- Email: [email protected]
Serum level and prognostic value of neopterin in patients after ischemic stroke
Clinical Biochemistry 45 (2012) 1596–1601
Contents lists available at SciVerse ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Serum level and prognostic value of neopterin in patients after ischemic stroke Hung-Sheng Lin a, Tzu-Hsien Tsai b, d, 1, Chu-Feng Liu c, Cheng-Hsien Lu a, Wen-Neng Chang a, Shu-Feng Chen a, Chi-Wei Huang a, Chi-Ren Huang a, Nai-Wen Tsai a, Chih-Cheng Huang a, Chia-Wei Liou a, Tsu-Kung Lin a, Min-Yu Lan a, Hon-Kan Yip b,⁎ a
Department of Neurology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan Division of Cardiology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan Department of Emergency Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan d Graduate Institute of Clinical Medical Sciences, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan b c
a r t i c l e
i n f o
Article history: Received 10 August 2011 Received in revised form 24 July 2012 Accepted 31 July 2012 Available online 8 August 2012 Keywords: Ischemic stroke Neopterin
a b s t r a c t Background: We hypothesized that serum level of neopterin is significantly predictive of prognostic outcome in patients after acute ischemic stroke (IS). Methods: Between November 2008 and May 2010, serum levels of neopterin were prospectively collected at 48 h after acute IS in 157 patients. Results: Serum neopterin levels were substantially higher in patients with severe neurological impairment [National institutes of Health Stroke Scale (NIHSS) score ≥12] than in those with NIHSS b 12 (pb 0.008). Furthermore, Spearman's test showed a strongly positive correlation between neopterin level and NIHSS (p=0.003). Multiple logistic regression analysis demonstrated that serum neopterin level was strongly and independently predictive of NIHSS ≥12 (p=0.002) at 48 h after acute IS and 90-day major adverse clinical outcome (defined as NIHSS≥12, recurrent stroke or death) (p=0.003). Conclusion: Serum level of neopterin was notably increased after acute IS. This biomarker was strongly and independently predictive of 90-day unfavorable clinical outcome in patients after acute IS. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Abundant data support the proposal that inflammatory processes and immune responses are pivotal in the pathogenesis of atherosclerotic plaque [1–5]. Histopathological findings have further shown that complex interaction and activation of monocytes, macrophages, and lymphocytes within the plaque play a key role in the inflammatory process associated with atherosclerosis and plaque vulnerability [1,2,4,6]. This complex interaction finally causes atheroembolic plaque disruption and acute coronary syndrome (ACS) [1,2,4,6–8]. However, atherosclerotic plaque instability and disruption may not be confined only in epicardial coronary arteries but may also involve other arterial systems, including carotid arteries and cerebral arteries [6,9,10]. Thus, coronary artery disease (CAD) and cerebral vascular disease (CVD) are two sides of the same coin [11]. Besides, the activated inflammatory cells within atherosclerotic plaques are capable of producing a range of proteases that lead to
⁎ Corresponding author at: Division of Cardiology, Department of Internal Medicine, Chang Gung Memorial Hospital, Kaohsiung 123, Ta Pei Road, Niao Sung Hsiang, Kaohsiung City 83301, Taiwan. Fax: +886 7 7322402. E-mail address: [email protected] (H.-K. Yip). 1 Indicates equal contribution in this study compared with the first author.
proteolytic destruction of the extra-cellular matrix [12,13] and production of interferon-γ, both of which subsequently activate macrophages within the plaque and interfere with matrix collagen synthesis within [14]. These enzyme and cytokine productions, which contribute to a persistent inflammatory reaction within the atherosclerotic plaque, may also play a crucial role in the disruption of the unstable plaque [6,12–14]. Neopterin, a pteridine derivative and a side-product of the guanosine triphosphate–biopterin pathway [15] secreted by activated macrophages after stimulation by IFN-γ [16], has been suggested to be a biomarker of immune activation and macrophage activity [16]. Additionally, neopterin has been found to participate in enhancing inflammatory processes within vulnerable plaques [15,16]. Besides, neopterin has been reported to be increased in the serum of patients with ACS and associated with coronary artery complex lesions [17–19]. Increased serum level of neopterin, therefore, has been suggested to be a biomarker of acute widespread atherothrombotic plaque inflammation and CAD activity [17–19]. Surprisingly, while an association of elevated serum neopterin level with ACS and coronary artery complex lesion has been extensively investigated [17–20], the correlation of serum neopterin level with carotid artery disease [6,10] and acute ischemic stroke (IS) has seldom been reported [21]. Therefore, the potential of serum neopterin level as a clinical biomarker for predicting prognostic outcome after acute IS remains unclear. Accordingly, this study assessed the serum neopterin
0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2012.07.113
H.-S. Lin et al. / Clinical Biochemistry 45 (2012) 1596–1601
level in patients after acute IS to evaluate its clinical value as a biomarker for predicting 90-day unfavorable clinical outcome following the cerebrovascular episode.
Materials and methods Definition, exclusion criteria, and patient enrollment This study was approved by the Institutional Review Committee on Human Research of Chang Gung Memorial Hospital (No. 96-1381A) in 2007 and conducted at Kaohsiung Chang Gung Memorial Hospital. Acute IS was defined as sudden onset of loss of global or focal cerebral function persisting for more than 24 h. The radiological criteria for diagnosis of acute IS included a new finding of low attenuation density in focal or diffuse brain area on computed tomography of the brain; or appearance of area(s) of high intensity (bright spots) on diffusion weighted image (DWI) magnetic resonance imaging (MRI) and lower intensity on apparent diffusion coefficient (ADC) value MRI. Patients of all ages with acute IS were eligible for enrollment in the current study. Inclusion criteria included a scoring > 2 on the National Institutes of Health Stroke Scale (NIHSS) and a time window of ≤48 h from onset of symptoms to collection of blood samples (at 48 h after IS). Patients who had a history of one or more of the following were excluded from the current study: major surgery or trauma within the preceding 3 months, liver function abnormality, hematological disorders, renal insufficiency (defined as serum creatinine> 1.5 mg/dL), malignancy, febrile disorders, acute or chronic inflammatory disease at study entry, atrial fibrillation, congestive heart failure, contraindications for MRI examination, no evidence of acute IS by MRI study, pregnancy, or tPA therapy for acute IS. From October 2008 through March 2010, consecutive patients with acute IS were enrolled by the responsible neurologists at the institute. Over a period of 17 months, 157 consecutive patients who experienced acute IS occurring less than 48 h and fitted the inclusion criteria were recruited for blood sampling. Thirty age- and gender-matched healthy subjects were also studied for measuring the serum level of neopterin. Informed consent was obtained from all study subjects.
1597
Blood sampling and assessment of serum level of neopterin by ELISA Blood samples were obtained once at 48 h (acute phase) after IS for assessment of the serum level of neopterin, WBC count, and biochemical measurement. Blood samples were also obtained in control subjects who participated in a health screening program in our Health Clinic once. The blood samples were stored at −80 °C until measurement of neopterin was performed in batches at the end of recruitment. Serum neopterin concentration was assessed by duplicated determination with a commercially available ELISA kit (B & D systems, Inc., Minneapolis, MN). The lower detection limit was 0.7 nmol/L. Intraindividual variability in neopterin level was assessed in study patients and control subjects. The mean intra-assay coefficients of variance were all less than 5.0% (3.6%–6.8%). All other biochemical measurements were performed by the Clinical-Pathological Analytical Unit at our institution. Medications Aspirin was the first choice for acute IS patients unless they were allergic or intolerant to aspirin, such as having a history of peptic ulcer or upper gastro-intestinal tract bleeding during aspirin therapy. Clopidogrel was used in patients who were intolerant to aspirin treatment. Other commonly used drugs for the patients included statins, angiotensin converting enzyme inhibitors, calcium channel blocking agents, and beta blockers. Definition of extra-cranial carotid artery stenosis Carotid Doppler was routinely utilized for determining the degree of stenosis of extra-cranial carotid artery (ECCA), including common carotid artery, external and internal carotid arteries in each patient. The findings included (1) normal or mild atherosclerosis (defined as normal or obstruction of any one of ECCA b30%), (2) mild to moderate ECCA stenosis (defined as obstruction of any one of ECCA ≥30% b50%), and (3) significant ECCA stenosis (defined as obstruction of any one of ECCA ≥50%). When the number of the obstructed vessel was greater than one, only the most severely obstructed vessel was utilized for analysis in the present study. Statistical analysis
Neurological assessment Assessment of the physical function and degree of neurological impairment in the stroke patients was according to the NIHSS [22] during the acute (at 48 h) and chronic (day 90) phases of IS by neurologists. Severe neurological impairment (alive in care) was defined as a score of ≥12 on the NIHSS according to the previous reported studies [11,23]. This model has been reported to give a sensitivity of 0.76 and a specificity of 0.89 at 30-day follow-up. Therefore, the IS patients with NIHSS ≥ 12 were categorized into group 1 based on our previous study [11] and those with NIHSS b 12 (defined as having less severe neurological impairment) served as group 2.
