(If 4- CORNELL UNIVERSITY. THE THE GIFT OF ROSWELL P. FLOWER FOR THE USE OF THE N. Y. STATE VETERINARY COLLEGE 1897 RB 53.c94"'"""''"'"^">"-"'™'T Clinical urinology, 3 1924 000 886 535 u DATE DUE 1 _ _ ^^ 1 j CAYLORD PRINTEDIMU.S.A L^l Cornell University Library The original of tiiis book is in tine Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924000886535 Plate. Sun Spectrum, I BCD Eh T 0x1/ -Hemoglobin n ..... Acid Mefkemoglohin Reduced Hemo(flobm Reduced Hematinr (Hemochamwgml 1 Acid Hemattn i Alkaline ffematin Acid Hematoporphyrin m. -mn Alkaline llematoporphyrin "1 ■ Urobilin Spectra of Urinary Pigments. CLINICAL URINOLOGY ' ■ _;BRA^^V I ALFRED C. CROFTAN X^jJ Professor of Medicine, Chicago Post Graduate Medical College and Hospital Physicinn-in-Chief to St. Mary's Hospital Pathologist to St. Luke's Hospital SIIuisttateD NEW YORK "« WILLIAM WOOD & COMPANY MDCCCCXV r aV Rid C ^4 COPTKIGHT, 1904 By WILLIAM WOOD & COMPANY 53 C9^ mount pifaaant %rtB» J. Horace McFarland Co. Harrigbure, Fn, PREFACE This book, as the title "Clinical Urinology" indicates, is a treatise on the urinary aspect of disease. It is in no sense meant to be merely a laboratory guide to the analysis of urine; nor is it intended to be a purely clinical disquisition on the disorders that produce uri- nary abnormalities. Its purpose is to describe the borderland that lies between the laboratory and the clinic. The practitioner of medicine is not satisfied with cold chemical facts; he needs more; viz., the biological and clinical interpretation of these facts, and an elucidation of their relationship to normal physiology and to physi- ology perverted by disease. Our knowledge of urinary pathology and of clinical urinology, while still woefully incomplete, has neverthe- less advanced so rapidly within the last few years that I feel justified in divorcing myself completely from prece- dent in many directions. I refer to the arrangement of the subject-matter in this volume and, in particular, to the interpretation of the pathogenesis and of the clin- ical significance of many of the uiinary ingredients. I am fully aware of the fact that a few of the statements made in the text will appear iconoclastic to the casual reader, but it is well that false idols, ancient atid vener- (iii) iv PREFACE ated though they may be, should be overthrown. The incessant advance of science alone can corroborate or refute the facts of Clinical Urinology that we believe to be true today. ALFRED C. CROFTAN. Chicago, May, 1904. TABLE OF CONTENTS CHAPTER I P4flH The Albumens ot- the Urine 1 Albuminuria — Physiological Albuminuria, Albuminuria Minima, Intermittent and Cyelio Albuminuria, Postural Albuminuria, Extra Renal Albuminuria, Renal Albuminuria. The different albumens of the urine and their clinical significance — Serum Albumen, Serum Globulin, Nuoleo -Albumen (Mucin), Albu- moses and Peptone, "Benee-Jones Albumen," Fibrin (Fi- brinogen), Histon and Nucleo-Histon. The qualitative and quantitative determination of the different albumens of the urine. CHAPTER II The Purin Bodies op the Urine; Uric Acid and Its Chemical Congeners 26 Nomenclature and Definition of the Members of the Purin Group (Uric Acid, Xanthin, Hypoxanthin, etc.). The Factors Deter- mining the Excretion of Uric Acid and its Chemical Con- geners. The Pathogenetic Role of the Purin Bodies. Uric Acid — Its Clinical Significance and Estimation. The Purin Bases — Their Clinical Significance and Estimation. Other Bodies Allied to Uric Acid and the Purin Bases; viz., Nu- cleinic Acids and Allantoin. CHAPTER III This Total Urinary Nitrogen and Urea 39 The Nitrogenous Constituents of the Urine. The Laws Govern- ing '^Nitrogen Equilibrium." The Factors Determining the Urinary Excretion of Nitrogenous Bodies in Health and in Disease. The Clinical Significance of Fluctuations in the Urea Output and in the Output of the Total Urinary Nitrogen. Some Fallacies of Urea Determinations as an Index of Renal Inadequacy. The Quantitative Estimation of Urea. The De- termination of the Total Urinary Nitrogen. Miscellaneous Nitrogenous Constituents of the Urine. Creatinin — Its Clini- cal Significance and Determination. (V) vi TABLE OF CONTENTS CHAPTER IV PAGB The Carbohydrates of the Urine 58 Note on the Physiological Chemistry of the Urinary Carbohydrates — Monosaccharides ; Disaccharides ; Polysaccharides ; GIuoo- sides; Glycoproteids. The Carbohydrates of the Urine as a Group — The Unfermentable Carbohydrates. Glycosuria — Phy- siological and Alimentary Glycosuria; Glycosuria e saecharo and e amylo. Toxic Glycosuria after Drugs and Poisons, Organ Extracts, Bacterial Poisons, in Auto-intoxication, Psychic Glycosuria and Glycosuria in Diseases of the Nervous Sys- tem. Eenal Glycosuria. Glycosuria in Obesity, Gout and Arteriosclerosis. Diabetic Glycosuria, the Three Degrees of Diabetic Glycosuria and the Mathematics of the Diabetic Diet. Tlie Different Carbohydrates of the Urine, their Clinical Signifi- cance, Detection and Determination — Pentoses (Pentosuria). Dextrose, Levulose, Laiose (Leo's Sugar), Isomaltose, Lactose. Animal Gum, Glycogen. CHAPTER V The Acetone Bodies op the Urine; /S-Oxybutyric Acid, Diacetic Acid and Acetone 98 Note on the Physiological Chemistry of the Acetone Bodies of the Urine — The Factors Determining the Excretion of the Ace- tone Bodies in Various Morbid States. The Clinical Signifi- cance of the Excretion of Acetone Bodies. Qualitative Tests and Quantitative Determination of jS-Oxybutyric Acid, Dia- cetic Acid and Acetone. CHAPTER VI The Blood- and Bile-pigments op the Urine lU The Chemical Relationship Between Blood- and Bile-pigments. The Blood-pigments of the Urine — Hemoglobin (Hemoglobi- nuria and Hematuria), Hematin, Hematoporphyrin, their Clini- cal Significance, Detection and Quantitative Estimation. The Bile-pigments ( Choluria ) — ^The Factors Determining their Excretion, their Clinical Significance (hematogenous and hepatogenous icterus), their Detection and Quantitative Esti- mation. The Bile Acids of the Urine — Their Clinical Signifi- cance, Detection and Quantitative Estimation. Urobilin (Urobilinuria) — Its Formation, Clinical Significance and De- termination. Meli^nin (Phymatorhusin) — Its Clinical Signifi- cance and Detection. TABLE OF CONTENTS vii CHAPTER VII PAWK The Aromatic Constituents of the Urine 135 Classification, According to Pathogenesis and Chemical Constitu- tion, into Four Groups. 1. Ttie Conjugate Sulphates — Phe- noles. Carbolic Aeid,Cresole, Pyroeateehin, Hydroquinon, Indol (Indoxyl), Indicanuria, Skatol; Their Clinical Significance, Detection and Quantitative Estimation. 2. The Conjugate Glyeuronates — Their Physiological and Pathological Chemis- try,^ Clinical Significance, Detection and Estimation. 3. The Compound Glycoeolls — Hippuric Acid and Phenaeeturic Acid; Their Clinical Significance, Determination and Detec- tion. 4. T)ie Aromatic Oxyadds — The Alkaptonic Acids (Homogentisic and Uroleucinic Acids), Alkaptonuria, Its Pathogenesis and Clinical Significance. CHAPTER VIII Miscellaneous Fatty Acids of the Urine 159 Oxalic Add. Volatile Fatty Adds — Formic, Acetic, Propionic, Butyric, Valerianic and Oleic Acids. Lactic Add. Leucin and Tyrosin. Their Pathogenesis, Clinical Significance, Detection and Determination. CHAPTER IX The Inorganic Constituents of the Urine 172 The Factors Determining the Excretion of the Mineral Salts of the Urine. The Total Urinary Ash. The Inorganic Adds of the Urine — Hydrochloric Acid (Chlorides) — Hydrofluoric Acid (Fluorides) — Sulphuric Acid (Sulphates), Preformed or Min- eral Sulphates, Conjugate or Aromatic or Ethereal Sulphates, Neutral Sulphur Compounds (Cystinuria and Diaminuria) — Sulphureted Hydrogen (Sulphides) — Phosphoric Acid (Phos- phates) — Carbonic Acid (Carbonates) — Silicic Acid (Silicates) — Nitrates and Nitrites — Peroxide of Hydrogen. The Inorganic Bases of the ZTrine — Potassium and Sodium — Calcium and Magnesium — Ammonium — Iron. CHAPTER X Urinary Sediments 219 The Factors Determining the Formation of Urhinry Setliiiienta. Unorganized Sediments, (1) in Acid Urine, (2) in Alkaline viii TABLE OF CONTENTS PAGE Urine. The Macroscopic Appearance of Certain Inorganic Sediments. The Different Inorganic Sediments in Detail ; Uric Acid and Urates; Calcium Oxalate; Calcium Sulphate; Calcium Carbonate; Phosphates; Cystin; Xanthin, Hippuric Acid; Bilirubin and Hematoidin; Indigo-blue and Indigo-red; Hetero-albumose; Leuein and Tyrosin. Organized Sediments. Blood-corpuscles; Pus; Epithelia; Casts — the Pathogenesis of Casts — Their Classification — the Different Casts in Detail and Their Clinical Significance; Spermatozoa. Micro-organ- isms. Saprophytic Bacteria. Pathogenic Bacteria: Bacillus Coli — Eberth Bacillus — Pus Germs — Gonoooecus — Tubercle Bacillus — Actinomycosis. Moulds. AnimM Parasites — Filaria Sanguinis Hominis ( Chyluria ) — Distoma Hematobium — Echinococcus — Eustrongylus Gigas — Feecal and Vaginal Para- sites in the Urine. CHAPTER XI The Concretions of the Ukine, Their Description and Analysis . 259 Uratio Concretions; Phosphate Concretions; Calcium Oxalate Con- cretions; Cystin Concretions; Xanthin Concretions; Mixed Con- cretions — Calcium Carbonate, Calcium Sulphate, Leuein, Tyrosin, Bilirubin, Hippuric Acid, Cholesterin, Pat. — Gen- eral Preliminary Analysis. CHAPTER XII The Physical Properties of the Urine ~ 264 Quantity. Specific Gravity. Reaction. Optical Properties: Color; Fluorescence; Behavior toward Polarized Light; Spectrum; Transparency. Odor. Freezing Point {Cryoscopy) . Electric Conductivity . CHAPTER XIII The Determination of the Renal Function 273 The Facts and Principles Underlying the Different Methods for Determining the Renal Function ; The Kidneys Considered as Osmo- regulatory Apparatus, as Selective Filters, as Glands. Cryoscopy. The Methylene Blue Test. The Phlorizin Method. The Toxicity of the Urine as an Index of the Renal Function. INTRODUCTION Under normal conditions, that is, in health, and on an average mixed diet, the urine contains a fairly constant mixture of highly oxidized terminal products that are derived from the metabolism of the food constituents and of the body tissues. Numerous morbific influences can pervert this normal metabolism in different directions, so that in disease the urine contains a large variety of bodies that are either altogether foreign to normal urine or that are present in normal urine in very much smaller quantities^ frlie character of the urine in disease depends chiefly on certain general factors, as the character and the quan- tity of the diet, the condition of the digestive and assimi- lative functions, the-pfeserre6"Df absence of fever, the in- volvement of various organs in the disease process, cer- tain neurosal tendencies and many other elements. ^ Only in rare cases do specific factors become operative that cause the excretion in the urine of bodies that can be con- sidered pathognomonic for any one disease. The formulation of urinary paradigmata for different diseases, that many authors attempt is, therefore, mani- festly neither accurate nor practical. It is simpler and more useful, even from the clinical standpoint, to classify the different normal and pathological ingredients of the ui*ine, according to chemical groups ; for it will be found that in the majority of instances the urinary bodies that X INTBOBUGTION are chemically allied are also clinically, scil., pathogeneti- cally related. The plan of the book, therefore, as far as the dis- cussion of the chemical ingredients of the urine is con- cerned, is to group these bodies according to their chem- ical relationship, to discuss in broad outlines the factors that determine their excretion in health and in disease, to relate what is known in regard to the effect exercised on their excretion by qualitative and quantitative changes in food and drink, by fever, by specific bacteria and poi- sons, and by impairment of the function of various organs, including the kidneys and the lower portions of the uropoietic apparatus. Inversely, the clinical inter- pretation of certain groups of urinary bodies is attempted whenever it is possible in the present state of our knowl- edge to do so. In a general sense, therefore, the book covers the pathology of metabolism in so far as perver- sions of metabolism due to whatever cause become mani- fest in the urine and determine its character. I have devoted a long chapter to the inorganic con- stituents of the urine, because I believe that future clinical studies will have to occupy themselves much inore assid- uously than in the past with these cinders of metabolism. The day is over when the inorganic salts circulating through and around living protoplasm could be disre- garded as "the ash, the unessential, the clinkers clogging the grates of the protoplasmic fires." I need only call attention to the role of small quantities of calcium in the clotting of the blood, the effect of different salts ("ions") of sodium, potassium, calcium, magnesium, etc., upon the rhythmical action of the heai-t, upon INTRODUCTION xi muscle contraction, upon nerve stimulation and inhibi- tion, upon intracellular oxidation, upon the power of the circulating ferments, and of the ferments poured into the gastro- enteric tract, upon parthogenesis, etc. The inorganic salts of the body fluids, while constituting only about one -twentieth of the total solids in solution, nevertheless play a most important part in the regula- tion of those osmotic phenomena that govern many life processes; they are, moreover, vitally concerned in the generation and transmission of the electrical charges and discharges that accompany all the activities of living protoplasm. As variations in the electrolytic components, or ions, of the inorganic salts determine changes in the protoplasmic function, it is of paramount importance that the relations of the different ions should remain constant. The maintenance of this constancy is largely relegated to the kidneys. They promptly eliminate a surplus and refuse to eliminate when there is a deficit. This activity of the kidneys becomes mani- fest in the urine, and I will have occasion to explain the important clinical conclusions that can often be drawn from fluctuations in the excretion of the differ- ent inorganic constituents of the urine. As the excretion of urinary bodies is directly depen- dent upon the state of the kidneys, a special chapter upon the determination of the renal function has been added. For it is clear that inadequacy of the kidneys to excrete certain effete products that are carried to them in the renal blood, and that clamor for elimination, as well as abnormal permeability on the part of the kid- neys for circulating bodies that should properly be xii INTRODUCTION retained, must produce corresponding deviations from the normal in the composition of the urine. In this chapter some of the generalizations of physical chem- istry are brought to bear upon this clinical problem; for our chief means for determining the state of the renal function is to study the relative amounts of inorganic ingredients in solution in the blood and in the urine. Here, then, the modern clinician must fa- miliarize himself with the technique and interpretation of freezing-point determinations (cryoscopy), and of the measurement of the electric conductivity of blood and urine. In addition, the method of experimental elimination ("elimination provoquee") of bodies arti- ficially introduced will be studied as an index of renal permeability. Finally, the power of the kidneys to perform certain chemical reactions (cleavage and syn- thesis) with various bodies that are carried to them in the renal blood will be discussed, because this, too, gives us valuable information in regard to the state of the renal function. In the chapter on urinary sediments^ particular empha- sis has been placed upon the origin of these bodies. In the case of the inorganic sediments, the factors that determine their precipitation are fully discussed, and in the case of the morphotic elements, the pathological pro- cesses that lead to their appearance in the urine are carefully described. All the important sediments are depicted in drawings, many of them original, and rela- tively little space is given to descriptions of their appear- ance under the microscope ; for a single glance at a good illustration conveys more to the mind than the most INTRODUCTION xiii exhaustive word -picture. The clinical significance of the different sediments is conservatively interpreted, and many popular misconceptions in regard to the diagnostic importance of many of these bodies, I trust convincingly, corrected. In the case of the vegetable and animal para- sites of the urine, only those have been described that possess some pathological importance, or that might lead to confusion with such organisms. The concretions of the urine, their pathogenesis, differentiation and clinical significance have been described in a separate chapter. A description of the physical 'properties of the urine is given in a special chapter. Here will be found in a very concise form merely what we need know from a clinical standpoint in regai'd to the quantity, the reaction, the specific gravity, the optical properties, the odor, the freezing point, the electric conductivity, etc., of the urine. This chapter, however, represents only a con- densed summary of our knowledge of these facts. Scat- tered throughout the book will be found, in their proper places, discussions of these various physical properties of the urine, and of the factors that influence them, and of the clinical interpretation of deviations from the normal. I have exercised much care in the selection of the qualitative and quantitative methods that I describe. It is, of course, an impossible task within the narrow limits of a book of this character to give all the methods. In fact, from the standpoint of the clinician, i. e., the stu- dent of medicine and the practician, it is not desirable that every method that has evier been employed should be described. A clinical work should, above all things, be practical and convenient for ready reference. I have. xiv INTRODUCTION therefore, chosen those methods only that, in my experi- ence, are particularly adapted for clinical work, that require the smallest sacrifice of time and the minimum of apparatus, that do not require expert skill of a very high order, and that at the same time yield results that are sufficiently reliable for clinical purposes. Here and there I have even considered it expedient to give a method because it is simple in preference to a somewhat more accurate, but at the same time much more complicated one. I presuppose a knowledge in my readers of the ordi- nary chemical and histological manipulations of the ana- lytical, microscopical and bacteriological laboratory. Very few practicing physicians will employ the spectro- scope and the polariscope extensively ; those that do have been instructed in their use, or have access to text- books describing them. I have refrained, therefore, from de- voting valuable space to a discussion of the principles underlying the use of these apparatus, and to the descrip- tion of the technique employed in working with them. The conclusions, on the other hand, that can be drawn from polariscopic and spectroscopic examinations are fully discussed. A brief description of the cryoscope for performing freezing point determinations is given, because this apparatus is not so well known nor so simply described in the average work on Physics. A large number of text-books and original articles were consulted in gathering the material for this book. To all these sources I herewith collectively acknowledge my indebtedness. I have omitted specific references to these books and monographs, for literature references at best INTRODUCTION xv are of value only to the original investigator, and not to the student and practician, for whom this book is pri- marily written. Here and there I was forced to refer to some of my experimental contributions to the subject of clinical urinology when bestowing an interpretation upon certain phenomena, that was based exclusively on the results of my own researches. No claim to completeness is made in this little volume. Everything that I consider of clinical value is given, nothing that I believe to be of practical use is omitted. If such subjects as Ehrlich's diazo- reaction, the "uro- logical coefficients" and the "coefficient of demineraliza- tion" of French authors, the discussion of the fluctuating ratios between uric acid and urea, between the phos- phates and urea or the total nitrogen, etc., are not incor- porated in this book, it is because I do not think that they have so far vindicated their claim to recognition as valuable adjuvants to urinary diagnosis. CLINICAL URmOLOGY CHAPTER I THE ALBUMENS OF THE TJEINE Albuminuria — Physiological Albuminuria, Albuminuria Minima, Intermit- tent and Cyclic Albuminuria, Postural Albuminuria, Extra Renal Albu- minuria, Renal Albuminuria. The different albumens of the urine and their clinical significance — Serum Albumen, Serum Globulin, Nueleo- Albumen (Mucin), Albumoses and Peptone, "Benee- Jones Albumen,'-' Fibrin (Fibrinogen), Histon and Nuoleo-Histou. The qualitative and quantitative determination of the different albumens of the urine. ALBUMINURIA Physiological Albuminuria. — The urine always con- tains cellular elements that are derived from the urinary- passages. The albumen contained in these desquamated epithelia is not altogether insoluble in the urinary water, particularly if the urine is alkaline; consequently every normal urine contain traces of albumen in solution. The latter, however, is not serum albumen, but nucleo-albu- men (see below), and is present, moreover, in such minimal quantities that it escapes detection by the ordi- nary clinical tests ; in order to positively demonstrate its presence, it must be isolated from large quantities of urine and identified by complicated chemical manipula- tions. In addition to this nucleo- albumen (mucin), the urine of apparently healthy subjects often contains traces of serum albumen. It is established that this albuminuria may be transitory, and may lead to no serious conse- quences. Hence, this condition has been christened with the name of physiological albuminuria. I consider this A (1) 2 CLINICAL URINOLOGY term a misnomer, for a biologic phenomenon, because it is innocent, need not, therefore, be physiological. It is better always to regard the excretion of serum albumen as a pathological phenomenon. In many of these cases, it is true, evidences of organic renal changes are absent and even remote manifestations of renal involvement fail to appear. The only symptom is albuminuria; but this transudation of albumen is invariably caused by nutri- tional impairment of the renal epithelia, due to intoxica- tion or to insufficient supply of oxygen. The determin- ing factors may be excessive muscular exercise, cold bathing, psychic shock, slight digestive disorders, epilep- tic seizures, dyspnoea, etc., — in short, any agency that can cause either circulatory disturbances in the kidneys or transitory flooding of the blood- stream with abnormal and poisonous products of metabolism. In addition to these acute and very evanescent so- called physiological albuminurias or "albuminurias of healthy subjects," there are a variety of recurring transi- tory albuminurias that are chronic and still not due to organic renal changes, i. e., nephritis. True, it is often difficult to determine the boundary between functional disorders of the kidneys and organic changes in these organs, and as a matter of fact some of these recurrent forms of transitory albuminuria, on the one hand, follow acute infectious nephritides, and on the other hand ulti- mately develop into true nephritis. Pathologically, there- fore, these forms are not clearly differentiated; clinically, however, they form distinct and important entities that are important, chiefly, because they are so often overlooked, or, if discovered, are, as a rule, grossly misinterpreted. It is convenient to group these cases under the ortho- dox heading of albuminuria minima, with two subdivi- sions; viz., intermittent albuminuria and cydlic albumi- nuria. The terms intermittent and cyclic albuminuria are often employed synonymously; they are not, how- TEE ALBUMENS OF TEE URINE 3 ever, interchangeable, for intermittent albuminuria need by no means be cyclic in character. Albuminuria Minima. — This form of albuminuria is most commonly discovered by chance, as, for instance, during examinations for life insurance in subjects who are supposedly healthy, it is prevalent, according to our statistics, particularly among physicians, druggists and others who possess the technical skill and the facilities to frequently analyze their own urine. The pathogenesis is altogether unclear, because no autopsy reports are on record, as no one dies of the disease, and because sub- jects afflicted with albuminuria minima are not sufficiently sick to place themselves in the hands of a physician for long periods of time. The quantity of albumen passed is very small, rarely exceeding 0.5 to 1 pro mille a day; at times the albumen disappears completely, at other times, again, larger quan- tities are excreted. This condition of aflEairs persists for years without, in the majority of cases, entailing any serious consequences; per contra these individuals fre- quently present a most flourishing appearance and enjoy the best of health. Nevertheless, a certain not incon- siderable proportion of the cases finally terminate in true diffuse nephritis, so that the serious character of minimal albuminuria should never be underestimated. One should, therefore, constantly in these eases be on guard for the appearance of renal elements, evidence of renal inadequacy and the development of cardio- vascular changes. Intermittent Albuminuria.— The most common factors that determine the intermittent and transitory appear- ance of albumen are: (1) Nervous influences, (2) ex- posure to cold, (3) the diet, (4) physical overexertion. Many sufferers fi'om intermittent albuminuria are neuras- thenic; it is difficult to determine, however, whether the neurasthenia is a cause of the albuminuria, or whether 4 CLINICAL URINOLOGY both states are manifestations of a primary vaso-motor disorder afifecting simultaneously the nervous system and the renal epithelia. Sometimes psychic and emotional shock, mental depression, chorea, epilepsy, etc., cause transitory albuminuria, but the determining influence of the nervous factor cannot always be definitely established in all of these cases. Cold bathing presumably also causes transitory minimal albuminuria in predisposed subjects by vaso-motor and nervous influences. The albuminuria after cold bathing is usually very slight (0.2 to 0.3 pro mille), and is not dependent on the polyuria that follows the exposure to cold. The influence of the diet, of the time of eating, on the appearance of albumen in the urine is important. There are subjects with perfectly normal digestion who excrete a little albumen after each meal, others who excrete it only after eating certain articles of food. In digestive disorders this phenomenon is still more common, and there is, I believe, a distinct dyspeptic albuminuria that is very common and very important; it is seen in dilata- tion of the stomach and in many intestinal disorders. This form of albuminuria may be due to incomplete disassimila- tion of the food albumen, with entrance into the blood of proteids that are abnormal to the tissue fluids, and that are consequently at once eliminated through the kidneys ; or it may be due to the absorption of toxic products that irritate the renal epithelia in their passage ; this form, at all events, is almost invariably coupled with great indican- uria and, as a rule, yields promptly when the gastro-intes- tinal disorder is corrected. If allowed to persist it may lead to true nephritis. That excessive physical exertion may finally produce transitory albuminuria is a well-established fact. Ordi- narily the albumen excreted is nucleo- albumen (mucin) ; in predisposed subjects, however, serum - albumen may also appear. The albuminuria is here due either to cir- THE ALBUMENS OF THE UBINE 5 culatory disturbances in the kidneys, or to the flooding of the blood with catabolic poisons. The occurrence of this form is clinically important, because the element of over-exertion should always be ruled out in interpreting albuminuria. Cyclic Albuminuria. — This form of albuminuria is char- acterized by periodic fluctuations in the excretion of albu- men in the twenty-four hours period; there are, there- fore, each day periods in which albumen appears in the urine, and periods in which it is absent. Occasionally no albumen appears for days, or weeks, or months, and then the daily cycle is resumed; such forms may fitly be called intermittent cyclic albuminuria. I wish to clearly exclude from the category of cyclic albuminuria minima those cases of true nephritis in which more or less regu- lar fluctuations in the amount of albumen recur each day ; there we are dealing with definite factors (to be discussed below) that determine these fiuctuations; I include only those forms in which no evidence of nephritis can be discovered. The most common cycle is the following one: On rising in the morning, no albumen; first appear- ance of albumen between 9 and 12 a.m., reaching its maximum early in the afternoon and gradually disappear- ing again toward evening. The quantity of albumen may fluctuate on different days. In the majority of cases the albumen is nucleo-albumen, although cases also oc- cur in which serum-albumen aloneis excreted, and others in which serum-albumen with nucleo-albumen, or with albumose, hemoglobin, etc., is excreted. All the factors that are incriminated with producing intermittent (not cyclic) albuminuria have also been made responsible for cyclic albuminuria. A careful sift- ing of the casuistic material, however, shows that only two elements enter into the pathogenesis of this peculiar anomaly; viz., — changes in the position of the body and muscular fatigue. 6 CLINICAL URINOLOGY The influence of posture upon this form of albu- minuria is so well recognized that a number of terms, that are synonymous with cyclic albuminuria, are em- ployed to designate this relation; viz., — "postural albu- minuria," "orthotic or orthostatic albuminuria." Numerous explanations have been proffered to explain this pecu- liarity, but none of them is altogether satisfactory. The most plausible assumption is the one that cyclic albu- minuria is due to a certain reactive insufficiency of the circulatory apparatus. In subjects afflicted with cyclic albuminuria, it appears, the blood pressure becomes lower after exercise than in normal subjects; the circula- tion in the kidneys is impaired as a result, and albu- minuria follows. On this basis the fluctuations at ditferent periods of the day must be explained by differences in the reaction of the blood pressure to certain bodily activities, the reaction being less energetic in the early morning hours, i. e., at the time of albuminuria, than late in the day. Cyclic albuminuria, according to this very seductive theory, is hence to be considered a mani- festation of vaso-motor fatigue. Heredity and family predisposition seem to play a role in the production of this state. The theory has finally been advanced that cyclic albuminuria is a light form of paroxysmal hemo- globinuria (see Chapter VI on Blood and Bile Pigments) , in which the blood pigments are retained in the liver and spleen and the albuminous residue is excreted; cyclic albuminuria has, in fact, been known to precede, accom- pany and follow paroxysmal hemoglobinuria. On the basis of the vaso-motor fatigue theory, cardiac tonics are indicated, and good results have actually been obtained by this therapy. In rendering a diagnosis of cyclic albuminuria the presence of tumors that can compress one kidney or its vessels (splenic tumor, e. g.) and abnormal motility or dislocation of one or both kidneys must be excluded, THE ALBUMENS OF THE URIXE 7 for it is clear that these factors by interfering with the renal circulation when the patient occupies certain posi- tions may well simulate the picture of postural albu- minui'ia. Extrarenal Albuminuria.— {^yn. Pseudo- albuminuria, albuminuria spurea). The urine may contain albumen from the admixture of albuminous fluids (blood, pus, sperma, tumor- juice, etc.) that enter the urinary pas- sages below the kidneys. As a rule, it is easy to deter- mine that the albumen is not derived from the kidneys if the urine is allowed to stand for a time; the heavy albuminoixs admixture, that usually contains many mor- photic elements, settles at the bottom of the vessel and the supernatant clear, or filtered, urine contains only traces of albumen as compared with the number of cellu- lar elements in the sediment. If the latter consists of pus alone, Groldberg's rule applies; viz., the proportion of albumen in the filtrate (expressed in pro mille according to Esbach) compared with the number of pus-cells in 1 cmm. of the sediment must be less than 1:50,000; or, expressed differently, if a cubic millimeter of the sedi- ment contains 50,000 pus-cells, then the albumen in the filtrate must not exceed one-tenth of one per cent; if it is more, then some of the albumen is derived from other sources than the pus. When interstitial nephritis with a small excretion of albumen is complicated, e. g., with catarrh of the bladder, the interpretation of the albumi- nuria may be difficult; here the remote cardiac and ocu- lar symptoms of the nephritis, the low specific gravity, the increased quantity of the urine, the presence of casts, etc., must determine the diagnosis. Renal Albuminuria (albuminuria vera). — This term was originally employed to designate the entrance of albumen into the urine somewhere within the glomeruli, or the uriniferous tubules of the kidneys. It included not only the excretion of the normal albumens of the 8 CLINICAL URINOLOGY blood serum, but also the excretion of albumoses, pep- tones, hemoglobin, fibrin and nucleo-albumen. It is more correct to limit the use of the term to the excretion of the albumen and globulin of the blood-plasma, and to designate the other forms of albuminuria as albumosuria, peptonuria, fibrinuria, etc. The term also broadly covers albuminuria minima, i. e., intermittent and cyclic albu- minuria as discussed above. I propose to limit the term renal albuminuria to the excretion of serum-albumen and serum-globulin in organic lesions of the glomeruli and canals of the kidneys. The factors that determine degenerative and destruc- tive lesions of the renal epithelia with resulting diapedesis of plasma albumen are, in the overwhelming majority of cases, toxic; occasionally the mechanical effect of bacteria acting as foreign bodies or certain circulatory distur- bances of extra-renal origin may exercise the same influ- ence. The character of the lesions will depend on the virulence and the intensity of the poison, its selective affini- ties for certain portions of the renal structures (epithelia, blood-vessels, interstitial tissues) and the length of time during which it exercises its effect. In view of the fact that the renal epithelia react at once to the slightest changes in the qualitative and quantitative composition of the blood, it is difficult to determine where so-called functional perversions stop and where organic lesions be- gin. The strict differentiation of so-called dyscrasic albu- minuria (anaemia, cachexia, etc.) and albuminuria due to nephritis, is therefore difficult; the albuminurias de- scribed above as transitory albuminurias are transitory only because the intoxication of the epithelia does not persist for a sufficient length of time to cause irremedi- able degenerative or destructive lesions of the epithelia. If we accept the above etiology, a strict classification of the different forms of nephritis becomes impossible ; the cumbersome nomenclature that has been invented is of THE ALBUMENS OF TEE UBINE 9 no value in advancing our clinical understanding, it is confusing to a degree and largely theoretically constructed. As a matter of fact every nephritis is " mixed " ; there is no parenchymatous nephritis without some interstitial changes, no primarily interstitial nephritis without some secondary parenchymatous lesions. There is a- nephritis of acute onset and short duration that may or may not lead to chronic changes and the acute infection may affect primarily the glomerular or tubular epithelia. There are a variety of chronic forms of nephritis that may follow an acute nephritis, or may develop insidiously, the dam- age occurring first in the secreting epithelia or in the in- terstitial tissues ; in the latter case usually as the result of circulatory changes of manifold origin. As far as the albuminuria in nephritis is concerned, all that we can say is that in acute nephritis the percentage of albumen is high — as high as 5 per cent, although usually not above 1 per cent; that in certain chronic forms that incline more to the parenchymatous type, the percentage of albumen may be equally high, although as a rule it is lower ; that the more chronic the nephritis and the more it approaches the interstitial type the smaller the percentage of albumen; until finally we reach certain very chronic forms in which, for long periods of time, there may be only traces of albumen or none at all. The last-named observation teaches that nephritis should not be excluded from the absence of albumen in isolated specimens of urine; in very early (transitory) irritation of the kidneys and in very late stages of nephritis, when albuminuria may be absent at times, the diagnosis must be attempted from the ocular and the cardio-vascular symptoms and from certain urinary phenomena, other than albuminuria, that will be discussed in subsequent chapters. 10 CLINICAL URINOLOGY THE DIFFERENT ALBUMENS OP THE URINE AND THEIR CLINICAL SIGNIFICANCE The urine may contain the following albumens, singly or in combination: (1) the albumens of the normal blood- plasma, i. e., serum-albumen (serin) and serum -globulin (paraglobulin) ; (2) nucleo- albumen (mucin); (3) albu- moses and peptone; (4) "Bence- Jones albumose"; (5) fibrin (fibrinogen); (6) histon and nucleo -histon; (7) hemoglobin. The latter will be discussed in the chapter on the blood- and bile -pigments, of the urine. 1. Serum - albumen and Serum - globulin. — These two albumens almost invariably occur together; a few cases are on record of pure " serinuria " in subacute and chronic nephritis, and in one case of gastic carcinoma; and one case of "globulinuria" is reported in a patient with leucaemia. The serum- albumen always predominates. The proportion |fj^ has been called the "albumen quo- tient " ; one would imagine it to be fairly uniform and to correspond to the albumen quotient of the blood- plasma of the patient; as a matter of fact, this is not the case. Clinical Significance. — The appearance of serum- albumen and serum-globulin alone or in combination with other albumens is always pathological. Minimal quan- tities when unaccompanied by other urinary evidence of nephritis indicate one of the forms of minimal albu- minuria described above; the appearance of minimal quantities urgently calls for frequent analyses of the urine not only each day but several times a day ; in this way alone can the character of the -minimal albuminuria, i. e., whether it be permanent, transitory, intermittent, cyclic or cyclic -intermittent, be determined. Every case of minimal albuminuria calls for a guarded prognosis, and one should constantly be on the lookout for the development of true nephritis. Serum -albumen and serum -globulin in quantities of over 1 pro mille always J'HE ALBUMENS OF THE URINE 11 render the diagnosis nephritis justified even though un- accompanied at times by renal elements and unless extra-renal in origin; if renal elements are present the case is one of nephritis. The albumen quotient is of little clinical value; rel- atively large quantities of globulin are excreted in acute forms of nephritis, in the albuminuria of pregnancy and in amyloid kidney; improvement of nephritic processes is usually accompanied by a decrease of the urinary globulin; the determination of the quotient may there- fore be said to possess some value in prognosis. 2. Nucleo - albumen (mucin). — The mucin of the older writers is a complex body consisting of three distinct combinations of serum- albumen with three different acids; these acids are nucleinic acid, chondroitin- sul- phuric acid and taurocholic acid; the first two are always present in minimal quantities in normal urine. If albu- men enters such urine a portion of it unites with these acids to form "mucin." Taurocholic acid is not found in normal urine ; it is a bile-derivative and is poured into the urine in large quantities in icterus. The character of the mucin varies according to the relative proportion of the three acids present in the urine at any given time. Acetic acid precipitates the three compounds as nucleo- albumen, chondro- albumen and albumen taurocholate. In desquamation of epithelia, with resulting degeneration of cell-nuclei, the nucleinic acid of the urine is increased ; as destruction of renal epithelia is invariably accom- panied by the exudation of some plasma albumen, we find the nucleo -albumen of the urine increased in all desquamative catarrhs of the kidneys. There is also an extra-renal form of nucleo-albuminuria due to desquama- tion of ureteral, vesical and urethral epithelia ; only in the latter form do we find nucleo -albumen alone, in all other states it occurs together with serum -albumen and serum-globulin. 12 CLINICAL URINOLOGY Clinical Significance. — Nucleo-albuminuria (mucinuria) is important clinically chiefly because it indicates degen- eration of epithelia. As these epithelia may be extra- renal, it is necessary in each case of mucin excretion to determine whether or not serum -albumen and globulin, renal elements and other signs of nephritis appear; if these signs are absent, then one is dealing with a catarrh of the lower urinary passages. As mucin gives several of the plasma-albumen reactions (see below) , particular care should be exercised not to confound the two. Nucleo- albumen is common in cyclic albuminuria; a few cases are recorded of pure cyclic (?) nucleo-albuminuria that were presumably, however, extra-renal in origin ; as a rule, in cyclic albuminuria both varieties of albumen appear together. In many infectious and toxic forms of nephritis, after chloroform anaesthesia, in croupous pneu- monia (disappearing as a rule within twenty-four to forty- eight hours after the crisis) and, above all, in icterus, the excretion of mucins, together with other albumens, is common. 3. Albumoses and Peptone. — In addition to the albu- mens of the blood-plasma and mucin, certain degra- dation products of native albumen, that are not coagulable by heat, occasionally appear in the urine, either alone or in combination with other albumens. As the occurrence of these bodies in the urine was described before the finer chemical differentiation of this group of albumens was understood, the clinical nomenclature that is generally used does not correspond to the more exact chemical nomenclature that we have since learned to adopt. This means confusion, and it is often difficult to determine just which albumen the older authors referred to when describing cases of albumosuria and peptonuria. The present status of this important inquiry may be briefly expressed as follows: The gastro-intestinal secretions contain ferments that THU ALBUMENS OF THE UBINE 33 convert native albumen (i. e., the ordinary food albu- men) into more soluble disassimilation products ; • chief among the latter are the albumoses (hetero-albumose, proto-albumose, deutero-albumose) and pepton. These bodies are absorbed from the bowel, and somewhere in the intestinal wall undergo reconversion into more com- plex albumens, chiefly serum- albumen. Normally, there- fore, the blood contains neither albumoses nor peptones. In fact, these bodies are known to be very poisonous and when injected directly into the blood-stream are immedi- ately excreted through the kidneys. In a variety of lesions of the digestive tract the normal reconversion in the bowel-wall does not occur, and albumoses and pep- tones enter the blood and thence the urine. Contrary to older teachings, very little peptone is formed in the intes- tine, the disassimilation of the food albumens stopping short at the first albumoses ; only in cases of intestinal stagnation and in infection of the bowel contents with certain bacteria that secrete proteolytic enzymes of great power is the degradation of the albumens carried to the peptone stage. As a result, true peptone is hardly ever absorbed from the bowel, even under the pathological conditions outlined; albumoses, on the other hand, are not infrequently absorbed. Albumen - destroying ferments (proteases) are not found in the stomach and bowel alone, but also in the blood and lymph. Whether or not their normal function is to destroy the circulating albumen remains undeter- mined; we do know, however, that they promote the dis- assimilation, and hence the liquefaction (scil. autolysis) of degenerating and dead albuminous tissues and fluids wherever these may occur in the organism, and hence pro- mote their absorption and removal from the body. This, then, is another source of albumoses and peptones that must enter the blood and hence ultimately appear in the urine. Here, again, we obtain chiefly albumoses 14 CLINICAL URINOLOGY and only rarely peptone— then, namely, when there is prolonged stagnation in and around the degenerating or necrotic focus, with impaired circulation in the vicinity. Such conditions are rare, consequently albumoses are much more commonly derived from this source, too, than peptone. The identification of a pathological non - coagulable albumen in the urine as peptone is often impossible. Albumoses and peptones have many reactions in common, and the distinction between the two by differential pre- cipitation with ammonium - sulphate is never free from ambiguity; one should properly speak of peptonuria only when the urine contains an albumen that is not coagula- ble by heat, that is not precipitated by the ordinary albu- men precipitants (see below), and that gives the biuret reaction. As a matter of fact, such an albumen is very rarely found in the urine, so that nearly all the cases of peptonuria that have been described in the literature should properly be termed albumosuria. As the clinical significance of albumosuria and (true) peptonuria is the same, nothing is detracted from the value of the older observations on peptonuria. "We need only change the name, i. e., discard the term peptonuria and employ albumosuria in its place. Clinical Significance.— It is impossible to enumerate the many diseases in which albumoses (peptone) have been found in the urine. If one will remember what has been said above in regard to the origin of the urinary albumoses and peptone; viz., that they may enter the blood- stream and thence the urine, either when the bowel- wall is diseased in such a way that its function to recon- vert albumoses (and peptone) into serum-albumen is per- verted, or when degenerating or necrotic tissues undergo proteolytic intracellular digestion (autolysis), one need not burden one's memory with remembering a variety of so-called clinical forms of albumosuria (peptonuria) that THE ALBUMENS OF THE URINE 15 have been formulated by different writers. Such terms as enterogenous, puerperal, pyogenous, hematogenous "peptonuria," are intended to designate the tissues from which the "pepton" is derived and the conditions under which it is formed. Thus we know that in many diseases of the liver, ac- companied by gastro- intestinal disturbances of the char- acter described above, albumoses enter the blood-stream; that in carcinoma of the stomach and in atrophic pro- cesses of the stomach and bowel, albumoses may appear in the urine; this is the enterogenous form. During the first four days of the puerperium albumose is commonly found in the urine — here it is derived from the subinvolution of the uterus and the absorption of dis- assimilating dead albuminous debris. To the same cate- gory belongs that form of albumosuria that accompanies the absorption of hemorrhagic or fibrinous exudates; it is seen after ecchymoses, croupous pneumonia, apo- plexy, etc. The most important form, from a practical point of view, is the pyogenic form. The presence of pus in an inclosed cavity almost invariably determines the appear- ance of albumose in the urine; the explanation for this is apparent from what has been said above, for in a pus- focus we have a mass of degenerating albuminous mate- rial. Combined with a polynuclear leucocytosis this albu- mosuria is of the greatest value in establishing the diag- nosis of "pus somewhere." It is often possible by this means to decide with a reasonable amount of certainty between an empyema on the one hand and a simple serous or tuberculous pleuritis on the other, between a pus-tube and an ovarian pregnancy or hematoma, between a puru- lent or a serous arthritis, a purulent or a tuberculous men- ingitis, etc. Whenever, finally, there is much destruction of blood corpuscles, either red or white (cytolysJs, leucocytosis) , as 16 CLINICAL URINOLOGY in severe intoxication or infections (septicsBmia, phos- phorus poisoning, leucaemia, scurvy, intermittent fever, malaria, after tuberculin injections, etc.), albumose ap- pears in the urine ; — this is the so-called hematogenous "peptonuria." It will be seen, therefore, that albumosuria is a very common urinary symptom. If one will remember that in pure nephritis albumose never appears in the urine, the importance of identifying the urinary albumen as albu- mose becomes apparent, for nephritis should never be diagnosed from albumosuria alpne. It is important, finally, to remember that the urine normally contains small quantities of the digestive fer- ments, among them proteases (pepsin, trypsin ?) , and that the latter under favorable conditions may digest any serum- albumen that may be present in the urine and convert it into albumoses or peptone. Such conditions are stagnation of the urine, as in hydronephrosis, renal or ureteral calculus, retention of urine in the bladder from a variety of possible causes — for in all these states time is given for proteolytic digestion ; if the urine is kept for some time after it is voided, particularly if the tempera- ture conditions are favorable, the same may occur. This excretion of urinary albumose and peptone may fitly be called extra-renal albumosuria; it should always be thought of in interpreting albumosuria. 4. "Bence- Jones Albumen."— This body was first discovered in the urine by Bence- Jones in 1848. There is much diversity of opinion in regard to its mode of origin and its chemical constitution. A careful study of the cases reported seems to show that there is some doubt in regard to the uniformity of the albumen de- scribed by different clinicians under the name of Bence - Jones albumen. The body described partakes of some of the chemical characteristics of the albumoses, but is not identical with any of the albumoses normally formed by TEE ALBUMENS OF THE URINE 17 peptic or tryptic digestion in the gastro- intestinal tract; by artificial peptic digestion, however, it can be con- verted into the latter. It differs from all known albu- moses in that it is coagulable by heat (see below) . Clinical Significance. — The occurrence of this albumose in the urine is rare. In a large proportion of the cases it was associated wdth certain bone lesions (osteomalacia, multiple myelomata) ; in a considerable number of the cases, however, no bone disease was discovered. It is not impossible that this body is formed by intracellular digestion of degenerating bone -marrow — at least the ap- pearance in the urine at the same time of certain nucleins and of iron -containing bodies bears out this hypothesis. The appearance of Bence- Jones albumose in the urine should always direct attention to bone lesions and, in particular to osteomalacia and myelomata. The body is also of clinical interest, because it has been known to crystallize out of the urine in the form of spheroliths of very small size; in order that this should occur, the reaction of the urine must be acid. It is also important to remember that this albumose, in contradistinction to the other albumoses described, may appear in the urine in large quantities and for long periods of time. It almost invariably occurs alone — although one case is on record in which it appeared in association with other albumens. 5. Fibrin ( Fibrinogen ). — Pathological urine occa- sionally coagulates spontaneously. If the coagulation occurs after the urine is voided the whole contents of the vessel may be converted into a jelly, or shreds of fibrin may appear; if the coagulation occurs in the bladder, ureters or pelvis, then shreds and chunks of fibrin are passed with the urine, often with symptoms of colic or obstruction. This coagulation is due to the passage of fibrinogen into the urine; the latter, under the influence of the fibrin ferment that is generally present in the urine, 18 CLINICAL URINOLOGY is split into fibrin and other substances. Occasionally the urine coagulates on slight heating (56° C.) (i. e., be- low the coagulation temperature of the ordinary coag- ulable albumens) ; in such a case the fibrinous charac- ter of the coagulate should always be suspected. Much depends on the character and the quantity of neutral salts present in the urine, as these exercise a distinct influence on the coagulation temperature. Clinical Significance. — In hematuria fibrin coagulates are very often found in the urine; here the clinical inter- pretation of the fibrinuria is self-evident; as a rule, these coagula are red from the enclosure of red blood-cells. In chyluria fibrin is also often passed — the character of the affection can here easily be determined from the exami- nation of the sediment. In addition, however, there are cases of fibrinuria that are due to violent inflammation of the kidneys, ureters or bladder; just what factors determine the excretion of fibrinogen (fibrin), scil. the spontaneous coagulation of the urine in certain forms of renal and cystic inflammation and not in others, remains undetermined. In two cases of cantharidal nephritis in patients in whom cantharides plasters had been applied for acute rheumatic polyarthri- tis, the urine contained fibrin coagula; in several cases of nephritis the urine has been known to coagulate as a whole, and in other instances to deposit fibrin coagula. In one case of renal abscess of unknown etiology the urine contained fibrinous casts of the pelvis and ureter. Fibrinuria, therefore, unless associated with hematuria or chyluria, always indicates violent inflammation of the lower urinary passages. 6. Histon and Nucleo - histon. — These two albumens are derivatives of the cell-nuclei and are concerned in the coagulation of the blood. They partake of the chemical characteristics of the albumoses. (Special tests, see below.) THE ALBUMENS OF THE URINE 19 Clinical Significance. — These bodies have been found in the urine in diseases accompanied by great destruction of white blood -corpuscles. As histon is a splitting pro- duct of nucleo- histon, the former is more commonly found than the latter. Nucleo -histon has been found in one case of pseudo- leucaemia, histon in a number of cases of lymphaemia, and in many acute febrile diseases that are accompanied by leucocytosis, e. g., in general peritonitis, in erysipelas, in scarlatina and in the later stages of pneumonia. THE QUALITATIVE AND QUANTITATIVE DETERMINATION OF THE DIFFERENT ALBUMENS OP THE URINE Of the many tests for albumen that are known, I have selected the following four because they are particularly adapted for use in the clinical laboratory ; they are simple and rapid of execution and at the same time permit a ready differentiation of the most important urinary albu- mens. These four tests are: (1) the boiling test; (2) the nitric acid test; (3) the potassium -ferrocyanide test; (4) the biuret reaction test. (1) The Boiling Test. — This test, while the most popu- lar of all, is the least reliable. The urine should always be slightly acidulated, preferably with nitric acid; a floc- culent precipitate appearing on boiling indicates the pres- ence of serum-albumen or serum -globulin. If the urine is not acidulated a flocculent precipitate of normal calcium phosphate may appear; this, however, disappears on the addition of nitric acid, whereas an albumen precipitate persists or becomes more intense. Not more and not less than one or two drops of nitric acid (sp. gr. 1.2) should be added to each cubic centimeter of urine; if more is added albumen nitrate may form and go into solution; if less is added, only a portion of the basic phosphate is converted into soluble acid phosphate, and the albumen 20 CLINICAL URINOLOGY in combination with a base (alkali albuminate) may again remain in solution. It will be seen, therefore, that on the one hand small quantities of albumen can readily escape detection by the boiling test, and that on the other hand precipitates may be formed that can be confounded with albumen; for, in addition to the basic phosphates already described, uric acid and urates may precipitate on boiling urine contain- ing much neutral salt, and bile -pigments and certain uri- nary products excreted after the administration of certain balsams, resins and petroleum may form very misleading deposits. As the alhumoses and nucleo- albumens finally escape detection altogether by this test, I do not consider it a safe reaction for albumen in the urine, particularly in unskilled hands and when it is the only test performed. Bence-Jones albumen coagulates on heating the acidified urine but at a low temperature (50° C.) on boiling, this precipitate disappears again. Histon is not precipitated. (2) The Nitric Acid Test.