Folate and vitamin B12 are both part of the B-complex group of vitamins and are required for biological methylation and DNA synthesis. Deficiency in these vitamins can lead to several adverse effects on health, including anaemia, neuropathy and birth defects( Reference Mahmood 1 , Reference Shane 2 ).
There are several known causes of folate and vitamin B12 deficiencies. The most common cause of folate deficiency is low intake of sources rich in this vitamin, such as green leafy vegetables and legumes, but it can also be caused by alcoholism and lactation. Vitamin B12 deficiency, in turn, can be caused by low intake of this vitamin which is naturally present in animal-source foods, malabsorption, parasitic infections, medications and polymorphisms( Reference Allen 3 ).
To prevent folic acid-sensitive neural tube defects, several countries have adopted mandatory fortification of wheat flour and maize meal with folic acid, a synthetic form of folate, as a public health intervention to improve the folate status of women of reproductive age( Reference Garrett and Bailey 4 ). In Brazil, this intervention became mandatory in 2004 and consists of the addition of 140 to 220 μg of folic acid per 100 g of maize and wheat flours marketed in the country.
Epidemiological studies have shown a low prevalence of folate deficiency in children from countries where folic acid fortification is in place( Reference Ng’eno, Perrine and Whitehead 5 – Reference Villamor, Mora-Plazas and Forero 7 ); however, the same might not be observed for vitamin B12 deficiency, which still varies greatly according to the child’s age and eating practices( Reference Bjørke-Monsen and Ueland 8 ).
In Nepal, for example, the prevalence of vitamin B12 deficiency in children aged 6–23 months was 30·2 %, while no children presented with folate deficiency( Reference Ng’eno, Perrine and Whitehead 5 ). In Mexico, 7·7 and 3·2 % of children aged 1–6 years had vitamin B12 and folate deficiencies, respectively( Reference Cuevas-Nasu, Mundo-Rosas and Shamah-Levy 6 ); while in Colombia, 1·6 % of school-age children presented with vitamin B12 deficiency and less than 1 % with folate deficiency( Reference Villamor, Mora-Plazas and Forero 7 ). In Brazil, a study conducted with 106 children (≤6 years old) attending at a health-care centre in a south-western city of Brazil identified that no children had folate or vitamin B12 deficiency( Reference Barnabé, Aléssio and Bittar 9 ); however, a low prevalence of folate deficiency (4·1 %) with 13·6 % of vitamin B12 deficiency was found in a study conducted with 169 Amazonian children younger than 24 months( Reference Cobayashi, Tomita and Augusto 10 ).
The mandatory flour fortification with folic acid in Brazil has proved to be effective in increasing serum folate concentrations in children, adolescents and healthy adults( Reference Britto, Cançado and Guerra-Shinohara 11 ). However, some international researchers raise questions about the harmful effects of excessive folic acid intake on one’s health, especially in people with low vitamin B12 status( Reference Selhub and Rosenberg 12 , Reference Paul and Selhub 13 ).
Among older adults, some evidence has shown that the imbalance of serum folate status and low vitamin B12 status was associated with increased prevalence of anaemia and cognitive impairment. During pregnancy, this combination may increase women’s predisposition to gestational diabetes and may cause insulin resistance, adiposity and low birth weight in their offspring( Reference Paul and Selhub 13 ). In school-age children, a cohort conducted in Bogotá, Colombia identified that Hb concentration was inversely associated with red blood cell (RBC) folate, especially in children with marginal and low vitamin B12 status( Reference Arsenault, Mora-Plazas and Forero 14 ). The mechanisms that explain this association are unclear. Some researchers speculate that excess folate may interfere with vitamin B12 metabolism and may worsen the functional consequences of impaired vitamin B12 deficiency( Reference Paul and Selhub 13 ).
To our knowledge, few studies have assessed the distribution of folate and vitamin B12 nutritional status in young children; they tend to focus on rates of deficiency instead. In addition, no studies have assessed the association between high folate and low vitamin B12 status in this age group. Thus, the objectives of the present study were to assess the nutritional status of folate and vitamin B12 and to explore their combined association with anaemia in young Brazilian children.
Methods
Study design, settings and participants
The present cross-sectional study is part of the Estudo Nacional de Fortificação caseira da Alimentação Complementar (ENFAC Study). This study was a pragmatic controlled clinical trial designed to assess the impact of multiple micronutrient powder on anaemia and nutritional deficiencies of infants seen at primary health-care centres in four Brazilian cities. A detailed description of the study design, sampling methodology and field procedures can be found elsewhere( Reference Cardoso, Augusto and Bortolini 15 ).
