(Stroke. 1997;28:2425-2428.)
© 1997 American Heart Association, Inc.
Articles |
From the Department of Neurology, University of Heidelberg (Germany).
Correspondence to Matthias Spranger, MD, Department of Neurology, University of Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany.
| Abstract |
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Methods We measured SOD activity in the serum of 41 patients with acute ischemic stroke with a chemiluminometric assay based on the generation of oxygen free radicals by xanthine and xanthine oxidase.
Results SOD activity was significantly lower in patients with ischemic stroke than in age-matched control patients with nonvascular, neurological illnesses (n=24; P<.034, Wilcoxon rank test). The activity was inversely correlated with the size of infarction on CT (P=.01, Spearman correlation) and the severity of neurological deficits (P<.001, Spearman correlation). The decreased SOD activity recovered within 5 days after stroke to values found in serum of control patients.
Conclusions Our data suggest that the SOD activity in serum is reduced in stroke patients, and replacement of antioxidative activity could be beneficial in the acute treatment of cerebral ischemia.
Key Words: central nervous system free radicals stroke, acute superoxide dismutase
| Introduction |
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These constantly produced superoxide radicals are scavenged by a number of antioxidant enzymes, including superoxide dismutase (SOD), glutathione reductase, and catalase. Additionally, chemical antioxidants such as glutathione, ascorbic acid, and vitamin E are also likely to be involved in the detoxification of free radicals. During reperfusion after ischemia, perturbation of the antioxidative defense mechanisms is a result of the overproduction of oxygen radicals, inactivation of detoxification systems, and consumption of antioxidants.
Investigations in animal models of cerebral ischemia suggest a particular role of SOD in the reperfusion injury.4 5 6 However, the reports of the effect of cerebral ischemia on SOD expression and activity in vivo are contradictory. A small decrease in SOD activity was observed 7 days after middle cerebral artery occlusion in a rat model of focal ischemia.7 This was thought to be the consequence of ongoing additional damage to the peri-infarct tissue. Also, in gerbil focal ischemia/reperfusion and global ischemia models, SOD activity in the cerebral tissues decreased by approximately 20% when assayed by the chemiluminescence method.8 Other investigators found an increased immunoreactivity against mitochondrial Mn-SOD and cytosolic CuZn-SOD after transient forebrain ischemia in neurons of the gerbil hippocampus9 and increased SOD activity in the rat after global ischemia.10 Matsumiya and collaborators11 found increased mRNA levels but decreased protein concentrations of CuZn-SOD in the CA1 region of the hippocampus after transient forebrain ischemia in the cat, which was thought to reflect a less functional antioxidant system in the vulnerable CA1 neurons. These contradictory results might be caused by the different SOD isoforms, methods, and animal models used to investigate the impact of cerebral ischemia on SOD levels and activity.
In the present study we measured the SOD activity in the serum of patients with stroke and correlated the findings with both functional outcome and infarct size.
| Subjects and Methods |
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The study was approved by the local ethics committee. Patients with (1) concomitant cardiac, renal, hepatic, or cancerous disease; (2) recent head trauma; (3) recent history of transient ischemic attacks; or (4) CT and/or MRI results inconclusive for the location of the ischemic lesion were excluded from this study. All patients were evaluated by CT and/or MRI on day 1 and on day 4 after stroke.
Clinical examination was performed on admission and daily thereafter and was scored according to the 58-point Scandinavian Stroke Scale (SSS).12 13 On admission and 10 days after stroke, the neurological deficit was assessed by the SSS.
Blood samples were drawn between 8 AM and noon from indwelling venous catheters within the first 24 hours after stroke and again 72 hours, 5 days, and 10 days thereafter. Samples were immediately centrifuged (1500g, 10 minutes), and serum was stored at -80°C until assayed. Serum SOD activity was measured with a chemiluminometric method described in detail before.14 Briefly, xanthine oxidase (grade III from buttermilk, 420 mU/mL, Sigma), 0.1 mmol/L lucigenin (Sigma), and serum samples were diluted in 50 mmol/L potassium phosphate buffer, pH 10.0. Serum samples were diluted 1:10.4 At this concentration, the influence of natural reducing agents such as ascorbate or epinephrine is excluded in the assay,14 and the inhibition of chemiluminescence is caused by SOD. Mn-SOD (Sigma) dilutions or buffer was used as control. The superoxide-producing reaction was initiated by the automated dispensing of 60 µL of 1.45 mmol/L xanthine. All reagents were freshly prepared in 50 mmol/L potassium phosphate buffer, pH 10.0. The buffer and xanthine solutions were kept at room temperature, the other solutions on ice. Chemiluminescence was measured for 20 minutes in 1-minute cycles in BioLumat LB 9501. Mean blank values given by cuvettes without xanthine oxidase were subtracted from the peak counts per minute. Results are expressed as micrograms per milliliter SOD according to a standard curve with bovine Mn-SOD.
Statistical Analysis
For statistical analysis we used the Wilcoxon
rank test and Spearman correlation, as appropriate. P<.05
was assigned as statistically significant. Data are given as
mean±SEM.
| Results |
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The chemiluminescence triggered by xanthine/xanthine oxidase was
significantly less inhibited by serum of patients after acute stroke
compared with age-matched control patients (1489.12±28.7 versus
1335.9±32.6, mean±SEM; P<.034, Wilcoxon rank
test), indicating less SOD activity in their serum. SOD activity was
decreased most profoundly within 24 hours after onset of neurological
deficits and recovered to values not different from control subjects
within 5 days after stroke (Fig 1
). We found
a significant correlation between the infarct volume on the cranial CT
scan and the SOD activity in the serum obtained within 24 hours after
stroke (P=.01, Spearman correlation). Patients with large
infarcts (>150 cm3) had a higher chemiluminescence and
thus a lower SOD activity than those with small infarcts
(P<.001, Wilcoxon rank test) (Fig 2
). Accordingly, the degree of neurological
deficit of stroke patients scored by SSS on admission correlated well
with the SOD activity (r=.599, P<.001, Spearman
correlation). In patients with low SSS scores on admission a higher
chemiluminescence was observed (Fig 3
).
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| Discussion |
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| Acknowledgments |
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Received July 29, 1997; revision received September 11, 1997; accepted September 16, 1997.
| References |
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