Original Contribution

ischemia-modified albumin levels in cerebrovascular accidents

Abdulkadir Gunduz MD^a,?, Suleyman Turedi MD^a, Ahmet Mentese MS^b, Vildan Altunayoglu MD^c, Ibrahim Turan BS^b, Suleyman Caner Karahan MD^b, Murat Topbas MD^d, Murat Aydin MD^a, Ismet Eraydin MD^a, Buket Akcan BS^b

^aDepartment of Emergency Medicine, Karadeniz Technical University Faculty of Medicine, Turkey ^bDepartment of Biochemistry, Karadeniz Technical University Faculty of Medicine, Turkey ^cDepartment of Neurology, Karadeniz Technical University Faculty of Medicine, Turkey ^dDepartment of Public Health, Karadeniz Technical University Faculty of Medicine, Turkey

Received 12 September 2007; revised 16 November 2007; accepted 19 November 2007

Abstract

Background: Previous studies have demonstrated that ischemia-modified albumin is a useful marker for the diagnosis of Ischemic events. It was also recently demonstrated that IMA levels increase in the acute phase of cerebrovascular diseases. Yet the data regarding IMA levels in various types of Cerebrovascular events are insufficient. The aim of this study was to evaluate IMA levels in various types of cerebrovascular events such as ischemic stroke, subarachnoid hemorrhage , and intracranial hemorrhage.

Methods: This case-controlled study consisted of 106 consecutive patients, 43 with Brain infarction (BI), 11 with Brain hemorrhage (ICH), 52 with SAH, and a 43-member control group. We investigated whether there was a statistical correlation between these 3 groups and the control group. The relations among the 3 groups were also examined. Comparisons among groups were done with analysis of variance.

Results: Mean serum IMA levels were 0.280 +- 0.045 absorbance units (ABSU) for BI patients,

0.259 +- 0.053 ABSU for ICH patients, 0.243 +- 0.061 ABSU for SAH patients, and 0.172 +- 0.045 ABSU for the control group.There was a statistically significant difference between the mean IMA levels of BI, ICH, and SAH patients and the mean control patient IMA levels (P b .0001). Conclusions: Ischemia-modified Albumin levels are high in cerebrovascular diseases. Ischemia- modified albumin measurement can also be used to distinguish SAH from BI during the acute phase of cerebrovascular event in the emergency department.

(C) 2008

Introduction

* Corresponding address. Acil Tip AD, Karadeniz Teknik Universitesi Tip Fakultesi Hastanesi, Trabzon, 61080, Turkey. Tel.: +90 0462 377 5715;

fax: +90 0462 325 12 46.

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

A number of difficulties may be experienced in the diagnosis of patients with acute stroke and acute subar- achnoid hemorrhage (SAH) presenting to the Emergency Department (ED). Acute BI generally exhibits no radiologi- cal findings in the early stage. Patients presenting within 3

0735-6757/$ - see front matter (C) 2008 doi:10.1016/j.ajem.2007.11.023

hours of an acute ischemic stroke often have a normal computed tomographic (CT) scan result; but diffusion- weightED magnetic resonance imaging (MRI), CT perfusion of brain, and cerebral angiography can confirm the diagnosis at this early stage [1]. Similarly, there may be no radiological signs in some SAH patients [2].

Another approach to the diagnosis of cerebrovascular accident would be to use biochemical markers. Measurement of brain-specific proteins in peripheral blood may overcome these concerns. Serum levels of glial and neuronal proteins are elevated after stroke, traumatic brain injury, and SAH [3- 5]. During acute ischemic conditions, the metal-binding capacity of albumin for transition metals, such as copper, nickel, and cobalt, is reduced, generating a metabolic variant of the protein, commonly known as ischemia-modified albumin (IMA) [6]. Ischemia-modified albumin is a sensitive marker of myocardial ischemia, skeletal muscle ischemia, pulmonary embolism, mesenteric ischemia, and stroke [7- 12]. It is a new marker used to rule out myocardial ischemia and has been approved by the US Food and Drug Administration [13]. Recently, it was also found to be a biomarker for early identification of acute stroke [12]. However, the usefulness of IMA measurement in clinical practice and diagnosis of certain type of cerebrovascular event is still uncertain.

The aim of this study was to compare IMA levels in various types of cerebrovascular diseases such as brain hemorrhage (ICH), SAH, and ischemic stroke to case-control patients presenting to the ED.

Patients and methods

Laboratory analysis“>This case-controlled study consisted of 106 consecutive patients, 43 with BI, 11 with ICH, 52 with SAH, and a 43- member control group. It was performed in the ED of the Karadeniz Technical University Hospital, Turkey. The protocol for the study was approved by the hospital’s ethics committee, and the study period ran from May 2006 through June 2007.

