- Email: [email protected]
Cellular oxygen utilization in health and sepsis
Cellular oxygen utilization in health and sepsis GI van Boxel PhD BSc BMBCh WL Doherty BMSc MBChB FRCA FFICM M Parmar BSc MBBS MRCP FRCA
The mitochondrion contains the components for the electron transport chain and is the location of cellular respiration. Molecular oxygen is the terminal electron acceptor of the mitochondrial electron transport chain performing aerobic respiration. Mitochondrial dysfunction and hibernation is the potential driving force in sepsis-associated multiorgan dysfunction syndrome. Organ support during sepsis-associated mitochondrial hibernation may permit complete cellular recovery. Novel therapeutics designed to optimize mitochondrial function have shown to improve survival in animal models, but clinical trials remain scarce. GI van Boxel PhD BSc BMBCh Foundation Year 2 Doctor Cheltenham General Hospital Cheltenham, UK WL Doherty BMSc MBChB FRCA FFICM Consultant in Anaesthesia and Intensive Care Cheltenham General Hospital Cheltenham, UK Fax: 08454 224013 E-mail: [email protected] (for correspondence) M Parmar BSc MBBS MRCP FRCA Consultant in Anaesthesia Cheltenham General Hospital Cheltenham, UK
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The importance of ‘Airway Breathing Circulation’ in delivering oxygen to the tissues is well established and understood. Within the tissues, the mitochondrion is responsible for the utilization of oxygen in the cell. The molecular dynamics that drive the cellular utilization of oxygen, however, is less well described but has distinct applications to clinical practice, especially in sepsis. This article describes the fate of molecular oxygen as it passes from the airway through the circulation into the cell. Particular focus will be placed on the cellular location where oxygen is ultimately utilized, the mitochondrion. Here, oxygen serves as an electron acceptor in the electron transport chain—analogues to the requirement of air in a combustion engine. This is particularly relevant to multiorgan distress syndrome (MODS) in sepsis, where increasing evidence suggests a central role for mitochondrial dysfunction and, in particular, the mitochondrion’s ability to utilize oxygen effectively.
The journey of oxygen to the cell The details of the process of oxygen absorption through the lungs into the circulation were recently described in this journal.1 Briefly, oxygen travels down a concentration gradient, freely diffusing across alveolar cell membranes into the blood. Here, it is mostly (.99%) bound to haemoglobin where each gram of haemoglobin carries 1.39 ml of molecular oxygen (Huffner’s constant). In the context of a non-anaemic (Hb .15 g dl21) adult, this equates to 1000 ml of oxygen gas for a circulating volume of 5 litre. Oxygen is delivered to the cell, bound to haemoglobin (HbO2), where it dissociates due to a change in its affinity; the molecular structure of haemoglobin changes in the presence of acid altering its affinity for
oxygen—the Bohr effect (Christian Bohr; 1855–1911). Since cellular respiration generates CO2, an acid in solution by forming carbonic acid, the oxygen-dissociation constant shifts allowing dissociation. Dissociation is further aided by the co-operative nature of the haemoglobin tetramer resulting in the sigmoid nature of the oxygen binding curve.
The eukaryotic cell and the mitochondrion Eukaryotic cells are defined by the presence of a membrane-bound nucleus, specialized organelles such as mitochondria and functionally by an elaborate system of division by mitosis. Figure 1 depicts the simplified organization of a eukaryotic cell, some of its constituent organelles, and the surrounding vasculature. Molecular oxygen readily dissociates from red cell haemoglobin in the capillary into the cell where it enters the mitochondrion by simple diffusion. All cells of the human body contain mitochondria, although in different numbers depending on metabolic requirements. Red blood cells are the exception—as all internal organelles are lost during maturation. The mitochondrion has many important cellular functions such as lipid metabolism, calcium homeostasis, steroid synthesis, and elements of regulating apoptosis ( programmed cell death). Its primary function, however, is supplying the cell with readily available energy in the form of adenosine triphosphate (ATP). Mitochondria can divide independently of cell division or in contrast be removed by autolysis and aoutophagy (mitophagy). Mitochondrial numbers may therefore alter during the lifespan of the cell. Separating the lifespan of the mitochondrion from that of the cell is therefore a mechanism for the dynamic regulation of ATP provision. This is particularly relevant to
doi:10.1093/bjaceaccp/mks023 Advance Access publication 7 May, 2012 Continuing Education in Anaesthesia, Critical Care & Pain | Volume 12 Number 4 2012 & The Author [2012]. Published by Oxford University Press on behalf of the British Journal of Anaesthesia. All rights reserved. For Permissions, please email: [email protected]
Downloaded from http://ceaccp.oxfordjournals.org/ at Grand Valley State University on November 1, 2013
Key points
Matrix reference 1A01,1A02,2C02
Cellular oxygen utilization
sepsis-induced MODS, where mitochondrial numbers alter parallel to organ function.
