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Article

Caco-2 Cells Can Be Used to Assess Human Iron Bioavailability from a Semipurified Meal¹

Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 50011

²To whom correspondence should be addressed.

	ABSTRACT

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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.

KEY WORDS: • nonheme iron • bioavailability • Caco-2 cells

	INTRODUCTION

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Several decades ago, dietary components that enhanced or inhibited nonheme iron absorption were identified (Carpenter and Mahoney 1992 ) mainly by the application of extrinsic radioiron labeling in humans. The important enhancing factors are ascorbic acid (Hallberg et al. 1989 , Lynch and Cook 1980 ) and animal tissues (Cook and Monsen 1976 , Hurrell et al. 1988 ), whereas inhibitors are numerous. Those inhibitory factors include phytate (Gillooly et al. 1983 , Hallberg et al. 1987 ), polyphenols (Disler et al. 1975 , Gillooly et al. 1983 , Tuntawiroon et al. 1991 ), calcium (Cook et al. 1991a , Hallberg et al. 1991 ), some plant proteins (Cook et al. 1981 , Hurrell et al. 1992 , Steinke and Hopkins 1978 ), wheat bran (Hallberg et al. 1987 ) and fiber (Reinhold et al. 1982 ).

The enhancing or inhibiting effects of the above dietary factors in humans have been mostly examined singly and some in combinations. Human studies relying on isotopic measurement of iron absorption are impractical because of the risks of radioactivity exposure, cost and complexity. Animal studies, especially rodents, were used to test iron availability of many food items. However, rats were found to be less sensitive to dietary factors than humans, and the behavior of the rats’ iron absorption might be different from that of humans (Reddy and Cook 1991 ).

Recently, cell culture has been used extensively as an in vitro method to assess human iron bioavailability. Caco-2 cells, a human colon adenocarcinoma cell line, have demonstrated numerous morphological and biochemical characteristics of enterocytes. These cells spontaneously differentiate into polarized monolayers with a well-developed brush border and associated enzymes (Pinto et al. 1983 ). This cell model has been used in a wide variety of nutritional studies, particularly in the study of mechanisms (Han et al. 1995 , Nuñez et al. 1994 ) and regulation of iron absorption (Alvarez-Hernandez et al. 1994 , Gangloff et al. 1996 , Tapia and Nuñez 1999 ) and iron bioavailability studies (Garcia et al. 1996 , Glahn et al. 1996 , Glahn and Van Campen 1997 , Glahn et al. 1998a , 1998b ). The application of a Caco-2 cell model appears to be promising as a physiological means of measuring mucosal cell iron uptake.

Although many studies have utilized this model to investigate the factors affecting iron bioavailability, no study has ever been reported to compare iron uptake by Caco-2 cells with human absorption data. The aim of this study was to test the applicability of Caco-2 cells to assess nonheme iron bioavailability by a direct comparison of cell uptake results with published human absorption data (Cook et al. 1981 , Hurrell et al. 1988 , 1989 , Reddy and Cook 1991 , Reddy et al. 1996 ) using exactly the same meal composition. The results of our study will strengthen the use of this model to study human iron bioavailability from a number of food combinations.

	MATERIALS AND METHODS

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Cell culture.

Caco-2 cells were obtained at passage 18 from an American Type Culture Collection (Rockville, MD). All the experiments were conducted at passages 35–43. The cells were grown in Dulbecco’s Modified Eagle Medium (Sigma, St. Louis, MO) with 16% fetal bovine serum (Sigma), 1% nonessential amino acids (Gibco BRL, Grand Island, NY) and 1% antibiotic-antimycotic solution (Gibco BRL). They were maintained at 37°C in an incubator with 95% atmospheric O₂ and 5% CO₂. At 80–100% confluency, the cells were trypsinized and seeded in a cell culture flask of 75-cm² area (Corning Costar Corporation, Cambridge, MA) at a density of 1.68 x 10⁷ cells/L or 5,600 cells/cm² for continuing growth. For iron uptake experiments, the cells were seeded onto collagen-treated membrane (polytetrafluororethylene) of the inserts fitted in bicameral chambers (Transwell-COL, six-well, 24-mm-diameter, 0.4-µm pore size; Corning Costar Corporation) at a density of 1.65 x10⁸ cells/L or 55,000 cells/cm². Phenol red testing (Garcia et al. 1996 ) indicated that 12–14 d after seeding, cells reached 90–100% confluency with tight intercellular junctions, and the confluent cell layers were used for iron uptake experiments.

