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Carotenoid, tocopherol, and retinol

concentrations in elderly human brain

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The Journal of Nutrition, Health & Aging
Volume , Number , 2004

THE JOURNAL OF NUTRITION, HEALTH & AGING

CAROTENOID, TOCOPHEROL, AND RETINOL CONCENTRATIONS

IN ELDERLY HUMAN BRAIN

N.E. CRAFT1, T.B. HAITEMA1, K.M. GARNETT2, K.A. FITCH3, C.K.DOREY4

1. Craft Technologies, Inc., 4344 Frank Price Church Road, Wilson, NC. 2. Applied Food Biotechnology, Inc., OFallon, MO; 3. Alzheimers Disease Research Center, Massachusetts
General Hospital, Boston, MA; 4. Dept. Biomedical Sciences, Florida Atlantic Univ. Boca Raton, FL Correspondence to: Neal E. Craft, Ph.D. Craft Technologies, Inc., 4344 Frank Price
Church Road, Wilson, NC 27893, Telephone: (252) 206-7071, FAX: (252) 206-1305, Email: ncraft@crafttechnologies.com

Abstract: Background: Antioxidants, such as tocopherols and carotenoids, have been implicated in the
prevention of degenerative diseases. Although correlations have been made between diseases and tissue levels of
antioxidants, to date there are no reports of individual carotenoid concentrations in human brain. Objective: To
measure the major carotenoids, tocopherols, and retinol in frontal and occipital regions of human brain. Design:
Ten samples of brain tissue from frontal lobe cortex and occipital cortex of five cadavers were examined.
Sections were dissected into gray and white matter, extracted with organic solvents, and analyzed by HPLC.
Results: At least 16 carotenoids, 3 tocopherols, and retinol were present in human brain. Major carotenoids were
identified as lutein, zeaxanthin, anhydrolutein, - cryptoxanthin, - cryptoxanthin, -carotene, cis- and trans--
carotene, and cis- and trans-lycopene. Xanthophylls (oxygenated carotenoids) accounted for 66-77% of total
carotenoids in all brain regions examined. Similar to neural retina, the ratio of zeaxanthin to lutein was high and
these two xanthophylls were significantly correlated (p<0.0001). The tocopherol isomers occurred in the brain
over a wider range of mean concentrations (0.11-17.9 nmol/g) than either retinol (87.8 - 163.3 pmol/g) or the
identified carotenoids (1.8-23.0 pmol/g). Conclusions: The frontal cortex, generally vulnerable in Alzheimers
disease, had higher concentrations of all analytes than the occipital cortex which is generally unaffected.
Moreover, frontal lobes, but not occipital lobes, exhibited an age-related decline in retinol, total tocopherols, total
xanthophylls and total carotenoids. The importance of these differences and the role(s) of these antioxidants in
the brain remain to be determined.

Key words: Antioxidant, human brain, carotenoid, tocopherol, retinol, xanthophyll, Alzheimers disease, vitamin
A, vitamin E, tissue, carotene

