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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 55:B144-B151 (2000)
© 2000 The Gerontological Society of America

Effect of Fruits, Vegetables, or Vitamin E–Rich Diet on Vitamins E and C Distribution in Peripheral and Brain Tissues

Implications for Brain Function

Antonio Martina, Ronald Priora, Barbara Shukitt-Halea, Guohua Caoa and James A. Josepha

a Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts

Antonio Martin, USDA-Neuroscience Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, MA 02111 E-mail: amartin{at}hnrc.tufts.edu.

Jay Roberts, PhD


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Age-related neurodegenerative conditions are the principal cause of declining cognitive and motor function during aging. Evidence support that fruits and vegetables containing generous amounts of antioxidant nutrients are important for neurological function. We investigated the effect of diets enriched with fruits or vegetables but low in vitamin E and a diet high in vitamin E on the distribution of vitamins C and E in the brain and dopamine release of Fischer 344 rat model, over an 8-month period. The low–vitamin E diet resulted in lowered {alpha}-tocopherol levels in brain and peripheral tissues, whereas the animals that received a diet enriched in vitamin E showed a significant increase, between 500–900%. Vitamin C concentration in plasma, heart, and liver was reduced in the vitamin E–supplemented group. It is concluded that supplementation or depletion of {alpha}-tocopherol for 8 months results in marked changes in vitamin E levels in brain tissue and peripheral tissues, and varied distribution of {alpha}-tocopherol throughout the different brain regions examined. In addition, compared to control group, rats supplemented with strawberry, spinach, or vitamin E showed a significant enhancement in striatal dopamine release. These findings suggest that other nutrients present in fruits and vegetables, in addition to the well-known antioxidants, may be important for brain function.

AGE-RELATED neurological deterioration is accompanied by a significant decrease of transmitter levels as well as activity of neurotransmitter-synthesizing enzymes (1)(2). Little is known about the mechanisms behind these neurological declines, but toxic species evolved from inflammatory processes and metabolism of cathechols may be involved in its etiology (3)(4)(5). The recognition that oxidative stress may be an important etiological factor in the pathogenesis of various degenerative diseases has sparked interest in the role(s) that antioxidants may play in preventing oxidative damage, and their application in the prevention of neurodegenerative diseases. Moreover, the consumption of fruits and vegetables has been associated with a decreased risk of different chronic pathologies (6). Fruits and vegetables contain large amounts of vitamin C, carotenoids, some vitamin E, and other phytochemicals such as flavonoids. These nutrients prevent or diminish degenerative diseases associated with aging (4)(7)(8)(9).

Vitamin E is an essential fat-soluble vitamin, which includes different naturally occurring isomers ({gamma}, {delta}, ß). Among them, d-{alpha}-tocopherol has the highest biological activity and is the most abundant form in food. This nutrient is the most effective chain-breaking lipid soluble antioxidant in the biological membrane, where it prevents the propagation of free-radical damage, and contributes to membrane stability (10). Recent evidence indicates, however, that vitamin E may have structure-specific roles in addition to its antioxidant function, through its modulation of signal transduction pathways (11)(12)(13), and participation in biochemical processes involving synthesis and distribution of neurotransmitters. Interestingly, there is significant evidence showing neuropathological abnormalities during vitamin E deficiency, and that central nervous system requirements may change during aging (14)(15)(16). In addition, several studies have shown evidence indicating the importance of vitamin E for normal neurological function (17)(18). During aging a decline in both cognitive and motor functions has been observed, but the mechanisms involved in these declines are not well understood. Changes related to the alteration in neurotransmitter receptor sensitivity, such as dopaminergic receptors, have been observed, as have changes related to a reduction of dopamine (DA) release (19)(20). Therefore, in an effort to understand the basis of neuropathology of vitamin E, diverse studies have examined the pattern of vitamin E distribution in different regions of the brain (21)(22). Interestingly, although there is unanimous accord on the positive effect of dietary vitamin E intake on {alpha}-tocopherol concentration in the central nervous system (CNS), brain endogenous levels of lipid peroxidation have not been found to reflect levels of vitamin E intake (22).

