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Research ArticleArticle

Simultaneous Absolute Protein Quantification of Transporters, Cytochromes P450, and UDP-Glucuronosyltransferases as a Novel Approach for the Characterization of Individual Human Liver: Comparison with mRNA Levels and Activities

Sumio Ohtsuki, Olaf Schaefer, Hirotaka Kawakami, Tae Inoue, Stephanie Liehner, Asami Saito, Naoki Ishiguro, Wataru Kishimoto, Eva Ludwig-Schwellinger, Thomas Ebner and Tetsuya Terasaki
Drug Metabolism and Disposition January 2012, 40 (1) 83-92; DOI: https://doi.org/10.1124/dmd.111.042259
Sumio Ohtsuki
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Olaf Schaefer
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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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Naoki Ishiguro
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Wataru Kishimoto
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Eva Ludwig-Schwellinger
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Thomas Ebner
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Tetsuya Terasaki
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Abstract

The purpose of the present study was to determine the absolute protein expression levels of multiple drug-metabolizing enzymes and transporters in 17 human liver biopsies, and to compare them with the mRNA expression levels and functional activities to evaluate the suitability of the three measures as parameters of hepatic metabolism. Absolute protein expression levels of 13 cytochrome P450 (P450) enzymes, NADPH-P450 reductase (P450R) and 6 UDP-glucuronosyltransferase (UGT) enzymes in microsomal fraction, and 22 transporters in plasma membrane fraction were determined using liquid chromatography/tandem mass spectrometry. CYP2C9, CYP2E1, CYP3A4, CYP2A6, UGT1A6, UGT2B7, UGT2B15, and P450R were abundantly expressed (more than 50 pmol/mg protein) in human liver microsomes. The protein expression levels of CYP3A4, CYP2B6, and CYP2C8 were each highly correlated with the corresponding enzyme activity and mRNA expression levels, whereas for other P450s, the protein expression levels were better correlated with the enzyme activities than the mRNA expression levels were. Among transporters, the protein expression level of organic anion-transporting polypeptide 1B1 was relatively highly correlated with the mRNA expression level. However, other transporters showed almost no correlation. These findings indicate that protein expression levels determined by the present simultaneous quantification method are a useful parameter to assess differences of hepatic function between individuals.

Footnotes

  • This study was supported in part by grants for Development of Creative Technology Seeds Supporting Program for Creating University Ventures from Japan Science and Technology Agency (JST); Strategy Promotion of Innovative Research and Development from JST; and the Industrial Technology Research Grant Program from New Energy and the Industrial Technology Development Organization of Japan.

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

    http://dx.doi.org/10.1124/dmd.111.042259.

  • ↵Embedded Image The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.

  • ABBREVIATIONS:

    P450
    cytochrome P450
    ABC
    ATP-binding cassette
    BCRP
    breast cancer resistance protein
    CNT
    concentrative nucleoside transporter
    HPLC
    high-performance liquid chromatography
    LC/MS/MS
    liquid chromatography/tandem mass spectrometry
    LOQ
    lower limit of quantification
    MRM
    multiple reaction monitoring
    MRP
    multidrug resistance-associated protein
    NTCP
    sodium/taurocholating polypeptide
    OATP
    organic anion-transporting polypeptide
    OCT
    organic cation transporter
    OST
    organic solute transporter
    P450R
    NADPH-P450 reductase
    PCR
    polymerase chain reaction
    RT-PCR
    reverse transcription-PCR
    UGT
    UDP-glucuronosyltransferase
    MDR
    multidrug resistance protein
    BSEP
    bile salt export pump
    MATE
    multidrug and toxin extrusion
    Ct
    cycle threshold.

  • Received August 8, 2011.
  • Accepted October 12, 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

PROTEIN LEVELS OF P450, UGT, AND TRANSPORTERS IN HUMAN LIVER

Sumio Ohtsuki, Olaf Schaefer, Hirotaka Kawakami, Tae Inoue, Stephanie Liehner, Asami Saito, Naoki Ishiguro, Wataru Kishimoto, Eva Ludwig-Schwellinger, Thomas Ebner and Tetsuya Terasaki
Drug Metabolism and Disposition January 1, 2012, 40 (1) 83-92; DOI: https://doi.org/10.1124/dmd.111.042259

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Research ArticleArticle

PROTEIN LEVELS OF P450, UGT, AND TRANSPORTERS IN HUMAN LIVER

Sumio Ohtsuki, Olaf Schaefer, Hirotaka Kawakami, Tae Inoue, Stephanie Liehner, Asami Saito, Naoki Ishiguro, Wataru Kishimoto, Eva Ludwig-Schwellinger, Thomas Ebner and Tetsuya Terasaki
Drug Metabolism and Disposition January 1, 2012, 40 (1) 83-92; DOI: https://doi.org/10.1124/dmd.111.042259
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