— A few cubic centimeters of cold filtered urine a,re poured into a test-tube and about one cubic centimeter of officinal nitric acid (sp. gr. 1.2) allowed to carefully flow down the sides of the tube ; the .acid, being heavier than the urine, will form a separate layer below the urine. If considerable quantities of serum- albumen or -globulin are present, a white opaque zone wUl form at the plane of contact; if the amount of albumen is small, one or two minutes may elapse before the ring appears. Other urinary bodies may give rise to the formation of a nitric acid ring; with care they can, however, be dis- tinguished from the albumen ring. Thus urates in very concentrated urine may form a ring; in contradistinction, however, to the albumen ring, which begins at the plane of contact and extends upward, the urate ring usually begins above the plane of contact and extends downward toward the acid. Occasionally the particles of the urate THE ALBUMENS OF TEE URINE 21 cloud can, moreover, be seen to scintillate; this is due to their crystalline structure. To remove all doubt, the test should be repeated with diluted urine; if the ring was a urate ring it will not appear. The urate ring, moreover, disappears on heating. Urea, if present in large quanti- ties, may form urea nitrate and crystallize out in a zone above the acid ; a microscopical examination will at once reveal the typical crystals. Balsams and resins also form a nitric acid ring, but the cloudiness disappears at once if a little ether is added to the urine. Nucleo- albumin also forms a ring with nitric acid; the test should be repeated with diluted urine, and if the ring then becomes thicker the presence of this albumen should be suspected. Alhumoses form a nitric acid ring that disappears on heating the mixture and reappears on cooling; if the urine is heated to boiling the mixture may turn bright yellow. If both serum- albumen and albumoses are pres- ent, the urine will not become elear on heating but the cloudiness will increase on cooling. Bence-Jones albumen and histon act like the albumoses. If the urine contains much pigment, multi-colored rings may appear below the albumen ring. (3) The Potassium Ferrocyanide Test. — For this test the urine should be perfectly clear ; if, owing to the pres- ence of microbes, the specimen cannot be rendered quite clear, then a tube of the untreated urine should always be compared with the tube to which the reagents have been added. The urine is rendered acid with acetic acid and a 10 per cent solution of potassium ferrocyanide added drop by drop. Very small quantities of serum- al- bumen can be detected in this way by the appearance of a faint cloudiness; if much serum- albumen is present a thick, flocculent precipitate forms at once. It is well to dilute the urine for this test. In performing this test, a precipitate often forms on the addition of the acetic acid 22 CLINICAL URINOLOGY alone; this may be due to the presence of nucleo-alburaen, globulin or urates, and should be filtered off before adding the potassium ferrocyanide. Albumoses also gives a cloudiness with this reagent, but the precipitate disappears promptly on heating, to re- appear on cooling. Globulin and nucleo-albumen, as stated above, are precipitated on the addition of acetic acid — the two may be differentiated by adding more acetic acid, the former promptly going into solution, the latter not. (4) The Biuret Meaction. — The urine is treated with a 10 per cent solution of potassium hydrate and a 10 per cent solution of cupric sulphate, added drop by drop. In the presence of serum-albumen and -globulin alone the liquid turns pure violet; in the presence of albumoses or peptone alone it turns rose; if several of the albumens are present together the urine assumes tints intermediary be- tween violet and rose. If small quantities of albumen are present, care should be exercised not to add too much cupric sulphate, as otherwise the blue of the copper solution will cover the violet or rose tint of the biuret reaction. The Differentiation of the Urinary Albumens with the Aid of the above Tests. — With the aid of these four tests, the detection of the five important urinary albumens is possible; viz., serumulbumen gives tests 1, 2, 3 and 4. Globulin also gives all four tests and is, moreover, pre- cipitated on the addition of acetic acid in test 3, but redis- solves on the addition of more. Nucleo-albumen does not give test 1, but gives tests 2, 3 and 4; like globulin, it is precipitated by acetic acid in test 3, but, in contradis- tinction to globulin, it does not redissolve on the addition of more acid. Albumoses do not give test 1, but give tests 2, 3 and 4; the precipitates formed in the cold in tests 2 and 3 disappear on heating, only to reappear on cooling. Fepton finally gives neither tests 1, 2 nor 3, but gives a biuret reaction (test 4) of characteristic color. THE ALBUMENS OF THE URINE 23 If, as is commonly the case, several of the albumens are present together, it often becomes necessary to sepa- rate them and to perform special tests. The most impor- tant of these may be briefly given. Special Test for Globulin. — The urine is mixed with an equal volume of a saturated ammonium sulphate solution ; the globulins and albumoses are precipitated while the serum -albumen remains in solution; the precipitate is dissolved in a 1 per cent soda solution and the liquid acidified with acetic acid; the globulin is precipitated while the albumoses remain in solution. The globulin precipitate must be insoluble in a solution of sodium chloride. Special Test for Nucleo- albumen. — The urine is freed from serum-albumen and serum-globulin by boiling and filtering off the coagulate; the filtrate is treated with acetic acid, as in test 3; a precipitate indicates the pres- ence of nucleo-albumen. The chemical differentiation of the three mucins, nucleo- albumen, chondro-albumen and taurocholate of albumen is too complicated and too unimportant, clinically, to warrant description in this volume. Special Test for Albumoses. — The urine is acidified with acetic acid and mixed with an equal quantity of a satu- rated solution of common salt; the mixture is heated to boiling and filtered hot; any coagulable albumen or nucleo-albumen that may have been present remains on the filter while the hot filtrate contains the albumoses; on cooling, the latter should precipitate from the filtrate; the filtrate should give the biuret reaction. Special Test for Peptone (Salkowski). — Fifty ccm. of the urine are poured into a beaker and acidified with 5 ccm. of hydrochloric acid, the mixture precipitated with a 10 per cent solution of phosphotungstic acid and heated. The supernatant fluid is poured off from the resinous pre- cipitate and the latter dissolved in 8 ccm. of water, to 24 CLINICAL URINOLOGY which are added 0.5 ccm. of soda lye (sp. gr. 1.16). The blue solution is heated until it turns yellow. After cool- ing, the addition of a few drops of a very dilute cupric sulphate solution should give a red color (biuret reaction, test 4). Special Test for Bence-Jones Albumen. — If the urine gives a very marked albumose reaction, the presence of Bence-Jones albumose may be suspected, for in no other form of albumosuria are such large quantities of albumose excreted. The Bence-Jones albumose may be precipi- tated from the urine by adding an equal volume of satu- rated ammonium sulphate solution; the precipitate is taken up in hot 3 per cent sodium chloride solution and the solution treated with hydrochloric acid until every- thing is dissolved; on cooling the albumose precipitates. The precipitate is freed from the adherent salt solution by washing and centrif ugation and finally by dialysis ; as the salt diffuses out the albumose partially precipitates; finally the balance of the albumose is precipitated by alcohol, filtered off, dried and subjected to organic analy- sis; it must contain no phosphorus. For the details of this complicated method, I must refer to text-books of physiological chemistry. The Quantitative Determination of the Coagulahle Albu- mens. — The quantitative determination of the albumoses, nucleo-albumens, Bence-Jones albumen, nucleo-histon and histon, is of such subordinate clinical importance that it need not be described in this book. The determination of the coagulable albumens, serum-albumen and globu- lin, however, is important. Weighing Method. — The only absolutely accurate method for determining the coagulable albumens is to precipitate the albumens as in test 1, to collect the pre- cipitate on a weighed filter, to wash with water, alcohol and ether, to dry to a constant weight and to weigh. A nitrogen determination is then made with a portion of TSE ALBUMENS OF TEE URINE 25 this residue (Kjehldahl method, see Chapter III) , and the value obtained multiplied by 6.25; the figure obtained by this multiplication indicates in grams the amount of albumen present in the quantity of residue used for the determination ; from this the amount present in the whole residue can be calculated. For ordinary clinical work the most rapid and the simplest method is that of Esbach: it is sufficiently accurate for clinical purposes of comparison. The Method of Esbach. — The method is based on the precipitation of albumen by picric acid. The apparatus employed is called Esbach's Albumenometer (Fig. 1) . The tube is filled with urine to the mark U and with reagent to the mark E. The two liquids are thoroughly mixed by inverting the tube several times. The ap- paratus is then allowed to stand for twenty - four hours and the height of the column of precipitate read off on the scale. The figures % to 7 below U indicate the amount of albumen t-ig. i. in grams per liter of urine ; if, therefore, e. g. , the j^il^^eno- height of the column of coagulate is 3, then the ™«*«''- urine contains three grams of albumen to the' liter (1,000 ccm.), or 0.3 per cent of albumen. Esbach's reagent consists of 10 grams of picric acid and 20 grams of citric acid dissolved in one liter of water. In this determina- tion the reaction of the urine should be acid, its specific gravity not above 1010, and it should not contain more than 0.4 per cent of albumen. As a rule, therefore, the urine will have to be acidified and diluted and the dilu- tion included in the calculation. Occasionally the picric acid precipitate of albumen does not settle properly, then the above weighing method will have to be employed. The presence of quinine or antipyrin in the urine also in- terferes with this determination. CHAPTER II THE PUBIN BODIES OF THE UBINE ; URIC ACID AND ITS CHEMICAL CONGENERS Nomenclature and Definition of the Members of the Purin Group (Uric Acid, Xanthin, Hypoxanthin, etc. ) . The Factors Determining the Excretion of Uric Acid and its Chemical Congeners. The Pathogenetic Role of the Purin Bodies. Uric Acid — Its Clinical Significance and Estimation. The Purin Bases — Their Clinical Significance and Estimation. Other Bodies Allied to Uric Acid and the Purin Bases; viz., Nucleinic Acids and AUantoin. Nomenclature and Definition. — Uric acid and a number of basic bodies, of which xanthin and hypoxanthin are the most important members, form a chemical group called the purin group, or the purin bodies. The modern no- menclature employed in speaking of these compounds individually and collectively is involved and confusing, because a variety of recently constructed terms are used synonymously by different writers to designate the same thing. The term purin bodies is derived from an hypo- thetical chemical molecule containing carbon and hydro- gen atoms arranged in a ring structure; this "ring" is called purin ; it is contained in uric acid and all its chemi- cal congeners — the difference between the members of the group lying in the substitution of one or more hydro- gen atoms of the ring by different organic radicles. Uric acid is the only member of the group that possesses faint acid properties, all the other members are basic; hence, the latter are often called purin bases. The purin bases form salt-like compounds with phosphoric acid and a pro- teid molecule, and these compounds are the nucleins; hence, the purin bases are also called nuclein bases. Some writers also designate them as xanthin bases, from (26) THE PUEIN BODIES OF THE URINE 27 their chief i-epresentative, xanthin. Finally, uric acid and its congeners are also called alloxuric bodies and the bases alone alloxuric bases. To summarize, therefore, uric acid plus its chemical congeners forms the group of purin bodies, or alloxuric bodies; the bases xanthin, hypoxanthin, etc., are known by four different terms, i. e. (1) purin bases, (2) nuclein bases, (3) xanthin bases, (4) alloxuric bases. The terms purin bodies and purin bases are the most modern ones, and will presumably hereafter figure exclusively in medi- cal literature. The Factors Determining the Urinary Excretion of Uric Acid and Its Congeners. — The excretion of the purin bodies is subject to many fluctuations in health and in disease. The chief, and probably the only source of the purin bodies, are the nucleins (see above) , the chief chemi- cal constituent of all cell-nuclei. As these nucleins may be derived either from the food or from the cells of the tissues of the organism, the excretion of the purin bodies is primarily determined by the amount of nuelein-contain- ing food that is eaten, and by the catabolism of the proper tissues of the body. The old idea that uric acid is an oxidation product of albumen in general is wrong; the administration of enor- mous quantities of albuminous food, containing no nuclein, is not followed by an increase of uric acid in the urine; the administration, on the other hand, of pabulum con- taining many nucleated cells (i. e., internal organs, young germinating plants, etc.), is invariably followed by a great outpouring of uric acid or its congeners. Purin bodies derived from this source are called eomgenous purin bodies. If a subject is fed for a long time on pabulum con- taining no nuclein, or if a body is starved for several days, the urine nevertheless contains considerable quan- tities of purin bodies; the latter are derived from the 28 CLINICAL URINOLOOY cells of the body proper and are called endogenous purin bodies. Whereas the excretion of exogenous purin bodies is independent of the individual and constant, in the sense, namely, that a definite quantity of food nuclein leads to the excretion of a definite and calculable quantity of exogenous purin bodies, the excretion of the endogenous purin bodies varies in different individuals, is inconstant and cannot be calculated in advance; it varies from 0.1 to 0.2 grams a day in normal adults. These facts make it clear that all attempts to find "normal" values for the uric acid excretion or to deter- mine the significance o.f fiuctuatipns in the daily uric acid and purin bases, output aire altogether fictitious so long as the nuclein content, of the food is not included in the cal- culation; hence the great diversity of opinion among different authors in regard to the effect of albuminous foods on the excretion of uric acid. In addition to the diet, two other factors influence the urinary excretion of uric acid and the purin bases; viz., (1) the state of intracellular oxidation, (2) the state of the renal function. Of uric acid introduced by the mouth only a small portion reappears in the urine, and of the uric acid that can be formed from a given quantity of nuclein or nuclein-containing pabulum that is eaten, also only a frac- tion is excreted via the kidneys. This may be due to one of three factors, i. e. (1) the uric acid (scil. nuclein) may not be absorbed from the gastro- intestinal tract and hence be voided in the faeces, (2) the uric acid may be retained in the tissues, (3) the uric acid may be in part destroyed en route from the intestinal mucosa to the kidneys. Without entering into the experimental evidence, the statement may be made that the first two possibilities can be readily excluded, and that a large por- tion of the uric acid entering the blood- stream, either THE PUBIN BODIES OF THE URINE 29 from without or within — i. e., of the exogenous and the endogenous circulating uric acid — is destroyed (trans- formed, oxidized) in the tissues. I have recently shown (Medical Eecord, July 4, 1903) that all the organs of the body possess the power of destroying uric acid and of transforming it in large part into urea and oxalic acid, and that, bulk for bulk, the kidneys possess the greatest power in this direction; next the liver, and next the mus- cles. The transformation of uric acid proceeds with the aid of certain oxidizing ferments that are universally present. The fact that the kidneys can destroy uric acid teaches that the urinary uric acid cannot be considered an index of the circulating uric acid; it merely represents the alge- braic sum of the uric acid circulating in the blood and the uric acid destroyed in the kidneys. The formation of urea from uric acid finally demon- strates that uric acid is by no means a terminal product, but, instead, an intermediary product between nucleins and urea; it is clear from this that much that has been writ- ten in regard to the urea-uric acid ratio is sheer nonsense. The state of the renal epithelium has little to do with the excretion of uric acid, for we know that uric acid and its salts are excreted without difficulty when the kidneys are diseased, as in nephritis; this does not, of course, apply to very acute forms of nephritis or ter- minal stages of chronic nephritis in which all solids are retained. The Pathogenetic Role of the Purin Bodies. — The chemical interrelationship existing between uric acid and its congeners, the purin bases, is of some clinical inter- est. Both, as I have stated, are derived from nucleins but under slightly different conditions. If nuclein or nuclein-containing organs (spleen pulp, for example) are allowed to stand at body-temperature with free access of oxygen, uric acid is formed; if oxygen is withheld the 30 CLINICAL URINOLOGY purin bases are formed instead. It is possible also to convert the purin bases into uric acid by oxidation with the aid of the oxidizing ferments spoken of above; and uric acid, therefore, may be considered an oxidation pro- duct of the purin bases. This is clearly demonstrated by comparing the empiric formula of xanthin, e. g., with that of uric acid, the latter, as will be seen, containing one atom of oxygen more than the former: CsHiNiOs + O = C5H4N4O3 Xanthin Urie Aeid As a matter of fact, most of the purin bases when ad- ministered by mouth are partly excreted as uric acid, partly as urea and partly as unchanged purin bases. These chemical facts have some clinical application; viz., one can assume that, if the oxidation processes going on in the organism proceed in a normal manner, all the nucleins (of the food and tissues) undergo oxidation to uric acid (and in part via uric acid to urea) , and that if these oxidative processes are interfered with a portion of the nucleins is converted into purin bases instead of uric acid. Now, as I have shown (Am. Journ. of Med. Sciences, 1900) that the purin bases are toxic (in contradistinction to uric acid that is quite innocuous even when introduced in large quantities directly into the circulation), and that they are capable of producing certain renal and cardio- vascular changes that are identical with those seen in the so-called uric acid diathesis (gout and goutiness), I am inclined, therefore, to the belief that interference with normal oxidation, by leading to the formation of poison- ous purin bases from nuclein instead of harmless uric acid, may be in part incriminated with producing the lesions characteristic of goutiness. Uric acid itself pre- sumably acts pathologically in the uric acid diathesis, only from its tendency to form concretions. Uric Acid. — Uric acid, as explained above, is a normal THE PUBIN BODIES OF THE TJRINE 31 constituent of the urine. The average daily excretion of a healthy adult on an ordinary mixed diet varies from 0.2 to 1.25 grams; this constitutes from 1 to 2 per cent of the average daily output of nitrogen. Pathologically, the uric acid output is increased in febrile disorders, and in all diseases in which there is rapid loss of flesh; here the uric acid is endogenous, i. e., it is derived from the catabolism of the cell-nuclei of the body tissues. Whenever there is a leucocytosis the urinary uric acid is also increased, again from disintegration of leucocytic nuclei; hence the increase of uric acid after a full meal (digestion leucocytosis), in septic disorders and notably in leucaemia. In gout and the so-called uric acid diathesis (lithemia, goutiness) , there is much diversity of opinion among dif- ferent writers in regard to the uric acid output. This is due to neglect on the part of most investigators- to include the nuclein content of the food in their calculations — a gouty subject, like a well subject, excreting much uric acid whenever much nuclein- containing pabulum is in- gested and little when the diet contains no nucleins. The consensus of opinions among modern authors who looked to a constant diet of known nuclein content during the periods of observation, is that the uric acid output is somewhat increased during the gouty attack, and that immediately preceding the attack it is slightly decreased; in the intervals between the attacks the uric acid excre- tion in gouty subjects differs in no way from that in healthy persons. Estimation of Uric Acid. — An increase of the urinary uric acid should never be assumed from the precipitation of an abundant uric acid or urate sediment (see Urinary Sediments). The urine from day to day may contain approximately the same amount of uric acid and still form a heavy precipitate on one day and a light one or 82 CLINICAL URINOLOGY none at all on another; it may contain less uric acid on days in which much uric acid sediment forms than on days in which little uric acid deposit occurs. The pre- cipitation of the uric acid from the urine is exclusively a question of the urinary reaction and the proportion of basic and acid salts dissolved in the urine. Basic phos- phates hold uric acid in solution, acid phosphates precipi- tate it; an acid urine, therefore, containing an excess of acid over basic phosphate, will precipitate the bulk of the uric acid it incorporates; whereas an alkaline or am- photeric urine that contains an excess of basic phos- phates will keep the uric acid in solution. (See Chapter X, page 224.) The Method of Heintz. — This method may be de- scribed because it is so popular and so easy of execution, and because — in view of the relatively small clinical value of fluctuations in the urinary uric acid excretion anyhow — it furnishes results that are sufficiently useful for com- parative estimation. This method gives values that are invariably too low. 200 cc. of the urine are treated with 10 cc. of concentrated hydrochloric acid; the mixture is allowed to stand for twenty -four to forty -eight hours, and the crystalline deposit of uric acid gathered on a weighed filter, washed with alcohol and ether, dried at 110° C. to constant weight and weighed. The Method of Ludwig-SalkowsM.— This method is very accurate, and when the solutions are ready and the technical details are once mastered it is very simple and rapid. Other methods, notably those of Hopkins and Folin, are probably just as good, but I favor the Ludwig method as modified by Salkowski, and will describe it alone. The technique that I have learned to adopt is the following: Three solutions are required; viz. — (1) An ammoniacal silver solution. Twenty grains of silver nitrate are dissolved in water and so much am- monia added that the brown precipitate that forms dis- THE PURIN BODIES OF THE URINE 33 solves in the excess of ammonia. The solution is filled up to 1,000 cc. and kept in a dark place. (2) Magnesia mixture. One hundred grams of crys- tallized magnesium chloride are dissolved in water, and a large excess of ammonia added — this leads to the pre- cipitation of magnesium hydroxide. A cold saturated solution of ammonium chloride is now added in such quantity that the above precipitate goes into solution. The moderately clear liquid is filled up to 1,000 cc. (3) Sodium - monosulphide solution. Ten grams of sodium -hydrate (or 15 grains of potassium hydrate) are dissolved in 1,000 cc. of water. 500 cc. of this solution are saturated with sulphuretted hydrogen gas and then added to the remaining 500 cc. of the soda lye. The concentration of these three solutions is such that 10 cc. of each suffice to precipitate and later redissolve (see below) any uric acid that may be present in 100 cc. of urine. The principle underlying the method is the fol- lowing : In ammoniacal solution silver salts precipitate uric acid in the presence of magnesium salts as silver- magnesium urate. If this precipitate is treated with alkali sulphide the silver is precipitated as silver-sulphide, while the uric acid goes into solution as alkali urate. On acid- ulating the urate solution, uric acid crystallizes out and can be weighed as in Heintz' method. 100 cc. of urine are poured into a beaker. 10 cc. of solution 1 and 10 cc. of solution 2 are mixed in another vessel and treated with so much ammonia that the pre- cipitate of silver chloride goes into solution — a floc- culent precipitate of magnesium hydrate that does not dissolve in an excess of ammonia does not interfere with the reaction. The reagent thus prepared is slowly poured into the urine, and the gelatinous precipitate allowed to settle, then filtered off and repeatedly washed with weak ammonia water ; it is finally transferred to a beaker and treated with a boiling mixture of 10 cc. of solution 3 and 34 CLINICAL URINOLOGY 10 cc. of water. This mixture is preferably poured through the filter from which the precipitate was removed. (I usually punch a hole through the bottom of the filter and wash the precipitate through with a stream of water from a wash bottle ; if any traces of precipitate remain behind, their presence is revealed as soon as solution 3 is poured through, for the resulting silver -sulphide is conspicuous by its black color.) The contents of the beaker is now slowly heated to boiling, the precipitate of silver- sulphide filtered off, repeatedly washed with hot water, washings and filtrate acidulated with dilute hydrochloric acid (5 cc. of HCL, sp. gr. 1.12, diluted four times are sufficient) and evaporated down to about 20 cc. On cooling, the uric acid crystallizes out; it should be pure white. The solution should be allowed to stand at least six hours before the crystals are filtered off. I do not believe that crystalliza- tion is complete before that time. The crystals are gathered on a weighed filter, washed with very little water, then alcohol, then ether, dried at 110° C. and weighed. For every 10 cc. of the mother liquor and water washings, 0.0005 grams of uric acid should be added, for this small amount of uric acid is soluble in 10 cc. of acid- ulated water. Salkowski's modification of, this method consists in the use of a stream of sulphureted hydrogen gas instead of Solution 3 containing the alkali sulphide. It is a little more rapid but requires more skill. If many estimations have to be made, and if a saving of a few hours' time is an object, this method may be employed to advantage. Albumen and sugar must always first be removed from the urine. The urine is treated with a concentrated solu- tion of sodium chloride (15 cc. to each 100 cc. of tirine), acidulated with acetic acid and boiled, the coagulate filtered off, washed with hot water and the filtrate and washings filled up to the original volume of urine. Sugar can be removed by fermentation. If the urine contains uric THE PUBIN BODIES OF THE UBINE 35 acid or urate crystals, these must be dissolved by warm- ing and alkalinizing the urine. The addition of a little sodium carbonate to the urine when it is freshly voided will prevent urate deposits. The Purin Bases (syn., alloxuric, nuclein, xanthin bases). — To this group belong many bodies, of which the following have been found in the urine: xanthin, heteroxanthin, hypoxanthin, paraxanthin, guanin, ade- nin, episarcin, carnin, epiguanin. The most important of these substances is xanthin. These bodies are con- tained in many articles of food, notably in meat extracts, broths, bouillon, etc., forming the so-called extractives, and in tea, coflEee and cocoa. Some of them given by mouth appear in the urine as purin bases, notably if they contain methyl-groups, others are converted into uric acid and excreted as such, or after further oxidation as urea. The chemical relationship of the purin bases to uric acid and their role in the pathogenesis of the so- called uric acid diathesis have already been discussed. Normal urine contains only small quantities of the purin bases; their excretion fluctuates with the character of the diet, so that absolute "normal" values cannot be given. On an ordinary mixed diet, a healthy adult ex- cretes some 50 to 90 mg, of the purin bases in the twenty- four hours. Pathologically, an increase of the purin basis has been described in leucaemia and in the uric acid diathesis, no- tably in gout. The idea was advanced that in gout the nor- mal conversion of nuclein to uric acid was perverted, and that the purin bases were formed instead ; as a matter of fact, some experimental investigations were published by different authors, including myself, that showed an in- crease of the total urinary purin bodies in gout, due, in great part, to an abnormal increase of the purin bases. Unfortunately, the method employed at that time for esti- mating the urinary purin bases has since been shown to 36 CLINICAL URINOLOGY be inaccurate, in the sense of giving too large values for the purin basis ; the validity of these interesting results is hence somewhat impaired, and future investigations with improved methods will have to show whether or not these suggestive findings are correct. The only exact method for estimating the purin bases of the urine is that of Salkowski. This method is com- plicated and tedious, and will not become popular in or- dinary clinical work. It gives very accurate results. A measured quantity of urine is treated with ammonia- eal magnesia mixture and ammoniacal silver solution, as described above (Ludwig - Salkowski method ) under "Ui'ic Acid." The uric acid is removed as in the Ludwig method, and the final filtrate, which contains the purin bases, together with the washings, treated with ammonia- cal silver solution. This repreeipitates the purin bases. The precipitate is collected on a filter, washed, dried, in- cinerated. The ash is dissolved in dilute nitric acid and the silver estimated in this solution by titration. The best method of titration is with potassium sulphocyanide, using ammonio-ferric alum as an indicator. In a com- pound of the silver salts of xanthin, hypoxanthin, etc., one atom of silver represents 0.277 gm. of nitrogen, or 0.7381 gm, of the bases. Consequently 1 cc. of the sulphocyanide solution will correspond to 0.002 gm. of nitrogen, or 0.00542 gm. of the bases. The sulphocya- nide solution is made up as follows : The solution should contain 12.9849 gm. to a liter. The salt is very hygro- scopic, so that it cannot be weighed accurately. There- fore a concentrated solution is made and standardized against a silver solution containing 29.042 of silver nitrate to the liter, using ammonio-ferric alum as an indicator. The separation and identification of the different mem- bers of the group of purin bases is possible, but it re- quires very much time and considerable chemical skill. As this separation is of no clinical value, but merely of THE PUniN BODIES OF THE URINE 37 physiological interest, it will not be described in this book. Closely related to the purin bodies are two substances that merit brief mention in this place, as they occasion- ally appear in the urine and possess some clinical impor- tance; they are nucleinic acids and allantoin. Nucleinic Acids. — The nucleinic acids are peculiar com- binations of phosphoric acid with the purin bases and a nitrogen - free molecule (possibly pentoses); the term nucleinic acid is frequently used synonymously with nuclein. These nucleins sometimes appear in small quantities in the urine, either free or in combina- tion with albumen; in the latter combination they are called nucleo-albumens, and form the chief constituent of "mucin" (see chapter on Albuminuria). These nucleins and nucleo-albumens contain much phosphorus, the former as much as 9.5 per cent, the latter 1.5 per cent or more. The significance and the origin of nucleo-albumen has already been discussed ; it is probable that the free nu- cleins are derived from the nucleo-albumens by disinte- gration and splitting off of the albumen radicle. The nucleins in their turn may, by disintegration, as already set forth, become a source of uric acid or of purin bases. This possible origin of purin bodies should, therefore, always be considered in studying urines containing much nucleo-albumen (mucin), i. e., urines derived from pa- tients afflicted with catarrhal afflictions of the lower genito- urinary tract. Allantoin. — This body is an intermediary product in the oxidation of uric acid to urea. It appears in cats' and dogs' urine after feeding nucleins or uric acid, but it does not appear in human urine after such a diet. Occasionally it is found in the arine of new-born children (during the first week of life) , and is here presumably, like the uric acid infarcts in the kidneys of the new-born, a product of the excessive degradation of the nuclei of nucleated red corpuscles; the latter, as is well known, circulate 38 CLINICAL URINOLOGY for the first days of extra-uterine life and then rapidly disappear. In the urine of pregnant women allantoiu is also occasionally found; here it is probably absorbed from the amniotic fluid that always contains allantoin; possible, too, that the urinary allantoin of the new-born is sometimes derived from the same source, the foetus having swallowed some of the liquor. In male adults allantoin is a rare urinary constituent; when found it indicates interference with the normal conversion of uric acid to urea, presumably' due to deficient intracellular oxidation. CHAPTER III THE TOTAL UEINART NITROGEN AND UREA The Nitrogenous Constituents of the Urine. The Laws Governing "Nitro- gen Equilibrium." The Factors Determining the Urinary Excretion of Nitrogenous Bodies in Health and in Disease. The Clinical Signifi- cance of Fluctuations in the Urea Output and in the Output of the Total Urinary Nitrogen. Some Fallacies of Urea Determinations as an Index of Renal Inadequacy. The Quantitative Estimation of Urea. The De- termination of the Total Urinary Nitrogen. Miscellaneous Nitrogenous Constituents of the Urine. Creatinin — Its Clinical Significance and Determination . The output of urinary nitrogen is dependent on two factors, i. e., (1) the amount of nitrogenous food (chiefly albumen) ingested; (2) the eatabolism of the tissues of the organism proper (chiefly the organized albumen). From 83 per cent to 93 per cent of the urinary nitrogen is excreted as urea; the remaining 7 per cent to 17 per cent appears in the urine in the form of purin bodies (uric acid and its congeners), certain aromatic compounds, creatin and creatinin, nueleinic acid, allantoin, oxaluric acid, diamins, amido acids and preformed ammonia. If an individual is fed on a constant diet sufficiently abundant to maintain full nutrition, then the urinary nitrogen excretion also becomes constant at the expiration of from two to four days. At this time the nitrogen out- put equals the nitrogen intake, and we say that the subject is in a state of ^^ nitrogen equilibrium." The organism, however, also has a tendency to accommodate the nitro- gen output during one period of twenty-four hours to the nitrogen intake during the preceding period of twenty - four hours. If the nitrogen intake is suddenly altered, then in the beginning the latter factor predominates; (39) 40 CLINICAL URINOLOGY after a number of days, however, the former factor de- termines the nitrogen output. Even if, therefore, the nitrogen intake fluctuates from day to day, the sum of the total urinary nitrogen excreted during a given period will nevertheless equal the sum of the nitrogen intake during this time, but for periods of twenty -four hours great differences in the nitrogen output will become ap- parent. It is clear, therefore, that any calculations based on an observation extending only over twenty-four hours are of very little value in determining the level of nitro- gen equilibrium. A healthy adult can maintain nitrogen equilibrium on 0.6 grains of albumen pro die per kilo of body weight — or on more. If less nitrogenous material is ingested nitrogen equilibrium can no longer be maintained, for then the organism begins at once to disassimilate the albumen of its proper tissues in order to cover the deficit. The upper boundary of nitrogen equilibrium has so far never been determined. The Factors Determining the Excretion of Urea. — As the bulk of the urinary nitrogen is excreted as urea, it has become fashionable of late years to pay particular attention to the determination of the urinary urea. Nearly every practitioner conscientiously performs his urea de- terminations, and, I am convinced, honestly believes that he is thereby gaining valuable information in regard to the state of the nitrogen economy of the body, and, above all, in regard to the functional powers of the kidneys. The determination of the urea, however, in isolated specimens of urine, or even in a mixture of the total twenty-four hours' quantity, is essentially valueless un- less the nitrogen content of the diet is included in the calculation; and it is, moreover, not suflBcient to know the nitrogen intake alone of the period of twenty-four hours preceding the urea determination, but one should know the nitrogen intake and nitrogen output of several periods TOTAL UBINABY NITBOGEN AND UBEA 41 of twenty-four hours preceding; in other words, the patient should be in a state of nitrogen- equilibrium be- fore changes in the nitrogen intake are made, and before corresponding changes in the nitrogen output are esti- mated and utilized as the basis of conclusions in regard to the nitrogen metabolism and the renal functions. Even if the diet were carefully, mathematically regu- lated, even if the observation extended over several days, even if the methods of determination were altogether accurate, still other factors, that are as important as the diet, would have to be considered, because they are capa- ble of exercising a colossal influence on the plus or minus of urea and nitrogen excretion, I refer to the element of exercise, the amount of water ingested, the presence or absence of fever, the state of the gastro- intestinal tract, the existence of cachexias or of other wasting dis- eases, the reaction of the blood, etc. The more a man exercises the more does he disassimi- late the albumens of his own tissues and the more urea must he excrete. Abundant water-drinking, or diuresis stimulated in other ways, invariably leads to the elimina- tion in the urine of large quantities of nitrogenous waste products, chiefly urea, while, inversely, restriction of water or loss of water through other channels than the kidneys (sweating, profuse vomiting, diarrhoea, etc.) leads to a decrease of the urinary nitrogen and urea. In febrile disorders there is great destruction of albumen, and hence increased excretion of urea; the same applies to cachectic states of all kinds — the urea is increased. If the food is poorly assimilated, as in catarrh or atrophy of the stomach or intestine, then the nitrogen ingested, how- ever abundant it may be, reappears in the faeces and does not enter the urine, so that here again we find the urinary urea decreased. In acidosis, i. e., in any condi- tion in which the alkalinity of the blood is reduced (scil. the acidity increased), and such changes in the blood- 42 CLINICAL URINOLOGY reaction may be physiological or may be due to the entrance into the blood of abnormal acid products of per- verted metabolism (oxybutyria acid, diacetic acid, lactic acid, uric acid, phosphates, etc. —in diabetes, gout, obesity), the urea output is decreased; this is due to the fact that the blood jealously guards its alkaline re- action by splitting off ammonia radicles from more com- plex compounds and neutralizing the abnormal acids with it. Whenever this occurs, the ammonium-nitrogen bound to acids can no longer form urea but is excreted in the urine in the form of ammonia salts, while the urinary urea is correspondingly decreased. In many disorders of the liver, finally, both organic and functional, the manufac- ture of urea is reduced and its excretion in the urine decreased. Some Fallacies of Urea Determinations as an Index of Renal Inadequacy. — It will be seen, therefore, how pre- carious a matter it is to draw conclusions from fluctuations in the urea output. The chief errors are unquestionably committed when attempts are made to estimate the state of the renal function from the urea percentage of the urine. As this practice is so common, I will attempt to point out some of the fundamental fallacies underlying this idea. In the first place, all that has been said in regard to the multitudinous factors that determine the urea output must be carefully considered; in the second place, the patient must be in a state of nitrogen-equilibrium before the study of the renal functions is begun; in the third place, not the urea excretion alone, but the total urinary nitrogen must be determined, for the urea forms only an indefinite and varying portion of the nitrogen output; in the fourth place, an accurate method for determining the urea and the nitrogen must be employed instead of the crude and inexact so-called clinical methods that are in almost universal use; in the fifth place, the nitrogen or urea determination must be made with the twenty -four TOTAL URINARY NITROGEN AND UREA 43 hours' quantity of urine on several successive days; lastly, it is often necessary that the urine should he collected separately from each kidney (this applies particularly to cases in which surgical procedures are contemplated) , as in unilateral kidney disease, the healthy organ may under- go compensatory hypertrophy and vicariously assume the urea eliminating function of the diseased kidney. It is clear that for practical work these accurate methods are altogether too complicated, and the question arises, is it worth while to proceed with ordinary clinical methods or even with the pedantic accuracy of a meta- bolic experiment in any case ? In other words, does the plus or minus of urea excretion ever give us absolutely reliable information in regard to the excretory powers of the kidneys ? The answer to this important question is uncertain. There are, of course, cases of acute nephritis with almost total suppression of urine, cases of anuria in surgical affections of the kidney and terminal forms of interstitial nephritis in which the renal function is almost completely arrested; here the perversion of the kidney function is so apparent that urea determinations are altogether super- fluous, for in such cases it is clear that neither urea nor any of the other urinary solids are properly ex- creted. There are also cases of acute exacerbation of parenchymatous and interstitial nephritis, and cases of true Bright's disease in which some retention of urea temporarily occurs, but such cases are comparatively rare. In many cases of nephritis, moreover, in which nitrogen and urea estimations are made, even with all the precau- tions outlined above, some decrease of the nitrogen out- put will be observed, but at the same time it will be found that the patients gain in weight. Here, then, the nitrogen, it is true, is retained, but not in the form of urea or of other nitrogenous waste product that should properly be eliminated through the kidneys, but in the 44 CLINICAL URINOLOGY form of organized albumen ; the nitrogen is built up into "flesh and blood." Far from being a bad omen, this is a very happy event, particularly in cases convalescing from acute nephritis, or from an acute exacerbation of a chronic nephritis. Only, therefore, if we find by accurate metabolic studies that less nitrogen is excreted in the urine than is ingested by mouth (after deduction of the faeces nitrogen) and if at the same time the patient does not gain in weight, can we speak of nitrogen retention, due to renal inadequacy. Assuming, now, that deficient elimination of urea had been determined in some chronic case of nephritis, does this finding always denote renal inadequacy? This is questionable; for, in the first place, cases are on record of perfectly healthy subjects, i. e., of subjects whose kid- neys were in no way diseased, in whom from time true retention of nitrogen (urea) occurred. Here the fluctua- tions in the urinarj'' excretion of nitrogen could in no way be interpreted to signify renal inadequacy, as all the other tests performed for renal insufficiency gave negative re- sults. A retention of urea at best indicates renal inade- quacy only for urea and not necessarily for the numerous other urinary bodies that are carried to the kidneys in the renal blood. In the second place, von Noorden and his pupils have shown, and I have been able repeatedly to convince myself of the truth of their demonstration, that in many cases of nephritis the relation between the nitro- gen intake and the nitrogen output may be altogether changeable ; that periods of good elimination may alternate with periods of bad elimination without any other evidence of renal inadequacy. Some clinicians go so far as to say that these fluctuations in the urea and nitrogen output must be considered a characteristic feature of Bright' s disease, and that they are due to certain metabolic changes, and not to changes in the functional powers of the kidneys. It is clear, therefore, that great care must be exercised in TOTAL UBINARY NITROGEN AND UREA 45 interpreting the pins or minus of urea excretion as an index of the state of the renal function in kidney dis- eases, even if all the common sources of error are eliminated. Much will depend upon the exact period in the disease when the determination is made; and even if the ebb happens to be low one cannot always conclude that there is renal inadequacy. We possess, fortunately, other more accurate and more reliable means than urea determinations for estimating the functional powers of the kidneys; these will be discussed in a special chapter. One other source of error may finally be mentioned. Assuming the kidneys to be really insufiBcient, and this insufficiency to be manifested by a partial retention of the urea that is clamoring for elimination through the kidneys ; assuming that as a result an abnormally large amount of retained urea were circulating in the blood, or that possibly the patient were suffering from some febrile or wasting disease (malignant tumor, diabetes, etc.), causing an abnormally large amount of urea to be poured into the blood- stream and carried to the kidneys, the result would be a partial retention of an abnormally large amount of urea, and consequently the elimination of a quantity of urea that would approximate normal average figures. Here, then, real renal inadequacy would be masked if one relied upon the elimination of urea as an index. To summarize what has been said: Urea and total nitrogen determinations, unless performed with the pain- ful accuracy of a metabolic experiment, and unless cov- ering several days, are of very small value. Performed in a proper manner they are of inestimable value, but should be interpreted with care and conservatism, par- ticularly in drawing conclusions in regard to renal inade- quacy. The ordinary urea determinations as performed by practicians in general with the inaccurate Doremus 46 CLINICAL URINOLOGY apparatus are useless ; they are often worse than value- less, for they may give rise to serious misinterpretation, causing, on the one hand, alarm when there is no danger, lulling the physician into a false sense of security, on the other, when serious danger is impending. Combined with the crude ideas on under- feeding nephritics (exclu- sive milk diet, withdrawal of meat, etc.), that are still largely in vogue, I think that these urea determinations are the chief reason why so many sufferers from nephritis are literally starved to death. A pity that so much time is wasted on these rough estimations and that so much ink is spilled in recording them. The Quantitative Estimation of Urea. — In view of the fact that the excretion of urea is dependent on so many different factors, no "normal" values for the urea excre- tion can be given. A healthy adult of 75 kilos weight living on an average mixed diet excretes about 33 grams of urea in the twenty four hours. According to the char- acter of the diet, the amount of exercise, the quantity of water taken and the method of water-drinking, etc., the urea output may normally fluctuate from 20 to 45 grams a day and still be strictly within physiological limits. The amount of urea in single specimens of the urine may, of course, fluctuate within still wider limits and still indi- cate nothing pathological . Percentic urea determinations in single specimens are, therefore, a waste of time. If we assume J ,500 cc. to be the normal daily average quantity of urine, then the normal average urea excretion under the conditions outlined would be from 2 to 3 per cent. By designating the urea excretion in per cent we merely in- troduce another labile quantity into the equation, i. e., the amount of urine, so that it is by all means preferable to designate the urea excretion in grams pro die. There are relatively few accurate methods for deter- mining the urinary urea. In nearly all of the older methods other nitrogenous constituents of the urine that TOTAL UBINAEY NITROGEN AND UREA 47 are closely related to urea were determined as urea. The old titration method of Liebig was popular for many years and is often referred to; it is very inaccurate, giv- ing values for urea that are invariably too high. The best method of all is the method of Morner and Sjoqvist; it is based on the following principle: If urine is treated with a mixture of barium chloride and barium hydrate (saturated barium chloride solution with 5 per cent barium hydrate), and then allowed to stand under ether- alcohol for twenty-four hours, all the nitrogenous constituents of the urine are precipitated, while the urea is dissolved in the ether-alcohol ; the urea solution is fil- tered off, the nitrogen content of the filtrate determined after Kjeldahl (see below), and the urea calculated from the amount of nitrogen found by multiplying the latter with 2.143. This method is too complicated for clinical work; for metabolic work it is the best. The most convenient and, relatively, the most accu- rate clinical method is: The Hypobromite Method of Hiifner (Knop).— This method is based on the fact that sodium hypobromite in alkaline solution decomposes urea into nitrogen, carbon dioxide and water, according to the formula: CO{NH2)2 + SNaOBr = 3NaBr -|- CO2 + 2H2O -|- 2N Urea Sodium Sodinin Carbon Water Nitrogen hypobromite bromide dioxide The carbon dioxide that develops is absorbed by the soda lye, while the nitrogen passes through the lye and can be measured ; from the volume of nitrogen liberated the amount of urea can be calculated. The method is slightly inaccurate in two respects; viz., (1) the amount of nitrogen liberated is always' somewhat smaller than the calculated amount, (2) hypobromite solution also liberates nitrogen from certain other nitrogenous constituents of the urine. In a measure these two errors compensate each other, the first error reducing the nitrogen volume. 48 CLINICAL URINOLOGY the second increasing it. At all events, the method is sufficiently accurate for clinical use. The Apparatus of Hilfner (see Fig. 2).— The appara- tus consists of three parts; viz. (1) a cylindrical vessel, A, connected by a glass cock, B, with a small cylindrical recep- tacle, C; (2) a dish, D, that is connected with the upper end of A by a rubber cork (as shown in the figure) in such a manner that the upper end of A protrudes into D; (3) an eudiometer tube, E, from 30 to 40 cm. long, 2 cm. wide and graduated for 0.2 cm. The eudiometer tube is held inverted over the upper end of A by a clamp, F, attached to a stand, GG. The Hypohromite Solution. — 70 cc. of a 30 per cent sodium hydrate solution are diluted with 180 cc. of distilled water; the solu- tion is mixed with 5 cc. of bro- mine; the bromine dissolves in the lye and sodium- bromide and -hypobromite are formed accord- ing to the formula: 2NaOH 4- Brs = NaBr + NaBrO -f- H2O The hypobromite solution should be kept in a cool dark place in a tightly stoppered bottle. Fig. 2. Hiifner's Apparatus for The SOlutioU should bC freshly the Determination of Urea. prepared every few days, as the sodium hypo-bromite has a tendency to decompose, forming sodium bromide. TOTAL URINARY NITROGEN AND UREA 49 Method of Performing the Determination. — The urea content of the urine is first roughly estimated from the specific gravity, as follows: "We know empirically that urine of a specific gravity of 1014 contains about one per cent of urea, of 1014 to 1020 about 1.5 per cent, of 1020 to 1024 about 2 to 2% per cent, of 1028 about 3 per cent; we can estimate the urea thus crudely because urea is the chief solid constituent of the urine, and hence largely determines its specific gravity. The urine is, if necessary, diluted so as not to contain more than one per cent of urea. Of this diluted urine exactly 5 cc. are allowed to flow from a long pipette into the small recep- tacle C, care being taken not to spill any of the urine in- to A; the pipette is carefully rinsed out with water and the rinsings also poured into C until the latter receptacle is filled to the cock, B. The cock is now closed, A filled to overflowing with hypobromite solution, the dish, D, and the eudiometer tube, E, filled with concentrated sodium chloride solution and E inverted in D and fixed over the upper opening of A with the clamp, F. Now the cock, B, is opened. As the hypobromite solution is heavier than the urine, it runs from A into C and, mixing with the urine, at once starts a lively development of gas (CO2 and N). The CO2 is absorbed in the sodium hydrate solution that forms part of the hypobromite reagent in A, while the nitrogen passes through A into the tube, E. The reac- tion is completed in about twenty minutes. The eudi- ometer is then carefully held shut with the thumb im- mersed into D and transferred to a deep vessel filled with distilled water of room temperature ; here it is allowed to stand fifteen minutes. The final reading is made by im- mersing the tube so far that the level of the fluid in the outer vessel and in the eudiometer is equal. At the same time the temperature of the water and the barometric pressure are noted. In ordinary clinical work, pi'ovided the room temperature is fairly constant from day to day, 50 CLINICAL URINOLOGY the reading can be made directly from the eudiometer while it is still inverted over A; for exact work (and I have always found it quite as convenient to work with precision, and certainly more gratifying in the end) , the volume of gas must be reduced to 0°C., 760 mm. baro- metric pressure and absolute dryness with the aid of the formula: V fB — W) V' = 760 (1 + 0.0036 1). 354.3 V = Volume of nitrogen (corrected). V = Volume of nitrogen as read on the eudiometer. B == Barometric pressure. W*^ Tension of water vapors at temperature t. t = Temperature of water at time of reading. 0.0036 = Coefficient of expansion of gases for 1° C. As urea when it is disassociated with hypobromite solution only yields 354.3 cc. of nitrogen, instead of the theoretical amount of 372.7 cc, V^ must be divided by 354.3 in order to obtain the correct amount of nitrogen developed, for 354.3 : 1 = V : X 354.3 If 5 CC. of urine were employed for the determination, then yi multiplied by 20 gives the percentage of urea, for v^ 1 on 5 : V' = 100 : X ; x = -!Lj_J^" = y' .20 5 The Apparatus of Doremus (ureometer) (Fig. 3) is based on a similar principle as the apparatus of Hiifner- Knop. I have already spoken of it, to condemn its use. One should either work accurately or not at all. As the apparatus is in such universal use, however, it merits mention. *The values for W at ordinary temperatures are the following. 10° C = 9,126 14° C = 11,882 18° C = 15,351 22° C = 19,675 11° C = 9,751 15° C = 12,667 19° C = 16,345 23° C = 20,909 12° C = 10,421 16° C = 13,519 20° C = 17,396 24° C = 22,211 13° C = 11,130 17° C = 14,009 21° C = 18,505 25° = 23,582 TOTAL URINARY NITROGEN AND UREA 51 . The apparatus consists of a graduated tube with a bulb attachment as shown in Fig. 3, and a 1 cc. nipple pipette. The tube and bulb are filled with a hypobromite solution made by dissolving 100 grams of sodium hydrate in 250 cc. of water and treating this solution with 25 cc. of bromine. It is advised to make the solution fresh each time by adding 5 cc. of bromine to 50 cc. of the above solution of sodium hydrate immediately before the deter- mination. 1 cc. of urine is then allowed slowly to flow from the nipple pipette into the appara- tus as shown in the figure. After fifteen min- utes the volume of gas is read off. This tube is graduated so as to indicate the grams and fractions of grams of urea in each cubic centi- meter of the urine ; by moving the de- Pig. 3. X^ ^^ cimal two figures to the right the urea percentage is given. Thus a reading, e. g., of 0.017 indicates that each cubic centimeter of the urine contains 0.017 grams of urea, and that the specimen of urine has a urea content of 1.7 per cent. Determination of the Total Urinary Nitrogen. — As con- siderable nitrogen is eliminated in the fseces, in part de- rived from the gastro- intestinal secretions and from desquamating epithelia, in part from food-residues, this amount must be determined separately and included in the calculation when making metabolic studies; for it is clear that nitrogen equilibrium is only established when Pood -Nitrogen — Fseees-Nitrogen = Urine -Nitrogen. The estimation of the faecal nitrogen is performed in the same way as the estimation of the urinary nitrogen 52 CLINICAL URINOLOGY to be presently described, with the difference merely that the dried fseees are weighed, whereas the urine is measured. The urine should be gathered from one morning (be- fore breakfast) to the following morning, and not from night to night. The nitrogen found in urine gathered in this way correctly indicates the total urinary nitrogen ex- creted in twenty-four hours, if, first, the last nitrogenous food is eaten eight or ten hours prior to the collecting of the last urine (hence the advantage of collecting the urine from early morning to early morning) and, second, if the amount of water taken during the period of observation is not much more, nor much less, than the water ingested for some days before ; for abundant water-drinking always flushes out an abnormally large amount of residual ni- trogenous waste, whereas restriction of water exercises the reverse effect and favors the retention of nitroge- nous material. The Method of Kjeldahl for Determining the Urinary Nitrogen.— The method is based on the following princi- ple: The nitrogenous constituents of the urine, on boiling the urine with concentrated sulphuric acid, are destroyed and all the nitrogen that is not in direct combination with oxygen is converted into ammonia; the latter combines with the sulphuric acid and goes into solution as sulphate of ammonium. The bodies that contain nitrogen in direct combination with oxygen are present in such very small quantities (and occasionally not at all) that they may be neglected for all practical purposes. The acid solution of ammonium sulphate is treated with hot soda lye and the ammonium thus liberated caught in a meas- ured quantity of normal acid and the excess of acid titrated back ; from the amount of ammonia formed the nitrogen of the urine can readily be calculated. There are many methods for performing the Kjeldahl determination. The following technique is probably the TOTAL URINARY NITROGEN AND UREA 53 most simple and satisfactory one; in my hands it has proven altogether reliable. When the necessary solutions are once made up and the apparatus properly installed, a number of determinations can easily be made in a day. According to the concentration of the urine, 5 or 10 cc. are poured into a so-called Kjeldahl flask, i. e., a flask of hard glass with a round bottom and a long neck, hold- ing from 200 cc. to 300 cc. ; to the urine are added 20 cc. of concentrated sulphuric acid and a small quantity, about one- third of a gram, of yellow oxide of mercury; the latter aids in the destruction of the organic material in the urine. The mixture is boiled over a small flame until the solution becomes quite colorless, care being taken not to heat too rapidly, as otherwise some nitrogenous vapors may escape. On cooling, the contents of the flask (that may have become solid from the crystallization of mercury sulphates) is transferred to a large distilling flask by repeated rinsing out with water. To the liquid in this retort are added 40 cc. of a solution of sodium sulphide (40 grams to 1,000 cc. of water), 160 cc. of a solution of sodium hydrate (270 grams to 1,000 cc. of water) and a small quantity of talcum. The sulphide decomposes the amido compounds of mercury and liberates the nitrogen (ammonia) from them, the talcum renders ebullition more gentle and prevents bumping. The distiUing flask is now rapidly connected with a condenser and the distilla- tion begun. The water and the ammonia vapors are col- lected in a flask containing 40 cc. of a one- fourth normal sulphuric acid solution. The distillation is interrupted when about two-thirds of the liquid have gone over; the condenser is rinsed with water and the rinsings added to the distillate. The acid is now retitrated with a one- fourth normal sodium hydrate solution, using rosolic acid as an indicator. The difference indicates the amount of acid neutralized by the ammonia. As 1 cc. of the one- fourth normal solution represents 0.0035 grams of nitro- 54 CLINICAL URINOLOGY gen, the number of cubic centimeters of acid neutralized by the ammonia must be multiplied by this figure in order to determine the quantity of nitrogen contained in the 5 or 10 cc. of urine used. From this figure the amount of nitrogen contained in the total twenty-four hours' quan- tity can easily be calculated. Certain organic bodies that are chemically and gen- etically related to urea may be briefly discussed in this place. Only a small proportion of the urinary nitrogen is excreted in the form of these bodies, the bulk, as already stated, appearing as urea and as uric acid and its chemical congeners. The exact clinical significance of many of these bodies is not understood ; their appearance in greatly increased quantities, if not dependent on changes in the diet, always, however, indicates some per- version of proteid-metabolism, and hence commands in- terest in this sense. The most important of these bodies is creatinin (creatin) ; other members of the group that. merit mention are certain diamins (putrescin and cada- verin) (see also pages 190, 191) tliat are highly toxic, and certain amido- acids, including such important bodies as leucin, tyrosin and cystin. As the latter substance is rarely discovered unless it appears in the form of a crystalline deposit, its pathogenesis and chemistry will be discussed in the chapter on Unorganized Urinary Sediments and urinary concretions. Leucin and tyrosin are discussed in the chapter on Miscellaneous Fatty Acids of the Urine. There remains, therefore, among these unclassified nitro- genous urinary bodies only creatinin. Creatinin. — The mother substance of creatinin is creatin, and the latter on rare occasions has been found in the urine. Creatinin itself is a normal urinary constituent, and appears in quantities varying from 0.6 to 1.3 grams in the twenty -four hours' quantity. Creatin and creatinin are always derived from muscle tissue, and the latter may TOTAL UBWABY NITROGEN AND UBEA 55 either be muscle that is eaten or the muscle structures of the organism. The excretion of creatinin is, therefore, largely dependent on the diet — much meat, much crea- tinin. But even if all meat is excluded from the diet, or if muscle meat is first carefully leached out with water, some creatinin nevertheless continues to be excreted. The blood and tissue juices, as well as the muscles themselves, contain chiefly creatin, whereas the urine contains almost exclusively creatinin ; just where the con- version of the former into the latter occurs remains undetermined; it is probable that the kidneys are con- cerned in this process. Pathologically, the urinary creatinin has been found increased in nearly every disease in which there is much rapid wasting of muscle tissue, as, for instance, in acute febrile diseases, in rapid cachexias and in diabetes. Much care must be exercised in interpreting a plus or minus of the creatinin excretion, particularly in such a disease as diabetes, in which patients are so commonly placed upon a diet containing abnormally large quantities of meat. Here the excretion of creatinin is largely alimentary, to borrow a term from our glycosuria nomenclature, or ex- ogenous, to borrow a term from our uric acid nomencla- ture. It is only the endogenous excretion of creatinin that promises to be of clinical importance, i. e., the excre- tion of creatinin that is derived from the proper muscle tissues of the body. So far we have not learned to clinically interpret the plus or minus of creatinin excretion, but this field is very promising and for that reason I give the quantitative method of determining creatinin, hoping that some will feel stimulated to perform creatinin determinations in different diseases, thereby contributing to our scanty casuistic material on this subject. Such isolated state- ments as that creatinin has been found decreased in chronic nephritis, diabetes insipidus, anemia, chlorosis 56 CLINICAL URINOLOGY and tuberculosis are of little value unless the elements of reduced meat-eating, lack of appetite, deficient assimila- tion, etc. are carefully excluded. Creatinin determina- tions may prove to be of value in muscular dystrophies and, one might imagine, in the diagnosis of myositis and parasitic affections of muscle tissue accompanied by ab- normal destruction of muscle. Qualitative Test.