Introduction particularly in smokers (13). A case-control study by Zaman et

Common fruits and vegetables in the human diet contain 20-
30 carotenoids which are absorbed into the plasma.
Carotenoids and tocopherols are of particular interest to
medical researchers due to their putative antimutagenic,
anticarcinogenic character (1), their association with longevity
(2), and their association in epidemiologic studies with reduced
risk for cancer and cardiovascular diseases (3,4). Although
supplementation with -carotene has not been associated with
benefit in reducing risk for cardiovascular disease or cancers
(5), recent reports have indicated that higher dietary or plasma
lutein and/or zeaxanthin were associated with reduced
progression of atherosclerotic lesions (6), and lower risk for
cataracts (7,8).
Both carotenoids and tocopherols have been reported in the
brain (9-11), and several lines of evidence indicate that
carotenoids and/or tocopherols (both antioxidants) may slow
degenerative changes in the brain, and/or decrease risk for
Alzheimers disease (AD). Among 1166 elderly people, the
highest risk for cognitive decline during a 4 year study was in
those with the highest serum index of oxidation and lowest
serum antioxidants (12). A recent study of non-demented
elderly people found that higher serum carotenoids were
associated with less severe periventricular white matter lesions,
al identified significantly lower concentrations of serum -
carotene, vitamin E, and vitamin A in patients with AD (14).
Retention of better memory performance in 432 elderly
subjects was associated with higher dietary intake of vitamin C
and -carotene (15).
There is substantial evidence that carotenoids and
tocopherols protect another neural tissue, the retina. The multi-
center Age-Related Eye Disease Study found that treatment
with high doses of -tocopherol, vitamin C, -carotene and
zinc slowed progression of age-related macular degeneration
(AMD), a common cause of blindness (16). The neuroretina
selectively accumulates high concentrations of lutein and
zeaxanthin, and excludes almost all other carotenoids (17,18).
Higher concentrations of dietary or plasma lutein/zeaxanthin
were associated with reduced risk for AMD (19,20), and higher
retinal zeaxanthin reduced light-damage to retina in
experimental animals (21,22).
Despite growing evidence that dietary antioxidants play a
pivotal role in prevention of neurodegenerative diseases, the
distributions of individual carotenoids and tocopherol isomers
in different brain regions and gray and white matter have not
been reported. This lack of information prompted the present
analysis of the distribution of individual carotenoids, retinol,
and -, - and -tocopherols in frontal lobe and occipital cortex

