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Plasma levels of adrenomedullin in patients with traumatic brain injury: Potential contribution to prognosis
Peptides 56 (2014) 146–150
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Plasma levels of adrenomedullin in patients with traumatic brain injury: Potential contribution to prognosis Tie-Jiang Chen ∗ , Qing-Yang Fu, Wu-Quan Wu Department of Emergency Surgery, Yiwu Central Hospital, 699 Jiangdong Road, Yiwu 322000, Zhejiang Province, China
a r t i c l e
i n f o
Article history: Received 26 March 2014 Received in revised form 4 April 2014 Accepted 4 April 2014 Available online 18 April 2014 Keywords: Adrenomedullin Traumatic brain injury Mortality Functional outcome
a b s t r a c t High plasma levels of adrenomedullin have been associated with stroke severity and clinical outcomes. This study aimed to analyze plasma levels of adrenomedullin in traumatic brain injury and their association with prognosis. One hundred and forty-eight acute severe traumatic brain injury and 148 sexand age-matched healthy controls were recruited in this study. Plasma adrenomedullin concentration was measured by enzyme-linked immunosorbent assay. Unfavorable outcome was defined as Glasgow Outcome Scale score of 1–3. Compared to controls, the patients had significantly higher plasma concentrations of adrenomedullin, which were also highly associated negatively with Glasgow Coma Scale score. Plasma adrenomedullin level was proved to be an independent predictor for 6-month mortality and unfavorable outcome of patients in a multivariate analysis. A receiver operating characteristic curve was configured to show that a baseline plasma adrenomedullin level predicted 6-month mortality and unfavorable outcome of patients with high area under curve. The predictive performance of the plasma adrenomedullin concentration was also similar to that of Glasgow Coma Scale score for the prediction of 6-month mortality and unfavorable outcome of patients. In a combined logistic-regression model, adrenomedullin improved the area under curve of Glasgow Coma Scale score for the prediction of 6-month mortality and unfavorable outcome of patients, but the differences did not appear to be statistically significant. Thus, high plasma levels of adrenomedullin are associated with head trauma severity, and may independently predict long-term clinical outcomes of traumatic brain injury. © 2014 Elsevier Inc. All rights reserved.
Introduction Traumatic Brain Injury (TBI) is known to represent a major public health concern potentially resulting in death or neurological impairment [6,17]. The pathophysiological mechanisms implicated in the cellular and molecular change following TBI remains unclear [1]. Reliable biomarkers for early prediction of prognosis and functional recovery are very few [13]. Adrenomedullin (AM) is a vasoactive peptide first isolated from pheochromocytoma [12]. Its gene expression is promoted by various stimuli, including inflammation, hypoxia, oxidative stress, mechanical stress and activation of the renin–angiotensin and sympathetic nervous systems [22]. AM possesses neuroprotection in experimental brain disease models including ischemic stroke [28] and traumatic brain injury [2], as well as cardioprotection in human myocardial infarction [11]. Blood levels of AM have been associated with prognosis of plenty of diseases including myocardial infarction, heart failure
∗ Corresponding author. Tel.: +86 0579 85209666. E-mail address: [email protected] (T.-J. Chen). http://dx.doi.org/10.1016/j.peptides.2014.04.005 0196-9781/© 2014 Elsevier Inc. All rights reserved.
and pulmonary hypertension [7]. AM has also been reported to be present in neurons and glia in the central nervous system [8]. Recently, circulating AM has been demonstrated to be of prognostic importance in ischemic or hemorrhagic stroke [27,29]. The present study aimed to further investigate the ability of plasma AM to predict long-term clinical outcomes in a group of patients with acute severe TBI. Materials and methods Study population This study included severe isolated head trauma patients with postresuscitation Glasgow Coma Scale (GCS) score of 8 or less from Yiwu Central Hospital between April 2010 and April 2013. Nonselection criteria involved less than 18 years of age, admission time > 6 h, previous head trauma, neurological disease including ischemic or hemorrhagic stroke, use of antiplatelet or anticoagulant medication, diabetes mellitus, hypertension or presence of other prior systemic diseases including uremia, liver cirrhosis, malignancy, chronic heart or lung disease. Healthy age- and sex-matched
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volunteers were recruited as control group. At study entrance, all participants or their legal representatives gave their informed consent, and study protocol was approved by the Ethics Committee of Yiwu Central Hospital before implementation. Clinical and radiological assessment Information regarding the following variables: postresuscitation GCS score, systolic blood pressure, blood oxygen saturation, blood glucose and papillary reactivity was obtained at admission. Head trauma severity was assessed using initial postresuscitation GCS score. Shock was defined as systolic blood pressure less than 90 mmHg [10]. Hypoxia was defined as blood oxygen saturation less than 85% [10]. Hyperglycemia was defined as blood glucose more than 11.1 mmol/L [16]. Hypoglycemia was defined as blood glucose less than 2.2 mmol/L [26]. Neurology deterioration was defined as occurring in patients who manifested clinically identified episodes of one or more of the following: (1) a spontaneous decrease in GCS motor scores of 2 points or more from the previous examination; (2) a further loss of papillary reactivity; (3) development of papillary asymmetry greater than 1 mm; or (4) deterioration in neurological status