Continuous variables with normal distribution were expressed as mean values ± SD and those with non-normal distribution were presented as median values (interquartile interval). Categorical data were analyzed by Chi-square test, whereas continuous variables were analyzed using unpaired t test and the Mann–Whitney U test where appropriate. The Spearman's test was used to assess the relation between two quantitative variables with non-normal distribution. Statistical analysis was performed using SPSS statistical software for Windows version 13 (SPSS for Windows, version 13; SPSS Inc., IL, U.S.A.). A p value of b 0.05 was considered statistically significant. Results
Imaging studies and laboratory investigations In addition to complete clinical assessment, other studies were performed including chest X-ray film, routine brain computed tomography, magnetic resonance imaging if non-contraindication, duplex scanning of the carotid arteries, and routine cardiac analysis by 12-lead electrocardiogram and echocardiography. Moreover, white blood cell (WBC) count and biochemical data were acquired on admission.
The baseline characteristics of study patients and control subjects (Table 1) Table 1 shows the baseline characteristics of the study patients and normal control subjects. The age, gender, systolic blood pressure and diastolic blood pressure upon presentation did not differ between the two groups. In addition, the serum levels of total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and creatinine were similar. Moreover, the WBC count and serum level
1598
H.-S. Lin et al. / Clinical Biochemistry 45 (2012) 1596–1601
Table 1 Baseline characteristics of ischemic stroke and normal control groups. Variables
Study patients (n = 157)
Normal control (n = 30)
Age (yrs) Male gender WBC count (×103/μL) Total cholesterol level HDL (mg/dL) LDL (mg/dL) Creatinine (mg/dL) SBP (mm Hg) DBP (mm Hg) Significant ECCA stenosis (%)b Statin therapy ACEI/ARB therapy Neopterin (nmol/L)c Recurrent stroke 90-day mortality
65.4 ± 12.4 73.9% (116) 8.1 ± 3.3 183 ± 42 45.2 ± 14.3 113 ± 31 1.1 ± 0.4 144 ± 22 82 ± 13 22.3% (35) 38.2% (60) 36.3% (57) 14.5 (9.4–19.5) 9 (5.7%) 3 (1.9%)
62.8 ± 6.1 76.7% (23) 6.5 ± 2.2 191 ± 37 53.8 ± 15.4 115 ± 34 1.0 ± 0.3 138 ± 19 85 ± 11 – – – 8.4 (6.3–9.9) – –
p Valuea 0.259 0.927 0.01 0.309 0.003 0.898 0.501 0.237 0.417
b0.001
Data are expressed as mean ± SD or % (no.) of patients. ACEI/ARB = angiotensin converting enzyme inhibitor/angiotensin II type I receptor blocker; DBP = diastolic blood pressure; ECCA = extra-cranial carotid artery; HDL = high-density lipoprotein; LDL = low-density lipoprotein; WBC = white blood cell. a By Chi-square test or Fisher's exact test for categorical data; by t-test or Mann Whitney U test for continuous data. b Defined as ECCA stenosis ≥50% regardless of anatomical locations by carotid Doppler examination. c Presented as median (interquartile range) level.
Comparison of baseline characteristics, laboratory findings and clinical outcome between patients with NIHSS ≥ 12 and patients with NIHSS b 12 at 48 h after IS (Table 3) To determine whether the serum level of neopterin was different between patients with severe neurological impairment (NIHSS ≥ 12) and those in less severe condition (NIHSS b 12), the patients were categorized into group 1 (NIHSS ≥ 12) and group 2 (NIHSS b 12). The results of Table 2 demonstrated that there were no significant differences in terms of gender, hypertension, diabetes mellitus, current smoking, systolic or diastolic blood pressure, and previous stroke by both history taking and MRI findings in group 1 and group 2. In addition, the serum levels of total cholesterol, HDL, LDL, and creatinine level were similar between group 1 and group 2. Moreover, the incidence of statin or angiotensin converting enzyme inhibitor (ACEI)/angiotensin II type I receptor blocker (ARB) therapy did not differ between group 1 and group 2. Furthermore, there was also no significant difference in the incidence of ECCA stenosis between the two groups. However, group 1 patients were older than those in group 2. Additionally, the WBC count and serum level of neopterin were substantially higher in group 1 compared with those in group 2. Besides, the incidences of NIHSS ≥ 12 and mortality by day 90 after acute IS were notably higher in group 1 than in group 2. However, the incidence of recurrent stroke at the end of 90 days after acute IS was similar between the two groups. Correlation between serum level of neopterin and extra-cranial carotid artery stenosis (Fig. 1)
of neopterin were remarkably higher in patients compared with those in normal subjects.
To assess the impact of an increased serum level of neopterin on the severity of ECCA stenosis, Mann Whitney U test was utilized for the assessment. The results showed that the serum level of neopterin
The predictors for increasing circulating level of neopterin (Table 2) Univariate and multivariate analyses were used respectively for determining the predictors of increased circulating level of neopterin. The results showed that only old age (≥ 65 years old) was the significant and independent predictor of increased level of neopterin in circulation (p b 0.04).
Table 2 A) Univariate analysis of predictors for elevation of circulating level of neopterin. B) Multiple stepwise logistic regression analysis of predictors based on the results of Table 2A. Variables
Odds ratio
95% CI
p Value
A Male vs. female With vs. without smoking With versus without DM With vs. without ACEI/ARB use With vs. without statin use Age (≧65 vs. b65 yrs) WBC count (>10,000 vs. ≦10,000)/mL Aspirin vs. clopidogrel
0.979 0.963 0.999 0.992 1.004 1.041 1.004 0.998
0.947–1.012 0.923–1.004 0.966–1.032 0.960–1.026 0.973–1.034 1.003–1.080 0.963–1.048 0.948–1.051
0.203 0.076 0.932 0.652 0.783 0.032 0.844 0.947
B Male vs. female With vs. without smoking With versus without DM With vs. without ACEI/ARB use With vs. without statin use Age (≧65 vs. b65 yrs) WBC count (>10,000 vs. ≦10,000)/mL Aspirin vs. clopidogrel
0.654 0.1874 0.634 1.190 0.564 2.323 2.141 0.635
0.295–1.446 0.835–4.204 0.305–1.318 0.563–2.515 0.274–1.159 1.168–4.620 0.823–5.568 0.282–1.431
0.294 0.128 0.223 0.648 0.119 0.016 0.118 0.273
ACEI/ARB = angiotensin converting enzyme inhibitor/angiotensin II type I receptor blocker; DM = diabetes mellitus; CI = confidence interval; WBC = white blood cell count.
Table 3 Comparison of baseline characteristics between patients with NIHSS ≥12 and patients with NIHSS b 12 at 48 h after acute IS. Variables
NIHSS ≥ 12 (n = 31)
NIHSS b 12 (n = 126)
p Valuea
Age (yrs) Male gender Hypertension Diabetes mellitus Current smoking Previous stroke by history Previous stroke by MRI WBC count (×103/mL) Total cholesterol level HDL (mg/dL) LDL (mg/dL) Creatinine (mg/dL) BMI (kg/m2) HbA1c (%) SBP (mm Hg) DBP (mm Hg) Significant ECCA stenosisb Statin therapy ACEI/ARB therapy Neopterin (nmol/L)c Day-90 NIHSS ≥ 12 Recurrent stroke 90-day mortality
70.0 ± 14.1 61.3% (19) 71.0% (22) 35.5% (11) 22.6% (7) 32.3% (10) 64.5% (20) 9.5 ± 2.6 177.9 ± 51.1 46.6 ± 18.4 116.2 ± 35.7 1.09 ± 0.5 23.7 ± 4.4 7.0 ± 2.2 142 ± 24 79 ± 14 29.0% (9) 32.3% (10) 32.3% (10) 20.1 (16.1–26.0) 58.1% (18) 3.2% (1) 6.5% (2)
64.3 ± 11.7 77.0% (97) 65.9% (83) 33.3% (42) 34.1% (43) 24.6% (31) 59.5% (75) 7.8 ± 3.3 184.4 ± 42.4 44.9 ± 13.2 115.5 ± 39.3 1.15 ± 1.0 24.1 ± 4.6 7.0 ± 1.8 144 ± 25 82 ± 13 20.6% (26) 39.7% (50) 37.3% (47) 13.6 (9.0–17.8) 4.0% (5) 6.3% (8) 0.8% (1)
0.020 0.120 0.744 0.988 0.307 0.522 0.761 0.007 0.474 0.560 0.890 0.766 0.609 0.675 0.693 0.246 0.444 0.578 0.753 0.001 b0.001 0.690 0.039
Data are expressed as mean ± SD or % (no.) of patients. ACEI/ARB = angiotensin converting enzyme inhibitor/angiotensin II type I receptor blocker; BMI = body mass index; DBP = diastolic blood pressure; ECCA = extra-cranial carotid artery; HbA1c = hemoglobin A1c; HDL = high-density lipoprotein; LDL = low-density lipoprotein; NIHSS = National institutes of Health Stroke Scale; WBC = white blood cell. a By Chi-square test or Fisher's exact test for categorical data; by t-test or Mann Whitney U test for continuous data. b Defined as ECCA stenosis ≥50% regardless of anatomical locations by carotid Doppler examination. c Presented as median (interquartile range) level.
