Alpha-tocopherol improves biochemical and dynamic parameters in cryopreserved boar semen

Cryopreservation is associated with the production of reactive oxygen species which lead to lipid peroxidation of sperm membranes. The objective was to determine an alpha-tocopherol concentration capable of improving the quality of cryopreserved porcine semen. Boar spermatozoa frozen with 200, 500 or 1000 microg/mL alpha-tocopherol were thawed and incubated at 37 degrees C for 4 h. Routine parameters of semen quality, susceptibility to lipid peroxidation 2-thiobarbituric acid (TBARS) and oxygen uptake were evaluated. Motility was higher (P<0.05) in samples treated with different concentrations of alpha-tocopherol up to 2 h of incubation. Viability and acrosome integrity significantly decreased during incubation (no significant differences between treatments). Two hundred micrograms per milliliter alpha-tocopherol protected spermatozoa against lipid peroxidation during incubation, but 1000 microg/mL failed to protect after 2 h of incubation. There was a negative association between TBARS and motility, suggesting that lipid peroxidation affected sperm motility. Both control and 200 microg/mL alpha-tocopherol samples preserved the capacity to generate oxidative energy up to 1 h of incubation. The addition of 200 microg/mL alpha-tocopherol in the semen extender could be useful to preserve boar spermatozoa against the oxidative stress generated by cryopreservation.     

Effect of alpha-tocopherol on adrenal cortex functions under stress]

alpha-Tocopherol has been studied for its effect on lipid peroxidation and steroidogenesis in the adrenal cortices of rat and rabbit under stress. The vitamin is shown to exert an inhibitory effect on the lipid peroxidation developing under chronic stress. A biphasic pattern of the alpha-tocopherol effect on the steroidogenesis in the adrenal cortex is established: a decrease in the release of the steroids under the acute stress and maintaining of their levels under the chronic stress. A conclusion is drawn about a potential alpha-tocopherol application to correct the adrenal cortex function under stress.     

The effect of dietary supplementation with blueberry, alpha-tocopherol or astaxanthin on oxidative stability of Arctic char (Salvelinus alpinus) semen

The objective was to determine the oxidative stability of Arctic char (Salvelinus alpinus) semen following dietary supplementation with lowbush blueberry (Vaccinium angustifolium) product, alpha-tocopherol, alpha-tocopherol+blueberry product, or alpha-tocopherol+astaxanthin. Sperm lipid peroxidation was initiated by challenging with ferrous sulphate/ascorbic acid (Fe(++)/Asc) at level of 0.04/0.2 mmol/L. Addition of blueberry, alpha-tocopherol, or both to char diets inhibited semen lipid peroxidation by: (a) decreasing the rate of sperm lipid peroxidation, an effect which was more pronounced with alpha-tocopherol treatments; and (b) increasing the antioxidant potential of seminal plasma, based on the lipid peroxidation process of sperm and an in vitro chicken brain tissue model. Dietary supplementation with astaxanthin and alpha-tocopherol had the same effect as the supplementation with alpha-tocopherol alone on inhibiting the lipid peroxidation process of sperm and chicken brain. Catalase-like activity increased significantly in sperm of fish fed alpha-tocopherol, blueberry, or both. There was a negative correlation (r= -0.397, P < 0.05) between catalase-like activity in sperm cells and the rate of sperm lipid peroxidation. Seminal plasma alpha-tocopherol levels increased significantly in fish supplemented with alpha-tocopherol alone or in combination with blueberry or astaxanthin. There were negative correlations between seminal plasma alpha-tocopherol levels and lipid peroxidation rates of sperm cells (r= -0.625, P < 0.01) and brain tissue (r= -0.606, P < 0.01). In conclusion, dietary supplementation of blueberry product or alpha-tocopherol inhibited lipid peroxidation in Arctic char semen. Further experiments are needed to test the effect of dietary blueberry and antioxidants on Arctic char semen quality during liquid and cryopreserved storage.     

Glutathione disulfide enhances the reduced glutathione inhibition of lipid peroxidation in rat liver microsomes

Experiments were undertaken to examine the effects of reduced (GSH) and oxidized (GSSG) glutathione on lipid peroxidation of rat liver microsomes. Dependence on microsomal alpha-tocopherol was shown for the GSH inhibition of lipid peroxidation. However, when GSH (5 mM) and GSSG (2.5 mM) were combined in the assay system, inhibition of lipid peroxidation was enhanced markedly over that with GSH alone in microsomes containing alpha-tocopherol. Surprisingly, the synergistic inhibitory effect of GSH and GSSG was also observed for microsomes that were deficient in alpha-tocopherol. These data suggest that there may be more than one factor responsible for the glutathione-dependent inhibition of lipid peroxidation. The first is dependent upon microsomal alpha-tocopherol and likely requires GSH for alpha-tocopherol regeneration from the alpha-tocopheroxyl radical during lipid peroxidation. The second factor appears to be independent of alpha-tocopherol and may involve the reduction of lipid hydroperoxides to their corresponding alcohols. One, or possibly both, of these factors may be activated by GSSG through thiol/disulfide exchange with a protein sulfhydryl moiety.     