Test meals.

The composition of the meals that we utilized in this study was based on the meals used in earlier human studies (Cook and Monsen 1977 , Hurrell et al. 1988 , 1989 , Reddy and Cook 1991 , Reddy et al. 1996 ), with the exception that only one-eighth of the total meal was used for in vitro digestion. Cook and Monsen (1975) developed this semipurified meal containing 30 g protein, 68 g carbohydrate and 35 g fat, and it was fed to humans. In this study, the semipurified meals contained 3.8 g protein and 8.4 g dextrimaltose (Amaizo; American Maize-Products Company, Hammond, IN) dissolved in 45 mL deionized (DI)³ water, and 4.4 g corn oil (Mazola, Engelwood Cliffs, NJ). The ingredients were blended with 30 mL DI water. Composition of each meal is shown in Table 1 .

In Study 1, 4.17 g of egg albumen (Monark Egg Corporation, Kansas City, MO) was used as the protein source for the control meal (Meal A). The test meals contained the same protein, fat and carbohydrate sources but with added ascorbic acid, bran, phytate and tea, which were represented as Meals B, C, D and E, respectively. Meal B contained 12.5 mg ascorbic acid (Sigma Chemical Company). Meal C contained 2 g hard wheat red bran (AACC, St. Paul, MN). To Meal D was added 37.5 mg phytic acid (10.8 mg phytate-P) as dodecyl sodium phytate (Sigma Chemical Company). Tea was prepared by using 0.22 g tea leaves (Lipton, Englewood Cliffs, NJ) steeped in 31 mL boiling water for 4 min, then strained. Prepared tea was substituted for DI water in preparing Meal E.

In Study 2, protein sources were substituted for egg albumen of the control to study the effect of proteins on iron uptake by Caco-2 cells. Meal A containing egg albumen served as a control, and Meals F, G, H and I, containing 3.91 g bovine serum albumin (BSA) fraction V lyophilized powder (Sigma Chemical Company), 4.06 g casein (Vitafree; United States Biochemical, Cleveland, OH), 4.55 g cooked freeze-dried beef powder and 4.17 g soy protein isolates (Archer Daniels Midland Company, Decatur, IL), respectively, served as test meals. Freeze-dried beef was prepared from lean beef purchased locally. Prior to cooking, all visible fat from meat was trimmed off, and the meat was cut into small cubes. A total of 2.6 kg of lean beef was simmered in 700 mL distilled DI water for 1.5 h. Then the cooked beef was refrigerated overnight, and visible fat floating on top was removed. Homogenization with DI water was carried out with Polytron (Brinkmann, Westbury, NY). The slurry was frozen at -20°C, freeze-dried in ziploc bags, ground into powder using a coffee grinder (Regal Ware, Kewaskum, WI) with precaution taken not to overheat the proteins and stored at -20°C until use. Each semipurified meal was prepared in duplicates for in vitro digestion. Iron uptake for each duplicate digested sample was measured in 3–4 inserts of six-well plates.

In Vitro digestion.

All the enzymes and other chemicals were obtained from Sigma Chemical Company. All the reagents were prepared fresh before each experiment. A total of nine samples (A-I) were prepared. Prior to adding test factors, protein sources, dextrimaltose and corn oil were blended (Osterizer, Bay Springs, MS) with 30 mL DI water or tea before being homogenized with Polytron (Brinkmann) at 18,000 rpm for 45 s. For Study 1, ascorbic acid, bran phytate and tea were added to Meals B, C, D and E, respectively, then mixed well. An appropriate amount of FeCl₃ in 0.01 mol/L HCl was added to give a total of 0.51 mg iron for each meal; then in vitro digestion was carried out. First, pH of the mixture was brought to 2 with 5 mol/L HCl before adding 1 mL pepsin solution [0.08 g (2,500 U/mg protein) per mL 0.1 mol/L HCl] and then incubated for 1 h at 37°C in a shaking water bath at a speed of 90 rpm to simulate gastric digestion. Following incubation, pH was raised to 6 with slow addition of 1 mol/L NaHCO₃ dropwise, and 5 mL pancreatin-bile mixture [0.12 g bile extract and 0.02 g pancreatin (4 x U.S.P. activity) in 5 mL 0.1 mol/L NaHCO₃] was added. Incubation was continued for 30 min more at 37°C to mimic duodenal digestion. Shortly after removal from the shaking water bath, each sample was placed in an ice bucket in order to stop pancreatic activity. The digests were subjected to centrifugation at 5,000 x g for 30 min. Supernatants were assayed for protein (Lowry et al. 1951 ), and nonheme iron was determined by a slight modification of the method of Torrance and Bothwell (1968) using ferrozine instead of bathophenanthroline disulfonic acid as the chromogen (Chidambaram et al. 1989 ).

