Acute Ischemic Stroke (AIS) is a major cause of morbidity and mortality worldwide and accounts for nearly 85% of all stroke cases (1). The global burden continues to rise, especially in developing countries, due to increasing prevalence of vascular risk factors such as hypertension, diabetes mellitus, and smoking (2). Early identification of prognostic indicators in AIS is essential for guiding treatment strategies, predicting clinical outcomes, and reducing long-term disability (3). Although clinical assessment tools like the National Institutes of Health Stroke Scale (NIHSS) and the modified Rankin Scale (mRS) are widely used, there is a growing interest in biochemical markers that may serve as reliable severity predictors.

Serum bilirubin, typically a marker of liver function, has gained scientific attention because of its antioxidant, anti-inflammatory, and cytoprotective properties (4). Emerging evidence suggests that bilirubin levels may influence vascular function and oxidative stress, both of which are implicated in the pathogenesis of ischemic stroke (5). However, studies examining bilirubin in AIS have yielded conflicting results. Some report that elevated bilirubin is associated with greater stroke severity and worse outcomes (6,7), whereas others suggest a protective antioxidant role (8).

Given these inconsistent findings and the limited data available from South Indian populations, further research is necessary. Therefore, this one-year prospective observational study aimed to assess the role of serum bilirubin (total, direct, and indirect) as a severity and prognostic indicator in AIS. The study evaluated correlations between bilirubin levels and NIHSS scores, mRS outcomes, and determined diagnostic accuracy using ROC curve analysis. Multivariate logistic regression was performed to identify bilirubin as an independent predictor of AIS outcome.

MATERIALS AND METHODS

Study Design and Setting

This prospective observational study was conducted in the Emergency Department of Villupuram Medical College, a tertiary care centre in Tamil Nadu.

Study Duration

The study was conducted over a period of one year, from April 2024 to April 2025.

Study Population

The study included newly diagnosed adult patients presenting with Acute Ischemic Stroke (AIS).

Inclusion Criteria

• Patients aged ≥18 years.
• Newly diagnosed AIS presenting within 48 hours of symptom onset.

Exclusion Criteria

• Haemorrhagic stroke.
• Post-traumatic stroke.
• Post-surgical stroke.
• Transient Ischaemic Attack (TIA).
• Patients/attenders unwilling to provide consent.

Sample Size Calculation

Based on a study by Ushalakshmi et al., where the mean ± SD serum bilirubin level among AIS patients was 0.6 ± 0.249 mg/dL, the sample size was calculated using the formula:

Where:

• (95% confidence),
• (absolute precision),
• .

The calculated sample size was 96.

Ethical Approval

Approval was obtained from the Institutional Ethics Committee (IEC) of Government Medical College, Villupuram. The study details were explained to the patients or their attenders in their native Tamil language, and written informed consent was obtained. Confidentiality and anonymity were strictly maintained.

Sampling Technique

A consecutive sampling technique was used. All AIS patients presenting to the emergency department and meeting the eligibility criteria were enrolled until the sample size of 96 was achieved.

Study Procedure

Following ethical clearance, 96 newly diagnosed AIS patients were recruited. A predesigned semi-structured pro forma (Annexure) was used to collect sociodemographic details, clinical history, examination findings, laboratory parameters, and imaging results. All patients received treatment as per standard management protocols.

• Stroke severity was assessed at admission using the National Institutes of Health Stroke Scale (NIHSS).
• Functional outcome was assessed at discharge using the modified Rankin Scale (mRS).

Data Collection Steps

1. Obtained IEC approval prior to study initiation.
2. Written informed consent was collected from patient attenders.
3. Semi-structured pro forma was completed in Tamil for all participants.
4. Each patient underwent clinical evaluation, laboratory investigations, and radiological imaging.
5. Treatment was provided according to clinical condition and institutional protocol.
6. Stroke severity (NIHSS) and functional outcome (mRS) were recorded.

Potential Risks

No risks were anticipated for the study participants.

Data Analysis

Data were entered into Microsoft Excel and analysed using SPSS version 16.0. Missing or invalid entries were removed prior to analysis to ensure accuracy. Categorical variables such as gender and smoking status were summarized using frequencies and percentages, while continuous variables, including age and serum bilirubin, were expressed as mean ± SD for normally distributed data or as median values for non-normally distributed data. The Kolmogorov–Smirnov test was used to assess the normality of continuous variables. Depending on the number of groups and distribution of data, comparisons of continuous variables were performed using the independent t-test or ANOVA. Pearson’s correlation coefficient was applied to evaluate the linear relationship between continuous variables. Associations between categorical variables were examined using the chi-square test or Fisher’s exact test as appropriate. Multivariate logistic regression analysis was carried out to identify predictors, such as serum bilirubin, influencing Acute Ischaemic Stroke outcomes measured by the modified Rankin Scale (mRS). A p-value of <0.05 was considered statistically significant.

