Dietary vitamin E deficiency increases anxiety-like behavior in juvenile and adult rats

Vitamin E deficiency from birth or infancy has recently been found to increase anxiety-like behavior in rodents. The present study was undertaken to elucidate the effect of dietary vitamin E deficiency on anxiety in adult rats in comparison with juvenile rats. Male Wistar rats, 3 or 10 weeks old, were divided into two groups and fed a control or vitamin E-deficient diet for 4 weeks. The results of behavioral analysis revealed that vitamin E-deficiency increased anxiety in both juvenile and adult rats. Plasma, liver, and brain α-tocopherol concentrations decreased significantly due to vitamin E deficiency in both age groups. Plasma corticosterone concentrations were higher in the vitamin E-deficient rats in response to the stress of a behavioral test. Based on these results, we conclude that dietary vitamin-E deficiency induces anxiety in adult rats as well as juvenile rats. This might be due to an elevated plasma corticosterone concentration.     

Dietary Vitamin E Deficiency Increases Anxiety-Like Behavior in Juvenile and Adult Rats

Vitamin E deficiency from birth or infancy has recently been found to increase anxiety-like behavior in rodents. The present study was undertaken to elucidate the effect of dietary vitamin E deficiency on anxiety in adult rats in comparison with juvenile rats. Male Wistar rats, 3 or 10 weeks old, were divided into two groups and fed a control or vitamin E-deficient diet for 4 weeks. The results of behavioral analysis revealed that vitamin E-deficiency increased anxiety in both juvenile and adult rats. Plasma, liver, and brain α-tocopherol concentrations decreased significantly due to vitamin E deficiency in both age groups. Plasma corticosterone concentrations were higher in the vitamin E-deficient rats in response to the stress of a behavioral test. Based on these results, we conclude that dietary vitamin-E deficiency induces anxiety in adult rats as well as juvenile rats. This might be due to an elevated plasma corticosterone concentration.     

Vitamin E regulates mitochondrial hydrogen peroxide generation.

The mitochondrial electron transport system consumes more than 85% of all oxygen used by the cells, and up to 5% of the oxygen consumed by mitochondria is converted to superoxide, hydrogen peroxide, and other reactive oxygen species (ROS) under normal physiologic conditions. Disruption of mitochondrial ultrastructure is one of the earliest pathologic events during vitamin E depletion. The present studies were undertaken to test whether a direct link exists between vitamin E and the production of hydrogen peroxide in the mitochondria. In the first experiment, mice were fed a vitamin E-deficient or-sufficient diet for 15 weeks, after which the mitochondria from liver and skeletal muscle were isolated to determine the rates of hydrogen peroxide production. Deprivation of vitamin E resulted in an approximately 5-fold increase of mitochondrial hydrogen peroxide production in skeletal muscle and a 1-fold increase in liver when compared with the vitamin E-supplemented group. To determine whether vitamin E can dose-dependently influence the production of hydrogen peroxide, four groups of male and female rats were fed diets containing 0, 20, 200, or 2000 lU/kg vitamin E for 90 d. Results showed that dietary vitamin E dose-dependently attenuated hydrogen peroxide production in mitochondria isolated from liver and skeletal muscle of male and female rats. Female rats, however, were more profoundly affected by dietary vitamin E than male rats in the suppression of mitochondrial hydrogen peroxide production in both organs studied. These results showed that vitamin E can directly regulate hydrogen peroxide production in mitochondria and suggest that the overproduction of mitochondrial ROS is the first event leading to the tissue damage observed in vitamin E-deficiency syndromes. Data further suggested that by regulating mitochondrial production of ROS, vitamin E modulates the expression and activation of signal transduction pathways and other redox-sensitive biologic modifiers, and thereby delays or prevents degenerative tissue changes.     

