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 language | English (US) |
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Pages (from-to) | 192-197 |
Number of pages | 6 |
Journal | Methods in enzymology |
Volume | 77 |
Issue number | C |
DOIs | |
State | Published - Jan 1 1981 |
Externally published | Yes |
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology