Practical Examination of Urine Tyson Ninth Edition. EXAMINATION OF URINE. BY THE SAME AUTHOR. BRIGHTS DISEASE AND DIABETES. A treatise on Bright’s Disease and Diabetes. With Especial Reference to Pathology and Therapeutics. With colored plates and many wood engravings. 8vo. Price, net, $2.50. “ Dr. Tyson has presented his subjects so clearly and concisely, and has selected his material with such sound judgment, that his work cannot fail to be useful to practitioners and students.”—The American Journal of the Medical Sciences. “The symptoms are clearly defined, and the treatment is exceedingly well described, so that every one reading the book must be profited.”—Cincinnati Lancet and Clinic. THE CELL DOCTRINE: Its history and present state, together with a copious Bibliography of the Subject. With a colored plate and other illustrations. Second revised edition. Cloth, price, net, $1.50. “ The first edition of Dr. Tyson’s work appeared in 1870, and at once received the favorable reception from the profession to which it was entitled by its merits. The present edition shows an increase in size of about fifty pages, and almost every page furnishes evidence of careful revision.”—American Journal of the Medical Sciences. “ The author in this work presents to medical students a concise and instructive resume of the origin and advance of the doctrine of cell evolution. The work is calculated to be of decided benefit to all who desire a general knowledge of the history, progress, and peculiar phases of the cell doctrine.”—American Prac- titioner. A MANUAL OF PHYSICAL DIAGNOSIS. For the use of Stu- dents and Physicians. Second Edition. Cloth, price, net, $1.25. “ The present volume is by a clinical teacher of much experience and a practi- tioner of well-known ability, so that his work has a value outside of a class of undergraduates.”—American Journal of the Medical Sciences. IN PREPARATION. A TEXT-BOOK of the PRACTICE of MEDICINE, FOR THE USE OF PHYSICIANS AND STUDENTS. 4. PcdeYcllow. 2. LighiYcllow. 3. Ydlow. 4. Reddish, Pellow. 5. Yellowish Red. 6. Red. 7. Brownish Red. 8. ReddishBromi. 9. BrownisJiBlack Bromldature byDr. JVogcZ. F. MORAS. LITH PH!LA VOGEL'S SCALE OF URINE TINTS. A GUIDE TO THE PRACTICAL EXAMINATION OF URINE FOR THE USE OF PHYSICIANS AND STUDENTS. JAMES TYSON, M.D., 15Y PROFESSOR OF CLINICAL MEDICINE IN TfTE UNIVERSITY OF PENNSYLVANIA, AND PHYSICIAN TO THE HOSPITAL OF THE UNIVERSITY; PHYSICIAN TO THE PHILADELPHIA HOSPITAL; FELLOW OF THE COLLEGE OF PHYSICIANS OF PHILADELPHIA, ETC., ETC., ETC. NINTH EDITION. REVISED AND CORRECTED. WITH A COLORED PLATE AND WOOD ENGRAVINGS. PHILADELPHIA : P. BLAKISTON, SON & CO., IOI2 WALNUT STREET, 1895. Entered according to Act of Congress, in the year 1895, by JAMES TYSON, M.D., In the Office of the Librarian of Congress, at Washington, D. C. PRESS OF WM. F. FFLL & CO., 1220-24 Sansom Street, PHILADELPHIA. PREFACE TO NINTH EDITION. No large additions have been made to the previous edition in preparing the present. On the other hand some less important paragraphs have been omitted, so that I have been able to keep the book to its moderate size. The usual care has been taken to secure accuracy, and some alterations have been made to this end. Acknowl- edgment is due my son, Dr. T. Mellor Tyson, for material assistance. James Tyson. 1506 Spruce Street, December /, 1893. VII LIST OF ILLUSTRATIONS. COLORED PLATE Vogel’s scale of urine tests, Frontispiece. WOOD ENGRAVINGS. FIG. PAGE 1. Measuring-glasses for measuring urine, 19 2. Apparatus for determining specific gravity, 27 3. Small urinometer, 28 4. Dr. Squibb’s new urinometer and floating cylinder, .... 30 5- Testing for albumin by nitric acid, 40 6. Esbach’s albuminometer, 55 7. Burette stand with different forms of burette, 80 8. Dr. Johnson’s picro-saccharimeter 90 9. Laurent’s polarizing saccharimeter 101 10. Illustration of the use of Laurent’s saccharimeter, 102 11,12. Illustrations of the use of Fleischl’s saccharimeter, . . . 103 13. Crystals of nitrate of urea 153 14. Burette stand with two forms of burette, 158 15. Marshall’s apparatus for estimation of urea by the hypobro- mite process, 163 16. Ureometer of Doremus, 166 17. Prismatic crystals of sodium urate, spherules of ammonium urate, and amorphous urates, with octahedral crystals of oxa- late of lime, 190 18. Spiculated spherules of ammonium urate along with triple (ammonio-magnesian) phosphate and octahedral crystals of the oxalate of lime, 192 19. More usual forms of uric acid crystals, 196 IX X LIST OF ILLUSTRATIONS. FIG. PAGE 20. More unusual forms of uric acid crystals, 197 21. Other unusual forrps of uric acid, not unlike crystals of the triple phosphate of ammonium and magnesium. X 150. • • 198 22. Spherules and spiculated spherules of urate of ammonium (sodium ?) ; amorphous granular urates 200 23. Prismatic crystals of acid-sodium urate, spherules of ammo- nium urate, and amorphous urates with octahedral crystals of oxalate of lime, 201 24. Dumb-bell and octahedral crystals of oxalate of lime, .... 204 25. Prismatic crystals of triple phosphate (ammonio-magnesian phosphate), 209 26. Stellate feathery crystals of triple phosphate, 210 27. Crystalline and amorphous phosphate of lime, 211 28. Crystals of calcium sulphate, 213 29. Leucin spheres and tyrosin needles, 214 30. Cystin, 216 31. Mucus- and pus corpuscles before and after the addition of acetic acid, 220 32. Round epithelial cells from the convoluted tubules of the kid- ney found in urine from a case of acute nephritis. X 42°> • 224 33. Epithelial cells from different parts of the genito-urinary tract, 226 34. Blood-disks, 228 35. Epithelial casts and compound granule cells, 229 36,37. Pus-casts, blood casts, 230 38. Hyaline casts. X 2IO> 23° 39. Hyaline and granular casts, 231 40. Dark granular casts. X 225, 232 41. Waxy casts. X I5°> 233 42. Oil casts and fatty epithelium. X 200> 233 43. Cylindroids or mucus-casts. X 200, 234 44. The Purdy electric centrifuge, 237 45. The Daland centrifuge 238 46. Human spermatozoids, 240 47. Tubercle bacilli from urine. X 75°» 242 48 Yeast fungus, 243 TABLE OF CONTENTS. PAGE Secretion of Urine, Reagents and Apparatus Required for Examination of Urine, . 16 Selecting a Specimen of Urine, Xg General Physical and Chemical Characters of the Urine, .... 20 To Determine the Amount of Solid Matters in the Twenty-four hours’ Urine, INTRODUCTION. PART I. Albumin and Tests therefor, Quantitative Estimation of Albumin, 54 Other Proteids in Urine ry Serum-globulin, Globulin or Paraglobulin ry Nucleo albumin (Mucin), 58 Peptone gj Hemialbumose or Propeptone, 67 Fibrin» 68 Sugars found in Urine, yQ Glucose, y 0 Other Saccharine Substances found in Urine, 107 Inosite, Fruit-Sugar or Lsevulose, 1Qg Sugar of Milk or Lactose, IOg Aceton and Acetone Producing Substances, no Coloring Matters, Normal Coloring Matters, 121 Abnormal Coloring Matters, 130 Biliary Acids, Leucin and Tyrosin, t^g Fatty Matters, Urea I52 Uric Acid jg9 XI XII TABLE OF CONTENTS. PAGE Urates, 174 Chlorides, 176 Phosphates, ... 179 Sulphates 185 Urinary Deposits, 188 Unorganized Sediments, 195 Uric Acid, 195 Uric Acid Compounds, 199 Oxalate of Lime, 203 Earthy Phosphates, 208 Calcium Carbonate, 213 Calcium Sulphate, 213 Leucin and Tyrosin, 213 Cystin, 215 Organized Sediments, 217 Mucus and Pus, 217 Epithelium, 223 Blood-corpuscles, 225 Tube-casts, 228 Spermatozoids, 239 Fungi 241 The Elements of Morbid Growths, 244 Entozoa, 244 The Preservation of Organized Urinary Sediments for Sub- sequent Examination, 245 Differential Diagnosis of Renal Diseases, 247 PART II. Urinary Calculi, 254 To Determine the Composition of Calculi, 255 PART III. APPENDIX. Mode of Recording an Examination, 261 Tables for Reducing the Metric System into the English and vice versa, and for Converting Degrees Centigrade to Degrees Fahrenheit and vice versa 263 Index, 267 PRACTICAL EXAMINATION OF THE URINE. INTRODUCTION. SECRETION OF URINE. The theory which explains the secretion of urine most consistently with the facts, is one which, while it makes the process partly physical, requires also something of the nature of elaboration in the office of the kidney. Nothing can be more attractive at first thought than the theory of Ludwig, according to which the process is purely physical —partly a filtration and partly a diffusion, or osmosis. It is true that in the capillaries of the Malpighian tuft the blood-pressure is relatively greater on account of the resist- ance to the exit of the blood through the efferent vessel. As the result of this, a filtration of the watery constituents of the blood, with some dissolved salts, takes place into the Malpighian capsule. Thus the blood is greatly thickened when it reaches the second capillary network embracing the convoluted tubules into which has descended the thin, aqueous filtrate from the Malpighian bodies. Here are the essential elements of a complete osmometer—an animal 13 14 PRACTICAL EXAMINATION OF THE URINE. membrane formed by the thin wall of the capillary and the delicate basement membrane of the tubule, having a dense fluid, the blood, on one side, and a thin saline solution on the other. An interchange now takes place, as the result of which a current sets in, of liquid from the tubules to the blood, and of the products of regressive metamorphosis— urea and salts—to the tubules, concentrating the fluid in the latter, making it, in a word, urine ; while the albumin- ous constituents of the blood are retained in it because of their well-known indisposition to osmosis. The objection formerly made to the physical nature of the act of secretion of urine, on the ground that we cannot by this method account for the formation of an acid fluid from an alkaline one, no longer holds, since Charles Henry Ralfe has shown this to be quite possible. Into one limb of a small, U-shaped tube, fitted with a membranous diaphragm at the bend, he introduced an alkaline solution of sodium bicarbonate, and into the other limb a solution of neutral sodium phosphate. He then passed a weak electric current through the solutions. In a short time the fluid in the limb connected with the positive pole became acid from the formation of acid sodium phosphate, while the fluid in the limb connected with the negative pole increased in alka- linity. The changes are represented by the following formula :— Sodium bicarbonate. Neutral sodium phosphate. Sodium carbonate. Acid sodium phosphate. NallCOj + Na2HP04 = Na2COs + NaH2PQ4. One important fact, however, remains unaccounted for by this theory, clear and simple as it is. This is, that if * Medical News and Library, October, 1871, fiom London Lancet, July 4, 1871. the tubules are stripped of their epithelium, as they often are in disease, urea and other products of regressive meta- morphosis are no longer so thoroughly removed, but accu- mulate in the blood, producing the phenomena of the condition known as urcemia. We must therefore admit some elaborating action on the part of the epithelium, as originally suggested by Bowman. Doubtless, however, a part of the act is physical—a process of transudation or filtration, and of diffusion or osmosis. The experimental researches of Heidenhain* settled the question in favor of an active elaborating office on the part of the epithelium of the kidney. Heidenhain injected into the blood of animals indigo-carmine, a substance which is promptly separated by the kidneys. He removed these organs at suitable intervals after the operation and examined them minutely. In no instance did he find any of the indigo-carmine in the Malpighian capsules, but the cells lining the convoluted tubules and the looped tubes of Henle were filled with it, as was also the lumen of the tubes, if*the animal was killed sufficiently long after the injection. Similar experiments with urate of sodium showed that it is secreted at the same place and in the same manner. More recent studies of Heidenhainf and J. G. Adami J go to diminish still further the importance of the “ mechan- ical ” factor in the secretion of urine and to assign a selective or secretory office even to the cells of the glom- SECRETION OF URINE. 15 * Max Schultze’s Archiv, vol. x, 1874, p. 1, and Pfliiger’s Arc/iiv, vol. ix, 1874, p. 1. f Heidenhain, Hermann’s Handbuch der Physiologie, vol. v, Leip- zig, 1880. J J. G. Adami, On the Functions of the Glomeruli of the Kidney, The Practitioner, London, 1888. 16 eruli, in accordance with which they permit certain sub- stances to pass through and prevent others, among the latter albumin. On the other hand, they indicate that even the passage of fluid through the glomeruli is not a mere transudation, but a matter of selection also, though it is influenced greatly also by the rate of flow of blood through them, which again depends (1) on the general blood pressure and (2) inversely on the resistance against which the blood is forced through the kidney. PRACTICAL EXAMINATION OF THE URINE. REAGENTS AND APPARATUS REQUIRED FOR QUALITATIVE AND APPROXIMATE ANALYSIS.* It is not a matter of very great importance in what form of bottles reagents are kept. They should hold enough— four ounces is a convenient quantity—and be provided with ground-glass stoppers for the acids, but the alkalies are better kept in bottles with rubber stoppers. Those required are as follows :— 1. Pure colorless nitric acid (HNOs). 2. Nitroso-nitric acid, the brown, fuming nitrous acid of commerce, —nitric acid containing nitrogen tetroxide (HNOs-|- N204, or NO,). 3. Pure hydrochloric acid (HC1). 4. Pure colorless sulphuric acid (H2S04). 5. Pure acetic acid (C2H402). 6. Liquor potassce, U. S. P. The sp. gr. is 1065, and it contains .058 of potassium hydroxide (HKO). 7. Solution of caustic potash, or caustic soda, I part to 2 of distilled water, sp. gr. 1330 to be spoken of in the text as the “ stronger solution of potash.” It is the aetzkalilauge (or * All reagents and apparatus suitable for urinalysis may be obtained of Bullock & Crenshaw, 528 Arch Street, Philadelphia. REAGENTS AND APPARATUS. 17 aetznatronlauge, if soda) of the German Pharmacopoeia, and con- tains from .30 to .31 of the hydrate of potassium (or of sodium). 8. Solution of sodium carbonate, 1 part water and 2 parts of the crys- tallized salt. 9. Solution of barium chloride, 4 parts crystallized barium chloride, 16 of distilled water, and I of hydrochloric acid. 10. Liquor ammoniae, U. S. P. 11. The magnesian fluid, containing of magnesium sulphate and pure ammonium chloride, each 1 part, distilled water 8 parts, and pure liquor ammoniae I part. 12. Solution of copper sulphate, say 1 gram to 30 c.c., or 15 grs. to 13. Pavy’s or Fehling’s copper solutions, made as directed under volu- metric analysis for sugar. 14. Solution of silver nitrate, 1 part to 8 of distilled water. 15. Solution of neutral lead acetate (sugar of lead), I part to 4 of dis- tilled water. 16. Solution of basic lead acetate, 1 part to 4 of distilled water. 17. Distilled water, a litre or a quart. 18. Alcohol, 95 per cent., half a litre or a pint. Other solutions as required. Apparatus. A note and drawing-book. 2 dozen test-tubes, assorted sizes, some narrow. Some test-tubes with bases, so that they may stand on a shelf or table, are convenient and desirable ; see Fig. 5. Some tubes should be graduated in divisions of a centimetre and fractions thereof. They may be used as fluid measures, and to determine the proportion of a sediment, or of albumin after its precipitation by heat. Test-tube rack and drainer. 4 conical glasses. (Observe that there is not a convexity at the bottom, and the edge should be ground so that they may be covered with ground-glass covers and thus made air-tight.) 2 or 3 smooth wineglasses, with broad bottoms, of the kind sometimes known as “ collamore” wineglasses. Red and blue litmus paper ; Swedish filtering paper. 18 PRACTICAL EXAMINATION OF THE URINE. Urinometer and urinometer glass. 4 ground-glass covers, assorted sizes. Spirit-lamp, or Bunsen burner. 3 porcelain capsules. 6 beaker glasses, small and medium sizes. 6 watch-glasses. 3 glass funnels, assorted sizes. i long narrow funnel tube 12 inches long and I inch wide, for filtering through animal charcoal. Glass stirring-rods and plain glass pipettes. I large receiving-glass to measure twenty-four hours’ urine, with capa- city of 2000 cubic centimetres or more, i graduated measuring glass holding 500 c.c. 1 wash-bottle with distilled water. 1 retort stand ; water-bath. I or 2 sheet-iron tripods with wire gauze to cover. 1 100-minim pipette; 1 volume pipette for 5 c.c., another for 10 c.c. Platinum spoon ; tongs. Blowpipe. Swabs for cleaning test-tubes, etc. A microscope with two object-glasses, a \ or £ inch, and a I inch or inch; stage micrometer ; camera lucida for drawing; glass slides, thin covers, shallow cells ; a double nose-piece for quickly chang- ing objectives is very convenient. For volumetric analysis are required in addition :— A full set of volume pipettes, 5, 10, 15, 20, 30, 50 c.c. X dropping pipette holding 1 c.c., graduated in and fractions thereof. 2 burettes of 50 c.c. capacity; burette stand. A half-litre flask. Volumetric solutions as directed under Volumetric Analysis. If the solutions are made by the physician himself, as they may be, he should be provided with a balance which will turn with a milligram, or with fa of a grain if the English system is used. SELECTING A SPECIMEN OF URINE. 19 SELECTING A SPECIMEN OF URINE. In obtaining a specimen of urine for chemical examina- tion, it should, as far as possible, be a part of the whole twenty-four hours’ urine, as the specific gravity, reaction, and other properties are well known to vary during the twenty-four hours, and the only accurate method is, therefore, to take a part of the total. Circum- stances, however, constantly demand a modification of this course. Thus, when a small quantity of albumin is present in urine, it is often increased after a meal, and sometimes when there is no trace apparent in the morning urine passed on rising, a little will be detect- able after the patient has been up for a time or has taken food. The same is true of sugar, and a very good plan in all cases is to ask for two specimens, one passed on retiring or about an hour after dinner, and the other passed on rising. For micro- scopical examination, the importance of having a fresh specimen which has not undergone decomposition or change of reaction is paramount. In Fig. i are repre- sented forms of glass vessels used for measuring large quantities of urine. In warm weather urine decomposes rapidly, becoming alkaline, and it is especially important where microscopic examination is to be made that such decomposition should be obviated, as certain casts and crystalline sediments of uric acid are often dissolved in alkaline urine. Where urine Fig. i. 20 PRACTICAL EXAMINATION OF THE URINE. cannot be examined in the fresh state, and especially when it has to be sent any distance, decomposition may be averted by adding five grains of salicylic acid or ten grains of salicylate of sodium to each four ounces of urine. GENERAL PHYSICAL AND CHEMICAL CHARACTERS OF URINE. Normal urine may be described as a transparent, aqueous fluid, of a pale lemon-yellow hue, acid reaction, specific gravity of about 1020 when passed in the average quantity of 1500 cubic centimetres (50 ounces) in the twenty-four hours, and possessing an odor which can only be indicated as “ characteristic ” or “ urinous.” The odor is sometimes spoken of as “ aromatic.” Each one of these characters is, however, liable to some variations within the limits of health, as well as in disease, and with these variations we should be thoroughly familiar before interpreting a given specimen. I. As to Transparency.—This, although quite a con- stant, can scarcely be considered an essential character of normal urine, while, on the other hand, it by no means follows that because a given specimen of urine is trans- parent it is therefore normal. Causes of Diminished Transparency.—Dimin- ished transparency may be due to one of three causes. 1. Even urine which is apparently perfectly transparent when passed, commonly exhibits, a few minutes after stand- ing, floating somewhere between the top and bottom, a faint cloud, which is said to be mucus derived from the genito- urinary tract. In the urine of females this cloud is apt to be more distinctly visible, in consequence of a larger amount of epithelium from the vagina and adjacent mucous surfaces PHYSICAL AND CHEMICAL CHARACTERS. in this sex. There is nothing abnormal in the presence of such an amount of mucus as is covered by the above descrip- tion. The effect of alkalies, heat, and strong acids is to leave the appearance unchanged, but acetic acid may pro- duce a slight increase of the opacity by coagulating the mucin. Mucus can be filtered out, leaving the urine clear. 2. Normal, slightly acid urine may be turbid at the moment when passed, by reason of the presence of the earthy phosphates of calcium and magnesium. These, shortly after passing, begin to subside, and, within half an hour, present an appearance not unlike that of mucus,—that of a floccu- lent mass, floating somewhere between the top and bottom of the vessel. But still later, generally within an hour, they have approached the bottom, and become a sediment, cloudy and bulky, leaving a transparent supernatant fluid. To test the nature of such sediment add a few drops of any acid, as nitric, which will cause the prompt disappear- ance of any earthy phosphate, while the application of heat will increase the deposit, such increase being also rapidly dissipated on adding an acid. The more or less constant presence of the earthy phos- phates above mentioned cannot be considered abnormal. Requiring an acid urine to keep them in solution, a dimi- nution of the degree of acidity may result in their precipi- tation, which is further increased by an alkaline reaction. Such diminished acidity and substitution of alkalinity always takes place from two to four hours after a meal, and the deposit is therefore commonly observed at such time. 3. Urine is sometimes rendered turbid by the presence of the so-called mixed urates of sodium, potassium, calcium, 21 22 and magnesium. The most frequent cause of this precipi- tation in normal urine is a reduction in the temperature of the urine after being passed. Although highly soluble in water at the temperature of the body, the urates drop promptly from a cold urine, of a temperature such as would prevail in a room without fire in winter. As in the case of earthy phosphates, such opacity soon diminishes by subsidence of the disseminated urates, which becomes a white or pink deposit, less bulky than that of phosphates; urates are also apt to be precipitated on the side of the vessel. To test the nature of this deposit apply heat, which quickly causes the dissipation of urates, while a precipitate of phosphates is increased by it. 1. Pathologically, urine may be opaque or semi-opaque from abnormal degrees of the above conditions, or from the presence of pus, which also subsides with a rapidity in- versely as the quantity of mucus. If the latter is absent, or present in small quantity, the subsidence is rapid; if, on the other hand, it is large, subsidence is slow, often requiring several hours. The turbidity of such urine is increased by the application of heat and acids, in conse- quence of the precipitation of albumin, which is always a constituent of liquorpuris. 2. The presence of fat in a state of minute subdivision, as in the so-called chylous urine, produces a degree of tur- bidity ranging from mere cloudiness to absolute milkiness. In such urine the fatty matter is disposed to rise and form a whitish, creamy layer on top of a less turbid fluid. Such urine, not uncommon in tropical countries, is also some- times met in temperate climates. II. As to Consistence.—In health, urine is never PRACTICAL EXAMINATION OF THE URINE. PHYSICAL AND CHEMICAL CHARACTERS. 23 anything else but aqueous, that is, it drops and flows readily, like water. Pathologically, it often becomes viscid, glutinous, and separable with difficulty into drops, or not at all. Such state may be due to the presence of an excess of pure mucus, or of a mixture of mucus and pus, and very frequently it is caused by the action upon pus of an alkalinity due to the presence of ammonium carbonate. This will be again alluded to. In the chylous urine above referred to, the presence of the molecular fat also increases the consistence of the urine. III. As to Color.—While normal urine may be char- acterized in general terms as pale-yellow, lemon-yellow, or amber-hued, there may be considerable variation in health. Due to the presence, in solution, of the normal coloring matters, the color is deeper or paler according to the pro- portion of water dissolving them. After copious libations of beer or water, the quantity of urine discharged being large, it is very pale. On the other hand, circumstances which diminish the proportion of water within the limits of health deepen the color. The complemental relation of the skin and kidneys is well understood. Given the same amount of water ingested, under the influence of warmth, when the skin is acting freely, the quantity of urine is smaller, and it is darker; in winter, the skin being less active, the quantity of urine is larger, and its color less deep. In persons from whom the respiratory exhalation is greater, the urine is likewise less abundant, darker, and vice versa. Pathologically, the color of urine may be altered, 24 PRACTICAL EXAMINATION OF THE URINE. first, by increase or diminution of the normal coloring matters, or, second, by the addition of abnormal ones. 1. The former is generally due to a change in the pro- portion of the coloring matters to the watery constituent. Thus we have almost an absence of color in the copious urines of diabetes, hysteria, and convulsions, while the urine of fevers and febrile states is high colored, chiefly because the quantity of water is diminished, but in the latter instance also because of the addition of an abnormal coloring matter known as uroerylhrin. 2. (a) The addition of abnormal coloring matters is seen in the instances just mentioned—fevers, in urines contain- ing blood or blood-coloring matters, and bile pigment; and in the blue and brown urines of which instances have been reported. (b\) The urine is also colored after the ingestion of certain vegetable matters eliminated by the kidneys, as santonin, which imparts a yellow color. IV. The reaction of normal mixed urine, that is, the urine of the entire twenty-four hours, is always acid. And, usually, specimens of urine passed at any time of day ex- hibit this reaction, though there is a difference in its degree, while three or four hours after a meal the urine may become neutral or even alkaline. The cause of this change in the reaction is still disputed. Roberts believes that it is due to an admixture with the blood of the elements of food, which are largely alkaline, and that the resulting increased alkalinity affects the reac- tion of the urine secreted. Bence Jones contended that it is the demand made on the blood for the elements of the acid gastric juice, which thus affects the reaction of the PHYSICAL AND CHEMICAL CHARACTERS. 25 urine secreted during digestion. Neither explanation is altogether satisfactory. In health, urine passed on rising after the night’s sleep is always acid. The cause of the acid reaction of the urine is mainly acid sodic phosphate, though the reaction is probably also slightly contributed by other acid constituents, as uric and hippuric acids, and, under certain circumstances, also by lactic and acetic acids. There is often observed in urine which has been standing for a short time, especially at a moderate temperature, an increased degree of acidity, which sometimes results in a decomposition of urates, and a precipitation, first of acid urates, and later of uric acid crystals. This has been ascribed by Scherer to an acid fermentation, in which, the mucus acting as a ferment, lactic and acetic acids are formed by the decomposition of the coloring matters of the urine. This has not been satisfactorily proven, while the increased acidity is by no means constant. It is certain, however, that acid urine which has stood for some time, especially in hot weather, acquires an ammoniacal odor, and becomes alkaline in its reaction ; attending this change of reaction is a turbidity with a pre- cipitation of a white amorphous and crystalline sediment, and often also with the formation of an iridescent pellicle on the surface. The cause of these changes has been well determined, and has already been alluded to. Through the action of mucus and other organic matters, acting in their decomposition as a ferment, the urea is converted into ammonium carbonate by the addition of two equivalents of water. Thus: — CH4N20 +2 H20 = (NH4)2C03, 26 PRACTICAL EXAMINATION OF THE URINE. which gives the odor, of ammonia and the alkaline reac- tion.* The turbidity and deposits are due to the precipitation of the crystalline triple phosphate of ammonium and mag- nesium, the amorphous phosphate of lime, urate of ammo- nium, and to living vegetable organisms known as bacteria. The alkalinity thus resulting from the presence of 7>olatile alkali (ammonia) is easily distinguished from that due to fixed alkali (potash or soda). On drying the test-paper, which has been rendered blue by volatile alkali, the blue color disappears, and the paper resumes its original red or violet tint; if due to fixed alkali the blue remains after desiccation. V. The specific gravity of normal urine may be put down at 1020 for an average amount of 1500 c.c. (50 oz.) in the twenty-four hours. But as this quantity is by no means fixed, while that of solid matter remains about the same, the specific gravity must vary accordingly. When, from the action of cold or other cause, the skin is not acting, and after copious use of water and diuretics, the specific gravity may descend to 1010, and even lower, within the limits of health. But, when perspiration is copious, or a drain of water from the economy takes place through some other channel, the urine becomes concentrated, and may be 1030 or higher in specific gravity. Pathologically, the specific gravity of urine is increased or diminished, but to be quite reliable, observation should * Normal urine may have an amphoteric reaction, i. e., it may change blue litmus paper to red and red litmus blue, because of the simultaneous presence in the urine of acid and neutral phosphates. SPECIFIC GRAVITY. 27 be made on the entire quantity parsed in the twenty-four hours. The specific gravity is increased in diabetes inellitus, where it sometimes reaches 1050. A specific gravity of more than 1028, if it attend a copious urine, should excite suspicion of diabetes, and calls for sugar tests. It not infrequently happens that urines with a specific gravity Fig. 2.—(From Harley.) of ioio, and even lower, contain glucose, and it is not safe to infer from a low specific gravity alone the absence of sugar. The specific gravity is also increased in the first stage of acute fevers, in consequence of the increased amount of solid matters excreted and diminished water separation ; and in the first stage of acute Bright’s disease, from the presence 28 PRACTICAL EXAMINATION OF THE URINE. of blood, the higher specific gravity of the latter and the diminished secretion raising that of the mixed fluid. The specific gravity is lowered in hysterical and spasmodic hydruria, though here it attends a proportionate increase of water, and is not of much practical significance. In all forms of Bright's disease, except the stage of acute nephritis referred to, and in the condition known as cya- notic induration of the kidney, which often attends heart disease, there is a tendency to lowering of specific gravity owing to the diminished proportion of urea. Particularly is such reduction of specific gravity significant when it attends diminished secretion of urine. In a general way, the presence of albumin and sugar being eliminated, varia- tions in the specific gravity point to variations in the amount of urea present; lower specific gravity of mixed uritie generally means less urea. To determine specific gravity the so-called uri- nometer is almost invariably used, and though less accurate than the picnometer (e, Fig. 2) and balance, it is still suf- ficiently so when carefully constructed. Every urinometer should first be tested with distilled water at 6o° F. (15.540 C.), into which it should sink to the mark o, or 1000. In its graduation the lines indicating the degree should gradually approach each other as the bulb is reached, because allowance must be made for the weight of the stem above water. The English-made urinometers, about five inches long (Fig. 2, c), are generally accu- rate, but the short German instruments (three inches) are very convenient for small quantities of urine. In the Fig. 3. SPECIFIC GRAVITY. little urinometer of Heller (Fig. 3), in which the “sink ” consists of leaden shot, the graduation of Baumd is retained, where one degree corresponds with seven of the ordinary scale. Thus, 1001 — 1007, 1002 = 1014, and so on. Especial care should be taken in testing these instruments, as a slight variation in them indicates a large one by the ordinary scale. The writer has in his possession an instrument of this kind which recorded the specific gravity of a given specimen of urine 1004, that is, 1028 by the ordinary scale, of which the specific gravity by a long-tried English instrument was found to be 1019. And on testing the former with distilled water it was found to sink, not to 1000, but to 1001 proving its inaccuracy. Another German urinometer, even slightly shorter than the original of Heller, has the ordinary scale on an ivory stem within the tube, and the “ sink ” contains mercury instead of shot. It is apparently altogether more carefully made, and I have found it accurate. There is great convenience in a short urinometer, as much smaller quantities of urine can be tested by it. Dr. E. R. Squibb, of Brooklyn, N. Y., has recently sug- gested a small urinometer standardized for 250 C., or 770 F., a temperature much more usual than 6o° F., and at three points, 1000, or 1030, and 1060, with the variations marked, whence corrections are easily made. The glass jar supplied with it is fluted, and in this way the instru- ment is kept from clinging to the side of the glass. This object is further secured by making the air-chamber a double cone, base to base, as seen in a, Fig. 4, instead of a cylinder, as in c. In this way, also, there is obtained a single point of contact between the urinometer and the jar. Finally, the instrument is provided with a thermom- 29 30 PRACTICAL EXAMINATION OF THE URINE. eter to secure greater accuracy, but as 4, if water be put at 1000, is about the maximum error which can occur at any temperature at which urine is likely to be tested, it is not really necessary. This is certainly the most accurate urinometer I have seen ; and by reason of the fluting of the glass, a smaller amount of urine is required than in the ordinary cylindrical jar. Fig. 4.