Relations Between Tocopherol Depletion and Coenzyme Q During Lipid Peroxidation in rat Liver Mitochondria

In order to evaluate different mitochondrial antioxidant systems, the depletion of alpha-tocopherol and the levels of the reduced and oxidized forms of CoQ were measured in rat liver mitochondria during Fe⁺⁺/ascorbate and NADPH/ADP/Fe⁺⁺ induced lipid peroxidation. During the induction phase of malondialdehyde formation, alpha-tocopherol declined moderately to about 80% of initial contents, whereas the total CoQ pool remained nearly unchanged, but reduced CoQ9 continuously declined. At the start of massive malondialdehyde formation, CoQ9 reaches its fully oxidized state. At the same time alpha-tocopherol starts to decline steeply, but never becomes fully exhausted in both experimental systems. Evidently the oxidation of the CoQ9 pool constitutes a prerequisite for the onset of massive lipid peroxidation in mitochondria and for the subsequent depletion of alpha-tocopherol. Trapping of the GSH by addition of dinitrochlorbenzene (a substrate of the GSH transferase), results in a moderate acceleration of lipid peroxidation, but alpha-tocopherol and ubiquinol levels remained unchanged when compared with the controls. Addition of succinate to GSH depleted mitochondria effectively suppressed MDA formation as well as alpha-tocopherol and ubiquinol depletion. The data support the assumption that the protective effect of respiratory substrates against lipid peroxidation in the absence of mitochondrial GSH is mediated by the regeneration of the lipid soluble antioxidants CoQ and alpha-tocopherol.     

Lipid peroxidation in the adrenal cortex during exhausting stress

Under prolonged stress which is connected with exhaustion of functional resources of adrenal cortex the activation of lipid peroxidation processes in this gland was found. It is possible that the reason for such lipid peroxidation activation is the decrease in the content of adrenal cortex ascorbic acid and alpha-tocopherol.     

Relations Between Tocopherol Depletion and Coenzyme Q During Lipid Peroxidation in rat Liver Mitochondria

In order to evaluate different mitochondrial antioxidant systems, the depletion of alpha-tocopherol and the levels of the reduced and oxidized forms of CoQ were measured in rat liver mitochondria during Fe⁺⁺/ascorbate and NADPH/ADP/Fe⁺⁺ induced lipid peroxidation. During the induction phase of malondialdehyde formation, alpha-tocopherol declined moderately to about 80% of initial contents, whereas the total CoQ pool remained nearly unchanged, but reduced CoQ9 continuously declined. At the start of massive malondialdehyde formation, CoQ9 reaches its fully oxidized state. At the same time alpha-tocopherol starts to decline steeply, but never becomes fully exhausted in both experimental systems. Evidently the oxidation of the CoQ9 pool constitutes a prerequisite for the onset of massive lipid peroxidation in mitochondria and for the subsequent depletion of alpha-tocopherol. Trapping of the GSH by addition of dinitrochlorbenzene (a substrate of the GSH transferase), results in a moderate acceleration of lipid peroxidation, but alpha-tocopherol and ubiquinol levels remained unchanged when compared with the controls. Addition of succinate to GSH depleted mitochondria effectively suppressed MDA formation as well as alpha-tocopherol and ubiquinol depletion. The data support the assumption that the protective effect of respiratory substrates against lipid peroxidation in the absence of mitochondrial GSH is mediated by the regeneration of the lipid soluble antioxidants CoQ and alpha-tocopherol.     

