2004 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 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 - Milestone 1 - Based on greenhouse trials, select kale line(s) with high lutein and flavonol content for more detailed studies. FY 2005 - Milestone 2 - Prepare/submit manuscript comparing ultraviolet light responses of kale lines under contrasting greenhouse light conditions (winter vs. summer). FY 2005 - Milestone 3 - In controlled environment chambers, determine conditions to optimize taproot growth and content of pigments in purple carrots. FY 2005 - Milestone 4 - Initiate cell culture studies with purple carrot and tomato fruit explants. FY 2005 - Milestone 5 - Complete pigment-rich fruit and vegetable juicing and analysis. FY 2005 - Milestone 6 - Prepare/submit manuscript on partitioning of pigments into juice and pulp. FY 2005 - Milestone 7 - Complete carrot juice pigment dose response study. FY 2005 - Milestone 8 - Complete plasma/urine analysis from carrot juice pigment dose response study. FY 2005 - Milestone 9 - Complete bioactivity assessment of different doses of pigments from purple carrot juice. FY 2005 - Milestone 10 - Purify and isolate tagged tomato pigment from synthesis by-products. FY 2005 - Milestone 11 - Complete modeling of tagged carotenoid time course in plasma of subjects who consumed specially tagged kale. FY 2006 - Milestone 1 - Label purple carrots with special isotopic tags. FY 2006 - Milestone 2 - Prepare and submit manuscript on carrot growth and labeling. FY 2006 - Milestone 3 - Initiate controlled environment chamber studies to optimize content of lutein and flavonoids in kale by manipulating light period duration, irradiance, spectral quality, UV radiation, carbon dioxide, and temperature. FY 2006 - Milestone 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. FY 2006 - Milestone 5 - Continue cell culture studies with purple carrot and tomato fruit explants. FY 2006 - Milestone 6 - Prepare/submit manuscript on the dose response of pigments from purple carrot juice. FY 2006 - Milestone 7 - Prepare/submit manuscript on the bioactivity measures related to dose of pigments from purple carrot juice. FY 2006 - Milestone 8 - Complete carotenoid-rich fruit and vegetable juicing and analysis. FY 2006 - Milestone 9 - Prepare/submit manuscript on partitioning of pigments into juice and pulp. FY 2006 - Milestone 10 - Complete tomato pigment dose-response study. FY 2006 - Milestone 11 - Complete tomato pigment analysis in blood samples from tagged tomato pigment dose-response study. FY 2006 - Milestone 12 - Complete bioactivity measures related to dose of tomato pigment. FY 2006 - Milestone 13 - Complete modeling of tagged beta-carotene and vitamin A time course in plasma after subjects consumed different doses of tagged beta-carotene. FY 2006 - Milestone 14 - Prepare/submit manuscript describing the kinetics of carotenoids absorbed from kale FY 2007 - Milestone 1 - Continue kale studies in controlled environment chambers. FY 2007 - Milestone 2 - Prepare and submit manuscript describing environmental effects on lutein and flavonol content in kale. FY 2007 - Milestone 3 - Continue tomato studies in greenhouse; test gas exchange apparatus for isolated leaf-fruit system. FY 2007 - Milestone 4 - Initiate growth chamber studies with girdled tomato plants; test response to day-length, irradiance, temperature, carbon dioxide, and UV radiation. FY 2007 - Milestone 5 - Continue cell culture studies with purple carrot and tomato fruit explants. FY 2007 - Milestone 6 - Label tomatoes using tagged carbon dioxide (pending availability of tagged carbon dioxide). FY 2007 - Milestone 7 - Complete clinical study of tagged tomato pigments to determine absorption of different forms. FY 2007 - Milestone 8 - Complete tagged tomato pigment analysis in plasma samples from clinical study to determine absorption of different forms. FY 2007 - Milestone 9 - Complete bioactivity measures related to forms of tomato pigments. FY 2007 - Milestone 10 - Prepare/submit manuscript on tomato pigment dose-response. FY 2007 - Milestone 11 - Prepare/submit manuscript on tomato pigment bioactivity as related to dose. FY 2007 - Milestone 12 - Complete analysis of tagged carotenoids in lipoprotein fractions of subjects who consumed tagged kale. FY 2007 - Milestone 13 - Complete modeling of tagged