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
(1,2). Little is known about the mechanisms behind these
neurological declines, but toxic species evolved from in-
flammatory processes and metabolism of cathechols may be
involved in its etiology (35). The recognition that oxida-
tive 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 prevent-
ing oxidative damage, and their application in the preven-
tion of neurodegenerative diseases. Moreover, the con-
sumption 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, carot-
enoids, some vitamin E, and other phytochemicals such as
flavonoids. These nutrients prevent or diminish degenera-
tive diseases associated with aging (4,79).
Vitamin E is an essential fat-soluble vitamin, which in-
cludes different naturally occurring isomers (, , ). Among
them, d--tocopherol has the highest biological activity and is
the most abundant form in food. This nutrient is the most ef-
fective chain-breaking lipid soluble antioxidant in the bio-
logical 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
13), and participation in biochemical processes involving
synthesis and distribution of neurotransmitters. Interestingly,
there is significant evidence showing neuropathological ab-
normalities during vitamin E deficiency, and that central ner-
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
(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). There-
fore, 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 -tocopherol
concentration in the central nervous system (CNS), brain en-
dogenous 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-
-hydroxylase, or mediating glutamate uptake, are well
documented (2325). 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

B144
 EFFECT OF DIET ON DISTRIBUTION OF VITAMINS E AND C IN BRAIN
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 objec-
tives 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 brains 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
Male Fischer 344 rats (Harlan Sprague Dawley, India-
napolis, 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--tocopheryl acetate/kg diet) on vitamins E and C tis-
sue 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 Labora-
tory Animals. The Animal Care and Use Committee of our
Center approved this study. Following a 12-day acclimati-
zation 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 min-
utes. The homogenate was then centrifuged at 13,000 g
for 15 minutes at 4C (to separate just the fiber). The super-
natant 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 antioxi-
dant level. Each of the strawberry and spinach diets pro-
vided 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 con-
tained 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, distribu-
B145
tion of vitamins E and C, and muscarinic receptor sensitiv-
ity (as assessed via oxotremorine enhancement of DA re-
lease from striatal slices); cerebellar GABAergic receptors
(as assessed via isoproterenol facilitation of GABA inhibi-
tion of cerebellar Purkinje cell firing), calcium release, and
behavior response were also evaluated (29).
Quantification of Vitamin E
Vitamin E (- and -tocopherol) content of plasma or tis-
sues was measured by reverse-phase high performance liq-
uid chromatography (HPLC). Briefly, 100 L of plasma
sample or 100 L of homogenized tissue were mixed with
one 100 L ethanol; after vortexing, tocopherols were ex-
tracted into 500 L 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 4C. The su-
pernatant was collected and dried under a stream of nitrogen
gas, and reconstituted in 100 L of methanol. Tocopherols
were separated by HPLC using a 3 m C18 reverse-phase
column (Perkin-Elmer, Norwalk, CT). The mobile phase,
delivered at a flow rate of 1.2 mL/min, consisted of 1% wa-
ter 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 am-
perometric electrochemical detector (Bioanalytical Sys-
tems, West Lafayette, IN). Tocopherols eluted as well-sepa-
rated peaks with a retention time between 2 to 6 minutes.
Peaks were integrated with a ChemStation (Hewlett Pack-
ard), -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
L 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 40100 mg of tissue homogenized in 500 L of
the same solution, and centrifuged to remove the precipi-
tated proteins. An aliquot of the supernatant was chromato-
graphed on a LC8 column (150 mm 4.6 mm, i.d., 3 m
particle size) (Supelco, Bellefonte, PA) using 99% deion-
ized water and 1% methanol containing 40 mmol/L sodium
acetate and 1.5 mmol/L dodecyltriethylammonium phos-
phate (Q12 ion pair cocktail, Regis, Morton Grove, IL) as
the mobile phase. Samples were injected with an autosam-
pler, 1100 series (Hewlett Packard). Ascorbate was detected
at an applied potential of 0.6 V by a LC 4B amperometric
electrochemical detector (Bioanalytical Systems). Ascor-
bate eluted as a single peak with a retention time of 5.5 min-
utes. 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).
