Characterization of Caco-2 and HT29-MTX cocultures in an in vitro digestion/cell culture model used to predict iron bioavailability

Cocultures of two human cell lines, Caco-2 and HT29-MTX mucus-producing cells, have been incorporated into an in vitro digestion/cell culture model used to predict iron bioavailability. A range of different foods were subjected to in vitro digestion, and iron bioavailability from digests was assessed with Caco-2, Caco-2 overlaid with porcine mucin, HT29-MTX or cocultures of Caco-2 and HT29-MTX at varying ratios. It was found that increasing the ratio of HT29-MTX cells decreased the amount of ferritin formed and resulted in an overall decline in the ability of the model to detect differences in iron bioavailability. At the physiologically relevant ratios of 90% Caco-2/10% HT29-MTX and 75% Caco-2/25% HT29-MTX, however, a mucus layer completely covered the cell monolayer and the in vitro digestion model was nearly as responsive to changes in sample iron bioavailability as pure Caco-2 cultures. The in vitro digestion/Caco-2 cell culture model correlates well with human iron bioavailability studies, but, as mucus appears to play a role in iron absorption, the addition of a physiologically realistic mucus layer and goblet-type cells to this model may give more accurate iron bioavailability predictions.     

Preparation and characterization of iron-containing liposomes: their effect on soluble iron uptake by Caco-2 cells

The aim of this work was to study the iron uptake of Caco-2 cells incubated with five different formulations of liposomes containing iron. The vesicles were also characterized before, during, and after in vitro digestion. Caco-2 cells were incubated with digested and nondigested liposomes, and soluble iron uptake was determined. Nondigested liposomes made with chitosan (CHI) or the cationic lipid, DC-Cholesterol (DC-CHOL), generated the highest iron uptake. However, these two formulations were highly unstable under in vitro digestion, resulting in nonmeasurable iron uptake. Digested conventional liposomes composed of soybean phosphatidylcholine (SPC), hydrogentated phosphatidylcholine (HSPC), or HSPC and cholesterol (CHOL) presented the highest iron-uptake values. These liposomal formulations protected iron from oxidation and improved iron uptake from intestinal cells, compared to an aqueous solution of ferrous sulphate.     

Alginate Inhibits Iron Absorption from Ferrous Gluconate in a Randomized Controlled Trial and Reduces Iron Uptake into Caco-2 Cells

Previous in vitro results indicated that alginate beads might be a useful vehicle for food iron fortification. A human study was undertaken to test the hypothesis that alginate enhances iron absorption. A randomised, single blinded, cross-over trial was carried out in which iron absorption was measured from serum iron appearance after a test meal. Overnight-fasted volunteers (n = 15) were given a test meal of 200 g cola-flavoured jelly plus 21 mg iron as ferrous gluconate, either in alginate beads mixed into the jelly or in a capsule. Iron absorption was lower from the alginate beads than from ferrous gluconate (8.5% and 12.6% respectively, p = 0.003). Sub-group B (n = 9) consumed the test meals together with 600 mg calcium to determine whether alginate modified the inhibitory effect of calcium. Calcium reduced iron absorption from ferrous gluconate by 51%, from 11.5% to 5.6% (p = 0.014), and from alginate beads by 37%, from 8.3% to 5.2% (p = 0.009). In vitro studies using Caco-2 cells were designed to explore the reasons for the difference between the previous in vitro findings and the human study; confirmed the inhibitory effect of alginate. Beads similar to those used in the human study were subjected to simulated gastrointestinal digestion, with and without cola jelly, and the digestate applied to Caco-2 cells. Both alginate and cola jelly significantly reduced iron uptake into the cells, by 34% (p = 0.009) and 35% (p = 0.003) respectively. The combination of cola jelly and calcium produced a very low ferritin response, 16.5% (p<0.001) of that observed with ferrous gluconate alone. The results of these studies demonstrate that alginate beads are not a useful delivery system for soluble salts of iron for the purpose of food fortification.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01528644","term_id":"NCT01528644"}}NCT01528644     