A sample size of at least 105 children in each control and intervention group by study city was deemed minimally necessary for the trial and then increased by 30 % to account for dropouts. This resulted in an anticipated 540 children in each group. Eligibility criteria comprised parental approval to participate and absence of current treatment for anaemia. Exclusion criteria included premature births (<37 weeks’ gestation), twins, reported cases of HIV infection, malaria, tuberculosis or genetic Hb disorders, as well as fever (>39°C) on the day of blood sampling.
All children receiving routine paediatric care at primary health-care units during data collection who met the inclusion and exclusion criteria of the study were invited to participate as a convenience study population. For the present analysis, the study sample was composed of all children from the ENFAC control group, aged 11–15 months. Overall, 543 children were invited to participate; parents of twenty-two children (4·1 %) declined participation; one child did not provide a blood sample and the blood samples of sixty children were insufficient for folate and vitamin B12 laboratory analysis, resulting in a final sample of 460 children.
Data collection and laboratory procedures
From June 2012 to January 2013, caregivers of children attending at primary health-care centres were invited to participate in the present study. At enrolment, trained fieldworkers applied a structured questionnaire through a face-to-face interview with each child’s caregiver, to collect data on socio-economic, environmental, demographic and maternal variables, as well as to gain information on the child’s birth, infant feeding practices, supplementation and morbidities.
Each child’s anthropometric measurements were obtained in duplicate directly by trained research assistants following standardized procedures and using calibrated equipment( Reference Lohman, Roche and Martorel 16 ). Weight was measured with an electronic scale (model UM-061; Tanita, Tokyo, Japan), accurate to 10 g, and recumbent length was ascertained to the nearest 0·1 cm on a flat surface with a portable infant measuring board (model ES-2000; Sanny, Los Angeles, CA, USA). The mean value of both measurements was analysed. BMI was computed as [weight (kg)]/[length (m)]2. Z-scores for length-for-age and BMI-for-age were calculated according to WHO guidelines( 17 ). The cut-off defined for stunting was length-for-age Z-score<−2 and that for overweight was BMI-for-age Z-score >1( Reference de Onis and Lobstein 18 ).
Venous blood samples were collected by trained technicians up to a week after the interview. They were collected in the morning on a day previously scheduled with the caregivers, after at least 3 h of fasting. At the field laboratory, Hb concentrations were determined on portable haemoglobinometers (Hb301; HemoCue®, Angelholm, Sweden). A separate blood sample was protected from light and centrifuged within 1 h of collection; serum and plasma samples were separated in micro tubes and frozen at −20ºC before being shipped to São Paulo on dry ice and maintained at −70ºC until further analysis.
Serum folate and vitamin B12 concentrations were measured by fluoroimmunoassay (PerkinElmer; Wallac Oy, Turku, Finland). Plasma ferritin and soluble transferrin receptor concentrations were measured using commercially available enzyme immunoassays (Ramco, Houston, TX, USA) and serum concentrations of retinol by HPLC methods (HP-1100 HPLC system; Hewlett Packard, Palo Alto, CA, USA) as previously described( Reference Cardoso, Augusto and Bortolini 15 ). C-reactive protein and α1-acid glycoprotein were determined using an IMMAGE Immunochemistry System (Beckman Coulter, Brea, CA, USA). The laboratory assayed internal and external blinded quality control specimens in each run. Based on the control specimens, the accuracy and inter-assay CV for these analyses were within 7 %.
Folate deficiency was defined as serum folate concentration <10 nmol/l( Reference Benoist 19 ) and high folate concentration as serum folate ≥50·1 nmol/l (lower bound of highest tertile). Vitamin B12 status was defined as normal (>221 pmol/l), marginal (221–148 pmol/l) or low (<148 pmol/l)( Reference Green, Allen and Bjørke-Monsen 20 ). Anaemia was defined as Hb concentration <110 g/l as established by WHO( 21 ). Iron deficiency was defined as plasma ferritin concentration <12 µg/l and/or soluble transferrin receptor concentration >8·3 mg/l( 22 ). Children with serum retinol concentration <1·05 µmol/l were considered as having marginal vitamin A status. C-reactive protein concentration >5 mg/l and α1-acid glycoprotein concentration >1 g/l were considered as presence of inflammation( Reference Thurnham, McCabe and Haldar 23 ).
