Stroke is a major cause of morbidity and mortality worldwide [
Oxidative stress has been defined as “an imbalance between oxidants and antioxidants in favor of oxidants, potentially leading to damage” [
On the other hand, the antioxidant defense system has been studied in stroke patients as regards enzymes, including superoxide dismutase (SOD) and glutathione peroxidase [
Under the hypothesis that the level of oxidative stress is increased and may be diverse in different subtypes of stroke, this study evaluated longitudinal changes in serum oxidant and antioxidant levels after ischemic stroke to determine their value in predicting short-term outcome. Serial changes of serum TBARS and free thiol were measured in different subtypes during the first month after stroke and the possibility of using these markers for predicting three-month outcome was assessed.
From August 2010 to July 2012, consecutive patients with AIS who were admitted to the Neurology Department of Chang Gung Memorial Hospital-Kaohsiung were evaluated. Acute ischemic stroke was defined as an acute onset loss of focal cerebral function persisting for at least 24 hours. Diagnosis was based on clinical presentation, neurologic examination, and results of brain magnetic resonance imaging (MRI) with diffusion-weighted imaging (DWI). Patients aged 18–80 years with acute noncardioembolic ischemic stroke were included and divided into two major etiologic subtypes (i.e., large-artery atherosclerosis and small-artery occlusion) according to the TOAST (Trial of Org 10172 in Acute Stroke Treatment) classification [
Patients with cardioembolic stroke, other determined causes and undetermined causes of stroke, and those with underlying neoplasm, end-stage renal disease, liver cirrhosis, and congestive heart failure were excluded. Clinical examination, electrocardiography, and cardiac ultrasound were conducted to exclude cardioembolic stroke. Patients with fever or any infectious disorder within the first week after acute stroke were also excluded.
All of the participants underwent complete neurologic examination. Brain MRI with DWI, extracranial carotid sonography, and transcranial color-coded sonography were performed during the hospitalization. The therapeutic regimens for AIS were based on the American Heart Association (AHA)/American Stroke Association (ASA) guidelines [
Blood samples were collected by venipuncture of forearm veins from patients within 48 hours of the stroke (presented as day 1) and on days 7 and 30 after stroke. Serum MDA was measured using the TBARS assay. The concentration of TBARS was assessed based on the method of Huang et al. [
The ability of antioxidative defense in response to increased oxidative damage was evaluated by measuring the serum level of total reduced thiols because thiols were physiologic free radical scavengers. Serum free thiols were determined by directly reacting thiols with 5,5-dithiobis 2-nitrobenzoic acid (DTNB) to form 5-thio-2-nitrobenzoic acid (TNB). The amount of thiols in the sample was calculated from absorbance, as determined using the extinction coefficient of TNB (A412 = 13,600 M−1 cm−1).
Data were presented as mean ± SEM. Continuous variables, including age, cell count, lipid profile, hemoglobin A1c (HbA1c), blood pressure, and serum free thiol and TBARS, were analyzed by independent
Scheffe’s multiple comparison was used to analyze the intraindividual courses of parameters over time. These were then compared among patients with small-vessel and large-vessel diseases. Multiple logistic regression analyses determined the independent influence of different predictive variables on clinical outcome. Statistical significance was set at
Of the 120 patients with acute noncardioembolic ischemic stroke, 20 were excluded for various infections or fever in the first week after acute stroke (
Baseline characteristics and laboratory data between small-vessel and large-vessel disease.
Small vessel disease | Large vessel disease |
| |
---|---|---|---|
( |
( | ||
Age (y) (mean ± SD) | 61.1 ± 11.5 | 64.6 ± 8.8 | 0.17 |
Sex (male) ( |
60 | 17 | 0.27 |
Hypertension ( |
56 | 22 | 0.26 |
Diabetes mellitus ( |
32 | 10 | 0.82 |
Dyslipidemia ( |
37 | 12 | 0.91 |
Coronary artery disease ( |
5 | 2 | 0.82 |
White blood cells (×103/mL) | 7.4 ± 0.3 | 8.1 ± 0.5 | 0.22 |
Red blood cells (×106/mL) | 4.8 ± 0.1 | 4.5 ± 0.1 | 0.11 |
Platelet counts (×104/mL) | 21.1 ± 0.7 | 19.8 ± 1.2 | 0.30 |
Total cholesterol (mg/dL) | 181.2 ± 3.8 | 194.6 ± 11.6 | 0.15 |
LDL-cholesterol (mg/dL) | 109.9 ± 3.7 | 123.4 ± 9.9 | 0.12 |
Triglyceride (mg/dL) | 134.7 ± 7.7 | 134.9 ± 13.3 | 0.99 |
HbA1c (%) | 7.0 ± 0.2 | 6.4 ± 0.3 | 0.15 |
Free thiol ( |
0.90 ± 0.03 | 0.81 ± 0.06 | 0.19 |
TBARS ( |
19.7 ± 1.2 | 20.7 ± 2.6 | 0.85 |
Serial changes in serum concentration of TBARS among patients groups and in the controls (Figure
Serial changes in serum TBARS among patients with small-vessel and large-vessel diseases and in the controls at various time points after stroke.