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The Journal of Nutrition, Health & Aging
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CAROTENOIDS IN HUMAN BRAIN
from elderly human brains.
Methods
Preparation of Donor Brains
Frozen samples of brain from each of five elderly donors
who had died of causes unrelated to dementia were obtained
from the Massachusetts Alzheimers Disease Research Center
at Massachusetts General Hospital. The frontal and occipital
lobes were examined to form a basis for a subsequent study
comparing antioxidant nutrients in brain tissue of normal
elderly subjects with those in brains with AD. The donors of
the brain specimens included two females, ages 85 and 90, and
three males, ages 67, 73 and 77. Upon arrival at the brain bank,
fresh donor brains were dissected, and 0.5 cm sections of each
brain region were placed on cards, frozen on dry ice and
maintained at -80 C. The brain bank had previously examined
microscopic sections (stained with silver stain and Luxol blue,
Hematoxylin and Eosin) from every brain area to confirm the
brains were free of identifiable neuropathology. The 10
individual samples of frontal and occipital cortex ranged in
weight from approximately 1 g to over 100 g. Samples were
maintained at -70 C prior to extraction.
Reagents
Sodium sulfate, ethyl acetate, hexane, ethanol, pyrogallol,
potassium hydroxide, acetonitrile, dioxane, methanol, isopropyl
alcohol, triethylamine and ammonium acetate of reagent grade
or higher were purchased from various sources. Lycopene and
-carotene standards were purchased from Sigma Chemical Co.
(St. Louis, MO). Retinol and -tocopherol were purchased
from US Biochemical (Cleveland, OH). Tocol, -carotene, -
cryptoxanthin, and zeaxanthin were gifts from Hoffman- La
Roche (Basel, Switzerland). The lutein standard was a gift
from Kemin Industries (Des Moines, IA). The - and -
tocopherols were a gift from Henkel Corporation (Chicago, IL).
Sample processing
While still partially frozen, each section of brain was
dissected into white and gray matter. A 1 to 3 g sample was
weighed and placed in a mortar. Approximately 0.5 g of
sodium sulfate was placed on the tissue and ground with the
pestle. A 7-ml portion of hexane:ethyl acetate (90:10) was
added to the tissue that was ground further. The solvent was
carefully decanted into a glass funnel containing a glass fiber
filter and the filtrate collected in a 25ml volumetric flask. This
extraction was repeated until the volumetric was full.
A 10-ml portion of the extract was placed in a 20-ml screw-
cap test tube and dried under nitrogen gas. The dried extract
was dissolved in 1 ml of ethanol, and 500 l of 10% pyrogallol
in ethanol solution and 1 ml of 40% potassium hydroxide in
methanol were added. The saponification mixture was
sonicated for 30 minutes then allowed to stand at room
temperature for 30 minutes. After the addition of 2.5 ml of
2
water, 5 ml of hexane:ethyl acetate solution was added and the
mixture was vortexed for 45 seconds. The organic layer was
removed and placed in a clean 20-ml screw-cap test tube. This
extraction was repeated two additional times. The extract was
washed twice with 5 ml of water and then dried under nitrogen
gas. Analytes in the residue were dissolved by adding 25 l of
ethyl acetate and vortexed for 20 s; diluted with 75 l of mobile
phase, vortexed again for 15 s, and finally sonicated for 15 s.
The reconstituted extract was transferred to a conical vial insert
and centrifuged prior to HPLC analysis.
HPLC analysis
Extracts were analyzed by an HPLC method described
previously (23). A Spherisorb ODS2, 3 m, 4 x 250 mm
column with titanium frits protected by a Javelin guard column
containing the same packing from Keystone Scientific
(Bellefonte, PA) was used to separate the analytes. The mobile
phase was 80% acetonitrile/15% dioxane/2.5% methanol/2.5%
isopropyl alcohol/ 0.1% triethylamine. Ammonium acetate
(150 mM) was added separately to the alcohol components of
the mobile phase. Solvents were degassed using a vacuum
degasser prior to the HPLC pump. The flow rate was 1.2
ml/minute and the column was maintained at 31 C. The HPLC
components were all ThermoSeparation Products (San Jose,
CA). Carotenoids and retinyl palmitate were monitored using a
programmable UV/VIS detector equipped with both tungsten
and deuterium lamps. The wavelength was programmed at 450
nm until 18.8 minutes then switched to 325 nm until the end of
