Biochemical measurements

Iron level: Iron level was assessed in the brain hippocampal homogenates using an iron colorimetric assay kit according to the manufacturer’s instructions (Biodiagnostic Company, Dokki, Giza, Egypt, Cat No. IR 15 10).

Assessment of oxidative stress in brain hippocampal tissue homogenates of mice was carried out through measurement of nitric oxide (NO) level, malondialdehyde (MDA) level, and glutathione peroxidase activity (GPX).

NO activity in brain tissue was measured directly by colorimetric method using an NO assay kit according to the manufacturer protocol (Biodiagnostic Company, Giza, Egypt. Cat No. NO 25 33). This assay was performed according to the Greiss method in which nitrite forms nitrous acid diazotize sulphanilamide in an acidic medium, which further can be coupled with N-(1–naphthyl) ethylenediamine resulting in the formation of a bright reddish–purple color azo dye. The color intensity can be measured at 540 nm. It is directly proportional to NO concentration.

The lipid Peroxide Malondialdehyde (MDA) was measured according to the MDA assay kit in compliance with the manufacturer’s instructions (Biodiagnostic Company, Giza, Egypt. Cat No. MD 25 29). This assay is based on the reaction between thiobarbituric acid (TBA) and MDA in an acidic medium to form thiobarbituric acid, which is a pink reactive product proportional to the MDA level in the sample.

The activity of GPX in the brain was measured according to the manufacturer protocol of the Biodiagnostic Company kit, Dokki, Giza, Egypt, Cat No GR 25 24. The kit employs UV method procedures that detect the change in absorbance per minute over 3 min at a wavelength of 340 nm (A₃₄₀ / min). The GPX activity in the sample is positively proportional to the rate of decrease in the A₃₄₀.

Beta-amyloid (Aβ) and P53 measurement: The brain hippocampal tissue levels of Aβ 42 were measured using ELISA kits according to the manufacturer instructions (BioSource Company, San Diego, CA, USA) with a sensitivity level of 1.0 pg/ml. P53 level was assessed using ELISA kits according to the manufacturer’s instructions (RayBiotech Company, 3607 Parkway Lane, Suite 100, USA, Cat No. ELR-p53) with a sensitivity level of 0.32 ng/ml. The Aβ 42 and P53 concentrations were determined based on standard and reported as pg/mg and ng/mg protein, respectively.