PMID: 6408946Apr 15, 1983Paper

A novel assay of glucuronidation of C- and N-hydroxylated metabolites of the carcinogen N-2-fluorenylacetamide

Analytical Biochemistry
C N Ryzewski, D Malejka-Giganti

Abstract

A method for the assay of glucuronidation of C- and N-hydroxylated metabolites of the carcinogen N-2-fluorenylacetamide is described. The method employs UDP-[U-14C ))glucuronic acid and Baker C18 extraction columns for separation of the glucuronides from their aglycones and from the glucuronic acid. The 14C-labeled glucuronides, generated by rat liver microsomes, are eluted from the columns with 30% (v/v) methanol after prewashing the columns and elution of the radioactivity of 14C-glucuronic acid with 1 mM ammonium acetate, pH 6.9. The radioactivity of the eluates is measured by scintillation counting. The method is modified for assays of glucuronidation of alpha-naphthol and p-nitrophenol in that 1 mM phosphoric acid is used instead of 1 mM ammonium acetate, and the method is potentially adaptable to other aglycones. By monitoring radioactivity or uv absorbance of the column eluates, it is shown that all aglycones, except p-nitrophenol, are retained on the columns during elution of their glucuronides with 30% (v/v) methanol and are eluted only when absolute methanol is used. The identity of the glucuronides is shown by their response to hydrolysis by beta-glucuronidase in the presence and absence of D-saccharic acid-1,4-lacto...Continue Reading

References

Jul 15, 1975·Biochemical Pharmacology·J M ZieglerG Siest
Mar 15, 1975·Biochemical Pharmacology·M M JacobsonA H Conney
Aug 1, 1974·Biochemical Pharmacology·A Aitio
Sep 15, 1973·Biochemical Pharmacology·K W MillerJ V Dingell
Nov 1, 1971·The Biochemical Journal·K P Wong
Nov 4, 1969·Biochimica Et Biophysica Acta·A Winsnes
Oct 1, 1981·Xenobiotica; the Fate of Foreign Compounds in Biological Systems·H P Illing, E S House

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