The Journal of Nutrition
Community and International Nutrition
Iron Fortification of Whole Wheat Flour Reduces
Iron Deficiency and Iron Deficiency Anemia
and Increases Body Iron Stores in Indian
Sumithra Muthayya,5,9* Prashanth Thankachan,5Siddhivinayak Hirve,6Vani Amalrajan,5Tinku Thomas,5
Himangi Lubree,7Dhiraj Agarwal,6Krishnamachari Srinivasan,5Richard F. Hurrell,8
Chittaranjan S. Yajnik,7and Anura V. Kurpad5
5Division of Nutrition, St. John?s Research Institute, St. John?s National Academy of Health Sciences, Bangalore, India;6Vadu Rural
Health Program, and7Diabetes Unit, King Edward Memorial Hospital Research Centre, Pune, India; and8Human Nutrition Laboratory,
Institute of Food Science and Nutrition, Swiss Federal Institute of Technology, Zurich, Switzerland
Wheat is the primary staple food for nearly one-third of the world?s population. NaFeEDTA is the only iron (Fe) compound
suitable for fortifying high extraction flours. We tested the hypothesis that NaFeEDTA-fortified, whole wheat flour reduces
Fe deficiency (ID) and improves body Fe stores (BIS) and cognitive performance in Indian children. In a randomized,
double-blind, controlled, school feeding trial, 6- to 15-y-old, Fe-depleted children (n = 401) were randomly assigned to
either a daily wheat-based lunch meal fortified with 6 mg of Fe as NaFeEDTA or an otherwise identical unfortified control
meal.Hemoglobin (Hb) andFestatusweremeasured atbaseline,3.5mo, and7 mo.Cognitiveperformance wasevaluated
at baseline and 7 mo in children (n = 170) at one of the study sites. After 7 mo, the prevalence of ID and ID anemia in the
treatment group significantly decreased from 62 to 21% and 18 to 9%, respectively. There was a time x treatment
interactionfor Hb, serum ferritin,transferrinreceptor,zinc protoporphyrin, and BIS (all P < 0.0001). Changesin BIS differed
between the groups; it increased in the treatment group (0.04 6 0.04 mmol/kg body weight) and decreased in the control
group (20.02 6 0.04 mmol/kg body weight) (P < 0.0001). In sensory tests, NaFeEDTA-fortified flour could not be
differentiated from unfortified flour. There were no significant differences in cognitive performance tests between the
groups. NaFeEDTA-fortified wheat flour markedly improved BIS and reduced ID in Fe-depleted children. It may be
recommended for wider use in national school feeding programs.J. Nutr. doi: 10.3945/jn.111.155135.
Iron (Fe) deficiency (ID)10and ID anemia (IDA) are widespread
globally. Forty percent of the world?s children in their school-
going years are reported to be anemic (1,2). Cereal flour
fortification with Fe is the most cost-effective and sustainable
way to improve its status in deficient populations (3). Wheat is
currently the primary staple food for nearly one-third of the
world?s population (4), providing >50% of the total energy
intake of people living in northern India.
Twomajorconcerns when considering flour fortification with
Fe have been the poor absorption of elemental Fe compounds
when added to whole-grain flour and the abundant phytic acid
present in the flour that hinders Fe absorption (5–7). NaFeEDTA
protects Fe from the phytic acid present in foods by binding
more strongly to ferric Fe at the pH of the gastric juice in the
stomach and then exchanging the ferric Fe for other metals in the
duodenum as the pH rises (8). It is 2- to 4-fold more bioavailable
than ferrous sulfate, particularly in meals with a high-phytate
content, thereby making it ideal for use in whole wheat flour (9–
11). Its efficacy as a fortificant has been demonstrated in food
vehicles such as curry powder, sugar, fish sauce, and maize flour
(12–15). In a recent directive, the WHO and partner organiza-
tions, while providing guidance on national fortification of
wheat and maize flours, have endorsed NaFeEDTA to be the
1Supported by the Department of Biotechnology, Ministry of Science and
Technology, Government of India; AkzoNobel, India; and St. John?s National
Academy of Health Sciences, Bangalore, India.
