Purification of Conjugated Bilirubin: A New Approach Utilizing Albumin-Agarose Gel Affinity Chromatography

Bilirubin, an organic anion and breakdown product of heme, is excreted chiefly by the liver by a process involving uptake by the hepatocyte and subsequent conjugation to form a more polar compound (1). The availability of purified preparations of bilirubin, especially of radiolabeled bilirubin, has greatly facilitated studies of its metabolism (1-5). Study of the transport of bilirubin from blood to bile, however, must also include consideration of the mechanisms for excretion of bilirubin conjugates. Such study has been limited because of difficulty in obtaining conjugated bilirubin free of significant quantities of contaminants (6-19).Recent studies of albumin-agarose gel affinity chromatography have revealed that the gels have a high affinity for bilirubin and a relatively low affinity and capacity for tri-and dihydroxy bile acids (20, 21), suggesting that this method might effectively separate bilirubin conjugates from other components in bile. We describe here, a rapid and relatively simple method for purification of conjugated bilirubin from bile.Materials and methods.Albumin-agarose gel was prepared by a modification of the cyanogen bromide method (20, 22), and packed in small columns of 0.9-cm i.d. Bile acids were quantitated in bile and column eluates by gas-liquid chromatography (23), cholesterol by the method of Zak et al.(24), and phospholipids by a modification of the method of Chen et al.(25, 26).Absorption spectra were obtained with a Beckman Acta III spectrophotometer, and fluorimetric analyses with a Farrand Mark I spectrofluorimeter.Tritium was quantitated by adding up to 0.5 ml of sample to 15 ml of Aquasol (New England Nuclear) and by counting in a liquid scintillation counter (Nuclear Chicago).All samples were recounted after addition of [3H]toluene (New England Nuclear) and results were corrected for background and quenching.