Iron from Co-Encapsulation of Defatted Nannochloropsis Oceanica with Inulin Is Highly Bioavailable and Does Not Impact Wheat Flour Shelf Life or Sensorial Attributes.

Defatted green microalgae Nannochloropsis oceanica (DGM) is a rich source of bioavailable iron. However, its use in foods results in unacceptable color and taste development. Therefore, the purpose of this study was to investigate strategies to enhance the use of DGM in foods. DGM and inulin were encapsulated (EC) in an oil-in-water emulsion using high-pressure homogenization. To confirm iron bioavailability, C57BL/6 mice were fed an iron-deficient diet (ID) for 2 weeks. The mice were then fed one of the four diets: ID, ID + DGM (DGM), ID + EC (EC50 or EC100) for 4 weeks. To test the stability of DGM as an iron fortificant at two different fortification rates of 17.5 mg Fe/kg (50%) or 35 mg Fe/kg (100%), whole (DGM50/DGM100), encapsulated (EC50/EC100) and color-masked (CM50/CM100) DGM were added to wheat flour (WF) at two different temperatures: 20 °C and 45 °C and were examined for 30 days. Acceptability studies were conducted to determine sensory differences between rotis (Indian flat bread) prepared from WF/EC50/CM50/EC100. The mice consuming EC50/EC100 diets showed comparable iron status to DGM-fed mice, suggesting that encapsulation did not negatively impact iron bioavailability. Addition of EC to wheat flour resulted in the lowest Fe2+ oxidation and color change amongst treatments, when stored for 30 days. There were no differences in the overall liking and product acceptance of rotis amongst treatments at both day 0 and day 21 samples. Our results suggest that EC50 can be effectively used as an iron fortificant in WF to deliver highly bioavailable iron without experiencing any stability or sensory defects, at least until 30 days of storage.

Supplemental dietary inulin of variable chain lengths alters intestinal bacterial populations in young pigs.

Previously, we showed that supplementation of diets with short-chain inulin (P95), long-chain inulin (HP), and a 50:50 mixture of both (Synergy 1) improved body iron status and altered expression of the genes involved in iron homeostasis and inflammation in young pigs. However, the effects of these 3 types of inulin on intestinal bacteria remain unknown. Applying terminal restriction fragment length polymorphism analysis, we determined the abundances of luminal and adherent bacterial populations from 6 segments of the small and large intestines of pigs (n = 4 for each group) fed an iron-deficient basal diet (BD) or the BD supplemented with 4% of P95, Synergy 1, or HP for 5 wk. Compared with BD, all 3 types of inulin enhanced (P < 0.05) the abundance of beneficial bifidobacteria and lactobacilli in the microbiota adherent to intestinal mucus of various gut segments of pigs. These changes were seen as proximal as in the jejunum with P95 but did not appear until the distal ileum or cecum with HP. Similar effects of inulin on bacterial populations in the lumen contents were found. Meanwhile, all 3 types of inulin suppressed the less desirable bacteria Clostridium spp. and members of the Enterobacteriaceae in the lumen and mucosa of various gut segments. Our findings suggest that the ability of dietary inulin to alter intestinal bacterial populations may partially account for its iron bioavailability-promoting effect and possibly other health benefits.

Supplemental Microalgal Iron Helps Replete Blood Hemoglobin in Moderately Anemic Mice Fed a Rice-Based Diet.

Iron deficiency anemia affects 1.2 billion people globally. Our objectives were to determine if (1) supplemental iron extracted from defatted microalgae (Nannochloropsis oceanica, DGM) and (2) a combination of minute amount of plant phytase and inulin could help replete hemoglobin in anemic mice. Mice (7 weeks old) were fed a control diet (6 mg Fe/kg). After 10 weeks, the mice were assigned to three treatments: control, control + DGM iron (Fe-DGM, 39 mg Fe/kg), or control + 1% inulin + 250 units of phytase/kg (INU-PHY, 6 mg Fe/kg). The mice had free access to diets and water for 6 weeks. The Fe-DGM group had elevated blood hemoglobin (p < 0.01) and a two-fold greater (p < 0.0001) liver non-heme iron over the control. Strikingly, the INU-PHY group had 34% greater non-heme iron than the control, despite the same concentrations of iron in their diets. Fe-DGM group had altered (p < 0.05) mRNA levels of hepcidin, divalent metal transporter 1, transferrin and transferrin receptor 1. Iron extracted from defatted microalgae seemed to be effective in alleviating moderate anemia, and INU-PHY enhanced utilization of intrinsic iron present in the rice diet. Our findings may lead to a novel formulation of these ingredients to develop safer and bioavailable iron supplements for iron-deficient populations.

