Peptides 59 (2014) 9–13
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Identiﬁcation of plasma adrenomedullin as a possible prognostic biomarker for aneurysmal subarachnoid hemorrhage Jian-Yong Cai, Xian-Dong Chen, Hua-Jun Ba, Jian-Hu Lin, Chuan Lu, Mao-Hua Chen, Jun Sun ∗ Department of Neurosurgery, The Central Hospital of Wenzhou City, 32 Dajian Lane, Wenzhou 325000, China
a r t i c l e
i n f o
Article history: Received 9 June 2014 Received in revised form 26 June 2014 Accepted 26 June 2014 Available online 5 July 2014 Keywords: Adrenomedullin Intracranial aneurysmal Subarachnoid hemorrhage Functional outcome Mortality Biomarker
a b s t r a c t Increased plasma adrenomedullin levels have been reported in critically ill patients. This study tested the hypothesis that plasma adrenomedullin levels are signiﬁcantly increased in patients with acute spontaneous aneurysmal subarachnoid hemorrhage, and are predictive of clinical outcomes. Plasma adrenomedullin levels from 120 adult patients with spontaneous aneurysmal subarachnoid hemorrhage and 120 healthy volunteers during the study period were evaluated. Mortality and poor long-term outcome (Glasgow Outcome Scale score of 1–3) at 6 months were recorded. Data showed that circulating plasma adrenomedullin levels signiﬁcantly increased in patients on admission compared with the volunteers. In patients who died or had poor outcome at 6 months, plasma adrenomedullin levels were signiﬁcantly higher compared with survivors and patients with good outcome. Plasma adrenomedullin levels on presentation were highly associated with clinical severity assessed using World Federation of Neurological Surgeons score and Fisher score, emerged as the independent risk factor of 6-month mortality and poor outcome, and possessed similar predictive value to World Federation of Neurological Surgeons score and Fisher score based on receiver operating characteristic curves. A combined logisticregression model did not demonstrate the additive beneﬁt of adrenomedullin to World Federation of Neurological Surgeons score and Fisher score. Thus, higher plasma adrenomedullin levels on presentation are associated with clinical severity and worse outcomes in patients with acute spontaneous aneurysmal subarachnoid hemorrhage. © 2014 Elsevier Inc. All rights reserved.
Introduction Intracranial aneurysms are the most common cause of spontaneous subarachnoid hemorrhage. Global incidence of aneurysmal subarachnoid hemorrhage (aSAH) is estimated at approximately 10 per 100,000. Despite comprising only 5% of strokes, aSAH accounts for a signiﬁcant proportion of stroke-related morbidity and mortality . The World Federation of Neurological Surgeons (WFNS) grade and Fisher grade are commonly used to assess the severity and the amount of bleeding of aSAH . However, prediction of outcome remains difﬁcult and complicates decision-making for active treatment in aSAH . Adrenomedullin (AM) is a 52-amino-acid peptide belonging to the calcitonin gene-related peptide family and identiﬁed as a multifunctional peptide which exerts, through an autocrine/paracrine
∗ Corresponding author. Tel.: +86 0577 88070335; fax: +86 0577 88070335. E-mail address: [email protected]
(J. Sun). http://dx.doi.org/10.1016/j.peptides.2014.06.014 0196-9781/© 2014 Elsevier Inc. All rights reserved.
mode of action, multiple biological effects , including the regulation of blood pressure, cell growth and differentiation, modulation of hormone secretion, central nervous system functions and the potentiation of host defences against microbes [6,12]. Its gene expression is promoted by various stimuli, including inﬂammation, hypoxia, oxidative stress, mechanical stress and activation of the renin–angiotensin and sympathetic nervous systems [26,31]. AM appears to be a promising therapeutic tool for human diseases including ischemic stroke [7,24], traumatic brain injury , myocardial infarction  and inﬂammatory bowel disease . Blood levels of AM were independently correlated with prognosis of ischemic or hemorrhagic stroke [30,33] and traumatic brain injury . Cerebrospinal ﬂuid AM concentration correlates with delayed ischemic neurological deﬁcits after aSAH [19,20]. However, plasma AM concentrations are not associated with angiographic vasospasm, but reﬂect the severity of hemorrhage . Thus, in this study, we aimed to ﬁnd out the relationship between AM and clinical outcome by determining the plasma levels of AM in samples obtained from patients with aSAH and healthy volunteers.
