Liquid chromatographic assay for the measurement of glucuronidation of arylcarboxylic acids using uridine diphospho-[U-14C] glucuronic acid.
A general method for the assay of UDP-glucuronosyltransferase activity towards arylcarboxylic acids (clofibric acid, 1- and 2-naphthylacetic acid) using UDP-[U-14C] glucuronic acid in liver microsomes is described. The 14C-labelled glucuronide was separated by high-performance liquid chromatography, identified by hydrolysis by beta-glucuronidase, characterized by laser desorption mass spectrometry and quantified by scintillation counting. The coefficient of variation of the enzyme activity for the inter-assay repeatability was below 4.5%. As little as 2.5 nmol of the arylcarboxylic acid glucuronides could be detected and precisely quantified. The method was applied to the determination of the apparent kinetic constants for glucuronidation of the acids. Clofibric acid was the best substrate for UDP-glucuronosyltransferase (Vmax/KM, the ratio of the maximum initial velocity and the Michaelis-Menten constant, is 12.3). The two isomers, 1- and 2-naphthylacetic acids, were transformed at a similar rate. However, they exhibited different enzymatic affinities, as the KM values were 1.0 mM and 5.6 mM for 1- and 2-naphthylacetic acid, respectively. This indicates that the spatial organization of the substrates played a critical role in this acyl glucuronoconjugation.
D000818:Animals; D002264:Carboxylic Acids / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D014453:Glucuronosyltransferase / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000201:enzymology; D009284:Naphthols; D051381:Rats; D011919:Rats, Inbred Strains; D014532:Uridine Diphosphate Glucose / Q000032:analysis*; D014539:Uridine Diphosphate Sugars / Q000032:analysis*
D002264:Carboxylic Acids; D009284:Naphthols; D014539:Uridine Diphosphate Sugars; C005366:naphthyl acetate; D014453:Glucuronosyltransferase; D014532:Uridine Diphosphate Glucose