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Journal of Gerontology: BIOLOGICAL SCIENCES Copyright 2000 by The Gerontological Society of America

2000, Vol. 55A, No. 3, B144B151

Effect of Fruits, Vegetables, or Vitamin ERich Diet


on Vitamins E and C Distribution in Peripheral and
Brain Tissues: Implications for Brain Function
Antonio Martin, Ronald Prior, Barbara Shukitt-Hale, Guohua Cao, and James A. Joseph

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

Age-related neurodegenerative conditions are the principal cause of declining cognitive and motor function dur-
ing 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 re-
lease of Fischer 344 rat model, over an 8-month period. The lowvitamin E diet resulted in lowered -tocopherol
levels in brain and peripheral tissues, whereas the animals that received a diet enriched in vitamin E showed a
significant increase, between 500900%. Vitamin C concentration in plasma, heart, and liver was reduced in the
vitamin Esupplemented group. It is concluded that supplementation or depletion of -tocopherol for 8 months
results in marked changes in vitamin E levels in brain tissue and peripheral tissues, and varied distribution of
-tocopherol throughout the different brain regions examined. In addition, compared to control group, rats sup-
plemented 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 antiox-
idants, may be important for brain function.

A GE-RELATED neurological deterioration is accompa-


nied by a significant decrease of transmitter levels as
well as activity of neurotransmitter-synthesizing enzymes
vous system requirements may change during aging (1416).
In addition, several studies have shown evidence indicating the
importance of vitamin E for normal neurological function
(1,2). Little is known about the mechanisms behind these (17,18). During aging a decline in both cognitive and motor
neurological declines, but toxic species evolved from in- functions has been observed, but the mechanisms involved
flammatory processes and metabolism of cathechols may be in these declines are not well understood. Changes related to
involved in its etiology (35). The recognition that oxida- the alteration in neurotransmitter receptor sensitivity, such as
tive stress may be an important etiological factor in the dopaminergic receptors, have been observed, as have changes
pathogenesis of various degenerative diseases has sparked related to a reduction of dopamine (DA) release (19,20). There-
interest in the role(s) that antioxidants may play in prevent- fore, in an effort to understand the basis of neuropathology
ing oxidative damage, and their application in the preven- of vitamin E, diverse studies have examined the pattern of
tion of neurodegenerative diseases. Moreover, the con- vitamin E distribution in different regions of the brain (21,22).
sumption of fruits and vegetables has been associated with a Interestingly, although there is unanimous accord on the
decreased risk of different chronic pathologies (6). Fruits positive effect of dietary vitamin E intake on -tocopherol
and vegetables contain large amounts of vitamin C, carot- concentration in the central nervous system (CNS), brain en-
enoids, some vitamin E, and other phytochemicals such as dogenous levels of lipid peroxidation have not been found to
flavonoids. These nutrients prevent or diminish degenera- reflect levels of vitamin E intake (22).
tive diseases associated with aging (4,79). Ascorbate (reduced vitamin C) is another important body
Vitamin E is an essential fat-soluble vitamin, which in- nutrient, stored in high concentrations (millimolar) in the
cludes different naturally occurring isomers (, , ). Among adrenal gland and the brain (23). However, in spite of the
them, d--tocopherol has the highest biological activity and is fact that this nutrient is widely distributed in the brain, only
the most abundant form in food. This nutrient is the most ef- a few of its roles, such as acting as a cofactor for dopamine-
fective chain-breaking lipid soluble antioxidant in the bio- -hydroxylase, or mediating glutamate uptake, are well
logical membrane, where it prevents the propagation of free- documented (2325). In addition, vitamin C exerts a large
radical damage, and contributes to membrane stability (10). scope of important antioxidant activities due to its ability to
Recent evidence indicates, however, that vitamin E may have react with numerous aqueous free radicals and oxygen species.
structure-specific roles in addition to its antioxidant function, Flavonoids are a family of biochemical compounds found
through its modulation of signal transduction pathways (11 in fruits and vegetables and consumed in the human diet.
13), and participation in biochemical processes involving These nutrients retain a high reductant capacity, have anti-
synthesis and distribution of neurotransmitters. Interestingly, inflammatory and immunomodulatory activities, and reduce
there is significant evidence showing neuropathological ab- histamine release from mast cells (26). The documented
normalities during vitamin E deficiency, and that central ner- role of these nutrients raises new questions and represents