—Squibb’s Urinometer and Jar, shown also in section at b. The cylindrical glass vessel usually supplied with the urinometer, or a sufficiently large test-tube, should be about three-fourths filled, the urinometer introduced, and when at rest the specific gravity read off. The cylinder or test-tube should not be too small in relation to the uri- nometer, lest, in consequence of the capillary attraction between the latter and the walls of the cylinder, the urinometer should not sink as low as it ought. For the SPECIFIC GRAVITY. same reason the urinometer should not be allowed to impinge against one side of the glass. All these dif- ficulties are provided against in Squibb’s urinometer. The scale should be read from above, not below the fluid. If the quantity of urine be too small sufficiently to fill the cylinder, it may be diluted with enough distilled water to fill the cylinder to the required height, noting the volume added. From the specific gravity of this mixture may be calculated that of the urine. Thus, suppose it is necessary to add four times as much water as urine to enable us to use the urinometer, that is, to make five volumes, and the specific gravity of the mixed fluid is 1004, then that of the urine will be 1000 -f (4 X 5) =1020. Although the principle of this method is correct, and the results must be, if the data are, the urinometers in use are not usually so nicely graduated that absolute accuracy in reading is secured : while any error in reading is multiplied by the number of volumes used.' Hence, it is desirable to use this method as rarely as possible, especially with urines of low specific gravity. VI. Quantity.—The average amount of urine in the twenty-four hours is usually put down at 1500 c.c., or about 50 fluidounces. I am inclined to think this is a little more than the average; perhaps it would be nearly correct to say between 40 and 50 ounces, or 1200 to 1500 c.c. But enough has already been said to allow the infer- ence that there is also much variation within the limits of health. All that has been said of color and specific gravity in this respect is true of the quantity of urine, though in an inverse ratio. That is, in health, diminished intensity of color and diminished specific gravity correspond with increased quantity of urine. It is with regard to quantity 31 32 that the complemental relation so well known to exist between the skin and kidneys most palpably shows itself, the increased activity of the former causing diminished water separation by the latter, and vice versa. In deranged conditions, it is the absence of this relation of color and specific gravity to quantity which gives significance to either. Pathologically, the quantity of urine is increased in diabetes mellitus and insipidus, and hysterical and convul- sive conditions; in the first with increased specific gravity, and in the remainder with diminished. In cardiac hy- pertrophy, in common with all conditions which cause increased blood-pressure, including the ingestion of large amounts of water, the peripheral action of cold, etc., there is an increase of water, and a corresponding reduction in specific gravity and color. In all forms of Bright’s disease, except the cirrhotic and lardaceous kidneys, there is a tendency to diminished secre- tion of urine. Toward the fatal termination, however, it is diminished even in these affections. Any marked dimi- nution of urine in these diseases, particularly if attended by low specific gravity, which means diminished urea, becomes a grave symptom. In acute fevers and inflammatory affections, the quantity of urine is very constantly diminished until convalescence sets in, when there is generally observed a marked increase, which, in common with the profuse perspiration often ob- served at the same time, was long ago characterized by the word “ critical.” VII. Odor.—Of the odor, little more can be said than that, in health, it is “peculiar” or “characteristic.” It is by some spoken of as “aromatic.” There is, however, PRACTICAL EXAMINATION OF THE URINE. SPECIFIC GRAVITY. 33 appreciable difference in its intensity, as most have observed in their own cases. Concentrated urines always exhibit what is described in common language as a “ strong odor. * ’ This is, undoubtedly, due to urea, though the characteristic odor of urine is not ascribed to urea, but rather to the minute quantities of phenylic, taurylic, and damoluric acids found in it. Urine which has been standing exposed in warm weather acquires an odor which is at once putrescent and ammoni- acal, the former from decomposition of mucus and other organic matters, the latter from the ammonium carbonate derived from the urea. The former is predominant when a large amount of organic matter is present, and is often observed in destructive disease of the kidney or its pelvis, and especially of the bladder. The odor of urine is very promptly influenced by that of substances separated by the kidney from the blood, illus- trated by the well-known odor of violets in the urine of persons taking turpentine. The odor of cubebs, copaiba, and sandal-wood oil is promptly communicated to the urine of persons taking them. So, too, the use of certain vege- table foods promptly influences the odor of the urine. Among these asparagus is conspicuous. Pathologically, except the increased intensity of the characteristic odor of concentrated urines, the putridity alluded to, and sweetish and fruity smell which often attends the presence of sugar in the urine, there seem to be no modifications of the “ characteristic” odor of urine except in those extremely rare instances where sulphuretted hydro- gen has been found in it. 34 PRACTICAL EXAMINATION OF THE URINE. TO DETERMINE THE AMOUNT OF SOLIDS IN THE TWENTY- FOUR HOURS’ URINE. Knowing the quantity of urine passed in the twenty-four hours, and its specific gravity, an approximation to the quantity of solid matters, and thence that of water, may be readily obtained by multiplying the last two figures of the specific gravity by the coefficient of Trapp—which is 2— or that of Hseser, 2.33. This will give approximately the number of grams of solid matters in the 1000 c.c. (33.8 f. oz.). Thus, suppose the twenty-four hours’ urine to be 1200 c.c., the specific gravity to be 1022, then, using Haeser’s coefficient, But the total quantity of urine in twenty-four hours is 1200 c.c., therefore it will contain more than 1000 c.c. contain. Hence, 22 X 2-33 — 51.26 grams in 1000 c.c. Cj 26 V 1200 xooo : 1200 : : 51.26 : x = J * . -P-—— = 61.51 grms. (948.09 grs.) 1000 Now,estimating the twenty-four hours’ urine at 1500 c.c., the normal amount of solid matters is about 70 grams (1080.1 grs.), showing that, in this instance, rather less than the normal quantity was separated. In this manner, valuable information, bearing upon diagnosis and progno- sis, may be obtained in a few seconds. The most striking variations are observed in diabetes and Bright’s disease. In the former the solids are increased by the addition of sugar, in the latter they are diminished by loss of urea. While this method of arriving at the solids is not suffi- ciently accurate for scientific uses, it answers for ordinary clinical purposes. PART I. THE DIFFERENT CONSTITUENTS OF URINE IN HEALTH AND DISEASE. In the examination of a specimen of urine, the following are the steps which will be found most convenient in actual practice. Observe— I. The quantity passed in twenty-four hours. II. Color and transparency. III. Odor. IV. Reaction. V. Specific gravity. VI. Presence or absence of sediment, its quantity, and characters. In all cases, whether the sediment be appreciable or not, a portion of the fluid should be set aside in a conical glass vessel for twelve hours, in order to collect it for microscopical examination. Or the sediment should be condensed by means of the centrifugal apparatus. The remaining or supernatant fluid, filtered if necessary, should then be further examined for certain organic and inorganic constituents. Organic Constituents. VII. Presence or absence of albumin and other proteid substances. VIII. Presence or absence of the different varieties of sugar. IX. Other saccharine substances. 35 36 PRACTICAL EXAMINATION OF THE URINE. X. Presence or absence of acetone and diacetic acid. XI. Coloring matters. ’ Normal. Abnormal. XII. The biliary acids. XIII. Leucin and tyrosin. XIV. Fatty matters. XV. Urea. XVI. Uric acid. XVII. Urates. XVIII. Chlorides. Inorganic Constituents. XIX. Phosphates. a. Earthy phosphates. b. Alkaline “ XX. Sulphates. Examination of Sediment Microscopically and Chemically. I. Unorganized deposits, including crystals and amor- phous deposits. II. Organized deposits, including anatomical elements, such as tube-casts, epithelium, pus, blood-corpuscles, etc. III. Other morphological elements, as fungi, granular matter, extraneous substances, etc. Nos. I, II, III, IV, V, VI require no further consider- ation than is involved in the description of the “general physical and chemical characters.” VII. Albumin and other Proteids. Organic Constituents. Albumin. The albumin, usually found in urine, is serum-albumin. It and serum-globulin are precipitated from their solutions ALBUMIN TESTING. 37 at a temperature of 730 to 750 C. (163.4° to 167° F.). This is true of no other of the proteids which occur in urine. Other agencies, however, also throw down serum- albumin, and become equally delicate tests for it, although less reliable, because of their precipitating other substances. In all instances, where the urine used for testing is not perfectly clear, it should be filtered before applying the tests. This may be done in a few minutes by means of a filtering-paper and a funnel. (a) The Test by Heat. A test-tube is filled to to its depth with perfectly clear urine, and heat applied until boiling occurs. Or a test-tube may be half-filled with clear urine, and heat ap- plied only to the upper part, when a resulting diminished transparency can be very easily reeognized on comparing the two portions of the tube. If a turbidity result, the slightest degree of which becomes visible in an otherwise clear urine held in a good light, it is due either to albumin or earthy phosphates. If the latter, it promptly disappears on the addition of a few drops of nitric or acetic acid ; if albumin it is permanent. If the urine has not been filtered, and is opaque from the presence of amorphous urates, the first effect of the application of heat is to clear up the fluid, and, as the temperature is increased, albumin, if present, is precipitated. I am satisfied, after long experience, that the more reliable method is to boil the urine first, and add the acid afterward, lest there be produced by the addition of the acid, acid-albumin. For it is to be remembered that, if a drop or two of nitric acid be added to a specimen of albu- minous urine so as to render it distinctly acid, it may happen 38 PRACTICAL EXAMINATION OF THE URINE. on boiling the urine that no precipitate whatever will appear, although much albumin is present. This is because the serum-albumin has been converted into acid-albumin or syntonin, which is not coagulated by heat. In like manner and for the same reason, albuminous urine boiled in a test-tube in which a drop of nitric acid happens to be present may fail to precipitate its albumin. The same thing may happen when urine is very highly acid from the natural causes of its acidity. Acetic acid, also, may produce out of serum-albumin a soluble acid-albumin not precipitable by heat. Whatever be its origin, the acid- albumin may be readily precipitated by the further addition of nitric acid or on neutralizing with liquor potassae. With the least excess of alkali the precipitate redissolves, being converted into alkali-albumin. It sometimes happens, also, when the precipitate obtained by boiling is albumin, that the addition of two or three drops of nitric acid will be followed by a partial disappear- ance of the turbidity, but if a few more drops be added, the full amount is again thrown down. We should, there- fore, continue the addition of the acid until io or 15 drops have been used. On the other hand, should the quantity of albumin be very small, too much acid will dissolve it. It is also to be remembered that two or three drops of nitric acid may be added to a specimen of cold or boiled albuminous urine, and although a little cloud of albumin may follow the entrance of each drop into the urine, it will be quickly redissolved, and on the application of heat no precipitate may take place, or a small quantity only will fall. If now more acid be added, after a little time more albumin will come down until all is precipitated. If nitric acid is used in this way it should be slowly added in con- siderable excess, yet short of an amount sufficient to dissolve the albumin. It also occasionally happens, when nitric or acetic acid is added to urine naturally acid, and at the same time albuminous, that the albumin is at first only partially pre- cipitated on the application of heat, there being a mere opalescence when the quantity of albumin may equal one- half the bulk of urine tested. After waiting a little while, however, the full amount of albumin is thrown down. Furthermore, serum-albumin is converted by the con- tinued action of an alkali into alkali-albumin, which is also non-coagulable by heat. This may occur in highly alkaline urines. But the alkali-albumin is promptly converted into serum-albumin by the addition of a drop or two of dilute acid. Heat, under favorable conditions, will show one part of albumin to 100,000 of urine. Mehu* calls attention to the fact that the urine charged with oxalate of lime becomes slightly turbid when heated, even after all the lime possible has been filtered out, and that the turbidity thus produced is not removed by the addition of a few drops of concentrated acetic acid. I have never encountered this source of error. Dr. D. D. Stewart, in his experiments with artificial solutions of nucleo-albumin (formerly regarded as mucin), also found that an albumin-free urine, rich in earthy phosphates and charged with nucleo-albumin, threw down a pre- cipitate on heating which was not dissipated by acetic acid. As such urines are passed sometimes after food ingestion, a source of error is here present which may be obviated by first removing the phosphates. ALBUMIN TESTING. 39 (b) The Nitric Acid Test. This is best applied by the contact- or Heller’s method. Upon a convenient quantity of pure, colorless nitric acid * L’Urine, Normale et Pathologique, Paris, 1880, p. 326. 40 PRACTICAL EXAMINATION OF THE URINE. in a small test-tube (one of those with a foot, seen in Fig. 5, is very convenient), allow to trickle from a pipette down the side of the inclined glass an equal amount of clear urine, which will thus overlie the acid. If albumin is pre- sent, there appears at the point of contact, between the Fig. 5.—Testing for Albumin by Nitric Acid. urine and nitric acid, a sharp whi/e band or zone of varying thickness, according to the quantity of albumin present. The urine may be put into the glass first, if preferred, and the acid may then be allowed to pass down the side and under the urine. The result is the same, but the former is somewhat more easily practiced. ALBUMIN TESTING. 41 When nitric acid is allowed to underlie normal urine, there appears between the urine and the acid a brown ring which grows in intensity on standing, and is due to the action of the acid on the coloring matters. In consequence of this fact, when the urine is highly charged with coloring matters, as it often is in fever cases, the albumin precipi- tated at the same place is similarly tinted. If there is much indican present in the urine, a rose-red or violet tint may be communicated to the albumin ; if much blood- coloring matter, a brownish-red, and if undecomposed biliary coloring matters, a green hue. Nitric acid, in any strength, does not precipitate pep- tones or proteid bodies, while strong nitric acid does not precipitate nucleo-albumin or mucin. Nitric acid is said also to show one part of albumin in 100,000 of urine, but in my hands it is decidedly less delicate than heat properly used. This test is, however, rendered very much more delicate by submerging the tube containing the overlaid urine in boiling water in a porce- lain capsule or beaker. Precautions.—I. Much difficulty is often experienced in causing the urine to flow from the pipette with sufficient slowness—that is, it will either not flow at all, or the finger, in the effort to cause it to flow, is raised so much as to permit a sudden fall of the urine into the acid, which interferes with the success of the test. This difficulty is readily overcome by rotating the pipette, under the end of the index-finger, between the middle finger and the thumb, whereby the flow maybe easily controlled; the process is further facilitated if the upper end of the pipette is slightly roughened. 2. A somewhat similar white zone is formed by the action of nitric acid on the mixed urates if present in excess, by which the more insol- uble acid urates are thrown down. This zone might be mistaken for that of albumin, but the acid urates begin to appear not so much at the 42 PRACTICAL EXAMINATION OF THE URINE. border between the urine and acid as higher up; nor does the zone on its upper surface remain so sharply defined, but while under examina- tion is seen to diffuse itself into the urine above. Further, this layer, if caused by urates, is easily dissipated on the application of heat, although some care is necessary in this application lest in ebullition the ring be commingled with the entire mass of fluid and thus lost to view, although not actually dissolved. After some hours have elapsed these amorphous acid urates are completely decomposed by a further action of the nitric acid, and uric acid is then deposited as a characteristic crystalline sedi- ment. Further difficulty arises where, as occasionally happens in very severe cases of fever, a small quantity of albumin coexists with an excess of acid urates. In these cases the urine is of high specific gravity, and the line of albumin, lying immediately on the acid, maybe obscured by the broader band and cloud of urates. The difficulty from this source is diminished if the urine is diluted with two or three parts of water, or if the nitric acid is used warm, while if the method laid down on page 52 is carefully carried out, a mistake is scarcely possible. It should be added that Thudichum considers that the “ cloud of acid urates here referred to is not urates, but hydrate of uric acid.”* This “ diffused haze” brought into view toward the upper part of the column of urine is regarded by Dr. Roberts f as mucin. Whether it be acid urates or hydrate of uric acid or mucin, its behavior is very different from that of albumin, which appears just above the line of junction of the two fluids. 3. This method of performing the nitric acid test operates equally well with serum-albumin, acid-albumin, and alkali-albumin, and there- fore obviates the possibility of the source of error referred to on page 37, originally pointed out by Bence Jones—first, that if albuminous urine be acidified by a small quantity of acid, as a drop or two, no pre- cipitation of albumin takes place; also, that arising from the addition of too large a quantity, as an equal bulk, of acid, when the mixture may in like manner remain perfectly clear. Roberts says he has known the * Thudichum, J. L. W., “ Pathology of the Urine,” 2d ed., London, 1877, p. 377. f Roberts on “ Tests for Albumin in Urine,” Medical Chronicle, Oct., 1884, p. 1. ALBUMIN TESTING. latter fallacy to cause the concealment of albumin in urine for months, in a case of Bright’s disease. 4. Occasionally, also, it happens that a urine is so highly concen- trated—so highly charged with urea—that the simple addition of nitric acid causes a precipitation of crystals of nitrate of urea at the junction of the two fluids. But these are readily distinguished from albumin by their solution on the application of heat, and by their appearance under the microscope, which exhibits them made up of six-sided rhombic tab- lets. Such urine is always of high specific gravity, while albuminous urine, except in cases of acute Bright’s disease, is apt to be of low specific gravity. 5. If carbonic acid be abundantly present in urine, either free or combined with ammonium, as after the alkaline fermentation, or with sodium or potassium, during the administration of alkaline carbonates or salts of the vegetable acids, an added acid liberates it with efferves- cence. Under ordinary circumstances, this does not interfere with the test; but if the quantity of carbonate of ammonium be very large, as is the case with some old urines, and that of albumin small, the effer- vescence is so great as to make the nitric acid test impossible ; while the amount of acetic acid required to secure an acidity sufficient to permit the use of the heat test may be so great as to completely hold in solu- tion the small quantity of albumin. Such difficulty is further increased by the fact that these alkaline urines are always more or less cloudy, from the presence of amorphous phosphates and of bacteria, and cannot be cleared up by ordinary filtration. Under these circumstances the fol- lowing method recommended by Hofmann and Ultzmann must be pursued. Add to the urine about a fourth part of its volume of liquor potassae, warm the mixture, and filter. If the filtrate is still not quite clear, add one or two drops of the magnesian fluid (11, p. 17), warm again, and filter. The fluid is then always clear and transparent, and albumin, if present, may be revealed by Heller’s nitric acid test, or by the cautious addition of acetic acid. 6. Occasionally, after the administration of turpentine or balsam copaibse, resinous matters are found in the urine. These are precipi- tated by nitric acid in the shape of a yellowish-white cloud, which is, however, redissolved on the addition of alcohol. 43 44 PRACTICAL EXAMINATION OF THE URINE. Other Tests for Albumin. It has long been known that other agents besides heat and nitric acid coagulate albumin. Some of these have lately claimed a large amount of attention, and been found to be extremely delicate tests; some more delicate even than the heat test applied in the most careful manner, and very much more delicate than the nitric acid test. An objection, however, which holds against the most delicate of the reagents is, that they precipitate other substances besides albumin, and although these substances are gener- ally distinguishable from albumin by the aid of certain precautions or additional steps, it dare not be said that the reliability of the tests is not weakened thereby. As the result of a careful study of these tests, based upon experiment and clinical application, I have come to the conclusion that, for the present at least, it is safer to rely upon them in conjunction with the heat and acid tests, with a view to confirming or extending results' attained by the latter. Such use is, however, of the greatest importance ; and I shall, therefore, now consider the most delicate of them, including the method of their application, and the precautions to be observed in rendering them reliable. Those I deem most worthy of consideration are in the order in which I have found them most delicate: Picric acid,so- dium tungstate with citric acid, potassio-mercuric iodide,* * The first three named of these agents are so nearly equal in deli- cacy that one arranges them in any order at some risk, and I must confess to not invariably having found them delicate in the order named. The experiments of Dr. George Oliver, of London, show that any one of these used in solution by the contact method, will precipi- tate i part of albumin in 20,000 of urine. Dr. Henry B. Millard's ALBUMIN TESTING. 45 ferrocyanide of potassium, and Dr. Roberts’s nitric-mag- nesian solution. A very important practical fact to be remembered in connection with these tests is that urine, when filtered through the best Swedish filtering paper, extracts enough vegetable albumin to give a distinct line with any of them, used with the contact method. The heat test does not show albumin in the filter paper filtrate. Picric Acid.—This has its ablest and most enthusiastic exponent in Dr. George Johnson, of London, who says, “ There is no known sub- stance occurring in either normal or abnormal urine, except albumin, which gives a precipitate with picric acid insoluble by the subsequent application of heat.” * An ounce of water at 6o° F. retains in solution 5.3 grains of the dry acid. A saturated solution may be made by dissolving 6 or 7 grains of the powder in an ounce of boiling distilled or rain water. A portion of the acid will crystallize out on cooling, leaving a transparent, yellow, supernatant liquid. Such a solution has a specific gravity of 1005. Dr. Johnson’s mode of applying the test for the detection of a very minute trace of albumin is as follows: Into a test-tube six inches long pour a four-inch column of urine ; then, holding the tube in a slanting position, pour gently an inch of the picric acid solution on the surface of the urine, where, in consequence of its low specific gravity, it mixes only with the upper layer of the urine. As far as the yellow color of the picric acid solution extends, the coagulated albumin renders the liquid turbid, contrasting with the transparent urine below. For the action of the test, there must be an actual mixture, and not a mere sur- face contact. When, in consequence of the scantiness of the albumin, experiments go to show that Tanret’s test will show very easily i part of albumin in 200,000 parts of urine; that his own test of phenic acid and potash will show the same even more easily; that the nitric- magnesian test will exhibit 1 in 150,000, and that heat and nitric acid will each show I part in 100,000, heat being more sensitive than nitric acid. (Millard, “ Bright’s Disease,” 3d ed., 1892, p. 85.) * Johnson, “ Albumin and Sugar Testing,” London, 1884, p. 11. 46 PR ACT J CAL EXAMINATION OF THE URINE. the turbidity is very slight, the application of heat to the upper part of the turbid column increases it. Then, if the tube be placed in a stand the coagulated albumin will gradually subside, and, in the course of an hour or so, forms a delicate, horizontal film at the junction of the col- ored and unstained strata of urine. No previous acidulation of the urine is usually required, as the picric acid accomplishes this, if needed. If, however, a specimen be highly alkaline and ammoniacal, Dr. Johnson says the safest method is by acetic or citric acid ; then filter and add picric acid to the filtrate. The precipitated mucin will remain on the filter. Precautions.— 1. The urine to be tested should be perfectly clear, and if not clear when obtained should be rendered so by filtration, or the process described on p. 42. 2. Urates, peptones, vegetable alkaloids, as quinine, morphia, etc., are all precipitated by picric acid solution at the point of contact, but are promptly redissolved by a degree of heat much lower than that of the boiling-point. Quinine promptly appears in the urine after the administration of 10 grains of this drug. Dr. Oliver correctly claims that the addition of citric acid in the pro- portion of two drachms to an ounce of the picric solution makes a reagent which gives a more distinct reaction than the plain picric acid solution ; but Dr. Johnson says that this is because the citric acid pre- cipitates also the mucin which exists in all urines. Dr. Johnson also insists that mucin (nucleo-albumin) is not precipi- tated from urine by picric acid, and that albumin is the only substance found in the urine which gives with picric acid an opalescence or pre- cipitate insoluble in heat. He is not sustained in this view by others, notably by Roberts,* Oliver,f and Millard.J In my experience nucleo- albumin is precipitated by picric acid, but less decidedly than by acetic acid. Sodium Tungstate with Citric Acid.—This solution is made by mixing equal parts of a saturated solution of sodium tungstate (1 to 4) and a saturated solution of citric acid (10 to 6). It has a specific gravity * Loc. cit., p. 3. fOn “Bedside Urine Testing,” 3d ed., London, 1885, p. in, note. J Op. citat., p. 63. ALBUMIN TESTING. 47 of 1214, and is best used by the overlaying method. It is a test of extreme delicacy, quite as delicate in my hands as picric acid, and has the advantage over picric acid of not precipitating quinine from its solution, but, like the picric acid, precipitates acid urates, peptones, and mucin, which are also promptly dissipated by heat. The Potassio-Mercuric Iodide.—This test was suggested by M. Charles Tanret, of Paris, and is regarded by Dr. Oliver as the most sen- sitive test known, discovering, like the picric acid and sodium tungstate, i part of albumin in 20,000 of urine, while Millard says it will recog- nize 1 part in 200,000 of urine. In my own experiments, however, I have several times failed with the mercuric iodide when I succeeded both with picric acid and sodium tungstate. I am inclined to believe that the age of the preparation and the mode of compounding it have something to do with the results. The solution is prepared by M. Tanret as follows : Bichloride of mer- cury, 1.35 grams; iodide of potassium, 3.32 grams; acetic acid, 20 cubic centimetres; distilled water, enough to make too cubic centi- metres. The bichloride of mercury and iodide of potassium should be separately dissolved in portions of the water, which are then united. The resulting reagent is the double iodide of mercury and potassium, the chloride of potassium being without effect. It is also a heavy fluid, having a specific gravity of 1040 +, and is used by the contact method. The urine requires no previous acidulation. It coagulates the same substances as picric acid, including nucleo- albumin, which are likewise dissipated by heat, or alcohol. On cool- ing they reappear. With regard to nucleo-albumin, however, Dr. Oliver says it is not dissipated by heat if a large excess of the reagent be em- ployed, the mercuric salt apparently preventing solution. Tanret also suggests a quantitative method, which is, however, not accurate.* Ferrocyanide of Potassium.—This test, used in saturated solu- tion, while less delicate than any of the three previously considered, has the advantage over them of not precipitating peptones or the alka- loids, but it may throw down acid urates. It is fully as delicate as * Recherche de 1’ albumine dans 1’ urine; Bulletin General de Th6ra- peutique, tom. 92, 1887, p. 308. 48 PRACTICAL EXAMINATION OF THE URINE. nitric acid, but less so than heat. It requires for its operation that the urine shall be acid. It also reacts slightly with artificial solutions of nucleo-albumin. Dr. Roberts’s Acidulated Brine Solution.—This solution, which consists of a pint of a saturated solution of common salt, to which is •added an ounce of hydrochloric acid, and the whole filtered, is about equal in delicacy to nitric acid, but much less so than heat. It has a high specific gravity, and is used by the contact method. It has the great advantage over nitric acid in that it is less caustic and corrosive, and therefore much pleasanter to work with, but recent ex- periments have shown it to be fallacious, and it is now, I believe, re- garded by Roberts himself as unsatisfactory. Roberts’s Nitric Magnesian Test.—Dr. Roberts has suggested* another modification of the nitric acid test, really a modification of a test suggested by C. Gerhardt in 1856, which consisted in saturating the urine with magnesium sulphate, filtering, and then testing the liquid for albumin with nitric acid. The test fluid consists of 1 volume of strong nitric acid and 5 of a saturated solution of sulphate of magnesium. This, he says, is more prompt and sensitive than pure nitric acid, and its reaction in regard to albumin, mucin, and peptone is similar. It forms a watery, clear solution, which does not fume, nor stain, nor burn the fingers, acts less strongly than the pure acid on the coloring matter of the urine, and may be carried in a corked bottle with less risk of acci- dent. It has received the highest praise from Dr. Henry B. Millard, who says that, as regards delicacy, accuracy, and facility of employment, it is among the most satisfactory tests he has used, detecting less than 1 part in 150,000 of water, but he regards as more sensitive his own test of phenic and acetic acids and Tanret’s mercuric iodide. The nitric magnesian fluid has a specific gravity of 1240, and is used by the con- tact method. It cannot be used with urine clarified by liquor potassse, on account of decomposition induced. My own experience with it is limited. Millard’s Phenic Acetic Acid Test.