Patients with documented or clinical evidence of BI, ICH, or SAH were included in the study. Ischemic stroke was defined as focal neurologic deficit lasting longer than 24 hours with ischemic lesion at CT/MRI. Patients whose focal neurologic deficit recovered in 24 hours in the absence of CT/MRI findings in the appropriate region were regarded as transient ischemic attack and excluded from study. Patients whose focal neurologic deficit lasted longer than 24 hours and whose CT scan did not reveal ischemia in the appropriate brain region were further investigated with MRI; and in case of MRI evidence of BI, patients were included in the study. Intraparenchymal hemorrhage was diagnosed by performing CT scan on admission. Patient with SAH was diagnosed with the determination at CT of hemorrhage in the subarachnoid region. Diagnosis of SAH was confirmed by performing

lumbar puncture, but not all patients had invasive or noninvasive angiography. Patient with Normal CT scan but hemorrhagic cerebrospinal fluid (CSF) at lumbar puncture were not excluded in the study. The presence of any sta- tistical correlation among the 3 patient groups and the control was investigated. The relations among all 3 patient groups were also examined.

Demographic characteristics, vascular risk factors in patients’ histories (diabetes mellitus, hypertension, coronary artery disease), systolic and diastolic blood pressure values, and biochemical blood values (full blood count, Blood sugar level, kidney function tests, total protein, serum albumin) on admission were recorded in detail. Blood samples for IMA determination were taken within 24 hours of symptom onset. Neurologic examinations were performed on the day of admission. The National Institutes of Health Stroke Scale was used to determine Stroke severity and patients’ neurologic deficits. Blood specimens for the determination of IMA levels were taken at the same time

as neurologic examinations.

Patients were excluded from the study if they had other ischemic diseases, such as acute coronary syndrome, acute myocardial infarction, pulmonary embolism, or an abnormal Serum albumin level. Abnormal serum albumin levels (level b3.5, N5.5 mg/dL) made the determination of IMA levels impossible. Patients were also excluded because of advanced hepatic, renal, or cardiac insufficiency; or troponin T and electrocardiogram abnormalities suggestive of acute coron- ary syndrome. Patients were further excluded if age was b18 years or if they refused to participate in the study. A control group of 43 nonhospitalized ED patients (mean +- SD age, 57 +- 8 years) served as a reference for Biochemical parameters. The exclusion criteria for the control group were the same as those for the patient group.

Laboratory analysis

Blood samples were drawn on admission. serum samples were prepared by 15 minutes of centrifugation at 3000 rpm. Specimens to be used for measuring IMA serum concentra- tions were pipetted into Eppendorf tubes and stored at -80?C. Reduced cobalt to albumin binding capacity (IMA level) was analyzed using the rapid and colorimetric method of Bar-Or et al [7]. Two hundred microliters of patient serum was placed into glass tubes, and 50 uL of 0.1% CoCl₂ 6H₂O (Sigma, St Louis, MO) was added. After gentle shaking, the solution was left for 10 minutes to ensure sufficient cobalt-albumin binding. Afterward, 50 uL of

1.5 mg/mL dithiothreitol (DTT) was added as a coloring agent. After waiting for 2 minutes, 1 mL of 0.9% NaCl was added to halt the cobalt-albumin binding process. A colorimetric control specimen was prepared for every Limb ischemia and control patient specimen. For the colorimetric control samples, 50 uL of distilled water was substituted for 50 uL of 1.5 mg/mL DTT. Specimen absorbencies were analyzed at 470 nm using a spectro-

photometer (Shimadzu UV1601, Tokyo, Japan). The color of the DTT-containing specimens was compared with that of the colorimetric control tubes. The results were reported as absorbance units (ABSU).

Statistical analysis

Statistical analysis was performed using SPSS for Windows, release 11.0 (SPSS, Chicago, IL). Results are expressed as mean +- SD.

Data normality was assessed by the Kolmogorov- Smirnov test. Comparisons among groups (SAH, BI, ICH, and control) were done with analysis of variance (Bonferroni test as post hoc test) for IMA level normally. Statistical significance was assumed at a level of P b .05.

Receiver operating characteristic curve analysis was done for determination of specificity and sensitivity of the cutoff values of all patients.

Results

One hundred six patients presenting to the ED with BI, ICH, or SAH were included in the study. Ten patients were excluded on the basis of predefined criteria: acute coronary syndrome (n = 1), advanced kidney insufficiency (n = 3), advanced heart insufficiency (n = 1), and low albumin levels (n = 5).

The general clinical characteristics of the 106 patients in the SAH, BI, and ICH groups are shown in Table 1.

Serum samples were collected from the 43 healthy (control) individuals and the 43 patients with BI, 11 with ICH, and 52 with SAH.

