384 CHEMISTR Y 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 1 as hceinatoidin, are usually bilirubin ; this 

 shows that the bile pigments are probably products of disintegration of 

 haemoglobin. 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 carbon 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 16 H l7 ]Sr 2 3 ) 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 

 formulae have been proposed for bilirubin ; 2 the most generally accepted is 

 that of Maly (C 16 H 18 ISr 2 3 ). 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. 



Ehrlich's 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 Virchow' 's Archiv, 1847, Bd. i. S. 379, 407. See also Robin, Compt. rend. Acad. d. sc., 

 Paris, 1855, tome xli. p. 506 ; Jaffa", Virclioufs Archiv, 1862, Bd. xxiii. S. 192 ; E. Salkowski, 

 Hoppe-Seylers Med.-chem. Untersuch., Berlin, 1868, S. 436. 



2 Stadeler, Ann. d. Chem., Leipzig, 1864, Bd. cxxxii. S. 323 ; Thudichum, 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 : Nencki u. Sieber, JBer. d. deutsch. chem. Gesellsch., 

 Berlin, 1884, Bd. xvii. S. 2275. 



3 Centralbl. f. Tclin. Med., Bonn, 1883, Bd. iv. S. 722; Krukenberg, "Chem. Unter- 

 such.," 1886, S. 77. 