Serial changes in serum concentration of free thiol among patients with small-vessel and large-vessel diseases and in the controls (Figure
Serial changes in serum free thiol among patients with small-vessel and large-vessel diseases and in the controls at various time points after stroke.
Potential prognostic factors of the 100 stroke patients were listed in Table
Prognostic factors in patients with acute ischemic stroke.
Good outcome |
Poor outcome |
Crude OR |
|
Adjusted OR |
| |
---|---|---|---|---|---|---|
Age (year) | 61.2 ± 11.5 | 65.1 ± 8.0 | 1.04 (0.99–1.09) | 0.16 | — | — |
Sex (male) ( |
62 | 15 | 0.87 (0.28–2.72) | 0.81 | — | — |
Hypertension ( |
61 | 17 | 1.77 (0.47–6.68) | 0.55 | — | — |
Diabetes mellitus ( |
32 | 10 | 1.50 (0.56–4.01) | 0.46 | — | — |
Hyperlipidemia ( |
38 | 11 | 1.35 (0.51–3.61) | 0.62 | — | — |
Cardiac disease ( |
5 | 2 | 1.67 (0.30–9.30) | 0.80 | — | — |
NIHSS score on admission | 1.35 (1.16–1.58) | <0.001 | 1.55 (1.11–2.16) | 0.01 | ||
Stroke subtype (large/small) | 21.0 (6.26–70.4) | <0.001 | 0.01 (0.001–0.33) | 0.008 | ||
Small vessel disease | 70 | 5 | — | — | ||
Large vessel disease | 10 | 15 | — | — | ||
With statin therapy | 35 | 11 | 1.49 (0.56–4.00) | 0.46 | — | — |
Laboratory data on admission | — | — | ||||
White blood cells (×103/mL) | 7.4 ± 0.2 | 8.3 ± 0.6 | 1.17 (0.96–1.43) | 0.12 | — | — |
Hemoglobin (g/dL) | 13.8 ± 0.2 | 13.6 ± 0.3 | 0.91 (0.69–1.22) | 0.53 | ||
Red blood cells (×106/mL) | 4.8 ± 0.1 | 4.5 ± 0.1 | 0.50 (0.24–1.07) | 0.07 | — | — |
Platelet counts (×104/mL) | 21.2 ± 0.6 | 18.9 ± 1.4 | 0.99 (0.98–1.00) | 0.12 | — | — |
Total cholesterol (mg/dL) | 185.7 ± 4.6 | 180.0 ± 8.6 | 0.99 (0.98–1.01) | 0.58 | — | — |
LDL-cholesterol (mg/dL) | 114.2 ± 4.2 | 109.6 ± 7.7 | 0.99 (0.98–1.01) | 0.62 | — | — |
HDL-cholesterol (mg/dL) | 43.8 ± 1.0 | 45.6 ± 3.0 | 1.02 (0.97–1.07) | 0.48 | — | — |
Triglyceride (mg/dL) | 136.3 ± 7.6 | 128.3 ± 13.8 | 0.99 (0.99–1.01) | 0.63 | — | — |
HbA1c (%) | 6.8 ± 0.2 | 7.3 ± 0.6 | 1.14 (0.90–1.45) | 0.28 | — | — |
Systolic BP (mmHg) | 147.1 ± 2.8 | 140.1 ± 4.4 | 0.99 (0.97–1.01) | 0.28 | — | — |
Diastolic BP (mmHg) | 84.5 ± 1.5 | 81.1 ± 2.8 | 0.98 (0.94–1.02) | 0.31 | — | — |
Free thiol on admission ( |
0.92 ± 0.03 | 0.70 ± 0.07 | 0.04 (0.01–0.32) | 0.002 | — | — |
TBARS on admission ( |
19.9 ± 0.72 | 24.1 ± 3.0 | 1.05 (0.99–1.11) | 0.06 | — | — |
Free thiol on day 7 ( |
0.96 ± 0.04 | 0.66 ± 0.08 | 0.12 (0.03–0.52) | 0.005 | — | — |
TBARS on day 7 ( |
18.4 ± 0.77 | 29.1 ± 2.8 | 1.14 (1.07–1.22) | <0.001 | 1.37 (1.14–1.65) | 0.001 |
Free thiol on day 30 ( |
1.00 ± 0.04 | 0.88 ± 0.11 | 0.29 (0.05–1.75) | 0.78 | — | — |
TBARS on day 30 ( |
19.7 ± 1.24 | 20.4 ± 2.4 | 1.01 (0.95–1.07) | 0.82 | — | — |
Abbreviations: BP: blood pressure; HbA1c: hemoglobin A1c.
