Effectiveness of Weekly and Daily Iron Supplementation for the Prevention of Iron-deficiency Anemia in Infants. Impact on Genomic Stability
Phase: PHASE4
Conditions: Anemia, Iron-deficiency
Sponsor: Instituto de Desarrollo e Investigaciones Pediátricas Prof. Dr. Fernando E. Viteri

 
Study Protocol 
 
 
 
OFFICIAL TITLE 
Effectiveness of weekly and daily iron supplementation for the prevention of iron-deficiency anemia in 
infants. Impact on genomic stability. 
 
 
 
NCT ID not yet assigned 
 
03/01/2017 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

BRIEF TITLE  
Iron deficiency anemia, iron supplementation and genomic stability in infants. 
 
BRIEF SUMMARY AND DETAILED DESCRIPTION  
Iron deficiency is the most prevalent nutritional deficiency and the main cause of anemia. Globally, 43% 
of pre-school children have anemia. In Argentina, the National Nutrition and Health Survey reported that 
34.5% of children less than two years of age and 50.8% of 6 to 9-month-old infants were anemic. 
Although daily iron supplementation is supported as a preventive strategy, adherence is low in our 
country due to adverse effects and low prescription rates. Likewise, excess iron may cause genomic 
instability and thereby structural and functional alterations in proteins, lipids and DNA. Such damage 
may be permanent and lead to the development of neoplastic processes, diabetes and cardiovascular 
disease. Weekly iron supplementation is proposed as an effective alternative for older children and 
pregnant women, but evidence of such an effect in infants is scarce. The aim of this study is to compare 
the effectiveness of weekly and daily (current prescription) iron supplementation to prevent anemia in 
infants and to analyze its impact on genomic stability. We will perform a randomized, controlled clinical 
trial in infants that attend IDIP´s Health Observatory for periodic health controls. Infants with exclusively 
breastfeeding (EBF) will be randomized into three groups: daily and weekly iron supplementation and no 
supplementation. Infants receiving both breastfeeding and infant formula i.e. mixed feeding (MF) will be 
randomized into two groups: daily and weekly iron supplementation. We will study anemia, iron status 
and genomic stability before supplementation (at 3 months) and at 6 months. Weekly iron 
supplementation is expected to be more effective than daily supplementation for the prevention of infant 
iron-deficiency anemia due to a higher adherence and a lower incidence of adverse effects. Further, 
genomic stability is also expected to be lower. We expect that our results will allow to revise the current 
supplementation schedules (start, frequency, dose, EBF) on scientific grounds and improve the economic 
resources devoted to anemia prevention (lowering medication expenses). 
 
TECHNICAL DESCRIPTION  
Research problem and clinical-sanitary relevance 
Iron deficiency (ID) is the most prevalent nutritional deficiency and the main cause of anemia worldwide 
(WHO 2001), infants less than 2 years and pregnant women being the most vulnerable groups. Iron 
deficiency anemia (IRA) may impact at early stages of life by causing growth retardation, lower cognitive 
abilities, lethargy and poor attention (Clark 2008, Beard 2008, Carter 2010, Lozoff 2007). This public 
health concern has stimulated the research for the development of alternative preventive solutions.  
The overall prevalence of anemia among preschool children is 43% (Stevens 2013). In Argentina, the 
National Nutrition and Health Survey (ENNyS by its Spanish acronym) reported that such prevalence was 
34.5% in children less than 2 years of age and 50.8% in 6-9-month-old infants (ENNyS 2007). A study 
performed at the Health Observatory of IDIP (La Plata Children´s Hospital) showed that one out of three 
clinically healthy infants less than 6 months of age had anemia, regardless of their being exclusively 
breastfed or receiving mixed feeding (MF) (Iannicelli 2012). According to a survey performed with 
clinical record data of the period 2013-2015, anemia affects more than half of infants that attend the 
Health Observatory (unpublished data). 
Iron supplementation is recommended as a priority strategy and has been endorsed by the World Health 
Organization (WHO) when ID prevalence is high in a specific population. In our country, the Argentine 
Pediatrics Society recommends daily ferrous sulfate supplementation in infants less than 4 months of age 
receiving MF and from 6 months onwards in EBF (SAP 2009). Despite this recommendation and the free 
supply of ferrous sulfate through the health program Remediar, adherence is low, mainly due to 
gastrointestinal adverse effects, mothers´ neglect and low rate of prescription by healthcare team 
members (Christensen 2013, Bernztein 2008). Thus, since the nineties, weekly ferrous sulfate 
supplementation has been proposed as an alternative preventive treatment for IDA. However, most 
studies are focused on pregnant women and children older than 1 year (WHO 2011, De Regil 2011, Peña-

