CHAP. IV.] BILIVERDIN. 323 



It has already been stated that when the reddish- 

 ' ' yellow bile of carnivorous animals is exposed to the air, 

 it gradually acquires a green hue which resembles that of the 

 secretion of the herbivora. This change in colour is associated with 

 an absorption of oxygen and depends upon the conversion of bili- 

 rubin into biliverdin ; it occurs all the more readily if the bile be 

 rendered decidedly alkaline by the addition of a caustic alkali and 

 then exposed in thin layers to the action of the air. The same 

 change is observed, though it proceeds less rapidly, if the bile be 

 acidified by means of acetic, hydrochloric or sulphuric acids, and then 

 exposed to air ; the presence of oxygen being under these circum- 

 stances absolutely necessary to the production of the colour. The 

 body which is the cause of the beautiful green tint developed by the 

 action of nitric and nitrous acids upon bile or bilirubin is, unques- 

 tionably, biliverdin, which is, in this case, produced by the oxidising 

 action of the acids employed, quite independently of the atmo- 

 spheric oxygen. 



Biliverdin is most readily prepared in a state of purity (Stadeler) 

 by dissolving pure bilirubin in a diluted solution of sodium hydrate 

 and either exposing the liquid to air or causing a stream of oxygen 

 gas to pass through it. When the solution has acquired a bright 

 green colour an excess of dilute hydrochloric acid is added; this 

 precipitates the biliverdin which is washed with water, until the 

 washings contain no trace of chlorine. It is then dried, dissolved in 

 absolute alcohol, the alcoholic solution is filtered and precipitated by 

 the addition of water. 



Biliverdin is readily produced when bilirubin is heated to 100 C. 

 in sealed tubes containing a mixture of chloroform and glacial acetic 

 acid, care being taken that a considerable space filled with air is left 

 in the tubes (Heynsius and Campbell). The objection to this process 

 is that water very imperfectly precipitates the biliverdin which has 

 been formed. The process was modified in an important manner by 

 Maly 1 who found that at moderate temperatures and in the absence 

 of chloroform, monochloracetic acid readily leads to the absorption 

 of oxygen by bilirubin and its conversion into biliverdin. Mono- 

 chloracetic acid, the melting point of which is 62 C. is rendered 

 fluid by warming it in a beaker ; powdered bilirubin is then 

 digested in it with the aid of a gentle heat, on the water bath 

 After a couple of days, water is added to the dark green solution, 

 the whole of the biliverdin being precipitated. In this process, again, 

 the production of the body is associated with, and dependent upon, 

 the atmospheric oxygen. 



Maly 2 also converted bilirubin into biliverdin by the oxidising 



1 Rich. Maly, ' Ueber Biliverdin,' Aus Untersuchungen iiber die Gallenfarbstoffe, 

 iv. Abhandl., Sitzungsber. d. Wiener Akademie, Vol. LXX. (1874). The Author has 

 not seen this paper but quotes from the careful abstract in Maly's Jahresbericht, 

 Vol. iv. (1875), pp. 302304. 



2 Maly, Sitzungsber. d. Wiener Akad., Vol. LVII. (1868). Quoted by Maly in 

 Hermann's Handbuch, Bd. vn. p. 158. 



212 