Ascorbate (reduced vitamin C) is another important body nutrient, stored in high concentrations (millimolar) in the adrenal gland and the brain (23). However, in spite of the fact that this nutrient is widely distributed in the brain, only a few of its roles, such as acting as a cofactor for dopamine-ß-hydroxylase, or mediating glutamate uptake, are well documented (23)(24)(25). In addition, vitamin C exerts a large scope of important antioxidant activities due to its ability to react with numerous aqueous free radicals and oxygen species.

Flavonoids are a family of biochemical compounds found in fruits and vegetables and consumed in the human diet. These nutrients retain a high reductant capacity, have anti-inflammatory and immunomodulatory activities, and reduce histamine release from mast cells (26). The documented role of these nutrients raises new questions and represents new and exciting areas for research in nutrition-related cell regulation, with important physiological implications.

We were interested in examining the effect of low and high vitamin E intakes on the distribution of vitamins E and C in brain regions after long dietary intervention. Our objectives were: (i) to analyze the long-term (8 months) effect of low vitamin E intake on vitamin E levels in brain and other tissues; (ii) to examine the brain's vitamin E distribution following dietary vitamin E treatment; (iii) to determine if low vitamin E intake could affect vitamin C synthesis to compensate for the vitamin E deficit; and (iv) to determine if a high intake of fruits and vegetables may affect brain function and attenuate the deleterious effects associated with aging without affecting the concentrations of vitamins E and C.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Male Fischer 344 rats (Harlan Sprague Dawley, Indianapolis, IN) were used to investigate the long-term (8 months) effect of a control diet (modified AIN-93) (27) or a diet containing extracts of either strawberries (9.5 g/kg diet), spinach (6.4 g/kg diet), or vitamin E (with 500 mg all-rac-{alpha}-tocopheryl acetate/kg diet) on vitamins E and C tissue distribution. Eighty animals were used in these studies. The rats were individually housed in stainless steel mesh suspended cages, provided food and water ad libitum and maintained on a 12-hour light/dark cycle. All animals were observed daily for clinical signs of disease. These animals were utilized in compliance with all applicable laws and regulations as well as principles expressed in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The Animal Care and Use Committee of our Center approved this study. Following a 12-day acclimatization period to the facility, the 6-month-old rats were weight-matched and given 2 weeks on the control diet. They were then divided into four diet groups (20 animals/group). Monthly weights and food intakes over a 48-hour period were recorded for all diet groups.

The strawberry and spinach extracts were prepared by adding 400 g of the strawberry or spinach foods to water in the ratio of 2:1, and homogenized in a blender for 2 minutes. The homogenate was then centrifuged at 13,000 x g for 15 minutes at 4°C (to separate just the fiber). The supernatant was recovered and combined in freezer bags, 500 mL per bag. Frozen extracts were placed in a freeze drier until dry, which usually required about 7 days. The freeze-dried extracts were then shipped to Research Diets Inc. (New Brunswick, NJ) where they were mixed with the control diet. The amounts of strawberry or spinach extracts added into the diets were equivalent in terms of their total antioxidant level. Each of the strawberry and spinach diets provided about 1.4 mmol Trolox equivalent and the vitamin E diet 1.2 mmol Trolox equivalent per kg diet, based on the ORAC assay (28); therefore, the supplemented groups contained 1.4 or 1.2 mmol Trolox equivalent per kg diet more than the control group. At 15 months of age, the various diet groups were examined for differences in various indexes to indicate different responses induced by the different dietary treatments. These include indices for body weight, distribution of vitamins E and C, and muscarinic receptor sensitivity (as assessed via oxotremorine enhancement of DA release from striatal slices); cerebellar GABAergic receptors (as assessed via isoproterenol facilitation of GABA inhibition of cerebellar Purkinje cell firing), calcium release, and behavior response were also evaluated (29).

