2005 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? The Dietary Antioxidants, Aging and Oxidative Stress Status CRIS is comprised of the Antioxidants Research Laboratory (ARL) and the Carotenoids and Health Laboratory (CHL). The generation of oxygen free radicals during cellular metabolism and by certain environmental factors, including lifestyle, appear to play a critical role in the aging process. High dietary intakes of antioxidant vitamins and phytochemicals are associated with better maintenance of physiologic function and a lower prevalence of many degenerative conditions in older adults. Understanding how antioxidants, especially the phytochemical carotenoids and flavonoids, reduce oxidative stress and impact the pathogenesis of chronic disease present opportunities for health promotion and alternative therapeutic modalities for an aging population. The ARL conducts studies to understand the role of dietary antioxidants, particularly the flavonoids in conjunction with vitamins C and E, on free radical reactions and changes in oxidative stress status during aging. Lipid, protein, nucleic acid and other cellular targets of oxidative damage are assessed in experimental models and in healthy and other human populations to elucidate antioxidant requirements for optimal health. These projects contribute to the description and prediction of the impact of antioxidants and oxyradicals on age-related changes in nutrient requirements and chronic degenerative conditions. The CHL investigates dietary carotenoids and healthy aging. There is a lack of important data concerning key aspects of absorption and metabolism in humans of carotenoids contained in food. These areas include:. 1)the bioavailability of carotenoids from different food matrices and the efficiency of conversion of carotenoids to vitamin A;. 2)the function of carotenoids as antioxidants in both lipid and aqueous compartments of serum; and. 3)the distribution of absorbed carotenoids, such as lutein, in humans with various levels of fat tissue. This research on the bioavailability, bioconversion, antioxidant function, and tissue distribution of dietary carotenoids will continue to improve our understanding of dietary carotenoids and human health. It will furnish information regarding which plant foods can provide nutrients that might help to prevent or delay certain chronic diseases common to the elderly. There is a compelling body of scientific evidence that suggests that free radical pathology is associated with many of the chronic diseases that are common among older adults, e.g., cancer, heart disease, and degenerative eye disorders such as cataracts and macular degeneration. Though not so extensively explored, several recent studies suggest antioxidants may also play a preventive or adjunctive role in conditions like arthritis, diabetes, and Parkinson’s disease. However, definitive evidence is absent in all of these cases. Quantitative information linking dietary requirements for antioxidants, validated biomarkers of intermediary oxidative stress events and status, and health and aging outcomes is lacking. Such information is necessary to the development of scientifically-based recommended dietary allowances, including tolerable upper limits of intake, for antioxidant nutrients across the lifespan and particularly for the elderly. Further, the conversion efficiency of ß-carotene to vitamin A in humans needs to be comprehensively quantified. Research within these laboratories falls under National Program 107 – Human Nutrition, components 2: Bioavailability of nutrients and food components; 4: Nutrient requirements; and 5: Health promoting properties of plant and animal foods. This research is related to Performance Measure 4.1.2: Improve Human Health by Better Understanding the Nutrient Requirements of Individuals and the Nutritional Value of Foods. 2.List the milestones (indicators of progress) from your Project Plan. ARL: Objective 1. 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. Objective 2. To determine the molecular and biochemical mechanisms of flavonoids and their interactions within the antioxidant defense network. FY2005 1. Determine in vitamin E deficient rats if quercetin bioavailability is enhanced and serves as an antioxidant. (Objective. 1) 2. Characterize in hamsters the bioavailability, metabolism, and antioxidant action of flavonoids from almond skins. (Objective. 1) 3. Determine in older adults the bioavailability and antioxidant actions of flavanols alone and with N-acetylcysteine. (Objective. 1) 4. Characterize the effect of flavonoids from almond skins on lag time to Cu2+-induced oxidation in human low-density lipoproteins. (Objective. 2) 5. Determine in retinal pigment epithelium if anthocyanins are taken up, transported, and metabolized. (Objective. 2) 6. Determine if iron-induced oxidative stress accelerates colon tumorigenesis and epigallocatechin gallate slows this process by modulating oxidative injury and/or oxidative stress responses. (Objective. 