RESULT AND OBSERVATION

Table 1 Age and Sex Distribution of Study Participants (N = 96)

Table:2Sociodemographic and Clinical Characteristics of Study Participants (N = 96)

Figure 1: Mean Serum Bilirubin

Table:3 NIHSS and mRS Severity Scores and Outcome Distribution Among Study Participants (N = 96)

Table 4: Correlation of Serum bilirubin and AIS outcome: (N = 96)

Figure 2: ROC curve of serum bilirubin as a predictor AIS outcome severity

Table 5: Area Under the Curve

Table: 6ROC Curve Characteristics and Cutoff-wise Distribution of Serum Bilirubin Parameters (N = 96)

Table 7: Association of serum bilirubin with AIS outcome (mRS): N = 96

Table 8: Multinomial Logistic Regression of Serum Bilirubin with mRS severity: (N = 96)

DISCUSSION

This prospective observational study demonstrated a significant association between serum bilirubin levels and both stroke severity and functional outcomes in patients with Acute Ischemic Stroke (AIS). Total, direct, and indirect bilirubin showed strong positive correlations with NIHSS and mRS scores, indicating that higher bilirubin levels were associated with more severe neurological deficits and poorer outcomes. These findings support earlier research conducted by Ushalakshmi et al. (6) and Vala et al. (7), who also reported a similar pattern of association between elevated bilirubin and increased stroke severity.

The elevation of serum bilirubin in AIS may reflect the underlying oxidative stress occurring during ischemic neuronal injury. Activation of heme oxygenase-1 during ischemia leads to increased heme breakdown and subsequent bilirubin production, which may serve as a biomarker of cellular oxidative burden (9). Therefore, although bilirubin possesses antioxidant properties, its elevated levels in AIS likely represent a compensatory response to severe oxidative and inflammatory stress rather than a protective mechanism. This biological explanation aligns with the significant correlations observed in the present study.

The ROC analysis further revealed that total bilirubin had excellent predictive accuracy for determining severe mRS outcomes, with an AUC of 0.798, followed by direct bilirubin with an AUC of 0.785. These diagnostic characteristics are comparable to the findings of Zhai et al. (10), who demonstrated the utility of bilirubin as a predictor of stroke prognosis. The cutoff value of ≥2 mg/dL for total bilirubin achieved high sensitivity and specificity, supporting its clinical relevance in early risk stratification.

Multivariate logistic regression analysis showed that total bilirubin <2 mg/dL was independently associated with better functional outcomes (Adjusted OR = 9, p = 0.012), while direct and indirect bilirubin did not remain significant predictors after adjustment. This emphasizes that total bilirubin, reflecting the combined effect of both its fractions, is likely the most reliable prognostic marker.

The findings of this study are largely consistent with the majority of available literature, which supports the association between higher bilirubin levels and poor AIS outcomes (6,7,10). However, some studies propose a protective antioxidant role of bilirubin (8), leading to conflicting interpretations. These discrepancies may be attributed to differences in genetic factors, baseline bilirubin levels, patient demographics, sampling times, and variations in stroke severity across study populations.

The study has several strengths, including its prospective design, standardized assessment tools, and robust statistical analyses incorporating ROC characteristics and multivariate regression. However, certain limitations should be considered. This was a single-center study with a relatively small sample size, and bilirubin levels were measured only once, preventing evaluation of dynamic changes during the clinical course. Additionally, radiological parameters such as infarct volume were not included, which could further strengthen prognostic assessment.

Despite these limitations, the study highlights a practical and low-cost biomarker that is routinely measured in clinical laboratories. The strong association between elevated bilirubin and poorer outcomes suggests that serum bilirubin, particularly total bilirubin ≥2 mg/dL, may serve as a valuable early prognostic indicator in AIS, aiding in risk stratification and guiding treatment planning.

CONCLUSION

Serum bilirubin, especially total bilirubin ≥2 mg/dL, is strongly associated with greater stroke severity and poorer functional outcomes in Acute Ischemic Stroke. It demonstrated good diagnostic accuracy and remained an independent predictor of prognosis. As a simple and routinely available marker, serum bilirubin can aid early risk stratification in AIS patients.

REFERENCES

1. World Stroke Organisation. Global Stroke Fact Sheet 2023. World Stroke Organisation; 2023.
2. Easton JD, Saver JL, Albers GW, Alberts MJ, Chaturvedi S, Feldmann E, et al. Definition and evaluation of transient ischemic attack: a scientific statement for healthcare professionals from the American Heart Association/American Stroke Association Stroke Council. Stroke. 2009;40(6):2276–2293.
3. Goldstein LB, Bertels C, Davis JN. Interrater reliability of the NIH Stroke Scale. Arch Neurol. 1989;46(6):660–662.
4. Kim SY, Kim HJ, Kim JH, Huh J, Kim H. Association between bilirubin and cardiovascular disease: A systematic review and meta-analysis. Atherosclerosis.2017;265:50–58.
5. Wang J, Doré S. Heme oxygenase-1 exacerbates early brain injury after intracerebralhaemorrhage. J NeurolNeurosurg Psychiatry. 2007;78(3):257–260.
6. Ushalakshmi K, Sandhya Rani TS, Ramesh Kumar G. Study of serum bilirubin levels in acute ischemic stroke and its correlation with severity and functional outcome. J ClinDiagn Res. 2017;11(9):OC38–OC41.
7. Vala M, Parikh N, Sharma H, Patel D. Serum bilirubin: a potential biomarker in acute ischemic stroke. Neurol India. 2017;65(2):393–397.
8. Perlstein TS, Pande RL, Beckman JA, Creager MA. Serum total bilirubin level and prevalent lower-extremity peripheral arterial disease: a cross-sectional study. Circulation. 2008;117(14):1784–1791.
9. Huang J, Upadhyay UM, Tamargo RJ. Inflammation in stroke and the role of the hemeoxygenase pathway. J Cereb Blood Flow Metab. 2005;25(12):1556–1570.
10. Zhai H, Zhou H, Du Y, Wang D, Zhang R. Prognostic value of serum bilirubin in patients with acute ischemic stroke. Stroke. 2017;48(2):470–476.