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Community and International Nutrition

Weekly Iron Supplementation Does Not Block Increases in Serum Zinc Due to Weekly Zinc Supplementation in Bangladeshi Infants¹

Abdullah H. Baqui^*,,2, Christa L. Fischer Walker^*, K. Zaman, Shams El Arifeen, Hafizur Rahman Chowdhury, Mohammed A. Wahed, Robert E. Black^* and Laura E. Caulfield^*

^* Johns Hopkins Bloomberg School of Public Health, Department of International Health, Baltimore, MD and ICDDR,B: Centre for Health and Population Research, Dhaka, Bangladesh

²To whom correspondence should be addressed. E-mail: abaqui{at}jhsph.edu.

	ABSTRACT

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Because infants and young children in many developing countries are deficient in both iron and zinc, and zinc can affect iron metabolism, evaluation of optimum strategies to simultaneously supplement iron and zinc is an important public health priority. This study evaluated the efficacy of weekly supplementation of iron or zinc or both on iron, zinc, and copper status in Bangladeshi infants. In a double-blind, randomized, controlled community trial, 6-mo-old infants were assigned to receive weekly supplements of 1 mg riboflavin (control, n = 82) or 1 mg riboflavin + 20 mg iron (n = 83), 20 mg zinc (n = 83), or both (n = 85) for 6 mo. Hemoglobin, serum ferritin, transferrin receptor, zinc, and copper concentrations were measured at baseline and at the end of intervention. Serum Zn increased in both groups receiving zinc; the increase was greatest among children with low baseline serum zinc concentration. Iron status indicators did not differ among the groups before or after 6 mo of supplementation. Supplementation with either zinc or iron decreased serum copper after 6 mo. Joint supplementation did not alter the individual effects of iron or zinc supplementation in these Bangladeshi children. However, the dosing regimen may not have been adequate to achieve the desired biochemical effects.

KEY WORDS: • supplementation • iron • zinc • hemoglobin • copper

Deficiencies of both iron and zinc are highly prevalent, especially in developing countries (1). An estimated 2 billion people are iron deficient (2) and an estimated 1.2 billion are at risk of inadequate zinc intake (3) with the majority of both of these being women and children. The diets of infants and children, especially during the weaning period, do not contain adequate amounts of animal foods, which are good sources of iron and zinc; thus, over time, deficiencies of both iron and zinc develop. Iron deficiency in children can impair sensorimotor development, decrease physical activity, lower resistance to infection, increase rates of iron-deficiency anemia, and in the case of severe anemia, increase risk of mortality (4). Zinc deficiency leads to growth retardation and a decrease in immune function, impairing the prevention of and recovery from infectious diseases (5–7).

Daily iron supplementation is recommended for children living in regions characterized by iron-deficiency anemia beginning at 6 mo of age (2). However, some have argued that intermittent supplementation (e.g., weekly) might effectively prevent anemia and be more feasible and acceptable (8). Randomized trials demonstrated that supplemental zinc can reduce the duration of diarrhea and prolong the time to the next episode, leading to the speculation that intermittent supplementation with zinc might also prove effective in preventing morbidity in young children. Combining iron and zinc supplements, although a pragmatic need, may not be effective if they interfere with each other (9,10). Iron and zinc have similar absorption mechanisms and may compete for absorption (11). Iron and copper also compete for absorptive pathways, and there may be biochemical or functional consequences when supplementation levels are excessive (9). High levels of zinc supplementation were shown to decrease plasma copper concentration in animals and adults but only 1 of 4 studies of zinc supplementation in children showed a decrease in plasma copper (12–15).

We conducted a randomized controlled trial of weekly iron and zinc supplementation for 6 mo among 6-mo-old Bangladeshi infants and examined their single and combined effects on morbidity, child development, and biochemical outcomes. Previously, we reported the effects of supplementation on morbidity (16) and development (17), and briefly reported on overall biochemical outcomes (16). This paper presents the effects of iron and/or zinc supplementation on iron, zinc, and copper status indicators in greater detail. Here, we describe the prevalence of iron and zinc deficiency in young Bangladeshi infants and compare the changes in biochemical status among the treated groups and with the control group after 6 mo of supplementation. We present the individual effects and potential interactions of joint supplementation.