Iron uptake by Caco-2 cells.

At 12–14 d postseeding, cells reached 90–100% confluency and were used for iron uptake experiments. Protein in supernatants of each meal was diluted to a concentration of 1 g/L using EBSS (Earle’s Balanced Salt Solution, Gibco BRL) with 25 mmol/L HEPES (Sigma Chemical Company) at pH 6.8. Radioactive tracer Fe-59 (Dupont, NEN products, Boston, MA) in 0.01 mol/L HCl was added to each 4 mL diluted supernatant to provide 0.74 kBq, which contained a very negligible amount of iron (1 ng). Before placing samples (1.5 mL) onto the cell monolayers in each insert, growth medium was removed and 1 mL EBSS was used to rinse both apical and basal chambers. Separate aliquots of 1.5 mL extrinsically labeled solutions were set aside for measurement of initial radioactivity. An aliquot of 1.5 mL EBSS/HEPES was placed in each basal chamber. Samples were incubated at 37°C with 95% O₂-5% CO₂ for 1 h. After incubation, solutions were collected from apical chambers, and then cell layers were rinsed with 1 mL EBSS to remove any nonspecific-bound iron. Collagen membranes with cell monolayer and EBSS/HEPES in basal chamber also were subsequently removed. Total radioactivity was measured in solutions that were removed from apical and basal chambers, washings and cell monolayers, and the percentage recovery was calculated with respect to initial radioactivity. Samples were not used if the radioactivity recovery was not close to 100%. Radioactivity in the cell monolayers and in the basal chamber solution for each well provided the percentage radioiron cell uptake and transport, respectively. Radioactivity was counted by using a gamma scintillation counter (Packard Instrument Company, Meriden, CT). One insert in each six-well plate was used for ferrous ascorbate (Fe²⁺-AA) as a positive control that contained 10 µmol/L ferrous sulfate and 200 µmol/L ascorbic acid. Cell monolayers were solubilized in 0.5 mol/L sodium hydroxide (Fisher Scientific, Fair Lawn, NJ) and were sonicated by using a sonic dismembrator (Fisher Scientific, Pittsburgh, PA) at a setting of 2 before protein determination (Lowry et al. 1951 ) of the sonicated solution. Results were expressed as total cell iron uptake, which was calculated based on the amount of nonheme iron present in the uptake solutions and percentage of total radioiron taken up by the cells plus the iron transported into the basal chambers. Comparisons between meals were made using the amount of nonheme iron absorbed rather than the percentage of radioiron absorption assuming radioiron exchanged completely with the intrinsic nonheme iron. To obtain consistency among experiments, total cell iron uptake results were normalized by dividing each value with Fe²⁺-AA iron uptake value obtained from the same plate and multiplying it by an average Fe²⁺-AA uptake value obtained from all the plates we have used in this study. Cell uptake ratios were calculated using the normalized values for test/control meals.

Statistical analysis.

Differences in mean concentrations of soluble protein and nonheme iron, percentage of and the amount of cell iron uptake were compared for test meals (B-I) with the control meal (A) using ANOVA with Dunnett’s Multiple Comparison Test. Differences in mean concentrations of protein in the cells were compared using ANOVA followed by Tukey’s Multiple Comparison Test. The differences were considered significant at P 0.05.

