Branched-Chain Keto-Acids and Pyruvate in Blood: Measurement by HPLC with Fluorimetric Detection and Changes in Older Subjects

Abstract

AbstractBackground: Measurement of keto-acids is important in various clinical situations. The aim of the present work was to develop a rapid HPLC method for the determination of keto-acids in human serum and to assess the concentrations of these acids in young adults and institutionalized elderly adults. This method was applied to the determination of blood keto-acid concentrations of young adults and institutionalized elderly people, divided into age groupsMethods: Four keto-acids (α-ketoisocaproate, α-ketoisovalerate, α-keto-β-methylvalerate, and pyruvate) were derivatized with o-phenylenediamine to give fluorescent derivatives. After the sample preparation step (75 min to prepare 20 samples), the derivatives were separated chromatographically on a reversed-phase column using a binary gradient.Results: The fluorometric detection of the four keto-acids was rapid, <12 min. The method is repeatable and reproducible: the CVs were <6% and <11%, respectively, for each of the keto-acids. We found no significant difference between males and females. Concentrations of the branched-chain keto-acids decreased after age 60 years, especially α-ketoisocaproate, which decreased ∼40%.Conclusions: The proposed method allows rapid and reliable measurement of keto-acids. The data demonstrate that changes in branched-chain keto-acids concentrations in serum occur with age.