Statistical analysis
Descriptive statistics were calculated as numbers and percentages for the categorical variables and as means and standard deviations, or medians and interquartile ranges, for the continuous variables. The differences in the prevalence rates between groups were assessed using Pearson’s χ 2 and Fisher’s exact tests and the differences in the medians of the continuous variables were assessed using the Kruskal–Wallis test.
To evaluate the combined association of vitamin B12 status and serum folate in relation to anaemia frequency, prevalence ratios (PR) were estimated using random multilevel mixed-effects Poisson regression models adjusted for city as clustering variable. For the present study, a hierarchical conceptual framework model with three levels of determination was used for selecting independent variables, as proposed by Cardoso et al.( Reference Cardoso, Scopel and Muniz 24 ). The most distal level included demographic, socio-economic, environmental and maternal characteristics; the intermediate level comprised birth variables, infant feeding practices and use of supplements; and the proximal level comprised anthropometric variables, reported morbidity and biochemical indicators. First, in model 1, analyses were conducted for selecting variables to be tested in multiple models (P<0·20). Then, at each level of determination, variables were retained in the model if they were associated with the outcome at P<0·10 (model 2).
In further analyses, PR were also estimated to assess the factors associated with high concentration of serum folate using random multilevel mixed-effects Poisson regression models adjusted for city as clustering variable, following the same hierarchical approach for selection of independent variables. The main dichotomous dependent variable was high serum folate and the independent variables were: child’s sex and age; child’s ethnicity (classified according to skin colour as used in the Brazilian census); maternal schooling, age and marital status; sanitary sewer (categorized as no public sewage or public sewage); number of children aged less than 5 years living in the child’s house; child’s birth weight; duration of breast-feeding; timing of introduction of fruits and/or vegetables, meat, beans and cereals, defined as early (<6 months), adequate (≥6–<8 months) or late (≥8 months); iron, vitamin A plus D and multivitamin supplementation; stunting; overweight; hospitalization at least once in life; presence of diarrhoea, fever and wheezing in the past 15d; and presence of vitamin B12 deficiency, inflammation, anaemia, iron deficiency and marginal vitamin A status. The predictors of high serum folate were considered if, after adjustment for potential factors at the same level and hierarchically superior levels, they presented P<0·05 in the final model.
Missing observations were included in the multiple models by creating missing-value categories. All analyses were performed using the statistical software package Stata version 12.0.
Results
Of the 460 children, the mean age was 13·51 (sd 1·03) months and 47·6 % were female. More than half of children’s mothers had more than 9 years of education. About 65 % of children were born with adequate weight and about a third of them were weaned before age 6 months. Few children were stunted (4·7 %) but 37·1 % already were overweight. Further characteristics are presented in Table 1.
Table 1 Characteristics of the young Brazilian children (n 460) included in the study, June 2012–January 2013
* Totals differ from the total number of study children due to missing values.
† Defined as length-for-age Z-score<−2.
‡ Overweight was defined as BMI-for-age Z-score>1 (including ‘at risk of overweight’, overweight and obesity).
§ Defined as plasma ferritin<12 μg/l and/or soluble transferrin receptor >8·3 mg/l.
║ Defined as vitamin A<1·05 µmol/l.
¶ Defined as α1-acid glycoprotein>1 g/l and C-reactive protein>5 mg/l.
Overall, the median of serum folate was 39·7 (IQR 28·8–55·3) nmol/l and only four children (0·9 %) presented with folate deficiency (<10 nmol/l; Table 2). Surprisingly, 142 (30·9 %) children presented with serum folate concentrations above the upper limit of detectable values by the commercial kit used for biochemical analysis. In total, 15·0 % of children had vitamin B12 deficiency (<148 pmol/l) and it was inversely associated with the highest tertile of serum folate concentrations (P<0·001). The same was noted for anaemia: the overall frequency of anaemia (Hb<110 g/l) was 23·0 % and it decreased in the highest tertile of folate concentrations (P<0·001). Six children (3·9 %) presented with an imbalance of high folate status (lower bound of highest tertile) and low vitamin B12 status (<148 pmol/l; Table 2).
Table 2 Vitamin B12 and Hb status by serum folate concentration in young Brazilian children (n 460), June 2012–January 2013
* Kruskal–Wallis or Pearson χ 2 tests were used to examine differences in continuous variables and in proportions, respectively, between serum folate status groups.
