Dismutation of Bilirubin Monoglucuronide

J. Roy Chowdhury, Irwin M. Arias

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

Bilirubin monoglucuronide is the major pigment in the human and rat bile. The dismutation of bilirubin monoglucuronide occurs at a normal rate in vitro in the liver of uridine diphosphate glucuronosyltransferase deficient man and rat. This chapter presents a procedure for the isolation of azopigmcnts. Preparations procedure involves the preparation of rat liver microsomes; the biosynthesis of bilirubin monoglucuronide; and the preparation of ethyl anthranilate diazo reagent. In the assay, the enzyme suspension is incubated with sodium phosphate at pH 6.6 containing glucaro-l,4-lactonc. Bilirubin monoglucuronide is dissolved in Tris-HCl at pH 7.8 and 0.05 ml is added to the enzyme–buffer mixture. After incubation at 37 ° for 3 min, the reaction is stopped with 2 ml ice-cold ethyl anthranilate diazo reagent. After incubation at 25 ° for 30min, 1ml of 20% ascorbic acid is added and azopigmcnts are extracted in 0.5 ml of 2-pentanone : butyl acetate. The organic solvent extract is separated by centrifugation and azopigments arc analyzed by thin-layer chromatography or high performance liquid chromatography.

Original languageEnglish (US)
Pages (from-to)192-197
Number of pages6
JournalMethods in Enzymology
Volume77
Issue numberC
DOIs
Publication statusPublished - Jan 1 1981
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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