384 CHEMISTRY OF THE DIGESTIVE PROCESSES. 
human serum and that of the ox. Most important, from the point of 
view of the origin of the bile pigments, is the discovery that the 
microscopic crystals often found in old blood clots and extravasations, 
and described by Virchow x as hsematoidin, are usually bilirubin ; this 
shows that the bile pigments are probably products of disintegration of 
hemoglobin. Here it is needful to guard against mistaking lutein for 
bilirubin. The two substances may be distinguished by their solubilities. 
Both are soluble in chloroform, but bilirubin is thrown out of solution 
on the addition of an alkali (from the formation of a compound with the 
alkali insoluble in chloroform), while lutein is not so precipitated. 
Bilirubin is also found, in cases of jaundice, in the urine and in the 
tissues. 
Bilirubin is best prepared from the gallstones of the ox, which are very 
common and easily procurable. The gallstones are washed, dried, powdered, 
and then extracted in turn with ether, boiling alcohol, and boiling water, to 
remove cholesterin (which is, however, rarely present in appreciable quantity 
in ox gallstones) and bile acids. The residue is treated with dilute hydro- 
chloric acid, to set free the bilirubin from its calcium combination, washed 
with water and alcohol, and finally extracted with boiling chloroform, in which 
the bilirubin dissolves. 
The chloroform is distilled from the extract, and the impure bilirubin is 
freed from an accompanying substance, bilifuscin, by digesting with absolute 
alcohol, in which this substance dissolves, and is then redissolved in chloro- 
form. It is purified further by throwing out of concentrated solution in 
chloroform, by the addition of absolute alcohol, redissolving and reprecipitating 
repeatedly. It is lastly dissolved in as little as possible of boiling chloroform, 
from which it crystallises on cooling. 
In amorphous condition, as when precipitated by alcohol and dried, 
bilirubin is an orange-coloured powder ; when crystalline, it is of a dark red 
or reddish-brown colour, resembling chromic acid. The crystals are rhombic 
plates with rounded-off angles; they are more easily soluble in chloroform 
than in any other solvent ; somewhat soluble in car Don bisulphide and amyl 
alcohol ; nearly insoluble in ether, alcohol, turpentine, benzol, and glacial acetic 
acid. Bilirubin is soluble easily in alkalies and their carbonates, combining 
with them to form salts. Calcium chloride, added to these solutions, pre- 
cipitates the calcium compound (C 1(3 H 17 ]Sr 2 8 ). 2 Ca as a rust-coloured precipitate. 
Treated with sodium amalgam, bilirubin yields hydrobilirubin ; on oxidation 
it passes into biliverdin and more highly oxygenated compounds. Several 
formulas have been proposed for bilirubin ; 2 the most generally accepted is 
that of Maly (C 16 H 1S 1S' 03). Bilirubin is oxidised in alkaline solution in the 
air to biliverdin in the same manner as bile, which owes this reaction to the 
bilirubin it contains. 
Elirliclxh test. 3 — Ehrlich describes a colour test for bilirubin, which is not 
given by biliverdin. To a solution of bilirubin in chloroform an equal volume 
of a watery solution of diazobenzolsulphonic acid is added, and just enough 
alcohol to cause the two fluids to mix, when the fluid turns a beautiful red 
colour ; on adding, drop by drop, concentrated hydrochloric acid, the colour of 
1 Virchmvs Arcliiv, 1847, Bd. i. S. 379, 407. See also Robin, Compt. rend. Acad. d. so., 
Paris, 1855, tome xli. p. 506 ; Jafte, Virchoiv's Archiv, 1862, Bd. xxiii. S. 192 ; E. Salkowski, 
Hoppe-Sey/er's Med. -chem. Untersuch., Berlin, 1868, S. 436. 
- Stadeler, Ann. d. Chem., Leipzig, 1864, Bd. cxxxii. S. 323 ; Tlmdiclmm, Journ. f. 
prakt. Chem., Leipzig, 1868, Bd. civ. S. 401 ; Maly, ibid., Bd. civ. S. 28 ; Maly, Ann. d. 
Chem., Leipzig, 1876, Bd. clxxxi. S. 106 : Nenckiu. Sieber, Ber. d. deutsch. chem. Geselhch., 
Berlin, 1884, Bd. xvii. S. 2275. 
3 Centmlbl. f. Jclin. Med., Bonn, 1883, Bd. iv. S. 722; Krukenberg, "Chem. Unter- 
such.," 1886, S. 77. 
