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.     

In vitro iron availability from iron-fortified whole-grain wheat flour

Iron deficiency is the most common nutritional disorder worldwide. Iron fortification of foods is considered to be the most cost-effective long-term approach to reduce iron deficiency. However, for fortified foods to be effective in reducing iron deficiency, the added iron must be sufficiently bioavailable. In this study, fortification of whole-grain wheat flour with different sources of iron was evaluated in vitro by measuring the amount of dialyzable iron after simulated gastrointestinal digestion of flour baked into chapatis and subsequent intestinal absorption of the released iron using Caco-2 cell layers. The dialyzability of iron from iron-fortified wheat flour was extremely low. Additions of 50 mg/kg iron to the flour in the form of ferrous sulfate, Ferrochel amino acid chelate, ferric amino acid chelate taste free (TF), Lipofer, ferrous lactate, ferrous fumarate, ferric pyrophosphate, carbonyl iron, or electrolytic iron did not significantly increase the amount of in vitro dialyzable iron after simulated gastrointestinal digestion. In contrast, fortification of flour with SunActive Fe or NaFeEDTA resulted in a significant increase in the amount of in vitro dialyzable iron. Relative to iron from ferrous sulfate, iron from SunActive Fe and NaFeEDTA appeared to be 2 and 7 times more available in the in vitro assay, respectively. Caco-2 cell iron absorption from digested chapatis fortified with NaFeEDTA, but not from those fortified with SunActive Fe, was significantly higher than from digested chapatis fortified with ferrous sulfate. On the basis of these results it appears that fortification with NaFeEDTA may result in whole-grain wheat flour that effectively improves the iron status.     

Characterization of commercial iron chelates and their behavior in an alkaline and calcareous soil

Iron deficiency is a common problem for many plants grown in alkaline and calcareous soils. To correct this problem, iron is supplied to plants as chelates. Several iron chelates are sold under diverse trademarks with different characteristics. This work evaluated 18 commercial products containing the most representative chelated iron sources used in agricultural practice in Spain when the study was done, namely the ferric chelates of EDDHA, EDDHMA, EDDCHA, EDDHSA, EDTA, and DTPA. The chelates were comprehensively characterized and quantitated by several techniques, including several chromatographic methods. Iron and chelate dynamics in soil were also studied in a model alkaline and calcareous soil. Results indicate that, in this model soil, among the different iron compounds studied only FeEDDHA and analogues have the capacity to maintain soluble iron in soil solution over time. These results are in agreement with general experience under field conditions. Furthermore, among the different ortho-ortho isomers of FeEDDHA's, FeEDDHSA and FeEDDCHA showed greater capacity than FeEDDHA and FeEDDHMA to maintain the chelated iron in soil solution over time.     

Combined impact of pH and organic acids on iron uptake by Caco-2 cells

Previous studies have shown that organic acids have an impact on both Fe(II) and Fe(III) uptake in Caco-2 cell. However, to what extent this effect is correlated with the anion of organic acids per se, or with the resulting decrease in pH, has not yet been clarified. Therefore, we studied the effect of five organic acids (tartaric, succinic, citric, oxalic, and propionic acid) on the absorption of Fe(II) and Fe(III) in Caco-2 cells and compared this with sample solutions without organic acids but set to equivalent pH by HCl. The results showed that the mechanisms behind the enhancing effect of organic acids differed for the two forms of iron. For ferric iron the organic acids promoted uptake both by chelation and by lowering the pH, whereas for ferrous iron the promoting effect was caused only by the lowered pH.     

Comparison of iron bioavailability from 15 rice genotypes: studies using an in vitro digestion/caco-2 cell culture model

An in vitro digestion/Caco-2 model was used to compare iron bioavailability from 15 selected Fe-dense and normal genotypes of unpolished rice from the International Rice Research Institute. Iron uptake was determined using Caco-2 cell ferritin formation in response to exposure to a digest of the cooked rice. Iron bioavailabilities from all rice genotypes were ranked as a percent relative to a control variety (Nishiki). Iron concentration in the rice samples ranged from 14 to 39 microg/g. No correlation was observed between Fe uptake and grain-Fe concentration. Furthermore, phytic acid levels were not correlated with Fe bioavailability. Genotypes with low Fe bioavailability (Tong Lan Mo Mi, Zuchein, Heibao, and Xua Bue Nuo) were noticeably more brown to purple in color. The results suggest that certain unknown compounds related to rice grain color may be a major factor limiting Fe bioavailability from unpolished rice.     

