The global incidence for out-of-hospital cardiac arrest (OHCA) varies between 36 and 55 adults per 100,000 person-years (1). Patients with shockable initial rhythm (pulseless ventricular tachycardia (VT) or ventricular fibrillation (VF)) have the best chance of survival (2). During cardiac arrest (CA) blood flow is disrupted and patients are subjected to brain injury, myocardial dysfunction, and global ischemia (3). After successful resuscitation reperfusion injury follows, that leads to cell death and organ dysfunction. Heme oxygenase- 1 (HO-1) is an endogenous stress response enzyme that is ubiquitously expressed among different cells and tissues. It degrades heme into carbon monoxide (CO), free iron, and biliverdin, which is rapidly converted into bilirubin by biliverdin reductase. These by-products mediate anti-apoptotic, anti-inflammatory, anti-oxidative, and vasoactive effects of HO-1 (4). In addition, overexpression of HO-1 associates with a decreased incidence of ischemia/reperfusion-induced VF in isolated rat and mouse hearts (5–7). While HO-1 is normally expressed in low levels, it is highly inducible in different pathologic conditions, especially in response to oxidative stress, ischemia-reperfusion, and inflammation (8). Liu et al. (9) demonstrated that HO-1 expression is increased in brain microglia/macrophage cells in a rat traumatic brain injury model. In addition, HO-1 gene expression has been shown to increase in peripheral blood mononuclear cells in an experimental swine CA model (10). However, the role of HO-1 in human CA has not been studied earlier. In our previous study, we found that plasma HO-1 associated with more severe disease and multiple organ dysfunction in critically ill patients (11). Accordingly, we hypothesized here that higher plasma HO-1 would be associated with longer ROSC, higher 90-mortality, and poor 12-month neurological outcome after OCHA.
Patients and blood sampling
We conducted a substudy of the 12-month prospective observational multicenter cohort study (FINNRESUSCI) in 21 Finnish ICU's between March 2010 and February 2011. The main study focused on post-resuscitation care in therapeutic hypothermia treated unconscious patients with shockable initial rhythm (12). The inclusion criteria for the study were out-of-Hospital Cardiac Arrest (OHCA), successful resuscitation, age over 18, and admission to a participating ICU. Written informed consent from the patients’ next of kin was obtained for data collection and blood sampling. The study was approved by the Ethics Committee of Helsinki University Hospital.
Plasma samples were collected after consent from the next-of-kin at admission and at 1, 2, and 4 days after the arrest, and stored at −70°C for subsequent analysis. To obtain a homogeneous population for this substudy, we included only patients with shockable initial rhythm (VF/VT). Based on experimental studies, maximum HO-1 levels are usually found at 24 h after the onset of stress or administration of HO-1 inducers (13, 14). Therefore, admission and day 1 samples were analyzed from the whole study population. In addition, the full time course including all plasma samples from admission to day 4 was analyzed from 30 randomly selected patients to evaluate the kinetics of plasma HO-1 in cardiac arrest. The primary outcomes of this study were 90-day mortality, and 12-month neurological outcome.
A neurologist (MT) evaluated the neurological outcome by telephone interviewing either the patient, the patient's next-of-kin or health care facility staff 12 months after the incident. Patient's neurological functional status was categorized according to the Pittsburgh Cerebral Performance Categories (CPC). A CPC 1 (normal cerebral performance) or CPC 2 (moderate cerebral disability) was considered good neurological outcome, and CPC 3 (severe cerebral disability), CPC 4 (coma or vegetative state), and CPC 5 (brain death or death) as a poor neurological outcome.
HO-1 plasma concentration
Human HO-1 ELISA kit (Enzo Life Sciences, Farmingdale, New York) was used for the analysis of HO-1 plasma concentrations. The coefficient of variation (CV%) was determined for precision of the analysis and yielded 5.4% for intra-assay and 7.2% for inter-assay CV. The reference range for HO-1 (0.66–2.39 ng/mL) was determined in our previous study from 58 healthy volunteers (28 women and 30 men) (11). The same reference population was used as a control group in this study.
