Abstract
Background:
The 2006 Mexican National Health and Nutrition Survey documented a prevalence of zinc deficiency of almost 30% in children under 2 years of age.
Objective:
We sought to validate a food frequency questionnaire (FFQ) for quantifying dietary bioavailable zinc intake in 2-year-old Mexican children accounting for phytic acid intake and using serum zinc as a reference.
Methods:
This cross-sectional study was nested within a longitudinal birth cohort of 333 young children in Mexico City. Nonfasting serum zinc concentration was measured and dietary zinc intake was calculated on the basis of a semiquantitative FFQ administered to their mothers. The relationship between dietary zinc intake and serum zinc was assessed using linear regression, adjusting for phytic acid intake, and analyzed according to two distinct international criteria to estimate bioavailable zinc. Models were stratified by zinc deficiency status.
Results:
Dietary zinc, adjusted for phytic acid intake, explained the greatest proportion of the variance of serum zinc. For each milligram of dietary zinc intake, serum zinc increased on average by 0.95 μg/dL (0.15 μmol/L) (p = .06). When stratified by zinc status, this increase was 0.74 μg/dL (p = .12) for each milligram of zinc consumed among children with adequate serum zinc (n = 276), whereas among those children with zinc deficiency (n = 57), serum zinc increased by only 0.11 μg/dL (p = .82).
Conclusions:
A semiquantitative FFQ can be used for predicting serum zinc in relation to dietary intake in young children, particularly among those who are zinc-replete, and when phytic acid or phytate intake is considered. Future studies should be conducted accounting for both zinc status and dietary zinc inhibitors to further elucidate and validate these findings.
Introduction
The 1999 National Nutrition Survey reported a 34% prevalence of low serum zinc levels among Mexican children under 2 years of age
Zinc, an essential nutrient that participates in numerous enzyme systems 5,6 and contributes to the regulation of cell proliferation, plays a key role as a necessary nutrient for growth, brain development, and immunity. 7,8 Moderate zinc deficiency in preschool-aged children is common in developing countries 8,9 and can delay linear growth, inhibit the immune response, suppress appetite, and reduce the capacity to detect flavors. 10 -12
The Food and Agriculture Organization (FAO) and the World Health Organization (WHO) have classified diets into three categories depending on whether zinc bioavailability is low, moderate, or high. These categories take into account absorption enhancers, absorption inhibitors, and the phytic acid: zinc ratio in the diet. 13 Primary zinc deficiency is common in young children 14 , as zinc concentrations in breastmilk reach >3 mg/L at the beginning of lactation but drop to < 1 mg/L throughout the first 6 months of life. 15 Infants of this age also depend on other foods in order to satisfy their physiological requirements; thus, it is important to provide them with quality complementary feeding. Foods with low zinc and high phytate levels will lower zinc absorption to less than two-thirds of intake and are likely to result in moderate zinc deficiency. 14
Evaluating the risk of zinc deficiency in children requires analysis of their dietary histories (breast and complementary feeding patterns) and gastrointestinal pathology reports. 16 Despite the widely documented lack of adequate biomarkers for measuring zinc, serum zinc has been found to be the most suitable for evaluating zinc status in infants and young children 17 and is recommended by both WHO and the International Zinc Consultative Group (IZiNCG). 18,19
Given the public health importance of zinc in Mexican children and considering that food frequency questionnaires (FFQs) offer a low-cost, easily applicable tool for estimating usual zinc intake, this study was aimed at validating an FFQ to estimate the dietary zinc intake of young children using serum zinc as a reference biomarker for zinc concentration. The association between serum zinc and dietary zinc parameters was analyzed to account for bioavailable zinc, adjusted for phytic acid intake, and compared with recognized international criteria.
Methods
Study Population
This cross-sectional study was nested in the second birth cohort (1999–2001) of the Early Life Exposure in Mexico to Environmental Toxicants (ELEMENT) Project. We evaluated 333 2-year-old children affiliated with the Mexican Social Security Institute in Mexico City, who attended study visits at the National Institute of Perinatology Isidro Espinosa de los Reyes (INPer) between 2001 and 2003. 20,21 During morning visits, fasting venous blood samples were drawn from children and mothers answered questions regarding their children’s usual dietary intake during the previous 3 months. This study was evaluated and approved by the Research, Ethics and Biosafety Committees of the National Institute of Public Health of Mexico (INSP) and participating institutions. Mothers of the cohort-enrolled children were informed of the study procedures and objectives and signed an informed consent prior to participation.
