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ORIGINAL RESEARCH COMMUNICATION |
1 From the Facultad de Ciencias Naturales, Universidad Autónoma de Querétaro, Querétaro, México (MD and JLR); the Department of Nutrition, University of California, Davis (LHA and OPG); and the US Department of Agriculture, Agricultural Research Service Childrens Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston (SA).
2 Supported by a grant from USAID-OMNI.
3 Reprints not available. Address correspondence to JL Rosado, Facultad de Ciencias Naturales, Universidad Autónoma de Querétaro, Clavel no. 200, Prados de la Capilla, Querétaro, Querétaro. CP 76176, México. E-mail: jlrosado{at}avantel.net.
| ABSTRACT |
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Objective: The study, conducted in rural Mexico, measured stable isotopes of iron to evaluate the effect on iron absorption of the addition of 25 mg ascorbic acid as agua de limón (limeade) to 2 typical meals per day for 2 wk.
Design: Fifteen nonpregnant, nonlactating, iron-deficient (ferritin < 12 µg/L) women (
± SD age: 28.3 ± 7.7 y) fasted overnight and were brought to a community clinic. After an initial blood sample, subjects consumed 0.25 mg 57Fe with both breakfast and lunch for 14 d. On day 29, another blood sample was taken, and a reference dose of 2.7 mg 58Fe with 25 mg ascorbic acid was given. For the following 15 d, participants consumed 0.25 mg 57Fe added to both breakfast and lunch with 25 mg ascorbic acid added to each meal as limeade. A final blood sample was taken on day 59.
Results: Iron absorption was calculated from recovery of isotopes in blood obtained 14 d after administration of each isotope. When 25 mg ascorbic acid as limeade was added to test meals twice a day for 2 wk, iron absorption increased significantly (P < 0.001) in every subject: the mean absorption rose from 6.6 ± 3.0% to 22.9 ± 12.6%.
Conclusions: The consumption of 25 mg ascorbic acid as limeade twice daily with meals substantially improved iron absorption and may improve the iron status of nonpregnant, nonlactating, iron-deficient women.
Key Words: Iron absorption bioavailability ascorbic acid stable isotopes Mexican diet
| INTRODUCTION |
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Populations such as those in rural Mexico, which have a high intake of cereals (particularly maize) and legumes as staples and a low intake of meat, poultry, and fish, may be iron deficient because of poor dietary iron bioavailability (57). Whereas the total iron content of the traditional Mexican diet is quite high, almost all of this iron is in the nonheme form and is predominantly from beans and maize (8), which contain large amounts of phytate. Intakes of nonheme-iron absorption enhancers such as ascorbic acid are low in Mexico (4, 9). Thus, increasing the intake of ascorbic acid from local foods may be a sustainable approach to improving iron absorption and status in rural Mexico.
Published studies showed that ascorbic acid increases iron absorption with a linear dose-related response of up
200 mg/d (10). Several investigators concluded that, in the long term, ascorbic acidinduced increases in nonheme-iron bioavailability from whole diets might be less than that observed from ascorbic acidrich foods or from foods to which ascorbic acid was added. In iron-sufficient Mexican boys 1215 y old, feeding ascorbic acid as 300 mL orange juice for 2 wk increased nonheme-iron bioavailability to a greater extent than did feeding a meal to which synthetic ascorbic acid had been added in the same amount (11). Likewise, when nonheme-iron absorption was measured from the whole diet over a 2-wk period, there was a 2.5-fold difference between a diet containing foods that enhance iron absorption and a diet containing foods that inhibit iron absorption, but there was a 5.9-fold difference in iron absorption from foods to which ascorbic acid was added and foods containing inhibitors (12).
Before starting a community trial of the effect on iron status of increasing the intake of ascorbic acid as agua de limón (limeade), the present study was designed to test the effect on iron absorption from typical Mexican diets of consuming limeade that provides 25 mg ascorbic acid with both breakfast and lunch over a period of 2 wk. The study was conducted with typical diets in the field on iron-deficient (ID) women. Iron absorption was assessed by measuring the stable isotopes of iron, which were previously used in the field (13). The hypothesis to be tested was that the consumption of habitual doses of limeade significantly increases iron absorption.
| SUBJECTS AND METHODS |
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2 h). This solution was transferred to a beaker, and the vial was rinsed with 41 mL of 0.5 mol H2SO4/L. The solution was dried in a muffle furnace at 120 °C for 23 h, at 230 °C for 30 min, and at 500 °C for 30 min. The powder obtained was reconstituted with 50 mL of 0.2 mol H2SO4/L, filtered through a 0.5-µm Millex filter (Millipore), and brought to a final volume of 308 mL. The final iron concentration was checked with the use of atomic absorption spectrometry. All acids used for the preparation of the isotope solutions were Ultrex II Ultrapure Reagent (JT Baker Inc, Pillsburg, NJ). Final solutions were stored with protection against light at 5 °C. 57Fe was enriched to 95.7% 57Fe and 58Fe was enriched to 93% 58Fe.
