1. If the urine gives no precipitate on boiling after acidulation, albumin and globulin are absent. If a precipitate occurs, albumin or globulin or both are present. 2. If the urine after neutralisation gives no precipitate on saturation with magnesium sulphate, globulin and hetero-proteose are absent. If such a precipitate occurs, one or other is present. 3. If the urine be saturated with ammonium sulphate and filtered, and the filtrate gives no xanthoproteic or biuret reaction (a large excess of potash must always be added), peptone is absent. 4. If the urine gives no precipitate on boiling after acidulation, no precipitate with nitric acid, and no precipitate on adding ammonium sulphate to saturation peptone can be the only proteid present. Confirm this by the biuret reaction. 5. If all proteids are present they may be separated as follows:-Saturate the urine (faintly acidified with acetic acid) with ammonium sulphate. A precipitate is produced. Filter. a. Precipitate b. Filtrate Contains peptone. Contains albumin, globulin, heteroand deutero-proteose. Collect the precipitate on a filter, wash it with saturated solution of ammonium sulphate, and redissolve it by adding a small quantity of water. To this solution add ten times its volume of alcohol; a precipitate is formed; collect this, and let it stand in absolute alcohol for from seven to fourteen days. Then filter off the alcohol, dry the precipitate at 40° C., extract it with water, and filter. An insoluble residue is left. a. Residue This consists of albumin and globulin coagulated by the alcohol. b. Extract This contains the proteoses in solution. Hetero-caseose is precipitated by heating the solution to 65° C., or by saturating a portion of the extract with magnesium sulphate. Deutero-proteose remains in solution. Take another portion of urine, neutralise it, and saturate with magnesium sulphate. A precipitate is produced. a. Precipitate This consists of globulin and heteroproteose, which may be separated by the prolonged use of alcohol, as above. Filter. b. Filtrate This contains albumin, deutero-proteose, and peptone. Add alcohol as above; albumin is rendered in seven to ten days insoluble in water. The deutero-proteose and peptone are soluble, and may then be separated by ammonium sulphate. THE URINE IN DIABETES Diabetes insipidus is a disease in which there is a very abundant secretion of very watery urine, and is probably dependent on a derangement of the vaso-motor nerves of the kidney or their centre. The disease which, however, we have now to deal with is called, in contradistinction to the above, diabetes mellitus, and is characterised by a very abundant secretion of urine with a high specific gravity 1 The quantity in the twenty-four hours rarely falls below two litres; it may rise to eight and even ten litres. (1030 to 1050), and containing dextrose. Sugar is present in both blood and urine normally in traces. The excess that occurs in this disease in both fluids, passing from the blood into the urine probably at the glomeruli, is due to a disordered state of the metabolic functions of the liver or of the muscles. Injury to the floor of the fourth ventricle produced artificially in animals (Bernard 2), or by disease in man, produces glycosuria, probably by interference with the centre of the vaso-motor nerves of the liver. Complete extirpation of the pancreas also produces glycosuria (see p. 663). Transitory glycosuria (sugar in the urine) may occur in cholera, ague, cerebro-spinal meningitis, liver cirrhosis, and gout. Certain poisons (morphia, curare, chloroform, &c.) were said formerly to produce glycosuria; we now know that the substance in the urine in these cases which reduces Fehling's solution is not sugar, but glycuronic acid. The presence of other substances in the urine besides sugar that reduce Fehling's solution is a great source of error when only a small amount of reduction occurs: these substances are uric acid, hippuric acid, creatinine, pyrocatechin, and glycuronic acid. The fermentation test is the best means of distinguishing sugar from these other bodies. Sugar alone is transformed into alcohol and carbonic acid under the influence of yeast. In addition to dextrose, urine may in this disease contain small quantities of inosite (p. 100), of levulose,3 and in a few cases glycogen itself has been found (Leube 1). Milk sugar, which occurs often in the urine of nursing mothers, is also apt to be mistaken for grape sugar. It undergoes the alcoholic fermentation slowly, and can only be distinguished with certainty from grape sugar by separating it out from the urine and examining its properties (see p. 757). In these cases, however, the symptoms of diabetes are absent. The quantity of grape sugar in diabetic urine is, as a rule, over 4 per cent., rising to 7, 9, or even 12 per cent. The quantity varies considerably with the diet; carbohydrate diet increases it; the withdrawal of carbohydrate food, especially if combined with the adminis 1 The reader will find an account of recent researches on this question in Bunge's Physiol. Chem. translated by Wooldridge. It, however, appears to me that Bunge is inclined to lay too much stress on the muscular glycogen as a source of diabetic sugar. See also pp. 314 and 540 et seq. of this book. Phloridzin diabetes referred to on p. 544 has been the subject of several researches since that page was written. The chief memoirs on the subject are: v. Mering, Verhandl. V. and VI., Cong. inn. Med. Zeit. klin. Med. xiv. 415, xvi. 431. Moritz and Prausnitz, Zeit. Biol. xxvii. 