Effect of Iron Source on Color and Appearance of Micronutrient‐Fortified Corn Flour Tortillas

Iron deficiency anemia is a widespread occurrence. Consequently, iron is commonly added in cereal fortification programs. However, many iron sources cause undesirable sensory changes, especially color changes, in the food being fortified. This study evaluated the effect of different iron sources on CIE L*a*b* color values and sensory color perception in fortified corn tortillas. Corn masa flour was fortified with micronutrient premix containing vitamins, zinc, and one of eight iron compounds. Iron sources included ferrous fumarate (F), ferrous sulfate (S), ferric orthophosphate (OP), ferrous lactate (L), ferrous gluconate (G), ferric pyrophosphate (PP), sodium iron (III)‐EDTA, and A‐131 electrolytic iron (E), with addition levels adjusted based on bioavailability. Control (Ct) samples were prepared with all micronutrients except iron. All iron‐fortified tortillas had lower L* values and were significantly darker than control tortillas. Based on instrumental color values and Mexican regulatory recommendations, five treatments were selected for further testing. A difference‐from‐control sensory test was conducted comparing PP, E, OP, F, and S with Ct tortillas. Sensory rankings were C t > E = PP > OP > F > S. A‐131 electrolytic iron is recommended for fortification of corn tortillas due to minimal effect on color and significantly lower cost than other iron sources evaluated.     

Comparison of iron uptake from reduced iron powder and FeSO4 using the Caco-2 cell model: effects of ascorbic acid, phytic acid, and pH

The reduced iron powder has considerable potential for use as an iron fortificant because it does not change organoleptically during storage or food preparation for cereal flour, and its bioavailability is scarcely influenced by iron absorption inhibitors in foods. The objective of this article is to study the effects of ascorbic acid, phytic acid, and pH on iron uptake from reduced iron powder (43 microm) and FeSO 4, and to compare iron bioavailability of reduced iron powders among four selected granularity levels. The cell ferritin formation is used as a marker of iron uptake. Obviously, iron uptake of reduced iron powder is increased with decreasing of powder granularity and is much lower than FeSO 4 when the size is above 43 microm, but significantly higher at 40-60 nm. In the presence of ascorbic acid or phytic acid, Caco-2 cell iron absorption from reduced iron powder (43 microm) is significantly higher than that from FeSO 4. And iron uptake of Caco-2 cells is decreased with increasing of pH from 5.5 to 7.5. Moreover, the decrease trend is more obvious for reduced iron powder than for FeSO 4. Our results indicated that iron bioavailability of reduced iron powder by intestinal enterocytes is similar to that of iron salts, and reduced iron powder is more excellent than FeSO 4 as food fortificant, especially at ultramicroscopic granularity.     

Visualisation of oxidizing power of rice roots and of possible participation of bacteria in iron deposition

The oxidizing power of rice roots was observed in narrow transparent root boxes containing different media. Plants precultivated in nutrient solution were embedded in semisolid agar medium to observe oxidation of ferrous iron cations and leuco methylene blue as well as solubilization of ferrous sulfide. In the presence of ferrous sulfate reddish brown coloration due to formation of ferric oxide/hydroxide was observed around the roots and on the root surface during one day of incubation. When agar medium blackened by ferrous sulfide was used, the root zone became transparent. Within a few hours leuco methylene blue was oxidized to methylene blue on and near the roots. Furthermore, seedlings were grown in agar medium containing ferrous sulfide inoculated with soil filtrate. Besides diffuse ferric iron precipitation, iron was also deposited on spherically shaped structures in the rhizosphere and near the agar surface as well as in slimy layers appearing on the root surface. The spherical structures and slimy layers were obviously bacterial colonies extending with time. As the roots grew old, parts of them turned black. In the rhizosphere, black spots occurred resembling colonies of sulfate-reducing bacteria. Rice was also grown in sand supplemented with nutrients and iron sulfide. While root growth was straight in agar, it was twisted in the sand medium. Again, heavy ferric iron deposition occurred on the root surface. On older root parts the lateral roots became blackish. The results suggest participation of bacteria in ferric iron deposition in the rhizosphere of rice.     

