Excretion in dog bile of glucose and xylose conjugates of bilirubin

1. T.l.c. with neutral solvent systems of ethyl anthranilate azopigments derived from bile of man, dog and rat revealed pronounced species variation. The less polar components (α-group) could be separated conveniently by development with chloroform–methanol (17:3, v/v). 2. The azopigment material derived from gallbladder bile of dog contained about 10% of azobilirubin β-d-monoxyloside (azopigment α₂) and 30% of azobilirubin β-d-monoglucoside (azopigment α₃). The sugar moieties were identified by t.l.c. with acidic, neutral and basic solvent systems and by anion-exchange column chromatography of their boric acid complexes. Treatment of the purified azopigments with ammonia vapour led to the formation of the amide of azobilirubin, indicating that both pigments are ester glycosides. The β-d configuration was demonstrated by enzymic studies with emulsin (an adequate source of β-glucosidase activity) and with Mylase-P (an adequate source of β-glucosidase and β-xylosidase activities). 3. Hydrolysis studies with model substrates and with the α₂- and α₃-azopigments suggested that in Mylase-P the β-glucosidase and β-xylosidase activities reside in separate enzymes. 4. Compared with the accepted conjugation with glucuronic acid as a major route of detoxication in mammals, the detection of large amounts of xylose and glucose conjugates of bilirubin in dog bile suggests that the underlying biosynthetic pathways may be important alternative routes of detoxication.