—This consists of glacial phenic acid (95 per cent.), 5 ij; ac. acet. pur. sjvij. Mix, and add liquor potassm, 5 ij, g vj; then filter. Millard claims the test is equally * Loc. cit., p. x. ALBUMIN TESTING. delicate with Tanret’s and the picric acid test, and, like them, precip- itates mucin, peptone, and the alkaloids, and requires the same precau- tions. Millard also claims that it will detect I part albumin in 200,000 more clearly than Tanret’s double iodide of mercury and potassium test, as it precipitates fewer of the alkaloids. Trichlor-acetic Acid.—This reagent is regarded by many as a delicate test, and esteemed accordingly. A saturated solution ( 25 ss to 3 'j of water) of the crystals is made, and it is used by the contact method. Its reaction with mucin is so delicate that it cannot be re- garded as a reliable albumin test. 49 Albumin Test-Papers.—All of these regeants, except the acid brine solution, may be used in the shape of the test-papers suggested by Dr. Oliver, which are more espe- cially useful for bedside testing, although Dr. Oliver claims for them some advantages over the solutions even when used in the laboratory.* Most recently, f Dr. Oliver has rejected all of the albu- min papers except the “ mercuric” and “ ferrocyanic,” and recommends these to be used as follows :— To Use the Papers.—A mercuric or ferrocyanic, and a citric acid paper are dropped into the test-tube, and water added to 60 minims. After gentle agitation for half a minute or so, the test-papers are removed, and the trans- parent solution is ready for testing. The pipette containing the suspected urine is held in a vertical position over the tube, and the urine is delivered in drops. If four drops of urine added to the mercuric *The different forms of test-papers suggested by Dr. Oliver, origi- nally made for him by Wilson & Son, of Harrowgate, London, are now furnished by Parke, Davis & Co., of Detroit, Mich., as are also, in one case with the test papers, the suitably graduated test-tubes, j- “ Bedside Urine Testing.” London, 1885. 50 PRACTICAL EXAMINATION OF THE URINE. solution, and six to the ferrocyanic, * do not produce a trace of milkiness when the contents of the tube are viewed against a dark background, it may safely be inferred that if albumin is present it is in so small a quantity that nitric acid, applied after the contact method for one minute, will not discover it. If a slight milkiness is apparent, it will represent a trace of albumin detectable by nitric acid. If there is no response, the dropping should be contin- ued, and if instead of four drops of the mercuric and six of the ferrocyanic solution there be required io of the former and 15 of the latter to produce an appreciable opacity, it will indicate a quantity which can readily be shown by heat and acidulation. If 20 of the former and 30 of the latter are required, it indicates a trace of albumin which can be shown only by careful acidulation and sub- sequent boiling. In the case of the mercuric test, if a reaction occurs, the solution should be boiled so as to prove the presence or absence of one of the diffusible proteids, peptone, or hemi- albumose. If the opacity is unaffected by heat, or is intensified by it, it is caused by albumin, but if it is diminished or entirely removed, presumptive evidence is afforded of the presence of a peptoid body, either alone or in conjunction with albumin. The mercuric test, supple- mented by heat, may be said, therefore, to provide a fuller knowledge of the proteids which may appear in the urine than the ferrocyanic, which precipitates albumin only. *The ferrocyanic solution should be allowed a minute in which to develop the reaction from a trace of albumin. ALBUMIN TESTING. 51 Remarks on Testing for Small Quantities of Albumin. The Author's Method. To determine the presence of albumin in urine when it is abundantly present is usually a very simple matter. The application of heat will throw down albumin even from an alkaline solution, if the latter is highly charged with it, while the addition of a few drops of acid removes all possi- bility of error. But it is well known that small quantities of albumin, the significance of which in diagnosis and prognosis is sometimes greater than that of large amounts, often escape detection; while large quantities are some- times obscured in consequence of peculiarities of combina- tion between albumin and acids, and albumin and alkalies, resulting in the formation of the so-called acid- and alkali- albumins. It is with a view to pointing out the way to avoid such errors that the following paragraphs are written. Under all ordinary circumstances by far the most striking test for small quantities of albumin is that form of the nitric acid test described as the contact method (p. 39), and in the majority of cases, this test, carefully carried out, even in the hands of the inexperienced, will exhibit the presence of albumin when it would have been overlooked in the ordinary mode of application of heat and nitric acid. But it is not so delicate a test as the latter applied in the manner to be described. Many who have tested urine for albumin by the ordinary heat and acid test will have observed that after boiling the flear urine and adding a few drops of nitric acid, the resulting fluid will be apparently clear, but upon setting aside the urine thus treated, say for twelve hours, or until 52 PRACTICAL EXAMINATION OF THE URINE. the next morning, there will sometimes be found a small deposit. Supposing the urine before testing to have been carefully filtered, this deposit is either, ist, acid urates ; 2d, uric acid ; 3d, nitrate of urea; 4th, nucleo-albumin or mucin ; 5th, albumin. The first arise from a partial de- composition of the neutral urates by the nitric acid added ; the second by a further action of the acid upon the acid urates, and a resulting complete separation of the uric acid from the sodium, potassium, etc., with which it was com- bined ; the third is found only when the urine happens to be highly concentrated and contains an unusual proportion of urea ; the fourth where there is irritation of the urinary passages insufficient to produce albuminurea. The second and third have well-known forms of crystallization by which they can be easily recognized under the microscope, but the acid urates and albumin are both amorphous and cannot therefore be thus distinguished. All, however, except albumin, disappear on the reapplication of heat. In ■sail instances, therefore, urine which has been tried by heat and nitric acid, in which, after cooling and standing from six to twelve hours, a sediment is present, should be boiled again ; and if the sediment is not redissolved by such ebul- lition, it is albumin. Author’s Method.—My own method, therefore, of examining a specimen of urine for albumin is invariably as follows:— 1. Unless perfectly clear, it is first filtered, and if not rendered clear by filtration, it is clarified by strong alka- lies, or by the magnesian fluid, according to the directions on page 43. A portion of the filtered fluid is then boiled, being carefully watched in a good light for detection of the least diminution of transparency. A drop or two of ALBUMIN TESTING. 53 nitric acid is then added, and if a turbidity which has ensued upon the action of the heat disappears, it is caused by phosphates of lime and magnesium, and not albumin. The addition of the nitric acid should be cautiously con- tinued until a decided excess is added—io to 15 drops— but not more, lest a small amount of albumin present be redissolved by the excess of acid. If any degree of turbidity remains it is caused by albumin, since the nucleo-albumin (mucin) of normal urine will not cause turbidity under these circumstances, and the test may end here—although it is well to put the tube aside, in order that the albumin may subside and be approximately estimated. If, however, there is the least doubt about the presence of albumin, the tube must be set away, carefully protected from dust, for six to twelve hours, in order that any appreciable sediment may subside, and be subsequently again tried with heat. II. A test-tube is now filled to the depth of half an inch with colorless nitric acid. About as much urine is then allowed to fall gently upon it in the manner described on page 39, and the point of junction of the two fluids care- fully examined for the white line. This is best observed by holding the tube against a dark ground, produced by a book, pamphlet, or coat sleeve, so that the light may fall obliquely upon the line of junction of the two fluids, while at the same time it is seen against the dark ground. When this double test is carefully applied as above de- scribed, it is scarcely possible to err as to the presence of albumin. Where it is abundantly present, it is, of course, unnecessary to use either the modified heat and acid test or the contact method, although the latter is always useful 54 PRACTICAL EXAMINATION OF THE URINE. in that it affords one means of approximately estimating the amount of albumin.* Quantitative Estimation of Albumin. Gravimetric Method.—It is a matter of extreme im- portance in the course of Bright’s disease that we should be able to compare the quantity of albumin contained in the urine from day to day. The only accurate method is by precipitation by acetic acid and boiling, separation by filtration, drying, and weighing by delicately accurate balances, the weight of the filter having been previously determined. This, however, involves too much time for *The following observation by Dr. C. E. Brown-Sequard, in the first number of his Archives of Scientific and Practical Medicine (1873), emphasizes some of the difficulties occasionally encountered in this ordi- narily very simple process of testing for albumin : “ If we first test, by heat, urine containing albumin (after having ascertained that it is natu- rally acid), we may not find the least precipitate; and if we add nitric acid to it after it has boiled and become somewhat cold, we may yet not find precipitation of albumin. But if we boil a second time that now acidified urine, the solidification of albumin quickly takes place, and a precipitate soon appears." He further says: “ In three cases in which the microscope showed tubular casts in the urine, the albumin contained by this fluid was so modified by the heat that if the urine (which was naturally acid) was boiled first, the addition of nitric acid in small or in large quantity, at a low temperature or at the degree of boiling, produced no solidification of that proteid substance. But when I added either a small or a large quantity of nitric acid to the fresh unboiled urine and then boiled it, the ordinary coagulation took place, and after some time of rest, the ordi- nary precipitate appeared. It is evident, therefore, that there is some- times in the urine a kind of albumin which loses its coagulability by boiling.” QUANTITATIVE ESTIMATION OF ALBUMIN. 55 the busy practitioner, and we must fall back on one of the approximative methods. Approximate Estimation with Boil- ing.—The easiest of these is to boil a given quantity of urine in a test-tube, add a few drops of nitric acid, and set aside for at least twelve hours—shaking the urine once or twice in this period in order to secure a uniform subdivision and precipitation of the particles of albumin. The proportion of bulk occupied—one-fourth, one-eighth, a trace, etc.—is used to indicate the quantity of albumin. Greater accuracy is obtained by previously filtering the urine of urates, epithelium, or extraneous matter, which might unduly increase the bulk of deposit on standing. Nothing more than a refinement of this is the Estimation with Esbach’s Albumin- ometer.—This albuminometer consists of a graduated glass tube like that shown in Figure 6. To use it, fill with urine to U, and to R with the test solution, consisting of picric acid, io grams; citric acid, 20 grams, and water sufficient to make a litre. Close the tube by a rubber stop- per, and mix the contents cautiously, allow the mixture to remain undisturbed for twenty-four hours, and then read off the quantity of precipitated albumin. Each of the main lines to which the precipitate reaches indicates one gram of albumin in one litre of urine. Approximate Estimation with Nitric Acid.—The following method of approximate quantitative estimation by means of Heller’s nitric acid method is given by Hofmann and Ultzmann. According Fig 6.—Es- bach’s Albu- minometer. 56 PRACTICAL EXAMINATION OF THE URINE. to them, if the white zone of albumin has the depth of from 2 to 3 mm. (y to | inch), is delicate and faintly white in color, has no granular appearance, and appears clearly defined only when placed against a dark background, the quantity is less than \per cent., usually •Jjj- per cent. If the zone is 4 to 6 mm. to i inch) in depth, granular, white, opaque, and perceptible without a dark background, the quantity is considerable, j to | per cent. If, however, the zone of albumin appears granular and flocculent, and sinks in more or less lumpy masses to the bottom, and when by stirring the albumin by means of a glass rod the mixture assumes the consistence and appear- ance of sour cream, then the quantity is very large, 1 to 2 per cent. The Proportion of Albumin Found in Urine.— There is much carelessness of expression among physicians in speaking of the quantity of albumin found in a given specimen of urine. Thus we often read that a specimen contains 25 per cent., or even 50 per cent., of albumin. The proportion in bulk is of course intended, but no indi- cation given that this is what is meant. In point of fact 3 or 4 per cent, is probably the maximum amount of albumin which urine can contain, since Hammarstein has shown the total albumin of the blood-serum to be only 7 to 8 per cent., of which 4.5 per cent, is serum-albumin and 2.5 to 3.5 globulin or serum-globulin or paraglobulin. So that a 2 per cent, albuminuria is a very large one. A half per cent, is much more common, and many albuminuric urines contain much less than a half per cent, of the proteid. It is not unusual, also, to over-estimate the amount of albumin passed in the twenty-four hours, and thence to ex- aggerate the drain upon the system. Suppose, for example, the percentage of albumin by weight is .5 of 1 per cent., and the quantity of urine is 50 ounces in the twenty- four hours, then, there being 455.7 grains in an ounce, 455.7 X -o°5 X 50= 113.9 grains, the amount of daily GLOBULIN. 57 discharge, or about one-quarter of an ounce. Supposing there be 2 per cent., which is a very large amount of albumin, and 40 ounces of urine. Then 455.7 X -02 X 40 = 364.5 grains, or little more than three-fourths of an ounce. Of such a loss Senator well says that half a pound of beef will more than make up the loss of a week. Other Proteids Found in Urine. It has long been recognized that modifications of albu- min occur in urine, either alone or in association with it, but since the introduction of the delicate tests, more attention has been paid the subject. Among the most constant of these is:— Serum-globulin, Globulin, or Paraglobulin.— Globulin is almost always associated with serum-albumin, from which it may be separated by Poehl’s method as fol- lows : Render the urine slightly alkaline by ammonium hydrate, and after several hours filter to separate the phos- phates. Then add saturated solution of ammonium sul- phate in the proportion of one volume to one volume of the filtrate. If a precipitate forms it is globulin. Globulin is also separated by diluting the urine, after filtration, until the specific gravity is 1003 or 1002. Occa- sionally a cloudiness appears immediately, due to the sepa- ration of some of the globulin. But the globulin is com- pletely separated by passing a stream of carbonic acid through the dilute fluid for from two to four hours. In from twenty-four to forty-eight hours the globulin falls to the bottom as a milk-white, flocculent substance. The supernatant fluid contains the albumin. Should the urine be neutral or alkaline, it must be rendered slightly acid by a few drops of dilute acetic acid. 58 PRACTICAL EXAMINATION OF I'HE URINE. This test is based upon the fact that globulin is held in solution by the sodium chloride and other neutral salts always present in the urine. When urines are largely diluted with moderately pure water, the percentage of neutral salts is so reduced that the globulin falls out of solution. Dr. Roberts* suggests the following simple modification of the test: Fill a urine-glass or test-tube with water, and let fall into it a succession of drops of albuminous urine. In many cases, each drop as it falls is followed by a milky train, and when a sufficient number of drops has been added, the water assumes throughout an opalescent appear- ance, as if a few drops of milk had been added to it. The addition of acetic acid causes the opalescence to disappear ; for globulin is soluble in concentrated acetic acid, and also in a one per cent, solution of hydrochloric acid. From its solution in common salt it is completely separated on heating. Occurrence.—Not only does globulin accompany serum- albumin in most instances, but, it is said, sometimes ex- ceeds it decidedly, although the proportion in the blood is much less, being to serum-albumin as i to 1.5. It may even occur, although very rarely, without serum-albumin. According to Senator, it is most abundant in the urine of lardaceous disease of the kidney. It also occurs in acute nephritis, in the hyperaemia of cantharides poisoning, and in albuminuria associated with deranged digestion. Nucleo-albumin (Mucin).—This proteid, heretofore * Discussion on Albuminuria before the Glasgow Pathological and Clinical Society, p. 17. Reprinted from the Glasgow Medical Journal, 1884. MUCIN. 59 known as mucin, is not true mucin. The substance is iden- tical with the nucleo-albumin of bile. It is abundant in urine which has passed over irritated urinary passages, and is said to be present, to some extent, in all urines. It is certainly present in a very large number. * It is not pre- cipitated from solutions by boiling, but is precipitated by alcohol, by dilute mineral acids, and by all vegetable acids, including picric acid. To Test for Nucleo-albumin.—The tests usually employed for mucin are citric and acetic acids used by the contact method, the acid being introduced first. It is also precipitated by dilute nitric acid. Just above the point of contact a cloud-like coagulum gradually makes its ap- pearance, contrasting with the opaque white coagulum of albumin. When albumin and mucin are both present, the latter appears at the upper part of the column of urine, while the albumin is confined to the point of contact be- tween the two fluids. Or the following method may be followed :— To one volume of urine add three of strong alcohol and allow to stand for several hours, when mucin and all albu- minous bodies will have precipitated. Filter and wash the precipitate with alcohol, then treat it with warm water. The filtrate, which should contain the mucin, is then strongly acidified with acetic acid, and if turbidity results, it is due to mucin. Or the contact method may be used with acetic or citric acids. *, See an admirable paper by Dr. D. D. Stewart, of Philadelphia, on “ The Reactions of Nucleo-albumin (erroneously styled Mucin) with the Commonly Employed Urinary-albumin Tests.”—Medical News, July 14, 1894. 60 PRACTICAL EXAMINATION OF THE URINE. The Nucleo-albumin or Mucin Reaction of Normal Urine.—Dr. George Oliver,* in 1885, called attention to the fact that even when a normal urine is under- laid by citric acid solution, there appears in the course of several minutes, along the plane of contact of the two fluids, a delicate whitish zone which becomes gradually more pronounced, and which is mucin.f This reaction, concentrated by the contact method, becomes, when the precipitate is diffused throughout the urine, either totally inappreciable, or appreciable only in the slightest degree. It may be studied by adding a citric acid test-paper to 60 minims of transparent urine. If mucin is present in larger quantity than usual, a slight milkiness appears. If, however, to the normal urine, acidified by citric acid, a mercuric test paper be added, a delicate haze may be de- tected on holding the urine to the light, and against a dark background. This haziness disappears on applying heat to near the boiling point, and reappears on cooling, to revanish on reheating. If, on the other hand, a solution of the potassio-mercuric iodide is used by the contact method, the opacity thus produced by mucin does not disappear when heat is applied. J Thus, heat removes all * “On Bedside Urine Testing,” 3d ed., London, 1885, p. no. f The recent experiments of Dr. D. D. Stewart, referred to, entirely confirm this observation of Dr. Oliver’s. J The student is strongly recommended to study the reactions of mucin by impregnating normal urine with saliva, as directed by Dr. Oliver. The clear saliva and a solution of salt, say 20 grains to the ounce, should be mixed in equal parts, and one drop of acetic acid, or a citric acid test-paper, added to a 4-inch column, which should be thoroughly boiled, when the milkiness produced by a trace of albumin will appear and should be filtered out. This highly muciparous solution PEPTONE. 61 sources of error in testing for albumin with the potassio- mercuric iodide, when used in the shape of test-paper. This, therefore, Dr. Oliver regards as possessing a distinct clinical advantage over the solution. Peptone.—The frequent occurrence of peptone in urine has rendered it one of the most important of the possible constituents demanding attention. Much has been lately added to our knowledge of peptonuria, but it is possible that further changes will be made in such knowledge before it is complete. It is well known that peptone is a proteid substance which is the final result of gastric and pancreatic digestion. It may also be produced from albumin by the continued action of acids and alkalies, and it is said, also, by the decomposing action of bacteria, as well as the long- continued operation of a temperature of 268° to 290° F. Between albumin at the beginning and peptone at the end of the process of albuminous digestion there are certain intermediate bodies collectively known as albumoses, of which the most important are syntonin (acid albumin), the is added to albumin-free urine, I to I or I to 2 according as the observer may wish to charge it with mucin. In any case the urine will then become more highly muciparous than is likely to be met with in prac- tice. A citric acid and a mercuric test-paper, added to 60 minims, produces an opacity exactly like that produced by a small quantity of albumin, but it differs from it in completely vanishing when heated. The opacity returns as the temperature of the solution falls, and in the cold it greatly exceeds the original amount. Heat will again disperse it asj before. If a trace of albumin be added to the mucin-charged urine, the test-papers will produce an opacity which heat will clear up only to a certain degree, that which remains over being due to albumin. 62 product of neutralization, and propeptone, or hemialbu- mose. These substances may likewise be the product of bacterial organisms, and it is through these agencies, as, for example, the pyogenic organisms, that they may be pro- duced in the urine. They may doubtless, however, also be secreted by the kidneys from the blood, into which they are introduced in various morbid states, especially suppu- rative affections, phosphorus poisoning and typhus fever, tuberculosis, and other diseases named below under “ clini- cal significance of peptonuria.” Peptone differs from albumin and propeptone in that it is not precipitated by heat or nitric acid, by acetic acid combined with chloride of sodium, or with cyanide of potassium. Like albumin and propeptone, it is precipi- tated by tannin, corrosive sublimate, phosphortungstic acid, Millon’s reagent,* and by sodium tungstate, potassio- mercuric iodide and picric acid, the last three being among the recently suggested delicate tests for albumin ; but when precipitated by these last three reagents it is re-soluble by heating. It is further characterized by the purple reaction its solutions strike with sodic hydrate and solution of a little salt of copper. The sensitiveness of this so-called “ biuret ” reaction is increased by slight coloration of the fluid, especially a yellow. So delicate is it that a solution as weak as i part in 1000 responds promptly. This re- action is shared by propeptone, while albumin produces only a blue coloration—under no circumstances a red or PRACTICAL EXAMINATION OF THE URINE. * To make Millon’s reagent, dissolve one part by weight of quick- silver, by the aid of warmth, in one part by weight of concentrated nitric acid, and dilute with an equal volume of water. PEPTONE. 63 violet. It has been suggested that peptone bears the same relation to albumin as grape sugar to starch—that is, that it is a hydrate of albumin. Peptone is not present in healthy blood or normal urine, and even during digestion the portal vein contains but traces of it. Injected into the blood, it disappears from it very quickly, a certain proportion reappearing in the urine, while some is taken up by various organs of the body. These organs also appear to retain any small excess which may have been introduced into the blood from over-inges- tion of albuminous food. It would seem, therefore, that it is converted into albumin at the moment it is absorbed into the circulation, in the very act of absorption, and is incapable of taking the place of albumin in the blood, although the reverse has been alleged by Plosz, Maly, and Adamkiewicz. During health, too, the various organs of the body contain traces of it, and it is a constant con- stituent of pus, as was first shown by Hofmeister. Tests for Peptone.—The best tests for peptone are those which require some preliminary treatment of urine suspected to contain it. For these we are chiefly indebted to Hofmeister.* The simplest is the Phosphor-Tungstate Test.—(i) The urine is decolor- ized and freed from mucin and phosphates by treating say half a litre or about a pint with solution of neutral acetate of lead until the thick, flocculent precipitate ceases to form. It is then filtered. (2) To a portion of the filtrate add acetic acid and a few cjrops of a solution of ferrocyanide of potassium. Should * Neubauer and Vogel, 8th ed., 1881, p. 138, and Zeitschrift fib Physiol. Chetnie, 5, 73. 64 PRACTICAL EXAMINATION OF THE URINE. there be a cloudiness or precipitate it is due to albumin, and the addition should be continued as long as a precipi- tate occurs. The albumin must be removed by filtration. (3) Add to a portion of the filtrate about one-fifth its bulk of concentrated acetic acid, and then phosphortungstic acid acidulated with acetic acid.* If the fluid remains clear after standing some time, it does not contain pep- tone. Should, however, after the lapse of ten minutes, a cloudiness appear, peptone is present. The Biuret Test.—Treat the urine with acetate of lead, filter, and add to the entire filtrate concentrated solution of tannic acid, so long as a precipitate takes place. Filter and treat the lead-filtrate with half a volume or its own bulk of concentrated hydrochloric acid, and then with an acid solution of phosphortungstic acid, until there is no longer a pre- cipitate. The latter is then promptly filtered off, because, if allowed to stand, there appears on the surface a second reddish precipitate, which interferes with the subsequent demonstration of the peptone. The pre- cipitate is washed on the filter with a solution of sulphuric acid, three to five per cent, strong, until the filtrate passes through colorless, then turned into a capsule, intimately mixed with baryta in substance, the mixture treated with a little water, and, after being warmed for a short time, filtered. If too strongly heated, a dark-hued filtrate is obtained, which is to be avoided. To this filtrate the “biuret” test is again applied. Or throw the lead-filtrate, after twenty-four hours, on a filter, and wash with water in which tannic acid and magnesium sulphate are dissolved. The precipitate is now thoroughly rubbed up in a capsule with saturated bartya water, and, after the addition of a fragment of solid baryta, re- tained at a boiling temperature for a few minutes. If care is not taken to mix intimately the precipitate with the baryta, resinous masses form * The solution of phosphortungstic acid is made by adding to a boil- ing watery solution of tungstate of sodium enough phosphoric acid to produce an acid reaction. After cooling, the fluid is to be made strongly acid with acetic or hydrochloric acid, and, after standing one day, fil- tered. TESTING FOR PEPTONE. 65 during the heating, which interfere with the proper action of the baryta. After a few minutes the mixture is filtered, baryta water again added, and thoroughly shaken with it until the fluid filters off from the dark- colored precipitate, either colorless or slightly yellow. In the filtrate thus obtained the “ biuret ” reaction is now sought by adding first liquor sodas or potassas until a decided alkalinity is produced, and then, drop by drop, a very weak solution of sulphate of copper. If a reddish color appears, the addition of the copper solution is to be con- tinued until the reddish-violet has reached its greatest intensity. If no peptone is present the fluid becomes simply green, or bluish-green. The simultaneous baryta precipitate does not interfere with the test, as it rapidly subsides while the supernatant fluid retains the color. The first method is both shorter and more delicate, detecting, accord- ing to Neubauer, peptone in solutions containing but .1 gram to the litre, while the tannic acid method requires .15 to .2 gram. It is the one preferred by v. Jaksch, to whom we are indebted for so much of our knowledge of peptonuria and its clinical significance. Ralfe's Test.—A rough test for peptone, which answers very well when a considerable quantity is present, may be made by placing adrachm, or 3.5 c.c., of Fehling’ssolution in a test-tube and gently overlaying it with an equal bulk of urine. At the point of contact a zone of phosphates forms, while above this, if peptones are present, a delicate rose-colored halo will float. Should the peptones be mixed with albumin, the halo will be purple. Randolph's Test with Acid Mercuric Nitrate and Potas- sium Iodide.—This test, suggested by the late Dr. N. Archer Randolph, of Philadelphia, is based upon the fact, that if Millon’s reagent * be added to an aqueous solution of iodide of potassium, a red precipitate of mercuric iodide results, but if peptone or bile acids are present, the precipi- tate is yellow. To 5 c.c. of urine, which must be cold and * See note on p. 62. 66 PRACTICAL EXAMINATION OF THE URINE. but faintly acid, add two drops of a saturated solution of iodide of potassium and then 3 or 4 drops of Millon’s reagent, when, if peptones or bile acids are present, a yellow precipitate falls. Then the question as to whether it be bile acids or peptones must be settled by the tests for the former. The Clinical Significance of Peptonuria.—The instances in which peptonuria occurs are numerous. The best determined fact with regard to it is that discovered by Maixner, that it is always present when pus corpuscles are disintegrating somewhere in the body, and in most of the diseased states in which it has been found, such a condition of affairs has been probable. It has been especially studied by Maixner, v. Jaksch, Fenomenow, and Pacancowski. Among the diseases in which it has been found may be named typhoid fever, variola, scarlatina, miliary tubercu- losis, erysipelas, acute arthritis, pulmonary tuberculosis, gangrene of the lungs, croupous pneumonia, purulent pleurisy, embolism, carcinoma of the gastro-intestinal tract and of the liver, catarrhal jaundice, parametritis, cerebral apoplexy, parotitis, abscess. As above mentioned, it may be the product of the pyogenic organisms, as the pyogenes aureus, or of the pneumonococcus and the strepto- coccus pyogenes. Exception to the above explanation may have to be made in some cases of carcinoma of the gastro-intestinal tract, and cancer of the liver and uterus. In cancer of the stomach and small intestine, Maixner early ascribed the peptonuria to absorption by the ulcerated surfaces, of the peptone of digestion, but in cancer of the oesophagus, rectum, and uterus, we must have recourse to disintegration of new-formed tissue as the sole source. The almost 67 HEMIALBUMOSE. invariable association of peptonuria with cancer of the liver, Pacancowski thinks, compels the conclusion that the liver in health has something to do with the conversion of peptone into albumin, an office that in cancer is interfered with. Although Senator, Petri, and Poehl assert that albumin- uria and peptonuria coexist, Maixner, and more recently Pacankowski, fail to confirm this. The latter failed to find it in four cases of chronic nephritis and one of acute. Their possible coexistence is now, however, generally admitted. It has also been held that peptone may originate from the conversion of albumin by reason of a sort of fermenta- tive action of the cellular elements of urine, as well as by the agency of bacteria. Hemialbumose, or Propeptone.