To compare our results with human absorption data, absorption values for each meal were obtained from published human studies (Cook and Monsen 1977 , Hurrell et al. 1988 , 1989 , Reddy and Cook 1991 , Reddy et al. 1996 ). Since each human study utilized different groups of subjects with different mean serum ferritin values, the absorption values were adjusted to a serum ferritin value of 40 µg/L using the equation reported by Cook et al. (1991b) , which is as follows: Log A_c = Log A_o + Log F_o – Log 40, where A_c is adjusted absorption, A_o is observed absorption, and F_o is observed serum ferritin (Table 2 ). Absorption ratios were calculated for each test meal in relationship to the control meal (test/control). Pearson correlation was employed to correlate Caco-2 cell iron uptake ratios with human absorption ratios. Statistical software, GraphPad Prism (1994 San Diego, CA), was used to perform statistical analyses.

	RESULTS

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Percentages of radioiron taken up by the cells and transported into the basal chambers were combined and expressed as the percentage of radioiron uptake (Table 4 ). Because there were significant differences in iron concentrations among iron uptake solutions for Meals A-I, the cell iron uptake values were expressed as ng/insert (Table 3) and then normalized to the Fe²⁺-AA control (Table 4) for statistical analysis. The data were normalized due to the variation of Fe²⁺-AA uptake from plate to plate (9.16–13.37%). Uptake ratios were determined by using total cell iron uptake from test/control meals and are shown in Figure 1 . For example, for tea, the percentage uptake of radioiron was as high as the control, but it contained less soluble nonheme iron than the control (0.17 vs. 0.03 µg). Such low iron content in tea accounted for 1.3 ng of iron uptake, indicating an 80% reduction from the control (P < 0.01). As expected, in addition to tea, bran and phytate also decreased iron uptake significantly by 83 (P < 0.01) and 49% (P < 0.01), respectively (Table 4 and Fig. 1 ). Ascorbic acid had no enhancing effect on total iron uptake. BSA also did not show any significant effect on enhancing iron bioavailability with an absorption ratio of 1.1. However, casein and soy inhibited iron absorption markedly by 78 (P < 0.01) and 66% (P < 0.01), respectively. A 100% (P < 0.01) increase in iron uptake also was observed with beef.

View larger version (49K): [in this window] [in a new window]   Figure 1. Effects of dietary factors on iron uptake in Caco-2 cells from semipurified meals. The results were expressed as iron uptake ratios between the test meals and the control meal. Iron uptake ratios were calculated based on average iron uptake values from test/control meals. The absorption ratio of 1.0 indicates no effect. BSA, bovine serum albumin.

With all the semipurified meals evaluated (A-I), an excellent correlation (r = 0.97; P < 0.0001) was obtained between uptake ratios in Caco-2 cells and absorption ratios in humans (Fig. 2 ). This correlation was best illustrated by beef and several inhibitors including bran, tea and soy, as these dietary factors showed strong enhancement and inhibition on Caco-2 cell iron uptake.

View larger version (16K): [in this window] [in a new window]   Figure 2. Correlation (r = 0.97; P < 0.0001) between iron uptake ratios in Caco-2 cells and absorption ratios in humans. Iron uptake ratios in Caco-2 cells were calculated using iron uptake from test/control meals. Since the iron content was the same in all the meals, percentage absorption from test/control meals was used to calculate human iron absorption ratios. Human absorption values were adjusted to a serum ferritin value of 40 µg/L (Cook et al. 1991b ) before calculating the ratios. References for human studies are listed in Table 2 .

