Studies on Bile Pigments II. Seperation of Natural Direct Bilirubins

Separation of both forms of the direct bilirubin were carried out from the dog's gallbladder bile, and further isolations of them were also done. 1. The natural salt-form bilirubin was isolated after separation on the column of aluminium oxide with a n-propanolic aqueous solution. 2. The natural salt-form bilirubin was obtained in amorphous yellow powders which were strongly hygroscopic and easily soluble in water and methanol but not in chloroform or carbon tetrachloride. An aqueous solution of these powders showed both the direct diazo and Gmelin reaction, but neither Ehrlich's aldehyde nor Schlesinger reaction. The salt-form bilirubin was transferred into chloroform only when some quantities of hydrochloric acid were added to a mixture of chloroform and an aqueous solution of it. 3. The absorption maxima of the natural salt-form bilirubin existed at 420 to 430 mμ in a methanolic solution and at 425 or 435mμ in 50% or 10% n-propanol. 4. The natural ester-form bilirubin was isolated after separating on the column of silica gel with a chloroformethanolic mixture. 5. The natural ester-form bilirubin was obtained in amorphous greenish yellow powders. It was further hygroscopic and easily soluble in water and methanol but not in chloroform or carbon tetrachloride. An aqueous solution of it showed the direct diazo and Gmelin reaction, but neither Ehrlich's aldehyde nor Schlesinger's reaction. No pigment was transferred into chloroform even if some quantities of hydrochloric acid were added to a mixture of chloroform and an aqueous solution of it, but did by saponification with 5% methanolic potash. 6. The absorption maxima of the natural ester-form bilirubin existed at 415 mμ in both methanolic and aqueous solutions.