水稻土的氧化还原过程及其与水稻生长的关系

水稻土的主要特点是在一年之中有季节性的淹水和落干,而各种水稻土的干湿程度和时间都有不同,因而影响土壤性质发生复杂的变化。在这些性质中,氧化还原状况是变化最剧烈而指示最明显的性质,并且直接或间接影响水稻的生长。为此,作者等从1954年起,开始了这方面的研究工作,下面是关于几个主要简题的筒单概括。     

Nitrite stability influenced by iron compounds

The decomposition of nitrite was studied in the presence of (1) different amounts of ferrous iron and (2) an amorphous and a crystalline (haematite) iron product at different pH and Eh conditions. It was found that ferrous iron positively influenced the nitrite decomposition. Even at pH 6, where self-decomposition is excluded, some nitrite was decomposed. It was shown that at all studied pH values the second order decomposition rate increased as the amount of ferrous iron increased. From the calculation of the activation energy it was found that the dependence of the rate constant on temperature increased when the medium was more acid, or when the amount of Fe²⁺ increased at the same pH. The nitrite half-life was longest at pH 6, 25°C and 200 mg Fe²⁺ l^?1; it was shortest at pH 4, 30°C and 800 mg Fe²⁺ l^?1. The experiments with Fe²⁺ derived from solid iron compounds showed that all conditions favouring a high amount of ferrous iron in solution, such as low redox potential, low pH, amorphous or less crystalline material, enhanced nitrite decomposition.     

FORMATION OF IRON OXIDES BY DECOMPOSITION OF IRON-PHENOLIC CHELATES

Iron chelates of some simple di-and trihydric phenols can be decomposed by peroxide treatment or by hydrolysis to yield crystalline ferric oxides. The species of ferric oxide obtained depends upon the precipitating conditions and the nature of the chelating ligand. In oxidative virtually anion-free conditions, decomposition of all aged chelates yields a precipitate of disordered ferric oxide, which is referred to as‘protohaematite’, since it ages to haematite in water even at room temperature. Protohaematite is considered to be a discrete form of ferric oxide similar in structure to δ-FeOOH but devoid of hydroxyl groups and may be present in freely drained soils as a precursor of haematite. Hydrolysis of all unaged chelates, except that of iron protocatechuic acid, yields lepidocrocite. The redox cycle undergone by iron in this reaction may be analogous to one prevailing in gley soils. In the presence of montmorillonite, the iron-catechol system forms a clay-metal-organic complex, which also decomposes to yield lepidocrocite provided chloride ions are present on the clay surface. The fact that various inorganic gels amorphous to X-rays can also deplete the ligand content of the chelates indicates a possible inorganic decomposition mechanism for metal-organic chelates in soils.     

不同铁形态对水稻根表铁膜及铁吸收的影响

通过溶液培养试验研究了FeCl2?4H2O和FeCl3?6H2O对水稻根表铁膜数量及铁吸收的影响。结果表明,FeCl2处理时水稻根表铁膜浓度是FeCl3处理的197%～233%。利用EDTA-BPDS对铁膜形态分析看出,根表铁膜中Fe3+占85%～92%,Fe2+占8%～15%。水稻天优998根表铁膜数量显著高于培杂泰丰,其铁吸收是培杂泰丰的115%～138%。两种铁形态处理明显提高水稻的根系活力,其中,FeCl2处理时水稻根系活力增加24%～69%,FeCl3为16%～54%。FeCl2处理时水稻根系SOD、POD和CAT活性分别增加11%～32%、15%～30%和30%～31%,但FeCl3处理没有明显影响。上述结果表明一定浓度铁处理明显增加水稻根表铁浓度和铁吸收;与FeCl3处理相比,FeCl2处理能提高根系抗氧化酶活性,增加水稻的铁吸收和根表铁膜数量。     

The influence of vegetation and soil type on the speciation of iron in soil water

Soluble organic matter derived from exotic Pinus species has been shown to form stronger complexes with iron (Fe) than that derived from most native Australian species. It has also been proposed that the establishment of exotic Pinus plantations in coastal southeast Queensland may have enhanced the solubility of Fe in soils by increasing the amount of organically complexed Fe, but this remains inconclusive. In this study we test whether the concentration and speciation of Fe in soil water from Pinus plantations differs significantly from soil water from native vegetation areas. Both Fe redox speciation and the interaction between Fe and dissolved organic matter (DOM) were considered; Fe – DOM interaction was assessed using the Stockholm Humic Model. Iron concentrations (mainly Fe²⁺) were greatest in the soil waters with the greatest DOM content collected from sandy podosols (Podzols), where they are largely controlled by redox potential. Iron concentrations were small in soil waters from clay and iron oxide‐rich soils, in spite of similar redox potentials. This condition is related to stronger sorption on to the reactive clay and iron oxide mineral surfaces in these soils, which reduces the amount of DOM available for electron shuttling and microbial metabolism, restricting reductive dissolution of Fe. Vegetation type had no significant influence on the concentration and speciation of iron in soil waters, although DOM from Pinus sites had greater acidic functional group site densities than DOM from native vegetation sites. This is because Fe is mainly in the ferrous form, even in samples from the relatively well‐drained podosols. However, modelling suggests that Pinus DOM can significantly increase the amount of truly dissolved ferric iron remaining in solution in oxic conditions. Therefore, the input of ferrous iron together with Pinus DOM to surface waters may reduce precipitation of hydrous ferric oxides (ferrihydrite) and increase the flux of dissolved Fe out of the catchment. Such inputs of iron are most probably derived from podosols planted with Pinus.     

