Abstract. Key words: Anemia, day care, fortification, iron, preschool. Introduction

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1 Effectiveness of fortification of drinking water with iron and vitamin C in the reduction of anemia and improvement of nutritional status in children attending day-care centers in Belo Horizonte, Brazil Daniela da Silva Rocha, Flávio Diniz Capanema, Michele Pereira Netto, Carlos Alberto Nogueira de Almeida, Sylvia do Carmo Castro Franceschini, and Joel Alves Lamounier Abstract Background. Because of the high prevalence of irondeficiency anemia in Brazil, individual control measures tend to be ineffective, and fortification of foods with iron is considered the most effective method to fight anemia. Objective. To evaluate the effectiveness of fortification of drinking water with iron and vitamin C in the reduction of anemia in children in day-care centers in Belo Horizonte, Brazil. Methods. This before-and-after study evaluated 318 children aged 6 to 74 months. Identification data and data on socioeconomic variables were collected; anthropometric and biochemical measurements were performed before and after 5 months of fortification of water with 5 mg of elemental iron and 50 mg of ascorbic acid per liter. The fortified water was used for drinking and cooking at the day-care center. Wilcoxon s nonparametric test was used to evaluate the differences in continuous variables, and McNemar s test was used to compare the prevalence rates of anemia. Results. The prevalence of anemia decreased significantly from 29.3% before fortification to 7.9% at the end of the study, with a significant increase in hemoglobin levels. Reductions in the prevalence rates of stunting and underweight were observed. Conclusions. Fortification of water with iron and Daniela da Silva Rocha is affiliated with Universidade Federal da Bahia, Vitória da Conquista, Bahia, Brazil; Flávio Diniz Capanema is affiliated with Fundação Hospitalar do Estado de Minas Gerais, Minas Gerais, Brazil; Michele Pereira Netto is affiliated with Universidade Federal de Juiz de Fora, Minas Gerais, Brazil; Carlos Alberto Nogueira de Almeida is affiliated with Universidade de Ribeirão Preto, São Paulo, Brazil; Sylvia do Carmo Castro Franceschini is affiliated with Universidade Federal de Viçosa, Minas Gerais, Brazil; Joel Alves Lamounier is affiliated with Faculdade de Medicina da Universidade Federal de Minas Gerais, Minas Gerais, Brazil. Please direct queries to the corresponding author: Daniela da Silva Rocha, Instituto Multidisciplinar em Saúde, Campus Anísio Teixeira, Universidade Federal da Bahia, Rua Rio de Contas, 58 quadra 17 Lote 58, Vitória da Conquista, BA, Brazil ; danisr_nutricao@yahoo.com.br. vitamin C significantly reduced the prevalence of anemia and improved nutritional status among children attending day-care centers. Key words: Anemia, day care, fortification, iron, preschool Introduction Iron deficiency is the result of a negative balance between the amount of biologically available iron consumed and the body s iron requirements over a long period of time. Iron-deficiency anemia is the last stage of this deficiency [1]. Lack of iron and iron-deficiency anemia are among the main public health problems in developing countries and are a challenge due to their economic, social, and health implications [2]. The prevalence of anemia among preschool children is 47.4% worldwide and 39.5% in Latin America and the Caribbean. In Brazil, according to a recent meta-analysis, anemia is considered to be a serious public health problem, affecting 53% of children under 5 years of age [3]. Regional studies in Brazil confirm the high prevalence of anemia; a study conducted in day-care centers of the city of Cuiabá among 271 children younger than 36 months found a prevalence of anemia of 63% [4], and a study conducted in day-care centers of São Paulo among 330 children aged between 6 and 75 months found a prevalence of 68.8% [5]. The typical dietary patterns and the high physiological iron requirements of children under 5 years of age are the main factors responsible for the greater vulnerability to anemia of children in this age group. The amount of bioavailable iron in the regular diet of children in developing countries is frequently insufficient to maintain adequate nutritional status of this mineral, thus resulting in anemia, especially in children younger than 2 years [6, 7]. Iron deficiency, even without anemia, can affect neuropsychomotor and 340 Food and Nutrition Bulletin, vol. 32, no , The United Nations University.

2 Fortification of drinking water with iron and vitamin C behavioral development in the long term, and some of the changes are irreversible [8]. According to the World Health Organization (WHO), the basic measures for the prevention and control of anemia are nutrition education aimed at increasing the consumption of iron-rich foods, programs for the control of parasitic diseases, drug supplementation, and fortification of foods with iron [1]. Increasing the consumption of iron-rich foods through dietary changes is considered a preferred method, but it has major practical limitations, because it is difficult to change food habits and because foods rich in bioavailable iron are expensive [9]. Prophylactic iron supplementation can be directed toward populations at a higher risk for anemia, such as children younger than 24 months, and has the advantage of producing fast results. However, this method requires an efficient healthcare system and is costly [10]. Furthermore, supplemental iron may cause side effects leading to low adherence to treatment when the parents or other responsible adults fail to administer iron to the children [9]. Because of the high prevalence of iron-deficiency anemia in Brazil, individual control measures tend to be ineffective. Thus, iron fortification of foods is considered the most effective method to fight anemia; it is the least expensive method, does not depend on individual decisions, and can be aimed at all segments of the population [10, 11]. Drinking water is a potential vehicle for iron fortification to reduce the prevalence of anemia among children attending day-care centers. Studies conducted in southeastern Brazil showed promising results [12 14]. The advantage of drinking water as a vehicle for iron fortification is its widespread consumption and its use in the preparation of foods frequently consumed in day-care centers [15, 16]. The present study aimed to evaluate the effectiveness of fortification of drinking water with iron and vitamin C for the reduction of anemia, using doses of iron lower than those used in previous studies. Methods A longitudinal, before-and-after study was conducted among children aged between 6 and 74 months in day-care centers of the Eastern Sanitary District of the city of Belo Horizonte who were given drinking water fortified with iron and vitamin C during a period of five consecutive months. At the time of the study, there were 26 day-care centers in this district, of which 24 were affiliated with and 2 were owned by the city of Belo Horizonte municipal government; all of these centers participated in the study except for 2 that were affiliated with the municipal government. This study was approved by the Universidade Federal de Minas 341 Gerais Research Ethics Committee (Official Opinion ETIC 273/04) and City of Belo Horizonte Department of Health Research Ethics Committee (Protocol ). The study sample was taken from the 2,344 children cared for on a full-time basis in the 24 participating day-care centers. The sample size was calculated on the assumption of a significance level of 1% and a power of It was assumed that the magnitude of the effect studied was the difference in the average amount of hemoglobin before and after fortification (0.5 g/dl) and the value of 1.4 g/dl as the variability of outcome variable. Thus, the minimum number of children studied would be 217. To minimize errors from dropouts, we chose a minimum sample size of 300 children. The children were randomly selected. The sample was taken in proportion to the total number of children in each daycare center. Initially, 379 children 6 to 74 months of age whose parents signed an informed consent form were considered eligible for the study. After examination, the following categories of children were excluded from the study: those with some manifestation of acute illness, such as fever, diarrhea, or vomiting (n = 2), or any chronic disease (n = 1), because the presence of inflammatory processes may cause changes in hemoglobin levels; children using medications containing iron (n = 2); children with other types of anemia according to reports from parents or guardians (n = 0); and children with hemoglobin levels 9.0 g/dl, who were treated medically with supplementary iron (n = 2). After application of the exclusion criteria, 372 children entered the study. Of these, 24 left the day-care centers for unspecified reasons, 14 were not present on the examination day, and 16 were treated with a medication containing iron during the intervention period. Thus, there was a loss of 54 children (14.5%), while 318 remained in the study. Child identification data were obtained from the day-care centers medical records. Socioeconomic and family data were obtained by interviews conducted with the use of questionnaires with the parents or responsible adults when the children were brought to or picked up from their day-care centers. Anthropometric evaluation and anemia testing were performed at baseline and after 5 months of fortification. Anthropometric data (weight and height) were collected according to the techniques described by WHO [17]. The children were weighed on an electronic scale with a capacity of 150 kg and an accuracy of 10 g while wearing light clothing. The length or height of children younger than 24 months was measured with an anthropometer (Altura Exata). The height of children older than 24 months was measured with the child standing barefoot, after the anthropometer was adapted to function as a stadiometer. The children s nutritional

3 342 D. da Silva Rocha et al. status was evaluated by weight-for-age, height-for-age, and weight-for-height z-scores. Children with z-scores of less than 2 were classified as malnourished or stunted, those with z-scores of 2 or greater but less than 1 were classified as at risk for malnutrition or stunting, and those with z-scores of 1 or greater were classified as without nutritional deficit. The WHO growth curves were adopted as a benchmark, using WHO Anthro software, version 3.0.1, for children younger than 60 months, and WHO Anthro software, version 1.0.2, for children aged 5 years or older. Blood samples were collected with disposable microcuvettes by capillary puncture of the finger, and the hemoglobin concentration was measured with a portable hemoglobinometer (Hemocue). This method of measuring hemoglobin has been recommended for population studies because it requires only a small amount of blood and thus is a less invasive technique. Children aged between 6 and 59 months were considered anemic when they had a hemoglobin concentration lower than 11.0 g/dl, while those aged between 60 and 74 months were considered anemic when this value was lower than 11.5 g/dl [1]. Although we did not study the children s iron status, the high prevalence of iron-deficiency anemia and the relative unimportance of other causes of anemia in Brazil [3] led us to consider, for the purposes of the study, all the anemic children as suffering from iron-deficiency anemia. The children were provided exclusively with fortified drinking water in the day-care centers from July to December Fortified water was also used in the preparation of juices, infant formulas, and foods for lunches and dinners. To prepare the fortified water, a 500-mg packet of hepta-hydrated ferrous sulfate plus 1,000 mg of ascorbic acid was added to 20 L of water. The resulting solution contained 5 mg of elemental iron and 50 mg of ascorbic acid per liter. The water containers were protected from sunlight because of the photosensitivity of ascorbic acid. To ensure quality control of the water consumed by the children, the team of researchers conducted training sessions on the preparation and use of the fortified water in all day-care centers. In addition, weekly visits were made to the centers during which the number of packets consumed was recorded and questions were answered. Statistical analyses were performed with Epi Info, version 6.04, and SPSS. The Shapiro-Wilk and Kolmogorov-Smirnov tests were used to test the normality of distribution of numerical data. Since the variables hemoglobin after fortification and difference in hemoglobin before and after fortification did not have a normal distribution, Wilcoxon s nonparametric test was used to evaluate the differences in continuous variables before and after fortification, and McNemar s test was used to compare the prevalence rates of anemia before and after intervention. Results A total of 318 children were evaluated before and after fortification, of which 52.2% were males. The children s age at baseline ranged from 8 to 74 months, with a mean of 45.4 ± 15.8 months. The mothers age ranged from 19 to 48 years, with a mean of 29.3 ± 6.0 years. The majority of the mothers were married, and 59.2% of them had 8 years or less of education. The mean monthly household income was R$ ± (US$ ± ). The characteristics of the children evaluated are shown in table 1. There was a significant increase in mean hemoglobin level in all age groups, with an overall mean increase of 9.62%. The increase was greater (11.5%) in children younger than 24 months; the mean hemoglobin level in this age group before fortification was below the cutoff point for anemia (11.0 g/dl) and reached a normal level after fortification (table 2). There was a global reduction of 73% in the prevalence of anemia after 5 months, from 29.3% before fortification to 7.9% after fortification (fig. 1). The reduction was 62.5%, 75%, and 78.8% in children aged 24 months or less, between 24 and 48 months, and greater than 48 months, respectively. TABLE 1. Baseline demographic and socioeconomic characteristics of children Characteristic No. Percent Sex Female Male Age (mo) < > Mother s age (yr) < > Mother s education (yr) > Mother s marital status Married Separated Single Widowed Household income ( minimum wage) < >

4 Fortification of drinking water with iron and vitamin C 343 The prevalence rates of stunting (height-for-age z-score) and underweight (weight-for-age z-score) were reduced after 5 months, although only the former change was statistically significant (table 3). Corroborating previous data on nutritional status, an increase of 20.5% in the mean height-for-age z-score was observed after fortification (p <.001). The mean weight-for-age z-score also increased by 12.5%, although the change was not statistically significant. In contrast, the mean weight-for-height z-score decreased significantly (table 4). Discussion The present study had a before-after design, i.e., the children served as their own control group, without a separate control group. Despite this limitation, this type of study was selected for ethical reasons. Thus, children with a diagnosis