Effects of long-term vitamin E deficiency and restoration on rat hepatic peroxisomes

Effects of vitamin E deficiency and its restoration on biochemical characteristics of hepatic peroxisomes were studied. Rats were maintained on the vitamin E-deficient diet for 25 weeks and then on a diet supplemented with vitamin E for 5 weeks. Blood hemolysis by hydrogen peroxide and lipid peroxidation in the liver increased markedly in vitamin E-deficient rats. The former returned to the control level after the resupplying of vitamin E, but the latter did not. Of liver peroxisomal enzymes, the activities of catalase, D-amino-acid oxidase and urate oxidase decreased in vitamin E-deficient rats. On the other hand, activities of fatty acyl-CoA oxidase and carnitine acetyltransferase increased significantly in vitamin E-deficient rats. All activities of these peroxisomal enzymes were restored to the control levels in vitamin E-supplemented rats. The activities of the mitochondrial, lysosomal and microsomal enzymes tested showed no apparent change except that the change of mitochondrial palmitoyltransferase was shown to be similar to that of peroxisomal fatty acid oxidation. These results were also supported by cell fractionation techniques. Following the methods of aqueous polymer two-phase systems, the characteristics of peroxisomal surface membranes altered in respect of their hydrophobicity, but not in respect of the surface charge of peroxisomal membranes. These results indicate that peroxisomal functions, especially those of the fatty acid oxidation system, change their activities more sensitively than other intracellular organelles in response to the condition of vitamin E deficiency.     

Decreased citrate synthesis: possible indication of early degenerative changes in testes of vitamin E-deficient rats.

We have shown that intravenously administered glucose disappears from the blood of E-deficient rats at different rates compared to that of the control rats and that this difference could possibly be explained by membrane permeability changes in E-deficiency. We have also shown that the ability of E-deficient rat testis tissue to synthesize citrate is decreased, and that this decrease is probably an early manifestation of testicular degeneration.     

Complement-component-C3-opsonized immunoglobulin G anti-DNA antibodies do not bind effectively to red blood cells unless aggregated on a high-Mr DNA matrix.

The significance of microsomal vitamin E in protecting against the free-radical process of lipid peroxidation was evaluated with the low-level-chemiluminescence technique in microsomal fractions from vitamin E-deficient and control rats. The induction period that normally precedes the ascorbate/ADP/Fe3+-induced lipid peroxidation was taken as reflecting the microsomal vitamin E content and was found to be 5-6-fold decreased in microsomal fractions from vitamin E-deficient rats. Supplementation of microsomal fractions from vitamin E-deficient rats with exogenous vitamin E partially restores the induction period observed in that from control rats. The decrease in chemiluminescence intensity and the increase in the induction period both correlate linearly with the amount of vitamin E added. However, the efficiency of exogenous vitamin E is about 50-fold lower than that exerted by the naturally occurring vitamin E in microsomal membranes. These observations are discussed in terms of the process of re-incorporation of vitamin E into membranes, the experimental model for lipid peroxidation selected, and the method to evaluate lipid peroxidation, namely low-level chemiluminescence.     

The Effect of Vitamin E-Deficiency in Isolated Rat Heart on the Cellular Defence System Against Free Radicals During Normal Reperfusion After Hypoxic, Ischemic and Ca2+-Free Perfusion

We investigated whether vitamin E plays a role in the protection against potential free radical formation and related biochemical changes in hypoxic, ischemic and Ca²⁺-depleted rat heart upon normal reperfusion.

In the heart of normally fed rats a decrease in the activity of superoxide dismutase and the capacity of the glutathione system, factors of the cellular protective mechanisms against free radicals, occurred upon exposure to the above mentioned treatments. This decrease was not further enhanced if vitamin E-deficient rat hearts were treated. Vitamin E-deficiency, however, led to detectable peroxidation of lipids if Ca²⁺-depleted or hypoxic hearts were reperfused. Lipid peroxidation was measured as the formation of thiobarbituric acid reactive material, which is readily formed during this process. Reflow after ischemia did not induce lipid peroxidation either in normal or in vitamin E-deficient rat heart.

