Testing for hyperhomocysteinemia in subjects with a history of thromboembolic events using HPLC technique

Abstract

An elevated homocysteine level is a well-known thrombophilic risk factor. Determination of total plasma homocysteine therefore is an integrated part of the diagnostic setting after thromboembolic events; about 5-7% of the population do have elevated homocysteine levels. Some forms of hyperhomocysteinemia are treatable; thus a standardized and reliable diagnostic setting has to be at hand. HPLC analysis is widely available in routine diagnostic laboratories. We use the fluorogenic reagent SBD-F to derivate with plasma homocysteine after release of the amino acid from homo- and heterodimers and protein bond using TBP. Separation is performed using a c18 reverse-phase column with aqua and acetonitrile as solvent. Due to continuous release of homocysteine from blood cells centrifugation and separation of plasma within 30 min after venous puncture are crucial for reproducible results.