greatest secretion occurs after eleven to twelve hours, and with one of albumin and carbohydrates after from nine to fourteen hours. These figures have reference to observations which were made after the administration of only one meal in the twenty- four hours. With two meals analogous results are obtained. The individual periods, however, are shorter; and as the number of feedings is increased the secretion becomes more uniform, so that with a meal every two hours no variations of moment can be discerned. Amount. — The amount of bile which is eliminated in the twenty- four hours is variable even under normal conditions. In dogs from 2.9 to 36.4 grammes can be obtained pro kilogramme of weight of the animal. In man the secretion apparently varies between 400 and 800 grammes ; but it is possible that these figures do not repre- sent normal values. Ranke has estimated that a man weighing 75 kilogrammes secretes about 1050 grammes even in health. To a certain extent the amount is influenced by the character of the food, and it appears to be quite generally accepted that a diet rich in albumins will call forth a greater secretion of bile, while the carbo- hydrates are though! to diminish its amount, or are at least incapable of increasing this, like the albumins. The fats, on the other hand, are probably without effect in either direction. It was formerly thought that a number of drugs could increase the How of the' bile, and physicians were wont to administer cholagogues when they supposed that the secretion of bile was deficient. This view has now been abandoned, as it has been defi- nitely established that drugs are without effect in this direction. The only cholagogue, indeed, if it may so be termed, is the bile itself. This is readily understood, if we bear in mind that the bile i- essentially an excretory product. General Properties.— The color of the bile differs m different animals, and may vary from a bright yellow to an intense grass- green, with various shades of brown and blue, [n man it is usually of a golden-yellow color, but it may at time- appear bright green even when perfectly fresh. 144 THE DIGESTIVE FLUIDS. While pure bile, uncontaminated with mucus, is a thin transparent fluid, that which is eliminated into the intestinal tract and is found in the gall-bladder is markedly viscid and more or less turbid. Its taste is intensely bitter, with a sweetish and nauseating after-taste. The odor is in many animals peculiar, and at times suggestive of musk. The reaction is normally alkaline. After exposure to the air, however, it soon becomes acid, but becomes alkaline again owing to the development of trimethylamin and ammonia. The specific gravity varies between 1.010 and 1.033. In the gall-bladder it is higher (1.026-1.033), where a constant resorption of water is going on, and where considerable amounts of mucus are added to the bile, than in the bile proper, which is secreted by the liver (1.010-1.012). We accordingly find that the amount of solids is greater in the bladder-bile than in that which may be termed the hepatic bile. Chemical Composition. — The following analyses show the gen- eral chemical composition of the bile in different animals, and also illustrate the differences which exist between bladder-bile and hepatic bile : Human bladder-bile Human hepatic bile (normal), (normal). (Frerichs.) (Hammarsten, 1 to 3; Zeynek, 4.) 1. ' 2. 1. 2. 3. 4. Water 860.0 859.2 974.80 964.74 974.60 989.24 Solids 140.0 140.8 25.20 35.26 25.40 30.76 JJucm \ 26.6 29.8 5.29 4.29 5.15 2.08 -Pigments .... J Salts of bile acids . . 72.2 91.4 9.31 18.24 9.04 18.31 Taurocholates 3.03 2.17 2.18 Glycocholates 6.27 16.16 6.86 . . Fatty acids (soaps) . . : . . 1.23 1.36 1.01 2.08 Cholesterin .... 1.6 2.6 0.63 1.60 1.50 2.30 J Lecithin \ ft 99 / 0.57 0.65 0.73 Fat 3.2 9.2/ u,zz \ 0.95 0.61 . . Soluble salts - . . \ . , 77 J 8.07 6.76 7.25 9.10 Insoluble salts . . / °'° ' \ 0.2o 0.49 0.21 0.81 Analyses of the Beadder-bile of Animaes. Dog. Pig. Ox. Birds. Shad. (Hoppe-Seyler.) (Grundelach- (Berzelius.) (Marsson.) (Schloss- Strecker.) berger.) Water 813.56 888.0 904.4 800.2 944.8 Solids 186.44 112.0 95.6 199.8 55.2 1 170.6 36.3 Sodium glycocholate • • \ 000 Sodium taurocholate 122.8 f M'° Cholesterin .... 2.91") Fats 15.11 I Soaps 16.03 ( Lecithin 18.11 J 22.3 80.0 I }■ 3.6 J Mucin 3.49 5.9 \ f 25.6 14.8 Other organic solids; \ „ r. Y 3.0 ] ' ' insoluble in alcohol I J I Inorganic solids ... . . . . 12.6 21.0 • • Analyses of the inorganic salts have given the following results, which are taken from Jacobsen and Hoppe-Seyler, respectively. The figures have reference to 100 parts by weight of mineral ash : > Including fat. THE BILE. 145 Man (hepatic bile). Sodium chloride 65.16 Potassium chloride 3.39 Sodium carbonate 11.16 Trisodium phosphate 15.90 Tricalcium phosphate 4.44 Calcium carbonate Potassium sulphate variable Sodium sulphate . . Iron, Silica 1 4 Ar ' . > traces Magnesium, copper J Ox (bladder-bile). 7.50 l 2.50 40.0 9.50 2.0 25.0 traces The Mucinous Body of the Biles. The mucinous body which is found in the bladder-bile of all animals is apparently of a different nature in different animals. The mucus of the human bile thus largely consists of true mucin, while in ox-bile a mucinous nucleo-albumin is principally found. To isolate this latter, the bile is precipitated with 5 times its volume of absolute alcohol and immediately centrifugalized. The super- natant liquid is poured off, and the sediment rapidly dried with filter-paper and dissolved in water. By repeating this process the substance can be obtained in a fairly pure form. Its character as a nucleo-albumin becomes apparent on treating its neutral solutions with a small amount of hydrochloric acid. A flocculent precipitate then develops, which readily dissolves upon the further addition of hydrochloric acid to the extent of 0.3 per cent. This solution re- mains clear for a long time even when kept at the temperature of the body. If a small amount of pepsin, however, is added, a sepa- ration of nuclein occurs. On fusing the dried substance, moreover, with potassium hydrate and sodium nitrate, an amount of phosphoric acid is obtained which is greatly in excess of the amount required to saturate all of the mineral ash that is present when calculated as tricalcium phosphate. On boiling with a dilute mineral acid no reducing-substance is formed, as in the case of true mucin. Acetic acid precipitates the substance from its solutions, in the absence of biliary acids ; but this precipitate, unlike that of mucin, is soluble in an excess of the acid. The mucin proper which is found in human bile may be isolated, as already described (page 113). The Biliary Acids. The biliary acids which are normally found only in the bile arc essentially compound amido-acids, which arc formed through the union of glycocoll on the one hand, and tanrin on the Other, with a cholalic acid. In the bile of sharks Elammarsten discovered the ex- istence of a third group of biliary acids, which are rich in sulphur, and, like the conjugate sulphates of the urine, yield sulphuric acid ' Tbi- figure '- too low, owing to the farl thai Hoppe Seyler'i analysis lias reference t'> i be Inorganic salts, which werenol dissolved by alcohol. 10 146 THE DIGESTIVE FLUIDS. on boiling with hydrochloric acid. Traces of these acids are said to occur also in human bile. Of their chemical nature, however, nothing is known. Under pathologic conditions, when the free outflow of bile is impeded, owing to a swelling of the mucous membrane of the common duct or to the presence of a calculus, resorption of the bile takes place through the lymph-channels, and the secretion thus finds its way into the blood. Jaundice then results, and in such cases not only the bile-pigments, but also the bile-acids can be demonstrated in the general circulation. To the presence of the latter is due the slow pulse which is so constantly observed in icterus. At the same time the bile-acids bring about a dissolution of the red corpuscles, and thus manifest their true nature as excretory products. Regarding the origin of the biliary acids, we know only that they are formed exclusively in the liver, but of the manner in which their formation takes place we are ignorant. The amido-radicles of the bile-acids are unquestionably formed in the general nitrogenous metabolism of the body, and, as such, are found in other organs besides the liver. Glycocoll is then to a cer- tain extent further oxidized, and contributes to the formation of urea. Of the ultimate fate of taurin, we know that its sulphur can be oxidized to sulphuric acid and be eliminated in the urine in this form. In the human being traces are further excreted as tauro- carbaminic acid ; and in rabbits, at least, its hypodermic injection leads to the elimination of the body as such. It is thus difficult to understand why two substances like these, for the removal of which the animal body has manifestly other means at its disposal than their elimination through the bile, should here appear. We may imagine, however, that the formation of the bile-acids in the liver represents a conservation of energy on the part of the body, and further constitutes a reserve mechanism by which Avaste-products can be removed in a state of incomplete oxidation. Of the origin of the non-nitrogenous acids, which combine with glycocoll and taurin to form the biliary acids, we know nothing. The synthesis, however, manifestly occurs in the liver, and here only, as after extirpation of the organ in birds and frogs bile-acids are not found in the blood. In mammals, also, neither bile- pigments nor bile-acids can here be demonstrated after ligating the gall-duct and the thoracic duct. In the bile of most animals the biliary acids occur in combination with sodium, while in sea-fish they are, curiously enough, present as potassium salts. As regards the relative quantity of the two principal biliary acids which are found in the bile, it is to be noted that in man glycocholic acid is usually more abundant than taurocholic acid. In strictly carnivorous animals, on the other hand, the latter only is found ; but the same also holds good for certain herbivora, such as the sheep THE BILE. 147 and the goat. In other animals the relation is variable, and in some, it is stated, glycocholic acid only is found. Isolation. — Collectively, the biliary aeids, in the form of their salts, can be obtained in the following manner : the bile is mixed with animal charcoal, evaporated to dryness, and the residue ex- tracted with absolute alcohol. This extract, which contains the biliary acid salts, cholesterin, fats, soaps, and lecithin, is then filtered, concentrated, and treated with a large excess of ether. In this manner the salts of the bile-acids are precipitated, while the other substances remain in solution. On standing, the precipitate gradually becomes crystalline, and is then spoken of as Planner's crystallized bile. In this form the biliary acids are conveniently estimated as a whole. If then it is desired to determine the rela- tive amount of the two principal acids present, it is only necessary to estimate the sulphur in Platner's bile, from which the corre- sponding amount of taurocholic acid can be calculated. To separate the two acids from each other, Platner's bile is dis- solved in water and completely precipitated with a solution of neutral acetate of lead. The corresponding lead salts are thus formed, and ■can now be readily separated from each other, as the glycocholate is thrown down, while the taurocholate remains in solution. In the filtrate the latter is then precipitated with ammonia. To obtain the free acid from the glycocholate, the precipitated lead salt is sus- pended in water and evaporated to dryness in the presence of sodium carbonate. The sodium salt is thus obtained and extracted with alcohol. The alcohol is evaporated off, the residue dissolved in water and treated with hydrochloric acid, when the glycocholic acid will separate out. The taurocholic acid can be similarly obtained from its lead salt by decomposing the solution with hydrogen sul- phide. The resulting plumbic sulphide is filtered off, the filtrate evaporated to dryness, the residue dissolved with a small amount of alcohol, and then precipitated with an excess of ether, when the free acid is thrown down. Tests for the Bile-acids. — Pettenkofer's Test. — On treating the biliary acids in aqueous or alcoholic solution with a few drops of an 0.1 per cent, solution of furfurol and 1 or 2 c.c. of concentrated, chemically pure sulphuric acid, a beautiful purple color develops. AjB furfurol results when concentrated sulphuric acid is added to a carbohydrate, the test may also be conducted by treating the solu- tion of the biliary acids (1 or 2 c.c) with a few drops of a 10 per cent, solution of cane-SUgar and then with sulphuric acid. In either case, however, care should be hail that the temperature which results during the- reaction does not exceed 70° ( '., as otherwise the result- ing pigment i- destroyed. This may be obviated by substituting -tron_' phosphoric acid for the sulphuric acid, and placing the solu- tion in boiling water. The resulting pigment shows two bands of absorption on spectroscopic examination. One of these is situated at F; the other between I) and E, near E. On diluting with 148 THE DIGESTIVE FLUIDS. alcohol a green fluorescence is observed. In the presence of an excess the red pigment disappears, but reappears upon the further addition of sulphuric acid. On standing, the color gradually turns to a bluish violet. As Pettenkofer's reaction can also be obtained with other sub- stances, such as the phenols, the higher alcohols, certain bases of the aromatic series, the higher hydrocarbons, and acids of the fatty series and the benzol series, it is always necessary to isolate previ- ously the biliary acids as Platner's bile, before drawing conclusions from the reaction. Albumins, if present, must in every case first be removed. For, as has been stated, many of these contain carbo- hydrate groups, which on contact with concentrated sulphuric acid give rise to the formation of furfurol. This in turn combines with the aromatic oxy-acids and phenols that result from decomposition of the amido-acids to form colored compounds, which are either identical with or very similar to those obtained in the case of the biliary acids (^see the reaction of Adamkiewicz and the hydrochloric acid test for albumins). Pettenkofer's reaction is, in the case of the biliary acids, referable to the non-nitrogenous acid constituents of these acids, viz., to cholalic acid or one of its congeners. Physiological Test. — Aside from their reaction with furfurol, the bile-acids, or their salts, may also be identified as such from their effect upon the action of the heart. To this end, the heart of a cura- rized frog is exposed and moistened with a small drop of a 1 per cent, solution of atropin, so as to eliminate the action of the vagus. A few drops of an aqueous solution of the bile-acids are then placed upon the heart, when their retarding action can readily be demonstrated. Glycocholic Acid. — As has been stated, glycocholic acid is the principal biliary acid that is found in the bile of man. It is formed through the union of glycocoll and cholalic acid, as represented by the equation : C2H5N02 + C2.H40O5 = C26H43N06 + H20 Glycocoll. Cholalic Glycocholic acid. acid. It is accordingly decomposed into these constituents when treated with dilute acids or alkalies under the application of heat. In the pure state glycocholic acid occurs in the form of fine, silk- like needles, which are readily soluble in alcohol, less readily so in water, and insoluble in ether. From its aqueous solutions it is readily precipitated on adding a small amount of a mineral acid. The crystals melt at 133° C. It is a monobasic acid, and forms salts which, with the exception of the lead and silver compounds, are all soluble in water and alcohol. The free acid and its salts give Pettenkofer's reaction, and turn the plane of polarization to the right. On heating glycocholic acid with concentrated sulphuric acid the THE BILE. 149 anhvdride of glycocholic acid is precipitated in the form of oily- droplets, which subsequently tend to coalesce. This anhydride is termed cholonic acid, and has the composition C20H41NO5. According to Michailoff, glycocholic acid when treated with con- centrated sulphuric acid in the presence of acetic acid is said to yield an orange color with a green fluorescence. On salting with ammo- nium sulphate a precipitate is formed which in its reactions is identical with biliverdin. Urobilin is said to remain in solution. This observation is of special interest, as showing the possible rela- tionship which may exist between the biliary acids and the bile-, pigments. We find, as a matter of fact, that an increase in the production of bile-pigments on the part of the liver is associated with a diminished formation of biliary acids. Others have con- cluded from this observation that a connection between the produc- tion of bile-acids and bile-pigments does not exist, and that the origin of the two classes of substances must be referred to a separate activity on the part of the hepatic cells. It appears to me, however, that this inference is not altogether justifiable. Closely related to the common glycocholic acid is the so-called hyo- glycocholic acid, which has been found in small amounts in the bile of the pig. On decomposition it yields glycocoll and hyocholalic acid, as shown in the equation : C27H43X05 + H20 = C2H5N02 + C25H40O4 Hyoglycocholic Glycocoll. Hyocholalic acid. acid. The substance itself is almost insoluble in water, but soluble in alcohol. It is crystallizable, but usually obtained as a resinous mass. Its salts are precipitated from their solutions by calcium chloride, barium chloride, and magnesium chloride. By salting with sodium sulphate they separate out like soaps. Like the com- mon glycocholates, they give Pettenkofer's reaction. In addition to these two forms still other glycocholates apparently exist. In the bile of rodents a glycocholate is thus found, which cannot be salted out with sodium sulphate, but which is also precipi- tated by the salts of the alkaline earths. Of its nature, however, as also of the so-called guano-bUiary acid3 which apparently belongs to this order, nothing is known. Isolation. — The common glycocholic acid is most conveniently obtained by starting with Platner's bile, that has been prepared from human bile or from that of the ox, as already described. Hyoglycocholic acid fan be isolated from the bile of the pig by fir-' decolorizing with animal charcoal and then salting with sodium sulphate in substance. The acid is thus precipitated, ami can then he filtered off. It IB washed with a solution of the salt, dissolved in water, and precipitated in the form of the free acid by means of hydrochloric acid. Taurocholic Acid. — TaurochollC acid, as has been slated, is the only biliary acid that is found in the bile of the purely carnivor- 150 THE DIGESTIVE FLUIDS. ous animals, but it also occurs in the bile of man and most herbiv- orous animals. Among these, the sheep and goat are especially noteworthy, as in these, like the pure carnivora, taurocholic acid only is found. It is formed synthetically in the liver from taurin aud cholalic acid, and is accordingly resolved into its components by treating with dilute acids or alkalies under the application of heat. The same result indeed is reached by evaporating the bile together with water or allowing it to undergo putrefaction. The chemical change is represented by the equation : C26H45NS07 + H20 = C2H7NS03 + C24H40O5 Taurocholic Taurin. Cholalic acid. acid. In the pure state taurocholic acid occurs in the form of fine deli- quescent needles, which are soluble in water and alcohol, but insoluble in ether. It is capable of maintaining glycocholic acid in solution, and it is for this reason that the latter acid cannot be pre- cipitated from the bile by adding a mineral acid when taurocholic acid is at the same time present in sufficient amount. Like glyco- cholic acid, it is a monobasic acid, and forms salts which for the most part are soluble in water and in alcohol. Its salts with the alkalies are precipitated from their solutions by subacetate of lead and ammoniacal subacetate of lead, but not by the neutral acetate, by copper sulphate, or silver nitrate. Curiously enough, the sodium salt obtained from the bile of the ox is dextrorotatory, while the same salt which is found in that of the dog turns the plane of polarization to the left. An isomerism thus apparently exists which is analogous to that observed in the case of the tartaric acids. Taurocholic acid forms emulsions with the peptones, but does not precipitate them, as is generally stated ; but it does precipitate albu- mins, syntonins, and albumoses. Hyotaurocholic acid is the biliary acid which, as a sodium salt, is found in the bile of pigs, and is analogous to the hyoglycocholic acid already described. Its amount, however, is small. On decomposi- tion it yields taurin and hyocholalic acid, as shown in the equation : C26H45NSOfi + H20 = C^EUO, + C2H7NS03 Hyotaurocholic Hyocholalic Taurin. acid. acid. Chenotaurocholic acid is the most important biliary acid, which is found in the bile of geese. It is indistinctly crystalline, and is soluble in water and alcohol. Like the acids already described, it gives Pettenkofer's reaction. On prolonged boiling with alkalies it is decomposed into taurin and chenocholalic acid, as shown in the equation : C29H49NS06 + H20 = C27H4404 + C2H7NS03 Chenotaurocholic Chenochola- Taurin. acid. lie acid. Isolation. — Taurocholic acid is most conveniently obtained from Platner's bile of man or the ox, as already described. To isolate THE BILK 151 it from the bile of dogs, the fluid is shaken with animal charcoal and alcohol, then decanted and filtered. The filtrate is evaporated to dryness, the residue taken up with a small amount of warm absolute alcohol, and filtered. The clear solution is then treated with ether until it becomes cloudy. On standing, the taurocholate of -odium separates out in the form of fine crystals. These are dissolved in water and treated with ammoniacal subacetate of lead. The resulting precipitate is suspended in alcohol and decomposed with hydrogen sulphide. The free acid i- thus liberated, and can he obtained as such by evaporating the filtrate to dryness or by diluting with ether. To isolate chenotaurocholic acid, the bile of geese is first treated with strong alcohol, to remove the mucus. The filtrate is mixed with ether and set aside, when the biliary salts separate out as a glutinous mass, which is then washed, dried, redissolved in 99 per cent, alcohol, and again precipitated with ether. The crystalline deposit which is formed is dissolved in strong alcohol anil decom- posed with hydrogen sulphide. On evaporation the free acid is obtained. Cholalic Acid. — Cholalic acid is the principal biliary acid which is formed in the liver, and to its presence in the molecule ot glyoo- cholic acid and taurocholic acid Pettenkofer's reaction is due. It is a product of the specific activity of the liver-cells, and is normally not found in any other tissues or organs of the animal body. In the intestinal contents, however, it may be encountered, as such, even in health, and in cases of jaundice it has been observed also in the urine. A- T have already shown, cholalic acid is liberated when glyco- cholic acid or taurocholic acid i- decomposed with alkalies or acids, under the application of heat. Its crystalline form differs accord- ing to the medium in which crystallization has taken place. From it- alcoholic solution it separates out in the form of rhombic tetrahedra or octahedra, which contain one molecule i>f alcohol as li,,uid of crystallization, C^H^Oj 0aH6O. On prolonged boiling with water the crystal-alcohol can be removed. When dissolved in dilute boiling acetic acid cholalic acid takes up on.' molecule of water, and can be obtained from such solution- in the form of rhombic plates or prisms, < '..,1 1 ,,< >,II.O. On exposure to the ait- tin' crystals in either case Boon become opaque. They melt al a temperature of 195 < '. The free acid is readily soluble in alcohol, with difficulty bo in water, and is almosl insoluble in ether. According to Mylius, it is a monobasic alcoholic acid, and contains one secondary and two primary alcohol groups, as represented by CH.OH the formula: C II MII..OH).. It combines with alkalies and cool I alkaline earths, a- also with the heavy metals, to form salts. Its compounds with the alkalies are readily soluble in water, but with 152 THE DIGESTIVE FLUIDS. some difficulty in alcohol. The barium salt is somewhat soluble in water, and, like the lead salt, soluble in hot alcohol. The calcium salt is slightly soluble in boiling alcohol. Concentrated solutions of the alkaline hydrates and carbonates precipitate the alkaline salts from their solutions in the form of an oily material which becomes crystalline on cooling. The salts, like the free acid, are dextro- rotatory. On prolonged boiling with acids or at a temperature of 200° C. cholalic acid loses two molecules of water and is transformed into dyslysin, as shown in the equation : C24H40O5 = C24H3603 + 2H20 Cholalic Dyslysin. acid. The same result is brought about through the influence of various bacteria, and there can be no doubt that the dyslysin which is con- stantly encountered in the stools is referable to the normal processes of putrefaction, which take place in the intestinal canal. Choloidinic acid, C^H^Oj, probably represents an intermediary product which is formed during this process, and may be regarded as a primary anhydride of cholalic acid. The common dyslysin which is met with in the feces is amorphous, and is insoluble in water and in dilute solutions of the alkalies. On oxidation with permanganate cholalic acid first yields dehydro- cholalic acid, and then bilianic acid, together with isobilianic acid. These changes may be represented by the equations : (1) C24H40O5 + 30 = C24H3(05 + 3H20 Cholalic Dehydrocholalic acid. acid. (2) C24H3405 + 30 = C24H3408 Dehydrochol- Bilianic alic acid. acid. On further oxidation another acid has been obtained, which is termed cilianic acid, and which is said to have the composition Dehydrocholalic acid also results on oxidizing cholalic acid with nitric acid ; but it is to be noted that on further oxidation a new acid results, which is known as choleocamphoric acid, and is thought to be isomeric with camphoric acid. Its formula is given as CI0H16O4. On oxidation with potassium bichromate and sulphuric acid, on the other hand, Tappeiner claims to have obtained cholesteric acid (not to be confounded with cholesterinic acid, see below), C12H1607 ; 'pyrocholesteric acid, C,,H1607; cholanic acid, C20H28O6; as also palmitic, stearic, and acetic acids. On reduction, as during the process of putrefaction, cholalic acid may give rise to the formation of desoxy cholalic acid, C24H40O4. On boiling with concentrated solutions of the alkaline hydrates a mixt- ure of the corresponding salts of formic acid, acetic acid, propionic acid, and palmitic acid is obtained, and it is interesting to note that the latter, like cholalic acid, gives Pettenkofer's reaction. THE BILE. 153 Hyocholalic acid and ehenocholalic acid, which result from the decomposition of hyotaurocholic acid, hyoglycocholic acid, and chenotaurocholic acid, respectively, in their general properties and reactions are closely analogous to the common form of cholalic acid that has just been considered. Like the latter, they are trans- formed in the intestinal tract into the corresponding dyslysins, and these in turn yield the original acids on heating with sodium hydrate solution. Both are soluble in alcohol and ether, but insoluble in water. Isolation of Cholalic Acid from the Bile. — Platner's bile, obtained from the ox, is boiled for twenty-four hours with barium hydrate. The cholalic acid which is thus set free is precipitated by adding an excess of hydrochloric acid. It is then washed with water, dissolved in a dilute solution of sodium carbonate, and repre- cipitated with hydrochloric acid. The precipitate is covered with ether and allowed to stand, when crystallization will gradually occur. The crvstals are freed from liquid as far as possible by nitration with the suction-pump, and are redissolved in hot alcohol. The solution is diluted with water until a persisting turbidity develops, and is then set aside in a cold place until crystallization is complete. Choleic Acid. — When the biliary acids of ox-bile are decomposed, as just described, a small amount of choleic acid, C24H4u04, is always found associated with common cholalic acid. On oxidation it fii'st yields drln/drocholeic acid, C24H3404, and then eholanie acid, C24H34Or It is possibly identical with desoxycholalic acid (see above). The substance has also been found in human bile. Fellic Acid. — This acid has been obtained together with common cholalic acid and choleic acid from human bile, where it possibly exists in combination with glycocoll and taurin. Its amount is always small. With Pettenkofer's test it gives a reddish-blue color. On heating, it is decomposed, with the formation of vapors which are stronglv suggestive of turpentine. Its formula is given as a{H,„04. Lithofellic Acid. — Lithofellic acid is a substance which is closely related to the cholalic acids just described, and is found in certain gastro-intestinal concretions of various ruminants. It forms the greater portion of the oriental Bezaar stones, which are obtained from the stomach of the wild goat and antelope. It gives Petten- kof'er'- reaction, and is said to have the formula C2(JII.ir04. Taurin. — As has been pointed out, taurin is not exclusively found in the bile, but occurs also in the lungs and kidneys and in the muscle-tissue of many animals, and notably in that of the verte- brates. While it is unquestionably of albuminous origin, and prob- ably represents an intermediary product in the katalysis of the organic sulphur compounds of the body, it has thus far not been obtained from tin- source by artificial means. Unlike the loosely combined sulphur of the nlbiiminoiis molecule, the sulphur which is present in the taurin molecule cannot be split oil' on boiling with 154 THE DIGESTIVE FLUIDS. dilute alkalies. Its separation, indeed, necessitates the complete destruction of the taurin, viz., the albuminous material, by boiling with a concentrated solution of sodium hydrate or by fusing with potassium hydrate and potassium nitrate. Potassium sulphite, acetic acid, ammonia, and hydrogen thus result in the first case, while in the latter sulphates, carbon dioxide, ammonia, and water are obtained. Taurin can be formed synthetically by heating ammonium-ox v- ethyl-sulphonate to a temperature of 230° C, or from ammonia and chlor-ethyl-sulphonic acid, as represented by the equations : /C2H4C1 /C2H4.NH2 NH3 + S02< = SO/ + HC1 xOH X)H Chlor-ethyl-sul- Taurin. phonic acid. /C2H4.OH /C2H4.NH2 S02< = S02< + H20 xO.NH4 X)H Ammonium oxy- Taurin. ethyl-sulphonic acid. It can hence be regarded as amido-ethyl-sulphonic acid (viz.r amido-isethionic acid). It crystallizes in the form of four- or six- sided prisms, and is fairly soluble in hot water, slightly soluble in common alcohol, and insoluble in absolute alcohol and ether. It has a markedly acid character, and accordingly does not combine with acids, but with alkalies, to form salts. Its compound with mercuric oxide is quite insoluble. Whether or not a relation exists between taurin and cystin is as- yet unknown. The latter has thus far only been observed under conditions which must be regarded as abnormal, and it would be interesting to ascertain whether in such cases the formation of taurin is possibly diminished or even suspended. Isolation of Taurin. — Taurin is most conveniently prepared from the bile of animals in Which taurocholic acid is present. To this end, the fluid is boiled for several hours with hydrochloric acid. The dy sly sin and choloidinic acid are filtered off. The filtrate is concentrated on a water-bath to a small volume, and freed from the sodium chloride and other substances that have separated out, by filtering while still -warm. The liquid is then evaporated to dryness and the residue extracted with strong alcohol, which dissolves any glycocoll hydrochlorate that may be present, while the taurin re- mains behind. This is then dissolved with a little warm water, fil- tered while still warm, and treated with an excess of warm alcohol. The resulting precipitate is immediately filtered off. In the filtrate the taurin crystallizes out on cooling, and can be identified by the form of its crystals, their solubility in water and insolubility in alcohol,. and the formation of potassium sulphate when fused with potassium hydrate and potassium nitrate. Should glycocholic acid be present in the bile at the same time, this is likewise decomposed on boiling with hydrochloric acid. The THE BILE. 155 hydrochlorate of glycocoll which thus results is found in the first alcoholic extract. To isolate the glvcocoll as such, the solution is evaporated to dryness, the residue dissolved in water and treated with plumbic hydrate. On filtering, the solution, which contains the lead salt of glvcocoll, is decomposed with hydrogen sulphide. The resulting lead sulphide is filtered off', and the filtrate concen- trated until crystallization occurs. The crystals are then dissolved in water and decolorized with animal charcoal, and the solution is evaporated until the crystals again separate out. Glycocoll. — Glvcocoll is now known to be a constant decomposi- tion-product of most albumins, but is formed in especially large amounts during the hydrolytic decomposition of collagen and spongin. From this fact and its sweetish taste it is also known as glucin or collagen-sugar (Leimzucker). It is one of the most important decomposition-products which are formed in the nitro- genous metabolism of the animal body, and in part at least gives rise to the formation of urea in mammals, and to uric acid in birds and reptiles. Another portion, as we have seen, is eliminated in the bile in combination with cholalic acid; while a third portion appears in the urine in combination with benzoic acid and phenyl- acetic acid, as hippuric acid and phenaceturic acid, respectively (which see). As we have seen, glvcocoll is amido-acetic acid. The pure sub- stance crystallizes in the form of colorless rhombohedra or of four- sided prisms, which are readily soluble in water, with difficulty so in warm alcohol, and insoluble in absolute alcohol and ether. It combines with acids and alkalies to form salts. The most important of these are the hydrochlorate, which is soluble in water and alcohol, and the copper salt, which results when a boiling solution of glycocoll is added t<> freshly precipitated cupric hydrate; the hydrate is thus dissolved, and after concentrating the solution blue needles of the copper salt separate out on cooling. Isolation. — Glvcocoll can be obtained from the bile of those animals in which glycocholic acid is found, as described above; or it may be prepared from hippuric acid by decomposing this by boil- ing with dilute anlphuric acid. On cooling, the benzoic acid that has separated out i- filtered oil', the filtrate is concentrated and ex- tracted with ether to remove such benzoic acid as still remains in solution ; the sulphuric acid is removed with barium carbonate, and the filtrate i- evaporated until crystals of glycocoll begin to separate out (see al-o pages 192 and 259). The Bile-pigments. The bile-pigments which have thus Car been obtained from the bile it-elf or from biliary concretion- are bilirubin, biliverdin, bili- prasin, bilifuscin, and other- which are less well known. 156 THE DIGESTIVE FLUIDS. Perfectly fresh hepatic bile, in contradistinction to that which is found in the gall-bladder, contains only one pigment, bilirubin, from which all other forms are derived. Such bile is of a golden-yellow color, while bladder-bile usually presents an olive-brown color, owing to the simultaneous presence of its nearest oxidation-product, bili- verdin. A grass-green color is observed when the latter predomi- nates or is exclusively present. Bilirubin. — Bilirubin is now known to result from the decompo- sition of haematin, and normally constitutes a specific product of the activity of the hepatic cells. It appears, however, that the power of transforming the blood-pigment into bilirubin is common to other tissues as well, if we regard the haematoidin of Virchow, which is so often found in old extravasations of blood, as identical with bili- rubin. That this is actually the case seems now undoubted. Under normal conditions, however, the liver is apparently the only organ of the body in which the formation of bilirubin takes place. "Whether or not the final dissolution of disintegrating red corpuscles also occurs at this place has not been decided, but the liver is manifestly capable of retaining the haemoglobin which is thus set free. If a moderate amount of a solution of haemoglobin is injected into the bloodvessels of an animal, an increased elimination of bilirubin results, while the blood-pigment does not appear in the urine. If larger amounts are injected, the resulting bilirubin ap- parently cannot be removed with sufficient rapidity through the biliary ducts. Resorption of the pigment then takes place through the lymph-vessels, jaundice results, and the bile-pigment appears in the urine. If excessive amounts of haemoglobin finally are injected, the liver is manifestly incapable of retaining all the pigment which reaches the organ, jaundice results, and both the bile-pigment and the blood-pigment appear in the urine. Even in such extreme cases the remaining tissues of the body do not participate in the formation of bilirubin. This has been conclusively demonstrated by Minkowski and Xaunyn. These observers have shown that while in normal geese poisoning with hydrogen sulphide, which causes an extensive dissolution of the red corpuscles, invariably leads to jaundice and the appearance of bile-pigment in the urine, the previous removal of the liver prevents such an occurrence, and results in simple hemoglobinuria. In mammals such crucial tests unfortunately cannot be applied, but there is no reason to suppose that different conditions there exist. The possible occurrence of a haematogenic icterus, in contradistinction to a hepatogenic icterus, is thus rendered extremely improbable, and it is scarcely warrantable to point to the development of bilirubin from blood-pigment in the tissues of the organs, as indicating the possibility of such a trans- formation in the circulating blood. Of the manner in which this transformation is effected m the liver we know nothing. "We may imagine, however, that the oxyhemo- globin is here first decomposed into its albuminous component — THE BILE. 157 globin — and into hsematin, which latter then passes over into bili- rubin, as shown in the equation : LVJI3.N AFe + 2H20 — Fe = C32H36N4< >6 Haematin. Bilirubin. This reaction, it will be noted, is also supposed to express the formation of haematoporphyrin from hsematin (see page 837). The two substances, indeed, are now quite generally regarded as isomeric. As regards the size of the molecule, however, opinions differ, and it is possible, as Nencki and Sieber suppose, that the molecule of bili- rubin, as well as of haematoporphyrin, is only half as large as expressed above. In that case, of course, the equation would have to be written : C32H32X AFe + 2H20 — Fe = 2C16H18N2Os On reduction with nascent hydrogen bilirubin is transformed into hydrobilirubin, as is shown by the equation : C32H36X405(viz., 2C16H18N203) + H20 + 2H = C32H40N4O7 Uydrobilirubin. This actually takes place in the intestinal canal, where nascent hydrogen is constantly being formed through the action of various bacteria, as during the process of lactic acid fermentation. During intra-uterine life, however, where no bacteria are found in the intestinal tract, bilirubin appears in the feces as such (meconium). According to some observers, hydrobilirubin is thought to be identical with the febrile urobilin of Jaffe, which is met with in the urine in various febrile diseases, and exceptionally also under normal conditions, but it is said to differ from the normal urobilin which represents the principal coloring-matter of the urine in health. This question, however, still awaits solution. The possible identity also of hydrobilirubin and stercobilin, which is the principal pig- ment that is found in the feces, has not been definitely established (see also pages 207 and 291). In the crystalline state bilirubin occurs in the form of reddish- yellow rhombic platelets with rounded angles, which arc soluble in benzol, carbon disulphide, amyl alcohol, glycerin, the fatty oils, and especially in warm chloroform. In alcohol and ether they are but little soluble, and in water, as also in Platner's bile, they are insol- uble On spectroscopic examination its solutions do not give rise to any specific bands of absorption, but to a continuous absorption which extend- from the red to the violet end. Bilirubin as such is a weak acid, bilirubinic arid, and combines with bases to form salts, which for the raosl part are either insoluble or only slightly Boluble in water and insoluble in chloroform. Its salts with the alkalies, however, arc soluble in solution- of the alka- line hydrates and carbonates. In the bile bilirubin i- largely present as neutral bilirubinate of sodium, and is held in solution owing to the presence of alkaline carbonates. 158 THE DIGESTIVE FLUIDS. When perfectly fresh bile of a golden-yellow or olive-brown color is exposed to the air for a while, it will be noted that the fluid grad- ually assumes a bright-green color, owing to a transformation of the bilirubinate into bidverdinate of sodium. Free bilirubin, according to Dastre and Floresco, does not absorb oxygen and is thereby transformed into biliverdin, as has been supposed. The same observers state that on careful oxidation bilirubin can be trans- formed into biliprasin, which presents a green color as such, while its sodium salt, sodium biliprasinate, is yellowish brown. On further oxidation the biliprasin then yields biliverdin, viz., its cor- responding salt. According to former views, the relation existing between bilirubin and biliprasin were represented by the equations given below ; but it is, of course, manifest that these are no longer tenable if the work of Dastre and Floresco should be confirmed. C16H18N203 + 0 = C18H18N204 Bilirubin. Biliverdin. C16H18N,03 + H20 = C16H20NA Bilirubin. Bilifuscin. ClfiH20N2O4 + H20 + O = ClfiH22N206 Bilifuscin. Biliprasin. The formula of biliprasin, as here indicated, would, moreover, be an impossibility. As a matter of fact, these formulae cannot be regarded as definitely established ; and according to some observers, the molecule of biliverdin is only one-half as large as represented above. Much work still remains to be done in this connection, but we know at least that biliverdin constitutes a normal oxidation- product of bilirubin. From biliverdin Kiister claims to have obtained a new oxidation-product, which he termed biliverdinic acid, but which must not be confounded with the substance of the same name referred to above. This body has the formula C8H9N04, and is apparently identical with the dibasic hsematinic acid, which results from haematin directly, and, like this, yields a substance of ihe com- position C8H805, viz., the anhydride of the dibasic hsematinic acid C8H10O6. In this manner the origin of bilirubin from the coloring- matter of the blood is still further shown. If bile containing bilirubin is filtered through Swedish filter- paper, and a drop of concentrated nitric acid containing a trace of nitrous acid, is placed upon the paper, which is colored a bright yellow, a play of colors will be observed, in which the yellow first turns to green, then to blue, to violet, to red, and ultimately to orange. This reaction is commonly referred to the formation of various oxidation-products of bilirubin, and is very characteristic. The individual products, however, which thus result are mostly but imperfectly known. The green color, of course, is referable to the biliverdin. The blue color is ascribed to bilicyanin or cholecyanin, and the final orange to choletelin. Tests for Bilirubin. — Gmelin's Test. — The fluid to be examined THE BILE. is treated with an amount of concentrated nitric acid, containing a trace or nitrous acid, sufficient to form a layer beneath the liquid to be tested, when in the presence of bilirubin the color-play referred to will be observed at the zone of contact ; the green will be noticed nearest the bile-cuntaining solution, and the orange in the upper portion of the nitric acid. Various modifications of this reaction have been proposed, such as the one described above. The test is exceedingly sensitive, and is said to indicate the | ence of bilirubin in a dilution of 1 v \ The green color which develops is the most characteristic, but a reddish violet must also occur. Huppeet'- Test. — A few cubic centimeters of the solution to lie examined are precipitated with barium chloride and ammonia. The precipitate is washed with water and suspended in a small amount of alcohol that has been acidulated with sulphuric acid. This mixture is then boiled for a few minutes, when in the presence of bilirubin a bright emerald-ofreen color develop- . Smith's Te-t. — A -mall amount of the fluid is placed in a test- tube and treated with a few cubic centimeters of tincture of iodine which has been diluted with alcohol in the proportion of 1 : 10, so as to form a layer above the fluid to be examined. In the presence of bilirubin a distinct emerald-green ring will develop at the zone of contact. According to some observers, the green color which thus results when bilirubin is treated with inline is not referable to the forma- tion of biliverdin, but to a substitution- or addition-product of bili- verdin with iodine. This, however, is denied by others, and Jolles has recently shown that the iodine merely acts as an oxidizinsr agent, and that true biliverdin is thus formed, as indicated by the equation : C^H^A - -21 - H,0 = C16H„XA - 2HI. Spectroscopic- Tebt. — If a dilute solution of sodium bilirubi- nate in water is treated with an exces- of ammonia and a small amount of a solution of chloride of zinc, the liquid at first turns a deep orange, but subsequently becomes olive brown, and finally green. If this solution i- examined spectroscopically, it will be •1 that the violet and blue portions of the spectrum are at first quite dark, but subsequently the bands presented by an alkaline -olution of bilicyanin become apparent, and notably the one between I md D, near C see below). The test i- said to be very g 1 1 immarsten). Isolation of Bilirubin. — Bilirubin i- most conveniently obtained from the biliary concretions which an- -<> often found in the srall- Madder -.f cattle, and which consist almost entirely of the calcium -alt of the pigment. They ;ire finely powdered and extracted with ether and then with hot water, bo a- to remove the cholesterin and the biliary acids which an- present. The remaining material i- treated with hydrochloric acid, soaa to liberate die pigment. It i- 160 THE DIGESTIVE FLUIDS. then washed free from acid with water, and subsequently with abso- lute alcohol, to remove the water and any biliverdin that may be present. The pigment remains, and is now dissolved with boiling chloroform. From this solution the chloroform is distilled off, the resklue extracted with absolute alcohol, so as to remove any bili- fuscin, and the remaining bilirubin dissolved in a small amount of chloroform and precipitated with alcohol. This procedure is repeated until the substance has been obtained in pure form ; it is then allowed to crystallize out from its chloroform solution on cooling. Biliverdin. — Biliverdin is found in the bladder-bile of many ani- mals together with bilirubin, and is especially abundant in certain herbivora, where the bile frequently presents a bright grass-green color. It is said to occur also in the placenta of the bitch, in the shells of certain mollusks, and in birds' eggs. Its relation to bili- rubin has already been considered. In the bile it is present princi- pally in the form of its sodium salt, and, like bilirubin, the free pigment possesses acid properties ; this is termed biliverdinic acid, but should not be confounded with the acid of the same name which Kuster obtained from biliverdin on oxidation with sodium chromate. In acid bile biliverdin is found as such. Unlike bilirubin, the free pigment is readily soluble in normal as well as in neutral and acid bile. It is insoluble in water, ether, and chloro- form, but dissolves in alcohol, glacial acetic acid, and solutions of the alkalies. From the latter it is precipitated by the salts of the alkaline earths and the heavy metals, as also by acids. On treating an alcoholic solution of biliverdin with ammoniacal chloride of zinc solution the fluid exhibits a green fluorescence. The green color of the pigment is changed to yellow if its solution in acid alcohol is treated with zinc. If chlorine-water is added instead, a blue color develops at the bottom of the liquid, and above it layers present- ing a violet, a red and a yellow color will be observed. On adding an excess of chlorine-water the solution is decolorized. Pure biliverdin, like bilirubin, gives no characteristic band of absorption in alkaline solution. In acid solution, however, or in pure alcoholic solution, an indistinct band is observed at D, and one that is more pronounced near F. The substance is amorphous, or at least cannot be obtained in a pronounced crystalline form. On reduction, biliverdin is supposedly transformed into bilirubin, though this is denied by some observers. On oxidation with nitric acid biliverdin gives rise to the various colors which have already been described, beginning with blue. It gives Huppert's reaction directly. Isolation. — To prepare biliverdin, it is most convenient to start with a solution of bilirubin in the form of its sodium salt which has been exposed to the air until the original golden-yellow color has changed to a brownish green. The biliverdin is then precipitated THE BILE. 161 by adding an excess of hydrochloric acid. It is filtered off, washed free from all acid, dissolved in absolute alcohol, and precipitated by copiously diluting with water. Any bilirubin that may be present is removed by extracting with chloroform. Biliprasin.— Biliprasin as such, and biliprasinate of sodium, 'ac- cording to Dastre and Floresco, are also found in normal bladder- bile, and, as has been indicated, represent intermediary products of oxidation between bilirubin and biliverdin. The sodium salt, according to these observers, is a yellowish-brown pigment, and can be transformed into biliprasin through the agency of mineral acids, of acetic acid, and even of carbonic acid. On ex- posure to the air it passes over into the corresponding biliverdinate. To its presence the yellow color of the bile of calves and of other animals is supposedly due. Biliprasin itself is green, but can be transformed into the yellow salt by adding a few drops of an alkali, and this in turn vields the green pigment on treating with an acid. This reaction, according to Dastre and Floresco, explains the fact that yellow bile can become green without oxidation, viz., without the formation of biliverdin. According to older ideas, however, biliprasin is an oxidation-product of biliverdin, and is supjjosed to result from this with the inter- mediary formation of bilifuscin, as has alreadv been outlined. Bilifuscin is said to occur in gall-stones together with bilirubin. The formula which has been ascribed to it is that of bilirubin plus one or two molecules of water, viz., C82HwN"408 or C1HH20N,O4. It is of a dark-brown color, and is soluble in alcohol and the solutions of the alkaline hydrates, but insoluble in water and chloroform. In pure form it does not give Gmelin's reaction. Bilicyanin, or cholecyanin, is the blue pigment -which is formed during the oxidation of bilirubin arid biliverdin with nitric acid. It has been found together with the common bile-pigments in gall- 81 « taken from man. Its neutral and alkaline solutions give rise to three bands of absorption. One of these is located between C and I), near C; another about D; and a third very faint band midway between Hand E. In acid solutions two bands are seen between C and E. On treating the alcoholic solution of the pig- menl with an ammoniacal solution of zinc chloride a distinct fluor- escence is obtained. Its formula has not as yet been determined, and, according to some observers, indeed, bilicyanin does nut repre- sent a separate substance. Bilipurpurin.— This term has been applied to the red pigment which is formed from bilirubin and biliverdin on treating with nitric acid. Nothing is known of its properties or chemical com- position. Choletelin, or bilixanthin, is generally regarded as the final oxidation-product of the common bile-pigments. It is an amorphous brown substance, which i- soluble in alcohol, ether, chloroform, and 1,1 solutions of the alkaline hydrates, from which latter it can be n 162 THE DIGESTIVE FLUIDS. precipitated by the addition of acids. Its formula is given as C16H1?NA; Bilihumin is a pigment of unknown composition that has been found in gall-stones. It is insoluble in all organic solvents. Cholesterin. Cholesterin is not exclusively a product of the activity of the hepatic cells, but is found in other tissues as well. It has thus been encountered in the red corpuscles of the blood, in the plasma, in the yolk of eggs, in the semen, and in the secretion of the sebaceous glands, and is especially abundant in nerve-tissue. In the vegetable world also cholesterin is distributed. The liver is probably the organ through which the substance, wherever formed, is eliminated. Ultimately it appears in the feces. In the urine it is found only under exceptional conditions, and then only in very small amounts. Of its mode of formation nothing is known, but it is interesting to note that wherever cholesterin is found lecithin is likewise observed. In the brain a considerable amount of the substance occurs in com- bination with a fatty acid, cholic acid, from which it can only be separated by saponification. The amount of cholesterin which is found in the bile represents about 2 per cent, of the total solids. Normally it is held in solution owing to the presence of the biliary acids, but under pathologic con- ditions it may separate out in crystalline form, either in the gall- bladder itself or in the larger hepatic ducts, and then gives rise to the formation of stones. Of the origin of these concretions we know little. Very often they contain a nucleus of epithelial cells or of bacteria, around which the cholesterin, together with a variable amount of bile-pigment and mineral salts, becomes deposited. It is possible that they result owing to a temporary absence of the biliary acids, but this is only a supposition. The stones which are usually found in man are for the most part very rich in cholesterin, while the pigment-stones which are so common in cattle are less frequently seen in the human being. The common cholesterin which is found in the animal body has the composition C27H45.OH (Obermiiller), and is usually regarded as a monatomic alcohol. Of its structure, however, nothing is known. It combines with fatty acids to form compound ethers which are analogous to the fats, and in this form also, as has been shown (page" 67) cholesterin occurs widely distributed in the animal world. The common lanolin of wool-fat thus contains large amounts of such compound ethers, both of cholesterin and its isomeric compound isocholesterin. On treating cholesterin with concentrated sulphuric acid the substance gives rise to the formation of certain hydrocarbons, which are termed cholesterilins, and which are supposed to stand in a close relation to the terpene group. With iodine these bodies give a blue color. THE BILE. 163 Cholesterin usually occurs in the form of colorless, transparent plates, with ragged margins and angles, which are very characteristic. It is practically insoluble in water, dilute acids and alkalies, but dis- solves with ease in ether, chloroform, benzol, and in boiling alcohol. From its ethereal solutions it crystallizes out in the form of fine needles. It is further soluble in the essential and fatty oils, as also in the presence of biliary acids. Its crystals melt at 145° C. Tests. — Salkowski's Test. — A few crystals of cholesterin are dissolved in a small amount of chloroform and treated with an equal volume of concentrated sulphuric acid. The solution of cholesterin then first assumes a blood-red color, and then gradually turns to a violet red, while the sulphuric acid appears dark red and shows a green fluorescence. On pouring the chloroform into a shallow porcelain dish it turns violet, then green, and finally becomes yellow. The Test of Liebermann-Burckhard. — If a small amount of cholesterin is dissolved in about 2 c.c. of chloroform and is treated with 10 drops of acetic acid anhydride, and subsequently with concentrated sulphuric acid, the solution at first assumes a red, then a blue, and finally a green color. The latter develops at once if cholesterin is present only in traces. Isolation. — To prepare cholesterin for purposes of study, it is most convenient to isolate the substance from cholesterin stones. To this end, the concretions are finely pulverized and extracted with boiling water and then with boiling alcohol. From the alcoholic extract the cholesterin crystallizes out on cooling, and is then boiled with an alcoholic solution of sodium hydroxid, so as to saponify the fats which are at the same time present. The alcohol is then distilled off and the residue extracted with ether, which removes the cholesterin and leaves the soaps behind. On evaporat- ing this extract, after filtration, the substance is obtained in crystal- line form, and can be further purified by recrystallization from a mixture of alcohol and ether. Other Organic Constituents of the Bile. — In addition to the bodies already described, the bile contains also small amounts of lecithin, of palmitin, stearin, olein, and the soaps of the correspond- ing fatty acids. In ox-bile Lassar-Cohn found also traces of myristinic acid, CuH28021, which has heretofore only been observed in the spermaceti of whales. We further find traces of urea, and occasionally a diastatic ferment, which is by some observers regarded as identical with ptyalin. Its presence, however, is by no means constant, and it can scarcely be regarded as playing a rdle in the process of intestinal digestion. Larger amounts of urea, according to Hammai-ten, are found in the bile of the shark and the sturgeon. In decomposing bile cholin, glycerin-phosphoric acid and tri- incthvlaniin mav be observed, and are referable to the decomposition of lecithin (see pages 65 and 66). 164 THE DIGESTIVE FLUIDS. The Biliary Iron. If we recall the origin of bilirubin from hsematin, we should expect to find in the bile the iron which is liberated during the decomposition of the latter. Traces of iron, indeed, are constantly present, principally in combination with phosphoric acid. The amount, however, which is thus eliminated is far too small to repre- sent that which must of necessity be set free. Kunkel thus found that while 100 parts of hsematin correspond to 9 parts of iron, only 1.4—1.5 parts of iron appear in the urine for every 100 parts of bilirubin. But even if we add to this the amount which is eliminated in the urine and that which is excreted through the intestinal mucosa, we still find a very large deficit, and we are accordingly forced to the conclusion that the greater portion of the iron must be retained in the liver. But while such a retention must of necessity occur, we are profoundly ignorant of the manner in which it is accomplished. Of the form, also, in which the iron exists in the liver we know but little. That it is subsequently utilized in the construction of haemo- globin is quite likely, but not proved. Naunyn and Minkowski have observed that following poisoning with arseniuretted hydrogen, iron- containing pigments can be demonstrated in the liver. Latschen- berger speaks of the formation of choleglobins as antecedents of bili- rubin, and of the simultaneous appearance of iron-containing melanins in the liver; and Neumann has demonstrated the presence of an organic iron pigment, hsemosiderin, in old extravasations of blood and in thrombi together with hsematoidin ; but of the true nature of all these substances we practically know nothing. It is possible that the globin, which must of necessity be liberated during the decomposition of hsematin, takes up the iron which is set free from the latter ; but this also is a supposition, and further researches in this direction are urgently needed. CHAPTER VIII. THE PROCESSES OF DIGESTION AND RESORPTION. In the preceding chapter we have considered the various digestive fluids which are concerned in the transformation of those food- stuffs that are incapable of resorption as such into material which the body can utilize for purposes of nutrition, and we have seen that the most important agents which are here concerned belong to the class of the non-organized ferments. In the present chapter we shall study the action of these various substances upon the different classes of food-stuffs collectively and in somewhat greater detail, ami shall incidentally also consider the resorption of the final prod- ucts of digestiou from the gastro-intestinal canal. THE DIGESTION OF THE CARBOHYDRATES. The digestion of the carbohydrates is essentially effected in the small intestine through the agency of the amylolytic ferment of the pancreas, ptyalin. and the inverting ferments maltase, lactase, and invertin, which are in part also furnished by the pancreas, but are principally found in the enteric juice. In those animals in which ptyalin occurs in the saliva, amylolysis to a certain degree also takes place in the mouth and continues in the stomach until hydrochloric acid appears in the free state, when the ferment is rapidly destroyed. In man, however, the salivary digestion only plays a secondary rdle. it is true that the end-product of amylo- lytic digestion, viz., maltose, can probably always be demonstrated in the gastric contents, even after a few minutes following the ingestion of starch ; but it must be borne in mind that the trans- formation of starch into sugar docs not take place in distinct phases, but that one molecule of the substance may have already been changed to maltose, while another is as yet unaffected. In determining the intensity and the extent of amylolytic activity it i- hence unwarrantable to draw conclusions from mere qualitative tests, and it i- necessary to compare the amount of sugar which is actually formed with the amount of starch thai lias been ingested. in the majority of the purely carnivorous animals, as has been pointed out. the saliva contain- no digestive ferments, and, in such, carbohydrate digestion takes place exclusively in the small intestine. Through the action of the ptyalin of the pancreatic juice or of the saliva, as the case may be, the insoluble starch is firsl transformed into soluble starch or amidulin (amylodextrin), and ie then succes- I6fi 166 THE PROCESSES OF DIGESTION AND RESORPTION. sively decomposed by hydrolysis into erythrodextrin, achroodextrin, isomaltose, and finally into maltose, as shown by the equations : (1) (C12H O10)54 +3H20 =3[(C12H20O10)17.C12H22Ou] Amiduhn. Erythrodextrin. (2) 3[(C12H20O10)17.C12H22On] + 6H20 =9[(C12H20O10)5.C12H22Ou] Erythrodextrin. Achroodextrin. (3) 9[(C12H20O10)5.C12H22Ou] + 45H20 ■= 54C12H22Ou = 54C12H2„Ou Achroodextrin. Isomaltose. Maltose. Glycogen is similarly decomposed, and, like starch, gives rise to the formation of isomaltose and maltose. The celluloses, on the other hand, are not affected by ptyalin, nor, indeed, by any of the digestive fluids. As we shall see, however, they undergo a certain kind of fermentation under the influence of various bacteria, and as a result we find that in herbivorous animals, at least, only a frac- tion of the ingested cellulose reappears in the feces. Thus far a transformation into maltose or glucose has not been observed in the intestinal tract. It is stated that through the activity of the bacteria of the intesti- nal canal, viz., their ferments, a certain amount of starch is trans- formed into maltose, and that inversion of disaccharides to mono- saccharides is likewise brought about in this manner. To what extent this bacterial action can be regarded as representing an actual digestion is, however, an open question. The presence of bacteria in the intestinal canal is certainly not imperative, as has been con- clusively established by JNuttall and Thierfelder; and wre know, moreover, that bacterial action extends far beyond the action of digestive ferments, and that the further changes that can thus be effected do not serve a useful purpose. The normal inversion of the disaccharides is unquestionably brought about by the invertin, maltase, and lactase, which, as has been indicated, are partly fur- nished by the pancreas, but principally by the enteric juice. This inversion is likewise of the nature of a hydrolytic process, and may be represented by the general equation : Ci2H22On + H20 = 2C6H1206 Disaccharide. Monosaccharide. To judge from certain experiments which have been performed on animals, it appears, however, that amylolysis at least can also take place in the absence of the principal gland by which the ptyalin is formed, viz., the pancreas. For we find that following the extirpation of this organ or ligation of the pancreatic duct dogs are still capable of utilizing as much as from 47 to 71 per cent, of the starch ingested. As the dog's saliva contains no ptyalin, the amylolysis cannot be referable to a converting activity of the salivary glands. Whether in such cases the small amount of the ferment which is also furnished by the enteric juice is sufficient to transform the ingested starch into maltose is questionable, and there is some reason for supposing that the epithelial cells of the small intestine DIGESTION OF THE CARBOHYDRATES. 167 are likewise capable not only of causing the transformation of disac- charides into monosaccharides, but also of inverting dextrin to maltose. It has been shown that in animals with Thiry-Vella fistulse injected solutions of starch and cane-sugar rapidly disappear, although maltose cannot at times be demonstrated in the fluid. In what manner this change is effected by the epithelial cells is not known. In any event, however, it is necessary that the polysac- charides should be inverted to monosaccharides before passing beyond the mucous membrane of the gastro-intestinal tract. Resorption takes place primarily through the specific activity of the epithelial lining of the gastro-intestinal mucosa. The monosaccharides then enter the blood-current and are carried to the muscles and the liver, where they are transformed into glycogen and stored in a manner analogous to the reserve starch of the plant. This transformation, however, as well as the subsequent fate of the sugar, we shall have occasion to study in greater detail in a subsequent chapter. Neither the polysaccharides nor the disaccharides when introduced into the blood-current directly can be utilized by the body as such, and they are accordingly eliminated in the urine as foreign matter. The extent to which amylolysis can occur in the intestinal canal is remarkable, and far exceeds the ability of the liver and the muscle- tissue to transform the corresponding amount of monosaccharides into glycogen. As a consequence, the percentage of circulating sugar rises beyond the normal and glucosuria results. That disac- charides may pass the intestinal mucosa without being inverted is possible, but is certainly of exceptional occurrence. In such cases we must imagine that the intestinal epithelium has lost its specific power as a barrier to the passage of the sugars, as well as its ability to cause their inversion. As a result they pass this barrier by diffu- sion, and probably enter both the blood- and the lymph-current, and are then eliminated in the urine. A formation of glycogen from di- saccharides directly is apparently not possible. The rapidity with which resorption takes place in the small intes- tine seems t<> vary with the character of the sugar. In dogs Alber- tini tli us found that of 100 grammes of glucose, 60 grammes are absorbed in the course of the first hour, while of maltose and cane- sugar from 70 to 80 grammes and of lactose only 20 to 40 grammes disappear within the same period of time. The ingestion of very large amounts of disaccharides and mono- saccharides Leads to a general disturbance of intestinal digestion and results in diarrhoea. A corresponding amount of starch, on the other hand, is without effect in this respect. This is nodoubtowing to the fact that in the latter case inversion and resorption proceed paripa88U, so that the bacteria have hut little chance of setting up fermentative changes, which lead to the formation of substances that directly increase the peristalsis owing to their irritating prop- erties. In the presence of abnormally large amounts of sugars as such, on the other hand, resorption is not sufficiently rapid, and in 168 THE PROCESSES OF DIGESTION AND RESORPTION. the presence of the increased amount of pabulum an increase of bacterial fermentation beyond the normal takes place. As a result the various acid decomposition-products of the carbohydrates such as lactic acid, butyric acid, acetic acid, formic acid, succinic acid, together with carbon dioxide, methane, and hydrogen, are formed in increased amounts, and are responsible for the resulting pathological conditions. DIGESTION OF THE ALBUMINS. The digestion of the albumins takes place in the stomach and in the small intestines under the influence of the pepsin and the hy- drochloric acid of the gastric juice, and the trypsin of the alkaline pancreatic juice, respectively. We know that the presence of the former is not altogether necessary, however, and that the pancreatic juice is in itself quite sufficient to accomplish the digestion of the albumins alone, but under normal conditions the gastric juice also plays a part. Of the relative extent to which the one or the other enters into this process our knowledge is not complete. We may imagine that in the stomach a primary dissolution of the solid con- stituents of the food takes place, and that the soluble products which are thus formed are further digested by the pancreatic juice. This actually occurs to a certain extent, but we further know that in the stomach certain albuminous food-stuffs are decomposed, with the liberation of constituents which are insoluble in the gastric juice, and which pass the pylorus as such and are then modified by the pancreatic juice. Tryptic digestion, moreover, is far more extensive than peptic digestion, so that we may well conclude that the latter essentially represents a preliminary phase of digestion; and that the digestion proper, viz., the transformation of the albumins into those final products which can be directly utilized by the body, occurs under the influence of the trypsin of the pancreatic juice. For convenience' sake, we shall study the action of the gastric juice and of the pancreatic juice separately upon the various classes of albumins, as the digestive products which are formed are somewhat different in the different classes. In every case we shall follow the fate of these various substances to the final products, as we obtain them artificially in digestive experiments in vitro ; but we must bear in mind that such experiments cannot reproduce what actually takes place in the living body, where resorption is constantly going on, and where the various digestive processes in a manner supple- ment each other, and conditions overlap. Whenever possible we shall attempt to point out where gastric digestion probably ceases and pancreatic digestion begins, but such an attempt must of necessity be more or less crude. Digestion of the Native Albumins. Gastric Digestion. — In the stomach the native albumins, if in- troduced in the coagulated state, are first transformed into a soluble DIGESTION OF THE ALBUMINS. 169 form, and at the same time or immediately following their dissolu- tion they undergo the process of denaturization — i. e., they are trans- formed into syntonins or acid albumins, and as a consequence all individual characteristics which previously existed are lost. This transformation is essentially referable to the hydrochloric acid of the gastric juice, and can be brought about artificially in the absence of pepsin. In such an event, however, a higher grade of acidity and a higher temperature are required. The presence of the pepsin ob- viates such a necessity. A possible explanation of this phenomenon is afforded by the modern doctrine which teaches that the action of enzymes merely consists in hastening the rapidity of reaction. On continued exposure to the acid gastric juice the syntonins are decomposed by hydrolysis into albumoses, and finally into peptones, and it is to be noted that this result also can be effected by hydro- chloric acid alone ; but here again the presence of the pepsin does away with the necessity of employing stronger solutions of the acid and a higher temperature. This decomposition may be compared to the inversion of the poly- saccharides to monosaccharides, and here, as there, intermediary prod- ucts are formed which differ not only from the syntonins, but also from each other. Kuhne and his school, who have largely contribu- ted to our knowledge of these products, have suggested their divi- sion into two classes, viz., the primary and secondary albumoses, according to their nearer or more distant relationship to the original albumins. This division has been generally accepted. Further researches have shown that two distinct varieties of the primary albumoses exist, namely, a proto-albumose and a hetero-albumose, each of which on further decomposition is supposed to give rise to a secondary albumose or deutero-albumose, from which in turn a pep- tone is derived. According to Neumeister, the digestion of the native albumins may accordingly be represented by the following schema : Native albumin. I . Syntonin. Proto-albumose. Hetero-albumose. I I Deutero-albumose. Deutero-albumose. I I Peptone Peptone. With the formation of peptones peptic digestion, according to Kuhne, comes to an end. Supposing this to be the case, and bear- ing in mind that the albuminous molecule contains certain atomic; groups — the hemi-groups — which can be readily broken down with trypsin, with tin- formation of amido-acids, while others — the anti- groups — are more resistant, the conclusion suggests itself that in the albumoses and the peptones which result from the action of pep-in both of these groups musl still be united. Such peptones Kuhne 170 THE PROCESSES OF DIGESTION AND RESORPTION. has accordingly termed amphopeptone. It has further been observed that during peptic digestion a certain amount of an insoluble albu- minous substance usually remains behind, which is characterized by its great resistance to further decomposition by means of pepsin and hydrochloric acid. This substance Kiihne has termed anti-albumid, and he supposed that in it certain anti-groups were already isolated. Within recent years our conception of the decomposition of the albumins as just outlined has undergone certain modifications, and through the researches of the Strassburg school, and notably those of Pick, Zuntz, and others, our knowledge of the products of diges- tion has been much extended. It has thus been shown that Kiihne' s views, as regards the formation of the primary albumoses from the syntonins, were in the main correct, and that proto-albumose as well as hetero-albumose develop from the latter simultaneously, and are not derived the one from the other, as was once supposed. These observers have further shown, however, that one deutero-albumose at least is also formed at the same time, and which, in contradistinc- tion to the other two primary albumoses, contains the carbohydrate group of the original albumin. This deutero-albumose is spoken of as the deutero-albumose-B. Whether still other primary albumoses exist is as yet an open question, but there is reason to suppose that this may be the case, and it appears, moreover, that not all albumins give rise to the same primary albumoses, and that distinct quantita- tive differences further exist. These primary albumoses on further digestion with pepsin give rise to secondary albumoses. But, while according to Neumeister's schema one deutero-albumose only results from every primary albumose, the studies of Pick clearly show that proto-albumose yields two secondary albumoses, which have been termed albumoses A and B. This B-albumose, how- ever, differs from the primary albumose which is designated by the same letter in containing no carbohydrate group, and for conveni- ence' sake we shall speak of the secondary product as albumose-B'. The hetero-albumose similarly yields three secondary albumoses — that is A, B', and C. The further decomposition of the primary albumose B finally has not been studied in detail, and it may indeed be questionable whether it actually represents one single substance. On prolonged digestion with pepsin it yields a peptone, A, which is insoluble in alcohol, and an albumose which is manifestly identical with the secondary albumose B'. Of the subsequent fate of the secondary albumoses we know little, as these bodies have not as yet been studied in this direction. On prolonged digestion of the primary albumoses with pepsin substances are obtained which, ac- cording to Kiihne' s definition, would correspond to peptones. In the mixture of the secondary albumoses, which Pick obtained from hetero-albumose, peptone was found, which was soluble in alcohol, and which he termed peptone-B. From proto-albumose, on the other hand, peptone-like bodies were obtained which gave an intense biuret reaction, and which, of course, could not correspond to the DIGESTION OF THE ALBUMINS. 171 peptone-A. For the present we shall speak of this also as peptone B. According to the above considerations, we may possibly represent the process of peptic digestion by the following schema, which, how- ever, is only provisional, and merely represents the facts as just outlined : Native albumin I Syntonin Proto-albumose Hetero-albuinose Deutero-alburuose-B Anti-albumid I II II Deutero-albuinoses Deutero-albumoses Deutero-albumose-B' I I I I I A W A B' C Proto-albumose. Contains 25 per cent, of total nitrogen in basic form. Yields much tyrosin, viz., indol and skatol. Peptone-B Peptone-B Peptone-B Peptone-A Whether or not the different albumoses which arc thus formed during the process of peptic digestion are qualitatively the same, irrespective of their origin, we do not know, but it is likely that certain differences exist. Quantitative variations also occur without doubt, as is a priori suggested by the varying amounts of the amido- acids of the fatty series and of the aromatic group which are con- tained in the original albuminous molecule, as also by the absence of a carbohydrate group in some of the albumins, the digestion of which is quite analogous to that of the native albumins. The principal points of difference between proto-albumose and hetero- albumose are here given. Hetero-ai.bumose. Contains 39 per cent, of the total nitrogen in basic form. The aromatic group is present only to a slight extent in a form which can give rise to tyrosin or indol. Yields much leucin and glycocoll. | Yields but little leucin and glycocoll. It is thus manifest that those albumins which are especially rich in aromatic groups will furnish a correspondingly larger amount of proto-albumose than those in which the fatty acid radicles arc prin- cipally found, and which accordingly yield a' large amount of hetero- albumose. That these differences will further become manifesl in the secondary product- of decomposition i-, of course, apparent. Of the character of the final products of peptic digestion, in the sense of Ktihne, viz., the peptones, our knowledge is very imperfect. While according to olaer views amphopeptone was' thought to represent a chemical unity, Pick and others have Bhown thai two peptones at least resull from the action of pepsin on albumin, one of which is, soluble in alcohol, while the other is insoluble. These bodies, however, are most likely n,,t units in themselves, but prob- ably represent mixture- of other substances, which for the most part are as yet unknown. 172 THE PROCESSES OE DIGESTION AND RESORPTION. Until quite recently it was thought that peptic activity ceased with the formation of amphopeptone, in the sense of Kiihne, and that other nitrogenous decomposition-products beyond those already considered were not formed during this process. In the light of modern investigations, however, this view can no longer be upheld, for it has been shown that in a very early stage of digestion already, and before the formation of peptones and the deutero- albumose-C, at least, a very considerable portion of the albuminous nitrogen is split oif in the form of substances which no longer give the biuret reaction. It is known, moreover, that the greater portion of the final decomposition-products which result from the action of pepsin do not consist of either albumoses or peptones, but appar- ently of these same bodies which do not give the biuret reaction. Of their nature, however, and their mode of origin, nothing is known. From what has been said, it is thus clear that the peptic disinte- gration of the albuminous molecule is very much more complicated than was formerly supposed, and much work must still be done before we can form a clear idea of the entire process. As regards the extent to which peptic digestion is carried in the stomach, our knowledge is likewise not complete, but there is reason for the assumption that ordinarily the process does not extend beyond the formation of the primary albumoses. We find, as a matter of fact, that while these appear within the first half-hour of digestion, the secondary albumoses, in experiments in vitro at least, are not formed until after the second hour, beyond traces, and it is to be noted that a more energetic formation in fact does not occur within the period of time during which remnants of an ordinary meal are usually found in the stomach. We are thus forced to the conclusion that if a more extensive decomposition of the albumins is necessary before resorption can take place, this must occur in the small intestine under the influence of the pancreatic juice. But we may also suppose that the epithelial lining of the gastric mucosa can bring about the further transformation of the primary albumoses of itself. We know, as a matter of fact, that in the resorption of the albumins, as in that of the carbohydrates, the epithelial cells play an active part, and that absorption by osmosis under normal conditions can scarcely enter into consideration. We have seen, moreover, that an inversion of polysaccharides to monosaccharides can thus be effected, and we have reason to suppose that a digestion of albumins also can be accomplished in the same manner. It is even claimed that a resorption of native albumins can take place in the absence of the proteolytic ferments, and that, unlike the poly- saccharides, solutions of such albumins can be injected directly into the ' blood-current without causing albuminuria. We have seen that the presence of the disaccharides in the blood at once leads to their elimination, and that the body is manifestly incapable of caus- ing their inversion and further transformation into glycogen when they are present as such. As the introduction into the blood-current DIGESTION OF THE ALBUMINS. 