Progress 10/01/99 to 09/30/09
Outputs
OUTPUTS: We completed two major studies using our in vitro Caco-2 cell system and a broiler chicken model for assessing iron bioavailability, focusing on crops that have been selected for biofortification. Biofortification is a strategy for preventing micronutrient malnutrition in resource poor populations by increasing the concentration and/or bioavailability of micronutrients in these crops, using plant breeding methods. The first study was designed to evaluate a biofortified maize line. We identified a line that was substantially higher in bioavailable iron, as determined using our Caco-2 model, than the rest even though the seed iron concentrations were similar. This suggests that the high bioavailable iron line either contained higher concentrations of an iron absorption enhancer or lower concentrations of an iron absorption inhibitor. To confirm this finding in an in vivo system, we used our chicken model to compare the iron bioavailability of the high bioavailable iron ("High") line with a line that showed low iron bioavailability ("Low"). Diets were formulated to contain 75% maize. The iron concentrations of the 2 diets were not different. Chicks were fed the maize-based diets for 4 weeks. Hemoglobin concentration (Hb), body weight, feed consumption, liver ferritin concentration, and gene expression (using semi-quantitative PCR) were measured. Hemoglobin maintenance efficiency (HME) was defined as the gain in total hemoglobin iron divided by the total iron intake. Duodenal DMT1, Dcytb, and ferroportin were higher (P<0.05) in the "Low" group vs. the "High" group, indicating adaptation to the "Low" diet. Hb, HME, Hb-Fe and liver ferritin were higher in the "High" group vs. the "Low" group (P<0.05), indicating greater Fe absorption from the diet and improved Fe status. We conclude that the "High" variety contains more bioavailable Fe than the "Low." Our second study focused on the common bean, also a targeted crop for iron biofortification. Colored beans are high in polyphenols that may inhibit Fe absorption. In vitro studies consistently show that Fe bioavailability from white beans is higher than colored beans. However, last year we reported that there was no difference in iron bioavailability between white and colored beans when compared in an anemic piglet model. Therefore, our objective was to use the chicken model to compare iron bioavailability between white and colored beans. We formulated 4 diets containing either white (Matterhorn) or red (Merlot) beans which differ in polyphenol content: 1. "WB": 40% white bean diet; 2. "RB": 40% red bean diet; 3."WB+Fe": 40% white bean diet; 4."RB+Fe": 40% red bean diet (51;47;179 and 175ppm Fe in diets, respectively). Corn comprised 35% of the diets. Chicks were divided into 4 groups and fed the respective diets for 8 wks. Hb, feed consumption and body weights were measured. DMT1, DcytB and ferroportin expressions were higher and villus surface area was greater in the "RB" group vs. other groups. Hb, Hb-Fe and BW were lower in the "RB" group vs. other groups (P<0.05). We conclude that white beans contain more bioavailable Fe than red beans. PARTICIPANTS: Dr. Elad Tako was a post-doctoral associate on the project and received training in designing scientific studies. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our discovery of a high bioavailable iron line of maize is novel and potentially very important. Maize is a staple food crop in many regions of the developing world where iron deficiency is highly prevalent. Attempts to breed for higher iron concentrations in maize have been disappointing. This means that breeding for enhanced bioavailability may be the only way that biofortification can work in maize. If the genes encoding for this high iron bioavailbility trait can be transferred into maize lines grown in Africa, Latin America, and other resource poor regions, then there is potential for a real positive impact on improving iron status among populations living in these areas.
Publications
- Tako E, Glahn RP, Laparra JM, Welch RM, Lei XG, Kelly JD, Rutzke MA, Miller DD. Iron and Zinc Bioavailabilities to Pigs from Red and White Beans (Phaseolus vulgaris L.) Are Similar. J Agric Food Chem. 2009; 57:3134-3140.
