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.     

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.     

Bioavailability of calcium from milk-based formulas and fruit juices containing milk and cereals estimated by in vitro methods (solubility, dialyzability, and uptake and transport by caco-2 cells)

An adequate calcium intake during the first years of life is needed for normal growth and development and to prevent rickets. The bioavailability of calcium from infant foods (milk-based formulas and fruit juices containing milk and cereals, FMC), the dietary sources of calcium in these stages of life, has been estimated on the basis of simulated gastrointestinal digestion and calcium solubility and dialyzability values and on the efficiency of transport and uptake by Caco-2 cells. The ranking of samples according to calcium bioavailability depends on the use of solubility or dialyzability as criterion. On the basis of the former, the highest value corresponded to adapted formulas and the lowest to fruit juices. However, when using percentage dialysis, the highest value corresponded to fruit juices and the lowest to follow-up formulas. The highest percentages of transport efficiency and uptake by Caco-2 cells corresponded to calcium from the analyzed fruit juices, followed by toddler, follow-up, and adapted formulas.     

Inositol phosphates influence iron uptake in Caco-2 cells.

Phytate, inositol hexaphosphate (InsP(6)), may be hydrolyzed to inositol phosphates with lower degree of phosphorylation, i.e., inositol penta- to monophosphates (InsP(5)-InsP(1)), during food processing. Each of these lower inositol phosphates exists in different isomeric forms. The objective of this study was to determine if different isomers of InsP(3)-InsP(5) (Ins(1,2,4)P(3), Ins(1,2,3)P(3), Ins(1,2,6)P(3), Ins(1,3,4)P(3), Ins(1,2,3,4)P(4), Ins(1,2,5,6)P(4), Ins(1,2,4,5,6)P(5), and Ins(1,3,4,5,6)P(5)) and InsP(6) affect the uptake of iron. We studied the iron absorption in vitro using the human intestinal epithelial cell line, Caco-2. Addition of a 2-fold molar excess of InsP(6) or InsP(5) in proportion to Fe (1 h incubation at 37 degrees C) reduced iron uptake by 46-52% (p < 0.001). Neither InsP(4) isomers nor InsP(3) isomers affected iron uptake significantly at 1 h incubation with a molar InsP:Fe level of 2:1. Iron uptake was shown to not be a function of the isomeric form of inositol phosphates. The inositol phosphate isomers did not seem likely to interact with each other through iron to form more stable iron complexes. At a molar InsP:Fe level of 20:1 an inhibitory effect of InsP(4) was found, while InsP(3) did not affect the iron absorption even at a 20-fold molar excess.     

Influence of Added Enzymes and Bran Particle Size on Bread Quality and Iron Availability

The aim of this investigation was to study the influence of different bran proportions and particle sizes, addition of fungal phytase, and α‐amylase addition on bread quality and phytate levels, and how these treatments affect availability of iron to intestinal epithelial (Caco‐2) cells. Potential mineral contributions to dietary reference intakes and phytate‐to‐mineral molar ratios were also evaluated. Wheat bran supplementation significantly affected bread quality. Smaller bran particle size affected crumb firmness negatively, whereas the use of α‐amylase, in some cases in combination with phytase, could improve technological bread quality. The use of phytase in the formulation significantly reduced the level of phytates, and phytate hydrolysis also led to smaller bran particle size. Increasing the bran proportion used in the bread formulation increased the iron concentration in bread samples by 18.9%. Phytase addition proved to be a useful strategy to improve iron dialyzability; however, incomplete dephytinization still had an inhibitory effect on iron uptake, with the exception of samples formulated with 10% bran. The inhibitory effect of phytate could be predicted from the values of the phytate‐to‐iron ratios. Reduction of particle size did not improve iron availability or uptake by Caco‐2 cells.     

