352 ABSTRACTS OF CHEMICAL PAPERS BACTERIOLOGICAL, PHYSIOLOGICAL, ETC. Estimation of Small Quantities of Bilirubin. E. Herzfeld. (Zeitsch. physiol. Chem., 1912, 77, 280-284.) - When an alcoholic solution of bilirubin is treated with a few drops of a solution of p-dimethylaminobenzaldehyde (prepared by dissolving 20 grms. of this substance in 500 C.C. of hydrochloric acid and adding 500 C.C. of water) a green coloration is produced, the intensity of which is propor- tional to the amount of bilirubin present, and this amount may be estimated byBACTERIOLOGICAL, PHYSIOLOGICAL, ETC.353 examining the mixture with the spectrophotometer. For the estimation of bilirubin in blood, 20 C.C. of the latter are mixed in a mortar with absolute alcohol, then transferred to a 100 C.C.flask, diluted to about 75 C.C. with alcohol, and 2 C.C. of the reagent are added. The mixture is heated on a water-bath until the green coloration has developed, then cooled, and diluted with alcohol to the mark. The solution is now examined with the spectrophotometer. The quantity of bilirubin, x, is calculated from the extinction coefficient, e, observed, according to the formulae : x = 0.00132.e e w. P. s. Mechanism of Coagulation. J. Duclaux. (Comptes rend., 1912, 154,1426- 1429.)-A theory of coagulation by osmotic action is based upon the following hypotheses : (1) The osmotic pressure of a solution of several substances is the sum of the partial osmotic pressures ; and (2) in a state of equilibrium the relative propor- tions of different substances that a membrane will allow to pass will be the same on each side of the membrane, the only differences being in the proportions of substances that cannot pass through the membrane.If, for example, to alcohol containing one substance in solution a second soluble substance be added, the alteration in the osmotic pressure will depend upon the size of the molecule of the added substance.If the molecules are very small, the increase of volume caused by their addition will be negligible ; whereas, if they are large, there will be considerable increase in volume and a corresponding decrease in the pressure. If, further, two vessels, A and B, are charged with the same alcoholic solution, and separated by a membrane porous to water and alcohol, and a substance capable of reducing the osmotic pressure in B and of not traversing the membrane, be added, water will pass from B into A.But since, according to the second hypothesis, the relative proportions of water and alcohol must always be the same in A and B to obfain equilibrium, alcohol must also pass from B into A in such quantity that the composition of the liquid in A remains constant in so far as concerns the relative proportions of water and alcohol.But under such conditions the osmotic pressure in B muet remain lower than in A , and consequently the osmosis will only cease when the whole of the original liquid in B has passed through the membrane into A. The added substance will separate spon- taneously. If the original solution contained one grm.-molecule per litre, it is necessary that the grm.-molecule of the added substance should occupy in solution a volume greater than 1 litre before such conditions of osmosis are brought about ; and this is usually the case with colloidal substances.In colloidal solutions the place of the porous membrane is assumed to be taken by the free space between the particles of the colloidal substance, and this space will be indefinitely enlarged by the force of osmotic action, until separation of the colloid occurs spontaneously. C.A. M. Studies on Enzyme Action. 11. Hydrolytic Action of Some Amino-Acids and Polypeptides on Certain Esters. K. G. Falk and J. M. Nelson. (J. Amer. Chein. SOL, 1912, 34, 828-845.)