2007 Annual Report 1a.Objectives (from AD-416) ANTIOXIDANTS RESEARCH LAB To determine if flavonoids are bioavailable as aglycones and glycosides and both parent compounds and their metabolites act in vitro and in vivo as antioxidants. To determine molecular and biochemical mechanisms of flavonoids and their interactions within the antioxidant defense network. CAROTENOIDS AND HEALTH LAB To determine bioavailability and vitamin A equivalents of plant-carotene. To determine antioxidant functions of lutein and tea polyphenols in relation to Age-related Macular degeneration (AMD). To determine the role of body fat on tissue uptake of carotenoids. 1b.Approach (from AD-416) ANTIOXIDANTS RESEARCH LAB 1(A)Weanling rats will be divided into groups fed vitamin E replete, depleted or supplemented diets with or without different doses of quercetin. Blood and tissues will be obtained at intervals over 14 wk for analysis of antioxidant status and metabolism and biomarkers of oxidative stress. (B)Hamsters fed extracts of oat bran, almond skin or bilberry and their blood/tissue collected to determine flavonoid pharmacokinetics and distribution and antioxidant actions. Randomized clinical trials with a cross-over design in healthy older adults will test the bioavailability and antioxidant capacity of these same plant extracts. (C)A pilot clinical trial will test the adequacy of dose and formulation of a manufactured beverage containing catechins and/or N-acetylcysteine. 2(A)Human LDL will be incubated with different flavonoids alone or in combination with other antioxidants to determine their impact on the resistance of nLDL and LDL to oxidation induced by various radical generators. (B)A hemochromatosis mouse model will be used to test the preventive actions of the green tea flavonoid. (C)The effect of bilberry anthocyanins alone or together with xanthophyll carotenoids will be tested in humans for their uptake and metabolism in retina cells and subsequent action on biomarkers of oxidative stress and apoptosis following challenges with hydrogen peroxide or glucose. The impact of anthocyanins on the expression of stress and apoptosis-related proteins and genes will be investigated utilizing mRNA expression gels and GeneChip arrays. CAROTENOIDS AND HEALTH LAB 1. Hydroponically grown deuterium labeled vegetables and Golden Rice will be used to determine bioavailability of beta-carotene, lutein, and zeazanthin, and vitamin A equivalence of provitamin A carotenoids. Labeled food will be given to volunteers, blood samples at different time points will be collected and analyzed using advanced high-performance liquid chromatography (HPLC) gas chromatography mass spectrometry (GC/MS) and liguid chromatography mass spectrometry (LC/MS). 2. Examine the absorption kinetics of major carotenoids, lutein, beta-carotene and lycopene, and measure the functional bioavailability of carotenoids in healthy women in response to an 8-week period of carotenoid supplementation. The antioxidant capacity, DNA oxidation, gene expression profile, lipid peroxidation, and antioxidant nutrient levels in the circulation will be determined in response to a combination or single dose of carotenoids. B.The alteration of macular pigment density, oxidizability both in blood lipid and aqueous compartments, lipid peroxidation and nitric oxide levels following the consumption of a fat-soluble antioxidant, lutein or a combination dose of lutein and water-soluble antioxidant, green tea polyphenols will be determined. 3. Daily supplementation of lutein will be given to subjects of varying body weights (normal, overweight, obese). Changes in carotenoid concentrations will be determined in serum, adipose tissue and macula using HPLC and psychophysical techniques. 4.Accomplishments ANTIOXIDANTS RESEARCH LAB 1)Anthocyanins may promote eye health. Anthocyanins are a class of polyphenolic flavonoids responsible for the color in berry fruits like blueberry and bilberry. The putative health properties of anthocyanins are often attributed to their antioxidant capacity but their cellular concentration is quite low compared to antioxidants like vitamins C and E. Using retinal pigment epithelial cells in culture, we found anthocyanins and related polyphenols in bilberries can quench intracellular radicals, they do not prevent or diminish cell death following an oxidative stress. However, these nutrients and their metabolites did increase the production of messenger RNA for proteins (heme oxygenase-1 and glutathione S-transferase) that could lead to subsequent cell protection and repair activity. These results suggest that anthocyanins and related phytonutrients can influence gene expression in the retina in a manner that could promote eye health. This work is aligned with NP107 Human Nutrition Component 5-Health Promoting Properties of Plant and Animal Foods. 