Caco-2 Cell Ferritin Formation Research Articles

In Vitro Iron Bioavailability of Brazilian Food-Based by-Products.

Background: Iron deficiency is a public health problem in many low- and middle-income countries. Introduction of agro-industrial food by-products, as additional source of nutrients, could help alleviate this micronutrient deficiency, provide alternative sources of nutrients and calories in developed countries, and be a partial solution for disposal of agro-industry by-products. Methods: The aim of this study was to determine iron bioavailability of 5 by-products from Brazilian agro-industry (peels from cucumber, pumpkin, and jackfruit, cupuaçu seed peel, and rice bran), using the in vitro digestion/ Caco-2 cell model; with Caco-2 cell ferritin formation as a surrogate marker of iron bioavailability. Total and dialyzable Fe, macronutrients, the concentrations of iron-uptake inhibitors (phytic acid, tannins, fiber) and their correlation with iron bioavailability were also evaluated. Results: The iron content of all by-products was high, but the concentration of iron and predicted bioavailability were not related. Rice bran and cupuaçu seed peel had the highest amount of phytic acid and tannins, and lowest iron bioavailability. Cucumber peels alone, and with added extrinsic Fe, and pumpkin peels with extrinsic added iron, had the highest iron bioavailability. Conclusion: The results suggest that cucumber and pumpkin peel could be valuable alternative sources of bioavailable Fe to reduce iron deficiency in at-risk populations.

The Manteca Yellow Bean: A Genetic Resource of Fast Cooking and High Iron Bioavailability Phenotypes for the Next Generation of Andean Dry Beans (Phaseolus vulgaris L.)

Dry beans (Phaseolus vulgaris L.) are a nutrient dense food produced globally as a major pulse crop for direct human consumption. Despite being rich in protein and micronutrients, long cooking times limit the use of dry beans worldwide, especially in regions relying on wood and charcoal as the primary sources of fuel for cooking, such as Sub‐Sahara Africa and the Caribbean. These same regions also have populations at risk for iron deficiency. There is need for a fast cooking bean, which can positively impact consumers by reducing fuel cost and preparation time. To help accelerate a reliable increase in dry bean production for Sub‐Saharan Africa, the Andean Bean Diversity Panel (ADP) was assembled as a genetic resource in the development of fast cooking, nutritional improved, biotic/abiotic resistant varieties (http://arsftfbean.uprm.edu/bean/). Six genotypes were identified as fast cooking (<25 min @ 100°C) from a germplasm screening for atmospheric cooking time of over 200 bean genotypes from the ADP. Three of these genotypes had a pale ‘Manteca’ yellow seed color (Entry name: Ervilha; Cebo Cela; Mantega, Kibala). Yellow beans of various shades are important in Eastern and Southern Africa. Their popularity has increased in recent years and they often fetch the highest prices at the marketplace. There is evidence to suggest that Manteca yellow beans have a unique nutritional profile when compared to other yellow seed types; with more soluble dietary fiber, less indigestible protein and starch and are free of condensed tannins. The hypothesis was tested that this unique composition would also have a positive influence on the bioavailability of iron in an in vitro digestion/Caco‐2 cell culture bioassay. A subset of 16 yellow bean genotypes entries selected from ADP that varied in color, appearance and cooking time (named the Yellow Bean Collection; YBC) were planted in a Randomized‐Complete‐Block Design with 2 field replicates at the Michigan State University, Montcalm Research Farm near Entrican, MI for 2 field seasons and evaluated 3 months after harvest for iron concentration and bioavailability (Figure 1). Significant variation (P < 0.0001) in iron bioavailability was measured in cooked/lyophilized/milled seed of the YBC, when expressed as the percentage of Caco‐2 cell ferritin formation (ng ferritin/mg total cell protein) relative to a high bioavailable Fe white navy bean (cv. Merlin) control with each assay (Table 1). Significantly greater Fe uptake was observed from the fast cooking Manteca yellow genotypes (17–23 min; 90 – 110% of control) when compared to slower cooking (38 – 48 min) Njano and Canary yellow genotypes (32 – 42% of control). These data suggest that genotypes within the Manteca yellow market class have enhanced nutritional quality (ie. more absorbable Fe) and fast cooking properties. Such traits can be promoted to increase bean consumption and improve Fe status of those who rely on beans as a major component of their diet.Support or Funding InformationThis research funded by USDA‐ARS. Iron concentrations, iron bioavailability, phytate to iron molar ratios and cooking times among yellow genotypes of the Andean Diversity Panel. Entry/Seed TypeParameter1 ErvilhaManteca Uyole 98Yellow Brown CanarioCanary PI527538Njano Iron (ppm) 74a 70b 79a 79a Iron bioavailability (%)2 106a 62b 47b 32c Phytate:iron ratio3 16a 15a 12b 14ab Cooking time (min) 22d 35c 38b 48a Values are presented as means of two field replicates per entry measured over 2 field seasons at Montcalm Research Farm, MI. Means sharing the same letter in each row are not significantly different at P ≤ 0.05. To compare between field seasons in vitro iron bioavailability is expressed as the percentage of Caco‐2 cell ferritin formation (ng ferritin/mg total cell protein) relative to a navy bean control with each assay. The molar ratio between concentrations of phytate and iron in lyophilized cooked whole seed (dry weight).

