2.7.2. Antioxidant Enzymes and Glutathione

Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) were assessed in erythrocytes, adipose tissue, and liver. The total SOD and CAT activities were measured according to the methods developed by Mirsa and Fridovich [27] and Cohen et al. [28], respectively, using a Lambda 25 UV-Vis spectrophotometer (Perkin Elmer, Shelton, CT, USA). GPx and GR activities were measured according to the method developed by Wheeler et al. [29] using a COBAS MIRA autoanalyzer (Roche Diagnostics System, Madrid, Spain).

Reduced glutathione (GSH) and oxidized glutathione (GSSG) in plasma, erythrocytes, adipose tissue, and liver were measured according to the method developed by Hissin and Hilf [30] using an LS55 fluorescence spectrophotometer (Perkin Elmer, Shelton, CT, USA) and the corresponding standard curves (Sigma-Aldrich, Madrid, Spain). In addition, the GSSG/GSH ratio was calculated as a biomarker of the redox state.

Measurements in erythrocyte samples were normalized to hemoglobin (Hb) concentration in total blood. Hb concentration was measured according to the Drabkin method [31] using a Lambda 25 UV-Vis spectrophotometer (Perkin Elmer, Shelton, CT, USA), and an Hb standard (Spinreact, Girona, Spain).