sufficient to warrant immediate medical or surgical intervention [10]. All computerized tomography (CT) scans were performed according to the neuroradiology department protocol. Investigators who read them were blinded to clinical information. Abnormal cisterns, midline shift >5 mm and subarachnoid hemorrhage were recorded on initial CT scan. CT classification was performed using Traumatic Coma Data Bank criteria on initial postresuscitation CT scan according to the method of Marshall et al. [19]. Participants were followed up until death or completion of 6 months after head trauma. The functional outcome was defined by Glasgow outcome scale (GOS) score. GOS was defined as follows: 1 = death; 2 = persistent vegetative state; 3 = severe disability; 4 = moderate disability; and 5 = good recovery [9]. Unfavorable outcome was defined as GOS of 1–3. For follow-up, we used structure telephone interviews performed by 1 doctor, blinded to clinical information and AM levels. Determination of plasma AM levels The informed consents were obtained from all participants or their legal representatives before the blood were collected. Venous blood was drawn at study entry in the control group and on admission in the patients. The blood samples were immediately placed into sterile EDTA test tubes and centrifuged at 1500 × g for 20 min at 4 ◦ C to collect plasma. Plasma was stored at −70◦ C until assayed. Plasma AM concentration was analyzed by enzyme-linked immunosorbent assay using commercial kits (R&D Systems, Heidelberg, Germany) in accordance with the manufactures’ instructions. The blood samples were run in duplicate. Researchers running enzyme-linked immunosorbent assays were blinded to all patient details. Statistical analysis Statistical analysis was performed with SPSS 19.0 (SPSS Inc., Chicago, IL, USA) and MedCalc 9.6.4.0. (MedCalc Software, Mariakerke, Belgium). All values are expressed as mean ± standard deviation or counts (percentage) unless otherwise specified. Comparisons were made by using (1) Chi-square test or Fisher exact test for categorical data, (2) Student t test for continuous distributed variables. The association of plasma AM levels with GCS scores was analyzed using Spearman correlation coefficient. The relations of AM to 6-month mortality and unfavorable outcome were assessed in a logistic-regression model with odds ratio (OR)
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Table 1 The demographic data, clinical and biochemical characteristics of 148 patients. Characteristics Sex (male/female) Age (y) GCS score on admission Shock on admission Hyperglycemia on admission Hypoglycemia on admission Hypoxia on admission Pupils unreactive on admission CT classification 5 or 6 Abnormal cisterns on initial CT scan Midline shift > 5 mm on initial CT scan Presence of traumatic SAH on initial CT scan Neurological deterioration Mechanical ventilation Intracranial surgery in 1st 24 h Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Mean arterial pressure (mmHg) Heart rate (beats/min) Body temperature (◦ C) Respiratory rate (respirations/min) Blood oxygen saturation (%) Blood white blood cell count (×109 /L) Blood hemoglobin level (g/L) Blood platelet count (×109 /L) Blood glucose level (mmol/L) Blood sodium level (mmol/L) Blood potassium level (mmol/L) Prothrombin time (s) Thrombin time (s) Partial thromboplastin time (s) Plasma C-reactive protein level (mg/L) Plasma fibrinogen level (g/L) Plasma d-dimer level (mg/L) Plasma adrenomedullin level (pg/mL)
98/50 43.6 ± 17.6 5.4 ± 1.8 29 (19.6%) 36 (24.3%) 8 (5.4%) 15 (10.1%) 75 (50.7%) 76 (51.4%) 72 (48.7%) 74 (50.0%) 78 (52.7%) 30 (20.3%) 125 (84.5%) 70 (47.3%) 2.1 ± 1.2 3.0 ± 1.3 119.5 ± 32.6 72.4 ± 20.9 88.1 ± 23.9 86.9 ± 21.9 36.6 ± 0.8 18.8 ± 3.9 91.2 ± 5.9 7.7 ± 2.8 124.5 ± 24.2 169.3 ± 40.1 11.5 ± 3.6 142.5 ± 8.6 4.4 ± 0.7 14.7 ± 2.4 18.3 ± 2.7 38.9 ± 6.4 11.0 ± 3.4 4.3 ± 1.9 2.8 ± 1.3 114.2 ± 44.1
Numerical variables were presented as mean ± standard deviation. Categorical variables were expressed as counts (percentage). GCS, Glasgow Coma Scale; CT, computerized tomography; SAH, subarachnoid hemorrhage.
and 95% confidence interval (CI). The receiver operating characteristic (ROC) curves was used to determine the best threshold of AM values to predict 6-month clinical outcomes with calculated area under curve (AUC). In a combined logistic-regression model, the additive benefit of AM to GCS score was estimated. A P value of <0.05 was considered significant for all test. Results Study population characteristics This study finally assessed one hundred and forty-eight severe isolated head trauma patients and 148 sex- and age-matched healthy controls. Table 1 summarized the demographic data, clinical and biochemical characteristics of patients at baseline. Compared to controls, the patients had significantly higher plasma concentrations of AM (114.2 ± 44.1 pg/mL vs. 46.6 ± 16.0 pg/mL, P < 0.0001). Fig. 1 showed that plasma concentrations of AM were also highly associated negatively with GCS scores (r = −0.568, P < 0.0001). Functional outcome prediction Sixty-six patients (44.6%) had unfavorable outcome at 6 months after head trauma. In Table 2, a univariate analysis found that plasma AM levels were markedly higher in patients with unfavorable outcome than favorable outcome. When configured was
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Fig. 1. Graph showing the relationship between plasma adrenomedullin concentration and Glasgow Coma Scale score using Spearman correlation coefficient.
a logistic-regression model which included the significant variables in the univariate analysis, it was demonstrated that GCS score (OR 0.300, 95% CI 0.193–0.466, P < 0.001) and plasma AM level (OR 1.232, 95% CI 1.112–2.351, P < 0.001) appeared to be the independent predictors of 6-month unfavorable outcome in the patients. Table 2 The factors associated with 6-month unfavorable outcome.