The role of reduced iron powder in the fermentative production of tetanus toxin

When tetanus toxin is made by fermentation with Clostridium tetani, the traditional source of iron is an insoluble preparation called reduced iron powder. This material removes oxygen from the system by forming FeO₂ (rust). When inoculated in a newly developed medium lacking animal and dairy products and containing glucose, soy-peptone, and inorganic salts, growth and toxin production were poor without reduced iron powder. The optimum concentration of reduced iron powder for toxin production was found to be 0.5 g/l. Growth was further increased by higher concentrations, but toxin production decreased. Inorganic iron sources failed to replace reduced iron powder for growth or toxin formation. The iron source that came closest was ferrous ammonium sulfate. The organic iron sources ferric citrate and ferrous gluconate were more active than the inorganic compounds but could not replace reduced iron powder. Insoluble iron sources, such as iron wire, iron foil, and activated charcoal, were surprisingly active. Combinations of activated charcoal with soluble iron sources such as ferrous sulfate, ferric citrate, and ferrous gluconate showed increased activity, and the ferrous gluconate combination almost replaced reduced iron powder. It thus appears that the traditional iron source, reduced iron powder, plays a double role in supporting tetanus toxin formation, i.e., releasing soluble sources of iron and providing an insoluble surface.     

Calcification in insects: The dwelling-tube and midgut of machaerotid larvae (Homoptera)

The dwelling-tubes of machaerotid larvae consist of a mineralized organic scaffolding of mucofibrils. The mineral component accounts for 85 per cent of the dry weight and is composed of calcium, ferrous iron, manganese, magnesium, potassium, sodium, phosphate, carbonate, and chloride and of these the major ions are calcium and carbonate. Ferric iron in the form of ferritin is probably also present.Calcium, manganese, magnesium, and phosphate are derived from spherites secreted by a specialized region of the midgut. Calcium and phosphate are present in the spherites, probably as amorphous tricalcium phosphate. Subsequent to secretion the spherites are slowly dissolved and the calcium is incorporated into the dwelling-tube as calcium carbonate. Thus it appears that within the dwelling-tube calcium phosphate is converted to calcium carbonate.Ferritin and ferrous iron are secreted by another specialized region of the midgut and are also incorporated into the dwelling-tube.     

Absorption of therapeutic preparations of iron measured with a whole body counter

The Oxford Whole Body Counter was used to measure absorption from various therapeutic preparations of iron in five groups of subjects. Succinic acid enhanced absorption of iron when added to a solution of ferrous fumarate, but not when given with tablets of ferrous fumarate or ferrous sulphate. Ferrous fumarate plus ascorbic acid was absorbed better than ferrous fumarate alone but no better than ferrous sulphate. The addition of ascorbic acid and succinic acid to tablets of ferrous sulphate did not enhance absorption significantly.     

Impact of iron and vitamin C-containing supplements on preterm human milk: in vitro

Stress due to reactive oxygen species (ROS) may lead to neonatal diseases, such as necrotizing enterocolitis and respiratory distress. Enteral supplements for premature infants (PREM) added to human milk (HM) to increase nutrient content may induce lipid oxidation due to free radical formation via Fenton chemistry. We hypothesized that ferrous iron and vitamin C-containing supplements added to HM in vitro cause oxidation of milk fats, affect intracellular redox balance, and induce DNA damage. Lipid peroxidation in HM was measured by FOX-2 and TBARS assays; fatty acid composition of supplemented HM was measured by gas chromatography. Two cell culture bioassays were used for assessing either intracellular oxidative stress or DNA damage: the former involved Caco-2BBe cells, a secondary differentiated cell line, and the latter utilized FHS-74 Int cells, a primary fetal small intestinal culture. Lipid oxidation products of HM increased after the addition of iron alone, iron and vitamin C, or iron and a vitamin C-containing supplement (Trivisol, TVS). A reduced content of mono and polyunsaturated fatty acids in HM was also observed. Iron, not iron+vitamin C, but iron+TVS induced significant intracellular oxidative stress in FHS-74 Int cells. In contrast, iron, either alone or in combination with TVS or vitamin C, increased DNA damage in Caco-2BBE cells. Iron supplementation may increase oxidative stress in PREM infants and should be given separately from vitamin C-containing supplements.     

Effect of cooking and legume species upon calcium, iron and zinc uptake by Caco-2 cells

An in vitro system, consisting of simulated gastrointestinal digestion and Caco-2 cell culture, was used to estimate the uptake of calcium, iron and zinc from white beans, chickpeas and lentils, and the effect of cooking upon uptake, with the ultimate aim of evaluating legumes as a dietary source of the aforementioned minerals. In raw products, differences were observed in the uptake percentages by Caco-2 cells of a same mineral from different legumes, although these were not related to the total mineral content. In the three elements studied, the highest uptake values corresponded to chickpeas. Traditional cooking significantly (p<0.05) increased the uptake (%) of calcium, iron and zinc from white beans, and of calcium from lentils. This effect can be partially ascribed to the conversion of inositol hexaphosphate to its lower phosphate forms. When mineral uptakes from raw, traditionally cooked, and ready-to-eat lentils were compared, the highest uptake values corresponded to the ready-to-eat product, which could be attributed to the combined effect of EDTA soaking, the cooking under pressure process, and citric and ascorbic acid addition.     