—This substance is an intermediate product in the conversion of albumin into peptone during gastric and pancreatic digestion. It is, therefore, abundantly present along with albumin in the gastric and intestinal contents, and, unlike peptone, is also found in the blood during digestion. It was first found by Bence Jones in the urine in a case of osteomalacia, and later by Kiihne in another case of the same disease. Hemialbumose, like albumin, is insoluble in alcohol; sparingly soluble in cold water, but very easily in hot water and water containing only traces of acids, alkalies, or salts. It is not, therefore, precipitated by heat from its watery solution, as is albumin, but if the solution be made strongly acid and concentrated salt solution be added thereto, hemialbumose is precipitated. If the cloudy fluid be now heated, it becomes transparent, but again turbid on cooling. A further large addition of salt maintains the 68 PRACTICAL EXAMINATION OF THE URINE. precipitate in spite of heating. Hemialbumose is precipi- tated by pure nitric acid, but redissolved with the produc- tion of an intense yellow color on being heated, and reprecipitates on cooling. An excess of nitric acid redis- solves the precipitate even in the cold, with the production of the same orange-red color. In these respects hemialbumose differs strikingly from both peptone and albumin. It is like peptone in that it exhibits the biuret reaction with an alkali and salt of copper. Like albumin, it is precipitated by adding first acetic acid and then ferrocyanide of potassium ; also by phosphor-molybdanic, phosphor-wolframic, tannic, and picric acids, the precipitate, except that with picric acid, being undissolved by warmth. To test the presence of Hemialbumose, the albu- min must first be removed. This is accomplished by acidi- fying the urine with a few drops of acetic acid, adding about one-sixth its volume of concentrated salt solution, boiling, and filtering off the precipitate. Albumin and globulin remain upon the filter. The filtrate is then allowed to cool, and if a turbidity arises or is produced by the further addition of salt solution, which disappears by heating, and reappears with cold, propeptone is present. If desired, the precipitate can be filtered off the filtrate, redissolved in a little water, and reprecipitated by acetic acid and ferrocyanide of potassium. Hemialbumose is removed from fluids by adding acetate of iron and boiling, or by boiling with hydrated lead oxide. Fibrin.—Fibrin is found in the urine when there are hemorrhages from the genito-urinary passages, and in in- tense inflammation of these passages and of the kidneys ; PROTEIDS CONTRASTED. 69 also in a condition of fibrinuria which occurs in the Isle of France; finally, in chylous urine fibrin is present. Recognition.—It is recognized by its spontaneous coagulation, the product of which is, however, not to be confounded with mucus or the glairy substance formed by the action of ammonium carbonate on pus ; also by its fibrillar structure as shown by the microscope. Coagula may be filtered out from urine by means of muslin, and washed with water to free them from urinary constituents. If insoluble in dilute alkalies and in 5 to 10 per cent, solution of sodium chloride, they are fibrin. The presence of Pepsin and Trypsin in urine has been lately asserted. Proteids Contrasted. Albumin, hemialbumose or propeptone, and peptone, haemoglobin, and methsemoglobin are soluble in water ; serum-globulin and nucleo- albumin are not, or at most very sparingly, soluble. All of these pro- teids are soluble in alkalies and strong acids, forming with them soluble binary combinations, behaving to the alkalies as acids and to acids as bases. They are soluble also in basic salts (phosphates and carbonates of the alkalies), since they take away from these salts the bases. The combination of albumin with a base is called an albumi- nate, that of albumin with an acid, acid-albumin (syntonin). No spe- cial term is applied to the combinations of the other proteids with bases or with acids. Globulin and nucleo-albumin are both sparingly soluble in water and easily soluble in weak neutral salt solutions, as chloride of sodium. Globulin is also soluble in organic acids; nucleo-albumin is soluble in alkalies, but not in dilute acids, organic or mineral. If the acid be withdrawn from an acid-albumin in solution, by neu- tralization with an alkali, the insoluble proteid is liberated, and falls as a precipitate. The same thing happens when a dissolved albuminate is neutralized by an acid. 70 PRACTICAL EXAMINATION OF THE URINE. Hemialbumose, soluble in water, is even more easily soluble than globulin in alkalies and acids, as well as in neutral salts. Albumin and peptone differ from each other also in that albumin is easily converted into precipitable albumin, while peptone is not. Fibrin is soluble neither in water, like albumin, hemialbumose, and peptone, nor in dilute cold acids or alkalies, like precipitable albumin, nor in salt solutions, like globulin. Both nucleo-albumin and serum-globulin are precipitated by magne- sium sulphate and ammonium sulphate. Not much is known about true mucin, but the chief difference between it and nucleo-albumin seems to be that while both are precipitated by acetic acid, mucin is not soluble in an excess of the acid while nucleo-albumin is; also that mucin, after prolonged boiling with dilute mineral acids, yields a re- ducing substance, and nucleo-albumin does not. Haemoglobin and methaemoglobin are recognizable by their color. VIII. Sugars Found in Urine. Glucose, C6Hi206. While the assertion of Briicke and Bence Jones that glu- cose is present to a slight degree even in normal urine has been quite generally accepted, and has apparently been confirmed by the more recent researches of Dr. Pavy,* of London, it has been contradicted by Seegen, and very careful investigations by my colleague, Professor Wormley, confirm the results of Seegen. f Dr. George Johnson, J and his son, G. Stillingfleet Johnson, have also lately joined the group who deny the presence of glucose in norbial urine. It does not follow, however, that instances * Pavy, “Points Connected with Diabetes,” London, 1879. f Seegen, “ Der Diabetes Mellitus,” 2 Aufl., s. 224. X Johnson, G., Brit. Med. Jour., Jan. 8, 1887. GLUCOSE. 71 may not occur in which small quantities of sugar, barely, if at all, recognizable by the ordinary tests, are present in urine. Of the large number of tests for the detection of sugar, only those will be given which have borne the trial of experience, or, being new, are so highly commended that some notice is demanded; and it is suggested that for practical purposes the student should select some one or more of these and accustom himself to their use, and to the modifications in results to which all are more or less subject. I am confident that much of the difference of opinion as to the value of the different tests is due to the unequal experience of different observers with a particular test. Mistakes are less likely to be made by a beginner with a freshly made Fehling’s solution than with Trom- mer’s test, but if the latter is done as directed below a tolerably constant result obtains. It is necessary, however, to be familiar with more than one test, because cases of doubt constantly arise where the evidence of one is insuffi- cient. (See especially remarks on qualitative testing, p. 104.) Specific Gravity and Quantity.—The specific gravity alone, when 1030 or more, affords a presumption of the presence of sugar, and if at the same time the urine is very pale, and far exceeds 1500 c. c. (50 fluidounces) in twenty-four hours, the probabilities are much increased. These facts at least call for the use of other tests to deter- mine the question. The Copper Tests. The copper tests depend upon the power which grape- sugar possesses of reducing the oxide of copper to a lower 72 PRACTICAL EXAMINATION OF THE URINE. state of oxidation. The oldest of these tests is Troramer’s, in which the oxide of copper is set free at the time of its application by liquor potassse or sodas, in excess. Trommer’s Test.—i. To 4 or 5 c.c. of the suspected urine, add one quarter its bulk of liquor potassae or sodae. Then add drop by drop of a 10 per cent, solution of cupric oxide. On adding the first drop, a blue precipi- tate of hydrated cupric protoxide occurs, which, if sugar is present, is redissolved on agitating the mixture. This is presumptive evidence of the presence of sugar, but is not relied upon. The addition of the copper solution is continued until a slight excess has been added and the precipitate is no longer dissolved. The mixture is then boiled for a few seconds, and if sugar is present, a copious precipitate of yellow cuprous hydroxide takes place. This subsequently loses its water and becomes the red cuprous oxide which falls to the bottom or sides of the test-tube, to which it often closely adheres. If more than 5 to 10 drops of the cupric oxide are required to produce the turbidity, dilute a portion of the urine with 4 or 9 volumes of water and reapply the test as before. The precise reaction is not known. 2. A second similarly prepared mixture of these ingredi- ents may be made and set aside without the addition of heat for from six to twenty-four hours. If sugar is present a similar precipitate of suboxide of copper will take place. If the reaction is at all doubtful it is important that this check-test should be made, since, as Neubauer points out, most of the other organic substances which reduce the salts of copper do so only when heated or after long boiling. Precautions.—These apply to all the copper tests and trommer’s test. 73 should be carefully studied, i. Albumin must always be removed, as it interferes with the reduction of the cupric oxide. 2. Too much of the solution of cupric sulphate or too strong a solution should not be used, because the prolonged boiling of any urine with a large excess of copper will pro- duce a yellow or greenish-yellow color, which may not appear until the mixture cools off. 3. While the fluid must be made to boil for perhaps half a minute, the precipitate should take place without pro- longed boiling, as a reduction by other organic substances is induced by prolonged boiling. 4. The flocculent precipitate of earthy phosphates should ?iot be mistaken for the suboxide of copper; it is either trans- parent or of a pale grayish hue. On the other hand, a mere change of color is not sufficient. Strictly normal urine almot always has a decolorizing effect. There must be an actual yellow or red precipitate. If it be desired to eliminate altogether any error due to the precipitation of the earthy phosphates, it may be done by adding the potash solution and filtering before adding the copper. 5. As already stated, cupric protoxide is sometimes reduced by other organic matters found in urine, especially uric acid, by creatinin, hippuric acid, the urates, hypoxan- thin, mucus, indican, urochloralic acid found in the urine when chloral is administered internally, turpenoglycuronic acid when turpentine is taken, etc. Creatinin has also the property of redissolving cuprous oxide. Practically, uric acid is almost the sole source of error, and this may be suspected when the urine is dark-colored or scanty, saccha- rine urines being for the most part light - hue d and copious. On the other hand, a small amount of sugar may be present 74 PRACTICAL EXAMINATION OF THE URINE. in urine and fail to reduce the oxide in the presence of certain other substances. Dr. Beale* has shown that am- monium chloride, ammonium urate, and other ammoniacal compounds have this latter effect; and not only albumin but organic substances generally, including creatin, creat- inin, pepsin, peptones, urinary coloring matters, etc., act similarly. When these partial reductions occur, a yellowish- green precipitate results. Attention should be paid to the specific gravity, to the fact that a precipitate of the phos phates always takes place which must not be mistaken for the suboxide, while the disappearance of the blue color and the substitution of a yellowish tinge is also not to be mistaken for a precipitate. A yellowish precipitate, how- ever, does not indicate a partial reduction either by some other organic substance or by the sugar itself, and demands that the urine should be subjected to the bismuth or fermen- tation test, or, if absolute accuracy is required, to Briicke’s process, described on page 87. When proper precautions are observed, reliable results may be expected with Trommer’s test with saccharine urines containing y2 of 1 per cent. Salkowski’s Modification of Trommer’s Test.—Solution i.— Dissolve ioo grams of sodic hydrate, purified in sticks, in 300 c.c. dis- tilled water. If there is any sediment carefully decant the solution. Solution 2.—Cupric sulphate, chemically pure, 10 grams in 100 c.c. distilled water. To 9 c.c. urine add 3 c.c. of solution 1 in an eight-inch test-tube, then the solution 2, drop by drop, shaking the mixture after each until the copper ceases to dissolve. Slowly heat to a little below the boiling point (be especially careful as to this), and if glucose be present the usual greenish, or yellow, or reddish hydroxide of copper will be seen, first in the upper stratum, then throughout the * “ Kidney Diseases and Urinary Deposits,” p. 246. fehling’s solution. 75 liquid. Where only a minute quantity of glucose is present the test- tube should be set aside for five or ten minutes, to allow the subsidence of the flocculent phosphates. Two minutes’ maintenance near (but avoiding the actual) boiling point will suffice in all cases. It is said that this test in experienced, careful hands, will demonstrate 0.05 per cent, of glucose. Other Copper Test Solutions—Fehling's and Pavy's Fluid. It has been stated that when an alkali is added to a solution of sulphate of copper an abundant precipitate of hydrated cupric protoxide is thrown down. This is not dissolved by any excess of alkali added, but if some organic matter is present, or is added, an excess of alkali dissolves the protoxide. It is for this reason that, if sugar happens to be present in a suspected fluid to which these have been added, the precipitated protoxide is dissolved and a clear blue fluid results. These facts enable us to construct a fluid which will hold the protoxide of copper in solution ; but, in selecting an organic substance, one must be chosen which will not reduce the oxide of copper as does sugar, else it will make our test inoperative. Such substances are tartaric acid, mannite, and glycerine, all of which are employed. Fehling’s Solution.—Dissolve 34.652 grams pure crystallized sulphate of copper in 200 grams distilled water ; 175 grams chemically pure crystallized neutral sodic tar- trate are then dissolved in 480 grams solution of caustic soda of specific gravity 1.14 and into this basic solution the copper solution is poured, a little at a time. The clear mixed fluid is diluted to 1 litre, or 1000 c.c. Ten c.c. of this solution will be reduced by .05 gram, or 76 PRACTICAL EXAMINATION OF THE URINE. 50 milligrams, of diabetic sugar. If the Fehling’s solution is to be kept some time, it is absolutely essential that it should be placed in smaller bottles holding 40-80 grams, sealed, and kept in the cellar. Still greater security may be obtained by dissolving the cupric sulphate in 500 c.c. water, diluting the tartrate salt and potash to 500 c.c., and keeping the two solutions separately in rubber-stoppered bottles. Equal volumes of the two solutions are united when needed for use. With a view to avoiding the well-known defect of Fehl- ing’s solution—its tendency to spoil on keeping—Schmiede- burg suggested the substitution of 15 grams pure mannite for the sodic tartrate. The mannite should first be dis- solved in 100 c.c. of water and 500 grams solution caustic soda, sp. gr. 1145, and the solution completed as above. My experience with this solution fully confirms Schmiede- burg’s statement, and I strongly commend it. Fehling’s Solution with Glycerine.—With the same end in view, glycerine is used as follows : Dissolve 34.652 grams of pure cupric sulphate in 200 c.c. of pure water, add 175 c.c. of pure glycerine, and mix thoroughly. 2. Dissolve 130 grams KHO in 500 c.c. water. Then add slowly solution x to solution 2, and dilute the whole to 1000 c.c. The solution is used as is Fehling’s. Pavy’s Solution consists of— Cupric sulphate, 320 grains. Neutral potassic tartrate, 640 grains. Caustic potash, 1280 grains. Distilled water, 20 fluidaunces. The solution is made in the same manner as Fehling’s, and ioo minims correspond to y2 grain of grape-sugar, the QUALITATIVE TESTING. 77 formula for grape-sugar being here taken C6H1407, while by Fehling it is taken C6H1206.* To Use Fehling’s and Pavy’s Solutions for Qualitative Testing.— The same precautions laid down for the use of Trommer's test are to be observed, since the Fehling's and Pavy's solutions are simple modifications of Trommer s test and subject to the same sources of error. In using either of the above solutions for qualitative test- ing, a small quantity, say i c.c., should be placed in a test- tube, and diluted with about four times its bulk of water. The mixture should then be boiled for a few seconds. If the solution remains clear after this boiling, add the suspected urine drop by drop. If sugar is present in any quantity, the first few drops will usually cause the yel- low precipitate; but the dropping may be continued if no precipitate occurs, reapplying the heat occasionally, until an equal volume of the urine has been added. If no pre- cipitate occurs, sugar is absent, clinically speaking. If a precipitate occurs on boiling the test fluid alone, a new supply must be obtained, or a little more soda or pot- ash may be added, and the fluid filtered, when it is again fit for use. The precipitate referred to is a cuprous oxide, the result of a reduction of the protoxide, which sometimes occurs when Fehling’s or Pavy’s solution is kept for some time. It is said to be due to racemic acid, into which tar- taric acid is convertible on exposure. Under the influence of heat this acid oxidizes at the expense of the protoxide of copper, and the suboxide is precipitated ; hence the * This should be remembered, as, in consequence of it, the same urine in the hands of different observers would yield slightly different results, according as one or the other solution is used. 78 PRACTICAL EXAMINATION OF THE URINE. necessity of boiling a solution before adding the suspected fluid. I have sometimes noted that boiling a spoiled undi- luted Fehling solution does not reduce the copper, while from the diluted solution, when boiled, the suboxide is thrown down ; so that boiling a diluted Fehling’s solution becomes a more delicate test of its quality than boiling the undiluted solution. All possibility of such source of error may be avoided by keeping the solution of copper separate from that of the potassic tartrate in the caustic soda solu- tion, and mixing them at the moment they are required for use. The delicacy of Fehling’s test is said to be o.ooi per cent, glucose in water. With glucose in urine it is less delicate, but still in my hands it is the most delicate and reliable test available. It will be noted that in the use of Fehling’s and Pavy’s copper solutions an excess of the test fluid is always used, while in the method described as Trommer’s, the effect of adding too much may be to produce a yellow sediment and coloration on boiling with any urine, especially on cooling. It occasionally happens, also, when Fehling’s solution is used, that no reaction occurs until considerable urine has been added and the mixture cools down after the boil- ing, when a yellowness or milkiness makes its appearance. Dr. Roberts* believes this reaction due to sugar. But this is at least doubtful, for I have known it to occur in urine which, when treated by Briicke’s process, presently to be described, was found to be without a trace of sugar. It may be due to sugar, but it is as likely to be due to uric acid or to some other of the reducing substances contained *“ Urinary and Renal Diseases,” Amer. ed., 1879, P- r90' QUANTITATIVE GLUCOSE-TESTING. 79 in urine. In such a case the only way to settle the ques- tion is to resort to Briicke’s process, described on p. 87. But this may be said, that the quantity of sugar which could occasion such a reaction is of doubtful clinical significance. Filtering through animal charcoal is a less troublesome expedient than the lead process. This gives a perfectly clear fluid to work with, but in such filtration a small quantity of sugar is retained by the charcoal and must be washed out with distilled water. Again, it is almost impossible to obtain charcoal sufficiently free from impuri- ties, even when specially prepared for the purpose. Quantitative Analysis by Fehling’s or Favy’s Solutions. Volumetric Process. The simplest method of analysis by Fehling’s or Pavy’s solutions, and one which may be used in the consulting room as easily as the laboratory, is the following : One cubic centimetre of Fehling’s solution is diluted in a large test-tube with four cubic centimetres of distilled water, and boiled as described for qualitative testing. Its purity being thus ascertained, y cubic centimetre of the suspected urine is added from a suitably graduated pipette. Heat is then reapplied, the precipitate watched, and then another y added, the heat again reapplied, until it is found, after proper subsidence, that all the blue color is removed from the cubic centimetre of Fehling’s solution. If in doing this, i c.c. of urine has been added, it will have contained just half of i per cent, of sugar. If more than i c.c., it will have contained less than one-half per cent., but more than one-quarter per cent. If exactly 2 c.c. are used, it will have contained exactly one-quarter per cent. If, on 80 PRACTICAL EXAMINATION OF THE URINE. the other hand, but half a cubic centimetre is used, it will have contained i per cent., one-quarter of a cubic centi- metre, 2 per cent., and so on. If the proportion of sugar is large, as indicated by the Fig. 7.—Varieties of Burette Stand. specific gravity or qualitative test, the urine should be diluted with nine parts of water, and the result multiplied by io. In point of fact, most saccharine urines met with in practice require this dilution. PERCENTAGE OF GLUCOSE. 81 The following table, prepared by Prof. Wormley, will greatly aid in the estimation of glucose by this method :— Percentage of glucose in tirine as indicated by the quantity of urine required to exactly decolorize 1 c.c. (16 minims') of Fehling's solution. Of Undiluted Urine. C. C. Urine Glucose Per cent. °-i [5 -+- I], . . 5.0 0.12, . . .... 4.2 0.14, . . .... 3.5 0.16, . . .... 3.1 0.18, . . ... 2.7 0.2, . . .... 2.5 0.25, . . . . . 2.0 0-3, .... i 66 o-35, • • . ... 1.4 0.4, . . . . 1.25 0-45. • • . . 1.10 0.5, • • . . . .1.0 0.6, . . .... 0.83 0.7, . . . . . .0.71 0.8, . . 0.9, • • ■ • 0 55 1.0, .... 0.5 Of Diluted Urine, i in io. Glucose C. C. Urine Per cent. + O »-o Tt* o • • 12-5 0.5, . . . . . . 10.0 0.6, . . . . ■ ■ 8.33 0.7, . . . . • • 7-14 0.8, .... • • 6.25 0.9, . . . . • ■ 5-55 1.0, . . . . • • 5-o 12, .... . . 4.2 1.4, . • • . • • 3-5 1.6 • • 3-i 1.8, . . . . . . 2.7 2.0, . . . . • ■ 2.5 2.25 . . 22 2.50, . . . . . 2.0 2.75, .... . . 1.8 3.00, .... . . 1.6 3-5, .... . . 1.4 4.0, .... 1.25 4 5, .... 1.1 5.0, .... 1.0 6.0, .... . . 0.83 7.0, ... . . 0.7 8.0 . . 0.6 9-0 • • 0.55 10.0, ... • . 0.5 If it is desired to determine the quantity in English measures, Pavy’s solution may be used instead of Fehling’s, and ioo mimims measured off into the test-tube, diluted with four times its bulk of water, and boiled as before. Then the urine, diluted if necessary, is allowed to fall into the liquid,drop by drop, the heat being constantly renewed, until all the blue color has disappeared ; and when this has 82 PRACTICAL EXAMINATION OF THE URINE. happened the quantity of urine used will have contained just half a grain of sugar. Greater precision may be obtained by the use of the burette and stand shown in Fig. 7. Ten cubic centi- metres of Fehling’s solution are placed in a porcelain cap- sule and diluted with 40 c.c. of distilled water. Fill the burette to 50 c.c. with urine diluted x to 9. The capsule con- taining the diluted Fehling’s solution should be placed on a wire gauze and heated with a gas flame or spirit lamp, when half a c.c. of urine is allowed to fall into the hot so- lution from the burette. Immediately a yellow or red pre- cipitate will fall. This is allowed to subside, and if any blue color remains the urine is cautiously added, the solu- tion being kept hot, until all the blue color disappears. The titration must be repeated, if necessary, until the exact point of disappearance is ascertained and noted. If 5 c.c. of the urine are used to decolorize 10 c.c. of Fehling’s solution, the 5 c.c. of urine will have contained exactly this quantity, that is, .05 gram, since 10 c.c. of Fehling’s solu- tion corresponds to .05 gram of sugar. If now the urine has been diluted 10 times, the 5 c.c. will contain .5 gram sugar, and 100 c.c. will contain 10 grams or 10 per cent. Pavy’s solution may be used similarly. Cupric Test Pellets.—These were first suggested by Dr. Pavy at a meeting of the Clinical Society of London, in January, 1880, and were first made in this country by McKelway, of Philadelphia, at the suggestion of Dr. Joseph Neff.* They contain the elements of Feh- ling’s solution in solid form, and are very neatly made in the shape of a compressed pill. As made by Mr. McKelway, each pellet when dis * New York Medical Record, March 23, 1880. AMMONIATED CUPRIC TEST. 83 solved in distilled water represents 5 milligrams of diabetic sugar, and they may be used for approximate quantitative as well as qualitative testing. To use them, dissolve one in a small quantity of water in a test-tube and boil the solution. The purity of the pellet is thus tested, for if it has spoiled, the cupric oxide is reduced spontaneously. If the solution remains clear, the urine is added drop by drop, the temperature being kept up, and if sugar is present the usual precipitate occurs. The quantity of urine which just removes the blue color of the solution in which one pellet is dissolved contains 5 milligrams of sugar. These pellets are very convenient to carry, but in my experience they are liable to become unfit for use sooner than a Fehling’s solution, and unless very carefully made they keep but a short time. Pavy’s Ammoniated Cupric Test.—Dr. Pavy has suggested, in lieu of the usual form of Fehling’s solution, an ammoniated cupric test solution which has the advantage of obviating the precipitate of sub- oxide of copper, which is held in solution by the ammonia, so that a simple decolorization takes place as the cupric oxide is reduced ; the complete reduction being indicated by the total disappearance of the blue color. The proportions are as follows :— Cupric sulphate, .4.158 grams. Potassic sodic tartrate, ..... 20.400 “ Potash, caustic, 20.400 “ Strong ammonia (sp. gr. 0.880), . 300 c. c. Water to 1 litre. METRIC SYSTEM. ENGLISH SYSTEM. Cupric sulphate, grains. Potassic sodic tartrate, 178 “ Potash, caustic, 178 “ Strong ammonia (sp. gr. 0.880), . 6 fluidounces. Water to one pint. Dissolve the potassic sodic tartrate and potash together in a portion of the water, and the cupric sulphate with the aid of heat in another 84 PRACTICAL EXAMINATION OF THE URINE. portion ; pour the solution of cupric sulphate into the mixture of potassic sodic tartrate and potash ; when cold add the ammonia, and finally, with water, bring the volume of liquid to the bulk specified. Ten c. c. of the liquid, corresponding to .05 gram or 50 milligrams of sugar, is a convenient quantity to work with, and does not call for the employment of any appliance to obviate the inconvenience arising from the evolved ammonia. In performing the analysis twice the volume of water is added to the 10 c. c. of test employed. It should be stated that Hehner, Yeshida, and Sutton assert that by this method the ratio of reduction is seriously influenced by the amount of fixed alkali present and by the strength of the ammonia. Purdy’s Modification.—Dr. Charles W. Purdy substitutes in his formula 50 c. c. of glycerin for the sodic tartrate, dissolving the cupric sulphate in a part of the water and adding the glycerin, while the caustic potash is dissolved in another portion. The two solutions are then mixed, the ammonia added to make one litre, the whole filtered, and the volume increased. Ten c. c. of this solution correspond to 0.5 gram or 50 milligrams of sugar. If the English system is used the sodic tartrate is substituted by half an ounce of glycerin and the ammonia is reduced to five and a half ounces. One ounce of this solution is reduced by one-fourth of a grain of grape sugar. Cupric Test Paper.—Dr. George Oliver has also constructed a cupric test-paper with tartrate of cuprammonium, this salt being selected as the only one which is permanent on exposure to the air, and the only one that is stable when boiled with an alkaline carbonate as well as with a caustic alkali. The test-paper is a compound one, consisting of one charged with the reagent and the other with sodium carbonate, the two being united by a thin layer of rubber. To Use.—1. Drop a test-paper into 60 minims of soft or distilled water. 2. Boil for a few seconds until the water assumes a greenish tint. 3. Remove the paper. 4. Reboil the solution, and then add one drop of the suspected urine. 5. If glucose be present, reduction will take place without further application of heat, though, if preferred, the boiling may be continued and the reaction hastened. In any case, if the solution remains transparent for a quarter of a minute, heat should be applied to the boiling point for one minute. If then no opacity what- FERMENTATION TEST. 85 ever appears, it may be safely inferred that glucose is not present in pathological amount. The Fermentation Test. An excellent test for the presence of sugar is the fermen- tation test. The most convenient method of its applica- tion is as follows: a small quantity of ordinary baker’s or brewer’s yeast (about a fluidrachm, or 3 to 4 c.c.) is added to about 4 ounces of urine in a 6-ounce vial, which is lightly corked and subjected to a temperature of 15-250 C. (59-77° F.). If sugar is present evidences of fermen- tation will present themselves, generally within twelve hours, by the formation of carbonic acid gas, which passes off, leaving the fluid lighter and reduced in specific gravity in proportion to the quantity of sugar present. Dr. Roberts early announced that urine containing less than 0.5 per cent., or 2*4 grains to the ounce, yields no sign to the fermentation test. Claims to much greater delicacy are made, but it is evidently impossible to dis- cover a much smaller proportion, because water will absorb an equal bulk of carbon dioxide, so that all the gas generated in a solution containing T4y per cent, must disappear. Dr. Roberts has made use of the fact of the lowering of the specific gravity in devising a quantitative method. He has shown by careful experiments that every “ degree” in specific gravity lost in fermentation corresponds to one grain of sugar per fluidounce. Thus, if before fermenta- tion the specific gravity of a given specimen is 1050, and after fermentation it is 1020, it will have contained 30 grains to the fluidounce. The method recommended by Dr. Roberts is as follows: Four ounces of the saccharine 86 PRACTICAL EXAMINATION OF THE URINE. urine are put in a 12-ounce bottle and a piece of com- pressed yeast, as large as a small walnut, is added. The bottle is then covered with a nicked cork to permit the escape of the carbonic acid, and set aside on a mantelpiece or other warm place. Beside it is placed a tightly-corked 4-ounce vial, filled with the same urine, but without any yeast. In eighteen to twenty-four hours fermentation will be complete, and the scum cleared off or subsided. The specific gravity of the decanted fermented urine is then taken ; at the same time, that of the unfermented urine, and a comparison made. While some time is required to complete the fermentation, yet, as Dr. Roberts says, the preparation can be made by the patient himself or friends, and each day, when the physician makes his visit, he has only to make the comparison. The percentage may be roughly arrived at by multiply- ing the number of degrees lost by .23. Appliances have been suggested within the past year by Max Einhorn and S. P. Kramer with the object of securing greater accuracy in the quantitative estimation by fermentation; but, as the results are at best but approximate and the apparatus even less convenient than that sug- gested by Roberts, I omit them. They may be purchased, with direc- tions for their use, from Eimer & Amend, New York City. Bo tiger's Bismuth Test. To a given quantity of urine add an equal volume of a concentrated solution of sodium carbonate (crystals i part, distilled water 3 parts) or of the liquor potassae (U. S. P.), then a little subnitrate of bismuth. Shake and boil for a couple of minutes. The sugar possesses the power of reduc- ing the salts of bismuth, and if present, the black metallic BISMUTH TEST. 87 bismuth will shortly be deposited on the side of the test- tube. If the quantity of sugar is small, the bismuth will assume a grayish hue; hence, when this is the case a very small amount of bismuth should be used in making the test. This is a brilliant test, and except albumin or other sub- stance containing sulphur, nothing but sugar is supposed to reduce bismuth salts. I do not, however, think as highly of it as I formerly did, as the dirty gray hue said to be characteristic of very small quantities of glucose sometimes presents itself when no sugar is present. Especial care must be taken to remove the albumin before applying the bismuth test, or we may use— Briicke’s Modification of the Bismuth Test.