Vitamin B12 (cobalamin) is essential for brain development, and deficiency is common in many low- and middle-income countries(Reference Black1). Vitamin B12 status depends largely on the amount of animal source foods in the diet(Reference Murphy and Allen2), and in South Asian countries, where diets are predominantly vegetarian, poor vitamin B12 status is common(Reference Taneja, Bhandari and Strand3,Reference Ulak, Chandyo and Adhikari4,Reference Ulak, Chandyo and Thorne-Lyman5) . Breastfed infants have a higher risk of poor vitamin B12 indices than weaned infants(Reference Taneja, Bhandari and Strand3,Reference Ulak, Chandyo and Adhikari4) . In Nepal, consumption of animal products particularly among children is very low(Reference Shrestha, Parmar and Kulig6).
Vitamin B12 is required for cell division and differentiation, energy production and the generation of methionine, which is needed to produce neurotransmitters and myelin in the brain(Reference Dror and Allen7,Reference Finkelstein, Layden and Stover8) . Subclinical vitamin B12 deficiency is associated with poor cognitive performance in both the elderly and children(Reference Black1,Reference Strand, Taneja and Ueland9,Reference van de Rest, van Hooijdonk and Doets10) . Results from an Indian cohort study suggested that children with inadequate vitamin B12 status had lower scores on the Bayley Scales of Infant and Toddler Development 2nd edition (Bayley-II) than replete children(Reference Strand, Taneja and Ueland9). Moreover, in a cohort of Nepalese children, we found that vitamin B12 status in infancy was positively associated with cognitive function when the children were 5 years old(Reference Kvestad, Hysing and Shrestha11). In two randomised-controlled trials (RCT) in Norwegian infants with signs and symptoms of vitamin B12 deficiency, a high dose of vitamin B12 improved short-term motor functioning(Reference Torsvik, Ueland and Markestad12,Reference Torsvik, Ueland and Markestad13) . Another RCT in North India reported a small borderline significant beneficial effect of 1·8 µg vitamin B12 supplementation given daily for 6 months on neurodevelopment in young children, with the most pronounced effect among children who had evidence of vitamin B12 deficiency and who were stunted at study start(Reference Kvestad, Taneja and Kumar14).
Whether the results from these aforementioned RCTs can be generalised to populations without overt vitamin B12 deficiency is not known. We recently undertook an RCT in mildly stunted Nepalese infants receiving 2 µg of vitamin B12 or placebo for one year starting when they were 6–11 months old(Reference Strand, Ulak and Hysing15). This amount of vitamin B12 corresponds to approximately 2–3 recommended daily allowances depending on the age of the child(16). We did not find any effect of vitamin B12 supplmentation on growth or neurodevelopment after one year of supplementation despite an excellent compliance to the supplements and a metabolic response reflecting improved vitamin B12 status in the intervention group(Reference Strand, Ulak and Hysing15). The reliability of neurodevelopmental measures during the first years of life is limited because children develop rapidly and at an uneven pace during this period(Reference Brito, Fifer and Amso17). This may affect the ability to detect differences between groups in young children. The reliability of the tools improves with age(Reference Rubio-Codina and Grantham-McGregor18) and measures taken when the children are older could accordingly be better suited to identify changes to the developing brain that started during infancy. The objective of the current study was to measure the effects of vitamin B12 supplementation that started in infancy on neurodevelopment at 30–47 months of age, with the Wechsler Preschool and Primary Scale of Intelligence 4th edition (WPPSI-IV) full scale intelligence quotient (FSIQ) at 42–47 months as the primary outcome.