Biofortified black beans in a maize and bean diet provide more bioavailable iron to piglets than standard black beans.

Our objective was to compare the capacities of biofortified and standard black beans (Phaseolus vulgaris L.) to deliver iron (Fe) for hemoglobin (Hb) synthesis. Two lines of black beans, one standard and the other biofortified (high) in Fe (71 and 106 microg Fe/g, respectively), were used. Maize-based diets containing the beans were formulated to meet the nutrient requirements for swine except for Fe (Fe concentrations in the 2 diets were 42.9 +/- 1.2 and 54.6 +/- 0.9 mg/kg). At birth, pigs were injected with 50 mg of Fe as Fe dextran. At age 28 d, pigs were allocated to the experimental diets (n = 10). They were fed 2 times per day for 5 wk and given free access to water at all times. Body weights and Hb concentrations were measured weekly. Hb repletion efficiencies (means +/- SEM) did not differ between groups and, after 5 wk, were 20.8 +/- 2.1% for the standard Fe group and 20.9 +/- 2.1% for the high Fe group. Final total body Hb Fe contents did not differ between the standard [539 +/- 39 mg (9.7 +/- 0.7 micromol)] and high Fe [592 +/- 28 mg (10.6 +/- 0.5 micromol)] bean groups (P = 0.15). The increase in total body Hb Fe over the 5-wk feeding period was greater in the high Fe bean group [429 +/- 24 mg (7.7 +/- 0.4 micromol)] than in the standard Fe bean group [361 +/- 23 mg (6.4 +/- 0.4 micromol)] (P = 0.034). We conclude that the biofortified beans are a promising vehicle for increasing intakes of bioavailable Fe in human populations that consume beans as a dietary staple.

Supplemental dietary inulin influences expression of iron and inflammation related genes in young pigs.

We have previously shown improved hemoglobin (Hb) repletion efficiency by supplementing a 50:50 mixture of short (P95) and long-chain (HP) inulin (Synergy 1, BENEO-Orafti) into a corn-soybean meal-basal diet (BD) for young pigs. In this study, weanling pigs (5 or 6 wk old) were fed the BD or the BD + 4% of P95, HP, or Synergy 1 (50:50 mixtures of HP and P95) for 5-7 wk. Blood Hb concentrations of pigs were measured weekly and digesta samples were collected at the end of the trial. In a replicate experiment, total RNA was isolated from the liver and mucosa of duodenum, ileum, cecum, and colon of all pigs at the end of the trial. Relative mRNA expression of 27 genes, including iron and inflammation-related genes, was quantified using real-time quantitative-PCR. Although all 3 types of inulin resulted in similar improvements (P < 0.05) in blood Hb concentration and liver ferritin protein amount, neither type of inulin was detectable in the digesta of cecum or colon. Supplemental inulin enhanced the expression of iron-storing protein genes but decreased that of inflammation-related genes. Such effects were more pronounced (P < 0.05) in the mucosa of the lower than the upper gut and were seen on 7 genes in liver. In conclusion, all 3 types of inulin shared similar efficacy and possibly similar modes of action in improving dietary iron utilization by young pigs. Suppressing inflammation-induced genes that can negatively influence iron metabolism might help explain the benefit of inulin.

Different responses of Fe transporters in Caco-2/HT29-MTX cocultures than in independent Caco-2 cell cultures.

