214 Dr. C. A. MacMunn. Researches into the [Dec. 16,. 



of that band, urobilin might be present — for its band is invisible in 

 slightly alkaline or neutral solution, and even after shaking with 

 chloroform it may still be invisible until after an acid has been added 

 — the conclusion follows that what has been supposed to be due to the 

 transformation of bilirubin into urobilin may, after all, be nothing 

 more than the gradual appearance under oxidation of the band of a 

 pigment already existing in the solution. The behaviour of even 

 impure solutions such as these is, however, very instructive. When 

 a chloroformic solution is put into a stoppered bottle, so as to fill it 

 about one-third, and the bottle is shaken from time to time, the fluid 

 gradually gets light in colour. The general absorption of the violet 

 end of the spectrum characteristic of bilirubin gradually gives way to 

 an interesting special absorption, which is characterised by the appear- 

 ance of a band on each side of D, which is soon followed by the 

 appearance of one at F. Then the former bands fade gradually 

 away, leaving the band at F. This change was found to have been com- 

 pleted at the end of three weeks, and the colour of the fluid was then a 

 pale brownish-yellow with transmitted daylight. The same series of 

 changes in the spectrum accompanies the play of colour got by the 

 action of nitric acid on bilirubin, and at the penultimate stage of the 

 reaction, at the brown-yellow stage, we can isolate the pigment giving 

 the band at F. But if the reaction be allowed to go on further, the 

 fluid becomes almost colourless, and no longer can the band at F be 

 seen. Isolated at the penultimate stage, the pigment is found to be 

 choletelin, but it is evident that if the bilirubin thus treated contained 

 urobilin, accurate inferences cannot be drawn from the characters of the 

 pigment isolated, as it might be not only an oxidised pigment derived 

 from bilirubin, but also a decomposition product of urobilin. 



Preparation of Pure Bilirubin. — Accordingly, it became necessary to 

 procure pure bilirubin, which was done according to the directions 

 given in the excellent " Handbook for the Physiological Laboratory" of 

 Professor Burdon- Sanderson. Brown human gall-stones were powdered, 

 extracted with ether, the residue boiled with water and treated with 

 diluted hydrochloric acid. After washing and drying, the mass was 

 boiled with chloroform ; the chloroform distilled off over the water- 

 bath ; the residue treated with absolute alcohol. It was then treated 

 with ether and alcohol repeatedly, and again dissolved in chloroform,, 

 from which it was precipitated by absolute alcohol. The pigment 

 thus obtained was an amorphous orange-coloured powder. 



It is not possible in the limits of this paper to describe all the 

 reactions and the spectra of the solutions obtained by this treatment 

 of the gall-stones, so that I shall only refer briefly to them as they 

 bear upon the subjects discussed here. The first ether extraction of 

 the gall-stones gave two bands, which are evidently those of lutein — 

 7 from wave-length 482 to 469, and B from wave-length 459 to 442. 