B144
EFFECT OF DIET ON DISTRIBUTION OF VITAMINS E AND C IN BRAIN B145

new and exciting areas for research in nutrition-related cell tion of vitamins E and C, and muscarinic receptor sensitiv-
regulation, with important physiological implications. ity (as assessed via oxotremorine enhancement of DA re-
We were interested in examining the effect of low and lease from striatal slices); cerebellar GABAergic receptors
high vitamin E intakes on the distribution of vitamins E and (as assessed via isoproterenol facilitation of GABA inhibi-
C in brain regions after long dietary intervention. Our objec- tion of cerebellar Purkinje cell firing), calcium release, and
tives were: (i) to analyze the long-term (8 months) effect of behavior response were also evaluated (29).
low vitamin E intake on vitamin E levels in brain and other
tissues; (ii) to examine the brains vitamin E distribution
Quantification of Vitamin E
following dietary vitamin E treatment; (iii) to determine if
Vitamin E (- and -tocopherol) content of plasma or tis-
low vitamin E intake could affect vitamin C synthesis to
compensate for the vitamin E deficit; and (iv) to determine sues was measured by reverse-phase high performance liq-
if a high intake of fruits and vegetables may affect brain uid chromatography (HPLC). Briefly, 100 L of plasma
sample or 100 L of homogenized tissue were mixed with
function and attenuate the deleterious effects associated
one 100 L ethanol; after vortexing, tocopherols were ex-
with aging without affecting the concentrations of vitamins
tracted into 500 L hexane containing 0.002% butylated
E and C.
hydroxyl toluene (BHT) (Sigma Chemical Co., St Louis,
MO). Tocol (a gift from Hoffmann-La Roche, Nutley, NJ)
MATERIALS AND METHODS was added to the mixture as an internal standard. Samples
Male Fischer 344 rats (Harlan Sprague Dawley, India- were centrifuged at 800 rpm for 5 minutes at 4C. The su-
napolis, IN) were used to investigate the long-term (8 pernatant was collected and dried under a stream of nitrogen
months) effect of a control diet (modified AIN-93) (27) or a gas, and reconstituted in 100 L of methanol. Tocopherols
diet containing extracts of either strawberries (9.5 g/kg were separated by HPLC using a 3 m C18 reverse-phase
diet), spinach (6.4 g/kg diet), or vitamin E (with 500 mg all- column (Perkin-Elmer, Norwalk, CT). The mobile phase,
rac--tocopheryl acetate/kg diet) on vitamins E and C tis- delivered at a flow rate of 1.2 mL/min, consisted of 1% wa-
sue distribution. Eighty animals were used in these studies. ter in methanol, containing 20 mM lithium perchlorate.
The rats were individually housed in stainless steel mesh Samples were injected with an autosampler (1100 series,
suspended cages, provided food and water ad libitum and Hewlett Packard Co, Wilmington, DE). Eluted peaks were
maintained on a 12-hour light/dark cycle. All animals were detected at an applied potential of 0.6 V by a LC 4B am-
observed daily for clinical signs of disease. These animals perometric electrochemical detector (Bioanalytical Sys-
were utilized in compliance with all applicable laws and tems, West Lafayette, IN). Tocopherols eluted as well-sepa-
regulations as well as principles expressed in the National rated peaks with a retention time between 2 to 6 minutes.
Institutes of Health Guide for the Care and Use of Labora- Peaks were integrated with a ChemStation (Hewlett Pack-
tory Animals. The Animal Care and Use Committee of our ard), -tocopherol concentration was expressed in pmol/mg
Center approved this study. Following a 12-day acclimati- protein (12). Protein was measured by the method of Lowry
zation period to the facility, the 6-month-old rats were and colleagues (30).
weight-matched and given 2 weeks on the control diet. They
were then divided into four diet groups (20 animals/group). Quantification of Ascorbate
Monthly weights and food intakes over a 48-hour period Ascorbate was analyzed by paired-ion, reversed-phase
were recorded for all diet groups. HPLC coupled with electrochemical detection. In brief, 100
The strawberry and spinach extracts were prepared by L of plasma sample was mixed with an equal volume of
adding 400 g of the strawberry or spinach foods to water in cold 5% (w/v) metaphosphoric acid containing 1 mmol/L of
the ratio of 2:1, and homogenized in a blender for 2 min- the metal ion chelator diethylenetriaminepentaacetic acid
utes. The homogenate was then centrifuged at 13,000 g (Sigma), or 40100 mg of tissue homogenized in 500 L of
for 15 minutes at 4C (to separate just the fiber). The super- the same solution, and centrifuged to remove the precipi-
natant was recovered and combined in freezer bags, 500 mL tated proteins. An aliquot of the supernatant was chromato-
per bag. Frozen extracts were placed in a freeze drier until graphed on a LC8 column (150 mm 4.6 mm, i.d., 3 m
dry, which usually required about 7 days. The freeze-dried particle size) (Supelco, Bellefonte, PA) using 99% deion-
extracts were then shipped to Research Diets Inc. (New ized water and 1% methanol containing 40 mmol/L sodium
Brunswick, NJ) where they were mixed with the control acetate and 1.5 mmol/L dodecyltriethylammonium phos-
diet. The amounts of strawberry or spinach extracts added phate (Q12 ion pair cocktail, Regis, Morton Grove, IL) as
into the diets were equivalent in terms of their total antioxi- the mobile phase. Samples were injected with an autosam-
dant level. Each of the strawberry and spinach diets pro- pler, 1100 series (Hewlett Packard). Ascorbate was detected
vided about 1.4 mmol Trolox equivalent and the vitamin E at an applied potential of 0.6 V by a LC 4B amperometric
diet 1.2 mmol Trolox equivalent per kg diet, based on the electrochemical detector (Bioanalytical Systems). Ascor-
ORAC assay (28); therefore, the supplemented groups con- bate eluted as a single peak with a retention time of 5.5 min-
tained 1.4 or 1.2 mmol Trolox equivalent per kg diet more utes. Peaks were integrated with a ChemStation (Hewlett
than the control group. At 15 months of age, the various diet Packard). Ascorbate concentration was calculated based on
groups were examined for differences in various indexes to a calibration curve, and its concentration was expressed in
indicate different responses induced by the different dietary nmol/mg protein (31). Protein was measured by the method
treatments. These include indices for body weight, distribu- of Lowry and colleagues (30).
B146 MARTIN ET AL.

