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

Anal Biochem. 2001 May 15;292(2):178-87. doi: 10.1006/abio.2001.5092.


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.

MeSH terms

  • Aminobiphenyl Compounds / metabolism
  • Amitriptyline / chemistry
  • Amitriptyline / metabolism
  • Chromatography, High Pressure Liquid / methods*
  • Glucuronides / metabolism
  • Glucuronosyltransferase / analysis*
  • Glucuronosyltransferase / chemistry
  • Glucuronosyltransferase / metabolism*
  • Humans
  • Imidazoles / metabolism
  • Kinetics
  • Medetomidine / metabolism
  • Microsomes, Liver / enzymology
  • Nitrophenols / metabolism
  • Radiochemistry*
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Spectrometry, Mass, Electrospray Ionization
  • Substrate Specificity
  • Time Factors
  • Ultraviolet Rays


  • Aminobiphenyl Compounds
  • Glucuronides
  • Imidazoles
  • Nitrophenols
  • atipamezole
  • 4-biphenylamine
  • Amitriptyline
  • 4(5)-(2,6-dimethylbenzyl)imidazole
  • 4(5)-2-(2,6-dimethylphenyl)ethylimidazole
  • detomidine
  • Glucuronosyltransferase
  • Medetomidine
  • 4-nitrophenol