2) FY2006 1. Determine in rats chronically fed almond skins or sweet potato leaves the bioavailability and metabolism of the constituent flavonoids. (Objective. 1) 2. Characterize the effect of flavonoids from almond skin on Cu2+-induced changes to particle fluidity and protein oxidation of human low-density lipoproteins. (Objective. 2) 3. Determine in retinal pigment epithelium the expression of stress- and apoptosis-related proteins following treatment with anthocyanins. (Objective 2) FY2007 1. Determine in rats deficient in vitamins C and E the antioxidant action and metabolism of quercetin. (Objective 1) 2. Determine in a clinical trial of older adults the bioavailability and antioxidant capacity of flavonoids from almond skins, bilberry, and oat bran. (Objective. 1) 3. Determine the effect of flavonoids from almond skin on myeloperoxidase and other pro-oxidants on particle fluidity and oxidation potential of human low-density lipoproteins. (Objective 2) 4. Characterize in retinal pigment epithelium interactions between anthocyanins and xanthophylls on biomarkers of oxidative stress, mitochondrial integrity, and apoptosis. (Objective 2) FY2008 1. Determine in rats deficient in vitamins C and E the antioxidant action and metabolism of quercetin plus epigallocatechin gallate. (Objective 1) 2. Determine in hamsters the effect of age on flavonoid bioavailability, metabolism, and antioxidant activity. (Objective. 1) 3. Characterize in human low density lipoproteins treated with flavonoids from almond skins the independent and interactive actions of flavonoids, carotenoids, and vitamins C and E. (Objective 2) FY2009 1. Determine in hamsters the effect of age and exercise exertion on flavonoid bioavailability, metabolism, and antioxidant activity. (Objective. 1) 2. Determine in retinal pigment epithelium the effect of flavonoids on age-related declines in the transcriptional activity of Nrf2 and glutathione synthesis. (Objective 2) 3. Determine in retinal pigment epithelium the expression of genes (using GeneChip® arrays) following treatment with anthocyanins and flavanols. (Objective. 2) CHL: Objective 1: To determine bioavailability and vitamin A equivalents of plant beta-carotene. Objective 2: To determine antioxidant functions of lutein and tea polyphenols in relation to Age-related Macular Degeneration (AMD). Objective 3: To determine role of body fat on tissue uptake of carotenoids FY2005 1. Determine the bioavailability of carotenoids (ß-carotene, lycopene, lutein, and zeaxanthin) and vitamin A equivalence of provitamin A carotenoids using intrinsically labeled plants. (Objective. 1) 2. The development and application of a biomarker assay of total antioxidant capacity in both the lipid and aqueous compartments of biological samples, and the interaction between various antioxidants. (Objective. 2) FY2006 1. Analyze samples by high performance liquid chromatography (HPLC) and gas chromotography/mass spectrometry (GCMS) collected a pilot study of the bioavailability of labeled dietary carotenoids. (Objective 1) 2. Prepare a field intervention project to determine the vitamin A values of spinach and Golden Rice. (Objective 1) 3. Prepare deuterium labeled peppers for human investigations. (Objective 1) 4. Recruit volunteers for the project of examining lutein and tea polyphenols in relation to AMD. (Objective 2) 5. Initiate a protocol to recruit volunteers for the project designed to determine the role of body fat on tissue uptake of carotenoids. (Objective 3) FY2007 1. Conduct and complete the field study on the vitamin A value of plant carotenoids. (Objective 1) 2: Recruit volunteers for the project on the bioavailability of pepper lutein and zeaxanthin. (Objective 1) 3. Continue recruitment for the project of examining lutein and tea polyphenols in relation to AMD. (Objective. 2) 4. Continue subject recruitment for the project on the role of body fat on tissue uptake of carotenoids. (Objective 3) FY2008 1. Perform sample analysis from the field study on the vitamin A value of labeled dietary provitamin A carotenoids, spinach, Golden Rice. (Objective 1) 2. Analyze samples from the study on peppers carotenoids. (Objective 1) 3. Perform analyses of antioxidant capacity and lipid peroxidation in samples collected from the subjects in the lutein and tea and AMD study. (Objective 2) 4. Sample and data analysis of carotenoid content in diet, serum, and tissues (including macula) collected from the project on the role of body fat on tissue uptake of carotenoids. (Objective 3) FY2009 1. Perform data analysis on the vitamin A value of labeled dietary provitamin A carotenoids, spinach, and Golden Rice. (Objective 1) 2. Perform data analysis on the bioavailability of pepper carotenoids. (Objective 1) 3. Perform data analyses on the antioxidant capacity and lipid peroxidation from the project. (Objective 2) 4a.What was the single most significant accomplishment this past year? Carotenoid