The human intestinal epithelium is composed of several cell types, mainly enterocytes and goblet (mucin-secreting) cells. This study compares the cellular response of Fe transporters in Caco-2, HT29-MTX, and Caco-2/HT29-MTX co-culture models for Fe bioavailability. Caco-2 cells in vitro differentiate into enterocyte-like cells and HT29-MTX cell lineage into a mucin-secreting cellular population. Cell cultures were exposed to digests of Fe+3, Fe+3/ascorbic acid, cooked fish (high-available Fe) or white beans (low-available Fe). Cell responses as shown by mRNA expression of the main Fe transporters, DMT1 and DcytB, and cell ferritin formation were monitored. In Caco-2/HT29-MTX co-cultures, the mucin layer lowered the pool of free Fe to diffuse towards the cell brush border membrane of enterocytes, which was accompanied of an upregulation of DMT1 mRNA expression. In contrast, cultures exposed to digests of fish or white beans showed no significant differences in the regulation of Fe transporters.

The pig as an experimental model for elucidating the mechanisms governing dietary influence on mineral absorption.

This review highlights the similarities between pigs and humans and thereby the value of the porcine human nutritional model, and reviews some of the more recent applications of this model for nutritional research.

Inulin affects iron dialyzability from FeSO4 and FeEDTA solutions but does not alter Fe uptake by Caco-2 cells.

The in vitro effects of inulin on the fluxes of Fe (F(Fe)) and uptake by Caco-2 cells from FeSO4 and FeEDTA were evaluated. Cell ferritin formation was used as a measure of Fe uptake. Mitochondrial (MTT test) and lysosomal activities were monitored as biomarkers of the changes of cellular metabolism. Changes in mRNA expression of Fe transporters, DMT1 and Dcytb, were evaluated. Inulin decreased dialyzability and F(Fe) from FeSO4 solution, suggesting a mineral binding effect, but increased those from FeEDTA. Cultures exposed to FeEDTA solutions exhibited higher ferritin values and MTT conversion percentages. Regardless of Fe source, cell Fe uptake and mRNA expression of Fe transporters were similar with or without inulin, suggesting that inulin did not impair Fe uptake. These observations might indicate a faster cellular Fe internalization from FeEDTA solutions. From a physiological perspective, the decreased F(Fe) from FeSO4 might be reflected in a decreased Fe uptake.

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.

The in vitro effects of supplemental inulin (4%) on iron (Fe) availability in two different probiotic-containing yogurts were examined. Milk or soy-based yogurts, with and without inulin, were incubated (37°C) for 48h or without any incubation before comparison by an in vitro gastrointestinal digestion/Caco-2 cell culture model was used to assess iron bioavailability. The dialysable Fe fraction, cell ferritin formation, and cell associated Fe were monitored. Supplemental inulin decreased dialysable Fe only in non-incubated milk-based yogurt. In both yogurts incubation by itself increased dialysable Fe, and inulin increased the latter only in soy-based yogurt. Cellular ferritin concentration were higher after exposure to non-incubated milk-based than soy-based yogurt, although, after incubation the latter induced the highest ferritin formation. These data suggest that inulin does not have a direct effect on Fe bioavailability in the small intestine, and that probiotic bacteria play an enhancing role on Fe bioavailability.

Tissue iron distribution and urinary mineral excretion vary depending on the form of iron (FeSO4 or NaFeEDTA) and the route of administration (oral or subcutaneous) in rats given high doses of iron.

