510 On Urobilin from Bilirubin and Biliverdin. tympani permanently increases in area or relaxes under the atmos- pheric pressure, so it shortens permanently under the long-continued contraction of collodion. I have, perhaps, in the opinion of some, entered upon this subject too fully. I do not think so, however. I have endeavoured to avoid a mistake too common in medicine and surgery--of hastily con- cluding that a certain effect was owing to a particular cause, when it might be really owing to another cause or combination of causes. I am the more particular in dealing with this question in a strictly logical way, as I desire that a method of treatment which has been so successful in my hands should be adopted by my professional brethren, not simply on my representations, but because it is rational. ART. XXIV.--0n Urobilin and its production from Bilirubin and Biliverdin. By C. A. MACMU~N, B.A., M.D., Univ. Dubl. ; Wolverhampton. ACCORDING to M'Kendrick's Physiology,a urobilin and indican are the principal urinary pigments. I have shown elsewhere b that urobilin is constantly present in healthy human urine, and so far as the researches which I am about to describe have gone, I have no reason to change this opinion. Urobilin is, therefore, a pigment of very great importance to the physician, and the study of its chemical and optical characters, and of its parentage, if I may be allowed to use this term, will help us to form more accurate ideas as to the value which its absence from, or its increase in, urine may possess. Moreover, as I shall endeavour to explain, the urobilin of health differs from urobilin excreted in some diseases in certain particulars, the reason of which, I think, can be explained. Method of obtaining Urobilin from Urine.--In a paper read before the Royal Society,c I have described a method for the separation of urobilin from urine, which was arrived at after many unsuccess- ful attempts had been made to isolate urobilin by other methods. The urine is precipitated with neutral and basic acetate of lead, and filtered--the precipitate in the filter is washed with water; it is then extracted with alcohol acidulated with sulphuric or hydro- AOutlines of Physiology. 1878. b Spectroscope in Medicine. 1880. Proceedings Royal Society,No. 202. 1880. [By DR. MAcMuNN. 511 chlorie acid, and again filtered. The filtrate, in small quantities at a time, is put into a separating funnel, a large quantity of water added, and then pure chloroform; the whole is repeatedly shaken, and then allowed to stand. The red chloroform layer is separated off and filtered, the chloroform driven off, and the residue repeat- edly dissolved in chlorofbrm, or preferably in absolute alcohol; finally, on evaporation we obtain a brown-red, amorphous, shiny residue, which is perfectly soluble in alcohol, chloroform, certain acids, and acidulated water, partially soluble in ether, in water, and in benzol, but quite insoluble in bisulphide of carbon. Uro- bilin is found, when thus prepared, to contain carbon, hydrogen, oxygen, and nitrogen. Its various solutions all show a black band at F. This band can be made to disappear by adding to these solutions ammonia in excess, and is replaced by another band nearer the red end of the spectrum on the addition of caustic soda or caustic potash--in fact, the solutions of urobilin thus obtained behave, as regards their spectra, in the same manner as urine itself which contains enough urobilin to allow of thin layers of it to be examined satisfactorily by means of the spectroscope. If a solution of urobilin be first treated with caustic soda, and then with ammonia, the band nearer the red produced by the caustic soda will not disappear. The pigment itself gets redder when exposed to the air. I have not examined its solutions for fluorescence s on the addition of chloride of zinc, but the characters given above are quite sufficient to enable the pigment to be diagnosed from others giving a band in the same part of the spectrum. The urobilin which I obtained from the urine of a case of phthisis, in which large portions of the lungs had been destroyed, presented in deep layers of its chloro- tbrmie and alcoholic solutions a feeble band on each side of D, in addition to the band at F, It was suggested to me by a well- known physiological chemist that these two bands indicated an impurity in the pigment obtained--indeed he suggested that, by the adoption of my method, "omicholine" had been separated--so I set to work to find out the cause of the appearance of these bands. The result was very interesting. I found not only that these bands were not due to the presence of an impurity, but that Hoppe-Seyler. Handb. d. Phy. und Path. Chem. Analy. 