Cayman kit

To determine the activities of the following antioxidant enzymes: superoxide dismutase (SOD) and catalase (CAT), Cayman kits (Cayman Chemical MI, USA) were used. The activities of SOD and CAT were expressed in U or nanomole/min, respectively, per milligram of protein.
The method of Ohkawa et al. [27 (link)] was used to determine the content of TBARS (thiobarbituric acid reactive substances) in the total homogenate of the lenses. This method is based on the reaction between lipid peroxidation products and thiobarbituric acid. TBARS content is expressed in nanomole per gram of the lens. The intensity of the obtained color was determined spectrophotometrically at the wavelength of 535 nm. To establish a standard curve, 1,1,3,3-tetraethoxypropane (Sigma-Aldrich, St. Louis, MO, USA) was used.
The concentration of advanced oxidation protein products (AOPP) in the lens homogenate was determined using spectrophotometric method described by Witko-Sarsat et al. [28 (link)]. The calibration curve was established using chloramine T (Sigma-Aldrich, St. Louis, MO, USA), while the absorbance was measured at the wavelength of 340 nm. The content of AOPP was expressed in nanomole chloramine T equivalents per milligram of protein.

Glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were assessed in seminal plasma using Cayman kits (ref. 703102 and SD125, respectively). GPx activity was determined at 340 nm, and SOD activity was assayed at 505 nm [34 (link)]. Reactions were performed using technical triplicates. A BioTek Gen 5 microplate reader at room temperature was used to assess all enzymatic activities.

The activities of CAT and SOD enzymes were measured using Cayman kits (Cayman Chemical, Ann Arbor, MI, USA), according to the manufacturer`s instructions [23 (link)]. The Varioskan™ LUX multimode microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) was used for the measurement of the absorbance. The measurements were performed in triplicate.
The total antioxidant capacity (TAC) was determined using a method described by Marin et al. [23 (link)]. The results are expressed as μmol TEAC (Trolox equivalent antioxidant capacity)/ml of cell culture supernatant. Three independent replicates were performed for TCA determination.

The activities of CAT, GPx and SOD enzymes were measured using Cayman kits (Cayman Chemical, Ann Arbor, MI, USA), according to the manufacturer`s instructions [23 (link)]. Also, the methods described by Marin et al. [23 (link)] were used for evaluation of the TAC and thiobarbituric acid reactive substances (TBARS). The results are expressed as nmol TEAC (Trolox equivalent antioxidant capacity)/g of tissue for TAC and nmol/g sample for TBARS. The analysis of DNA/RNA oxidative damage and of protein carbonyl were performed as described by [25 (link)] and carried out in triplicate.

The total protein and soluble protein levels were evaluated according to Lowry’s method [33 (link)] in total and centrifuged homogenates, respectively.
Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) activities were measured with Cayman kits (Cayman Chemical, Ann Arbor, MI, USA). The activity of glucose-6-phosphate dehydrogenase (G6PD) was assessed with a Pointe Sci. kit (Pointe Scientific, Inc., Canton, MI, USA). Reduced (GSH), oxidized (GSSG) and total (total GSH) glutathione levels were determined with the Cayman kit. The assay proposed by Jagota and Dani [34 (link)] was used in order to evaluate the level of vitamin C.

The measurement of the antioxidant enzymes superoxide dismutase (SOD, EC 1.15.1.1), glutathione S-transferase (GSH S-T, EC 2.5.1.18), glutathione peroxidase (GSH-Px, EC 1.11.1), and glutathione reductase (GSH-R, EC 1.6.4.2) were performed with the Sigma-Aldrich Determination Kit (SOD, Cat. 19160, Sigma, St. Louis, MO, USA) and Cayman Kits (GSH S-T, 703302; GSH-Px, 703102; GSH-R, 703202, Cayman Chemical, Ann Arbor, MI, USA), following the manufacturer’s instructions. The catalytical activity of catalase (EC 1.11.1.6) was determined according to Aebi method (1984) [21 (link)], by the spectrometric analysis of the H₂O₂ consumption at 240 nm. Results are expressed as enzymatic activity units (U) in relation to the total protein of the samples.
The total protein content in liver (μg/mL) was determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA) based on the Bradford dye-binding method.