— As creatinin is always present in the urine, a qualitative examination of the urine for its presence is rarely called for. Of the numerous tests described, the following one is the simplest and the most reliable. Jaffe's Test. — The urine is treated with a few drops of a dilute sodium hydrate solution (10 per cent) and a few drops of a 10 per cent picric acid solution; if appreciable quantities of creatinin are present the liquid turns red at once, even in the cold. This reaction can be obtained in' the presence of 1 part of creatinin to 5,000 parts of water. No other urinary constituent gives this color reaction. Quantitative Determination. — The principle underlying the method is the following: Creatinin forms a double compound with chloride of zinc that is essentially insolu- ble in alcohol (1 to 9,217). This compound is prepared and weighed, and the creatinin calculated from the amount of double salt formed. Execution. — 200 ce. of the urine are rendered alka- line with linie-water, and treated with a 10 per cent watery solution of calcium chloride so long as a pre- cipitate continues to form ; the mixture is allowed to stand for two hours and filtered, the filtrate acidulated with dilute sulphuric acid and evaporated to a syrupy consis- tency. While still warm the syrup is treated with 50 cc. of 95 per cent alcohol, the mass thoroughly mixed with the alcohol and allowed to stand for eight hours in a cool place. The sediment is filtered off, washed with 95 per cent alcohol, and the filtrate and alcoholic washings TOTAL URINARY NITROGEN AND UREA 57 united and evaporated down to about 50 cc. This solu- tion is now treated with an alcoholic chloride of zinc solution prepared by treating a saturated watery solution of zinc chloride with absolute alcohol until the mixture . has a specific gravity of 1200. Of this solution 0.5 cc. are added to the liquid containing the creatinin, and the mixture allowed to stand for two days. The crystals of creatinin - zinc chloride that form are gathered on a weighed filter, washed with alcohol until the filtrate is chlorin - free (no clouding with silver nitrate) , dried and weighed. One gram of creatinin -zinc chloride repre- sents 0.6242 grams of creatinin. In order, therefore, to determine the amount of creatinin that was present in the quantity of urine employed for the determination (200 cc), the figure obtained by weighing the creatinin- zinc compound must be multiplied by 0.6242. CHAPTER IV THE CAEBORYDBATES OF TBE UBINE Note OH the Physiological Chemistry of the Urinary Carbohydrates— Monosae- oharides; Disaooharides ; Polysaocliarides ; Glneosides; Glyooproteids. The Carbohydrates of the Vrine as a Group — The Unfermentable Carbo- hydrates. Glycosuria — Physiological and Alimentary Glycosuria; Gly- cosuria e saccharo and e amylo; Toxic Glycosuria after Drugs and Poisons, Organ Extracts, Bacterial Poisons, in Auto -intoxication; Psy- chic Glycosuria and Glycosuria in Diseases of the Nervous System. Renal Glycosuria; Glycosuria in Obesity, Gout and Arteriosclerosis; Diabetic Glycosuria, the Three Degrees of Diabetic Glycosuria and the Mathematics of the Diabetic Diet. The Different Carbohydrates of the Urine, their Clinical Significance, Detection and Determination — Pentoses (Pentosuria). Dextrose, Levulose, Laiose (Leo's Sugar), Isomaltose, Lactose. Animal Gum, Glycogen. NOTE ON THE PHYSIOLOGICAL CHEMISTRY OP THE URINARY CARBOHYDRATES The name " carbohydrate " is intended to signify that this group of bodies contains carbon and the atoms of hydrogen and oxygen in the proportion of two to one, i. e., in the same proportion as water, HgO. As a matter of fact this does not apply to the carbohydrates alone, for numerous other organic bodies, e. g., acetic acid, C2H4O2, have a similar arrangement of hydrogen and oxygen atoms. Carbohydrates may contain three, four, five, six or more carbon atoms, and they are called, accordingly, trioses, tetroses, pentoses, hexoses, etc. Of these only the pentoses and hexoses are of importance in the human economy. None of the others if administered by mouth are utilized but appear unchanged in the urine ; they are not, moreover, contained in the normal food elements of man. A few cases of pentose excretion (pentosuria) are on record and the clinical significance of this urinary (58) THE GABBOHYDBATHS OF THE UBINE 59 abnormality will be briefly discussed; otherwise, in speak- ing of the carbohydrates in the following, the hexoses alone are meant. Hexoses. — The hexoses are conveniently, both for chemical and for clinical purposes, divided into three groups; viz.: (1) Monosaccharides (or glucoses), containing six car- bon atoms (OeHiaOg) . (2) Disaecharides (or saccharoses), containing twice six, i. e., twelve carbon atoms (C12H22OU). (3) Polysaccharides (or amyloses), containing three times six carbon atoms or more (CeHioOs)^. The disaecharides and the polysaccharides must be regarded as anhydric condensation products of the mono- saccharides, i. e., as combinations of two or more mono- saccharides with loss of water, thus : CeHiaOu + CoHi20(i — HsO = C12H22O11 disacoharide, (C6Hi206)n — (H20)n = (CeHioOs)]! polysaccharide. The most important members of these three groups, from a clinical standpoint, are the following: Monosaccharides: dextrose (glucose), and levulose (fructose). — The former rotates the plane of polarized light to the right, the latter to the left, hence their names ; both ferment with yeast, both reduce alkaline copper solu- tions and both give osazons with phenylhydrazin. Disaecharides: Cane-sugar (saccharose), milk-sugar (lactose), malt-sugar (maltose, and its isomer isomal- tose). — All three are dextrorotatory. Certain ferments split them into their component monosaccharides (cane- sugar into dextrose and levulose, milk-sugar into dextrose and galactose, malt -sugar into two molecules of dex- trose) ; they are, therefore, not directly fermentable with yeast, but must first be split into fermentable monosac- charides, a process that is called inversion. Milk-sugar and malt-sugar reduce alkaline copper solutions; cane- sugar does not possess" this power. 60 CLINICAL URINOLOGY Polysaccharides.— To this group belong ordinary starch (vegetable amylum), glycogen (animal amylum), various dextrines and "animal gum." All these bodies are dextro- rotatory; none of them reduce alkaline copper solutions and none of them are directly fermentable ; they can be split into their component monosaccharides by certain vegetable ferments and by boiling with dilute mineral acids, all the above polysaccharides furnishing dextrose. Glucosides and Glycoproteids. — The carbohydrates form complex compounds (1) with certain organic bodies of an alcohol structure, and (2) with proteids; the former com- pounds are called glucosides, the latter glycoproteids. Both occur in the human body and may occasionally ap- pear in the urine. As they split off sugars on boiling with dilute mineral acids, or by the action of certain fer- ments occasionally found in the urine, they may simulate sugar excretion. It is important to remember that many drugs, e. g., digitalin, arbutin, salicin, phlorizin, etc., have a glucoside structure and may split off sugar in their transit through the organism. The most important urinary glycoproteids are found in mucin (i. e., nucleo- albumen and chondro- albumen; see chapter on Albumi- nuria) ; many internal organs also contain such glycopro- teids (pancreas, thymus, spleen, thyroid, brain) ; the lat- ter split off pentoses. THE CAKBOHYDRATES OF THE URINE AS A GROUP Normal urine always contains small quantities of carbo- hydrates, and consequently possesses some cupric reducing power; this corresponds to a dextrose solution of about 0.3 to 0.4 per cent strength — other bodies, however, than carbohydrates (notably uric acid, creatinin and glyeuro- nates) participate in this reduction. The most important carbohydrates of normal urine are traces of glucose (dex- trose) , isomaltose and animal gum; in addition certain TEE GABBOUYDBATES OF THE UBINE Gl glucosides (contained in mucin, i. e., chondroitin-sulphuric and nucleinic acids ; see also chapter on Albuminuria) . Normal urine also sometimes contains pentoses. Pathological urine may contain pentoses and of the hexoses the monosaccharides dextrose, levulose and laiose, the disaccharides isomaltose and lactose, the polysac- charides glycogen (erythrodextrin?) and animal gum (achroodextrin). Of all these dextrose is the most im- portant clinically. The carbohydrates as a group are important, clinically, chiefly because recent investigations seem to show that in a variety of obscure metabolic disorders the carbo- hydrate mechanism, as a whole, is perverted. It appears, for instance, that in diabetes the quantity of unferment- ahle carbohydrates of the urine (i. e., of those carbohy- drates of the urine that remain behind after the dextrose is removed by fermentation) is increased, and that furthermore, the amount of carbohydrates other than dextrose and levulose is often increased in disorders that later determine toward diabetes. The increase of the total carbohydrates of the urine must therefore, it ap- pears, always be considered a pathological phenomenon that should put us on our guard for the development of diabetes. The best quantitative method for determining the total carbohydrates (or, after deduction of the fer- mentable carbohydrate, the unfermentable carbohydrates) is Baumann's method. Baumann^s Method for Determining the Total Carbo- hydrates. — The earthy phosphates of the urine are re- moved by treating it with soda lye and allowing the mix- ture to stand for twelve hours. The precipitate of phos- phates is removed by filtration and the filtrate treated with 4 to 5 cc. of benzoyl chloride (that should be free from chlorine -benzoyl chloride and benzaldehyde) and 40 cc. of 10 per cent soda lye for every 100 ec. of the fil- trate; (it is necessary to add as much as ten times as 62 CLINICAL URINOLOGY much soda lye as benzoyl chloride, for otherwise the ben- zoeesters form a sticky mass that cannot be separated by filtration) . The mixture is thoroughly shaken for fifteen minutes, or preferably longer, i. e., until no odor of ben- zoyl chloride is any longer preceptible ; the liquid is then neutralized with HCl and the precipitate allowed to settle at the bottom of the flask. The benzoeesters are filtered off, washed with ether, dried and weighed. Normal urine may contain from 2 to 3 g. of these benzoeesters. NON- DIABETIC GLYCOSUEIAS Physiological Glycosuria. — The blood always contains appreciable quantities of dextrose (about 0.2 per cent). Under normal conditions, only traces of this sugar ap- pear in the urine; it is even questionable whether the minimal amounts of dextrose that are found in every normal urine are derived from the blood -sugar at all; it is more probable that this urinary sugar is a splitting product of certain glycoproteids (nucleo albumen, chondro albumen) that enter the urine as mucin (see above). Other carbohydrates found in normal urine have already been mentioned. Whenever the blood- sugar is increased considerably above 0.2 per cent (hyperglycsemia) then dextrose ap- pears in the urine. The hyperglycaemia may be due to a great variety of causes, and hence glycosuria, too, may be the result of manifold factors. The blood -sugar is principally derived from two sources; viz., the carbohy- di-ates of the food and the proteids (and possibly fats) of our own tissues. The food carbohydrates (the starches after splitting, cane-sugar, presumably, after inversion) are in great part absorbed into the portal blood and carried to the liver; here the bulk of the sugar is stored as glycogen, while a certain proportion passes through the liver into the blood- stream beyond, either to be con- TRE CARBOHYDRATES OF THE URINE 63 sumed at once or to be stored for future use as glycogen or fat in the muscles, glands and other working organs of the body. The organism jealously guards the sugar content of the blood and endeavors to hold the sugar percentage at a certain height; as soon, therefore, as the blood-sugar is used up new sugar is poured into the blood from the glycogen reservoirs, and if these become emptied before more, sugar, or sugar-forming pabulum is introduced with the food, then sugar is split off from the body -albumen (and fat?) ; if, on the other hand, the blood- sugar is in- creased while the glycogen reservoirs are full, then the excess is at once gotten rid of in the urine. The increase of the blood-sugar may be caused (1) by excessive inges- tion of sugar or sugar forming pabulum; for then the glycogen reservoirs become insufficient to hold the sugar, and the extra sugar enters the blood -stream; (2), by deficient destruction of blood-sugar ; for if less sugar than normally is destroyed, then, other things being equal, the sugar must, accumulate in the blood; (3), by sudden emptying of the glycogen resex'voirs, or by inability of the glycogen reservoirs to hold and to store the sugar that is carried to them. All these different factors may be operative to cause hyperglycaemia, and nearly all the clinical and experimental forms of glycosuria may be attributed to one or several of them. Glycosuria, then, is a symptom of a manifold variety of possible perversions. The term glycosuria, properly speaking, means the excretion in the urine of glycose (syn. dextrose) ; in clinical parlance it is used, however, to designate the excretion of any sugar in the urine. The terms pentosuria, levulosuria, lactosuria, maltosuria are more correct, but the general term glycosuria may be employed in clinical writing if for no other reason than that it possesses historical dignity, and that dextrose is by far the most important and the most common of the urinary sugars. 64 CLINICAL URINOLOGY The following are the most important clinical forms of glycosuria : — Alimentary Glycosuria. — There are two forms of ali- mentary glycosuria that must be clinically differen- tiated; they are, first, glycosuria following the ingestion of large quantities of sugar — so-called glycosuria e sac- char o, — second, glycosuria following the ingestion of large quantities of starchy foods — so-called glycosuria e amylo. The latter is a more serious condition than the former; it always indicates serious perversion of the car- bohydrate metabolism, i. e., it is diabetic; glycosuria e saccharo, on the other hand, may be altogether transi- tory, need not indicate diabetes, and is frequently seen in patients who never develop diabetes; it may, of course, and often does occur in diabetes. The difference between the two is really one of degree, and not of kind. In the glycosuria following sugar eating the perversion may be very slight, and the appearance of sugar in the urine may indicate merely that a portion of the sugar escaped absorp- tion by the portal blood-vessels and was absorbed by the lacteals instead, so that entering the systemic blood-stream via the thoracic duct directly, it caused sudden hyper- glycaemia followed by glycosuria. In the glycosuria fol- lowing a starch diet, on the other hand, the organism is altogether incapable of assimilating even those small quan- tities of sugar that are slowly formed in the bowel from the amylaceous food, so that here the power to assimilate sugars must be seriously deranged. Any normal, healthy individual may and does develop glycosuria e saccharo if sufficient sugar is given at once; no healthy, normal in- dividual ever develops glycosuria e amylo, however, large the quantities of starchy food administered. A sufferer from glycosuria e saccharo may develop glycosuria e amylo, but need not, whereas, a sufferer from the latter form of glycosuria is always also afflicted with the former. THE CABBOHYDBATES OF THE UBINE 65 There is an alimentary glycosuria for dextrose, levu- lose, lactose, saccharose, etc., each form following the ingestion of the particular sugar, a few cases are on record of alimentary levulosuria following the ingestion of dextrose. A very important clinical test is the determination of the "boundary of assimilation" (see below), i. e., the de- termination of the amount of sugar that must be eaten before sugar appears in the urine; this boundary varies for the different sugars, and also varies somewhat in dif- ferent individuals. The following are average values in healthy subjects: Glycosuria appears if more than 150- 200 grams of dextrose, 150 grams of levulose, 150-200 grams of saccharose (cane-sugar), 125 grams of lactose (milk-sugar) are taken in one dose on an empty stomach. In some normal individuals alimentary glycosuria may follow much smaller quantities of dextrose (as small as 50 grams) , in others up to 350 grams have been given without producing glycosuria; such exceptions, however, are rare and glycosuria following the ingestion of less sugar than above, must as a rule, be considered abnormal. • The first sugar usually appears within an hour after the sugar test- meal, and the excretion of sugar as a rule persists for two or three hours thereafter. Of the total sugar ingested only about two or three per cent reappears in the urine; if more appears this is abnormal (see diabetic glycosuria) . Alimentary glycosuria, that is not due to diabetes, has often been noted in cirrhosis of the liver. Whether the occurrence of glycosuria e saccharo in this disease is due to the formation of anastomoses between the portal vein , and the vena cava, with admission of portal blood carrying sugar directly into the systemic blood, or whether it is due to injury of the liver parenchyma with loss of glyeo- gen-holding powers, remains undertermined. The glyco- suria following the use of very sweet alcoholic beverages has been attributed to similar causes, i. e., to functional 66 CLINICAL URINOLOGY impairment of the liver cells from the alcoholic intoxica- tion with passage through the liver of the sugar these liquors incorporate. In many nervous disorders, traumatic neurosis, neura- sthenia, hysteria, chronic plumbism, delirium tremens, etc., alimentary glycosuria is not unusual; to an extent it may here be due to the increased diuresis so common in all of these states. I consider all these forms danger- ously near the borderline of true diabetes, for the neurosal element in the latter affection is very apparent. Pos- sible too, that the anemia that many of these nervous cases suffer from is at the bottom of the trouble ; this con- dition may very well cause a variety of functional perver- sions in organs concerned in the metabolism of sugar. In a number of infectious diseases and in Basedow's disease (exophthalmic goitre, Graves' disease) glycosuria e sac- charo is by no means rare. Toxic Glycosuria. — To this form belong all those gly- cosurias that follow the exhibition of certain drugs and poisons, the injection of certain organ extracts, the in- toxication of the organism with certain bacterial poisons and autointoxication with certain intestinal poisons and poisonous products of perverted metabolism. Among the drugs and poisons that may produce gly- cosuria are acids (here the acidulation of the blood and organs and no specific action of the different acids must be made responsible), uranium salts and corrosive sub- limate. A group of drugs comprising carbon monoxide (CO), amylnitrate, curare, methyldelphinin, strychnine, morphine, chloroform, ether and other narcotics produces what may be called cellular asphyxia, and hence, by interference with the normal oxydative destruction of sugar, glycosuria. Another group of drugs, i. e., chlor- alamid, chloral, nitrobenzol and nitro toluol leads to the excretion in the urine of certain reducing bodies that may erroneously be taken for sugar; the urine, how- TEE CARBOHYDRATES OF THE URINE 67 ever, will be found to rotate polarized light to the left and not to ferment, and this excludes dextrose and levulose ; the reducing bodies are compound glycuronates that will be discussed in another chapter. A special form of toxic glycosuria is phlorizin glyco- suria. Phlorizin produces glycosuria differently from any of the other poisons; for, whereas in all the other toxic forms the blood-sugar is increased, this is not the case in phlorizin glycosuria. This form is due to some renal pro- cess, as is clearly shown by the absence of hyperglycas- mia even after ligation of the ureters or ablation of the kidneys; in fact, phlorizin injected into one renal artery leads to sugar excretion, at first only on that side. The pathogenesis of this form is presumably the following: Phlorizin is a glucoside that is split into phloretin and phlorose, the latter being a sugar closely allied to dex- trose; this splitting we must assume occurs in the kid- neys. The phlorose (sugar) is excreted while the phloretin returns into the circulation, recombines with sugar to phlorizin, is again disintegrated in the kidneys and so on until, finally, all the phloretin is either destroyed or gradually eliminated. An argument in favor of this view is that phloretin itself injected into the circulation can produce glycosuria. Changes in the renal epithelia have never been observed in phlorizin glycosuria, so that in- jury to the kidneys by phlorizin, permitting diapedesis of blood-sugar can probably be excluded. The glycosuria in this form is considerable; two grams of phlorizin in one instance injected daily for thirty consecutive days led to the excretion of more than one hundred grams of dex- trose a day. (For the uses of phlorizin in determining the renal function, see Chapter XIII.) Extracts made from several of the ductless glands, notably the thyroid and the adrenals, when injected into the circulation are capable of producing glycosuria. Both dry thyroid and the active principle of the gland, thyreo- 68 CLINICAL URINOLOGY iodine can produce this effect; glycosuria is not, how- ever, invariably produced by thyroid ; the genesis of the sugar here is presumably the accelerated metabolism of the body proteids that leads to the liberation and incom- plete destruction of the carbohydrate group. Suprarenal extract and adrenalin can both produce glycosuria; here the adrenal substance seems to exercise a specific action on the pancreas, or the liver, leading to hyperglycsemia and glycosuria. In Basedow'' s disease (hyperthyroidism) glycosuria is not uncommon; here, however, we again approach neurosal diabetes, the connection between Base- dow's disease and true diabetes that is occasionally seen being only slightly understood. In acromegaly finally glycosuria has been known to appear and to persist throughout the course of the disease. In epidemic cholera, in malaria, typhoid, scarlatina, pertussis, measles and ««/?wew^a transitory glycosuria has been described. Glycosuria is also quite common "after" carbuncle, gangrene, erysipelas, furunculosis, noma, etc.; it is more than probable, however, that in all of these cases the patients were diabetic, developed these skin lesions, and that then only was the sugar discovered in the urine. In syphilis, finally, glycosuria is not at all rare — here, however, we are probably dealing with syphi- litic lesions of the nervous system, the liver or the pan- creas and not with any specific effect of the syphilitic virus. Injections, finally, of dialysed bowel- contents from diabetics and of diabetic urine have been known to pro- duce glycosuria. Indirectly, certain acid products of per- verted metabolism, as diacetic acid, oxybutyric acid, lactic acid, possibly uric acid, can produce glycosuria, owing to their power to acidulate the blood and tissues (see above) . Experimental Glycosuria. — These forms are of inter- est to the clinician, chiefly, because they throw much light upon the pathogenesis of glycosuria and diabetes in THE CARBOHYDRATES OF THE URINE 69 spontaneous degeneration and in traumatic lesions of different organs in man. For the sake of completeness the chief experimental forms of glycosuria may be named. First and foremost, the glycosuria (and diabetes) follow- ing complete (n. b. ! ) removal of the pancreas, or follow- ing manipulations that lead to complete degeneration of this organ; second, the glycosuria following the classi- cal "piqure" of the floor of the fourth ventricle; third, glycosuria following the intravenous infusion of salt solu- tions that can produce profuse diuresis. These three forms have their clinical counterpart in certain pancreatic forms of diabetes, in glycosuria following trauma or de- generation of the region of the fourth ventricle, in the occasional glycosuria of polyuria in many functional neuroses, diabetes insipidus, etc. To the experimental glycosurias also belong many of the toxic forms already mentioned. Psychic Glycosuria and Glycosuria in Diseases of the Nervous System. — Psychic and emotional shock may pro- duce glycosuria; in many diabetics, moreover, the sugar excretion will be found to fluctuate with the mental and emotional state of the patient, exultation and joyful emo- tions being often followed by a decrease of the sugar, depression and worry leading to increased glycosuria. Here we may be dealing with nervous or vasomotor influ- ences governing glycogenesis in the liver and muscles or determining acceleration or retardation of sugar combus- tion. In various traumata and degenerations of the cerebro-spinal axis, principally in lesions in the region of the fourth ventricle (see above), we often see glycosuria; for instance in apoplexy, encephalomalacia, multiple sclerosis, paralytic dementia, cerebral tumor, gumma, etc. The relation of these lesions to the piqure experi- ment is apparent, and we must imagine that in all such cases, possibly, the normal nerve impulses governing the glyeogen-holding function of the liver are perverted. 70 CLINICAL URINOLOGY Renal Glycosuria. — I have already spoken of phlorizin glycosuria, one form of renal glycosuria. Cases are re- corded of glycosuria without hyperglycsemia, and with certain renal disorders that must also be considered as renal glycosuria, for they never develop into true diabetes. On the other hand, granular atrophy of the kidneys is not uncommon in diabetes; in such cases the sugar for some mysterious reason often disappears from the urine; this cannot, however, be considered a favorable omen, because the renal atrophy is usually very complete and rapidly precipitates a fatal issue. In renal hemorrhages, in chy- luria (particularly the tropical variety) and occasionally in nephritis sugar appears in the urine. Care must of course always be exercised in interpreting these cases in order to determine whether the patients are suffering from diabetes or whether the kidneys have merely become abnormally permeable to sugar. Glycosuria in increased diuresis from various causes has already been mentioned. Glycosuria in Obesity, Gout and Arteriosclerosis. — Obesity and glycosuria are often associated. The obesity, as a rule, precedes the appearance of sugar in the urine. This form of diabetes (diabete gras of the French, lipogener Diabetes of the Cermans) is usually mild, particularly in older subjects; in young subjects it is generally grave, and death ensues within a short time. Theoretically, this combination is very interesting; obesity and diabetes run together in families, some of the members becoming obese, others developing diabetes and still others both obesity and glycosuria ; this in itself seems to indicate an intimate rela- tion between the two diseases. The combination of obe- sity and diabetes can be explained on the basis that sugar may normally either be completely oxidized or may be con- verted into fat and deposited in the tissues ; if the sugar destruction is only slightly impaired, then the conversion into fat may still occur so that the sugar, instead of escap- ing combustion altogether and appearing in the urine as THE CARBOHYDRATES OF THE URINE 71 such, is deposited in the tissues as fat; if the perversion becomes more severe, then some of the sugar is wasted in the urine ; and, finally, in still more advanced degrees the fat deposits themselves are made to give up sugar molecules which are excreted in the urine and lost. Clini- cally, these three forms are characterized as follows (von Noorden) : (1) cases in which both the combustion of the sugar and its conversion into fat are impaired (ordinary diabetes, with emaciation) ; (2) cases in which the com- bustion of sugar is impaired, but in which the conversion into fat is unimpaired (obesity without glycosuria, "masked diabetes"). These cases show a tendency to alimentary glycosuria and later to develop into (3) cases in which the combustion of sugar is again interfered with, while at the same time there is some loss of the power to store sugar as fat (the ordinary diabetes of obese subjects, "diabete gras"). Gout and glycosuria (diabetes) are of ten seen together ; here, again, we have hereditary tendencies, some mem- bers of a family developing gout, others diabetes, still others gout and diabetes. The two appear simultane- ously or consecutively, the gout as a rule preceding the diabetes. Obesity, gout and diabetes also occur together or alternately or in different members of the same family. Glycosuria may complicate any of the manifestations of goutiness (uric acid diathesis) , but is in no way dependent on gouty attacks ; the glycosuria is usually slight, the dia- betes of a mild type. Glycosuria is particularly common in urolithiasis, and here it may be of renal origin (see above) . Arteriosclei'osis is often a precursor of glycosuria, and is common in diabetes (and obesity and gout) . This con- dition of the arterial system may, in fact, be considered the connecting link between diabetes, obesity and gout, in- asmuch as the cardiovascular disturbances incident to arteriosclerosis determine certain hepatic, renal, pancre- atic disorders that in their turn interfere with the proper 72 CLINICAL URINOLOGY utilization of the sugars, the fats, the puriii bodies; in addition, certain cerebral lesions, both functional and organic, may develop in arteriosclerosis that may deter- mine glycosuria as outlined in another paragraph. It is well to remember that arteriosclerosis, aside from being a cause of these different disorders of metabolism, may also be either the resuU of the flooding of the blood and tissues with abnormal products (sugar, fat, uric acid and its congeners) or may be one of the manifestations of some primary intoxication affecting simultaneously the catabolic processes of the organism and the cardio- vascular system. DIABETIC GLYCOSURIA The discussion of glycosuria in obesity, gout and arteriosclerosis leads us directly to the glycosuria of true diabetes; this glycosuria has been incidentally referred to in several of the previous paragraphs. Glycosuria is the most important symptom of diabetes, in fact, the symptom that alone determines the diagnosis ; at the same time, as we have seen, glycosuria by no means always means diabetes. Unfortunately, many still con- tinue to use the terms diabetes and glycosuria synony- mously. This is radically wrong. In the treatment of diabetes the reduction of the glycosuria (with certain limitations, see below) should be the chief aim, not only because this symptom is the best index of the progress or regress of the disease, but also because the inhibition of the loss of sugar is a distinct gain to the patient. In view of the great clinical importance of careful metabolic studies in diabetes, using the urinary sugar and nitrogen excretion as an index, the following rationale of practical dietetics in diabetic glycosuria may be given in this place* : •Quoted in part from an article by the author on "The Mathematics of the Diabetic Diet" (Journ. Am. Medl. Ass'n., March 26, J904).' TEE CABBOHYDBATES OF THE UBINE 73 The Caloric Deficit in Diabetic Glycosuria.— In order to fully appreciate the beautiful accuracy that can be adopted in feeding diabetics, it is necessary to appreciate the significance of the laws of nutrition, as we understand them today. Basing on the knowledge of metabolism that has been evolved from latter-day researches in physio- logic chemistry, we can almost mathematically regulate the diet in diabetes. The gratifying results obtained from this practice fully justify the pains that must be expended in studying the problem carefully in each case that comes under observation. For measuring the nutritive value of the different classes of foods, the term "calorie" has been imported from the realm of physics. A calorie is that amount of heat that is needed to raise the temperature of 1 kilo of water 1 degree (Celsius). Each article of food, it has been found, in process of metabolism (i. e., of assimila- tion, oxidative disassimilation and elimination) in the body generates a definite quantity of heat, or the me- chanical equivalent of this heat in labor. Expressed in calories : 1 gr. of pvoteid furnishes 4.1 eal. 1 gr. of carbohydrate furnishes 4.1 cal. 1 gr. of fat furnishes 9.3 cal. It has further been determined that a normal adult requires from 30 to 35 calories a day per kilo of body weight, and that these calories can be furnished by any one or all of the three classes of food vicariously. If this calorie value is not supplied, the body must consume its own tissues, and as a result emaciates. In the case of diabetes, with the loss of valuable un- consumed sugar in the urine, the average diet dpes not furnish sufficient caloric value, as may be seen from the following example that I quote from my case book: 74 CLINICAL URINOLOGY Case 21. — Mrs. W. P. Weight 60 kilo. Calories required for adequate nutrition, 60x35 = 2100. Average diet on six successive days: Proteids 150g. x4.1= 615.0 eal. Carbohydrates 190g. x4.1= 779.0 eal. Fat 110 g. X 9.3 = 1023.0 eal. 2417.0 eal. Average daily sugar excretion . . . 160 g. x4.1= 656.0 eal. Resulting food value only ^1761.0 eal. Instead of receiving, therefore, the full caloric value required, i. e., 2100 eal., the patient, owing to the loss of sugar, received only 1761.0 eal., although the diet repre- sents 2417.0 eal. This represents a deficit of 2100 — 1761 = 339 eal. And these deficient calories must be supplied from the destruction of the patient's proper tissues. One can further readily calculate what proportion of this deficit is made good from the albumen of the patient's tissues, which from the fat and the diet can be regulated accordingly. All one has to do is to determine the out- put of nitrogen in the urine and fseces, and compare it with the nitrogen intake (food nitrogen) . This patient, for instance, received in the daily diet 150 grams of albumen, and as albumen contains 16 per cent of nitrogen, this amount contained 24.0 grams of nitrogen. On this diet the patient excreted a daily aver- age of 23.7 grams of nitrogen in the urine, and of 3.1 grams of nitrogen in the faeces, making the total nitrogen output 23.7 + 3.1 = 26.8 grams of nitrogen. The nitrogen output, therefore, is greater by 2.8 grams (26.8 — 24) than the nitrogen intake, and this excess must have been derived from the patient's own albumen. As 2.8 grains of .nitrogen are contained in 17.5 grams of albumen (^^^^" = 17.5) and 17.5 grams of albumen can produce 71.75 calories (17.5X4.1=71.75), there re- main only 267.25 (339 — 71.75 = 267.25) of the 339 de- ficient calories to be accounted for. As these must be derived from the patient's fat, one can readily determine THE GARBOHYBBATES OF TEE URINE 75 by dividing 267.25 by 9.3 (the caloric value of one gram of fat) that 28.8 grams of the patient's fat were con- sumed. The patient, therefore, on a diet valued at 2417 calories, i. e., considerably more than the calculated value neces- sary to nourish a normal subject of 60 kilo (2100 cal.), lost 17.5 grams of her own albumen and 28.8 grams of her own fat. Hence the polyphagia that is so common in this dis- ease. The patients eat as much as a normal subject, but the diet does not adequately nourish them. "Stomach hunger" may be appeased, because the stomach is filled, but "tissue hunger" soon appears. This hunger only dis- appears when the meat and fat are increased. The Boundary of Sugar Assimilation in Diabetes. — If it were absolutely true that diabetics could use none of the sugar that enters the blood- stream, the question of feeding would be theoretically a very simple one; viz., one would have to exclude carbohydrates and replace them with meats and fats of sufficient caloric value to make up the caloric deficit. As a matter of fact, this is not the case. Only a small minority of cases are altogether unable to utilize any 6i. the sugar. These are the very grave cases that are, fortunately, rare, and would be still less frequent if many milder cases were not transformed into grave cases by injudicious dieting. In the great majority of cases the patients can utilize some of the sugar, and it is absolutely bad practice to withold this food permanently; it is equally bad practice, however, to give such cases too much sugar, for this is apt, by overtaxing of the sugar-destroy- ing function, to "fatigue" this function and to lead to in- creased inability on the part of the body to utilize sugar. It is necessary, therefore, in each case, to determine the exact tolerance of the body, the so-called boundary of as- similation, and to feed the patients accordingly. 76 CLINICAL URINOLOGY The Three Degrees of Diabetic Glycosuria. — It is con- ventional to distinguish several degrees of diabetic gly- cosuria that vary in degree but not in kind. In the mild kind all the sugar disappears from the urine in a few days if the carbohydrates of the food are withheld. In such cases the addition of small quantities of carbo- hydrate to the diet may not even cause glycosuria; as soon as more than a certain quantity (less, however, than the amount required to produce alimentary gly- cosuria in a normal subject) is given, sugar invariably appears in the urine. In some of the cases, further, no sugar appears if a considerable quantity of carbohydrate is given in small, divided doses, whereas the ingestion of the same quantity at once is followed by glycosuria. In these cases, then, the power to destroy sugar is by no means completely lost; for even when we withdraw all carbohydrates from the food, a certain amount of sugar nevertheless enters the blood-stream "from within," i. e., from the degradation of the circulating and organized albumen molecules that split off a sugar group. This sugar the organism can completely utilize in the mild form, and in addition it can utilize a certain portion of the food sugar. In the severe form of diabetic glycosuria, we encounter altogether different conditions. Here the sugar never dis- appears completely from the urine, even if all carbo- hydrates are withheld for days or weeks. This indicates that not even the sugar that is derived from the intracel- lular disassimilation of albumen, and from the disassimi- lation of the food albumen, can be utilized. In extreme cases, even complete withdrawal of all food does not stop the sugar excretion, indicating that even the small quantities of sugar derived from the destruction of the patient's proper albuminous tissues are only incompletely utilized. In still other cases, the carbohydrates must be ex- THE GABB0HYDBATE8 OF THE URINE 77 eluded from the diet and the albumen considerably re- duced before the glycosuria is inhibited. These cases properly constitute a third group, i. e., diabetic glycosuria of medium severity. The boundaries of this group are not clearly defined, and many transition forms between it and the mild cases on the one hand, the severe cases on the other, can be arranged. With proper dieting, cases of medium severity can usually be converted into the mild type ; with impi'oper dieting, they almost invariably develop into the severe type. To summarize: One may speak of the mild form of glycosuria if the sugar excretion stops at once after with- drawal of carbohydrates, if it does not reappear when small quantities of carbohydrates are administered, and if at no time the amount of food albumen need be reduced. One may speak of the medium form if not only the carbohydrates must be withdrawn completely before sugar excretion cases, but the food albumen must also be re- duced to such a point that less than 18 grams but more than 10 grams of nitrogen appear in the urine. One may speak of the severe form if, in order to stop the glycosuria, the carbohydrates must be stopped, and in addition so much albumen must be withdrawn that less than 10 grams of nitrogen appear in the urine. Cases in which even this deprivation of food fails to reduce the glycosuria are, of course, also to be included under the severe form. The Determination of the Degree of Glycosuria. — A popular method for determining the degree of gly- cosuria is the following: The patient is given what may be called the "diabetic test meal." This consists of a carbohydrate- free portion and a weighed portion of some carbohydrate food. The former may be composed of meats (about 350 g.), eggs, cream, cheese, spinach, as- paragus, salad with oil dressing, meat broths, tea, cofEee, 78 CLINICAL URINOLOGY clavet. The latter consists of 100 grams of white bread, preferably administered in two portions of 50 grams each, in the forenoon and afternoon. At times it may be nec- essary to administer other carbohydrate food instead of white bread, because it may be of practical importance to determine the tolerance of the organism for other starchy foods and for the different sugars. If the patient 6n this diet (carbohydrate-free meal +100 g. of white bread) excretes no sugar, then we are dealing with a very mild form of glycosuria; the amount of bread should then very gradually be increased on suc- cessive days until sugar finally appears in the urine. Thus, if the patient on one day excretes no sugar after eating 3X50 grams=150 grams of bread, and on the next day passes sugar on 4X50=200 grams of bread, then the "boundary of assimilation" (see above) lies between 150 and 200 grams of white bread. If the patient excretes sugar on the test diet, then we are dealing either with the mild or the medium form. If after withdrawal of the 100 grams of bread the glycosuria stops, then it is a mild form. If the sugar secretion still persists, then the case is medium or severe. The food albumen must now be reduced. If the glycosuria stops after the albumen is reduced to such a point that less than 18 grams of nitrogen appear in the urine, then the case is one of medium severity. If the albumen must be reduced so much that less than 10 grams of nitrogen appear in the urine, or if it does not stop after the with- drawal of all food, then we are dealing with a case of severe diabetic glycosuria. I need hardly emphasize that one single determination of this kind is of little value in so chronic a disease as diabetes; consequently the above tests should be per- formed several times during the year. Only in this way can the success of the dietetic treatment instituted on the basis of such tests be gauged. One must, moreover, TEE CABBOEYDRATES OF TEE URINE 79 never forget that incalculable fluctuations in the sugar ex- cretion and in the tolerance of these patients for sugar occur in nearly all the cases, and that a single determina- tion of the boundary of assimilation may hence lead to wrong conclusions. Practical Dietetics of Diahetic Glycosuria.— Within the narrow scope of this work, only the broad principles underlying the feeding of diabetics can be declared. The fundamental postulate is to maintain the patient's nutrition, and this is essentially synonymous with main- taining what is called "the nitrogen equilibrium" (see Chapter III); i. e., the nitrogen output must never ex- ceed the nitrogen intake; in other words, the albumen content of the patient must be jealously maintained. The patient, therefore, must receive food that, after the deduction of the sugar wasted in the urine, allows him to utilize at least 35 calories per kilo of body weight pro die. The amount of carbohydrate permitted must be deter- mined according to the methods described above, i. e., the boundary of assimilation must be established. Theo- retically, this is a very simple matter. The physiologic chemist may be guided exclusively by the results of these tests. The physician should, however, take into consideration certain clinical aspects of the question that may induce him to modify according to his best judgment the mathematical conclusions that are forced on him. Thus, on the one hand, it may become necessary to reduce the carbohydrates considerably below the estab- lished boundary of assimilation, or even to exclude them altogether for a time, if it is found that the tolerance of the patient is growing less; if this precaution is not ob- served, the mild form may be rapidly transformed into a severe one. One may say axiomatically that the reduc- tion of the carbohydrates considerably below the tolerance 80 CLINICAL URINOLOGY of the patient places the sugar-destroying function at rest and thus enables it to regain its old power, and that, inversely, crowding of the carbohydrates to the limit of tolerance engenders fatigue of the already impaired func- tion, and hence reduces the tolerance of the organism for sugar. In addition, we know from experience that the reduction or withdrawal of the carbohydrates greatly aids in combating certain complications that may endanger the life of the patient. Occasionally these only yieM if carbohydrates are withdrawn or reduced to a minimum for a time. I refer, e. g., to furunculosis, pruritus, neu- ralgias, retinitis, etc. On the other hand, it may occasionally be good prac- tice to give more carbohydrates than the mathematical tests indicate. The removal of the glycosuria is not the only aim, for the excretion of sugar is, after all, merely a symptom. True, it is our chief index of the progress or regress of the disease, and the loss of sugar constitutes the chief danger to the patient. Nevertheless, the loss of some sugar in the urine may occasionally be less dangerous to the patient than the withdrawal of all carbohydrates from the food. Rather prolong the patient's life, even though glycosuria persists, than stop the glycosuria and incidentally hasten the patient's death. Two reasons chiefly should induce us to give carbohy- drates to patients with glycosuria, even at the risk of in- creasing the excretion of sugar; viz. (1) the impossibil- ity of adequately nourishing most patients on a meat-fat diet alone; (2) the increased danger of acidosis and coma incident to the withdrawal of all carbohydrate. A meat-fat diet would soon become utterly disgusting to a well person, and he could ill tolerate the withdrawal of carbohydrate. A diabetic, moreover, has an abnormal craving for carbohydrates, particularly bread. Albuminous foods alone can not be given in sufficient THE GABBOHYDBATES OF THE UBINE 81 quantities to furnish enough calories. The addition of fat may make up the deficit, but it is a peculiar fact that one can eat much more fat with relish if some carbohy- drate is given, than without it; here also alcohol, with its 7 calories per gram, often helps out. If the patients do not relish their food, the normal psychic stimulus so nec- essary to normal gastro- intestinal digestion is sacrificed ; the poor appetite induces the sufferer to eat too little ; the perverted gastro - intestinal secretions only inade- quately prepare the food for assimilation, and the inev- itable result is starvation, and the development of the manifold complications incident to malnutrition. As far as the development of acidosis finally is con- cerned, we know that a certain amount of carbohydrate is necessary to promote the oxidation of the members of the acetone group (oxybiityric acid, diacetic acid and acetone) that escape destruction and thus accumulate in diabetics (see Chapter V) . If all carbohydrate is perma- nently withdrawn, this oxidation is particularly difiicult. It is a well-known fact that in diabetics the administra- tion of a little carbohydrate will greatly reduce the acetone excretion and will often cause the rapid disappearance of oxybutyric acid and diacetic acid from the urine. Hence, in acidosis and impending coma, the administration of carbohydrates, if necessary dextrose per rectum or lev- ulose intravenously, is good practice; and in the same sense the occasional administration of moderate quanti- ties of carbohydrate pabulum is good preventive treat- ment. I make an earnest plea for more careful, more mathe- matical regulation of the diet in this class of sufferers, and I emphatically decry the slovenly, the criminal rou- tine habit so universally adopted in this country, of hand- ing every patient with sugar in his urine a diet-list on which is found printed a list of articles that contain no carbohydrates. 82 CLINICAL URINOLOGY THE DIFFERENT CARBOHYDRATES OP THE URINE, THEIR CLINI- CAL SIGNIFICANCE, DETECTION AND DETERMINATION Pentoses (pentosuria). — As tea, coflEee, certain wines, milk, cherries, plums, certain drugs (see above) , contain pentoses, as furthermore many internal organs (thyroid, spleen, brain, thymus, pancreas) contain glycoproteids that readily split off pentoses, one need not be surprised occasionally to find pentoses in the urine. It is question- able, moreover, whether pentoses introduced into the body are assimilated; it is probable per contra that they are at once excreted in the urine. Furthermore, a few cases are on record in which pentoses were excreted after prolonged periods of fasting — it seems probable, there- fore, that these sugars can also be formed within the body, presumably from the above-named glycoproteids of our internal organs. In grave diabetes and in depancreatized dogs with ex- perimental diabetes pentosuria is not uncommon; several cases of morphine poisoning are also on record in which pentoses were excreted and in which the pentosuria ceased within a few days after the morphine was with- drawn. Clinical Significance. — The clinical significance of pen- tosuria is not understood; in the majority of the cases one is presumably dealing with an alimentary excretion of pentoses — this applies particularly to those cases in which the urine is otherwise found normal; in diabetes the appearance of pentoses simply indicates the perver- sion of the total carbohydrate mechanism; whether or not pentosuria in such cases must be considered a bad prognostic omen is not sufliciently established. Pentose excretion is especially important for the present, because it may lead to confusion and may simulate, on superficial examination , the excretion of other clinically more impor- tant sugars. THE CABBOHYVBATES OF THE UBINE 83 Properties of Pentoses. — The pentoses reduce alkaline copper solutions very energetically; they are not directly fermentable with pure yeast; they form osazons with phenylhydrazin that possess a melting point considerably below (159°C) that of the osazons of the hexoses. Most pentoses are optically inactive, one of them (xylose) is slightly dextrorotatory. Recognition of Pentoses in the Urine. — When pentoses appear in the urine alone their recognition is easy. The urine will reduce alkaline copper solution very energeti- cally; at the same time it will possess no rotatory power, or only very slight dextrorotatory power, it will not fer- ment, it will give an osazon of low melting point (159°C) and, finally, it will give the phloroglucin reaction of Tollens that may be considered characteristic for pentoses (see below) . True, milk-sugar (lactose) also reduces cupric solutions and is also unfermentable, and may even give the Tollens reaction, but the occurrence of the latter sugar is very rare as compared to the occurrence of pen- tose. If necessary the two can readily be differentiated by spectroscopic examination of the urine in which the phloroglucin test has been made ; if the reaction was due to the presence of pentoses, absorption bands in D and E will be seen, while these are absent in the case of milk- sugar (galactose reaction). In diabetic urine the recognition of pentoses is also simple. Here again a lack of correspondence between the cupric- reducing powers and the dextrorotation will be found, indicating that the reduction cannot be due to the dextrose alone, but that certain other reducing bodies that are optically inactive, or less dextrorotatory than dex- trose, or levorotatory, must be present. (Such bodies may be pentose, levulose, /8-oxybutyric acid or glycuronates, see pages 89, 90). This finding calls for a phloroglucin test; if this is positive, the urme contains pentoses. The detection and differentiation of the other bodies mentioned 84 CLINICAL URINOLOGY will be discussed under appropriate headings in the fol- lowing paragraphs. Tollens'' Phloroglucin Reaction for Pentoses. — A little powdered phloroglucin is dropped into a test-tube contain- ing 5-6 cc. of fuming hydrochloric acid, and so much phloroglucin is added that some of it remains undissolved on heating the mixture. Three to four cubic centimeters of this reagent are treated with about half a cubic centi- meter of filtered urine (it is well to decolorize high-col- ored urine by boiling it with animal charcoal and then filtering) . The tube is then heated in a beaker contain- ing boiling water; if pentoses are present the urine will rapidly assume a dark red color. The red liquid may be examined spectroscopically — it must show absorption- bands at D and E (see above) . Dextrose (syn., glycose, glucose, grape-sugar).— Dex- trose being the most important sugar of the urine, nearly all that has been said under the caption of glycosuria properly pertains to dextrosuria. The clinical significance of dextrosuria is, therefore, essentially synonymous with that of glycosuria and will not be further discussed in this place. Qualitative and Quantitative Determination of Dextrose. — Of the many tests for dextrose that are in use, only those few will be described that are rapid and simple of execution and that are at the same time sufficiently relia- ble for clinical purposes. For the many tests that are in use in strictly scientific work where the greatest accuracy is demanded, where time is no object and where much technical skill is presupposed, I refer to text -books of physiological chemistry. The following tests I particu- larly recommend for clinical work: FeUing^s Test (Trommer's test modified).— Two solu- tions are required; viz. (1) a solution of Eochelle salts and sodium hydrate, (2) a solution of copper sulphate. The alkaline solution (1) contains 173 grams of Eochelle THE GARB0HYDBATE8 OF THE URINE 85 salts (potassium-sodium tartrate) and 120 grams of sodium hydrate in 500 cc. of water. The copper solution (2) contains 34.639 grams of copper sulphate in 500 cc. of water. In Trommer's original test the alkaline solution contained no Eochelle salts. In performing the test, equal parts of the two solutions are mixed and diluted with 5 parts of water, — this is the reagent. Equal parts of this and of the urine are poured into two test-tubes and each tube heated to boiling; both tubes are then allowed to cool off a little and the reagent poured into the urine ; if sugar is present, the yellow or red oxide of copper will appear almost instantaneously and through the whole liquid at once. The advantage of performing the test in this way is that the reduction of the alkaline copper solu- tion that may be brought about by numerous other uri- nary bodies, as uric acid, creatinin, allantoin, nucleo- albumen, conjugate glycuronates, bile pigments, homo- gentisinic acid, etc., all substances that reduce alkaline copper solutions on boiling, is ruled out; a positive Fehling's test occurring below the boiling point of the mixture of urine and reagent may be said always to in- dicate the presence, of dextrose. Haines^ Test. — A convenient and popular modification of the cupric reducing test is the one described by Haines. The reagent is prepared as follows : Pure copper sulphate 2 grams, pure glycerine 15 cc, liquor potassse (U. S. P., sp. grav. about 1,036) 150 cc, water enough to make 200 cc Dissolve the copper sulphate in about 20 cc. of water, add the glycerine, then the liquor potassse and agitate thoroughly. Make up to 200 cc. This solution has the great advantage of being very stable. The test is carried out as follows : Half a test-tube of the solution is heated to boiling (no color change should take place) ; add, not to exceed, 6 to 8 drops of the urine and again heat to boiling. If sugar is present an abundant yellow or yellowish red precipitate appears. 