Subjects and methods
Study setting and participants
The study is a follow-up of a recently completed double-blind randomised placebo-controlled trial conducted in Bhaktapur municipality, Nepal and surrounding areas(Reference Strand, Ulak and Hysing15). Enrollment in the original study took place from April 2015 to February 2018 where 600 children aged 6 to 11 months with a length-for-age < −1 Z-score and who planned to reside in the field site at least for the next 12 months were included(Reference Strand, Ulak and Hysing15). Children were excluded in case of acute or chronic illness, severe malnutrition (weight-for-length Z-score <-3), severe anaemia (Hb<7 g/dl), if they were taking multivitamin supplements containing vitamin B12, if they required treatment with vitamin B12 or if parents or caretakers did not consent to participate in the study. Details on study protocol and the random selection procedure for the study have been published elsewhere(Reference Strand, Ulak and Hysing15). The assignment to intervention groups was concealed and blinded to all study personnel and participants. The intervention consisted of daily administration of a paste containing either 2 µg (2–3 recommended daily allowances) of vitamin B12 (cyancobalamin) or a placebo over a period of 12 months. The supplement was delivered in sachets (eeZee20) containing 20 g of a lipid-based, 108 kcal nutrient paste produced by GC Rieber Compact (http://www.gcriebercompact.com). The sachets contained either vitamin B12 or placebo, and in addition an equal quantity of other multivitamins and minerals at approximately 1 recommended daily allowance. The main outcomes of the trial were growth, neurodevelopment and Hb concentration measured after 12 months of supplementation(Reference Strand, Ulak and Hysing15).
Between April 2017 and February 2020, we approached all participating children who had not dropped out of the main study (n 574), 1 and 2 years after the end of supplementation (i.e., children aged 30–35 months and 42–47 months, respectively) for follow-up assessments. Field workers contacted the families either via phone or through a visit to their homes. We also informed families who had moved outside of the area about the extended follow-up and requested them to visit the study clinic according to the scheduled activities. On the days of assessment, the study psychologist obtained written informed consent from the children’s caregiver for the purpose of the present follow-up study.
Data collection
Trained field workers collected information on the family’s demographics and socio-economic situation, as well as the child’s morbidity history at enrollment. They also measured length and weight and collected blood samples. Weight was measured to the nearest 50 g using Seca scales. Length was measured by wooden infantometer (UNICEF), reading to the nearest 0·1 cm. Blood was collected up to 4 ml into a polypropylene tube containing EDTA (Sarstedt, Germany). Immediately after the blood sampling, Hb was measured by Hemocue (Ångelholm, Sweden), and plasma was separated by centrifugation at room temperature (for 10 min at 700 g), transferred into storage vials and stored at < -80°C until analysis. The plasma concentrations of vitamin B12 and the metabolic markers of total homocysteine and methylmalonic acid (MMA) were analysed at Bevital laboratory, Bergen, Norway. We also calculated a combined indicator of vitamin B12 status (3cb12) based on the three biomarkers vitamin B12, total homocysteine and MMA. For the follow-ups, field workers completed a questionnaire concerning morbidity, schooling and growth of the participant children and the study psychologists performed neurodevelopmental assessments.
Neurodevelopmental outcomes
In the first follow-up, when the children were 30–35 months old, they were assessed with the Bayley-III. In the second follow-up at 42–47 months, children were assessed using the WPPSI-IV and the Neuropsychological Assessment, 2nd edition (NEPSY-II). The neurodevelopmental assessments for both follow-ups were conducted at the study clinic in well-lit rooms with minimal distractions. The children were accompanied by their caregivers who had the opportunity to sit in the back of the room during the assessments. Three study psychologists performed all assessments and quality control. Standardisation exercises were performed in twenty children per tester for each test. Double scoring was done throughout the study in 10 % of all assessments by two psychologists. We attained an inter-correlation coefficient >98 % for both standardisation and double scoring indicating excellent inter-rater agreement.
Bayley-III is a standard assessment tool of developmental functioning in infants and toddlers aged 1–42 months. It is one of the most widely used tools worldwide for research purposes in this age group(Reference Bayley19). The Bayley-III consists of a series of developmental tasks and takes between 45 and 60 min to administer. For the current study, we used both the four subscale composite scores (i.e. Cognitive, Language, Motor and Socio-emotional) and the six subtest scaled scores (i.e. Cognitive, Receptive and Expressive language, Fine and Gross motor and Socio-emotional). The raw scores of each subscales were converted into scaled and composite scores based on American norms(Reference Bayley19). The Bayley-III scaled scores have an expected mean (standard deviation (sd)) of 10 (3), and the composite scores have a expected mean (sd) of 100 (15)(Reference Bayley19). The Bayley-III scales was adapted and translated for the Nepalese context, and its feasibility and reliability for use in Nepalese children have been discussed previously(Reference Murray-Kolb, Rasmussen and Scharf20,Reference Ranjitkar, Kvestad and Strand21) . Translation of the test instructions was done according to standard procedures, and small adaptions to the materials (i.e. changing pictures and drawings in the picture and stimulus books) were done to improve the acceptability for the Nepalese setting.
