Total-Homocysteine Enzymatic Assay

Abstract

McCully (1) in 1969 first observed that plasma hyperhomocysteinemia was involved in the pathogenesis of arteriosclerosis. More studies have reported that total serum or plasma homocysteine (tHCY) is an independent risk factor for cardiovascular diseases, including arteriosclerosis, coronary artery disease, cerebrovascular disease, and myocardial infarction (2)(3)(4)(5)(6)(7). Furthermore, plasma tHCY is a strong predictor of mortality in patients with existing coronary artery disease (8). A major basis for hyperhomocysteinemia is insufficient intake of vitamins B6, B12, and folic acid, which are necessary for homocysteine metabolism and which lower the risk for homocysteine-associated diseases (9)(10)(11). Homocystinuria can result from inborn errors, such as deficiencies in the enzymes cystathionine β-synthase, methionine synthase, and methylenetetrahydrofolate reductase (12). These patients have premature vascular disease, ∼50% of untreated patients have thromboembolic events, and the mortality rate is ∼20% before the age of 30 (12). Many methods have been developed to measure tHCY, such as the tHCY enzyme conversion immunoassay designed for the Abbott IMx analyzer (13), the microtiter-plate tHCY enzymatic immunoassay kit (14), and HPLC (15)(16)(17). However, these methods are relatively complex and require highly specialized equipment. To meet the need for a widely available homocysteine assay, we have developed and describe here a simple single-enzyme tHCY assay based on a highly specific homocysteine α,γ-lyase (rHCYase) (18). l-Homocystine, l-cysteine (l-cys), l-methionine (l-MET), dithiothreitol (DTT), Triton X-100, citric acid, potassium ferricyanide, and other common reagents for the tHCY enzymatic assay were obtained from Sigma. 4-Fluoro-7-sulfobenzofurazan ammonium salt was obtained from Wako Fine Chemicals, and N,N -dibutylaniline was obtained from Acros Organics. N,N -Dibutyl phenylene diamine (DBPDA) was synthesized in our laboratory (L. Tang and Y. Tan, unpublished …