- Tako E, Laparra JM, Glahn RP, Welch RM, Lei XG, Beebe S, Miller DD. Biofortified black beans in a maize and bean diet provide more bioavailable iron to piglets than standard black beans. J Nutr. 2009 Feb;139(2):305-9.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: During the past year we continued to explore the interaction of Fe and polyphenols in foods such as beans. The major focus of this project during the past year was the interaction of iron and polyphenols in foods, using beans as the source of both iron and polyphenols. Polyphenols in foods may chelate dietary Fe and lower its bioavailability. Concentrations of phenols are substantially higher in red, pinto or black beans than in white beans. In many studies, using an in vitro digestion/Caco-2 cell culture model, we have observed that the colored beans have significantly less bioavailable Fe. We recently compared Fe availability from white beans versus red beans in piglet model. In the piglet model, there was no difference in Fe bioavailability between the beans. At present we are testing these diets in poultry and are finding a significant difference in Fe absorption, with the white bean diet providing more bioavailable Fe to the birds. This difference in result between the piglet and poultry model may be due to differences in intestine length, microflora and the Fe status of the animals at the start of the feeding trial. These effects will need to be investigated in future studies. We also investigated the potential benefits of having higher levels of Fe in beans, to determine if higher bean Fe concentrations would significantly improve Fe status of piglets. In this study on Fe concentration, we utilized black beans of approximately 70 and 100 ppm Fe formulated into a swine ration. In the piglet model, Fe deficient cross bred piglets (Hampshire*Landrace*Yorkshire) were used. Nutritionally balanced diets (except for Fe) were formulated to contain 35% precooked, dehydrated black beans (either low or high Fe). At age 5 wk, the piglets were assigned to 2 groups and fed diets containing either red or white beans for 4 wks. Weight and hemoglobin (Hb) concentrations were monitored weekly. Feed intakes were measured daily. Hemoglobin repletion efficiency (HRE) was calculated as the gain in total body hemoglobin Fe (Hb-Fe) divided by Fe intake. The results indicated that high Fe blacks can provide more Fe to Fe deficient piglets and thus have significant nutritional benefit. We also investigated the effects of inulin, a prebiotic compound, on Fe dialyzability and uptake via Caco-2 cells. In these studies, an in vitro digestion/Caco-2 cell model was used. We observed that inulin affects iron dialyzability from FeSO4 and FeEDTA solutions but does not alter Fe uptake by Caco-2 Cells. Hence, these results indicate that the beneficial effects of inulin on Fe absorption (observed previously in a piglet study) may be indirect. PARTICIPANTS: Dr. Moises Laparra, a Fulbright Postdoctoral Fellow from Spain, worked on this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our results are important because they demonstrate clearly that the common bean provides significant amounts of bioavailable iron to iron deficient piglets when fed over an extended period of time. Poultry are also able to show reasonably high absorption of Fe from diets rich in inhibitors. This is in contrast to several in vitro and human studies that showed that the bioavailability of iron from beans is extremely low. The in vitro and human studies were all short-term trials with no time for the cells or human subjects to adapt to the high levels of polyphenols present in the beans. It is possible that animals, and by extrapolation, humans adapt to the inhibitory effects of polyphenols on iron absorption by secreting proline rich proteins in the saliva. These proteins form complexes with the polyphenols and thereby prevent them from interacting with the iron. This adaptation takes time because it involves the up-regulation of genes encoding for the proteins. More studies are needed to explore this hypothesis. Our studies with inulin indicate that the beneficial effects of this prebiotic on Fe absorption appear to be indirect, and are related to proliferation of probiotic bacteria and possibly their metabolic byproducts. Certainly additional research is needed to define the role of inulin in Fe absorption as it may be a significant enhancer of Fe bioavailability.
Publications
- Tan SY, Yeung CK, Tako E, Glahn RP, Welch RM, Lei X, Miller DD. Iron Bioavailability to Piglets from Red and White Common Beans (Phaseolus vulgaris.J Agric Food Chem. 2008. 56 (13):5008-5014.