Dopamine Release cerebellum were not significantly different, with an average


Measurement of DA release was conducted as previously of 20 7 nmol/mg protein (Table 1). The striatum, how-
described by Joseph and colleagues (32,33). DA release ever, showed a trend towards lower concentrations of C
from striatal slices obtained from the animals maintained on with an average of 13.5 4 nmol/mg protein, and no signif-
the different diets following stimulation with 0 or 500 M icant differences among groups (Table 1). As previously de-
oxotremorin was quantitated by HPLC coupled with elec- scribed, brain tissues from animals fed strawberry, spinach,
tronic detection (33). and control diets very nicely reflected their lowvitamin E
intake, compared to highvitamin E treatment. Interestingly,
Statistic Analysis diets enriched with extracts of strawberry or spinach, or vi-
Results were expressed as mean SD. - and -tocoph- tamin E, showed enhanced dopamine release from striatal
erol in the different brain regions, heart, and plasma; con- slices following oxotremorine stimulation by 100%, 300%,
centrations of vitamin C in the different brain regions, heart, and 150%, respectively, compared to control (p .05).
and plasma; and the releases of dopamine were analyzed by
ANOVA. The Tukey test was used for multiple comparison Vitamins E and C in Plasma
post hoc analyses. We applied the Kruskal-Wallis ANOVA Levels of vitamin E and C in plasma were significantly
with Bonferroni-corrected Mann-Whitney to compare the affected after 8 months of dietary intervention among the
concentration of vitamin E in the liver in the different di- various diet groups (p .001). After 8 months of dietary in-
etary groups, because the SD is larger in the vitamin E-fed tervention the levels of -tocopherol in plasma were 2.2
group than the other groups. 1.5, 2.5 2.6, 1.9 0.4 M, respectively, for control, con-
trol plus strawberry, and control plus spinach diets. Rats fed
RESULTS the vitamin Eenriched diet had a plasma -tocopherol con-
centration of 73.4 23.8 M, which was significantly
Weights and Food Intakes higher (p .0001) than all other diets (Figure 2A). As com-
The rats significantly increased their weight from an av- pared to the high vitamin Esupplemented diet group, the
erage of 356.0 0.4 g (6 months) to 473.4 3.4 g (15 ratio between -tocopherol and -tocopherol in plasma was
months) ( p .001). However, there were no differences in strikingly reduced in all the lowvitamin E dietfed ani-
weights between the different dietary treatments over time mals. However, because these diets are an important source
or at the age of 15 months. There were also no differences in of -tocopherol and rats absorb -tocopherol well (34) the
food intakes between the dietary treatments over the course concentration of -tocopherol in plasma increased to similar
of the study, as previously reported (29). or higher concentrations than -tocopherol.
Plasma vitamin C concentrations were similar in rats fed
Vitamins E and C in Brain (Cortex, Hippocampus, the control, strawberry, or spinach diets with values of 18.6
Cerebellum, and Striatum), Functional Implications 6, 20.1 8, 21.3 7 M respectively. However, in the
Levels of vitamin E in brain were significantly affected group fed the vitamin Eenriched diet, plasma C levels de-
after 8 months of dietary intervention among the various creased significantly to 9 3 (p .05) (Figure 2B).
diet groups ( p .001). In this regard, concentrations of
-tocopherol in the animals fed with a control, strawberry, Vitamins E and C in Liver
or spinach diet were 100 81, 154 105, and 123 56 Animals fed the vitamin Eenriched diet showed a signif-
pmol/mg protein respectively. In contrast, -tocopherol lev- icant increase in the concentration of -tocopherol in liver
els in the highvitamin E group were 911 560 pmol/mg (4172 2700 pmol/mg protein) compared to control, con-
protein ( p .0001) in cortex (Figure 1A); 132 85, 137 trol plus strawberry, and control plus spinach groups, which
67, 222 164 versus 1077 511 pmol/mg protein respec- had concentrations of 23 13, 23 13, 26 25 pmol/mg
tively ( p .0001) in the hippocampus (Figure 1B); 83 protein respectively (p .0001) (Figure 3A). The ability of
78, 55 33, 115 98 versus 424 262 pmol/mg protein the liver to uptake -tocopherol increases when the -tocoph-
respectively ( p .001) in the cerebellum (Figure 1C); and erol present in the diet decreases. Thus, when a diet is low in
26 23, 14 7 versus 212 101 pmol/mg protein respec- -tocopherol, the liver becomes more active in incorporat-
tively ( p .01) in the striatum (Figure 1D). Data from the ing other tocopherols into lipoprotein fractions and releas-
striatum region in the spinach group are absent because ing them into the blood stream (35,36). This may explain
samples were used for other assays. Thus, the different re- the relatively high concentrations of -tocopherol observed
gions of the brain accumulated distinct amounts of -tocoph-
erol following supplementation with vitamin E (Figure 1E). Table 1. Ascorbate Concentration (nmol/mg protein)
The striatum and cerebellum were the regions that had sig- in Different Regions of Brain
nificantly lower concentrations compared to cortex or hip-
pocampus ( p .05). In addition to -tocopherol, other vita- Group

