- Email: [email protected]
Serum Malondialdehyde Levels and Mortality in Patients with Spontaneous Intracerebral Hemorrhage
Accepted Manuscript Serum malondialdehyde levels and mortality in patients with spontaneous intracerebral hemorrhage Leonardo Lorente, María M. Martín, Pedro Abreu-González, Rafael Sabatel, Luis Ramos, Mónica Argueso, Jordi Solé-Violán, Marta Riaño-Ruiz, Alejandro Jiménez, Victor García-Marín PII:
S1878-8750(18)30356-5
DOI:
10.1016/j.wneu.2018.02.085
Reference:
WNEU 7500
To appear in:
World Neurosurgery
Received Date: 8 August 2017 Revised Date:
12 February 2018
Accepted Date: 14 February 2018
Please cite this article as: Lorente L, Martín MM, Abreu-González P, Sabatel R, Ramos L, Argueso M, Solé-Violán J, Riaño-Ruiz M, Jiménez A, García-Marín V, Serum malondialdehyde levels and mortality in patients with spontaneous intracerebral hemorrhage, World Neurosurgery (2018), doi: 10.1016/ j.wneu.2018.02.085. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Serum malondialdehyde levels and mortality in patients with spontaneous intracerebral hemorrhage Authors
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1. Leonardo Lorente Intensive Care Unit. Hospital Universitario de Canarias. Ofra, s/n. La Laguna - 38320. Santa Cruz de Tenerife. Spain. (Email: [email protected]) 2. María M. Martín Intensive Care Unit. Hospital Universitario Nuestra Señora de Candelaria. Crta del Rosario s/n. Santa Cruz de Tenerife - 38010. Spain. (Email: [email protected]) 3. Pedro Abreu-González Deparment of Phisiology. Faculty of Medicine. University of the La Laguna. La Laguna – 38320. Santa Cruz de Tenerife. Spain. (Email: [email protected]) 4. Rafael Sabatel Deparment of Radiology. Hospital Universitario de Canarias. Ofra, s/n. La Laguna - 38320. Santa Cruz de Tenerife. Spain. (Email: [email protected]) 5. Luis Ramos Intensive Care Unit. Hospital General La Palma. Buenavista de Arriba s/n, Breña Alta, La Palma- 38713. Spain. (Email: [email protected]) 6. Mónica Argueso Intensive Care Unit. Hospital Clínico Universitario de Valencia. Avda. Blasco Ibáñez nº17-19. Valencia - 46004. Spain. (Email: [email protected]) 7. Jordi Solé-Violán Intensive Care Unit. Hospital Universitario Dr. Negrín. CIBERES. Barranco de la Ballena s/n. Las Palmas de Gran Canaria - 35010. Spain. (Email: [email protected]) 8. Marta Riaño-Ruiz Servicio de Bioquímica Clínica. Complejo Hospitalario Universitario Insular Materno-Infantil. Plaza Dr. Pasteur s/n. Las Palmas de Gran Canaria - 35016. Spain. (Email: [email protected]) 9. Alejandro Jiménez Research Unit. Hospital Universitario de Canarias. Ofra, s/n. La Laguna - 38320. Santa Cruz de Tenerife. Spain. (Email: [email protected]) 10. Victor García-Marín Deparment of Neurosurgery. Hospital Universitario de Canarias. Ofra, s/n. La Laguna - 38320. Santa Cruz de Tenerife. Spain. (Email: [email protected])
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Corresponding author: Leonardo Lorente. e-mail: [email protected]
Fundings: This study was supported by a grant from Instituto de Salud Carlos III (INT16/00165) (Madrid, Spain) co-financed by Fondo Europeo de Desarrollo Regional (FEDER), and by a grant from Grupo de Expertos Neurológicos de Canarias (GEN-Canarias. Santa Cruz de Tenerife. Spain). Short Title: Serum malondialdehyde levels and spontaneous intracerebral hemorrhage Abstract Words: 195 Text Words: 1632 Key words: malondialdehyde; spontaneous; intracerebral hemorrhage; patients; mortality Competing interests: none declared
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ACCEPTED MANUSCRIPT ABSTRACT
Objective: Oxidative stress has been associated with secondary brain injury after spontaneous intracerebral hemorrhage (SIH). Malondialdehyde (MDA) appears in blood during lipid oxidation. Higher serum MDA levels have been found in patients with SIH than in healthy
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controls. However, we have not found data indicating an association between elevated serum MDA and early mortality in this population. This was the main objective of our study.
Methods: MDA levels were measured in serum samples obtained from 100 patients at
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diagnosis of severe SIH (Glasgow Coma Scale ≤8) and 80 healthy controls. The end-point of the study was mortality at 30 days.
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Results: Serum MDA levels were significantly higher in patients with severe SIH than in healthy controls (1.46 (1.18-2.24) vs 1.11 (0.72-1.51); p<0.001), and in non-surviving (n=46) than in surviving (n=54) patients (1.68 (1.23-4.02) vs 1.37 (0.99-1.92); p=0.002). The area under the ROC curve of serum MDA levels to predict 30-day mortality was 0.68 (95% CI=0.580.77; p<0.001). Serum MDA levels were associated with 30-day mortality (OR=6.279; 95%
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CI=1.940-20.319; p=0.002).
Conclusions: The most important new finding of our study was that there is an association
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between serum MDA levels at diagnosis of severe SIH and early mortality.