86 CLINICAL URINOLOGY Nylander^s Bismuth Test. — This test is also based on the property of dextrose to reduce salts of the heavy metals on heating in alkaline solution; in this test the subnitrate of bismuth is reduced instead of the sulphate of copper. Uric acid, ereatinin, hemogentisinic acid do not reduce bismuth subnitrate as they do Fehling's solu- tion; uroerythrin and hematoporphyrin may, however, simulate a reduction. After the administration of rheum, senna, salol, antipyrin and quinine, the urine always gives the test, so that this source of error must be ruled out ; if, finally, the urine contains a great excess of ammonia salts, as, for instance, in diabetic acidosis, the test may fail, even if the urine contains considerable dextrose. Nylander's reagent consists of 4 grams of sodium- potassium tartrate, 2 grams of bismuth subnitrate and 10 grams of sodium hydrate dissolved in 90 cc. of boiling water. The solution, after cooling, must be filtered and pre- served in a dark bottle. Ten parts of the urine are mixed with one part of the reagent and boiled for two or three minutes ; it is necessary to boil for several minutes, as the precipitate of bismuth, or its oxide, may not form for some time. If dextrose is present a gray or dark brown precipitate of bismuthous oxide or a black precipitate of metallic bismuth will form. The Fermentation Test. — Yeast splits dextrose into alcohol and carbon dioxide (and glycerin, succinic acid, etc.), according to the formula: C0H12O0 = 2C2H|,0 + 2CO2 Dextrose Alcohol Carbon dioxide According to this formula, which represents the chief reaction, 100 parts of dextrose should yield 51,1 parts of alcohol and 48.9 parts of carbon dioxide; as a matter of fact, only 48.67 parts of alcohol and 46.54 parts of carbon dioxide are formed, the deficit of 4.79 parts being made up of 3.85 parts of glycerin and succinic acid and 0.94 THE CARBOHYDRATES OF THE URINE 87 parts of unknown bodies. It. will be seen, however, that the proportion between the two main products of the fer- mentation remains the same; viz., 1.045 : 1 as called for by the above equation. The temperature optimum for the alcoholic fermentation of sugar lies at 34° C. ; below 15° C. the process is not completed, and above 45° C. it does not take place at all . Sugar solutions of the strength of 4 per cent to 8 per cent ferment more rapidly than more, or less, concentrated solutions. The proper pro- portion of CO2 (i. e., 46.54 per cent) is only obtained if not more than half of one part of fresh doughy yeast is used for each part of dextrose; more yeast leads to the development of more CO2, owing to the autofermentation (budding) of the yeast. The test is performed as follows: A piece of fresh yeast about as large as a split pea is shaken with about 25 cc. of urine and the mixture placed into a saccharome- ter (Einhorn's saccharometer is the most popular appa- ratus for clinical work) . If dextrose is present, CO2 will be developed at room temperature or, more rapidly, at 34°C. in the incubator. At least six hours should be allowed to elapse before the presence of dextrose is ex- cluded from failure of the solution to develop gas. Be- fore performing the test, a control tube should be arranged containing a 4 to 8 per cent solution of dextrose ; in this way one can determine whether the yeast is active. A very small bubble of gas should not lead to the diagnosis glycosuria, for normal urine frequently ferments a little. The presence of dextrose should never be diagnosed from a positive fermentation test alone; it should be supple- mented by either Fehling's or Nylander's test. The Fhenylhydrazin Test. — It is claimed that normal urine, when treated with phenylhydrazin-HCl, as de- scribed below, occasionally precipitates crystals that can be mistaken for typical osazons. I have never seen this and consequently am not inclined, as some authors are, 88 CLINICAL URINOLOGY to declare the phenylhydrazin test not pathognomonic for pathological sugars. The test is performed as follows: 10 ce. of urine are poured into a test-tube and to it are added X to % grams of phenylhydrazin- HCl, and not quite twice as much (1 to IX grams) of sodium acetate ; with a little practice the proper amounts of the two salts can be estimated with sufficient accuracy. The mixture is heated to boiling, and if the salts do not dissolve readily a little more water is added. The tube is placed in boiling water for from twenty to thirty minutes and then allowed to cool slowly. At the expiration of half an hour a yellow crys- talline precipitate of phenylglucosazon should appear; occasionally the sediment appears amorphous to the naked eye, but is found to present the characteristic crystallized appearance under the microscope. The microscopic ap- pearance is quite typical; viz., fine, bright yellow needles arranged in bundles, sheaths or rosettes. Yellow flakes or plates prove nothing in regard to the presence of sugar. A number of rapid tests by this method have been de- scribed, but I do not consider any of these "five-minute tests" reliable, nor do I think one should exclude the presence of dextrose from failure of these quick tests to give a positive reaction. The phenylhydrazin test can be successfully performed even if the urine contains small quantities of albumen; large quantities of albumen are preferably first removed by boiling. Very small quanti- ties of dextrose (as little as 0.1 per cent) can easily be detected by this method. The precipitate formed in the test is chemically charac- terized as an osazon, and many sugars form these com- pounds (compare also paragraphs on "Physiological Chem- istry of the Carbohydrates," in this chapter) . The osazons of the different sugars can readily be differentiated by their melting points, the melting points of the most im- portant osazons being the following: dextrose 204-205° C, THE CABBORYDBATES OF THE UBINE 89 levulose 204-205° C, galactose 193° C, maltose 206° C, isomaltose 150-153° C, lactose 200° C. The osazons of the different sugars are termed dextrosazon, lactosazon, maltosazon, etc. In addition to hexosws, pentoses also give pentosazons and glyeuronic acid forms an osazon compound after treating with phenylhydrazin ; aceton and diacetic acid give a hydrazon that is characterized by its insolubility; oxalic acid forms a compound that is also quite difficult to dissolve in water. Urea in concentrated urines forms a peculiar compound with phenylhydrazin that may lead to confusion with osazons unless the characteristic proper- ties of the latter are determined. These possible sources of error should always be borne in mind in interpreting a precipitate formed with phenylhydrazin. Polarimetric Examination. — For the clinical determina- tion of dextrose the polariscopic examination is super- fluous, as any of the above tests reveals the presence of dextrose with certainty; for scientific investigations re- quiring a high degree of accuracy, particularly for comparative quantitative work, this method is, however, indispensable. As a costly and complicated apparatus is required, and much technical skill is needed to manipulate it properly, this method will probably never become popular for ordinary clinical work. For a description of the polariscope, for the principles underlying its use, for the technique of polarimetric examinations, I refer, therefore, to text- books of physics. Aside from dextrose, certain other urinary bodies turn the plane of polarized light to the right, e. g., the biliary acids. Other bodies turn it to the left, as levulose, con- jugate glycuronates and /8-oxybutyric acid. Albumen, too, is levorotary. Dextrorotation in itself is, therefore, not conclusive evidence of the presence of dextrose, nor is optical inactivity or even levorotation conclusive evi- dence of the absence of dextrose; for while any dextrose 90 CLINICAL URINOLOGY that may be present may turn the plane to the right, cer- tain levorotatory bodies that may be present at the same time may neutralize the dextrorotation, or may even de- termine levorotation. Albumen and bile acids can easily be removed by precipitation with lead acetate ; occasionally urine that was optically inactive or levorotatory before treatment with lead acetate will become dextrorotatory, thias revealing the presence of dextrose. If the urine reduces Fehling's solution and is also dextrorotatory, then quantitative estimations should be made by both methods. If the cupric reducing powers and the degree of dextrorotation, calculated for dextrose, both agree, then the urine contains only dextrose. If polarization is less than reduction, then the urine must contain certain other reducing bodies that are either optically inactive or less dextrorotatory than dextrose, or levorotatory. This is a frequent occurrence in diabetic urine. The bodies in question may be pentoses (optically inactive or slightly dextrorotatory) or glycuronates, or levu- lose or p-oxyhuty^ic acid. The presence or absence of pentoses can be established by performing Tollens' re- action (see pentoses) . In order to detect the presence of the other three levorotatory bodies, the urine must be fer- mented. If the fermented urine no longer reduces, nor shows levorotation, then levulose was present; if the urine no longer reduces but still shows considerable levorota- tion, then it presumably contained /3-oxybutyric acid; if, finally, the fermented urine still reduces and still shows much levorotation, then it contained glycuronates. Quantitative Estimation of Dextrose. — Of the many quantitative tests for dextrose in the urine, only three will be given; viz., Fehling's, Pavy's and Haines' method. The method of Knapp is probably the best for very accu- rate work; it is based on the reduction of mercuric cyanide by dextrose in alkaline solution; the resulting mercury settles to the bottom and the supernatant fluid THE CARBOHYDRATES OF THE URINE 91 becomes clear; the precipitation of all the mercury is preferably determined by the addition of stannous oxide as an indicator, the presence of mercury being revealed by the formation of a gray precipitate. The chief dis- advantage of this method is that the urine always con- tains bodies other than dextrose that can reduce Knapp's reagent, hence a second determination of the reducing power of the urine should be made after removal of the dextrose by fermentation and the value thus obtained sub- tracted from the reduction index of the unfermented urihe ; this makes the method too complicated for clinical work. Quantitative dextrose determinations by fermenta- tion are inaccurate, even in complicated apparatus; for purposes of comparison, using an Binhorn saccharom- eter, the method, nevertheless, is useful. The estima- tion of the dextrose by differential density determinations before and after fermentation may be mentioned; it is not practical for clinical work. The polarimetric method finally gives excellent results, provided all the possible sources of error enumerated above are carefully excluded, and provided it is combined with a quantitative Fehling's test and the elimination of disturbing bodies by fermen- tation (see also qualitative dextrose determinations by polarimetric analysis) . Fehling^s Method.— The solution required is the one described under "qualitative tests" by Fehling's method. The urine should not contain more than 0.5 per cent of sugar; the specific gravity of the urine should, therefore, first be taken and the sugar percentage roughly calculated from it, as described under " specific gravity " in another chapter. It is good practice to always dilute diabetic urine five or ten times. 5 cc. of the alkaline solution and 5 cc. of the cuprie solution are mixed with 40 cc. of water, poured into a beaker and heated just to the boiling point. The diluted urine is allowed to flow into this boiling liquid from a graduated burette. The urine should flow 92 CLINICAL. URINOLOGY very slowly, preferably drop by drop, and the boiling mixture be thoroughly agitated before more urine is allowed to flow in. Gradually, if the urine contains dex- trose, the precipitation of yellow and red oxides of copper will begin, while at the same time the liquid in which this precipitate is suspended becomes discolored. The reaction is complete, i. e., all the copper sulphate is reduced, when the liquid becomes white and clear. This point is not always easy to determine ; in observing the color change, the beaker must be allowed to stand for a little while, in order to give the precipitate time to settle; soon a clear zone will appear at the top of the liquid, in which the color of the solution can be observed. One must not wait too long, however, for otherwise some of the cuprous oxide that is held in solution (by traces of ammonia that develop when urine is boiled with alkali) will become reoxidized to cupric sulphate. The deter- mination should be repeated a number of times and the average of the readings taken. The amount of urine re- quired should be determined accurately to one tenth of a cubic centimeter. The calculation of the sugar percentage in the urine is performed as follows: As each cubic centimeter of the Fehling's solution used corresponds to 5 milligrams (0.005 g.) of dextrose, the 10 cc. of. Fehling's solution that were employed required 10 times 0.005 grams, or 0.05 grams, of dextrose for their reduction. This amount of sugar was contained in the number of cubic centimeters of the diluted urine that were required to decolorize the Fehling's solution. The quantity of undiluted urine that contained 0.05 grams of sugar can be determined by dividing the cubic centimeters of diluted urine by the figure indicating how often the urine was diluted. Know- ing this, one can easily calculate how much sugar, in grams, is contained in 100 cc. of urine, and this, inciden- tally, gives the sugar percentage. TEE GABBOHYBBATES OF THE UBINE 93 Example. — 8.4 cc. of urine diluted six times are re- quired to decolorize the 10 cc. of Fehling's solution; 0.05 grams of sugar are, therefore, contained in 8.4 cc. of the diluted urine and ^ = 1.4cc. of the original urine, 100 cc. of the urine contain '-^^^ = 3.57 grams of dex- trose; in other words, the urine contained 3,57 per cent of dextrose. Pavy^s Method. — It is not, as stated above, always an easy matter to determine the exact point at which the blue of the copper solution completely disappears ; this is the chief objection to Fehling's quantitative method. Pavy has suggested adding ammonia to Fehling's solution, for the latter keeps the cuprous oxide in solution and thus prevents the masking of the color change by the precipi- tate in suspension. The reagent is made by dissolving 20.4 grams of potas- sium hydrate, 20.4 grams of Eochelle salts, 300 cc. of am- monia (sp. gr. 0.88) and 4.158 of copper sulphate in one liter of water. Of this mixture 10 cc. is reduced by 0.5 mg. of dextrose like the original Fehling's solution. In performing this titration the air should be excluded ; a small flask holding 80 cc. is closed with a rubber stop- per with two perforations; through the one hole passes the burette, through the other a glass tube connected with a U tube filled with pieces of pumice stone saturated with dilute sulphuric acid; the purpose of the latter is to bind the escaping fumes of ammonia. 10 cc. of the reagent are diluted with an equal volume of water poured into the flask, and boiled for a few minutes until all the air is driven out of the flask; then the diluted urine is allowed to flow in slowly from the burette until the discolorization of the liquid is complete. The percentage of sugar in the urine is calculated as in Fehling's quantitative method. Haines' Method. — A very stable solution finally is the one given by Haines. Dissolve 8.314 grams of pure copper sulphate in about 400 cc. of water. Add 40 cc. of 9i CLINICAL URINOLOGY pure glycerine, then add 500 cc. of liquor potasse (U. S. P.) ; mix thoroughly and make up to a liter with water. 10 cc. of this liquid when mixed with 50 cc. of ammonia water are reduced, when heated, by 0.01 gram of dex- trose. The solution, if required for accurate work, should be standardized before use by titration with a solution of pure dextrose of known strength. Levulose (levulosuria). — Two levorotatory monosaccha- rides occur in the urine; viz., levulose (fructose) and laiose (Leo's sugar) . The former is the more important of the two. It appears from recent investigations that levulose is excreted together with dextrose in a consider- able number of cases of diabetes; there are also some cases on record, and their number is rapidly growing, of pure levulosuria (fructosuria) or of levulosuria with rela- tively minimal dextrosuria. The exact clinical signifi- cance of levulosuria is not understood ; as the levulose in the blood is always increased (hyper- levulosemia) in levulosuria, we must be dealing with a perversion of the carbohydrate metabolism, and not with a renal or extra- renal process. Alimentary levulosuria (see page 65) is said to be an index of hepatic insufficiency. Qualitative and Quantitative Determination. — If the urine contains much dextrose, the discovery of levulose may not be easy. The urine should always be acid (and diabetic urine that has not undergone decomposition usually is acid), for in alkaline urine dextrose seems occasionally to undergo spontaneous metamorphosis into levulose and mannose, so that here the appearance of levulose may be due to extrarenal processes. Urine con- taining levulose gives a typical reaction called, 8eliwanoff''s Reaction for Levulose. — Equal parts of urine and of fuming hydrochloric acid are mixed in a test- tube ; to the liquid are added a few grains of resorcin ; the mixture is then rapidly heated. If levulose is present the liquid turns deep red and precipitates a dark sediment THE GABBOMYDRATEti OF THE URINE 95 that is soluble in alcohol with a bright red color. Cer- tain rare urinary bodies of a ketose structure give the same reaction, but, clinically speaking, these may be con- sidered negligible quantities. The quantity of levulose can be determined with abso- lute accuracy only by isolating this body from the urine. In clinical work this is virtually an impossible under- taking; the best method is that of Neuburg by precipita- tion of the levulose with methyl -phenylhydrazin and weighing the methyl-levulosazon that is formed. By determining both the cupric reducing powers of the urine and its optical activity, the amount of levorotatory bodies can be estimated with a sufficient degree of accu- ra,cy (see paragraph on polarimetric examination of the urine, on pages 89, 90) . If the urine gives SeliwanofE's re- action before fermentation and not after fermentation, and if it loses both its reducing powers and its levorotatory powers after fermentation, then we may be certain that the levorotation (i. e., the reduction of the dextrorotation as calculated for dextrose) is due to the presence of levu- lose. While pentoses, ^-oxybutyric acid and glycuronates also rotate to the left, they never occur in so large amounts as levulose, so that any considerable differences between the cupric reducing powers of the urine and the dextrorotation in a urine that gives Seliwanoff's reaction may, in general, be attributed to levulose. The following example (quoted from a case report by Rosin and Labaud, Zeitschf. f. Klin. Med. 1902, Vol. 74, p. 182) may illustrate this method of calculating the amount of levulose: Case IV, urine gives marked Seli- wanoff reaction. Titration (Fehling) indicates 7.1 per cent, polarization 6.4 per cent. After fermentation : Seli- wanoff reaction negative, polarization 0. Titration less than 0.1 per cent dextrose. Difference between titration and polarization 0.7 per cent. The urine, therefore, con- tained 6.4 per cent of dextrose and 0.7 per cent of levulose. 96 CLINICAL URINOLOGY Laiose (Leo's Sugar).— This body can be isolated from many diabetic urines ; it is not identical with any of the known sugars ; it is still doubtful whether it is a hexose or a pentose. It is not so sweet as dextrose, has smaller dextrorotary powers and does not ferment so actively ; it has a specific levorotatory index and forms a well-charac- terized compound with phenylhydrazin. In order to iden- tify this interesting body, it must be isolated from the urine (method, see Leo. Virchow's Archiv, Vol. 107, p. 108, 1887) . It is clinically important chiefly because it may lead to confusion with levulose, for when dextrose and laiose are present in the urine together, from 1.2 to 1.8 per cent more sugar (calculated for dextrose) is in- dicated by titration than by polarization. Laiose does not give Seliwanoff's levulose reaction. Isomaltose. — This substance is precipitated from the urine together with other carbohydrates as a benzoeester by Baumann's method (see above). It can be isolated from the urine by a complicated method. Possible that it is formed from urinary dextrose when the latter body is isolated. Clinically, isomaltose is of no importance. Lactose (Milk-sugar), Lactosuria. — This sugar is found in nursing women when there is stasis of the milk. It has also been found in the urine of new-born children with gastric catarrh. There is an alimentary lactosuria of normal subjects (see page 65) following the ingestion of 125 grams of lactose or more in one dose on an empty stomach. In diabetics the ingestion of less lactose leads to the excretion of dextrose. In nursing women large quantities of dextrose (150 grams and more) given in one dose may lead to the excretion of lactose. Lactose reduces metal oxides, forms an osazon (melting point 200° C.) and rotates the plane of polarized light to the right; it does not ferment. In a puerperal woman, therefore, or in a subject living on an exclusive milk diet, the pres- ence in the urine of a reducing dextrorotatory body form- THE GABB0HYDBATE8 OF TEE URINE 97 ing an osazon melting at 200° C. after the urine has been fermented indicates lactose. Buhner'' s Test for Lactose. — The urine is boiled for two or three minutes with an excess of lead acetate ; if lactose is present the liquid turns yellowish brown; the precipi- tate dissolves in ammonia with a brick- red color; on standing, a cherry-red or copper-colored sediment settles at the bottom while the supernatant liquid becomes clear and colorless. Dextrose interferes with this test because it gives the same reaction; any dextrose that may be pre- sent' should, therefore, first be removed by fermentation. Animal Gum (Achrooglycogen, Urinary Dextrin). — This body is always present in the urine. It is precipitated by Baumann's method (see page 61), with benzoyl- chloride as a benzoeester. It is the chief member of a group of bodies called the " unfermentable carbohydrates " that have been mentioned under the caption of "The Uri- nary Carbohydrates as a Group " ; the clinical significance of these bodies has already been discussed. The isola- tion of animal gum is complicated and unnecessary for clinical purposes. Glycogen (Erythrodextrin ? ) .— The urine of diabetics occasionally contains a body that turns mahogany brown with iodine; after removal of the dextrose, by fermenta- tion, such urines on prolonged boiling again reduce Feh- ling's solution. This substance has never so far been found in healthy urine. The urine can be tested for this body by treating it with 5 volumes of alcohol, repeatedly washing the precipitate with alcohol until all the dextrose is removed and performing the iodine reaction with the residue. The residue boiled for half an hour with 10 per cent sulphuric acid should yield dextrose, i. e., should reduce Fehling's solution, ferment, rotate to the right and give a glucosazon melting at 204 to 205° C. The clinical significance of glycogen in the iirine is not under- stood. CHAPTER V TME ACETONE BODIES OF TEE UBINE: ^-OXTBUTYBIC ACID, DI ACETIC ACID AND ACETONE Note on the Physiological Chemistry of the Acetone Bodies of the Urine --The Factors Determining the Excretion of the Acetone Bodies in Various Morbid States. The Clinical Significance of the Excretion of Acetone Bodies. Qualitative Tests and Quantitative Determination of j3-0xy- butyric Acid, Diaoetio Acid and Acetone. NOTE ON THE PHYSIOLOGICAL CHEMISTRY OF THE ACETONE BODIES OB' THE UBINE The mother substance of this group is probably oxy- butyria acid, for this body can be oxidized to diacetic acid and the latter to acetone. The administration, more- over, of /3-oxybutyric acid by mouth is followed in normal subjects by the excretion of diacetic acid and acetone, whereas in abnormal subjects suffering from disorders in which intracellular oxidation is interfered with (diabetes, asphyxia, carbon monoxide poisoning) , the administration of ^-oxybutyric acid is followed by the reappearance of this acid unchanged in the urine. The administration, on the other hand, of acetone is never followed by the excretion of diacetic acid, nor of /3-oxybutyric acid. The interrelationship of the three bodies can be appreciated by studying their empiric formulas: CH3. CH3 CH3 I I I COOH CO CO I I I CHj.COOH CH2.COOH CH3 P-oxybutyrie Acid Diacetic Acid Acetone Under normal conditions the oxidation of yS-oxybutyric acid via diacetic acid via acetone to carbon dioxide and (98) THE ACETONE BODIES OF THE URINE 99 water is complete, so that none of these bodies, or at best only traces of acetone, appear in the urine. Whenever intracellular oxidation, however, is interfered with, there is a copious excretion of acetone, and if the interference with oxidation is very severe, of all three members of this group. It is clear from what has been said above that )8-oxybutyric acid and diacetic acid very rarely ap- pear without acetone ; but acetone may very well appear without its precursors (possible that a portion of the )8-oxybutyric acid and diacetic acid is always artificially converted into acetone in process of determination). The presence of acetone alone indicates a less serious per- version of oxidation than the appearance in the urine of all three members of the acetone group. TTie Factors Determining the Excretion of the Acetone Bodies. — The excretion of acetone cannot always be con- sidered a morbid phenomenon; only when very large quantities are excreted can we speak of pathological ace- tonuria. A normal subject, living on an ordinary mixed diet, excretes small quantities of acetone in the urine every twenty-four hours; the gastro- intestinal secretions also normally contain some acetone (whether this is formed by fermentative splitting of some of the food sugars, or whether this acetone is poured into the stomach and bowel as an excretory product, is not established) ; the expired air always contains some acetone; and finally, every tissue of the animal body (chiefiy the muscles and digestive glands), if submitted to distillation furnishes some acetone. It is a remarkable fact that acetone is excreted in large quantities as soon as the carbohydrates of the food are reduced below certain limits; this interesting discovery aids us in explaining many of the so-called pathological acetonurias on a uniform basis. We know, for instance, that a healthy subject that undergoes a course of fasting soon excretes large quantities of acetone ; this acetonuria, 100 CLINICAL URINOLOGY however, is not due to the withdrawal of fats and meats, i. e., not to the insufficient nutrition of the patient |)er se, but to the withdrawal of carbohydrates. For a patient living on a meat-fat diet alone invariably excretes acetone and often diacetic acid in considerable quantities, and the addition of even small quantities of carbohydrate food to this diet soon stops the acetonuria; in a subject, more- over, that is fasting the administration of carbohydrates in quantities, of course, that are altogether inadequate to furnish enough calories to nourish the patient, suffices to stop the excretion of acetone. A great many different varieties of pathological excre- tion of the acetone bodies have been formulated; as a matter of fact, a critical review of all these varieties seems to show that they can all be attributed to insufficient ad- ministration of carbohydrates, or insufficent utilization of the latter in intracellular metabolism, with resulting inter- ference with the intracellular oxidation of /8-oxybutyric acid, diacetic acid and acetone; for if the latter bodies are not oxidized they must accumulate in the blood and hence appear in the urine. One must imagine that the oxygen of the carbohydrates is required to promote intracellular oxidation, and that withdrawal of carbohydrates by de- priving the cells of this supply of nascent oxygen leads to cellular asphyxia. The source of /3-oxybutyric acid is obscure. All the facts seem to indicate that this body is built up by syn- thesis from certain fragments of disintegrating fat and albumen molecules. The carbohydrates of the food and the carbohydrate radicles of the proteids can certainly not be considered the mother- substance of j8-oxybutyric acid. The Clinical Significance of the Excretion of Acetone Bodies. — Of the pathological acetonurias (including the excretion of diacetic and ^-oxybutyric acids), the most important is diabetic acetonuria. In no other condition are such large quantities of acetone and its congeners ex- THE ACETONE BODIES OF THE URINE 101 creted. There are, of course, many cases of diabetes in which only very small quantities of acetone appear in the urine, i. e., quantities that are no larger than the amounts often appearing in normal urine. There is a certain numerical relationship between the quantity of acetone and of the other members of this group; thus we rarely find diacetic acid unless at least half a gram of acetone is excreted in the course of twenty- four hours; we can also say that ^-oxy butyric acid (and diacetic acid) is almost invariably present if more than two centigrams of acetone are excreted in twenty-four hours. In diabetes, we may find intracellular oxidation reduced to such a point that j8-oxybutyric acid (or diacetic acid) alone is excreted; such cases are, however, exceedingly rare. Diabetic aeetonuria (including the excretion of dia- cetic and oxybutyric acids) is generally influenced by car- bohydrates in the same way as aeetonuria in normal sub- jects, in the sense, namely, that the administration of carbohydrate food has a tendency to reduce or completely stop the excretion of these bodies. There is one excep- tion to this rule, i. e., those severe cases of diabetes in which none of the food -sugar is utilized; here carbohy- drates given by mouth merely increase the glycosuria with- out exercising any effect on the acfetonuria. Possible that in such cases reserve carbohydrate resources are brought into play, and that other bodies vicariously assume the role of the food carbohydrates in inti'acellular oxidation. At all events it is well to remember that, uninfluenced by the diet, the mode of life or medicines, unforeseen varia- tions in the acetone bodies excretion may occur in severe diabetes, and that such variations should be interpreted very guardedly in rendering a prognosis or in instituting treatment. The other pathologic forms of aeetonuria are presumably purely symptomatic and due to insufficient carbohydrate 102 CLINICAL URINOLOGY feeding. Whereas, in the diabetic form there is, in addi- tion to a quantitative perversion, some qualitative per- version of the carbohydrate metabolism, in all the other forms there is merely a quantitative perversion, i. e., an insuflBcient supply of carbohydrate followed by the con- sequences already enumerated ; viz., deficient intracellular oxidation with resulting excretion of acetone bodies. It will be readily understood that in all of the following forms there is insufficient ingestion of food, including car- bohydrates, and that as a result acetonuria develops; the enumeration of all these so-called clinical varieties of acetonuria has some historical interest but nothing more. Different authors have described and named the follow- ing array of acetonurias: acetonuria in fasting ("hunger- acetonuria"), febrile acetonuria, carcinomatous aceto- nuria, gastro-intestinal acetonuria, acetonuria in certain psychoses, epileptic acetonuria, asthmatic acetonuria, acetonuria in eclampsia, acetonuria in poisoning (toxic acetonuria), with many different poisons, acetonuria after narcosis, etc. It is interesting to note that the acetone excretion can he arrested in nearly all of these cases, and — with one excep- tion, that has been spolcen of above— also in diabetic aceto- nuria if carbohydrate pabulum is administered in sufficient quantity. The clinical significance of the acetone bodies, except- ing in diabetes, is not, therefore, so great as one was formerly inclined to believe. The appearance of acetone and its chemical congeners in the urine invariably indi- cates interference with intracellular oxidation, scil. cellu- lar asphyxia, and this suboxidation in the majority of cases is due to insufficient ingestion of carbohydrate pabulum. Even in diabetes in which the formation and excretion of the acetone bodies is in part due to insufficient car bo- THE ACETONE BODIES OF THE URINE 103 hydrate feeding (coupled with inadequate utilization of the carbohydrate administered) , in part to some qualita- tive perversion of metabolism that is probably specific to diabetes, the appearance of acetone bodies in the urine must be interpreted with care. Above all, it is clear that the element of deficient carbohydrate ingestion must always be carefully considered in basing prognostic con- clusions on acetonuria and the excretion of diacetic and j8-oxybutyric acid. As a rule, even in diabetes, the acetone bodies can be made to disappear if more carbo- hydrates are fed; if they do not disappear on this regime then the prognosis is more grave and the danger of im- pending coma may be considered greater. The appearance of large quantities of /3-oxybutyric acid and of diacetic acid in the urine invariably indicates acidosis and calls for energetic alkali- therapy ; one should not forget that acidosis aiid coma in diabetes are pre- sumably not due to any specific toxic effect of these two acids, but are due only to their acid character. The appearance of much acetone, particularly when associated with the excretion of much ammonia (the direct result of the acidulation of the blood -stream, see Chapter IX) is also a bad prognostic sign, and in most cases calls for careful supervision of the case ; in particular it should put us on our guard for coma. Acetone alone may appear in diabetic urine in large quantities for years without coma; on the other hand, there are numerous cases of coma on record in which the urinary acetone excretion was small throughout, or in which acetone disappeared completely from the urine immediately before and dur- ing the coma; here, however, the excretion of the other acetone bodies was increased, and it is probable that their conversion to acetone was arrested owing to sud- den inadequacy of oxidation, and that for this reason the acetone excretion was reduced. In my experience, the rapid disappearance or reduction 104 CLINICAL URINOLOGY of the acetone coupled with a corresponding! 5' rapid in- crease of )8-oxy butyric acid and of diacetic acid (and of ammonia) is a particularly bad omen. I recall no case in which this phenomenon was observed that did not develop coma within forty-eight hours after the decrease in the acetone, and that did not rapidly terminate fatally. THE DETECTION AND THE ESTIMATION OP THE ACETONE BODIES /3-oxybutyric Acid should be looked for only in urine that contains diacetic acid (i. e., gives the ferric chloride reaction to be described below) . Every urine, however, that contains diacetic acid does not, therefore, necessarily contain /3-oxy butyric acid. The detection of very small quantities of this acid is very difficult and uncertain, for in process of determination a portion is always converted into diacetic acid. The presence of the acid can be sus- pected (in diabetic urine — and here alone it is of clinical importance) when the dextrose determination by circum- polarization gives smaller values for dextrose than by titration, for this finding indicates the presence in the urine of some levorotatory substance that reduces the dex- trorotatory index of the dextrose. This levorotation may, of course, also be due to the presence of other bodies, notably pentoses, levulose and glycuronic acid com- pounds ; the differentiation of these various bodies should, therefore, be carried out as indicated in the chapter on the carbohydrates of the urine on pages 89, 90. If oxy butyric acid is present alone, then the quantity can be calculated from its levorotatory power after the dextrose has been removed by fermentation. For ordinary clinical purposes one may accept the presence of j8-oxybutyric acid in the urine if the urine after fermentation is levorotatory, and if it contains considerable quantities of diacetic acid. In order to be sure of the presence of ;8-oxybutyric acid it is necessary to proceed as follows: THE ACETONE BODIES OF THE URINE 105 Method of Kiils.— The urine, after removal of the dex- trose by fermentation, is evaporated to a syrupy con- sistency, treated with an equal volume of concentrated sulphuric acid and distilled. The distillate is collected in a test-tube and allowed to stand for several hours. If /S-oxybutyric acid was present in the urine, crystals of a-crotonic acid will form, for the heating with sulphuric acid withdraws one molecule of water from the /3-oxybuty- ric acid and converts it into a-crotonic acid, according to the formula: CH3.CH{OH).CH2.COOH — HjO = CH3.CH : CH.COOH P-Oxybutyric Acid tt-Crotonic Acid The crystals should have a melting point of 71° to 72° C. Small quantities of a-erotonic acid may not crystallize out; in order to detect them the distillate should be ex- tracted with ether, the ethereal extract poured off, evap- orated to dryness and the melting point of the residue determined. The quantitative determination is difficult. As a rule, the detection of the presence of ^-oxybutyric acid is suffi- cient for clinical purposes and the quantity can be esti- mated according to the above test of Kiilz; this method at least suffices for comparative studies of the urine from the same patient. Unfortunately, we possess no simple clinical method for the determination of j8-oxybutyric acid. The most accurate method employed in scientific work is the following one: One liter of the urine is ex- tracted with alcohol, the alcoholic extract treated with sulphuric acid and ether, the ethereal extract poured off, evaporated, the residue dissolved in water, the solution precipitated with lead acetate, the lead removed by sul- phuric acid, the filtrate brought to a definite volume (20 to 25 cc.) and submitted to polarimetric analysis (Coeffi- cient [8]n = 20°). (See alsoWolpe, Arch. f. exp. Path, n. Pharm. Vol. xxi, p. 138, 1886.) 106 CLINICAL URINOLOGY When the excretion of yS-oxybutyric acid has once be- gun, it rarely disappears again. It is not at all uncommon to find many grams of sodium-oxybutyrate in the urine, as much as 225 grams in the twenty-four hours' quantity having been found. Diacetic Acid.— The detection of this body in the urine is fortunately much more simple than the detection of y3-oxybutyi-ic acid. As diacetic is decomposed within twenty-four hours on standing, the specimen should al- ways be fresh. The most popular test is Gerhardt's ferric chloride reaction, the most accurate one that of Arnold, for the latter responds neither to acetone nor to /3-oxy butyric acid. GerhardVs Ferric Chloride Reaction. — The urine is treated with a 10 per cent solution of ferric chloride as long as a precipitate (consisting largely of phosphates) continues to form. If diacetic acid is present, the addi- tion of ferric chloride beyond this point should produce a typical Bordeaux red color. It is advantageous in doubtful cases to filter off the precipitate that forms and to add the excess of ferric chloride solution to the filtrate. Unfortunately, many other bodies give the same reaction, chief among them salicylic acid, antipyrin, acetates, phenoles, skatole-sulphuric acid, etc., so that a positive test is not always indicative of the presence of diacetic acid. It is well, therefore, always to eliminate the con- tamination of the urine with any of the above drugs be- fore concluding that diacetic acid is being excreted. This is particularly important in view of the deserved popular- ity of salicylates (aspirin, etc.), in the treatment of dia- betes. With practice, certain characteristic features of the reaction can be distinguished that may lead one to decide in favor of diacetic acid; thus the color produced by the salicylates is more violet than Bordeaux-red; that produced by antipyrin more purple ; the latter, moreover, does not appear at once as the diacetic acid color, but THE ACETONE BODIES OF THE URINE 107 requires two or three minutes to appear. The diacetic acid color, moreover, disappears very quickly, whereas the color produced by the other bodies is permanent. Sometimes it is of advantage to isolate some of the dia- cetic acid by acidifying the urine with sulphuric acid and extracting the mixture with ether; the ethereal extract then contains any diacetic acid that may be present. On addition of a ferric chloride solution to the ether extract the Bordeaux-red color should appear, and it should promptly disappear on boiling. Arnold'' s Test. — This test is the more useful of the two, because it does not respond to y8-oxybutyric acid nor to acetone, nor to any of the bodies mentioned above that give Gerhardt's test. The reagents required are a 1 per cent solution of sodium nitrate and a solution of 1 gram of para-amido- acetophenone in 100 cc. of water and acidulated with HCl until the yellow color disappears ; one part of the sodium nitrate solution is mixed with two parts of the acetophe- none solution. Equal parts of this mixture and of the filtered urine are treated with a few drops of ammonia. An amorphous reddish brown precipitate forms, that in itself does not denote the presence of diacetic acid ; if on addition of a large excess of concentrated HCl (10 to 1) a violet purplish color appears, then the urine contained diacetic acid. Quantitative Estimation. — This can only be performed for diacetic acid and acetone together. See, therefore, the quantitative estimation of acetone. Acetone: LegaVs Test. — This is the only test that can be performed with the urine itself. In all the other tests 200 or 300 ce. of urine, after the addition of a little phos- phoric acid, must be distilled and the tests performed with the first 20 or 30 cc. of distillate that pass over. Of the numerous tests that have been described for deter- mining the presence of small quantities of acetone in this 108 CLINICAL URINOLOGY distillate, only the iodoform test of Lieben and Gunning's modification of this test will be described. For clinical work Legal's and Lieben's tests are altogether adequate. To perform Legal's test, the urine is treated with a few drops of a freshly prepared 20 per cent solution of sodium nitroprusside and a few drops of a 10 per cent sodium hydrate solution. The urine will turn red immaterial whether acetone is present or not, and this red color soon fades. If a few drops of dilute acetic acid are now added, the urine turns purple or violet-red, provided acetone is present; if no acetone is present the liquid remains colorless. Lieben^s Iodoform Test.— A few cubic centimeters of the distillate described above are treated with a few drops of strong potassium hydrate solution and iodopotassic iodide (made by dissolving iodine in a strong solution of iodide of potash — the exact concentration is immaterial). If acetone is present a yellow precipitate of iodoform de- velops that can be identified by its characteristic odor, and by the typical microscopical appearance of the iodo- form crystals, i. e., yellow, thin hexagonal plates. This test is very delicate. Unfortunately, other bodies that may occur in the urine also give it, notably all sub- stances of an alcohol or aldehyde structure. For clinical purposes, however, a very strong iodoform reaction may be interpreted to signify the presence of acetone. Gunning, moreover, in order to exclude confusion with alcohols and aldehydes has modified the test as follows : He adds to the urine an excess of an alcoholic iodine solution and of ammonia; if acetone is present, iodoform is again formed. At first the liqviid often turns black from the precipitation of iodine-nitrogen; as this precipi- tate settles and disappears, however, the yellow iodo- form deposit is brought into view. The test is not quite so sensitive as Lieben's test. Quantitative Estimation.— ^ovrasl urine, as already THE ACETONE BODIES OF THE URINE 109 mentioned, always contains traces of acetone — usually about 0.001 grams. In many pathological conditions, and in diabetes particularly, very large quantities of ace- tone may appear in the urine. As much as 6.9 grams, and in one case in the terminal stage of diabetes over 10 grams of acetone have been found in the twenty -four hours' quantity of urine. Diacetic acid as stated above is determined as acetone, for diacetic acid in all the manipulations incident to its quantitative determination is promptly converted into acetone ; even in exact scientific work this conversion is by preference brought about, and the diacetic acid esti- mated as acetone. For clinical purposes it is superfluous to attempt a careful determination of the diacetic acid, our index for a plus or minus of diacetic acid excretion being the relative intensity of the qualitative color tests described above. This is not a very satisfactory state of affairs, but for lack of a simple and accurate clinical method for determining diacetic acid quantitatively we are forced to have recourse to this expedient. The quantitative estimation of acetone is based on the conversion of acetone to iodoform by the action of iodo- potassic iodide. The reaction proceeds in two stages, that may be illustrated as follows : — (1) CH3.CO.CH3 + 3K0I = CH3.CO.CI3 + 3K0H Acetone Tri- iodine-acetone (2) CH3.CO.CI3 + KOH = CH3.COOK + CHI3 Acetate of potassium Iodoform The amount of acetone present in a definite quantity of the urine is determined by the amount of iodine con- sumed in forming iodoform. The method is performed as follows: — Execution. — 100 ec. of urine are distilled. As the pres- ence of ammonia, or of nitrous or formic acids in the distillate interferes with subsequent manipulations (i. e., 110 CLINICAL URINOLOGY the development of iodoform and the titration) , the pas- sage of these bodies into the distillate must be prevented. This may be accomplished by adding a little calcium car- bonate to the urine. It is a little more diflBcult to prevent the passage of ammonia; this can be accomplished, how- ever, by distilling twice, the first time after the addition to the urine of 2 cc. of 50 per cent acetic acid, the second time after the addition to the distillate of 1 cc. of 12 per cent sulphuric acid. To the second distillate is added an excess of a -fV normal iodine solution (12.685 grams of iodine to 1,000 cc. of water) and then drop by drop so much of a 50 per cent solution of sodium hydrate that the color of the iodine disappears completely, and the yellow iodo- form precipitate appears. The mixture is thoroughly shaken and allowed to stand for five minutes. The liquid is now acidified with concentrated hydrochloric acid. The fluid turns brown from the iodine in excess that is liberated. This excess is titrated with a solution of so- dium thiosulphate (iV normal, i. e., containing 24.8 grams of the salt to 1,000 cc. of water), using a 1 per cent starch solution as an indicator. The starch should not be added until the liquid has become faint yellow on addi- tion of a sufficient quantity of the thiosulphate. As soon as the mixture turns blue, the thiosulphate is slowly added drop by drop until the blue color disappears. Every cubic centimeter of the rs normal sodium thiosul- phate solution corresponds to a cubic centimeter of the -iV normal iodine solution. The number of cubic centi- meters of the former employed in the titration must, therefore, be subtracted from the number of cubic centi- meters of iodine solution added to the distillate in order to give the amount of iodine consumed in the reaction. The figure obtained multiplied by 0.967 indicates the number of milligrams of acetone present in the amount of urine employed for the estimation. CHAPTER VI THE BLOOD- AND BILE-PIGMENTS OF THE URINE The Chemical Relationship Between Blood- and Bile-pigments. The Blood- Pigments of the Urine — Hemoglobin (Hemoglobinuria and Hematuria), Hematin, Hematoporphyrin, their Clinical Significance, Detection and Quantitative Estimation. The Bile-pigments (Choluria) — The Factors Determining their Excretion, their Clinical Significance (hematogenous and hepatogenous icterus), their Detection and Quantitative Estima- tion. The Bile Acids of the Urine — Their Clinical Significance, Detec- tion and Quantitative Estimation. Urobilin (Urobilinuria) — Its Forma- tion, Clinical Significance and Determination. Melanin (Phymato- rhusin) — Its Clinical Significance and Detection. The urinary pigments derived from the blood and the bile may be discussed together, because the latter are derived from the former, hemoglobin being the mother substance of the bile-pigments. Hemoglobin itself is, properly speaking, an albumen, and it gives many of the tests described under serum albumen. It occurs in the blood, and occasionally in the tu'ine, as oxyhemoglobin, a body that on heating to 80° C. coagulates and is decomposed into an albuminous body and an iron - containing pigment, hematin. The latter body when treated with concentrated sulphuric acid splits ofE iron and is converted into a body called hematopor- phyrin, and this substance is an isomer of the most im- portant bile-pigment; viz., bilirubin. From bilirubin, finally, are derived the various bile -pigments, including urobilin, that are often formed in the urine. The chemical relationship of the blood- and bile-pigments is, therefore, readily established. THE BLOOD -PIGMENTS The most important blood -pigments that appear in the urine are hemoglobin, hematin and hematoporphyrin; the (111) 112 CLINICAL UBINOLOGY most important bile-pigments are bilirubin, biliverdin and urobilin. The less important blood-pigments urorubro- hematin, urofuseohematin, and the unimportant bile-pig- ments biliprasin, bilifuscin and bilihumin, are of no clin- ical significance and will not be discussed. The bile acids are so closely related to the blood- and bile- pigments that they must properly be included in this chapter.* Hemoglobin (Methemoglobin).— When blood enters the urine somewhere in the urinary passages, we speak of hematuria. Hematuria may be renal or extrarenal, and these terms explain themselves. Hematuria is com- mon in acute inflammatory affections of the urinary pas- sages, in neoplasm, ulcerations, traumata, etc., of the kidneys, ureters, bladder or urethra. In hematuria, if the urine is fresh and if it is filtered at once, the filtrate may be quite colorless ; if the urine is allowed to stand, hemo- lysis of red blood - corpuscles occurs, some hemoglobin goes into solution and the filtrate is hence colored red. In hematuria, needless to say, the sediment always con- tains large numbers of red and white blood - corpuscles (see Urinary Sediments) , and if the admixture of blood is considerable, or if the urine has stagnated, coagulates of fibrin (see Fibrinuria) that may be white or red. The urine in hematuria presents certain macroscopic characteristics that differ according to the quantity of blood voided, the time that has elapsed since the hemor- rhage occurred, and the origin of the blood. Recent and profuse hemorrhages from any part of the urinary tract color the urine uniformly pink or red ; if the blood stagnates for a time in the lower urinary passages then the hemoglobin undergoes degeneration, the urine ap- pears darker, usually reddish brown or brown if the hemorrhage was profuse, smoky or even greenish if it was slight. *See a monograph by the author on the conversion of hemoglobin into bile-pig- ments and bile acids, Phila. Med. Journ. Jan. 11 and 18, 1902. THE BLOOD- AND BILE-PIGMENTS 113 The admixture of coagula, calculi, parasites, etc., to the bloody urine often aids in localizing the hemorrhage and determining its origin. Thus microscopic coagula representing molds of the uriniferous tubules, in other words, casts covered with blood-corpuscles or blood-cor- puscle derivatives indicate a renal hemorrhage, whereas large, massive clots indicate vesical hemorrhage; these clots usually become infected with pus-germs, so that hematuria is almost invariably accompanied by some de- gree of pyuria. In chronic vesical hemorrhages (para- sites, calculus, papilloma, etc.), the elements character- istic of chronic cystitis (pus, desquamating epithelia, pathogenic germs, etc.) usually accompany the blood and clots. In hemorrhaga of the neck of the bladder and the prostate the first urine passed is often free from blood, whereas the last portion contains it; and in ureteral hemorrhage precisely the reverse is the rule. In Jiemoglobinuria, blood-pigment alone passes through the renal epithelia and enters the urine; this may occur in any disorder accompanied by great hemolysis, i. e., disintegration of circulating red blood-cells with libera- tion of hemoglobin. If hemolysis is slight, hematuria does not occur. Hemolysis is a normal process and constantly occurs during life; the quantities of hemoglo- bin liberated at one time are, however, so small that they are arrested in the liver (and presumably also in other tissues), undergo conversion to bile-pigment and are ex- creted through the bile channels into the bowel or the gall bladder. If, in certain pathological conditions, much disintegration of red blood-corpuscles occurs in a closed cavity so that the liberated hemoglobin cannot at once enter the blood-stream, then again we have a conversion of blood - pigment into bile - pigments or their isomers, in situ, and the urine remains free from hemoglobin; here, also, we have an increased excretion of bile-pig- ments through the liver, and occasionally through the H 114 CLINICAL URINOLOGY kidneys (see below, under hepatogenous and hematoge- nous icterus). Excessive hemolysis of circulating red blood-corpuscles with hemoglobinuria is seen in certain forms of poisoning, notably with potassium chlorate, pyrogallic acid, some mushrooms, sulphureted hydrogen, arseniated hydrogen, etc.; it may also follow the transfusion of blood of one species into the veins of another; it is seen in severe cutaneous burns and in certain infectious diseases. In addition, there are two disorders sui generis of un- known etiology, known as paroxysmal hemoglobinuria and epidemic hemoglobinuria of children in which the urine paroxysmally contains large quantities of hemoglobin in solution. The excretion of hemoglobin in paroxysmal hemoglobinuria maybe precipitated by a variety of factors, chief among them cold, next postural changes, and finally a variety of apparently insignificant agencies, as tying a ligature around one finger, psychic shocks, a slight blow, etc. Hemoglobinuria of this character is seen only in predisposed subjects. The disorder seems to be hereditary and often runs in families. There is a certain resem- blance between paroxysmal hemoglobinuria and cyclic (intermittent) albuminuria (see Chapter I) , and occasion- ally attacks of hemoglobinuria alternate with attacks of transitory albuminuria; in the latter case one might sup- pose that the pouring of free hemoglobin into the blood was so slight that the hematin radicle was arrested (to be ultimately excreted as bile-pigment) , whereas the albumi- nous radicle entering the blood as a foreign body was at once excreted through the kidneys. In both conditions the urine may at times contain renal elements. In hemoglobinuria the filtered urine is always red and the sediment may contain none of the morphological ele- ments of the blood. In severe cases the blood-red color of the urine is unmistakable while the urine is fresh* if only small quantities of hemoglobin are excreted, so that THE BLOOD- AND BILE-PIGMENTS 115 the urine is colored pink, the nature of the pigment must be carefully determined, for other bodies may impart a similar color to the urine; if the urine is old, disintegra- tion of the blood-pigment may occur and the blood-red color of the urine be obliterated. In either case it be- comes necessary to perform certain chemical tests for the detection of hemoglobin, of whicih the following are the most simple and the most reliable for clinical purposes. Tests foe Hemoglobin. — (1) Boiling.— li the urine contains hemoglobin, a brown coagulate forms on boiling; this usually floats on the surface of the urine; it can be decolorized by shaking the urine with sulphuric acid- alcohol. (2) Heller's Test. — Five drops of a 10 per cent sodium hydrate solution are added to half a test tube of urine, and the mixture boiled. If hemoglobin is present it will be converted into hemochromogen, a brown-red pigment, and will hence color the precipitate of earthy phosphates and carbonates that forms brown, brown -red or blood- red. In alkaline urine this test may fail, as the phos- phates may already be precipitated. (3) Almen's Test. — 5 cc. of old oil of turpentine and 5 cc. of a fresh tincture of guaiac are added to 10 cc. of urine, the mixture shaken and extracted with 5 cc. of ether; if hemoglobin was present the ethereal layer will be colored blue. If the urine contains many pus-cells the same reaction may be obtained. Occasionally a white ring forms at the plane of contact between the turpen- tine-guaiac and the urine ; this ring, if the urine is allowed to stand for a time without shaking, turns blue if hemo- globin is present; on shaking the mixture the whole liquid turns blue. (4) Teichmann's Test. — This is the most reliable and the most delicate test of all. The hemoglobin is first pre- cipitated by boiling, or, as in Heller's test, by boiling the urine with alkali; better still, by treating the urine with a 116 CLINICAL URINOLOGY 5 per cent tannin solution until a drop of the tannin pro- duces no: further precipitation. The precipitate is filtered off, washed with water and allowed to dry in the air. A small portion of the dry residue is placed on a glass slide together with a small crystal of sodium chloride and a drop of glacial acetic acid ; a cover slip is then placed on top of the mixture and the slide held over a small flame until everything is dissolved; then the liquid is boiled for about a minute, care being taken that the evaporating fluid is constantly replaced by glacial acetic acid, added drop by drop; (all that is needed is to place a drop of the acetic acid at the edge of the cover slip — it will at once be drawn underneath) . As soon as the fluid turns brown, if hemoglobin is present, in about one minute at the longest, it is allowed to cool slowly, and the slide examined uiider the microscope. If hemoglobin is present crys- tals of hemin, so-called Teichmann's crystals, of char- acteristic form and arrangement will be seen. (5) Spectroscopic Test. — The urine is rendered acid and diluted ; on direct spectroscopic examination it shows the spectrum of oxyhemoglobin (Plate, No. 1) or of methe- moglohin (Plate, No. 2) . If now ammonium sulphide is added to the urine the spectrum of gas -free, reduced hemoglobin appears (Plate, No. 3). If the urine is first treated with caustic alkali, as described in Heller's test, the spectrum of hemochromogen appears (Plate, No. 4) . Hematin. — This derivative of hemoglobin is occasion- ally found in the urine ; it is presumably derived from the disintegration of hemoglobin in the urinary passages, or after the urine has been voided; it, therefore, partakes of the same clinical significance, as far as we know, as hemoglobin, and it is usually found in old urine or in bloody urine that has been stagnating in the renal pelvis or the bladder. When bloody urine is heated the hemo- globin often splits up into hematin and an albuminous radicle, the latter coagulating and tearing the hematin THE BLOOD- AND BILE-PIGMENTS 117 down with it. Hematin has also been described in the urine in a case of sulphuric acid poisoning. The presence of hematin is recognized by the spectro- scopic examination of the urine. The spectra of hematin and of methemoglobin (see above) are very similar (Plate, Nos. 2 and 5) ; in order to distinguish the two spectroscopically, the urine should first be treated with ammonia, and the filtrate examined with the spectro- scope, then ammonium sulphide should be added and the mixture examined .again; if hematin is present, spectra that are characteristic for alkaline hematin (Plate, No. 6) and for reduced hematin (Plate, No. 4) will appear in suc- cession on the addition of these reagents. In view of the subordinate clinical importance of this body, other tests and the methods of quantitative estima- tion will not be given. Hematoporphyrin. — Normal urine, it appears, always contains minimal quantities of this pigment. The exact source and mode of formation of hematoporphyrin are not known, for it does not always appear in the urine when there is widespread destruction of red blood -cor- puscles with liberation of hemoglobin; certain special factors, it seems, must therefore be operative that lead to its excretion in certain definite morbid states. At one time the theory was widely accepted that hematoporphy- rin was formed from hemoglobin in the bowel by the action of certain intestinal bacteria, and that the pig- ment was subsequently absorbed into the circulation and excreted in the urine; it has been shown, however, that this at all events is not the common mode of formation and that hematoporphyrin in those diseases in which it is excreted in large quantities in the urine is formed in the blood. The largest quantities of hematoporphyrin are excreted in acute poisoning with sul phonal, trional and tetronal, or after the prolonged use of these drugs. I (together 118 CLINICAL URINOLOGY with J. Tyson)* reported a case of hematoporphyrin- uria following sulphoual poisoning, iu which on three successive days 1.683, 1.013 and 0.098 grams of hem- atoporphyrin were passed in the urine. As fully 35.1 grams of hemoglobin must be disintegrated to furnish 1.683 grams of hematoporphyrin (for this calculation see the original article) , and as an average adult individual weighing from 60 to 70 kilograms contains about 600 grams of hemoglobin (Salkowski), one can say that in this patient fully one-seventeenth of the body hemoglobin was destroyed and wasted in the urine in the form of hematoporphyrin . Hematoporphyrin has also been found increased in a variety of disorders that are characterized by functional disorders of certain ductless glands and of the blood- forming organs, i. e., in Addison's disease, in Graves' disease, in primary anemias, and also in gout and gouti- ness, and in lead-poisoning. (Here we must remember the fact that chronic lead -poisoning produces a disease pic- ture that is often indistinguishable from gout.) In all of these states, then, we have peculiar metabolic anomalies, and we may hence be justified in regarding the appear- ance of hematoporphyrin in the urine as an evidence of abnormal hemoglobin catabolism. The clinical significance of hematoporphyrinuria is hence twofold; in the first place, it should always direct our attention to the possibility of sulphonal, trional or tetronal poisoning; if this cause can be excluded we should look for evidence of thyroidism or adrenal gland disease, we should carefully examine the blood for evi- dence of a primary anemia, we should look for the clini- cal evidence of goutiness and for possible exposure to lead intoxication. If none of these causes can be dis- covered, we should be on our guard for other evidence of obscure metabolic disorders. •Transactions of the Association of American Physicians, 1902. THE BLOOD- AND BILE-PIGMENTS 119 Qualitative Tests for Hematoporphyrin. — Urine contain- iug hematoporphyrin has a typical Burgundy-red color. On standing it becomes darker and may turn almost black. In order to determine that the pigment is hema- toporphyrin it must be isolated as follows : The urine is precipitated with barium mixture (containing one part of saturated barium-nitrate solution and two parts of con- centrated baryta water) until no further precipitate forms on the addition of the reagent. The sediment now con- tains the bulk of the hematoporphyrin, together with any other pigments that may be present; the precipitate is filtered off, washed and extracted with dilute (3 per cent) hydrochloric acid alcohol. This extract should be red- dish or pink in color, should fluoresce and should turn dark on heating. The most characteristic feature is the spectroscopic appearance (see Plate, Nos. 5, 7 and 8) ; hematoporphyrin in acid alcoholic solution showing a narrow absorption band in the yellow between C and D, and a second broader band between the yellow and green between D and E (Plate, No. 7) . If the solution is now rendered alkaline, four bands will appear (see Plate, No. 5), one between C and D, two between D and E, and a fourth very dark band between C and F, i. e., between the green and the blue (Plate, No. 8) . Quantitative Estimation. — 100 cc. of urine are rendered alkaline with a few drops of a 10 per cent sodium hydrate solution, treated with a 10 per cent solution of calcium chloride until no further precipitate forms, the reaction of the liquid being kept alkaline by the addition of soda solution. The dark red precipitate is filtered off, re- peatedly washed with water until the washings are free from chlorides (i. e., no longer give a clouding with dilute silver nitrate solution) , then washed with absolute alcohol to get rid of the water, and, finally, extracted with dilute HCl-aleohol at a temperature of about 40° C. From the pink alcoholic extract the pigment is precipi- 120 CLINICAL URINOLOGY tated with water after the liquid is first carefully neutral- ized with dilute ammonia. The flocculent precipitate is gathered on a filter, washed free from chlorides, the water removed with alcohol, the alcohol with ether, the residue dried at 115° to a constant weight and the weight deter- mined. In addition to hematoporphyrin, this precipitate contains certain inorganic salts. In order to determine the quantity of the latter, a weighed portion of the total precipitate is incinerated and the amount of ash deter- mined. The difference between the total weight of the precipitate and the calculated amount of total ash (as determined in a portion of the precipitate) represents organic matter, i. e., hematoporphyrin, BILE -PIGMENTS (cHOLURIa) The appearance of bile constituents in the urine is always pathdlogical . The most common cause of cho- luria is stasis of bile in the liver from stenosis or occlu- sion of bile-channels with diapedesis of bile from the bile- channels into the lymph -spaces and blood-vessels of the liver. In addition, as I have shown (1. c), bile-pig- ments and bile-acids can be formed in any other parts of the organism than the liver, so that icterus need by no means always be of hepatic origin. The controversy in regard to the existence or non-existence of so-called "hematogenous" icterus in contradistinction to "hepatog- enous" icterus has been waging for many years. Some writers claim that icterus with the passage of icteric urine, etc., is always of hepatic origin, even in those cases in which large quantities of hemoglobin are liberated else- where in the body (hematomata, bruises and extravasa- tions, ecchymoses, sanguinolent exudates, etc.) ; for, they argue, the disintegrating blood-pigment is carried to the liver and is there, and there alone, converted into bile- pigment; in cases, then, in which very much hemoglobin THU BLOOD- AND BILE-PIGMENTS 121 is brought to the liver at once, the bile becomes sticky and is viscid from the large excess of liver excretion, occludes the bile' channels owing to its changed physical properties and in this way favors diapedesis of bile with icterus and choluria. The adherents of this view claim that all icterus is hepatogenous. Other authors cannot reconcile the appearance of yellow and green discolorations in bruises and ecchymoses, the appearance of pigments that are isomeric, if not identical with bile -pigments, in hematomata, etc., with this view. They claim that some of the bile-pigments and bile-acids may be formed in situ, and hence they speak of "hematogenous" icterus. The clinical interpretation of choluria must needs vary according to the views of the observer in regard to the existence or non-existence of hematogenous, scil., non- hepatogenous icterus. For if one does not concede that bile- constituents can be formed in other portions of the body tha,n the liver, then the appearance of bile -pig- ments and bile -acids .must invariably denote some de- rangement, mechanical or functional, of the liver. If, on the other hand, onecdncedes that bile constituents can be formed in the tissues at large, then choluria indicates nothing more than excessive disintegration of hemoglobin somewhere in the body, and the diagnosis of a liver affection can only be made from other direct and remote signs that point to the liver or its channels as the seat of the trouble. In view of the fact that the degree of icterus and the quantity of bile-pigments and bile-acids is never so great as in biliary obstruction, the appearance of large quanti- ties of bile constituents will almost invariably signify liver trouble ; reasonable doubt should be entertained only when small quantities of bile-pigment or bile-acids appear in the urine and when there is reason to suspect from other urinary signs (see preceding paragraphs) that somewhere in the body red blood -corpuscles are under- going disintegration. 