Supplementation Decreases DNA Damage in Humans The CHL confirmed that carotenoid supplementation decreases DNA damage in humans, and that a combination of carotenoids (4 mg each of lutein, beta-carotene, and lycopene), which can be easily achieved by diet, or a larger dose (12 mg) of individual carotenoids can protect humans against in vivo DNA damage. Oxidative stress has been implicated in the etiology of chronic diseases of aging, such as cancer and cardiovascular disease. Carotenoids could be a part of a protective strategy to minimize the oxidative damage in a vulnerable population such as the elderly. To determine the protective effect of carotenoids against DNA damage, a randomized double-blind placebo controlled intervention study was conducted. Thirty-seven healthy non-smoking post- menopausal women (50-70 yrs) were randomly assigned to one of 5 groups, and were instructed to consume a daily dose of either mixed carotenoids (beta-carotene, lutein and lycopene, 4 mg each), 12 mg of a single carotenoid (beta-carotene, lutein or lycopene), or a placebo for 56 days. Plasma carotenoid concentrations were analyzed by HPLC, and lymphocyte DNA damage was determined by a single cell gel electrophoresis (comet) assay. On day 56, all carotenoid supplemented groups showed significantly lower endogenous DNA damage than that of baseline (P<0.01), while the placebo group did not show any significant change. The results indicate that carotenoid supplementation of a combination of carotenoids (4 mg each of lutein, beta-carotene, & lycopene) or a larger dose (12 mg) of individual carotenoids provides protection against DNA damage. This project relates to the Human Nutrition Program - 107 Component 6: Health promoting properties of plant and animal foods and Component. 4b.List other significant accomplishments, if any. ARL: 1. Quercetin is extensively metabolized in the GI tract. Quercetin and quercetin glycosides from food or dietary supplements appear in body tissues almost exclusively as glucuronated, sulfated and methylated quercetin conjugates, suggesting the in vivo bioactivity of quercetin may be due to its metabolites. To test the hypothesis that the GI tract is the major site of quercetin metabolism in vivo, and not the liver as commonly assumed, quercetin metabolites were extracted from liver, kidney and plasma, identified and quantified, and compared with metabolite patterns in GI tissues and contents. Following a feasibility study of the selectivity and sensitivity of our analytical methods, pre- and post-absorptive metabolism of orally ingested quercetin was examined by comparing the metabolite pattern in gastrointestinal (GI) tissues, contents and internal tissues. Rats were fed for 6 wk a diet containing either no quercetin or 0.45% quercetin and the metabolite patterns were determined in the tissues and contents of stomach, small intestine, cecum, colon, and in liver, kidney, and plasma using LC-MS/MS. GI contents contained predominantly un-metabolized quercetin at 91 - 100%, while quercetin in GI tissues was present as eleven different sulfated, glucuronated, and methylated metabolites at 46% in stomach, 93% in small intestine, 36% in cecum, and 57% in colon. Quercetin was further metabolized post-absorption and found in liver, kidney and plasma almost exclusively as sulfated methyl-quercetin glucuronide. Metabolite identity was further confirmed by comparison with tissue extracts from control animals and their disappearance upon hydrolysis after treatment with glucuronidase and sulfatase. The unique pattern of quercetin metabolites in each GI tissue indicates extensive biotransformation prior to absorption and distribution in the rat. 2. Almond skin flavonoids are antioxidants in vitro and bioavailable in vivo. Consumption of tree nuts such as almonds has been associated with a reduced risk of coronary heart disease. Flavonoids, found predominantly in the skin, may contribute to this putative health benefit of almonds. Using acidified methanol extraction and HPLC with electrochemical detection, almond skin flavonoids (ASF), including catechins and flavonols, were identified. ASF in physiologic concentrations enhanced the resistance of human low-density lipoprotein (LDL) against copper-induced oxidation in vitro in a dose-dependent manner. Combining ASF with vitamins C or E increased the antioxidant activity by 2-fold more than the expected additive value. The bioavailability and in vivo antioxidant activity of ASF were examined in an acute administration model in hamsters. Flavan-3-ols (catechin, epicatechin) and flavonols (quercetin, kaempferol, isorhamnetin) were detected in plasma and liver. Absorbed ASF significantly enhanced the ex vivo resistance of hamster LDL to oxidation by 18% and the in vitro addition of vitamin E synergistically extended the lag time by 53%. Thus, ASF possess antioxidant capacity in vitro and are bioavailable and act in synergy with vitamins C and E to protect LDL against oxidation in hamsters. 