Ferrous Fumarate and Ferric Pyrophosphate as Food Fortificants in Developing Countries
Condition: Healthy Participants; Non-anemic Children; Helicobacter Pylori Infection
Study type: Interventional
Phase: Phase 1

Summary: 
      Iron fortification of foods is usually considered the most cost-effective approach to prevent
      iron deficiency. However, iron is the most difficult mineral to add to foods. When added as
      water-soluble, highly bioavailable compounds such as ferrous sulfate, the soluble iron
      rapidly catalyzes fat oxidation resulting in rancid products. In addition, water-soluble iron
      compounds can cause unacceptable color reactions during storage and food preparation. Thus,
      food manufacturers are often obliged to use water-insoluble iron compounds to fortify foods
      and fortification compounds such as elemental Fe powder and ferric pyrophosphate are widely
      used to fortify cereal flours and infant cereals. However, these compounds never dissolve
      completely in the gastric juice and are usually far less well absorbed than ferrous sulfate
      (Hurrell 1997). Ferrous fumarate on the other hand, although almost insoluble in water,
      readily dissolves in the gastric juice and has been shown to have an equivalent absorption to
      ferrous sulfate in healthy, Western adults (Hurrell et al. 1989, 2000). Because it is
      non-water soluble, it causes relatively few sensory problems in the fortified foods and is
      therefore an interesting food fortificant. Iron absorption from ferrous fumarate has been
      demonstrated to be significantly higher than from ferric pyrophosphate in European infants
      (Davidsson et al. 2000) and this compound is currently used to fortify blended cereal flours
      for food aid programs and commercial infant cereals in Europe. However, based on our recent
      study in Bangladeshi children, there is now concern that due to lower gastric acid output,
      young children in developing countries may not be able to absorb ferrous fumarate as well as
      Western adults (Davidsson et al. 2001a, Sarker et al. 2001, 2003). Clearly, there is a need
      to evaluate the efficacy of water insoluble iron compounds to prevent the development of iron
      deficiency/iron deficiency anemia in infants and young children living in developing
      countries. The aim of this study is to evaluate the efficacy of ferrous fumarate and ferric
      pyrophosphate, as compared to ferrous sulfate, as food fortificants in preventing development
      of anemia/IDA in Bangladeshi infants and young children (part I).

      A potential cause of low gastric acid secretion in Bangladesh and many developing countries
      is Helicobacter pylori infection. Although H. pylori-infection appeared to have no influence
      on absorption of ferrous fumarate in children, the impact of chronic H. pylori infection in
      adults could be expected to be more pronounced due to long time effects on the gastric
      mucosa, resulting in reduced gastric acid output. The other aim of the study is therefore, to
      assess of iron absorption and gastric acid output in adult women of child-bearing age with H.
      pylori infection (part II).

      Two hundred and forty non-anemic Children (Hb>105 g/L) will be randomized to three study
      groups; ferrous fumarate, ferric pyrophosphate or ferrous sulfate (n=80 per group) in wheat
      flour- and cow milk-based infant formula and will be fed for 9 months. Hemoglobin, serum
      ferritin, and transferin receptor will be analyzed at baseline and after 4.5 and 9 months of
      intervention. Prevalence of anemia and iron deficiency during and after the intervention
      among the three groups will be compared (part I). We furthermore propose a complementary
      study to determine the relative absorption of ferrous fumarate (relative to ferrous sulfate)
      in H. pylori infected and non-infected adult Bangladeshi women (15 each) of 20-40 year of age
      with IDA using stable isotope technique based on the incorporation of iron stable isotopes
      into erythrocytes 14 days after administration. Assessment of gastric acid output will also
      be performed. Iron stature and absorption, and assessment of gastric acid output will be
      compared before and after therapy in H. pylori infected women (part II). The results of this
      study are expected to have implications in the prevention and treatment of iron deficiency
      anemia in developing countries
    

Detailed description: 
      The efficacy study will test the ability of ferrous fumarate and ferric pyrophosphate to
      prevent anemia/IDA in non-anemic Bangladeshi infants/young children in comparison to ferrous
      sulfate during a 9 month feeding trial. Hemoglobin and indicators of Fe status (ferritin and
      circulating transferrin receptor) and an acute phase reactant (C-reactive protein) will be
      monitored at baseline and after 4.5 and 9 months of intervention.