122 CLINICAL URINOLOGY Urine containing much bile-pigment has a characteris- tic greenish yellow color that is particularly marked in the foam that forms on shaking the tube. The urine may show no characteristic icteric color and the foam, nevertheless, appear distinctly yellowish green. Urine containing urobilin is usually brownish green or even dark brown (see below). Many drugs containing chryso- phanic acid, as, for instance, rhubarb, senna, etc, color the urine greenish brown; such urine, of course, does not give bile-pigment reactions. The addition of caustic alkali solution to such urine, moreover, colors it intensely red. After the ingestion of santonin the urine is always very yellow ; this yellow color can also readily be changed to red by caustic alkali. Tests foe Bile -pigments in the Ueine. — Only fresh urine should be examined, for if the urine is allowed to stand the bilirubin may readily undergo oxidation and form products that do not give the reactions to be presently described. Bilirubin is the only really important bile^ pigment of the urine, for biliverdin, bilifuscin and bilipra- sin are derivatives of bilirubin that develop secondarily. Bile-pigments are occasionally also found in the sediment and in some cases it is expedient to submit the urinary sediment to bile-pigment tests. From what has been said above, it is apparent that the chemical differentiation of the differe^nt bile-pigments of the urine is of no clinical significance whatsoever. Of the many bile-pigment reactions that have been described, only two will be given; viz., the test of Gme- lin and the test of Huppert, They are the most reliable and the most practical of all, (1) Gmelin^s Test. A few cubic centimeters of strong nitric acid are poured into a test-tube and about three times as much urine allowed slowly to flow down the sides of the inclined test-tube. If bile-pigment is pres- ent in the urine, a number of multi-colored rings will form THE BLOOD- AND. BILE-PIGMENTS 123 in the urine immediately above the plane of contact be- tween the urine and the acid. The lowest ring is green (biliverdin) , and this green ring alone is pathognomonic for bile -pigments; unless it is present the presence of bile-pigments should not be diagnosed. Above the green ring is usually seen a yellowish ring, and above this a reddish one. Occasionally a fourth bluish ring is seen over the reddish ring, but only if the urine contains much indican, the coloring matter in the last ring being indi- gotine. The nitric acid employed in this test should be yellow rather than white; it is well, therefore, to use an old acid that has been exposed to the light. A mixture of equal parts of nitric and sulphuric acid may also be used to advantage. Gmelin's test is not very sensitive; while a positive test, therefore, always indicates the presence of bile-pig- ment in the urine, a negative test does not necessarily exclude the presence of small quantities of bile-pigment. The presence of iodin (in patients taking iodide of potassium) or of urobilin in the urine may mask the color of the rings, for on addition of nitric acid to such urine the whole liquid is liable to turn dark brown. The presence of much albumen by causing the formation of a thick albumen ring also masks the reaction; albumen should, therefore, first be removed. Great care should, finally, be exercised not to contaminate the urine with alcohol or ether, for even a few drops of these bodies lead to the formation of multi-colored rings, in perform- ing Gmelin's test, one of which is green. Bosenbach has devised a very convenient modification of Gmelin^s tests. A piece of filter paper is moistened with the urine to be tested and a drop of yellow nitric acid placed on the moistened area; a number of multi- colored concentric rings form around the drop, and again the green ring alone is characteristic for bile-pigment; 124 CLINICAL URINOLOGY (2) HupperVs Test.— This method is more tedious and probably not so well adapted for clinical purposes as the above, but it is more sensitive and reveals the presence of much smaller quantities of the bile-pigments. It is carried out as follows: ; Ten cubic centimeters of the urine are treated with lime-water, the precipitate separated by filtration and brought from the filter into a test-tube, containing 5 per cent sulphuric acid- alcohol; the mixture is heated to boiling. If bile-pigments are present the liquid will turn green. Here again the production of a green color is important; one must remember that indican mny pro- duce a yellowish or reddish color, 'whereas bile- pigments invariably produce a green tint. THE BILE -ACIDS OP THE URINE Closely related to bile-pigments pathogeneticallyand, as we have seen, chemically and clinically, are the bile-acids. They can, therefore, properly be discussed in this place. Bile-acids always appear together with bile-pigments, and their excretion in the urine possesses the same clini- cal significance as the excretion of the latter. They, like the bile-pigments, are in great part formed in the liver, but they can also be formed elsewhere in the organism when hemolysis of blood-corpuscles occurs and the lib- erated hemoglobin stagnates (compare page 112) . For- merly the appearance of bile-acids in the urine was always interpreted to signify some affection of the liver or its ducts leading to stasis of bile an(i dispedesis of bile-pro- ducts into the blood. The excretion of bile-acids was signalized as the chief index of the hepatogenous charac- ter of all forms of icterus. Since I was able to show, however, that bile-acids are always present in the nor- mal blood * and, since it has become recognized that the ♦"Some Experiments on the Intermediary Circulation of the Bile-acids," Am. Journ. of Med. Sciences, Jan., 1902, and Pflueger's Archiv fin Physiologie, 1902. THE BLOOD- AND BILE-PIGMENTS 125 bile-acids normally perform an intermediary eiiculation from the bile-ducts via the intestine, the lymphatics, the systemic blood back to the liver, since, finally, the extra- hepatic origin of bile-acids under stated conditions (see above) has become recognized, the appearance of bile- acids in the urine can no longer be interpreted to indicate hepatic disease alone. As a matter of fact, bile-acids are excreted in numerous conditions in which the liver is not at all affected. The appearance of bile-acids in the urine, like the appearance of bile-pigments, is always pathological. The two chief bile-acids that appear in the urine are taurocholic and glycocholic acids; the former containing sulphur, the latter not. Both are derivatives of cholalic acid that combines with taurine to form the one, with glycocoll to form the other bile-acid. The two acids are present in bile in the form of their sodium salts. Tauro- cholic acid predominates in human bile. The role of the bile-acids in the formation of certain urinary nucleo-albu- mens ("mucin") has been discussed on page 11. Their role in the development of cystinuria is discussed under the heading "Cystin" on page 191. Until recently the recognition of the bile -acids in the urine direct was uncertain, as the most classical reaction for bile -acids; viz., Pettenkofer's reaction (see below) is given by a number of other normal ingredients of the urine. It was necessary, therefore, to isolate the bile- acids from the urine. Of late years, however, a method for detecting bile -acids that was described many years ago by Hay has acquired the prominence it deserves ; it is so simple and so delicate, and can be performed with such facility in the urine, that it promises to become the most popular clinical method for detecting bile -acids in the urine. Hay^s Test.— li a pinch of sublimed sulphur is placed upon the surface of normal urine it will float on top ; if 126 CLINICAL UBiNOLOGY the urine contains even minimal quantities of bile -acids, the sulphur powder will almost instantaneously drop to the bottom of the test tube in the form of a powdery sedi- ment, at the same time the balance of the sulphur float- ing on top of the liquid will coalesce to form a thin, granular, pellicle. If the tube is lightly tapped with the finger more of the surface sulphur will drop to the bot- tom in the form of a fine powder. The more bile- acids the urine contains, the more pronounced and the more prolonged this reaction. Even if the urine contains only traces of bile-acids, the pellicle will form within a min- ute, and, on shaking, a fine pulverulent precipitate will settle. In doubtful cases an inexperienced observer may to advantage compare a tube of the pathological urine with a tube of normal urine or of water, and observe the difference in the behavior of these tubes when sulphur is sprinkled on top of the liquid. The only other urinary bodies that ^ive a similar re- action are certain medicaments that may perchance enter the urine after their administration by mouth, chief among them the balsams (notably terebinth), chloroform and carbolic acid. If a patient, therefore, is known to have taken any of these drugs or their derivatives, a positive Hay reaction is not pathognomonic for bile-acids. In such a case and for purposes of more accurate study, the bile-acids must be isolated from the urine and one of the following two tests may be performed : — Isolation of the Bile-acids from the Urine. — Albu- men, if present, is removed by coagulation, the coagulate filtered off, the filtrate evaporated to dryness, the residue extracted with absolute alcohol, the alcoholic extract diluted with water and precipitated with basic lead ace- tate and ammonia. The precipitate is filtered off, ex- tracted with absolute alcohol and the extract filtered while still hot. This solution contains any bile-acids that may have been present in the urine. THE BLOOD- AND BILE-PIGMENTS 127 In order to identify bile-acids Hay's test may now be performed, or, if larger quantities are suspected to be present, Fettenkofer^ s test; finally an attempt may be made to cause crystallization of the bile- acid sodium salts as so-called Platner^s crystals. Pettenkofer^s Test (as modified by Udranszky). In the original Pettenkofer test cane-sugar and sulphuric acid were employed. Since it was shown that the sugar was converted into furfural by the action of the sulphuric acid, and that the furfurol gave rise to the typical color reaction with bile-acids, Udranszky advised the use of furfurol in the first place. One cubic centimeter of the urine is treated with one drop of a -h per cent solution of furfurol and slowly superposed upon 1 cc. of concen- trated sulphuric acid, care being taken by immersing the tube in cold water to prevent too great heating of the mixture. A purple color appears at the plane of contact, that gi'adually extends upward into the superposed solu- tion; on standing, the color turns bluish. In alcoholic solution a green fluorescence is seen. The pigment gives a typical spectrum; viz., two bands, the one near F, the other between D and E, near E. Platner^s Crystals. — An attempt may be made to obtain crystals of the sodium salts of the bile- acids by evaporating the solution with soda, extracting the residue with absolute alcohol, and adding enough ether to pro- duce a slight clouding. On standing, Platner's crystals of the sodium salts of the bile-acids form. These may further be identified as above or by testing their effect on the heart of a curarized frog (retardation) . UROBILIN This pigment is derived directly from bile -pigments, and hence, from blood pigments. In the great majority of cases urobilin is formed from the bile -pigments that 128 CLINICAL URINOLOGY are poured into the bowel by the action of intestinal bacteria; occasionally some of this urobilin of enteric origin is absorbed into the blood- stream and subsequently excreted in the urine. Certain authors in addition claim another origin for this pigment, namely, that it may be formed anywhere in the organism where hemolysis occurs with liberation and stagnation of hemoglobin. Some claim that urobilin is formed under these circumstances, via the bile -pigments, whereas others argue that it is formed in place of the ordinary bile -pigments. This con- troversy cannot be considered settled for the present. So much is known, that fresh normal urine rarely con- tains much urobilin. It does, however, contain urobilin- ogen, the chromogen from which urobilin is formed by the action of sunlight. Normal urine that has been allowed to stand exposed to light may contain from mere traces to 20 or 30 milligrams of urobilin in the total twenty -four hours' quantity. Pathologically, the excretion of urobilin may be great. As urobilin is largely formed in the bowel by the reduc- ing action of certain putrefactive bacteria, the amount excreted in intestinal putrefaction is usually considerable. In the same sense it is always completely absent from the urine of the new-born, because the enteric tract is normally sterile for the first few days of life. As the mother- substance of the urobilin of enteric origin are the bile -pigments, urobilin is often completely absent from the urine when there is complete occlusion of the common duct from whatever cause with exclusion of bile- pigments from the bowel. As soon as the bile -ducts again become permeable and bile can flow into the intes- tine, urobilin is formed and appears in the urine; this is particularly apt to happen after the sudden removal of an obturator, when large quantities of retained bile suddenly gush into the bowel. Simple stagnation of bowel contents (chronic constipa- THE BLOOD- AND BILE-PIGMENTS 129 tion, intestinal stenosis and occlusion) , some of the older writers to the contrary notwithstanding, is not followed by increased urobilinuria, excepting, peculiar to say, in constipation occuring in typhoid fever and after opium. Possible that the typhoid poison and the opium render the bowel wall abnormally permeable to the fsecal urobilin. In all cases of pleiochromia, i. e., in conditions in which there is an abnormally large formation of bile-pig- ments, as in cardiac lesions with hepatic stasis, the uri- nary urobilin is augmented. In many febrile disorders the excretion of urobilin is greatly increased; here it is difficult to decide what the biogenesis of the urobilin may be. Whether one is deal- ing with abnormally increased urobilin -formation from bile-pigment in the bowel, or with abnormal toxic perme- ability of the bowel wall or with some perversion of the bile -pigment -forming function of the organism, so that urobilin is formed in place of bilirubin, remains altogether a matter of theory. The clinical fact remains that in sepsis, in scarlatina, in pyemia, in acute articular rheu- matism, in pneumonia, in phthisis, erysipelas, malaria, typhoid, the excretion of urobilin in the urine is often enormously increased. In many disorders that are accompanied by great de- struction of red blood-corpuscles, as in scurvy, purpura, intoxication with hemolytic poisons (antifebrin, antipyrin, pyrodin) ; in disorders in which blood is extravasated into the tissues (cerebral apoplexy, hemorrhagic pleuritis, peritonitis, hematoma, ecchymoses, hemorrhagic infarct, etc.) , and in pernicious anaemia, urobilinuria is a common symptom. Here one is, in all probability, dealing with a hematogenous and not an enteric urobilinui'ia ; for one is almost forced to the conclusion that in all such cases urobilin is formed in the tissues, and not in the bowel, by the reducing action of the body-cells, or of the intra- cellular ferments they secrete. 130 CLINICAL URINOLOGY Both the atrophic and the hypertrophic forms of he- patic cirrhosis are almost invariably accompanied by'flood- ing of the blood and tissues with urobilin, so-called "urobilin icterus," and the excretion of urobilin in the urine. In such cases one must assume that the bile-pig- ment-forming function of the liver is perverted in the sense that urobilin is formed in place of bile-pigments; or one may assume that the bile-pigments that enter the blood-stream owing to the obstruction of biliary channels within the liver are converted into urobilin in the tissues by the same process of cellular reduction that has been postulated above. Whereas urobilinuria is a very com- mon symptom in certain liver affections, one is, neverthe- less, not justified in diagnosing an hepatic lesion from urobilinuria alone without other clinical evidence of a liver lesion; for urobilinuria, as we have seen, may also be due to a great variety of other causes. Urobilin has so far never been found in the blood, even in cases of so-called urobilin icterus. It is possible that this is due to technical difficulties that render the detec- tion of minute quantities of urobilin in the relatively small amounts of blood that can at best be removed intra vitam impossible; possible, however, also (as some experiments with kidney-pulp seem to show), that uro- bilin is formed in the kidneys from circulating bile -pig- ments. This might be considered a disintoxication pro- cess, for urobilin is less toxic and more readily diffusible than bile-pigments proper. If urobilin is really formed in the kidneys and not in the tissues— and I, personally, am inclined to the belief that it may be formed both in the bowel and the tissues at large, including the kidneys —then urobilinuria would be a symptom merely of a general cholemia. Clinical Significance of UroUlinuria.— At best, there- fore, as far as I am able to conclude, the symptom urobi- linuria possesses essentially the same clinical significance THE BLOOD- AND BILE-PIGMENTS 131 as the excretion of bile-pigments in the urine, provided one is willing to accept the facts as true that indicate the formation of bile-pigments, including their derivative by reduction urobilin, anywhere in the body and not in the liver alone. The factors that would determine the excre- tion of urobilin would then differ in degree and not in kind from the factors that cause the excretion of the other bile -pigments. The whole subject of urobilinuria, it will be seen, is an obscure one and the precise significance of urobilin is not understood. The clinical importance of this body has been greatly overestimated. It is important to recognize it in urine that does not give the ordinary bile-pigment reactions; but one should be guarded, in my belief, in drawing other clinical conclusions from its presence in the urine than one would draw from the appearance of the ordinary bile-pigments giving Gmelin's test. Detection of Urobilin. — Urine containing urobilin is usually very dark and occasionally, although not always, furnishes a brownish green or very yellow foam on shak- ing. The dark color of the urine alone is, of course, no indication of the presence of urobilin, as many other ' bodies, notably indican derivatives, may also darken the urine. If the freshly voided urine contains much of the chromogen of urobilin (urobilinogen) it may be very light at first; such urine, however, will soon turn dark on stand- ing exposed to light; this property is not pathognomonic for urobilin either, as other urinary bodies can produce the same color change (see pages 133, 140, 156) . If Gmelin's test is performed in urobilin urine, a brown or orange band is usually formed at the plane of contact between the nitric acid and the urine, but no green zone will appear. The appearance of a brown ring can hardly be considered proof of the presence of uro- bilin, but rather proof of the absence of bile-pigments 132 CLINICAL URINOLOGY proper; occasionally the brown ring is not formed at all even if the urine contains much urobilin. In urine of a greenish brown or dark brown hue known not to contain bile-pigments the presence of urobilin can often be determined direct; occasionally the direct tests fail and then it may become necessary to first isolate the urobilin and to perform one of the tests given below with the isolated product. To test the urine direct for urobilin we have two useful clinical tests; viz. — Fluorescence. — The urine is treated with an excess of ammonia and a few drops of a 1 per cent alcoholic solution of chloride of zinc. If the urine contains con- siderable quantities of urobilin a beautiful green fluo- rescence will be seen. Another simple method to produce fluorescence in urobilin urine is to treat the urine with a solution of potassium iodide containing some free iodin, and then rendering the urine alkaline with a solution of potassium hydrate. Spectroscopic Examination, — Urine containing other pigments than urobilin is not well suited for this test. It is always best to employ urine that has been exposed to light for a few hours, for such urine usually gives a much more definite urobilin spectrum than fresh urine, owing to the fact that the urobilinogen of the fresh urine is con- verted into urobilin by light. If the urine contains blood- or bile -pigments in considerable quantities, and one does not wish to attempt the tedious isolation of any urobilin the urine may contain, then the other pigments can be precipitated from the urine by mercuric sulphate and the spectroscopic test for urobilin performed with the filtrate. For this purpose 10 cc. of urine may be treated with 5 cc. of a solution of mercuric sulphate, made by dissolving 5 grams of mercuric oxide in a mixture of 20 cc. of H2SO4 and 100 cc. of water; the urine thus treated is allowed to stand for five minutes, the precipitate filtered THE BLOOD- AND BILE-PIGMENTS 133 off, and the spectroscopic test made with the filtrate, (absorption spectrum of mercuri - m'obilin : band to the right of E in the green) . If the urine itself is examined direct, preferably after acidulation, the spectrum of acid urobilin depicted on Plate, No. 9, will appear. If the urine is very dark it should be diluted. If the urine contains only very little urobilin so that the above tests are not positive, the urobilin should be extracted and the tests be performed with the extract. Urobilin can be extracted from urine by amyl- alcohol, chloroform, phenol, ether or acetic ether. The most popular and probably the best solvent is amyl -alcohol. Extraction of urobilin with amyl- alcohol: Ten to twenty cubic centimeters of the urine are treated with a few drops of hydrochloric acid and vigorously shaken with 10 cc. of amyl-alcohol. The tube is allowed to stand for a few minutes in a vertical position and the layer of amyl-alcohol decanted off. The alcoholic extract is sub- mitted to spectroscopic analysis. It is then treated with a few drops of a 10 per cent zinc chloride solution in ammonia -alcohol; if urobilin is present a green fluo- rescence will appear. MELANIN (SYN. PHYM ATORHUSIN ) This is a pigment of unknown origin ; it is presumably derived from blood-pigment and may hence properly be discussed in this chapter. It is of subordinate clinical importance, occurring only occasionally in the urine of patients suffering from melanotic neoplasms. Such pa- tients sometimes void urine that turns dark on standing, and the addition of certain oxidizing agents as nitric acid, chromic acid, bromine, ferric chloride, etc., to the urine often brings out the dark color more rapidly ; the dark color may inversely often be made to disappear by the addi- tion to the urine of reducing agents. If the urine is kept 134 CLINICAL URINOLOGY in an air-tight vessel darkening may be altogether pre- vented. The dark pigment may be confounded with indi- can or with urobilin, as urine containing the chromogens of these pigments also turns darker on standing and when it is treated with oxidizing agents. In order to rule out this error the urine must be tested for these two pigments as described elsewhere. Alkapton urine must also be ruled out (see alkaptonuria on page 156). If the presence of all these bodies can be ruled out, and if the urine turns very dark, even black, on addition of the oxidizing agents mentioned above, or on standing in contact with the air, then the presence of melanin in the urine becomes very probable. Special tests for melanin are the following: The addi- tion of bromine to urine containing melanin causes the precipitation of a yellow sediment that gradually turns black. On addition of a concentrated solution of ferric chloride to a few cubic centimeters of the urine, the liquid turns gray; on adding more of the reagent a precipitate of phosphates and pigment occurs that is resoluble in an excess of the concentrated ferric chloride solution. Clinical Significance.— Th.Q appearance of melanin in the urine must be interpreted with care; it is by no means pathognomonic for melanotic neoplasms, for in marantic and cachectic individuals who are not afflicted with such tumors melanin may occasionally appear in the urine, and inversely patients may be afflicted with melan- otic sarcomas or carcinomas and still not void melanin in the urine. In combination with the clinical evidence of sarcoma of the skin or the eye, or of carcinoma, the ex- cretion of melanin in the urine may, nevertheless, be con- sidered valuable corroborative evidence of the melanotic character of the neoplasm. CHAPTER VII TSJS AROMATIC CONSTITUENTS OF THE URINE Classiflcation, According to Pathogenesis and Chemical Constitution, into Four Groups. 1. The Conjugate Sulphates — Phenoles, Carbolic Acid, Cresole, Pyroeateohin, Hydroquinon, Indol (Indoxyl), Indicanuria, Skatol ; Their Clinical Significance, Detection and Quantitative Estima- tion. 2. The Conjugate Glycuronates — Their Physiological and Patho- logical Chemistry, Clinical Significance, Detection and Estimation. 3. The Compound Glycocolls — Hippurie Acid and Phenaceturic Acid; Their Clinical Significance, Determination and Detection. 4. Tlie Aro- matic Oxyacids — The Alkaptonic Acids (Homogentisic and Uroleucinic Acids), Alkaptonuria, Its pathogenesis and Clinical Significance. When albumen undergoes disassimilation a number of aromatic bodies, i. e., substances containing the benzene ring (CeHe) are formed. These bodies may be contained preformed in the food, they may be formed in the gastro- intestinal tract by the action of the digestive ferments, or by the action of bacteria, or they may be formed by intracellular digestion of the body albumen in the tissues. Normally only small quantities of these bodies are found in the urine ; pathologically they are often in- creased, particularly when the disassimilation of the albu- mens proceeds along abnormal channels. Hence the aromatic constituents of the urine are found increased chiefly in gastro- intestinal putrefaction and in perverted albumen catabolism due either to bacterial invasion, gan- grene, intoxication by different poisons or in metabolic disorders sui generis of unknown origin. Occasionally these aromatic bodies appear in the urine as such, e. g., in the form of certain aromatic oxyacids (alkaptonuria, see below) ; as a rule, however, they ap- pear combined with one of three other substances; viz., (1) sulphuric acid, forming the group of conjugate sul- (135) 136 CLINICAL URINOLOGY pJiates, chief among them iiidol and its derivative indican; (2) glyeuronic acid, forming the group of conjugate gly- curonates (see also "Mucin"), a class of substances that are related to the carbohydrates and that, as set forth at length in discussing glycosuria, may lead to confusion with sugars; (3) glycocoll, or amido-acetic acid, form-, ing the group of compound glycocolls, chief among them hippuric and phenaeeturic acid. One can conveniently, therefore, divide the aromatic bodies of the urine into four groups; viz, in the order of their clinical importance: (1) The conjugate (or "aro- matic" or "ethereal" sulphates; (2) the conjugate gly- curonates; (3) the compound glycocolls; (4) the aromatic oxyacids. (1) THE CONJUGATE SULPHATES The chemical constitution of this important group of urinary bodies may be illustrated diagrammatically by the following structural formulte: — „;^0H c,;^OH SO2 SO2 —OH —OR (Hi,S04) Sulphuric Acid Conjugate Sulphate R = aromatic radicle The conjugate sulphates include certain phenoles (cre- sole, pyrocatechin, hydroquinone) , indol (and its important derivative indican) and skatol. As a rule, these bodies appear in the urine as the potassium or sodium salt of the ethereal sulphate ; in other words , the free hydrogen atom in the above structural formula is substituted by K or Na. Indol and skatol, as will be presently shown, are not eliminated as indol- or skatol -sulphuric acid salt, but undergo preliminary oxidation to indoxyl and skatoxyl and appear in the urine as the potassium or sodium salts of indoxyl- sulphuric acid (indican) or of skatoxyl-sul- phuric acid. The quantity of conjugate sulphates excreted in the AROMATIC CONSTITUENTS OF THE URINE 137 urine varies according to the chavacter of the food, the ingestion of certain drugs (e. g., carbolic acid) that are excreted as ethereal sulphates, the degree of intestinal putrefaction, the character of the putrefactive bacteria and the existence of putrid pus cavities, gangrenous tissues, etc., in the body. On an ordinary mixed diet a healthy adult excretes an amount of conjugate sulphates that represents about one -tenth of the total sulphate ex- cretion, the other nine-tenths being furnished by mineral sulphates (see chapter on the sulphates of the urine). Expressed absolutely, this corresponds to from 0.15 to 0.3 grams of SO3. Exactly where in the body the synthesis of the aro- matic conjugate sulphates occurs we do not know. It is very interesting to note that large quantities of aromatic bodies, e. g., indol or phenole, when they enter the blood- stream first combine with all the available sulphuric acid, and that later, when the supply of sulphuric acid is exhausted, they combine with glyeuronic acid; it is clear from this that in excessive putrefaction not only the con- jugate sulphates but also the conjugate glycuronates are increased. Detection of Aromatic Sulphates.* — The presence of aromatic sulphates in the urine can be detected as follows: The aromatic (ethereal) sulphates, in contra- distinction to the mineral sulphates, are not precipitable by barium chloride. The urine is, therefore, rendered acid with acetic acid and treated with a 10 per cent solu- tion of barium chloride; the precipitate of barium sul- phate derived from the mineral sulphates is filtered off; the filtrate now contains any aromatic sulphates that may be present. The latter can be split into sulphuric acid and the aromatic radicle by boiling with hydrochloric acid, and the liberated sulphuric acid can be re- *Por the quantitative estimation of the conjugate sulphates, see the chapter on "Inorganic Constituents of the Urine" under "Sulphates," on page ]87. 138 CLINICAL URINOLOGY moved from the mixture by precipitation as barium sul- phate with barium chloride. The resulting liquid now contains the different alcohols. The formation of a ba- rium sulphate precipitate after the preliminary removal of the mineral sulphates, as described above, indicates the presence of aromatic sulphates; from the bulk of the precipitate certain rough conclusions can be drawn in regard to the quantity of aromatic sulphates ; this applies particularly to comparative work. Phenoles.— The most important member of this group is cresole, i. e., methylphenole. CeHjOH C0H4.CH3.OH Phenole Cresole Phenole itself is less abundant than cresole; the other urinary phenoles, pyrocatechin and hydroquinone, are of small clinical importance, and are, moreover, never pres- ent in large quantities. Their empiric formula is the same; viz., C6H4(OH)2, with the difference that the hydroxyl groups occupy different relative positions in the benzene ring. Phenole and Cresole. — These two bodies always occur together, the cresole preponderating. The chemical dif- ferentiation of cresole from phenole is possible, but clini- cally unimportant. Of the three possible isomeric cre- soles, para-, ortho- and meta- cresole, the para- cresole is the most abundant in human urine; ortho -cresole is usually excreted in minimal quantities and meta-eresole does not appear at all ; the latter has been isolated from the urine of horses. Phenole and cresole are never found in the urine of the new-born, an indication that the intestine is sterile, nor are they found in the urine of animals which are fed on sterile food from birth ; these facts point to the intestinal origin of a part of these bodies. The factors that other- wise determine the excretion of these aromatic bodies have already been enumerated; viz., coprostasis, particu- AROMATIC CONSTITUENTS OF TEE URINE 139 larly in the lower portion of the small intestine and in the colon, as in obstructive lesions of the bowel and in peri- tonitis. In pyemia, gangrene, in phosphorus poisoning and in diabetes the excretion of phenole and cresole is increased; the genesis here, as in all other cases in which aromatic bodies appear in the urine, is abnormal degra- dation of albumen. Benzole given by mouth increases the excretion of phenole and cresole; the same applies to tyrosin given per OS. The excretion of these bodies seems to be greater on a vegetable than on an animal diet. When the urine contains much indican it also always contains much phenole and cresole; the" reverse, however, is not the case. The average daily excretion of these bodies on an ordinary mixed diet does not exceed 0.03 grams. In the morbid conditions outlined above it may become enor- mous, as much as 0.5 grams of phenole having been isolated from the urine in twenty-four hours. The detection of phenole and cresole necessitates pre- liminary splitting of the phenole-sulphate salt and the liberation of the aromatic radicle. This is done by dis- tilling a large quantity of the urine (500 or 1,000 cc.) with enough sulphuric acid to make a 5 per cent solution; the distillate contains the phenoles and should respond to the following tests: (1) On addition of so much bromine that the liquid remains yellow, a yellow crystalline precipitate of tribromphenole should form on standing; (2) Millon's reaction should be positive, i. e., on addition of Millon's reagent* a rose-red color should appear; (3) the addition of a few drops of sodium hypochlorite should develop a blue color; (4) heating with iodine and sodium hydrate solution to 60° C. should cause the precipitation of dark red amorphous triiodine phenole. * Millon's Reagent.— Het&Wia mercury is dissolved in an equal portion, by- weight, ot 63 per cent nitric acid ; one volume of this solution is diluted with two volumes of water, and the precipitate that forms filtered off. The clear filtrate is the reagent. 140 CLINICAL URINOLOGY The latter reaction can also be utilized for a fairly accurate quantitative method of determining the phenoles as a group, the amount of phenoles being calculated from the amount of iodine required. The method is compli- cated. The reaction is illustrated by the equation : CeHsOH + 61 = CsHjIsOH + 3HI Phenole Iodine Triiodine phenole Hydroiodie Acid Pyrocatechin and Hydroquinone. — Urine containing either of these bodies turns dark on standing. Both bodies are excreted if carbolic acid (phenole) is given by mouth, Pyrocatechin is a normal constituent of the urine, as it is derived from pyrocatechuic acid, a substance that is con- tained in many of the vegetables we eat; thQ quantities present in normal urine are, however, so small that they do not markedly influence the color of the urine, even if it is alkaline and is allowed to stand a long time. Ben- zole given by mouth also leads to the excretion both of pyrocatechin and of hydroquinone. The two bodies never appear as such, but always in the form of the conjugate sulphates. If urine, therefore, turns dark on standing, the pres- ence of pyrocatechin or of hydroquinone in abnormally large quantities may be suspected. This phenomenon is usually indicative of carbolic acid poisoning, or of the accidental swallowing of benzole or of pyrocatechin or hydroquinone. One other aromatic body that is excreted in the form of a compound glycocoll, viz., homogentisic acid (see alkaptonuria below), also causes the urine to turn dark on standing. The genesis of this body is, how- ever, altogether different from that of the bodies under discussion, and the differentiation of the two not difficult. The detection of "alkaptonic acids" will be discussed below. Detection and Isolation. — If pyrocatechin is present the urine turns green on the addition of a few drops of ferric chloride solution; the addition of ammonia and a little AROMATIC CONSTITUENTS OF THE URINE 141 tartaric acid causes the urine to turn violet, and the addi- tion of more ammonia cherry-red. Hydroquinone can be recognized by boiling the urine with a dilute solution of ferric chloride; this liberates quinone, that can readily be recognized by its character- istic odor. The great medico-legal importance of these bodies in suspected carbolic acid (phenole) poisoning renders it occasionally necessary to isolate them from the urine. The urine for this purpose is acidulated with HCl, heated for half an hour on the water bath, and after cooling extracted with ether. The ethereal extract is shaken with a sodium-carbonate solution, the ether separated from the watery liquid with a separating funnel, the ether evapo- rated and the residue treated with small quantities of a saturated sodium- chloride solution. This salt solution takes up any pyrocatechin or hydroquinone that may have been present in the urine. The salt solution is diluted with water and distilled until all volatile phenoles are removed, i. e., until about two-thirds of the water have distilled over. The residue is again extracted with ether, the ethereal solution separated, the ether evaporated, the residue dissolved in water and precipi- tated with lead acetate. This precipitate contains the pyro- catechin, while the hydroquinone remains in the solution. The precipitate is decomposed with sulphuric acid, the lead sulphate filtered off, the filtrate extracted with ether, the ether separated. From this ethereal extract pyro- catechin crystallizes in prisms. The solution containing the hydroquinone is treated with sulphuric acid to remove surplus lead, the lead sulphate filtered off, then heated with barium carbonate to remove the excess of sulphuric acid, the barium sulphate filtered off, the filtrate extracted with ether and the extract allowed to stand. Hydroquinone crys- tallizes out in the form of rhomboid crystals that can be re- crystallized from benzole and have a melting point of 160°. 142 CLINICAL URINOLOGY Indol (indoxyl, indican). — Indol, like the phenoles just described, is a product of the putrefaction of albumen somewhere in the body. Whenever indican appears in the urine there is consequently also always much phenole (see above). In the new-born, indol (indican) is always absent from the urine; this is presumably due to the fact that the intestine remains sterile during the first days of life. After the first few days of life, however, indol (indi- can) is never absent from human urine. The quantity varies within wide limits even in health, much depending upon the character and quantity of the diet and the char- acter of the gastro-intestinal bacterial flora. A meat diet invariably increases the urinary indol (indican) , whereas a vegetable diet, other things being equal, decreases it. On a mixed diet, from 2 to 50 milligrams of indican may be isolated from the urine in twenty- four hours. Indicanuria. — Pathologically, the indol excretion (Indi- canuria) is increased in all conditions that favor abnor- mal intestinal putrefaction, particularly if there is at the same time some obstruction to the normal passage of the bowel contents onward through the intestine, — in other words, if there is coprostasis from any cause. Thus indi- canuria is common in mechanical obstruction of the bowel (incarceration, invagination, knuckling, stenosis) , in ileus, in acute and chronic peritonitis, in ulcerative processes leading to cicatrization or functional stenosis (typhoid ulceration, amoebic, tuberculous lesions of the bowel, etc.) , in gastrectasis, in gastroptosis and enter op tosis, in lead colic, in perityphlitis and appendicitis. In certain lesions not affecting the bowel contents, indican may also often be found increased, for instance, in empyema, in gan- grene, in putrid bronchiectasis, in breaking-down carci- noma of different organs, — in short, wherever there is putrid decomposition of body albumen and entrance into the blood-stream of the products of this decomposition; for indol, the mother- substance of indican, is one of them. AROMATIC CONSTITUENTS OF THE URINE 143 It will be seen, therefore, that the excretion of con- siderable quantities of indiean in the urine is a very com- mon phenomenon, that considerable quantities may appear in health, and that very many different pathological con- ditions may determine excessive indicanuria. All that one can conclude from a great increase of the urinary indiean is that there is abnormal putrefactive degradation of albumen somewhere in the body. In the overwhelming majority of cases the indol is formed in the bowel, and the administration of a purge will usually cause a diminution of the urinaiy indiean. One must never forget that a simple coprostasis may lead to copious indicanuria, and one should be very careful, indeed, in interpreting this sign. As the entrance of indol into the blood- stream is almost invariably accompanied by the entrance of other putrefactive products of albumen decomposition, and as indol and its congeners are toxic, and, it appears, particu- larly irritating to the renal epithelia, the fluctuations in the excretion of indiean may, under carefully defined conditions, be considered a valuable index of one form of auto -intoxication. Indicanuria in this sense may become a guide for treatment and an index of the result of treat- ment directed toward a correction of the abnormal putre- faction and its consequences. Herein lies the chief clinical value of a study of the indiean excretion. Indiean is the sulphate of indoxyl in combination with potassium or sodium; indoxyl in its turn is derived from indol by intracellular oxidation. The relation of the three bodies may be illustrated by the following scheme : CH C(OH) CCO.SOaK) / \ O / \ H2SO4 / \ CeHi CH ► C„H, CH »- CcH, CH \ / \ / K \ / NH NH , NH CxHtN CjHoN.OH CsHoN.O SOaK Indol Indoxyl ' Indiean 144 CLINICAL VRINOLOOY By referring to the schematic formula of all the con- jugate sulphates given above, it will be seen that the aromatic radicle (R) in this case is indoxyl, while the H of the other hydroxyl group is substituted by K or Na; thus OH O.K / / SO2 SO2 O.R O.CsHuN Conjugate sulphate Indiean General formula Indoxylpotassium sulphate R = aromatic radicle Other products of indol that often appear in the urine are indoxyl in combination with glycuronic acid (see below) and indoxyl in combination with isatin forming in- digo red and a number of pigments of indefinite chemical constitution, but all containing the indol -ring and known by the names of urohsematin, urorubin, urrhodin, indiru- bin, indigo-purpurin, etc. The clinical significance of all these bodies is nil. Qualitative Tests for Indican. — JaiWs Test. — This test and Obermeyer's test (see below) are both based on the principle that indican on oxidation is converted into a blue pigment; viz., indigo,* that is soluble in chloroform. In Jaffe's test, equal parts of urine and of concentrated hydrochloric acid are mixed in a test-tube; to the mix- ture are added a few cubic centimeters of chloroform, and, drop by drop, a 10 per cent solution of sodium-hypo- chloi'ite. (The ordinary "chlorinated lime" of commerce, if it is fresh, contains enough hypochlorite to perform the oxidation of indican; if no pure sodium hypochlorite is readily available this preparation may, therefore, conveni- ently be employed instead.) On the addition of each drop of hypochlorite the liquid should be thoroughly shaken. If indican is present, the layer of chloroform *The indigo derived from the oxidation of indican was formerly believed to be identical with vegetable indigo; the latter, however, is a glucoside of the formula C20H31NO17. AROMATIC CONSTITUENTS OF THE URINE 145 that settles at the bottom of the vessel will turn blue. Care must be taken not to add too much hypochlorite, as otherwise the oxidation is carried too far and the indigo is converted into a colorless compound. If the urine contains albumen the latter must first be precipitated with lead acetate and the test be performed with the fil- trate ; if the urine is very dark it may also first be decol- orized by treating it with lead acetate and removing the lead -pigment precipitate by filtration. Obermeyer^s Test. — Chloroform often forms an emul- sion with the urine, so that the droplets of chloroform that may contain the indigo in solution remain in suspen- sion and the blue color is masked. Obermeyer, there- fore, advises always to precipitate the urine with lead acetate and to perform the test with the filtrate that now no longer contains any of the disturbing substances. He uses a solution of ferric chloride in concentrated hydro- chloric acid as the oxidizing reagent. His reagent con- tains 3 grams of ferric chloride to one liter of concen- trated fuming HCl. The filtrate from the lead acetate precipitate is shaken vigorously with an equal volume of the above reagent, and the mixture then shaken with a few cubic centimeters of chloroform; this extracts any indigo that may be present. The blue chloroform solu- tion promptly settles at the bottom of the test-tube. Care should be taken not to add too much lead acetate, as otherwise lead chloride forms on addition of the reagent to the filtrate, and the lead chloride settling to the bottom renders the layer of chloroform cloudy and may hence obscure the blue color. Instead of using a hypochlorite solution or Obermeyer's reagent, one can also oxidize the indican with dilute chlorine or bromine water. Quantitative Estimation of Indican. — Obermeyer's test can be utilized for the quantitative determination of indi- can. A definite quantity of urine is treated with Ober- 146 CLINICAL URINOLOGY meyer's reagent as above and repeatedly extracted with chloroform. In the chloroform solution the amount of indigo can be determined (a) by weighing, (6) colorimet- rically, (c) spectrophotometrically, (a) By weighing. — The chloroform extract is poured into a weighed porcelain dish, the chloroform driven off on the water-bath, the residue dried at 110° C to constant weight and weighed. For clinical purposes this method is sufficiently accurate, particularly if daily fluctuations in the indican excretion are to be estimated. (&) Colorimetrically. — A number of solutions of indigo in chloroform of known percentage are prepared and dis- tributed in tubes of equal caliber. By comparing the urinary chloroform extract, after it has been brought up to the volume of the control solutions, with the standard tubes the amount of indigo-blue in the urine can be esti- mated by a simple calculation. (c) Spectrophotometrically (F. Miiller). — This method requires a complicated apparatus, a spectrophotometer, and considerable practice. It is very accurate, — more accurate than necessary for clinical work and too time- consuming to warrant its application in the clinical lab- oratory. A good description of the method will be found in Huppert, "Harnanalyse," 1898, p. 692 ff.). Skatol (skatoxyl) .— Skatol is indol in which a hydro- gen atom of one of the CH groups is substituted by a methyl radicle, CH3, thus: CH CCCHs) / \ / \ C6H4 CH C6H4 CH \ / \ / NH NH Indol Skatol As indoxyl is formed from indol by intracellular oxida- tion, so skatoxyl is formed from skatol; like indoxyl, it combines with sulphuric acid and appears in the urine as the sodium or potassium salt of skatoxyl-sulphuric acid ; AROMATIC CONSTITUENTS OF THE URINE 147 like indoxyl, an excess of skatoxyl combines with glycu- I'onic acid and appears in the urine as conjugate glycu- ronate (see below). Skatol is a normal constituent of the bowel contents and imparts to the stools their charac- teristic faecal odor. It is probable that its conjugate sul- phate is also always present in the urine like indican, but this proposition is difficult to prove, because its presence is revealed by the appearance of a red pigment when the indican tests are performed, and this red pigment may be . indigo-red and due to the presence of indican. In diabetes, it appears, skatoxyl-sulphuric acid is excreted in large quantities. The clinical significance of this body is virtually the same as that of indican (see above) . Detection.— The presence of much skatol may be sus- pected if the urine turns dark red or violet on standing. In making Jaffe's indican test the urine turns dark red or violet as soon as the HCl is added. Nitric acid alone, or with the addition of a little potassium nitrate, produces a cherry-red color; ferric chloride with or without hydro- chloric acid also a cherry -red color — the urine, therefore, turns cherry-red on performing Obermeyer's test. On boiling the urine, the pigment separates out and can be taken up with ether. Unfortunately, all these tests are not characteristic for skatol derivatives, for they are also given by indigo-red, an indol derivative. In order to differentiate the two, the urine must be treated as in Jaffe's test, the chloro- form extract evaporated, the residue treated with zinc powder and indol or skatol liberated; if enough indol or skatol are present a few crystals may be obtained that differ as to melting point, indol melting at 52° C, skatol at 95° ; if both are present the melting point will lie be- tween 52° and 95° C. More exact and delicate methods for recognizing the presence of skatol derivatives are known ; in view of the small clinical importance of this 148 CLINICAL URINOLOGY body they will not be given in this place ; they can be found in Huppert "Harnanalyse," pp. 169 and 170. (2) THE CONJUGATE GLYCURONATES This group of urinary compounds is attracting much attention of late years and promises to acquire consider- able clinical significance. For this reason a brief discus- sion of the physiological chemistry of glycuronic acid and its compounds appears useful to me. Note on the Physiological Chemistry of the Glycuro- nates. — Glycuronic acid maybe considered an oxidation product of dextrose, the relation between the two being illustrated by the following structural formulae: — CH2OH COOH I +0 I (CH0H)4 *■ {CH0H)4 + H2O COH COH Dextrose Glycuronic acid The chief source of glycuronic acid is the glycogen of the muscles, liver, etc., and it seems probable that the glycogen is first converted into dextrose, this in part into glycuronic acid, and the latter finally by further oxida- tion into carbon dioxide and water. At all events, the administration of glycuronic acid to animals seems to spare the liver glycogen. Grlycuronic acid, therefore, is an intermediary product between glycogen (dextrose) and the terminal products of its disassimilation ; viz., CO2 and H2O. The appearance of glycuronic acid in the urine of diabetics has hence been interpreted to signify the incomplete oxidation of the blood-sugar. Some authors go so far as to claim that a "delayed Fehling test" in urine that is free from dextrose, but that contains glycuronic acid, is evidence of deficient sugar destruction and should be considered a warning of impending gly- cosuria and diabetes. The excretion of glycuronic acid AROMATIC CONSTITUENTS OF THE URINE 149 would then be a precursor of glycosuria. If the other factors that can determine the excretion of glycuronic acid and the glycuronates, to be discussed below, can be ruled out in such a case, then this view has much in its favor. Another possible source of glycuronic acid is cartilage (chondroitin) and chondroitin- sulphuric acid (see under " Mucin " ) , for the latter can be split into glycuronic acid and glycosamiu via chondrosin by a process of hydrolytic disassociation. Glycuronic acid does not appear free in the urine, but only in combination with certain aromatic bodies, phe- noles, indoxyl and skatoxyl (see conjugate sulphates) , and certain drugs and poisons, as naphthol, camphor, toluol, menthol, oil of turpentine, morphine, chloral and others. Some of these bodies appear only in combination with glycuronic acid; others, like the phenoles, indoxyl and skatoxyl, combine first with all the available sulphuric acid to conjugate sulphates and with glycuronic acid, only after the supply of sulphuric acid is exhausted. The resulting conjugate glycuronates partake of the character of glucosides (see chapter on "Carbohydrates of the Urine"). The conjugate glycuronates are monobasic acids. They all rotate the plane of polarized light to the left. The individual compounds of this group, however, show in- dividual peculiarities in this respect. By hydrolysis the glycuronates can be split into free glycuronic acid ^,nd the component alcohol. In some cases this disassociation occurs on gentle heating or even at ordinary temperature, in others boiling with dilute acids, or even heating with superheated steam is necessary. Some of the conjugate glycuronates reduce cupric oxide in alkaline solution; others do not. Some are precipitable by lead acetate, and others are not. These differences are determined by the character of the alcohol in combination with the glycu- ronic acid molecule. 