The effect of oxidant-antioxidant balance on the one-year prognosis of patients with acute ischemic stroke: a case-control study

Stroke is a major cause of mortality and morbidity. Also, free radicals and oxidative stress are deleterious factor in the stroke progression. We aimed to evaluate the association between oxidative stress markers and odds of having risk factor for stroke or developing stroke. The present case control study conducted on 556 participants in Imam-Reza hospital, Tabriz, Iran. Subjects were divided into three group, including individuals with acute ischemic stroke, at risk of stroke, and healthy controls. All enrolled participants except for controls underwent neurological examinations and brain magnetic resonance imaging (MRI). Stroke-related disability and stroke severity were evaluated by modied Rankin Scale (mRS) and National Institutes of Health Stroke Scale (NIHSS), respectively. Serum malondialdehyde (MDA) level and total antioxidant capacity (TAC) were measured within 48 hours of stroke. One-way ANOVA and Chi-square tests for comparing characteristics between groups, multivariable logistic regression for odds of stroke based on MDA and TAC quartiles, and Spearman’s correlation were used.


Background
According to the ndings Global Burden of Disease (GBD) study, the disability-adjusted life-years (DALYs) attributable to stroke was 116.4 million (95% uncertainty interval (UI): 111.4, 121.4) in 2016 globally, which hemorrhagic stroke had a higher proportion than ischemic one (1). It was estimated that 80.1 million prevalent cases of stroke in 2016 worldwide (1). Over 1990-2016, the age-standardized incidence and mortality rate of stroke declined by 36.2% and 8.1%, respectively, however, it is stroke is the second cause of leading death in the world after cardiovascular diseases (2)(3)(4). The incidence of stroke initiated to continuously increase form the age of 30 and the it is higher in men, whereas it is not prominent (1).
Oxidative stress is de ned as an imbalance between pro-and anti-oxidants which has implicated in the pathogenesis of several chronic diseases such as stroke (5). It plays an important role in the central nervous system and can directly cause tissues damage through several mechanisms (6). The brain uses glucose almost exclusively as its source of energy, and due to low capacity of energy storage in the brain, it requires a steady ow of blood and glucose (7). The low blood ow decreases the amount of oxygen and glucose, which follows a cascade of events that leads to production of reactive oxygen species (ROSs) and free oxygen radicals (7,8). ROSs are necessary for various functions such as a vascular tunic, oxygen pressure monitoring, and erythropoietin production in low concentrations. In contrast, excessive amounts of oxidants may irreversibly oxidize macromolecules and cause severe cell injury (9). Antioxidant defense system is a special mechanism of dealing with damages induced by free radicals in the body. Healthy persons have a balance between the production of free radicals and antioxidant defense system, but a disruption in this balance, induces the oxidative stress that contribute to progression of stroke (10,11).
The oxidative stress can be measured using the oxidized products of macromolecules such as nucleic acids, lipids, proteins, and deoxyribonucleic acid (DNA). Lipid peroxides are unstable lipid radicals, which are derived from the oxidation of polyunsaturated fatty acids and can be converted to a different composition such as malondialdehyde (MDA) (12). MDA can cause irreversible disruption of the enzymes, receptors and membrane transfer mechanisms (13). In addition, it has been shown a direct correlation between increases in MDA and poor functional recovery in acute ischemic stroke (14). Total antioxidant capacity (TAC) measurement is a useful tool for the evaluation of the antioxidant capacity to prevent and protect against oxidative damage to membranes and other cellular components (15).
In the present study, the importance of oxidative stress role in the pathogenesis of acute ischemic stroke is taken into account. To our best of knowledge, no study has examined the long-term effects of oxidative stress on the clinical outcomes of stroke patients. The aim of this study was to investigate changing in markers of oxidative stress and antioxidant capacity to nd if there is any correlation between those and risk of stroke. Also, the correlation between severity and disability of stroke and biochemical markers were assessed.

Subjects and Design
The present case control study was conducted in Imam-Reza hospital in Tabriz, Iran from March 2017 to June 2019. Subsequently, 216 patients with stroke, 152 patients at risk of stroke, and 188 healthy controls matched for age and sex were included. Patients with stroke needed to have a de nite diagnosis of stroke by a physician using magnetic resonance imaging (MRI) with diffusion-weighted imaging (DWI). Inclusion criteria for group of at risk population was to have at least one of the underlying diseases, including hypertension, type diabetes, and hyperlipidemia. Exclusion criteria were history of hemorrhagic infarction, nervous system diseases, chronic diseases such as chronic kidney, liver, and biliary tract diseases, infectious and autoimmune diseases, antioxidant intake over the past three months, and smoking.
Written informed consent was obtained from all of the participants at the beginning of the study. The study protocol was approved by the ethics committee of Tabriz University of Medical Sciences (Ethics number: TBZMED 94/3-4/3).

