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Centro de Investigación en Nutrición y Salud, Instituto Nacional de Salud Pública, Cuernavaca, Morelos, México;
* U.S. Department of Agriculture Western Human Nutrition Research Center, University of California Davis, Davis, CA; and
Children’s Hospital Research Center at Oakland, Oakland, CA
2To whom correspondence should be addressed. E-mail: chotz{at}insp.mx.
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ABSTRACT |
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 TOP ABSTRACT SUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • zinc • absorption • maize • fortificants • stable isotopes
In recent years, the importance of zinc for human health and development (1–3) and the possible widespread occurrence of suboptimal zinc intakes (3,4) have gained recognition. The fortification of staple foods with zinc may play an important role in achieving adequate zinc intakes in at-risk populations. Although the relative absorption of iron varies substantially depending on the chemical form of the iron compound used as a fortificant (5), the relative bioavailability of different zinc compounds that may be used in food fortification is not well known, and hence the efficacy of such compounds to improve zinc status.
Two of the most commonly used zinc fortificants to date are zinc oxide and zinc sulfate (6,7). A few earlier studies compared the absorption of zinc between these chemical forms using the oral zinc tolerance test as a measure of bioavailability, but the results were conflicting (8,9). One of those studies reported a much lower plasma appearance from zinc oxide preparations compared with zinc sulfate (9), whereas the other reported no difference (8). A more recent study used stable isotope tracer techniques to measure zinc absorption in a group of Indonesian children; in that study, wheat flour dumplings were fortified with either zinc oxide or zinc sulfate (10) and zinc absorption did not differ between groups.
EDTA is a metal-chelating compound that may facilitate the absorption of nutritionally important minerals by preventing their binding with other compounds that inhibit mineral absorption (e.g., phytate). Various studies showed that iron is better absorbed from NaFeEDTA than from other compounds (11–14). It is also possible that EDTA would have an enhancing effect on zinc bioavailability, as was shown previously in some animal studies (15,16). To date, there are no reports from human studies of the effect of EDTA compounds in improving the absorption of zinc fortificants added to staple foods.
In Mexico, biochemical evidence suggests that the risk of zinc deficiency is high; approximately one third of women and preschool children have low serum zinc concentrations (<9.9 µmol/L) (17). The voluntary fortification of maize flour with zinc oxide and other nutrients was initiated through an agreement between the Mexican federal government and the major maize flour manufacturers (18). The effectiveness of zinc-fortified maize to prevent zinc deficiency may be improved if zinc compounds other than zinc oxide are found to be more efficiently absorbed from maize-based meals. Therefore, the objective of this study was to compare the absorption of zinc from zinc oxide, zinc oxide plus EDTA, prechelated sodium-zinc EDTA, and zinc sulfate, when added to maize flour and used to prepare a typical Mexican, maize-based meal.
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SUBJECTS AND METHODS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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5 and 
25 µg/L) were invited to participate. Although iron status is not expected to affect zinc absorption, we selected nonanemic women with low iron stores because we planned to repeat the study measuring iron absorption from iron and zinc–cofortified diets, in which case it would be desirable to limit the range of iron status among participants. Details of the study and requirements for participation were explained to the women and written consent was obtained from those who volunteered. Human subjects’ approval for this study was obtained from the Ethics Committee of the Instituto Nacional de Salud Pública (INSP; Cuernavaca, Morelos, Mexico) and from the University of California Davis Institutional Review Board (Davis, CA).
It was initially estimated that a group difference of 8% zinc absorption could be detected with 15 subjects/group, based on previous studies (19,20) (2-tailed test, SD = 10.5, 
= 0.05, 80% power). However, a lower SD was observed for the first subjects completing this study in 2 groups (ZnO and ZnO + EDTA), indicating that fewer subjects were needed.
Preparation of isotopes and study diet. We used the previously validated double isotopic tracer ratio method to measure fractional zinc absorption (21). Zinc oxide, highly enriched with 70Zn, was purchased from Oak Ridge National Laboratory (88.2% enrichment) and from ISOFLEX USA (91.2% enrichment). The orally administered 67Zn compounds were all prepared in powdered form; 67ZnO was purchased from Trace Sciences International (88.6% 67Zn enrichment) and the 67ZnSO4 and 67Na2ZnEDTA were purchased from ISOFLEX USA.
