2.5. Evaluation of Cardiac Oxidative and Nitrosative Stress Markers

Oxidative stress markers were determined in a cardiac homogenate where NOX-2, MDA, NO, and GSH levels as well as SOD activity were evaluated. For quantitative determination of NOX-2, the Rat NOX-2 ELISA Kit (Bioassay Technology Laboratory, Shanghai, China) was used according to the manufacturer's instructions. The MDA level was determined by the assessment of thiobarbituric acid reacting substance through spectrophotometric measurement of color at 535 nm, and the results were expressed as nmol/g tissue [28]. NO determination, an indicator of nitrosative stress, was done chemically by the Griess method, which estimates the stable oxidation end products of NO, namely, nitrite (NO₂⁻) and nitrate (NO₃⁻), as indicators of NO production. The principle of the assay is the reduction of NO₃⁻ to NO₂⁻ by copperized cadmium granules, followed by color development with a Griess reagent in acidic medium that is measured spectrophotometrically at 540 nm; the results are expressed as nmol/g tissue [29]. The GSH determination method is based on the reduction of Ellman's reagent by thiol groups of GSH to produce 5-thio-2-nitrobenzoic acid that has a yellow color, which was measured spectrophotometrically at 412 nm and expressed as nmol/g tissue [30]. Cardiac SOD activity was measured colormetrically at 420 nm by the method that is based on the inhibition of pyrogallol autoxidation by SOD, and the results were expressed as U/g tissue [31].