In vitro assessment of iron availability from commercial Young Child Formulae supplemented with prebiotics

PurposeIron is essential for development and growth in young children; unfortunately, iron deficiency (ID) is a significant public health problem in this population. Young Child Formulae (YCF), milk-derived products fortified with iron and ascorbic acid (AA, an enhancer of iron absorption) may be good sources of iron to help prevent ID. Furthermore, some YCF are supplemented with prebiotics, non-digestible carbohydrates suggested to enhance iron bioavailability. The aim of our study was to evaluate iron bioavailability of YCF relative to prebiotic and AA concentrations. We hypothesised that YCF with the highest levels of prebiotics and AA would have the most bioavailable iron.MethodsWe used the in vitro digestion/Caco-2 cell model to measure iron bioavailability from 4 commercially available YCF with approximately equal amounts of iron, but varying amounts of: AA and the prebiotics fructo- and galacto-oligosaccharides. Caco-2 cell ferritin formation was used as a surrogate marker for iron bioavailability.ResultsThe YCF with the highest concentration of prebiotics and AA had the highest iron bioavailability; conversely, the YCF with the lowest concentration of prebiotics and AA had the lowest. After the addition of exogenous prebiotics, so that all tested YCF had equivalent amounts, there was no longer a significant difference between YCF iron bioavailability.ConclusionOur results suggest that ascorbic acid and prebiotics in YCF improve iron bioavailability. Ensuring that iron is delivered in a bioavailable form would improve the nutritional benefits of YCF in relation to ID/IDA amongst young children; therefore, further exploration of our findings in vivo is warranted.

Ascorbyl palmitate/DSPE-PEG nanocarriers for oral iron delivery: Preparation, characterisation and in vitro evaluation

The objective of this study was to encapsulate iron in nanocarriers formulated with ascorbyl palmitate and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine polyethylene glycol (DSPE-PEG) for oral delivery. Blank and iron (Fe) loaded nanocarriers were prepared by a modified thin film method using ascorbyl palmitate and DSPE-PEG. Surface charge of the nanocarriers was modified by the inclusion of chitosan (CHI) during the formulation process. Blank and iron loaded ascorbyl palmitate/DSPE nanocarriers were visualised by transmission electron microscopy (TEM) and physiochemical characterisations of the nanocarriers carried out to determine the mean particle size and zeta potential. Inclusion of chitosan imparted a net positive charge on the nanocarrier surface and also led to an increase in mean particle size. Iron entrapment in ascorbyl palmitate-Fe and ascorbyl palmitate-CHI-Fe nanocarriers was 67% and 76% respectively, suggesting a beneficial effect of chitosan on nanocarrier Fe entrapment. Iron absorption was estimated by measuring Caco-2 cell ferritin formation using ferrous sulphate as a reference standard. Iron absorption from ascorbyl palmitate-Fe (592.17±21.12ng/mg cell protein) and ascorbyl palmitate-CHI-Fe (800.12±47.6ng/mg, cell protein) nanocarriers was 1.35-fold and 1.5-fold higher than that from free ferrous sulphate, respectively (505.74±23.73ng/mg cell protein) (n=6, p<0.05). This study demonstrates for the first time preparation and characterisation of iron loaded ascorbyl palmitate/DSPE PEG nanocarriers, and that engineering of the nanocarriers with chitosan leads to a significant augmentation of iron absorption.