Porewater oxidation,dissolved phosphate and the iron curtain

The process of dissolved phosphate removal from aqueous solution, which occurs during oxidation of soluble ferrous compounds to insoluble ferric forms, was examined in soils of two tidal freshwater marshes. Sites of amorphous iron deposition and sorption or co-precipitation of phosphate were found to be in surface soils and along creekbanks, where both ion diffusion and porewater advection move dissolved iron and phosphate from reduced to oxidized regions. Profiles of extractable iron and total phosphorus from creekbank and interior soils were consistent with hypothesized differences between a high and a low marsh. Porewater concentrations of dissolved phosphate were higher in creekbank soils of the high marsh, compared with water actually discharging from the creekbank during tidal exposure. We propose that an iron curtain of ferric hydroxides functions as a barrier to diffusive and advective movement of dissolved phosphate along surfaces of tidal freshwater marshes, and has important implications for the distribution and availability of phosphorus in other types of wetlands and aqueous systems.     

Effects of dietary factors on iron uptake from ferritin by Caco-2 cells

Biofortification of staple foods with iron (Fe) in the form of ferritin (Ft) is now possible, both by conventional plant breeding methods and transgenic approaches. Ft-Fe from plants and animals is absorbed well (25-30%) by human subjects, but little is known about dietary factors affecting its absorption. We used human intestinal Caco-2 cells and compared Fe absorption from animal Ft and FeSO4 to determine the effects of inhibitors and enhancers, such as phytic acid, ascorbic acid, tannic acid, calcium and heme. When postconfluent cells were coincubated with 59Fe-labeled (1 microM) FeSO4 and dietary factors, at different molar ratios of dietary factor to Fe (phytic acid:Fe, 10:1; ascorbic acid:Fe, 50:1; tannic acid:Fe, 50:1; calcium:Fe, 10:1 and hemin:Fe, 10:1), all inhibited uptake from FeSO4, except ascorbate, confirming earlier studies. In contrast, these dietary factors had little or no effect on Fe uptake from undigested Ft or Ft digested in vitro at pH 4, except tannins. However, results after in vitro digestion of Ft at pH 2 were similar to those obtained for FeSO4. These results suggest that Fe uptake occurs from both undigested as well as digested Ft but, possibly, via different mechanisms. The Fe-Ft stability shown here could minimize Fe-induced oxidation of Fe-supplemented food products.     

Respiratory enzymes of Thiobacillus ferrooxidans. A kinetic study of electron transfer between iron and rusticyanin in sulfate media

Thiobacillus ferrooxidans is a chemolithotrophic bacterium capable of fulfilling all of its energy requirements from the oxidation of soluble ferrous sulfate. Rusticyanin is a soluble blue copper protein found in abundance in the periplasmic space of this bacterium. The one-electron transfer reaction between soluble iron and purified rusticyanin has been studied by stopped flow spectrophotometry in acidic solutions containing sulfate. Second order rate constants for the reduction of rusticyanin by Fe2+, FeHSO4+, and FeSO4(0) were 0.022, 0.73, and 2.30 M-1 s-1, respectively. The pseudo-first order rate constant for the reduction of rusticyanin exhibited substrate saturation when the concentration of the total ferrous ion was varied in solutions of limiting sulfate. This saturation behavior was quantitatively described using the values of the second order rate constants listed above and the distribution of the total ferrous ion into its water-, bisulfate-, and sulfate-coordinated forms. Second order rate constants for the oxidation of rusticyanin by Fe3+ and FeSO4+ were 0.73 and 0.26 M-1 s-1, respectively. The electron transfer reactions between iron and rusticyanin monitored in vitro were far too slow to support the hypothesis that rusticyanin is the primary oxidant of ferrous ions in the iron-dependent respiratory electron transport chain of T. ferrooxidans.     

