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Peptides 56 (2014) 111–115

Contents lists available at ScienceDirect

Peptides

j ourna l h o mepa ge: www.elsev ier .com/ locate /pept ides

irculating levels of neuropeptide proenkephalin A predict outcomen patients with aneurysmal subarachnoid hemorrhage

iang-Lin Chena, Bing-Jian Yua, Mao-Hua Chenb,∗

The Second Zone, Department of Brain, The People’s Hospital of Qingyuan, The Affiliated Qingyuan Hospital of Jinan University, B24 Xinchengyinquanoad, Qingcheng District, Qingyuan 511518, Guangdong Province, ChinaDepartment of Neurosurgery, The Central Hospital of Wenzhou City, 32 Dajian Lane, Wenzhou 325000, Zhejiang Province, China

r t i c l e i n f o

rticle history:eceived 19 March 2014eceived in revised form 1 April 2014ccepted 1 April 2014vailable online 12 April 2014

eywords:roenkephalin Aneurysmal subarachnoid hemorrhageortality

unctional outcome

a b s t r a c t

High plasma proenkephalin A level has been associated with ischemic stroke severity and clinical out-comes. This study aimed to assess the relationship between proenkephalin A and disease severity aswell as to investigate its ability to predict long-term clinical outcome in patients with aneurysmal sub-arachnoid hemorrhage. Plasma proenkephalin A concentrations of one hundred and eighty patientsand 180 sex- and age-matched healthy controls were measured by chemoluminescence sandwichimmunoassay. Plasma proenkephalin A level was substantially higher in patients than in healthy con-trols (205.5 ± 41.6 pmol/L vs. 90.8 ± 21.1 pmol/L, P < 0.001), was highly associated with World Federationof Neurological Surgeons (WFNS) score (r = 0.470, P < 0.001) and Fisher score (r = 0.488, P < 0.001), wasan independent predictor for 6-month mortality [odds ratio (OR), 1.183; 95% confidence interval (CI),1.067–1.339; P = 0.004] and unfavorable outcome (Glasgow Outcome Scale score of 1–3) (OR, 1.119;95% CI, 1.046–1.332; P = 0.005) using multivariate analysis, and had high area under receiver operat-ing characteristic curve (AUC) for prediction of 6-month mortality (AUC, 0.831; 95% CI, 0.768–0.883) andunfavorable outcome (AUC, 0.821; 95% CI, 0.757–0.874). The predictive value of the plasma proenkephalin

A concentration was also similar to those of WFNS score and Fisher score (both P > 0.05). In a combinedlogistic-regression model, proenkephalin A improved the AUCs of WFNS score and Fisher score, but thedifferences were not significant (both P > 0.05). Thus, proenkephalin A level may be a useful, comple-mentary tool to predict mortality and functional outcome at 6 months after aneurysmal subarachnoidhemorrhage.

© 2014 Elsevier Inc. All rights reserved.

ntroduction

Aneurysmal subarachnoid hemorrhage (aSAH) is known toepresent a major public health concern potentially resulting ineath or neurological impairment [4,14,17]. The pathophysiologi-al mechanisms implicated in the cellular and molecular changeollowing aSAH remains unclear [2,13,20]. For assessment of theeverity of aSAH, the World Federation of Neurological SurgeonsWFNS) grade is the current gold standard [15]. The condition ofatients with aSAH may however be confounded by seizure, hydro-ephalus or sedation [1]. Treatment decisions such as occlusion of

n aneurysm are based on clinical grading [15]. Prediction of out-ome remains difficult and complicates decision making for activereatment [10].

∗ Corresponding author. Tel.: +86 0577 88070335; fax: +86 0577 88070335.E-mail address: zhanghaiwangew@126.com (M.-H. Chen).

ttp://dx.doi.org/10.1016/j.peptides.2014.04.001196-9781/© 2014 Elsevier Inc. All rights reserved.