Cooperation of antioxidants in protection against photosensitized oxidation

The aim of this study was to determine whether alpha-tocopherol and zeaxanthin offer synergistic protection against photosensitized lipid peroxidation mediated by singlet oxygen and free radicals. The antioxidant action of zeaxanthin and alpha-tocopherol was studied in liposomes made of phosphatidylcholine and cholesterol. Progress of lipid peroxidation, induced by aerobic photoexcitation of rose bengal, was monitored by the detection of lipid hydroperoxides and by electron spin resonance oximetry. In addition, cholesterol was employed as a mechanistic reporter molecule, which forms characteristic products of the interaction with singlet oxygen or free radicals. Cholesterol hydroperoxides were quantitatively determined by HPLC/electrochemical detection. HPLC/ultraviolet-visible (UV-VIS) absorption detection was used to measure concentrations of zeaxanthin and alpha-tocopherol. Zeaxanthin, even at concentrations of 2.5 microM, effectively protected against singlet oxygen-mediated lipid peroxidation but was rapidly consumed due to interaction with free radicals. alpha-Tocopherol alone was not effective in protecting against lipid peroxidation, even at concentration of 0.1 mM. Combinations of zeaxanthin and alpha-tocopherol exerted a synergistic protection against lipid peroxidation. The synergistic effect may be explained in terms of prevention of carotenoid consumption by effective scavenging of free radicals by alpha-tocopherol therefore allowing zeaxanthing to quench the primary oxidant-singlet oxygen effectively.     

Role of plasma membrane coenzyme Q on the regulation of apoptosis.

Serum withdrawal is a model to study the mechanisms involved in the induction of apoptosis caused by mild oxidative stress. Apoptosis induced by growth factors removal was prevented by the external addition of antioxidants such as ascorbate, alpha-tocopherol and coenzyme Q (CoQ). CoQ is a lipophilic antioxidant which prevents oxidative stress and participates in the regeneration of alpha-tocopherol and ascorbate in the plasma membrane. We have found an inverse relationship between CoQ content in plasma membrane and lipid peroxidation rates in leukaemic cells. CoQ10 addition to serum-free culture media prevented both lipid peroxidation and cell death. Also, CoQ10 addition decreased ceramide release after serum withdrawal by inhibition of magnesium-dependent plasma membrane neutral-sphingomyelinase. Moreover, CoQ10 addition partially blocked activation of CPP32/caspase-3. These results suggest CoQ of the plasma membrane as a regulator of initiation phase of oxidative stress-mediated serum withdrawal-induced apoptosis.     

The effect of concentration on the antioxidant effectiveness of alpha-tocopherol in lipid peroxidation induced by superoxide free radicals

Egg yolk phosphatidylcholine liposomes were rapidly oxidized in the presence of chelated iron and a superoxide-generating system. alpha-Tocopherol incorporated in the bilayer was oxidized at the same time. No lipid or alpha-tocopherol oxidation occurred in liposomes composed of dimyristoyl phosphatidylcholine. The antioxidant did not inhibit lipid peroxidation until its concentration reached a critical level, which depended on the effectiveness of the oxidative stress. Beyond this level, peroxidation was inhibited completely and, simultaneously, the rate of oxidation of tocopherol was lowered. The results suggest that the antioxidant efficiency of alpha-tocopherol depends on its ability to react mainly with the chain-initiating or chain-propagating lipid radicals. This, in turn, is closely tied to the tocopherol content of the membrane. Ascorbate inhibited the consumption of alpha-tocopherol, possibly by regenerating its reduced form.     

Effects of glutathione on the alpha-tocopherol-dependent inhibition of nuclear lipid peroxidation

Endogenous alpha-tocopherol levels in isolated rat liver nuclei were determined to be 0.045 mol% (mol alpha-tocopherol per mol phospholipid x 100). This value corresponds to 970 polyunsaturated fatty acid (PUFA) moieties to one molecule of alpha-tocopherol in the nuclear membrane. Isolated nuclei, when incubated with various concentrations of exogenous alpha-tocopherol, took up only a small percent of initial levels of alpha-tocopherol present in the incubation media. Exogenous alpha-tocopherol, when incorporated in isolated nuclei above a threshold value of 0.085 mol%, effectively inhibited NADPH-induced lipid peroxidation. The addition of 1 mM glutathione lowered the threshold levels of alpha-tocopherol needed to inhibit lipid peroxidation to about 0.040 mol%. We suggest the data indicate a glutathione-dependent enhancement of the ability of alpha-tocopherol to inhibit nuclear lipid peroxidation.     

Regulation of superoxide dismutase activity during deep hypothermia by simultaneous administration of water and lipid soluble antioxidants

Alongside anti-hypoxia activity, the method of deep hypothermia causes discoordination of metabolism in the heart. This is due to increased secretion of catecholamines in the process of cooling, to activation in free radical generation and lipid peroxidation. Pantethine and alpha-tocopherol were used. Pantethine reduced lipid peroxidation, preserved reaction activity of catalyzing resyntheses and transport of high energetic compounds in the heart, while alpha-tocopherol prevented lipid peroxidation activation and decrease in SOD. Simultaneous use of pantethine and alpha-tocopherol caused increase in SOD and normalization of heart metabolism. Thus, for protection of the heart against excessive free radical generation under deep hypothermia simultaneous use of antioxidants like pantethine and alpha-tocopherol is necessary.     

