Analysis of intracellular α-keto acids by HPLC with fluorescence detection

Abstract

Branched-chain keto acids and branched-chain amino acids are metabolites of branched-chain amino acid aminotransferases (BCATs), which catalyzes reversible transamination between them. We found that BCAT1 plays an important role in the progression of myeloid leukaemia, and a method for the analysis of intracellular α-keto acids including branched-chain keto acids was necessary to further investigate their role. In this study, we developed a method to analyze six α-keto acids (α-ketoglutaric acid (KG), pyruvic acid, α-ketobutyric acid, α-ketoisovaleric acid, α-ketoisocaproic acid, and α-keto-β-methylvaleric acid) in K562 cells by HPLC with fluorescence detection, using 1,2-diamino-4,5-methylenedioxybenzene (DMB) as a derivatization reagent. Because split peaks of DMB-KG were observed when injection samples were too acidic, the derivatization solution was diluted with NaOH solution to obtain a single peak. Limits of detection and limits of quantification were 1.3–5.4 nM and 4.2–18 nM, respectively. Intracellular concentrations of α-keto acids were 1.55–316 pmol/1 × 10⁶ K562 cells. The developed method realized reproducible and sensitive analysis of intracellular α-keto acids. Thus, the method could be used to elucidate the role of BCAT in myeloid leukaemia.