386 CHEMISTR Y OF THE DIGESTIVE PROCESSES. 



poured on the surface of fuming nitric acid in a test tube, so as not 

 to mix the two liquids, a series of coloured zones appears in the 

 lower part of the column of bile above the acid ; next to the acid is the 

 most oxidized product (choletelin), represented by a yellow-red zone ; 

 above this is a purer red, passing into a purple, which is replaced by a 

 blue zone (bilicyanin) ; and, lastly, there is a very broad green zone, 

 corresponding to the least oxidized product (biliverdin). The test may 

 also be made by spreading out the bile in a thin film over the inside 

 of a porcelain capsule and placing a drop of fuming nitric acid in the 

 centre of this film, when a series of colours develop in the above order 

 around the drop ; or perhaps, most conveniently of all, according to 

 Eosenbach's modification, by moistening a piece of filter paper in the 

 suspected fluid, and placing a drop of fuming nitric acid in the 

 centre. 



Huppert's test. Huppert's test consists in precipitating calcium 

 bilirubinate, by the addition of milk of lime, or calcium chloride and 

 ammonia, to a solution of an alkaline bilirubinate (or alkaline bile) ; 

 after washing with water, the precipitate is boiled for some minutes 

 with alcohol acidified with sulphuric acid, when, in presence of bile 

 pigments, the solution develops an emerald-green or blue -green colour. 



Bilicyanin is the name given to the substance present at that stage of 

 oxidation of bilirubin by artificial means, such as fuming nitric acid, when 

 the solution has a blue colour. The stage is a very transient one, and, though 

 many have worked at the subject, no one has yet succeeded in isolating the 

 substance to which the blue colour is due. It is probably an unstable 

 oxidation product, intermediate between biliverdin and choletelin. A blue 

 solution, which keeps for some hours, may be obtained by adding to a solution 

 of bilirubin in chloroform a little nitric acid, and shaking till a violet tint 

 first appears. Rectified spirit is then quickly added ; this very much slows the 

 completion of the oxidation, so that the blue colour is preserved for some 

 time. If an ammoniacal solution of bilirubin be mixed with strong fuming 

 nitric acid, a little at a time, and excess of acid removed each time by addition 

 of ammonia, a dark flocky precipitate is obtained, from which biliverdin can 

 be removed by alcohol, leaving behind a deep dark blue powder. 1 Heynsius 

 and Campbell 2 have found that certain gall stones in man, after extraction 

 with alcohol and ether, yield to dilute acids a violet-brown pigment, which 

 they identified as bilicyanin spectroscopically. 



Jaffe 3 first observed that the blue stage of the oxidation process 

 gave an absorption spectrum ; in strong solution, it shows a wide band 

 beginning to the red side of D, and ending between D and E ; on dilution 

 this band resolves itself into two dimmer bands ( and /3). As oxida- 

 tion proceeds, a third band (7) appears between I and F, whilst the two 

 first mentioned gradually become fainter and disappear. This third 

 band does not belong to the blue stage (bilicyanin), but to the substance 

 formed in the final stage of the oxidation (choletelin). The violet 

 colour obtained before the final permanent reddish brown is probably 

 due to an admixture of the latter colour with blue. 



Bilifuscin is a substance separated in the preparation of bilirubin from 

 gall stones ; very little is known of its properties or chemical relationships. 



1 Jaffe, Centralbl. f. d. med. Wissensch., Berlin, 1868, Bd. vi., S. 241 ; Journ. /. 

 prakt. Chem., Leipzig, 1868, Bd. civ. S. 401. 



2 Arch.f. d. ges. PhysioL, Bonn, 1871, Bd. iv. S. 537. 3 Loc. cit. 