150 CLINICAL URINOLOGY Free glycuronic acid and its salts rotate the plane of polarized light to the right, whereas, as stated above, the aromatic conjugate glycuronates are levorotatory. As the free acid, its salts and esters, can reduce metal oxides, glycuronates may readily be taken for sugar. This con- fusion may be still more enhanced if phenylhydrazin compounds are made, for the free acid forms a crystal- line phenylhydrazin compound that melts between 114° and 115° C. Glycui'onic acid is not, however, fermentable. The clinical significance of the conjugate glycuronates is chiefly this. In the first place, they may indicate, like the conjugate sulphates, that there is abnormal disassimi- lation of albumen somewhere, that this disassimllation is putrid in character, and presumably due to the invasion of the gastro- intestinal tract or of the tissues with putre- factive germs that lead to the formation of aromatic degradation products. The appearance of much glycuro- nate together with much conjugate sulphate indicates more absorption of putrefactive products than the appearance of conjugate aromatic sulphates alone. In diabetes the appearance of glycuronates may be interpreted to signify deficient sugar destruction, even in the absence of dextrose, care being taken always to ex- clude excessive intestinal putrefaction or stasis, or the ingestion of the drugs and poisons enumerated above as a possible cause of the urinary glycuronate excretion. In a negative sense, the excretion of conjugate glycuro- nates may lead to confusion with sugar excretion, for, as shown above, free glycuronic acid and its salts as well as the conjugate glycuronates are optically active, reduce Fehling's solution and form crystalline phenylhydrazin compounds. With ordinary care, of course, this error can be avoided; at the same time, I believe that the simple browning of Fehling's solution or a "delayed Fehling reaction" must often be attributed to the presence of glycuronates and not of sugars in the urine. AROMATIC CONSTITUENTS OF THE UEINE 151 Indirectly the excretion of large quantities of glycuro- nates may indicate that such poisons as naphthol, cam- phor, turpentine, chloral, etc., have been swallowed; in a sense, therefore, the excretion of these bodies has some toxicological and medico-legal significance. Finally, the presence of glycuronates should always be thought of in interpreting discrepancies between the cupric reducing power of urine and its power to rotate the plane of polarized light. For the levorotatory gly- curonates will reduce the dextrorotation of any dextrose that may be present. The other levorotatory bodies that must be considered in the same connection are pentoses, )3-oxybutyric acid and levulose. The detection and differ- entiation of these bodies has been discussed at length in the chapter on "The Urinary Carbohydrates," pages 89, 90. Detection of the Conjugate Glycuronates. — Unless the urine contains dextrose, glycuronates should only be looked for in urine that is levorotatory. If the urine con- tains dextrose the latter must first be removed by fer- mentation. In order to demonstrate the presence of glycuronie acid, the latter must be liberated from its compounds. This can be done as follows: The Orcin Test. — 50 ee. of urine are acidified with enough dilute sulphuric acid to make about a one per cent sulphuric acid solution and boiled for two or three minutes in a porcelain dish. The mixture, while still warm, is treated with a few drops of an over-saturated solution of orcin in concentrated HCl and boiled again for one or two minutes. If glycuronates are present the liquid should turn red. If the orcin test is not positive after boiling two minutes it is useless to boil longer, for any glycuronie acid that might be present would be de- composed by prolonged boiling with HCl. To be quite positive that the red color is due to the presence of gly- 152 CLINICAL URINOLOGY curonates the liquid should be examined spectroscopically, when certain characteristic absorption bands will be found in the spectrum (see Salkowski, Zeitschr. f . phys. Chemie, 1899, No. 27). For clinical work the spectroscopic iden- tification is superfluous, as no other important bodies give a positive orcin reaction. In diabetic urine, if the urine is levorotatory after the dextrose is removed by fermentation, one may be dealing with /3-oxybutyrie acid or glycuronates. If the urine still reduces Fehling's or Nylander's solution, and if it gives a positive orcin reaction, the presence of glycuronic acid compounds may be considered demonstrated. (3) THE COMPOUND GLYCOCOLLS Glycocoll is acetic acid CH3.COOH in which one H of the methyl group is replaced by the amido radicle-NH2, thus, CH2.NH2.COOH. Like sulphuric acid and glycuro- nic acid, it combines with certain aromatic disassimilation products of albumen to form the compound glycocolls. Certain drugs and poisons that belong to the group of aromatic bodies also combine with glycocoll, and when given by mouth appear in the urine as compound glyco- colls. Thus, to name only the most important of the latter substances, salicylic acid is excreted as salicyluric acid, nitrobenzoic acid as nitrohippuric acid, furfurol as furfuracryluric acid, toluol as toluric acid, etc. Clini- cally these bodies are only important inasmuch as the ap- pearance of the compound glycocolls in the urine always denotes the previous ingestion of the drug from which they are derived, and in this sense may possess medico- legal significance. Chemically the synthesis described is very interesting; it probably occurs in the kidneys. The most important compound glycocolls that are excreted in health on an ordinary mixed diet and that are occasionally found increased in coprostasis, and on a one- AROMATIC CONSTITUENTS OF THE VEINE 153 sided diet containing an excess of certain vegetables, are hippuric acid and phenaceturic acid. Hippuric Acid is benzoyl-glycocoU and is derived from combination of benzoic acid with glycocoll, as illustrated by the equation; CHs.COOH + CH2.NH2.COOH = (CoH5.CO)NH.CH2.COOH + H2O Benzoic Acid Glycocoll Hippuric Acid The benzoic acid may be derived from phenylpropionic acid, a product of albumen degradation by oxidation, thus: o C0H5.CH2.CH2.COOH f CoHs.COOH Phenylpropionic Acid Benzoic Acid The exact source of the glycocoll is not known; it may be formed from albuminous putrefaction, for all albumens contain a glycocoll radicle. Certain articles of diet, particularly many vegetables containing cinnamic acid, quinic acid, toluol, may lead to the formation of benzoic acid in the bowel, and hence to the elimination in the urine of hippuric acid. The hippuric acid excretion is, therefore, always greater on a vegetable diet than on a meat diet, and is always much greater in herbivorous animals living on certain herbs, grasses and fruits that contain the above mother sub- stances of benzoic acid than in human subjects who only occasionally eat those substances (cranberries, plums, prunes, etc.) . Benzoic acid or phenylpropionic acid given by mouth, of course, lead to a copious excretion of hip- puric acid. When hippuric acid is excreted on a meat diet exclu- sively, as in carnivorous animals, then the benzoic acid is derived from the putrefaction of albumen in the bowel. Hippuric acid occasionally appears in the urine as a sedi- ment of fine needles or of rhomboid prisms, particularly after eating copiously of cranberries. (See Chapter X.) On a mixed diet the hippuric acid excretion rarely ex- 15i CLINICAL URINOLOGY ceeds 0.7 grams in twenty-four hours; if much fruit is eaten, or if benzoic acid or its compounds are introduced for medicinal purposes, very much more may be excreted. The clinical significance of hippuric acid and of its chemical congener phenaceturie acid, to be presently dis- cussed, is small. In a subject living on an ordinary mixed diet, the excretion of more than 0.7 grams of hip- puric acid indicates abnormal disassimilation of albumen in the bowel, and hence ^arto/fces of the same significance as the excretion of the conjugate sulphates and glycuro- nates. It is interesting to note that whereas sulphuric and glyeuronic acid combine with a large variety of aro- matic alcohols and their derivatives (phenoles, indol, skatol, camphor, chloral, etc.), glycocoll only combines with non- hydroxy lized aromatic acids; viz., phenylpro- pionic and phenylacetic acid. Test for Hippuric Acid. — 500 cc. of urine are rendered alkaline, evaporated almost to dryness, and the residue re- peatedly extracted with alcohol ; the alcohol is driven off, the residue acidified with HCl and extracted with acetic ether, the extract evaporated at a low temperature and the residue extracted with petroleum ether. The hippuric acid remains behind, while any phenaceturie acid, fat, or benzoic acid that may be present is dissolved. The hip- puric acid can then be crystallized from warm water. The crystals are milk-white, semi-transparent, quadrilateral prisms and columns that may be united to rosettes (see Chapter X, page 232). On heating the crystals in a dry test tube -they first melt, forming an oily fluid (that recrystallizes on cooling) ; this turns red and finally gives off benzoic acid that sublimes on the walls of the tube and emits an agreeable aromatic odor, first of hay, later of bitter almonds. The crystals of hippuric acid are very soluble in alcohol and in hot water, and melt at 187.5° C. Phenaceturie Acid is derived from phenylacetic acid. AROMATIC CONSTITUENTS OF THE URINE 155 as hippuric acid is derived from phenylpropionic acid; whereas, in the case of hippuric acid, however, the aro- matic acid first undergoes oxidation to benzoic acid before it combines with glycocoll (see above), in the case of phenaceturie acid the combination occurs directly with- out previous oxidation of the acid, thus: — CoHa.CHzCOOH + CH2.NH2.COOH = Phenylacetic acid Glycocoll (CoH5CH2CO)NH.CH2.COOH + H2O Phenaceturie acid Phenaceturie acid, therefore, is phenylacetylglycocolL It always appears in small quantities, together with hip- puric acid, when there is much intestinal putrefaction and coprostasis. It can be isolated from the urine together with hippuric acid and separated from the latter by ex- traction with petroleum ether. It crystallizes in rhombic plates with rounded angles, that melt at 143° C. and assume a reddish color and emit an aromatic odor when heated above this point. (4) THE AROMATIC OXYACIDS The acids of this group are derived from the disinte- gration of albumen, like all the other aromatic bodies of the urine just described. It is probable that they are generated from albumen by way of tyrosin; feeding tyrosin or leucin to animals at all events causes an in- crease of the aromatic oxyacids of the urine. In acute phosphorus poisoning and in acute yellow atrophy of the liver, — both lesions that are accompanied by great destruc- tion of tissue albumen, and that lead to the appearance of much tyrosin and leucin in the urine, — the aromatic oxyacids are also found increased. In carbolic acid poison- ing they are also increased. Normally, a liter of human urine does not contain more than 0.01 to 0.02 grams of these acids, and the 156 CLINICAL URINOLOGY clinical importance of this whole group is very small; their appearance in the urine signifies abnormal or sud- denly increased albumen catabolism, either in the gastro- enteric tract or in the tissues. Possible that the appear- ance of much- ai'omatic oxyaeid, together with much tyrosin and leucin in the urine, may be utilized in deducing phosphorus poisoning or acute yellow atrophy of the liver. The acids may appear in the urine free (i. e., as alkali salts) or combined with sulphuric acid as conjugate sul- phates. The two principal acids that appear in the urine are para -oxyphenyl- acetic acid and para-oxyphenyl-pro- pionic acid. In view of the subordinate clinical im- portance of these acids, the methods for isolating and identifying them will not be given (see for these methods Huppert, "Harnanalyse," page 237, ff.). In acute yellow atrophy of the liver and in phosphorus poisoning two other acids appear in addition that belong to the group of aromatic oxyacids; viz., para-oxyphenyl-lactoic acid and oxyamygdalic acid. Two other aromatic oxyacids, finally, merit particular mention; true, their appearance in the urine is rare, but when they do appear they indicate an important and highly interesting but obscure metabolic perversion. I refer to homogentisic and uroleucinic acids, known collec- tively under the name of alkaptonic acids, and the condi- tion known as Alkaptonuria. — The characteristic features of the urine in alkaptonuria are that the urine, while quite normal in appearance when freshly voided, soon becomes deep brown, and finally black. If an alkali is added to such urine the darkening is hastened and the black color be- comes more intense. The only other bodies that produce this spontaneous darkening of the urine are urobilin and pyrocatechin and hydroquinone, urinary bodies that are excreted in carbolic acid poisoning, and in certain other AROMATIC CONSTITUENTS OF THE URINE 157 conditions that have been discussed above; the method of identfying these bodies, and of isolating them so that they can be distinguished from the alkaptonie acids have ah'eady been described. Alkaptonie acids when boiled with Fehling's solution color the liquid black; they also reduce ammoniacal sil- ver solutions in the cold. They do not give Nylander's bismuth test, nor do they ferment. They are optically inactive. They give a lemon- yellow precipitate with Millon's reagent (see foot-note to page 139) in the cold that gradually turns orange and, on heating, brick-red. Alkaptonie urine stains linen a dark brown color, and often the condition is first discovered in this way. Only a limited number of cases of alkaptonuria are on record. More males than females are reported. Heredity seems to play a role and the condition is probably always congenital; the abnormality persists during the life of the subject without apparently in any way interfering with his well-being. ' In some groups that are recorded two or more brothers or sisters were afflicted with alkap- tonuria without any history of alkaptonuria in parents or grandparents. Only one case is recorded of alkaptonuria occurring in father and son. It appears that in several of the cases the parents of the subject were first cousins; here, then, is a resemblance with albinism and cystinuria, both congenital anomalies that are common in the off- springs of such consanguineous marriages. The ingenu- ous theory has been advanced that in alkaptonuria we are dealing with a "chemical sport," or alternative mode of metabolism. Sufferers from alkaptonuria excrete from 2.6 grams (in a child) to 5.9 grams of alkaptonie acids (chiefly homogentisic acid) in a day, and it is interesting to note that the quantity excreted fluctuates within very narrow limits from day to day; the plus or minus of alkaptonie acid excretion being apparently chiefly dependent upon 158 CLINICAL URINOLOGY the amount of albumen eaten. On a vegetable diet these subjects excrete less alkaptonic acid than on a meat diet. No observations are on record in regard to the excretion of alkaptonic acids in sufferers from alkaptonuria aflflicted with fever, diabetes, yellow atrophy of the liver, phos- phorus poisoning or other conditions in which there is much destruction of tissue albumen. Starving such sub- jects for a number of days should throw much light upon the mooted question whether the abnormal formation of the alkaptonic acids from albumen via tyrosin and leucin (see above) occurs in the gastro-enteric tract or in the tissues proper, or in both locations. The separation of the two alkaptonic acids is of no clinical value. The diagnosis of alkaptonuria can be made from the above urinary signs; the presence of pyrocatechin and of hydroquinone should, of course, be excluded. Much more homogentisic than uroleucinic acid is always excreted. A convenient qualitative test for homogentisic acid is the following one: The urine is heated nearly to boiling; to each 100 cc. of the urine are added 6 grams of neutral lead acetate and the liquid is allowed to cool and stand in a quiet place for twenty- four hours. If homogentisic acid is present, brown star-shaped pointed crystals of lead homogentisate will form. The latter may be filtered off and dissolved in water; the solution should give all the reactions described above for alkaptonic urine. A more exact method for isolating and identifying homogentisic acid and for differentiating it from its con- gener uroleucinic acid, will be found in my -article on "Urine" in Buck's Reference Handbook, Vol. VIII, p. 47. For ordinary clinical purposes this complicated manipu- lation is altogether superfluous. CHAPTER VIII MISCELLANEOUS FATTY ACIDS OF THE URINE Oxalic Acid. Volatile Fatty Acids — Pormie, Acetic, Propionic, Butyric, Valerianic and Oleic Acids. Lactic Acid. Leucin and Tyrosin. Their Pathogenesis, Clinical Significance, Detection and Determination. The most important organic acids of the urine have already been discussed in previous chapters; viz., the aromatic acid, as well as glycuronic acid and their com- pounds, in the chapter on "Aromatic Constituents of the Urine"; uric acid in the chapter on "The Purin Bodies of the Urine"; diacetic and oxy butyric acid, in the chap- ter on "The Acetone Bodies of the Urine," and the bile acids in the chapter on "Blood- and Bile-pigments of the Urine." There remain a number of organic acids of some clinical significance that cannot be properly classified under any of these headings, that are neither clinically nor chemically related to each other, and that may, therefore, be discussed separately under the above caption. Only a few of these miscellaneous urinary acids merit discussion in a work on Clinical Urinology; these are oxalic acid, the volatile fatty acids (formic, acetic, propionic, butyric, valerianic and oleic acids) , lactic acid, leucin and tyrosin. Oxalic Acid. — Small quantities of oxalic acid (0.02 to 0.05 grams in twenty-four hours) may be found in every urine. It usually occurs in the form of the calcium salt that is held in solution by diacid sodium phosphate. On standing, calcium oxalate usually crystallizes out in char- acteristic forms (see chapter on Urinary Sediments). The presence of oxalic acid in the urine should never, however, be excluded from the absence of oxalate crystals (159) 160 CLINICAL URINOLOGY in the sediment. Oxalate of calcium octaeders should be insoluble in acetic acid, but readily soluble in hydro- chloric acid. The oxalic acid of the urine may be derived from numerous sources; its precise genesis is not completely understood. One source is unquestionably uric acid, for it has been shown that the administration of uric acid leads to an increased excretion of oxalic acid and urea, probably by way of oxaluric acid; the latter body occa- sionally appears in the urine and readily splits into oxalic acid and urea according to the formula: CO— NH COOH NH2 \ / CO + HoO = + CO / \ COOHNH2 COOH NH. Oj caluric Acid Oxalic Acid Urea It is probable, therefore, that oxalic acid is a product of the incomplete oxidation of uric acid, and, as the latter is derived from nuclein, one may say that oxalic acid ex- cretion may be due to incomplete nuclein catabolism. There are, however, other sources of oxalic acid, chief among them certain vegetable articles of the diet, as apples, grapes, asparagus and spinach, and the abundant ingestion of these articles is, as a rule, followed by an increased excretion of oxalic acid; here, then, we are dealing with an alimentary oxaluria. Oxalic acid may further be derived from dextrose ; and from the experimental and clinical evidence at hand it appears that oxalic acid appears in the urine when the degradation of the blood-sugar proceeds along abnormal channels. Experimentally, finally, the excretion of oxalic acid can be increased by interference with the supply of oxy- gen; it is probable that here the proper transformation of uric acid, or of sugar, is interfered with and that hence oxaluria occurs. FATTY ACIDS OF TEE URINE 161 Pathologically oxalic acid has been found in large quantities in the urine in diabetes and in icterus. The pathogenesis of the latter form of oxaluria is not at all understood, unless one would assume that both the icterus and the oxaluria were symptoms of some derangement of the liver function leading to bile absorption and per- verted catabolism of dextrose or of uric acid. In diabetes we are almost forced to assume that the excretion of oxalic acid is a manifestation of the interference with normal sugar destruction, and this view is borne out by the observation that the sugar excretion may occasionally decrease while the oxalic acid excretion seems to vicari- ously increase. Oxaluria has finally been vested with the dignity of an independent clinical entity {oxalic acid diathesis, idio- pathic oxaluria) . The clinical syndrome of this condition is vague and indefinite; there may be a variety of purely subjective manifestations, as malaise, headache, muscular soreness, there may be emaciation, or, again, there may be no other sign than an abnormal increase in the oxalic acid excretion, as much as 0.5 grams of the acid having been recovered from 1,000 cc. of the urine. The clinical significance ot oxaluria in the light of our present knowledge is slight. When oxaluria is not ali- mentaiy it presumably indicates some oxidative perver- sion, leading, to incomplete metabolism of uric acid and of blood-sugar; occasionally it may indicate derangement of the liver function. Tests for Oxalic Acid. — There is no convenient qualita- tive test for oxalic acid in solution. For clinical purposes no such test, moreover, is required, as the urine always contains some oxalic acid. It is important merely to de- termine whether the oxalic acid of the urine is increased and to what extent. Quantitative Determination of Oxalic Acid. — All the methods described indicate too little oxalic acid, and in K 162 CLINICAL URINOLOGY view of the minimal quantities of oxalic acid found in normal urine these small absolute errors become rela- tively large. In pathological increase of the oxalic acid excretion, all that we want to determine for clinical pur- poses is that the oxalic acid excretion is considerably greater than normal, and slight inaccuracies in the deter- mination of the absolute quantity are unimportant. One should never, of course, assume an increase of the oxalic acid excretion from the appearance of a copious oxalate sediment, for, as stated above, the urine may contain much oxalic acid and still precipitate only very few calcium oxalate crystals or none at all. The best quantitative method for estimating the urinary oxalic acid is, in my experience, the following: — Method of Neuhauer. — The total twenty-four hours' urine is gathered, rendered alkaline with an excess of ammonia and treated with calcium chloride (10 per cent solution) ; it is then acidulated with acetic acid and allowed to stand for several hours until the precipitate settles at the bottom of the vessel. The addition of a few drops of an alcoholic thymol solution to the mixture is advisable in order to check bacterial growth, for the presence of many microorganisms in the liquid may render it difficult to procure a clear filtrate. The precipi- tate is gathered on a small filter, and precipitate and filter together placed in a beaker containing warm di- lute hydrochloric acid; the liquid is heated for a few minutes on the water -bath and filtered until the liquid is no longer acid; the filter is then repeatedly washed with warm water, washings and filtrate united and evaporated to a small volume, and while still hot oversaturated with ammonia. The solution is allowed to stand for twenty- four hours and the crystals of calcium oxalate that form gathered on a weighed ash-free filter. The precipitate is freed from adherent chlorides by repeated washing, then dried and incinerated in a platinum crucible and FATTY ACIDS OF THE URINE 163 glowed to a constant weight. This converts the calcium oxalate into calcium oxide. As 56 parts of calcium oxide correspond to 90 parts of oxalic acid, the weight of the calcium oxide must be multiplied by H = 1.607, in order to give the amount of oxalic acid present in the quantity of urine used for the estimation. The Volatile Fatty Acids.* — Normal urine always con- tains traces of these acids, chiefly formic, acetic, pro- pionic and butyric acids. The average daily excretion of volatile fatty acids normally does not exceed 0.06 grams. In decomposing urine, chiefly in urine undergoing ammo- niacal fermentation, large quantities of these acids are formed; here they are presumably derived from the fer- mentation of urinary carbohydrates. In fever they are increased; the source and origin of the fatty acids in fever is not thoroughly understood; they may be derived from carbohydrates, but are presumably the product of the perverted disassimilation of the body fats and proteids. In typhoid fever, in variola and in acute yellow at- rophy of the liver, valerianic acid has been found in the urine; here this fatty acid is presumably derived from the putrefactive decomposition of leucin. In a case of phosphorus poisoning and in several cases of hematoporphyrinuria, oleic acid and certain solid fatty acids high in the series and melting at 49° to 51° C. have been recovered from the urine. In diabetic urine, in addition to oxybutyric acid and diacetic acid, simple acetic, butyric and propionic acid are occasionally encountered; they may partake of the same clinical significance as the acids of the acetone group. In the urine of herbivorous animals and in subjects living on a vegetable diet the excretion of fatty acids is greatly increased above average values. *To this group properly belong ;8oxy butyric acid and diacetic acid, but they have already been discussed, together with acetone, in the chapter on "The Ace- tone Bodies of the Urine." 164 CLINICAL URINOLOGY The clinical significance of these acids is slight ; in vegetarians the excretion of these bodies is alimentary ; in diabetes, as already said, the volatile fatty acids as a group are important inasmuch as they indicate acidosis (see chapter on "The Acetone Bodies of the Urine") ; in fevers they denote perverted disassimilation of the tissues of the body proper, under the influence of the high tem- perature or possibly of bacterial toxins; in acute yellow atrophy of the liver valerianic acid may possess the same significance as leucin inasmuch as it is derived from this body by putrefaction. In all the other states an increase of volatile fatty acids in the urine must, for the present, be considered an accidental finding. Isolation and Identification of the Volatile Fatty Acids . — Large quantities of the urine are acidulated with phos- phoric or sulphuric acid, and distilled in a current of steam until the distillate is no longer acid. The distillate is then neutralized with soda solution, evaporated to dry- ness and the residue repeatedly extracted with absolute alcohol. The alcoholic extract contains sodium salts of the fatty acids, sodium benzoate and para-cresole. The benzoate can be removed by evaporating the alcoholic solution to dryness, treating the residiie with cold sul- phuric acid, filtering and allowing the filtrate to stand until all the benzoic acid crystallizes out. The crystals can be filtered off and the filtrate neutralized with soda and shaken out with ether; the latter takes up the para- cresole. The ethereal solution of para-cresole is removed in a separating funnel, the watery residue heated to drive off the last traces of ether, and the fatty acids determined in the watery solution that remains behind. This is best done by treating the watery solution with barium hydrate to the point of neutralization, evaporating to dryness, weighing the residue of barium salts of fatty acids and some barium hydrate in excess, redissolving in water, determining the barium by precipitating it with sulphuric FATTY ACIDS OF THE URINE 165 acid as sulphate, and deducting the weight of the barium from the weight of the above dry residue; the difference indicates the amount of volatile fatty acids present. The method is sufficiently accurate for clinical purposes. In view of the small importance of this group, the deter- mination will not frequently be undertaken. The determi- nation of the most important members of this group; viz., /8-oxy butyric and diacetic acid, has already been discussed in the chapter on "The Acetone Bodies of the Urine." The isolation and identification of the different other volatile fatty acids is possible, but very difficult. It is clinically superfluous. The methods for performing these tests can be found in Huppert, "Harnanalyse," p. 178. Lactic Acid (Sarcolactic, paralactic, dextrorotatory lactic acid) . — Normal urine never contains lactic acid. Patho- logically it appears in certain disorders in which intra- cellular oxidation is perverted or in which the supply of oxygen is reduced. Thus, in experimental dyspnoea produced by ligation of the trachea, by asphyxiation with carbon- monoxide gas, etc., lactic acid always appears in the urine. Clini- cally it has also been discovered in patients suffering from acute larynx stenosis and in cases suffering from carbon -monoxide poisoning. The lactic acid excretion following epileptic seizures may possibly be explained on similar grounds, for it may be due to the interference with the respiratory excursions of the lungs resulting from tonic fixation of the thoracic muscles and the diaphragm during the epileptic spasm. In this disorder the origin of the urinary lactic acid may, however, also be attributed to increased muscular catabolism resulting from the violent convulsive muscular movements, for it is well known that lactic acid is always formed in muscle that contracts. After violent muscular exercise of any kind, lactic acid always appears in the urine; one must assume that here 166 CLINICAL URINOLOGY an unusual amount of the normal product of muscular catabolism; viz., lactic acid, is suddenly poured into the circulation, so that the further disassimilation of this in- termediary body to urea (see below) cannot be accom- plished as rapidly as it should be; hence lactic acid as such is excreted in the urine. In trichinosis, in which the nutrition of the muscle and its catabolism are interfered with, lactic acid also occasionally appears in the urine. Lactic acid, as mentioned above, is a normal inter- mediary body of metabolism; if too much of it is not thrown into the circulation at once, it combines with ammonia radicles and enters the liver as ammonium- lactate ; the latter, in its passage through the liver, is con- verted into urea and leaves the body as such. Whenever the urea-forming function of the liver is deranged, this conversion cannot occur and lactic acid (in the form of ammonium-lactate) appears in the urine. This event occurs in most destructive lesions of the liver parenchyma, notably in acute yellow atrophy and phosphorus atrophy of the liver, also in certain functional derangements of the organ consecutive to biliary obstruction with diapede- sis of bile. Extirpation of the liver in birds is invariably followed by a large excretion of lactic acid. In diabetep the appearance of lactic acid in the urine is not at all uncommon; in fact, it is probable that lactic acid in this disease plays an important part, together with the acids of the acetone group and the volatile fatty acids, in the production of diabetic acidosis. It is more than probable that lactic acid is derived from the body-glyeo- gen via dextrose through a process of anaerobic splitting. In diabetes one must assume that the dextrose disassimi- lation is partially arrested at the lactic acid stage ; or one may assume that normally, i. e., when intracellular oxi- dation is normal, dextrose is destroyed via alcohol and that as soon as intracellular oxidation becomes inadequate the sugar molecule splits into lactic acid and other prod- FATTY ACIDS OF THE URINE 167 ucts. This chapter of metabolism is still obscure. It seems very likely, however, that the muscular lactic acid that has been discussed above is derived from the glyco- gen depots that the muscles contain. It is possible that lactic acid (i. e., ammonium-lactate) is also derived from the albuminous tissues of the body, but I am inclined to favor the view that it is derived from the carbohydrates, as the experimental evidence in favor of the albuminous origin is scanty, insufficient and by no means convincing. Aside from phosphorus and carbon-monoxide, certain other poisons, notably strychnine, curare, morphine, amyl- nitrite, veratrine and arsenious acid can produce lactic acid excretion ; it is probable that these poisons act either like phosphorus, i. e., by causing interference with the liver function, or, like carbon-monoxide, by causing oxy- gen hunger and intracellular asphyxia. The clinical significance of lactic acid in the urine is, therefore, broadly speaking, the following: It usually denotes interference with intracellular oxidation superin- duced by a variety of possible factors that have just been enumerated. These factors may be operative either to cause dyspncea from an insufficient supply of atmospheri- cal oxygen, or they may be operative to produce interfer- ence with intracellular oxidation either in the cells of the liver or of the tissues of the organism at large. The appearance of lactic acid in the urine, as far as our ex- perimental and clinical knowledge goes, usually denotes perverted catabolism of the glycogen and dextrose of the blood and tissues — due either to excessive destruction of , glycogen with normal, but relatively inadequate oxidation, or to abnormal glycogen and dextrose destruction. The Detection and Estimation of Lactic Acid. — In order to detect the presence of lactic acid in the urine, the acid must be isolated in the form of one of its salts, and the latter analyzed. The ordinary qualitative tests for lactic acid that are so convenient in the analysis of the stomach 168 CLINICAL URINOLOGY contents cannot be employed in the case of the urine. Thus Uffelmann's reaction (yellow coloration of a blue carbolic acid -ferric chloride solution) gives doubtful results in urine, because phosphates, dextrose and other urinary bodies produce the same discoloration. The most convenient and most rapid method of deter- mining the presence of lactic acid in the urine is to manu- facture the zinc salt. For this purpose the urine is treated with a large excess of phosphoric acid and the mixture repeatedly extracted with ether; the ethereal extract evaporated down to a thick syrup, the syrup dissolved in water and the watery solution boiled with lead hydrate. This removes any phenoles, urea or hip- puric acid that may have been extracted from the urine by the ether. The lead precipitate is filtered off, the lead in the filtrate precipitated as lead sulphide by a stream of sulphureted hydrogen, the lead sulphide filtered off, the filtrate boiled in order to drive off all the sulphureted hydrogen, and then boiled with carbonate of zinc. The solution is then evaporated to a small volume and allowed to stand in a cool place until any zinc lactate that may be present crystallizes out. The crystals are gathered on a weighed filter, washed with absolute alcohol, dried in the air and weighed. They are then dried at 110° C. to con- stant weight; this drives off the water of crystallization. The loss of weight before and after drying at 110° C. cor- responds to the amount of water of crystallization that the crystals incorporated. If the crystals consisted of the para-lactate of zinc they should contain 14.58 per cent of water of crystallization. Inspection of the crys- tals under the microscope and determination of their crystal form may often aid in determining the character of the crystals, the zinc lactate appearing as small spheroids or prisms The identification of lactic acid, finally, is not com- plete without a determination of the zinc content of the FATTY ACIDS OF THE VBINF 169 crystals. This is carried out as follows: The dried and weighed crystals are treated with concentrated nitric acid, the nitric acid driven off by gentle heat in a porcelain crucible, the residue evaporated to dryness and glowed; this generates zinc oxide. The latter is weighed. Dried lactate of zinc should contain 33.43 per cent of zinc oxide (ZnO), or 26.84 per cent of zinc. Leucin and Tyrosin. — These two bodies properly belong to the group of fatty acids that are being discussed in this chapter because they are derivatives of acetic and of propionic acid; leucin being a-amido-isobutyl-acetic acid (a-amido-caproic acid) ; viz., (CH3)2 : CH.CH2.CH(NH2). COOH, tyrosin being para-oxyphenyl-a-amido-propionic acid; viz., HO.CeH*— CH2 — CH.NH2 — COOH. Leucin and tyrosin are formed from the disintegration of albumen. Whether or not they are normally formed in the tissues as an intermediary product, and are at once con- verted further into ammonia compounds and then to urea remains undetermined. I do not think that this idea is ten- able, for otherwise we should more often find tyrosin and leucin, that have partially escaped this conversion, in the urine. Some authors claim that leucin and tyrosin are often found in normal urine, others deny it. Albuminous urine, in particular, is said to contain these bodies; in this case one must think of an extra -renal origin of leucin and tyrosin by putrefactive decomposition of the urinary albumen or disintegration of this albumen by the tryptic ferments that the urine, as a rule, contains. The same explanation might be proffered for the genesis of leucin and tyrosin (in traces) in normal urine, for it always contains some albuminoid bodies; it is probable, how- ever, that some of the " physiological "(? ) urinary tyrosin and leucin is absorbed from the bowel where these bodies are continuously formed from albumen that is undergoing tryptic and bacterial decomposition. Pathologically, the greatest quantities of leucin and 170 CLINICAL URINOLOGY tyrosin are excreted in acute yellow atrophy of the liver. Many hypotheses have been advanced to explain this phenomenon, but none are satisfactory, none are dem- onstrated to be correct; it is needless, therefore, to dis- cuss them here. Leucin and tyrosin are not found in every case of acute hepatic atrophy, so that the existence of. this disease should not be excluded from the absence of these bodies from the urine. In phosphorus poisoning leucin and tyrosin are also often excreted, although not so often as in acute yellow atrophy of the liver, nor so copiously. Here, too, the explanation of the peculiar metabolic perversion that leads to the formation of leucin and tyrosin from albumen is still forthcoming. In cirrhosis of the liver, cancer of the liver, biliary lithiasis, in fact in almost every organic disease of the liver, leucin and tyrosin have been found increased in the urine. In a variety of intestinal diseases, particularly in severe typhoid and in tuberculosis of the bowel, leucin and tyrosin have been found in the urine; whether in these eases abnormal bacterial decomposition of albumen in the bowel occurs or whether the bowel -wall becomes abnor- mally permeable to these bodies is uncertain. The latter view appears to be the more rational of the two, for leucin and tyrosin, as stated above, are normally formed in the bowel, but do not appear in the urine in appreciable quantities unless the intestinal wall is diseased.* Leucin and tyrosin have also been found in consider- able quantities in severe smallpox. Detection and Determination of Leucin and Tyrosin. — Leucin and tyrosin when present in considerable quanti- ties in the urine are apt to crystallize out so that they appear in the sediment. For the identification of leucin * Feeding leucin and tyrosin to normal subjects is not followed by the ap- pearance of these bodies in the urine. FATTY ACIDS OF THE URINE 171 and tyrosin crystals in the urine, see the chapter on "Uri- nary Sediments," page 234. The absence of leucin and tyrosin from the sediment does not, however, exclude their presence in the urine in solution. In order to determine the presence of leucin and tyro- sin in the urine, they must be isolated as follows : — The urine is treated with lead acetate until no further precipitate forms, the precipitate is filtered off, the excess of lead removed from the filtrate by a stream of H2S, the lead sulphide filtered off, the filtrate evaporated to a small volume of syrupy consistency and allowed to crys- tallize. The crystals are examined under the microscope and identified further as given under "Urinary Sediments," on page 23 1. The separation of the two bodies can be performed; but this procedure is unnecessary for clinical pui'poses. CHAPTER IX THE INORGANIC CONSTITUENTS OF THE URINE The Factors Determining the Excretion of the Mineral Salts of the Urine. The Total Urinary Ash. The Inorganic Acids of the Pi-me— Hydro- chloric Acid (Chlorides) — HydroBuoric Acid (Fluorides) — Sulphuric Acid (Sulphates), Preformed or Mineral Sulphates, Conjugate or Aro- matic or Ethereal Sulphates, Neutral Sulphur Compounds Cystinuria and Diaminuria. Sulphureted Hydrogen (Sulphides), Phosphoric Acid (Phosphates^, Carbonic Acid (Carbonates), Silicic Acid (Sili- cates), Nitrates and Nitrites, Peroxide of Hydrogen. The Inorganic Bases of the J7jwe — Potassium and Sodium — Calcium and Magne- sium — Ammonium — Iron. The inorganic constituents of the urine are derived from the food and from the proper tissues of the body. Their excretion is dependent on the character of the food, the degree of tissue eatabolism and, to a certain extent, upon the condition of the kidneys. Until recently the urinary inorganic solids were considered to be merely the residue of the food that passed unchanged from the gas- tro-enteric tract to the kidneys ; of late years we have, how- ever, learned to appreciate that the mineral constituents of the body form an integral part of protoplasm, that their presence is essential to the carrying on of all life processes, and that the excretion of inorganic solids in the urine is merely an attempt on the part of the organ- ism to maintain the proper proportion of mineral salts in the blood- and tissue-juices. As soon as too much mineral matter enters the blood- and lymph -stream from without (i. e., from the ingesta), or from within (i. e., from the eatabolism of the tissues of the organism) , the excess is at once eliminated in the urine (and a part in other excreta of the body) ; as soon, on the other hand, as the (172) INORGANIC CONSTITUENTS OF THE URINE 173 supply of mineral salts becomes insufficient, then the body- jealously retains many of the bases and acids, and the urinary ash is consequently decreased. The biological and clinical significance of the different bases and acids constituting the salts of the urine will be discussed below. The Total Urinary Ash. — The inorganic constituents of the normal urine are the bases potassium, sodium, ammonium, calcium, magnesium and iron, the acids hydrochloric acid, sulphuric acid, phosphoric acid, car- bonic acid and traces of hydrofluoric, silicic, nitric and nitrous acid; in addition, one encounters "neutral sul- phur compounds," and occasionally sulphureted hydrogen and peroxide of hydrogen. The different acids are dis- tributed in varying proportions over the different bases, forming mono-, di- and tri-basic salts and double salts. The composition and the quantity of the urinary ash depend largely upon the character of the diet. As a rule, a large excess of inorganic salts is eaten and the body endeavors to rid itself of the surplus as rapidly as possible. The power of the organism, however, to elimi- nate different salts varies for each salt, so that certain salts are eliminated more rapidly than others; in addition, certain individuals, according to their taste, eat more so- dium chloride than others, and hence continuously elimi- nate more of this body. It is difficult, for this reason, to give any definite standard figures that indicate the pro- portion of inorganic bases and acids and the proportion of the different salts that a normal healthy adult elimi- nates in the urine on an average mixed diet. Von Noorden has established the following approximate values: — K2O 2-3 grams, Na20 4-6 grams, CaO 0.15-0.35 grams, MgO 0.2-0.3 grams, Fe traces, CI 6-8 grams, P2O5 2.0-3.5 grams, S03 2.0-3.5 grams. A healthy adult on an ordinary mixed diet excretes from 9 to 25 grams of urinary ash in twenty -four hours. 174 CLINICAL URINOLOGY Quantitative Determination of the Urinary Ash. — A definite quantity of the urine voided in twenty -four hours is evaporated to dryness and the residue earbonified. It is very important that the dry residue should not be inciner- ated at once at glow temperature, for if this is done the alkaline chlorides volatilize and are in great part lost ; in addition, the sulphates are partly reduced to sulphides by the carbonaceous material present; the mono -phosphates, too, undergo decomposition with loss of phosphorus. After the urinary residue is earbonified it is first, therefore, leached out with water, the residue again dried on a water- bath and finally incinerated over the direct flame; the watery extract, in its turn, is evaporated to dryness and incinerated; this may be done in the crucible contain- ing the first ash. The whole ash is then weighed and the total quantity of urinary ash determined in this way. The electrical conductivity of the urine and its freezing point are other physical methods for determining the total mineral ash of the urine. (See Chapters XII and XIII.) THK INORGANIC ACIDS OF THE URINE 1. Hydrochloric Acid (Chlorides). — Hydrochloric acid (HCl) appears in the urine as sodium, potassium, ammo- nium, calcium and magnesium chloride. Of these salts the most important and the most abundant is sodium chloride ; in fact, the fluctuations in the chlorine excretion may be said to be largely dependent on the fluctuations in the sodium chloride excretion. The chlorides, as stated above, are largely found preformed in the food, and whenever they are ingested in excess they at once appear in the urine. The organism jealously guards its sodium chloride content, so that a starving individual may excrete essentially no sodium chloride in the urine, or at least so little that a silver nitrate solution only produces a barely perceptible clouding of the urine. The food, in addition INORGANIC CONSTITUENTS OF THE URINE 175 to sodium chloride, usually contains small quantities of potassium and magnesium chloride. These chlorides when given by mouth are almost completely absorbed un- less they are given in such quantities or in such concentra- tion as to irritate the gastro- intestinal mucosa, cause diarrhoea and to be carried off in the bowel discharges. As the stools normally contain only very little chlorine (NaCl), one must assume that the gastric hydrochloric acid and the sodium chloride of the intestinal secretions are reabsorbed from the bowel. Whereas the blood- and tissue-juices seem always to contain an excess of sodium chloride, and whereas the so-called circulating albumen always contains much chlo- ride, the albumen of the tissues proper (i. e., "living" albumen) contains very little chlorine. Hence, in fast- ing when no chlorides are ingested the kidneys first eliminate the excess of circulating chlorine, so that for a time, a day or two, the urinary chlorine excretion remains considerable ; then, however, the chloride excretion drops ta very low figures (considerably less than one gram in the twenty-four hours), for now the body is destroying tissue-albumen and it contains little chlorine. This point is of some medico legal importance in cases that feign inaniti6n or claim to be starving, for in those persons the excretion of several grams of NaCl in the twenty-four hours' urine indicates that some food was eaten in the twenty-four hours' period preceding. The normal excretion of chlorides fluctuates within wide boundaries and is largely dependent on the palate of the individual, some subjects craving more salt than others. Usually from 12 to 15 grams are excreted a day. As a rule, the proportion of NaCl to urea is as 1 : 2. In chronic inanition and underfeeding this proportion is usually maintained, showing that the salt excretion to some extent parallels the eatabolism of the albumens and hence the nitrogen excretion. As soon, however, as all food 176 CLINICAL URINOLOGY is suddenly refused, then we witness the clinical phenome- non of a rapid drop in the sodium chloride excretion ; if, in addition, there should be profuse vomiting in such a case, with additional loss of chlorine in the vomit, then a condition of chlorine hunger may appear despite the fran- tic efforts of the body to maintain its chlorine content. As sodium chloride (see above) is necessary for ther main- tenance of life, such cases should receive salt hypodermi- cally; it will be found that they retain all the salt admin- istered in this way and waste none of it in the urine. The chlorine excretion in febrile disorders present some features of interest. It appears that during most fevers the chloride excretion is reduced and that it rises after the temperature drops; this is particularly striking in pneumonia, the chloride excretion being markedly reduced during the florid stage of the disease, and rapidly increas- ing just before and after the crisis; in fact, the sudden increase of the urinary chlorides may, with some reserve, be considered a forerunner of a crisis. Whether or not in this disease the decrease of urinary chlorides is due to retention of chlorides in the exudate is not positively demonstrated; it is possible. The retention of chlo- rides is seen only in acute febrile disorders of short dura- tion; in long-lasting fevers the chlorine excretion is ap- proximately normal. The exact explanation of this febrile diminution of the urinary chlorides is still forthcoming; it may in part be due to the decreased ingestion of food, it may be partially attributed to intoxication of the kidneys or to binding of the chlorine by disintegrating tissue albumen that is thrown into the circulation, or it may be due to deficient absorption of chlorides from the stomach and bowel. Several of these factors, not to speak of the increased water- drinking and the sweating, may be concerned in producing this phenomenon ; but no uniform interpreta- tion that would apply to all cases can be given. IFOBGANIG C0i!^8TITUENT8 OF THE URINE 177 The chloride excretion in diseases of the stomach is important. In sufferers from gastric disease the chloride excretion is, of course, primarily dependent upon the diet; in such cases, therefore, the NaCl excretion is apt to be reduced because they eat little and are not particu- larly apt to salt their food. In mild gastric disorders in which considerable food is taken the chloride excretion is, however, only slightly reduced. In severe gastric disorders, on the other hand, as in pyloric stenosis with dilatation, in ulcer, in carcinoma In which very little food can be taken per os, the chloride excretion sinks to a minimum (see chloride excretion in inanition, above) . In these cases it is clear that an in- crease of the chloride excretion must be considered a good prognostic omen, for it demonstrates that more nourishment than before is being absorbed. In combination with nitrogen determinations, the deter- mination of the urinary chlorides may even aid in mak- ing a differential diagnosis between simple dilatation of the stomach or dilatation with carcinoma ; for the appear- ance of very little chloride and very little nitrogen in the urine indicates simple inanition, whereas the appearance of very little chloride with a relatively large proportion of nitrogen (more than 1 CI to 2 urea) indicates that much tissue albumen (that, as stated above, contains little chlorine) is being destroyed, and this is apt to be the case in cancer. Theoretically, one might argue that in pylorus stenosis with hyperchlorhydria less chloride would appear in the urine than in pylorus stenosis without hyperchlorhydria, poor absorption being present in both cases. As a matter of fact this is not the case, for the reduction of urinary chlorides is already so great in advanced cases of pylorus stenosis (see above) that the outpouring of HCl into the stomach can not depress it much further; practically, therefore, this apparently rational reasoning is not borne 178 CLINICAL URINOLOGY out by facts. If there is much vomiting with loss of gastric HCl in such cases, the organism, of course, must lose chlorine, and this loss, as stated in a preceding para- graph, should be replaced by injections of chlorides. Certain nervous phenomena, tetanus, tremor, etc., that are not uncommon in such cases are probably due to chlorine hunger. In anaemia due to sudden loss of much blood, the uri- nary chlorides are always temporarily reduced. In chronic anaemias the chloride excretion fluctuates as in healthy subjects with the chloride ingestion. In advanced cases the same rules obtain as in gastric cancer; the chlorides are reduced from inanition, while the nitrogen excretion is relatively high; the latter finding being due to destruction of tissue- albumen that, as repeatedly stated, contains little chlorine. In nephritis the chloride excretion, as a rule, follows the same course as the urea excretion (I refer to the chapter on Urea), both being largely dependent on the food. Wherever urea is retained, as in acute nephritis and in very late stages of chronic nephritis, chlorides are, as a rule, also retained, although not to the same degree; sometimes the kidneys may be quite permeable for chlo- rides, and at the same time be much less permeable for urea. Much has been written on the retention of chlo- rides in the different forms of nephritis, but, as in the case of urea, the element of diet has not been sufficiently included in the calculation ; until this is done in many cases I consider it a precarious proceeding indeed to draw prognostic or diagnostic conclusions from fluctuations in the chloride excretion in nephritis. The statements made above probably cover all that we actually know of this matter today. It is interesting to note the clinical fact that withdrawal of chlorides from the diet seems in some cases to reduce the albuminuria, and that a reduction of the food chlorides in some patients INORGANIC CONSTITUENTS OF THE URINE 179 seems to cause the disappearance of nephritic cedemas. Possible that in oedema we are dealing with osmotic phe- nomena, scil., an occasional retention of chlorides in the tissues and an attraction of water from a less concen- trated chloride solution (the blood) into a more concen- trated one (the tissue-juices) . All this is for the present, however, theoretical. In diabetes the urinary chlorides are usually increased ; this is probably due to the bulimia and the ingestion of large quantities of proteid pabulum; the proportion of chlorides to nitrogen is maintained throughout in this disease. After chloroform ancesthesia the chloride excretion is always increased; this is, of course, due to the elimination in the urine of the chlorine absorbed from the chloro- form. Thyroid preparations , it appears, also increase the chloride excretion. Estimation of the Urinary Chlorides. — The urine is acidulated with a few drops of dilute nitric acid and a 10 per cent silver nitrate solution added. A cheesy white precipitate forms that should be readily soluble in an ex- cess of ammonia. The iodides, bromides and cyanides give the same reaction; they are, however, rarely present in the urine, so that for practical purposes this qualita- tive test for chlorides is sufficient. As the urine always, with very rare exceptions, contains small quantities of chlo- rides, the test is nearly always positive; if daily tests aro made and the same quantities of reagent and urine are used throughout, some information in regard to a relative increase or decrease of the chloride excretion from day to day may be gained from this test. The silver chloride precipitate need not always be snow-white; in fact, it is often colored in various hues ; when exposed to the light the precipitate rapidly turns dark. Quantitative Test.— Of all the methods for estimating the chlorides of the urine, I have found the method of 180 CLINICAL URINOLOGY Volhard to be the most accurate and the most convenient one for clinical purposes ; when all the solutions are pre- pared the method is simple. Other methods (Mohr, Zuelzer, Q-ayLussac, etc.) have no advantages over Volhard 's method; they are, moreover, not so rapid of execution. All the methods are based on the same prin- ciple; viz., the titration of the urinary chlorine with a standardized silver nitrate solution. A full description of these methods will be found in Huppert,"Harnanalyse," 1898, p. 705 ff. In this place I limit myself to a descrip- tion of Volhard's method. Method of FoZAarcZ.— Solutions required. (1) 29.042 grams of pure silver nitrate dissolved in ],000 ce. of water. (2) A concentrated solution of ferric-ammonium alum (chlorine-free!) . (3) Nitric acid of 1.200 specific gravity. (4) A solution of ammonium sulphoeyanide. The manufacture of the latter solution requires con- siderable care, and it is time-consuming; when a large quantity is once made up, however, it serves for a great many determinations. Theoretically, it should contain 12.984 grams of ammonium sulphoeyanide to the liter, for then 25 cc. would correspond to 10 cc. of the silver solution (1); as the salt is, however, very hygroscopic, it cannot be weighed with sufficient accuracy and the strength of the solution must, therefore, be determined by titration against the silver solution, as follows: 6 to 7 grams of ammonium sulphoeyanide are dissolved in 400 cc. of water, and some of this solution poured into a burette; exactly 10 cc. of the silver nitrate solution (1) are then diluted with 100 cc. of water and 4 cc. of the nitric acid (3) and 5 cc. of the alum solution (2) added. So much of the sulphoeyanide solution is now allowed to flow into this mixture that the liquid turns reddish, and remains so ; this manipulation is performed several times and the average number of cubic centimeters required to bring about the reaction calculated. Assuming that 22 cc. INORGANIC CONSTITUENTS OF THE URINE 181 of the sulphocyanide solution were required to bring about the permanent color change, then the proper dilution of the sulphocyanide solution, 25 ce. of which are to corre- spond to 10 cc. of the silver solution, can be calculated according to the formula: 22 : 25 = 1,000 : x; x = 1136.3. This means that the solution prepared must be filled up to 1136.3 cc. With the four solutions the chloride determination is performed as follows : 10 cc. of the urine are poured into a small flask with a mark indicating 100 cc. ; to the urine are added 4 ce. of the nitric acid, 15 cc. of the silver solution and 50 cc. of water. A precipitate forms that is allowed to settle to the bottom ; the supernatant fluid must be quite clear; the flask is now filled to the 100 cc. mark. The contents of the flask is then filtered and 80 ce. of the fil- ti-ate poured into a flask and mixed with 5 cc. of the alum solution (2) and with enough of the sulphocyanide solu- tion (4) to cause the liquid to turn reddish. The sulpho- cyanide solution is allowed to flow in from a burette, and the amount of the reagent required to bring about the color change read off; the titration should be performed two or three times, and the average number of cubic centimeters determined. It is assumed (and this assumption is based on innumerable empiric determinations) that 15 cc. of the silver solution suffice not only to precipitate all the chlorides that may be present in 10 cc. of urine, but also to leave an excess of silver nitrate in solution. This excess precisely is determined by the above titration and the percentage of chlorides in the urine calculated from this deficit according to the formula : — X = (37.5 — IE) .1% X = Amount of sodium chloride contained in one liter of the urine (expressed in grams). E = Number of cubic centimeters of the sulphocyanide solution re- quired to bring about the end reaction. 182 CLINICAL URINOLOGY The rationale of this formula is the following: As 10 cc. of the silver nitrate solution correspond to 25 cc. of the sulphocyanide solution, 15 cc. of the silver nitrate solu- tion correspond to 37.5 cc. of the sulphocyanide solution. As only 80 cc. of the 100 cc. of test solution were used in the reaction, 37.5 cc. of sulphocyanide solution less igiui = I of the number of cubic centimeters of sulpho- cyanide solution brought about the color change with 10 cc. of silver solution. As 25 cc. of the sulphocyanide solution correspond to 10 cc. of the silver solution, 1 cc. of the former corresponds to 0.4 cc. of the latter; and as the silver solution is originally prepared of such a strength that 1 cc. corresponds to 0.01 grams of sodium chloride, 0.4 cc. of the silver solution correspond to 0.004 grams of sodium chloride. Hence, in order to obtain the amount of chlorides present in 10 cc. of urine, the figure (37.5 — f R) must be multiplied by 0.004. In order to determine x, i. e., the grams of sodium chloride in one liter (1,000 cc.) the figure must be multiplied by QA = to instead. In this test the chlorides of the urine are all deter- mined as sodium chloride, and the fact that some of the HCl is bound to other bases is ignored; this source of error, however, is' very slight and for clinical purposes altogether negligible. One great advantage of this method is that the presence of neither albumen nor sugar in the urine interferes with the reaction. 2. Hydrofluoric Acid (Fluorides).