RT Journal Article
SR Electronic
T1 Glucuronidation of 1-Hydroxypyrene by Human Liver Microsomes and Human UDP-Glucuronosyltransferases UGT1A6, UGT1A7, and UGT1A9: Development of a High-Sensitivity Glucuronidation Assay for Human Tissue
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP 1096
OP 1101
VO 29
IS 8
A1 Leena Luukkanen
A1 Jouni Mikkola
A1 Tarja Forsman
A1 Päivi Taavitsainen
A1 Jyrki Taskinen
A1 Eivor Elovaara
YR 2001
UL http://dmd.aspetjournals.org/content/29/8/1096.abstract
AB Human UDP-glucuronosyltransferases (UGT, EC 2.4.1.17) involved in the biotransformation of pyrene were investigated by a sensitive fluorometric high-performance liquid chromatography (HPLC)method developed for determining activities toward 1-hydroxypyrene. The endpoint metabolite of pyrene, 1-pyrenylglucuronide, is a well-known urinary biomarker for the assessment of human exposure to polycyclic aromatic hydrocarbons. 1-Pyrenylglucuronide was synthesized using rat liver microsomes as biocatalyst. The yield was satisfactory, 22%. 1-Pyrenylglucuronide, identified by 1H NMR and by electrospray mass spectrometry, was used for method validation and calibration. The HPLC assay was very sensitive with a quantitation limit of 3 pg (8 fmol) for 1-pyrenylglucuronide. The assay was precise, showing a relative standard deviation of 5% or less at 0.1 to 300 μM 1-hydroxypyrene. Only 2 μg of microsomal protein was required for the assay in human liver. The glucuronidation of 1-hydroxypyrene was catalyzed at high rates in microsomes from pooled or three individual liver samples, showing comparable apparentKm values. The formation of 1-pyrenylglucuronide was catalyzed by recombinant human UGT1A6, UGT1A7, and UGT1A9, the Km values being 45, 12, and 1 μM, respectively. The apparent Km values in human liver microsomes, ranging from 6.9 to 8.6 μM, agreed well with these results. The method provides a sensitive tool for measuring extremely low UGT activities and a specific means for assessing interindividual differences in 1-hydroxypyrene-metabolizing UGT activities in human liver and other tissues. The American Society for Pharmacology and Experimental Therapeutics