432 THE LIVER. 



from bilirubin by oxidation under special conditions (KUSTER). A green 

 coloring-matter similar in appearance is formed by the action of other 

 reagents such as Cl, Br, and I. According to JOLLES/, biliverdin is 

 produced by the use of HUBL'S iodine solution, while according to others 

 (THUDICHUM, MALT 2 ) substitution products of bilirubin are formed. 



GMELIN'S Reaction for Bile-pigments. If one carefully pours nitric 

 acid, containing some nitrous acid, under an aqueous solution of alkali 

 bilirubin, there is obtained a series of colored layers at the juncture of the 

 two liquids in the following order from above downward: 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 reac- 

 tion 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. 



HAMMARSTEN'S Reaction. An acid is first prepared consisting of 1 

 vol. nitric acid and 19 vols. hydrochloric acid (each acid being about 

 25 per cent). One volume of this acid mixture, which can be kept for 

 at least a year, is, when it has become yellow by standing, mixed with 

 4 vols. alcohol. If a drop of bilirubin solution is added to a few cubic 

 centimeters of this colorless mixture a permanent beautiful green color 

 is obtained immediately. On the further addition of the acid mixture 

 to the green liquid all the colors of GMELIN'S scale, as far as choletelin, 

 can be produced consecutively. 



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

 milk of lime or with calcium chloride and ammonia, a precipitate is 

 produced consisting of calcium bilirubin. If this moist precipitate, which 

 has been washed with water, is placed in a test-tube and the tube half 

 filled with alcohol which has been acidified with hydrochloric acid, and 

 heated to boiling for some time, the liquid becomes emerald-green or 

 bluish-green in color. 



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 



1 Kttster, Ber. d. d. chem. Gesellsch., 35 and 59; Jolles, Journ. f .prakt. Chem. (N.F.), 

 59, and Pfliiger's Arch., 75. 



2 Thudichum, Journ. of Chem. Soc. (2)^13, and Journ. f. prakt. Chem. (N.F.), 

 53; Valy, Wien. Sitzungsber., 72. 