The neuropeptide enkephalin are active as neurotransmitters,are involved in nociception and immune stimulation and also havebeen implicated in the pathophysiology of subarachnoid hemor-rhage [5,8]. Measurement of the mature neuropeptide enkephalinin plasma is, however, complicated by rapid physiologic degra-dation of the bioactive peptides and by low in vitro stability. Bycontrast, assessment of stable precursor fragments of the neu-ropeptide enkephalin (proenkephalin A [PENK-A]) has been shownto circumvent this problem as they adequately reflect neuropeptideenkephalin production and show high stability in human plasmafor more than 48 h [6]. Recent study demonstrated plasma PENK-A levels were highly associated with severity of cerebral injury,and may have prognostic value for fatal and nonfatal events inischemic stroke [3]. The present study aimed to further investi-

gate the ability of plasma PENK-A to predict long-term clinicaloutcomes and to assess the relationship between plasma PENK-Aconcentrations and disease severity in such a group of patients withaSAH.

112 X.-L. Chen et al. / Peptides 56 (2014) 111–115

Table 1The characteristics in patients with aneurysmal subarachnoid hemorrhage and the factors associated with 6-month clinical outcomes.

Total 6-month mortality 6-month functional outcome

Non-survivors Survivors P value Unfavorable outcome Favorable outcome P value

Cases 180 22 158 50 130Male 102 (56.7%) 13 (59.1%) 89 (56.3%) 0.807 31 (62.0%) 71 (54.6%) 0.371Age (y) 44.0 ± 11.2 44.6 ± 12.4 43.9 ± 11.0 0.767 45.5 ± 10.3 43.4 ± 11.5 0.269WFNS score on admission 2 (1) 3 (1) 2 (2) <0.001 3 (1) 2 (1) <0.001Fisher score on admission 2 (1) 3 (1) 2 (1) <0.001 3 (1) 2 (1) <0.001Surgery 104 (57.8%) 14 (63.6%) 90 (57.0%) 0.553 30 (60.0%) 74 (56.9%) 0.708Acute hydrocephalus 45 (25.0%) 12 (54.6%) 33 (20.9%) 0.001 20 (40.0%) 25 (19.2%) 0.004Intraventricular hemorrhage 33 (18.3%) 9 (40.9%) 24 (15.2%) 0.007 16 (32.0%) 17 (13.1%) 0.003External ventricular drain 48 (26.7%) 14 (63.6%) 34 (21.5%) <0.001 21 (42.0%) 27 (20.8%) 0.004Vasospasm 62 (34.4%) 13 (59.1%) 49 (31.0%) 0.009 26 (52.0%) 36 (27.7%) 0.002Brain ischemic lesion 24 (13.3%) 8 (36.4%) 16 (10.1%) 0.001 12 (24.0%) 12 (9.2%) 0.009Admission time (h) 8.9 ± 3.5 9.3 ± 3.5 8.9 ± 3.5 0.592 8.3 ± 3.0 9.2 ± 3.7 0.134Plasma-sampling time (h) 10.7 ± 4.1 11.7 ± 4.0 10.6 ± 4.1 0.239 10.2 ± 3.8 10.9 ± 4.2 0.306Systolic arterial pressure (mmHg) 134.3 ± 23.3 141.1 ± 17.2 133.4 ± 23.9 0.146 138.6 ± 21.0 132.7 ± 23.9 0.128Diastolic arterial pressure (mmHg) 83.3 ± 13.0 85.8 ± 12.2 82.9 ± 13.1 0.339 84.6 ± 11.4 82.8 ± 13.5 0.394Blood WBC count (×109/L) 8.4 ± 2.9 8.8 ± 3.5 8.3 ± 2.9 0.471 8.9 ± 3.8 8.2 ± 2.5 0.210Blood hemoglobin level (g/L) 128.3 ± 22.4 132.8 ± 13.2 127.7 ± 23.4 0.317 128.1 ± 21.3 128.3 ± 23.0 0.949Blood platelet count (×109/L) 174.5 ± 40.9 186.2 ± 33.6 172.9 ± 41.7 0.101 172.8 ± 39.7 175.2 ± 41.5 0.724Prothrombin time (s) 16.0 ± 2.2 16.5 ± 1.2 16.0 ± 2.3 0.115 15.9 ± 2.0 16.1 ± 2.3 0.718Thrombin time (s) 19.1 ± 2.9 19.0 ± 1.9 19.2 ± 3.0 0.790 18.8 ± 2.7 19.3 ± 3.0 0.286Partial thromboplastin time (s) 39.9 ± 5.4 39.9 ± 4.8 39.9 ± 5.5 0.950 39.6 ± 5.2 40.0 ± 5.5 0.623Blood glucose level (mmol/L) 13.8 ± 4.4 16.7 ± 4.3 13.4 ± 4.3 0.001 15.5 ± 4.6 13.1 ± 4.2 0.001Plasma CRP level (mg/L) 10.8 ± 2.9 12.1 ± 3.3 10.6 ± 2.9 0.026 11.7 ± 3.1 10.5 ± 2.8 0.009Plasma fibrinogen level (g/L) 3.9 ± 1.2 4.1 ± 0.8 3.9 ± 1.2 0.593 4.0 ± 0.9 3.9 ± 1.3 0.849Plasma D-dimer level (mg/L) 2.7 ± 1.2 3.5 ± 1.2 2.6 ± 1.2 0.001 3.2 ± 1.3 2.5 ± 1.2 0.004Plasma proenkephalin A level (pmol/L) 205.5 ± 41.6 256.5 ± 53.3 198.4 ± 34.3 <0.001 238.2 ± 42.7 193.0 ± 33.6 <0.001

