The Effect of Human Milk Oligosaccharides and Galacto-oligosaccharides on Iron Absorption in Kenyan Infants

Study Overview
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Brief Summary
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Effective and safe strategies to deliver iron to infants and young children in Sub-Saharan Africa are urgently needed. One potential strategy to improve safety of iron fortification is to limit the total amount of unabsorbed iron entering the colon by lowering the daily iron dose but at the same time ensure efficacy by maximizing absorption from this lower dose. In Kenyan infants, the investigators have recently shown that consumption of 7.5 g of the prebiotic galacto-oligosaccharides (GOS) compared to no GOS consumption increased iron absorption from an iron containing micronutrient powder by ≈60%. It is uncertain whether a lower dose of GOS can also enhance iron absorption. Another question is whether HMOs, 'natural prebiotics' found in high concentration in human breast milk, can also increase iron absorption similar to GOS. Therefore, the aim of this study is to measure fractional iron absorption from a maize-based porridge fortified with A) iron as ferrous fumarate, B) iron as ferrous fumarate and GOS and C) iron ferrous fumarate and HMOs, using an established stable iron isotope technique in 55 infants aged 8-12 months living in Msambweni and surrounding rural communities, Kwale County of southern coastal Kenya. Assessing the effect of a low dose of GOS and of HMOs on iron absorption will provide valuable information towards the development of new, highly bioavailable iron formulations for African infants. As per the local standard of care, the participants who will be iron-deficient anemic at the end of the study will be treated with oral iron supplements. To evaluate the effects of iron supplementation on iron and anemia status and to estimate obligatory iron losses in the gastrointestinal tract, blood and fecal samples will be collected before, during and fourteen days after the beginning of the treatment with oral iron supplements. Data about the efficacy of current supplementation strategies in iron-deficient anemic children and obligatory iron losses would provide additional evidence for the optimization of iron supplementation regimens.

Detailed Description
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Iron absorption from differently labelled iron-fortified maize-based test meals will be measured in 55 infants. At baseline a venipuncture blood sample will be collected from all infants for the determination of the following iron and inflammation status parameters: hemoglobin (Hb), plasma ferritin (PF), soluble transferrin receptor (sTfR), C-reactive protein (CRP) and alpha-1-acid glycoprotein (AGP), and anti-oligosaccharide immunoglobulins. Anthropometrics will be measured; demographics, the medical history and the feeding habits will be assessed using a questionnaire. A breast milk sample from all mothers will be collected for determination of HMO profile and maternal secretor status. After baseline, 30 infants will consume three different test meals on alternate days (day 1, day 3, and day 5) and 25 infants will consume two different test meals on alternate test meal days (day 1, day 3 and day 5). The order of consumption of the three test meals will be randomly assigned. Test meal A will contain 5 mg of iron as ferrous fumarate given as 2.5 mg Fe-56 and 2.5 mg Fe-54 (control test meal). Test meal B will contain 5 mg of iron as ferrous fumarate given as 2.5 mg Fe-56 and 2.5 mg Fe-58 and 4 g of GOS-75 (≈ 3 g GOS) (GOS test meal).Test meal C will contain 5 mg of iron as ferrous fumarate given as 2.5 mg Fe-56 and 2.5 mg Fe-57 and 2.0 g 2'-fucosyllactose (2'-FL) and 1.0 g lacto-N-neotetraose (LNnT) (HMO test meal). The test meals will be based on maize porridge, consisting of refined maize flour, sugar and mineral water, and will be administered between 0700 and 0900. Overnight, only breast milk will be allowed to the infant and no breast milk and no other food will be given at least 3 h before test meal administration. Test meals plus mineral water will be consumed completely in the presence of the investigators, and the infant will not be allowed to eat or drink for 2 h after the test meal. Fourteen days after the third test meal administration (day 17 and 19, respectively) a whole blood sample will be collected by venipuncture for analysis of the ratios of the different molecular weight iron incorporation into red blood cells and determination of iron and inflammation status (Hb, PF, sTfR, CRP and AGP). Furthermore, anthropometrics and some parts of the baseline questionnaire will be repeated. At endpoint (day 17 and 19, respectively), if the infant will be diagnosed with iron-deficiency anemia (Hb concentration below 110 g/l and low red blood cells mean corpuscular volume), the caregiver will be instructed to give the infant 4mg/kg iron in the form of oral syrup, daily. Compliance during the follow-up will be assessed by weighting the iron syrup containers before and after 14 days of treatment with the iron syrup. Collection of fecal samples will be performed over 3 time periods of 72h each. The first time period will start the 3 days prior of beginning of oral iron supplementation, the second will take place on day 4, 5 and 6 of oral iron supplementation and the last on day 15, 16 and 17 of oral iron supplementation. A venepuncture blood sample will be collected in the morning after the last day of fecal sample collection (day 18 of oral iron supplementation). Furthermore, some parts of the baseline questionnaire will be repeated. Adverse events (AEs) will be assessed throughout the entire study period.