min E isomers such as -tocopherol were also present in Brain Region Control Strawberry Spinach High Vitamin E
brain in significant concentrations, with an average across all Cortex 20.4 7.2 (14) 13.6 6.2 (18) 19.2 8.5 (14) 19.0 7.7 (16)
groups of about 30 18 pmol/mg protein. No differences Cerebellum 16.5 5.7 (16) 19.4 5.2 (14) 21.3 6.3 (16) 14.2 5.1 (18)
were observed among groups, although the highvitamin E Hippocampus 20.5 6.3 (14) 19.5 5.5 (18) 21.7 5.5 (16) 19.9 8.7 (18)
Striatum 14.0 5.4 (4) 13.7 4.8 (4) 12.8 2.0 (4)
diet tended to have lower concentration of this vitamin E
isomer. Ascorbate concentrations in cortex, hippocampus, and Note: Each value represents mean SD. The number of animals tested are in parentheses.
EFFECT OF DIET ON DISTRIBUTION OF VITAMINS E AND C IN BRAIN B147

Figure 1. -Tocopherol and -tocopherol concentration (mean


SD) following long-term (8 months) dietary intervention with a con-
trol 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--tocopheryl acetate/kg diet), in the cortex (A), hip-
pocampus (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 -tocopherol concentration in the different regions of the brain in
the vitamin Esupplemented group (E); *significantly lower than ei-
ther cortex or hippocampus (p .05) (n 1418, and spinach group,
n 4, per group). Each point represents the mean SD.
B148 MARTIN ET AL.

Figure 3. -Tocopherol and -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 ei-
Figure 2. -Tocopherol and -tocopherol concentration (mean ther strawberries, spinach, or vitamin E (A). Significantly higher
SD) in plasma following long-term (8 months) dietary intervention -tocopherol than either control, strawberries, or spinach (p .01).
with a control diet (modified AIN-93) or a diet containing extracts of #Significantly higher -tocopherol than either control, strawberries,
either strawberries (9.5 g/kg diet), spinach (6.4 g/kg diet), or vitamin or spinach (p .05). (B) *The high vitamin E group had significantly
E (with 500 mg all-rac--tocopheryl acetate/kg diet) (A). Signifi- lower (p .05) ascorbate concentration than the other groups (con-
cantly higher than either control, strawberries, or spinach (p .05). trol, strawberries, or spinach). n 1418 per group. Each point rep-
(B) *The high vitamin E group had significantly lower (p .05) resents the mean SD.
plasma ascorbate concentration than the other groups (control,
strawberries, or spinach). n 1418 per group. Each point represents
the mean SD.

in plasma of rats fed a lowvitamin E diet. However, the


levels of -tocopherol observed in liver were significantly
higher in the vitamin Eenriched diet as compared to the
other groups (p .05) (Figure 3A). Perhaps the amount of
-tocopherol present in the highvitamin E diet was consid-
erably elevated, and therefore its absorption and incorpora-
tion into the liver was higher. Liver vitamin C concentration
was significantly lower ( p .05) in rats fed the vitamin
Eenriched 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) (Figure 3B).
Figure 4. -Tocopherol and -tocopherol concentration (mean
Vitamins E and C in Heart SD) in heart following long-term (8 months) dietary intervention
Levels of vitamin E in heart were significantly affected 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
after 8 months of dietary intervention among the various E (with 500-mg all-rac--tocopheryl acetate/kg diet). Significantly
diet groups (p .0001). The concentration of -tocopherol higher than either control, strawberries, or spinach (p .05). n 14
in the heart tissue of animals fed the vitamin Eenriched 18 per group. Each point represents the mean SD.
EFFECT OF DIET ON DISTRIBUTION OF VITAMINS E AND C IN BRAIN B149