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ACCEPTED MANUSCRIPT INTRODUCTION
Spontaneous intracerebral hemorrhage (SIH) is responsible for high rates of mortality and morbidity, as well as high resource consumption [1-3]. Oxidative stress has been associated
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with secondary brain injury after SIH [4-6]. Malondialdehyde (MDA) is a highly prevalent byproduct of lipid peroxidation of cellular membrane phospholipids during oxidative stress. Initially released into extracellular space, it finally appears in the blood. Thus, the level of serum MDA has been used as a biomarker of lipid oxidation [7,8].
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Circulating MDA in patients with SIH has not been extensively explored [9-13]. MDA levels differ between SIH patients and healthy controls according to some studies [11,12], but
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not others [9,10]. And one study has reported higher MDA levels in neonates with peri/intraventricular hemorrhage than in those without such hemorrhage [13]. Given the uncertainty about the clinical relevance of MDA determination in this population, we designed a prospective study to establish whether MDA levels differ between SIH survivors and non-
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METHODS
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predictor of survival.
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survivors, whether they are associated with early mortality, and whether they could serve as a
We conducted a prospective, observational study of patients admitted to the intensive
care units (ICUs) of six hospitals: Hospital Insular de Las Palmas de Gran Canaria, Hospital Universitario Nuestra Señora de Candelaria, Santa Cruz de Tenerife, Hospital General de La Palma, Hospital Universitario Dr. Negrín de Las Palmas de Gran Canaria, Hospital Clínico Universitario de Valencia, and Hospital Universitario de Canarias, La Laguna. The Clinical Research Ethics Committee of each participating hospital approved the study protocol. Informed consent for inclusion in the study was obtained from the legal guardians of the
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ACCEPTED MANUSCRIPT patients. The study adheres to the World Medical Association Declaration of Helsinki regarding ethical conduct of research involving human subjects. The study included patients with severe supratentorial SIH. Glasgow Coma Scale (GCS) [14] was used to classify the severity of SIH; and we considered that was severe when
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GCS≤8. We excluded patients with inflammatory or malignant disease, infratentorial or traumatic hemorrhage, hemorrhagic transformation of cerebral infarction, pregnancy and age <18 years.
The following variables were recorded for each patient: age, sex, cause, site and volume
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of SIH, midline, presence of intraventricular hemorrhage or hydrocephalus or transtentorial herniation, early evacuation of SIH (within the first 24 h of severe SIH diagnosis), intracerebral
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hemorrhage (ICH) score [15], temperature, sodium, platelets, pressure of arterial oxygen (paO2), fraction of inspired oxygen (FIO2), lactic acid, international normalised ratio (INR), glycaemia, GCS, fibrinogen, creatinine, activated partial thromboplastin time (aPTT), Acute Physiology and Chronic Health Evaluation II (APACHE II) score [16]. The end-point of our
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study was mortality at 30 days from the diagnosis of SIH.
Blood samples from 100 patients with severe SIH (taken within the first 4 hours of diagnosis) and from 80 healthy controls were collected to measure serum MDA levels, using the
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thiobarbituric acid-reactive substance (TBARS) method [17]. In previous studies by our group, we also determined circulating MDA levels using the same method, but in different populations
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of ICU patients, with other clinical situations such as traumatic brain injury [18], brain infarction [19], sepsis [20], and liver transplantation for hepatocellular carcinoma [21]. In this study, we explored the clinical usefulness of serum MDA levels in SIH patients. All statistical analyses were performed with SPSS 17.0 (SPSS Inc., Chicago, IL, USA).
Categorical variables are presented as frequencies and percentages, and chi-square test was used for comparison between groups. We used the Kolmogorov-Smirnov test to assess the normality assumption; and as serum MDA levels differed of normal distribution, then we used median and percentile 25-75 to describe that variable. Continuous variables are presented as medians and interquartile ranges (IQR), and Mann-Whitney U-test was used to compare the groups. Multiple
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ACCEPTED MANUSCRIPT logistic regression analysis was used to determine the independent contribution of serum MDA levels to 30-day mortality, after controlling for transtentorial herniation, ICH score, midline shift, intraventricular hemorrhage and early evacuation of SIH. To determine the capacity of serum MDA levels to predict 30-day mortality, we used receiver operating characteristic (ROC)
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curve analysis. Thirty-day Kaplan-Meier survival analysis was performed with serum MDA levels above or below 2.48 nmol/mL (Youden J index was used to select the optimal cut-off value of serum MDA levels), and we compared the survival time between both curves using
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log-rank test. Differences with a p value <0.05 were considered statistically significant.
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RESULTS
Patients with severe SIH showed significantly higher levels of serum MDA than healthy controls (p<0.001) (Table 1). In addition, both survivors and non-survivors had higher MDA levels than controls (p=0.004 and <0.001, respectively), as shown in Figure 1. Compared to
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surviving SIH patients (n=46), non-survivors (n=54) were older (p=0.006), had greater volume of intracerebral hemorrhage (p=0.02), greater midline shift (p=0.005), higher APACHE-II score (p<0.001), higher serum MDA levels (p=0.002), and lower GCS scores (p<0.001) (Table 2).
