The Journal of Nutrition Vol. 128 No. 7 July 1998, pp. 1139-1143

Sodium Iron EDTA [NaFe(III)EDTA] as a Food Fortificant Does Not Influence Absorption and Urinary Excretion of Manganese in Healthy Adults¹

Lena Davidsson², Annette Almgren^*, and Richard F. Hurrell

Laboratory for Human Nutrition, Swiss Federal Institute of Technology, ETH Zürich, CH-8803 Rüschlikon, Switzerland and ^* Department of Clinical Nutrition, Gothenburg University, Annedalsklinikerna, S-413 45 Göteborg, Sweden

	ABSTRACT

Abstract Introduction Methods Results Discussion References

NaFe(III)EDTA is a promising iron (Fe) compound for food fortification programs because of its high Fe bioavailability from meals containing dietary inhibitors of Fe absorption such as phytic acid. However, this Fe compound is not currently used in any large-scale fortification program because of concern over its possible negative influence on the metabolism of other essential minerals or its possible influence on the absorption of potentially toxic elements, such as manganese (Mn). In this study, Mn absorption and urinary excretion were studied in adults after intake of an Fe-fortified weaning cereal labeled with ⁵⁴Mn. In a crossover design, the fortification of the weaning cereal with Fe as NaFeEDTA was compared with ferrous sulfate. Manganese absorption was measured by extrapolation from whole-body retention data 10-30 d after intake, and urinary excretion of ⁵⁴Mn was measured over 7 d. No significant differences in ⁵⁴Mn absorption or urinary excretion were found; 1.1 ± 0.15 and 0.91 ± 0.35% of the ingested dose was absorbed from the cereal fortified with NaFe(III)EDTA and FeSO₄, respectively. Urinary excretion of ⁵⁴Mn was very low; the total radioactivity in urine represented 1.1 ± 0.55% of the absorbed dose with NaFe(III)EDTA and 0.72 ± 0.53% of the absorbed dose with FeSO₄. Until now, Fe-fortification programs have met with only limited success. The introduction of NaFeEDTA as a food fortificant could be a useful tool to provide bioavailable Fe to vulnerable groups in the population and thus aid in combating Fe deficiency.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Iron (Fe) deficiency is one of the major nutritional problems in the world among women of childbearing age, children and infants (Bothwell et al. 1979). The major causative factor in many developing countries is the poor bioavailability of Fe in the mainly cereal-based diets (Charlton and Bothwell 1983, Layrisse et al. 1990). One way to increase the Fe intake in the population is via Fe fortification of commonly consumed foods (Cook and Reusser 1983, INACG 1977). The main advantage of food-fortification programs, compared with supplementation with medicinal Fe, is that they do not require any compliance by the consumer and that they are relatively cheap to install and maintain. Industrially produced infant foods, e.g., infant formulas and cereals, are commonly Fe-fortified and staple foods, such as wheat flour, have been used as a vehicle for Fe fortification in several countries.

Bioavailability is a major concern related to Fe-fortification programs is that some of the Fe compounds added to foods have a low bioavailability, and technological problems occur during storage of fortified products containing more soluble and more bioavailable Fe compounds (Hurrell et al. 1989, Hurrell 1992). Inert Fe compounds, such as ferric pyrophosphate and elemental Fe, do not lead to organoleptic problems such as rancidity and color changes in the products, but these compounds are usually poorly absorbed and would not be expected to improve the Fe status of the consumers. An additional problem is that the bioavailability of Fe compounds used to fortify foods is dependent on the presence of enhancers and inhibitors in the diet. Thus Fe absorption even from Fe salts that are potentially highly bioavailable can be low from products based on cereals or legumes due to the presence of dietary inhibitors such as phytic acid. Ascorbic acid can be added as an absorption enhancer (Davidsson et al. 1994a), but this vitamin is subject to considerable losses during processing and storage of foods, especially in the hot and humid climate of many developing countries.

