Jawahar Kalra

Oxygen free radicals and hypercholesterolemic atherosclerosis: Effect of vitamin E☆

We investigated the effects of a high-cholesterol diet in the presence and absence of vitamin E on the lipid peroxidation product malondialdehyde of blood and aortic tissue, the oxygen-free-radical-producing activity of polymorphonuclear leukocytes (PMNs) (PMN chemiluminescence), and the blood lipid profile in rabbits. The animals were divided into four groups each of which comprised 10 rabbits. Rabbits in group I received a regular rabbit chow diet; those in group II received vitamin E; those in group III received high cholesterol + vitamin E; and those in group IV received a high-cholesterol diet. Blood concentrations of triglycerides, total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), very low-density lipoprotein cholesterol (VLDL-C), malondialdehyde, and PMN chemiluminescence were measured. The aorta of each rabbit was removed at the end of the protocol for assessment of atherosclerotic changes (gross and microscopic) and malondialdehyde. Serum triglycerides, total cholesterol, HDL-C, LDL-C, and VLDL-C increased while HDL/LDL ratio decreased in groups III and IV but remained unchanged in group I. There was an increase in the HDL-C component and HDL/LDL ratio and a decrease in the LDL-C component and triglycerides in group II. Blood and aortic tissue malondialdehyde increased in group IV but decreased in groups II and III. PMN chemiluminescence increased in groups III and IV. Atherosclerotic changes were marked in group IV as compared with those in group III. However, histologic changes in the aortas were similar in groups III and IV. The increased levels of blood and aortic tissue malondialdehyde and PMN chemiluminescence, which were associated with development of atherosclerosis, suggest a role of oxygen free radicals in the pathogenesis of hypercholesterolemia-induced atherosclerosis. The protection afforded by vitamin E, which was associated with a decrease in blood and aortic tissue malondialdehyde concentration in spite of hypercholesterolemia, supports the hypothesis that oxygen free radicals are involved in the development of hypercholesterolemic atherosclerosis.

Lipid peroxidation and activity of antioxidant enzymes in diabetic rats

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 animals 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.

ANTIOXIDANT DEFENSE SYSTEM IN DIABETIC KIDNEY : A TIME COURSE STUDY

Oxygen free radicals (OFRs) have been suggested to be a contributory factor in complications of diabetes mellitus. In the present study, we investigated the lipid peroxide level measured as thiobarbituric acid reactive substances (TBARS) and activities of antioxidant enzymes viz., [superoxide dismutase (SOD), catalase (CAT) and glutathione-peroxidase (GSH-Px)] in the kidney of streptozotocin induced diabetic rats at various stages of development of diabetes. Sprague Dawley rats were divided into two groups: group I, control (n = 42) and group II, diabetic (n = 42). Each group was further subdivided into seven groups each consisting of six rats. Rats in subgroups were studied at weekly intervals (0 to 6 weeks). Blood glucose levels were estimated at the time of sacrifice. TBARS levels and activity of antioxidant enzymes were measured in kidney. The levels of TBARS in the diabetic group increased initially, dropped to baseline level after 2 weeks and then progressively increased at 5th and 6th week (p < 0.05). There was an increase in catalase activity at first week after that it decreased as compared to control group. However, GSH-Px activity in the diabetic group increased after 1 week and then remained at the same level except a small drop in the 2nd week. Total SOD and CuZn-SOD activity increased significantly in diabetic kidney as compared to controls at all time intervals, while Mn-SOD activity showed no change. The present findings suggest that oxidative stress accompanies at early onset of diabetes mellitus and the susceptibility of the kidney to oxidative stress during the early stages may be an important factor in the development of diabetic nephropathy.