- Laparra JM, Tako E; Glahn RP, Miller DD. Inulin Affects Iron Dialyzability from FeSO4 and FeEDTA Solutions but Does Not Alter Fe Uptake by Caco-2 Cells. J Agric Food Chem. 2008; 56(8): 2846-2851.
- Laparra JM, Tako E, Glahn RP,Miller DD. Supplemental inulin does not enhance iron bioavailability to Caco-2 cells from milk- or soy-based, probiotic-containing, yogurts but incubation at 37 C does. Food Chemistry. 2008; 109:122-128.
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Progress 10/01/06 to 09/30/07
Outputs
The major focus of this project during the past year was the interaction of iron and polyphenols in foods, using beans as the source of both iron and polyphenols. Polyphenols in foods may chelate dietary Fe and lower its bioavailability. Concentrations of phenols are substantially higher in red beans than in white beans. We compared iron bioavailabilities from red and white beans using an in vitro digestion/Caco-2 cell culture model and a piglet hemoglobin repletion model. With the in vitro model, beans and bean-containing diets were subjected to in vitro gastro-intestinal digestion in the presence of a Caco-2 cell culture. Formation of ferritin by the cells was used as an index of Fe uptake and bioavailability. In the piglet model, Fe deficient cross bred piglets (Hampshire*Landrace*Yorkshire) were used. Nutritionally balanced diets (except for Fe) were formulated to contain 50% precooked, dehydrated beans (either small red or Great Northern white). At age 5 wk, the
piglets were assigned to 2 groups and fed diets containing either red or white beans for 4 wks. Weight and hemoglobin (Hb) concentrations were monitored weekly. Feed intakes were measured daily. Hemoglobin repletion efficiency (HRE) was calculated as the gain in total body hemoglobin Fe (Hb-Fe) divided by Fe intake. In the in vitro model, ferritin formation was higher in the cells exposed to white beans and white bean diets (p<0.05) compared to the red beans and red bean diets, suggesting that the higher concentration of polyphenols in the red beans was inhibiting Fe bioavailability. With the piglet model, in contrast, Hb concentrations, Hb-Fe gains and HRE were not different between the groups at any time point (p > 0.05). HRE values in the red bean group were 50% in the 1st week and 30% over the entire 4 week period. In the white bean group they were 56% and 26% respectively. Proline-rich protein mRNA concentrations in parotid glands were higher in the red bean group compared to the
white bean group. These results show that iron bioavailabilities from red and white beans are similar and suggest that pigs adapt to the inhibitory effects of polyphenols on iron absorption by increasing the secretion of protective proline-rich proteins in the saliva.
Impacts
Our results are important because they demonstrate clearly that the common bean provides significant amounts of bioavailable iron to iron deficient piglets when fed over an extended period of time. This is in contrast to several in vitro and human studies that showed that the bioavailability of iron from beans is extremely low. The in vitro and human studies were all short-term trials with no time for the cells or human subjects to adapt to the high levels of polyphenols present in the beans. We hypothesize that animals, and by extrapolation, humans adapt to the inhibitory effects of polyphenols on iron absorption by secreting proline rich proteins in the saliva. These proteins form complexes with the polyphenols and thereby prevent them from interacting with the iron. This adaptation takes time because it involves the up-regulation of genes encoding for the proteins.
Publications
- Hu Y, Cheng Z, Heller LI, Krasnoff SB, Glahn RP, Welch RM. 2006. Kaempferol in red bean seed (phaseolus vulgaris L) coats inhibits iron bioavailability using an in vitro digestion/human Caco-2 cell model. J Agric Food Chem 54:9254-9261.
- Jin F, Welch R, Glahn R. 2006. Moving toward a more physiological model: Application of mucin ot refine the in vitro digestion/Caco-2 cell culture system. J Agric Food Chem. 8962-8967.
