Rakesh Kakkar, Jawahar Kalra, Subrahmanyam V. Mantha and Kailash. Prasad. Departments of Pathology and Physiology, College of Medicine, University of ...
Molecularand CellularBiochemistry151:113-119, 1995. 9 1995KluwerAcademicPublishers.Printedin the Netherlands.
Lipid peroxidation and activity of antioxidant enzymes in diabetic rats Rakesh Kakkar, Jawahar Kalra, Subrahmanyam V. Mantha and Kailash Prasad Departments of Pathology and Physiology, College of Medicine, University of Saskatchewan and Royal University Hospital, Saskatoon, Saskatchewan, S7N OW8, Canada Received 1 February 1995; accepted 19May 1995
Abstract We hypothesized that oxygen free radicals (OFRs) may be involved in pathogenesis of diabetic complications. We therefore investigated the levels of lipid peroxidation by measuring thiobarbituric acid reactive substances (TBARS) and activity of antioxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT)] in tissues and blood of streptozotocin (STZ)-induced diabetic rats. The animals were divided into two groups: control and diabetic. After 10 weeks (wks) of diabetes the ~mimals were sacrificed and liver, heart, pancreas, kidney and blood were collected for measurement of various biochemical parameters. Diabetes was associated with a significant increase in TBARS in pancreas, heart and blood. The activity of CAT increased in liver, heart and blood but decreased in kidney. GSH-Px activity increased in pancreas and kidney while SOD activity increased in liver, heart and pancreas. Our findings suggest that oxidative stress occurs in diabetic state and that oxidative damage to tissues may be a contributory factor in complications associated with diabetes. (Mol Cell Biochem 151: 113-119, 1995) Key words: diabetes, lipid-peroxidation, thiobarbituric acid reactive substances, catalase, glutathione peroxidase, superoxide dismutase, oxidative stress
Introduction Insulin dependent diabetes mellitus (IDDM) is characterized by a series of complications that affect many organs. Oxygen free radicals (OFRs) have been implicated in the pathogenesis of diabetes mellitus [ I-4]. During diabetes persistent hyperglycemia causes increased production of OFRs through autoxidation of glucose [5] and also by non-enzymatic protein glycation [6]. OFRs exert their cytotoxic effects on membrane phospholipids resulting in the formation of malondialdehyde (MDA). Peroxidation of membranes increases its fluidity and permeability with loss of membrane integrity [7, 8]. Antioxidant enzymes [Catalase (CAT), Glutathione peroxidase (GSH-Px) and Superoxide dismutase (SOD)] offer protection to cells and tissues against oxidative injury [9]. Increases in levels of OFRs could be due to their increased production and/or decreased destruction. Ineffec-
tive scavenging of OFRs in certain pathological states may play a role in determining tissue injury [10]. OFRs are implicated in the pathophysiology of ischemia/ reperfusion injury and atherosclerosis [ 11, 12]. In diabetes significant changes in lipid metabolism occur leading to vascular complications [13]. Oxidation of lipids in plasma lipoproteins and in cellular membranes is associated with the development of vascular disease in diabetes [5, 14]. Much of the experimental evidence suggests that diabetes and hyperlipidemia alone are not sufficient to induce vascular disease but oxidative stress may be an important and independent risk factor in the development of vascular disease [5]. Apart from increased non-enzymatic glycosylation and autoxidative glycosylation, metabolic stress resulting from changes in status of antioxidant defense systems could lead to oxidative stress in diabetes. There is evidence that there are alterations in free radical
Addressfor offprints: J. Kal[ra,Departmentof Pathology,RoyalUniversityHospital, Saskatoon, Saskatchewan, S7N 0W8, Canada
114 metabolism during diabetes in various tissues [3, 4, 15] but the alterations are quite heterogenous. It is not clear how free radicals may be involved in organ specific complications in diabetes. Hence, the objective of the present study was to measure the levels of TBARS and activity of antioxidant enzymes in various tissues in experimental diabetes.
Materials and methods Induction of diabetes Diabetes was induced in overnight fasted male Fisher rats weighing between 155-185 gm using streptozotocin (STZ) by previously described method [ 16]. STZ was administered intraperitonealy (IP) in the dose of 40 mg/kg dissolved in citrate buffer (0.1 M, pH 4.5). Control rats received citrate buffer intraperitonealy. STZ-induced rats were allowed to drink 5% glucose solution overnight to overcome drug induced hypoglycaemia. Control and STZ-induced animals had free access to food and water ad libitum. Periodic testing (once in 3 days) was done for presence of glucosuria with Ames Multi stix (Miles Canada Incorporated Etobicoke, Ontario) during the 10 week period and body weight changes were also recorded.
Tissue collection and processing At the end of 10 weeks, the animals were anaesthetized with ether, blood was collected by cardiac puncture into heparinized tubes and various organs (liver, heart, pancreas and kidney) were removed and processed for biochemical measurement [12, 17]. Tissues were homogenized in 10 volumes of 50 mM phosphate buffer (pH 7.4) using a Polytron homogenizer (Brinkmann Instruments, Westburry, NewYork) at 4~ twice each for 15 sec. Homogenate was filtered through cheese cloth and filtrate was centrifuged at 3000 rpm for 5 min. Supernatant was used for various measurements. The erythrocyte lysate was prepared for the measurement of biochemical parameters in blood [12].