	SUBJECTS AND METHODS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Study design, site, and population.

We conducted a community-based, prospective, randomized, controlled trial in the Matlab field research area of ICDDR,B: Center for Health and Population Research. The details of the study population and methods were described previously (16). Briefly, 799 infants aged 5–6 mo were enrolled from participating villages in the Matlab area. An infant was excluded if he/she was fed infant formula, presented with severe malnutrition [mid-upper arm circumference (MUAC)³ < 110 mm], severe anemia [hemoglobin (Hb) concentration < 90 g/L], signs of neurological disorders, physical disability, or chronic illness that might affect feeding, activity, or cognitive development.

Enrolled infants were randomly assigned to 1 of 5 study groups: 1) 1 mg riboflavin alone (control), 2) 20 mg iron and 1 mg riboflavin, 3) 20 mg zinc and 1 mg riboflavin, 4) 20 mg iron, 20 mg zinc, and 1 mg riboflavin, or 5) 20 mg iron, 20 mg zinc, and 1 mg riboflavin plus twice the WHO recommended intake of other micronutrients (18). All infants were administered a weekly dose of their assigned micronutrient syrup directly from a trained community health worker during the weekly visit over the 6-mo period. Compliance was categorized as full, partial, or no dose by the community health worker each week. The ethical committees of ICDDR,B approved the study procedures. All parents of study infants signed written informed consent before participation.

Biochemical substudy.

A subsample of 417 infants was enrolled in the biochemical portion of the study [iron (n = 83), zinc (n = 83), iron and zinc (n = 85), multiple micronutrients (n = 84), or control (n = 82)]. Every other infant recruited into the larger study was asked to be part of this biochemical substudy. The substudy was designed to provide a sample of 60 in each treatment group, which was required to determine an 18% difference in serum zinc concentrations between treatment groups and control and assuming a 10% loss to follow-up. All calculations were made to determine a difference with 80% power at the 95% significance level. Here we excluded children (n = 84) receiving the multiple micronutrient supplements to be able to focus on the single and combined effects of iron and zinc supplementation on iron, zinc, and copper status.

The baseline blood sample was drawn before the start of supplementation and the final blood sample was drawn between 1 and 6 d after supplementation ended. Blood samples were collected in the morning at the ICDDR,B Matlab hospital. All infants were given a meal immediately after the blood draw.

Hb concentration was measured at baseline and after 6 mo of supplementation using HemoCue B (HemoCue AB). Venous blood samples (3 mL) were collected at these time points using nonheparin-treated, trace element–free tubes and disposable plastic syringes. They were collected and allowed to clot for 30 min. Serum was then kept on ice, and centrifuged at 2000 x g for 10 min within 4 h at the Matlab hospital laboratory. Serum was then stored in a trace element–free cryovial at –20°C. The specimens were then transported to the ICDDR,B Dhaka laboratory in cold boxes for biochemical analysis. Serum ferritin (SF) was assessed immunoturbidimetrically using a commercial kit (Roche Diagnostics) in a semiautoanalyzer (HITACHI 902, Roche Diagnostics). Serum transferrin receptor (sTfr) was assessed using the enzyme immunoassay double antibody sandwich method (Ramco Laboratories). Serum zinc (SZn) and copper (SCu) concentrations were analyzed in duplicate by flame atomic absorption spectroscopy (Atomic Absorption Spectrophotometer). Both commercial standards and pooled serum were used for quality control.

It was not possible to obtain 2 blood samples from all children and some samples were found to be hemolyzed or insufficient in quantity. These factors resulted in differing sample sizes across the different biochemical indicators within the substudy. There were 163 children contributing both 6- and 12-mo SZn samples [iron (n = 42), zinc (n = 42), both (n = 41), control (n = 38)], 249 children contributing both 6- and 12-mo Hb samples [iron (n = 68), zinc (n = 64), both (n = 57), control (n = 60)], 156 children contributing both 6- and 12-mo SF samples [iron (n = 38), zinc (n = 40), both (n = 41), control (n = 37)], 168 children contributing both 6- and 12-mo sTfr samples [iron (n = 42), zinc (n = 42), both (n = 41), control (n = 38)], and 162 children contributing both 6- and 12-mo SCu samples [iron (n = 41), zinc (n = 42), both (n = 41), control (n = 38). Overall, children with incomplete blood data were compared with children with complete blood data, and no differences were noted in baseline characteristics. All further analyses were done on children with both baseline and follow-up data for each biochemical outcome of interest.