Mitochondrial architecture and function The mitochondrion is an organelle measuring 1– 10 mm, equating to roughly the size of a bacterium. In contrast to most biological compartments, the mitochondrion has two membranes, an inner and an outer membrane. As a result, there are two distinct compartments within each mitochondrion, the intermembrane space
Fig 1 Schematic of the cell and some of its constituent organelles. Molecular oxygen is released from haemoglobin due to changes in pH and CO2 concentrations (the Bohr effect) and freely diffuses into the cell and the mitochondrion.
and the matrix (Fig. 2). The matrix is the space which, among many proteins and macromolecules, contains the enzymes for the tricarboxylic acid (TCA) cycle (Fig. 3). The substrate for the TCA cycle is acetyl-CoA which is derived from carbohydrates, longchain fatty acids, and proteins in the cytosol of the cell (not the mitochondrion). Acetyl-CoA enters the mitochondrial matrix by means of the carnitine shuttle—an enzyme-driven exchange mechanism. Once inside the mitochondrial matrix, acetyl-CoA combines with oxaloacetate to form citrate. Citrate is oxidized in sequential steps producing CO2. The overall effect of these multiple steps is to produce succinate and transfer electrons to nicotinamide adenosine diphosphate (NADþ) to produce the reducing agent NADH both of which are then used in the electron transport chain.
Fig 3 The TCA cycle. Number of carbon atoms are indicated in parentheses.
Fig 2 Schematic representation of the mitochondrion. Detail of the electron transport chain within the inner mitochondrial membrane is shown.
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Continuing Education in Anaesthesia, Critical Care & Pain j Volume 12 Number 4 2012
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motor known to men. Although the proton motive force is largely consumed by the F1Fo-ATP synthase to generate ATP, there are other proteins that consume it. For example, uncoupling proteins can generate heat in brown adipose tissue, useful in hibernating animals, and proton translocating transhydrogenase uses the proton motive force to shift hydride ions between NADH and NADPþ, fine-tuning the processes of anabolism and catabolism in the process. Also, a host of transport proteins rely on the proton motive force to regulate cellular metabolism.
The electron transport chain The electron transport chain is a series of protein complexes, residing in or near the inner mitochondrial membrane. Its components are complexes I –IV, which are membrane proteins, and ubiquinone and cytochrome c, the soluble components (Table 1 and Fig. 2). All the protein complexes contain redox centres which are able to accept and donate electrons (for definitions of redox terminology, see Table 2). Electrons originate from the TCA cycle in the form of NADH and succinate from which they flow either into complex I or into complex II, respectively. From there, the electrons flow down the chain according to the redox potential of the acceptor. The terminal electron acceptor in the chain is molecular oxygen, which combines with electrons and protons to form water. It is important to realize that this is where the molecular oxygen that we breathe in acts, without oxygen the electron transport chain grinds to a halt! The effect of electrons flowing down this chain is to cause conformational changes in complexes I, III, and IV, which result in the translocation of protons from the mitochondrial matrix to the inter membrane space. The resulting difference in both charge and proton concentration in the two compartments is called the proton motive force. The proton motive force is in essence a potential difference across a membrane which can be used to do work—not dissimilar to a battery.
Mitochondrial function in sepsis Mitochondrial function is paramount to a functional cell, especially when a cell is under stress, such as during sepsis. Sepsis, defined as the systemic inflammatory response syndrome (SIRS) in the presence of known or suspected infection, is the most common cause for intensive care admission (29% of UK ITU admissions in 2004 were for severe sepsis; ICNARC) and has poor clinical outcome. The initial management of sepsis is targeted at avoiding tissue hypoperfusion and hypoxia by optimizing oxygen delivery. This approach, involving aggressive fluid resuscitation, ionotropic, and vasoactive agents, can improve outcome.4 Severe sepsis and septic shock are often associated with cardiac, renal, or pulmonary failure, a process more commonly referred to as MODS. The underlying pathology for MODS has been the subject of intense study. For many years, the prevailing theory was that hypoxia, through inadequate perfusion, was the driving force behind MODS.5 However, many experiments since have shown normal or
Utilizing the proton motive force The function of generating a proton motive force is to convert an intermittent supply of fuel (carbohydrates, fatty acids. and proteins) into a constant supply of universally available energy. The vast majority of the proton motive force is utilized by the F1Fo-ATP synthase—the enzyme responsible for ATP synthesis. The enzyme couples the flux of protons down the charge and concentration gradient (i.e. in the opposite direction of proton flux created by complexes I, III, and IV) to the phosphorylation of ADP to generate ATP (Fig. 2). One molecule of glucose produces a net of 32 molecules of ATP, as six ATP molecules are used in the process. The most remarkable fact about the F1Fo-ATP synthase is that the F1 part physically rotates during this process,2,3 making it the smallest
Table 2 Definitions of redox terms Reduction Oxidation Redox potential Redox couple
Redox centre
The gain of electrons or the decrease in oxidation number The loss of electrons or the increase in oxidation number The tendency of a chemical species to acquire electrons measured in volts. The more positive the potential, the higher the tendency of the chemical species to be reduced A pair of molecules involved in the transfer of electrons, where one is oxidized and the other is reduced depending on the redox potential of each An active centre within a protein or macromolecule which can be sequentially reduced and oxidized
Table 1 The components of the electron transport chain Name
Other names
Substrate
Hþ translocated
Disease implication
Complex I
NADH dehydrogenase; NADH: ubiquinone oxidoreductase Succinate dehydrogenase
NADH
4
Succinate
0
Leigh syndrome; Parkinson’s disease; Leber’s hereditary optic neuropathy Leigh syndrome; optic atrophy; hereditary paraganglioma; hereditary pheochromocytoma
2 electrons from Complex I and/or II Reduced Coenzyme Q
0
Complex II Ubiquinone Complex III Cytochrome c Complex IV
Coenzyme Q; Coenzyme Q10; ubidecaranone Cytochrome bc1 complex; cytochrome c-oxidoreductase Cyt c Cytochrome c oxidase
1 electron from Complex III Reduced cytochrome c; O2
2 from the matrixþ2 from Coenzyme Q 0 4
Septo-optic dysplasia; Bjo¨rnstad syndrome; GRACILE syndrome Leigh syndrome; infantile hypertrophic cardiomyopathy; neonatal-onset hepatic failure and encephalopathy; leukodystrophy
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even high PO2 levels in the microcirculation, raising the possibility that the cellular utilization of oxygen may be impaired rather than oxygen delivery. When Mela and colleagues6 showed ultrastructural damage to the mitochondrion with the inhibition of its respiration in rodent models post-sepsis; the question was whether this was cause or effect? These changes will lead to disordered use of oxygen. While the topic still raises debate, there is increasing evidence to support the ‘cytopathic hypoxia’ hypothesis that mitochondrial dysfunction is a crucial factor in MODS rather than failure to deliver oxygen to the cell. The alternative view is that MODS is a result of a combination of microcirculatory and mitochondrial failure—the ‘microcirculatory and mitochondrial distress syndrome’ (MMDS) theory. Both these theories will be briefly reviewed.