An Fe compound that has a high bioavailability and whose absorption is not susceptible to the negative effects of inhibitory ligands would be an ideal way of administering Fe via foods. To date, only one compound fulfilling these criteria has been proposed for food fortification programs, NaFe(III)EDTA [International Nutritional Anemia Consultative Group (INACG)³ 1993]. However, the use of NaFe(III)EDTA is currently restricted to experimental trials because of concern over its possible negative influence on the metabolism of other essential minerals such as zinc, copper, magnesium and calcium and its influence on the absorption of toxic or potentially toxic elements, e.g., manganese (Mn), lead, cadmium or aluminum. Clearly, these issues must be carefully studied before recommendations on the use of NaFe(III)EDTA can be given.

The aim of this study was to investigate the influence of NaFe(III)EDTA added to a weaning cereal on the absorption and urinary excretion of Mn in adult humans. A radioisotopic technique based on the measurement of whole-body retention of ⁵⁴Mn during a period of ~30 d after intake of a labeled test meal was used to estimate absorption (Davidsson et al. 1989); urinary excretion of newly absorbed ⁵⁴Mn was monitored for 7 d after the administration of the labeled test meals. For comparison, a highly bioavailable iron salt (ferrous sulfate) was studied in a cross-over study design.

	SUBJECTS AND METHODS

Abstract Introduction Methods Results Discussion References

Subjects.  Ten healthy adult volunteers (1 man and 9 women; mean age 30 y, range 24-56 y) were recruited for the study. One woman (aged 56 y) was postmenopausal. Subjects were fully informed about the aims and procedures of the study and informed consent was obtained from all volunteers. The protocol was approved by the Ethical Committee and the Isotope Committee at the Sahlgren's Hospital, Göteborg, Sweden.

Test meal.  A weaning cereal, based on white wheat flour and soy, was prepared without added iron or ascorbic acid at a Nestlé Product Development Center (Linor, Orbe, Switzerland). The labeled test meals were prepared by mixing 50 g dry weaning cereal with 150 g hot deionized distilled water. Subjects were randomly assigned to start with the test meal fortified with NaFeEDTA or with FeSO₄. The labeled test meals were administered by one of the investigators after subjects fasted overnight. No food or fluid was allowed for 3 h after intake of the test meals. The test meals were extrinsically labeled with 0.2 MBq ⁵⁴Mn (first test meal) or 0.4 MBq ⁵⁴Mn (second test meal) by the addition of ⁵⁴MnCl₂ (almost carrier free; >3.7 MBq/µg Mn, Amersham International, Buckinghamshire, UK). The exact activity of each dose was measured in a Radio Isotope Calibrator (Capintec CRC 120, Scanflec Medical AB, Täby, Sweden). Food-grade Fe compounds [NaFe(III)EDTA and FeSO₄; P. Lohmann, Emmerthal, Germany and Merck, Darmstadt, Germany] were added at a concentration equivalent to 100 mg Fe/kg dry cereal product. The test meal that was fortified with FeSO₄ contained added ascorbic acid [L(+)ascorbic acid, Merck] at an Fe:ascorbic acid ratio of 1:5 (wt/wt). No ascorbic acid was added to the test meal with added NaFeEDTA.

Analysis.  An aliquot of the weaning cereal was mineralized in concentrated HNO₃ with the use of a microwave system (CEM, MDS-2000, Matthews, NC) and analyzed for Mn by atomic absorption spectrometry (AAS; GBC 932AA, GBC Scientific Equipment, Victoria, Australia). Iron was measured by AAS (SpectrAA 400, Varian, Mulgrave, Australia) after microwave digestion in a HNO₃/H₂O₂ mixture (MLS 1200, MLS, Leutkirch, Switzerland). A standard addition technique was used to minimize matrix effects. Total diet reference materials (Agriculture Research Center of Finland, Jokioinen, Finland and Standard Reference Material 1548, National Institute of Standards and Technology, Gaithersburg, MD) were analyzed together with the cereal for quality control of Mn and Fe analysis, respectively. Phytic acid was analyzed according to the method described by Makower (1970), which was modified by the use of cerium sulfate instead of ferric chloride for a more selective phytic acid precipitation.

