Download 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
Key points
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
Matrix reference 1A01,1A02,2C02
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]
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
Cellular oxygen utilization
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; Björnstad syndrome; GRACILE
syndrome
Leigh syndrome; infantile hypertrophic cardiomyopathy;
neonatal-onset hepatic failure and encephalopathy;
leukodystrophy
Continuing Education in Anaesthesia, Critical Care & Pain j Volume 12 Number 4 2012
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Cellular oxygen utilization
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,
210
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.
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Please see multiple choice questions 33 –36.
Continuing Education in Anaesthesia, Critical Care & Pain j Volume 12 Number 4 2012