Plasma glucose assay Plasma glucose levels were measured by oxygen rate method ofKadish et al. [18].
Malondialdehyde (Thiobarbituric acid reactive substances) Malondialdehyde (MDA), an end product of lipid peroxidation reacts with thiobarbituric acid (TBA) to form a coloured substance. Measurement of MDA by TBA reactivity is the most widely used method for assessing lipid peroxidation. Tissue and blood TBARS were estimated by the method of Yagi [19] and Prasad et al. [17]. Blood (0.2 ml) was added to 2 ml of freshly prepared isotonic (0.9%) saline. It was centrifuged at 3000 rpm for 10 min and supernatant was used for assay. Tissue supernatant (0.2 ml) and blood supernatant (0.75 ml) was added to test tubes containing 4.0 ml of 0.08N sulphuric acid (HzSO4). To this 10% phosphotungstic acid (PTA, 0.6 ml)was added and the tubes were vortexed and centrifuged at 3000 rpm for 6 min. The supernatant was discarded and to pellet 2.0 ml ofH2SO 4 and 0.3 ml of PTA was added. The tubes were vortexed and centrifuged again and supernatant was discarded. The pellet was suspended in 4.0 ml of distilled water, 1.0 ml of TBA reagent (pH 3.4, mixture of equal volumes of 0.67% thiobarbituric acid aqueous solution and glacial acetic acid) was added, and the reaction mixture was heated at 95~ for 60 rain.After cooling, 5.0 ml of n-butanol was added to each tube and were vortexed for 20 sec and centrifuged at 3000 rpm for 15 min and the n-butanol layer was used for fluorometric measurement at 553 nm emission and 515 nm excitation.) Tetraethoxypropane was used as a standard and the results are expressed as nmol MDA(TBARS)/mg protein for tissue and nmol MDA (TBARS)/ml blood.
Antioxidant enzymes Catalase measurement Catalase activity of tissues and erythrocyte lysate was measured by the method of Aebi [20] and as described by Mantha et al. [ 12]. Tissue supernatant and lysate ( 10 ~tl,dilution (1:10 v/v) with double distilled water was added to a cuvette containing 50 mM phosphate buffer (pH 7.0) to make final volume of 2 ml. The reaction was started by the addition of 1.0 ml of freshly prepared 30 mM H202. The rate of decomposition of H202 was measured spectrophotometrically from changes in absorbency at 240 nm. The activity of various tissues is expressed as k/sec/mg protein and for blood as k/ sec/gm Hb, where k is the first order rate constant. Glutathione-peroxidase measurement The method of Paglia and Valentine [21 ], modified by Lawrence and Burk [22] was used to measure GSH-Px activity. Assay mixture (2.4 ml) was taken in each tube. It consisted of 2.0 ml of 75 mM phosphate buffer (pH 7.0); 50 ~tl of 60 mM glutathione, 0.1 ml of 30 U/ml glutathione reductase, 0.1
115 ml of 15 mM disodiurn salt ofEDTA, 0.1 ml of 3 mM reduced nicotinamide adenine dinucleotide phosphate (NADPH). Tissue supernatant and erythrocyte lysate 10 ~tl (1:1 dilution with double distilled water) and 0.44).5 ml of H20 was added to make 3.0 ml, the final volume of reaction mixture. The reaction was started by addition of 0.1 ml of 7.5 mM H20 z. The rate o f change o f absorbance during the conversion o f NADPH to NADP was recorded using PU 3000 UV/VIS spectrophotometer at 340 nm for 3 min. GSH-Px activity was expressed as ~tmol of NADPH oxidized to NADP/min/mg protein for tissues and ~tmoles/min/gm Hb for blood using an extinction coefficient (6.22 x 10 6) for NADPH.
Superoxide dismutase measurement The method o f Sun et al. E23] and Prasad et al. [ 17] was used for measuring SOD activity in tissue supernatants and blood. The assay mixture contained (per litre) : xanthine solution (0.3 mM), diethylenetriamine-penta acetic acid (DETAPAC) (0.6 mM, prepared in 50 mM phosphate buffer, pH 7.0), bovine serum albumin (1 g/L), 50 mg nitroblue tetrazolium (NBT) 0.15 M, sodium carbonate (0.4 M, pH 10.2). The SOD activity was determined by adding 2.35 ml of assay mixture to each tube followed by 0.1 ml of 1.67 mM bathocuproinedi-sulfonic acid (BCS) solution. Water (0.5 and 0.55 ml) was added to the blank and reagent blank tubes, respectively. 10--500 ~tl sample was added to sample tubes. 50 I.tl ofxanthine oxidase (20 U/ml) was added to each tube except the reagent blank at an interval of 20 sees to start the reaction. After incubation at 25~ for 20 min, the reaction was terminated by the addition of 1 ml of 0.8 mmol/L cupric chloride. The formazon produced was measured spectrophotometrically at 560 nm. The percent inhibition was calculated and plotted against protein content of the sample. From this plot, the value of SOD was calculated in term of units defined as the amount of SOD that inhibits the reduction of NBT by 50%. Protein content of tissue supernatants was determined by Biuret method [24] and hemoglobin content by Eilers method [25]. Statistical analysis The results are expressed as Mean • S.E. of mean with n = 5. The comparison between control and diabetic groups was made using unpaired t test (BMDP statistical software, University o f California, lqlerkeley). Statistical significance was considered at p < 0.05.