H.-S. Lin et al. / Clinical Biochemistry 45 (2012) 1596–1601
1599
Univariate and multivariate analyses of the predictors of severe neurological impairment (NIHSS≥12) at 48 h after acute IS (Tables 4 and 5) Univariate analysis (Table 4) demonstrated a strong positive correlation between serum level of neopterin and NIHSS ≥ 12 at 48 h after acute IS. Additionally, the age and WBC were also significantly associated with NIHSS ≥ 12 at 48 h after acute IS. Of importance is that multiple stepwise logistic regression analysis revealed that serum neopterin level, age, and WBC count were significantly and independently predictive of severe neurological impairment at 48 h after acute IS (Table 5). This finding and those in Fig. 2 indicate that serum level of neopterin may be a valuable biomarker for risk stratification in patients after acute IS. Univariate and multivariate analyses of predictors for 90-day combined major adverse clinical outcome (MACO) (Tables 6 and 7)
Fig. 1. Correlation between serum level of neopterin and severity of extra-cranial carotid artery stenosis (statistical analysis by Mann Whitney U test). Group I = normal or mild atherosclerosis (defined as normal or obstruction of ECCA b30% regardless of anatomical locations); Group II = mild to moderate ECCA stenosis (defined as obstruction of ECCA ≥30% b50% regardless of anatomical locations); Group III = significant ECCA stenosis (defined as obstruction of ECCA ≥50% regardless of anatomical locations).
did not differ between patients with normal ECCA and those with non-significant ECCA stenosis. In contrast, serum neopterin level was remarkably higher in patients with significant ECCA stenosis than in those without or only with non-significant ECCA stenosis. These findings imply that increased serum level of neopterin may be a useful biomarker for predicting the severity of ECCA stenosis in CVD patients.
To evaluate whether serum neopterin level was a significant predictor of 90-day MACO (defined as 90-day NIHSS ≥ 12, recurrent stroke, or death), univariate analysis (Table 6) and further multiple stepwise logistic regression analysis (Table 7) were performed in the current study. As expected, univariate analysis identified serum neopterin level as a predictor strongly associated with 90-day MACO in patients after IS. Moreover, history of previous stroke and elevated WBC count were also significantly related to 90-day MACO in the study patients. Importantly, multiple stepwise logistic regression analysis showed that serum neopterin level was the strongest independent predictor of 90-day MACO in patients after acute IS. Furthermore, previous stroke and WBC count were also significantly and independently predictive of 90-day MACO in this patient population. This finding suggests that serum level of neopterin at 48 h after acute IS could be a useful biomarker for predicting clinical outcome in patients after acute IS. Discussion
Link between serum level of neopterin and NIHSS at 48 h after acute IS (Fig. 2) To determine the correlation between serum level of neopterin and NIHSS upon presentation (at 48 h after acute IS), Spearman's test was utilized for the analysis. As we expected, the results demonstrated a highly significant positive correlation (r = 0.345, p = 0.001) between elevation of this biomarker and NIHSS.
The present study, which investigated the correlation between serum level of neopterin and prognostic outcome of patients after acute IS, provided several striking clinical implications. First, the serum level of neopterin was markedly increased in patients after acute IS. Second, an increased serum neopterin level was strongly associated with significant ECCA stenosis. Third, an elevated neopterin serum level was significantly and independently predictive of NIHSS≥12 at 48 h after acute IS. Fourth, an important finding in the present study is that an increased serum level of neopterin at 48 h after acute IS was strongly and independently predictive of 90-day unfavorable clinical outcome. Previous studies have demonstrated that serum level of neopterin is markedly increased in patients with stable angina pectoris and ACS [6,18,20]. Surprisingly, despite the understanding that CAD and CVD share similar risk factors for developing acute vascular obstruction, an association between serum level of neopterin and acute IS has seldom been investigated [21]. One important finding in the current study is that, as compared with normal subjects, serum neopterin level was remarkably increased in patients after acute IS. A previous study on a relatively small patient population has also revealed that serum neopterin was significantly increased in acute IS patients. To the best of our knowledge, the current study is the largest cohort
Table 4 Univariate analysis of predictors for NIHSS ≥ 12 at 48 h after acute ischemic stroke.
Fig. 2. Correlation between serum neopterin level and NIHSS at 48 h after acute ischemic stroke. NIHSS = National Institutes of Health Stroke Scale.
Variables
Odds ratio
95% CI
p Value
Age (yrs) Neopterin (nmol/L) WBC count (×103/μL)
1.041 1.067 1.168
1.006–1.077 1.027–1.108 1.012–1.347
0.022 0.001 0.034
CI = confidence interval; WBC = white blood cell.
1600
H.-S. Lin et al. / Clinical Biochemistry 45 (2012) 1596–1601
Table 5 Multiple stepwise logistic regression analysis of independent predictors for NIHSS ≥ 12 at 48 h after acute ischemic stroke.
Table 7 Multiple stepwise logistic regression analysis of predictors for combined MACOa on day 90 after ischemic stroke.
Variables
Odds ratio
95% CI
p Value
Variables
Odds ratio
95% CI
p Value
Age (yrs) Neopterin (nmol/L) WBC count (×103/μL)
1.045 1.065 1.211
1.007–1.084 1.023–1.108 1.038–1.413
0.021 0.002 0.015
WBC count (×103/μL) Neopterin (nmol/L) Previous stroke by history
1.233 1.061 3.219
1.052–1.447 1.021–1.104 1.310–7.910
0.01 0.003 0.011
CI = confidence interval; WBC = white blood cell count. a MACO = combined major adverse clinical outcome (defined as recurrent stroke, 90-day NIHSS ≥12, or death).
CI = confidence interval; WBC = white blood cell.
study to address the issue of serum level of neopterin in patients after acute IS and our results support the findings of the previous study [21]. Another important finding is that elevated serum neopterin was significantly associated with the severity of ECCA obstruction. Interestingly, the link between increased serum level of neopterin and complicity of angiographic coronary artery lesions has been well reported in previous clinical observational studies [17–19]. Consistently, an association between increased circulating neopterin level and complex morphology of carotid plaque has also been documented in a previous pathological study [6]. Thus, our finding reinforces those of previous studies [6,17–19]. One principal finding in the current study is that serum neopterin level was strongly correlated with NIHSS at 48 h in patients after acute IS. Besides, elevated circulating neopterin level was significantly and independently predictive of severe neurological impairment (i.e. NIHSS ≥ 12 at 48 h after IS). To the best of our knowledge, this is the first time that circulating level of neopterin was identified as a useful biomarker for predicting severe neurological impairment in patients after an acute IS. Not only has an increased circulating level of neopterin been reported to be predictive of ACS and complex coronary artery lesions [17–19], but this biomarker has also been found to be a valuable biomarker for predicting future major adverse coronary events in patients with chronic stable angina pectoris [20]. The most important finding in the present study is that elevated circulating neopterin level was significantly and independently predictive of 90-day MACO in patients after acute IS. Surprisingly, no previous study has investigated the validity of using this biomarker as a predictor of clinical outcome after IS. The current study, therefore, helps in filling in the puzzle in the setting of acute IS. The attractive and promising findings of the present study encourage the use of this biomarker not only for risk stratification of patients into lower- and higher-risk subgroups after acute IS, but also for predicting long-term clinical outcome in patients with acute IS. Old age and a history of stroke have been well recognized as traditional risk factors for predicting unfavorable clinical outcome in patients after acute IS stroke [24–27]. The link between old age and severe neurological impairment has also been reported in previous clinical observational studies [24,25]. In the present study, we found that age was independently predictive of NIHSS ≥ 12 at 48 h after acute IS. Furthermore, previous stroke was found to be an independent predictor of MACO in the present study. Therefore, our finding strengthens those of previous studies [24–27]. The WBC count, a common index of inflammation, was found to be markedly increased in patients compared to that in normal control, Table 6 Univariate analysis of predictors for combined MACOa on day 90 after ischemic stroke. Variables 3
WBC count (×10 /μL) Neopterin (nmol/L) Previous stroke by history
Odds ratio
95% CI
p Value
1.205 1.061 2.823
1.036–1.403 1.022–1.101 1.246–6.398
0.016 0.002 0.013
CI = confidence interval; WBC =white blood cell. a MACO = combined major adverse clinical outcome (defined as recurrent stroke, 90-day NIHSS ≥ 12, or death).
