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? Epidemiological studies have repeatedly shown that diets high in fruits and vegetables are associated with decreased risk of chronic diseases, such as cancer and cardiovascular disease, which ultimately strike most Americans. However, the scientific foundation necessary to translate these epidemiological findings into dietary recommendations is weak. Research is needed to determine the how well key nutrients are absorbed by the body and how quickly they are utilized, thus needing replacement. This project aims to provide sound scientific data on which to base improved recommendations for nutrient intakes. The establishment of reliable recommendations for nutrient intake levels is extremely important and has the potential for widespread impact in many ways. First, consumers rely on intake recommendations to guide them in their food intake. Second, Government-approved food programs are required to meet the appropriate level of nutrients. Third, national surveys of food intake are compared to nutrient intake recommendations to identify nutrition problems in the United States, and the discrepancies between nutrient intakes and recommended intakes are used to guide nutrition education programs and policy. For these reasons, nutrient recommendations are very influential, and reliable information on which to base these recommendations is vital. 2.List the milestones (indicators of progress) from your Project Plan. FY 2005 1 - Based on greenhouse trials, select kale line(s) with high lutein and flavonol content for more detailed studies. 2 - Prepare/submit manuscript comparing ultraviolet light responses of kale lines under contrasting greenhouse light conditions (winter vs. summer). 3 - In controlled environment chambers, determine conditions to optimize taproot growth and content of pigments in purple carrots. 4 - Initiate cell culture studies with purple carrot and tomato fruit explants. 5 - Complete pigment-rich fruit and vegetable juicing and analysis. 6 - Prepare/submit manuscript on partitioning of pigments into juice and pulp. 7 - Complete carrot juice pigment dose response study. 8 - Complete plasma/urine analysis from carrot juice pigment dose response study. 9 - Complete bioactivity assessment of different doses of pigments from purple carrot juice. 10 - Purify and isolate tagged tomato pigment from synthesis by-products. 11 - Complete modeling of tagged carotenoid time course in plasma of subjects who consumed specially tagged kale. FY 2006 1 - Label purple carrots with special isotopic tags. 2 - Prepare and submit manuscript on carrot growth and labeling. 3 - Initiate controlled environment chamber studies to optimize content of lutein and flavonoids in kale by manipulating light period duration, irradiance, spectral quality, ultraviolet (UV) radiation, carbon dioxide, and temperature. 4 - Initiate greenhouse studies on girdled tomato leaf-fruit systems; determine ability to block phloem transport long-term and impact of treatment on fruit development and red pigment content. 5 - Continue cell culture studies with purple carrot and tomato fruit explants. 6 - Prepare/submit manuscript on the dose response of pigments from purple carrot juice. 7 - Prepare/submit manuscript on the bioactivity measures related to dose of pigments from purple carrot juice. 8 - Complete carotenoid-rich fruit and vegetable juicing and analysis. 9 - Prepare/submit manuscript on partitioning of pigments into juice and pulp. 10 - Complete tomato pigment dose-response study. 11 - Complete tomato pigment analysis in blood samples from tagged tomato pigment dose-response study. 12 - Complete bioactivity measures related to dose of tomato pigment. 13 - Complete modeling of tagged beta-carotene and vitamin A time course in plasma after subjects consumed different doses of tagged beta-carotene. 14 - Prepare/submit manuscript describing the kinetics of carotenoids absorbed from kale. FY 2007 1 - Continue kale studies in controlled environment chambers. 2 - Prepare and submit manuscript describing environmental effects on lutein and flavonol content in kale. 3 - Continue tomato studies in greenhouse; test gas exchange apparatus for isolated leaf-fruit system. 4 - Initiate growth chamber studies with girdled tomato plants; test response to day-length, irradiance, temperature, carbon dioxide, and UV radiation. 5 - Continue cell culture studies with purple carrot and tomato fruit explants. 6 - Label tomatoes using tagged carbon dioxide (pending availability of tagged carbon dioxide). 7 - Complete clinical study of tagged tomato pigments to determine absorption of different forms. 8 - Complete tagged tomato pigment analysis in plasma samples from clinical study to determine absorption of different forms. 