2Author disclosures: S. Muthayya, P. Thankachan, S. Hirve, V. Amalrajan, T.
Thomas, H. Lubree, D. Agarwal, K. Srinivasan, R. F. Hurrell, C. S. Yajnik, and A. V.
Kurpad, no conflicts of interest.
3This trial was registered at www.clinicaltrials.gov as NCT00741143.
4Supplemental Figure 1 and Supplemental Table 1are available from the ‘‘Online
Supporting Material’’ link in the online posting of the article and from the same
link in the online table of contents at http://jn.nutrition.org.
9Present address: Centre for Health Innovation and Partnership, NSW Health,
Cumberland Hospital, North Parramatta, NSW 2151, Australia.
10Abbreviations used: BIS, body iron store; ID, iron deficiency; IDA, iron
deficiency anemia; Hb, hemoglobin; SF, serum ferritin; TfR, serum transferrin
receptor; ZnPP, whole blood zinc protoporphyrin.
* To whom correspondence should be addressed. E-mail: sumi.muthayya@
ã 2012 American Society for Nutrition.
Manuscript received January 17, 2012. Initial review completed April 2, 2012. Revision accepted September 3, 2012.
Copyright (C) 2012 by the American Society for Nutrition
1 of 7
The Journal of Nutrition. First published ahead of print September 26, 2012 as doi: 10.3945/jn.111.155135.
only Fe fortificant suitable for use in high-extraction flours (16).
Information on the efficacy of NaFeEDTA-fortified, whole
wheat flour in improving Fe status is lacking and therefore much
The impact of Fe fortification on the cognitive functioning of
Fe-deficient, school-aged children has not been adequately
studied. Reports of modest improvements in mental develop-
ment scores in children older than 7 y following Fe supplemen-
tation have been mostly limited to initially anemic or IDA
children (17). However, ID results in decreased brain Fe even
before RBC production is affected (18), suggesting that cognitive
effects may precede hematological changes. Data from the
NHANES surveys (1988–1994) have revealed lower math scores
among Fe-deficient, school-aged children, including those with-
out anemia (19). We therefore hypothesized that fortification of
whole wheat flour with Fe as NaFeEDTA would improve Fe
status and body Fe stores (BIS) and would be beneficial for
cognitive performance in Fe-depleted school children.
Study children and sites. This study was carried out between July
2007 and May 2008 among school children located in 2 sites in India: an
urban primary school in Bangalore city, Karnataka state, and 2 primary
schools in rural Vadu in Maharashtra state. These schools serve the
educational needs of nearby poor communities at their respective
locations. Neither region is endemic for malaria and the presence of
intestinal parasitic infestation in this population is low (20,21). Children
attending these schools were taught in Kannada and Marathi, the local
languages spoken in Bangalore and Vadu, respectively. Informed, written
consent was obtained from the parents of the children and oral assent
was obtained from the children. The protocol of the study was approved
by the ethical committees at St. John?s National Academy of Health
Sciences, Bangalore and the King Edward Memorial Hospital Research
Centre, Pune, India.
Fortification of wheat flour with NaFeEDTA. Whole wheat flour
manufactured by Christy Fried Gram Industry was used for this study.
The whole wheat flour was produced by pulverizing dried and cleaned
wheat kernels to a particle size of 500 mm. The resulting homogenous,
whole wheat flour with a maximum moisture content of 6% was
fortified with NaFeEDTA (AkzoNobel). The fortification level was set at
6 mg Fe/0.1 kg flour, well below the recommended maximum daily safe
consumption level in humans of 0.37 mg Fe/kg body for NaFeEDTA and
of 0.8 mg Fe/kg body for Fe from all sources present in the diet (22). The
mixing of whole wheat flour with NaFeEDTAwas carried out under the
direct supervision of S. Muthayya and P. Thankachan. A total of 4000 kg
of fortified flour was prepared. Because the ribbon blender available had
only a 150-kg capacity, a premix of 20 kg was initially prepared and to
each kilogram of premix, 100 kg of unfortified flour was added.