Rosas, 2012), and evidence in infants is scarce (Yurdakov 2004). 
Iron is involved in intracellular redox reactions, where reactive-oxygen species (ROS) are formed. As 
iron is not easily eliminated from the body, it may adversely affect proteins, lipids and DNA. Such 
damage may be permanent and increase the risk of developing neoplastic processes, diabetes and 
cardiovascular and neurological diseases (Evans 2004, Liu 2009, Toxqui 2010). Furthermore, reports in 
the literature suggest that the iron dose used for the treatment of anemia in children may affect genomic 
stability (Aksu 2010). However, such effect has not been demonstrated in infants. The advantages of 
weekly iron supplementation regarding adherence and the probably lower damage on the genome could 
support the need of reconsidering the current IDA preventive guidelines on time and manner of 
administration and further design health promotion and disease prevention policies. 
 
Theoretical framework and empirical evidence  
ID is the most prevalent nutritional deficiency and the main cause of anemia worldwide. Infants less than 
2 years and pregnant women are among the most vulnerable groups. In developing countries, ID coexists 
with other conditions such as protein-energy malnutrition, vitamin A and folic acid deficiency, and 
infectious diseases (Olivares, 1999). In children, the most common cause of ID is an increased iron 
requirement, which is related to higher growth development rates (Clark, 2008).  
In 2009, the prevalence of anemia reported by the WHO was 47.4% in pre-school children (McLean, 
2009). More recently, the global estimation of such prevalence was 43% (Stevens 2013). In Argentina, 
the ENNyS reported that anemia affected 34.1% of infants between 6 and 23 months of age; this figure 
was higher in households with unsatisfied basic needs (Durán, 2009).  
Iron reserves in babies with normal gestational age and birth weight from well-nourished mothers and 
delayed umbilical cord clamping (> 2 min) should be enough to meet iron requirements until 6 months of 
age (Dewey, 2007). However, different studies report a high prevalence of anemia at early ages. In Latin 
America, around 60% of infants less than 6 months have anemia: 71.4% in Bolivia, 63.5% in Honduras 
and 59.4% in Peru (Lutter, 2008). Likewise, studies performed in other countries have also reported a 
high prevalence of anemia in EBF infants (Yang, 2006; Torres, 2006; Marques, 2014). A study performed 
in Argentina showed that the prevalence of anemia in EBF infants was comparable to that found in 
formula-fed infants, affecting 28.9% of infants less than 6 months assisted at the public healthcare sector 
(Ianicelli, 2012). 
Iron-deficiency anemia (IDA) is a pathologic condition characterized by the shortage of iron for the 
synthesis of iron-containing proteins and enzymes such as hemoglobin, myoglobin, cytochromes, 
catalases and various peroxidases (Archer, 2015; Dallman, 1986). Besides the classical symptoms, other 
non-hematological manifestations have been described, namely, decreased capacity for physical work and 
spontaneous motor activity, impaired cell immunity and bactericidal capacity of  neutrophils, decreased 
thermogenesis, 
functional 
and 
histological 
impairment 
of 
the 
gastrointestinal 
tract, 
defective mobilization of liver vitamin A, decreased growth development rate, behavioral, cognitive and 
motor impairment, lower sensory, auditory and visual conduction velocity, and reduced muscle tone. 
During brain development, ID causes the reduction of brain iron content, which is irreversible when 
treatment is not timely (Algarin, 2003; Beard, 2003 y 2008; Carter, 2010; Lozoff, 2011). 
At early life stages, IDA impacts strongly on public health. Therefore, research on alternative preventive 
solutions has been encouraged. The WHO endorses the general agreement that infant and pregnant 
women iron supplementation is a priority strategy when IDA prevalence in a particular group of 
individuals is higher than 40% (WHO, 2001). In Argentina, the Argentine Pediatrics Society recommends 
daily ferrous sulfate supplementation in infants less than 4 months of age receiving MF and from 6 
months onwards in infants with EBF (SAP 2009). Despite ferrous sulfate is prescribed and freely supplied 
through the health program Remediar, adherence is low due to gastrointestinal adverse effects, mothers´ 
neglect and low rate of prescription by healthcare team members (Christensen 2013, Bernztein 2008).  
On the other hand, it has been pointed out that daily oral iron supplementation reduces iron absorption a 
few days after treatment initiation, whereas intermittent doses maximize absorption because iron 

