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-glucuronosyltransfe rase activities (EC 2.4.1.17) toward the major endogenous substrates h yodeoxycholic and hyocholic acids, bilirubin, estriol, androsterone, a nd testosterone has been developed. In this assay, C-14-labeled glucur onides are formed from the enzyme-catalyzed reaction of C-14-labeled U DP-glucuronic acid with the unlabeled aglycones. Following incubation, the C-14-labeled glucuronides are separated under acidic conditions f rom the unreacted C-14-labeled UDP-glucuronic acid by a single extract ion with ethyl acetate. The recovery of glucuronides into ethyl acetat e was greater than 90%, whereas the carryover of unreacted UDP-glucuro nic acid into the organic phase was approximately 0.2%. The reaction p roducts extracted into ethyl acetate were characterized by their mobil ities in thin-layer chromatography and identified as glucuronides by t heir 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 microsom al protein are sufficient for enzyme estimation. The assay is applicab le to biochemical studies of UDP-glucuronosyltransferases, as well as to measurement of these enzyme activities from small amounts of clinic al liver specimens. (C) 1994 Academic Press,Inc.