Cytopathic hypoxia or cellular metabolic derangement Fink7 first used the term cytopathic hypoxia to describe the dysregulation of oxygen metabolism during sepsis; cells fail to produce adequate amounts of ATP in the presence of sufficient molecular oxygen. These terms reflect the fact that it may not be the availability of oxygen, but its usability by mitochondria within the cell which may be paramount in sepsis. In essence, the hypothesis proposes that inflammatory mediators, in the context of SIRS, directly cause mitochondria to fail which manifests itself as MODS. There are a number of mechanisms proposed to account for mitochondrial dysfunction. In trauma patients, the activation of the SIRS response has been associated with the release of mitochondrial damage-associated molecular patterns (DAMPs). The common endpoint is the uncoupling of electron transport from ATP production, down-regulation of mitochondrial electron transport proteins, mitochondrial membrane damage, and decreased lifespan of the mitochondrion. This directly translates into cellular dysfunction and potentially apoptosis. In the acute phase of sepsis, tumour necrosis factor (TNF)-a, interleukin (IL)-1, and IL-6 are released in major organ systems such as the kidneys, lungs, liver, and brain.8 These mediators can effect the mitochondrion profoundly. For example, TNF-a has the ability to cause direct cytopathic changes through binding the TNF receptor 1 (TNFR-1). The resulting intracellular signals can trigger mitochondrial permeability transition (MPT); the collapse of the proton motive force through increased solute permeability of the mitochondrial membranes. The resulting release of cytochrome c initiates cellular apoptosis. Furthermore, mitochondrial electron transport is itself directly inhibited, increasing the concentration of reactive oxygen species (ROS). Examples of ROS are superoxide, hydrogen peroxide, and hydroxyl radicals . (O2 2 , H2O2, and OH , respectively). ROS are an unavoidable byproduct of electron transport, through the leaking of electrons from complex I or complex III to molecular oxygen, and, in normal physiology, are thought to act as signalling molecules. At higher production rates however, such as in sepsis, ROS cause oxidative damage to membranes, proteins, and DNA. The combination of increased ROS production, due to a block of electron transport,
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and increased nitric oxide (NO), due to the activation of inducible nitric oxide synthase (iNOS), results in peroxinitrite (ONO2 2 ). Peroxinitrite is a highly reactive compound that readily oxidizes electron transport complexes and membrane components, further compromising mitochondrial function. Brearley and colleagues9 demonstrated an association between the inhibition of complex I and lower ATP synthesis with worse outcome in septic patients which may indeed be due to ROS-induced damage. In summary, the cytopathic hypoxia theory proposes that inflammatory mediators have a direct effect on the ability of the mitochondrion to utilize oxygen effectively, which can result in cellular hibernation or cell death. Altered metabolism within the cell is also demonstrated by down-regulation of the enzymes pyruvate dehydrogenase (PDH) and carnitine palmitoyl-transferase—this will lead to a decrease in the supply of energy to the cell and is thought to be a key element of sepsis-induced cardiac dysfunction. Decreasing tissue carnitine levels in the myocardium and endothelium result in increased plasma levels and increased renal loss. Infusing carnitine may represent a novel metabolic treatment alternative to conventional vasoactive therapy, which increases cardiac work, as by stimulating pyruvate oxidation recouping glycolysis and oxidation of pyruvate will lead to a more efficient cardiac muscle contraction.
Microcirculatory and mitochondrial distress syndrome An alternative hypothesis is the concept of MMDS.10 Traditionally, there has been a focus on macrocirculatory haemodynamics to restore delivery to the tissues. However, there is now an increasing focus on the interface between the tissue and the macrocirculation. In sepsis, one of the critical steps may be an alteration in the flow through the microcirculation, which is regulated in health by pressure (myogenic), neurohormonal, and metabolic factors such as CO2, lactate, and O2 to meet the oxygen needs of the cell. In sepsis, these autoregulatory mechanisms are disrupted, resulting in a mismatch between the requirements of the tissue for oxygen and its availability. Further to this, the inducible form of nitric oxide synthase (iNOS) is switched on in response to inflammatory mediators, causing shunting of flow to those areas where there is most nitric oxide. Coupled to the activation of the coagulation pathway and red cell sequestration, the net effect is MMDS. New imaging techniques that allow visualization of the microcirculation have demonstrated improved microcirculatory parameters after administration of fluids for resuscitation in the early stages of sepsis.
