Determination of bilirubin glucuronide and assay of glucuronyltransferase with bilirubin as acceptor

Abstract

1. Conjugated bilirubin is conveniently determined by coupling with the diazonium salt of ethyl anthranilate. 2. This method has been used in the development of assays for UDP-glucuronyltransferase (EC 2.4.1.17), with bilirubin as substrate, in rat liver homogenates, microsomal preparations and partly purified fractions. 3. Chromatographic analysis suggests that bilirubin monoglucuronide is the product of the enzyme systems studied.

Heterogeneity of bile pigment conjugates as revealed by chromatography of their anthranilate azopigments

“…The present paper extends previous work with the diazonium salt of ethyl anthranilate (Van Roy & Heirwegh, 1968;Heirwegh, Meuwissen & Jansen, 1969;Black, Billing & Heirwegh, 1970) to the analysis of bile pigments in biological fluids. The coupling reaction, which proceeds at room temperature in aqueous medium at the less acid pH 2.7, offers maximal opportunity to detect acid-labile conjugates.…”

“…Bilirubin glucuronide (crude) was prepared from human fistula bile by the method of Lucassen (1961). Azobilirubin was prepared by coupling unconjugated bilirubin in chloroform with diazotized ethyl anthranilate in the presence of propan-1-ol or methanol (Van Roy & Heirwegh, 1968).…”

“…Concentrations of conjugated bilirubin (expressed in bilirubin equiv./l) are determined from the E530 values, determined on the organic phases, and 6530 44.4 x 103 litremol-h cm-' (Van Roy & Heirwegh, 1968). Blanks are prepared in parallel with the unknowns by omitting NaNO2 from the reaction mixtures.…”

“…Azopigment fractions were concentrated in vacuo and the E530 values determined. The concentrations in azobilirubin equiv./l were then calculated from 6530 22.2 x 103 litremol-1 cm-' (Van Roy & Heirwegh, 1968). Known volumes of the concentrates were further evaporated to dryness.…”

“…The oc0/S ratio remained approximately constant when the treatment was prolonged for 15-30min. In protein-containing systems coupling is slower but was found to be complete at 30min, when pigment was present that yielded equal amounts of azopigments a0 and 8 (Van Roy & Heirwegh, 1968). Colour development with human bile and with icteric urine is more gradual than with rat bile and reaches stable values only after 30-40 min.…”