2.6. Estimation of SOD, CAT and GPx Activity

First, the blood samples were centrifuged at 16,000× g for 10 min at 4 °C. The plasma was removed, and the red blood cells were gently mixed with triple-deionized water (1:4, v/v). Second, the samples were centrifuged at 16,000× g for 10 min at 4 °C. The supernatant was quantified using GPx, CAT and SOD assay kits, according to the manufacturers’ instructions.

Evaluation of SOD activity was performed using superoxide dismutase assay kit (Cayman, MI, USA). The kit was used to measure all three types of SOD. These SOD have been characterized according to their Fe, Mn and Cu/Zn contents. The kit uses a tetrazolium salt for the determination of superoxide radicals generated by xanthine oxidase. One unit of SOD was defined as the amount of enzyme needed to dismutate 50% of the superoxide radicals. Use a Tecan Infinite 200 Microplate Reader (Männedorf, Switzerland), the absorbance was read at 450 nm. [27].

The methods described in previous studies were modified to evaluate CAT activity [28]. A catalase assay kit (Cayman, MI, USA) was used to evaluate CAT activity through the decomposition of hydrogen peroxide. Using a Tecan Infinite 200 Microplate Reader (Männedorf, Switzerland), the absorbance was read at 540 nm.

A GPx assay kit (Cayman, MI, USA) was used to determine GPx activity. The method converts reduced glutathione into oxidized glutathione by GPx and oxidizes NADPH into NADP⁺. Using a Tecan Infinite 200 Microplate Reader (Männedorf, Switzerland), the absorbance was read at 340 nm. [29].