3. N-acetylcysteine may promote catechin bioavailability. Antioxidants provide a network of defense against oxidative stress and studies suggest they may interact in a synergistic manner. We examined this relationship between flavonoids and N-acetylcysteine (NAC), a precursor of glutathione (GSH), in a randomized, double-blind, cross-over trial with healthy postmenopausal women consuming beverages formulated with: [1] flavonoids extracted from white tea and grape seeds, [2] NAC or [3] both formulations together. Following a low flavonoid diet run-in, subjects consumed 500 mL of the test beverage at 0, 2, 4, and 6 h. After consumption of beverages 1 and 3, plasma total catechins (TC) and epigallocatechin gallate (EGCG) increased over 4 h, reached a plateau, and declined after 8 h. Area under the time curve of plasma TC and EGCG were slightly higher after beverage 3 than 1. NAC increased plasma GSH by 10-50% during the 10 h period following consumption without influence by the flavonoids. No intervention modified antioxidant capacity determined by plasma Oxygen Radical Absorbance Capacity and malondialdehyde. Thus, NAC may act to promote flavonoid status, though its mechanism of action was not revealed in this study. 4. Almond skin flavonoids promote lipoprotein stability and integrity during oxidative stress. Low density lipoprotein (LDL), the major transport vehicle for cholesterol in human plasma, consists of an apolar core of cholesteryl esters and triglycerides surrounded by a monolayer of amphiphilic phospholipids and unesterified cholesterol, and one molecule of apolipoprotein B-100 (apoB-100). LDL oxidation is a key initiating event during development of atherosclerosis. The increased resistance of LDL to oxidation induced by dietary antioxidants may slow the initiation and progression of atherosclerosis. We have shown the antioxidant activity of ASF can diminish the rate of copper-induced LDL oxidation in a synergistic manner with vitamins C and E and are working to determine the mechanism of action. We have begun examining changes in polarity and the order of lipids in LDL by establishing new assays in the laboratory for generalized polarization (Gp) and tryptophan (Trp) fluorescence. An increase in the Gp value of Laurdan, a membrane probe with little affinity for proteins and with no appreciable fluorescence when in aqueous environments, is observed during and after lipid peroxidation. Trp, an amino acid in apoB-100, is sensitive to oxidative challenges and serves as a marker of protein oxidation. Preliminary experiments suggest ASF can slow changes in Gp during LDL oxidation and the loss of Trp fluorescence occurring prior to lipid peroxidation is inhibited by ASF in physiologic concentrations. 5. Anthocyanins may increase antioxidant defenses in retinal pigment epithelium. The uptake of anthocyanins from bilberry was determined in human retinal pigment epithelial (RPE) cells in culture via their identification in the cytosolic fraction following incubation with physiologically relevant concentrations. Further, phase II metabolites of anthocyanins were measured in the culture medium; these anthocyanin conjugates can only have been produced within the RPE cell and, thus, further substantiate the absorption of these flavonoids by RPE cells. When RPE cells were exposed to hydrogen peroxide, an oxidant produced in RPE as a by-product of photo-sensing metabolic processes, a decline in intracellular glutathione concentration was noted as well as a reduction in the pathway toward programmed cell death, measured by loss of ATP production, alteration in BCL2/Bax protein ratios, and impaired cell viability via the loss of mitochondrial reducing potential. Bilberry anthocyanins administered concurrently with hydrogen peroxide reduced oxidative stress and apoptosis via direct radical quenching, but the effective concentrations were too high to be achieved through normal physiological function. Preliminary evidence suggests anthocyanins may exert their putative health benefits via up-regulation of phase II metabolism and related endogenous antioxidant defenses. For the CHL: 1. Development of Using Intrinsic Labeled Tomato to Assess Lycopene Bioavailability Current knowledge of the bioavailability of lycopene in humans is limited by the inability to distinguish newly administered lycopene from the body reserves of lycopene. A quantitative method to assess the absorption and relative bioavailability of newly absorbed synthetic or natural lycopene was developed using two deuterated lycopene sources and advanced liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS) to analyze newly absorbed lycopene in blood samples of study subjects. Two subjects consumed hydroponically grown tomatoes containing deuterium-enriched lycopene and two subjects consumed synthetic lycopene in 6 grams of corn oil. Tomatoes were steamed and pureed. The doses were given together with a liquid formulated drink with 25 percent of the energy from fat. From these preliminary investigations, we found that the bioavailability of synthetic lycopene in oil appears to be about three times higher than that of lycopene from steamed and pureed tomatoes. This study provides evidence that the absorption of physiological levels of lycopene in intrinsically labeled tomatoes can be studied in humans. 