Degradation of phytate by high-phytase Saccharomyces cerevisiae strains during simulated gastrointestinal digestion

Hydrolysis of extracellular phytate (InsP(6)) by high-phytase yeast strains and survival of yeast cells were studied at simulated digestive conditions using yeast peptone dextrose growth medium and wheat gruel as model meals. An in vitro digestion method was modified to better correlate with the gastric pH gradient following food intake in vivo. High-phytase yeast gave a strong reduction of InsP(6) (up to 60%) in the early gastric phase, as compared to no degradation by wild-type strains. The degree of InsP(6) degradation during digestion was influenced by the type of yeast strain, cell density, and InsP(6) concentration. Despite high InsP(6) solubility, high resistance against proteolysis by pepsin, and high cell survival, degradation in the late gastric and early intestinal phases was insignificant. Dependency on pH for phytase expression and/or activity seemed thus to be an important limiting factor. Although further studies are needed, our results show the potential of using yeast as a phytase carrier in the gastrointestinal tract.     

Biofortification of rice with zinc: assessment of the relative bioavailability of zinc in a Caco-2 cell model and suckling rat pups

Staple foods, such as rice, can now be enriched in micronutrients through conventional breeding (i.e., biofortification) to enhance dietary intake of vulnerable populations. The objectives of this study were (1) to establish a rapid, high capacity Caco-2 cell model to determine the relative bioavailability of zinc (Zn) from samples of staple food breeding lines for potential use as a guideline for selection/breeding and (2) to determine the relative bioavailability of Zn from conventional rice varieties and one Zn-biofortified type. Polished or undermilled, parboiled rice samples were digested in vitro with pepsin and pH adjustment, and by pancreatic enzymes. Zn uptake from digested samples was measured in Caco-2 cells in culture. A previously validated rat pup model was also used to assess Zn absorption in vivo, using gastric intubation and (65)Zn labeling. Pups were killed after 6 h, and radioactivity in tissues and in small intestine perfusate and cecum-colon contents was used to measure Zn bioavailability. A biofortified rice variety contained substantially more Zn than conventional varieties, with no change in phytate content. Absorbed Zn (μg/g rice) was significantly higher from the new variety in both the in vitro Caco-2 cell model (2.1-fold) and the rat pup model (2.0-fold). Results from the two models were highly correlated, particularly for the polished samples. Biofortification of rice with Zn results in significantly increased Zn uptake in both models. Since results from the Caco-2 cell model correlated well with those from rat pups, this cell model is likely to predict results in human populations and can be used for screening purposes.     

Isolated glycosaminoglycans from cooked haddock enhance nonheme iron uptake by Caco-2 cells

This study continues previous research to confirm that glycosaminoglycans (GAGs) exert a positive effect on promoting iron uptake by Caco-2 cells. Cooked haddock was digested with papain, and GAGs were further purified on the basis of their sulfur content. Reverse phase chromatography (RP-HPLC) and digestion with chondroitinase ABC (Chase) (50 mU/mg) were used to approach the identification of the GAGs. FeCl 3 was mixed with the purified GAGs, and Fe uptake was measured by ferritin formation using an in vitro digestion/Caco-2 cell model. The identificative analyses suggest that chondroitin/dermatan sulfate-related structures promote Fe uptake by Caco-2 cells; however, this effect was lower (40%) than that observed with whole fish muscle. Chase eliminated the positive effect on Fe uptake. These results indicate that specific GAGs may contribute to the enhancing effect of meat on Fe absorption. Further in vivo studies addressing these aspects of the meat factor are needed.     

Effect of natural and controlled fermentation on chemical composition and nutrient dialyzability from beans (Phaseolus vulgaris L.)

The effect of natural and controlled fermentation with an inoculum of Lactobacillus plantarum and additional thermal treatment (dry heat at 120 degrees C for 20 min) on the availability of N, P, Fe, Cu, Zn, Ca, and Mg from Phaseolus vulgaris L. var. carrilla was estimated using an in vitro method based on equilibrium dialysis. Natural and controlled fermentations caused significant reductions in the pH and phytate content (36%) of the bean flours, with a concomitant increase in the titratable acidity and free phosphorus content, and had no effect on the other nutrients studied. The percentage of dialyzable N, P, Cu, and Mg was significantly improved by both types of fermentation, whereas Zn dialyzability was significantly reduced. The greatest reduction was observed for the bean flour fermented with an inoculum of L. plantarum. The percentage of dialyzable Fe improved significantly as a result of natural fermentation but was not affected by controlled fermentation. The application of dry heat at 120 degrees C for 20 min caused a significant increase in Fe dialyzability and a further reduction in the percentage of dialyzable Zn in fermented bean flours but did not affect the dialyzability of the other nutrients studied.     

