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Micronutrient Laboratory and Hematology Unit, Institute of Nutrition and Food Technology (INTA), University of Chile, Santiago, POB 138–11, Chile
2To whom correspondence and reprint requests should be addressed. E-mail: twalter{at}uec.inta.cl.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1%). NaFeEDTA had the highest bioavailability (5.3%). The bioavailability of all iron forms improved significantly when tested with Na2EDTA (<0.05). Adding Na2EDTA to ferrous fumarate increased bioavailability from 0.87% to 2.9% (P < 0.001). We conclude that NaFeEDTA is the form of iron best absorbed, but alternatively, ferrous fumarate plus Na2EDTA comprises a feasible option as a fortificant.
KEY WORDS: • iron fortification • iron bioavailability • corn-masa flour • NaFeEDTA • Na2EDTA
Iron fortification of foods continues to be one of the preferred ways of improving the iron status of the population. When intended for the population at large, this usually involves the fortification of cereal flours. In the case of Mexico and most of Central America, industrially processed, precooked, lime-treated (nixtamalized) corn-masa flour is undoubtedly the ideal vehicle for this purpose. The average consumption of this staple by the general population is 200 g/d. The poorer sectors of the community commonly consume >300 g/d, comprising 80% of their energy intake (1).
Once the vehicle has been chosen, the selection of the fortificant is a critical issue. Corn-masa flour contains an abundance of potent inhibitors of iron absorption mainly in the form of calcium and 
550 mg/100 g of phytic acid (2–4). Iron absorption from maize has been shown to be lower than absorption from other cereals (5,6). This lack of absorption and the rigorous preparation of tortillas that includes kneading and grilling for several minutes at 160–180°C on a hot steel plate must be evaluated in human bioavailability studies. The development of iron chelates with glycine (ferrous bis-glycinate), iron-sodium EDTA (NaFeEDTA, also called iron-EDTA) and others such as Na2EDTA, may improve iron bioavailability; these chelates have been tested favorably in other matrices (7,8). Assays of solubility were done in vitro for preliminary selection of iron forms and other combinations. The present study was designed to identify the best fortificant to overcome the inhibitory effects of corn-masa flour.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Three groups of women (n = 15/group) were invited to participate in separate studies. They were multiparous, used intrauterine anticonceptive devices and tested negative in a pregnancy test. Their median age was 40 y and ranged from 31 to 50 y (Table 1). Serum ferritin concentrations ranged from 6 to 150 µg/L. A physician ascertained that they were clinically healthy. They were informed orally and in writing about the purpose and risks of the study, signed informed consent agreements and were compensated for their transportation, meals and for their participation (5 d of work). The protocol was accepted by INTA’s Institutional Review Board for human experimentation and the Chilean Nuclear Energy Commission approved the radioiron doses.
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The addition of iron was designed to provide native iron (15 mg/kg) from corn-masa flour plus the added 30 mg elemental iron/kg of flour, resulting in 45 mg total iron/kg of flour. Ferrous fumarate was tested with and without Na2EDTA at a 2:1 mol/kg iron:EDTA ratio. Absorption from the native iron in the corn-masa flour was measured alone and with Na2EDTA in a meal with 90 g of refried black beans, 100 g of white polished rice containing an additional 2 mg of iron plus 5 mL of a commercial chili sauce. Native iron enters the iron pool freely (9,10), exchanging fully with the radioiron tag. Bis-glycinate iron was labeled intrinsically with radioiron with reagents following instructions from Albion Laboratories (Clearfield, UT). To allow for valid comparisons between subjects in separate experiments, bioavailability was normalized to 40% absorption of the reference dose of ferrous ascorbate (3 mg of 55Fe ferrous sulfate and ascorbic acid at a 1:2 mol/L ratio). All of these bioavailabilities were established using double radioactive iron isotopes as described by Eakins and Brown (11). In brief, after consumption of the test meals on d 1 using tracers (Du Pont, Wilmington, DE) of 59Fe Cl3 (37 kBq) and on d 2 with 55FeCl3 (111 kBq), a third test meal with 59FeCl3 (37 kBq) was given on d 15. Finally, the reference dose of 55ferrous ascorbate (111 kBq) was administered on d 16. On d 15 and 29, venous blood samples were obtained to determine iron status measures and the radioactivity incorporated into erythrocytes. Duplicates of 10-mL blood samples and triplicate aliquots of the labeled tortillas and reference dose ingested were prepared for counting as controls. The activity of radioisotopes in the processed samples was determined using a liquid scintillation counter (Beckman Model LS 5000 TD, Chicago, IL). Calculations assumed that 80% of the absorbed radioactivity would be incorporated into the hemoglobin of circulating erythrocytes. Blood volume was estimated on the basis of sex, weight and height (12). Hemoglobin and mean cell volumes were determined with a Cell-Dyn Model 1700 (Abbott Diagnostics, Abbott Park, IL). Serum iron and iron binding capacity were measured by the Fischer and Price technique (13). Erythrocyte protoporphyrin was measured with a hematofluorometer (Helena Laboratories, Beaumont, TX). Serum ferritin was quantified using an ELISA technique (14).