Quantification of Vitamin E
Vitamin E ({alpha}- and {gamma}-tocopherol) content of plasma or tissues was measured by reverse-phase high performance liquid chromatography (HPLC). Briefly, 100 µL of plasma sample or 100 µL of homogenized tissue were mixed with one 100 µL ethanol; after vortexing, tocopherols were extracted into 500 µL hexane containing 0.002% butylated hydroxyl toluene (BHT) (Sigma Chemical Co., St Louis, MO). Tocol (a gift from Hoffmann-La Roche, Nutley, NJ) was added to the mixture as an internal standard. Samples were centrifuged at 800 rpm for 5 minutes at 4°C. The supernatant was collected and dried under a stream of nitrogen gas, and reconstituted in 100 µL of methanol. Tocopherols were separated by HPLC using a 3 µm C18 reverse-phase column (Perkin-Elmer, Norwalk, CT). The mobile phase, delivered at a flow rate of 1.2 mL/min, consisted of 1% water in methanol, containing 20 mM lithium perchlorate. Samples were injected with an autosampler (1100 series, Hewlett Packard Co, Wilmington, DE). Eluted peaks were detected at an applied potential of +0.6 V by a LC 4B amperometric electrochemical detector (Bioanalytical Systems, West Lafayette, IN). Tocopherols eluted as well-separated peaks with a retention time between 2 to 6 minutes. Peaks were integrated with a ChemStation (Hewlett Packard), {alpha}-tocopherol concentration was expressed in pmol/mg protein (12). Protein was measured by the method of Lowry and colleagues (30).

Quantification of Ascorbate
Ascorbate was analyzed by paired-ion, reversed-phase HPLC coupled with electrochemical detection. In brief, 100 µL of plasma sample was mixed with an equal volume of cold 5% (w/v) metaphosphoric acid containing 1 mmol/L of the metal ion chelator diethylenetriaminepentaacetic acid (Sigma), or 40–100 mg of tissue homogenized in 500 µL of the same solution, and centrifuged to remove the precipitated proteins. An aliquot of the supernatant was chromatographed on a LC8 column (150 mm x 4.6 mm, i.d., 3 µm particle size) (Supelco, Bellefonte, PA) using 99% deionized water and 1% methanol containing 40 mmol/L sodium acetate and 1.5 mmol/L dodecyltriethylammonium phosphate (Q12 ion pair cocktail, Regis, Morton Grove, IL) as the mobile phase. Samples were injected with an autosampler, 1100 series (Hewlett Packard). Ascorbate was detected at an applied potential of +0.6 V by a LC 4B amperometric electrochemical detector (Bioanalytical Systems). Ascorbate eluted as a single peak with a retention time of 5.5 minutes. Peaks were integrated with a ChemStation (Hewlett Packard). Ascorbate concentration was calculated based on a calibration curve, and its concentration was expressed in nmol/mg protein (31). Protein was measured by the method of Lowry and colleagues (30).

Dopamine Release
Measurement of DA release was conducted as previously described by Joseph and colleagues (32)(33). DA release from striatal slices obtained from the animals maintained on the different diets following stimulation with 0 or 500 µM oxotremorin was quantitated by HPLC coupled with electronic detection (33).

Statistic Analysis
Results were expressed as mean ± SD. {alpha}- and {gamma}-tocopherol in the different brain regions, heart, and plasma; concentrations of vitamin C in the different brain regions, heart, and plasma; and the releases of dopamine were analyzed by ANOVA. The Tukey test was used for multiple comparison post hoc analyses. We applied the Kruskal-Wallis ANOVA with Bonferroni-corrected Mann-Whitney to compare the concentration of vitamin E in the liver in the different dietary groups, because the SD is larger in the vitamin E-fed group than the other groups.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Weights and Food Intakes
The rats significantly increased their weight from an average of 356.0 ± 0.4 g (6 months) to 473.4 ± 3.4 g (15 months) (p < .001). However, there were no differences in weights between the different dietary treatments over time or at the age of 15 months. There were also no differences in food intakes between the dietary treatments over the course of the study, as previously reported (29).