Ascorbic acid spares alpha-tocopherol and decreases lipid peroxidation in neuronal cells

Ascorbic acid is considered an antioxidant in the central nervous system, but direct evidence that ascorbate protects neuronal cells from oxidant stress is lacking. Differentiated SH-SY5Y cells in culture took up ascorbic acid on the sodium-dependent vitamin C transporter Type 2 and retained it much more effectively than dehydroascorbic acid. Intracellular ascorbate spared alpha-tocopherol, both in cells loaded with alpha-tocopherol in culture and in cells under oxidant stress due to extracellular ferricyanide. Sparing of alpha-tocopherol in response to ferricyanide was associated with protection against lipid peroxidation in cell membranes. These results show that neuronal cells concentrate ascorbate, and that intracellular ascorbate, either directly or through sparing of alpha-tocopherol, protects them against oxidant stress.     

Effect of alpha-tocopherol on peroxidative membrane damage caused by doxorubicin: an in vitro study in human erythrocytes

Level of lipid peroxidation in doxorubicin treated human erythrocytes was studied and compared with that of cells pretreated with alpha-tocopherol. Erythrocytes treated with alpha-tocopherol had reduced level of lipid peroxidation with concomitantly lowered membrane damage. The membrane damage was monitored by the levels of conjugated diene absorption, lipid hydroperoxides and lipid peroxides. alpha-tocopherol was not effective in inhibiting the conjugated diene formation, but the lipid hydroperoxides and the lipid peroxide levels were significantly decreased. Methemoglobin level was found to be increased in alpha-tocopherol pretreated cells, which protects the membrane from damage. Erythrocyte membrane lipids were found to be decreased during doxorubicin treatment and alpha-tocopherol significantly reduced the membrane lipid breakdown. Level of reduced glutathione was maintained in alpha-tocopherol pretreated cells. These results are discussed with reference to the antioxidant property of alpha-tocopherol.     

Cold-stress-induced modulation of antioxidant defence: role of stressed conditions in tissue injury followed by protein oxidation and lipid peroxidation

The aim of this study was to determine the effects of cold stress on antioxidant enzyme activities and examine protein oxidation and lipid peroxidation in various tissues (brain, liver, kidney, heart and stomach). Twenty male Wistar rats (3 months old) weighing 220 ± 20 g were used. The rats were randomly divided into two groups of ten: the control group and the cold stress group. Cold stress was applied to the animals by maintaining them in a cold room (5 °C) for 15 min/day for 15 days. Blood samples were taken for measuring plasma corticosterone levels. Tissues were obtained from each rat for measuring the antioxidant enzyme activities, protein oxidation and lipid peroxidation. Corticosterone levels were increased in the cold stress group. Copper, zinc superoxide dismutase activities were increased in the brains, livers and kidneys, whereas they decreased in the hearts and stomachs of rats in the cold stress group. Catalase activities were increased in the brains, livers, kidneys and hearts, whereas they decreased in the stomachs of rats in the cold stress group. Selenium-dependent glutathione peroxidase activities were increased in the brain, liver, heart and stomach. Reduced glutathione levels were decreased, while levels of protein carbonyl, conjugated diene and thiobarbituric-acid-reactive substances were increased in all tissues of the cold stress group. These results lead us to conclude that cold stress can disrupt the balance in an oxidant/antioxidant system and cause oxidative damage to several tissues by altering the enzymatic and non-enzymatic antioxidant status, protein oxidation and lipid peroxidation.     

Relationship between mitochondrial lipid peroxidation and alpha-tocopherol levels in the guinea-pig adrenal cortex

Lipid peroxidation in mitochondria from the functionally distinct inner (zona reticularis) and outer (zona fasciculata + zona glomerulosa) zones of the guinea-pig adrenal cortex was investigated. Ferrous ion (Fe2+)-induced lipid peroxidation was far greater in inner than outer zone mitochondria. Ascorbic acid similarly initiated lipid peroxidation to a greater extent in inner zone mitochondrial preparations. Differences in the unsaturated fatty acid content of inner and outer zone mitochondria could not account for the regional differences in lipid peroxidation. Total fatty acid concentrations were greater in the outer than in the inner zone, and the relative amounts of each fatty acid were similar in the two zones. However, mitochondrial concentrations of alpha-tocopherol, an antioxidant known to inhibit lipid peroxidation, were approx. 5-times greater in the outer than inner zone. The results demonstrate that there are regional differences in mitochondrial lipid peroxidation in the adrenal cortex which may be attributable to differences in alpha-tocopherol content. Thus, alpha-tocopherol may serve to protect outer zone mitochondrial enzymes from the consequences of lipid peroxidation and thereby contribute to some of the functional differences between the zones of the adrenal cortex.     