173 of certain albumins, such as the syntonins, obtained from myosin, fibrin, etc., does not lead to their elimination in the urine, and as the blood manifests a very strong tendency to maintain its composition uniform, it has been argued that these albumins are probably re- tained in some organ of the body, such as the liver, and may here be transformed into material which can be utilized for purposes of nutrition. Such an assumption seems to me entirely unwarrantable, however, as it is solely based upon the non-appearance of these albumins in the urine in the state in which they were introduced. Egg-albumin, it is true, is immediately eliminated under such con- ditions, and it might be concluded that the kidneys could also eliminate those other forms, if the body were incapable of utiliz- ing them as such. This, however, does not follow, for we know that the introduction of egg-albumin in large amounts into the stomach leads to its appearance in the blood as such, so that the conditions here are reversed ; but it would be manifestly inadmis- sible to conclude from this observation that because egg-albumin can pass the epithelial barrier, as such, it cannot be utilized within the body itself or cannot be digested in the stomach and intestines. That the introduction of such albumins which are normally present in the blood does not lead to albuminuria is, of course, not sur- prising ; but to conclude that syntonin can be utilized by the body directly because it is not eliminated in the urine in the same form is, as I have said, unjustifiable. On the other hand, we must admit that the resorption of native albumin can occur in the absence of the proteolytic ferments, and it is more than probable that during this resorption the epithelial cells bring about a rearrangement of the atomic groups of the albu- minous molecule, so that the ultimate result is the same as though the hydrolytic decomposition had proceeded further under the influ- ence of the ferments and resorption had then occurred. Of the extent to which epithelial activity enters into the process of diges- tion, however, Ave know nothing, but it is likely that under normal conditions fermentative digestion prevails, and that the function of the epithelial cells principally consists in transforming the albumins and peptones into material which can be utilized by the body for purposes of nutrition. That this transformation actually takes place within the epithelial cells which line the gastro-intestinal mucosa is now established beyond a doubt. Formerly it was sup- posed that this change occurred within the liver, especially as neither albumoses nor peptones can normally be found in the periph- eral circulation. lint this ha- since been disproved in many ways. It ha- thus been found that in animals which are killed by bleeding from the portal vein ;it a time when peptones arc abundantly present in the intestines no peptones can be found in the blood. Whenever peptones are introduced into the blood artificially, or whenever they are formed beyond the intestinal mucous membrane, as under certain pathologic conditions, they are invariably eliminated in the urine as 174 THE PROCESSES OF DIGESTION AND RESORPTION. foreign material. The same indeed holds good of the albumoses, and both are known to be distinctly toxic. This fact in itself shows that after the stage of denaturization has been passed a direct resorp- tion of the resulting products of digestion cannot take place by simple osmosis, and that certain changes must occur in the gastro- intestinal walls whereby these products are deprived of their toxicity. Ludwig and Salvioli, moreover, have shown that by sub- stituting an artificial circulation in an isolated intestinal loop for the normal, no peptones or albumoses could be found either in the blood returning from the loop nor after some time in the gut itself, although an abundant amount had been previously introduced. The same result may be demonstrated by finely hashing a piece of care- fully cleansed and perfectly fresh intestine, and placing this in a solution of peptone in the defibrinated blood of the same animal. It can then be demonstrated that after a relatively short time already a considerable amount of the peptones has disappeared, and is, moreover, not stored as such in the tissue. While it is thus definitely proved that albumoses and peptones are transformed into material which the body can utilize for pur- poses of nutrition, we know absolutely nothing of the manner in which this transformation is effected, nor of the products which thus result. According to some observers, a further decomposition of the albuminous molecule into smaller atomic groups takes place, but thus far no substances have been found in the tissues and fluids of the body in sufficient amount to favor such an explanation. On the other hand, it is possible, as in the case of the carbohydrates, that a polymerization of peptone radicles occurs, and that albumins again result which are comparable to those from which they are derived. As I have stated, there can be no doubt that the epithelial cells which line the gastro-intestinal tract are capable of effecting the transformation of syntonin, and possibly even of native albumins into forms which can be utilized by the body directly ; but under normal conditions it appears that the action of the epithelium scarcely begins before the albumins have been digested by the fer- ments to the stage of albumoses. We have seen that in the stomach the process of digestion scarcely goes further than the formation of the primary albumoses, and the question naturally suggests itself: Are the primary albumoses resorbed in the stomach already or is it necessary that they be further exposed to the action of the pan- creatic juice? To this question an ultimate answer cannot be given, but it is likely that resorption takes place in the stomach to a limited extent only, and that the greater portion of the primary albumoses is further decomposed in the small intestine, where the resorption processes are most active. Much work remains to be done in this direction. . 2. Tryptic Digestion of the Native Albumins. — Upon enter- ing the small intestine the acid gastric contents are rendered alkaline, the pepsin is destroyed, and tryptic digestion begins. DIGESTION OF THE ALBUMINS. 175 The material which is exposed to the action of the pancreatic juice consists in part of the primary albumoses which were formed in the stomach, in part of Kiihne's anti-albumid, and in part of -vntonin and of native albumins, in soluble or insoluble form, which have escaped the action of the gastric juice. The latter are first dissolved, and together with the syntonins transformed into alkaline albuminate. This result, analogous to the formation of the syntonin in acid solution, as well as the further decomposition of the alkaline albuminate, is no doubt primarily referable to the action of the alkalies of the pancreatic juice, and merely hastened by the ferment which is at the same time present. But unlike the action of the gastric juice, tryptic digestion immediately leads to the formation of deutero-albumoses without the intermediary production of primary albumoses in the sense of Kiihne. According to older views, amphopeptone then results. Subsecpnently the anti- and the hemi- groups become separated with the formation of antipeptone and hemipeptone, respectively. Hemipeptone, hoAvever, is manifestly only a hypothetical substance, as, in experiments in vitro at least, no substance of this character can be obtained. Instead we find amido-acids, tryptophan, and other substances, which are as yet imperfectly known whenever the process of digestion has extended beyond the formation of deutero-albumoses. In the living organ- isms, it is true, these products are found only in traces, and we might hence imagine that hemipeptone is here resorbed as soon as formed. But beyond the absence of amido-acids as just stated, we have no actual proof of such an occurrence. Antipeptone, on the other hand, viz., a substance or substances which still give the biuret reaction, but which, in contradistinction to the albumoses, cannot be precipitated by salting with ammonium sulphate, can always be obtained in experiments in vitro. As regards the chemical nature of the antipeptone, however, opinions differ. Kiihne and Chittenden long ago questioned the chemical unity of the body, and Kutscher has recently announced that he WES able to isolate the three known liexon bases, as also small amounts of leucin, tyrosin, and asparaginic acid from the substance. In addition, >till other products of digestion were found, which were not identified, however. In my own laboratory I have attempted to repeal Kutscher's work, together with Dr. Amberg, and we have found that, as a matter of fact, Kiihne's antipeptone does not represent a chemical unity, but owing to Kutscher's insuffi- cient working directions we were unable to confirm his results in detail. It manifestly consists of two portions however, one of which can be precipitated with phosphotungstic acid. This portion repre- —*-iit — about 30 per cent, of Kiihne's antipeptone, and according to Kutscher consists to the extent of 30—31 per cent, of hexon bases. Siegfried's claim that antipeptone is identical with his carnic acid, and may be represented by the formula C10H18N8Ow is, in view of Kutscher's work and my own experience, altogether untenable. 176 THE PROCESSES OF DIGESTION AND RESORPTION. That it may constitute a fraction of Kuhne's antipeptone, however, and perhaps even the greater portion, on the other hand, is quite possible. We have seen in the preceding section that in the stomach the digestion of the native albumins scarcely extends beyond the forma- tion of the primary albumoses. Their subsequent fate, on exposure to tryptic digestion, has recently been studied by Pick. From his researches it appears that the proto-albumose here apparently yields the same deutero-albumoses as those which were obtained on peptic digestion, and here as there the secondary albumose-C was want- ing. Hetero-albumose gives rise to the formation of the deutero- albumoses A and B', but curiously enough the C-albumose does not appear. The fate of the primary deutero-albumose-B, on the other hand, as also of the deutero-albumose-C, is as yet unknown, but it is likely that the former is here also transformed into the deutero- albumose-B' and into peptone-A. Especially interesting further is the observation of Pick that the proto-albumose, as also the hetero- albumose, rapidly disappears, so that after twenty-four to thirty-six hours traces only can be found in vitro, while with peptic digestion a complete transformation into secondary albumoses is scarcely effected even after several weeks. Of the subsequent change which Kuhne's anti-albumid under- goes little is known, but it is possible that the substance is first transformed into anti-deutero-albumose, and then contributes to the formation of antipeptone. As regards the distribution of anti- and hemi-groups in the orig- inal albuminous molecule, it appears that both are present in about the. same proportion. With beginning digestion in the stomach, however, this relation is disturbed, and we find that the hetero- albumose contains rather more nitrogen in the basic form — 39 per cent. — than the proto-albumose — 25 per cent. Of the further sepa- ration of the two groups, we know nothing, and it is indeed a matter of doubt whether a complete separation occurs at any time preceding the formation of antipeptone, and, as has been seen, this consists in part at least of amido-acids, which, however, are here present in the free state. I have pointed out that in the small intestine amido-acids arc found only in traces, and that the existence of a hemipeptone is extremely doubtful. The question hence arises : In what form are the digestive products of the albumins here absorbed ? As anti- peptone, or its decomposition-products, so far as we know, are not eliminated in the feces, we may conclude that if this complex of digestive products is formed at all in the living body, its absorption must take place in the intestinal canal. But as Ellinger has shown that it is impossible to maintain the nitrogenous equilibrium by the administration of hexon bases alone, this portion of the antipep- tone can scarcely enter into consideration from the standpoint of nutrition. Whether or not those other bodies, which appear to DIGESTION OF THE ALBUMINS. 177 constitute the greater portion of the antipeptone and of the nature of which nothing is as yet known, can serve as food-stuffs in the narrower sense of the term, remains to be seen. Under such con- ditions it is perhaps wiser to conclude that the deutero-albumoses represent those products of tryptic digestion which actually play a role in the process of nutrition, and to suppose that a formation of antipeptone is essentially a process which takes place in vitro, and normally occurs only to a slight extent in the living organism. This, however, is a mere assumption and lacks experimental proof. In the account of the process of albuminous digestion, as out- lined in the foregoing pages, it has in a manner been assumed that the digestive products which are thus formed are identical, irrespective of their origin. Strictly speaking, this is not the case, however, as we know as a matter of fact that the distribution of the nitrogen in the original albuminous molecule differs in the different albumins. The amount of hexon bases, moreover, which may be obtained from the various albumins is not constant, and we have reason to think that the number of carbohydrate groups also is more or less variable. Such differences, it is true, have thus far been mainly established for the original substances, but it is, of course, manifest that the corresponding products of digestion cannot be the same. With the usual analytical methods, however, these differences are scarcely apparent, and ammonium sulphate, which is now so extensively uti- lized in separating the various albumins from each other, apparently acts in the same manner with these products, no matter what their origin may have been. In the accompanying tables I have collected various data from the literature to show the difference in the distribu- tion of the nitrogen and the corresponding amount of arginin which can be obtained from some of the more important albumins. Amido- Diamino- Monamino- nitrogen. nitrogen, nitrogen. Crystallized egg-albumin (Hansmann) 8.53 21.33 67.80 Crystallized serum-albumin (Hausmann) ■ . . . 6.34 Serum-globulin (Hausmann) 8.90 24.95 68.28 Casein [Hausmann) 13.38 11.71 75.98 Gelatin (Hausmann) 1.61 35.83 62.56 Proto-albnmoae of fibrin (Pick) 7.14 25.42 68.17 Hetero-albumose of fibrin (Pick) 6.45 38.93 57.40 Arginin. Keratin 2.25 per eent. Clutin 2.6 '' " Conglutin 2.75 •' " Albumin (yolk) 2.3 " " Albumin (white of egg) 0.8 " " Dried Mood-serum 0.7 " " Casein 0.25 " " To distinguish the different albumoses which arc derived from the true albumins, including those which result on the decomposition of the proteida from the albuminoid albumoses, Chittenden has in- 12 178 THE PROCESSES OF DIGESTION AND RESORPTION troduced the generic term proteoses, and according to their individual origin divides the proteoses into globulinoses, vitelloses, fibrinoses, myosinoses, seroses, etc. Their stages of digestion are further indi- cated by the prefixes proto-, hetero-, and deutero, so that we speak of a proto- and a hetero-vitellose, of deutero-caseoses, etc. Digestion of the Proteids. The digestion of the proteids, or of the nucleo-albumins, the glucoproteids, and the haemoglobins at least, like that of the native albumins, begins in the stomach. Here the separation of the non- albuminous pairling is first effected, and is then followed by the digestion of the liberated albumins. This digestion is in all respects analogous to that of the native albumins proper. Syntonins are first formed, then primary albumoses, subsequently secondary albumoses, and finally peptones — i. e., bodies which still give the biuret reac- tion, but which in contradistinction to the albumoses are not pre- cipitated by salting with ammonium sulphate. The individual products which thus result from the proteids have not as yet been studied with the same care as those which are derived from the native albumins, but it is likely that here also Kuhne's schema of digestion does not apply in its original form. Individual differ- ences also no doubt exist between the various digestive products according to their origin, but of these also we know but little. Of special interest are the earlier phases of digestion of the casein of milk. This normally exists in the milk in solution as a neutral calcium salt. In the stomach a transformation into the corresponding acid salt is then first effected by the hydrochloric acid of the gastric juice, and followed by the action of the chymosin. According to Hammarsten, this effects a partial decomposition of the soluble acid salt with the formation of calcium-paracasein, and a small amount of an albumose-like posset albumin. The paracasein is then precipitated and decomposed, with the formation of the cor- responding paranuclein and the albuminous pairling. Of the fate of the non-albuminous components of the proteids but little is known. The paranuclein of casein, it is stated, undergoes solution on continued digestion in vitro, but is at the same time de- composed with the formation of a small amount of orthophosphoric acid and an organic acid, which likewise contains phosphorus. Of this, however, nothing further is known (see also page 77). The nucleins proper are not digested in the stomach and remain undissolved. Under the influence of the pancreatic juice casein is digested in very much the same manner as with the gastric juice, but in this case the transformation into paracasein is brought about through the influence of the chymosin of the pancreas in an alkaline medium. Caseoses then result as with the common native albumins, and finally peptone is formed. DIGESTION OF THE ALBUMINS. 179 Aside from its proteid character, casein differs from the common albumins in one very important particular, namely, in the exceed- ingly small number of hexon groups which the substance apparently contains. In its antipeptone, therefore, which has not as yet been studied in this direction, however, we can hence not expect these bodies beyond traces. The glucoproteids and the haemoglobins are decomposed as in the case of the gastric juice, and the albuminous components further digested like the native albumins. The individual products, how- ever, which are thus formed have not as yet been studied in detail. The true nucleins, which, as we have seen, escape gastric diges- tion and which do not undergo solution in the stomach, are dissolved by the pancreatic juice and are decomposed with the liberation of the contained nucleinic acids and the albuminous radicles. The latter are further digested in the usual manner. The paranucleins similarly undergo dissolution, and are probably decomposed as already indicated. Of the subsequent fate of the non-albuminous pairlings of the proteids in general, however, but little is known. Digestion of the Albuminoids. The only albuminoids which are digested in the stomach in the case of the higher vertebrate animals are collagen and elastin. Both then give rise to protogelatose and proto-elastose, respectively, while curiously enough hetero-albumoses are not formed. The cor- responding deutero-albumoses then result. But while the deutero- gelatose subsequently gives rise to the formation of peptone — the BO-called glutin-peptone — a similar transformation of the deutero- elastose apparently does not occur. In the small intestine, under the influence of the pancreatic juice, collagen and elastin can also be digested, and it is note- worthy that the transformation of the gelatins into glutin-peptone is apparently more readily effected than that of any other albumin- ous substance. Unlike the gastric juice, however, the pancreatic secretion i- in itself not capable of transforming the native collagen into gelatin. This change must hence be first effected artificially or in the stomach before its further digestion can occur. The peptonization of elastin in the pancreatic juice likewise ceases with the formation of deutero-elastose, while gelatin is transformed /'// vitro, &\ least, into glutin-peptone. According to Chittenden, this i- not further decomposed by the trypsin, and amido-acids are hence not formed. This i- rather remarkable, as on hydrolytic decom- position with mineral acids gelatin yields lencin, asparaginic acid, glutarainic acid, and considerable amount- ofglycocoll. The existence of aromatic groups in the original molecule, on the other hand, is very doubtful, and as a matter of fid it i- impossible to obtain either tyrosin, or indol, or skatol, from the substance, even on bac- terial decomposition. Hexon groups, however, are largely present. 180 THE PROCESSES OF DIGESTION AND RESORPTION. The fact that neither elastin nor collagen (gelatin) gives rise to the formation of hetero-albumoses is of special interest in view of the fact that both substances cannot be regarded as true food-stuffs, viz., thev are unable to maintain the nitrogenous equilibrium of the higher animals when exclusively used. Whether or not this is due tothe absence of the aromatic group in the gelatin, and its presence only in small amounts in elastin, is not decided. The hexon bases are manifestly of no moment in this connection, as gelatin, at least, yields rather more arginin than any of the common albuminous food- stuffs, and, as I have pointed out, Ellinger has shown that the hexon bases alone are likewise not capable of maintaining nitro- genous equilibrium. Future researches, no doubt, will explain this essential difference between the albumins proper, including the proteids and the albuminoids. A digestion of other albuminoids, notably of the keratins, does not take place in the small intestine of the higher animals, while some of the invertebrates, such as the common house moth, are manifestly capable of utilizing these also for purposes of nutrition. In contradistinction to collagen and elastin, the keratins yield a relatively large amount of tyrosin, in addition to leucin, aspara- ginic acid, and glutaminic acid, on hydrolytic decomposition. DIGESTION OF THE FATS. Notwithstanding innumerable researches in this direction, our knowledge of the digestion of tats and their subsequent absorption is still imperfect. This is true more especially of the role which the pancreatic juice and the bile play in the process. That both secretions are actively concerned in the digestion of the tats can- not be doubted. Minkowski and Abelmann have thus shown that following extirpation of the pancreas in dogs the absorption of fats ceases altogether, if we except the fat of butter, of which from 28 to 53 per cent, can still be utilized. Other observers, it is true, obtained results which differ somewhat from those of Minkowski ; but in all cases it could at least be demonstrated that in the absence of the pancreatice juice the absorption of fats is impeded. In other experiments in which the bile was prevented from entering the intestinal tract it was similarly demonstrated that only one-seventh to one-half of the fat was resorbed. while the remainder, princi- pally in the form of fatty acids, appeared in the feces. This is the more remarkable, since Munk has shown that the fatty acids can be absorbed as such, and are retransformed into neutral tats in the intestinal mucous membrane. While the importance of the pancreatic juice and the bile in the digestion of the fats is thus manifest, we have no clear conception of the manner in which their presence favors their resorption. It is generally stated that this is primarly dependent upon a previous emulsitication, which is supposedly effected through the activity of DIGESTION OF THE FATS. 181 the steapsin of the pancreatic juice. This, us we have seen, brings about a partial decomposition of the neutral fat, and it is thought that the resulting fatty acids combine with the alkalies of the pan- creatic juice and the bile to form soaps, and that these in turn emulsify the neutral fats. We might hence conclude that the pres- ence of the alkali in these secretions is the essential factor which renders the absorption of the fats possible. I have shown that the succus entericus is manifestly incapable of furnishing this in suffi- cient amount, as large quantities of the fatty acids appear in the feces as such when either the bile or the pancreatic juice is pre- vented from entering the intestinal canal. It has been found, on the other hand, that even in the absence of the pancreas the absorp- tion of fats may be fairly normal providing that fresh pancreas, finely hashed, is given the animal together with the fatty food. Kiihne, moreover, has demonstrated that pancreatic juice, even after having been rendered feebly acid, is still capable of emulsifying fats. He also pointed out long ago that while the secretion from permanent pancreatic fistula? can bring about the emulsification of fats, the juice obtained from temporary fistulse is much more potent in this respect. He accordingly concluded that this property is essentially referable to albumins which are present in solution, and he showed, moreover, that these are capable of bringing about the emulsification of fats even in feebly acid solution. This opin- ion is shared by Minkowski and others, and the fact that the fat of milk can be resorbed much more readily than other fats is now generally explained upon this basis. However this may be, the fact remains that resorption of fats can only proceed in a normal manner if emulsification has previously taken place. At present there is a tendency among physiologists to assume that the digestion of the fats presupposes their decomposition into fatty acids and glycerin. The former, in the form of soaps, are then supposedly absorbed and reconstructed into neutral fats in the epi- thelial cells, which possibly obtain the requisite amount of glycerin from the intestinal lymph-glands. It must be admitted that this view has much in its favor, but it cannot as yet be regarded as an established fact. Of the manner in which resorption occurs, we now know that, contrary to the former supposition, according to which the leucocytes play an active part in this process, the epithelial cells are of prime importance, and it seems that even though the neutral fats may be absorbed directly, a synthesis of fats from fatty acids or their soaps can here also take place. This indeed is the prevailing idea at the present time, and, as I have said, the glycerin which is necessary to effect this synthesis is in all probability derived from the lymph- glands of the intestinal tract, but it is also possible that it may be formed in the cells themselves or may be absorbed together with the soaps. It i- stated that the bile assists in the resorption of fat from the 182 THE PROCESSES OF DIGESTION AND RESORPTION. intestinal tract, but of the manner of its action in this respect we know little that is definite. After resorption from the intestinal canal the fats are transferred from the epithelial cells to the lymph- vessels, and subsequently reach the general circulation through the thoracic duct. The lecithins, like the fats, are decomposed by steapsin into their components, viz., into glycerin-phosphoric acid, the corresponding fatty acids, and cholin. The former is then absorbed, and appears in part at least in the urine as such. The fatty acids after saponifi- cation are then similarly absorbed and reconstructed into neutral fats, while cholin is decomposed by the bacteria which are present in the intestines, with the formation of carbon dioxide, methane, and ammonia. CHAPTEK IX. ANALYSIS OF THE PRODUCTS OF ALBUMINOUS DIGESTION. To demonstrate the formation of the various products of albu- minous digestion and to separate the individual substances from each other, the following plan of work may be adopted : THE PRODUCTS OF PEPTIC DIGESTION. One hundred grammes of moist fibrin that has been thoroughly washed in running water are placed in 1000 c.c. of an 0.3 per cent, solution of hydrochloric acid, to which a few grammes of pepsin have been added. The mixture is kept at a temperature of 40° C, and may be examined for the primary products of digestion after about two hours, while the secondary products can only be demon- strated after a longer period of time. From time to time it is then necessary to ascertain whether free hydrochloric acid is still present, and to add an additional amount whenever it is wanting, so that the acidity referable to free acid remains about the same during the entire period of digestion. The occasional addition of a little pepsin is also advisable. The first specimen for examina- tion is taken after two hours. The liquid is filtered and neutral- ized with a dilute solution of sodium hydrate. The precipitate which thus forms consists of syntonin and is filtered off. The fil- trate is rendered feebly acid with very dilute acetic acid, treated with an equal volume of a saturated solution of common salt, and boiled. Any native coagulable albumin that may be present is thus precipitated and is filtered off on cooling. The solution is again made neutral and treated with an equal volume of a satu- rated solution of ammonium sulphate. In this manner the primary albumoses of Kiihne are precipitated, and are filtered off after stand- ing for about one-half hour. To separate the proto-albumose from the hetero-albumose, the precipitate is dissolved in hot water and treated with an equal volume of 95 per cent, alcohol. On standing, the hetero-albumose separates out, while the proto-albumose is found in tin; aleoholic filtrate. It is purified by repeated solution in hot water and precipitation with alcohol. To isolate the proto-albumose, the alcoholic filtrate is evaporated to dryness on a water-bath, or the alcohol h distilled off in the vacuum. The remaining material is repeatedly dissolved in water ami treated with alcohol until the alcoholic solution remains clear on standing. To this end, it is u-ually necessary to repeat the solution in water and the treatment with alcohol five or six times. 183 184 THE PRODUCTS OF ALBUMINOUS DIGESTION. The further steps in the digestion of fibrin may be studied in a specimen which has been kept at a temperature of 40° C. for two to three weeks. Syntonin or native soluble albumin that may still be present, as well as the primary albumoses, are removed as just described. The neutral solution is then treated with one-half its volume of a saturated solution of ammonium sulphate. In this manner a two-thirds saturation of the solution is effected, and on standing the deutero-albumose-A separates out. This is filtered off, and the solution saturated with ammonium sulphate in sub- stance. As a result the deutero-albumose-B' is thrown down, and on acidifying the filtrate with one-tenth its volume of a solution of sulphuric acid that has been saturated with ammonium sul- phate, aud of which 10 c.c correspond in strength to 17 c.c. of a one-tenth normal solution of sodium hydrate, the deutero-albu- mose-C finally separates out on standing. The resulting filtrate is then free from albumoses, and should contain the amphopeptone of Kuhne. But, as I have indicated, two additional fractions can be obtained from the final solution. To this end, a solution of iodo- potassic iodide, containing two parts of the iodide to one part of iodine, which has been saturated with ammonium sulphate, is added until precipitation is complete. The material which is thus thrown down is placed in 96 per cent, alcohol. Peptone-B then passes into solution, while peptone-A remains undissolved. This portion is dis- solved in a little warm water, the solution saturated with ammonium sulphate, and reprecipitated with the iodine solution. The peptone is then redissolved in warm water, reprecipitated with alcohol, and freed from any remaining iodine by shaking with ether. Peptone-B, on the other hand, is obtained by evaporating its alcoholic solution to dryness, when the residue is dissolved in water and freed from iodine by shaking with ether. Pick's deutero-albumose-B, which, in contradistinction to the B'-albumose, is said to contain carbohydrate groups, has not as yet been accounted for in the above analytical schema. This is owing to the fact that Pick has not indicated the exact manner in which the substance can be isolated. In his latest publication he merely states that on careful purification of the deutero-albumoses which can be obtained from Witte's peptone (this is largely a mixture of albumoses derived from fibrin) it was noted that the deutero- albumose-B showed in gradually increasing degree the existence of carbohydrate radicles, while A and C in pure form were free from these groups. But, as we have seen, both the proto- and the hetero- albumose on further digestion yield a deutero-albumose-B7 which manifestly contains no carbohydrate groups. It is possible that the B-albumose is hence precipitated together with the B'-albumose ; but if it is found in this fraction, it should be possible to isolate the substance in the earlier stages of digestion already, as it is stated that its formation coincides in point of time with that of Kiihne's primary albumoses. THE PRODUCTS OF TBYPTIC DIGESTION. 185 The general reactions of the various albumoses and the two pep- tone fractions which can thus be obtained from fibrin are shown in the accompanying table (pages 186 and 187). But while the albu- moses, of whatever origin, apparently behave toward ammonium sulphate in the same manner, some of these at least ditfer from the fibrinoses in other respects. The deviations, however, are on the whole but slight, and may well be disregarded at this place. THE PRODUCTS OF TRYPTIC DIGESTION. One hundred grammes of moist fibrin, as in the above experi- ments, are placed in a liter of an 0.25 per cent, solution of sodium carbonate, to which a few grammes of commercial pancreatin have been added. Putrefaction is guarded against by the addition of chloroform and thymol. The mixture is kept at a temperature of 40° C, and can be examined after twenty-four to thirty-six hours. For the preparation of antipeptone, however, in amounts which oan be utilized to demonstrate the presence of the hexon bases, it is necessary to take a much larger quantity of fibrin and to extend the period of digestion over several weeks. From 1410 grammes Kutscher claims to have obtainetl as much as 200 grammes, but I have personally not been so successful. The filtered fluid is first neutralized with dilute sulphuric acid, which causes the separation of any alkaline albuminate that may be present. Coagulable albumins are removed by acidifying the solu- tion with acetic acid and boiling. The solution is then treated with one and one-half times its volume of a saturated solution of ammo- nium sulphate. On standing, the deutero-albumose-A separates out. On complete saturation with the salt in substance the deutero- alburnose-B' is obtained ; a C-albumose is not formed on tryptic digestion. On acidifying with sulphuric acid, however, as in the study of peptic digestion, it may happen that a turbidity appears, which is probably due to the presence of Neumeister's antideutero- albumose. The final filtrate then contains the common amido- a«'ids, antipeptone, tryptophan, and probably other substances also which are as yet but imperfectly known. Leucin. — Aside from its formation during the process of pancreatic digestion or on artificial decomposition of albumin with dilute mineral acids and alkalies, leucin has been demonstrated in the spleen, in the Lymph-glands, in the thyroid, the kidneys, the liver, and the brain, though mostly under pathological conditions, when it may also appear in the urine. It is further found in sheep's wool, in decom- posing epithelial structures, as in the desquamated material which is found between the toes, etc Its presence in the intestine may also be due to the action of bacteria upon the albuminous products of digestion. Jn pure form leucin crystallizes in extremely thin white lustrous platelets; but more commonly it i- seen in the form of spherules of 186 THE PRODUCTS OF ALBUMINOUS DIGESTION. 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S 2 oj.a'd £ 2 * " # ,r ® £ 2 o £.— — > s = ■2 - t: ." as jh t-.z 3ft o »ft o ? J= . -• o 5o«o o'S ■f ft' - - -.: Ms 6- a 3=-,- S -£^ = « s-a-c aj A £ x P u 2 ^-o|g S ►SSI! = - S 2 3 t> of** c^ft*S c a -3 .0 g 2 ass's « c «s o ? 53 5 ~ tc 3 ggsgio* = 3-S2 £§ d c — _ = "■ B r. o >rt ~ "312*8*5 "&"S3 ~ o " o "5 0) 00 £ qo £ S M r— © "^ o 5 5 E £ e -3 s ? '3 w s c d tllg-l » p a u -3"^c^" 188 THE PRODUCTS OF ALBUMINOUS DIGESTION. variable size, which closely resemble globules of fat. In these, con- centric striatums, as well as very fine radiating lines, can at times be made out on careful examination. Several leucins apparently exist. One form, which can be pro- duced synthetically from hydrocyanic acid and ammonium-iso- valerianic aldehyde, is optically inactive. The common leucin, on the other hand, which is formed during tryptic digestion or on decomposition of the native albumins with hydrochloric acid, is dextrorotatory. A third form results from the action of Penicilium glaucum upon the inactive substance, and is said to be lsevorotatory. According to Cohn, moreover, several isomeric, optically active leucins exist. The common form is easily soluble in water, in alkalies and acids, as also in hot alcohol ; in ether it is insoluble. It combines with acids, alkalies, and the oxides of some of the heavy metals to form salts. On boiling a solution of leucin with subacetate of lead the corresponding compound of lead oxide can thus be obtained if ammonia is carefully added to the cooled solution. A copper salt is similarly formed if leucin in aqueous solution and con- taining a small amount of alkali is treated with a solution of cupric sulphate, care being taken not to add an excess. On standing, the compound separates out in the form of clusters of blue needles, which are characterized by their pronounced insolubility. When carefully heated to a temperature of 170° C. leucin melts and sublimes in the form of white flakes, which are deposited on the cooler portion of the tube. At the same time the odor of amylamin develops. On evaporating a small amount of leucin upon platinum foil with nitric acid a colorless residue is formed. If to this a drop of sodium hydrate solution is added and heat is carefully applied, a yellowish or brownish color develops, and on further heating an oil-like droplet is obtained, which rolls about upon the platinum without adhering (Scherer's test). On decomposition with an alkali or during the process of putre- faction leucin yields ammonia and valerianic acid. On oxidation leucinic acid results. As has been indicated, leucin is an amido-capronic acid of the formula (CH3)2.CH.CH2.CH(NH2).COOH, and may hence also be regarded as a-amido-isobutyl-acetic acid. Tyrosin. — Tyrosln can be obtained on tryptic digestion from all those albumins in which aromatic groups exist. Collagen, in which this is absent, accordingly yields no tyrosin, and very small amounts only are obtained from elastin. In the animal body it is practically found as such only under pathological conditions if Ave disregard the minute quantity which is formed in the intestinal canal. Like leucin, it is also formed during the process of albuminous putrefaction, and can be obtained artificially by decomposing albuminous sub- stances with dilute mineral acids or alkalies. While impure tyrosin may occur in the form of spherules similar THE PRODUCTS OF TRYPT1C DIGESTION. 18$ to those of leucin, the pure substance crystallizes in delicate, silky needles, which are often grouped in sheaves and rosettes. Accord- ing to its mode of formation, the substance is optically inactive, as when formed synthetically or by decomposition with baryta-water, or it is lsevorotatory when derived from albumins on boiling with acids. In cold water it is only slightly soluble, while in boiling water it dissolves in the proportion of 1 to 154. Its solubility is increased in the presence of alkalies or mineral acids. In alcohol and ether it is insoluble. Tvrosin may be regarded as para-oxy-phenvl-propionic acid, and has the formula Ct;H4(OH).CH2.CH(NH2).COOH. It may be formed synthetically from ethylene oxide and para-am ido-benzoic acid, and can also be obtained from para-amido-phenyl alanin and para-nitro- phenyl alanin. On bacterial decomposition it yields hydroparacu- maric acid (para-oxy-phenvl-propionic acid), which can be further transformed into para-oxy-phenyl-acetic acid, and this into para- cresol, as has been shown (page 89.) On fusion with caustic alkali, on the other hand, it gives rise to the formation of para-oxy-benzoic acid, acetic acid, and ammonia, as is shown in the equation : X)H .OH C,;Ht + H20 + O = C6H4 + CH3.COOH + NH3. eiL.CH(XH,).COOH XSOOH On oxidation with potassium bichromate and sulphuric acid hydro- cyanic acid, benzoic acid, acetic acid, and formic acid result. - With acids and alkalies, as also with certain salts of the heavy metal-, tyrosin combines with difficulty to form salt-like bodies. Tests for Tyrosin. — Hoffmann's Test. — With Millon's reagent tj rosin gives the well-known reaction of those albumins in which a.'/omatic groups arc present, but, as would be expected, in a degree n,uch more intense. The reaction is due primarily to the formation 0 .' oxy-benzoic acid (salicylic acid). Pikia's Test. — A. i'cw crystals of tyrosin are dissolved in con- centrated sulphuric acid and heated to about 100° C, when the sub- stance dissolves. Tyrosin-sulphuric acid is thus formed, and gives rise to a red color. ( )n cooling, the liquid is diluted, and treated with barium carbonate while heating until the reaction becomes just alkaline. Tyrosin-sulphate of barium tlms results, which gives rise to a dark-violet color on treating with a very dilute solution of sesquichloride of iron. An c\i-c<> ul' iron, however, must be care- fully avoided. Oxy-benzoic acid gives the same reaction. Scheber's Test.— On evaporating tyrosin with a few drops of nitric acid on platinum foil a yellow, transparent residue is obtained, which turn- ro\ on moistening the substance with a drop of sodium hydrate solution, and becomes brown on further evaporation. The reaction is due to the formation of nitro-tyrosin nitrate, but is not characteristic, as other bodies behave in a similar manner. Isolation of Leucin and Tyrosin. — To isolate Leucin and tyrosin 190 THE PRODUCTS OF ALBUMINOUS DIGESTION. among the final products of tryptic digestion, and to separate the two from each other, the digestive mixture is first freed from alkaline albuminate, coagulable albumins, and the albumoses, as already described (page 185). The final filtrate is concentrated to a syrupy consistence, when on cooling leucin and tyrosin crystallize out. The mass of crystals is then boiled with a large quantity of water, to which a sufficient amount of ammonia is added to insure solution of the substances. The boiling solution is treated with subacetate of lead until the resulting precipitate appears almost white. The fil- trate is brought to the boiling-point, neutralized with sulphuric acid, and filtered while boiling hot. On cooling, the tyrosin crystallizes out, while the leucin remains in solution. The former can then be purified by recrystallization from boiling water or from very dilute ammonia. The solution which contains the leucin is freed from lead with hydrogen sulphide, and the filtrate is concentrated and boiled with an excess of freshly precipitated cupric, hydrate. A portion of the leucin is thus precipitated, while the rest remains in solution, but partly crystallizes out on cooling as the corresponding copper compound. The precipitate is placed in the copper-containing solution, and is freed from copper with hydrogen sulphide ; the filtrate is then decolorized with animal charcoal, strongly concen- trated, and set aside for crystallization. Asparaginic Acid. — While asparaginic acid is apparently formed from all albuminous substances on digestion with trypsin, the largest amounts are obtained from fibrin and gelatin. Like leucin and tyrosin, it likewise results on artificial decomposition of the albu- mins with dilute mineral acids and alkalies, and is also formed during the process of albuminous putrefaction. Outside the in- testinal canal asparaginic acid has not been found in the animal body. In the form of its amide asparagin, however, it is widely distributed in the vegetable world, and supposedly plays an im- portant role in the synthesis of the vegetable albumins. The substance crystallizes in rhombic prisms, which are soluble with difficulty in cold water, but are quite soluble in hot water. In absolute alcohol it is insoluble. Its aqueous solutions are lsevorota- tory, while in the presence of nitric acid dextrorotation is observed. As has been shown (page 86), asparaginic acid is a dibasic acid of the fatty series. It is amido-succinic acid, and is represented by the formula CH2.CH(NH2).(COOH)2. It can be obtained from asparagin on boiling with hydrochloric acid, as shown in the equation : /CONH2 /COOH CH2.CH(NH„)< + H20 = CH2.CH(NH2)< + NH3. " \COOH \COOH Asparagin. It has also been produced synthetically. On reduction it yields succinic acid, as has been shown. With cupric oxide asparaginic acid forms a crystalline compound THE PRODUCTS OF TRYPTIC DIGESTION. 191 "which is almost insoluble in cold water, but dissolves in boiling "water with comparative ease. This property is utilized for the pur- pose of isolating the substance from the mixture of digestive products. Glutaminic Acid. — Whether or not glutaminic acid is formed during the trvptic digestion of the albumins in general has not as yet been ascertained. Kutscher claims to have found it in the so-called antipeptone of Kuhne, which was obtained from fibrin. On boiling with strong mineral acids, however, it is constantly formed. But it is noteworthy that much larger quantities are found if the decomposition of the albumins is effected with hydrochloric acid than with sulphuric acid. Kutscher thus found only 1.8 per cent, among the decomposition-products of casein when using sul- phuric acid, while Hlasiwez and Habermann obtained as much as 29 per cent, when hydrochloric acid was used. This is, of course, remarkable, and it would be exceedingly interesting to ascertain the fate of those radicles which can yield so large an amount of gluta- minic acid when decomposition is effected by hydrochloric acid. Glutaminic acid crystallizes in small glistening crystals, whi^h are soluble with difficulty in cold water, while in boiling water they dissolve with greater ease, but separate out on cooling. With acids and alkalies it combines to form salt-like products, among which t/ie hydrochlorate is conveniently utilized for the purpose of identifying the substance. The melting-point of this compound is 193° C. The composition of glutaminic acid is expressed by the formula CH2.CH2.CH(XH2).(COOH)2. It is thus amido-glutaric acid, and bears the same relation to glutamin as that which exists between asparaginic acid and asparagin. This is represented in the equation : /conii2 /cooh €h2.ch2.chi\h + h20 = ch2.ch2.ch(nh2)< + nt13> oooh x:ooh Glutamin. On reduction it yields glutaric acid. Isolation of Asparaginic Acid and Glutaminic Acid. — To isolate the two acids in question among the products of tryptic digestion, the mixture must first be freed from albumins and albumoses, as has been described. The remaining solution is acidified with sulphuric acid and precipitated with phosphotungstic acid. The filtrate is freed from sulphuric acid and any excess of the phosphotungstic acid by means of barium hydrate. From the resulting filtrate leucin and tyrosin are then removed by concentration. The mother- liquor contains the glutaminic acid and asparaginic acid. These are now separated from each other ill the following manner: the diluted solution i- brought to the boiling-point and digested with carbonate of copper. It IS filtered while still hot, and precipitated with subacetate of lead, care being taken to avoid an excess. This precipitate i- decomposed with hydrogen sulphide, and the filtrate concentrated to a -mall volume. On standing, a crystalline mass is 192 THE PRODUCTS OF ALBUMINOUS DIGESTION. obtained, which is then dissolved in boiling water and digested with an excess of carbonate of copper, as before. The hot filtrate is again concentrated, when on standing the copper salt of asparaginic acid separates out in characteristic groups of needles. The nitrate is freed from copper by means of hydrogen sulphide, concentrated,, and set aside, when the glutaminic acid crystallizes out. Glycocoll. — While it is generally known that glycocoll plays an important part in the nitrogenous metabolism of the animal body, and is intimately concerned in the formation of urea, hippuric acid, phenaceturic acid, certain biliary acids, and in birds and reptiles of uric acid, it is of interest to note that the substance has thus far not been found as such among the products of pancreatic digestion, although its radicle is manifestly present in certain albumoses. On hydrolytic decomposition with mineral acids, on the other hand, glycocoll can be obtained from most albumins, but is especially abundant in collagen, viz., gelatin. Two exceptions to this general rule, however, are noted, viz., casein and (according to Magnus- Levy) the peculiar albuminous substance which is known as the Bence Jones' body, and from either of these it is also impossible to obtain a hetero-albumose. The hetero-albumose of fibrin, according to Spiro, yields a considerable amount of glycocoll, while from the proto-albumose it cannot be obtained. Heretofore the isolation of glycocoll and its recognition as such, were attended with great difficulties. A somewhat simpler pro- cedure, however, has recently been suggested by Baum, and with its aid Spiro was able to show that, contrary to former views, the substance can be obtained not only from the albuminoids, but also from the native albumins, with the exceptions indicated. t The method is based upon the observation that glycocoll can be trans- formed into hippuric acid in the test-tube by treating with benzoyl chloride in the presence of sodium hydrate, and that the formation of the resulting hippuric acid can be readily demonstrated by con- densing this with benzaldehyde in the presence of sodium acetate and acetic anhydride. The lactimide of benzoyl-amido-cinnamic acicl is thus formed. On decomposition with sodium hydrate this yields phenyl-pyro-racemic acid, which in ethereal solution gives a. green color on treating with chloride of iron. With phenyl- bydrazin, moreover, it forms an osazon which melts at 161° C. These changes may be represented by the equations : (1) CH2.(NH2).COOH + C6H5.C0C1 = CH2.NH(C6H5.CO).COOH + HC1 Glycocoll. Benzoyl chloride. Hippuric acid. (2) CH2.NHfC,H5.CO).COOH 4- C6H5COH = C6H5.CO.N.C:CH.C6H5 + 2H20 Hippuric acid. Benzaldehyde. | / CO Lactimide. (3) C6H5.CO.N.C:CH.C6H5 + H20 = C6H5.CO.NH2.C.C6H5.CH.COOH \/ Benzoyl-amido-cinnamic p,,-. acid. Lactimide. THE PRODUCTS OF TRYPTIC DIGESTION. 193 (4) C6H3CO.XH2.C.C6H5.CH.COOH + H20 = Benzoyl-amido-ciunamic acid. , the solution is poured into absolute alcohol while stirring. The antipeptone is then precipitated and allowed to settle, when the supernatant fluid is siphoned off and the antipeptone col- lected on a filter with the aid of a suction-pump. It is finally washed with absolute alcohol, then with ether, and rapidly placed in a desiccator over sulphuric acid. From this material Kutscher claims that the three hexon bases can then be isolated. To demonstrate that these bodies actually result from the albuminson hydrolytic decomposition, it is more con- venient, however, to effect this by boiling with dilute acids. To this end, commercial gelatin is conveniently utilized as a starting material, a- larger amounts of* arginin at least can thus be obtained, The method, however, is quite complicated and scarcely requires consideration at this place. CHAPTEK X. BACTERIAL ACTION IN THE INTESTINAL TRACT. I have pointed out in a preceding chapter that the gastric juice possesses marked germicidal and antiseptic properties, so that a large number of bacteria which are constantly swallowed with the saliva and the food are subsequently destroyed in the stomach. A perfect barrier to the invasion of micro-organisms, however, does not exist, and after having passed the pylorus they are placed in surround- ings which are in all respects most favorable to their develop- ment. Here they take an active part in the decomposition of the various food-stuffs which have escaped digestion in the stomach, and further modify the digestive products which have already been formed, as also those which result from the action of the various intestinal ferments. The greater portion of the products of normal digestion, however, escapes the specific activity of the bacteria, and is absorbed in a form which can be utilized by the body for purposes of nutrition. Formerly it was supposed that the biliary acids played an important part in preventing undue activity on the part of the bacteria, but this view has now been largely abandoned, and we are totally ignorant as to the manner in which the body here protects itself against excessive bacterial action. It has been argued that an accumulation of the decomposition-products which result from the action of bacteria upon the various food-stuffs in itself inhibits the further activity of the organisms, but we can hardly regard such an explanation as valid in view of the fact that in the intestines these decomposition-products are to a large extent absorbed, and it seems more probable that a vital activity of the epithelial cells is here of prime importance. In the small intestine at least, where peri- stalsis is extremely active, and where the intestinal contents are churned in such a manner that the individual particles are almost constantly in contact with the intestinal walls, we accordingly find that bacterial action is not nearly so extensive as in the large intes- tine, where the opposite conditions prevail. In the clinical labo- ratory we find, as a matter of fact, that the degree of intestinal putrefaction increases at once when the peristalsis of the small intestine is impeded, and reaches its greatest height if the secretion of hydrochloric acid becomes arrested at the same time. In former years a tendency existed among physiologists to regard bacterial action in the intestine as serving a useful purpose, and it was even supposed that, as in the case of plants, animal life could not go on in the absence of micro-organisms from the alimentary 196 BACTERIAL ACTIOS IN THE INTESTINAL TRACT. 197 canal. This view has now been abandoned, however, especially since Thierfelder and Xuttall were able to demonstrate that guinea- pigs, after removal from the uterus of the mother by Csesarean section, can be maintained in perfect condition as to health and body-weight when fed on sterile food and when furnished with sterile air exclusively. On subsequent examination it was shown that the intestinal contents of these animals were also sterile. We may thus conclude that the presence of bacteria in the intestinal contents is at best unnecessary, and it is doubtful, indeed, whether they serve a useful purpose at any time. The action of bacteria upon the food-stuffs is in certain respects quite analogous to that of the digestive ferments which are fur- nished by the digestive glands of the animal body. The primary digestion of the original material, however, does not cease with the production of substances which the animal can subsequently utilize for the purpose of replacing tissue, but is, on the whole, far more extensive. Polysaccharides and disaccharides are thus not only inverted to monosaccharides, but the latter are subsequently further decomposed into material in which but little potential energy, if any, remains stored. Albumins are similarly decomposed, with the ultimate formation of substances which in part at least are dis- tinctly toxic ; and the fats are divided into their components, which are then further broken down, with the final formation of fattv acids of the lowest order, etc. A great variety of decomposition- products thus result from the normal food-stuffs, which are further increased by those arising from material which the ferments of the animal itself are incapable of digesting. To these are added the decomposition-products of the various biliary constituents and of the albuminous secretions which are poured into the intestinal canal by the digestive glands themselves. A- has been pointed out, the most intense degree of bacterial action is observed in the large intestine, and it is interesting to note that while albuminous putrefaction here prevails, the fermenta- tive processes in the more restricted sense of the term, viz., the decom]K>sition of carbohydrates and fats, occur almost exclusively in the small intestine. This difference may be dependent to a certain degree upon the difference in the reaction of the intestinal contents in the two sections of the gut — that of the small intestine in its lower portion at least being acid, while the reaction of the contents of the large intestine is usually alkaline. But it is also possible that other and -till unknown factors determine this difference, ami that the varying reaction is primarily due to the decomposition-products directly which result from the action of the bacteria. Among these factors tli'' relative amount of water may be of importance. Nencki, MacFadyen, and Sieber, who had occasion to study the chemical composition of the intestinal contents in ;i patient in whom an artificial ami- had been established at the distal end of the ileum, give the following account of their observations : The reaction was 198 BACTERIAL ACTION IN THE INTESTINAL TRACT. quite constantly acid, owing to the presence of organic acids, and notably of acetic acid. Other acids that were present were lactic acid, paralactic acid, various volatile fatty acids, succinic acid, and the biliary acids. The odor but rarely suggested the existence of putrefactive changes. Indol, skatol, and phenol could not be demonstrated as such, although the urine contained indican on several occasions. Leucin and tyrosin were not found. Alcohol could always be demonstrated. Of gases, carbon dioxide was ob- served, as also faint traces of hydrogen sulphide, while methyl- mercaptan was absent. Carbohydrate fermentation thus manifestly stands in the fore- ground, and is exemplified in various types by the equations : (1) C6H1206 = 2C2H5.OH + 2C02, alcoholic fermentation. (2) C2H5.OH + 20 = CHg.COOH + H20, acetic acid fermentation. (3) C6HI206 = 2CH3.CH2(OH)COOH, lactic acid fermentation. (4) 2C3H603 = C3H7.COOH + 2C02 + 4H, butyric acid fermentation. The products of albuminous putrefaction, on the other hand, are almost exclusively formed in the large intestine. Primarily they are in part at least the same as those which result from the action of trypsin on albumins, and in experiments in vitro we thus find albumoses, peptone-like bodies, tryptophan, leucin, tyrosin, aspara- ginic acid, and glutaminic acid. In the contents of the large intes- tine, however, these substances are found only in traces, so that we are forced to the conclusion that they are either absorbed as soon as formed or that they are further decomposed. Both, no doubt, occurs, and related bodies are, as a matter of fact, encountered in the feces. As a result of bacterial activity still other substances are formed, however, which are apparently not derived from the final products of digestion, but which are formed from the more or less intact albuminous molecule directly. The more important decomposition-products which result from the action of bacteria upon the products of albuminous digestion are here considered. Indol. — We have seen that on decomposition of the albumins with trypsin, as also with boiling mineral acids, the aromatic groups of the albuminous molecule are split off in the form of tyrosin — i. e., a body belonging to the para-series. Indol, on the other hand, belongs to the ortho-series, and cannot be obtained in this manner. It is a specific product of albuminous putrefaction, and it would, of course, be interesting to ascertain why the aromatic groups in the one case are set free exclusively in the form of tyrosin, while in the other both result side by side. At present we are unable to offer an adequate explanation of this phenomenon, but it is possible, as Neumeister suggests, that certain bacteria produce indol synthetically from simpler aromatic groups. We find, as a SKATOL. 199 matter of fact, that under the influence of certain organisms, such as the Proteus vulgaris, indol is formed almost exclusively. Structurally indol is closely related to indigo, and according to Xeneki this transformation can be effected through the action of ozone. It is represented by the equation : 2C6H4< >CH + 40 = C6H / >C = C< }C,H4 + 2H20. XII XNH7 XNHX Indol. Iudigo. ( inversely, indigo can be transformed into indol on reduction. From the albumins the substance can also be obtained on fusion with potassium hydroxide (see page 38). The greater portion of the indol that is formed in the large in- testine is no doubt eliminated in the feces. A certain amount, however, is absorbed, and after oxidation to indoxyl appears in the urine in combination with sulphuric acid as so-called indican (pages 90 and 250). If larger quantities are formed, a variable fraction is further eliminated in the urine as an indoxyl compound of glucuronic acid. Indol crystallizes in small platelets, which melt at 52° C, and are soluble in hot water, ether, alcohol, and benzol. Its odor is feculent ; it is quite volatile, and when boiled with water passes over into the distillate. With picric acid it forms a beautifully red crys- talline compound, which is readily decomposed, however, on boil- ing with dilute ammonia, and the liberated indol is then found in the distillate. On distilling in the presence of sodium hydrate, on the other hand, the indol is decomposed. Tests. — When treated in aqueous solution with nitric acid and a trace of sodium nitrite a red precipitate of the nitrate of nitroso- indol is formed. This is soluble in alcohol and crystallizes out upon the addition of ether. If a small piece of pine wood is moistened with strong hydro- chloric acid and then placed in a watery solution of indol, it gradu- ally assumes a cherry-red color. An aqueous solution of indol is treated with a small amount of a solution of sodium nitroprusside until a brownish-yellow color develops. If now a dilute solution of sodium hydrate is added drop by drop, the color changes to violet. Upon the further addi- tion of a little dilute hydrochloric acid this becomes a deep blue, while an exce88 of the ;teid destroys the blue color. Por the isolation of indol, see page 206). Skatol. — Skatol, like indol, belongs to the ortho-series, and is likewise formed during the process of albuminous putrefaction. It i- a methylated indol, and may !"■ represented by the formula: C(CH MI By combining with carbon dioxide it gives rise to the formation "MM '•,.11, CH. MI 200 BACTERIAL ACTION IN THE INTESTINAL TRACT. of skatol-carbonic acid, which is also found in the contents of the large intestine, and belongs to the ortho-series. Its formula is /C(CH3U C6H4< ^C.COOH. Like indol, skatol is also formed on fusing albumins with caustic soda, and can be obtained from indigo on reduction with tin and hydrochloric acid. When passed through a red-hot tube it yields indol. On absorption, it is oxidized to skatoxyl and is eliminated in the urine in combination with sulphuric acid and glucuronic acid, as in the case of indol (see pages 90 and 250). Skatol-carbonic acid, on the other hand, appears in the urine as such. Skatol crystallizes in fine platelets, which melt at 95° C. and are readily soluble in ether, alcohol, and benzol ; in hot water it is soluble with greater difficulty than indol. Its odor is exceedingly offensive. Like indol, it is volatile, and combines with picric acid to form a red crystalline compound. On distilling this in ammo- niacal solution or in the presence of sodium hydrate the skatol passes over as such, while indol in the latter instance is decomposed. On distilling a mixture of indol and skatol in aqueous solution the skatol passes over first, and it is thus possible to separate the two substances from each other. Tests. — From its aqueous solutions skatol is precipitated by yellow nitric acid as a white substance — skatol nitrate. If a small piece of pine wood is moistened with an alcoholic solu- tion of skatol and then placed in strong hydrochloric acid, it assumes a red color. If, on the other hand, the test is conducted as with indol, no reaction is obtained. With nitric acid of a specific gravity of 1.2 skatol gives a marked xanthoproteic reaction on boiling — i.e., a yellow color which changes to orange when ammonia is added in excess. The substance does not give the reaction with sodium nitro- prusside. Isolation (see page 206). Phenol. — The phenol which is formed during the process of intestinal putrefaction is derived from tyrosin. As has been shown, this is first reduced to hydroparacumaric acid. This in turn is oxidized to para-oxy-phenyl-acetic acid. Paracresol then is formed through a splitting off of carbon dioxide, and on subsequent oxida- tion phenol results (see page 96). To a certain extent this is elimi- nated in the feces, but a variable amount is always absorbed, and subsequently oxidized in part to hydroquinon or pyrocatechin. These three' bodies then combine with sulphuric acid and are elimi- nated through the urine in this form. A certain amount of para- cresol, moreover, is absorbed as such, and likewise appears in the urine as a conjugate sulphate. According to some observers, indeed, a larger quantity of paracresol is here encountered than of phenol. PHENOL. 201 Tests. — An aqueous solution of phenol when treated with a few drops of a solution of the sesquichloride of iron assumes an amethyst color, which becomes especially apparent on further dilution with water if much phenol is present. With bromine-water a crystalline precipitate of tribromophenol is obtained. With Millon's reagent a red color develops, which, however, is common to other bodies of this series as well (see page 34). Isolation (see page 206). In addition to phenol, indol, skatol, and skatol-carbonic acid, as also the two hvdroxylated benzol-derivatives of tyrosin, viz., para- oxy-phenyl-propionic acid (hydroparacumaric acid) and para-oxy- phenyl-acetic acid, we further meet with two non-hydroxylated aromatic acids, which are homologous with benzoic acid, viz., phenyl- propionic or hydrocinnamie acid and phenyl-acetic acid. Accord- ing to Salkowski, these may develop directly from the albuminous molecule, but may also result from tyrosin (see page 97). The non-nitrogenous aromatic acids are in part eliminated in the feces. To some extent, however, they are also absorbed. The hvdroxylated acids are then eliminated in the urine either as such, or, like phenol, indol, and skatoxyl, in combination with sulphuric acid, while the non-hydroxylated acids combine with glycocoll, and are eliminated as hippuric acid and phenaceturic acid, as already described (page 97). As regards the fate of the small amounts of leucin, asparaginic acid, and glutaminic acid which are also formed during the process of albuminous putrefaction, it seems that they are usually absorbed, and are then further decomposed within the body of the animal. To a slight extent, however, this decomposition also takes place within the large intestine, and we accordingly meet with small amounts of -uccinic acid, glutaric acid, eapronic acid, valerianic acid, butyric acid, and acetic acid. The sulphur of the albuminous molecule is usually set free in the form of hydrogen sulphide, but traces of methyl-mereaptan are also frequently observed, and still further contribute to the offensive odor of the feces. Whether these sulphur bodies result from decomposition of the tryptophan, is not known. Of the gases which are constantly present in the contents of the large intestine, methane further deserves especial mention. It is to a great extent, in, doubt, referable to the peculiar form of fermenta- tion to which the celluloses are subject. But in part at least it prob- ably also results from the decomposition of the fattv acids and of cholin. Ptomains are normally not found in the intestinal contents, but may l»e encountered under certain pathological conditions. In Asiatic cholera and in cases ofcystinuria putrescin and cadaverin have thus been isolated, and in other diseases, no doubt, they also occur. Th" methods which are employed fir the purpose of isolating the 202 BACTERIAL ACTION IN THE INTESTINAL TRACT. more important products of albuminous putrefaction are described in the chapter on the Feces. BACTERIAL DECOMPOSITION OF THE FATS. As in the case of the carbohydrates and albumins, a comparatively small portion of the fats only undergoes bacterial decomposition, and it appears that this principally occurs in the lower portion of the small intestine. As in the case of the steapsin of the pancreatic juice, the neutral fats are thus first decomposed into glycerin aud the corresponding fatty acids, but the process extends further, and as a result a gradual reduction of the higher acids to the lowest forms takes place. To a certain extent these are then absorbed and further decomposed in the body, but a not inconsiderable portion is directly eliminated in the feces, and we accordingly find here repre- sentatives of the group, from palmitic, stearic, and oleic acids down to butyric acid and acetic acid. The glycerin is absorbed, and is to a certain extent no doubt utilized by the epithelial cells in the syn- thesis of fats. The lecithins are decomposed in the same manner as under the influence of steapsin, with the formation of glycerin-phosphoric acid, fatty acids, and cholin. Whether or not the latter may then be transformed into neurin is not known, but under normal condi- tions this probably does not occur. The glycerin-phosphoric acid is subsequently no doubt absorbed together with some of the fatty acids, and appears in the urine as such. The cholin, on the other hand, is further decomposed, with the formation of ammonia, carbon dioxide, and methane. BACTERIAL DECOMPOSITION OF THE BILIARY CON- STITUENTS. In former years it was generally supposed that the biliary acids after their elimination into the intestinal canal were there absorbed to a large extent and returned to the liver, while a smaller portion was decomposed and eliminated in the feces. Some observers even now maintain the occurrence of such a circulation of the bile-acids, but there is a strong tendency among physiologists at present to deny its existence. As a matter of fact, bile-acids are not found in the blood or in the urine under normal conditions. In the human being, moreover, dyslysins are found only in the feces, while the amido-radicles have apparently been decomposed. In other animals glycocholic acid has been found, but taurocholic acid apparently always succumbs to the action of the bacteria. Of the fate of the amido-radicles we know little, but it is possible that both are in part further decomposed and in part absorbed. Taurin may then appear in the urine either as such or as tauro- carbaminic acid; but it may, on the other hand, again combine with cholalic acid and reappear in the bile. The glycocoll similarly may in part be transformed into urea ; or it may combine with the DECOMPOSITION OF THE BILIARY CONSTITUENTS. 203 non-hydroxylated aromatic acids which are also formed during the process of intestinal putrefaction, and appear in the urine as hip- puric acid and phenaceturic acid; or it may find its way to the liver and be re-eliminated into the intestinal tract as glycocholic acid. Bilirubin is reduced to hydrobilirubin during the process of intestinal putrefaction and largely eliminated in the feces as such. This reduction, according to Nencki, MacFadyen, and Sieber. occurs in man, in the large intestine. A portion, however, is prob- ably absorbed and eliminated in the urine as urobilin. CHAPTER XI. THE FECES. * I have shown in the preceding chapters that the greater portion of the ingested food is transformed in the gastro-intestinal canal into material which can be utilized by the body for purposes of nutrition, and is there absorbed. A certain proportion, however, invariably escapes digestion, and is partly decomposed by the bacteria of the intestinal canal into the various substances which have been considered in the preceding chapter. These substances in turn are in part absorbed, and are partly eliminated in the feces, together with particles of undigested food and undigestible material which have passed through the digestive tract as such. In addition we find here the various native and decomposition -products of the bile, the pancreatic juice, the enteric juice, in so far as they have not been absorbed, together with intestinal mucus, desquamated epithe- lial cells, and bacteria. Consistence and Form. — The consistence and form1 of the feces are principally dependent upon the amount of water that is present, and vary in different animals. Generally speaking, they are softer in the herbivorous animals than in the carnivora. In man they usually occur in the characteristic plastic, cylindrical form, but they may at times be mushy, or round and hard, even in health. Amount. — The amount of fecal material which is eliminated in the twenty-four hours depends primarily upon the amount and the character of the food that has been ingested. In man it normally varies between 100 and 200 grammes, but may diminish to 60 grammes or rise to 250 grammes, even in health, according to the preponderance of animal food or of vegetable material, which has entered into the composition of the diet. Odor. — The disagreeable odor of the feces is largely due to indol and skatol, but may be further increased by the presence of hydro- gen sulphide, methane, and methyl-mercaptan. Color. — The color varies with the character of the food ingested, and is usually but little influenced by the decomposition-products of the biliary pigments. In carnivorous animals the feces are almost black, owing to the presence of hsematin and sulphide of iron. In adult man the color normally varies from a light to a dark brown. In infants in which the bile-pigments appear as such the feces are of a bright-yellow or a greenish-yellow color. At times and apparently under normal conditions stools are also passed which are grayish white in color and closely resemble the so-called acholic stools which are observed in cases of biliary ob- 204 GENERAL CHEMICAL COMPOSITION. 205 struction. Fats, however, are not necessarily present in increased amounts, and there is no reason to assume that the biliary passages are not patent or that no bile is being secreted. Possibly the lack of color in such stools is referable to the formation of colorless decomposition-products of bilirubin, such as the leuko-urobilin of Nencki, and in the last instance to the presence in the intestinal canal of micro-organisms which are usually absent. Nothing defi- nite is, however, as yet known of the conditions which favor the for- mation of such products. Macroscopic Constituents. — On macroscopic examination of the feces we frequently find undigested particles of food, such as skins of berries, large pieces of connective tissue, woody vegetable fibres, undigested pieces of apples, pears, potatoes, grains of corn, flakes of casein, etc. Microscopic Constituents. — On microscopic examination we usually find undigested bits of muscle-fibre, connective-tissue of the white fibrous variety, fragments of the framework of vegetable matter, often still enclosing cells with starch-granules, flakes of casein, globules of fat, fatty acid needles, crystals of calcium oxalate, neutral calcium phosphate, ammonio-magnesium phosphate, calcium lactate (these are seen especially in children on a milk diet), and more rarely of calcium carbonate, calcium sulphate, and cholesterin. So-called Charcot-Leyden crystals, which consist of the phosphate of spermin,are in my experience only found under pathological con- ditions. We further meet with more or less disintegrated epithelial cells, a few leucocvtes, bits of mucus, and, above all, with innumer- able micro-organisms. Often, indeed, it appears as though the stools consist of these exclusively. Their number, even in health, is enormous. Sucksdorff thus found in his own person that on an average 53,124,000,000 were eliminated in the twenty-four hours. Reaction. — In adult man the reaction of the stools is usually alkaline, sometimes neutral, and but rarely acid. Acid stools, on the other hand, are the rule in infants. General Chemical Composition. — A general idea of the average composition of the human feces may be formed from the following analyses, which are taken from Gautier, and have reference to 1000 parte by weight of the fresh material: Adult man. Snclding. Water 744.00 Ml.:; Solids 267.00 14S.7 Total organic matter 208.75 137.1 ' Total mineral matter 10.95 2 13.6 Alimentary residue 84.00 The organic material yielded ■ Aqueous extract 53.40 53.5 Alcoholic extract 41.65 8.2 Ethereal extracl 30.70 17.0 » uding -I pari "i mucus, epithelium, and calcareous Milts. a Not ates. ' >! this, 1 part 206 THE FECES. The individual constituents of the feces may be grouped as follows : 1. Food-material which has escaped the process of digestion, and of bacterial decomposition, such as starches, muscle-tissue, connective- tissue, fats, etc. -. Vndigestible material, which lias been ingested as such, or which has resulted from the decomposition of complex substances which are partly digestible, such as gums, pectins, resins, ehitin, chlorophyl. hamiatin. and insoluble silicates, sulphates, phosphates, etc. o. Derivatives of the bile, such as the dyslvsins, cholesterin, and exceptionally the native biliary acids as such, and further hydrobili- rubin, stereobilin. etc. 4. Intestinal mucus. 5, Products of albuminous digestion, such as albumoses. peptone- like bodies, leuein, ty rosin, asparaginic acid, and glutaminie acid. (i. Products of bacterial action. These comprise the entire series of tatty acids from acetic acid to palmitic acid ; further, lactic acid, succinic acid, glutarie acid, leuein. tyrosin, hydroparaeumarie acid, para-oxY-phenyl-aeetic acid, phenyl-propionic acid, phenyl-aeetie acid, phenol, paraeresol, indol, skatol, skatol-earbonie acid, ammo- nium carbonate, ammonium sulphide, etc. 7. Products of metabolism, which are in part eliminated through the intestines, such as uric acid, urea, xanthin bases, etc. S. Water. 9. Gases, which are in part referable to the various fermentative and putrefactive processes which take place in the intestinal canal, such as carbon dioxide, methane, hydrogen, hydrogen sulphide, methvl-mereaptan, and phosphin. The nitrogen, on the other hand, which is also constantly met with, is probably derived from the blood, and has in part been swallowed. Many of these substances have already been considered in detail, and it will suffice at this place to indicate the manner in which the most important products of albuminous putrefaction can be isolated from the feces. ANALYSIS OF THE PRODUCTS OF ALBUMINOUS PUTREFACTION. The feces are diluted with water, passed through a muslin filter to remove particles of food-material, and distilled until about fonr- fifths of the entire volume have passed over. The distillate B eon- tains indol. skatol, phenol, paraeresol, and the volatile acids which are present in the free state, while the remaining products of putre- faction are found in the residual solution A. The distillate B is neutralized with sodium carbonate and redistilled. This second dis- tillate. C, contains indol, skatol, phenol, and paraeresol, while the volatile acids remain behind as sodium salts, and can be sepa- PRODUCTS OF ALBUMINOUS PUTREFACTION. 207 rated from each other according to the usual analytical methods (see page 264). Distillate C is now rendered alkaline with sodium hydrate and extracted with ether by shaking. This takes up the indol and skatol, which may be obtained in crystalline Conn on evaporation of the ether, and can be separated from each other by fractional distillation with steam, when the skatol passes into the distillate first. The residual solution of C contains phenol and paracresol as sodium compounds. This is now acidified with sul- phuric acid and distilled, when the phenols pass over, and may then be separated from each other as described elsewhere. The residual solution A is now concentrated and treated with a large excess of alcohol, in order to precipitate any albumins and mineral salts that may be present. The alcoholic filtrate is then transformed into an aqueous solution and aeiditied with sulphuric acid. The aromatic acids are thus set free and are extracted with ether by shaking. The ethereal solution is evaporated to dryness, the residue dissolved in a small amount of a dilute solution of sodium hydrate, and precipitated with barium chloride. The fatty acids are thus obtained as barium soaps, and are filtered off". They are placed in water, which dissolves the salts of the aromatic acids. In this solution the acids are then set free by acidifying with sul- phuric acid, and are extracted with ether. The ethereal extract is now evaporated to dryness and the free acids dissolved in water. On distillation in a current of steam, phenyl-propionic acid and phenyl-acetic acid pass over, and can be subsequently separated from each other by fractional distillation. The hydroxylated oxy- acids and skatol-carbonic acid remain behind. The latter separates out, on further concentration of the solution and cooling, in the form of white wart-like granules, while the oxy-acids remain behind, ami can be separated from each other by means of their varying solubility in benzol. They can be recognized by applying Millon's test. Skatol-carbonic acid, on the other hand, reacts in very much tin- same manner with yellow nitric acid as does indol, but the red color is in this ease referable to a different pigment (see also page 204). Hydrobilirubin. — [t has been stated that bilirubin under the influence of bacterial action supposedly undergoes a process of reduc- tion in the intestinal canal and is transformed into hydrobilirubin. It i- assumed that as such it is then in part absorbed and possibly appears in the urine, while the remaining portion is eliminated in the feces. A.CCOrding to some observers, it is identical with the etercobilin of Vanlair and Masius. The spectrum of the two bodies i- very similar, but while solutions of hydrobilirubin on treatment with chloride of zinc and ammonia show three bands of absorption, stercobilin i-> said to give rise to four bands. Garrod claims that stercobilin is identical with the urobilin of the urine, and differs from hydrobilirubin in containing a much smaller percentage of nitrogen, viz., 1.1 I, as compared with \):l-l. According to the same observer, 208 THE FECES. hydrobilirubin is a laboratory product, and is met with neither in the feces nor the urine. Excretin. — This is a substance which was first isolated by Marcet from the feces of herbivorous animals, but is said to occur also in human stools. According to Hinterberger, it has the formula C20H36O, and is thus closely related to cholesterin. Stercorin. — Stercorin, or serolin, as it has been called, is a sub- stance which Flint obtained from the feces of man, but which is probably an impure form of cholesterin, both having the same general reactions. MECONIUM. The term meconium has been applied to the material which accu- mulates in the intestinal tract during foetal life, and which is expelled soon after birth. Food-products are here, of course, wanting, and as the intestinal tract of the foetus is free from bacteria the meconium consists essentially of mucus, desquamated epithelial cells, and the normal biliary constituents which are present in the intestinal tract before birth. We accordingly find bilirubin and biliverdin, the former often in crystalline form, the native biliary acids, a small amount of fatty acids, cholesterin, and mineral salts, while hydro- bilirubin, the dyslysins, leucin, tyrosin, indol, skatol, lactic acid, albumoses, etc., are absent. According to Zweifel, it contains from 79.8 to 80.5 per cent, of water and from 19.5 to 20.2 per cent, of solids, of which 0.978 is referable to mineral ash, 0.797 to choles- terin, and 0.772 to fatty acids. Its color is a dark brownish-green, and the reaction usually acid. In general appearance it resembles pitch, and is hence also spoken of by the Germans as Kindspech (infant pitch). CHAPTER XII. THE URINE. The urine is by far the most important excretory product of the animal body, and the medium through which the end-products of nitrogenous metabolism and soluble mineral salts are almost exclu- sively eliminated under normal conditions. Abnormal products of metabolism also, and many substances that have found their way into the circulation from without, and which are foreign to the body, are likewise removed in this manner, either as such or in a more or less modified form. All these substances are found in the urine in aqueous solution, and it is to be noted that of the total amount of water which is daily excreted at least 50 per cent, appears in this form. Formerly, it was supposed that the various elements which are found in the urine, and notably the mineral salts and water, were eliminated by a simple process of osmosis. Later it was shown, however, that in the elimination of the organic constituents at least the renal epithelium of the uriniferous tubules plays an active part, and it now appears, indeed, that all the substances which occur in the urine, including a certain amount of water even, are removed from the blood, viz., the lymph, through the intervention of the epithelial cells. It is supposed, moreover, that these structures possess certain selective properties, and we can accordingly under- stand why the composition of the blood always remains constant. The kidneys cannot he regarded as simple excretory organs, how- ever, for we know that important synthetic processes also take place in them, the object of which is to transform certain substances which may occur in the circulating blood into compounds that can be more readily eliminated. The most important synthesis of this kind is that of glycocoll and benzoic acid, which results in the formation of hippuric acid (see page 258). GENERAL CHARACTERISTICS OF THE URINE. The general appearance of the urine varies in different animals. In man it i- perfectly transparent when recently passed, but soon becomes turbid, and on standing deposits a light, flocculent sediment, which consists of :i mucinous body and a few epithelial cells and leucocytes thai are derived from the urinary passages. In addition, ;i -mull number of crystals of uric acid or of oxalate of calcium may also be seen. The Bupernatanl fluid is then per- 14 209 210 THE URINE. fectly clear, and remains so if care is taken to prevent the access of micro-organisms. If left exposed to the air, however, bacterial decomposition soon takes place. Ammonia appears in the free state, and as a consequence of the change in reaction certain constituents of the urine are precipitated and render the liquid turbid. Sooner or later they settle to the bottom, but owing to the presence of innumerable micro-organisms the supernatant fluid remains cloudy. Such urine is said to have undergone ammoniaeal decomposition. A formation of sediments, aside from the light cloud which de- velops in every urine on standing for a short while, may, however, also occur in the absence of micro-organisms. In the winter-time it is a common experience to see the entire volume of urine become turbid when kept in a cold room. This is owing to the fact that the urates of the urine are very much less soluble in cold than in warm water, and are hence thrown down. On standing, they soon settle to the bottom, and the supernatant liquid remains clear .so long as bacterial decomposition does not occur. A similar forma- tion of sediments is observed if the reaction of the urine is alkaline, owing to the presence of fixed alkali in contradistinction to free ammonia. This may at times be observed after a large meal, or after the administration of sufficiently large amounts of alkalies as such, or of substances which are oxidized to alkaline carbonates within the body. In such an event the urine may be clear when first passed, but after standing a short time it becomes turbid, and deposits a sediment of phosphates and carbonates of the alka- line earths. The change is, no doubt, due to an escape of the carbon dioxide which was present in solution. But here also the supernatant liquid is clear. In herbivorous animals, by which an alkaline urine is passed habitually, the liquid is turbid when discharged. In man the pas- sage of a turbid urine is always abnormal, excepting during the first days of life, when cloudy urine is the rule. This is largely referable to desquamated epithelial cells and relatively large amounts of urates. While the urine of all mammalian animals is liquid, the lower animals excrete a urine that is more or less solid. In birds and reptiles, for example, in which the ureters end in a common cloaca with the rectum, the excrements appear in the form of a whit- ish pasty material. A gelatinous urine is observed in turtles. The color of the urine in man normally varies from light yellow to dark amber, and is largely influenced by the concentration of the secretion and its reaction. Acid urine is thus always darker than an alkaline urine, and the color is naturally lighter when the secretion is abundant than when scanty. A gradual darkening of the urine is observed when the material is kept for some time and access of micro-organisms is prevented. Deviation from the normal color is notably observed in disease, or following the administration of various drugs, but may also occur m GENERAL CHARACTERISTICS OF THE URIXE. 