Number Sex (male/female) Age (y) GCS score on admission Shock on admission Hyperglycemia on admission Hypoglycemia on admission Hypoxia on admission Pupils unreactive on admission CT classification 5 or 6 Abnormal cisterns on initial CT scan Midline shift > 5 mm on initial CT scan Traumatic SAH on initial CT scan Neurological deterioration Mechanical ventilation Intracranial surgery in 1st 24 h Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Mean arterial pressure (mmHg) Heart rate (beats/min) Body temperature (◦ C) Respiratory rate (respirations/min) Blood oxygen saturation (%) Blood white blood cell count (×109 /L) Blood hemoglobin level (g/L) Blood platelet count (×109 /L) Blood glucose level (mmol/L) Blood sodium level (mmol/L) Blood potassium level (mmol/L) Prothrombin time (s) Thrombin time (s) Partial thromboplastin time (s) Plasma C-reactive protein level (mg/L) Plasma fibrinogen level (g/L) Plasma d-dimer level (mg/L) Plasma adrenomedullin level (pg/mL)
Unfavorable outcome
Favorable outcome
66 (44.6%) 41/25 44.0 ± 17.1 4.0 ± 0.8 19 (28.8%) 22 (33.3%) 4 (6.1%) 8 (12.1%) 45 (68.2%) 41 (62.1%) 42 (63.3%) 45 (68.2%) 42 (63.3%) 19 (28.8%) 66 (100.0%) 34 (51.5%) 1.9 ± 0.8 2.8 ± 0.9 114.1 ± 36.6 69.9 ± 24.2 84.7 ± 27.1 84.1 ± 20.5 36.6 ± 0.7 18.4 ± 4.0 90.2 ± 7.7 7.4 ± 3.2 123.3 ± 25.0 166.3 ± 38.5 12.4 ± 4.2 144.0 ± 9.6 4.4 ± 0.9 14.9 ± 2.4 18.7 ± 2.5 38.9 ± 6.9 12.0 ± 4.1 4.6 ± 1.8 3.1 ± 1.4 142.6 ± 42.1
82 (55.4%) 57/25 43.3 ± 18.1 6.6 ± 1.4 10 (12.2%) 14(17.1%) 4 (4.9%) 7 (8.5%) 30 (36.6%) 35 (42.7%) 30 (36.6%) 29 (35.4%) 36 (43.9%) 11 (13.4%) 59 (72.0%) 36 (43.9%) 2.2 ± 1.4 3.1 ± 1.6 123.9 ± 28.6 74.5 ± 17.7 90.9 ± 20.7 89.1 ± 22.8 36.7 ± 0.8 19.2 ± 3.8 92.0 ± 3.6 7.9 ± 2.4 125.4 ± 23.7 171.7 ± 41.5 10.8 ± 2.8 141.4 ± 7.7 4.3 ± 0.6 14.6 ± 2.5 18.0 ± 2.8 39.0 ± 5.9 10.2 ± 2.5 4.0 ± 2.0 2.6 ± 1.3 91.3 ± 30.3
P value
0.345 0.817 <0.001 0.011 0.022 0.752 0.473 <0.001 0.019 0.001 <0.001 0.017 0.021 <0.001 0.357 0.091 0.159 0.076 0.208 0.123 0.170 0.561 0.250 0.094 0.230 0.595 0.426 0.007 0.060 0.319 0.465 0.123 0.928 0.002 0.037 0.014 <0.001
Numerical variables were presented as mean ± standard deviation and analyzed by unpaired student t test. Categorical variables were expressed as counts (percentage) and analyzed by Chi-square test or Fisher exact test. GCS, Glasgow Coma Scale; CT, computerized tomography; SAH, subarachnoid hemorrhage.
Fig. 2. Graph showing receiver operating characteristic (ROC) curve analysis of plasma adrenomedullin concentration for identifying patients with 6-month unfavorable outcome. ROC curves were constructed based on the sensitivity and specificity of the plasma adrenomedullin concentration for identifying 6-month unfavorable outcome. The area under curve (AUC) was calculated based on the ROC curves and expressed as 95% confidence interval (CI). AUC ranges from 0.5 to 1.0. An AUC closer to 1 indicates a higher predictive power.
A ROC curve analysis found that a baseline plasma AM level predicted 6-month unfavorable outcome of patients with high AUC in Fig. 2. The AUC of the AM concentration was similar to that of GCS scores (AUC, 0.914; 95% CI, 0.857–0.954) (P = 0.103). When a combined logistic-regression model was configured, AM improved the AUC of GCS score to 0.940 (95% CI, 0.889–0.972), but the difference did not appear to be statistically significant (P = 0.134). Mortality prediction Forty-two patients (28.4%) died within 6 months after head trauma. In Table 3, a univariate analysis found that plasma AM levels were markedly higher in non-survivors than survivors. When configured was a logistic-regression model which included the significant variables in the univariate analysis, it was demonstrated that GCS score (OR 0.285, 95% CI 0.184–0.410, P < 0.001) and plasma AM level (OR 1.302, 95% CI 1.119–3.042, P < 0.001) appeared to be the independent predictors of 6-month mortality in the patients. A ROC curve analysis found that a baseline plasma AM level predicted 6-month mortality of patients with high AUC in Fig. 3. The AUC of the AM concentration was similar to that of GCS scores (AUC, 0.924; 95% CI, 0.868–0.961) (P = 0.123). When a combined logistic-regression model was configured, AM improved the AUC of GCS score to 0.954 (95% CI, 0.907–0.982), but the difference did not appear to be statistically significant (P = 0.113). Discussion In agreement with previous reports that high plasma AM levels are highly associated with severity of ischemic or hemorrhagic stroke and also their clinical outcomes [27,29], The current study further determined the plasma levels of AM in such a group of patients with severe TBI and verified that plasma AM level was statistically significantly enhanced compared with healthy control and in addition, was negatively associated with GCS score; moreover, a multivariate logistic regression model identified it as an independent predictor of long-term clinical outcomes; in the other hand, ROC curve actually found its high predictive performance for long-term clinical outcome, suggesting that AM level may be associated with disease severity and become a good biomarker
T.-J. Chen et al. / Peptides 56 (2014) 146–150 Table 3 The factors associated with 6-month mortality.
