The usefulness of in vitro models to predict the bioavailability of iron and zinc: a consensus statement from the HarvestPlus expert consultation

A combination of dietary and host-related factors determines iron and zinc absorption, and several in vitro methods have been developed as preliminary screening tools for assessing bioavailability. An expert committee has reviewed evidence for their usefulness and reached a consensus. Dialyzability (with and without simulated digestion) gives some useful information but cannot predict the correct magnitude of response and may sometimes predict the wrong direction of response. Caco-2 cell systems (with and without simulated digestion) have been developed for iron availability, but the magnitude of different effects does not always agree with results obtained in human volunteers, and the data for zinc are too limited to draw conclusions about the validity of the method. Caco-2 methodologies vary significantly between laboratories and require experienced technicians and good quality cell culture facilities to obtain reproducible results. Algorithms can provide semi-quantitative information enabling diets to be classified as high, moderate, or low bioavailability. While in vitro methods can be used to generate ideas and develop hypotheses, they cannot be used alone for important decisions concerning food fortification policy, selection of varieties for plant breeding programs, or for new product development in the food industry. Ultimately human studies are required for such determinations.     

The use of caco-2 cells to estimate fe absorption in humans--a critical appraisal

The Caco-2 cell model is widely used to assess the bioaccessibility/availability of iron from foods and diets. Analysis of iron uptake in this human epithelial cell line is usually preceded by a two-step digestion to simulate the conditions in the stomach and small intestine. Moreover, culturing the cells on inserts permits the measurement of iron transport. The cellular iron uptake is determined by direct measurements using radioisotopes, or indirectly by measurement of ferritin, the intracellular storage form of iron. There is a good correlation between Caco-2 cell uptake and human iron bioavailability for a number of dietary factors known to affect iron absorption. However, recent data suggest that in some cases there is no correlation. Possible reasons for such discrepancies, the benefits, and limitations of the Caco-2 cell model are discussed. In conclusion, in vitro experiments with Caco-2 cells are important tools for ranking foods with respect to bioavailability, for mechanistic studies of iron absorption, and for studies of dietary factors influencing absorption. The results need to be confirmed in humans.     

The use of Caco-2 cells as an in vitro method to study bioavailability of iron

Iron absorption is essential for the maintenance of iron levels in the body, since excretion is poorly regulated. Dietary factors can influence iron absorption including low molecular weight substances such as ascorbic acid which has been shown to enhance iron transport across mucosal cell monolayers. Both in vivo and in vitro work may be carried out to study iron absorption. Studies in vivo have the drawback of dealing with a complex system in which it is difficult to determine the relative importance of different factors. In vitro cell culture models could overcome this difficulty but attempts to establish differentiated enterocyte cell lines in culture have not been successful. However the Caco-2 line, derived from a colon carcinoma, is able to differentiate spontaneously when grown in standard culture conditions. The differentiated cells polarized, formed microvilli and T-junctions associated with the duodenal enterocytes brush border. This cell line thus represents an appropriate model for the study of transport mechanisms related to the intestinal barrier and can be used to study the absorption of nutrients especially iron in relation to dietary intake in particular pertaining to dietary factors that may affect absorption. In this work we have therefore used differentiated Caco-2 cells grown in bicameral chambers as a intestinal cell model to study the absorption of iron from different sources and compared it with INT 407 cells. Transfer of iron across the monolayers in the apical-to-basolateral direction has been found to be greater from feric lactoferrin than from iron citrate, while very little transport occurred from Fe-transferrin. It is concluded that in this in vitro study lactoferrin but not transferrin enhances mucosal iron transport. More importanty this study has also shown that Caco-2 can be used as an in vitro method to investigate not only iron bioavailability but can be applied to other minerals as well.     

