Resuscitation 85 (2014) 595–601
Contents lists available at ScienceDirect
Resuscitation journal homepage: www.elsevier.com/locate/resuscitation
Review article 1
H NMR-metabolomics: Can they be a useful tool in our understanding of cardiac arrest?夽 Athanasios Chalkias a,b,∗ , Vassilios Fanos c , Antonio Noto c , Maaret Castrén d , Anil Gulati e , Hildigunnur Svavarsdóttir f , Nicoletta Iacovidou b,g , Theodoros Xanthos a,b a
MSc “Cardiopulmonary Resuscitation”, Medical School, National and Kapodistrian University of Athens, Athens, Greece Hellenic Society of Cardiopulmonary Resuscitation, Athens, Greece c Neonatal Intensive Care Unit, Puericulture Institute and Neonatal Section, AOU and University of Cagliari, Cagliari, Italy d Karolinska Institutet, Department of Clinical Science and Education, Södersjukhuset and Section of Emergency Medicine, Södersjukhuset, Stockholm, Sweden e Midwestern University, Downers Grove, IL, USA f School of Health Sciences, University of Akureyri, Akureyri, Iceland g 2nd Department of Obstetrics and Gynecology, Neonatal Division, Medical School, National and Kapodistrian University of Athens, Athens, Greece b
a r t i c l e
i n f o
Article history: Received 14 October 2013 Received in revised form 12 December 2013 Accepted 26 January 2014 Keywords: Cardiac arrest Cardiopulmonary resuscitation Metabolomics
a b s t r a c t Objective: This review focuses on the presentation of the emerging technology of metabolomics, a promising tool for the detection of identifying the unrevealed biological pathways that lead to cardiac arrest. Data sources: The electronic bases of PubMed, Scopus, and EMBASE were searched. Research terms were identified using the MESH database and were combined thereafter. Initial search terms were “cardiac arrest”, “cardiopulmonary resuscitation”, “post-cardiac arrest syndrome” combined with “metabolomics”. Results: Metabolomics allow the monitoring of hundreds of metabolites from tissues or body fluids and already influence research in the field of cardiac metabolism. This approach has elucidated several pathophysiological mechanisms and identified profiles of metabolic changes that can be used to follow the disease processes occurring in the peri-arrest period. This can be achieved through leveraging the strengths of unbiased metabolome-wide scans, which include thousands of final downstream products of gene transcription, enzyme activity and metabolic products of extraneously administered substances, in order to identify a metabolomic fingerprint associated with an increased risk of cardiac arrest. Conclusion: Although this technology is still under development, metabolomics is a promising tool for elucidating biological pathways and discovering clinical biomarkers, strengthening the efforts for optimizing both the prevention and treatment of cardiac arrest. © 2014 Elsevier Ireland Ltd. All rights reserved.
