Introduction
Stroke, or cerebrovascular accident, poses a major global public health challenge, ranking as the second leading cause of death and third for combined death and disability (measured by disability-adjusted life years). Age-standardized incidence rates ranged from approximately 140-150 per 100,000 population in recent global assessments [1]. The World Health Organization defines it as rapidly developing clinical signs of focal (or global) disturbance of cerebral function, lasting more than 24 hours or leading to death, with no apparent cause other than vascular origin [2].
Ischemic stroke stems from non-modifiable risk factors (age, race/ethnicity, genetics) and modifiable ones (hypertension, diabetes mellitus, dyslipidemia, smoking, abdominal obesity, poor diet, physical inactivity, psychosocial stress). These often interact, hindering precise risk prediction. Vitamin B12 deficiency emerges as a key modifiable factor, vital for one-carbon metabolism including homocysteine re-methylation to methionine and central nervous system function [3].
Deficient vitamin B12 traps folate as methyl tetrahydrofolate, elevating homocysteine a prothrombotic, atherogenic stroke risk factor linked to neuronal apoptosis and mitochondrial dysfunction, with worse outcomes in the elderly due to impaired absorption [3].
Combined folic acid and vitamin B12 supplementation reduces total homocysteine in hyper-homocysteinemia, potentially curbing ischemic stroke risk, as seen in folate fortification programs lowering homocysteine and stroke mortality [4-5]. However, the VITATOPS trial found no reduction in recurrent stroke or vascular events with B vitamins (folic acid, B6, B12) post-stroke or transient ischemic attack. Similarly, the HOPE-2 trial reported no cardiovascular event benefit from these vitamins in high-risk patients. Such discrepancies highlight the need to investigate serum vitamin B12 associations with acute ischemic stroke and underlying mechanisms [6].
This study aims to estimate serum vitamin B12 levels in patients with acute ischemic stroke, determine these levels compared to healthy controls, and explore their potential as a modifiable risk factor for prevention and improved outcomes.
Materials and Methods
Study Design and Setting
This cross-sectional study was conducted at Tertiary care hospital from July 2024 to December 2025. Patients aged 18-70 years presenting to the Medicine Department (inpatient, outpatient, and emergency services) with acute ischemic stroke confirmed clinically per WHO criteria and by MRI brain comprised the case group (Group A). Age- and sex-matched healthy controls (Group B) were recruited from patient attendants or volunteer staff without cerebrovascular disease history. Participants were selected via convenience sampling.
Inclusion criteria (cases)
Acute ischemic stroke (WHO definition), MRI confirmation, NIHSS assessment for severity.
Exclusion criteria
Hemorrhagic stroke; prior vitamin B12 supplementation; liver/renal disease; major gastrointestinal surgery or malabsorption; pregnancy; age<18 years; prior stroke episodes. A total of 130 participants (80 cases, 50 controls) were enrolled.
Clinical Assessment
Stroke severity was quantified using the validated National Institutes of Health Stroke Scale (NIHSS):
| NIHSS Score | Severity Category |
| 0 | No stroke symptoms |
| 1-4 | Minor stroke |
| 5-15 | Moderate stroke |
| 16-20 | Moderate-severe stroke |
| 21-42 | Severe stroke |
Ethical clearance
The present research study initiated after approval from the Institutional Ethics Committee and the Institutional Research Committee (SGRD/IEC/2024-375). Written Informed consent was obtained in the local vernacular language. Detailed history, physical examination, and diagnostic investigations confirmed eligibility and stroke diagnosis.
Laboratory Measurements
Fasting venous blood (5 ml) was collected from cases and controls. Serum vitamin B12 was quantified via chemiluminescent immunoassay on the VITROS 5600 analyzer (Ortho Clinical Diagnostics). Reference range: 239-931 pg/ml. (VITROS 5600 Analyzer) Additional biochemical parameters (lipid profile, renal function) were assessed per standard protocols.
Statistical Analysis
Data were analyzed using Microsoft Excel. Continuous variables were expressed as mean ± standard deviation; categorical variables as frequencies/percentages. Chi-square test assessed categorical associations (gender, diet, comorbidities, B12 status); Fisher's exact test applied when expected frequencies <5. Statistical significance was set at p<0.05.
Results
The current case-control study (80 acute ischemic stroke cases vs 50 age-/sex-matched controls) reveals balanced baseline demographics that minimize confounding, with mean ages closely aligned at 59.82±8.73 versus 57.38±9.89 years across comparable age strata (55% and 50% aged 61–70 years; ~28–29% aged 51–60), and near-identical male predominance (58.75% vs 60%). Notably, cases demonstrated strikingly high exposure to modifiable risk factors: 75% adhered to a vegetarian diet prevalent in Punjab yet potentially linked to B12 deficiency alongside hypertension (51.25%) and type 2 diabetes mellitus (33.75%), both established cerebrovascular triggers (Table 1). These prevalence patterns, exceeding regional norms, underscore the urgent need for targeted screening and risk modification in similar South Asian cohorts to curb stroke burden.
