Antioxidants alleviate nicotine-induced platelet aggregation in cerebral arterioles of mice in vivo

Experimental data on the effect of nicotine on cerebral microvessel thrombosis is lacking. Therefore, this study was carried out to elucidate the effects of nicotine on platelet aggregation in cerebral (pial) microcirculation of the mouse, and the possible protective effect of vitamins C and E. Male TO mice were divided into six groups, and injected i.p. with saline as a control, nicotine (1 mg/kg), vitamin C alone (100 mg/kg), vitamin E alone (100 mg/kg), nicotine plus vitamin C or nicotine plus vitamin E, all for one week before the experiment. After one week, platelet aggregation in cerebral microvessels of these groups of mice were studied in vivo. The appearance of the first platelet aggregation and total blood flow stop in arterioles and venules were timed in seconds. In the animals treated with nicotine, venules did not show any alteration in the platelet aggregation time in comparison to the control animals. However, in arterioles platelet aggregation time was significantly accelerated (p<0.001) in nicotine-treated animals as compared to controls. Both vitamins C and E prevented the shortening of arteriolar platelet aggregation time significantly (p<0.001) when applied with nicotine but not alone. It can be concluded that nicotine enhances the susceptibility to thrombosis in the cerebral arterioles in vivo and that vitamins C and E have alleviating effect on nicotine-induced thrombotic events in mice pial microvessels.     

Dietary selenium- and vitamin E-induced alterations in some rabbit tissues

The present study was designed to investigate and compare the effects of dietary selenium (Se) and vitamin E on some physiological parameters and histological changes in liver, heart, and skin tissues, as well as the blood parameters and the related enzymes. Both sex young rabbits were fed with deficient (9.8 μg/kg diet), adequate (225 μg/kg diet), and rich (4.2 mg/kg diet) Se and vitamin E diets for 12–15 wk for this purpose. As the plasma Se levels and the erythrocyte glutathione (GSH) peroxidase activity decreased (79.8±9.4 ng/ml and 2.0±0.3 U/g Hb, respectively) in the deficient group, these values increased (100.4±2.7 ng/mL and 14.5±4.3 U/g Hb) in the rich group significantly with respect to the control group. The other antioxidant enzyme activities and the related element levels did not change significantly in either one of the experimental groups. Although the platelet counts of the two experimental groups were not different from the control values, the collagen and the adenosine diphosphate (ADP) stimulated platelet aggregation rate and intensity increased in the deficient group (p<0.05) and decreased very significantly (p<0.001) in the rich group. In both of the experimental groups, as the percentage values of the neutrophils decreased, the lymphocytes and the eosinophils increased significantly. According to the light microscopic investigations, the observed lesions of considerable intensity within the tissues that elicit cell degenerations were more pronounced in the animals fed with the rich diet than in those fed with the deficient diet. The deficiency as well as toxicity of Se and the deficiency of vitamin E caused several alterations in the physiological functions of the tissues, and these alterations were supported by the histological lesions within these tissues.     

In vitro and in vivo effects of selenium and selenium with vitamin E on platelet functions in diabetic rats relationship to platelet sorbitol and fatty acid distribution

In vitro 30 min of incubation with selenomethionine (Sm)+vitamin E multiplied by about five platelet selenium (Se) decreased significantly platelet thrombin and ADP-induced aggregation decrease. Four groups of streptozotocin-induced diabetic rats were fed with a supplemented purified diet with an Se-rich yeast (Selenion): DSel, Sm: DSm, Sm α-tocopherol: DSmE or unsupplemented diet: D. After 24 wk of supplementation, only a decrease in thrombin-induced aggregation in group DSel compared to DSm and DSmE and D was observed. However, after 24 wk of diet compared to 14 wk, in group D and DSm, a significant increase in thrombin-induced aggregation occurred (p<0.0001), whereas a significant decrease in groups DSel and DSmE (p<0.0001,p<0.03) was noted. After 21 wk of diet, in DSmE, platelet adhesion to fibronectin was significantly decreased compared to group D (p<0.05). These changes in DSmE were associated with a significant decrease in platelet sorbitol (p<0.02) and a very significant increase in platelet Se (p<0.0005). Sm associated with vitamin E would appear more efficient to prevent oxidative damage of diabetic platelet membrane and thus to modulate its hyperactivity.     