- Beiseigel JM, Hunt JR, Glahn RP, Welch RM, Menkir A, Maziya-Dixon BB. 2007. Iron bioavailability from maize and beans: a comparison of human measurements with Caco-2 cell and algorithm predictions. Am J Clin Nutr. 86:388-396.
- Tako E, Glahn RP, Welch RM, Lei X, Yasuda K, Miller DD. 2007. Dietary inulin affects the expression of intestinal enterocyte iron transporters, receptors and storage protein and alters the microbiota in the pig intestine. Br J Nutr. Sep 10;:1-9 [Epub ahead of print]
- Tan, Siow Ying. 2007. Effects of common beans and by-products of fermentation of prebiotics on iron bioavailability in pig and Caco-2 cell models. Ph.D. Dissertation. Cornell University.
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Progress 01/01/06 to 12/31/06
Outputs
Inulin, a linear beta fructan, is present in a variety of plants including chicory root, onions, garlic, and wheat. It exhibits prebiotic properties and has been shown to enhance calcium and iron absorption and increase beneficial bacteria in the colon, including Bifidobacteria and Lactobacilli. The aim of this study was to assess the effect of dietary inulin on the expression of genes in intestinal enterocytes that encode for selected iron and zinc transporters and binding proteins. Anemic piglets at age 5 weeks were allocated to a standard corn-soy diet (control) or the same diet supplemented with inulin at a level of 4 percent. After 7 weeks, the animals were killed and cecum contents and sections of the small intestine and colon and were quickly removed. The tissues samples were immediately frozen in liquid nitrogen for RNA extraction. Segments of the genes encoding for the pig divalent metal transporter 1 (DMT1) and duodenal Cytochrome-b reductase (Dcytb) were
isolated and sequenced (gi86197473, gi105295521 respectively). Using primers designed to represent DMT1, Dcytb, ferroportin, ferritin, transferrin receptor, semi quantitative RT-PCR analyses were performed to quantify the expression of the respective genes encoding for these proteins. Concentrations of mRNA encoding for DMT1, Dcytb, ferroportin, ferritin, and transferrin receptor in duodenal samples were significantly higher in the inulin group (P less than or equal to 0.05) compared to the control group. In colon samples, DMT1, transferrin receptor and ferritin were significantly up-regulated in the inulin group. Additionally, the intestinal microflora were examined using 16S ribosomal DNA (rDNA) targeted probes from bacterial DNA isolated from cecal contents. The ribosomal gene sequence was amplified using PCR with universal primers to determine total bacterial DNA and specific primers directed at 4 bacterial genera: Lactobacillus, Bifidobacterium, Escherichia coli, and Clostridium.
The use of universal primers extends these methods to allow determination of relative proportions of different bacterial species. The Lactobacillus and Bifidobacterium populations were significantly increased in the inulin group (P less than or equal to 0.05) compared to the control group. These results indicate that dietary inulin triggers an up-regulation of genes encoding for iron transporters in intestinal enterocytes. The specific mechanism for this effect remains to be elucidated.
Impacts
Inulin is a form of soluble dietary fiber that exhibits prebiotic properties when added to diets. These properties include improved 'intestinal health', enhanced resistance to pathogenic organisms, and increased iron bioavailability. This enhancing effect on iron bioavailability has been observed in several studies with animal models but the mechanism of the effect is unknown. These results show for the first time that dietary inulin may alter the expression of genes that encode for iron transport proteins in intestinal epithelial cells.
Publications
- Yasuda K, Roneker KR, Miller DD, Welch RM, Lei XG. 2006. Supplemental dietary inulin affects the bioavailability of iron in corn and soybean meal to young pigs. J Nutr 136:3033-3038.