*—To meet the difficulty referred to in the above paragraph, due to sulphur occasion- ally present in the urine causing a black precipitate of sulphide of bis- muth, Briicke recommends the use of Frohn’sf reagent—iodide of bismuth and potassium—to remove the disturbing elements, as fol- lows:—Pour into a test-tube a certain quantity of water, say 10 c.c., and fill another tube to the same level with urine. To the first add a drop of the Frohn’s reagent, which will cause a decided cloudiness. Then add, drop by drop, hydrochloric acid until the precipitate is re- dissolved. In this way we ascertain approximately how much HC1 must be added to the urine. Acidify the urine with such quantity; treat it with the reagent until precipitation is complete and filter. The filtrate, which should not now become cloudy on adding HC1 or the * Proceedings of American Pharmaceutical Association, 1887, p. 287. Also Hofmann and Ultzmann, “Analysis of Urine,” American transla- tion. New York, 1879, P- 93- j- 1.5 gram freshly precipitated subnitrate of bismuth is mixed with 20 grams water and heated to boiling; then 7 grams iodide of potas- sium and 20 drops of hydrochloric acid are added. The reagent is orange red. 88 PRACTICAL EXAMINATION OF THE URINE. reagent, is boiled for a few minutes with an excess of a concentraied solution of caustic soda or potash, as in Bottger’s test; if a gray or black color results, or such a precipitate is formed, the presence of sugar is proven beyond a doubt. The delicacy of this bismuth test is said to be 0.4 per cent, glucose in water. Nylander’s Modification of the Bismuth Test.—The solution for this test, also known as Almen’s solution, is made as follows: Basic bismuth nitrate, 2 grams; sodic potassic tartrate, 4 grams; sodic hydrate (purified sticks), 8 grams; distilled water, 100 c.c. Rub the solids to a coarse powder, add a little water to form a paste, then, while stirring briskly, gradually add the balance of the water. Let stand for five minutes to complete the solution. Filtration gives a perfectly clear solution. Add 1 c.c. of this to 10 c.c. of urine, boil for two minutes in an eight-inch test-tube. If a light gray to black precipi- tate form, it may be, usually is, due to the presence of glucose. If albumin is present, it must first be removed. This test, it is claimed, will detect .025 per cent, of glucose, while smaller quantities give a grayish tint to the flocculent precipitate. Urines of high specific gravity also reduce the bismuth oxide. This test, originally suggested in 1865 by C. D. Braun,* and revived by Dr. George Johnson f in 1882, is based upon the fact that grape-sugar, when boiled with picric acid and potash, reduces the yellow picric acid to the deep red picramic acid, the depth of color depending upon the amount of sugar present. For qualitative testing, to a fluidrachm of the sus- The Picric Acid and Potash Test. * Ueber die Einwandlung der Pikrinsaure in Pikraminsaure, und iiber die Nachweisung der Trauben Zucker.—Zeitschrift fur Chimie, 1865. f Lancet, November 18, 1882. PICRIC ACID TEST. 89 pected urine, add 40 minims of a saturated solution of picric acid, and half a drachm of liquor potassse.* If albumin is present, a turbidity will develop on the addition of picric acid, and thus the presence of this substance be recog- nized ; but it does not interfere with the test. Boil the mixture, and if sugar is present, a dark mahogany-red color will be produced. If normal urine be treated in the same way, a somewhat darker hue is also developed, but not nearly so marked as when sugar is present. Or the test may be applied, especially at the bedside, in the following manner:— Put into a test-tube about one-third of a grain of picric acid—as much as can be carried on the point of a pen- knife ; then add half a drachm of water ; dissolve the acid in the water by the heat of a lamp. Now, add half a drachm of urine, and the presence of albumin is ascer- tained ; next add a grain lump of caustic potash, and boil the liquid for a few seconds, and the dark coloration appears. A delicacy of 0.01 per cent, of glucose in water is claimed for picric acid. For quantitative testing Dr. Johnson directs a standard solution made by boiling together a fluidrachm of a solution of grape-sugar of the strength of one grain to the fluidounce, half a drachm of liquor potassse, and forty minims of a saturated solution of picric acid, the whole increased to four drachms with distilled water. The mixture is conveniently made in a large test-tube, which should be marked at four drachms. The liquid is kept boiling for sixty seconds, during * Dr. Charles F. Adams has altered the strength of the solutions so that the same result is obtained by taking 5 c.c. of each. Thus, of urine 5 c.c.; liq. potassse, sp. gr. 1.036, 5 c.c.; sol. picric acid (gr. 3.5 to fg) 5 c.c.; water 5 c.c. 90 PRACTICAL EXAMINATION OF THE URINE, which its pale-yellow becomes a beautiful claret- red. It is cooled by cautiously immersing the tube in cold water, and if the level of the liquid is not that of the four-drachm mark, it is raised to it by adding distilled water. The color thus obtained is that which results from the decom- position of 40 minims of a saturated solution of picric acid by a grain of sugar to the ounce, four times diluted. The color of this solution is, how- ever, not permanent, and Dr. Johnson imitates it with a solution of ferric acetate made by mixing thoroughly of solution of perchloride of iron, sp. gr. 1.44; giv of solution of acetate of ammo- nium ; 3 iv of glacial acetic acid, sp. gr. 1065; adding gj of liquor ammoniae, and diluting with distilled water up to iv. The ingredients are all of the strength of the British Pharmacopoeia.* This solution, corresponding to a grain of sugar to the ounce, four times diluted, retains its color unchanged, at least when kept in the dark, and is used for comparison. In testing for sugar, treat a fluidraclim of urine as indicated for qualitative testing, and dilute the mixture with distilled water up to four fluidrachms. The test-tube, which should be a large one, may be conveniently marked at four fluidrachms. Raise the liquid to the boil- ing point and keep it boiling for sixty seconds. Cool the liquid by carefully immersing in cold water, and if below the four-drachm mark it is to be again raised to it by the addition of distilled water. A comparison is then made between the Fig. 8. * Dr. Adams has also substituted the ingredients by those of the United States Pharmacopoeia, much more convenient to the American worker, as follows:— Liquor ferri perchlor. f 3 j; liq. ammon. acetat. f 25 ss; ac. acet. glacial, liq. ammon. fgj; aqua destil. ad f 2pv. PICRIC ACID TEST. 91 resulting fluid and the standard solution by means of the picro- saccharimeter devised by Dr. Johnson and illustrated by Figure 8. The stoppered tube on the left contains the standard solution. Into the graduated tube on the right, having the same diameter as the standard tube, the boiled mixture is to be introduced. Should the two agree precisely in color, the suspected urine will have contained one grain of sugar to the ounce. If, on the other hand, the mixture requires to be diluted in order to make it correspond with the standard solution, this should be done with distilled water, and if enough must be added to raise the level of the fluid, say from the io-division mark, at which it stood, to the 20-mark, the quantity of sugar would be two grains; if to 40, four grains ; to 45, four and a half grains.* In making an analysis, the picric acid must be added in proportion to the amount of sugar. If the proportion of sugar be as high as six grains per ounce, a drachm of the picric acid solution will be required. If it is higher than this, the urine should be diluted with distilled water in a definite proportion before commencing the analysis, and the dilution borne in mind in making the calculation. When the urine has been diluted ten times, the figures on the saccharimeter indicate the number of grains per ounce. Thus, when the ten-times diluted urine, after boiling with picric acid and potash, is further diluted from 10 divisions to 35, to obtain the standard color, the amount of sugar is 35 grains to the ounce.f We may reduce the amount of sugar per ounce to the pro- * A more exact comparison of the saccharine liquid with the standard can be made by pouring into a flat-bottomed colorless tube, about six inches long and an inch in diameter, as much of the standard as will form a column of liquid about an inch in height, and an exactly equal column of the saccharine liquid in a precisely similar tube. Looking down on the surface of a white porcelain slab or white paper through both tubes at once, a slight difference in tint is easily recognized ; and if the liquid to be analyzed be found darker than the standard, it is returned to the saccharimeter and diluted until the two fluids are found to be identical, when the final reading is made. •f Distilled or clear rain-water should be used, because the turbidity resulting from the action of potash on the salts of lime interferes with the exact estimation of the depth of color. 92 PRACTICAL EXAMINATION OF THE URINE. portion per cent, by a simple proportion, in which the first term is 455-7 grs.,the weight of an ounce of water at o°Cent.,* ioo the second term, and the quantity of sugar per ounce the third. Thus, if there be 20 grs. of sugar to the ounce, then as 455.7 : 100 : : 20 : 4.3. It has been said that when normal urine is treated with picric acid, potash, and heat, a slight coloration also takes place, which Dr. Johnson estimates as about equal to the change which would be produced by a solution of glucose containing 0.5 to 0.7 grains to the fluidounce. Dr. John- son, in common with others, now ascribes this reaction to creatinin. Dr. Johnson, after much experimental research, made in association with his son, G. Stillingfleet Johnson, claims that this method is “as accurate as any other,” and that for the estimation of sugar in the urine it is even more accurate than either Fehling’s or Pavy’s process, because the picric acid is not acted on by uric acid or urates, which reduce the oxide of copper. He also claims that the method by the picro-saccharimeter is more speedy than any other, the materials and apparatus inexpensive, and not liable to undergo rapid change. The Indigo-carmine Test. When a solution of indigo-carmine alkalized by sodium carbonate is boiled and kept heated, the blue color remains, but when a drop of a solution of glucose or saccharine urine is added to the hot solution, a beautiful play of colors occurs, terminating in pale yellow. Mulder first suggested this reaction as a test for sugar. Dr. Oliver has revived it for both qualitative and quantitative analysis. The indigo carmine *This is the weight of the ounce of distilled water according to the United States Pharmacopoeia. The English fluidounce weighs 437.5 grains. INDIGO-CARMINE TEST. solution is not available, because when the carmine and sodium car- bonate are present in solution the fluid undergoes a gradual change, the indigo-blue color gradually pasing into a pale green. Nor is it conve- nient to keep the solution separate. Dr. Oliver has, however, constructed a stable test-paper, charged with a definite quantity of the two reagents, indigo-carmine and sodium carbonate; while increased sensitiveness is produced by the use of an additional paper charged with sodium car- bonate. In Testing.—I. Unless very recently prepared, one of the carmine test papers should first be gently heated, say on a knife blade or spatula, by being held for a few seconds just above a flame.* It should then be dropped into a half-inch test tube, and water should be poured in to the amount of 60 minims. 2. Heat is applied, the tube being gently shaken, and boiling kept up for a second or two. The solution will then be quite blue, and, if the water added was soft or distilled, it will be perfectly transparent. Any turbidity observed will arise from the use of hard water, in which case a sodium carbonate paper should be dropped into the solution. The test- paper may now be removed or allowed to remain. 3. Not more than one drop of the suspected urine is let fall into the tube from the pipette held in an upright position. 4. The contents of the tube are again boiled for a few seconds; then the tube should be raised an inch or two above the flame, and held without shaking while the solution is kept quite hot, but without ebulli- tion, for exactly one minute. If glucose is present “ in abnormal amount,” says Dr. Oliver, “ the soft rich blue will be seen, first of all, to darken into violet; then, according to the quantity of sugar, there will appear in succession purple, red, reddish-yellow, and, finally, straw-yellow.” When the last color has developed, agitation will cause the return of purple and violet, and, finally, of the original blue. The time required for the reaction to commence, after boiling, varies inversely with the amount of glucose present. When the latter is large —over 20 grs. to the ounce—it will amount only to a few seconds. 93 * This is necessary, because the test-paper, on being kept for some time, loses its sensitiveness for detecting small quantities of sugar, say I to 2 grs. per ounce. 94 PRACTICAL EXAMINATION OF THE URINE. When small—say from 2 to 3 grs. to the ounce—it may require from 30 to 60 seconds. If the urine contains less than ]/2 a grain to the ounce, the color of the solution at the end of one minute will be unchanged. Precautions.—1. Care should be taken during the heating not to shake the tube, or to keep up active ebullition. 2. While keeping the contents of the tube hot, it should not be held between the eye and the sky, for then the early color changes may escape detection. The tube should be kept below the eye level, and its contents viewed by the reflected light of some bright object—as a sheet of white paper propped up an inch or two beyond the tube. The test is as available by artificial as by daylight. 3. Any caustic alkali, as liquor potassae or sodae, will discharge the blue color of carmine. Hence, care should be taken not to use a test- tube containing a trace of either of these substances or of Fehling’s solution or the alkaline picric solution, lest a deceptive reaction occurs. No amount of agitation will restore the blue removed by these agents. Dr. Oliver, comparing the results of experiments with the indigo- carmine and Fehling’s solution, found that whenever one drop was sub- mitted to the indigo test and the presence of sugar shown, confirmation was invariably provided by Fehling’s solution used in the ordinary way. On the other hand, whenever one drop of urine gave no reaction with the indigo, Fehling’s solution also gave negative results. In further experimental testings, Dr. Oliver found that none of the ordinary constituents of urine affect the carmine test, but all the free acids of the urine, utic, oxalic, lactic, etc., reduce Fehling’s solution. Of substances apt to appear in urine in disease, albumin, peptone,* pus, mucus, blood, bile, leucin, tyrosin do not react with either test; nor does one drop of ammoniacal or decomposing albuminous urine, or weak solution of ammonium sulphide; f but dextrin and milk-sugar as well as glucose reduce both. Inosit reacts with the carmine, and turns Fehling’s solution green—a green precipitate falling, leaving the supernatant fluid blue, which, however, becomes green on reheating. * In a letter from Parke, Davis & Co , I am informed that peptone decolorizes the solution just as does iodized starch. f Even a weak solution of ammonium sulphide will reduce Fehling’s solution. PHENYL-HYDRAZIN TEST. 95 Of medicinal agents likely to find their way into the urine, iron sulphate, gallic and tannic acids alone react with carmine, as do they, also, with Fehling’s solution. Comparative experiments by Oliver with picric acid and potash showed that whenever the indigo-carmine test-paper afforded a reaction, a correspondent reaction was obtained with the picric solution. Dr. Oliver has also applied his test to quantitative analysis, but it is rather cumbersome and not likely to be availed of. It is therefore omitted from this edition. The Phenyl-hydrazin Test.—The true value of this test, suggested by Emil Fischer, is as yet undetermined, although highly estimated by some. Most recently, in a noteworthy paper, Dr. Frank Dudley Blane says,* as modified by Ultzmann and Bond, “ it is the best single test known,” detecting .025 per cent, of sugar in urine. I regard it, however, too troublesome a test to be ever largely availed of. The test was origi- nally directed as follows: To 50 c.c. (13.5 f!J ) of the suspected urine add 2 grms. (30.8 grains) of phenyl-hydrazin hydrochlorate, and 1.5 grm. (23 grains) sodium acetate, or 1 grm. (15.4 grains) of the latter if the urine is not decidedly acid. Add also 20 c.c. (5.4 f3) of water unless the urine is nearly colorless. The capsule or beaker containing the urine and reagents is then placed in a water-bath and gently warmed for one hour. If sugar is present, needle-shaped crystals of phenyl- glucosazon will have appeared, which must be recognized by the micro- scope. But this is not sufficient. Their nature must be confirmed by determining their melting point, which is 204 to 205° C. For this purpose the dry crystals are obtained by filtering them off the urine, washing with a small quantity of water, dissolving in a small amount of dilute alcohol, and recrystallizing by evaporating at a low temperature. After repeating this two or three times the crystals are collected in a desiccator over sulphuric acid. Then draw out a piece of thin glass tubing in a Bunsen or spirit lamp flame, so that the sealed extremity is 2 or 3 cm. (.8 or 1.2 in.) from where the tube is of original diameter. The tube is broken by a file near where the contraction begins, and a small quantity of the dry body is introduced into the sealed extremity. The piece of tubing is now * IV. Y. Med Jour., Jan. 7, 1893. 96 PRACTICAL EXAMINATION OF THE URINE. attached to a thermometer by a small rubber band. The capillary end of the tube containing the substance is placed adjacent to the bulb of the thermometer, and the tube with the bulb is placed into concentrated sulphuric acid in a beaker which is gradually heated. As the mercury ascends to 204° C., the substance will begin to show evidence of fusion, providing the increase in temperature be gradual and the heat be equally diffused by stirring the acid with a glass rod. Ultzmann’s Modification.—Dr. A. K. Bond, of Baltimore, pub- lishes* the following simple modification of this test, suggested by Ultzmann, of Vienna: — Pour into a test-tube five inches long the phenyl salt to the depth of about T4ff of an inch, and add crystals of sodium acetate, ground fine, to an equal height. Upon this pour the urine—clear or cloudy—until the tube is half full. This gives in a test-tube five inches long about the following proportions in weight: I part phenyl salt, 2 parts sodium acetate, 15 parts urine. Shake the tube until the crystals of sodium acetate are dissolved; heat gently over a low flame until the mixture boils, and continue boiling for about half a minute—whether it becomes clear or not makes no difference. Then cover the tube and let it stand, and, after a proper interval, three to twelve hours, examine the sediment with the microscope. If sugar is present, there will be seen first fine, bright yellow needles of phenyl-glucosazon, which branch out or are joined by others as they are formed, until the field is dotted with groups like delicate sprays or sheaves, or radiating from a centre. A magnify- ing power of 200 diameters is sufficient for this study. That the phenyl salt is always in excess when the test is made in this way is shown by the constant presence of reddish globules in the field. My friend Dr. Purdy informs me that phenyl-hydrazin, either the salt or solution, is apt, when brought in contact with the fingers, to produce a painful eczema. The Alphanaphthol and Thymol Test.—'This recent test for sugar as directed by Molisch is made by adding to 1 c.c. of the fluid to be tested 2 drops of a 15 to 20 per cent, solution of alphanaphthol, and after mixing an excess of concentrated sulphuric acid. Upon shaking, if sugar is present, a deep violet color is developed, and on * Medical News, Aug. 6, 1887. ALPHANAPHTHOL TEST. dilution with water a violet blue precipitate occurs, soluble in alcohol and ether with a yellow color, and in potassium hydrate with a deep yellow. If the alphanaphthol is replaced with thymol a deep red color is produced, and on dilution with water a carmine-red flocculent pre- cipitate, soluble with a pale yellow color, in alcohol, ether, and potassium hydrate, but with a bright yellow in ammonium hydrate. These tests, according to Molisch, are the most delicate known, detecting sugar in solutions containing .0001 per cent. It is claimed that most sugars, to which inosit is an exception, respond to these tests, as do most glucosides except indican. Urea, creatinin, xanthin, uric acid, allantoin, hippuric and succinic acids, phenol and pyrocatechin all give negative results. Normal urine, however, responds when diluted 300 times, from which Molisch concludes it contains sugar. He gives the following method of distinguishing normal from diabetic urine. I. Dilute a specimen of normal urine, and one of the urine to be tested for sugar with 100 volumes of water, and compare the colors resulting from the application of the tests. 2. Dilute two similar speci- mens with 300 or 400 volumes of water. The diabetic urine will still respond to the lest while normal urine fails. Seegen has carefully examined these tests and declares them less sensitive than Trommers, while various animal substances and secre- tions and chemically pure albuminous bodies, as peptone, serum albu- min, egg-albumin, and casein all reacted. Hence he says they are of no value as tests for sugar alone, and Molisch’s conclusion that glucose is a constituent of normal urine is not justified. In reply to Seegen, Molisch reasserts his original claim, and says that in dilute solutions it is necessary to employ a small quantity of solid alphanaphthol in place of naphthol solution. With reference to albu- minous bodies, Molisch says that while these may give results resembling somewhat those obtained with sugar solutions, still, the precipitates obtained upon dilution with water are, excepting in the case of peptone, all of a different color (dirty yellow or yellowish brown) from those produced with sugar solutions. Besides, they are all soluble in hydro- chloric acid with a carmine-red or reddish-violet color, while the precipitate obtained with sugar solutions is insoluble in hydrochloric acid. Molisch also states that in place of sulphuric acid, hydrochloric acid 97 98 PRACTICAL EXAMINATION OF THE URINE. may be employed in these tests, the mixture being subsequently boiled for a minute. Fibrine, vitellin, serum albumin, egg-albumin, and peptone do not give the reaction when hydrochloric acid is used. Normal urine gives the reaction when boiled with alphanaphthol and hydrochloric acid even if diluted ten times. Molisch still asserts that normal urine must con- tain sugar, or otherwise some body as yet unknown. Moore's Test. Moore’s test depends upon the fact that grape-sugar, with which diabetic sugar is identical, becomes oxidized when boiled in contact with caustic alkali, taking the oxygen from the atmosphere. To a small quantity of urine in a test-tube, add half as much liquor potassse or liquor sodas and boil. If sugar is present a yellowish-brown color soon makes its appearance, which becomes more intense as the boiling is continued,and which will be the deeper the larger the propor- tion of sugar, becoming finally almost black if the quantity is very large. The coloration is due to the formation, first, of glucic, and finally of melassic acid, both of which remain in solution. The flaky precipitate which is observed after the addition of the alkali, and is increased on the application of heat, consists of the earthy phosphates, which, if very abundant, may be filtered off before the heat is applied. If, now, to the coloredfluid, a few drops of nitric acid be added, the brown discoloration disappears, and the odor of caramel or of burnt molasses is developed. Precautions.—I. Solutions of soda and potash are liable to become impregnated with lead, either from being kept in flint glass bottles, or from the glazed earthenware vessels in which, during preparation, they are evaporated. Such contamination always causes the production of a brown and black color when boiled with organic matter containing sul- phur, due to the formation of sulphuret of lead. This error may be avoided by first ascertaining the purity of the alkaline solutions, and afterward keeping them in green glass bottles. 2. If the urine exhibits already a high color, which is, however, very rare with diabetic urines, the coloring matter may be precipitated by solution of acetate (sugar) of lead, or by filtering through animal char- coal. The former throws down a small quantity of sugar, and the latter retains a little. 3. The coloring matters of bile in urine, either when pure or decom- posed (that is, when they respond neither to Gmelin’snor Heller’s test), produce a brown color with liquor potassa or soda without the applica- tion of heat. 4. Bodeker found in the urine of an adult a substance which he calls alkapton, which, when strong solutions of alkali are added, produces a brown discoloration from above downward. This, according to him, also reduces the salts of copper, but does not affect the bismuth salts. The delicacy of the test is put down at 0.3 per cent, glucose. POLARIMETRY. 99 The most convenient and the quickest of the methods for the quantitative determination of glucose, when the quantity exceeds i per cent., and when all the appliances are at hand, is by polarimetry. Smaller amounts can of course be detected—it is said .01 of 1 per cent, with the best instruments by those who are expert. The costliness of the apparatus, however, will probably always be in the way of its general use. The process is based upon the fact that glucose rotates polarized light toward the right, and the proportion of sugar in solution is determined by the degree of deviation noted. The best instrument, probably, is the shadow ap- paratus of Laurent, shown in Fig. 9. Light is admitted through a Nichols prism or polarizer in the tube RB, and falls on another prism, the analyzer in the eye-piece OH. Polarimetry. A ray of light entering the polarizer is divided into two parts vibrat- ing at right angles to each other, but the construction of the prism is such that the ray vibrating in one plane is absorbed while the other passes through. The latter is therefore polarized. Now if the second prism or analyzer in the eye-piece OH is so placed in relation to the 100 PRACTICAL EXAMINATION OF THE URINE. polarizer that their oblique ends are parallel, the polarized ray will pass through the analyzer without obstruction. If, however, the analyzer be rotated on its axis, the light gradually diminishes, until the rotation reaches 90°, when it is totally cut off. After 90° is passed, the light begins to return in increased quantity until 180° is reached, when it again passes unobstructed. Continuing the rotation, it is again gradu- ally diminished until 270° is reached, when it is again totally absent. After 270° it again returns until 360°, or the starting-point, is reached. Now if between the prisms so arranged that light passes through unobstructed a column of glucose is placed, the plane of vibration of light is rotated by the sugar so that when it reaches the second prism it is partly obstructed. By turning this prism on its axis the light may again be transmitted. The quantity of sugar and the length of the column determine the degree of deviation. Hence, knowing the specific rotary power of glucose, and the length of the tube, together with the angle of deviation, it is easy to calculate the percentage of sugar. If the tube is 10 cm. long, we have simply to multiply the degrees of rotation by 100 and divide by 53.1, the specific rotation of diabetic sugar. In most polarizing saccharimeters the analyzing prism is attached either to the graduated arc of the circle or to the vernier, so that when one or the other is turned the prism is turned. But the difficulty of determining the precise moment at which the maximum quantity of light is passing through is so great that some special means is provided for recognizing it. In Laurent’s so called “ shadow ” instrument, a quartz plate is so interposed that when the prisms are placed in certain rela- tion, half of the field of view is dark and the other light; in other positions both halves are equally light, while in intermediate relation- ships intermediate shades appear, as in Figure 10. In using Laurent’s instrument the two prisms are ad- justed by means of the screw F, which moves the analyzer until the field is uniformly illuminated on both sides. The vernier should then read o. The suspected wine, freed from albumin if this should be present, is treated with a solution of basic acetate of lead in the proportion of i to io 101 POLARIMETRY Fig. 9—The Laurent Shadow Polarizing Saccharimeter. 102 PRACTICAL EXAMINATION OF THE URINE. of urine, and filtered. The tube of known length is then filled with the mixture, and placed in position between the two prisms. Immediately light passes through the analyzer in OH, and this must be again turned by means of the screw F, until the light is again stopped and the field is uniformly illuminated. The angle is now read off on the dial-plate C and the percentage calculated—if the tube is 10 cm. long, by multiplying the degrees of rotation by 100 and dividing by 53.1, the specific rotation of diabetic sugar. To this result should be added -Jg- to allow for the 10 c.c. of lead solution* used. Fig. 10. Until recently a sodium flame has been the source of light, but an ordinary gas-flame now suffices. A new vernier is interposed between the analyzer in OH and the dial-plate C, Fig. 9, and we have simply to multiply the reading increased by Jg- (allowance for the lead solution) by the figures .2051, a factor arrived at by experiment as that which multiplied by the reading gives the percent- age when a tube 200 millimetres long is used. Thus, suppose the reading to be 24.9, then 24.9 -f 2.49 = 27.39, and 27.39 X -2051 = 5.61 per cent. * The urine may also be clarified by filtering through animal char- coal, in which event, of course, no allowance of this kind need be made. POLARIMETRY. 103 In the instrument of von Fleischel, in use by the writer, and with which a condensed kerosene or gas flame is used, when adjusted and the vernier reads o, two spectra are Fig. 11. placed one above the other, with the colors in each exactly continuous, the dark band proceeding through the centre of both, as shown in Fig. 11. Fig. 12. When the column of sugar is interposed the relationship of the spectra is at once altered, as shown in Fig. 12. 104 PRACTICAL EXAMINATION OF THE URINE. The proper relationship is again restored by moving the vernier to which the analyzing prism is attached, and the percentage of glucose is at once read off. For qualitative testing the polarizing saccharimeter is less convenient than Fehling’s solution—indeed, will not detect as small quantities. Its greatest convenience ap- pears in quantitative determinations where quantities of over one per cent, are to be measured. This instrument is less costly than Laurent’s. Remarks on the Qualitative Testing of Urine suspected to contain Sugar. There has been more criticism of the methods employed in testing urine for sugar and the results obtained from them than is justified. It arises partly from the fact that the chemist and the clinical physician view the subject from different standpoints. The former makes his experiments with test-fluids upon pure aqueous solutions of sugar, the latter upon a fluid containing numerous other organic con- stituents, more than one of which is capable of influencing certain test-solutions, though it may be in a less degree than sugar. Let us suppose the chemist to have made solu- tions of grape-sugar of different strengths which he is test- ing with Fehling’s solution diluted in the manner indicated on p. 77. He tests weaker and weaker solutions, and finally reaches one by which the test fluid is just decolorized while pure water will have no such effect. The chemist knows that it is sugar which thus reacts, because he has nothing but the test fluid, sugar, and water in combination. But it is very different with the clinician who tests urine suspected to contain sugar. There is no urine which will not partially decolorize a Fehling’s solution sufficiently REMARKS ON SUGAR TESTING. 105 dilute when it is boiled with it. Yet no one can claim that it is sugar which produces the decoloration when there may be several organic substances in the urine which can do it. On the other hand, no one can deny that it is sugar, because a very small amount of sugar will do the same thing. Further, it is easy to show that a quantity of sugar so small that it cannot be detected in urine can be readily detected in water, though it is also true that minute traces of sugar can be extracted from urine by certain pro- cesses, one of which will be detailed below. But these processes are not often available for the practical physician, nor is it necessary that they should be. For such amounts of sugar have no more clinical significance than has the normal proportion of urea in a specimen. It is only tan- gible amounts of sugar which are significant, and these are ordinarily recognizable by any one of the tests named, practiced with due precaution. With all tests, there are certain very evident reactions and certain doubtful ones, and to interpret these last experience must often be relied upon.* Exact Qualitative Method of Testing for Glucose—Briicke’s Lead Process. While any one of the methods just detailed suffices, if carefully used, to determine qualitatively, with sufficient * A correspondent calls my attention to the fact that he introduced a drop of glucose into a non-saccharine urine and failed to obtain a reac- tion with Fehling’s test. He then treated pure water in precisely the same way, adding a drop of glucose, and secured a beautiful reaction with Fehling’s solution. He asks an explanation. It is possible that the sugar is held in solution by creatinin, but more likely that there was an excess of phosphates in the specimen; that cupric phosphate was formed, which is not reduced by glucose. 