Clinical assessments
An expert neurologist underwent neurological examinations for all enrolled cases, and further evaluation by brain MRI with DWI in order to con rm an acute stroke. Ischemic stroke was de ned as focal neurologic de cits due to vascular causes lasting more than 24 hours and could not be explained by other causes (16). Stroke-related disability and stroke severity were evaluated by modi ed Rankin Scale (mRS) and National Institutes of Health Stroke Scale (NIHSS), respectively (17,18).

Biochemical assessments
Blood samples were collected from participants within 48 hours following the stroke. Serum MDA level was measured using the thiobarbituric acid reactive substance (TBARS) assay (Radioimmunoassay Kit). TAC was measured by the values extracted from ferric-reducing antioxidant power (FRAP) assay that was adjusted based on Iranian foods. The ability of dietary antioxidants for reducing ferric to ferrous ion is calculated by FRAP and are expressed by mmol per 100 g of foods (19).

Statistical Analysis
The included participants were classi ed into quartiles based on their TAC and MDA. One-way analysis of variance (ANOVA) and Chi-square test were used to compare general and demographic characteristics between the three groups for continuous and categorical variables, respectively. Multivariable logistic regression in two different models, without adjustment and with adjustment for age, sex, and body mass index (BMI), were used in order to evaluate the relationship between TAC and MDA and odds of stroke or having risk factors for stroke. Correlation between all the variables were assessed with the Spearman correlation coe cient. P values less than 0.05 were considered as statistical signi cance. The Statistical Package for the Social Sciences (SPSS) version 22.0 (SPSS Inc., Chicago, IL, USA) was used for performing statistical analysis.

Baseline characteristics
A total of 556 participants were included in this study (216 in stroke group; 152 in group at risk of stroke; and 188 individuals in control group). Overall, the mean age of participants was 72.32 years and 41.01% were females. Moreover, we found a higher mean serum concentration of TAC in the patients at risk of stroke compared to healthy and stroke patients (3909.29 µmol/L in at risk group vs. 3905.98 µmol/L in controls and 3909.29 µmol/L in stroke group). Nevertheless, the mean serum MDA level was lower in the at risk of stroke group than the control group (1.75 vs. 1.85 µmol/L) ( Table 1). The baseline characteristics of participants in each group are presented in Table 1. A signi cant difference was shown among patients with stroke and control group in term of baseline systolic blood pressure (SBP) and diastolic blood pressure (DBP), which were signi cantly higher in both stroke group and at risk of stroke group than healthy controls. In addition, we found signi cantly increased levels of triglyceride (TG) and total cholesterol in the stroke group than healthy ones (P < 0.001) ( Table 2).

Predictors of stroke development
No signi cant association was found between quartiles of serum TAC and odds of stroke neither without adjustment for confounding factors nor after adjustment for them (P = 0.12 without adjustment; P = 0.14 after adjustment). While, levels of serum MDA were signi cantly associated with development of stroke before and after adjustment for confounding factors. Looking at the quartiles of dietary TCA showed that participants in the second quartile had a reduced odds of stroke by 71% (odds ratio (OR) = 0.29; 95% con dence interval (CI): 0.09-0.94)). Moreover, increasing the levels of serum MDA was associated with increasing risk of stroke development. In this regard, after adjustment for age, sex, and BMI, the third and fourth quartile had ORs of 7.98 (95% CI: 1.94, 32.80) and 11.97 (95% CI: 2.74, 52.35), respectively (Table 3).

