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

机译：HPLC-荧光检测法分析细胞提取物中的支链酮酸

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摘要

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 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.
机译：一种定量分析细胞中支链酮酸（BCKA）的分析方法，即α-酮异戊酸（KIV），α-酮异己酸（KIC）和α-酮-β-甲基戊酸（KMV）开发了提取物并将其应用于人慢性骨髓性白血病细胞系。通过与邻苯二胺反应衍生为荧光喹喔啉后，进行反相液相色谱分离BCKA。校准曲线在0.5至50μM范围内呈线性。日内和日间分析的精确度值分别小于3.1％和5.6％（n = 4）。 K562细胞中KIV，KIC和KMV的细胞内浓度分别为47.6±10.2、312.4±40.6和282.4±71.6 pmol / 1 x 10〜（6）细胞（每个BCKA的n = 5）。此外，该方法还用于研究加巴喷丁（支链氨基酸氨基转移酶1的抑制剂）对细胞内BCKA水平的影响。

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