211 apparently healthy individuals in consequence of certain abnormali- ties of metabolism (see page 261). The urine may then be of a normal color when recently passed, but soon darkens on standing and finally appears almost black. In diabetes a light color may be associated with a high specific gravity. The odor of recently passed urine is peculiarly aromatic, and is probably referable to the presence of several volatile acids. Decom- posing urine has a characteristic odor, which is in part due to ammonia. l Amount— The amount of urine eliminated in the twenty-four hours is quite variable even under normal conditions. It is of course, primarily dependent upon the amount of water ingested, but is also influenced by the character and the quantity of the food the process of digestion, the blood-pressure, the surrounding tempera- ture the emotions, sleep, exercise, body-weight, sex, age, etc! It must hence differ in different countries, according to the habits of the people, the climate, etc., and we accordingly find that different observers give different figures. In Germany and Austria, where much beer is consumed, from 1500 to 2000 c.c. are regarded as average amounts. In England 1000 to 1500 c.c. are regarded as normal; in France, 1250 to 1300 c.c. In this country I have found that the average daily amount is innnT TonnVer' and am T1^1 t0 reSard an elimination of from 100 to 1200 c.c.^ as normal for men, while in women a somewhat smaller quantity is normally passed. Children pass absolutely less adults m°re UYme' US °0mparecl with their body-weight, than In the summer-time, when the sweat-glands are especially active and when larger amounts of water are eliminated through the lungs f'n i i "^n sccre;ion of urine » proportionately less, but rarely tails below 800 c.c. unless active exercise is indulged in at the same During repose, moreover, much less urine is voided than when exercise is taken and we hence find a smaller secretion of urine during the night than during the day. The maximum secretion is usually observed a few hours after the midday meal ArtilH-.allv .I,,, secretion can be increased by the ingestion of tnosearticles of food which tend to increase the blood-pressure such as coffee, tea and alcohol. Many drugs also bring about the 'same effect, though the modus operandi of each is no, known. The raosl important medicinal diuretics are digitalis, squill, broom, juniper nitrous ether, urea, etc. Distilled water also has distinct diuretic properties. In disease, and notably in diabetes mellitus, diabetes insipidus :i1"' chronic interstitial nephritis the amount of urine may far sur- 1,;|" ,,|r nsnal quantity, and may indeed exceed 10,000 c.c in the twenty-four hours (polyuria\ Abnormally small amounts, on the other band (oliguria), are observed in the acute febrile diseases in 212 THE URINE. various diseases of the circulatory apparatus, in certain diseases of the kidneys and liver, etc. Complete anuria may indeed occur. Specific Gravity. — The specific gravity of the total amount passed in twenty-four hours normally varies between 1.015 and 1.025. Generally speaking, it increases with the solids, the amount of water remaining the same, and diminishes as the amount of fluid increases while the solids remain constant. Under pathological conditions, however, deviations from this rule are not uncommon. The specific gravity may then fall as low as 1.000 and 1.002, or may be increased to 1.050 and even higher. Reaction. — The reaction of the twenty-four hours' urine is, in man, normally acid, sometimes amphoteric, and more rarely alka- line. The normal acidity is, however, not due to the presence of a free acid, but to acid salts, and in the first instance to acid phos- phates. As the reaction of the blood is alkaline, the question naturally arises : How is it that an acid secretion can be derived from an alkaline fluid? In the case of the gastric juice we have met with a very similar phenomenon, and it was explained that the free acid in that case most likely resulted through a mass-action, on the part of carbonic acid, upon sodium chloride within the oxyntic cells, the hydrochloric acid being then secreted into the lumen of the glandular ducts, while the resulting alkaline carbonate is returned to the blood. Similar conditions probably exist in the kidneys, where, as has been mentioned, the mineral salts are also secreted into the uriniferous tubules through the specific activity of the renal epithelial cells. AVe may imagine that here also a mass- action on the part of carbonic acid takes place, which in this case, however, is directed toward the alkaline phosphates of the blood, as is shown in the equation : Na2HP04 + H2C03 = NaH2P04 + NaHC03. We may then imagine that the resulting alkaline carbonate is returned to the blood, while the acid phosphate appears in the urine. The acidity of the urine, however, is primarily clue to the character of the diet. In man and the carnivorous animals this is especially rich in albumins, and contains a comparatively small amount of alkaline salts or of organic acids which could be trans- formed into alkaline carbonates in the body. During the process of metabolism, then, the ingested albumins are broken down, and uric acid, hippuric acid, phenaceturic acid, oxalic acid, aromatic oxy- acids, and notably sulphuric acid and phosphoric acid result. These acids, however, are immediately transformed into neutral salts by combining with the available alkaline carbonates which are present in the lymph and the blood. As a consequence the alkalinity of the blood must of necessity diminish. But as such a change would give rise to serious disturbances, and as there is a strong tendency on the part of the body to maintain the composition of the blood, GENERAL CHARACTERISTICS OF THE URINE. 213 and particularly its alkalinity, constant, a loss of alkali is guarded against by subjecting the various neutral salts to the specific activity of the renal epithelial cells. As a result a portion of the alkali is returned to the blood, and acid salts hence appear in the urine. In the herbivorous animals, on the other hand, in which a super- abundance of alkaline salts is either directly ingested or is formed within the body from salts of organic acids which have been taken with the food, an alkaline urine is eliminated. In this case a formation of acid salts and a return of alkali to the blood are unnecessary. Similar conditions at times occur in man, and the elimination of an alkaline urine, the alkalinity being due to a fixed alkali, cannot hence be regarded as pathological. During the process of digestion, indeed, when an additional amount of alkaline salts finds its way into the blood in consequence of the formation of hydrochloric acid, an increased alkalinity would result. This, however, is prevented by the excretion of a urine which, if not alkaline, is at least less acid. It has been stated above that the organic acids which are formed during the nitrogenous metabolism of the body combine with the alkaline carbonates of the lymph and blood-plasma to form neutral salts. This statement requires modification in so far as it conveys the idea that the acids in question are eliminated in the urine in combination with fixed alkalies only. As a matter of fact, this is true only in part, and a certain proportion of the acid is eliminated in combination with ammonia. Generally speaking, the ammonium salts which are formed within the body appear in the urine as urea, but aside from their impor- tance in this respect they represent a reserve of alkali which is capa- ble of preventing an undue diminution in the alkalinity of the blood by vicariously taking the place of the fixed alkalies. This vicarious action is normally also at work, but is then comparatively insi intimately related to the normal blood-pig- ment. In addition, traces of various other substances may be encountered, the origin of which is obscure. Under pathological Conditions we meet with certain normal constituents of the blood which generally do not appear in the urine as such, or occur in infinitesimally small amount-, and also with various abnormal products of metabolism, all of which will be considered in detail. 222 THE URINE. The Nitrogenous Constituents of the Urine. Urea. While in birds and reptiles the greater portion of the urinary nitrogen is excreted in the form of uric acid, urea constitutes the most important end-product of the nitrogenous metabolism of the remaining groups of vertebrate animals. In man, 86 per cent, of the total nitrogen eliminated in the urine appears in this form. Origin. — Formerly it was supposed that urea resulted from uric acid through a process of oxidation, and that this was its only source. We have seen that the formation of urea from uric acid is possible, and we cannot deny that a certain proportion of the sub- stance may be derived in this manner. Modern researches, how- ever, have shown that in man and the mammalian animals uric acid is largely derived from a destruction of the nucleins within the body, and results from oxidation of the xanthin bases, which are thus set free. In birds and reptiles, on the other hand, the greater portion of the uric acid is formed synthetically from simpler sub- stances, and is hence not directly comparable to the form which is found in the higher animals. In these a synthetic formation is also possible, but probably does not occur under normal conditions. As we can therefore recognize one origin of uric acid only in the mammal, and as this source of the nitrogen is insignificant when compared with the large amount of urea actually found, we are forced to the conclusion that the greater portion of the urea must originate in a different Avay. It has been repeatedly shown that during the decomposition of the albumins by means of acids and alkalies, as also during the process of tryptic digestion and albuminous putrefaction, a large amount of mono-amido-acids results. It has hence been supposed that these bodies probably represent intermediary products in the transformation of the albuminous nitrogen into urea, and it has actually been demonstrated that in mammals — and to these I shall confine my remarks for the present — the administration of such acids in the food is followed by a corresponding increase in the amount of urea. Under certain pathological conditions, moreover, these acids appear in the urine as such, and it is then noted that the elimination of urea is much diminished. In health, however, this does not occur, and on examination of the different tissues and organs of the body such acids are found only in traces. We must hence assume that these acids, supposing them to occur as primary products of albuminous decomposition within the body, are trans- formed at once into other substances, which in turn give rise to urea. As all these bodies on oxidation yield ammonium carbonate, this substance would hence suggest itself as a probable antecedent of urea. We find, as a matter of fact, that ammonium carbonate when ingested by the mouth, or otherwise introduced into the body, THE ORGANIC CONSTITUENTS OF THE URINE. 223 appears in the urine as urea. This transformation of mono-arnido- acids into urea may be represented by the following equations : (1) CH2(NH,).COOH + 20 = NH4.COOH + C02 Glycocoll. Ammonium formate. (2) 2NH4.COOH + 20 = (NH4)2.C03 + H20 + CO, VIT (3) (XH4)2.C03 = CO<^ ' + 2H20 \NH2 Urea. Drechsel has further shown that the amido-acids yield carbamic acid on oxidation, and that through alternate oxidation and reduction urea can result from the ammonium salt, as shown in the equations : NH2 NH2 (1) CO' + O = C0< + H20 X).NH4 X).NH2 Ammonium Carbamic acid. carbamate. /NH, .NH2 (2) C0< + 2H = C0< + H20 xO.XH, \NH2 Carbamic acid. Urea. That carbamic acid is present in the normal acid urine of man and the dog has been proved. Nencki and Halin, moreover, observed that in dogs in which the liver was temporarily excluded from the general circulation larger amounts of carbamic acid appeared in the urine than under normal conditions, and that the animals showed symptoms of intoxication identical with those observed when carbamates are directly introduced into the blood- current. These symptoms were also present when carbamates were introduced into the stomach, in which case normal dogs show no signs of poisoning. While it has been assumed above, that urea is largely referable to a transformation of mono-amido-acids into ammonium carbonate or carbamate, as the case may be, and while it has been shown that such a transformation actually does occur, we must yet remember tli.it only traces of amido-acids are normally found in the tissues. There is reason to believe that the greater portion of the albuminous nitrogen is normally sel free from the various organs of the body in the form of the ammonium salt of paralactic acid, and there is a tendency among physiologists at the present time to regard this -alt a- the- common antecedent of urea. It has been demonstrated, a- a matter of fact, that urea results when ammonium lactate is passed through the isolated liver of a dog; and clinically, also, we observe that both ammonia and lactic acid appear in the urine in increased amounts when the liver is extensively diseased. Similar results are obtained in birds, in which uric acid represents the principal end-producl of nitrogenous metabolism. In <^'v^v it is thus noted thai after extirpation of the liver the greater portion of the urinary nitrogen appears in the form of ammonium lactate. 224 THE URINE. Under normal conditions it is assumed that the lactate is trans- formed into ammonium carbonate, which in turn yields the carba- mate, and the urea finally results through a synthetic process, which is probably effected through the agency of a certain ferment. We may further imagine that the paralactic acid in the last instance may result from a decomposition of the mono-amido-radicles of the albu- minous molecules, and in this form the theory would embrace the two outlined above. The various changes may be represented by the equations : (1) 2NH4.C3H503 + 120 = (NH4)2C03 + 5C02 + 5H20 Ammonium lactate. jra2 (2) (KE4)2C03 = C0< + H20 X).NH4 Ammonium carbamate. ,NH2 NH2 (3) C0( = C0( + H20 XO.NH4 XNH2 Urea. While we have seen that urea may originate in the animal body through a process of oxidation only, as also synthetically through alternate reductions and oxidations, there is still another possibility, viz., that it may be derived from the albumins by hydrolysis only. We know, as a matter of fact, that a number of nitrogenous substances are found in the body, such as kreatin, kreatinin, oxaluric acid, and others, which on hydrolytic decomposition give rise to the formation of urea, and it is quite possible that a certain proportion may be referable to this source. We have also seen that on hydrolytic decomposition all albumins which have been examined in this direction yield comparatively large amounts of arginin, and that this can be further decomposed in the same manner into ornithin and guanidin, which latter then yields urea. As arginin is now known to occur in the animal body as such, there is no reason for supposing that a certain fraction of the urea may not be formed as just indicated, and Drechsel indeed has expressed the opinion that 10 per cent, of the total amount may thus result through hydrolytic processes only. Hoppe-Seyler has suggested that in the transformation of the mono-amido acids into urea cyanic acid may be produced as an inter- mediary product, and that urea then results through the interaction of two molecules of this acid, as is shown in the equation : /NH2 CONH + CONH + H20 = CO< + C02. XNH2 In all probability a certain amount of urea is produced in the animal body in different ways, and there is reason to believe, more- over, that its formation is not confined to one single organ. The greater portion, no doubt, is formed synthetically in the liver. Of THE ORGANIC CONSTITUENTS OF THE URINE. 225 this, indeed, we have abundant proof. It has been shown that in diseases of this organ which are associated with an extensive destruc- tion of the glandular elements a diminished amount of urea is found in the urine, while ammonia and lactic acid are present in increased quantity, and the mono-amido acids may appear as such. In cases of this kind as much as 37 per cent, of the total amount of urinary nitrogen has been found in the form of ammonia. In the mammal, moreover, symptoms of carbamic acid poisoning are observed when the liver is excluded from the general circulation, and, as has been shown, the formation of urea from ammonium lactate or carbonate may be demonstrated in the isolated livers of dogs. As negative results were obtained by von Schroeder when blood containing am- monium carbonate was passed through the kidneys and through the isolated hind-quarters of dogs, the conclusion suggests itself that in these organs a formation of urea does not occur/ This inference is, however, not admissible in the light of our modern knowledge of the origin of urea, for Ave can readily conceive that although a syn- thetic formation from ammonium carbonate may not occur in these organs, it is nevertheless possible that it may originate in a different manner. The transfusion experiment, after all, only shows whether or not a new body can be formed in the organ under investigation from other substances which are passed through it as such. Before deciding that urea cannot be produced in these parts it would hence be neces- sity to experiment with all those substances which can be made to yield urea in the test-tube, and which we know to occur in the ani- mal body. In the spleen, where arginin, for example, is known to occur, it would be of interest to ascertain whether urea is produced here when blood containing arginin is passed through the organ. If we accept the modern doctrine that urea not only originates in the animal body in different ways, but that it may also be formed in other organs besides the liver, we can also understand why it is that in certain diseases of the liver the diminution in the formation of area is not always proportionate to the extent of parenchymatous degeneration, and that no case has been reported in which the formation of urea ceased altogether. It is also made clear why una is found in the urine of birds and reptiles, although a synthetic production of the substance manifestly does not occur in these animals. Its origin is here no doubt to be sought in its formation from -uch bodies as kreatin, kreatinin, arginin, and the like. Nitrogenous Equilibrium. — The albumins, of course, are the ulti- mate source of the urea. According to Pettenkofer, they exist in the body in two forms, viz., :i^ organized albumin which is built up into tissues, and n- so-called circulating albumin which is present in js of what i- actually required, and is broken down directly and eliminated in the urine without having entered into the con- struction of the body proper. This portion of the albumin furnishes the greater portion of the urea, while the organized albumin repre- 15 226 THE URINE. sents a minor but more constant source. The actual amount that is eliminated is thus primarily dependent upon the amount ingested. The total urinary nitrogen is under normal conditions practically equivalent to the quantity ingested, barring the small fraction which escapes digestion in the feces. Such a condition is spoken of as the nitrogenous equilibrium of the body. Of this, however, different levels may exist, which may vary in the same individual. If the amount of nitrogenous food is thus diminished, the amount of urinary nitrogen will also decrease ; and if the amount of food then remains constant, the nitrogenous output will likewise remain the same. If, on the other hand, more nitrogen is now ingested, an increased elimination will result ; but a certain fraction is retained by the body, and gradually a higher level of equilibrium becomes established. There are natural limits to this power of accommodation, how- ever, and we finally reach a point which varies in different indi- viduals, where a further increase in the amount of nitrogen that is ingested does not lead to a higher level of equilibrium, and where consequently a further retention of nitrogen does not occur. Over- feeding then results in various digestive disturbances — diarrhoea and vomiting occur, and the body. thus protects itself against an undue accumulation of circulating albumin which it would not be able to dispose of in a normal manner. Underfeeding, on the other hand, gradually leads to an increased destruction of the organized albumins. For a while the reserve of fats and carbohydrates is still capable of protecting the body against an unduly rapid loss of nitrogen from this source, but death finally results. From the fact that the level of nitrogenous equilibrium is different in different people and may vary in one and the same individual, it follows that the amount of urea also must vary. Any figures indi- cating the amount of urea that is eliminated in the urine can there- fore be of little value unless we are acquainted with the actual state of health of the individual, his body-weight, his habits of life as regards exercise, the amount of nitrogenous food ingested, etc. Having a knowledge of all these factors, however, we may be able to sav whether the "amount of urea is normal or not. Certain figures have'been given by physiologists to indicate the amount of nitrogen- ous food which should enter into the composition of the diet, and from these we can approximately calculate the amount of urea that should appear in the urine. By estimating this in turn, or still better, of course, the total amount of nitrogen, we can accordingly decide whether or not the individual is consuming a sufficient amount of nitrogen in his food. The figures, however, have been constructed withoutdue regard to the factors above indicated, and are in my opinion, at least, too high as averages. I am willing to admit that an elimination of 40-50 grammes of urea may be normal in certain cases, as in soldiers on forced marches, THE ORG A SIC CONSTITUENTS OF THE URINE. 'I'll among the laboring-classes, etc., but I should certainly look upon the average merchant or student, who leads a sedentary life, as an overfed individual if his daily elimination of urea should exceed 30 grammes in the twenty-four hours. Among the well-to-do classes I find that an elimination of from 20 to 25 grammes is probably normal, taking the body-weight of the person into due considera- tion. A smaller amount even is not infrequently met with in people of sedentary habits who are in perfect health, but I should scarcely regard such a quantity as normal for the average laboring-man. While extensive variations in the amount of urea are thus observed in health, still greater deviations from average figures are noted in disease, but here as there we must always take into account the amount of nitrogen that is ingested and the body weight. An increase in the elimination of urea, referable to the destruction of organized albumins, is here frequently observed, but may be obscured, owing to a deficient ingestion of nitrogen, unless the amount of the latter is known. At this place, however, it is scarcely neces- sary to enter into pathological considerations, and I must refer the reader to other works for detailed information on such questions. But I may briefly recall that in certain diseases of the liver in which an extensive destruction of the parenchyma is taking place the amount of urea may be greatly diminished, although a fairly abundant supply of nitrogenous food is ingested. As has been shown, the synthetic formation of urea is here seriously impeded, and as a result we find that a considerable proportion of the urinary nitrogen then appears in the form of ammonium salts of paralactic acid, of carbamic acid and carbonic acid, and in extreme cases, indeed, mono-amido acids, such as leucin and tyrosin, may be found. Properties of Urea. — Urea crystallizes in two forms, viz., in loiv/, i'uiQ white needles if rapidly formed, or in long, colorless rhombic prisms when allowed to crystallize more slowly from its solutions. It melts at 130° to 132° C, but is probably decomposed already at a temperature of 100° C. It is readily soluble in water and alcohol, but is insoluble in anhydrous ether, chloroform, and benzol. A- the substance is an acid amide, its solutions present a neutral ion. In accordance with its character as an unsaturated amide of carbonic acid, however, it combines with acids to form crystalline, salt-like compounds. The most important of these are the nitrate and the oxalate. Urea nitrate, ( K )( X I L)_,. II .V ).., crystallizes in two forms, viz., in delicate rhombic, horizontal platelet-, which :ire commonly arranged overlapping in a shingle-like manner when rapidly formed, or as thicker rhombic columns or plate- when allowed to crystallize more .-lowly. Qrea nitrate is readily soluble in distilled water, but dissolves with difficulty if this is acidulated with nitric acid, and also in 228 THE URINE. alcohol. Its formation is frequently observed when urine contain- ing much urea is examined tor albumin in the cold with nitric acid. On standing, the nitrate may then separate out in crystalline form. On heating, the substance is decomposed without leaving a residue. Urea oxalate, CO(NH2)2.C2H204, crystallizes in rhombic plates, or hexagonal prisms, and is less soluble in water than the nitrate ; in alcohol and in dilute solutions of oxalic acid it is nearly insoluble. The substance is obtained in crystalline form on adding a saturated solution of oxalic acid to a concentrated solution of urea. Urea also combines with various neutral salts, such as sodium chloride and ammonium chloride, and also with the nitrates of sodium and the oxides of silver and mercury, to form double salts. With mercuric nitrate three different compounds result, according to the concentration of the two solutions, viz., CO(NH2)2.Hg2(N03)4, CO(NH2)2.Hg3(N03)6, and . [CO(NH2)2]2.Hg(N03)2 + 3HgO. The latter compound is of special interest, as Liebig's quantitative esti- mation of urea, which was formerly much employed, was based upon its formation. It results when a 2 per cent, solution of urea is treated with a feebly acid solution of mercuric nitrate, and the mixture is subsequently neutralized. Mercuric chloride precipitates urea in alkaline, but not in neutral solutions. Very important, further, is the behavior of urea toward sodium hypochlorite or hypobromite, as the most usual method of esti- mating urea in the clinical laboratory is based upon the reaction which here takes place. This reaction may be represented by the equation : CO(NH2)2 + SNaOBr = 3NaBr + C02 + 2H20 + 2N. In practical work an alkaline solution of the hypobromite is employed, so that the carbon dioxide which is liberated is at once absorbed, while the nitrogen remains. It is to be noted, however, that while 1 gramme of urea should theoretically give rise to the formation of 372.7 c.c. of nitrogen, 354.3 c.c. are at best obtained at 0° C. and a pressure of 760 Hgmm. In clinical work this difference is unimportant, and it is in a measure equalized by the evolution of a small amount of nitrogen from some of the other nitrogenous constituents which are at the same time present. On hydrolysis urea is transformed into ammonium carbonate : CO(NH2)2 + 2H20 = (NH4)2C03. This occurs during the process of ammoniacal fermentation, which results when urine is exposed to the air, and is referable, as I have pointed out, to the action of a specific bacterial enzyme. On boiling with acids or alkalies the same result is primarily obtained, but the salt is then further decomposed, with the liberation of carbon dioxide THE ORGANIC CONSTITUENTS OF THE URINE. 229 and ammonia, respectively. This decomposition is, however, also noted during the process of ammoniacal fermentation. On heating the substance in aqueous solution in a sealed tube to a temperature of 100° C. ammonia and carbon dioxide likewise result. Nitrous acid when added in excess decomposes urea, with the formation of nitrogen, carbon dioxide, and water, but the acid is at the same time decomposed, as is seen in the equation : CO(NH2)2 + 2HN02 = C02 + 4N + 3H20. This reaction is utilized when it is desired to remove nitrous acid from a solution. On heating the dry substance in a test-tube to a temperature of about 150°-i70° C, fumes of ammonia are freely evolved, owing to the decomposition of the urea with the formation of biuret, as shown in the equation : /NH2 CCK 2CO(NH2)2 = NH3 + NNH XNH2 Biuret. On further heating, more ammonia is given off; the melted mass finally solidifies, and may be shown to contain both biuret and cyanuric acid. The reaction which takes place may be represented as follows : (1) 3CO(NH2)2 == 3CONH + 3NH3 Cyanic acid. (2) 3CONH = C3N3(OH)3 Cyanuric acid. To demonstrate the presence of the biuret, the residue is dis- solved in a dilute solution of sodium hydrate, when upon the care- ful addition of a dilute solution of copper sulphate a beautiful, purple-red color develops (see also page 34). A very delicate test also is the following : 2 c.c. of a concentrated solution of furfurol arc- treated with 4-b' drops of strong hydro- chloric acid. If to this mixture, which should not present a red color, a small crystal of urea is then added, a deep violet develops in the course of a few minutes. Synthetic Formation. — As has been mentioned, urea was the first organic substance formed in the animal body which was made syn- thetically in the chemical laboratory. Wohler in 1828 produced the substance artificially by heating ammonium cyanate to a temperature of loo- C., when a rearrangemenl of atoms occurs and urea results : MI, (NH4)CN0 CO Other methods now exisl by which urea can also be made syn- 230 ' THE URINE. thetically, but they are all more or less modifications of the one just described. Isolation from the Urine. — To isolate urea on a small scale, 50-100 c.c. of urine are evaporated to a syrup on a water-bath and ex- tracted with 150 c.c. of strong alcohol by rubbing in a mortar. The alcoholic extract is filtered, the alcohol distilled off, and the syrupy residue treated with concentrated nitric acid in the cold. The urea nitrate which crystallizes out on standing is filtered off with a suction-pump, dissolved in hot water, and the aqueous solution decolorized by gently heating with animal charcoal. The colorless filtrate is then treated with barium carbonate in substance so long as carbon dioxide is evolved, and finally rendered alkaline with barium hydrate solution. The urea is thus liberated according to the equation : 2CO(NH2)2.HN03 + BaC03 = Ba(N03)2 + 2CO(NH2)2 + H20 + C02. The solution is now evaporated to dryness and the residue extracted with absolute alcohol. On concentrating this extract the urea crys- tallizes out in colorless prisms, which may then be treated as above indicated. Quantitative Estimation. — In the clinical laboratory the old method of Knop and Hufner is almost exclusively employed. This is based upon the decomposition of urea with sodium hypobromite in alkaline solution, as already described. The nitrogen which is thus liber- ated is measured and the corresponding amount of urea determined by calculation. For scientific purposes, however, this method in any one of its numerous modifications is not sufficiently accurate, as the actual volume of nitrogen which is obtained always falls somewhat short of the theoretical amount. The sodium hypobromite, moreover, also causes a partial decomposition of other nitrogenous constituents of the urine, and as the resulting amount of nitrogen is not con- stant, a further error is incurred. In scientific research we are hence forced to resort to some other procedure, such as that proposed by Morner and Sjoquist, or the simpler method which has recently been suggested by Folin. Method of Morner and Sjoquist. — This is based upon the fact that the organic nitrogenous constituents of the urine, with the exception of urea and ammonia, are precipitated by an alkaline barium chloride mixture. This precipitate is insoluble in ether- alcohol, while urea and ammoniacal salts are dissolved together with a small amount of barium hydrate. In the concentrated ether- alcoholic filtrate the nitrogen is then determined according to Kjel- dahl's method, after the ammoniacal salts have been decomposed, and the resulting ammonia has been driven on0. From the per- centage of nitrogen the corresponding amount of urea is then cal- culated by multiplying by 2.14. Five c.c. of urine are treated with an equal volume of baryta THE ORGANIC CONSTITUENTS OF THE URINE. 231 mixture, which contains 250 grammes of barium chloride in 1000 c.c. of a 5 per cent, solution of barium hydrate. To this are added 100 c.c. of a mixture of two parts of 97' per cent, alcohol and one part of ether. After twenty-four hours the precipitate is filtered off and treated with 100 c.c. of ether-alcohol. Filtrate and wash- ings are then concentrated at a temperature not exceeding 60° C. to about 20 c.c, or to a point where ammonia is no longer evolved, which can be recognized by testing the vapor with litmus-paper. A small amount of water may be added if the solution should become too concentrated. It is then washed into a Kjeldahl digest- ing flask with as little water as possible, to which a few drops of concentrated sulphuric acid have been added. The solution is now concentrated to a very small volume, upon a water-bath, and treated with 20 c.c. of a mixture of two parts of concentrated sulphuric acid and one part of the fuming acid. A pinch of yellow oxide of mercury (about 0.3 gramme) is further added, when the process is continued according to Kjeldahl, as described below. Method of Folix. — This is based upon the following considera- tions : crystallized magnesium chloride, MgCl2.6H20 boils in its water of crystallization at a temperature of about 160° C. Urea is quantitatively decomposed in such a solution into ammonia and carbon dioxide within one-half hour. If the process is carried out in acid solution, the ammonia can subsequently be distilled off after rendering the mixture alkaline, and is then titrated. The cor- responding amount of urea is ascertained by calculation. At the same time, however, the preformed ammonia is obtained, and it is hence necessary to eliminate this source of error by a separate estimation of this form. This is conveniently done "according to the method which has likewise been suggested by Folin (sec below). Method. — Three c.c. of urine are placed in an Erlenmeyer flask of 200 c.c. capacity, together with 20 grammes of magnesium chloride and 2 c.c. of concentrated hydrochloric acid. (The magne- sium chloride usually contains a small amount of ammonia, which must be separately determined.) The flask is closed with a per- forated stopper through which a straight glass tube passes, measur- ing 200 mm. in length, with a diameter of 10 nun. The mixture is now boiled until the drops flowing back through the tube produce a hissing sound on coming in contact with the solution. Alter this point has been reached, the boiling is continued more moderately for twenty-five to thirty minutes. The solution while still hot is care- fully diluted to about 500c.C. — at first by allowing the water to How drop by drop through the tube; it is then transferred to a ]()()() c.c. retort, treated with about 7 or 8 <•.<■. of* a 20 per cent, solnti. f -odium hydrate, and the ammonia distilled oil' into a measured amount, of a decinormal solution of sulphuric acid. The distillation may be interrupted when about 350 c.c. have passed over (viz., after about sixty minutes). The distillate is boned lor ,-i moment to remove any carbon dioxide which may he presenl in solution, and 232 THE URINE. on cooling is titrated to determine the excess of acid. Each cubic centimeter of the decinormal ammonia present in the distillate cor- responds to 0.003 gramme, viz., to 0.1 per cent of urea. From this result the amount of preformed ammonia and that present in the 20 grammes of magnesium chloride must be deducted. If desired, the estimation can also be made with the urea-con- taining nitrate obtained with Morner and Sjoquist's method, but Folin states that the previous isolation of the urea in such manner is probably not necessary. Estimation of the Preformed Ammonia (according to Folin). — Ten c.c. of urine are diluted to about 450 c.c, treated with a small amount of burnt magnesia (0.5 gramme) and boiled for forty-five minutes, the distillate being received in decinormal sul- phuric acid. The ammonia is then determined by titration as above. As a small amount of urea, however, is decomposed during the prolonged ebullition, it is necessary to ascertain separately the quantity of ammonia which is referable to this source. To this end, the retort is opened at the expiration of forty-five minutes, and an amount of water added which is approximately equivalent to that of the distillate. The distillation is then continued for another period of forty-five minutes, the distillate received in decinormal sulphuric acid, and the ammonia referable to decomposition of the urea estimated as before. The difference between the two results indicates the amount of preformed ammonia that was originally present. Estimation of the Total Urinary Nitrogen. — Kjeldahl's Method. — The method is based upon the observation that on treating urine with a mixture of two parts of concentrated sulphuric acid and one part of fuming sulphuric acid and boiling, the entire amount of nitrogen can be transformed into ammonium sulphate. This is then decomposed with an excess of sodium hydrate and the liberated ammonia estimated by distilling into a known amount of dilute acid, and retitrating the excess of acid. Five c.c. of urine are treated in a Kjeldahl digesting flask with a pinch of yellow oxide of mercury (about 0.3 gramme) and 20 c.c. of the sulphuric acid solution. The mixture is boiled until a per- fectly colorless solution is obtained. Vigorous ebullition, how- ever, must be avoided, and the flask should be placed at an angle of about 45 degrees, so as to prevent loss from spurting. The milder the ebullition the better. On cooling, the contents of the flask are transferred to a Kjeldahl retort, with the aid of a little distilled water. Sodium hydrate solution (27 per cent.) is then added until the greater portion of the acid is neutralized. The fluid is allowed to cool again, and a few pieces of granulated zinc or a little talcum is thrown in, when a mixture of the hydrate solution and of a 4 per cent, solution of potassium sulphide is further added in excess. Of either solution, 40 c.c. are added in all. The addition of the latter is necessary, as the mixture not only contains ammonium sulphate, but also amido- THE ORGANIC CONSTITUENTS OF THE URINE. 233 ■compounds of mercury, which latter would not give up their entire amount of nitrogen if sodium hydrate alone were present. The talcum or zinc merely prevents an unduly violent bumping when boiling. The retort is then immediately connected with a condenser through the intervention of a Kjeldahl distilling tube. The condensing tube dips into a nitrogen bulb, which contains a carefully measured amount of a one-fourth normal solution of sulphuric aeid ; 30 c.c. are usually sufficient. The mixture is now distilled until about two-thirds have passed over. The condenser is rinsed with a little distilled water, which is added to the distillate. After the addition of a few drops of tincture of cochineal the excess of acid is retitrated with a one-fourth normal solution of sodium hydrate. The difference indicates the amount of acid which was consumed in uniting with the liberated ammonia. As 1 c.c. of the one-fourth normal solution represents 0.0035 gramme of nitrogen, the amount contained in the 5 c.c. of urine is ascertained by multi- plying the number of cubic centimeters employed by this figure, from which the total amount of twenty-four hours is then readily calculated. The corresponding amount of albumin is obtained by multiplying this figure by 6.25. The method, as just described, appears simple enough, but in reality requires a considerable amount of experience to obtain figures that are reliable. With experience, however, the method is exceedingly accurate. In every case, of course, chemically pure reagents are necessary, and it is well to test these with care before proceeding. Uric Acid. Whereas in mammals, the amphibia, and fishes urea is the most important end-product of nitrogenous metabolism, the greater por- tion of the urinary nitrogen in birds and reptiles is eliminated as uric aeid. Origin. — The formation of uric acid in birds and reptiles is analo- gous t<> the formation of urea in the mammal. It is derived in thi' last instance from the albumins of the tissues and from the ing -I'd food, and, like urea, is formed synthetically in the liver. Tin- i- true at leasl of the greater portion ; while a variable frac- tion originates from the nucleins, viz., the xanthin bases. Organic ammonium -alt-, amido-acids, urea, and ammonium carbonate, when CO+0 co — nh/ Alloxan. CO — NH CO — NH^ (3) | >CO + H20 = | >CO CO — NHX COOH-NH/ Parabanic acid. Oxaluric acid. CO - NH^ CO — OH 7NH2 (4) I ^CO + H20= I +CO< COOH-NH/ CO -oh \nh2 Oxaluric acid. Oxalic acid. Urea. As oxalic acid on further oxidation is decomposed into water and carbon dioxide, it would thus appear that both oxaluric acid and oxalic acid may be regarded as complete oxidation-products of uric acid. We find, as a matter of fact, that oxalic acid is increased in various diseases in which the oxidation-processes are manifestly impaired, such as diabetes mellitus, various diseases of the circulatory apparatus when associated with deficient oxygenation of the blood, in obesity, etc. I have frequently observed, moreover, that an increased elimination of oxalic acid is associated with an increased excretion of uric acid in young, more or less ansemic individuals of a neurotic type. Whether or not oxalic acid may further be derived from carbohydrates is as yet unknown, but is rather improbable. CO- 1 ■NHv 1 CO 1 \C0 + CO(NH2). co — nh/ Alloxan. CO — NH, | >CO + C02 CO — NH^ Parabanic acid. THE ORGANIC CONSTITUENTS OF THE URINE. 243 Aside from its occurrence in solution and in urinary sediments, oxalic acid is also not infrequently found to constitute the greater portion of renal and vesical calculi. Quantitative Estimation of Oxalic Acid. — Dunlop's Method (modified by Baldwin). — Five hundred cc. of a well-mixed specimen of the twenty-four hours' urine are treated with 150 cc. of over 90 per cent, alcohol, to precipitate the oxalate of calcium. After forty- eight hours the crystals are collected on a filter, thoroughly washed with hot and cold water and with dilute acetic acid (1 per cent.). The filter is placed in a small beaker and soaked in a small amount of dilute hydrochloric acid. It is then washed with hot water until there is no further acid reaction. The washings are filtered and evaporated to about 20 cc. A very little calcium chloride solution is added to insure an excess of calcium. The hydrochloric acid is neutralized with ammonia and the solution then rendered slightly acid with acetic acid. Strong alcohol is now added to the amount of 50 per cent, of the volume of the fluid and the solution set aside for forty-eight hours. The sediment is collected on a filter that is free from ash, and washed with cold water and dilute acetic acid until free from chlorides. (Hot water should here be avoided, as it carries the finely divided precipitate through the pores of the filter.) The filter is incinerated over a Bunsen burner, and afterward heated in the blowpipe-flame. The residue is allowed to cool over sulphuric acid and weighed. The ash is calcium oxide, each gramme of which corresponds to 1.6 grammes of oxalic acid. The urine in every case should be thymolized as soon as possible, to prevent fermentation and the precipitation of phosphates. If the specimen is alkaline, it is rendered slightly acid with acetic acid. The method is applicable in the case of human urine, but in that of dog- with a high specific gravity it is very difficult to remove the phosphate-. In such an event Salkowski's method is best employed. OALKOWSKl's METHOD. — If the urine is concentrated (sp. gr. 1.040-1.050), it is treated with 20 cc of hydrochloric acid (sp. gr. 1.12) fin' 200-250 cc, and extracted in a separating funnel three times with alcoholic ether (5-10 per cent, alcohol). Tlje ethereal extract is filtered through a dry filter, the ether is distilled off, the remaining fluid evaporated to 20 cc, and filtered on cooling. The filtrate i- rendered alkaline with ammonia, and is then treated with 1-2 e.e. of a 10 per cent, solution of calcium chloride and acetic acid. The process is continued as described. With human urine Larger quantities, such as 500 e.e., are em- ployed, which an; first concent ruled to about one-third of their original volume. Allantoin. Allantoic i- a normal constituent of the urine of man, as also of various animals, but i- usually present only in traces in the 244 ■ THE URINE. adult, while during the first weeks of life it is more abundant. Larger amounts also occur during pregnancy. Aside from the urine, it is found in the amniotic fluid and in the allantoic fluid of cows. Like oxaluric acid, it is an oxidation-product of uric acid, and Salkowski demonstrated that an increased elimination occurred in dogs when uric acid was ingested. An artificial increase is similarly observed in poisoning with hydrazin, and we can readily understand that in consequence of the degenerative changes which are thus produced in the liver the oxidation-processes are accordingly diminished, and allantoin results. The formation of allantoin from uric acid may be represented by the equation : .NH.CH.NH.CO.NH2 C5H4NA + H20 + O = CO'' + C02 xNH.CO Uric acid. Allantoin. It is a glyoxyl-diureid, and may be produced artificially by heating glyoxylic acid and urea together at a temperature of 100° C. The reaction then takes place according to the equation : /NH2 COH ,NH2 /NH.CH.NH.CO.NH2 CO< + | + CO< ' = CO( + 2H20 \NH2 COOH XNH2 xNH.CO Urea. Glyoxylic Urea. Allantoin. acid. The substance crystallizes in colorless rhombic prisms, which are frequently grouped in the form of stars and rosettes. It is soluble with some difficulty in cold water, more readily in hot water and hot alcohol, while in cold alcohol and ether it is insoluble. On pro- longed boiling, it reduces Fehling's solution, and it is owing to the formation of this substance, as already indicated, that uric acid can reduce cupric oxide when boiled in alkaline solution (see page 237). With silver nitrate allantoin forms a crystalline precipitate of allan- toin silver, if ammonia is cautiously added to the solution, but it is soluble in an excess of the alkalies. Isolation. — Allantoin is most conveniently obtained from the urine of calves. To this end, the urine is evaporated on a water- bath to a thick syrup and allowed to stand for several days in the cold. The crystalline constituents which have formed are then separated mechanically from the mother-liquor and the gelatinous material which is present, and dissolved in a small amount of hot water. The solution is decolorized with animal charcoal, filtered while hot, and the filtrate acidified slightly with hydrochloric acid. On standing, the allantoin separates out. Kreatinin. The kreatinin which is found in the urine of man and all mammals is in all probability derived from the kreatin of the muscle-tissue, and is thus in part referable to the ingested animal THE ORGANIC CONSTITUENTS OF THE URINE. 245 food, and in part to the wear and tear which constantly goes on in the muscular structures of the body. The latter source, as com- pared with the former, is normally, however, of secondary impor- tance, as the greater amount of kreatin which originates in the muscles is, in health at least, transformed into urea. We accord- ingly find that the elimination of kreatin is much reduced when the animal is placed on a diet of milk exclusively, while it is increased if a liberal amount of meat is ingested, or kreatin is administered as such. On the other hand, we observe that muscular exercise in itself does not call forth an increased excretion, although this has recently been denied. Under pathological conditions, however, in which an increased destruction of the albuminous constituents of the body occurs, abnormally large quantities may be found in the urine, although the patient receives food which is free from kreatin. In such cases we may assume that the muscles have lost to a greater or less degree the power of transforming kreatin into urea. Kreatin itself has thus far not been found in perfectly fresh urine, but is formed from the kreatinin on the occurrence of bacterial decomposi- tion. The transformation of kreatin into its anhydride supposedly occurs in the kidneys, and we accordingly find that in extensive disease of these organs the elimination of kreatinin is diminished. Of late, it has been claimed that the kreatinin which is found in muscle-tissue is not identical with the urinary kreatinin, and it has even been suggested that the source of the latter may have to be sought in other organs of the body, and notably in the thyroid gland, in which kreatinin has been found in considerable amount. It is, of course, possible that the substance may be formed in other organs as well, but the evidence is conclusive that the urinary form is largely derived from muscle-tissue. But even supposing that future researches should show that the two bodies are isomeric, but not identical, we could even then imagine that both originate from the same substance. The amount of kreatinin which is normally found in the urine of man is approximately 1 gramme, but varies, of course, with the character of the food. Properties. — Kreatinin crystallizes in colorless, highly refractive monoclinic prisms, and is quite soluble in hot and cold water, less readily so in alcohol, and is insoluble in ether. In aqueous solution it i- gradually transformed into kreatin. The same transformation results more rapidly when the substance i< heated in alkaline solu- tion. It combines with acids and various salts to form crystalline compounds, some of which are characteristic. This is true especially of the chlorozincatt — (C^HyNjOg^.Zni !12 — which results when a con- centrated alcoholic solution of kreatinin is treated with a solution of zinc chloride, which should be as little acid as possible. The crystalline form of this compound depends very much upon its purity. As firsi obtained from the urine, it occurs in the form of varicose conglomerations, which usually adhere firmly to the walls 246 THE URINE. of the vessel. In pure form it crystallizes in fine needles, which are commonly grouped in sheaves and stars. The salt is almost insoluble in alcohol, and nearly so in water, while free mineral acids dissolve it. Kreatinin is further precipitated by mercuric nitrate, notably in the presence of sodium carbonate, by silver nitrate, platinum chloride, stannous chloride, mercuric chloride, picric acid, phospho- tungstic acid, etc. With all these substances it forms well-charac- terized crystalline salts. In alkaline solution it reduces cupric hydroxide, and it is for this reason that urines which contain much kreatinin but no sugar give a positive reaction with Fehling's solution. The separation of the cupric oxide, however, only occurs on prolonged boiling. An alkaline solution of bismuth, on the other hand, is not affected. Tests. — Weyl's Test. — A few cubic centimeters of urine are treated with a few drops of a freshly prepared, very dilute solution of sodium nitroprusside, and then drop by drop with a solution of sodium hydrate, when in the presence of kreatinin a ruby-red color develops, which is especially pronounced in the lower portion of the tube. After a few minutes the color disappears. If now acetic acid is added in excess and the mixture heated, it assumes a greenish color, then turns blue, and on standing deposits a sediment of Prussian blue. Acetone and diacetic acid give a similar reaction if ammonia is added instead of sodium hydrate ; but with kreatinin no red color is thus obtained. Jaffe's Test. — If a few cubic centimeters of urine are treated with a dilute aqueous solution of picric acid, the corresponding compound of kreatinin is precipitated. On adding a few drops of a dilute solution of sodium hydrate a red color develops, which per- sists for several hours, and is changed to yellow upon the addition of an acid. With glucose a red color also is obtained, but only on heating, while the reaction in the case of kreatinin takes place only at ordinary temperatures. Acetone gives a reddish-orange color. Synthesis of Kreatinin. — Kreatinin can be formed synthetically from methyl-glycocoll and cyanamide. Kreatin is first produced, and then yields the anhydride, as shown in the equations : NH (1) N=C — NH2 + XH(CH3)CH2.COOH = NH=C< X(CH3).CH2.COOH Cyanamide. Methyl-glycocoll. Kreatin. XNH2 yNH CO (2) NH=C< = NH=C< | + H20 XN(CH3).CH2.COOH XN(CH3).CH2 Kreatin. Kreatinin. Isolation and Quantitative Estimation. — The quantitative estima- tion of kreatinin in the urine is based upon the formation of the chlorozincate, which is almost insoluble in alcohol : 240 c.c. of urine, which should be free from albumin and sugar, are rendered THE AROMATIC CONSTITUENTS OE THE URINE. 247 alkaline with milk of lime, and treated with a solution of calcium chloride so long as a precipitate forms. Water is then added to the .')()<) c.c. mark. After standing for a while the phosphates are tillered off. The precipitate is washed with a little water. Filtrate and washings are rendered slightly aeid with acetie acid, and are then evaporated to a syrup. This is stirred while still warm with about 25 to 30 C.C. of absolute alcohol, transferred to a glass- stoppered flask, and diluted with absolute alcohol to 100 c.c. After twenty-four hours the mixture is filtered. The filtrate is treated with a small amount of sodium acetate in solution, and is con- centrated to about 50 c.c. To this is added 0.5 c.c. of a concentrated alcoholic solution of zinc chloride, which is prepared by dissolving a small amount of the salt in SO per cent, alcohol, and diluting with 95 per cent, alcohol to a specific gravity of 1.2. The mixture is then well stirred and set aside in a cool place for several days. The crystals of the chlorozincate of kreatinin are now collected on a previously weighed filter and washed with alcohol until free from chlorides, when they are dried at 100° 0. and weighed. The corresponding amount of kreatinin is ascertained by multiply- ing the weight by 0.6243. The material which is thus obtained is, however, always impure, owing to an admixture of various pig- ments and traces of chlorides. If greater accuracy is required, it is hence necessary to determine the amount of zinc in the crystals, which may be done as follows: the material is covered with a little nitric acid ; the solution is evaporated, the residue incinerated, extracted with water, ami the aqueous solution evaporated, the residue ignited, and finally weighed. The zinc is thus obtained as oxide, from which the corresponding amount of kreatinin is cal- culated by multiplying by 2.77DO. To isolate the kreatinin from the chlorozincate, the latter is dis- solved in a small amount of hot water and boiled for ten minutes with well-washed plumbic hydrate. After filtering off the insoluble oxide of zinc and the chloride of lead the filtrate is evaporated to dryness and extracted with cold absolute alcohol. This takes up the kreatinin, while a small amount of kreatin formed during the process of boiling remains. On evaporation the kreatinin is obtained in crystalline form, and can be further purified by recrystallization from water. THE AROMATIC CONSTITUENTS OF THE URINE. It has been pointed out in a preceding chapter that (luring the process of intestinal putrefaction various aromatic bodies are formed from the albumins of the food or their products of digestion, and are then absorbed and eliminated in the urine, either as such or in combination with sulphuric acid, glucuronic acid, or glycocoll. Some of i Ik-,, bodies, Buch :i^ indol and skatol, may be regarded as specific product- of putrefaction ; while others or closely related 248 THE URINE. substances occur preformed also in many articles of food. We con- sequently recognize two sources of the aromatic bodies which are found in the urine, viz., the aromatic bodies which enter into the composition of our diet as such, and those which result from the destruction of albumins through the activity of micro-organisms. Under certain pathological conditions, further, substances of this character may also be formed in the body proper, owing to degenera- tive changes, which may or may not be the result of bacterial action. Under normal conditions, however, this source scarcely enters into consideration. The Conjugate Sulphates. Paracresol, phenol, hydroquinon, pyrocatechin, indol, and skatol are largely eliminated in the urine in combination with sulphuric acid as sodium or potassium salts. But while paracresol and its derivatives combine with sulphuric acid directly, indol and skatol are previously oxidized to indoxyl and skatoxyl, as has been shown. Conjointly the resulting compounds are spoken of as the conjugate or ethereal sulphates of the urine. Their daily excretion in man corresponds to about one-tenth of the mineral sulphates, viz., from 0.094 to 0.620 gramme, under normal conditions. Increased amounts are observed when from whatever cause the intestinal putrefaction is increased. It is to be noted, however, that the ratio between the individual substances is even normally not constant, and it seems that the relative preponderance of the one over the other is primarily referable to the extent to which individual groups of micro-organisms are active. In some instances we may thus find that the increase in the conjugate sulphates is referable to an increased production of indol and .skatol, while in others phenols are largely formed. Aside from an increase in the degree of intestinal putrefaction, larger amounts of the conjugate sulphates may also be observed if putrefactive processes are taking place within the body proper, pro- viding that active resorption occurs from the diseased area. An increased elimination is also noted when any of the aromatic sub- stances mentioned are ingested as such or otherwise introduced into the circulation from without. A notable increase is thus observed in poisoning with carbolic acid or its congeners, and is then, of course, principally owing to an increased formation of phenol sul- phates. The ingestion of ortho-nitro-phenyl-propiolic acid, which is reduced to indoxyl within the body, similarly leads to an increased elimination of indoxyl sulphate. The synthesis of the various conjugate sulphates is probably effected within the liver, but may also occur in other organs of the body. Their quantitative estimation in toto has been described. The Phenols. — Of the phenols which occur in the urine, para- cresol is the most abundant ; next in order comes phenol, while pyrocatechin and hydroquinon are found only in traces. Besides THE AROMATIC CONSTITUENTS OF THE URINE. 249 paracresol, the normal urine of man is said also to contain minute amounts of meta- and ortho-cresols. The total elimination of cresols and phenols, however, normally corresponds to only about 0.03 gramme in the twenty-four hours. Urine which contain- much hydroquinon or pyrocatechin gradually assumes a dark-brown color on standing if the reaction is alkaline, and it is noted that this change in color begins in the upper layers and gradually extends downward. Ultimately the urine becomes almost black. This change is referable to oxidation of the dioxy- benzols, but of the resulting compounds nothing is known. Urines of this character are mostly observed in poisoning with carbolic acid, following the administration of benzol, of phenol sulphates, etc. To demonstrate the presence of phenol or of paracresol, 1000 c.c. of urine are treated with 70 c.c. of concentrated hydrochloric acid, and distilled until about one-fourth of the total amount has passed over. The conjugate sulphates are thus decomposed, and phenol and cresol are found in the distillate. Their presence can here be demonstrated by testing with Millon's reagent or by adding bromine-water, when tribromophenol crystallizes out on standing. If much phenol is present, it may further be possible to obtain a positive reaction with ferric chloride solution if this is added drop by drop in very dilute solution. Hydroquinon and pyrocatechin remain in the acid solution. To demonstrate their pres- ence, the solution is evaporated to about 100 c.c, and on cooling is extracted with an equal volume of ether. Hydroquinon and pyro- catechin together with the aromatic oxy-acids are thus removed. On adding a dilute solution of sodium carbonate to the ethereal solu- tion the aromatic oxy-acids are transformed into the corresponding sodium salts. The ethereal extract, which now contains only the dioxy-benzols, i- evaporated to dryness ; the residue is dissolved in a little water and examined as follows: one portion is treated drop by drop with a dilute solution of ferric chloride, when in the pres- ence of pyrocatechin a green color develops, which turns to violet upon the addition of a small amount of tartaric acid and ammonia. Tin- remainder of the solution is precipitated with lead acetate and filtered. The filtrate contains the hydroquinon, while the pyro- catechin is in the precipitate. The hydroquinon can then lie isolated by acidifying and extracting with ether, when the substance crys- tallizes out on evaporation. It is dissolved in a little water, and treated drop by drop with the dilute iron solution. Quinon, C I !/>.. results, and may be recognized by it- penetrating odor. Quantitative Estimation. — METHOD of KOSSLEB and PENNY, modified by Neuberg. — This method is based upon the precipi- tation of phenol and paracresol, by mean- of iodine, a- tri-iodo- phenol. prom the amount of iodine which is thus used the corre- sponding amount of monoxy-benzols can be calculated, and is expressed either in terms of phenol or cresol, as the method does not indicate the separate amount of the individual bodies that are 250 THE URINE. present. The reaction which takes place may be represented by the equation : C6H5.OH + 61 = C6H2.I3OH + 3HI. Five hundred c.c. of urine are rendered feebly alkaline an.d evaporated to about 100 c.c. Any acetone which may have been present is thus removed. The residual fluid is acidified with sul- phuric acid, so as to contain 5 per cent, of the original volume, and is then repeatedly distilled. The individual portions thus obtained are shaken with calcium carbonate until the acid reaction has dis- appeared, so as to remove any nitrous or formic acid that may be present. The fluid is now again distilled, and the distillate treated with a solution of 1 gramme of caustic soda and 6 grammes of lead acetate in substance. The mixture is kept on a boiling water-bath for about fifteen minutes. A portion of the lead oxide is thus dis- solved by the phenol to form basic phenolates, while any aldehydes or ketones that may have been formed from the small amount of carbohydrates that are present in every urine escape. To remove these entirely, the mixture is heated over a free flame connected with a condenser until a few cubic centimeters of the distillate no longer reduce an alkaline solution of silver nitrate. After five minutes this point is usually reached. The fluid is then acidified with sul- phuric acid as before, and is distilled, water being added from time to time. The distillate is placed in a glass-stoppered bottle, treated with a deci normal solution of sodium hydrate until the reaction is markedly alkaline, and immersed in hot water. To the hot fluid a decinormal solution of iodine is added in an amount which should exceed that of the alkali solution by 15 to 25 c.c. The bottle is now closed at once, shaken, and set aside until cool. The solution is then acidified with dilute sulphuric acid, and the excess of iodine, which was not used in the formation of tri-iodophenols, retitrated with a decinormal solution of sodium thiosulphate. One c.c. of the iodine solution represents 1.567 milligramme of phenol, or 1.8018 milli- gramme of cresol. As the latter predominates in the urine, it is best to express the results in terms of cresol. Thus modified, the method is also applicable in the presence of sugar. The older gravimetric method, by which the phenols were isolated as bromine substitution- products, has now been largely abandoned, as it has been shown that the resulting precipitate is not of constant composition, and contains variable amounts of dibromocresol, besides tribromophenol and bromo-tribromophenol. Indoxyl Sulphate. — The incloxyl sulphate which occurs in the urine in combination with potassium and sodium is usually spoken of as indican, but should not be confounded with the vegetable indi- can, which, as has been shown, is a glucoside of the composition C26H31NO,7. The amount which is daily excreted by man is nor- mally small, and corresponds to about 0.0066 gramme. Larger quantities are observed when from any cause intestinal putrefaction THE AROMATIC CONSTITUENTS OF THE URINE. 251 is increased, or in cases in which putrefactive changes are taking place in the body proper, as in empyema, providing that active resorption can occur. In herbivorous animals larger amounts are found than in the carnivora. Artificially an increased elimination can be effected by feeding animals with ortho-nitro-phenyl-propiolic acid, which is reduced in the body to indoxyl, according to the equation : /C=COOH /C.OH=CH C6H4; +8H = C6H/ +3H20 \no2 xnh Ortho-nitro-phenyl- Indoxyl. propiolic acid. Indican crystallizes in colorless platelets, which are readily soluble in water and hot alcohol, while in cold alcohol they dissolve with great difficulty. On decomposition with hydrochloric acid the indoxyl is obtained in the form of oily droplets of an exceedingly offensive, feculent odor. On oxidation this is then transformed into indigo-blue, as is shown in the equation : /C.OH = CH ,CO v /CO N 2C6H^ +20 = C6H4< >C=C< >C6H4 + 2H20 \NH \nH/ xNH/ Indoxyl. Indigo-blue. On heating an aqueous solution of indoxyl to a temperature of 130° C. indoxyl-red also results. This is a brown amorphous sub- stance, which is insoluble in water, but dissolves with ease in alcohol, ether, and chloroform, with a beautiful red color. When Jaffe's test for indican is applied to the urine, or when this is boiled and treated drop by drop with concentrated nitric acid (Rosenbach's reaction), a mixture of a blue and a red pigment is not infrequently obtained, and it is quite likely that the latter is in part at least refer- able to the formation of the indoxyl-red. According to some observ- ers, the chromogen of this substance is identical with the so-called uroluematin, and the pigment is probably the same as the red pig- ment of Scherer, the urrhodin of Heller, the urorubin of Plosz, the indirubin of Schunk, the indigo-purpurin of Bayer, the pigment of Giacosa and others. The blue pigment which is found together with the red pigment when urine i- treated with a strong mineral acid and an oxidizing agent is, :i- lias been indicated, indigo-blue, and is identical with urocyanin, cyanurin, Harnblau, uroglaucin, etc., of former observers. A- ;i general rule, its amount is far greater than that of the red pig- ment, and is at times the only one that is obtained. In other cases, however, the red seems to prevail, and in still others both are appar- ently present in aboul equal proportion. The cause of these varia- tions i- a- vet not understood, but probably rests upon variations in bacterial action in the intestinal tract. As a general rule, indeed, notable quantities of the red pigment are observed only under patho- logical condition-. 252 THE URINE. Tests for Indican. — All the tests employed for the purpose of demonstrating the presence of indican in the urine are essentially based upon the decomposition of the substance, with the liberation of indoxyl and its oxidation to indigo-blue. Jaffe's Test, as modified by Stokvis. — A few cubic cen- timeters of urine are treated with an equal volume of concentrated hydrochloric acid, and two or three drops of a strong solution of sodium hypochlorite. The indigo-blue which thus results is then extracted by shaking with a little chloroform. If red pigment has been formed at the same time, the color varies from a violet to a purplish red. If it is desired to separate the two pigments, the chloroform extract is evaporated to dryness and the residue washed with a mixture of equal parts of 96 per cent, alcohol, ether, and water. This dissolves the red pigment and leaves the indigo-blue behind. Care must be had, however, not to add too much of the hypochlorite solution, as otherwise the indigo-blue is oxidized to indigo-white, and no color at all is obtained. Should this happen after the addition of only one or two drops, the following test had better be employed, as a further oxidation is here not effected : Obeema yer's Test. — A few cubic centimeters of urine are treated with an equal volume of a 2 pro mille solution of ferric chloride in concentrated hydrochloric acid. The indigo-blue is extracted, as above, by shaking with a little chloroform. As in the above test, indoxyl-red may thus also be obtained, and is separated from the blue pigment as just described. Test for Urohaematin (so-called). — A small amount of urine is thor- oughly agitated with chloroform and allowed to stand for a few days. The chromogen of indoxyl-red is thus extracted, for on adding a drop of concentrated hydrochloric acid to the chloroform extract a beautiful rose-color appears, which varies in intensity with the amount of the chromogen present. In normal urine a faint reaction only is usually seen ; but in disease, and notably in ileus, peritonitis, and cancer of the stomach, I have repeatedly met with more indigo- red than indigo-blue. Rosenbach's Reaction. — This reaction is mostly obtained under pathological conditions, and indicates the existence of greatly in- creased intestinal putrefaction. It is referable to the simultaneous formation of indigo-red and indigo-blue. While boiling, a few cubic centimeters of urine are treated drop by drop with concentrated nitric acid containing a little nitrous acid, when in the presence of much indigo-red, viz., its chromogen, the urine assumes a dark Burgundy color, and usually shows a bluish tint when held to the light. On standing, the red pigment is precipitated. If much indigo-blue is present at the same time, as is usual, the foam of the liquid is colored blue. On adding an excess of the acid the color often disappears and the urine turns yellow. THE AROMATIC CONSTITUENTS OF THE URINE. 253 The isolation of indican from urine as such is a rather complicated process, and need not be described at this place. Quantitative Estimation. — Wang's Method. — This method is based upon the decomposition of the indican by strong hydrochloric acid, and the ox illation of the resulting: indoxyl to indigo-blue. This is then transformed into indigo-sulphuric acid, and estimated as such by titration with a solution of potassium permanganate of known strength. To this end, a stock solution of the salt is kept on hand, which contains about 3 grammes to the liter. Five c.c. of this solution are diluted with 195 c.c. of water, when the titre is ascertained before each titration by comparing it with a dilute solu- tion of oxalic acid. The amount of indigo-blue which each cubic centimeter represents is ascertained by multiplying the correspond- ing amount of oxalic acid by 1.04. The amount of urine which is necessary varies with the amount of indican present. If a preliminary test gives an intense reaction, from 25 to 50 c.c. are sufficient ; otherwise it is better to use larger amounts, as from 200 to 250 c.c. The urine is then precipitated with a 20 per cent, solution of basic lead acetate, care being taken to avoid an excess. A large portion of the filtrate, representing a known amount of urine, is then treated with an equal volume of Obermayer's reagent, and extracted with chloroform by shaking. This is continued with portions of 30 c.c. until the chloroform takes up no more coloring-matter. The combined extracts are freed from chloroform by distillation. The residue is dried for a few minutes on a water-bath, and is then washed with a mixture of equal parts of water, ether, and alcohol (96 per cent.), to remove the reddish- brown pigment which is present together with the indigo-blue. The solution is passed through a small filter, so as to collect any particles of the blue pigment which may be present in suspension. The filter is dried, extracted with boiling chloroform, and the resulting solution filtered into the flask containing the residual indigo-blue. The chloroform is distilled off, and the residue treated with 3 or 4 c.c. of concentrated sulphuric acid, while still warm. This solution is allowed to stand for twenty-four hours. It is then poured into 100 c.c. of water, the bottle is washed out with a little more water, when the solution and washings are filtered and titrated with the permanganate solution. The color at first changes to green, and finally becomes yellowish or colorless. The calculation 18 conducted as outlined above. Skatoxyl Sulphate. — Skatoxy] sulphate, like indoxyl sulphate, occurs in the urine in combination with potassium and sodium. Its amount, however, i< normally small, and it may at times be absent altogether. Larger quantities are found under pathological condi- tion-, associated with an increased degree of intestinal putrefac- tion, and it may then happen thai more skatoxy] sulphate is found than indican. This, however, is uncommon, and in disease also more indican i- usually present. Like indican, it is decomposed 254 THE URINE. on treating with concentrated hydrochloric acid, and on subsequent oxidation the liberated skatoxyl yields pigments which are for the most part of a red color. Of their chemical nature, however, nothing is known. One of these may possibly be identical with Rosin's urorosein. Rosin, to be sure, claims that the chromogen of urorosein is not a conjugate sulphate but we know that a portion of the skatol also appears in combination with glucuronic acid in the urine, and it is hence possible that his pigment may be derived from this source. Urines containing notable quantities of skatoxyl become darker on exposure to the air, and may gradually turn a reddish-violet or almost a black color. This change, as in the phenol urines, begins at the surface and gradually extends down- ward. Tests. — To demonstrate the presence of skatoxyl in urine, this is strongly acidified with hydrochloric acid and extracted with amyl alcohol, which takes up the coloring-matter. Chloroform and ether do not dissolve this in acid solution, but do so in neutral or alkaline solution, providing that the pigment has been freshly formed. Test for Urorosein (so-called). — A few cubic centimeters of urine are treated with an equal amount of concentrated hydrochloric acid and one or two drops of a strong solution of calcium hypochlorite. The indigo-blue is extracted with chloroform, and it will now be observed that the supernatant fluid presents a red color. On shaking with amyl alcohol this takes up the red pigment, which is thus manifestly not identical with indigo-red. Upon the addition of sodium hydrate to the alcoholic solution the color disappears, but reappears upon the subsequent addition of hydrochloric acid. On standing, the color gradually disappears. Under normal conditions this reaction is not well marked, but becomes quite distinct in cases in which intestinal putrefaction is much increased. A portion of the skatol, as I have already stated, appears in the urine as skatol-carbonic acid: /C(CH3k QH4< >C.COOH. The substance is usually present in exceedingly small amounts, however, and has not been isolated in substance. To demonstrate its presence even, it is necessary to work with several liters of urine. To this end, the fluid (5000-6000 c.c.) is evaporated to a syrup ; the residue is extracted with absolute alcohol, the alcoholic solution is evaporated, and the remaining material extracted with ether after acidifying strongly with sulphuric acid. From the ethereal solution the substance is obtained in aqueous solution by shaking with a dilute solution of sodium hydrate. The alkaline solution is then evaporated and the residue extracted with alcohol. This extract is now concentrated to about 100 c.c. and precipitated THE AROMATIC CONSTITUENTS OF THE URINE. 255 with an equal volume of ether. The filtrate is evaporated to dry- ness, the residue acidified with hydrochloric acid, and extracted with ether. The residue of the ethereal extract is then finally dissolved in hot water. To remove the remaining hydrochloric acid, this solution, after filtering and cooling, is again evaporated to dryness and redissolved in hot water. To demonstrate the presence of skatol-carbonic acid, a portion of this final solution is then treated with a few drops of pure nitric acid and a trace of potassium nitrite. A red color thus develops, which may be extracted with amvl alcohol or acetic ether. This solution shows a band of absorp- tion in the green portion of the spectrum. On adding a solution of sodium hydrate to the ethereal solution the red color disappears, but reappears on the subsequent addition of hydrochloric acid. In addition to the aromatic bodies which have thus far been con- sidered, traces of the aromatic oxy-acids may also appear in the urine in combination with sulphuric acid, but the amount is exceed- ingly small, and may well be ignored. The Conjugate Glucuronates. Glucuronic acid as such does not occur in the urine. The sub- stance can combine with various aromatic bodies, however, and may in this manner escape further oxidation in the body. Normally, it is found only in traces, in combination with indoxyl, skatoxyl, phenol, and paracresol, while the greater portion of these substances is eliminated in the form of conjugate sulphates, as has been pointed out. Larger quantities are found in the urine after the administra- tion of chloral, camphor, naphtol, oil of turpentine, menthol, toluol, euxanthin, morphin, etc. With these it forms compounds which are closely related to the glucosides. According to the character of the aromatic component, the resulting glucuronates have been termed campho-glucuronic acid, menthol-glucuronic acid, urochloralic acid, phenyl-indoxyl-skatoxyl-glucuronic acid, etc. Of the origin of the glucuronic acid, and its fate under normal conditions, we know little. That it is formed in the tissues of the body is apparent from the fact that even in a starving animal the administration of camphor, chloral, etc., leads to elimination of these substances in combination with the acid in question. As glucuronic acid is a derivative of glucose, we may thus imagine that during starvation it is derived from glycogen, or even from the albumins, through a splitting off of the carbohydrate group. \t has been demonstrated, a- a matter of fact, that the formation of glycogen in the liver can be artificially increased by introducing glucuronic acid with the food. The chemical relation of glucuronic acid to glucose ha- already been considered. On oxidation glucose thus first yields the monobasic glucuronic acid, and then the dibasic saccharinic acid. The latter in turn may be transformed into saccharo-lactonic acid, which on reduction yields glucuronic acid, so that this stands mid- 256 THE URINE. way between gluconic acid and saccharinic acid. These relations are shown by the formulas : CH2.OH — (CH.OH)4 — COH, glucose. CH2.OH — (CH.OH)4 — COOH, gluconic acid. COOH — (CH.OH)4 — COH, glucuronic acid. COOH. — (CH.OH)4 — COOH, saccharinic acid. On boiling with water glucuronic acid is, in part at least, trans- formed into its anhydride, glucuron, C6H806. Aside from its probable origin from glycogen, glucuronic acid may also be derived from chondroitin-sulphuric acid, which has also been observed in normal urine. We have seen, as a matter of fact, that this acid occurs in the cartilaginous structures of the higher animals, and that its hyalin component chondroitin first yields chondrosin on hydrolytic decomposition, which is then further decomposed into glucuronic acid and glucosamin, as shown in the equations : (1) C18H27NOu + 3H20 = C12H21NOu + 3CH3.COOH Chondroitin. Chondrosin. Acetic acid. (2) C12H21NO„ + H20 = COOH.(CHOH)4.COH + C6Hn06.NH2 Chondrosin. Glucuronic acid. Glucosamin. Schmiedeberg, indeed, regards the chrondroitin-sulphuric acid as the normal source of the glucuronic acid. Glucuronic acid has thus far not been obtained in crystalline form. It is a syrupy substance which is readily soluble in water and alcohol. Its anhydride, however, is a crystalline body, and is likewise soluble in water but insoluble in alcohol. The free acid and its alkaline salts are dextrorotatory, while the conjugate glucuro- nates turn the polarized light to the left. The free acid, moreover, as well as its salts and most of its compound ethers, reduces the oxides of copper, bismuth, and silver in alkaline solution, and it is thus possible to confound them with glucose if reliance is placed upon the corresponding tests alone. "With phenyl-byclrazin the free acid is said to form a crystalline compound with a melting-point of 114°— 115° C. Unlike glucose, it is non-fermentable. It gives the fur- furol reaction and simulates the pentoses in reacting with phloro- glucin hydrochlorate, but not with orcin (see page 284). To demonstrate the presence of glucuronic acid in the urine, it is necessary to isolate its compound ethers and to decompose these with superheated steam. Under normal conditions this is practi- cally impossible unless very large quantities of urine are employed. Its presence may, however, be suspected if a urine reduces Fehling's and Nylander's solution, if it is lsevorotatory, and gives the phloro- glucin hydrochlorate reaction, while sugar is absent. The Compound Glycocolls. As has been pointed out, phenyl-propionic acid and phenyl-acetic acid, which are both formed from albuminous material during the THE AROMATIC CONSTITUENTS OF THE URINE. 