      Subjects Non-anemic infants and young children (6-24 months; n=240) (Hb >105g/L) will be
      recruited for the study from the population at Nandipara, a peri-urban community near Dhaka
      city. All children will be randomized to one of three study groups; ferrous fumarate, ferric
      pyrophosphate or ferrous sulfate (n=80 per group), and stratified based on H. pylori
      infection (based on H. pylori stool antigen test, positive or negative) at base line. All
      infants/young children will be retested for H. pylori infection at the end of the study.

      All parents or guardians will be fully informed about the aim and procedures of the study and
      informed consent will be obtained from at least one parent/guardian per child.

      Exclusion criteria Exclusion criteria include children with anemia (Hb<105 g/L), systemic
      infection or apparent inflammatory process or weight for age of < 70% of NCHS median. The
      children with exclusion criteria will be appropriately treated or if needed, or will be
      referred to appropriate health center for treatment.

      Sample size:

      The sample size is based on the expected anemia prevalence after the intervention, i.e.,
      children with anemia after 9 months of intake of fortified infant cereal. We assume that the
      prevalence of anemia will be 10% in infants consuming cereal fortified with ferrous sulfate,
      30% in infants consuming cereal fortified with ferrous fumarate and 50% anemia in infants
      consuming cereal fortified with ferric pyrophosphate. To detect a difference of 20% points
      ((P1 - P2); sulfate versus fumarate and sulfate versus pyrophosphate) with 95% confidence and
      power=0.80, the sample size is calculated to 62 infants/young children per group.

      Assuming a drop out rate of about 25 %, we will recruit 80 infants/young children per group
      i.e. 240 non-anemic infants/young children.

      We assume a high prevalence of anemia (70-80%) in the study population and will therefore
      need to screen approximately 1000-1200 children.

      Fortified food Each serving will consist of 25 g infant cereal based on wheat or rice flour
      and cow milk (produced especially for the study; 9.3 mg Fe as ferrous fumarate, ferric
      pyrophosphate or ferrous sulfate/25 g dry cereal will be added during manufacture. Ascorbic
      acid will be added during manufacture at a molar ratio 3:1 relative to added Fe. Fortified
      infant cereals will be administered by health workers, one serving per day, 6 days per week,
      under close supervision by the study physician/investigators. Intake will be monitored weekly
      and, if needed, fed during 7 days per week to compensate for days when the child was sick or
      absent.

      The fortified food will provide the Recommended Nutrient Intake of iron for infants 7-12
      months according to WHO/FAO 2002 (in press; please see PAHO/FNP/USAID consultation 2001) per
      serving, i.e., 9.3 mg Fe. Fortified infant cereal will be fed once per day, 6 days per week,
      for 9 months.

      Dietary information by simplified food frequency questionnaire (SFFQ), validated and
      currently being used by the Bangladesh Integrated Nutrition Project (BINP), will also be
      obtained twice a month. Anthropometrical measurement e.g. weight, height, and mid upper arm
      circumference (MUAC) will be measured at baseline, at 4.5 months and 9 months of
      intervention.

      All parents or guardians will be fully informed about the aim and procedures of the study and
      informed consent will be obtained from at least one parent/guardian per child. All anemic
      infants/young children (diagnosed during the screening study) will be treated with medicinal
      Fe according to standard treatment practice at ICDDR.B; (ferrous sulfate drops; (2 mg Fe/kg
      BW/d) for 2 months. Infants/young children with anemia at 4.5 and 9 months (efficacy study)
      will be treated with medicinal Fe (ferrous sulfate drops; (2 mg Fe/kg BW/d) for 2 months and
      excluded from the study. Medicinal Fe will be administered by health workers.