106 PRACTICAL EXAMINATION OP THE URINE. accuracy for clinical purposes, the presence of sugar in a given specimen of suspected urine, it sometimes happens, especially in watching the course of a case of diabetes mel- litus under treatment, that the physician desires to know with absolute certainty whether there is even a trace of sugar present. This can be done by the following method, which is a modification of the one originally proposed by Briicke, and used by Pavy to demonstrate the presence of sugar in normal urine : — Take 50 c.c. of urine and add 60 c.c. of a 10 per cent, solution of neutral acetate of lead. The precipitate which takes place includes sulphates, phosphates, carbonates, col- oring matters, and part of the uric acid and creatinine, while the sugar remains in solution. Separate the precipi- tate on a filter, and treat the filtrate with an excess of ammonium hydroxide. A further precipitate occurs, which contains the sugar in combination with lead as a plumbic saccharate (Pb0)3(C6H1206)2. This precipitate is then collected and washed, great care being taken to remove all the ammonia, which is best effected by repeating several times the subsidence and de- cantation before throwing the precipitate on the filter; after which water is passed through until red litmus paper is no longer turned blue by the filtrate.* The precipitate, suspended in about 100 c.c. of water, is decomposed by passing through it a stream of sulphur- etted hydrogen as long as a precipitate of black plumbic * Dr. Pavy cautions against washing with hot water. Although it expedites the process, there is danger of breaking up the combination between the plumbic oxide and the sugar, thereby losing some of the latter. BRUCKE’s LEAD PROCESS—INOSITE. 107 oxide is produced. Filtration is then performed and the excess of sulphuretted hydrogen expelled by heat. The filtrate, a colorless fluid, is then evaporated over a water- bath to a volume equal to that of the original urine oper- ated upon, that is, 50 c.c. Dr. Pavy recommends the testing of the fluid thus obtained by the Fehling’s solution, but Prof. Wormley has discovered that it still contains uric acid which is capable of producing a neat reaction with the cupric test. This uric acid he removes by simply allowing the fluid to stand twenty-four hours or longer, at the end of which time a considerable sediment of uric acid has fallen to the bottom of the vessel; and it is this acid which produces the reac- tion in urine in the hands of Briicke, Bence Jones, and Pavy, and ascribed by them to sugar. When the uric acid is wholly removed from normal urine, no reaction occurs ; but if the slightest trace of sugar is present, it occurs, and the sugar is detected. About 50 per cent, of the glucose is lost in the course of the lead process. IX. Other Saccharine Substances. Inosite. Inosite or muscle sugar is sometimes found in the albuminuria of nephritis as well as in diabetes mellitus, and in the latter disease it has been found occasionally to substitute glucose, especially during conva- lescence ; also in phthisis, the syphilitic cachexia, and in typhus fever. Gallois examined the urine of 102 patients for inosite and found it in seven only,—five times in 30 cases of diabetes along with sugar in variable quantity, and twice in 25 cases of albuminuria. 108 PRACTICAL EXAMINATION OF THE URINE. Recognition.—Inosite is obtained from urine as follows : After any albumin that may be present is removed, the urine is treated with neutral acetate of lead until precipitation ceases. It is then filtered, and the warmed filtrate treated with basic acetate of lead so long as a precipitate occurs. It is better, however, to concentrate the urine to one fourth its bulk over a water-bath before precipitation. The lead precipitate which contains the inosite combined with the lead oxide is collected after twelve hours, washed, suspended in water, and decom- posed with sulphuretted hydrogen. Out of the filtrate there is sepa- rated, after standing some time, a small quantity of uric acid. This is filtered out, the fluid concentrated as far as possible and treated while boiling with three to four times its volume of alcohol. If there arises a heavy precipitate adhering to the bottom of the glass, the hot alcoholic solution is simply poured off. But if a flocculent non-adhesive precipi- tate occurs, the hot solution is filtered through a hot funnel and allowed to cool. If, at the end of twenty-four hours, groups of inosite crystals appear, they are separated and washed with a little cool alcohol. In this case it is advisable, in order that there may be no loss of inosite, to dissolve the precipitate obtained by the addition of hot alco- hol in as small a quantity as possible of hot water, and precipitate a second time with three or four times its volume of hot water. If no crystals separate, ether is added to the clear, cold alcoholic filtrate, until on shaking thoroughly a milky cloudiness appears, and then the fluid is permitted to stand in the cold twenty-four hours. If not too small an amount of ether is added (an excess does no harm), all the inosite present is precipitated in pearl-like, shiny plates. Inosite differs from cane-sugar in not undergoing vinous fermentation when treated with yeast, although its solutions readily take on the lactic fermentation when brought in contact with putrefying cheese. It does not reduce cupric tartrate in solution with potassic hydrate, but changes it to an olive-green, and after a while a flocculent precipitate falls, and the supernatant fluid becomes blue, but on again heating the solution the olive-green color is redeveloped. This reaction is sometimes observed in treating urine with Fehling’s solution, as was originally pointed out by Dr. Ralfe, and it has been suggested by Dr. Oliver that it may be due to inosite. Should this be true, inosite would appear to be less rare than has been heretofore supposed, but it is doubtful whether a reaction so indistinctive should be allowed much stress as compared with the exact chemical process above described. FRUIT SUGAR—SUGAR OF MILK. 109 Fruit-sugar, or Leevulose. Fruit-sugar sometimes occurs in urine, but only in association with diabetic sugar. It is characterized by being non-crystallizable, and turning the plane of polarized light to the left instead of to the right. The specific rotary power diminishes as the temperature rises, while that of grape sugar is independent. It is -108.3 at °° C.; -99.44 at 140; -97.1 at 17.5° ; -52 5 at 87.2°, according to Tuchschmied. It reduces the salts of copper, as does glucose, but in a less degree. Recognition.—Since lsevulose is always associated with glucose when it occurs in urine, and both reduce the salts of copper, it is only by their opposite action on polarized light that they can be distin- guished. If, therefore, a sugar-holding urine deflects polarized light strongly to the left, or if, after allowing for the fact that normal urine turns it to the left 10, and that this property is increased by the inges- tion of large doses of benzol, phenol, bromo- and nitro-benzol, chloral, and camphor, polarized light is not deflected at all, we may conclude that lsevulose is present. It is to be remembered that these substances also reduce copper salts. Other substances turning polarized light to the left must likewise be excluded. Sugar of Milk, or Lactose. C12H22Ou -j- H20. Lactose is sometimes found in small quantity in the urine of nursing women and females of animals. Dr. Ralfe refers to a case under his observation in the London Hospital, of a young married woman, aged 29, who was suckling an infant, and suffering with debility and frequent micturition, whose urine contained as much as 3 per cent, of sugar. This occurred in three successive confinements, there being no sugar during pregnancy. Lactose is characterized by crystallizing in white, or colorless, four- sided prisms with acuminated ends bounded by four triangles, by its turning polarized light to the right with a rotating power of 59.30, PRACTICAL EXAMINATION OF THE URINE. 110 varying also with the degree of concentration ; while that of grape sugar is -)- 53. i° ; by the fact that it reduces the salts of copperas does grape- sugar, and that it does not undergo the alcoholic fermentation with yeast. The fungi which convert milk-sugar into alcohol are cleft fungi. On the other hand, the lactic acid and butyric acid fermentation are readily entered upon. Recognition.—A very strong reducing power and an otherwise in- explicable deflection to the right suggest milk-sugar. Especially is this suspicion justified if the urine is that of a nursing woman. It can be recognized with certainty only by isolating it with the urine, for the details of which the student is referred to the works on physiological chemistry by Gorup-Bezanez or Hoppe-Seyler, or the elaborate treatises on urine analysis by Neubauer and Vogel or Salkowski. X. Aceton and Acetone-producing Substances ; Diacetic Acid. Often closely, though not necessarily associated with glycosuria, are those conditions which respond to the tests for acetone. A peculiar fruity odor of the urine was occasionally noted by the earlier students of diabetes, who were ignorant, however, of its cause, and it was not until 1857 that Petters* recognized in certain urines, by its peculiar chemical behavior, the substance known as aceton, to which this odor is probably due. Kaulich f extended and completed the work of Petters. Numerous other studies followed, the resultant of which was the fact above men- Aceton. * Fetters, “ Untersuchungen iiber die Honigharnruhr.” Prager Vierteljahrschrift, 1857, xiv, Band 3, S. 81. f Kaulich, “ Ueber Acetonbildung in thiereschen Organismus.” Prager Vierteljahrschrift, i860, xvii, Band 3, S. 58. ACETON—LEGAL’S TEST. 111 tioned, that while in diabetes mellitus aceton often occurs in the urine, it is found also in the urine in other diseases and in small amount even in health. Further, not all urines containing aceton exhibit the fruity odor. Con- trary to what was at first thought, aceton does not arise from the glycogen in the blood or urine, but from albumin disintegration.* Such, of course, could only be its origin where there is no glycemia or glycosuria. In confirmation of this is the fact that physiological acetonuria is enor- mously increased by a meat diet. Tests for Aceton.—The accurate study of aceton is best secured by working with the distillate from the urine. By Legal’s test, however, even moderate quantities may be recognized in the urine itself, to which, indeed, the test is better adapted than to the distillate, because parakresol, which also passes over with the latter, strikes a similar reaction. For this reason it is also employed as a prelimi- nary test, even when the distillate is afterward employed. Legal’s Test.—A fresh, rather strong solution of sodium nitro-prusside is made by dissolving a few frag- ments in a little water in a test-tube. To 3 or 4 c.c. of the suspected urine add enough liquor sodse or liquor po- tassse to secure a distinct alkaline reaction. To the mix- ture then add a few drops of the nitro-prusside solution, when promptly the whole assumes a red color whether aceton is present or not, said to be produced by creatinine even more rapidly than by aceton. In any event the red color disappears, but if aceton is present, the addition of a few drops of concentrated acetic acid causes a purple or * V. Engel, “ Mengenverhaltnisse des Acetons unter Physiol, und Pathol.” Bedingungen, Zeitschrift fur klin. Med., Band xx, S. 521 1892. 112 PRACTICAL EXAMINATION OF THE URINE. violet red. If there is no aceton, this final change does not occur. The purple color also fades in a little while, even if caused by aceton. On standing a dark blue precipitate may fall, and may be hastened if, after the addition of acetic acid, the solu- tion is warmed. Legal’s test may also be applied to the distillate, but for reasons named is less satisfactory. To Test for Aceton in the Distillate.—For the study of aceton in the distillate, treat a half litre or a litre of urine with phosphoric or hydrochloric acid in the proportion of 3 cubic centimetres of the acid to each ioo c. c. of urine. Thus treated, the urine is partially evaporated in a distilling apparatus, and the process completed in a retort. The addition of the acid is made to prevent the evolution of gases. Von Jaksch prefers phosphoric acid. From io to 30 cm. of the distillate are thus obtained, and to it may be applied the following tests :— Lieben’s Iodoform Test.—To a portion of the dis- tillate add a small quantity of liquor potassae, then a few drops of a solution of iodine and iodide of potassium.* If aceton is present a yellowish precipitate of iodoform is produced at once. Should alcohol happen to be present in the distillate, the reaction takes place also, but more slowly, but with aceton it is immediate. Gunning's Modification of Lieben's Iodoform Test.—The latter source of error may be altogether avoided by the modification suggested by Gunning, who uses ammonium hydrate and tincture of iodine. With these, alcohol causes * Friedlander’s formula for this solution is, iodine i.o; potassium iodide 2 ; distilled water 50. Reynolds’ and the indigo test. 113 no precipitate, while aceton does produce one of iodoform in addition to iodine, which falls as a black sediment, even though there be no aceton. The precipitated iodine is soon redissolved if the urine contains much aceton ; if it contain but little, the iodoform crystals may be seen in twenty-four or forty-eight hours, resting in a thin layer upon the black precipitate of iodine. This test may also be used with the urine itself, but with satisfaction only if the quantity of aceton is large. Reynolds’ Test,—This test depends on the power of aceton to dissolve freshly precipitated mercuric oxide. It is performed as follows: From a solution of mercuric nitrate in a test-tube yellow mercuric oxide is precipitated by an alcoholic solution of potassium hydroxide. The solution supposed to contain aceton is added and the mixture shaken. The liquid is filtered, taking care that the filtrate passes through clear. To the latter, ammonium sulphide is now carefully added. If aceton is present some mercuric oxide is dissolved, which passes over in the filtrate and shows itself as a dark ring of mercuric sulphide at the point of contact of the two liquids. The Indigo Reaction of Bayer and Drewsen.— Heat a few crystals of nitro-benzaldehyde until dissolved. Allow the solution to cool, when the aldehyde separates as a white cloud. Then add the suspected fluid or preferably its distillate, and make the mixture distinctly alkaline with dilute caustic soda. If aceton is present, there appears first a yellow, then a green color, followed by an indigo- blue, in the course of ten minutes. If only traces of aceton are present, the yellow fluid is shaken with a few drops of chloroform, when a distinct blue coloration of the chloro- form takes place. 114 PRACTICAL EXAMINATION OF THE URINE. By this method, it is said, aceton can be easily detected in a dilution of 1 part to 2500 if the distillate is used. Even with undistilled urine, by the aid of chloroform, it can be detected if present in the proportion of 1 to 1000. Pyroracemic acid, aldehyde, and acetophenon are the only other substances producing the indigo reaction, and these have not as yet been found in urine. Quantitative Estimation of Aceton.—More than one method of quan- titative estimation for aceton has been practiced, but the most satisfac- tory is that of Messinger, applied to urine by Huppert and further de- veloped by Engel and Devoto. Process.—The urine for the twenty is collected, accurately measured, specific gravity and reaction determined. A preliminary test is made by Legal’s method, when, according to the result, 20 to 50 c.c., or at most 100 c.c., of urine are placed in a boiling flask and in- creased up to 100 c.c. by the addition of distilled water. Two c.c. of a fifty per cent, solution of acetic acid are added and the flask connected by a long glass tube with the cooler, in front of which a distilling flask is placed, and in front of that a bullet apparatus filled with urine. All the attachments must, of course, be closely made. The distillation is con tinued until nine-tenths of the original volume is carried over. A por- tion of the residue is tried with Lieben’s test, and if it shows the pres- ence of aceton, the result is rejected and the process recommenced after the addition of more distilled water. The distillate is then treated with 1 c.c. of a diluted solution of sulphuric acid (1 to 8) and submit- ted to redistillation. The second distillate is poured into a flask of one-litre capacity, pro- vided with a ground-glass stopper, but filled during distillation with a doubled perforated cork, and having also a bullet apparatus full of water in front of it. Immediately after the distillation the flask is closed with its glass stopper and titrated with the one tenth normal iodide solution and the one-tenth hyposulphite solution. One c.c. of the iodo solution corresponds to 0.967 milligram aceton. The addition of the 2 c.c. of fifty per cent, acetic acid solution is made to prevent the passing over of phenol, and the later addition of the DIACETIC ACID—CHLORIDE OF IRON TEST. 115 1 c.c. of the I to 8 sulphuric acid solution to prevent the transit of ammonia, both of which are disposed to take up iodine themselves. Diacetic Acid. In 1865 Gerhardt* discovered that the urine in certain cases of diabetes mellitus struck a red reaction with a solution of ferric chloride. This reaction was ascribed to diacetic ether or ethyl diacetate, a substance which splits up readily into aceton, alcohol, and carbonic acid. This appeared reasonable, because both alcohol and aceton had been found in diabetic urine. Deichmuller f and Tollens,J however, eventually showed this substance to be diacetic acid, also obtainable from acidulated urine by the action of ether. Other substances, not usually constituents of urine, which strike the same reaction are said to be formic § and acetic acids and the cinchona salts; also carbolic and salicylic acids, decomposition products of antipyrin, kairin, and thallin. Aceton itself does not respond to the chloride of iron test, although diacetic acid responds to the different tests for aceton. The Chloride of Iron Test for Diacetic Acid.— It is plain, from the above, that while the striking of a red reaction on the addition of chloride of iron to urine * Gerhardt, “ Diabetes Mellitus und Aceton.” Wiener medizinische Presse, 1868, Band vi, No. 28. f Deichrauller, “Ueber diabetische Acetonurie.” Liebig’s Annalen der Chemie, Band 209, S. 22. J Tollens, “ Ueber Eisenchlorid roth farbende Harne.” Liebig’s Annalen, Band 209, S. 30. | Le Noble, who also found formic acid in three out of seven cases of diabetes. American Journal of the Medical Sciences, January, 1887. 116 PRACTICAL EXAMINATION OF THE URINE. is presumptive evidence of the presence of diacetic acid, it is not conclusive. To secure greater certainty, this test is directed by von Jaksch as follows : To urine, as fresh as possible, add cautiously a few drops of a moderately strong watery solution of chloride of iron. If a precipitate of phosphates is produced, remove it by filtration, and to the filtrate again add more of the chloride of iron solution. If a Bordeaux red color develops, heat a portion of the original urine to boiling, and acidulate a second portion with sulphuric acid and extract with ether. If the urine which has been boiled shows little or no change, while the chloride of iron reaction with the ethereal extract pales after twenty-four to forty-eight hours, and the urine as well as the distillate is found to contain large quantities of aceton, diacetic acid is present. The precautions described are necessary because of the possible presence of the above-named substances, which exhibit the same reaction. At the same time, if the reac- tion is very vivid, and the conditions favorable to the presence of diacetic acid are present, its presence may be inferred from the primary iron reaction alone. It is fur- ther important, however, that the urine should be fresh or decomposition be prevented, because diacetic acid is so quickly converted into aceton. If the color reaction with the ethereal extract does not fade away, the substance, according to Minkowsky, is /?-oxybutyric acid, of which diacetic acid is said to be a further oxidation. Clinical Significance of Aceton and Diacetic Acid.—We are indebted to von Jaksch* for most of our * “ Acetonurie and Diaceturie,” Berlin, 1885. SIGNIFICANCE OF ACETON AND DIACETIC ACID. 117 knowledge of the difference in the clinical significance of these two substances, which were formerly thought to be of identical import. Nor do the very recent observations of von Engel referred to, while thorough and exhaustive, much more than confirm and extend von Jaksch’s results. Acetonuria.—It has already been said that traces of aceton may be found in normal urine, constituting physio- logical acetonuria. Further, aceton is found in cases whose course is usually favorable, and its presence may have little or no significance. Diacetic acid, on the other hand, never occurs in normal urine, and diaceturia is a most dan- gerous complication. Acetonuria seems to be a result of continued high tem- perature, or at least accompanies diseases attended with such temperature, and a lowering of temperature is fol- lowed by a fall in the quantity of aceton, while a re-de- velopment of high temperature is followed by an increase in aceton. Such acetonuria is known as febrile. Acetonuria often accompanies diabetes, constituting dia- betic acetonuria, but is not necessarily associated with it or with glycosuria, and while the development of acetonuria in diabetes is sometimes accompanied by very unpleasant symptoms, as headache, loss of appetite, and deranged digestion, all of short duration, it is otherwise of little significance except as a possible precursor of the diaceturia which often succeeds it in this disease. Among other diseases with which aceton has been found associated are certain forms of carcinoma, which have not yet led to inanition, cerebral psychoses accompanied by mental excitement and derangements of digestion. Very rarely aceton itself is responsible for a set of symp- toms in which restlessness, excitement, and delirium are 118 PRACTICAL EXAMINATION OF THE URINE. the most conspicuous, constituting a sort of auto-intoxica- tion, the symptoms of which pass away entirely with the disappearance of the aceton. . Diaceturia.—On the other hand, what is commonly known as diabetic coma is the result, according to von Jaksch, not of aceton in the blood, but of diacetic acid, although it is true that it is often preceded by a long-con- tinued acetonuria. Diacetic acid is never found in normal urine. It is commonly observed in cases of far-advanced diabetes, constituting diabeiic diaceturia. There is added to the usual feeling of weakness and depression, drowsiness, which may deepen into coma or pass away, the diaceturia continuing. Sometimes these symptoms are preceded by excruciating muscular pain. As a rule, there is no relation between the amount of sugar and diacetic acid eliminated, although a sudden diminution of glycosuria is sometimes followed by the appearance of a large amount of diacetic acid, by coma and death. There is also a febrile diaceturia which accompanies feb- rile diseases, among which are the acute exanthemata, peri- carditis, pleuritis, perityphlitis, typhoid fever, miliary tuberculosis, tubercular phthisis, and pneumonia. It is to be remembered, however, that symptoms of diabetic coma may occur without either acetonuria or diaceturia. Von Jaksch proposes to do away with the term “ diabetic coma ” and substitute “ coma diaceticum ” for all of those cases of coma, from whatever remote cause, accompanied by diaceturia. Finally, there appears also to be an idiopathic diacetemia, or auto-intoxication by diacetic acid, unattended by other grave disease, such as pneumonia or diabetes, and mani- fested by vomiting, dyspnoea, and jactitation, which soon DIACETURIA—/J-OXYBUTYR1C ACID. 119 terminates in coma and death. This condition, very grave, but rare in adults, is said by von Jaksch to be much more frequent in children and correspondingly less serious. In such cases the child feels weak, has a thickly-coated tongue, often slight conjunctival catarrh, there is sometimes vomit- ing, usually constipation, and very little or no fever. In two or three days all of these symptoms, together with the diaceturia, disappear. In other cases nervous symptoms are more marked. Yon Jaksch believes that all of these, as well as a certain number of other convulsive attacks in children, are the result of auto-intoxication with diacetic acid. It is evident that our knowledge of these substances, aceton and diacetic acid, as well as that of the symptoms developed by their presence, is not yet complete; but the statements just made may be considered as representing as definitely as possible our present information. /3-Oxybutyric Acid.—The above statements are not seriously affected by the studies of Hugounenq,* Lepine, Stadelmann, and Minkowski, to the effect that this sub- stance and not diacetic acid is the cause of diabetic coma, and which are apparently confirmed by more recent obser- vations. Beta-oxybutyric acid is found in the blood of diabetic patients, is the homologous superior of lactic acid, and is formed from diseased muscle, just as lactic acid is from healthy muscle. Diacetic acid is only a further oxi- dation of it. Just by what steps it is formed within the body we are unable to discover. The method by which the changes may be brought about outside of the body is, however, very simple; the series then running as follows: * American Journal of the Medical Sciences, October, 1887. glucose, alcohol, aldehyde, aldol, /9-oxybutyric acid, dia- cetic acid, aceton. The recommendation of Hugounenq to use hypodermic injections of alkaline solutions to neu- tralize the acid in the blood has been followed without per- manent benefit. Lepine also advises an alkaline treatment, and says an exclusive meat diet is to be avoided as favor- ing the development of coma. These observers say, too, the acid found in the urine in many cases of diabetes is not diacetic acid, but oxybutyric acid. As to the relation between the two substances, aceton and diacetic acid, the conclusions of von Jaksch, published in 1885,* are essentially true to-day, viz., that (1) aceton is an oxidation product of albuminous substances; (2) it is found largely increased in the organism under certain pathological states and then may cause toxic symptoms. If the quantity of aceton produced is enormously large, it unites with certain acids produced in retrograde albumin- ous metamorphoses, perhaps with formic acid only, produc- ing diacetic acid, perhaps also in part with another series of similar acids, which may even be such volatile substances as are contained in evaporated urine and out of which by oxidation aceton may arise. 120 PRACTICAL EXAMINATION OF THE URINE. The pathological significance of all the coloring matters has not, as yet, been determined. Many of them are, how- ever, of such importance that their consideration commands interest next to that of the proteids and sugars. XI. Coloring Matters. * “ Ueber Acetonurie und Diaceturie,” Berlin, 1885. NORMAL COLORING MATTERS. 121 I. Normal Coloring Matters. Notwithstanding the very considerable study given to this subject of late years, there is still much confusion as to the normal coloring matters of urine. The two as to the existence of which there is most unanimity are the chro- mogen* of Urobilin, and Urine-Indican, the chromogen of indigo, the latter being the uroxanthin of Heller. Thudichum f makes a single coloring matter, which he calls tirochrome. To this matter, according to Thudichum, the urine owes the whole or greater part of its yellow color, while numerous other coloring matters, including the urrhodin of Heller, Scherer’s urohsematin, and the urohse- matin of Harley, he considers mixtures of the products of decomposition of this yellow pigment. The urohsematin of Scherer and that of Harley are probably identical, Scherer $ admitting that urohsematin contains iron, and approving of the use of the term by Harley for his coloring matter. The urophain of Heller is also probably the same thing. It will at any rate here be so considered. Finally, all of these are probably modifica- tions, due to different methods of treating urine, of the substance known as urobilin, now generally acknowledged to be the most important coloring matter of the urine. Upon the presence of indican (Heller’s uroxanthin) in * Chromogens are substances which develop a color on the addition to the fluid of some oxidizing agent. f Tbudichum, “ A Treatise on the Pathology of the Urine,” 2d edi- tion, London, 1877. J Harley, “ The Urine and its Derangements.” Philadelphia, 1872, from London edition, 1871. 122 PRACTICAL EXAMINATION OF THE URINE. most normal urines, all are agreed, although Thudichum prefers not to consider it a coloring matter, but a chromo- gen or color generator. For the present I shall retain it among the normal coloring matters, treating, therefore, chiefly of two, viz. :— 1. Urobilin (Jaffe), hydrobilirubin of Maly, and its modifications; urohtzmatin of Harley and Scherer; urophain of Heller. 2. Urine-indican, or the uroxanthin of Heller; to which will be added a short account of urochrome as described by Thudichum. i. Urobilin—Hydrobilirubin— Urophain— Uroluematin. Urobilin, first extracted by Jaffe, and further studied by Maly, is believed to be the bilirubin or normal coloring matter of the bile—altered after passing into the small intestine by absorbing water and hydrogen—in a word, reduced bilirubin. Hence MaLy names it hydrobilirubin. The reaction may be expressed as follows :— 2(C16H18N203) + H20 + hb= c32h44n4o7. Bilirubin. Urobilin. As such it is reabsorbed and excreted by the kidneys. As bilirubin is itself the hsematin of the blood, reduced by the action of the bile acids, the direct descent of urobilin from the blood is established. Obtained most readily from high-colored fever urines by processes described in the larger works on urinalysis, urobilin is a brown, resinous mass, easily soluble in water, but more readily in alcohol, ether, and chloroform. It gives no play of colors with nitric acid. Its concentrated solutions are brown; more diluted they are yellow, and still more, rose-red. When concentrated they exhibit UROBILIN. 123 peculiar spectroscopic properties and a beautiful green fluorescence by reflected light. The spectrum is a dark absorption band between Fraunhofer’s lines b and F. Both properties become more distinct by the addition of solution of ammonia and a drop of chloride of calcium, while the fluorescence ceases on adding hydrochloric acid, and the absorption band recedes toward F and becomes more indistinct. Jaffe has inferred from the absence of these peculiar re- actions of urobilin in fresh urine that urobilin is not at first present, but is preceded by a chromogen, which is con- verted into urobilin on exposure, by absorbing oxygen. Test for Urobilin or Hydrobilirubin.—Normal Coloring Matter.—Add ammonia until distinctly alkaline, filter, and to the filtrate add a little chloride of zinc solu- tion. The appearance of a green fluorescence and the characteristic absorption band indicates the presence of a considerable amount of bilirubin. It is especially abundant in the high-colored urine of fever cases, heart and liver diseases, and after sweating. Heller’s test for urophain is as follows : About 2 c.c. (32.4 minims) of colorless sulphuric acid are poured into a small beaker-glass, or, better, a “ collamore ” wineglass (p. 17), and upon it in a fine stream, from a height of about four inches, twice as much urine is allowed to fall. The urine mingles intimately with the sulphuric acid, and in normal urine, of which the specific gravity is 1020 and the quantity 1500 c.c. in the twenty-four hours, produces a deep garnet-red coloration. If the coloring matter is increased, the coloration is no longer garnet-red, but is black and opaque; whereas, if the 124 PRACTICAL EXAMINATION OF THE URINE. coloring matter is diminished, the mixture appears pale garnet-red and transparent. Precautions.—Unfortunately, other conditions than that of increased amount of coloring matter produce the increased intensity of the uro- phain reaction. Thus diabetic urine produces the same dark opacity through carbonization of the sugar by the sulphuric acid. In like manner, urine containing blood, biliary coloring matters, and uroerythrin (an abnormal coloring matter), gives the same reaction with sulphuric acid. Before relying, therefore, upon this reaction, the above substances must be carefully excluded. Dr. Harley’s test for urohaematin is as follows: Dilute the twenty-four hours’ urine with water till it measures 60 ounces (1800 c.c.), or if the quantity exceeds 60 ounces, concentrate it to this amount; then to about 2 drachms (7.4 c.c.) of it, in a test-tube, add half a drachm (1.8 c.c.) of pure nitric acid, and allow the mixture to stand for some minutes. If the quantity of urohaematin is normal, the mixture will alter but slightly in tint; whereas, if there be an excess, it will become pink, red, crimson, or purple, according to the amount present. Heating the mixture hastens the change in color, but it is better to do this experiment in the cold, and, if necessary, allow plenty of time for the change to take place. The acid is added to liberate the coloring matter, which may be so thoroughly concealed that a pale urine often con- tains a large amount of urohcematin. Harley gives a second method, also easy of application, for determining an excess of urohasmatin in cases of destructive diseases of the blood. Boil 4 ounces (120 c.c.) of urine, and add nitric acid to set the coloring matter free. When cool, put the urine in a six-ounce bottle along with an ounce of ether. Cork the bottle, thoroughly shake UROCHROME. 