B146 MARTIN ET AL.
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 M
oxotremorin was quantitated by HPLC coupled with elec-
tronic detection (33).
Statistic Analysis
Results were expressed as mean SD. - and -tocoph-
erol in the different brain regions, heart, and plasma; con-
centrations 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 di-
etary groups, because the SD is larger in the vitamin E-fed
group than the other groups.
RESULTS
Weights and Food Intakes
The rats significantly increased their weight from an av-
erage 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
-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, -tocopherol lev-
els in the highvitamin E group were 911 560 pmol/mg
protein ( p .0001) in cortex (Figure 1A); 132 85, 137
67, 222 164 versus 1077 511 pmol/mg protein respec-
tively ( p .0001) in the hippocampus (Figure 1B); 83
78, 55 33, 115 98 versus 424 262 pmol/mg protein
respectively ( p .001) in the cerebellum (Figure 1C); and
26 23, 14 7 versus 212 101 pmol/mg protein respec-
tively ( p .01) in the striatum (Figure 1D). Data from the
striatum region in the spinach group are absent because
samples were used for other assays. Thus, the different re-
gions of the brain accumulated distinct amounts of -tocoph-
erol following supplementation with vitamin E (Figure 1E).
The striatum and cerebellum were the regions that had sig-
nificantly lower concentrations compared to cortex or hip-
pocampus ( p .05). In addition to -tocopherol, other vita-
min E isomers such as -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 highvitamin 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, how-
ever, showed a trend towards lower concentrations of C
with an average of 13.5 4 nmol/mg protein, and no signif-
icant differences among groups (Table 1). As previously de-
scribed, brain tissues from animals fed strawberry, spinach,
and control diets very nicely reflected their lowvitamin E
intake, compared to highvitamin E treatment. Interestingly,
diets enriched with extracts of strawberry or spinach, or vi-
tamin E, showed enhanced dopamine release from striatal
slices following oxotremorine stimulation by 100%, 300%,
and 150%, respectively, compared to control (p .05).
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 in-
tervention the levels of -tocopherol in plasma were 2.2
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
the vitamin Eenriched diet had a plasma -tocopherol con-
centration of 73.4 23.8 M, which was significantly
higher (p .0001) than all other diets (Figure 2A). As com-
pared to the high vitamin Esupplemented diet group, the
ratio between -tocopherol and -tocopherol in plasma was
strikingly reduced in all the lowvitamin E dietfed ani-
mals. However, because these diets are an important source
of -tocopherol and rats absorb -tocopherol well (34) the
concentration of -tocopherol in plasma increased to similar
or higher concentrations than -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 M respectively. However, in the
group fed the vitamin Eenriched diet, plasma C levels de-
creased significantly to 9 3 (p .05) (Figure 2B).
Vitamins E and C in Liver
Animals fed the vitamin Eenriched diet showed a signif-
icant increase in the concentration of -tocopherol in liver
(4172 2700 pmol/mg protein) compared to control, con-
trol plus strawberry, and control plus spinach groups, which
had concentrations of 23 13, 23 13, 26 25 pmol/mg
protein respectively (p .0001) (Figure 3A). The ability of
the liver to uptake -tocopherol increases when the -tocoph-
erol present in the diet decreases. Thus, when a diet is low in
-tocopherol, the liver becomes more active in incorporat-
ing other tocopherols into lipoprotein fractions and releas-
ing them into the blood stream (35,36). This may explain
the relatively high concentrations of -tocopherol observed
Table 1. Ascorbate Concentration (nmol/mg protein)
in Different Regions of Brain
Group
Brain Region Control Strawberry Spinach High Vitamin E
Cortex 20.4 7.2 (14) 13.6 6.2 (18) 19.2 8.5 (14) 19.0 7.7 (16)
Cerebellum 16.5 5.7 (16) 19.4 5.2 (14) 21.3 6.3 (16) 14.2 5.1 (18)
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)
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

Figure 2. -Tocopherol and -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--tocopheryl acetate/kg diet) (A). Signifi-
cantly 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). n 1418 per group. Each point represents
the mean SD.