2. Assessment of Antioxidant Capacity of Lipophilic Antioxidants Scientists developed a method to determine the antioxidant capacity of both the lipid and the aqueous compartments. Using this method, we can now measure the changes of the antioxidant capacity of serum before and after the physiological doses of either single or mixed carotenoids supplements in diets. This will enable us to study the functional bioavailability of carotenoids and to address the question as to whether absorbed carotenoids act as antioxidants in vivo. 3. The Effect of Lutein and Zeaxanthin Supplementation on Macular Pigment Macular pigment is composed of the xanthophylls, lutein and zeaxanthin (Z), and may help to prevent age-related macular degeneration or retard its progression. The laboratory conducted a study to investigate the effects of lutein or zeaxanthin supplementation on carotenoid levels in serum, adipose tissue, and retina of rhesus monkeys with no previous intake of xanthophyll. From birth to 7 to 16 years of age, 18 rhesus monkeys were fed semipurified diets containing all essential nutrients but no xanthophylls. Six were supplemented with pure lutein and 6 with pure zeaxanthin for 24 to 101 weeks. Our results showed that in lutein or zeaxanthin supplemented monkeys, serum and adipose tissue concentrations of carotenoids were significantly increased as compared with the control monkeys fed xanthophyll-free diets that had no lutein or zeaxanthin in serum or tissues. Both lutein and the zeaxanthin isomers 3R and 3’S-Z were incorporated into retinas of monkeys supplemented with lutein, with RSZ present only in the macula, as the isomers RSZ and meso-Z are not present in the diet. All-trans zeaxanthin, but not RSZ, accumulated in retinas of monkeys supplemented with zeaxanthin. This investigation demonstrated that lutein is the precursor of RSZ, a major component of macular pigment. It also demonstrated that xanthophyll-free monkeys can accumulate retinal xanthophylls and can provide a valuable model for examining their uptake and conversion. 4c.List any significant activities that support special target populations. None. 4d.Progress report. See progress report for 51000-065-03R High-Throughput In Vitro Screening of Botanical Products for Antioxidant Capacity and Bioactivity. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. The ARL is characterizing the bioavailability, metabolism, antioxidant activity, and interactions of flavonoids, along and together with other dietary antioxidants. We are creating information regarding the application of biomarkers of oxidative stress as they are used to define dietary antioxidants and applying them to the examination of flavonoids. By providing quantitative data on biomarkers of oxidative stress as measures of dietary antioxidant requirements, the proposed research will help substantiate and improve the scientific basis for the Dietary Guidelines for Americans and help provide a basis for Dietary Reference Intakes (DRI) for flavonoids, particularly with regard to the needs of older adults. Furthermore, our research on flavonoids will provide new information useful for the selection and production of plant cultivars rich in those compounds found to be most bioavailable and bioactive. The results of this research program are of primary interest to nutrition scientists and should prove of use to those making revisions to the Dietary Guidelines for Americans and the DRI. Agricultural and food scientists will be able to employ our research data toward the selection and production of plant cultivars and/or functional foods rich in those flavonoids found most bioavailable and bioactive. Examples of major accomplishments over the life of the project include: 1. Consumption of nuts has been associated with a reduced risk of coronary heart disease. We have created a database of the flavonoid content of almonds and provided it to the USDA for incorporation into its Database for the Flavonoid Content of Selected Foods. We have demonstrated that almond flavonoids and related polyphenolics are antioxidants in vitro, in animal models, and in humans and work in synergy with other dietary antioxidants. This work is related to National Program 107 – Human Nutrition, components 2: Bioavailability of nutrients and food components; and 5: Health promoting properties of plant and animal foods and to Performance Measure 4.1.2: Improve Human Health by Better Understanding the Nutrient Requirements of Individuals and the Nutritional Value of Foods. 