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European Journal of Clinical Nutrition (2009) 63, 842–849

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ORIGINAL ARTICLE

Hemoglobin concentration is inversely associated with erythrocyte folate concentrations in Colombian school-age children, especially among children with low vitamin B12 status JE Arsenault1, M Mora-Plazas2, Y Forero3, S Lopez-Arana4, A Baylin5 and E Villamor1,6 1 Department of Nutrition, Harvard School of Public Health, Boston, MA, USA; 2Department of Nutrition, National University of Colombia, Bogota, Colombia; 3Nutrition Group, National Institute of Health, Bogota, Colombia; 4San Rafael Clinic, Bogota, Colombia; 5Department of Community Health, Brown University, Providence, RI, USA and 6Department of Epidemiology, Harvard School of Public Health, Boston, MA, USA

Background: While the majority of cases of nutritional anemia in developing countries are caused by iron deficiency, other micronutrient deficiencies may also be involved. In Colombia, it was recently reported that 38% of school children were anemic; yet, the rate of iron deficiency was only 3.6%. Objective: To determine if micronutrients other than iron were responsible for low hemoglobin concentrations in Colombian school children. Methods: We examined hemoglobin concentrations in relation to plasma ferritin, vitamin A, vitamin B12, and erythrocyte folate levels in a representative sample of 2812 low- and middle-income children (5–12 years) from Bogota´, Colombia. Results: In multivariate analysis, hemoglobin concentration was positively associated with child’s age, mother’s age, household’s socioeconomic stratum, and family income. Low ferritin was related to 3.6 g/l lower hemoglobin concentration (95% confidence interval ¼ 6.0, 1.3). Unexpectedly, we found an inverse trend in hemoglobin concentration by quartiles of erythrocyte folate; the adjusted hemoglobin concentration difference between the highest and lowest folate quartiles was 6.0 g/l (95% confidence interval ¼ 7.2, 4.9; P for trend o0.0001). This difference was greatest among children with vitamin B12 concentration o148 pmol/l (11.5 g/l), followed by children with vitamin B12 concentration 148–221 pmol/l (7.7 g/l), and smallest in children with vitamin B12 concentration 4221 pmol/l (5.7 g/l); P for interaction ¼ 0.04. Conclusions: Hemoglobin concentration is inversely related to erythrocyte folate concentrations in a setting where folate fortification was adopted more than a decade ago. The impact of improving vitamin B12 status on this inverse relationship should be examined.

European Journal of Clinical Nutrition (2009) 63, 842–849; doi:10.1038/ejcn.2008.50; published online 29 October 2008 Keywords: hemoglobin; folate; vitamin B12; school children

Introduction Correspondence: Dr E Villamor, Department of Nutrition, Harvard School of Public Health, 655 Huntington Avenue, Building 2, Rm 333, Boston, MA 02115, USA. E-mail: [email protected] Contributors: JEA carried out the data analyses, interpreted the results, and wrote the initial draft of the manuscript. MMP participated in the study design and implementation in the field. YF contributed to the study implementation and carried out the laboratory analyses. SLA contributed to the study implementation and data management. AB contributed to the study design and interpretation of data. EV designed the study and contributed to data analyses and interpretation. All authors participated in the writing of the final draft of the manuscript. Received 10 March 2008; revised 18 August 2008; accepted 18 September 2008; published online 29 October 2008

Anemia is a widespread problem in developing countries, affecting approximately 50% of school children (WHO/ UNICEF/UNU, 2001). The consequences of anemia include poor school performance, reduced work capacity, increased susceptibility to infections, and stunted growth (Stoltzfus, 2001). While iron deficiency is the cause of approximately one-half of all cases of anemia (Zimmermann and Hurrell, 2007), deficiencies of other micronutrients, including folate, vitamin B12, and vitamin A, have also been identified as causes of low hemoglobin concentrations and anemia (Majia et al., 1977; Villalpando et al., 2006; Jones et al., 2007)