257 process of intestinal putrefaction, are in part absorbed in the intestinal tract, and are eliminated in the urine in combination with glycocoll as hippuric acid and phenaceturic acid, respectively. But while phenvl-acetic acid unites with glycocoll directly, phenyl-propionic acid is usually first oxidized to benzoic acid (see page 97). Hippuric Acid. — While a certain amount of the benzoic acid which enters into the construction of the hippuric acid molecule is derived from the phenyl-propionic acid which results during the process of intestinal putrefaction, another portion is ingested as such, or in the form of other aromatic substances which can be transformed into benzoic acid in the animal body. We can thus understand why larger amounts of hippuric acid are encountered in the urine of herbivorous animals than in that of the carnivora, as the food of the former always contains very considerable amounts of toluol, cinnamic acid, quinic acid, etc., all of which may give rise to benzoic acid. In man the daily elimination corresponds to about 0.7 gramme, but may be increased by the ingestion of such articles of food as cranberries, primes, reine-claudes, etc. In a few instances the substance has been found in urinary sediments. While the source of the aromatic component of hippuric acid is thus quite well understood, we know but little of the origin of glycocoll. To a certain extent this may also be formed during the process of albuminous putrefaction, as we know that all albumins contain a glycocoll radicle, but there is reason to believe that it may likewise originate within the tissues of the body. It mani- festly plays an important role in the process of metabolism, for we have seen that to a certain extent, no doubt, it is concerned in the formation of urea, and it has also been noted that in man and in other animals a very considerable proportion of cholalic acid is elimi- nated from the body in combination with glycocoll. Through the interesting researches of Sehmiedeberg we know that the synthesis of glycocoll and benzoic acid is in dogs effected in the kidneys exclusively, in other animals, however, this process may occur in other organs as well, for it has been shown that after removal of the kidneys hippuric acid can be isolated from the liver and the muscles, at least, if benzoic acid has been previously administered. Properties. — Hippuric acid crystallizes in long rhombic prisms when allowed to separate from its solutions slowly, while when rapidly formed, and especially if the amount is small, it occurs in long neend rosettes. The melting-point of the substance is 1*7. o ( !. It is i table with great difficulty in cold water and ether, while in hot water and alcohol it dissolves with comparative ease. In aqueous solutions of the alkaline hydrates and carbonates it dissolves with the formation of the corresponding -:ilt-, from which the free acid may again be obtained by acidifying with a mineral acid. 17 258 THE URINE. On boiling with dilute mineral acids or alkalies hippuric acid is decomposed into its components. The same result is reached if ammoniacal decomposition is allowed to occur, and in such urines benzoic acid only is found. In traces, benzoic acid is said to occur in every urine together with hippuric acid, and it is thought that its presence under normal conditions may be due to the action of a ferment, the so-called histenzyme of Schmiedeberg, which has been found in the kidneys, and which is known to be capable of effecting the decomposition of hippuric acid as outlined. On heating hippuric acid in a dry test-tube it melts, and is then decomposed with the formation of benzoic acid, which sublimes in the upper portion of the tube. The liquid mass at the same time assumes a red color, and develops an odor which at first is suggestive of hay, but subsequently resembles that of hydrocyanic acid. This reaction, together with the form of the crystals and their insolubility in petroleum-ether, serves to distinguish hippuric acid from benzoic acid. But like this, it develops a marked odor of bitter almonds when it is evaporated with nitric acid and the residue is then heated. The reaction is due to the formation of nitrobenzol. In the urine hippuric acid is, of course, not present in the free state, but in combination with alkalies, and notably potassium and sodium. Synthesis of Hippuric Acid. — Hippuric acid can be formed syn- thetically in vitro also from benzoic acid and glycocoll by heating the two substances together at a temperature of 160° C. in a sealed tube. In a similar manner it is obtained from benzamide and monochlor-acetic acid. The reactions which take place may be represented by the equations : C6H5.COOH + CH2(NH2).COOH = CH2.NH(C6H5.CO).COOH + H20 Benzoic Glycocoll. Hippuric acid, acid. C6H5.CO.NH2 + CH,.Cl.COOH = CH2.NH(C6H5.CO).COOH + HC1 Benzamide. Monochlor- Hippuric acid. acetic acid. Isolation of Hippuric Acid. — Hippuric acid is most conveniently isolated from the urine of herbivorous animals, in which, as has been stated, it is present in much greater amounts than in that of man and the carnivorous animals. To this end, 1000 c.c. of fresh urine are rendered feebly alkaline with milk of lime and boiled for a few minutes. The liquid is filtered while still warm, concentrated, and on cooling acidified with hydrochloric acid added in moderate excess. After twenty-four hours the crystals of hippuric acid which have separated out are filtered off, and are freed from adhering pig- ments as follows : they are dissolved in hot water, and treated with alum and then with an amount of sodium carbonate sufficient to cause the formation of an abundant precipitate ; the reaction, how- ever, should remain still acid. The pigments are retained by the aluminous precipitate. The filtrate is then strongly concentrated, THE AROMATIC CONSTITUENTS OF THE URINE. 259 acidified with hydrochloric acid, and allowed to stand. In this manner the hippuric acid is obtained in fairly pure form, and can be further purified, if desired, by recrystallization from hot water. Isolation of Glycocoll and Benzoic Acid from Hippuric Acid. — As stated before, glycocoll is most conveniently obtained from hippuric arid. To effect the decomposition, this is boiled for ten to twelve hours with four parts of diluted sulphuric acid. On cooling-, the benzoic acid which has separated out is filtered off. The filtrate is concentrated and extracted with ether, which takes up the remain- ing benzoic acid. The sulphuric acid is now removed by means of barium hydrate and the excess of barium precipitated with carbon dioxide. The filtrate is concentrated, when on cooling the glycocoll is obtained in crystalline form. Isolation and Quantitative Estimation. — Five hundred to one thou- sand c.c. of urine are rendered slightly alkaline with sodium car- bonate, and are evaporated to a thick syrup, taking care that the reaction remains alkaline. It is then extracted with cold strong alcohol (90 to 95 per cent.) by adding about one-half the volume of the original solution, and allowing the mixture to stand for twenty- four hours. It is then filtered and the alcohol distilled off. The remaining aqueous solution is acidified with dilute sulphuric acid, and the liberated hippuric acid extracted with several portions of acetic ether The ethereal solution is evaporated to dryness, when the remaining impurities, such as phenols, aromatic oxy-acids, benzoic acid, and fat, are removed by washing with cold petroleum- ether, in which hippuric acid is insoluble. It is then dissolved in warm water, and the solution evaporated at a temperature of from 50° to 60° C. until crystallization occurs. On cooling, the crystals are filtered off and weighed. The mother-liquor is extracted with acetic ether, the ethereal solution is evaporated to dryness, and the weight of the residue added to that of the crystals. Phenaceturic Acid. — In small amounts phenaceturic acid has been repeatedly obtained from the urine of man, but is principally met with in that of herbivorous animals, in which the putrefactive processes, owing to the greater length of the intestinal tract and the character <>(' the food, are more extensive. On boiling with dilute mineral acids it is decomposed into its components, as shown in the equation : < :i Nil < II .< M ,<<>, .COOH H,o rn,(Cfiir-,).n>(>ir | CH2(NHaVCOOH Phenaceturic acid. Phenyl-acetic acid. Glj cocoll. Properties. — Phenaceturic acid crystallizes in small rhombicqdatos with rounded angles, which are very similar to the corresponding crystals of uric acid. Isolation. — Phenaceturic acid may be isolated from the urine of the horse after separation of the hippuric acid, as shown above. The mother-liquor i- then extracted with acetic ether, which takes id th<- acid. On evaporation the residue is dissolved in a dilute 260 THE URINE. solution of sodium hydrate. From this solution the substance is again extracted with ether, after acidifying with hydrochloric acid, and thus purified, and finally allowed to crystallize from its aqueous solution. In this connection brief reference may be made to omithuric acid, which may be obtained from the urine of birds when these are fed with benzoic acid, and which probably also represents a normal con- stituent of such urine. Its formation is analogous to that of hip- puric acid in mammals, but the benzoic acid here combines with a diamido-acid, ornithin, which is a, o-diamido-valerianic acid, as has been shown before. The relation of ornithin to arginin has already been considered. THE AROMATIC OXY-ACIDS. The aromatic oxy-acids which may be found in the urine under normal conditions are para-oxy phenyl-propionic acid or hydro- paracumaric acid, and para-oxyphenyl-acetic acid, which results from the former on oxidation. Both are derivatives of tyrosin, and are formed during the process of intestinal putrefaction, as has been shown. Para-oxy phenyl-lactic acid may further be encountered when tyrosin has been administered to animals in large amounts. Under pathological conditions, as in acute yellow atrophy, where leucm and tyrosin may appear in the urine as such, para-oxyphenyl-glycolic acid or oxy-amygdalic acid has also been found. Of special interest finally is the fact that in some instances dioxyphenyl-acetic acid (homogentisinic acid) and trioxyphenyl-pro- pionic acid or uroleucinic acid also may occur in the urine. The chemical relationship which exists between these various substances and tyrosin, from which they are all probably derived, is apparent from their formula? : CfiH 6-LJ-4 C6H4< C H /OH(l) CH2.CH(NH).COOH(4) para-oxyphenyl-amido-propionic acid (tyrosin). X>H(1) vCH2.CH2.COOH para-oxyphenyl-propionic acid. ,(OH)3 vCH2.CH2.COOH trioxyphenyl-propionic acid. C H 'XCH2.CH(OH).COOH para-oxyphenyl-lactic acid. .OH C6H4< Nm^COOH para-oxyphenyl-acetic acid. THE AROMATIC OXY- ACIDS. 261 /(OH), xCH,.COOH dioxyphenyl-acetic acid. .OH c«hZ CH(OHj.COOH para-o.xyphenyl-glycolic acid. As has been pointed out, the two aromatic oxy-acids which are usually found in the urine originate in the intestinal tract during the process of albuminous putrefaction. There is reason to believe, moreover, that homogentisinic acid and uroleucinic acid are likewise of intestinal origin, and are referable to the activity of a special micro-organism, which is usually not found. But as para-oxy- phenyl-glyeolic acid manifestly originates in the tissues of the body, we must admit that the other members of the group may also be formed beyond the intestinal tract. To what extent this occurs, however, we do not know. _ A certain fraction of these bodies appears in the urine in com- bination with sulphuric acid, but the greater portion is eliminated as such, viz., as potassium and sodium salts. The amount of the common oxy-acids, however, is always small, and rarely exceeds 0.03 gramme in the twenty-four hours. To demonstrate the presence of the common oxy-acids in the urine, we proceed as follows : 500 c.c. of urine are strongly acidified with hydrochloric acid and distilled until the phenols, viz., phenol and paracresol, have passed over. This can be recognized by test- ing the distillate from time to time with Millon's reagent, On cooling, the remaining fluid is thoroughly extracted with ether, which takes up the oxy-acids as well as pyrocatechin and hydro- quinon. To separate the acids from the latter, the ethereal extract is shaken with a dilute solution of sodium carbonate. The acids are thus transformed into the corresponding salts and are found in the alkaline solution. After separation from the ether this is acidified with dilute sulphuric acid and extracted with ether. The ethereal solution contain-; the free oxy-acids. Their presence can be demon- strated by evaporating to dryness, when the residue is dissolved in a little water and tested with Millon's reagent. To isolate the individual acid-, much larger Quantities of urine are necessary. We may then proceed as described in the section on ih- Feces. Homogentisinic Acid.— The presence of homogentisinic acid may he suspected if a urine, on being rendered alkaline, turns a dark reddish-brown on standing, and ultimately becomes black. At the same time a positive reaction with Fehlingpa solution is obtained, while polar i metric examination shows that the urine is optically inactive. \vland< ■!■'- solution is not reduced. Upon the addition of a small amount of a dilute solution of ferric chloride a greenish- blue color develop-, which is only of momentary occurrence, how- ever. 262 THE URINE. Boedeker was the first to describe a urine of this kind, and termed the substance giving rise to the above reaction alkapton. Subse- quently, however, he expressed the opinion that his alkapton may have been pyrocatechin. Other investigators have isolated sub- stances from such urines, which have been variously termed pyro- catechuic acid, urrhodinic acid, glycosuric acid, uroxanthinic acid, and uroleucinic acid, but there is reason to suppose that, with the possible exception of the last mentioned, all these substances are identical with homogentisinic acid. This was first isolated from an " alkapton " urine by Baumann and Wolkow, and has since been found in every case that has been examined in this direction. Alkaptonuria, though it may occur in disease, is generally regarded as the expression of an unusual form of intestinal putrefaction which in no way affects the health of the individual. Some ob- servers, on the other hand, look upon it as a metabolic abnormality, and it must be confessed that micro-organisms have thus far not been isolated from the intestinal contents of such cases which are capable of effecting the transformation of tyrosin to homogentisinic acid in vitro. That tyrosin, indeed, is its ultimate source cannot be doubted, and it has been shown, as a matter of fact, that following the administration of this substance homogentisinic acid appears in the urine in greatly increased amount. Baumann thus noted that while the average elimination in one of his cases amounted to 4.6 grammes, 14 grammes were once extracted in the twenty-four hours after tyrosin had been ingested. Isolation. — Homogentisinic acid may be conveniently isolated from the urine according to the method suggested by Garrod. The collected urine of twenty-four hours is heated nearly to the boiling- point, and then treated with 5 or 6 grammes of neutral lead acetate in substance for every 100 c.c. of the urine. As soon as the salt is dissolved, the resulting precipitate is filtered off and the filtrate set aside in the cold for twenty-four hours. The crystals of lead homo- gentisinate are then collected on a filter and dissolved in hot water. This solution gives the various reactions described above. To isolate the free acid, the lead compound is decomposed with hydrogen sul- phide and the filtrate carefully evaporated on a water-bath until the. fluid begins to darken, when it is further concentrated in a vacuum to the point of crystallization. The resulting crystals are soluble in water, alcohol, and ether, but are insoluble in chloroform, benzol, and toluol. They melt at 146.5°-147° C. Uroleucinic acid has been found only once in an alkapton urine. In its general reactions it resembles homogentisinic acid, but does not give the iron reaction described above. Unlike the latter, it reduces Nylander's solution when present to the extent of 0.5 per cent, or more. Inosit. — The origin and chemical constitution of inosit will be considered elsewhere. According to Hoppe-Seyler, it may occur in the urine under normal conditions, but more commonly it is found THE AROMATIC OXY- ACIDS. 263 in diseases which are associated with a high grade of polyuria, such as diabetes insipidus, diabetes mellitus, in chronic interstitial neph- ritis, etc. To demonstrate its presence in the urine large amounts are con- centrated to a syrup, which is then extracted with four times its volume of alcohol by boiling. On cooling, this extract is treated with an excess of ether, when the inosit gradually crystallizes out and may be recognized by its special tests (see page 360). The remaining aromatic substances which have been found in the urine are the kynurenic acid and urocaninic acid of the urine of dogs, and the so-called lithuric acid, which has been obtained from the urine of the ox. The two latter have thus far been found in only one instance and need not( be considered at this place. Their formulae are given as : CjjH^X/)^ urocaninic acid. C15H,9X09, lithuric acid. The so-called damalic acid and damaluric acid, which are obtained from the urine of the horse and the cow, probably represent a mixt- ure of benzoic acid and volatile fatty acids. Kynurenic Acid. — Kynurenic acid is said to be a constant con- stituent of the urine of dogs, and is supposedly formed during the process of intestinal putrefaction. Its mother-snbstance is mani- festly an albuminous derivative, as the amount which appears in the urine is largely dependent upon the quantity of albuminous food ingested. Of its chemical nature and immediate antecedents, how- ever, nothing is known. Kynurenic acid is now regarded as oxyquinolin-carbonic acid, and is decomposed by heat, with the formation of carbon dioxide and a basic substance, kynurin. On reduction the latter is transformed into quinolin. These changes are represented by the equations : (1) CH.C9H5N.COOH = C9IIftNO + C02. Kynurenic acid. Kynurin. (2) C9II7NO I _>II =C9HTN +H20. Kynurin. Quinolin. Isolation. — To isolate kynurenic acid from the urine, 500 c.c. are treated with hydrochloric acid in the proportion of 4 c.c. for every loo p.c. of lb'' urine. On standing for forty-eight hours the sub- stance in question crystallizes out together with uric acid. To sepa- rate it from tin- latter, dilute ammonia i~ added to the crystalline precipitate. The uric acid remains, and from its ammoniacal solu- tion the kynurenic acid is then precipitated by acidifying with hydro- chloric acid. The crystals are soluble in alcohol mid melt at 253° C. On porating a bit of the materia] with hydrochloric acid and potas- sium chlorate on a porcelain plate a reddish residue is obtained, 264 THE URINE. which principally consists of tetrachloro-oxykynurin. When this is moistened with ammonia a brownish-green color develops, and on standing this soon passes into a fine emerald green. The Fatty Acids. The Volatile Fatty Acids. — The volatile fatty acids which may be isolated from any urine, and which are especially abundant in that of herbivorous animals, are normally derived from the intestinal tract, where they are principally formed during the process of carbo- hydrate fermentation ; a certain fraction, however, is also referable to albuminous putrefaction. These acids are acetic acid, formic acid, propionic acid, and butyric acid. The non-volatile acids, capric acid and caprylic acid, have further been found in the urine of herbivorous animals. In man about 0.05 gramme of volatile fatty acids is excreted in twenty-four hours. Especially large amounts, such as 3 grammes pro die, have been found in the urine of the goat. In various diseases larger amounts have also been encountered in man, but it is still an open question whether they are then derived from the intestinal tract exclusively. From decomposing urine a larger quantity can be obtained than from fresh urine. This is no doubt owing to the fact that every urine contains a small amount of carbohydrates, which yield fatty acids on bacterial decomposition. Old diabetic urine is hence especially rich in such acids. The higher fatty acids are normally not observed in the urine, but traces may appear under certain pathological conditions. Isolation and Quantitative Estimation. — To isolate the volatile fatty acids the collected urine of twenty-four hours is acidified with phosphoric acid in the proportion of 10 : 100, and distilled in a cur- rent of steam so long as the distillate shows an acid reaction. This is then neutralized, evaporated to dryness, and the residue extracted with alcohol. Traces of sodium chloride, which are formed on the addition of the alkali, owing to the presence of a little hydrochloric acid that has passed over, remain behind. The alcoholic solution is then evaporated to dryness, the residue is dissolved in a little water, acidified with sulphuric acid, and set aside in the cold, when traces of benzoic acid are precipitated. The filtrate is neutralized with a solution of sodium carbonate and extracted with ether, which removes the phenols. The solution is now acidified with sulphuric acid and is again distilled in a current of steam, when the volatile fatty acids pass over. Their presence can be established according to the common methods of analysis. To estimate the amount, the final distillate is neutralized with barium hydrate, evaporated to dry- ness, and the residue weighed. This weight less that of the barium, which is in combination, and which can be determined as barium sulphate, after incineration and extraction with dilute hydrochloric acid, indicates the amount of the fatty acids in general. THE AROMATIC OXY- ACIDS. 265 ^-oxybutyric Acid. — This acid is never found in the urine under normal conditions, and is principally met with in the severer forms of diabetes, when it is associated with the presence of diacetic acid and acetone. It is supposedly derived from the albumins of the tissues, and may accordingly also appear in the urine in various cachectic conditions, in the continued fevers, and during starvation. As a general rule it is found in combination with the common alkalies of the blood, but when it is produced in especially large amounts a corresponding quantity of ammonia is furnished by the body to effect its neutralization. It may happen, however, that the acid formation exceeds that of ammonia, and in such cases the free acid occurs in the urine, and can also be demonstrated in the blood as such. Symptoms of acid intoxication then exist, and it is noteworthy that in such cases the amount of carbonic acid in the blood has been found markedly diminished, showing that the alkaline salts are not present in sufficient amount to remove the carbonic acid from the tissues. Of the immediate antecedents of the acid nothing is known, and if formed at all under normal conditions it is manifestly oxidized at once beyond the stage of diacetic acid, as this substance also is only found under pathological conditions. Traces of acetone, however, which is likewise derived from oxybutyric acid, are found in every urine. The amount of oxybutyric acid which may occur in the urine is extremely variable. In the milder cases of diabetes it is usually absent ; in the severer forms, however, large quantities may be found, and Kiilz reports that in three cases a daily elimination of 67, 100, and 226 grammes, respectively, was observed. The chemical relation which exists between /?-oxybutyric acid, diacetic acid, and acetone is seen from the equations : (1) CH3.CH(OII).CH2.COOH + O = (CII3 CO) ClI,,COOH + H20. 0-oxy butyric acid. Diacelic acid. (2) (CIL.Co MU'OOII =CO(CIT3)2 +co2. Diacetic acid. Acetone. We can thus readily understand that in certain conditions acetone may be found in the urine alone, while in others diacetic acid, and in -till others ^-oxybutyric acid may be present as well. On boiling ,9-ox ybntyrie, acid in aqueous solution with dilute mineral acids, a-crotonic acid results, and it is thus apparent that this acid is also found in the distillate, when urine containing the lir-t i- distilled with sulphuric acid. Otherwise, however, it does CCUr. The reaction which takes place may be represented by the equation : Ml MI nil ( ll.COOir MI,.MI CHOOOH | 11./) ixybutyric acid. o-crotontic acid. Test. — A- the presence of oxybutyric arid presupposes that of diacetic acid, and as the presence of the latter can much more readily be demonstrated than that of oxvbutvric acid, a tesl in this 266 THE URINE. direction should always precede a more detailed examination (see below). If a positive reaction is thus obtained, any sugar that may be present is removed by fermentation. The liquid is cleared by adding neutral acetate of lead, when the filtrate is examined with the polarimeter. Should lsevorotation now be observed, the presence of oxybutyric acid is rendered very probable. To demonstrate this beyond a doubt, the liquid is evaporated to a syrup, treated with an equal volume of concentrated sulphuric acid and distilled, without cooling. In this manner the oxybutyric acid is decomposed with the formation of a-crotonic acid, which is accordingly found in the distillate. If this is present in larger amounts, it crystallizes out in the distillate, when this is strongly cooled, and may be identified by its melting-point, 72° C. Should smaller amounts, however, be present, crystallization does not occur. In this case the distillate is extracted with ether by shaking. Traces of benzoic acid and the phenols are thus likewise extracted, but if then the residue of the ethereal solution is washed with water other impurities are removed, and the crotonic acid remains. Diacetic Acid. — From what has been said above, it is clear that every urine which contains /3-oxybutyric acid must also contain diacetic acid, and in extreme cases both may be found in the blood as such. On the other hand, it will also be understood that diacetic acid may occur in the urine in the absence of ^-oxybutyric acid, and this is indeed more common. Tests. — As diacetic acid is rapidly decomposed on standing, it is necessary that the urine should be as fresh as possible, when it is to be examined in this direction. To this end, several direct tests are available. Arnold's Test. — This test is the most reliable, as it does not respond to acetone and /9-oxybutyric acid, nor to the common anti- pyretics, salicylic acid, or bile-pigment. Highly colored urines, however, should first be filtered through animal charcoal. Two solutions are employed, viz., a 1 per cent, solution of sodium nitrite, and a solution of para-amido-acetophenon. The latter is prepared as follows : 1 gramme of the acetophenon is dissolved in from 80 to 100 c.c. of distilled water, and treated drop by drop with hydrochloric acid until the solution, which at first is yellow, becomes entirely colorless ; an excess, however, should be carefully avoided. Before using, the two solutions are mixed in the propor- tion of one part of the nitrite to two of the acetophenon solution. A, few cubic centimeters of the urine are then treated with an equal volume of the reagent, and a few drops of ammonia. All urines thus give a more or less well-marked brownish-red color on shaking, and in the presence of much diacetic acid an amorphous precipitate of the same color is formed. If now a small amount of the colored solution is treated with an excess of concentrated hydrochloric acid (10-12 c.c. for every 1 c.c), a beautiful purplish-violet color de- velops if diacetic acid is present. THE AROMATIC OXY- ACIDS. 267 Gerhakdt's Test. — In its original form this test also reacts with the common antipyretics, and it is hence necessary to isolate the diacetic acid to a certain degree. To this end, a few cubic centim- eters of the urine are strongly acidified with sulphuric acid and extracted with ether, which takes np the acid. The extract is then shaken with a few cubic centimeters of a dilute solution of the chloride of iron, when in the presence of diacetic acid the aqueous layer assumes a violet or Bordeaux-red color. This, however, is not permanent, and soon fades on boiling the solution. Acetone. — Traces of acetone, varying between 0.008 and 0.027 gramme in the twenty-four hours are normally found in the urine. Larger quantities are met with if the carbohydrates are withdrawn from the diet, and in such cases from 0.2 to 0.7 gramme may be excreted after the sixth day. If then carbohydrates are again ingested, the elimination of the acetone rapidly reaches its origi- nal figure ; the ingestion of fats, on the other hand, is without effect in this respect. Acetonuria, however, is essentially a patho- logical phenomonen, and is observed in its most pronounced form in severe cases of diabetes, in which, as I have stated, it is fre- quently met with in association with /3-oxybutyric acid and diacetic acid. Like diacetic acid, however, it may occur in the absence of oxybutyria acid, and in the milder forms of diabetes, as also under normal conditions, it may be present alone. But while its import is generally the same as that of its immediate antecedents, it appears that it may originate also in the gastro-intestinal tract as the result of some abnormal form of albuminous putrefaction, and it is possi- ble, indeed, that the so-called asthma-acetonicnm may be of this origin. That small amounts are formed also during the hydro- lytic decomposition of the albumins with boiling mineral acids or the caustic alkalies, has been mentioned, v. Jaksch, further, has shown that acetone may be formed as a by-product during the process of lactic acid fermentation, and it has been suggested that the small amounts which are normally met with in the urine may originate in this manner. At present, however, we are not in a position to speak authoritatively of the origin of these normal trace-, and, pathologically at least, we can thus far acknowledge but our- source of the acetone, viz., the albumins of the body tissues, ami secondarily perhaps the circulating albumins. Tests. — Should diacetic acid be demonstrated in the urine, the simultaneous presence of acetone may be directly inferred. If this i- not the case, it is best to distill from 250 <<• 500 c.c. of the urine, after the addition of a small amount of phosphoric acid, and to apply the following tests to the firsi 15 or 30 c.c. (,f the distillate thai has passed over. Legal's Test. — A few cubic centimeters of the distillate are treated with several drops of a freshly prepared, concentrated solu- tion of -odium nitroprusside, and a small amount of a dilute solu- 268 THE URINE. tion of sodium hydrate. In the presence of acetone a red color develops, which rapidly fades however, but is replaced by a beau- tiful carmin or purple red if the solution is treated with acetic acid in excess ; on standing, this turns to a bluish violet. Lieben's Test. — On adding a few drops of a dilute solution of iodopotassic iodide to a small amount of the distillate that has been rendered alkaline with sodium hydrate solution, a precipitate of iodoform develops in the presence of acetone, which may readily be recognized by its odor on warming the mixture. This test, how- ever, is not conclusive, as other substances, such as alcohol, give the same reaction. Gunning's Test, as modified by Salkowski. — A small amount of the distillate is treated drop by drop with freshly pre- cipitated and well-washed mercuric oxide in alcoholic suspension until a portion of the oxide remains undissolved. The liquid is then filtered, and the filtrate superposed with a solution of ammo- nium sulphide, when in the presence of acetone the zone of contact assumes a grayish-black color, owing to the formation of mercuric sulphide. Quantitative Estimation. — The quantitative estimation of acetone is best conducted according to the method of Messinger, as modified by Huppert. It is based upon the principle underlying Lieben's test, viz., the formation of iodoform when a dilute solution of iodo- potassic iodide is added to an alkaline solution of acetone. By determining the amount of iodine which is consumed in this reac- tion the corresponding amount of acetone can then be calculated. One hundred c.c. of urine, or less if much acetone is present, as determined by Legal's test applied directly to the urine, are treated with 2 c.c. of a 50 per cent, solution of acetic acid and distilled until all the acetone has passed over. The distillate is received in a bulb-tube containing water. The solution which thus results is treated with 1 c.c. of a 12 per cent, solution of sulphuric acid and redistilled. The second distillate is free from phenols. To it a carefully measured quantity of a one-tenth normal solution of iodine is added (10 c.c. for every 100 c.c. of urine), together with a 50 per cent, solution of sodium hydrate, until the iodoform separates out. After shaking, the mixture is set aside for a few minutes, and then acidified with concentrated hydrochloric acid. If iodine is present in excess, a brown color thus develops. This excess is then titrated with a decinormal solution of sodium thiosulphate, using starch solution as a final indicator. The number of cubic centim- eters emyloyed in this titration is deducted from the amount of the iodine solution added. The difference multiplied by 0.967 then indicates' the amount of acetone in the 100 c.c. of urine, in milli- grammes. Ehrlich's Reaction. — When normal urine is treated with an equal volume of a saturated solution of sulphanilic acid in 5 per cent. THE AROMATIC OXY-ACIDS. 269 hydrochloric acid to which a 0.5 per cent, solution of sodium nitrite has been added in the proportion of 1 : 40, and the mixture is then rendered strongly alkaline with ammonia, a more or less well- marked orange color develops. Under pathological conditions, on the other hand, and notably in typhoid fever, a red color results which varies in intensity from a light carmin to a deep garnet-red. This reaction is known as Ehrlich's diazo-reaction, as it is dependent upon the presence of a diazo-compound of the nature of diazo- benzene-sulphonic acid in the reagent. This results through an interaction between the sodium nitrite and the sulphanilic acid, as represented in the equations : (1) NaN02 +HC1 =HN02 + NaCl. XNH2 . N (2) C6H4< +HNOa = C6H4( >N + 2H20 / Sulphanilic Diazo-benzene- acid. sulphonic acid. I briefly refer to the reaction at this place, as v. Jaksch has expressed the opinion that in " most " cases it is referable to the presence of acetone, and in reality represents only an uncertain test for this substance. This statement I wish to contradict most emphatically, as I have been able to show beyond a doubt that Ehrlich's reaction quite commonly occurs at a time when abnormally large amounts of acetone cannot be demonstrated in the urine, and is absent in many diseases in which a marked degree of acetonuria exists. Normal urines, moreover, in which traces of acetone are (••instantly found do not give the red color. At the present time we are in total ignorance of the nature of the substance that is so com- monly present in typhoid urines, and which reacts with diazo- benzene- sulphonic acid in the manner indicated. Paralactic Acid. — As I have shown, there is reason to believe that the greater portion of the nitrogen which is set free in the katabolism of the various tissues appears in the form of the am- monium salt of paralactic acid and is transformed into urea in the liver. Normally, indeed, paralactic acid is not found in the urine. It occurs, however, whenever the further transformation of the ammonium salt is impeded, and is hence met with in various diseases of the liver which arc associated with an extensive destruc- tion of the hepatic parenchyma, as also in conditions in which the oxidation-processes of the body arc impaired in general. It is thus notably met with in acute yellow atrophy, in poisoning with phos- phorus and carbon monoxide, in long-continued anaemic conditions, see also page 223). Smaller amounts have; been found in soldier after forced marches, and in epileptic patients after severe convulsive seizures. Isolation. — To isolate the substance from the urine the following method may he employed as suggested by Araki : The collected urine of twenty-four hours is evaporated to 210 THE UH1SE. about 50 or 60 c.c, treated with ten times as much of 95 per cent, alcohol, and set aside for twelve hours. It is then filtered and freed from the alcohol by distillation. The residual fluid is acidified with phosphoric acid, and repeatedly extracted with five times its volume of ether. The ethereal extract is evaporated and the remaining yellow syrup dissolved in a little water. Any hippuric acid which may be present thus separates out and is filtered off. The filtrate is now treated with pure lead carbonate in substance, heated on a water-bath for thirty minutes, and filtered on cooling. From the filtrate the lead is removed Joy means of hydrogen sulphide, and the excess of the latter by gently warming on a water-bath. The fluid is then concentrated to a thick syrup and extracted with ether. The ethereal solution is evaporated and the residue boiled for some time with water and an excess of zinc carbonate. The mixture is filtered while hot, concentrated to a small volume, and then set aside in the cold after adding a little alcohol. The zinc salts of both paralactic acid and the common optically inactive lactic acid which may also be present in traces, then crystallize out. They can be separated from each other by treating with absolute alcohol, in which the latter is insoluble. It must be noted that the solu- bility of the paralactate is also slight (1 : 1100), so that it is necessary to add a large amount of alcohol. In order to prevent confusion with the aromatic oxy-acids of the urine, the lactate crystals should now be further identified, which is most conveniently done by estimating the water of crystallization. The paralactate contains two molecules of this, which escapes at 105° C, and at this temperature the weight of the crystals should therefore diminish 12.9 per cent. The salt, moreover, like its acid, is laevorotatory, while the common lactate is optically inactive. Leucin and Tyrosin. — Leucin and tyrosin, according to some observers, are normally present in the urine in traces. By others this is denied, and I must admit that I have never found either of the substances under normal conditions. In certain diseases of the liver, however, in which extensive destruction of the hepatic cells is going on, both may be found. But it is noteworthy that while in acute yellow atrophy this is a common occurrence after the first week of the disease, in acute phosphorus poisoning they are usually not found. Thus far we have no adequate explana- tion to offer for this difference, and we are in ignorance, more- over, of the origin of the bodies in question in the former disease. "We have seen that as a general rule at least the greater portion of the tissue nitrogen which is set free during the process of metabolism is carried to the liver in the form of ammonium paralactate, and there is no evidence to show that this may be transformed into either leucin or tyrosin. We see, in fact, that in extensive hepatic disease ammonium lactate appears in the urine. Whether or not in acute yellow atrophy leucin and tyrosin are also set free in the tissues in general, we do not know. In the liver, it is THE NEUTRAL SULPHUR OF THE URINE. 271 true, both are then met with in large amount, but we may readily suppose that the substances may have originated here directly, and possibly as a result of some fermentative action. This, indeed, appears the most likely explanation. Isolation. — If leiicin and tyrosin are present in the urine in small amounts, they are held in solution. In the presence of larger amounts the tyrosin may separate out, and can then be isolated from the sediment and identified as described. Leucin, however, is rarely found in this manner, and remains in solution even though very large quantities are eliminated. To demonstrate both when they are held in solution, it is some- times only necessary to concentrate a small amount of urine on a water-bath and to examine the residual syrup with the microscope. Otherwise it is advisable to precipitate the collected urine of twenty-four hours, after removing any albumin that may be present, with basic lead acetate. The filtrate is then freed from lead with hydrogen sulphide, evaporated to a thick syrup, and set aside for crystallization. Tyrosin and leucin can then be demonstrated by a microscopical examination and identified in the usual manner (see page 188). THE NEUTRAL SULPHUR BODIES OF THE URINE. In the section on the mineral constituents of the urine I pointed out that the greater portion of the sulphur which is set free during the metabolism of the nitrogenous constituents of the body is elimi- nated in the urine in a completely oxidized form. A much smaller fraction, however, escapes oxidation, and appears in the urine as so-called neutral sulphur. Normally this constitutes about 12-15 per cent, of the total amount. Of the individual components which go to make up this neutral sulphur comparatively little is known, ami it appears, moreover, that their character may be different in different animals. In the urine of eats, and less constantly in that of dogs, traces of thiosulphatea are thus found, while in man this is normally absent, ami in disease even thiosulphuric acid has been found in only one instance— in typhoid fever. Ethyl sulphide, or a body which gives rise to its formation when the urine is treated with lime-water, is thus similarly not found in the urine of man, while it is apparently a constant constituent of that of dogs. Ol the normal constituents of the neutral sulphur which is found in human urine, only two are actually known. These are the sulpho- cyanides, which are found in small quantities in tin- saliva and the trie juice, and cystein, or a body which is closely related to it. Whether or not taurocarbaminie acid is also constantly present has not as* yet been determined. I have shown, however, that to a certain extent at least tanrin is eliminated in this form when given by the month. In cases of obstructive jaundice, moreover, or after ligation of the common dud in dogs, the neutral sulphur may increase 272 THE URINE. to 40 per cent, of the total amount, and it is known that in such cases taurocarbaminic acid is constantly present. Its formation may be represented by the equation : /C2H4.NH2 yNH2 /NH2 S02< +CO< =CO< xOH \NH, \NH.C2H4.S02.O.NH4. Taurin. Urea. Ammonium taurocarbamate. In all probability this synthesis is effected in the kidneys. In rabbits, on the other hand, taurin is largely oxidized to sulphuric acid, while a small portion appears as thiosulphuric acid. Of the origin of taurin, as I have stated, nothing definite is known. Cystein, on the other hand, is apparently derived from the loosely combined sulphur of the albumins, and probably represents an intermediary product of oxidation, which is normally further oxidized to sulphuric acid. Traces, however, apparently escape this destruction and normally appear in the urine. As a matter of fact, cystein is not readily oxidized within the body, and in dogs one- third of the ingested amount reappears as such. Following the administration of chlorine, bromine, or iodine substitution-products of the benzols, moreover, a diminished elimination of sulphuric acid occurs, and in place of this we meet with a conjugate glucuronate, which contains the greater portion of the lacking sulphur. The product which thus results can readily be decomposed, with the formation of glucuronic acid and chlorophenyl-mercapturic acid, which latter manifestly contains the cystein group, as is seen from the formulae : CH3 CH3 NH2X | CH3.CO.NHv | C6H4C1.S^^ | COOH COOH On decomposition it yields acetic acid and chlorophenyl-cystein, as shown in the equation : CHS CH3 CH3.CO.NH | NH2v | C6H4C1.S/ | >C + H20 = C6H4C1.S/ | = CH3.COOH + COOH COOH The amount of cystein which is normally present in the urine probably does not exceed 0.015 gramme in the twenty-four hours. Larger quantities are found in phosphorus poisoning, which further suggests the occurrence of the substance as an intermediary product in the normal metabolism of the organic sulphur, as we know that in such cases the oxidation-processes of the body, as a whole, are much impaired. THE NEUTRAL SULPHUR OF THE URISE. 273 On exposure to the air cystei'n is transformed into cystin, as is shown in the equation : Cystin. This transformation is of special interest, owing to the fact that under certain conditions cystin also may appear in the urine, while normally it is absent. It is noteworthy, moreover, that cystinuria can scarcely be regarded as a pathological phenomenon, although it mav occur in association with a definite disease. More commonly it is observed in otherwise normal individuals, and, like alkaptonuria, it may persist for a lifetime, and may occur in families. It may hence be regarded as a metabolic anomaly in which the oxidation of the loosely combined sulphur is diminished, or perhaps even suspended. Very curiously, it has further been observed that cystin uria is quite constantly accompanied by the appearance of cadaverin in the urine, while in two instances at least putrescin also was found. Some observers have hence suggested that a genetic relationship may exist between the two conditions, and that the formation of the diamins may be the primary factor. But while most observers assume that the diaminuria is referable to the presence of certain specific organisms in the intestinal canal, which are usually absent, I am personally inclined to regard the formation of the diamins also as a metabolic anomaly, and suppose that both conditions are the outcome of a third factor, as yet un- known, but which no doubt operates within the tissues directly. The possible formation of diamins in the absence of micro-organ- isms can indeed no longer be doubted (see page 73). The amount of cystin which may be met with in the urine is extremely variable. On some days traces only are found, while on others the elimination may exceed 1 gramme in the twenty-four hours. That the total amount of the neutral sulphur is then also proportionately increased is of course, self-evident. Outside of the urine cystin has been encountered in onlv a few instances. Cloetta thus ''hums to have obtained the substance from the kidneys of the ox. Seherer found it once in the liver of a typhoid-fever patient, and Drechsel isolated the body from th'- liver of a horse and a porpoise. Kiilz further claims to have found cystin among the decomposition-products of fibrin on one ision where the digestion was effected with pancreas. Together with Dr. Amberg, however, 1 have been unable to confirm Kiilz's temenl in a series of experiments undertaken in my laboratory. Of late, Morner has shown that cystin results on decomposing the keratine of horn shavings with mineral acid-. [Jnlese the cystin i- found directly in a urinary sediment, its pres- 18 274 THE UEINE. ence will scarcely be suspected. If, however, a urine develops a marked odor of hydrogen sulphide on standing, it is well to add an excess of acetic acid and to examine the sediment somewhat later. The characteristic hexagonal platelets of cystin may then at times be found, and cau be recognized from their solubility in ammonia and hydrochloric acid, while they are insoluble in acetic acid, water, alcohol, and ether. But sometimes this procedure does not lead to the desired end, even though a decided increase in the amount of neutral sulphur is observed, and hydrogen sulphide is formed in abundance on standing. Whether or not it may then be justifiable to refer this increase of the neutral sulphur to the presence of cystin, is questionable. Clinically cystin is of interest in so far as its continued appear- ance in the urine may be regarded as a probable precursor of the formation of cystin gravel or calculi ; and we find, as a matter of fact, that this occurs in a very considerable proportion of all cases. A quantitative estimation of the cystin, isolated as such, is not as yet possible. When it is found in a sediment the crystals may be col- lected and weighed. But as a variable amount remains in solution, even after the addition of much acetic acid to the urine, it is further necessary to estimate the total amount of neutral sulphur that re- mains, when an excess beyond the average figures may be referred to cystin, and the result added to that obtained directly. Isolation. — As the synthesis of cystin has not as yet been effected, we are generally obliged to rely upon cystin concretions for purposes of study. If such material is inaccessible, we may prepare the sub- stance from horn shavings by decomposing the contained keratins with mineral acids. For the isolation of the body from the result- ing decomposition-products, however, I must refer the reader to Morner's article. Properties. — Several varieties of cystin apparently exist, of which one islsevorotatory, another dextrorotatory, while a third is optically inactive. The common cystin which is found in the urine belongs to the lsevorotatory type. It crystallizes in colorless, hexagonal platelets, which are quite characteristic. They are soluble in solu- tions of the alkaline hydrates, in ammonia, and the mineral acids. In water, alcohol, ether, and acetic acid the substance is insoluble, as also in solutions of ammonium carbonate, and it is for this reason that cystin is apt to crystallize out from decomposing urines if it was previously present in solution only. Structurally, cystin is the disulphide of cystei'n, which in turn is «-amido-thioiactic acid. On reduction it is transformed into cyste'in, as shown in the equation : CH3 CH3 CH3 | /NH2 NH2. | NH2X I c/ \C +2H=2 >C | \S S/ | SH / I COOH COOH COOH Cystin. Cyste'in. THE CARBOHYDRATES. 275 On heating the substance on platinum foil it does not melt, but ignites and burns with a bluish-green flame ; at the same time a peculiar, penetrating odor develops. It does not give the murexid reaction. When boiled with caustic alkali it is decomposed and the sulphur liberated as a sulphide. With benzoyl chloride, in the presence of an excess of caustic alkali, it forms benzoyl-cystin, and is thus precipitated as a sodium salt in the form of fine lustrous platelets, which are readily soluble in water, but insoluble in solu- tions of the caustic alkalies. Upon the addition of an acid to such a solution, benzoyl-cystin separates out as such. It is soluble in alcohol and alcohol-ether, slightly so in pure ether, and almost insolu- ble in water. Its needle-like crystals melt at 156°-158° C. The formation of benzoyl-cystin may be expressed by the equation : CH3 CH3 2C,-H5.C0C1 + C( >C = Benzoyl I \S S' I •« chloride. coofj C00H Cystin. CH3 CH3 I /NH(C6H5.CO) (C6H5.CO)NHN | C< >C + 2HC1 I S B/ I CUOH COOH Benzoyl-cystin. On boiling with concentrated hydrochloric acid, benzoyl-cystin is decomposed with the formation of benzoic acid and cystin. Quantitative Estimation of the Neutral Sulphur — In one portion of the urine the oxidized sulphur, viz., the mineral and the conjugate sulphates, is estimated as has been described. In a second portion the total sulphur is then ascertained as follows: 100 c.c. of urine are treated with 12 grammes of a mixture of sodium and potassium carbonate (11 : 14), and evaporated to dryness in a platinum dish. The residue is thoroughly fused, and on cooling extracted with hot water. The carbonaceous residue is filtered off, washed with hot water, and filtrate and washings treated with a few crystals of potassium permanganate. After heating for fifteen minutes (a little more of the permanganate must be added if the solution becomes decolorized), and concentrated hydrochloric acid is added until the liquid is distinctly acid. It is then brought to the boiling-point, treated with _!<> c.c. of a hot saturated solution of barium chloride, when the barium sulphate which is thus formed Is estimated as usual (see page 220). The difference between the two results indicates the amount of the neutral sulphur. THE CARBOHYDRATES. The carbohydrates which may be found in the urine comprise glucose, laevulose, laiose, maltose, lactose, dextrin, animal gum, and certain pentoses. Of these, truce- of glucose, dextrin, animal gum, 276 THE URINE. and possibly also pentoses, may be found at all times. Their amount, however, is normally so small that their presence cannot be recognized by the common tests. Larger amounts of carbohydrates are found in health only during the puerperal state, and in the course of lactation, when lactose is commonly present. Otherwise the elimination of sugar in amounts which can be demonstrated by the ordinary tests must be regarded as abnormal. Glucose. — As I have indicated, glucose appears in the urine whenever its amount in the blood exceeds 3 pro mille. This, however, occurs only under abnormal conditions, and in the pres- ence of small amounts the kidneys are manifestly capable of preventing its passage into the urine. Under certain conditions, however, this power is apparently lost, and we find, as a matter of fact, that following the administration of phlorhizin glucosuria occurs, although the percentage of sugar is not increased in the blood. Whether or not such an insufficiency on the part of the kidneys may also occur spontaneously we do not know. As a gen- eral rule, however, glucosuria is associated with a hypergluchsemia. This may result if unduly large amounts of sugar reach the liver, so that the organ is incapable of transforming the entire quantity into glycogen, and I have pointed out that the functional capacity of the liver in this respect is of a much lower order than the ability of the intestinal epithelium to transform polysaccharides and disaccharides into glucose. The extent to which the liver can normally transform glucose into glycogen seems to vary with different individuals. Generally, glucosuria occurs when the amount of sugar exceeds 200 grammes. There are many individuals, however, in which this occurs following the admin- istration of only 150 grammes, and there are others in which the ingestion of 250 grammes does not cause glucose to appear in the urine. Glucosuria following the ingestion of 100 grammes of grape-sugar is now regarded as abnormal, and there is reason to believe that the hepatic insufficiency thus manifested may be of the type of a mild form of diabetes. The amount of sugar which then appears in the urine rarely exceeds 3 per cent. The glucosuria, moreover, is only temporary, and disappears as soon as the ingestion of sugar (viz., starches) is diminished. Between this form of glu- cosuria and the common form of diabetes, in which practically no sugar can be utilized by the body, but in which the elimination ceases as soon as carbohydrates are withdrawn from the diet, all gradations may occur. These forms are now generally regarded as referable to a hepatic insufficiency of whatever origin. Quite differ- ent from diabetes of this character, on the other hand, is the type in which the glucosuria continues although no sugars are ingested. In such cases a hepatic insufficiency need not necessarily exist, and there is evidence to show that in these forms the formation of glycogen may still occur. We must therefore assume that other organs are primarily involved, and there is every reason to THE CARBOHYDRATES. 277 suppose that the metabolism of muscle-tissue is here principally at fault, and that the tissue has lost the power of decomposing the sugar which reaches it from the liver. As a consequence, increased destruction of muscle-tissue occurs, as the inability on the part of these structures to decompose sugar amounts to the same as though no sugar were present at all. The body therefore liberates the carbohydrate groups of its albumins to supply the apparent deficit, and thus further increases the hypergluchsernia and the resulting glucosuria. We accordingly find that even though the carbohydrates have been withdrawn from the food sugar still appears in the urine. The increased destruction of the tissue albumins is in such cases sufficiently apparent from the pro- gressive loss of flesh which is so constantly observed. Of the causes which are operative in bringing about the muscular insufficiency as regards the decomposition of sugar, we know but little. That cer- tain nervous influences may here be at work is probable, and we know, as a matter of fact, that injury to a certain region in the floor of the fourth ventricle is invariably followed by the appearance of glucose in the urine. But, on the other hand, we may also imagine that the normal decomposition of the sugar is prevented owing to the absence of some such ferment as the glucolytic ferment of Lepine, and, as has been pointed out, this ferment is in all likelihood formed in the pancreas. In support of this view is the fact that after extirpation of the pancreas death invariably results with symptoms whicli are practically identical with those seen in the gravest types of diabetes. Ligation of the duct does not produce this effect ; and it is noteworthy, moreover, that the glucosuria disappears when pieces of the pancreas are transplanted under the skin or when fresh raw pancreas is given with the food. Within the past ten years it has been found that in a not inconsiderable number of cases of diabetes degenerative lesions can be demonstrated in the pancreas, and there can be no doubt at the present time that a certain percentage of cases are directly referable to such origin. In the milder forms, on the other hand, an insufficiency on the part of the muscle-tissue manifestly does not exist, as it is possible to prevent the occurrence of glucosuria, temporarily at least, if the demand for sugar is increased by abundant muscular exercise. That a hepatogenic diabetes finally may coexist with a myogenic form, cannot be- doubted. This i-. however, not the plaee to enter into a detailed account of the mechanism by which glucosuria is produced, and for further information, and lor a consideration of the various pathological con- dition- under which BUgar may be found in the urine, the reader is referred to other works. The amount of* sugar which may be present in the urine under pathological condition- i- exceedingly variable. On the one hand, traces only may he found, which may be normal ; while, on the other hand, the daily excretion may exceed 1000 grammes. In diabetes 278 , THE URINE. an elimination of from 3 to 6 per cent, in an amount of urine varying between 3000 and 6000 c.c. may be regarded as moderate. Tests for Sugar. — Simple tests by means of which glucose can be demonstrated directly in the urine as such are, unfortunately, not available. Other sugars, it is true, enter into consideration only under exceptional conditions, but if it is desired to prove that the substance which gives the common sugar reactions is actually glu- cose, a more detailed examination is necessary. Some of these tests, moreover, may be simulated by substances which are not carbo- hydrates, and deductions as to the presence or absence of sugar are hence only warrantable when these can be excluded. If albumins are present, they must first be removed. Nylander's Test. — This test is to be preferred to the more common one of Trommer, as the reagent does not react with uric acid, kreatinin, or homogentisinic acid. With many of the conju- gate glucuronates, however, a reduction is observed, and it is hence necessary to eliminate this source of error when a positive reac- tion is obtained, or to apply additional tests in which this possi- bility does not enter into consideration. The reagent is prepared as follows : 4 grammes of the tartrate of potassium and sodium, together with 2 grammes of subnitrate of bismuth and 10 grammes of sodium hydrate are placed in 90 c.c. of water. The solution is heated to the boiling-point, filtered on cooling, and is then ready for use. It is kept in a dark-colored bottle. A few cubic centimeters of the urine are treated with the reagent, in the proportion of 11 : 1, and boiled, when in the presence of sugar a reduction of the subnitrate of bismuth to bismuthous oxide, or even to the metallic form, occurs. As a consequence the mixture assumes a grayish, dark-brown, or black color, and on standing the precipitated oxide or metal settles to the bottom together with the earthy phosphates. Fehling's Test. — This is merely a modification of the older Trommer's test. The reagent consists of two solutions, viz., one containing 34.64 grammes of copper sulphate in 500 c.c. of water, while the other is prepared by dissolving 173 grammes of tartrate of potassium and sodium and 125 grammes of caustic soda in a like amount of water. Before using, equal parts of the two solutions are mixed and diluted with four times as much water. A few cubic centimeters of the resulting reagent are boiled and treated with a small amount of the urine, when in the presence of sugar yellow cuprous hydroxide or red cuprous oxide separates out, and on stand- ing settles to the bottom. After the addition of the urine the solu- tions should no longer be boiled, but may be held near the flame for a few moments. Unless this precaution is taken, fallacious results are often obtained, as uric acid, and kreatinin more especially, may cause a partial reduction of the copper solution on prolonged boiling. The test at best is open to many objections. Conjugate THE CARBOHYDRATES. 279 glucuronates and homogentisinic acid likewise give a positive reac- tion, and ammonia, if present beyond traces, may hold in solution any cuprous oxide that may be referable to very small amounts of sugar. Fermentation Test. — This test, when controlled by Nylander's test, is the most satisfactory one. To this end, a little compressed yeast is shaken with about 20 c.c. of urine, and the mixture is placed in a saccharimetric tube, such as that devised by Lohnstein or Einhorn. On standing at the ordinary temperature of the room, or, still better, at 37° C, fermentation occurs if glucose is present, and the liberated carbon dioxide collects at the top of the tube. In any case, however, two controls should be made, viz., one to determine that the yeast is active, and another with normal urine. If a small amount of sugar is present, it may happen that the resulting carbon dioxide is absorbed. If in such a case Nylander's test first gave a positive reaction, but no longer reacts after fermenta- tion is complete (in twelve to twenty-four hours), the presence of sugar may be inferred. If, on the other hand, no fermentation occurs, and Xylander's test still gives a positive result, we may con- clude that the reaction is due to the presence of a non-fermentable reducing substance. Phenylhydrazin Test. — As has been pointed out, all mono- saccharides and some of the disaccharides, such as maltose, isomal- fcose, and lactose, form compounds with phenylhydrazin which are known as osazons (see page 55). The resulting bodies are all very similar, but may be distinguished from each other by the melting- point of their crystals, and to some extent also by their microscopical appearance. With free glucuronic acid a similar compound may be obtained, according to Thierfelder, which may also be recognized by its melting-point, while the conjugate glucuronates are inactive in this respect. Pentoses likewise give rise to the formation of osa- zons, but the melting-point of the resulting crystals serves to distin- guish these also from the osazons of the hexoses. As a general rule, however, neither the pentoses nor glucuronic acid interferes with the reliability of the test. If doubt should arise, a special examination should be made to ascertain whether pentoses or glu- curonates arc present in amounts sufficient to react with the reagent. A further objection to the phenylhydrazin test has been urged on the basis that its delicacy is such that a positive reaction is obtained even under normal condition-. This, however, I must deny. The tes< is conveniently conducted as follows; 5 drops of pure phenylhydrazin are mixed in a test-tube with 10 drops of glacial acetic acid and 1 C.C. of a saturated solution of common salt. To this are added :; c.C. of urine, when the mixture is boiled for two minutes and is then s,.( aside to cool. in the presence of more than 0.5 per cent, of glucose, crystals of phenyl-glucosazon begin to Separate out after one or two minute-. Should smaller amounts be present, it is necessary to wait. The sedimenl is then exam- 280 THE URINE. ined microscopically. As we are generally only dealing with glu- cose in the urine, a further examination is usually not necessary, especially if the substance crystallizes out in large needles, which are. often collected in stars and sheaves. To identify these further, how- ever, their melting-point must be determined. As has been stated, this differs in the different osazons, with the exception of lsevulose and glucose, which have the same melting-point. Lsevulose, how- ever, is found only under exceptional conditions. Its presence may be established as shown below. The melting-points of the various osazons which may be encountered are as follows : Glucose 204°-205° C. Lsevulose 204°-205° C. Galactose - 193° C. Maltose 206° C. Isomaltose 150°-153° C. Lactose 200° C. Arabinose 159° C. Xvlose 159° C. Glucuronic acid 114°-115° C. The glucosazon is insoluble in water, but dissolves with ease in hot alcohol, from which it can be precipitated on cooling in crystal- line form, by diluting with water. The crystals are then collected on a filter, dried over sulphuric acid, and further examined if desired. Polarimetric Examination. — The polarimetric examination for the presence of sugar should always be controlled by one or more of the tests that have just been described. Dextrorotation, unless biliary acids are present, can be directly referred to the presence of sugar, and usually to glucose. Lsevorotation, however, may be referable to other reducing substances besides lsevulose, such as the conjugate glucuronates, /3-oxy butyric acid, and others. If such substances, moreover, are present in larger amounts, traces of dextrose may be overlookod. It is hence advisable to examine the urine both before and after treatment with yeast, and in doubt- ful cases to control the quantitative results, which are obtained by the polarimeter, by some other method. For a detailed description of this method I must refer the reader to special works. In every case the urine must be perfectly clear and free from albumin. If highly colored, it should be treated with lead acetate solution and then filtered, in which case allowance must be made for the degree of dilution if quantitative results are desired. Quantitative Estimation. — Differential Density Method. — The methods available for the quantitative estimation of glucose are, like the common tests, on the whole most unsatisfactory. The least objectionable perhaps is that based upon the determination of the difference in the specific gravity before and after fermentation. It has been found that a diminution by 0.001 corresponds to the previous presence of 0.230 per cent, of sugar. THE CARBOHYDRATES. 281 The specific gravity is first determined in the fresh urine, after adding 2 grammes of tartrate of potassium and sodium, and 2 grammes of diacid sodium phosphate to every 100 c.c. To about 200 c.c. which have thus been prepared, from 5 to 10 grammes of fresh yeast are added, and the mixture is set aside at a tem- perature of from 20° to 25° C. until fermentation is completed. If but little sugar is present, two or three hours will suffice; otherwise the mixture is allowed to stand for twelve hours. Evaporation is guarded against by closing the bottle with a perforated stopper through which a finely drawn out tube passes, which is open at the distal end. The specific gravity is then again determined at the same time as before, and the difference multiplied by 0.230. The result indicates the amount of sugar in per cent. The method yields good results unless very small amounts of sugar are present, viz., less than 0.5 per cent. In such an event, the reducing power of the urine is first ascertained according to Knapp's method. It is then fermented, when the remaining re- ducing substances are again determined. The difference may be referred to sugar. Fermentation Method. — In the clinical laboratory especially constructed saccharimetric tubes are used, of which Lohnstein's is probably the best. These are provided with a scale which enables the percentage of sugar to be read off directly from the amount of carbonic acid that has gathered in the upper end of the tube. The instruments are accompanied by printed instructions for use, which need not be considered at this place. Kxapp's Method. — This method is to be preferred to the older method of Fehling, which furnishes results of value only in espe- cially experienced hands. The method is based upon the observation that mercuric cyanide in alkaline solution is reduced by sugar to metallic mercury. If urine is then added to a solution containing a known amount of the cyanide until this is entirely reduced, the corresponding amount of sugar can be directly ascertained. The solution which is generally employed for this purpose con- tains 10 grammes of the chemically pure cyanide, and 100 c.c. of a solution of sodium hydrate (sp. gr. 1.145) in the liter: 20 c.c. cor- respond to 0.05 gramme of glucose. \\v urine must be free from albumin and should contain not more than 0.5 to 1 per cent, of sugar. This should first be ascer- tained by a preliminary test. If more is present, the urine should be correspondingly diluted. Twenty e.e. of the reagent are diluted with 80 c.c. of distilled water, or with less if ;i smaller amount of sugar than 0.5 per cent, i- present. The solution is heated to the boiling-point, and then titrated with the diluted urine, boiling for one-half minute after the addition of every 2 e.e. or Less of the urine. As the end-reaction is approached, the mercury together with the phosphates settles to 282 . THE URINE. the bottom and the supernatant fluid becomes clear. The final point is reached when a drop of the liquid, placed upon filter-paper, and successively held over the mouth of a bottle containing fuming hydrochloric acid and over that of one containing a strong solution of hydrogen sulphide, is no longer colored yellow. The results are then calculated on the basis outlined above. Fehling's Method. — For a consideration of this method, which at best is open to numerous objections, and which sometimes leads to no end whatever, the reader is referred to other works. At this place it may well be omitted. Lactose. — The presence of lactose in the urine is a normal occurrence in nursing women, and it is at times found also imme- diately preceding confinement. Its appearance in the urine is un- doubtedly referable to absorption, owing to the fact that a super- abundance of milk is being produced, and we accordingly also find the substance in the urine when for any reason lactation is sup- pressed. Once it has found its way into the circulation, its elimina- tion through the kidneys necessarily follows, as the body is incapable of inverting the disaccharides to monosaccharides. Aside from its occurrence in connection with lactation, lactose is found in the urine only if abnormally large amounts have been ingested. In such an event, as has been stated, a certain proportion of the sugar escapes inversion in the epithelial cells which line the intestinal tract, and on entering the general circulation it is elimi- nated as such. The amount of lactose which may be found in the urine of nursing women varies between 0.013 and 0.438 per cent. Its presence in the urine may be suspected if the reduction test and the phenylhydrazin test yield a positive result, while the fermenta- tion test, as usually conducted, is negative. Like glucose, the sub- stance is dextrorotatory. To identify the sugar positively as lactose, however, it is necessary to isolate it as such. Isolation. — The collected urine of twenty-four hours is precipi- tated with lead subacetate and filtered. After washing with water the filtrate and washings are mixed and treated with ammonia. The resulting precipitate is filtered off and the filtrate again pre- cipitated with lead subacetate and ammonia, and so on until the final filtrate is optically inactive. The precipitates, with the excep- tion of the first, are then mixed, washed with water, decomposed with hydrogen sulphide, and filtered. In the filtrate the excess of hydrogen sulphide is removed by a current of air, and- freed from any acids that have been liberated by shaking with argentic oxide. The mixture is filtered, freed from soluble silver with hydrogen sul- phide, treated with barium carbonate, and concentrated to a small volume ; 90 per cent, alcohol is then added, which causes the formation of a flocculent precipitate. This is filtered off. The filtrate is placed in the desiccator, when on standing crystals of lactose gradually separate out. These may be purified by recrys- THE CARBOHYDRATES. 283 tallization, decolorization with animal charcoal, and extraction with 60-70 per cent, alcohol. Laevulose. — The occurrence of a laevorotatory sugar has been at times, though rarely, observed in the urine of diabetic patients, where it was present either alone or in association with glucose. Like dextrose, the substance reduced Fehling's and Nylander's solu- tion, and formed an osazon with phenylhydrazin, with a melting- point of 205° C. It was fermentable, but, unlike true laevulose, could be precipitated with basic lead acetate. Leo's laiose, which was also obtained from a diabetic urine on one occasion, was very similar to the body just described, but, unlike this, could not be fermented. With phenylhydrazin, moreover, it formed a yellowish-brown non-crystallizable oil. Of the nature of these bodies nothing further is known. The presence of a laevorotatory sugar can, of course, readily be established by the common tests, supplemented by a polarimetric examination, if it is present alone. If glucose, however, also is con- tained in the urine in amounts sufficient to counteract the laevorota- tion, the matter is more difficult. In such an event, however, it will be observed that higher values are obtained in estimating the sugar with Knapp's method, or according to the differential density method, than with the polarimeter, for reasons which are self-evident. Maltose. — Maltose together with glucose was found on one occa- sion in the urine of a patient supposedly the subject of pancreatic disease. Its recognition is essentially dependent upon the forma- tion of its osazon, and the identification of the latter by its melting- point (see also page 59). Dextrin. — That traces of dextrin are found in the urine under normal conditions has been pointed out. Larger amounts have been observed in the case of a diabetic patient, where the substance apparently took the place of glucose. Of its origin nothing definite is known, but it is likely that in health the substance gains entrance to the circulation in a more or less accidental way, and is then, of course, eliminated at once. It is now regarded as identical with the animal gum of Landwehr. To demonstrate the presence in normal urine of carbohydrates of this character, the urine is boiled witli dilute sulphuric acid for about thirty minutes, and after being rendered alkaline with sodium hydrate is examined with Nylander's lest. A positive reaction may now be obtained, while previously no reduction occurred. For the isolation of these normal carbohydrates the reader is referred to special works. Pentoses. — Thai traces of pentoses may occur in the urine under normal conditions has been stated. They are due then, no doubt, to the ingestion of such articles of food as prunes, cherries, grapes, beer, wine, etc. It will be shown, ii loreo ver, t hat the peculiar gluco- proteid which occur- in the pancreas yields a pentose on decom- position, and it i- possible (hat this also may at times be a source 284 THE URINE. of the pentoses which are found in the urine. As a general rule, it is true, ingested pentoses are mostly decomposed within the body ; but in chickens and rabbits, in which a more marked ability exists to effect their oxidation than in man, this is never complete. As in the case of glucose, the power to assimilate pentoses seems to vary with different individuals, and here, as there, a digestive pentosuria can artificially be produced. In diabetes also the power to oxidize the pentoses may be much impaired, and, very curiously, the largest quantities have thus far been observed in morphin habitues. The individual pentoses which have thus far been encountered in the urine are arabinose, xylose, and rhamnose. They all reduce Fehling's solution, and give rise to osazons with phenylhydrazin. The melting-points of the resulting compounds, however, are differ- ent from those of the common hexosazons (see page 280). In the amounts, however, in which they are usually present no reactions are obtained in this manner. The fermentation test is not obtained. Xylose and rhamnose turn the plane of polarization to the right, while arabinose is optically inactive. To demonstrate the presence of pentoses in the urine the following test of Tollens is employed : A saturated solution of orcin in concentrated hydrochloric acid is first prepared, and should contain a slight excess of the substance. Six c.c. of this are divided into two equal parts and are allowed to cool. To one portion 0.5 c.c. of the urine under examination is added, while the other is treated with the same amount of normal urine of a like specific gravity. In either case it is well first to decolorize the urine with animal charcoal. Both specimens are then placed in a beaker with boiling water, when the urine contain- ing pentoses gradually assumes a green color, which begins in the surface layers, while the color of the normal urine is scarcely changed. One-tenth per cent, of pentoses can thus be demonstrated. With Tollens' phloroglucin test, which is conducted in the same manner, a deep-red color develops instead ; but this reaction is also common to the glucuronates. (The reagent is prepared in the same manner as in the case of the orcin reagent, phloroglucin being simply substituted for the orcin.) THE ALBUMINS. As every urine contains a small number of cellular elements which are derived from the urinary tract, it can readily be under- stood that even under normal conditions albumin can be demon- strated with suitable methods. The amount, however, is exceed- ingly small, and with the common tests a positive reaction cannot be obtained unless the substance in question has been previously isolated from a large quantity of urine. In such an event a trace of a nucleo-albumin can be demonstrated. The occurrence of the common albumins of the blood, on the other hand, is always a THE ALBUM IS S. 285 pathological phenomenon. Some writers, it is true, speak of a physiological albuminuria, which may be observed after severe muscular exercise, following cold baths, during pregnancy, etc., and it is even claimed that the elimination of albumin in young persons, which is so commonly observed in neurotic and anremic individuals, in association with an increased amount of uric acid and oxalic acid, belongs to this order. There is an increasing tendency among pathologists, however, to doubt the physiological character of such forms of albuminuria, and personally I main- tain that every albuminuria of a hajmatogenic type is a pathological phenomenon/ We may, in fact, go further, and assume that the appearance of nucleo-albumin also in amounts which can be de- monstrated by ordinary tests is abnormal, as such an occurrence must of necessity be associated with an increased desquamation of epithelial cells, which in itself is evidence of a pathological process. This, however, is hardly the place to enter upon a detailed con- >ideration of the various morbid conditions in which albuminuria may occur, and it will suffice to state that any disturbance in the nutrition of the glandular elements of the kidney, from whatever cause, will at once find expression in the appearance of albumin in the urine. The albumins which are then eliminated are the common albumins of the blood, and notably serum-albumin and serum- globulin. Fibrinogen, on the other hand, is usually not found. In cases of hematuria and chyluria, however, its presence may be inferred from the formation of coagula of fibrin. This may occur in the urinary passages already, but more commonly it is observed after the urine has been voided. Other albumins besides those which are normally found in the blood are encountered only exceptionally in the urine ; but it may be stated that whenever such substances find their way into the general circulation their elimination at once follows. Formerly it was taught that peptones could thus appear, and for many years various types of peptonuria were described. More recent investiga- tion-, however, have shown that the substances in question were in realitv no peptones in the sense of Kiilme, but albumoses. Some of these are, no doubt, identical with the common digestive albu- moses, and find their way into the blood, when their further trans- formation into native albumins does not occur in the epithelial ce|]s ,,f the digestive tract. Others again are probably formed in the body proper in diseases which are associated with suppurative processes, and in which the formation of albumoses occurs at the expense of the tissue albumins under the influence of various micro- organisms, ruder still other conditions, as in the various non- septic fevers, in phosphorus poisoning, etc., the albumosuria may We the expression of ;i metabolic abnormality {><■/■ se, and is possibly dependent upon the action of the various tissue ferments. Of special interest, further, i- the appearance in the urine of the so-called albumin of Bence done-, which has been repeatedly ob- 286 THE URINE. served in association with multiple myelomata of the bones. Of its chemical nature, however, little is known. By some it is regarded as an albumose, but, according to Neumeister, it is not identical with any of the known digestive albumoses. I shall revert to it later. In diseases, finally, in which an increased destruction of leuco- cytes is taking place, both histon and nucleohiston have been found. Of other albumins which are foreign to the blood, only egg- albumin has been encountered, following the ingestion of excessive amounts of the substance. Tests for the Common Albumins of the Blood. — The Niteic Acid Test. — A small amount of urine is placed in a conical glass and is underlaid with a few cubic centimeters of concentrated nitric acid, when in the presence of serum-albumin and serum-globulin a white, opaque disk of coagulated albumin is formed at the zone of con- tact, which varies in intensity and extent with the amount of albumin present. Immediately below this variously colored rings also are observed, which are in part referable to the decomposition of indoxyl and skatoxyl sulphate, and the oxidation of the liberated indoxyl and skatoxyl to blue and red pigments. In the presence of bile-pigment a green color will then also be noted. If much urea be present at the same time, it may happen that after a few minutes a dense disk of urea nitrate crystals separates out in the lower pigmented layer, but more commonly these are formed throughout the mixture on standing, and gradually settle to the bottom. Should uric acid, further, be present in increased amount, a white disk develops higher up in the urine, and separated from that referable to albumins by a layer of clear urine. This may at times be quite marked, and may extend downward toward the nitric acid so rapidly that it is difficult to say whether it is referable to albumin or a large excess of uric acid. Should this occur, it is best to dilute the urine with an equal volume of water, or, even more strongly, when a portion of the uric acid at least is prevented from separating out or is held in solution altogether. As nucleo-albumins, when present beyond traces, can simulate the true albumin reaction, it is well to dilute the urine with water and to examine again when its presence is suspected. If then the reaction is more pronounced than before, the precipitate may, in part at least, be referable to this source. This possibility should be con- sidered if the urine contains an increased number of morphological elements, and if the reaction is slight. Other tests should then also be employed. Albumoses, if present beyond traces, also react with nitric acid, but it is to be noted that in such cases the precipitate disappears on heating and reappears on cooling, while the liquid at the same time assumes an intensely yellow color. Should a mixed albuminuria exist — i. e., should albumoses and albumin be present simultane- ously— the clearing of the urine is only partial. THE ALBUM IX S. 287 As nitric acid also precipitates certain resins which may have been administered for medicinal purposes, it is at times necessary to eliminate this possibility of error. Their presence is indicated if the precipitate disappears on shaking the mixture with ether. The Boiling Test. — The urine should present a feebly acid or neutral reaction. If alkaline, it is rendered nearly neutral by adding a drop or two <»f dilute acetic acid. A few cubic centimeters are then boiled in a test-tube, when in the presence of coagulable albumins the liquid becomes turbid, and on standing a flocculent precipitate gathers at the bottom of the tube. The turbidity may, however, at times be due to a precipitation of neutral earthy phos- phates. To distinguish between the two, one or two drops of a 25 per cent, solution of nitric acid are now added for every 1 c.c. of the urine. The earthy phosphates are thus dissolved, while the pre- cipitate of albumin remains unaffected. If more than traces of albumin are present, this test is very reliable ; otherwise there is danger of dissolving the small amount of albumin. If nitric acid is used instead of acetic acid, this danger is generally small, how- ever, but the possibility exists, nevertheless. Hence in doubtful cases it is always best to resort to the nitric acid test as well. If the albumin of Bence Jones should be present, coagulation occurs at a temperature of 50° C. already, but it will be noted that the precipitate disappears on subsequent boiling and reappears on cooling. The common albumoses, as well as nucleo-albumin, are not thrown down. The presence of the former may be inferred if after the addition of the acid and subsequent cooling a white pre- cipitate is formed, which dissolves upon the application of heat and reappears on cooling. If acetic acid is to be employed instead of nitric acid, it is best to treat the urine with one-sixth of its volume of a saturated solu- tion of common salt, after having rendered it distinctly acid. It is then boiled as before. In this case the danger of dissolving the precipitated albumins is much lessened. The Potassium Ferrocyanide Test. — A few cubic centimeters of urine are strongly acidified with acetic acid, and treated with a 10 per cent, solution of potassium ferrocyanide drop by drop, when in the presence of albumin a precipitation occurs which varies in intensity with the amount present. Concentrated urines should first be diluted. Albumoses are also thrown down, but are redis- solved on boiling and reappear on cooling. Should nucleo-albu- mins be present beyond traces, a precipitate develops upon the addition of the acetic acid. This may also occur if urates are pre-,. nt in large amounts. But in this event the precipitate clears upon warming the solution ; and if the urine, moreover, is previously diluted, ii doe-; not occur at all, while the separation of nucleo-albu- min takes place more rapidly in the latter ease than before. Still other teste exisl which are equally good, but for practical 288 THE URINE. purposes the three just described will suffice. In every case, however, it is necessary that the urine should be perfectly clear. If turbid, owing to precipitation of any of the normal constit- uents of the urine, simple filtration generally suffices ; but if refer- able to bacteria, it is best shaken with powdered talcum and then filtered. Special Test for Serum-albumin. — If it is desired to demonstrate the. presence of serum-albumin by itself, the urine is rendered amphoteric or faintly alkaline with sodium hydrate, and satu- rated with magnesium sulphate to remove any globulins that may be present. The filtrate is then acidified with acetic acid and boiled, when in the presence of serum-albumin precipitation occurs. Special Test for Serum-globulin. — This is conducted as above, or by adding an equal volume of a saturated solution of ammo- nium sulphate to the amphoteric urine, when the globulins are thrown down. Urates may then also separate out, but this always occurs later. The precipitated globulins are soluble in acetic acid. As I have stated before, there is one instance of globulinuria on record in which the substance was found in the sediment in crystal- line form. Test for Nucleo-albumin. — A small amount of urine is diluted with water and then treated drop by drop with strong acetic acid. In this manner the precipitation of urates is prevented, while the nucleo-albumin separates out. To identify it as such, however, it is necessary to isolate the substance in larger amounts. To this end, the collected urine of twenty-four hours is carefully neutralized and concentrated to about 1000 c.c. at a temperature of 60°-70° C. On filtering, it is saturated with ammonium sulphate in substance and the precipitate collected on a filter. This is dissolved in a little water and freed from salts by dialysis. Should hetero-albumosebe present, this separates out and is removed by filtration. A portion of the remaining solution is then tested with acetic acid, as described ; the precipitate should be soluble in mineral acids. In the remaining solution the body is completely thrown down with acetic acid. The precipitate is filtered off, washed with dilute acetic acid, and dried. On fusion with caustic alkali and potassium nitrate, phosphoric acid should then be liberated if the substance is a nucleo-albumim If this reaction is not obtained, but if on boiling with dilute mineral acids a reducing substance is set free, it may be assumed that the body in question is mucin. Test for Albumoses. — To test for albumoses in general, a small amount of the urine is acidified with acetic acid and treated with an equal volume of a saturated solution of common salt. The solution is then boiled and filtered while still hot, so as to remove any eoagu- lable albumins that may be present. On cooling, the albumoses separate out, but redissolve on boiling. In such an event, the solu- tion also gives the biuret reaction and that of Millon. Safer, however, is the following method, which should always be THE ALBUMINS. 