The isolation and characterization of bilirubin diglucuronide, the major bilirubin conjugate in dog and human bile

Abstract

The chemical structure of the major conjugate of bilirubin was unequivocally elucidated by structural analysis. The conjugated bilirubins were first separated from the lipid components of human duodenal aspirates or dog gall-bladder bile, and then resolved by TLC into a series of tetrapyrroles. The major tetrapyrrole was then converted into its more stable dipyrrolic azo derivative for further analysis. The conjugated moiety of the azopigment was characterized after methanolysis with sodium methoxide. This reaction yields 2 products, those soluble in H2O and those soluble in organic solvents. The organic-soluble fraction was shown by TLC and mass spectrometry to contain the methyl esters of the dipyrrolic azo derivatives of bilirubin. The water-soluble materials were analyzed by enzymic procedures, TLC, NMR spectrometry and combined gas-liquid chromaotgraphy (g.l.c.) and mass spectrometry. This analysis showed that the only water-soluble product resulting from the methanolysis was glucuronic acid. The structure was identical with that of pure standards, on both mass spectrometry and NMR spectroscopy. No contaminating moieties were found. Quantitative measurement indicated that the glucuronic acid was released in a 1:1 molar ratio with the resulting methyl esters of the dipyrrolic azo derivatives of bilirubin. This unequivocally establishes bilirubin diglucuronide as the major pigment present in bile. Past problems with identification of bilirubin diglucuronide were shown to originate from procedures which resulted in incomplete separation and isolation of the azopigments of the conjugated bilirubins, owing to contamination by biliary lipids.