Determination of hepatic uridine 5′-diphosphoglucuronic acid concentration by conjugation with diethylstilbestrol

J. B. Watkins, C. D. Klaassen

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Abstract

A sensitive and reliable assay for uridine 5′-diphosphoglucuronic acid (UDPGA) was developed that involved conjugation of diethylstilbestrol (DES) in vitro. This conjugation reaction is solely dependent upon UDPGA concentration. The assay uses 0.13 M Tris-HCl, pH 7.4, 6.7 mM MgCl2, 0.05% Brig 58, 0.25 mg guinea pig liver microsomal protein, 0.13 mM 3H-DES (0.2 μCi/ml), and 200 μl of boiled 10% liver homogenate in a total volume of 0.5 ml. After a 60-min incubation at 37°C, unconjugated DES is extracted into 5 ml of chloroform and the residual metabolized 3H-DES in the aqueous phase is determined by liquid scintillation spectrometry. After addition of ß-glucuronidase to the aqueous phase, about 90% of the radioactivity could be extracted into chloroform, demonstrating that DES-glucuronic acid is the primary metabolite. Thus, this method easily permits quantitation of UDPGA in rat liver in the 1-10 nmol range.

Original languageEnglish (US)
Pages (from-to)145-151
Number of pages7
JournalJournal of Pharmacological Methods
Volume7
Issue number2
DOIs
StatePublished - Mar 1982

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Keywords

  • Glucuronidation of diethylstilbestrol
  • Glucuronyltransferase
  • Uridine 5′-diphosphoglucuronic acid determination

ASJC Scopus subject areas

  • Pharmacology

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