of anemia were not deprived of a proven and effective intervention by being placed in a separate control group. This decision was even % < 24 Before fortification > 48 Child s age (mo) After fortification Total FIG. 1. Prevalence of anemia in children attending day-care centers in Belo Horizonte, Minas Gerais, Brazil, before and after receiving water fortified with iron and vitamin C more appropriate in a population such as the one in this study with a high prevalence of anemia. The absence of a control group in such cases was recommended by the Conselho Nacional de Saúde (Brazilian Health TABLE 2. Hemoglobin level (g/dl) of children at baseline and after 5 months of receiving water fortified with iron and vitamin C Age (mo) Baseline 5 mo Mean ± SD Median (min, max) Mean ± SD Median (min, max) Mean variation (%) All ages ± (9.10, 17.70) ± (8.90, 16.60) < ± (9.30, 13.20) ± (9.10, 14.80) ± (9.10, 17.70) ± (8.90, 15.80) > ± (9.10, 16.30) ± (10.20, 16.60) a. Wilcoxon test. TABLE 3. Nutritional status of children at baseline and after 5 months of receiving water fortified with iron and vitamin C number (percent of children) Index TABLE 4. Height-for-age, weight-for-height, and weight-for-age z-scores of children at baseline and after 5 months of receiving water fortified with iron and vitamin C Index Mean ± SD Baseline Baseline Median (min, max) Mean ± SD 5 mo Median (min, max) Mean variation (%) HAZ 0.39 ± ( 3.31, 2.44) 0.31 ± ( 3.07, 2.57) <.0001 WAZ 0.08 ± ( 3.44, 3.17) 0.07 ± ( 3.42, 3.57) WHZ 0.28 ± ( 2.72, 3.87) 0.16 ± ( 2.92, 3.92) 42.7 <.0001 HAZ, height-for-age z-score; WAZ, weight-for-age z-score; WHZ, weight-for-height z-score a. Wilcoxon test. 5 mo < 2 2 to 1 > 1 < 2 2 to 1 > 1 HAZ 19 (5.99) 72 (22.71) 226 (71.29) 13 (4.10) 65 (20.50) 239 (75.39).017 WHZ 2 (0.9) 19 (8.56) ) 4 (1.80) 16 (7.20) 202 (90.99).475 WAZ 9 (2.83) 48 (15.09) ) 8 (2.51) 51 (16.04) 259 (81.45).324 HAZ, height-for-age z-score; WAZ, weight-for-age z-score; WHZ, weight-for-height z-score a. McNemar test. p a p a p a

5 344 D. da Silva Rocha et al. Council), and by the Conselho Federal de Medicina [18, 19]. Other studies have also used this methodology [14]. It is well known that before-after studies are prone to error of measurement of the true effect, However, we have sought to minimize any errors, for example by excluding children who used iron during the study. Moreover, there was no change in the food served in the nursery, which also could have affected the results. Nevertheless, the consensus is that the randomized clinical trial is the best method because it is able to provide the best evidence. According to WHO, iron-deficiency anemia is a public health problem of varying severity, which mainly affects children younger than 5 years [1]. Moreover, the effects of this deficiency on psychomotor development, in both the short and the long term, are well known, with sequelae that can remain even after treatment of the deficiency [8, 20, 21]. The prevalence of anemia in the present study (29.3%) was similar to that found by Almeida et al. [22] in 184 children aged between 12 and 59.9 months who were cared for on a full-time basis in five day-care centers in the city of Jardinópolis, state of São Paulo, but different from that found in other studies of children in day-care centers [4, 5, 14, 23]. For an intervention to be effective, it is important to take into consideration not only the fortified food, but also the use of such food by the population at a higher risk for anemia [24, 25]. Researchers have used several types of foods, such as rice, milk, cookies, bread, juice, and cassava flour, in addition to different iron salts (ferrous sulfate, iron amino acid chelate, ferric pyrophosphate) to fight this nutritional deficiency [16, 23, 25-28]. In the present study, water was selected for fortification because it has the advantage of being consumed both directly by drinking and indirectly in food, so that an extra amount of iron could be incorporated into the daily diet of the children. The first study that used water as a vehicle for fortification was conducted by Dutra de Oliveira et al. [13], who followed 31 preschool children, aged between 2 and 6 years, attending day-care centers in the city of Ribeirão Preto, São Paulo. These children drank water fortified with iron (20 mg of iron per liter) over a period of 8 months. A significant reduction in the prevalence of anemia was observed from 58% before fortification to 16% after 4 months of fortification to 3% at the end of the study. The mean hemoglobin level rose from 10.6 ± 1.1 g/dl before fortification to 12.1 ± 1.4 g/dl in the fourth month and to 13.0 ± 1.1 g/dl in the eighth month of the study. Subsequently, Almeida et al. [12] used a lower dose of iron (10 mg of iron per liter), with the inclusion of 100 mg of vitamin C, in 150 children attending six day-care centers of the city of Ribeirão Preto. After 6 months of intervention, the prevalence of anemia had decreased from 45.9% to 31.1%, while the mean hemoglobin level significantly increased from ± 1.66 to ± 1.35 g/dl. Beinner et al. [14] also evaluated the effectiveness of water fortified with 12 mg of iron and 90 mg of vitamin C per liter. This study was conducted in the city of Diamantina, state of Minas Gerais, on 160 children attending day-care centers, during a period of 8 months. A reduction in