Contents 1. 2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathophysiology of cardiac arrest and resuscitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Cardiac arrest – the onset of the ischemic cascade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Cardiopulmonary resuscitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Post-resuscitation period . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. The need for a better understanding of the pathophysiology of cardiac arrest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The metabolomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. The metabolomics workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
596 596 596 596 597 597 597 598
夽 A Spanish translated version of the abstract of this article appears as Appendix in the final online version at http://dx.doi.org/10.1016/j.resuscitation.2014.01.025. ∗ Corresponding author at: National and Kapodistrian University of Athens, Medical School, MSc “Cardiopulmonary Resuscitation”, Hospital “Henry Dunant”, 107 Mesogion Av., 115 26 Athens, Greece. E-mail address: [email protected] (A. Chalkias). 0300-9572/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.resuscitation.2014.01.025
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A. Chalkias et al. / Resuscitation 85 (2014) 595–601
3.2. The technology of 1 H NMR/metabolomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Metabolomics data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Metabolomics and cardiac arrest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Metabolomics in the prevention of cardiac arrest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix A. Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Cardiac arrest is a leading cause of death, affecting more than one million individuals worldwide every year. Patients who restore spontaneous circulation (ROSC) have a long way to go until recovery, as they have to pass through the “Clashing Rocks” of postcardiac arrest syndrome. Indeed, following successful resuscitation from cardiac arrest, neurological impairment as well as other types of organ dysfunction still cause significant morbidity and mortality. Research so far has shown that the best chance of survival with good neurological outcome is gained by strengthening the links of the Chain of Survival, i.e. early recognition of cardiac arrest, highquality cardiopulmonary resuscitation (CPR), early defibrillation, and subsequent care in a specialist center.1 In fact, prognostication for cardiac arrest victims remains dismal, as only about 17% survive to hospital.1,2 In this regard the scientific world has improved the knowledge on cardiopulmonary diseases, especially regarding risk factors and early diagnosis of the diseases; however, the management of the risk stratification and complications need more extensive study. Apart from the reduced financial resources, the lack of research may also be due to the complexities on the study of cardiopulmonary patients. The advent of metabolomics may be useful because the study of the metabolome is comparable to observe a snapshot of a particular biological sample in a particular moment, which may reveal biochemical pattern that could be correlated with the diagnosis and classification of diseases. From the clinical point of view, the metabolomics approach, offers unique insight into small molecule regulation and signaling, by providing access to a portion of biomolecular space not covered by other approaches such as genomics and proteomics.
2. Pathophysiology of cardiac arrest and resuscitation 2.1. Cardiac arrest – the onset of the ischemic cascade With the onset of cardiac arrest the effective blood flow stops and noradrenaline (norepinephrine) and neuropeptide Y are released from the cardiac sympathetic nerve terminals, while the release of acetylcholine diminishes.3 The acute onset of ischemia activates the adrenal glands which release noradrenaline, but despite the 1- to 100-fold elevation in endogenous plasma catecholamines, tissue perfusion remains poor. The impaired coronary flow diminishes the removal of noradrenaline from the interstitial spaces, resulting in prolonged vasoconstriction and myocardial hypoperfusion. Interestingly, the adrenal blood flow further worsens due to microvessel contraction, a phenomenon which is partly mediated by adrenomedullin.4 At the same time, various cytokines, complement components, and other molecules are synthesized and released in response to global hypoxia, such as tumor necrosis factor-a (TNF-a), interleukin 1b (IL-1b), C3, C4, C5, C5b-9, P-selectin, and intercellular adhesion molecule-1, and reactive oxygen species (ROS) are released from activated polymorphonuclear leukocytes (PMN).3 The cellular
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response to hypoxia is coordinated by the hypoxia-inducible factor (HIF) and its regulator, the Von Hippel–Lindau tumor suppressor protein.5 HIF 1-a is accumulated under hypoxic conditions and activates the transcription of endothelial nitric oxide (NO) synthase. The formation of NO involves arginine and its metabolites, ornithine and citrulline, along with enzymes that induce or inhibit NO synthase such as asymmetric dimethylarginine or l-NGmonomethylarginine.6 In addition, the activated platelets release 5-hydroxytryptamine and the expression of cyclooxygenase-2 is induced, enhancing the contractile response and the development of intracellular acidosis which increases the concentration of inorganic phosphate, pyruvate, hydrogen ions, and lactate.4 Furthermore, the ROS-mediated damage of the fatty acids of membrane phospholipids increases cell membrane permeability, diminishing the concentration of intracellular K+ and Mg2+ and increases that of Na+ and Ca2+ which together with the depletion of adenosine triphosphate (ATP) and the increased oxidative stress form the mitochondrial permeability transition pore (MTP).4,7 Of note, the mitochondrial metabolism is impaired