In vitro studies.
To determine the extent of dialysis equilibrium solubility, the fortificant-vehicle preparations were subjected to conditions simulating the gastrointestinal environment during digestion and absorption. This estimated the potential extent of their participation in the "common pool" of nonheme iron in the diet. These data are useful for comparing fortificants and allow the selection of the best mixtures for human studies, thus saving resources. We used a procedure of Kapsokefalau and Miller (15) with modifications. In brief, previously minced fortified tortilla and pepsin were digested enzymatically (pH 2.0) by continuous agitation in a water bath at 37°C for 2 h, mimicking the gastric contents. Then the conditions in the duodenum were met by a change to alkaline pH 6–7 and the addion of pancreatin and bile for 2 more hours (all reagents obtained from Sigma, St. Louis, MO). At this time, a dialysis bag with a cut-off of 6000–8000 (Spectra/molecular porous membrane tubing MWCO 6000–8000, Spectrum, Gardena, CA) filled with HEPES buffer was placed in the reaction flask and stirred with the digest. After equilibrium was reached, the amount of Fe transferred into the bags was measured by atomic absorption spectrometry (Perkin-Elmer model 2280, Chicago, IL) This soluble, dialyzable iron was expressed as a percentage of total iron. We measured the dializability of elemental reduced iron, a commonly used fortificant; however, because it cannot be labeled radioactively, it could not be tested for bioavailability in humans.
Preparation of meals for bioavailability studies.
Each of the three groups of subjects were given three test meals and a reference dose. Three of the meals were used for a different purpose; thus we present only six in this study. Meals tested are those numbered 1–6 and are detailed in Table 2. All meals contained native iron (15 mg/kg) from corn-masa flour plus the added 30 mg elemental iron/kg of flour except meal 1, which contained native iron only, and meal 5, which contained native iron plus Na2EDTA and frijoles, rice and chili, thereby contributing 2 mg of nonheme iron. The tortillas were prepared with fresh, industrial, precooked, nonfortified, nixtamalized corn-masa flour provided by MINSA S.A de C.V. Mexico, D.F. In brief, 1000 g of flour was placed in a stainless steel mixing drum to which the fortificants were added and mixed for 1 h; they were then placed in a bowl to which 1200 mL of deionized water was added. At this point radioisotopes were added diluted in 0.1 mol/L HCl. Kneading was performed until the dough was sufficiently consistent and elastic, after which it was allowed to rest for 20–30 min. Then, 30-g portions were patted and grilled on a griddle for 1 min per side and for another minute to seal. The women consumed three tortillas at each test meal. Tortillas from the same batch were used as standards and controls of radioiron measurements.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Native iron (meal 1), fumarate (meal 2) and bis-glycinate iron (meal 5) had low bioavailabilities, ranging from 0.69 to 1.27%. These were lower than those from the same meals with Na2EDTA (P < 0.05). When Na2EDTA was added to fumarate in a 2:1 mol/L ratio of iron:EDTA (meal 4), its bioavailability increased from 0.87 to 2.98% (P < 0.001). Similarly, native iron absorption rose upon addition of Na2EDTA in a 2:1 mol/L ratio from 0.69% (alone in meal 1) to 3.19% (in meal 5 with black beans, rice and chili), a 3.6-fold increase (P < 0.001). The latter occurred even in the presence of black beans, which contributed to the inhibitors and the quantity of iron. Thus, the addition of Na2EDTA to the corn-masa flour increased bioavailabilities in all meals tested (P < 0.05). The greatest bioavailability (5.3%) occurred with NaFeEDTA (meal 6), which was >4 times that of any form of iron without EDTA (P < 0.001); however, it was not higher than fumarate or native iron plus Na2EDTA (Table 2).