125 it, and place aside for twenty-four hours. At the end of that time the ether will be found to be like a red, tremulous jelly. Such a case, however, he admits to be a bad one. There is every reason to believe that urohsematin represents the disintegration of red blood corpuscles, and that it fluctuates, therefore, with the rate of their destruc- tion. Urochrome of Thudichum.—Thudichum terms the substance, to which he considers the whole or greater part of the yellow color of the urine is due, urochrome. It is an alkaloid, but not of pronounced basic properties. It has been isolated, but not finally analyzed. Its principal characteristic is that on chemolysis with acids it is split up into several bodies of smaller atomic weight, one of which —uromelanin—seems to be derived from the coloring ingredient of the blood. Urochrome does not show any specific absorption band before the spectroscope when strongly acidified, but by chemolysis probably gives rise to two or three substances having distinct spectrum phe- nomena which greatly aid in their diagnosis. It is not the chromogen of urobilin. Thudichum gives (op. citf several methods of isolating urochrome, the briefest of which consists in precipitating fresh urine with neutral and basic lead acetate, decompos- ing the precipitate with sulphuric acid, and precipitating the urochrome and some xanthin-like body from the filtrate by phosphomolybdic acid. 2. Urine in die an—Uroxanthin of Heller—Indigogen of Thudichum. Indican, C52H62N2034, or uroxanthin, is itself a color- less substance, separable from urine in the shape of a clear 126 brown syrup, easily soluble in water, alcohol, and ether. It has a bitter taste, and is easily converted by treatment with acids under warmth into indigo-blue (the uroglaucine of Heller), and a red coloring matter (urrhodine of Heller) said by Kletzinsky to be identical with indigo red ; but this is denied by Thudichum. This conversion is said to occur also as the result of putrefaction. Formerly one of these products was said to be indigo-glucin, a saccharine substance which is said to respond to Trommer’s test, but not to the fermentation test. This is now denied. Accord- ing to Thudichum, urrhodin is the result of chemolysis by acids of a separate chromogen which he calls urrhodinogen Uroxanthin or urine-indican was formerly thought to be identical with plant-indican, but more recent investigations tend to show a difference. Heller’s test is performed as follows : 4 c.c. or (5j of pure hydrochloric acid are poured into a smooth wine- or a small beaker-glass, and into the same while stirring 10 to 20 drops of urine are dropped. Under the normal con- ditions indican is present in urine in so small quantity that the acid to which the urine is added is colored pale yellow- ish-red. If indican is present in larger amount, the color- ation is violet ox blue. The more abundant the indican the more rapidly does the violet or blue coloration take place, and often 1 to 2 drops of urine are sufficient to color fjj or 4 c.c. hydrochloric acid. The blue color does not always make its appearance immediately. It is well then to wait ten or fifteen minutes, but the reaction which appears aftersuch an interval indicates buta small quantity of indican. The addition of 2 or 3 drops of pure nitric acid makes the test more delicate, and small amounts of indican are thus recognized. If it is desired to test urine PRACTICAL EXAMINATION OF THE URINE. TESTS FOR INDICAN. 127 containing the biliary coloring matters for indican, the former must be precipitated by solution of lead acetate and filtered out. Jaffe’s method is more striking in its results, and is even approximately quantitative. To io or 15 c.c. (2.7 or 2.24 (3) of urine in a large test-tube add an equal amount of fuming hydrochloric acid, and then, with constant shaking, a perfectly fresh saturated solution of calcic hypo- chlorite (chloride of lime) drop by drop, until the greatest intensity of the blue color is reached. This is then shaken with chloroform, which readily dissolves the freshly formed indigo, and separates from the aqueous solution as a blue fluid, the color being more or less deep according to the amount of indican present. In pale urines, often very rich in indican, this method will serve to determine its amount with sufficient accuracy for clinical purposes. Dark urines, whose other coloring matters are also decom- posed by hydrochloric acid and calcic hypochlorite, should first be decolorized by a solution of basic acetate of lead, avoiding a great excess of the latter, when, if indican is present, a good indigo extract can be obtained in this way. Weber’s test is commended by v. Jaksch. To 30 c.c. of urine add an equal quantity of hydrochloric acid, 1 to 3 drops of dilute nitric acid, and heat to boiling. The mix- ture becomes dark; after cooling, add ether and shake. If indican is present a blue froth appears on the surface, while the ether itself assumes a rose to violet color. Precaution.—Albumin must always be separated before performing these tests, as it develops a blue color with hydrochloric acid after standing a long time. If the resulting color is red instead of blue, iodine is present, and thus the absorption of iodides ascertained. 128 PRACTICAL EXAMINATION OF THE URINE. Clinical Significance of Indican in the Urine.— Normal urine, according to Jaffe, coniains 4.5 to 19.5 mil- ligrams in 1500 c.c., or about 6.6 in 1000 c.c. It is in- creased by a meat diet, in obstructive diseases of the bowel, in pyelitis, diseases of the spinal cord and its membranes, and especially derangements of the entire central and peri- pheral nervous system, in urina spastica, after coitus, and in hot weather, probably from concentration of the urine. It is also especially abundant in the urine secreted during the reaction from cholera (Wyss). It has been found by Neftel in cases of cancer of the liver; and its presence in large quantities in persons af- fected with malignant tumors he considered pathognomonic of cancer of the liver; by Hoppe-Seyler, in a case of mela- notic cancer of the orbit. Jaffe finds indican increased in all diseases attended by intestinal obstruction, cancer of the stomach, lymphoma and lympho-sarcoma in the abdo- men, purulent peritonitis, certain forms of diarrhoea, and in various diseases where the latter is a symptom. Rosen- stein found indican increased eleven to twelve times in Addison’s disease. I found it markedly increased in two cases of cirrhosis of the liver confirmed by post-mortem examination, and in one of evident malignant disease of some abdominal organ, probably the liver ; but the diagnosis was not certain and there was no autopsy. M. Robin has recently announced that he considers the presence of indican a valuable diag- nostic sign in typhoid fever.* From these facts it is evident that it is difficult to asso- ciate it pathognomonically with any disease. But recent * Philadelphia Medical Times, October 22, 1881, p. 63. UROGLAUCINE. 129 physiological observations afford a rational explanation for its increase, which is strikingly confirmed by the clinical observations above noted. Indican is increased when a substance known as indol (C8H9N), first discovered by Baeyer, is introduced into the blood. It was found by Ktihne (Virchow’s Archiv, vol. xxxix) that during the artificial fermentation of albumin in the presence of minced pancreas, indol was produced. Jaffe suggested that the indol thus produced during digestion is absorbed and converted in the blood into urine-indican. Now it is supposed that in ordinary normal intestinal digestion very little indol is produced ; but wherever digestion is inter- fered with or delayed, as is evidently likely to be the case in almost all of the conditions above instanced, more is pro- duced, absorbed, oxidized, and excreted as indican, thus accounting for its presence in increased amount under the circumstances. Uroglaucine.—Apery has * recently announced that he has found uroglaucine in every one of twelve cases of scarlet fever, deposited in small, blue masses so distinctive that they can scarcely be confounded with any other substance. Both uroglaucine and urrhodine are some- times found in the sediment of cystitis and Bright’s disease. To Obtain Uroglaucine.—Filter the urine and sediments. Dry the filtrate and treat with boiling alcohol, which dissolves out the blue substance, and on evaporation the uroglaucine is left with certain other matters which are washed off with cold water. The uroglaucine is again treated with boiling alcohol and the blue crystals are obtained by careful evaporation. Dr. Harley believes that all the various colored urine- pigments are but different grades of oxidation of urohsema- * Apery, “ Les Nouveaux November I, 1885. 130 PRACTICAL EXAMINATION OF THE URINE. tin,* and thus accounts for the various cases of blue, green, brown, and black urines which have been at different times reported, a most important fact with regard to which is that they never exhibit these colors at the moment the urine is passed, but acquire them after exposure to the air or the action of chemical reagents. He considers these changes which occur in urohaematin out of the body due to prior changes in the body caused by disease. He admits, however, in common with others, that some portion of the coloring matter of the urine comes from the food, chiefly vegetable food.f My friend, Dr. S. Weir Mitchell, has called attention to a peculiar greenish or yellowish-green coloration exhibited by the urine of those who are upon a diet of skimmed milk alone. This coloration is probably such as would be expected when the coloring matter derived from the haemoglobin of the red blood-corpuscles is uninfluenced by coloring matters contained in food, but it is a subject which requires to be in- vestigated. II. Abnormal Coloring Matters. Under abnormal coloring matters are included those which never enter into the composition of normal urine, whether found elsewhere in the body or not. They include (a) the coloring matters of blood, haemo- globin or oxyhaemoglobin, methaemoglobin, and haematin. Haematin is a deoxygenated haemoglobin, into which and a coagulated albuminous substance the latter is converted by the action of heat. Methaemoglobin is an intermediate condition, approaching, however, nearer to haematin, and gives the same absorption band, in the yellow of the spec- * Op. cit., p. I io. -j- Op. cit., p. ioi, ad. fin. COLORING MATTERS OF THE BLOOD. 131 trum between Fraunhofer’s lines C and D, but nearer to D, while haemoglobin gives one band in the yellow and one in the green between D and E. In fresh urine containing blood-coloring matters the prevailing one is haemoglobin ; but if a specimen of such urine be treated by sulphuret of ammonium, it becomes reduced haemoglobin by loss of its oxygen. Of this, "the spectrum gives a single broad band between the lines D and E. Shaking with oxygen or atmospheric air again restores the reduced haemoglobin to oxyhaemoglobin. (b) The uroerythrin of Heller. (c) Vegetable coloring matters. (d) Biliary coloring matters. (a) The Coloring Matters of the Blood—Hcemoglobin, Meth cem agio bin, and Hcematin. These substances can enter the urine either by direct transudation, or arise from the dissolution of blood-cor- puscles themselves, after they have entered the urine in different ways. They may be present in the urine in very small quantities without being accompanied by albumin, as was first shown by Dr. F. A. Mahomed.* The color of the urine is different according as it con- tains more haemoglobin or methaemoglobin, the former being brighter, the latter darker, brownish-red. Hemor- rhages from the larger vessels produce more haemoglobin ; capillary hemorrhages, on the other hand, more methaemo- globin. Heller proposes to account for the difference by *“ Transactions of the Royal Medico-Chirurgical Society of Lon- don,” vol. lvii, 1874, p. 196. the fact that in the hemorrhages which take place from the capillaries in renal disease, the blood is much more slowly and more intimately commingled with the urine, and there- fore longer retained with it at the normal temperature of the body. Temperature, the presence of carbonic acid, and the absence of oxygen, may favor the change of hae- moglobin to methaemoglobin. 132 PRACTICAL EXAMINATION OF THE URINE. Detection of Blood- Coloring Matters. i. Mahomed’s Test for Small Quantities of Hemo- globin Unaccompanied by Albumin.—Dr. Mahomed (op. cit.) directs as follows: One end of a small slip of white blotting-paper is dipped in the urine and dried over the flame of a spirit-lamp ; by this means the dilute solution of the crystalloid is concentrated by evaporation ; two drops of the tincture of guaiacum are then dropped on the paper, and after a minute or so, allowed for the spirit to evapor- ate, a single drop of ozonic ether is let fall in the centre of the guaiacum strain. A blue color appears if haemoglobin is present. Some time, perhaps a quarter of an hour, will elapse before the reaction becomes visible, especially if it be slight; when it appears it is not permanent; it will begin to fade in a few hours, and will have disappeared in a day or two. The advantage of this test lies in the fact that the physi- cian can carry a few slips of blotting-paper in his pocket- book, dip one in the urine during his visit, allow it to dry, and make the test at home. Dr. Stevenson’s modification of Dr. Mahomed’s test, acknowledged by the latter to be far more brilliant, is as follows : To a drop or two of urine in a small test-tube HEMOGLOBINURIA. add one drop of the tincture of guaiacum and a few drops of ozonized ether ; agitate and allow the ether to collect at the top, forming an upper layer of fluid. If haemoglobin be present the ether carries up with it the blue color that is produced, leaving the urine colorless below. In this method the blotting-paper, which is somehow the source of fallacy, is not required. 133 Precautions.—Saliva, nasal mucus, and a salt of iodine (as hap- pens when the patient is taking iodide of potassium) all strike a blue color with tincture of guaiacum, some without and some after the addi- tion of ozonic ether. Clinical Application.—By this test, according to Dr. Mahomed, infinitesimal traces of haemoglobin can be de- tected in urine which, to the naked eye, the microscope, the spectroscope, and even to the nitric acid test for albu- min, affords no indication whatever of abnormality. In- deed, the presence of albumin in any quantity interferes with the test, and it is in the prealbuminuric stages of scarlatina, or just after it has disappeared, and where there is a high state of vascular tension, that it is serviceable. It will respond in chronic albuminuria also, where minute traces of blood are present. Where the response precedes the appearance of albuminuria, it fades when the albumin becomes copious, and reappears again as it diminishes or after it disappears. The most useful application of the test, according to Mahomed, is in the prealbuminuric stage of scarlatina, where it will give us information of a state of affairs in the kidney previous to actual inflammation of the organ, when a brisk purge or copious sweat may avert more serious mischief. In cases of albuminuria produced by intense 134 PRACTICAL EXAMINATION OF THE URINE. fever and due to venous congestion, as in enteric fever, pneumonia, and sometimes in the febrile stage of scarlatina, when the fever is intense and the albuminuria only slight, no reaction showing the transudation of the haemoglobin can be obtained. 2. The presence of haemoglobinuria as distinguished from hsematuria is determined by the absence of blood disks and the presence of a smaller quantity of albumin, derived from the decomposition of the haemoglobin. When a solution of haemoglobin is heated in a test-tube it breaks up into a coagulated albuminous substance, haematin and methaemoglobin. The former is precipitated not in flakes which quickly coalesce and form a large, white, bulky pre- cipitate, as does coagulated serum-albumin, but forms a small, brownish, coherent coagulum which floats upon the surface. The color may be removed from the washed coagulum by boiling with alcohol containing sulphuric acid, the fluid becoming tinted reddish to reddish-brown, and giving the spectrum of haematuria. Again, the color of such urine, although dark red in bulk, is yellowish and more transparent in thin layers than urine containing blood-corpuscles. It is of lower specific gravity than such blood, and deposits a less copious sediment. It must not be concluded that, because blood-corpuscles are absent from a given specimen of urine containing haemoglobin, they have never been present; for they are sometimes rapidly dissolved, especially in alkaline urine. In such event we must depend upon the smaller amount of albumin just alluded to as characteristic of simple haemo- globinuria, and the smaller sediment. The urine contain- ing the dissolved corpuscles is more apt to be alkaline, while the urine of haemoglobin is acid in reaction. Should there be a transudation of serum at the same time with the haemoglobin it would, of course, be impossible to distin- guish the two. To Prove the Presence of the Blood-Coloring Matters Hcemoglobin or Hcematin in the Absence of Red Blood- Discs.—This may be done by Heller’s test for haematin, and by making haemin crystals. Heller’s test for hsematin is as follows: Precipitate from urine in a test-tube the earthy phosphates by caustic potash and gentle heat, over a flame. The earthy phos- phates carry with them, as they sink, the blood-coloring matters, and appear, therefore, not white, as in normal urine, but blood-red. When the quantity of coloring matter in urine is very small, the earthy phosphates appear dichroic. If the urine is already alkaline, and no precipi- tate of earthy phosphate appears on the addition of liquor potassae and heat, a precipitate can be artificially produced by the addition of one or two drops of the magnesium fluid, which, with the application of heat, carries down the coloring matters; whence it is possible. To Prepare Haemin Crystals.—If the precipitated earthy phosphates are filtered out and placed on an object- glass, and carefully warmed until the phosphates are com- pletely dry, Teichmann’s haemin crystals can be produced therefrom. For this purpose a minute granule of common salt is carried on the point of a knife to the dried haematin and earthy phosphate, and thoroughly mixed with it. Any excess of salt is then removed, the mixture is covered with a thin glass cover, a hair interposed, and a drop or two of glacial acetic acid allowed to pass under. The slide is then carefully warmed until bubbles begin to make their appear- ance. After cooling, haemin crystals can be seen by aid of H^MATIN. 135 136 PRACTICAL EXAMINATION OF THE URINE, the microscope. These, though often very small and in- completely crystallized, are easily recognizable by an ampli- fication of 300 diameters. They are chemically hydro- chlorate of haematin. Precautions.—Care must, however, be taken to apply only a gentle heat in precipitating the earthy phosphates with caustic potash solution, and to filter quickly, lest the haematin may decomposed. It sometimes happens, also, that vesicles develop under the thin glass cover, after the addition of acetic acid, even before heat has been applied. These are carbonic acid. They should be allowed to pass away, and the slide then warmed until the formation of vesicles, that is, to the boiling-point of acetic acid. Occurrence.—Haetnoglobinuria, that is, the direct pas- sage into the urine from the blood of the coloring matters unaccompanied by the corpuscular element, occurs in cer- tain general diseases, as scurvy, purpura, scarlatina, pro- found malarial poisoning, etc. Hsematuric or bloody urine results from the above and from a variety of other causes which require no special mention. Melanin is sometimes found in the urine of persons having melanotic cancer or sarcoma. It is deposited from urine in the shape of granular particles. These are soluble in liquor potassse, and their solution is decolorized by pass- ing chlorine through it. Melanin differs from carbon in being soluble in potash, while carbon is not. (b) Uroerythrin. Heller ascribes the well-known dark, reddish yellow, or “ high ” color of all fever urines to the presence of a sub- stance which he calls uroerythrin, as well as to an increase of the normal coloring matters. Except that it contains UROERYTHRIN. 137 iron, little else that is certain is known with regard to uro- erythrin. To it he ascribes the reddish color which so often characterizes the deposits of urates known as “ lateritious ;” if the supernatant urine in such cases be treated with solu- tion of neutral acetate of lead, the precipitate presents a simi- lar “ rosy red” or “ flesh color,” which he attributes to the same substance. It is doubtless a modified hsematin, being found especially in diseases where there is evident blood dyscrasia, as in low fevers, septic conditions, etc. It so far at least corresponds with the urohsematin of Harley that it is a measure of the destruction of the blood-corpuscles, though it will be remembered that the urohsematin of Har- ley is looked upon as a normal constituent of urine which may be abnormally increased, while uroerythrin, although a modified haematic, is still not considered identical by its discoverer. Neubauer includes uroerythrin among the normal color- ing matters, while Hofmann and Ultzmann, following Hel- ler, treat it as abnormal. Detection.—Uroerythrin is known to be present by its pink coloration of the “lateritious” sediment, or by its precipitation by solution of neutral acetate of lead. Too much lead solution must not be added lest the precipitate be too abundant, and the coloring matter rendered less dis- tinct by its being disseminated over a large amount of de- posit. If the urine contains hsematin, or the coloring mat- ter of blood, it must be first removed. Precautions.— X. The froth of a urine highly charged with uroery- thrin may appear yellow, as that of urine containing biliary coloring matter, but the precipitate of the latter by acetate of lead is also yellow and not pink, as is uroerythrin. 2. The earthy phosphates, which are precipitated on heating the urine 138 with caustic potash, are “dirty-gray” when the urine contains uroery- thrin, while in urine containing hsematin they are “ blood-red ” or di- chroic. The absence of albumin from the urine, the gray coloration of the earthy phosphates, and the red precipitate with solutions of lead, serve as points in the differential diagnosis between uroerythrin and the coloring matter of the blood. PRACTICAL EXAMINATION OF THE URINE. Clinical Significance.—Uroerythrin is found in the urine in all febrile affections, howaver slight; also, it is said, in pyaemia, diseases of the liver, and lead colic. All urine, according to Heller, which contains uroerythrin must be abnormal. (c) Vegetable Coloring Matters. The coloring matter of plants, especially chrysophanic acid, found in rhubarb and senna leaves, contributes to alkaline urine a reddish-yellow to a deep-red color. It can be recognized by the fact that the red alkaline urine on adding an acid becomes yellow, and on the addition of an excess of ammonia again takes on the red color. Precautions.—Such precipitation by heat and potash solution might possibly be taken for blood-coloring matters. But the absence of albu- min from the urine, the production of the red color by addition of an excess of ammonia, and its paling on the further addition of an excess of acid, serve to distinguish the vegetable-coloring matter from blood- coloring matter and uroerythrin. Numerous other vegetable matters color the urine, among which santonin is conspicuous for the bright yellow color it produces in acid urine, while the staining of linen by it closely resembles that of biliary coloring matter. Dr. W. G. Smith (Dublin Quarterly Journal of Medical Science, November, 1870) has investigated the subject, and found that the addition of alkali causes the development of fine BILIARY COLORING MATTERS. 139 cherry-red or crimson color, according to the amount of santonin present; but it will be observed that this reaction is that of the vegetable coloring matters generally, as above described. Madder, indigo, gamboge, rhubarb, logwood, carrots, whortleberries, etc., give to urine more or less of their peculiar colors. Salicylic acid, when administered in sufficient doses, gives a smoky hue to the urine, and the urine strikes a blue color when a few drops of a solution of ferric chlo- ride are added. Carbolic acid introduced into the system in sufficient quantity causes a dark and even black discoloration of urine, due probably to its disintegrating effect on blood discs, whose haemoglobin is discharged with the urine. (d) Biliary Coloring Matters—The Detection of Bile in the Urine. The biliary coloring matters are chiefly bilirubin (C16H18N203), biliverdin (C16H20N2O5), and bilifuscin (Ci6H22N206), the last two being derivatives by oxidation of the former. The last is found as such in herbivorous bile, and bilifuscin can be obtained from human gall-stones. None of these give any spectrum unless acted upon by re- agents. We have seen that urobilin, the normal coloring matter of urine, is bilirubin altered by taking up, while in the small intestine, water and hydrogen, as the result of which it acquires the spectrum described on page 123. From the intestine it is absorbed, and excreted by the kid- neys as the normal coloring matter of urine. When bile is abundantly present in urine, the yellow PRACTICAL EXAMINATION OF THE URINE. 140 color of the fluid, and especially of the froth or foam pro- duced by shaking, is sufficient to excite suspicion. Further, if a piece of filtering-paper or a piece of linen be moistened with such urine, it retains a permanent yellow color on drying. The only positive proof of the presence of the coloring matters of bile in the urine is found in Gmelin’s or Heller’s test for the unaltered coloring matters. Gmelin’s nitrous acid test is performed in two ways:— First. A quantity of urine is placed in a test-tube, and a small quantity of fuming nitric acid (nitrous acid of com- merce) i£ allowed to pass carefully down the sides of the test-tube to underlie the urine, as described in Heller’s test for albumin. If biliary coloring matters are present, at the point of union between the urine and the acid will very soon be seen a set of colors, which, if typical, should be green, blue, violet, red, and yellow, or yellowish-green again, in the order named from above downward. Often, how- ever, one or more colors are wanting. The green is most constant and the first green indispensable to prove the pres- ence of bile ; but violet, shading into red and yellow, is also very constantly seen. The other colors may be pro- duced by other coloring matters, especially indican. Second. Equally satisfactory is the test if a few drops of the urine are placed upon a porcelain plate, and as much of the fuming acid is placed adjacent and allowed gradually to approach the urine. The same play of colors occurs. E. Fleischl’s Test.*—A modification of Gmelin’s * Boston Med. and Surg. Journal, Jan. 13, 1876, from Cenlralblatt fur die medicinischen Wissenschaften, 1875, No. 34. heller’s test for bile pigment. 141 test, by which it is made more delicate. Instead of having impure nitric acid added in such a way that it will form a separate layer at the bottom, the urine should be thoroughly mixed with pure nitric acid, or, still better, with a concen- trated solution of the nitrate of sodium, and then concen- trated sulphuric acid should be carefully added so as to form a separate layer at the bottom. The play of colors forms at the junction of the urine and the sulphuric acid, the green appearing first above the acid, but rising gradu- ally and giving place to the blue, violet-blue, and yellow. The advantage of this modification is that the pigment is not oxidized so rapidly, and, therefore, the color is not changed so quickly, remaining often half an hour or longer. Heller’s Test for Bile Pigment.—Pour into a test-tube about 6 c.c. (1.6 f of pure hydrochloric acid, and add to it, drop by drop, just sufficient urine to distinctly color it. The two are mixed and “ under- laid” as before with pure nitric acid, and at the point of contact between the mixture and the colorless nitric acid a handsome play of colors ap- pears. If the “underlaid” nitric acid is now stirred with a glass rod, the set of colors which were superimposed upon one another now ap- pear alongside of each other in the entire mixture, and should be studied by transmitted light. Heller further says, if the hydrochloric acid on addition of the biliary urine is colored reddish-yellow, the coloring mat- ter is bilirubin ; on the other hand, if it is colored green, it is biliverdin. If the amount of coloring matter is very small, a large quantity of urine should be shaken with chloroform; the chloroform allowed to separate at the bottom of the vessel in large drops. The yellow-colored chloroform is then removed by means of a pipette, washed with distilled water, and poured into a beaker-glass containing hydrochloric acid. The yellow drops of chloroform sink to the bottom. If now, while diligently shaking the glass, nitric acid is added, the changes of color can be distinctly observed in the chloroform. In consequence of the 142 PRACTICAL EXAMINATION OF THE URINE. slower action of the acid upon the coloring matters dissolved in the urine and the consequent slower transition of colors, this method is peculiarly adapted for demonstration. Precautions.—I. With neither test should toodark-hued a urine be employed. Very dark urines should first be diluted with water. 2. Should albumin be present, the opaque zone at the point of con- tact between the urine and acid imbibes the coloring matters and exhibits a green coloration, so that the test is in no way interfered with. 3. Urine rich in indican may, however, deceive, forming at the point of contact a blue layer of indigo, which, along with the yellow urine, in reflected light, may appear green. In these doubtful cases the chloro- form modification of the test should be used, or the urine may be pre- cipitated with solution of acetate of lead, and the filtrate examined for indican. 4. The earthy phosphates, precipitated from biliary urine by liquor potassse and heat, exhibit a brown coloration. Ultzmann’s Test.—Add to 10 c.c. of urine 3 or 4 c.c. of pure caustic potash solution (1 part of KOH to 3 of H20), then shake and add an excess of pure hydrochloric acid. The mixture assumes a beautiful emerald-green color. Marechalt’s Test.—Upon a specimen of urine in a test-tube allow a few drops of tincture of iodine to fall carefully. If biliary pigments are present a green color appears at the point of contact between the two fluids, and remains for some time, even twenty-four hours. In this test the possibility of confounding indican is said to be excluded. Test for Decomposed Biliary Coloring Matters. —Should the urine contain only altered biliary color- ing matters which respond to neither Gmelin’s nor Heller’s test, Hofmann and Ultzmann recommend the following: — A piece of white linen or filtering-paper is immersed in the suspected urine, and allowed to dry, when it will appear colored brown. A further confirmation that the decomposed coloring matters are present will be found in DECOMPOSED BILIARY COLORING MATTERS. 143 a low specific gravity and a dark urophain reaction.* If, moreover, the urine be treated with liquor potassae and heat, to precipitate the earthy phosphates, it becomes darker than before and the phosphates are precipitated brown. Bile-pigments have a property of adhering to precipitates much more tenaciously than other pigments, and therefore sometimes cannot be detected in fluid urine when they maybe in precipitates. Hence Dr. J. F. Tarchanoff (Cen- tralblattfilr die medicinischen Wissenschaften, 1875, No. 6) recommends, in order to separate with certainty the biliary from the urinary pigments, precipitating the urine with milk of lime, freeing from excess of lime by a current of carbonic acid gas, allowing the whole to stand a few hours, filtering, and washing the precipitate with water. The bile-pigments are contained in the precipitate, while the indican, haemoglobin, and methaemoglobin are in the fil- trate. The precipitate is then dissolved in acetic acid and tested by Gmelin’s test. From a perusal of most of the text-books on physiology, and even of numerous manuals on the examination of urine, the student is led to suppose that the detection of bile acids, if present in urine, by means of what is called Pettenkofer’s test, is one of the easiest possible. This is, however, far from being the case, and the fact is that such detection by the direct application of the elements of Petten- XII. The Biliary Acids. * It should be remembered that this dark urophain reaction is also produced by sugar and blood-coloring matters. These causes should, therefore, be eliminated. 