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Research ArticleArticle

Optimized Methods for Targeted Peptide-Based Quantification of Human Uridine 5′-Diphosphate-Glucuronosyltransferases in Biological Specimens Using Liquid Chromatography–Tandem Mass Spectrometry

Yuichiro Sato, Masanori Nagata, Kazuhiro Tetsuka, Kouichi Tamura, Aiji Miyashita, Akio Kawamura and Takashi Usui
Drug Metabolism and Disposition May 2014, 42 (5) 885-889; DOI: https://doi.org/10.1124/dmd.113.056291
Yuichiro Sato
Drug Metabolism Research Laboratories, Astellas Pharma Inc., Yodogawa-ku, Osaka, Japan (Y.S., M.N., A.M., A.K., T.U.); Astellas Research Institute of America LLC., Skokie, Illinois (K.Te., K.Ta.)
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Masanori Nagata
Drug Metabolism Research Laboratories, Astellas Pharma Inc., Yodogawa-ku, Osaka, Japan (Y.S., M.N., A.M., A.K., T.U.); Astellas Research Institute of America LLC., Skokie, Illinois (K.Te., K.Ta.)
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Kazuhiro Tetsuka
Drug Metabolism Research Laboratories, Astellas Pharma Inc., Yodogawa-ku, Osaka, Japan (Y.S., M.N., A.M., A.K., T.U.); Astellas Research Institute of America LLC., Skokie, Illinois (K.Te., K.Ta.)
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Kouichi Tamura
Drug Metabolism Research Laboratories, Astellas Pharma Inc., Yodogawa-ku, Osaka, Japan (Y.S., M.N., A.M., A.K., T.U.); Astellas Research Institute of America LLC., Skokie, Illinois (K.Te., K.Ta.)
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Aiji Miyashita
Drug Metabolism Research Laboratories, Astellas Pharma Inc., Yodogawa-ku, Osaka, Japan (Y.S., M.N., A.M., A.K., T.U.); Astellas Research Institute of America LLC., Skokie, Illinois (K.Te., K.Ta.)
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Akio Kawamura
Drug Metabolism Research Laboratories, Astellas Pharma Inc., Yodogawa-ku, Osaka, Japan (Y.S., M.N., A.M., A.K., T.U.); Astellas Research Institute of America LLC., Skokie, Illinois (K.Te., K.Ta.)
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Takashi Usui
Drug Metabolism Research Laboratories, Astellas Pharma Inc., Yodogawa-ku, Osaka, Japan (Y.S., M.N., A.M., A.K., T.U.); Astellas Research Institute of America LLC., Skokie, Illinois (K.Te., K.Ta.)
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Abstract

The aim of this study was to optimize methods for quantifying 13 uridine 5′-diphosphate-glucuronosyltransferase (UGT) isoforms (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B15, and 2B17) in human liver, intestinal, and kidney microsomes, and in recombinant human UGT-expressing insect cell membranes (rhUGTs) by targeted peptide–based quantification using liquid chromatography–tandem mass spectrometry. Production of targeted peptides was compared by combining three denaturing agents (urea, sodium deoxycholate, and octyl glucoside) and three denaturing temperatures (37°C, 60°C, and 95°C) followed by tryptic digestion for 2–20 hours. Denaturing conditions and digestion times yielding high production efficiency varied markedly among isoforms and specimens, indicating the importance of specific optimization. Each UGT isoform was quantified using the methods found to be optimal. The expression of 10 (1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B10, 2B15, and 2B17), 6 (1A1, 1A3, 1A4, 1A10, 2B7, and 2B17), and 3 (1A6, 1A9, and 2B7) isoforms was detected in human liver, intestinal, and kidney microsomes, respectively, and levels were reproducible using multiple protocols. All isoforms were quantified in rhUGTs. Determining the levels of UGTs in human tissue specimens and those in rhUGTs is important for estimating the contribution of glucuronidation to body clearance based on in vitro–in vivo extrapolation.

Footnotes

    • Received November 28, 2013.
    • Accepted March 4, 2014.
  • This study was supported by Astellas Pharma Inc. and Astellas Research Institute of America, LLC.

  • dx.doi.org/10.1124/dmd.113.056291.

  • Embedded ImageThis article has supplemental material available at dmd.aspetjournals.org.

  • Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 42 (5)
Drug Metabolism and Disposition
Vol. 42, Issue 5
1 May 2014
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Research ArticleArticle

Quantification of UGTs in Biological Specimens

Yuichiro Sato, Masanori Nagata, Kazuhiro Tetsuka, Kouichi Tamura, Aiji Miyashita, Akio Kawamura and Takashi Usui
Drug Metabolism and Disposition May 1, 2014, 42 (5) 885-889; DOI: https://doi.org/10.1124/dmd.113.056291

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Research ArticleArticle

Quantification of UGTs in Biological Specimens

Yuichiro Sato, Masanori Nagata, Kazuhiro Tetsuka, Kouichi Tamura, Aiji Miyashita, Akio Kawamura and Takashi Usui
Drug Metabolism and Disposition May 1, 2014, 42 (5) 885-889; DOI: https://doi.org/10.1124/dmd.113.056291
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