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umerical variables were presented as median (interquartile range) or mean ±stanariables were analyzed by Mann–Whitney U-test or unpaired Student’s t test. Catorld Federation of Neurological Surgeons; CRP, C-reactive protein; WBC, white bl

aterials and methods

tudy population

This study included aSAH patients confirmed by computerizedomography (CT) angiography with or without digital subtrac-ion angiography from The People’s Hospital of Qingyuan betweenanuary 2011 and April 2013. Patients with clinical history of SAH

ithin the last 24 h before admission and the treatment by surgeryr coiling within the 48 h after admission were included. Patientsith rebleeding after admission, less than 18 years of age, exist-

ng previous head trauma, neurological disease, use of antiplateletr anticoagulant medication, presence of other prior systemic dis-ases including uremia, liver cirrhosis, malignancy, chronic heartr lung disease, diabetes mellitus and hypertension were excluded.ealthy age- and sex-matched volunteers were recruited as con-

rol group. Patients or healthy controls were included in this studyfter they or their relatives provided written informed consent. Thisrotocol was approved by the Ethics Committee of The People’sospital of Qingyuan before implementation.

linical and radiological assessment

At admission, we collected demographic, clinical, radiologicalnd outcome data for all patients. For assessment of the sever-ty of aSAH, the WFNS grade is used. Imaging at admission wassed to determine Fisher grade, intraventricular hemorrhage andydrocephalus. Admission CT angiography or digital subtractionngiography was used for aneurysm detection. Cerebral vasospasmnd ischemic lesions were defined using the CONSCIOUS criteria12]. All CT scans were performed according to the neuroradiology

epartment protocol. Investigators who read them were blindedo clinical information. Participants were followed up until deathr completion of 6 months after aSAH. Unfavorable outcome wasefined as Glasgow outcome scale score of 1–3. For follow-up,

deviation. Categorical variables were expressed as counts (percentage). Numericalal variables were analyzed by chi-square test or Fisher exact test. WFNS indicatesll.

structure telephone interviews were performed by one doctor whowas blinded to clinical information and PENK-A levels.

Immunoassay methods

Venous blood of patients was drawn on admission, and those ofcontrol group were drawn at study entry. Samples were placed onice, centrifuged at 3000 × g, aliquoted and frozen at −70 ◦C. PENK-A119-159 was assessed by a newly developed chemoluminescencesandwich immunoassay, using a chemiluminescence platform andcoated tube technique, as described previously (ImmunochemicalIntelligence GmbH, Berlin, Germany) [6]. All assessments were runin duplicates. The person carrying out the assays was completelyblinded to the clinical information.