and free fatty acids, long-chain acyl CoA, and acylcarnitine accumulate and are incorporated into membranes impairing their function. Shortly after the development of cardiac arrest, the blood–brain barrier is disrupted, allowing serum proteins, Na+ , and water to enter the brain microfluid environment.8–10 Brain edema and intracranial pressure increase and neuronal damage occurs, although mitochondrial swelling is not observed. Nevertheless, mitochondria can kill neurons by releasing apoptotic factors into cytosol, by releasing Ca2+ , and/or by generating ROS.11 Meanwhile, disturbances in Ca2+ homeostasis, inhibition of glycolysis, and oxidative stress triggers the release of signaling proteins and the activation of apoptotic mechanisms, such as the apoptotic pathway of stress-activated protein kinases (SAPKs) 1 and 2.12,13 Also, MTP opening can lead to apoptosis via cytochrome c release and/or may promote autophagy,14 while apoptosis may be also initiated by hypoxia-induced p53 accumulation which, in turn, it directly interacts with HIF 1-a and limits its expression.15 2.2. Cardiopulmonary resuscitation With the onset of chest compressions blood flow increases, although during optimal CPR, the cardiac output is between 25 and 40% of pre-arrest values.3 During the CPR interval, the coronary blood flow is low and cannot maintain aerobic myocardial metabolism, but it is sufficient enough to promote the deleterious effects of reperfusion. At this stage, generation of ROS, further activation of PMN, exacerbation of intracellular Ca2+ concentration, and the production of small amounts of ATP in myocardium lead to the formation of ischemic contracture,8 although, the badrenergic action of exogenous adrenaline [epinephrine] decrease the myocardial ATP content and increases the lactate content.16 The reperfusion-induced activation of blood coagulation which leads to the formation of microthrombi, together with the microvessel accumulation of activated PMN and platelets contribute to microvascular obstruction and to the onset of “no-reflow” phenomenon.3 Of note, microvascular obstruction in adrenal
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glands seems to be partly responsible for the low concentration of serum cortisol which is often observed after cardiac arrest.17 On the other hand, the electrical shock during CPR cause myocardial injury that is proportional to the amount of energy delivered. It increases intramyocardial temperature, potentiates excitation–contraction uncoupling, and is linked with dose-dependent release of free radicals and mitochondrial dysfunction.18 After countershocks, the oxidative metabolism is depressed and lactate is produced, while cell membrane permeability further increases, converting myocardium into a depressed and unexcitable tissue. The stunned myocardium is characterized by significant amounts of NO which were generated shortly after the onset of cardiac arrest and may exert protective as well as deleterious effects. During CPR, NO participates in mitochondrial oxygen sensing and inhibits cytochrome c oxidase, while it can interact with superoxide to form peroxynitrate, exacerbating oxidative stress. 2.3. Post-resuscitation period With the onset of ROSC, the automaticity of the heart is restored, although its mechanical function is impaired.3 The transient increase of catecholamines results in normal or elevated blood pressure, decreased microcirculatory blood flow, and increased Ca2+ overload.19 Despite the fact that ROSC is characterized by the upregulation and release of several cytokines and especially TNF-a and IL-8,20 targeted temperature management suppresses the production of proinflammatory cytokines and may block other manifestations associated with post-cardiac arrest syndrome, such as the increase of intracellular Ca2+ and glutamate which is observed after exposure to excitotoxin.11 The chemical changes that occur during cardiac arrest predispose to a massive burst of ROS production during the first minutes after ROSC, although free-radical production and vascular permeability may be attenuated by induced hypothermia.21 The endothelium becomes more dysfunctional and NO formation decreases, resulting in impaired vasodilation, further activation of PMN and platelets and extend tissue injury.22 However, the already increased levels of NO depress cardiac contractility and exacerbate post-resuscitation myocardial stunning.6 Although anaerobic metabolism is impaired, metabolism of lipids, glutamate, ␥-aminobutyric acid, and glutamate accelerates.23 The oxidation of fatty acids inhibits glucose oxidation rates and glycolysis continuous uncoupled from the oxidative process resulting in a net increase in cytosolic H+ concentration.24 The low levels of ATP and the increased amounts of ROS preserve the intracellular influx of Ca2+ which is further exacerbated by the increased activation of renin–angiotensin system and the production of angiotensin II.3 Approximately 2.5 h after ROSC, the levels of the soluble receptors for tumor necrosis factor type II (sTNFII) and other interleukins reach their peak,25 while cortisol concentration increases.17 Ischemia/reperfusion triggers apoptotic cell death through the protein kinase C family pathway, the Fas/Fas ligand pathway, and the caspase pathway which is activated by TNF-a and Fas receptors.26 In addition, the caspase cascade is activated by the increased release of cytochrome c from mitochondria. Nevertheless, cooling reduces delayed cell death including apoptosis and inhibits the neuroexcitatory cascade. During the recovery phase, the pathophysiological changes subside, although delayed neuronal degeneration may occur, as morphological changes in brain tissue reach maximum levels only after three weeks.27 Regarding the outcome, the post-resuscitation myocardial stunning is important in the early phases of ROSC, but thereafter, multi-organ dysfunction, irreversible myocardial necrosis, and irreversible cerebral injury play major roles.
