A total of 40 male Wistar albino rats, 200-250 g, were randomly divided into 4 groups, two diabetic and two control. They were kept at a constant temperature (22 ± 1°C) with 12 h light and dark cycles. The diabetic groups were rendered diabetic at day 0 by intraperitoneal injection of STZ (65 mg/kg, citrate buffer pH 4.5)
14. Two days later than the STZ injection blood glucose levels were determined. The rats with blood glucose below 150 mg/dL were discarded from the experiment. After diabet induction one of the control and one of the diabetic groups were given 80 mg/kg vitamin C (Roche, Turkey) by the intraperitoneal injection daily until the end of the experiment at day 8. Saline solution was given to the rest of control and diabetic groups intraperitoneally. On day 8, cardiac blood samples were taken from all rats under ether anaesthesia. All experiments were carried out in accordance with the guidelines of the Animal Care and Use Committee of Istanbul University, The Institute of Experimental Medicine (DETAE).
The rats were then killed by administering excess ether and their lenses extracted intracapsularily. The right and left lenses were both homogenized together with physiological saline. Skin samples were taken from the back of each rat after removing local fur. After the epidermis was removed, the skin samples were homogenized in physiological saline. Blood glucose was measured by the RANDOX glucose kit (RANDOX, GL3981, United Kingdom).
Protein assay
In alkali medium, proteins are reacted with cupper ions than reduced by pholine reactive (phosphomobydic-phosphotungstic acid). The absorbance of the blue colored product at 500 nm was evaluated Bovine serum albumin was used as a standard. Total protein level was expressed as % mg15.
Nonenzymatic glycation assay
Protein glycation was assed by the 2-thiobarbituric acid method. The latter involved hydrolysing each 0,5 ml homogenate with 0.5 ml of 0.5 M oxalic acid in an autoclave for 1 h at 124±1 ºC. To this, 0,5 ml 40% trichloracetic acid (w/v) was added, mixed, centrifuged at 1500x g for 10 min, and filtered using fitler paper. Absorbance at 443 nm was recorded. Then 0.75 ml of supernatant was incubated in 0.25 ml of 0.05 M 2-thiobarbituric acid at 37 oC for 30 min. After standing for 15 min at room temperature, absorbance was again measured at 443 nm and the differences between the first and second absorbances were calculated. The protein glycation values were expressed as nmol of fructose per mg protein. Commercial fructose (Sigma) was used as a standard16.
Glutathione assay
The GSH levels were determined according to Beutler's method using Ellman's reagent. The procedure is based on the reduction of Ellman's reagent by SH groups to form 5,5′-dithiobis (2-nitrobenzoic acid) with an intense yellow color, measured spectrophotometrically at 412 nm using a Shimadzu spectrophotometer. Results were expressed as µmol GSH/g tissue17.
Lipidperoxidation assay
Tissue samples were homogenized with ice-cold saline solution (0.9 %) for the determination of malondialdehyde (MDA) and glutathione levels. LPO levels were estimated by Ledwozyw's method18. In brief, the adducts formed following boiled tissue sample with thiobarbutiric acid is extracted with n-butanol. The difference in optical density at 532 nm is measured in terms of the tisuue malondialdehyde (MDA) content, also of TBARS, which is undertaken as an index of lipid peroxidation. Results were expressed as µmol MDA g protein−1.
SDS-Polyacrlyamide gel electrophoresis
Protein electrophoresis was carried out as described by Laemmli19. Schleicher and Schueller Profile System mini-electrophoresis was performed with Sigma low molecular and high molecular weight protein standards (SDS-7,Dalton Mark VII-L and Hemocyanin crosslinked standart, Sigma) After electrophoresis, scans of Coomassie blue stained protein bands were obtained using a densitometer (Helena Laboratories TCL plus). Peak areas were measured with a plannimeter (Placom-Sokkisha, Kp-90N, digital) and the protein percentage calculated in each band.
Statistical Analysis
Statistical analysis was carried out using GraphPad Prism 3.0 (GraphPad Software, San Diego, CA, USA). Groups of data were compared with an analysis of variance (ANOVA) followed by Tukey's multiple comparison tests. Values of P < 0.05 were regarded as significant.
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