Sodium iron ethylenediaminetetraacetate (NaFeEDTA) has considerable promise as an iron fortificant in food. However, effects of administering high levels of NaFeEDTA on tissue iron distribution and mineral excretion are not well understood. The objectives of this study were to assess nonheme iron distribution in the body and urinary excretion of Ca, Mg, Cu, Fe, and Zn after daily administration of high levels of iron to rats over 21 days. Iron was either given orally with food or injected subcutaneously, as either FeSO 4 or NaFeEDTA. Selected tissues were collected for nonheme iron analysis. Estimated total body nonheme iron levels were similar in rats fed NaFeEDTA or FeSO 4, but the tissue distribution was different: it was 53% lower in the liver and 86% higher in the kidneys among rats fed NaFeEDTA than among those fed FeSO 4. In contrast, body nonheme iron was 3.2-fold higher in rats injected with FeSO 4 than in rats injected with NaFeEDTA. Administering NaFeEDTA orally elevated urinary Cu, Fe, and Zn excretion compared with FeSO 4 (1.41-, 11.9-, and 13.9-fold higher, respectively). We conclude that iron is dissociated from the EDTA complex prior to or during intestinal absorption. A portion of intact FeEDTA may be absorbed via a paracellular route at high levels of intake but is mostly excreted in the urine. Metal-free EDTA may be absorbed and cause elevated urinary excretion of Fe, Cu, and Zn.

Peptides isolated from in vitro digests of milk enhance iron uptake by caco-2 cells.

Milk proteins, during digestion, produce a range of biologically active peptides. Among those are peptides that may enhance iron absorption. The objective of this project was to investigate the effect of isolated milk peptides on iron uptake. Cow's milk, 0% fat, was subjected to a modified in vitro digestion process. The milk digest was further fractionated by gel filtration. All eluted fractions as well as beta-casein synthetic peptides (a tripeptide and a hexapeptide) were subsequently tested for effects on iron uptake with Caco-2 cell monolayers. Fractions of milk digests obtained through Sephadex G-25 gel filtration had a significant enhancing effect on iron uptake in Caco-2 cells compared to nonfractionated milk digests. Two fractions (P = 0) and the hexapeptide (P < 0.0001) enhanced iron uptake by up to 3-fold, whereas others and the tripeptide had no effect. These results suggest that selected peptides produced during the in vitro digestion of milk may enhance iron absorption; however, it remains to be demonstrated whether this effect may be nutritionally significant.

Iron uptake by Caco-2 cells from NaFeEDTA and FeSO4: Effects of ascorbic acid, pH, and a Fe(II) chelating agent.

Sodium iron(III) ethylenediaminetetraacetate (NaFeEDTA) has considerable promise as an iron fortificant because of its high bioavailability in foods containing iron absorption inhibitors. In this study, uptakes of iron from NaFeEDTA, FeSO4, and FeCl3 by Caco-2 cells were compared in the absence or presence of ascorbic acid (AA), an iron absorption enhancer; at selected pH levels; and in the absence or presence of an iron absorption inhibitor, bathophenanthroline disulfonic acid (BPDS). Ferritin formation in the cells was used as the indicator of iron uptake. Uptake from all three Fe sources was similar in the absence of AA. Adding AA at a 5:1 molar excess as compared to Fe increased uptake by 5.4-, 5.1-, and 2.8-fold for FeSO4, FeCl3, and NaFeEDTA, respectively. The smaller effect of AA on uptake from NaFeEDTA may be related to the higher solubility of NaFeEDTA and/or the strong binding affinity of EDTA for Fe3+, which may prevent AA and duodenal cytochrome b from effectively reducing EDTA-bound Fe. Uptake was inversely related to the pH of the media over a range of 5.8-7.2. Because uptake by DMT-1 is proton-coupled, the inverse relationship between pH and Fe uptake in all three iron sources suggests that they all follow the DMT-1 pathway into the cell. Adding BPDS to the media inhibited uptake from all three iron compounds equally. Because BPDS binds Fe2+ but not Fe3+ and because only Fe2+ is transported by DMT-1, the finding that BPDS inhibited uptake from NaFeEDTA suggests that at least some iron dissociates from EDTA and is reduced just as simple inorganic iron at the brush border membrane of the enterocyte. Taken together, these results suggest that uptake of iron from NaFeEDTA by intestinal enterocytes is regulated similarly to uptake from iron salts.

Iron dissociates from the NaFeEDTA complex prior to or during intestinal absorption in rats.

