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ARS Home » Northeast Area » Ithaca, New York » Robert W. Holley Center for Agriculture & Health » Plant, Soil and Nutrition Research » Research » Research Project #426380

Research Project: Bioavailability of Iron, Zinc and Select Phytochemicals for Improved Health

Location: Plant, Soil and Nutrition Research

2018 Annual Report

1. In collaboration with international teams of plant breeders, we will develop lines of staple food crops such as beans, pearl millet, sorghum, rice, lentils and maize with improved Fe and Zn content and bioavailability. 1.A. Identify molecular markers and ultimately the genes that influence iron level and bioavailability in maize seed. Quantitative trait loci (QTL) mapping and an in-vitro digestion/Caco-2 cell culture model system will be used for this research sub-objective. 1.B. Identify molecular markers and ultimately the genes that influence iron level and bioavailability in lentils. Quantitative trait loci (QTL) mapping and an in-vitro digestion/Caco-2 cell culture model system will be used for this research sub-objective. 1.C. Identify agricultural and processing practices that enhance the nutritional quality of Fe in staple food crops. 1.D. Continue our work to identify compounds that promote or inhibit Fe bioavailability in staple food crops. 2. Evaluate a surgical duodenal loop model in poultry as a tool to measure bioavailability of health promoting phytochemicals. 2.A. Determine if absorption of selected phytochemicals can be measured in the poultry duodenal loop model. 2.B. Define bioavailability of specific phytochemicals from select foods using the validated duodenal loop model.

This project has two major objectives. The first is to develop lines of staple food crops with improved bioavailability and content of iron (Fe). To do so we will employ our established screening tools of an iron bioavailability bioassay and an animal model. In conjunction with these tools, we will make use of mass spectroscopy and marker assisted molecular breeding techniques. With these approaches, we will be able to identify and utilize regions of the plant genome that harbor genes associated with the enhanced food crop nutritional quality for Fe. To further complement the above approaches, we will continue our work on the identification of agricultural and food processing practices that enhance the bioavailability and content of Fe and Zn in staple food crops. The second major objective of this project is to develop a method to measure and evaluate the bioavailability of functional phytochemicals from plant foods. To do so, we will assess the use of a surgical model utilizing the unique intestinal anatomy of the broiler chicken to measure absorption of compounds from the duodenal segment of the intestine. This approach uses an established surgical preparation involving anesthetized animals. Our initial efforts with this model will focus on compounds that are common to many staple food crops, fruits and vegetables. These phytochemicals will include the flavonoids epicatechin, quercitin, caffeic acid, chlorogenic acid and kaempferol. Enhancing our knowledge of bioavailability of health promoting phytochemicals will significantly improve efforts to develop more nutritious plant foods.

Progress Report
Maize Research: All milestones from the planned maize research, Hypotheses 1.A.1, 1.A.2, and 1.A.3 are no longer applicable. This was primarily due to the departure of our in-house maize breeder associated with this project within the first year of this project plan. In an attempt to recover from this setback, we established a collaboration with another maize breeder who provided a mapping population from which we identified several QTL for Fe concentration and Fe bioavailability. However, the maize breeder was unable to follow up on this information and perform additional breeding. Given the apparent difficulties we have abandoned this approach for improving the nutritional quality of Fe in maize. As an alternative approach to improving the nutritional quality of Fe in maize, we conducted studies to determine the concentration and bioavailability of Fe from the endosperm and the germ fractions of maize. We observed that the germ fraction was quite dense in Fe that was poorly available for absorption. We also observed that the Fe in the endosperm can be highly available, particularly when the germ fraction was not present in the meal. These observations indicate that the germ fraction inhibits Fe absorption from the maize kernel, presumably due to high levels of phytate, an inhibitor of Fe bioavailability, known to be present in the germ fraction. In addition these observations suggest that breeding for enhanced Fe in the endosperm fraction represents a strategy to improve the nutritional quality of maize for populations at risk of Fe deficiency. The above observations are significant as they suggest that consuming maize without the germ fraction is a better source of Fe relative to consuming the whole kernel. Degermed corn products are indeed in the market place and thus can and may already be providing enhanced Fe nutrition. Indeed we have analyzed such products in our lab and found the degermed meal to exhibit higher Fe bioavailability. This is a new and exciting observation as degerming corn meal could therefore represent a ready to go nutritional intervention that could improve Fe nutrition from populations consuming maize as a major portion of their diet. In addition, this observation could lead to a new and easily measured maize breeding objective of increasing the endosperm Fe density. Bean Research: In collaboration with the ARS-Sugarbeet and Bean Research Unit located at Michigan State University, a Yellow Bean Panel (YBP) was assembled to explore the cooking time and health benefits of the five major seed types within the yellow bean market class (Amarillo, Canary, Manteca, Mayocoba, Soya Njano) over the course of two field seasons. This study shows how the Manteca yellow bean possess a fast cooking phenotype, which could serve a genetic resource for introducing fast cooking properties into a new generation of dry beans with cooking times. Nutritional evaluation revealed fast cooking yellow beans have the advantage of high iron and zinc retention (>80%) after boiling. An in vitro digestion/Caco-2 cell culture bioassay revealed a strong negative association between cooking time and iron bioavailability in the YBP (r values > -0.73). When either pre-soaked or left unsoaked the highest iron bioavailability scores were measured in the fast cooking Manteca genotypes providing evidence that these unique landraces are worthy of germplasm enhancement through the added benefit of improved iron quality after cooking. Bean samples were assembled from marketplaces and bean breeders of multiple countries of east Africa. These samples were measured for Fe content to document the average bean Fe concentration in the African marketplace, and from CIAT (Center for International Tropical Agriculture) bean breeders. Additionally, this work sought to determine if the lines released as “high Fe” varieties by bean breeders maintained their original Fe levels after being released to farmers. The results indicate that the “high Fe” varieties do not maintain their original Fe levels. These results are significant as they suggest that breeding for high Fe content in beans may not be a sustainable approach to improving beans as a source of dietary Fe. In collaboration with scientists from Brazil, we demonstrated in our cell culture model and in an animal feeding trial that certain carioca color bean varieties can provide enhanced Fe nutrition when consumed alone or as part of a typical Brazilian vegetarian diet. This work is significant as it is an excellent example of how the application of these model systems can be applied to improve the nutritional quality of Fe in the human food supply. Yellow beans are a developing dry bean market class in the U.S. and abroad. In Africa, they are more established and are quite popular in certain regions. Our research involving yellow beans shows that it may be the one color class that can have a profile of compounds (polyphenols) in the seed coat that can promote the absorption (bioavailability) of Fe from the bean. This research is significant as it identifies yellow bean varieties that can be advanced for improved Fe nutrition. Progress was made with the further characterization and identification of a specific intestinal microbiome profile under various dietary conditions. For example, a recent comparison between birds receiving a standard wheat based diet relative to subjects receiving a Zn biofortified wheat based diet. Results indicated that dietary Zn content affected the relative abundances of intestinal microbial species. Through metagenomic analysis, we showed that predicted Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways responsible for macro- and micronutrient uptake are significantly depleted under Zn deficiency; along with concomitant decreases in beneficial short chain fatty acids, such depletions may further preclude optimal host Zn availability. We also identified several candidate microbes that may play a significant role in modulating the bioavailability and utilization of dietary Zn during prolonged deficiency. Our results are the first to characterize a unique and dysbiotic cecal microbiota during Zn deficiency, and provide evidence for such microbial perturbations as potential effectors of the Zn deficient phenotype. Further, and by using the red blood cell fatty acid ratio as a novel biomarker for Zn status, we were able to validate its reactiveness to dietary Zn in wheat based diets. This is the first demonstration of Zn biofortified wheat to improve Zn status in vivo. Overall, these findings will allow better understanding of the potential effects that dietary Zn has on various physiological process and on dietary Zn bioavailability and absorption.