In vitro studies.
Dializability of iron ranged from 1 to 19.4%, with the lowest values associated with ferrous sulfate, ferrous fumarate, bis-glycinate, all without Na2EDTA, and the highest with NaFeEDTA. Solubilities for reduced iron alone, reduced iron-Na2EDTA, ferrous fumarate Na2EDTA and native iron plus Na2EDTA were intermediate at 8.8, 15.3, 10.2 and 18.2%, respectively. Due to these results, we pursued ferrous sulfate no further because it negatively affects organoleptic characteristics and is not a realistic option for fortification. Native iron from corn-masa flour had a solubility of 1.4%, but upon addition of Na2EDTA, it increased to 18.2%. Elemental reduced iron was not tested alone but, in combination with Na2EDTA, solubility ranged from 8.8 to 15.3%. The human bioavailability results closely paralleled the ranks obtained in the dialysis studies. The solubility in vitro and the bioavailability in vivo were highly correlated (r = 0. 89, P < 0.001). The correlation between bioavailability of the reference dose and serum ferritin concentration was weaker (r = -0.50, P < 0.05).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1%. All of the forms of iron tested (except ferrous sulfate, excluded from human studies) in the presence of Na2EDTA in molar ratios of iron:EDTA of 2:1 increased their bioavailability 2–4 times. Iron-EDTA had the highest bioavailability (5.3%), similar to recent studies in nixtamalized corn porridge (16) perhaps because it is the product with the highest molar ratio of iron to EDTA. Fumarate-Na2EDTA is also a reasonable alternative at an absorption of nearly 3%, which is not lower than that of NaFeEDTA. Ferrous bis-glycinate is a molecule that allegedly decreases inhibitory interactions. We and others have established that ferrous bis-glycine in water and cereals is absorbed similarly to ferrous sulfate (17) and is inhibited by phytates to the same degree (18). These observations prompted our studies presented here showing disappointingly low bioavailability in humans in agreement with the solubility results.
Another interesting chelate is iron-EDTA (NaFeEDTA). It should be most efficacious when added to highly inhibitory foods such as corn-masa flour due to the properties discussed below. The JECFA (Joint Expert Committee for Food Additives of FAO-WHO) recently concluded that NaFeEDTA is acceptable as long as it is used in supervised food fortification programs in iron-deficient populations (19). This approval has cleared the way for large-scale fortification programs. A comprehensive report on the properties of iron EDTA has been issued under the auspices of The International Nutritional Anemia Consultative Group (7). Several intervention studies, although limited and short-lived, demonstrated improved iron bioavailability of fish sauce in Thailand (20), sugar in Guatemala (21) and curry powder in South Africa (22) with NaFeEDTA fortification.
Like ascorbic acid, disodium EDTA (Na2EDTA) may combine with the iron fortificant, thereby enhancing bioavailability, with the advantage that it is stable during storage and processing. It is best when added in lower than 1:1 mol/L ratios to iron in the meal. El-Guindi and Lynch (23) added equimolar quantities of ferrous sulfate and Na2EDTA to Egyptian Baladi bread, thereby increasing absorption from 2.1 to 5.3%. Studies in South Africa (24,25) showed that adding Na2EDTA to a rice meal containing ferrous sulfate increased absorption significantly. They used iron to EDTA ratios of 4:1 to 1:1, with the best effect obtained at a ratio of 2:1, similar to what we used in this study
Most of the previous iron bioavailability studies of nonnixtamalized corn flour have been performed with ferrous sulfate in a porridge; absorption averaged 3.7% in iron-sufficient individuals (26–28). Lower absorptions occurred in a recent study with nixtamalized corn porridge (1.1–1.9%) (16). Only three studies examined tortillas prepared with nixtamalized corn flour in a meal with beans and rice characterized as a typical Mexican combination (1,27,29). In the sudy of Hallberg and Rossander (27), the addition of 50 mg of ascorbic acid increased iron absorption twofold; however, the addition of ascorbic acid to a fortification mix is impracticable. In the second study (1), ferrous sulfate in a tortilla prepared with whole maize flour and consumed with frijoles and rice had a bioavailability of 2.8 ± 1.2%, i.e., in the same range as found in our study. A more recent study in 12- to 13-y-old girls (29) showed bioavailabilities of 5.5% for ferrous sulfate and 5.5–6.2% for ferrous fumarate, both as stable isotopes. Inexplicably, the addition of Na2EDTA in a 1:1 mol/L ratio did not improve the absorption of iron in the ferrous fumarate–fortified tortillas, whereas in the case of ferrous sulfate, Na2EDTA, it did raise iron bioavailability. Again, a reference dose was not used, precluding direct comparisons with our data. As in our study, NaFeEDTA consistently showed the highest bioavailability (9%). In Guatemala, Davidsson et al. (29) added iron stable isotopes to the tortillas after they were grilled. In contrast, we added the Na2EDTA during the flour mixing step, as would occur in a commercial mill. It is conceivable that most of the interaction between Na2EDTA and iron occurred in the stage at which the flour is humidified, kneaded and allowed to rest 20–30 min before grilling.