Sodium iron ethylenediaminetetraacetate (NaFeEDTA) has superior iron bioavailability especially in foods containing iron absorption inhibitors. However, mechanisms involved in the absorption and subsequent partitioning of iron complexed with EDTA are poorly understood. Our objectives were to compare retention and tissue distribution of iron administered to rats either as FeSO4 or NaFeEDTA, either orally (OR) or subcutaneously (SC). Weanling rats were fed semipurified diets supplemented with either FeSO4 or NaFeEDTA for 7 days. They were then given a meal containing 59Fe-labeled FeSO4 or NaFeEDTA, or they were injected SC with these two forms of radiolabeled Fe. 59Fe retention was measured by whole body counting. Urine was collected and counted at 24 h intervals throughout the counting period. Tissue samples were analyzed for nonheme iron and 59Fe activity. Absorption of iron from FeSO4 or NaFeEDTA was similar (57.7 and 53.4%, respectively). Seventy-seven percent of the injected Na59FeEDTA was excreted in the urine within 24 h, whereas only 0.5, 0.8, and 1.4% of the injected 59FeSO4, oral 59FeSO4, and oral Na59FeEDTA, respectively, was excreted in the urine. The nonheme iron content was lower in the liver and spleen, by 56.8 and 28.4%, respectively, among rats consuming the NaFeEDTA diet as compared to rats fed FeSO4. We conclude that iron is dissociated from EDTA prior to or during intestinal absorption and that some fraction of the dissociated EDTA is absorbed separately from the iron.

Effect of bread baking on the bioavailability of hydrogen-reduced iron powder added to unenriched refined wheat flour.

Elemental iron powders are widely used to fortify flour and other cereal products. Our objective was to test the hypothesis that baking enhances the bioavailability of elemental iron powders by oxidizing Fe(0) to Fe(2+) or Fe(3+). An in vitro digestion/Caco-2 cell culture model and a piglet model were used to measure bioavailability. Bread flour, either unfortified or fortified with hydrogen-reduced (HR) iron powder or FeSO(4) (300 mg Fe/kg flour), was baked into bread. For the in vitro studies, bread samples were treated with pepsin at pH 2, 3, 4, 5, 6, or 7 and subsequently incubated with pancreatic enzymes at pH 7 in a chamber positioned above monolayers of cultured Caco-2 cells. Ferritin formation in the cells was used as an index of iron bioavailability. Ferritin formation in cells fed HR Fe bread was similar to cells fed FeSO(4) bread when the peptic digestion was conducted at a pH 2 but lower when the peptic phase was conducted at pH 3, 4, 5, 6, or 7 (P < 0.05). Pig diets containing 35% dried bread were prepared and fed to cross-bred (Hampshire x Landrace x Yorkshire) anemic pigs in two studies. The rate of increase in hemoglobin Fe over the feeding period was used to calculate relative biological value (RBV), an index of iron bioavailability. In the first pig study, RBV of HR Fe added to flour prior to baking was 47.9% when compared to FeSO(4) fortified flour (P < 0.05). In the second pig study, a third treatment consisting of unfortified bread with HR iron added during diet mixing (after bread baking) was included. RBVs of the HR Fe diet (Fe added after baking) and HR Fe diet (Fe added before baking) were 40.1% and 53.5%, respectively, compared to the FeSO(4) diet. Differences in RBV between the HR Fe (before and after baking) and FeSO(4) (before baking) treatment groups were significant, but the difference between the before and after HR treatment groups was not significant. We conclude that bread baking does not enhance the bioavailability of elemental iron powders.

Tissue iron distribution and adaptation of iron absorption in rats exposed to a high dietary level of NaFeEDTA.