Nonfortified maize flour was obtained from a local producer (Maseca) and was mixed with water to form a soft dough. Individual oral doses of the 67Zn-enriched powders were weighed accurately (±0.03 mg) on a microbalance (Sartorius Micro M3P) and placed in the center of 4-g dough balls, which were folded and stored frozen until use. For the group administered ZnO + Na2EDTA, 2.71 mg of Na2EDTA (Sigma Chemical) was added to the dough ball to achieve a 0.5:1 mol/L ratio of EDTA:Zn. Additional doses of each isotope were also weighed out and used to verify the zinc content.
For the test diet, the maize flour was mixed with water, and tortillas were formed and cooked on a nonstick pan. For the tortilla that was to include the isotope, the labeled dough ball was thawed slightly, placed in the center of a fresh dough ball, and made into a tortilla. The test meal also included cooked yellow beans, a tomato and chili sauce, and milk with instant coffee (Table 1). These foods were chosen because they are representative of a typical rural Mexican meal (17). The quantity of zinc added as 67Zn enriched powder to the flour was equivalent to adding fortificant at a concentration of 40 mg zinc/kg flour. The test meal, including the added zinc, would provide about one third of the current U.S. Recommended Dietary Allowance of zinc for adult women (8 mg/d) (22).
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Sample processing and analysis. The urine samples were purified using ion exchange columns (24), and isotopic ratios were measured by inductively coupled plasma MS using the methods and equipment described by Pinna et al. (25). The fractional absorption of the 67Zn dose from the test meal was calculated by the tracer-to tracee method (21,24). If the intrasubject CV for these 3 data points was >20%, purification and enrichment analysis was repeated for urine samples with aberrant results, or the aberrant point was removed. The overall dose of zinc from the 67Zn enriched compounds was (mean ± SD) 0.96 ± 0.05 mg as determined by analysis of the duplicate doses.
Biochemical analyses. Serum zinc was measured by flame atomic absorption spectrophotometry (AAnalyst 300, Perkin-Elmer) and hemoglobin was measured using a portable Hemocue. Serum ferritin and C-reactive protein were measured by nephelometry following specifications by the equipment manufacturer (Nephelometer 100, Dade-Behring).
Data management and statistical analysis. Statistical analyses were performed using SPSS for Windows version 10.1. Data are presented as means ± SD, unless indicated otherwise. Differences among the 4 study groups for baseline characteristics and the percentage absorption of zinc were determined by ANOVA and Tukey’s post hoc analysis; the analyses of serum zinc and serum ferritin at baseline were controlled for C-reactive protein. Associations between study variables were tested using Pearson correlations. Group differences or associations were considered to be significant when P < 0.05.
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RESULTS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The estimated, daily intakes of energy, zinc, iron, and phytate of the subjects, as derived from the FFQ, were [median (25th, 75th percentile)] 7.5 (5.8, 9.4) MJ/d, 8.9 (6.7, 10.9) mg zinc/d, 11.2 (9.3, 14.0) mg iron/d, and 1564 (1150, 2000) mg phytate/d, respectively, and the phytate:zinc molar ratio was 17. Baseline characteristics of participants did not differ among study groups except for serum ferritin concentration, which was significantly higher in the zinc oxide + EDTA group than in the zinc sulfate group (Table 2).
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0.05) to detect a difference in fractional absorption of zinc of 0.016 (i.e., 1.6% absorption) or less between any 2 test groups. Also, there was no significant correlation between any biochemical indicator and fractional zinc absorption. |
DISCUSSION |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Our results comparing the absorption of zinc from tortillas fortified with either zinc oxide or zinc sulfate are consistent with results of similar studies reported since we initiated the present study. A study by Lopez de Romaña et al. (28) measured zinc absorption from both bread and porridge prepared from wheat using a 65Zn label and whole-body counting. Zinc absorption did not differ between bread fortified with either zinc oxide or zinc sulfate (14 vs. 14%, respectively) or between porridge fortified with either zinc oxide or zinc sulfate (6 vs. 7%, respectively). Preliminary results reported by Rosado et al. (7) indicated no difference in zinc absorption from tortillas fortified with zinc oxide (37%) or zinc sulfate (37%) using a single zinc stable isotope method and fecal monitoring. Herman et al. (10) also reported no difference in zinc absorption by children from wheat dumplings fortified with either zinc oxide (24%) or zinc sulfate (24%).