SD) in liver following long-term (8 months) dietary intervention with
a control diet (modified AIN-93) or a diet containing extracts of ei-
ther strawberries, spinach, or vitamin E (A). Significantly higher
-tocopherol than either control, strawberries, or spinach (p .01).
#Significantly higher -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 (con-
trol, strawberries, or spinach). n 1418 per group. Each point rep-
resents 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
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 -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).
However, the concentration of -tocopherol was signifi-
cantly higher in the control, strawberry, and spinach groups
than in the highvitamin E diet (p .05) (Figure 4). Vita-
min 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 highvitamin 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.
DISCUSSION
These data are in agreement with the scientific literature
on the relationship between vegetables and fruit consump-
tion 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 signifi-
cant improvements in neurological markers were observed
by our group in the strawberry, spinach, and highvitamin E
groups (29). Even though the total antioxidant capacity was
equivalent in three of the diets (strawberry, spinach, or
highvitamin 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 -tocopherol and 0.12 0.6 mg
-tocopherol per 100 g wet weight. Comparatively, straw-
berry contains only 0.4 0.2 mg -tocopherol and 0.13
0.8 mg -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 highvita-
min E diet. Therefore, the observation made by our group
that the spinach, highvitamin E, and, in lesser degree,
strawberry groups were able to reverse some of the neuro-
logical decline associated with aging (29) is important and
clearly indicates in this model that other nutrients, in addi-
tion 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 di-
ets containing strawberry, spinach, or vitamin E, was signif-
icantly 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 highvita-
min E group, suggesting that other nutrients may be impor-
tant for maintaining brains function. We have previously
demonstrated that the ability of brain tissue to uptake vita-
min 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
B149
mg all-rac--tocopheryl acetate/kg diet for 8 months, the
brains vitamin E concentration was equivalent to the con-
centration achieved when the rats are fed a 80 mg/kg diet
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 150200 mg vitamin E/day
(equivalent to 23 mg/kg body weight/day) is sufficient to
increase maximally the -tocopherol effect on immune re-
sponse (13,37) and decrease the risk of cardiovascular dis-
eases (38).
On the other hand, highvitamin E intakes for long peri-
ods 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 anti-
oxidants such as vitamin E can modulate gene expression
(39), raising the possibility that vitamin E may be interact-
ing with cells regulatory signal(s). It is possible that high
amounts of -tocopherol in liver may be downregulating
the hepatic expression of l-gulonolactone oxidase, the en-
zyme 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 con-
sumes some of the C. However, we think that this possibil-
ity is remote because the vitamin Efed 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 in-
duced by highvitamin E intakes.
The range of -tocopherol concentrations selected in this
study to feed the rats has been shown to significantly reduce
and increase the levels of -tocopherol in plasma and tis-
sues (21,22,42). Tissues such as plasma and liver incorpo-
rate -tocopherol almost in a dose-response manner, reach-
ing 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 accumu-
late 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--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 at-
tained 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 con-
tained 100 mg/kg diet versus 20 mg/kg. In these studies no
further differences were observed when the vitamin E in-
take was above100 mg/kg diet (43). However, in studies
using diets enriched with large concentrations of -toco-
pherol for several weeks, -tocopherol concentration in
liver and plasma generously augmented (43,44).
Given the prominent role that -tocopherol seems to play
in brain function (17,45), our findings that different regions
of the brain accumulate different concentrations of -tocoph-
erol and that the central nervous system does not accumu-
late -tocopherol above a certain concentration in spite of
increasing levels in plasma are remarkable. These results
B150 MARTIN ET AL.