provision is concomitant with intestinal cell turnover (Anderson 2005; Frazer 2003a; Frazer 2003b). It is 
postulated that daily iron doses saturate the transport mechanisms, and that this correlates with intolerance 
symptoms, whilst weekly doses optimize absorption through transport mechanisms, decreasing 
intolerance symptoms (Viteri 1997, Srigiridhar 2001). On this basis, weekly iron supplementation is 
proposed as an effective IDA preventive alternative for older children and pregnant women (WHO 2011, 
De Regil 2011, Peña- Rosas 2012). Although different studies report the use of weekly iron 
supplementation, age-groups are dissimilar and include anemic patients; therefore, treatment instead of 
prophylactic doses are used (Coutinho 2008, Lima 2006). Meanwhile, evidence of weekly preventive 
supplementation in infants less than 6 months is scarce (Yurdakov 2004). 
Finally, micronutrient deficiencies and excess may cause genomic damage, which could be in the same 
range of magnitude, or even higher, than genomic damage caused by exposure to high doses of 
environmental genotoxic agents such as carcinogenic agents, ultraviolet radiation and ionizing radiation 
(Fenech, 2010). Furthermore, since iron is not easily eliminated from the body and participates in the cell 
redox reactions, where ROS generate through Fenton reactions, increased iron concentrations could 
damage proteins, lipids and DNA permanently, causing structural and functional impairments. High 
plasma iron concentrations may amplify the effect of free radicals (Aksu, 2010); although cells have 
developed different mechanisms to reduce iron toxicity, including the regulation of iron transport through 
cell membranes and the production of ROS-eliminating enzymes. An imbalance of these systems caused 
by excess iron could increase oxidative damage susceptibility that could cause an increase in the mutation 
rate. These could increase the risk of developing neoplastic processes, diabetes and cardiovascular and 
neurological disease (Evans, 2004; Liu, 2009; Toxqui, 2010). 
The effect of treatment with iron on DNA oxidation has been investigated in children with IDA, 
observing a significant increase of DNA strand breaks and baseline oxidative DNA damage as compared 
with children not receiving treatment (Aksu, 2010). However, we have not found evidence in the 
literature about the effect of preventive doses of iron supplementation on DNA. Therefore, we propose to 
compare the effectiveness of weekly and daily supplementation for the prevention of anemia in infants 
less than six months of life and to analyze their impact on genomic stability. 
 
Objectives  
 
General: To compare the effectiveness of weekly and daily iron supplementation for the prevention of 
anemia in infants. 
 
Specific: To compare:  
- To compare the effectiveness of weekly, daily and no iron supplementation at six months of age in EBF 
infants. 
- To compare the effectiveness of weekly and daily iron supplementation in six-month-old infants 
receiving MF. 
- To compare the incidence of adverse effects for weekly and daily iron supplementation at six months of 
age. 
- To compare the adherence to treatment of weekly and daily iron supplementation at six months of age. 
- To compare the concentration of oxidative stress biomarkers for both types of iron supplementation.  
- To compare the genomic damage in both types of iron supplementation. 
 