      Hookworm infection is not prevalent in this community but all children participating in the
      study will be screened for hookworm infection at the time each blood sampling. Infected
      children will be treated with Albendazole 400 mg single dose for two times (2 weeks apart)
      but will not be excluded from the intervention study.

      Blood analysis Venous blood samples will be drawn (2 ml in EDTA treated tubes) during the
      screening study to identify non-anemic infants/young children and at baseline and after 4.5
      and 9 months of intervention. Hb will be analyzed using the cyanomethemoglobin method (Sigma
      kit, Sigma, St Louis, MO). Plasma ferritin and plasma circulating transferrin receptor will
      be measured by ELISA technique by using commercial kits during the initial screening study
      (Ramco, Houston, TX). Samples drawn at baseline and after 4.5 and 9 months of intervention
      will be analyzed at KUMC for plasma ferritin and plasma circulating transferrin receptor by
      ELISA assays based on monoclonal antibodies (Flowers et al. 1986)/dual monoclonal antibodies
      (Flowers et al. 1989). Plasma samples will also be analyzed for C-reactive protein (CRP) at
      KUMC. CRP will be monitored as an acute phase reactant. Dr. Cook and Ms. Flowers are
      currently developing a monoclonal ELISA technique for CRP, which will be used in this study.
      Commercial quality control materials (DiaMed, Cressier sur Morat, Switzerland, Sigma and
      Ramco) will be analyzed together with all series of samples analyzed for Hb and ferritin.

      Food analysis Infant cereals will be analyzed for Fe and calcium (Ca) by electrothermal/flame
      atomic absorption spectroscopy (SpectrAA 400, Varian, Mulgrave, Australia) after
      mineralization by microwave digestion (MLS 1200) using a HNO3/H2O2 mixture, using standard
      addition technique to minimize matrix effects. The phytic acid content will be determined by
      HPLC technique (Sandberg & Ahderinne 1986, Sandberg et al. 1989

      Fecal samples; screening for H. pylori infection and hookworm infection Stool specimens will
      be collected and tested for the presence of H. pylori antigen (HpSA) by a newly developed
      enzyme immunoassay (EIA). A polyclonal commercial kit (H. pylori antigen EIA; Cat# 740096,
      Novitech, Freiburg, Germany) will be used. The HpSA test has been demonstrated to be
      sensitive (>80 %) and specific (>95 %) for the detection of H.pylori infection (Braden et al.
      2000, Vakil et al. 2000, Sarker et al. personal communication 2003). Stool samples will also
      be screened for the presence of parasite eggs, using standard microscopic technique.

      Statistics ANOVA will be used to compare variables within each study group as well as between
      the 3 study groups. Results with significant F ratio (p<0.05) will be further analyzed by
      Student's t-test using multiple comparison procedure. Different outcome variables, i.e., Hb,
      ferritin, and TfR concentrations before and after intervention in each group will be compared
      by paired t-test. Data with skewed distribution will be normalized by log transformation
      before statistical analysis. Chi-squared test will be used to evaluate binary variables at
      different time points during the study. Odds Ratios and confidence intervals will be
      calculated. Multivariate analysis (logistic regression and multiple regression) will also be
      performed to adjust for confounders, e.g. age, nutritional status.
    

Intervention name(s): Ferrous fumarate; Ferric pyrophosphate; Placebo
Intervention type(s): Drug; Drug; Drug
Intervention description(s): 9.3 mg once per day, 6 days per week, for 9 months; 9.3 mg once per day, 6 days per week, for 9 months; 9.3 mg once per day, 6 days per week, for 9 months
Intervention MeSH terms: Ferrous fumarate
Allocation: Randomized
Intervention model: Single Group Assignment
Masking: Quadruple (Participant, Care Provider, Investigator, Outcomes Assessor)
Primary purpose: Prevention
Keywords: Iron deficiency anemia; Helicobacter pylori; Iron absorption; Iron fortification; ferrous sulphate; ferrous fumarate