HYPERHOMOCYSTEINEMIA INDUCED BY FOLIC-ACID DEFICIENCY AND METHIONINE LOAD - APPLICATIONS OF A MODIFIED HPLC METHOD

The increasing possibility that homocysteine might be involved in athe rosclerosis in non-homocysteinuric subjects has required the measureme nt of low concentrations of this aminothiol in biological samples. The procedure described here represents an improvement of different HPLC methods. We utilized an isocratic HPLC system with fluorescence detect ion of plasma total homocysteine derivatized after reaction with ammon ium 7-fluoro-benzo-2-oxa-1,3-diazole-4-sulphonate. With the help of th e rapidly eluting internal standard N-acetyl-cysteine, the method ensu res very good recovery (similar to 100%), reproducibility and precisio n (within-assay: 2.31%; day-to-day: 2.8%) in the physiological concent ration range. This procedure allowed us to validate various animal mod els of hyperhomocysteinemia such as dietary folic acid deficiency in r at and acute methionine loads in rat and hamster. Using this method, w e also confirmed that men have higher plasma total homocysteine levels than women. Due to its simplicity and reliability, our procedure is s uitable for routine analysis of total homocysteine and other aminothio ls (cysteine, cysteinyl-glycine and glutathione) in biological samples , as required in clinical and research laboratories.