The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is ...

Robert Holley Center for Agriculture and Health

Despite widespread consumption of soil among animals, the role of geophagy in health maintenance remains an enigma. It has been hypothesized that animals consume soil for supplementation of minerals and protection against toxins. Most studies determine only the total elemental composition of soil, which may not reflect the amount of minerals available to the consumer. Our aim was to test these hypotheses by evaluating the bioavailability of iron in soil consumed by chacma baboons, using a technique that simulates digestion and adsorption. Our results indicate that, despite variation in absolute iron concentration of soil samples, actual iron bioavailability was low while clay content was quite high. This suggests that iron supplementation is unlikely to be the primary motivation for geophagy in this population, and that detoxification is a plausible explanation. This study demonstrates that more research on bioavailability and clay composition is needed to determine the role geophagy plays in health maintenance.

Soil consumed by chacma baboons is low in bioavailable iron and high in clay.

Extrinsic isotopic labeling of food Fe has been used for over 50 years to measure Fe absorption. This method assumes that complete equilibration occurs between the extrinsic and the intrinsic Fe prior to intestinal absorption. The present study tested this assumption via in vitro digestion of varieties of maize, white beans, black beans, red beans, and lentils. Prior to digestion, foods were extrinsically labeled with (58)Fe at concentrations of 1, 10, 50, and 100% of the intrinsic (56)Fe. Following an established in vitro digestion protocol, the digest was centrifuged and the Fe solubilities of the extrinsic (58)Fe and the intrinsic (56)Fe were compared as a measure of extrinsic/intrinsic equilibration. In the beans, significantly more of the extrinsic Fe (up to 2-3 times, p < 0.001) partitioned into the supernatant. The effect varied depending upon the seed coat color, the harvest, and the concentration of the extrinsic Fe. For lentils and maize the extrinsic Fe tended to partition into the insoluble fraction and also varied depending on variety and harvest. There was no crop that consistently demonstrated full equilibration of the extrinsic Fe with the intrinsic Fe. These observations challenge the accuracy of Fe absorption studies in which isotopic extrinsic Fe was used to evaluate Fe absorption and bioavailability. 

Extrinsic Labeling of Staple Food Crops with Isotopic Iron Does Not Consistently Result in Full Equilibration: Revisiting the Methodology.

Strategies that enhance the Fe bioavailability of the bean are of keen interest to nutritionists, bean breeders and growers. In beans, the cotyledons contain 75-80% of the total seed Fe, most of which appears to be located within the cotyledon cells. The cotyledon cell walls are known to be resistant to digestion in the stomach and the upper small intestine. Therefore, given the above and the general belief that the primary site for human Fe absorption is the upper small intestine, the present study was designed to determine if the cotyledon cell walls represent a barrier to Fe absorption from the bean. To do so, we utilized high pressure to rupture bean cotyledon cells. The iron bioavailability of cooked bean samples was assessed using an in vitro digestion/Caco-2 cell culture model. Microscopy analyses confirmed that the cotyledon cell walls are highly resistant to pepsin, the low pH of the stomach, and the pancreatic enzymes, indicating that the walls are a barrier to Fe absorption from the bean. Relatively high intracellular pressure (>4000 psi) was required to initiate cell wall rupture. Surprisingly, the lysis of cotyledon cells did not result in a consistent or strong enhancement of bioavailable Fe, suggesting that the liberated intracellular starch and protein influenced the Fe bioavailability by creating a matrix that inhibited the exchange of Fe with the cell transport mechanism. Such observations warrant further pursuit in vivo as the confirmation of these effects would reshape strategies to enhance Fe absorption from beans. 

The cotyledon cell wall and intracellular matrix are factors that limit iron bioavailability of the common bean (Phaseolus vulgaris).

The predominant bean iron (Fe) biofortification approach is to breed for high Fe concentration and assumes the average Fe concentration is 50 μg/g. This approach also assumes that a 40 μg/g increase is sustainable and Fe bioavailability will not decrease to negate the increase in Fe.

Iron Concentrations in Biofortified Beans and Nonbiofortified Marketplace Varieties in East Africa Are Similar.

A new iron-casein complex (ICC) has been developed for iron (Fe) fortification of dairy matrices. The objective was to assess the impact of ascorbic acid (AA) on its in vitro bioavailability in comparison with ferrous sulfate (FeSO) and ferric pyrophosphate (FePP). A simulated digestion coupled with the Caco-2 cell culture model was used in parallel with solubility and dissociation tests. Under diluted acidic conditions, the ICC was as soluble as FeSO, but only part of the iron was found to dissociate from the caseins, indicating that the ICC was an iron chelate. The Caco-2 cell results in milk showed that the addition of AA (2:1 molar ratio) enhanced iron uptake from the ICCs and FeSO to a similar level ( = 0.582;  = 0.852) and to a significantly higher level than that from FePP ( < 0.01). This translated into a relative in vitro bioavailability to FeSO of 36% for FePP and 114 and 104% for the two ICCs. Similar results were obtained from water. Increasing the AA to iron molar ratio (4:1 molar ratio) had no additional effect on the ICCs and FePP. However, ICC absorption remained similar to that from FeSO ( = 0.666;  = 0.113), and was still significantly higher than that from FePP ( < 0.003). Therefore, even though iron from ICC does not fully dissociate under gastric digestion, iron uptake suggested that ICCs are absorbed to a similar amount as FeSO in the presence of AA and thus provide an excellent source of iron.

Impact of Ascorbic Acid on the In Vitro Iron Bioavailability of a Casein-Based Iron Fortificant.

Inadequate nutritional status contributes to substantial losses in human health and productivity globally. A multiple biofortified food crop trial targeting iron, zinc, and vitamin A deficiencies among young children and their breastfeeding mothers is being conducted in India.

Iron Bioavailability from Multiple Biofortified Foods Using an In Vitro Digestion, Caco-2 Assay for Optimizing a Cyclical Menu for a Randomized Efficacy Trial.

Raymond P. Glahn

United States Department of Agriculture,
Agricultural Research Service,
United States Department of Agriculture, Agricultural Research Service

Robert Holley Center for Agriculture and Health

The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability

Raymond P. Glahn

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Robert Holley Center for Agriculture and Health

The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability

United States Department of Agriculture,
Agricultural Research Service,
United States Department of Agriculture, Agricultural Research Service