Modulation of the platelet thromboxane A2 and aortic prostacyclin synthesis by dietary selenium and vitamin E

Vitamin E and selenium (Se) interact synergistically as an important antioxidant defense mechanism. Se, an essential component of glutathione peroxidase (GSH-Px) and vitamin E decompose fatty acid hydroperoxides and hydrogen peroxides generated by free radical reactions. Vitamin E and GSH-Px may modulate arachidonic acid metabolism and the activity of cyclooxygenase enzymes by affecting peroxide concentration. The balance between arterial wall prostacyclin (PGI₂) production and platelet thromboxane (TX)A₂ directly influences platelet activity. In order to elucidate the differential role of dietary vitamin E and Se in aortic PGI₂ and platelet TXA₂ synthesis, 1-mo-old F344 rats were fed semipurified diets containing different levels of vitamin E (0, 30, 200 ppm) and Se (0, 0.1, 0.2 ppm) for 2 mo. Thromboxane B₂ (TXB₂) and 6-keto-PGF₁α, were measured by radioimmunoassay (RIA) after incubation of whole blood and aortic rings at 37°C for 10 and 30 min, respectively. Vitamin E deficiency reduced plasma vitamin E to 5–17% of control-fed rats, and supplementation increased it to 53% of the control-fed rats. Se supplementation in vitamin E-supplemented animals increased plasma GSH-Px by 17%, compared to vitamin E-deficient rats. Se and vitamin E supplementation did not have a similar effect on TXB₂ and PGI₂ synthesis. Se deficiency did not alter platelet TXB₂ synthesis, but significantly decreased aortic PGI₂ synthesis. It was necessary to supplement with both antioxidants in order to increase, PGI₂ synthesis. Se and vitamin E deficient groups had a higher TXB₂/PGI₂ ratio (0.17±0.08) compared to Se- and vitamin E-supplemented groups (0.03±0.01). These results confirm previous reports in humans and animals and are in accordance with epidemiological data indicating an inverse relationship between plasma Se and platelet aggregation. Thus, further suggesting that vitamin E and Se may have a specific role in controlling TXA₂ and PGI₂ synthesis.     

Influence of dietary vitamin E on prostaglandin biosynthesis in rat blood.

A vitamin E (alpha-tocopherol) deficient diet stimulated prostaglandin biosynthesis in coagulating rat blood. Prostaglandins were extracted from serum, purified and bioassayed. The identity of prostaglandin E2 was confirmed by gas chromatography-mass spectrometry. Withholding vitamin E from the diet caused a marked increase in PGE2 and a lesser increase in PGF2alpha production in serum. In rats maintained on diets containing different concentrations of vitamin E, serum concentrations of PGE2 and PGF2alpha were inversely related to serum concentrations of alpha-tocopherol. These data suggest that in vitro alpha-tocopherol inhibits the endogenous conversion of arachidonic acid into PGE2 and PGF2alpha. The possibility that alpha-tocopherol may inhibit the formation of endoperoxide intermediates of PGE2 and PGF2alpha biosynthesis and subsequent induction of platelet aggregation is discussed.     

Synthetic byproducts of tocopherol oxidation as inhibitors of platelet function

Vitamin E and its fully oxidized form tocopherol quinone are known inhibitors of platelet aggregation. Previous results from our laboratory have shown that the quinone was approximately equal in effectiveness to vitamin E. A recent report of a far greater inhibitory activity of the quinone produced by nitric acid oxidation of vitamin E prompted this investigation. Our studies show that the unusually high inhibition of platelet aggregation, release and cyclooxygenase activity associated with nitric acid oxidized vitamin E is due to byproducts of the oxidation process and not due to tocopherol quinone. Treatment of vitamin E acetate, a substance of mild effect on aggregation and arachidonate metabolism of platelets, with nitric acid did not produce tocopherol quinone but exerted as potent an inhibition as oxidized vitamin E. We conclude that nitric acid oxidation is unsuitable for preparation of tocopherol quinone unless the latter is carefully isolated. Oxidation with permanganate proved to be an alternate method without these difficulties.     