Iron status variables were measured at an accredited local clinical laboratory by using autoanalyzer systems (Bayer Technicon System H2, Bayer Diagnostics, Tarrytown, NY) for hemoglobin (Hb) and (BM/Hitachi 917, Boehringer-Mannheim, Mannheim, Germany) for serum-Fe (S-Fe) and serum-total iron binding capacity (TIBC) (Mohandas et al. 1986, Siedel et al. 1984). Quality control materials were analyzed together with the samples for Hb (Cal-Chex S21-103, Streck Laboratories, Omaha, NB), S-Fe and TIBC (Seronorm, Nycomed S/A, Nyegaard & Co., Oslo, Norway). Serum-ferritin (S-ferritin) was measured by a double-antibody ¹²⁵I RIA (Diagnostic Products, Los Angeles, CA). Manganese in whole blood was measured as an indicator of Mn status (Clegg et al. 1986). Analyses were done by AAS according to the method described earlier (Davidsson et al. 1995).

Study design and measurements of whole-body retention and ⁵⁴Mn in urine.  A blood sample was drawn before the administration of each labeled test meal for analysis of Fe-status indices and for determination of whole-blood Mn. Urine was collected in polyethylene containers in 24-h portions for 7 d, starting immediately after intake of each labeled test meal. ⁵⁴Mn in individual 24-h urinary samples was measured using a NaI(TI) detector placed in a lead cave (Davidsson et al. 1989). Urinary excretion of ⁵⁴Mn was calculated as a percentage of total absorbed radioactivity.

Whole-body retention was measured in a detector system equipped with four large plastic scintillator blocks in a floor-roof configuration with the subject lying on a stretcher between the detectors. The technique has been described in detail earlier (Davidsson et al. 1989). Whole-body measurements were made before the start of the study and on every weekday during the first 7 d after intake of the labeled test meals. Thereafter, whole-body retention was measured on d 10, 14, 17, 21, 24 and 28. Initial Mn absorption was calculated by extrapolation from whole-body retention measurements on d 10-30, back to d 0 (Davidsson et al. 1989).

On d 28, the second labeled test meal was fed. The study protocol for this part of the study was identical to the first 4 wk of the study.

Statistics.  Paired t tests were used for the statistical evaluation of data on Mn absorption and urinary excretion. The correlations between indicators of Fe status and Mn absorption and urinary excretion of ⁵⁴Mn were evaluated by Spearman's rank correlation. Statistical significance was defined as P < 0.05. Values are means ± SD.

	RESULTS

Abstract Introduction Methods Results Discussion References

Manganese, iron and phytic acid contents of the weaning cereal were 7.7 mg, 19.7 mg and 3.9 g/kg dry cereal product, respectively. Individual data on sex, age and blood analysis are presented in Table 1. Iron status variables in the nine women showed that one subject was anemic (Hb <120 g/L), one had low S-Fe (<10 µmol/L), four women had elevated TIBC (>70 µmol/L) and one low S-ferritin (<12 µg/L). Whole-blood Mn values were in the normal range (0.11-0.29 µg/L) according to our reference values.

No significant difference in ⁵⁴Mn absorption was found; 1.1 ± 0.15 and 0.91 ± 0.35% was absorbed from the weaning cereal fortified with NaFe(III)EDTA and FeSO₄, respectively (Table 2). Urinary excretion of newly absorbed ⁵⁴Mn was below the detection limit (<2.5 Bq) in 54 (37%) of a total of 145 samples. The total radioactivity in urine collected during the two study periods was 1.1 ± 0.55 and 0.72 ± 0.53% of absorbed dose from the weaning cereal fortified with NaFe (III)-EDTA and FeSO₄, respectively (Table 2). Values were not significantly different between the two periods.