Meat and ascorbic acid can promote Fe availability from Fe-phytate but not from Fe-tannic acid complexes

This study utilized an in vitro digestion/Caco-2 cell model to determine the levels of ascorbic acid (AA) and "meat factor" needed to promote Fe absorption from Fe complexed with phytic acid (PA) or tannic acid (TA). AA reversed the inhibition of Fe absorption by PA beginning at a molar ratio of 1:20:1 (Fe:PA:AA) but essentially had no effect on the Fe complexed with TA. Fish also reversed the inhibition of Fe uptake by PA but not by TA. TA and fish decreased total Fe solubility. Iron in the presence of PA was highly soluble. AA, but not fish, increased the percentage of soluble Fe as Fe2+ in the presence of both inhibitors. The results indicate that monoferric phytate is a form of Fe that can be available for absorption in the presence of uptake promoters. In contrast, a TA-Fe complex is much less soluble and unavailable in the presence of promoters.     

Effect of dietary ligands and food matrices on zinc uptake in Caco-2 cells: implications in assessing zinc bioavailability

The kinetics, depletion/repletion of zinc, and effects of dietary ligands/food matrices on (65)Zn uptake was studied in Caco-2 cells. The uptake of zinc showed a saturable and nonsaturable component, depending upon the media zinc concentrations. Intracellular depletion increased zinc uptake, whereas zinc loading did not. Phytic acid and histidine inhibited zinc uptake, while tannic acid, tartaric acid, arginine, and methionine increased zinc uptake. Tannic acid at a 1:50 molar ratio promoted zinc uptake from wheat- and rice-based food matrices. Further, Caco-2 cells responded similarly with zinc and iron uptake when fed Indian bread prepared from low- and high-extraction wheat flour, representing low and high phytate content. However, inclusion of tea extract or red grape juice as a source of polyphenols enhanced the uptake of zinc while decreasing that of iron. These results suggest that the Caco-2 cells predict the correct direction of response to dietary ligands even from complex foods.     

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.     

Nutritional potential of raw and free alpha-galactosides lupin (Lupinus albus Var. multolupa) seed flours. Effect of phytase treatment on nitrogen and mineral dialyzability

The effect of the removal of alpha-galactosides from Lupinus albus L. var. multolupa on the chemical composition of the prepared flour and the dialyzability of N, total P, Ca, Mg, Fe, Zn, and Mn was studied. The extraction process caused a significant increase in total and insoluble nitrogen contents and decreased the amount of soluble protein nitrogen. However, neither these changes nor treatment with phytase seemed to considerably affect in vitro protein digestibility. Except for Ca and Cu, total mineral contents were significantly reduced by the extraction process. The process also caused a significant reduction in the dialyzability of all the minerals studied except P. The decrease in mineral dialyzability was partially counteracted by phytase treatment in amounts of 250-500 phytase units/kg of lupin flour. In the case of Fe, mineral dialyzability did not differ significantly between the two lupin flours studied with treatment with 500 phytase units/kg. Zinc dialyzability was the most efficiently improved by phytase treatment (P < 0.0001), followed by P, Fe, and Mn, and finally by Ca and Mg (P < 0.05).     

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.     

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.     

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.     

Phytate Quantification using HPIC in the Presence of Iron and Aluminum

Phytate is an organic form of P that is difficult to analyze in complex matrices. To test if high concentrations of aluminum (Al) and iron (Fe) hinder accurate quantification of phytate in dairy manure and broiler litter when measured by high-performance ion chromatography (HPIC), researchers spiked dairy manure and broiler litter samples with Al, Fe, and phytate. Samples were alkaline extracted, acidified, and filtered, and then phytate spike recovery was analyzed with HPIC. High concentrations of Fe did not hinder phytate recovery in manure or litter samples. While phytate recovery was complete at typical manure and litter Al concentrations, high concentrations of Al inhibited phytate recovery in litter samples and in some manure samples. Overall, alkaline extraction of dairy manure and broiler litter and analysis with HPIC proved to be relatively accurate, fast, and cheap within normal Al and Fe ranges, compared to the commonly used nuclear magnetic resonance (NMR) method.     