Number Sex (male/female) Age (y) GCS score on admission Shock on admission Hyperglycemia on admission Hypoglycemia on admission Hypoxia on admission Pupils unreactive on admission CT classification 5 or 6 Abnormal cisterns on initial CT scan Midline shift > 5 mm on initial CT scan Traumatic SAH on initial CT scan Neurological deterioration Mechanical ventilation Intracranial surgery in 1st 24 h Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Mean arterial pressure (mmHg) Heart rate (beats/min) Body temperature (◦ C) Respiratory rate (respirations/min) Blood oxygen saturation (%) Blood white blood cell count (×109 /L) Blood hemoglobin level (g/L) Blood platelet count (×109 /L) Blood glucose level (mmol/L) Blood sodium level (mmol/L) Blood potassium level (mmol/L) Prothrombin time (s) Thrombin time (s) Partial thromboplastin time (s) Plasma C-reactive protein level (mg/L) Plasma fibrinogen level (g/L) Plasma d-dimer level (mg/L) Plasma adrenomedullin level (pg/mL)
Non-survival group
Survival group
42 (28.4%) 26/16 41.7 ± 17.8 3.6 ± 0.6 13 (31.0%) 16 (38.1%) 3 (7.1%) 6 (14.3%) 32 (76.2%) 28 (66.7%) 28 (66.7%) 29 (69.1%) 29 (69.1%) 13 (31.0%) 42 (100.0%) 22 (52.4%) 2.0 ± 0.8 3.0 ± 0.8 120.5 ± 37.6 73.3 ± 22.2 89.1 ± 25.8 84.3 ± 21.4 36.5 ± 0.7 19.8 ± 3.5 89.3 ± 9.3 8.0 ± 3.7 128.4 ± 28.2 170.6 ± 45.6 12.9 ± 4.3 144.2 ± 11.2 4.5 ± 0.9 15.1 ± 2.7 18.7 ± 2.5 39.1 ± 5.4 12.3 ± 4.1 4.8 ± 1.8 3.3 ± 1.6 155.2 ± 43.9
106 (71.6%) 72/34 44.4 ± 17.6 6.1 ± 1.5 16 (15.1%) 20 (18.9%) 5 (4.7%) 9 (8.5%) 43 (40.6%) 48 (45.3%) 44 (41.5%) 45 (42.5%) 49 (46.2%) 17 (16.0%) 83 (78.3%) 48 (45.3%) 2.1 ± 1.3 3.0 ± 1.5 119.1 ± 30.6 72.1 ± 20.5 87.8 ± 23.2 87.9 ± 22.1 36.7 ± 0.8 18.5 ± 4.0 91.9 ± 3.5 7.6 ± 2.4 123.0 ± 22.5 168.8 ± 38.0 11.0 ± 3.1 142.0 ± 7.3 4.3 ± 0.6 14.5 ± 2.3 18.1 ± 2.7 38.9 ± 6.7 10.5 ± 3.0 4.1 ± 1.9 2.7 ± 1.2 97.9 ± 32.0
P value
0.486 0.408 <0.001 0.028 0.014 0.556 0.292 <0.001 0.019 0.006 0.004 0.012 0.042 0.001 0.436 0.854 0.891 0.818 0.744 0.768 0.371 0.174 0.064 0.081 0.520 0.222 0.797 0.009 0.231 0.145 0.181 0.268 0.840 0.014 0.039 0.030 <0.001
Numerical variables were presented as mean ± standard deviation and analyzed by unpaired student t test. Categorical variables were expressed as counts (percentage) and analyzed by Chi-square test or Fisher exact test. GCS, Glasgow Coma Scale; CT, computerized tomography; SAH, subarachnoid hemorrhage.
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for the prediction of long-term clinical outcomes of patients with acute severe TBI. AM expression is up-regulated by ischemia–reperfusion in the cerebral cortex of the adult rat [25] and neurotoxicant in hippocampus and glia cultures [8]. In addition, AM levels in cerebrospinal fluid were increased obviously in patients with TBI [23,24] and aneurysmal subarachnoid hemorrhage [14,15]. Therefore, AM may be released into the cerebrospinal fluid from damaged brain tissue and afterwards escapes into the circulation in TBI. However, it is recently found that AM gene expression in peripheral blood leukocytes is enhanced after ischemic stroke [18]. Thus, AM in the peripheral blood is also proposed to be partly produced by peripheral blood cells. Although the precise reason for the increased plasma AM concentrations in TBI patients is not well understood, subsequent release of AM from injured cerebral tissues is a possible explanation. AM, as a biologically active peptide with potent vasodilating action, is now known to exert a wide range of physiological effects, including cardiovascular protection, neovascularization, and apoptosis suppression [3,4]. In a mouse brain-specific AM knockout model, lack of AM in the brain was associated with tubulin hyperpolymerization and decreased resistance to hypobaric hypoxia [5]. Similarly, AM heterozygous knockout led to accumulation of reactive oxygen species and increase in infarct size and neurological deficits after experimental stroke [20]. In contrast, AM overexpression mice were shown to be protected from ischemic stroke [21]. In addition, administration of exogenous AM has shown neuroprotection in experimental models of stroke [28] and traumatic brain injury [2]. Thus, the increased plasma AM level may be beneficial in suppressing brain injury after TBI. In this study, there was a close relationship between plasma AM levels and GCS scores, suggesting plasma AM levels should reflect head trauma severity. A ROC curve showed that plasma AM level on admission could obviously predict long-term unfavorable outcome and mortality; and its predictive value was similar to GCS score’s. Yet, a combined logistic-regression model did not verify that AM statistically significantly improved the predictive performance of GCS score. Therefore, the determination of AM in the plasma of head trauma patients on admission provides the ability to identify TBI patients at risk of 6-month bad outcome. Conclusions In this study, high plasma levels of AM are associated with head trauma severity, and may independently predict long-term clinical outcomes of TBI. Competing interests The authors declare that they have no competing interests. References
Fig. 3. Graph showing receiver operating characteristic (ROC) curve analysis of plasma adrenomedullin concentration for identifying patients with 6-month mortality. ROC curves were constructed based on the sensitivity and specificity of the plasma adrenomedullin concentration for identifying 6-month mortality. The area under curve (AUC) was calculated based on the ROC curves and expressed as 95% confidence interval (CI). AUC ranges from 0.5 to 1.0. An AUC closer to 1 indicates a higher predictive power.