Since changes in Ca²⁺ -homeostasis are thought to be primarily responsible for the Ca²⁺-reperfusion injury, a role for Ca²⁺-ions in lipid peroxidative processes, either directly or indirectly, seems indicated. Furthermore the results imply that even a sharp and extensive decrease of reduced glutathione, as seen upon Ca²⁺ -repletion after a period of Ca²⁺ -depletion, does not necessarily induce peroxidation of lipids in heart tissue. Obviously, vitamin E is very important in the protection of cardiac membranes. Replenishment of the water-soluble protective factors in the heart seems, however, more important during above mentioned treatments, especially since repair of the vitamin E-free radical is dependent on water-soluble factors.     

Free Radical-Induced Liver Injury. I. Effects of Dietary Vitamin E Deficiency on Triacylglycerol Level and its Fatty Acid Profile in Rat Liver

Effects of dietary vitamin E deficiency on the fatty acid compositions of total lipids and phospholipids were studied in several tissues of rats fed a vitamin E-deficient diet for 4, 6, and 9 months. No significant differences were observed between the vitamin E deficiency and controls except in the fatty acid profiles of liver total lipids. Triacylglycerol (TAG) accumulation was found in the liver of rats fed a vitamin E-deficient diet. The levels of TAG-palmitate and -oleate increased particularly in the liver from such animals. The fatty acid compositions of hepatic phospholipids were not affected by the diet. Increased TAG observed in the liver of rats fed a vitamin E-deficient diet was restored to normal when the diet was supplemented with 20 mg α-tocopheryl acetate/kg diet. These findings indicate that dietary vitamin E deficiency causes TAG accumulation in the liver and that the antioxidant, vitamin E, is capable of preventing free radical-induced liver injury.     

Modification of hepatic vitamin E stores in vivo. II. Alterations in plasma and liver vitamin E content by 1,2-dibromoethane

Previous studies with methyl ethyl ketone peroxide (MEKP), a radical generator, showed depletion of plasma vitamin E and liver glutathione (GSH) levels prior to a decrease of liver vitamin E levels. Since hepatic pools of this vitamin may serve to maintain circulating levels of vitamin E under conditions of oxidative challenge, we have evaluated the similarity of response after treatment with 1,2-dibromoethane (DBE), a compound that is not known to generate oxyradicals or to induce lipid peroxidation in vivo. Treatment of normal rats with DBE caused a depletion in hepatic vitamin E levels 1 day after treatment; however, in contrast to our prior findings with MEKP this depletion after DBE treatment was observed in tandem with elevations in the plasma content of vitamin E. Liver vitamin E depletion was neither dependent upon a sustained liver GSH depletion nor upon hepatocellular death. Mobilization and export of hepatic vitamin E did not result in an immediate whole body redistribution of this vitamin in that pulmonary and renal levels of vitamin E remained normal under conditions of liver vitamin E depletion. Moreover, the stimulus that resulted in exportation of liver vitamin E was maintained by daily treatments with DBE. DBE caused a substantial elevation above control values in liver GSH content and these elevations were also maintained by daily DBE treatments. In experiments to assess the influence of prandial replacement of vitamin E on the extent of depletion in response to DBE treatment, rats were fed a vitamin E-deficient diet for 2 days prior to treatment. This short pulse of a vitamin E-deficient diet delayed (to 2 days) both the elevation in liver GSH content and the depletion of liver vitamin E and hastened (to 1 day) the elevation in plasma vitamin E concentration. These observations suggest the presence of at least two pools of liver vitamin E and that one of these pools, which comprises at least 30% of the total hepatic vitamin E content, is able to be mobilized and exported in response to chemical challenge. The stimulus that resulted in liver vitamin E exportation in response to DBE treatment seems to result from wholly intrahepatic processes and may not be a direct response to lipid peroxidation. Moreover, the similarity between the time-course and the extent of hepatic vitamin E depletion observed after treatment with either MEKP or DBE suggests a similarity in physiochemical processes that function to mobilize hepatic vitamin E stores.     