and significantly increased in patients with NIHSS ≥ 12 than in those with NIHSS b 12 after acute IS. Importantly, the results of our study demonstrated that not only was an increased WBC count significantly and independently predictive of severe neurological impairment, but it was also strongly and independently predictive of 90-day MACO. An association between increased WBC count and adverse clinical outcome after acute IS has been reported previously [28–30]. Our findings, therefore, corroborate those of previous studies. This study has limitations. First, this study did not measure the serial changes in serum neopterin level after acute IS. The results of the present study, therefore, cannot provide information on the timing of the peak serum level of neopterin after acute IS. Second, only WBC count served as a common inflammatory marker to be assessed in the current study. Therefore, we did not know whether other inflammatory biomarkers, such as high-sensitivity C-reactive protein, also played an important role for the prediction of short-term and long-term prognostic outcome in patients after acute IS. In conclusion, circulating neopterin level was markedly increased after acute IS. Elevated circulating level of neopterin was a strong independent predictor of severe neurological impairment on admission and 90-day MACO in patients after acute IS. The findings of the current study encourage the use of neopterin as a reliable biomarker for risk stratification in patients after acute IS. References [1] Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med 1999;340:115-26. [2] Yip HK, Sun CK, Chang LT, Wu CJ. Strong correlation between serum levels of inflammatory mediators and their distribution in infarct coronary. Circ J 2006;70: 838-45. [3] Yip HK, Wang PW, Chang LT, Youssef AA, Sheu JJ, Lee FY, et al. Cytotoxic T lymphocyte antigen 4 gene polymorphism associated with ST-segment elevation acute myocardial infarction. Circ J 2007;71:1213-8. [4] Van der Wall AC, Becker AE, van der Loos CM, Das PK. Site of intimal rupture or erosion of thrombosed coronary atherosclerotic plaques is characterized by an inflammatory process irrespective of the dominant plaque morphology. Circulation 1994;89:36-44. [5] Yip HK, Wu CJ, Yang CH, Chang HW, Fang CY, Hung WC, et al. Serial changes in circulating concentrations of soluble CD40 ligand and C-reactive protein in patients with unstable angina undergoing coronary stenting: role of inflammatory mediators in predicting late restenosis. Circ J 2005;69:890-5. [6] Sugioka K, Naruko T, Hozumi T, Nakagawa M, Kitabayashi C, Ikura Y, et al. Elevated levels of neopterin are associated with carotid plaques with complex morphology in patients with stable angina pectoris. Atherosclerosis 2010;280:524-30. [7] Goldstein JA, Demetriou D, Grines CL, Pica M, Shoukfeh M, O'Neill WW. Multiple complex coronary plaques in patients with acute myocardial infarction. N Engl J Med 2000;343:915-22. [8] Rioufol G, Finet G, Ginon I, Andre-Fouet X, Rossi R, Vialle E, et al. Multiple atherosclerotic plaque rupture in acute coronary syndrome: a three-vessel intravascular ultrasound study. Circulation 2002;106:804-8. [9] Golledge J, Greenhalg RM, Davies AH. The symptomatic carotid plaque. Stroke 2000;31:774-81. [10] Jander S, Sitzer M, Schumann R, Schroeter M, Siebler M, Steinmetz H, et al. Inflammation in high-grade carotid stenosis: a possible role for macrophages and T cells in plaque destabilization. Stroke 1998;29:1625-30. [11] Yip HK, Chang LT, Chang WN, Lu CH, Liou CW, Lan MY, et al. Level and value of circulating endothelial progenitor cells in patients after acute ischemic stroke. Stroke 2008;39:69-74. [12] Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest 1994;94:2493-503. [13] Dollery CM, McEwan JR, Henney A. Matrix metalloproteinases and cardiovascular disease. Circ Res 1995;77:863-8.
H.-S. Lin et al. / Clinical Biochemistry 45 (2012) 1596–1601 [14] Neri Serneri GG, Abbate R, Gori AM, Attanasio M, Martini F, Giusti B, et al. Transient intermittent lymphocyte activation is responsible for the instability of angina. Circulation 1992;86:790-7. [15] Werner-Felmayer G, Werner ER, Fuchs D, Hausen A, Reibnegger G, Schmidt K, et al. Pteridine biosynthesis in human endothelial cells. Impact on nitric oxide-mediated formation of cyclic GMP. J Biol Chem 1993;268:1842-6. [16] Huber C, Batchelor JR, Fuchs D, Hausen A, Lang A, Niederwieser D, et al. Immune response‐associated production of neopterin: release from macrophages primarily under control of interferon-gamma. J Exp Med 1984;160:310-6. [17] Gupta S, Fredericks S, Schwartzman RA, Holt DW, Kaski JC. Serum neopterin in acute coronary syndromes (letter). Lancet 1997;349:1252-3. [18] Schumacher M, Halwachs G, Tatzber F, Fruhwald FM, Zweiker R, Watzinger N, et al. Increased neopterin in patients with chronic and acute coronary syndromes. J Am Coll Cardiol 1997;30:703-7. [19] Garcia-Moll X, Coccolo F, Cole D, Kaski JC. Serum neopterin and complex stenosis morphology in patients with unstable angina. J Am Coll Cardiol 2000;35:956-62. [20] Avanzas P, Arroyo-Espliguero R, Quiles J, Roy D, Kaski JC. Elevated serum neopterin predicts future adverse cardiac events in patients with chronic stable angina pectoris. Eur Heart J 2005;26:457-63. [21] Grau AJ, Reis A, Buggle F, Al-Khalaf A, Werle E, Valois N, et al. Monocyte function and plasma levels of interleukin-8 in acute ischemic stroke. J Neurol Sci 2001;192: 41-7. [22] Goldstein LB, Bertels C, Davis JN. Interrater reliability of the NIH stroke scale. Arch Neurol 1989;46:660-2.
1601
[23] Muir KW, Weir CJ, Murray GD, Povey C, Lees KR. Comparison of neurological scales and scoring systems for acute stroke prognosis. Stroke 1996;27:1817-20. [24] Howard G, Walker MD, Becker C, Coull B, Feibel J, McLeroy K, et al. Community hospital-based stroke programs: North Carolina, Oregon, and New York, III: factors influencing survival after stroke: proportional hazards analysis of 4219 patients. Stroke 1986;17:294-9. [25] Chambers BR, Norris JW, Shurvell BL, Hachinski VC. Prognosis of acute stroke. Neurology 1987;37:221-5. [26] Petty GW, Brown RD, Whisnant JP, Sicks JD, O'Fallon WM, Wiebers DO. Survival and recurrence after first cerebral infarction: a population-based study in Rochester, Minnesota, 1975 through 1989. Neurology 1998;50:208-16. [27] Hénon H, Godefroy O, Leys D, Mounier-Vehier F, Lucas C, Rondepierre P, et al. Early predictors of death and disability after acute cerebral ischemic event. Stroke 1995;26:392-8. [28] Czlonkowska A, Ryglewicz D, Lechowicz W. Basic analytical parameters as predictive factors for 30-day case fatality rate in stroke. Acta Neurol Scand 1997;95:121-4. [29] Kazmierski R, Guzik P, Ambrosius W, Ciesielska A, Moskal J, Kozubski W. Predictive value of white blood cell count on admission for in-hospital mortality in acute stroke patients. Clin Neurol Neurosurg 2004;107:38-43. [30] Tuttolomondo A, Pedone C, Pinto A, Di Raimondo D, Fernandez P, Di Sciacca R, et al. Gruppo Italiano di Farmacoepidemiologia dell'Anziano (GIFA) researchers: predictors of outcome in acute ischemic cerebrovascular syndromes: the GIFA study. Int J Cardiol 2008;125:391-6.