Vitamins E and C in Brain (Cortex, Hippocampus, Cerebellum, and Striatum), Functional Implications
Levels of vitamin E in brain were significantly affected after 8 months of dietary intervention among the various diet groups (p < .001). In this regard, concentrations of {alpha}-tocopherol in the animals fed with a control, strawberry, or spinach diet were 100 ± 81, 154 ± 105, and 123 ± 56 pmol/mg protein respectively. In contrast, {alpha}-tocopherol levels in the high–vitamin E group were 911 ± 560 pmol/mg protein (p < .0001) in cortex (Fig. 1a); 132 ± 85, 137 ± 67, 222 ± 164 versus 1077 ± 511 pmol/mg protein respectively (p < .0001) in the hippocampus (Fig. 1b); 83 ± 78, 55 ± 33, 115 ± 98 versus 424 ± 262 pmol/mg protein respectively (p < .001) in the cerebellum (Fig. 1c); and 26 ± 23, 14 ± 7 versus 212 ± 101 pmol/mg protein respectively (p < .01) in the striatum (Fig. 1d). Data from the striatum region in the spinach group are absent because samples were used for other assays. Thus, the different regions of the brain accumulated distinct amounts of {alpha}-tocopherol following supplementation with vitamin E (Fig. 1e). The striatum and cerebellum were the regions that had significantly lower concentrations compared to cortex or hippocampus (p < .05). In addition to {alpha}-tocopherol, other vitamin E isomers such as {gamma}-tocopherol were also present in brain in significant concentrations, with an average across all groups of about 30 ± 18 pmol/mg protein. No differences were observed among groups, although the high–vitamin E diet tended to have lower concentration of this vitamin E isomer. Ascorbate concentrations in cortex, hippocampus, and cerebellum were not significantly different, with an average of 20 ± 7 nmol/mg protein (Table 1 ). The striatum, however, showed a trend towards lower concentrations of C with an average of 13.5 ± 4 nmol/mg protein, and no significant differences among groups (Table 1 ). As previously described, brain tissues from animals fed strawberry, spinach, and control diets very nicely reflected their low–vitamin E intake, compared to high–vitamin E treatment. Interestingly, diets enriched with extracts of strawberry or spinach, or vitamin E, showed enhanced dopamine release from striatal slices following oxotremorine stimulation by 100%, 300%, and 150%, respectively, compared to control (p < .05).


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  		Table 1. Ascorbate Concentration (nmol/mg protein) in Different Regions of Brain

 
Vitamins E and C in Plasma
Levels of vitamin E and C in plasma were significantly affected after 8 months of dietary intervention among the various diet groups (p < .001). After 8 months of dietary intervention the levels of {alpha}-tocopherol in plasma were 2.2 ± 1.5, 2.5 ± 2.6, 1.9 ± 0.4 µM, respectively, for control, control plus strawberry, and control plus spinach diets. Rats fed the vitamin E–enriched diet had a plasma {alpha}-tocopherol concentration of 73.4 ± 23.8 µM, which was significantly higher (p < .0001) than all other diets (Fig. 2a). As compared to the high vitamin E–supplemented diet group, the ratio between {alpha}-tocopherol and {gamma}-tocopherol in plasma was strikingly reduced in all the low–vitamin E diet–fed animals. However, because these diets are an important source of {gamma}-tocopherol and rats absorb {gamma}-tocopherol well (34) the concentration of {gamma}-tocopherol in plasma increased to similar or higher concentrations than {alpha}-tocopherol.

Plasma vitamin C concentrations were similar in rats fed the control, strawberry, or spinach diets with values of 18.6 ± 6, 20.1 ± 8, 21.3 ± 7 µM respectively. However, in the group fed the vitamin E–enriched diet, plasma C levels decreased significantly to 9 ± 3 (p < .05) (Fig. 2b).