Potential variables such as age, sex, stroke subtype, blood pressure, HbA1C, total cholesterol, HDL, LDL, and TBARS and free thiol levels on admission were analyzed using a stepwise logistic regression model. Only the stroke subtype (OR: 0.014, 95% CI: 0.001–0.325;
Changes in serum TBARS between groups with good and poor outcomes (Figure
Serial changes in serum TBARS among patients with good and poor outcomes and in the controls at various time points after stroke.
Changes in serum concentration of free thiol among patients with good and poor outcomes, and in the controls (Figure
Serial changes in serum free thiol among patients with good and poor outcomes and in the controls at various time points after stroke.
The present study has four major findings. First, patients with AIS in the acute phase had significantly higher TBARS and lower free thiol levels than the controls. Second, the level of free thiol is significantly lower in patients with large-vessel disease than in those with small-vessel disease on day 7 after stroke. Third, the higher TBARS and lower free thiol levels in the acute phase of AIS is associated with poor outcome. Lastly, the most important finding in this study is that TBARS level on day 7 after stroke is an independent predictive factor of three-month outcome.
The results on the MDA level changes over time in stroke patients are controversial. Some researchers observed higher erythrocyte MDA levels in the very early phase of stroke, with subsequent decline in the levels of the controls [
Furthermore, the current study demonstrates that antioxidant levels, measured by free thiol, are much lower in patients with large-vessel cerebral infarction than in those with small-vessel infarction on day 7 after stroke. The level of free thiol gradually increases until the difference is no longer significant one month after the stroke. These suggest that oxidant/antioxidant balance is related to the different pathogenesis in the two major subtypes of noncardioembolic stroke. The pathogenesis of small-vessel infarction is lipohyalinosis [
Oxidative stress is an important contributor to the pathophysiologic sequelae of stroke. A correlation of MDA level with infarct size, clinical stroke severity, and patient outcome has been observed [
On the other hand, antioxidant vitamin concentrations are associated with neurologic damage and stroke prognosis [
Although other inflammatory biomarkers like high-sensitivity C-reactive protein (hs-CRP) and leukocytes have been reported to be useful in predicting clinical outcome after stroke [
Some limitations of this study should be acknowledged. First, the measurement of only few biomarkers of oxidative damage cannot be considered a valid tool for exploring a multifaceted, complex oxidant/antioxidant imbalance after acute stroke. Second, the oxidant/antioxidant balance of stroke patients may be influenced by a multitude of parameters, including age, sex, smoking habit, alcohol consumption, physical activity, and vitamin supplementation. Third, oxidative stress may be influenced by other drugs (e.g., antiplatelet, angiotensin II type 1 receptor blockers, and antidiabetics). Since the use of these drugs depends on the preference of the attending physician, this may cause potential bias in statistical analysis and in drawing conclusions. Nonetheless, the sample size is not large and the number of variables considered in the stepwise logistic regression analysis is small. Hence, the maximum likelihood estimates of the coefficients are valid in the analysis.
In conclusion, patients with AIS have significantly higher TBARS and lower free thiol levels than healthy controls. The level of free thiol is significantly lower in patients with large-vessel disease than in those with small-vessel disease in the acute phase of stroke. Serum TBARS on day 7 after stroke is an independent predictive biomarker of three-month stroke outcome.
All of the authors declared that they have no conflict of interests.
This study was supported by grants from the National Science Council Research Project (99-2314-B-182-055-MY2). The authors wish to thank Doctor Gene Alzona Nisperos who provided language help and writing assistance for this paper.