Official Title
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The Effect of Human Milk Oligosaccharides (HMOs) (2'-Fucosyllactose (2'-FL) and Lacto-N-neotetraose (LNnT)) and Galacto-oligosaccharides (GOS) on Iron Absorption From a Maize-based Porridge in Kenyan Infants

Conditions
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Anemia, Iron-deficiency

Intervention / Treatment
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* Dietary Supplement: ferrous fumarate
* Dietary Supplement: ferrous fumarate + GOS
* Dietary Supplement: ferrous fumarate + HMOs


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Age of 8-12 months at baseline Assessment of good health as assessed by professional staff at Msambweni District Hospital The caregiver is willing to participate in the study The informed consent form has been read and signed by the caregiver (or has been read out to the caregiver in case of illiteracy) Residence in the study area for the period of the study Willingness of the caregiver to provide 2 blood samples from their child and 1 breast milk sample from the mother Exclusion Criteria: Hb <70 g/L Severe wasting (Z-score weight-for-height <-3) Chronic or acute illness or other conditions that in the opinion of the Principle Investigator (PI) or co-researchers would jeopardize the safety or rights of a participant in the trial or would render the participant unable to comply with the protocol Participants taking part in other studies requiring the drawing of blood Regular intake (>2 days) of iron-containing mineral and vitamin supplements or fortified foods within the last 2 months

Ages Eligible for Study
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Minimum Age: 8 Months
Maximum Age: 12 Months

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Treatment
Allocation: Randomized
Intervention Model: Crossover Assignment
Masking: Double


