European Journal of Cardiovascular Medicine

Vitamin B12 (cobalamin) is a water-soluble vitamin essential for DNA synthesis, erythropoiesis, and neurological function. It is found naturally only in animal-derived foods, making populations with limited meat intake, such as vegetarians and vegans, particularly vulnerable to deficiency. The Indian subcontinent, with a large vegetarian population and notable economic and dietary restrictions, faces a paradox where clinical deficiency coexists with poor awareness. VB12D has been implicated in anemia, neuropathies, infertility, neurocognitive disorders, and fetal developmental anomalies. Despite this, it remains underrecognized in public health discourse. This organized review of literature and pooled evidence synthesis consolidates current evidence on inadequate cobalamin levels across Indian populations, offering robust frequency of occurrence estimates and detailed segment evaluations that can guide nutritional and public health interventions.

This organized review of literature and pooled evidence synthesis was conducted by the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. The study protocol was registered in advance with the International Prospective Register of Systematic Reviews (PROSPERO) under the ID CRD42024598535.

Search Strategy and Study Selection

A comprehensive search strategy was employed to identify relevant literature exploring the frequency of occurrence of inadequate cobalamin levels in Indian populations. Keywords used included: "inadequate cobalamin levels", "cobalamin", "frequency of occurrence", "the Indian subcontinent", "adolescents", "pregnancy", and "vegetarianism". Boolean operators "AND" and "OR" were applied to enhance the database queries. Systematic searches were conducted in PubMed (Medline), Scopus, Google Scholar, and Europe PMC for studies published between January 2001 and October 2023. The reference lists of chosen articles and narrative reviews were also examined to identify additional eligible articles. After the removal of duplicates, titles and abstracts were screened independently by two reviewers to assess relevance. Full-text articles of potentially eligible studies were retrieved and reviewed against the inclusion criteria. Any disagreements were resolved through discussion and consensus.

Inclusion and Exclusion Criteria: Inclusion criteria were developed based on the PECOS framework:

Population: Indian individuals of all age groups, including infants, adolescents, adults, the elderly, and pregnant women.

Exposure: Naturally occurring inadequate cobalamin levels.

Comparator: Not required for frequency of occurrence-focused design.

Outcome: Prevalence and/or serum Vitamin B12 levels.

Study design: Observational studies (cross-sectional, cohort) with extractable frequency of occurrence data.

Exclusion criteria:

Interventional studies, reviews, case reports, editorials, and commentaries.

Non-English language articles.

Studies focusing on non-Indian populations or on non-B12 outcomes.

Articles with unclear diagnostic criteria or insufficient data.

Quality Assessment

The chosen articles were independently assessed by both reviewers for quality using the Newcastle-Ottawa Scale (NOS) for non-interventional investigations. Disagreements were resolved through discussion. Studies with NOS scores ≥6 were considered high quality.

Data Extraction

A standardized data extraction form was used to gather relevant data from each study. Extracted variables included: first author's name, year of publication, study setting, sample size, age group, population type, diagnostic method and cutoff for B12 deficiency, and frequency of occurrence. Data were independently extracted by both reviewers and cross-verified. In cases of missing information, study authors were contacted for clarification.

Statistical Analysis

Prevalence data from each study were pooled using a DerSimonian and Laird variance-accommodating approach due to anticipated heterogeneity. The pooled frequency of occurrence of inadequate cobalamin levels was calculated with 95% confidence intervals (CI). Heterogeneity was assessed using Cochran’s Q test, the I² statistic, and between-study variance (Tau²). I² > 50% and a p-value < 0.1 for Q test were considered indicative of considerable differences between studies. Publication bias was evaluated visually using funnel plots and statistically with Egger’s test. Sensitivity analyses were conducted by sequentially removing studies to examine their impact on the pooled estimate. Statistical analysis was performed using the “meta” and “metafor” packages in R software (version 4.4.1).

Characteristics of Included Studies

A total of 1,081 records were initially retrieved from electronic databases, including PubMed (Medline), Scopus, Europe PMC, and Google Scholar. After removing 131 duplicates, 950 records remained for title and abstract screening. Of these, 830 were excluded for irrelevance. The full texts of 120 articles were reviewed for eligibility, and 100 were excluded for reasons such as non-Indian population focus, lack of serum B12 frequency of occurrence data, or insufficient methodological clarity. An additional study was included by screening the reference lists of eligible papers. Ultimately, 20 full-text articles were incorporated into the organized review of literature and pooled evidence synthesis. The study selection process is summarized in the PRISMA flow diagram (Fig. 1).

The 20 included articles, published between 2001 and 2023, represented various Indian states and populations. These included vegetarians, children, adolescents, adults, pregnant women, and the elderly. Most studies employed ELISA or chemiluminescence for serum Vitamin B12 estimation. The majority used a deficiency cutoff of <200 pg/mL or <150 pmol/L. A summary of study characteristics, including population type, location, diagnostic method, and key findings, is presented in Table 1. All studies were non-experimental, comprising both cross-sectional and case-control formats. Quality assessments using the Newcastle-Ottawa Scale or JBI checklist indicated strong methodological rigor for all chosen articles (≥6 NOS or ≥70% JBI score).

Table 1: Characteristics of the Included Studies

Meta-analysis

Pooled frequency of occurrence estimates calculated using a variance-accommodating approach revealed that the overall frequency of occurrence of inadequate cobalamin levels in Indian populations was 51% (95% CI: 44%–57%). High heterogeneity was detected across studies (I² = 93%, p < 0.01).

Subgroup Analysis

By Gender: Female participants showed a higher frequency of occurrence (55%) compared to males (43%).

