a b s t r a c t

Background: The aims were to investigate the role of serum Ischemia-modified albumin , tumor necrosis factor ? (TNF-?), and Myeloperoxidase and to evaluate the relationship between IMA and cardiac markers (creatine kinase myocardial isoenzyme [CK-MB] and cardiac troponin I [cTnI]) related to cardiac abnor- malities in adult patients after nontraumatic subarachnoid hemorrhage .

Methods: Twenty-nine patients with nonTraumatic SAH admitted to the emergency department and 20 Healthy adults as the control group were included in the study. Ischemia-modified albumin, TNF-?, MPO, CK-MB, cTnI, and leukocyte count (white blood cell [WBC]) in the circulation were measured on admission.

Results: Ischemia-modified albumin, TNF-?, and MPO levels were higher by mean values of 11.6%, 9.5%, and 2.9%, respectively, in patients with SAH compared with control group. However, levels of these parameters were not statistically different between the groups (P N .05). However, WBC, CK-MB, and cTnI values were significantly higher in patients with SAH compared with healthy control (P b .001, P b .01, and P b .05, respectively). White blood cell and cTnI levels in the circulation were positively correlated with patients’ clinical severity (r = 0.598, P = .001 and r = 0.461, P = .012, respectively). Ischemia-modified albumin has a poor diagnostic value in comparison with WBC, CK-MB, and cTnI tests to differentiate between patients after SAH and controls according to receiver operating characteristic curve.

Conclusions: The results suggest that IMA is not better than CK-MB and cTnI in predicting a cardiac injury in patients after nontraumatic SAH.

(C) 2014

Introduction

nontraumatic subarachnoid hemorrhage (SAH) is a neurologic emer- gency that still carries unacceptably high Morbidity and mortality rates [1,2]. The primary and secondary intracerebral injuries and nonneurologic Medical complications have been significantly associated with SAH. One of these medical complications is neurocardiogenic injury, and Cardiac manifestations are most common in SAH [3-5]. Subarachnoid hemor- rhage diagnosis currently relies on clinical symptoms and neuroimaging; however, Molecular biomarkers that can improve diagnosis are required to reduce SAH mortality in the early stage.

Scientific reports suggest that the increased release in catechol- amines is the most probably underlying cause of Cardiac damage or

? Declaration of conflict of interest: The authors have no conflicts of interest to declare.

* Corresponding author at: Department of Emergency Medicine, Faculty of Medicine, Ondokuz Mayis University, Kurupelit, 55139 Samsun, Turkey. Tel.: +90 362 3121919×2096; fax: +90 362 4576041.

E-mail address: [email protected] (A. Baydin).

neurocardiogenic injury after SAH [3-6]. The degree of cardiac dys- function is highly variable in patients with SAH [6,7]. The clinical signifi- cance of acutely increased Cardiac troponin I and ischemia- modified albumin (IMA) that imply to be an early indicator of myocardial ischemia (MI) after SAH [8-11], especially when mildly positive cTnI and IMA elevation, was not well understood. Accordingly, our objective was to test the hypothesis that rises in cTnI and IMA in patients after SAH; we also evaluate the role of white blood cell (WBC), tumor necrosis factor ? (TNF-?), and Myeloperoxidase in patients after SAH according to the Hunt & Hess score to classify SAH severity using clinical symptoms on admission.

