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 de La Palma from Breña Alta, H. Universitario Dr. Negrín from Las Palmas de Gran Canaria, H. Insular from Las Palmas de Gran Canaria, H. Universitario Nuestra Señora de Candelaria from Santa Cruz de Tenerife, H. Clínico Universitario de Valencia from Valencia, and H. Universitario de Canarias from La Laguna.
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)  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 . In this current work, we determine serum malondialdehyde concentrations at days 1, and also at days 4 and 8.
Clinical and demographic variables
We recorded the following variables from the patients: age, arterial hypertension, sex, chronic obstructive pulmonary disease (COPD), diabetes mellitus, heart failure, chronic renal failure, temperature, CGS, lactic acid, sodium, bilirubin, creatinine, glycaemia, pressure of arterial oxygen (PaO2), fraction inspired oxygen (FI02), PaO2/FIO2 ratio, platelets, leukocytes, haemoglobin, fibrinogen, international normalized ratio (INR), activated partial thromboplastin time (aPTT), Acute Physiology and Chronic Health Evaluation II (APACHE II) score , thrombolysis, volumen infarction, haemorrhagic transformation, midline shift, and descompresive craniectomy. Thirty-day mortality was considered as the end-point study.
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. . 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.
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.