N U TR ITI O N RE S EA R CH 3 4 ( 2 01 4 ) 7 4 –8 4 Available online at www.sciencedirect.com ScienceDirect www.nrjournal.com Children's nutrient intake variability is affected by age and body weight status according to results from a Brazilian multicenter study Michelle A. de Castro a,⁎, Eliseu Verly-Jr. b , Mauro Fisberg c , Regina M. Fisberg d a Department of Nutrition, School of Public Health, University of São Paulo, São Paulo, Brazil Department of Epidemiology, Institute of Social Medicine, State University of Rio de Janeiro, Rio de Janeiro, Brazil c Department of Pediatrics, Federal University of São Paulo, São Paulo, Brazil d Department of Nutrition, School of Public Health, University de São Paulo, Sao Paulo, Brazil b ARTI CLE I NFO A BS TRACT Article history: A major challenge in nutritional studies focusing on children is estimating “true” intake Received 21 May 2013 because the type and amount of foods eaten change throughout growth and development, Revised 18 September 2013 thereby affecting the variability of intake. The present study investigated the hypothesis Accepted 20 September 2013 that age and body weight status affect the ratio of the within- and between-subject variation of intakes (VR) as well as the number of days of dietary assessment (D) of energy and Keywords: nutrients. A total of 2,981 Brazilian preschoolers aged 1–6 years were evaluated in a cross- Children sectional study. Weighed food records and estimated food records were used to assess Diet dietary intake inside and outside of school. Within- and between-subject variations of Nutrition assessment intakes were estimated by multilevel regression models. VR and D were calculated Variation according to age group and body weight status. VR ranged from 1.17 (calcium) to 8.70 (fat) Multilevel analysis in the 1- to 2-year-old group, and from 1.47 (calcium) to 8.95 (fat) in the 3- to 6-year-old group. Fat, fiber, riboflavin, folate, calcium, phosphorus, and iron exhibited greater VR and D in the 3- to 6-year-old group. For energy, carbohydrates, and protein, both within- and between-subject variation increased with increasing age. In both body weight groups, calcium showed the lowest VR. Fat showed the highest VR in nonoverweight/obese children (9.47), and fiber showed the highest VR in overweight/obese children (8.74). For most nutrients, D = 7 was sufficient to correctly rank preschoolers into tertiles of intake. In conclusion, age and body weight status affected the within- and between-subject variation and the VR of energy and nutrient intakes among Brazilian preschool children. © 2014 Elsevier Inc. All rights reserved. Abbreviations: BMI, body mass index; CVb, between-subject coefficient of variation; CVw, within-subject coefficient of variation; D, number of days of dietary assessment; EFR, estimated food record; r, correlation coefficient; Sb2, between-subject variation of intake; SDw, within-subject SD; SDb, between-subject SD; Sw2, within-person variation of intake; WFR, weighed food record; VR, ratio of the within- to between-subject variation of intake. ⁎ Corresponding author. School of Public Health, University of São Paulo, Av Dr Arnaldo, 715 Cerqueira Cesar, 01246-90 São Paulo, SP, Brazil. Tel.: +55 11 3061 7869; fax: +55 11 3061 7130. E-mail addresses: [email protected] (M.A. Castro), [email protected] (E. Verly-Jr), [email protected] (M. Fisberg), [email protected] (R.M. Fisberg). 0271-5317/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nutres.2013.09.006 N U TR IT ION RE S EA R CH 3 4 ( 2 01 4 ) 7 4 –8 4 1. Introduction Dietary intake in infancy and childhood is of scientific interest because of an increasing amount of evidence suggesting that there is a relationship between early exposure to dietary factors and the risk of developing noncommunicable chronic diseases such as obesity, diabetes mellitus, and cardiovascular diseases [1-3]. A major challenge in assessing dietary intake in infants and children is estimating “true” food and nutrient intakes because the type and amount of foods eaten change considerably during growth and development, thus affecting the overall variability of dietary intake. Dietary variability mainly arises from changes in day-today food and nutrient intakes by an individual (withinsubject variation) as well as from differences in usual intake among individuals (between-subject variation) [4]. From a statistical standpoint, within-subject variation is an important source of error in the analysis and interpretation of data [5] because it attenuates measures of association, such as regression coefficients and relative risks, and introduces bias in the proportion of individuals below or above the requirements of intake [6-9]. Within- and between-subject variation estimates can be useful in identifying the number of days of dietary assessment (D) needed to evaluate energy and nutrient intakes in groups of individuals at a given degree of precision [10-12]. For a particular dietary factor, the greater the variance ratio (VR), that is, the ratio of the within-subject variation to the between-subject variation, the greater D is needed to evaluate the intake of the dietary factor with some degree of precision [10,11]. Observational studies conducted in developed countries have shown the influence of age, sex, culture, and socioeconomic status of the child on the magnitude of VR of energy and nutrient intakes, as well as on D [9,12-15]. However, it remains unknown whether the body weight status of a child may influence