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© 2004 The American Society for Nutritional Sciences J. Nutr. 134:395-398, February 2004

Nutrient Metabolism
Research Communication

The Mechanisms for Regulating Absorption of Fe Bis-Glycine Chelate and Fe-Ascorbate in Caco-2 Cells Are Similar1

Dora Inés Mazariegos, Fernando Pizarro, Manuel Olivares, Marco T. Nuñez* and Miguel Arredondo2

Micronutrients Laboratory, Institute of Nutrition and Food Technology (INTA) and * Millenium Institute for Advanced Studies in Cell Biology and Biotechnology and Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile

2To whom correspondence should be addressed. E-mail: marredon{at}inta.cl.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Inorganic iron (Fe) absorption from the diet is controlled mainly in the intestinal tract where apical Fe uptake is inversely related to the Fe content in the enterocyte. Iron bis-glycine chelate is an iron compound that may be absorbed by a mechanism different from the regulated nonheme Fe pathway. Because Fe bis-glycine chelate is used increasingly as an Fe fortificant in foods, the critical question is whether this compound is a safe Fe supplement. We compared apical Fe uptake and transepithelial transport offered either as 59Fe bis-glycine chelate or a 59Fe-ascorbate (Fe-AA) complex in Caco-2 cells, as a model of human intestinal epithelia, grown in different Fe concentrations in the media (0.5, 5 and 20 µmol/L Fe). Apical Fe uptake from 59Fe-AA and 59Fe bis-glycine chelate did not differ nor did transepithelial transport rates. The rate of 59Fe uptake decreased with increasing intracellular Fe concentration (P < 0.001), an indication of a common absorption regulatory mechanism. We also evaluated the effect of an excess of Fe (100 µmol/L) provided as Fe bis-glycine chelate or Fe-AA on the incorporation of 1 µmol/L 55Fe-AA into Fe-replete Caco-2 cells. The inhibition of Fe bis-glycine chelate on the absorption of the extrinsic tag of 55Fe-AA (87.5%) did not differ from that of Fe added as Fe-AA (86.8%). These results suggest that Fe derived from Fe bis-glycine chelate and Fe-AA have similar regulatory absorption mechanisms.

KEY WORDS: • Fe absorption • bioavailability • Fe bis-glycine chelate • Caco-2 cells

Iron (Fe) deficiency is often associated with poor Fe bioavailability in foods, predominantly cereals, with high fiber and phytate content, which are inhibitors of Fe absorption (1,2). Fe fortification is a strategy employed in countries in which Fe deficiency anemia is still highly prevalent (3). Iron compounds used in food fortification should be readily available for absorption but they also should not induce organoleptic changes in the food vehicle chosen. Compared with ferrous sulfate, Fe offered as Fe bis-glycine chelate is better absorbed from maize-based Venezuelan breakfasts, even in the presence of polyphenols from coffee and tea (4) and from a whole maize–based porridge (5). However, Fe absorption from Fe bis-glycine chelate did not differ from that from ferrous sulfate when these compounds were added to a vegetable infant weaning food (6). The absorption of Fe bis-glycine chelate was the same as ferrous ascorbate (Fe-AA),3 when given alone with water (7). Fe bis-glycine chelate appears to be at least as well absorbed as Fe-AA and it has certain advantages in stability and organoleptic properties.

Inorganic Fe is absorbed in the intestine by a process that includes solubilization in the acid milieu of the stomach, entrance to a common nonheme Fe pool, reduction of Fe3+ to Fe2+ and transport of Fe2+ into the absorptive duodenal cells. Fe absorption is a process regulated by the levels of intracellular Fe (8,9), through the transcriptional and translational regulation of the apical Fe divalent metal transporter 1 (DMT1) (10–12). Phytates and polyphenols decrease Fe bis-glycine chelate absorption (6). This observation supports the hypothesis of Fe bis-glycine chelate entering at least partially into the nonheme Fe pool, where it is subject to the formation of irreversible ligands with phytate, thus reducing its bioavailability. Another indirect observation supporting this hypothesis is the fact that Fe bis-glycine chelate absorption is regulated by Fe stores (13,14). Unregulated absorption of Fe bis-glycine chelate could cause Fe overload. Therefore, we investigated whether the Fe bis-glycine chelate has the same regulated pathway of absorption as Fe-AA in Caco-2 cell cultures.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Cells. The Caco-2 cell line (American Type Collection, HTB-37, Rockville, MD) provides a model with which to study intestinal Fe absorption, metabolism and regulation (15) and has been used extensively as an in vitro model to estimate Fe bioavailability (16–19). Caco-2 cells were cultured in DME Medium (Gibco BRL, Carlsbad, CA), 10% fetal bovine serum (FBS), 10 kU/L penicillin/streptomycin and 25 mg/L fungizone. Cells were seeded in 25-cm2 flasks and incubated at 37°C, 5% CO2. After 1 wk, the cells were trypsinized, reseeded at a density of 1 x 105 cells/flask and cultured to reach 80% confluence. FBS, MOPS buffer pH 7.4 (mmol/L: 50 MOPS, 94 NaCl, 7.4 KCl, 0.74 MgCl2, 1.5 CaCl2 and 5 glucose) and PBS were filtered through Chelex-100 (Bio-Rad Laboratories, Hercules, CA) to minimize contamination from Fe (8). Fe concentration was measured in all solutions by atomic absorption spectrometry (Perkin Elmer, SIMAA 6100, Shelton, CT).