Statistical analysis

Statistical analysis was performed with SPSS 19.0 (SPSS Inc.,Chicago, IL, USA) and MedCalc 9.6.4.0. (MedCalc Software, Mari-akerke, Belgium). The results were reported as counts (percentage)for the categorical variables, mean ± standard deviation if normallydistributed and median (interquartile range) if not normally dis-tributed for the continuous variables. Comparisons were made byusing (1) chi-square test or Fisher exact test for categorical data,(2) Student’s t test for continuous normally distributed variables,and (3) the Mann–Whitney U-test for continuous non-normallydistributed variables. The association of plasma PENK-A level withWFNS score and Fisher score was analyzed using Spearman correla-tion coefficient. The relations of PENK-A to 6-month mortality andunfavorable outcome were assessed in a logistic-regression modelwith odds ratio (OR) and 95% confidence interval (CI). The receiver

operating characteristic (ROC) curves were used to determine thebest threshold of PENK-A values to predict 6-month clinical out-comes with calculated area under curve (AUC). In a combinedlogistic-regression model, the additive benefit of PENK-A to WFNS

X.-L. Chen et al. / Peptides 56 (2014) 111–115 113

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Fig. 1. The change of plasma proenkephalin

core and Fisher score was estimated. A 2-tailed probability valuef <0.05 was considered as statistically significant.

esults

tudy population characteristics

This study included one hundred and eighty aSAH patients and80 sex- and age-matched healthy controls. Table 1 shows the mainemographic, clinical, radiological and laboratory characteristics ofhese patients. Fig. 1 shows plasma PENK-A levels were statisticallyignificantly higher in patients than in healthy controls (P < 0.0001).ig. 2 shows that plasma PENK-A levels were obviously associatedith WFNS score (P < 0.0001) and Fisher score (P < 0.0001).

ortality prediction

Twenty-two patients (12.2%) died within 6 months after aSAH.able 1 found that plasma PENK-A levels and other variablesere highly associated with 6-month mortality. When the above

ariables found to be significant in the univariate analysis werentroduced into the logistic model, a multivariate analysis selected

FNS score (OR, 6.135; 95% CI, 3.593–12.738; P < 0.001), Fishercore (OR, 5.136; 95% CI, 3.351–9.279; P < 0.001) and plasmaENK-A level (OR, 1.183; 95% CI, 1.067–1.339; P = 0.004) as thendependent predictors for 6-month mortality of patients.

Just as shown in Fig. 3, a ROC curve identified that a base-

ine plasma PENK-A level predicted 6-month mortality of patients

ith high AUC. The predictive value of the PENK-A concentra-ion was thus similar to those of WFNS score (AUC, 0.884; 95%I, 0.828–0.927) (P = 0.427) and Fisher score (AUC, 0.881; 95% CI,

ig. 2. The correlative analysis of plasma proenkephalin A levels with WFNS scores anederation of Neurological Surgeons.

ls in aneurysmal subarachnoid hemorrhage.

0.825–0.925) (P = 0.392). In a combined logistic-regression model,PENK-A improved the AUCs of WFNS score and Fisher score to 0.920(95% CI, 0.870–0.955) and 0.908 (95% CI, 0.856–0.946) respectively,but the differences were not significant (P = 0.238 and 0.388).

Functional outcome prediction

Fifty patients (27.8%) had unfavorable outcome at 6 months afteraSAH. Table 1 found that plasma PENK-A levels and other variableswere highly associated with 6-month unfavorable outcome. Whenthe above variables found to be significant in the univariate analy-sis were introduced into the logistic model, a multivariate analysisselected WFNS score (OR, 5.328; 95% CI, 3.290–10.555; P < 0.001),Fisher score (OR, 4.378; 95% CI, 2.751–7.571; P < 0.001) and plasmaPENK-A level (OR, 1.119; 95% CI, 1.046–1.332; P = 0.005) as the inde-pendent predictors for 6-month unfavorable outcome of patients.

Just as shown in Fig. 4, a ROC curve identified that a baselineplasma PENK-A level predicted 6-month unfavorable outcome ofpatients with high AUC. The predictive value of the PENK-A concen-tration was thus similar to those of WFNS score (AUC, 0.869; 95%CI, 0.811–0.915) (P = 0.288) and Fisher score (AUC, 0.859; 95% CI,0.800–0.907) (P = 0.400). In a combined logistic-regression model,PENK-A improved the AUCs of WFNS score and Fisher score to 0.896(95% CI, 0.842–0.936) and 0.894 (95% CI, 0.840–0.935) respectively,but the differences were not significant (P = 0.197 and 0.084).

