RADIOASSAY OF UDP-GLUCURONOSYLTRANSFERASE ACTIVITIES TOWARD ENDOGENOUS SUBSTRATES USING LABELED UDP-GLUCURONIC ACID AND AN ORGANIC-SOLVENT EXTRACTION PROCEDURE

A rapid and sensitive radioassay for measuring UDP-glucuronosyltransferase activities (EC 2.4.1.17) toward the major endogenous substrates hyodeoxycholic and hyocholic acids, bilirubin, estriol, androsterone, and testosterone has been developed. In this assay, C-14-labeled glucuronides are formed from the enzyme-catalyzed reaction of C-14-labeled UDP-glucuronic acid with the unlabeled aglycones. Following incubation, the C-14-labeled glucuronides are separated under acidic conditions from the unreacted C-14-labeled UDP-glucuronic acid by a single extraction with ethyl acetate. The recovery of glucuronides into ethyl acetate was greater than 90%, whereas the carryover of unreacted UDP-glucuronic acid into the organic phase was approximately 0.2%. The reaction products extracted into ethyl acetate were characterized by their mobilities in thin-layer chromatography and identified as glucuronides by their sensitivity to hydrolysis with beta-glucuronidase and inhibition of hydrolysis by the specific beta-glucuronidase inhibitor D-saccharic acid-1,4-lactone. The optimal conditions of enzyme reactions with the individual aglycones have been defined with human liver microsomes as enzyme source. For all aglycones investigated, 10-30 mu g of microsomal protein are sufficient for enzyme estimation. The assay is applicable to biochemical studies of UDP-glucuronosyltransferases, as well as to measurement of these enzyme activities from small amounts of clinical liver specimens. (C) 1994 Academic Press,Inc.