The isolation of an azobilirubin β-d-monoglucoside from dog gall-bladder bile

An ethyl anthranilate azopigment of bilirubin conjugated to β-d-monoglucoside was isolated from dog gall-bladder bile. Glucose was cleaved from the azopigment by treatment with β-glucosidase and β-glucuronidase. Mild alkaline hydrolysis of the compound by sodium methoxide yielded two kinds of compounds, water-soluble and organic-soluble. The former were shown, by enzymic analysis, t.l.c., nuclear magnetic resonance, and combined g.l.c. and mass spectrometry, to contain glucose. No evidence was obtained from these data that a disaccharide was present in this fraction. The organic-soluble compounds formed during this methanolysis were shown, by t.l.c. and mass spectrometry, to be the isomeric dipyrrole azopigments of bilirubin. These findings contribute further evidence to the controversy surrounding the nature of conjugated bilirubin.