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Progress 01/01/05 to 12/31/05
Outputs
Mineral bioavailabilities from fortified bread and human milk fortified with human milk fortifiers (HMF) were studied. Iron fortification of staple foods is arguably the most widely used strategy for increasing the iron intake of populations. Although FeSO4 is a bioavailable form of iron, elemental iron powders are often used to fortify products with a long shelf-life, such as wheat flours, to avoid problems associated with the reactive nature of FeSO4. Therefore, the objectives of this study were to compare the bioavailabilities of elemental iron powders manufactured with different production methods in wheat flour breads and to determine the effects of added ascorbic acid and baking, using an in vitro digestion/Caco-2 cell culture model. Two types of wheat flour (low-extraction and high-extraction) were fortified with 10 different commercial elemental iron powders and baked into breads. Iron bioavailabilities from the resulting breads, with and without added
ascorbic acid, were evaluated using FeSO4 as the control. Depending on the type of wheat flour, bioavailabilities of several powders were comparable to FeSO4, but there was no consistent trend as to which production method produced the most bioavailable powder. In general, ascorbic acid enhanced, whereas the baking process reduced iron bioavailability from bread. Our results suggest that some elemental iron powders are potential alternatives to FeSO4. Human studies are warranted before any of these powders are selected for national fortification programs. The calcium, zinc, and iron bioavailabilities of human milk with commercial and noncommercial human milk fortifiers (HMFs) were evaluated under a variety of conditions: peptic digestion at pH 2 and pH 4, supplementation of ascorbic acid, and addition of three calcium salts. The noncommercial HMFs consisted of casein phosphopeptides (CPPs), alpha-lactalbumin, colostrum, and hydrolyzed whey protein concentrate (WPC). They were mixed
with human milk (HM) and calcium, zinc, and iron were added. Ascorbic acid (AA) was added in certain studies. The commercial HMFs were Nestle FM-85, Similac HMF (SHMF), and Enfamil HMF (EHMF). All HMFs were compared to S-26/SMA HMF. Results showed that the peptic pH (2 vs. 4) had no effect on mineral bioavailability. Addition of different calcium salts had no effect on calcium cell uptake and cell ferritin levels (an indicator of iron uptake), however, the addition of calcium glycerophosphate/gluconate increased zinc uptake by Caco-2 cells. Addition of AA significantly increased ferritin levels, with no effect on calcium or zinc uptake. Among the commercial HMFs, FM-85 was significantly lower in zinc uptake than S-26/SMA, and HM+EHMF was significantly higher than HM+S-26/SMA. Cell ferritin levels were significantly higher for HM+S-26/SMA than for all other commercial fortifiers. None of the commercial HMFs were different from HM+S-26/SMA in calcium uptake.
Impacts
Mineral deficiencies are a problem among many populations. Iron deficiency affects more than 2 billion people. Women, children, and especially premature infants are at high risk for these nutrient deficiencies. Fortification of foods with minerals is one strategy for reducing the risk for mineral malnutrition. These studies provide evidence that will be useful to food technologists working to improve the nutritional quality of fortified foods and human milk fortifiers.
Publications
- Etcheverry P, Wallingford JC, Miller DD, Glahn RP. 2005. The effect of calcium salts, ascorbic acid and peptic pH on calcium, zinc and iron bioavailabilities from fortified human milk using an in vitro digestion/Caco-2 cell model. International Journal for Vitamin and Nutrition Research. 75 (3): 171-178.
- Yeung CK, Miller DD, Cheng Z, Glahn RP. 2005. Bioavailability of elemental iron powders in bread assessed with an in vitro digestion/Caco-2 cell culture model. J Food Sci. 70(3):199-203.