289 employed when there is reason to believe that albumoses are present only in traces. The collected twenty-four hours' urine is carefully neutralized, concentrated to about 1000 c.c. at 60°-70° C, filtered, and saturated with ammonium sulphate in substance. The pre- cipitate is collected on a filter, and dissolved in a little water, when a small portion is treated with an equal volume of a saturated solu- tion of common salt, and with acetic acid or nitric acid drop by drop so long as any precipitate that has formed is thus increased. The solution is then boiled. If coagulable albumins are present, these are precipitated, and are filtered off from the hot solution. If the filtrate becomes turbid again on cooling, and clears upon sub- sequent boiling, the presence of albumoses may be inferred. To determine the character of the albumoses in question, the remaining liquid is dialyzed (see above), freed from nucleo-albumin by means of acetic acid, neutralize,!, concentrated on a water-bath, and saturated with rock-salt. If primary albumoses are present, they are thus precipitated and filtered off. When acetic acid that has been saturated with common salt is added to the filtrate the deutero- albumoses are thrown down. This test should be applied in the preliminary examination also if no reaction is obtained. Instead of using sodium chloride to precipitate the albumoses, ammonium sulphate can, of course, also be used, as has been described on page 184. True peptones, in the sense of Kiihne, do not occur in the urine, and it is hence unnecessary to describe the older and more complicated methods which formerly were employed in their search. Bence Jones' Albumin. — This body, as has been pointed out, has repeatedly been encountered in the urine in association with the existence of multiple myelomata of the bones. Of its nature, however, little is known. Most observers have regarded it as an albumose, but it is admitted that it is not identical with any of the known digestive albumoses. Like the globulin described bv Paton, it has been found in crystalline form in the urinarv sediment. Magnus- Levy, who has recently studied the bodv in question, while likewise unable to identify it with any of the known albumins or albumoses, points out that it has in realitv only one propertv in common with the albumoses, viz., the solubility of its precipitate on boiling. Be points out, however, that this is only apparent, and that under suitable conditions the body is coagulated on heating to 100° C, like the native albumins. He further noted that, like the true albumins, the substance yields the common digestive products of these bodies, viz., primary and secondary albumoses; but, as in the case of casein, no hetero-group could be demonstrated. These results T have personally confirmed, and it is thus conclusively iblished thai the body cannot be an albumose. Pending further investigations, it is hence advisable to term the substance the albumin of Bence Jones. Of its origin nothing definite is known. The amount whieh i- often found, however, is so large that the con- 19 290 THE URINE. elusion suggests itself that the substance may be derived from the ingested albumins, and is formed in the intestinal canal or its walls as a result of some abnormal digestive process. The pres- ence of the albumin may be suspected if a urine gives the usual albumose reaction to a marked degree, as the disease in question is in reality the only one in which larger amounts of an " albumose"- like body are obtained. It can then be isolated by treating the neutralized urine with double its volume of a saturated solution of ammonium sulphate. To identify the substance, it is advisable to digest the body with pepsin, to demonstrate the formation of proto- albumose, and to show that no hetero-albumose is produced. For further details the reader is referred to Magnus-Levy's work. Test foe Fibriist. — When fibrin is present in the urine it usually occurs in the form of distinct clots, the nature of which is commonly apparent without chemical examination. If it is to be identified in this manner, however, the clots are washed with water until free from blood-pigments. They are then placed in a 5 per cent, solu- tion of sodium chloride containing an excess of thymol, to guard against putrefactive changes. It will be observed that the substance does not dissolve, while in a 0.3 per cent, solution of hydrochloric acid it rapidly swells and is digested after the addition of a little pepsin. Test for Histon. — The twenty-four hours' urine is first freed from coagulable albumins by boiling. It is then precipitated with a large excess of 94 per cent, alcohol. The precipitate is washed with hot alcohol and dissolved in boiling water. On cooling, the solution is acidified with hydrochloric acid and allowed to stand for a number of hours. Any uric acid that has separated out is removed by filtration, when the filtrate is precipitated with ammonia. The collected material is washed with ammoniacal water until the washings no longer give the biuret reaction. It is then dissolved in dilute acetic acid. If histon is present, the solution coagulates on boiling and gives the biuret reaction. The coagulated material dis- solves in mineral acids. Quantitative Estimation of the Coagulable Albumins. — In the clinical laboratory the so-called albuminimeters of Esbach are conveniently employed for this purpose. The method is exceedingly simple, and gives results which are sufficiently accurate for ordinary purposes. To this end, the tube is filled with urine to the mark U. Esbach's reagent, which consists of an aqueous solution containing 10 grammes of picric acid and 20 grammes of citric acid to the liter, is then added to the mark R. The tube is closed, inverted a number of times, and set aside for twenty-four hours. The number of the scale which corresponds to the height of the precipitated albumins indicates the amount in grammes in 1000 c.c. of urine. Care must be had, however, that the urine is acid, that the density does not exceed 1.006-1.008, and that the temperature remains at about 15° C. THE PIGMENTS OF THE URINE. 291 If more accurate results are desired, a known volume of urine, feebly acidified with nitric acid if necessary, is heated, first on a water-bath and then over a free flame, until coagulation is com- plete. The precipitate is eollected on a small filter, and washed with water, alcohol, and ether. The contained nitrogen is now esti- mated according to Kjeldahl's method, when the result multiplied by 6.3 will indicate the corresponding amount of albumin. If it is desired to estimate the amount of the individual albumins separately, they are first isolated, as has been described, and are then subjected to Kjeldahl's process. In this case, however, ammonium sulphate cannot be used for purposes of salting. THE PIGMENTS OF THE URINE. Of the chemical nature of the pigments of normal urine little is known that is definite. According to some observers, the yellow color is due, in part at least, to the presence of so-called urochrome, which in turn is regarded as identical with the normal urobilin of MacMunn. Others, again, claim that there is no reason to .- u ] > j >■ . - . that a difference exists between this normal urobilin and the urobilin of Jaffe, which is mostly observed under pathological conditions, but which may occur also in health. Jaffe's urobilin, further, is held by some to be identical with the hydrobilirubin which results from bilirubin through the action of sodium amalgam. Of late, how- ever, this view has been questioned, especially as bilirubin on oxida- tion furnishes a substance, choletelin, which cannot be distinguished from hydrobilirubin on the one hand, or urobilin on the other. A similar pigment, or one which is identical with urobilin, has further been obtained from hsematoporphyrin. That the urobilin which is notably observed under pathological conditions can lie formed within the body in the absence of micro-organisms is now a well-established feet. We thus find that in diseases in which the elimination of bile through the usual channels is prevented, urobilin may occur in the urine, nevertheless; and it has further been noted that both at the beginning and at the end of jaundice increased amount-; are found. Similar results have been obtained when from any cause an increased destruction of blood-pigmeni occurs. We may thus imagine that in such cases the urobilin results from bilirubin through an extensive oxidation to choletelin. This view of the origin of urobilin, of '•our-'', does not necessarily preclude the possibility that a certain amount of the pigment, which, as I have said, may normally also occur in tin- urine, may be derived from bilirubin through a process of reduction in the intestinal tract. But, as is apparent from the considerations jusl related, we are scarcely in a position to speak authoritatively of the origin of the normal urinary pigments. The chemical position of the colorless mother-substance of urobilin, more- over, which i- spoken of n- urobilinogen, and which can usually be demonstrated whenever urobilin also is present, is thufi far not clear. 292 THE URINE. According to Garrod, the urobilin of the urine is identical with the stercobilin of the feces, both in composition and properties, but differs conspicuously from hydrobilirubin, especially in the much smaller percentage of nitrogen which it contains, viz., 4.11 per cent., as compared with 9.22. The elementary composition of urobilin is given by Garrod and Hopkins as : C, 63.58 per cent. ; H, 7.84 ; N, 4.11, and O. 24.47. Garrod further states that by acting upon urochrome with acids he never succeeded in obtaining any prod- uct showing the urobilin band, or yielding the well-known fluores- cence with zinc chloride and ammonia, as Thudichum. claimed. But he found that a substance having both these properties is readily obtained by the action of aldehyde upon an alcoholic solu- tion of urochrome. In a short time — shorter still when the liquid is warmed — an absorption-band appears like that of urobilin, and the tint of the solution deepens to a rich orange-yellow* With zinc chloride and ammonia a brilliant green fluorescence occurs, and the band is shifted toward red, as that of urobilin is under like conditions. Isolation of Urochrome. — To demonstrate the presence of so-called urochrome in normal urine, the fluid is acidulated with 1 or 2 c.c. of dilute sulphuric acid pro liter. On filtering, it is saturated with ammonium sulphate. The resulting precipitate is dried and ex- tracted with warm and slightly ammoniacal alcohol. The pigment passes into solution, and is obtained on evaporation of the alcohol as an amorphous reddish-brown substance, which is readily soluble in acidulated water, chloroform, and common alcohol, but is practically insoluble in ether and benzol. According to Garrod, its solutions do not give rise to any bands of absorption, and do not fluoresce upon the addition of ammonia and zinc chloride. Gautier, on the other hand, states that its acidulated solutions show a band of absorption about F, and that the remainder of the spectrum from about G on to the right is obscured. He adds that in this respect urochrome and choletelin are alike. Garrod regards the action of aldehyde upon an alcoholic solution of urochrome, outlined above, as a very delicate test for the pigment. The process can be stopped then by simple dilution with water, as aldehyde has no such action upon aqueous solutions of urochrome. If, however, the action is allowed to continue, a further change ensues. The liquid reddens and a second band appears in the violet. The fluorescence can still be obtained with zinc chloride and ammonia, and both bands are shifted toward red and are closer together than before. The term uroerythrin has been applied to the pigment which imparts the salmon-red color to sediments which are composed of urates or uric acid. Of its chemical nature, however, nothing defi- nite is known, but there is evidence to show that it also is a deriva- tive of the normal coloring-matter of the blood. It contains 62.51 per cent, of carbon and 5.79 per cent, of hydrogen. Its THE PIGMENTS OF THE URINE. 2lJ3 amount is noticeably increased in extensive disease of the liver, as also in conditions associated with an increased destruction of red corpuscles. Isolation of Uroerythrin. — As has been stated, the salmon color of sediments of urates and uric acid is due to this pigment. In their absence the urine is precipitated with neutral acetate of lead or barium chloride. If uroerythrin is present beyond traces, it is thrown down, and colors the resulting precipitate a more or less intense salmon red. The pigment is soluble in boiling alcohol, and may thus be extracted. Its solutions are said to give rise to two bands of absorption to the left of F. Urobilin. — Urines which contain much urobilin, viz., the patho- logical urobilin of Jaffe, present a dark-yellow color, which may be imparted to the foam on shaking. They are thus quite similar to icteric urines. Tests. — To identify the substance, the urine is precipitated with a mixture of barium hydrate and barium chloride. If notable quanti- ties of urobilin are present, the precipitate is thus colored a more or less intense brownish-red. On boiling with acidulated alcohol the pigment is then extracted, and imparts a brownish or pomegranate- red color to the alcoholic solution (v. Jaksch). Gerhardt's test also is very serviceable. To this end, 10-20 c.c. of urine are extracted with chloroform by shaking. A few drops of a dilute solution of iodopotassic iodide are added to the extract, when, upon the further addition of a dilute solution of sodium hydrate, the solution is colored yellow or yellowish brown and ex- hibits a beautiful greenish fluorescence. If the substance cannot be demonstrated with these tests, the urine is acidulated with hydrochloric acid and allowed to stand exposed to the air, so that any urobilinogen that mav be present is transformed into the free pigment. The fluid is then examined with the spectroscope, when in the presence of urobilin a distinct band of absorption is obtained between b and F, extending bevond F to the right. A similar band is also obtained in alkaline solu- tion, but is not so intense and does not extend bevond F. Isolation. — To isolate the pigment, if present in large amounts, the urine is directly precipitated with ammonia and chloride of zinc. The precipitate is thoroughly washed with water, extracted with alcohol bv boiling, dried, and then dissolved in ammonia. The resulting solution ia precipitated with subacetate of lead, the precipi- tate washed with water, and extracted with boiling alcohol as before, and decomposed with acid alcohol. The filtered alcoholic solution is treated with one-half its volume of chloroform and diluted with water; the urobilin passes into the chloroform on moderate agita- tion. The chloroform solution is then washed with water. On in! distillation the pigmenl remains as an amorphous reddish mntc- rial, which can be further purified by washing with ether, which takes up contaminating red pigments. The substance is readily soluble 294 THE URINE. in alcohol, amyl alcohol, and chloroform, less readily so in water and ether. Under pathological conditions still other pigments may be found in the urine. These comprise haemoglobin and its derivatives, hsematin, methaemoglobin, and haematoporphyrin ; further, also uro- rubrohaematin and urofuscohasmatiu, which are also undoubtedly derived from haemoglobin, but which have thus far been found in the urine on only one occasion ; further, the common pigments of the bile ; and, finally, substances which belong to the class of the so-called melanins. For a consideration of the various pathological conditions under which the bodies may be met with, however, the reader is referred to special works on diagnosis. At this place I shall merely describe the more common tests by which their presence can be demonstrated. The Blood-pigments. — If the microscopical examination of the urine shows the presence of red blood-corpuscles in the sediment, further chemical examination is, of course, unnecessary. Cases of simple haemoglobinuria, in contradistinction to haematuria, may occur, however, in which dissolution of the haemoglobin has taken place in the circulation already, and in which blood-corpuscles do not appear in the urine. In such an event the demonstration of blood-pig- ment can be made only by chemical methods. Its presence, it is true, is usually indicated by the color of the urine, but this may be simulated by other substances as well. Heller's Test. — This is the most convenient test for demon- strating the presence of blood-pigment in the urine, and, in the modi- fication here given, exceedingly sensitive. It is based upon the decomposition of the pigment in question by means of caustic alkali and the resulting formation of haemochromogen. To this end, a small amount of the urine, or, still better, of the sediment, is rendered strongly alkaline with caustic alkali and boiled. On stand- ing, the precipitated earthy phosphates settle to the bottom, and are colored a more or less intense carmin by the haemochromogen, which has likewise separated out. That the pigment is in reality haemo- chromogen can be readily demonstrated on spectroscopic exami- nation (see page 330). When controlled in this manner, the test is exceedingly sensitive, and may still yield a positive result even when the chemical test by itself does not give a well-pronounced reaction. Spectroscopic Examination. — On direct spectroscopic exam- ination the spectrum of methaemoglobin is usually obtained. The urine should first be acid, and if" necessary a little acetic acid is added. On the addition of a little ammonia and ammonium sul- phide and subsequent filtration the broad band of haemoglobin is then obtained. With oxyhaemoglobin, on the other hand, the two bands between T> and E are observed ; and upon the subsequent addition of ammonia and ammonium sulphide and filtration the spectrum of reduced haemoglobin results. If this does not appear distinctly, the THE PIGMENTS OF THE URIXE. 295 solution is treated with an excess of sodium hydrate solution, and will then give the spectrum of haemochromogen. Haematin. — Haematin is very rarely found in the urine. Its presence as such can be determined only by spectroscopic examina- tion. Like haemoglobin and methaemoglobin, it gives Heller's reaction. Haematoporphyrin. — According to Garrod, traces of haematopor- phvrin may be found in every urine. Larger quantities are observed in a number of diseases, but even in these the amount is usually so small that its presence will scarcely be suspected from simple inspection. Typical haematoporphyrinuria, on_ the other hand, may be observed following the prolonged administra- tion of sulphonal, trional, and tetronal, or in cases of poisoning with the substances in question. The urine then appears dark red in color, and on standing may turn almost black. As Hammarsten has pointed out, this change in color is only in part due to haemato- porphyrin, and is largely referable to other red and reddish-brown pigments of unknown character. Whether or not different haemato- porphyrins exist has not been definitely determined, but is probable. In freshly voided urines haematoporphyrin probably exists in com- bination with some other, still unknown body, with which it forms a colorless chromogen. From this the free pigment then develops on exposure to the air. Like the common blood-pigments and haematin, haematoporphyrin also reacts with Heller's test. To prove its presence, however, as such, a spectroscopic examination is necessary. To this end, the urine is precipitated with barium hydrate and barium chloride. The precipitate is washed and allowed to stand in contact with acidulated alcohol, which extracts the pigment. After filtering, the solution is examined with the spectroscope; if subsequently the solution is rendered alkaline with ammonia, the spectrum of haematoporphyrin in alkaline solution is obtained. To isolate the substance as such, the acid solution is mixed with a little chloro- form and diluted with water. On gentle agitation the chloroform take-; up the greater portion of the haematoporphyrin, while a -mill fraction and other pigments remain in the diluted alcoholic solution. On evaporating the chloroform extract the substance is obtained in comparatively pure form. Neumeister states that besides haematoporphyrin another deriva- tive of the blood-pigment may be observed in cases of poisoning with sulphonal, which, in contradistinction to the first, contains iron. This does not read with Heller's lest, however, while the color of the urine is the same as in typical haematoporphyrinuria. The pigment is precipitated by an alkaline barium chloride solution, and can be subsequently dissolved in acid alcohol. This solution presents ;i reddish-violel color, and shows one broad band of absorp- tion in the bine portion of the spectrum immediately bordering on the green. 296 THE URINE. On rendering the solution alkaline with ammonia the pigment is thrown down. On adding an excess of sodium hydrate solution, on the other hand, it is dissolved, while the liquid assumes a yellow color. This solution then shows a sharp, narrow line in the green, near the blue portion of the spectrum, but disappears after the solution has stood for some time. Urorubrohsematin and Urofuscohssmatin. — These pigments were isolated by Baumstark from the urine of a leprosy patient, but have not been encountered since. Their relation to hseniatin is apparent from the formula? : C32H32]Sr+04Fe, hsematin (Nencki and Sieber). C34H35N405Fe, bsematin (Hoppe-Seyler). C68PI94X8O30Fe, urorubrohsematin. CG8H106N8O26, iirofuscohaematin. The pigments were isolated as follows : the urine, which presented a color varying from a dark red to a brownish red, was dialyzed and the final contents of the dialyzer dissolved in sodium hydrate solu- tion ; upon the addition of hydrochloric acid to this solution urofuscoheematin separated out in brown flakes, while the second pigment remained in solution, coloring this a beautiful red. After filtering off the first, the solution was again dialyzed, when the second pigment separated out. Whether or not any relation exists between these two bodies and hsematoporphyrin in impure form, as Hammarsten suggests, must remain an open question. Melanins. — Notably in association with the existence of melanotic tumors, but at times also in other diseases which are associated with an increased destruction of red blood-corpuscles, urines are met with which gradually turn a dark brown or black on standing. When freshly voided, however, they commonly present a normal color. The pigment or pigments which are thus formed belong to the class of melanins, and are identical with those which can be obtained from the pigmented growths. They are probably eliminated in combination with some other substance which is as yet unknown, as colorless melanoc/ens and from which the free pigments are obtained on oxidation. They are unquestionably derived from the common pigments of the blood, but are individually little known. To prove that the change in the color of the urine is referable to melanins, a fresh specimen should be procured, and treated with bromine-water. If the chromogens in question are present, the resulting precipitate, which is yellow at first, turns black on standing. On the addition of a few drops of a strong solution of ferric chloride a similar reaction is obtained. To isolate the pigments from the urine, the fluid is first precipi- tated with an alkaline solution of barium chloride. From the resulting precipitate the pigments are extracted with a concentrated THE PIGMENTS OF THE URINE. 297 solution of sodium carbonate, and are then precipitated by adding an excess of sulphuric acid. By redissolution in a dilute solution of sodium hydrate and reprecipitation with acetic acid they can be obtained in comparatively pure form. But it "will be noted that a certain fraction remains in the acetic acid solution, which indicates the existence of at least two different pigments. The soluble form has been termed phymatorhusin, and, according to Nencki and Sieber. contains no iron, while Morner claims that this is present. Elementary analysis of this pigment has given the fol- lowing results (Morner) : From growth. From urine. Carbon 55.32 to 56.13 per cent. 55.76 per cent. Hvdrogen 5.65 to 6.33 " " 5.95 " " Nitrogen 12.30 " " 12.27 " " Sulphur 7.97 " " 9.01 " " Iron 0.063 to 0.081 " " 0.20 " From melanotic growths in horses a hippomelanin has been obtained, which, in contradistinction to the first, is soluble in solu- tions of the alkalies with difficulty. The Bile-pigments. — Bile-pigments are never found in the urine under normal conditions. As a rule, freshly voided urine contains only bilirubin. If a complicating cystitis, however, exists, the common derivatives of bilirubin, viz., biliverdin, bilifuscin, biliprasin, and bilihumin, may also be encountered. Bile-containing urines present a very characteristic color, which may vary from a bright golden-yellow to a greenish brown, and on microscopical examination it is common to find the morphological elements stained an intense yellow. This color is further imparted to the foam on shaking. But as urobilin when present in large amounts may impart a similar color to the urine, it is always better to resort to chemical tests. These have been described in detail in the section on the Bile, and are directly applicable also to the urine (see page 158). Oilier pigments also may occur in the urine after the ingestion of various drugs, but as the products thus formed are of no special interesl from the standpoint of animal chemistry, they are not con- sidered at this place. The Bile-acids. — The occurrence of bile-acids in the urine is solely a pathological phenomenon. In normal urines they arc never found, and even in complete obstruction of the common duct their amount i- quite .-mall. To demonstrate their presence, they must firsl be isolated as Platner'a bile, and can then he identified by polarimetric examination, their action upon the frog's heart, etc. [see pages 117 and 148). 298 THE URINE. Fats, Cholesterin, and Lecithins. Fats. — Traces of fat may be observed in the urine under normal conditions when excessive amounts have been ingested. During pregnancy also a lipuria has been noted, and is probably associated with the development of the function of lactation. Otherwise the condition is essentially a pathological phenomenon, and is notably observed in acute yellow atrophy, in phosphorus poisoning, follow- ing fracture of the long bones, etc. The largest amounts, however,, are found in cases of so-called chyluria. Owing to the existence of the fats in fine emulsions, such urines may resemble milk in their general appearance, and on standing a layer of "cream" forms at the top. To establish the presence of fats, the surface layer of the urine is extracted with ether, the ether is evaporated, and the residue brought in contact with a piece of paper, when characteristic stains result. Cholesterin. — Cholesterin is very rarely found in the urine, and has thus far been encountered only under pathological conditions. It probably always occurs in crystalline form, and is thus readily recognized. If any doubt exists, the substance in question is examined as has been described (page 163). Lecithins. — Lecithins as such have been observed in the urine only in chyluria, where they are commonly present in association with cholesterin and fat. One of the derivatives of lecithin, how- ever, is found also in the urine under normal conditions, though in very small amounts. This is glycerin-phosphoric acid. It is no doubt- referable to decomposition of the lecithins of the food in the intestinal canal, but may at times also be derived from the lecithins of the tissues. To demonstrate its presence, several liters of urine are freed from the common phosphates by rendering the urine alkaline with barium hydrate and precipitating the heated mixture with barium chloride. The excess of barium is removed with carbonic acid and the filtrate evaporated to a syrup. This is ex- tracted with absolute alcohol, when the remaining material is dis- solved in a little water and boiled with hydrochloric acid. The glycerin-phosphoric acid is thus decomposed, with the liberation of glycerin and phosphoric acid. On evaporating to dryness, the residue is extracted with water, and the presence of phosphoric acid demonstrated in the aqueous solution by the usual tests. Ferments. Every urine contains ferments which are thought to be identical with the pepsin, ptyalin, and chymosin of the digestive fluids. It can be shoAvn, as a matter of fact, that substances are present which are capable of digesting fibrin in acid solution, of inverting starch to maltose, and of coagulating milk. There is no proof, however, that these bodies are derived from the digestive glands, as Neumeister and others claim. THE PTOMAINS OF THE URINE. 299 Gases. In normal urine a certain amount of oxygen, nitrogen, and nota- bly of carbon dioxide is found in solution, and can be withdrawn by the air-pump. Under pathological conditions, further, a variable amount of hydrogen sulphide may be encountered. This notably occurs in cases of cystitis, in which the decomposition of albumin and sulphur bodies may already take place within the bladder, owing to the activity of various micro-organisms. But in a few isolated cases the gas was apparently derived from the intestinal tract, and absorbed either directly from the rectum or indirectly from the blood. All urines when exposed to the air sooner or later contain hydro- gen sulphide in the free state, which is referable, as stated above, to the action of certain micro-organisms. Especially large amounts are observed when cystin-containing urines are thus allowed to undergo decomposition. To test for hydrogen sulphide, a strip of filter-paper is moistened with a few drops of a solution of sodium hydrate and one of lead acetate, and is then clamped in the neck of the bottle containing the urine. If the gas in question is present, the paper is colored a grayish brown or black, owing to the formation of lead sulphide. When present in large amounts it is detected also by its odor. Ptomains. So far as known, ptomains are not found in the urine under normal conditions. In disease, however, various basic substances have been encountered which supposedly belong to this class. But with the exception of cadaverin and putrescin, which, as has been stilted, may occur in association with cystinuria, these bodies have l)icii isolated in amounts scarcely sufficient to establish their chemical nature. This holds good more especially of the bodies which Griffith claims to have isolated from the urine of patients suffering from scarlatina, measles, mumps, carcinoma, etc. A- regards the origin of putrescin and cadaverin in cystinuria, the opinion prevails that they are due to a specific form of intestinal putrefaction. This is, however, not necessarily the case, and in my opinion the diaminuria, like the cystinuria, is the expression of a distincl metabolic disturbance. I have pointed out that both diamins can l»e derived from arginin and lysin in the laboratory, and there is every reason to suppose that the same transformation can also occur in the living organism. That arginin at least actually occurs in the tissues of the body has been demonstrated by Gulewitch, who found the substance in the spleen. The- quantity of the diamins which may be eliminated in the mine in cases of cystinuria is quite variable. On some days traces only or none ;it nil i- found, while at other times very considerable amount- may be obtained. In one of mycases I was able to isolate 300 THE URINE. 1.6 grammes of the benzoylated cadaverin from the collected urine of twenty-four hours. To demonstrate the presence of diamins, the method of Bau- mann and v. Udranszky is most conveniently employed. To this end, the collected urine of twenty-four hours or more is benzoylated by shaking with benzoyl chloride in the presence of sodium hydrate. As a general rule, 25 c.c. of the chloride and 200 c.c. of a 10 per cent, solution of sodium hydrate are used for 1500 c.c. of the urine. The resulting precipitate, which contains the earthy phosphates, the benzoylated carbohydrates which are normally present in every urine, and the greater portion of the benzoylated diamins, is then filtered oiF, extracted with boiling alcohol, filtered, and the alcoholic extract concentrated on a water-bath. This solution is then poured into thirty times its volume of water. On standing, the benzoy- lated diamins separate out in crystalline form, and are then freed from adhering carbohydrates by repeated solution in alcohol and precipitation in water. The process is continued until the desired degree of purity is obtained. The resulting crystals are finally filtered off, dried over sulphuric acid, and identified by their melting-point and the contained amount of nitrogen. If both diamins are present, the crystals lose their water of crystalliza- tion at 120° C, and melt at 140° C. To separate them from each other, they are dissolved in a little warm alcohol, and are treated with twenty times as much ether. Benzoyl putrescin is thus thrown down, while the cadaverin compound remains in solu- tion. The crystals of the former melt at 175°-176° C, while the melting-point of the latter lies between 129° and 130° C. A small portion of the diamins remains in the first filtrate. To isolate these, the liquid is acidified with sulphuric acid and ex- tracted with ether. The ethereal extract is evaporated, and the final solution, before congealing, placed in as much of a 12 per cent, solution of sodium hydrate as is required for its neutralization. From three to four times as much of the alkali solution is then added. On standing in the cold, sodium benzoyl-cystin separates out, together with the benzoylated diamins. The crystals are fil- tered off and placed in cold water. This dissolves the cystin com- pound, while the diamins remain undissolved. They are soluble in warm alcohol, and can then be separated from each other, as has been described. CHAPTER XIII. THE ANIMAL CELL. The cell constitutes the morphological unit of all animal and vegetable life, and as such is capable of manifesting those peculiar activities which we regard as characteristic of living matter. In its simplest form it represents a tiny bit of a more or less granular, gelatinous substance — the so-called protoplasm — in the interior of which a somewhat more solid-looking body can be made out, which is termed the nucleus. Such simple cells exist in nature, either as such or as conglomerations of many cells which represent the higher forms of animal and vegetable life. All living matter, however, whether simple or complex, lias for its origin the single cell. But while in the lowest forms of life the single cell is capable of per- forming all those functions which are characteristic of living matter by itself, we find, as we ascend in the scale of animal and vegetable life, that certain groups of cells are here set aside for the purpose of executing separate functions. Such groups of cells we speak of a-5 tissues, and we accordingly find in the highly organized mammal a differentiation of the entire body into tissues, which according to their functions may be grouped as tissues of locomotion, of re- production, of digestion, of excretion, etc. With such a differentia- tion of cells into tissues, however, the original aspect of the cell is m >re or less changed. The highly differentiated voluntary muscle- cell would thus at first sight scarcely be recognized as being in any way related to the oval cell from which it is primarily derived. On careful examination, however, we find that, no matter how unlike its ancestral cell such a specialized cell may appear, the dif- ference is only apparent, for all cells of the body consist at one time of i heir existence at least of protoplasm and nucleus. The striated portion of the muscle-cell is thus nothing more than the protoplasm of the original cell, differentiated and modified in accord- ance with the function which the cell is to perform. In some cells, however, such as those of the adipose tissue, the original differentia- tion into protoplasm and nucleus is apparently lost, and on ordinary microscopical examination it appears that such cells are nothing but large globules of fat. Bui with special methods of staining we can demonstrate even here that there area nucleus and protoplasm. The only cells, in fact, in which a nucleus cannot always be demonstrated are tin- red corpuscles of the circulating blood of man and the anthropoid ape-. We find, however, that even in adult man all 301 302 THE ANIMAL CELL. red corpuscles at one period of their existence, viz., in their juve- nile form, are nucleated, and that under certain conditions, as after copious hemorrhages, such nucleated corpuscles may occur in the circulating blood in large numbers. In the bone-marrow, where they are apparently formed, they are always present. As all manifestations of life are intimately associated with chemical changes which bring about a transformation of potential into kinetic energy, such changes must of necessity occur in every living cell. These changes, moreover, must vary with the func- tion which the cell is to perform, and will hence differ, to a certain extent at least, with the different tissues of the complex organism. In the monocellular organisms, where all the various functions are performed by the single cell, all these varying changes must hence be represented. But it is to be inferred that in accord- ance with the greater simplicity in structure the chemical changes also must be of a simpler character. We should hence expect that a study of the chemical processes which take place in such low forms of life would furnish us with a better insight into the metabolism of the complex organism than could be attained from an investiga- tion of the higher forms. Unfortunately, however, this is almost an impossibility with the usual chemical and physical methods, for in attempting such a study we are met with a most serious obstacle, viz., our inability to maintain the life of the individual cell during such an investigation. We would consequently have no proof that those products which we could isolate from the dead cells were present as such in the living organism. The technical difficulties, moreover, which stand in the way of such a study are almost insur- mountable. With microchemical methods, it is true, something more definite might be accomplished, and although this branch of investigation is still in its infancy, it has already furnished us with a certain amount of valuable information. The great advances which have thus been made in our knowledge of the structure of cells have largely been accomplished in this manner. Upon the chemical processes themselves, however, which take place in the cell, not much light has as yet been thrown in this manner. We are consequently dependent for our knowledge of the metabolic processes which take place in the living body upon a study of the individual tissues as such, and the changes which result in certain substances when introduced from without. " An analysis of these tells us in what form the various food-stuffs are represented in the individual cells. By then studying the vari- ous decomposition-products which can be isolated from the tissues, we can in a measure form an idea of the manner in which these products were produced and of the form in which they were represented in the original and more complex molecule. With some tissues, however, this is more difficult than with others. The most satisfactory results, on the whole, regarding the chemical structure of the individual cell have thus far been obtained from THE ANIMAL CELL. 303 an investigation of those tissues which are especially rich in cells, and in which the cells can he more or less completely separated from the underlying matrix and from other components which may be present at the same time. This is especially true of the leucocytes of the blood. As these bodies, moreover, are but little differentiated, they may well serve as types of primitive cells. They are all nucleated, and contain a varying amount of protoplasm, which in some is capable of progressive movement. A limiting membrane, as in most animal cells, does not exist. But it is generally supposed that a meshwork of fine fibrils pervades the protoplasm, and that in the meshes a more liquid portion is contained. This is termed the hyaloplasm, in contradistinction to the more solid spongioplasm. In some forms the protoplasm is apparently perfectly homogeneous, while in others it is studded with numerous granules of variable size, which execute more or less active oscillatory movements, which are spoken of as the molecular movements of Brown. The reaction of the protoplasm is alkaline, while the nucleus apparently contains no free alkali. This may be shown by staining dried blood films with a solution of acid erythrosin in chloroform, when it will be seen that the body of the cell is colored a bright red, while the nucleus is not stained. The most intense reaction is obtained with the protoplasm of the so-called lymphocytes. The granules which are found in certain forms of leucocytes are apparently of an albuminous nature. According to their affinity for acid, basic, or neutral dyes, they are termed oxyphilic, basophilic, ami neutrophilic, respectively. Fatty, mineral, or pigment granules, which may be found in other animal cells, are usually not seen in leucocytes. In the eosinophilic leucocytes, however, the presence of iron can readily be demonstrated by microchemical methods. In another form it seems to be present in all varieties of cells, and is especially abundant in the nuclei. In the mineral ash we further find potassium, sodium, calcium, magnesium, phosphorus, and chlorine, and it is to be noted that, in contradistinction to the animal fluids, the cell contains a relatively larger amount of potassium and phosphorus, while sodium and chlorine are more abundant in the fluids. That the phosphates are of prime importance in the life of the cell is now definitely established, and Loew showed that in the spirogyra, for example, growth and cellular division are greatly interfered with by their absence. The importance of the phosphates is without doubt con- nected with the presence of the nucleins in the nuclei — i. e., of albuminous substances which, as we have seen, contain a relatively large amount of phosphorus in organic combination. The protoplasm of the cell is very rich in water, and, in addition to small an nt- of mineral -alt;-, consists essentially of albumins. Some of these are albumins proper, but the greater portion by far i- represented 1a- substances which belong to the proteid group. It appears, moreover, that the traces of serum-albumin and 304 THE ANIMAL CELL. globulin which are present do not represent integral constituents of the living protoplasm, but are merely to be regarded as food-stuffs, or possibly even as decomposition-products of the protoplasmic molecule. The proteids which are here found principally belong to the nucleo-albumins, and it is to be noted that, in contradistinction to those which occur in the nucleus, these nucleo-albumins contain relatively but little phosphorus. The albuminous radicle in one of them at least is quite constantly a vitellin. Gluco proteids may also be present, but are not so constant as the nucleo-albumins. Of other constituents of the protoplasm, lecithin is the most constant. In addition we find certain protagons, glycogen, choles- terins, and in dead cells also paralactic acid, to which the acid reaction of dead protoplasm is due. The total amount of solids, including the mineral salts, which are thus found in protoplasm, is always small, and probably never exceeds 15—20 per cent., while the remaining weight is referable to water. In some instances indeed almost the entire cell is taken up by the nucleus, and in the lymphocytes, for example, only 1.76 per cent, of the total 79.21 per cent, of albumins, as calculated for the dry material, is present in the protoplasm. The nucleus may be regarded as the essential living part of all animal and vegetable cells, and from it, no doubt, the various func- tions of the cell as a whole are directed. It is intimately connected with the-process of reproduction, and during this process it under- goes a series of most remarkable changes, which are collectively termed the karyokinesis or karyomitosis of the nucleus. Micro- scopically the quiescent nucleus represents a round or oval little body, which usually occupies an excentric position within the cell. It is surrounded by a nuclear membrane, and contains a meshwork of extremely fine fibrils, and one or more nucleoli. Both fibrils and nucleoli possess a marked affinity for anilin dyes, while the nuclear membrane and the more liquid hyaloplasm within the nuclear meshes are scarcely stained at all. We therefore recognize in the nucleus the existence of chromatic and achromatic substances, which are usually spoken of as the nuclear chromatins and achromatins. During the process of division a peculiar spinclle-like body is also observed in the nucleus, which, like the nuclear hyaloplasm, is achromatic. In contradistinction to the cellular protoplasm, 'the nucleus con- tains a much larger quantity of solids, but here as there the albu- mins stand in the foreground. Whether or not native albumins also occur in the nucleus is not definitely known, but it is generally assumed that this is not the case. The proteids, on the other hand, are abundant and largely represented by the nucleins and the nucleo-albumins. The nucleins indeed are thought to con- stitute the greater portion of the chromatic constituents of the nucleus, and among them the so-called plastin apparently occupies THE ANIMAL CELL. 305 a prominent position. This substance, while not definitely known, is usually classed as a nuelein, but differs from the more common forms in being soluble with difficulty. Especially abundant also is a nucleo-alburnin, which Kossel and Lilienfeld first obtained from the thymus gland of the calf. This they termed nucleohiston, from the fact that on treatment with hydrochloric acid it is decomposed into a nuelein — leukonuclein, and a special albumose-like substance — biston, which differs from other albumins in being insoluble in am- monium hydrate (see page 322). This substance is probably iden- tical with "the so-called tissue fibrinogen and cellular fibrinogen of other observers, and, no doubt, is closely related to the cytoglobin and prseglobulin of Alexander Schmidt. In addition to these substances, we further meet with nucleinic acids in the free state, and in some cells also with the basic radicles of the nucleinic acids — i. e., the xanthin bases, as such. Whether lecithins, protagon, and glycogen, which are constantly found in the cellular protoplasm, likewise occur in the nucleus, is not known. Of mineral constituents, iron is constantly present, and appar- ently occurs in combination with the nucleins in organic form. CHAPTER XIV. THE BLOOD. General Considerations. — The blood of man and of almost all vertebrate animals is a slightly viscid, somewhat opaque-looking fluid, which, according to its origin from an artery or a vein, pre- sents a color that varies from a bright scarlet to a dark bluish-red. On microscopical examination it is seen to contain a large number of cellular elements, which are partly colored and partly colorless. The former, which greatly predominate, are the red corpuscles, or erythrocytes of the blood. In man they are homogeneous, normally non-nucleated, circular, biconcave disks, measuring on an average 7.5 p. in diameter. When viewed through the microscope they are of a faint greenish-yellow color, while en masse they present the ordinary color of the blood. The colorless bodies, or leucocytes, on the other hand, are all nucleated and in part capable of executing amoeboid movements. Some of them are of about the same size as the red corpuscles, while others are larger. The nucleus may be single or multiple, and it will be noted that in the mononuclear forms the surrounding protoplasm is more or less homogeneous, while in the polynuclear varieties it is distinctly granular. The total number of the leucocytes per cubic millimeter varies under normal conditions between 3000 and 10,000, and is thus much smaller than the number of the red corpuscles, which is generally placed at between 4,000,000 and 5,000,000 in the same volume of blood. In addition to the red corpuscles and the leucocytes, we further find a large number of minute colorless disks, measuring less than one-half the diameter of a red corpuscle, and usually occurring in bunches of from six to a dozen or more. They are termed the plaques or blood-plates of Bizzozero. On an average, about 635,000 are found in the cbmm. Other morphological elements are not found in the blood under normal conditions, while in disease nucleated red corpuscles, both of the adult and the embryonic type, as well as other forms of leucocytes, may be encountered. When blood is drawn from the living body and is allowed to stand, it will be noted that after a variable length of time the entire mass is transformed into a semisolid, jelly-like material, wrhich is termed the -placenta sanguinis or blood-clot. On microscopical ex- amination this will be seen to consist of a dense network of fibres, in the meshes of which the corpuscles of the blood are found. If the clot is now carefully separated from the walls of the vessel, it 306 GENERAL CONSIDERATIONS. 307 undergoes shrinkage, and presses out from its meshes a clear, straw- colored fluid, which is termed the blood-serum. This gradually increases in amount, while the size of the clot diminishes, and may then be utilized for the purpose of chemical examination. The fibrous material which is formed during the process of clotting is termed fibrin. Its formation is intimately associated with the death of the organism, either local or general, and is dependent in the first instance upon the presence of a specific ferment — the so-called fibrin ferment, or thrombin of Alexander Schmidt. In the circulating blood fibrin is not found, but we here meet with its mother-substance, fibrinogen, which is not present in the blood-serum. The blood- plasma, viz., the fluid, non-cellular portion of the circulating blood, thus differs from the blood-serum in containing fibrinogenic material, but not the fibrin ferment, which is found in the serum. The fer- ment itself results from decomposition of the cellular elements of the blood, notably of the blood-plates. This may be seen when the process of coagulation is observed through a microscope. After a variable length of time, more rapidly when the blood-drop is not too small and when the surface of the glass is a little uneven, fine filaments of fibrin thus begin to appear, which usually have for their starting-point those bunch-like conglomerations of the plaques which have already been described. In these bodies the pro-enzyme of the ferment, the so-called prothrombin of Alexander Schmidt, is proba- bly contained, and gives rise to the ferment itself when the death of the cell occurs. It should be stated, however, that the fibrin fer- ment is not only derived from the plaques, but may also be formed during decomposition of the remaining cellular elements of the blood, and, to judge from recent observations, from protoplasmic material in general (see page 325). PHYSICAL CHARACTERISTICS OF THE BLOOD. Color. — The color of normal blood is referable to the presence of a peculiar albuminous substance in the red corpuscles belong- ing to tin.' class of proteids which is termed haemoglobin. In arterial Mood this is principally found in combination with oxy- gen as oxyhemoglobin, while in venous blood a mixture of both occurs. With a preponderance of oxyhemoglobin over hemoglobin the color of the blood tends toward a bright scarlet-red. In its >nce ii assumes a dark-bluish color, and we accordingly find all gradationa in shade between the two. When venous blond is exposed to the air the hemoglobin immediately absorbs oxygen, and ie transformed into oxyhemoglobin. This actually takes place in the alveoli of the lungs, and explain- the difference in color between the blood of ih<- right and the l saltrjplasma, which results when blood is received in a solution of a neutral salt, whereby coagulation is also prevented. To this end it is best to employ a saturated solution of sodium sulphate or a 10 per cent, solution of sodium chloride, to which the blood i- added in an equal amount. A saturated solution of inagne- sium sulphate may likewise be used, in the proportion of one part for three parts of blood, but is not so satisfactory, as it causes pre- cipitation of certain albumins which are essential to coagulation. After standing tor twenty-four hours the plasma may be siphoned off, or may lie separated from the corpuscles at once by centrifugation. As coagulation of the blood is apparently dependent upon the presence of soluble calcium salts, coagulation may also be prevented by precipitating with ammonium oxalate those which are present in the blood. To this end the blood is received in a solution of am- monium oxalate, such that the quantity of the latter present in the mixture amounts to about 0.1 per cent. This constitutes oxalate-plasma. Of especial value in these examinations is the blood of the horse, in which coagulation occurs much more slowly than in that of mammals. If this is available, it is only necessary to receive it in a narrow cylinder surrounded with a freezing-mixture. Kept in this manner it will remain liquid for several days. Separated from the corpuscles, the plasma is a clear, straw-colored, slightly viscid fluid, of alkaline reaction, and a specific gravity varying between 1.026 and 1.029 in man. It is capable of under- going coagulation, like the native blood, and is thus converted into blood-serum. Its general chemical composition has already been considered. It contains but 8.2 per cent, of solids, of which 6.9 per cent, is represented by albumins. These are serum-albumin, serum-fflobulin, and fibrinogen. The relation between these bodies i- subject to cousiderable variations. In all animals, however, the globulins predominate, and in some indeed, as in snakes, serum- albumin is apparently absent. In the horse the globulins con- stitute about 64.6 per cent, of the total amount of albumins. In 1000 parts by weight ilanmiarsten thus found 38.4 parts of serum- globulin, 6.5 parts of fibrinogen, and 24.6 parts of serum-albumin. Of these albumins, fibrinogen is of especial interest, as it represents the mother-substance of fibrin, and is thus intimately connected with the process of coagulation. Fibrinogen. — Isolation. — Fibrinogen is most conveniently ob- tained from the plasma by half-saturation with sodium chloride — i. e.f by treating oik; volume of the plasma with an equal volume of a saturated solution of common salt. The resulting precipitate of fibrinogen is filtered off, washed with a half-saturated solution of sodium chloride, and dissolved in an 8 per cent, solution of the salt. To further purify the Bubstance, this solution is reprecipitated, redis- ed, and the process repeated twice. The i\w.\\ precipitate is 314 ' THE BLOOD. pressed between filter-paper and suspended in water, in which it readily dissolves owing to the small amount of salt that still remains. This may be removed by dialysis. The purified substance, in a moist state, appears in the form of white flocculi, which readily coalesce to form a tough elastic mass. The isolation of the fibrinogen must be performed rapidly, as pro- longed exposure to the half-saturated salt solution tends to render the substance insoluble. Properties. — Fibrinogen belongs to the class of globulins. It is insoluble in distilled water, but soluble in dilute solutions of the neutral salts. From these solutions it may be precipitated by dial- ysis, by increasing the amount of the salt, and by passing a stream of carbon dioxide through the solution. When kept under water for a comparatively short time it is rendered insoluble. When heated to 56° C. coagulation occurs, but it appears that the fibrinogen is at the same time decomposed into two other globulins, one of which coagulates at the temperature just mentioned, while the other remains in solution until the temperature reaches 65° C. Of the nature of these two substances, however, we know but little ; it is possible that one is the so-called fibrinoglobulin, which, as we shall see later, is formed during coagulation of the blood. Fibrinogen turns the plane of polarized light to the left ; its rotation for the yellow D line corresponds to — 52.5 degrees. It consists of carbon, hydrogen, nitrogen, sulphur, and oxygen, in the proportion of 52.93, 6.9, 16.6, 1.25, and 22.26, respectively. Its most characteristic property is its tendency to the formation of fibrin, and upon this its specific test and quantitative estimation are based. This transforma- tion will be considered in detail later (see Coagulation). In addition to the blood-plasma, fibrinogen has been found in the chyle, the lymph, and various exudates and transudates. Serum-globulin. — This substance has also been termed para- globulin, Alexander Schmidt's fibrinoplastic substance, and serum- casein. Like fibrinogen, it is found in the plasma of the blood, in the lymph, in various exudates and transudates ; but it likewise occurs in the serum, in the white and red corpuscles of the blood, and in traces at least in all cellular elements of the animal body. In the urine it has been encountered in association with serum- albumin under various pathological conditions. Isolation. — Serum-globulin is most conveniently obtained from blood-serum by half-saturation with ammonium sulphate — i. e., by treating a given volume of the serum with the same amount of a saturated solution of the salt. Saturation with magnesium sulphate in substance may also be employed. In either case the precipitated serum-globulin is filtered off, washed with the corresponding salt solution, dried at 115° C, then washed with boiling water to remove the remaining salts, extracted with alcohol, then with ether, and finally dried and weighed. In any case the original solution should be nearly neutral in reaction. CHEMICAL EXAMINATION OF THE BLOOD. 315 Properties. — As the term indicates, serum-globulin belongs to the class of globulins. In its moist state it represents a snowy-white, finelv flocculent mass, which is not tough and elastic like fibrinogen. From its solutions the substance can be precipitated, as already in- dicated, by saturation with magnesium sulphate or by half-saturation with ammonium sulphate. Sodium chloride causes only an incom- plete separation of the substance when added to saturation, while half-saturation is of no effect at all. It is thus an easy matter to isolate serum-globulin when fibrinogen is present. An incomplete separation also occurs when its neutral or feebly acid solutions (using acetic acid) are diluted from ten to twenty times with distilled water, or by passing a stream of carbon dioxide through such dilute solutions. In the presence of from 5 to 10 per cent, of sodium chlo- ride its solutions coagulate at 75° C. They cause a rotation of the polarized light to the left, the specific degree corresponding to 47.8. According to Morner, serum-globulin yields a reducing substance when boiled with dilute mineral acids. It might thus be supposed that serum-globulin is in reality a glucoproteid, and not a natiye albumin, but it is possible also that the reaction is due to the acci- dental presence of a glucoproteid, which is thrown down together with the globulin. Though serum-globulin is usually spoken of as a unity — that is, an individual substance — it appears likely that the compound which is thrown down upon saturation with magnesium sulphate repre- sents a mixture of seyeral globulins. If this material is thus dis- solved in dilute saline solution and subjected to dialysis, a precipitate forms which possesses all the properties of serum-globulin which we regard as characteristic of globulins. The remaining solution, how- ever, contains an albuminous substance which may be thrown down by magnesium sulphate, and which when isolated in this manner differs only from the first precipitate in being soluble in water. Formerly it was thought that all globulins are insoluble in water, but it is thus shown that at least one form differs in this respect. The globulin which is obtained from blood-plasma is also spoken of as plasma-globulin, in contradistinction to the so-called cell- globulin; and Bammarsten's secondary globulins, which are found in the serum together with plasma-globulin, are thought to result from disintegration of the leucocytes and the fibrinogen molecule, respectively. A.ccordingly we also find more serum-globulin in the --'Turn 1 1 1 : m in tin- plasma. Chemically, these various globulins are not sufficiently characterized to warrant a separate description, and as they are all thrown down together with our present methods of isolation, it follows that the numerical data regarding the elementary composition of serum-globulin as unity must also be more of less at fault. We find, a- a matter ,,{' fact, that these data are by no mean- constant. The value of carbon thus varies between 52.32 and 53.3, and that of nitrogen between 15.61 and 16.25, which would represent a difference of nearly 1 and 0.64 per cent., respectively 316 THE BLOOD. — that is, amounts which could scarcely be owing to technical errors. Serum-albumin. — Serum-albumin is found in the plasma, the serum, the lymph, in exudates and transudates, and under certain pathological conditions also in the urine, where it usually occurs in association with serum-globulin. It is most conveniently obtained from blood-serum after removal of the serum-globulin by saturation with magnesium sulphate at a temperature of 30° C. The nitrate is saturated with sodium sulphate or ammonium sulphate at 40° C, or treated with acetic acid, so that the solution contains about 1 per cent. In either case the precipitated serum-albumin is filtered off, pressed between layers of filter-paper, dissolved in water (the reac- tion should be neutral), and separated from the remaining salt by dialysis. From its aqueous solutions it is finally obtained by evaporation at a low temperature or by precipitation with alcohol, which must be rapidly removed, however, as otherwise it will cause coagulation of the albumin. In the dry state serum-albumin is a transparent, gum-like, brittle, hygroscopic mass, or a white powder, which can be heated to 100° C. without undergoing decomposition. Solutions of the pure substance in distilled water coagulate at 50° C, while in the presence of salts a higher temperature is necessary. This varies with the amount of salt present, as also with the concentration of the albumin. A 1 to 2 per cent, solution containing 5 per cent, of sodium chloride coagulates between 75° and 90° C. From its salt solutions serum- albumin may be obtained in crystalline form. Its specific rotation in distilled water varies between 62.6° and 64.6° — a. [D]. According to Halliburton, the serum-albumin of mammalian blood-serum is not a single substance, but consists of three distinct albumins, which he terms a-, /?-, and ^-serum-albumin. They are said to coagulate at 73° C, 77° C, and 84° C, respectively. In cold-blooded animals, a-serum-albumin only is said to occur. Separation of the Albumins of the Blood-plasma from Each Other. — To isolate the fibrinogen, the plasma is treated with an equal volume of a saturated solution of sodium chloride. The result- ing precipitate is filtered off and purified as described. The filtrate contains serum-globulin and serum-albumin. The globulin is pre- cipitated by saturation with magnesium sulphate, filtered off, and likewise purified. The filtrate contains only the serum-albumin, which may be obtained, as just described, by saturation with sodium or ammonium sulphate. Quantitative Estimation of the Total Albumin of the Plasma. — The albumins are most conveniently estimated by treating a care- fully measured and weighed amount of the plasma, after neutraliza- tion with acetic acid, with five times its volume of alcohol. After standing for twenty-four hours the solution is boiled for several minutes, and the resulting precipitate collected on a weighed filter, washed with hot alcohol, then with ether, dried at 115° C, weighed, CHEMICAL EXAMINATION OF THE BLOOD. 317 and incinerated. The weight of the ash is deducted from the weight of the precipitate. More accurate is the following method : a carefully measured and weighed amount of the plasma is treated with one-half its volume of a saturated solution of sodium chloride and a slight excess of tannic acid. In the resulting precipitate the nitrogen is then esti- mated according to Kjeldahl's method. When multiplied by 6.37 the corresponding amount of albumin is obtained. To remove all albumins from the blood, Cavazzani's method may be employed. To this end, 20-30 c.c. of blood are added to 200 c.c. of distilled water, and treated with five or six drops of a solution consisting of ten parts of acetic acid (sp. gr. 1.040) and one part of lactic acid. The mixture is boiled for about ten minutes, filtered, and the precipitate washed separately with hot water, and finally pressed in a piece of muslin. The filtrate and washings, which are practically colorless, are then concentrated to a small volume. Any traces of albumin which may still be present thus separate out and are filtered off. If too much of the acid solution has been added, the mixture may not clear on boiling. In that event a few crystals of sodium carbonate are added, when coagulation promptly occurs. On the other hand, it may at times be necessary to add a few drops more of the acid solution. The remaining constituents of the plasma are also found in the serum, and will be considered in that connection. The Serum. The serum results from the blood-plasma during the process of coagulation. It is most conveniently obtained by whipping blood immediately after being shed, whereby the greater portion of the fibrin is removed and the formation of large clots prevented. The corpuscles and smaller pieces of fibrin are separated by centrifuga- tion or by allowing the fluid to stand in the cold until sedimen- tation has occurred. The serum is then siphoned off and filtered. It thus appears as a slightly viscid, fairly transparent fluid of a light straw color, which presents a feebly alkaline reaction and a specific gravity varying between 1.026 and 1.029 in man. In its chemical composition serum differs from plasma principally in the presence of the fibrin ferment and in the absence of fibrinogen. In its place, however, traces of two other globulins, which arc not present in the plasma, arc found. One of these is termed fibrinoglobulin, and is thought to result during the formation of fibrin from fibrinogen. The .,ther ifl the BO-Called Cell-globulin, and i< supposedly referable to the decomposition of leucocytes during the process of coagulation. The remaining constituents are qualitatively the same in both fluids. Slight quantitative differences, however, exist. A portion of the calcium, magnesium, and phosphoric acid is thus eliminated 318 THE BLOOD. together with the fibrin, and accordingly lower values are found in the serum than in the plasma. An idea of the mineral constituents of the serum, and their quantitative relations, may be had from the accompanying table : Man. Potassium oxide 0.387 — 0.401 pro mille. Sodium oxide 4.290 " Chlorine 3.565—3.659 " Calcium oxide 0.155 " Magnesium oxide 0.101 " From this it will be seen that sodium in the form of the chloride largely predominates in the serum, while potassium occurs only in small amounts. This is exactly the reverse of what is seen in the morphological elements of the body, of which potassium compounds are the principal salts present. It is noteworthy, moreover, that the amount of sodium chloride is practically constant in the blood, no matter whether large quantities are ingested or the salt is given in only small amounts. During starvation even, or when the potassium salt is artificially substituted, the amount present in the blood remains practically constant. Apparently it occurs only in solution, and does not form an integral part of the albuminous molecule, as is the case with the phosphates of the blood. Of these, traces only are present in solution, while the greater portion is more or less intimately com- bined with the albumins. As the lecithins which are found in the blood also contain phosphorus, it follows that these figures, which are obtained by incinerating a given amount of serum or plasma and determining the phosphoric acid in the ash, are too high. In serum which had been freed from lecithin Sertoli and Mroczkowski found amounts varying between 0.02 and 0.09 pro mille, calculated as di- sodium phosphate. In the estimation of the sulphates we meet with still greater difficulties, as the sulphur of the albumins is included in the determination. The amount which is present in solution, however, is certainly very small. The iron which is at times met with in the serum is unquestionably derived from the leucocytes, and is an accidental constituent. The amount which may be obtained is always exceedingly small. Of other elements, traces of silicon, fluorine, copper, and man- ganese have at times been observed. The coloring-matter of the serum and plasma is supposedly due to a substance belonging to the class of lipochromes or luteins. The albumins of the serum which also occur in the plasma, viz., serum-albumin and serum-globulin, have been considered. Of the fibrinoglobulin which is formed during coagulation of the blood, and which is thought to result from decomposition of fibrinogen, comparatively little is known. It coagulates at 64° C, and appar- ently represents about one-third of the fibrinogen molecule. Of the so-called cell-globulin, still less is known, and both are found only in traces. More important is the presence of the fibrin ferment, CHEMICAL EXAMINATION OF THE BLOOD. 319 which is considered at this place, as it is usually regarded as an albuminous substance. The Fibrin Ferment. — The fibrin ferment, or thrombin of Alex- ander Schmidt, is thought to result from decomposition of the cellu- lar elements of the blood, and notably the leucocytes and blood- plates, in which it is supposedly present in the form of a pro- enzvme, the so-called prothrombin of Alexander Schmidt. As a matter of fact, it is possible to prevent the formation of the ferment by allowing blood to flow directly from the vessel into a solution of one of the neutral salts. In the circulating blood the ferment is manifestly not present, as its solution when injected into the blood- vessels of a living animal will cause almost instantaneous death from thrombosis. Isolation. — The isolation of the fibrin ferment is most conveni- ently accomplished in the following manner: taking the serum of the ox, the globulins are first precipitated by saturation with magnesium sulphate. The filtrate is then diluted with water, and treated while stirring with a very dilute solution of sodium hydrate until an abundant and flocculent precipitation of magnesium hydroxide has been brought about. This precipitate, which contains a large pro- portion of the ferment, is washed with water, pressed between filter- paper, and dissolved in water by neutralizing the solution with diluted acetic acid. The salts are then removed by dialysis, when the ferment can be precipitated by a suitable addition of acetic acid. Properties. — Of the nature of the product which can thus be ob- tained little is known. By some observers it is regarded as a globu- lin, while others class it as a nucleo-albumin. On digestion with pepsin it is said to yield a nuclein or a pseudonuclein. As is the case witli all ferments, its solutions are rendered inactive by exposure to a moderately high temperature (70°-75° C), while the various antiseptic substances which do not interfere with the activity of other ferments are likewise without effect upon the fibrin ferment. It- specific activity is manifested when brought into contact with fibrinogen, which is apparently decomposed by hydrolysis into fibrin and fibrinoglobulin. To elfect this change, however, the presence of a neutral -alt anil of a soluble calcium salt is essential. In their absence coagulation does not take place. According to Pekelharing, the fibrin ferment is a calcium compound of the pro-enzvme, and it is supposed that during the process of coagulation the calcium is transferred to the fibrinogen, whereby this is transformed in part into the insoluble calcium-containing fibrin. At the same time the ferment is retransformed into its pro-enzyme, which again combines with calcium to form the ferment ; this, in turn, deposits its calcium on the fibrinogen, and is thus changed back into the pro-enzyme, and BO on. Fibrin. — Fibrin is formed during the spontaneous coagulation of all albuminous solutions which contain fibrinogen and cellular ele- ment- that can give rise to the fibrin ferment. It is most conveni- 320 THE BLOOD. ently obtained by whipping freshly shed blood with a suitable instru- ment, when the fibrin is deposited as an elastic, stringy material, which may be freed from adhering corpuscles by thorough washing and kneading in running water. Such fibrin, however, is still con- taminated with serum-globulin and certain phosphorus-containing substances which have resulted from the decomposition of leucocytes. The serum-globulin may be removed by separate washing and knead- ing in a 5 per cent, solution of common salt ; but the other products, as well as the remains of the corpuscles of the blood, can scarcely be removed. To obtain pure fibrin, therefore, it is necessary to start with filtered plasma or with filtered transudates, which are beaten with a piece of whalebone, after adding a little serum, if the fluid is not spontaneously coagulable. The resulting material is washed with water, then with a 5 per cent, solution of sodium chloride, and finally extracted with alcohol and ether. The fibrin then appears as a white stringy substance, which is somewhat elastic, but is easily rendered brittle on contact with alcohol or on warming the substance in water to a temperature of 75° C. It is closely related to the coagulated albumins, and ac- cordingly is soluble only with difficulty. It is questionable, more- over, whether solution of the substance can be accomplished without causing its decomposition. If fibrin is thus placed in a 5 to 10 per cent, solution of sodium chloride, or a 6 per cent, solution of sodium nitrate, and kept at a temperature of 40° C, it first swells up and gradually disappears as such. In its place two globulins or related substances are then said to be found. This transformation, accord- ing to some observers, is referable to adherent bacterial enzymes. In dilute alkalies and acids it likewise dissolves. Stronger acids, as also the proteolytic ferments, dissolve the fibrin, but at the same time cause its transformation into acid albumin and albumoses. In water, alcohol, and ether it is entirely insoluble. The elementary analysis of fibrin gives 52.68 parts of carbon, 6.83 of hydrogen, 16.41 of nitrogen, 1.1 of sulphur, and 22.48 of oxygen. It is to be noted, further, that in addition to these elements calcium is constantly present, and, as has been seen, its formation is largely dependent upon the presence of a soluble calcium salt. According to Lilienfeld, the fibrin is not formed directly during the decomposition of fibrinogen, but in its stead we obtain another substance, thrombosin, which as an alkali compound is soluble in water, but immediately combines with calcium, and then sejuarates out as calcium thrombosin or insoluble fibrin. This idea, however, is not generally accepted, and it is thought to be more likely, as Pekelharing suggests, that the calcium merely transforms the pro- enzyme into the fibrin ferment proper, and is then mechanically carried down during the separation of the fibrin. The amount of fibrin which may be obtained from the blood, not- withstanding its bulk, does not exceed 0.1-0.4 per cent. CHEMICAL EXAMINATION OF THE BLOOD. 321 Estimation. — In order to determine the amount of fibrin in a given volume of blood, from 30 to 40 c.c. are placed in a previously- weighed I (Laker, which is closed with an India-rubber cap. Through the centre of this passes a piece of whalebone that is firmly fixed and provided with a rudder-like end, which dips into the blood. This is now defibrinated by beating with the whalebone paddle, when the beaker is again weighed with its contents. The differ- ence, as compared with the first weight, indicates the weight of the blood. The beaker is then filled with water and the mixture again beaten. The fibrin is allowed to settle, and after being washed by decantation with normal salt solution it is collected on a filter of known weight. On this it is further washed with normal salt solu- tion until free from coloring-matter; it is then extracted with boiling alcohol, then with ether, and finally dried at 115° C. and weighed. Other albumins in addition to those already considered are not found in normal serum. Under pathological conditions, however, nueleo-albumins and deutero-albumoses may be encountered. They will be considered in future chapters. The remaining solids which are found in both plasma and serum are, as has been pointed out, present in only very small amounts. The most important of these is glucose. Its amount varies between 1 and 1.5 pro mille, and is but little influenced by the character of the food unless a large excess of carbohvdrates has been ingested. In such an event the amount may increase to 3 pro mille, or even higher, but it then appears also in the urine, whereby a further increase is prevented. Larger amounts, such as 9 pro mille, are found only under pathological conditions. It is interesting to note that after blood has been shed the glucose rapidly diminishes in amount. This phenomenon is thought by some to be due to the action of a special ferment, the c/lucolytic ferment of Lepine. It is supposedly derived from the leucocytes, and, according to Lepine, is eliminated into the blood by the pan- ereas. Arthus and others regard this process of glucolysis as a post-mortem change, but there is reason to suppose that the activity of the glueolytic ferment is exercised also during the life of the animal, and is possibly of fundamental importance in the metabo- lism of glycogen. Whether Lepine's ferment is identical with another ferment which was found in the blood, and has been studied by Rdhmann and Bial, and which is said to transform starch and glycogen into glucose, remains to be seen. In addition to glucose, another reducing substance is found in the blood, which to a certain degree is fermentable and is soluble in ether. From the- researches of P. .Mayer, it appears that this sub- stance is a conjugate glucuronate. The presence of jecorin, on the other hand, which has repeatedly been reported, is doubtful. . 1 nimalgum has also been found in small amounts (0.01 5 per cent.). Glycogen. — Glycogen is constantly present in normal blood. Its 21 322 THE BLOOD. amount, however, is subject to great variations. As a general rule, traces only are found, but it may increase at times to 1.56 per cent., as calculated for the blood as a whole. Larger amounts are seen under pathological conditions. Fat is normally found to the extent of from 0.2 to 0.3 per cent., but may be greatly increased by the ingestion of much fatty food, as also in various pathological conditions. Urea is likewise found in only very small amounts under normal conditions (0.016 to 0.020 per cent.), while in disease much greater quantities may be encountered. Ammonia is said to be present in normal blood to the amount of 0.001 per cent. The further occurrence in the blood of soaps, cholesterin, and lecithins, as also of uric acid, kreatin, carbaminic acid, paralactic acid, hippuric acid, etc., has been mentioned. All these bodies are found in only extremely small amounts, and need not be con- sidered at this place. The pathological constituents of blood, such as leucin, tyrosin, acetone, bilirubin, etc., will be considered in future chapters. Of gases, finally, we find in the plasma and serum small amounts -of nitrogen and oxygen, which are present simply in solution, and somewhat larger amounts of carbon dioxide, which in part at least is more or less firmly combined with albumins. The Leucocytes. The morphological characteristics and general chemical composi- tion of the leucocytes have already been considered (see pages 303 and 306). At this place I wish merely to draw attention to one substance which, according to Lilienfeld, is found in special abundance in the nuclei of these bodies, and which has been termed nucleohiston. Nucleohiston. — This substance was first isolated by Kossel and Lilienfeld from the thymus gland of the calf, but has since been obtained from the leucocytes of the lymph-glands, as also from the splenic cells, the testicular cells, the spermatozoa, and from the epithelial lining of the small intestine. In all probability it repre- sents an important constituent of all cellular nuclei. Isolation. — Nucleohiston is most conveniently obtained from the leucocytes of the thymus gland. To this end, the gland is carefully dissected free from fat and all larger bloodvessels, and finely hashed. This mass is extracted with cold water, passed through muslin, and centrifugalized. The aqueous extract is further filtered, and the nucleohiston precipitated by the careful addition of dilute acetic acid. It is filtered off, dissolved in water with the aid of a small amount of a dilute solution of sodium carbonate, reprecipitated with acetic acid, and purified by a repetition of this process. It is then washed with acetic water, extracted with alcohol and ether, and finally dried at a temperature of from 110° to 115° C. CHEMICAL EXAMINATION OF THE BLOOD. 