Determination of hepatic uridine 5'-diphosphoglucuronic acid concentration by conjugation with diethylstilbestrol.
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 microCi/ml), and 200 microliters of boiled 10% liver homogenate in a total volume of 0.5 ml. After a 60-min incubation at 37 degrees 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 beta-glucuronidase to the aqueous phase, about 90% of the radioactivity could be extracted into chloroform, demonstrating the DES-glucuronic acid is the primary metabolite. Thus, this method easily permits quantitation of UDPGA in rat liver in the 1-10 nmol range.
D000818:Animals; D004054:Diethylstilbestrol / Q000378:metabolism*; D005965:Glucuronates / Q000378:metabolism; D006168:Guinea Pigs; D008099:Liver / Q000032:analysis*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D014535:Uridine Diphosphate Glucuronic Acid / Q000032:analysis*; D014539:Uridine Diphosphate Sugars / Q000032:analysis*
D005965:Glucuronates; D014539:Uridine Diphosphate Sugars; D014535:Uridine Diphosphate Glucuronic Acid; D004054:Diethylstilbestrol