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*
Unidad de Investigación Médica en Nutrición. Centro Médico Nacional Siglo XXI, Mexico City;
Coordinación de Investigación y Escuela de Ciencias Químicas de la Universidad La Salle, Mexico City;
**
Instituto Nacional de Salud Pública, Cuernavaca, Mexico
2To whom correspondence should be addressed. E-mail: marsau2{at}prodigy.net.mx.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • lime-treated corn flour • wheat flour • iron utilization • iron bioavailability • rats
Food iron fortification is a strategy used worldwide in public nutrition intervention to fight iron deficiency. Iron deficiency, with and without anemia, is the most prevalent nutritional problem globally, but particularly in less developed nations where food consumption is based on cereals and legumes. These foods have a high phytate content (1
,2
), which is a strong inhibitor of nonheme iron absorption (3
,4
). Wheat flour and its products are the more frequently fortified foods, mainly with reduced iron, which has low bioavailability (5
,6
). Studies in Venezuela have reported that fortification of wheat and corn flour with ferrous fumarate is more successful than with other iron sources (7
,8
).
Recently, the Mexican federal government launched a fortification program of wheat flour and lime-treated corn flour with reduced iron. Lime-treated corn flour is used to make tortillas, the most frequently consumed staple in Mexico (9
). Although both wheat and corn are cereals, they go through different processes before they are ready for consumption. Wheat flour is obtained by grinding the grain after losing its external husk, where most of the phytate is contained (10
). Then, it is supplemented with ascorbic acid to improve its baking quality, thus improving iron absorption (11
). In contrast, lime-treated corn flour is obtained after maize is heated in an alkali solution (calcium hydroxide) at 90–92°C until soft (12
). Although this procedure reduces its native phytate content by 16–28% (537–812 mg/100 g), it remains high (420–700 mg/100 g) (13
,14
). Although calcium concentrations increase to values higher than 200 mg/100 g (13
), a concentration that is lower than values reported by others, it still might inhibit iron absorption (15
–17
).
On the basis of previous measurements of phytate found in lime-treated corn flour in our laboratory, we predict that the above-mentioned public nutrition intervention in Mexico would have a poor effect on iron status of the population because of low bioavailability of reduced iron. This investigation compares the bioavailability and utilization of several chemical compounds of iron added to lime-treated corn flour and to wheat flour. Thus, this information will help to identify an iron fortifier with better bioavailability than reduced iron.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Diets were based on commercially available wheat and lime-treated corn flours with and without fortification with hydrogen-reduced iron, 325 mesh. Similarly, wheat and lime-treated corn flours were fortified in our laboratory by adding 30 mg iron/kg flour to unfortified flours in the form of ferrous sulfate, ferrous fumarate and iron ammonia citrate to match the iron content to that of commercial flours fortified with reduced iron. Table 1
shows the composition of the diets. Casein, free of vitamins (Sigma, St. Louis MO), was used as the protein source to prepare a low iron diet. Diets were prepared with 95% lime-treated corn flour and 82% wheat flour (70–72% extraction), with protein contents of 8 and 9%, respectively. This concentration of protein was similar to that of the low iron, casein diet. The proximate composition of flours was assessed using techniques described by the AOAC (18
). Diet samples were ashed by calcination to determine iron content by atomic absorption spectrometry (Perkin Elmer Analyst 300, Norwalk, CT), and the phytate content by the method described by Frübek et al. (19
). All experimental procedures involving laboratory animals were performed in accordance to the Mexican Official Regulations NOM-062-Z0–1999, which are essentially in line with NIH guidelines for experimentation in rats.
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Indices of iron bioavailability and utilization.
Hemoglobin and iron determination were used to estimate the following variables (20
–22
).
1) Percentage of bioavailability, calculated as hemoglobin regeneration efficiency (HRE):
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2) Iron utilization:
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3) Iron content of Hb. Calculated assuming a total blood volume of 6.7% of the rat body weight, and an average iron content of Hb of 0.335 (21
)
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4) Relative biological value (RBV):
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5) Iron absorption:
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6) Protein efficiency ratio (PER) (23
)
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Statistical analysis.
The Minitab statistical software (Minitab 12, State College, PA) was used for statistical analyses. Data are presented as means ± SD, n = 6. The General Linear Model and Bonferroni’s test were used for comparisons among groups. A correlation between the iron content of diets and iron bioavailability was determined with Pearson correlation analysis. Differences were considered significant at P < 0.05 (24
).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). In contrast, the fiber and phytate contents in lime-treated corn flour were almost three- to fourfold higher than those values in wheat flour.
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, P < 0.05), but the iron source added to the diets did not influence the PER. There were no differences in food intake among groups fed wheat and lime-treated corn flours. The food intake of rats fed flours fortified with ferric citrate was higher than that of groups fed unfortified diets and those fortified with reduced iron (P < 0.05).
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). Iron balance in the groups that consumed diets supplemented with iron sulfate, fumarate and citrate was higher than that of rats fed flours supplemented with reduced iron (P < 0.01). The percentage of iron absorbed was higher for the groups fed wheat flour than for those fed lime-treated corn flour. The highest absorption was observed in the group of rats fed the unfortified wheat flour diet and the lowest in the groups fed lime-treated corn flour with and without fortification with reduced iron. Analyzing separately the groups fed lime-treated corn flour, iron absorption was higher for the rats that consumed diets fortified with iron sulfate, fumarate and citrate than for rats that consumed unfortified diets and those fortified with reduced iron.
