Nx 2332

Measurement of TAS in blood serum was performed by a colorimetric method using the kit NX 2332 (Randox Laboratories, Crumlin, UK). The principle of the method is based on the incubation of ABTS (2,2’-Azino-di-[3-ethylbenzthiazoline sulfonate]) with peroxidase (metmyoglobin) and hydrogen peroxide. The resulting ABTS radical-cation has a green-blue colour, the intensity of which is inversely proportional to antioxidant concentration [34 (link)]. Absorbance was measured at 600 nm. Randox Total Antioxidant Control (Cat. No. NX 2331) was used.

The GPX activity in whole blood was measured with a RANSEL kit (RANDOX Laboratories, UK, Cat No. RS 505) using a UV method at 340 nm wavelength^{43 (link)}. Whole blood was prepared according to the manufacturer’s guidelines (RANDOX Laboratories, UK, Cat No. SD125) and analyzed to determine SOD activity at 505 nm wavelength. Serum TAC concentration was measured using standardized kit (RANDOX Laboratories, UK, Cat No. NX2332) at 600 nm wavelength. An automatic COBAS MIRA PLUS biochemical analyzer (Roche, Switzerland) was used for the measurement of the three indices. Intra- and inter assay coefficients of variation were 4.52 and 4.12% respectively. The detection limits for SOD, GPX, and TAC were 0.1, 40, and 0.05 U/mL, respectively, and the lowest measured concentrations were 0.35, 120, and 0.22 U/mL, respectively. The concentration of malondialdehyde (MDA) was estimated in serum according to the method of Placer et al.^{44 (link)}.

To measure antioxidant and prooxidant balance, cells were lysed by 0.5 ml of ice-cold lysis buffer (Cat No: FNN001; Invitrogen) and the bradford method were done to measure the total protein content. Then commercially available colorimetric kit was used to determine the malondialdehyde (MDA) (ZellBio; MDA48), total antioxidant capacity (TAC) (Randox; NX2332) based on ABTS (Azino ethylbenzthiazoline sulphonate) oxidation, super oxide dismutase (SOD) (ZellBio; SOD48), glutathione peroxidase (GPX) (ZellBio; GPX48), catalase (CAT) (ZellBio; CAT48), total glutathione (GSH) (Sigma; CS0260) and H2O2 (Sigma, MAK165) assay according to the manufacturer’s instruction.