Materials and methods

Study design and setting

The study was approved by the Hospital Research Ethics Committee (ethics approval no. OMU KAEK 2014/673) and was conducted in the

http://dx.doi.org/10.1016/j.ajem.2014.10.007

0735-6757/(C) 2014

Department of Emergency Medicine, Samsun, Turkey. In addition, written informed consent was obtained from the patients themselves or their next of kin before enrolling in the study. From March 2014 to September 2014, we prospectively enrolled consecutive patients admitted to the emergency department (ED) within 24 hours of the first clinical symptoms of SAH, with a diagnosis of SAH documented using brain computed tomographic scanning, and angiography subsequently confirms a ruptured aneurysm. Control subjects (12 females and 8 males; 47.3+- 8.6 years) with no history of neurologic or cardiovascular disease and with electrocardiographic and routine serum bio- chemistry within the normal range and all patients with spontaneous aneurysmal and nonaneurysmal SAH were adult individuals (age, 18-76 years) included in the study. The exclusion criteria of this study included the following: (1) younger than 18 years; (2) traumatic SAH;

(3) cardiac surgery; (4) cardiac pacing; (5) trauma within the previous 3 months; (6) known arrhythmia, acute coronary syndromes, and myo- cardial infarction (receiving the past medical chart or by the statements of the patients themselves); (7) pheochromocytoma; or (8) an abnor- mal serum albumin level, Renal impairment defined as serum creatinine more than 1.4 mg/dL.

Demographic data and clinical variables were recorded at the admission time for all patients: age, sex, vital signs, laboratory data, the severity of neurologic injury on admission was graded clinically using the Hunt & Hess scale to classify SAH severity using clinical symptoms, defined as grade I, asymptomatic or mild headache; grade II, moderate or Severe headache, Nuchal rigidity, can have oculomotor palsy; grade III, confused, drowsiness, or mild focal signs; grade IV, stupor or hemiparesis; and grade V, coma, moribund, and/or extensor posturing [12]. Electrocardiographic changes were examined by a cardiologist, ECG findings: heart rate and heart rate-Corrected QT interval (QTc) using the Bazett formula. The morphologic abnormalities were diagnosed as the presence of 1 more of following 6 variables, which are commonly noted after SAH, in at least 2 leads: (1) ST elevation (ST elevation

>=0.1 mV), (2) ST depression (ST depression >=0.1 mV), (3) inverted T wave, (4) abnormal Q or QS wave, (5) QTc prolongation, or (6) sinus tachycardia or bradycardia.

Blood samples and measurements

Blood samples for the detection of IMA, MPO, and TNF-? were taken from patients who have the first clinical symptoms of SAH upon arrival to the ED. Serum was separated and stored at - 70?C until analysis. Ischemia-modified albumin, MPO, and TNF-? levels were measured by enzyme-linked immunosorbent assay using a kit from SunRed Biotechnology Company (cat. no. 201-12-1173), Boster Biological Technology Co, Ltd (cat. no. EKO850), and DIAsource ImmunoAssays S.A. (cat. no. KAP1751), respectively. Creatine kinase-MB (CK-MB) and cTnI levels were measured in serum by Simens ADVIA Centaur Cp analyzer, and leukocyte count was measured in the whole blood by Simens Advia 2120i analyzer and the other analyses were determined by Roche Cobas Integra 800 analyzer in emergency laboratory.

Statistical analysis

The statistical analyses were performed with use of a SPSS 15.0 for Windows (SPSS, Chicago, IL). All data were checked for the normality of distribution by Shapiro-Wilk test. None of the data distributed normally. Mann-Whitney U, Kruskal-Wallis analysis of variance, Bonferroni-corrected Mann-Whitney U, and Spearman Correlation analyses were used. P b .05 is accepted significantly for Mann-Whitney U, Kruskal-Wallis analysis of variance, and spearman correlation analyses, and P b .0125 is accepted significantly for Bonferroni-corrected Mann-Whitney U test.