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Plasma levels of adrenomedullin in patients with traumatic brain injury: Potential contribution to prognosis Tie-Jiang Chen ∗ , Qing-Yang Fu, Wu-Quan Wu Department of Emergency Surgery, Yiwu Central Hospital, 699 Jiangdong Road, Yiwu 322000, Zhejiang Province, China
a r t i c l e
i n f o
Article history: Received 26 March 2014 Received in revised form 4 April 2014 Accepted 4 April 2014 Available online 18 April 2014 Keywords: Adrenomedullin Traumatic brain injury Mortality Functional outcome
a b s t r a c t High plasma levels of adrenomedullin have been associated with stroke severity and clinical outcomes. This study aimed to analyze plasma levels of adrenomedullin in traumatic brain injury and their association with prognosis. One hundred and forty-eight acute severe traumatic brain injury and 148 sexand age-matched healthy controls were recruited in this study. Plasma adrenomedullin concentration was measured by enzyme-linked immunosorbent assay. Unfavorable outcome was defined as Glasgow Outcome Scale score of 1–3. Compared to controls, the patients had significantly higher plasma concentrations of adrenomedullin, which were also highly associated negatively with Glasgow Coma Scale score. Plasma adrenomedullin level was proved to be an independent predictor for 6-month mortality and unfavorable outcome of patients in a multivariate analysis. A receiver operating characteristic curve was configured to show that a baseline plasma adrenomedullin level predicted 6-month mortality and unfavorable outcome of patients with high area under curve. The predictive performance of the plasma adrenomedullin concentration was also similar to that of Glasgow Coma Scale score for the prediction of 6-month mortality and unfavorable outcome of patients. In a combined logistic-regression model, adrenomedullin improved the area under curve of Glasgow Coma Scale score for the prediction of 6-month mortality and unfavorable outcome of patients, but the differences did not appear to be statistically significant. Thus, high plasma levels of adrenomedullin are associated with head trauma severity, and may independently predict long-term clinical outcomes of traumatic brain injury. © 2014 Elsevier Inc. All rights reserved.
Introduction Traumatic Brain Injury (TBI) is known to represent a major public health concern potentially resulting in death or neurological impairment [6,17]. The pathophysiological mechanisms implicated in the cellular and molecular change following TBI remains unclear [1]. Reliable biomarkers for early prediction of prognosis and functional recovery are very few [13]. Adrenomedullin (AM) is a vasoactive peptide first isolated from pheochromocytoma [12]. Its gene expression is promoted by various stimuli, including inflammation, hypoxia, oxidative stress, mechanical stress and activation of the renin–angiotensin and sympathetic nervous systems [22]. AM possesses neuroprotection in experimental brain disease models including ischemic stroke [28] and traumatic brain injury [2], as well as cardioprotection in human myocardial infarction [11]. Blood levels of AM have been associated with prognosis of plenty of diseases including myocardial infarction, heart failure
∗ Corresponding author. Tel.: +86 0579 85209666. E-mail address: [email protected] (T.-J. Chen). http://dx.doi.org/10.1016/j.peptides.2014.04.005 0196-9781/© 2014 Elsevier Inc. All rights reserved.
and pulmonary hypertension [7]. AM has also been reported to be present in neurons and glia in the central nervous system [8]. Recently, circulating AM has been demonstrated to be of prognostic importance in ischemic or hemorrhagic stroke [27,29]. The present study aimed to further investigate the ability of plasma AM to predict long-term clinical outcomes in a group of patients with acute severe TBI. Materials and methods Study population This study included severe isolated head trauma patients with postresuscitation Glasgow Coma Scale (GCS) score of 8 or less from Yiwu Central Hospital between April 2010 and April 2013. Nonselection criteria involved less than 18 years of age, admission time > 6 h, previous head trauma, neurological disease including ischemic or hemorrhagic stroke, use of antiplatelet or anticoagulant medication, diabetes mellitus, hypertension or presence of other prior systemic diseases including uremia, liver cirrhosis, malignancy, chronic heart or lung disease. Healthy age- and sex-matched
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volunteers were recruited as control group. At study entrance, all participants or their legal representatives gave their informed consent, and study protocol was approved by the Ethics Committee of Yiwu Central Hospital before implementation. Clinical and radiological assessment Information regarding the following variables: postresuscitation GCS score, systolic blood pressure, blood oxygen saturation, blood glucose and papillary reactivity was obtained at admission. Head trauma severity was assessed using initial postresuscitation GCS score. Shock was defined as systolic blood pressure less than 90 mmHg [10]. Hypoxia was defined as blood oxygen saturation less than 85% [10]. Hyperglycemia was defined as blood glucose more than 11.1 mmol/L [16]. Hypoglycemia was defined as blood glucose less than 2.2 mmol/L [26]. Neurology deterioration was defined as occurring in patients who manifested clinically identified episodes of one or more of the following: (1) a spontaneous decrease in GCS motor scores of 2 points or more from the previous examination; (2) a further loss of papillary reactivity; (3) development of papillary asymmetry greater than 1 mm; or (4) deterioration in neurological status sufficient to warrant immediate medical or surgical intervention [10]. All computerized