Hemoglobin and folate in school children JE Arsenault et al

843 through diverse mechanisms. Folate is a carbon donor for purine and pyrimidine synthesis, which are needed for the rapidly developing erythroid cells (Scott, 1999). Impaired DNA synthesis, a result of folate deficiency, leads to erythroid cell apoptosis and anemia (Koury and Ponka, 2004). The vitamin B12-dependent enzyme methionine synthase is involved in folate metabolism. Deficiency of vitamin B12 results in a pseudo-folate deficiency, with impaired DNA synthesis and anemia (Koury and Ponka, 2004). Vitamin A may also modulate erythropoiesis. The erythropoietin gene contains a response element in the enhancer region that is regulated by retinoic acid (Evans, 2005). In addition, vitamin A appears to modulate the mobilization of iron stores from tissues (Staab et al., 1984). The 2005 National Nutrition Survey of Colombia reported that 38% of school children were anemic; yet, the rate of iron deficiency (ferritin o12 mg/l) was only 3.6% (Instituto Colombiano de Bienestar Familiar, 2005). A policy of wheat flour fortification was mandated in Colombia in 1996. Fortification includes folate (1.54 mg/kg), thiamin (6 mg/ kg), riboflavin (4 mg/kg), niacin (55 mg/kg), and iron (44 mg/ kg). A child consuming two slices of bread per day is expected to consume an additional B60 mg folic acid from the fortified flour. The impact of folate fortification on folate status in Colombia has not been reported, although increases in blood folate concentrations were reported after folate fortification in the United States (Pfeiffer et al., 2005), Chile (Hertrampf and Cortes, 2004), and Costa Rica (Chen and Rivera, 2004). Some recent evidence suggests that high folate status is associated with anemia in older individuals with vitamin B12 deficiency (Morris et al., 2007). We aimed to determine if micronutrient status was responsible for low hemoglobin concentrations in Colombian school children. We examined the cross-sectional associations between hemoglobin concentrations and plasma ferritin, retinol, vitamin B12, and erythrocyte folate concentrations in a representative sample of children from public schools in Bogota, Colombia. We hypothesized that concentrations of these micronutrients would be positively associated with hemoglobin concentration.

Subjects and methods Study population This study is part of a project on children’s health and nutritional status in primary public schools of Bogota, Colombia, that we initiated in 2006. Details of the study designed have been reported earlier (Isanaka et al., 2007). In brief, we selected a representative sample of 3202 primary school children of age 5–12 years from 3032 households, using a cluster sampling strategy in which all primary school grades 1–5 of all 361 public schools in the city were included. The sampling units were the classrooms. Recruitment was conducted at the beginning of the school year, in February 2006. The sample is representative of low- and middle-

income families from Bogota, as the public primary school system enrolls the majority of school-age children and almost 90% of them belong to low- and middle-socioeconomic strata (Alcaldı´a Mayor de Bogota´, 2006).

Field procedures At the time of enrollment, we obtained information on the parents’ sociodemographic characteristics, including age, marital status, education level, self-reported height and weight, and indicators of the household socioeconomic status, using a self-administered questionnaire that was completed and returned by 2466 households (2637 children). During the 3 weeks after recruitment, teams of trained research assistants visited the schools to obtain a fasting blood specimen by venipuncture from enrolled children and to collect anthropometric measurements using standardized techniques (Lohman et al., 1988). Weight was measured to the nearest 0.1 kg on Tanita HS301 electronic scales (Tanita, Tokyo, Japan), while height was measured to the nearest 1 mm using wall-mounted portable Seca 202 stadiometers (Seca, Hamburg, Germany).

Laboratory methods An aliquot of approximately 4 ml blood was drawn into an EDTA tube. The tubes were inverted gently to avoid clotting and transported on the same day on ice and protected from sunlight to the National Institute of Health in Bogota, where all biochemistry analyses were carried out. We carried out a complete blood count and determined hemoglobin concentrations with the hemiglobincyanide method. We separated plasma in one aliquot, where we measured ferritin and vitamin B12 concentrations using competitive chemiluminescent immunoassay in an ADVIA Centaur analyzer (Bayer Diagnostics, Tarrytown, NY, USA). C-reactive protein level was measured using a turbidimetric immunoassay on an ACS180 analyzer (Bayer Diagnostics, Tarrytown, NY). The packed red cell volume was hemolyzed by dilution in a hypotonic aqueous solution of 1% ascorbic acid. Erythrocyte folate was measured on the red blood cell lysates using chemiluminescent immunoassay. Plasma retinol was quantified using high-performance liquid chromatography on a Waters 600 System (Waters Corporation, Milford, MA, USA).