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The area under the ROC curve of serum MDA levels to predict 30-day mortality was 0.68 (95% CI = 0.58-0.77; p<0.001), as shown in Figure 2. Elevated serum MDA levels were
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associated with 30-day mortality (OR=6.279; 95% CI=1.940-20.319; p=0.002) after controlling for after controlling for transtentorial herniation, ICH score, midline shift, intraventricular hemorrhage and early evacuation of SIH. (Table 3). SIH patients with serum MDA above 2.48 nmol/mL (n=22) presented higher 30-day mortality (hazard ratio=3.0; 95% CI=1.41-6.44; p<0.001) than those with lower levels (n=78) (Figure 3).
DISCUSSION
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ACCEPTED MANUSCRIPT The major finding of the present study was that elevated serum MDA levels measured at diagnosis of severe SIH were associated with 30-day mortality. Some authors have reported higher serum MDA in patients with SIH than in healthy controls [11,12], and in neonates with peri/intraventricular hemorrhage than in those without it
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[13]; however, others found no difference in plasma MDA between SIH patients and healthy controls [9,10]. In our severe SIH patients, serum MDA levels determined at the time of diagnosis were significantly higher than in healthy controls; thus, our findings are consistent with those of some previous studies [11-13].
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A novel finding of our study was the presence of higher serum MDA levels in nonsurviving severe SIH patients compared to survivors. In addition, we found for the first time that
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there is an association between elevated serum MDA levels at diagnosis of severe SIH and early mortality, after controlling for age, volume of intracerebral hemorrhage, midline shift, intraventricular hemorrhage and GCS score. Also, another new finding was that serum MDA levels at diagnosis of severe SIH could be used as a biomarker to predict early mortality.
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Other variable that has been found to be related with 30-day mortality in our SIH patients was ICH score, which include age, GCS, haemorrhage volume, infratentorial origin, and intraventricular haemorrhage, and all those variables have been associated with a poor
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prognosis in SIH patients [1-3].
In addition, we also found that early evacuation of SIH was associated with a better
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prognosis in our series. Now a day, there is disagreement about when to perform surgery in SIH. In randomised trial published in 2005 with 1033 patients with SIH from 83 centres in 27 countries was not found a benefit from early surgery when compared with initial conservative treatment [22]. In a review published in 2008 of ten randomised trials comparing routine medical treatment plus intracranial surgery compared with routine medical treatment alone in 2059 patients with SIH was found that surgery was associated with statistically significant reduction in dead or dependent at final follow up with no significant heterogeneity among the study results, and with significant reduction in death at final follow up with significant heterogeneity among the study results [23]. In last guidelines for the management of SIH was
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ACCEPTED MANUSCRIPT recommended the evacuation of supratentorial hematoma in patients in coma (class IIb, level C of evidence) [3]. The finding of our study about the association between serum MDA levels at diagnosis of severe SIH and early mortality is in line with the results of previous work by our group where
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the same association was found in patients with traumatic brain injury [18], brain infarction [19], sepsis [20], and liver transplantation for hepatocellular carcinoma [21].
Different antioxidant agents have been administered to animals with SIH and beneficial effects have been reported [24-32], such as greater recovery of motor function and memory.
beneficial effect of antioxidant agents in SIH patients.
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Thus, the findings of the current study could stimulate interest in research on the potential
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Our study has several limitations. First, we did not determine serum levels of other compounds produced by oxidative stress. Second, we have not recollected the exact time when blood samples were recollected and could be different between patients. Third, we have not recollected cerebrospinal fluid samples, for example in patients with an external ventricular
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drain or in those undergoing neurosurgery, to explore a possible association between cerebrospinal fluid and serum MDA levels. Fourth, we found statistically significant differences in serum MDA values levels between healthy controls, and survivor and non-survivor patients;
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however, there was overlap in serum MDA values levels between the three groups and therefore their clinical utility for the prognostic prediction of an individual patient should be taken with
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caution. Although the sample size of our study was enough to find an association between serum MDA levels and mortality in logistic regression analysis; and we think that could help in the mortality prediction along with other markers (as ICH score, age, GCS, volumen of SIH, infratentorial origin, intraventricular haemorrhage). Thus, patients with high ICH score and serum MDA above 2.48 nmol/mL (according to Kaplan-Meier survival analysis) have a higher risk of death. Fifth, serum MDA levels were only determined at the time of severe SIH diagnosis; thus, we can not know as are the levels during evolution and as varied in basis to therapy response
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CONCLUSIONS
The most important new finding of our study was that there is an association between
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serum MDA levels at diagnosis of severe SIH and early mortality.
Acknowledgments
This study was supported by a grant from Instituto de Salud Carlos III (INT16/00165)
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(Madrid, Spain) co-financed by Fondo Europeo de Desarrollo Regional (FEDER), and by a grant from Grupo de Expertos Neurológicos de Canarias (GEN-Canarias. Santa Cruz de
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Tenerife. Spain).
List of abbreviations
- APACHE: Acute Physiology and Chronic Health Evaluation
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- aPTT: activated partial thromboplastin time - FIO2: fraction inspired of oxygen - GCS: Glasgow Coma Scale
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- ICU: Intensive Care Unit
- INR: international normalized ratio
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- MDA: malondialdehyde
- PaO2: pressure of arterial oxygen - SIH: spontaneous intracerebral hemorrhage
Competing interests The authors declare that they have no competing interests.
Author Contributions
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ACCEPTED MANUSCRIPT Conceived and designed the experiments: LL Acquired the data: LL, MMM, PAG, RS, LR, MA, JSV, MRR, VGM Serum MDA analysis: PAG Analyzed the data: LL, AJ
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Wrote the paper: LL All authors revised the manuscript critically for important intellectual content and approved the
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final version to be published.
