2009 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.
The combined use of molecular genetic mapping for maize seed Fe bioavailability and a simulated digestion/intestinal cell uptake model for Fe bioavailability enabled us to develop identify regions of the maize genome where genes reside that influence maize seed Fe bioavailability. This information was used to breed for maize varieties with either high or low bioavailable Fe in the seed. These varieties were tested in poultry and were found to give the expected results. 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. These maize varieties differ only in 3 genetic regions and are otherwise identical genetically. If these lines can be consistently reproduced and shown to have nutritional benefit then, this is a major advancement in adding nutritional value to staple foods such as maize.
Prebiotics (ie. nondigestible carbohydrates) which are known to stimulate probiotic or beneficial intestinal bacteria were not found to have direct effects on Fe biovailability. Thus, it appears the potential benefits of prebiotics on Fe absorption are entirely indirect via the generation of probiotic bacteria. 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.
Our long term (ie. 4-6 week) feeding trials in poultry indicate that bean polyphenols do indeed inhibit Fe absorption and thus additional efforts should continue to determine the specific polyphenols in staple food crops that inhibit Fe bioavailability.
The development of a poultry model to assess Fe absorption at the level of the intestine has been developed. It has also been developed for long term feeding trials as reported above in our studies on maize. This model could be extremely useful in determining the factors and mechanisms that influence Fe bioavailability. It may also be useful in studying other nutrients. Further development and application of this model will occur in the coming year.
In the past year, numerous lines of wheat were screened for high and low inulin content to test the hypothesis whether non-digestible carbohydrates such as inulin increase the availability of Fe for animals and humans. Two varieties were planted and grown, however the expected differences did not occur; hence, no animal studies were performed to assess the effects of higher dietary inulin on Fe bioavailability.
Developed a Poultry Model to Assess Fe Bioavailability. Due to the complex interactions of Fe with factors in foods that affect the absorption of this essential nutrient, tools that enable scientists to isolate these interactions and factors are in high demand. We have developed surgical and feeding protocols that enable the use of the modern broiler chicken to assess Fe bioavailability from foods. This surgical model incorporates the unique anatomy of the chicken intestine allowing test substances (containing Fe) to be infused into the intestinal lumen and the measurement of absorption of that Fe via blood sampling from the duodenal vein. Essentially, these techniques enable measurement of Fe absorption from an “isolated” segment of live intestine under controlled conditions on an animal whose size and blood volume make it practical for repeated measurement of Fe uptake over a prolonged period of time (ie. 2 hours). In complement with the surgical techniques, we have developed the poultry model for “long term” measurement of Fe bioavailability. We expect this model to be extremely useful in development of staple food crops such as maize, wheat, lentils and beans for improved Fe biovailability.
Identified regions in the maize genome that influence 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. We have identified three genetic regions in maize that influence Fe bioavailability and have confirmed these effects in an animal feeding trial where the animals fed the “low” bioavailable Fe maize became anemic and those fed the “high” bioavailable Fe maize maintained their iron status. This is the first study to identify specific genomics regions harboring genes that control the bioavailability of iron in a staple food crop. This research has the potential to significantly improve Fe bioavailability to both humans and animals consuming maize as a major part of their diet.
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Tako, E., Glahn, R.P., Welch, R.M., Lei, X., Yasuda, K., Miller, D. 2008. Dietary inulin affects the expression of intestinal enterocyte iron transporters, receptors and storage protein and alters the microbiota in the pig intestine. British Journal of Nutrition. 99:472-480.
Lung'Aho, M.G., Glahn, R.P. 2009. Micronutrient Sprinkles Add More Bioavailable Iron to some Kenyan Complementary Foods: Studies Using an In Vitro Digestion/Caco-2 Cell Culture Model. Maternal and Child Nutrition. 5 (2):151-158.
