M.A. Villalobos

Lipid peroxidation and glutathione system in hyperlipemic rabbits: influence of olive oil administration

We studied the effect of supplementation (10% w/w) of a hyperlipemic diet (1% cholesterol) with olive oil (OLIV) for 6 weeks in four groups of 10 rabbits each. At the end of this period, we determined lipid peroxidation, glutathione content, and glutathione peroxidase, reductase and transferase activities in liver, brain, heart, aorta and platelets. The atherogenic diet increased tissue lipid peroxidation and decreased the protective antioxidant effect of glutathione. Dietary supplementation with olive oil reduced tissue lipid peroxidation by 71.6% in liver, 20.3% in brain, 84.5% in heart, 63.6% in aorta, 72% in platelets. The ratios total/oxidized glutathione were increased in all tissues (49% in liver, 48% in brain, 45% in heart, 83% in aorta, 70% in platelets). Olive oil increased glutathione peroxidase and transferase activities in all tissues. We conclude that in rabbits made hyperlipemic with a diet rich in saturated fatty acids, olive oil decreased tissue oxidative stress.

Effect of propofol on oxidative stress in an in vitro model of anoxia-reoxygenation in the rat brain

Propofol, an intravenous anaesthetic, is similar in chemical structure to the active nucleus of antioxidant substances such as alpha-tocopherol (vitamin E). The present study was designed to test whether propofol had antioxidant effects in an in vitro model of anoxia-reoxygenation in slices of rat brain. We used seven experimental groups: (1) control oxygenated tissue; (2) tissue subjected to anoxia for 20 min and reoxygenation for 3 h; and tissues processed as described and incubated with (3) Intralipid (commercial solvent for propofol), or propofol at a concentration of (4) 10 micromol/l, (5) 50 micromol/l, (6) 150 micromol/l or (7) 300 micromol/l. The production of lipid peroxides was quantified as thiobarbituric acid reactive substances (TBARS); tissular glutathione production and the activities of glutathione peroxidase (GSHpx), glutathione reductase (GSSGrd) and glutathione transferase (GSHtf) were also measured. Reoxygenation led to tissular oxidative stress, which was curtailed by propofol. The anaesthetic led to a 47% reduction in TBARS, a 165% increase in the reperfusion-inhibiting glutathione content, a 47% decrease in GSHpx activity, and an 87% increase in GSHtf activity. Intralipid had no effect on any of the parameters studied here. We conclude that propofol has a clear antioxidant effect in rat brain tissue subjected to anoxia-reoxygenation.

Antithrombotic Potential of Olive Oil Administration in Rabbits with Elevated Cholesterol

Olive oil is the main source of dietary fatty acids in the Mediterranean region. The objective of this study was to evaluate the effect of dietary supplementation with virgin olive oil in an experimental model with rabbits fed an atherogenic diet (saturated fat 48% of total fat). Four different groups of 10 animals each were studied: (1) normolipemic diet (NLD), (2) atherogenic diet or saturated fatty acid-enriched diet (SFAED), (3) NLD with 15% olive oil (NLD+OLIV), and (4) SFAED with 15% virgin olive oil (SFAED+OLIV). The animals were fed the experimental diets for 6 weeks, after which we determined serum lipid profile (total cholesterol, HDL-cholesterol, and triglycerides), platelet aggregation, platelet thromboxane B(2), aortic prostacyclin, and platelet and vascular lipid peroxidation. Scanning electron microscopic images of the vascular endothelium were studied, as were morphometric parameters in the arterial wall and thrombogenicity of the subendothelium (annular perfusion chamber). Animals fed the SFAED showed platelet hyperactivity and increased subendothelial thrombogenicity. Animals fed the SFAED+OLIV showed, compared with the SFAED group, an improved lipid profile with decreased platelet hyperactivity and subendothelial thrombogenicity and less severe morphological lesions of the endothelium and vascular wall. We conclude that supplementation of the SFAED with 15% olive oil reduced vascular thrombogenicity and platelet activation in rabbits. Although the percentage of olive oil in the diet was higher than the amount in the human diet, these results may be helpful in determining the effect of olive oil in the human thrombogenic system.

In vitro effects of Clopidogrel on the platelet-subendothelium interaction, platelet thromboxane and endothelial prostacyclin production, and nitric oxide synthesis

Clopidogrel is an antiplatelet drug that belongs to the group of thienopyridines. Because of its main mechanism of action most studies of clopidogrel have centered on the platelet ADP pathway. The aim of the present study was to compare the effects of clopidogrel, ticlopidine, and aspirin, on platelet activation by collagen (the main inducer of platelet activation in vivo), prostanoid, and NO production, and the effects on blood perfusion experiments. Clopidogrel inhibited platelet aggregation induced in whole blood by collagen and TxB2 production to a greater extent than did ticlopidine. Prostacyclin synthesis did not change after incubation with thienopyridines, whereas aspirin inhibited synthesis in a dose-dependent manner. Thienopyridines increased NO production to a greater extent than did aspirin. All three drugs impaired the platelet-subendothelium interaction under flow conditions. With thienopyridines, the presence of endothelium did not modify the percentage of the surface coated by platelets.