diet was 825 456 pmol/mg protein, as compared to the mg all-rac--tocopheryl acetate/kg diet for 8 months, the
animals fed the control diet, or the diets enriched with brains vitamin E concentration was equivalent to the con-
strawberry or spinach extract, which had significantly lower centration achieved when the rats are fed a 80 mg/kg diet
(p .0001) concentrations of -tocopherol (35 49, 21 for 2 months (2.4 mg/kg body weight per day). This finding
18, and 26 18 pmol/mg protein, respectively (Figure 4). indicates that the amount of vitamin E that the brain regions
However, the concentration of -tocopherol was signifi- can incorporate following long dietary supplementation is
cantly higher in the control, strawberry, and spinach groups limited, even when the intake of this nutrient is high, and
than in the highvitamin E diet (p .05) (Figure 4). Vita- the time of supplementation prolonged (35,36). Curiously,
min C concentration in the heart of the rat is normally less in humans, supplementation with 150200 mg vitamin E/day
than 1 nmol/mg protein; this decreased even further to 0.16 (equivalent to 23 mg/kg body weight/day) is sufficient to
0.12 nmol/mg protein in animals receiving a highvitamin E increase maximally the -tocopherol effect on immune re-
diet. Comparatively, the control and strawberry groups had sponse (13,37) and decrease the risk of cardiovascular dis-
concentrations of 0.3 0.2 nmol/mg protein, and the spinach eases (38).
group had a concentration of 0.7 0.4 nmol/mg protein. On the other hand, highvitamin E intakes for long peri-
ods of time may alter tissue function as the animals fed with
DISCUSSION high levels of vitamin E showed a decreased concentration
These data are in agreement with the scientific literature of C in liver, and diminished concentration in plasma and
on the relationship between vegetables and fruit consump- heart in a parallel fashion. Recent studies showed that anti-
tion and risk of degenerative disease, demonstrating that oxidants such as vitamin E can modulate gene expression
other nutrients in addition to the well-known antioxidants (39), raising the possibility that vitamin E may be interact-
may play important roles in the CNS. In fact, some signifi- ing with cells regulatory signal(s). It is possible that high
cant improvements in neurological markers were observed amounts of -tocopherol in liver may be downregulating
by our group in the strawberry, spinach, and highvitamin E the hepatic expression of l-gulonolactone oxidase, the en-
groups (29). Even though the total antioxidant capacity was zyme that, in mammals such as rats, regulates the synthesis
equivalent in three of the diets (strawberry, spinach, or of vitamin C by the liver (25,40,41). It is possible that high
highvitamin E), their capacity to delay the effect of aging vitamin E may be acting as a pro-oxidant and thus con-
on the sensitivity of muscarinic receptors, calcium flux, and sumes some of the C. However, we think that this possibil-
ultimately cognitive behavior was different. ity is remote because the vitamin Efed group showed an
HPLC analysis of the spinach showed that, in addition to enhancement in the neurological functions examined (33).
a large array of lipophilic compounds, including vitamin K, However, this finding raises the possibility of using this
it contains 1.8 0.3 mg -tocopherol and 0.12 0.6 mg model to further investigate the potential toxic effects in-
-tocopherol per 100 g wet weight. Comparatively, straw- duced by highvitamin E intakes.
berry contains only 0.4 0.2 mg -tocopherol and 0.13 The range of -tocopherol concentrations selected in this
0.8 mg -tocopherol per 100 g wet weight. Additionally, study to feed the rats has been shown to significantly reduce
compared to control and strawberry groups, the spinach and increase the levels of -tocopherol in plasma and tis-
group showed a slight but a significant increase in vitamin E sues (21,22,42). Tissues such as plasma and liver incorpo-
concentration in the hippocampus and cortex. However, the rate -tocopherol almost in a dose-response manner, reach-
concentration of vitamin E in the brain of the spinach-fed ing a vitamin E concentration that is proportional to its
group was remarkably lower than those fed the highvita- concentration in food. In agreement with previous studies,
min E diet. Therefore, the observation made by our group our results also demonstrate that liver and plasma accumu-
that the spinach, highvitamin E, and, in lesser degree, late large amounts of vitamin E when it is present in the diet
strawberry groups were able to reverse some of the neuro- in high concentrations. However, we observed that after
logical decline associated with aging (29) is important and feeding rats with 500 mg all-rac--tocopheryl acetate/kg
clearly indicates in this model that other nutrients, in addi- diet for 8 months, the brain was only able to accumulate
tion to vitamin E, may be important. Additionally, these limited amounts of vitamin E, similar to those that can be at-
findings suggest that oxidative stress may not be the main tained with a diet containing 80 mg of vitamin E/kg diet.
etiologic factor in the neurological decline associated with Other studies have also observed differences in the amount
aging. of vitamin E incorporated into the brain when the diets con-
The observation that dopamine release induced by the di- tained 100 mg/kg diet versus 20 mg/kg. In these studies no
ets containing strawberry, spinach, or vitamin E, was signif- further differences were observed when the vitamin E in-
icantly enhanced compared to control diet is outstanding take was above100 mg/kg diet (43). However, in studies
(29). These results are important because brain tissues from using diets enriched with large concentrations of -toco-
control and animals fed diets enriched with strawberry or pherol for several weeks, -tocopherol concentration in
spinach had very low vitamin E, compared to the highvita- liver and plasma generously augmented (43,44).
min E group, suggesting that other nutrients may be impor- Given the prominent role that -tocopherol seems to play
tant for maintaining brains function. We have previously in brain function (17,45), our findings that different regions
demonstrated that the ability of brain tissue to uptake vita- of the brain accumulate different concentrations of -tocoph-
min E reaches a maximum when the intake of vitamin E is erol and that the central nervous system does not accumu-
about 80 mg vitamin E/kg diet (Martin and colleagues, late -tocopherol above a certain concentration in spite of
Brain Res, in press). Following supplementation with 500 increasing levels in plasma are remarkable. These results
B150 MARTIN ET AL.