Contents lists available at SciVerse ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Serum level and prognostic value of neopterin in patients after ischemic stroke Hung-Sheng Lin a, Tzu-Hsien Tsai b, d, 1, Chu-Feng Liu c, Cheng-Hsien Lu a, Wen-Neng Chang a, Shu-Feng Chen a, Chi-Wei Huang a, Chi-Ren Huang a, Nai-Wen Tsai a, Chih-Cheng Huang a, Chia-Wei Liou a, Tsu-Kung Lin a, Min-Yu Lan a, Hon-Kan Yip b,⁎ a
Department of Neurology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan Division of Cardiology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan Department of Emergency Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan d Graduate Institute of Clinical Medical Sciences, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan b c
a r t i c l e
i n f o
Article history: Received 10 August 2011 Received in revised form 24 July 2012 Accepted 31 July 2012 Available online 8 August 2012 Keywords: Ischemic stroke Neopterin
a b s t r a c t Background: We hypothesized that serum level of neopterin is significantly predictive of prognostic outcome in patients after acute ischemic stroke (IS). Methods: Between November 2008 and May 2010, serum levels of neopterin were prospectively collected at 48 h after acute IS in 157 patients. Results: Serum neopterin levels were substantially higher in patients with severe neurological impairment [National institutes of Health Stroke Scale (NIHSS) score ≥12] than in those with NIHSS b 12 (pb 0.008). Furthermore, Spearman's test showed a strongly positive correlation between neopterin level and NIHSS (p=0.003). Multiple logistic regression analysis demonstrated that serum neopterin level was strongly and independently predictive of NIHSS ≥12 (p=0.002) at 48 h after acute IS and 90-day major adverse clinical outcome (defined as NIHSS≥12, recurrent stroke or death) (p=0.003). Conclusion: Serum level of neopterin was notably increased after acute IS. This biomarker was strongly and independently predictive of 90-day unfavorable clinical outcome in patients after acute IS. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Abundant data support the proposal that inflammatory processes and immune responses are pivotal in the pathogenesis of atherosclerotic plaque [1–5]. Histopathological findings have further shown that complex interaction and activation of monocytes, macrophages, and lymphocytes within the plaque play a key role in the inflammatory process associated with atherosclerosis and plaque vulnerability [1,2,4,6]. This complex interaction finally causes atheroembolic plaque disruption and acute coronary syndrome (ACS) [1,2,4,6–8]. However, atherosclerotic plaque instability and disruption may not be confined only in epicardial coronary arteries but may also involve other arterial systems, including carotid arteries and cerebral arteries [6,9,10]. Thus, coronary artery disease (CAD) and cerebral vascular disease (CVD) are two sides of the same coin [11]. Besides, the activated inflammatory cells within atherosclerotic plaques are capable of producing a range of proteases that lead to
⁎ Corresponding author at: Division of Cardiology, Department of Internal Medicine, Chang Gung Memorial Hospital, Kaohsiung 123, Ta Pei Road, Niao Sung Hsiang, Kaohsiung City 83301, Taiwan. Fax: +886 7 7322402. E-mail address: [email protected] (H.-K. Yip). 1 Indicates equal contribution in this study compared with the first author.
proteolytic destruction of the extra-cellular matrix [12,13] and production of interferon-γ, both of which subsequently activate macrophages within the plaque and interfere with matrix collagen synthesis within [14]. These enzyme and cytokine productions, which contribute to a persistent inflammatory reaction within the atherosclerotic plaque, may also play a crucial role in the disruption of the unstable plaque [6,12–14]. Neopterin, a pteridine derivative and a side-product of the guanosine triphosphate–biopterin pathway [15] secreted by activated macrophages after stimulation by IFN-γ [16], has been suggested to be a biomarker of immune activation and macrophage activity [16]. Additionally, neopterin has been found to participate in enhancing inflammatory processes within vulnerable plaques [15,16]. Besides, neopterin has been reported to be increased in the serum of patients with ACS and associated with coronary artery complex lesions [17–19]. Increased serum level of neopterin, therefore, has been suggested to be a biomarker of acute widespread atherothrombotic plaque inflammation and CAD activity [17–19]. Surprisingly, while an association of elevated serum neopterin level with ACS and coronary artery complex lesion has been extensively investigated [17–20], the correlation of serum neopterin level with carotid artery disease [6,10] and acute ischemic stroke (IS) has seldom been reported [21]. Therefore, the potential of serum neopterin level as a clinical biomarker for predicting prognostic outcome after acute IS remains unclear. Accordingly, this study assessed the serum neopterin
0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2012.07.113
H.-S. Lin et al. / Clinical Biochemistry 45 (2012) 1596–1601
level in patients after acute IS to evaluate its clinical value as a biomarker for predicting 90-day unfavorable clinical outcome following the cerebrovascular episode.
Materials and methods Definition, exclusion criteria, and patient enrollment This study was approved by the Institutional Review Committee on Human Research of Chang Gung Memorial Hospital (No. 96-1381A) in 2007 and conducted at Kaohsiung Chang Gung Memorial Hospital. Acute IS was defined as sudden onset of loss of global or focal cerebral function persisting for more than 24 h. The radiological criteria for diagnosis of acute IS included a new finding of low attenuation density in focal or diffuse brain area on computed tomography of the brain; or appearance of area(s) of high intensity (bright spots) on diffusion weighted image (DWI) magnetic resonance imaging (MRI) and lower intensity on apparent diffusion coefficient (ADC) value MRI. Patients of all ages with acute IS were eligible for enrollment in the current study. Inclusion criteria included a scoring > 2 on the National Institutes of Health Stroke Scale (NIHSS) and a time window of ≤48 h from onset of symptoms to collection of blood samples (at 48 h after IS). Patients who had a history of one or more of the following were excluded from the current study: major surgery or trauma within the preceding 3 months, liver function abnormality, hematological disorders, renal insufficiency (defined as serum creatinine> 1.5 mg/dL), malignancy, febrile disorders, acute or chronic inflammatory disease at study entry, atrial fibrillation, congestive heart failure, contraindications for MRI examination, no evidence of acute IS by MRI study, pregnancy, or tPA therapy for acute IS. From October 2008 through March 2010, consecutive patients with acute IS were enrolled by the responsible neurologists at the institute. Over a period of 17 months, 157 consecutive patients who experienced acute IS occurring less than 48 h and fitted the inclusion criteria were recruited for blood sampling. Thirty age- and gender-matched healthy subjects were also studied for measuring the serum level of neopterin. Informed consent was obtained from all study subjects.
1597
Blood sampling and assessment of serum level of neopterin by ELISA Blood samples were obtained once at 48 h (acute phase) after IS for assessment of the serum level of neopterin, WBC count, and biochemical measurement. Blood samples were also obtained in control subjects who participated in a health screening program in our Health Clinic once. The blood samples were stored at −80 °C until measurement of neopterin was performed in batches at the end of recruitment. Serum neopterin concentration was assessed by duplicated determination with a commercially available ELISA kit (B & D systems, Inc., Minneapolis, MN). The lower detection limit was 0.7 nmol/L. Intraindividual variability in neopterin level was assessed in study patients and control subjects. The mean intra-assay coefficients of variance were all less than 5.0% (3.6%–6.8%). All other biochemical measurements were performed by the Clinical-Pathological Analytical Unit at our institution. Medications Aspirin was the first choice for acute IS patients unless they were allergic or intolerant to aspirin, such as having a history of peptic ulcer or upper gastro-intestinal tract bleeding during aspirin therapy. Clopidogrel was used in patients who were intolerant to aspirin treatment. Other commonly used drugs for the patients included statins, angiotensin converting enzyme inhibitors, calcium channel blocking agents, and beta blockers. Definition of extra-cranial carotid artery stenosis Carotid Doppler was routinely utilized for determining the degree of stenosis of extra-cranial carotid artery (ECCA), including common carotid artery, external and internal carotid arteries in each patient. The findings included (1) normal or mild atherosclerosis (defined as normal or obstruction of any one of ECCA b30%), (2) mild to moderate ECCA stenosis (defined as obstruction of any one of ECCA ≥30% b50%), and (3) significant ECCA stenosis (defined as obstruction of any one of ECCA ≥50%). When the number of the obstructed vessel was greater than one, only the most severely obstructed vessel was utilized for analysis in the present study. Statistical analysis
Neurological assessment Assessment of the physical function and degree of neurological impairment in the stroke patients was according to the NIHSS [22] during the acute (at 48 h) and chronic (day 90) phases of IS by neurologists. Severe neurological impairment (alive in care) was defined as a score of ≥12 on the NIHSS according to the previous reported studies [11,23]. This model has been reported to give a sensitivity of 0.76 and a specificity of 0.89 at 30-day follow-up. Therefore, the IS patients with NIHSS ≥ 12 were categorized into group 1 based on our previous study [11] and those with NIHSS b 12 (defined as having less severe neurological impairment) served as group 2.