Adaptation in human zinc absorption as influenced by dietary zinc and bioavailability

BACKGROUND: An understanding of the relations among dietary zinc intake, bioavailability, and absorption is necessary for making dietary intake recommendations. OBJECTIVES: We aimed to assess adaptation in human zinc absorption to controlled differences in zinc and phytate intakes and to apply the results to predictive models. DESIGN: In 3 experiments, radiotracers were used to assess zinc absorption by healthy adults (n = 109) from controlled diets, before and after 4 or 8 wk of dietary equilibration. Subjects consumed 4-29 mg Zn/d from 1 of 10 diets, 5 with molar ratios of phytate to zinc from 2 to 7 and 5 with ratios from 15 to 23. RESULTS: Absorptive efficiency was inversely related to dietary zinc from both low- and high-phytate diets. In response to low zinc intakes (<11 mg/d) for 4-8 wk, zinc absorption was up-regulated to as high as 92%, but only if the diets were low in phytate. The results help validate and refine a published saturable transport model that predicts zinc absorption from dietary zinc and phytate. Possible biomarkers of impaired zinc status, including erythrocyte osmotic fragility, in vitro erythrocyte (65)Zn uptake, and leukocyte expression of the zinc transport proteins Zip1 and ZnT1, were unresponsive to dietary zinc content. CONCLUSIONS: Humans absorbed zinc more efficiently from low-zinc diets and adapted to further increase zinc absorption after consuming low-zinc, low-phytate diets for several weeks. Such adaptation did not occur with higher phytate diets. Zinc absorption can be predicted from dietary zinc and phytate after allowing for dietary equilibration.     

Phytate:zinc and phytate X calcium:zinc millimolar ratios in self-selected diets of Americans, Asian Indians, and Nepalese

The literature indicates that phytate:zinc and phytate X calcium:zinc millimolar ratios of a diet may provide useful indexes of the bioavailability of dietary zinc. However, there is little information on the phytate:zinc and phytate X calcium:zinc millimolar ratios for total human diets. We have therefore determined the phytate:zinc and phytate X calcium:zinc millimolar ratios of self-selected diets of 29 American omnivores, 23 American vegetarians, 30 Asian Indian immigrant vegetarians, and 26 Nepalese lactating vegetarians. Criteria for selection of subjects were: good health, no extreme dietary habits, and no intake of nutrient supplements. According to the limited literature, the suggested critical values for phytate:zinc and phytate X calcium:zinc millimolar ratios in animal diets and retrospective calculations from human diets are greater than 10 and greater than 200, respectively. The mean phytate:zinc and phytate X calcium:zinc millimolar ratios of American omnivorous diets in the present study were less than 10 and 200, respectively. Confirmation of the critical molar ratios as indexes of the bioavailability of zinc in human diets has not been established by experimentation. However, if the data from animal studies are applicable to human diets, the present study suggests that phytate has little influence on zinc bioavailability of most American diets. In contrast, the mean phytate:zinc and phytate X calcium:zinc millimolar ratios of all vegetarian diets were above the proposed critical levels. Those data, therefore, suggest that phytate might increase the risk of impaired zinc bioavailability for vegetarians consuming a relatively high level of calcium.     

The precision of in vitro methods and algorithms for predicting the bioavailability of dietary iron

Three factors determine how much iron will be absorbed from a meal. They are the physiological mechanisms that regulate uptake by and transfer through the enterocytes in the upper small intestine, the quantity of iron in the meal, and its availability to the cellular iron transporters. Established methods exist for predicting the effect of physiological regulation and for measuring or estimating meal iron content. Three approaches to estimating bioavailability have been advocated. Two are in vitro screening procedures: measurement of dialyzable iron and Caco-2 cell uptake, both carried out after in vitro simulated gastric and pancreatic digestion. The third is the use of algorithms based on the predicted effects of specific meal components on absorption derived from isotopic studies in human volunteers. The in vitro procedures have been very useful for identifying and characterizing factors that affect non-heme iron absorption, but direct comparisons between absorption predicted from the in vitro tests and measurements in human volunteers have only been made in a limited number of published studies. The available data indicate that dialysis and Caco-2 cell uptake are useful for ranking meals and single food items in terms of predicted iron bioavailability, but may not reflect the magnitudes of the effects of factors that influence absorption accurately. Algorithms based on estimates of the amounts of heme iron and of enhancers and inhibitors of non-heme iron absorption in foods make it possible to classify meals or diets as being of high, medium, or low bioavailability. The precision with which meal iron bioavailability can be predicted in a population, for which a specific algorithm has been developed, is improved by measuring the content of the most important enhancers and inhibitors. However, the accuracy of such predictions appears to be much lower when the algorithm is applied to meals eaten by different populations.     