Immediately after, the Fe-fortified and unfortified wheat flour were
assigned color codes, and packed in color-coded, identical, 20-kg
polyethylene bags under the supervision of an investigator not involved
in the study; the bags were then dispatched to the study sites. The study
investigators, assessors of cognitive tests, and study children were all
unaware of the group assignments until the study was completed, all
data were entered, and the analyses were performed.
Cooking of meals. The lunch meals using the NaFeEDTA-fortified and
unfortified whole wheat flour were prepared daily at both study sites.
Study research assistants, who were responsible for the meals each day,
weighed out edible portions of all foods according to standard local
recipes using electronic food scales (Soenle-Waagen; precision, 1 g) and
supervised the cooking. The chapathi preparation was standardized such
that each child received 3 standard-sized chapathis made from 100 g of
wheat flour daily. The nutrient composition of the meals was calculated
using Indian food conversion tables (23).
The meals were then transported to the school and individual
portions of the lunch meal were served in color-coded plates, 6 d/wk
(except on school holidays). At the school, the group assignment of the
participating children was identified by using a color-coded personal
badge. Three or 4 local recipes for vegetable or lentil dishes with
different seasoning ingredients were presented along with the chapathis
in a repeating sequence to maintain interest. The research staff ensured
that the study children consumed their standard meals (3 chapathis and
vegetable/lentil accompaniments) under their direct supervision. The
staff at both the study sites were given adequate training on the
measurement of leftovers on a visual scale to ensure standardization. At
the end of the meal, the percentage leftover of chapathis was estimated in
individual children using visual scales as percentages of 25, 50, 75, or
100 and recorded. A total 131 and 138 d of study intervention were
completed at the Bangalore and Vadu sites, respectively.
Sensory testing. At the Bangalore site, triangle tests (24) were
performed to determine whether local women could distinguish the
Fe-fortified flour from unfortified flour. The panel was composed of 18
middle-class Indian women. Three local dishes made of wheat flour
(chapathi, poori, and dosa) and uncooked wheat flour were tested. The
food samples were presented in a randomized block design. The cooked
wheat dishes (from 30 g flour) and uncooked wheat flour (30 g flour)
were presented on coded,polyethyleneplates; 3 coded samples of each of
the 4 dishes were given in random order in a semiprivate setting. The
panelists determined which among the 3 samples differed from the other
2 samples and described how it differed in taste, smell, or appearance.
The women were informed about the procedures of the test only after
completion of the entire study.
Efficacy trial. A total of 756 children aged between 6 and 13 y at
Bangalore and 561 children between 7 and 15 y at Vadu were invited to
participate in the baseline screening of their anthropometric measure-
ments, clinical health, and biochemical status for anemia, Fe status, and
inflammation. Measurements of body weight and height and a brief
medical history were obtained from each child. Five milliliters of whole
blood was collected by veni-puncture for the determination of hemo-
globin (Hb), serum ferritin (SF), serum transferrin receptor (TfR), whole
blood zinc protoporphyrin (ZnPP), and serum C-reactive protein (CRP).
Children were eligible for inclusion into the study if they were: 1)
apparently healthy, without any chronic illness and physical/mental
handicaps; 2) not severely anemic (Hb <80 g/L); 3) Fe depleted (SF <20
mg/L or TfR >7.6 mg/L and ZnPP concentration >40 mmol/molheme); 4)
not intending to use micronutrient supplements during the study; and 5)
planning to reside in the study area during the next 12 mo. In total, 1317
children from both sites were screened, of whom 916 children were not
eligible, because they did not fulfill the inclusion criteria (Fig. 1). The
remaining 401 children (194 children in Bangalore and 207 children in
Vadu)were separatelyrandomized ateachsite into2 groupsandenrolled
into the trial. They were individually allocated into 1 of 2 groups to
receive either an NaFeEDTA-fortified, wheat-based meal (treatment
group) or an otherwise identical, wheat-based control meal with no
fortificant Fe (control group). Randomization was performed by means
of a computer-generated list in blocks of 8. The enrolled children, who
were arranged in ascending order by grade at school and age in years,
were assigned intervention codes in sequence. All measurements done at
baseline were repeated at 3.5 mo (midpoint) and at 7 mo (endpoint) into
the study. Severely anemic children received supervised treatment with
oral Fe tablets [60 mg Fe (as ferrous sulfate) 4 d/wk for 12 wk]. Others
who were diagnosed with illnesses were referred to a physician.
