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United States Department of Agriculture

Agricultural Research Service

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Location: Plant, Soil and Nutrition Research

2010 Annual Report

1a. Objectives (from AD-416)
Objective 1. Determine the genes affecting iron level and bioavailability in edible portions of maize by utilizing quantitative trait loci (QTL) mapping and an in-vitro digestion/Caco-2 cell culture model system. Confirm this QTL information via in-vitro and in-vivo studies and transfer this information to plant breeders. Objective 2. Investigate the role and mechanisms of action of prebiotic compounds, beneficial bacteria, and plant phenolic constituents on the bioavailability of iron and zinc from the diet.

1b. Approach (from AD-416)
This project seeks to improve the bioavailability of iron (Fe) and zinc (Zn) in plant foods by two distinct strategies. First, a multidisciplinary, genomics-based approach will be used to identify the genes and chemical factors that modulate Fe bioavailability in maize grain, using maize as the model to demonstrate proof of concept for this work. The second approach defines how prebiotic (non-digestible carbohydrates) and polyphenolic compounds in plant foods influence Fe and Zn bioavailability and thereby enhance absorption of Fe and Zn from plant foods in the intestine. Maize Fe Bioavailability: The goal here is to improve the nutritional quality of maize with respect to Fe via identifying the genetic factors that contribute to improved seed-iron concentration and bioavailability. This will be accomplished by building on current genetic mapping of maize seed-Fe bioavailability using a simulated intestinal digestion/Caco-2 cell assay. Animal models will then be used to confirm the cell culture results before definitive testing in humans. By identifying the genes that contribute to seed-iron accumulation and bioavailability, plant breeders will be able to improve the nutritional quality of maize. Prebiotics, Beneficial Bacteria, Polyphenols and Fe/Zn Bioavailability: The gastrointestinal microflora is increasingly becoming recognized as a major factor in human health via systemic effects on gut health and nutrient absorption. Prebiotic compounds such as inulin dramatically alter the profile of intestinal microflora and this alteration may enhance absorption of Fe and Zn. Therefore, one of the goals here is to determine if prebiotics enhance Fe and Zn absorption via changes in gut microflora. The second subobjective is to determine if polyphenolic compounds play a significant role in Fe absorption in long term feeding trials. This will be accomplished via animal feeding trials and cell culture models, whereby foods will be compared that have low and high amounts of prebiotics (eg. inulin, raffinose, stachyose) and polyphenols (e.g., kaempferol, quercitin, chlorogenic acid, ferulic acid). In these studies Fe and Zn absorption from these foods will be measured, as well as their effect on the bacterial profile. By understanding the role of prebiotics and polyphenols in the absorption of these two essential minerals, plant foods and food products can be improved to provide more optimal nutrition.

3. Progress Report
In the previous year and this year, we used a combination of molecular genetic mapping for maize seed Fe bioavailability and a simulated digestion/intestinal cell uptake model for Fe bioavailability to identify regions of the maize genome where genes reside that influence maize seed Fe bioavailability. We developed maize with high and low bioavailable Fe in the seed (ie. kernels). These varieties have now been tested twice in poultry and were found to give the expected results on both occasions. That is the birds fed the “high” bioavailable Fe maize maintained their Fe status whereas the birds on the “low” bioavailable Fe maize became anemic. We have therefore demonstrated some consistency in our ability to reproduce these lines and that we can produce in relatively large quantities (ie. several tons of each). These varieties now warrant testing in human subjects for final definitive confirmation that the nutritional value of iron in the maize has been enhanced. We are moving towards identifying the compounds that account for the differences in the above-mentioned maize lines. However, this research is impeded by the lack of a critical piece of equipment, specifically an LC mass spectrometer with state-of-the-art software. This equipment will enable us to identify the compounds or metabolites in the maize seed that are related to Fe bioavailability. In previous years and in this year our research showed that consumption of prebiotics (ie. nondigestible carbohydrates) such as inulin enhances Fe absorption from foods. These prebiotics are known to stimulate probiotic or beneficial intestinal bacteria but have not been shown to have direct effects on Fe biovailability. Moreover, the effects do not appear to be due to enhanced uptake of Fe from the colon, but more likely to enhanced uptake from the small intestine. Thus, it appears the potential benefits of prebiotics on Fe absorption are mostly indirect via the generation of probiotic bacteria, which probably allow for a healthier intestine. A common prebiotic, inulin, does not appear to be present in sufficient amounts in staple food crops such as wheat or maize to promote Fe absorption; hence we are working with one of our collaborators to see if similar compounds (ie. fructans) which are present in wheat at levels of 3-6% have prebiotic effects (ie. promotion of Fe absorption).