Study hypothesis 
Weekly ferrous sulfate supplementation will be more effective in the prevention of IDA in infants than 
daily supplementation due to a higher adherence and a lower incidence of adverse effects. Weekly iron 
supplementation will cause less genomic instability as compared with daily iron supplementation. 
 
Study design 

Randomized controlled clinical trial. 
 
Study sample  
Eligibility 
Infants performing monthly health controls at the Pediatric Health Observatory of IDIP, La Plata 
Children´s Hospital, from birth to six months of age will be included in the study. 
 
Inclusion, exclusion and elimination criteria 
Inclusion criteria: Three-month-old clinically healthy breastfeeding infants with birth age > 37 weeks, 
birth weight between 2500 and 4000 g, a normal fetal and neonatal history, that attend IDIP´s Health 
Observatory and whose parents/guardians express the will to participate in the study and sign a written 
informed consent form.  
Exclusion criteria: Anemic and/or iron deficient infants or with chronic diseases or acute infections or 
under antibiotic/vitamin treatment within 15 days prior to the study.  
Elimination criteria: infants with severe intolerance to iron preventive supplementation, who will receive 
the alternative preventive treatment with ferrous fumarate or iron polymaltose complex). 
 
Unit of analysis: Three to six month-old infants. 
 
Sample size and selection 
According to ENNyS, the prevalence of anemia in 6-9 months old infants is 50.8%. Our controlled 
interventions would reduce such prevalence to 30%. Sample size was estimated with 0.95 significance 
level and 0.80 power, resulting in 174 infants (n= 87 for each supplementation group). This number was 
adjusted considering a 15% dropout, thus enrolling 204 infants. 
 
Methods  
Operational definition of variables 
Main variables 
-Anemia is defined as the “decrease in the total amount of blood cells or a blood hemoglobin (Hb) 
concentration more than 2 standard deviations below the mean of an age- and sex-matched reference 
range”. We will define the presence of anemia when Hb < 9.5 g/dL at 3 months of age and Hb <11.0 g/dL 
at six months of age (CDC, 2007). 
-Iron deficiency is defined as insufficient iron in the body. It will be considered when blood ferritin <12 
ng/ml (WHO 2001). Since ferritin is an acute phase reactant, we will measure C-reactive protein (CRP) to 
assess an eventual inflammatory state. When CRP is ≥5 mg/L, ID will be defined by a blood ferritin <30 
ng/ml. The soluble transferrin receptor (sTfR) which is not influenced by inflammation will also be used 
(cut-off value, sTfR 8.3 mg/L) (Grant 2012). 
Secondary variables 
-Adverse effects: presence (or absence) and frequency of one or more of the following effects during 
ferrous sulfate supplementation (rejection, constipation, vomiting, diarrhea, abdominal pain).  
-Adherence to treatment: is the rate of adherence to medication intake, dietary plan or lifestyle changes 
according to recommendations agreed on with healthcare professionals (OMS, 2003).  
Adherence will be measured with the formula: 
 
(ml SO4Fe received - ml SO4Fe remaining)/days between the medication vial delivery and its return x100, 
  
where ml SO4Fe received is the volume of ferrous sulfate delivered in a vial, sufficient to cover the 
prescription until the infants' next control (1 month), and ml SO4Fe remaining is the residual volume in 
the vial at the moment of the health control. 
We will consider low adherence the consumption of less than 50% of the amount of medication 