	DISCUSSION

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Addition of enhancers and inhibitors to egg albumen-containing meals in Study 1 resulted in less variation in the soluble protein concentration than was found with different protein sources in Study 2. Although not significant, protein concentration in Study 2 varied, which might be attributed to the differences in protein digestibilities with gastrointestinal enzymes. Strong reduction of iron solubility and absorption was seen with meals containing casein, phytate, bran, tea and soy. In general, the inhibitory effects observed in this study were consistent with those of previous human absorption studies. The effect of casein in this study was more pronounced in our study than in humans (Hurrell et al. 1989 ). A reduction of 78% in iron uptake observed in this study compared with a 33% reduction of iron absorption in the human studies. One possible explanation is that the casein used in our study is different from that used in the human studies (Hurrell et al. 1989 ). Nevertheless, the inhibitory effect of casein was evident in both studies. A similar reduction of iron uptake by bran was observed in this study and in humans (Reddy and Cook 1991 ). The inhibitory effect of phytate observed in our study was less marked than that observed in human studies. Phytate decreased iron uptake by 49% as compared with 78% reduction in human studies (Reddy et al. 1996 ). Phytate, due to its six phosphate groups, binds cations and proteins strongly at acidic pH by virtue of its strong negative charge, thus decreasing iron solubility and absorption through the formation of insoluble iron complexes (Cheryan 1980 ). The pH of 6.8 that we used in the present study may be higher than the pH in the early part of the human duodenum, which might have caused phytate to bind iron less strongly. Thus, phytate resulted in a less inhibitory effect on iron uptake in Caco-2 cells. The observed inhibitory effect of tea was similar (73 vs. 78%) to that observed in humans (Reddy and Cook 1991 ), which was attributed to tannins and phenolic compounds. Presumably, phenolic compounds form insoluble complexes with iron within the intestinal lumen (Disler et al. 1975 ). Soy not only decreased iron solubility but also inhibited cell uptake, consistent with human studies (Cook et al. 1981 , Reddy and Cook 1991 ). The inhibitory effect was attributed to the high phytate content of soy proteins or protein itself (Cook et al. 1981 , Hurrell et al. 1992 ). In contrast, BSA, which has an enhancing effect on human absorption (Hurrell et al. 1988 ), had no effect in our study.

The 100% enhancement by beef was significant (P < 0.01) and was consistent with a previous human study (Reddy et al. 1996 ). The mechanism of enhancement remains unclear, but it is believed that a factor in meat is responsible for increasing nonheme iron absorption by forming soluble iron complexes. However, ascorbic acid, which enhances human iron absorption due to its reducing capacity (Cook and Monsen 1977 , Hallberg et al. 1989 , Lynch and Cook 1980 ), had no effect in our study of Caco-2 cells. The enhancing effect of ascorbic acid also was lower in the human absorption data that we referenced because we combined the percentages of absorption of the control meals from different studies (Cook and Monsen 1977 , Hurrell et al. 1988 , Reddy and Cook 1991 , Reddy et al. 1996 ). The reducing capacity of ascorbic acid is pH-dependent (Bothwell et al. 1979 ), with a maximum at pH 5.5. Therefore, under the pH conditions that we used, it may not have the strong capability to reduce Fe³⁺ to Fe²⁺. We have repeated cell uptake experiments with ascorbic acid and meat treatments at pH 6.0. As expected, compared with the control, a 6–7-fold increase was seen with ascorbic acid at this pH. Surprisingly, beef had no effect at this pH. These results suggest that when food enters the duodenum, ascorbic acid is effective in the proximal region where pH is low and beef is effective in the distal region where pH is high. Hence, future studies are warranted to modify the method by exposing the cell monolayers to a gradual pH change and measuring iron uptake simultaneously.

Indicating a strong correlation with those of human absorption studies, these results support the usefulness of this Caco-2 cell model human iron bioavailability study. Because of the unreliability of rodent models (Reddy and Cook 1991 ) and of the risks involved in human studies using radioisotopic methods, in vitro models appear to be useful tools in assessing human nonheme iron availability. To our knowledge, this is the first study using the same meals that were used in humans and directly comparing the effect of a wide range of dietary factors on iron bioavailabilty in Caco-2 cells with human data. If conditions of pH, protein and iron concentration are optimized, this model can be utilized to study many combinations of dietary factors in complex meals that are not feasible to study in human subjects.

	FOOTNOTES

 
¹ Journal Paper No. J-18541 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, project no. 3432, and supported by Hatch Act and State of Iowa funds.

³ Abbreviations used: BSA, bovine serum albumin; EBSS, Earle’s Balanced Salt Solution.