Although it has been shown that iron absorption from NaFeEDTA, a promising iron fortificant, is effectively down-regulated in iron-loaded rats, effects of prolonged exposure to high dietary levels of NaFeEDTA are not well understood. The objectives of this study were to determine whether rats can adapt to a high dietary level of NaFeEDTA by down-regulating iron absorption, and to determine effects on tissue iron distribution, with or without an iron absorption inhibitor. Male Sprague-Dawley rats were exposed to diets supplemented with FeSO4 or NaFeEDTA at 1200 mg of Fe/kg of diet, with or without tea, for 27 days. Iron absorption measured by whole-body counting before and after exposure showed that rats adapted to the high dietary level of FeSO4 or NaFeEDTA by down-regulating iron absorption to a similar extent. However, nonheme iron concentrations in liver and spleen were about 35-50% lower, whereas the concentration in kidney was about 300% higher in rats fed NaFeEDTA, compared to rats fed FeSO4. Tea had no major impact on iron absorption or iron status, regardless of iron source. Our results showed that although iron absorption was down-regulated similarly, body iron distribution was markedly different between rats exposed to FeSO4 and those exposed to NaFeEDTA. Further studies are warranted to determine the effects of prolonged exposure to dietary NaFeEDTA on kidney iron accumulation and kidney function.

Inhibition of iron uptake from iron salts and chelates by divalent metal cations in intestinal epithelial cells.

Iron chelates, namely, ferrous bisglycinate and ferric EDTA, are promising alternatives to iron salts for food fortification. The objectives of this study were to compare iron uptake from radiolabeled ferrous sulfate, ferrous ascorbate, ferrous bisglycinate, ferric chloride, ferric citrate, and ferric EDTA by Caco-2 cells with different iron status and in the presence of divalent metal cations. Iron-loaded Caco-2 cells, with reduced DMT-1 and elevated HFE mRNA levels, down-regulated uptake from ferrous ascorbate and bisglycinate but not from ferric compounds. Nevertheless, iron uptake from all compounds was markedly inhibited in the presence of 100-fold molar excess of Co2+ and Mn2+ cations, with ferrous compounds showing a greater percent reduction. Our results suggest that ferrous iron is the predominant form of iron taken up by intestinal epithelial cells and the DMT-1 pathway is the major pathway for uptake. Iron uptake from chelates appears to follow the same pathway as uptake from salts.

Simultaneous determination of (45)calcium and (65)zinc uptake by caco-2 cells.

A simple method for simultaneously determining cell-associated Ca and Zn in Caco-2 cells is described. Calcium and zinc uptake was measured via radioisotopes (45)Ca and (65)Zn. Preliminary studies revealed that (65)Zn, a positron (beta(+)) and gamma emitter, contributed to (45)Ca counts in a liquid scintillation counter (LSC). However, (45)Ca, being a true beta emitter, did not contribute to the counts in a gamma counter (gammaC). To differentiate the counts of (45)Ca from those of (65)Zn, first a (65)Zn-labeled cell suspension was read in a gammaC and an LSC, thus obtaining the relationship between the radioactive counts obtained from the gammaC and LSC. This information defined the linear relationship between gammaC (65)Zn counts per minute (CPM) and LSC (65)Zn CPM. Because the (45)Ca and (65)Zn counts obtained in the LSC are additive, giving total LSC CPM, the value of LSC (45)Ca CPM was obtained by subtracting LSC (65)Zn CPM from total LSC CPM for the dual-labeled cell sample, obtaining then LSC (45)Ca CPM. To determine the absolute activity or disintegrations per minute (DPM) of each isotope in the dual-labeled sample, the linear relationship between DPM and CPM was determined for each isotope. The method is simple and straightforward for the determination of (45)Ca counts from a sample also containing (65)Zn, using gamma and liquid scintillation counters.

Inhibition of iron uptake by phytic acid, tannic acid, and ZnCl2: studies using an in vitro digestion/Caco-2 cell model.

The objective of this study was to document the effects of phytic acid, tannic acid, and zinc on iron uptake in an in vitro digestion/Caco-2 cell culture model. The effects of phytic acid and tannic acid on iron uptake were measured at increasing molar ratios of FeCl3 to phytic acid or tannic acid. Maximal inhibition of iron uptake by phytic acid occurred at a 1:10 ratio of Fe to phytic acid. Dialyzable Fe decreased with a low Fe to phytic acid ratio but increased with Fe:phytic acid ratios greater than 1:3 indicating that more iron was soluble at higher phytic acid levels but less available. As in human studies, heme iron was less inhibited by phytic acid than nonheme iron. Tannic acid was a more potent inhibitor of nonheme iron uptake, as maximal inhibition (97.5%) of iron uptake occurred at a ratio of 1:1 or less. The addition of ZnCl2 to the digest at ratios of 1:0.5 and 1:1 decreased iron uptake by 57 and 80%, respectively. Overall, the results agree qualitatively with studies in humans and demonstrate the relative effects of these compounds on iron uptake in this model system. This study provides key information for determining iron availability under more complex meal conditions.