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2.4. The need for a better understanding of the pathophysiology of cardiac arrest The increased quantity and quality of research efforts during the last decades have led the scientific community to the conclusion that only a better understanding of the pathophysiology of cardiac arrest and resuscitation will lead to the optimization of survival rates. Despite the recent progress in CPR, it was only during the recent years that we began to deepen into the pathophysiological mechanisms governing cardiac arrest and post-resuscitation syndrome. Throughout the evolution of resuscitation knowledge, however, research has shown an abundance of metabolites in several biological fluids or tissues whose presence has not been adequately explained. Today, the emerging technology of proton nuclear magnetic resonance (1 H NMR)/metabolomics might help us to identify and study the biomarker profile of metabolic systems and metabolic perturbations that occur in response to cardiac arrest and resuscitation and to establish the unrevealed pathways and mechanisms of post-resuscitation syndrome. 3. The metabolomics The metabolomics approach is based on the quantitative analysis of a large number of small metabolites (
Contents lists available at ScienceDirect
Resuscitation journal homepage: www.elsevier.com/locate/resuscitation
Review article 1
H NMR-metabolomics: Can they be a useful tool in our understanding of cardiac arrest?夽 Athanasios Chalkias a,b,∗ , Vassilios Fanos c , Antonio Noto c , Maaret Castrén d , Anil Gulati e , Hildigunnur Svavarsdóttir f , Nicoletta Iacovidou b,g , Theodoros Xanthos a,b a
MSc “Cardiopulmonary Resuscitation”, Medical School, National and Kapodistrian University of Athens, Athens, Greece Hellenic Society of Cardiopulmonary Resuscitation, Athens, Greece c Neonatal Intensive Care Unit, Puericulture Institute and Neonatal Section, AOU and University of Cagliari, Cagliari, Italy d Karolinska Institutet, Department of Clinical Science and Education, Södersjukhuset and Section of Emergency Medicine, Södersjukhuset, Stockholm, Sweden e Midwestern University, Downers Grove, IL, USA f School of Health Sciences, University of Akureyri, Akureyri, Iceland g 2nd Department of Obstetrics and Gynecology, Neonatal Division, Medical School, National and Kapodistrian University of Athens, Athens, Greece b
a r t i c l e
i n f o
Article history: Received 14 October 2013 Received in revised form 12 December 2013 Accepted 26 January 2014 Keywords: Cardiac arrest Cardiopulmonary resuscitation Metabolomics
a b s t r a c t Objective: This review focuses on the presentation of the emerging technology of metabolomics, a promising tool for the detection of identifying the unrevealed biological pathways that lead to cardiac arrest. Data sources: The electronic bases of PubMed, Scopus, and EMBASE were searched. Research terms were identified using the MESH database and were combined thereafter. Initial search terms were “cardiac arrest”, “cardiopulmonary resuscitation”, “post-cardiac arrest syndrome” combined with “metabolomics”. Results: Metabolomics allow the monitoring of hundreds of metabolites from tissues or body fluids and already influence research in the field of cardiac metabolism. This approach has elucidated several pathophysiological mechanisms and identified profiles of metabolic changes that can be used to follow the disease processes occurring in the peri-arrest period. This can be achieved through leveraging the strengths of unbiased metabolome-wide scans, which include thousands of final downstream products of gene transcription, enzyme activity and metabolic products of extraneously administered substances, in order to identify a metabolomic fingerprint associated with an increased risk of cardiac arrest. Conclusion: Although this technology is still under development, metabolomics is a promising tool for elucidating biological pathways and discovering clinical biomarkers, strengthening the efforts for optimizing both the prevention and treatment of cardiac arrest. © 2014 Elsevier Ireland Ltd. All rights reserved.