Bioavailability of Iron from Wheat Aegilops Derivatives Selected for High Grain Iron and Protein Contents

A coupled in vitro digestion/Caco-2 model was employed to assess iron bioavailability from wheat Aegilops derivatives selected for high iron and protein contents. The iron content in wheat genotypes used in this study correlated to a great extent with both protein (r = 0.80) and phytate (r = 0.68) contents. The iron bioavailability was based on Caco-2 cell ferritin formation from cooked digests of these derivatives (relative to WL711 control) and correlated positively with dialyzable iron (r = 0.63) and total iron content (r = 0.38) but not with the phytate content. The apparently decreased phytate/iron molar ratios, however, correlated negatively (r = -0.42) with the iron bioavailability, justifying the utilization of these parameters in biofortification programs. Iron bioavailability in the derivatives increased up to 1.5-fold, corresponding to a 1.5-2.2-fold increase observed in iron content over control. These data suggest that biofortification for iron proportionately leading to higher iron bioavailability will be the most feasible and cost-effective approach to combat micronutrient deficiency.

Micronutrient sprinkles add more bioavailable iron to some Kenyan complementary foods: studies using an in vitro digestion/Caco-2 cell culture model.

Iron deficiency anaemia is arguably the most important public health problem in developing countries. In Sub-Saharan Africa, iron deficiency anaemia has largely been attributed to poor infant feeding practices where complementary foods low in iron bioavailability are offered to at-risk infants. Home fortification of complementary foods using micronutrient Sprinkles has been shown to reduce iron deficiency anaemia in many resource-poor settings. In this study, the benefit of using the micronutrient Sprinkles as a home fortificant for some Kenyan complementary foods was assessed using an in vitro Caco-2 cell model. In each case when micronutrient Sprinkles were added to the complementary food, the amount of Caco-2 cell ferritin formation increased. For example, the addition of Sprinkles to corn porridge increased ferritin formation 5-fold from 5.8 to 31.8 ng mg(-1). Therefore, as indicated by the results, micronutrient Sprinkles would be a suitable form of home fortification for Kenyan complementary foods. Their use should be encouraged so as to improve infant feeding practices in Kenya.

Bioaccessibility of Phenols in Common Beans (Phaseolus vulgaris L.) and Iron (Fe) Availability to Caco-2 Cells

Samples of common and biofortified beans ( Phaseolus vulgaris ), both raw and cooked (autoclaved at 120 degrees C for 20 min) were analyzed for their polyphenol composition. Polyphenols were identified via HPLC-UV/diode array detection. Cooking favored the extraction of polyphenols without the need of a hydrolysis step, a fact that is of interest because this is the usual form in which beans are consumed. The main differences between white and colored beans were the presence of free kaempferol (13.5-29.9 microg g(-1)) and derivatives (kaempferol-3-O-glucoside) (12.5-167.5 microg g(-1)), only in red and black beans. An in vitro digestion (pepsin, pH2; pancreatin-bile extract, pH 7) was applied to beans to estimate bioaccessibility of individual polyphenols. Kaempferol from seed coats exhibited high bioaccessibility (45.4-62.1%) and a potent inhibitor effect on Fe uptake at concentrations ranging from 0.37 to 1.30 microM. Caco-2 cell ferritin formation was used to evaluate Fe uptake. Cell Fe uptake was significant only from white beans.

Gastric digestion of pea ferritin and modulation of its iron bioavailability by ascorbic and phytic acids in caco-2 cells.

