20 A. Walczewska et al. Table 2. Composition and energy density of the purified diets Low-fat diet High-fat diet g/kg Casein Sucrose Corn starch1 Fat2 Cellulose3 AIN-93 Mineral Mix4 AIN-93 Vitamin Mix4 Choline bitartrate5 L-Cystine5 Energy density (kcal/g) 194.4 291.5 364.4 52.5 48.6 34.0 9.7 1.9 2.9 3.92 228.6 201.1 252.6 203.4 57.1 40.0 11.4 2.3 3.4 4.62 1 Stobimyl XMH 042 (Stockmeier Food, Germany); 2 Lard, (Pamso Com. Inc, Pabianice, Poland), Menhaden Fish Oil (Omega Protein, Inc. Hammond, LA), Sunflower Oil (Fat Processing Com. Inc, Warsaw; Poland); 3 Arbocel® (J. Rettenmaier & Söhne Gmbh + Co Faserstoff-Werke, Rosenberg, Germany); 4 Research Diets, Inc (New Brunswick, NJ); 5 Sigma-Aldrich Ltd (Poznan, Poland). 6 weeks of feeding followed by overnight fasting, the rats were anesthetized with ketamine and xylazine (i.p. 20 and 10 mg/kg, respectively), blood was withdrawn by way of heart puncture, and then the animals were decapitated. Chemiluminescence assay Luminol-dependent whole blood chemiluminescence (CL) was measured according to Kukovetz et al. [19] with some modifications [20]. Tubes with three microlitres of fresh blood, and 947 μl of the mixture solution were placed into the thermostatically controlled 1251 luminometer (Bio- Orbit®, Turku, Finland) and incubated at 37°C. After 30 min of incubation the resting CL (Baseline) was recorded for 1 min. Then 50 μl of phorbol-12-myristate-13-acetate (PMA) in sterile 0.9% NaCl was added by an automatic dispenser to a final concentration of 10−5 M and CL was measured continuously for 20 min. CL peak intensity (Peak) and total CL in response to PMA (Total) measured as the area under the CL intensity curve until returning to baseline were calculated by MultiUse v. 2.01 software. CL signal in all samples returned to a baseline between 6th to 9th minute of recording. The mixture solution containing 0.025% w/v luminol and 5% w/v glucose in deionized pyrogen-free water was freshly prepared. The experiment was run in triplicate. The baseline CL and CL Peak were expressed in arbitrary units corresponding to milivoltage potential (aU) per 104 leukocytes in the blood sample (aU/104 WBC). The Total CL was expressed in arbitrary unit × seconds per 104 WBC (aU × s/104 WBC). Erythrocyte hemolysis The hemoglobin concentration and hematocrit were measured using hematological apparatus (ABX Micros, Montpellier, France) calibrated for the rat blood. Packed red blood cells were washed three times with buffered saline solution, pH 7.4, hemolysed with 5 volumes of ice-cold distillated water, deproteinized with 10% 5-sulphosalicilic acid, and centrifuged at 4°C. GSH and GSSG measurement The Tietze’s GSH recycling method based on the sequential GSH oxidation by 5,5'-dithiobis-2-nitrobenzoic acid (DTNB), and reduction by the glutathione reductase in the presence of NADPH was applied [21]. The formation of 2-nitro-5-thiobenzoic acid was measured spectrophoto- metrically at 412 nm for 5 min. The total glutathione con- centration was calculated on the basis of a calibration curve obtained with authentic GSH in range 1.5–50 μM. Briefly, the samples were added to 115 mM phosphate buffer, pH 7.4, containing EDTA (5 mM), NADPH (0.2 mM), and DTNB (0.6 mM). The reaction was initiated by addition of glutathione reductase (0.8 U/L). Oxidized glutathione was determined by the same method after derivatization of native GSH with 2-vinylpiridyne (185 mM) [22]. The lower detection limit for GSSG concentration was 0.1 μM. All measurements were performed in duplicate. Ferric reducing ability of plasma (FRAP) This method is based upon the reduction of ferric tripyridyltriazine Fe (III)-TPTZ to coloured ferrous tripy- ridyltriazine Fe (II)-TPTZ at low pH resulting in an increase in absorbance at 593 nm. This assay was performed according to Benzie and Strain [23]. The FRAP solution consisted of 300 mM acetate buffer, pH 3.6, 10 mM TPTZ in 40 mM HCl, and 20 mM FeCL3 in the ratio 10:1:1 was prepared freshly. The FRAP solution (900 μl) was pre- warmed to 37°C, next 50 μl of plasma and 50 μl of deionized water were added, and all reagents were incubated at 37°C for 20 min. Then, the absorbance was measured in spectrophotometer (Ultrospec III, Pharmacia LKB, Cambridge, England) in duplicate. The results were expressed in Fe (II) ions using the calibration curve prepared with the FRAP reagent containing Fe2SO4 in range 0.5–50 mM. Plasma radical scavenging activity DPPH test based on decomposition and discolouring of red stable 1,1-diphenyl-2-picrylhydrazyl radicals by a variety of plasma antioxidants acting as electron donors was assayed as Takao et al. [24]. Briefly, 970 μl of 50% methanol, 5 μl of 10 mM in methanol were combined and the control absorbance was measured at 520 nm (A520). The samples were incubated at 20°C for 3 min, then 25 μl of deproteinized with acetonitryl plasma was added, shaken, J. Clin. Biochem. Nutr.