2. Observational studies suggest increased consumption of dietary flavonoids may be inversely related to risk of coronary heart disease. We have found that dark chocolate, a rich source of cocoa flavanols and procyanidins, increases vascular responsiveness in healthy adults and reduces blood pressure and insulin resistance in people with essential hypertension. This work is related to National Program 107 – Human Nutrition, component 5: Health promoting properties of plant and animal foods and to Performance Measure 4.1.2: Improve Human Health by Better Understanding the Nutrient Requirements of Individuals and the Nutritional Value of Foods. 3. Muscle damage resulting from eccentric exercise provides a useful model of free radical-induced injury and age-related responses to oxidative stress. We reported that vitamin E supplementation prevents exercise-induced increases in selected biomarkers of oxidative stress in a complex relationship dependent upon age. In contrast, we found no benefit of vitamin E supplementation in older adults with rheumatoid arthritis. This work is related to National Program 107 – Human Nutrition, component 5: Health promoting properties of plant and animal food and to Performance Measure 4.1.2: Improve Human Health by Better Understanding the Nutrient Requirements of Individuals and the Nutritional Value of Foods. 4. Ischemia-reperfusion events are associated with acute ischemic stroke and may promote oxidative stress and contribute to brain injury. We found serum vitamins C and E and total antioxidant capacity is reduced during early post-stroke phases and returns toward baseline during the recovery period and concluded that our observations were consistent with increased antioxidant consumption during early ischemic brain injury in humans. This work is related to National Program 107 – Human Nutrition, component 5: Health promoting properties of plant and animal foods and to Performance Measure 4.1.2: Improve Human Health by Better Understanding the Nutrient Requirements of Individuals and the Nutritional Value of Foods. 5. Antioxidants such as vitamins C and E show radioprotective effects on normal cells in vitro but limited evidence in this regard is available in healthy tissue or tumor cells in vivo. We observed dietary vitamin C and epigallocatechin gallate slowed the growth of tumors in mice, but had modest radioprotective effects even as they reduced adverse soft-tissue reactions. This work is related to National Program 107 – Human Nutrition, component 4: Nutrient requirements and to Performance Measure 4.1.2: Improve Human Health by Better Understanding the Nutrient Requirements of Individuals and the Nutritional Value of Foods. 6. Cases of enhanced anticoagulant effect have been reported among patients taking oral anticoagulants in response to high dose vitamin E supplementation; however, vitamin E-vitamin K interactions have not been investigated in adults with normal baseline coagulation status. We found high dose vitamin E supplementation reduced the g-carboxylation of the vitamin K-dependent protein prothrombin among adults not receiving oral anticoagulant therapy. The clinical significance of these changes warrants further investigation, but it is possible that high doses of vitamin E antagonize vitamin K. This work is related to National Program 107 – Human Nutrition, component 5: Health promoting properties of plant and animal foods and to Performance Measure 4.1.2: Improve Human Health by Better Understanding the Nutrient Requirements of Individuals and the Nutritional Value of Foods. The Carotenoids and Health Laboratory conducts studies that compare the differences in the absorption of intact carotenoids and the conversion of provitamin A carotenoids to vitamin A from different plant sources and beta-carotene in oil capsules. Scientists are evaluating the effects of different plant foods and *-carotene in oil capsules on the bioavailability of provitamin A carotenoids and their conversion to retinol in subjects with or without vitamin A malnutrition. The major accomplishments during the life of this project include: Completion and data analysis of a study conducted during the previous CRIS cycle where the absorption and conversion to vitamin A of labeled provitamin A carotenes from a commonly consumed vegetable (spinach), and pure beta-carotene in oil capsules was measured in children with lower or normal vitamin A status. This study provides a model for evaluating the vitamin A value of plant foods at one or two blood sampling time points to be used in future studies to determine the vitamin A value of plant foods in children. These studies, along with our previous studies in well-nourished adults using carrots and green leafy vegetables, allow us to compare the conversion efficiency of beta-carotene to vitamin A of various plant foods. Information on the conversion of plant provitamin A carotenoids to vitamin A may provide a scientific basis for choosing alternative food sources of vitamin A for the general population. This may be of greater concern for the elderly population