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Materials and methods Study population This prospective observatory study initially assessed the patients with ﬁrst-ever non-traumatic SAH admitted to the Department of Neurosurgery, Central Hospital of Wenzhou City, China during the period of January 2011–May 2013. Only patients who had clinical history of SAH within the last 24 h before admission, suffered from aSAH conﬁrmed by computerized tomography (CT) angiography with or without digital subtraction angiography and received the treatment by surgery or coiling within the 48 h after admission were include in this study. Patients were excluded if they had (1) rebleeding after admission, (2) less than 18 years of age, (3) previous head trauma, (4) previous neurological disease, (5) previous use of antiplatelet or anticoagulant medication, and (6) other prior systemic diseases including uremia, liver cirrhosis, malignancy, chronic heart disease, chronic lung disease, diabetes mellitus and hypertension. We also excluded patients with (1) unavailable biomarker measurements, (2) refusal of participation and (3) loss of follow-up. Finally, One hundred and twenty aSAH patients were enrolled in this study. The mean age of the patients with aSAH, a group consisting of 71 men and 49 women, was 41.1 ± 11.5 years. A control group consisted of 120 healthy subjects. The healthy volunteers had a mean age of 42.1 ± 13.5 years and included 65 men and 55 women. Intergroup differences in sex and age did not appear statistically signiﬁcant. The study was conducted in accordance with the guidelines approved by the Human Research Ethics Committee at our hospital. Written informed consent was obtained from the patients or their relatives. Clinical and radiological assessment At admission, we collected information on demographic, clinical, radiological and outcome data for all patients. The radiological severity of SAH on admission was classiﬁed according to the Fisher grading system . Clinical severity of SAH was classiﬁed according to the WFNS system . Whenever clinical deterioration occurred, CT was performed to search for secondary complications such as hydrocephalus or ischemia. Clinical onset of cerebral vasospasm was deﬁned as the acute onset of a focal neurologic deﬁcit or a change in the Glasgow Coma Scale score of 2 or more
points. All suspected cases of cerebral vasospasms were conﬁrmed by CT angiography. All CT scans were performed according to the neuroradiology department protocol. Investigators who read them were blinded to clinical information. Outcome at 6 months was classiﬁed according to the Glasgow Outcome Scale, as previously reported : Scores 1–3, poor outcome; Scores 4–5, favorable outcome. For follow-up, structure telephone interviews were performed by one doctor who was blinded to clinical information and AM levels. Immunoassay methods The informed consents were obtained from all participants or their legal representatives before the blood were collected. Venous blood of patients was drawn on admission, and those of control group were drawn at study entry. Samples were placed on ice, centrifuged at 3000 × g, and plasma aliquoted and frozen at −70 ◦ C. Plasma AM concentration was analyzed by enzyme-linked immunosorbent assay using commercial kits (R&D Systems, Heidelberg, Germany) in accordance with the manufactures’ instructions. The blood samples were run in duplicate. Researchers running enzyme-linked immunosorbent assays were blinded to all patient details. Statistical analysis All statistical analyses were performed with the use of computer software SPSS 19.0 (SPSS Inc., Chicago, IL, USA). The results were reported as counts (percentage) for the categorical variables and mean ± standard deviation for the continuous variables. Comparisons were made using Chi-square test or Fisher exact test for categorical data, and unpaired Student’s t-test for the continuous variables. Correlations of AM with WFNS score and Fisher score were assessed by Spearman’s or Pearson’s correlation coefﬁcient and followed by a multivariate linear regression. A logistic-regression model was constructed to identify independent outcome predictors. The logistic regression results were presented as odds ratio (OR) and 95% conﬁdence interval (CI). A receiver operating characteristic (ROC) curve was conﬁgured to determine the cutoff point of the AM concentration to differentiate poor outcome. The results were estimated with calculated area under curve (AUC) and 95% CI. A combined logistic-regression model was conﬁgured
Table 1 The characteristics in patients with aneurysmal subarachnoid hemorrhage and the factors associated with 6-month clinical outcomes. Total