Results

Demographic and clinical characteristics of the patients are shown in Table 1. Upon measuring the serum IMA, TNF-?, and MPO levels as well as ECG abnormalities of patients with SAH, the mean level of IMA, TNF-?, and MPO was higher by 11.6%, 9.5%, and 2.9% in patients with SAH compared with control subjects, respectively. However, no statistical difference was found between the groups (P N .05). However, WBC, CK-MB, and cTnI values were significantly higher in patients with SAH compared with healthy control group (P b .001, P b .01, and P b .05, respectively) as presented in Table 2. To determine whether the Hunt & Hess grades in patients is associated with the observed changes in IMA, TNF-?, MPO, WBC, cTnI, and CK-MB levels, the Spearman correlation was used. White blood cell and cTnI levels in the circulation were positively correlated with patients’ the Hunt & Hess grade (r = 0.598, P = .001 and r = 0.461, P = .012, respectively). diagnostic values for IMA, CK-MB, cTnI, WBC, TNF-?, and MPO tests to differentiate between SAH patients and control group were shown in Fig. 2 and its legend. The cutoff value for WBC was found 10 100/mm³ with a sensitivity of 89.7% and a specificity of 100%. The predictive accuracy was 93.9%; positive and negative predictive values for WBC were accounted for 100% and 87.0%, respectively.

Table 1

Demographic and clinical characteristics of the patients with SAH

Variable Patients Reference range

Age, y 58.8 +- 13.0

Sex, n (%)

Female 17 (59%)

Male 12 (41%)

Initial SAH symptoms, n (%)

Headache	10 (34.5%)
Impairment of consciousness	9 (11.3%)
Nausea and vomiting	6 (20.7%)
Focal neurologic deficit	4 (13.8%)
Aneurysm location, n (%) Anterior communicating artery	10 (34.5%)
Posterior communicating artery	2 (6.9%)
Middle cerebral artery	2 (6.9%)
Other locations	6 (20.7%)
No aneurysm at CT angiography	3 (10.3%)
Not having CT angiography	6 (20.7%)
Systolic blood pressure (mm Hg)	139 +- 30	(b120)
Diastolic blood pressure (mm Hg)	85 +- 16	(b90)
Pulse rate (beats per minute)	78 +- 24	(60-100/min)
Respiratory rate	16 +- 6	(12-20/min)
Complete blood count
WBC count (/mm3)	15 030 +- 5651	(4100-11200)
Hemoglobin level (g/dL)	14 +- 2	(11.7-15.5)
Thrombocyte (1000/uL)	251 690 +- 62 254	(159 000-388 000)
Biochemical features Sodium (mEq/L)	138.1 +- 4.1	(135-145)
Potassium (mEq/L)	3.9 +- 0.5	(3.5-5.5)
Chloride (mEq/L)	103.3 +- 5.4	(99-110)
Calcium (mg/dL)	8.4 +- 0.8	(8.1-10.7)
Glucose (mg/dL)	198.1 +- 79.3	(70-110)
BUN (mg/dL)	16.9 +- 7.1	(5-24)
Creatinine (mg/dL)	0.9 +- 0.3	(0.4-1.4)
CK-MB (ng/mL)	4.9 +- 5.6	(0-3.23)
cTnI (ng/mL)	0.5 +- 1.6	(0-1)
AST (U/L)	34.5 +- 19.9	(8-46)
ALT (U/L)	31.6 +- 23.5	(7-46)
Admission Hunt & Hess grade, n (%)
Grade 1	6 (20.7%)
Grade 2	10 (34.5%)
Grade 3	6 (20.7%)
Grade 4	3 (10.3%)
Grade 5	4 (13.8%)
Final outcome, n (%) Discharged	15 (51.7%)

Exitus 14 (48.3%)

Data are presented as mean +- SD or frequency. Abbreviations: BUN, blood urea nitrogen; AST, aspartate aminotransferase; ALT, alanine aminotransferase.