Assessment of Dietary Zinc
To estimate dietary intake in children, ELEMENT used a semiquantitative FFQ that was originally validated by Hernández-Ávila et al. 22 , using the Willett methodology 23 , for adult women of low to medium socioeconomic level residing in Mexico City.
Administered by trained, standardized personnel, the FFQ included 116 foods grouped into 10 categories (dairy products, fruits, vegetables, legumes, cereals, sweets, beverages, fats, snacks, and eggs and meats).
The zinc content of each item on the list (for a standard portion size of one customary unit, cup, slice, piece, etc.) was obtained mainly from food composition tables supplied by two sources: 25 the US Department of Agriculture (USDA) and the Mexican National Institute of Nutrition and Medical Sciences Salvador Zubirán (INNCMNSZ). 26 The data were compiled by specialized personnel at INSP.
Average daily dietary nutrient (e.g., zinc, phytic acid) intakes were calculated with a computer program developed at INSP. Predetermined food portion sizes were applied to mothers’ FFQ responses. The following values were used to estimate the equivalent frequencies consumed per day: 0, never; 0.016, less than once a month; 0.08, two or three times a month; 0.14, once a week; 0.43, two to four times a week; 0.8, five or six times a week; 1, once a day; 2.5, two or three times a day; 4.5, four or five times a day; and 6, six or more times a day. Next, these frequencies were multiplied by the nutrient content in each food item and summed over all food sources of the nutrient.
Several validation studies recommend the incorporation of data on multivitamin intake. 27 However, only three children in our study (< 1%) had taken zinc-containing vitamins and the doses were minimal. Therefore, we did not include a multivitamin-use variable in our analyses.
Estimation of Bioavailable Zinc
Phytate:zinc molar ratios were calculated using the IZiNCG equation (phytate/660)/(zinc/65.4) 19 , which takes into account the molecular weight of both components. Bioavailable zinc in the children’s diets was then estimated using two international criteria. Bioavailable zinc (mg/day) according to WHO criteria: 50% zinc absorption where phytate:zinc molar ratios fall under 5; 30% absorption where they range from 5 to 15; and 15% absorption where they exceed 15. 28 Bioavailable zinc (mg/day) according to IZiNCG criteria: 31% zinc absorption in children where phytate:zinc molar ratios range from 5 to 18 (corresponding to mixed diets), and 23% absorption where they exceed 18 (corresponding to vegetarian and whole-grain diets). 18
Serum Zinc Levels
Whole blood samples were centrifuged and serum was isolated at the INPer laboratory, where zinc concentrations were determined by flame atomic absorption spectrometry (FAAS), the technique deemed most suitable for this procedure. 29 Serum zinc values were used as the criterion measure, and children with values < 65 μg/dL were classified as having zinc deficiency. 30
Covariates
Maternal characteristics, such as mother’s age and education at delivery, and infant weight and length at birth were recorded. Weight and length were also measured at 24 months. WHO Anthro software, version 3.2.2 (January 2011), was used to evaluate the nutritional status of the participants.
Statistical Analysis
Descriptive statistics (means and standard deviations) were computed for zinc and phytate levels, and the rates of low zinc intake (below the EAR of 2.5 mg/day)
Additionally, models were stratified by zinc status to assess whether the FFQ predicted the relationship between dietary and serum zinc differently among children who were zinc replete compared with those who were zinc deficient. All statistical analyses were performed with STATA, version 11.0.