323 Properties. — Thus obtained, nucleohiston represents a snowy-white, fine powder, which is insoluble in benzol, alcohol, chloroform, methyl alcohol, ether, and acetic acid, but is soluble in water, glacial acetic acid, concentrated nitric acid, and hydrochloric acid, in solutions of -odium carbonate, sodium hydrate, ammonia, and, when freshly pre- cipitated, also in solutions of sodium chloride and magnesium sul- phate, especially in the presence of a little acetic acid. On boiling with water, or on treating with baryta water or dilute hydrochloric acid, nucleohiston is decomposed into a nuclear nuclein, the so-called leuconuclein, and an albumose-like substance, liiston, which Kossel first obtained from the nuclei of the red corpuscles of the goose. It may therefore be regarded as a nucleo-albumin, but differs from most of the other representatives of this group in the large amount of phosphorus — 3.025 percent. — which it contains. The histon radicle possesses marked basic properties, and readily combines with acids. From its acid solutions it is precipitated by ammonia, and it is insoluble in an excess of the reagent. The leuconuclein, on the other hand, has markedly acid properties. On treating with an alcoholic alkali solution, it is decomposed into an albuminous sub- stance and a nucleinic acid, which is termed thymonueleinic acid. On further decomposition this yields an acid phosphoric radicle, and nucleinic bases, among which adenin and guanin prevail. On boiling with water the acid radicle is transformed into so-called thyminic acid, which on treating with strong sulphuric acid gives rise to thymin. If, on the other hand, the nucleinic acid is treated with dilute boiling mineral acids, or with superheated steam, adenin, thymin, and another basic substance, cytosin, are obtained, as also ammonia, formic acid, phosphoric acid, and lsevulinic acid; the presence of the latter suggests that in the nucleinic acid radicle a carbohydrate group also exists. The nucleohiston, as such, may be regarded as an acid salt, as it i- capable of binding a certain amount of calcium and sodium. Its elementary analysis lias given the following results : carbon, 48.46 ; hydrogen, 7; nitrogen, 16.86; phosphorus, 3.025 ; sulphur, 0.701; and oxygen, 23.95 per cent. The chemical analysis which Lilienfeld made of the leucocytes of the thymus gland, and which probably expresses the constitution of all leucocytes, gave the following results : Water 88.51 per cent. ids 11.49 " " Ubumine 1.76 '' " Leuconuclein 68.78 " " Burton 8.67 " " Lecithin 7.51 " " 4.02 " " Cholecterin 4.40 " " Glycogen 0.80 " " Nuclein baset ai -ilver salts 15.17 " " 1 .ill phosphorus ."..01 " " Total nitrogen 15.03 " " 324 THE BLOOD. Of albumins proper, there are said to be present in the leucocytes the so-called cell-globulins, of which Halliburton recognizes two — one coagulating at 50° C, the other at 73° C. — and one of which is by some thought to be identical with the fibrin ferment ; then serum-albumin, and a mucinous body, the so-called hyalin sub- stance of Rovida. These bodies, however, represent only a very small portion of the solids of the leucocytes, as is seen from Lilien- felcl's table, and it is doubtful indeed whether their true chemical nature has been sufficiently established. The Plaques. Of the chemical composition of the plaques little is known. According to Lilienfeld, they contain an albuminous substance and a nuclein ; for on treatment with artificial gastric juice they can be differentiated into a homogeneous portion, which is subsequently dissolved, and an insoluble granular portion, which gives the vari- ous reactions of .the nucleins, and may be shown to contain phos- phorus. In the plaques the albumin is probably combined with the nuclein to form a nucleo-albumin, which may be identical with the nucleohiston, which has just been described. According to Lilien- feld, indeed the plaques must be regarded as nuclear derivatives, and he has accordingly termed them the nuclein platelets of the blood. The Coagulation of the Blood. According to modern ideas, the coagulation of the blood is ref- erable in the first instance to the decomposition of fibrinogen into fibrin and fibrinoglobulin. This decomposition, is thought to be effected through the activity of a special ferment, the fibrin ferment, which is supposedly contained in the cellular ele- ments of the blood in the form of a pro-enzyme, and is set free during the death of these elements as a calcium compound of the pro-enzyme. According to Pekelharing, the ferment then trans- fers its calcium to the fibrinogen, which is thus decomposed, and is itself reconverted into the pro-enzyme, and as such imme- diately combines with calcium, which is again transferred to an- other portion of fibrinogen, and so on. This theory is in all like- lihood correct in its general features, but has probably not yet assumed its ultimate form. That other substances, besides fibrin- ogen, a small amount of a neutral and a soluble calcium salt, and the fibrin ferment, need not be present to effect coagulation, is cer- tainly beyond dispute. But, on the other hand, we must admit that our knowledge of the fibrin ferment itself and the mode of action of the calcium salt is still imperfect. According to Halliburton, the fibrin ferment is a cell-globulin and is contained as such in the bodies of the leucocytes. Pekelharing, on the other hand, regards the ferment as a calcium compound of a nucleo-albumin. Lilienfeld, CHEMICAL EXAMINATION OF THE BLOOD. 325 while not denying the existence of the ferment, regards it as a de- composition-product which is formed during the coagulation of the blood According to his ideas, it cannot very well be concerned in this process, and he assumes that the coagulation of the blood is normally referable to the acid radicle of the nucleohiston, which, as we have seen, is present in the nuclei of the leucocytes in large amounts. _ , The leuconuclein, however, does not cause the coagulation ot the blood at once, but first effects the decomposition of fibrinogen with the formation of thrombosin and an albumose-like body, which may be a derivative of Hammarsten's fibrinoglobulin. The thrombosin is soluble in dilute alkalies. Upon the addition of a soluble calcium salt, however, such solutions coagulate, and the thrombosin is thus transformed into the insoluble fibrin. Fibrin, according to this theory, is thus a calcium compound of thrombosin. The active principle of the leuconuclein is unquestionably its nucleinic acid radicle, and, as a matter of fact, the same changes can be brought about by using this directly. This explains the observation which has been repeatedly made, that the coagulation of albuminous fluids which are not spontaneously coagulable can also be effected by the addition of almost any cellular formation, such as yeast cells', various bacteria arid moulds, spermatozoa and pro- tozoa, etc.—/. e., bodies which all contain fairly large amounts of nuclei ns. It is to be noted, moreover, that the intensity of action of these various substances is intimately dependent upon the amount of nuclein present, and we accordingly find that of aH the cellular elements of the body the spermatozoa are the most active. While this portion of Lilienfeld's theory is practically unassailable, some doubt has been raised as to the existence of his soluble thrombosin, and Hammarsten thus states that the thrombosin is no decomposi- tion-product of fibrinogen, but fibrinogen itself which has been pre- cipitated with nucleinic acid. He has shown, moreover, that the formation of fibrin can take place in the absence of calcium salts, providing that a sufficient amount of the fibrin ferment is present. If we are to accept Lilienfeld's theory then, we may imagine that through the influence of the soluble calcium salt the nucleohiston is decomposed, and that the leuconuclein is thus enabled to exercise its special activity. Future researches, however, will be necessary to aettle this question definitely, and to determine whether the coagu- lation of the blood is normally referable only to the action of nucleins, and whether the fibrin* ferment is in reality a product of coagulation. The question, why the blood does not coagulate within the vessels of the body where fibrinogenic material is available, and where leuco- cytes no doubt undergo degeneration, has been variously answered. On the one hand, it is stated thai the integrity of the endothelial lining i- hen- of prime importance, ami thai coagulation will occur whenever this ie impaired. As a matter of fact, we find that coagu- 326 THE BLOOD. lation takes place after ligation of an artery, and that the coagulum invariably extends as far as the next collateral vessel. That the nutrition of the intima is here seriously interfered with cannot be doubted. Similarly we find a more or less extensive thrombosis in atheromatous vessels, not to speak of the process of clotting in association with wounds. In such cases it appears that owing to the lesion of the endothelial coat an aggregation of leucocytes occurs in the affected parts, which in turn results in the death and disso- lution of many of the cells at these places. Contact with a foreign substance, and as such diseased or dying endothelial cells must be viewed, in some manner brings about the early dissolution of the leucocytes, and we find accordingly that on introducing a silk thread into the bloodvessel of a living animal coagulation takes place around the foreign body. Similarly, it may be observed that when blood is received in a vessel, the walls of which have been carefully lubricated with vaselin, coagulation is greatly delayed, but may be brought about at once on introducing bits of foreign material, such as dust or ashes and the like. While we have thus seen that coagulation will occur within the living body whenever foreign material is present, we have not as yet offered an explanation for the non-occurrence of coagulation when such influences are not at work. That leucocytes are constantly broken down in the living organism cannot be questioned. It is possible, however, that the amount of nucleohiston, or fibrin ferment, as the case may be, which is thus formed, is too small at any one time to exert its special activity. On the other hand, it is conceiv- able that such decomposition may take place within certain organs of the body, such as the spleen and the liver, and that the nucle- histon, which is here set free, is again utilized in the construction of new cells. The nucleohiston, moreover, contains another radicle, the albumose-like histon, which has exactly the opposite effect upon coagulation. For whereas the injection of leuconuclein into the cir- culation of living animals rapidly brings about the death of the animal from thrombosis, histon injections render the blood refractory to coagulation. Such plasma is termed histon-plasma, and it is to be noted that this coagulation can later be brought about only through the addition of the leuconuclein or nucleinic acid. It is thus possi- ble that during the dissolution of the leucocytes in the living animal the leuconuclein is in some manner prevented from exercising its special activity, while the histon further prevents coagulation in itself. The primary factor, however, upon which the occurrence or non-occurrence of coagulation in the living body probably depends, is the extent to which the leucocytes are undergoing decomposition. Rapidity of Coagulation. — The rapidity with which coagu- lation of the blood occurs after being shed varies with different animals, with the districts from which the blood is taken, etc. In birds it thus occurs after one and a half minutes ; in man after from three to four minutes ; while in cold-blooded animals CHEMICAL EXAMINATION OF THE BLOOD. 327 it begins onlv after a quarter of an hour. In the horse, in which coagulation is Likewise delayed, the corpuscles of the blood have time to settle and form two distinct layers— the red at the bottom and the white on top, where they appear as a grayish-white zone, and con- stitute the so-called crusta phhgistica or infiammatoria. Above this we then see the plasma, which has undergone coagulation, and on top of it the serum that has been squeezed out from the clot. The same phenomenon may be observed in human blood when cooled to about 0° C, and from the extent of the crusta phlogistica in such blood the older physicians were wont to draw prognostic conclusions as to the course of the disease. The rapidity of coagulation can be artificially increased and diminished. Bv beating the blood, by increasing its temperature a little beyond that of the body, and by diluting with water it is increased ;" while exposure to a low temperature, the presence of much carbon dioxide in the blood, the careful lubrication of the vessel with vaselin or similar unguents, cause a retardation of coagu- lation. Its prevention finally may be brought about through influ- ences already mentioned, viz., by salting with the neutral salts, following the previous injection into the body of albumoses or of histon, of diastatic ferments, of extracts of the mouth-parts of the leech, after elimination of the intestinal bloodvessels by ligation, etc. The Red Corpuscles. The red corpuscles of the blood, as has been mentioned, owe their color to the presence of haemoglobin or its oxygen compound, oxy- hemoglobin. This may be extracted by diluting with water, by alternate freezing and thawing, by shaking with ether, chloroform, etc. The blood is thereby rendered lake-colored, and on microscopi- cal examination it will be observed that instead of the original cor- puscles, BO-called blood-shadows are now found. These are colorless ring-like bodies, and constitute the stroma of the red corpuscles. In the circulating blood the dissolution of the haemoglobin is pre- vented by the presence of large amounts of sodium chloride. Such blood is -aid to be hyperi&otonic — i. e., it contains more sodium chloride than is necessary to prevent the dissolution of the coloring- matter from its corpuscles. Within the corpuscles the haemoglobin i-, however, supposedly not present in the free state, but in com- bination with some other substance, such as lecithin; and Hoppe-: Sevier accordingly distinguishes between the so-called arterin and phlebin, which represents the lecithin compound of oxyhemoglobin and haemoglobin respectively. A- has been mentioned, the red corpuscles represent nearly one- half of the liquid blood. They contain aboul o7.7 per cent, of water and 10.5 per cent, of oxyhemoglobin, while tin' constituents of the stroma inclusive of mineral salts amounl only to about 1.9 per cent. A.mone these constituents Halliburton's cell-globulin is said 328 THE BLOOD. to be most abundant ; in addition we find traces of lecithin, choles- terin, and nucleo-albumin, while serum-albumin and albumoses are apparently absent. In the nucleated red corpuscles of birds we further meet with the integral constituents of the nuclei, among which Lilienfeld's nucleohiston probably always prevails. Its basic constituent, histon, was first discovered by Kossel in the red cor- puscles of the goose. An idea of the mineral constituents of the red corpuscles, viz., their stroma, may be had from the following table, which is taken from C. Schmidt, and calculated for 100 parts of the moist cor- puscles. Potassium chloride 3.68 Sodium chloride traces. Potassium sulphate 0.13 Potassium phosjmate 2.34 Sodium phosphate 0.63 Calcium phosphate 0.09 Magnesium phosphate 0.06 The iron of the haemoglobin is not included in this table ; in man it varies between 0.506 and 0.537 pro mille. In addition traces of copper are not infrequently met with, and, as will be seen later, this element in some of the invertebrate animals apparently takes the place of iron in the coloring-matter of the blood. Isolation of the Red Corpuscles of the Blood. — To isolate the red corpuscles, the blood is defibrinated by beating, diluted with ten times its volume of a 1 per cent, solution of sodium chloride, and passed through a muslin filter. On subsequent centrifugation and repeated washing with the salt solution, they are then freed from the serum, and may now be collected on a paper filter, after the previous addition of a large amount of alcohol. Any fats, lecithins, or cholesterins that may be present are extracted with warm alcohol and ether, when the corpuscles can be dried and weighed. To isolate the stromata, the mass of corpuscles, when freed from serum, is shaken with five or six times its volume of water and a small amount of ether. The mixture is then centrifugalized, and is thus separated from the leucocytes. The stromata are now precipitated by adding a few drops of a 1 per cent, solution of acid sodium phosphate, until the liquid has almost assumed the consistence of the original blood. They are then collected on a filter, quickly washed with water, and may now be dissolved in a 5 per cent, solu- tion of magnesium sulphate. Haemoglobin and Its Derivatives. Haemoglobin. — The hsemoglobin is the coloring-matter of the red corpuscles, and is present in these in combination with an- other body, which may be a lecithin, as the phlebin of Hoppe- Seyler, while the corresponding compound of oxyhemoglobin is termed arterin. Of the nature of these compounds, however, CHEMICAL EXAMINATION OF THE BLOOD. 329 but little is known, and it has not even been definitely ascertained that the pairling of the. coloring-matter is really a lecithin. Hemoglobin or its oxy-compound occurs widely distributed in the animal world, and is found not only in the vertebrates, but also in many of the invertebrates. But while in the former it is con- tained in definite cellular elements of the blood it may also occur as such among certain invertebrate animals. Closely related to it is the so-called oxyhcemocyanin, which is found in certain arthropods and molluscs, and in which the iron is apparently replaced by copper Then again we find among invertebrate animals various violet and purplish-red pigments, the so-called floridins, which are likewise to be classed with haemoglobin, and as we have previously seen a genetic relationship apparently also exists between haemo- globin and the chlorophyl of plants. These various pigments are collectively spoken of as respiratory pigments, as they are intimately concerned" in the transportation of the oxygen of the air to the various tissues of the body, and in the removal of the carbon dioxide which results as a product of cellular metabolism. Outside of the blood haemoglobin is found also in striped and un- striped muscle-tissues, and under pathological conditions it may appear in the urine as such. Different haemoglobins apparently exist It is hence impossible to give a definite formula which ex- presses the constitution of all. An idea of their quantitative ele- mentary composition may be had from the accompanying table : Carbon. Hydrogen. Nitrogen. Oxygen. Sulphur. Phosphorus. Iron. Horse 54.87 6.97 17.31 19.730 0.650 . . 0.470 Sof ..... 54.57 7.22 16.38 20.930 0.568 . . 0.336 P& 54.71 7.38 17.43 19.602 0.479 . . 0.399 Guinea-pi" . . • -54.12 7.36 16.78 20.680 0.580 . . 0.480 Sau"rrel . . .54.09 7.39 16.09 21.440 0.400 . . 0.590 hquirrei o. 0 540 Q 770 0 430 aSkea' : : : : :ili? f.lS &3 22.500 0.857 0.197 0.335 The size of the hamoglobin molecule is, like that of all albu- minous substances, very Targe. For that of dog's blood Hiifner obtained the figure 14,129, which would correspond to the formula C II X, FeS3Owl. It thus contains three atoms of sulphur for one atom of iron, while the haemoglobin of the horse and pig has only two atoms of sulphur for one atom of iron. Of interest further is the presence of phosphorus in the hasmoglobin of the goose and chicken. Whether this forms an integral component of the bsemoglobiii molecule, however, is questionable, and it is quite possible thai its presence is owing to a contamination of the coloring- matter with nucleinic acid derived from the nuclei of the red corpuscles. Structurally, haemoglobin must be regarded as a proteid, viz., as a compound of an albuminous radicle with another complex organic radicle. This other radicle U here an iron-containing pigment, which may be separated from its albuminous pairling, and is termed 330 THE BLOOD. hcemochromogen (see below), while the albuminous substance is known as globin. These two substances are apparently united in the haemoglobin molecule, through an additional radicle, which is as yet unknown. Globin, is closely related to histon, and, like it, presents the fol- lowing characteristic reactions : it is precipitated by ammonia from its solutions in dilute hydrochloric acid, and is insoluble in an excess of the reagent. With concentrated nitric acid it is thrown down in the cold, but not from its heated solutions. Under certain condi- tions it can be coagulated on boiling, but, unlike the other coagula- ble albumins, its coagulate is readily soluble in acids. It contains 54.97 per cent, of carbon, 7.2 per cent, of hydrogen, 16.89 per cent, of nitrogen, and 0.42 per cent, of sulphur. The amount of globin which can be obtained from the haemoglobin molecule is quite large, and according to Schultz amounts to 86.5 per cent., while 4.2 per cent, only is represented by the pigment itself. Isolation. — A solution of oxyhemoglobin in water, prepared at a temperature of 40° C, is treated with dilute hydrochloric acid until the red color changes to brown. This mixture is extracted with 80 per cent, alcohol (one-fifth volume) and ether (one-half volume) until the ether takes up no more coloring-matter. The resulting aqueous-alcoholic solution is precipitated with ammonia, filtered, and the precipitate dissolved in very dilute acetic acid. On filtering, the globin is again precipitated with ammonia and col- lected on a silk filter. After washing with absolute alcohol, then with water, again with alcohol, and finally with ether, the substance is dried first in the air and then at a temperature of 100° C. The resulting material constitutes pure globin, as a yellowish loose powder, which is not especially hygroscopic. Haemochromogen. — The isolation of haemochromogen is rather difficult, owing to the avidity with which it combines with oxygen to form hcematin in alkaline solution. Hoppe-Seyler, however, suc- ceeded in obtaining the substance in crystalline form, by heating haemoglobin with sodium hydrate solution in an atmosphere of hy- drogen. In acid solutions haemochromogen gradually loses its iron and is converted into hcematoporphyrin. In alkaline solution it presents a beautiful cherry-red color, and on spectroscopic examina- tion gives two bands of absorption. One of these is very intense, and located between D and E, nearer D, while the other is not so dark, but wider, and found about E and extending beyond b. To demonstrate the spectrum of hsemochromogen, bloody fluid is mixed with a solution of sodium hydrate, when the resulting haematin is reduced with ammonium sulphide, or Stokes reagent, viz., an am- moniacal solution of ferrous tartrate, or stannous chloride. The haemochromogen radicle, as has been stated, represents the pigmented group of the haemoglobin molecule, and to its presence the power of haemoglobin to combine with oxygen, carbon dioxide, and other gases, is unquestionably due. CHEMICAL EXAMINATION OF THE BLOOD. 331 Hemoglobin itself can be obtained in crystalline form, and is characterized by the great resistance which it offers to putrefaction and tryptic decomposition. It is stated that even after the lapse of years decomposed blood contains its haemoglobin as such, and that on shaking with air it may again be transformed into pure oxy- hemoglobin. Its solutions present a beautiful purplish-red color, and on spectroscopic examination gives rise to a single band of absorption, which lies between D and E and extends slightly to the left beyond D. This is most conveniently shown by taking a solu- tion of oxyhemoglobin, and reducing this with ammonium sulphide or Stokes' fluid, as directed above. Most important is the avidity with which haemoglobin combines with various gases, and upon this characteristic indeed its chief function, as a respiratory pigment, is based. This property, as has been stated, is referable to the chromogen radicle, and more partic- ularly to the iron, which it contains. Every atom of this is capable of combining with one molecule of oxygen or of carbon dioxide, and in this form largely the oxygen of the air is carried to the vari- ous tissues of the body, and the carbon dioxide removed. In the circulating blood we accordingly find only relatively small amounts of free haemoglobin, and in arterial blood its oxy-compound is almost exclusively encountered. The amount of haemoglobin which is contained in human blood, either as such or in combination with oxygen or carbon dioxide, is about 14 per cent., but subject to certain variations, even in health, while in disease still greater deviations from the average normal amount are observed. A great diminution may here occur, and is most marked in chlorosis and pernicious anaemia, in which the per- centage may fall as low as 2.35. As the isolation of the haemoglobin from the blood resolves itself into the isolation of its oxy-compound, this will be considered together with its quantitative estimation under that heading. Oxyhaemoglobin. — Oxyhaemoglobin is the oxy-compound of haemo- globin, and differs from its mother-substance in containing two at'. ni~ more of oxygen, which are bound to the one atom of iron, than are present in the haemochromogen radicle. In this manner, however, the haemochromogen is converted into haematin, and we may therefore say that oxyhaemoglobin, in contradistinction to haemoglobin, consists of the globin radicle united by some unknown group to a haematin radicle (see also page 333). Haematin. — In accordance with the above considerations, we find that on decomposition of oxyhaemoglobin haematin is obtained i r i - 1 * -; i « I of haemochromogen. This latter, indeed, is at once trans- formed into haematin on exposure to oxygen, and, as we have seen, the haematin is correspondingly reconverted into haemochromogen by treating with reducing agents. The decomposition of oxynaemo- globin with th<' formation of* haematin can be readily effected by Beating its solutions to a temperature of 80" C, by treating with 332 THE BLOOD. dilute mineral acids, or with stronger solutions of the alkaline hydrates, as also by peptic or tryptic digestion. To obtain the sub- stance in a pure state, however, it is best to start with its hydro- chlorate anhydride, hcemin, which can be readily obtained in crystal- line form. To this end, oxyhemoglobin is treated with a trace of sodium chloride and dissolved in glacial acetic acid. On heating, the hsemin is precipitated in the form of very characteristic, drawn- out, rhombic platelets, which are insoluble in water, alcohol, and ether, with difficulty so in glacial acetic acid and in dilute mineral acids, but are readily soluble in dilute alkaline solutions and acid alcohol. The crystals are collected on a filter, washed with alcohol and ether, and are thus obtained in pure form. To prepare the hsematin, the hsemin is dissolved in a dilute solution of sodium hydrate and supersaturated with dilute hydrochloric acid. The sub- stance is thus precipitated in the form of brownish flakes, which are washed free from chlorides and dried at 120° C. Hsematin, in con- tradistinction to oxyhemoglobin and hsemin, is non-crystallizable. Its solubility is essentially the same as that of hsemin. In acid solu- tion both hsematin and hsemin show a well-defined spectral band "between C and D. Between D and F a second band is seen, which is much wider but less sharply defined than the first. By diluting the solution this band may be resolved into three bands, of which one is located between b and F, near F ; another, between D and E, nearer E ; and a third faint band between D and E, nearer D. As a rule, however, only two bands are seen. The alkaline solutions of hsematin are distinctly dichrotic and give only one band of absorp- tion, the greater portion of which lies between C and D, and extends slightly beyond D. On careful oxidation hsematin may give rise to an iron-containing body and dibasic hsematinic acid, C8H10O5, which can be further transformed into the tribasic acid, C8H10O6. Of the nature of these substances, however, but little is known. Of greater interest is the fact that on treatment with concentrated sulphuric acid, with hydro- chloric acid, or with glacial acetic acid and hydrobroinic acid, hsematin as well as hsemin is freed from iron, and on the subsequent addition of water is transformed into hsematoporphyrin, C32H36N406, which is isomeric with bilirubin (see below). Hsematin is thus a decomposition-product of oxyhsemoglobin, and is not found as such in the circulating blood. It is said to occur in the urine, however, in cases . of poisoning with arsenious hydride. In the stools it is found after hemorrhages into the stomach or the upper portion of the small intestine, and also after the ingestion of large amounts of red meats. In such cases, of course, its origin is referable to the decomposition of hsemoglobin through the agency of the gastric and pancreatic juices. Isolation of Oxyhsemoglobin. — Oxyhsemoglobin in crystalline form is best obtained from the red corpuscles of the horse or dog, according to the following method : the blood is first rendered CHEMICAL EXAMINATION OF THE BLOOD. 333 uncoacmlable by treating with ammonium oxalate (0.06-0.1 per cent.),°and is then centrifugalized to effect the separation of the cor- puscles. This mass, when freed from plasma by siphonage, is treated with twice its volume of water and placed on ice. The resulting fluid is now mixed with an equal volume of a saturated solution oi ammonium sulphate that has likewise been cooled to a low tempera- ture. This mixture is also placed on ice until the precipitate of globulins, which is referable to remaining plasma, has settled. On filtering in the refrigerator a perfectly clear dark-red filtrate is obtained, which contains the greater portion of the coloring-matter. If now the solution is brought to the temperature of the room, the separation of crystalline oxyhemoglobin begins, and may be hastened, if necessary, by the further addition of a small amount of the ammonium sulphate solution. After a few days the process is completed, when the crystals are filtered off through a Biichner filter bv the aid of a suction pump, and are partially freed from the mother-liquor bv pressing between filter-paper. The substance is then purified by recrystallization. To this end, the crystalline mass is dissolved in water, reprecipitated by the addition of an equal volume of the ammonium sulphate solution, and so on, until the required degree of purity has been attained. Adhering ammonium sulphate is finally removed mechanically. To preserve the sub- stance, it is dried in a vacuum or at a low temperature over sulphuric acid, and is then quite stable. The ease with which oxyhemoglobin can be brought to crystal- lization differs with different animals. In guinea-pigs, squirrels, and rats it is most pronounced ; and it is here only necessary to mix a few drops of the blood with an equal amount of water, when the process may be directly observed with the microscope. Human blood, as also that of the pig and the ox, is much more refractory, and is not well adapted for the preparation of the pigment in its crystalline state. The form of the crystals varies in different animals. We thus find hexagonal platelets in the squirrel, rhombic tetrahedra in the guinea-pig and various birds, rhombic needles in man, etc. In" its chemical behavior oxyhemoglobin manifests its albuminous nature. It is thus coagulated on boiling, and on treating with absolute alcohol and most of the salts of the heavy metals. It is to be noted, however, that the process of coagulation is associated with the decomposition of the compound into hematin and globin, which latter is precipitated in its coagulated state. From its solutions the substance IS thrown down by half-saturation with ammonium Sulphate. On reduction with ammonium sulphide, with Stokes' reagent, or during the process of putrefaction, it is transformed into haemoglobin. Other reducing agents, such as sodium hydro- Bulphite, give rise to the formation of so-called pseudohaemoglobin, which apparently Btanda midway between oxyhemoglobin and hemoglobin in containing less oxygen than the former, but more 334 THE BLOOD. than the latter. Its spectrum, however, is the same as that of the completely reduced haemoglobin. In sufficiently dilute solution oxyhemoglobin shows two bands of absorption between D and E. The one to the left, which is not so wide as the other, but darker and more sharply denned, borders on D, while the second lies at E. The Quantitative Estimation of Oxyhemoglobin. — This is best accomplished by Hoppe-Seyler's method, which is based upon the comparison of a given amount of diluted blood with a stand- ard solution of crystallized oxyhemoglobin. This solution is prepared by dissolving 2 grammes of the pure coloring-matter in 50 c.c. of distilled water. The oxyhemoglobin is then transformed into carbon monoxide hemoglobin by passing a current of the gas through the solution to saturation. It is then stored in drawn- out and sealed glass tubes, such that each tube contains about 6 c.c. The contents of each tube, when diluted with ten times its volume of water, will then represent a 0.2 per cent, solution of the oxyhemoglobin. A carefully measured or weighed amount of blood, not exceed- ing 0.5 c.c, is now diluted with water that has been saturated with carbon monoxide to exactly 5 c.c. A small drop of a very dilute solution of sodium hydrate is added if necessary to remove any turbidity that may exist. This solution is further saturated with car- bon monoxide, and freed from fibrin by filtration. The filtrate should measure exactly 4 c.c. The comparison of the two solutions, and the further dilution of the blood with carbon monoxide water then takes place in the so-called double pipette of Hoppe-Seyler. The color of the two solutions is here equalized, and the amount of hemoglobin present in the specimen of blood calculated from the degree of dilution. Example. — Suppose that we started with 0.5 gramme of blood, and that the standard solution contained 0.002 gramme of oxyhemoglo- bin in the cubic centimeter. The 4 c.c. of the diluted and filtered blood are further diluted in the pipette to 22 c.c, which corresponds to a total solution of 27.5 c.c. for the total 5 c.c. of the first dilution. In these 27.5 c.c, which represent the original 0.5 gramme of blood, there will consequently be 27.5 X 0.002 = 0.0550 gramme of hemoglobin. The percentage will accordingly be 11 per cent. In the clinical laboratory other forms of apparatus are in use, such as the hosmometer of Fleischl and the hcemoglobinometer of G-owers. In the first the color of the diluted blood is compared with that of a glass wedge that has been stained with the golden purple of Cassius. In the second a standard solution of carmin and picric acid is employed. But as these colors do not represent the exact shade of oxyhemoglobin, the results must of necessity be less accurate. For clinical purposes, however, these methods are sufficiently exact. The spectro-photometric determination of the blood coloring- CHEMICAL EXAMINATION OF THE BLOOD. 335 mutter is not described at this place. It is undoubtedly the most exact, but necessitates the use of costly apparatus, which will be found in only few laboratories. It has been pointed out that haemoglobin is characterized by the readiness with which it combines with certain gases, and we have just considered the most important of these compounds. Other compounds of this character are carbon dioxide haemoglobin, carbon monoxide haemoglobin, and nitric oxide haemoglobin. Carbon Dioxide Haemoglobin. — Three different forms are said to exist, which have been respectively termed a, ft, and y earbohcemo- globin, but they are comparatively little known. According to Bohr, the carbon dioxide in these compounds is united with the albuminous radicle of the haemoglobin, while the oxygen of oxy- haemoglobin is combined with the pigmented group. He accordingly finds that when a solution of haemoglobin is shaken with a mixture of oxygen and carbon dioxide both of these gases are taken up inde- pendently of each other. Carbon Monoxide Haemoglobin. — This compound results from the union of one molecule of haemoglobin with one molecule of carbon monoxide, and is characterized by its greater stability as compared with oxvhaemoglobin. The carbon monoxide is in this case united with the pigmented radicle of the haemoglobin, and may be split off in this combination as carbon monoxide luemochromogen. Like the native luemochromogen, the carbon monoxide compound can be obtained in crystalline form, and on exposure to the air is likewise transformed into haematin. Under the same conditions the haemo- globin compound is gradually reconverted into oxyhemoglobin. Blood containing carbon monoxide haemoglobin is characterized by its cherry-rod color, its resistance to putrefactive changes in the absence of oxygen, and by its spectrum. This is similar to that of oxvhaemoglobin, but its two bands of absorption, between D and E, are placed rather nearer the violet end of the spectrum. Unlike the spectrum of oxyhemoglobin, however, that of the carbon monoxide compound is not changed to the haemoglobin spectrum on treating with reducing agents. Should oxyhemoglobin be simultaneously present, a mixed spectrum of the two substances is obtained. Such blood, moreover, when treated with double its volume of a solution of sodium hydrate (sp. gr. 1.3), is not changed to a dirty brownish mass, with a tint of green, as with normal blood, but presents a beautiful red color, which changes to brown only on standing. In it- crystalline state carbon monoxide haemoglobin may be obtained by saturating a sufficiently concentrated solution with car- l.on monoxide and cooling the mixture to 0° C, when one-fourth of it- volume of cooled alcohol i- added. On standing in the refrigera- tor the substance separates out in the form of bluish-red crystals, which are isomorphous with those of oxyhemoglobin, but much more stable. 336 THE BLOOD. Nitric Oxide Haemoglobin. — This compound is more stable even than carbon monoxide haemoglobin, and, like this, may be obtained in crystalline form. Its spectrum is similar to that of carbon mon- oxide haemoglobin. The bands, however, are less sharply defined and paler than those of that compound, and, like these, do not dis- appear on the addition of a reducing agent. The substance is met with in poisoning with the gas in question. Methaemoglobin. — Methaemoglobin is a pigment which normally does not occur in the blood, but is found after the ingestion of large amounts of potassium chlorate, antifebrin, potassium permanganate, turpentine, kairin, thallin, following the inhalation of nitrite of amyl, ether, etc. It is encountered also in hemorrhagic transudates and cystic fluids, and may occur in the urine when methaemoglobin- aernia exists. The elementary composition of methaemoglobin is the same as that of oxyhaemoglobin, but its molecular structure is manifestly different, as in a vacuum it does not give up its oxygen. On treating with reducing agents or on exposure to putrefactive organ- isms, in the absence of oxygen, it is converted into haemoglobin. When oxyhaemoglobin is decomposed with dilute acids or alkalies, methaemoglobin is formed at some stage of the process, and precedes the formation of haematin. During the preservation of oxyhaemo- globin in the dry state, moreover, a partial transformation into methaemoglobin is very likely to occur. The substance is crystal- lizable, and may be obtained in this form by treating a concentrated solution of oxyhaemoglobin with a saturated solution of potassium ferricyanide until the color has changed to a port-brown. The mixture is cooled to 0° C, and treated, with one-quarter of its volume of cooled alcohol. When kept in the refrigerator crystal- lization takes place in the course of a few days. The crystals are of a brown color, and occur as needles, prisms, or hexagonal plate- lets. They may be purified by recrystallization from water in the presence of alcohol. An aqueous solution of the substance is brown, while its alkaline solutions are beautifully red. On exposure to sunlight its neutral and dilute solutions gradually assume a dark-red color, which is thought to be referable to a transformation of methaemoglobin into photomethcemoglobin. On spectroscopic examination such solutions show one broad band of absorption in the green portion of the spectrum no matter whether the reaction is alkaline, neutral, or acid. Methaemoglobin proper under the same conditions gives a fairly broad band of absorption between C and D, nearer C, which is characteristic, and disappears on the addition of sodium hydrate solution. In addition, two different bands may at times be seen between D and E, which are thought to be referable, however, to a contamination of the substance with haemoglobin. Some observers further speak of an additional band near F, but this is not characteristic. On reduction of the alkalinized solution with ammonium sulphide the spectrum of haemochromogen results. CHEMICAL EXAMINATION OF THE BLOOD. 337 Like oxyhemoglobin, methsemoglobin is capable of combining with certain gases to form molecular compounds. Of these, a carbon dioxide methoewioglobin, a methcvmoylobin sulphide, and a cyan- methcemoglobin have been described. Acetylene also is said to enter into combination with the coloring-matter of the blood. These compounds, however, are but little known. The methsemoglobin sulphide results when hydrogen sulphide and air are simultaneously passed through lake-colored blood. It gives rise to a greenish-red color, and it is thought that the greenish discoloration of decom- posing bodies is referable to its presence. On spectroscopic exami- nation its neutral solutions give two bands of absorption between C and D, of which one is brighter and located near C, while the other and darker band occupies the middle portion between C and D. The two are united by a diffuse shadow. On adding a strong solution of sodium hydrate the darker band disappears, and if now the solution is heated and treated with a reducing agent, the spectrum of hoemochromogen results. The substance itself has not been isolated. Haematoporphyrin. — This substance, as has been indicated, results from haematin when this is treated with concentrated sulphuric acid that has been saturated with hydrobromic acid. During this process the iron of the haematin is split off, and a new pigment, haematoporphyrin, is formed. In the circulating blood of the verte- brate animals it is not found under normal conditions, but is appar- ently formed in certain diseases, and during the long-continued administration of sulphonal and related bodies, as also in lead poisoning and following intestinal hemorrhages, when it may also be found in the urine. Among invertebrate animals it is said to occur in the integument of the star-fish, in certain snails, in the earth- worm, in various sponges, etc. Hsematoporphyrin is thought to be isomeric with bilirubin, and is thus represented by the formula C,2Hji;N40(;. On reduction it yields a pigment which is possibly identical with hydrobilirubin, or very closely related to it. It may be obtained in crystalline form as a hydrochlorate, while the pigment itself is amorphous. Its solu- tions in acid alcohol present a beautiful purple color, which is changed to a violet blue on adding an excess of the acid. It is most conveniently obtained by starting with haemin and decomposing tlii-i with glacial acetic acid that has been saturated with hydro- bromic acid. Its solutions in acid alcohol give two bands of absorp- tion. One of these is located between C and I), while the second band, which is much darker and more strongly defined, occupies a position midway between I) and E, and extends as a shadow toward I). In dilute alkaline solutions, on the other hand, we find four band-: one between C and D; a second one, which is broader than the first, between I) and E and about 1); a third band, between I> and E, near I']; ami finally a further band between band F, which LS the widest and much darker than the rest. On treating 22 338 THE BLOOD. with an alkaline solution of zinc chloride this spectrum gradually passes into a new spectrum with only two bands, of which one is seen about D, and the other between I) and E. Closely related to haematoporphyrin, apparently, is the phyllopor- phyrin which may be obtained from the chlorophyl of plants. From its formula, C32H34jNT402, and that of haematoporphyrin anhy- dride, C32H34N405, it is suggested that both are different oxidation- products of one and the same substance, which is still unknown, but undoubtedly represents the mother-substance of the respiratory pig- ment of both animals and plants. The spectrum of both is practi- cally identical. On distillation with zinc dust phylloporphyrin gives the pyrrol reaction, which is also obtained with the coloring-matter of the blood. Haematoidin. — This pigment, which was first observed by Vir- chow in old extravasations of blood, in which it may occur in crystal- line form, is now known to be identical with bilirubin. As a separate substance it therefore no longer merits consideration. Its develop- ment from blood-pigment, however, demonstrates the close relation existing between it and the coloring-matter of the bile (which see). I have pointed out that in some of the lower animals haemoglobin is also found, and may occur in the blood either as such or bound to certain cellular elements which may be compared to the red cor- puscles of the vertebrates. In other invertebrate animals we find no haemoglobin, but related respiratory pigments, which are partly violet or purplish red in color, and partly blue. The former com- prise the so-called floridins, of which little is known, while the latter group is represented by the oxy-compound of haemocyanin. Haemocyanin is of special interest, as it is apparently closely related to haemoglobin, but contains copper in its molecule in the place of iron. Unlike haemoglobin, however, hsemocyanin is itself colorless, while its oxy-compound, oxy haemocyanin, presents a beau- tiful blue color. On decomposition oxyhaemocyanin yields an albu- minous substance, which may be compared to globin, and a copper- containing pigment which corresponds to haematin. On reduction with ammonium sulphide or on exposure to an atmosphere of carbon dioxide it yields the colorless haemocyanin. On spectroscopic exami- nation haemocyanin and oxyhaemocyanin show a shadow at both ends of the spectrum, which is more marked in the latter ; true absorp- tion-bands, however, are not observed. Neither substance has been obtained in crystalline form, and of their elementary composition we are also in ignorance. Other invertebrate animals contain only lipochromic pigments in their haemolymph, which probably do not possess a respiratory func- tion however, and in the lowest forms of life, of course, special oxygen-carriers are not required. CHAPTER XV. THE LYMPH. In its course through the blood-capillaries a portion of the plasma passes out through the vessel-walls and enters a system of irregular interfascicular clefts, which are bounded by bundles of fibrous tissue and constitute the radicles of the lymphatic system. Through these clefts the plasma reaches the individual cells of the various tissues and organs of the body, and supplies these with the requisite nourishment, while at the same time it takes up the waste matter that is formed in the metabolism of the cells, and through the lymph-vessels carries these into the venous current of the blood. This fluid-, which thus contains the various constituents of the blood-plasma and the decomposition-products of the cells, is termed the lymph. In addition to the lymph-vessels proper and their radicles, the lymph-clefts, this fluid is also found in the so-called serous cavities of the body, viz., the pleura, the peritoneal and pericardial cavities, in the ventricles of the brain and the spinal cord, in the communi- cating subarachnoid space, and also in the anterior chamber of the eye. In health, however, these cavities contain but little fluid, and quantities sufficient for analytical purposes can normally be obtained only from the pericardial sac, and at times from the subarachnoid space. Under pathological conditions, however, large accumulations of fluid may be observed, and not only in the serous cavities of the body, but also in the areolar connective tissue, beneath the skin, and beneath the muscles. When due to circulatory disturbances, a hydrsemic condition of the blood, or an insufficient elimination of water through the kidneys, such accumulations of fluid are spoken of as transudates, while the term exudates is applied to similar accumulations of inflammatory origin. Formerly it was supposed that the lymph resulted from the blood- plasma through a simple process of lilt ration or transudation only, and in accordance with this view we find that in the various accumu- tion- of lymph the salts and extractives are presenl in about the same amount as in the blood-plasma. Ileidenhain, however, has shown that the flow of the lymph-current is far too sluggish to supply th" various organs of the body with the proper amount of nourishment, supposing it- composition to be everywnere the same as that of the blood-plasma. We are hence forced to the conclusion that the endothelial cells of the capillaries possess a selective secre- 339 340 THE LYMPH. tory power similar to that of the renal epithelium, and are thus capable of furnishing to each tissue its proper amount and kind of food. This view, however, does not preclude the possibility that some of the constituents of the plasma may pass over into the lymph by a simple process of filtration, and it is likely that this actually occurs with the water and most of the mineral salts. According to its origin, then, we may expect to find certain dif- ferences in the chemical composition of the lymph, and we find, as a matter of fact, that such differences exist. These are, however, essentially of a quantitative kind, and qualitatively we find the same constituents in the lymph from the various districts as compared with each other and with the blood-plasma. Like the blood-plasma, the lymph consists of a liquid portion, the lymph-plasma, and cellular elements, the lymph-corpuscles. These latter are essentially mononuclear leucocytes, and are largely derived from the lymphoid tissue which abounds in the course of the lymph- vessels. Red corpuscles are either lacking entirely or they are pre- sent in very small numbers. They are of much darker color than those of the blood, but on exposure to the air they take on the bright-red color of oxyhemoglobin. According to some observers, they represent transition-forms between the leucocytes and the normal red corpuscles of the blood. In various inflammatory diseases of the serous cavities the leuco- cytes may be present in very large numbers, and in extreme cases, indeed, they predominate to such an extent that the liquid character of the lymph may be almost entirely lost. The appearance of the lymph is dependent upon the number of the leucocytes and the amount of fat present. As obtained from fasting animals, it represents a slightly viscid, straw- or rose-colored, transparent fluid. During the process of digestion, on the other hand, and especially after the ingestion of much fatty food, it becomes more or less opaque, owing to the admixture of the fat, which is carried into the general lymph-current through the chyle, viz., the lymph coming from the intestinal canal. The odor of the lymph, like that of the blood, is different in different animals. Its taste is salty, and the reaction slightly alka- line. The specific gravity varies between 1.015 and 1.021. The amount of lymph which is produced in the twenty-four hours is largely influenced by the process of digestion. During starvation a smaller amount is thus found than after the ingestion of food, and it appears, moreover, that an albuminous diet causes a much greater increase than one of carbohydrates. Active muscular exercise has a similar stimulating effect upon its formation. Artificially the amount of lymph can be increased by the intravenous injection of so-called lymphagogues, of which Heidenhain recognizes two classes, viz., those which merely increase the amount of ■ water in the lymph and those which also bring about an increase of the organic solids. The former include such crystalline substances like sugar, urea, THE LYMPH. 341 sodium chloride, etc. ; and Heidenhain supposes that their action is dependent upon their passage into the lymph, where they exert a stimulating effect upon the cells of the tissues and cause an absorption of cellular water. The latter, on the other hand, are in part unknown substances which can be extracted from the muscles of the crab, from the head and body of leeches, from the bodies of anodonts, from the liver and intestines of the dog, and also com- prise the peptones and egg-albumin. The influence of these sub- stances is apparently exerted upon the endothelial cells of the blood-capillaries, whereby the secretory power is increased, and we accordingly find more albumin in the lymph than in the remain- ing blood-plasma. Such lymph then, in contradistinction to the cellular lymph which is found in the first instance, may be termed blood-lymph. The state of the blood-pressure, according to Heiden- hain, is of no moment in bringing about these changes, and he found, as a matter of fact, that variations between 10 and 20 Hgmm. on the one hand, and 150 to 200 Hgmm. on the other hand, are of little influence upon the amount of lymph that is produced. This view, however, is in all probability not final. According to Bunge, the amount of lymph that is formed in the twenty-four hours by the human being amounts to about 4000 c.c. A general idea of the chemical composition of lymph may be formed from the following analysis of Munck and Rosenstein. The material was obtained from a fistula in the thigh of a young woman. Accompanying this is an analysis of the blood-plasma (taken from Hammarsten) for comparison : Lymph. Blood-plasma. Water 96.5 -94.5 per cent. 91.8 per cent. Solids 3.7 -5.5 " " 8.2 " " Albumins 3.4 -4.1 " " 6.9 " " Ethereal extract 0.06 -0.13 " " Sugar 0.1 " " 0.46 " " Salts 0.8 -0.9 " " 0.84 " " Sodium chloride . . . 0.55 -0.58 " " Sodium carbonate . . 0.24 " " Disodic phosphate . . 0.028 " " Of these constituents, the fat is subject to the greatest variations, and is. of course, always more abundant during the process of diges- tion in the chyle than in any other lymphatic district. In Munck's case the amount rose to 4.7 per cent, after the ingestion of a large amount of fatty food, and decreased to 0.06-0.26 per cent, when food was withheld for twenty-four hours. The (at i- present in the lymph to the greatest extent as neutral fat, and it i- to be noted that it bere exists in a state of emulsion, so that upon microscopical examination the chyle more especially will be seen to contain innumerable fat droplets, which vary but little in Bire and have no tendency to flow together, as in the case of milk. 342 TEE LYMPH. Why this is we do not know, but it has been suggested that each fat droplet is surrounded by a delicate albuminous envelope, which is derived from the normal albumins of the lymph. On the other hand, it is conceivable that the surface layer of each droplet con- sists of modified fat or of a denser layer of the fluid in which it is suspended. The fats which are found in the lymph are always identical with those of the food, and can be recovered for analytical purposes by extracting with ether. In its course through tissues which are rich in fat no fat is absorbed. Soaps are present in the lymph in only very small amounts. The albumins which are found in the lymph are the same as those of the blood, and are present in the same ratio to each other. The amount varies somewhat with the character of the lymph, but is normally always smaller than that of the blood-plasma. Like the blood-plasma, so also does the lymph coagulate on standing. The coagula, however, are very delicate and tend to separate out in frac- tions. Some transudates, indeed, such as pericardial effusions and hydrocele fluid, do not coagulate spontaneously at all, while coagula- tion occurs at once if leucocytes or blood is added. The peculiar behavior of such lymph is no doubt due to the absence of cellular elements. It is to be noted also that following the injection of those substances which prevent coagulation of the blood coagula- tion of the lymph similarly does not occur. Other albumins besides serum-albumin, serum -globulin, and fibrin- ogen are not found in normal lymph, such as we obtain from the thoracic duct, for example. Under pathological conditions, how- ever, the exudates more particularly may contain the various albu- minous derivatives of the leucocytes, such as nucleins, nucleo- albumins, etc. In cysts of the ovaries and their appendages met- albumin or paralbumin may further be found. The amount of sugar which is found in normal lymph is fairly constant, and is derived from the hepatic lymph. It can be increased artificially by ligating the ureters and then injecting glucose into the blood. It is noteworthy that under such conditions the lymph may contain a larger percentage of sugar than the blood itself. This further shows that the formation of lymph cannot be explained upon the basis of filtration and osmosis only, and demonstrates the specific activity of the endothelial lining of the capillaries. Under pathological conditions, it is claimed, sugar may be altogether absent. The extractives of the lymph are essentially the same as those of the plasma. In certain districts, however, the one or the other will be found to preponderate, and in some localities we further meet with extractives which are peculiar to that particular region. In the cerebrospinal fluid, for example, pyrocatechin has been found ; allantoin is present in the allantoic fluid and in ascitic accumula- tions ; succinic acid and inosit may be obtained from hydrocele THE LYMPH. 343 fluid, in which cholesterin may also be present in very considerable amount. . . , . . Traces of urea, uric acid, lecithin, xanthm, kreatin, and lactic acid are commonlv found. . Of ferments, we find a diastatic ferment and the glucolytic ter- raent of Lepine. . The gases of the lymph differ from those of the blood m the presence of larger amounts of carbon dioxide— 35 to 45 per cent.— as compared with arterial blood, and smaller amounts than are found in venous blood. The tension of the carbon dioxide, as compared with venous blood, is lower, which suggests that a por- tion of the gas enters the blood from the lymph through a reverse secretorv activity of the capillary endothelium. Oxygen is present only in 'traces, while the amount of nitrogen is the same in both, viz., 1.6 per cent. For purposes of comparison and reference, a few analyses ot some of the more important normal and pathological varieties of lymph are appended. Some of these will be considered in greater detail in subsequent chapters. Analysis of Human Pericardial Fluid (Hammarsten). Water *%*» Solid 39.14 Albumins 2^° Fibrin V^i (ilobulins • kg Serum-albumin "no Extractives --^ Soluble salts . . _ °™ Sodium ebloride '■-- Insoluble salts °-10 Analysis of Dog's Chyle (Hoppe-Seyler). Water 906.77 Solids ^.23 Fibrin }•" Albumins and jrlobulms -i.uo lilthin . I 64.8G < ii'.lcsterin j Fatty acids and soaps 1 2.34 Other organic bodies j Mineral -alts '••'"- Anai.v-i- OF AqTTBOUS HlTMOB OF CAM? (Halliburton). Water 986.87 &£ • : : : i«> Albumins , ,7 Extractives 4.21 Inorganic salts '■'" - dium chloride °-8y 344 THE LYMPH. Analysis of Cerebrospinal Fluid (Gautier). Water 987.00 Solids 10.59 Albumins 1.10 Fats 0.09 Cholesterin 0.21 Alcoholic and aqueous extracts, minus salts, but including sodium lactate 2.75 Salts 6.44 Chlorides 6.14 Sulphates 0.20 Earthy phosphates 0.10 Ammonia traces. Analyses of Pleural Effusions (Gautier). Acute pleurisy. Water 937.60 Organic matter 54.40 Fibrin 0.09 Mineral matter 8.00 Chronic C Hydrothora pleurisy. (cardiac). 933.80 958.70 58.20 32.30 0.00 0.19 8.00 9.00 •rivon and Sch> erer) . Chronic Hepatic nephritis. cirrhosis. 978.00 9S4.50 Ovarian cancer. Water 946.50 Serum-albumin 19.40 Serum-globulin 18.581 Q 4A ftl7 Mucin (?) 0.95/ 8>4U b-1' Fats 1 , q . Cholesterin [ 1.25 *' 00 } ' ' Extractives Soluble salts 5.521 Insoluble salts 7.53 J 1.9 .0C .00 8.46 Analysis of Hydrocele Fluid (Hammarsten). Water 938.85 Solids 61.15 Fibrin 0.59 Globulins 13.25 Serum-albumin 35.94 Ethereal extract 4.02 Soluble salts 8.60 Insoluble salts 0.66 Analysis of Amniotic Fluid (Labroche). Human. Water 987.300 Solids 16.700 Serum-albumin 2.590 Mucin 1 Albumoses j- 1.604 Glucose J Urea 0.450 Fats 0.356 Mineral salts 7.695 Sodium chloride 6.071 Disodium phosphate 1.621 Sulphates traces. Salts of calcium and potassium none. Til E LYMPH. 345 j Analysis of Lymph from Cellular Tissue ((Edema). Water 975.20 Albumins 5.42 Fats and extractives 3.76 Mineral salts * 15.62 Analysis of Pus. — The composition of the leucocytes which v enter into the formation of pus has been considered (pages 303 and 322). An analysis of pus-serum is here given, which is taken from Robin : Water 937.90 Metalbumin ~) Serum-albumin [■ 11.00 Serum-globulin J Lecithin 6.00 Fats and soaps 10.00 Cholesterin 3.50 Serolin 1.00 Leucin, tyrosin, and extractives in general 15.00 Salts of organic acids traces. Sodium chloride 3.11 Sodium phosphate traces. Phosphates of calcium and magnesium 0.50 Sulphates 1.87 Salts of iron and silica 0.16 THE SYNOVIAL FLUID. The synovial fluid, though not a lymph in the narrower sense of the term, is for convenience' sake briefly described at this place. It is the specific secretion of the synovial membrane of the joints, and constitutes a strongly alkaline, viscid, yellowish, somewhat cloudy fluid. In addition to the common albumins, fats, salts, and extractives of the lymph, it contains also a peculiar mucinous body, which is termed synovin, and apparently belongs neither to the nucleo-albumins nor the mucins or mucoids. It can be precipitated with acetic acid and coagulates on the application of heat. Of its nature, however, nothing further is known. In addition, another mncin-like body is found which is rich in phosphorus, and probably belongs to the ouoleo-albumins. Quantitatively the composition of the synovial fluid varies with exercise and pest in such a manner that on motion the mucinous body, as also the albumins and extractives, increase, while the salts diminish. This is shown in the appended analyses, which are taken from Frerichs : Stalled ox. Ox on pasture. Water 960.90 948.54 Solids 30.10 51.50 Mucin (?) 2.40 5.60 Albumins and extractives 15.76 35.12 0.62 0.76 11.32 9.98 CHAPTER XVI. THE MUSCLE-TISSUE. I have pointed out that while in the monocellular organisms the various functions of the body are carried on by the single cell, a gradual division of labor occurs as we ascend in the scale of both animal and vegetable life, where groups of cells are set aside for the performance of certain special functions. Structurally this division of labor finds its expression in a more or less well-marked deviation from the original type, as has been shown. At first sight, it is. thus difficult to connect the highly differentiated muscle-cell with the apparently much more simple ovum from which it has originated. The element of reproduction and secretion is here manifestly placed in the background, while in its co-ordinate and rapid contraction on stimulation we have abundant evidence of its highly specialized function. That this should further be expressed in the chemical composition of the cells suggests itself at once. As a matter of fact, we here find substances which may be regarded as specific muscle components, and it seems warrantable to assume that a definite connection exists between these bodies and the special func- tion of the cell. On chemical examination we may then further expect to meet with the various products of katabolism, so far as these are found in the muscle-tissue proper, and have not as yet been removed by the blood or the lymph. Before proceeding to a study of these various substances in detail, a few analyses of muscle-tissue are here introduced, which will furnish a general idea of its chemical composition. Qualitatively this is fairly constant, but quantitative variations occur which are often very marked. In preparing the tissue for analytical purposes, the blood should first be washed out entirely with dilute saline solution (0.6 per cent.). Fibrous tissue and fat must be dissected away as far as possible and all larger bloodvessels removed. The material is then further scraped, so as to get rid of as much of the connective tissue as pos- sible which binds the individual fibres together, and is now ready for examination. Analyses of Fresh Muscle-tissue (Neumeister). — The figures represent average values, which have been collected from various sources, and have reference to mammalian muscle-tissue in general, unless otherwise stated. 346 THE MUSCLE-ALBUMINS. 347 Per cent. Water *. 75.50 Solids 24.50 Organic constituents 23.50 Myosin (?) 7.74 Xncleins 0.37 Albumins, proteids, and albuminoids (insoluble in neutral solution) 15.25 Collagen (referable to interiibrillary connective tissue) 3.16 Fats 3.71 Glycogen 0.7-1.0 Lactic acid 0.1-1.0 Inosit 0.003 Kreatin 0.21-0.28 Xanthin 0.01-0.11 Hypoxantbin 0.04-0.12 Guanin 0.005 Inorganic constituents 1.000 Phosphoric acid 0.4674 Chlorine 0.0672 Potassium oxide 0.4654 Sodium oxide 0.0770 Calcium oxide 0.0086 Magnesium oxide 0.0412 Iron oxide 0.0057 In studying this analysis we observe that, aside from the mineral constituents, various bodies are encountered here which represent distinct products of katabolism, and which are hence most likely not concerned in the specific function of the muscle-tissue. These com- prise the common extractives, viz., xanthin, hypoxanthin, guanin, kreatin, lactic acid, and possibly also inosit. They are no doubt formed as a consequence of cellular activity, but play no role in the function of the muscle proper. On the other hand, we meet with food-stuffs proper, viz., albumins, carbohydrates, and fats, and we may a priori expect that all these substances, conjointly or in- dividually, are directly concerned in the contractile function of the cell. THE MUSCLE-ALBUMINS. An in the case of all tissues of the body, the muscle-tissue also consists of a liquid portion, the so-called muscle-plasma, and a more solid portion, which may be termed the muscle-stroma. Muscle-plasma may be obtained in the following manner : while the animal is -till living the blood is thoroughly washed from the large skeletal muscles by injecting into the larger arteries a dilute saline solution that has been wanned to the temperature of the body, and allowing the fluid to escape from the corresponding veins. This i- continued after death until the outflowing water is colorless. The muscles are then rapidly dissected oil', ground to a pulp together with pumice-stone, and passed through a filter-press. The resulting liquid i- tli'- muscle-plasma. 1 In birds 348 THE MUSCLE-TISSUE. While this procedure is applicable in the case of mammalian muscle-tissue in general, special precautions are necessary if the muscles of the lower animals are to be studied. In the frog, for example, the tissue, after removal of the blood, must be frozen in a gradual manner, after which the entire process is continued at a temperature below — 3° C. A snow-like mass is finally obtained, which melts at — 3° C. The color of the muscle-plasma varies with the color of the muscles from which it has been obtained. In the case of the dog it is of a brownish color, in rabbits it is yellowish red, and in frogs a light yellow. The particular shade of color, as will be seen later, is a direct expression of the degree of functional activity of the individual muscles, and is in part due to haemoglobin and in part to certain lipochromes. The reaction of the plasma is neutral or slightly alkaline. On standing for a length of time mammalian muscle-plasma gradually undergoes a process of coagulation, but it is to be noted that the coagulum M-hich separates out is slight in amount. In the case of the frog, on the other hand, the entire bulk of the plasma becomes gelatinous, and, in contradistinction to the mam- malian plasma, this process begins at a temperature of 0° C. As in the case of the blood-plasma, the coagulum gradually contracts, and the liquid which remains is then spoken of as muscle-serum. The reac- tion is then acid. The substance which composes the clot is termed my og en-fibrin. The behavior of muscle-plasma is thus quite simi- lar to that of blood-plasma, and here, as there, the resulting fibrin is derived from an albuminous substance which was previously pre- sent in solution. This substance is here termed myogen. In addi- tion the muscle-plasma contains another albuminous body, myosin ; and it appears from the researches of v. Furth that these two sub- stances are the only soluble albumins which are contained in muscle- tissue, if we disregard a variable amount of a soluble myogen-fibrin, which is itself a derivative of myogen. Myogen. — Isolation. — Myogen is most conveniently obtained from muscle-plasma after the myosin has been removed by the previous addition of ammonium sulphate to the extent of 28 per cent. The resulting precipitate is removed and the filtrate saturated with the same salt in substance. The myogen is thus thrown down together with the soluble myogen-fibrin. It is washed with a satu- rated solution of ammonium sulphate, dissolved in water, and freed from the soluble myogen-fibrin by heating to 40° C, when this is transformed into the insoluble form and is filtered off. The remain- ing solution contains the myogen in pure form. In its general properties it resembles the albumins proper, in con- tradistinction to the globulins. It is soluble in water, and can be precipitated by alcohol, by salting with ammonium sulphate, sodium chloride, and magnesium sulphate. The two latter salts, however, do not cause a complete precipitation. Alcohol (92 per cent.) THE M I 'SCLE-A LB CMIXS. 34 9 renders tbe substance insoluble to a slight extent, but the greater portion is refractory in this respect. Bv acetic acid myogen is precipitated only in the presence of a neutral salt, but redissolves in an excess of the acid, with the forma- tion of syntonin. The tendency to the transformation into albu- minates is indeed more marked in the case of the soluble muscle- albumins, in general, than with any other forms. Mineral acid- are in this respect still more active than acetic acid, and as a conse- quence a precipitation of myogen is observed only when such acids are present in certain proportion. Carbonic acid and the salts of the heavy metals precipitate myo- gen only in the presence of a neutral salt. Myogen coagulates at a temperature of from 55° to 65° C. It is not precipitated on dialysis. When solutions of myogen are kept at a certain temperature, and in the presence of a definite amount of a neutral salt, the substance is gradually transformed into a soluble form of myogen-fibrin, which differs from myogen in the fact that it is throwwdown on dialysis, and in its point of coagulation, which lies at 40° C. As has been stated, a certain amount of soluble myogen-fibrin seems to occur pre- formed in the muscle-tissue, and separates out gradually on standing. At 40° C, however, this occurs instantaneously. By coagulation the soluble myogen-fibrin is transformed into the insoluble form, the myogen-fibrin pr< >per. The relative amount of myogen, as compared with myosin (see below), which is found in muscle-tissue varies in all probability with different animals. In rabbits, v. Fiirth observed that myosin repre- sented about 80 per cent, of the total amount of soluble albumin-. The amount of soluble myogen-fibrin which is included in the above figures is in mammals apparently very small, as only slight coagula are formed when the plasma is heated to 40° C. But in the frog large amounts are manifestly present. In some animals, on the other hand, it is apparently absent. Myosin. — Myosin i- conveniently isolated from muscle-plasma by salting with ammonium sulphate to the extent of 2X per cent. Sodium chloride and magnesium sulphate may also be employed, but it i- then necessary to add the salt to saturation. Tli" substance is a globulin, and, curiously, contains a consider- able amount of calcium. It is soluble in dilute saline solution-, and i- precipitated from these solutions by Baiting, as just indicated, by passing n stream of carbon dioxide through its solutions, by diluting with water, and on dialysis. It i- characterized bv its pronounced tendency to coagulate, ami, unlike myogen, i- rendered almost entirely insoluble on precipitation with alcohol. Like tlii-. it i< also readily transformed into syntonin or alkaline albuminate on treating with acids or alkalies ; and here, as there, a precipitation results only if very dilute acids are used. In an excess the precipitate rapidly dissolves. On heating solutions of myosin to 35 ( '. 1 1 1<- 350 THE MUSCLE-TISSUE. substance is gradually coagulated, while this occurs at once at a tem- perature of 50° C. In its insoluble form myosin is termed myosin-fibrin, which, like the insoluble myogen-fibrin, belongs to the class of the coagulated albumins. Of special interest, further, is the fact that on evaporating a few drops of a solution of myosin in soda solution on a slide, at a low temperature, a jelly-like material is obtained, which on polariscopic examination is seen to be doubly refracting. In this respect it behaves exactly as the anisotropic material which is found in the dark bands of the voluntary muscle-fibres. The amount of myosin found in the muscle-tissue of the rabbit is much less than that of myogen, and, according to v. Furth, corre- sponds to only 20 per cent, of the total amount of soluble albumins. Significance of the Common Muscle-albumins. — Of the part which the common muscle-albumins take in the function of the cell little is known that is definite. From the researches of some ob- servers, it appears that the nitrogenous components of the albumins, at least, do not furnish the energy which is here required. Petten- kofer and Voit have thus shown that an increase in the amount of muscular work does not lead to an increased elimination of nitrogen or to an increase which is insignificant. This view is now gener- ally held ; but it must be admitted that evidence is not lacking which suggests that an increased albuminous destruction may occur nevertheless when the amount of work is increased. It has been shown, as a matter of fact, that the total elimination of sulphur, which usually follows that of the nitrogen quite closely, is increased by muscular exercise and diminished thereafter. Bat while we may admit that the nitrogenous components of albumin may furnish a certain fraction of the energy which is required in muscular work, this is, after all, but slight, and there is abundant evidence to show that by far the greater amount of energy must be referable to the decomposition of non-nitrogenous material. The question, of course, suggests itself, Do the soluble albumins of the muscle-plasma represent the contractile element of the muscle-tissue? but to this question no answer can as yet be given. We might imagine that in some manner a transformation of the soluble albumins into the fibrin form occurs, and vice versa ; but of this we have no evidence in the living tissue. On the other hand, we know that rigor mortis, as well as the rigor which results from ex- posure of muscle-tissue to a temperature of 47° C, is owing to such a change, and it is quite probable that in either event both myosin and myogen pass over into the coagulated state. The subsequent relaxa- tion is then no doubt referable to the formation of syntonin, which may be effected by the lactic acid that is then ahvays found in consid- erable amount, and may be aided by the presence of certain ferments. Whether or not a myosin-ferment exists which is responsible for the transformation of the soluble albumins into the insoluble form, THE MUSCLE-ALBUMINS. 351 has not been ascertained, but is very probable. Some writers indeed regard the coagulation of muscle-plasma which occurs on standing as being referable to the presence of such an enzyme. Other Albumins. — Besides myosin and myogen, which latter was formerly termed myosinogen, muscle-plasma was also supposed to contain' traces of serum-albumin, myoglobulin, and myo-albumose. v. Fiirth, however, has shown that any trace of serum-albumin that may be found is referable to the presence of small amounts of lymph or blood that have not been removed by washing, and that if this is dune with special care no serum-albumin can be demonstrated. Halliburton's myoglobulin, moreover, he regards as identical with myogen, while the existence of a myo-albumose in muscle-plasma has been disproved by more recent investigations. That substances belonging to the albumoses may be found in muscle-tissue after death, when syntonin also is found, is, of course, likely, but in the living tissue their presence can hardly be expected under normal conditions. Myoproteid is a substance which v. Fiirth obtained from the muscle-plasma of fish. Of its chemical nature, however, nothing further is known than the fact that it apparently does not belong to the commonly recognized classes of albumins. It is neither a nucleo-albumin nor a glucoproteid. Nucleins are not found in the muscle-plasma, but can be isolated from the muscle-tissue as a whole or from the insoluble material which remains in the filter-press after separation from the plasma. Their amount is small, and in accordance with the slight degree to which the nuclei enter into the structural composition of the muscle- cell. Larger amounts arc obtained from embryonic muscle, where cellular reproduction is, of course, more active. From the tissue of an adult dog Pekelharing obtained about 0.37 per cent. These nucleins must be regarded as the material from which the xanthin- bases that can always be demonstrated in muscle-tissue are derived. These will be considered in detail later. Phosphor-carnic Acid. — A few years ago Siegfried announced that after removing the phosphates from extracts of muscle-tissue, and treating with ferric chloride, under the application of heat, a phosphorus-containing iron compound is obtained, which is insolu- ble in water, but easily soluble in solutions of the alkalies. This ~nl )~t an**- he regards as the irou salt of an organic acid, which he term- phosphor-carnic acid ; the salt he speaks of as carniferrin. On decomposition with barium hydrate he then obtained tin; barium -alt of ;i cry-tiilli/able acid, ctiriiic CH HN— CO — CO N^ N C N^ GASES. 367 It is obtained in indistinctly crystalline form. In hot water it is readily soluble, while in cold water it dissolves with great difficulty, and is insoluble in alcohol and ether. It is neutral in reaction, and combines with acids and alkalies to form salts. These salts are not decomposed by water. Its hydrochlorate, which results when adenin is dissolved in warm hydrochloric acid, crystallizes out on cooling, and combines with platinum chloride to form a double salt. Silver nitrate precipitates it from its aqueous solutions as a silver salt, and it is to be noted that this compound is insoluble both in ammonia and nitric acid. Subacetate of lead precipitates adenin as a lead salt ; this is soluble in boiling water. Acetate of copper produces no precipitate. The substance gives none of the common reactions of the xanthin -bases, and is best identified by the behavior of its lead and silver salts. Still other nitrogenous extractives may be obtained from muscle- tissue, but, with the exception of taurin and inosinic acid, they are scarcely known. For a description of taurin see page 153. Inosinic Acid. — Inosinic acid is apparently a constant con- stituent of muscle-tissue, but is most abundantly encountered in the muscles of ducks, from which Creite was able to isolate as much as 0.2b' per cent., calculated as barium salt. The substance has the composition CU)H13X4POs, and is com- monly regarded as a nucleinic acid. On decomposition with boiling water it is -aid to yield hypoxanthin, trioxy-valerianic acid, and phosphoric acid. Whether or not a relationship exists between inosinic acid and phosphor-carnic acid is as yet unknown. GASES. Both, when at rest as also during its activity, the muscle-tissue is constantly taking up oxygen from the blood and the lymph. This \- stored in the cells proper, and is extensively utilized in the oxida- tion-processea which are constantly going on, but which occur with increased intensity when the muscle is at work. Carbon dioxide is similarly given oil*, and it can be readily proved that the oxygen which is utilized in its formation is in part at least stored within the tissue. Carbon dioxide is thus -till given off, even when a muscle i- removed from the body and worked in an atmosphere which is free from oxygen. It has been noted, moreover, that the amount which is then gel i'vr \< the same as that which results when the muscle is worked in the presence of an abundance of oxygen. Of the form, however, in which the gas exists in the muscle we know nothing, bul it is manifestly not present in the free state, as no Oxygen at all. or very -mall amounts only, can be extracted by a vacuum pump. With increasing activity larger amounts of oxygen ■'ire taken up, while larger amounts of carbon dioxide are being given off. This difference is well shown in the following table, 368 THE MUSCLE-TISSUE. which is taken from Gautier. The figures have reference to 100 volumes of blood, calculated at 0° C. and 1000 Hgmm. pressure: Carbon Oxygen, dioxide. Arterial blood from muscle-tissue 15.25 26.71 Venous blood from muscle-tissue while at rest . . 6.70 33.20 Venous blood from muscle-tissue while at work . . 2.97 36.38 In addition to carbonic acid, small amounts of nitrogen can fur- ther be obtained from muscle-tissue, which are manifestly absorbed from the blood and apparently exist in a state of solution. As in other tissues and fluids of the body where nitrogen is also found, its presence is probably of no significance. The quantity that can be obtained by the vacuum pump is essentially the same as that which is found in the lymph and in the blood. FAT. The amount of fat which is found in muscle-tissue varies not only with different animals but also in one and the same individual at different periods of life. Some of the analytical results which have been obtained are shown below : Pro mille. Lean beef 6.1-7.6 Eabbit 10.7 Partridge 14.3 Pig 40.0-90.0 Salmon 100.0 Mackerel ■ 164.0 Eel 329.0 The fat is deposited not only in the interfibrillary connective tissue, but also in the sarcoplasm proper, and is apparently more abundant in the red meat, which contains more sarcoplasm than in white meat. Like glycogen, it here represents a reserve source of muscular energy, but is apparently utilized more especially when a sufficient supply of the former or of grape-sugar, as such, is not available. While it is ordinarily derived from the ingested fats or from carbo- hydrates, there can be no doubt that under certain pathological condi- tions, which are associated with an increased destruction of tissue albumins, it can also' originate from these. This question, however, we shall not consider in detail at this place, but shall revert to it in a future section. In addition to fats, muscle-tissue also contains a small amount of cholesterin, fats, and fatty acids, and at times considerable quanti- ties of lecithins (0.69 per cent.). The chemical composition of involuntary muscle-tissue is essen- tially the same as that of the striped variety. CHAPTER XVII. THE NERVE-TISSUE. Owixg to the difficulty which attends the separation of the vari- ous morphological components of nerve-tissue, and the peculiar prop- erties of some of its most important chemical constituents, it is as yet impossible to give an account of its chemical composition which is at all satisfactory. Here, as in the other organs of the body, we meet with certain substances which are generally spoken of as ex- tractives, and which are manifestly katabolic products that result during the functional activity of the tissue in question. These are in no sense specific of nerve-tissue, however. They comprise kreatin, uric acid, urea, xanthin, hypoxanthin, guanin, adenin, inosit, and lactic acid — viz., substances which, as we have just seen, are also found in muscle-tissue. In addition, we find certain albumins which in part belong to the native albumins and in part to the globu- lins; further, nucleins and albuminoids, among which the so-called neurokeratin is of special interest, as it largely enters into the com- position of the supporting structure of the nerve-tissue, and is char- acteristic of this. Besides these various substances nerve-tissue contains especially large amounts of so-called myelins — viz., lecithin, cholesterin, and protagon. A certain amount of mineral salts and a large quantity of water constitute the remaining known components of the tissue. As regards the distribution of these substances among the gangli- onic cells and nerve-fibres more especially, our knowledge is as yet quite incomplete; but it appears from the analyses which are avail- able that the gray substance of the brain in the dried state consists to the extent of one-half at least of albumins, while the substances which arc soluble in ether amount to only about one-quarter of the total quantity. Protagon is here present in only very small quantity. In the white substance of the brain, on the other hand, it is found in considerable amount, while the albumins constitute only one- quarter of the dry material. In embryonic! brains, in which the medullary sheaths are not us yet developed, much smaller amounts of lecithins are found than in the adult brain ; and it is noteworthy, moreover, that protagon and neurokeratin are hen; both absent. It may thus be concluded that these substances are essentially com- ponent- of the medullary nerve-fibres. According to Hoppe-Seyler, indeed, the small amount of |>rota<_roii which is found in the gray Substance of the brain i- entirely referable to this source. The 21 36S 370 THE NERVE-TISSUE. presence of smaller amounts of lecithins in the embryonic brain, as compared with the adult brain, and notably the gray matter, is now generally explained upon the assumption that the material in question is in some manner intimately concerned in the growth of the cellular elements proper. Analysis of Brain-tissue (Baumstark). White matter.1 Gray matter.1 Water 69.53 77.00 Solids 30.47 23.00 Insoluble albumins and connective-tissue . . 