Highlights
malondialdehyde levels than survivor.
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* Non-survivor patients with spontaneous intracerebral hemorrhage showed higher serum
* There is an association between serum malondialdehyde levels and 30-day mortality in spontaneous intracerebral hemorrhage.
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* Serum malondialdehyde levels at moment of severe spontaneous intracerebral hemorrhage
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diagnosis could be used as a prognostic biomarker.
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Figure 1. Serum malondialdehyde levels in healthy controls and patients with spontaneous intracerebral hemorrhage.
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Figure 2. Receiver operator characteristic curve analysis using serum malondialdehyde level to predict mortality at 30 days
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Figure 3. Survival curves at 30 days using serum malondialdehyde levels higher or lower than 2.48 nmol/mL
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ACCEPTED MANUSCRIPT Serum malondialdehyde levels and mortality in patients with spontaneous intracerebral hemorrhage
Patients (n=100)
p-value
28 (35.0) 64 (55-72) 1.11 (0.72-1.51)
34 (34.0) 62 (54-73) 1.46 (1.18-2.24)
0.99 0.69 <0.001
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Healthy controls (n=80)
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Gender female – n (%) Age - median years (p 25-75) MDA - median nmol/mL(p 25-75)
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Table 1. Characteristics of healthy controls and patients with spontaneous intracerebral hemorrhage.
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ACCEPTED MANUSCRIPT Table 2. Clinical and biochemical characteristics of 30-day surviving and non-surviving patients with spontaneous intracerebral hemorrhage (SIH).
59 (52-67) 17 (31.5)
68 (57-74) 17 (37.0)
0.006 0.67 0.07
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P value
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30 (65.2) 4 (8.7) 0 0 6 (13.0) 6 (13.0) 0
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41 (75.9) 3 (5.6) 5 (9.3) 5 (9.3) 38 (17-62) 1 (0-7) 17 (31.5) 21 (38.9) 2 (3.7) 2 (1-2) 18 (33.3) 36.9 (36.0-37.4) 139 (137-142) 193 (145-252) 270 (189-350) 1.70 (1.00-2.51) 1.10 (1.00-1.31) 141 (118-190) 8 (6-8) 390 (280-493) 0.77 (0.68-0.90) 29 (27-32) 18 (14-20) 1.37 (0.99-1.92)
33 (71.7) 4 (8.7) 3 (6.5) 6 (13.0) 68 (29-99) 5 (0-11) 23 (50.0) 26 (56.5) 1 (2.2) 3 (2-3) 9 (19.6) 36.5 (35.0-37.0) 139 (135-143) 198 (159-270) 289 (215-397) 1.80 (1.30-2.55) 1.14 (1.02-1.87) 170 (141-216) 4 (3-6) 382 (350-510) 0.80 (0.60-1.01) 30 (24-34) 24 (20-26) 1.68 (1.23-4.02)
0.81
0.02 0.005 0.07 0.11 0.99 <0.001 0.18 0.10 0.93 0.57 0.40 0.23 0.34 0.01 <0.001 0.34 0.36 0.68 <0.001 0.002
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Non-survivors (n=46)
37 (68.5) 2 (3.7) 3 (5.6) 5 (9.3) 3 (5.6) 3 (5.6) 1 (1.9)
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Age (years) - median (p 25-75) Gender female – n (%) Cause of SIH– n (%) * Hypertension * Amyloid angiopathy * Aneurysm * Arteriovenous malformation * OAT in therapeutic range * OAT out of therapeutic range * Fibrinolytic treatment Site of SIH– n (%) * Lobar * Basal ganglia * Thalamus * Periventricular Volume of SIH (cc) - median (p 25-75) Midline shift (mm) - median (p 25-75) Intraventricular hemorrhage– n (%) Hydrocephalus– n (%) Transtentorial herniation - n (%) ICH score - median (p 25-75) Early evacuation of SIH– n (%) Temperature (ºC) - median (p 25-75) Sodium (mEq/L)- median (p 25-75) Platelets - median*103/mm3 (p 25-75) PaO2/FI02 ratio - median (p 25-75) Lactic acid (mmol/L)-median (p 25-75) INR - median (p 25-75) Glycemia (g/dL) - median (p 25-75) GCS score - median (p 25-75) Fibrinogen (mg/dl) - median (p 25-75) Creatinine (mg/dl) - median (p 25-75) aPTT (seconds) - median (p 25-75) APACHE-II score - median (p 25-75) MDA (nmol/mL) - median (p 25-75)
Survivors (n=54)
P 25-75= 25th-75th percentile; OAT= Oral anticoagulant treatment; ICH= intracerebral hemorrhage; PaO2= pressure of arterial oxygen/fraction inspired oxygen; FIO2= pressure of arterial oxygen/fraction inspired oxygen; INR= international normalized ratio; GCS= Glasgow Coma Scale; aPTT= activated partial thromboplastin time; APACHE II= Acute Physiology and Chronic Health Evaluation; MDA= Malondialdehyde
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ACCEPTED MANUSCRIPT Table 3. Multiple logistic regression analysis to predict 30-day mortality. Variable
Odds
95% Confidence
Ratio
Interval
P
0.272
0.013-5.782
0.40
ICH score (points)
3.723
1.538-9.016
0.004
Midline shift (mm)
1.118
0.971-1.286
0.12
Intraventricular hemorrhage (yes)
0.870
0.147-5.146
0.88
Early evacuation of SIH (yes)
0.124
0.020-0.755
0.02
Serum malondialdehyde levels (nmol/mL)
2.352