Rat lung microsomal lipid peroxidation: effects of vitamin E and reduced glutathione

Lung microsomal membranes that contain the redox active components associated with the mixed-function oxidase system can be peroxidized in vitro. To investigate the characteristics of rat lung microsomal lipid peroxidation, we performed experiments using a variety of peroxidation initiators and microsomes obtained from normal and vitamin E-deficient rats. We found that lung microsomes obtained from normal rats are peroxidized much less than liver microsomes obtained from the same animals. Only initiation systems using very high concentrations of ferrous iron produced any significant peroxidation of normal rat lung microsomes. Lung microsomes obtained from vitamin E-deficient rats were found to be much more susceptible to peroxidation. Glutathione (GSH) was effective in inhibiting peroxidation when lung microsomes from normal rats were peroxidized. GSH was not effective in decreasing peroxidation when microsomes from vitamin E-deficient rats were peroxidized in the same system. We conclude that both GSH and vitamin E protect lung microsomal membranes from peroxidation. Glutathione protection appears to be related to the presence of a sulfhydryl group.     

Vitamin E deficiency and vitamin C supplements: exercise and mitochondrial oxidation

The effects of dietary antioxidant vitamins E and C on exercise endurance capacity and mitochondrial oxidation were investigated in rats. The endurance capacity of both vitamin E-deficient and vitamin C-supplemented, E-deficient rats was significantly (P less than 0.05) lower (38.1 and 33.6%, respectively) than control animals. Compared with the normal and vitamin E-deficient rats, there was a significant (P less than 0.05) increase in the concentration of vitamin C in blood and liver of the vitamin E-deficient, C-supplemented animals. Hence dietary vitamin C supplementation does not prevent the inhibition of exercise endurance capacity or increased hemolysis seen in vitamin E deficiency. The mitochondrial activities for the oxidation of palmitoyl carnitine and alpha-ketoglutarate were significantly (P less than 0.05) decreased by a single bout of exercise in brown adipose tissue but not in muscle, heart, or liver from vitamin C-supplemented, E-deficient groups of rats when compared with the activities in the tissue from the same group of rats killed at rest. Similar results were also seen in brown adipose tissue from vitamin E-deficient rats. The results suggest a tissue-specific role for vitamins E and C in substrate oxidation and show that the poor endurance capacity of vitamin E-deficient rats cannot be attributed to any changes in the mitochondrial activity in skeletal or cardiac muscles. It is also concluded that vitamin C supplementation, at least at the dose employed in the present study, cannot counteract the detrimental effects associated with vitamin E deficiency.     

Effect of vitamin E on transcription in isolated nuclei and rat liver chromatin in normal status and in E-hypovitaminosis]

The effect of alpha-tocopherol on the RNA-polymerase activity in isolated rat nuclei and chromatin from normal and E-deficient rats and the possible role of tocopherol-binding proteins in this process were studied. Some differences in the RNA-polymerase activities of the nuclei were found; however, in vitro added alpha-tocopherol had no effect on the level of the label incorporation into RNA. No effect of alpha-tocopherol on this process was observed after addition of cytosol either. Analysis of chromatins from normal and E-deficient rats revealed no differences in their RNA-polymerase activities. In vitro added alpha-tocopherol increased the RNA-polymerase activity of normal (but not of vitamin E-deficient) rats. Some differences in the RNA-polymerase activities were noted after addition to the incubation medium of the Triton X-100-solubilized nuclear fraction specifically binding alpha-tocopherol. This effect was enhanced in the presence of exogenous alpha-tocopherol. The susceptibility of chromatin from normal and E-deficient rats to DNAse I hydrolysis was also found to be different. It was concluded that vitamin E can influence the RNA-polymerase activity of the nuclei and chromatin as well as the chromatin structure and that alpha-tocopherol-binding proteins are necessary for the vitamin E effect on the RNA-polymerase activity to be manifested.     