    Intrinsic labeling of iron compounds. The day before the uptake experiment, the radioactive Fe compounds were prepared from 59FeCl3/HCl or 55FeCl3/HCl 0.5 mol/L (NEN, Life Science Products, Boston, MA). Fe concentration was estimated from the specific activity supplied by the producer and analyzed in 100 µmol/L Fe solutions. Unlabeled and radioactive stock of FeCl3 solutions were combined in a Fe to ascorbic acid (AA) molar ratio of 1:2. 59Fe bis-glycine chelate (100 µmol Fe/L; 3.7 kBq) and 55Fe bis-glycine chelate (100 µmol Fe/L; 3.7 kBq) were prepared according to Olivares et al. (7). 59Fe bis-glycine or 55Fe bis-glycine chelate intrinsically labeled were diluted to 100 µmol/L Fe with 0.5 mol/L HCl, and then to 1 µmol/L with MOPS buffer.

    Equilibrium Fe loading of Caco-2 cells. Caco-2 cells (1 x 105) from the cultures prepared as above were incubated for 7 d in Iscove’s medium, 10% low-Fe FBS, supplemented with different Fe concentrations (0.5, 5 or 20 µmol/L 55Fe-nitrilotriacetate (NTA) in a 1:2.2 Fe:NTA molar ratio or 55Fe bis-glycine chelate). Equilibrium Fe loading was performed according to Arredondo et al. (8). Inserts were used when they attained stable resistance values between 240 and 280 {Omega} · cm2. Inserts with <240 {Omega} · cm2 were discarded.

    Uptake and transepithelial transports studies in Caco-2 cells. The day of the experiment, Caco-2 cells cultured in inserts as above, were incubated with 700 µL of MOPS and 50 µmol/L desferrioxamine on the basolateral side. Fe uptake was started by the addition of 200 µL of 59Fe-AA or 1 µmol/L 59Fe bis-glycine chelate in MOPS buffer in the apical side with incubation for 5, 15, 30 and 60 min at 37°C. The uptake was stopped by washing the inserts three times with ice-cold 1 mmol/L EDTA in PBS. Cell-associated radioactivity (apical 59Fe uptake and intracellular 55Fe content), apical and basolateral media and apical transport solutions were measured in a liquid scintillation dual channel counter (Beckman LS 5000 TD. Beckman Instruments, Fullerton, CA). Kinetic data were expressed as nmol Fe/(mg protein · h).

    Competition studies of Fe bis-glycine chelate and Fe-ascorbate in Caco-2 cells. Caco-2 cells were seeded onto 24-well tissue culture plates (Costar, MA), as above for 10–12 d. Each well was washed with warmed MOPS buffer. Fe-AA (1 µmol/L), 100 µmol/L Fe-AA or 100 µmol/L Fe bis-glycine chelate uptake was started by the addition of 1 mL of transport solution to each well with incubation at 37°C. Uptake was stopped as above, and a cellular extract was prepared with lysis buffer (mmol/L: 10 Hepes, pH 7.5,3 MgCl3, 40 KCl, 1 phenylmethylsulfonyl fluoride; 1 dithiothreitol, and 10 mg/L leupeptin, 0.5 mg/L aprotinin, 0.7 mg/L pepstatin A, 5% glycerol, 0.5% Triton X-100). Protein determination (20) and cell associated radioactivity were measured in the cellular extract.

    Statistical analysis. Values in the text, table and figures are given as the mean ± SEM of three independent measures. Statistical analysis included two-way ANOVA for repeated measures (main effects tested were Fe concentrations and type of Fe compounds) and Sheffé’s post-hoc test when the F-test was significant (Student’s t test and Pearson correlations). Differences with P < 0.05 were considered significant. Statistical analysis was performed by the program SAS (SAS Online Doc 8.0, SAS Institute, Cary, NC).


