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 floeky 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 
floeky 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 X. 2 O 5 , from analyses 
by Heintz. Heintz himself 3 gives C^H^X.^Cv. This formula assumes that 
in passing from bilirubin to biliverdin, the molecnle takes up water as well as 
oxygen (C ir ,H ls X 2 3 + H.,0 + = C lt ;H 20 X 2 O 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 ls X n 4 , and this result is confirmed by Thudichum, 5 except 
that the latter halves the formula, giving C 8 H 9 X0 2 . 
Gmeliris 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 Joum. Physiol., Cambridge and London, 1885, vol. vi. p. 25. 
2 Ann. d. Chem., Leipzig, 1864, Bd. exxxii. S. 323. 
3 Jahresb. u. d. Fortschr. d. ges. Med., Erlangen, 1851, Bd. ii. S: 59 ; Ann. d. Phys. 
u. Chem., Leipzig, 1851, Bd. lxxxiv. S. 106. 
4 Jahresb. il. d. Fortschr. d. Thier-Chem., Wiesbaden, 1874, Bd. iv. S. 302 ; and Her- 
mann's "Handbuoh," Bd. v. (2), S. 159. 
5 Joum. f. prakt. Chem., Leipzig, 1868, Bd. civ. S. 220. 
VOL. I.— 25 
