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The Journal of Nutrition Vol. 127 No. 7 July 1997, pp. 1407-1411
Copyright ©1997 by the American Society for Nutritional Sciences

Milk Inhibits and Ascorbic Acid Favors Ferrous Bis-Glycine Chelate Bioavailability in Humans1,2

Manuel Olivares3, Fernando Pizarro, Oscar Pineda*, José J. Name*, Eva Hertrampf, and Tomás Walter

Hematology Unit, Institute of Nutrition and Food Technology (INTA), University of Chile, Santiago 11, Chile and * Latin American Center for Nutrition and Metabolic Studies (CELANEM), Antigua, Guatemala 03001

ABSTRACT
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
LITERATURE CITED

ABSTRACT

The chemical properties of ferrous bis-glycine chelate allow for its use as a fortificant in fluid, high fat vehicles. This chemical form may also protect iron from the inhibitory or enhancing effects of the diet on iron absorption. Alternatively, iron bis-glycine chelate may be absorbed by a mechanism independent of an individual's iron stores. To test these hypotheses, the bioavailability of iron bis-glycine chelate added to water and milk was studied using a double-isotopic method in two groups of 14 women. Iron absorption from aqueous solutions of 0.27 mmol/L (15 mg/L) of elemental iron as either iron bis-glycine or ferrous ascorbate was not significantly different (34.6 and 29.9%, respectively). There were significant correlations between (log) iron absorption of iron bis-glycine with (log) serum ferritin (r = -0.60, P < 0.03) and with (log) iron absorption from ferrous ascorbate (r = 0.71, P < 0.006), suggesting that iron bis-glycine chelate bioavailability is indeed affected by iron stores. Iron absorption of iron bis-glycine given in milk was significantly lower (P < 0.002) than when given in water, with values of 11.1 and 46.3%, respectively (standardized to 40% absorption of the reference dose). With the addition of 0.57 mmol/L ascorbic acid (100 mg/L), iron absorption of iron bis-glycine given in milk increased significantly from 11.1 to 15.4% (P < 0.05). These findings show that milk and ascorbic acid affect iron bis-glycine chelate bioavailability and also demonstrate that iron stores may influence its bioavailability as well. The good bioavailability of iron bis-glycine makes this compound a suitable alternative to be considered in iron fortification programs.

KEY WORDS: iron absorption · iron bis-glycine chelate · milk · ascorbic acid · humans

INTRODUCTION

Iron deficiency continues to be one of the most prevalent nutritional deficiencies in the world. For physiological reasons, the most commonly affected groups are infants, children, adolescents and women of childbearing age (DeMayer and Adiels-Tegman 1985).

Fortification of food with iron is considered the best sustainable way of preventing iron deficiency (International Anemia Consultive Group 1977). The sequential steps that should be followed in establishing an iron fortification program have been well defined. Perhaps the most difficult technical hurdle is finding the adequate combination of iron fortification compound and food vehicle. Most foods consumed in undeveloped countries have a predominance of inhibitors of nonheme iron absorption. Therefore, strategies to improve iron bioavailability are needed. Fortification of foods with an iron form that is less influenced by inhibiting dietary ligands is an appealing strategy.

When milk is selected as the vehicle, iron salts can be added safely only to powdered milk sealed in airtight containers. If iron salts are added to fluid, high fat milk, lipoperoxidation occurs within hours. Thus, other fortificants must be considered when fluid milk is the selected vehicle. Fluid milk is preferred in many areas of the world such as Brazil and Argentina where powdered milk has lower appeal. Iron bis-glycine chelate is a fortificant that can be added to the fluid phase of high fat compounds such as milk or milk products without inducing prompt peroxidation or rancidity (Galdi et al. 1989a and 1989b, Name 1995).