the VR and D of energy and nutrient intakes. It has been suggested that overweight and obese children have a lower ability to regulate the amount of foods consumed during the day compared with normalweight children [16], which could affect the variation of energy and nutrient intakes. Considering this, our initial hypothesis is that the VR and D of energy and nutrient intakes of Brazilian preschool children would be different between overweight/obese children and nonoverweight children, even after controlling for age and sex effects. In addition, we also hypothesized that older children (3-6 years) will exhibit greater VR and D than younger ones (1-2 years). To investigate our hypotheses, we sought to estimate the within- and between-subject variation of intakes through a multilevel model approach and to calculate the VR and number of days of dietary assessment of energy and nutrients, according to age group and body weight status. With this study, we expect to advance the current knowledge regarding factors related to variability of intake in pediatric populations to help researchers obtain more precise estimates of dietary intake in this group. 2. Methods and materials 2.1. Study population 75 Data were gathered from a large multicenter cross-sectional survey titled “Nutri-Brasil Infância” that was conducted from February to December 2007. The survey was designed to evaluate the nutritional risk by estimating the prevalence of inadequate nutrient intake among children from daycare centers and preschools living in Brazil [17]. The cities investigated were as follows: Manaus (northern region), Recife and Natal (northeast region), Brasília and Cuiabá (eastern-central region), Caxias do Sul (southern region), and Belo Horizonte, Rio de Janeiro, and São Paulo (southeast region). For a total of 3150 children, 350 male and female children between 1 and 6 years of age who attended public and private daycare centers and preschools were invited from each of the 9 cities. Of the 350 children from each city, 250 were from public institutions and 100 were from private ones, in accordance with data from the National Scholar Census conducted in 2005 by the Brazilian Ministry of Education. The number of children invited in each city was based on an estimated 65% prevalence of inadequate intake with a margin of error of 5% and a confidence level of 95%. Owing to the lack of national data regarding the prevalence of inadequate nutrient intake in children and considering the great vulnerability of this life stage group to nutritional deficiencies, we anticipated that 60% to 70% of the Brazilian children would exhibit an intake inadequacy of at least 1 nutrient. A total of 85 daycare centers and preschools were invited and agreed to participate. Both public (n = 54) and private schools (n = 31) were not randomly selected; hence, this study used a nonrepresentative sample. The selection criteria for schools were geographic location (ie, schools located in urban areas), full-time attendance (7-8 h/weekday), and a conventional food service system (ie, the portioning of all foods and beverages at school/center should be performed by the same food service attendant who was trained to portion the same amount of food). Of the 3150 children invited, 92 (3%) were not evaluated because of the lack of parental consent for data collection; hence 3058 children were evaluated overall. This study was conducted according to the guidelines of the Declaration of Helsinki, and all procedures involving human subjects were approved by the Research Ethics Committees at the Federal University of São Paulo and the School of Public Health, University of São Paulo. Written, informed consent was obtained from the parents or guardians of the children evaluated. 2.2. Data collection Data collection occurred on weekdays (Monday to Friday) and was performed by undergraduate nutrition students who were trained by nutrition researchers from local universities before the onset of the study. Structured forms and manuals with instructions for data collection were developed by these researchers, who also closely supervised data collection. 76 2.2.1. N U TR ITI O N RE S EA R CH 3 4 ( 2 01 4 ) 7 4 –8 4 Anthropometric measures Each child's weight and height were measured in duplicate using a calibrated digital scale [G-Tech (Zhongshan Camry Electronic Co., Ltd., China), model Glass-6, accuracy: 100 g)] and a portable wall-mounted stadiometer [Seca (Seca GmBh &., Germany) model 206, accuracy: 1 mm]. The measurement of weight (in kilograms) and height (in centimeters) was performed with the child standing upright on a firm level surface while wearing light clothes without shoes, and internationally accepted techniques were followed [18]. For children younger than 2 years, before calculating body mass index (BMI), 0.7 cm was added to the measurement of height to convert to the length as recommended by the World Health Organization (WHO) [19]. Arithmetic means of weight and height/length were used to calculate BMI values (kg/m2). Based on z-score values of BMI-for-age and cutoffs proposed by WHO [19] (<−2 SD, = −2 SD and ≤+1 SD, >+1 SD and ≤+2 SD, and >+2 SD, respectively), children were categorized as underweight, normal weight, overweight, or obese. 