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Transtentorial herniation (yes)
0.01
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1.198-4.617
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S1878-8750(18)30356-5
DOI:
10.1016/j.wneu.2018.02.085
Reference:
WNEU 7500
To appear in:
World Neurosurgery
Received Date: 8 August 2017 Revised Date:
12 February 2018
Accepted Date: 14 February 2018
Please cite this article as: Lorente L, Martín MM, Abreu-González P, Sabatel R, Ramos L, Argueso M, Solé-Violán J, Riaño-Ruiz M, Jiménez A, García-Marín V, Serum malondialdehyde levels and mortality in patients with spontaneous intracerebral hemorrhage, World Neurosurgery (2018), doi: 10.1016/ j.wneu.2018.02.085. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Serum malondialdehyde levels and mortality in patients with spontaneous intracerebral hemorrhage Authors
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1. Leonardo Lorente Intensive Care Unit. Hospital Universitario de Canarias. Ofra, s/n. La Laguna - 38320. Santa Cruz de Tenerife. Spain. (Email: [email protected]) 2. María M. Martín Intensive Care Unit. Hospital Universitario Nuestra Señora de Candelaria. Crta del Rosario s/n. Santa Cruz de Tenerife - 38010. Spain. (Email: [email protected]) 3. Pedro Abreu-González Deparment of Phisiology. Faculty of Medicine. University of the La Laguna. La Laguna – 38320. Santa Cruz de Tenerife. Spain. (Email: [email protected]) 4. Rafael Sabatel Deparment of Radiology. Hospital Universitario de Canarias. Ofra, s/n. La Laguna - 38320. Santa Cruz de Tenerife. Spain. (Email: [email protected]) 5. Luis Ramos Intensive Care Unit. Hospital General La Palma. Buenavista de Arriba s/n, Breña Alta, La Palma- 38713. Spain. (Email: [email protected]) 6. Mónica Argueso Intensive Care Unit. Hospital Clínico Universitario de Valencia. Avda. Blasco Ibáñez nº17-19. Valencia - 46004. Spain. (Email: [email protected]) 7. Jordi Solé-Violán Intensive Care Unit. Hospital Universitario Dr. Negrín. CIBERES. Barranco de la Ballena s/n. Las Palmas de Gran Canaria - 35010. Spain. (Email: [email protected]) 8. Marta Riaño-Ruiz Servicio de Bioquímica Clínica. Complejo Hospitalario Universitario Insular Materno-Infantil. Plaza Dr. Pasteur s/n. Las Palmas de Gran Canaria - 35016. Spain. (Email: [email protected]) 9. Alejandro Jiménez Research Unit. Hospital Universitario de Canarias. Ofra, s/n. La Laguna - 38320. Santa Cruz de Tenerife. Spain. (Email: [email protected]) 10. Victor García-Marín Deparment of Neurosurgery. Hospital Universitario de Canarias. Ofra, s/n. La Laguna - 38320. Santa Cruz de Tenerife. Spain. (Email: [email protected])
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Corresponding author: Leonardo Lorente. e-mail: [email protected]
Fundings: This study was supported by a grant from Instituto de Salud Carlos III (INT16/00165) (Madrid, Spain) co-financed by Fondo Europeo de Desarrollo Regional (FEDER), and by a grant from Grupo de Expertos Neurológicos de Canarias (GEN-Canarias. Santa Cruz de Tenerife. Spain). Short Title: Serum malondialdehyde levels and spontaneous intracerebral hemorrhage Abstract Words: 195 Text Words: 1632 Key words: malondialdehyde; spontaneous; intracerebral hemorrhage; patients; mortality Competing interests: none declared
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ACCEPTED MANUSCRIPT ABSTRACT
Objective: Oxidative stress has been associated with secondary brain injury after spontaneous intracerebral hemorrhage (SIH). Malondialdehyde (MDA) appears in blood during lipid oxidation. Higher serum MDA levels have been found in patients with SIH than in healthy
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controls. However, we have not found data indicating an association between elevated serum MDA and early mortality in this population. This was the main objective of our study.
Methods: MDA levels were measured in serum samples obtained from 100 patients at
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diagnosis of severe SIH (Glasgow Coma Scale ≤8) and 80 healthy controls. The end-point of the study was mortality at 30 days.
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Results: Serum MDA levels were significantly higher in patients with severe SIH than in healthy controls (1.46 (1.18-2.24) vs 1.11 (0.72-1.51); p<0.001), and in non-surviving (n=46) than in surviving (n=54) patients (1.68 (1.23-4.02) vs 1.37 (0.99-1.92); p=0.002). The area under the ROC curve of serum MDA levels to predict 30-day mortality was 0.68 (95% CI=0.580.77; p<0.001). Serum MDA levels were associated with 30-day mortality (OR=6.279; 95%
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CI=1.940-20.319; p=0.002).
Conclusions: The most important new finding of our study was that there is an association
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between serum MDA levels at diagnosis of severe SIH and early mortality.