Possible therapies The proposal that mitochondrial dysfunction is the driving force behind MODS opens up a large avenue for potential pharmacological treatments. Therapeutic agents have targeted specific pathways or functions of the mitochondrion; mitochondrial substrate and co-factor provision, antioxidants, ROS scavengers, and
Continuing Education in Anaesthesia, Critical Care & Pain j Volume 12 Number 4 2012
Cellular oxygen utilization
Table 3 Experimental therapies based on the cytopathic hypoxia theory Type
Name
Improved survival
Substrate provision
ATP-MgCl2 Succinate L-carnitine Caffeine Cytochrome c Coenzyme Q10 a-Lipoic acid MitoQ SS peptide N-acetyl cysteine Melatonin SS peptide Tempol 4NH2-Tempo Ethyl pyruvate Melatonin Cyclosporin A NIM811 Bcl over-expression L-NAME 6 L-N -(1-imminoethyl)lysine Aminoguanide
Y Y Y N Y Y N N N N Y N Y Y Y N Y Y Y N N N
Co-factor provision
Antioxidants
ROS scavengers
Membrane stabilisers
iNOS inhibitor
with histopathological abnormalities at autopsy, ‘failure’ in MODS can often be reversible and shows little necrosis or apoptosis at autopsy. Instead, it appears as though the cells are able to adopt a state of hibernation. Down-regulating energy consumption maintains the ‘normal’ intracellular store of ATP, therefore allowing full recovery of cells that would usually be very sensitive to hypoxic injury. This observation has led to the proposal that MODS is, potentially, a protective mechanism to preserve cellular integrity during stressful times.13 Furthermore, the ability of the mitochondrion to divide, increasing its numbers per cell, allows sufficient levels of ATP when recovering from this hibernating state. Of course, the consequences of this protective mechanism, as is seen all too often in intensive care units, may be death. This leaves a therapeutic conundrum: if MODS is a protective mechanism in the face of overwhelming sepsis, should we aim to prevent it with agents that improve mitochondrial function? Or should we focus on optimizing our ability to assist failing organs and await resolution of the initial insult and then target mitochondrial regeneration?
Conclusion membrane stabilizers. Table 3 lists the experimental treatments (adapted from Dare and colleagues).11 Improvements in mitochondrial function, measured by ATP production and a reduction in markers of oxidative stress, were commonly associated with improvements in haemodynamics parameters, organ function, and, crucially, survival. Although many agents have shown benefit in animal models of MODS, none has yet become part of the standard regime for the treatment or prevention of MODS thus far. Administration of cytochrome c in animals has been shown to resolve some of the abnormalities in the mitochondrial electron chain with enhanced cardiac function and decreased mortality. Clinical trials of these agents would be the next step. In accordance with the Surviving Sepsis guidelines, antibiotic therapy should be administered early for bacterial-induced sepsis. However, the role of antibiotics in sepsis-induced MODS is potentially more complex. The potent ability of certain bacteriostatic antibiotics (e.g. chloramphenicol) to inhibit mitochondrial regeneration is a characteristic to be cautiously aware of.12 Considering the evolutionary origin of mitochondria (i.e. bacterial), this perhaps is no surprise. The difficult question therefore arises as to whether we should be concerned about prolonged antibiotic therapy in sepsis which may indeed be delaying or inhibiting recovery from MODS.
Mitochondrial regeneration and MODS During sepsis, all cells in the body experience a certain degree of stress through systemic inflammatory mediators, metabolic changes, and stress hormones (such as cortisol and catecholamines), which may lead to MODS. In contrast to conventional organ failure (e.g. liver failure secondary to alcohol), which is an irreversible process
Oxygen serves as the terminal electron acceptor in cellular respiration, allowing the mitochondrion to generate the proton motive force. The proton motive force is predominantly utilized by F1Fo-ATP synthase to generate readily available energy in the form of ATP. In sepsis, a common and potentially fatal condition, mitochondrial function is severely impaired. In vitro and in vivo studies now point to a causative role of mitochondrial dysfunction in MODS.
Declaration of interest None declared.
References 1. Sirian R, Wills J. Physiology of apnoea and the benefits of preoxygenation. Contin Educ Anaesth Crit Care Pain 2009; 9: 105– 8 2. Abrahams J, Leslie A, Lutter R et al. Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria. Nature 1994; 370: 621–8 3. Noji H, Yasuda R, Yoshida M et al. Direct observation of the rotation of F1-ATPase. Nature 1997; 386: 299–302 4. Rivers E, Nguyen B, Havstad S. Early goal-directed therapy in the treatment of severe sepsis and septic shock. N Engl J Med 2001; 345: 1368– 77 5. Broder G, Weil M. Excess lactate: an index of reversibility of shock in human patients. Science 1964; 143: 1457–9 6. Mela L, Bacalzo L, Miller L. Defective oxidative metabolism of rat liver mitochondria in hemorrhagic and endotoxin shock. Am J Physiol 1971; 220: 571–7 7. Fink M. Cytopathic hypoxia: mitochondrial dysfunction as mechanism contributing to organ dysfunction in sepsis. Crit Care Clin 2001; 17: 219–37 8. Chinnaiyan A, Huber-Lang M, Kumar-Shina C et al. Molecular signature of sepsis: multiorgan gene expression profiles of systemic inflammation. Am J Pathol 2001; 159: 1199– 209
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9. Brearley D, Brand M, Hargreaves I et al. Association between mitochondrial dysfunction and severity and outcome of septic shock. Lancet 2002; 360: 219– 23
12. Riesbeck K, Bredberg A, Forsgren A. Ciprofloxacin does not inhibit mitochondrial functions but other antibiotics do. Antimicrob Agents Chemother 1990; 34: 167 –9
10. Freelander S, Lenhart C. Clinical observations on the capillary circulation. Arch Intern Med 1922; 29: 12– 32
13. Singer M, De Santis V, Vitale D et al. Multiorgan failure is an adaptive, endocrine-mediated, metabolic response to overwhelming systemic inflammation. Lancet 2004; 364: 545– 8
11. Dare A, Phillips A, Hickey A et al. A systematic review of experimental treatments for mitochondrial dysfunction in sepsis and multiple organ dysfunction syndrome. Free Rad Biol Med 2009; 47: 1517 – 25
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Please see multiple choice questions 33 –36.
