Stability of Blood Homocysteine and Other Thiols: EDTA or Acidic Citrate?

Abstract

Homocysteine (Hcy), a thiol-containing amino acid resulting from demethylation of methionine (Met), is relevant to the risk of vascular diseases (1). In plasma, total homocysteine (tHcy) includes the free reduced and oxidized forms as well as the Hcy bound by disulfide bonds in proteins. tHcy is frequently increased in patients with coronary, cerebrovascular, or peripheral arterial diseases; the association is independent of most other risk factors for atherosclerosis (2). The simultaneous measurement of other thiols is of interest because most of them are metabolically related and disturbances of their concentrations can correspond to disorders of metabolism. Hcy may either be catabolized to cysteine (Cys) or remethylated to Met (3). In addition, Cys and γ-glutamylcysteine are precursors to glutathione (GSH), and cysteinylglycine (CysGly) is a breakdown product of GSH; this latter plays a major role in defense against oxidative and free radical-mediated cell injury, and its measurement permits the evaluation of oxidative status of cells and tissues (4). It should be expected that less rigorous blood sampling and treatment conditions are needed than those involved for thiol redox status evaluation (5). For plasma, storage conditions have little influence on tHcy values: tHcy in plasma is stable at −20 °C for at least 3 months and after nine freeze/thaw cycles (6). In contrast, in whole blood, an increase of tHcy is observed after collection because of ongoing metabolism and time-dependent release from erythrocytes (7). The artificial increase in plasma tHcy occurs at a rate of 1 μmol/L per hour at room temperature (6)(7). In most studies, blood is drawn in tubes containing potassium EDTA, which are put immediately in crushed ice and centrifuged “as soon as possible”, and then the plasma is frozen. These conditions prevent increases in tHcy in whole blood after collection, but they are not always …