Cases Male Age (year) WFNS score on admission Fisher score on admission Surgery Acute hydrocephalus Intraventricular hemorrhage External ventricular drain Vasospasm Computed tomography ischemia Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Blood glucose level (mmol/L) Plasma CRP level (mg/L) Plasma adrenomedullin level (pg/mL)
120 71 (59.2%) 41.1 ± 11.5 2.6 ± 1.1 2.8 ± 0.9 68 (56.7%) 26 (21.7%) 23 (19.2%) 30 (25.0%) 36 (30.0%) 15 (12.5%) 6.4 ± 4.1 8.2 ± 4.5 130.7 ± 23.7 80.7 ± 13.6 13.2 ± 4.8 11.1 ± 3.3 109.1 ± 34.1
6-Month functional outcome
16 9 (56.3%) 41.9 ± 11.8 3.9 ± 0.7 3.9 ± 0.6 10 (62.5%) 8 (50.0%) 7 (43.8%) 8 (50.0%) 9 (56.3%) 6 (37.5%) 6.3 ± 6.0 8.0 ± 6.1 137.5 ± 18.8 85.4 ± 10.9 16.5 ± 4.7 13.7 ± 3.3 146.4 ± 32.6
104 62 (59.6%) 40.9 ± 11.5 2.4 ± 1.0 2.6 ± 0.8 58 (55.8%) 18 (17.3%) 16 (15.4%) 22 (21.2%) 27 (26.0%) 9 (8.7%) 6.4 ± 3.8 8.3 ± 4.3 129.7 ± 24.2 79.9 ± 13.8 12.8 ± 4.6 10.8 ± 3.1 103.4 ± 30.6
0.799 0.741 <0.001 <0.001 0.613 0.003 0.007 0.026 0.020 0.005 0.964 0.880 0.220 0.137 0.003 0.001 <0.001
35 20 (57.1%) 41.3 ± 10.7 3.6 ± 0.7 3.6 ± 0.8 22 (62.9%) 13 (37.1%) 12 (34.3%) 14 (40.0%) 16 (45.7%) 9 (25.7%) 5.6 ± 4.3 7.5 ± 4.5 134.7 ± 21.6 80.8 ± 12.3 15.0 ± 4.9 12.6 ± 3.3 135.4 ± 30.4
85 51 (60.0%) 40.9 ± 11.9 2.2 ± 1.0 2.5 ± 0.7 46 (54.1%) 13 (15.3%) 11 (12.9%) 16 (18.8%) 20 (23.5%) 6 (7.1%) 6.7 ± 4.0 8.5 ± 4.5 129.1 ± 24.4 80.6 ± 14.1 12.5 ± 4.5 10.5 ± 3.1 98.3 ± 29.4
0.772 0.858 <0.001 <0.001 0.380 0.008 0.007 0.015 0.016 0.012 0.170 0.238 0.244 0.945 0.009 0.002 <0.001
Numerical variables were presented as mean ± standard deviation. Categorical variables were expressed as counts (percentage). Numerical variables were analyzed by unpaired Student’s t-test. Categorical variables were analyzed by chi-square test or Fisher exact test. WFNS indicates World Federation of Neurological Surgeons; CRP, C-reactive protein.
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to evaluate the additive beneﬁt of AM to WFNS score and Fisher score. P < 0.05 was considered signiﬁcant. Results Study population characteristics The demographic, clinical and laboratory data of aSAH patients are shown in Table 1. Sixteen patients (13.3%) died and 35 patients (29.2%) had worse outcome at 6 months after aSAH. The plasma AM concentrations in all patients (109.1 ± 34.1 pg/mL) were substantially high compared with healthy controls (54.8 ± 12.8 pg/mL, P < 0.001). Correlations of plasma AM levels with disease severity A signiﬁcant correlation emerged between WFNS scores and plasma AM levels (r = 0.565, P < 0.001), as well as between Fisher scores and plasma AM levels (r = 0.561, P < 0.001), as well as between plasma AM levels and other variables shown in Table 2. When the above variables were introduced into the linear regression model, plasma AM levels remained positively associated with WFNS scores (t = 6.409, P < 0.001) and Fisher scores (t = 5.902, P < 0.001). Mortality prediction Tables 1 and 3 show the variables associated with death at 6 months in patients. When was conﬁgured a logistic-regression model which included the signiﬁcant variables in the univariate analysis, it was demonstrated that WFNS score (OR, 4.702; 95% CI, 1.798–12.291; P = 0.002), Fisher score (OR, 3.802; 95% CI, 1.834–7.882; P = 0.007) and plasma AM level (OR, 1.035; 95% CI, 1.018–1.061; P = 0.008) were the independent predictors for 6month mortality of patients. A ROC curve analysis was performed to determine the cutoff point to differentiate patients with aSAH according to their death based on their AM concentrations at admission. This analysis showed that a plasma AM level >116.8 pg/mL predicted 6-month mortality of patients with 87.5% sensitivity and 70.2% speciﬁcity (AUC, 0.837; 95%CI, 0.758–0.898) in Fig. 1A. Based on AUC, its predictive value was similar to WFNS score’s (AUC, 0.882; 95% CI, 0.810–0.934; P = 0.512) and Fisher score’s (AUC, 0.865; 95% CI, 0.791–0.921; P = 0.690). When a combined logistic-regression Table 2 Clinical, radiologic and laboratory factors correlated with plasma adrenomedullin levels. Characteristics
Gender (Male/Female) Age (year) WFNS score on admission Fisher score on admission Surgery Acute hydrocephalus Intraventricular hemorrhage External ventricular drain Vasospasm Computed tomography ischemia Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Blood glucose level (mmol/L) Plasma CRP level (mg/L)
0.048 0.051 0.565 0.561 0.058 0.224 0.263 0.181 0.267 0.255 0.143 0.133 0.103 0.107 0.280 0.401
0.603 0.581 <0.001 <0.001 0.526 0.014 0.004 0.048 0.003 0.005 0.119 0.146 0.263 0.245 0.002 <0.001
Correlations of plasma adrenomedullin levels with other variables were assessed by Spearman’s or Pearson’s correlation coefﬁcient. WFNS indicates World Federation of Neurological Surgeons; CRP, C-reactive protein.