Legumes and Mineral Absorption,With Special Reference to Soybean Proteins

Among the legume foods, soy proteins are finding increased use in human diets. Hence, selected aspects of dietary mineral absorption and availability in reference to soy proteins are reviewed, with particular attention being given to the human nutritional context. Data from animal feeding studies are considered first as a basis for indicating the value, as well as limitations, of data from animal bioassays on mineral absorption as they may relate directly to human feeding. It is apparent that the availability of zinc and iron in soy-based diets given to animals is variable and it is difficult to draw generalized conclusions about legumes and mineral absorption in reference to man. The limited data obtained in human metabolic studies are reviewed and they lead to the conclusion that well-processed soy concentrates and isolates do not impair zinc absorption or nutriture. The effects of diets based on significant intakes of soy foods on the absorption of iron are still unclear although the major available evidence does not suggest significant untoward consequences of increased soy-protein intake on body iron nutriture. This problem deserves more attention, and not only with respect to iron but also other trace elements for which data in human subjects are limited or entirely lacking. Preliminary results of studies of MIT experiments, in which stable isotope probes are being developed to quantify mineral bioavailability, arc reviewed as a basis for indicating possible novel and useful approaches for exploring quantitative aspects of nutrient bioavailability in diets consumed directly by man.     

Methods and options for estimating iron and zinc bioavailability using Caco-2 cell models: benefits and limitations

Micronutrient deficiencies are prevalent worldwide and have detrimental effects on human health. Complex interactions between micronutrients and other dietary components largely determine micronutrient bioavailability, and understanding these interactions is key to improving micronutrient status. A number of in vitro and in vivo methodologies are available for assessing micronutrient bioavailability. The purpose of this review is to highlight the usefulness of one of the in vitro models, the Caco-2 cell, as a predictive tool for human micronutrient bioavailability. The review focuses on current methods used with the Caco-2 cell line, their benefits and limitations, and the possibilities for the future development of this model.     

The use of solubility, dialyzability, and Caco-2 cell methods to predict iron bioavailability

An update on in vitro methods employed to assess iron bioavailability is presented. Solubility and dialyzability are not useful predictors of iron absorption, whereas Caco-2 cell models provide useful comparisons on the availability of iron from different sources. Strengths and weaknesses of in vitro approaches are briefly described, including the need to characterize Caco-2 cells in order to interpret results correctly. Further developments are required to refine Caco-2 model systems, including optimization and standardization of methods.     

In vitro screening of relative bioaccessibility of carotenoids from foods

Carotenoids are lipophilic pigments in plant foods that are of particular interest as precursors of vitamin A, a nutrient required for vision, cell differentiation, and the immune system. In order to mediate such activities, carotenoids and their metabolites must be absorbed for delivery to tissues. Unlike many other dietary lipids, the efficiency of carotenoid absorption is typically inefficient, being affected by food matrix, style of processing, other dietary components, and nutritional and physiological status. Thus, reliable prediction of carotenoid bioavailability is problematic. We have developed a relatively simple and cost effective procedure to study the potential bioavailability, i.e., the bioaccessibility, of carotenoids. The method involves simulated oral, gastric and small intestinal digestion of test samples to access the efficiency of incorporation into micelles, an obligatory step for absorption of lipophilic compounds. The model can be further expanded by adding micelles generated during small intestinal phase of digestion to monolayers of Caco-2 human intestinal epithelial cells to investigate apical uptake, cellular metabolism and transepithelial transport of carotenoids. Recent work by Borel and associates has demonstrated that the relative bioaccessibility of carotenoids observed in vitro is highly correlated with in vivo observations and results from bioavailability trials with human subjects. Results from recent studies using the in vitro model to screen relative bioaccessibility of beta-carotene in various cultivars of cassava, impact of amount and types of fatty acyl groups in triglycerides on micellarization of carotenoids, and the mechanism of digestion and intestinal cell uptake of xanthophyll esters are presented.     

