Serum Prolidase Enzyme Activity as a Diagnostic Marker for Acute Ischemic Stroke

Ischemic stroke constitutes about 80-85% of all stroke cases and is caused by the interruption of cerebral blood flow due to a blood clot. Because of reactive oxygen species (ROS) production and its oxidative metabolite activity, the brain is highly sensitive to oxidative stress. This characteristic plays an important role in the pathogenesis of ischemic and hemorrhagic brain injuries .Prolidase, which is a cytosolic exopeptidase and a member of the matrix metalloproteinase (MMP) family, cleaves iminodipeptides from carboxy-terminal ends of proline or hydroxyproline and is actively involved in collagen metabolism. It has been shown that brain MMP activity is correlated with nutritive/oxidative stress and increases during reperfusion. Furthermore, in stroke patients, elevated serum MMP levels have been reported. Biomarkers that predict the outcome and occurrence of ischemic stroke are critically important for prevention and treatment .However, serum prolidase activity (SPA) has not been previously assessed in acute ischemic stroke (AIS) patients to our knowledge. Therefore, in this study, it's aimed to investigate whether SPA levels in AIS patients can be used as a potential diagnostic and prognostic marker.In the study, 37 patients aged between 20 and 85 years who were admitted within 24 hours of the onset of AIS were prospectively evaluated. The control group consisted of 37 healthy volunteers of similar age without any disease. A comprehensive physical examination was performed consisting of a neurological examination, blood biochemistry and blood count tests, electrocardiography, and a posterior-anterior chest X-ray for all patients. AIS patients underwent transthoracic echocardiography, multi-slice computed tomography (CT), and bilateral carotid-vertebral artery Doppler ultrasonography. National Institutes of Health Stroke Scale (NIHSS) scores, measured at 24 hours, 48 hours, and 28 days after stroke, were used to determine stroke severity.Ischemic stroke subtypes classification was done according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria, as cardioembolism, large-artery atherosclerosis, small artery occlusion, stroke of other determined cause, or undetermined cause. The measurement method for SPA was defined by Myara et al. The optimized method of Özcan et al. was used. Prolidase activity was evaluated with a spectrophotometric method, by measuring the proline levels. Briefly, 500 μL pre-incubation solution (50 mmol/L Tris hydrochloride buffer at power of hydrogen (pH) 7.8, with 1 mmol/L endogenous antioxidant glutathione (GSH), 5 mmol/L manganese(II) chloride (MnCl2), and 0.1% Triton X-100) and 100 μL serum were mixed; this mixture was then pre-incubated for 3 h at 37°C. A 100-μL volume of pre-incubation serum was added to 100 μL 144 mmol/L Gly-Pro solution, and this mixture was incubated for 30 min at 37°C. After the incubation, 1 ml 0.45 mol/L Trichloroacetic acid solution was added quickly to the incubation tube, and the incubation reaction was stopped. This mixture was centrifuged at 1500 rpm for 5 min, and 500 μL supernatant was removed.