Caco-2 Cell Acquisition of Dietary Iron(III) Invokes a Nanoparticulate Endocytic Pathway

Dietary non-heme iron contains ferrous [Fe(II)] and ferric [Fe(III)] iron fractions and the latter should hydrolyze, forming Fe(III) oxo-hydroxide particles, on passing from the acidic stomach to less acidic duodenum. Using conditions to mimic the in vivo hydrolytic environment we confirmed the formation of nanodisperse fine ferrihydrite-like particles. Synthetic analogues of these (~ 10 nm hydrodynamic diameter) were readily adherent to the cell membrane of differentiated Caco-2 cells and internalization was visualized using transmission electron microscopy. Moreover, Caco-2 exposure to these nanoparticles led to ferritin formation (i.e., iron utilization) by the cells, which, unlike for soluble forms of iron, was reduced (p=0.02) by inhibition of clathrin-mediated endocytosis. Simulated lysosomal digestion indicated that the nanoparticles are readily dissolved under mildly acidic conditions with the lysosomal ligand, citrate. This was confirmed in cell culture as monensin inhibited Caco-2 utilization of iron from this source in a dose dependent fashion (p<0.05) whilet soluble iron was again unaffected. Our findings reveal the possibility of an endocytic pathway for acquisition of dietary Fe(III) by the small intestinal epithelium, which would complement the established DMT-1 pathway for soluble Fe(II).     

Promotion and nucleation of carbonate precipitation during microbial iron reduction

Iron‐bearing early diagenetic carbonate cements are common in sedimentary rocks, where they are thought to be associated with microbial iron reduction. However, little is yet known about how local environments around actively iron‐reducing cells affect carbonate mineral precipitation rates and compositions. Precipitation experiments with the iron‐reducing bacterium Shewanella oneidensis MR‐1 were conducted to examine the potential role of cells in promoting precipitation and to explore the possible range of precipitate compositions generated in varying fluid compositions. Actively iron‐reducing cells induced increased carbonate mineral saturation and nucleated precipitation on their poles. However, precipitation only occurred when calcium was present in solution, suggesting that cell surfaces lowered local ferrous iron concentrations by adsorption or intracellular iron oxide precipitation even as they locally raised pH. Resultant precipitates were a range of thermodynamically unstable calcium‐rich siderites that would likely act as precursors to siderite, calcite, or even dolomite in nature. By modifying local pH, providing nucleation sites, and altering metal ion concentrations around cell surfaces, iron‐reducing micro‐organisms could produce a wide range of carbonate cements in natural sediments.     

A Model for Calcium Permeation into Small Intestine

An in vitro model was used to simulate the intestinal permeation of calcium ions depending on the type of salt (carbonate, fumarate, citrate, or gluconate), its concentration (1.0, 2.5, 5.0, or 10 mM/l), and pH (1.3, 4.2, 6.2, or 7.5). To simulate the conditions for calcium permeation in a patient in a fasting state, the solutions were placed in contact with segments of small intestine of pig: stomach, duodenum, jejunum, and ileum. The percent permeation, its rate, and half-time were measured in each case. In all cases, the maximum permeation was seen at 1 mM concentration, depending on pH: 100% for carbonate at pH 1.3; 82% for fumarate, pH 6.2; 79.5% for citrate at pH 4.2, and 81% for gluconate at pH 7.4. The maximum rate of permeation (% h⁻¹) was also observed at 1 mM: 2.16 for carbonate at pH 1.3, 0.29 for fumarate at pH 6.2, 0.26 for citrate at pH 4.2, and 0.28 for gluconate at pH 7.4. The shortest half-time permeation (t _1/2, h) for 1 mM solutions depended also on pH (in parentheses): carbonate 0.3 (1.3), fumarate 2.4 (6.2), citrate 2.6 (4.2), and gluconate 2.5 (7.4). The results suggest that calcium carbonate and citrate can be recommended to patients with normal gastric acidity and hyperacidity while fumarate and gluconate to patients with hypoacidity.     

Biochemistry of nonheme iron in man. II. Absorption of iron

The currently accepted concept of iron absorption proposes first the entry of iron into the intestinal mucosal cell through the brush border membrane. It is a relatively slow process. In the cell, the iron may be transferred to plasma or become sequestered by ferritin. The latter becomes unavailable for transfer to plasma and is exfoliated and excreted. In iron deficiency and idiopathic hemochromatosis, the rate of iron uptake into the intestinal mucosal cell is increased and entry into ferritin is decreased, whereas the rate of transfer to plasma remains constant. The reverse occurs in case of secondary iron overload. It is currently accepted that a transferrin, whose levels increase in iron deficiency, enters the intestinal lumen from the liver via bile, where it may sequester iron and bring it into the cells by the process of endocytosis. Iron presented as inorganic ferric or ferrous salts may also be absorbed, though the more soluble ferrous salts are adsorbed much more rapidly. Heme iron is absorbed very effectively, though it is not subject to regulation by the individual's iron status to the same extent as is inorganic iron absorption. Brush border membranes apparently contain saturable iron receptors for inorganic iron, but whether or not the absorption process requires energy is an open question. Absorption of iron may also be affected by its availability; different food components affect iron absorbability to a different extent.     