Production and supply of high-quality food protein for human consumption: sustainability, challenges, and innovations.

The Food and Agriculture Organization of the United Nations estimates that 843 million people worldwide are hungry and a greater number suffer from nutrient deficiencies. Approximately one billion people have inadequate protein intake. The challenge of preventing hunger and malnutrition will become even greater as the global population grows from the current 7.2 billion people to 9.6 billion by 2050. With increases in income, population, and demand for more nutrient-dense foods, global meat production is projected to increase by 206 million tons per year during the next 35 years. These changes in population and dietary practices have led to a tremendous rise in the demand for food protein, especially animal-source protein. Consuming the required amounts of protein is fundamental to human growth and health. Protein needs can be met through intakes of animal and plant-source foods. Increased consumption of food proteins is associated with increased greenhouse gas emissions and overutilization of water. Consequently, concerns exist regarding impacts of agricultural production, processing and distribution of food protein on the environment, ecosystem, and sustainability. To address these challenging issues, the New York Academy of Sciences organized the conference "Frontiers in Agricultural Sustainability: Studying the Protein Supply Chain to Improve Dietary Quality" to explore sustainable innovations in food science and programming aimed at producing the required quality and quantity of protein through improved supply chains worldwide. This report provides an extensive discussion of these issues and summaries of the presentations from the conference.

Bioavailability of iron in geophagic earths and clay minerals, and their effect on dietary iron absorption using an in vitro digestion/Caco-2 cell model.

Geophagy, the deliberate consumption of earth, is strongly associated with iron (Fe) deficiency. It has been proposed that geophagy may be practiced as a means to improve Fe status by increasing Fe intakes and, conversely, that geophagy may cause Fe deficiency by inhibiting Fe absorption. We tested these hypotheses by measuring Fe concentration and relative bioavailable Fe content of 12 samples of geophagic earth and 4 samples of pure clay minerals. Further, we assessed the impact of these samples on the bioavailability of Fe from an Fe-rich test meal (cooked white beans, WB). Fe concentrations were measured with inductively coupled plasma atomic emission spectroscopy. Fe bioavailability was determined using an in vitro digestion/Caco-2 cell model in which ferritin formation was used as an index of Fe bioavailability. Geophagic earth and clay mineral samples were evaluated with this model, both alone and in combination with WB (1 : 16 ratio, sample : WB). Median Fe concentration of the geophagic earth was 3485 (IQR 2462, 14 ,571) µg g⁻¹ and mean Fe concentration in the clay minerals was 2791 (±1782) µg g⁻¹. All specimens had Fe concentrations significantly higher (p ≤ 0.005) than the Fe concentration of WB (77 µg g⁻¹). Ferritin formation (i.e. Fe uptake) in cells exposed to geophagic earths and clay minerals was significantly lower than in cells exposed to WB (p ≤ 0.05) and Fe uptake responses of 11 of the 16 samples were not significantly different from the blank, indicating no bioavailable Fe. When samples were combined with WB, 5 of 16 had mean ferritin levels that were significantly lower (p ≤ 0.05, one tail) than the WB alone, indicating that the samples inhibited Fe uptake from the WB. None of the ferritin responses of cells exposed to both WB and earth/clay were significantly higher than WB alone. Thus, although geophagic earths and mineral clays are high in total Fe, very little of this Fe is bioavailable. Further, some geophagic earth and clay mineral samples inhibit Fe absorption from foods. In vivo research is warranted to confirm these observations and to determine if geophagic earth samples can be a source of Fe and/or inhibit Fe absorption.