Experimental Anaphylaxis in Pigs

An anaphylactic reaction was provoked in pigs by two intravenous injections of chicken egg albumin given approx. 2 weeks apart; some of the animals were in a state of vitamin E deficiency. The second injection of albumin was followed by vomiting, forced respiration, severe cyanosis and distinct edema in the stomach, larynx and gall bladder. These pathomorphological changes are in accordance with those regarded as typical for edema disease. Both the clinical symptoms and the pathomorphological alterations were most evident in animals with vitamin E supplementation, whereas vitamin E deficient animals showed a more moderate response. The anaphylactic reaction was not accompanied by evident changes resembling Mulberry heart disease, either in normal pigs or in vitamin E deficient animals.     

Dietary polyenoic fatty acids change the response of cat blood platelets to inductions of aggregation by ADP

Dietary polyenoic fatty acid influence on platelet aggregation has been difficult to assess in mammalian species, as endogenous polyenoic acid production from plant precursors has clouded the effects of diet. The cat is incapable of such endogenous production, and thus is a good experimental model. Adult and juvenile cats dietarily deficient in post delta-6-denaturation fatty acids (PDFA), were supplemented with different PDFA and the ADP-induced platelet aggregation assessed. At the same time, the effects of oral dosage with aspirin, and the exchange of stimulated and non-stimulated plasma were investigated. Animals deficient in all PDFA showed high levels of aggregation, irrespective of aspirin dosage. Feeding only n-6 or n-3 PDFA induced low levels of aggregation, which was reversed by aspirin dosage. Both n-6 and n-3 dietary PDFA together produced very low levels of aggregation, whether aspirin-dosed or not. Thus, ADP-induced platelet aggregation is dependent on the nature of dietary PDFA, and is partially dependent on the presence or absence of aspirin, at least in cases of PDFA imbalance. Non-ADP-stimulated plasma caused only slight changes in aggregation profiles. However, ADP-stimulated plasma from fully sufficient animals produced marked changes in the aggregation pattern of deficient platelets. This indicated the release of an anti-aggregatory substance by normal platelets, but not those of PDFA deficient animals.     

Influence of dietary vitamin E on prostaglandin biosynthesis in rat blood

A vitamin E (-tocopherol) deficient diet stimulated prostaglandin biosynthesis in coagulating rat blood. Prostaglandins were extracted from serum, purified and bioassayed. The identity of prostaglandin E₂ was confirmed by gas chromatography-mass spectrometry. Withholding vitamin E from the diet caused a marked increase in PGE₂ and a lesser increase in PGF₂ production in serum. In rats maintained on diets containing different concentrations of vitamin E, serum concentrations of PGE₂ and PGF₂ were inversely related to serum concentrations of -tocopherol. These data suggest that in vivo -tocopherol inhibits the endogenous conversion of arachidonic acid into PGE₂ and PGF₂. The possibility that -tocopherol may inhibit the formation of endoperoxide intermediates of PGE₂ and PGF₂ biosynthesis and subsequent induction of platelet aggregation is discussed.     

Effect of vitamin E on the function of blood platelets in patients with diabetes mellitus

An effect of vitamin E on blood platelets functioning was studied in 39 patients with diabetes mellitus type 1. Control group included 20 healthy blood donors. Vitamin E in a daily dose of 1000 mg produced statistically significant decrease in platelets aggregation, number of circulating platelet aggregates and release of the platelet factory 4 in diabetics after 7 days of treatment. No adverse reactions were seen in any patient treated with vitamin E. The obtained results indicate that vitamin E inhibits increased platelets activity in the patients with diabetes mellitus type 1 and does not exert toxic reactions during the treatment.     