The effectiveness of iron bis-glycine chelate in the treatment of iron deficiency anemia has been shown in humans (Pineda et al. 1994). This chelate is formed by two glycine molecules bound to a ferrous cation, resulting in a double heterocyclic ring compound. The carboxyl group of glycine is linked with iron by an ionic bond, whereas the alpha -amino group is joined with the metal by a coordinate covalent bond (Ashmead et al. 1985). It has been proposed that this configuration protects the iron from dietary inhibitors and intestinal interactions (Ashmead et al. 1985) and that it has a different route of absorption than nonheme iron that is independent of iron stores.

To test whether an inhibitor (milk) and an enhancer (ascorbic acid) have an effect on the bioavailability or iron bis-glycine chelate, we studied the absorption of iron bis-glycine chelate given in water, in milk and in milk with ascorbic acid. To test the effect of iron stores, we compared the absorption of iron bis-glycine chelate in water with the absorption of ferrous ascorbate in water.

SUBJECTS AND METHODS

Subjects. Iron absorption studies were performed in two groups of 14 women between the ages of 33 and 51 y. None were pregnant, all used contraceptive intrauterine devices and were in apparent good health. Written, informed consent was obtained from each volunteer before participation in the study. The protocol was reviewed and was in accordance with the standards set by the Institute of Nutrition and Food Technology's Ethics Committee on Human Research; radioactive doses were approved by the Chilean Commission of Nuclear Energy.

Isotope studies. Iron isotopes (55Fe and 59Fe) of high specific activity were used as tracers; both isotopes were iron(III) chlorides as purchased (Du Pont de Nemours, Wilmington, DE). Isotopes were mixed with water and milk immediately before administration. Iron bis-glycine chelate (Ferrochel, Albion Laboratories, Clearfield, Utah) was labeled intrinsically during the synthesis of amino acid chelate. This process was performed by the manufactuter.4 In brief, the iron was added to distilled water as FeCl3·6H2O carrier (Merck, Darmstadt, Germany) and a trace amount of 55FeCl3 (Du Pont de Nemours) dissolved in 0.01 mol HCl. Then, the iron was reduced to the ferrous state adding sodium borohydride (Sigma, St. Louis, MO). Glycine (Sigma) was slowly added with constant stirring to the beaker containing the Fe solution in a molecular ratio of elemental iron to glycine of 1:2. The beaker was heated at 50°C until all of the precipitate was dissolved; it was then allowed to cool to room temperature. This solution was kept refrigerated at 4°C in an amber airtight container previously flushed with nitrogen. The structure of the labeled compound was verified by comparing the mid-infrared spectrograph of the reactants to the finished product using the band assignments established by Nakamoto et al. (1961) and Nakamoto (1978). Previous testing using infrared spectroscopy of similarly nonradioactive amino acid chelate iron has demonstrated that the percentage of iron chelation obtained is over 97% (Fujita et al. 1982). The specific activity of the labeled iron bis-glycine chelate was 37 kBq of 55Fe/mg elemental iron. A solution containing 0.27 mmol/L (15 mg/L) of elemental iron, as ferrous sulfate, and 95 mg/L (0.54 mmol/L) of ascorbic acid was labeled extrinsically with tracer amount of 59Fe chloride (Du Pont de Nemours). The specific radioactivity of this aqueous solution (reference dose) was 12.3 kBq of 59Fe/mg elemental iron. The preparations were consumed after an overnight fast, and no food or beverages other than water were permitted during the following 4 h. The amounts of aqueous solution and milk ingested were calculated by differential weight of the glasses. For the calculation of total radioactivity ingested, aliquots of the aqueous solution and milk were counted in sextuplicate as standards. Measurement of blood radioactivity was performed in duplicate venous samples according to the method of Eakins and Brown (1966); they were counted a sufficient number of times to allow <3% counting error. A liquid-scintillation counter (Beckman LS 5000 TD, Beckman Instuments, Fullerton, CA) was used for the double isotope measurements. The percentage of absorption was calculated based on the blood volume estimated from height and weight and assuming an 80% red cell utilization of radio iron.