-The authors suggest as a tentative hypothesis that354 ABSTRACTS OF CHEMICAL PAPERS the hydrolytic action of lipase is due to an optically active substance of protein character, readily hydrolysed in aqueous solution to form lipolytically inactive Substances.The characteristic groups of protein substances are the amino and carboxyl groups, free or combined. The possible hydrolytic actions of some simple amino-acids and polypeptides were therefore studied by observing the quantity of acid produced at 38" C., when methyl acetate, ethyl butyrate, and olive oil were dissolved or suspended in aqueous solutions containing glycin, alanin, phenylalanin, leucin, glycylgl ycin, leucylglycin, glycylleucin, diglycylgl ycin, aspartic acid, and glutamic acid.Three sets of measurements were necessary for each experiment : with solutions containing amino-acid or polypeptide alone, ester alone, and with a mixture containing botb.Sorensen's formol method (ANALYST, 1908, 33, 19) was used for the estimation of amino-acids and polypeptides, except that Tc soda was used for the titrations in place of p; phenolphthalein was used as indicator through- out, most of the end-points being a distinct pink colour, and not the definite red recommended by Sorensen.Many hundreds of determinations are tabulated, and there was found to be a satisfactory agreement between the results obtained and those already published by Sorensen, in so far as the work covered the same ground. Glycin and alanin showed the greatest amount of action with ethyl butyrate, and least with methyl acetate. Phenylalanin showed a markedly greater action with methyl acetate, less with ethyl butyrate, and least with olive oil.Leucin gave practically no action with the three esters. Glycylglycin gave the same slight action with methyl acetate and ethyl butyrate, but none with olive oil. With leucylglycin, glycylleucin, and diglycylglycin, maximum, though small, actions were obtained with ethyl butyrate, very slight but distinct with olive oil, while with glycylleucin and diglycylglycin and methyl acetate, negative values were obtained.With aspartic and glutamic acids the order of magnitude of action was methyl acetate, methyl butyrate, olive oil, considerably greater action being caused by the former, as was to be expected from the greater ionisation constant of the former.The selective action with different esters is strongly suggestive of the selective action of lipases from different sources with different esters. I t seems probable that many of these selective actions of the lipases may be reproduced with amino-acids and polypeptides of varying structure or in the presence of other substances. There is no evidence, however, that the hydrolytic action of lipase is to be attributed to amino- acids or polypeptides.The specific groupings present in the amino-acids or polypeptides which show this activity may be present in more complex substances, such as the proteins, and from this point of view the stirdy of the hydrolytic actions of the decomposition products, such as the amino-acids from preparations possessing lipolytic activity, and of the more complex polypeptides or other substances synthesised from them, may throw light upon the substances capable of causing such lipolytic act ion.H. F. E. H. Kumagawa-Suto Method of Estimating Fat in Animal Substances. R. Watanabe. (Biochem. Zeitsch., 1912,41,71-77.)-As the result of a, critical exami- nation of this method (ANALYST, 1908, 33, 362), the author finds that it is quite reliable for the direct estimation of fat in the following animal substances withoutBACTERIOLOGICAL, PHYSIOLOGICAL, ETC.355 drying the latter before saponification : Flesh, heart, liver, pancreas, kidneys, intestines, bones, skin, ascitic and pleural fluids, and frogs. In the case of blood, defibrinated blood, blood-plasma, blood-serum, and brains, better results are obtained when the substances are extracted with alcohol, and the extract used for the estimation of the fat (cf.ANALYST, 1910, 35, 527). Suprarenal glands yielded, after saponification, 27 per cent. of matter soluble in petroleum spirit, but more than one-half of this consisted of unsaponifiable substances. w. P. s. Loss of Fat during the Drying of Animal Tissues.M. Tamura. (Biochem. Zeitsch., 1912, 41, 78-lOl.)-Shimidzu has already drawn attention to the fact that an apparent loss of fat takes place when animal tissues are dried before the fat is estimated by the Kumagawa-Suto process (cf. ANALYST, 1910, 35, 527), and this is confirmed by the result of an extended investigation carried out by the author. If, however, the substance be dried as quickly as possible in quantities of from 100 to 200 grms., with the addition of an equal quantity of alcohol, the loss of fat due to oxidation does not usually exceed 0.5 per cent.of the total quantity of fat present. w. P. s. Blue Colour-Reaction of Phosphotungstic Acid with Uric Acid and Other Substances. 