A simple procedure for the isolation of bilirubin monoglucuronide and diglucuronide from bile

Isolation of bilirubin monoglucuronide and diglucuronide from rat and human bile has been accomplished by affinity chromatography over albumin agarose, followed by reverse-phase adsorption chromatography over siliconized kieselguhr. The glucuronide nature of the conjugating saccharide was confirmed by TLC, employing two different solvent systems, of the sugar released by either alkaline hydrolysis or ammonolysis. The bilirubin diglucuronide preparation migrated as a single polar band on direct silica gel TLC, yielded exclusively the delta ethyl anthranilate azopigment, and had a sugar:bile pigment molar ratio of 2.01 +/- 0.03 (S.E.M.). The monoglucuronide migrated as a single, less polar band on TLC, yielded equimolar quantities of alpha and delta azopigments, and had a sugar:bile pigment ratio of 1.02 +/0- 0.04. Both conjugates were preparable in milligram quantities, free of phospholipid, bile acid, and cholesterol, at yields averaging 40% to 60%, and could be isolated with high specific activity by injection of pheonbarbital-penetrated rats with delta-aminolevulinic acid-4- 14C or -2,3-3H. Preparations of pure bilirubin conjugates should greatly facilitate studies of bilirubin metabolism.