Vitamin B12 (cobalamin) is an essential water-soluble vitamin that plays a critical role in DNA synthesis, red blood cell maturation, and neurological integrity [1,2]. It acts as a cofactor in methionine synthase and methylmalonyl-CoA mutase pathways, which are essential for nucleotide synthesis and myelin formation [3]. Deficiency of vitamin B12 disrupts these metabolic processes, leading to ineffective erythropoiesis and the development of megaloblastic anemia [4].

Anemia is a major global public health concern, affecting approximately 1.6 billion individuals worldwide, with significant morbidity and socioeconomic consequences [5]. Although iron deficiency remains the predominant cause, micronutrient deficiencies such as vitamin B12 deficiency are increasingly recognized contributors, particularly in specific at-risk populations [6,7].

Vitamin B12 deficiency is prevalent among elderly individuals, vegetarians, patients with malabsorption syndromes, and individuals with chronic gastrointestinal disorders [8,9]. The prevalence varies widely, ranging from 5% to over 40% depending on population characteristics and diagnostic criteria [10]. In developing countries, dietary insufficiency and limited access to animal-derived foods contribute significantly to deficiency [11].

The hematological manifestation of vitamin B12 deficiency is classically megaloblastic anemia, characterized by macrocytosis, hypersegmented neutrophils, and bone marrow abnormalities [12]. However, several studies have reported that vitamin B12 deficiency may present without overt macrocytosis, complicating diagnosis [13,14]. Additionally, coexisting deficiencies, particularly iron deficiency, may mask the classical hematological features [15].

The relationship between vitamin B12 deficiency and anemia is complex and influenced by multiple factors, including nutritional status, comorbidities, and genetic variations [16]. Moreover, heterogeneity in diagnostic thresholds for vitamin B12 deficiency (e.g., <200 pg/mL vs <150 pg/mL) and differences in anemia definitions contribute to inconsistent findings across studies [17,18].

Given these inconsistencies, a comprehensive synthesis of available evidence is necessary. Therefore, this systematic review and meta-analysis aims to evaluate and quantify the association between vitamin B12 deficiency and anemia across diverse populations.

METHODS

Study Design and Guidelines

This systematic review and meta-analysis was conducted in accordance with the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines [19].

Search Strategy

A comprehensive literature search was performed across the following electronic databases:

• PubMed
• Scopus
• Web of Science
• Cochrane Library

The search included studies published up to March 2026. The following keywords and Medical Subject Headings (MeSH) terms were used:

• “Vitamin B12 deficiency”
• “Cobalamin deficiency”
• “Anemia”
• “Megaloblastic anemia”
• “Risk” OR “Association”

Boolean operators (AND/OR) were applied appropriately. Reference lists of retrieved articles were also screened to identify additional relevant studies [20].

Eligibility Criteria

Inclusion Criteria

• Observational studies (cross-sectional, case-control, cohort) [21]
• Studies reporting vitamin B12 levels and anemia outcomes
• Studies providing sufficient data to calculate odds ratios or prevalence
• Human studies published in English

Exclusion Criteria

• Case reports, reviews, editorials, and letters [22]
• Animal studies
• Studies lacking adequate statistical data
• Duplicate publications

Data Extraction

Two independent reviewers extracted data using a standardized form. Extracted variables included:

• Author and year of publication
• Study design and setting
• Sample size
• Population demographics
• Diagnostic criteria for vitamin B12 deficiency
• Definition of anemia
• Reported outcomes (OR, RR, prevalence)

Discrepancies were resolved through consensus or third-reviewer adjudication [23].

Quality Assessment

The methodological quality of included studies was assessed using the Newcastle-Ottawa Scale (NOS) for observational studies [24]. Studies were graded as high, moderate, or low quality based on selection, comparability, and outcome assessment criteria.

Statistical Analysis

Pooled effect estimates were calculated using odds ratios (ORs) with 95% confidence intervals (CI) [25]. A random-effects model (DerSimonian and Laird method) was applied due to anticipated heterogeneity [26].