Continuing Education in Anaesthesia, Critical Care & Pain j Volume 12 Number 4 2012
The mitochondrion contains the components for the electron transport chain and is the location of cellular respiration. Molecular oxygen is the terminal electron acceptor of the mitochondrial electron transport chain performing aerobic respiration. Mitochondrial dysfunction and hibernation is the potential driving force in sepsis-associated multiorgan dysfunction syndrome. Organ support during sepsis-associated mitochondrial hibernation may permit complete cellular recovery. Novel therapeutics designed to optimize mitochondrial function have shown to improve survival in animal models, but clinical trials remain scarce. GI van Boxel PhD BSc BMBCh Foundation Year 2 Doctor Cheltenham General Hospital Cheltenham, UK WL Doherty BMSc MBChB FRCA FFICM Consultant in Anaesthesia and Intensive Care Cheltenham General Hospital Cheltenham, UK Fax: 08454 224013 E-mail: [email protected] (for correspondence) M Parmar BSc MBBS MRCP FRCA Consultant in Anaesthesia Cheltenham General Hospital Cheltenham, UK
207
The importance of ‘Airway Breathing Circulation’ in delivering oxygen to the tissues is well established and understood. Within the tissues, the mitochondrion is responsible for the utilization of oxygen in the cell. The molecular dynamics that drive the cellular utilization of oxygen, however, is less well described but has distinct applications to clinical practice, especially in sepsis. This article describes the fate of molecular oxygen as it passes from the airway through the circulation into the cell. Particular focus will be placed on the cellular location where oxygen is ultimately utilized, the mitochondrion. Here, oxygen serves as an electron acceptor in the electron transport chain—analogues to the requirement of air in a combustion engine. This is particularly relevant to multiorgan distress syndrome (MODS) in sepsis, where increasing evidence suggests a central role for mitochondrial dysfunction and, in particular, the mitochondrion’s ability to utilize oxygen effectively.
The journey of oxygen to the cell The details of the process of oxygen absorption through the lungs into the circulation were recently described in this journal.1 Briefly, oxygen travels down a concentration gradient, freely diffusing across alveolar cell membranes into the blood. Here, it is mostly (.99%) bound to haemoglobin where each gram of haemoglobin carries 1.39 ml of molecular oxygen (Huffner’s constant). In the context of a non-anaemic (Hb .15 g dl21) adult, this equates to 1000 ml of oxygen gas for a circulating volume of 5 litre. Oxygen is delivered to the cell, bound to haemoglobin (HbO2), where it dissociates due to a change in its affinity; the molecular structure of haemoglobin changes in the presence of acid altering its affinity for
oxygen—the Bohr effect (Christian Bohr; 1855–1911). Since cellular respiration generates CO2, an acid in solution by forming carbonic acid, the oxygen-dissociation constant shifts allowing dissociation. Dissociation is further aided by the co-operative nature of the haemoglobin tetramer resulting in the sigmoid nature of the oxygen binding curve.
The eukaryotic cell and the mitochondrion Eukaryotic cells are defined by the presence of a membrane-bound nucleus, specialized organelles such as mitochondria and functionally by an elaborate system of division by mitosis. Figure 1 depicts the simplified organization of a eukaryotic cell, some of its constituent organelles, and the surrounding vasculature. Molecular oxygen readily dissociates from red cell haemoglobin in the capillary into the cell where it enters the mitochondrion by simple diffusion. All cells of the human body contain mitochondria, although in different numbers depending on metabolic requirements. Red blood cells are the exception—as all internal organelles are lost during maturation. The mitochondrion has many important cellular functions such as lipid metabolism, calcium homeostasis, steroid synthesis, and elements of regulating apoptosis ( programmed cell death). Its primary function, however, is supplying the cell with readily available energy in the form of adenosine triphosphate (ATP). Mitochondria can divide independently of cell division or in contrast be removed by autolysis and aoutophagy (mitophagy). Mitochondrial numbers may therefore alter during the lifespan of the cell. Separating the lifespan of the mitochondrion from that of the cell is therefore a mechanism for the dynamic regulation of ATP provision. This is particularly relevant to
doi:10.1093/bjaceaccp/mks023 Advance Access publication 7 May, 2012 Continuing Education in Anaesthesia, Critical Care & Pain | Volume 12 Number 4 2012 & The Author [2012]. Published by Oxford University Press on behalf of the British Journal of Anaesthesia. All rights reserved. For Permissions, please email: [email protected]
Downloaded from http://ceaccp.oxfordjournals.org/ at Grand Valley State University on November 1, 2013
Key points
Matrix reference 1A01,1A02,2C02
Cellular oxygen utilization
sepsis-induced MODS, where mitochondrial numbers alter parallel to organ function.