Fig. 1. The analysis of predictive value of plasma adrenomedullin level for 6-month mortality (1A) and poor outcome (1B) in aneurysmal subarachnoid hemorrhage. Receiver operating characteristic curves were constructed based on the sensitivity and speciﬁcity of the plasma adrenomedullin concentration for identifying 6-month mortality and poor outcome. The area under receiver operating characteristic curve ranges from 0.5 to 1.0. An area under curve closer to 1 indicates a higher predictive power.
model was constructed, AM did not enhance AUCs of WFNS score (AUC, 0.931; 95% CI, 0.870–0.969; P = 0.181) and Fisher score (AUC, 0.896; 95% CI, 0.827–0.944; P = 0.393). Poor outcome prediction Tables 1 and 3 show the variables associated with poor outcome at 6 months in patients. When was conﬁgured a logistic-regression model which included the signiﬁcant variables in the univariate analysis, it was demonstrated that WFNS score (OR, 4.575; 95% CI, 2.072–10.100; P = 0.001), Fisher score (OR, 3.365; 95% CI, 1.401–8.080; P = 0.001) and plasma AM level (OR, 1.033; 95% CI, 1.014–1.053; P = 0.003) were the independent predictors for 6month poor outcome of patients. A ROC curve analysis was performed to determine the cutoff point to differentiate patients with aSAH according to their poor outcome based on their AM concentrations at admission. This analysis showed that a plasma AM level >116.8 pg/mL predicted 6month poor outcome of patients with 77.1% sensitivity and 78.8%
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Table 3 The characteristics associated with 6-month clinical outcomes of patients using univariate logistic-regression analysis. Characteristics
Male Age (year) WFNS score on admission Fisher score on admission Surgery Acute hydrocephalus Intraventricular hemorrhage External ventricular drain Vasospasm Computed tomography ischemia Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Blood glucose level (mmol/L) Plasma CRP level (mg/L) Plasma adrenomedullin level (pg/mL)
6-Month functional outcome
Odds ratio (95% conﬁdence interval)
Odds ratio (95% conﬁdence interval)
0.871 (0.301–2.520) 1.008 (0.963–1.055) 6.133 (2.612–14.396) 5.026 (2.366–10.675) 1.322 (0.447–3.906) 4.778 (1.584–14.408) 3.980 (1.304–12.152) 3.727 (1.257–11.054) 3.667 (1.244–10.804) 6.333 (1.867–21.481) 0.996 (0.875–1.133) 0.988 (0.877–1.112) 1.014 (0.992–1.037) 1.033 (0.989–1.079) 1.216 (1.063–1.392) 1.309 (1.103–1.552) 1.036 (1.018–1.054)
0.799 0.739 <0.001 <0.001 0.614 0.005 0.015 0.018 0.018 0.003 0.948 0.840 0.220 0.140 0.004 0.002 <0.001
0.889 (0.400–1.974) 1.003 (0.969–1.038) 6.491 (3.150–13.376) 5.942 (3.111–11.347) 1.435 (0.640–3.217) 3.273 (1.324–8.090) 3.975 (1.563–10.110) 2.875 (1.207–6.847) 2.737 (1.190–6.294) 4.558 (1.481–14.025) 0.930 (0.838–1.032) 0.946 (0.863–1.037) 1.010 (0.993–1.027) 1.001 (0.972–1.031) 1.134 (1.034–1.244) 1.228 (1.079–1.398) 1.038 (1.022–1.055)
0.772 0.857 <0.001 <0.001 0.381 0.010 0.004 0.017 0.018 0.008 0.172 0.238 0.243 0.945 0.008 0.002 <0.001
Univariate logistic-regression analysis was used to calculate the odds ratio and 95% conﬁdence interval. WFNS indicates World Federation of Neurological Surgeons; CRP, C-reactive protein.