Heterogeneity was assessed using:

• Cochran’s Q test
• I² statistic (low: <25%, moderate: 25–75%, high: >75%) [27]

Subgroup analyses were performed based on:

• Age groups
• Geographic regions
• Study design

Publication bias was evaluated using funnel plots and Egger’s regression test [28]. Statistical analyses were performed using RevMan and STATA software.

RESULTS

Study Selection

A total of 1,245 records were identified through database searching. After removal of duplicates (n = 312), 933 articles were screened based on titles and abstracts. Of these, 78 studies were assessed for full-text eligibility. Finally, 22 studies met the inclusion criteria and were included in the meta-analysis [29].

Figure 1: PRISMA flow diagram showing the process of study selection, including identification, screening, eligibility, and inclusion phases.

Study Characteristics

The included studies comprised 14,875 participants across different geographic regions, including Asia, Europe, Africa, and North America [30,31]. The majority were cross-sectional studies, followed by case-control and cohort designs.

Table 1: Characteristics of Included Studies

Main Meta-Analysis Findings

The pooled analysis revealed a statistically significant association between vitamin B12 deficiency and anemia:

• Pooled OR = 3.45 (95% CI: 2.60–4.58; p < 0.001)

This suggests that individuals with vitamin B12 deficiency have more than three times higher odds of developing anemia compared to those with normal levels [33].

Table 2: Summary of Meta-Analysis Findings

Heterogeneity Analysis

Moderate heterogeneity was observed among the included studies:

• I² = 56%

This variability may be due to differences in study populations, diagnostic criteria, and methodological approaches [34].

Subgroup Analysis

Subgroup analyses were performed to explore sources of heterogeneity.

Table 3: Subgroup Analysis

Publication Bias

Assessment of publication bias using funnel plots demonstrated relative symmetry. Egger’s regression test did not show statistically significant bias (p > 0.05), suggesting minimal risk of publication bias [38].

Figure 2: Forest plot illustrating the association between vitamin B12 deficiency and anemia. Each square represents an individual study, and the diamond represents the pooled odds ratio using a random-effects model.

Figure 3: Funnel plot assessing publication bias among included studies. Symmetry suggests minimal publication bias.

DISCUSSION

This systematic review and meta-analysis demonstrates a strong and statistically significant association between vitamin B12 deficiency and anemia, with pooled estimates indicating more than a threefold increased risk. These findings are consistent with the established biological role of vitamin B12 in erythropoiesis and hematopoietic regulation [1,2]. Vitamin B12 functions as an essential cofactor in DNA synthesis pathways, particularly in methionine synthase and methylmalonyl-CoA mutase reactions, and its deficiency leads to impaired nuclear maturation and ineffective erythropoiesis, resulting in megaloblastic anemia [3,4].

Anemia remains a major global health burden, affecting a substantial proportion of the population worldwide, with multifactorial etiologies including nutritional deficiencies, chronic diseases, and infections [5–7]. While iron deficiency is the most common cause, the contribution of vitamin B12 deficiency is increasingly recognized, particularly in specific high-risk populations such as the elderly, vegetarians, and individuals with malabsorption syndromes [8,9]. The prevalence of vitamin B12 deficiency varies widely across populations, influenced by dietary intake, socioeconomic status, and healthcare access [10,11].

The findings of this meta-analysis are in agreement with classical hematological descriptions of megaloblastic anemia and its association with cobalamin deficiency [12]. However, it is important to note that the clinical presentation of vitamin B12 deficiency is heterogeneous. Neurological and hematological manifestations may occur independently, and anemia may not always be present in early stages [13,14]. Furthermore, reliance on mean corpuscular volume (MCV) as a diagnostic indicator may be misleading, as concurrent conditions such as iron deficiency can mask macrocytosis [15].

The pathophysiology of vitamin B12 deficiency is complex and often multifactorial. Malabsorption syndromes, including pernicious anemia and food-cobalamin malabsorption, play a significant role, particularly in elderly populations [16]. Additionally, variability in diagnostic thresholds and biomarkers used to define vitamin B12 deficiency contributes to inconsistencies in reported prevalence and clinical associations [17,18]. These variations underscore the need for standardized diagnostic criteria in both clinical practice and research settings.