Discussion
Findings of this hospital-based case control study demonstrated that TAC levels despite MDA levels, which were higher in patients with stroke, were lower in this group. Also, it showed that MDA levels is a better predictor of stroke development than TCA, while none of these measures was signi cantly associated with having risk factors for stroke. Furthermore, we found a negative correlation between clinical tools, NIHSS and mRS, and chemical measures, TAC and MDA.
Numerous studies have investigated the TAC and MDA levels in stroke patients and showed that serum TAC levels in stroke cases were signi cantly lower (20,21) and MDA levels were higher than the control group (22,23). A case-control study on 195 hospitalized cases with stroke and 195 healthy controls in Iranian populations which were categorized into three groups showed that the top tertile of dietary TAC had lower chance to have stroke than the bottom tertile (OR = 0.49 (95% CI: 0.23, 1.00)), although our study revealed a signi cant protective association between the bottom quartile of TCA and stroke (OR = 0.29 (95% CI: 0.09-0.94)) (24). The discrepancy might be due to different methods for determining categories. Moreover, the article by Guldiken et al. which categorized participants into diabetic stroke, nom-diabetic stroke, and healthy controls showed that TAC levels were signi cantly higher in diabetic acute stroke patients than in non-diabetic ones (10.03 vs. 5.97 mM; P < 0.001) and was higher in diabetic patients with stroke compared to control group (10.03 vs. 5.44 mM; P < 0.001) (25). Opara et al. found that the total antioxidant capacity was depleted in the diabetic patients compared to normal subjects (26). On the contrary, Savu et al. showed that the TAC of plasma, despite of high oxidative stress levels, was increased in patients with uncomplicated type diabetes (27). In the present study, the TAC levels of the patient at risk of stroke did not show any signi cant difference from control group, whereas it was higher than the stroke patients and healthy group, which might be due to the different assays for determination of TAC.
The study by Al-Rawi et al. conducted on 50 patients with ischemic stroke, 75 participants with a risk factor for stroke, including diabetes, hypertension, and ischemic heart disease, and 25 healthy individuals. MDA levels were measured in the serum and saliva of subjects and showed that MDA levels in both groups were signi cantly higher than the healthy group (P < 0.001) (28). Our ndings con rmed that MDA has a signi cant increasing association with stroke occurrence, while this association was not signi cant in patients who were at risk of stroke. We propose that signi cant increase in MDA level in stroke is a re ection of increased MDA production and oxidative stress in cerebral ischemia since the top quartile of MDA was in higher risk of stroke compared to the bottom quartile, although they were not signi cant.
A case-control study on 50 patients with stroke and 50 healthy controls represented higher levels of MDA in cases than controls (3.31 vs. 1.62 nmol/ml; P < 0.0001) (29). In this regard, the article by Bir et al. showed signi cantly greater MDA values in both atherothrombotic ischemic stroke and with lacunar infarction compared to healthy controls (P < 0.001) (30). It has been suggested that blood or neural lipids may be the source of lipid peroxidation caused by ischemia. In addition, during ischemia, increased cytosolic calcium leads to the activation of phospholipases and proteases, which leading to conversion of xanthine dehydrogenase to xanthine oxidase or activation of protein kinase. Consequently, these activated enzymes can also be the cause of the increased free radicals (31).
This study also compared the correlation between TAC or MDA with NIHSS-baseline, NIHSS-follow-up, mRS-discharge, and mRS-follow-up. A cohort study on 42 patients with acute ischemic stroke found no signi cant association between severity of stroke based on baseline NIHSS and level of MDA (P = 0.60), whereas there was a signi cant positive correlation between level of MDA and mRS after three months of follow-up (r = 0.54; P = 0.001) (32). In addition, Yaseen et al. revealed that level of MDA on the 7th day had a positive correlation with NIHSS and mRS scores at 7th day (r = 0.335; P = 0.024 for NIHSS and r = 0.342; P = 0.022) (33). We found a negative correlation between levels of MDA and TAC and NIHSS and mRS. Our ndings is in accordance with a study on 34 ischemic stroke patients and 34 healthy controls that showed a negative correlation between total antioxidant status (TAS) and NIHSS values, even though it was not signi cant (r= -0.17; P = 0.34) (34). Moreover, another study on acute ischemic stroke patients and healthy controls showed that TAC levels were negatively correlated with NIHSS scores (r= -0.38; P = 0.02) (21). The differences in results of our study with mentioned articles can be due to differences in methods of measurement of factors, especially oxidative stress parameters, time to assess the values, and study participants.
The strength of this study is that it is among pioneer studies which included a group of participants who were potentially at risk of having stroke, while several previous studies compared serum levels of oxidative markers only between stroke cases and healthy controls. However, our study had some limitations. First, we adjusted multiple logistic regression test by age, sex, and BMI, while other potential confounding and risk factors, especially atrial brillation for stroke were not included in our analysis (35). Second, selection and recall bias could have in uenced the results because of susceptibility of casecontrol studies. Third, we could not reach to a cause-effect relationship because of observational design of this study. Fourth, body composition might have effects on in ammatory factors (36), while the study did not include data on some body composition components measures such as fat mass.

Conclusions
In the light of present ndings, it seems that MDA is a better predictor of stroke than TCA, while both TCA and MDA might not be recommended to use for prediction of having stroke risk factors. In addition, TCA and MDA had negative association with severity and disability of stroke. Altogether, it will be worthwhile to pursue oxidative stress role in stroke pathogenesis and it is needed to be design further large-scale studies to investigate TAC and MDA role in stroke patient clearly.

Declarations
Ethics approval and consent to participate The study was approved by the ethics committee of Tabriz University of Medical Sciences (Ethics number: TBZMED 94/3-4/3). Our study was implemented in accordance with the ethical standards of the 1964 Declaration of Helsinki and its lateral amendments. All participants had been given the written informed consent.

Consent for publication
Written informed consent was obtained from all of the participants at the beginning of the study.

Availability of data and materials
The datasets generated and/or analyzed during the current study are not publicly available due for they are personal data but are available from the corresponding author on reasonable request.