Subjects and location
The research was conducted in a rural area in Mexico, and a metabolic unit and community clinic were used for the study. Thirty-four nonpregnant, nonlactating (NPNL) women were evaluated for hemoglobin and plasma ferritin concentrations; from these 34, 16 ID (plasma ferritin concentration: < 12 µg/L) subjects were identified. One of the subjects became pregnant and was subsequently removed from the study. The purpose of the study was described to the 15 women (
± SD age: 28.3 ± 7.7 y), who gave written informed consent. The project was approved by the Human Subjects Review Committee of the University of California, Davis. Results for 4 subjects were not included in the statistical analyses because of laboratory error in handling samples; the statistical analyses were based on data from the remaining 11 subjects.
Study design and test meal
The test meal, based on the model rural diet described by Rosado et al (5, 15), was maize tortillas and beans with vegetables and small amounts of salsa. Three vegetables [cactus (Opuntia sp), green snap beans, and squash] were selected to add variety to the meals. The amounts of each vegetable were equivalent in terms of their fiber content (16). Thus, 3 test meals were given on a 3-d rotating basis to the subjects, each served twice daily (Table 1
). The WORLDFOOD software program from the University of California was used to calculate the energy, phytate, ascorbic acid, and iron content of the test meals (17). The molar ratio of phytate to iron for each meal was 21.9, 20.1, and 18.9, respectively.
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4 h to ensure that they did not consume other foods during this time. They then had the second meal at the community clinic and returned to their homes for the remainder of the day, having been instructed not to eat anything for the next 4 h.
Preparation of limeade and measurement of ascorbic acid content
The ascorbic acid content of freshly squeezed lime juice was measured by using the 2,6-dichlorophenolindophenol method twice every day immediately before each meal was served (19). The limeade was prepared by dissolving 1.1 kg sugar in 8 L water at breakfast and at lunch and pouring it into the subjects glasses. Then, the amount of lime juice needed to provide 25 mg ascorbic acid per meal was added separately to each glass.
Biochemical measurements
Hemoglobin and plasma ferritin concentrations were measured in the blood samples taken at 0, 29, and 59 d. The hemoglobin concentration was measured in one drop of venous blood by using the HemoCue (HemoCue Inc, Mission Viejo, CA). Plasma ferritin concentrations were measured in duplicate by immunoradiometric assay (Coat-A-Count Ferritin IRMA; Diagnostic Products Corp, Los Angeles). Cutoffs for anemia and iron deficiency were a hemoglobin concentration of
120 g/L (adjusted for the altitude of 2300 m) (20) and a plasma ferritin concentration of
12 µg/L (21).
Estimation of iron absorption
The stable isotopes of iron were measured in blood by using a magnetic-sector thermal-ionization mass spectrometer (22). Each blood sample (0.10.5 mL) was digested in 210 mL concentrated HNO3 in a titration flask on a hot plate at a subboiling temperature for 24 h. Once dried, the sample was redissolved in 12 mL of a 6-mol HCl/L solution. A polyethylene column 0.4 cm in diameter and 8 cm in length, with a 4-mL reservoir on top, was filled with anion exchange resin and cleaned with 4 mL of an ultraclean 6-mol HCl/L solution and 4 mL ultrapure H2O. The column was then reconditioned in a 6-mol HCl/L solution, and each sample solution was loaded. After the sample solution had passed through, the column was washed with 6 mL of a 6-mol HCl/L solution, and then iron was extracted from the column by using 1 mL of a 0.5-mol HCl/L solution. The collected solution was dried, resuspended in 3050 mL 3% HNO3, and loaded onto the filament for mass spectrometric analysis.
All samples were analyzed for isotopic enrichment by using a thermal-ionization mass spectrometer (model 261; Finnigan MAT, Bremen, Germany). Each sample was manually heated for 1015 min to
3 A to obtain a total ion current of 58 x 10-11 of the iron isotopes on the axial Faraday detector.
Red blood cell iron incorporation was determined by evaluating the recovery of the orally administered isotopes in blood obtained 14 d after isotope administration, as previously described (22). Circulating iron was calculated with the use of a mean blood volume of 65 mL/kg, the measured hemoglobin concentration, and the concentration of iron in hemoglobin (3.47 mg/g).