given that the serum vitamin A levels increase with age, which may indicate an increased risk of hypervitaminosis A. This work is related to Objective 1, Milestone 1: The bioavailability of carotenoids (ß-carotene, lycopene, lutein, and zeaxanthin) and vitamin A equivalence of provitamin A carotenoids using intrinsically labeled plants, such as, spinach and Golden Rice. We have completed study to further our understanding of the methodology and the application of antioxidant capacity measures in biological samples. This work is related to Objective 2, Milestone 2: The development and application of a biomarker assay of total antioxidant capacity in both the lipid and aqueous compartments of biological samples, and the interaction between various antioxidants. The application of these assays to older populations as well as the general population undertook various antioxidant food components will facilitate our investigation greatly. These accomplishments are related to National Program 107 – Human Nutrition program components 2: Bioavailability of nutrients and food components; and 5: Health promoting properties of plant and animal foods; and to Performance Measure 4.1.2 Improve Human Health by Better Understanding the Nutrient Requirements of Individuals and the Nutritional Value of Foods. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The research accomplishments of the ARL have been presented at several forums to research scientists, healthcare providers, and policy makers including: American Chemical Society, San Diego, CA; 5th International Conference and Exhibition on Nutraceuticals and Functional Foods, San Francisco, CA; Korean Society of Food and Nutrition Science, Jeju Island, South Korea; SupplySide West International Trade Show and Conference, Las Vegas, NV; California Avocado Commission, Laguna Beach, CA; Almond Board of California, Modesto, CA; Cranberry Institute and Cranberry Marketing Committee, San Diego, CA; Unilever Health Institute, Rotterdam, The Netherlands; as well as at Harvard Medical School, University of Southern California, University of British Columbia, Oregon State University, and University of Arizona. The research accomplishments of the CHL have been presented at several forums to research scientists, healthcare providers, and policy makers including: “Carotenoids and Human Health” Zhejiang Academy of Medical Sciences, Hangzhou, China, 2004; “Dietary Supplementation and Macular Pigmentation: What should be considered?” Macula and Nutrition Group, Association for Research in Vision and Ophthalmology Meeting, Fort Lauderdale, Florida, 2005 and Bausch & Lomb, Rochester, NY, 2005; “Synergistic interactions of antioxidant nutrients” School of Medicine, Yonsei University, Seoul, Korea, 2004; “Modification of Lymphocyte DNA damage by a physiologic dose of mixed carotenoids in Humans” Annual Meeting of French Free Radical Society, Paris, France, 2005. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). ARL research was presented in stories by several media outlets, including: Print media – Alternative Medicine, Shape, U.S. News & World Report, AARP Magazine, Consumer Reports on Health, Environmental Nutrition, USA Today, Wall Street Journal, Prevention, Detroit News, USA Weekend, Kansas City Star, Baltimore Sun, Cincinnati Enquirer, Los Angeles Times, Miami Herald, San Francisco Examiner, Washington Post; Radio - KGO (San Francisco, CA); Television - WCVB-TV (Boston, MA) ; Websites - WebMDHealth.com, TheBostonChannel.com. CHL research was highlighted on the website http://nutrition.tufts.edu/magazine/ 2004fall/vitamina3.html; and in www.foodproduction daily.com/news/news-NG.asp?id= 51668. “The egg and eye” and “The many faces of vitamin A” reported our work in Tufts Nutrition, volume 6, No. 1, Fall, 2004. The article “Biomarkers of antioxidant capacity in the hydrophilic and lipophilic compartments of human plasma” was one of the top 20 downloaded articles from the journal of Arch Biochem Biophys in 2004. Review Publications Chen, C., Blumberg, J.B. 2004. Use of biomarkers of oxidative stress in human studies [expanded abstract]. Journal of Nutrition. 134:3188S-3189S. Chen, C., Milbury, P., Lapsley, K., Blumberg, J. 2005. Flavonoids from almond skins are bioavailable and act synergistically with vitamins C and E to enhance hamster and human LDL resistance to oxidation. Journal of Nutrition. 135(6):1366-1373. Hathcock, J., Azzi, A., Blumberg, J., Bray, T., Dickinson, A., Frei, B., Jialal, I., Johnston, C.S., Kelly, F.J., Kraemer, K., Packer, L., Parthasarathy, S., Sies, H., Traber, M.G. 2005. Vitamins E and C are safe across a broad range of intakes. American Journal of Clinical Nutrition. 81:736-745. Ilsley, J.N., Belinsky, G.S., Guda, K., Zhang, Q., Blumberg, J.B., Milbury, P.E., Roberts, J., Stevens, R.G., Rosenberg, D.W., Huang, X. 2004. Dietary iron promotes azoxymethane-induced colon tumors in mice. Nutrition and Cancer. 