WPPSI-IV is a clinical assessment tool of intellectual ability in children aged 2 years and 6 months through 7 years and 7 months (2:6 to 7:7)(Reference Wechsler22). We used six subtests; Receptive vocabulary, Block Design, Picture Memory, Object Assembly, Zoo Locations and Information to generate standardised composite scores for the FSIQ. In addition, we used three indexes: Verbal Comprehension Index, Visual Spatial Index and Working Memory Index. The raw scores of each subscales were converted into index scores and FSIQ scores with an expected mean (sd) of 100 (15). The calculation of the FSIQ scores was based on American norms, as there were no Nepalese norms(Reference Bayley19). The WPPSI-IV takes 45–60 min to administer(Reference Colom and Spielberger23).
NEPSY-II is a neuropsychological cognitive assessment tool that can be tailored for children aged 3–16 years(Reference Korkman, Kirk and Kemp24). The NEPSY-II consists of thirty-two subtests in six functional domains. The following three age appropriate subtests were administered in this study: Affect Recognition, Statue and Geometric Puzzles from the Social Perception, Executive Functioning and Visual-Spatial Processing domains, respectively. The subscale raw scores were converted into scaled scores for the subtest by age group based on American norms. For the geometric puzzles, there were no norms for this age range, so we used raw scores(Reference Brooks, Sherman and Strauss25). The NEPSY-II scaled scores have a mean (sd) of 10 (3). After careful consideration of the test material, no changes were made to the WPPSI and NEPSY test for the current study.
Sample size
Of the original sample of 600, we were able to measure neurodevelopment in at least 533 children for both of the time points during the extended follow-up. This follow-up rate ensured a statistical power of 82 and 93 % to detect minimal meaningful differences of 0·25 and 0·3 standardised effect sizes, respectively. These calculations assumed equal variances between the groups and an α of 0·05. For details regarding the sample size estimations, please see the protocol paper(Reference Strand, Ulak and Chandyo26).
Ethics
The study obtained approval from the Nepal Health Research Council (Reg.no.73/2017) in Nepal and from REC Norway (2014/1528). The clinical trials register number is NCT02272842.
Statistical analysis
All forms were manually checked for inconsistencies by field supervisors, and data were entered in computerised databases; discrepancies and completeness were checked by the data entry supervisor. Analyses were performed using Stata® version 16 (Stata Corp.)(27). Proportions and mean (sd) for all baseline categorical and continuous variables were calculated for each intervention group. Children’s weight-for-age, weight-for-length/height and length/height-for-age Z-score were calculated based on the WHO growth standards(Reference Mercedes28). Vitamin B12 status at baseline and at the end of the study was described in the manuscript reporting the main outcomes(Reference Strand, Ulak and Hysing15,Reference Chandyo, Ulak and Kvestad29) . Means (sd) and mean differences (95 % CI) for the cognitive outcomes between intervention groups were calculated. In linear regression analyses, we examined the effect of the intervention on the primary outcome (WPPSI-IV FSIQ) in pre-defined subgroups, which were the same as reported in the main paper(Reference Strand, Ulak and Chandyo26). These subgroups were defined according to baseline vitamin B12 status (i.e., plasma concentration of vitamin B12, total homocysteine, MMA and 3cb12), as well as according to baseline anaemia status, birth weight, stunting, wasting and underweight. The subgroup effects are presented in forest plots (metan function in Stata® version 16).