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. The Hb concentration after depletion was 57 ± 17 g/L. After repletion, rats fed wheat flours gained more Hb than the groups fed lime-treated corn flours (P = 0.02, Table 6
). Rats fed diets supplemented with iron sulfate, fumarate and citrate gained more Hb than those fed unfortified diets or diets fortified with reduced iron (P < 0.05, Table 6
). The percentage of iron bioavailability, expressed as HRE, was higher for unfortified flours than for fortified lime-treated corn flour and wheat flour (P < 0.01). The highest bioavailability was in the rats fed unfortified wheat flour, and the lowest in rats fed lime-treated corn flour fortified with reduced iron. In general, iron utilization was lower in rats fed lime-treated corn flours than in rats fed wheat flours. Rats fed flours fortified with iron sulfate, fumarate or citrate had higher utilization than rats fed unfortified flours or flours fortified with reduced iron (Table 6)
.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). This difference may be explained by the manner in which iron bioavailability was evaluated. As suggested by Layrisse et al. (8
), the percentage of iron absorbed decreases as iron intake increases, but the total iron absorbed is higher. Similar results were reported by Cook et al. (26
), who found in humans that those fed bread fortified with increasing amounts of iron (1, 3 and 5 mg) had lower percentages of iron absorbed, but their absolute absorption increased in response to increasing iron intakes. In our study, we also found an inverse correlation between diet iron content and bioavailability (r = -0.83, P < 0.05), and a direct relationship between the gain in Hb and iron utilization (r = 0.93, P < 0.05). These results suggest that this method is adequate for determining the effect of food fortification because the rats that were fed wheat flour fortified with ferrous sulfate had low iron bioavailability, but significantly higher utilization compared with rats fed unfortified flour. Further, most of the rats recovered their Hb status after repletion (Hb >120 g/L) except for those fed unfortified flours or flour fortified with reduced iron.
We confirmed that iron absorption and bioavailability do not reflect appropriately the effect of iron supplementation. Thus, we decided to use iron utilization as the outcome variable because it evaluates the efficiency of Hb regeneration (see Methods section). Iron utilization from unfortified lime-treated corn flour or this flour fortified with reduced iron was not different (RBV = 51 and 55% respectively). These values are slightly higher than those found by Fritz et al. (5
) in nonlime-treated corn flour added with reduced iron. This is important because reduced iron is currently used to fortify lime-treated corn flour in public nutrition programs of Mexico to decrease the prevalence of iron deficiency anemia. In contrast, lime-treated corn flour fortified with citrate had a RBV of 81%, and with fumarate of 70% relative to wheat flour fortified with ferrous sulfate. Therefore, it is clear that reduced iron is of little use as a fortifier of lime-treated corn flour because corn flour fortified with reduced iron that is not lime treated also has low iron utilization (5
). There is enough evidence to suggest a change of iron compound to ferrous sulfate, ferrous fumarate or ferric citrate. Effects of the fortifier on the organoleptic characteristics of the product during the required shelf life must be assessed before any decision is made.
Our results confirmed the findings reported by others that ferrous sulfate is well utilized when added to wheat flour (5
). However, it is not a suitable source of iron fortification because it easily oxidizes the food matrix, affecting its shelf-life and acceptability in storage (27
). Fortunately, we found that flours fortified with iron fumarate and citrate were also well utilized and are less likely to oxidize the food matrix before use.
The validity of the depletion/repletion method was verified by Forbes et al. (6
) and by Hurrell et al. (27
) in rats to predict iron bioavailability. In these experiments, the depletion/repletion model defined the adequacy of the level of depletion when Hb was reduced at least 50%, which, in our case occurred in 10 d. There is great variability in the duration of such a period in the literature (7–40 d), (5
,6
,21
,22
,25
,27
), and none of the studies used indices of iron status such as serum iron, ferritin or others as indicators of depletion. Further, the vitamin mix was calculated to ensure that folic acid and vitamin B-12 intakes met recommendations. Thus, such a reduction in Hb ensures a robust indicator of iron deficiency and provides enough sensitivity to detect changes during the repletion period.
We demonstrated a better iron absorption from wheat flour diets than from lime-treated corn flour diets. The difference may be explained by the high phytate content in lime-treated corn flour (threefold greater than in wheat flour). As previously reported, phytate is one of the most potent inhibitors of intestinal iron absorption (11
,28
). A note of caution about the interpretation of these results is in order: the diets used here were based on raw flours. Thus, we did not take into account the decrease in phytate activity caused by higher temperatures during baking or cooking. Future experiments using bread and tortillas baked with these flours are warranted.
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FOOTNOTES |
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3 Abbreviations used: Hb, hemoglobin; HRE, hemoglobin regeneration efficiency; PER, protein efficiency ratio; RBV, relative biological value.
Manuscript received 26 June 2002. Initial review completed 6 August 2002. Revision accepted 2 October 2002.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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