3 S6 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 yell< >w-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 
Eosenbaeh's modification, by moistening a piece of filter paper in the 
suspected fluid, and placing a drop of fuming nitric acid in the 
centre. 
Hwppert'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. Eectified 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 - 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 (a and /3). As oxida- 
tion proceeds, a third band (7) appears between b 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 knoAvn of its properties or chemical relationships. 
1 Jaffe, Centralbl. f. d. med. IVissmsch., Berlin, 1S6S, Bd. vi., S. 241; Journ. f. 
l)rakt. Ohem., Leipzig, 186S, Bd. civ. S. 401. 
2 Arch. f. d. ges. Physiol., Bonn, 1871, Bd. iv. S. 537. 3 Loc. cit. 