Antioxidant enzymes in hypercholesterolemia and effects of vitamin E in rabbits

We investigated the effects of high cholesterol diet in the absence and presence of vitamin E on the activity of antioxidant enzymes [superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px)] in rabbits. The animals were divided into 4 groups each comprising of 10 rabbits. Group I, regular rabbit chow diet; Group II, regular rabbit chow diet with added vitamin E; Group III, high cholesterol diet; and Group IV, high cholesterol diet+vitamin E. Antioxidant enzymes of blood were measured in each group before and after 1, 2, 3, and 4 months on the experimental diets. The aorta was removed at the end of the protocol for measurement of antioxidant enzymes. There was a decrease in activity of SOD and GSH-Px and an increase in activity of catalase in blood of Group III. Vitamin E produced a decrease in blood SOD, catalase and GSH-Px activity in Group II and prevented the decrease in SOD and GSH-Px activity in Group IV but did not affect the changes in the catalase activity. SOD, catalase and GSH-Px activity of aortae from Group III increased significantly, while catalase activity increased and GSH-Px activity decreased in those from Group II. Vitamin E prevented the cholesterol-induced rise in catalase and GSH-Px activity in aorta but did not prevent the rise in SOD activity. These results suggest that the activity of antioxidant enzymes in blood is affected differently from that in aortic tissue. There appears to be a mutually supportive interaction among the antioxidant enzymes which provide defense against oxidant injury. The protective effects of vitamin E against hypercholesterolemic atherosclerosis may not be due to changes in the antioxidant enzymes but may be mainly mediated through its chain-breaking antioxidant activity.

Effect of polymorphonuclear leukocyte-derived oxygen free radicals and hypochlorous acid on cardiac function and some biochemical parameters

Polymorphonuclear (PMN) leukocyte activation is known to result in the production and release of oxygen free radicals and hypochlorous acid. Various clinical conditions are associated with PMN leukocyte stimulation. The present investigation deals with the effects of stimulated PMN leukocytes in the absence and in the presence of scavengers of oxygen free radicals (superoxide dismutase, catalase), hypochlorous acid quencher (methionine), and myeloperoxidase inhibitor (azide) on cardiac function and contractility; blood lactate, gases, and pH levels, blood and cardiac tissue malondialdehyde; and PMN leukocyte chemiluminescence activity in anesthetized dogs. Opsonised zymosan was used for stimulation of PMN leukocytes, and the effects were observed for 2 hours. The dogs were divided into four groups: group I, zymosan; group II, superoxide dismutase + catalase + zymosan; group III, methionine + zymosan; group IV, azide + methionine + zymosan. Zymosan produced a decrease in cardiac function and in indices of myocardial contractility and an increase in systemic and pulmonary vascular resistance. There was a decrease in blood pH and in PMN leukocyte chemiluminescense and an increase in the blood lactate and malondialdehyde. Superoxide dismutase plus catalase and methionine reduced the effect of zymosan on cardiac function and contractility and on blood malondialdehyde, lactate, and pH. The combination of azide and methionine did not prevent the deleterious effects of zymosan on cardiac function and contractility. Cardiac tissue malondialdehyde levels were lower in groups III and IV than in groups I and II which had values similar to each other. Methionine was superior to superoxide dismutase plus catalase in the prevention of the deleterious effects of PMN leukocyte stimulation on the various measured parameters. These results suggest that oxygen free radicals and hypochlorous acid are cardiac depressants and increase systemic and pulmonary vascular resistance in addition to causing tissue damage. Clinical situations with PMN stimulation may result in cardiac depression. The oxygen free radical scavenger and hypochlorous acid quencher may be beneficial in the counteraction of the deleterious effects of PMN leukocyte stimulation on the hemodynamic parameters and cellular integrity.

Increased oxygen free radical activity in patients on cardiopulmonary bypass undergoing aortocoronary bypass surgery