2.2.2. Dietary intake assessment Weighed food records (WFRs) were used to measure children's dietary intake at school mealtime. For this process, 3 samples of each food and beverage offered at the school meal were portioned by the food service attendant and weighed by the undergraduate student on a calibrated electronic scale [Plenna (Plebal Plenna Balanças Comércio Importação e Exportação LTDA., Brazil), model MEA06030, 3 kilogram maximum capacity, accurate to 1 g]. This procedure was performed to standardize the portion size of the foods and beverages portioned by the attendant and to minimize the influence of the attendant's variability in portioning the foods offered at the meal. Arithmetic means of the 3 samples were calculated and considered as the amount of foods and beverages offered to all children. After all foods and beverages were sampled and weighed, the same attendant portioned the foods and beverages and offered them to each child. If a child received a second portion of any food or beverage, this was added to the averaged portion offered. All leftovers/spillages from each plate were collected in individual bags and weighed. The beverages were weighed separately. A proportional estimate of the contribution of individual foods to the total plate waste was calculated for each child. The amounts of foods and beverages eaten at school mealtime were estimated by the difference between the portion offered and the individual plate waste of each child. Dietary intake away from school was evaluated using the estimated food record (EFR) method that was completed by 1 parent, mainly the mother, on the same day that WFR was performed. If the mother was illiterate, the child's father or another adult family member completed the EFR. Instructions for recording in real time were given to the parents. Detailed descriptions of all foods and beverages, such as recipes, cooking methods, household measures, condiments, and brand names, were collected. Quality control of the EFR was conducted during data collection to identify and correct reporting errors. All household measures were quantified in grams or milliliters based on the standardized methods of Pinheiro et al [20] and Fisberg and Villar [21]. Daily energy and nutrient intakes were calculated using the Nutrition Data System for Research software (database version 2007; Nutrition Coordinating Center, University of Minnesota, MN, USA), whose main database is the food composition table that was developed by the US Department of Agriculture. Regional food preparations not included in the database software, such as mixed dishes, were obtained from national publications [20,21] and entered into the program as standard recipes. Nutritional values of regional foods were obtained from the national food composition table (Tabela Brasileira de Composição de Alimentos). A second dietary measurement was performed in about 25% of the sample (n = 788) to allow the estimation of the within-subject variation of nutrient intakes. The second dietary measurement occurred with at least 1 day apart from the first measurement (ie, nonconsecutive days), to obtain more reliable estimates of the within-subject variation [4]. In each city, a maximum of 87 children were randomly selected for reevaluation. Dietary data collection was the same as the first data collection. Therefore, the sample comprised 2270 children with 1 day of dietary intake data and 788 children with 2 days of dietary intake data. Owing to incomplete dietary data (dietary data provided only by the WFR), 77 children (2.5%) were excluded from the analysis. For the present analysis, 2981 children between 1 and 6 years of age were included (1525 boys and 1456 girls), with 2231 having 1 day of dietary measurement and 750 having 2 nonconsecutive days of dietary measurement. 2.3. Statistical analyses Descriptive analysis of means and SD of energy and nutrient intakes were estimated for each age evaluated. To estimate the within- and between-subject variation of intake as well as the number of days of dietary assessment, a Box-Cox power transformation was applied for each nutrient to correct the skewness of the data. After transformation, cholesterol and vitamins A, C, D, E, and K, as well as copper and selenium, did not follow a symmetrical distribution (normal distribution) and could not be further analyzed. The within- and between-subject variation of energy and nutrients was estimated by a multilevel regression model. This model was chosen because it is suitable for nested or grouped data, thus allowing for correlation between units or observations at the same level, that is, for nonindependent data [22]. In this study, dietary data (level 1) were assessed in children (level 2) “nested” within schools (level 3), which were nested within cities (level 4). For each nutrient, the model included the age of the child, as a fixed and a random effect and adjusted for the fixed effect of sex [14]. The multilevel model equation used was developed based on the notation of Goldstein [22]: γijkl ¼ β 0ijkl þ β 1 x jkl þ β 2jkl β 0ijkl ¼ β 0 þ f 0l þ v 0kl þ u 0jkl þ e0ijkl ð1Þ ð2Þ where yijkl is the nutrient intake estimated by the ith dietary measurement of the jth child belonging to the kth daycare/ preschool from the lth city of the study and β0ijkl is the random intercept. The fixed part of the model is represented by β1, the regression coefficient of the variable sex (x), and β2, the regression coefficient of the variable age (z), in years. The terms f0l, ν0kl, u0jkl, N U TR IT ION RE S EA R CH 3 4 ( 2 01 4 ) 7 4 –8 4 and e0ijkl are the random effects of the intercept at each hierarchical level: level 4 (fl), cities; level 3 (νkl), daycare/preschools; level 2 (ujkl), children; and level 1 (eijkl), dietary