Determination of homocysteine levels in cells and
serum is important because high homocysteine is a risk factor for cardiovascular disease. The currently used methods for homocysteine analysis either are time consuming or rely on the use of expensive equipment. Described in this study is an enzymatic assay that determines levels of homocysteine in multiple samples in less than 30 min at levels from 5 to 50 pmol using only a spectrophotometer. The reproducibility of the assay is consistent with the other methods currently used. A second assay, that is about 5-fold more sensitive, follows the enzymatic catalyzed solvent exchange of protons on glycine, which requires a scintillation counter. Both the spectrophotometric and the radiometric methods are based on the conversion of 5-methyltetrahydrofolate to tetrahydrofolate by methionine synthase. The tetrahydrofolate is formed in stoichiometric amounts to the homocysteine in the sample. In the spectrophotometric method the tetrahydrofolate is used at catalytic levels by three enzymes to form a metabolic cycle that generates NADPH from NADP Ψ . In the radiometric assay tetrahydrofolate is required for the enzymatic exchange of the pro 2S proton of glycine with solvent. L-Cysteine, at levels more than 30-fold higher than the upper level of homocysteine used in these assays, does not give any measurable response.