the run at 23 minutes. Retinol and tocopherols were monitored
using a programmable fluorescence detector at 326 nm
excitation and 460 nm emission for retinol until 4 minutes, then
296 nm excitation and 340 nm emission for tocopherols until
11 minutes. Quantitation of all the analytes was by external
standards. The unidentified xanthophylls and 2,3-
anhydrolutein were quantified using the response factor for
lutein while -cryptoxanthin was quantified using a response
factor midway between the response factors for lutein and -
carotene. The cis isomers of lycopene and -carotene were
quantified using response factors for their respective trans
isomers. Craft Technologies, Inc. was a regular participant in
the National Institute of Standards and Technology (NIST)
Micronutrient Quality Assurance Program for fat-soluble
vitamins. Our performance for the analysis of retinol,
tocopherol and -carotene was consistently within 2 SD of the
NIST assigned values.
Statistics
Since there were only 5 donor brains, gender was
disregarded in analysis. Because of potential relevance to
neurodegeneration, samples were grouped into those from
frontal regions and vulnerable to Alzheimers disease, and
those from less vulnerable occipital regions (24). Statistical
analyses were performed using commercial software (Statview,
Abacus Concepts, Cupertino CA). Due to the non-normal
distribution of the population and small sample size, Spearman
The Journal of Nutrition, Health & Aging
Volume , Number , 2004
THE JOURNAL OF NUTRITION, HEALTH & AGING
Rank Correlations test was used to assess the correlation
between age and antioxidant concentrations. Wilcoxon
analysis of variance, Mann-Whitney U-Tests and Paired Sign
Test (all non-parametric tests), were used to compare frontal
and occipital regions, and gray and white matter. P-values =
0.05 were considered significant.
Figure 1
Representative chromatogram of gray matter from the brain of
a 76 year old male. Carotenoids, shown in panel A, were
monitored at 450 nm. Peaks eluting before LYC are
xanthophylls, those eluting after -CRYPT are carotenes.
Retinol and tocopherols, shown in panel B, were monitored by
fluorescence.
3
Results
-Cryptoxanthin was the major carotenoid in brain, with
lutein, zeaxanthin, anhydrolutein, and -cryptoxanthin present
in significant quantities (Figure 1). The total concentration of
carotenes (lycopene, cis-lycopene, -carotene, -carotene, cis-
-carotene) was significantly less (P<0.0001; Wilcoxon
Analysis) than the total concentration of the xanthophyll
carotenoids, which constituted 66-77% of the total carotenoids
in both frontal and occipital regions (Figure 2). Measurable
levels of retinol, -cryptoxanthin, lutein, zeaxanthin, and -
tocopherol were present in every sample of brain tissue tested.
The typical detection limits were 0.3 pmol/g for retinol, 2
pmol/g for the tocopherols, and 0.06 pmol/g for most
carotenoids, but varied in proportion to the weight of the
sample extracted.
Figure 2
Total concentrations of carotenoids, xanthophylls and carotenes
(mean S.E.) in human brain. Regional differences are
indicated above the bar, group differences indicated by bracket.
T = 0.06<P<0.12; * = P<0.05; **** = P<0.0001;
The mean concentrations of the major xanthophylls in the
frontal and occipital regions and in white and gray matter of
these 5 normal brains are presented in Table 1; concentrations
of retinol, tocopherols, and total carotenoids are found in Table
2. Tocopherols were found in greater abundance than
carotenoids in all brain regions (Figure 1, Tables 1, 2).
-Tocopherol was the analyte present at highest concentration
in these brain samples, followed by -tocopherol, -tocopherol,
retinol, -cryptoxanthin, and lutein. Concentrations of lutein
were significantly greater than zeaxanthin (P<0.02). Reported
lutein/zeaxanthin ratios in human serum range from 3-9 (25).
The mean lutein/zeaxanthin ratio of 1.39 in brain was
significantly less than 3 (P<0.0001; one sample sign test).
In addition to the identifiable compounds, eight unidentified
peaks were consistently detected at 450 nm (See Fig. 1). These
have been tentatively identified as xanthophylls, and further
identification of these unknown peaks is underway. Most of
the unknowns occurred at concentrations that were 10-50%
lower than anhydrolutein; unknown 8 occurred in the highest
The Journal of Nutrition, Health & Aging
Volume, Number, 2004