Results
Table 1 shows a list of the principal sources of zinc (≥
Foods With Zinc Content
Table 2 describes the relevant demographic characteristics and dietary parameters of the sample population. Mean (± SD) zinc intake was 8.0 ± 2.3 mg/day, with only one child below the EAR of 2.5 mg/day. 31 Phytic acid intake was 545.2 ± 190.1 mg/day, with an average phytate:zinc molar ratio of 7 (interquartile range, 5.4 to 8.4). According to IZiNCG criteria, only 31% of the zinc consumed was absorbed, and the average amount of bioavailable zinc consumed was 2.5 ± 0.7 mg/day. By WHO criteria, 97% of the children had zinc absorption of 50% (phytate:zinc molar ratio < 5). The variance in mean bioavailable zinc estimated by the two criteria was significantly different (p = .02). The average serum zinc level was 79.6 ± 16.1 μg/dL (12.2 ± 2.5 μmol/L), and the level was higher, though not significantly, in females (80.3 ± 16.3 μg/dL, 12.3 ± 2.5 μmol/L) than in males (78.9 ± 16.3 mg/dL, 12.1 ± 2.5 μmol/L) (p = .41). Serum zinc deficiency was present in 17% of children, with no difference by sex (females 15.5% and males 18.7%, p = .43).
Characteristics and Dietary Parameters Estimated by a Semiquantitative Food Frequency Questionnaire Among Mexican Children at 2 Years of Age (n = 333).
Table 3 shows the results of linear regression models used to compare various methods of dietary zinc estimation as predictors of serum zinc. Total dietary zinc (mg/day), as estimated directly from the FFQ, was not a statistically significant predictor of serum zinc in the full sample or when stratified by zinc status. After adjustment for phytic acid intake, for every additional milligram of zinc consumed, serum zinc increased on average by 0.95 μg/dL (0.15 μmol/L) (p = .06). Among children without zinc deficiency (n = 276), the increase was 0.74 μg/dL (p = .12) for each milligram of zinc consumed, whereas among children with zinc deficiency (n = 57), serum zinc increased by only 0.11 μg/dL (p = .82).
Association Between Serum Zinc and Dietary Zinc Parameters, Stratified by Zinc Status, Among Mexican Children at 2 Years of Age.
For each unit increase in the phytate:zinc molar ratio, serum zinc decreased on average by 0.69 μg/dL (0.11 μmol/L) (p = .13), indicating that for the same amount of zinc intake, higher phytic acid intake may decrease zinc bioavailability. In the models stratified by zinc status, this negative relationship was present in children without zinc deficiency (β = –0.74, p = .1) not in children with zinc deficiency (β = 0.04, p = .9).
According to WHO and IZiNCG criteria, serum zinc increased by an average of 1.40 (95% CI, –0.26 to 3.08) and 0.68 (95% CI, –1.77 to 3.14) μg/dL, or 0.21 (95% CI, –0.04 to 0.47) and 0.10 (95% CI, –0.27 to 0.48) μmol/L, respectively, for each additional milligram of bioavailable zinc in the diet.
Discussion
This is the first study to validate the use of an FFQ for estimating zinc consumption using a serum zinc reference biomarker in young Mexican children, and it provides the first comparison of zinc bioavailability criteria used by international organizations. In addition, this study offers evidence that phytic acid intake should be accounted for when estimating dietary zinc intake. This is important for populations consuming non-Westernized diets and in developing countries, where diets tend to be more plant-based, as well as for plant-based or vegetarian eaters in developed countries, who tend to consume more phytic acid than omnivores. 32
As this study includes a sample of children from Mexico City, the prevalence of zinc deficiency is lower (17%) than the previously reported national prevalence (34%) because it is expected that urban children would have better nutrition status than children in rural areas of Mexico. Also, even though the mean dietary zinc intake of the sample (8 mg/day) is 1 mg higher than the upper tolerable intake level (UL) of 7 mg/day, this does not reflect an overconsumption of zinc, because the high intake of phytic acid in the Mexican population inhibits the absorption of the majority of the zinc consumed. 3
Earlier studies have validated the use of diverse dietary assessment instruments for dietary zinc evaluation 33 ; of these, the semiquantitative FFQ is the most commonly used in epidemiological studies to evaluate dietary intake, especially long-term intake (> 7 days). Few studies have demonstrated the effects of zinc intake, absorption-related factors, and diet quality on serum zinc concentration. 34,35 This may be due to the fact that few studies in children have characterized usual zinc intake accurately.