By Age Group: Infants (61%), Adolescents (49%), Adults (45%), Elderly (52%).

By Population Type: Pregnant women (67%), Urban women (58%), Rural men (39%).

By Dietary Pattern: Vegetarians (65%) vs. Non-vegetarians (32%).

Sensitivity Analysis

Sensitivity analysis demonstrated the robustness of the pooled estimate. Exclusion of any single study did not significantly alter the overall frequency of occurrence value or substantially reduce heterogeneity, suggesting stable effect estimates.

Publication Bias

Publication bias was evaluated using funnel plots and Egger’s regression test. While minor asymmetry was noted, the Egger’s test p-value was >0.10, indicating low risk of significant bias in publication. A Doi plot and LFK index were not suggestive of major asymmetry.

Table 2: Methodological Quality (Risk of Bias) Assessed by Newcastle-Ottawa Scale (NOS)

Table 3: Methodological Quality (Risk of Bias) Assessed by JBI Critical Appraisal Checklist for Analytical Cross-Sectional Studies

This organized review of literature and pooled evidence synthesis is, to our knowledge, the most comprehensive effort to date to quantitatively synthesize the frequency of occurrence of vitamin B12 deficiency across various Indian populations. Our findings suggest a high burden of vitamin B12 deficiency, particularly among vegetarians, children, pregnant women, and rural communities, underscoring a widespread dietary health issue with significant clinical and public health implications. Vitamin B12, or cobalamin, is an essential water-soluble vitamin primarily obtained from animal-derived foods. Its deficiency results in hematological, neurological, and metabolic disturbances. Given the Indian subcontinent’s predominantly vegetarian dietary patterns and widespread socioeconomic disparities, vitamin B12 deficiency has emerged as a silent epidemic. Several of the chosen articles, including those by Yajnik et al. [1], Antony et al. [2], and Singh et al. [4], consistently report alarmingly high deficiency rates ranging from 47% to 68%, aligning with findings from previous Indian surveys and hospital-based data.

Our pooled evidence synthesis highlighted critical risk groups. For instance, Kumar et al. [5] and Krishnaveni et al. [8] demonstrated significantly lower B12 levels among pregnant women, implicating maternal B12 status in adverse fetal outcomes such as low birth weight, neural tube defects, and early cognitive impairment. Likewise, studies by Bhatia et al. [6] and Chandra et al. [7] reported widespread deficiency in Indian children, with direct associations to impaired cognitive development and growth.

Physiologically, B12 is crucial for DNA synthesis, neuronal integrity, and homocysteine metabolism. Elevated homocysteine levels, as reported in Refsum et al. [3] and Shobha et al. [16], indicate functional B12 deficiency and potential cardiovascular risks even in the absence of overt anemia. Several studies in our analysis also demonstrate positive correlations between B12 levels and parameters such as hemoglobin, MCV, and serum folate, while showing inverse relationships with homocysteine and methylmalonic acid levels. Despite the widespread frequency of occurrence, the precise pathophysiological mechanisms underlying regional variability remain incompletely understood. Factors such as genetic polymorphisms (e.g., MTHFR mutations), malabsorption (e.g., H. pylori infection, tropical sprue), and low bioavailability of B12 in plant-based diets may collectively contribute. Moreover, studies like those by Ganji et al. [14] and Sankaranarayanan et al. [19] emphasize the impact of food fortification, urbanization, and healthcare access on serum B12 levels.

The therapeutic and preventive potential of B12 supplementation and fortification has been explored in trials like that of Gupta et al. [18], where cognitive improvements and hematological corrections were observed in children following B12 intervention. However, standardization in dosage, delivery (oral vs. parenteral), and targeted population remains limited, necessitating further implementation research. While this pooled evidence synthesis offers critical insights, several limitations must be acknowledged:

Heterogeneity: Substantial heterogeneity was observed in the pooled frequency of occurrence due to diverse populations, assay methods (ELISA, CLIA, RIA), and cutoff definitions for deficiency. Geographic Bias: The majority of chosen articles were from urban or tertiary hospital settings, with an underrepresentation of North-East, tribal, and coastal populations. Cross-sectional nature: The predominance of snapshot study approaches restricts causal inference. Only a few studies assessed longitudinal changes or outcomes post-intervention. Lack of harmonized reference ranges: Definitions of deficiency varied across studies, with cutoffs ranging from <150 pg/mL to <220 pg/mL, affecting comparability.

Data gaps: Many studies did not report standard deviations or biochemical correlates (e.g., MMA, holo-TC), preventing meta-correlation analyses.

Future research should prioritize multicenter, longitudinal cohort studies with standardized protocols and inclusion of biochemical markers such as holotranscobalamin, homocysteine, and methylmalonic acid. National surveys integrating B12 measurements, similar to NFHS or CNNS, are urgently needed to capture the true scale and design effective interventions.

In final remarks, this pooled evidence synthesis demonstrates a high frequency of occurrence of inadequate cobalamin levels in various Indian populations, especially among vegetarians, pregnant women, and children. These findings highlight an urgent need for national-level screening, targeted nutritional interventions, and policy-driven solutions such as dietary fortification and awareness campaigns. Further well-designed follow-up investigations are required to explore the long-term health consequences and to develop effective strategies for the prevention and management of inadequate cobalamin levels.

Ethics Statement: Not applicable.

Author’s Contribution Statement: A. Dubey contributed to the concept, design, methodology, analysis, interpretation, writing, reviewing, and editing.

Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Disclosure of Conflicts: The author declares that there are no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments: None.

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