Influence of dietary vitamin E on the intermembrane transfer of alpha-tocopherol as mediated by an alpha-tocopherol binding protein

The effect of dietary vitamin E on the intermembrane transfer of (3R)-alpha-tocopherol, a spontaneous process accelerated in the presence of an alpha-tocopherol binding protein (alpha TBP), was examined. The transfer activity of this cytosolic liver protein was assayed via in vitro transfer of (3R)-alpha-[3H]tocopherol (alpha[3H]T) from egg lecithin liposomes to human erythrocyte ghosts (EG). Male Fisher 344 rats (1 and 20 months old) were fed diets containing 0, 30, and 500 mg/kg vitamin E (dl-alpha-tocopheryl acetate) for 15 weeks. Liver cytosol fractions were assayed for alpha[3H]T transfer activity (alpha TTA). Among young rats, those fed vitamin E-deficient diets had the highest alpha TTA, 5.02 +/- 3.10 pmole alpha[3H]T/min (mean +/- SD), which was different (P less than 0.05) from the spontaneous transfer rate of 2.10 pmole/min. Neither young rats fed 30 and 500 mg/kg vitamin E diets nor any of the aged rats showed alpha TTA which differed significantly from the spontaneous transfer rate. To examine the relationship between hepatic alpha-tocopherol levels and alpha TTA, alpha-tocopherol concentration per gram of wet liver was assayed by HPLC. A steep positive slope (6.39 +/- 1.46 pmole min-1 nmole g-1) and strong correlation (r = 0.873) between hepatic alpha-tocopherol and alpha TTA were observed (P less than 0.005) among young vitamin E-deficient rats. The data indicates that alpha TTA varies directly with hepatic alpha-tocopherol concentration when total liver vitamin E stores are very low. Thus, alpha TBP-mediated transfer of alpha-tocopherol may be manifest only when vitamin E status is compromised.     

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.     

Effect of vitamin E-deficiency on the activity of some lysosomal and non-lysosomal proteases in rabbit muscles.

The activity of different cathepsins and neutral proteinases was measured in normal and vitamin E-deficient rabbit muscles using specific substrates. Among the changes of enzyme activities in dystrophy caused by vitamin E-deficiency the increase in the activity of cathepsin B is the most striking. The activity of cathepsin H, both in the fast and slow muscles and that of MMP-ase in the slow muscle remains practically unchanged. Activities of other proteases significantly increase. The change in the activity of proteolytic enzymes in striated muscle of vitamin E-deficient rabbits seems to be selective. As a rule the increase in the activity is higher in fast than in slow muscles.     

Effect of vitamin E on adjuvant arthritis in rats

Adjuvant arthritis was induced in rats fed a diet deficient in or supplemented with vitamin E, and its severity was scored according to the macroscopic findings of their legs, tails, and ears. The average score so obtained was higher in the vitamin E-deficient diet group than in the group of rats supplemented with vitamin E. Whereas the A/G ratio remained depressed in vitamin E-deficient rats, rats on a vitamin E-supplemented diet showed a fast recovery from A/G-ratio depression. The serum levels of beta-glucuronidase and acid phosphatase were elevated after administration of an adjuvant. The serum levels of these lysosomal enzymes showed a remarkable increase in rats fed a vitamin E-deficient diet, while the elevation in lysosomal enzyme levels in rats fed a vitamin E-supplemented diet was inhibited. The levels of thiobarbituric acid (TBA) reactants in the synovia were elevated at 2 weeks after exposure to the adjuvant and were decreased thereafter. In rats maintained on a diet supplemented with vitamin E, on the other hand, the increase in synovial level of TBA reactive substances was inhibited. These observations suggest that the aggravation of adjuvant arthritis may be associated with lipid peroxidation and that antioxidants, such as vitamin E, may be beneficial for arthritis.     