1. High Fe carioca beans from Brazil provide more bioavailable Fe. In Brazil, the prevalence of Fe deficiency is high in populations that rely primarily on staple foods such as beans. To combat Fe deficiency, bean breeders and nutritionists in Brazil have developed high Fe common beans of the “carioca” color class. In collaboration with Brazilian scientists, we utilized our cell culture model and our poultry model to demonstrate that high Fe carioca beans would provide improved Fe nutrition when consumed in a typical Brazilian diet. This research is significant as it confirms this bean variety as a “biofortified” line that can be released with confidence of nutritional benefit. Overall, this work demonstrates the culmination of our bioavailability models as valuable tools for enhancing human Fe nutrition.

2. Development of a novel zinc (Zn) status physiological biomarker system to assess Zn nutritional status. Dietary Zn deficiency affects 20% of the world’s population. The paucity of reliable Zn biomarkers, and a representative animal model in which to test them, has made the assessment of human Zn deficiency difficult. Studies have shown plasma/serum Zn to be insensitive indicators of Zn status. The need to develop robust indicators of Zn status is prioritized by the World’s Health Organization. This accomplishment by ARS researchers in Ithaca, New York, included the identification, confirmation and implementation of a novel physiological biomarker of Zn status pertaining to red blood cell fatty acids ratio. The assay proved to be sensitive to Zn depletion over time demonstrating the assay’s robustness. The Zn biomarker was validated in a human efficacy study, allowing establishment of a platform for reliable Zn status assessment. This represents a significant step forward enabling more detailed studies of Zn nutrition.

Review Publications
Glahn, R.P., Tako, E., Hart, J.J., Haas, J., Lung'Aho, M., Beebe, S. 2017. Iron bioavailability studies of the first generation of iron-biofortified beans released in Rwanda. Nutrients. 9(7):787.
Hou, T., Tako, E.N. 2018. The in ovo feeding administration (gallus gallus)-an emerging in vivo approach to assess bioactive compounds with potential nutritional benefits. Nutrients. 10(4):418.
Knez, M., Stangoulis, J., Tako, E. 2017. The linoleic acid: dihomo-y-linolenic acid ratio (LA:DGLA)— an emerging biomarker of Zn status. Nutrients. 9(8):825.
Dias, D., Costa, N., Nutti, M., Tako, E.N., Martino, H. 2017. Advantages and limitations of in vitro and in vivo methods of iron and zinc bioavailability evaluation in the assessment of biofortification program effectiveness. Critical Reviews in Food Science and Nutrition.
Podder, R., Dellavalle, D., Tyler, R., Glahn, R.P., Tako, E.N., Vandenberg, A. 2018. Relative bioavailability of iron in Bangledeshi traditional meals prepared with iron-fortified lentil dal. Nutrients. 10(3).
Fleige, L., Bhoite, R., Marwaha, A., Glahn, R.P. 2018. Development of a fortified juice beverage: assessment of iron bioavailability using an established caco-2 cell bioassay. Journal of Nutrition & Food Sciences. 6(3):1-7.
Hart, J.J., Tako, E.N., Glahn, R.P. 2017. Characterization of polyphenol effects on inhibition and promotion or iron update by caco-2 cells. Journal of Agricultural and Food Chemistry. 65(16):3285-3294.
Reed, S., Knez, M., Uzan, A., Stangoulis, J., Glahn, R.P., Koren, O., Tako, E.N. 2018. Alterations in the gut (Gallus gallus) microbiota following the consumption of zinc biofortified wheat (Triticum aestivum) -based diet. Journal of Agricultural and Food Chemistry. 66(25):6291-6299.