Mitochondrial architecture and function The mitochondrion is an organelle measuring 1– 10 mm, equating to roughly the size of a bacterium. In contrast to most biological compartments, the mitochondrion has two membranes, an inner and an outer membrane. As a result, there are two distinct compartments within each mitochondrion, the intermembrane space
Fig 1 Schematic of the cell and some of its constituent organelles. Molecular oxygen is released from haemoglobin due to changes in pH and CO2 concentrations (the Bohr effect) and freely diffuses into the cell and the mitochondrion.
and the matrix (Fig. 2). The matrix is the space which, among many proteins and macromolecules, contains the enzymes for the tricarboxylic acid (TCA) cycle (Fig. 3). The substrate for the TCA cycle is acetyl-CoA which is derived from carbohydrates, longchain fatty acids, and proteins in the cytosol of the cell (not the mitochondrion). Acetyl-CoA enters the mitochondrial matrix by means of the carnitine shuttle—an enzyme-driven exchange mechanism. Once inside the mitochondrial matrix, acetyl-CoA combines with oxaloacetate to form citrate. Citrate is oxidized in sequential steps producing CO2. The overall effect of these multiple steps is to produce succinate and transfer electrons to nicotinamide adenosine diphosphate (NADþ) to produce the reducing agent NADH both of which are then used in the electron transport chain.
Fig 3 The TCA cycle. Number of carbon atoms are indicated in parentheses.
Fig 2 Schematic representation of the mitochondrion. Detail of the electron transport chain within the inner mitochondrial membrane is shown.
208
Continuing Education in Anaesthesia, Critical Care & Pain j Volume 12 Number 4 2012
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motor known to men. Although the proton motive force is largely consumed by the F1Fo-ATP synthase to generate ATP, there are other proteins that consume it. For example, uncoupling proteins can generate heat in brown adipose tissue, useful in hibernating animals, and proton translocating transhydrogenase uses the proton motive force to shift hydride ions between NADH and NADPþ, fine-tuning the processes of anabolism and catabolism in the process. Also, a host of transport proteins rely on the proton motive force to regulate cellular metabolism.
The electron transport chain The electron transport chain is a series of protein complexes, residing in or near the inner mitochondrial membrane. Its components are complexes I –IV, which are membrane proteins, and ubiquinone and cytochrome c, the soluble components (Table 1 and Fig. 2). All the protein complexes contain redox centres which are able to accept and donate electrons (for definitions of redox terminology, see Table 2). Electrons originate from the TCA cycle in the form of NADH and succinate from which they flow either into complex I or into complex II, respectively. From there, the electrons flow down the chain according to the redox potential of the acceptor. The terminal electron acceptor in the chain is molecular oxygen, which combines with electrons and protons to form water. It is important to realize that this is where the molecular oxygen that we breathe in acts, without oxygen the electron transport chain grinds to a halt! The effect of electrons flowing down this chain is to cause conformational changes in complexes I, III, and IV, which result in the translocation of protons from the mitochondrial matrix to the inter membrane space. The resulting difference in both charge and proton concentration in the two compartments is called the proton motive force. The proton motive force is in essence a potential difference across a membrane which can be used to do work—not dissimilar to a battery.
Mitochondrial function in sepsis Mitochondrial function is paramount to a functional cell, especially when a cell is under stress, such as during sepsis. Sepsis, defined as the systemic inflammatory response syndrome (SIRS) in the presence of known or suspected infection, is the most common cause for intensive care admission (29% of UK ITU admissions in 2004 were for severe sepsis; ICNARC) and has poor clinical outcome. The initial management of sepsis is targeted at avoiding tissue hypoperfusion and hypoxia by optimizing oxygen delivery. This approach, involving aggressive fluid resuscitation, ionotropic, and vasoactive agents, can improve outcome.4 Severe sepsis and septic shock are often associated with cardiac, renal, or pulmonary failure, a process more commonly referred to as MODS. The underlying pathology for MODS has been the subject of intense study. For many years, the prevailing theory was that hypoxia, through inadequate perfusion, was the driving force behind MODS.5 However, many experiments since have shown normal or
Utilizing the proton motive force The function of generating a proton motive force is to convert an intermittent supply of fuel (carbohydrates, fatty acids. and proteins) into a constant supply of universally available energy. The vast majority of the proton motive force is utilized by the F1Fo-ATP synthase—the enzyme responsible for ATP synthesis. The enzyme couples the flux of protons down the charge and concentration gradient (i.e. in the opposite direction of proton flux created by complexes I, III, and IV) to the phosphorylation of ADP to generate ATP (Fig. 2). One molecule of glucose produces a net of 32 molecules of ATP, as six ATP molecules are used in the process. The most remarkable fact about the F1Fo-ATP synthase is that the F1 part physically rotates during this process,2,3 making it the smallest
Table 2 Definitions of redox terms Reduction Oxidation Redox potential Redox couple
Redox centre
