Determination of UDP-glucuronosyltransferase UGT1A6 activity in human and rat liver microsomes by HPLC with UV detection.
A simple and sensitive method for the determination of UDP-glucuronosyltransferase UGT1A6 activity using 4-methylumbelliferone (4-MU) and 4-nitrophenol (4-NP) as substrates in human and rat liver microsomes by high-performance liquid chromatography (HPLC) with uv detection is reported. The method was validated for the determination of 4-methylumbelliferyl beta-D-glucuronide (4-MUG) and 4-nitrophenyl beta-D-glucuronide (4-NPG) with respect to specificity, linearity, detection limit, recovery, stability, precision and accuracy. There was no interference from matrix and non-enzymatic reactions. Calibration curves for 4-MUG and 4-NPG are linear from 0.5 to 500 microM. Average recoveries ranged from 98 to 100% in spiked liver microsomes samples. 4-MUG and 4-NPG were stable at 4 degrees C for at least 72 h in spiked liver microsomes samples. The method was found to be more sensitive than previous methods using a spectrophotometer, a spectrofluorometer and HPLC. The detection limit for 4-MUG and 4-NPG (signal-to-noise ratio of 3) was 14 and 23 nM, respectively. The intra- and inter-day precision (relative S.D. (RSD)) and accuracy (relative mean error (RME)) was <5 and 9%, respectively. The intra- and inter-day reproducibility (RSD) of UGT1A6 enzyme assay in liver microsomes was <6%. With this improved sensitivity, the kinetics of UGT activities toward 4-MU and 4-NP in human and rat liver microsomes could be determined more precisely. In addition, the method could determine the non-inducible, and 3-methylcholanthrene- and phenobarbital-inducible activities of UGT1A6 in rat liver microsomes under the same assay conditions. Therefore, this method is applicable to in vivo and in vitro studies on the interaction of xenobiotic chemicals with UGT1A6 isoform in mammals using small amounts of biological samples.
Division of Environmental Chemistry, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. hanioka@nihs.go.jp
N
N
Hanioka
H
H
Jinno
T
T
Tanaka-Kagawa
T
T
Nishimura
M
M
Ando
D000818:Animals; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004790:Enzyme Induction; D004795:Enzyme Stability; D005965:Glucuronates / Q000737:chemistry; D014453:Glucuronosyltransferase / Q000032:analysis*; D006801:Humans; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D009596:Nitrophenols / Q000378:metabolism; D011786:Quality Control; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
D005965:Glucuronates; D009596:Nitrophenols; C110655:UDP-glucuronosyltransferase, UGT1A6; D014453:Glucuronosyltransferase; C024836:4-nitrophenol