The use of NaFeEDTA has raised concern due to potential interference with absorption of water-soluble minerals or a greater loss of these minerals bound to the EDTA molecule. However, adult women consuming wheat rolls fortified with NaFeEDTA had almost doubled zinc absorption with no effect on calcium or other heavy metals (30,31). On the other hand, countries using large amounts of EDTA calcium or sodium salts as preservatives in foods for many years have not reported any evidence of toxicity. There are no restrictions by JECFA on the use of sodium or calcium EDTA except for the total EDTA dose per person of 2.5 mg/(kg · d), a figure difficult to reach, particularly in developing countries where the use of these preservatives is relatively low.
Despite the above considerations, the most widely used fortificant is elemental iron, which cannot be evaluated with these methods. Stable isotopes of reduced iron may eventually clarify this issue.
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FOOTNOTES |
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Manuscript received 14 April 2003. Revision accepted 26 June 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Fossi, M., Mendez-Castellano, H., Jaffe, W. G., Martinez-Torres, C., Leets, I., Taylor, P. & Layrisse, M. (1987) Hematologic profile and absorption of iron from diets consumed by a population of low socioeconomic level of 2 Venezuelan states. Arch. Latinoam. Nutr. 37:23-35.[Medline]
2. Reddy, M. B., Hurrell, R. F. & Cook, J. D. (2000) Estimation of nonheme-iron bioavailability from meal composition. Am. J. Clin. Nutr. 71:937-943.
3. Hurrell, R. F., Juillerat, M. A., Reddy, M. B., Lynch, S. R., Dassenko, S. A. & Cook, J. D. (1992) Soy protein, phytate, and iron absorption in humans. Am. J. Clin. Nutr. 56:573-578.
4. Cook, J. D., Reddy, M. B., Burri, J., Juillerat, M. A. & Hurrell, R. F. (1997) The influence of different cereal grains on iron absorption from infant cereal foods. Am. J. Clin. Nutr. 65:964-969.
5. Martinez-Torres, C. & Layrisse, M. (1974) Interest for the study of dietary absorption and iron fortification. World Rev. Nutr. Diet. 19:51-70.[Medline]
6. International Nutritional Anemia Consultative Group (INACG) (1982) The Effects of Cereals and Legumes on Iron Availability 1982:23-38 The Nutrition Foundation/ILSI Washington, D.C.
7. International Nutritional Anemia Consultative Group (INACG) and INAC Group (1993) Iron EDTA for Food Fortification. 1993 The Nutrition Foundation/ILSI Washington, D.C.
8. Hurrell, R. F., Reddy, M. B., Burri, J. & Cook, J. D. (2000) An evaluation of EDTA compounds for iron fortification of cereal-based foods. Br. J. Nutr. 84:903-910.[Medline]
9. Cook, J., Layrisse, M., Martínez-Torres, C., Monsen, E. & Finch, C. (1972) Food iron absorption measured by an extrinsic tag. J. Clin. Investig. 51:805-815.
10. Layrisse, M., Martinez-Torres, C., Cook, J. D., Walker, R. & Finch, C. A. (1973) Iron fortification of food: its measurement by the extrinsic tag method. Blood 41:333-352.
11. Eakins, J. D. & Brown, D. A. (1966) An improved method for simultaneous determination of iron-55 and iron-59 in blood by liquid scintillation counting. Int. J. Appl. Radiat. Isot. 17:191-197.
12. Nadler, S., Hidalgo, I. & Block, T. (1962) The Tulane table for Blood volume in normal men. Surgeon 51:224-232.
13. Fischer, D. S. & Price, D. C. (1964) A simple serum iron method using the new sensitive chromogen tripyridyl-triazine. Clin. Chem. 10:21-31.[Abstract]
14. Arredondo, M., Pizarro, F., Walter, T. & Hertrampf, E. (1991) Determinacion de ferritina serica por ELISA. Rev. Chil. Nutr. :20-24.