9 - Complete bioactivity measures related to forms of tomato pigments. 10 - Prepare/submit manuscript on tomato pigment dose-response. 11 - Prepare/submit manuscript on tomato pigment bioactivity as related to dose. 12 - Complete analysis of tagged carotenoids in lipoprotein fractions of subjects who consumed tagged kale. 13 - Complete modeling of tagged carotenoid time course in lipoprotein fractions of subjects who consumed tagged labeled kale. 14 - Complete modeling of tagged tomato pigment time course in blood of subjects who consumed different doses of tagged tomato pigment. 15 - Prepare/submit manuscript describing the kinetics of beta-carotene and vitamin A absorbed from different doses of labeled beta-carotene. FY 2008 1 - Prepare/submit manuscript on production of labeled tomato (or identify and discuss strategies to do so). 2 - Label kale with carbon tag (pending successful enhancement of lutein and flavonols and availability of carbon tag). 3 - Test potential impact of isotope discrimination on content of lutein and flavonols in kale (contingency). 4 - Evaluate suitability of in vitro systems as candidates for labeling using carbon-tagged sugars or phenylalanine (contingency). 5 - Complete clinical study of chylomicron carotenoid response from carrot/spinach/yellow potato. 6 - Complete analysis of plasma samples from carrot/spinach/yellow potato chylomicron carotenoid response study. 7 - Complete relative bioactivity of carotenoids from carrot, spinach, and yellow potato. 8 - Prepare/submit manuscript on relative absorption of different forms of tomato pigment. 9 - Prepare/submit manuscript on relative bioactivity of different forms of tomato pigment. 10 - Complete modeling of tomato pigment time course in plasma of subjects after consumption of different forms of tomato pigment. 11 - Complete modeling of purple pigment time course in plasma and urine after subjects consumed different doses of purple pigments in purple carrots. 12 - Complete manuscript on the kinetics of carotenoids in lipoprotein fractions of subjects who consumed carbon-tagged kale. 13 - Complete manuscript on the kinetics of tomato pigment in blood of subjects who consumed different doses of tomato pigment. FY 2009 1 - Prepare/submit manuscript on relative chylomicron response from carrots/spinach/yellow potato. 2 - Prepare/submit manuscript on relative bioactivity of carotenoids from carrots, spinach, and yellow potato. 3 - Complete clinical study of pigment dose-response from red potatoes. 4 - Complete analysis of plasma samples from clinical study of pigment dose-response from blue potatoes. 5 - Complete bioactivity measures as related to dose of pigment from red potatoes. 6 - Prepare/submit manuscript on dose-response of pigment from red potatoes. 7 - Prepare/submit manuscript on bioactivity as related to dose of pigment from red potatoes. 8 - Complete modeling of pigment time course in plasma and urine after subjects consumed different doses of pigment in red potatoes. 9 - Prepare/submit manuscript(s) on the kinetics of pigment from purple carrots and red potatoes. 10 - Prepare/submit manuscript on the kinetics of different forms of tomato pigment. 4a.What was the single most significant accomplishment this past year? We showed that one class of red-purple antioxidant pigments (non-acylated anthocyanins) are significantly more bioavailable than another class (acylated anthocyanins), that cooking improved absorption of some but not all of these antioxidant pigments, and that the body appears to have a limit in its ability to absorb these healthful pigments. Consumers are seeking more ways to incorporate healthful antioxidants into their diets, and our study has provided information about how to do that effectively. We conducted a clinical feeding study in which purple carrots were fed to volunteers. The carrots were fed raw or cooked and in two dose levels. Blood and urine from study volunteers was analyzed for pigment appearance over time after the carrot doses. These results will benefit consumers and health professionals as diets are planned. 4b.List other significant accomplishments, if any. We showed that the yellow pigment lutein (which reduces risk of age-related blindness) is better absorbed from kale than orange pigment beta-carotene (an important source of vitamin A), that the vitamin A formed from the kale beta-carotene was related to circulating vitamin A levels, and that lutein absorption may be related to beta-carotene absorption. Consumers are seeking more ways reduce risk of chronic disease through diet, and our study has provided information about how to do that effectively. We conducted a clinical feeding study in which specially tagged kale was fed to volunteers. Blood samples from study volunteers were analyzed for appearance of kale nutrients over time after the kale was consumed. These results will benefit consumers and health professionals as diets are planned. 