Radioassay of UDP-glucuronosyltransferase activities toward endogenous substrates using labeled UDP-glucuronic acid and an organic solvent extraction procedure

Anal Biochem. 1994 Jun;219(2):182-8. doi: 10.1006/abio.1994.1255.


A rapid and sensitive radioassay for measuring UDP-glucuronosyltransferase activities (EC 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autoradiography / methods*
  • Carbon Radioisotopes
  • Glucuronosyltransferase / analysis*
  • Glucuronosyltransferase / metabolism
  • Hydrogen-Ion Concentration
  • Kinetics
  • Sensitivity and Specificity
  • Solvents
  • Substrate Specificity
  • Uridine Diphosphate Glucuronic Acid / metabolism*


  • Carbon Radioisotopes
  • Solvents
  • Uridine Diphosphate Glucuronic Acid
  • Glucuronosyltransferase