Effects of triflusal and its main metabolite HTB on platelet interaction with subendothelium in healthy volunteers

The ex vivo effect of triflusal and acetylsalicylic acid (ASA) on platelet interaction with the subendothelium using the Baumgartner perfusion system (wall shear rate 350 s−1) was assessed in blood from 10 healthy volunteers who given a 15-day course of triflusal 600 mg per day and ASA 400 mg per day in a crossover trial.The percentage of platelets on the subendothelium showed a decrease of 62% in samples from subjects on ASA and a decrease of 93% in those from subjects on triflusal (P<0.005). The percentage of the subendothelial surface covered by platelets was reduced by 23.3% after treatment with ASA, mainly due to inhibition of aggregates (75.2%), and by 29.9% after treatment with triflusal, mainly due to inhibition of aggregates (89.6%) and of adhesion (25%). The subendothelial surface covered by activated platelets (adhesions and thrombi) showed 32.5% inhibition after treatment with triflusal and 11.6% after treatment with ASA (P<0.043 vs. triflusal). In the in vitro experiments, 10 μmol·l−1 triflusal did not modify the percentage of the subendothelium covered by platelets. HTB 1 mmol·l−1 inhibited adhesion (26%) and aggregates (18%).We conclude that HTB participates in the ex vivo effects of triflusal on the platelet-subendothelium interaction.The ex vivo effect of triflusal and acetylsalicylic acid (ASA) on platelet interaction with the subendothelium using the Baumgartner perfusion system (wall shear rate 350 s−1) was assessed in blood from 10 healthy volunteers who given a 15-day course of triflusal 600 mg per day and ASA 400 mg per day in a crossover trial. The percentage of platelets on the subendothelium showed a decrease of 62% in samples from subjects on ASA and a decrease of 93% in those from subjects on triflusal (P<0.005). The percentage of the subendothelial surface covered by platelets was reduced by 23.3% after treatment with ASA, mainly due to inhibition of aggregates (75.2%), and by 29.9% after treatment with triflusal, mainly due to inhibition of aggregates (89.6%) and of adhesion (25%). The subendothelial surface covered by activated platelets (adhesions and thrombi) showed 32.5% inhibition after treatment with triflusal and 11.6% after treatment with ASA (P<0.043 vs. triflusal). In the in vitro experiments, 10 μmol·l−1 triflusal did not modify the percentage of the subendothelium covered by platelets. HTB 1 mmol·l−1 inhibited adhesion (26%) and aggregates (18%). We conclude that HTB participates in the ex vivo effects of triflusal on the platelet-subendothelium interaction.

Effects of α-tocopherol on lipid peroxidation and mitochondrial reduction of tetraphenyl tetrazolium in the rat brain

The antioxidant effect of alpha-tocopherol was assessed in a model of ischemia-reperfusion in the rat brain. In this model, permanent ischemia of the cortical branches of the middle cerebral artery was combined with bilateral occlusion of the common carotid arteries for 1 h and restoration of circulation for a period of 2 h. Lipid peroxidation and mitochondrial reduction of tetraphenyl tetrazolium (TPT) were determined in both untreated and d-alpha-tocopherol treated rats. Ferrous sulfate and ascorbic acid (FeAs) were used to induce lipid peroxidation via the formation of hydroxyl anions. Malondialdehyde (MDA) increased in the ischemia-reperfusion areas (+101%), but FeAs-induced MDA did not vary in the area of permanent ischemia. Brain tissue undergoing ischemia-reperfusion was about 50% less sensitive to the antioxidant effect of ascorbic acid. The reduction of TPT showed 52% mitochondrial damage in the area of ischemia-reperfusion, whereas mitochondrial activity in the area of permanent ischemia was 177 times lower as compared to controls. d-alpha-tocopherol caused a 40% inhibition of MDA production and 16.5% and 21.5% decrease in mitochondrial activity in the areas of ischemia-reperfusion and permanent ischemia, respectively.

