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Analysis of Branched-Chain Keto Acids in Cell Extracts by HPLC-Fluorescence Detection
Ayuna HATTORITakahiro ITOMakoto TSUNODA
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Volume 38 (2017) Issue 3 Pages 129-133

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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.

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© 2017 The Society for Chromatographic Sciences
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