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Active Comparator: ferrous fumarate<br>Maize-based porridge fortified with iron (5mg) as ferrous fumarate | Dietary Supplement: ferrous fumarate<br>* Maize-based porridge fortified with iron (5mg) in form of ferrous fumarate<br>|
| Active Comparator: ferrous fumarate + GOS<br>Maize-based porridge fortified with iron (5mg) as ferrous fumarate + GOS (3g) | Dietary Supplement: ferrous fumarate + GOS<br>* Maize-based porridge fortified with iron (5mg) in form of ferrous fumarate and GOS (3g)<br>|
| Active Comparator: ferrous fumarate + HMOs<br>Maize-based porridge fortified with iron (5mg) as ferrous fumarate + HMOs (2'-FL (2g) + LNnT (1g)) | Dietary Supplement: ferrous fumarate + HMOs<br>* Maize-based porridge fortified with iron (5mg) in form of ferrous fumarate and HMOs (2'-FL (2g) + LNnT(1g))<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Fractional iron absorption in % | Fractional iron absorption (%), measured as erythrocyte incorporation of stable iron isotopes at day 19 | Day 19 | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Hemoglobin (Hb) | Iron status will be determined at baseline | Baseline | 
| Hemoglobin (Hb) | Iron status will be determined at day 19 | Day 19 | 
| Plasma Ferritin (PF) | Iron status will be determined at baseline | Baseline | 
| Plasma Ferritin (PF) | Iron status will be determined at day 19 | Day 19 | 
| Soluble Transferrin Receptor (sTfR) | Iron status will be determined at baseline | Baseline | 
| Soluble Transferrin Receptor (sTfR) | Iron status will be determined at day 19 | Day 19 | 
| C-reactive protein (CRP) | Inflammation status will be determined at baseline | Baseline | 
| C-reactive protein (CRP) | Inflammation status will be determined at day 19 | Day 19 | 
| Alpha-1-acid glycoprotein (AGP) | Inflammation status will be determined at baseline | Baseline | 
| Alpha-1-acid glycoprotein (AGP) | Inflammation status will be determined at day 19 | Day 19 | 
| Human Milk Oligosaccharides concentrations in breast milk | Human Milk Oligosaccharides concentrations in breast milk of the mothers of the participating infants will be measured at baseline to determine maternal secretor status. | Baseline | 
| Human Milk Oligosaccharides concentrations in breast milk | Human Milk Oligosaccharides concentrations in the breast milk of the mothers of the participating infants will be measured at Day 19 to determine maternal secretor status. | Day 19 | 
| Anti-oligosaccharide immunoglobulins | Infant blood serum immunoglobulins toward mucosal oligosaccharide antigens and microbial carbohydrate antigens will be measured at baseline | Baseline | 
| Intestinal Fatty Acid Binding Protein (I-FABP) in infants diagnosed with iron deficiency anemia | I-FABP will be assessed in infants diagnosed with iron-deficiency anaemia and receiving supplementation with oral iron syrup as per local standard of care. | Day 19 | 
| Fecal calprotectin in infants diagnosed with iron deficiency anemia | Fecal calprotectin will be assessed in infants diagnosed with iron-deficiency anaemia and receiving supplementation with oral iron syrup as per local standard of care. Fecal calprotectin will be measured 3 days before beginning of oral iron supplementation. The sampling period will last for 72 hours. | 3 days before oral iron supplementation | 
| Fecal calprotectin in infants diagnosed with iron deficiency anemia | Fecal calprotectin will be assessed in infants diagnosed with iron-deficiency anaemia and receiving supplementation with oral iron syrup as per local standard of care. Fecal calprotectin will be measured on day 4 of oral iron supplementation. The sampling period will last for 72 hours. | Day 4 of oral iron supplementation | 
| Fecal calprotectin in infants diagnosed with iron deficiency anemia | Fecal calprotectin will be assessed in infants diagnosed with iron-deficiency anaemia and receiving supplementation with oral iron syrup as per local standard of care. Fecal calprotectin will be measured on day 15 of oral iron supplementation. The sampling period will last for 72 hours. | Day 15 of oral iron supplementation | 
| Hemoglobin (Hb) in infants diagnosed with iron deficiency anemia | Iron status will be assessed in infants diagnosed with iron-deficiency anaemia and receiving supplementation with oral iron syrup as per local standard of care | Day 18 of oral iron supplementation | 
| Plasma Ferritin (PF) in infants diagnosed with iron deficiency anemia | Iron status will be assessed in infants diagnosed with iron-deficiency anaemia and receiving supplementation with oral iron syrup as per local standard of care | Day 18 of oral iron supplementation | 
| Soluble Transferrin Receptor (sTfR) in infants diagnosed with iron deficiency anemia | Iron status will be assessed in infants diagnosed with iron-deficiency anaemia and receiving supplementation with oral iron syrup as per local standard of care | Day 18 of oral iron supplementation | 
| C-reactive protein (CRP) in infants diagnosed with iron deficiency anemia | Inflammation status will be assessed in infants diagnosed with iron-deficiency anaemia and receiving supplementation with oral iron syrup as per local standard of care | Day 18 of oral iron supplementation | 
| Alpha-1-acid glycoprotein (AGP) in infants diagnosed with iron deficiency anemia | Inflammation status will be assessed in infants diagnosed with iron-deficiency anaemia and receiving supplementation with oral iron syrup as per local standard of care | Day 18 of oral iron supplementation | 
| Hemoglobin in stool from infants diagnosed with iron deficiency anemia | Hemoglobin concentration will be assessed in stools from infants diagnosed with iron-deficiency anaemia and receiving supplementation with oral iron syrup as per local standard of care. Hemoglobin concentration in stools will be measured 3 days before beginning of oral iron supplementation. The sampling period will last for 72 hours. | 3 days before oral iron supplementation | 
| Hemoglobin in stool from infants diagnosed with iron deficiency anemia | Hemoglobin concentration will be assessed in stools from infants diagnosed with iron-deficiency anaemia and receiving supplementation with oral iron syrup as per local standard of care. Hemoglobin concentration in stools will be measured on day 4 of oral iron supplementation. The sampling period will last for 72 hours. | Day 4 of oral iron supplementation | 
| Hemoglobin in stool from infants diagnosed with iron deficiency anemia | Hemoglobin concentration will be assessed in stools from infants diagnosed with iron-deficiency anaemia and receiving supplementation with oral iron syrup as per local standard of care. Hemoglobin concentration in stools will be measured on day 15 of oral iron supplementation. The sampling period will last for 72 hours. | Day 15 of oral iron supplementation. | 
| Intestinal Fatty Acid Binding Protein (I-FABP) in infants diagnosed with iron deficiency anemia | I-FABP will be assessed in infants diagnosed with iron-deficiency anaemia and receiving supplementation with oral iron syrup as per local standard of care. | Day 18 of oral iron supplementation | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Anemia, Iron-deficiency, Iron absorption, Prebiotic, Galacto-oligosaccharides, Human milk oligosaccharides, Infants, Kenya