*2.4. Brain Enzymes*

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured with UV-kinetic diagnostic kits according to the kit protocol of the manufacturer (United Diagnostics Industry (UDI, Dammam, Saudi Arabia). The method was based on the oxidation of NADH, and the rate of decrease in absorbance at 340 nm is proportional to the ALT activity of the sample. ALT and AST reagents were reconstituted with 3 mL of distilled water, then 1000 µLof reconstituted reagent were pre-warmed at 37 ◦C for 2 min followed by mixing with 100 µLof the sample. The mixture was allowed to stand for 60 s for temperature equilibrium. ALT absorbance was measured every 60 s within 3 min at 340 nm against distilled water, and AST absorbance was measured every 60 s within a 2-min interval at 340 nm against distilled water. Eventually, ∆A/min was determined. Each unit of AST and ALT enzyme activity was defined as micromoles of NADH decomposed per minute using a molar absorbance of 6.22 <sup>×</sup> <sup>10</sup><sup>3</sup> <sup>×</sup> <sup>M</sup>−<sup>1</sup> Cm−<sup>1</sup> .

Superoxide dismutase was determined by the method of Nishikimi et al. [21]. Briefly, the reaction mixture contained 0.1 mL of sodium pyrophosphate (0.1 mM), 0.1 mL of NBT (0.3 mM), 0.1 mL of NADH (0.47 mM), 0.05 mL of PMS (0.93 µM), and 0.1 mL of enzyme (homogenized brain tissue) in a total volume of 1 mL. The rate of change of absorbance was measured at 560 nm. Values were expressed as units of enzyme min−<sup>1</sup> mg protein. Catalase activity was estimated in the whole homogenized brain tissue by the method of Aebie, 1984 [22]. The reaction mixture in a total volume of 3 mL contained 0.4 M sodium phosphate buffer pH 7.2, 1.2 mL of H2O2, and a suitably diluted enzyme. The reaction was started by adding H2O<sup>2</sup> and reading the change in absorbance at 240 nm for two minutes. One unit of CAT activity was defined as micromoles of H2O<sup>2</sup> decomposed per min using molar absorbance of H2O<sup>2</sup> (43.6 M−<sup>1</sup> Cm−<sup>1</sup> ).