The gain of electrons or the decrease in oxidation number The loss of electrons or the increase in oxidation number The tendency of a chemical species to acquire electrons measured in volts. The more positive the potential, the higher the tendency of the chemical species to be reduced A pair of molecules involved in the transfer of electrons, where one is oxidized and the other is reduced depending on the redox potential of each An active centre within a protein or macromolecule which can be sequentially reduced and oxidized
Table 1 The components of the electron transport chain Name
Other names
Substrate
Hþ translocated
Disease implication
Complex I
NADH dehydrogenase; NADH: ubiquinone oxidoreductase Succinate dehydrogenase
NADH
4
Succinate
0
Leigh syndrome; Parkinson’s disease; Leber’s hereditary optic neuropathy Leigh syndrome; optic atrophy; hereditary paraganglioma; hereditary pheochromocytoma
2 electrons from Complex I and/or II Reduced Coenzyme Q
0
Complex II Ubiquinone Complex III Cytochrome c Complex IV
Coenzyme Q; Coenzyme Q10; ubidecaranone Cytochrome bc1 complex; cytochrome c-oxidoreductase Cyt c Cytochrome c oxidase
1 electron from Complex III Reduced cytochrome c; O2
2 from the matrixþ2 from Coenzyme Q 0 4
Septo-optic dysplasia; Bjo¨rnstad syndrome; GRACILE syndrome Leigh syndrome; infantile hypertrophic cardiomyopathy; neonatal-onset hepatic failure and encephalopathy; leukodystrophy
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even high PO2 levels in the microcirculation, raising the possibility that the cellular utilization of oxygen may be impaired rather than oxygen delivery. When Mela and colleagues6 showed ultrastructural damage to the mitochondrion with the inhibition of its respiration in rodent models post-sepsis; the question was whether this was cause or effect? These changes will lead to disordered use of oxygen. While the topic still raises debate, there is increasing evidence to support the ‘cytopathic hypoxia’ hypothesis that mitochondrial dysfunction is a crucial factor in MODS rather than failure to deliver oxygen to the cell. The alternative view is that MODS is a result of a combination of microcirculatory and mitochondrial failure—the ‘microcirculatory and mitochondrial distress syndrome’ (MMDS) theory. Both these theories will be briefly reviewed.
Cytopathic hypoxia or cellular metabolic derangement Fink7 first used the term cytopathic hypoxia to describe the dysregulation of oxygen metabolism during sepsis; cells fail to produce adequate amounts of ATP in the presence of sufficient molecular oxygen. These terms reflect the fact that it may not be the availability of oxygen, but its usability by mitochondria within the cell which may be paramount in sepsis. In essence, the hypothesis proposes that inflammatory mediators, in the context of SIRS, directly cause mitochondria to fail which manifests itself as MODS. There are a number of mechanisms proposed to account for mitochondrial dysfunction. In trauma patients, the activation of the SIRS response has been associated with the release of mitochondrial damage-associated molecular patterns (DAMPs). The common endpoint is the uncoupling of electron transport from ATP production, down-regulation of mitochondrial electron transport proteins, mitochondrial membrane damage, and decreased lifespan of the mitochondrion. This directly translates into cellular dysfunction and potentially apoptosis. In the acute phase of sepsis, tumour necrosis factor (TNF)-a, interleukin (IL)-1, and IL-6 are released in major organ systems such as the kidneys, lungs, liver, and brain.8 These mediators can effect the mitochondrion profoundly. For example, TNF-a has the ability to cause direct cytopathic changes through binding the TNF receptor 1 (TNFR-1). The resulting intracellular signals can trigger mitochondrial permeability transition (MPT); the collapse of the proton motive force through increased solute permeability of the mitochondrial membranes. The resulting release of cytochrome c initiates cellular apoptosis. Furthermore, mitochondrial electron transport is itself directly inhibited, increasing the concentration of reactive oxygen species (ROS). Examples of ROS are superoxide, hydrogen peroxide, and hydroxyl radicals . (O2 2 , H2O2, and OH , respectively). ROS are an unavoidable byproduct of electron transport, through the leaking of electrons from complex I or complex III to molecular oxygen, and, in normal physiology, are thought to act as signalling molecules. At higher production rates however, such as in sepsis, ROS cause oxidative damage to membranes, proteins, and DNA. The combination of increased ROS production, due to a block of electron transport,
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and increased nitric oxide (NO), due to the activation of inducible nitric oxide synthase (iNOS), results in peroxinitrite (ONO2 2 ). Peroxinitrite is a highly reactive compound that readily oxidizes electron transport complexes and membrane components, further compromising mitochondrial function. Brearley and colleagues9 demonstrated an association between the inhibition of complex I and lower ATP synthesis with worse outcome in septic patients which may indeed be due to ROS-induced damage. In summary, the cytopathic hypoxia theory proposes that inflammatory mediators have a direct effect on the ability of the mitochondrion to utilize oxygen effectively, which can result in cellular hibernation or cell death. Altered metabolism within the cell is also demonstrated by down-regulation of the enzymes pyruvate dehydrogenase (PDH) and carnitine palmitoyl-transferase—this will lead to a decrease in the supply of energy to the cell and is thought to be a key element of sepsis-induced cardiac dysfunction. Decreasing tissue carnitine levels in the myocardium and endothelium result in increased plasma levels and increased renal loss. Infusing carnitine may represent a novel metabolic treatment alternative to conventional vasoactive therapy, which increases cardiac work, as by stimulating pyruvate oxidation recouping glycolysis and oxidation of pyruvate will lead to a more efficient cardiac muscle contraction.