Result General characteristics of diabetic rats Diabetes was confirmed by the presence of glycosuria after 36 h of injection of streptozotocin. More than 75% of the animals that received streptozotocin developed diabetes. Urinary glucose level was greater than 56 mmol/L in diabetic rats. However no glucose was observed in urine of control rats. Plasma glucose level was higher in diabetic rats than in controls (Fig. 1). Body weight of diabetic animals was lower as compared to controls.
Malondialdehyde (Thlobarbituric acid reactive substances) The changes in TBARS of tissues and blood are summarized in Fig. 2 and Table 1 respectively. There was a significant increase in TBARS of heart (37.9%) and pancreas (69.4%) in diabetic rats as compared to controls. TBARS of liver and kidney were similar in both groups. TBARS of blood was higher in diabetic (65.0%) as compared to control group.
Antioxidant enzymes Catalase The changes in catalase activity of the various tissues are summarized in Fig. 3. In control group, CAT activity was lowest in heart and pancreas and highest in liver. CAT activity was higher in liver (51.7%), heart (57.7%) and lower in kidney of diabetic rats as compared to controls. CAT activity of blood in diabetic rats was approximately twice than in controls (Table 1). 30" 4t
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Fig. 1. Changesin the plasma glucose levels in controland diabetic rats. Results are expressedas mean + S.E. *p < 0.05 Controlvs Diabetic.
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Fig. 2. Malondialdehyde content (TBARS) of different organs in control and diabetic rats. Results are expressed as mean • S.E. *p < 0.05 Control vs Diabetic.
Table 1. Malondialdehyde content (TBARS) and antioxidant enzymes in blood of control and diabetic rats
Discussion
Parameters
In the present study the levels of TBARS increased in heart, pancreas and blood of diabetic rats. The highest increase in TBARS was observed in pancreas and blood. The increase in the MDA of pancreas in alloxan induced diabetic rats has been reported previously [26]. The increase in the MDA content of plasma in diabetic patients has also been reported [27]. The increased levels of TBARS suggest an increased levels of OFRs which could be due to their increased production or decreased destruction. Increase in OFRs levels in diabetes could be due to increase in blood glucose levels. Glucose can increase OFRs through autoxidation and through nonenzymatic protein glycation [5,6]. STZ can also give rise to OFRs [4]. Especially in pancreas increase in TBARS could be due to effect of STZ. It has been demonstrated that STZ stimulates H 2 0 2 generation in vitro as well as in vivo in pancreatic [3-cells which causes damage to DNA by .02- and .OH radicals [28]. It has also been reported that STZ accumulates in pancreatic islets [29]. The low levels of antioxidant enzymes observed in the heart and pancreas may make these tissues susceptible to oxidative attack. The increase in antioxidant enzyme activity in various tissues could be due to oxidative stress. Catalase activity was elevated in liver, heart and blood. Our findings in control animals that pancreas contains relatively low CAT activity agrees with Wohaeib and Godin [30]. The increase observed in CAT activity could be due to higher production of H20 r Hypoinsulinemia increases the activity of enzyme fatty acylCo-A oxidase that initiates [3-oxidation of fatty acids resulting in production of H202 [31].
BloodTBARS (nmole/ml blood) Catalase (k/sec/gm Hb) Glutathione-Peroxidase (gmoles/min/gm Hb) Superoxide dismutase (U/gin Hb)
Control
Diabetic
0.409 + 0.042
0.675 • 0.021"
0.115 • 0.003
0.219 • 0.010"
0.374 • 0.013
0.288 • 0.071
79358.00 • 3211
89019.00 • 4547
Values are Mean • S.E. *Significantly different from control at p < 0.05.
Glutathione-peroxidase The changes in GSH-Px activity in various tissues of the two groups are summarized in Fig. 4. In control group GSH-Px activity was highest in liver and lowest in pancreas. In diabetic rats, activity was higher in pancreas and kidney as compared to controls. Diabetic pancreas showed more than two fold increase in GSH-Px activity. No significant change in GSH-Px activity of blood was observed (Table 1). Superoxide-dismutase The changes in SOD activity in various tissues of the two groups are summarized in Fig. 5. In control group, SOD activity was highest in liver and kidney and lowest in pancreas. SOD activity of diabetic rats increased significantly in liver, heart and pancreas as compared to controls. However, no significant change was observed in kidney and blood of diabetic rats (Table 1).
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Fig. 3. Catalase activity of different organs in control and diabetic rats. Results are expressed as mean + S.E. *p < 0.05 Control vs Diabetic.
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Fig. 4. Glutathione peroxidase activity of different organs in control and diabetic rats. Results are expressed as mean + S.E. *p
