A general assay for UDPglucuronosyltransferase activity using polar amino-cyano stationary phase HPLC and UDP[U-14C]glucuronic acid.
The assay of UDPglucuronosyltransferase activity toward various substrates using UDP[U-14C]glucuronic acid is described. HPLC on a polar amino-cyano bonded phase column was used to separate radioactive glucuronides from unmetabolized UDP[U-14C]glucuronic acid and other labeled reaction products. Radioactivity was measured using flow-through scintillation counting. All the glucuronides analyzed, with one exception, chromatographed with the same retention time (9.0-9.6 min) under the conditions described. Glucuronide conjugates were identified by comparison with retention times of commercial glucuronide standards, using radioactive aglycones, or hydrolysis with beta-glucuronidase. The method provides a unified, sensitive (100-200 pmol of glucuronide product) and reproducible assay for a wide variety of UDPglucuronosyltransferase substrates, and could be extended to include many others.
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005965:Glucuronates; D014453:Glucuronosyltransferase / Q000032:analysis*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D014535:Uridine Diphosphate Glucuronic Acid
D005965:Glucuronates; D014535:Uridine Diphosphate Glucuronic Acid; D014453:Glucuronosyltransferase