CAROTENOIDS IN HUMAN BRAIN

concentrations. Zeaxanthin and lutein were highly correlated
with each other ( = 0.93; P<0.0001) and with anhydrolutein,
-cryptoxanthin, -cryptoxanthin, Peak 1 and Peak 7 ( > 0.79;
P = 0.0006 for all). Both were also correlated with Peak 3 ( >
0.52; P = 0.008 for both) and Peak 4 ( > 0.76; P = 0.001 for
both), but neither was correlated with Peak 5, 6, or 8. Peak 8
was strongly correlated with - and -tocopherol ( = 0.76 and
0.91, respectively; P<0.0001 for both).
difference between white and gray matter in the occipital cortex
may be underestimated.
Frontal versus Occipital
Antioxidant concentrations in frontal lobes--a brain region
affected early in AD--were compared with those in occipital
lobes, a region not normally affected until late in AD. The
frontal lobes had significantly higher -tocopherol, total
tocopherol and total xanthophyll concentrations, and tended to

Table 1
Xanthophylls in Elderly Human Brain (Means S.E.)
Brain Region ZEA LUT Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 ANLUT Peak 7 Peak 8 -CRYPT -CRYPT Tot. XAN

Frontal
Gray 9.2 2.3 11.8 2.6 1.3 0.2 2.3 0.7 0.9 0.2 1.0 0.3 0.8 0.3 0.2 0.1 4.1 0.8 2.4 0.5 9.7 4.4 3.9 0.8 17.5 3.2 65.1 10.5
White 7.8 2.8 8.7 3.2 1.5 0.6 4.1 2.6 1.5 0.7 2.1 1.3 1.9 1.2 0.6 0.3 5.8 2.1 3.0 1.2 21.8 12.0 6.9 2.5 23.0 8.5 88.6 32.3