prescribed for the study period, and less than 50% of intakes prescribed for the study period.  
We will consider high adherence consumption of more than 80% of the amount prescribed and more than 
80% of the intakes prescribed. 
All combinations not covered by low and high adherence will be considered as moderate adherence. 
-Genomic instability: Chromosome, DNA and cell damage caused by certain agents. We will evaluate 
oxidative stress and genomic damage at cytomolecular level and at DNA base level. 
- Oxidative stress: imbalance between reactive oxygen species (ROS) production and accumulation and 
the counteracting enzymatic/non-enzymatic antioxidant mechanisms. We will measure catalase and 
superoxide-dismutase enzymatic activity and evaluate cell damage by lipid peroxidation through 
thiobarbituric acid-reactive substances (TBARS) assessment. 
- Genomic damage: DNA damage assessed by cytomolecular techniques using the Comet assay (damage 
index assessment in 200 cells, quantifying damage by Tail moment and Olive Tail moment). The 
frequency of oxidized DNA bases will be determined by competitive 8-oxo-dG Elisa. 
Other variables: 
- Sex, weeks of gestation, umbilical cord clamping. 
- Infant nutritional condition will be assessed by anthropometry (weight, height, head circumference) at 
three and six months. 
- Sociodemographic data of infants and relatives, including place of residence, unsatisfied basic needs, 
maternal education. 
- Maternal nutritional condition will be evaluated with anthropometry and iron status. 
- Environmental exposure to genotoxic agents (antibiotics, radiation (X-rays) and chemical substances 
like pesticides, solvents and industrial waste) will be assessed by questionnaire. 
 
Data collection 
-Patient enrolment 
During health control visits to IDIP Health Observatory on the first and second month of life, parents and 
tutors will be invited to participate in the study; the study's protocol, its aims and benefits will be 
explained in a plain language. They will be asked to sign a written informed consent form in case they 
accept their children be incorporated to the study. Infants meeting the inclusion criteria with exclusively 
breastfeeding (EBF) will be randomly divided into three groups: weekly, daily and no supplementation. 
Those with mixed feeding (breastfeeding and formula; MF) will be randomly divided into two groups: 
weekly and daily supplementation.  
- Intervention 
Weekly supplementation (EBF or MF): infants will receive a weekly dose of ferrous sulfate 
(4mg/kg/week).   
Daily supplementation (EBF or MF): infants will receive a daily dose of ferrous sulfate (1 mg/kg/day).   
Without intervention (only EBF): infants will receive no supplementation, following the current SAP 
guidelines. Ferrous sulfate will be supplied by pediatricians.  
- Randomization  
Randomization will be performed by computer-generated random sequences of numbers in blocks of 
variable length. Randomization will be stratified by type of intake (EBF or MF). 
Treatment assignments will be placed in sequentially numbered, opaque and sealed envelopes which will 
be consecutively put in a box and later removed one at a time. The person in charge of the random 
allocation process will not take part in the enrollment process. 
- Blinding 
Pediatricians in charge of supplementation will know the assigned intervention only when the envelopes 
are open. Professionals in charge of performing laboratory tests will not know intervention assignments. 
-Sample collection. Surveys 
Before supplementation, 3-ml blood samples from infants will be drawn to assess anemia, ID, and 
genomic stability. All non-anemic/iron deficient infants will be recruited and will be supplemented 