Table 3 summarizes the vitamin B12 status, serum folate status and the combined association between these two vitamins in relation to frequency of anaemia. It was observed that having low vitamin B12 alone was significantly associated with higher frequency of anaemia in these children (PR=1·78; 95 % CI 1·10, 2·88), while having high serum folate concentration alone was significantly associated with lower frequency of this condition (PR= 0·53; 95 % CI 0·31, 0·88). In the combined analysis, compared with having normal status for both vitamins, having high serum folate status alone was significantly associated with lower frequency of anaemia (PR=0·53; 95 % CI 0·30, 0·92). No significant association was found for a combination of low vitamin B12 status and normal serum folate with anaemia (PR=1·48; 95 % CI 0·89, 2·46), or for low vitamin B12 status and high serum folate with anaemia (PR=1·83; 95% CI 0·42, 7·92; data not shown in tables), probably due to the low number of observations.
Table 3 Combined association of vitamin B12 status and serum folate in relation to anaemia risk in young Brazilian children (n 460), June 2012–January 2013
PR, prevalence ratio; ref., reference category.
* Normal B12 (vitamin B12≥148 pmol/l); low B12 (vitamin B12<148 pmol/l); normal folate (serum folate<50·1 nmol/l); high folate (serum folate≥50·1 nmol/l, representing the lower bound of highest tertile of folate concentration).
† Defined as Hb concentration<110 g/l.
‡ Pearson’s χ 2 or Fisher’s exact tests.
§ Multilevel Poisson regression models adjusted for city as clustering variable.
║ Multilevel Poisson regression models adjusted for city (clustering variable), maternal schooling, more than one child aged <5 years in the house, age of introduction of fruits and/or vegetables, age of introduction of grains, use of multivitamin supplements, stunting, previous hospitalization, inflammation, iron deficiency and marginal vitamin A status (vitamin A<1·05 µmol/l).
The factors associated with high serum folate are presented in Table 4. In the multiple analysis, vitamin B12 deficiency was associated with lower frequency of high folate concentration.
Table 4 Factors associated with high concentration of serum folate (≥50·1 nmol/l) among young Brazilian children (n 460), June 2012–January 2013
PR, prevalence ratio; ref., reference category.
* Totals differ from the total number of study children due to missing values.
† Multilevel Poisson regression model adjusted for city (clustering variable).
‡ Multilevel Poisson regression model adjusted for city (clustering variable) and variables that were associated with the outcome at P<0·10 following a hierarchical conceptual framework model.
§ Defined as Hb<110 g/l.
|| Defined as vitamin A<1·05 µmol/l.
¶ Defined as vitamin B12<148 pmol/l.
Discussion
In the present study, 15 % of children had vitamin B12 deficiency and one-third of them had marginal vitamin B12 status. Comparing these findings with other studies, similar prevalence to this deficiency was reported among Amazonian children aged 6–24 months (13·6 % defined as vitamin B12<150 pmol/l)( Reference Cobayashi, Tomita and Augusto 10 ) and among 1-year-old Mexican children (9·1 % deficiency defined as vitamin B12<203 pg/ml (equivalent to 150 pmol/l))( Reference Cuevas-Nasu, Mundo-Rosas and Shamah-Levy 6 ). A higher prevalence of vitamin B12 deficiency was found in Nepalese children aged 6–23 months (30·2 %)( Reference Ng’eno, Perrine and Whitehead 5 ).
In infants and young children, vitamin B12 deficiency is mainly due to maternal low vitamin B12 status during pregnancy and lactation that influences the infant’s stores of this vitamin and causes a lower vitamin B12 concentration from breast milk. In addition, low intake of animal-source foods in complementary feeding and malabsorption of vitamin B12 due to intestinal parasite infection can also cause deficiency in young children( Reference Bjørke-Monsen and Ueland 8 , Reference Jones, Ramirez-Zea and Zuleta 25 ).
