Total antioxidant status kit

TAS in plasma was determined using the Randox Total Antioxidant Status kit with colorimetric detection at 600 nm. Concentrations were calculated in mmol/L. TBARS were estimated in plasma after reaction with thiobarbituric acid (TBA), quantified spectrophotometrically at 532 nm and expressed in μmol/L [36 (link)].

Total antioxidant capacity (TAC) was measured using a Total Antioxidant Status kit from Randox (Crumlin, United Kingdom). All reagents were prepared according to the manufacturer’s instructions. In the first step, peroxidase catalyses one-electron oxidation of ABTS by hydrogen peroxide which results in the formation of a green ABTS+ radical cation. In this method, ABTS® (2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) is incubated with peroxidase (metmyoglobin) and H₂O₂, which leads to the formation of the cationic form ABTS®+ and blue-green colouration of the test solution. Colour intensity is measured at a wavelength of 600 nm. The degree to which antioxidants inhibit the formation of a coloured compound is determined by their concentration in the sample.

Mice spleens were dissected at 7 days after radiation exposure and assessed for various oxidative stress parameters. Spleens were homogenized in ice-cold potassium phosphate buffer solution (0.1 mol/l, pH 7.4) using a Potter-Elvehjem homogenizer to give a 10% W/V homogenate. Spleen tissue homogenates were prepared and used to obtain glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and total antioxidant capacity (TAC) level estimations. GSH content was determined as previously described by Beutler et al. [21 (link)], SOD activity as in Minami and Yoshikawa [22 (link)] and CAT activity according to Luck [23 ]. TAC level was measured using Randox total antioxidant status kit (UK) according to Rice-Evans and Miller [24 (link)]. LPx product malondialdehyde (MDA) was measured as described by Yoshioka et al. [25 (link)]. Nitric oxide (NO) and protein carbonyl (PCO) in spleen homogenates were estimated according to Miranda et al. [26 (link)] and Levine et al. [27 (link)], respectively. Reactive oxygen species (ROS) was assessed by Rat ROS ELISA Kit (Catalogue Number SL1189Ra, Sun Long Biotech Co., LTD, China). 8-hydroxydeoxyguanosine (8-OHdG), the DNA adduct for oxidative damage, was estimated using Rat 8-OHdG ELISA Kit (Catalog Number CSB-E10526r, Cusabio Technology, LLC, USA).

Total Antioxidant Status (TAS) in plasma was determined using Total Antioxidant Status Kit (Randox, County Antrim, United Kingdom). Lipid peroxidation level was determined as the concentration of thiobarbituric acid reactive substances (TBARS) according to Ohkawa et al. [21 (link)]. The concentration of TBARS expressed as malonic aldehyde (MDA) was calculated from the standard curve.

The antioxidative system in seminal plasma was monitored by determining the superoxide dismutase, glutathione peroxidase, and total antioxidant capacities. The superoxide dismutase activity, glutathione peroxidase activity, and total antioxidant capacity of the seminal plasma samples were each determined spectrophotometrically on an Olympus AU 400 automatic biochemistry analyzer (Olympus, Tokyo, Japan) using commercial Randox ^® kits (Randox Laboratories Ltd., London, UK). Namely, the superoxide dismutase activity was determined by a commercial Ransod ^® kit (Cat. No. SD124; Ransod Control reference material SD126, Randox Laboratories Ltd., London, UK), the activity of glutathione peroxidase by a commercial Ransel ^® kit (Cat. No. RS504; Ransel Control reference material SC692, Randox Laboratories Ltd., London, UK), and the total antioxidant capacity concentration was determined with a commercial Total Antioxidant Status ^® kit (Cat. No. NX 2332; Randox Total Antioxidant Control Cat. No. NX 2331, Randox Laboratories Ltd., London, UK). Superoxide dismutase activity was expressed in U/mL, glutathione peroxidase activity in IU/L, and the concentration of total antioxidant capacity in mmoL/L of seminal plasma.

Cardiac levels of oxidative stress indices including SOD, GPx, and TAC were measured by biochemical kits, following the manufacturer's instruction (Randox Company, England). TAC was determined by Randox's total antioxidant status kit, as described by Miller et al. [38 (link)]. Cardiac SOD was measured, according to Breinholt et al., using a RANSOD kit (Randox Labs Crumlin, UK) [39 (link)]. Also, cardiac GPx was assayed by a RANSEL Kit (Randox Labs Crumlin, UK), as described by Paglia and Valentine [40 (link)]. Thiobarbituric acid was used to measure the MDA level; TBARS were determined by Esterbauer and Cheeseman [41 (link)]. In addition, serum glucose was evaluated spectrophotometrically using a diagnostic reagent kit (Pars Azmoon Company, Tehran, Iran). Furthermore, serum concentration of insulin was determined by the ELISA method (Shanghai Crystal Day Biotech Co., Ltd., China).