2) Oat avenanthramides are absorbed and increase antioxidant defenses. Increased consumption of polyphenolic phytonutrients correlates with a decreased risk of cardiovascular disease. Found only in oats, the polyphenol avenanthramides may contribute to the heart health benefits associated with this whole grain. However, no information had been available regarding the absorption and antioxidant function of avenanthramides in humans. We characterized in healthy older adults the bioavailability, excretion, and antioxidant action of two doses avenanthramides extracted from oats in a controlled clinical trial. We found these compounds were quickly absorbed, reached their maximum concentration in plasma after 2 hours, and were eliminated from the circulation by 10 hours. Avenanthramides increased the antioxidant capacity of blood. Thus, oat avenanthramides are absorbed and increase antioxidant defenses in healthy older adults. This work is aligned with NP107 Human Nutrition Component 5-Health Promoting Properties of Plant and Animal Foods. 3) Molecular mechanisms of almond polyphenols are characterized. Oxidation of low density lipoproteins (LDL, the “bad cholesterol”) is a key biochemical step in the development of cardiovascular disease. Previously, we found that combining polyphenolic phytonutrients, including flavonoids, from almond skin (ASP) with vitamins C or E inhibited LDL oxidation in a synergistic manner. However, the mechanism(s) by which this synergy occurred was unknown. So we examined the effect of ASP plus antioxidant vitamins on the oxidation of protein moiety as well as on the structure (conformation) and chemical (electronegativity, LDL-) properties of this particle. In a dose-dependent manner, ASP decreased LDL protein oxidation, stabilized LDL conformation, and reduced LDL- formation. The actions of ASP on these parameters were generally additive to those of vitamins C and E. However, ASP plus vitamin C showed a synergy in protecting the protein moiety from oxidation. ASP and vitamin E acted in synergy to reduce LDL- formation. This information helps us to understand at a molecular level how antioxidant nutrients may reduce the risk for cardiovascular disease. This work is aligned with NP107 Human Nutrition Component 5-Health Promoting Properties of Plant and Animal Foods. 4)Vitamin E affects quercetin metabolism. From studies in test tubes, we know dietary antioxidants interact in a dynamic fashion, including recycling and sparing one another, to decrease oxidative stress. Limited information is available regarding the interrelationships between the flavonoid quercetin and vitamin E in the body, so we investigated the antioxidant activity and metabolism of quercetin in rats fed with vitamin E sufficient and deficient diets. The vitamin E deficient diet decreased serum vitamin E and led to muscle damage in a time-dependent manner. Vitamin E deficiency increased plasma protein oxidation but did not affect lipid peroxidation. Dietary quercetin increased the concentration of quercetin and its metabolites in plasma and liver but did not affect vitamin E deficiency-induced changes in plasma vitamin E, muscle damage or protein oxidation. Plasma isorhamnetin, a metabolite of quercetin, and its disposition in skeletal muscle were enhanced by vitamin E deficiency. Conversely, disposition of another quercetin metabolite, tamarixetin, in muscle was decreased by vitamin E deficiency. Thus, quercetin did not slow the loss vitamin E status in the deficient group or substitute for the loss of its antioxidant activity. However, vitamin E status appears to modify the concentration of plasma isorhamnetin and its disposition in muscle. This work is aligned with NP107 Human Nutrition Component 5-Health Promoting Properties of Plant and Animal Foods. CAROTENOIDS AND HEALTH LAB 1)The correlation between dietary intakes and total-body vitamin A stores assessed by the labeled-retinol-dilution method in American adults. For well-nourished adults, data on the correlation between dietary nutrient intakes and total-body vitamin A stores are limited. This study was