0. Folin and B. Macallum. (J. Biol. Chem. 1912,11, 265; through Chenz.Zentralb., 1912, I, 1928.)-Phosphotungstic acid in presence of saturated sodium carbonate solution gives a blue coloration not only with uric acid, but also with phenol, tyrosin, tannic acid, thymol, orcinol, resorcinol, vanillin, and phloro- glucinol. The reaction appears to be characteristic of aromatic compounds containing a hydroxyl-group in thep-position. Moreover, it appears to be due to an impurity in the phosphotungstic acid, and not to the acid itself.0. E. M. Refractive Indices of Solutions of Certain Proteins. VI. The Proteins of Ox Serum: a New Optical Method for the Estimation of the Various Proteins in Ox Serum. T. B. Robertson. (J. Biol. Chem. 1912, 11, 179; through Chem. Zeiztmlbl. 1912, I, 1929.)-The alteration a in the refractive index of a solvent produced by dissolving in it 1 grm.of protein was the same, whether the proteins were dissolved in the native serum, or whether they were precipitated with alcohol, washed with alcohol and ether, dried, and dissolved in & potassium hydroxide solution. The value of a was also independent of the dilution, and was not altered when the serum was made acid.For the proteins of ox serum the value of a is 0.00195. The refractive power of the mixed proteins in ox serum represents the sum of the refractive powers of tho individual proteins. For refractometric purposes the non-proteins of the serum may be regarded as a &-grm. molecular sodium chloride solution. The value of a for the albumins of ox serum dissolved in Q saturated or weaker ammonium sulphate solution is identical with the value for solutions in distilled water : it is 0.00177.The discovery of the additive character of the refractive indices rendered it possible to modify the method of Reiss so that the individual proteins hitherto identified in blood serum could be estimated356 ABSTRACTS OF CHEMICAL PAPERS separately. insoluble globulins, and 5.4 per cent.total albumins. Ox serum was found to contain 2.34 per cent. soluble globulins, 0.76 0. E. M. Estimation of Sugar in Blood and Urine. E. Frank. (Zeitsch. phzysiol. Chem., 1912,78,165-166.)-The author replies to criticisms of his method for the estimation of sugar in blood (ANALYST, 1910, 35, 260). w. P. s. Estimation of Benzoic Acid, Hippuric Acid, and Phenaceturic Acid in Urine.H. Steenbock. ( J . Biol. Chem., 1912, 11, 201; through Chem. Zentralbl., 1912, I , 1930.)-For the estimation of hippuric acid 100 C.C. of urine are boiled for two hours with 10 grms. sodium hydroxide and 25 C.C. of hydrogen peroxide ; the hippuric acid is thus converted into benzoic acid, and the colouring matter of the urine is oxidised. The liquid is rendered acid with sulphuric acid, bromine water is added and the liquid made up to 200 C.C.and filtered; 50 C.C. of the filtrate are extracted with ether, the ether evaporated off, and the residue sublimed and weighed. To estimate the phenaceturic acid, the sublimate is titrated with & sodium hydri- oxide, and the proportion of the benzoic acid to the phenylacetic acid formed from the phenaceturic acid calculated from this titration and the weight of the dry sublimate.0. E. M. New Method for the Estimation of Hippuric Acid in Urine. 0. Folin and F. F. Flanders. (J. Biol. Chem., 1912,11, 257; through Chem. Zentralbl, 1912, I, 1930.)-A mixture of 100 C.C. urine and 10 C.C. of 5 per cent. sodium hydroxide solution is evaporated to dryness on the water-bath ; the hippuric acid in the urine is thus converted into benzoic acid.The residue is transferred by means of 25 C.C. of water and 25 C.C. of concentrated nitric acid to a flask fitted with a reflux condenser, and boiled for four and a half hours with 0-2 grm. copper nitrate to destroy colouring matter and similar substances in the urine. The contents of the flask are saturated with ammonium sulphate in a separating funnel, and extracted with chloroform, and the united chloroform extracts washed once by shaking with a solution of sodium chloride containing hydrochloric acid, and the benzoic acid in the chloroform extract titrated with TG sodium ethoxide and phenolphthalein. 