Fr. Versus Occ.(1) T T 0.02 NS NS NS NS NS NS NS NS T T 0.03

Occipital
Gray(2) 6.7 2.4 8.3 2.3 0.7 0.1 1.5 0.3 0.6 0.2 0.7 0.1 0.8 0.2 0.3 0.0 3.4 0.8 2.3 0.7 4.3 1.8 3.7 0.9 14.6 3.3 47.9 8.1
White 1.8 0.6 2.8 1.2 0.3 0.2 0.6 0.3 0.3 0.1 0.4 0.1 0.5 0.2 0.2 0.2 1.9 0.4 0.9 0.1 9.3 4.8 2.1 0.4 7.8 0.9 23.9 7.5

Gray>White (2) 0.03 0.03 NS 0.03 NS NS NS NS NS NS NS NS T T

Concentrations (expressed in pmol/gram) of analytes found in gray and white matter of the frontal (FR) and occipital (OCC) lobes of 5 normal human brains ranging in age from 67-90
years old. Abbbreviations: ZEA=zeaxanthin, LUT=lutein, ANLUT=anhydrolutein, CRYPT = cryptoxanthin, Tot.XAN = total xanthophylls, (1) P-Values resulting from Mann-Whitney
comparisons of concentrations in frontal and occipital lobes. (2) P-Values resulting from Mann-Whitney comparisons of concentrations in gray and white matter of occipital and frontal
regions; NS = Not Significant; T = 0.06 < P< 0.12* = P < 0.05;
Table 2
Carotenes, Retinol and Tocopherols in Elderly Human Brain (Means S.E.)

Brain Region -CAR -CAR LYC Tot.CARS Retinol -TOC -TOC -TOC Tot.TOC Tot.CAROTS

Frontal
Gray 3.6 1.2 7.6 3.2 7.9 3.8 19.1 7.0 163.3 74.5 17928 3214 186.7 83.3 632.7 199.3 18748 8317 84.2 12.7
White 6.3 2.8 15.2 7.0 11.1 9.0 32.7 17.2 142.0 47.0 24681 7988 252.1 135.4 864.7 317.2 25798 8114 121.2 48.5

Fr vrs Occ (1) NS NS NS NS NS 0.03 T NS 0.02 T

Occipital
Gray 3.3 1.2 7.8 2.3 6.0 1.8 17.1 3.2 101.8 40.9 10780 1894 113.1 50.1 415.6 145.5 11309 1999 65.0 9.6
White 2.0 0.4 9.8 3.2 3.4 1.8 15.1 3.6 87.8 44.8 9640 1906 113.1 57.3 337.7 177.8 10091 2033 44.0 10.7
Gr vrs Wh (2) NS NS NS NS NS NS NS NS NS NS

Concentrations (expressed in pmol/gram) of analytes found in gray (Gr) and White (Wh) matter of frontal (Fr) and Occipital (Occ) lobes of 5 normal human brains. Abbreviations:
CAR=Carotene; LYC=lycopene; Tot.=total; CARS=carotenes; TOC=Tocopherol, CAROTS=Carotenoids, vrs=versus. (1) P-Values resulting from Mann-Whitney comparisons of
concentrations in frontal and occipital lobes. (2) P-Values resulting from Mann-Whitney comparisons of concentrations in gray and white matter of both frontal and occipital regions. NS
= Not Significant; T = 0.06 < P < 0.12, * = P < 0.05