Manuscript received August 10, 1999. Initial review completed October 13, 1999. Revision accepted January 4, 1999.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Alvarez-Hernandez X., Smith M., Glass J. Regulation of iron uptake and transport by transferrin in Caco-2 cells, an intestinal cell line. Biochim. Biophys. Acta. 1994;1192:215-222[Medline]

2. Bothwell T., Charlton R., Cook J. D., Finch C. Iron Metabolism in Man 1979 Blackwell Scientific Oxford, UK.

3. Carpenter C., Mahoney A. Contributions of heme and nonheme iron to human nutrition. Crit. Rev. Food Sci. Nutr. 1992;31:333-367[Medline]

4. Chaney A., Marbach E. Modified reagents for determination of urea and ammonia. Clinical Chemistry 1962;8:130-132[Abstract]

5. Cheryan M. Phytic acid interactions in food systems. CRC Crit. Rev. Food Sci. Nutr. 1980;13:297-335

6. Chidambaram M., Reddy M. B., Thompson J., Bates G. W. In vitro studies of iron bioavailability: Probing the concentration and oxidation reduction reactivity of pinto bean iron with ferrous chromogens. Biolog. Trace Element Res. 1989;19:25-40

7. Cook J. D., Dassenko S. S., Lynch S. R. Assessment of the role of nonheme-iron availability in iron balance. Am. J. Clin. Nutr. 1991b;54:717-722[Abstract/Free Full Text]

8. Cook J. D., Dassenko S. S., Whittaker P. Calcium supplementation: effect on iron absorption. Am. J. Clin. Nutr. 1991a;53:106-111[Abstract/Free Full Text]

9. Cook J. D., Monsen E. R. Food iron absorption I. Use of a semisynthetic diet to study absorption of nonheme iron. Am. J. Clin. Nutr. 1975;28:1289-1295[Abstract/Free Full Text]

10. Cook J. D., Monsen E. R. Food iron absorption in human subjects III. Comparison of the effect of animal proteins on nonheme iron absorption. Am. J. Clin. Nutr. 1976;29:859-867[Abstract/Free Full Text]

11. Cook J. D., Monsen E. R. Vitamin C, the common cold, and iron absorption. Am. J. Clin. Nutr. 1977;30:235-241[Abstract/Free Full Text]

12. Cook J. D., Morck T. A., Lynch S. R. The inhibitory effect of soy products on nonheme iron absorption in man. Am. J. Clin. Nutr. 1981;34:2622-2629[Abstract/Free Full Text]

13. Disler P. B., Lynch S. R., Charlton R. W., Torrance J. D., Bothwell T.H., Walker R. B., Mayet R. The effect of tea on iron absorption. Gut 1975;16:193-200[Abstract/Free Full Text]

14. Gangloff M., Lai C., Van Campen D. R., Miller D. D., Glahn R. P. Caco-2 cell ferrous iron uptake but not transfer is down-regulated in cells grown in high iron serum-free medium. J. Nutr. 1996;126:3118-3127

15. Garcia M. N., Flowers C., Cook J. D. The Caco-2 cell culture system can be used as a model to study food iron availability. J. Nutr. 1996;126:251-258

16. Gillooly M., Bothwell T. H., Torrance J. D., MacPhail A., Derman D., Bezwoda W., Mills W., Charlton R., Mayet F. The effect of organic acids, phytates and polyphenols on the absorption of iron from vegetables. Br. J. Nutr. 1983;49:331-336[Medline]

17. Glahn R. P., Lai C., Hsu J., Thompson J. F., Guo M., Van Campen D. R. Decreased citrate improves iron availability from infant formula: application of an in vitro digestion/Caco-2 cell culture model. J. Nutr. 1998a;128:257-264[Abstract/Free Full Text]

18. Glahn R. P., Lee O. A., Yeung A., Goldman M. I., Miller D. D. Caco-2 cell ferritin formation predicts nonradiolabeled food iron availability in an in vitro digestion/Caco-2 cell culture model. J. Nutr. 1998b;128:1555-1561[Abstract/Free Full Text]

19. Glahn R. P., Van Campen D. R. Iron uptake is enhanced in Caco-2 cell monolayers by cysteine and reduced cysteinyl glycine. J. Nutr. 1997;127:642-647[Abstract/Free Full Text]

20. Glahn R. P., Wien E. M., Van Campen D. R., Miller D. D. Caco-2 cell iron uptake from meat and casein digests parallels in vivo studies: Use of a novel in vitro method for rapid estimation of iron bioavailability. J. Nutr. 1996;126:332-339