Contents 1. 2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathophysiology of cardiac arrest and resuscitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Cardiac arrest – the onset of the ischemic cascade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Cardiopulmonary resuscitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Post-resuscitation period . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. The need for a better understanding of the pathophysiology of cardiac arrest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The metabolomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. The metabolomics workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
596 596 596 596 597 597 597 598
夽 A Spanish translated version of the abstract of this article appears as Appendix in the final online version at http://dx.doi.org/10.1016/j.resuscitation.2014.01.025. ∗ Corresponding author at: National and Kapodistrian University of Athens, Medical School, MSc “Cardiopulmonary Resuscitation”, Hospital “Henry Dunant”, 107 Mesogion Av., 115 26 Athens, Greece. E-mail address: [email protected] (A. Chalkias). 0300-9572/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.resuscitation.2014.01.025
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A. Chalkias et al. / Resuscitation 85 (2014) 595–601
3.2. The technology of 1 H NMR/metabolomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Metabolomics data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Metabolomics and cardiac arrest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Metabolomics in the prevention of cardiac arrest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix A. Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Cardiac arrest is a leading cause of death, affecting more than one million individuals worldwide every year. Patients who restore spontaneous circulation (ROSC) have a long way to go until recovery, as they have to pass through the “Clashing Rocks” of postcardiac arrest syndrome. Indeed, following successful resuscitation from cardiac arrest, neurological impairment as well as other types of organ dysfunction still cause significant morbidity and mortality. Research so far has shown that the best chance of survival with good neurological outcome is gained by strengthening the links of the Chain of Survival, i.e. early recognition of cardiac arrest, highquality cardiopulmonary resuscitation (CPR), early defibrillation, and subsequent care in a specialist center.1 In fact, prognostication for cardiac arrest victims remains dismal, as only about 17% survive to hospital.1,2 In this regard the scientific world has improved the knowledge on cardiopulmonary diseases, especially regarding risk factors and early diagnosis of the diseases; however, the management of the risk stratification and complications need more extensive study. Apart from the reduced financial resources, the lack of research may also be due to the complexities on the study of cardiopulmonary patients. The advent of metabolomics may be useful because the study of the metabolome is comparable to observe a snapshot of a particular biological sample in a particular moment, which may reveal biochemical pattern that could be correlated with the diagnosis and classification of diseases. From the clinical point of view, the metabolomics approach, offers unique insight into small molecule regulation and signaling, by providing access to a portion of biomolecular space not covered by other approaches such as genomics and proteomics.