No significant correlations were found between indicators of Fe status (Hb, S-Fe, TIBC or S-ferritin) and Mn absorption or urinary excretion of ⁵⁴Mn after intake of test meals fortified with NaFeEDTA or FeSO₄.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

The usefulness of NaFeEDTA as a food fortificant has been demonstrated earlier in studies in which significantly increased (two- to threefold) Fe absorption has been found from meals fortified with NaFeEDTA, compared with FeSO₄, from a range foods with low bioavailability, such as cereals, legumes and milk (Layrisse and Martinez-Torres 1977, MacPhail et al. 1981, Martinez-Torres et al. 1979, Viteri et al. 1978). In addition, a recent study showed that Na₂EDTA added at molar ratios of EDTA to Fe between 1.0 and 0.25 significantly increased Fe absorption in women from meals with low bioavailability based on rice and fortified with FeSO₄ (MacPhail et al. 1994). Furthermore, the efficacy of NaFeEDTA in food-fortification programs has been evaluated in pilot studies in which foods fortified with NaFeEDTA have shown positive results on improving Fe status (Ballot et al. 1989, Garby and Areekul 1974, Viteri et al. 1983 and 1995).

Although other EDTA chelates (Na₂EDTA, CaNa₂EDTA) have been approved as food additives and used for many years to protect the color, flavor and texture of food (INACG 1993), NaFeEDTA was only recently provisionally considered by the Joint FAO/WHO Expert Committee on Food Additives (JECFA, 1993) to be safe for food fortification in populations in which Fe deficiency anemia is endemic. The increased interest in the use of NaFeEDTA is also demonstrated by the inclusion of a discussion on the recent provisional approval by JECFA and the opportunities for the use of this Fe compound in the draft report of the WHO/UNICEF/UNU consultation (1994) on indicators and strategies for iron deficiency and anemia programs.

To date, the use of NaFeEDTA has been restricted to experimental trials because of concern over its possible influence on the metabolism of other essential nutrients; EDTA is a strong metal chelator and the EDTA moiety might form other complexes in the gastrointestinal tract that could reduce or increase mineral absorption. After digestion, a small fraction (~5%) is absorbed and excreted in urine, whereas the majority is lost via the gastrointestinal tract (Candela et al. 1984); a major concern is that the absorbed EDTA moiety could potentially cause negative effects on the metabolism of minerals and trace elements by increased urinary excretion. The binding of EDTA to minerals can be evaluated from data on the stability constants (log K) at the pH optimum. These constants are relatively high for Cu²⁺ (18.4, pH 3) and Zn²⁺ (16.1, pH 4) (West and Sykes 1960). Thus, it could be assumed that these minerals would form complexes with EDTA and that the complex formation would take place in the gastrointestinal tract under normal physiologic conditions.

The potential problem of increased urinary excretion has recently been investigated in an animal model as well as in adult women. Although the mean urinary excretion of newly absorbed ⁷⁰Zn increased from 0.29 to 0.91% of administered dose when FeSO₄ was replaced by NaFeEDTA in the women's diet (Davidsson et al. 1994b), and mean urinary excretion of zinc increased 7.8-fold in rats with the inclusion of 1000 mg EDTA/kg diet zinc-deficient diet (Hurrell et al. 1994), this had little effect on overall zinc metabolism because the absorption of zinc increased to a far greater extent from the EDTA-containing diets (Davidsson et al. 1994b, Hurrell et al. 1994). No significant influence on the absorption, excretion or retention of calcium (Davidsson et al. 1994b, Hurrell et al. 1994) or copper (Hurrell et al. 1994) was found, although some indications of a positive effect on copper absorption were observed (Hurrell et al. 1994). Thus the results from these two studies demonstrate that NaFeEDTA added to foods containing dietary inhibitors might have an additional benefit in increasing zinc absorption from the diet and in providing Fe with high bioavailability.