Cardiac dysfunction after cardiopulmonary bypass (CPB) has been reported by various investigators. Oxygen free radicals have been shown to depress cardiac function and contractility. To evaluate the possible role of oxygen free radicals (OFR) in post-pump cardiac dysfunction, measurements of cardiac function, OFR producing activity of polymorphonuclear (PMN) leukocytes (PMN chemiluminescence) and malondialdehyde (MDA), a lipid peroxidation product, in blood were made at induction of anesthesia (T1), before cross clamping of the aorta (T2), after closure of the chest (T3), and 24 hours postoperatively (T4) in 21 patients undergoing aortocoronary bypass surgery. The total OFR-derived chemiluminescence at T1, T2, T3, and T4 was 1590 +/- 156, 3169 +/- 338, 1972 +/- 214, and 2614 +/- 366 mv.min.10(6) PMN-1, respectively. Superoxide dismutase (SOD)-inhibitable chemiluminescence at T1, T2, T3, and T4 was 1214 +/- 129, 2674 +/- 328, 1752 +/- 215, and 2139 +/- 292 mv.min.10(6) PMN-1, respectively. Superoxide anion at T1, T2, T3, and T4 was 0.99 +/- 0.14, 1.30 +/- 0.17, 1.07 +/- 0.14, and 1.19 +/- 0.12 nmol.10(6) PMN-1.30 min-1, respectively. Blood MDA at T1, T2, T3, and T4 was 0.17 +/- 0.02, 0.25 +/- 0.03, 0.20 +/- 0.03, and 0.23 +/- 0.02 nmol/ml, respectively. OFR-derived and SOD inhibitable chemiluminescence, superoxide anion, and blood MDA increased significantly during CPB and postoperatively. There were decreases in the blood pressure and stroke volume, and increases in the central venous pressure, capillary wedge pressure, and heart rate during CPB and postoperatively. Cardiac output remained unchanged during this procedure. There was leukopenia during CPB.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of oxygen free radicals on cardiovascular function at organ and cellular levels

Oxygen free radicals (OFR) have been implicated as a causative factor of cell damage in several pathologic conditions. It is possible that OFR could have effects on cardiac function and contractility. The present investigation deals with the effects of OFR in the absence and in the presence of scavangers of OFR (superoxide dismutase and catalase) on cardiac function, index of cardiac contractility, serum creatine kinase (CK), and blood lactate, PO2 and pH in the anesthetized dogs. The hemodynamic measurements and collection of blood samples for measurement of CK, lactate, PO2 and pH were made before and at various intervals after administration of OFR for 1 hour. Xanthine and xanthine oxidase were used to generate OFR. OFR produced a decrease in cardiac function and indices of myocardial contractility and an increase in the serum CK. OFR produced an increase in the systemic and pulmonary vascular resistance. Although there was a tendency for an increase in the blood lactate, the increase was not significant. The blood PO2 and pH were not affected. Superoxide dismutase (SOD), alone or in combination with catalase, tended to protect cardiac function against the deleterious effects of OFR. Scavangers of OFR prevented the OFR-induced rise in serum CK. Although the protective effect of SOD plus catalase was slightly better than SOD alone, the results were not significantly different from each other. These results suggest that OFR are cardiac depressant and increase the peripheral vascular resistance besides causing cellular damage. Scavangers of OFR may be beneficial in counteracting the deleterious effects of OFR on hemodynamic parameters and cellular integrity.

Oxygen free radicals as a mechanism of hypercholesterolemic atherosclerosis: Effects of probucol

We investigated the effects of high cholesterol diet in the presence and absence of probucol on the genesis of atherosclerosis, the blood lipid profile, aortic tissue lipid peroxidation product malondialdehyde (MDA), and aortic tissue chemiluminescence (CL) a marker for antioxidant reserve in rabbits. Five groups each of 10 rabbits were studied: group I, regular rabbit chow; group II, as I + cholesterol (0.5%); group III, as I + cholesterol (0.5%) and probucol (0.5 gm/kg/day); group IV, as I + cholesterol (1%), and group V, as I + cholesterol (1%) and probucol (0.5 gm/kg/day). Blood concentrations of triglyceride (TG), total cholesterol (TC), high density lipoproteincholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), very low density lipoprotein-cholesterol (VLDL-C) were measured at monthly intervals for 4 months. The aorta was removed at the end of the protocol for assessment of atherosclerotic changes (gross and microscopic), MDA concentration and CL. TC, LDL-C, LDL-C/HDL-C ratio increased in all the groups except group I, while VLDL-C increased in group II only. HDL-C decreased in groups III, IV and V but remained unchanged in groups I and II. There was a decrease in HDL-C and VLDL-C components and an increase in LDL-C components of total cholesterol in all the groups II, III, IV and V, the changes being greater in group IV than in group II. Probucol did not appreciably affect the changes in lipid profile except that it decreased HDL-C significantly. Aortic tissue MDA increased in groups II, IV and V to a similar extent. Aortic CL which was measured only in groups I, IV and V increased to similar extent in the latter two groups. Atherosclerotic changes were greater in group II than in group III but similar to that in groups IV and V. Histological changes were practically similar in groups II, III, IV and V. The increased levels of aortic MDA and CL, which were associated with development of atherosclerosis, suggest a role for oxygen free radicals in the pathogenesis of hypercholesterolemia-induced atherosclerosis. Protection afforded by probucol was associated with a decrease in aortic MDA in spite of hypercholesterolemia. Ineffectiveness of probucol in 1% cholesterolfed rabbits was associated with its inability to reduce MDA and increase antioxidant reserve. These findings further support for the hypothesis that oxygen free radicals are involved in the genesis and maintenance of hypercholesterolemic atherosclerosis.