We used SPSS 22.0 to analyze the data (SPSS Inc, Chicago, IL). Data are presented as medians and interquartile ranges (IQR), quartiles (Q1–Q4), percentages, or absolute values. The non-parametric data were analyzed using Fisher's exact for categorical variables and Mann–Whitney U test for continuous variables. Correlations between continuous variables were calculated with Spearman rank correlation. To assess the role of different independent factors predicting poor neurological outcome (CPC3-5) or mortality, a multivariate logistic regression model was performed. Variables included in the analysis were age, time to ROSC, witnessed CA, APACHE II, and HO-1 concentration at admission or day 1. The predictive value of HO-1 plasma concentration was analyzed with receiver-operating characteristics (ROC) and area under the curves (AUCs). Sensitivity, specificity, Youden index, likelihood ratios (LR +/−), and predictive values (PPV, NPV) for poor neurological outcome and mortality were calculated. Kaplan–Meier curve with log rank test was used to evaluate mortality between HO-1 quartiles (Q1–Q4). A P value <0.05 was considered significant.
The study included a total of 311 patients with a shockable initial rhythm after OHCA. One hundred sixty-eight patients were excluded for missing consent or for missing blood samples. Missing sample from either admission or day 1 resulted in exclusion from this substudy and therefore the final study sample included a total of 143 patients (Fig. 1). Median age of the patients was 62 (56–72) years and 85% (121 of 143) of the patients were male. The total mortality rate was 33% (47 of 143) at 90 days after OHCA. Poor neurological outcome (CPC 3-5) at 12 months was 41% (58 of 143). The characteristics of poor outcome patients compared with good outcome patients are presented in Table 1. Higher age, coronary artery disease, heart failure, longer time to ROSC, and more frequently unwitnessed cardiac arrest, as well as higher APACHE II, SAPS II and SOFA scores were associated with increased mortality and poor neurological outcome (CPC 3-5). No statistically significant difference was found between patients excluded from the study and study group (Supplemental Table 2, Supplemental Digital Content 1, at https://links.lww.com/SHK/A342).
HO-1 plasma concentrations
HO-1 plasma concentration increased significantly after OHCA and remained higher up to 4 days of observation compared with healthy individuals (admission 2.75 ng/mL; day 1 2.76 ng/mL; day 2 2.87 ng/mL; day 4 3.18 ng/mL vs. healthy controls 1.47 ng/mL; P < 0.0001 each; Supplemental Figure 1, Supplemental Digital Content 1, at https://links.lww.com/SHK/A342). No significant differences between time points after admission were observed. HO-1 concentration did not change markedly in individual patients over time.
HO-1 concentrations were significantly higher in 90-day non-survivors compared with survivors at admission (3.09 (2.34–4.13) ng/mL vs. 2.56 (2.09–3.20) ng/mL, P = 0.017) and day 1 (2.99 (2.35–4.11) ng/mL vs. 2.58 (1.97–3.49) ng/mL, P = 0.026) (Fig. 2). Admission HO-1 concentrations were also higher in patients with poor 12-month neurological outcome compared with good outcome (3.07 (2.22–4.14) ng/mL vs. 2.56 (2.09–3.20) ng/mL, P = 0.024) (Fig. 2). In addition, higher HO-1 levels at admission and day 1 correlated significantly with time to ROSC (Rho admission 0.256, P = 0.002; day 1 0.246, P = 0.003), APACHE II (Rho admission 0.212, P = 0.011; day 1 0.249, P = 0.003) and SAPS II (Rho admission 0.157, P = 0.062; day 1 0.191, P = 0.022) scores. We found no correlation between age, weight, creatinine levels, or other disease states and HO-1 concentrations.
Predictive value of plasma HO-1
Admission plasma HO-1 levels had an AUC of 0.623 to predict 90-day mortality and an AUC of 0.611 to predict poor neurological outcome at 12 months. The corresponding AUC values of day 1 plasma HO-1 levels were 0.615 and 0.585 to predict 90-day mortality and poor neurological outcome. AUC values of SAPS II and SOFA were superior to HO-1 for predicting both mortality (SAPS II 0.765, SOFA 0.719) and poor neurological outcome (SAPS II 0.770, SOFA 0.702).
The optimal cut-off values of admission and day 1 HO-1, and the diagnostic performance of HO-1 are shown in Table 2. In a multivariate logistic regression model factors included were age, time to ROSC, witnessed CA, APACHE II, and HO-1 concentration at admission or day 1 (Supplemental Table 1, Supplemental Digital Content 1, at https://links.lww.com/SHK/A342). In this model, age, time to ROSC, and APACHE II were independent predictors of both mortality and poor neurological outcome, whereas HO-1 was not an independent predictor of outcome. HO-1 did not significantly improve AUC when combined to other factors (mortality admission: AUC with HO-1 = 0.856 vs. AUC without HO-1 = 0.855, P = 0.985; mortality day 1: AUC with HO-1 = 0.857 vs. AUC without HO-1 = 0.855 P = 0.970; poor neurological outcome admission: AUC with HO-1 = 0.848 vs. AUC without HO-1 = 0.847, P = 0.984; poor neurological outcome day 1: AUC with HO-1 = 0.848 vs. AUC without HO-1 = 0.847, P = 0.984). The factors chosen to regression model had no significant interaction.
Kaplan–Meier survival curves according to quartiles (Q1–Q4) of admission HO-1 concentrations are shown in Figure 3. Mortality was significantly higher with higher HO-1 concentrations (Q1 vs. Q4 P = 0.046; Q2 vs. Q4 P = 0.007).
In this prospective multicenter observational study we found that HO-1 plasma concentrations are significantly elevated after OHCA compared with controls, and higher HO-1 plasma concentrations at admission are associated with longer time to ROSC, increased 90-day mortality and poor 12-month neurological outcome.
The results of this study are in broad agreement with our previous study showing increased HO-1 plasma concentrations in a heterogeneous group of critically ill patients and association of higher HO-1 concentrations with more severe disease and multiple organ dysfunction (11). Increased HO-1 plasma levels have also been shown in patients with sepsis compared with healthy controls (15). Similarly, Takaki et al. (16) showed that arterial blood CO levels and HO-1 protein expression of peripheral blood monocytes are increased in patients with severe sepsis compared with non-septic patients. In recent studies, increased plasma HO-1 concentrations have also been shown to be associated with acute kidney injury and type 2 diabetes (17–19). However, to the best of our knowledge, this study is the first to confirm increased HO-1 levels among OHCA patients.
During CA a whole-body ischemia and reperfusion occurs due to dramatically decreased cardiac output leading to oxygen debt. After successful resuscitation the reperfusion injury follows. The pathophysiology of CA comprises increased apoptosis, impaired vasoregulation, increased blood coagulation, extensive inflammation reaction followed by activation of immunologic pathways, and oxidative stress due to increased ROS and free radical production (20). Thus, the detected higher HO-1 levels are biologically plausible.
HO-1 has been implicated in cardioprotection (5, 21, 22), and neuroprotection (23, 24), but also in protection of other organs such as lung (25, 26), kidney (27), and liver (28), all vulnerable to ischemia-reperfusion injury after CA. HO-1 is strongly induced in ischemic conditions and in response to oxidative stress, suggesting that it may regulate the development of ischemia-reperfusion injury.
The beneficial effects of HO-1 (antiapoptotic, antiinflammatory, antioxidative, and vasoactive effects) have been widely evaluated in numerous experimental studies (8). HO-1 knockout mice hearts are more vulnerable to ischemic injury in hypoxic conditions (29, 30). In a global ischemia-reperfusion injury model in rabbits induction of HO-1 was shown to decrease the number of apoptotic cardiomyocytes, caspase-3, Akt, and level of nuclear NF-κB and AP-1 (31). Juhasz et al. (7) showed in an ischemia-reperfusion injury model that overexpression of HO-1 improves hemodynamics and decreases infarct size in isolated mouse hearts, whereas HO-1 inhibitor SnPPIX exacerbates these effects. We have described earlier that HO-1 induction reduced apoptosis and increased proliferation and repair of cardiomyocytes after experimental myocardial infarction (14). Heme degradation by HO-1 is the major source of endogenous CO. Yao et al. (32) 2014 showed that CORM2, a CO-releasing molecule, protected the heart in a rat cardiac arrest model. They suggested a mechanism where CO attenuates mitochondrial injury and dysfunction. Pretreatment with CO has also been shown to protect the heart against reperfusion injury after cardiopulmonary bypass in pigs (33).