2. Pathophysiology of cardiac arrest and resuscitation 2.1. Cardiac arrest – the onset of the ischemic cascade With the onset of cardiac arrest the effective blood flow stops and noradrenaline (norepinephrine) and neuropeptide Y are released from the cardiac sympathetic nerve terminals, while the release of acetylcholine diminishes.3 The acute onset of ischemia activates the adrenal glands which release noradrenaline, but despite the 1- to 100-fold elevation in endogenous plasma catecholamines, tissue perfusion remains poor. The impaired coronary flow diminishes the removal of noradrenaline from the interstitial spaces, resulting in prolonged vasoconstriction and myocardial hypoperfusion. Interestingly, the adrenal blood flow further worsens due to microvessel contraction, a phenomenon which is partly mediated by adrenomedullin.4 At the same time, various cytokines, complement components, and other molecules are synthesized and released in response to global hypoxia, such as tumor necrosis factor-a (TNF-a), interleukin 1b (IL-1b), C3, C4, C5, C5b-9, P-selectin, and intercellular adhesion molecule-1, and reactive oxygen species (ROS) are released from activated polymorphonuclear leukocytes (PMN).3 The cellular
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response to hypoxia is coordinated by the hypoxia-inducible factor (HIF) and its regulator, the Von Hippel–Lindau tumor suppressor protein.5 HIF 1-a is accumulated under hypoxic conditions and activates the transcription of endothelial nitric oxide (NO) synthase. The formation of NO involves arginine and its metabolites, ornithine and citrulline, along with enzymes that induce or inhibit NO synthase such as asymmetric dimethylarginine or l-NGmonomethylarginine.6 In addition, the activated platelets release 5-hydroxytryptamine and the expression of cyclooxygenase-2 is induced, enhancing the contractile response and the development of intracellular acidosis which increases the concentration of inorganic phosphate, pyruvate, hydrogen ions, and lactate.4 Furthermore, the ROS-mediated damage of the fatty acids of membrane phospholipids increases cell membrane permeability, diminishing the concentration of intracellular K+ and Mg2+ and increases that of Na+ and Ca2+ which together with the depletion of adenosine triphosphate (ATP) and the increased oxidative stress form the mitochondrial permeability transition pore (MTP).4,7 Of note, the mitochondrial metabolism is impaired and free fatty acids, long-chain acyl CoA, and acylcarnitine accumulate and are incorporated into membranes impairing their function. Shortly after the development of cardiac arrest, the blood–brain barrier is disrupted, allowing serum proteins, Na+ , and water to enter the brain microfluid environment.8–10 Brain edema and intracranial pressure increase and neuronal damage occurs, although mitochondrial swelling is not observed. Nevertheless, mitochondria can kill neurons by releasing apoptotic factors into cytosol, by releasing Ca2+ , and/or by generating ROS.11 Meanwhile, disturbances in Ca2+ homeostasis, inhibition of glycolysis, and oxidative stress triggers the release of signaling proteins and the activation of apoptotic mechanisms, such as the apoptotic pathway of stress-activated protein kinases (SAPKs) 1 and 2.12,13 Also, MTP opening can lead to apoptosis via cytochrome c release and/or may promote autophagy,14 while apoptosis may be also initiated by hypoxia-induced p53 accumulation which, in turn, it directly interacts with HIF 1-a and limits its expression.15 2.2. Cardiopulmonary resuscitation With the onset of chest compressions blood flow increases, although during optimal CPR, the cardiac output is between 25 and 40% of pre-arrest values.3 During the CPR interval, the coronary blood flow is low and cannot maintain aerobic myocardial metabolism, but it is sufficient enough to promote the deleterious effects of reperfusion. At this stage, generation of ROS, further activation of PMN, exacerbation of intracellular Ca2+ concentration, and the production of small amounts of ATP in myocardium lead to the formation of ischemic contracture,8 although, the badrenergic action of exogenous adrenaline [epinephrine] decrease the myocardial ATP content and increases the lactate content.16 The reperfusion-induced activation of blood coagulation which leads to the formation of microthrombi, together with the microvessel accumulation of activated PMN and platelets contribute to microvascular obstruction and to the onset of “no-reflow” phenomenon.3 Of note, microvascular obstruction in adrenal