Cognitive measurements. Prior to start and at the end of the
intervention, all randomized children at the Bangalore site were
subjected to a battery of tests to assess cognitive performance. These
tests were carried out in sound-protected cubicles at the St. John?s
Medical College premises, Bangalore. A few days prior to testing, the
children were brought to these cubicles to orient them to the environ-
ment and the psychologists administering the tests. The cognitive
measures consisted of a series of neuropsychological tests applicable for
2 of 7Muthayya et al.
use in school-aged children related to specific cognitive domains (short-
term memory and retrieval ability, cognitive speed, and fluid reasoning)
consistent with the Carroll model (25). The cognitive battery included 3
core tests from the Kaufman Assessment Battery for Children (26) and
additional tests (27–29) that underwent an extensive adaptation process
to ensure their applicability in the local cultural context (30). The specific
tests used were Atlantis (learning ability/long-term storage and retrieval
scale), KOH?S BlockDesign (visuo-spatialability),Word Order(sequential
processing/short-term memory scale), Pattern Reasoning (planning/
fluid reasoning scale), Verbal Fluency (broad retrieval ability), and Coding-
WISC-III (cognitive speed). The tests were adapted for use in 7- to 15-y-old
Kannada-speaking children of low socioeconomic status in Bangalore,
India through an iterative process of translating, piloting, and modifying
(30,31).These cognitive measureswere previouslyshown tobesensitiveto
the effects of nutritional interventions (32) and were administered
by trained masters-level psychologists in the local Kannada language.
A team of 5 psychologists were extensively trained during 3 wk prior to
the study to ensure standardization in the test administration and scoring
procedures with retraining as needed throughout the study period. The
training was repeated in the week prior to the cognitive assessments at
7 mo. To exclude individual variation, cognitive assessments for
each child were conducted by the same psychologist at both baseline and
the end of study. The tests were administered to each child during the
morning hours between 0900 and 1100 h for a 25-min period. Care was
taken to ensure that all children had breakfast before testing began in the
morning, because omitting breakfast is known to impair cognitive
Biochemical analysis. At baseline, midpoint, and endpoint, Hb was
Bangalore, and an ABX Micros 60 (ABX Diagnostics) at Vadu. All other
biochemical analyses for SF, TfR, ZnPP, and CRP were carried out at the
Core Biochemistry Laboratory Facility at St John?s Research Institute,
where these assays have been established and validated against external
control materials. SF was measured by electro-chemiluminescence on an
Elecsys 2010 analyzer (Roche Diagnostics). External 3-level control
method. TfR and CRP were measured using immunoassays on a Hitachi
902 analyzer (Roche Diagnostics). The TfR values obtained from the
Hitachi 902 analyzer were converted to results corresponding to the
Ramco assay using the regression equation: Ramco TfR value = (Roche
Tfr value 20.299) 3 0.63121(34). ZnPP was measured on washed RBC
with a hematofluorometer (Aviv Biomedical). Serum samples were ali-
quotedand frozen at 220?C until analysis. Anemia was defined as an Hb
concentration <120 g/L in children aged $12 y and <115 g/L in children
aged 5–11 y (1). SF values of children with elevated CRP ($5 mg/L) were
excluded from analysis (n = 5, 16, and 17 each at baseline, 3.5 mo, and
7 mo, respectively). ID was defined as having an SF <15 mg/L (35) or as
TfR >7.6 mg/L plus a ZnPP concentration >40 mmol/mol heme (35–37).
IDA was defined as anemia with ID using the above-mentioned criteria.
BIS was calculated from the ratio of TfR:SF by using the method of Cook
et al. (38). To convert the BIS values from mg/kg to mmol/kg, they were
divided by 55.847. Only children with normal CRP concentrations were
included in the calculation of BIS.