Effect of bioactive dietary polyphenols on zinc transport across the intestinal Caco-2 cell monolayers

Polyphenolic compounds are known to possess many beneficial health effects, including the antioxidative activities of scavenging reactive oxygen species and chelating metals, such as iron and zinc. Tea and red wine are thought to be important sources of these compounds. However, some polyphenolic compounds can also reduce the absorption of iron, and possibly other trace metals, when included in a diet. There is very little information on the effect of dietary polyphenolic compounds on the status of trace elements other than iron. The effects of epigallocatechin-3-gallate (EGCG), green tea extract (GT), and grape seed extract (GSE) on the absorption of (65)Zn were examined and compared with their effects on (55)Fe absorption in human intestinal Caco-2 cells grown on microporous membrane inserts. The levels of EGCG, GT, and GSE used in this study were within physiological ranges and did not affect the integrity of the Caco-2 cell monolayers. GSE significantly (P < 0.05) reduced zinc transport across the cell monolayer, and the decreased zinc transport was associated with a reduction in apical zinc uptake. However, EGCG and GT did not alter zinc absorption. In contrast, the polyphenolic compounds in EGCG, GT, and GSE almost completely blocked transepithelial iron transport across the cell monolayer. The effect of GSE on zinc absorption was very different from that on iron absorption. Whereas GSE decreased zinc absorption by reducing apical zinc uptake, the polyphenolic compounds inhibited iron absorption by enhancing apical iron uptake. GSE inhibited zinc absorption similarly to that observed for phytate. Phytate significantly (P < 0.05) decreased transepithelial zinc transport by reducing apical zinc uptake. The inhibition of zinc absorption may be due to the presence of procyanidins in GSE, which bind zinc with high affinity and block the transport of zinc across the apical membrane of enterocytes. Further research on the absorption of zinc as zinc-polyphenol complexes and free zinc should provide further insight into the process of dietary zinc absorption in the presence of GSE and other bioactive dietary polyphenols. The present study suggests that some individuals should consider their zinc status if they regularly consume procyanidin-containing foods in their diet. However, further studies, especially in vivo studies, are needed to confirm these results.     

Mineral dialyzability in milk and fermented dairy products fortified with FeNaEDTA

Iron, zinc, and calcium dialyzability and ascorbic acid (AA) concentrations were evaluated in milk and yogurt fortified with FeNaEDTA (FE) or ferrous sulfate (FS) as a control, with or without AA addition. The values obtained for FE iron dialyzability in milk were much higher than those obtained for FS. The addition of AA to milk improved Fe dialyzability when using FS and slightly decreased Fe dialyzability in the FE-fortified nonfermented samples. Milk fermentation increased iron availability from both iron sources. Zinc and calcium dialyzability in products containing any of the two iron sources was increased in fermented milks. EDTA improved Zn dialyzability from intrinsic zinc in every manufactured dairy product. Whereas for milks fortified with FS and stored at 4 degrees C for 24 h, the AA content remained close to the original concentration, a higher AA degradation was observed when milks were fortified with FE.     

Ion chromatography of phytate in roots and tubers

The ion chromatographic method for the quantification of phytate (InsP(6)) in foods was adapted for the analysis of roots and tubers. To maximize sensitivity, ultraviolet (UV) detection following postcolumn derivatization was compared with evaporative light-scattering detection (ELSD). Detection limits for phytate were 0.5 and 1 microg for UV and ELSD, respectively. Unidentified peaks eluting close to and after InsP(6) were removed by solid-phase extraction. Phytate was detected in 11 of 15 roots or tubers. The highest phytate levels were 0.169 and 0.133% of the fresh weight of taro (Colocasia esculenta) and yuca (Manihot esculenta), respectively. Potatoes (Solanum tuberosum) contained 0.035-0.073% phytate, whereas no phytate at a detection limit of 0.003% of fresh weight was observed in sweet potatoes (Ipomoea batatas).