Non-survivor patients with malignant middle cerebral artery infarction showed persistently high serum malondialdehyde levels

Objective Previously there have been found higher circulating malondialdehyde levels during the first week of ischemic stroke in patients with worst neurological functional outcome, and at moment of ischemic stroke in non-survivor patients. Thus, the aim of our study was to determine the potential role of serum malondialdehyde levels during the first week of a severe cerebral infarction to mortality prediction. Methods This study was observational, prospective, and multicenter. We included patients with a severe malignant middle cerebral artery infarction (MMCAI) defined as patients with computed tomography showing acute infarction in more than of 50% of the territory and Glasgow Coma Scale (GCS) lower than 9. We determined serum concentrations of malondialdehyde on days 1, 4 and 8 of MMCAI. Results Serum malondialdehyde concentrations at days 1 (p < 0.001), 4 (p < 0.001), and 8 (p = 0.001) of MMCAI in non-survivor patients (n = 34) were higher than in survivor patients (n = 34). ROC curve analyses showed that serum malondialdehyde concentrations at days 1, 4, and 8 of MMCAI had an AUC (95% CI) to predict 30-day mortality of 0.77 (0.65–0.86; p < 0.001), 0.82 (0.69–0.91; p < 0.001) and 0.84 (0.70–0.93; p < 0.001) respectively. Conclusions The new findings of our study were that serum malondialdehyde levels during the first week of MMCAI could be used as biomarkers to mortality prediction.


Introduction
A large quantity of disabilities, deaths and resources consumption are generated by ischemic stroke [1]. In ischemic stroke, in addition to cell death produced by brain vasculature obstruction that causes a reduction of blood containing oxygen and substrates to neurons, could appears a secondary brain injury mediated by oxidative stress [2][3][4][5][6]. Different end-products could appear during lipid peroxidation such as malondialdehyde, which is formed during cellular membrane phospholipids degradation [3,4]. Afterwards malondialdehyde could be released to extracellular space and appears in the blood; and circulating malondialdehyde levels have been used as lipid oxidation biomarker [7,8].
Previously have been found higher circulating malondialdehyde levels during the first week of ischemic stroke in patients with worst neurological functional outcome [9][10][11][12], and at moment of ischemic stroke in nonsurvivor patients [13,14]. Thus, the aim of our study was to determine the potential role of serum malondialdehyde levels during the first week of a severe cerebral infarction to mortality prediction.

Design and subjects
This study was observational and prospective. This multicentre study was performed with the Institutional Review Board approval of the six participating hospitals and with the written informed consent of patient legal guardians. This study was carried out in the Intensive Care Units of the following hospitals: H. General  We included patients with a severe malignant middle cerebral artery infarction (MMCAI), defined as computed tomography showing acute infarction in more than of 50% of the territory and Glasgow Coma Scale (GCS) [15] lower than 9; and there were excluded patients with brain hemorrhage, less than 18 years of age, inflammatory or malignant disease, or pregnancy.
Previously, our team determined serum malondialdehyde concentrations in some of those patients in the day of a severe MMCAI [14]. In this current work, we determine serum malondialdehyde concentrations at days 1, and also at days 4 and 8.

Meassure of serum malondialdehyde concentrations
We obtained serum blood samples on days 1, 4 and 8 of MMCAI and were frozen at − 80°C until the determination of serum malondialdehyde concentrations. All assays for the meassure of malondialdehyde concentrations were carried out in the Physiology Department of Medicine Faculty of La Laguna University (Tenerife, Spain). The meassure of malondialdehyde concentrations was performed according to thiobarbituric acid-reactive substance (TBARS) method by Kikugawa et al. [17]. We mixed serum (200 μL), thiobarbituric acid (2.5 mL at 0.8%), sodium dodecyl sulfate (200 μL at 8.1%), trichloroacetic acid (1.5 mL with pH 3.5) and butylated hydroxytoluene (50 μL at 0.8%). We kept the mixture during 1 h at 5°C and later it was heated during 1 h at 100°C. Afterwards, n-butanol was extracted. Finally, the sample was placed doubly in a 96-well plate and read at 535 nm with a spectrophotometer reader (Benchmark Plus, Bio-Rad, Hercules, CA, USA). The assay detection limit, intra-assay coefficient variation, and inter-assay coefficient variation were of 0.08 nmol/ml, 1.82, and 4.01% respectively.

Statistical methods
Medians (and interquartile ranges) were used to describe continuous variables, and frequencies (and percentages) to describe categorical variables. Wilcoxon-Mann-Whitney test was used to compare continuous variables between survivor and non-survivor patient groups, and chi-square test to compare categorical variables between patient groups. Receiver operating characteristic (ROC) analyses were used to determine the capacity for 30-day mortality prediction by serum malondialdehyde levels at day 1, 4 and 8 of MMCAI. Area under curve (AUC), and sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, positive predicted value and negative predicted value of serum malondialdehyde levels cut-offs for mortality prediction are showed with its 95% confidence intervals (CI). Optimal cut-off values at days 1, 4 and 8 were selected according to Youden J index. Multiple logistic regression was carried out to determine whether exists an association between serum malondialdehyde levels and 30-day mortality after to control for platelet count, lactic acid and GCS. P-values< 0.05 was the point considered to determine as statistically significant. SPSS 17.0 (SPSS Inc., Chicago, IL, USA), LogXact 4.1 (Cytel Co., Cambridge, MA), and NCSS 2000 (Kaysville, Utah) were the programs used for statistical analyses.

Discussion
The new findings of our study were that serum malondialdehyde levels during the first week of MMCAI could be used as biomarkers to mortality prediction.
Previously have been found higher circulating malondialdehyde levels during the first week of ischemic stroke in patients with worst neurological functional outcome   [9][10][11][12], and at moment of ischemic stroke in nonsurvivor patients [13,14]. Thus, the higher serum MDA levels during the first week of MMCAI observed in nonsurvivor patients in respect to survivor patients, and that those levels could be used as mortality prediction are two novel findings of our study. We believed that those higher concentrations of serum malondialdehyde during the first week of MMCAI in non-survivor patients reflects a higher ROS production and lipid peroxidation in comparison to survivor patients, and the use of antioxidant agents could be a new therapeutic to explore in MMCAI patients. The administration of melatonin in animal models of ischemic stroke has been associated with a reduction of oxidation [18][19][20][21][22], specifically a reduction of malondialdehyde levels [21,22], and even an increase of survival. In patients with ischemic stroke, the oral administration of different antioxidant vitamins (B2, B6, B12, C, E) during the first 14 days of stroke has been associated with lower plasma levels of malondialdehyde [23][24][25]. Thus, although we recognize that our study has the limitation that other oxidant state compounds were not reported, we think that all those findings could open the interest for study in patients with ischemic strike the oxidative stress and the potential role of antioxidant agents in your treatment.

Conclusions
The new findings of our study were that serum malondialdehyde levels during the first week of MMCAI could be used as biomarkers to mortality prediction.