Vitamin E deficiency accentuates adriamycin-induced cardiomyopathy and cell surface changes

Free radicals have been suggested to play a role in adriamycin-induced cardiomyopathy. Adriamycin-induced myocardial effects were examined in rats maintained on a vitamin E deficient diet. Animals were divided into four groups: I, control; II, adriamycin-treated; III, vitamin E deficient diet; IV, vitamin E deficient diet plus adriamycin treatment. Adriamycin-treated animals (groups II and IV) were given six injections (i.p.) over two weeks for producing a cumulative dose of 15 mg/kg. Animals in groups III and IV were placed on vitamin E deficient diet starting two weeks prior to the first injection of adriamycin or vehicle. Myocardial tissue analysis were performed on animals sacrificed 1 week after the last injection. Mortality was significantly higher in group IV which also showed doubling of myocardial malondialdehyde content relative to the non-adriamycin-treated vitamin E deficient group (III). Myocardial cell damage in group IV was characterized by separation of the external lamina, subsarcolemmal changes, mitochondrial swelling and myofibril dropout. Group II hearts showed only a mild dilation of the sarcotubules and swelling of the mitochondria. Total sialic acid content of the sarcolemma in groups II, III and IV was 55, 90 and 24% of the control values in group I. These data show a characteristic sarcolemmal injury produced by adriamycin in hearts of animals with reduced antioxidant capacity which is probably mediated by increased free radical activity as well as lipid peroxidation.     

Combining Drosophila melanogaster somatic-mutation-recombination and electron-spin-resonance-spectroscopy data to interpret epidemiologic observations on chromium carcinogenicity

The aim of this study was to investigate the relationship between endothelial dysfunction and low density lipoprotein (LDL) size and susceptibility to oxidation in nephrotic rats with or without deficiency of vitamin E and selenium. Four groups of male Wistar rats were studied: control (C), vitamin E and selenium deficient control (DefC), nephrotic (NS), and vitamin E and selenium deficient NS (DefNS). Nephrotic syndrome was induced by puromycin aminonucleoside. The molar ratio of vitamin E/LDL-cholesterol was significantly lower in DefNS, DefC rats, and NS vs. C rats. In comparison with control animals, vasodilation and LDL oxidability were significantly lower in nephrotic animals. LDL size was similar in all groups. Abnormal endothelial function in response to acetylcholine and carbachol was observed in NS animals compared to control rats. Relaxation response was inversely associated with an increase in LDL susceptibility to oxidation and with a lower molar ratio of vitamin E/LDL-c. LDL oxidability and LDL-c were the only variables independently associated with vasodilation. These results suggest that endothelial dysfunction of NS may be a consequence of the increased LDL susceptibility to oxidation, secondary to antioxidant deficiency.     

Effect of vitamin E-deficiency on regeneration of the sciatic nerve

The regeneration of the sciatic nerve fibres was studied in both normal and vitamin E-deficient rats at 30 and 60 days after crush. The vitamin E is involved in one of the most important mechanisms of protection against peroxidation of plasma membrane lipids; the plasma membrane plays certainly a role in nerve regeneration. Both the diameter and the total number of myelinated nerve fibres was calculated at different times. The number of myelinated fibres in the undenervated deficient animals was lower than that found in the undenervated normals animals. Following the nerve crush, in normal animals after two months the number of myelinated fibres exceeded the number found in undenervated normal animals, whereas in the deficient rat nerves it was significantly lower than in the corresponding controls and moreover it did not even reach the number found in the nerves of undenervated deficient rats. Finally, the caliber distribution of myelinated fibres in undenervated and denervated deficient rats shows a relative percent increase in the number of greatest axons and a decrease in smaller axons. This result confirm the vitamin E to be an important factor of the normal process of nerve regeneration.     