measurement. All random effects were assumed to follow a normal distribution with a mean equal to 0 and variances represented by σ2f0l, σ2v0kl, σ2u0jkl, and σ2e0ijkl, respectively. In this model, the between-subject variation is the variance at level 2 (Var(ujkl)) and the within-subject variation is the variance at level 1 (Var(eijkl)). Both variances were estimated for each child as a function of the age: Var u jkl ¼ σ2 u0 þ 2z ijkl σu02 ð3Þ Var eijkl ¼ σ2 e0 þ 2z ijkl σe02 ð4Þ where σ2u0 is the variance of the intercept at level 2, z is the age of the child, and σu02 is the covariance between intercept (β0) and slope (β2; ie, regression coefficient of the variable age) at level 2. The term σ2e0 is the variance of the intercept at level 1, and σe02 is the covariance between intercept and slope at this level. Positive values of covariance suggest an increase in within- or between-subject variation of intake with the age of the child, whereas negative values suggest a decrease. The multilevel models used the restricted iterative generalized least squares parameter estimation, which is suitable for unbalanced data sets, that is, for data sets with a variable number of units or observations across levels (eg, number of days of dietary measurements per children). The restricted iterative generalized least squares also accounts for the loss of degrees of freedom in fixed effects estimation and provides unbiased estimates of the random effects [23]. The number of days of dietary assessment (D) to correctly rank individuals into tertiles, quartiles, or quintiles of the distribution of the nutrient intake was calculated for each nutrient using the equation proposed by Black et al [11], which is based on the ratio of the within-subject variation (Sw2) to between-subject variation (Sb2), as well as on the hypothetical correlation coefficient (r) between observed and true intakes: D¼ r2 Sw2 1−r 2 Sb2 ð4Þ The correlation coefficient can be interpreted as a measure of how correctly individuals can be ranked, such as into tertiles, quartiles, and quintiles of nutrient intakes [12]. In this study, the ratio of the within- to between-subject variation (ie, the VR) was calculated according to age group (1-2 years; 3-6 years) and body weight status (nonoverweight/obese; overweight/obese). The age group was defined according to the Brazilian Ministry of Health, which proposes specific food guidelines for children up to 2 years of age. The arithmetic means of Sw2 and Sb2 were used to calculate VR, and r = 0.8 and r = 0.9 were considered in our calculations. These values of correlation were chosen because they correctly classify a high proportion of children into tertiles of the distributions (72% and 80%, respectively) and grossly misclassify a small proportion of them (3% and <1%, respectively) [12]. In this article, the VR, the within-subject coefficient of variation (CVw), and the between-subject coefficient of variation (CVb) were presented. Coefficients of variation were calculated as: CVw = (SDw/mean) × 100; CVb = (SDb/ 77 mean) × 100, where SDw is the within-subject SD and SDb is the between-subject SD. In addition, 4 sets of graphs were constructed plotting the within- and between-subject variation of energy and macronutrients (carbohydrates, protein, and total fat), according to the age of the child. Descriptive analyses were performed using Stata (Statistics/Data Analysis, version 10.0; Stata, College Station, TX, USA), and the multilevel models were fitted by MLwiN (Centre for Multilevel Modeling, version 2.16; University of Bristol, Bristol, United Kingdom). 3. Results The distribution of children according to sociodemographic and anthropometric variables is presented in Table 1. On average (SD), the children were 3.9 (1.0) years of age, and had BMI values of 16.1 (1.7) kg/m2. Boys and girls were almost equally represented within age groups. A total of 68.4% of the children were normal weight, 29.5% were overweight/obese, and 2.0% were underweight. In addition, 74% of the children belonged to low-income families (ie, families with <R$1000.00 Brazilian reals per month; equivalent to 500 US dollars per month). Small differences were observed in the average intake of energy and most nutrients, among children aged 1 to 6 years (Table 2). The average intake of energy increased only about 10% in children aged 1 to 6 years (from 1528 to 1683 kcal/d), whereas the average intake of carbohydrates, protein, and total fat increased about 6% (from 228.60 to 243.90 g/d), 9% (from 55.71 to 60.82 g/d), and 18% (from 45.28 to 53.73 g/d), respectively. The largest increases were observed for copper (40%; from 1.05 to 1.47 mg/d), vitamin B12 (36%; from 4.18 to 5.67 mg/d), and folate (33%; from 414.54 to 550.39 μg/d). Conversely, the SD of energy and nutrients increased substantially in children aged 1 to 6 years, with the exception of carbohydrates and vitamin C. In the youngest group (1–2 years), CVw was quite large compared with CVb, especially for total fat, total fiber, and sodium. Regarding the VR, the values ranged widely from 1.17 for calcium to 8.70 for total fat. Riboflavin had the second and pantothenic acid the third lowest VR (1.73 and 1.83, respectively), whereas zinc exhibited the second and total fiber the third highest VR (7.36 and 6.60, respectively). Overall, 7 days of dietary assessment would be sufficient to achieve r = 0.8 and 16 days to achieve r = 0.9 in the assessment of all but 5 nutrients (total fat, fiber, thiamine, sodium, and zinc), in