0.E. M. Estimation of Phenol in Urine. M. Hensel. (Zeitsch. physioZ. Chem., 1912, 78, 371-381.)-The method proposed depends on the fact that phenol is dis- solved from its acid aqueous solution by ether, and that it can be extracted from the ethereal solution by means of sodium hydroxide, but not by sodium hydrogen carbonate.Five hundred C.C. of the urine are rendered alkaline and evaporated to 100 c.c., the solution being then acidified with phosphoric acid and distilled with frequent additions of water, until all the phenol has passed over, as is shown by the distillate ceasing to give a reaction with Millon’s reagent.The distillate is then extracted four successive times with ether, the united ethereal extracts are shaken four times with a 4 per cent. sodium hydrogen carbonate solution, and afterwards the same number of times with The sodium hydroxide extracts are united, acid added until the alkalinity of the solution is equivalent to sodium hydroxide solution.BACTERIOLOGICAL, PHYSIOLOGICAL, ETC.357 20 C.C. of & solution, and the phenol in the alkaline solution is titrated by Penny and Kossler's method. The method yields trustworthy results in the case of urines containing large quantities of carbohydrates. A method described by Mooser (Zeitsch.physiol. Chem., 1908, 63, 155), in which the distillate from the urine, acidified with phosphoric acid, is redistilled from calcium carbonate in a current of carbon dioxide, and the phenol titrated in this distillate, was found to yield accurate results. w. P. s. Estimation of Urea in Urine by Riegler's Method. A Correetion Factor.T. Ekeerantz and S. Eriekson. (Zeitsch. physzol. Chem., 1912,79,171-176.)-1n the estimation of urea in urine by Riegler's method (c.f. ANALYST, 1912, loo), substances other than urea, such as uric acid, purine bases, creatinine, neucleo-albumin, etc., are decomposed and yield carbon dioxide and nitrogen, and the results are, consequently, too high. Those substances which interfere may be removed by treating the urine with twice its volume of a solution containing 10 per cent. of phosphotungstic acid and 1 per cent.of hydrochloric acid of sp. gr. 1.12, and removing the precipitate by filtration after the lapse of four hours; the urea is then estimated in the filtrate. In most cases, however, sufficiently accurate results for all practical purposes may be obtained by treating the urine directly and multiplying the result obtained by 0.926 in order to correct for the amount of nitrogen liberated by the substances other than urea.w. P. s. Detection of Savin Oil in Toxicological Cases. J. Hamalainen. (Biochem. Zeitsch., 1912,41, 241-246.)-Savin oil contains about 50 per cent. of sabinol acetate, which constitutes the toxic principle of the oil; this acetate on hydrolysis yields sabinol, which, in the body, combines in part with glycuronic acid, and is excreted in the urine as sabinol-glycuronic acid.When the latter is boiled with a, mineral acid a cymene is formed, and the identification of the latter has been suggested as a means of detecting savin oil in urine. Other oils, however, such as turpentine, thuja oil, etc., when administered to animals, pass into the urine in the form of derivatives which yield cymene.Sabinol-glycuronic acid may be identified by means of the characteristic crystalline salts it yields with certain bases, particularly with strychnine, and the following method is described by the author for the detection of savin oil in cases of suspected poisoning by this substance : The urine is treated with neutral lead acetate in neutral or feebly acid solution, the precipitate formed is separated by filtration and washed, and, after the filtrate has.been rendered ammoniacal, basic lead acetate is added as long as a precipitate is produced. The basic lead salt is collected on a filter, washed with water, decomposed with cold 5 per cent, sulphuric acid, the lead sulphate is separated, and the solution