15. Kapsokefalou, M. & Miller, D. D. (1991) Effects of meat and selected food components on the valence of nonheme iron during in vitro digestion. J. Food Sci. 56:352-358.
16. Mendoza, C., Viteri, F. E., Lonnerdal, B., Raboy, V., Young, K. A. & Brown, K. H. (2001) Absorption of iron from unmodified maize and genetically altered, low-phytate maize fortified with ferrous sulfate or sodium iron EDTA. Am. J. Clin. Nutr. 73:80-85.
17. Olivares, M., Pizarro, F., Pineda, O., Name, J. J., Hertrampf, E. & Walter, T. (1997) Milk inhibits and ascorbic acid favors ferrous bis-glycine chelate bioavailability in humans. J. Nutr. 127:1407-1411.
18. Fox, T. E., Eagles, J. & Fairweather-Tait, S. J. (1998) Bioavailability of iron glycine as a fortificant in infant foods [see comments]. Am. J. Clin. Nutr. 67:664-668.[Abstract]
19. FOA/WHO (1999) 1999 Joint FAO/WHO Expert Committee on Food Additives Rome, Italy.
20. Garby, L. & Areekul, S. (1974) Iron supplementation in Thai fish-sauce. Ann. Trop. Med. Parasitol. 68:467-476.[Medline]
21. Viteri, F. E., Alvarez, E., Batres, R., Torun, B., Pineda, O., Mejia, L. A. & Sylvi, J. (1995) Fortification of sugar with iron sodium ethylenediaminotetraacetate (FeNaEDTA) improves iron status in semirural Guatemalan populations [see comments]. Am. J. Clin. Nutr. 61:1153-1163.
22. Ballot, D., MacPhail, A., Bothwell, T., Gillooly, M. & Mayet, F. (1989) Fortification of curry powder with NaFe(III)EDTA: report of a controlled iron fortification trial. Am. J. Clin. Nutr. 49:162-169.
23. El-Guindi, M., Lynch, S. & Cook, J. (1988) Iron fortification from fortified flat bread. Br. J. Nutr. 59:205-213.[Medline]
24. MacPhail, A. P., Bothwell, T. H., Torrance, J. D., Derman, D. P., Bezwoda, W. R., Charlton, R. W. & Mayet, F. (1981) Factors affecting the absorption of iron from Fe(III)EDTA. Br. J. Nutr. 45:215-227.[Medline]
25. MacPhail, A. P., Patel, R. C., Bothwell, T. H. & Lamparelli, R. D. (1994) EDTA and the absorption of iron from food. Am. J. Clin. Nutr. 59:644-648.
26. Bjorn-Rasmussen, E. & Hallberg, L. (1974) Iron absorption from Maize: Effect of ascorbic acid on iron absorption from maize supplemented with ferrous sulfate. Nutr. Metab. 23:192-202.
27. Hallberg, L. & Rossander, L. (1984) Improvement of iron nutrition in developing countries: comparison of adding meat, soy protein, ascorbic acid, citric acid, and ferrous sulfate on iron absorption from a simple Latin American-type of meal. Am. J. Clin. Nutr. 39:577-583.
28. Martinez-Torres, C., Romano, E. L., Renzi, M. & Layrisse, M. (1979) Fe(III)-EDTA complex as iron fortification. Further studies. Am. J. Clin. Nutr. 32:809-816.
29. Davidsson, L., Dimitriou, T., Boy, E., Walczyk, T. & Hurrell, R. F. (2002) Iron bioavailability from iron-fortified Guatemalan meals based on corn tortillas and black bean paste. Am. J. Clin. Nutr. 75:535-539.
30. Davidsson, L., Kastenmayer, P. & Hurrell, R. F. (1994) Sodium iron EDTA [NaFe(III)EDTA] as a food fortificant: the effect on the absorption and retention of zinc and calcium in women. Am. J. Clin. Nutr. 60:231-237.
31. Davidsson, L., Almgren, A. & Hurrell, R. F. (1998) Sodium iron EDTA [NaFe(III)EDTA] as a food fortificant does not influence absorption and urinary excretion of manganese in healthy adults. J. Nutr. 128:1139-1143.
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