Effects of inadequate vitamin E and/or selenium nutrition on the release of arachidonic acid metabolites in rat alveolar macrophages

Effects of vitamin E and/or selenium (Se) deficiency on the secretion of arachidonic acid metabolites by zymosan-stimulated pulmonary alveolar macrophages (AM) were examined using cells from male Long-Evans hooded rats fed torula-yeast based diets with or without the supplementation of vitamin E (150 IU/kg) or Se (0.5 mg/kg). Alveolar macrophages obtained by lavage were purified by adherence and cultured for 4 h in Hank's balanced salt solution containing bovine serum albumin (0.1%) and zymosan (300 micrograms/ml). The arachidonic acid metabolites present in the culture supernatant were measured by radioimmunoassay. Altered vitamin E and Se nutrition had no effect on the number of cells or cell types recovered from the pulmonary airways. Alveolar macrophages derived from animals fed on diets deficient in vitamin E or Se or both nutrients secreted higher levels of prostaglandin E2 and thromboxane B2. Levels of both 5-hydroxyeicosatetraenoic acid and leukotriene B4 were significantly increased only in the group fed the diet adequate in Se but deficient in vitamin E. Our data suggest that vitamin E and Se might play an important role to control the levels of several physiologically and pathologically important arachidonic acid metabolites.     

Effects of inadequate vitamin E and/or selenium nutrition on the release of arachidonic acid metabolites in rat alveolar macrophages

Effects of vitamin E and/or selenium (Se) deficiency on the secretion of arachidonic acid metabolites by zymosan-stimulated pulmonary alveolar macrophages (AM) were examined using cells from male Long-Evans hooded rats fed torula-yeast based diets with or without the supplementation of vitamin E (150 IU/kg) or Se (0.5 mg/kg). Alveolar macrophages obtained by lavage were purified by adherence and cultured for 4 h in Hankś balanced salt solution containing bovine serum albumin (0.1%) and zymosan (300 μg/ml). The arachidonic acid metabolites present in the culture supernatant were measured by radioimmunoassay. Altered vitamin E and Se nutrition had no effect on the number of cells or cell types recovered from the pulmonary airways. Alveolar macrophages derived from animals fed on diets deficient in vitamin E or Se or both nutrients secreted higher levels of prostaglandins E₂ and thromboxane B₂. Levels of both 5-hydroxyeicosatetraenoic acid and leukotriene B₄ were significantly increased only in the group fed the diet adequate in Se but deficient in vitamin E. Our data suggest that vitamin E and Se might play an important role to control the levels of several physiologically and pathologically important arachidonic acid metabolites.     

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.     

Vitamin E regulates acetylcholine receptor function of molluscan neurons.

1. Intracellular recordings were made from identified LP11, RBc4, D1 and E4 neurons in perioesophageal ganglionic ring with buccal ganglia of the mollusc Helix pomatia. 2. The modulations of acetylcholine (ACh)-induced current by vitamin E in these neurons were investigated using two-microelectrode intracellular recording and voltage-clamp techniques. 3. ACh receptors function on LP11 and RBc4 neurons was strongly regulated by intracellular calcium ions. For these ACh receptors application of 10(-6) to 10(-4) M vitamin E and calcium influx both induced an enhancement of the ACh-induced chloride current. Application of 10(-5) to 5.10(-5) M arachidonic acid on the same identified LP11 and RBc4 neurons was shown to evoke a decrease of the ACh-induced chloride current. 4. The elevation of calcium levels into D1 and E4 neurons induced a faint decrease of ACh-induced chloride current, but vitamin E and arachidonic acid were ineffective. 5. The calmodulin inhibitor, chloropromazine (6.10(-5) M), strongly inhibited the enhancing effect of calcium influx on ACh-induced chloride current in LP11 and RBc4 neurons, but it had a weak influence on the effect of vitamin E. 6. The effect of vitamin E on surface distribution of functional ACh receptors in LP11 and RBc4 neurons was found. 7. Application of 10(-4) to 10(-6) M vitamin E (DL-alpha-tocopherol) triggered mechanisms, which after a 5 to 45-min period lead to appearance of functional ACh receptors on the parts of neuronal soma, which were further from the axon. 8. Arachidonic acid (vitamin F) evoked a disappearance of functional ACh receptors, which were activated by vitamin E.     

