Diabetes mellitus (DM) is a chronic, multifactorial metabolic disorder characterized by persistent hyperglycemia resulting from defects in insulin secretion, insulin action, or both. It represents a major global public health challenge due to its rising prevalence, long-term complications, and substantial socioeconomic burden. According to the International Diabetes Federation (IDF), approximately 537 million adults worldwide were living with diabetes in 2021, with projections estimating a rise to 783 million by 2045, largely driven by urbanization, sedentary lifestyles, dietary transitions, and population aging [1]. Type 2 diabetes mellitus (T2DM) accounts for nearly 90–95% of all diabetes cases and is particularly prevalent in low- and middle-income countries, including India, where early onset and rapid disease progression are increasingly observed [2].

Metformin, a biguanide antihyperglycemic agent, is widely recommended as the first-line pharmacological therapy for T2DM due to its proven efficacy, safety profile, affordability, and cardiovascular benefits. It primarily reduces hepatic gluconeogenesis and improves peripheral insulin sensitivity through activation of adenosine monophosphate–activated protein kinase (AMPK), thereby lowering fasting and postprandial glucose levels without causing hypoglycemia or weight gain [3,4]. Owing to these advantages, metformin remains the cornerstone of T2DM management worldwide and is often prescribed for long-term use.

Despite its favorable therapeutic profile, prolonged metformin therapy has been increasingly associated with vitamin B₁₂ deficiency, an adverse effect that has gained growing clinical attention. Vitamin B₁₂ (cobalamin) is an essential water-soluble vitamin required for DNA synthesis, erythropoiesis, and maintenance of neurological function. Deficiency may lead to megaloblastic anemia, peripheral neuropathy, cognitive impairment, and neuropsychiatric manifestations, many of which may overlap with complications of diabetes itself, resulting in underdiagnosis or delayed treatment [5].

Several observational studies, systematic reviews, and meta-analyses have demonstrated a significant association between metformin use and reduced serum vitamin B₁₂ levels, with reported prevalence of deficiency ranging from 15% to over 50% among long-term users [6–8]. The risk appears to be dose- and duration-dependent, with higher daily doses and prolonged therapy conferring greater susceptibility [9]. The proposed mechanisms include impaired calcium-dependent absorption of the vitamin B₁₂–intrinsic factor complex in the terminal ileum, altered intestinal motility, and modulation of gut microbiota leading to reduced bioavailability of cobalamin [10,11].

The clinical relevance of metformin-induced vitamin B₁₂ deficiency is particularly significant in the Indian population, where vegetarian dietary practices are common and baseline cobalamin intake may already be marginal. Furthermore, early-onset T2DM in Indian adults results in prolonged exposure to metformin, increasing the cumulative risk of deficiency and its neurological and hematological consequences. Despite this, routine screening for vitamin B₁₂ deficiency is not consistently implemented in clinical practice, and region-specific longitudinal data remain limited [12].

Therefore, the present study was undertaken to evaluate the effect of metformin therapy on serum vitamin B₁₂ levels in patients with Type 2 diabetes mellitus and to determine the prevalence of vitamin B₁₂ deficiency during the course of treatment.

MATERIAL AND METHODS

Study Design and Setting

This study was a cross-sectional observational study conducted in the Department of Pharmacology in collaboration with the Department of General Medicine at the Career Institute of Medical Sciences & Hospital, Lucknow, Uttar Pradesh, India.

Study Population

Patients diagnosed with Type 2 diabetes mellitus (T2DM) and receiving metformin monotherapy were recruited from the outpatient department (OPD) after obtaining written informed consent. Eligible participants included individuals aged 35–45 years with a confirmed diagnosis of T2DM who had been receiving metformin monotherapy for a minimum duration of six months and were willing to participate in the study. Patients were excluded if they did not provide informed consent, were using medications known to affect serum vitamin B₁₂ levels (such as corticosteroids, phenytoin, proton pump inhibitors, colchicine, or neomycin), had a history of malabsorption disorders (including celiac disease, chronic pancreatitis, or lactose intolerance), had known hematological disorders such as pernicious anemia or pre-existing vitamin B₁₂ deficiency, were pregnant or lactating, had major chronic illnesses other than T2DM (including congestive heart failure or severe pulmonary disease), or had experienced recent trauma or undergone surgical intervention.

Sample Size Calculation

The sample size was calculated using the prevalence-based formula for cross-sectional studies, based on a previously published study reporting a vitamin B₁₂ deficiency prevalence of 17.5% among metformin-treated diabetic patients. With a 95% confidence level and an absolute precision of 5%, the minimum required sample size was calculated to be 221 participants. Accordingly, 221 eligible patients were enrolled in the study.

Data Collection and Clinical Assessment

A detailed medical history, including duration of diabetes, metformin dosage, dietary pattern, and lifestyle habits (alcohol consumption, smoking, tobacco use), was recorded for each participant. A general physical and systemic examination was conducted at baseline.