Study 1. On d 1, the subjects received 200 mL of an aqueous solution of 0.27 mmol/L (15 mg/L) of elemental iron as iron bis-glycine labeled with 111 kBq of 55Fe; on d 2, they received 200 mL of a reference dose of 0.27 mmol/L (15 mg/L) of elemental iron as ferrous ascorbate (molar ratio 1:2 iron to ascorbic acid) labeled with 37 kBq of 59FeCl3 . A venous blood sample was obtained 2 wk later (d 16) to measure the circulating radioactivity and to determine the iron status of the subjects. On d 16, subjects were given 200 mL of whole cow's milk fortified with 0.27 mmol/L (15 mg/L) of iron as iron bis-glycine chelate labeled with 111 kBq 55Fe. A final venous sample was obtained on d 31 to determine the increase in red blood cell radioactivity.

Study 2. On d 1, the subjects received 200 mL of a reference dose of 0.27 mmol/L (15 mg/L) of elemental iron as ferrous ascorbate labeled with 37 kBq of 59FeCl3 ; on d 2, they received 200 mL of whole cow's milk fortified with 0.27 mmol/L (15 mg/L) of iron as iron bis-glycine chelate labeled with 111 kBq 55Fe and 0.57 mmol/L (100 mg/L) of ascorbic acid. A venous blood sample was obtained on d 16 to measure the circulating radioactivity and to determine the iron status of the subjects.

In both studies, hemoglobin, mean cell volume, free erythrocyte protoporphyrin, serum iron, total iron binding capacity and serum ferritin were determined in venous blood obtained on d 16 (International Anemia Consultive Group 1985).

For comparative studies of iron bioavailability, the absorption of 0.05 mmol (3 mg) of elemental iron as ferrous ascorbate is used to offset the effect of differences in iron status among individuals (Cook and Bothwell 1984). For purposes of comparison, all studies currently refer to a 40% absorption of the reference dose of ferrous ascorbate. This absorption percentage is used because it corresponds to that which is obtained in borderline iron-deficient populations.

Because the percentages of iron absorption and serum ferritin concentrations have a skewed distribution, these values were converted to logarithms before performing mean and SD analysis; the results were retransformed to antilogarithms to recover the original units and expressed as geometric means and ±1 SD ranges (Cook et al. 1969). Statistical analyses included Wilcoxon and Mann-Whitney tests, for comparison within and between groups, respectively, Pearson correlation and covariate analysis (Colton 1974). Pearson correlation and covariate analysis were calculated on logarithmically transformed data. Statistical analyses were performed by the program SPSS for Windows, release 6.0, SPSS, Chicago, IL.

RESULTS

Study 1. The iron bioavailability of the aqueous solutions of iron bis-glycine and ferrous ascorbate were not significantly different (34.6 and 29.9%, respectively). Iron absorption of iron bis-glycine given with milk was significantly lower (Wilcoxon test, P < 0.002) than that observed when this compound was administered as an aqueous solution (8.3 and 34.6%, respectively). When iron absorption was standardized to 40% absorption of the reference dose, the corresponding percentages of iron absorption were 11.1 and 46.3% for the iron bis-glycine chelate given in milk and water, respectively (Table 1).

Table 1. Iron absorption by women of iron bis-glycine chelate given in water and milk compared with the reference dose of ferrous ascorbate1

[View Table]

Study 2. The absorption of iron bis-glycine chelate given in milk with ascorbic acid was 10.7 or, when adjusted to 40% absorption of the reference dose, 15.4% (Table 2). This is significantly higher (Mann-Whitney test, P < 0.05) than when milk was given alone (11.1%) as in Study 1. 

Table 2. Iron absorption by women of a milk fortified with iron bis-glycine chelate plus ascorbic acid and the reference dose of ferrous ascorbate1

[View Table]

Study 1 vs. Study 2. To offset differences in iron status between groups, we calculated the ratio (I) between the absorptions of iron bis-glycinate chelate given in milk and the reference dose (A/C, Table 1), and the ratio (II) between the absorption of iron bis-glycinate chelate given in milk plus ascorbic acid and the reference dose of the corresponding group (A/B, Table 2). Ratios I and II were significantly different (Mann-Whitney test, P < 0.05 ), with ratio II (0.38) higher than ratio I (0.28) (milk plus ascorbic acid vs. milk alone, respectively).

