2.3. Blood Antioxidant Methods

Three redox biomarkers, namely, total antioxidant capacity (TAC) as a crude index of the antioxidant potential of the samples, thiobarbituric acid reactive substances (TBARS) as a biomarker of lipid peroxidation and reduced glutathione (GSH) as the most important endogenous antioxidant molecule, were evaluated in all blood samples. All the methods were based on the protocols described by Veskoukis et al. [30,31,32,33].

In brief for the TAC assay, 20 μL of each plasma sample was mixed with 10 mM sodium phosphate buffer pH = 7.4 (480 μL) and 0.1 mM 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) solution (500 μL), and then incubated for 1 h in the dark at room temperature (RT), centrifuged (20,000× g for 3 min at 4 °C) and the absorbance was measured at 517 nm in a spectrophotometer (U-1900; Hitachi, Ltd., Tokyo, Japan). TAC was calculated on the basis of the mmol DPPH• reduced by the antioxidants present in the samples. For the TBARS assay, 100 μL of plasma was mixed with 35% trichloroacetic acid (TCA) (500 μL) and 200 mM Tris-HCl pH = 7.4 (500 μL), then incubated for 10 min at RT and 1 mL of 2 M Na₂SO₄ and 55 mM of thiobarbituric acid (TBA) were added. Following 45-min incubation at 95 °C, 1 mL of 70% TCA followed. The samples were centrifuged (15,000× g for 3 min at 20 °C) and the absorbance was measured at 520 nm in a spectrophotometer (U-1900; Hitachi, Ltd., Tokyo, Japan). The concentration of TBARS was calculated on the basis of the millimolar extinction coefficient of malonyldialdehyde (156 L/mmol/cm). Finally, for GSH assay 20 μL of erythrocyte lysate treated with TCA was mixed with 67 mM phosphate buffer (pH = 7.95) (660 μL) and 1 mM 5. 5-dithiobis (2 nitrobenzoic acid) (DTNB) (30 μL), then incubated for 45 min in the dark at RT and the absorbance was measured at 412 nm in a spectrophotometer (U-1900; Hitachi, Ltd., Tokyo, Japan). GSH concentration was calculated on the basis of the millimolar extinction coefficient of DTNB (13.6 L/mmol/cm). Haemoglobin concentration of erythrocyte lysate was measured using a commercially available kit.

All reagents were purchased from Sigma-Aldrich. Each assay was performed in triplicate and within 3 months of collection.