tomography (CT) scans were performed according to the neuroradiology department protocol. Investigators who read them were blinded to clinical information. Abnormal cisterns, midline shift >5 mm and subarachnoid hemorrhage were recorded on initial CT scan. CT classification was performed using Traumatic Coma Data Bank criteria on initial postresuscitation CT scan according to the method of Marshall et al. [19]. Participants were followed up until death or completion of 6 months after head trauma. The functional outcome was defined by Glasgow outcome scale (GOS) score. GOS was defined as follows: 1 = death; 2 = persistent vegetative state; 3 = severe disability; 4 = moderate disability; and 5 = good recovery [9]. Unfavorable outcome was defined as GOS of 1–3. For follow-up, we used structure telephone interviews performed by 1 doctor, blinded to clinical information and AM levels. Determination of plasma AM levels The informed consents were obtained from all participants or their legal representatives before the blood were collected. Venous blood was drawn at study entry in the control group and on admission in the patients. The blood samples were immediately placed into sterile EDTA test tubes and centrifuged at 1500 × g for 20 min at 4 ◦ C to collect plasma. Plasma was stored at −70◦ C until assayed. Plasma AM concentration was analyzed by enzyme-linked immunosorbent assay using commercial kits (R&D Systems, Heidelberg, Germany) in accordance with the manufactures’ instructions. The blood samples were run in duplicate. Researchers running enzyme-linked immunosorbent assays were blinded to all patient details. Statistical analysis Statistical analysis was performed with SPSS 19.0 (SPSS Inc., Chicago, IL, USA) and MedCalc 9.6.4.0. (MedCalc Software, Mariakerke, Belgium). All values are expressed as mean ± standard deviation or counts (percentage) unless otherwise specified. Comparisons were made by using (1) Chi-square test or Fisher exact test for categorical data, (2) Student t test for continuous distributed variables. The association of plasma AM levels with GCS scores was analyzed using Spearman correlation coefficient. The relations of AM to 6-month mortality and unfavorable outcome were assessed in a logistic-regression model with odds ratio (OR)
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Table 1 The demographic data, clinical and biochemical characteristics of 148 patients. Characteristics Sex (male/female) Age (y) GCS score on admission Shock on admission Hyperglycemia on admission Hypoglycemia on admission Hypoxia on admission Pupils unreactive on admission CT classification 5 or 6 Abnormal cisterns on initial CT scan Midline shift > 5 mm on initial CT scan Presence of traumatic SAH on initial CT scan Neurological deterioration Mechanical ventilation Intracranial surgery in 1st 24 h Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Mean arterial pressure (mmHg) Heart rate (beats/min) Body temperature (◦ C) Respiratory rate (respirations/min) Blood oxygen saturation (%) Blood white blood cell count (×109 /L) Blood hemoglobin level (g/L) Blood platelet count (×109 /L) Blood glucose level (mmol/L) Blood sodium level (mmol/L) Blood potassium level (mmol/L) Prothrombin time (s) Thrombin time (s) Partial thromboplastin time (s) Plasma C-reactive protein level (mg/L) Plasma fibrinogen level (g/L) Plasma d-dimer level (mg/L) Plasma adrenomedullin level (pg/mL)
98/50 43.6 ± 17.6 5.4 ± 1.8 29 (19.6%) 36 (24.3%) 8 (5.4%) 15 (10.1%) 75 (50.7%) 76 (51.4%) 72 (48.7%) 74 (50.0%) 78 (52.7%) 30 (20.3%) 125 (84.5%) 70 (47.3%) 2.1 ± 1.2 3.0 ± 1.3 119.5 ± 32.6 72.4 ± 20.9 88.1 ± 23.9 86.9 ± 21.9 36.6 ± 0.8 18.8 ± 3.9 91.2 ± 5.9 7.7 ± 2.8 124.5 ± 24.2 169.3 ± 40.1 11.5 ± 3.6 142.5 ± 8.6 4.4 ± 0.7 14.7 ± 2.4 18.3 ± 2.7 38.9 ± 6.4 11.0 ± 3.4 4.3 ± 1.9 2.8 ± 1.3 114.2 ± 44.1
Numerical variables were presented as mean ± standard deviation. Categorical variables were expressed as counts (percentage). GCS, Glasgow Coma Scale; CT, computerized tomography; SAH, subarachnoid hemorrhage.
and 95% confidence interval (CI). The receiver operating characteristic (ROC) curves was used to determine the best threshold of AM values to predict 6-month clinical outcomes with calculated area under curve (AUC). In a combined logistic-regression model, the additive benefit of AM to GCS score was estimated. A P value of <0.05 was considered significant for all test. Results Study population characteristics This study finally assessed one hundred and forty-eight severe isolated head trauma patients and 148 sex- and age-matched healthy controls. Table 1 summarized the demographic data, clinical and biochemical characteristics of patients at baseline. Compared to controls, the patients had significantly higher plasma concentrations of AM (114.2 ± 44.1 pg/mL vs. 46.6 ± 16.0 pg/mL, P < 0.0001). Fig. 1 showed that plasma concentrations of AM were also highly associated negatively with GCS scores (r = −0.568, P < 0.0001). Functional outcome prediction Sixty-six patients (44.6%) had unfavorable outcome at 6 months after head trauma. In Table 2, a univariate analysis found that plasma AM levels were markedly higher in patients with unfavorable outcome than favorable outcome. When configured was
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Fig. 1. Graph showing the relationship between plasma adrenomedullin concentration and Glasgow Coma Scale score using Spearman correlation coefficient.
a logistic-regression model which included the significant variables in the univariate analysis, it was demonstrated that GCS score (OR 0.300, 95% CI 0.193–0.466, P < 0.001) and plasma AM level (OR 1.232, 95% CI 1.112–2.351, P < 0.001) appeared to be the independent predictors of 6-month unfavorable outcome in the patients. Table 2 The factors associated with 6-month unfavorable outcome.
