Novel approach for the determination of the redox status of homocysteine and other aminothiols in plasma from healthy subjects and patients with ischernic stroke

Abstract

Background: Plasma "redox" status can be assessed by measurements of reduced (r)-, free (f)-, oxidized (ox)-, and protein-bound (b)-homocysteine (Hcy) plus the related aminothiols cysteine, cysteinylglycine (CysGly), and glutathione (GSH), but sample collection has been complex. The redox status has not been determined in ischemic stroke patients and may provide increased understanding of its role in pathogenesis. We wished to examine the feasibility of this measurement in samples collected in readily available acidic sodium citrate. Methods: We measured aminothiols and their stability in stabilized protein-free filtrate using acidic sodium citrate (BioPool® Stabilyte™, pH 4.3) vs EDTA whole blood. Before analysis, plasma samples were also ultra-filtered to obtain a protein-free filtrate. The concentrations of total Hcy (tHcy), fHcy, and rHcy and their related aminothiols, cysteine, cysteinylglycine, and glutathione were simultaneously determined on acidic sodium-citrated blood using reversed-phase HPLC with fluorescence detection. Bound and oxidized aminothiols were calculated by difference using the concentrations of the total, free, and reduced fractions. Using this approach, we compared the redox status in newly diagnosed ischemic stroke patients (n = 20) and healthy age- and sex-matched subjects (n = 20). Results: tHcy, tCys, tCysGly, and tGSH concentrations in whole blood with Stabilyte were stable for 8 h; the reduced fraction of each aminothiol was stable for 4 h. Recovery in the protein-free filtrate was 90-100% for all reduced thiols in acidified sodium-citrated blood. Patients with ischemic stroke had higher plasma tHcy, fHcy, bHcy, rHcy, and oxHcy (P <0.0005) and higher plasma t-, f-, r-, and oxCys (P <0.05). t-, b-, and rCysGly concentrations were lower in the stroke patients (P <0.05), as were t-, b-, and oxGSH (P <0.005). Conclusions: Collection of blood in acidic sodium citrate (BioPool Stabilyte) permits the determination of the redox status of Hcy and its related aminothiols, which may add to the understanding of their relationship to the etiology of cerebrovascular disease. © 2001 American Association for Clinical Chemistry.