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Progress 01/01/04 to 12/31/04
Outputs
We developed a rapid in vitro digestion/Caco-2 cell culture model for assessing relative bioavailabilities of iron in foods and meals. The objective of the present study was to determine how closely our Caco-2 model reflects the human response. Meals described in published reports of studies on effects of varying levels of ascorbic acid (AA) and tannic acid (TA) on iron absorption by human subjects were carefully replicated. Iron absorption ratios (iron absorption from meals containing AA or TA divided by iron absorption from identical meals without these enhancers or inhibitors) were determined using the Caco-2 model. Ferritin formation by the Caco-2 cells was used as an indicator of iron absorption. Response patterns of effects of AA and TA on absorption ratios (AR) calculated from Caco-2 and human data were very similar: AA increased ARs in a dose-response manner and TAs decreased AR. The natural logs of the ARs determined in Caco-2 and human studies were
correlated: R = 0.935 (P = 0.012) and 0.927 (P = 0.007) for AA and TA, respectively. When results from meals with AA and TA were pooled, a linear relation between the natural logs of ARs from Caco-2 and human studies was observed (R = 0.968, P less than 0.001). We conclude that our Caco-2 model accurately predicts the human response to AA and TA in the meals we tested. If future work reproduces the precision and accuracy shown in this paper for predicting iron bioavailability to humans, then the implications for saving time and resources in iron bioavailability measurements are considerable.
Impacts
We have developed a rapid and inexpensive method for assessing iron bioavailability from foods and meals. Our method makes it possible to screen large numbers of foods for iron bioavailability efficiently and rapidly. Therefore, our method has the potential to expand our understanding of factors that inhibit iron bioavailability and thereby provide clues for strategies for improving iron bioavailability of foods. Given that a major factor contributing to iron deficiency is poor iron bioavailability and that more than 2 billion people in the world suffer from iron deficiency, this project could help improve the lives of millions of people.
Publications
- Yun, S., Habicht, J.-P., Miller, D.D., and Glahn, R.P. 2004. An in vitro digestion/Caco-2 cell culture system accurately predicts the effects of ascorbic acid and polyphenolic compounds on iron bioavailability in humans. J. Nutr. 134:2717-2721.
- Etcheverry, P., Wallingford, J., Miller, D.D., and Glahn, R.P. 2004. Calcium, zinc, and iron bioavailabilities from a commercial human milk fortifier: A comparison study. J. Dairy Sci. 87:3629-3637.
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Progress 01/01/03 to 12/31/03
Outputs
The iron bioavailability of human milk (HM) is substantially greater than that of cow milk (CM), but the factor responsible for this high bioavailability is unknown. This study evaluated the effects of various HM and CM fractions on iron bioavailability. Milk was separated into fat, casein and whey fractions by ultracentrifugation. Whey was further fractionated by ultrafiltration with a 10-kDa membrane to produce a 10-kDa retentate (10kR) and a 10-kDa filtrate (10kF). Samples were prepared by mixing various combinations of the fractions, bringing the samples to prefractionation weight with minimum essential medium (MEM), and adding iron (10 micromol/L) as ferrous sulfate. Samples were divided into two aliquots: one was subjected to in vitro digestion, the other was not. Bioavailability was assessed by applying the samples to Caco-2 cell monolayers and incubating for 24 h. Ferritin formation in the cells was used as an index of iron uptake. Removing the fat from
undigested HM samples doubled the ferritin formation, but removing the whey or casein had no effect. Results with digested HM samples were similar, except that removing the whey decreased ferritin formation by 48 percent. Removing the fat from digested CM samples had no effect, but removing the casein doubled the ferritin formation. Removing the 10kF from HM reduced ferritin formation by 60 percent, but removing the 10kR had no effect. These data suggest that a low-molecular-weight factor (less than 10 kDa) in human milk enhances iron absorption.