also indicate that an optimum intake of this nutrient on a on Aging at Tufts University, 711 Washington Street, Boston, MA 02111.
daily basis may be crucial to maintain maximum levels of E-mail: amartin@hnrc.tufts.edu
vitamin E in different regions of the brain. In addition, we
found that the striatum contains the lowest amount of vita- References
min E. Perhaps this region is particularly active in the me- 1. Carlsson A. Neurotransmitter changes in the aging brain. Danish Med
tabolism of vitamin E and consequently may be more sensi- Bulletin. 1985;32(Suppl):4043.
2. Roth G. Changes in tissue responsiveness to hormones and neurotrans-
tive to oxidative damage, and thus to vitamin E changes mitters during aging. Exp Gerontol. 1995;30:361368.
than other brain areas. This hypothesis appears to be well 3. Cohen G. The pathobiology of Parkinsons disease: biochemical as-
supported in general. Some authors have not been able to pects of dopamine neuron senescence. J Neural Transm Suppl. 1983;
detect any distribution pattern of vitamin E in the brain (21); 19:89103.
4. Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants, and the
however, other studies are in agreement with our results and degenerative disease of aging. Proc Natl Acad Sci USA. 1993;90:
have also shown differences in its distribution among some 79157922.
of the brain regions (46,47). 5. Mazzoccoli G, Correra M, Bianco G, et al. Age-related changes of
Vitamin E is generally considered to be a relatively non- neuro-endocrine-immune interactions in healthy humans. J Biol Regul
toxic nutrient in adults, and it has been used for therapeutic Homeostatic Agents. 1997;11:143147.
6. Potter JD, Steinmetz K. Vegetables, fruits and phytoestrogens as pre-
and prophylactic purposes in a variety of disorders. How- ventive agents. IARC Sci Publ. 1996;139:6190.
ever, there have been warnings about the toxic effect of 7. Steinmetz KA, Potter JD. Vegetables, fruits, and cancer prevention: a
megadosages of this nutrient because it is a fat-soluble vita- review. J Am Diet Assoc. 1996;96:10271039.
min and may be difficult to eliminate from the body, thus 8. Gey KF, Brubacher GB, Stahelin HB. Plasma levels of antioxidant vi-
tamins in relation to ischemic heart disease and cancer. Am J Clin
leading to various undesirable effects (48,49). Because Nutr. 1987;45:13681377.
some human studies use high concentrations (2000 IU vita- 9. Gey KF, Puska P, Jordan P, Moser KU. Inverse correlation between
min E/day), it is important to consider possible functional plasma vitamin E and mortality from ischemic heart disease in cross-
interactions induced by an excess of -tocopherol deliber- cultural epidemiology. Am J Clin Nutr. 1991;53:326S334S.
ated to some tissues without augmenting the benefit(s) ac- 10. Niki E, Yamamoto Y, Takahashi M, Komuro E, Miyama Y. Inhibition
of oxidation of biomembranes by alpha-tocopherol. Ann NY Acad Sci.
complished by using a smaller supplement. 1989;570:2331.
Fruits and vegetables are rich in antioxidant nutrients in- 11. Azzi A, Boscobonik D, Hensey C. The protein kinase C family. Eur J
cluding vitamin C and flavonoids, which appear to protect Biochem. 1992;208:547557.
against chronic disease (50). Interestingly, the concentration 12. Martin A, Foxal T, Blumberg JF, Meydani M. Vitamin E (-T) de-
creases intracellular adhesion molecule (ICAM-1) expression and
of vitamin C in the extracelluar compartment of the rat brain monocyte adhesion to human aortic endothelial cells. Arterioscl
cerebral cortex, corpus striatum and hippocampus is esti- Thromb Vasc Biol. 1997;17:429438.
mated to vary between 100 and 500 M, representing a bal- 13. Meydani S, Meydani M, Blumberg J, et al. Vitamin E supplementation