Statistical analysis. Thebaselinebiochemicalstatusofchildren(Hband
Fe indices), the main outcome variables for the study, were comparable
between the sites (data not shown). Data from both sites were therefore
combined for analysis. Data processing and statistical analysis were
performed with SPSS (version 13.0, 2004) and with Microsoft Excel (XP
2006). The normality of the data was checked before analysis using the
Shapiro-Wilk?s test and by graphically evaluating Q-Q plots. Normally
distributed data were expressed as mean 6 SD and as median (quartile 1,
quartile 3), if otherwise. Variables that were not normally distributed
variables were compared between the treatment and control groups
at baseline using the independent sample t tests. Repeated-measures
Hb,SF, TfR,and BISat the3 timepointsby considering the group3time
interaction effect. Bonferroni-adjusted post hoc comparisons were done
for the significant main and interaction effects. The time effect for
the binary variables of anemia, ID, and IDA was tested by using the
McNemar?s test and the group effect by using the Pearson x2test. Results
of the sensory study were evaluated using the binomial test, with an
expected probability of correct detection in the triangle test to be 0.3. An
ANCOVA was performed for the comparison of raw scores for all
cognitive variables between the study groups at the end of study after
baseline and endpoint within each group using paired t test with
Bonferroni correction for multiple testing. Separate models with gender,
greater or equal to median), and BIS group (group 1 with BIS values less
than median; group 2 with BIS values greater or equal to median) as
additional factors were performed to examine the interaction of the
treatment effect with these variables. Log-transformed values were used
when data did not follow normal distribution. Differences were consid-
ered significant at P < 0.05.
Of the 401 children (n = 200 in the treatment group and 201 in
the control group) who participated in the study, 379 children
(n = 186 in the treatment group and n = 193 in the control group)
who received wheat-based meals that were or were not fortified with
NaFeEDTA at baseline, 3.5 mo, and 7 mo. *Different from control, P ,
0.05 (Pearson?s x2test). Percentages within each group with a
superscript without a common letter differ, P , 0.05 (McNemar?s
test). ID, iron deficiency; IDA, iron deficiency anemia.
Prevalence of anemia (A), ID (B), and IDA (C) in children
Iron fortification of wheat flour with NaFeEDTA3 of 7
completed the study. The dropout rate was similar in the 2
groups (Supplemental Fig. 1). There were no significant be-
tween-group differences for any of the baseline characteristics
(Table 1). The mean age of all the children in the study was
10.4 6 2.6 y. Their overall prevalences of anemia, IDA, and ID
were 20.4, 18, and 63.5%, respectively.
The study children consumed meals containing 100 g wheat
flour/meal daily (Table 2). The daily lunch meals provided
11.2 6 0.7 mg Fe in the treatment and 5.1 6 0.6 mg Fe in the
control groups. In the treatment group, the chapathis contained
6 mg of added fortificant Fe in the form of NaFeEDTA. The Fe
content in the unfortified chapathi meal served daily with the
added vegetable and/or lentil gravy is summarized in the table.
On any given day, ;80% of the children at both sites had no
leftovers on their plate after the meal. Compliance was estimated
based on the mean consumption of the cooked meal per day per
child throughout the study period. The mean compliance with
the intervention in the Bangalore and Vadu sites was estimated
to be 85 and 78%, respectively. While compliance in the treat-
ment and control groups at the Bangalore site was 84.3 and
85.7% respectively, the figures for Vadu were 78.7 and 76.5%,
respectively. The level of compliance between the intervention
groups was comparable throughout the study period.
The sensory testsindicated that at2 levels offortification, i.e.,
6.0 and 10 mg Fe/100 g flour, NaFeEDTA-fortified and un-
fortified wheat flour in the raw form were comparable in taste,
flavor, color, and odor (data not shown). Similarly, in all of the
cooked recipes, namely chapathi, poori, and dosa, the meals
containing wheat fortified flour at both 6 and 10 mg Fe/100 g
were indistinguishable from the respective meals containing
unfortified wheat flour.
Both the treatment and control groups showed similar,
marked gains in weight and height after 7 mo of intervention
when compared with their baseline values (Table 3). However,
gains in body weight of 3.2 6 2.1 kg (n = 371) and height of
4.5 6 2.0 cm (n = 364)did not differ between the groupsover the
duration of the study.