4. Accomplishments
1. Produced maize with improved iron (Fe) bioavailability. Iron bioavailability in maize is relatively low; hence populations that consume maize as a major staple in their diet are at risk for Fe deficiency. In previous years ARS scientists at the Robert W. Holley Center for Agriculture & Health in Ithaca, NY identified regions in the maize genome that influence Fe bioavailability, and produced relatively small amounts of a high and low bioavailable Fe maize. In the past year we have demonstated the ability to produce this maize in relatively large quantities (ie. approximately 3 tons). Recent completion of a poultry feeding trial using maize from this large harvest has confirmed the nutritional benefits of the “high” Fe maize. This is the first series of studies to identify specific genomics regions harboring genes that control the bioavailability of iron in a staple food crop, and to produce this crop consistently in a large quantity. This research has the potential to significantly improve Fe bioavailability to both humans and animals consuming maize as a major part of their diet, and thus alleviate iron deficiency.

Review Publications
Argyri, K., Tako, E., Miller, D., Glahn, R.P., Komaitis, M., Kapsokefalou, M. 2009. Milk peptides increase iron solubility in water but do not affect DMT-1 expression in Caco-2 cells. Journal of Agricultural and Food Chemistry. 57(4):1538-1543.

Patterson, J., Rutzke, M.A., Fubini, S.L., Glahn, R.P., Welch, R.M., Lei, X., Miller, D.D. 2009. Dietary inulin supplementation does not promote colonic iron absorption in a porcine model. Journal of Agricultural and Food Chemistry. 57(12):5250-5256.

Laparra, J., Barbera, R., Alegria, A., Glahn, R.P., Miller, D.D. 2009. Purified glycosaminoglycans from cooked haddock may enhance Fe uptake via endocytosis in a Caco-2 cell culture model. Journal of Food Science. 74(6):H168-173.

Zhu, L., Glahn, R.P., Nelson, D., Miller, D.D. 2009. Comparing soluble ferric pyrophosphate to common iron salts and chelates as sources of bioavailable iron in a caco-2 cell culture model. Journal of Agricultural and Food Chemistry. 57(11):5014-5019.

Mahler, G., Esch, M.B., Glahn, R.P., Shuler, M.L. 2009. Characterization of a gastrointestinal tract microscale cell culture analog used to predict drug toxicity. Biotechnology and Bioengineering. 104(1):193-205.

Tako, E., Rutzke, M.A., Glahn, R.P. 2010. Using the domestic chicken (Gallus gallus) as an in vivo model for iron bioavailability. Poultry Science. 89:514:521.

Lung'Aho, M.G., Glahn, R.P. 2009. In vitro estimates of iron bioavailability in some Kenyan complementary foods. Food and Nutrition Bulletin. 30(2):145-152.

Laparra, J., Glahn, R.P., Miller, D.D. 2009. Effects of Tea Phenolics on Iron Uptake from Different Fortificants by Caco-2 cells. Food Chemistry. 115(3):974-981.

Laparra, J., Glahn, R.P., Miller, D. 2009. Different responses of Fe transporters in Caco2/HT29-MTX cocultures than in independent Caco-2 cell cultures. Cell Biology International. 33(9):971-977.

Tako, E., Glahn, R.P., Laparra, J.M., Welch, R.M., Lei, X., Kelly, J.D., Rutzke, M.A., Miller, D.D. 2009. Iron and zinc bioavailabilities to pigs from red and white beans (Phaseolus vulgaris L.) are similar. Journal of Agricultural and Food Chemistry. 57(8):3134-3140.

Young, M.F., Glahn, R.P., Ariza-Nieto, M. 2009. Serum hepcidin is significantly associated with iron absorption from food and supplemental sources in healthy young woman. American Journal of Clinical Nutrition. 89(2):533-538.

Last Modified: 2/23/2016
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