In contrast to the observations of vitamin B12 deficiency in the present study, less than 1 % of children had folate deficiency. This low frequency of folate deficiency is consistent with other studies carried out in Brazil( Reference Barnabé, Aléssio and Bittar 9 , Reference Cobayashi, Tomita and Augusto 10 ) and in other developing countries, such as Nepal, Mexico and Colombia( Reference Ng’eno, Perrine and Whitehead 5 – Reference Villamor, Mora-Plazas and Forero 7 ). The lack of folate deficiency in these studies could be related for the most part to the folic acid fortification of staple foods in place in these countries. Several studies have shown that this strategy has the potential to increase folate intake in the entire population( Reference Britto, Cançado and Guerra-Shinohara 11 , Reference Crider, Bailey and Berry 26 ) and, consequently, serum folate concentrations( Reference Britto, Cançado and Guerra-Shinohara 11 ). A review of forty-seven Brazilian studies showed that serum folate concentrations increased by 57 % in healthy children and adolescents and by 174 % in adults after the implementation of flour fortification with folic acid in Brazil( Reference Britto, Cançado and Guerra-Shinohara 11 ).
Surprisingly, the median of serum folate concentration found in the present study (39·7 nmol/l) was much higher than that found in other studies: 22·7 nmol/l in Amazonian children below 24 months of age( Reference Cobayashi, Tomita and Augusto 10 ) and 12·4 ng/ml (28·1 nmol/l) in children younger than 6 years( Reference Barnabé, Aléssio and Bittar 9 ). In addition, almost one-third of children from the present study presented serum folate concentrations above the upper limit of detectable values by the commercial kit used for biochemical analysis.
We also found that early introduction of cereals (<8 months of age) was positively associated with having high folate concentration in these children, although this finding was only marginally significant (P=0·073). Although fortification of wheat and corn flours with folic acid is mandatory in Brazil and is effective for increasing serum folate concentrations in the Brazilian population( Reference Britto, Cançado and Guerra-Shinohara 11 ), this association was undesired because of the children’s age. At 1 year, children should not consume a considerable amount of farinaceous products that supposedly are increasing the folate concentrations in this population. This hypothesis corroborates a study conducted among 446 children aged 2–3 years in Brazil, which identified that 88·1% of children consumed fortified foods and the percentage of folate intake provided by consuming ultra-processed foods was 11·3 %. Moreover, 70·2 % of the children presented with consumption above the RDA value for this vitamin( Reference Sangalli, Rauber and Vitolo 27 ).
Despite the known beneficial effects of high acid folic intake in reducing the risk of neural tube defects and other congenital anomalies, and (less conclusively) reducing the risk of CVD and cancer( Reference Colapinto, O’Connor and Sampson 28 ), an increasing amount of evidence has raised concerns regarding the adverse effects of high folate concentrations on one’s health, especially in individuals with marginal or deficient vitamin B12 status( Reference Paul and Selhub 13 ).
In school-age children, two studies conducted in Colombia and Guatemala have found an inverse association between Hb concentration and folate status( Reference Arsenault, Mora-Plazas and Forero 14 , Reference Rogers, Boy and Miller 29 ). In addition, the Colombian study showed that Hb concentration was inversely associated with RBC folate, especially in children with marginal or deficient vitamin B12 status( Reference Arsenault, Mora-Plazas and Forero 14 ). In our study, contrary to these studies, Hb concentration was positively associated with serum folate concentration and, unfortunately, our data have insufficient power to assess the interaction between low vitamin B12 and high folate status in relation to anaemia risk. Arsenault et al. ( Reference Arsenault, Mora-Plazas and Forero 14 ) discussed in their paper that those studies were characterized by the absence of folate deficiency in the children studied, but this explanation does not apply to our study because the frequency of folate deficiency in our children was less than 1 %. However, a comparison of these findings should be made with caution for some reasons: (i) the children in the present study are younger than those in previous studies; (ii) different indicators to assess folate status were used; and (iii) a higher frequency of anaemia was found in the present study (23% v. <7% in those studies).
In our analysis, compared with having normal status for both vitamins, high serum folate status alone was significantly associated with lower frequency of anaemia. The literature clearly presents the mechanisms by which low concentrations of folate as well as vitamin B12 lead to anaemia. These vitamins are metabolically linked and the deficiency of any of these vitamins results in an inhibition of DNA synthesis within RBC production leading to megaloblastic anaemia( Reference Mahmood 1 ).
It is important to mention that vitamin B12 status can influence folate metabolism. This vitamin serves as a coenzyme in the methionine synthase reaction required for the conversion of 5-methyltetrahydrofolate to tetrahydrofolate. Although Bruyn et al. ( Reference Bruyn, Gulbis and Cotton 30 ) found in their study a positive correlation between RBC or serum folate and serum vitamin B12, there was an exception in cases where vitamin B12 values were lower than 200 ng/l or 150 pmol/l, when a negative correlation with serum folate was observed, a phenomenon known as folate trapping( Reference Bruyn, Gulbis and Cotton 30 ).
