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

Abstract

An analytical method for quantifying branched-chain keto acids (BCKAs) - namely alpha-keto-isovaleric acid (KIV), alpha-keto-isocaproic acid (KIC), and alpha-keto-beta-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 mu M. 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 10(6) 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.