2.2. Induction of Mild Hyperhomocysteinemia (hHcy) by Met Enriched Diet

Rats underwent Met diet in the duration of 28 days before the experiment. Met (L-methionine, Sigma-Aldrich, Taufkirchen, Germany) was given in drinking water at a dose of 2 g/kg of animal weight per day in accordance with Xu et al. [1]. The daily volume of water intake was established at 46.53 ± 8.34 mL for rats in all experimental groups. After this treatment, a mild hHcy was evoked in the animals. Determination of plasma Hcy concentration followed. At day 29, from the naïve controls and controls treated by the Met diet, peripheral blood samples (1.5 mL) were collected from the retro-orbital venous plexus. The blood was immediately cooled on ice and centrifuged. The supernatant was collected and plasma was stored at −80 °C. The plasma Hcy levels were measured by enzymatic assay with a Hcy Liquid Stable Reagent Kit (Axis-Shield Diagnostics, Dundee, Scotland) in accordance with the instructions of the manufacturer and analysed with an automatic biochemical analyser (Siemens ADVIA 1650).