High-performance liquid chromatographic method combining radiochemical andultraviolet detection for determination of low activities of uridine 5 '-diphosphate-glucuronosyltransferase

A novel reversed-phase high-performance liquid chromatographic method was d eveloped to measure UDP-glucuronosyltransferase (UGT) activity. Radiochemic al and UV detection were combined in this UDP-[C-14]glucuronic acid-utilizi ng method which was especially aimed at determination of low activities typ ical of N-glucuronidation of various amines and heterocycles. 4-Nitrophenol and levomedetomidine were used as substrates to validate this method, and applicability was tested with commonly used model substrates of N-glucuroni dation, 4-aminobiphenyl and amitriptyline, and several 4-arylalkyl-1H-imida zole compounds. Detection limits were very low, 0.5-10 pmol, corresponding to UGT activities from 0.04 to 0.8 pmol/min/mg protein depending on UV abso rbance of the glucuronide conjugate. The sensitivity was 10- to 100-fold co mpared with earlier HPLC assays using radiochemical detection. This method enabled quantitation without a reference glucuronide, and its high sensitiv ity allows for characterization of N-glucuronidation kinetics of various su bstrates. Using this method, human liver microsomal UGT activity was determ ined for a series of 4-arylalkyl-1H-imidazoles. Of these compounds, levomed etomidine was glucuronidated at the highest rate, 1.69 nmol/min/mg protein, using a 500 muM substrate concentration. In comparison, activities for the commonly used UGT substrates, 4-nitrophenol, 4-aminobiphenyl, and amitript yline were 18.80, 3.23, and 0.23 nmol/min/mg protein, respectively, (C) 200 1 Academic Press.