Effect of dietary vitamin E on platelet tocopherol values and 12-lipoxygenase activity

This study was designed to evaluate the effects of different amounts of dietary vitamin E on platelet tocopherol levels and 12-lipoxygenase activity when exogenous arachidonic acid was used as substrate. Weanling male Sprague-Dawley rats were fed diets containing 0, 50, and 5000 ppm of D-alpha-tocopherol acetate for 4 months. Platelet tocopherol was increased with increasing concentrations of dietary vitamin E; however, the conversion of exogenously added arachidonate by platelet to 12-HETE (12-hydroxyeicosatetraenoic acid) and thromboxane B2 from these three dietary groups was essentially the same. This study provides direct evidence that platelet 12-lipoxygenase activity is independent of its vitamin E content when exogenously added arachidonate was used as substrate.     

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 fo thromboxane A₂ (TxA₂) when they are incubated with unesterified arachidonic acid. Platelets from vitamin E-deficient rats produce 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, $\text{in}$$\text{vivo}$, inhibits platelet aggregation both by lowering platelet TxA₂ and by raising arterial PGI₂.     

Effects of tocopherol derivatives on arachidonic acid-dependent acetylcholine-induced current in molluscan neurons

-Tocopherol (vitamin E) and some of its derivatives have been found to exert modulatory actions, opposite to each other, on acetylcholine-induced current in identified molluscan neurons. A comparison of the infrared absorption spectra of arachidonic acid obtained in the presence of vitamin E, its analog, and some of its derivatives with the results of electrophysiological experiments allows us to suggest that vitamin E and its derivatives are catalysts either slowing down or accelerating the arachidonic acid metabolism, correspondingly.Neirofiziologiya/Neurophysiology, Vol. 25, No. 3, pp. 216–218, May–June, 1993.     

Impaired resistance to oxidation of low density lipoprotein in cystic fibrosis: Improvement during vitamin E supplementation

Antioxidants such as vitamin E protect unsaturated fatty acids of LDL against oxidation. In the ex vivo model used, LDL was exposed to Cu²⁺ ions, a potent prooxidant capable of initiating the oxidation of LDL. The lag time, indicating the delay of conjugated diene formation in LDL due to antioxidant protection, was measured in 54 cystic fibrosis (CF) patients with plasma -tocopherol levels below (Group A, n = 30) or above (Group B, n = 24) 15.9 μmol/L (mean - 2 SD of Swiss population). Patients were reevaluated after 2 months on 400 IU/d of oral RRR--tocopherol. In group A, -tocopherol concentrations in LDL increased significantly from 3.2 ± 1.6 mol/mol LDL to 8.2 ± 2.8 mol/mol (P < 0.001) and lag times increased from 79 ± 33, min to 126 ± 48 min (P < 0.001), whereas in the vitamin E sufficient group B no further increase neither in LDL -tocopherol concentrations or in lag times was observed. LDL oleic acid concentrations were higher, and linoleic acid concentrations were lower in patients than in controls. After efficient vitamin E supplementation, lag times were positively related to LDL -tocopherol (P < 0.01) and negatively to LDL linoleic and arachidonic acid content (P < 0.001). The maximum rate of oxidation correlated positively with linoleic and arachidonic acid concentrations, as did the maximum conjugated diene absorbance. These results indicate that LDL resistance to oxidation is impaired in vitamin E deficient CF patients but can be normalized within 2 months when -tocopherol is given in sufficient amounts. Linoleic and arachidonic acid content exhibit a major influence on the LDL resistance to oxidation.     

Changes in n-6 and n-3 polyunsaturated fatty acids during lipid-peroxidation of mitochondria obtained from rat liver and several brain regions: effect of alpha-tocopherol