Lipid peroxidation, antioxidant status and survival in institutionalised elderly: a five-year longitudinal study

Oxidative stress has been related to ageing and risk of death. To determine whether oxidative status was associated with all-cause risk of death we carried out a prospective study in 154 non-smoking Spanish elderly without major illness. Baseline glutathione peroxidase (GPx) and superoxide dismutase (SOD) were analysed in plasma and erythrocytes. alpha-tocopherol, beta-carotene, lycopene and retinol were determined in serum samples and malondialdehyde (MDA), as a lipid peroxidation marker, in plasma. Mean survival time was 4.3 years. A total of 31 death cases (20.1%) occurred during the follow-up. Plasma-MDA predicted mortality independently of all other variables, while erythrocyte-SOD (e-SOD), beta-carotene and alpha-tocopherol were positively associated with survival. alpha-tocopherol and MDA were revealed as independent predictors in a joint survival model, being the group with low MDA and high alpha-tocopherol that with the lowest mortality. In conclusion, a higher risk of death was associated with increased lipid peroxidation and lower antioxidant defenses.     

Interaction of nitrogen dioxide with human plasma. Antioxidant depletion and oxidative damage.

Nitrogen dioxide (NO2.) is often present in inhaled air and may be generated in vivo from nitric oxide. Exposure of human blood plasma to NO2. caused rapid losses of ascorbic acid, uric acid and protein thiol groups, as well as lipid peroxidation and depletions of alpha-tocopherol, bilirubin and ubiquinol-10. No increase in protein carbonyls was detected. Supplementation of plasma with ascorbate decreased the rates of lipid peroxidation, alpha-tocopherol depletion and loss of uric acid. Uric acid supplementation decreased rates of lipid peroxidation but not the loss of alpha-tocopherol. We conclude that ascorbic acid, protein -SH groups, uric acid and alpha-tocopherol may be important agents protecting against NO2. in vivo. If these antioxidants are depleted, peroxidation of lipids occurs and might contribute to the toxicity of NO2..     

Effect of alpha-tocopheryl acetate on blood biochemical parameters in albino rats as affected by acoustic stress

Experiments on random-bred white male rats have demonstrated the activation of induced lipid peroxidation in red cell membranes, elevation on the basal level of plasma lipid peroxides, a decrease in the content of alpha-tocopherol in plasma and red blood cell membranes, considerable shifts in the content of esterified and free cholesterol in plasma and red blood cell membranes under prolonged acoustic stress (91 dB). Administration of alpha-tocopheryl acetate in a dose of 1 mg/kg exerted a beneficial effect on the test parameters under prolonged acoustic stress.     

Resveratrol inhibition of lipid peroxidation

To define the molecular mechanism(s) of resveratrol inhibition of lipid peroxidation we have utilized model systems that allow us to study the different reactions involved in this complex process. Resveratrol proved (a) to inhibit more efficiently than either Trolox or ascorbate the Fe2+ catalyzed lipid hydroperoxide-dependent peroxidation of sonicated phosphatidylcholine liposomes; (b) to be less effective than Trolox in inhibiting lipid peroxidation initiated by the water soluble AAPH peroxyl radicals; (c) when exogenously added to liposomes, to be more potent than alpha-tocopherol and Trolox, in the inhibition of peroxidation initiated by the lipid soluble AMVN peroxyl radicals; (d) when incorporated within liposomes, to be a less potent chain-breaking antioxidant than alpha-tocopherol; (e) to be a weaker antiradical than alpha-tocopherol in the reduction of the stable radical DPPH*. Resveratrol reduced Fe3+ but its reduction rate was much slower than that observed in the presence of either ascorbate or Trolox. However, at the concentration inhibiting iron catalyzed lipid peroxidation, resveratrol did not significantly reduce Fe3+, contrary to ascorbate. In their complex, our data indicate that resveratrol inhibits lipid peroxidation mainly by scavenging lipid peroxyl radicals within the membrane, like alpha-tocopherol. Although it is less effective, its capacity of spontaneously entering the lipid environment confers on it great antioxidant potential.