this age group (Table 3). In the oldest group (3–6 years), CVw was larger than CVb for all nutrients, notably for total fat, total fiber, and zinc. The VR ranged from 1.47 for calcium to 8.95 for total fat. Pantothenic acid exhibited the second and phosphorus the third lowest VR (1.92 and 2.15, respectively), whereas total fiber had the second and zinc the third highest VR (8.41 and 5.52, respectively). A total of 7 days of dietary assessment would be sufficient to achieve r = 0.8 and 18 days to achieve r = 0.9 in the assessment of all but 4 of the nutrients evaluated (total fat, fiber, folate, and zinc; Table 3). Compared with the youngest group, the oldest one showed higher CVw in 72% of the nutrients evaluated. Differences in 78 N U TR ITI O N RE S EA R CH 3 4 ( 2 01 4 ) 7 4 –8 4 Table 1 – Sociodemographic and anthropometric characteristics of children from daycare centers and preschools according to age group Age group a 1-2 y (n = 715) Age (y), mean (SD) Weight (kg), mean (SD) Height (cm), mean (SD) BMI (kg/m2), mean (SD) Sex, n (%) Male Female Body weight status, n (%) c Underweight Normal weight Overweight Obese Family Income (reals/mo), n (%) d <1000.00 ≥1000.00 Dietary assessment, n (%) 1d 2d a b c d 2.5 13.7 90.9 16.5 Total b 3-6 y (n = 2266) (0.3) (1.8) (5.4) (1.6) 4.3 17.2 103.1 16.1 (0.8) (3.4) (7.4) (1.8) 3.9 16.3 100.2 16.1 (1.0) (3.4) (8.6) (1.7) 367 (51.3) 348 (48.7) 1158 (51.1) 1108 (48.9) 1525 (51.2) 1456 (48.8) 16 470 155 74 44 1570 471 181 60 2040 626 255 (2.2) (65.7) (21.7) (10.4) (1.9) (69.3) (20.8) (8.0) (2.0) (68.4) (21.0) (8.6) 501 (70.1) 214 (29.9) 1702 (75.1) 564 (24.9) 2203 (73.9) 778 (26.1) 552 (77.2) 163 (22.8) 1679 (74.1) 587 (25.9) 2231 (74.8) 750 (25.2) One year old (n = 58); 2 years old (n = 657). Three years old (n = 974); 4 years old (n = 836); 5 years old (n = 398), and 6 years old (n = 58). According to the BMI cutoff points for age and sex proposed by WHO [19]. US $1 ≈ 2.5 reals. VR were found between age groups for all nutrients. Total fat, total fiber, riboflavin, folate, calcium, phosphorus, and iron exhibited greater VR and D in the oldest group. Conversely, energy, carbohydrates, protein, thiamine, magnesium, sodium, potassium, and zinc showed greater VR and D in the youngest group. Pantothenic acid, vitamin B6, and niacin showed slight differences in VR but not in the number of days of dietary assessment between age groups. Changes in within- and between-subject variation of energy, carbohydrates, protein, and total fat, according to the age of the child, are illustrated in Fig. For energy, carbohydrates, and protein, both within- and betweensubject variation increased with increasing age. The greatest relative increase was observed for the between-subject variation of energy (58%), carbohydrates (29%), and protein (29%). For total fat, however, the within-subject variation increased from 1 to 6 years, whereas the between-subject variation decreased until around 3.5 years and then increased from this age onward. Higher CVw and lower CVb were observed in overweight/ obese children for around 61% of the nutrients investigated (Table 4). Lower VR was observed among overweight/obese children for energy, carbohydrates, protein, total fat, thiamine, niacin, magnesium, sodium, potassium, and zinc. Calcium had the lowest VR in both body weight status groups, whereas total fat had the highest VR in nonoverweight/obese children and total fiber had the highest in overweight/obese children. In nonoverweight/obese children, all but 5 nutrients (total fat, total fiber, folate, sodium, and zinc) required a total of 7 days of dietary assessment to achieve r = 0.8 and 17 days to achieve r = 0.9. In overweight/ obese children, all but 4 nutrients (total fat, total fiber, folate, and zinc) required 7 days to ensure r = 0.8 and 16 days to ensure r = 0.9 between observed and true intakes. 4. Discussion This was the first study to investigate the influence of age and body weight status on VR of energy and nutrient intakes as well as D among children from daycare centers and preschools living in Brazil. For all nutrients, the VRs were larger than 1.0, indicating that the within-subject variation was higher than the between-subject variation of intake. In fact, the VR was larger for total fat, total fiber, and zinc and smaller for calcium, phosphorus, riboflavin, and pantothenic acid. These findings indicate that Brazilian children consume, on a daily basis, less regular amounts of the main dietary sources of fat (oils and fatty foods), fiber (leafy vegetables and wholegrain cereals), and zinc (meat products and seafood) than the main dietary sources of calcium, phosphorus, riboflavin, and pantothenic acid (namely, milk and dairy products). For most nutrients, the average of intake increased less from 1 to 6 years than the SD of intake. This suggests that the effects of increasing age were less expressive in increasing food consumption than in increasing the variation of intake. It is possible that young children have a low ability to selfregulate food intake, especially when they are fed by caregivers and that this effect progressively declines as children get older and more autonomous in controlling their own intakes. Caregivers' attitudes that encourage children to clean their plate or stimulate food consumption through 79 N U TR IT ION RE S EA R CH 3 4 ( 2 01 4 ) 7 4 –8 4 Table 2 – Energy and nutrient intakes according to children's age Nutrients Age (y) 1 (n = 58) Mean SD 2 (n = 657) Mean SD 3 (n = 974) Mean SD 