| Age (Years) | Cases (n=80) (%) | Controls (n=50) (%) |
| 30–40 | 4 (5.00%) | 5 (10.00%) |
| 41–50 | 9 (11.25%) | 6 (12.00%) |
| 51–60 | 23 (28.75%) | 14 (28.00%) |
| 61–70 | 44 (55.00%) | 25 (50.00%) |
| Mean ± SD | 59.82 ± 8.73 | 57.38 ± 9.89 |
| Gender | ||
| Female | 33 (41.25%) | 20 (40.00%) |
| Male | 47 (58.75%) | 30 (60.00%) |
| Dietary | ||
| Vegetarian | 60 (75.00%) | |
| Non-Vegetarian | 20 (25.00%) | |
| Comorbidities | (n, %) | |
| Hypertension | 41 (51.25%) | |
| Type 2 Diabetes Mellitus | 27 (33.75%) |
In table 2 cohort of 80 acute ischemic stroke patients, NIHSS scores revealed predominantly mild-moderate severity (93.75% combined: 42.50% minor, 51.25% moderate), with rare severe cases (6.25%). Dyslipidemia patterns were alarming: only 60% had desirable total cholesterol (<200 mg/dL), while 17.50% showed high levels (≥240 mg/dL); triglycerides exceeded 200 mg/dL in 33.75% despite 43.75% desirable; LDL was suboptimal in over half (21.25% high/very high ≥160 mg/dL vs 43.75% desirable); VLDL rose >34 mg/dL in 37.50%; and low HDL afflicted 43.75% males (<40 mg/dL) and 35% females (<50 mg/dL). These lipid derangements, alongside moderate stroke predominance, highlight aggressive dyslipidemia management as a critical secondary prevention target.
| Parameter | Category | n (%) |
| Stroke Severity (NIHSS) | Minor (1–4) | 34 (42.50%) |
| Moderate (5–15) | 41 (51.25%) | |
| Moderate-Severe (16–20) | 2 (2.50%) | |
| Severe (21–42) | 3 (3.75%) | |
| Serum Cholesterol | Desirable (<200 mg/dl) | 48 (60.00%) |
| High (≥240 mg/dl) | 14 (17.50%) | |
| Serum Triglycerides | Desirable (<150 mg/dl) | 35 (43.75%) |
| High (≥200 mg/dl) | 27 (33.75%) | |
| LDL Cholesterol | Desirable (<100 mg/dl) | 35 (43.75%) |
| High/Very High (≥160 mg/dl) | 17 (21.25%) | |
| VLDL Cholesterol | >34 mg/dl | 30 (37.50%) |
| Low HDL | Male (<40 mg/dl) | 35 (43.75%) |
| Female (<50 mg/dl) | 28 (35.00%) |
Vitamin B12 deficiency emerges as a striking differentiator in the case-control analysis of 80 acute ischemic stroke cases versus 50 matched controls (Table 3). Low serum B12 (<239 pg/ml) afflicted 63.75% of cases nearly 5-fold higher than the 14% in controls yielding a highly significant chi-square statistic (χ²=34.37, p<0.001). Normal levels (239-931 pg/ml) were roughly balanced (28.75% vs 50%), while high B12 (>931 pg/ml) was markedly underrepresented in cases (7.5% vs 36%). This dramatic skew toward deficiency among stroke patients strongly implicates suboptimal B12 status as a potential modifiable risk factor, warranting urgent integration into cerebrovascular risk stratification and supplementation protocols in high-prevalence regions like Punjab.
| Vitamin B12 Status (pg/ml) | Cases (n=80) | Controls (n=50) | χ2 | p-value |
| Low (<239) | 51 (63.75%) | 7 (14.00%) | 34.37 | <0.001 |
| Normal (239–931) | 23 (28.75%) | 25 (50.00%) | ||
| High (>931) | 6 (7.50%) | 18 (36.00%) | ||
| Total | 80 (100%) | 50 (100%) |
The analysis unveils nuanced patterns in serum vitamin B12 across key stroke risk factors and severity strata. Type 2 diabetes patients showed markedly elevated median B12 (379.5 pg/ml) versus non-diabetics (301.5 pg/ml; p=0.001), suggesting hyperglycemia-driven inflammation or supplementation effects (Table 4). Hypertension and vegetarian diet yielded non-significant differences (p=0.842, 0.479), with broad IQRs reflecting heterogeneity. Stroke severity trended toward depletion in severe cases (NIHSS 16-42: medians 195.0-190.0 pg/ml vs 322.8-386.8 pg/ml in milder groups; p=0.068), hinting at consumption during acute neuroinflammation. These gradients spotlight diabetes as a B12 modulator and severe stroke as a deficiency state, guiding precision supplementation strategies in high-risk cohorts.