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ACCEPTED MANUSCRIPT INTRODUCTION
Spontaneous intracerebral hemorrhage (SIH) is responsible for high rates of mortality and morbidity, as well as high resource consumption [1-3]. Oxidative stress has been associated
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with secondary brain injury after SIH [4-6]. Malondialdehyde (MDA) is a highly prevalent byproduct of lipid peroxidation of cellular membrane phospholipids during oxidative stress. Initially released into extracellular space, it finally appears in the blood. Thus, the level of serum MDA has been used as a biomarker of lipid oxidation [7,8].
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Circulating MDA in patients with SIH has not been extensively explored [9-13]. MDA levels differ between SIH patients and healthy controls according to some studies [11,12], but
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not others [9,10]. And one study has reported higher MDA levels in neonates with peri/intraventricular hemorrhage than in those without such hemorrhage [13]. Given the uncertainty about the clinical relevance of MDA determination in this population, we designed a prospective study to establish whether MDA levels differ between SIH survivors and non-
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METHODS
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predictor of survival.
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survivors, whether they are associated with early mortality, and whether they could serve as a
We conducted a prospective, observational study of patients admitted to the intensive
care units (ICUs) of six hospitals: Hospital Insular de Las Palmas de Gran Canaria, Hospital Universitario Nuestra Señora de Candelaria, Santa Cruz de Tenerife, Hospital General de La Palma, Hospital Universitario Dr. Negrín de Las Palmas de Gran Canaria, Hospital Clínico Universitario de Valencia, and Hospital Universitario de Canarias, La Laguna. The Clinical Research Ethics Committee of each participating hospital approved the study protocol. Informed consent for inclusion in the study was obtained from the legal guardians of the
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ACCEPTED MANUSCRIPT patients. The study adheres to the World Medical Association Declaration of Helsinki regarding ethical conduct of research involving human subjects. The study included patients with severe supratentorial SIH. Glasgow Coma Scale (GCS) [14] was used to classify the severity of SIH; and we considered that was severe when
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GCS≤8. We excluded patients with inflammatory or malignant disease, infratentorial or traumatic hemorrhage, hemorrhagic transformation of cerebral infarction, pregnancy and age <18 years.
The following variables were recorded for each patient: age, sex, cause, site and volume
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of SIH, midline, presence of intraventricular hemorrhage or hydrocephalus or transtentorial herniation, early evacuation of SIH (within the first 24 h of severe SIH diagnosis), intracerebral
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hemorrhage (ICH) score [15], temperature, sodium, platelets, pressure of arterial oxygen (paO2), fraction of inspired oxygen (FIO2), lactic acid, international normalised ratio (INR), glycaemia, GCS, fibrinogen, creatinine, activated partial thromboplastin time (aPTT), Acute Physiology and Chronic Health Evaluation II (APACHE II) score [16]. The end-point of our
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study was mortality at 30 days from the diagnosis of SIH.
Blood samples from 100 patients with severe SIH (taken within the first 4 hours of diagnosis) and from 80 healthy controls were collected to measure serum MDA levels, using the
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thiobarbituric acid-reactive substance (TBARS) method [17]. In previous studies by our group, we also determined circulating MDA levels using the same method, but in different populations
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of ICU patients, with other clinical situations such as traumatic brain injury [18], brain infarction [19], sepsis [20], and liver transplantation for hepatocellular carcinoma [21]. In this study, we explored the clinical usefulness of serum MDA levels in SIH patients. All statistical analyses were performed with SPSS 17.0 (SPSS Inc., Chicago, IL, USA).
Categorical variables are presented as frequencies and percentages, and chi-square test was used for comparison between groups. We used the Kolmogorov-Smirnov test to assess the normality assumption; and as serum MDA levels differed of normal distribution, then we used median and percentile 25-75 to describe that variable. Continuous variables are presented as medians and interquartile ranges (IQR), and Mann-Whitney U-test was used to compare the groups. Multiple
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ACCEPTED MANUSCRIPT logistic regression analysis was used to determine the independent contribution of serum MDA levels to 30-day mortality, after controlling for transtentorial herniation, ICH score, midline shift, intraventricular hemorrhage and early evacuation of SIH. To determine the capacity of serum MDA levels to predict 30-day mortality, we used receiver operating characteristic (ROC)
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curve analysis. Thirty-day Kaplan-Meier survival analysis was performed with serum MDA levels above or below 2.48 nmol/mL (Youden J index was used to select the optimal cut-off value of serum MDA levels), and we compared the survival time between both curves using
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log-rank test. Differences with a p value <0.05 were considered statistically significant.
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RESULTS
Patients with severe SIH showed significantly higher levels of serum MDA than healthy controls (p<0.001) (Table 1). In addition, both survivors and non-survivors had higher MDA levels than controls (p=0.004 and <0.001, respectively), as shown in Figure 1. Compared to
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surviving SIH patients (n=46), non-survivors (n=54) were older (p=0.006), had greater volume of intracerebral hemorrhage (p=0.02), greater midline shift (p=0.005), higher APACHE-II score (p<0.001), higher serum MDA levels (p=0.002), and lower GCS scores (p<0.001) (Table 2).
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The area under the ROC curve of serum MDA levels to predict 30-day mortality was 0.68 (95% CI = 0.58-0.77; p<0.001), as shown in Figure 2. Elevated serum MDA levels were
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associated with 30-day mortality (OR=6.279; 95% CI=1.940-20.319; p=0.002) after controlling for after controlling for transtentorial herniation, ICH score, midline shift, intraventricular hemorrhage and early evacuation of SIH. (Table 3). SIH patients with serum MDA above 2.48 nmol/mL (n=22) presented higher 30-day mortality (hazard ratio=3.0; 95% CI=1.41-6.44; p<0.001) than those with lower levels (n=78) (Figure 3).