There was a time 3 treatment interaction for the prevalence
of anemia, ID, and IDA (P < 0.01) (Fig. 1). The prevalence of
anemia, ID, and IDA significantly decreased from baseline at 3.5
and 7 mo in the treatment group. During the 7 mo, the anemia
prevalence in the treatment group decreased (20.5 to 14.1%; P <
0.05), whereas it increased in the control group (19.2 to 24.4%;
P = 0.05). The prevalence of ID decreased in the treatment group
(62.5 to 20.5%; P < 0.001) but did not change in the control
group. Fe fortification resulted in a decrease in IDA prevalence
from 17.7 to 8.6% (P < 0.001), whereas in the control group
there was no change.
There was a progressive change inHb, SF, TfR, ZnPP,and BIS
concentrations after 7 mo (Table 3). A time 3 treatment
interaction was observed for Hb, SF, TfR, ZnPP, and BIS (all P <
0.0001). Changes in BIS differed between the groups; it
increased in the treatment group (0.04 6 0.04 mmol/kg body
weight) and decreased in the control group (20.02 6 0.04
mmol/kg body weight) (P < 0.0001). Blood Hb, SF, ZnPP, and
TfR (all P < 0.0001) significantly differed between groups after 7
mo. There was no significant change in Hb, SF, TfR, and BIS in
the control group at the end of study at 7 mo.
There was no overall effect of treatment on cognitive
performance at the end of the intervention period after adjusting
for baseline scores for each of the tests (Supplemental Table 1).
The interaction effects of treatment with either gender or
grouping by SF or BIS status were also not significant for any of
the cognition variables.
Whole-grain wheat flour is one of the most inhibitory food
vehicles for Fe absorption. Through fortification with Na-
FeEDTA, in this controlled study, whole wheat flour has been
demonstrated to be suitable as a highly effective vehicle for Fe
fortification in markedly reducing anemia (35%), IDA (51%),
and ID (67%) in Fe-depleted, school-going Indian children.
These figures remained unchanged, except for a slight increase in
anemia prevalence in the control group. Consumption of whole
wheat flour fortified with NaFeEDTA providing 6 mg Fe/ 100 g
flour resulted in a marked and sustained improvement in Fe
status, as indicated by their increases in SF and BIS. A similar
study with school children in Kenya (15) consuming fortified
whole-grain corn flour showed that whereas the addition of
56 mg/kg of elemental Fe did not result in a reduction in the
prevalence of IDA or ID and an improvement in Fe status any
better than in the control group, adding 28 or 56 mg/kg of Fe as
NaFeEDTA did significantly reduce ID among these school
children by 70 and 91%, respectively. Ferrous sulfate due to
both its affordability and high bioavailability is often the
preferred choice for fortification. However, Fe absorption from
FeSO4 used to fortify cereal foods has been shown to be
unacceptably low due to the natural presence of phytic acid (39).
Reductions in ID and IDA in the present study were of a
similar magnitude, as observed earlier in a 7-mo, randomized,
controlled study of Fe-fortified rice, where Indian school
children received nearly 3 times the quantity of Fe as micronized
ferric pyrophosphate than the Fe in the NaFeEDTA-fortified
flour meal (40). Clearly, Fe present in NaFeEDTA is better
absorbed by at least 3 times that of other Fe fortificants (10–12).
In communities that typically have a low consumption of meat
and meat products that are rich in the highly bioavailable heme
Fe and use wheat flour as a major staple in their diet such as in
North India, NaFeEDTA added as a Fe fortificant may be the
best alternative for improving Fe status. The drop in anemia
prevalence was more pronounced in the first half of the
characteristics of the children who received wheat-based meals
that were or were not fortified with NaFeEDTA at baseline1
Age, gender, and anthropometric and hematological
Treatment groupControl group
Child gender, %
10.8 6 2.6
10.7 6 2.7
26.5 6 8.0
1.34 6 0.15
125 6 13
5.8 6 3.4
0.03 6 0.05
26.2 6 7.8
1.33 6 0.15
125 6 12
5.8 6 2.8
0.03 6 0.05
BIS, mmol/kg body weight
1Data are mean 6 SD, geometric mean (6 SD), or percentage. There were no
differences between groups. BIS, estimated body iron store; ID, iron deficiency IDA,
iron deficiency anemia; Hb, hemoglobin; SF, serum ferritin; sTfR, serum transferrin
4 of 7 Muthayya et al.
intervention, after which it remained unchanged inthe treatment
group. The improving Fe status and/or a possible simultaneous
worsening of micronutrient deficiencies (riboflavin, folate, and/
or vitamin B-12) that may have impaired Fe utilization over the
7-mo period could have resulted in the less-pronounced drop in
anemia prevalence observed in the latter part of the study.