Table 2

Ischemia-modified albumin, CK-MB, cTnI, TNF-?, MPO, and WBC levels in SAH patients on admission and healthy control group

Parameters Groups P

SAH patients (n = 29) healthy controls (n = 20)

IMA (ng/mL)	37.1 (19.2-331.6)	32.8 (10.5-80.1)	N.05
TNF-? (pg/mL)	7.4 (1.6-26.3)	6.7 (2.4-9.7)	N.05
MPO (pg/mL)	2598.4 (1874.5-3308.8)	2523.1 (2179.6-2960.1)	N.05
CK-MB (ng/mL)	3.0 (1.1-26.0)	2.2 (1.1-3.1)	b.01
cTnI (ng/mL)	0.03 (0.01-8.60)	0.01 (0.01-0.03)	b.05
WBC (/mm3)	14 500 (5800-30 500)	5800 (3500-10 000)	b.001

Data are presented as median (minimum-maximum).

Discussion

Many factors such as cerebral injuries and vasospasm, inflammation, Coronary vasospasm, hypoxia, electrolyte imbalance, and sudden increase in intracranial pressure triggering a sympathetic or vagal discharge due to damage or compression of brain structures may have a role in the development of ECG abnormalities in the patients with SAH [3-6,13]. Recently, neurocardiogenic injury after SAH has been further explained; it includes ECG abnormalities, arrhythmias, MI/myocardial infarction, left ventricular dysfunction, elevations in serum cardiac injury biomarkers, and even cardiac arrest [3-5]. Cardiac injury induced by SAH is associated with an increased risk of heart failure and death [14,15]. In addition to these, ECG abnormalities and elevation of cardiac injury markers such as CK-MB and cTnI were observed in patients after SAH as shown in Fig. 1 and Table 1.

electrocardiographic abnormalities and laboratory evidence of cardiac injury complicate rarely the management of SAH patients in the ED. For this reason, the newest biomarkers are required in early SAH diagnosis and in predicting a cardiac injury in patients after SAH. In relation to the issue, IMA, modified from Human serum albumin, is a biomarker for acute ischemia that is approved by the US Food and Drug Administration [16]. Recent reports focus large interest in new biomarker IMA for an earlier diagnosis of SAH and detection of myocardial injury after SAH. In a study on 18 patients, IMA concentration was significantly high measured in serum samples taken within the first 3 hours in SAH patients according to healthy control [10]. Similarly, in a study on 52 patients presenting to the ED, its level was significantly high determined in blood samples taken within 24 hours of symptom onset in SAH patients

[17]. In contrast to 2 reports, no significant difference was observed for serum IMA elevation at days 1 and 2 after SAH, except for day 7 in relation to cardiac damage after experimental SAH [11]. In our study on 29 patients, no significant difference was observed for IMA values between patients with SAH and control group; however, its levels were mildly high determined in serum specimens taken within 24 hours after SAH. In addition, serum IMA levels in patients were not significantly correlated with the severity of SAH (Hunt & Hess scale), whereas cTnI correlated positively with the Hunt & Hess grade on admission. In addition, CK-MB and cTnI values were elevated in patients with SAH in a statistically significant manner when compared with control group. Actually, CK-MB and cTnI supported by clinical history, physical, and ECG findings are the criterion standard biomarkers for identifying myocardial injury to clinicians, and the latter is used commonly in conjunction with CK-MB and myoglobin to enable a more rapid diagnosis of MI [16,18]. Thus, cTnI is a highly sensitive and specific indicator of myocardial dysfunction in association with SAH [8,19]. As a result of above state- ments, IMA may be considered as a marker of acute Ischemic events not specific for cardiac ischemia or injury, indicating that it is a poor discriminator between ischemic individuals with and without MI in relation to SAH, as IMA has a poor diagnostic value in comparison with CK-MB, cTnI, and WBC tests to differentiate between SAH patients and healthy control as shown in Fig. 2 and its legend.