ance between uptake and secretion (5154). A gradient of and in vivo immune response in healthy elderly subjects. A random-
vitamin C has been demonstrated within some brain tissues ized controlled trial. JAMA. 1997;277:13801386.
14. Schocken DD, Roth GS. Reduced beta-adrenergic receptor concentra-
such as the thalamus, suggesting that there may be an asso- tions in ageing man. Nature. 1977;267:856858.
ciation between the distribution of vitamin C in brain tissue 15. Misra CH, Shelat HS, Smith RC. Effect of age on adrenergic and
and the distribution of catecholamines and peptide neu- dopaminergic receptor binding in rat brain. Life Sci. 1980;27:521526.
rotransmitters. On the other hand, there is a direct demon- 16. Noda Y, McGeer PL, McGeer EG. Lipid peroxides in brain during ag-
ing and vitamin E deficiency: possible relations to changes in
stration of the formation of metabolites derived from the neurotransmitter indices. Neurobiol Aging. 1982;3:173178.
metabolism of dopamine, such as quinones in the central 17. Muller D, Lloyd J, Wolff O. Vitamin E and neurological function.
nervous system, where they will remain in this form (55). Lancet. 1983;i:225228.
Quinones are very reactive with protein thiol groups; thus, 18. Sano M, Ernesto C, Thomas R, et al. A controlled trial of selegiline, al-
they may conjugate with these moieties on the receptor pro- pha-tocopherol, or both as treatment for Alzheimers disease. N Engl J
Med. 1997;336:12161222.
tein via a nucleophilic reaction (55). The presence of power- 19. Joseph JA, Berger RE, Engel BT, Roth GS. Age-related changes in the
ful reducing systems in vivo, such as high concentrations of nigrostriatum: a behavioral and biochemical analysis. J Gerontol.
vitamin C in the brain, and perhaps the presence of fla- 1978;33:643649.
vonoids, may well serve this purpose, preventing the irre- 20. Roth GS, Joseph JA. Cellular and molecular mechanisms of impaired
dopaminergic function during aging. Ann NY Acad Sci. 1994;719:129
versible binding of the reactive quinones to specific recep- 135.
tor proteins such as the dopamine D2 subclass. Therefore, 21. Vatassery GT, Angerhofer CK, Knox CA, Deshmukh DS. Concentra-
the presence in brain of nutrients with powerful reducing tion of vitamin E in various neuroanatomical regions and subcellular
capacities (such as vitamin C and flavonoids), or with the fractions, and the uptake of vitamin E by specific areas, of rat brain.
ability to maintain membrane function and integrity (such Biochem Biophys Acta. 1984;792:118122.
22. Meydani M, Macauley J, Blumberg J. Influence of dietary vitamin E,
as vitamin E), may provide important clues to the etiology selenium and age on regional distribution of -tocopherol in the rat.
of different neurodegenerative processes. Lipids. 1986;21:786791.
23. Ghasemzadeh B, Cammack J, Adams RN. Dynamic changes in extra-
Acknowledgments cellular fluid ascorbic acid monitored by in vivo electrochemistry.
Brain Res. 1991;547:162166.
Mention of trade name, proprietary product, or specific equipment does 24. Grunewald RA, Fillenz M. Release of ascorbate from a synaptosomal
not constitute a guarantee by the U.S. Department of Agriculture and does fraction of rat brain. Neurochem Int. 1984;6:491500.
not imply its approval to the exclusion of other products that may be suitable. 25. Padh H. Vitamin C: newer insights into its biochemical functions. Nutr
Address correspondence to Antonio Martin, MD, PhD, USDA-Neuro- Rev. 1991;49:6570.
science Laboratory, Jean Mayer USDA Human Nutrition Research Center 26. Mookerjee B, Lee T, Logue G, Lippes H, Middleton E. The effects of
EFFECT OF DIET ON DISTRIBUTION OF VITAMINS E AND C IN BRAIN B151