144 PRACTICAL EXAMINATION OF THE URINE. kofer's test in urine, or any other animal fluid, is practically impossible, even if the bile acids are present in considerable amount. Nor have any of the modifications of Petten- kofer’s test, recently announced as clinically available, proved such in my hands, even where the elements of bile have been added to the urine, except where inspissated ox-bile has been used. The results of a complete investi- gation of this subject in its practical bearings will be found in a clinical lecture by the writer, in the Philadelphia Medical Times for July 5, 1873, “ 0° a Case of Jaundice, with Remarks on the Availability of Pettenkofer’s Test,” to which the reader is referred. In these experiments the simplest method of obtaining the biliary acids was found to be as follows: Six or eight ounces (180-240 c.c.) of the suspected urine are evaporated to dryness over a water- bath. The residue thus obtained is treated with an excess of absolute alcohol, filtered, and the filtrate treated with an excess of ether (12 to 24 times its bulk), by which the bile-acids, if present, are precipitated. These are then removed by filtration and redissolved in distilled water. The solution is then decolorized by passing through animal charcoal, and the resulting colorless fluid tried by Petten- kofer’s test as follows: A single drop of a 20 per cent, solution of cane-sugar (simple syrup of the Pharmacopoeia is many times too strong) is then added to a drachm or two (3.7-7.4 c. c.) in a test-tube or a porcelain capsule. Sul- phuric acid is then added drop by drop, while the test- tube is kept in a vessel of cold water, to prevent too great a rise in temperature, which should not exceed 5o°-7o° C. (i22°-i58° F.). As the quantity added approaches a bulk equal to that of the fluid to be tested, a beautiful cheny-red or purple-violet color should make its appear- BILIARY ACIDS. 145 ance. So soon as a yellow color appears, then the sul- phuric acid is acting on the sugar, and the cherry-red can no longer be looked for. This carbonizing of the sugar is obviated by keeping the temperature down to the degree mentioned. Even this method involves more time than is often avail- able to the active practitioner, but there is none more simple, and there is really rarely any necessity for any other than the color test, for the presence of the biliary acids, although undoubtedly occurring, is very rare, and the circumstances under which they occur are illy deter- mined. It is not true, as was once supposed, that they are always present in the urine in cases of jaundice from obstruction and subsequent reabsorption of bile (hepato- genous jaundice), and absent in cases of jaundice from dissolution of the blood (hrematogenous jaundice) else would the determination of their presence be of real value in diagnosis. The only circumstances under which they are undoubtedly present in the urine are rapidly destructive diseases of the liver, as acute yellow atrophy and phosphorus poisoning. On the other hand, traces of the bile acids are said to be present in normal urine, Dragendorf having found .7 to .8 gram in 100 litres.* The bile acids yield a spectrum, which MacMunn has investigated. It gives a band outside D, and a broad band at E. Dr. Oliver’s New Peptone Test for the Bile- Acids.—This is founded on the physiological fact that when the products of gastric digestion, peptone and para- * The presence of bile-acids in normal urine is also asserted by Dr. Oliver, this assertion being based on experience with his new peptone test considered in the ensuing section. 146 PRACTICAL EXAMINATION OF THE URINE. peptone, which leave the stomach in an acid solution, meet with the bile, they are thrown down in a tenacious layer over the entire mucous membrane of the duodenum. So, too, albuminous urine or urine charged with peptone is precipitated by a solution of bile salts or of their deriva- tive, cholate of sodium. Hence acidified albuminous urine becomes a test for bile salts, but an acidulated antiseptic solution of peptone is a readier and more delicate reagent. Such a solution is made by Dr. Oliver as follows : — Pulverized peptone (Savary and Moore), . . . . gr. xxx Salicylic acid, gr. iv Acetic acid, Ttpxxx Distilled water, tof^viij. Perfect transparency is secured by repeated filtration. Application.—The urine should be perfectly clear, ren- dered so by filtration if necessary, boiled and filtered if bloody, rendered normally acid if alkaline, and finally reduced to a specific gravity of 1008. Twenty minims should then be run into 60 minims of the test solution, and if the proportion of bile salts is normal or subnormal there is no immediate reaction, but in a little while there is a mere tinge of milkiness. If, however, the bile salts are present in excess, a distinct milkiness promptly appears, becoming more intense in a minute or two, the degree of opacity being directly proportionate to the amount of bile derivatives. On agitation, the opalescence diminishes and perhaps finally vanishes, but is restored on adding more of the test solution. The precipitate differs from all other urinary precipitates induced by an acidified reagent, in dissolving completely on adding a drop or two of acetic acid or a citric acid test-paper, and by diminishing but not disappear- Oliver’s test for bile-acids. 147 mg when boiled, but the opacity is not affected by such a degree of warmth as is sufficient to dissolve urates. Further, an insufficiency, as well as an excess of acid, interferes with the reaction, as also does an excess of proteids, or of the salts themselves. Hence the importance of securing the proper proportions, as in Dr. Oliver’s formula, and of di- luting the urine to be operated upon to a specific gravity of 1008. By the dilution is also secured such a solution of the urates as avoids their precipitation and also any error con- sequent thereon. The reduction in specific gravity also obviates another source of error, in that concentrated urines often simulate an excess, while urines of low specific gravity, though affording a reaction similar to normal urine, may actually contain more than the normal amount of bile salts. The test may be used in the contact method, by running the solution over the urine reduced in specific gravity to 1008. If the bile salts are present in normal amount or less, there is again no immediate response, but in the course of a minute, a delicate, thread-like line makes its appearance, which may increase slightly. If the bile salts are abnormally increased an immediate reaction takes place. This test, according to Dr. Oliver, is so delicate that there can readily be detected one part of bile salts in at least 18,000 to 20,000 parts of a solution of chloride of sodium. So far, he has been unable to find any other con- stituent of urine which reacts similarly, and, although it is true that a concentrated solution of chloride of sodium in the presence of an acid will precipitate a proteid, experi- ment shows that when the peptone solution is run upon a solution of salt of any specific gravity below 1050, no pre- cipitation takes place. Hence there can be no error from this source in urine. 148 PRACTICAL EXAMINATION OK THE URINE. Mucin may be eliminated as a source of error, because this substance in acid solution is not precipitated by add- ing more acid, and when it is thrown down in urine of acid reaction, it is highly probable that the acid is not the reagent producing it, but merely supplies the requisite degree of acidity to enable the precipitant already present to operate, and in that event the mucin would only indi- cate the presence of bile salts. Quantitative Estimation.—This is based upon a permanent stand- ard of opacity provided by mixing together, in equal proportions, the test solution and normal urine reduced to the specific gravity of 1008. To 60 minims of the test solution add the suspected urine reduced to a specific gravity of 1008, usually 10 to 20 minims at a time, allowing a minute to elapse after each addition, until the opacity induced is ex- actly equal to or slightly exceeds that of the standard, the tubes being held to the light, shaded by a dark background, such as a coat sleeve. If 50 or 60 minims bring up the opacity to that of the standard, the proportion of bile salts does not exceed the normal amount. Any smaller quantity required indicates an excess, while the smaller the amount needed, the larger the proportion of bile salts present. Dr. Oliver has constructed a table showing the percentage of increase indicated by a varying number of drops :— Minims. I or Drops. 2 Percentage of Increase on the Normal Standard. 6000 2 or 4 = 3000 3 or 6 = 2000 4 or 8 = 1500 5 or IO = 1200 IO or 20 = 600 i5 or 30 = 400 20 or 40 = 300 25 or 5° = 24O 30 or 60 = IOO 35 or 70 = 83 40 or 80 = 66 45 or 90 — 50 LEUCIN AND TYROSIN. 149 An increase beyond 700 per cent, over the normal is rarely met, al- though Dr. Oliver mentions an instance of non-jaundiced urine which showed an increase’of over 1500 per cent , which afforded at once a beautiful reaction with Pettenkofer’s test. Peptone Test-Paper.—Dr. Oliver has also constructed a peptone test-paper which he considers permanent and reliable, and best used as follows : — The peptone paper with half a citric paper is dropped into 60 minims water in a test-tube or wineglass. After the lapse of a minute the solution is slightly agitated, and on being set aside for another minute is ready for use. The solution thus prepared is taken up by the pipette and carefully run over the transparent urine. If bile salts are present in larger amount than the normal average, an immediate reaction is observed as a pearly-white thread or band. In urine in which there is no such ex- cess, a delicate zone may appear, but only in the course of one or two minutes. Leucin and tyrosin, products of a retrograde metamor- phosis of nitrogenous substances, are found physiologically only in certain fetid secretions, as those of the axilla and between the toes, but can be produced by chemical means from some glands, as the liver, pancreas, and spleen, where they also occur in certain pathological states. They are found in urine, chiefly in rapidly destructive diseases of the liver, as acute yellow atrophy or phosphorus poisoning, but occasionally also in typhus and smallpox. They always accompany a large amount of biliary coloring matter and the presence of albumin. When at all abundant, as they generally are in acute yellow atrophy, they are deposited from urine and are found in the sediment, the former in the shape of centrically marked spheres, arranged in XIII. Leucin (C6H13N02) and Tyrosin (C9HuN03). 150 PRACTICAL EXAMINATION OF THE URINE. warty masses, or druses, the latter in needles. (Fig. 27.) Schultzen has shown * that in animals poisoned by phos- phorus “ urea disappears from the urine and is replaced by leucin and tyrosin, which in the healthy organism are converted into urea.” A similar substitution takes place in cases of acute atrophy of the liver, the retained urea accounting for the convulsive attacks which usually pre- cede death in these cases. Detection.—If the crystals—to be more fully described in treating of sediments—do not present themselves in the spontaneous deposit of such cases, the evaporation of a small quantity of the urine will generally promptly dis- play them. If they are not sufficiently abundant to be thus demon- strated, the method of Frerichs must be pursued to sepa- rate them. A large amount of urine is precipitated with basic acetate of lead, filtered, the excess of lead removed from the filtrate by sulphuretted hydrogen, and the clear fluid evaporated to a small volume over a water-bath. In twenty-four hours tyrosin needles will be found to have crystallized out, but leucin spheres will not appear until later, because of the great solubility of the leucin.f * Boston Medical and Surgical Journal, July 23, 1884, from Zeit- schrift fur Biologie, viii, 124, and Berliner klin. Wochenschrift, 1872, p. 417. f Leucin and tyrosin are more fully treated by the writer in the American Journal of ihe Medical Sciences for January, 1872. The above is believed to be sufficient for practical purposes. FATTY MATTERS. 151 XIV. Fatty Matters. That a trace of fat exists dissolved in normal urine has been shown by Schunk ; while the list of reported cases in which fat is present in abnormal quantity is gradually increasing. In such cases are, of course, not included those* in which fatty epithelium, fatty casts, and free oil drops are present,as the result of chronic Bright’s disease ; nor those in which fatty epithelium from the bladder or vagina occurs. The oil may be present in urine in a state of minute sub- division into small drops and molecules, as in the so-called chylous urine, or in the form of clear fluid oil. In the former instance the admixture probably results from the leakage of a lymph vessel into some part of the urinary tract. In the latter its source has been, in one instance* at least, traced to an abscess in the left lumbar region com- municating with the left ureter ; and such possible source should always be remembered. It is not impossible also that it may come directly from the kidney in cases of cystic cheesy degeneration of that organ, instances of which I have seen where there has been considerable free oil with compound granule cells and cholesterin plates among the cheesy matter. Dr. Roberts f refers to three cases in which pure yellow oil was present in the urine, in two during the administration of cod-liver oil, and in the third during the use of an emulsion. Dr. Henderson J reported three cases * Dr. E. W. Cushing, in Boston Medical and Surgical Journal, vol. 104, 1880, p. 242. f “ Urinary and Renal Diseases,” Am. ed., 1879, P- I25* | British Medical Journal, May 22, 1858. 152 PRACTICAL EXAMINATION OF THE URINE. of heart disease in which free oil globules were suspended through the urine. I have recently had under my care a young woman in whose urine, always free from albumin, free oil drops and aggregates of oil drops could be found at almost any time, and at times tube-casts containing oil drops, the most scrupulous care being taken to avoid error. Fat has been found in the urine from cases of calculous disease of the pancreas, and in one case referred to by Dr. George W. Johnston,* fat made its appearance in the urine one month before it was detected in the alvine dejecta, and in such quantity as to float, when cool, in greasy flakes on the surface. The presence of cholesterin in the urine is also a pos- sible but very rare occurrence, and may be conceived to occur in such a case of cheesy cystic kidney above alluded to. The only well-authenticated case I have ever seen reported is that given by Dr. Roberts.f Dr. Beale has shown that cholesterin may be obtained by treating large quantities of urine from cases of chronic Bright’s disease, but this is a different matter from its being contained in urine in the free state. XV. Urea ( CN2H40 ). Urea is the chief organic constituent of urine and the index of nitrogenous excretion. Its quantity fluctuates with changes in the quantity and composition of ingesta, * Inaugural thesis for the degree of Doctor of Medicine in the Uni- versity of Pennsylvania, 1882. Published in the A7nerican Jour, of the Medical Sciences, October, 1883, of which see p. 427. f Op. cit., p. 125. and with the rapidity of tissue metamorphosis in health and disease. A range of at least from 20 to 40 grams (308.6 to 617.2 grains) in twenty-four hours must be ad- mitted in adults, or 1.5 to 2.5 per cent. Detection and Estimation.—The odor of urine highly charged with urea may be said to be characteristic, but certain evidence of its presence can only be obtained by treating the solution suspected to contain it with nitric or oxalic acid. Though crystallizing itself in glistening UREA. 153 Fig. 13.—Crystals of Nitrate of Urea. (After Beale.) needles, it is too soluble to permit of easy detection by its own form. If it be desired to detect its presence in a sus- pected fluid, a drop or two is placed upon a glass slide, a drop of nitric acid added, the slide carefully warmed over a spirit-lamp, and placed aside to crystallize. If urea is present, the microscope will reveal, singly or in strata, six- sided and quadrilateral plates of nitrate of urea (Fig. 13 ). The crystals have acute angles measuring about 82°, and 154 are so characteristic as to be easily recognizable ; they often overlap each other like the shingles of a roof. Solution of oxalic acid produces similar but less regular crystals of oxalate of urea. In ordinary normal urine, this crystallization does not take place unless the urine is concentrated by evaporation. But in some urines highly charged with urea, it is simply necessary to add nitric acid to produce the crystals, and thus is arrived at a rough quantitative estimation of urea. As urea is by far the most abundant solid constituent of the urine, it follows that the specific gravity may become a means of approximately estimating its amount, especially when there is no sugar present, if the quantity of albumin is small and that of the chlorides is normal. A specimen of urine neither albuminous nor saccharine, containing a normal proportion of chlorides, and having a specific gravity of 1020-4 t0 a quantity of 1500 c.c. (50 oz.) in twenty-four hours, may be taken as a standard normal specimen containing 2 per cent, to 2y2 per cent, of urea. These conditions being observed, a higher specific gravity would indicate an increased proportion, and a lower, a diminished proportion. Under these circumstances, a specific gravity of 1014 indicates about 1 per cent, of urea and of 1028 to 1030 about 3 per cent. But the chlorides fluctuate markedly in some diseases, and by far the largest proportion of urines in which a knowledge of the amount of urea is important contain albumin. Next to urea, supposing albumin and sugar absent, the chlorides most affect the specific gravity, being separated to the amount of 10 to 16 grams (154 to 247 grains), or to 1 per cent, in the twenty-four hours. If these are totally absent, as they often are in pneumonia PRACTICAL EXAMINATION OF THE URINE. UREA. 155 and other febrile diseases, accompanied by an increase in the elimination of urea, then must a specific gravity of 1020 indicate more than 2*4 per cent, of urea, or, if the percentage of chlorides replaced by urea be added, per cent. This is on the supposition, of course, that the remaining constituents, uric acid, creatinin, phosphates, sulphates, etc., have little influence on the specific gravity —which is the fact. If albumin is present in small quantity, not exceeding y percent., it has little effect, and it can be thrown out of the question. If, however, the albumin be more abundant, 1 to 2 per cent., it must first be removed by coagulation and filtration, and the approximate estimation be made from the specific gravity of the filtrate after cooling. Care must, of course, be taken to wash the coagulum by further addition of water until the quantity of fluid originally operated with is restored. After such removal of albumin, if not before, the specific gravity will generally be found lower than in health, showing—what volumetric analysis has determined more precisely—that in chronic albumi- nuria, at least, the quantity of urea is generally diminished. Where sugar is present, the percentage of urea is also generally less, though with increased specific gravity, while the large total quantity of urine in the twenty-four hours may show an increase in the total urea for the day. There is no way of allowing here for the specific gravity due to the presence of sugar, and the only way to arrive at a knowledge of the amount of urea is by volumetric analysis. Volumetric Analysis for Urea. Under any circumstances, when an accurate estimation of urea is required, we must have recourse to volumetric 156 analysis. Several methods of volumetric analysis for urea have been suggested, of which that of Liebig, with the nitrate of mercury solution, was for a long time solely employed, but of late has been largely superseded by the hypobromite and hypochlorite processes. Liebig’s method is based upon the fact that urea produces a precipitate with mercuric nitrate. The following test solutions are required:— 1. The baryta solution, consisting of one volume of cold saturated solution of barium nitrate with two volumes of cold saturated solution of caustic baryta (barium hydrate). 2. A saturated solution of sodium carbonate to be used as an indicator. 3. A standard solution of mercuric nitrate of such strength that 1 c.c. is precisely equivalent to .010 gram, or 10 milli- grams, of urea. PRACTICAL EXAMINATION OF THE URINE. To Prepare the Standard Solution of Mercuric Nitrate.—i. Dissolve 71.48 grams of pure mercury or 77.2 grams of the mercuric oxide in nitric acid by the aid of heat. The acid fluid is concentrated by evaporating over a water-bath to a syrupy consistence, and then diluted with distilled water to a volume somewhat less than a litre. If on dilution a white precipitate of basic nitrate of mercury fall, allow it to settle, and decant the clear liquid. Then add to the residue a few drops of nitric acid to dissolve the precipitate. Add the solution thus obtained to the former decanted liquid and dilute to exactly one litre. Thus the solution will contain 77.2 grams mercuric oxide to the litre, or 77.2 milligrams to the c. c. The solution requires to be graduated by 2. The Standard Solution of Urea.—Two grams of pure Urea should now be dissolved in 100 c.c. of distilled water, of which 10 c.c. will then contain 0.2 gram, or 200 milligrams. The solution is a 2 per cent, solution. Ten c.c. of the standard solution, containing 200 milligrams of urea, are now placed in a beaker-glass. A burette is then filled to o with liebig’s process for urea. 157 the solution of mercuric nitrate (taking care that the lower edge of the meniscus which forms the upper surface of the liquid corresponds with the arrow on the burette), which is then allowed to drop into the beaker, where it will quickly form a dense precipitate. When the pre- cipitation seems about complete, a drop of the fluid containing it is allowed to fall on a drop of the solution of sodium carbonate, of which several are previously ready on a piece of glass on a dark ground. If the urea is not completely precipitated, no change of color takes place. The cautious addition of the mercuric nitrate is continued, also the process of testing with the Na2C03, until finally a yellow color appears. This proves that the mercuric nitrate has been added in excess—con- sumed all the urea in combination and left some mercuric nitrate to react with the sodic carbonate, which it does by forming sodic nitrate and the yellow oxide of mercury. If now the mercuric solution is correct, it will require exactly 20 c.c. of it to precipitate the whole of the urea in the 10 c.c. of the standard solution of urea, and enough more to react with the sodium carbonate. If the yellow coloration does not occur under these circumstances, there has been some impurity in the mercury compounds employed, or some error in making up the solution. Process.—Take 40 c.c. urine and 20 c.c of the baryta solution and throw them into a beaker-glass. By this means the phosphates, sulphates, and carbonates are pre- cipitated. They are removed by filtration through a dry filter, and if the filtrate happens not to be quite clear, it may be passed through a second time.* While this is taking place, the burette is filled to o with the mercuric nitrate solution, and 15 c.c. of the filtrate from the mixed baryta fluid and urine, containing of course * If the filtrate is not alkaline, the precipitation of the phosphates and sulphates may not have been complete. This may be determined by adding a drop or two of the baryta mixture to the filtrate, when, if a precipitate appears, a fresh quantity of urine must be taken, and a larger proportion of the baryta solution added. 158 PRACTICAL EXAMINATION OF THE URINE. 10 c.c. of pure urine, are measured off into a small beaker- glass. Into this the mercuric nitrate solution is allowed to fall from the burette, first, a number of cubic centimetres approaching the last two figures of the specific gravity (that is, if the specific gravity is 1017, drop say 15 c.c.) Fig. 14-—Burette Stand with Two Forms of Burette. before testing with the sodium solution. If no yellow coloration appears on such testing, then proceed cautiously, adding a fractional part of a cubic centimetre at a time, and testing with the Na2C03 until the yellow coloration liebig’s process for urea. is obtained. When that point is reached, read off the number of cubic centimetres employed.* The number of cubic centimetres of mercury solution thus used, minus 2, multiplied by .010 gram, gives the amount of urea in frac- tions of a gram contained in 10 c.c. of the urine, when the latter is of average composition—that is, when it con- tains no abnormal constituent, and the amount of chlo- rides is nearly normal. Corrections Explained.— The two cubic centimetres are first subtracted because it takes about this quantity of the reagent to convert the chloride of sodium into the nitrate, and until this combination is complete, the combination with the urea does not begin. Hence this amount must first be subtracted. 159 If, however, the chlorides are not of average amount, but dimin- ished or increased, and we wish to be accurate, we must first estimate the amount of chlorides calculated as NaCl in io c.c. of the urine, by the process to be explained under chlorides, and from a fresh quantity of urine remove the whole of the chlorides by a standard solution of silver nitrate. For this purpose a solution of nitrate of silver is required, of such strength that I c.c. will precipitate io milligrams sodium chloride. 29.059 grams of fused nitrate of silver, dissolved in distilled water and diluted to a litre, will be such a fluid. In 10 c.c. of the original urine we determine with the nitrate of silver solution the chloride of sodium by the method for the determination of the chlorides, p. 178. Suppose there are required for this 17.5 c.c. of the silver solution, this indicates 175 milligrams sodium chloride. * The tinge of yellow at which we cease the titration must of course be the same as that at which in originally standardizing the nitrate of mercury solution the titration was stopped. It is evident that ceasing the titration, now at a slight tinge and again at a marked yellow colora- tion, must give rise to an error, which practice will soon teach the stu- dent to avoid. 160 Take now 30 c.c. (containing 20 c.c. of urine) of the filtrate from the mixture of baryta fluid and urine, add a drop of nitric acid, and then 17.5 X 2 c.c. = 35 c.c. °f the nitrate of silver solution. This will pre- cipitate all the chlorides, which should be removed by filtration, and the filtrate may be now estimated for urea. It is important always to bear in mind the exact amount of urine operated with after adding the nitrate of silver solution to a mixture of baryta solution and urine, of which only two-thirds are urine. Thus, if 35 c.c. of the silver solution are added to 30 c.c. of the filtered mixture of urine and baryta fluid, of the resulting 65 c.c. only 20 would be urine minus the chlorine, or out of 32.5 c.c. 10 would be urine minus the chlorine. If the case be one of inflammation, as pneumonia, where there is a total or almost total absence of chlorides, they may be thrown out of the question altogether. PRACTICAL EXAMINATION OF THE URINE. If the undiluted urine under examination contains over 3 per cent, of urea, then on mixing it with the baryta fluid in the proportion of 10 c.c. of the former to 5 of the latter, the resulting mixture will contain over 2 per cent, of urea. Under these circumstances 15 c.c., or say one volume of the urine mix- ture, will require over 30 c.c., or two volumes, of the mercuric nitrate re- agent for complete precipitation of the urea. Consequently the excess of mercuric oxide originally present in the reagent for the purpose of acting upon the indicator (sodium carbonate) will be under a less degree of dilution than was present when the reagent was standardized ; and hence a portion of this excess will be consumed in the precipitation of urea, there still being left an excess of mercuric oxide equal to that originally present when the reagent was standardized, namely, 3.466 milligr. per c.c. of the mixture.* The quantity of the excess of mercuric oxide thus consumed by urea can be accurately compensated for by adding to the reading of the amount Correction for Varying Quantities of Urea. *0n standardizing used 20 c. c. reagent of 5.2 mgs. HgO excess per c. c. -f- 10 “ of 2 per cent, urea solution. = 30 “ mixture, So 5.2 X 20 = io4 in 30 “ “ = 104 -5- 30 = 3.466 per c.c. QUANTITATIVE CORRECTIONS. 161 of reagent employed o.l c.c. for every 4 c.c. of reagent required above 30 c.c. Thus if 34 c.c. of the reagent are required this should be read 34.1 c.c., which would indicate that the undiluted urine contained 3.41 per cent, of urea. On the other hand, should the undiluted urine contain less than 3 per cent, of urea, on mixing 10 c.c. of the urine with 5 c.c. of the baryta fluid, the resulting mixture will contain less than 2 per cent, of urea, and conse- quently 15 c.c. of the mixture will require less than 30 c.c. of the standard mercuric nitrate solution for complete precipitation of the urea. Under these conditions the normal excess of mercuric oxide in the re- agent for the purpose of acting upon the indicator will be under a greater degree of dilution than was present when the reagent was standardized; and hence a portion of the mercuric oxide intended for the precipitation of urea will be consumed as the indicator. Therefore, when less than 30. c.c. of the reagent are required for 15 c.c. of the urine mixture, deduct in the reading o.l c.c. for every 4 c.c. of reagent required less than 30 c.c. Thus, if 26 c.c. are required read 25.9 c.c., which would indicate that the undiluted urine contained 2.59 per cent, of urea. In other words, these corrections may be stated as follows : For every c.c. of reagent required above two volumes of reagent for one volume of the urine mixture, add 0.025 c.c. to the reading : whereas, for every c.c. of reagent less than two volumes of reagent for one volume of the urine mixture deduct 0.025 cc- fr°m the reading.* * Calculation explaining above correction : — 432 : 60. Precipitate —- 2 HgO : urea; Urea. HgO Urea. HgO Hence 60 : 432 : : 10 : 72 grams. And 72 HgO per liter. = 10 mgs. urea per c.c. But each c.c. of reagent contains :— 72 mgs. HgO for urea ; and 5.2 “ “ “ indicator. 77.2 72 mgs. Hg O per c.c. = 10 mgs. urea per c.c. The reagent when standardized contained 3.466 excess HgO per c.c. to act on the indicator. 162 PRACTICAL EXAMINATION OF THE URINE. Estimation of Urea by the Hypobromite Pro- cess.—The principle on which this process is based—that urea, when brought in contact with hypochlorite of cal- cium, is decomposed into nitrogen, carbonic anhydride, and water—was suggested many years ago by Davy,* and at the same time by LeConte.f In 1874, Messrs. Russell and West £ again directed attention to the subject, substi- tuting an alkaline solution of hypobromite of sodium and caustic soda, which yields similar products, the carbonic anhydride being absorbed by the caustic alkali. The fob lowing is the reaction :— CON2H4 + 3(NaBrO) = 3(NaBr) -f C02 -f 2H20 + N2; the volume of nitrogen disengaged being the measure of the urea. Many forms of apparatus have been suggested by different experimenters, all based upon the assumption that one gram of urea contains 372 c.c. nitrogen, measured at o° C. and 760 mm. barometric pressure ; or that each c.c. of nitrogen evolved, measured under the conditions stated, represents 0.00282 gram urea.§ Urine. BaO Mixt. Reagt. Hence if mix. 10 c.c. -f 5 c.c. + 40 c.c. = 55 c.c. mixt. 40-c.c. reagent contains 77.2 X 4° = 3088.00 HgO and 55 c.c. mixt. if 3.466 per c.c. = 3.466 X 55 = 190.63 “ Leaving for precipitating urea 2897.37 “ So if 10 mgs. urea per c.c. requires 72 HgO per c.c., then 2897.37 -5- 72 = 40.25 c.c. of 10 mgs. urea per c.c., and if excess of 10 over 30 = .25 excess, then “ “ 1 “ “ = .025 “ and “ “4 “ 30 = .1 “ * Philosoph. Mag., 1854, p. 345. f Comptes Rendus, xlvii, 237. X Journal of the Chetnical Society (London), August, 1874. \ See note to p. 165. HYPOBROMITE PROCESS FOR UREA. 163 The simple apparatus shown in Fig. 15, devised by Dr. John Marshall,* is that in use in the chemical laboratory of the University of Pennsylvania. It consists of a grad- uated measuring tube, a, a graduated pipette, c, a funnel tube, d, and a saucer-like apparatus, b, which serves as a Fig. 15.—Marshall’s Apparatus. stand and also as a receptacle for the hypobromite solution which in the operation overflows from the measuring tube. The measuring tube, including the bulbous part, holds about 77 c.c., and can, by means of a perforated cork, be fastened to the saucer-like vessel. The apparatus can * Zeit. f Physiolog. Chemie, Bd. xi, p. 179. 164 PRACTICAL EXAMINATION OF THE URINE. easily be taken apart and cleansed of the grease-like material which, in the performance of the hypobromite method, usually accumulates in the measuring tube. The alkaline hypobromite solution is made by dissolving ioo grams of caustic soda in 250 c.c. of water, and adding 25 c.c. of bromine to the solution thus produced. 2NaH0 -f Br.2 = NaBr -j- H.20 -f- NaBrO. Process.—The thumb is placed over the opening a, and the hypobromite solution is poured in through b. The latter open- ing, b, is then closed by a rubber stopper, and any air bubbles in the tube are allowed to pass out at the opening a. The gradu- ated tube is now reversed and the closed oval end of the tube is fastened in the opening in the saucer-like vessel. A measured quantity of urine (one cubic centimetre generally suffices) is allowed to pass from the pipette C into the hypobromite solution through the opening a. Decomposition of the urea immediately occurs, and the evolved nitrogen collects in the upper part of the graduated tube. The carbon dioxide evolved in the decomposi- tion of the urea is absorbed by the excess of caustic soda in the hypobromite solution. About 20 minutes are required for the com- plete decomposition of the urea. When the decomposition is com- pleted and all the gas bubbles have collected with the gas in the upper part of the tube, the atmospheric pressure is equalized by attaching the funnel tube (