— Traces of hydro- fluoride are occasionally voided in the urine; this salt possesses no known clinical significance whatever. Its presence can be detected by precipitating a large volume of the urine with ammonia, calcining the precipitate, treating it with sulphuric acid and heating in a platinum crucible. A glass plate is held over the crucible and if hydrofluorides are present hydrofluoric acid will be liber- ated and will etch the glass. INORGANIC CONSTITUENTS OF THE URINE 183 3. Sulphuric Acid (Sulphates). — Sulphuric acid appears in the urine either iu combination with alkali or with cer- tain aromatic constituents of the urine (see Chapter VII). In the former case we speak of mineral or preformed sul- phates, in the latter of conjugate or aromatic or ethereal sulphates. The conjugate sulphates nominally constitute about one-tenth of the total sulphates; in certain patho- logical conditions, that have been fully discussed in the chapter on "Aromatic Constituents of the Urine," they may be greatly increased so that they constitute much more than one-tenth of the total urinary sulphates. The sulphuric acid that forms the sulphates of the urine is derived from two sources; viz., the food and the tissues of the body itself. Preformed sulphates in small quantities are often ingested with the food; the bulk of the sulphuric acid is, however, derived from the catabo- lism of food- or tissue albumen ; combining with circulat- ing alkali or circulating aromatic bodies, it appears in the urine as mineral or conjugate sulphate. The bulk of the aromatic radicles in the latter, however, as stated else- where, are derived from the putrefaction of albumen in the bowel. The sulphur of the albumens is not all oxidized to sul- phuric acid, but appears in part as so-called "neutral sul- phur compounds " ; these will be discussed below. The preformed mineral sulphates of the food are prob- ably only in small part absorbed from the bowel, because they are apt to form insoluble sulphate of calcium in the bowel. A portion of the sulphate derived from food- albumens by splitting (proteolysis and bacteria) probably also remains behind in the form of organic sulphate. Finally, a portion of the circulating sulphate is excreted into the bowel and not through the kidneys, and hence does not appear in the urine. The bowel contents, in addition, contains much sulphur in the form of sulphides, sulphocyanides, taurin, etc., that is derived from the de- 18i CLINICAL URINOLOGY composition of food -albumens, from desquamating epi- thelia, from the digestive secretions (KCNS in saliva) and from the bile. A portion of this latter sulphur is, however, absorbed from the bowel and, finally, excreted in the urine, partly oxidized as sulphate. The mineral sulphates of the urine are the sulphates of sodium, potassium, magnesium and calcium. The com- position of the aromatic, or conjugate, sulphates has been discussed in another chapter; the composition of the neutral sulphur compounds will be discussed below. Clinical Significance of the Sulphates as a Group. — As the bulk of the urinary sulphates is derived from the metabolism of albumen, the sulphate excretion gives us some information in regard to the degree of disassimi- lation of albumen in the body; the sulphur of the urine consequently partakes of the same significance as the urinary nitrogen. Whereas, however, all albumens con- tain the same amount of nitrogen, they do not all contain the same amount of sulphur (0.8-2.1 per cent) ; con- sequently a change in the kind of albuminous food eaten will determine a change in the sulphur excretion but not in the nitrogen excretion, provided the quantity of albu- men remains the same. The average proportion of nitro- gen to sulphur is as 16 : 1 ; the proportion of nitrogen to H2SO4 is 5 : 1 (Voit) . In febrile disorders the sulphate excretion approxi- mately parallels the nitrogen excretion, for both are de- pendent on the degree of albumen destruction. Occa- sionally more nitrogen is excreted than sulphate; this is probably due to the fact that the kidneys are more per- meable for urea than for sulphates. If the total sulphur, i. e., the sulphates and the neutral sulphur compounds, are included in the calculation, then fewer deviations from the parallelism will probably be found. As a matter of fact, as I will show below, the neutral sulphur excretion is generally increased in febrile diseases. INORGANIC CONSTITUENTS OF THE URINE 185 In gastric disorders the sulphate excretion rises and falls with the ingestion of albuminous food, and parallels the nitrogen excretion. It is interesting to note that in achylia gastrica the conjugate sulphate excretion is no greater than in hyperchlorhydria, for this demonstrates, when we remember the chief origin of the conjugate sul- phates from putrefying albumen in the bowel, that, many older writers to the contrary notwithstanding, the gastric HCl in no way influences intestinal bacterial putrefaction. In icterus with diapedesis of bile the neutral sulphur of the urine is generally increased (taurin) , whereas the sulphates fluctuate with the amount of albumen that is eaten and maintain the same proportion to the urinary nitrogen that we see in normal subjects. After the icterus has persisted for some time the urinary neutral sulphur again deei-eases. In nephritis the sulphate excretion runs parallel to the urea excretion; occasionally exceptions are noted, chiefly in very acute forms of nephritis and in amyloid kidney, sometimes in chronic parenchymatous nephritis. The exact rules that govern the relative permeability of the diseased kidneys for sulphates and urea are not clear; we must wait until we are in possession of more casuistic material before drawing conclusions. Much has been written in regard to the clinical signifi- cance of changes in the ratio between conjugate and total sulphates, and the ratio between conjugate sulphates and the total sulphur. When one considers, however, that the conjugate sulphates are largely derived from intes- tinal putrefaction, whereas the excretion of the total sul- phates and the total sulphur is largely dependent on the degi'ee of intracellular disassimilation of albumen, and is in no way dependent on intestinal putrefaction, it will be seen that the two values are not comparable at all, and that changes in the ratio between the two are no index of normal or abnormal function. It is still more unreason- 186 CLINICAL URINOLOGY able to compare the excretion of the conjugate sulphates with the excretion of preformed sulphates, for the latter merely indicate the amount of sulphate that remains after the aromatic radicles have combined with all the circiilat- ing sulphuric acid they require (see chapter on "Aromatic Constituents of the Urine"). The total sulphate excretion, then, gives us some in- formation in regard to the degree of albumen metabolism and is of clinical importance in certain pathological states that have been enumerated above. It normally approxi- mates from 1.5 to 3 grams in twenty-four hours in a healthy adult living on an average mixed diet. The conjugate sulphate excretion informs us in regard to the degree of intestinal putrefaction and certain other pathological conditions that have been described in the chapter on "The Aromatic Constituents of the Urine." The preformed sulphates, finally, indirectly indicate the amount of conjugate sulphates when subtracted from the total sulphates, but taken alone they have virtuallv no clinical significance. The method of estimating their quantity may, however, be given because occasionally the differential determination of the conjugate sulphates (by subtracting preformed from total sulphates) is more convenient and accurate than the direct determination of this important group of bodies. Quantitative Determination of the Total Sulphates. — The urine is treated with several cubic centimeters of con- centrated HCl and boiled ; this decomposes the conjugate sulphates into aromatic constituents and sulphuric acid. The liquid is now treated with a slight excess of barium chloride ; this precipitates all the sulphuric acid as barium sulphate. The mixture is allowed to stand for twenty- four hours, the precipitate filtered off, washed with cold water, alcohol and ether, and dried; the filter with the precipitate is then incinerated and weighed. One hun- dred parts of barium sulphate correspond to 34.28 parts INORGANIC CONSTITUENTS OF THE URINE 187 of SO3, or 41.43 parts of SO4, or 41.99 parts of H2SO4, and the result may, therefore, be expressed as grams, either of SO3, SO4 or H2SO4 by multiplying the weight of the barium sulphate precipitate with 0.3428, 0.4143 or 0.4199. Quantitative Determination of the Preformed Sulphates. — The barium precipitate -obtained from the urine direct by the addition of barium chloride contains oxalates and phosphates; it is not, therefore, barium sulphate alone; in order to get rid of the oxalates and phosphates the precipitate must be repeatedly treated with HCl in a platinum crucible. This is a very tedious and somewhat complicated procedure and cannot be recommended for clinical work. If it is desired, therefore, to determine the preformed sulphates at all (see above) the convenient titration method of Freund may be employed to advan- tage. 50 cc. of the urine are treated with 10 drops of a one per cent solution of alizarin red; to the red mixture is added five per cent acetic acid drop by drop until the red color disappears. When this point is reached, 5 cc. more of acetic acid are added and the urine heated almost to boiling and a solution of barium acetate added from a burette until the urine again turns red. The barium acetate solution is of such a strength that 24 cc. corre- spond to 0.2101 grams of BaS04, i. e., it contains 9.579 grams of anhydrous barium acetate to 1,000 cc. of water. Knowing that 24 cc. of the barium acetate solution cor- respond to 0.2101 grams of barium sulphate, the calcula- tion of the preformed sulphate present in the 50 cc. of urine used for the determination is simple Quantitative Determination of the Conjugate Sulphates (Method of Baumann) . — 50 cc. of urine are rendered acid with acetic acid, diluted one-half and precipitated with barium chloride in excess, heated on the water-bath until the barium sulphate settles, and the latter filtered ofE and 188 CLINICAL URINOLOGY repeatedly washed out with cold water. In this way the preformed sulphates are removed. The filtrate and wash- ings are united and the conjugate sulphates they contain decomposed with strong HCl, as described under total sul- phates. The liberated sulphuric acid is then determined as barium sulphate, as described above. 5. The "Neutral Sulphur Compounds" of the Urine. — In the preceding paragraphs the "neutral sulphur com- pounds " of the urine have been repeatedly mentioned. The bulk of the sulphur that is split off when albumen under- goes disassimilation appears in the urine in a highly oxi- dized form, i. e., as sulphate, either in combination with certain inorganic bases and ammonia, or in combination with certain aromatic radicles that are largely derived from the putrefaction of albumen in the bowel ; a portion of the sulphur, however, seems to escape oxidation and appears in the urine in the form of a variety of complex compounds that are grouped under the name of "neutral" sulphur compounds, in contradistinction to the above "acid" sulphur compounds. The relation between the total sulphur, the neutral and the acid sulphur compounds of the urine may be illus- trated by the following diagram: Acid Sulphur Com- f i. Preformed (mineral) sulphates. on'^r^^ u p es I 2 Conjugate (aromatic, ethereal) 80-86 per cent. o i >. ^ Total '- bulphates. Sulphur I Neutral Sulphur Com- r pounds ( Suboxi- I cystin and Taurin, Thiosulphates, dized Sulphur) 14- \ Sulphocyanides, etc. 20 per cent. (^ From 14 to 20 per cent of the total urinary sulphur consists of such neutral compounds. The exact reason why this portion of the sulphur does not undergo com- plete oxidation remains for the present obscure. A large proportion of the neutral sulphur compounds are certainly derivatives of the taurocholic acid of the bile, a portion INORGANIC CONSTITUENTS OF THE URINE 189 of which is always reabsorbed from the bowel ; possible that this portion is not reeliminated into the bowel, but passes into the urine instead. This view is strengthened by the discovery I made some years ago (1. c. page 124) that the leucocytes of normal blood always carry some bile-acids. A certain part of the neutral sulphur compounds, then, is derived from the taurocholic acid of the hile^ this taurin derivative appearing in the urine in the form of taurocarbaminic acid. In patients with biliary fistula the amount of neutral sulphur compounds in the urine is consequently reduced, whereas, per contra, in biliary ob- struction the urinary neutral sulphur is increased. A second part of the neutral sulphur compounds of the urine consists of sulphocyanides, and these are derived directly from the saliva. The salivary glands, it is well known, excrete some potassium sulphocyanide (KCNS) ; this is swallowed, reabsorbed from the bowel and excreted in the urine. A third part of the neutral sulphur compounds may, under pathological conditions, consist of thiosulphates, i. e., salts of thiosulphuric acid (S02ZsS). This body is never present in normal urine, but has occasionally been found in typhoid urine. In cats and dogs this body is a common urinary constitutent, and its excretion can be greatly increased in these mammals by feeding sub- limed sulphur ; in man the latter is often completely oxi- dized and appears in the urine as sulphate. There seems, therefore, to be a considerable difference in different ani- mals in regard to the oxidative energy toward sulphur. The occasional appearance of thiosulphates in fever urine must, presumably, be attributed to some perversion of oxidation. The clinical significance of this urinary body is for the present nil. The urine finally seems to contain a neutral sulphur compound in solution that is related to cystin. This body 190 CLINICAL URINOLOGY is presumably an intermediary complex lying between the proteids and the highly oxidized sulphates that are ulti- mately split off from them. Under certain conditions, the cystin of the urine may be greatly increased, and we then have an anomaly of considerable clinical importance ; viz., Cystinuria. Cystinuria.— Theoretically, cystin should appear in the urine whenever the oxidation of the sulphur- containing radicle of albumen is interfered with. Normally, it ap- pears, the oxidation of cystin is, as a rule, complete, so that normal urine contains only traces of this body or none at all. Occasionally, however, the urine may con- tain so much cystin that the neutral sulphur constitutes nearly 50 per cent of the total sulphur excretion. Cystinuria, like alkaptonuria, is apparently a congeni- tal chemical anomaly, a "metabolic sport," for the disease seems to occur in several members of the same family and to persist with fluctuations during the lifetime of the patient. Many explanations have been vouchsafed. Some authors lay particular stress upon the fact that cystinuria is often accompanied "by the excretion of diamins, i. e., certain ptomains (putrescin, cadaverin, neuridin, saprin) that are unquestionably of gastrointestinal origin. The theory has been advanced that these diamins when they ai-e absorbed into the circulation form compounds with the normal intermediary cystin complex, and thus protect it from further oxidation. The diamin- cystin compound when it reaches the kidneys is then split into its compo- nent parts and we have diaminuria and cystinuria. In support of this view, attention is called to analogous conditions existing in regard to glycuronic acid and gly- cocoU (see Chapter VII) , two bodies that combine with certain aromatic bodies of gastrointestinal origin and thus escape further oxidation, appearing in the urine as compound glycuronates and glycocolls. Further attention INORGANIC CONSTITUENTS OF THE URINE 191 is directed to the fact that as camphor, for instance, can bind glycuronic acid, and benzoic acid can bind glycocoll (see Chapter VII), so brom-bensole can bind cystin, pro- tect it from further destruction and cause the excretion of a cystin compound. The diamins are believed to act hke brom- benzole. At all events, these diamins must be formed by some peculiar and pathological form of intes- tinal putrefaction, for the urine does not normally contain them. Hence, cystinuria when combined with diaminuria has been attributed to some special form of intestinal mycosis. There are, however, also cases of cystinuria on record in which no diamins appeared in the urine. Here we must either assume some obscure arrest of the oxidation of the tissue -albumens, or we must have recourse to still another theory that has recently been advanced and that sounds plausible. According to this idea, cystin is normally transformed into taurin, and the latter, combining with cholalic acid, forms the taurocholic acid of the bile men- tioned above. In case the transformation of cystin to taurin (for unknown causes) is impeded, or in case a sufficient quantity of cholalic acid (again for unknown causes) is not formed, then taurocholic acid, the normal product, cannot be formed, and cystin is poured into the circulation instead, and hence appears in the urine. It will be seen from the above that the pathogenesis of cystinuria is very obscure. I have sketched the various theories that have been advanced for completeness sake, and because they suggest important and fruitful researches in this unknown field. The chief clinical significance of an increased cystin excretion is the following: When cystinuria persists for years, it merely indicates a peculiar metabolic anomaly that may in no way impair the patient's health and need not shorten his life. The chief danger lies in the possible formation of cystin gravel and cystin calculi (see Sedi- 192 CLINICAL URINOLOGY merits and Concretions) ; one should, therefore, always be on guard for this occurrence, Cystinuria with diami- nuria presumably indicates the invasion of the bowel by abnormal parasites, and, as the diamins are toxic, sig- nifies a form of self -poisoning ; in such cases the toxicity of the urine should be tested and proper measures insti- tuted to clean out the bowel and to keep it clean. Clinical Significance of the Neutral Sulphur Compounds. — Normally, the small quantities of neutral sulphur that are excreted in the urine are largely derived from the saliva and the bile; a small portion is derived from the putrefaction of albumen in the bowel and from the incom- plete oxidation of tissue-albumens. Pathologically, the neutral sulphur is increased in inanition; this finding is diflficult to interpret, for one should imagine that the sulphur excretion would be de- creased, inasmuch as less albumen than normal is dis- assimilated, and less saliva and bile are secreted during fasting than during full meals. Possible that the reduc- tion may be attributed to sluggish metabolism and defi- cient oxygenation of the sulphur compounds that are poured into the blood-stream. In certain febrile disorders, particularly in pneumonia and in typhoid, large quantities of the total sulphur appear in the form of neutral sulphur; in typhoid, as stated above, some thiosulphates also occasionally appear in the urine. In disorders of the liver accompanied by biliary stasis and diapedesis of bile, the neutral sulphur is always in- creased. This is clear when we consider that a large pro- portion of the neutral sulphur of the urine is derived from taurocholic acid. Some clinicians subdivide the neutral sulphur com- pounds of the urine into two groups; viz., such com- pounds that can readily be oxidized (chiefly the salivary sulphocyanides), and such compounds that are oxidized only with difficulty (chiefly the biliary sulphur bodies). INORGANIC CONSTITUENTS OF THE UBINE 193 In bile stasis, particularly in the beginning, the latter are chiefly increased; gradually the neutral sulphur excretion decreases, and in chronic icterus it reaches normal values. In biliary fistula exactly the reverse is seen, because less taurocholic acid, or none at all, enters the bowel and is reabsorbed into the blood. After violent muscular exercise, in asphyxia and dysp- noeic states, after the administration of chloroform and of chloral, after the ingestion of sublimed sulphur the neu- tral sulphur compounds of the urine may be increased. Determination of Neutral Sulphur Compounds. — The preformed and conjugate sulphates are first precipitated as barium sulphate (see determination of total sulphates above), the barium sulphate filtered off, the excess of barium chloride removed from the filtrate by soda, the fil- trate evaporated to dryness and the residue oxidized. Various oxidizing agents have been recommended, the most simple one being a mixture of four parts of niter and one part of soda. The residue is fused with this mixture in a platinum crucible, the residue taken up in about 100 cc. of dilute hydrochloric acid and transferred to a porcelain dish. The solution is evaporated to dry- ness, again dissolved in 100 cc. of HCl, evaporated, then dissolved in 100 cc. of HCl a third time and evaporated to dryness; all this is necessary to drive off nitric acid. The residue is then dissolved in water, filtered, and the sulphate that has been formed by the oxidation of the neutral sulphur precipitated with barium chloride, and the barium sulphate precipitate weighed as described in another paragraph. Sometimes it is desirable to determine the readily oxi- dizable neutral sulphur ("salivary sulphur") and the sul- phur that is difficult to oxidize ("biliary sulphur," see above) separately. To do this the sulphates are first removed, the filtrate acidulated and treated with bromine water; on addition of barium chloride, any sulphur that M 194 CLINICAL URINOLOGY may have been oxidized to sulphuric acid by this mild oxidation will be precipitated as barium sulphate. By subtracting this amount from the total neutral sulphur determined as above, the amount of sulphur that is oxi- dized with difficulty can be determined. Qualitative tests for thiosulphates and sulphocyanides will not be given, because these bodies possess no clinical importance. If the urine contains much sulphocyanide (rhodanate), the addition of a ferric chloride solution should produce a red color. Cystin may be precipitated from the urine with ben- zoyl -chloride, and the sulphur determined in the precipi- tate. 200 cc. of the urine are treated with 10 cc. of ben- zoyl -chloride and 70 cc. of a 10 per cent solution of sodium hydrate, and the mixture shaken until the odor of ben- zoyl -chloride disappears. The precipitate of benzoyl- cystin can be separated by filtration and used directly for a sulphur determination. The benzoyl- cystin is oxidized as above, the sulphuric acid precipitated as barium sul- phate, weighed, and the sulphur calculated. From the sulphur the amount of cystin can readily be estimated, the empiric formula of cystin being (C3H6NS03)2- The benzoyl -chloride precipitate may be tested for benzoyl- cystin by boiling it with a solution of lead oxide in soda lye. A black sediment of lead sulphide should form. A more complicated method for determining the cystin as benzoyl-cystin has been devised by Udransky and Baumann (see Zeitschr. f. jphys. Chem. vol. 13, p. 564). The above method, however, is sufficiently accurate for all clinical purposes. 6. Sulphuretted Hydrogen (H2S) (Sulphides). — This sulphur compound properly belongs to none of the above groups of sulphur bodies; it is a neutral sulphur com- pound, but it is not genetically related to the other bodies included under that category; it may, therefore, be briefly discussed separately. INORGANIC CONSTITUENTS OF THE UBINE 195 Sulphuretted hydrogen or its salts the sulphides are rarely found in fresh urine, but nearly every old urine contains these bodies and emits a disagreeable odor of H2S when treated with mineral acids. Sulphuretted hydrogen and sulphides are the products of decomposi- tion of the urinary sulphur bodies. Occasionally certain bacteria that can generate H2S infect the bladder so that the gas is generated in this viscus and then the freshly voided urine emits an odor of rotten eggs, that is inten- sified on the addition of mineral acids. If an abnormal communication exists between the rectum and the bladder, H2S may pass into the urine from the fasces. Intestinal putrefaction or putrefaction in the tissues do not lead to the passage of sulphides into the urine, nor are sulphides excreted in the urine after the ingestion for medicinal purposes of waters containing the alkali sulphides ; nor does bathing in such waters lead to uri- nary sulphide excretion. Tests for H2S and Sulphides in the Urine.— The urine should, of course, be fresh. The odor may in itself reveal the presence of H2S, particularly after the addition of mineral acids. A piece of paper saturated with lead acetate may be held over the urine and air blown through ; if the urine contains II2S the gas will be driven off and will blacken the paper. 7. Phosphoric Acid (Phosphates). — Phosphoric acid forms four different salts with the bases that it combines with; all four of these salts may appear in the urine. Using the sodium salts of phosphoric acid as a prototype, we have the following four compounds, viz.: H3PO4 = Phosphoric Acid. (1) NaH2P04 = Mono- or primary sodium phosphate.. (2) Na2HP04 = Di- or secondary sodium phosphate. (3) Na3P04 = Tri- or tertiary* sodium phosphate. (4) Na3P04 (+NaOH) = Basic sodium phosphate t. * Also called "normal" sodium phosphate. t Sodium salts of this type do not actually occur in the urine, but calcium and magnesium phosphates do. 196 CLINICAL URINOLOGY Salts (1), (2) and (3) are also spoken of as acid, neu- tral and basic phosphates and salt (4) as "over basic" phosphate. The mono -phosphates can be converted into di- and tri-phosphates by the addition to the urine of alkali hydrates or alkali carbonates. Inversely, the tri- and di-phosphates can be converted into mono-phosphates by the addition of mineral acids to the urine. When phosphoric acid is combined with the alkalis Na and Ka, one speaks of alkaline phosphates ; when combined with Ca and Mg, of earthy phosphates. Small quantities of the urinary phosphoric acid finally are combined with organic radicles. The phosphoric acid excretion is usually expressed in terms of P2O5, i. e., phosphoric acid anhydride; the average P2O5 excretion in a normal adult is 3.5 grams pro die ; of this amount about 60 per cent appears as mono- phosphate, 40 per cent as di-phosphate. The phosphoric acid excretion is dependent on the composition of the food and on the catabolism of the tissues of the body, chiefly of those tissues that contain nuclein. The former source is by far the greater. The food always contains some preformed mineral phosphates, as calcium and potassium phosphates in bone, meat and cereals; in addition, it contains considerable quantities of organic phosphorus compounds that undergo oxidation in transit through the body and appear in the urine as mineral phosphates. The two chief organic phosphorus compounds of the food are the nueleo- albu- mens and the lecithins ; the former being present in yolk of eggs, in casein, in milk, and in all organs containing nuclei, the latter being the principal constituent of brain- and nerve -tissues, and of yolk of egg, young germinating plants, etc. It is for the present undecided whether the nueleo -albumens and lecithins are, in part, absorbed as such and directly utilized by the organism or whether they are split into their component parts in the gastro- IN0R6ANIG CONSTITUENTS OF THE URINE 197 enteric tract, to be subsequently built up into living nuclein and lecithin; in either event the phosphorus they incorporate ultimately appears in the urine (and in part in the faeces) as highly oxidized phosphate. It is an interesting and an important clinical fact that the urinary phosphate excretion is largely dependent on the calcium content of the ingesta; the more calcium the food contains, the less phosphoric acid appears in the urine, and the more in the faeces. This is due, on the one hand, to the tendency on the part of calcium to form in- soluble calcium phosphates in the gastro- enteric tract and in this way to prevent the absorption of the food phosphates; on the other hand, to the well-established tendency of calcium salts to be excreted into the bowel and not into the bladder; one must imagine in the latter case that calcium salts circulating in the blood combine with circulating phosphoric acid and bear the latter with them into the bowel. This fact is of some therapeutic importance in the treatment of nephrolithiasis due to uric acid calculi, for the administration of calcium salts in this affection by tearing much phosphoric acid into the bowel, leads to the excretion in the urine of less phosphoric acid, and hence of normal and basic instead of acid phosphates; and as the latter precipitate and the former dissolve uric acid, it will be seen that by giving calcium we prevent the pre- cipitation of crystalline uric acid and urate deposits in the urinary passages. (See my monograph on "The Admin- istration of Calcium Salts in Nephi'olithiasis Due to Uric Acid Calculi," Journ. Am. Med. Assn., March 28, 1903.) The minimum phosphate excretion occurs in the urine if the food contains considerable calcium and if besides the urine is alkalinized by the administration of sodium or po- tassium salts. In herbivorous animals, who eat enormous quantities of phosphorus-containing food, but whose urine is alkaline, very little phosphate is excreted in the urine. 198 CLINICAL URINOLOGY From all these facts it will be understood that, in pass- ing judgment on the phosphorus metabolism of the or- ganism, the phosphate excretion in the faeces must always be included in the calculation. Clinical Significance of the Urinary Phosphate Excre- tion. — It is precisely owing to failure to do this that much of the work that has been published on the urinary phos- phate excretion in various disorders is of little clinical value, for the amount of urinary phosphates alone is no index of the catabolism of phosphorus -containing foods and tissues, but represents merely the difference between the total phosphate excretion and the phosphate excre- tion in the stools; as the latter is, as we have seen, in a large measure dependent on the character of the phos- phorus-bearing compounds of the food, on the amount of calcium salts ingested, and on the alkalinity of the blood and lymph, it will readily be understood that the urinary phosphate excretion can be utilized only as a basis for clinical conclusions if all these factors are considered and if the faeces-phosphates are determined at the same time as the urine-phosphates. In disorders accompanied by loss of flesh, the attempt has often been made to draw conclusions in regard to the particular tissues that are wasting from the relative excre- tion of nitrogen and phosphorus in the urine. This can actually be done if the faeces-phosphorus is also deter- mined; without the latter determination, however, such calculations are foolish. In inanition and chronic under-nutrition the relation of nitrogen to phosphorus in the urine remains approxi- mately the same as in normal feeding; in fevers, with rapid loss of flesh, there is no uniformity — some authors claim a constant increase, others a constant decrease of the phosphate excretion in the urine. The problem in fevers is so complex that the factors influencing the urinary phosphate excretion must be carefully analyzed INORGANIC CONSTITUENTS OF THE UEINE 199 in each case. For (1) the ingestion of food and hence of food-phosphorus is, as a rule, decreased in fevers; (2) the catabolism of phosphorus-holding tissues (glands, nervous tissues) is usually increased; (3) there is often renal irritation, even nephritis, with occasional retention of phosphates; (4) many white blood-corpuscles (leuco- cytes) with phosphorus -containing nuclei are manufac- tured and later destroyed, so that at first they would retain phosphorus, later pqur phosphates into the blood- stream. The problem, it will be seen, is chaotic, and I doubt whether any of the statements made with so much assurance by different writers in regard to the retention or the increased outpouring of urinary phosphates in fevers can stand the light of critical illumination. One should expect that in leucaemia the urinary phos- phates would be greatly increased owing to the destruc- tion of many leucoeytic nuclei; as a matter of fact, the phosphate excretion is often relatively increased, but the proportion of phosphorus to nitrogen remains the same as in normal subjects. In advanced stages of any form of ancemia, however, the P2O5 excretion is greatly in- creased, showing that some tissue containing much phos- phorus is undergoing disassimilation ; presumably this is bone. There is a popular prejudice to the effect that in cer- tain functional and organic nervous disorders the urinary phosphate excretion is increased. This is false, for assum- ing even that there were increased catabolism of nerve - tissue in these disorders, then the amount of phosphorus poured into the circulation and eliminated in the urine would be so small, as compared to the quantities derived from the food or the tissues at large, that the increase would be hardly perceptible. In nephritis, phosphates are occasionally retained, so that the urinary phosphate excretion reaches remarkably low figures. This may occur even when the nitrogen 200 CLINICAL URINOLOGY excretion is not reduced. In some cases, therefore, the kidneys seem to become especially impermeable for phos- phates; as a rule, however, this impermeability soon yields to permeability and phosphates are again elimi- nated in a broad stream. The phosphate excretion in nephritis, therefore, in general follows the same rule as the excretion of all other urinary solids; viz., there is retention during the early stages of acute nephritis, the terminal stages of destructive nephritis and the acute exacerbations of chronic forms of renal inflammation and degeneration ; in other forms the phosphate excretion fluctuates within wide boundaries, and these fluctuations, as far as we know today, follow the same rules as similar fluctuations in individuals with healthy kidneys. In diabetes the phosphate excretion is, as a rule, great, and corresponds to the increased nitrogen excretion; the increase is due to the ingestion of large quantities of food in this disease. As the total calcium excretion also seems to be inci'eased in diabetes, it is not impossible that some bone is destroyed in this disorder and that some of the urinary phosphate is derived from this source. In gout and goutiness the phosphate excretion seems to follow the same fluctuations as the uric acid excretion between, during and after attacks. The origin of both uric acid and phosphates from nuclein readily explains this parallelism. The existence of digestive or renal complications may, of course, readily upset this relation. Tests for Phosphates in the Urine. — As the urine al- ways contains phosphates, it is superfluous to perform special qualitative tests. Alkaline urine or urine that becomes alkaline through bacterial decomposition, or that is made alkaline by the addition of an alkaline re- agent, always precipitates a sediment of earthy phos- phates, while the alkaline phosphates remain in solu- tion. This sediment may consist of the di- or tri-phos- phate of the earthy alkalies, or, if ammonia or ammonia INORGANIC CONSTITUENTS OF THE UBINE 201 salts are present in sufficient quantity, of ammonium-mag- nesium phosphate. It is clear from all this that the quantity of total urinary phosphates should not be esti- mated from the bulk of the phosphate sediment. In testing urine for phosphates, some ammonia should be added; this causes a precipitation of earthy phosphates, while the filtrate contains normal alkali phosphates. The latter when treated with magnesia mixture (100 grams of magnesium chloride dissolved in water, precipitated with strong ammonia, the precipitate redissolved by adding a strong ammonium chloride solution and the liquid filled up to 1,000 cc) gives a precipitate of triple phosphate (see chapter on Urinary Sediments) . Or the filtrate may be acidulated with acetic acid and tested for phosphoric acid with uranium nitrate or ferric chloride; the former producing a yellowish white precipitate, the latter a white precipitate that stains yellow if an excess of ferric chloride solution is added. On boiling urine that is faintly acid, a flocculent sedi- ment of calcium phosphate often forms* that may be mistaken for albumen (see boiling test for serum albu- men). If the sediment is phosphate it will dissolve on the addition- of a few drops of dilute mineral acid, whereas an albumen coagulate will remain or become thicker. Occasionally it is necessary to remove all the phos- phates from the urine in order to perform certain tests. This can best be done by precipitating the urine with neutral or basic lead- acetate and filtering off the sedi- ment; or the urinary phosphates are all converted into normal phosphates by treating the urine with alkali hydrate and the solution precipitated with calcium or barium chloride. The filtrate will be free from phos- phates. Quantitative Estimation of Urinary Phosphates. — The separate estimation of the different urinary phosphates is •See, also, under Calcium, page 210. 202 CLINICAL URINOLOGY of no clinical importance and will hence not be given in this book. The estimation of the different bases will be given below. The estimation of the total phosphoric acid, expressed, as explained above, as P2O5 is performed as follows: Solutions: (1) Solution of uranium nitrate containing 35.461 grams of the salt to a liter. Uranium nitrate is dissolved in a little less than 1,000 cc. of water and titrated against a solution of disodium phosphate (10.0845 grams of Na2HP04,12H20 to 1 liter), 50 cc. of which should correspond to 0.1 gram of P2O5. In order to bind the nitric acid developed in this titration, 3 grams of sodium acetate are added to the uranium nitrate solution. The results of the titration indicate how much the ura- nium nitrate solution must be diluted to correspond to the phosphate solution. (2) A solution of 100 grams of sodium acetate and 30 grams of acetic acid to a liter; 5 cc. of this mixture are used for every 5 cc. of urine. The solution contains enough acetic acid to convert all the urinary phosphates into mono -phosphate. (3) Tincture of cochineal to be used as an indicator, 5 grams of cochineal are dissolved in the cold in 500 cc. of a mixture of 4 parts of water and 1 part of alcohol. Any undissolved residue is removed by filtration. The uranium solution is now standardized; 20 cc. should exactly neutralize 50 cc. of the phosphate solution; 50 cc. of the latter solution are mixed with 5 cc. of the acetate solution (2) and a few drops of the indicator (3) added. The mixture is heated to boiling and uranium solution added, drop by drop. The appearance and per- sistence of a green- colored precipitate indicates the end reaction. If in this titration only 18 cc. of uranium solu- tion were required to neutralize the 50 cc. of phosphate, then the uranium nitrate solution must be diluted so that 2 cc. of water are added to every 18 cc. of the solution. INORGANIC CONSTITUENTS OF THE URINE 203 Execution: Fifty cubic centimeters of urine are treated exactly as the 50 cc. of phosphate solution above. As 20 ec. of the uranium nitrate solution correspond to 50 cc. of the phosphate solution, and as these 50 ec. represent exactly 0.1 grams of P2O5, the phosphate content of the urine can be estimated by simple calculation from the number of cubic centimeters of the uranium solution required to bring about the end reaction in 50 cc. of urine. Carbonic Acid (Carbonates). — The fluctuations in the urinary carbonate excretion are of subordinate clinical, but of considerable chemical interest. The urinary car- bonic acid and carbonates are largely derived from in- gested carbonates (taken as medicines or in mineral waters) and from the vegetable acids of the food (as citric, acetic, tartaric acids, etc.), the latter being oxi- dized to carbonic acid and combining with fixed alkalis or ammonia in transit through the body. The urine of herbivorous animals consequently contains large quanti- ties of alkaline and earthy carbonates, the latter often forming a sediment. Cows' or horses' urine may contain so much carbonate that it foams on addition of a mineral acid. Carbonic acid is present in the urine as free carbonic acid gas, or as alkaline or earthy carbonate in solution or as a sediment. The free carbonic acid gas can be driven off by physical means (boiling, evacuation, pas- sage of an air- current), the bound carbonic acid only by chemical means, i. e., the disassociation of the carbon- ates with some mineral acid. The proportion of the free to the bound carbonic acid of the urine is largely dependent on the amount of mono- phosphates present in the urine; the latter possessing the power of liberating CO2 from the alkali -carbonates; if the gas escapes or is driven off, more alkali- carbonate is disassociated until finally only traces of it are left. 204 CLINICAL URINOLOGY Carbonic acid forms mono- (acid) and di- (normal, neutral) carbonates of the types NaHCOa and Na2C03. The urine may contain the carbonates of K, Na and NH3, and of Ca and Mg. The latter are less soluble by far than the former, and hence have a tendency to precipi- tate.* (See "Urinary Sediments.") Normal urine of the specific gravity of 1020 and of an acid reaction contains an average of 50 cc. of free car- bonic acid gas when fresh ; urine of an alkaline reaction usually contains more, 100 cc. or over. The total quan- tity of carbonic acid fluctuates with the diet; on an ordinary mixed diet the average lies between 250 and 400 cc, on a vegetarian diet the urine contains more, i. e., 450 to 600 cc. It appears that in febrile disorders the urinary carbonic acid is often found increased. Tests foe Carbonic Acid. — (1) Free Carbonic Acid. — A current of air is first passed through a sodium hydrate solution (to remove the CO2 it contains), then through the urine and finally through a solution of barium hydrate (baryta water). The CO2 in the urine will be expelled by the air-current and passing through the baryta water will cause clouding of the liquid (formation of barium carbonate). (2) Mineral Carbonates. — The urine is treated with mineral acids and the liberated CO2 deter- mined as above. The quantitative estimation of the urinary carbonic acid is performed according to the same principle with the difference that the vessel containing the baryta water is weighed before and after the air- current loaded with CO2 passes through it. For the details of this simple determination, I refer to text-books of analytical chem- istry. The estimation of CO2 is of too small clinical significance to warrant a full description of the method in this place. *See also under Calcium, p. 210. INORGANIC CONSTITUENTS OF THE URINE 205 Silicic Acid (Silicates), Nitrates and Nitrites, Peroxide of Hydrogen are all occasionally encountered in traces in human urine. The silicates, nitrates and nitrites are derived from the drinking water ; the source of the hydro- gen peroxide is unknown. None of these inorganic bodies possess any clinical significance whatsoever, so that, fol- lowing the general plan of this book, the methods for their detection and estimation in the urine will not be described. THE INORGANIC BASES OP THE URINE The inorganic bases of the urine can be conveniently discussed in three groups; viz., (1) the group of fixed aSksli&^potassiwm and sodium; (2) the group of earthy alkalis — calcium and magnesium^ and (3) ammonia. The urine also contains iron, that under certain conditions, to be related below, acquires some clinical significance. Many facts relating to the excretion of these bases have been given under inorganic acids of the urine and their salts ; this information may be supplemented by the following facts. Potassium and Sodium. — The proportion of K and Na normally excreted on an average mixed diet corresponds to the proportion of these two elements in the food; viz., it is approximately as 2 : 3. As meat contains relatively much K and relatively little Na, a meat diet leads to a relatively increased excretion of K, so that, then, the two are eliminated in approximately equal quantities. Many vegetables, too, contain much K; the excess of Na in the urine on a mixed diet is, therefore, largely due to the ad- mixture to our food of the NaCl, that our palate craves. Clinical Significance. — In starvation the proportion of K to Na is reversed, more K being excreted thanNa. This is due to the fact that (1) the excretion of circulating NaCl stops as set forth at length under "Chlorides," the or- ganism jealously guarding its NaCl- content when no NaCl 206 CLINICAL URINOLOGY is ingested; (2) the tissues of the body proper, that con- tain much K (as phosphate) and little Na (the NaCl being chiefly found in the circulating fluids of the body and not in the tissues) undergo disassimilation. This reversal of the K-Na-quotient must be considered characteristic for all diseased states in which the body consumes its own tissues. In acute febrile diseases this reversal is particularly strik- ing, because here, as a rule, little food (i. e. , NaCl) is eaten, and the body also consumes much of its proper substance. In convalescence from fevers the relative excretion of K- salts decreases again, and this signifies retention of food pabulum and reconstruction of body-tissues. The excretion of sodium as chloride in nephritis has already been discussed under chlorides, the excretion of potassium as phosphate under phosphates. The ingestion of certain K-salts as the citrate and the phosphate increases the excretion of sodium (as chloride) , and, vice versa, the ingestion of sodium citrate and phos- phate increases the excretion of potassiurri, though not to such a marked degree. Excessive muscular exercise, by causing catabolism of muscle tissue holding much potas- sium phdsphate, seems to increase the K-excretion. The urine voided in twenty -four hours by a healthy adult on a mixed diet contains from 2 to 4 grams of potas- sium (expressed as K2O) and from 4 to 8 grams of sodium (expressed as Na20). Estimation of Sodium and Potassium in the Urine. — As the urine always contains K and Na, qualitative tests for these metals are superfluous. The presence of Na can be determined by spectroscopic examination of the urinary ash (line D in the spectrum) and by the yellow flame produced on glowing the ash. The presence of potassium is made known by the precipitation of yellow octahedral crystals of potassium platinum chloride when the urine is acidulated with HCl and treated with two volumes of an alcohol-ether solution of platinum chloride. INORGANIC CONSTITUENTS OF THE URINE 207 The best methods for determining the quantities of Na and K in the urine are based on the following principle: The Na and K salts of the urine are converted into chlo- rides and isolated as such; the NaCl and KCl are then weighed together and the K determined as K-platinum chloride; the amount of K is calculated as chloride, de- ducted from the sum of NaCl and KCl obtained before, and the NaCl (i. e., Na) calculated from the difference. The quickest method, and the one that in my hands has given altogether reliable results, is the following one: 100 cc. of urine are treated with baryta water and evaporated to dryness, the dry residue incinerated at red heat, the carbonaceous residue extracted with hot water and repeatedly washed with water on a filter. The filtrate is evaporated to di-yness and redissolved in hot water; this causes a precipitation of some more of the earthy alkalis ; the latter are filtered off, the filtrate evaporated to dryness with HCl, and the residue of alkali chlorides glowed at low temperature. These are weighed. The residue is now dissolved in a little water, poured into a platinum crucible and treated with an excess of platinum chloride solution ; the liquid is slowly evaporated on a water -bath until a crystalline sediment begins to form on cooling. After crystallization is complete, a mix- ture of one part of ether and four parts of absolute alcohol is poured into the dish and the mixture allowed to stand for two hours. The supernatant liquor is poured off through a small weighed filter, the crystals of potassium-platinum chloride then washed onto the filter with ether -alcohol mixture and repeatedly washed with the same liquid. The filter is then dried in a vacuum desiccator and weighed. This weight less the weight of the filter indicates the amount of K-platinum chloride. As 100 parts of the lat- ter salt correspond to 30.69 parts of KCl, the amount of KCl can readily be calculated. This subtracted from the weight of the total alkali chlorides obtained before gives 208 CLINICAL URINOLOGY the amount of NaCl. By multiplying the KCl with 0.6317 the amount of K2O will be known; by multiplying the NaCl with 0.5302 the amount of NagO is given. Numerous other methods for determing the K and Na in the urine have been described. They have no advan- tages over the above procedure and need not, therefore, be given. Calcium and Magnesium. — The urinary Ca and Mg are principally derived from the ingesta; both animal and vegetable foods (chiefly milk, yolk of egg, seeds and ger- minating plants, bone) containing considerable quantities of these metals. A vegetable diet contains more of them than an animal diet, unless bone is eaten. The food contains both mineral salts of calcium and magnesium and organic compounds of these metals. Many waters, too, contain considerable quantities of Ca (lime) and of magnesium. All the inorganic salts of calcium that are taken with food or drink are converted into calcium chloride or cal- cium mono -phosphate in the stomach (unless exception- ally large quantities of Ca salts are given; e. g., for medicinal purposes) . It is a remai-kable fact, and one that I have repeatedly had occasion to mention, that only a small proportion (5 to 10 per cent) of the calcium given by mouth appears in the urine; the bulk appears in the faeces. This is due to the fact that much of the calcium remains unabsorbed in the bowel, and that, in addition, a large proportion of the circulating Ca is excreted into the bowel (presumably as phosphate) and not into the bladder; it appears that this excretion occurs chiefly through the intestinal wall and only in small part through the liver. It is clear from all this that the urinary Ca excretion is no true index of the Ca-economy of the organ- ism, and that the excretion of a certain amount of Ca in the urine justifles us in concluding that a much larger proportion of this base is leaving the body in the fseees. We know, empirically, that the administration of HCl INORGANIC CONSTITUENTS OF THE tfRINE 209 (or hyperchlorhydria) and abundant water -drinking in- crease the urinary Ca excretion and that the administra- tion of alkalies or of sodium phosphate diminish it; that more of the Ca contained in animal food appears in the urine than of the Ca contained in vegetable food. Magnesium is absorbed with greater ease than calcium, and a much larger proportion of ingested magnesium appears in the urine than of calcium. A portion of the Mg, too, however, leaves the body through the bowel wall, so that the urinary Mg excretion is no true index of the total Mg excretion. Acids and alkalies seem to exer- cise no appreciable effect upon the amount of Mg appear- ing in the urine. The urine, as a rule, contains about twice as much Mg as Ca. This is due to the fact that most articles of diet (with the exception of eggs and milk) contain more Mg than Ca, that more of the Mg that is absorbed is excreted in the urine than of the Ca, and that more of the food Mg is absorbed from the bowel than of the food Ca. In in- anition the proportion of urinary Ca and Mg is, as a rule, reversed; this is due to the withdrawal of the excess of Mg of the food. The average daily excretion of calcium (CaO) fluctu- ates from 0.15 to 0.25 grams; of magnesium (MgO) from 0.18 to 0.33 grams. The bulk of the urinary Ca in the acid urine of man and of carnivorous animals presumably appears in the form of the mono-phosphate, a salt that is readily soluble in water. In neutral or only slightly acid urine some of the Ca must be present aS di-phosphate, a salt that is not readily soluble in water but that is distinctly soluble in liquids containing alkali mono-phosphates and sodium chloride. On boiling such a solution, neutral Ca phos- phate is formed that, being insoluble, precipitates; this is often seen on boiliug slightly acid urine or urine that has been rendered almost neutral by the addition of N 210 CLINICAL URINOLOGY dilute alkali. The formation of insoluble Ca phosphate on boiling acid urine may, however, also occasionally be due to the presence of much CO2 in the urine, for the latter can hold the Ca phosphate in solution; when the urine is boiled the CO2 is driven off and the Ca phosphate is precipitated; that this actually occurs may be demon- strated by testing the reaction of the urine before and after boiling — it will be found that urine that was acid before boiling is alkaline afterwards. The urine of herbivorous animals and of vegetarians usually also contains Ca carbonate as a sediment; this insoluble compound is presumably formed in the bladder by the action of alkali carbonates on the soluble mono- carbonate of Ca. Occasionally the urine of man contains calcium sulphate, and an abundant sediment of crystals of this salt (gypsum) have occasionally been found (see Sediments). As the total sulphate excretion in these cases is not increased, one must postulate that less alkali than normal was excreted in the urine, and hence the excess of sulphuric acid combined with Ca. Clinical Significance. — The fact that so much of the Ca and Mg of the body leave the organism via the bowel, and the fact, moreover, that so many accidental factors (water- drinking, exercise, ingestion of acids or alkalies, etc.) can influence the excretion of these bases through the urine, render the interpretation of the figures for the urinary Ca and Mg that have been obtained in almost every variety of disease very difficult. So much can be said that in complete inanition the urinary Ca and Mg excretion is increased, due presumably to the consumption of bone tissue. In many diseases in which the patients are chronically under- fed, the Ca and Mg excretion has been reported high; whether this is due in these disorders to some specific morbific influence or to the under-feeding with resulting consumption of the patients' proper tissues, including hone, is altogether indefinite. In all forms of debility and INORGANIC CONSTITUENTS OF THE URINE 211 wasting, at all events, the excretion of Ca (and Mg) as phosphates in the urine is high. This applies particu- larly to many febrile disorders. I have shown* that in tuberculosis the urinary calcium excretion is quite re- markably increased. In diabetes the excretion of Ca is often found increased; this is presumably due to the ingestion of abnormally large quantitities of food; possible, however, that the acidosis of diabetes has something to do with the in- creased Ca (and Mg) excretion, inasmuch as the abnor- mal acid circulating in the blood may possibly dissolve Ca and Mg out of bone tissue. Whether or not this explains the fact that in diabetics fractures heal with exceptional difficulty, remains to be determined. In osteomalacia one should expect a great urinary Ca and Mg excretion. As a matter of fact, the Ca excretion is often very great in this disease ; occasionally, however, it is normal and often below normal. Until many more determinations of the Ca excretion, both in the urine and the faeces, are made in this disease, this point must re- main uncertain. In rhachitis the urinary Ca and Mg are not increased. Determination of Calcium and Magnesium in the Urine. — Calcium. — The urinary calcium can be deter- mined by weighing or by titi-ation. I consider the former method more useful for clinical purposes. It is based on the precipitation of the calcium as oxalate, conversion of the latter into carbonate and then oxide by glowing, and weighing as CaO. This method indicates not quite all the Ca, but is sufficiently accurate for clinical purposes. 200 cc. of the filtered urine are treated with ammonia until a precipitate forms ; the latter is redissolved in the smallest possible quantity of HCl, and the liquid treated with ammonium oxalate in excess and sodium acetate * ''The Urinary Calcium Excretion in Tuberculosis," Journal of Tuberculosis, January, 1903. 212 CLINICAL URINOLOGY solution. The mixture is allowed to stand for twelve hours on the water-bath. The precipitate of calcium oxalate that forms is gathered on a small filter of known ash weight and washed free from chlorides (the filtrate must give no clouding with silver nitrate) . The filtrate and washings should be kept for the determination of the magnesia. The filter holding the calcium oxalate is dried and transferred to a weighed platinum crucible, inciner- ated over a small flame until it is white and then glowed to a white heat for ten minutes over a blow-flame, allowed to cool and weighed. The difference between this weight and the weight of the platinum crucible indicates the amount of CaO. Magnesium. — The above filtrate and washings from the calcium oxalate precipitate are treated with X volume of ammonia of 10 per cent (sp. gr. 0.96) ; this precipitates all the magnesia as ammonium -magnesium phosphate. The precipitate is allowed to settle for two or three hours and is then gathered on a filter of known ash weight and repeatedly washed with a mixture of % ammonia and % water. Filter and precipitate are now incinerated in a platinum dish (the precipitate being first transferred to the dish, and the filter incinerated over it separately according to the method commonly adopted in analytical chemistry. It is well to add a small piece of nitrate of ammonia to the ash in order to promote the combustion of the organic material it contains; in this way a pure white ash is obtained. By incineration the ammonium- magnesium phosphate MgH4NP04 is converted into magnesium -pyrophosphate Mg2P207; 100 parts of the latter correspond to 36.208 parts of MgO. Ammonium (NH4). — The ammonium of the urine is largely derived from the catabolism of albumen. It is probable that albumen normally splits off compounds con- taining substituted ammonia radicles, and that the latter are normally in great part converted into urea in the liver; INORGANIC CONSTITUENTS OF THE URINE 213 the portion that escapes this conversion appears in the urine as NH4 salt. As NH4 has a great affinity for acids, the acidity of the blood- and tissue-juices, in a measure, determines the amount of ammonia that appears in the urine; for circulating NH4, when combined with acids (certain organic acids excepted) , escapes conversion into urea. For this reason, too, the administration of acids increases, the administration of alkalies, other things be- ing equal, decreases the urinary NH4 excretion. For when alkalies are given, these bind the circulating acids instead of the ammonia. As a meat diet leads to the acidulation of the body by formation of sulphuric and phosphoric acid from the albumen the meat contains, the NH4 excretion is increased on such a regime; and as, inversely, vegetables and fruits reduce the blood acidity, the NH4 excretion is decreased on a vegetarian diet. A portion of the urinary NH4 is derived from other sources than the albumens of the proper tissues; thus some ammonia compounds are formed in the upper por- tion of the bowel, either by the action of the proteolytic enzymes or of bacteria ; this NH4 is absorbed and rapidly excreted in the urine. The stomach -contents, moreover, always contain some ammonia as chloride derived, in all probability, from the food (radishes, tobacco smoke, drinking water). Traces of ammonia may, finally, be absorbed from the inspired air in the lungs. Clinical Significance. — Pathologically, the excretion of ammonia is increased whenever there is increased cata- bolism of the tissue albumen with resulting acidulation of the blood-stream (sulphuric and phosphoric acid, see above) . Thus after violent muscular exercise, during com- plete inanition, in febrile disorders, the urinary ammonium excretion is increased and, as far as one is able to judge, the urea excretion correspondingly reduced. In febrile disorders the ammonia increase may be due to the inani- 214 CLINICAL URINOLOGY tion, or to reduced feeding, or to specific action of the high temperature that causes rapid breaking-down of tissue albumen, or to all of these factors combined. In convalescence from fevers the NH* excretion usually de- creases rapidly, and soon reaches normal figures when sufficient food is taken and the wasting is inhibited. In serious functional disorders of the liver the excretion of ammonia is increased. This is due to the fact that the conversion of NH4 to urea, that, as stated above, occurs in the liver is inhibited. In simple biliary stasis no marked departures from the normal NH4 excretion ai*e noted. In cirrhosis of the liver the NH4 excretion is always considerably increased ; this is due, in partj to in- adequacy of the liver to form urea, in part to the flooding of the blood with abnormal acids (acetic, valerianic, pro- pionic, butyric). In acute yellow atrophy of the liver, and in phosphorus liver, in which the excretion of urea is greatly reduced, one should expect a corresponding in- crease in the NH4 excretion, particularly as in this dis- order much lactic acid is poured into the blood. As a matter of fact, many data show that the NH4 excretion is great in this disorder ; a few cases are, however, also recorded, particularly by older authors, in which the uri- nary NH4 was not increased. Most modern writers on the subject, however, mention a great increase. In amy- loid, syphilis and carcinosis of the liver and in other dis- eases of the organ in which much of the parenchyma is destroyed the NH4 excretion is increased. In nephritis, retention of ammonia salts does not seem to occur, excepting in the beginning of acute nephritis, when all urinary solids are retained, and in terminal stages of nephritis when much renal epithelium is gone. The ammonia excretion in nephritis seems, in general, to fol- low the same rules as the urea excretion (see chapter on " Total Nitrogen and Urea"). In diabetes the excretion of NH4 is higher than in any INORGANIC CONSTITUENTS OF THE URINE 2] 5 other pathological condition. This is due, in the first place, to the large amount of albumen that diabetics eat when they are placed on a meat- fat diet ; in the second place, to the acidosis that is so common in this disease. The great excretion of NH* in diabetes is an index of the endeavors on the part of the organism to maintain the blood alkalinity. The appearance of large quantities of NH4 in the urine of diabetics must, therefore, be consid- ered a bad prognostic omen, inasmuch as it indicates acidosis and often impending coma. The reverse, how- ever, is not the case, for the excretion of NH4 may be low and still coma be impending; it is possible that in many diabetics the power to split off NH4 for the purpose of neutralizing the circulating acids is lost, and that hence in such cases the acids appear in the urine in combination with other bases than NH4 ; here the NH4 excretion would remain low while the acidosis and the danger of coma would be great, particularly as the acids must tear K and Na out of their organic combinations and in this way must produce dangerous "demineralization" of protoplasm and interference with its functions. In many cases of dia- betes living on an ordinary mixed diet the NH4 excretion may occasionally be quite normal. The normal daily ammonia excretion in an adult living on a mixed diet fluctuates from 0.3 to 1.2 grams. In diabetes as much as 6 grams a day has been found. From 4>2 to 7 per cent of the total urinary N normally appear as NH4 in the urine. No definite ratio of NH4 to urea can be given. Determination of Ammonia in the Urine. — As the urea of the urine is readily converted into ammonia by micro- organisms, the urine must be quite fresh. The most popular and the most simple method for determining the urinaiy ammonia is that of Schloesing, to be presently described. Other methods are perhaps more accurate and consequently better suited for exact metabolic work. 216 CLINICAL URINOLOGY For clinical determinations the Schloesing method, owing to its simplicity, is the best. It is executed as follows: — 25 cc. of the filtered urine are poured into a shallow crystallizing dish; upon this is placed a metal triangle that serves to support a second flat dish containing 20 ce. of X normal H2SO4. To the urine are added 10 cc. of lime water. The two vessels are at once covered with a bell- jar; the latter has ground edges that are smeared with vaseline and pressed down tightly on the ground- glass plate on which the two dishes are standing. The lime-water drives all the ammonia out of its compounds in the urine and the vapors rising from the urine dish are greedily absorbed by the sulphuric acid; in this way a part of the acid is neutralized. The process requires about two days. At the expiration of this time the free acid remaining is determined by titration with -n,- normal sodium hydrate solution, using methyl orange as an indicator. The amount of ammonia absorbed by the sulphuric acid, i, e., the amount of ammonia originally contained in the urine, can readily be calculated from the number of cubic centimeters of the alkaline solution re- quired to neutralize the free acid remaining as follows : — the number of cubic centimeters of to normal NaOH sub- tracted from 50 and multiplied by 1.7 indicates the num- ber of milligrams of ammonia present in the 25 cc. of urine used for the determination. It is claimed that the addition of the lime-water alone prevents the develop- ment of bacteria in the urine and the formation of ammonia from urea. I consider it safer, however, to add carbolic acid (enough to make a 2 per cent solution) or chloroform to the urine. Iron. — Only very small quantities of iron appear in the urine, and the determination of the urinary iron is a task of very subordinate clinical value. The urinary iron ex- cretion, moreover, is no index of the iron metabolism of the body, for the bulk of the circulating iron compounds INORGANIC CONSTITUENTS OF THE URINE 217 are eliminated from the body through the intestinal wall and the bile, or they are stored, after they have served their usefulness, in the liver and spleen; here they under- go complicated metamorphoses, and ultimately either reenter the circulation in a changed form or leave the body as excreta in the faeces. The proof of this peculiar bowel elimination and storage of iron compounds can readily be furnished by injecting iron-salts into the circu- lation; the bulk of this iron will be found in the faeces, a small quantity in the liver and spleen and only traces in the urine. Unless the circulation is suddenly flooded with enormous quantities of iron compounds that can no longer be utilized, only minimal traces appear in the urine. In certain qualitative perversions of the iron nietabolism, as in pernicious ancemia with rapid destruc- tion of hemoglobin, and in certain forms of intoxication and infections (fevers) that are accompanied by hemolysis, the iron content of the urine may be increased. The administration by mouth of large quantities of certain iron-containing nucleo-albumens also leads to an increased excretion of iron in the urine ; finally, in parenchymatous nephritis in which the renal filter is damaged, and in diabetes in which colossal quantities of water are elimi- nated, an increase of the urinary iron is sometimes noted. The appearance of more iron in the urine than is nor- mally excreted partakes, therefore, of a similar character as the appearance of albumen and sugar, inasmuch as this increase is presumably the result of some deviation from the normal iron metabolism, or of certain mechani- cal factors that force some of the iron from the blood through damaged or overstrained kidneys into the urine. Possible that the traces of iron that are normally excreted are physiological only in the sense that traces of albumen and of sugar are physiological. The excretion of iron as blood- and bile-pigments in hemoglobinuria, hematuria and choluria is, of course, a thing by itself. 