Our study has limitations that deserve mention. First, the cross-sectional nature of the study is a potential limitation because this kind of design does not allow one to infer causality; the directions of the relationship between variables are unclear and, for this reason, results must thus be interpreted carefully. Second, the present study lacked a sufficient number of individuals with high folate levels and low vitamin B12 status to have statistical power to detect a difference between the groups. Despite that, the present study is one of the most comprehensive ones on folate and vitamin B12 in young children carried out in Brazil. It is important to highlight that folate status can be assessed by serum concentration or RBC folate. Both indicators are recommended by WHO( 31 ) as an accurate biomarker for large-scale assessment of folate status. In our study, we chose to use serum folate concentrations because is cheaper and faster to perform than RBC folate. Although RBC folate reflects the long-term folate status, since we assessed children around 1 year old, the use of serum folate could be considered a good proxy for folate status.
We suggest more studies assessing the distribution of folate status, rather than focusing on rates of deficiency, in young children and further research to clarify the short- and long-term effects of high intake of folic acid in infants and young children to elucidate these complex relationships with potential adverse effects.
Conclusion
A high serum folate concentration was found among 1-year-old children in Brazil and 15 % of them had vitamin B12 deficiency. The combination of high serum folate status and normal vitamin B12 status was associated with lower frequency of anaemia. Adequate complementary feeding and periodic monitoring of nutritional status of these vitamins in this age group are recommended to avoid possible side-effects of these conditions in the future.
Acknowledgements
Acknowledgements: The authors thank the families and health professionals as well as their research team members for their collaboration in the present study. Financial support: The present study was funded by the Ministry of Health of Brazil with administrative and financial management by the Brazilian National Council of Technological and Scientific Development, CNPq (grant number 552747/2011-4). L.L.S.S. received a sandwich doctorate scholarship from the CNPq (grant number 200487/2015-9) and a doctoral scholarship from the Coordination for the Improvement of Higher Education Personnel, CAPES. The views expressed in the present article are those of the authors and not necessarily those of any funding agencies or others whose support is acknowledged. The funders had no role in the design, analysis or writing of this article. Conflict of interest: None. Authorship: L.L.S.S. contributed to the data collection, conducted data analyses, participated in data interpretation, and wrote the initial draft of the article; W.W.F. participated in data analysis and interpretation, and was involved in the review of the article; M.A.C. supervised all study protocols, was responsible for project management, participated in data analysis and interpretation, and was involved in the writing of the article. All authors have critically reviewed the manuscript content and have approved the final version submitted for publication. Ethics of human subject participation: This study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures involving human subjects were approved by the Human Ethical Review Board of the School of Public Health, University of São Paulo, Brazil. Written informed consent was obtained from the caregivers of all children after they had been informed of the objectives, benefits and possible risks of this study. Children who were anaemic or had any nutritional deficiency diagnosed during the study were referred for treatment in the health centres. This study was registered at www.ensaiosclinicos.gov.br as RBR-5ktv6b.
Appendix
Members of the ENFAC Working Group
Marly Augusto Cardoso, Rosângela Aparecida Augusto, Fernanda Cobayashi (Departament of Nutrition, University of São Paulo, São Paulo, Brazil); Maria Claret C.M. Hadler, Maria do Rosário G. Peixoto (School of Nutrition, Federal University of Goiás, Goiânia, Brazil); Pedro Israel C. Lira, Leopoldina Augusta S. Sequeira (Department of Nutrition, Federal University of Pernambuco, Recife, Brazil); Pascoal Torres Muniz, Cristiéli Sérgio de Menezes Oliveira (Department of Health Sciences, Federal University of Acre, Rio Branco, Brazil); Márcia Regina Vitolo, Daniela Cardoso Tietzmann (Federal University of Health Science of Porto Alegre, Porto Alegre, Brazil); Márcia Maria Tavares Machado (Department of Preventive Medicine, Federal University of Ceará, Fortaleza, Brazil); Patrícia Constante Jaime, Eduardo Augusto Fernandes Nilson, Gisele Ane Bortolini, Sara Araújo da Silva (Coordenação Geral de Alimentação e Nutrição, Ministry of Health, Brasília, Brazil).