the prevalence of anemia was observed from 43.2% to 21% at the end of this study. A significant increase in mean hemoglobin level from 11.8 ± 1.3 to 12.4 ± 0.93 g/dl was found in the eighth month. In addition, there were significant improvements in anthropometric indices (weight-for-age, height-for-age, and weight-for-height z-scores) and in child growth after the intervention (p <.05). In a more recent double-blind, randomized study conducted by Arcanjo et al. [29], whose goal was to evaluate the effect of drinking water with different concentrations of iron on hemoglobin and hematocrit level in children attending four schools, the consumption of drinking water fortified with 7.5 mg of elemental iron per liter resulted in increases in hemoglobin values and a reduction in the prevalence of anemia. In the present study, we chose to work with drinking water, which is used not only for drinking but also in food preparation. Thus, all meals offered in the daycare centers were made with the use of fortified water. In addition, the consumption of fortified water was ad libitum, from a single source. This extra amount of iron was incorporated into the diet of children daily. The amounts of iron and vitamin C used were based on previous studies by Dutra de Oliveira et al. [13], Almeida et al. [12], and Beinner et al. [14]; however, the concentrations used in our study, 5 mg of elemental iron and 50 mg of ascorbic acid per liter of water, were lower than those used in the previous studies. There was a 73% reduction in the prevalence of anemia in the total population, and the reduction was greater than 60% in all age groups. Similarly, there were significant increases in hemoglobin values in all age groups, in particular in children younger than 2 years, who had mean values lower than the cutoff point for anemia before fortification. This situation was modified after the intervention period (fortification) due to the reduction of the prevalence of anemia and increases in hemoglobin values at this age. With regard to nutritional status, the mean heightfor-age and weight-for-age z-scores increased, although only the former change was statistically significant. In contrast, the weight-for-height z-score decreased significantly. The increase in height is expected in populations with iron deficiency, since this mineral is essential for cell multiplication and potential gene expression may be restricted by the presence of such deficiency [30, 31]. Some studies of iron fortification have shown that the weight-for-height z-score is maintained after the intervention period [16, 28], whereas others [12, 32]

6 Fortification of drinking water with iron and vitamin C have found a reduction after intervention, as observed in the present study. The evaluation of factors leading to a decrease in the weight-for-height z-score is complex, because growth is a process involving several factors, such as nutrition and disease, in addition to social and psychological factors. Thus, it is difficult to explain this result. One possibility is that the children reduced their food intake because fortification of the water changed its taste. At the beginning of the study, the daycare personnel reported that the children complained about the taste of the water served, although this was not observed after some time, probably because they became accustomed to the change. In addition, in several day-care centers, there were reports of an increase in the appetite of the children after fortification, which could be associated with the recovery of body iron status. The most probable explanation is that the reduction in the weight-for-height z-score was due to an increase in the rate of growth, with an increase in final height, which influenced this index, rather than a decrease in the rate of weight gain. However, the height velocity was not measured in the present study. Finally, the increase found in the height-for-age z-score, which 345 is the index least subject to acute variations, is a relevant factor to show the positive effect of fortification on child nutritional status. In conclusion, consumption of water fortified with doses of iron and vitamin C lower than those used in previous studies resulted in improvement in hemoglobin levels and, consequently, reduction of the prevalence of anemia in children attending day-care centers. This study also showed an improved cost benefit ratio compared with previous studies, since a lower concentration of iron and vitamin C had the same level of efficacy at a lower cost, with a mean cost of fortification of approximately R$1.50 (US$0.85) per child per month. Thus, these data point to a viable strategy for the prevention and control of anemia in children attending day-care centers. Acknowledgments The authors thank FAPEMIG (Fundação de Amparo a Pesquisa do Estado de Minas Gerais) and Centrais Elétricas de Furnas for financial support. References 1. World Health Organization. Iron deficiency anaemia. Assessment, prevention and control: a guide for programme managers. Geneva: WHO, World Health Organization. The World Health Report 2002: reducing risks, promoting healthy life. Geneva: WHO, Jordão REB, Filho AAB. Prevalence of iron-deficiency anemia in Brazil: a systematic review. Rev Paul Pediatr 2009;27: Brunken GS, Guimarães LV, Fisberg M. 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