Vitamins E and C in Liver
Animals fed the vitamin E–enriched diet showed a significant increase in the concentration of {alpha}-tocopherol in liver (4172 ± 2700 pmol/mg protein) compared to control, control plus strawberry, and control plus spinach groups, which had concentrations of 23 ± 13, 23 ± 13, 26 ± 25 pmol/mg protein respectively (p < .0001) (Fig. 3a). The ability of the liver to uptake {gamma}-tocopherol increases when the {alpha}-tocopherol present in the diet decreases. Thus, when a diet is low in {alpha}-tocopherol, the liver becomes more active in incorporating other tocopherols into lipoprotein fractions and releasing them into the blood stream (35)(36). This may explain the relatively high concentrations of {gamma}-tocopherol observed in plasma of rats fed a low–vitamin E diet. However, the levels of {gamma}-tocopherol observed in liver were significantly higher in the vitamin E–enriched diet as compared to the other groups (p < .05) (Fig. 3a). Perhaps the amount of {gamma}-tocopherol present in the high–vitamin E diet was considerably elevated, and therefore its absorption and incorporation into the liver was higher. Liver vitamin C concentration was significantly lower (p < .05) in rats fed the vitamin E–enriched diet (2.3 ± 0.6 nmol/mg protein) compared to the rats fed the control, strawberry, or spinach diets (3.6 ± 1.8, 2.9 ± 1, 3.1 ± 1.2 nmol/mg protein, respectively) (Fig. 3b).

Vitamins E and C in Heart
Levels of vitamin E in heart were significantly affected after 8 months of dietary intervention among the various diet groups (p < .0001). The concentration of {alpha}-tocopherol in the heart tissue of animals fed the vitamin E–enriched diet was 825 ± 456 pmol/mg protein, as compared to the animals fed the control diet, or the diets enriched with strawberry or spinach extract, which had significantly lower (p < .0001) concentrations of {alpha}-tocopherol (35 ± 49, 21 ± 18, and 26 ± 18 pmol/mg protein, respectively (Fig. 4). However, the concentration of {gamma}-tocopherol was significantly higher in the control, strawberry, and spinach groups than in the high–vitamin E diet (p < .05) (Fig. 4). Vitamin C concentration in the heart of the rat is normally less than 1 nmol/mg protein; this decreased even further to 0.16 ± 0.12 nmol/mg protein in animals receiving a high–vitamin E diet. Comparatively, the control and strawberry groups had concentrations of 0.3 ± 0.2 nmol/mg protein, and the spinach group had a concentration of 0.7 ± 0.4 nmol/mg protein.



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  		Figure 4. {alpha}-Tocopherol and {gamma}-tocopherol concentration (mean ± SD) in heart following long-term (8 months) dietary intervention with a control diet (modified AIN-93) or a diet containing extracts of either strawberries (9.5 g/kg diet), spinach (6.4 g/kg diet), or vitamin E (with 500-mg all-rac-{alpha}-tocopheryl acetate/kg diet). *Significantly higher than either control, strawberries, or spinach (p < .05). . Each point represents the mean ± SD.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
These data are in agreement with the scientific literature on the relationship between vegetables and fruit consumption and risk of degenerative disease, demonstrating that other nutrients in addition to the well-known antioxidants may play important roles in the CNS. In fact, some significant improvements in neurological markers were observed by our group in the strawberry, spinach, and high–vitamin E groups (29). Even though the total antioxidant capacity was equivalent in three of the diets (strawberry, spinach, or high–vitamin E), their capacity to delay the effect of aging on the sensitivity of muscarinic receptors, calcium flux, and ultimately cognitive behavior was different.

HPLC analysis of the spinach showed that, in addition to a large array of lipophilic compounds, including vitamin K, it contains 1.8 ± 0.3 mg {alpha}-tocopherol and 0.12 ± 0.6 mg {gamma}-tocopherol per 100 g wet weight. Comparatively, strawberry contains only 0.4 ± 0.2 mg {alpha}-tocopherol and 0.13 ± 0.8 mg {gamma}-tocopherol per 100 g wet weight. Additionally, compared to control and strawberry groups, the spinach group showed a slight but a significant increase in vitamin E concentration in the hippocampus and cortex. However, the concentration of vitamin E in the brain of the spinach-fed group was remarkably lower than those fed the high–vitamin E diet. Therefore, the observation made by our group that the spinach, high–vitamin E, and, in lesser degree, strawberry groups were able to reverse some of the neurological decline associated with aging (29) is important and clearly indicates in this model that other nutrients, in addition to vitamin E, may be important. Additionally, these findings suggest that oxidative stress may not be the main etiologic factor in the neurological decline associated with aging.

