Elevated levels of plasma homocysteine (Hcy) are associated with increased risk of cardiovascular disease though it is uncertain whether increases in Hcy represent a cause or a consequence of the disease process. Plasma Hcy exists in reduced, free oxidized, and protein-bound forms, that together comprise total Hcy (tHcy). Free reduced Hcy is thought to be the atherogenic, though minor, sub-fraction of tHcy.

We present a highly selective and sensitive method for the determination of total and free reduced homocysteine and related aminothiols that play important roles in health and disease. The key step in the analysis is derivatization with 2-chloro-1-propylpyridinium iodide followed by separation from other thiols derivatives by high-performance liquid chromatography (HPLC) with detection at 312 nm.

Homocysteine is clearly separated from other thiols, the retention time being 6,23 min, total analysis time is 10 min. To determine total homocysteine it is necessary to cleave disulphide bonds by the use of tris(2-carboxyethyl)phosphine hydrochloride in order to form free sulfhydryl group.

The described method has several advantages: simple sample preparation procedure, simultaneous determination of low-molecular-mass aminothiols during the procedure, fast chromatography procedure, simple and prevalent mobile phases .

Our method was used in routine clinical analyses of homocysteine in plasma in patients with acute myocardial infarction.

Keywords: L-homocysteine; L-cysteine; gluthatione; HPLC; UV; CMPI;

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