carotenoid time course in lipoprotein fractions of subjects who consumed tagged labeled kale. FY 2007 - Milestone 14 - Complete modeling of tagged tomato pigment time course in blood of subjects who consumed different doses of tagged tomato pigment. FY 2007 - Milestone 15 - Prepare/submit manuscript describing the kinetics of beta-carotene and vitamin A absorbed from different doses of labeled beta-carotene FY 2008 - Milestone 1 - Prepare/submit manuscript on production of labeled tomato (or identify and discuss strategies to do so). FY 2008 - Milestone 2 - Label kale with carbon tag (pending successful enhancement of lutein and flavonols and availability of carbon tag). FY 2008 - Milestone 3 - Test potential impact of isotope discrimination on content of lutein and flavonols in kale (contingency). FY 2008 - Milestone 4 - Evaluate suitability of in vitro systems for as candidates for labeling using carbon-tagged sugars or phenylalanine (contingency). FY 2008 - Milestone 5 - Complete clinical study of chylomicron carotenoid response from carrot/spinach/yellow potato. FY 2008 - Milestone 6 - Complete analysis of plasma samples from carrot/spinach/yellow potato chylomicron carotenoid response study. FY 2008 - Milestone 7 - Complete relative bioactivity of carotenoids from carrot, spinach, and yellow potato. FY 2008 - Milestone 8 - Prepare/submit manuscript on relative absorption of different forms of tomato pigment. FY 2008 - Milestone 9 - Prepare/submit manuscript on relative bioactivity of different forms of tomato pigment. FY 2008 - Milestone 10 - Complete modeling of tomato pigment time course in plasma of subjects after consumption of different forms of tomato pigment. FY 2008 - Milestone 11 - Complete modeling of purple pigment time course in plasma and urine after subjects consumed different doses of purple pigments in purple carrots. FY 2008 - Milestone 12 - Complete manuscript on the kinetics of carotenoids in lipoprotein fractions of subjects who consumed carbon-tagged kale. FY 2008 - Milestone 13 - Complete manuscript on the kinetics of tomato pigment in blood of subjects who consumed different doses of tomato pigment. FY 2009 - Milestone 1 - Prepare/submit manuscript on relative chylomicron response from carrots/spinach/yellow potato. FY 2009 - Milestone 2 - Prepare/submit manuscript on relative bioactivity of carotenoids from carrots, spinach, and yellow potato. FY 2009 - Milestone 3 - Complete clinical study of pigment dose-response from red potatoes. FY 2009 - Milestone 4 - Complete analysis of plasma samples from clinical study of pigment dose-response from blue potatoes. FY 2009 - Milestone 5 - Complete bioactivity measures as related to dose of pigment from red potatoes. FY 2009 - Milestone 6 - Prepare/submit manuscript on dose-response of pigment from red potatoes. FY 2009 - Milestone 7 - Prepare/submit manuscript on bioactivity as related to dose of pigment from red potatoes. FY 2009 - Milestone 8 - Complete modeling of pigment time course in plasma and urine after subjects consumed different doses of pigment in red potatoes. FY 2009 - Milestone 9 - Prepare/submit manuscript(s) on the kinetics of pigment from purple carrots and red potatoes. FY 2009 - Milestone 10 - Prepare/submit manuscript on the kinetics of different forms of tomato pigment. 3.Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. CRIS 107-1235-51000-046-00D is new, having been initiated on January 14, 2004. The first milestones are scheduled to be completed during FY 2005. Progress has been made on the following milestones: FY 2005 - Milestone 5 - Complete pigment-rich fruit and vegetable juicing and analysis: This work has been initiated with purple carrots. FY 2005 - Milestone 7 - Complete carrot juice pigment dose response study: We have recruited subjects, conducted the clinical trial, and collected blood and urine samples from the volunteers after they consumed the carrot juice. FY2005 Milestone 8 - Complete plasma/urine analysis from carrot juice pigment dose response study: Sample analysis is underway. FY 2005 - Milestone 10 - Purify and isolate tagged tomato pigment from synthesis by-products: Completed. B. List the milestones that you expect to address over the next 3 years (FY 2005, 2006, & 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY 2005 - Milestone 1 - Based on greenhouse trials, select kale