A method for the determination of UDP-glucuronosyltransferase activity toward arylcarboxylic acids.
A fast and reliable method for the assay of UDP-glucuronosyltransferase (UGT) activity toward aglycones containing a carboxylic acid function is described. The procedure involves incubation with UDP-[U-14C]-glucuronic acid, the common substrate for the reaction, solid-phase separation of the radiolabeled acylglucuronides and unreacted cofactor, and quantification by liquid scintillation counting. The technique was validated for each of the seven substrates tested by reversed-phase HPLC, and was then applied successfully to the determination of optimal conditions for the activation of the carboxylic acid-UGT, and the estimation of kinetic constants for the glucuronidation of clofibric acid, 2-naphthylacetic acid, naproxen, and 4,4,4-triphenylbutanoic acid in rat liver microsomes. From the results obtained, we believe that this is an assay which, with only minor modification, could be applied to a wide range of carboxylic acid substrates for which, until now, specific and sensitive assays have been largely unavailable.
Pritchard
M
M
Centre du Médicament, URA CNRS 597, Faculté des Sciences, Pharmaceutiques et Biologiques, Nancy, France.
Fournel-Gigleux
S
S
Siest
G
G
Magdalou
J
J
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000378:metabolism; D002250:Carbon Radioisotopes; D002264:Carboxylic Acids / Q000378:metabolism*; D002853:Chromatography, Liquid; D002995:Clofibric Acid / Q000378:metabolism; D004789:Enzyme Activation; D005965:Glucuronates / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000378:metabolism*; D007052:Ibuprofen / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008722:Methods; D009288:Naproxen / Q000378:metabolism; D010666:Phenylpropionates / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D014535:Uridine Diphosphate Glucuronic Acid / Q000378:metabolism
D000894:Anti-Inflammatory Agents, Non-Steroidal; D002250:Carbon Radioisotopes; D002264:Carboxylic Acids; D005965:Glucuronates; D010666:Phenylpropionates; D014535:Uridine Diphosphate Glucuronic Acid; D002995:Clofibric Acid; D009288:Naproxen; D014453:Glucuronosyltransferase; C008924:pirprofen; D007052:Ibuprofen