Results
Among 600 participant infants, we were unable to follow-up on twenty-six participants from the original study mainly due to migration or refusal to participate. Among the 574 children who completed the intervention, we were able to re-consent 555 (96·7 %) at the first scheduled time point and 533 (92·8 %) at the second scheduled time point. The main reason for not being re-enrolled in the follow-up rounds was migration (Fig. 1).
Fig. 1. Trial profile of a randomised control trial on vitamin B12 in Nepalese infants and a follow-up study 1 and 2 years after end of the intervention. *Number of lost to follow-up of the 1st and 2nd /follow-ups were from the total number of 574 participants who completed the one year supplementation.
Demographic characteristics among the intervention and placebo group at enrollment were similar (Table 1). The mean age of the children at enrollment was 8 months (sd 1·8). Almost one-half of the participants lived in joint families and half of the participants in rented houses. One in every five children was born with low birth weight and one-third were stunted at enrollment (LAZ < -2 Z score).
Table 1. Baseline information of the 6–11-month-old Nepalese infants in the original randomised-controlled trail (Numbers and percentages; mean values and standard deviations, n 600)
n, number.
* Among 579 infants from whom birth weight was recorded.
† Among 487 fathers who were available.
Means (sd) and the mean differences of the cognitive outcomes by intervention group are shown in Table 2. For the primary outcome (i.e., WPPSI-IV FSIQ), the mean (sd) score for the vitamin B12 and placebo group were 84·4 (8·4) and 85·0 (8·6), respectively, with a mean difference of −0·5 (95 % CI -1·97, 0·94). Likewise, there were no significant differences between the intervention groups in any of the other study outcomes.
Table 2. Mean (sd) neurodevelopmental scores by study group at the follow-ups in Nepalese children aged 30 to 47 months old in Bhaktapur (Numbers and percentages; mean values and standard deviations)
WPPSI-IV, Wechsler Preschool and Primary Scale of Intelligence fourth edition; NEPSY-II, Neuropsychological Assessment, 2nd edition; Bayley-III, Bayley Scales of Infant and Toddler Development, 3rd edition.
* Mean (sd) of FSIQ and index scores 100 (15).
† Mean (sd) of scaled score 10 (3).
‡ Mean (sd) of composite and Index score 100 (15), the scaled scores and composite scores were calculated based on American norms.
The effects in the different subgroups are shown in Fig. 2. There were no significant differences between the intervention groups in any of the subgroups on the WPPSI-IV FSIQ, except for those born at low birth weight. In this subgroup, vitamin B12 supplementation leads to 4·54 (95 % CI 1·20, 7·88) lower FSIQ score. The interaction between intervention group and birth weight was statistically significant (interaction term regression coefficient: 4·02 (95 % CI 1·52, 6·51), P = 0·002).
Fig. 2. The effect of vitamin B12 supplementation in infancy in pre-defined subgroups on the Wechsler Preschool and Primary Scale of Intelligence (WPPSI-IV), Full Scale Intelligence Quotient. MMA, methylmalonic acid; tHcy, total homocysteine; 3cb12, combined indicator of vitamin B12 status, calculated based on vitamin B12, MMA and tHcy; LAZ, length for age, WAZ, weight for age; P-value for interaction between birthweight and intervention group: 0·002; all other P-values for interaction are not significant (>0·05).
Discussion
In this follow-up study, our objective was to examine potential changes to the developing brain from 1 year vitamin B12 supplementation during infancy, on neurodevelopment measured beyond 30 months of child age when these measures are considered more reliable. We were not able to demonstrate any effect from the 1 year vitamin B12 supplementation on neurodevelopment at this age overall or in pre-defined sub-groups.
