Multicenter Analytical Evaluation of an Enzymatic Method for the Measurement of Plasma Homocysteine and Comparison with HPLC and Immunochemistry

Homocysteine (Hcy) is a sulfur amino acid and a metabolite of the amino acid methionine. Hcy is very reactive because it contains a free sulfhydryl (thiol) group, and it readily oxidizes to form various disulfides. The fraction of free Hcy in plasma is therefore <2% of total plasma Hcy (tHcy) (1).Increased Hcy has been associated with cardiovascular, cerebrovascular, and peripheral vascular disease (2)(3)(4) and is recognized as an independent risk factor. The need for a simple automated assay is therefore increasing, and various analytical methods have become available. Currently the two most widely used techniques are HPLC and immunochemistry.We implemented a new automated enzymatic method for the measurement of tHcy on two different routine clinical chemistry analyzers and compared these assays with HPLC and the AxSYM immunoassay.Reagents and calibrators for the enzymatic method were obtained from Catch Inc. In this method Hcy and l-serine form cystathionine, which is then converted to Hcy, pyruvate, and ammonia. The enzymes involved are cystathionine β-synthase and cystathionine β-lyase, respectively. tHcy is measured by the production rate of pyruvate by inclusion of lactate dehydrogenase and NADH in the reaction mixture. Reagent 1 contains serine, NADH, and lactate dehydrogenase; reagent 2 contains the reducing substance; and reagent 3 contains cystathionine β-synthase and cystathionine β-lyase. The calibrators have Hcy concentrations of 0.0 and 26.5 μmol/L (5). The enzymatic assay was implemented on two Synchron LX-20 and CX-5 analyzers (Beckman Coulter, Inc.).The HPLC method is a modification from Spaapen et al. (6), as obtained from Instruchemie. Plasma samples were pretreated with tri- n -butylphosphine for reduction and deproteinized with trichloroacetic acid. Derivatization was performed with 7-fluorobenzo-2-oxal,3-diazole-4-sulfonic acid. 2-Mercaptoethylamine was used as an internal standard. Separation was obtained by use of an Inertisil ODS-3 column with sodium acetate buffer …