The consistency among the above-mentioned studies strongly suggests that the water-insoluble zinc oxide is not absorbed to a lesser degree than the water-soluble zinc sulfate, when added to staple food products. Because zinc oxide is soluble in dilute acid, it likely dissolves in the hydrochloric acid secretions of the stomach. One study demonstrated that the intestinal absorption of zinc from zinc oxide was reduced with an experimental reduction in gastric acid secretion and a resultant increased pH (5), compared with that at the lower, normal pH of the stomach (29). No such inhibition by reduced stomach acidity was found with water-soluble zinc acetate (29). It was therefore suggested that there may be an advantage to using zinc sulfate as a fortificant among populations in which hypochlorydria, a condition that occurs frequently among the elderly and with some bacterial infections, is widespread. Therefore, although it appears that either zinc oxide or zinc sulfate can be recommended for food fortification programs among healthy populations, further epidemiologic research is required to determine the extent of hypochlorydria and whether it poses a concern with regard to selection of a soluble zinc salt over zinc oxide.
EDTA appeared to confer no advantage to the absorption of zinc from the test meals in this study. This is in contrast to the ample evidence that EDTA enhances the absorption of iron in humans (11–14). The discrepancy may be related to differences in stability constants and optimal pH for the binding of different minerals with EDTA. For example, EDTA complexes of Fe3+ and Cu2+ have a higher stability constant than with Zn2+. However, stability of mineral chelation with EDTA is also dependent on pH. It is possible that at the pH (6–7) of the upper small intestine, where the majority of mineral absorption occurs, EDTA may chelate more readily with Ca2+, whose optimum pH for chelation is 7.5, than with Zn2+, whose optimum pH for chelation is 4.0 (30). Because our test meal included milk and a total of 345 mg of calcium compared with only 3.3 mg of zinc, preferential binding with calcium may have occurred. On the other hand, a study of zinc and calcium absorption among women demonstrated an increase in zinc absorption, but no change in calcium absorption, from a test diet when bread was fortified with iron EDTA (31). The interaction of EDTA with minerals and its ultimate effect on intestinal mineral absorption is complex and will depend on the concentration of other ions, other competing mineral ligands, as well as pH. The effect of EDTA on zinc fortificant absorption may thus be dependent on the composition of the diet.
Some studies showed that high ratios of EDTA:zinc may have an inhibitory effect on zinc absorption, such that the enhancing effect diminishes at higher EDTA:zinc molar ratios. The inhibitory effect shown by Hempe and Cousins (32) in their rat study occurred with an EDTA:zinc ratio of 0.43:1. On the other hand, Hurrell et al. (16) did not observe an inhibitory effect of EDTA on zinc retention in rats with EDTA:zinc molar ratios of up to 37:1. Solomons et al. (33) suggested that the inhibitory effect of EDTA on zinc absorption from aqueous solutions occurred when the EDTA:zinc ratio was > 0.33:1, as determined by oral zinc tolerance tests. However, in the latter study, EDTA was provided in the form of iron-EDTA and all tests were done using aqueous solutions with pharmacologic doses; thus, the nature of the inhibitory effect is difficult to interpret. In our study the EDTA:zinc molar ratio of 0.5:1 in the test diet did not appear to inhibit zinc absorption. Dose-response trials of increasing EDTA:zinc molar ratios on zinc absorption in humans using isotopic tracer techniques and standard test meals would be useful to further understand this relation.
In conclusion, our study suggests that zinc oxide and zinc sulfate are equally well absorbed when added as fortificants to a maize-based diet and that EDTA did not increase zinc absorption in this diet type. The possibility that EDTA could enhance zinc absorption in other diet types requires further investigation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received 11 August 2004. Initial review completed 12 September 2004. Revision accepted 8 February 2005.
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LITERATURE CITED |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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