Introduction

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., O’Fallon, MO; 3. Alzheimer’s 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, α- cryptoxanthin, ß- cryptoxanthin, α-carotene, cis- and trans-ßcarotene, 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 Alzheimer’s 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, Alzheimer’s disease, vitamin A, vitamin E, tissue, carotene Introduction particularly in smokers (13). A case-control study by Zaman et al identified significantly lower concentrations of serum ßcarotene, 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 ß-carotene (15). There is substantial evidence that carotenoids and tocopherols protect another neural tissue, the retina. The multicenter Age-Related Eye Disease Study found that treatment with high doses of α-tocopherol, vitamin C, ß-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 α-, γ- and δ-tocopherols in frontal lobe and occipital cortex Common fruits and vegetables in the human diet contain 2030 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 ß-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 Alzheimer’s 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, 1 The Journal of Nutrition, Health & Aging© Volume, Number, 2004 CAROTENOIDS IN HUMAN BRAIN from elderly human brains. 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 µl of ethyl acetate and vortexed for 20 s; diluted with 75 µl 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. 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 Alzheimer’s 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 -70º C prior to extraction. HPLC analysis Extracts were analyzed by an HPLC method described previously (23). A Spherisorb ODS2, 3 µm, 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 31º 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 α-cryptoxanthin was quantified using a response factor midway between the response factors for lutein and αcarotene. The cis isomers of lycopene and ß-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 ß-carotene was consistently within 2 SD of the NIST assigned values. 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 ß-carotene standards were purchased from Sigma Chemical Co. (St. Louis, MO). Retinol and α-tocopherol were purchased from US Biochemical (Cleveland, OH). Tocol, α-carotene, ßcryptoxanthin, and zeaxanthin were gifts from Hoffman- La Roche (Basel, Switzerland). The lutein standard was a gift from Kemin Industries (Des Moines, IA). The δ- and γtocopherols 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 screwcap test tube and dried under nitrogen gas. The dried extract was dissolved in 1 ml of ethanol, and 500 µl 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 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 Alzheimer’s 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 2 The Journal of Nutrition, Health & Aging© Volume , Number , 2004 THE JOURNAL OF NUTRITION, HEALTH & AGING© Results 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. ß-Cryptoxanthin was the major carotenoid in brain, with lutein, zeaxanthin, anhydrolutein, and α-cryptoxanthin present in significant quantities (Figure 1). The total concentration of carotenes (lycopene, cis-lycopene, α-carotene, ß-carotene, cisß-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, ß-cryptoxanthin, lutein, zeaxanthin, and αtocopherol 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 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 ß-CRYPT are carotenes. Retinol and tocopherols, shown in panel B, were monitored by fluorescence. 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). α-Tocopherol was the analyte present at highest concentration in these brain samples, followed by γ-tocopherol, δ-tocopherol, retinol, ß-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 3 The Journal of Nutrition, Health & Aging© Volume, Number, 2004 CAROTENOIDS IN HUMAN BRAIN concentrations. Zeaxanthin and lutein were highly correlated with each other (ρ = 0.93; P<0.0001) and with anhydrolutein, α-cryptoxanthin, ß-cryptoxanthin, Peak 1 and Peak 7 (ρ > 0.79; P = 0.0006 for all). Both were also correlated with Peak 3 (ρ > 0.52; P = 0.008 for both) and Peak 4 (ρ > 0.76; P = 0.001 for both), but neither was correlated with Peak 5, 6, or 8. Peak 8 was strongly correlated with α- and γ-tocopherol (ρ = 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 α-tocopherol, total tocopherol and total xanthophyll concentrations, and tended to Table 1 Xanthophylls in Elderly Human Brain (Means ± S.E.) Brain Region Frontal Gray White ZEA LUT Peak 1 Peak 2 1.3 ± 0.2 1.5 ± 0.6 2.3 ± 0.7 4.1 ± 2.6 T 0.02 NS 8.3 ± 2.3 2.8 ± 1.2 0.7 ± 0.1 0.3 ± 0.2 1.5 ± 0.3 0.6 ± 0.3 0.03 NS 0.03 9.2 ± 2.3 11.8 ± 2.6 7.8 ± 2.8 8.7 ± 3.2 Fr. Versus Occ.(1) T Occipital Gray(2) White 6.7 ± 2.4 1.8 ± 0.6 Gray>White (2) 0.03 Peak 3 Peak 4 0.9 ± 0.2 1.0 ± 0.3 1.5 ± 0.7 2.1 ± 1.3 NS NS 0.6 ± 0.2 0.7 ± 0.1 0.3 ± 0.1 0.4 ± 0.1 NS NS Peak 7 Peak 8 α-CRYPT ß-CRYPT Tot. XAN Peak 5 Peak 6 ANLUT 0.8 ± 0.3 1.9 ± 1.2 0.2 ± 0.1 0.6 ± 0.3 4.1 ± 0.8 5.8 ± 2.1 NS NS NS NS NS T 0.8 ± 0.2 0.5 ± 0.2 0.3 ± 0.0 0.2 ± 0.2 3.4 ± 0.8 1.9 ± 0.4 2.3 ± 0.7 0.9 ± 0.1 4.3 ± 1.8 9.3 ± 4.8 3.7 ± 0.9 2.1 ± 0.4 NS NS NS NS NS NS 2.4 ± 0.5 9.7 ± 4.4 3.9 ± 0.8 3.0 ± 1.2 21.8 ± 12.0 6.9 ± 2.5 17.5 ± 3.2 65.1 ± 10.5 23.0 ± 8.5 88.6 ± 32.3 T 0.03 14.6 ± 3.3 47.9 ± 8.1 7.8 ± 0.9 23.9 ± 7.5 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 α-CAR Frontal Gray White 3.6 ± 1.2 7.6 ± 3.2 7.9 ± 3.8 19.1 ± 7.0 6.3 ± 2.8 15.2 ± 7.0 11.1 ± 9.0 32.7 ± 17.2 Fr vrs Occ (1) Occipital Gray White Gr vrs Wh (2) ß-CAR LYC Tot.CARS NS NS NS NS 3.3 ± 1.2 2.0 ± 0.4 7.8 ± 2.3 9.8 ± 3.2 6.0 ± 1.8 3.4 ± 1.8 17.1 ± 3.2 15.1 ± 3.6 NS NS NS NS α-TOC Retinol 163.3 ± 74.5 17928 ± 3214 142.0 ± 47.0 24681 ± 7988 NS 0.03 101.8 ± 40.9 10780 ± 1894 87.8 ± 44.8 9640 ± 1906 NS NS γ-TOC δ-TOC 186.7 ± 83.3 632.7 ± 199.3 252.1 ± 135.4 864.7 ± 317.2 T NS 113.1 ± 50.1 415.6 ± 145.5 113.1 ± 57.3 337.7 ± 177.8 NS NS Tot.TOC Tot.CAROTS 18748 ± 8317 84.2 ± 12.7 25798 ± 8114 121.2 ± 48.5 0.02 T 11309 ± 1999 10091 ± 2033 65.0 ± 9.6 44.0 ± 10.7 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 have higher concentrations of zeaxanthin, lutein, α- and ßcryptoxanthin (Tables 1 and 2). Comparison of white matter in frontal and occipital regions showed that the more vulnerable frontal regions also had 4 times more zeaxanthin and 3 times more lutein, anhydrolutein, ß-cryptoxanthin, total xanthophylls, and total carotenoids than white matter in occipital regions, but the differences did not achieve statistical significance (Tables 1 and 2). Gray and White Matter The total concentration of xanthophylls significantly exceeded that of carotenes in both gray matter and in white matter (P<0.005 for both; Wilcoxon Analysis; Figure 3). The mean concentrations of lutein and zeaxanthin in gray matter of the occipital region were nearly double those in white matter (P<0.03 for both), and the concentrations of ß-cryptoxanthin and total xanthophylls also tended to be greater in gray matter 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 4 The Journal of Nutrition, Health & Aging© Volume , Number , 2004 THE JOURNAL OF NUTRITION, HEALTH & AGING© Figure 3 Total xanthophyll and total carotene concentrations (mean ± S.E.) in gray and white matter isolated from frontal and occipital lobes of 5 normal human brains. *** = P<0.005. Discussion The role of antioxidants in chronic disease prevention remains a topic of great debate and an area of intense scientific research. Evidence that supplemental vitamin E slows the progression of Alzheimer’s disease (AD) (26) and that serum concentrations of zeaxanthin, ß-cryptoxanthin, lycopene, lutein, and α- and ß-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 α-tocopherol, total tocopherol and total xanthophylls were significantly higher in frontal cortex suggests that regional differences may exist in factors regulating the distribution of these antioxidants in the brain. Decline in Antioxidants with Age With only 5 specimens between ages of 67 and 90, it was surprising to observe correlations with age. The frontal-- but not the occipital--lobes exhibited significant age-related declines in retinol, α- and γ-tocopherols, total tocopherols, total carotenoids, and total xanthophylls (Table 3). The most significant declines occurred in α-cryptoxanthin and in several of the unknown peaks. There were no age-related losses in zeaxanthin or lutein (Table 3) in either brain region. Further analysis revealed significant declines in α-tocopherol in white matter (ρ= -0.69; P<0.04) but not in gray matter (ρ= -0.42; P = 0.18; data not shown). In general, carotenes did not change with age, but there was a significant decline in ß-carotene in white matter (ρ= -0.66; P<0.04). Tocopherols These data represent the first examination of the distributions of individual tocopherol isomers in the human brain, and in gray and white matter of frontal lobe cortex and occipital cortex. Our examination indicated no significant differences in tocopherol contents of gray and white matter. Dju et al. reported a tocopherol concentration of 2.5 µg/g (5.7 nmol/g) in fetal brain, 2-5 times lower than in other tissues (e.g., liver with 13.8 µg/g, 31nmol/g) (28). The authors did not quantify δ-, γ- and α-tocopherols. The mean (total) tocopherol concentrations that we observed in the adult brains ranged from 4.4 - 11.2 µg/g (10.1 - 25.8 nmol/g), but the lowest tocopherol measured in our elderly donors was 1.3 µg/g (2.98 nmol/g). These observations thus encompass the value reported by Dju et al (28). The absolute concentration of tocopherols in the brain was less than 20% of those found in many other tissues, e.g. heart, kidney, and liver (9,10). Podda et al. reported that the brains of hairless mice contained 99.8% α-tocopherol, whereas other tissues contained about 1% each of γ-tocopherol, and α- and γ-tocotrienols (29). The tocopherol isomer concentration in human brain followed the pattern α > γ > δ, with α-tocopherol constituting 95.5% of the total, and γ- and δ constituting 3.5% and 1%, respectively. The recent report by Williamson and colleagues (30) that 5nitro-γ-tocopherol is elevated in AD brain suggests that γtocopherol plays a significant role in quenching peroxynitrite. Table 3 Age Related Declines in Antioxidants in Human Brain Frontal P-Value Occipital Rho P-Value Analyte Rho Retinol - 0.83 0.01 - 0.03 NS Carotenoids - 0.74 0.02 0.06 NS Xanthophylls α-CRYPT β-CRYPT ANLUT Peak 2 Peak 3 Peak 5 Peak 8 Zeaxanthin Lutein - 0.72 - 0.62 - 0.53 - 0.59 - 0.81 - 0.74 - 0.78 - 0.64 + 0.08 -0.44 0.03 0.06 0.09 0.06 0.01 0.02 0.02 0.05 NS NS 0.09 0.31 - 0.25 - 0.08 - 0.14 - 0.27 - 0.11 - 0.19 +0.13 -0.09 NS NS NS NS NS NS NS NS NS NS Carotenes ß-Carotene - 0.45 NS - 0.40 NS Tocopherols α-Tocopherol γ-Tocopherol - 0.86 - 0.86 - 0.64 0.008 0.008 0.05 - 0.28 - 0.28 - 0.08 NS NS NS Carotenoids This is also the first report of individual carotenoids in nondiseased human brain and their distributions in gray and white matter of frontal lobe cortex and occipital cortex. MathewsRoth et al. reported total carotenoid concentrations of 13 ng/g (24 pmol/g) and 27 ng/g (50 pmol/g) in the cerebrum of a 4 year-old girl and a 22 year-old woman, both of whom had been Concentrations of brain micronutrients in frontal, but not occipital lobes, were negatively correlated with the age of 5 normal human brains whose age ranged between 67 and 90 years old. (Spearman Rank Correlations; rho is the correlation coefficient). 5 The Journal of Nutrition, Health & Aging© Volume, Number, 2004 CAROTENOIDS IN HUMAN BRAIN receiving ß-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 µg/g (0.8 nmol/g) in the thyroid to a high of 18.3 µg/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). 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 Alzheimer’s 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 ß-amyloid (41). Neural damage in Parkinson’s 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. 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 ß-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, ß-cryptoxanthin was the dominant xanthophyll in these brain samples, whereas it is present in only trace amounts in the human retina (23). 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 agerelated 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. 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 Acknowledgments: This study was supported by funds from Applied Food Biotechnology Inc, O’Fallon, Missouri. Brain tissue was obtained from the Massachusetts Alzheimer’s 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. 6 The Journal of Nutrition, Health & Aging© Volume , Number , 2004 THE JOURNAL OF NUTRITION, HEALTH & AGING© References 23. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. Bertram JS, Bortkiewicz H. Dietary carotenoids inhibit neoplastic transformation and modulate gene expression in mouse and human cells. 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