Copper and zinc absorption in the rat: mechanism of mutual antagonism

The influence that copper and zinc exert on each other's absorption was studied by using the isolated, vascularly perfused rat-intestine system. In the first series of experiments, rats were fed for 1 wk one of nine diets, with different copper and zinc concentrations representing low, adequate and high dietary metal intakes. Copper concentrations were 1, 6 and 36 mg/kg diet and zinc concentrations were 5, 30 and 180 mg/kg. The small intestine was perfused with M199 tissue culture medium containing 6 mg/L copper and 30 mg/L zinc. Neither metal was found to significantly alter the other's absorption. High dietary zinc increased metallothionein-bound copper but did not change the intracellular copper concentration. In the second series of experiments, the dietary copper and zinc concentrations were held at 6 mg/kg and 30 mg/kg, respectively, while the metal concentrations in the luminal perfusate were changed (from 1 to 36 mg/L and from 5 to 180 mg/L for copper and zinc, respectively). The higher copper concentrations in the perfusate increased zinc accumulation in mucosal cells and decreased the zinc transferred to the portal perfusate at the highest luminal zinc concentration. These data indicate that a competition and/or inhibition of a pathway for zinc out of the mucosal cell occurs at high luminal copper concentrations. High luminal zinc concentrations in the perfusate decreased the copper concentration in the mucosal cell cytosol and the amount transferred to the portal effluent. These results taken together indicate that a competition and/or inhibition of copper or zinc intake into intestinal cells occurs when the luminal concentration of the respective congener is very high.     

Caco-2 cells can be used to assess human iron bioavailability from a semipurified meal

A Caco-2 cell model with extrinsic radioiron was used to evaluate the effect of dietary factors on nonheme iron bioavailability from a semipurified meal. Study 1 was conducted to evaluate the effect of enhancers (ascorbic acid) and inhibitors (bran, phytate and tea) on iron bioavailability when added to semipurified meal containing egg albumen as a protein source. The effect of various proteins [bovine serum albumin (BSA), casein, beef and soy] on iron bioavailability was evaluated in Study 2 by substituting the above protein sources for egg albumen. Protein solubilization following in vitro digestion for individual test meals was not significantly different from the control. On the other hand, nonheme iron solubilization (0.8+/-0.0 to 5.9+/-0.3 vs. 4.9+/-0.8 mg/L) varied significantly. The total iron uptake for each meal was calculated based on the percentage of radioiron taken up and transported by Caco-2 cells and the amount of nonheme iron present in uptake solutions. Iron uptake ratios represent test/control values. With the exception of BSA and ascorbic acid, the effect of dietary factors was similar to that found in humans reported in the literature. A significant correlation (r = 0.97; P<0.0001) was found between the published human absorption data and the iron uptake by the Caco-2 cells. The results of our study indicate the usefulness of Caco-2 cells in assessing human iron absorption and the feasibility of this cell model in studying iron bioavailability from various food combinations, otherwise not easily performed in humans.     

Interaction of iron, zinc and copper in the body of animals and humans

In the light of the pertinent literature the sites and mechanisms of interactions of iron, zinc and copper in the human and animal organism are discussed. Both low and high intake of any of them may influence the utilization and metabolism of both remaining ones. E.G. dietary copper deficiency impairs the mobilization of iron reserves from the liver, and its excess inhibits the intestinal absorption of iron and zinc. Iron in excess may antagonize copper and zinc in the intestinal mucosa. Zinc excess has an unfavourable influence on iron balance by reducing the bioavailability of copper, and zinc deficiency reduces the rate of protein synthesis impairing, among others, the transport of these elements with blood. The occurrence of interaction depends on the mutual proportions of components and their sum in the diet. Attention is called to these interrelationships which are of practical importance in human nutrition.     

Benzo[a]pyrene bioavailability from pristine soil and contaminated sediment assessed using two in vitro models

A major route of exposure to hydrophobic organic contaminants (HOCs), such as benzo[a]pyrene (BaP), is ingestion. Matrix-bound HOCs may become bioavailable after mobilization by the gastrointestinal fluids followed by sorption to the intestinal epithelium. The purpose of this research was to measure the bioavailability of [14C]-BaP bound to pristine soils or field-contaminated sediment using an in vitro model of gastrointestinal digestion followed by sorption to human enterocytes (Caco-2 cells) or to a surrogate membrane, ethylene vinyl acetate (EVA) thin film. Although Caco-2 cells had a twofold higher lipid-normalized fugacity capacity than EVA, [14C]-BaP uptake by Caco-2 lipids and EVA thin film demonstrated a linear relationship within the range of BaP concentrations tested. These results suggest that EVA thin film is a good membrane surrogate for passive uptake of BaP. The in vitro system provided enough sensitivity to detect matrix effects on bioavailability; after 5 h, significantly lower concentrations of [14C]-BaP were sorbed into Caco-2 cells from soil containing a higher percentage of organic matter compared to soil with a lower percentage of organic matter. The [14C]-BaP desorption rate from Caco-2 lipids consistently was twofold higher than from EVA thin film for all matrices tested. The more rapid kinetics observed with Caco-2 cells probably were due to the greater surface area available for absorption/desorption in the cells. After 5 h, the uptake of BaP into Caco-2 lipid was similar in live and metabolically inert Caco-2 cells, suggesting that the primary route of BaP uptake is by passive diffusion. Moreover, the driving force for uptake is the fugacity gradient that exists between the gastrointestinal fluid and the membrane.     