There was a significant inverse correlation between (log) serum ferritin and (log) iron absorption from iron bis-glycine given with water (r = -0.60, P < 0.03). The correlation between (log) serum ferritin and (log) absorption from ferrous ascorbate in water was r = -0.62 (P < 0.02). Slopes and intercepts of the former two regressions were not significantly different (covariance analysis; F = 2.498 and 0.532, respectively). There was a direct correlation between the absorptions of (log) ferrous ascorbate and (log) iron bis-glycine chelate given with water (r = 0.71, P < 0.006). The absorption of (log) ferrous ascorbate was significantly correlated with the absorption of (log) iron bis-glycine chelate plus ascorbic acid given with milk (r = 0.87, P < 0.001). A borderline significant correlation between (log) serum ferritin and (log) absorption of iron bis-glycine chelate plus ascorbic acid given with milk was found (r = -0.53, P = 0.066). The absorption of iron bis-glycine chelate in milk without ascorbic acid was not correlated with either serum ferritin or iron ascorbate absorption (r = -0.32 and 0.39, respectively).

DISCUSSION

The efficacy of an iron supplementation or fortification intervention can be predicted from iron bioavailability studies of the supplement or fortified food. Bioavailability of iron is influenced by the properties of the iron compound, total amount of iron in the diet, iron status of the individual, rate of erythropoiesis and inhibitors or enhancers of iron absorption present in the intestinal lumen or in the diet (Bothwell 1983, Cook and Bothwell 1984). However, the presence or absence of inhibiting or enhancing factors of nonheme iron absorption is the main determinant in the feasibility of meeting iron requirements of people whose diet contains little heme iron (NRC 1989). If soluble inorganic iron salts are given with water solutions without food, absorption of iron is high, but absorption of iron salts decreases markedly when given with foods (Hurrell 1984).

Highly modified milk formulas fortified with iron have been used mainly in the prevention of iron deficiency in infancy (American Academy of Pediatrics 1989). However, in the undeveloped world, powdered or fluid cow's milk with little modification is commonly used primarily because of its lower cost. Unmodified cow's milk has a marked inhibitory effect on the absorption of nonheme iron as a result of its high concentration of inhibitors of iron absorption (Hurrell 1984, Cook and Bothwell 1984). The main inhibitors existing in cow's milk are casein, calcium, whey protein and phosphates (Hallberg et al. 1991, Hurrell et al. 1989, Peters et al. 1971). When 0.18-0.27 mmol/L (10-15 mg/L) of iron, as ferrous sulfate, is added to unmodified cow's milk, only 4-5% is absorbed (Heinrich et al. 1975, Stekel et al. 1986). However, this absorption can be doubled by the addition of 0.57 mmol (100 mg) of ascorbic acid (Stekel et al. 1986). An alternate strategy is the fortification of milk with an iron compound, such as an iron amino acid chelate, that might be less influenced by the inhibitors present in milk.

Our results showed that iron bioavailability of iron bis-glycine chelate given with water is high, compared with the absorption of the reference dose of ferrous ascorbate. The iron absorption of this compound was calculated to be 46.3% in a population with low iron stores (a population absorbing 40% of the reference dose of iron ascorbate).

It has been proposed that iron amino acid chelates are absorbed like peptides in the jejunum rather than as inorganic iron in the duodenum (Ashmead et al. 1985). Therefore, there has been concern about the role of iron stores on the regulation of iron absorption from the chelate. From results of this study, we suggest that the absorption of the iron amino acid chelate is likely controlled by the iron stores of the subjects. There was an inverse relationship between the iron stores of the body, as reflected by serum ferritin, and the absorption of the iron amino acid chelate. This holds true as well when we compare the absorption of the chelate with that of ferrous ascorbate which showed an excellent correlation (r = 0.71). However, this correlation does not necessarily prove causality. If confirmed, the suggestion that regulation of iron absorption by iron stores occurs with this iron chelate should dispel the notion of a risk of iron overload if this compound were to be used in fortification programs of the population at large.