Laparra, J., Glahn, R.P., Miller, D. 2009. Assesing potential effects of inulin and probiotic bacteria on Fe bioavailability from common beans (Phaseolus vulgaris L.) to Caco-2 cells. Journal of Food Science. 74(2):H40-46.
Laparra, J., Glahn, R.P., Miller, D.D. 2008. Bioaccessibility of phenols in common beans (Phaseolus vulgaris L.) and iron (Fe) availability to Caco-2 cells. Journal of Agricultural and Food Chemistry. 56(22):10999-11005.
Jin, F., Glahn, R.P., Rutzke, M., Zhiqiang, C., Welch, R.M. 2008. Extrinsic Labeling Method May Not Accurately Measure Fe Absorption from Cooked Pinto Beans (Phaseolus vulgaris):Comparison of Extrinsic and Intrinsic labeling of Beans. Journal of Agricultural and Food Chemistry. 56(16):6881-6885.
Prom-U-Thai, C., Glahn, R.P., Cheng, Z., Fukai, S., Rerkasem, B., Huang, L. 2009. The bioavailability of iron fortified in whole grain parboiled rice. Food Chemistry. 112(4):982-986.
Jin, F., Welch, R.M., Glahn, R.P. 2009. Effects of Ascorbic Acid, Phytic Acid and Tannic Acid on Ferritin-Iron Bioavailability as Determined Using an In Vitro Digestion/Caco-2 Cell Model. British Journal of Nutrition. 101(7):972-981.
Laparra, J., Tako, E., Glahn, R.P., Miller, D. 2008. Inulin affects iron dialyzability from FeSO4 and FeEDTA solutions but does not alter Fe uptake by Caco-2 cells. Journal of Agricultural and Food Chemistry. 56:2846-2851.
Mahler, G.J., Shuler, M.L., Glahn, R.P. 2008. Characterization of Caco-2 and HT29-MTX co-cultures in an in vitro digestion/cell culture model used to predict iron bioavailability. Journal of Nutritional Biochemistry. 20(7):485-562.
Laparra, J., Tako, E., Glahn, R.P., Miller, D. 2008. Supplemental inulin does not enhance iron bioavailability to Caco-2 cells from milk- or soy-based, probiotic-containing, yogurts but incubation at 37 oC does. Food Chemistry. 109:122-128.
Tan, S., Yeung, C., Glahn, R.P., Welch, R.M., Lei, X., Miller, D.D. 2008. Iron bioavailability to piglets from red and white common beans (Phaseolus vulgaris L.). Journal of Agriculture and Food Chemistry. 56(13):5008-14.
Laparra, J.M., Tako, E., Glahn, R.P., Miller, D. 2008. Isolated Glycosaminoglycans from Cooked Haddock Enhance Nonheme Iron Uptake by Caco-2 cells. Journal of Agricultural and Food Chemistry. 56(21):10346-10351.
Tako, E., Laparra, J., Glahn, R.P., Welch, R.M., Lei, X., Beebe, S., Miller, D.D. 2009. Biofortified black beans (Phaseolus vulgaris L.) in a maize/bean diet provide more bioavailable iron to piglets than standard black beans. Journal of Nutrition. 139(2):417-422.
Pachon, H., Stoltzfus, R., Glahn, R.P. 2009. HOMOGENIZATION, LYOPHILIZATION OR ACID-EXTRACTION OF MEAT PRODUCTS IMPROVES IRON UPTAKE FROM CEREAL-MEAT PRODUCT COMBINATIONS IN AN IN VITRO DIGESTION/CACO-2 CELL MODEL. British Journal of Nutrition. 101(6):816-821.
Pachon, H., Stoltzfus, R., Glahn, R.P. 2008. CHICKEN THIGH, CHICKEN LIVER AND IRON-FORTIFIED WHEAT FLOUR OPTIMIZE IRON UPTAKE IN AN INVITRO DIGESTION/CACO-2 CELL MODEL. Nutrition Research. 28(12):851-858.