49(2):162-169. Mayne, S., Walter, M., Goodwin, W.J., Blumberg, J. 2004. Supplemental beta-carotene, smoking, and urinary F2-isoprostane excretion in patients with prior early stage head and neck cancer. Nutrition and Cancer. 49(1):1-6. Ribaya-Mercado, J.D., Blumberg, J.B. 2004. The potential health benefits of lutein and zeaxanthin. The World of Food Ingredients: The Journal of the Practicing Food Technologist. July/August:p.44-46. Ribaya-Mercado, J.D., Blumberg, J.B. 2004. Lutein and zeaxanthin and their potential roles in disease prevention. Journal of American College of Nutrition. Dec;23(6 Suppl):567S-587S. Ribaya-Mercado, J.D., Solomons, N.W., Medrano, Y., Bulux, J., Dolnikowski, G.G., Russell, R.M., Wallace, C.B. 2004. Use of the deuterated-retinol-dilution technique to monitor vitamin a status of Nicaraguan school children one year after initiation of the Nicaraguan national program of sugar fortification with vitamin A. American Journal of Clinical Nutrition. 80:1291-1298. Shi, H., Paolucci, U., Milbury, P.E., Vigneau-Callahan, K.E., Matson, W.R., Krista, B.I. 2004. Development of biomarkers based on diet-dependent metabolic serotypes: Practical issues in development of expert system-based classification models in metabolomic studies. Omics - A Journal Of Integrative Biology. 8(3):197-208. Blumberg, J. 2004. Do dietary antioxidants really help prevent or treat cancer? [Commentary]In: Bagchi, D. Editor. Phytopharmaceuticals in Cancer Prevention. Boca Raton, Florida: CRC Press. p.639-642. Chen, C., Milbury, P., Li, T., O'Leary, J., Blumberg, J. 2005. Antioxidant capability and bioavailability of oat avenanthramides [abstract]. Journal of Federation of American Societies for Experimental Biology. 19(5):A1477. Graf, B.A., Milbury, P.E., Ameho, C.K., Li, T., Dolnikowski, G.G., Blumberg, J.B. 2004. Gastrointestinal tissues of the rat metabolize dietary quercetin [abstract]. Experimental Biology 2004:Meeting Abstracts. p. A516. Graf, B.A., Milbury, P., Ameho, C., Dolnikowski, G.G., Blumberg, J.B. 2005. Metabolism of dietary quercetin in the gastrointestinal tract, liver and kidney of the rat [abstract]. IIn The Cornucopia. The 229th National Meeting of the American Chemisal Society. March 3-17, 2005, San Diego, California. Paper No. 184. Chung, H., Rasmussen, H.M., Johnson, E.J. 2004. Lutein bioavailability is higher from lutein-enriched eggs than from supplements and spinach in men. Journal of Nutrition. 134(8):1887-93 Neuringer, M., Sandstrom, M.M., Johnson, E.J., Snodderly, D.M. 2004. Nutritional manipulation of primate retina, I: Effects of lutein or zeaxanthin supplements on serum and macular pigment in xanthophyll-free rhesus monkeys. Investigative Ophthalmology and Visual Science. 45(9):3234-3243. Yeum, K.J., Russell, R.M., Krinsky, N.I., Ginacarlo, A., 2004. Biomarkers of antioxidant capacity in the hydrophilic and lipophilic compartments of human plasma. Archives Of Biochemistry and Biophysics. 430(1):97-103. Johnson, E.J., Neuringer, M., Russell, R.M., Schalch, W., Snodderly, D.M. 2005. Nutritional manipulation of primate retinas. III: Effects of lutein or zeaxanthin supplementation on adipose tissue and retina of xanthophylls-free monkeys. Investigative Ophthalmology and Visual Science. 46(2):692-702. Putzbach, K., Krucker, M., Albert, K., Grusak, M.A., Tang, G., Dolnikowski, G.G. 2005. Structure determination of partially deuterated carotenoids from intrinsically labeled vegetables by hplc-ms and proton-nmr. Journal of Agriculture and Food Chemistry. 53 (3), 671 -677. Available at http://pubs.acs.org/cgi-bin/article.cgi/jafcau/2005/53/i03/pdf/jf0487506.pdf Preston, I.R., Tang, G., Tilan, J.U., Hill, N.S., Suzuki, Y.J. 2005. Retinoids and pulmonary hypertension. Circulation. 111:782-790. Ferreira, A.L., Ladeira, M.S., Salvadori, D.M., Yeum, K.J., Tang, G.W., Russell, R.M., Krinsky, N.I., Matsubara, L.S., Matsubara, B.B. 2004. Effect of lycopene on myocardial dysfunction and on dna damage induced by adriamycin in rats [abstract]. Federation of American Societies for Experimental Biology Conference. 18(4):A534. Johnson, E.J., Russell, R.M. 2004. Beta-carotene. In: Coates, P., Blackman, M., Cragg, G.M., Levine, M.A., Moss, J., White, J.D. Editors. Encyclopedia of Dietary Supplements. New York, NY: Marcel Dekker. p. 81-87. Snodderly, D.M., Chung, H., Caldarella, S., Johnson, E. 2005. The influence of supplemental lutein and docosahexaenoic acid on their serum levels and on macular pigment [abstract]. Investigative Ophthalmology and Visual Science. 2005;46: E-Abstract 1766¿B535. Available at http://abstracts.iovs.org YEUM, K.J., RUSSELL, R.M. CAROTENOID BIOAVAILABILITY AND BIOCONVERSION. ANNUAL REVIEW OF NUTRITION. 2002;22:483-504. Milbury, P.E., Graf, B.A., Blumberg, J.B., Curran-Celentano, J.M., Mcdonald, J., Vinqvist, M., Kalt, W. 2005. Anthocyanins cross the blood brain barrier: effects on oxidative stress-induced apoptosis [abstract]. In The Cornucopia. The 229th National Meeting of the American Chemical Society. March 3-17, 2005, San Diego, California. Paper No. 171.