Number Sex (male/female) Age (y) GCS score on admission Shock on admission Hyperglycemia on admission Hypoglycemia on admission Hypoxia on admission Pupils unreactive on admission CT classification 5 or 6 Abnormal cisterns on initial CT scan Midline shift > 5 mm on initial CT scan Traumatic SAH on initial CT scan Neurological deterioration Mechanical ventilation Intracranial surgery in 1st 24 h Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Mean arterial pressure (mmHg) Heart rate (beats/min) Body temperature (◦ C) Respiratory rate (respirations/min) Blood oxygen saturation (%) Blood white blood cell count (×109 /L) Blood hemoglobin level (g/L) Blood platelet count (×109 /L) Blood glucose level (mmol/L) Blood sodium level (mmol/L) Blood potassium level (mmol/L) Prothrombin time (s) Thrombin time (s) Partial thromboplastin time (s) Plasma C-reactive protein level (mg/L) Plasma fibrinogen level (g/L) Plasma d-dimer level (mg/L) Plasma adrenomedullin level (pg/mL)
Unfavorable outcome
Favorable outcome
66 (44.6%) 41/25 44.0 ± 17.1 4.0 ± 0.8 19 (28.8%) 22 (33.3%) 4 (6.1%) 8 (12.1%) 45 (68.2%) 41 (62.1%) 42 (63.3%) 45 (68.2%) 42 (63.3%) 19 (28.8%) 66 (100.0%) 34 (51.5%) 1.9 ± 0.8 2.8 ± 0.9 114.1 ± 36.6 69.9 ± 24.2 84.7 ± 27.1 84.1 ± 20.5 36.6 ± 0.7 18.4 ± 4.0 90.2 ± 7.7 7.4 ± 3.2 123.3 ± 25.0 166.3 ± 38.5 12.4 ± 4.2 144.0 ± 9.6 4.4 ± 0.9 14.9 ± 2.4 18.7 ± 2.5 38.9 ± 6.9 12.0 ± 4.1 4.6 ± 1.8 3.1 ± 1.4 142.6 ± 42.1
82 (55.4%) 57/25 43.3 ± 18.1 6.6 ± 1.4 10 (12.2%) 14(17.1%) 4 (4.9%) 7 (8.5%) 30 (36.6%) 35 (42.7%) 30 (36.6%) 29 (35.4%) 36 (43.9%) 11 (13.4%) 59 (72.0%) 36 (43.9%) 2.2 ± 1.4 3.1 ± 1.6 123.9 ± 28.6 74.5 ± 17.7 90.9 ± 20.7 89.1 ± 22.8 36.7 ± 0.8 19.2 ± 3.8 92.0 ± 3.6 7.9 ± 2.4 125.4 ± 23.7 171.7 ± 41.5 10.8 ± 2.8 141.4 ± 7.7 4.3 ± 0.6 14.6 ± 2.5 18.0 ± 2.8 39.0 ± 5.9 10.2 ± 2.5 4.0 ± 2.0 2.6 ± 1.3 91.3 ± 30.3
P value
0.345 0.817 <0.001 0.011 0.022 0.752 0.473 <0.001 0.019 0.001 <0.001 0.017 0.021 <0.001 0.357 0.091 0.159 0.076 0.208 0.123 0.170 0.561 0.250 0.094 0.230 0.595 0.426 0.007 0.060 0.319 0.465 0.123 0.928 0.002 0.037 0.014 <0.001
Numerical variables were presented as mean ± standard deviation and analyzed by unpaired student t test. Categorical variables were expressed as counts (percentage) and analyzed by Chi-square test or Fisher exact test. GCS, Glasgow Coma Scale; CT, computerized tomography; SAH, subarachnoid hemorrhage.
Fig. 2. Graph showing receiver operating characteristic (ROC) curve analysis of plasma adrenomedullin concentration for identifying patients with 6-month unfavorable outcome. ROC curves were constructed based on the sensitivity and specificity of the plasma adrenomedullin concentration for identifying 6-month unfavorable outcome. The area under curve (AUC) was calculated based on the ROC curves and expressed as 95% confidence interval (CI). AUC ranges from 0.5 to 1.0. An AUC closer to 1 indicates a higher predictive power.
A ROC curve analysis found that a baseline plasma AM level predicted 6-month unfavorable outcome of patients with high AUC in Fig. 2. The AUC of the AM concentration was similar to that of GCS scores (AUC, 0.914; 95% CI, 0.857–0.954) (P = 0.103). When a combined logistic-regression model was configured, AM improved the AUC of GCS score to 0.940 (95% CI, 0.889–0.972), but the difference did not appear to be statistically significant (P = 0.134). Mortality prediction Forty-two patients (28.4%) died within 6 months after head trauma. In Table 3, a univariate analysis found that plasma AM levels were markedly higher in non-survivors than survivors. When configured was a logistic-regression model which included the significant variables in the univariate analysis, it was demonstrated that GCS score (OR 0.285, 95% CI 0.184–0.410, P < 0.001) and plasma AM level (OR 1.302, 95% CI 1.119–3.042, P < 0.001) appeared to be the independent predictors of 6-month mortality in the patients. A ROC curve analysis found that a baseline plasma AM level predicted 6-month mortality of patients with high AUC in Fig. 3. The AUC of the AM concentration was similar to that of GCS scores (AUC, 0.924; 95% CI, 0.868–0.961) (P = 0.123). When a combined logistic-regression model was configured, AM improved the AUC of GCS score to 0.954 (95% CI, 0.907–0.982), but the difference did not appear to be statistically significant (P = 0.113). Discussion In agreement with previous reports that high plasma AM levels are highly associated with severity of ischemic or hemorrhagic stroke and also their clinical outcomes [27,29], The current study further determined the plasma levels of AM in such a group of patients with severe TBI and verified that plasma AM level was statistically significantly enhanced compared with healthy control and in addition, was negatively associated with GCS score; moreover, a multivariate logistic regression model identified it as an independent predictor of long-term clinical outcomes; in the other hand, ROC curve actually found its high predictive performance for long-term clinical outcome, suggesting that AM level may be associated with disease severity and become a good biomarker
T.-J. Chen et al. / Peptides 56 (2014) 146–150 Table 3 The factors associated with 6-month mortality.