Effect of dietary lipid and vitamin E on mitochondrial lipid peroxidation and hepatic injury in the bile duct-ligated rat.

To assess whether lipid peroxidation of hepatic mitochondria is associated with cholestatic hepatic injury we examined the effect of bile duct ligation (BDL) versus sham surgery on mitochondrial lipids of rats maintained on one of seven diets. Diets included vitamin E-deficient (E-) and vitamin E-sufficient (E+) combined with normal lipid (11.9% calories as stripped corn oil), high lipid (35% calories as stripped corn oil), or n-3 fatty acid (fish oil) supplementation. Rats were killed 17 days after surgery, mitochondria were isolated by differential centrifugation, and lipid-conjugated dienes and thiobarbituric acid-reacting substances (TBARS) were measured in mitochondrial lipids as indices of lipid peroxidation. BDL resulted in significant increases in lipid peroxidation in all dietary groups. The E- high lipid diets (with either corn oil or fish oil) were associated with higher lipid peroxide and serum bilirubin values in BDL rats compared to the normal lipid diets. Fish oil supplementation did not ameliorate cholestatic or oxidative injury. Serum alanine aminotransferase, bilirubin, alkaline phosphatase, and cholylglycine levels correlated significantly with levels of mitochondrial conjugated dienes and TBARS. These data suggest that free radical stress occurs during BDL in the rat and may result in mitochondrial lipid peroxidation, and that diets high in lipid may increase free radical damage to hepatic mitochondria. The role of free radicals in cholestatic hepatic injury requires further investigation.     

Iron uncouples oxidative phosphorylation in brain mitochondria isolated from vitamin E-deficient rats

Few, if any, studies have examined the effect of vitamin E deficiency on brain mitochondrial oxidative phosphorylation. The latter was studied using brain mitochondria isolated from control and vitamin E-deficient rats (13 months of deficiency) after exposure to iron, an inducer of oxidative stress. Mitochondria were treated with iron (2 to 50 microM) added as ferrous ammonium sulfate. Rates of state 3 and state 4 respiration, respiratory control ratios, and ADP/O ratios were not affected by vitamin E deficiency alone. However, iron uncoupled oxidative phosphorylation in vitamin E-deficient mitochondria, but not in controls. In vitamin E-deficient mitochondria, iron decreased ADP/O ratios and markedly stimulated state 4 respiration; iron had only a modest effect on these parameters in control mitochondria. Thus, vitamin E may have an important role in sustaining oxidative phosphorylation. Low concentrations of iron (2 to 5 microM) oxidized mitochondrial tocopherol that exists in two pools. The release of iron in brain may impair oxidative phosphorylation, which would be exacerbated by vitamin E deficiency. The results are important for understanding the pathogenesis of human brain disorders known to be associated with abnormalities in mitochondrial function as well as iron homeostasis (e.g., Parkinson's disease).     

Ca2+-activated Protease Activity in Vitamin E-Deficient Rats

The mechanism and control of protein degradation in cells are quite mysterious. We investigated the change of protease activities in animals fed a vitamin E-deficient diet. The Ca²⁺-activated protease activity was not significantly changed in vitamin E-deficient rats during the 45 weeks of the experiment. The cathepsin B activity was increased in those animals. Electron microscopic observation on the muscle of the vitamin E-deficient rats showed destruction of myofibrils at the Z-line, narrowness of myofibrils, and dispersed myofibrils. The M-line, which is known to disappear with cathepsin L treatment, was clearly observed. The phagocytosis of muscle cells by macrophages was also observed. These results show that the abnormal myofibril protein degradation in muscle tissue of vitamin E-deficient rats is not only due to the activation of macrophages and the increment of lysosomes in muscle cells, but also due to the protease which can destroy the myofibril at the Z-line. It may be a Ca²⁺-activated protease.