NaFeEDTA also has excellent organoleptic properties as has
been shown in the results of the triangle tests. Women in the
present study rated wheat flour (in cooked and raw form)
fortified with 6 and 10 mg Fe/100 g as comparable with un-
fortified wheat flour. In addition, children participating in the
study did not react unfavorably to the color or taste of the
wheat-based meals during the course of the study. Addition of
NaFeEDTA increases intrinsic Fe absorption and improves the
apparent absorption of zinc in the diet (41,42). It has no effect,
however, on copper, calcium, or magnesium absorption (43) or
of heavy metals (41). It has also been reported not to cause fat
oxidation during long periods of storage at ambient tempera-
ture, unlike ferrous sulfate, when added to wheat flour (42). A
limitation of our study is that we did not evaluate the population
acceptability and the long-term stability of the fortified wheat
flour and cooked, wheat-based meals. The long-term potential
of NaFeEDTA fortification to induce Fe overload has also been a
concern, but the available evidence suggests that homeostatic
controls would prevent excess Fe accumulation in the normal
population (41). Like most EDTA-metal complexes, NaFeEDTA
also dissociates in the gut to a bioavailable form of Fe and an
EDTA salt, with almost all the EDTA excreted in the stool (44).
In ratstudies, NaFeEDTA has been demonstrated tohave an oral
toxicity similar to that of ferrous sulfate (45).
At 7 mo, both groups showed impressive, significant in-
creases in body weight (3.2 kg) and height (4.5 cm) from their
baseline values. The observed changes exceeding that of Indian
growth norms (46) may possibly be attributed to the additional
energy and protein in the lunch meal. Although the provision
of Fe to Fe-deficient children has been shown to improve
growth (47–50), our study did not demonstrate any effects of Fe
on growth. A possibility, however, for the significant changes in
both weight and linear growth in all the study children could be
attributed to the extra protein consumed in the meal that con-
tained lentils (which are high in lysine content). Because most
protein in the diet of the study children comes from rice at
Bangalore and pearl millet at Vadu, the protein quality of their
habitual diet could have been suboptimal. Lysine is the
limiting amino acid in a cereal-based diet and the intake of
35% of the children in our study at Bangalore did not meet
the estimated average requirements of lysine (S. Muthayya,
unpublished observations). Where undernutrition, namely
underweight, stunting, and ID often coexist, adding Fe to a
food vehicle that is rich in lysine could be considered a public
health strategy to address these problems among marginally
deficient school children.
Although Fe fortification resulted in significant increases in
Fe status indicators among Fe-depleted children in the present
study, it did not reflect concurrent, significant improvements in
their cognitive performance. There may be several reasons for
the lack of effect of Fe fortification on cognitive measures. First,
as suggested by 2 recent reviews, which include data from
India, beneficial effects of Fe treatment on cognition were
particularly apparent in children that were anemic or had IDA
(17,51). Second, in many studies, the intervention occurred
during infancy (52–54). There are suggestions that a Fe-deficient
state during early life is associated with delayed neuronal
Nutrient composition of the wheat-based chapathi meals per daily serving
Bangalore siteVadu site
Meal 1 +
Meal 2 +
green pea curry
Meal 3 +
chick pea curry
Meal 1 +
Meal 2 +
Meal 3 +
Meal 4 +
7780 8997 99
4.744.84 5.844.56 4.92
received wheat-based meals that were or were not fortified with
NaFeEDTA at baseline, 3.5 mo, and 7 mo1
Anthropometry and Fe status indices in children who