Data analyses We defined the primary outcome of interest, anemia, as hemoglobin concentration o127 g/l, based on the recommended definition of anemia in this age group (o115 g/l) plus a 12 g/l adjustment for an altitude of 2500 m (Nestle, 2002). Given that we found low prevalence of anemia, we decided to treat the outcome as a continuous variable (hemoglobin concentration). The main exposures of interest were the concentrations of plasma ferritin, retinol, vitamin B12, and erythrocyte folate. Low ferritin was defined as European Journal of Clinical Nutrition

Hemoglobin and folate in school children JE Arsenault et al

844 o15 mg/l if C-reactive protein concentration was p10 mg/l or as o30 mg/l if it was 410 mg/l (Zimmermann and Hurrell, 2007). Retinol was categorized as X1.05, 0.70–1.04, 0.35– 0.69, or o0.35 mmol/l (de Pee and Dary, 2002). Vitamin B12 status was defined as normal (4221 pmol/l), marginal (148–221 pmol/l), or low (o148 pmol/l) (Jones et al., 2007), and erythrocyte folate concentrations were categorized in quartiles as only two children were below 305 nmol/l, a cutoff point suggested to define deficiency (Institute of Medicine, 1998). We considered child and maternal characteristics as covariates in the analysis. Child characteristics included age, sex, stunting, and thinness. Child stunting was defined as height-for-age o2 standard deviations from the sex and age-specific median of the NCHS/WHO reference population (World Health Organization, 1983). Child thinness was the grade 1 definition based on body mass index-for-age and sex as proposed by Cole et al. (2007). Maternal characteristics included age, marital status, education level, body mass index, and parity. Body mass index was calculated as kg/m2 from self-reported maternal weight and height and categorized according to WHO recommendations (WHO Expert Committee on Physical Status, 1995). Household socioeconomic indices included the daily income per capita (household income divided by the household size), money spent on food per capita (amount of money spent on food divided by the number of people in household), home ownership, and the household socioeconomic stratum according to the city’s classification of the neighborhoods’ public service fees. We first compared the distribution of hemoglobin concentrations by categories of each sociodemographic characteristic using univariate linear regression models in which hemoglobin concentration was introduced as the outcome and each characteristic as the predictor. For ordinal predictors, we obtained a test for trend by introducing a variable representing the ordinal categories of the predictor in the linear model as a continuous covariate. We specified an exchangeable correlation matrix in these models (PROC GENMOD, SAS Institute, Cary, NC, USA) to account for potential correlations within households among siblings (Fitzmaurice et al., 2004). The effect of clustering from the sampling strategy was also considered in the models, but it was negligible and was excluded henceforth. To identify sociodemographic covariates that were independently associated with hemoglobin concentration and thus could potentially confound the associations between micronutrient concentrations and hemoglobin levels, we constructed a multivariate model considering the variables that were significantly associated with hemoglobin concentration at Po0.10 in univariate analyses. We retained in the model variables that were statistically significant at Po0.05 or were considered to be relevant from a mechanistic viewpoint. We calculated adjusted differences in hemoglobin concentration and 95% confidence intervals between categories of predictors from the final multivariate linear regression model. European Journal of Clinical Nutrition

Next, we examined the associations between the concentrations of the micronutrients of interest (ferritin, retinol, vitamin B12, and folate) and that of hemoglobin. We compared the distributions of hemoglobin concentration by categories of each micronutrient by following a similar procedure as described above for sociodemographic variables. We estimated adjusted hemoglobin concentration differences and 95% confidence interval by levels of each micronutrient after adjusting for the sociodemographic covariates that were retained in the final multivariate model. Finally, we assessed whether there were interactions between ferritin, folate, and vitamin B12 concentrations on hemoglobin concentration by introducing cross-product terms into the model and testing them with the likelihood ratio test. All tests were double-sided and considered to be statistically significant if Pp0.05. Analyses were performed using the Statistical Analyses System (release 9.1; SAS Institute, Cary, NC, USA).

Ethical considerations We obtained written informed consent from the children’s primary care providers before enrollment. The research protocol was approved by the Ethics Committee of the National University of Colombia Medical School. The Human Subjects Committee at the Harvard School of Public Health approved the use of data from the study.