Experimental Atherosclerosis and Oxygen Free Radicals

Oxygen free radicals are known to produce cellular injury by peroxidation of phospholipids in the cell membrane. These free radicals might damage the endothelial cell and thus set the stage for atherosclerosis. The authors studied the effect of high-cholesterol diets on the genesis of atherosclerosis and lipid peroxidation products, malondialdehyde (MDA) in rabbits. The animals were divided into four groups each comprising 5 rabbits, on the basis of their diets. Group I, control diet; group II, cholesterol; group III, coconut oil; group IV, a mixture of cholesterol, coconut oil, and cholic acid. Rabbits were sacrificed five months after being on the respective diets. Blood samples were obtained for the measurements of total cholesterol, high density lipoprotein cholesterol (HDL- C), low density lipoprotein cholesterol (LDL-C), very low density lipoprotein cholesterol (VLDL-C), triglycerides, and MDA at the end of the protocol. The aortas were removed from different animals for the identification of athero sclerotic plaques. Plaques were detected in all the animals in group II and group IV. The serum total cholesterol, LDL-C, and VLDL-C were significantly higher in animals of group II and IV than in those of group I. The values for serum total cholesterol, HDL-C, LDL-C, and VLDL-C in group III were not signifi cantly different from those in group I. The blood MDA and serum triglycerides were also higher in animals of group II and IV than in those of group I. There were, however, no significant differences in these parameters in group III as compared with those in group I. This study showed that there was an increase in the lipid peroxidation product (MDA) in all those rabbits that had atheroscler otic changes in the aorta. This increase might be due to an increase in oxygen free radicals, which may be involved in the genesis and maintenance of choles terol-induced atherosclerosis.

Detection of ischemia-reperfusion cardiac injury by cardiac muscle chemiluminescence

Various methods have been used in the past to assess the implication of oxygen free radicals (OFR) in ischemia-reperfusion-induced cardiac injury. Luminol-enhanced tert-butyl-initiated chemiluminescence in cardiac tissue reflects oxidative stress and is a very sensitive method. It was used to elucidate the role of OFR in cardiac injury due to ischemia and reperfusion. Studies were conducted on perfused isolated rabbit hearts in three groups (n = 8 in each): I, control; II, submitted to global ischemia for 30 min; III, submitted to ischemia for 30 min followed by reperfusion for 60 min. The heart tissue was then assayed for chemiluminescence (CL); content of malondialdehyde (MDA), an indicator of OFR-induced cardiac injury; and activity of tissue levels of antioxidants [superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px)].The control values for left and right ventricular CL and malondialdehyde were 81.1 ± 15.4 (S.E.) and 182.4 ± 50.3 (S.E.), mv-min-mg protein−1; and 0.024 ± 0.006 (S.E.) and 0.324 ± 0.005 (S.E.) nmoles-mg protein−1 respectively. Ischemia produced an increase in the cardiac CL (3.3 to 4.4 fold) and MDA content (2 to 2.6 fold). Reperfusion following ischemia also produced similar changes in CL and MDA content. The control values for activity of left ventricular SOD, catalase, and GSH-Px were 45.77 ± 1.73 (S.E.) U-mg protein−1 5.35 ± 0.51 (S.E.) K-10−3-sec−1-mg protein−1, and 77.50 ± 7.70 (S.E.) nmoles NADPH-min−1-mg protein−1 respectively. Activities of SOD and catalase decreased during ischemia but were similar to control values in ischemic-reperfused hearts. The GSH-Px activity of left ventricle was unaffected by ischemia, and ischemia-reperfusion. GSH-Px activity of the right ventricle increased with ischemia, and ischemic-reperfusion.These results indicate that cardiac tissue chemiluminescence would be a useful and sensitive tool for the detection of oxygen free radical-induced cardiac injury.