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High-Performance Liquid Chromatographic Method Combining Radiochemical and Ultraviolet Detection for Determination of Low Activities of Uridine 5′-Diphosphate-Glucuronosyltransferase by Sanna Kaivosaari; Jarmo S. Salonen; Jonna Mortensen; Jyrki Taskinen is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

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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, levomed

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Author

Sanna Kaivosaari; Jarmo S. Salonen; Jonna Mortensen; Jyrki Taskinen

Publisher

Elsevier Science; Elsevier ; Elsevier Inc.; Elsevier BV (ISSN 0003-2697)

Published

2001

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)