21. GraphPad Prism User’s Guide 1994 San Diego, CA.

22. Hallberg L., Brune M., Erlandsson M., Sandberg A.-S., Rossander-Hulten L. Calcium: Effect of different amounts on nonheme- and heme-iron absorption in humans. Am. J. Clin. Nutr. 1991;53:112-119[Abstract/Free Full Text]

23. Hallberg L., Brune M., Rossander L. The role of vitamin C in iron absorption. Int. J. Vitam. Nutr. Res. Suppl. 1989;30:103-108[Medline]

24. Hallberg L., Rossander L., Skanberg A. Phytate and the inhibitory effect of bran on iron absorption in man. Am. J. Clin. Nutr. 1987;45:988-996[Abstract/Free Full Text]

25. Han O., Failla M., Hill A., Morris E., Smith J. Jr Reduction of Fe(III) is required for uptake of nonheme iron by Caco-2 cells. J. Nutr. 1995;125:1291-1299

26. Hurrell R., Jillerat M., Reddy M. B. Soy protein, phytate, and iron absorption in humans. Am. J. Clin. Nutr. 1992;56:573-578[Abstract/Free Full Text]

27. Hurrell R., Lynch S. R., Trinidad T., Dassenko S. S., Cook J. D. Iron absorption in humans: bovine serum albumin compared with beef muscle and egg white. Am. J. Clin. Nutr. 1988;47:102-107[Abstract/Free Full Text]

28. Hurrell R., Lynch S. R., Trinidad T., Dassenko S. S., Cook J. D. Iron absorption in humans as influenced by bovine milk proteins. Am. J. Clin. Nutr. 1989;49:546-552[Abstract/Free Full Text]

29. Lowry O., Rosebrough N., Farr A., Randall R. Protein measurement with the folin phenol reagent. J. Biol. Chem. 1951;193:265-275[Free Full Text]

30. Lynch S. R., Cook J. D. Interaction of vitamin C and iron. Ann. N. Y. Acad. Sci. 1980;355:32-44[Medline]

31. Nuñez M., Alvarez X., Smith M., Tapia V., Glass J. Role of redox systems on Fe³⁺ uptake by transformed human intestinal epithelial (Caco-2) cells. Am. J. Physiol. 1994;267(Cell:Physiol.36):C1582-C1588[Abstract/Free Full Text]

32. Pinto M., Robine-Leon S., Appay M.-D., Kedinger M., Triadou N., Dussaulx E., LaCroix B., Simon-Assmann P., Haffen K., Fogh J., Zweibaum A. Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol. Cell. 1983;47:323-330

33. Reddy M. B., Cook J. D. Assessment of dietary determinants of nonheme-iron absorption in humans and rats. Am. J. Clin. Nutr. 1991;54:723-728[Abstract/Free Full Text]

34. Reddy M. B., Hurrell R., Juillerat M., Cook J. D. The influence of different protein sources on phytate inhibition of nonheme-iron absorption in humans. Am. J. Clin. Nutr. 1996;63:203-207[Abstract/Free Full Text]

35. Reinhold J., Garcia L., P.M.,Arias-Amando L., Garyon P. Dietary fiber-iron Interactions: fiber-modified uptakes of iron by segments of rat intestine. Vahouny G. Kritchevsky D. eds. Dietary Fiber in Health and Disease 1982:117-132 Plenum Press New York, NY.

36. Stahr H. eds. Analytical Methods in Toxicology 1991:71-73 John Wiley & Son New York, NY.

37. Steinke F., Hopkins D. Biological availability to the rat of intrinsic and extrinsic iron with soybean protein isolates. J. Nutr. 1978;108:481-489

38. Tapia V., Nuñez M.T. Transferrin stimulates iron absorption, exocytosis and secretion in cultured intestinal cells. Am. J. Physiol. 1999;276:C1085-C1090[Abstract/Free Full Text]

39. Torrance J. D., Bothwell T. H. A simple technique for measuring storage iron concentrations in formalinised liver samples. S. Afr. J. Med. 1968;33:9-11

40. Tuntawiroon M., Sritongkul N., Brune M., Rosssander-Hulten L., Pleehachinda R., Suwanik R., Hallberg L. Dose-dependent inhibitory effect of phenolic compounds in foods on nonheme-iron absorption in men. Am. J. Clin. Nutr. 1991;53:554-557[Abstract/Free Full Text]

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