Impacts
The iron in human milk is highly bioavailable but the identity of the factor or factors responsible for this high bioavailability remain elusive. We found that the factor is a small molecule present in the whey fraction of human milk. It appears to be a product of the digestion of whey protein but the exact identity is not yet known. Identification of the factor may lead to the development of infant formulas with improved iron bioavailability.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
The long-term goal of this project is to improve the bioavailability of mineral nutrients in foods. Previously, we developed an in vitro digestion/dialyzable iron method and an in vitro digestion/Caco-2 cell culture model system for studying factors and mechanisms that affect mineral bioavailabilites in foods. We are currently collecting data to validate the model system as a predictor of iron bioavailability to humans and to study factors in foods that may affect iron bioavailability. In one study, we assessed the bioavailabilities of selected iron fortificants (electrolytic iron, ferrous sulfate, ferrous fumarate, ferrous bis-glycinate, and NaFe(III)EDTA) in bread and milk. In bread, the bioavailability of electrolytic iron was lower than the other fortificants but all fortificants had similar bioavailabilities when added to milk. In another study, we measured the effects of phytic acid, tannic acid, and zinc on iron uptake in our in vitro digestion/Caco-2 cell
model. Phytic acid was a potent inhibitor of nonheme iron uptake with maximum inhibition occurring at a 1:10 molar ration of iron to phytic acid. Phytic acid also inhibited heme iron uptake but to a lesser degree. Tannic acid was an even more potent inhibitor of iron uptake than phytic acid. Zinc, at a Zn:Fe molar ratio of 1:1, decreased iron uptake by 80 percent. We also developed a method for the simultaneous determination of the uptake of 45Ca and 65Zn by Caco-2 cells. This method allows us to simultaneously assess the bioavailabilities of iron, calcium, and zinc with our in vitro digestion/Caco-2 cell culture model.
Impacts
The in vitro digestion/Caco-2 cell model we have developed allows us to rapidly and inexpensively screen foods and diets for the bioavailabilities of iron, zinc, and calcium. This will improve our ability to develop effective strategies for improving the nutritional status of populations, especially in developing regions of the world.
Publications
- Glahn, R.P., Wortley, G.M., South, P.K., and Miller, D.D. 2002. Inhibition of iron uptake by phytic acid, tannic acid, and ZnCl2: Studies using an in vitro digestion/Caco-2 cell model. J. Agric. Food Chem. 50:390-395.
- Etcheverry, P., Wallingford, J.C., Miller, D.D., and Glahn, R.P. 2002. Simultaneous determination of 45Calcium and 65Zinc uptake by Caco-2 cells. J. Agric. Food Chem. 50:6287-6294.
- Yeung, A.C., Glahn, R.P., and Miller, D.D. 2002. Comparison of athe availability of varous iron fortificants in bread and milk using an in vitro digestion/Caco-2 cell culture model. J. Food Sci. 67(6):2357-2361.
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Progress 01/01/01 to 12/31/01
Outputs
The long-term goal of this project is to improve the bioavailability of mineral nutrients in foods. Previously, we developed an in vitro digestion/dialyzable iron method and an in vitro digestion/Caco-2 cell culture model system for studying factors and mechanisms that affect mineral bioavailabilites in foods. We are currently collecting data to validate the model system as a predictor of iron bioavailability to humans and to study factors in foods that may affect iron bioavailability. In one study, we assessed the effects of adding selected phytases to whole wheat bread on phytate-phosphorous release and iron dialyzability in the bread. Selected microbial phytases and citric acid were added to water used in dough preparation. Breads were baked from the dough and evaluated for iron dialyzability and phytate and free phosphorous content. Addition of citric acid at 6.25 g/kg flour enhanced phytate degradation by phytases intrinsic to the flour. Addition of microbial
phytases further enhanced phytate reduction up to 85%. Addition of citric acid and phytase improved iron dialyzability from the bread by up to 15 fold. These results suggest that addition of citric acid and microbial phytases during bread baking could significantly enhance the amount of bioavailable iron in bread. In addition, we developed a simple, rapid method for determining the iron content of fortified and unfortified foods. Food samples are mixed with an iron extracting solution, heated to boiling, cooled and filtered. A colorimetric assay is used to quantify the iron content in the food. The method is simple, rapid, and inexpensive. It is suitable for monitoring the iron concentration in fortified foods in settings where expensive analytical instruments and highly trained laboratory personnel are not available.
