B1LIVERDIN. 409 



Bilirubin is best prepared from gall-stones of oxen, these concretions 

 being very rich in calcium bilirubin. The finely powdered concrement is 

 first exhausted with ether and then with boiling water, so as to remove 

 the cholesterin and bile-acids. In order to remove the mineral con- 

 stituents it is better to use 10-per cent acetic acid instead of hydrochloric 

 acid (KtiSTER 1 ). A green pigment is now removed by extraction with 

 alcohol, and the choleprasin is extracted with hot glacial acetic acid. 

 After washing with water it is dried, and extracted repeatedly with boiling 

 chloroform. The bilirubin separates from the chloroform as crusts, 

 which are treated once or twice in the above manner. It is then extracted 

 with alcohol and precipitated from its chloroform solution by alcohol, or 

 crystallized from boiling dimethylaniline. Further details are given by 



KtJSTER. 2 



The quantitative estimation of bilirubin may be made by the spectro- 

 photometric method, according to the steps suggested for the blood- 

 coloring matters. 



Biliverdin, Ci 6 Hi 8 N 2 04 or C32H 36 N 4 8 . This body, which is formed 

 by the oxidation of bilirubin, occurs in the bile of many animals, in 

 vomited matter, in the placenta of the bitch (?), in the shells of birds' 

 eggs, in the urine in icterus, and sometimes in gall-stones, although in 

 very small quantities. 



Biliverdin is amorphous; at least it has not been obtained in well- 

 defined crystals. It is insoluble in water, ether, and chloroform (this is 

 true at least for the artificially prepared biliverdin), but is soluble in 

 alcohol or glacial acetic acid, showing a beautiful green color. It is dis- 

 solved by alkalies, giving a brownish-green color, and this solution 

 is precipitated by acids, as well as by calcium, barium, and lead salts. 

 Biliverdin gives HUPPERT'S, GMELIN'S, and HAMMARSTEN'S reactions, 

 commencing with the blue color. It is converted into hydrobilirubin 

 by nascent hydrogen. On allowing the green bile to stand, also by the 

 action of ammonium sulphide, the biliverdin may be reduced to bilirubin 

 (HAYCRAFT and SCOFIELD 3 ). 



Biliverdin is most simply prepared by allowing a thin layer of an 

 alkaline solution of bilirubin to stand exposed to the air in a dish until 

 the color is brownish green. The solution is then precipitated by hydro- 

 chloric acid, the precipitate washed with water until no HC1 reaction is 

 obtained, then dissolved in alcohol and the pigment again separated by 

 the addition of water. Any contaminating bilirubin may be removed 

 by means of chloroform. KUSTER has shown that the biliverdin is only 

 formed by the oxygen of the air from bilirubin under certain conditions: 

 The presence of 2 molecules caustic alkali with the addition of water so 

 that the solution contains 0.2 per cent and, a temperature not above 5 C. 



1 Zeitschr. f. physiol. Chem., 47. 



2 Zeitschr. f. physiol. Chem., 59. 



3 Centralbl. f. Physiol., 3, 222, and Zeitschr. f. physiol. Chem., 14. 