4c.List any significant activities that support special target populations. None. 4d.Progress report. This report serves to document research conducted under a trust agreement with Cooperative State Research, Education, and Extension Service (CSREES.) Additional details of research can be found in the report for project 1235-51000-046-00D, Bioavailability and metabolism of phytochemicals and other micronutrients. Anthocyanins are purple pigments found in berries, purple carrots, and other blue, red, or purple foods, and these pigments have strong antioxidant capacities. Further, there is interest in developing these pigments into commercial food colorants, especially the acylated forms, which are more stable than the non-acylated forms. Purple carrots contain both acylated and non-acylated anthocyanins, so we used purple carrots as a vehicle to deliver these anthocyanins to humans for a bioavailability study. In addition, we tested whether cooking or dose size affected anthocyanin bioavailability, two parameters that have never been tested in the past for anthocyanins. Through a human feeding study, we showed that non-acylated anthocyanins are significantly more bioavailable than acylated anthocyanins, that cooking increased the bioavailability of non-acylated anthocyanins but not acylated antocyanins, and that anthocyanin absorption is saturable. These results will benefit consumers and health professionals as diets are planned. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. This project provides valuable information for the National Academy of Sciences to use in establishing intake recommendations. Specific information includes efficiency of absorption and metabolism of plant nutrients, including carotenoids, vitamins, minerals, and polyphenols, as well as new methodologies to assess these issues. 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? Science transferred to other scientists includes the following: information about the effect of cooking on carrot anthocyanin bioavailability, information about the effect of acylation on anthocyanin bioavailability, and information about the kinetics of carotenoid metabolism. There are no expected constraints related to the technology to be produced. 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). Quoted in Eating Well Magazine concerning synergistic effects among phytonutrients. August 2005. Quoted in Washington Post regarding the benefit of whole foods over nutrient supplements. August 2004. Presentation entitled "Phytonutrient Bioavailability Studies at the Beltsville Human Nutrition Research Center" for the American Chemical Society Washington Area meeting. May 2005. Presentation entitled "What can pharmacokinetic models tell us about lycopene disposition and its potential role in cancer prevention?" for the National Cancer Institute Workshop entitled Promises and Perils of Lycopene/Tomato Supplementation and Cancer Prevention. February 2005. Presentation entitled "Stable Isotopes and Compartmental Modeling to Understand Carotenoid Absorption in Humans" for the University of California Department of Nutrition. May 2005. Presentation entitled "Nutritional Science and the Food Pyramid" for the Rosemary Hills Primary School Career Series. April 2005. Presentation entitled "Tropical and Subtropical Fruits: Phytonutrients and Anticipated Health Benefits" at the Third International Symposium on Tropical and Subtropical Fruits, Fortaleza, Brazil, September 14, 2004. Presentation entitled "Lycopene: Factors Affecting Bioavailability" at the Tomato Breeders’ Roundtable. Annapolis, MD, October 20, 2004. Presentation entitled "Phytonutrients and Human Health" for the George Washington University. April 11, 2005. Presentation (at request of International Food Information Council) entitled "The Polyphenols: Science and Food Sources" at Institute of Food Technologists Meeting in session entitled "Functional Foods: Opportunities and Challenges." New Orleans, LA, July 16, 2005. Review Publications Clevidence, B.A., Kurilich, A.C., Britz, S., Simon, P.W., Novotny Dura, J. 2004. Bioavailability of anthocyanins from raw and cooked purple carrots. Proceedings of the 33rd U.S.-Japan Cooperative Program in Natural Resources. UJNR 33:171-174. Novotny Dura, J. 2005. Mathematical investigations of carotenoid kinetics in humans. Mathematical Modeling in Health and Toxicology. Athens, GA: Mathematical Biology Press. 189-204. Kurilich, A.C., Clevidence, B.A., Britz, S.J., Simon, P.W., Novotny Dura, J. 2005. Plasma and urine responses are lower for acylated vs nonacylated anthocyanins from raw and cooked purple carrot. Journal of Agricultural and Food Chemistry. 53:6537-6542.