Stabilization of Homocysteine Concentration in Whole Blood

Homocysteine (Hcy) is a cytotoxic ((1)) , sulfur-containing amino acid that increasingly appears to be an independent risk factor for coronary artery disease, stroke, and peripheral occlusive arterial disease (POAD) ((2)(3)(4)) when present at high plasma concentrations. In addition to several physiological factors that can increase Hcy concentration in blood such as vitamin B6, B12, or folate ((5)) deficiency and renal insufficiency ((6)) , there is an artificial increase of Hcy concentration in whole blood after blood collection. This severe preanalytical problem is due to the well-known, time-dependent release of Hcy from erythrocytes ((7)) in the isolated blood sample, even when stabilization is attempted with different additives such as EDTA, NaF, or heparin/NaF ((8)(9)(10)) . The result of this release is a falsely increased plasma Hcy concentration, unless plasma is separated from blood cells immediately after blood collection. We describe a method to overcome this problem for making the routine determination of Hcy possible for institutions where sample transport is critical. For this purpose, we used a specially prepared blood collecting system, where whole blood is lysed and Hcy generating and converting enzymes are inhibited at the time of venipuncture, resulting in a stabilization of Hcy for 2 days at room temperature. Preparation of the blood collecting system for Hcy determination in lysate was performed by adding Nonidet P40 (25 μL, pure), 50 μL of EDTA disodium salt dihydrate (75 g/L), and 25 μL of citric acid monohydrate (610 g/L) to a …