81; Külz and Wright, Ibid. p. 181. Leçons, Paris, 1855, pp. 288, 355. 5 For references see p. 120; to these may be added, Külz, 'On lævorotatory sugar in urine,' Zeit. Biol. xxvii. 228. 4 Virchow's Archiv, cxiii. 391. tration of morphia or codria, diminishes it, or may even cause the entire disappearance of sugar from the urine. In the most severe form of diabetes, however, both drugs and dieting are useless. The properties and reactions of dextrose have been already fully dealt with in Chapter IX; it is, therefore, only necessary here to briefly recapitulate those tests which are most suitable for its detection in urine. 1. The copper test.-Boiling witn Fehling's solution (which should itself be previously boiled to ensure that no reduction occurs in it without admixture with urine) produces a yellow or red precipitate of the cuprous hydrate or oxide. Trommer's test (the addition of copper sulphate to the urine followed by caustic potash) does not in my experience answer so well.' Fehling's solution in solid form may be applied to urine by means of Pavy's test-pellets, and Dr. Oliver has introduced cupric test-paper. 2. Bismuth test.-This consists in the black precipitate of metallic bismuth that occurs on boiling an alkaline solution of bismuth nitrate with the urine. For this purpose Bottger's solution (bismuth subnitrate 5 grammes, tartaric acid 5 grammes, 30 c.c. distilled water, and strong caustic soda added carefully till a clear solution is obtained), or Nylander's reagent (bismuth subnitrate 2 grammes. sodio-potassium tartrate 4 grammes, liquor sodæ 55 c.c., distilled water 47 c.c.) may be employed. As albuminous materials in the urine may also produce a black precipitate from the formation of bismuth sulphide, Brücke recommends the following method: Fröhn's reagent is made as follows: freshly precipitated bismuth subnitrate, 15 gramme, and 20 c.c. water are heated to boiling, and 7 grammes potassium iodide and 20 drops of hydrochloric acid are then added. Equal quantities of urine and water are put into two test-tubes; hydrochloric acid is added to the water till Fröhn's reagent no longer produces cloudiness. In this way the necessary quantity of hydrochloric acid is ascertained, and this quantity is added to the urine; the reagent is then added and the mixture filtered. The filtrate should not now become cloudy on adding either hydrochloric acid or the reagent. It is boiled with excess of caustic soda or potash; if a grey or black colour results sugar is present. 3. Moore's test. The urine containing sugar is heated with caustic potash solution, and the mixture becomes yellow, then brown. 4. Picric acid test.3-Heat the urine with a few drops of concentrated solution of picric acid, or a little solid picric acid may be used. Add caustic potash, and a brown red colour, due to puramic acid, is obtained. 5. Fermentation test.-A test-tube is half filled with the urine, and a little German yeast added; the tube is filled up with, and inverted over mercury, and left in a warm place for twenty-four hours. Carbonic acid collects in the tube, and may be tested for in two ways: (1) it is wholly absorbed by strong caustic potash solution; (2) it gives a white cloudy precipitate with lime or baryta-water. The liquid gives the tests for alcohol. A control experiment should be made in another test tube with yeast and 1 See also Lauder Brunton, 'Non-precipitation of Cuprous Oxide in certain cases of Diabetic Urine' (Bartholomew's Hosp. Reports, xvi. 235); a yellow solution instead of a yellow precipitate occasionally forms, the cuprous oxide being apparently held in solution by certain urinary constituents. 2 Wien. Akad. Sitzungsber. vol. lxii. 1875, 2. Abth. 3 G. Johnson, Lancet, November 18, 1882. water, as a small yield of carbonic acid is often obtained from impurities in the yeast. Another method of performing the test is to connect a flask containing the urine and yeast by a bent glass tube with another vessel containing lime-water; the gas passes into the second flask as it comes off, and gives a precipitate of calcium carbonate with the lime-water. In order to separate grape sugar from the urine the latter is evaporated to a syrup on the water-bath, and the residue extracted with absolute alcohol; evaporate the extract to dryness, and once more extract with absolute alcohol; add to this extract a solution of potash in 80 per cent. alcohol; a precipitate forms which after pouring off the alcohol is dissolved in water, neutralised with acetic acid, and precipitated with lead acetate, filtered, and the filtrate treated with sulphuretted hydrogen; the lead sulphide is removed by filtration, and the filtrate contains the grape sugar, which crystallises out on evaporation, and may be purified by recrystallisation (Salkowski and Leube). Another method consists in precipitating the urine with normal lead acetate, filtering, and treating the filtrate with basic lead acetate and ammonia. This precipitates the sugar; the precipitate suspended in water is decomposed with sulphuretted hydrogen and filtered. The filtrate is evaporated, and grape sugar crystallises out (Brücke). Hydroxybutyric acid in the urine.