The Influence of Condition on Permeation of Ca(II) Ions from Solutions of Selected Calcium’s Salts Through Model Membrane

The permeation of calcium’s ions from calcium solutions of fumarate, gluconate, and citrate through model membrane from the donor chamber to the acceptor chamber has been examined. Process was traced depending on the concentration of the appropriate calcium’s salts (1, 2.5, and 5 mmol/l) and pH value of acceptor environment (1.3, 6.2, and 7.4) which imitated natural conditions appearing in the digestive tract. The amount of permeating Ca(II) ions (percent) and their Ca(II) availability AUC (0–6 h) has been determined. In dependence on the conditions, penetration was as follows: 30.3–95.2% of calcium ions from fumarate solution; 73.0–90.1% of Ca(II) from citrate solution; and 19.0–95.0% of Ca from gluconate solution. The investigation indicates that the amount of permeated Ca(II) ions and their availability are connected with the concentration of the calcium salt and pH of acceptor environment. Fumarate and citrate are available at pH value of acceptor environment 1.3 and 6.2 and gluconate at the pH value of 6.2 and 7.4. These substances are practically unavailable from the acceptor environment at pH value 1.3 for gluconate and 7.4 for fumarate. Results suggest that calcium citrate can be available for organism independently from pH value of acceptor environment.     

Anaerobic and aerobic oxidation of ferrous iron at neutral pH by chemoheterotrophic nitrate-reducing bacteria

Nine out of ten anaerobic enrichment cultures inoculated with sediment samples from various freshwater, brackish-water, and marine sediments exhibited ferrous iron oxidation in mineral media with nitrate and an organic cosubstrate at pH 7.2 and 30° C. Anaerobic nitrate-dependent ferrous iron oxidation was a biological process. One strain isolated from brackish-water sediment (strain HidR2, a motile, nonsporeforming, gram-negative rod) was chosen for further investigation of ferrous iron oxidation in the presence of acetate as cosubstrate. Strain HidR2 oxidized between 0.7 and 4.9 mM ferrous iron aerobically and anaerobically at pH 7.2 and 30° C in the presence of small amounts of acetate (between 0.2 and 1.1 mM). The strain gained energy for growth from anaerobic ferrous iron oxidation with nitrate, and the ratio of iron oxidized to acetate provided was constant at limiting acetate supply. The ability to oxidize ferrous iron anaerobically with nitrate at approximately pH 7 appears to be a widespread capacity among mesophilic denitrifying bacteria. Since nitrate-dependent iron oxidation closes the iron cycle within the anoxic zone of sediments and aerobic iron oxidation enhances the reoxidation of ferrous to ferric iron in the oxic zone, both processes increase the importance of iron as a transient electron carrier in the turnover of organic matter in natural sediments. Received: 24 April 1997 / Accepted: 22 September 1997     

Physicochemical properties and inhibition effect on iron deficiency anemia of a novel polysaccharide-iron complex (LPPC)

Porphyran (P) was extracted from red algae Porphyra by boiling water. A novel polysaccharide-iron complex (LPPC) was prepared under the alkaline condition by adding a ferric chloride solution to the low molecular weight porphyran (LP) solution. Physicochemical properties and inhibition effect on iron deficiency anemia of this complex were studied. The content of iron(III) in the complex is 21.57% determined with iodometry. The results indicate that LPPC was product required. The complex can increase red blood cell count (RBC), hemoglobin (Hb), Serum iron (SI), spleen index, spleen mass and mass of mice with iron deficiency anemia (IDA). Although the structure and deeper mechanisms on hemolytic anemia of LPPC should be further studied, LPPC is hoped to be developed as a late-model iron supplement which has a synergism on anemia.     

Bile and the absorption of strontium and iron

1. Strontium absorption was studied in vivo with loops of ileum in rachitic chicks and found to be increased by vitamin D(3), bile salts and sodium lauryl sulphate. 2. Bile salts and sodium lauryl sulphate rendered strontium soluble in butanol-benzene (1:1, v/v). 3. Bile was not concerned in the absorption of iron in rats from its water-soluble form, ferrous sulphate. 4. Ligation of the bile ducts in rats caused a decrease in the absorption of iron presented as its sparingly soluble phosphate. 5. The effect of bile on cation absorption is discussed.