4 (n = 836) Mean SD 5 (n = 398) Mean SD 6 (n = 58) Mean SD Energy (kcal/d) 1528 456 1507 461 1588 479 1614 473 1635 490 1683 498 Carbohydrates (g/d) 228.60 81.21 219.34 74.46 230.79 79.25 232.49 71.33 240.53 76.97 243.09 69.92 Protein (g/d) 55.71 16.28 56.93 20.37 58.50 20.12 60.19 21.97 58.69 20.97 60.82 22.04 Total fat (g/d) 45.28 14.79 46.79 18.99 49.93 19.32 51.35 19.75 51.01 19.86 53.73 21.19 Total fiber (g/d) 16.05 7.50 16.30 7.47 17.19 7.59 18.51 8.05 18.81 8.13 19.50 7.80 Cholesterol (mg/d) 154.63 76.90 168.17 116.93 168.06 102.42 172.31 108.31 165.10 105.10 172.55 101.72 Thiamine (mg/d) 1.16 0.44 1.22 0.60 1.28 0.70 1.29 0.56 1.28 0.64 1.37 0.48 Riboflavin (mg/d) 1.48 0.51 1.71 0.88 1.66 0.85 1.64 0.70 1.58 0.64 1.68 0.69 Panthotenic acid (mg/d) 4.22 1.23 4.69 2.26 4.58 3.17 4.45 2.09 4.25 1.77 4.23 1.65 Vitamin B6 (mg/d) 1.20 0.35 1.30 0.52 1.34 0.74 1.35 0.60 1.35 0.61 1.32 0.55 Vitamin B12 (mg/d) 4.18 3.74 6.90 13.57 5.68 8.85 5.38 8.12 4.63 6.81 5.67 8.98 Vitamin C (mg/d) 374.33 1008.79 238.99 708.91 296.52 898.11 333.95 901.82 284.09 783.49 321.61 709.51 665.84 577.00 973.03 1836.44 791.82 1199.02 741.10 1114.95 645.03 943.67 704.57 1256.47 Vitamin A a (μg/d) 4.52 2.56 5.24 4.26 5.51 5.87 5.38 6.58 4.93 5.51 5.02 7.71 Vitamin D b (μg/d) Vitamin E c (mg/d) 4.16 1.55 4.76 2.77 4.89 4.31 4.81 2.81 4.66 2.45 4.53 1.90 46.46 24.92 54.30 49.74 58.18 55.55 64.60 71.27 63.76 72.92 56.32 41.98 Vitamin K d (μg/d) Niacin equivalents (mg/d) 26.57 7.40 27.15 10.24 27.83 11.07 28.22 10.66 27.55 9.71 28.10 9.36 Dietary folate equivalents (μg/d) 414.54 157.96 444.88 189.84 466.20 270.88 486.92 189.71 503.17 186.47 550.39 186.81 Calcium (mg/d) 692.42 322.64 766.36 362.04 747.38 357.02 720.06 358.47 700.13 338.62 723.27 334.53 Phosphorus (mg/d) 893.74 273.02 962.88 344.60 959.15 328.45 961.21 338.05 939.87 321.98 969.76 328.30 Magnesium (mg/d) 206.57 58.27 222.08 81.95 223.74 77.01 226.54 77.95 224.86 75.33 215.75 62.70 Iron (mg/d) 10.18 3.54 11.02 4.95 11.36 4.44 11.73 4.61 11.85 4.37 12.35 4.27 Sodium (mg/d) 2057.17 602.55 2062.45 785.83 2190.23 751.64 2317.42 875.44 2333.56 888.48 2530.07 897.38 Potassium (mg/d) 2110.14 592.60 2212.48 714.41 2235.38 736.22 2261.33 737.64 2271.60 746.09 2211.03 715.83 Zinc (mg/d) 8.21 2.98 8.64 3.56 8.81 3.46 9.05 3.97 8.69 3.77 9.05 3.93 Copper (mg/d) 1.05 0.77 1.59 2.68 1.38 1.80 1.36 1.65 1.26 1.41 1.47 1.87 Selenium (μg/d) 75.00 23.30 78.59 29.49 80.89 29.71 84.57 31.65 83.32 30.62 93.49 37.03 The total number of subjects was 2981. a As retinol equivalents activity. b As cholecalciferol. c As α-tocopherol. d As phylloquinone. rewards have been considered external cues that contribute to lack of self-regulation of intake in children [24]. In disagreement with our initial hypothesis, a noticeable finding was that the VRs were higher in the 3- to 6-year-old group for some but not all nutrients evaluated. Based on previous studies about nutrient intake variability among preschool children [9,15], we hypothesized that VR of energy and nutrient intakes would be consistently higher in the older age group in comparison with the younger age group. Instead, the influence of age on the VR among Brazilian children varied according to the nutrient under investigation. Hence, it seems that for energy, carbohydrates, protein, thiamine, magnesium, sodium, potassium, and zinc, the VR tended to decrease with age; whereas for total fat, total fiber, riboflavin, folate, calcium, phosphorus, and iron, the VR tended to increase with age. A more consistent pattern of increase in the VR, according to the age of the child, was observed by Huybrechts et al [9] in Belgian preschoolers aged 2.5 to 6.5 years and by Erkkola et al [15] in Finnish children aged 1, 3, and 6 years. The VR decrease for energy, carbohydrates, and protein with age may be explained by the larger increase in the betweensubject variation than in the within-subject variation of intake. However, for total fat, the VR increase may be explained by 2 different aspects: the increase in the within-subject variation in parallel with the reduction in the between-subject variation until around 3.5 years and the larger increase in the withincompared with the between-subject variation from 3.5 years on. These changes in within- and between-subject variation of energy and macronutrient intakes, especially of fat, occurred during a period of change in growth rate as well as in the body composition of the child [25]. During the first and the second years of life, children exhibit a rapid reduction in their weight and height growth rate, whereas from 3 to 6 years of age, their growth rate tends to stabilize [25]. Along this period of growth rate stabilization, the body fat mass increases as a physical adaptation to the pubertal growth spurt (adiposity rebound) [26-29]. The relation of growth and body composition with changes in the within- and between-subject variation of energy and macronutrients, however, could not be addressed in this study and should be further investigated. Another noticeable finding was that overweight/obese children showed lower VR of energy, carbohydrates, protein, and total fat than did their nonoverweight counterparts, which was in agreement with our initial hypothesis. Examining the within- and between-subject variation of intake, overweight/obese children presented lower within-subject 80 N U TR ITI O N RE S EA R CH 3 4 ( 2 01 4 ) 7 4 –8 4 Table 3 – Nutrients intake, variability estimates, and number of days of dietary assessment according to age