Discussion

Recent study has demonstrated that plasma PENK-A levels arehighly associated with severity of ischemic stroke and also theirclinical outcomes [3]. The current study continued to demonstrate

d Fisher scores in aneurysmal subarachnoid hemorrhage. WFNS indicates World

114 X.-L. Chen et al. / Peptides

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ig. 3. The receiver operating characteristic curve analysis of plasma proenkephalin level for 6-month mortality in aneurysmal subarachnoid hemorrhage.

hat plasma PENK-A level in the patients with aSAH were signif-cantly higher than those in healthy controls on admission; andn patients who died or suffered from unfavorable outcome in 6

onths, the PENK-A levels on admission were significantly higherompared with levels in survivors or those with favorable outcome.n addition, PENK-A levels were highly associated with aSAH sever-ty as assessed by WFNS score and amount of bleeding as evaluatedy Fisher score. Moreover, in multivariate logistic regression mod-ls of predictors of death and unfavorable outcome, the PENK-Aevels on admission were an independent predictor; and in ROCurve, the PENK-A levels actually had high predictive values for 6-onth mortality and unfavorable outcome of patients with aSAH.

herefore, PENK-A level may be associated with disease severitynd long-term clinical outcomes of aSAH patients.

Enkephalin, thought of originally as signal molecule confined tohe nervous system, also plays multiple roles in the coordinationf internal immunoregulatory processes [11]. Enkephalin levels inhe peripheral blood are associated with severity and prognosis ofome illness such as liver disease, human hepatic encephalopathy,nd primary biliary cirrhosis [7,16,18,19]. Plasma immunoreac-ive enkephalin concentration is markedly higher in patients with

upratentorial infarction than in control subjects. However, it isnrelated to infarct size or patient severity [9]. Recently, Ernstt al. [6] developed a newly sandwich immunoassay using anti-odies against the proenkephalin A 119-159 peptide and showed

ig. 4. The receiver operating characteristic curve analysis of plasma proenkephalin level for 6-month unfavorable outcome in aneurysmal subarachnoid hemorrhage.

56 (2014) 111–115

that PENK A 119-159 immunoreactivity was detectable in the cir-culation of human blood. Importantly, PENK A 119-159 is stablein vitro for at least 48 h at room temperature as compared to thelow stability of the peptides enkephalin [6]. More interestingly,it was also found that an elevated PENK-A 119-159 expression inplasma was associated with the clinical severity of ischemic stroke,with brain lesion size, and with a poor clinical outcome [3]. Thissuggests the use of PENK A 119-159 measurement as surrogatemolecule for the release of the mature peptide enkephalin derivedfrom proenkephalin A [6].

This study also determined PENK A 119-159 and showed thehigher plasma PENK A level in aSAH patients compared withhealthy controls. Moreover, there were close relationships betweenplasma PENK A levels and WFNS scores as well as between plasmaPENK A levels and Fisher scores, suggesting plasma PENK A levelsshould reflect aSAH severity. A ROC curve showed that plasma PENKA level on admission could obviously predict long-term unfavor-able outcome and mortality; and its predictive value was similar toWFNS score’s and Fisher score’s. Yet, a combined logistic-regressionmodel did not verify that PENK A statistically significantly improvedthe predictive performances of WFNS score and Fisher score. There-fore, the determination of PENK A in the plasma of aSAH patientson admission provides the ability to distinguish between aSAHpatients with 6-month good and bad outcome.

Conclusions

In this study, plasma PENK A levels may be associated with aSAHseverity and increased plasma PENK A levels can independentlyidentify aSAH patients at risk of long-term poor clinical outcomes.

Declaration of interest

Institution at which the work was performed: The People’s Hos-pital of Qingyuan.

Competing interests

The authors declare that they have no competing interests.

Acknowledgment

This work was financially supported by Zhejiang ProvincePublic Techology Applied Research Project (2010C33015), Zhe-jiang Province Medical and Health Platform Project (2014RCA026)and Qingyuan Science and Technology Research Fund Project(2011B011112009).

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