The Impact of Vitamin B12 Deficiency on Infant Gut Microbiota

Comparison of Gut Microbial Composition in Breastfed Infants With and Without Vitamin B12 Deficiency

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.

Study Overview

• Status: Completed
• Conditions: Vitamin B 12 Deficiency

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.

• Study Type: Observational
• Enrollment (Actual): 90

Contacts and Locations

Study Locations

• Turkey
  • Istanbul, Turkey
    • Marmara University School of Medicine

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 4 months to 6 months (CHILD)
• Accepts Healthy Volunteers: Yes
• Genders Eligible for Study: All

• Sampling Method: Probability Sample

Study Population

Healthy term exclusively breastfed infants aged between 4 to 6 months who fulfill the inclusion criteria are enrolled

Description

Inclusion Criteria:

• must be >37 gestational weeks, birth weight must be >2500 gr, must be exclusively breastfed there should be no congenital anomalies, hemoglobin should be ≥10 g/dL,

Exclusion Criteria:

• prematurity, low birth weight, intrapartum antibiotic use, current or previous use of infant formula, malnutrition, use of iron supplements, use of antibiotics, use of probiotics within the last 8 weeks before study entry, and being introduced to solid foods

Study Plan

How is the study designed?

Number of groups / cohorts

2

Cohorts and Interventions

Group / Cohort
Vitamin B12 deficiency; No intervention will be administered for this study. Serum vitamin B12 <203 pg/mL is considered as vitamin B12 deficiency. Fecal microbiota composition will be analyzed with 16S rRNA sequencing. In a subgroup of infants (n=11), fecal samples will be recollected after the treatment as usual
Vitamin B12 sufficient; Serum vitamin B12 ≥203 pg/mL is considered as vitamin B12 sufficient Fecal microbiota composition will be analyzed with 16S rRNA sequencing.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Gut microbial community differences between infants with vitamin B12 deficiency and infants with sufficient vitamin B12	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	DNA extraction and fecal microbiota composition with 16S rRNA sequencing is anticipated to be completed at June 2018

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Gut microbial community differences within subject	Fecal bacterial communities will be assessed before and after treatment as usual in a subgroup of vitamin B12 deficient infants	Recollection of stool samples one month after treatment as usual. DNA extraction and fecal microbiota composition with 16S rRNA sequencing is anticipated to be completed at June 2018

Collaborators and Investigators

• Sponsor: Marmara University

Study record dates

Study Major Dates

• Study Start (ACTUAL): March 3, 2017
• Primary Completion (ACTUAL): September 3, 2017
• Study Completion (ACTUAL): November 3, 2017

Study Registration Dates

• First Submitted: December 2, 2017
• First Submitted That Met QC Criteria: December 2, 2017
• First Posted (ACTUAL): December 7, 2017

Study Record Updates

• Last Update Posted (ACTUAL): March 6, 2019
• Last Update Submitted That Met QC Criteria: March 5, 2019
• Last Verified: March 1, 2019

More Information

Terms related to this study

• Keywords: Microbiome; Vitamin B12; infant
• Additional Relevant MeSH Terms: Nutrition Disorders; Avitaminosis; Deficiency Diseases; Malnutrition; Vitamin B Deficiency; Vitamin B 12 Deficiency
• Other Study ID Numbers: 09.2016.137

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No