DISCUSSION
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ACCEPTED MANUSCRIPT The major finding of the present study was that elevated serum MDA levels measured at diagnosis of severe SIH were associated with 30-day mortality. Some authors have reported higher serum MDA in patients with SIH than in healthy controls [11,12], and in neonates with peri/intraventricular hemorrhage than in those without it
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[13]; however, others found no difference in plasma MDA between SIH patients and healthy controls [9,10]. In our severe SIH patients, serum MDA levels determined at the time of diagnosis were significantly higher than in healthy controls; thus, our findings are consistent with those of some previous studies [11-13].
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A novel finding of our study was the presence of higher serum MDA levels in nonsurviving severe SIH patients compared to survivors. In addition, we found for the first time that
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there is an association between elevated serum MDA levels at diagnosis of severe SIH and early mortality, after controlling for age, volume of intracerebral hemorrhage, midline shift, intraventricular hemorrhage and GCS score. Also, another new finding was that serum MDA levels at diagnosis of severe SIH could be used as a biomarker to predict early mortality.
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Other variable that has been found to be related with 30-day mortality in our SIH patients was ICH score, which include age, GCS, haemorrhage volume, infratentorial origin, and intraventricular haemorrhage, and all those variables have been associated with a poor
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prognosis in SIH patients [1-3].
In addition, we also found that early evacuation of SIH was associated with a better
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prognosis in our series. Now a day, there is disagreement about when to perform surgery in SIH. In randomised trial published in 2005 with 1033 patients with SIH from 83 centres in 27 countries was not found a benefit from early surgery when compared with initial conservative treatment [22]. In a review published in 2008 of ten randomised trials comparing routine medical treatment plus intracranial surgery compared with routine medical treatment alone in 2059 patients with SIH was found that surgery was associated with statistically significant reduction in dead or dependent at final follow up with no significant heterogeneity among the study results, and with significant reduction in death at final follow up with significant heterogeneity among the study results [23]. In last guidelines for the management of SIH was
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the same association was found in patients with traumatic brain injury [18], brain infarction [19], sepsis [20], and liver transplantation for hepatocellular carcinoma [21].
Different antioxidant agents have been administered to animals with SIH and beneficial effects have been reported [24-32], such as greater recovery of motor function and memory.
beneficial effect of antioxidant agents in SIH patients.
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Thus, the findings of the current study could stimulate interest in research on the potential
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Our study has several limitations. First, we did not determine serum levels of other compounds produced by oxidative stress. Second, we have not recollected the exact time when blood samples were recollected and could be different between patients. Third, we have not recollected cerebrospinal fluid samples, for example in patients with an external ventricular
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drain or in those undergoing neurosurgery, to explore a possible association between cerebrospinal fluid and serum MDA levels. Fourth, we found statistically significant differences in serum MDA values levels between healthy controls, and survivor and non-survivor patients;
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however, there was overlap in serum MDA values levels between the three groups and therefore their clinical utility for the prognostic prediction of an individual patient should be taken with
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caution. Although the sample size of our study was enough to find an association between serum MDA levels and mortality in logistic regression analysis; and we think that could help in the mortality prediction along with other markers (as ICH score, age, GCS, volumen of SIH, infratentorial origin, intraventricular haemorrhage). Thus, patients with high ICH score and serum MDA above 2.48 nmol/mL (according to Kaplan-Meier survival analysis) have a higher risk of death. Fifth, serum MDA levels were only determined at the time of severe SIH diagnosis; thus, we can not know as are the levels during evolution and as varied in basis to therapy response
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CONCLUSIONS
The most important new finding of our study was that there is an association between
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serum MDA levels at diagnosis of severe SIH and early mortality.
Acknowledgments
This study was supported by a grant from Instituto de Salud Carlos III (INT16/00165)
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(Madrid, Spain) co-financed by Fondo Europeo de Desarrollo Regional (FEDER), and by a grant from Grupo de Expertos Neurológicos de Canarias (GEN-Canarias. Santa Cruz de
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Tenerife. Spain).
List of abbreviations
- APACHE: Acute Physiology and Chronic Health Evaluation
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- aPTT: activated partial thromboplastin time - FIO2: fraction inspired of oxygen - GCS: Glasgow Coma Scale
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- ICU: Intensive Care Unit
- INR: international normalized ratio
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- MDA: malondialdehyde
- PaO2: pressure of arterial oxygen - SIH: spontaneous intracerebral hemorrhage
Competing interests The authors declare that they have no competing interests.
Author Contributions
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ACCEPTED MANUSCRIPT Conceived and designed the experiments: LL Acquired the data: LL, MMM, PAG, RS, LR, MA, JSV, MRR, VGM Serum MDA analysis: PAG Analyzed the data: LL, AJ
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Wrote the paper: LL All authors revised the manuscript critically for important intellectual content and approved the
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final version to be published.
Highlights
malondialdehyde levels than survivor.
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* Non-survivor patients with spontaneous intracerebral hemorrhage showed higher serum
* There is an association between serum malondialdehyde levels and 30-day mortality in spontaneous intracerebral hemorrhage.