done to evaluate the relation between dietary intakes, serum retinol concentrations, total-body vitamin A stores and liver vitamin A concentrations in the American adults. Our results showed that subjects who took a multivitamin supplement showed significantly higher body stores than those who never took any multivitamin supplement (p< 0.05). Our results also proved that serum retinol is not a good indicator of vitamin A status for a well-nourished population. The doubled liver stores of subjects taken multivitamin supplement raised the concerns of possible over intake of vitamin A and its toxicity. From this result, the intake of plant foods with high provitamin A carotenoids should be encouraged. This accomplishment addresses NP107 Component 2-Bioavailability of Nutrients and Food Components. 2) Determinants of Macular Pigment Age-related macular degeneration (AMD) is a major cause of visual impairment in the United States. Lutein and zeaxanthin, the sole components of macular pigment, have been implicated in being protective against AMD. We have further refined the determinants of macular pigmentation through research in the carotenoid content in lipoproteins (sole transporters of carotenoids in the circulation) in health and AMD, cross sectional studies in healthy older adults and studies in the high-density lipoprotein deficient chicks. An understanding of factors related to macular pigmentation will aid in the prevention and treatment of AMD. This accomplishment addresses NP107 Component 2 - Bioavailability of Nutrients and Food Components. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of patent granted 2 Number of newspaper articles and other presentations for non-science audiences 19 Review Publications Chen, C., Blumberg, J. 2007. Oxidative stress status in humans with metabolic syndrome. Obesity Epidemiology, Inherent Mechanisms and Novel Ingredients in Weight Management. Boca Raton, FL: CRC Press. Volume 1. p.123-137. Mckay, D.L., Blumberg, J.B. 2006. A review of the bioactivity and potential health benefits of peppermint tea (mentha piperita l.). Phytotherapy Research. 20(8):619-33. Mckay, D.L., Blumberg, J. 2006. A review of the bioactivity of south african herbal teas: rooibos (aspalathus linearis) and honeybush (cyclopia intermedia). Critical Reviews in Food Science and Nutrition. 21:1-16. White, A.C., Sousa, A., Ryan, H.F., Blumberg, J.B., Fanburg, B.L., Kayyali, U.S. 2006. Plasma antioxidants in subjects prior to hematopoietic stem cell transplantation. Bone Marrow Transplantation. 38(7):513-520. Blumberg J.B., Milbury, P.E. 2006. Dietary Flavonoids. In: Bowman B.A., Russell, R.M., editors. Present Knowledge in Nutrition. 9th edition, Volume I. Washington, DC: International Life Sciences Institute. p. 361-70. Chen, C., Lapsley, K., Blumberg, J. 2006. A nutrition and health perspective on almonds. Journal of the Science of Food and Agriculture. 86(14):2245-2250. Kwak, H., Blumberg, J., Chen, C., Milbury, P. 2006. Microplate-based oxygen radical absorbance capacity (orac) assay of hydrophilic and lipophilic compartments in plasma. Nutritional Sciences. 9(1):48-54. Ribaya-Mercado, J., Maramag, C.C., Tengco, L.W., Dolnikowski, G., Blumberg, J., Solon, F. 2007. Carotene-Rich Plant Foods Ingested With Minimal Dietary Fat Enhance The Total-Body Vitamin A Pool Size In Filipino Schoolchildren As Assessed By Stable-Isotope-Dilution Methodology. American Journal of Clinical Nutrition. 85:1041-9. Milbury, P., Graf, B., Curran-Celentano, J.M., Blumberg, J. 2007. Bilberry (Vaccinium myrtillus) Anthocyanins Modulate Heme Oxygenase-1 and Glutathione S-Transferase-pi Expression in the ARPE-19 Cells. Investigative Opthalmology and Visual Science. 48(5):2343-2349. Chen, C., Milbury, P., Collins, F.W., Blumberg, J. 2007. Avenanthramides Are Bioavailable and Have Antioxidant Activity in Humans After Acute Consumption of an Enriched Mixture from Oats. Journal of Nutrition. 137:1375-1382. Maki, K.C., Galant, R., Samuel, P., Tesser, J., Witchger, M., Ribaya-Mercado, J., Blumberg, J., Geohas, J. 2006. Effects of consuming foods containing oat beta-glucan on blood pressure, carbohydrate metabolism, and biomarkers of oxidative stress in men and women with elevated blood pressure. European Journal of Clinical Nutrition. 61:786-795. Johnson, E., Schaefer, E. 2006. The potential role of dietary omega 3 fatty acids in the prevention of dementia and macular degeneration. American Journal of Clinical Nutrition. 83: S1494 - 1498S.