The observation that dopamine release induced by the diets containing strawberry, spinach, or vitamin E, was significantly enhanced compared to control diet is outstanding (29). These results are important because brain tissues from control and animals fed diets enriched with strawberry or spinach had very low vitamin E, compared to the high–vitamin E group, suggesting that other nutrients may be important for maintaining brain's function. We have previously demonstrated that the ability of brain tissue to uptake vitamin E reaches a maximum when the intake of vitamin E is about 80 mg vitamin E/kg diet (Martin and colleagues, Brain Res, in press). Following supplementation with 500 mg all-rac-{alpha}-tocopheryl acetate/kg diet for 8 months, the brain's vitamin E concentration was equivalent to the concentration achieved when the rats are fed a 80 mg/kg diet for 2 months (2.4 mg/kg body weight per day). This finding indicates that the amount of vitamin E that the brain regions can incorporate following long dietary supplementation is limited, even when the intake of this nutrient is high, and the time of supplementation prolonged (35)(36). Curiously, in humans, supplementation with 150–200 mg vitamin E/day (equivalent to 2–3 mg/kg body weight/day) is sufficient to increase maximally the {alpha}-tocopherol effect on immune response (13)(37) and decrease the risk of cardiovascular diseases (38).

On the other hand, high–vitamin E intakes for long periods of time may alter tissue function as the animals fed with high levels of vitamin E showed a decreased concentration of C in liver, and diminished concentration in plasma and heart in a parallel fashion. Recent studies showed that antioxidants such as vitamin E can modulate gene expression (39), raising the possibility that vitamin E may be interacting with cells' regulatory signal(s). It is possible that high amounts of {alpha}-tocopherol in liver may be downregulating the hepatic expression of L-gulonolactone oxidase, the enzyme that, in mammals such as rats, regulates the synthesis of vitamin C by the liver (25)(40)(41). It is possible that high vitamin E may be acting as a pro-oxidant and thus consumes some of the C. However, we think that this possibility is remote because the vitamin E–fed group showed an enhancement in the neurological functions examined (33). However, this finding raises the possibility of using this model to further investigate the potential toxic effects induced by high–vitamin E intakes.

The range of {alpha}-tocopherol concentrations selected in this study to feed the rats has been shown to significantly reduce and increase the levels of {alpha}-tocopherol in plasma and tissues (21)(22)(42). Tissues such as plasma and liver incorporate {alpha}-tocopherol almost in a dose-response manner, reaching a vitamin E concentration that is proportional to its concentration in food. In agreement with previous studies, our results also demonstrate that liver and plasma accumulate large amounts of vitamin E when it is present in the diet in high concentrations. However, we observed that after feeding rats with 500 mg all-rac-{alpha}-tocopheryl acetate/kg diet for 8 months, the brain was only able to accumulate limited amounts of vitamin E, similar to those that can be attained with a diet containing 80 mg of vitamin E/kg diet. Other studies have also observed differences in the amount of vitamin E incorporated into the brain when the diets contained 100 mg/kg diet versus 20 mg/kg. In these studies no further differences were observed when the vitamin E intake was above100 mg/kg diet (43). However, in studies using diets enriched with large concentrations of {alpha}-tocopherol for several weeks, {alpha}-tocopherol concentration in liver and plasma generously augmented (43)(44).

Given the prominent role that {alpha}-tocopherol seems to play in brain function (17)(45), our findings that different regions of the brain accumulate different concentrations of {alpha}-tocopherol and that the central nervous system does not accumulate {alpha}-tocopherol above a certain concentration in spite of increasing levels in plasma are remarkable. These results also indicate that an optimum intake of this nutrient on a daily basis may be crucial to maintain maximum levels of vitamin E in different regions of the brain. In addition, we found that the striatum contains the lowest amount of vitamin E. Perhaps this region is particularly active in the metabolism of vitamin E and consequently may be more sensitive to oxidative damage, and thus to vitamin E changes than other brain areas. This hypothesis appears to be well supported in general. Some authors have not been able to detect any distribution pattern of vitamin E in the brain (21); however, other studies are in agreement with our results and have also shown differences in its distribution among some of the brain regions (46)(47).

