Glucuronic acid conjugates of bilirubin-IXα in normal bile compared with post-obstructive bile. Transformation of the 1-O-acylglucuronide into 2-, 3-, and 4-O-acylglucuronides

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Abstract
Structures were determined for bilirubin-IX.alpha. conjugates in freshly collected bile of normal rats, dogs and man and in post-obstructive bile of man and rats. The originally secreted conjugate was characterized as azopigment (I), i.e., a 1-O-acyl-.beta.-D-glucopyranuronic acid glycoside. Conversion of the acetylated methyl ester of azopigment (I) into methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-.beta.-D-glucopyranurnate (V) indicates the pyranose ring structure for the carbohydrate and a C-1 attachment for the bilirubin-IX.alpha. acyl group. Alternative procedures for deconjugation of azopigment (I) and its derivatives are also described. In post-obstructuve bile, the 1-O-acylglucuronide is converted into 2-,3- and 4-O-acylglucuronides via sequential intramolecular migrations of the bilirubin acyl group. The following approach was utilized. The tetrapyrole conjugates were cleaved to dipyrrolic aniline and ethyl anthranilate azopigments, and the azopigments were separated as the acids or methyl esters. The isomeric methyl esters were characterized by mass spectral analysis of the acetates and silyl ethers. The free glycosidic function was demonstrated by 1-oxime and 1-methoxime derivative formation. The position of the dipyrrolic O-acyl group was determined for the methyl esters by protecting the free hydroxyl groups of the glucuronic acid moieties as the acetals formed with ethyl vinyl ether and by further conversion of the carbohydrates into partially methylated alditol acetates. These were analyzed by using GLC-mass spectrometry. The relevance of these results with regard previous reports on disaccharidic conjugates is discussed.