The Impact of Vitamin B12 Deficiency on Infant Gut Microbiota
Condition: Vitamin B 12 Deficiency
MeSH terms: Vitamin B 12 Deficiency
Study type: Observational

Summary: 
      Vitamin B12 plays important roles in DNA synthesis and neurological functions. Vitamin B12
      deficiency is one of the most common micronutrient deficiencies and it has become
      increasingly more frequent in infants. The deficiency develops more often between 4 to 12
      months of age, coinciding with the decreased fetal storage. The highest rates of vitamin B12
      deficiency were observed among infants who are breastfed by mothers receiving a diet low in
      vitamin B12.

      The main objective of this study is to compare the gut microbial composition of healthy term
      exclusively breastfed infants with or without vitamin B12 deficiency. We hypothesized that
      gut microbiome differs between vitamin B12 deficient and control infants; pathogenic bacteria
      are hypothesized to be higher, and beneficial bacteria to be lower in vitamin B12 deficient
      infants as compared to control.

      An observational study in the Marmara University Pendik Research and Training Hospital well
      child outpatient clinic is conducted in 90 infants. Participants aged between 4 to 6 months
      who fulfill the inclusion criteria are enrolled.

      Inclusion criteria are term birth (>37 gestational weeks), birth weight >2500 gr, absence of
      congenital anomalies, hemoglobin ≥10 g/dL, and exclusive breastfeeding.

      Exclusion criteria include prematurity, low birth weight, intrapartum antibiotic use, current
      or previous use of infant formula, malnutrition, use of iron supplements, infant use of
      antibiotics, use of probiotics within the last 8 weeks before study entry, and introduction
      to solid foods.

      Primary outcome measures:Microbial counts, and a comparison of type and abundance of species
      (predominant, pathogenic, and opportunistic bacteria, yeast, and parasites) between vitamin
      B12 deficient and sufficient subjects will be assessed.

      Secondary outcome measures: Fecal bacterial communities will be assessed before and after
      treatment in a subgroup of infants.

      Each stool sample is collected from cloth diapers, and placed in sterile collection tubes.
      Samples are kept at 4 C and in less than 5 hours, they are frozen at -80 C until further
      analysis. Specimens are transported on dry ice to the Diagen Inc for DNA extraction.

      For the extraction protocol, QuickGene (Kurabo, North America) extraction device will be
      used.

      The extracted DNA samples will be sent to Diversigen Inc (Houston, USA) for analysis. Fecal
      microbiota composition will be analyzed with 16S ribosomal ribonucleic acid (rRNA)
      sequencing.
    

Detailed description: 
      Vitamin B12 plays important roles in DNA synthesis and neurological functions. Vitamin B12
      deficiency is one of the most common micronutrient deficiencies and it has become
      increasingly more frequent in infants. The deficiency develops more often between 4 to 12
      months of age, coinciding with the decreased fetal storage. The highest rates of vitamin B12
      deficiency were observed among infants who are breastfed by mothers receiving a diet low in
      vitamin B12. The data on prevalence of vitamin B12 deficiency in Turkey mainly relate to
      pregnant women and varies between 34% to 72%. In our previous study, 36.3% of the infants
      (n=222) were vitamin B12 deficient. The prevalence was even higher (43%) in infants
      exclusively breastfed for 6 months.

      The physiologic and neurologic consequences of micronutrient deficiencies have been addressed
      extensively. Although it is known that individuals with high bacterial loads in their small
      intestines tend to have low cobalamin status, less is known about its effects on the
      developing gut microbiota. It is suggested that early environmental factors influencing gut
      microbiota during this crucial developmental period can lead to long term consequences on
      health and disease. The data available is mostly limited to the effects of iron. The review
      of the literature has not yielded any study, which investigates the effects of vitamin B12 on
      the developing gut microbiota.

      The study is conducted at Marmara University well child outpatient clinic. The clinic is
      hospital based and located in Pendik, Istanbul. The outpatient clinic provides well child
      care; and besides anthropometric measurements, infants are routinely screened for anemia at 4
      to 6 months of age.

      Ninety-two exclusively breastfed infants are approached for eligibility. Two infants are
      excluded due to prior antibiotic use and 90 infants are enrolled.

      Fecal collection tubes are given to the parents of infants who fulfill the inclusion criteria
      during routine blood withdrawal. For the reporting, serum vitamin B12 <203 pg/mL is
      considered as vitamin B12 deficiency, and ≥203 pg/mL as vitamin B12 sufficient.

      Infants with vitamin B12 deficiency are treated with intramuscular vitamin b12
      (hydroxycobalamin), 250-500 mcg twice weekly for a week, and then 500 mcg weekly for another
      3 weeks. One month after the supplementation in the recommended dose, stools are recollected
      in a subgroup of subjects (n=11) with vitamin B12 deficiency.

      Samples are kept at 4 C and in less than 5 hours, they are frozen at -80 C until further
      analysis.

      Questionnaires are completed, antropometric measures are taken and fecal samples are
      transported on dry ice to the Diagen Inc for DNA extraction DNA extraction will be performed
      by Diagen Inc. For the extraction protocol, QuickGene (Kurabo, North America) extraction
      device will be used.

      The extracted DNA samples will be sent to Diversigen Inc (Houston, USA) for 16S rRNA Gene V4
      region sequencing.
    

Observational model: Cohort
Time perspective: Cross-Sectional
Sampling method: Probability Sample
Study population: 
        Healthy term exclusively breastfed infants aged between 4 to 6 months who fulfill the
        inclusion criteria are enrolled
      
Biospecimen description: 
      Fecal samples of breastfed infants will be extracted for DNA
    
Keywords: Vitamin B12; Microbiome; infant