The effect of intraperitoneal administration of alpha-tocopherol (100 mg/kg weight/24 h) on ascorbate (0-0.4 mM) induced lipid peroxidation of mitochondria isolated from rat liver, cerebral hemispheres, brain stem and cerebellum was examined. The ascorbate induced light emission in hepatic mitochondria was nearly completely inhibited by alpha-tocopherol (control-group: 114.32+/-14.4; vitamin E-group: 17.45+/-2.84, c.p.m.x10(-4)). In brain mitochondria, 0.2 mM ascorbate produced the maximal chemiluminescence and significant differences among both groups were not observed. No significant differences in the chemiluminescence values between control and vitamin E treated groups were observed when the three brain regions were compared. The light emission produced by mitochondrial preparations was much higher in cerebral hemispheres than in brain stem and cerebellum. In liver and brain mitochondria from control group, the level of arachidonic acid (C20:4n6) and docosahexaenoic acid (C22:6n3) was profoundly affected. Docosahexaenoic in liver mitochondria from vitamin E group decreased by 30% upon treatment with ascorbic acid when compared with mitochondria lacking ascorbic acid. As a consequence of vitamin E treatment, a significant increase of C22:6n3 was detected in rat liver mitochondria (control-group: 6.42 +/-0.12; vitamin E-group: 10.52 +/-0.46). Ratios of the alpha-tocopherol concentrations in mitochondria from rats receiving vitamin E to those of control rats were as follows: liver, 7.79; cerebral hemispheres, 0.81; brain stem, 0.95; cerebellum, 1.05. In liver mitochondria, vitamin E shows a protector effect on oxidative damage. In addition, vitamin E concentration can be increased in hepatic but not in brain mitochondria. Lipid peroxidation mainly affected, arachidonic (C20:4n6) and docosahexaenoic (C22:6n3) acids.     

Vitamin e regulates acetylcholine receptor function of molluscan neurons

1. Intracellular recorclings were made from identified LP11, RBc4, D1 and E4 neurons in perioesophageal ganglionic ring with buccal ganglia of the mollusc Helix pomatia.2. The modulations of acetylcholine (ACh)-induced current by vitamin E in these neurons were investigated using two-microelectrode intracellular recorcling and voltage-clamp techniques.3. ACh receptors function on LP11 and RBc4 neurons was strongly regulated by intracellular calcium ions. For these ACh receptors application of 10⁻⁶ to 10⁻⁴ M vitamin E and calcium influx both induced an enhancement of the ACh-induced chloride current. Application of 10⁻⁵ to 5.10⁻⁵M arachidonic acid on the same identified LP11 and RBc4 neurons was shown to evoke a decrease of the ACh-induced chloride current.4. The elevation of calcium levels into D1 and E4 neurons induced a faint decrease of ACh-induced chloride current, but vitamin E and arachidonic acid were ineffective.5. The calmodulin inhibitor, chloropromazine (6.10^−-5M), strongly inhibited the enhancing effect of calcium influx on ACh-induced chloride current in LP11 and RBc4 neurons, but it had a weak influence on the effect of vitamin E.6. The effect of vitamin E on surface distribution of functional ACh receptors in LP11 and RBc4 neurons was found.7. Application of 10⁻⁴ to 10⁻⁶ M vitamin E (DL-α-tocopherol) triggered mechanisms, which after a 5 to 45-min period lead to appearance of functional ACh receptors on the parts of neuronal soma, which were further from the axon.8. Arachidonic acid (vitamin F) evoked a disappearance of functional ACh receptors, which were activated by vitamin E.     

Species differences in membrane susceptibility to lipid peroxidation

The susceptibility of liver microsomes to lipid peroxidation was evaluated in seven species: rat, rabbit, trout, mouse, pig, cow, and horse. Lipid peroxidation was measured as thiobarbituric acid reactive substances formed in the presence of either FeCl₃-ADP/ascorbate or FeCl₂/H₂O₂ initiating systems. For rat, rabbit, and trout microsomes, the order of susceptibility to peroxidation was rat > rabbit >> trout. The lack of peroxidation in trout microsomes could be explained by high microsomal vitamin E levels. Membrane fatty acid levels differed between species. Docosahexaenoic acid predominated in the trout, arachidonic acid in the rat, and linoleic acid in the rabbit. The contribution of individual fatty acids to lipid peroxidation reflected the degree of unsaturation with docosahexaenoic > arachidonic >>> linoleic. For all species except trout, the predicted susceptibility to peroxidation, based on the response of individual fatty acids, agreed well with directly measured microsomal peroxidation. With the exception of the trout, vitamin E content ranged from 0.083–0.311 nmol/mg microsomal protein between species, and low levels did not influence susceptibility to peroxidation. Trout microsomes peroxidized only after vitamin E depletion by prolonged incubation. The data indicate that below a vitamin E threshold, species differences in membrane susceptibility to peroxidation can be reasonably predicted based only on content of individual peroxidizable fatty acids.     