In contrast, very little is known about the effect of NaFeEDTA on the absorption and retention of other minerals and trace elements, and concern has been raised that EDTA would increase the absorption and retention of potentially toxic elements such as Mn, aluminium (Al), cadmium (Cd) and lead (Pb). The stability constants (log K) are relatively high for Al (15.5), Cd (15.0-16.1), Pb (16.8-17.7) and Mn²⁺ (13.5) (West and Sykes 1960). The optimum pH for chelate formation with Mn²⁺ is 5.5. Of special interest is the effect of EDTA on the absorption and retention of Mn²⁺ because this element is an essential trace element that is also a known toxic metal (Kawamura et al. 1941, Mena 1981, Papavasiliou 1978). The fractional absorption of dietary Mn is generally low, ~3-5% (Mena et al. 1969), and homeostasis is regulated mainly via excretion, primarily through bile (Britton and Cotzias 1966). Very little Mn is excreted in urine (Hurley and Keen 1987). Studies of Mn absorption and retention are thus complicated by the low fractional absorption and the substantial endogenous excretion of newly absorbed mineral. The use of a radioactive isotope of Mn (⁵⁴Mn) as a label can overcome several of these methodological problems (Davidsson et al. 1989). Manganese is a trace element of special interest in infant nutrition because fractional absorption in animals has been demonstrated to be significantly higher early in life (Keen et al. 1986). Higher retention of Mn has also been indicated in human infants (Dörner et al. 1987). Furthermore, the risk for Mn toxicity is assumed to be higher during infancy because bile flow is not fully established until 1 y of age (Watkins 1981).

If staple foods were fortified with Fe as NaFeEDTA, the molar excess EDTA to Mn in the diet can be calculated. For example, for a 0.5- to 1-y-old infant consuming 3.5 mg Fe as NaFeEDTA per day and 0.6-1 mg Mn (NRC 1989), the EDTA to Mn molar excess would be in the range of 3-6. In the diet of an adult human containing the Estimated Safe and Adequate Daily Dietary Intake of Mn (2-5 mg; NRC 1989), fortified with 10 mg Fe/d as NaFeEDTA, the corresponding values for the molar excess of EDTA to Mn would be in the range of 2-5.

This study was designed to study the fractional absorption of Mn from an Fe-fortified weaning cereal by using NaFeEDTA and FeSO₄ as the added Fe compounds and to measure urinary excretion of newly absorbed Mn in healthy adults. The administration of a radioactive isotope of Mn excluded, for ethical reasons, the possibility of studying the effect of NaFeEDTA on Mn metabolism in infants; a stable isotope technique was not an option because Mn has only one stable isotope. The results from this study clearly demonstrated that neither fractional Mn absorption nor urinary excretion of Mn was influenced by the addition of NaFeEDTA as an Fe fortificant at an EDTA to Mn molar excess of 12.7. Neither Mn absorption nor urinary excretion of ⁵⁴Mn was significantly correlated to any of the indicators of Fe status measured in this study.

Fractional mean Mn absorption from the weaning cereal was found to be low, 1.1% (range 0.81-1.3%, NaFeEDTA) and 0.91% (range 0.46-1.6%, FeSO₄). No comparable data on Mn absorption from weaning foods are available, but similarly low fractional Mn absorption has been reported from infant formula based on soy with the native phytic acid content (Davidsson et al. 1995). The weaning cereal used in this study contained a relatively high amount of phytic acid, which has been demonstrated to inhibit Mn absorption (Davidsson et al. 1995).

In conclusion, on the basis of the data from this study, Fe fortification in the form of NaFeEDTA does not influence the absorption and urinary excretion of Mn. The information gained from this study provides new essential data to support the use of NaFeEDTA in food-fortification programs. Until now, food-fortification programs have met with only limited success, and the introduction of NaFeEDTA could be a useful tool to provide bioavailable Fe to vulnerable groups in the population and thus aid in combating Fe deficiency.

	FOOTNOTES

Manuscript received 27 August 1997. Initial reviews completed 8 October 1997. Revision accepted 16 February 1988.

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