*Department of Pediatrics, Washington University School of Medicine, St Louis, USA
†University of Colorado, Denver, USA
‡College of Medicine, University of Malawi, Blantyre.
Received 4 February, 2010
Accepted 28 April, 2010
Address correspondence and reprint request to Dr Mark Manary, Department of Pediatrics, St Louis Children's Hospital, One Children's Place, St Louis, MO 63110 (e-mail: email@example.com).
The work was supported by the Allen Foundation.
The authors report no conflict of interest.
Whole-body zinc homeostasis is maintained primarily by short-term regulation of absorption of bioavailable ingested zinc and by both short- and long-term regulation of the quantity of endogenous zinc excreted via the intestine (1,2). The latter is frequently termed “endogenous fecal zinc” (EFZ), and this normally decreases when the quantity of absorbed zinc decreases and vice versa.
Phytate comprises 1% to 2% of total cereal and legume content and chelates divalent cations such as zinc. Phytate is not broken down in the digestive tract and forms insoluble complexes with zinc (3). Phytate limits absorption of dietary zinc and may limit the reabsorption of zinc endogenously secreted into the intestinal lumen. Malawian children, consuming a habitual high-phytate diet, were found to have large EFZs, and the large EFZ was perturbing zinc homeostasis rather than preserving it (4). In this study, the EFZ of the same children was measured after dietary phytate reduction to test the hypothesis that dietary phytate reduction would result in reduced EFZ in this population of children likely to be zinc deficient.
SUBJECTS AND METHODS
Subjects were 10 healthy rural Malawian children ages 2 to 5 years who presented at the Mpemba Health Center in southern Malawi for immunizations. Eligible children were those who reported consuming animal source foods less than once per month and resided in Kalisero in April to June 1999. All of the children approached were eligible; this dietary criterion was meant to exclude the few children consuming a diet atypical of the area. Mothers were subsistence farmers living in mud and thatch homes without electricity or access to transportation, which is typical of most rural Malawians.
This was a prospective observational study in 10 children who served as their own controls. The primary outcome was change in EFZ. The sample size was chosen to detect a 20% change in EFZ with 95% specificity and 80% power, assuming the variances were similar to those in a hospital-based study in Malawi (5).
Diet quality was determined with a food-frequency questionnaire, as previously described (4). The 6 most commonly consumed foods were unrefined maize flour, red kidney beans, peanut flour, tomatoes, bananas, and Chinese cabbage. These foods accounted for a majority of the dietary energy intake. Upon enrollment, mothers were provided with rations of these foods for 6 days to standardize the diet with minimal disruption of typical consumption habits.
After this 6-day period, each child attended the local health center for an entire day where he or she received 3 meals and 2 snacks. The meals were prepared from the 6 foods identified from the questionnaire. A weighed food record was kept for each of the children and food samples were analyzed for zinc content. At midday on day 1, each child received a precisely measured, sterile intravenous dose of ≈170 μg 70Zn (Martin Marietta Energy Systems, Oak Ridge, TN). A blood sample was obtained from each child just before administering the 70Zn. The subsequent days of the study were completed at home.
A blue fecal marker was given 72 hours after administration of the intravenous zinc and a second marker was given exactly 4 days later. All of the stools were collected between the markers in zinc-free plastic bags by mothers and caretakers. A clean-void midstream urine sample was also collected each day, and stool was collected by study staff. Specimens were frozen and transported unprocessed to the laboratories at the Center for Human Nutrition in Denver, Colorado. The study investigator and research nurse were continuously present in the village during specimen collection to ensure complete collection.
Following the completion of the sample collection, families continued to consume their habitual (standardized) diet for 40 days, except that food preparation techniques to reduce the phytate in the maize were practiced. After consuming a reduced-phytate diet for 40 days, the measurement of EFZ was repeated, keeping a weighed food record and administering the intravenous zinc and blue fecal markers as stated above.
To reduce phytate content of unrefined maize flour, 1 part flour was mixed with 4 parts water. One gram of phytase (BASF, Morristown, NJ) was added per 5-kg flour and the flour slurry set for 1 hour. The water was then decanted and the flour was either used immediately for cooking or sun-dried on mats and stored for later use. Phytate reduction, meal preparation, and meal consumption were under the direct supervision of our study team for 8 hours/day every day. Families used the same method of reducing the phytate content every time food was prepared. Forty samples of cooked food from a variety of mealtimes before and after phytate reduction were saved and analyzed for phytate content by the method of Hotz (6).
