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

¹ Biochem Laboratory, Emile Roux Hospital, AP-HP, 1 Avenue de Verdun, 94456 Limeil-Brévannes Cedex, France.

² Biology Laboratory, Joffre-Dupuytren Hospital, AP-HP, 91211 Draveil Cedex, France.

³ Biochem Laboratory, Hôtel-Dieu Hospital, AP-HP, 1 Place du Parvis Notre-Dame, 75181 Paris Cedex 04, France.

⁴ Nutrition Laboratory, EA 2498, Paris V University, 4 Avenue de l’Observatoire, 75006 Paris, France.
^a Address correspondence to this author at: Laboratoire de Biologie, Hôpital Joffre-Dupuytren, 1, Rue Louis Camatte, 91211 Draveil Cedex, France. Fax 33-1-69-83-6495; e-mail f.blonde-cynober{at}jfr.ap-hop-paris.fr

Background: 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 groups

Methods: Four keto-acids (-ketoisocaproate, -ketoisovalerate, -keto-ß-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 -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.

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