The mean serum IMA level was 0.280 +- 0.045 ABSU for BI patients, 0.259 +- 0.053 for ICH patients, and 0.243 +-

0.061 for SAH patients. The mean serum IMA level for the control patients was 0.172 +- 0.045. There was a statistically

Table 1 Clinical characteristics of BI, ICH, and SAH patients

Mean

3.90

3.78

3.95

Group	BI (n = 43)	ICH (n = 11)	SAH (n = 52)
Age (y)
Median	71	62	56
Minimum-maximum	44-87	48-83	36-84
Sex (%)
Male	23 (54%)	8 (73%)	24 (46%)
Female	20 (46%)	3 (27%)	28 (54%)
Hypertension	30 (69%)	7 (63%)	32 (61%)
Diabetes	5 (11%)	1 (9%)	7 (13%)
Vascular history	20 (46%)	1 (9%)	4 (8%)
NIHSS
Mean	15	23	9
Albumin level (g/dL)

Fig. 1 The ROC curves for all patients (SAH, BI, and ICH patients).

significant difference between the mean BI, ICH, and SAH patient group and mean control patient IMA levels (P b

.0001). There was also a statistically significant difference between mean SAH patient and BI patient levels (P b.0001). There was no statistically significant difference, however, between the mean BI and ICH group levels (P b .05).

For all of patients’ ROC curve analysis (SAH, BI, and ICH patients), the area under the curve for IMA was 0.845 (bootstrap 95% confidence interval [CI], 0.780-0.911). The optimum diagnostic cutoff point maximizing sensitivity and specificity was 0.190 ABSU, with a sensitivity of 86.8% and a specificity of 60.5% (Fig. 1). The serum absorbance values of BI, ICH, and SAH patients and healthy individuals are shown in Fig. 2.

Discussion

Ischemia-modified albumin is a nonspecific marker of tissue ischemia, which has previously been studied in patients with acute chest pain and has been shown to be higher in patients with myocardial ischemia, either sponta- neously or subsequent to percutaneous coronary intervention [6,7]. Zapico-Muniz et al [14] and Roy et al [15] have reported that despite the high negative predictive value of a negative IMA level in acute coronary syndrome, its cardiospecificity is so far unproven. They also determined normal IMA level as 85 and 101 kU/L, respectively.

Extracardiac oxidative stress may elevate IMA levels and, therefore, limit the usefulness of elevated IMA levels in the detection of cardiac ischemia [15]. Gunduz et al determined elevated IMA levels in mesenteric ischemia

[11]; and Turedi et al, in pulmonary embolism [10]. Ischemia-modified albumin is also elevated in patients with chronic claudication, aortic cross-clamping, and cross- clamping during arterial reconstruction [16]. In a study of

Fig. 2 Serum absorbance values of SAH, BI, and ICH patients and healthy individuals.

marathon runners, Apple et al determined no immediate change in serum IMA. However, they found an elevation of IMA at 24 to 48 hours [13]. Ischemia-modified albumin levels in acute stroke were recently studied by Abboud and colleagues. They found significantly higher IMA levels in intracerebral hemorrhage and BI patients without any difference between ischemia and hemorrhage groups [12]. They also found significant correlation between baseline IMA levels and NIHSS of patients. The results of our study were similar to this study. However, in addition to ICH and BI, we also studied SAH patients and compared IMA levels between the 3 different stroke subtypes. The highest mean IMA levels in our study were determined in patients with BI followed by those with intracerebral hemorrhage, the lowest levels being in those with SAH. There was a statistically significant difference found between IMA levels in BI and SAH groups. Ischemia-modified albumin levels had not previously been investigated in the SAH patient group. That there is relatively less cerebral parenchymal ischemia patients with SAH may explain the lower IMA levels in these patients.

During ischemia/reperfusion, modifications altering the binding capacity of albumin for cobalt may occur as the result of acidosis, reduced oxygen tension, and the genera- tion of free radicals [7]. Several studies revealed that free radical production increases in stroke, particularly with ischemia and reperfusion [17]. During the oxidative stress occurring in acute stroke, the combined production of Reactive oxygen species with free radicals and the passage through an impaired brain-blood barrier may partly account for IMA formation [17-19].

In conclusion, IMA levels are higher in patients with acute cerebrovascular diseases compared with healthy individuals, although IMA is not able to distinguish between intracerebral hemorrhage and ischemic stroke. Ischemia-

modified albumin levels may be used to differentiate SAH and BI especially for clinically challenging and CT-negative patients. This study also suggests that elevated IMA levels may help identify patients with SAH, a potentially devastat- ing condition without useful biomarkers that is often missed on initial presentation. Because IMA measurement can easily be performed and results can be quickly obtained, it can be of clinical use in the ED. Before interpretation of IMA levels, patient’s serum albumin levels should be determined. This study is the first to compare SAH, BI, and ICH patient groups and a healthy control group. There is now a need for a wider series of studies, particularly because of the low numbers of patients in the ICH group.

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