Continuous variables with normal distribution were expressed as mean values ± SD and those with non-normal distribution were presented as median values (interquartile interval). Categorical data were analyzed by Chi-square test, whereas continuous variables were analyzed using unpaired t test and the Mann–Whitney U test where appropriate. The Spearman's test was used to assess the relation between two quantitative variables with non-normal distribution. Statistical analysis was performed using SPSS statistical software for Windows version 13 (SPSS for Windows, version 13; SPSS Inc., IL, U.S.A.). A p value of b 0.05 was considered statistically significant. Results
Imaging studies and laboratory investigations In addition to complete clinical assessment, other studies were performed including chest X-ray film, routine brain computed tomography, magnetic resonance imaging if non-contraindication, duplex scanning of the carotid arteries, and routine cardiac analysis by 12-lead electrocardiogram and echocardiography. Moreover, white blood cell (WBC) count and biochemical data were acquired on admission.
The baseline characteristics of study patients and control subjects (Table 1) Table 1 shows the baseline characteristics of the study patients and normal control subjects. The age, gender, systolic blood pressure and diastolic blood pressure upon presentation did not differ between the two groups. In addition, the serum levels of total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and creatinine were similar. Moreover, the WBC count and serum level
1598
H.-S. Lin et al. / Clinical Biochemistry 45 (2012) 1596–1601
Table 1 Baseline characteristics of ischemic stroke and normal control groups. Variables
Study patients (n = 157)
Normal control (n = 30)
Age (yrs) Male gender WBC count (×103/μL) Total cholesterol level HDL (mg/dL) LDL (mg/dL) Creatinine (mg/dL) SBP (mm Hg) DBP (mm Hg) Significant ECCA stenosis (%)b Statin therapy ACEI/ARB therapy Neopterin (nmol/L)c Recurrent stroke 90-day mortality
65.4 ± 12.4 73.9% (116) 8.1 ± 3.3 183 ± 42 45.2 ± 14.3 113 ± 31 1.1 ± 0.4 144 ± 22 82 ± 13 22.3% (35) 38.2% (60) 36.3% (57) 14.5 (9.4–19.5) 9 (5.7%) 3 (1.9%)
62.8 ± 6.1 76.7% (23) 6.5 ± 2.2 191 ± 37 53.8 ± 15.4 115 ± 34 1.0 ± 0.3 138 ± 19 85 ± 11 – – – 8.4 (6.3–9.9) – –
p Valuea 0.259 0.927 0.01 0.309 0.003 0.898 0.501 0.237 0.417
b0.001
Data are expressed as mean ± SD or % (no.) of patients. ACEI/ARB = angiotensin converting enzyme inhibitor/angiotensin II type I receptor blocker; DBP = diastolic blood pressure; ECCA = extra-cranial carotid artery; HDL = high-density lipoprotein; LDL = low-density lipoprotein; WBC = white blood cell. a By Chi-square test or Fisher's exact test for categorical data; by t-test or Mann Whitney U test for continuous data. b Defined as ECCA stenosis ≥50% regardless of anatomical locations by carotid Doppler examination. c Presented as median (interquartile range) level.
Comparison of baseline characteristics, laboratory findings and clinical outcome between patients with NIHSS ≥ 12 and patients with NIHSS b 12 at 48 h after IS (Table 3) To determine whether the serum level of neopterin was different between patients with severe neurological impairment (NIHSS ≥ 12) and those in less severe condition (NIHSS b 12), the patients were categorized into group 1 (NIHSS ≥ 12) and group 2 (NIHSS b 12). The results of Table 2 demonstrated that there were no significant differences in terms of gender, hypertension, diabetes mellitus, current smoking, systolic or diastolic blood pressure, and previous stroke by both history taking and MRI findings in group 1 and group 2. In addition, the serum levels of total cholesterol, HDL, LDL, and creatinine level were similar between group 1 and group 2. Moreover, the incidence of statin or angiotensin converting enzyme inhibitor (ACEI)/angiotensin II type I receptor blocker (ARB) therapy did not differ between group 1 and group 2. Furthermore, there was also no significant difference in the incidence of ECCA stenosis between the two groups. However, group 1 patients were older than those in group 2. Additionally, the WBC count and serum level of neopterin were substantially higher in group 1 compared with those in group 2. Besides, the incidences of NIHSS ≥ 12 and mortality by day 90 after acute IS were notably higher in group 1 than in group 2. However, the incidence of recurrent stroke at the end of 90 days after acute IS was similar between the two groups. Correlation between serum level of neopterin and extra-cranial carotid artery stenosis (Fig. 1)
of neopterin were remarkably higher in patients compared with those in normal subjects.
To assess the impact of an increased serum level of neopterin on the severity of ECCA stenosis, Mann Whitney U test was utilized for the assessment. The results showed that the serum level of neopterin
The predictors for increasing circulating level of neopterin (Table 2) Univariate and multivariate analyses were used respectively for determining the predictors of increased circulating level of neopterin. The results showed that only old age (≥ 65 years old) was the significant and independent predictor of increased level of neopterin in circulation (p b 0.04).
Table 2 A) Univariate analysis of predictors for elevation of circulating level of neopterin. B) Multiple stepwise logistic regression analysis of predictors based on the results of Table 2A. Variables
Odds ratio
95% CI
p Value
A Male vs. female With vs. without smoking With versus without DM With vs. without ACEI/ARB use With vs. without statin use Age (≧65 vs. b65 yrs) WBC count (>10,000 vs. ≦10,000)/mL Aspirin vs. clopidogrel
0.979 0.963 0.999 0.992 1.004 1.041 1.004 0.998
0.947–1.012 0.923–1.004 0.966–1.032 0.960–1.026 0.973–1.034 1.003–1.080 0.963–1.048 0.948–1.051
0.203 0.076 0.932 0.652 0.783 0.032 0.844 0.947
B Male vs. female With vs. without smoking With versus without DM With vs. without ACEI/ARB use With vs. without statin use Age (≧65 vs. b65 yrs) WBC count (>10,000 vs. ≦10,000)/mL Aspirin vs. clopidogrel
0.654 0.1874 0.634 1.190 0.564 2.323 2.141 0.635
0.295–1.446 0.835–4.204 0.305–1.318 0.563–2.515 0.274–1.159 1.168–4.620 0.823–5.568 0.282–1.431
0.294 0.128 0.223 0.648 0.119 0.016 0.118 0.273
ACEI/ARB = angiotensin converting enzyme inhibitor/angiotensin II type I receptor blocker; DM = diabetes mellitus; CI = confidence interval; WBC = white blood cell count.
Table 3 Comparison of baseline characteristics between patients with NIHSS ≥12 and patients with NIHSS b 12 at 48 h after acute IS. Variables
NIHSS ≥ 12 (n = 31)
NIHSS b 12 (n = 126)
p Valuea
Age (yrs) Male gender Hypertension Diabetes mellitus Current smoking Previous stroke by history Previous stroke by MRI WBC count (×103/mL) Total cholesterol level HDL (mg/dL) LDL (mg/dL) Creatinine (mg/dL) BMI (kg/m2) HbA1c (%) SBP (mm Hg) DBP (mm Hg) Significant ECCA stenosisb Statin therapy ACEI/ARB therapy Neopterin (nmol/L)c Day-90 NIHSS ≥ 12 Recurrent stroke 90-day mortality
70.0 ± 14.1 61.3% (19) 71.0% (22) 35.5% (11) 22.6% (7) 32.3% (10) 64.5% (20) 9.5 ± 2.6 177.9 ± 51.1 46.6 ± 18.4 116.2 ± 35.7 1.09 ± 0.5 23.7 ± 4.4 7.0 ± 2.2 142 ± 24 79 ± 14 29.0% (9) 32.3% (10) 32.3% (10) 20.1 (16.1–26.0) 58.1% (18) 3.2% (1) 6.5% (2)
64.3 ± 11.7 77.0% (97) 65.9% (83) 33.3% (42) 34.1% (43) 24.6% (31) 59.5% (75) 7.8 ± 3.3 184.4 ± 42.4 44.9 ± 13.2 115.5 ± 39.3 1.15 ± 1.0 24.1 ± 4.6 7.0 ± 1.8 144 ± 25 82 ± 13 20.6% (26) 39.7% (50) 37.3% (47) 13.6 (9.0–17.8) 4.0% (5) 6.3% (8) 0.8% (1)
0.020 0.120 0.744 0.988 0.307 0.522 0.761 0.007 0.474 0.560 0.890 0.766 0.609 0.675 0.693 0.246 0.444 0.578 0.753 0.001 b0.001 0.690 0.039
Data are expressed as mean ± SD or % (no.) of patients. ACEI/ARB = angiotensin converting enzyme inhibitor/angiotensin II type I receptor blocker; BMI = body mass index; DBP = diastolic blood pressure; ECCA = extra-cranial carotid artery; HbA1c = hemoglobin A1c; HDL = high-density lipoprotein; LDL = low-density lipoprotein; NIHSS = National institutes of Health Stroke Scale; WBC = white blood cell. a By Chi-square test or Fisher's exact test for categorical data; by t-test or Mann Whitney U test for continuous data. b Defined as ECCA stenosis ≥50% regardless of anatomical locations by carotid Doppler examination. c Presented as median (interquartile range) level.