Statistical methods.

Data were entered using a customized data entry system with necessary range and logic checks. All analyses were done with STATA 8.0 statistical software (19). Baseline characteristics of enrolled children were described. The outcome variables Hb, Sf, sTfr, SZn, and SCu were assessed for normality. For statistical analyses, variables not normally distributed were transformed using a natural logarithm. All data are presented as the mean (or geometric mean if transformed) and SD (or 95% CI if transformed) by supplementation group and time point. All outcome variables were treated as continuous.

To illustrate the observed distribution of changes from baseline to follow-up in these children, we calculated the change in status for each child and each biochemical indicator. However, for statistical comparisons, we analyzed the data using analysis of covariance in which we adjusted each final biochemical marker for its baseline value. Because of the factorial design, our first analysis examined whether there was an interaction between the 2 treatments; we did this by fitting 3 indicator variables, 1 for each treatment and their product. If no interaction was present, we reduced the model and estimated the main effects of iron and zinc supplementation. Subgroup analyses were also performed for SZn, Hb, SF, and SCu in which we stratified the baseline values at the median. All statistical differences were assessed at P < 0.05.

	RESULTS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Baseline sociodemographic and anthropometric characteristics did not differ among children in the 4 groups (Table 1). The infants consumed 82% of the doses given: iron alone, 84%; zinc alone, 83%; iron + zinc, 77%; and control, 84%. Overall, 45% of the infants had low SZn (<9.9 µmol/L) and 50% had low Hb (<105 g/L) at baseline. All biochemical indicators were normally distributed except for SF concentrations; these values were transformed using a natural logarithm for all subsequent analyses. Interactions between zinc and iron were not significant (P > 0.20); therefore, we focused on the main effects of the 2 treatments.

SZn increased in both of the zinc supplementation groups after 6 mo (P < 0.05) (Table 2). This increase was greater among children with baseline serum zinc concentrations < 9.9 µmol/L compared with those with serum zinc concentrations greater than the cutoff point (P < 0.005).

Baseline and postsupplementation Hb concentrations did not differ in any of the groups (Table 3). Hb concentrations also did not change due to the treatments when the children were stratified by baseline Hb concentration.

Transferrin receptor concentration decreased in all groups, but after 6 mo, the concentration did not differ among the groups.

Serum copper.

SCu declined in all supplementation groups (Table 4). In the subgroup analysis by baseline Cu status, copper declined in all supplementation groups in children with higher levels at baseline and it increased among children with lower levels at baseline receiving iron and/or zinc supplementation.

	DISCUSSION

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

After 6 mo of weekly supplementation, zinc supplementation had a positive effect on zinc status, iron supplementation had no detectable effect on the iron status indicators studied, and copper status was negatively affected by supplementation with zinc and/or iron. There was no interaction of iron and zinc when assessed in combination. In the larger study, the combined iron and zinc supplement was associated with lower morbidity and improved child development (17,18). Taken together, the results indicate that the intermittent delivery of a combined iron and zinc supplement can positively affect health and developmental outcomes in late infancy in deficient populations.