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glands seems to be partly responsible for the low concentration of serum cortisol which is often observed after cardiac arrest.17 On the other hand, the electrical shock during CPR cause myocardial injury that is proportional to the amount of energy delivered. It increases intramyocardial temperature, potentiates excitation–contraction uncoupling, and is linked with dose-dependent release of free radicals and mitochondrial dysfunction.18 After countershocks, the oxidative metabolism is depressed and lactate is produced, while cell membrane permeability further increases, converting myocardium into a depressed and unexcitable tissue. The stunned myocardium is characterized by significant amounts of NO which were generated shortly after the onset of cardiac arrest and may exert protective as well as deleterious effects. During CPR, NO participates in mitochondrial oxygen sensing and inhibits cytochrome c oxidase, while it can interact with superoxide to form peroxynitrate, exacerbating oxidative stress. 2.3. Post-resuscitation period With the onset of ROSC, the automaticity of the heart is restored, although its mechanical function is impaired.3 The transient increase of catecholamines results in normal or elevated blood pressure, decreased microcirculatory blood flow, and increased Ca2+ overload.19 Despite the fact that ROSC is characterized by the upregulation and release of several cytokines and especially TNF-a and IL-8,20 targeted temperature management suppresses the production of proinflammatory cytokines and may block other manifestations associated with post-cardiac arrest syndrome, such as the increase of intracellular Ca2+ and glutamate which is observed after exposure to excitotoxin.11 The chemical changes that occur during cardiac arrest predispose to a massive burst of ROS production during the first minutes after ROSC, although free-radical production and vascular permeability may be attenuated by induced hypothermia.21 The endothelium becomes more dysfunctional and NO formation decreases, resulting in impaired vasodilation, further activation of PMN and platelets and extend tissue injury.22 However, the already increased levels of NO depress cardiac contractility and exacerbate post-resuscitation myocardial stunning.6 Although anaerobic metabolism is impaired, metabolism of lipids, glutamate, ␥-aminobutyric acid, and glutamate accelerates.23 The oxidation of fatty acids inhibits glucose oxidation rates and glycolysis continuous uncoupled from the oxidative process resulting in a net increase in cytosolic H+ concentration.24 The low levels of ATP and the increased amounts of ROS preserve the intracellular influx of Ca2+ which is further exacerbated by the increased activation of renin–angiotensin system and the production of angiotensin II.3 Approximately 2.5 h after ROSC, the levels of the soluble receptors for tumor necrosis factor type II (sTNFII) and other interleukins reach their peak,25 while cortisol concentration increases.17 Ischemia/reperfusion triggers apoptotic cell death through the protein kinase C family pathway, the Fas/Fas ligand pathway, and the caspase pathway which is activated by TNF-a and Fas receptors.26 In addition, the caspase cascade is activated by the increased release of cytochrome c from mitochondria. Nevertheless, cooling reduces delayed cell death including apoptosis and inhibits the neuroexcitatory cascade. During the recovery phase, the pathophysiological changes subside, although delayed neuronal degeneration may occur, as morphological changes in brain tissue reach maximum levels only after three weeks.27 Regarding the outcome, the post-resuscitation myocardial stunning is important in the early phases of ROSC, but thereafter, multi-organ dysfunction, irreversible myocardial necrosis, and irreversible cerebral injury play major roles.
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2.4. The need for a better understanding of the pathophysiology of cardiac arrest The increased quantity and quality of research efforts during the last decades have led the scientific community to the conclusion that only a better understanding of the pathophysiology of cardiac arrest and resuscitation will lead to the optimization of survival rates. Despite the recent progress in CPR, it was only during the recent years that we began to deepen into the pathophysiological mechanisms governing cardiac arrest and post-resuscitation syndrome. Throughout the evolution of resuscitation knowledge, however, research has shown an abundance of metabolites in several biological fluids or tissues whose presence has not been adequately explained. Today, the emerging technology of proton nuclear magnetic resonance (1 H NMR)/metabolomics might help us to identify and study the biomarker profile of metabolic systems and metabolic perturbations that occur in response to cardiac arrest and resuscitation and to establish the unrevealed pathways and mechanisms of post-resuscitation syndrome. 3. The metabolomics The metabolomics approach is based on the quantitative analysis of a large number of small metabolites (