The neuroprotective effect of HO-1 has also been widely studied both in vivo(24) and in vitro(23). Zhang et al. (34) found that HO-1 induction reduces brain edema, improves neurological outcome, and protects against neuronal apoptosis in a rat experimental CA model. In addition, inhaled CO has been shown protective against ischemic brain injury in vivo by altering tissue bioenergetics and preventing neuronal apoptosis (35, 36). Furthermore, metformin was shown to protect the brain by reducing levels of NF-κB, TNF-α, and COX2 inflammation pathways and increasing the level of HO-1 in brain tissue through Nrf2 pathway in a rat global brain ischemia model (37). Likewise, propofol attenuated brain ischemia-reperfusion injury partially through induction of HO-1 in the ischemic area (38).
The increase in HO-1 plasma concentration after OHCA is likely a physiological defense response against ischemia-reperfusion injury. We and others have shown earlier that HO-1 induction and CO-treatment decrease the incidence of ischemia-reperfusion induced VF in isolated and perfused mouse and rat hearts (5, 6). Although HO-1 is commonly considered to protect against I/R injury, in CA patients, higher HO-1 levels correlated significantly with time to ROSC and disease severity scores APACHE II and SAPS II. Longer time to ROSC and higher disease severity scores indicate worse injury and cell death. Therefore, it appears that increased plasma HO-1 is rather a measure of the degree of I/R- injury and/or the degree of oxidative stress, and it is most likely released from injured cells and organs. However, compared with organ specific markers, like neuron-specific S100 B, HO-1 is not applicable for diagnosing specific organ injury as it is ubiquitously expressed in all organs. Thus, the results of this study suggest that plasma HO-1 could be useful as a general marker of disease severity.
We found that higher HO-1 plasma concentrations were statistically related to 90-day mortality and poor 12-month neurological outcome. However, the sensitivity and specificity remained inadequate. In this homogenous patient group of shockable OHCA patients, the HO-1 AUC value was highest at admission yielding 0.623 for mortality and 0.611 for poor neurological outcome. Furthermore, HO-1 was not an independent predictor for either outcome. Our study suggests that the performance of plasma HO-1 is inadequate in clinical decision making or prediction of outcome in cardiac arrest patients either as an individual biomarker or in addition to ICU scores. However, plasma HO-1 may turn out to be useful in other conditions or in combination with other biomarkers in the future. Our findings are also significant concerning HO-1 in the post resuscitation pathophysiology but its role remains unclear.
The strength of this study is the homogenous group of OHCA patients with a shockable initial rhythm. As a limitation of the study, we did not obtain informed consent for the laboratory measurements from all patients. It is also possible that some patients with a short ROSC and stable hemodynamics were removed from the ICU before day 1 and thus discarded from the study. Some of the commonly prescribed drugs such as aspirin (39, 40), metformin (37), simvastatin (41), and ACE inhibitors (42) have been shown to induce HO-1 expression. In addition, other drugs might also have an effect on HO-1 plasma levels. Based on this study, it is not possible to determine whether HO-1 has a specific role regulating the development of I/R-injury in OCHA patients. Studying the association of HO-1 polymorphisms with the outcome of CA might shed a light on this in the future, as HO-1 polymorphisms affect HO-1 protein levels and the degree of HO-1 induction in response to stress. HO-1 pathway may also be a potential therapeutic target in cardiac arrest patients in the future as the protective properties of HO-1 induction against development of I/R-injury have been clearly demonstrated in experimental studies (7, 14, 32, 34). Furthermore, clinical trials, such as the StAamP trial that studies the induction of HO-1 with statins to protect against preeclampsia, are already conducted at the moment (43).
In conclusion, we found that higher HO-1 plasma concentrations associated with 90-day mortality and poor 12-month long-term neurological outcome. However, HO-1 plasma concentrations do not provide adequate additional value for clinical use.
The authors are grateful to the FINNRESUSCI study group for providing the data and blood samples for this study. The authors would also like to thank Riikka Kosonen for expert technical assistance. This study was supported by the Finnish Cardiac Society, Finnish Foundation for Laboratory Medicine, Finska Lakaresallskapet, the Liv och Halsa Foundation, the Finnish Foundation for Cardiovascular Research and Research Funding of the Hospital District of Helsinki and Uusimaa.
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