A novel assay of glucuronidation of C- and N-hydroxylated metabolites of the carcinogen N-2-fluorenylacetamide.
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-lactone and, in some instances, by chromatographic and spectral analyses of the released aglycones.
D015073:2-Acetylaminofluorene / Q000378:metabolism*; D000818:Animals; D003902:Detergents; D005260:Female; D005937:Glucaric Acid; D005965:Glucuronates / Q000302:isolation & purification*; D005966:Glucuronidase / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000378:metabolism; D006868:Hydrolysis; D006900:Hydroxylation; D066298:In Vitro Techniques; D007783:Lactones / Q000737:chemistry; D008862:Microsomes, Liver / Q000378:metabolism; D051381:Rats
C571832:D-Glucaric acid, 1,4-lactone; D003902:Detergents; D005965:Glucuronates; D007783:Lactones; D015073:2-Acetylaminofluorene; D014453:Glucuronosyltransferase; D005966:Glucuronidase; D005937:Glucaric Acid