5.00 6.08 Neurokeratin 1.89 1.04 Nucleins 0.29 0.20 Protagon 2^51 L08 Cholesterins, free 1.82 0.63 Cholesterins, combined 2.69 1.75 Mineral salts 0.52 0.56 Other substances, soluble in ether 30.47 23.00 Analysis of the Mineral Salts (Geoghegan). Chlorine 0.42 — 1.06 Phosphoric acid (P04) 0.85 — 1.39 Carbonic acid (C03) 0.25 — 0.33 Sulphuric acid (S04) 0.14 — 0.13 Phosphate of iron (Fe2(P04)2) 0.09 — 0.30 Calcium 0.02 Magnesium 0.06 — 0.07 Potassium 0.58 — 1.52 Sodium 0.45 — 0.78 Albumins. — Of the character of the individual albumins which occur in nerve-tissue very little is known. Baumstark states that they are essentially the same as those of muscle-tissue, and, to judge from the researches of Petrowski, it appears that one of these may be identical with myosin, as it is soluble in dilute saline solution, and can be precipitated by diluting Avith water or by salting with sodium chloride. The coagulation-point of the substance has, how- ever, not been determined. It is said to occur both in the gray and the white matter. More recently Halliburton claims to have isolated two neuro- globulins, both 'from the white and the gray matter, with a coagu- lation-point of 47° and 75° C, respectively. In addition he found a nucleo-albumin in the gray substance, with 0.5 per cent, of phos- phorus, which coagulated between 55° and 60° C. v. Jaksch further claims to have isolated a nuclein from the gray matter which contains but little phosphorus, and yields hypoxanthin, xanthin, phosphoric acid, and an albuminous body on decomposition. ^ The albumins, as I have indicated, are principally found in the gray matter of the brain, and constitute about one-half of the dried substance. In the white matter, however, they are also found, though in mnch smaller amount, and it is thought that the cylinder 1 The complete separation into gray and white matter is, of course, impossible. THE NERVE- TISSUE. 371 of the nerve-fibre is essentially of an albuminous nature. They are without doubt intimately concerned in the specific function of the nerve-tissue, but of the part which they take in such function noth- ing whatever is known. It is interesting to note, however, that the gray matter of the brain, as also of the spinal cord and groups of ganglionic cells outside of the central nervous system, always pre- sent an acid reaction, while the white matter of the brain and cord and the peripheral nerves is always neutral or slightly alkaline. The substance which produces the acid reaction of the gray matter is apparently the common, optically inactive lactic acid, and it is noteworthy that in the nerve-tissue also a lactic acid is encountered in those portions which are especially rich in albumins. But while the acid reaction of muscle-tissue becomes manifest only after death, it can be readily shown that in the case of the brain and spinal cord this is normal even during life. Whether or not other substances besides lactic acid contribute to the acid reaction of the gray matter has not been definitely established. But it is quite likely that this is the case, as in the presence of lactic acid a transformation of diphosphates to monophosphates would of necessity occur. Bibra and Midler, moreover, claim to have obtained traces of formic acid from the aqueous extract of the gray matter. Paralactic acid has not been found in nerve-tissue. Neurokeratin. — This substance, which was first isolated by Ki'ihne, forms the greater portion of the supporting tissue of the c sural nervous system, and is likewise found in the medullary fibres, where it constitutes the axilemma and outer sheath of the medullary substance. According to some observers, moreover, it tonus a tine reticulated network in the latter. Neurokeratin is an albuminoid and belongs to the group of the keratins, which are found widely distributed among the tissues of epiblastic origin. In the invertebrate animals, in which medullary fibres are not found, and chitinous substances largely enter into tli'' composition of the outer skeleton of the body, it is accordingly represented by a neurochitin. Neurokeratin is insoluble in water, ether, alcohol, in dilute solu- tion-of tin' alkaline hydrates, in gastric juice and pancreatic juice. To isolate th" substance from nerve-tissue, this is accordingly ex- tracted with alcohol and ether, to remove the myelin substances. The remaining material is freed from albumins and other albuminoids by digestion with i_ra- the decomposition of the glucosides just mentioned. Protagon is easily soluble in warm alcohol and ether, while in cold alcohol and cold ether it dissolves with difficulty. On cooling the substance crystallizes out in tine needles or in waxy masses, which can readily be broken up into a fine powder. On heating its alcoholic solutions to a temperature of 48° C. or on boiling its ethereal solutions the substance is readily decomposed into its com- ponents, a- indicated above. In the dry state it can be heated to a higher temperature, but it is then also decomposed before 100° C. is reached. The resulting products melt between 200° and 203° C, and begin to volatilize at 220°. When moistened with water, the substance swells and is partly decomposed, with the formation of so-called " myelin " droplets. If much water is added, an opaque fluid is obtained. Isolation. — To isolate protagon from brain-tissue this should be as fresh as possible, as otherwise partial decomposition occurs spon- taneously. The material is freed from its membranes and adhering blood, and is then stirred to a pulp, and extracted with 85 per cent. alcohol, at a temperature of 45° C.. using fresh portions of alcohol from time to time until a specimen no longer deposits a sediment when cooled to 0° C. The extracts are filtered at 45° C, and sub- sequently kept at 0° C. The resulting precipitates are extracted with cold ether to remove cholesterins and lecithins, when the remaining material is pressed between filter paper and dried over sulphuric acid. It is finally pulverized, again extracted with alcohol at 45° C, when the solution is filtered and cooled to 0° C. To purify the substance it is recrystaUized from warm alcohol or ether. Cerebrin. — Cerebrin, as I have stated, is a normal decomposition- product of protagon, but probably does not occur in the living nerve-tissue as such. Associated with lecithin, it is also found in the stroma of the red corpuscles of the blood, in leucocytes, in sper- matozoa, in the spleen, in the yolk of birds' eggs, etc. It is questionable, however, whether it actually exists in the free State, and the fact of its constant association with lecithin rather suggests that here also it is primarily present in the protagon molecule. Cerebrin is said to have the formula ('7„III)IIX;,()I... Elementary analysis ha- given the following results: C, . The isolation of free cholesterin has been described above. The Extractives. — The extractives of nerve-tissue, as I have already stated, are essentially the same as those which can be isolated from other organs of the body. They comprise traces of kreatin, uric acid, xanthin, hypoxanthin, guanin, adenin, inosit, volatile fatty acids (acetic acid and formic acid), lactic acid, glycogen, leucin (or rather a lower member of the homologous series C2„H2n + jNO.,), and urea. In addition, jecorin, cholin, and neu- ridin have also been found ; neurin, on the other hand, does not occur in the brain under normal conditions. Neuridin is of special interest, as the substance is constantly formed during the putrefaction of meat and gelatin, and has also been obtained from cultures of the typhoid organism. According to Brieger, who first isolated the body, it is also present in traces in the yolk of birds' eggs. It is a diamin of the composition C5H14N2. With the chlorides of gold and platinum it forms well-defined crystalline salts. On boiling with caustic alkalies it is decomposed into trimethyl-amin and dimethyl-amin. It is not toxic. Jecorin is a substance of unknown composition, but apparently contains both sulphur and phosphorus. It is not exclusively encoun- tered in nerve-tissue, but has also been found in traces in the liver, in the muscles, and, according to some observers, in the blood. It reduces cupric oxide in alkaline solution on boiling, and on cool- ing separates out in the form of a thick jelly. It is soluble in ether, and can be precipitated from its solutions by alcohol. CHAPTER XVIII. THE EYE AND THE EAR. THE EYE. In studying the chemical composition of the eye, we shall con- sider the most important parts of the organ in succession. It should be pointed out in advance, however, that the subject has thus far received but little attention, and our account must hence of necessity be very imperfect. The Cornea. — An analysis of the cornea of the ox has given the following results: Pro mille. Water 758.3 Solids 241.7 Collagen 203.8 Other organic substances 28.4 Mineral salts 9.2 The collagen, which forms the greater portion of the fibrous net- work of the cornea, is probably identical with the common form which can be isolated from cartilage, and, according to Morner, con- tains 16.95 per cent, of nitrogen. The semi liquid interfibrillary substance consists of a mucoid, which yields a reducing substance on boiling with dilute mineral acids. It contains about 2 per cent, of sulphur, and seems to be characteristic of corneal tissue. In addition, we find two globulins, which, according to Morner, do not belong to the cornea proper, however, but are contained in the epithelial layer. Nucleins have not been found. I)r.sf'f>inrt,.s membrane principally consists of a membranin, which contains 14.77 per cent, of nitrogen, and 0.90 per cent, of sulphur. The substance is a glucoproteid, and belongs to the group of hyalo- gens (which see). On boiling with dilute hydrochloric acid it yields a reducing substance. In ordinary boiling water it is insoluble, but dissolves under the action of superheated steam. It is digested by trypsin, while the gastric juice is without effect. The Sclerotic. — The composition of the sclerotic coat of the ey«- i- very much the same as thai of the cornea, but it appears that the quantity of the mucoid is here much less, while collagen repre- sents about seven-eighths of the entire amount of solids. The Aqueous Humor. — The aqueous humor is a clear fluid of an alkaline reaction and a specific gravity varying between 1.003 and 877 378 THE EYE AND THE EAR. 1.009. Its quantitative composition has already been given (page 343). According to Grunhagen, it contains traces of paralactic acid, a dextrorotatory body, and a reducing substance, which is not sugar. Both the latter are unknown. The albumins in question are serum- albumin, serum-globulin, and traces of fibrinogen. The Crystalline Lens. — The capsule of the crystalline lens, like Descemet's membrane, consists essentially of a membranin, which is not identical with that found in the latter, however, as it is less resistant to the action of boiling water and of acids and alka- lies. According to Morner, it contains 14.10 per cent, of nitrogen and 0.83 per cent, of sulphur. A general idea of the chemical composition of the lens itself may be formed from the following analysis, which I have taken from Neumeister : Per cent. Water 63.50 Solids 36.50 Albumins 35.00 Insoluble albuminoid 17.00 /3-crystalline 11.00 a-crystalline 6.80 Albumin 0.20 Fats 0.29 Lecitbins 0.23 Cholesterin 0.22 Salts 0.80 The albumins of the lens can be divided into two groups, viz.,, those which are soluble in dilute saline solution, and those which are insoluble. The latter group is represented by a substance which is spoken of as albumoid. It is manifestly a true albumin, as it is entirely dissolved by the gastric juice, and does not yield a reducing substance on boiling with mineral acids. It gives all the common color reactions of the true albumins, and has the same elementary composition. In dilute mineral acids and alkalies it dissolves with ease, and is reprecipitated on neutralization. Unlike the alkaline albuminates, however, its solution in dilute alkalies coagulates at 50° C, in the presence of 8 per cent, of sodium chloride. The sub- stance manifestly constitutes the greater portion of the lens-fibres, as the nitrogen and sulphur values of the two are practically the same, viz., N, 16.62 and S, 0.79 per cent, in the case of the albumoid, as compared with N, 16.61 and S, 0.77 of the fibres. It can be shown, moreover, that after extraction of the soluble constituents of the lens the fibrous framework remains and gives the same reactions as the isolated albumoid. Its amount increases from without inward, in accordance with the increasing age of the fibres. Aside from a very small amount of serum-albumin, the remaining soluble albumins of the lens are represented by two vitellins, which are termed a-crystalline and /9-crystalline, respectively. Of these, the a-body is notably found in the outer portion of the lens, while the /5-substance occurs in the inner portion more particularly, THE EYE. 379 and is apparently the only one that is found in the centre of the lens. The two substances can be isolated from an aqueous extract of the lens by saturating the solution with magnesium sulphate at a temperature of 30° C. The precipitate is then dissolved in water, dialvzed, and the resulting solution precipitated with acetic acid, which throws down the a-body, while the /3-crystalline remains in solution. A small amount of the /9-substance, it is true, is also pre- cipitated by the acetic acid, but can be separated from the a-body by a repetition of the process. Both substances are precipitated from their neutral solutions by carbon dioxide, but in the case of the ^-crystalline this precipitation is never complete. The latter coagulates at 63° C, and the a-crys- talline at 72° C. The ^-substance further differs from the a-body in containing much more sulphur, 1.27 per cent., as compared with 0.56 per cent., which is, moreover, in part at least, present in a loosely combined form, while the entire quantity that is found in the a-crystalline is firmly combined. That these bodies are intimately concerned in the concentration of the light cannot be doubted. The refractive index of the inner layer of the lens, in man, is given as 1.407, while that of the central portion is 1.456. Of the significance of the fats, lecithins and cholesterins in the lens, nothing is known, but it appears that the amount of the two latter, at least, is much increased in senile cataract, while the quantity of the albumins, as a whole, is diminished. The albumoid, however, is then possibly increased. The Vitreous Body. — The vitreous body of the eye is a jelly- like material, which consists of a fine framework of collagen, enclos- ing the liquid portion of the body proper. This presents an alka- line reaction, and contains only a very small amount of solids. Its general composition is seen below : Tro mille. Water 989.00 Solids 11.00 Albumin 0.70 Urea . 0.04 Paralactic acid traces. GrIucoBe traces. Mineral salts 9.00 Among tlic albumins present Morner claims to have found a hyalomucoid, which is closely related to the cornea] mucoid, but contains 12.^7 per cent of nitrogen and 1.19 per cent, of sulphur, a- compared with 12.79 per cent. <>(' nitrogen and 2.07 percent, of sulphur in the case of the latter. The Retina. — A general idea of the chemical composition of the retina may lie formed from the following analyses, which are taken from ( 'aim : 380 THE EYE AND THE EAR. Horse. Ox. Water 89.99 86.52-87.61 Solids 10.01 13.43-] 2.39 Soluble albumins 4.35 \ q ak 7 no Insoluble albumins 1.36/ Extractives 0.67 0.67- 1.07 Cholesterin ) 0.65-0.77 Lecithin [ 2.39 2.08- 2.89 Fats ) 0.00- 0.47 Soluble salts 1.11 0.67- 0.93 Insoluble salts 0.01 0.02- 0.27 Like the gray matter of the brain, from which the retina is essen- tially derived, the membrane presents an acid reaction when per- fectly fresh, but becomes alkaline soon after death. The albumins which are found in the retina appear to be identical with those of the brain substance, and here, as there, we also meet with neurokeratin. This apparently forms the sheath of the outer portion of the rods. In their interior we meet with protagon, lecith-albumins, and in many animals with a peculiar red pigment, which has been termed rhodopsin. Rhodopsin. — Of the significance of this pigment nothing is known. It occurs in the outer portion of the rods and is absent in the cones. As the macula lutea, viz., the point of clearest vision, is composed only of cones, we may conclude that its presence is not essential to sight, and, as I have just said, the pigment is not found in all animals. It is absent in chickens, pigeons, in certain reptiles, bats, etc., but is present in owls and deep-sea fishes. On exposure to daylight the pigment fades, and, to isolate the substance, it is necessary to work with sodium light. If the living retina, after having been kept in the dark for some time, is suddenly exposed to an intense light, which is broken in part through the interposition of some dark object, such as the framework of a window, and if the remaining pigment is then fixed with a 4 per cent, solution of alum, red pictures of the interposed object can be obtained on the retina, while the remaining portion has become decolorized. Such pictures are termed optograms. The regeneration of the pigment, which is constantly going on in the living animal, is apparently dependent upon the integral union of the layer of rods and cones with the pigmented epithelial layer of the retina ; but of the manner in which this restitution takes place, we know nothing. It appears, however, that its formation is preceded by the development of a yellow pigment, which is termed xanthopsin. Of the chemical nature of rhodopsin nothing is known. Be- sides daylight, it is decomposed by acids, alcohol, ether, chloroform, and solutions of the alkaline hydrates, by heating to a temperature of from 52° to 53° C. for several hours, or instantaneously at 76° C. Toward ammonia and a solution of alum it is refractory. It is easily soluble in water, containing from 2 to 5 per cent, of Platner's bile. From such a solution it is precipitated on dialysis THE EYE. 381 or by salting with ammonium sulphate or magnesium sulphate. The substance then appears as a violet amorphous material. On spec- troscopic examination no specific bands are observed, but merely a general absorption between D and C, which is especially marked about E. Chromophanes. — Chromophanes are pigments which apparently belong to the lipochromes, and are found in the retinal cones of reptiles and bin Is. They here occur in the form of red, green, and yellow oil globules, which are quite distinct, and are situated at the inner ends of the cones. The pigments in question are termed rhodo- phane, chlorophane, and xanthophane, respectively. Unlike the pigment of the rods, these chromophanes are apparently not affected by light, unless exposed for several days. Of their significance, nothing is known. The epithelial layer of the retina, which adjoins the choroid, con- tains a black pigment, which is probably identical with that of the choroid. This is termed fusdn, and belongs to the class of the mel- anins, which comprise the black pigments that are found in the hair, in the negro-skin, in melanotic tumors, etc. According to Lan- dolt, this particular form contains C, 54.48 per cent.; H, 5.36 ; X. 12.65; O, 27.52. In addition iron is also found, and the opinion has been expressed that the substance may be derived from the coloring-matter of the blood. This supposition is strengthened by the observation that the first appearance of the pigment in the embryo coincides in point of time writh the development of the choroidal bloodvessels. Besides fuscin, a pigment of a yellow color has also been found in the pigmented epithelial lining of the retina, which is termed lipochrin. It is apparently a yellow lipochrome. The Choroid. — Aside from the common components of connec- tive tissue, the choroid, as has just been mentioned, contains a black pigment, fuscin, which is probably identical with that found in the pigmented epithelial lining of the retina (see above). THE EAR. The chemical composition of the organic portion of the middle arid the internal ear has not as yet been studied. The perilymph and endolymph present an alkaline reaction, and, in addition to the common mineral -alt- of the' lymph, contain traces of albumin, and in aome animals a mucinous body of unknown character. CHAPTER* XIX THE SUPPOKTING TISSUES. In contradistinction to those tissues of the animal body which are essentially composed of cells, and in which the albumins proper con- stitute the greater portion of the organic solids, we find that in the so-called supporting tissues which comprise the common connective tissues, cartilage, and bone, the albuminoids stand in the foreground. Their preponderance here coincides with the extensive development of the matrix, while the cellular elements enter into the histological picture to a more or less insignificant extent. This statement, how- ever, holds good only for the higher animals, and more specifically for the fully developed animals. In lower forms of life, and during the embryonic stage of the development of the higher forms, these structures are rich in cells, and we find then an underlying matrix in which a differentiation into supporting tissue proper has not as yet occurred or exists to only a limited extent. Such tissue is termed embryonic connective tissue, and is also known as mucous tissue. In the adult animal it is found only in the vitreous humor of the eye. In typical form it is seen in the umbilical cord, in which it constitutes the so-called jelly of Wharton. The matrix is here very rich in water, and contains a mucinous substance, which is soluble in a 0.5 pro mille solution of hydrochloric acid. In addi- tion, traces of albumin are met with, while collagen is usually absent. Of the composition of the cells nothing specific is known, but it is quite likely that their processes consist of collagen. White Fibrous Tissue. — The fibrils of white fibrous tissue con- sist of collagen, and are bound together by a cement-substance, which represents the original undifferentiated matrix. As in the case of the embryonic connective tissue, this contains traces of the common albumins of the plasma, and a mucinous substance, which, in contradistinction to that of the umbilical cord, is insoluble in a 0.5 pro mille solution of hydrochloric acid. Analysis of this substance has given the following results: C, 48.30; H, 6.44; N, 11.75 ; S, 0.81 ; and O, 32.70 per cent. According to Lobisch, its formula is C160H256N32SO80. To isolate the body in question, liga- ments, such as the tendo Achillis, are cut into small pieces and first extracted with cold water, which dissolves the albumins and a small fraction of the mucin. The remaining material is then placed in a half-saturated solution of lime-water, in which the mucin is readily soluble. After filtering, the substance is precipitated by adding an excess of acetic acid (see page 113). The residual sub- 382 CARTILAGE. 383 stance, after removal of the mucin, consists of the collagen-fibrils, a few cellular elements, and quite commonly also contains isolated fibrils of the yellow or elastic variety, which can be readily recog- nized on microscopical examination by their higher power of refrac- tion. When plaeed in water, or, still better, in a dilute solution of acetic acid or caustic alkali, the white fibres swell, while solutions of some of the metallic salts, such as ferric sulphate and mercuric chloride, cause them to shrink. Tannic acid acts in a similar manner. Owing to the great stability of the compound of the latter with collagen, tannic acid is extensively utilized in the preparation of leather. On boiling white fibrous tissue in water the collagen dissolves, with thf formation of gelatin, which latter separates as a jelly-like mass on cooling. Yellow or Elastic Tissue. — In the yellow elastic fibres, elastin takes the place of the collagen of the white fibrous variety. For purposes of study, the substance is most conveniently obtained from the ligamentum nuchas of the ox, in which such fibres are almost exclusively found (see page 47). Reticulated Tissue. — In the reticulated tissue, which constitutes the fibrous framework of the lymph-glands of the body, but which is also found in the alveoli of the lungs, in the liver, the kidneys, and the intestinal mucous membrane, the fibres consist of reticulin. Reticulin is said to have the composition C, 52.88 ; H, 6.97 ; N, 15.63; S, 1.88; P, 0.34. It is insoluble in water, alcohol, ether, dilute mineral acids, lime-water, and solutions of sodium carbonate. It resists the action of pepsin and trypsin, and is dissolved only in cold .-odium hydrate solution on standing for several weeks. It docs not give Millon's reaction, and accordingly yields no ty rosin on hydrolytic decomposition. On prolonged boiling with water or dilute alkalies, its phosphorus is split off; the residual material is then Boluble in water, and can be precipitated from its solutions by means of acetic acid. CARTILAGE. Histologically considered, cartilage consists of a more or less hyalin matrix, in which a variable nimiber of cartilage-cells are found imbedded In certain localities, further, a differentiation of the mat- rix into fibres, both of the white and the yellow clastic variety, is observed. Such fibres, as in the case of the corresponding connec- tive tissue, COnsisf of collagen and elastin, respectively. Of the composition of the cells nothing specific is known. Appar- ently they contain a small amount of glycogen, which disappears during starvation. Traces of fat are also found. During embryonic life they are quite numerous, but later they diminish in number, and in the adult animal the matrix largely predominates. Embryonic cartilage doe- not yield gelatin on boiling with water, and it is quite likely that a- in the cage of the matrix of embryonic connective 384 THE SUPPORTING TISSUES. tissue the matrix here also consists essentially of water and some mucinous substance. Whether or not this is identical with the so- called chondromucoid, which can be obtained from the cartilage of the adult animal, is not known. A general idea of the chemical composition of cartilage may be formed from the following analyses, which are taken from His : Costal cartilage Articular cartilage (human). from knee-joint (human). Water 67. 67 per cent. 73.59 per cent. Solids 32.33 " " 26.41 " " Organic material .... 30.13 " " 24.87 " " Mineral salts 2.20 " " 1.54 " " Analysis of the mineral salts has given the following results (calculated for 100 parts of the mineral ash): Sodium chloride 6.11 per cent. 22.48 per cent. Sodium sulphate 44.81 " " 55.17 " " Potassium sulphate 26.66 " " . . Sodium phosphate 8.42 " " 7.39 " " Calcium phosphate 7.88 \ u u ..__..„ IC Magnesium phosphate . . . 4.55 j The organic constituents of the cartilaginous matrix are essentially represented by chondroitin-sulphuric acid as such, and its compounds with collagen and albumins. In addition, a small amount of soluble albumins is found, as also a peculiar insoluble albuminous sub- stance, which has been termed albumoid. Chondroitin-sulphuric Acid. — This substance is a conjugate sulphate, and, according to Schmiedeberg, has the composition C18H2rN014.S03. On hydrolytic decomposition it yields a hyalin, chondroitin, which in turn gives rise to the formation of chondrosin, and this to glucuronic acid and glucosamin, as represented by the equations : (1) C18H27NOu.S03 + H20 = C18H27NOu + H2S04 Chondroitin-sulphuric Chondroitin. acid. (2) C18H27NOM + 3H20 = C12H2,NOn + 3CH3.COOH Chondroitin. Chondrosin. Acetic acid. (3) Cl2H21NOH + H20 = COOH(CH.OH)4.COH + C6Hn05.NH2 Chondrosin. Glucuronic Glucos- acid. amin. Both chondroitin and chondrosin are monobasic acids. The latter reduces Fehling's solution directly, while in the case of chondroitin this occurs only after the substance has been decomposed. Chondroitin-sulphuric acid is an amorphous substance, and is soluble in water. A concentrated solution resembles mucilage in appearance and consistence. Its salts are also for the most part soluble in water. The sodium and potassium salts can be pre- cipitated by means of ferric chloride, lead subacetate, and alcohol, while silver nitrate, zinc chloride, tannic acid, and potassium ferro- cyanide, the latter in the presence of acetic acid, are without effect. CARTILAGE. 385 In the cartilage its potassium and sodium salts occur both as such and in combination with collagen and albumins. A mixture of these compounds, according to Schmiedeberg, constitutes the so-called chondromucoid of Morner. If a solution of gelatin is mixed with an acidified solution of the potassium or sodium salts of the acid, a precipitate occurs. This also results if cartilage is boiled with water and the resulting impure solution of gelatin, which was formerly termed chondrin, is acidified with a dilute mineral acid. The precipitate consists of the free chondroitin-sulphuric acid, and is soluble in an excess of the acid. Chondroitin-sulphuric acid, while essentially a constituent of car- tilage, has also been found in other organs of the body, as in the inner coats of the larger arteries, in the kidneys, and under patho- logical conditions in amyloid livers. Traces are likewise found in the urine. Isolation. — To isolate chondroitin-sulphuric acid from cartilage shavings, the material is boiled with a 5 per cent, solution of caustic alkali. The solution is neutralized, and freed by filtration from the alkaline albuminates which have been formed during the process of boiling. Albumoses are removed by means of tannic acid, the excess of the latter by means of lead subacetate, and the excess of lead with hydrogen sulphide. The acid is then precipitated with alcohol. To purify the substance, it is dissolved in water, and the solution dialyzed and reprecipitated with alcohol. The solution in water and precipitation with alcohol is repeated several times, when the acid is finally washed with alcoholic ether. Isolation of Chondromucoid. — To isolate the chondromucoid, viz., the compounds of chondroitin-sulphuric acid with collagen and albumins, the cartilage shavings are first extracted with water, which dissolves the free chondroitin-sulphuric acid and a small amount of the chondromucoid. On acidulating this solution with a •') pro mille solution of hydrochloric acid and heating on a water- bath, the chondromucoid is gradually precipitated, while the free acid remains in solution. The cartilaginous residue is then ex- tracted with a 2 to 3 pro mille solution of hydrochloric acid at a temperature ol* .'>o° to 40° C, which dissolves any collagen that may be presenl a- such. Alter washing with water the remaining material i- extracted with a 5 pro mille solution of caustic alkali. The chondromucoid i~ thus dissolved, and is then precipitated with an acid. After repeated solution in an alkali and precipitation with an acid it i- finally washed with alcohol and ether. Albumoid. — The albumoid which is found in the cartilage of adult animals is apparently closely related to elastin and keratin, but differs from the latter iii containing sulphur, and from the former in its digestibility by gastric juice. It gives the common color-reac- tions of the albumins, bul is insoluble in all neutral solvents, and dissolves in acid- and alkalies only with great difficulty. Isolation. — To isolate the Bubstance, cartilage shavings arc first 386 THE SUPPORTING TISSUES. extracted with a 0.5 per cent, solution of caustic alkali, to remove the chondromucoid, and the free chondroitin-sulphuric acid. The remaining material is then washed with water and boiled with water in a Papin digester. Any collagen that may be present is thus dissolved, while the albuminoid together with the cartilage-cells remains behind. Mineral Constituents.— Among the mineral constituents of car- tilage, the very large amount of alkaline sulphates is especially noteworthy. These are supposedly not present in the free state, however, beyond traces perhaps, but result from the chondroitin- sulphate on incineration. In the cartilage of the shark, very curi- ously, sodium chloride constitutes as much as 94.2 per cent, of the total amount of mineral ash. As this represents 17.7 per cent, of the moist material, the amount of sodium chloride would be suffi- cient to form a concentrated solution in the cartilage, which, of course, is scarcely conceivable as occurring in living tissue. It is hence assumed that the salt is present in organic combination, but of its pairling nothing definite is known. According to Bunge, such large amounts of sodium chloride are also found in mammals during the period of intra-uterine life and shortly after birth, and he be- lieves that this is in accordance with the biogenetic law Mrhich under- lies the development of the higher forms of life from those of a lower order. With the appearance of old age a gradual deposition of calcium salts occurs in the matrix of the cartilage, so that partial ossifica- tion takes place. This, of course, also occurs during the develop- ment of normal bone, but it is to be noted that, in contradistinction to true bone, the matrix of senile, ossified cartilage retains its original characteristics. BONE. The matrix of bone-tissue, like its contained fibrils, is com- posed of collagen, which is here termed ossein, and is supposedly identical with the common form that is obtained from connective tissue. Of the composition of the cells, viz., the so-called bone- corpuscles, nothing is known. With their processes they occupy the lacunae and canaliculi, and are separated from the bony structure proper by a layer of a very resistant albuminous substance of unknown character. In contradistinction to the other supporting tissues of the body which have thus far been considered, bone-tissue apparently con- tains no glucoproteids. The function of the bone-tissue, as the principal supporting tissue of the body, finds its expression in the preponderance of the mineral constituents over the organic solids, and it is interesting to note that the ratio between the two is fairly constant, not only in different bones, but also in different animals. These salts are largely repre- sented by calcium phosphate and carbonate, which impregnate the BONE. 387 entire matrix. In addition, we find magnesium phosphate and small amounts of calcium chloride, calcium fluoride, potassium and sodium salts, and a little iron. Of the manner in which the differ- ent -alt- are combined with each other, nothing definite is known, but we may possibly assume, with Gabriel, that the composition of the bone-ash, as well as the tooth-ash, can be represented by the formula [Ca,(P04)2 + Ca5HP3013 + H20], in which 2 to 3 per cent. of calcium is replaced by magnesium, potassium, and sodium, and 4 to b' per cent, of the phosphoric acid by carbonic acid, chlorine, and fluorine. Whether or not the mineral constituents of the bone exist in com- bination with the organic components of the tissue has not as yet been definitely ascertained, but does not appear improbable. An idea of the quantitative distribution of the different salts _ in different animals and bones may be formed from the accompanying analyses. The figures have reference to 100 parts of bone-ash (Zalefsky) : Calcium phosphate (Ca3(P04).2) . . Magnesium phosphate (Mg3(P04)2) . Calcium in combination with carbon dioxide, chlorine, and fluorine . . Carbon dioxide1 Chlorine Fluorine2 Small flounder (ash in general) . Man fash in general) .Man ( humerus) < )x I lemur) ash in general) Babbits, varying in age between one day and four years (general asb | Human. Ox. Turtle Guinea- pig- 83.89 86.09 85.98 87.38 1.04 1.02 1.36 1.05 7.65 7.36 6.32 7.03 5.73 6.20 5.27 1.80 2.00 . . 2.30 3.00 2.00 Calcium Phosphoric Magnesium oxide acid oxide (CaO). (PA). (MgO). 53.13 42.72 0.91 52.83 38.73 0.48 51.31 36.65 0.77 51.28 37.46 1.05 51.01 38.19 1.27 51.91-52.89 39.78-42.20 0.83-1.38 The variations in the amount of bone-ash, as a whole, in different bones of the same animal, are seen in the following table (Fremy): Per cent. Femur Humerus Tibia f 64.1-64.6 Occipital bone I Cranium J Scapula 63.3 Vertebrae 54.2 The ; 1 1 1 1 ' » 1 1 1 i 1 of water which is found in bones varies between 13.8 and 1 L3 per cent. It is greater in the spongy bones than in those of the compact variety, and gradually diminishes with age. 1 These figures are lomewhal too low, us a certain amount of carbon dioxide escapes during the incineration of the bone. - according to Uabrlel, the amount of fluorine does not exceed u.i percent., and is usually less than 0.5 per cent. 388 THE SUPPORTING TISSUES. The bone-marrow is pervaded by a network of connective tissue, which is partly of the white fibrous variety and partly reticulated. In its meshes we find the cellular elements of the marrow, viz., the so-called myeloplaxes, the juvenile forms of the polynuclear neutrophilic and eosinophilic leucocytes, viz., the myelocytes, red cor- puscles in various stages of development, and a variable number of fat cells. These latter are especially numerous in the so-called yellow marrow, where the amount of fat may represent as much as 96 per cent, of the entire substance. It consists of olein, palmitin, and stearin. The red marroiv, on the other hand, contains a much smaller amount of fat, and owes its color to large numbers of red corpuscles. It contains albumins, of which one is regarded as a globulin, and is said to coagulate at 50° C. But especially interesting is the pres- ence of peculiar iron compounds which are as yet but little known, but probably belong to the nucleo-albumins and iron-containing albuminates. Their presence is no doubt intimately associated with the formation of red corpuscles. Among the extractives of bone- marrow we notably find lactic acid and hypoxanthin. THE TEETH. The dentin of the teeth is a peculiarly modified form of bone- tissue, and is likewise composed of an organic matrix, which con- sists of collagen and is impregnated with mineral salts. The latter are here even more abundant than in true bone, and represent from 64 to 68 per cent, of the fresh tissue, while of organic matter we find between 26 and 28 per cent., thus leaving 10 per cent, for water. The relative distribution of the individual salts is about the same as in common bone. The dentinal tubules, like the lacunas and canaliculi, are appar- ently lined by the same albuminous substance. The cement which surrounds the dentin of the root as far as the neck of the tooth consists of true bone. The enamel, in accordance with its epithelial origin, contains no collagen. It is very rich in lime salts, and its mineral constituents represent as much as 96 per cent, of the total substance. Its organic components correspond to about 3.6 per cent., but are as yet unknown. Water is practically absent. Other tissues which are closely related to bone are ivory, tortoise- shell, and the scales of fishes. In the two former the mineral con- stituents predominate over the organic matter, as in bone, while in the latter more organic material is found. Ivory is especially rich in magnesium phosphate, of which it contains about 15.72 per cent. The outer surface of tortoise-shell is covered with a layer of kera- tinized epidermis. In the invertebrate animals, with the exception of the cephalopods, and possibly also the branchiopods, collagen is not found. The ADIPOSE TISSUE. 389 internal supporting structures are here represented by ingrowths of the cutieular formations, which are derived from the epidermal cells, and consist largely of skdetins and hyabgens which have become impregnated with lime salts. Closely related to the latter is chitin, which enters largely into the composition of the outer _ skeleton of the arthropods and their nerve-sheaths. Associated with it we find the so-called bmiein, which is regarded as a cellulose, and which is found in especial abundance in the mantle of the tunicates. ADIPOSE TISSUE. Adipose tissue may be regarded as a special form of connective tissue in which the cellular elements enclose globules of fat. AVhen fully developed, the individual cells appear as greatly distended vesicles, which are covered by a cell-membrane. The original proto- plasm has been almost entirely replaced by fat, and occurs merely as a thin laver beneath the membrane. The nucleus also has been displaced to the periphery, and can scarcely be discerned with- out special methods of staining. Such fat-cells usually occur in -roups, and are held together by delicate fibres of connective tissue, in the meshes of which a network of blood-capillaries is found surrounding each cell. When large numbers of fat-cells occur, the individual groups are gathered into lobules, and these into lobes. In the living tissue the contained fat exists in a liquid form, but congeals after death, and is then more or less solid accord- ing to the character of the individual fats. Stearin and palmitin then often separate out in crystalline form. Of the chemical cora- position of the original cells/ before their invasion with fat-globules, oothing definite is known. They contain albumin and are appar- ently rich in water. The cell-membrane is exceedingly resistant to solvents, but is digested by the gastric juice, and possibly consists of an elastin-like substance. The relative amount of water and fat which is found in adipose tissue varies primarily with the state of nutrition, and differs in ditl'Tent animal-. Tie- fats in question are principally the triglycerides of stearic acid, palmitic acid, and oleic acid. Others, such as the pjlycerides ofcapronic acid and valerianic acid,are ool constant constituents of adipose tissue, but are met with only exceptionally, and always in very small ai mts. In man, a comparatively large amount oi olein is found, but it is not BO abundant as in certain cold-blooded animals, in which it may form the greater portion of the fat. The quantitative relation between the three forms is by 00 means con- -mni in all parts of the body, so that the melting-poinl of the fats from differenl regions may be quite different. It differs, moreover, in different animals. This ie shown in the following table, which is taken fr<»m Gautier : 390 THE SUPPORTING TISSUES. Mutton (subcutaneous) 27°-31° C. Mutton (perirenal) 37°-43° C. Mutton (epiploic) 36°-39° C. Man (panniculus adiposus) 15°-22° C. Man (perirenal ) ' 25° C. Dog 20°-22.5° Ox 39° C. Bone-marrow of ox 45° C. Calf 52° -f C. Horse 31° + C. Pig 40° C. Duck 35° C. Of special interest is the fact that it is possible to replace the com- mon fats of one animal by those of another, and even by fats which are not found normally in the animal world. If dogs, in which the fats have been removed by starvation, are thus fed with vegetable fats, such as rape-oil, this is subsequently found in the tissues of the animal, and may be recognized by its low melting-point (23° C.) and the presence of the glyceride of erucic acid. In a similar manner a deposition of mutton tallow may be effected, which begins to melt at about 40° C, while the common fat of dogs melts at 20° C. In addition to the fats, small amounts of lecithin, cholesterin, and free fatty acids may also be isolated from adipose tissue. We further find a yellow lipochrome, to which the color of the fat is due. Analysis of Adipose Tissue. — The material in question is first dried, ground with a little sand, and extracted successively with ether and alcohol. The alcoholic extract is evaporated to dry- ness, the residue washed with water to remove the soluble salts, and is then extracted with ether. The ethereal extracts are united, and the ether is distilled off, when the fats, lecithins, cholesterins, free fatty acids, and the lipochromes remain. The fatty acids are transformed into their salts by adding a slight excess of sodium carbonate, and heating to a temperature of 100° C. The resulting soaps are ex- tracted with water. The insoluble portion is dissolved in ether, the ether is distilled off, and the residue is heated on a water-bath with an alcoholic solution of sodium hydrate, which saponifies the fats. The resulting material is extracted with ether, which dissolves the cholesterin. The insoluble residue is dissolved in water, and the solution is saturated with carbon dioxide and extracted with strong alcohol. This takes up the soaps and the glycerin. The alcoholic solution is transformed into an aqueous solution, in which the soaps are decomposed with a dilute acid. The free fatty acids are thus precipitated, and can then be separated from each other according to the usual methods. Aside from adipose tissue, fats are met with in all the organs of the body, but, with the exception of the mammary glands during their functional activity, they are found normally only in traces. Under pathological conditions, however, notable quantities of fat ADIPOSE TISSUE. 391 may be met with. AVe then speak of a fatty degeneration of the organs. This is especially observed in the liver in cases of acute yellow atrophy, and can also be brought about artificially by poison- ing with phosphorus, antimony, arsenic, etc. Among the fluids of the body, large quantities are normally found only in the milk, and in the chyle during the process of digestion. Origin of the Fats. — That a portion of the fat that is found in the animal body is directly referable to the fats which have been ingested as such cannot be doubted. This is proved not only by the observation that it is possible to replace the fats which are peculiar to a certain animal by those of another, or even by vegetable 'ats, as has been shown above, but also by the fact that a gradual deposition of fats occurs in dogs which have been starved and are then fed on very little albuminous material, but with much fat. 1.1 such cases it can easily be proved that the amount of albu- mins invested is far too small to be the source of the fat that has been stored. The ingested fats, however, are not the only source of the fats found in the tissues, and there is evidence to show that they may also be derived from the albumins and the carbohydrates. Their origin from the former is suggested by many observations. It is thus well known that the albuminous constituents of human bodies when buried in moist ground, and notably the muscle-tissue, may undergo a peculiar transformation, which is characterized by the disappearance of the albumins and their replacement by free fatty acids and the calcium and ammonium soaps of palmitic acid and -tearic acid, which constitute the so-called adipoaere, or Leichen, wax of the Germans. This transformation, however, is probably brought about through the activity of micro-organisms, and does not prove in itself that in the living animal an actual formation of neutral fats can occur from the albumins. But it shows, at all event-, that forces which are at work in the living world can bring about the formation of two of the higher fatty acids at least which enter into the composition of the common fat from albuminous material. In the laboratory such a transformation has not as vet been accomplished, if we disregard the observations of E. Voit, who claims to have noted the appearance of higher fatty acids, when 43 grammes of albumin were kept in milk of lime for twelve months. Proof of the possible origin of fats from albumins, on the other band, seems to be afforded l,v the phenomena of fatty degenera- tion, where an actual deposition of large amounts of fat can be demonstrated in the cell- of organs in which only traces are nor- mally found. It has been urged, however, thai the fat which is here encountered has not developed /'// situ, but has been carried to the organs in question from the adipose tissue proper. That such a transposition of Cat- may occur is indeed possible, bul it has been conclusively shown that large quantities of fin may be isolated from 392 THE SUPPORTING TISSUES. the liver of dogs which have been poisoned with phosphorus after having previously fasted for twelve days. In such an event it scarcely seems admissible to attempt to account for the presence of the large amounts of fat in the liver on the theory of a transposi- tion. Bauer, moreover, has pointed out that while in such cases a largely increased elimination of nitrogen occurs, there is evidence to show that a non-nitrogenous portion of the albuminous molecule is retained, as the absorption of oxygen and the elimination of carbon dioxide are decreased by one-half. A further proof of the possible origin of fats from albumins has been furnished by Hofmann. Experimenting with maggots of flies, he determined the amount of fat in one portion directly, and then permitted a second portion of the same weight to develop in defibrinated blood containing a known amount of fat. These were then killed and analyzed. The result showed that they contained an amount of fat which was from seven to eleven times as large as the total amount of fat in the blood, plus the amount which they originally contained. Of the manner in which the fat originates from albuminous material we know nothing definite, but we may assume that non- nitrogenous groups are here first split off, and that the fats are then formed from these synthetically. That an actual liberation of fatty radicles can occur directly appears very unlikely, as we have no evidence whatever to show that the albuminous molecule contains any radicles with more than six or nine atoms of carbon. Of the nature of these non-nitrogenous groups we know nothing. We have shown, however, that most albumins contain a carbohydrate group, and that glucose and glycogen can both be derived from that source. The question hence suggests itself, Is it possible that the formation of fats from albumins takes place with the inter- mediary formation of carbohydrates? As a matter of fact, there is evidence to show that this may occur, as the possible origin of fats from carbohydrates is now well established. This, transforma- tion represents one of the most important synthetic phenomena which occur in the animal world, and is of the nature of a synthetic reduction, in which the CHOH groups of the carbohydrates are transformed into CH2 groups. That fats may actually be formed from carbohydrates can be demonstrated in various ways. Two animals from the same litter and approximately of equal weight are starved until the stored fat has mostly disappeared. The one is then killed so as to ascertain the amount of albumins and fats which still remains. The other animal is now fed with a definite quantity of some cereal, the con- tained amounts of albumins, starch, and fat of which are known. The feces are carefully collected and the amount of non-resorbed fat and albumins ascertained. After a variable period of time this animal is also killed, and the amount of albumins and fat esti- mated. The increase in the amount of albumins must, of course, ADIPOSE TISSUE. 393 he referable to that ingested, while the increase in the fat may be in part due to the ingested fat, in part to albumins, and in part to carbohydrates. In such cases it has been found that the amount of both albumins and fat which were contained in the food are by no means sufficient to account for all the accumulated fat, so that the conclusion is unavoidable that a certain proportion of this must be referable to the ingested carbohydrates. Calculation has, indeed, shown that as much as 86.7 per cent, of the fat is of such origin. Significance of the Fats. — As regards the function of fat in the animal body, our knowledge is still very imperfect. Owing to its property as a poor conductor of heat, it is probably of moment in preventing an undue irradiation. Its principal significance, how- ever, is undoubtedly connected with its manifest value as a food-stuff and as a source of energy. But we do not know whether this is expressed in any specific function of the body beyond the produc- tion of heat in general. As the fat disappears during starvation before the albumins are attacked, we may assume that its presence under normal conditions prevents an undue destruction of those ele- ments which essentially represent the living tissue. In this respect, however, the fats are inferior to the carbohydrates, as is apparent from the fact that in starving animals the administration of fats does not lead to so marked a diminution in the elimination of nitrogen as is effected by a corresponding amount of carbohydrates. Upon this basis also Voit lias explained the well-known phenomenon that herbivorous animals are more likely to accumulate albumins than the carnivora, as the latter receive scarcely any carbohydrates in their food, while that of the former contains comparatively large amounts. CHAPTER XX. THE SKIN AND ITS APPENDAGES. In considering the chemical composition of the skin and its appendages, we shall deal more exclusively with those substances which are more or less peculiar to its epidermal structures. The remaining components have already been studied in detail and require no further consideration at this place. The epidermal structures in the case of the vertebrate animals comprise the epithelial lining of the skin, together with its sweat glands, the sebaceous glands and allied glands, the hair, the nails, the hoofs, the feathers, etc. On section of the epidermis we discern different layers of cells. The lowest of these, which is known as the Malpighian layer, is composed of the youngest cells, from which all others are derived. These are distinctly protoplasmic in character, but with increasing age they become dry and scaly, and are finally represented by fine lamellas of keratin, which are constantly thrown off and regenerated from below. Keratin, itself, however, is not found in the lower layers of the epidermis, as has been shown by Ernst, but appears only above the so-called stratum granulosum. In the latter we find peculiar granules, which are scattered about the nuclei of the cells, and which Ernst regards as derivatives of the nuclei. They are known as eleidin granules, and, according to some observers, represent intermediary products in the formation of keratin. Of their chemical nature, however, nothing is known. The transition of the soluble albumins of the lower strata of cells into the insoluble keratin also finds its expression in the greater resistance which the upper layers offer to the action of the caustic alkalies. For, while the lower cells are dissolved with comparative ease the upper strata are scarcely affected by the reagent. Pancreatic juice and gastric juice behave in the same manner, and it is thus possible to separate the insoluble keratin from the soluble albumins which may be present at the same time. Like the horny layer of the skin, so also are other epidermal structures, such as nails, hair, horn, feathers, etc., largely composed of keratin. In addi- tion, we find a variable amount of salts, among which the insoluble forms are especially important. Their presence manifestly serves the purpose of increasing the rigidity of these structures. Espe- cially noteworthy is the large amount of silicic acid which is found in the hair and in feathers. Besides this, we meet with variable 394 THE SKIN AXD ITS APPENDAGES. 395 amounts of phosphates and sulphates of the alkalies and alkaline earths, and very curiously also with iron salts. The fact that larger amounts of the latter are found in dark-colored hair than in hair of a lighter color suggests that their presence may be dependent upon these pigments. The black and brown pigments which are found in the hair and in the skin of the negro belong to the group of the mdanins. Individually these bodies are but little known, and it is an open question whether the iron that is found in the ash is present in these structures in molecular combination with the pigments. Unlike the fuscin of the choroid and the hippomelanin that has been obtained from melanotic tumors in horses, the melanins of the skin and the hair arc easily soluble in solutions of the alkaline hydrates. They contain sulphur (2 to 4 per cent.), but not in so large amounts as the phymatorhusin that has been isolated from melanotic growths of man and from the urine (8 to 10 per cent.). Into the various pigments which have been found in the skin of reptiles, in the scales of fishes, in the feathers of birds, etc., it is scarcely necessary to enter at this place. They partly belong to the melanin-, partly to the lipochromes ; others are classified as mela- noids, while still others are closely related to the haemoglobins. To a certain extent, an >reover, the colors of birds' feathers appear to be of a physical nature and referable to certain phenomena of interference. In the invertebrate animals various pigments are also observed, but are for the most part unknown. The keratin, as I have already stated, is here represented by other tegumentary substances, such as chitin, tuniciu, the hyalogens, and the skeletins (which see). The Sweat. — The sweat is the specific, secretory product of the corresponding glands, which are found imbedded in the lower portion of the dermis. In man, these glands rank next in order to the kidney.- in importance as excretory organs of water, and arc capa- ble, to a certain extent at least, of assuming a vicarious activity when the kidneys are diseased. In man their number is quite large, exceeding 2,000,000. Their distribution, however, is not uniform, and as a result there are certain portions of the body in which a more abundant secretion is noted than in others. Such ions an- the forehead, the armpits, the palms of the bands, the Bolea of the feet, etc. Anion'/ the mammalian animal-, however, some exist in which a secretion of sweat docs not occur. This is the case in many of the rodent- and the goat. The sheep, the horse, and the ape-. on the other hand, sweat over their entire body, while other animals, like the eat and the dog, BWeal only from the balls of the tor-. The amount ,,(" -went excreted in man is very variable. It differs in different individuals ; and i- dependent upon the surrounding temperature, the amount of water ingested, the temperature of the body, the amount of exercise taken, etc. It is manifestly under the control of* the central -nervous system, and is increased by painful 396 THE SKIN AND ITS APPENDAGES. sensations and emotions, by stimulation of the sciatic nerve, follow- ing the administration of pilocarpin, etc. Ordinarily the secretion of sweat is scarcely noticeable, as the droplets evaporate almost as rapidly as they appear on the sur- face of the skin. But even so, from 700 to 900 c.c. of water are daily eliminated by the body. Artificially this amount can be greatly increased, and it is stated that from 6000 to 8000 c.c. may be excreted in the twenty-four hours if the body is kept at a tem- perature of from 40° to 50° C, and large amounts of fluid are ingested. Sweat, recently secreted, is more or less turbid, owing to an admix- ture of desquamated epithelial cells, and droplets of fat, which are in part derived from the sebaceous glands, but are to some extent also referable to the sweat-glands proper. After filtration it appears as a clear, transparent, colorless fluid, of a salty, somewhat acrid taste, and a very characteristic odor, which differs somewhat according to the region of the body from which the sweat is derived. Its specific gravity varies between 1.004 and 1.005. At the beginning of its secretion the sweat presents an acid reac- tion, which is probably referable to an admixture of fatty acids derived from the sebaceous glands ; later, however, it is alkaline. Under normal conditions the sweat is essentially a very dilute aqueous solution of mineral salts, but in addition we also find small amounts of many urinary components, such as urea, uric acid, kreatinin, aromatic oxy-acids, volatile fatty acids, skatoxyl and phenol sulphate, besides fat, cholesterin, traces of albumin, salts of lactic acid, and so-called sudoric or hydratic acid, etc. Larger amounts of solids are principally met with in cases of renal insuffi- ciency, and it may then happen that the elimination of urea through the sweat increases to 10 grammes, as compared with 0.043—1.55 pro mille, which may be regarded as normal. In some cases of this kind the urea may actually be found in the form of a fine cry- stalline powder deposited all over the skin. Glucose has been observed in diabetes. Cystin has been noted in cases of cystinuria, and abnormally large amounts of uric acid have been found in gout. In jaundice bilirubin may color the sweat a bright yellow. A blue and red color, which is thought to be referable to the presence of indigo-blue and indigo-red, has also been observed (chromhidrosis or cyanhidrosis) ; and it is stated that in rare instances blood may appear in the sweat (hsemahidrosis). It is noteworthy, furthermore, that a number of foreign substances, when ingested by the mouth, such as quinin, the various salts of iodine, mercury, and arsenic, are in part eliminated in the sweat. A general idea of the quantitative composition of the sweat may be formed from the accompanying analyses, which are taken from Favre, Schottin, and Funke. THE SKIN AND ITS APPENDAGES. 397 Sweat in gen- eral (obtained by elevation of tempera- ture). Favre. Water 995.573 Solids . . 4.427 Soluble in water : Sodium chloride 'J. 2.30 Potassium chloride 0.244 Alkaline sulphates 0.012 \ Alkaline phosphates traces J Albuminates 0.005 Insoluble in water, but soluble in acidu- lated water : Earthy phosphates traces Soluble in alcohol : Alkaline lactates 0.3171 Alkaline sudorates 1.562 ! Urea 0.043 j Fats and fatty acids . ...... 0.014 J Insoluble in water and alcohol : Epithelium traces Sweat (from extremities). SchSttin. 977.40 22.60 3.6 1 1.31 Funke. 988.40 11.60 4.36 0.39 J 11.30 4.20 7.24, of which 1.55 urea. 2.49 Gases. — While in mammals and birds the respiratory function of the skin is insignificant as compared with that of the lungs, we find that in the amphibia life may persist for quite a while after removal of the lungs, and that during this time oxygen is actively taken up from the air and carbon dioxide eliminated in turn. If, however, the exchange of gases is impeded or prevented by covering the skin with a thin layer of varnish, death rapidly takes place. This also occurs, it is true, in some of the smaller mammals which have a delicate skin, but it is now known that the fatal end is here not referable to the impairment of the cutaneous respiration, nor to a retention of waste products, as was formerly supposed, but to a paresis of the cutaneous vasomotor nerves and a resulting dilatation of the bloodvessels. As a result an abnormally increased irradia- tion of heat occurs, which constitutes the direct cause of death. If tin- is prevented by placing the animal in a warm chamber or by surrounding it with cotton and the like, recovery may take place, in the larger mammals, including man, in which a coarser skin exists, no deleterious effects are noted even after ten days. The amounl of carbon dioxide which is given off by man through the skin is principally dependent upon the surrounding temperature, and varies between 8. } grammes at 29° to 33° C, and 28.8 grammes ai 38.5 C. The Sebum. — The sebum is the specific secretory product of the Bebaceous glands, and serves the purpose of a lubricant. Amounts sufficient foY analytical purposes can be obtained from newly born children, in which the secretion constitutes the so-called vernix case- osa. In the fresh state it is a semiliquid, oily material, in which on microscopical examination can be discerned desquamated epithe- lial cells in various stages of degeneration, fal droplets, fatty acid needle-, and quite constantly also plates of cholesterin. Almost 398 THE SKIN AND ITS APPENDAGES. immediately on exposure to the air it solidifies to a white, tallow-like material. Its reaction is alkaline. The most important constituents of the sebum, as also of the related secretion of the uropygian gland of birds, are the compound eholesterins (see page 67). Their presence has been demonstrated on the feathers and bills of birds, in the wool of sheep, in the hair of mammals, on the spikes of porcupines, etc. ; and as these bodies are remarkably resistant to the influence of putrefactive organisms, it has been supposed that their presence on the skin and its append- ages serves the purposes of protecting the exposed portions of the body against bacterial invasion. In addition to substances of this order, the sebum contains a vari- able amount of fats, fatty acids, soaps, lecithins, mineral salts, and at least two albumins, of which one is commonly regarded as casein. Closely related to the common sebum of the sebaceous glands of the skin proper is the secretion of the preputial gland — the so-called smegma prseputii and the cerumen of the ceruminous glands of the external ear. In addition to the common constituents of the sebum, the smegma also contains certain components of the urine and their decomposition-products, such as ammonium soaps, and in the horse hippuric acid, benzoic acid, and oxalate of lime. Its peculiar odor in man is no doubt due to the presence of certain fatty acids. In the secretion of the beaver — the castoreum of the shops — this is thought to be referable to a phenol-like body, while in the corre- sponding product of the musk-deer (muse) a volatile base is, accord- ing to Wohler, the active odorous principle. The cerumen differs from the common sebum in containing a very considerable proportion of potassium soaps. In addition, we also meet with a peculiar yellow pigment, which has an exceedingly bitter taste ; but of the composition of this nothing is known. In the skin glands of certain amphibia, finally, and notably the toad and the salamander, poisonous substances have been found which in their physiological effect closely resemble the action of digitalis and strychnin. They have been termed bufidin and sala- mandrin, respectively. In the secretion of the toad, moreover, methyl-carbylamin and isocyan-acetic acid have been found, of which the former is especially toxic. CHAPTER XXI. THE GLANDULAR ORGANS. THE LIVER. The functions of the liver, as is apparent from a survey of the foregoing chapters, are manifold. During embryonic life the organ is intimately concerned in the production of red corpuscles, and at this time already manifests its function as an excretory organ also in the production of bile. After birth its hsemapoietic activity ceases, but it continues important as an excretory organ through which the decomposition-products of haemoglobin, in so far as they are not retained and utilized in the formation of new corpuscles, are eliminated from the body in association with taurin, glycocoll, cholesterin, and the cholalic acids. At the same time, however, the liver is the seat of some of the most important syntheses which occur in the animal body, and in which both anabolic and katabolic prod- ucts of the metabolism are involved. We have thus seen that the greater portion of urea in mammals, and of uric acid in birds and reptiles, is here produced, and we have also pointed out that certain aromatic substances which are formed during the process of intes- tinal putrefaction or have been ingested as such are transformed in the liver into conjugate sulphates and glucuronates and are thus rendered innocuous. Still other substances, moreover, which are for- eign to the body, such as various metallic salts and certain alkaloids, are here removed from the general circulation when artificially intro- duced, and it is for this reason also that the hypodermic injection of such substances is much more efficacious than their administra- tion by the mouth. The subsequent elimination of the metallic salts then occurs in part through the bile, and to a great extent also through the intestinal epithelium. The alkaloids are similarly re- moved, and also appear in the urine in a more or less modified form. Formerly it was supposed that the retransformation of peptones into native albumins occurred in the liver, but, as I have shown, tlii- h not the case. On the other hand, we have seen that the carbo- hydrates after their transformation into monosaccharides arc carried to the liver, and arc here stored in the form of glycogen when an immediate demand for glucose does not exist on the part of the other organs and tissues <>f the body. This transformation of mono- saccharides into glycogen represents one of the vaoai important syn- theses which OCCUr in the animal body, and it is interesting to note that whereas glycogen on decomposition always gives rise to the for- mation of glucose, the liver is capable of transforming the other monosaccharides into glycogen as well (see also page 167.) 399 400 THE GLANDULAR ORGANS. Of the forces which are at work in bringing about these various changes in the liver we know very little, but to judge from recent observations it appears that certain tissue ferments are here primarily concerned. The reaction of the living liver-tissue is alkaline. After death,, however, it becomes acid, and there is reason to believe that, as in the case of the muscle-tissue, this acid reaction is essentially refer- able to the formation of lactic acid. At the same time the tissue becomes opaque, owing to a coagulation of the liver albumins. A general idea of the chemical composition of the liver may be formed from the following analyses, which are taken from v. Bibra : Man. Ox. Water 761.7 713.9 Solids 238.3 286.1 Soluble albumins 24.0 23.5 Albuminoids 33.7 62.5 Fats 25.0 328 Extractives 60.7 49.1 Insoluble portion 94.4 112.9 The mineral salts, according to v. Bibra, constitute about 1 per cent, of the fresh tissue, and are essentially represented by the phos- phates of potassium and sodium and a fairly large amount of iron. Traces of manganese, copper, and lead are also found. The Albumins. — The albumins of the liver-tissue have been notably studied by Halliburton and Plosz. The gland was freed from blood and bile by transfusion with ice-water containing 0.75 per cent, of common salt. The tissue was then cut into small pieces with cooled knives, frozen and placed under pressure. On thawing, an alkaline fluid could thus be obtained, which represents the liver- plasma. In this fluid a globulin exists which coagulates at 45° C, and is regarded by Halliburton as being possibly identical with one of his cell-globulins. It can be digested by gastric juice. In addi- tion a nucleo-albumin was found, which coagulated at 70° C, and which yielded an insoluble residue of nuclein on digestion. From the cells proper they extracted a globulin with a 10 per cent, solution of sodium chloride, which coagulated at 75° O, and which may also be identical with one of Halliburton's cell-globulins ; further, an albumin (coagulation -point 70°-73° C.) and an alkaline albuminate. In addition, a glucoproteid has also been demonstrated, which yields a reducing substance on boiling with dilute mineral acids, and which is probably of a mucinous character and derived from the connective tissue of the organ. The nuclei finally contain nucleins, and it is of special interest to note that at least two of these contain iron. The one is apparently identical with, or at least closely related to, the hwmatogen of birds' eggs, while in the other, which Zaleski terms hepatin, the iron is even more firmly combined. The occurrence of these iron-contain- ing nucleins is important in view of the fact that the iron which is furnished in the food can apparently be utilized only by the THE LIVER. 401 body in the formation of haemoglobin, when introduced in such form. It has hence been suggested that these nucleins after resorp- tion are temporarily deposited in the liver until required by the baemapoietic organs. The iron which is present in the liver in molecular combination with the nucleins can be demonstrated only after the isolation of the nucleins in question and their incineration. But in addition we also find iron in combination with albumins as so-called iron- albuminates, from which the metal can be split off by treating with acid alcohol. The presence of this form can be directly demonstrated by moistening a slice of the liver-tissue with hydro- chloric acid, and then with a solution of potassium ferrocyanide or p itassium sulphocyanide, when a blue, viz., a red color develops. As regards the origin of these iron-albuminates, the opinion prevails that they are formed within the tissues of the body, and are refer- able to the disintegration of red corpuscles. They are accordingly also found in the spleen and the bone-marrow, and are met with in increased amounts in conditions which are associated with an increased destruction of red corpuscles. Large quantities are thus especially noted in cases of pernicious anaemia, in acute infantile gastritis, and in poisoning with arsenious hydride, where their presence constitutes the phenomenon of so-called siderosis of the liver. According to Vay, the average quantity of iron-albuminate which can be isolated from the fresh organ under normal conditions amounts to from 0.15 to 0.3 per cent., corresponding to from 0.01 to 0.018 per cent, of iron. The occurrence of especially large amounts of iron in the liver of newly horn animals is probably referable to the haemapoietic activity of the organ during embryonic life. Isolation of the Iron-containing Nucleins. — To prevent any con- tamination with haemoglobin, it is necessary to remove all traces of blood from the liver. To this ondy Bunge has suggested the follow- ing method : in the living animal which has been anaesthetized with morphin and chloroform a cannula is tied into the portal vein. Through this a stream of a 1 per cent, solution of sodium chlo- ride heated to the body temperature is introduced under moderate pressure. As Boon as the solution begins to flow the hepatic artery and the hepatic veins are divided and the abdomen closed. A minute later it is reopened, the liver is dissected out and placed in a porcelain bowl, while the transfusion is continued. The bowls arc changed until perfectly clear saline solution flows from the veins. To attain thi- end, the transfusion need l»e carried on for only a few minutes. If successfully performed, the liver should present a uni- formly light-brown color, and a portion of the minced organ when placed in distilled water should leave this entirely uncolored. The gall-bladder i- now removed, the organ pressed between filter-paper, finely bashed, and enveloped in muslin. It is (hen thoroughly kneaded under water. The connective tissue and vessels :ire thus 2fi 402 THE GLANDULAR ORGANS. separated from the cellular elements and remain behind. The cells are thoroughly extracted with water and with dilute saline solution, by decantation, until all soluble substances have been removed. They are then digested with gastric juice. The non-digested residue is extracted with acidulated alcohol and subsequently with ether, to remove pigments, cholesterin, and fats. It is then treated with weak ammonia-water, which dissolves the iron-containing nucleins. From this solution they are precipitated with absolute alcohol when added in excess. The resulting material constitutes the hepatin of Zaleski. The other iron-containing nuclein is apparently present in the liver as a nucleo-albumin, and is found in this form in the saline extract of the cells. To demonstrate its presence, the previ- ous extraction with saline solution is omitted. If the residue of nucleins, which remains after digestion with gastric juice, is then placed in a solution of ammonium sulphide, a greenish color gradu- ally develops which ultimately turns black, owing to the forma- tion of sulphide of iron. The hepatin itself does not give this reaction. Neither substance gives up its iron, even when treated with acidulated alcohol1 for days, thus differing from the iron albu- minate, which behaves in this manner exactly like inorganic prep- arations of iron. Isolation of the Iron- containing Albuminates. — In this case it is not necessary previously to wash out the blood. The organ is minced without further preparation, and is placed in from three to four times its volume of water. The mixture is slowly heated, boiled for about fifteen minutes, and filtered on cooling. The filtrate is carefully precipitated with a 10 per cent, solution of tartaric acid. The resulting flocculent material, which presents a brown color, is collected on a filter, washed with a weak solution of tartaric acid, then with 50 per cent, alcohol, and finally with absolute alcohol. It contains about 6 per cent, of iron. It is soluble in solutions of the alkalies, and does not react with ammonium sulphide at once. After a few minutes, however, the solution becomes darker and gradually turns black. On treating with acid alcohol (see above) the iron is split off, and can be directly demonstrated by testing with potassium ferrocyanide or potassium sulphocyanide. Schmiedeberg has termed the substance in question ferratin, and regards it as a ferri-albuminic acid. Ferments. — The ferments which occur in the liver are as yet but little known. It appears that several varieties exist, and it is quite probable that they are intimately concerned in the various functions of the organ. Some of the ferments are oxydases, and one of these in turn is an aldehydase, viz., a ferment that is capable of oxidizing salicylic aldehyde to the corresponding acid. The existence of another ferment which is capable of transforming firmly combined nitrogen into amido-nitrogen seems to have been established by 'The acidulated alcohol contains 10 volumes of a 25 per cent, solution of hydro- chloric acid and 90 volumes of 96 per cent, alcohol (Range's fluid). THE LIVER. 403 Jacoby. To the presence of this latter autolytic phenomena, which have been described by Salkowski and his pupils, are possibly due. A urea-forming enzyme also is said to occur in the liver, but its existence has not as yet been satisfactorily demonstrated. Glycogen. — Amount. — The amount of glycogen which occurs in the liver is primarily dependent upon the state of nutrition of the animal and the amount of exercise that is taken. This is apparent from the fact that it is constantly consumed during the activity of the muscle-tissue more especially, but is also utilized in the regeneration <>f all cellular elements of the body. During starvation it rapidly disappears, but it is also rapidly formed if carbohydrates are then ingested. Maximal amounts, according to Kiilz, are found after from fourteen to sixteen hours following the administration of food. It has been calculated that in the liver of man 150 grammes can be stored at one time. This would correspond to about 10 per cent, for an or^an weigdiino- 1500 grammes. In dogs which have been fed on potatoes and bread Pavy claims to have found as much as 17 per cent. After death the transformation of glycogen into glucose continues as in the case of muscle-tissue, and in order to ascertain the exact amount which was present during life it is hence necessary to remove the organ at once and to prevent the further inversion of the material, by the living protoplasm or the contained ferments, by placing the tissue in boiling water. Properties. — The pure substance represents a white, amorphous powder, which is both odorless and tasteless. In water it forms an opalescent solution, from which it can be precipitated by the addi- tion of alcohol, after adding a little sodium chloride, or by means of lead subacetate. The substance is dextrorotatory. The specific degree of rotation, however, seems to be influenced by various factor-. In pure solution it is given as f- 196.63°. It does not reduce Fehlin^'s solution, but can maintain cupric hydroxide in solution. After the addition of a little sodium chloride its solutions an; colored red by treating with iodine. With benzoyl chloride, in the presence of sodium hydrate, it gives a granular precipitate of benzoyl-glycogen. On boiling with dilute mineral acids it is trans- formed into glucose. Ferments invert it to maltose or glucose, according to the nature of the enzymes at work. Isolation and Quantitative Estimation. — The perfectly fresh liver, immediately after removal from the animal, is placed in boiling water and divided into -mall pieces. After boiling for a few minute-, these are removed, ground to a pulp with sand or pulverized glass, and then boiled in a 1 per cent, solution of sodium hydrate, using 100 c.c. 1 or every 100 grammes of tissue. With liver-tissue two to three hour- suffice, while with muscle-tissue it is best to boil for from four to eighl hour-. Care must be h;i then united with the watery solution first obtained, 404 THE GLANDULAR ORGANS. and neutralized with hydrochloric acid. After concentrating the resulting solution, the remaining albumins, notably gelatin, are pre- cipitated on cooling by alternate treatment .with a solution of iodo- mercuric iodide and hydrochloric acid added drop by drop. In the filtrate the glycogen is precipitated with an excess of alcohol. It is collected on a filter, washed with 60 per cent, alcohol, then with absolute alcohol and ether, and is finally dried in a desic- cator over sulphuric acid. From the weight thus obtained, that of the combined mineral salts must be deducted after incineration. Glucose. — The amount of glucose in the perfectly fresh liver varies between 0.2 and 0.6 per cent., but rapidly increases at the expense of the glycogen after the removal of the organ from the body. To obtain results which represent the actual amount that is present during life, it is hence necessary to eliminate the inverting action of the living protoplasm and of ferments by placing the organ in boiling water immediately after the death of the animal. It is then finely minced, thoroughly extracted with boiling water, and the sugar determined in the filtrate according to the usual methods. Fat. — The amount of fat which is found in the liver is quite large, as compared with the other organs of the body, and normally varies between 2 and 3.5 per cent. It is deposited in the cells, and beginning along the periphery of the acini increases in amount toward the centre. It is most abundant after meals, and to a certain degree is dependent upon the amount of fat ingested. Under suitable condi- tions the infiltration may become so marked as to simulate fatty degeneration ; but, in contradistinction to fatty infiltration, we find that in fatty degeneration the amount of the solids is markedly diminished. The amount of water in fatty infiltration is diminished, while in degenerative changes it is perhaps slightly increased. These relations are exemplified by the following figures, which are taken from Hammarsten : Water. Fat. ^SSd?* Normal liver . . 770 pro mille 20-35 pro mille 207-195 pro mille. Fatty defeneration 816 " " 87 " " 97 " " Fatty infiltration 616-621 " " 195-240 " " 184-145 " " Extractives. — The extractives which are found in the liver, aside from glycogen and glucose, are notably xanthin-bases, which are derived from the nuclei. They comprise xanthin, hypoxanthin, guanin, and adenin. Conjointly they represent about 4.52 pro mille of the dried tissue. They can be isolated, according to the method described on page 363. In addition we find small amounts of urea, uric acid, paralactic acid, and jecorin. Cystin also has been isolated from the normal liver of a horse and from that of the porpoise, but it is questionable whether the substance can actually be regarded as a normal constituent of the gland. It has once been obtained from the liver of a patient who during life had eliminated cystin in the urine. Under pathological conditions, and especially in acute yellow THE LYMPH-GLANDS. 405 atrophy, large quantities of paralactie acid have been found, in addition to a notable amount of leucin and tyrosin. In amyloid degeneration of the organ chondroitin-sulphuric aeid has been observed. Biliary pigments are normally not encountered in the liver-cells, but quite commonly they stain these an intense yellow in cases of obstructive jaundice. These various constituents have been studied in the foregoing chapters, and need not be reconsidered at this place. THE DIGESTIVE GLANDS. Tin- chemical composition of the digestive glands, viz., the salivary glands, the pancreas, and the glands of the stomach and the intes- tinal canal, is essentially expressed in the composition of their specific secretions, bearing in mind, however, that the various ferments exist in the cells as pro-enzymes. The mucin which is furnished by the sublingual and submaxillary glands and the small mucous glands of tin- stomach and the intestinal tract is similarly present as a mucin- ogen. Of common components, we find a certain amount of mineral salts, traces of the common albumins, nucleo-albumins, and nucleinic bases, and in the pancreas, in addition, free fatty acids, small amounts of leucin. tyrosin, inosit, and paralactie acid. In the pancreas a highly complex nucleo-glucoproteid is also found, which yields a somewhat less complex substance of the same character, together with a eoagulable albumin, when the fresh gland is boiled with water. The proteid is held in solution owing to its combination with an alkali, but is precipitated as such on treating with a dilute acid. Its elementary analysis has given the following results : carbon, 43 per cent.; hydrogen, •"> ; nitrogen, 0.7; and phosphorus, 4.5. In addition, the substance contains a considerable amount of iron. On digestion with gastric juice a nuclein remains behind, which i- very rich in phosphorus. Among the decomposition- products which result on hydrolysis with boiling hydrochloric acid we find a pentose and a large amount of nucleinic bases, among which guanin is especially abundant. According to Bang, a nucleinic acid can be isolated from the proteid, as also from the pancreas di- rectly, which he terms guanylic ; nitrogen and phosphorus, 7.64, which would correspond to the formula r.,,ir,fXl(lP.,017. On decomposition the substance yielded at least .