Patients with SAH have cerebral and extracerebral complications/

symptoms that are correlated with an activated inflammatory response, such as fever, leukocytosis, and elevated levels of inflammatory cytokines [3,20-22]. By the way, TNF-? is a potent Proinflammatory cytokine produced upon stimulation by monocytes, macrophages, lymphocytes, neutrophils, endothelial, and smooth muscle cells in response to inflammatory in cerebral aneurysm formation and SAH [23,24]. Hopkins et al [25] found detectable levels of TNF-? in only 30% of patients’ cerebro- spinal fluids and 10% of patients’ plasma samples after SAH. In our study, TNF-? level in serum specimens taken within 24 hours after SAH was higher measured by mean values of 9.5% in SAH patients compared with control, but no significant difference was observed for its values between the groups. In this condition, it is most likely to increase concen- trations of TNF-? in the circulation at 2 to 5 days after SAH in the rate of the severity of SAH, as the TNF-? signaling pathway plays a major role in the pathogenesis of SAH and it mediates hemolysis-induced vasoconstriction and cerebral vasospasm evoked by SAH [23,26]. Myeloperoxidase is a lysosomal enzyme found in leukocytes and

Fig. 1. Neurocardiogenic injury findings in patients after SAH in the present study.

Fig. 2. Diagnostic values of IMA, CK-MB, cTnI, WBC, TNF-?, and MPO tests to differentiate between SAH patients and healthy control group.

Legend for Fig. 2

Test Result Variable(s)	Area Under the Curve	Std. Error	P(b)	Asymptotic 95% Confidence Interval Upper Bound Lower Bound
IMA	.602	.082	.230	.442	.762
CK-MB	.724	.071	.008	.584	.864
cTnI	.693	.075	.023	.546	.840
WBC	.972	.020	.000	.933	1.012
TNF-?	.505	.084	.951	.341	.670
MPO	.510	.083	.903	.348	.673

^bNull hypothesis: true area = 0.5

especially in neutrophils. Elevated MPO levels in serum have been presented to be correlated with cerebral vasospasm in patients after SAH [22]. Myeloperoxidase activity is also high measured in the meaning of an inflammatory biomarker in human unruptured intracranial aneurysm tissues by Gounis et al [27], indicating an association with risk of aneurysm ruptures. In the current study, we might not find a significant relationship with MPO levels in serum samples taken within 24 hours after SAH with respect to controls. Leukocyte count is a marker of systemic inflammation, and levels of WBC frequently elevate in the onset of many ischemic conditions such as MI, mesenteric, and brain ischemia. Likewise, high WBC at baseline was associated with increased incidence of SAH and leukocytosis implied as an independent risk factor for cerebral vasospasm after SAH [21,28]. White blood cell in the blood was higher measured in patients presenting SAH compared with control; related to this, WBC and cTnI were significantly correlated with the severity of patients’ SAH on admission in this study. According to receiver operating characteristic analysis results, WBC has also the highest diagnostic value for discriminating between individuals in SAH and healthy controls. As a result of these findings, WBC in the blood samples taken within 24 hours after SAH may be useful as diagnostic or severity indicator in patients; its high levels along with cTnI elevations in SAH might predict a cardiac injury in association with clinical severity of patients.

Limitations

The present study has some limitations. The study is limited by the small patients’ size, sitting on the fence our attention to declare strong conclusions. Our findings should be confirmed in a large prospective

cohort study on admission to the ED; investigation using different time intervals (eg, 0-4, 4-8, and 8-12 hours after SAH) would contribute to a more complete understanding of the role of IMA, TNF-?, and MPO in patients after SAH as well.

Conclusions

Ischemia-modified albumin, TNF-?, and MPO levels were not statis- tically different in patients with SAH compared with healthy controls. These parameters in the circulation were not correlated with CK-MB, cTnI, and the severity of SAH on admission; however, cTnI elevation after SAH is associated with an increased risk of cardiac manifestations. Therefore, IMA, TNF-?, and MPO are not a predictor of the ability to show a cardiac Ischemic injury in patients with SAH within 24 hours, except for high WBC along with cTnI elevation according to the severity of SAH.

Acknowledgments

The authors would like to thank Prof Dr A Tevfik Sunter for statistical advice and did not receive a financial support from any funding agency in the commercial and profit sectors for this research.

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