Plasma Homocysteine Determined by Capillary Electrophoresis with Laser-induced Fluorescence Detection

Abstract

Homocysteine (Hcy) has emerged as another risk factor for the development of coronary heart disease (1). Genetic abnormalities of the enzymes cystathionine-β-synthase and methylene tetrahydrofolate reductase (folic acid and vitamin B6 and B12 cofactors) can cause raised plasma Hcy concentrations. Deficiencies in folic acid and vitamins B6 and B12 may also contribute to this increased concentration (1)(2)(3). Consequently, patients with coronary heart disease have been treated successfully with folic acid, vitamin B12, or vitamin B6 (1), but the benefit of such treatment in reducing morbid cardiovascular endpoints is presently unknown (3). A widely used technique for measuring total plasma Hcy is reversed-phase HPLC with fluorescence detection after derivatization of plasma thiols (4)(5)(6). Some studies use gas chromatography–mass spectrometry techniques (7) with various derivatization protocols (8). The increasing demand for the determination of plasma total Hcy prompted us to develop a rapid, automated method based on capillary electrophoresis (CE) and laser-induced fluorescence detection (LIF) , in which all specific thiols are detected selectively. Blood was collected by venipuncture into evacuated tubes containing EDTA from patients with episodes of deep-vein thrombosis or vascular disease. The plasma was separated by centrifugation and was stored at −20 °C until analysis. Plasma samples (100 μL) were treated for 30 min at room temperature with 10 μL of a novel thiol-reducing agent [35 mmol/L tris(2-carboxyethyl)phosphine; Molecular Probes], as reported recently by Gilfix et al. (9). Compared with the widely employed tributyl phosphine, this new phosphine is nonvolatile, stable, soluble in water, and without disagreeable odor (5)(6). When we used this procedure, total Hcy (mixed and symmetric disulfides, including protein-bound Hcy) was analyzed in its reduced …