according to the group assigned during 12 weeks (three months). At the end of this period (six months of 
age), blood samples will be drawn again to measure the study variables. Infant and family 
sociodemographic data will be surveyed at the first visit (three months of age). Anthropometric 
measurements will be taken at three and six months. Nutritional assessment of the mothers will be 
performed at the beginning and at the end of the study, measuring weight and height and drawing blood 
samples for anemia and ID assessment. 
- Intervention progress monitoring 
Pediatricians will prescribe iron doses and manner of administration. Parents and/or tutors will receive 
one or more droppers with ferrous sulfate sufficient to complete one-month supplementation, considering 
that all infants will perform monthly health controls. The Telegram program “LactaPlusIDIPBOT”, 
designed at IDIP, will be used to send reminders for iron supplement intake. They will be also contacted 
weekly (Facebook messenger, Whatsapp, Telegram). To monitor adherence, we will use a daily/weekly 
consumption record form that will be completed by parents/tutors and delivered at each health control. To 
determine consumption, we will control de excess volume in droppers during each monthly control.  
- Record of adverse effects 
Parents and/or tutors will receive a form to register adverse effects which will be presented at each 
monthly control. They will register intake rejection, constipation, vomiting, diarrhea, and abdominal pain. 
Infants with intolerance to iron supplementation will be excluded from the study but will continue on an 
alternative treatment (1 mg/kg/day fumarate or iron polymaltose complex). 
- Laboratory tests 
Blood samples (3 ml) from infants will be drawn at IDIP´s laboratory by venipuncture and divided into 
two vials: 1) an EDTA tube containing 1 ml blood for complete blood count, and 2) lyophilized lithium 
heparin tube that will be centrifuged to obtain plasma for the assessment of ferritin, CPR, sTfR, 8-oxodG 
and TBARS, and erythrocytes for the assessment of CAT and SOD determinations and for the Comet 
assay. The following methods will be used: 
Hemoglobin: Modified Drabkin method (ABX Pentra DX 120 Horiba).  
Ferritin and sTfR: enzymatic immunoassay using a chemiluminescence substrate (Access-Beckman 
Coulter, Fullerton, US).  
C-reactive protein: turbidimetry (Autoanalyzer BT 3000 Plus Wiener).  
All these tests will be performed at the Children´s Hospital Central Laboratory.  
Cytomolecular genomic stability: alkaline version of the Comet assay (electrophoresis of a cell sample 
previously embedded in agarose, being migration proportional to the damage present (Sinh et al., 1988, 
Padula 2016).  
ROS-mediated DNA damage: ELISA to quantify 8-oxodG, the most frequently oxidized nucleotide 
(Rossner et al 2006; Rossner & Sram 2012).  
These tests will be performed at IGEVET. 
CAT activity: spectrophotometry using Aebi´s method.  
SOD activity: commercial kit (Ransod; RandoxLabs).  
TBARS: spectrophotometry according to Okhawa.  
These tests will be performed at IDIP and IGEVET. 
Iron nutritional status (hemoglobin and ferritin) of mothers: blood samples taken at three and six months 
after delivery. 
Data analysis 
The effectiveness of the interventions will be evaluated at six months of age by comparing the incidence 
of anemia in both study groups as a relative risk (RR) and its corresponding 95% confidence interval 
(95% CI). For the EBF group, no supplementation will be the reference category, whilst for the MF 
group, the reference category will be daily iron supplementation.  
Intention-to-treat analysis will be used to account for the possible changes in type of feeding and/or 
intervention arms. However, per-protocol analysis will be also performed and compared with intention-
to-treat analysis in order to determine the result robustness.  

Losses to follow-up will be censured since analyses will be performed in closed cohorts because we will 
not be able to estimate incidence density (we will only collect data on anemia and ID at three and six 
months).    
We will also determine the incidence of adverse effects and the incidence of low treatment adherence at 
six months of age for weekly vs. daily iron supplementation in both EBF and MF and calculate RR (95% 
CI) at six months of age.  
Means and differences of enzymatic SOD and CAT activity and TBARS concentration at three and six 
months will be compared in both study groups. The same will apply to the Comet and 8 oxodG assays.   
Differences between groups will be determined by Anova or Kruskal-Wallis test for quantitative variables 
with post-hoc comparisons and Chi-square for categorical variables. Within group comparisons will be 
analyzed using Student´s or Wilcoxon test. 
Risk of bias assessment 
Selection bias could be present since parents/tutors willing to participate could show higher interest in the 
research project than those who do not participate. Considering that the population sample attends for 
monthly controls, a trust relationship may be established between parent/tutor and pediatrician, resulting 
in a higher degree of adherence to supplementation than in the general population.  
 
Project Schedule 
Activity 
Month 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
Enrolment according to inclusion/exclusion criteria 
 
 
 
 
 
 
 
 
 
 
 
 
Intervention assignment 
 
 
 
 
 
 
 
 
 
 
 
 
Sampling for hemoglobin, ferritin, CPR and sTfR 
assessment. 
 