-In addition to grape sugar, diabetic urine may contain other substances. One of these is an acid first discovered in the urine by Minkowski,' which he found to be identical with the B-hydroxybutyric acid of Wislicenus, but differing from it in being optically active; (a)p = −23·4. Such acids are poisonous when introduced into the circulation. Its quantity in the urine is variable; it often occurs in the urine when acetone also is present, and diacetic acid, a substance allied to acetone, can be formed from it. Diacetic acid may also be found in the urine of diabetics; its quantity and that of the hydroxybutyric acid vary in a parallel degree (Wolpe 2). The presence of B-hydroxybutyric acid in diabetic urine has been also observed by Külz3 and by Stadelmann,' who give methods for its separation from urine, but we are not at present acquainted with the clinical significance of its appearance there. Acetone and ethyl-diacetic acid in the urine."-The appearance of these substances in the urine is full of clinical importance. Acetone, or dimethyl ketone (C,H,O), has been prepared artificially, and its chemical relationship to the alcohols has already been described (p. 66). It may be detected by the following tests: (1) In the pure state it forms, with excess of concentrated aqueous solution of sodium bisulphite, a crystalline compound (acetone + sodium bisulphite) which separates out in shining scales. (2) Lieben's iodoform test, as modified by Ralfe, may be used to detect acetone in urine: 20 grains of potassium iodide are dissolved in a drachm of liquor potassæ and boiled; the urine is then carefully floated on to its surface in a test-tube. At the point of contact a precipitation of phosphates occurs, which, if acetone is present, becomes yellow and studded with yellow points of iodoform. This test is much better obtained by distilling a small quantity of urine and applying it to 1 Arch. exp. Path. u. Pharmak. xviii. 41. 3 Zeit. Biol. xxiii. 329. 2 Chem. Centralbl. 1887, p. 277. I am indebted for many of my references on this subject to a chapter with the above heading in MacMunn's Clinical Chemistry of Urine. the distillate. This test has the disadvantage that lactic acid and ethyl alcohol behave similarly. (3) Le Nobel's test.'--On ad ding an alkaline solution of sodium nitro-prusside, so dilute as to have only a slight red tint, to a fluid containing acetone, a rubyred colour is produced, which in a few moments changes to yellow, and on boiling, after adding acid, to greenish blue or violet. A quarter of a milligramme of acetone can be thus detected. (4) Chautard's test.-A drop of aqueous solution of magenta decolourised by sulphurous acid gives with fluids containing over 0:01 per cent. of acetone a violet colour. This appears in dilute solutions after the lapse of four or five minutes. (5) Baeyer and Drewsen's indigo test.-A few crystals of nitro-benzaldehyde are dissolved by heat in the fluid suspected to contain acetone; on cooling, the aldehyde separates as a white cloud. The mixture is then made alkaline with dilute soda, and if acetone be present, first yellow, then green, followed by an indigo blue colour, appear within ten minutes. In cases where only traces of acetone are present, large amounts of urine (50 litres) are acidulated with sulphuric acid, and submitted to fractional distillation, the lighter volatile part being collected. Acetone in the distillate is recognised by its boiling point (56° to 58° C.), specific gravity (0-814 at 0° C.), odour, and the reactions just mentioned. Alcohol, which is sometimes present in the urine with acetone, may also distil over. To separate them the residue is treated with fused calcium chloride in excess, and distilled on the water-bath; the distillate treated with calcium chloride and again distilled; the process may be again repeated; acetone distils over. Alcohol remains with the calcium chloride residues, which give it off by distillation over the free flame. It may be detected by the indoform reaction and the formation of aldehyde and acetic acid on oxidation (Salkowski and Leube). Ethyldiacetic acid (CHO) strikes a Bordeaux red colour with a solution of ferric chloride; it has often been confused with and mistaken for acetone; but the reaction with ferric chloride distinguishes them, and from this test its presence in urine is generally inferred. Under the influence of alkalis it takes up water and splits into acetone, alcohol, and carbonic acid (CH10O+H2O=C2H¿O+C2H ̧O + CO2).' If this occurs in the blood (for Acetonæmia, see p. 314) or urine it is probably the sodium salt, which undergoes a similar decomposition. CH,NaO, +2H2O = C2H ̧O + C2H2O + NaHCO This view of the origin of acetone was supported by the fact that alcohol was found in the urine with it. Some observers, however, have noted certain facts which bear against this theory of the origin of acetone. There are other substances that may occur in the urine which give the ferric chloride reaction, namely, B-hydroxybutyric acid, sulpho- (thio-) cyanates, acetic acid, and formic acid; and, according to Legal, the urine of patients who have taken thalline, antipyrine, salicylic, and carbolic acids may also give it. If, however, the urine is 1 Chem. Centralbl. 1884, p. 626. Legal's test, Journ. Pharm. (5), xviii. 206, is almost the same as Le Nobel's. 2 Bull. soc. chim. xlv. 83. 3 This was first noted in urine by Gerhardt, Wien. med. Presse, 1871, No. 1. 4 Rupstein, Centralbl. med. Wiss. 1874, No. 55. 3 Loc. cit. |