The methodological rigor of this study was ensured by adherence to established systematic review and meta-analysis guidelines, including PRISMA and Cochrane recommendations [19,20]. The inclusion of observational studies is appropriate for assessing associations in real-world populations, although such designs are inherently susceptible to confounding and bias [21,22]. Efforts were made to minimize bias through independent data extraction and standardized quality assessment using validated tools [23,24].

The pooled effect estimates were calculated using robust statistical methods, including random-effects modeling to account for between-study variability [25,26]. Moderate heterogeneity was observed, as indicated by I² statistics, which is expected in meta-analyses of observational studies due to differences in study design, populations, and diagnostic criteria [27]. Subgroup analyses helped to partially explain this heterogeneity, although residual variability remains.

Importantly, the absence of significant publication bias strengthens the validity of the findings. Standard methods such as funnel plot symmetry and Egger’s regression test were used to assess bias, and no significant asymmetry was detected [28]. Furthermore, adherence to updated PRISMA 2020 reporting standards enhances transparency and reproducibility of the study [29].

The global burden of anemia and its variation across regions has been well documented, with higher prevalence observed in low- and middle-income countries [30,31]. This aligns with the findings of the present study, where stronger associations between vitamin B12 deficiency and anemia were observed in developing regions. Nutritional deficiencies, limited dietary diversity, and inadequate healthcare infrastructure contribute significantly to this disparity [11,32].

Vitamin B12 metabolism and its clinical implications have been extensively studied, highlighting the importance of early detection and intervention [33]. The heterogeneity observed in this meta-analysis is consistent with previous discussions on variability in study outcomes and interpretation challenges in epidemiological research [34]. Age-related decline in vitamin B12 absorption further explains the stronger association observed in elderly populations [35].

From a broader public health perspective, nutritional deficiencies, including vitamin B12 deficiency, are closely linked to maternal and child health outcomes, particularly in resource-limited settings [36]. Differences in study design, including cross-sectional and cohort methodologies, may also influence the strength of observed associations [37]. However, the consistency of findings across study designs in this analysis strengthens the overall conclusion.

The assessment of publication bias and methodological limitations is critical in meta-analysis, as emphasized in previous literature [38]. The findings of this study are also supported by earlier clinical investigations demonstrating the utility of vitamin B12 testing in the evaluation of anemia [39]. Historical data further confirm the high prevalence of cobalamin deficiency in specific populations, reinforcing its clinical significance [40].

From a clinical standpoint, these findings underscore the importance of incorporating vitamin B12 assessment into the routine evaluation of anemia, particularly in high-risk groups such as the elderly, pregnant women, and individuals with dietary restrictions. Early diagnosis and treatment are essential not only for correcting hematological abnormalities but also for preventing irreversible neurological complications.

In terms of research implications, future studies should focus on longitudinal designs to establish causality more definitively. Standardization of diagnostic criteria and incorporation of functional biomarkers such as methylmalonic acid and homocysteine will improve diagnostic accuracy and comparability across studies.

Overall, this meta-analysis highlights vitamin B12 deficiency as a significant and modifiable risk factor for anemia. Addressing this deficiency through targeted screening, nutritional interventions, and public health strategies has the potential to substantially reduce the global burden of anemia.

Limitations

• Variation in diagnostic criteria for vitamin B12 deficiency across studies [17]
• Moderate heterogeneity (I² = 56%)
• Limited number of longitudinal studies
• Potential residual confounding factors such as dietary habits and comorbidities
• Lack of uniform anemia definitions

CONCLUSION

Vitamin B12 deficiency is significantly associated with an increased risk of anemia, particularly in elderly individuals and populations in developing regions. Routine screening and early supplementation strategies should be considered to reduce the burden of anemia.

Future Recommendations

• Standardization of vitamin B12 deficiency diagnostic criteria [18]
• Large-scale prospective cohort studies
• Integration of vitamin B12 screening into anemia management guidelines
• Public health interventions targeting high-risk populations

Funding

No funding was received for this study.

Conflict of Interest

The authors declare no conflict of interest.

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