2.5.4. Estimation of Malondialdehyde (MDA) and Reduced Glutathione (GSH)

Tissue malondialdehyde (MDA) level was estimated spectrophotometrically according to method described by Ohkawa and coworkers [45]. The method depends on the reaction of MDA with thiobarbituric acid and 1,1,3,3-tetramethoxypropane as was employed as a standard. In brief, about 0.1 g of tissue was homogenized at 4 °C in 1 mL of sodium phosphate buffer (0.1 M, pH 7.4) containing EDTA (0.1 mM). Then, 1ml of the homogenate was added to 2ml of thiobarbituric acid (TBA) reagent (composed of 15% trichloroacetic acid (TCA), 0.375% TBA, and 0.25 N HCl). The mixture was boiled for 15 min then ice-cooled, and centrifuged for 10min. at 3500 rpm. Using a UV-visible spectrophotometer (UV-1601PC, Shimadzu, Tokyo, Japan) the color intensity of the supernatant was recorded.

Tissue reduced glutathione (GSH) contents were estimated according to the method adapted by Ellman [46]. Briefly, about 0.5 mL of tissue homogenate in phosphate buffer was added to an equal volume of 10% trichloroacetic acid. 6 mM EDTA and the mixture was shaken gently for 10–15 min then centrifuged at 2000 rpm for 5 min. Then 0.2 mL of the supernatant was added to 1.7 mL of phosphate buffer (0.1 M, pH 8) followed by the addition of 0.1 mL of Ellman’s reagent (0.039 g of 5,5′-dithio-bis-(2nitrobenzoic acid) dissolved in 10 mL Phosphate buffer) The absorbance of the reaction product was recorded after 5 min. at λ 412 nm using a Shimadzu spectrophotometer.