line(s) with high lutein and flavonol content for more detailed studies. FY 2005 - Milestone 2 - Prepare/submit manuscript comparing ultraviolet light responses of kale lines under contrasting greenhouse light conditions (winter vs. summer). FY 2005 - Milestone 3 - In controlled environment chambers, determine conditions to optimize taproot growth and content of pigments in purple carrots. FY 2005 - Milestone 4 - Initiate cell culture studies with purple carrot and tomato fruit explants. FY 2005 - Milestone 5 - Complete pigment-rich fruit and vegetable juicing and analysis. FY 2005 - Milestone 6 - Prepare/submit manuscript on partitioning of pigments into juice and pulp. FY 2005 - Milestone 7 - Complete carrot juice pigment dose response study. FY 2005 - Milestone 8 - Complete plasma/urine analysis from carrot juice pigment dose response study. FY 2005 - Milestone 9 - Complete bioactivity assessment of different doses of pigments from purple carrot juice. FY 2005 - Milestone 10 - Purify and isolate tagged tomato pigment from synthesis by-products. FY 2005 - Milestone 11 - Complete modeling of tagged carotenoid time course in plasma of subjects who consumed specially tagged kale. FY 2006 - Milestone 1 - Label purple carrots with special isotopic tags. FY 2006 - Milestone 2 - Prepare and submit manuscript on carrot growth and labeling. FY 2006 - Milestone 3 - Initiate controlled environment chamber studies to optimize content of lutein and flavonoids in kale by manipulating light period duration, irradiance, spectral quality, UV radiation, carbon dioxide, and temperature. FY 2006 - Milestone 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. FY 2006 - Milestone 5 - Continue cell culture studies with purple carrot and tomato fruit explants. FY 2006 - Milestone 6 - Prepare/submit manuscript on the dose response of pigments from purple carrot juice. FY 2006 - Milestone 7 - Prepare/submit manuscript on the bioactivity measures related to dose of pigments from purple carrot juice. FY 2006 - Milestone 8 - Complete carotenoid-rich fruit and vegetable juicing and analysis. FY 2006 - Milestone 9 - Prepare/submit manuscript on partitioning of pigments into juice and pulp. FY 2006 - Milestone 10 - Complete tomato pigment dose-response study. FY 2006 - Milestone 11 - Complete tomato pigment analysis in blood samples from tagged tomato pigment dose-response study. FY 2006 - Milestone 12 - Complete bioactivity measures related to dose of tomato pigment. FY 2006 - Milestone 13 - Complete modeling of tagged beta-carotene and vitamin A time course in plasma after subjects consumed different doses of tagged beta-carotene. FY 2006 - Milestone 14 - Prepare/submit manuscript describing the kinetics of carotenoids absorbed from kale. FY 2007 - Milestone 1 - Continue kale studies in controlled environment chambers. FY 2007 - Milestone 2 - Prepare and submit manuscript describing environmental effects on lutein and flavonol content in kale. FY 2007 - Milestone 3 - Continue tomato studies in greenhouse; test gas exchange apparatus for isolated leaf-fruit system. FY 2007 - Milestone 4 - Initiate growth chamber studies with girdled tomato plants; test response to day-length, irradiance, temperature, carbon dioxide, and UV radiation. FY 2007 - Milestone 5 - Continue cell culture studies with purple carrot and tomato fruit explants. FY 2007 - Milestone 6 - Label tomatoes using tagged carbon dioxide (pending availability of tagged carbon dioxide). FY 2007 - Milestone 7 - Complete clinical study of tagged tomato pigments to determine absorption of different forms. FY 2007 - Milestone 8 - Complete tagged tomato pigment analysis in plasma samples from clinical study to determine absorption of different forms. FY 2007 - Milestone 9 - Complete bioactivity measures related to forms of tomato pigments. FY 2007 - Milestone 10 - Prepare/submit manuscript on tomato pigment dose-response. FY 2007 - Milestone 11 - Prepare/submit manuscript on tomato pigment bioactivity as related to dose. FY 2007 - Milestone 12 - Complete analysis of tagged carotenoids in lipoprotein fractions of subjects who consumed tagged kale. FY 2007 - Milestone 13 - Complete modeling of tagged carotenoid time course in lipoprotein fractions of subjects who consumed tagged labeled kale. FY 2007 - Milestone 14 - Complete modeling of tagged tomato pigment time course in blood of subjects who consumed different doses of tagged tomato pigment. FY 2007 - Milestone 15 - Prepare/submit manuscript describing the kinetics of beta-carotene and vitamin A absorbed from different doses of labeled beta-carotene. Increased consumption of fruits and vegetables is associated with decreased risk of chronic disease, presumably due to healthful components of those fruits and vegetables. Thus, we expect the most healthful fruits and vegetables to be those rich in these special plant nutrients called phytonutrients. Our work (FY 2005 Milestones 1, 2, 11; FY 2006 Milestones 3, 14; FY 2007 Milestones 1,. 