Mitochondrial electron transport-linked tocopheroxyl radical reduction

alpha-Tocopherol (vitamin E) is a lipophilic chain-breaking antioxidant which inhibits lipid peroxidation in isolated mitochondrial membranes and protects membranes from oxidative damage. The primary oxidation product of vitamin E is the tocopheroxyl radical. Reduction of the tocopheroxyl radical can occur by reactions with water-soluble anti-oxidants such as ascorbate or glutathione, resulting in the recycling of vitamin E. Physiological concentrations of vitamin E are too low to allow detection of tocopheroxyl radical by ESR. After dietary supplementation with vitamin E, a 10-20-fold increase in the rat liver mitochondrial membrane content of vitamin E was achieved and this allowed for direct detection of the tocopheroxyl radical by ESR, after treatment with an oxidizing system composed of lipoxygenase and arachidonic acid. By using submitochondrial particle membranes, it was shown that NADH, succinate, and reduced cytochrome c-linked oxidation reduce the tocopheroxyl radical, preventing both accumulation of the radical and vitamin E consumption. As the electron transport chain can reduce tocopheroxyl radical it may have an important physiological role in recycling vitamin E.     

Effect of selenium and vitamin E supplementation on lipid abnormalities in plasma, aorta, and adipose tissue of Zucker rats

Twenty-nine obese female Zucker rats (fa / fa) were fed with a laboratory chow supplemented or not with a selenium-rich yeast (Selenion), or Selenion + vitamin E, or vitamin E alone. Twelve lean female Zucker rats (Fa / Fa) of the same littermates fed with the same diet were used as control. After 32 wk of diet, obesity induced a large increase in plasma insulin and lipid levels. A significant decrease in the plasma vitamin E/triglycerides ratio (p < 0.005) and an increase in plasma thiobarbituric reactive substances (TBARS) (p < 0.005) were also observed. Plasma selenium and vitamin E increased in all supplemented rats. The plasma insulin level was decreased by selenion supplementation and the vitamin E/triglycerides ratio was completely corrected by double supplementation with Selenion + vitamin E. TBARS were also efficiently decreased in two obese groups receiving vitamin E. In plasma, adipose tissue and aorta, obesity induced an increase in palmitic acid (C16:0), a very large increase in monounsaturated fatty acids (palmitoleic acid C16:l, stearic acid C18:l) associated with a decrease in polyunsaturated n-6 fatty acids (linoleic acid C18:2 n - 6, arachidonic C20:4 n - 6). These alterations in fatty acid distribution were only partly modulated by Se and vitamin E supplements. However, in the aorta, antioxidant treatment in obese rats significantly reduced the increase in C16:0 and C16:l (p < 0.05 andp < 0.01, respectively) and the decrease in arachidonic acid (p < 0.05). These changes could be beneficial in the reduction of insulin resistance and help to protect the vascular endothelium.     

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.     

Interrelated influence of superoxides and free fatty acids over mitochondrial uncoupling in skeletal muscle

Mitochondrial uncoupling protein 3 (UCP(3))-mediated uncoupling has been postulated to depend on several factors, including superoxides, free fatty acids (FFAs), and fatty acid hydroperoxides and/or their derivatives. We investigated whether there is an interrelation between endogenous mitochondrial superoxides and fatty acids in inducing skeletal muscle mitochondrial uncoupling, and we speculated on the possible involvement of UCP(3) in this process. In the absence of FFAs, no differences in proton-leak kinetic were detected between succinate-energized mitochondria respiring in the absence or presence of rotenone, despite a large difference in complex I superoxide production. The addition of either arachidic acid or arachidonic acid induced an increase in proton-leak kinetic, with arachidonic acid having the more marked effect. The uncoupling effect of arachidic acid was independent of the presence of GDP, rotenone and vitamin E, while that of arachidonic acid was dependent on these factors. These data demonstrate that FFA and O(2-) play interrelated roles in inducing mitochondrial uncoupling, and we hypothesize that a likely formation of mitochondrial fatty acid hydroperoxides is a key event in the arachidonic acid-induced GDP-dependent inhibition of mitochondrial uncoupling.     

