154 PHYSIOLOGICAL CHEMISTRY. 



in water are precipitated by the soluble salts of the alkaline earths 

 and also by metallic salts. 



If an alkaline solution of bilirubin be allowed to stand in contact 

 with the air, it gradually absorbs oxygen and green biliverdin is 

 formed. Biliverdin is also formed from bilirubin by 'oxidation under 

 other conditions. A green coloring matter similar in appearance is 

 formed by the action of other reagents such as 01, Br, and I. In these 

 cases it does not seem to be biliverdin, but a substitution product of 

 bilirubin (TnuDiCHUM, MALY) which is obtained. 



GMELIN'S Reaction for Bile-pigments. If we carefully pour under 

 a solution of bilirubin-alkali in water nitric acid containing some 

 nitrous acid, we obtain a series of colored layers at the juncture of 

 the two liquids, in the following order from above downwards: 

 green, blue, violet, red, and reddish yellow. This color reaction, 

 GMELIN'S test, is very delicate and serves to detect the presence of 

 one part bilirubin in 80,000 parts liquid. The green ring must 

 never be absent; and also the reddish violet must be present at the 

 same time, otherwise the reaction may be confused with that for 

 lutein, which gives a blue or greenish ring. The nitric acid must 

 not contain too much nitrous acid, for then the reaction takes place 

 too quickly and it does not become typical. Alcohol must not be 

 present in the liquid, because, as is well known, it gives a play of 

 colors, in green or blue, with the acid. 



HUPPERT'S Reaction. If a solution of bilirubin-alkali is treated 

 with milk of lime or with calcium chloride and ammonia, a precipi- 

 tate is produced consisting of bilirubin-calcium. If this moist pre- 

 cipitate, which has been washed with water, is placed in a test- 

 tube and the tube half filled with alcohol which has been acidified 

 with sulphuric acid, and heated to boiling for some time, the liquid 

 becomes emerald -green or bluish green in color. This reaction is a 

 good and easily-performed test for bile-pigments. 



In regard to the modifications of GMELIN'S test and certain 

 other reactions for bile-pigments, see Chapter XIV (Urine). 



That the characteristic play of colors in GMELIN'S test is the 

 result of an oxidation is generally admitted. The first oxidation 

 step is the green biliverdin. Then follows a blue coloring matter 

 which HEINSIUS and CAMPBELL call bilicyanin and STOKVIS calls 

 cliolecyanin, and which shows a characteristic absorption-spectrum. 



