Enzymatic and Oxidative Stress Analysis

For the third set of experiments, PBMCs were again centrifuged at 300× g for five minutes and were suspended in PBS supplemented by protease inhibitors to be sonicated twice for 10 s in ice, with a break of 30 s. The activity of H6PD and G6PD were assayed using an absorbance microplate reader (ELx808™, Winooski, VT, USA) to follow the reduction of NADP at 340 nm [24 (link),25 (link),26 (link),33 (link)]. H6PD enzymatic function was tested in the presence of Tris-HCl pH 7.4 100 mM, glucose 10 mM, and NADP 0.5 mM. By contrast, G6PD activity was assayed in the presence of Tris-HCl pH 7.4 100 mM, glucose-6-phosphate (G6P) 10 mM, and NADP 0.5 mM.
Malondialdehyde (MDA) levels were evaluated, by the thio-barbituric acid reactive substances assay, using a UV/visible spectrophotometer (Ultraspec 2000, Pharmacia Biotech, Erie, PA, USA) [26 (link),34 (link)]. In all cases, enzymatic activity was normalized for total protein concentrations tested using Bradford analysis [35 (link)].
Finally, total antioxidant capacity was evaluated following the manufacturer’s instructions of a dedicated kit (MAK187, Sigma, St. Louis, MO, USA) that provides a complete description of the total cell antioxidant power associated with the endogenous scavengers, expressed as Trolox equivalent antioxidant capacity content.