To our knowledge, this is one of the first studies that have examined the long-term relationship between vitamin B12 supplementation in infancy and neurodevelopmental outcomes after 3 years of age. Our findings are similar to the results from a long-term follow-up of a RCT in India, where 6 months of vitamin B12 and/or folic acid supplementation in early childhood was not associated with cognitive outcomes when the children reached school age(Reference Kvestad, Taneja and Upadhyay30). Notably, in this RCT, the dose of vitamin B12 was 2 to 3 recommended daily allowances, similar to the current trial. Our findings in this follow-up are also in accordance with the results from the original trial where there was no effect of vitamin B12 supplementation on neurodevelopment measured with Bayley-III immediately following the end of supplementation when the children were 18–23 months old(Reference Strand, Ulak and Hysing15). The lack of effect was in spite of an excellent compliance to the supplements, and a clear metabolic response reflecting improved vitamin B12 status. The aim of the current study was to examine potential long-term effects of the vitamin B12 intervention at ages when neurodevelopmental measures are considered to be more reliable. Given the null findings of the current follow-up, the results substantiate the fact that providing 2 µg vitamin B12 daily to children during infancy does not benefit neurodevelopment in children.
Our finding contradicts two Norwegian RCT where improved motor development was seen among mild vitamin B12-deficient infants following a high-dose vitamin B12 provided intramuscularly(Reference Torsvik, Ueland and Markestad12,Reference Torsvik, Ueland and Markestad13) . The contrasting findings could be due to poor absorption and/or the relatively low dose in the current study, and a higher dose of vitamin B12 provided intramuscularly could provide a more favourable impact on the neurodevelopmental outcomes. Moreover, although we targeted marginally stunted infants at enrollment with the presumption of prevalent vitamin B12 deficiency among these children(Reference Ulak, Chandyo and Thorne-Lyman5), only 11 % of the infants were vitamin B12 deficient (<148 pmol/l) and <2 % of the families were practicing a vegetarian diet. The low occurrence of vitamin B12 deficiency could be another explanation for the lack of an effect of the supplementation.
In the subgroup analyses, a surprising finding was a negative effect of vitamin B12 on WPPSI-IV FSIQ seen in low birth weight children. To the best of our knowledge, there are no previous reports of any adverse effect of vitamin B12 in low birth weight children. This is one out of many subgroup analyses and may be a chance finding. However, this apparent negative effect of vitamin B12 in low birth weight babies, needs to be explored in further studies.
Strengths of the study include the fact that we were able to re-enroll >93 % of the original study children after 2 years of the end of supplementation and the use of high quality, comprehensive and validated tools such as the Bayley-III, WIPPSI-IV and NEPSY-II when children were 3 years and above. There are also a few limitations of the study. We gave a peanut-based paste of 20 mg containing either 2 µg of vitamin B12 or placebo. Field workers did weekly visits, and we observed excellent compliance to the supplementation. However, in case of difficulties of feeding the peanut-based paste, social desirability might have led to over-reporting by the mothers. The neurodevelopmental tools were not validated for a Nepali context and as no local norms existed, we have used American norms when converting from raw scores to subscale and composite scores.
Conclusion
We found no long-term effect of vitamin B12 supplementation during early childhood on neurodevelopment in children at age 30–47 months. Our findings do not support supplementation of vitamin B12 around the recommended daily intake in infants from low- and middle-income countries to improve brain development. Further studies are needed to investigate the importance of vitamin B12 for childhood neurodevelopment.
Acknowledgements
We thank the psychologists Jaya Silpakar and Roshan Sintakala who performed the cognitive tests, supervisors, data management team and all field workers. We are grateful to all the participant children and their families for their valuable time to participate in the study. We are also grateful to founder Shyam Sunder Dhaubhadel and all staffs at Siddhi Memorial Hospital.
This work was supported by the Thrasher Research Fund (award # 11512), the GC Rieber foundation, the University of Bergen (UiB) and the Research Council of Norway through a grant to Centre for Intervention Science in Maternal and Child Health (CISMAC).
Conceptualization: T. A. S., I. K., M. H., R. K. C., M. U., M. S. and L. P. S.; Investigation: M. U., R. K. C., S. R., M. S. and L. P. S.; Formal analysis: M. U., I. K., T. A. S. and C. S.; Writing original draft: M. U., I. K., T. A. S. and C. S.; Writing – review & editing: S. B., M. H., R. K. C., M. S., S. R. and L. P. S.
There are no conflicts of interest.
Supplementary material
For supplementary material/s referred to in this article, please visit https://doi.org/10.1017/S0007114522000071
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