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 vita-
min E. Perhaps this region is particularly active in the me-
tabolism of vitamin E and consequently may be more sensi-
tive 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 non-
toxic nutrient in adults, and it has been used for therapeutic
and prophylactic purposes in a variety of disorders. How-
ever, there have been warnings about the toxic effect of
megadosages of this nutrient because it is a fat-soluble vita-
min 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 vita-
min E/day), it is important to consider possible functional
interactions induced by an excess of -tocopherol deliber-
ated to some tissues without augmenting the benefit(s) ac-
complished by using a smaller supplement.
Fruits and vegetables are rich in antioxidant nutrients in-
cluding 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 esti-
mated to vary between 100 and 500 M, representing a bal-
ance between uptake and secretion (5154). A gradient of
vitamin C has been demonstrated within some brain tissues
such as the thalamus, suggesting that there may be an asso-
ciation between the distribution of vitamin C in brain tissue
and the distribution of catecholamines and peptide neu-
rotransmitters. On the other hand, there is a direct demon-
stration 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 pro-
tein via a nucleophilic reaction (55). The presence of power-
ful reducing systems in vivo, such as high concentrations of
vitamin C in the brain, and perhaps the presence of fla-
vonoids, may well serve this purpose, preventing the irre-
versible binding of the reactive quinones to specific recep-
tor 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.
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.
Address correspondence to Antonio Martin, MD, PhD, USDA-Neuro-
science Laboratory, Jean Mayer USDA Human Nutrition Research Center
on Aging at Tufts University, 711 Washington Street, Boston, MA 02111.
E-mail: amartin@hnrc.tufts.edu
References
1. Carlsson A. Neurotransmitter changes in the aging brain. Danish Med
Bulletin. 1985;32(Suppl):4043.
2. Roth G. Changes in tissue responsiveness to hormones and neurotrans-
mitters during aging. Exp Gerontol. 1995;30:361368.
3. Cohen G. The pathobiology of Parkinsons disease: biochemical as-
pects of dopamine neuron senescence. J Neural Transm Suppl. 1983;
19:89103.
4. Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants, and the
degenerative disease of aging. Proc Natl Acad Sci USA. 1993;90:
79157922.
5. Mazzoccoli G, Correra M, Bianco G, et al. Age-related changes of
neuro-endocrine-immune interactions in healthy humans. J Biol Regul
Homeostatic Agents. 1997;11:143147.
6. Potter JD, Steinmetz K. Vegetables, fruits and phytoestrogens as pre-
ventive agents. IARC Sci Publ. 1996;139:6190.
7. Steinmetz KA, Potter JD. Vegetables, fruits, and cancer prevention: a
review. J Am Diet Assoc. 1996;96:10271039.
8. Gey KF, Brubacher GB, Stahelin HB. Plasma levels of antioxidant vi-
tamins in relation to ischemic heart disease and cancer. Am J Clin
Nutr. 1987;45:13681377.
9. Gey KF, Puska P, Jordan P, Moser KU. Inverse correlation between
plasma vitamin E and mortality from ischemic heart disease in cross-
cultural epidemiology. Am J Clin Nutr. 1991;53:326S334S.
10. Niki E, Yamamoto Y, Takahashi M, Komuro E, Miyama Y. Inhibition
of oxidation of biomembranes by alpha-tocopherol. Ann NY Acad Sci.
1989;570:2331.
11. Azzi A, Boscobonik D, Hensey C. The protein kinase C family. Eur J
Biochem. 1992;208:547557.
12. Martin A, Foxal T, Blumberg JF, Meydani M. Vitamin E (-T) de-
creases intracellular adhesion molecule (ICAM-1) expression and
monocyte adhesion to human aortic endothelial cells. Arterioscl
Thromb Vasc Biol. 1997;17:429438.
13. Meydani S, Meydani M, Blumberg J, et al. Vitamin E supplementation
and in vivo immune response in healthy elderly subjects. A random-
ized controlled trial. JAMA. 1997;277:13801386.
14. Schocken DD, Roth GS. Reduced beta-adrenergic receptor concentra-
tions in ageing man. Nature. 1977;267:856858.