flavonoids on human lymphocyte proliferative responses. Prog Clin 41. Kratzing CC, Kelly JD. Ascorbic acid synthesis by the mammalian fe-
Biol Res. 1986;213:511520. tus. Int J Vit Nutr Res. 1986;56:101113.
27. Reeves P, Nielsen F, Fahey G. AIN-93 purified diets for laboratory ro- 42. Vatassery G, Angerhofer C, Robertson R, Sabri M. Vitamin E concen-
dents: final report of the American Institute of Nutrition Ad Hoc trations in different regions of the spinal cord and sciatic nerve of the
Committee on the reformulation of the AIN-76A rodent diet. J Nutr. rat. Neurochem Res. 1986;11:14191424.
1993;123:19391951. 43. Bendich A, Machlin LJ. The safety of oral intake of vitamin E: data
28. Cao G, Verdon CP, Wu AHB, Wang H, Prior RL. Automated assay of from clinical studies from 1986 to 1991. In: Packer L, Fuchs J, eds. Vita-
oxygen radical absorbance capacity with the COBAS FARA II. Clin min E in Health and Disease. New York: Marcel Dekker; 1993:411416.
Chem. 1995;41:17381744. 44. Vatassery GT, Brin MF, Fahn S, Kayden HJ, Traber MG. Effect of
29. Joseph JA, Shukitt-Hale B, Denisova NA, et al. Long-term dietary high doses of dietary vitamin E on the concentrations of vitamin E in
strawberry, spinach, or vitamin E supplementation retards the onset of several brain regions, plasma, liver, and adipose tissue of rats. J Neu-
age-related neuronal signal-transduction and cognitive behavioral def- rochem. 1988;51:621623.
icits. J Neurosci. 1998;18:80478055. 45. Muller D, Gross-Sampson M, Burton G, Ingold K. Turnover of vitamin E
30. Lowry O, Rosebrough N, Farr A, Randall R. Protein measurement in neural and other tissues. Free Radic Res Commun. 1992;16:1015.
with the Folin phenol reagent. J Biol Chem. 1951;193:265269. 46. Clement M, Bourre J-M. Graded dietary levels of RRR--tocopherol
31. Martin A, Frei B. Both intracellular and extracellular vitamin C inhibit induce a marked increase in the concentrations of - and -tocopherol
atherogenic modification of LDL by human vascular endothelial cells. in nervous tissues, heart, liver and muscle of vitamin-E-deficient rats.
Arterioscler Thromb Vasc Biol. 1997;17:15831590. Biochem Biophys Acta. 1984;1334:173181.
32. Joseph JA, Dalton TK, Hunt WA. Age-related decrements in the mus- 47. Meydani M, Macauley JB, Blumberg JB. Effect of dietary vitamin E
carinic enhancement of K-evoked release of endogenous striatal and selenium on susceptibility of brain regions to lipid peroxidation.
dopamine: an indicator of altered cholinergic-dopaminergic reciprocal Lipids. 1988;23:405409.
inhibitory control in senescence. Brain Res. 1988;454:140148. 48. Hillman R. Tocopherol excess in man: creatinuria associated with pro-
33. Joseph JA, Dalton TK, Roth GS, Hunt WA. Alterations in muscarinic longed ingestion. Am J Clin Nutr. 1957;5:597600.
control of striatal dopamine autoreceptors in senescence: a deficit at 49. Briggs M. Vitamin E supplementation and fatigue. N Engl J Med.
the ligand-muscarinic receptor interface? Brain Res. 1988;454:149155. 1974;290:579580.
34. Bieri JG, Evarts RP, Gart JJ. Relative activity of alpha-tocopherol and 50. Block G, Patterson B, Subar A. Fruits, vegetables, and cancer prevention:
gamma-tocopherol in preventing oxidative red cell hemolysis. J Nutr. a review of the epidemiological evidence. Nutr Cancer. 1992;18:129.
1976;106:124127. 51. Gonon F, Buda M, Cespuglio R, Jouvet M, Pujol J. Voltammetry in
35. Traber M, Kayden H. Preferential incorporation of -tocopherol vs the striatum of chronic freely moving rats: detection of catechols and
-tocopherol in human lipoproteins. Am J Clin Nutr. 1989;49:517526. ascorbic acid. Brain Res. 1981;207:239244.
36. Traber MG, Rudel LL, Burton GW, Hughes L, Ingold KU, Kayden 52. ONeill R, Grunewald R, Fillenz M, Albery W. Linear sweep voltam-
HJ. Nascent VLDL from liver perfusions of cynomolgus monkeys are metry with carbo paste electrodes in the rat striatum. Neurosci. 1982;7:
preferentially enriched in RRR- compared with SRR--tocopherols: 19451954.
studies using deuterated tocopherols. J Lipid Res. 1990;31:687694. 53. Schenk J, Miller E, Gaddis R, Adams R. Homeostatic control of ascor-
37. Meydani S, Barklund P, Liu S. Effect of vitamin E supplementation on bate concentration in the CNS extracellular fluid. Brain Res. 1982;
immune responsiveness of healthy elderly subjects. Am J Clin Nutr. 253:353356.
1990;52:557563. 54. Grunewald RA. Ascorbic acid in the brain. Brain Res Rev. 1993;18:
38. Stampfer MJ, Hennekens CH, Manson JE, Colditz GA, Rosner B, Wil- 123133.
let WC. Vitamin E consumption and the risk of coronary disease in 55. Fornstedt B, Pileblad E, Carlsson A. In vivo autoxidation of dopamine in
women. N Engl J Med. 1993;328:14441449. guinea pig striatum increases with age. J Neurochem. 1990;55:655658.
39. Houglum K, Brenner DA, Chojkier M. d--tocopherol inhibits -1(I)
gene expression in cultured human fibroblasts. J Clin Invest. 1991;87:
22302235.
40. Yoshioka M, Aoyama K, Matsushita T. Effects of ascorbic acid on Received November 2, 1998
blood pressure and ascorbic acid metabolism in spontaneously hyper- Accepted August 3, 1999
tensive rats (SH rats). Int J Vit Nutr Res. 1985;55:301317. Decision Editor: Jay Roberts, PhD