Microcirculatory and mitochondrial distress syndrome An alternative hypothesis is the concept of MMDS.10 Traditionally, there has been a focus on macrocirculatory haemodynamics to restore delivery to the tissues. However, there is now an increasing focus on the interface between the tissue and the macrocirculation. In sepsis, one of the critical steps may be an alteration in the flow through the microcirculation, which is regulated in health by pressure (myogenic), neurohormonal, and metabolic factors such as CO2, lactate, and O2 to meet the oxygen needs of the cell. In sepsis, these autoregulatory mechanisms are disrupted, resulting in a mismatch between the requirements of the tissue for oxygen and its availability. Further to this, the inducible form of nitric oxide synthase (iNOS) is switched on in response to inflammatory mediators, causing shunting of flow to those areas where there is most nitric oxide. Coupled to the activation of the coagulation pathway and red cell sequestration, the net effect is MMDS. New imaging techniques that allow visualization of the microcirculation have demonstrated improved microcirculatory parameters after administration of fluids for resuscitation in the early stages of sepsis.
Possible therapies The proposal that mitochondrial dysfunction is the driving force behind MODS opens up a large avenue for potential pharmacological treatments. Therapeutic agents have targeted specific pathways or functions of the mitochondrion; mitochondrial substrate and co-factor provision, antioxidants, ROS scavengers, and
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Table 3 Experimental therapies based on the cytopathic hypoxia theory Type
Name
Improved survival
Substrate provision
ATP-MgCl2 Succinate L-carnitine Caffeine Cytochrome c Coenzyme Q10 a-Lipoic acid MitoQ SS peptide N-acetyl cysteine Melatonin SS peptide Tempol 4NH2-Tempo Ethyl pyruvate Melatonin Cyclosporin A NIM811 Bcl over-expression L-NAME 6 L-N -(1-imminoethyl)lysine Aminoguanide
Y Y Y N Y Y N N N N Y N Y Y Y N Y Y Y N N N
Co-factor provision
Antioxidants
ROS scavengers
Membrane stabilisers
iNOS inhibitor
with histopathological abnormalities at autopsy, ‘failure’ in MODS can often be reversible and shows little necrosis or apoptosis at autopsy. Instead, it appears as though the cells are able to adopt a state of hibernation. Down-regulating energy consumption maintains the ‘normal’ intracellular store of ATP, therefore allowing full recovery of cells that would usually be very sensitive to hypoxic injury. This observation has led to the proposal that MODS is, potentially, a protective mechanism to preserve cellular integrity during stressful times.13 Furthermore, the ability of the mitochondrion to divide, increasing its numbers per cell, allows sufficient levels of ATP when recovering from this hibernating state. Of course, the consequences of this protective mechanism, as is seen all too often in intensive care units, may be death. This leaves a therapeutic conundrum: if MODS is a protective mechanism in the face of overwhelming sepsis, should we aim to prevent it with agents that improve mitochondrial function? Or should we focus on optimizing our ability to assist failing organs and await resolution of the initial insult and then target mitochondrial regeneration?
Conclusion membrane stabilizers. Table 3 lists the experimental treatments (adapted from Dare and colleagues).11 Improvements in mitochondrial function, measured by ATP production and a reduction in markers of oxidative stress, were commonly associated with improvements in haemodynamics parameters, organ function, and, crucially, survival. Although many agents have shown benefit in animal models of MODS, none has yet become part of the standard regime for the treatment or prevention of MODS thus far. Administration of cytochrome c in animals has been shown to resolve some of the abnormalities in the mitochondrial electron chain with enhanced cardiac function and decreased mortality. Clinical trials of these agents would be the next step. In accordance with the Surviving Sepsis guidelines, antibiotic therapy should be administered early for bacterial-induced sepsis. However, the role of antibiotics in sepsis-induced MODS is potentially more complex. The potent ability of certain bacteriostatic antibiotics (e.g. chloramphenicol) to inhibit mitochondrial regeneration is a characteristic to be cautiously aware of.12 Considering the evolutionary origin of mitochondria (i.e. bacterial), this perhaps is no surprise. The difficult question therefore arises as to whether we should be concerned about prolonged antibiotic therapy in sepsis which may indeed be delaying or inhibiting recovery from MODS.
Mitochondrial regeneration and MODS During sepsis, all cells in the body experience a certain degree of stress through systemic inflammatory mediators, metabolic changes, and stress hormones (such as cortisol and catecholamines), which may lead to MODS. In contrast to conventional organ failure (e.g. liver failure secondary to alcohol), which is an irreversible process
Oxygen serves as the terminal electron acceptor in cellular respiration, allowing the mitochondrion to generate the proton motive force. The proton motive force is predominantly utilized by F1Fo-ATP synthase to generate readily available energy in the form of ATP. In sepsis, a common and potentially fatal condition, mitochondrial function is severely impaired. In vitro and in vivo studies now point to a causative role of mitochondrial dysfunction in MODS.
Declaration of interest None declared.
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9. Brearley D, Brand M, Hargreaves I et al. Association between mitochondrial dysfunction and severity and outcome of septic shock. Lancet 2002; 360: 219– 23
12. Riesbeck K, Bredberg A, Forsgren A. Ciprofloxacin does not inhibit mitochondrial functions but other antibiotics do. Antimicrob Agents Chemother 1990; 34: 167 –9
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Please see multiple choice questions 33 –36.
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