Impacts
Iron deficiency is the most prevelant nutrient deficiency in the world, affecting up to 2 billion people. A major cause of iron deficiency is low bioavailability of dietary iron. This project may provide the knowledge necesssary for improving iron bioavailabiliy of foods.
Publications
- Yeung AC, Glahn RP, Miller DD. 2001. Dephosphorylation of sodium caseinate, enzymatically hydrolyzed casein and casein phosphopeptides by intestinal alkaline phosphatase: implications for iron availability. J Nutr Biochem. 12(5):292-299.
- Kapsokefalou M, Miller DD. 2001. Large doses of ascorbic acid promote lipid peroxidation in the serum of iron loaded guinea pigs. British Journal of Nutrition. 85(6):681-687.
- Kosse J, Yeung A, Gil A, Miller DD. 2001. A rapid method for iron determination in fortified foods. Food Chemistry. 75:371-376.
- Porres JM, Etcheverry P, Miller DD, Lei XG. 2001. Phytase and citric acid supplementation in whole-wheat bread improves phytate-phosphorus release and iron dialyzabilitiy. J Food Science. 66(4):614-619.
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Progress 01/01/00 to 12/31/00
Outputs
The long-term goal of this project is to improve the bioavailability of mineral nutrients in foods. Our approach is to develop and apply in vitro digestion/Caco-2 cell culture model systems for studying factors and mechanisms the affect mineral bioavailabilites. A method was developed to simultaneously measure the bioavailability of iron, zinc and calcium using our in vitro digestion/Caco-2 cell model system. This is a significant advance since previously only iron availability could be measured with the system. Milk and dairy products inhibit iron absorption in human subjects. It is well established that clusters of phosphoserine residues in casein bind iron with high affinity but it is unclear whether this binding is a factor in the inhibition of iron absorption by dairy products. We compared iron bioavailability from various milk protein fractions to test the hypothesis that hydrolysis, by intestinal alkaline phosphatase, of phosphate groups on phosphoserine
residues in casein facilitates iron availability from casein-containing products. Iron availability from casein phosphopeptides (CPP) was higher than caseinate and similar to whey protein concentrate. However, in the presence of citrate, differences between CPP and caseinate disappeared. Added alkaline phosphatase did not affect iron availability but inhibition of membrane-bound alkaline phosphatase by Na2WO4 inhibited 59Fe uptake by 10%. These results suggest that alkaline phosphatase plays only a small role in the release of casein-bound iron during digestion. They also suggest that solubility of iron complexes play a major role in iron availability. Phytic acid, tannic acid and zinc are well-known inhibitors of iron bioavailability. We compared the effects of these 3 inhibitors. Tannic acid proved to be the most potent inhibitor, followed by phytic acid and zinc. We compared the bioavailability of 19 different forms of iron used as a fortificant in ready-to-eat cereal (Wheaties).
All forms of iron were compared relative to "reduced Fe" which is the form of Fe currently used in most cereals. The results indicate that several forms of FeEDTA were highly available, exhibiting iron availability values 200-300% greater than that produced by reduced Fe. Other forms of iron such as electrolytic, encapsulated Fe Fumarate, Sun Active and Ferrochel exhibited availability values 30-125% greater than that of reduced iron. Of these four forms and relative to the reduced Fe, the Ferrochel displayed the highest availability value (+125%), followed by Sun Active (+79%), Electrolytic (+55%) and encapsulated Fe Fumarate (+30-40%). Similar results have recently been observed in human trials for the FeEDTA and Ferrochel. Overall, this study demonstrates that the in vitro digestion/Caco-2 cell culture model is an effective tool for developing improved forms of iron fortificants in breakfast cereals.
Impacts
Iron deficiency is the most prevelant nutrient deficiency in the world, affecting up to 2 billion people. A major cause of iron deficiency is low bioavailability of dietary iron. This project may provide the knowledge necesssary for improving iron bioavailabiliy of foods.
Publications
- No publications reported this period
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