Deletion of apolipoprotein E gene modifies the rate of depletion of alpha tocopherol (vitamin E) from mice brains

Our previous reports show that apolipoprotein E (apoE) influences the dynamics of alpha tocopherol (vitamin E) in brain. In this investigation, the patterns of depletion of alpha tocopherol from tissues of apoE deficient and wild type mice were compared after the animals were fed vitamin E deficient diets. Alpha tocopherol concentrations in specific regions of the brain and peripheral tissues at different times were determined by HPLC with electrochemical detection. ApoE deficiency significantly retarded the rate of depletion of alpha tocopherol from all regions of the brain. In addition, comparison of the rates of depletion of alpha tocopherol in both apoE deficient and wild type animals showed that cerebellum behaved differently from other areas such as cortex, hippocampus and striatum. This reinforces the uniqueness of cerebellum with regard to vitamin E biology. Patterns of depletion of tocopherol from peripheral tissues were different from brain. Serum tocopherol was higher in apoE deficient animals and remained higher than wild type during E deficiency. Depletion of liver tocopherol also tended to be unaffected by apoE deficiency. Our current and previous observations strongly suggest that apoE has an important role in modulating tocopherol concentrations in brain, probably acting in concert with other proteins as well.     

Modulation of Platelet thromboxane A2 and arterial prostacyclin by dietary vitamin E

Platelets from vitamin E-deficient and vitamin E-supplemented rats generate the same amount of thromboxane A2 (TxA2) when they are incubated with unesterified arachidonic acid. Platelets from vitamin E-deficient rats produced more TxA2 than platelets from vitamin E-supplemented rats when the platelets are challenged with collagen. Arterial tissue from vitamin E-deficient rats generates less prostacyclin (PGI2) than arterial tissue from vitamin E- supplemented rats. The vitamin E effect with arterial tissue is observed when the tissue is incubated with and without added unesterified arachidonic acid. These data show that arterial prostacyclin synthesis is diminished in vitamin E-deficient rats. Vitamin E, in vivo, inhibits platelet aggregation both by lowering platelet TxA2 and by raising arterial PGI2.     

Relative resistance of the hamster to aortic atherosclerosis in spite of prolonged vitamin E deficiency and dietary hypercholesterolemia. Putative effect of increased HDL?

Male golden hamsters were rendered hypercholesterolemic by feeding diets enriched with cholesterol and fat. In the first series of experiments, 5% butter and 1% cholesterol were added to a chow diet and plasma cholesterol levels were maintained at 350–390 mg/dl over the entire experimental period. Groups of hamsters and their age controls consuming the chow diet, were killed after 7, 15 and 20 months when the aorta was examined for atherosclerosis by determination of cholesterol mass. In the controls, aortic total cholesterol (TC) increased with age by 28% and esterified cholesterol increased to 11% of TC. In the hypercholesterolemic animals aortic TC was only 28% higher than in the controls and cholesteryl ester was also 11.5% of TC. In the second series, one group of hamsters were fed a semi-purified diet deficient in vitamin E, containing 1% cholesterol and 10% lard; a second group received the same diet, but supplemented with vitamin E. Controls consumed local chow. After 7 months on the vitamin E deficient diet plasma α-tocopherol was 0.05 mg/l, in those supplemented with vitamin E it was 20 mg/l, while in the controls it was 3.3 mg/l. Plasma thiobarbituric acid reactive substances (TBARS) were higher in the vitamin E deficient group and there was a greater propensity of lipoproteins (d < 1.063 g/ml to peroxidation in vitro than in the vitamin E supplemented group. Plasma cholesterol was 366 mg/dl in the vitamin E deficient, 336 mg/dl in the vitamin E supplemented group, and 64 mg/dl in controls. Aortic cholesterol was 79.1 in vitamin E supplemented and 84.4 μg/ 10 mg dry weight in vitamin E deficient hamsters. In both series of experiments, HDL amounted to 36–41% of plasma TC in the hypercholesterolemic animals and 59–62% in the controls. In conclusion: the hamster appears to be quite resistant to atherosclerosis in face of sustained hypercholesterolemia, even in the presence of increased peroxidative stress caused by vitamin E deficiency. This relative resistance could be related to commensurate increase in plasma HDL which was observed in both series of experiments. Since vitamin E deficiency did not enhance aortic cholesteryl ester deposition, the protective effect of HDL seems to be related to its role in reverse cholesterol transport, rather than in prevention of peroxidation.     