H.-S. Lin et al. / Clinical Biochemistry 45 (2012) 1596–1601
1599
Univariate and multivariate analyses of the predictors of severe neurological impairment (NIHSS≥12) at 48 h after acute IS (Tables 4 and 5) Univariate analysis (Table 4) demonstrated a strong positive correlation between serum level of neopterin and NIHSS ≥ 12 at 48 h after acute IS. Additionally, the age and WBC were also significantly associated with NIHSS ≥ 12 at 48 h after acute IS. Of importance is that multiple stepwise logistic regression analysis revealed that serum neopterin level, age, and WBC count were significantly and independently predictive of severe neurological impairment at 48 h after acute IS (Table 5). This finding and those in Fig. 2 indicate that serum level of neopterin may be a valuable biomarker for risk stratification in patients after acute IS. Univariate and multivariate analyses of predictors for 90-day combined major adverse clinical outcome (MACO) (Tables 6 and 7)
Fig. 1. Correlation between serum level of neopterin and severity of extra-cranial carotid artery stenosis (statistical analysis by Mann Whitney U test). Group I = normal or mild atherosclerosis (defined as normal or obstruction of ECCA b30% regardless of anatomical locations); Group II = mild to moderate ECCA stenosis (defined as obstruction of ECCA ≥30% b50% regardless of anatomical locations); Group III = significant ECCA stenosis (defined as obstruction of ECCA ≥50% regardless of anatomical locations).
did not differ between patients with normal ECCA and those with non-significant ECCA stenosis. In contrast, serum neopterin level was remarkably higher in patients with significant ECCA stenosis than in those without or only with non-significant ECCA stenosis. These findings imply that increased serum level of neopterin may be a useful biomarker for predicting the severity of ECCA stenosis in CVD patients.
To evaluate whether serum neopterin level was a significant predictor of 90-day MACO (defined as 90-day NIHSS ≥ 12, recurrent stroke, or death), univariate analysis (Table 6) and further multiple stepwise logistic regression analysis (Table 7) were performed in the current study. As expected, univariate analysis identified serum neopterin level as a predictor strongly associated with 90-day MACO in patients after IS. Moreover, history of previous stroke and elevated WBC count were also significantly related to 90-day MACO in the study patients. Importantly, multiple stepwise logistic regression analysis showed that serum neopterin level was the strongest independent predictor of 90-day MACO in patients after acute IS. Furthermore, previous stroke and WBC count were also significantly and independently predictive of 90-day MACO in this patient population. This finding suggests that serum level of neopterin at 48 h after acute IS could be a useful biomarker for predicting clinical outcome in patients after acute IS. Discussion
Link between serum level of neopterin and NIHSS at 48 h after acute IS (Fig. 2) To determine the correlation between serum level of neopterin and NIHSS upon presentation (at 48 h after acute IS), Spearman's test was utilized for the analysis. As we expected, the results demonstrated a highly significant positive correlation (r = 0.345, p = 0.001) between elevation of this biomarker and NIHSS.
The present study, which investigated the correlation between serum level of neopterin and prognostic outcome of patients after acute IS, provided several striking clinical implications. First, the serum level of neopterin was markedly increased in patients after acute IS. Second, an increased serum neopterin level was strongly associated with significant ECCA stenosis. Third, an elevated neopterin serum level was significantly and independently predictive of NIHSS≥12 at 48 h after acute IS. Fourth, an important finding in the present study is that an increased serum level of neopterin at 48 h after acute IS was strongly and independently predictive of 90-day unfavorable clinical outcome. Previous studies have demonstrated that serum level of neopterin is markedly increased in patients with stable angina pectoris and ACS [6,18,20]. Surprisingly, despite the understanding that CAD and CVD share similar risk factors for developing acute vascular obstruction, an association between serum level of neopterin and acute IS has seldom been investigated [21]. One important finding in the current study is that, as compared with normal subjects, serum neopterin level was remarkably increased in patients after acute IS. A previous study on a relatively small patient population has also revealed that serum neopterin was significantly increased in acute IS patients. To the best of our knowledge, the current study is the largest cohort
Table 4 Univariate analysis of predictors for NIHSS ≥ 12 at 48 h after acute ischemic stroke.
Fig. 2. Correlation between serum neopterin level and NIHSS at 48 h after acute ischemic stroke. NIHSS = National Institutes of Health Stroke Scale.
Variables
Odds ratio
95% CI
p Value
Age (yrs) Neopterin (nmol/L) WBC count (×103/μL)
1.041 1.067 1.168
1.006–1.077 1.027–1.108 1.012–1.347
0.022 0.001 0.034
CI = confidence interval; WBC = white blood cell.
1600
H.-S. Lin et al. / Clinical Biochemistry 45 (2012) 1596–1601
Table 5 Multiple stepwise logistic regression analysis of independent predictors for NIHSS ≥ 12 at 48 h after acute ischemic stroke.
Table 7 Multiple stepwise logistic regression analysis of predictors for combined MACOa on day 90 after ischemic stroke.
Variables
Odds ratio
95% CI
p Value
Variables
Odds ratio
95% CI
p Value
Age (yrs) Neopterin (nmol/L) WBC count (×103/μL)
1.045 1.065 1.211
1.007–1.084 1.023–1.108 1.038–1.413
0.021 0.002 0.015
WBC count (×103/μL) Neopterin (nmol/L) Previous stroke by history
1.233 1.061 3.219
1.052–1.447 1.021–1.104 1.310–7.910
0.01 0.003 0.011
CI = confidence interval; WBC = white blood cell count. a MACO = combined major adverse clinical outcome (defined as recurrent stroke, 90-day NIHSS ≥12, or death).
CI = confidence interval; WBC = white blood cell.
study to address the issue of serum level of neopterin in patients after acute IS and our results support the findings of the previous study [21]. Another important finding is that elevated serum neopterin was significantly associated with the severity of ECCA obstruction. Interestingly, the link between increased serum level of neopterin and complicity of angiographic coronary artery lesions has been well reported in previous clinical observational studies [17–19]. Consistently, an association between increased circulating neopterin level and complex morphology of carotid plaque has also been documented in a previous pathological study [6]. Thus, our finding reinforces those of previous studies [6,17–19]. One principal finding in the current study is that serum neopterin level was strongly correlated with NIHSS at 48 h in patients after acute IS. Besides, elevated circulating neopterin level was significantly and independently predictive of severe neurological impairment (i.e. NIHSS ≥ 12 at 48 h after IS). To the best of our knowledge, this is the first time that circulating level of neopterin was identified as a useful biomarker for predicting severe neurological impairment in patients after an acute IS. Not only has an increased circulating level of neopterin been reported to be predictive of ACS and complex coronary artery lesions [17–19], but this biomarker has also been found to be a valuable biomarker for predicting future major adverse coronary events in patients with chronic stable angina pectoris [20]. The most important finding in the present study is that elevated circulating neopterin level was significantly and independently predictive of 90-day MACO in patients after acute IS. Surprisingly, no previous study has investigated the validity of using this biomarker as a predictor of clinical outcome after IS. The current study, therefore, helps in filling in the puzzle in the setting of acute IS. The attractive and promising findings of the present study encourage the use of this biomarker not only for risk stratification of patients into lower- and higher-risk subgroups after acute IS, but also for predicting long-term clinical outcome in patients with acute IS. Old age and a history of stroke have been well recognized as traditional risk factors for predicting unfavorable clinical outcome in patients after acute IS stroke [24–27]. The link between old age and severe neurological impairment has also been reported in previous clinical observational studies [24,25]. In the present study, we found that age was independently predictive of NIHSS ≥ 12 at 48 h after acute IS. Furthermore, previous stroke was found to be an independent predictor of MACO in the present study. Therefore, our finding strengthens those of previous studies [24–27]. The WBC count, a common index of inflammation, was found to be markedly increased in patients compared to that in normal control, Table 6 Univariate analysis of predictors for combined MACOa on day 90 after ischemic stroke. Variables 3
WBC count (×10 /μL) Neopterin (nmol/L) Previous stroke by history
Odds ratio
95% CI
p Value
1.205 1.061 2.823
1.036–1.403 1.022–1.101 1.246–6.398
0.016 0.002 0.013
CI = confidence interval; WBC =white blood cell. a MACO = combined major adverse clinical outcome (defined as recurrent stroke, 90-day NIHSS ≥ 12, or death).