Results Blood specimens were collected from 2816 of the 3202 children enrolled, and results on hemoglobin concentrations were available for 2812 children. This constituted the final sample size for this study. The proportion of girls was larger among children who did not provide a blood sample (69%) compared with children who provided a sample (49%); however, there were no differences with regard to child’s age, maternal characteristics, or indicators of the household socioeconomic status. The mean (±s.d.) hemoglobin concentration was 145±12 g/l and the prevalence of anemia was 3.7%. Low ferritin was found in 3.3% of the children, and the prevalence of vitamin A deficiency (o0.70 mmol/l) was 13.7%. Marginal or deficient vitamin B12 status was found in 16.6% of the children. In univariate analyses, child’s age, mother’s age, mother’s body mass index, mean household income, and socioeconomic stratum by city classification were positively associated with the child’s hemoglobin concentration (Table 1). Stunted children had significantly lower hemoglobin concentrations than non-stunted children. In multivariate analyses, child’s age, mother’s age, and socioeconomic stratum were positively and independently associated with hemoglobin concentration. Money spent on food and home ownership were not significantly related to hemoglobin concentration.

Hemoglobin and folate in school children JE Arsenault et al

845 Table 1 Child, maternal, and socioeconomic correlates of hemoglobin in Colombian schoolchildren n (%)

Univariate Mean hemoglobin concentration (g/l) (s.d.)

P-valuea

Mean difference in hemoglobin concentration (95% CI)b

o0.0001

Child’s age (years) 5–6 7–8 9–10 11–12

546 855 1090 263

Child’s sex Female Male

1387 (49.3) 1425 (50.7)

145 (11) 145 (12)

Child is stunted No Yes

2430 (90.3) 260 (9.7)

146 (12) 144 (12)

Child is thin No Yes

2445 (91.0) 242 (9.0)

145 (12) 144 (12)

Mother’s age (years) 20–29 30–34 35–39 X40

Multivariate

(19.8) (31.1) (39.6) (9.6)

142 145 147 148

(13) (11) (11) (12)

P-valuec

o0.0001 Ref 3.0 (1.7, 4.3) 5.3 (3.9, 6.6) 6.0 (4.2, 7.9)

0.72

0.69 Ref 0.2 (1.0, 0.7)

0.02 — — 0.10 — — o0.0001 601 662 568 615

(24.6) (27.1) (23.2) (25.1)

144 145 146 147

(11) (11) (12) (11)

Maternal BMI category Underweight (o18.5 kg/m2) Adequate (18.5–24.9 kg/m2) Overweight (25.0–29.9 kg/m2) Obese (X30 kg/m2)

98 1493 532 122

(4.4) (66.5) (23.7) (5.4)

143 145 146 147

(14) (12) (12) (10)

Mean income per person per dayd Q1: median 1880 pesos Q2: median 3289 pesos Q3: median 4386 pesos Q4: median 6579 pesos

525 545 534 538

(24.5) (25.4) (24.9) (25.1)

144 146 146 146

(11) (13) (10) (12)

Household socioeconomic stratume 1 (lowest) 2 3 4

234 903 1286 60

(9.4) (36.4) (51.8) (2.4)

143 145 146 145

(11) (11) (12) (17)

0.02 Ref 0.2 (1.5, 1.1) 0.2 (1.2, 1.6) 1.4 (0.0, 2.7)

0.02 — — — — 0.03

0.07 Ref 0.8 (0.6, 2.2) 1.4 (0.1, 2.6) 1.1 (0.3, 2.5)

0.004

0.006 Ref 1.7 (0.1, 3.3) 2.6 (1.1, 4.2) 1.7 (2.7, 6.2)

a

For ordinal predictors, P-value is for a test for trend when a covariate representing the ordinal categories was introduced as a continuous predictor in a univariate linear regression model with hemoglobin concentration as the outcome. For dichotomous predictors, P-value is from the Wald test. From a multivariate linear regression model with hemoglobin concentration as the outcome and predictors that include indicator variables for child’s age and sex, mother’s age, mean daily income per person in the household, and household socioeconomic stratum. c For child’s age, maternal age, income, and socioeconomic stratum, P-value is for an adjusted test for trend from a variable representing the ordinal categories introduced into the model as a continuous predictor. For child’s sex, the P-value corresponds to the Wald test. d At the time of the study, the mean exchange rate was 1 USD ¼ 2326 Colombian pesos. e According to the city’s classification of the neighborhood’s public services fees. b