Gray and White Matter have higher concentrations of zeaxanthin, lutein, - and -
The total concentration of xanthophylls significantly cryptoxanthin (Tables 1 and 2). Comparison of white matter in
exceeded that of carotenes in both gray matter and in white frontal and occipital regions showed that the more vulnerable
matter (P<0.005 for both; Wilcoxon Analysis; Figure 3). The frontal regions also had 4 times more zeaxanthin and 3 times
mean concentrations of lutein and zeaxanthin in gray matter of more lutein, anhydrolutein, -cryptoxanthin, total xanthophylls,
the occipital region were nearly double those in white matter and total carotenoids than white matter in occipital regions, but
(P<0.03 for both), and the concentrations of -cryptoxanthin the differences did not achieve statistical significance (Tables 1
and total xanthophylls also tended to be greater in gray matter and 2).
in the occipital regions, indicating that these xanthophylls were
selectively distributed in gray matter (Table 1). The
concentrations of tocopherols and carotenes in gray matter were
not significantly different from those in white matter (Figure 3).
It should be noted that stripes of myelinated fibers run through
the gray matter of the occipital cortex, suggesting that the true

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The Journal of Nutrition, Health & Aging
Volume , Number , 2004

THE JOURNAL OF NUTRITION, HEALTH & AGING

Figure 3 Discussion
Total xanthophyll and total carotene concentrations (mean
S.E.) in gray and white matter isolated from frontal and The role of antioxidants in chronic disease prevention
occipital lobes of 5 normal human brains. *** = P<0.005. remains a topic of great debate and an area of intense scientific
research. Evidence that supplemental vitamin E slows the
progression of Alzheimers disease (AD) (26) and that serum
concentrations of zeaxanthin, -cryptoxanthin, lycopene, lutein,
and - and -carotene are reduced in subjects with AD (27),
emphasizes the potential importance of these compounds in the
brain. While this report is on the concentrations of antioxidants
in normal elderly brain, we chose to study one brain region that
is generally affected (frontal cortex) and one that is generally
unaffected (occipital cortex) in early stages of AD. The
observation that the average concentrations of all analytes were
higher in the frontal lobe than in the occipital cortex, and that
-tocopherol, total tocopherol and total xanthophylls were
significantly higher in frontal cortex suggests that regional
differences may exist in factors regulating the distribution of
Decline in Antioxidants with Age these antioxidants in the brain.
With only 5 specimens between ages of 67 and 90, it was
surprising to observe correlations with age. The frontal-- but Tocopherols
not the occipital--lobes exhibited significant age-related These data represent the first examination of the
declines in retinol, - and -tocopherols, total tocopherols, total distributions of individual tocopherol isomers in the human
carotenoids, and total xanthophylls (Table 3). The most brain, and in gray and white matter of frontal lobe cortex and
significant declines occurred in -cryptoxanthin and in several occipital cortex. Our examination indicated no significant
of the unknown peaks. There were no age-related losses in differences in tocopherol contents of gray and white matter.
zeaxanthin or lutein (Table 3) in either brain region. Further Dju et al. reported a tocopherol concentration of 2.5 g/g (5.7
analysis revealed significant declines in -tocopherol in white nmol/g) in fetal brain, 2-5 times lower than in other tissues
matter (= -0.69; P<0.04) but not in gray matter (= -0.42; P = (e.g., liver with 13.8 g/g, 31nmol/g) (28). The authors did not
0.18; data not shown). In general, carotenes did not change with quantify -, - and -tocopherols. The mean (total) tocopherol
age, but there was a significant decline in -carotene in white concentrations that we observed in the adult brains ranged from
matter (= -0.66; P<0.04). 4.4 - 11.2 g/g (10.1 - 25.8 nmol/g), but the lowest tocopherol
measured in our elderly donors was 1.3 g/g (2.98 nmol/g).
Table 3
These observations thus encompass the value reported by Dju
Age Related Declines in Antioxidants in Human Brain
et al (28). The absolute concentration of tocopherols in the
Frontal Occipital brain was less than 20% of those found in many other tissues,
Analyte Rho P-Value Rho P-Value e.g. heart, kidney, and liver (9,10).
Retinol - 0.83 0.01 - 0.03 NS
Podda et al. reported that the brains of hairless mice
contained 99.8% -tocopherol, whereas other tissues contained
Carotenoids - 0.74 0.02 0.06 NS about 1% each of -tocopherol, and - and -tocotrienols (29).
Xanthophylls - 0.72 0.03 0.09 NS The tocopherol isomer concentration in human brain followed
-CRYPT - 0.62 0.06 0.31 NS the pattern > > , with -tocopherol constituting 95.5% of
-CRYPT - 0.53 0.09 - 0.25 NS
ANLUT - 0.59 0.06 - 0.08 NS the total, and - and constituting 3.5% and 1%, respectively.
Peak 2 - 0.81 0.01 - 0.14 NS The recent report by Williamson and colleagues (30) that 5-
Peak 3 - 0.74 0.02 - 0.27 NS
Peak 5 - 0.78 0.02 - 0.11 NS nitro--tocopherol is elevated in AD brain suggests that -
Peak 8 - 0.64 0.05 - 0.19 NS
Zeaxanthin + 0.08 NS +0.13 NS tocopherol plays a significant role in quenching peroxynitrite.
Lutein -0.44 NS -0.09 NS
Carotenes - 0.45 NS - 0.40 NS Carotenoids
-Carotene This is also the first report of individual carotenoids in non-
Tocopherols - 0.86 0.008 - 0.28 NS diseased human brain and their distributions in gray and white
-Tocopherol - 0.86 0.008 - 0.28 NS matter of frontal lobe cortex and occipital cortex. Mathews-
-Tocopherol - 0.64 0.05 - 0.08 NS
Roth et al. reported total carotenoid concentrations of 13 ng/g
Concentrations of brain micronutrients in frontal, but not occipital lobes, were negatively (24 pmol/g) and 27 ng/g (50 pmol/g) in the cerebrum of a 4
correlated with the age of 5 normal human brains whose age ranged between 67 and 90 year-old girl and a 22 year-old woman, both of whom had been
years old. (Spearman Rank Correlations; rho is the correlation coefficient).