Laboratory Investigations

Baseline laboratory investigations included estimation of serum vitamin B₁₂ levels, complete blood count (CBC) comprising hemoglobin concentration, red blood cell count, white blood cell count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC), along with fasting blood glucose (FBG), postprandial blood glucose (PPBG), and glycated hemoglobin (HbA₁c). Serum vitamin B₁₂ levels were reassessed at 6 months and 12 months using the same standardized biochemical methods to ensure analytical consistency. Vitamin B₁₂ status was subsequently categorized as normal, borderline, or deficient in accordance with established laboratory reference ranges

Statistical Analysis

All collected data were entered into Microsoft Excel and analyzed using SPSS version 25. Continuous variables were expressed as mean ± standard deviation, while categorical variables were presented as frequencies and percentages. Changes in serum vitamin B₁₂ levels over time were analyzed using paired t-tests and chi-square tests, as appropriate. Pearson correlation analysis was performed to assess associations between vitamin B₁₂ levels and clinical variables. Multivariate regression analysis was conducted to identify potential predictors of vitamin B₁₂ change. A p-value <0.02 was considered statistically significant.

Ethical Considerations

The study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Ethics Committee of the Career Institute of Medical Sciences & Hospital, Lucknow. Written informed consent was obtained from all participants prior to enrollment, and confidentiality of patient data was strictly maintained.

RESULTS

A total of 221 patients with Type 2 diabetes mellitus (T2DM) receiving metformin monotherapy were included in the study. The results are presented with respect to demographic characteristics, clinical profile, and changes in serum vitamin B₁₂ levels over time.

Table 1: Baseline Demographic and Clinical Characteristics of Study Participants (n = 221)

Table 1 summarizes the baseline demographic and clinical characteristics of the study population. Most participants were aged between 41–43 years (31.2%), with a mean age of 40.01 ± 3.17 years, indicating early-onset Type 2 diabetes mellitus. The gender distribution was nearly equal, with a slight female predominance (52.0%). A majority of participants (55.7%) followed a vegetarian diet, which may predispose to lower vitamin B₁₂ intake. Regarding disease duration, 42.1% of patients had diabetes for more than 24 months, with a mean duration of 21.33 ± 9.07 months, reflecting a cohort with moderate disease chronicity.

Table 2: Effect of Metformin on Vitamin B₁₂ Status Over Time

Table 2 demonstrates the effect of metformin therapy on vitamin B₁₂ status over time. At baseline, 87.3% of patients had normal vitamin B₁₂ levels, while only 0.9% were deficient. After 6 months of metformin therapy, there was a slight increase in borderline and deficient cases, although this change was not statistically significant (p = 0.151). However, at 12 months, a significant decline in vitamin B₁₂ status was observed, with deficiency increasing to 9.0% (χ² = 22.4, p < 0.001), indicating a time-dependent effect of metformin on vitamin B₁₂ levels.

Table 3: Mean Serum Vitamin B₁₂ Levels Over Time

Table 3 shows the progressive decline in mean serum vitamin B₁₂ levels during follow-up. Mean vitamin B₁₂ decreased significantly from 423.95 ± 102.71 pg/mL at baseline to 393.22 ± 103.24 pg/mL at 6 months and further to 356.07 ± 104.56 pg/mL at 12 months. The reductions at both follow-up points were highly statistically significant (p < 0.001), confirming a cumulative decline with prolonged metformin use.

Table 4: Correlation of Duration of Diabetes and Metformin Dose with Vitamin B₁₂ Levels and Other Parameters Pearson Correlation Analysis

Pearson correlation analysis showed no statistically significant association between duration of diabetes or metformin dose and vitamin B₁₂ levels, hematological indices, or glycemic parameters. All correlation coefficients were weak and non-significant (p > 0.05), indicating that neither duration of diabetes nor daily metformin dose had a measurable impact on vitamin B₁₂ levels or related parameters in this study population.

Table 5: Multivariate Regression Analysis Showing Vitamin B₁₂ Level Changes with Possible Predictors Change in Vitamin B₁₂ after 6 Months

Table 5 presents the multivariate regression analysis assessing the association between potential predictors and change in serum vitamin B₁₂ levels after 6 months of metformin therapy. The intercept was statistically significant (B = −25.79, p < 0.001), indicating an overall decline in vitamin B₁₂ levels during the first 6 months of treatment, independent of other variables included in the model. Alcohol consumption, smoking, and tobacco use did not show a significant association with vitamin B₁₂ change (p > 0.05), suggesting that lifestyle habits did not influence early vitamin B₁₂ decline. Duration of diabetes (B = −0.02, p = 0.871) and metformin dose (B = 0.00, p = 0.422) were also not significantly associated with vitamin B₁₂ change, indicating that early depletion was not dependent on disease duration or drug dosage. Dietary patterns, including non-vegetarian and ovo-vegetarian diets compared with vegetarian diet (reference), did not significantly affect vitamin B₁₂ levels. The effect sizes for all predictors were minimal, demonstrating negligible contribution of individual variables to the observed decline in vitamin B₁₂ levels at 6 months.