Protective effect of vitamin E on spinal cord injury by compression and concurrent lipid peroxidation

Studies were made on the influence of vitamin E on the effects of compression injury of the spinal cord associated with ischemia in rats. The motor disturbance induced by spinal cord injury was greatly reduced by vitamin E supplementation. After injury, the spinal cord evoked potentials showed greater recovery of both amplitude and latency in the vitamin E-supplemented group than in the control group. Spinal cord blood flow was promptly restored and remained normal after injury in the vitamin E-supplemented group, but was significantly decreased from 3 h after injury in the control group. Thiobarbituric acid (TBA)—reactive substances in the spinal cord was immediately increased by compression injury in both groups, and after injury it persisted at a high value for 24 h in the control group, but decreased within 1 h in the vitamin E-supplemented group. Pathological examination of the spinal cord showed less damage, such as bleeding and edema, in the vitamin E-supplemented group than in the control group. Vitamin E may have protective effects on the spinal cord by inhibiting damage induced by lipid peroxidation and/or by sustaining the blood flow by maintaining the normal metabolism of arachidonic acid.     

Lipid peroxide makes rabbit platelet hyperaggregable to agonists through phospholipase A2 activation

Treatment of rabbit platelets with tert-butyl hydroperoxide and Fe2+ caused increasing arachidonic acid release, lysophosphatidylcholine formation, and aggregation with increasing concentrations of Fe2+. A combination of tert-butyl hydroperoxide and a low concentration of Fe2+, which by itself causes slight or no such activation, elicited synergistic release of arachidonic acid and aggregation under stimulation with a suboptimal concentration of collagen or arachidonic acid as an agonist. These responses were inhibited by pretreatment of the platelets with vitamin E or mepacrine in a concentration-dependent manner, but not by uric acid. The arachidonic acid release was dependent on the presence of Ca2+ in the medium. Synergistic formation of lysophosphatidylcholine, but not diacylglycerol, was also observed under this condition. The aggregation was also inhibited by indomethacin, a cyclooxygenase inhibitor. Cyclooxygenase activity was not affected by the oxidative treatment. These results suggest that lipid peroxide formed in membranes causes phospholipase A2 to become hypersusceptible to the agonist used, making the platelets hyperaggregable.     

Peculiarities of leukotrienes metabolism in vitamin E deficiency in rats

Some changes take place in the spectrum of fatty acid, cholesterol and individual phospholipids' composition in the rat liver, under E-hypovitaminosis, that can play a considerable role in the cell damage. The level of cysteinyl leucotriene decreases in the blood and liver under E-hypovitaminosis and it rises to the control level under vitamin E correction. This demonstrates the influence of tocopherol on 5-LO pathway arachidonic acid.     

Effect of vitamin C and vitamin E on prostaglandin synthesis by fibroblasts and squamous carcinoma cells.

Dietary levels of vitamins C and E have been associated with cancer prevention and to a lesser extent with therapeutic enhancement of cancer treatment. Inhibition of prostaglandins (PGs) by pharmacological agents has been demonstrated to enhance immunocompetence, and to suppress growth of tumors in animals and humans. We report here on the effect of vitamins C and E on PGE2 production by human gingival fibroblasts and SCC-25 oral squamous carcinoma cells. The results indicate: 1. vitamins C and E exert a dose-dependent effect on arachidonic acid (AA) release and PGE2 synthesis; 2. vitamin E has a biphasic effect which is stimulatory at 1 and 10 microM and inhibitory at 100 microM; 3. vitamin E is considerably more potent than vitamin C in its inhibitory effect on AA and PGE2 in both cell types; 4. a combination of the two vitamins has a consistent dose-dependent inhibitory effect on AA and PGE2; 5. vitamin C stimulates PGE2 synthesis from exogenous AA in fibroblasts, and inhibits it in SCC-25 cells. The in vivo significance of these findings requires further investigation.     

Hemolysis and membrane lipid changes induced by xanthine oxidase in vitamin E deficient red cells

A procedure to induce homolysis by the hypoxanthine-xanthine oxidase reaction was developed and applied to vitamin E deficient red blood cells (RBCs) in rats. The reaction system was as follows: 0. 16 mM hypoxanthine, 0.05 U/ml xanthine oxidase in 2.5% RBC suspensions with an isotonic buffer (pH 7.4) containing 10 mM phosphate buffer and 125 mM saline (227 mOsm). Hemolysis was observed to depend on the vitamin E concentrations in the RBCs. Hemolysis was inhibited by catalase but not by SOD. After the reaction with vitamin E deficient RBCs, an increase in TBARS in the aqueous phase of the reaction mixture was observed. This accompanied the increase in fluorescent substances in the lipid extracts, in associatioon with a significant decrease in the PE and PS of the RBCs, and a decrease in arachidonic acid in membrane lipids. The above changes were almost completely inhibited by tocopherol incorporated into vitamin E deficient RBCs.