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The Journal of Nutrition, Health & Aging
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CAROTENOIDS IN HUMAN BRAIN
receiving -carotene supplements (31). Stahl et al. reported
individual carotenoid values for liver, kidney, adrenals, fat,
testes and ovary, but they listed carotenoids in four samples of
brain stem as below detection limits (32). Kaplan et al.
reported that the total carotenoid concentrations in 10 human
tissues ranged from a low of 0.43 g/g (0.8 nmol/g) in the
thyroid to a high of 18.3 g/g (34 nmol/g) in adrenal tissue, but
they did not report carotenoid concentrations in the brain (9).
In a study that reported carotenoid concentrations in plasma,
liver, spleen, and retina of lutein-supplemented monkeys,
Leung et al also reported that carotenoids were not detected in
brain (33).
We found measurable levels of individual carotenoids in all
human brain sections. The total of individual brain carotenoid
concentrations ranged from 14.3 pmol/g to 303 pmol/g;
encompassing the values reported by Matthews-Roth (31). It is
unlikely that the carotenoids we detected came from remnants
of blood remaining in the brain vasculature because the
distribution of carotenoids observed in the brains did not match
the distribution in typical blood samples, and the ratio of lutein
to zeaxanthin was significantly lower than that found in plasma.
Our ability to measure carotenoids was the result of
saponification of the brain samples (a procedure not universally
utilized (32,33)) and improved limits of detection and
sensitivity, derived from extracting larger samples and from
improvements in HPLC hardware (smaller diameter column,
high sensitivity detector, and low volume flow cell).
Preferential Accumulation of Xanthophylls
Less than 40% of the total carotenoids in plasma and most
tissues are reported to be xanthophylls (9,10,34), but ~65% of
total brain carotenoids were xanthophylls, with -cryptoxanthin
exceeding the concentrations of zeaxanthin and lutein. The ratio
of lutein to zeaxanthin was significantly lower in human brain
than reported in serum (25), suggesting that brain preferentially
accumulated zeaxanthin, as has been reported in the retina (23).
However, -cryptoxanthin was the dominant xanthophyll in
these brain samples, whereas it is present in only trace amounts
in the human retina (23).
Retinol
Retinoids play a well-documented role in the development
and plasticity of the brain but the distribution of retinol in the
brain is not well documented. Recently Connor and Sidell (11)
reported the average retinoid concentration in human
hippocampi to be 7.56 ng/g (26.3 pmol/g); below the 87.8 -
163.3 pmol/g range of mean concentrations in frontal and
occipital lobes reported in this study (Table 2) but close to the
lowest concentration found (25.5 pmol/g in white matter of 77
year old brain). Since they were also studying elderly subjects,
age is probably not a factor in the difference between our
means. However, the area of brain being analyzed may be a
factor. We observed an almost 5-fold variation within different
sections of the same brain, and the mean vitamin A
6
concentrations in the frontal gray and white matter were ~60%
higher than the means in the occipital regions.
Age-Related Loss of Antioxidants
Despite the small sample size and an age-range of only 3
decades, we observed strong inverse relationships between age
and brain concentrations of retinol, total carotenoids, total
xanthophylls and total tocopherols in the frontal, but not the
occipital regions. However, we cannot eliminate the possibility
that the decline was due to gender-related differences; the two
oldest subjects were female, and brain sections from males
tended to be higher in total carotenoids than females. Dramatic
age-related decline of carotenoids, retinol and tocopherols in
the frontal lobe, and/or their selective loss in frontal lobes of
aging women, may have important implications for the etiology
of Alzheimers disease. Recent evidence suggests retinoids may
reduce some types of memory deficits in aging animals (35).
Altered vitamin E status has been linked with AD in several
studies. Scores on cognitive function tests were lower in elderly
subjects who consumed less than 50% of the recommended
dietary intake of vitamin E and higher in subjects with higher
serum vitamin E (36). Low tocopherol concentrations have
been found in plasma and cerebral spinal fluid of subjects with
AD (37). Supplemental vitamin E has slowed the progression
of AD in several studies (38,39) but the role of vitamin E in AD
remains controversial (40). The potential importance of
vitamin E is emphasized by experimental evidence that dietary
tocopherol supplements prevented memory defects in rats given
continuous intraventricular infusion with -amyloid (41).
Neural damage in Parkinsons disease and in AD appears to be
mediated, at least in part, by oxidative damage (42,43), and
reduced by antioxidants (44,45). It is, therefore, important to
confirm the age-related loss of tocopherols, carotenoids and
retinol in a larger sample of normal brains.
Summary
While limited samples were tested, these data provide
information about the distributions of individual carotenoids,
tocopherols, and retinol in gray and white matter of the frontal
and occipital regions of elderly human brain. Concentrations of
these antioxidants tended to be higher in the frontal region than
in the occipital region. The data suggest a preferential age-
related loss of antioxidants in the frontal cortex. Understanding
the distribution of these antioxidants and how they change with
aging and pathology may lead to new hypotheses regarding the
role of these antioxidants in brain physiology and function. A
comparison of these antioxidants in donor brains with no
neuropathology and in brains with confirmed AD is
forthcoming.
Acknowledgments: This study was supported by funds from Applied Food
Biotechnology Inc, OFallon, Missouri. Brain tissue was obtained from the Massachusetts
Alzheimers Disease Research Center Brain Bank (Director: E. Tessa Hedley-Whyte). We
thank Hoffmann-La Roche, Henkel Corporation, and Kemin Industries for the generous
gifts of carotenoid and vitamin standards.
The Journal of Nutrition, Health & Aging
Volume , Number , 2004
THE JOURNAL OF NUTRITION, HEALTH & AGING
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