A new method for determining ferrous iron in paddy soils

Introduction

To make clear the chemical behavior of free iron compounds in paddy soils, it is necessary to find an accurate and rapid method for determining ferrous iron in waterlogged paddy soils. Several methods^1,2,3) have been proposed for this purpose, most of which use dilute acids, such as sulfuric and hydrochloric acid, as extracting reagents. According to the writers' experience, however, these methods seem to be inadequate. The reason is that the acid solutions extract not only ferrous iron, but also ferric iron and reducing substances from soils, and the latter two react immediately in the extract to produce ferrous iron, thus we obtain larger value for ferrous iron than the one actually existing in soils. The writers have found that acetate buffer of pH 2.8 is a very suitable extractant for ferrous iron, and they have been able to establish a new method for the determination of ferrous iron in soils using this buffer. The experimental details will be given in this paper.     

Removal of ferrous iron from field drainage waters by conifer bark

Iron ochre deposition in field drainage systems is produced as a result of chemical and microbiological oxidation of ferrous iron in soil solution to the insoluble ferric form. Conifer bark, from several plant species, will absorb ferrous iron from solution quickly and irreversibly, the bark changing colour from brown to blue in the process. This bark, unlike that from deciduous trees, contains only small quantities of soluble phenolic components so it does not create an environmental problem under field conditions. Trials carried out at a test farm, where bark was placed within nylon mesh sacks and incorporated into the drainage system, were very successful, and drainage pipes which had previously blocked twice yearly remained relatively free of ochre. The use of bark from conifers can offer an inexpensive method of ameliorating the problem of ochre formation without producing environmental pollution.     

Interaction of Different Iron Chelates with an Alkaline and Calcareous Soil: A Complementary Methodology to Evaluate the Performance of Iron Compounds in the Correction of Iron Chlorosis

Abstract

A great number of studies have shown that the stability of iron chelates as a function of pH is not the unique parameter that must be considered in order to evaluate the potential effectiveness of Fe‐chelates to correct iron chlorosis in plants cultivated in alkaline and calcareous soils. In fact, other factors, such as soil sorption on soil components or the competition among Fe and other metallic cations for the chelating agent in soil solution, have a considerable influence on the capacity of iron chelates to maintain iron in soil solution available to plants. In this context, the aim of this work is to study the variation in concentration of the main iron chelates employed by farmers under field conditions—Fe‐EDDHA (HA), Fe‐EDDHMA (MA), Fe‐EDDHSA (SA), Fe‐EDDCHA (CA), Fe‐EDTA (EDTA), and Fe‐DTPA (DTPA)—in the soil solution of a calcareous soil over time. To this end, soil incubations were carried out using a soil:Fe solution ratio corresponding to soil field capacity, at a temperature of 23°C. The soil used in the experiments was a calcareous soil with a very low organic matter content. The variation in concentration of Fe and Fe‐chelates in soil solution over time were obtained by measuring the evolution in soil solution of both the concentration of total Fe (measured by AAS), and the concentration of the ortho‐ortho isomers for Fe‐EDDHA and analogs or chelated Fe for Fe‐EDTA and Fe‐DTPA (measured by HPLC). The following chelate samples were used: a HA standard prepared in the laboratory and samples of HA, MA, SA, CA, Fe‐EDTA, and Fe‐DTPA obtained from commercial formulations present in the market. The percentage of iron chelated as ortho‐ortho isomers for HAs was: HA standard (100%); HA (51.78%); MA (60.06%); SA (22.50%); and CA (27.28%). In the case of Fe‐EDTA and Fe‐DTPA the percentages of chelated iron were 96.09 and 99.12, respectively. Results show that it is possible to classify the potential effectiveness of the different types of iron chelates used in our experiments as a function of two practical approaches: (i) considering the variation of total iron in soil solution over time, MA is the best performing product, followed by HA, CA, SA, DTPA, EDTA, and ferrous sulfate in the order listed and (ii) considering the capacity of the different iron chelates to maintain the fraction of chelated iron (ortho‐ortho isomers for HA, MA, SA, and CA and total chelated iron for EDTA and DTPA) in soil solution, the order is: SA > CA > HA > MA > EDTA ≈ DTPA. This result, that is related to the nature of the chelate and does not depend on the degree of chelated Fe in the products, indicates that SA and CA might be very efficient products to correct iron chlorosis. Finally, our results also indicate the suitability of this soil incubation methodology to evaluate the potential efficiency of iron compounds to correct iron chlorosis.     

