242 BILIKUBIN. 



and simplest source for the preparation of this substance. 1 The 

 stones are finely powdered, extracted with ether to remove any 

 cholesterin, then with water and treated with either strong acetic 

 acid or dilute hydrochloric acid. By -this means the bilirubin is 

 set free from its calcium compound, and after being washed with 

 water and alcohol is dissolved in chloroform, and finally separated 

 by precipitation with alcohol as already described. To obtain it 

 quite pure the dissolving in chloroform and precipitating by alco- 

 hol should be repeated several times. The final product is amor- 

 phous. Crystals are most readily obtained by slow evaporation 

 of the first and hence slightly impure solution in chloroform. 



When carnivorous bile is exposed to the air it turns more or 

 less rapidly green ; this is due to its oxidational conversion into 

 biliverdin, the normal pigment of herbivorous bile. A similar 

 change is at once produced by an oxidising agent such as nitric 

 acid containing nitrous acid, but in this case the change of colour 

 does not stop short with green, but passes successively through 

 blue, violet, and red to a final yellow. These later colours are 

 due to products of the progressive oxidation of the first formed 

 biliverdin, but with the exception of the final substance (cholete- 

 lin) are as yet but imperfectly characterised. The play of colours 

 observed when either bilirubin or biliverdin is oxidised, consti- 

 tutes the well-known Gmelin's reaction. 2 This is extremely 

 delicate and may be applied in either of the two following ways. 

 A few drops of the suspected solution are placed on a porcelain 

 slab and a drop of yellow fuming nitric acid is brought into con- 

 tact with it. A play of colours is observed at the junction of the 

 fluids if bile-pigments are present. Or on the other hand some 

 of the acid may be poured into the bottom of a test-tube and the 

 suspected fluid carefully added so as not to mix with the acid but 

 float on its surface. If bile-pigments are present coloured rings 

 (layers) appear at the junction of the two liquids, being yellow 

 nearest the acid and progressively red, violet, blue, and green 

 passing upwards. It is stated that this test will detect as little 

 as 1 part of bilirubin in 70,000 80,000 parts of solvent. 



Other tests have been recommended, but they are perhaps un- 

 necessary in view of the extreme delicacy of Gmelin's reaction when 

 properly applied. 3 The certain detection of minute amounts of bile- 

 pigments in urine is frequently of great clinical and physiological 

 importance. If any very appreciable quantity of the pigments are 

 present, Gmelin's reaction applied as above will \isually suffice to 



1 The coloured residue from human gall-stones left after the extraction of 

 cholesterin (p. 131) may also be used for the preparation of bilirubin. 



2 Tiedemann u. Gmelin. Die Verdauung nach ^ 7 ersuchen, 1826, S. 80. 



3 See more particularly Capranica, Gaz. chim. Ital. Vol. xi. (1881), p. 430. 

 Moleschott's Untersuch. z. Naturlehre, Bd. xin. (1882), S. 190. Ehrlich. Centralb. 

 f. klin. Med. 1884, No. 45, or Centralb. f. d. med. Wiss. 1884, S. 143. In the latter 

 case a solution of diazobenzosulphonic acid is employed, and is stated to discriminate 

 between bilirubin and other bile-pigments. 