Vitamin E is generally considered to be a relatively nontoxic nutrient in adults, and it has been used for therapeutic and prophylactic purposes in a variety of disorders. However, there have been warnings about the toxic effect of megadosages of this nutrient because it is a fat-soluble vitamin and may be difficult to eliminate from the body, thus leading to various undesirable effects (48)(49). Because some human studies use high concentrations (2000 IU vitamin E/day), it is important to consider possible functional interactions induced by an excess of {alpha}-tocopherol deliberated to some tissues without augmenting the benefit(s) accomplished by using a smaller supplement.

Fruits and vegetables are rich in antioxidant nutrients including vitamin C and flavonoids, which appear to protect against chronic disease (50). Interestingly, the concentration of vitamin C in the extracelluar compartment of the rat brain cerebral cortex, corpus striatum and hippocampus is estimated to vary between 100 and 500 µM, representing a balance between uptake and secretion (51)(52)(53)(54). A gradient of vitamin C has been demonstrated within some brain tissues such as the thalamus, suggesting that there may be an association between the distribution of vitamin C in brain tissue and the distribution of catecholamines and peptide neurotransmitters. On the other hand, there is a direct demonstration of the formation of metabolites derived from the metabolism of dopamine, such as quinones in the central nervous system, where they will remain in this form (55). Quinones are very reactive with protein thiol groups; thus, they may conjugate with these moieties on the receptor protein via a nucleophilic reaction (55). The presence of powerful reducing systems in vivo, such as high concentrations of vitamin C in the brain, and perhaps the presence of flavonoids, may well serve this purpose, preventing the irreversible binding of the reactive quinones to specific receptor proteins such as the dopamine D2 subclass. Therefore, the presence in brain of nutrients with powerful reducing capacities (such as vitamin C and flavonoids), or with the ability to maintain membrane function and integrity (such as vitamin E), may provide important clues to the etiology of different neurodegenerative processes.







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  		Figure 1. {alpha}-Tocopherol and {gamma}-tocopherol concentration (mean ± SD) following long-term (8 months) dietary intervention with a control diet (modified AIN-93) or a diet containing extracts of either strawberries (9.5 g/kg diet), spinach (6.4 g/kg diet), or vitamin E (with 500-mg all-rac-{alpha}-tocopheryl acetate/kg diet), in the cortex (A), hippocampus (B), cerebellum (C), in striatum (D). *Significantly higher than either control, strawberries, or spinach (p < .05); #significantly higher than either control or strawberries (p < .05). Comparison of the {alpha}-tocopherol concentration in the different regions of the brain in the vitamin E–supplemented group (E); *significantly lower than either cortex or hippocampus (p < .05) (n = 14–18, and spinach group, n = 4, per group). Each point represents the mean ± SD.

 



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  		Figure 2. {alpha}-Tocopherol and {gamma}-tocopherol concentration (mean ± SD) in plasma following long-term (8 months) dietary intervention with a control diet (modified AIN-93) or a diet containing extracts of either strawberries (9.5 g/kg diet), spinach (6.4 g/kg diet), or vitamin E (with 500 mg all-rac-{alpha}-tocopheryl acetate/kg diet) (A). *Significantly higher than either control, strawberries, or spinach (p < .05). (B) *The high vitamin E group had significantly lower (p < .05) plasma ascorbate concentration than the other groups (control, strawberries, or spinach). per group. Each point represents the mean ± SD.

 



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  		Figure 3. {alpha}-Tocopherol and {gamma}-tocopherol concentration (mean ± SD) in liver following long-term (8 months) dietary intervention with a control diet (modified AIN-93) or a diet containing extracts of either strawberries, spinach, or vitamin E (A). *Significantly higher {alpha}-tocopherol than either control, strawberries, or spinach (p < .01). #Significantly higher {gamma}-tocopherol than either control, strawberries, or spinach (p < .05). (B) *The high vitamin E group had significantly lower (p < .05) ascorbate concentration than the other groups (control, strawberries, or spinach). . Each point represents the mean ± SD.

 

   Acknowledgments
 
Mention of trade name, proprietary product, or specific equipment does not constitute a guarantee by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.

Received November 2, 1998

Accepted August 3, 1999


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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