2)will investigate factors that affect phytonutrient content of vegetables, using kale as a test vegetable. By identifying how to improve phytonutrient content of fruits and vegetables, we will provide a benefit to the consumer, who will receive improved protection from chronic disease, and we will provide benefit to the farmer, who sees an increased demand for his/her product. Our work (FY 2005 Milestones 3-9; FY 2006 Milestones 1, 2, 5-7, 9; FY 2007 Milestones. 5)will focus on specialty purple carrots that are rich in anthocyanins, a class of strong antioxidants. Our studies will clarify the potential health benefits of these specialty carrots and clarify how well the purple pigments are absorbed by the body. This will benefit small farmers who favor specialty crops by increasing demand for these carrots, and this will benefit the consumer by expanding the vegetable market with a healthful, good tasting product. Lycopene is the red pigment in tomatoes and has been repeatedly associated with decreased risk of prostate cancer. Yet the absorption of lycopene is poorly understood, thus making it difficult to make recommendations for daily intakes. Our studies (FY 2005 Milestones 4, 10; FY 2006 Milestones 4, 5, 10-12; FY 2007 Milestones 3-11, 14) will elucidate key issues surrounding lycopene absorption, thus benefiting the consumer, who can more effectively plan his/her diet. Tomato farmers will also benefit, as additional evidence will be generated to support the potential of lycopene to provide health benefits. Carotenoids are orange, yellow, and red pigments in fruits and vegetables. These compounds provide health benefits in several ways, including supplying vitamin A and preventing age-related blindness. Our studies (FY 2005 Milestone 11, FY2006 Milestone 8, FY 2007 Milestone 12, 13, 15) will clarify factors that affect absorption, thus providing health professionals and consumers with better information for planning diets to maximize health benefits. Moreover, we are poised to utilize a combination of specialty techniques of isotopic methodology, plant science, and chemistry to approach these problems in a unique way, such that our results will be especially pertinent to whole fruits and vegetables as they are actually consumed. 4.What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2004. Anthocyanins are purple pigments found in berries, purple carrots, and other blue, red, or purple foods, and these pigments have strong antioxidant capacities. However, little is known about the ability of the body to absorb these compounds. We have shown for the first time that anthocyanins can be absorbed from purple carrots, and we have shown the effect of dose and cooking on anthocyanin absorption. A human feeding study was conducted in which volunteers consumed raw or cooked purple carrots. Blood and urine samples were analyzed for the appearance of the purple pigments after carrot consumption. The demonstration that anthocyanins can be absorbed from purple carrots is expected to increase demand for purple carrots (thus, helping specialty crop farmers), and may benefit consumers and health professionals by supplying information about a source of healthful plant nutrients. The demonstration that absorption may be limited at high doses will be helpful to consumers and health professionals as diets are planned. B. Other significant accomplishments. Nothing to report. C. Accomplishments/activities that support special target populations. Nothing to report. D. Progress Report. Nothing to report. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. None. 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? None. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. Nothing to report.