Number Sex (male/female) Age (y) GCS score on admission Shock on admission Hyperglycemia on admission Hypoglycemia on admission Hypoxia on admission Pupils unreactive on admission CT classification 5 or 6 Abnormal cisterns on initial CT scan Midline shift > 5 mm on initial CT scan Traumatic SAH on initial CT scan Neurological deterioration Mechanical ventilation Intracranial surgery in 1st 24 h Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Mean arterial pressure (mmHg) Heart rate (beats/min) Body temperature (◦ C) Respiratory rate (respirations/min) Blood oxygen saturation (%) Blood white blood cell count (×109 /L) Blood hemoglobin level (g/L) Blood platelet count (×109 /L) Blood glucose level (mmol/L) Blood sodium level (mmol/L) Blood potassium level (mmol/L) Prothrombin time (s) Thrombin time (s) Partial thromboplastin time (s) Plasma C-reactive protein level (mg/L) Plasma fibrinogen level (g/L) Plasma d-dimer level (mg/L) Plasma adrenomedullin level (pg/mL)
Non-survival group
Survival group
42 (28.4%) 26/16 41.7 ± 17.8 3.6 ± 0.6 13 (31.0%) 16 (38.1%) 3 (7.1%) 6 (14.3%) 32 (76.2%) 28 (66.7%) 28 (66.7%) 29 (69.1%) 29 (69.1%) 13 (31.0%) 42 (100.0%) 22 (52.4%) 2.0 ± 0.8 3.0 ± 0.8 120.5 ± 37.6 73.3 ± 22.2 89.1 ± 25.8 84.3 ± 21.4 36.5 ± 0.7 19.8 ± 3.5 89.3 ± 9.3 8.0 ± 3.7 128.4 ± 28.2 170.6 ± 45.6 12.9 ± 4.3 144.2 ± 11.2 4.5 ± 0.9 15.1 ± 2.7 18.7 ± 2.5 39.1 ± 5.4 12.3 ± 4.1 4.8 ± 1.8 3.3 ± 1.6 155.2 ± 43.9
106 (71.6%) 72/34 44.4 ± 17.6 6.1 ± 1.5 16 (15.1%) 20 (18.9%) 5 (4.7%) 9 (8.5%) 43 (40.6%) 48 (45.3%) 44 (41.5%) 45 (42.5%) 49 (46.2%) 17 (16.0%) 83 (78.3%) 48 (45.3%) 2.1 ± 1.3 3.0 ± 1.5 119.1 ± 30.6 72.1 ± 20.5 87.8 ± 23.2 87.9 ± 22.1 36.7 ± 0.8 18.5 ± 4.0 91.9 ± 3.5 7.6 ± 2.4 123.0 ± 22.5 168.8 ± 38.0 11.0 ± 3.1 142.0 ± 7.3 4.3 ± 0.6 14.5 ± 2.3 18.1 ± 2.7 38.9 ± 6.7 10.5 ± 3.0 4.1 ± 1.9 2.7 ± 1.2 97.9 ± 32.0
P value
0.486 0.408 <0.001 0.028 0.014 0.556 0.292 <0.001 0.019 0.006 0.004 0.012 0.042 0.001 0.436 0.854 0.891 0.818 0.744 0.768 0.371 0.174 0.064 0.081 0.520 0.222 0.797 0.009 0.231 0.145 0.181 0.268 0.840 0.014 0.039 0.030 <0.001
Numerical variables were presented as mean ± standard deviation and analyzed by unpaired student t test. Categorical variables were expressed as counts (percentage) and analyzed by Chi-square test or Fisher exact test. GCS, Glasgow Coma Scale; CT, computerized tomography; SAH, subarachnoid hemorrhage.
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for the prediction of long-term clinical outcomes of patients with acute severe TBI. AM expression is up-regulated by ischemia–reperfusion in the cerebral cortex of the adult rat [25] and neurotoxicant in hippocampus and glia cultures [8]. In addition, AM levels in cerebrospinal fluid were increased obviously in patients with TBI [23,24] and aneurysmal subarachnoid hemorrhage [14,15]. Therefore, AM may be released into the cerebrospinal fluid from damaged brain tissue and afterwards escapes into the circulation in TBI. However, it is recently found that AM gene expression in peripheral blood leukocytes is enhanced after ischemic stroke [18]. Thus, AM in the peripheral blood is also proposed to be partly produced by peripheral blood cells. Although the precise reason for the increased plasma AM concentrations in TBI patients is not well understood, subsequent release of AM from injured cerebral tissues is a possible explanation. AM, as a biologically active peptide with potent vasodilating action, is now known to exert a wide range of physiological effects, including cardiovascular protection, neovascularization, and apoptosis suppression [3,4]. In a mouse brain-specific AM knockout model, lack of AM in the brain was associated with tubulin hyperpolymerization and decreased resistance to hypobaric hypoxia [5]. Similarly, AM heterozygous knockout led to accumulation of reactive oxygen species and increase in infarct size and neurological deficits after experimental stroke [20]. In contrast, AM overexpression mice were shown to be protected from ischemic stroke [21]. In addition, administration of exogenous AM has shown neuroprotection in experimental models of stroke [28] and traumatic brain injury [2]. Thus, the increased plasma AM level may be beneficial in suppressing brain injury after TBI. In this study, there was a close relationship between plasma AM levels and GCS scores, suggesting plasma AM levels should reflect head trauma severity. A ROC curve showed that plasma AM level on admission could obviously predict long-term unfavorable outcome and mortality; and its predictive value was similar to GCS score’s. Yet, a combined logistic-regression model did not verify that AM statistically significantly improved the predictive performance of GCS score. Therefore, the determination of AM in the plasma of head trauma patients on admission provides the ability to identify TBI patients at risk of 6-month bad outcome. Conclusions In this study, high plasma levels of AM are associated with head trauma severity, and may independently predict long-term clinical outcomes of TBI. Competing interests The authors declare that they have no competing interests. References
Fig. 3. Graph showing receiver operating characteristic (ROC) curve analysis of plasma adrenomedullin concentration for identifying patients with 6-month mortality. ROC curves were constructed based on the sensitivity and specificity of the plasma adrenomedullin concentration for identifying 6-month mortality. The area under curve (AUC) was calculated based on the ROC curves and expressed as 95% confidence interval (CI). AUC ranges from 0.5 to 1.0. An AUC closer to 1 indicates a higher predictive power.
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