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    Intracellular Fe content. Caco-2 cells grown in increasing concentrations of 55Fe bis-glycine chelate or Fe-AA acquired more Fe when the Fe in culture media increased from 0.5 to 20 µmol/L. (P < 0.001; Table 1). The relationship between Fe in culture media and intracellular 55Fe was linear (r = 0.998; P < 0.01), showing that incorporation of Fe into cells continues if available Fe is offered in the apical side. Iron uptake was significantly affected by the interaction between iron concentration and the type of ferrous compound (P < 0.004) but not by the type of ferrous compound per se.


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  		TABLE 1 Intracellular 55Fe content in Caco-2 cells cultured for 2 wk with different types and concentrations of radioactive Fe

 
    Apical iron uptake by Caco-2 cells. In a comparison of the uptakes at different extracellular Fe concentrations, the apical uptake from Fe:AA and Fe bis-glycine chelate did not differ (Fig. 1). In both cases, uptake decreased as the intracellular Fe increased (P < 0.05). In both, there was an inverse relationship between Fe uptake and intracellular Fe (r = -0.89, P < 0.03).



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  		FIGURE 1 Apical 59Fe uptake from Fe:AA and Fe bis-glycine chelate in Caco-2 cells grown in 0.5, 5 and 20 µmol/L Fe. Values are means ± SEM, n = 3. Means without a common letter differ, P < 0.05).

 
    Transepithelial iron transport by Caco-2 cells. Transepithelial transport rates were ~20% of the apical transport rates, consistent with the intracellular handling of Fe in these cells (Fig. 2). Transepithelial transport rates were faster in media with low intracellular Fe concentration and slower in media with high intracellular Fe concentration. At each media iron concentration, transepithelial transport rates did not differ between the two Fe sources. There was a negative correlation between intracellular Fe and transepithelial rates (r = -0.85; P < 0.03), for both Fe bis-glycine chelate and Fe-AA.



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  		FIGURE 2 59Fe transepithelial transport from Fe:AA and Fe bis-glycine chelate and in Caco-2 cells grown in 0.5, 5 and 20 µmol/L Fe. Values are means ± SEM, n = 3. Means without a common letter differ, P < 0.05).

 
    Competition of Fe bis-glycine chelate and Fe-AA for the absorption of 55Fe-ascorbate. Unlabeled Fe, added as Fe bis-glycine chelate or Fe-AA, competed with the labeled pool for incorporation into the cells, as shown by a lower uptake of 55Fe, at each time point measured (Fig. 3). When 1 µmol/L 55Fe-AA was offered to the cell, the uptake rate [92.5 ± 2.5 nmol Fe/(mg protein · h)] was greater than the rates of 10.6 ± 1.1 and 13.4 ± 1.5 in the presence of Fe-AA and Fe bis-glycine chelate, respectively (P < 0.01). The inhibition of unlabeled Fe bis-glycine chelate on the absorption of the extrinsic tag of 55Fe-AA (87.5%) did not differ from that of Fe added as Fe-AA (86.8%). The curves of Fe uptake for Fe bis-glycine chelate and Fe-AA were not different (Fig. 3).



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  		FIGURE 3 Competition of Fe bis-glycine chelate and Fe-AA for the absorption of 1 µmol/L 55Fe-AA in Caco-2 cells. Values are mean ± SEM, n = 3.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
This work was designed to compare Fe bis-glycine chelate and Fe:AA intestinal absorption using Caco-2 cells grown in bicameral inserts. This model allows us to eliminate the effect of some variables such as day-to-day and interindividual variations in gastric and intestinal processes, as well the effects related to interactions with food chelators. The apical Fe uptake from Fe bis-glycine chelate was similar to that of Fe-AA, when Caco-2 cells with similar intracellular Fe content were compared. The similarity of uptake rates suggests a common pathway of incorporation into the cell. One simple hypothesis would be that the Fe in the Fe-glycine complex is incorporated into the absorbable Fe iron pool and transported by DMT1. This seems a likely pathway because Fe is derived from Fe-AA chelate in a similar way.

Iron bis-glycine is well absorbed, as demonstrated by several absorption studies in humans. The bioavailability data give us an estimation of the total amount of Fe absorbed through the enterocyte and incorporated into RBC (4,21). However, absorption studies cannot give a mechanistic explanation of the process of absorption. Our hypothesis to explain the high bioavailability of the compound was that it represents a source of inorganic Fe, and as such, its absorption is regulated by corporal stores. This regulation was demonstrated in humans using a Fe bis-glycine chelate (7). We showed previously (22) that Fe bis-glycine competed for the absorption of ferrous sulfate. Human volunteers were given a labeled dose with 0.5 mg of nonheme Fe, and the absorption of this dose was measured in the same subjects with increasing amounts of unlabeled Fe given as Fe:AA or Fe bis-glycine chelate. The inhibition curves were similar, showing competition from both the nonheme Fe and from Fe bis-glycine chelate. Using this approach, we could not determine whether the competition between Fe bis-glycine chelate and nonheme Fe arose from a full exchange in the intestinal Fe pool or if some Fe escaped from the chelates as a result of hydrolysis.