There are no other published studies of intermittent zinc supplemented alone with which to compare our results For iron, there were 2 studies assessing weekly iron supplementation that included 6- to 12-mo-old infants (22,23). In a study by Kapur et al. (23) 9- to 36-mo-old Indian children were randomly assigned to receive 20 mg iron supplementation, nutrition education, both supplementation and education, or placebo (supplementation groups vs. placebo are discussed here). After 16 wk of weekly supplementation, children receiving iron supplementation maintained, but did not increase their Hb concentration, whereas children not receiving iron became more iron deficient (effect of iron, P < 0.05); however, SF declined in both iron groups, regardless of supplementation. In a study by Thu et al. (22) 6- to 24-mo-old children in Vietnam were randomly assigned to receive placebo or 20 mg iron, 17 mg zinc, 1700 µg retinol, and 20 mg vitamin C 1 time/wk for 12 wk (daily doses were assessed but not reported here). In that study, among infants 6–12 mo of age at baseline, both Hb and SZn concentrations increased significantly with 12 wk of supplementation (109.2 vs. 123.2 g/L, P < 0.05, and 13.21 vs. 17.49 µmol/L, P < 0.05, respectively); there was no effect in the placebo group. Because micronutrients were given together, individual effects could not be assessed.

Intermittent iron supplementation did not affect changes in iron status indicators over time, and the addition of zinc to the supplement had no differential effect. This was unexpected because weekly iron supplementation is efficacious. Overall compliance was good, and there is no evidence that absorption was limited; however, one limitation of the study is that we did not collect data on absorption. It is possible that this population was deficient in other nutrients that could be limiting the hematologic responses. More research is required to understand specifically which nutrient(s) may be limiting. There is much discussion about the cutoff points used for Hb to define anemia in infancy. Because our previously published work showed functional responses to supplementation, these data add to the growing body of literature that suggests additional work is required to define low iron status and anemia in y 1 of life (17). Our results are also limited by the biochemical indicators we used; additional indicators may have improved our understanding. One additional limitation of this study is that we did not have information on acute phase reactants for these infants. Although these data would be helpful to explain changes, or lack of changes, in individual SF levels, they are not compulsory for population-level interpretation (25).

There are no published studies of the effect of weekly supplementation on copper status among infants in this age group. Zinc supplementation for 14 d for the treatment of diarrhea did not decrease plasma copper concentrations in 3 of 4 published studies (12–15). The infants in our study had slightly lower serum copper concentrations than the children in 2 of the studies in which there was no effect of zinc supplementation on copper status, and copper status was similar to that reported by Bhutta et al. (15) in a study of very malnourished infants. A small decline in serum copper was observed in all infants, yet when stratified with baseline status, this was observed only among infants with higher SCu status at baseline. Although copper status may be negatively affected by iron and/or zinc supplementation for longer periods of time or at higher doses of the micronutrients, 6 mo of supplementation with 20 mg of iron and/or zinc does not appear to have a significant effect among infants with lower copper status.

Weekly iron supplementation in preschool age children was shown to be efficacious, but less effective than daily supplementation when compliance is a problem (8). Compliance was not a problem in this study, i.e., >80% of supplements were consumed by the children; however, this was an efficacy study and children were fed the supplements by a community health worker. In situations in which > 1 micronutrient may be administered in a supplement or simultaneously, it is important to confirm the safety and efficacy of this delivery modality. Further research assessing the efficacy and safety of different doses of both iron and zinc is needed to achieve the highest biochemical and functional responses in this and similar populations.

	ACKNOWLEDGMENTS

 
The authors acknowledge the assistance of Dr. M. Yunus, Head, Matlab Health Research Program, ICDDR,B for supervision of field work and Nazma Begum, Data Manager, Child Health Unit, ICDDR,B for assistance with data management and analysis.

	FOOTNOTES

 
¹ This study was conducted at ICDDR,B: Centre for Health and Population Research with funding from the U.S. Agency for International Development and the Nutricia Foundation.

³ Abbreviations used: Hb, hemoglobin; MUAC, mid- upper arm circumference; SCu, serum copper; SF, serum ferritin; sTfr, serum transferrin receptor; SZn, serum zinc.

Manuscript received 26 January 2005. Initial review completed 20 April 2005. Revision accepted 28 June 2005.

	LITERATURE CITED

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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19. STATA 8.0 8.0 ed. STATA Corporation College Station, TX.

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This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Baqui, A. H. Articles by Caulfield, L. E. Search for Related Content PubMed PubMed Citation Articles by Baqui, A. H. Articles by Caulfield, L. E. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB COPPER, ELEMENTAL IRON RIBOFLAVIN ZINC COMPOUNDS ZINC, ELEMENTAL Medline Plus Health Information Dietary Supplements