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Research ArticleArticle

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

Leena Luukkanen, Jouni Mikkola, Tarja Forsman, Päivi Taavitsainen, Jyrki Taskinen and Eivor Elovaara
Drug Metabolism and Disposition August 2001, 29 (8) 1096-1101;
Leena Luukkanen
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Jouni Mikkola
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Tarja Forsman
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Päivi Taavitsainen
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Jyrki Taskinen
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Eivor Elovaara
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Abstract

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.

Footnotes

  • ↵1 Present address: Viikki Drug Discovery Technology Center, Department of Pharmacy, P.O. Box 56 (Viikinkaari 5E), FIN-00014 University of Helsinki, Finland.

  • This work was supported by the Commission of the European Communities, Biomed 2 program (BMH4-CT97-2621).

  • Abbreviations used are::
    1-HP
    1-hydroxypyrene
    PAH
    polycyclic aromatic hydrocarbon
    UGT
    UDP-glucuronosyltransferase
    UDPGA
    UDP-glucuronic acid
    HPGA
    1-pyrenylglucuronide
    DMSO
    dimethyl sulfoxide
    BSA
    bovine serum albumin
    HPLC
    high-performance liquid chromatography
    SFV
    Semliki Forest virus
    RSD
    relative standard deviation
    • Received January 9, 2001.
    • Accepted April 26, 2001.
  • The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 29 (8)
Drug Metabolism and Disposition
Vol. 29, Issue 8
1 Aug 2001
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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
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Research ArticleArticle

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

Leena Luukkanen, Jouni Mikkola, Tarja Forsman, Päivi Taavitsainen, Jyrki Taskinen and Eivor Elovaara
Drug Metabolism and Disposition August 1, 2001, 29 (8) 1096-1101;

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Research ArticleArticle

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

Leena Luukkanen, Jouni Mikkola, Tarja Forsman, Päivi Taavitsainen, Jyrki Taskinen and Eivor Elovaara
Drug Metabolism and Disposition August 1, 2001, 29 (8) 1096-1101;
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