218 CLINICAL URINOLOGY The urinary iron appears only in organic combination. Some of the "normal" urinary pigments contain a little iron. The chemical character of the organic iron com- pounds of the urine is not known. The average normal daily excretion of iron probably does not exceed 2 to 3 mg in the whole twenty -four hours. The Determination of Iron in the Urine. — ^As iron oc- curs in the urine only in organic combinations, it cannot be detected by the ordinary qualitative tests for inorganic iron compounds. In order to demonstrate its presence, the urine is evaporated to dryness and the residue incin- erated. The ash is dissolved in a little HCl and boiled with one drop of HNO3. After cooling, the liquid is treated with potassium sulphoeyanide solution. If iron is present even in traces a few drops of this reagent will produce a reddish color; if much iron is present the liquid will turn dark red. Or the HOI-HNO3 solution of the ash may be treated with a few drops of potassium- ferrocyanide solution; if iron is present in appreciable quantities a blue flocculent precipitate will form. The quantitative determination of the urinary iron is for the present never performed in the clinical laboratory for purposes of diagnosis. In scientific work and in accurate metabolic and pharmacologic studies the determination may have to be performed; as this book, however, deals exclusively with the clinical aspects of urinology, the methods employed will not be described. They can be found in full in Huppert, "Harnanalyse," 1898, p. 750 ff. CHAPTER X URINARY SEDIMENTS The Factors Determining the Formation of Urinary Sediments. Unorganized Sediments, (1) in Aeid Urine, (2) in Alkaline Urine. The Macro- scopic Appearance of Certain Inorganic Sediments. The Different Inorganic Sediments in Detail; Uric Acid and Urates; Calcium Oxalate; Calcium Sulphate; Calcium Carbonate; Phosphates; Cystin; Xanthin; Hippurio Aeid; Bilirubin and Hematoidin; Indigo-blue and Indigo-red ; Hetero-albumose ; Leucin and Tyrosin. Organized Sediments. Blood -Corpuscles; Pus; Epithelia; Casts: the Pathogenesis of Casts — Their Classification — the Different Casts in Detail and Their Clinical Significance; Spermatozoa. Micro-organisms. Saprophytic Bacteria; Pathogenic Bacteria: Bacillus Coli — Eberth Bacillus — Pus Germs — Gonoeoecus — Tubercle Bacillus — Actinomycosis. Moulds. Ani- mal Parasites — Filaria Sanguinis Hominis (Chyluria) — Distoma Hematobium — Echinoeoccus — Eustrongylus Gigas — Feeeal and Vaginal Parasites. Normal urine is usually quite clear or only slightly cloudy when voided. On, cooling a small cloud, the "nubecula," forms that, on microscopic examination, is found to consist of a small number of epithelial cells in various stages of degeneration, derived from the genito- urinary passages, a few leucocytes, a scanty number of crystals, amorphous inorganic material and morphologically undefined organic debris. In addition, nearly every urine contains certain moulds and bacteria and various accidental admixtures that are derived from the skin around the urinary orifices and from the clothing. On standing, every urine precipitates a sediment. Its size and density varies greatly in health. Very much depends upon the mixture of salts that the ui'ine contains, the presence or absence of much free carbonic acid in solution, and other elements that will be considered below in discussing the factors that determine the precipitation (219) 220 CLINICAL URINOLOGY of inorganic urinary sediments. The chief constituents of the normal sediment are the urates, oxalates and phos- phates of the urinary bases K, Na, Ca, Mg and NH4, singly or in combination. One should never forget that the deposit of a large inorganic sediment per se need not be pathological, and that the urine from a perfectly healthy individual may precipitate a bulky sediment, particularly of urates and of phosphates. The formation of the sediment may occasionally take place ih the lower urinary tract or in the renal pelvis. As a rule, however, no sediment forms until after the urine has been voided. Pathological urine in general deposits a more profuse sediment than normal urine, and, moreover, deposits it sooner; this rule is, however, by no means absolute, for, as already stated, normal urine may deposit a very heavy precipitate within a short time after it is passed, whereas pathological urine may contain the salts in such combina- tion that no precipitate of the latter occurs, and morphotie elements in such scanty numbers that centrifugation of the urine is necessary before they can be discovered. The bulk of the sediment and the rapidity with which it forms are therefore no index whatsoever of the normal or ab- normal composition of the urine. It may seem a work of supererogation to lay particular stress upon this point. I do so merely in order to aid in dispelling the popular prejudice existing in the minds of many physicians and of the laity (fostered in the latter case by unscrupulous venders of kidney nostrums) that a large urinary sedi- ment invariably spells disease of the kidneys and abnor- mal urine. UNORGANIZED SEDIMENTS The character and the quantity of the unorganized sedi- ments are dependent (1) on the reaction of the urine, (2) on the concentration of the urine, (3) on the mixture of salts it contains. URINARY SEDIMENTS 221 Certain sediments form only in alkaline urine, others only in acid urine; a knowledge of the bodies that pre- cipitate only in alkaline or only in acid urine, therefore, frequently aids in the chemical or microscopical identifi- cation of the crystals or amorphous deposits seen. The foHowing scheme may aid as a guide in this direction: 1. Acid Urine. {A) An Amorphous Sediment. {a) The sediment is granular and soluble on slight heating; a drop of acetic acid applied to the edge of the cover -slip causes the amorphous granules to disappear. On standing, rhomboid crystals of uric acid appear. The sediment consisted of urates. (See Fig. 4.) (&) A sediment of dumb-bell shaped masses may con- sist of calcium oxalate or calcium sulphate. If it is soluble in concentrated HCl it is the former. (See Fig. 10.) (c) A yellow granular sediment consists of hematoidin or bilirubin. (d) A refractive, shiny, circular sediment consists of fat droplets. {B) A Crystalline Sediment. (a) Wedge-shaped crystals, yellow to brown, single or in heaps, alone or together with amorphous granules, soluble in sodium hydrate, formation of rhomboid crystals after the addition of HCl. This sediment is Uric Acid. (See Fig. 6.) (6) Small yellow rhomboid plates, often imbedded in organized tissues: Bilirubin or Hematoidin. (c) "Wedge-shaped crystals alone or in groups, soluble in acetic acid: Di- calcium phosphate. (See Fig. 12.) (d) Sheaths of fine needles, insoluble in acetic acid, soluble in ammonia and hydrochloric acid: Ty rosin. (See Fig. 17.) (e) Prisms, single or in rosettes; if soluble in ammonia hippuric acid (Fig. 15), if insoluble in ammonia calcium sulphate (Fig. 9). (/) Large, flat, refractive, rhomboid plates, soluble in acetic acid: Normal magnesium phosphate (Fig. 11). 222 CLINICAL UBLNOLOOY (g) Colorless, whetstone crystals, insoluble in acetic acid: Xanthin (Fig. 14). (h) Hexagonal plates, insoluble in acetic acid, soluble in ammonia: Cysim (Fig. 13). (i) Colorless, octahedral, refractive crystals (envelope shape) soluble in hydrochloric, insoluble in acetic acid: Calcium oxalate (Fig. 8) . (jfc) Coffin-lid crystals, hexagonal (only in urine that is faintly acid), soluble in acetic acid: Triple phosphate (Figs. 5 and 11). 2. Alkaline Urine.* (A) 4-n Amorphous Sediment. {a) Small granules, soluble in acetic acid without de- velopment of gas : Calcium and Magnesium phosphate (Fig. 11) ; soluble in acetic acid with development of gas bubbles: Calcium carbonate. (6) Dumb-bells, or large spheres, soluble in acetic acid with gas formation: Calcium carbonate (Fig. 10). (c) Large, dark spheres, in rows and mixed with crys- tals, soluble in acetic or hydrochloric acid with subsequent formation of rhomboid plates: Ammonium urate (Fig. 7). {B) A Crystalline Sediment. (a) Blue plates or rosettes of fine blue needles: Indigo (Fig. 16). (b) Violet-red rhomboid plates or needles: Indigo-red. (c) Large, colorless crystals, prismatic with broken edges (coffin-lid shape), readily soluble in acetic acid: Triple phosphate (Figs. 5 and 11). The Macroscopic Appearance of Certain Sediments. — Oc- casionally the naked eye appearance of certain urinary sediments is characteristic. The Brick-dust Sediment. — This sediment consists of uric acid, urates and often calcium oxalate; it occurs only in acid urine. Microscopically it presents the appearance *I£ the urine becomes alkaline after it is voided, then it may still contain some of the sediments found only in acid urine; e. g., uric acid, calcium oxalate, etc. The following, therefore, applies only to urine that is alkaline when voided or that does not deposit a sediment until it becomes alkaline. URINARY SEDIMENTS 223 depicted in Fig. 4. The large and thick rhomboid plates with rounded angles being uric acid, the fine granular masses quadriurates, the octahedral crystals with a shining cross in their center oxalate of calcium. The uric acid in this sediment is col- ored various shades of pink, brown and red, brick - red predomina- ting, hence the name brick -dust sediment. If the urine is allowed to stand in a pointed glass the heavier red brick- dust is apt to settle at the bottom of the sedi- ment layer, forming a ment dissolves on heating the urine. The %■: "-'? Fig. 4. Brick-dnst or Uratic Sediment from Aeid Urine. (After Punclie.) Tile large whetstone crystals are uric acid, tlie smaller octahedral crystals calcium oxa- late, the amorphous masses quadriurates. distinct red zone. This sedi- Vif^ jk ■rder to recognize tubercle bacilli in the urine, it is necessary to centrifuge the specimen several times. It is always well to take large quantities of virine, to centrifuge many single portions, to unite the precipitates and to centrifuge again. In the ultimate precipitate, tubercle bacilli are looked for as in sputum; it is always well, in Fig. 24. Gonococcus and Pus Cells. a. Pus cells; 6. Gonococcus. (After L^tienne and Masselin. ) URINARY SEDIMENTS 255 addition, to inject some of the sediment into the peri- toneal cavity of a number of guinea pigs and to watch for the development of tubercles within three weeks. Actinomycosis germs have been found in the urine in cases of actinomycosis of the urogenital tract and in cases of rupture of an actinomycotic abscess into the urinary passages. Moulds. — A great variety of moulds have been found in old urine ; the character of the vegetable organisms found depending largely on the reaction, the salt content and the presence or absence of sugar or albumen in the urine. These organisms possess very small clini- cal interest ; it is impor- tant merely to be able to recognize them in order not to confound them with structures of greater clinical impor- tance. Decomposing diabetic urine contains the greatest number of moulds ; they may be so numerous as to form a layer of several millimeters thickness on the surface of the liquid ; the appearance of such a layer, in fact, should always lead one to suspect sugar in the urine. The most common of these organisms are yeast (sac- charomyces cerevisiae) , penicillium glaucum, oidium albi- cans, mycelia and a large sarcina. The appearance of these different moulds is depicted on the accompanying drawing (Fig. 25). Fig. 25. Moulds and Infusoria. 1. Yeast cells; 2. Penicillium plaucum; 3. Oidium albicans; 4. Mycelial filaments; 5. Sarcina. (Original.) ANIMAL PARASITES ( VERMES ) The majority of the animal parasites found in the urine are of tropical origin. In temperate zones they are only 256 CLINICAL URINOLOGY sporadically encountered, chiefly in individuals who have lived in the tropics. For the life history and the semi- ology of the different parasites I refer to text- books on parasitology; here only the most important of these organisms will be briefly described. Filaria Sanguinis Hominis (Fig. 26, i). — A small col- orless worm, some forty millimeters in length. Human blood usually contains the embryo, a very small worm, with a very thin flagellated extremity. A mosquito. Pig. 26. Animal Parasites. 1. Pilaria sanguinis; 2. Distoma hematobium, a. male, 6. female, e. eggs; 3. Echinococcus booklets. (Original). Culex ciliaris, seems to be an intermediary host, and, according to some investigators, transmission of filaria to man can occur only through mosquito bites ; others claim that the parasite may gain an entrance with the food through the gastroenteric tract. The organism undergoes a cyclic development in the lymph and" blood. It should be looked for in the blood at night. In the kidneys it seems capable of producing dilatation and rupture of lymph- channels with excretion of lymph into the urine, thus causing Chylukia. — In this disorder the urine is milky, con- tains much fat, cholesterin, some albumen and sugar. URINARY SEDIMENTS 2bl some lymph-corpusclfes and usually some red blood-cells. On standing, the urine deposits coagula of fibrin, or it may coagulate as a whole, forming a gelatinous mass. The urine in true filaria chyluria contains no renal ele- ments, showing that the passage of albumen into the urine is not due to any inflammatory lesion of the kidneys. The attacks of chyluria are usually intermittent and ap- pear suddenly. In the tropical form of chyluria the uiine always con- tains specimens of filaria sanguinis hominis; occasionally, of course, chyluria may be produced by other agencies than filaria that lead to the formation of abnormal com- munications between the lymph- and blood -channels in various parts of the body or between the lymph- and urine -channels in the kidneys or below them. Filaria is also incriminated by some with causing a tropical form of hematuria. Distoma Hematobium (Bilharzia Hematobia) (Fig. 26, 2) , — This organism appears in the blood in two forms, a male and a female organism, the former 12-14 mm. long and broader than the female, that is from 16-19 mm. long. The worm invades many organs and deposits eggs (0.12 mm. long and 0.04 mm. broad) that are elliptoid in shape, with a sharp prong on one side or at one end. When these eggs invade the veins of the urinary mucosa they occude their lumen, and owing to their peculiar shape may cause rupture of some of the smaller vessels, possibly with ulceration of the mucosa. The result is hematuria. The urine in these cases may also occasion- ally contain fat, showing that possibly distoma sometimes causes rupture of lymph -vessels. As a rule, however, distoma urine contains no chylous elements. The hemor- rhages are usually fresh, the urine is passed with some sharp shooting pain, and the last few drops usually con- tain a small blood coagulate. Echinococcus (Fig. 26, 3).— Eupture of an hyatid cyst 258 CLINICAL URINOLOGY of the urinary passages is naturally followed by the pas- sage of echinoeoccus hooklets (and membranes), together with blood (often pus -cells) and cells derived from the aflEected part of the urinary tract in the urine. Echinococci may also appear in the urine in rupture of an echinoeoccus cyst of some neighboring organ into the urinary passages. Eustrongylus Gigas.— This worm is very rarely found in the urine ; some doubt, in fact, is cast upon all the re- ports describing the passage of this worm in human urine. It is not so rare in animals. It has been found occasionally, post-mortem, imbedded in the tissues of the kidney and the bladder wall. Faecal and Vaginal Parasites. — Ascaris lumbricoides, oxyuris vermicularis, trichomonas vaginalis and other animal parasites, or their eggs, derived from the bowel or vagina are sometimes found in the urine. They may have entered the urine through some abnormal communi- cation between bladder or urethra and the rectum or vagina, or may have accidentally contaminated the urine after it was voided. Their appearance in the urine may occasionally be utilized in the diagnosis of minute and tortuous fistulse that are otherwise difficult to detect. CHAPTER XI THE CONCRETIONS OF TSE URINE, THEIR DESCRIPTION AND ANALYSIS Uratic Concretions. Phosphate Concretions.. Calcium Oxalate Concretions. Cystin Concretions. Xanthin Concretions. Mixed Concretions — Calcium Carbonate, Calcium Sulphate, Leucin, Tyrosin, Bilirubin, Hippurio Acid, Cholesterin, Fat. — General Preliminary Analysis. Almost any of the bodies that form the microscopic sediments of the urine, may form larger conglomerates in different portions of the urinary tract that vary in size, shape and composition. Sometimes the concretions con- sist of a single substance, as uric acid, calcium oxalate, cystin or xanthin; more commonly, however, they consist of several bodies that may be intimately mixed or may be deposited one upon the other in concentric layers. Occasionally they contain some organic material, as a blood-clot, fibrin or mucus as a nucleus upon which the inorganic materials constituting the bulk of the calculus are deposited. The most important varieties of urinary concretions are the following ones: 1. Uratic Concretions, consisting of uric acid and urates, the uric acid, as a rule, predominating; others consist- ing of ammonium urate alone. The former are very hard, reddish to reddish brown in color, with a rough surface, whereas the latter are soft, light yellow in color and usually very small. They are often combined with cal- cium oxalate or triple phosphate. Analysis. — The powdered concretion should be insolu- ble in warm dilute hydrochloric acid, it should emit an odor of prussic acid (bitter almonds) when glowed on a (259) 260 CLINICAL URINOLOGY piece of platinum foil. It should give the murexid reac- tion, i. e., dissolved in nitric acid and evaporated to dry- ness the residue should be red; touched with a drop of ammonia the latter should .turn purple, and touched with a drop of sodium hydrate reddish blue. 2. Phosphate Concretions, consisting, as a rule, of nor- mal phosphates of calcium and magnesium and of triple phosphates; occasionally a concretion consisting of cal- cium mono-phosphate alone is encountered. The ordi- nary phosphate concretion is yellowish or white in color, brittle, fragile and flaky; the calcium mono-phosphate concretions are snow-white, of a crystalline structure and hard. Analysis. — The concretion is powdered, leached out with warm hydrochloric acid and the acid solution filtered off. It contains all the phosphates that may have been present in the concretion. The solution is carefully neutralized with sodium carbonate until a slight precipi- tate forms; this is redissolved in the smallest possible quantity of hydrochloric acid and the liquid treated with an excess of a 30 per cent solution of sodium acetate ; the precipitate that may form (calcium oxalate, cystin) is fil- tered off; the filtrate contains only phosphates. In order to test whether there are any phosphates present, the liquid is treated with ammonia; if no precipitate forms the concretion contained no phosphates; a cloudiness or a sediment indicates the presence of phosphates. Or the liquid may be treated with ferric chloride solution; a white or yellowish gelatinous precipitate indicates the presence of phosphates. Calcium is determined in the filtrate from the acetate solution (see above) by treating it with ammonium oxa- late; a fine white precipitate that is soluble in dilute hydrochloric acid indicates the presence of calcium, i. e., of calcium phosphate in the concretion. Magnesium is found by precipitating all the calcium THE CONCRETIONS OF THE URINE 261 with ammonium oxalate, evaporating the filtrate to a small volume and treating it with one-third its volume o^ 10 per cent ammonium. A crystalline precipitate of am- monium-magnesium phosphate indicates the presence of magnesium as phosphate in the concretion. Ammonium. — The original hydrochloric acid extract of the concretion is rendered strongly alkaline with soda lye ; a piece of moistened red litmus paper is held over the mouth of the tube; if it turns blue the concretion contained ammonium. 3. Calcium Oxalate Concretions appear in two forms, viz.: (1) small, white smooth stones and (2) large, white or yellowish stones, of a mulberry structure, often with angular protuberances, and not infrequently covered with blood -pigments or urinary pigments. Both varieties are very hard and show 'a crystalline structure if they are cracked or sawed in two. Calcium ox«,late and uric acid or urates are often found together in the same concretion. Analysis. — The concretion is treated as described under phosphate concretions and the precipitate formed on addition of sodium acetate filtered off; the latter should be insoluble in ammonium, (in contradistinction to cystin that is precipitated together with calcium oxa- late), insoluble in acetic acid, but soluble in hydrochloric acid; if the hydrochloric acid solution is over- saturated with sodium acetate a pulverulent precipitate of calcium oxalate should form again . This precipitate may be glowed on a piece of platinum foil, and then treated with acetic acid; it should develop CO2; the addition of ammonium oxalate to the acetic acid solution of the glowed precipitate should cause the formation of a calcium oxalate pre- cipitate. 4. Cystin Concretions are smooth or angular, not very hard, yellowish white in color, and show a crystalline structure when cracked or sawed in two. Analysis.— Hhe concretion is treated as described under 262 CLINICAL URINOLOGY phosphate concretions, and the precipitate formed on addition of sodium acetate filtered off; the latter is treated with ammonia; any eystin that may be present is solu- ble in the ammonia (whereas calcium oxalate, as shown above, remains behind); the addition of acetic acid to the ammoniacal solution causes the precipitation of a crystalline sediment of eystin. In order to further identify eystin, the sediment may be filtered off and treated with a solution of lead oxide in soda lye ; on boil- ing, the liquid turns black. The same test may be per- formed with the original concretion. An intense black- ening of the liquid (liberation of alkali sulphide and lead sulphide) is almost pathognomonic for eystin in a concre- tion. It is always safer, however, to isolate the eystin as described above. 5. Xanthin Concretions are usually of a cinnamon-brown color, waxy and shiny if rubbed with a cloth, and rather hard. They are composed of several layers of an amor- phous structure that can easily be peeled off in scales. Analysis,— The concretion is powdered and leached out with an excess of dilute warm hydrochloric acid; the solution contains any xanthin that may be present; the liquid is over- saturated with ammonia and treated with an ammoniacal silver nitrate solution ; a precipitate indicates the presence of xanthin (see also Chapter II, page 36) . 6. Mixed Concretions may contain, in addition to the substances enumerated above, calcium carbonate, calcium sulphate, leucin, tyrcsin, bilirubin, hippuric acid, choles- terin and fats. The recognition of carbonate in a con- cretion is simple, for when such a concretion is treated with dilute hydrochloric acid in the cold it effervesces and develops CO2. Sulphate can be recognized by the formation of a thick precipitate of barium chloride in the warm hydrochloric acid extract of the powdered concre- tion when a solution of barium chloride is added. Leucin, tyrosin, bilirubin and hippuric acid must be sought for THE CONGRDTIONS OF THE UEINE 263 according to the chemical methods described in previous chapters. Cholesterin and fats can be extracted from the finely powdered calculus with ether. On evaporation, the cholesterin forms large rhomboid plates or fine silky needles that turn red and blue when touched with con- centrated sulphuric acid. Fat can be recognized from its property to render paper translucent ("grease spot") and from the pungent odor of acrolein it emits when heated on a piece of platinum foil. General Preliminary Analysis of Urinary Concretions. — As a preliminary to every analysis of a concretion found in the urine, the stone or gravel should be pow- dered and glowed on a piece of platinum foil. If the powder emits an odor of prussic acid (bitter almonds), the concretion contains either uric acid or xanthin; if it emits an odor of SO2 (burning sulphur) , and burns with a bluish flame, it contains cystin; if it leaves an abundant residue of ash that emits a white glow when heated to a high temperature, the concretion contained phosphates. Much preliminary informa.tion can also be gained from inspection of the outer surface of the concretion and of the surfaces of the stone after it is cracked or sawed in two, for in this way one can determine its color and structure, whether it is homogeneous or mixed, inorganic or partly organized. The factors that determine the formation of urinary concretions have already been discussed at length in the preceding chapter, when describing the formation of the various sediments of the urine. CHAPTER XII THE PHrSICAL PROPERTIES OF THE URINE Quantity. Specific Gravity. Reaction. Optical Properties: Color; Fluores- eence; Behavior toward Polarized Light; Spectrum; Transparency. Odor. Freeeing Point {Cryoscopy). Electric Conductivity. Quantity. — The amount of urine voided is dependent (1) on the state of the renal epithelium ; (2) on the rapid- ity of the blood flow through the kidneys, and hence, in- directly on variable nervous influences. It is independent of the blood pressure. A normal healthy adult of the average weight of 75 kilograms passes from 1,500 to 2,000 cc. of urine in twenty-four hours. Men pass absolutely more urine than women; infants pass absolutely less, but, in proportion to their body weight, relatively more urine ; this is largely due to the liquid diet. Abundant ingestion of fluids in- creases diuresis (urina potus) ; sweating (violent exer- cise, hot weather) decreases it. Less urine is normally passed at night than during the day (in chronic nephritis this ratio may be reversed) . Destructive renal lesions or local circulatory distur- bances, in order to influence the flow of urine, must be bilateral; unilateral interference with diuresis is largely compensated by the healthy organ. Increased urination is called polyuria, decreased urination oliguria and sup- pression of urine anuria. Polyuria. — The more chronic the nephritis the greater the tendency to polyuria (contracted kidney, amyloid) ; this is chiefly due to the contraction of the renal tissues and the resulting acceleration of the blood flow. During convalescence from acute nephritis; in heart and lung (264) PHYSICAL PROPERTIES OF THE URINE 265 disease as soon as the circulatory disturbances begin to be compensated; in diabetes mellitus, and insipidus; after psychic shocks, and in various neuroses (urina spastica) ; after the exhibition of certain diuretic drugs, and after the ingestion of certain articles of food (tea, coffee, alcohol, etc.), the flow of urine is increased. Oliguria.— In acute nephritis, in acute exacerbations of chronic nephritis, and in heart and lung diseases lead- ing to stasis, the flow of urine is decreased. Anuria.— In uraemia (occasionally), in diseases caus- ing great loss of fluids through other emunctories than the kidneys (profuse sweating, acute gastritis and intesti- nal catarrh, with profuse vomiting and diarrhoea, cholera, dysentery) , in rapidly progressive forms of anaemia, and in hysteria, the urine may be altogether suppressed. Determination. — The urine should preferably be col- lected from morning to morning before eating; the blad- der should be emptied before the collection is begun; the patients should urinate before going to stool, as, particu- larly in old women, much urine may be lost during the act of defecation. The total quantity may be measured or determined by weighing; the weight divided by the specific gravity yielding the volume. For clinical pur- poses the former method is best ; for exact determinations the latter is to be preferred. Specific Gravity. — The specific gravity of the urine is, as a rule, high when little urine is voided and low when the flow of urine is abundant. The same factors, there- fore, that determine physiological fluctuations in the amount of urine also determine corresponding fluctuations in its specific gravity. As the normal total quantity varies from 1,500 to 2,000 cc, so the normal specific gravity varies correspondingly from 1.025 to 1.015. The specific gravity of the urine is an index of the ex- cretion of urinary solids. If the latter were all heavier than water, the last two figures of the specific gravity 266 CLINICAL URINOLOGY (expressed in four figures) would directly indicate the quantity by weight of urinary solids contained in one liter of urine. It has been found empirically that the last two figures of the specific gravity multiplied by 2.2337 give the weight in grams of the solids in one liter of the urine. [Example: Specific gravity = 1.018 ; 18 multi- plied by 2.2337 = 40.2066 grams of solids in one liter of urine.] This calculation, while not absolutely accurate, is useful for comparative studies. In severe febrile diseases the specific gravity is usually low without a corresponding increase in the quantity of urine as soon as the patient's vitality begins to flag and general met9,bolism is reduced. In acute and subacute nephritis the specific gravity is usually high, the water excretion correspondingly re- duced. In the healing stage of acute nephritis the flow often increases, whereas the specific gravity remains low; this indicates that the kidneys are regaining their power to excrete water, but not as yet to eliminate solids. In acute exacerbations of chronic nephritis and in the termi- nal stages of destructive inflammation and degeneration of the kidneys the specific gi-avity is reduced; when this occurs suddenly, this is a bad prognostic omen, and often indicates threatening uraemia. In contracted kidneys the specific gravity is low and the flow abundant. In diabetes mellitus the flow is great and the specific gravity very high, whereas in diabetes insipidus the flow is also great, but the specific gravity very low. Determination. — The specific gravity may be deter- mined {a) by weighing with a hydrostatic balance, (&) with a pyknometer, and (c) with an aerometer. The first method is useful only in very exact work, and requires a complicated balance; it will not be described here. The second and third methods are useful for clinical work. In ordinary comparative studies the aerometer is quite sufficient. PHYSICAL PROPERTIES OF THE URINE 267 Fig. 27. Pyknometer, The pyknometer is a flask with a long neck that is drawn out in one place (see Fig. 27) ; at the narrowest point is a mark; the flask is closed with a ground -glass stopper. The flask is first filled with dis- tilled water to the mark and weighed ; it is then filled with the filtered urine to the mark and weighed again. The weight of the urine divided by the weight of the water gives the specific gravity. It is very impor- tant that the temperature of the water and that of the urine should be exactly alike when the pkynometer is filled and when it is weighed. The aerometer (urometer). This instru- ment consists of a glass cylinder and a float (see Fig. 28) . The latter should be grad- uated from 1.000 to 1.040. It is better to have two floats, the one reading from 1.000 to 1.020 and the other from 1.020 to 1.040. The urine is poured into the cylinder, the float introduced into the urine, and the specific gravity read off di- rectly from the aerometer scale. Here, too, the temperature is important; it should be about 17.5° C, for the aerometers are grad- uated at this temperature. If the urine is warmer than 17.5° C, one-third of a urom- eter degree should be added for each degree of temperature ; if the urine is colder than 17.5° C, the reading should be cor- rected by subtracting one -third urometer degree for each degree of temperature. [Example: Urine 20.5° C, specific gravity 1.017. Corrected, 1.017 -f 3 X % = 1.018.] A temperature scale is found on many urometers. AsromeL. Reactloii.— Normal urine is never neutral 268 CLINICAL URINOLOGY to litmus; it is either acid, amphoteric or alkaline. The acidity is never due to the presence of free acid, but to the excess of acid salts over basic salts. When the two are mixed in certain definite proportions the reaction becomes amphoteric. Uric acid in solution never determines the urinary acidity directly; it may increase the acidity indi- rectly by transforming neutral phosphates into acid phos- phates. The urine becomes alkaline after eating (HCl secretion into the stomach) and after the ingestion of cer- tain salts of vegetable origin (citrates, tartrates, etc.), as the latter are excreted as carbonates (see "Carbonates of the Urine"). Old urine is usually alkaline, as the mi- crococcus urea generates ammonium carbonate. Pathologically, the acidity of the urine is always in- creased in febrile processes, for here increased catabolism of proteid with liberation of sulphuric and phosphoric acids from proteid- sulphur and proteid -phosphorus oc- curs. In leucaemia, scurvy and diabetes the urine is also in general acid. Increased alkalinity is a valuable sign only if the above-named physiological factors can all be excluded. It is important to determine whether the urinary alkalin- ity is due to the liberation of ammonia or to the presence in solution of fixed alkali. Free ammonia signifies fer- mentation of the urine either in the bladder or after the urine is voided. Fixed alkali appearing in excess in fresh urine may be due to the withdrawal of acid from the body (vomiting and frequent lavage), to the admixture of alkaline secretions from the urinary passages, or to the rapid absorption of exudates and transudates. Determination.— Vov clinical purposes the litmus test is sufficient: blue litmus paper turning red in acid urine, and red litmus paper turning blue in alkaline urine. If the blue color remains after the paper dries, the alkalinity is due to fixed alkali; if it vanishes, to free ammonia. In view of the many variable factors that determine PHYSICAL PROPERTIES OF THE URINE 269 the reaction of the urine, in view of the rapid change in reaction that occurs as soon as air-borne micro-organisms contaminate the urine, in view finally of the great diffi- culty encountered in interpreting fluctuations in the uri- nary acidity for clinical purposes, it is usually an altogether futile task to determine the urinary acidity by titration. Only when such determinations are performed with the mathematical accuracy of a metabolic experiment do they possess some value; at best, however, the results ob- tained from these determinations are clinically never quite free from ambiguity. I do not consider it necessary in this clinical treatise, therefore, to give a full description of the methods that are employed. The determinations can best be performed by titration of a mixture of the twenty -four hours' quantity (withdrawn and preserved under aseptic precautions) against -rs normal soda solu- tion, using phenole phthalein as an indicator; the acidity of the urine may then be conveniently expressed in terms of oxalic or sulphuric acid. The normal average acidity of the urine expressed in this way fluctuates from 1.75 to 2 grams of oxalic acid and from 1.35 to 1.55 grams of of sulphuric acid. Optical Properties. — Color. The color of the urine is dependent on its concentration, its reaction, and the pig- ments it contains. Normal urine may be from pale yel- low to reddish brown. The greater the concentration the darker the color. Acid urine becomes more pale when it is alkalinized, and alkaline urine darker when it is acidi- fied. The normal yellow pigment of the urine is known by various names, i. e., urochrome, urohematine, uro- erythrine, etc. The exact origin and the chemical con- stitution of this pigment are unknown. Certain pathologic pigments change the color of the urine. Bile-pigment colors it green or brown, blood-pig- ment red to brown-red, urobilin dark brown, and melanin brown to black. Certain aromatic decomposition products 270 CLINICAL URINOLOGY that are formed in the body (indican, phenols) cause the urine to turn dark on standing. Certain fruits (cherries, raspberries, etc.) and certain medicaments impart a char- acteristic color to the urine' (carbolic acid, coal-tar prepa- rations, resorcin, naphthol, salol, chrysarobin, rheum, senna, santonin, etc.) . (See also chapters on "Blood- and Bile-pigments of the Urine," and "Aromatic Constituents of the Urine.") Fluorescence. — Pale yellow normal urine shows a bluish, yellowish red urine a greenish or yellow fluorescence. Urine containing albumen shows more fluorescence than normal urine, and ammoniacal urine more than urine that is not decomposed. Behavior toward Polarised Light. — Normal urine is almost always slightly levorotatory ; it is never dextro- rotatory, and hardly ever optically inactive. The presence' of certain abnormal constituents (dextrose, levulose, gly- curonic acid, etc.) produces typical polarimetric phenom- ena that have been described above. (See chapters on "Carbohydrates of the Urine," particularly paragraphs on glycuronic acid compounds, on pages 89 and 90.) The Spectrum. — Normal urine shows no absorption bands, but the coefficient of light extinction varies in different regions of the spectrum. In pathologic urines, haematoporphyrin, uroerythrin, urobilin, etc., produce typical spectra. (See chapter on "Blood- and Bile-pig- ments of the Urine," and Plate of Spectra.) Transparency. — Normal acid urine is clear, for the normal acid and neutral salts of the urine are all readily soluble in water. Normal acid urine becomes cloudy if more quadriurate is excreted than can be dissolved in the urinary water (see page 224) . Normal alkaline urine is always cloudy, for the alkaline salts of the urine (i. e., normal phosphates and carbonates and di-phosphates of the alkaline earths) are essentially insoluble in water or in the mixture of neutral and acid salts in solution in the PHYSICAL PROPERTIES OF THE URINE 271 urine. The proteid constituents of clear urine are not in true solution, but in colloidal solution ; hence the tendency of the urine to filter with decreasing rapidity. The scanty epithelia, the mucin bodies, and the crystals in suspen- sion in normal urine form a fine cloud (nubecula) on standing. Odor. — Fresh normal urine has a characteristic odor that is not disagreeable. The disagreeable, so-called, urinous odor is due to the presence of bacterial decompo- sition products (probably ammonia and phenols) . Occa- sionally the urine emits an odor of H2S (hydrothionuria) (see page 271) . Certain substances impart a characteristic odor to the urine when taken by mouth (valerian, garlic, copaiba, cubebs, saffron, turpentine [violets], etc.). The peculiar odor of the urine after eating asparagus is due to methylmercaptan . Freezing Point. — The freezing point of the urine may be considered an index of the osmo-regulatory function of the kidneys and indirectly of the amount of electro- lytes in solution. Fluctuations in the freezing point of the urine (and the blood) are utilized in the determi- nation of the renal function. The method of freez- ing point determination is called cryoscopy (from Kpvos, ice and o-kottuv, to observe) and is described in detail in the chapter on "Determination of the Renal Function," on pages 276-284. Electric Conductivity, — The resistance offered by the urine to the passage of an electric current has been uti- lized in order to determine the quantity of mineral salts present in the urine. Only those urinary constituents can be estimated by determining the electrical conductivity that are electrolytes, and it might be imagined that the large number of non- electrolytes that may be present in normal and pathological urines would interfere with this determination. It has been shown, however (chiefly by Long) , that such bodies as urea, uric acid, even sugar and 272 CLINICAL URINOLOGY albumen in the quantities ordinarily present in pathologic urine, exercise only a very small effect upon the con- ductivity of the urine. The conductivity of the urine is essentially, therefore, a function of the 'total mineral content, and the determination has a significance similar to that of the determination of the specific gravity. It is, however, more accurate. This method, when simplified as to technique, will prove a valuable aid in the deter- mination of the eliminatory power of the kidneys for inorganic constituents. In combination with similar de- terminations with the blood serum, it promises to throw much light on the pathology of renal insufficiency and of a variety of metabolic disorders. For the present the methods are somewhat complicated, requiring also an expensive apparatus, and the results obtained too doubt- ful as far as their clinical application is concerned to warrant a description of the technique of determining the electric conductivity of the urine in this book. The method suffers from the same drawbacks as cryoscopy; both require expert skill of a higher order than that possessed by the average practician. In the hands of skilful operators, with a well-equipped laboratory and much time at their disposal, these methods, nevertheless, promise to be of great value. CHAPTER XIII THE DETERMINATION OF THE BENAL FUNCTION The Facts and Principles Underlying the Different Methods for Determining the Eenal Function. The Kidneys Considered as Osmo- regulatory Apparatus, as Selective Filters, as Glands. Cryoscopy. The Methylene Blue Test. Tlie Phlorizin Method. The Toxicity of the Urine as an Index of the Senal Function. Op the many methods that can be employed for study- ing the state of the renal function, only the most im- portant and the most practical ones will be described. Catheterization of the ureters, a procedure that is often indispensable in determining the renal function and that is of paramount diagnostic importance, particularly in unilateral diseases of the kidneys, is, properly speaking, a surgical procedure. A description, therefore, of the technique of ureteral catheterization, as well as of the other less satisfactory methods for segregating the urine from each kidney, is. beyond the scope of this book and should be sought for in text-books of surgery. The results ob- tained from the analysis of the urines collected from the two kidneys by this means, and the clinical interpretation of these results must, however, be included in this chapter. I have taken particular pains to emphasize, conserva- tively, the limitations of all the methods we possess to- day for determining the state of the renal function. We know too little as yet of the finer mechanism of urine formation to warrant our drawing far-reaching conclu- sions from anomalies in renal permeability for different substances. The kidneys are not a simple filter, for they exercise a K (273) 274 CLINICAL URINOLOGY selective affinity for certain constituents of the renal blood, allowing the passage of some, preventing the pas- sage of others; they act, moreover, as dialyzers in cer- tain portions, as secreting glands in others. The different methods for testing the renal function must, therefore, be directed towards determining the functional powers of the kidneys considered as selective filters for certain circulat- ing bodies, as dialyzers, i. e., as osmo- regulatory appara- tus, and as glands that pour a specific secretion into the urine. In order to gain information in regard to the osmo- regulatory powers of the kidneys, the analysis of the solids as a whole in solution in the blood and in the urine must be undertaken. This phase of the inquiry is discussed in the paragraphs on Cryoscopy of the blood and urine. Similar information can be obtained by measuring the electric conductivity of the blood and urine; but this method is more complicated, and, in the light of our present knowledge, less reliable for clinical work than cryoscopy. I have not, therefore, described it in this chapter. The principles underlying electric conductivity determinations as a measure of the molecular (ionic, electrolyte) concentration of a solution have, however, been discussed in the chapter on "Physical Properties of the Urine" (page 271). Whereas these physical methods are intended to give us information in regard to the relative amounts of urinary bodies as a whole in solution in the blood and urine, and hence in regard to the permeability of the kidneys for all these bodies collectively, certain chemical methods are intended to inform us in regard to the per- meability of the kidneys for single bodies that circu- late through them. Inasmuch as renal permeability is a relative conception, and it is important to determine what the kidneys fail to eliminate rather than what they, absolutely speaking, do eliminate, these chemical methods DETERMINATION OF RENAL FUNCTION 275 can be of value only if the amounts of the different sub- stances present in the blood and the urine are determined at the same time. Unfortunately, the chemical analysis of the blood is technically a very tedious and very difficult procedure, and one that is altogether impractical for ordinary clinical purposes. For this reason and for many other reasons that have been set forth at length in Chap- ter III, when discussing urea determinations as a measure of the renal function (page 42 ff) the discussion of the excretion and retention of normal products of metabolism (the urinary nitrogen, urea, uric acid, chlorides, phos- phates, etc.), as an index of renal permeability, will be omitted from this chapter. By injecting into the tissues certain indifferent but well- characterized /orei^rw substances that must ultimately be eliminated in the urine, by establishing certain normal values for the elimination of each of these bodies, more definite information in regard to the permeability of the kidneys can be obtained than from a study of the metia- bolic products that circulate in unknown and constantly varying quantities through the blood- stream. In select- ing such foreign bodies, care must be exercised not to inject toxic substances nor compounds that undergo dis- assimilation in their passage through the organism and hence escape elimination through the kidneys. The most popular of these bodies are methylene blue, iodide of potassium, rosanilin and acid fuchsin. I have described only the methylene blue test for renal perme- ability, because it is the most thoroughly studied and the most carefully interpreted method of all. Tests with the other bodies mentioned are no more simple than the methylene blue test and the results obtained not so free from ambiguity. As a test of the kidneys considered as secreting glands, the interesting and valuable phlorizin test is described. By employing this method we gain information not in 276 CLINICAL URINOLOGY regard to the permeability of the kidneys for bodies that are carried to them preformed in the renal blood, but in regard to the ability of the renal epithelia to elaborate a urinary product themselves from other blood constituents. This teaches us indirectly how much working epithelium is present in the kidneys. Here, again, certain normal values had first to be established and deviations from these values utilized as an index of abnormal function. The two determining factors in this test, as in all the chemical tests, are the amount of the substance present in the blood and the activity of the renal circulation. These varying elements must, therefore, always be in- cluded in the calculation. Finally, the toxicity test is described for completeness sake. It is, theoretically, of the greatest interest, for it gives us values that are not physical, not chemical, but physiological. Unfortunately, as will be shown below, the technical difficulties in performing this test are so great and the possible sources of error so many, that the clinical value of this test as a measure of the functional powers of the kidneys is exceedingly small. CRYOSCOPY* It is a well-known fact that the more concentrated a solution, the higher it boils and the lower it freezes. The boiling point or the freezing point of a solution, therefore, is an index of its molecular concentration. For clinical purposes the determination of the freezing point of the urine (and blood) with the apparatus to be described below is simpler and more practical than the exact deter- mination of the boiling point. The former method alone will, therefore, be described. Raoult was the first to show that the freezing point of a solution is lowered in proportion to the number of mole- * See also Chapter XII, page 271. DETERMINATION OF RENAL FUNCTION 277 cules it contains. The freezing point of a watery solu- tion is, conventionally, always compared with that of dis- tilled water, and the difference between the two points, i. e., the lowering of the freezing point, designated by the symbol A. If A is the same in two solutions, then they contain the same number of molecules, and two solutions containing the same number of molecules also develop the same osmotic pressure. Consequently, A is an index of the osmotic pressure of a solution. As the kidneys are intended to lower the osmotic pres- sure, i. e., the molecular concentration of the blood, it is clear that under normal conditions the molecular concen- tration of the urine is greater than that of the blood. The greater, therefore, the difference between the A of the urine and of the blood, the greater the permeability of the kidneys ; the more the A of the urine approaches that of the blood the less the permeability of the kidneys. By determining A in blood and urine one can, therefore, establish the relation between retained and eliminated substances. I refrain from entering into a detailed discussion of the physical principles underlying this method. Freezing point determinations are a most useful means, no doubt, for determining the molecular concentration of solutions, and they could not be dispensed with in pure physics and chemistry. Clinically, however, I consider the method for the present to be of subordinate importance. Cryos- copie examinations of the blood and urine give very little information that cannot also be obtained by the ordinary chemical and microscopical examination of the urine. The method requires great care, skill and experi- ence. The sources of error in performing the determina- tions themselves are many. The freezing point of the urine is influenced by many factors that must be carefully controlled if the results are to be properly interpreted; chief among these factors are the diet (a meat diet and 278 CLINICAL URINOLOGY starvation lowering the freezing point), the amount of liquid ingested and the amount lost through other emunc- tories of the body, the amount of exercise, the presence or absence of anemia (Koranyi) . The determination of A in a single specimen of urine is of no value whatsoever. The determination of A in the twenty-four hours' urine gives us no information not ob- tainable by other methods. If the urine is removed by catheterization from each kidney, if the freezing point of the blood is at the same time determined, and if all the sources of error enumerated above can be ruled out, then the cryoscopic examination of the urine may be of some prognostic, but rarely of diagnostic value. The great advantage of this physical method over the chemical methods heretofore in vogue to determine the eliminatory powers of the kidneys, is that it considers all the solids of the urine and not isolated urinary ingredi- ents. It also has this advantage over the determination of the specific gravity of the urine as an index of the mo- lecular concentration that it is not influenced by the presence of albumen in pathological urines. Normal urine has a freezing point of from — 0.9° to — 2.6°C. If the kidneys are diseased one should postu- late a slighter lowering of the freezing point (a smaller molecular concentration) and, as a matter of fact, it ap- pears that the average freezing point of the urine in suf- ferers from advanced kidney disease is, as a rule, less than — 1.0°C. This occurs only, however, if there is no com- pensatory hypertrophy of portions of the diseased kidney ; a freezing point, therefore, that lies within the normal limits does not exclude disease of a portion of a kidney. In cases in which there is true retention of excremen- titious urinary bodies the molecular concentration of the blood must increase, both absolutely and relatively to the molecular concentration of the urine. This is manifested by a lowering of the freezing point of the blood. Here, DETEBMIN^ATIOy OF RENAL FUNCTION 279 again, this physical means of analysis has the great ad- vantage over all quantitative chemical analyses of the blood, that it indicates the retention of the exere- mentitious bodies in their totality, and not of single urinary bodies. In fact, the technical difficulties of a complete chemical analysis of the blood are so great that such a determination would be altogether impractical. Cryoscopy of the blood, while by no means a convenient procedure, if for no other reason than that the patients usually object to the withdrawal of blood from a vein, is immeasurably more simple and more rapid. The molecular concentration of the blood is kept very constant in health, and its freezing point fluctuates within narrow limits ; i. e., between —0.56° and — 0.58°C. If metabolism is very active, so that many disintegrated molecules are poured into the blood, then the kidneys im- mediately eliminate the surplus and in this way maintain the constancy of the blood concentration; if one kidney is diseased this constancy, it appears, is nevertheless maintained, but if both kidneys become diseased then the molecular concentration of the blood rises and its freezing point is correspondingly lowered, approaching more and more that of the urine. Two factors, according to Ko- ranyi, the chief advocate of blood- and urine-cryoscopy, may, in a measure, vitiate this result; viz., a retention of water (dropsy, in kidney patients!) with resulting dilution of the concentrated blood and reduction of its molecular concentration, or an adaptation of proteid me- tabolism to the renal inadequacy, leading to a decreased breaking down of complex albumen molecules (anaemia, cachexia, malnutrition in nephritis) and a corresponding decrease in the molecular concentration of the blood. Water retention, and retardation of proteid metabolism must therefore always be included in the calculation in drawing conclusions in regard to renal inadequacy from a lowering of the freezing point of the blood. 280 CLINICAL UEINOLOGY One clinical point of great importance remains to be mentioned, for it should put us on our guard in inter- preting lowering of the freezing point of the blood in kid- ney cases; viz., that disturbances in the circulation of the kidneys and certain reflex influences can produce transitory elevations in the blood- concentration. This is seen, for instance, in tumors of one kidney, in attacks of renal colic, in pyonephrosis in which one kidney is organ- ically sound but functionally impaired. In such cases one might hesitate to operate on the diseased kidney when the blood concentration is high, for fear of inadequacy of the other "well" kidney. As a matter of fact, the removal of the tumor, the drainage of the abscess, the passage of the stone by permitting the reestablishment of normal circulation in the well kidney has been known to cause a rapid decrease in the blood concentration. Here, then, cryoscopic examination of the blood and urine might lead to wrong and dangerous surgical conservatism. Clinical Application. — Cryoseopy of the blood and urine for the present, therefore, has only the following clinical application: (1) In unilateral, lesions of the kidneys, cryoseopy of the urine withdrawn by catheterization from each kidney gives valuable information in regard to the competency of the well organ, inasmuch as it indicates the difference in the number of soluble molecules dissolved in the urine from each kidney. Even here, however, care must be exercised, for it appears that even normally differences in the excretory power of the two kidneys are noted. (2) Lowering of the freezing point of the blood below — 60°G. (provided all the extra-renal factors enumerated above can be excluded) should warn the surgeon against removing a kidney. This warning is not, however, abso- lute, for the incompetency of the well kidney may be due to circulatory or reflex disturbances that yield after the removal of the diseased kidney. On the other hand, a BEWERMINATION OF RENAL FUNCTION 281 competent kidney may become functionally inadequate after the operation as a result of surgical shock, manipu- lation or the effect of the anaesthetic. In emergencies that call for quick intervention, as renal abscess, pyo- nephrosis, malignant tumor, severe hemorrhage, the operation will have to be performed despite the lowering of the freezing point of the blood below — 60° C. (3) Generally speaking, therefore, the prognosis of a renal operation can be made according to the cryoscopic findings in the blood and urine, a low freezing point of the blood below — '■ 60° C. always rendering the prognosis gi-ave; a high freezing point, about — 56° to —58° C, of the blood combined with a low freezing point of the total twenty-four hours' urine or of the urine removed by catheterization from the well kidney, rendering the prognosis more favorable. TJie Method of Performing Freezing -Point Determina- tions. — The apparatus employed for freezing-point deter- minations is Beckmann's Cryoseope (Fig, 29) . It consists of a tube, B, that stands in a glass jar, D, containing the freezing mixture; a second tube, A, is placed into B and contains the liquid whose freezing point is to be deter- mined and the thermometer, T. R and Ei are stirring rods, intended to stir the freezing mixture bnd the liquid to be tested. Before making a series of determinations, the freezing point of distilled water must be determined on the thermometer scale. The thermometer is prepared for use as follows : The thermometer is dipped into ice- water ; if the column of mercury rises ^o the upper third of the thermometer, then no further adjiistment is needed ; if it does not rise above the lower third of the thermo- meter scale, then the thermometer is taiken out of the ice- water, the mercury bulb heated in the hand until the column of mercury shoots to the top into the bulb reser- voir; the thermometer is then grasped at its lower end and shaken until some of the mercury in the reservoir 282 CLINICAL URINOLOGY flies from b to a; the thermometer is then again dipped into ice- water and the stand of the mercury observed. If it still stands too low, the same manipulations are repeated. If the mercury stands too high the bulb is warmed in the hand until the column rises into the reservoir, and the upper part of the thermometer is then gently tapped until a little of the mercury drops from a to b. In this way the thermometer can be so regulated that the freezing - point of water is indicated somewhere in the upper third of the instrument. Now the exact freezing point of distilled water must be de- termined. For this purpose 15 cc. of distilled water are •v »