Dietary alpha-tocopherol affects differential gene expression in rat testes

Gene-chip technology was employed to study the effect of dietary vitamin E (VE) on gene expression in rat testes. Male albino rats were fed with either a diet deficient in VE or a standard diet containing VE. Differential gene expression was monitored at five individual time-points over a period of 14 months with all animals individually profiled. Low VE intake resulted in the consistent up-regulation of 7-dehydrocholesterol reductase and GATA binding protein 4, both involved in testosterone synthesis. Cyclin D3, important in cell cycle progression and Wilms tumor 1, related to cancer development, were also up-regulated in the vitamin E deficient animals. This study demonstrates that low dietary VE intake has long-term effects on gene expression in the testes. Our data provides insights into the possible molecular mechanisms underlying the beneficial effects of vitamin E on the male reproductive organ.     

Sheep deficient in vitamin E preferentially select for a feed with a higher concentration of vitamin E

Given the capacity of ruminants to modify diet selection based on metabolic needs, we hypothesised that, when given a choice, lambs experiencing a vitamin E deficiency would consume more of a vitamin E-enriched feed than lambs not deficient in vitamin E. Fifty-six Dohne Merino lambs were divided into two groups and fed either a vitamin E-deficient diet over 40 days to induce low plasma vitamin E or a vitamin E-enriched diet to induce high plasma vitamin E. The lambs were then offered a choice of vitamin E-enriched and vitamin E-deficient pellets. For half of the animals, the enriched diet was paired with strawberry flavour and the deficient diet was paired with orange flavour, while the reverse pairings were offered to the others. Lamb preference for the diets was measured daily for the following 15 days. There was a three-way interaction between the high and low vitamin E treatment groups×vitamin E content and type of flavour in the feed×time (days). The lambs preferred pellets flavoured with strawberry but this preference changed to orange flavour in vitamin E-deficient lambs if the orange flavour was paired with high vitamin E. Lambs without a deficiency continued to prefer strawberry-flavoured pellets, regardless of the vitamin E concentrations in the pellets. It is possible that self-learning contributed to the low vitamin E group of lambs changing preference to orange flavour in order to consume more vitamin E, presumably to remediate the deficiency.     

Role of antioxidant systems in induced nutritional pancreatic atrophy in chicken

One-day-old chicks were reared using diets differing in their vitamin E and/or selenium content. The purpose of this research was to detect any possible imbalance in the antioxidant defense system, which could be related to development of nutritional pancreatic atrophy. Mitochondrial membranes from animals deficient in both nutrients, or just vitamin E, submitted to peroxidizability ‘in vitro’ had the production of TBARS greatly enhanced. Measurements of the 2-GSH/GSSG ratio suggested that selenium and vitamin E, the latter in higher magnitude, were responsible for maintenance of the reducing capacity of the cell. Enzymatic defense systems against oxidative stress were also studied. The results indicated that the total antioxidant enzymatic activity of pancreatic cells was not sufficient to scavenge all the ROS generated in the nutritionally deficient animals. The present study suggests that nutritional deficiency of selenium and/or vitamin E generates one imbalance between pro-oxidant and antioxidant systems in chicken pancreas, leading to oxidative stress and pancreatic atrophy.     

Role of dietary lipids in arteriosclerosis in experimental animals

Effects of dietary fats include the development of arteriosclerosis in humans and experimental animals, in addition to hypercholesterolemia. None of the preceding studies explicitly compared the effects of individual fatty acids. To address these issues, we chose exogenously hypercholesterolemic (ExHC) rats and apolipoprotein (apo) E deficient mice as a model for atherosclerosis and assessed the individual role of fatty acids in animals' susceptibility to atherosclerosis. The rats fed on the diet containing DHA or EPA, compared with those fed on the safflower oil (SO) diet, lowered serum cholesterol concentration, prevented platelet aggregation and slowed thickening in the ascending aorta. Apo E deficient mice developed hypercholesterolemia and severe lesion area in aortic root and arch, to a similar extent when they received DHA or SO. These results suggest a direct action of polyunsaturated fatty acids in the arterial wall, in addition to their effects on hypocholesterolemic and haemodynamic action.