The association observed between the FFQ and the reference method in our population demonstrates that dietary zinc, adjusted for phytic acid consumption, is the most suitable predictor of serum zinc in young children. Zinc intake analyses that do not account for intake of phytic acid, the principal inhibitor of zinc absorption, may lead to less accurate estimates of dietary zinc intake, especially in populations with high phytic acid intake. This finding is consistent with a previous study that designed an FFQ to measure zinc intake and recognized that the lack of information on phytic acid intake was an important drawback. 33
The observed associations become more evident when analyzed according to zinc status. Higher zinc intake increased serum zinc only by 15% as much, on average, in the zinc-deficient children compared with the zinc-replete subgroup. Because of the limited sample size of the study, we cannot rule out chance as an explanation for these findings. In addition, apparent zinc deficiency (i.e., low zinc concentrations without necessarily low total body amounts) may be triggered by factors other than diet, including infection 36 , inflammatory response 37 , or other conditions that alter zinc metabolism. 12 In this respect, one of the shortcomings of our study is the lack of information on the children’s recent illnesses, immunological conditions, and inflammatory biomarkers, such as measurements of C-reactive protein. However, we had information on lymphocyte count, so we included this information as a proxy for inflammation in a sensitivity analysis, and the results were similar (data not shown).
Comparing different methods for estimation of zinc bioavailability in our study demonstrated that average intake varies considerably according to the method used, and none of the methods predict serum zinc levels adequately. This may arise from the fact that their cutoff values involve wide calculation ranges and their designs are aimed at adults, not children.
We are aware that FFQs have limitations for the calculation of nutrient intakes, since the respondents’ recall plays a major role in their responses. 27,38 In addition, dietary assessment and evaluation in young children is complicated, given that dietary habits change rapidly in the early years of life, parents share their feeding responsibilities with other adult caregivers and may not be completely aware of their children’s diets, and children do not necessarily consume all of the foods given to them. 39 Our study is not completely free of three well-recognized sources of FFQ-related measurement error: 1) The food portions in a semiquantitative FFQ are predetermined and may not have been recognized or remembered consistently and therefore may have distorted our estimated daily zinc intakes. 2) The instrument was originally designed for epidemiological studies on chronic diseases to evaluate the diets of adult females, a population group that consumes more varied foods than our sample subjects. 3) The FFQ does not measure certain types of foods consumed by children, such as a broadly distributed multifortified milk provided to low-income families (Liconsa fortified milk) and other processed infant foods that tend to be enriched with zinc. However, structured, interviewer-administered questionnaires were used by trained personnel to assist with portion size determination, recall, and capture of common foods not queried on standard forms by prompting for additional information in open-ended questions at the end of the survey.
Based on our findings, a semiquantitative FFQ can be used to predict serum zinc levels in relation to dietary intake of zinc in young children, particularly among those who are zinc-replete, and when phytic acid or phytate intake is considered. Future studies should be conducted accounting for both zinc status and dietary zinc inhibitors to elucidate and validate these findings.
Footnotes
Authors’ Contributions
Alejandra Cantoral and Martha Téllez-Rojo were responsible for the conception and design of the study; Lourdes Schnaas, Teresa Shamah-Levy, and Alejandra Cantoral for acquisition of the data; Alejandra Cantoral, Adrienne Ettinger, Karen Peterson, and Martha Téllez-Rojo for analysis and interpretation of the data; Alejandra Cantoral, Karen Peterson, and Adrienne Ettinger for drafting the manuscript; Karen Peterson and Adrienne Ettinger for critical revision and editing of the manuscript; Alejandra Cantoral and Martha Téllez-Rojo for statistical analysis; and Karen Peterson for obtaining funding.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grants from the National Institutes of Health/National Institute of Environmental Health Sciences (R01-ES007821 and P20-ES018171), the US Environmental Protection Agency (RD83480001), and the Consejo Nacional De Ciencia y Tecnología (CONACYT 41912-M). The contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health/National Institute of Environmental Health Sciences or the US Environmental Protection Agency.