BILE PIGMENTS AND THEIR DERIVATIVES. 385 



the solution changes through violet into blue ; if a layer of potassium hydrate 

 solution is now introduced beneath the blue solution, there develops an 

 (alkaline) bluish-green zone underneath, separated from the blue solution above 

 by a red band where the reaction is neutral. 



Biliverdin is present in all green-coloured biles, and may be obtained from 

 them, by adding a solution of barium chloride, as a dark green precipitate, 

 which may be washed with water and alcohol, and decomposed by dilute hydro- 

 chloric acid, when the biliverdin remains as a flocky precipitate ; this is freed 

 from fats by washing with ether, and is then dissolved in alcohol ; on evaporat- 

 ing the alcohol, the biliverdin is left behind as a dark green scale. Biliverdin 

 can best be prepared pure from an alkaline solution of bilirubin. This is 

 allowed to oxidise by exposing to the air in a shallow dish, until it turns a 

 brownish-green colour ; the solution is then precipitated with hydrochloric 

 acid, which sets free insoluble biliverdin from the soluble compound with the 

 alkali ; the precipitate is washed with water till free from hydrochloric acid, 

 dissolved in alcohol, and reprecipitated by the addition of water. This 

 precipitate is washed with chloroform to remove traces of bilirubin, and pure 

 biliverdin remains behind, being insoluble in chloroform. It forms a very 

 dark green amorphous powder, insoluble in water, ether, chloroform, carbon 

 bisulphide, or benzol ; but soluble with a fine green colour in alcohol, glacial 

 acetic acid, or concentrated sulphuric acid. According to MacMunn, 1 there is 

 a green pigment in ox bile which differs from that prepared as above, in being 

 soluble in chloroform. Biliverdin does not easily crystallise ; it is said to be 

 occasionally obtainable, by evaporating a solution in glacial acetic acid, in 

 rhombic plates with rounded angles. With alkalies, biliverdin forms soluble 

 compounds, giving brownish-green solutions, from which biliverdin falls as a 

 flocky precipitate on the addition of acids. Calcium, barium, and lead salts 

 form insoluble compounds with biliverdin ; these are thrown down as dark 

 green precipitates on addition of solutions of the corresponding salts to an 

 alkaline solution of biliverdin. By nascent hydrogen, biliverdin^is converted 

 through bilirubin into hydrobilirubin. Different formulae are given by different 

 authors for biliverdin. Stadeler 2 calculated it as C 16 H 20 N 2 5 , from analyses 

 by Heintz. Heintz himself 3 gives C 16 H 1S ]N" 2 5 . This formula assumes that 

 in passing from bilirubin to biliverdin, the molecule takes up water as well as 

 oxygen (C 10 H 18 N" 2 3 + H 2 + = C 16 H 20 N 2 5 ), but the accuracy of this is 

 denied by Maly, 4 both from analysis and the amount of increase in weight 

 observed in passing from bilirubin into biliverdin. He gives the formula of 

 biliverdin as C 16 H 1S N 2 4 , and this result is confirmed by Thudichum, 5 except 

 that the latter halves the formula, giving C 8 H 9 N0 2 . 



G-melins test for 'bile pigments. This very distinctive test for the bile 

 pigments has already been mentioned. It depends upon the remarkable 

 changes in colour accompanying the oxidation of bilirubin. In such 

 oxidation the other normal bile pigment, biliverdin, is first produced ; 

 and this in turn, by further oxidation, is converted into a blue pigment, 

 bilicyanin; after this follows, according to some, a purple pigment 

 (bilipurpurin) before the final stage of oxidation to a yellow compound, 

 choletelin. The production in series of these artificial products of 

 oxidation is what constitutes Gmelin's test. 



If either a solution of bilirubin or some diluted bile be carefully 



1 Journ. Physiol., Cambridge and London, 1885, vol. vi. p. 25. 



2 Ann. d. Chem., Leipzig, 1864, Bd. cxxxii. S. 323. 



3 Jahresb. il. d. Fortschr. d. ges. Med., Erlangen, 1851, Bd. ii. S. 59; Ann. d. Phys. 

 u. Chem., Leipzig, 1851, Bd. Ixxxiv. S. 106. 



4 Jahresb. il. d. Fortschr. d. Thier-Chem., Wiesbaden, 1874, Bd. iv. S. 302 ; and Her- 

 mann's "Handbuch," Bd. v. (2), S. 159. 



5 Journ. /. prakt. Chem., Leipzig, 1868, Bd. civ. S. 220. 



VOL. I. 25 