We first demonstrated that the dose-dependent competition shown in human studies (22) was also observed in the cell model. A baseline level was set by exposing Caco-2 cells grown in normal culture medium to 1 µmol/L Fe-AA labeled with 55Fe. Unlabeled Fe, added as Fe-AA, entered the common nonheme Fe pool and competed for the absorption of the labeled Fe. Fe bis-glycine chelate produced the same inhibiting effect. Thus, Fe bis-glycine chelate, presented intact to the brush border of the enterocyte, was as good a donor of nonheme Fe as Fe-AA. This suggests that Fe bis-glycine chelate also enters the common nonheme Fe pool, and as such, is taken by the cell through the same transporter. We do not yet understand where the Fe2+ breaks from the chelate. However, if the chelate behave similarly to nonheme Fe at the brush border, previous hydrolysis during digestion would not affect the degree of inhibition, as was previously described for the absorption in humans (22).

It is also worthwhile to note that in Fe-replete Caco-2 cells, more Fe was taken up by the cells as more Fe was offered in the apical medium. These cells retain a high capacity for Fe uptake; this points to an important role for them in the transepithelial transport because the regulatory mechanism that prevents Fe overload should include the inhibition of this pathway to lose excess Fe during the epithelial desquamation process. In this experiment, cells were exposed to a constant and high amount of Fe in the apical medium; thus, it is not possible to extrapolate to an in vivo situation. In that case, Fe absorption would clearly depend on other factors such as gastric transit time, solubility of the form of Fe and the presence of inhibitors.

We did not find differences between Fe:AA and Fe bis-glycine chelate uptake and transepithelial transport rates at various intracellular Fe concentrations. As we expected, apical uptake and transepithelial transport rates were higher in Fe-deficient cells and lower in Fe-sufficient and Fe-replete cells.

Better absorption of Fe bis-glycine chelate in diets with a high content of inhibitors can be explained by a protective effect of the glycine moiety, both chelating and protecting Fe from inhibitors of the diet, but keeping it soluble and readily available (7) for incorporation into the cell. Once liberated in the gut, despite different sources, the iron utilizes common pathways for absorption.

In summary, we presented strong evidence indicating that Fe bis-glycine chelate is absorbed as nonheme Fe. Fe bis-glycine chelate competes for the absorption of nonheme Fe from Fe-AA, and its uptake and transepithelial transport follow the same pattern as Fe-AA.


   FOOTNOTES
 
1 Funded in part by grant 1981217 from Fondo Nacional de Ciencia y Tecnología, Chile. Back

3 Abbreviations used: AA, ascorbate/ascorbic acid; DMT1, divalent metal transporter 1; FBS, fetal bovine serum; NTA, nitrilotriacetate. Back

Manuscript received 18 July 2003. Initial review completed 26 August 2003. Revision accepted 27 October 2003.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

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

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

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4. Layrisse, M., García-Casal, M. N., Solano, L., Barón, M., Arguello, F., Llovera, D., Ramírez, J., Leets, I. & Tropper, E. (2000) Iron bioavailability in humans from breakfasts enriched with iron bis-glycine chelate, phytates and polyphenols. J. Nutr. 130:2195-2199.[Abstract/Free Full Text]

5. Bovell-Benjamin, A., Viteri, F. & Allen, L. (2000) Iron absorption from ferrous bis-glycine and ferric trisglycine in whole maize is regulated by iron status. Am. J. Clin. Nutr. 71:1563-1569.[Abstract/Free Full Text]

6. Fox, T., Eagles, J. & Fairweather-Tait, S. (1998) Bioavailability of iron glycine as a fortificant in infant foods. Am. J. Clin. Nutr. 67:664-668.[Abstract]

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8. Arredondo, M., Orellana, A., Gárate, M. & Núñez, M. T. (1997) Intracellular iron regulates iron absorption and IRP activity in intestinal epithelial (Caco-2) cells. Am. J. Physiol. 273:G275-G280.

9. Gárate, M. & Núñez, M. T. (2000) Overexpression of the ferritin iron-responsive element decreases the labile iron pool and abolishes the regulation of iron absorption by intestinal epithelial (Caco-2) cells. J. Biol. Chem. 275:1651-1655.[Abstract/Free Full Text]

10. Gunshin, H., Mackenzie, B., Berger, U. V., Gunshin, Y., Romero, M. F., Boron, W. F., Nussberger, S., Golan, J. L. & Hediger, M. A. (1997) Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature (Lond.) 388:482-488.[Medline]

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

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