 
 
 
 
 
 
 
 
 
 
 
Oxidative stress and genomic damage assessment 
 
 
 
 
 
 
 
 
 
 
 
 
Anthropometric nutritional assessment 
 
 
 
 
 
 
 
 
 
 
 
 
Monitoring ( registry of drug administration, adverse effects) 
 
 
 
 
 
 
 
 
 
 
 
 
Data registry (data base entry) 
 
 
 
 
 
 
 
 
 
 
 
 
Data analysis and interpretation of results 
 
 
 
 
 
 
 
 
 
 
 
 
Conclusions. Final report  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Study flowchart 
 
 
Patient enrolment at IDIP´s Health Observatory 
One or Two-month-old healthy infants 
Three-month-old (±10 days) healthy infants 
Anemia, ID, genomic instability 
Anemic and/or ID 
infants are excluded 
and referred for 
treatment 
EBF infants 
MF infants 
Without 
supplementation 
Randomization 
Randomization 
Daily 
supplementation 
Weekly 
supplementation 
Weekly 
supplementation 
Daily 
supplementation 
INTERVENTION 
Daily: 1 mg/kg/day ferrous sulfate  
Weekly: 4 mg/kg/week ferrous sulfate 
Six-month-old (±10 days) healthy infants 
Anemia, ID, genomic instability 
Anemic and/or ID 
infants are referred for 
treatment 

Work plan  
 
SPECIFIC OBJECTIVE 1: Compare the effectiveness of weekly, daily and no iron 
supplementation in EBF infants.  
SPECIFIC OBJECTIVE 2: Compare the effectiveness of weekly, daily and no iron 
supplementation in MF infants. 
ACTIVITY 
RESPONSIBLE 
EXPECTED RESULT 
Enrollment of infants according 
to the inclusion criteria. 
Informed consent.  
Pediatricians 
Incorporate infants who comply with 
inclusion criteria 
Intervention assignment. 
Statistician 
Random allocation 
Blood sample collection. 
Laboratory technician 
Adequate specimen collection (enough 
volume, non clotted)  
Hemoglobin, ferritin, PCR and 
sTfR determinations. 
Biochemists 
Assessment of the prevalence of anemia 
and iron deficiency 
Indication for and monitoring 
of the intervention 
Pediatricians  
Complete registry of the intervention. 
Text message reminders  
Pediatricians 
Intervention adherence. 
Weight and height 
measurement. 
Pediatrician, Nutritionist 
Determine the anthropometric 
nutritional condition. 
Registry of data. 
Data entry 
Complete database. 
Data analysis and interpretation 
of results. 
Team, Statistician 
Report of results and conclusions. 
 
SPECIFIC OBJECTIVE 3: Compare the incidence of adverse effects on weekly vs. .daily iron 
supplementation for the prevention of anemia  
Monitoring of the record of 
adverse effects. 
Pediatricians 
Adequate record of the presence and 
frequency of adverse effects in both 
types of supplementation. 
Participant follow-up. 
Pediatricians 
Early detection of adverse effects that 
may prevent intervention progression. 
Control of data record forms 
Team 
Early detection of adverse effects. 
Data analysis and interpretation 
of results. 
Team, statistician 
Report of results and conclusions. 
 
SPECIFIC OBJECTIVE 4: Compare the degree of treatment adherence to weekly and daily iron 
supplementation at six months of age. 
Monthly control of droppers to 
calculate adherence 
Biochemist 
Correct administration of iron 
supplementation  
Monitoring of drug intake 
(registry form and phone calls) 
Pediatricians 
Adequate drug intake according to 
prescription. 
Data analysis and interpretation 
of results. 
Team, statistician 
Report of results and conclusions. 
SPECIFIC OBJECTIVE 5: Compare oxidative stress indicators resulting from both forms of iron 
supplementation. 
Blood sample collection 
Laboratory technician 
Adequate specimen collection 

(enough volume, non clotted)  
TBARS, CAT and SOD 
assessment 
Biochemist, IGEVET 
researchers 
Evaluation of oxidative stress 
Data analysis and interpretation 
of results. 
Team, statistician 
Report of results and conclusions. 
SPECIFIC OBJECTIVE 6: Compare genomic damage with both forms of iron supplementation 
Blood sample collection 
Laboratory technician  
Adequate specimen collection 
(enough volume, non clotted) 
Comet assay 
IGEVET researchers 
Cytomolecular damage evaluation 
8 oxo-dG assessment 
IGEVET researchers 
Quantification of oxidized bases 
Data analysis and interpretation 
of results. 
Team, statistician 
Report of results and conclusions. 
 
 
 

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