Dietary intake was determined using a 3-day, 24-hour recall method with food models and standard measurement vessels used as aids for the mothers (6), administered 20 to 25 days after the completion of the stool sample collection for habitual high-phytate diet. The procedures for analyses of isotopic enrichment in stool and urine specimens, as well as the calculations of EFZ, have been described previously (4,5).
Data were expressed as mean ± SD. The paired Student t test was used to compare EFZ before and after phytate reduction. Statistical differences were considered to be significant at P < 0.05. The study was approved by the College of Medicine Research and Ethics Committee of the University of Malawi and the Human Studies Committee of Washington University.
Ten children were enrolled (Table 1). No differences in taste or appearance of food prepared with phytate-reduced flour were noted by mothers or children. Children consumed 5.9 ± 2.1 and 1048 ± 325 kcal zinc per day at the midpoint of the study (repeat dietary recall). Meat, milk, or eggs were reported to be consumed less than once per month (food frequency questionnaire).
The mean phytate content of the maize used habitually was 1391 ± 78 mg/100 g flour and the phytate-reduced maize contained 50 ± 49 mg/100 g flour dry weight. This is equivalent to 3.5% of the phytate content of the unprocessed maize flour. Total dietary phytate was estimated to be 1390 mg/day on the habitual diet and 490 mg/day on the reduced phytate diet, a 65% reduction when compared to the habitual diet. The habitual diet had a phytate:zinc molar ratio of 23.0 ± 2.2, and the reduced phytate diet had a phytate:zinc molar ratio of 7.6 ± 1.4. There was no change in EFZ on the reduced phytate diet when compared to the habitual high-phytate diet (Table 1).
Dietary phytate reduction did not affect EFZ in Malawian children consuming a plant-based diet who were at risk for zinc deficiency. The primary limitation of the study is that concomitant dietary zinc absorption data are not available in these children, which would allow for interpretation of EFZ with respect to net zinc retention. The absolute value of EFZ found here is similar to that found in Malawian children previously on low- and high-phytate diets (4,5,7). In previous studies, EFZ was shown to perturb zinc homeostasis rather than maintain it. If perturbations of EFZ were the result of high dietary phytate, then we would have expected lower values of EFZ in these Malawian children, who were judged to be at risk for zinc deficiency on the basis of their modest plasma zinc measurements, short stature, and habitual diet with poor zinc bioavailability. However, because we did not measure dietary zinc absorption, we cannot be certain whether EFZ was inappropriately high. Because of this these results must be considered preliminary.
Because participation in this study required concerted commitment on the part of mothers and the study team, only a small number of children were included. A cross-over study design was not used because mothers were working daily with research staff to remove dietary phytate from maize and it would have been unacceptable for the mothers to remove dietary phytate from some children's diets, but not from others.
The value of EFZ found in these children was 85 μg/kg/day, compared to the estimate of 34 μg/kg/day used by the Institute of Medicine to determine the dietary recommended intakes for children 1 to 8 years (8), 2.5-fold greater. Given that this population was likely to be zinc deficient, the magnitude of EFZ measured on the reduced phytate diet supports the notion that impaired gut function was compromising normal zinc homeostatic mechanisms.
Our findings are consistent with a previous study in Guatemalan school-age children at risk for zinc deficiency, in which the consumption of reduced-phytate maize for 56 days showed no change in EFZ when compared to control children (9).
The 40-day period allowed adequate time for the body to habituate and adjust to the low-phytate diet. This strengthens the validity of the finding, suggesting that dietary phytate does not affect EFZ in this environment. By assessing the same child before and after the dietary intervention, we were better able to control for physiological determinants of EFZ that are poorly understood, such as factors determined by the host genetic background or local effects of the microbiota in the gut.
A role for EFZ in perturbing zinc homeostasis, and thereby contributing to zinc deficiency, has been suggested in this population recently (7). Given the findings presented in this study, as well as previous findings, it is unlikely that dietary phytate is limiting the conservation of endogenous secreted zinc in the gut in this population.
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