4th Edition. 1875. Urobilin contains, according to Ma]y, C3~H40N407 ; larobably it will be found nearer Ci6HlsN~O 6 or O e (?) 512 On Urobilin from Bilirubin and Biliverdin. the urobilin .found in this case of phthisis had not been completely oxidised in the body of the man from whom it was obtained. This statement may appear startling to those who believe that urobilin is produced by reduction, a and, on the other hand, to those who believe, with Rokitansky, that the blood is hyperoxidised in phthisis; but the experiments which I am about to relate will, I am sure, eonvinee the one, and the fact that half the lung- substance was destroyed will convince the other, that the deduction is legitimate. ~lT~e Spectrum of Gmelin's Reaction.--The well-known play of eolours which takes place when bile-pigment is treated with nitric acid, is accompanied by a series of alterations in the spectrum,b which may be thus summed up:--A black band at :F, and two bands on each side of D, at an intermediate stage of oxidation, but only the band at ]~" at the completion of the oxidation process. Therefore, the pigment which gives the bands near D, as well as that at :F, is produced by a less complete oxidation than that which gives the band at :F alone. We can isolate the pigment which gives the band at F. Thus I have isolated it by the following method :-- A chloroformie solution of bilirubin, which had become partially changed into biliverdin by exposure to the air, was evaporated over the water-bath; the residue, dissolved in water, was treated with nitric acid, and this solution was observed with the spectro- scope until the bands on each side of D disappeared, and that at F only was left. It was then put into a separating funnel, and shaken with chloroform; the chloroformic solution evaporated down, and after solution in chloroform and filtering, it was again evaporated over the water-bath. The pigment thus obtained was similar in every respect to the urobilin which I have got by the method mentioned above from healthy urine, the same solvents dissolved it, and re- agents produced the same changes in the respective spectra of its solutions. The urobilin obtained from healthy chrome-yellow urine gives, in solution, only a band at F, which disappears on adding ammorrla, and which is replaced by another band nearer the red when treated with excess of caustic soda; so also did this pigment. The best way of studying the spectrum of Gmelin's reaction is to shake up human bile with chloroform, and act upon the chloro- formic solution of the pigment in "t watch-glass with nitric acid. When this method is adopted, and the solution examined spectro- i Hoppe-Seyler. Loc. eit. b Spectroscopein Medicine. Chart 1L, Sp. 5. By DR. MACMUNN. 513 scoplcally, we'see, at a certain stage of the reaction, three bands-- a dark one at F, and two others, one on each side of D; and these bands are identical, not only in position, but also as regards their shading, with those got when the urobilin of the case of phthisis was dissolved in chloroform, and treated in the same way. Hydrochloric acid acts in a similar manner on the spectrum of a chloroformic solution of bile-pigment, but sulphuric acid produces an orange-red coloured fluid, which gives two bands between D and :F, and in deep layers another band on the red side of D ; this spectrum is remarkably like that which is fbund when a hot alcoholic solution of the brick-red urates of rheumatic fever is examined, and it also somewhat resembles the spectrum of a similar solution of pink urates, which owe their colour to urersthrin, a It also resembles a pigment which is got from the urine of the pig, when an acidulated alcoholic extract of the lead precipitate fi'om this urine is treated with nitric acid. But I need not here go further into the colouring matter of pig's urine; it will suffice to say that, so far as I have gone, I can find no urobilin in the urine of this animal. As urobilin, then, is produced by the action of hydrochloric acid as well as nitric acid, it becomes necessary to see whether we cannot get urobilin from bile by other methods wherein oxygen is the agent concerned. Action of Air alone on Bilirubin.--Human bile, obtained from the gall-bladder of a man who died from cerebral hmmorrhage, was treated with absolute alcohol to precipitate the mucus, &c. This method is preferable to the acetic acid one, as acetic acid is apt to slightly change bilirubin, as the spectroscope shows.