2.6. Assessment of GSH and GSSG
COLD
A colorimetric detection kit (Invitrogen by Thermofisher Scientific, ref: EIAGSHC, Madrid, Spain) was used to perform the GSH and GSSG determination assay. The erythrocyte samples were treated with sulfosalicylic acid to precipitate the proteins. Thereafter, 0.050 mL of the sample was placed in an Eppendorf tube and 0.150 mL of 5% sulfosalicylic acid was added. Preparation of sulfosalicylic acid: 1 g of sulfosalicylic acid was placed in a beaker and made up to 20 mL with distilled water. Samples were shaken and then incubated at room temperature for 10 min, then centrifuged at 14,000 rpm for 10 min at 4 °C. The supernatant was diluted with 1% sulfosalicylic acid. Then, 0.05 mL of the diluted sample was transferred to the test well together with a colorimetric detection reagent, glutathione reductase, and NADPH. The absorbance was measured at 405 nm in a microplate reader (Biostack neo. BiotTek. By Izasa Scientific, Madrid, Spain). An assay curve was also constructed and measured, which was then used to extrapolate the absorbance and obtain the concentration of the samples. To measure oxidized glutathione, the same procedure was followed by adding 2-vinylpyridine to the sample. Two quality controls from two known concentrations of the calibration curve were used. Samples from a temperature of −80 °C were kept cold and under the same conditions throughout the determination process by a researcher specialized in clinical analysis.