*2.4. Enzyme Analysis*

All antioxidant assays of plasma and animal tissues are described in more detail in the study by Cobanova [ ˇ 14]. The activity of superoxide dismutase (SOD, EC 1.15.1.1) in the erythrocytes was measured using the RANSOD kit (Randox, Crumlin, UK). The activity of SOD was expressed in U/g Hb for the specific activity. Total SOD and Cu/Zn superoxide dismutase (Cu/Zn SOD) activity in the liver, pancreas, and kidney cortex of each lamb was measured by means of spectrophotometric assay according to the procedure proposed by Marklund and Marklund [24], whereby one unit of enzyme activity was defined as the amount of enzyme required to inhibit pyrogallol autoxidation by 50%.

The activity of glutathione peroxidase (GPx, EC 1.11.1.9) in blood and hemoglobin (Hb) content were analyzed using commercial kits from Randox, Crumlin, UK, and GPx activity in lamb tissues was assayed by measuring the oxidation of nicotinamide adenine dinucleotide phosphate (NADPH) according to Paglia and Valentine [25]. The assays were carried out at 25 ◦C, and hydrogen peroxide was used as the substrate. The enzyme activities of SOD and GPx are expressed as units of enzyme per milligram and per gram of tissue protein, respectively.

The total antioxidant status (TAS) of the blood plasma and lamb tissues was determined by means of the ferric-reducing antioxidant power (FRAP) assay using the modified method of Benzie and Strain [26]. TAS is expressed as μmol Fe2+ per gram of tissue protein or mmol Fe2+ per liter of plasma.

Lipid oxidation in the serum and tissues was estimated using the modified fluorometric TBARS method according to Jo and Ahn [27]. Lipid peroxidation was expressed as μmol of malondialdehyde (MDA) formed per L of plasma or nmol MDA per gram of tissue protein. Total protein in the tissue homogenates was assayed using the Bradford method [28].

The total concentration of thiol groups (TSH) was found spectrophotometrically based on the reaction of 5,5-dithio-bis (2-nitrobenzoic acid) with protein thiol groups [29]. The concentrations of the TSH groups are expressed in mmol per liter of plasma or μmol per g of tissue.

Metallothionein 1 (MT1) plasma concentration was determined with the colorimetric method using commercial ELISA kits (Sheep Zn-MT ELISA kit, NeoScientific, London, UK) following the manufacturer's instructions. The optical density of the samples was measured using a microplate reader (Apollo 11 LB913, Berthold Technologies GmHB & Co., KG, Bad Wildbad, Germany).