To understand the digestive stability and mechanism of release and intestinal uptake of pea ferritin iron in caco-2 cell line model. Pea seed ferritin was purified using salt fractionation followed by gel filtration chromatography. The bioavailability of ferritin iron was assessed using coupled in vitro digestion/Caco-2 cell model in the presence or absence of ascorbic acid and phytic acid. Caco-2 cell ferritin formation was used as a surrogate marker of iron uptake. Structural changes of pea ferritin under simulated gastric pH were characterized using electrophoresis, gel filtration and circular dichroism spectroscopy. The caco-2 cell ferritin formation was significantly increased (P<0.001) with FeSO(4) (19.3 +/- 9.8 ng/mg protein) and pea ferritin (13.9 +/- 6.19 ng/mg protein) compared to the blank digest (3.7 +/- 1.8 ng/mg protein). Ascorbic acid enhanced while phytic acid decreased the pea ferritin iron bioavailability. However, either in the presence or absence of ascorbic acid, the ferritin content of caco-2 cells was significantly less with pea ferritin than with FeSO(4). At gastric pH, no band corresponding to ferritin was observed in the presence of pepsin either on native PAGE or SDS-PAGE. Gel filtration chromatography and circular dichroism spectroscopy revealed a pH dependent loss of quaternary and secondary structure. Under gastric conditions, the iron core of pea ferritin is released into the digestive medium due to acid induced structural alterations and dissociation of protein. The released iron interacts with dietary factors leading to modulation of pea ferritin iron bioavailability, resembling the typical characteristics of non-heme iron.

A comparison of iron availability from commercial iron preparations using an in vitro digestion/caco-2 cell culture model

The objectives of this study were to compare iron availability from commercial preparations of FeSO 4, ferrous gluconate, ferrous fumarate, and a polysaccharide-iron complex using an in vitro digestion/Caco-2 cell culture model. In addition, we sought to determine if calcium carbonate and calcium acetate (common phosphate binding agents) inhibited iron availability from an oral iron supplement when digested simultaneously. Caco-2 cell ferritin formation following exposure to simulated gastric and intestinal digests of the iron supplements was used as a measure of iron uptake and availability. Plates without cell monolayers were included in each replication of the experiment to measure the total amount of soluble iron that resulted from the in vitro digestion. Significantly more iron was taken up from the FeSO 4, ferrous gluconate, and ferrous fumarate than the polysaccharide-iron complex. Similar results comparing FeSO 4 and the polysaccharide-iron complex have been observed in humans. In addition, less iron was taken up from digests with calcium carbonate relative to calcium acetate even though similar amounts of soluble iron were observed in these experiments. The results indicate that when iron supplements and phosphate binders are consumed simultaneously, calcium acetate may be the preferred phosphate binder to maximize iron availability.

Caco-2 Cell Ferritin Formation Predicts Nonradiolabeled Food Iron Availability in an In Vitro Digestion/Caco-2 Cell Culture Model

We have adapted an in vitro digestion/Caco-2 cell model to assess Fe availability from foods, by using ferritin formation by Caco-2 cells as an indicator of Fe uptake. Ferritin formation by Caco-2 cells occurs in response to Fe uptake at concentrations of available Fe greater than that of the culture media to which the cells have been adapted. This methodology circumvents the need for using radioactive Fe and thus eliminates the costs and controversies associated with food radiolabeling. To validate this method, we measured ferritin formation in Caco-2 cells exposed to digests containing Fe of relatively high and low availability. Our objective was to determine if ferritin formation would be proportional to Fe uptake and sufficiently sensitive to be an indicator of Fe availability from food digests. Our model uses established in vitro digestion techniques coupled with uptake of Fe by Caco-2 cell monolayers. Measurement of cell ferritin was done by a commercially available RIA. Higher ferritin formation was observed in cells exposed to digests containing FeSO4 plus ascorbic acid vs, digests containing FeSO4 plus citric acid. Additional comparisons of Fe availability from digests of beef, fish, corn and green beans yielded results that demonstrate higher Fe availability (i.e., greater ferritin formation) from beef and fish digests than from digests of corn and green beans. Overall, the results document the promotional effects of ascorbic acid and animal tissue on Fe uptake as measured indirectly by ferritin formation. The results of this study indicate that ferritin formation by Caco-2 cell monolayers is highly sensitive and accurately measures food Fe availability in this in vitro system.