and significantly increased in patients with NIHSS ≥ 12 than in those with NIHSS b 12 after acute IS. Importantly, the results of our study demonstrated that not only was an increased WBC count significantly and independently predictive of severe neurological impairment, but it was also strongly and independently predictive of 90-day MACO. An association between increased WBC count and adverse clinical outcome after acute IS has been reported previously [28–30]. Our findings, therefore, corroborate those of previous studies. This study has limitations. First, this study did not measure the serial changes in serum neopterin level after acute IS. The results of the present study, therefore, cannot provide information on the timing of the peak serum level of neopterin after acute IS. Second, only WBC count served as a common inflammatory marker to be assessed in the current study. Therefore, we did not know whether other inflammatory biomarkers, such as high-sensitivity C-reactive protein, also played an important role for the prediction of short-term and long-term prognostic outcome in patients after acute IS. In conclusion, circulating neopterin level was markedly increased after acute IS. Elevated circulating level of neopterin was a strong independent predictor of severe neurological impairment on admission and 90-day MACO in patients after acute IS. The findings of the current study encourage the use of neopterin as a reliable biomarker for risk stratification in patients after acute IS. References [1] Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med 1999;340:115-26. [2] Yip HK, Sun CK, Chang LT, Wu CJ. Strong correlation between serum levels of inflammatory mediators and their distribution in infarct coronary. Circ J 2006;70: 838-45. [3] Yip HK, Wang PW, Chang LT, Youssef AA, Sheu JJ, Lee FY, et al. Cytotoxic T lymphocyte antigen 4 gene polymorphism associated with ST-segment elevation acute myocardial infarction. Circ J 2007;71:1213-8. [4] Van der Wall AC, Becker AE, van der Loos CM, Das PK. Site of intimal rupture or erosion of thrombosed coronary atherosclerotic plaques is characterized by an inflammatory process irrespective of the dominant plaque morphology. Circulation 1994;89:36-44. [5] Yip HK, Wu CJ, Yang CH, Chang HW, Fang CY, Hung WC, et al. Serial changes in circulating concentrations of soluble CD40 ligand and C-reactive protein in patients with unstable angina undergoing coronary stenting: role of inflammatory mediators in predicting late restenosis. Circ J 2005;69:890-5. [6] Sugioka K, Naruko T, Hozumi T, Nakagawa M, Kitabayashi C, Ikura Y, et al. Elevated levels of neopterin are associated with carotid plaques with complex morphology in patients with stable angina pectoris. Atherosclerosis 2010;280:524-30. [7] Goldstein JA, Demetriou D, Grines CL, Pica M, Shoukfeh M, O'Neill WW. Multiple complex coronary plaques in patients with acute myocardial infarction. N Engl J Med 2000;343:915-22. [8] Rioufol G, Finet G, Ginon I, Andre-Fouet X, Rossi R, Vialle E, et al. Multiple atherosclerotic plaque rupture in acute coronary syndrome: a three-vessel intravascular ultrasound study. Circulation 2002;106:804-8. [9] Golledge J, Greenhalg RM, Davies AH. The symptomatic carotid plaque. Stroke 2000;31:774-81. [10] Jander S, Sitzer M, Schumann R, Schroeter M, Siebler M, Steinmetz H, et al. Inflammation in high-grade carotid stenosis: a possible role for macrophages and T cells in plaque destabilization. Stroke 1998;29:1625-30. [11] Yip HK, Chang LT, Chang WN, Lu CH, Liou CW, Lan MY, et al. Level and value of circulating endothelial progenitor cells in patients after acute ischemic stroke. Stroke 2008;39:69-74. [12] Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest 1994;94:2493-503. [13] Dollery CM, McEwan JR, Henney A. Matrix metalloproteinases and cardiovascular disease. Circ Res 1995;77:863-8.
H.-S. Lin et al. / Clinical Biochemistry 45 (2012) 1596–1601 [14] Neri Serneri GG, Abbate R, Gori AM, Attanasio M, Martini F, Giusti B, et al. Transient intermittent lymphocyte activation is responsible for the instability of angina. Circulation 1992;86:790-7. [15] Werner-Felmayer G, Werner ER, Fuchs D, Hausen A, Reibnegger G, Schmidt K, et al. Pteridine biosynthesis in human endothelial cells. Impact on nitric oxide-mediated formation of cyclic GMP. J Biol Chem 1993;268:1842-6. [16] Huber C, Batchelor JR, Fuchs D, Hausen A, Lang A, Niederwieser D, et al. Immune response‐associated production of neopterin: release from macrophages primarily under control of interferon-gamma. J Exp Med 1984;160:310-6. [17] Gupta S, Fredericks S, Schwartzman RA, Holt DW, Kaski JC. Serum neopterin in acute coronary syndromes (letter). Lancet 1997;349:1252-3. [18] Schumacher M, Halwachs G, Tatzber F, Fruhwald FM, Zweiker R, Watzinger N, et al. Increased neopterin in patients with chronic and acute coronary syndromes. J Am Coll Cardiol 1997;30:703-7. [19] Garcia-Moll X, Coccolo F, Cole D, Kaski JC. Serum neopterin and complex stenosis morphology in patients with unstable angina. J Am Coll Cardiol 2000;35:956-62. [20] Avanzas P, Arroyo-Espliguero R, Quiles J, Roy D, Kaski JC. Elevated serum neopterin predicts future adverse cardiac events in patients with chronic stable angina pectoris. Eur Heart J 2005;26:457-63. [21] Grau AJ, Reis A, Buggle F, Al-Khalaf A, Werle E, Valois N, et al. Monocyte function and plasma levels of interleukin-8 in acute ischemic stroke. J Neurol Sci 2001;192: 41-7. [22] Goldstein LB, Bertels C, Davis JN. Interrater reliability of the NIH stroke scale. Arch Neurol 1989;46:660-2.
1601
[23] Muir KW, Weir CJ, Murray GD, Povey C, Lees KR. Comparison of neurological scales and scoring systems for acute stroke prognosis. Stroke 1996;27:1817-20. [24] Howard G, Walker MD, Becker C, Coull B, Feibel J, McLeroy K, et al. Community hospital-based stroke programs: North Carolina, Oregon, and New York, III: factors influencing survival after stroke: proportional hazards analysis of 4219 patients. Stroke 1986;17:294-9. [25] Chambers BR, Norris JW, Shurvell BL, Hachinski VC. Prognosis of acute stroke. Neurology 1987;37:221-5. [26] Petty GW, Brown RD, Whisnant JP, Sicks JD, O'Fallon WM, Wiebers DO. Survival and recurrence after first cerebral infarction: a population-based study in Rochester, Minnesota, 1975 through 1989. Neurology 1998;50:208-16. [27] Hénon H, Godefroy O, Leys D, Mounier-Vehier F, Lucas C, Rondepierre P, et al. Early predictors of death and disability after acute cerebral ischemic event. Stroke 1995;26:392-8. [28] Czlonkowska A, Ryglewicz D, Lechowicz W. Basic analytical parameters as predictive factors for 30-day case fatality rate in stroke. Acta Neurol Scand 1997;95:121-4. [29] Kazmierski R, Guzik P, Ambrosius W, Ciesielska A, Moskal J, Kozubski W. Predictive value of white blood cell count on admission for in-hospital mortality in acute stroke patients. Clin Neurol Neurosurg 2004;107:38-43. [30] Tuttolomondo A, Pedone C, Pinto A, Di Raimondo D, Fernandez P, Di Sciacca R, et al. Gruppo Italiano di Farmacoepidemiologia dell'Anziano (GIFA) researchers: predictors of outcome in acute ischemic cerebrovascular syndromes: the GIFA study. Int J Cardiol 2008;125:391-6.