Iron and Zinc Nutrition in the Economically-Developed World: A Review

This review compares iron and zinc food sources, dietary intakes, dietary recommendations, nutritional status, bioavailability and interactions, with a focus on adults in economically-developed countries. The main sources of iron and zinc are cereals and meat, with fortificant iron and zinc potentially making an important contribution. Current fortification practices are concerning as there is little regulation or monitoring of intakes. In the countries included in this review, the proportion of individuals with iron intakes below recommendations was similar to the proportion of individuals with suboptimal iron status. Due to a lack of population zinc status information, similar comparisons cannot be made for zinc intakes and status. Significant data indicate that inhibitors of iron absorption include phytate, polyphenols, soy protein and calcium, and enhancers include animal tissue and ascorbic acid. It appears that of these, only phytate and soy protein also inhibit zinc absorption. Most data are derived from single-meal studies, which tend to amplify impacts on iron absorption in contrast to studies that utilize a realistic food matrix. These interactions need to be substantiated by studies that account for whole diets, however in the interim, it may be prudent for those at risk of iron deficiency to maximize absorption by reducing consumption of inhibitors and including enhancers at mealtimes.     

Dietary protein, as egg albumen: effects on bone composition, zinc bioavailability and zinc requirements of rats, assessed by a modified broken-line model.

The effect of dietary protein concentration on zinc bioavailability, requirements and incorporation into bones was investigated in growing rats. Zinc requirements were determined by the broken-line method. Protein did not affect either absorption or biological half-life of 65Zn added to the diet. Zinc requirements based on weight gain or tibia zinc were generally greater when rats were fed 30% rather than 15% egg white. When fed 30% rather than 15% egg white, zinc-deficient rats gained less weight and tended to incorporate less zinc into bone, whereas zinc-adequate rats gained weight similarly and incorporated more zinc into bone. Dietary protein concentration apparently elevated the maximum amount of zinc incorporated into bones of rats fed adequate zinc. When dietary zinc was adequate (25 mg/kg diet), tibia zinc concentrations increased linearly with dietary concentrations of 15, 25, 35 and 45% egg white. When dietary zinc was adequate, higher protein diets resulted in lower tibia nitrogen, and higher tibia zinc, without substantial changes in tibia size or calcium concentration. These results indicate that high protein diets increase zinc requirements and bone zinc deposition, the latter being a consequence of altered bone zinc metabolism, rather than improved zinc bioavailability.     

Assessment of lutein bioavailability from meals and a supplement using simulated digestion and caco-2 human intestinal cells

Lutein and zeaxanthin are selectively accumulated in the lens and macular region of the retina. It was suggested that these xanthophylls protect ocular tissues from free-radical damage that can cause cataracts and age-related macular degeneration. Insights regarding the absorption of dietary xanthophylls for delivery to ocular tissues are limited. Our primary objective was to examine factors affecting the transfer of lutein from foods to absorptive intestinal epithelial cells during digestion. Lutein and other carotenoids present in spinach purée and lutein from a commercial supplement were relatively stable during in vitro digestion. Micellarization of lutein and zeaxanthin during the small intestinal phase of digestion exceeded that of beta-carotene and was greater for xanthophylls in oil-based supplements than in spinach. Apical uptake of lutein from micelles by Caco-2 human intestinal cells was linear for at least 8 h, and accumulation from synthetic micelles exceeded that from micelles generated during simulated digestion. Stimulation of chylomicron synthesis resulted in the secretion of 7.6 +/- 0.1% of cellular lutein into the triglyceride-rich fraction in the basolateral chamber. These data support the use of simulated digestion and the Caco-2 cell model as effective tools for identifying factors affecting absorption of dietary carotenoids.     

