THE BILE. 159 



is treated with an amount of concentrated nitric acid, containing a 

 trace of nitrous acid, sufficient to form a layer beneath the liquid to 

 be tested, when in the presence of bilirubin the color-play referred to 

 will be observed at the zone of contact ; the green will be noticed 

 nearest the bile-containing solution, and the orange in the upper 

 portion of the nitric acid. Various modifications of this reaction 

 have been proposed, such as the one described above. 



The test is exceedingly sensitive, and is said to indicate the pres- 

 ence of bilirubin in a dilution of 1 : 80,000. The green color which 

 develops is the most characteristic, but a reddish violet must also 

 occur. 



HUPPERT'S TEST. A few cubic centimeters of the solution to 

 be examined are precipitated with barium chloride and ammonia. 

 The precipitate is washed with water and suspended in a small 

 amount of alcohol that has been acidulated with sulphuric acid. 

 This mixture is then boiled for a few minutes, when in the presence 

 of bilirubin a bright emerald-green color develops. 



SMITH'S TEST. A small amount of the fluid is placed in a test- 

 tube and treated with a few cubic centimeters of tincture of iodine 

 which has been diluted with alcohol in the proportion of 1 : 10, so 

 as to form a layer above the fluid to be examined. In the presence 

 of bilirubin a distinct emerald-green ring will develop at the zone 

 of contact. 



According to some observers, the green color which thus results 

 when bilirubin is treated with iodine is not referable to the forma- 

 tion of biliverdin, but to a substitution- or addition-product of bili- 

 verdin with iodine. This, however, is denied by others, and Jolles 

 has recently shown that the iodine merely acts as an oxidizing agent, 

 and that true biliverdin is thus formed, as indicated by the equation : 



C 16 H 18 N 2 3 + 21 + H 2 = C 16 H 18 N 2 4 + 2HI. 



SPECTROSCOPIC TEST. If a dilute solution of sodium bilirubi- 

 nate in water is treated with an excess of ammonia and a small 

 amount of a solution of chloride of zinc, the liquid at first turns 

 a deep orange, but subsequently becomes olive brown, and finally 

 green. If this solution is examined spectroscopically, it will be 

 noted that the violet and blue portions of the spectrum are at first 

 quite dark, but subsequently the bands presented by an alkaline 

 solution of bilicyanin become apparent, and notably the one between 

 C and D, near C (see below). The test is said to be very good 

 (Hammarsten). 



Isolation of Bilirubin. Bilirubin is most conveniently obtained 

 from the biliary concretions which are so often found in the gall- 

 bladder of cattle, and which consist almost entirely of the calcium 

 salt of the pigment. They are finely powdered and extracted with 

 ether and then with hot water, so as to remove the cholesterin and 

 the biliary acids which are present. The remaining material is 

 treated with hydrochloric acid, so as to liberate the pigment. It is 