neutralised with barium carbonate.After filtration, the filtrate is concentrated, preferably under reduced pressure, and the barium is precipitated by treating the warm solution with hot strychnine sulphate solution. The mixture is filtered while hot, and the filtrate allowed to cool. Strychnine sabinol-glycuronate separates out in the form of brilliant needles which melt a t 196" C. with decomposition.This salt has 2 molecules of water of crystallisation; it is fairly soluble in hot water, and but358 ABSTRACTS OF CHEMICAL PAPERS slightly soluble in cold water, alcohol, ether, acetone, chloroform, benzene, petroleum spirit, or ethyl acetate. On concentrating the mother-liquor, a strychnine salt with 3 molecules of water crystallises out, melting at 193" C.It is more readily soluble than the first-mentioned salt, and on cooling its solution rapidly, the salt with 2 molecules of water is obtained. Urine from rabbits, which had received a dose each of 1.5 C.C. of savin oil, yielded crystals of strychnine sabinol-glycuronate when examined by the above method. w. P. s. Microchemical Reaction of Spermatic Fluid.P. Aleixandre. (Zeitsch. anal. Chem., 1912, 51, 473-475.)-A reaction proposed by De Dominicis (Resveglio Medico, May 15, 1910) consists in treating a trace of the fluid with a drop of a saturated gold tribromide solution on a microscope slide, heating the mixture to boiling, and examining it under the microscope. At a magnification of 300 diameters rectangular or cross-shaped crystals, having a garnet colour, are observed. The crystals are soluble in alcohol and in alkali solutions, but not in acids. The author has examined this test, and finds that it is not very sensitive; the crystals are rarely obtained unless the spermatic fluid is of a concentration of more than 2 per cent.Quadratic crystals are also frequently obtained, and are due to the presence of cholin.w. P. s. Colorimetric Method for the Estimation of Uric Acid. E. Riegler. (Zeitsch. anal. Chem., 1912, 51, 466-470.)-The method depends on the blue colora- tion which is obtained when uric acid is treated with phosphomolybdic acid and disodium phosphate. The coloration is not given by albumin, psptones, albumoses, creatine, creatinine, or sugar, although these substances yield a similar colour when treated with phosphomolybdic acid and potassium or sodium hydroxides.For the estimation of uric acid in urine, 1 C.C. of the sample is placed in a test-tube, and in two similar tubes are placed, respectively, 1 C.C. of a 0.1 per cent. uric acid solution and 1.2 C.C. of the same uriue from which the uric acid has been removed by means of ammonium chloride.(This iis effected by heating 10 C.C. of the urine with 3 grms. of ammonium chloride to 40" C,, and filtering the mixture after the lapse of thirty minutes; 1.2 C.C. of the filtrate is employed, in order to allow for the increase of volume due to the ammonium chloride.) To each of the iubes are then added 2 C.C. of a 10 per cent. phosphomolybdic acid solution, and a sufficient quantity of a 5 per cent. disodium phosphate solution to make the contents of each tube up to 10 c.c.; a mark may be provided on the tubes for this purpose. After the contents of the tubes have been mixed, they are heated over a flame until small bubbles of gas are evolved, then cooled, and the colorations compared in a colorimeter. The coloration yielded by the urine is first compared with that given by the known quantity of uric acid. The apparent quantity of uric acid in the 1.2 C.C. of urine from which the uric acid has been removed is also estimated by comparison with the standard, and its quantity deducted from the amount found in the urine itself. This correction has to be made, as urine contains substances other than uric acid, which give a coloration with the reagents. Should the urine contain albumin, this must be separated by boiling the urine and filtering it before the estimation is carried out. The standardORGANIC ANALYSIS 359 solution of uric acid is prepared by boiling 0.1 grm. of uric acid and 0.1 grm. of sodium hydrogen carbonate with 50 C.C. of water, cooling the solution, and diluting it with water to a volume of 100 C.C. w. P. s.