The use of iron chelates in compound fertilizers containing trace elements

Abstract

The chemical behaviour of iron chelates, incorporated in compound fertilizers with inorganic micronutrient compounds, was studied. Pot experiments were conducted to evaluate the effectiveness of these products in controlling iron deficiency. A commercial compound fertilizer, containing 0.09% Fe as Fe‐DTPA, 0.12% Cu, 0.16% Mn and 0.04% Zn as sulfates, proved to be ineffective in preventing iron chlorosis in Chamaecyparis lawsoniana ’Alumii’, growing on sphagnum peat at two lime levels. The same fertilizer formulation with 0.09%‐ Fe as Fe‐EDDHA did prevent iron chlorosis at both lime levels. Fe‐EDTA, incorporated in the compound fertilizer, gave good results at the lower but not at the higher lime level, due to the low stability of Fe‐EDTA at high pH. Data from laboratory experiments showed that copper replaced the chelated iron in the compound fertilizer containing Fe‐DTPA, causing the iron to precipitate. The strong competition between copper and iron for the organic ligand is due to a specific affinity of copper for DTPA, resulting in a copper chelate with high stability constant and a molar ratio of copper to chelating agent of 2 : 1.

In the case of Fe‐EDDHA and Fe‐EDTA the competition between iron and copper is much weaker. In contrast to Fe‐DTPA, these chelates remain rather stable when incorporated in fertilizers containing micronutrients.     

Coupled diffusion and oxidation of ferrous iron in soils. I. Kinetics of oxygenation of ferrous iron in soil suspension

The kinetics of oxidation of iron in an aqueous suspension of a thoroughly reduced low-humus tropical rice paddy soil were followed by measuring the extractable ferrous iron in the whole suspension and in the solution. Three-quarters of the initial ferrous iron was oxidized rapidly (first-order rate constant = 9.2 × 10^?5 s^?1). The subsequent reaction was slow (first-order rate constant = 9.4 × 10^?7 S^?1) and was not studied in detail. The pH fell from 6.6 to 4.9 over the course of the fast reaction. In further experiments the rate of oxidation was followed at constant pH values in the range 6.5 to 4.5. It was concluded that the oxidation of adsorbed iron was much faster than solution iron, and that the adsorbed iron was oxidized at a rate that was nearly independent of the pH. During the reaction some ferrous iron is adsorbed on the ferric hydroxide formed. The proportion of the remaining ferrous iron adsorbed on ferric hydroxide rather than the original exchange surfaces was high at pH > 6.0 and low at pH < 5.0. The rate of oxidation of the ferrous iron was similar whether it was adsorbed on exchange sites or on the ferric hydroxide formed. Since the rate of oxidation of the iron adsorbed on ferric hydroxide was very much slower than that on ferric hydroxide formed in the absence of soil, it is suggested that the rate in soil may be controlled by diffusion of oxygen to the adsorption sites.     

Prolonged transit time through the stomach and small intestine improves iron dialyzability and uptake in vitro

The iron dialyzability and uptake in relation to transit time through the stomach and small intestine was investigated using a dynamic in vitro gastrointestinal model in combination with Caco-2 cells. Three test meals were evaluated, consisting of lactic fermented vegetables with white (I) or whole meal bread (II) and of sourdough-fermented rye bread (III). Three transit times were tested (fast, medium, and slow transport). Iron dialyzability and absorption differed significantly between medium and slow transit time for meal I and between fast and medium transit time for meal III. For meal II, high in phytate, the iron dialyzability and absorption were low irrespective of transit time. The meals could be ranked with respect to iron dialyzability and uptake in the order I > III > II. Although the in vitro models used have limitations compared to in vivo experiments, the results suggest that an increased transit time may improve iron availability.     

Effect of carbon dioxide on the stability of iron chelates

The effect of carbon dioxide partial preassure on the stability of iron chelates in calcareous soils is often laid aside. Theoretical stability diagrams have been developed showing the large importance of this compound on total iron solubility from Fe‐EDTA and ‐DTPA chelates. As CO₂ increases free Ca²⁺ activity in solution decreases. Then chelation of iron increases. CO₂ partial preassure does not change the stability of Fe‐EDDHA since percentage of chelation is 100% in all conditions tested for this chelate.

Experimental data fit theoretical diagrams well when CO₂ partial preassure is high, but when CO₂ ^‐free air is used, chelation is greater than expected. Causes that can produce this difference are discussed.