°>5 percent, of guanin, about •';" per cent, of a pentose (calculated as glucose), and as a third product, ammonia. THE LYMPH-GLANDS. The lymph-glands comprise the lymph-glands proper, the thymus gland, and the spleen. Their fibrous framework, as T have pointed out (page 383), consists essentially of reticulin, but also contains fibres oi collagen and elastin. The composition of the cellular ele- 406 THE GLANDULAR ORGANS. ments of the glands has been considered in the section on the Animal Cell and in studying the leucocytes of the blood (pages 303 and 322). To recapitulate in brief, the cells contain small amounts of albumin, a very large proportion of nucleo-histon, besides lecithins, fats, cholesterins, traces of glycogen, succinic acid, and larger amounts of nucleinic bases, among which adenin predominates. In the spleen we also meet with uric acid, and, as in the liver, with iron-containing nucleins and iron albuminates, which may be isolated as there described. In addition, small amounts of inosit, jecorin, and cerebrosides may be encountered. Of special inter- est is the fact, which has been established by Gulewitscfh, that arginin is a normal constituent of the spleen. All these bodies have been described and require no further consideration at this place. THE KIDNEYS. In addition to the common albuminoids which enter into the com- position of the supporting tissue of the kidneys, we find the common extractives, viz., nucleinic bases, uric acid, urea, leucin, inosit, gly- cogen, fats, and at times taurin. All these substances, however, are present in only small amounts. On one occasion, in the ox, cystin has also been encountered, but it is questionable whether this is a constant constituent of the organs. Of albumins, Halliburton has isolated a globulin and a nucleo-albumin, with coagulation-points of 52° C. and 63° C, respectively. In addition, a mucin-like body has been found, which does not yield a reducing substance, however, on boiling with mineral acids, and which is probably a nucleo- albumin. It is notably found in the papillary portion of the kidneys, while the other nucleo-albumin is principally met with in the cortical portion. Serum-albumin is said to be absent. THE MAMMARY GLANDS. The chemical composition of the mammary glands has not been studied in detail. We know, however, that the protoplasm of the functionally active glands is rich in albumins, and it appears that, as in the .case of the pancreas, a very complex nucleo-gluco- proteid is here also present, and is probably intimately concerned in the formation of two of the most important constituents of the milk, viz., the casein and lactose. It may be obtained in solution by first washing the gland thoroughly in water, so as to free it from milk ; it is then extracted with a 0.5 pro mille solution of sodium hydrate at ordinary temperatures. Such solutions also contain the common albumins, and represent an exceedingly viscid, stringy fluid, from which the proteid in question can be precipitated by acidifying care- fully with dilute acetic acid. On boiling with dilute acids the sub- stance is decomposed into albumin, phosphoric acid, and a reducing substance of unknown composition. On digestion with gastric juice it yields a paranuclein. THE MILK. 407 V< in the case of the pancreas, the substance is decomposed by boiling the -land with water. A coagukble albumin and a nucleo- elucoproteid. which is somewhat less complex than the original sub- stance thus result. From this solution the proteid can be precipi- tated by the addition of a dilute acid. Like its niother-substance, it also yields a reducing substance on hydrolytic decomposition. Of the rclati.-n of the latter to lactose nothing is known but it is noteworthy that this is formed on standing if a functionally active u-land, while perfectly fresh, is ground to a pulp and kept in normal salt solution at the temperature of the body. An intermediary prod- uct is then also apparently formed, which is of a colloid nature but not identical with glycogen. In view of recent researches, which tend to show that the reducing group which is present in the gluco- proteids is. in the case of the mucins at least, not a true carbohydrate, but of the nature of chondroitin-sulphuric acid or an allied substance, it would be exceedingly interesting to ascertain whether the reduc- ing substance in the case of the mammary nucleo-glucoproteid also may not be of this order. . Of other constituents of the gland, we find various xanthin- bases and in the functionally actiye organ also a certain amount ot fat which is present in the form of globules of variable size, in the bodies of the cells. . ... The specific secretorv product of the mammary glands is the milk. The Milk.— The milk is the specific secretory product ot the mammary glands, and constitutes the natural food of all mammals in the early stages of their extra-uterine existence. It contains all those food-stuffs which are necessary for the maintenance ot lite, viz albumins, carbohydrates, and fats. The nutrient components of the milk, however, are more or less specific of the secretion in question, and are not found elsewhere in the body as such. They arc produced in the -land itself from the common constituents of the blood. Am ong these the albumins are the most important, alHl there can be little doubt at the present time that the fits of the milk also are largely referable to this source. This is appar- enl ,yom thefacl thai in the bitch, for example, the amount of tai increases with an increased ingestion of meat that is free from fats, while it is diminished when the animal is fed on fats only. # I lie 80-called milk-SUgar also is apparently derived from albumins, as the substance continues to be formed although no carbohydrates are ingested. It- amount, however, is then somewhat smaller, and increases if cane-sugar or starch i- added to the diet. General Characteristics.— Fresh milk is an opaque, white, yellow- ish-white, or bluish-White liquid, of a somewhat creamy consistence, a more or less sweetish taste, and an insipid odor which is peculiar to the particular animal from which the milk has been obtain..!. On microscopical examination it is seen that the opacity is largely due to the presence of fat-globules, wind, vary from 0.002 14 to 00046 ram. in diameter, and number from 200,000 to 5,000,000 408 THE GLANDULAR ORGANS. per cbmm.j with an average of about 1,050,000. The fat is thus present in a state of fine emulsion, but, in contradistinction to other emulsions, in feebly alkaline media, it cannot be extracted by shak- ing with ether directly, or at least only with much difficulty. If, on the other hand, an acid or a caustic alkali is previously added to the milk, this is readily accomplished. From this observation it has been concluded that each fat-globule is surrounded by an albumi- nous membrane, the haptogenic membrane of Ascherson, which is dissolved by acids and alkalies, but which normally prevents the solvent action of the ether upon the contained fat. Later investi- gations have rendered this view improbable, however, and, as a matter of fact, no one has ever succeeded in demonstrating the pres- ence of a special membrane. The normal resistance to the action of ether is now explained upon the assumption that each globule is surrounded by a delicate layer of albumin, which does not consti- tute a true membrane, however, but is formed as a result of molecu- lar attraction. It is possible, indeed, to prepare emulsions of fat artificially by shaking with albuminous solutions, which in their behavior to ether are quite similar to milk. As regards the char- acter of the particular albumin which forms this layer, our knowl- edge is not complete. It has been supposed by some that it is formed by casein, but there are reasons for believing that the albumins of the milk in general may here be concerned. On standing, the greater portion of the fat rises to the surface of the milk and forms its cream. On beating the milk for some time, the individual fat-globules are caused to coalesce, and sepa- rate out as a semisolid mass, which constitutes the butter. The remaining liquid is termed buttermilk, and still contains a consider- able amount of fat which has remained in emulsion. Besides the fat-globules the milk contains also innumerable gran- ules of calcium phosphate (probably a mixture of diphosphates and triphosphates) in suspension, which are visible only on microscopical examination and are said to number about 4,000,000 per cbmm. On filtration through a Chamberlain filter, under pressure, these remain behind together with the fat. But we then also find that one of the most important albuminous constituents of the milk, viz., casein, which is found in combination with lime, is likewise not present in solution, and is thus obtained in the form of a thin, jelly- like material. The filtrate constitutes the milk-serum and contains those components of the fluid which are present in a state of actual solution. Upon the addition of chymosin to fresh milk, at the temperature of the body, it coagulates almost at once. The resulting clot, which constitutes cheese, then contracts and a yellowish fluid gradu- ally appears, which is termed sweet whey. During this process the reaction of the milk is not changed. A similar coagulation is noted when fresh milk is allowed to stand exposed to the air. In this case, however, the reaction of the whey is acid, owing to the formation of THE MILK. 409 lactic acid from lactose in consequence of the activity of certain micro-organisms. Perfectly fresh milk does not coagulate on boiling-, but it will be noted that a skin forms on the surface of the milk, which is rapidly reformed when removed. This consists of coagulated casein in com- bination with mineral salts, and especially phosphates of calcium. Actual coagulation does not occur, even if a current of carbon dioxide has previously been passed through the liquid. If the milk has stood for some time, however, and lactic acid fermentation has begun, a tendency to coagulation soon becomes manifest, and at different stages this may then be effected by boiling after saturation with carbon dioxide, then by boiling alone, subsequently on treating with carbon dioxide without boiling ; and finally, as I have stated, it occurs spontaneously. Sterilization of the milk, with the subsequent exclu- sion of micro-organisms, as also the addition of preservatives, such as boric acid, salicylic acid, thymol, etc., will prevent lactic acid fer- mentation, and consequently also coagulation referable to this source. On exposure to the air, milk is said to absorb its own volume of oxygen within three days. Amount. — -The amount of milk furnished in the twenty-four hours is, of course, different in different animals. It is largely dependent upon the development of the glands, and accordingly is most abun- dant in those animals in which by artificial selection a marked hyper- trophy of the organs has been produced. Some cowrs may thus yield 24 liters of milk in the twenty-four hours. The amount is further influenced by the age, as also by the character of the diet, the amount of liquid ingested, etc. Especially important is the character of the diet, ami notably the amount of albuminous food that is ingested. Where this is deficient the amount of milk is diminished, while, •■ eteris paribus, larger amounts are furnished if an abundance of albumins is ingested. Women furnish from 900 to 1000 grammes on an average during the height of lactation ; loOO grammes probably represent the maxi- mum output. Good cows commonly yield from b' to 10 liters, goats and sheep about 1 liter, in the twenty-four hours. With the gradual cessation of lactation and the coincident atrophy of the mammary glands the amount decreases, until finally the secretion is arrested entirely. In women and cows the period of lactation usually lasts about ten months. Specific Gravity. — The specific gravity of the milk is largely de- pendent upon the amounl of fal present, and is much the same in different animals. Its normal variations are seen in the accom- panying table : Woman 1.028-1.034 1.029-1.034 1.030-1.034 MP 1.037 1.040 \ 1.029 1.035 Mara 1.028-1.034 Bitch 1.034 1.040 410 THE GLANDULAR ORGANS. Skimmed milk is, of course, specifically heavier than full milk, and a higher specific gravity is accordingly also noted in milk which is poor in fat than in rich milk. Reaction. — Woman's milk and that of most herbivorous animals is uniformly amphoteric, owing to the presence of diacid and mon- acid phosphates in association with the calcium compound of casein. The relative values of the acid and basic components in cows' milk and human milk are given below in terms of decinormal sodium hydrate and sulphuric acid solution. The figures have reference to 100 c.c. of milk and are average values (Courant) : TySTaOH i10H2S04 Ratio. Human milk 10.8 c.c. 3.6 c c. 3:1 Cows' milk 41.0 c.c. 19.5 c.c. 2:1 Human milk is thus relatively more alkaline than cows' milk, but is absolutely both less alkaline and less acid. Mares' milk is alkaline and that of the carnivorous animals acid. Chemical Composition. — A general idea of the chemical composi- tion of the milk of different animals and of woman may be formed from the following analyses, which are taken from Konig, Gorup- Besanez, Hoppe-Seyler, and others : Human. sCow. Water : 872.40-892.90 842.8-860.0 Solids 108.00-127.60 140.0-157.2 Albumins (total) 16.13-36.91 33.0-43.2 Albumin (proper) 3.50- 9.91 1.2- 2.8 Casein 12.80- 27.00 30.2- 42.0 Fats 25.60- 43.20 40.0- 64.7 Lactose 53.90- 60.90 43.4- 50.0 Salts 1.650- 4.200 6.3- 7.1 A survey of this table thus shows that human milk contains a smaller amount of albumins and fats but more lactose than cows' milk. In addition to the above components the milk contains traces of urea, kreatin, kreatinin, hypoxanthin, cholesterin, animal gum, and, curiously enough, citric acid, which is present as a calcium salt to the extent of from 0.18 to 0.25 per cent. Besides these, we find a small amount of lecithins and a yellow lipochrome. Goat. Sheep. Mare. Ass. Dog. Cat. Water 869.1 835.0 900.6 900.0 754.4 816.3 Solids 130.9 165.0 99.4 100.0 245.6 183.7 Albumins . . 36.9 57.4 1S.9 21.0 99.1 90.8 Fats .... 40.9 61.4 10.9 13.0 95.7 33.3 Lactose ... 445 39.6 66.5 63.0 31.9 49.1 Salts .... 8.6 6.Q 3.1 3.0 7.3 5.8 Analysis of the inorganic components of human milk has given the following results (Bunge) : the figures of the first column were obtained at a time when but little sodium chloride was ingested, while those of the second column were gotten while the woman ingested 30 grammes a day (the total ash is calculated as 1000 parts by weight) : THE MILK. 411 I. II. Potassium (K20) 0.780 0.703 Sodium (Xa.,U) 0.232 0.257 Calcium (OaO) 0.328 0.343 Magnesium (MgO) 0.064 0.065 Iron (Fe203) 0.004 0.006 Pbospborie acid (,P205) °-478 °-469 Chlorine (CI) 0.438 0.445 The differences which exist in the composition of full milk, as compared with skimmed milk, cream, buttermilk, and whey, are shown below : Full milk Shimmed c Buttermilk. Whey. (cows). milk. J Water 871.7 906.6 655.1 902.7 932.4 Solids 128.3 93.4 344.U 97.3 67.6 Albumins . . 35.5 31.1 35.5 35.5 8.5 Fats .... 36.9 7.4 267.5 9.3 2.3 Lactose . . . 48.8 47.5 35.2 37.3 47.0 Lactic acid . . none none none 3.4 3.3 Salts 7.4 7.4 6.1 6.7 6.5 Of gases, milk contains a small amount of oxygen and nitrogen, and from 5.8 to 7.5 per cent, of carbon dioxide, which can be removed with the exhaust pump. The Albumins. — The albumins which are found in milk are casein, lactalbumin, and so-called lactoglobulin, which is probably identical with the serum-globulin of the blood-plasma. Of these, casein is the most abundant and the most important. Casein. — Casein is a nudeo-albumin, and has the character of a dibasic acid. In the dry state it occurs as a white amorphous powder, which i- almost insoluble in water, in dilute acids, and solu- tion- of the neutral salts. In dilute solutions of the alkaline hydrates and in lime-water it dissolves with ease, at the same time forming gaits. Such solution- are neutral or slightly acid in reaction, accord- ing to the amount of alkali that lias been added, which is owing to the formation of neutral or acid salts, respectively. When triturated in water with calcium or sodium carbonate, the carbonates are decom- posed with the liberation of carbon dioxide; the same salts are then formed a- in the case of the alkaline hydrates. Soldner has isolated two calcium salts of casein, containing 1.55 and 2.36 per cent, of calcium oxide; according to Courant, these are dicalcium and tricalcium casein, respectively. The salt> of casein with the alkalies and alkaline earths are readily soluble in water, even in the absence of neutral -,-ilr-, and arc hence not precipitated on dialysis. On decomposition with dilute acids the free casein is obtained again in insoluble form. Suspended in water, the substance is coagulated on boiling, t ud rpU9cles, and cellular bodies enclosing lecithin granules, besides a large number of free granules, which arc apparently of an albumin- ous nature. In a fresh specimen the normal spermatozoa are ac- tively motile, and continue so for a variable length of time if evapo- ration is prevented Tin' movements are in all likelihood analogous to those of the cilia of certain epithelial elements of the body, and are arrested by the addition of water, dilute acids, alcohol, ether, strongly alkaline Solutions, etc. in dilute alkaline solutions, on the other hand, ami those of the neutral salts they continue for a long time. Semen i- heavier than water, and falls to the bottom as a jelly- like mass : at the same time a light flocculent precipitate develops, which consist* of the so-called fibrin of Eenle. On exposure to the air it is apparently coagulated, but later becomes Liquid, as before. It- reaction i- neutral or slightly alkaline. Testicular Bemen, in contradistinction to that which has been ejaculated, is said to be odorless. After emission, however, an odor develops which is suggestive of glutin, and is supposedly referable 420 THE GLANDULAR ORGANS. to the presence of an alkaloidal substance — spermin. In combina- tion with phosphoric acid, this is found in the secretion of the prostate gland, as phosphate of spermin, and is partly decomposed on exposure to the air, with the liberation of the free base (see below). The Spermatic Liquid. — The liquid in which the spermatozoa are suspended is nearly transparent. It contains a small amount of mucin (?) ; a nucleo-albumin, which has been termed spermatin, and which is precipitated by acetic acid, but is readily soluble in an excess of the reagent; also, cerebrin and lecithins, phosphate of spermin, and various mineral salts, among which sodium chloride and the phosphates of the alkaline earths predominate. Spermin. — Spermin, as stated above, occurs in the spermatic liquid in combination with phosphoric acid, as phosphate of spermin ; it is viewed as ethylenimin, C2H5N, and is manifestly closely related to the diethylene diamin (piperazin) of Ladenburg and Abel. To the free base the peculiar odor of the semen is, as I have said, supposedly due. This disappears after a short while, owing to a polymerization of the ethylenimin to diethylene diamin (piperazin) as shown in the equation : /H 7NHV 2Nf CH2 = C2HZ >C2H4. \CH2 NNH/ The phosphate can be readily obtained in crystalline form on slow evaporation of the semen, but may also separate out spontaneously on standing for about twenty-four hours. It occurs in the form of hexagonal pyramids, which appear under the microscope as flat needles. They are soluble in dilute acids and alkalies, as also in ammonia, less readily so in hot water, and are insoluble in alcohol, ether, and chloroform. These crystals are known as Bottcher's spermin-cry state, and are probably identical with- the so-called (Jliarcot-Leyden crystals, which are commonly found in asthmatic sputa, and also occur in the blood and lymph-gland of leukemic patients. They have likewise been observed in dried egg-albumin and in anatomical specimens preserved in alcohol, and also develop in red bone-marrow that has been exposed to the air for a few days. Heated to 100° C, the crystals turn yellow and melt near 170° C, but are at the same time decomposed. To isolate the free base, the semen is extracted with alcohol and then with dilute sulphuric acid. If the acid extract is then treated with baryta-water and evaporated at a low temperature, the free base is obtained. It can be precipitated from its solution by treating with auric chloride, platinum chloride, argentic nitrate, tannic acid, phosphotungstic acid, etc. Spermin has attracted much attention of late, owing to the stimulating effect which the substance is supposed to exert upon the oxidation processes of the body, the functions of the central nervous THE REPRODUCTIVE GLAXDS. 421 system, and the reproductive organs. It represents the active prin- ciple of Brown-Sequard's elixir. The Spermatozoa. — Our knowledge of the chemical composi- tion of the spermatozoa has been greatly extended within recent years through the researches of Kossel and his pupils, preceded by those of Miescher and Piccard. These observers were able to show that in certain fishes, such as the salmon, sturgeon, pike, shad, herring, and mackerel, substances can be isolated from the mature spermatozoa, which apparently represent the simplest forms of albumin, and are now collectively termed protamins. Their general characteristics have already been described (page 69), and I shall merely recall at this place that, according to Kossel, a protamin radicle is contained in all albumins and represents the fundamental nucleus of the albuminous molecule. These protamins, of which several varieties are described, and which yield the hexon-bases on hydrolytic decomposition, are supposedly combined with nucleinic acids to form nucleins. The individual nucleinic bases which further enter into the construction of the nucleinic acids are the common forms, which are also found elsewhere in the animal body. But it appears that the spermatozoa of different animals do not con- tain all forms. In the case of the salmon, Miescher and Piccard thus found guanin and hypoxanthin, while from the semen of the carp Kossel obtained adenin and hypoxanthin, as also small amounts of xanthin, but no guanin. Inoko, on the other hand, claims to have found all forms in the semen of the salmon, boar, and ox, but states that the relative amounts of the individual forms are not constant. Immature spermatozoa appai-cntly contain no protamins as such, and researches must be undertaken to ascertain whether the results which have thus far been obtained in the lower forms of animal life also hold good for the higher forms. But even so, it is apparent that the protamins play an important rdle in the process of reproduction, and a key may thus be furnished which will admit of an insight into the chemical basis of those mysterious morphological changes which find their expression in the development of the ovum. For there can be no doubt that in those animals in which the presence of protamins has been established in the spermatozoa they represent the essential reproductive elements on the part of lie- male. This suggests itself at once from a survey of the analysis of the spermatozoa of the salmon as given by Miescher: The nucleins, which are here referred to are, according to Kossel, nucleinic acids : Per Cent. NucleiM 48.68 Protamine (salmin) ii ' >.T * > Other albumiiu 10.32 !.«•< ithina 7.47 Cliolefterin 2:2\ Fata 4.53 422 THE GLANDULAR ORGANS. The albumins referred to in this table have not been studied in detail. One of them, according to Miescher, contains 4 per cent, of sulphur. In addition, the spermatozoa are said to contain a cere- broside, which is similar to cerebrin ; also a very considerable proportion of inorganic salts, which are essentially represented by phosphates. Detailed analyses of the spermatozoa of the higher animals and of man are not yet available. As regards the composition of the separate parts of the sperma- tozoa very little is known, bat it seems that the protamins, com- bined with nucleinic acids, are the most important components of the head. The tails are dissolved in gastric juice on prolonged digestion, and hence probably consist of albumins. As a whole, the spermatozoa are exceedingly resistant to ordinary solvents. They are soluble in boiling solutions of the caustic alkalies, while in concentrated sulphuric acid, nitric acid, acetic acid, and boiling- solutions of sodium carbonate they dissolve only in part. They are likewise resistant to putrefactive changes, and can be obtained from dried semen, with the preservation of their natural form, by placing the material in a 1 per cent, solution of sodium chloride. For a detailed description of the methods which are employed in the isolation of the individual protamins, I must refer the reader to the articles of Kossel, Kurajeff, and others. A satisfactory result may be expected only if the spermatozoa are mature, but even then no protamins may be found, as I have already indicated. Working with mature testicles of the sea-trout, I was unable to obtain a body of this order. The Ovaries. — Thus far a study of the chemical composition of the ovaries has not revealed any special points of interest. In addition to collagen and mucins, which enter into the con- struction of the supporting tissue of the organs, nucleins and true albumins have also been found, and are probably derived from the contained ova and other cellular elements. The most important constituents of the cortex of the gland, viz.r the Graafian follicles, which enclose the specific product of the func- tional activity of the ovaries, viz., the ova, have for obvious reasons not been open to a detailed investigation. The contained fluid is apparently serous in character. After the discharge of the ova the remaining follicles are first filled with blood from the torn vessels of the vesicle, and are subsequently transformed into the so-called corpora lutea. The yellow color of these is owing to lipo- chromes, or luteins, of which an amorphous and a crystalline form may be isolated (see also page 429). The Ovum. — Of the chemical composition of the ova of the human being and mammals in general, nothing definite is known, as it is impossible to collect them for purposes of analysis. The eggs of fishes, amphibia, reptiles, and especially of birds, on the THE REPRODUCTIVE GLANDS. 423 other hand, can readily be obtained and have been studied in greater detail. In the following pages we shall confine our attention to the composition of birds7 eggs, which is best understood. The egg proper is here surrounded by the so-called white of egg, which in turn is enclosed in a double membrane, and is covered by the shell. These additional structures, however, are not formed in the ovary, but are produced during the passage of the egg through the oviduct from material, which is -here secreted by the lining cells. The Shell. — The shell consists essentially of an organic matrix of the character of keratin, which is largely impregnated with lime salts. Of these, calcium carbonate is the most abundant, and con- stitutes about 90 per cent, of the weight of the entire shell. In addition, we find a small amount of magnesium carbonate, as also phosphates of both elements. Water is present to the extent of only about 1 per cent. The pigments met with in birds' eggs are closely related to the biliary pigments, and, like these, are derived from the common pigment of blood. The oorhodc'ln, which presents a reddish or brownish-red color, is supposedly identical with haematoporphyrin ; while the blue or green pigment, which is termed oocyanin, is composed partly of biliverdin, and is in part a blue derivative of bilirubin. The membranes of birds' eggs consist essentially of keratin, but contain also a small amount of mineral salts, of which calcium phosphate is the most abundant. In fishes and amphibia the egg envelope is represented by a trans- parent gelatinous material, which seems to consist almost exclusively of mucin. In the invertebrates chitin and skeletins take the place of the keratin of birds' egg*, but in some the latter also is found. The weight of the shell and membranes in the case of hens' eggs represents about 9 to 11 per cent, of the total weight of the egg, while the albumen constitutes about 60.5 per cent, and the yolk, viz., the ovum proper, the remaining 29 per cent. The total weight of hens' eggs may vary between 40 and 70 grammes. The Albumen. — The albumen or white of egg, as obtained directly from the raw egg, appeai-s as a faintly yellow, exceedingly viscid, semi liquid material. On microscopical examination this can be shown to consist of compartments, which are limited by very delicate membranes, and enclose the albumen proper. These membranes are continuous with the so-called chalazse and the membranes immediately beneath the shell, and are, like these, composed of keratin. The albumen proper may be separated from its membranous con- stituents by pressing the material through a cloth, and then appears :i- an opalescenl fluid, which is only slightly viscid, and can be filtered without much difficulty. It- reaction is distinctly alkaline, and the specific gravity about 1.045. On boiling, it coagulates to a compad ma--, which in the case of liens' eggs is entirely opaque. In some birds, however, such a- the SWalloW, the crow, the finch, 424 THE GLANDULAR ORGANS. etc. — i. e., in true nesting birds — the albumen remains transparent, owing to the formation of alkaline albuminates. Such albumen has been termed tata-albumen. It may be produced artificially by placing hens' eggs in a 10 per cent, solution of sodium hydrate for two or three days, when a gradual diffusion of alkali occurs into the albu- men. On subsequent boiling, this appears like true tata-albumen. Analysis of the albumen of hens' eggs has given the following results : Per cent. Water 80.00-86.68 Solids 13.32-20.00 Albumins 11.50-12.27 Extractives 0.38- 0.77 Glucose 0.10- 0.50 Fats and soaps traces Mineral salts 0.30- 0.66 Lecithins and cholesterin traces According to Poleck and Weber, the mineral ash has the follow- ing composition, calculated for 100 parts: Sodium (Na.,0) 23.56-32.93 Potassium (K.,0) 27.66-28.45 Calcium (CaO) 1.74- 2.90 Magnesium (MgO) 1.60- 3.17 Iron (Fe203) 0.44- 0.55 Chlorine (CI) 23.84-28.56 Phosphoric acid (P205) 3.16-4.83 Carbonic acid (C02) 9.67-11.60 Sulphuric acid (S()3) 1.32- 2.63 Silicic acid (Si02) 0 28-0.49 Fluorine (Fl) traces Of these constituents, the large amount of sodium chloride is espe- cially noteworthy, and shows in itself that the albumen is in reality a secretory product, and does not represent a mere transudation from the blood-plasma. One portion of the bases is in combination with the albumins of the albumen, while the remainder exists in the form of sulphates, phosphates, and notably carbonates. The slightly yellow color of albumen is referable to the presence of a lipochrome, which can be demonstrated on spectroscopic exam- ination. The Albumins. — The albumins of albumen are largely represented by the so-called ovalbumins ; in addition, globulins are found, as also a mucoid, which is known as ovomucoid. Ovalbumins. — According to Gautier and others, the albumen con- tains three ovalbumins, which are termed a-, /?-, and ^-ovalbumin, respectively. They are all closely related to the common serum- albumin of the blood-plasma, but differ from this in several impor- tant particulars. When introduced into the circulation as such, they are eliminated in the urine as foreign matter. They are pre- cipitated if a sufficient amount of hydrochloric acid is added, but are soluble in an excess with much greater difficulty than serum- THE REPRODUCTIVE GLANDS. 425 albumin. Alcohol and ether rapidly destroy their solubility. The difference in their degree of* rotation on polariscopic examination, as compared with serum-albumin, is seen below : Serum-albumin a ( I>) f>2.6°-64.6° Ovalbumin-a a (D) 33.1° Ovalbumin-/? all)) 53. 6° Ovalbumin-? a (D) 70.8° The coagulation-point of the three ovalbumins collectively, in a 0.3 per cent, solution, is at 56° C. That of the individual sub- stances is given as 72° C, 76° C, and 82° C, respectively. The elementary composition of the different forms is probably much the same, and is represented by the following figures (Hammarsten) : C, 52.25; H, 6.90; N, 15.25; S, 1.67-1.93; O, 23.67-23.93 percent. Hofmeister obtained the albumins in crystalline form by slow evaporation of their solution in a dilute solution of ammonium sul- phate. On fractional crystallization the different forms may then be obtained. The crystals contained 0.55 per cent, of calcium phos- phate, which is apparently present in molecular combination. Isolation". — To isolate the ovalbumins conjointly, the albumen, after separation from its membranes, is diluted with two and one-half times its volume of water. A slight turbidity thus results, which is filtered off. The liquid is saturated with magnesium sulphate in substance at a temperature of 20° C, which causes precipitation of the globulins. After filtration sodium sulphate is further added to saturation, at the same temperature. The ovalbumins separate out on standing. They are filtered off, dissolved in water, and freed from salts by dialysis. On evaporation in a vacuum, at a tempera- ture of from 40° to 50° C, they are obtained in pure form. To isolate tin; albumins in crystalline form, the albumen is beaten to a froth and allowed to drip. The drippings are treated with an equal volume of a saturated solution of ammonium sul- phate, and freed from globulins by filtration. The filtrate is then placed in a shallow vessel and is allowed to evaporate at the tem- perature of the room. The material, which thus separates out is dissolved in water, treated with a saturated solution of ammonium sulphate until the solution becomes turbid, and is allowed to stand. The resulting crystals can be purified by a repetition of the process. Globulins. — The globulins of the albumen represent only about 7 per cent, of* the total amount of albumin. Different forms appar- ently exist, of which one is said to coagulate at 47° C. and another at f) per cent, of the entire weight. Its actual weight may thus vary between 8.7 and 20.3 grammes. The general composition of the yolk is seen in the following analyses, which are taken from Gautier : Per cent. Water 47.19-51.49 Solids 48.51-42.81 Fats (olein, palmitin, and stearin) 21.30-22.84 Vitellin and other albumins 15.63-15.76 Lecithins 8.43-10.72 Cholesterin 0.44- 1.75 Cerebrin 0.30 Mineral salts 3.33- 0.36 Coloring-matter \ 0 553 (t hi cose I Analysis of the mineral salts, calculated for 100 parts of ash, has given the following results (Poleck and Weber) : Sodium (Na20) 5.12- 6.57 Potassium (K20) 8.05- 8.93 Calcium (CaO) 12.21-13.28 Magnesium (MgO) 2.07- 2.11 Iron (FeA) 1.19- 1.45 Phosphoric acid, free (P205) 5.72 Phosphoric acid, combined 63.81-66.70 Silicic acid (Si02) 0.55- 1.40 Chlorine traces Of the mineral constituents, the large amount of calcium and phosphoric acid is especially noteworthy. Soluble phosphates, however, are not found as such in the yolk. The amount of potas- sium and sodium, it will be observed, is much smaller than in the albumen. The Albumins. — Our knowledge of the individual albumins which arc found in the yolk is still very imperfect. But it appears from recent researches that they are represented notably by nucleo-albu- mins, which in turn may be combined with lecithins to form com- plex lecithalbumins. This, however, is not proved, and it is as- sumed by some that the lecithins which are obtained so commonly together with the albumins do not exist in chemical combination, but arc to l>c regarded as contaminations. The best known repre- sentative of the nucleo-albumins of the yolk is the so-called ovo- vitellin. True nucleins do not occur in the yolk. Ovovitellin. — Formerly this was regarded as a globulin, but it is now known to be a nudco-albumin in which an albuminous radicle \- combined with a paranuclein — /'. e.,a nuclein which does not yield DUcleinic bases on decomposition with mineral acids. The substance ha- thu- far not been obtained free from lecithins, and it is for this reason that the latter i- thought by some to be present ill chemical combimit ion. 428 THE GLANDULAR ORGANS. Of the character of the albuminous radicle which is present in combination with the paranuclein nothing is known. The paranuclein has recently been studied in detail by Levene and Alsberg. They term it avivitellinic acid, and give the following figures to express its elementary composition : C, 32.31 ; H, 5.58 ; N, 13.13; P, 9.88; S, 0.3266; O, 38.28 per cent, In addition they found 0.57 per cent, of iron, which is present in organic com- bination. This is especially interesting in view of the fact that Bunge also obtained a nuclein from the yolk of hens' eggs, which contained iron, and which he termed hcematogen, as the product must of necessity be concerned in the formation of the blood-coloring mat- ter of the developing animal. The elementary composition of Bunge's hsematogen, however, is different from Levene's avivitellinic acid, viz., C, 42.11 ; H, 6.08; N, 14.73; S, 0.55; P, 5.19; Fe, 0.29 ; and O, 31.05 per cent, Its relation to ovovitellin is at present not clear, but it is manifestly closely related to avivitellinic acid. The albuminous radicle of avivitellinic acid manifestly contains the protamin group, as Levene was able to isolate both arginin and histidin from its decomposition-products, which resulted on boiling the substance for seventy-two hours with a 20 per cent, solution of hydrochloric acid. Whether or not lysin is also present remains to be seen. The amount of arginin and histidin obtained was so small, however, that it is scarcely warrantable to assume that a protamin constitutes the entire albuminous radicle, as in the case of the nii- cleins which can be obtained from certain fishes. The substance gives Millon's reaction, moreover, which is not obtained with pro- tamins. The biuret reaction was positive. For a more detailed account of Levene's most interesting work, and a description of the method which was employed for isolating the avivitellinic acid, I must refer the reader to his article.1 The ovivitellin as it is obtained from the yolk contains about 25 per cent, of lecithin. It is soluble in dilute solutions of the neutral salts, and in very dilute (1 pro mille) solutions of hydrochloric acid, and the hydrates and the carbonates of the alkalies. In water it is insoluble, and accordingly is precipitated from its solutions on copious dilution. On prolonged contact with water its properties are changed, and it is converted gradually into an albuminate-like substance. Sodium chloride when added to saturation causes only a partial precipitation. When slowly heated in its solutions of neutral salts it coagulates between 70° and 75° C. ; when rapidly heated, coagulation is retarded until 80° C is reached. On diges- tion with gastric juice ovivitellin yields a paranuclein — avivitellinic acid. From the ovivitellin of the eggs of the bony fishes a gluco- paranuclein may be obtained. Elementary analysis of the ichthulin of carp eggs has given the following results : C, 53.52 ; H, 7.6 ; N, 15.63 ; O, 22.19 ; S, 0.41 ; P, 0.43 ; and Fe, 0.1 per cent. For the ichthulin of codfish eggs 1 Zeit. f. physiol. Cliem., vol. xxxi. pp. 543-556. THE REPRODUCTIVE GLANDS. 429 Levene found C, 52.44 ; H, 7.45 ; N, 15.96 ; S, 0.92 ; P, 0.65 j Fe and O, 22.58 per cent. On treating with alkalies a substance is obtained from this latter form, which is quite similar in compo- sition to avivitellinic acid, as is seen from the figures: C, 32.56; II, 6; X, 14.03; S, 0.146; P, 10.34 per cent. It is termed ich- thulinic aeid (see also page 428). Isolation. — To isolate the ovivitelliu from the yolk, it is well to employ a large number of eggs. The yolks are thoroughly mixed with an equal volume of a 10 per cent, solution of sodium chloride, and arc completely extracted with ether, by shaking, viz., until no more coloring-matter can be removed, and the sodium chloride solution has become perfectly transparent. This is then diluted with twenty times its volume of water, and the ovovitellin thus precipitated. To purify the substance further, it is dissolved re- peatedly in a 10 per cent, saline solution, and reprecipitated with water. It is washed finally with alcohol and ether, and dried over sulphuric acid. The Fats. — The fat of the yolk consists almost entirely of olein, palmitin, and stearin. As a whole, it contains a somewhat smaller amount of' carbon than ordinary fat, which may be due to the pres- ence of mono- and diglycerides, or to the presence of a fatty acid which contain- less carbon than usual. On saponification Lieber- mann obtained 40 per cent, of oleic acid, 38.04 per cent, of palmitic acid, and 15.21 per cent, of stearic acid. The lipochromes or luteins of the yolk can be isolated as follows : the fats of the yolk are saponified by boiling with an alcoholic solution of sodium hydrate. The alcohol is then evaporated. The remaining solution is treated with calcium chloride, which trans- forms the soluble sodium salts into the corresponding insoluble cal- cium salts. On cooling, the soaps are extracted with petroleum- ether, which takes up the lipochromes. On evaporation they are then obtained in pure form. The entire process of isolation must be carried on in the absence of daylight, as otherwise the pigments are decomposed after being separated from the fats. In birds' eggs a yellow lipochrome, vitettolutein, is notably found, but, in addi- tion, traces of a red pigment of the same order, which is termed vitellorubin, may also be encountered. This latter cannot well be obtained by extracting the soaps with petroleum-ether directly, but it i- necessary previously to decompose these with a mineral acid. Lecithins. — The general properties of the lecithins have been con- sidered in a previous chapter (page 65). Isolation. — To isolate the lecithins, the method of Ziilzer may be conveniently employed. To this end, the yolks of a large num- ber of eggs (fifty or more) arc first extracted with ether by shaking, until the ethereal solution takes up no more pigment. The ethereal extracts are united, the ether is distilled off, and the oil tillered oil" at the temperature of the body. This is best accomplished in a thermostat The yellow, somewhat frothy material which remains 430 THE GLANDULAR ORGANS. on the filter is dissolved in as little ether as possible, and precipi- tated with acetone. The precipitate is collected on a filter and washed with acetone until the wash-acetone dissolves no more cholesterin. The residue is again dissolved in a small amount of ether or benzol. To this solution an excess of absolute alcohol is added, when on standing a white amorphous substance separates out, which can be obtained in crystalline form by solution in hot alcohol and cooling ; this apparently consists of tripalmitin. After filtration the pure lecithin can then be obtained from the ether- alcoholic solution by precipitating with acetone, as before, or by dis- tilling off the alcohol and ether. The resulting material is dried in the vacuum: Its phosphorus varies between 3.7 and 4.1 per cent, in amount. Incubation. — Of the chemical changes which take place during the process of fertilization, and in which the nucleus of the ovum is primarily concerned, we know nothing. But there can be no doubt that, in fishes at least, the protamin radicle of the nucleins of the spermatozoa plays an important part. As a result, the reproductive function of the ovum, which previously has remained dormant, now manifests itself in the mysterious morphological changes which the cell undergoes, and which end in the production of an organism that is morphologically and chemically like its parents. In mammals the food-stuffs which are required by the devel- oping organism are constantly supplied through the blood of the mother-animal, but in the lower forms of life they are furnished directly in the egg itself. These products have been studied in some detail in the foregoing pages, and we have seen that they are in part, at least, specific of the egg, and do not occur elsewhere in the animal body. This holds good more especially of the albu- mins, and it follows that all those forms that enter into the com- position of the various tissues must of necessity be-produced from the pre-existing forms during the development of the young animal. The fats may, in part, be utilized directly in the construction of the fats of the embryo, but to a large extent, no doubt, they represent the principal form of energy which is placed at the disposal of the developing organism. Carbohydrates, as such, are practically lack- ing among the food-stuffs of the egg, and must hence be formed synthetically. That glycogen can be demonstrated in the tissues of the embryo at a very early date, has already been stated, which proves in itself that the animal organism is not dependent upon the ingested carbohydrates for its glycogen supply. As nuclear nucleins, moreover, do not occur in the egg, it follows that these also must be formed from other albuminous substances, and there can be little doubt that the paranucleins are here of prime impor- tance. The salts which are required by the developing organism are, as has been seen, present in the egg in abundance. The essential factor, however, which is necessary to development THE REPRODUCTIVE GLAXDS. 4.J1 after fertilization, is an abundant supply of oxygen, and a tempera- ture of about 40° C. The requisite amount of oxygen is obtained from the air by a process of diffusion through the shell. In return carbon dioxide is eliminated, together with a small amount of nitrogen. These respiratory changes are but slight in the begin- ning, but gradually increase. Water also is given off, and, as a result, the weight of the ego- diminishes. The increase of the solids in the developing animal is, of course, accompanied by a correspond- ing diminution of those of the egg itself. Systematic chemical examinations of the ovum in its various stages of development have thus far not been made. Liebermann appears to be the only one, indeed, who has attempted the problem. His principal results may be summarized as follows : during the first stage of development tissues are formed, which are very rich in water ; later, however, the amount of water decreases. The abso- lute amount of substances which are soluble in water steadily in- creases, while their relative amount diminishes as compared with the remaining solids. After the fourteenth day a large increase in the amount of fat is noted, while previously this remains fairly con- stant. The amount of soluble albumins and albuminoids increases steadily and in such a manner that the absolute quantity increases, while their relative amount remains nearly constant. Up to the tenth day no collagen is found, but after the fourteenth day a sub- stance i- present which on boiling with water yields a material sim- ilar to cartilaginous glutin. A mucinous substance is found about the sixth day, but it subsequently disappears. The amount of haemoglobin steadily increases in its relation to the body-weight. The chemical composition of the allantoic fluid and the amniotic fluid has already been considered (page 344). The placenta has not as yet been studied in detail, but it is likely that its greater portion consists of collagen, in accordance with its fibrous structnre. In its marginal zone two pigments have been encountered which apparently are related closely to bilirubin and biliverdin, and are derivatives of haemoglobin. The orange pigment may be obtained in crystalline form, while the green pigment, which has been termed fuematochlorine, is amorphous. CHAPTER XXII. THE DUCTLESS GLANDS. THE THYROID GLAND. Of the function of the thyroid gland very little is known. Its- removal leads sooner or later to the death of the animal. This is preceeded by various symptoms of nerve irritation, and in man by the development of marked anaemia, impairment of the mental powers, general prostration, and curious trophic disturbances of the skin, which are associated with an increased development of the subcutaneous connective tissue and a coincident increase in the amount of mucin. As a result the skin appears swollen and oedematous, constituting the condition known as myxoedema. Sim- ilar results occur if from any cause the gland atrophies. If, how- ever, the resection of the gland is partial, deleterious results do not necessarily follow. It is thus manifest that the function of the organ is a most important one, and it is interesting to note that the apparent antitoxic properties of the gland persist even after its removal from the body and subsequent desiccation. The various symptoms which have just been described as following extirpation of the organ may thus be prevented by administration of the dried gland, and in cases of atrophy a curative effect may similarly be obtained. It is noteworthy, moreover, that the administration of the substance has a marked effect upon the nitrogenous metabo- lism of the body, which is distinctly increased, and, if continued, emaciation results although an abundant amount of food is ingested, and digestion and resorption remain unimpaired. In some instances true diabetes develops. In addition, an increased pulse-rate and heightened blood-pressure are commonly observed, and even sud- den death may occur during the use of the substance. That these curious properties of the thyroid gland should also find expression in its chemical composition suggests itself at once. Numerous attempts have accordingly been made to isolate the "active principle" of the organ, and to a certain extent these at- tempts have been successful. Baumann and Roos thus succeeded in isolating from the gland a substance which has manifestly the same properties as the entire organ in preventing the deleterious results which follow its extirpation or atrophy, and which also has the same effect upon the circulation and the nitrogenous metabolism. This substance Baumann termed thyroiodine, from the fact that it contains iodine in organic combination. It is obtained by boiling the 432 THE THYROID GLASD. 433 gland for several hours with a 10 per cent, solution of sulphuric acid, and by subsequently extracting the insoluble residue with 90 per cent, alcohol. It is insoluble in water and acids, but readily dissolves' in dilute alkaline solutions, from which it is precipitated by adding an excess of an acid. The substance, as first obtained by Baumann, contained 9.3 per cent, of iodine and a small amount of phosphorus. This latter, however, he regarded as a contamination, and he expressed the opinion that future researches would show that the chemically pure body contained even more iodine than the crude product he obtained. Regarding the chemical nature of the thv- roiodine, which itself does not give the biuret reaction, Baumann supposed that it existed in the gland in combination with an albu- min, viz.. as a thvro-iodoglobulin- or albumin. This has since been proved, through the researches of Oswald, who succeeded in extracting from the gland a globulin which contains the entire amount of iodine, and which yields Baumann's thyroiodine on decomposition with mineral acids. This substance is termed thy- reoglobulin. Thyreoglobulin is found in the colloid material of the organ together with a nucleo-albumin, which latter, however, is present in" much smaller amounts. Its quantity is directly dependent upon the amount of the colloid, and is thus subject to variation. In the human gland it normally represents about one-third of the weight of the dried organ, viz., 1.6 grammes. In that of sheep_ it has been found in the proportion of 1 : 2, or 2 : 3, as compared with the total amount of solids, and similar results have been obtained in the pig. Its general elementary composition in animals of the -;iin( species is quite constant, and varies but little indeed in animals of different species. The amount of iodine, however, which i- present in organic combination is subject to fairly wide variations. This is shown in the following analyses, which are taken from Oswald: ox. n Man, Man, Pig. Sheep. Ox. normal. colloid goitre. C 52.21 52.32 52.45 51.85 52.02 II 6.83 7.02 6.93 6.88 6.91 N 16.59 15.90 15.92 15.49 15.32 I 0.46 0.39 0.86 0.34 0.07 S 1.86 1.95 1.83 1.87 1.93 O 22.15 22.42 22.01 23.57 23.75 it i- thus seen that in colloid goitres especially small amounts of iodine are apparently present. This, however, is only relatively tin- case, and in accordance with the presence of a larger quantity of colloid the total amount of the thyreoglobulin is increased, and we find thai the absolute amount of iodine is actually larger than normal. It- quantity can artificially be increased by the ingestion of iodine or iodide- ;i- such, so that it is apparent that the globulin i- capable of binding a certain amount that is intro- duced from without. 434 THE DUCTLESS GLANDS. The iodine, however, is manifestly not a constant component of the globulin, and may be absent altogether. This is especially interesting in view of the fact that, whereas the iodized globulin possesses all the specific properties of the entire gland, the non- iodized substance is inert. An adequate explanation of this curious phenomenon cannot at present be given, but we may imagine that in those instances in which the iodine is normally absent its place may be taken by some other halogen, or a compound halogen which has escaped observation. To conclude that the iodized substance is not the active principle of the gland, on the basis that the glands of some animals contain no iodine, and that its amount is more or less variable and can artificially be increased, is scarcely warrantable. It is conceivable that in sucklings, for example, in which iodine is commonly absent, a compound halogen takes its place, and is, for the time being at least, fully capable of preventing the development of the complex of symptoms which we term ca- chexia strumipriva. Further researches, however, are necessary to explain satisfactorily the apparent contradiction. The fact that the thyreoglobulin of those glands in which it is especially abundant contains a relatively smaller amount of iodine than others in which less globulin is present, can readily be explained on the basis that the total amount of iodine is here distributed over a larger quantity of the globulin, and, as has been stated, the absolute amount is here larger than in normal glands. In its general properties thyreoglobulin resembles the common globulin of the blood. It differs, from this, however, in the fact that it is precipitated from its saline solutions on the addition of dilute acids. In this respect it resembles the myosin of muscle- tissue. Its point of coagulation, in a 10 per cent, solution of mag- nesium sulphate, varies between 65° and 67° C. It is precipitated from its solutions by the addition of an equal volume of a saturated solution of ammonium sulphate. On decomposition with dilute acids it yields the thyroiodine of Baumann, but contains more iodine, viz., 14.29 per cent., and, like its mother-substance, it is free from phosphorus. Thyreo-nucleo-albumin. — The nucleo-albumin which, as I have stated, is found in association with thyreoglobulin in the colloid material of the gland, but is present in much smaller amounts, contains 0.16 per cent, of phosphorus. In a 10 per cent, solu- tion of magnesium sulphate it coagulates at 73° C. On digestion with gastric juice a nuclein is split off, which contains xanthin- bases. The substance is free from iodine, and is physiologically inert. It is precipitated from its solutions by salting with am- monium sulphate to saturation. For a further description of the two albumins, and of the methods which may be employed for their isolation, I must refer the reader to Oswald's paper.1 xZeit. f. pliysiol. Chem., vol. xxvii. p. 14. THE ADRENAL GLANDS. 435 The extractives of the thyroid gland are represented by traces of xanthin, hypoxantbin, lenein, succinic acid, and paralactic acid. In addition, notable quantities of kreatinin may be obtained. THE ADRENAL GLANDS. < >f the function of the adrenal glands, nothing definite is known- Their integrity, however, is essential to life, and, as in the case of the thyroid, their removal leads to the death of the animal. It has been noted, moreover, that the injection of blood from a dog which has died as a result of the operation, into the circulation of a second animal that has been operated in the same manner, will hasten the fatal end, while in normal dogs no deleterious results are observed. It has hence been concluded that the glands normally furnish a secretion which renders certain metabolic products innocuous, and that the fatal result which follows the removal of the organs is the result of an auto-intoxication. In man, disease of the adrenal glands leads to the complex of Bymptoms which is commonly known as Addison's disease, and like- wise results in death ; but as in the case of the thyroid gland, it has been observed that the fatal issue. may here also at least be retarded by the administration of an aqueous extract of the organs. Experi- ment- with such extracts have further shown that the gland contains a substance which lias a very marked effect upon the blood-pressure, raising this far beyond the normal. This substance is found in the medullary portion of the glands. Further investigations have then demonstrated the existence of a chromogen which on exposure to the air in aqueous solution yields a beautiful carmin-colored pigment, which, like its mother-substance, is soluble in water. The same result is reached at once on treating with chlorine-, bromine-, or iodine-water. This chromogen is present in the intracellular fluid of the medullary portion of the gland. When this is extracted with a dilute acid, a violet-red precipitate results on the addition of an - of ammonia, which suggests that the pigment is of a basic nature. \\ ith a solution of ferric chloride the juice that can be expressed from the glands gives a bright emerald-green color. This reaction has b.cn referred to the supposed presence of pyrocatechin, but thus for this has never I n isolated. Modern researches lead to the conclusion that the blood-pressure- raising constituent of the gland, as also the chromogen, which gives i'i-e to the carmin color ami the pyrocatechin reaction, are identical bodies. Abel, who claims to have isolated the blood-pressure-raising principle of the glands, states that this must in all probability be classed with the pyrrol compounds or with the pyridin bases or alka- loid-. II. • \\:1- unable, however, to obtain the -ub, bfVwlutionoMuteln (etheieal extract 437 INDEX. ACCIPENSERIN, 70 Acetic acid, 93 in stomach contents, 116 tests for, 123 Acetone, 94, 267 estimation of, 268 tests for, 267 Achromatin, 304 Achroodextrin, 61, 111, 166 Acid, acetic, 93 adenylic, 75 arachinic, 415 avivitellinic, 428 barbituric, 82 benzoic, 259 bilianic, 152 bilirnbinic, 157 biliverdinic, 158 bromo-phenyl-mercapturic, 88 butyric, 9.J, 116 capronic, 93 carbamic, 87 carnic, 175, 351 chenocholalic, 153 chenotaurocliolic, 150 chi tonic, 50 cholalic, 151 cliolanic, 152, 153 choleic, 153 choleo-camphoric, 152 cholesteric, 152 choloidinic, 1 ■">— cholonic, 1 19 chondroitin-eulphuric, 45, 47, 256, 348 citric, 117 damalic, 263 damaliiric, 263 dehydrocholalic, 152 dehydrocholeic, 153 deaoxycholalic, L52 diacetic, 94, 266 dialui dioxy-phenyl-aoetic, 260 eleidinic, 95 ethylene-succinic, 72 fatty, 93, 264 fellic, 163 ferri-albuminic, (02 formti galactonic, 55. 57 Acid, gluconic, 55, 256 glucuronic, 55, 91, 255, 321, 384 glutaminic, 37, 86, 191 glutaric, 95 glycerin-phosphoric, 65, 298 glycocholic, 89, 148 glycolic, 94 glycoluric, 82 glycosuric, 262 guanidin-butyric, 72 guano-biliary, 149 guanylic, 75, 405 haematinic, 158, 332 liippuric, 87, 97, 257 honiogentisinic, 261 hydantoic, 82, 85 hydantoin-hydroparacumaric, 88 hydrocinnamic, 97 hydroparacu marie, 95, 201, 260 hydurilic, 82 hyocholalic, 153 hyoglycocbolic, 149 hyotaurocholic, 150 icbthulinic, 429 inosinic, 75, 367 isobilianic, 152 kynurenic, 263 lactic, 94, 115, 356,359 lfflvulinic, 56, 323 laurinic, 415 leucinic, 94 lithofellic, 153 lithuric, 363 lysuric, 74 mannonic, 5 1 manno-saccharinic, 55 methyl-hydantoinic, 88 tnucinic, 55, 57 myristinic, 168, 415 nuclei 1 1 i<-, .'54, 74 oleic. 64 ornithuric, 74, 87, 260 oxalic, 95, 241 oxaluric, 82, 241 oxy-amygdalic, 260 oxy-hydroparacumaric, 95 oxy-protonic, 88 oxy-proto-Hulphonic, '-'>x palmitic. 64, 98 parabanic, ho, 82 paralactic, 269 130 440 INDEX. Acid, para-oxy-benzoic, 96 para-oxy-pbenyl-acetic, 95, 201, 260 para-oxy-phenyl-glycolie, 260 para-oxy-phenyl-lactic, 95, 260 para-oxy-phenyl-propionic, 95, 260 peroxy-pro tonic, 38 phenaceturic, 97, 259 phenyl-acetic, 97, 201 phenyl-propionic, 97, 201 phosphor-car nic, 351 phyllocyanic, 222 plasminic, 76, 77 propionic, 93 pyrocatecliuic, 262 pyrocliolesteric, 152 saccliarinic, 55, 91, 256 saccharo-lactonic, 55 sarcylic, 75 skatol-carbonic, 207, 254 sperma-nucleinic, 75 stearic, 93 tartronic, 83 taurocarbaminic, 88, 271 taurocholic, 88, 149 thyminic, 76, 77, 323 thymo-nucleinic, 75, 323 trioxy-phenyl-propionic, 260 tyrosin-hydantoinic, 88 uramic, 88 uramido-benzoic, 88 uric, 80, 81, 233 nrocaninic, 263 urochloralic, 255 uroleucinic, 262 uroxanthinic, 262 urrhodinic, 262 valerianic, 93 xanthylic, 75 yeast-nncleinic, 75 Acids, biliary, 145 Acrolein, 65 Adamkiewicz' reaction, 34 Addison's disease, 435 Adenin, 78, 240, 366 isolation of, 363 tests for, 366 Adenylic acid, 75 Adipocere, 391 Adipose tissue, 3S9 analysis of, 390 origin of, 391 significance of, 392 Adrenal glands, 435 Adrenalin, Takamine's, 436 Albumen of birds' eggs, 423 albumins of, 424 analysis of, 424 ovalbumins of, 424 ovomucoid of, 425 tata, 424 Albumin of Bence Jones, 289 Albuminates, 49 alkaline, 49 Albuminates of iron, 401 Albuminoids, 46 digestion of, 179 Albumins, 30, 41 behavior toward neutral salts and alcohol, 31 toward polarized light, 33 classification of, 40 coagulated, 49 coagulation of, 32 color-reactions of, 34 crystallization of, 30 decomposition of, 37 denaturization of, 33 derived, 48 digestion of, 168 elementary composition of, 30 estimation of, in milk, 415 in plasma, 316 in urine, 290 molecular size of, 38 native, 41 nitrogenous derivatives of, 69 of the albumen, 424 of the blood, 309 of the lymph, 342 of the milk, 411 of the muscle-tissue, 347 of the nerve-tissue, 370 of the urine, 284 of the yolk, 427 precipitation of, 36 solubility of, 31 structural composition of, 39 synthesis of, 26, 38 tests for, in the urine, 286 Albuminuria, 284 physiological, 285 Albumoid, 378, 385 isolation of, 385 Albumose-plasma, 313 Albumoses, 49, 169 analysis of, 183 in the urine, 285 primary, 50, 169 secondary, 50, 169 reactions of, 186, 187 tests for, in the urine, 288 Aldehydase, 106, 402 Aldoses, 54 Aleuron crystals, 30, 42 granules, 30, 42 Alkapton, 262 Allantoic acid, 82 Allantoin. 82, 83 in the urine, 243 isolation of, 244 Allanturic acid, 82 Allituric acid, 82 Alloxan, 80, 82, 83 Alloxanic acid, 82 Alloxantin, 82 Alloxuric bases, 43, 75, 77 INDEX. 441 Amido-acids, 85 Amido-nitrogen, 37 Amido-thio-lactic acid, 88 Aniidulin, 111, 166 Ammonia, estimation of, in the urine, 232 in the blood, 322 Ammonium purpurate, 82, 83 Amniotic fluid, analysis of, 344 Amphikreatin, 363 Amphikreatinin, 84 Amphopeptone, 51, 170 Amygdalin, 26 Amyiodextrin, 60 Amyloid, 48 Amylolytic ferment of pancreatic juice, 133 Amylopsin, 138 Amylum, 60 Animal cell, 301 fat, 389 gum, 44 sinistrin, 44 Anti-albumid, 170, 176 Anti-albumoses, 50 Antipeptone, 51, 175 preparation of, 195 Aqueous humor, analysis of, 343, 377 Arabinose, 62, 284 Arachinic acid, 415 Arbacin, 71 Arbutin, 25 Arginin, 37, 69, 72, 84 in the spleen, 406 Arnold's test for diacetic acid, 266 Aromatic constituents of the urine, 247 oxy-aci pigments of, 155, 156 in the urine, 297 Bile, secretion of, 142 significance of, 142 Biliauic acid, 152 Biliary acids, 145, 202, 297 formation of, 145 isolation of, 147 tests for, 147 constituents, bacterial decomposition of, 202 iron, 164 Bilicyanin, 161 Bilifuscin, 158, 161 Bilihumin, 162 Biliprasin, 158, 161 Bilipurpurin. 161 Bilirubin, 22, 156 _ isolation of, 159 tests for, 158 Bilirubinic acid, 157 Biliverdin, 158, 160 isolation of, 160 Biliverdinic acid, 158 Bilixanthin, 161 Biuret reaction, 34 Blood, 306 amount of, 309 chemical composition of, 310 examination of, 309 coagulation of, 324 color of, 307 extractives of, 322 fat in, 322 fibrin-ferment in, 319 fibrinogen in, 313 glucose in, 321 glycogen in, 321 leucocytes of, 322 odor of, 308 physical characteristics of, 307 pigments of, 328 plaques of, 324 reaction of, 309 red corpuscles of, 327 serum-albumin of, 316 serum-globulin of, 314 specific gravity of, 308 taste of, 308 urea in, 322 Blood-pigments in the urine, 294 Blood-plasma, 307, 312 Blood-serum, 307, 317 Boas' method of estimating lactic acid, 123 test, 120 for lactic acid, 122 Bone, 386 _ analysis of, 387 Bone-marrow, 388 Boucher's crystals, 420 Brain-tissue, analysis of, 370 Bromo-phenyl mercapturic acid, 88 Brown-Sequard's elixir, 421 Brucke's method of isolating pepsin, 128 442 INDEX. Brunner's glands, secretion of, 139 Bufidin, 398 Butter, 415 Buttermilk, 411 Butyric acid, 93 in stomach contents, 116 tests for, 123 nACHEXIA strumipriva, 434 \J Cadaverin, 74 in the urine, 299 Caffein, 78, 240 Calcium-thrombosin, 320 Cane-sugar, 58 Capronic acid, 93 Carbamic acid, 87 Carbohsemoglobin, 335 Carbohydrates, 53 fermentation of, 56 of the urine, 275 synthesis of, in plants, 24 Carbon dioxide haemoglobin, 335 methsemoglobin, 337 monoxide haemoglobin, 335 Carnic acid, 351 in antipeptone, 175 Carniferrin, 351 Carnin, 78, 240 in muscle-tissue, 366 isolation of, 363 properties of, 367 relation of, to hypoxanthin, 366 tests for, 367 Cartilage, 383 albumoid in, 385 analysis of, 384 chondroitin-sulphuric acid in, 384 chondromucoid in, 385 embryonic, 383 mineral salts in, 386 Casein, 411 digestion of, 178 estimation of, 415 isolation of, 414 origin of, 414 properties of, 411 Caseoses, 50 Castoreum, 398 Cavazzani's method to remove albumins from the blood, 317 Cell -globulins, 324 Celluloses, 60, 61 Cement-substance of teeth, 388 Cerebrin, 57, 373 isolation of, 374 properties of, 374 Cerebrosides in nerve-tissue, 372 Cerebrospinal fluid, analysis of, 344 Cerumen, 398 Cetin, 64 Cetylid, 373 Cliarcot-Leyden crystals, 420 Cheese, 412 Chenocholalic acid, 153 Chenotaurocholic acid, 150 Chitin, 46, 56, 389 Chitonic acid, 56 Chitose, 56 Chlorides, estimation of, in the urine, 219' Chlorine-hunger, 217 Chlorophane, 381 Chlorophyl, 19, 21 chemical nature of, 21 granules, 21 hydride, 24 Cholagogues, 143 Cholalic acid, 151 Cholanic acid, 152, 153 Cholecyanin, 161 Choieglobins, 164 Choleic acid, 153 Choleo-camphoric acid, 152 Cholesteric acid, 152 Cholesterilins, 162 Cholesterin, 67 in nerve-tissue, 376 in the bile, 162 in the urine, 298 isolation of, 163 tests for, 163 Choletelin, 161, 291 Cholin, 65 Choloidinic acid, 152 Cholonic acid, 149 Chondrin, 47, 385 Chondroitin, 46, 384_ Chondroitin-sulphuric acid, 45, 47 in cartilage, 384 isolation of, 385 properties of, 384 Chondromucoid, 385 isolation of, 385 Chond rosin, 45, 46, 384 Choroid, 381 Chromatin, 304 Chromogen of adrenal glands, 435 Chromophanes, 381 Chyle, analysis of, 343 Chymosin, 130, 139 estimation of, 132 isolation of, 131 properties of, 130 tests for, 131 Chymosinogen, 130 estimation of, 132 test for, 131 Citric acid in milk, 417 Clabber, 416 Clupein, 69 Coagulated albumins, 49 Coagulation of the blood, 324 rapidity of, 326 theories of, 324 Collagen, 47 Collagen-sugar, 155 Colloid, 45 INDEX. 443 Colloidal platinum, 20 Colostrum, 417 Compound glycocolls in the urine, 256 Conchiolin, 47, 48 Conjugate glucuronates, 96 in the urine, 255 sulphates, 96 estimation of, 220 in the urine, 248 Connective tissue, embryonic, 382 Copper test for uric acid, 237 Cornea, 377 Corpora lutea, 422 Cream, 411 a-Crotonic acid, 95 Crusokreatinin, 84, 363 Crusta phlogistica (seu inflammatoria), 327 Crystalline lens, 378 albumins of, 378 albumoid of, 378 analysis of, 378 crystallins of, 378 Crystallins, 378 Cyan-methaemoglobin, 337 Cyanurin, 251 Cyclopterin, 71 Cystei'n, 88 in the urine, 272 Cystin, 88 in the kidneys, 406 in the liver, 404 in the urine, 273 isolation of, 274 properties of, 274 Cystinuria associated with diaminuria, 299 Cytoglobin, 305 ( Vtosin, 75, 323 DAM A LIC acid, 263 Damaluric acid, 263 Dehydrocholalic acid, 152 Dehydrocholeic acid, 183 Denaturization, 169 Denniges' (est for uric acid, 237 Dentin I terived albumins, 48 met'a membrane, 377 I '• xycliolalic acid, 162 Deutero-albumoaes, 60, 169, 171, 186 l)extrins, UK, 6] in tin- arine, Dezi Diabetes, 277 Diacetic -Kid. '.» 1 in the arine, 266 for, 246 I >i;ilnrit acid, 82 Diamino-nitrogen, 88 I »i:imiiis in tin- urine, 299 isolation ol DiamiBuria, 27:; Dibutyl-diethylene diamin, 38 Diethylene diamin, 38 Differential density method of estimating sugar, 280 Digestion, 165 of albumins, 168 of albuminoids, 179 of carbohydrates, 165 of fats, 180 ofproteids, 178 Digestive fluids, 108 glands, 405 Dioxv-phenyl-acetic acid in the urine, 260 Disaccharides, 57 Diureids, 81 Dry pancreas, 138 Dulcite, 54 Dun lop's method of estimating oxalic acid, 243 Dysalbumose, 50 Dyslysin, 152 EAR, the, 381 Egg-albumin, 41 Eggs, 422 albumen of, 423 albumins of, 424 fats of, 429 incubation of, 430 lecithins of, 42y lipochromes of, 429 ovomucoid in, 425 ovovitellin in, 427 shell of, 423 yolk of, 426 Ehrlich's reaction, 268 Elastin, 47 Elastoidin, 47, 48 Elastoses, 50 Eleidin granules, 394 Eleidinic acid, 95 Emulsin, 105 Emydin, 427 Enamel of teeth, 388 Encephalin, 375 Enteric juice, 140 Enzymes. Sec Ferment*. Eosinophilic crystalloids, 30 Epiguanin, 78 Epinephrin, Abel's, 435 Episaroin, 78, 240 Erythrodextrin, 60, 111, 166 Esbach's reagent, 129, 290 Ethylene-succinic acid, 72 Ethylenimin, 120 Ethyl Sulphide in the urine, 271 Excretin in the feces, 208 Extractives "f nerve tissue, 376 of the blood, 822 Of the liver, Mil of the lymph, 848 of the milk, 417 444 INDEX. Extractives of the thyroid gland, 435 Eye, 377 FAT, 63 analysis of, 390 animal, 389 bacterial decomposition of, 202 chemistry of, 64 digestion of, 180 estimation of, in the milk, 416 melting-point of, 390 of birds' egg, 429 of the blood, 322 of the liver, 404 of the lymph, 341 of the milk, 415 of the muscle-tissue, 368 of the urine, 298 origin of, 63, 391 in milk, 415 significance of, 393 synthesis of, in plants, 26 Fatty acids, 93 estimation of, in the urine, 264 isolation of, from the urine, 264 degeneration, 391, 404 infiltration, 404 tissue (see also Adipose tissue), 389 Feces, 204 amount of, 204 bacteria in, 205 chemical composition of, 205 color of, 204 consistence of, 204 crystals in, 205 excretin in, 208 form of, 204 hydrobilirubin in, 207 macroscopical constituents of, 205 microscopical constituents of, 205 odor of, 204 reaction of, 205 serolin in, 208 stercobilin in, 207 stercorin in, 208 Fehling's test for sugar, 278 Fellic acid, 153 Fermentation, acetic, 198 alcoholic, 198 butvric, 198 lactic, 198 method of estimating sugar, 281 test for sugar, 279 Ferments, 20, 98 amylolytic, 105 chemical composition, 103 classification, 104 coagulating, 105 general properties of, 101 reactions of, 103 inverting, 105 mode of action, 104 of the liver, 402 Ferments of the lymph, 343 of the muscle-tissue, 352 of the pancreatic juice, 136 of the urine, 298 oxidizing, 106 proteolytic, 105 reversible action of, 21, 103 steatolytic, 105 tissue-, 106 Ferratin, 402 Ferri-albuminic acid, 402 Fertilization of eggs, 430 Fibrin, 48, 319 estimation of, 320 formation of, 319 isolation of, 320 properties of, 320 test for, in the urine, 290 Fibrin-ferment, 319, 324 chemical nature of, 319, 324 isolation of, 319 properties of, 319 Fibrin of Henle, 419 Fibrinogen, 41 isolation of, from the blood, 313 properties of, 314 tissue-, 305 Fibrinoglobulin, 41, 324 Fibrinoplastic substance of Schmidt, 314 Fibroin, 47, 48 Fibrous tissue, elastic, 383 reticulated, 383 white, 382 yellow, 383 Fish-scales, 388 Fleischl's ruemometer, 334 Floridins, 329, 338 _ Fluorides in the urine, 219 Folin's method of estimating urea, 231 uric acid, 238 Food-stuffs of plants, 23 Formic acid, 93 Freund's method of estimating the acid- ity of the urine, 215 Fructose. See Lcevulose. Fuscin, 381, 395 pADUShiston, 71 \J Galactonic acid, 55, 57 Galactose, 54, 57, 373 Gallois' test for inosit, 360 Gases of the blood, 312 of the intestinal contents, 201 of the lymph, 343 of the muscle-tissue, 367 of the stomach contents, 132 of the urine, 299 Gastric digestion, 168 juice, 114 acidity of, 115 amount of, 114 analysis of, 115 chemical composition of, 115 IXDEX. 445 Gastric juice, ferments of, 124 gases of, 132 hydrochloric acid of, 117 lactic acid in, 122 pro-enzymes of, 124 sulphocyanides in, 132 (Gelatin, 47 < relatoses, 50 Gerhardt's test for diacetic acid, 267 for urobilin, 293 Giacosa's pigment, 251 » < Hands, adrenal, 435 digestive, 405 ductless, 432 gastric, 405 intestinal, 405 lymph, 405 mammary, 406 reproductive, 419 salivary, 405 2 VKoSSIX, 45 ^\ Nerve-tissue, 369 albumins in. 370 cerebri n in, 373 cholesterin in, 376 encephalin in, 375 extractives of, 376 general chemical composition of, 369 homocerebrin in, 374 jecorin in, 376 lecithins in, 375 myelins in, 372 neuridin in, 376 neurokeratin in, 371 protagon in, 372 Neuridin in nerve-tissue. 376 Neu rin, 66 Neurokeratin, 371 Neutral oxygen, 19 sulphur, estimation of, 275 Nitrates in the saliva, 113 test for, 113 in the urine, 219 test for, 221 Nitric acid test lor albumin, 286 for xanthin, 365 oxide haemoglobin, 336 Nitrogen, estimation of, 232 Nitrogenous equilibrium, 225 Nuclear nucleins, 43. 71 Nucleinic acids, 43, 74 bases, 43, 77 Nuclein platelets of the Mood, 324 Nucleins, 11 digestion of, 178, 179 isolation of, 101 Of the liver. 400 of the muscle-tissue, 351 Nudeo -albumin-. 13 test for, in the urine, 288 Nucleo-gliicoproteid of the mammary gland. 406 of the pancre is, 105 Nucli-o-bi-ton, 305 isolation of, 322 properties of, 323 Surleons, v. l.uid'T'- test for - . 0BERM IYER'S test for indioan, 262 Oleic acid, 61 Oletn, 61 Onuphin, 16 nio, 12.; Oornodein, 123 Ornithin, 72, 88 Ornithuric acid. 74. 87, 260 Osazons, 55 ( )>« >ns, 55 Ossein, 3S6 Ovalbumins, 424 isolation of, 425 Ovaries, 422 Ovomucoid, 425 isolation of, 426 Ovovitellin, 427 isolation of, 429 properties of, 428 Ovum, 422 ( Palate-plasma, 313 ( txalic acid, 95 estimation of, 243 in the urine. 241 origin of, 241 Oxaluric aeid, 82 in the urine, 241 origin of, 241 Oxidations in animal life, 19 Oxy-acids, aromatic, 260 in the urine, 260 isolation of, 201 tests for, 261 < >xy-amygdalic acid in the urine, 260 /3-oxybutyric acid, 94, 265 relation to acetone, 265 to a-crotonic acid, 265 to diacetic acid, 265 tests for, 265 Oxydases, 106 in the liver, 402 in the saliva, 1 12 Oxyhsemocyanin, 329, 338 ( (xvluemoglobin, 331 estimation of, 334 isolation of, 33.2 properties of, 333 Oxy-nydroparacumaric acid, 95 < txyneurin, 66 < >xypiperidin, 73 < >xyprotonic acid, 38 ( >xyproto-8iil phonic acid, 38 PALMITIC acid, 64, 93 I'almitin. 6 I Pancreas, 405 Kubnc'sdiv, 138 Pancreatic juice, 133 amount of, 135 chemical composition of, 135 ferments of, 136 general properties of, 1 34 significance of, L33 specific gravity of, 134 zymogens of, 136 Parabanic acid, 80, 82 Paracasein, 1 12 Paracholesterin, 67 Paracresol, 89, 9C 450 INDEX. Paracresol in the urine, 248 Paraglobnlin, 314 Paralactic acid in the urine, 269 isolation of, 269 Paralbumin, 45 Paranucleus, 43, 74, 77 Para-oxy-benzoic acid, 96 Para-phenyl-acetic acid, 95, 201 in the urine, 260 isolation of, 207 Para-oxy-phenyl-glycolic acid, 95 in the urine, 260 Para-oxy-phenyl-lactic acid, 95 in the urine, 260 Para-oxy-phenyl-propionic acid, 95 in the urine, 260 Paraxanthin, 78, 240 Paton's globulin, 285, 289 Penta-methylene-diamin, 74 Pentoses, 54, 62 in the urine, 283 tests' for, 284 Pepsin, 124, 126 estimation of, 129 isolation of, 127 properties of, 126 Pepsinogen, 124 estimation of, 130 test for, 129 Peptic digestion, products of, 183 Peptone-plasma, 313 Peptones, 50, 169 Pericardial fluid, analysis of, 343 Peritoneal effusions, analysis of, 344 Peroxy-jarotonic acid. 38 Pettenkofer' s test, 147 Phenaceturic acid, 97, 259 isolation of, 259 origin of, 259 properties of, 259 Phenol, 89, 96 estimation of, in the urine, 249 in the intestinal contents, 200 in the urine, 248 tests for, 201 Phenyl-acetic acid, 97 in the intestinal contents, 201 isolation of. 207 Phenylhydrazin-test for sugar, 279 Phenyl-propionic acid, 97 in the intestinal contents, 201 isolation of, 207 Phlebin, 327 Phloretin, 25 Phloridzin, 25 Phosphates, estimation of. in the urine, 220 Phospho-gluco-proteids, 44, 46 Phosphor-carnic acid, 351 Photo-methsmoglobin, 336 Phyllocyanic acid, 22 Phylloporphyrin, 22, 338 Phylloxanth'in, 22 Phymatorhusin, 297, 395 Phytosterin, 67 Pigments of the blood, 328 of the muscle-tissue, 353 of the skin, 395 of the urine, 291 respiratory, 329 Pine-apple test for butyric acid, 123 Piperazin, 420 Piria's test for tyrosiri, 189 Placenta, 431 Plaques, 324 Plasma, albumose-, 313 blood-, 312 histon-, 326 muscle-, 347 oxalate-, 313 peptone-, 313 salt-, 313 Plasminic acid, 76, 77 Plastin, 304 Platner's bile, 147 Pleural effusions, analyses of, 344 Plosz' urorubrin, 251 Polysaccharides, 59 Prseglobulin, 305 Pro-enzymes, 101 Propionic acid, 93 Protagon, 372 isolation of, 373 properties of, 372 Protamins, 37, 69, 74 in the spermatozoa, 421 Proteids, 42 digestion of, 178 Proteinochromes, 193 Proteinochromogen, 193 Proteins, 30 Proteoses, 50 Prothrombin, 319 Proto-albumose, 50, 169 Protons, 71 Protophyllin, 25 Pseudomucin, 45 Pseudonucleins, 43, 74 Ptomains, 91, 100 acyclic, 91 cyclic, 91 in the intestinal contents, 201 in the urine, 299 Ptyaliu, 110 action of, 110 chemical nature of, 110 isolation of 111 Ptyalinogen, 110 Purin, 78 bases, 43, 75, 77 Pus, analysis of, 345 Putrefaction, analysis of products of, 206 Putrescin, 73 in the urine, 299 Pyogenin, 372 INDEX. 451 Pyosin, 372 Pyrocatechin, 96 in the urine, '24s Pyrocatecnin-reaction of adrenal glands, "435 Pyrocatechuic acid, 262 Pyrocholesteric acid, 152 1 ) KXXIX (see also Ohymosin), 130, 139 xi Reproductive glands, 419 Resorption of albumins, 172 of carbohydrates, 167 of fats, 181 Reticulin, 383 Retina, 379 analysis of, 380 chromophanes in, 381 rhodopsin in, 380 Rhamnose, 284 Rhodophane, 381 Rhodopsin, 380 Rosenbach's reaction, 252 SA.CCHARINIC acid, 55, 91, 256 Baccharo-lactonic acid, 55 Saccharose, 58 Salamandrin, 398 Salicin, 25 Saligenin, 25 Saliva, 108 amount of, 108 analysis of, 109, 110 chemical composition of, 109 chorda-, 109 digestive importance of, 112 extractives in, 114 ferments in, 1 12 s in, 114 general characteristics of, 108 mineral constituents of, 114 nitrite- in, 1 ]:; paralytic, 109 I iuii of, 108 sulphocyanides in, 1 13 sympathetic, LOfl Salivary glands, L08, 405 Salkow-ki - method of estimating oxalic acid, 2 13 holesterin, 163 Salkowski-Volhard method of estimating chlorides, 219 Salinin, 69 Salt-plasma, 813 Saponification, 65 s 1 1 • i n iee a I-., Hypoxanthin le-i for, 865 Sarcylic acid, 75 nk'i indirubin, 251 Scherer't red pigment of tic urine, 251 ■ tor inosit, 860 for leucin, 188 tor tjrrosin, I 39 Schifl i uric acid, 287 Schmiedeberg's histenzyme, 258 Schiitzenberger's hypothesis, 39 Sclerotic, 377 Scombrin, 69 Scombron, 71 Scyllite, 360 Sebum, 397 Semen, 419 general properties of, 419 Sericin, 47 Serolin in the feces, 208 Serum of the blood, 317 composition of, 317 Serum-albumin, 41 forms of, 316 in the urine, 288 isolation of, 316 of the blood, 316 test for, 288 Serum-casein, 314 Serum-globulin, 41 in the urine, 288 isolation of, 314 properties of, 315 test for, 288 Siegfried's carniferrin, 351 Silicates in the urine, 219 Silurin, 70 Skated, 90, 199 isolation of, 206 tests for, 200 Skatol-carbonie acid, 90 isolation of, 207 test for, 254 Skatoxyl, 90 sulphate in the urine, 253 tests for, 254 Skeletins, 47, 48, 389 Skin, 394 elei'din granules in, 394 keratin in, 394 Smegma, 398 Smith's test, 159 Soaps, Ii5 Solanidin, 26 Solanin, 26 Sorbite, 5 1 Spectroscopic lest for bilirubin, 159 Spermanucleinic acid, 75 Spermatic fluid, 420 Spermatin, 120 Spermatozoa, 421 analysis of, 121 chemical composition of, 421 protamine in, 421 Spermin, 420 phosphate of, 420 Spirographin, 15 Spleen. IMC, arginin in, I hi; Spongin, 17, Is Spongioplasm, 308 Starch* 452 INDEX. Steapsin, 138 Stearic acid, 64, 93 Stearin, 64 Stercobilin in the feces, 207 relation to urobilin, 292 Stercorin in the feces, 208 Stoke' s reagent, 330 Stools (see also Feces), 204 acholic, 204 Sturin, 69 Succinic acid, 95 Sugar in lymph, 342 Sulphates, estimation of, in the urine, 220 Sulphocyanides in the gastric juice, 132 in the saliva, 113 in the urine, 271 test for, 113 Sulphur, acid, of the urine, 218 neutral, of the urine, 218 Sulphur-test, 35 Supporting tissues, 382 Suprarenin, v. Fiirth's, 436 Sweat, 395 analyses of, 397 chemical composition of, 396 gases of, 397 general characteristics of, 396 significance of, 396 Synaptose, 105 Synovial fluid, 345 Synovin, 345 Syntonin, 49, 169 TAKTKONIC acid, 83 Tauriu, 88, 153 in muscle-tissue, 363 isolation of, 154 Taurocarbaminic acid, 88 in the urine, 271 Taurocholic acid, 88, 149 Teeth, 388 Testicles, 419 Tetra-hydro-dioxypyridin, 73 Tetra-methylene-diamin, 73 Theobromin, 78, 240 Theophyllin, 78 Thiosulphates in the urine, 271 Thrombin, 319 Thrombosin, 320, 325 -calcium, 320 Thymin, 76 Thyminic acid, 76, 77, 323 Thymo-nucleinic acid, 75, 323 Thymus gland, 405 Thyreoglobulin, 433 Thvreo-nucleo-albumin, 434 Thyroid gland, 432 Thyroiodine, 432 Tissue-fibrinogen, 305 Tollens' orcin test, 284- phloroglucin test, 284 Topfer's method of estimating hydro- chloric acid, 1 20 Topfer's test for hydrochloric acid, 1 1 9, 1 20 Toxalbumins, 100 Toxins, 100 Triglycerides, 64 Trioxy-phenyl-propionic acid in the urine, 260 Trypsin, 136 action of, 137 isolation of, 137 test for, 137 Trypsinogen, 136 Tryptic digestion, 174 products of, 185 Tryptophan, 193 tests for, 193 Tunicin, 389 Tyrosin, 37, 86, 188 in the urine, 270 isolation of, 189, 271 tests for, 189 Tyrosin-hydantoinic acid, 88 UFFELMANN'S test for!acticacid,122 Uramic acids, 88 Uramido- benzoic acid, 88 Urea, estimation of, 230 formation of, 86, 229 in the urine, 222 isolation of, from the urine, 230 origin of, 222 properties of, 227 Urea-nitrate, 227 Urea-oxalate, 228 Ureids, 81 Uric acid, 80, 81 estimation of, 238 in the urine, 233 isolation of, 237 origin of, 233 properties of, 236 tests for, 237 Urine, 209 acetone in, 267 acidity of, 215 albumins in, 284 allantoin in, 243 ammoniacal decomposition of, 210 amount of, 211 aromatic constituents of, 247 oxy-acids of, 260 carbohydrates of, 275 chemical composition of, 216 chlorides of, 216 cholesterin in, 298 color of, 210 compound glycocolls in, 256 conjugate glucuronates in, 255 sulphates in, 248 cy stein in, 272 cystin in, 273 dextrin in, 283 diacetic acid in, 266 fats in, 298 IXDEX. 453 Urine, fatty acids in, 264 ferments in, 298 gases in. 299 general characteristics of, 209 glucose in. 27»'> hippuric acid in. 257 homogentisinic acid in. 261 indican in, 230 inorganic constituents of, 216 inosit in. 262 kreatinin in, 244 kynurenie acid in, 263 lactose in, 282 lsevulose in, 2*3 lecithin in, 298 leucin in, 270 lithuric acid in, 263 maltose in, 283 neutral sulphur in, 271 nitrogenous constituents of, 222 odor of, 211 organic constituents of, 221 ornithuric acid in, 260 oxalic acid in, 241 oxaluric acid in, 241 /i-oxybutyrie acid in, 265 parallactic acid in, 269 pentoses in, 283 phenaceturic acid in, 259 phenols in, 248 phosphates in, 217 pigments in, 291 ptomains in, 299 reaction of, 212 skatol-carbonic acid in, 254 skatoxyl sulphate in, 253 specific gravity of, 212 sulphates in, 217 tyrosin in, 270 urea in, 222 uric acid in, 233 urocaninic acid in, 263 uroltucinic acid in, 262 volatile fatly acid- in, 264 xantbin-bases in, 240 I Frobilin, isolation of, 293 Jaffe's. 291, 293 MacMuni, for, 293 Urocaninic acid. 263 I Irochloralic acid, 2">"> I frochrotne, 293 isolation of. ■_! '. • - I fro< rythrin, 2'.<1 isolation ol 1'rofii-' <,|i;iiiiatii .in in, 261 l.anialiii, 'J". 1 for, 262 rjroleucinic acid in the urine, 262 I , :..,•'., I L'roi 26 1 Urorubin, 251 Urorubrohsematin, 296 Uroxanthinic acid, 262 Urrhodin, 251 Urrhodinic acid, 262 Uterine milk, 419 VALERIANIC acid, 93 Vegetable albumins. 41 amyloid, 61 globulins, 41 gums, 60 ViteUins, 42 Vitellolutein, 429 Vitellorubin, 429 Y helioses, 50 Vitreous body, 379 analysis of, 379 WALLEATH, 163 Wang's method of estimating indican, 253 Weidel's test for xanthin, 365 Welcker's method of estimating the total amount of blood, 309 Wharton's jelly, 382 Whey, acid, 416 sweet, 408 v. Wittich's method of isolating pepsin, 128 . XANTHIN, 78, 240 in muscle-tissue, 363 isolation of, 362 properties of, 364 tests for, 365 Xantbin-bases, 43, 75, 77 estimation of, 241 in muscle-tissue, 363 isolation of, 363 of the urine, 240 origin of, 241 Xanthinic leucomains, 79 Xanthokreatinin, 84, 363 Xanthophane, :>sl Xanthoproteic reaction, 34 Xantliopsin, 3SO Xanthylic acid, 75 Xylose, 62, 284 YEAST-NUCLEINIC acid, 75 folk of birds' eggs, 426 album inB of, 127 analysis of, 127 fats of, 129 bsematogen in, 428 lipochromes, of, 429 ovovitellin in, 427 white, 426 Yolk platelet-, 80, 42 / YMOGENS, I'd Catalogue of Books PUBLISHED BY Lea Brothers & Company, 706, 708 & 710 Sansom St., Philadelphia. Ill Fifth Avenue, New York. The books in the annexed list will be sent by mail, post-paid, to any Post-Office in the United States, on receipt of the printed prices. No risks of the mail, however, are assumed either on money or books. 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