15. Misra CH, Shelat HS, Smith RC. Effect of age on adrenergic and
dopaminergic receptor binding in rat brain. Life Sci. 1980;27:521526.
16. Noda Y, McGeer PL, McGeer EG. Lipid peroxides in brain during ag-
ing and vitamin E deficiency: possible relations to changes in
neurotransmitter indices. Neurobiol Aging. 1982;3:173178.
17. Muller D, Lloyd J, Wolff O. Vitamin E and neurological function.
Lancet. 1983;i:225228.
18. Sano M, Ernesto C, Thomas R, et al. A controlled trial of selegiline, al-
pha-tocopherol, or both as treatment for Alzheimers disease. N Engl J
Med. 1997;336:12161222.
19. Joseph JA, Berger RE, Engel BT, Roth GS. Age-related changes in the
nigrostriatum: a behavioral and biochemical analysis. J Gerontol.
1978;33:643649.
20. Roth GS, Joseph JA. Cellular and molecular mechanisms of impaired
dopaminergic function during aging. Ann NY Acad Sci. 1994;719:129
135.
21. Vatassery GT, Angerhofer CK, Knox CA, Deshmukh DS. Concentra-
tion of vitamin E in various neuroanatomical regions and subcellular
fractions, and the uptake of vitamin E by specific areas, of rat brain.
Biochem Biophys Acta. 1984;792:118122.
22. Meydani M, Macauley J, Blumberg J. Influence of dietary vitamin E,
selenium and age on regional distribution of -tocopherol in the rat.
Lipids. 1986;21:786791.
23. Ghasemzadeh B, Cammack J, Adams RN. Dynamic changes in extra-
cellular fluid ascorbic acid monitored by in vivo electrochemistry.
Brain Res. 1991;547:162166.
24. Grunewald RA, Fillenz M. Release of ascorbate from a synaptosomal
fraction of rat brain. Neurochem Int. 1984;6:491500.
25. Padh H. Vitamin C: newer insights into its biochemical functions. Nutr
Rev. 1991;49:6570.
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
flavonoids on human lymphocyte proliferative responses. Prog Clin
Biol Res. 1986;213:511520.
27. Reeves P, Nielsen F, Fahey G. AIN-93 purified diets for laboratory ro-
dents: final report of the American Institute of Nutrition Ad Hoc
Committee on the reformulation of the AIN-76A rodent diet. J Nutr.
1993;123:19391951.
28. Cao G, Verdon CP, Wu AHB, Wang H, Prior RL. Automated assay of
oxygen radical absorbance capacity with the COBAS FARA II. Clin
Chem. 1995;41:17381744.
29. Joseph JA, Shukitt-Hale B, Denisova NA, et al. Long-term dietary
strawberry, spinach, or vitamin E supplementation retards the onset of
age-related neuronal signal-transduction and cognitive behavioral def-
icits. J Neurosci. 1998;18:80478055.
30. Lowry O, Rosebrough N, Farr A, Randall R. Protein measurement
with the Folin phenol reagent. J Biol Chem. 1951;193:265269.
31. Martin A, Frei B. Both intracellular and extracellular vitamin C inhibit
atherogenic modification of LDL by human vascular endothelial cells.
Arterioscler Thromb Vasc Biol. 1997;17:15831590.
32. Joseph JA, Dalton TK, Hunt WA. Age-related decrements in the mus-
carinic enhancement of K-evoked release of endogenous striatal
dopamine: an indicator of altered cholinergic-dopaminergic reciprocal
inhibitory control in senescence. Brain Res. 1988;454:140148.
33. Joseph JA, Dalton TK, Roth GS, Hunt WA. Alterations in muscarinic
control of striatal dopamine autoreceptors in senescence: a deficit at
the ligand-muscarinic receptor interface? Brain Res. 1988;454:149155.
34. Bieri JG, Evarts RP, Gart JJ. Relative activity of alpha-tocopherol and
gamma-tocopherol in preventing oxidative red cell hemolysis. J Nutr.
1976;106:124127.