group Nutrients Mean Energy (kcal/d) 1-2 y 1507 3-6 y 1606 Carbohydrates (g/d) 1-2 y 219.75 3-6 y 233.41 Protein (g/d) 1-2 y 56.87 3-6 y 59.2 Total fat (g/d) 1-2 y 42.81 3-6 y 50.72 Total fiber (g/d) 1-2 y 16.29 3-6 y 18.01 Thiamine (mg/d) 1-2 y 1.22 3-6 y 1.28 Riboflavin (mg/d) 1-2 y 1.70 3-6 y 1.74 Pantothenic acid (mg/d) 1-2 y 4.67 3-6 y 4.77 Vitamin B6 (mg/d) 1-2 y 1.29 3-6 y 1.34 Niacin equivalents (mg/d) 1-2 y 27.12 3-6 y 27.93 Dietary folate equivalents (μg/d) 1-2 y 443.53 3-6 y 482.11 Calcium (mg/d) 1-2 y 763.08 3-6 y 728.43 Phosphorus (mg/d) 1-2 y 959.81 3-6 y 956.68 Magnesium (mg/d) 1-2 y 221.39 3-6 y 224.81 Iron (mg/d) 1-2 y 10.98 3-6 y 11.60 Sodium (mg/d) 1-2 y 2062.21 3-6 y 2269.38 Potassium (mg/d) 1-2 y 2207.93 3-6 y 2250.87 Zinc (mg/d) 1-2 y 8.62 3-6 y 8.88 CVw a CVb b VR Dc Dd 23.0 24.7 13.4 11.9 3.43 3.09 6 6 15 13 74.74 75.89 23.9 26.0 13.9 11.9 3.41 3.17 6 6 15 14 20.2 21.0 28.5 29.6 16.8 13.6 3.73 3.56 7 6 16 15 21.59 19.61 36.9 33.2 12.0 11.7 8.7 8.95 15 16 37 38 7.46 7.89 37.3 35.6 8.3 9.5 6.60 8.41 12 15 28 36 0.59 0.64 34.4 37.6 30.8 28.0 4.22 4.01 8 7 18 17 0.87 0.76 30.8 32.6 29.3 23.3 1.73 2.16 3 4 7 9 2.22 2.57 31.5 37.7 26.5 36.4 1.81 1.92 3 3 8 8 0.51 0.67 29.7 39.3 21.9 25.6 2.54 2.69 5 5 11 11 10.13 10.65 28.4 31.1 18.1 16.3 3.27 3.18 6 6 14 14 188.6 228.6 30.7 40.4 20.9 21.2 3.74 4.81 7 9 16 21 360.5 354.2 36.0 34.0 28.1 29.6 1.17 1.47 2 3 5 6 341.9 330.8 26.5 27.1 20.0 17.2 1.83 2.15 3 4 8 9 81.09 76.78 29.7 26.4 16.6 16.5 2.72 2.26 5 4 12 10 4.89 4.49 31.6 32.8 24.7 15.0 2.51 3.11 4 6 11 13 778.3 829.9 33.4 30.7 9.5 13.7 4.95 4.20 9 7 21 18 709.5 737.9 24.8 26.6 17.4 13.6 3.00 2.59 5 5 13 11 32.7 36.5 19.0 13.1 7.36 5.52 13 10 31 24 SD 460 479 3.54 3.72 The total number of subjects was 2981. CVw = (SDw/mean) × 100; estimated in a Box-Cox scale through a multilevel model. b CVb = (SDb/mean) × 100; estimated in a Box-Cox scale through a multilevel model. c D—considering r = 0.8. d D—considering r = 0.9. a variation and larger between-subject variation for these nutrients than nonoverweight/obese children. Considering the impaired control of food consumption as one of the main factors affecting the development and maintenance of obesity in childhood [30] and the influence of body weight status on children's ability to self-regulate energy intake [24], it is possible that overweight/obese children fail in compensating energy and macronutrient intakes between days, as do the 81 N U TR IT ION RE S EA R CH 3 4 ( 2 01 4 ) 7 4 –8 4 Table 4 – Nutrients intake, variability estimates, and number of days of dietary assessment according to body weight status Nutrients Energy (kcal/d) Nonoverweight/Obese Overweight/Obese Carbohydrates (g/d) Nonoverweight/Obese Overweight/Obese Protein (g/d) Nonoverweight/Obese Overweight/Obese Total fat (g/d) Nonoverweight/Obese Overweight/Obese Total fiber (g/d) Nonoverweight/Obese Overweight/Obese Thiamine (mg/d) Nonoverweight/Obese Overweight/Obese Riboflavin (mg/d) Nonoverweight/Obese Overweight/Obese Pantothenic acid (mg/d) Nonoverweight/Obese Overweight/Obese Vitamin B6 (mg/d) Nonoverweight/Obese Overweight/Obese Niacin equivalents (mg/d) Nonoverweight/Obese Overweight/Obese Dietary folate equivalents (μg/d) Nonoverweight/Obese Overweight/Obese Calcium (mg/d) Nonoverweight/Obese Overweight/Obese Phosphorus (mg/d) Nonoverweight/Obese Overweight/Obese Magnesium (mg/d) Nonoverweight/Obese Overweight/Obese Iron (mg/d) Nonoverweight/Obese Overweight/Obese Sodium (mg/d) Nonoverweight/Obese Overweight/Obese Potassium (mg/d) Nonoverweight/Obese Overweight/Obese Zinc (mg/d) Nonoverweight/Obese Overweight/Obese Mean 1524 1647 SD 459 464 CVw a CVb b VR Dc Dd 23.8 25.4 14.4 12.1 3.18 3.10 6 6 14 13 220.32 234.22 73.24 75.36 24.9 26.2 14.2 11.9 3.39 3.22 6 6 14 14 55.39 59.88 19.80 21.22 28.1 31.9 15.9 13.8 3.58 3.49 6 6 15 15 47.14 51.04 18.90 19.74 34.5 33.8 11.3 11.7 9.32 7.77 17 14 40 33 16.46 17.48 7.15 8.96 34.6 37.9 11.0 9.1 7.59 8.79 13 16 32 37 1.21 1.30 0.65 0.56 36.3 35.4 32.6 23.9 3.97 3.90 7 7 17 17 1.60 1.69 0.8 0.75 31.8 36.7 29.9 16.2 1.82 2.28 3 4 8 10 4.43 4.62 2.66 2.00 38.6 34.5 40.1 19.9 1.90 1.99 3 4 8 8 1.29 1.39 0.65 0.59 37.2 34.9 26.9 24.6 2.59 2.83 5 5 11 12 26.45 28.98 10.54 10.52 29.6 32.9 17.3 14.8 3.34 3.27 6 6 14 14 470.81 479.92 228.17 195.11 38.7 32.6 24.2 16.5 4.37 5.11 8 9 19 22 725.83 759.43 350.62 369.87 32.8 35.3 28.1 26.7 1.29 1.66 2 3 5 7 946.08 981.19 328.05 345.19 25.8 28.9 17.2 16.9 2.22 2.43 4 4 9 10 220.66 230.89 75.02 81.98 28.9 26.4 14.1 18.3 2.36 2.17 4 4 10 9 11.01 12.92 4.77 4.99 32.5 32 15.4 18.7 2.79 3.39 5 6 12 14 2147.29 2344.56 807.14 857.33 29.6 34 14.7 13.2 4.39 4.21 8 7 19 18 2169.35 2374.72 717.96 759.22 25.2 26.4 14.9 16.5 2.80 2.59 5 5 12 11 8.68 8.99 3.59 3.73 36.4 36.8 15.1 12.3 6.15 5.42 11 10 26 23 The total number of subjects was 2981. CVw = (SDw/mean) × 100; estimated in a Box-Cox scale through a multilevel model. b CVb = (SDb/mean) × 100; estimated in a Box-Cox scale through a multilevel model. c D—considering r = 0.8. d D—considering r = 0.9. a nonoverweight ones. Thus, this may contribute to lower within-subject variation of intake. In the study of Jansen et al [31], overweight children aged 8 to 12 years failed to regulate food intake after eating a small portion (preload) of appetizing food, whereas normal-weight children down-regulated their intake because of greater satiety responsiveness. 