Effects of dietary fibers on magnesium absorption in animals and humans

There is overwhelming evidence that dietary fibers are an important component of human and animal diets and play an important role in human health. Because dietary fibers and some associated substances, such as phytate, have in vitro mineral-binding capacities, they have been thought to impair absorption of minerals such as calcium, iron and zinc, although magnesium absorption seems to be less affected. Indeed, the effect of dietary fibers depends largely on their own nature and characteristics, and also on mineral homeostasis. In 1977 it was observed that resistant starch, a fermentable dietary fiber, could improve Mg absorption in rats. More recently, attention has been focused on other fermentable substrates such as inulin and oligo- or polysaccharides, for their potential prebiotic and health effects. Studies conducted on different types of fermentable carbohydrates have confirmed their beneficial effect on Mg absorption in different animal species. The majority of these studies have also sought to determine the effects of fibers on other minerals such as calcium, iron and zinc. In contrast to the studies with Mg, these studies did not show a consistent effect on the absorption of these minerals. This is due to the particularities of sites and mechanism of Mg absorption. To date, four human studies have been carried out that generally confirmed the enhancing effect of fermentable oligo- or polysaccharides on Mg absorption.     

The concept of iron bioavailability and its assessment

Summary In this review a broad overview of historical and current methods for the assessment of iron bioavailability was given. These methods can be divided into iron solubility studies, iron absorption studies, endpoint measures, and arithmetic models. The pros and cons of all methods were discussed. First, studies on in vitro and in vivo iron solubility have been described. The disadvantages of iron solubility include the impossibility of measuring absorption or incorporation of iron. Furthermore, only the solubility of nonheme iron, and not heme iron, can be studied. Second, we focused on iron absorption studies (either with the use of native iron, radioiron or stable iron isotopes), in which balance techniques, whole-body counting or postabsorption plasma iron measurements can be applied. In vitro determination of iron absorption using intestinal loops or cell lines, was also discussed in this part. As far as absorption studies using animals, duodenal loops, gut sacs or Caco-2 cells were concerned, the difficulty of extrapolating the results to the human situation seemed to be the major drawback. Chemical balance in man has been a good, but laborious and expensive, way to study iron absorption. Whole-body counting has the disadvantage of causing radiation exposure and it is based on a single meal. The measurement of plasma iron response did not seem to be of great value in determining nutritional iron bioavailability. The next part dealt with endpoint measures. According to the definition of iron bioavailability, these methods gave the best figure for it. In animals, the hemoglobin-repletion bioassay was most often used, whereas most studies in humans monitored the fate of radioisotopes or stable isotopes of iron in blood. Repletion bioassays using rats or other animals were of limited use because the accuracy of extrapolation to man is unknown. The use of the rat as a model for iron bioavailability seemed to be empirically based, and there were many reasons to consider the rat as an obsolete model in this respect. The double-isotope technique was probably the best predictor of iron bioavailability in humans. Disadvantages of this method are the single meal basis and the exposure to radiation (as far as radioisotopes were used). Finally, some arithmetic models were described. These models were based on data from iron bioavailability studies and could predict the bioavailability of iron from a meal. Received: 26 March 1997, Accepted: 9 October 1998     

An in vitro digestion/Caco-2 cell culture system accurately predicts the effects of ascorbic acid and polyphenolic compounds on iron bioavailability in humans

We developed a rapid in vitro digestion/Caco-2 cell culture model for assessing relative bioavailabilities of iron in foods and meals. The objective of the present study was to determine how closely our Caco-2 model reflects the human response. Meals described in published reports of studies on effects of varying levels of ascorbic acid (AA) and tannic acid (TA) on iron absorption by human subjects were carefully replicated. Iron absorption ratios (iron absorption from meals containing AA or TA divided by iron absorption from identical meals without these enhancers or inhibitors) were determined using the Caco-2 model. Ferritin formation by the Caco-2 cells was used as an indicator of iron absorption. Response patterns of effects of AA and TA on absorption ratios (AR) calculated from Caco-2 and human data were very similar: AA increased ARs in a dose-response manner and TAs decreased AR. The natural logs of the ARs determined in Caco-2 and human studies were correlated: R = 0.935 (P = 0.012) and 0.927 (P = 0.007) for AA and TA, respectively. When results from meals with AA and TA were pooled, a linear relation between the natural logs of ARs from Caco-2 and human studies was observed (R = 0.968, P < 0.001). We conclude that our Caco-2 model accurately predicts the human response to AA and TA in the meals we tested. If future work reproduces the precision and accuracy shown in this paper for predicting iron bioavailability to humans, then the implications for saving time and resources in iron bioavailability measurements are considerable.