Analysis of Branched-Chain Keto Acids in Cell Extracts by HPLC-Fluorescence Detection

• HATTORI Ayuna

  Department of Biochemistry and Molecular Biology, The University of Georgia

• ITO Takahiro

  Department of Biochemistry and Molecular Biology, The University of Georgia

• TSUNODA Makoto

  Graduate School of Pharmaceutical Sciences, The University of Tokyo

Abstract

An analytical method for quantifying branched-chain keto acids (BCKAs) - namely α-keto-isovaleric acid (KIV), α-keto-isocaproic acid (KIC), and α-keto-β-methylvaleric acid (KMV) - in cell extracts was developed and applied to a human chronic myelogenous leukemia cell line. Reversed-phase liquid chromatography was conducted for separation of the BCKAs after derivatization into fluorescent quinoxalines by reaction with o-phenylenediamine. The calibration curve was linear over the range from 0.5 to 50 μM. The precision values in intra- and inter-day assays were less than 3.1% and 5.6% (n = 4), respectively. Intracellular concentrations of KIV, KIC, and KMV in K562 cells were 47.6 ± 10.2, 312.4 ± 40.6, and 282.4 ± 71.6 pmol/1 x 10⁶ cells, respectively (n = 5 for each BCKAs). Furthermore, this method was applied to study the effect of gabapentin - an inhibitor of branched-chain amino acid aminotransferase 1 - on intracellular BCKA levels.

Journal

• CHROMATOGRAPHY

  CHROMATOGRAPHY 38 (3), 129-133, 2017

  The Society for Chromatographic Sciences

Keywords

• K562 cells
• <i>o</i>-Phenylenediamine
• Branched-chain amino acids
• Cancer

Details

• CRID: 1390001205744311808
• NII Article ID: 130007040468
• NII Book ID: AA1137755X
• DOI: 10.15583/jpchrom.2017.010
• ISSN: 13483315
• ISSN: 13428284
• NDL BIB ID: 028644699
• Web Site: http://id.ndl.go.jp/bib/028644699
• Web Site: https://ndlsearch.ndl.go.jp/books/R000000004-I028644699
• Web Site: https://www.jstage.jst.go.jp/article/jpchrom/38/3/38_2017.010/_pdf
• Text Lang: en
• Data Source:
  • JaLC
  • NDL
  • Crossref
  • CiNii Articles
  • KAKEN
• Abstract License Flag: Disallowed