Assessment of brain superoxide dismutase (SOD) activity

Whole brain SOD activity estimation helps in the assessment of brains’ endogenous antioxidant SOD levels. Brain SOD activity was measured using spectrophotometrically (UV-1800 ENG 240V, Shimadzu Corporation) at 560 nm [35]. The method is based on the formation of water insoluble blue from the NBT reduction. For the estimation of SOD activity, brain homogenate (0.5 ml) was mixed with 50 mM Na₂CO₃ (1 ml), 24 mm NBT (0.4 ml), and 0.1 mM EDTA (0.2 ml). To this mixture, 1 mM hydroxylamine hydrochloride (0.4 ml) was further added to begin the reaction. The blue colour thus developed in the reaction was observed at 560 nm. Absorbance, at 560 nm and at 25°C was recorded at Zero-time and then every 30 seconds during the span of 5 minutes. Control reading was obtained by performing the above reaction excluding the brain homogenate. The rate of absorbance units (A) increase per minute for the test and control sample(s) were recorded. The percentage inhibition of the NBT reduction served as a measure for the SOD presence and this was calculated with the help of the following formula:

where (ΔA560 nM at 5 minutes and 30 seconds − A560 nM at 30 seconds)/5 minutes = ΔA560 nM/minute.

SOD activity in units represented the quantity of enzyme required to inhibit 50% of the NBT reduction and was expressed as units per mg of protein.