Mean hemoglobin concentration among children with low ferritin was 4 g/l significantly lower than that of children with normal ferritin (Table 2). There was a slight inverse trend in hemoglobin concentrations by categories of retinol concentrations, but this trend did not remain after controlling for other factors. Hemoglobin concentration was not associated with vitamin B12 concentrations. Unexpectedly, we found an inverse trend in hemoglobin concentrations by

quartiles of erythrocyte folate concentration (Po0.0001). In a multivariate model controlling for other micronutrients and socioeconomic factors, hemoglobin concentrations were 6 g/l lower in the highest quartile of folate concentration compared with the lowest quartile. The prevalences of anemia from the highest to the lowest quartile of folate concentration were 5.3, 2.8, 2.5, and 1.0% (Po0.0001, test for trend). European Journal of Clinical Nutrition

Hemoglobin and folate in school children JE Arsenault et al

846 Table 2 Micronutrient correlates of hemoglobin in Colombian school children n (%)

Mean hemoglobin (g/l) (s.d.) Plasma ferritin (mg/l)d Normal Low

2700 (96.7) 91 (3.3)

146 (12) 142 (12)

Plasma retinol (mmol/l) X1.05 0.70–1.04 0.35–0.69 o0.35

1246 1178 347 35

146 145 145 144

Plasma vitamin B12 (pmol/l) Normal (4221) Marginal (148–221) Low (o148)

2264 (83.4) 408 (15.0) 43 (1.6)

Erythrocyte folate (nmol/l) Q1 (o700.5) Q2 (700.5–824.4) Q3 (824.5–976.0) Q4 (4976)

Multivariate model 1a

Univariate P-valuec

Mean difference (95% CI)

0.002

(12) (11) (13) (13)

(25.0) (25.0) (25.1) (24.9)

149 146 145 143

(11) (10) (10) (11)

0.40 Ref 0.3 (1.1, 0.5) 0.5 (1.7, 0.8) 0.4 (4.5, 3.7)

0.14 Ref 0.4 (1.4, 0.6) 2.3 (5.5, 0.8)

o0.0001 677 678 680 676

0.003

0.02

0.96

0.33 Ref 0.2 (1.2, 0.9) 2.0 (5.0, 1.1)

o0.0001 Ref 2.5 (3.6, 1.4) 3.0 (4.2, 1.9) 5.6 (6.7, 4.4)

P-valuec

Ref 3.6 (6.0, 1.3)

Ref 0.8 (1.7, 0.0) 1.2 (2.4, 0.1) 1.8 (6.2, 2.6)

146 (12) 146 (11) 144 (11)

Mean difference (95% CI)

0.0004 Ref 4.3 (6.7, 1.9)

0.07 (44.4) (42.0) (12.4) (1.3)

P-valuec

Multivariate model 2b

o0.0001 Ref 2.7 (3.8, 1.6) 3.3 (4.4, 2.2) 6.0 (7.2, 4.9)

a

Model 1 is a multivariate linear model with hemoglobin concentration as outcome and predictors that include indicator variables for plasma ferritin, retinol, vitamin B12, and erythrocyte folate concentrations. b Model 2 is a multivariate linear model with hemoglobin concentration as outcome and predictors that include indicator variables for plasma ferritin, retinol, vitamin B12, erythrocyte folate concentrations, child’s age and sex, mother’s age, household income, and household’s socioeconomic stratum. c For retinol, vitamin B12, and folate concentrations, P-value is for a test for trend when a covariate representing the ordinal categories was introduced as a continuous predictor in the regression models with hemoglobin concentration as the outcome. For ferritin, P-value is from the Wald test. d Low ferritin was defined as ferritin concentration o15 mg/l if the concentration of CRP was p10 mg/l or as ferritin concentration o30 mg/l if CRP concentration was 410 mg/l (Zimmermann and Hurrell, 2007).

The inverse association between folate and hemoglobin concentrations was significantly modified by vitamin B12 status (adjusted P for interaction ¼ 0.04). The greatest difference in hemoglobin concentration between the highest and lowest quartiles of erythrocyte folate concentrations was in children with low vitamin B12 concentrations (11.5 g/l), followed by children with marginal vitamin B12 concentration (7.7 g/l), and smallest in children with normal vitamin B12 status (5.7 g/l) (Table 3). There were no significant interactions between folate or vitamin B12 and ferritin concentrations on hemoglobin concentration.

