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Research ArticleArticle

Absolute Quantification and Differential Expression of Drug Transporters, Cytochrome P450 Enzymes, and UDP-Glucuronosyltransferases in Cultured Primary Human Hepatocytes

Olaf Schaefer, Sumio Ohtsuki, Hirotaka Kawakami, Tae Inoue, Stephanie Liehner, Asami Saito, Atsushi Sakamoto, Naoki Ishiguro, Takehisa Matsumaru, Tetsuya Terasaki and Thomas Ebner
Drug Metabolism and Disposition January 2012, 40 (1) 93-103; DOI: https://doi.org/10.1124/dmd.111.042275
Olaf Schaefer
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Sumio Ohtsuki
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Hirotaka Kawakami
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Tae Inoue
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Stephanie Liehner
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Asami Saito
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Atsushi Sakamoto
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Naoki Ishiguro
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Takehisa Matsumaru
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Tetsuya Terasaki
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Thomas Ebner
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Abstract

The levels of metabolizing enzymes and transporters expressed in hepatocytes are decisive factors for hepatobiliary disposition of most drugs. Induction via nuclear receptor activation can significantly alter those levels, with the coregulation of multiple enzymes and transporters occurring to different extents. Here, we report the use of a targeted liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method for concurrent quantification of multiple cytochrome P450 (P450), UDP-glucuronosyltransferase (UGT), and transporter proteins in cultured primary human hepatocytes. The effects of culture format (i.e., sandwich culture versus conventional culture) and of dexamethasone (DEX) media concentrations on mRNA, protein, and activity levels were determined for three donors, and protein expression was compared with that in liver. In general, P450 and UGT expression was lower in hepatocyte cultures than that in liver, and CYP2C9 was found to be the most abundant P450 isoform expressed in cultured hepatocytes. The sandwich culture format and 0.1 μM DEX in media retained the protein expression in the hepatocytes closest to the levels found in liver. However, higher in vitro expression was observed for drug transporters, especially for multidrug resistance protein 1 and breast cancer resistance protein. Direct protein quantification was applied successfully to study in vitro induction in sandwich cultured primary hepatocytes in a 24-well format using the prototypical inducers rifampicin, omeprazole, and phenobarbital. We conclude that targeted absolute LC-MS/MS quantification of drug-metabolizing enzymes and transporters can broaden the scope and significantly increase the impact of in vitro drug metabolism studies, such as induction, as an important supplement or future alternative to mRNA and activity data.

Footnotes

  • Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

    http://dx.doi.org/10.1124/dmd.111.042275.

  • ABBREVIATIONS:

    P450
    cytochrome P450
    ABC
    ATP-binding cassette protein
    BCRP
    breast cancer resistance protein
    BSEP
    bile salt export pump
    CNT
    concentrative nucleoside transporter
    DEX
    dexamethasone
    ECM
    extracellular matrix protein
    ENT
    equilibrative nucleoside transporter
    γ-GTP
    γ-glutamyl transpeptidase
    HuHep
    human hepatocyte preparation
    LC-MS/MS
    liquid chromatography coupled to tandem mass spectrometry
    LOQ
    limit of quantification
    MATE
    multidrug and toxin extrusion protein
    MCT
    monocarboxylate transporter
    mio
    million
    MDR
    multidrug resistance protein
    MRP
    multidrug resistance-associated protein
    MRM
    multiple reaction monitoring
    NTCP
    sodium/taurocholate cotransporting polypeptide
    OATP
    organic anion-transporting polypeptide
    OCT
    organic cation transporter
    OME
    omeprazole
    PB
    phenobarbital
    RIF
    rifampicin
    RT-PCR
    reverse transcription-polymerase chain reaction
    UGT
    UDP-glucuronosyltransferase
    WME
    Williams' medium E
    Ct
    cycle threshold.

  • Received August 8, 2011.
  • Accepted October 5, 2011.
  • Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 40 (1)
Drug Metabolism and Disposition
Vol. 40, Issue 1
1 Jan 2012
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Research ArticleArticle

ABSOLUTE PROTEIN EXPRESSION IN CULTURED HUMAN HEPATOCYTES

Olaf Schaefer, Sumio Ohtsuki, Hirotaka Kawakami, Tae Inoue, Stephanie Liehner, Asami Saito, Atsushi Sakamoto, Naoki Ishiguro, Takehisa Matsumaru, Tetsuya Terasaki and Thomas Ebner
Drug Metabolism and Disposition January 1, 2012, 40 (1) 93-103; DOI: https://doi.org/10.1124/dmd.111.042275

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Research ArticleArticle

ABSOLUTE PROTEIN EXPRESSION IN CULTURED HUMAN HEPATOCYTES

Olaf Schaefer, Sumio Ohtsuki, Hirotaka Kawakami, Tae Inoue, Stephanie Liehner, Asami Saito, Atsushi Sakamoto, Naoki Ishiguro, Takehisa Matsumaru, Tetsuya Terasaki and Thomas Ebner
Drug Metabolism and Disposition January 1, 2012, 40 (1) 93-103; DOI: https://doi.org/10.1124/dmd.111.042275
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