TY - JOUR 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 SP - 1096 LP - 1101 VL - 29 IS - 8 AU - Leena Luukkanen AU - Jouni Mikkola AU - Tarja Forsman AU - Päivi Taavitsainen AU - Jyrki Taskinen AU - Eivor Elovaara Y1 - 2001/08/01 UR - http://dmd.aspetjournals.org/content/29/8/1096.abstract N2 - 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 ER -