speciﬁcity (AUC, 0.827; 95%CI, 0.748–0.890) in Fig. 1B. Based on AUC, its predictive value was similar to WFNS score’s (AUC, 0.866; 95% CI, 0.792–0.921; P = 0.483) and Fisher score’s (AUC, 0.854; 95% CI, 0.778–0.912; P = 0.621). When a combined logistic-regression model was constructed, AM did not enhance AUCs of WFNS score (AUC, 0.899; 95% CI, 0.830–0.946; P = 0.147) and Fisher score (AUC, 0.892; 95% CI, 0.822–0.941; P = 0.133). Discussion Previous studies have demonstrated that enhanced AM levels in peripheral blood are highly associated with disease severity and clinical outcomes of patients with adult intracerebral hemorrhage, cerebral infarction and traumatic brain injury [5,30,33]. Another report has also found that the plasma AM concentration is increased after aSAH . To the best of our knowledge, no research analyzes the relationship between plasma AM levels and clinical outcome of patients with aSAH. The current study determined AM levels in plasma and further analyzed the association of plasma AM levels with risk of long-term clinical outcomes and severity of aSAH. This study showed plasma AM levels were positively correlated with disease severity and independently associated with 6-month clinical outcomes, suggesting AM could have potential as a biomarker for prognostic prediction of aSAH. AM is a multifunctional peptide that was discovered during high throughout screening of pheochromocytoma extracts for novel biologically active peptides . AM is later found to be widely distributed throughout mammalian tissues, including the brain [13,27]. Its well-known ability is to induce vasodilation . In vivo and in vitro studies have documented that AM causes vasodilation of cerebral arteries [3,7,21,33]. Marked increases in AM levels in the cerebrospinal ﬂuid in adult occur after aSAH, and delayed ischemic neurological deﬁcit was positively correlated with AM levels in the cerebrospinal ﬂuid [19,20]. Thus, elevated AM levels in cerebrospinal ﬂuid following aSAH may play a modulatory role in the development of symptomatic vasospasm and subsequent brain ischemia . However, the principal organs responsible for maintaining the plasma concentrations of AM have not been clearly identiﬁed . Endothelial cells and vascular smooth muscle cells have been reported to actively produce AM. The AM gene transcription level in these cells is much higher than that in other tissues, such as adrenal gland and lung [14,28]. Moreover, AM gene transcription has been demonstrated in intact rat aorta . These
vascular tissues are assumed to be one of the major sources of plasma AM. In addition, AM concentrations after aSAH were signiﬁcantly higher in the cerebrospinal ﬂuid than in the plasma . Therefore, in part, AM in peripheral blood may be derived from central nervous system . Recently, Wang et al. have reported plasma AM levels are associated with disease severity and 3-month clinical outcome of acute intracerebral hemorrhage. Our research, for the ﬁrst time, demonstrated the close relationships between plasma AM levels and disease severity and 6-month clinical outcome of aSAH. Importantly, the current study used a multivariate linear regression, but not a univariate analysis, to demonstrate the association of plasma AM levels with the severity of aSAH reﬂected by WFNS scores and Fisher scores. Moreover, discussion section in this study also analyzed the source of plasma AM referred to peripheral and central releasing, suggesting the role of AM in both central and peripheral system. Convincingly, in order to verify the relationship between plasma AM levels and long-term outcome of aSAH, this study extended the duration of following up from 3 months to 6 months compared with the Wang et al.’s report, and meantime, in terms of statistical methods, at ﬁrst there is univariate analysis including t-test, Chi-square test and a univariate logistic-regression analysis, and then a multivariate logistic-regression analysis was implemented, showing the statistical analysis was more systemic in this study. Thus, the data further conﬁrm the hypothesis that plasma AM may have high predictive power to identify the aSAH patients at risk of poor long-term outcome. Conclusions This study suggests that AM may indicate clinical severity of the initial bleeding and also have prognostic value for clinical outcomes in aSAH and may therefore help in guiding treatment decisions in the setting of aSAH. Conﬂict of interest The authors have no conﬂict of interest. Acknowledgements The authors thank all staffs in Department of Neurosurgery, The Central Hospital of Wenzhou City (Wenzhou, China) for their technical support. This study was supported by a grant from
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