Discussion In this study, we examined the cross-sectional associations between biomarkers of several micronutrients and hemoglobin concentrations in a large representative sample of lowand middle-income school children from Bogota, Colombia. We found low prevalences of anemia and iron deficiency. Iron deficiency, as suggested by low ferritin concentrations, was predictive of low hemoglobin concentrations. Neither vitamin A nor vitamin B12 concentrations were related to hemoglobin concentrations after adjusting for potential confounding factors. Unexpectedly, erythrocyte folate European Journal of Clinical Nutrition

concentrations were inversely associated with hemoglobin concentrations and this association was strongest among children with low vitamin B12 status. This population was not folate-deficient, probably because of a policy of wheat flour fortification that was mandated in Colombia in 1996. Although the prevalence of folate deficiency before 1996 is unknown, comparable levels of fortification in other countries including the United States (Pfeiffer et al., 2005), Chile (Hertrampf and Cortes, 2004), and Costa Rica (Chen and Rivera, 2004) have coincided with increases in the populations’ levels of folate. In adult women from Chile, for example, mean erythrocyte folate concentrations were 290 nmol/l before and 707 nmol/l after fortification of wheat flour with 2.2 mg folate/kg was introduced (Hertrampf and Cortes, 2004). Our finding of an inverse association between hemoglobin concentration and folate status is consistent with reports from two other studies conducted in countries where folic acid fortification is in place. In a sample of Guatemalan school children, none were folate-deficient and an inverse association between hemoglobin concentration and serum folate was reported (Rogers et al., 2003). Among older adults in the United States, high serum folate concentration was associated with anemia in those who had low vitamin B12 status (Morris et al., 2007). By contrast, in Mexico, where there was no folic acid

Hemoglobin and folate in school children JE Arsenault et al

847 Table 3 Stratified analysis of hemoglobin and erythrocyte folate concentrations by vitamin B12 status n (%)

Multivariatea

Univariate Mean hemoglobin (g/l) (s.d.)

Normal B12 (4221 pmol/l) Erythrocyte folate concentration (nmol/l) Q1 (o700.5) Q2 (700.5–824.4) Q3 (824.5–976.0) Q4 (4976)

526 537 571 555

(24.0) (24.5) (26.1) (25.4)

149 146 146 144

(11) (10) (10) (11)

Marginal B12 (148–221 pmol/l) Erythrocyte folate concentration (nmol/l) Q1 (o700.5) Q2 (700.5–824.4) Q3 (824.5–976.0) Q4 (4976)

124 99 84 88

(31.4) (25.1) (21.3) (22.3)

149 147 144 141

(11) (9) (7) (10)

Low B12 (o148 pmol/l) Erythrocyte folate concentration (nmol/l) Q1 (o700.5) Q2 (700.5–824.4) Q3 (824.5–976.0) Q4 (4976)

12 16 6 9

(27.9) (37.2) (14.0) (20.9)

148 145 142 137

(6) (10) (8) (17)

P-valueb

Mean difference (95% CI)

o0.0001

P-valueb

o0.0001 Ref 3.0 (4.2, 1.8) 3.2 (4.4, 2.0) 5.7 (6.9, 4.4)

o0.0001

o0.0001 Ref 1.2 (3.7, 1.4) 4.2 (6.7, 1.8) 7.7 (10.4, 5.0)

0.04

0.02 Ref 1.6 (7.2, 4.0) 3.4 (11.0, 4.1) 11.5 (21.8, 1.2)

a Multivariate linear model with hemoglobin as the outcome and predictors that included indicator variables for ferritin concentration, child’s age and sex, mother’s age, household income, and household’s socioeconomic stratum. P-value for interaction ¼ 0.04. b Test for trend for a variable representing quartiles of folate concentration introduced as a continuous predictor.

