Elsevier

Analytical Biochemistry

Volume 159, Issue 1, 15 November 1986, Pages 198-205
Analytical Biochemistry

A general assay for UDPglucuronosyltransferase activity using polar amino-cyano stationary phase HPLC and UDP[U-14C]glucuronic acid

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Abstract

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 (a) comparison with retention times of commercial glucuronide standards, (b) using radioactive aglycones, or (c) hydrolysis with β-glucuronidase. The method provides a unified, sensitive (100–200 pmol of glucuronide product) and reproducible assay for a wide variety of UDPglucurono-syltransferase substrates, and could be extended to include many others.

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    Present address: Department of Pharmacology and Toxicology, University of Western Ontario, London, Ontario, Canada N6A 5C1.

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