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* Serum malondialdehyde levels at moment of severe spontaneous intracerebral hemorrhage
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diagnosis could be used as a prognostic biomarker.
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Figure 1. Serum malondialdehyde levels in healthy controls and patients with spontaneous intracerebral hemorrhage.
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Figure 2. Receiver operator characteristic curve analysis using serum malondialdehyde level to predict mortality at 30 days
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Figure 3. Survival curves at 30 days using serum malondialdehyde levels higher or lower than 2.48 nmol/mL
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Patients (n=100)
p-value
28 (35.0) 64 (55-72) 1.11 (0.72-1.51)
34 (34.0) 62 (54-73) 1.46 (1.18-2.24)
0.99 0.69 <0.001
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Healthy controls (n=80)
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Gender female – n (%) Age - median years (p 25-75) MDA - median nmol/mL(p 25-75)
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Table 1. Characteristics of healthy controls and patients with spontaneous intracerebral hemorrhage.
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ACCEPTED MANUSCRIPT Table 2. Clinical and biochemical characteristics of 30-day surviving and non-surviving patients with spontaneous intracerebral hemorrhage (SIH).
59 (52-67) 17 (31.5)
68 (57-74) 17 (37.0)
0.006 0.67 0.07
RI PT
P value
SC
30 (65.2) 4 (8.7) 0 0 6 (13.0) 6 (13.0) 0
M AN U
41 (75.9) 3 (5.6) 5 (9.3) 5 (9.3) 38 (17-62) 1 (0-7) 17 (31.5) 21 (38.9) 2 (3.7) 2 (1-2) 18 (33.3) 36.9 (36.0-37.4) 139 (137-142) 193 (145-252) 270 (189-350) 1.70 (1.00-2.51) 1.10 (1.00-1.31) 141 (118-190) 8 (6-8) 390 (280-493) 0.77 (0.68-0.90) 29 (27-32) 18 (14-20) 1.37 (0.99-1.92)
33 (71.7) 4 (8.7) 3 (6.5) 6 (13.0) 68 (29-99) 5 (0-11) 23 (50.0) 26 (56.5) 1 (2.2) 3 (2-3) 9 (19.6) 36.5 (35.0-37.0) 139 (135-143) 198 (159-270) 289 (215-397) 1.80 (1.30-2.55) 1.14 (1.02-1.87) 170 (141-216) 4 (3-6) 382 (350-510) 0.80 (0.60-1.01) 30 (24-34) 24 (20-26) 1.68 (1.23-4.02)
0.81
0.02 0.005 0.07 0.11 0.99 <0.001 0.18 0.10 0.93 0.57 0.40 0.23 0.34 0.01 <0.001 0.34 0.36 0.68 <0.001 0.002
AC C
EP
Non-survivors (n=46)
37 (68.5) 2 (3.7) 3 (5.6) 5 (9.3) 3 (5.6) 3 (5.6) 1 (1.9)
TE D
Age (years) - median (p 25-75) Gender female – n (%) Cause of SIH– n (%) * Hypertension * Amyloid angiopathy * Aneurysm * Arteriovenous malformation * OAT in therapeutic range * OAT out of therapeutic range * Fibrinolytic treatment Site of SIH– n (%) * Lobar * Basal ganglia * Thalamus * Periventricular Volume of SIH (cc) - median (p 25-75) Midline shift (mm) - median (p 25-75) Intraventricular hemorrhage– n (%) Hydrocephalus– n (%) Transtentorial herniation - n (%) ICH score - median (p 25-75) Early evacuation of SIH– n (%) Temperature (ºC) - median (p 25-75) Sodium (mEq/L)- median (p 25-75) Platelets - median*103/mm3 (p 25-75) PaO2/FI02 ratio - median (p 25-75) Lactic acid (mmol/L)-median (p 25-75) INR - median (p 25-75) Glycemia (g/dL) - median (p 25-75) GCS score - median (p 25-75) Fibrinogen (mg/dl) - median (p 25-75) Creatinine (mg/dl) - median (p 25-75) aPTT (seconds) - median (p 25-75) APACHE-II score - median (p 25-75) MDA (nmol/mL) - median (p 25-75)
Survivors (n=54)
P 25-75= 25th-75th percentile; OAT= Oral anticoagulant treatment; ICH= intracerebral hemorrhage; PaO2= pressure of arterial oxygen/fraction inspired oxygen; FIO2= pressure of arterial oxygen/fraction inspired oxygen; INR= international normalized ratio; GCS= Glasgow Coma Scale; aPTT= activated partial thromboplastin time; APACHE II= Acute Physiology and Chronic Health Evaluation; MDA= Malondialdehyde
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ACCEPTED MANUSCRIPT Table 3. Multiple logistic regression analysis to predict 30-day mortality. Variable
Odds
95% Confidence
Ratio
Interval
P
0.272
0.013-5.782
0.40
ICH score (points)
3.723
1.538-9.016
0.004
Midline shift (mm)
1.118
0.971-1.286
0.12
Intraventricular hemorrhage (yes)
0.870
0.147-5.146
0.88
Early evacuation of SIH (yes)
0.124
0.020-0.755
0.02
Serum malondialdehyde levels (nmol/mL)
2.352
SC
RI PT
Transtentorial herniation (yes)
0.01
AC C
EP
TE D
M AN U
1.198-4.617
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AC C
EP
TE D
M AN U
SC
RI PT
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AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT