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. The bile was filtered, and then shaken with chloroform, and the solution thus obtained I may call, for the sake of avoiding repetition, the chlo- roformic solution ofbilirubin. Although the presence of alcohol, no doubt, would tend to keep any biliverdin formed by exposure to the air in solution, yet this is rather an advantage, as it enables us to dispense with the further use of solvents after biliverdin has begun to form. Such a solution was found at first to give no par- ticular absorption spectrum (except a shadow extending over the violet, blue, and green). After exposure to air in a corked bottle, and repeated shaking, the fluid became greenish, and still no well- marked bands were seen; but after forty-eight hours the fluid gave a I take this opportunity of correcting a mistake in the Spectroscope in Medicine, as the eolouring matter of pink urates is in no way apparently related to urobilin. 2L 514 On Urobilin from Bilirubin and Biliverdin. three bands--a feeble one on each side D, and a black one at :F. These bands were identical in position and in shading with those observed in the ehloroformie solution of the urobilin of phthisis, and with those of the chloroformic solution of biliverdin treated with nitric acid. Moreover, ammonia produced a band a covering D, identical with one produced by the same reagent when it was added to a chloroformie solution of the phthisical urobilin. At the end of three weeks the colour of this fluid was brown-yellow, and it then gave only a band at F. By mere exposure r the air, then, the same series of changes which take place by the action of nitric acid on bile-pigment was produced in the spectrum; and further, the fluid no longer gave the colour reaction with nitric acid which is charac- teristic of Gmelin's test. Action of Oxygen on Biliverdin.--A chloroformic solution of bilirubin was exposed to the air until it had changed to biliverdin ; it was then evaporated over the water-bath, and the residue dis- solved in water. The solution was sap-green in colour, and gave no absorption bands. A test-tube was half filled with this solution, and a stream of oxygen gas passed into it; the colour soon became darker, and coincidently a band appeared at F. On continuing the action of the oxygen the colour changed to a dirty green-blue, with reflected, but brown with transmitted, light; and three bands now appeared, one on each side of D, and one at F, which, allowing for the difference of refraction of the medium in which the pigment was dissolved, were found to be exactly the same bands which were seen in the solutions mentioned above. The colour then became purple, then a dirty lavender, with reflected light, and, lastly, yellow-brown, with transmitted light ; and at that stage only one band could be seen-- viz., that at F. :Nitric acid, which brought out a beautiful play of colours with the original solution, now no longer affected it, so that the result coincided with the former ones. Permanganate of potassium and peroxide of hydrogen were also added to solutions of biliverdin, and the result noted. I may here, however, content myself with stating that the former supported the other experiments, while the latter was almost inert in its action. Aetion of Sodium Amalgam on Biliverdin.--An aqueous solution of biliverdin was treated with sodium amalgam, before the addition of which it gave no bands. At the end of two hours the fluid had changed to a pale green-yellow, and gave the same band that is found when solutions of urobilin are treated with caustic soda. In thinner layers that at F had gone. By DR. MAcMu~N. 515 .After longer action no further change took place. Treated with hydrochloric or acetic acid a the fluid became reddish-brown, and now gave a black band at F, which was mueh more distinct after filtration. An aqueous solution of biliverdin was now treated with caustic soda, and the result was exactly the same as with the sodium amalgam. I think I am correct, therefore, in assuming that the change which took place was due to the formation of caustic soda, and therefore to oxidation; for we know that caustic alkalies exert an oxidising action on organic bases, especially unstable ones. So far, then, as Maly's assertion, to which Hoppe-Seyler assents, is concerned--to the effect that sodium amalgam converts bilirubiu into urobilin--I can support him; but I must say that my experi- ments all tend to show that the transformation is due to oxidation, not reduction. I have made many other experiments which all lead to the same conclusion, so that I think I may now lay down the following proposition :- That urobilin can be formed from bilirubin and billverdin by oxidation, and that in health this transformation takes place in the body, but that in certain diseases, when oxidation is impeded, the urobilin in the urine is found in a condition of incomplete oxidation. All the bilirubin which gets into the blood is oxidised and excreted in the urine as urobilin, under normal conditions. If a portion entirely escapes oxidation, it may appear in the urine as bilirubin, or, if in larger quantities, it may produce jaundice. Thus we can understand lmw jaundice could be produced by destruction of the oxygen-carriers--the red blood-corpuscles ; or by the presence in the blood of poisons, which prevent the exchange of oxygen between the blood and the tissues; or by breathing vitiated air or certain poisonous gases; or by diseases of the lungs--in fact, by anything which prevents oxidation, immediately or remotely. I will now attempt to explain more in detail how this theory accounts tbr certain facts which are still sub judice. Applications to Medicine.--The urine of jaundice accompanied by fever generally fails to give a reaction with nitric acid. The reason of this is that the bilirubin, although in excess in the blood, has already, owing to the increased oxidation going on in the tissues, become changed into a further oxidation product, and appears as such in the urine, where it is present as the urobilin of fever, and

I object to the use of hydrochloric acid in this case, as it alone will convert bili- rubin into urobilin. 516 On U~'obilin from Bilirubin and Biliverdin. will not undergo further change with nitric acid. Again, in fever unaccompanied by jaundice, we often find a great excess of urobilin. This is owing to the fact that the rapidity of the heart's action, and, consequently, increased circulation, hurries into the blood a larger amount of bilirubin than normally is present therein ; but, owing to the increased oxidation which accompanies the febrile state, the pigment is quickly changed into urobilin, and is excreted in the urine as such. I cannot believe that urobilin is entirely formed in the intestine, for if it were we ought to be able to obtain it from the liver, which I have failed to do, for it would be again carried into the liver by means of the circulation first discovered by Schiff ;~ and, although Hammersten b says that urobilin is present in human bile, I have always failed to find it there. Nor can one easily see how the intestine could possibly .contain enough hydrogen in the nascent condition to convert bilirubin into urobilin, even allowing that the latter can be formed by reduction; to me it appears much more probable that (apart altogether from the urobilin produced by oxidation) the stercobilin of Vaulair and Masius is formed by tile action of the hydrochloric acid of the gastric juice on the bile-pigments in the intestine, and that the greater part, if not all, of the urobilin (or stercobilin)thus formed passes out with the f~eces; for hydro- chloric acid brings about the same transformation with billrubin that oxygen does. Moreover, it is not compatible with a knowledge of physiological chemistry to believe that a series of oxidations, beginning with the transfbrmation of bilirubin into biliverdin, and down through all the pigments which Heynsius and Campbell ~ have described, should suddenly stop short at urobilin, and that this pigment should prove an exception to the general rule and be formed by reduction. If we now try, by means of the theory for which I contend, to explain the causation of some kinds of jaundice which are inde- pendent of any disease of the liver, and which are put down to suppression or perhaps called hmmatogenous, which is really only a term to cloak our ignorance, we find that it fits in very well with the symptoms and appearances observed in such cases. I may mention the jaundice due to pneumonia, some kinds of icterus neonatorum, jaundice from breathing vitiated air, probably the

a Giorn. di Scienze Naturali ed Econ. Vol. IV., p. 9. Palermo, 1869. b MaFy's Jahresb. u. d. Fortschritte d. Thierchemie f. 1878. Bd. VIII., p. 260. c Arch. f. d. ges. Phys. 1871. Bd. IV., p. 497. By DR. MACMUNN. 517 jaundice of phosphorus poisoning (and the general fatty condition of various organs in these last cases is in keeping with deficiency of' oxidation); that due to snake-bites (since Lauder Brunton has shown that the blood-corpuscles are altered in such cases, and probably rendered unfit for carrying oxygen); that produced by chlorofbrm and chloral (Leyden), and probably the jaundice of certain specific fevers--in fact, in most cases of jaundice which are not due to obstruction, we find some interference with the normal oxidation whereby bilirubin is converted into urobilin2 Again, experiments on dogs have shown that the injection of bile- salts into the blood has caused the appearance of bile-plgment in the urine; the reason of this is--according to the theory which I have adopted--that the blood-corpuscles being destroyed by the bile acids there is not enough oxygen supplied to convert the bilirubia into urobilin, and the former, therefore, appears in the urine, for the destructive action of bile acids on the blood-cor- puscles is well known. But it is unnecessary to give further instances of the application of the study of the spectra of bile-pigments, b and the transfbrmation which they undergo with reagents, to medicine, as such will present themselves to every thoughtful mind. I know that the right hypochondrium is the starting point for a good deal of unsound doctrine; but I have, I trust, appealed more to i~acts than fancy, and have not trusted too much to authority, knowing that " the mortallest enemy unto knowledge, and that which hath done the greatest execution upon truth, hath been a peremptory adhesion unto authority" (Sir T. Browne).

Jaff$ believed that he had detected urobilin in blood, but Hoppe-Seyler thinks that he mistook sero-lutein for it. I hope soon to be able to bring forward evidence of the presence of bilirubin in this fluid. The amount of either is so small that it is almost impossible to detect them in the presence of so many complex substances. b The appearance of a band at F in the bile of various animals~ after exposure to air, noticed by me, and recorded in the Spectroscope in Medicine~ is explained by the oxidation theory. The bile of the mouse appears to owe its colour to a pigment which is closely related to, if not identical with, urobilin--in other words, to a pigment pro- duced by oxidation from another bile-pigment.