New Enzymatic Colorimetric Assay for Total Homocysteine

Abstract

In 1969, McCully (1) observed that increased plasma homocysteine (Hcy) was linked with vascular disease. Subsequent studies demonstrated that even mild hyperhomocysteinemia is an independent risk factor for cardiovascular, cerebrovascular, and peripheral vascular disease (2)(3)(4)(5)(6)(7)(8). Increased plasma Hcy is associated with a thermolabile variant of 5,10-methylenetetrahydrofolate reductase, smoking, lack of exercise, and excessive use of alcohol and coffee. Plasma or serum total Hcy (tHcy) concentrations are most commonly measured by HPLC (9), which is time- consuming and expensive, and by immunochemical (10)(11)(12) or enzymatic (13) methods, which may not be applicable to all colorimetric-based clinical chemistry analyzers. The thiol group of Hcy allows it to form a disulfide bond with other thiol-containing molecules, such as Hcy itself, cysteine, and the cysteine residue of plasma proteins. Biologic fluids may often contain both reduced and oxidized species of Hcy, and the sum of all the forms of Hcy is usually called total Hcy (tHcy) (14). Most clinical studies concerning Hcy have relied on the measurement of tHcy. An initial chemical reduction step of the sample is inevitable in the tHcy assay. Because reducing agents can interfere with the oxidation of redox indicators, such as Trinder’s reagents and derivatives of methylene blue generally used in diagnostic reagents, methods for tHcy that use these indicators have not been developed. The present method is a new enzymatic colorimetric assay for tHcy in biologic samples. The principle is as follows. In the first step, samples are reduced by dithiothreitol …