High-performance liquid chromatographic method combining radiochemical and ultraviolet detection for determination of low activities of uridine 5'-diphosphate-glucuronosyltransferase.
A novel reversed-phase high-performance liquid chromatographic method was developed to measure UDP-glucuronosyltransferase (UGT) activity. Radiochemical and UV detection were combined in this UDP-[(14)C]glucuronic acid-utilizing method which was especially aimed at determination of low activities typical 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-glucuronidation, 4-aminobiphenyl and amitriptyline, and several 4-arylalkyl-1H-imidazole 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 absorbance of the glucuronide conjugate. The sensitivity was 10- to 100-fold compared with earlier HPLC assays using radiochemical detection. This method enabled quantitation without a reference glucuronide, and its high sensitivity allows for characterization of N-glucuronidation kinetics of various substrates. Using this method, human liver microsomal UGT activity was determined for a series of 4-arylalkyl-1H-imidazoles. Of these compounds, levomedetomidine was glucuronidated at the highest rate, 1.69 nmol/min/mg protein, using a 500 microM substrate concentration. In comparison, activities for the commonly used UGT substrates, 4-nitrophenol, 4-aminobiphenyl, and amitriptyline were 18.80, 3.23, and 0.23 nmol/min/mg protein, respectively.
Kaivosaari
S
S
Department of Pharmacy, University of Helsinki, Helsinki, FIN-00014, Finland.
Salonen
J S
JS
Mortensen
J
J
Taskinen
J
J
D000611:Aminobiphenyl Compounds / Q000378:metabolism; D000639:Amitriptyline / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D020719:Glucuronides / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007093:Imidazoles / Q000378:metabolism; D007700:Kinetics; D020926:Medetomidine / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology; D009596:Nitrophenols / Q000378:metabolism; D011854:Radiochemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D013997:Time Factors; D014466:Ultraviolet Rays
D000611:Aminobiphenyl Compounds; D020719:Glucuronides; D007093:Imidazoles; D009596:Nitrophenols; C050701:atipamezole; C006757:4-biphenylamine; D000639:Amitriptyline; C037818:4(5)-(2,6-dimethylbenzyl)imidazole; C038226:4(5)-2-(2,6-dimethylphenyl)ethylimidazole; C041255:detomidine; D014453:Glucuronosyltransferase; D020926:Medetomidine; C024836:4-nitrophenol