Iron absorption of the iron bis-glycine fortified milk was 11.1% when standardized to 40% of the reference dose of ferrous ascorbate. This absorption is 2- to 2.5-fold higher than the 4% bioavailability obtained previously by us in cow's milk fortified with ferrous sulfate (Stekel et al. 1986), suggesting that iron bis-glycine is influenced less than ferrous sulfate by the action of the inhibitory ligands present in cow's milk. The iron absorption from this milk fortified only with iron bis-glycine is comparable to that obtained in milk fortified with ferrous sulfate plus ascorbic acid (Stekel et al. 1986). Furthermore, bioavailability of iron bis-glycine in milk was improved by adding ascorbic acid. In fact, iron absorption of the milk fortified with 0.27 mmol/L of iron bis-glycine and 0.57 mmol/L of ascorbic acid was 15.4% when standardized to 40% of the reference dose of ferrous ascorbate. However, the enhancing effect of ascorbic acid on iron absorption of the milk fortified with iron bis-glycine chelate was proportionately lower than that obtained previously in a milk fortified with ferrous sulfate (11.1-15.4% vs. 4-11.1%), showing that iron amino acid chelate absorption is less influenced by the action of this vitamin.

Using the absorption figures obtained in the present study and assuming a daily consumption of 750 mL of milk fortified with 0.27 µmol/L (15 mg/L) of elemental iron as iron bis-glycine would provide 0.03 mmol/d (1.65 mg/d) of absorbed iron and 0.04 mmol/d (2.25 mg/d) if this milk were additionally fortified with 0.57 mmol/L (100 mg/L) ascorbic acid. These amounts of iron absorbed are in sufficient excess to meet iron requirements of infants (FAO/WHO 1988). Based on this calculation, it seems reasonable to use less than this amount of iron bis-glycine in the fortification of milk. Recently, Brazilian investigators (Queiroz and Torres 1995) have shown in a field trial the beneficial effect on iron status in infants of a fluid cow's milk enriched with 0.05 mmol/L (3 mg/L) of elemental iron, as iron bis-glycine, without ascorbic acid. A 40% iron absorption of this fortified milk was estimated indirectly from calculations of increases in hemoglobin and ferritin (Name 1995). This relatively high absorption may be explained by the high prevalence of anemia in the infant population studied and by the low dose of iron because the lower the dose of iron, the higher the percentage of absorption. However, direct isotopic bioavailability studies have not been reported in this setting.

Iron bis-glycine chelate has a bioavailability comparable to that of ferrous sulfate plus ascorbic acid in milk. However, its low prooxidant properties (Name 1995), makes this compound advantageous when it is used as a fortificant in fluid, high fat dairy products. Iron bis-glycine chelate is stable in the ferrous form when exposed to ambient air and temperature between pH 3 and 10 (Albion Laboratories 1995). When stored in tetra-pack containers, it has a shelf life of over 6 mo at room temperature.

Studies with the ferric form of iron glycine chelate (ferric glycinate) have also shown good bioavailability in animals and low prooxidant properties (Galdi et al. 1988, Langini et al. 1988).

At present, proprietary whole fluid milk and other dairy products fortified with iron bis-glycine are available in many countries of Latin America and Europe. In future studies, the effects of other known inhibitors and enhancers on the absorption of this iron amino acid chelate should be evaluated. Moreover, the regulation of absorption of this iron amino acid chelate by iron stores should be further confirmed, as well as the mechanism of absorption.

FOOTNOTES

1   Funded in part by Albion Laboratories, Inc., Clearfield, UT.
2   The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
3   To whom correspondence and reprint requests should be addressed.
4   Information on file at Albion Laboratories, Inc., Clearfield, UT.

Manuscript received 9 August 1996. Initial reviews completed 26 September 1996. Revision accepted 13 March 1997.

LITERATURE CITED

0022-3166/97 $3.00 ©1997 American Society for Nutritional Sciences



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