Table 6: Change in Vitamin B₁₂ after 12 Months

Table 6 shows the multivariate regression analysis evaluating predictors of change in serum vitamin B₁₂ levels after 12 months of metformin therapy. The intercept remained highly significant (B = −71.35, p < 0.001), reflecting a marked overall decline in vitamin B₁₂ levels over one year of treatment. Alcohol intake, smoking, and tobacco use were not significantly associated with vitamin B₁₂ changes (p > 0.05), indicating that lifestyle factors did not influence long-term vitamin B₁₂ decline. Similarly, duration of diabetes (B = 0.12, p = 0.608) and metformin dose (B = 0.00, p = 0.937) showed no significant effect, suggesting that prolonged vitamin B₁₂ reduction was independent of disease duration and medication dose. Dietary patterns, including non-vegetarian and ovo-vegetarian diets relative to vegetarian diet, also failed to demonstrate any significant association with vitamin B₁₂ change. Effect sizes for all predictors were extremely small, indicating minimal explanatory power of these variables.

DISCUSSION

The present study observed a significant, time-dependent decline in serum vitamin B₁₂ levels in patients with Type 2 diabetes mellitus receiving metformin therapy. Mean vitamin B₁₂ levels decreased from 423.95 ± 102.71 pg/mL at baseline to 356.07 ± 104.56 pg/mL at 12 months (p < 0.001), with a corresponding increase in vitamin B₁₂ deficiency prevalence from 0.9% at baseline to 9.0% at 12 months. These findings align with several previous studies reporting similar trends in metformin-induced vitamin B₁₂ depletion.

Ramirez et al. documented a significant reduction in serum vitamin B₁₂ concentrations in patients on long-term metformin, with prevalence of deficiency increasing with duration of therapy, supporting the time-dependent decline observed in our cohort [8]. Similarly, Aroda et al. reported that metformin therapy was associated with a 14% absolute increase in the incidence of vitamin B₁₂ deficiency compared to placebo over 4.3 years, further validating the progressive pattern seen in the present study [9].

Our study’s lack of significant correlation between vitamin B₁₂ levels and metformin dose is consistent with findings by de Jager et al., who reported that cumulative duration rather than daily dose was more predictive of vitamin B₁₂ reduction among metformin users [10]. In contrast, Liu et al. found a modest association between higher metformin doses and greater decline in serum B₁₂ levels; however, their cohort included a wider age range and greater variability in treatment duration, which may explain the discrepancy [11].

The dietary patterns in our population, with a high proportion of vegetarian participants (55.7%), did not show a statistically significant impact on vitamin B₁₂ decline. This finding is similar to the results of Bauman et al., who reported that although vegetarian diet can predispose to lower baseline B₁₂ stores, metformin-induced reduction occurred irrespective of dietary intake patterns [12]. However, Reinstatler et al. observed a stronger association between vegetarian diet and severity of vitamin B₁₂ deficiency, particularly in patients with poor dietary cobalamin intake, suggesting that diet may exert an additive effect in susceptible individuals [13].

In terms of hematological parameters, our study found that complete blood count indices remained within normal limits despite significant biochemical decline in vitamin B₁₂ levels. This observation is in concordance with Pouwels et al., who reported that hematological manifestations often lag behind biochemical depletion and may remain subclinical for extended periods [14]. Similarly, Bell et al. observed that early metformin-associated B₁₂ deficiency may not present with anemia or macrocytosis initially, emphasizing the need for biochemical screening rather than reliance on hematological changes alone [15].

Overall, the trajectory of vitamin B₁₂ depletion in our study is consistent with the broader evidence base indicating that long-term metformin therapy significantly influences cobalamin status. The variation in magnitude and predictors across studies highlights the influence of population characteristics, dietary practices, baseline vitamin B₁₂ status, and study methodology.

CONCLUSION

The present study demonstrates that long-term metformin therapy in patients with Type 2 diabetes mellitus is associated with a significant, progressive, and time-dependent reduction in serum vitamin B₁₂ levels. Although overt hematological abnormalities were not observed, the increasing prevalence of borderline and deficient vitamin B₁₂ status highlights the risk of subclinical deficiency, which may remain clinically silent in the early stages. The decline in vitamin B₁₂ levels appeared to be independent of metformin dose, duration of diabetes, dietary pattern, and lifestyle factors, suggesting a direct effect of prolonged metformin exposure. These findings underscore the importance of routine monitoring of serum vitamin B₁₂ levels in patients receiving long-term metformin therapy to enable early identification and timely intervention, thereby preventing potential neurological and hematological complications.

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