Branched-chain keto-acids and pyruvate in blood: Measurement by HPLC with fluorimetric detection and changes in older subjects

Background: Measurement of keto-acids is important in various clinical situ ations. 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 concentrat ions of these acids in young adults and institutionalized elderly adults. T his method was applied to the determination of blood keto-acid concentratio ns of young adults and institutionalized elderly people, divided into age g roups Methods: Four keto-acids (alpha-ketoisocaproate, alpha-ketoisovalerate, alp ha-keto-beta-methylvalerate, and pyruvate) were derivatized with o-phenylen ediamine to give fluorescent derivatives. After the sample preparation step (75 min to prepare 20 samples), the derivatives were separated chromatogra phically on a reversed-phase column using a binary gradient. Results: The fluorometric detection of the four ketoacids was rapid, <12 mi n, The method is repeatable and reproducible: the CVs were <6% and <11%, re spectively, 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 alpha-ketoisocaproate, which decre ased similar to 40%. Conclusions: The proposed method allows rapid and reliable measurement of k eto-acids. The data demonstrate that changes in branched-chain keto-acids c oncentrations in serum occur with age. (C) 2000 American Association far Cl inical Chemistry.