European Journal of Clinical Nutrition (2009) 63, 842–849
& 2009 Macmillan Publishers Limited All rights reserved 0954-3007/09 $32.00
www.nature.com/ejcn
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 Bogotá, 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 (Alcaldı́a Mayor de Bogotá, 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 (Rodrı́guez
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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