Bilirubin conjugates of human bile. Isolation of phenylazo derivatives of bile bilirubin

Abstract

A method is presented that allows the isolation of eight different phenylazo derivatives of bile bilirubin. In step I of the isolation procedure, three bilirubin fractions (bilirubin fractions 1, 2 and 3) from human hepatic bile are separated by reverse-phase partition chromatography on silicone-treated Celite with the use of a solvent system prepared from butan-1-ol and 5mm-phosphate buffer, pH6.0. Azo coupling is then performed with diazotized aniline. The three azo pigment mixtures are subjected to step II, in which the above chromatography system is used again. With each azo pigment mixture this step brings about the separation of a non-polar and a polar azo pigment fraction (azo 1A and azo 1B, azo 2A and azo 2B, and azo 3A and azo 3B from bilirubin fractions 1, 2 and 3 respectively). Approximately equal amounts of non-polar and polar pigments are obtained from bilirubin fractions 1 and 2, whereas bilirubin fraction 3 yields azo 3B almost exclusively. In step IIIA the non-polar azo pigment fractions are fractionated further by adsorption chromatography on anhydrous sodium sulphate with the use of chloroform followed by a gradient of ethyl acetate in chloroform. Three azo pigments are thus obtained from both azo 2A (azo 2A1, azo 2A2 and azo 2A3) and azo 3A (azo 3A1, azo 3A2 and azo 3A3). The 2A pigments occur in approximately the following proportions: azo 2A1, 90%; azo 2A2, 10%; azo 2A3, traces. The pigments are purified by crystallization, except for the A3 pigments, which are probably degradation products arising from the corresponding A2 pigments. In step IIIB the polar azo pigment fractions are subjected to reverse-phase partition chromatography on silicone-treated Celite with the use of a solvent system prepared from octan-1-ol–di-isopropyl ether–ethyl acetate–methanol–0.2m-acetic acid (1:2:2:3:4, by vol.). Azo pigment fractions 2B and 3B each yield six azo pigments (azo 2B1 to azo 2B6 and azo 3B1 to azo 3B6 respectively) together with small amounts of products of hydrolysis (azo 2AB and azo 3AB). Only one azo B pigment is obtained from bilirubin fraction 1, and this azo pigment is probably of the B2 type. The yields of the azo 3B pigments suggest that these pigments are present in approximately the following proportions: azo 3B1, 0–0.4%; azo 3B2, traces; azo 3B3, traces; azo 3B4, 10%; azo 3B5, 50%; azo 3B6, 40%. Azo pigments 2B1 to 2B6 are estimated to occur in similar proportions. Since pairs of correspondingly numbered azo pigments from bilirubin fractions 1, 2 and 3 do not separate on rechromatography together (e.g. azo 2A1 co-chromatographs with azo 3A1, and azo 2B6 co-chromatographs with azo 3B6), it is concluded that such pigments are chemically identical. The structures of the isolated phenylazo derivatives are discussed in an accompanying paper (Kuenzle 1970c).