| Subgroup | N (%) | Vit.B12 Median | IQR | p-value |
| Hypertension | ||||
| Yes | 41 | 331.4 | 210.5-452.2 | 0.842 |
| No | 39 | 422.5 | 180.25-664.7 | |
| Type 2 Diabetes mellitus | ||||
| Yes | 27 | 379.5 | 210.0-549.0 | 0.001 |
| No | 53 | 301.5 | 200.0-403.0 | |
| Diet | ||||
| Vegetarian | 60 | 315.5 | 206.0-425.0 | 0.479 |
| Non-Veg | 20 | 428.5 | 199.0-658.0 | |
| Stroke Severity | ||||
| Minor | 34 | 322.8 | 210.5-435.0 | 0.068 |
| Moderate | 41 | 386.8 | 205.5-568.0 | |
| Mod-Severe | 2 | 195.0 | 190.0-200.0 | |
| Severe | 3 | 190.0 | 175.0-205.0 |
Discussion
Present investigation establishes profound vitamin B12 hypovitaminosis as a cardinal, amenable determinant of acute ischemic stroke susceptibility and trajectory in a Punjabi vegetarian-dominant milieu, consonant with and intensifying contemporary empirical precedents. Profound depletion (<239 pg/ml) in 63.75% of cases eclipsed controls at 14% (χ²=34.37, p<0.001), exceeding mean deficits chronicled by Atam et al.,[4] across 75 cases (194.24 pmol/L), where diminished B12 tracked amplified NIHSS severity (r=-0.720, p<0.001) alongside inferior functional prognoses.
Normal Vitamin B12 levels (239-931 pg/mL) were present in 28.75% of cases and 50% of controls, while high levels (>931 pg/mL) were found in 7.50% of cases and 36% of controls (Table 3). Comparable findings have been reported in previous studies. Ahmed et al.,[5] observed a significantly higher prevalence of Vitamin B12 deficiency among stroke patients (approximately 58%) compared to controls (around 20%). Similarly, Zhou et al.,[7] reported that low Vitamin B12 levels were associated with an approximately 1.8-fold increased risk of stroke, while Zhang et al.,[3] demonstrated a strong statistical association between Vitamin B12 deficiency and stroke risk (p<0.01).
Conversely, evidence suggests that lower vitamin B12 concentrations correlate with a poorer prognosis in patients diagnosed with cardiovascular disease. While the medical literature frequently highlights the link between vitamin B complex deficiencies and stroke risk, the specific nature and consistency of this relationship remain debated [7].
Stroke severity correlated with a downward trend in B12 levels, particularly in severe cases (NIHSS 16–42), where medians ranged from 190.0 to 195.0 pg/ml compared to 322.8–386.8 pg/ml in milder cohorts (p=0.068; Table 4). This suggests potential B12 consumption during acute neuroinflammation. These findings identify diabetes as a key modulator of B12 levels and suggest that severe stroke may induce an acute deficiency state, supporting the need for precision supplementation in high-risk patients Significant findings include a 15-year longitudinal study of 43,732 men (aged 40–75) by He et al.,[8] which observed that supplementing with folic acid and vitamin B12 was associated with a 30% decline in ischemic stroke incidence [8]. This is supported by Jenkins et al.,[9] whose meta-analysis on the efficacy of micronutrient supplementation for cardiovascular health concluded that both folate and vitamin B12 play a role in lowering stroke risk.
However, other data presents a more complex picture. A Mendelian randomization analysis conducted by Yuan et al.,[10] suggested a genetic predisposition linking folate and vitamin B6 levels to ischemic stroke, yet failed to establish a similar genetic connection for vitamin B12. Furthermore, clinical trials have not always yielded positive results; for instance, Lonn et al.,[11] studied 5,522 patients over the age of 55 and found that vitamin B supplementation provided no significant clinical benefit regarding cardiovascular outcomes.
Conclusion
The current study identifies Vitamin B12 deficiency as a significant, independent predictor of acute ischemic stroke, with a nearly five-fold higher prevalence in patients compared to healthy controls. The correlation between lower B12 concentrations and increased NIHSS scores suggests that deficiency may not only predispose individuals to stroke but also influence clinical severity. Given the high prevalence of hypovitaminosis in this population, targeted Vitamin B12 screening and repletion offer a low-cost, high-impact strategy for primary and secondary stroke prevention.
Declarations
Ethics Approval and Consent to Participate
All procedures in this case series adhered to the ethical standards of the relevant institutional committees and the Declaration of Helsinki. The institutional review board granted formal ethical approval where applicable. Patients or their legal guardians provided written informed consent before any procedures were performed.
Consent for Publication
Written informed consent was obtained from all participants or their legal representatives for the publication of clinical information and accompanying images. To ensure patient privacy, all personal identifiers have been thoroughly anonymized.
Data Availability
The datasets analyzed during this study are available from the corresponding author upon reasonable request, provided the request aligns with institutional and ethical guidelines.
Competing Interests
The authors declare no financial or personal conflicts of interest regarding the content or publication of this manuscript.
Funding
This research did not receive any dedicated grants or financial support from public, commercial, or non-profit organizations.
Authors’ Contributions
Every author contributed significantly to the study’s design, data collection, and analysis. Each author participated in drafting and refining the manuscript for intellectual content, and all have reviewed and authorized the final submitted version.