Statistical analysis
It was estimated that 10 subjects would be a large enough sample for detection of a 2-fold expected difference in iron absorption between treatments with and without ascorbic acid, on the basis of an
of 0.05 and a ß of 0.20 and a variability in iron absorption reported in previous studies (12).
Statistical analyses were performed by using SAS software (release 6.04; SAS Institute Inc, Cary, NC). Values for age, weight, hemoglobin, and plasma ferritin are expressed as means ± SDs and were examined for differences at baseline and after 29 and 59 d. Plasma ferritin concentrations were transformed to their log form to conform to normal distribution. Each subject served as her own control. Differences in iron absorption among time points were tested for significance with the use of a repeated-measures analysis of covariance. Tukeys test was used to compare treatments; P < 0.05 was used to denote significance. Linear regression was calculated for the relation between the percentage change in iron absorption and the ferritin concentration.
| RESULTS |
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| DISCUSSION |
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Before conducting this long-term study with stable isotopes in the field, we used radioactive iron isotopes to show that ascorbic acid from limeade with a Mexican meal was effective at increasing iron absorption in iron-replete adult men. Adding 25 mg ascorbic acid once a day did not improve iron absorption significantly, but adding 25 mg ascorbic acid to each of 2 meals per day increased iron absorption from 3.1 ± 6.2% to 7.4 ± 8.2% (P < 0.05) (O García, LH Allen, JL Rosado, unpublished observations, 2001). In the present study, the addition of 2 daily doses of ascorbic acid as limeade to typical rural Mexican meals for 2 wk increased iron absorption in ID women from 6.6 ± 3.0% to 22.9 ± 12.6% (P < 0.001). The percentage change in iron absorption and the initial plasma ferritin values showed, as expected, that subjects with a low initial concentration of ferritin had a better response to ascorbic acid concentrations. Thus, the effect of ascorbic acid from limeade was stronger in the subjects with more severe iron deficiency and also when the limeade was given for 2 wk rather than in a single meal (P < 0.05). The long-term administration of ascorbic acid as limeade tripled iron absorption, whereas only a 2-fold increase was found in the 2 short-term studies. Our results do not agree with those of Cook et al (29), who found less effect after 2 wk. The meal in their study had a lower content of inhibitors (< 500 mg) than the meal used in the present study, and their subjects were not iron deficient.
Although ascorbic acid improves iron bioavailability, the effectiveness of increasing ascorbic acid intake to improve iron status has not been shown. Cook et al (29) found that providing 2 g synthetic ascorbic acid/d with meals for 16 d did not increase serum ferritin in non-ID volunteers eating self-selected diets. The lack of response was not shown to be due to an adaptation to the high ascorbic acid intake, because iron absorption from single meals could still be stimulated by ascorbic acid at the end of the 16 wk. Similarly, the consumption for 8 wk of 100 mg synthetic ascorbic acid/d, divided among 3 meals per day, by healthy female volunteers failed to increase serum ferritin (30). It is probable that the effect of ascorbic acid may be more evident in ID populations, such as the population of rural Mexico, who consume a diet high in iron absorption inhibitors and low in ascorbic acid (12). However, supplementation with 500 mg ascorbic acid, divided into 3 doses per day, for 5 wk failed to improve iron status in women with low iron stores as assessed by serum ferritin (31). Thus, the effectiveness of ascorbic acid in improving iron status over the long term is uncertain. The long-term effect of ascorbic acid on iron status should be more evident with the consumption of diets with a high content of inhibitors (10). We did see an effect of ascorbic acid on iron absorption, although we did not show an increase on plasma ferritin concentrations at the end of the 2 wk, probably because the study cases were too short for significant changes to develop. According to the percentage iron absorption determined in the present study, a span of
2 mo would be needed to show an increase in plasma ferritin.
In conclusion, the intake of 25 mg ascorbic acid as limeade twice a day for 2 wk more than doubles iron absorption from typical Mexican meals. The increase in the efficiency of iron absorption, combined with the high content of iron and iron inhibitors present in rural Mexican diets, justifies testing the effectiveness of increasing ascorbic acid intake in the form of limeade as a practical approach for improving iron status in ID populations, such as the population of rural Mexico.
| ACKNOWLEDGMENTS |
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| REFERENCES |
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