82 N U TR ITI O N RE S EA R CH 3 4 ( 2 01 4 ) 7 4 –8 4 Fig. – Within- and between-subject variation of energy, carbohydrates, protein, and total fat intakes estimated through a multilevel model according to the age of the child (n = 2981), Brazil, 2007. Moreover, Brazilian preschool children required more days of dietary assessment than did Finnish [15] and Belgian [9] preschoolers to ensure the same level of accuracy in ranking energy and nutrient intakes. In fact, Finnish preschoolers aged 1, 3, and 6 years evaluated in the Type 1 Diabetes Prediction and Prevention Project study [15] required a maximum of 8 days to achieve r = 0.8 and 18 days to achieve r = 0.9 in the simultaneous assessment of all nutrients evaluated in the present study. Likewise, Belgian preschoolers [9] aged between 2.5 and 6.5 years showed a lower number of days (considering r = 0.9), mainly for total fat (10–13 days), total fiber (6 days), and zinc (1–13 days) but a similar number of days for calcium (3–4 days). Differences in age group definitions, in dietary intake assessment methods, and in statistical procedures to estimate the within- and between-subject variation might have contributed to divergent results between studies. The number of days of dietary assessment was also higher than those obtained by Salles-Costa et al [32], who evaluated Brazilian infants aged 6 to 30 months in the metropolitan region of Rio de Janeiro. The authors found that a maximum of 6 days of dietary assessment was needed to achieve r = 0.9 for energy, carbohydrates, protein, total fat, calcium, iron, and ascorbic acid. It is possible that the inclusion of children not attending daycare or preschools and the high proportion of children living in households with food insecurity (77%) contributed to the differences in D between studies. In this study, 7 days of dietary assessment was sufficient to achieve r = 0.8 between observed and true intakes for most nutrients reported, irrespective of children's age and body weight status. This implies that around 72% of children will be correctly classified into tertiles of the distribution, whereas only 3% of them will be grossly misclassified. If a correlation coefficient of 0.9 were used, D would increase to 16 or more and the proportion of children correctly classified would increase only up to 80%. The advantage of increasing the correlation coefficient to 0.9 is mainly to reduce the proportion of children grossly misclassified (to <1%) rather than increase the proportion of children correctly classified. Our findings about D have important implications for planning studies and analyzing data about this population [33]. Too few days of dietary assessment can result in less precise estimates at individual levels [34]. However, the poor compliance of subjects and the high cost of studies that use multiple days of dietary assessment make it difficult to obtain reliable and precise estimates. To overcome this, many researchers advocate collection on at least 2 nonconsecutive days of short-term dietary measurement and apply statistical methods such as the National Cancer Institute Method (developed at National Cancer Institute) and the Web-based statistical technique Multiple Source Method (developed at German Institute of Human Nutrition Potsdam-Rehbrücke) to adjust data for the within-subject variation [33-37]. Another N U TR IT ION RE S EA R CH 3 4 ( 2 01 4 ) 7 4 –8 4 approach is to use external variance estimates (ie, VR) from similar populations when only 1 day of short-term dietary measurement is available [7,9,34,38-40]. The present study does have some limitations. First, our results may not be generalized to children who do not attend daycare or preschool because the repertoire of foods offered to the child may vary depending on the responsible caregiver for the child's diet, that is, family members or school staff. Second, it is conceivable that children from the same school had exhibited a low variation of the foods eaten, which could reduce the between-subject variation of intake and, consequently, increase the VR. However, the large number of schools evaluated, the large sample size, and the multilevel analysis contributed to mitigate this bias. Third, the study used 2 different methods to evaluate food intake: WFR and EFR. Errors commonly observed in the EFR collection, such as those in the recording process or in portion size quantification [41], can overestimate the within-subject variation of energy and nutrient intakes. We aimed to alleviate this by identifying and correcting reporting errors, using standardized data collection procedures, and issuing careful instructions to the parents to complete the EFR. Fourth, the second dietary measurement was collected in only a subsample of children from daycare centers and preschools (25% of the sample). It was demonstrated that the percentage of subjects with more than 1 dietary measurement in the sample (ie, the replication rate) may affect the precision of the usual intake estimates of episodically consumed foods by widening, in different extents, the confidence intervals of estimates [42]. However, the effects of different replication rates on the nutrient intake estimates remain unclear. In summary, the influence of age and body weight status on the VR among Brazilian children from daycare centers and preschools varied according to the nutrient under investigation. The elevated VR reinforces the need for about 7 days of dietary assessment to achieve a correlation coefficient equal to 0.8 between observed and true intakes among Brazilian preschool children. 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