fortification, 14% of children had low erythrocyte folate concentrations and anemic children had significantly lower folate concentrations compared with non-anemic children (Villalpando et al., 2006). The mechanisms to explain an inverse relation between folate and hemoglobin concentrations in the absence of folate deficiency, particularly in subjects with inadequate vitamin B12 status, are unclear. Since iron is essential for hemoglobin synthesis, it is tempting to speculate that high concentrations of folate might have an adverse effect on iron metabolism. The recently discovered intestinal membrane protein HCP1 transports both heme and folate in the brush border of duodenal cells (Shayeghi et al., 2005; Qiu et al., 2006) and, with a higher affinity for folate (Qiu et al., 2006), one could speculate that high folate intake may induce a competitive reduction of heme iron absorption leading to lower hemoglobin production. A recent in vitro study showed that exposure to high folic acid concentrations resulted in lower intracellular iron concentrations in HT29 colon cancer cells, suggesting an effect of folate on iron uptake or metabolism (Pellis et al., 2008). A dietary explanation for the inverse association could also be plausible. Children with higher folate and lower hemoglobin concentrations might represent a group with low intake of highly bioavailable heme-iron and elevated intake of folate-rich foods that are also rich in phytates (e.g. beans), which impair iron absorption. Children consuming a diet high in cereal may also have low intake of high-quality protein, which is needed for hemoglobin synthesis (Reissmann, 1964). Detailed

studies on dietary sources of iron, folate, and other micronutrients are needed in this population. Given the cross-sectional nature of the study, the inverse association between folate and hemoglobin concentrations might also represent an effect of iron on folate metabolism (Vitale et al., 1966; Woeller et al., 2007). In the course of iron deficiency, serum folate concentration has been shown to be reduced (Vitale et al., 1966), while erythrocyte folate concentration is increased (Omer et al., 1970; Saraya et al., 1973); in addition, treatment with iron could result in decreased erythrocyte folate concentration (Omer et al., 1970). These studies suggest that red cells might enhance the uptake of folate from serum in the presence of iron deficiency. Heavy-chain ferritin increases folate catabolism (Woeller et al., 2007); therefore, low ferritin in iron deficiency might spare folate by decreasing its catabolism (Suh et al., 2001). However, the prevalence of iron deficiency according to plasma ferritin concentration in our study was low, and we did not find any interactions between ferritin and folate concentrations in relation to hemoglobin concentrations. The combination of high blood folate and low vitamin B12 concentrations has been associated with cognitive impairment (Morris et al., 2007), elevated homocysteine and methylmalonic acid concentrations (Selhub et al., 2007), and insulin resistance (Yajnik et al., 2008), in addition to anemia. This may be an indication that excess folate worsens the functional consequences of impaired vitamin B12 status, and lends support to the proposal of adding vitamin B12 to European Journal of Clinical Nutrition

Hemoglobin and folate in school children JE Arsenault et al

848 foods that are routinely fortified with folic acid (Herbert and Bigaouette, 1997; Anonymous, 2004; Refsum and Smith, 2008). Nevertheless, limited evidence is available from intervention studies on the impact of improving vitamin B12 status on hematological or neurological outcomes. The prevalence of anemia in our study, 3.6%, was much lower than that reported in the National Nutrition Survey of 2005 among children 5–12 years of age from Bogota: 34.5% (Instituto Colombiano de Bienestar Familiar, 2005). By contrast, the prevalence of ferropenia in our study, 3.3%, was the same as that reported by the survey among children of the same age group who lived in urban areas. The survey analyzed hemoglobin concentrations using Hemocue, which can produce lower results than those using the hemiglobincyanide method (Neufeld et al., 2002); however, this does not explain the magnitude of the difference between our results and those of the national survey. Explanations for the high prevalence of anemia reported in the National Nutrition Survey are warranted. Despite the low prevalence of anemia, indicators of low socioeconomic status predicted lower hemoglobin concentrations, possibly through insufficient intake of highly bioavailable iron from animal food sources (Rodrı´guez et al., 2007). It is relevant to investigate whether hemoglobin concentrations are associated with functional outcomes in school children, including growth, morbidity from infections, and school performance, even in the absence of anemia. If this were the case, programs aimed at improving hemoglobin levels should be targeted at the poorest population groups. In conclusion, we found that low hemoglobin concentrations in Colombian school children were inversely associated with folate status, especially among those with low vitamin B12 status. Hemoglobin concentration was positively associated with age, socioeconomic status, and indicators of iron stores. Although the prevalence of anemia in this population was low, 17% of the children had inadequate vitamin B12 status. The impact of improving vitamin B12 status on this inverse relationship between folate and hemoglobin concentrations should be examined in populations that adopted folic acid fortification of foods.

Acknowledgements This research was supported by the Secretary of Education of Bogota, the David Rockefeller Center for Latin American Studies at Harvard University, the National University of Colombia, and the National Institute of Health of Colombia. Dr Arsenault is supported by the training grant T32DK07703 from the National Institutes of Health.

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