35. Traber M, Kayden H. Preferential incorporation of -tocopherol vs
-tocopherol in human lipoproteins. Am J Clin Nutr. 1989;49:517526.
36. Traber MG, Rudel LL, Burton GW, Hughes L, Ingold KU, Kayden
HJ. Nascent VLDL from liver perfusions of cynomolgus monkeys are
preferentially enriched in RRR- compared with SRR--tocopherols:
studies using deuterated tocopherols. J Lipid Res. 1990;31:687694.
37. Meydani S, Barklund P, Liu S. Effect of vitamin E supplementation on
immune responsiveness of healthy elderly subjects. Am J Clin Nutr.
1990;52:557563.
38. Stampfer MJ, Hennekens CH, Manson JE, Colditz GA, Rosner B, Wil-
let WC. Vitamin E consumption and the risk of coronary disease in
women. N Engl J Med. 1993;328:14441449.
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
blood pressure and ascorbic acid metabolism in spontaneously hyper-
tensive rats (SH rats). Int J Vit Nutr Res. 1985;55:301317.
B151
41. Kratzing CC, Kelly JD. Ascorbic acid synthesis by the mammalian fe-
tus. Int J Vit Nutr Res. 1986;56:101113.
42. Vatassery G, Angerhofer C, Robertson R, Sabri M. Vitamin E concen-
trations in different regions of the spinal cord and sciatic nerve of the
rat. Neurochem Res. 1986;11:14191424.
43. Bendich A, Machlin LJ. The safety of oral intake of vitamin E: data
from clinical studies from 1986 to 1991. In: Packer L, Fuchs J, eds. Vita-
min E in Health and Disease. New York: Marcel Dekker; 1993:411416.
44. Vatassery GT, Brin MF, Fahn S, Kayden HJ, Traber MG. Effect of
high doses of dietary vitamin E on the concentrations of vitamin E in
several brain regions, plasma, liver, and adipose tissue of rats. J Neu-
rochem. 1988;51:621623.
45. Muller D, Gross-Sampson M, Burton G, Ingold K. Turnover of vitamin E
in neural and other tissues. Free Radic Res Commun. 1992;16:1015.
46. Clement M, Bourre J-M. Graded dietary levels of RRR--tocopherol
induce a marked increase in the concentrations of - and -tocopherol
in nervous tissues, heart, liver and muscle of vitamin-E-deficient rats.
Biochem Biophys Acta. 1984;1334:173181.
47. Meydani M, Macauley JB, Blumberg JB. Effect of dietary vitamin E
and selenium on susceptibility of brain regions to lipid peroxidation.
Lipids. 1988;23:405409.
48. Hillman R. Tocopherol excess in man: creatinuria associated with pro-
longed ingestion. Am J Clin Nutr. 1957;5:597600.
49. Briggs M. Vitamin E supplementation and fatigue. N Engl J Med.
1974;290:579580.
50. Block G, Patterson B, Subar A. Fruits, vegetables, and cancer prevention:
a review of the epidemiological evidence. Nutr Cancer. 1992;18:129.
51. Gonon F, Buda M, Cespuglio R, Jouvet M, Pujol J. Voltammetry in
the striatum of chronic freely moving rats: detection of catechols and
ascorbic acid. Brain Res. 1981;207:239244.
52. ONeill R, Grunewald R, Fillenz M, Albery W. Linear sweep voltam-
metry with carbo paste electrodes in the rat striatum. Neurosci. 1982;7:
19451954.
53. Schenk J, Miller E, Gaddis R, Adams R. Homeostatic control of ascor-
bate concentration in the CNS extracellular fluid. Brain Res. 1982;
253:353356.
54. Grunewald RA. Ascorbic acid in the brain. Brain Res Rev. 1993;18:
123133.
55. Fornstedt B, Pileblad E, Carlsson A. In vivo autoxidation of dopamine in
guinea pig striatum increases with age. J Neurochem. 1990;55:655658.
Received November 2, 1998
Accepted August 3, 1999
Decision Editor: Jay Roberts, PhD