Effects of S-adenosyl-l-methionine on lipid peroxidation and glutathione levels in rat brain slices exposed to reoxygenation after oxygen-glucose deprivation

We analyzed the effects of S-adenosyl-L-methionine (AdoMet) on tissue oxidative stress in rat brain slices exposed to reoxygenation after oxygen-glucose deprivation. The thiobarbituric acid reactive substances (TBARS), total and oxidized glutathione, and lactate-dehydrogenase efflux (LDH) from tissue to the incubation medium, were measured. Brain slices were incubated without glucose and with N2, then glucose was added and O2 was perfused. After the anoxic-reoxygenation period, increase in TBARS, oxidized glutathione and LDH efflux, and decrease in total glutathione levels, were observed. The incubation with AdoMet before the anoxic period reduced TBARS (31-1000 micromol/l), glutathione production was increased (31-1000 micromol/l), LDH efflux decreased 6.41% with 15 micromol/l and 61.5% with 500 micromol/l). In the ex vivo experiments, we administered 50 mg/kg per day p.o., AdoMet for 3 days, then brain slices were collected and the anoxia-reoxygenation experiment was carried out. AdoMet led to the inhibition of brain lipid peroxidation and increased total glutathione production, after 3 h-reoxygenation. The increase of LDH efflux in non-treated rats was reduced by 77%. We conclude that AdoMet exerts citoprotective effects in an experimental model of brain slices reoxygenation after oxygen-glucose deprivation.

Effect of S-adenosyl-L-methionine on rat brain oxidative stress damage in a combined model of permanent focal ischemia and global ischemia-reperfusion.

We analyzed the effects of S-adenosyl-L-methionine (SAM) on tissue oxidative status in a combined model of permanent focal ischemia and global reperfusion in the rat brain. The production of thiobarbituric acid reactive substances (TBARS) was measured under basal conditions and after induction with ferrous salt as an indicator of brain lipid peroxidation. Total, oxidized and reduced glutathione were measured as indicators of the antioxidant defense capacity of brain tissue. Mitochondrial reduction of tetraphenyl tetrazolium (TPT) was quantified morphometrically. Results obtained in vitro showed that incubation with SAM reduced lipid peroxidation, with a maximum inhibition of 65.12+/-5.99% after incubation with 1 mmol/l; glutathione production was not significantly modified. In the brain ischemia-reperfusion model, TBARS production increased and glutathione content decreased, and mitochondrial reduction of TPT decreased significantly after ischemia-reperfusion in areas dependent on carotid circulation. The administration of 50 mg/kg SAM per day for 3 days led to the inhibition of brain lipid peroxidation and increased total glutathione production. These changes were accompanied by an increase in mitochondrial capacity to reduce TPT. We conclude that SAM reduces oxidative damage in the rat brain in an experimental model of ischemia-reperfusion.

Antioxidant potential of evening primrose oil administration in hyperlipemic rabbits

The dietary intake of saturated fatty acids affects arteriosclerosis. We studied the effect of supplementation (15% wt/wt) of a hyperlipemic diet (1.33% cholesterol) with evening primrose oil (EPO) (Oenothera biennis) for 6 weeks in four groups of 10 rabbits each. At the end of this period we determined lipid peroxidation, glutathione content, and glutathione peroxidase, reductase and transferase activities in liver, brain, heart, aorta and platelets. The atherogenic diet increased tissue lipid peroxidation and decreased the protective antioxidant effect of glutathione. Dietary supplementation with EPO reduced tissue lipid peroxidation (61% in liver, 57% in brain, 42% in heart, 24% in aorta, 33% in platelets). Total glutathione was increased, especially in the aorta (90%) and platelets (200%); however, in all tissues the percentage of oxidised glutathione decreased. Evening primrose oil reduced glutathione peroxidase activity and increased the activities of glutathione reductase and transferase. We conclude that in rabbits made hyperlipemic with a diet rich in saturated fatty acids, EPO decreased tissue oxidative stress.

EFFECT OF EVENING PRIMROSE OIL ON PLATELET AGGREGATION IN RABBITS FED AN ATHEROGENIC DIET

Evening primrose oil (Oenothera biennis) is a rich source of omega-6 series fatty acids. We report here the effects of dietary supplementation with evening primrose oil (EPO) on platelet aggregation as the main factor in arterial thrombus formation in an experimental model of atherogenesis in rabbits. A total of 40 male white New Zealand rabbits were divided into four groups (n = 10 animals/group): 1: normal diet, 2: atherogenic diet (ATD), 3: normal diet enriched with 15% EPO, 4: ATD + EPO. Each group was kept on the diet for 6 weeks. We determined serum lipid profile, platelet aggregation in whole blood, platelet thromboxane B2 production and platelet lipid peroxides. The atherogenic diet increased platelet aggregation (135% when ADP was used, and 185% when collagen was used as the inducer). Evening primrose oil reduced hyperaggregation to the values obtained in rabbits fed with the normal diet. Thromboxane synthesis was increased from 0.18 to 2.28 nmol/10(9) platelets); EPO reduced this value to 1.38 nmol/10(9) platelets. Lipid peroxides were increased by ATD from 0.27 to 0.81 nmol/10(8) platelets; EPO prevented this increase (0.35 nmol/10(8) platelets). In conclusion, EPO reduced platelet hyperaggregability in rabbits fed an atherogenic diet.