Indian Journal of Biochemistry & Biophysics Vol. 40, December 2003, pp. 447-450
Lipid peroxidation and scavenging enzyme levels in the liver of streptozotocin-induced diabetic rats Neslihan Bukan*, Banu Sancak1, Özlem Yavuz2, Cemile Koca 1, Funda Tutkun1, A Tanju Özçelikay 3 and Nilgün Altan1 1
Gazi University, School of Medicine, Department of Biochemistry, Ankara, Turkey 2 Abant İzzet Baysal University, Düzce School of Medicine, Department of Biochemistry, Düzce, Turkey 3 Ankara University, School of Pharmacy, Department of Pharmacology, Ankara, Turkey Received 28 March 2003; revised 28 August 2003 In this study, alterations in the liver antioxidant enzymes status and lipid peroxidation in short-term (8-weeks) and long-term (24-weeks) diabetic rats were examined. Glutathione peroxidase (GSH-Px) activity and malondialdehyde (MDA) levels were significantly increased, but superoxide dismutase (SOD) activity was significantly reduced in 8-weeks diabetic rats, compared to control. Catalase (CAT) activity, however, was found unchanged. In 24-weeks diabetic rats, while GSH-Px activity was unchanged, but SOD and CAT activities and MDA levels were significantly increased, compared to control. These results suggest that diabetes-induced alterations in tissue antioxidant system may reflect a generalized increase in tissue oxidative stress. It can be concluded that lipid peroxidation and antioxidant enzyme levels are elevated in diabetic condition. Hence, diabetes mellitus, if left untreated, may increase degenerative processes due to accumulation of oxidative free radicals. Key words: lipid peroxidation, antioxidant enzymes, diabetic rats
The pathological increase of oxygen free radical generation has been recognized in several diseases1-4. Free radical-mediated oxidative processes are also involved in the pathogenesis of diabetic complications5,6. During diabetes mellitus (DM), persistent hyperglycemia causes an increased production of free radicals via autoxidation of glucose7 and non-enzymatic protein glycation8 which may lead to disruption of cellular functions and oxidative damage to membranes9. Free radicals affect the cells components such as lipid, protein, DNA and ___________ *Corresponding author Tel: 00 90 312 2141000 / 5462 Fax: 00 90 312 2137237 E-mail:
[email protected]
carbohydrates, of which lipids are the most sensitive part. Hence, we determined the malondialdehyde (MDA) levels as the stable end product of lipid peroxidation. The levels of reactive oxygen species are controlled by antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and non-enzymatic scavengers, such as reduced glutathione (GSH). Earlier reports indicate increased or decreased lipid peroxidation, SOD, GSH-Px and CAT activities in various tissues, such as liver, kidney, blood vessels, heart, lymphoid organs, lungs and uterus10,11. Thus, the tissue antioxidant status seems to have an important role in the etiology of diabetic complications. If the diabetic state is associated with a generalized increase in tissue oxidative stress, it might well be reflected in the changes in tissue antioxidant system. Although, the effects of long-term diabetes on the antioxidant enzymes in the rat liver have been reported, the present study was aimed to test the antioxidant enzymes and lipid peroxidation profile in the liver of early and long term duration untreated diabetic rats. Materials and Methods Male albino rats (150-250 g) were used. The experimental group was injected with streptozotocin (STZ, freshly dissolved in citrate buffer, pH 4.5, 60 mg/kg) intraperitoneally, whereas the control group was injected with buffer only. All rats had free access to food and water. They were divided into four groups of 10 animals each: Group 1, 8-weeks control; group 2, 8-weeks DM; group 3, 24-weeks control; and group 4, 24-weeks DM rats. Body weights were obtained before treatment and prior to sacrifice. Rats were sacrificed at the end of 8th and 24th weeks. The 8weeks diabetic rats were considered as short-term diabetic condition and 24-weeks rats reflected the long-tem effects of untreated diabetes mellitus. We used the age-matched control groups to evaluate the changes only caused by diabetic condition. Blood samples were collected from the tail vein at the time of killing and blood glucose levels were determined using an Ames glucometer (Miles Laboratories Inc, Elkhart, IN, USA). Rats were anesthetized using ketamine hydrochloride (Ketalar, Eczacıbaşı, Turkiye). Tissues were
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INDIAN J. BIOCHEM. BIOPHYS., VOL. 40, DECEMBER 2003
briefly washed in ice-cold 0.9% saline (w/v), frozen in liquid nitrogen, weighed and stored at –70 oC, until the subsequent protein and enzyme assays. Protein concentration was measured in tissue homogenate by the method of Lowry12, using bovine serum albumin standard. For SOD assay, tissue samples were homogenized in the ratio of 1:10 in phosphate buffer (pH 7.4) and the supernatant was carefully separated and chloroform and ethanol (3:5 v/v) was added. This mixture was centrifuged at 5000 g for 2 hr. The supernatant was used for SOD assay, using xanthine oxidase as superoxide generator13. Protein concentration of supernatant was measured as described12 and the results were expressed as unit per mg protein tissue. One unit of SOD is defined as the amount of protein that inhibits the rate of nitro blue tetrazolium (NBT) reduction by 50%. For determination of GSH-Px activity, tissue samples were homogenized in the ratio of 1:10 in phosphate buffer (pH 7.0) containing 0.5 mM EDTA and centrifuged at 3500 rpm for 15 min. Protein concentration of supernatant was measured12 and GSH-Px activity was determined, as described14. The results were expressed as nmoles NADPH oxidized per min. per mg protein. Catalase (CAT) activity was measured as described15. Tissue samples were homogenized in phosphate buffer (pH 7.0) and then centrifuged at 3400 rpm for 15 min. H2O2 was added to supernatant and the decreasing of absorbance was measured at 240 nm for 3 min. Protein concentration of supernatant was measured12. The results were expressed as K/mg protein. The levels of malondialdehyde were determined in tissue samples homogenized in the ratio of 1:10 in 1.5% (w/v) cold KCl solution, using thiobarbituric acid method16 and the results were obtained in nmol/g tissue wt. Data are expressed as the mean ± S.D. KruskalWallis (nonparametric ANOVA) test was used for the statistical analysis and Dunn’s multiple comparison test was performed as post-hoc test. A p value of
