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, 14C-labeled glucuronides are formed from the enzyme-catalyzed reaction of 14C-labeled UDP-glucuronic acid with the unlabeled aglycones. Following incubation, the 14C-labeled glucuronides are separated under acidic conditions from the unreacted 14C-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 micrograms 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.
Matern
H
H
Department of Internal Medicine III, Aachen University of Technology, Germany.
Heinemann
H
H
Matern
S
S
D001345:Autoradiography / Q000379:methods*; D002250:Carbon Radioisotopes; D014453:Glucuronosyltransferase / Q000032:analysis* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D012680:Sensitivity and Specificity; D012997:Solvents; D013379:Substrate Specificity; D014535:Uridine Diphosphate Glucuronic Acid / Q000378:metabolism*
D002250:Carbon Radioisotopes; D012997:Solvents; D014535:Uridine Diphosphate Glucuronic Acid; D014453:Glucuronosyltransferase