nBaseline3.5 mo7 mo
Body weight, kg
ZnPP, mmol/mol heme
27.0 6 8.1
26.0 6 7.1
29.2 6 9.0
28.1 6 8.8
30.2 6 9.3
29.3 6 9.2
1.34 6 0.15
1.32 6 0.15
1.36 6 0.14
1.35 6 0.15
1.38 6 0.14
1.37 6 0.15
125 6 11a
124 6 11
128 6 10b*
125 6 12
129 6 11b*
123 6 13
166 14.6 (9.3, 17.9)a
180 13.9 (9.2, 17.6)
17.9 (11.8, 26.9)b* 26.6 (17.8, 35.5)c*
13.4 (8.7, 19.8)14.5 (9.3, 21.1)
5.57 6 2.36b
5.81 6 2.80
4.82 6 2.08a
5.46 6 2.61
5.59 6 2.03c*
6.70 6 3.05
186 61.0 (48.7, 80.0)b61.7 (48.0, 81.0)b
193 62.7 (49.2, 80.7)
54.3 (42.3, 69.7)a*
62.0 (47.0, 84.2)65.3 (50.0, 91.3)
0.0 (0.0, 0.2)
0.0 (0.0, 0.2)
0.0 (0.0, 0.4)
0.0 (0.0, 0.4)
0.1 (0.0, 0.7)
0.1 (0.0, 0.5)
0.03 6 0.05a
0.03 6 0.05
0.06 6 0.05b*
0.04 6 0.06
0.07 6 0.05b*
0.03 6 0.06
1Data are mean 6 SD, median (quartile 1, quartile 3). *Data different from control at
that time, P , 0.001. Means in a row with superscripts without a common letter differ,
P , 0.05. BIS, estimated body iron store; Hb, hemoglobin; SF, serum ferritin; sTfR,
serum transferrin receptor; ZnPP, erythrocyte zinc protoporphyrin.
Iron fortification of wheat flour with NaFeEDTA5 of 7
development that might persist despite correction of the Fe-
deficient state (55). Therefore, it is possible that the children
participating in this study may have been Fe deficient during
early life, with consequent irreversible changes in neural
mechanisms. Third, there is a possibility that the effect of Fe
fortification on cognitive measures was not strong enough to
overcome the adverse impact of poverty and poor socioeco-
nomic status on a child?s psychomotor development. Finally,
although the sample size in the present study was sufficient to
detect a significant change, it is possible that the study duration
of 7 mo was insufficient to significantly affect cognitive abilities
of the study children.
In summary, the findings of this study support the recent
fortification guidelines that suggestNaFeEDTAto be theideal Fe
compound for whole wheat flour fortification. Whole wheat
flour fortified with NaFeEDTA was efficacious in markedly
reducing ID prevalence and improving BIS and Fe status in
Fe-depleted Indian school children. This is particularly relevant
to the improvement of BIS in girls of school age in preparation
for their higher daily Fe requirements during puberty and later
during pregnancy. NaFeEDTA-fortified wheat flour may be
recommended for wider use in national school feeding programs
for children where there is a high prevalence of ID and in public
distribution systems among vulnerable populations.
The authors are most grateful to Sr. Lilly D?Souza, the Principal
of Franciscan Institute High School (Kannada Medium), for her
cooperation in the smooth conduct of the study at her school
premises. The authors express their thanks to all their
colleagues who were involved in data collection, biochemical
analyses, and the preparation and distribution of meals: Leena
Sebastian, Shanthi Chellan, Ajay Kumar, Juanita Amy Jones,
Tony Raj, and Uma Unni from St. John?s Medical College and
Research Institute, Bangalore, India; Sanjay Juvekar, from the
Vadu Rural Health Program, Vadu and Deepa A. Raut and
Pallavi S. Hardikar from the Diabetes Unit, KEM Hospital
Research Centre, Pune, Maharashtra, India. S.M., P.T., R.F.H.,
and A.V.K. were involved in the conception, design, and
interpretation of the results of the study; V.A., D.A., and H.L.
conducted research and assisted in manuscript preparation; K.S.
helped in designing cognitive function assessment and manu-
script preparation; S.H. and C.S.Y. provided critical comments
on the manuscript; T.T. and S.M. conducted the statistical
analyses; and S.M. wrote the manuscript and had primary
responsibility for final content. All authors read and approved
the final manuscript.
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