Surviving Anoxia
Introduction
Oxygen comprises a large proportion of our atmosphere (21%) and has become a vital gas for many organisms on Earth. Without it our ability to produce energy (as ATP) via aerobic respiration fails. Oxidative phosphorylation, the primary method of aerobic ATP production, produces large amounts of ATP (from our glycogen stores) just as long as we are able to continually supply O2. Without oxygen we resort to anaerobic respiration, where the primary method of ATP production is glycolysis. In comparison to oxidative phosphorylation, the amount of ATP produced is minimal.
Whilst we take oxygen for granted in our environment, there is a large variety of situations where oxygen levels are limited, or where oxygen is completely absent. Typically, such conditions arise in aquatic environments where the ability of oxygen to diffuse is greatly reduced and oxygen levels depend heavily on the ability of organisms to photosynthesise. Yet many respiring organisms are able to live in these environments due to adaptations to low oxygen/ zero oxygen situations.
When referring to the content of oxygen in an environment, we usually state its partial pressure i.e. the proportion of the total gas which comprises oxygen. At sea level, the total pressure of the gases in the environment is 101kPa (or 1 atmosphere), as oxygen makes up 21% of these gases, the partial pressure of oxygen at sea level is 21kPa.
From this we can define four terms:
Hyperoxia (>21kPa) – When oxygen levels are greater than normal
Normaxia (21kPa) – When oxygen levels are normal
Hypoxia (<21kPa) – When oxygen levels are below normal
Anoxia (0kPa) – When oxygen is completely absent
Why We Require Oxygen
Aerobic Respiration
The major role of oxygen is to provide the body with metabolic energy in the form of ATP. This is done during aerobic respiration via a process known as oxidative phosphorylation which occurs within the inner mitochondrial membranes. The role of oxygen in this process is to act as a terminal electron acceptor. The transfer of electrons to oxygen provides enough energy to phosphorylate ADP to ATP.
However, for oxygen to form ATP in this manner, there are a number of prerequisites. Glucose (a 6-carbon molecule) must be readily available, if it is not it must be mobilised from the body’s glycogen stores. There are 3 major steps which occur before oxidative phosphorylation:
Glycolysis – Glucose is metabolised into 2 molecules of pyruvate (a 3-carbon molecule). This provides a small amount of ATP as well (2 per glucose molecule).
Pyruvate is metabolised into 2 molecules of Acetyl CoA (a 2-carbon molecule). One molecule of carbon is lost as CO2.
The Krebs cycle – Each molecule of acetyl CoA undergoes a number of enzyme-catalysed reactions producing a number of highly energised electrons. These electrons are used in the electron transport chain to produce vast amounts of ATP.
Anaerobic respiration
However, oxygen isn’t always available. In this situation, respiration may continue but it can go no further than glycolysis. As a result, the ATP yield per molecule of glucose alters dramatically:
| Glycolysis | Krebs Cycle | Oxidative Phosphorylation | |
| Aerobic | 2 | 2 | 34 |
| Anaerobic | 2 | 0 | 0 |
Anaerobic Glycolysis
The efficiency of ATP production drops to around 5% during anaerobic reproduction. One way the body can cope with this is to increase the glycolytic rate (the rate of glycolysis). Increasing the glycolytic rate by about 20x returns the level of ATP production to that seen in aerobic respiration. This is known as the Pasteur Effect.
There are of course negative effects as a result of this:
Increasing the glycolytic rate causes a rapid drop in the body’s store of glycogen. Without glycogen, the body will be unable to perform any form or respiration – anaerobic or not, which will inevitably lead to death.
The Krebs cycle stops soon after oxygen becomes absent. This means there is no demand for acetyl CoA. The result of this is excess production of pyruvate. Lactate dehydrogenase converts the excess pyruvate to lactate (and also recycles NADH + H+ to NAD+ allowing glycolysis to continue). A build up of lactate is also undesirable however as it immediately dissociates into lactic acid. The low pH of lactic acid will begin to reduce cellular pH and denature sensitive enzymes.
Decreasing Cellular pH
As a result of decreased pH and ATP, maintaining the membrane potential becomes very difficult. The Na/K ATPase pump which actively removes N+ from the cell and uptakes K+ in order to maintain the correct (-70mV) membrane gradient, stops functioning when ATP production diminishes. The maintained -70mV gradient begins to equilibrate at 0mV, causing a breakdown of the transmembrane gradient within a matter of minutes.
This has dire consequences on the mammalian brain in particular; the change in the membrane potential allows calcium ions to enter the cell. Intracellular calcium causes cell damage and hastens enzyme breakdown. The breakdown of these important brain cells causes permanent brain damage and if not supplied with oxygen quickly enough, will lead to death.
Strategies to Survive Anoxia
To survive anoxic conditions, an organism must be able to deal with the problems associated with anaerobic respiration; keeping up ATP levels (particularly in the brain) and avoiding acidification and associated cellular damage. Two organisms which are highly adapted to surviving anoxic conditions are; the crucian carp and the painted turtle. Both have different strategies for maintaining the body without oxygen, but both do so by combating the associated problems with anaerobic respiration.
Glycogen Stores in the Crucian Carp
One of the problems with anaerobic respiration is that, after a short time in anoxia, glycogen levels decrease rapidly. The Pasteur effect (where glycolytic rate increases for a short period to accommodate the lack of ATP production from oxidative phosphorylation) is only temporary. All body stores of glycogen are quickly depleted and the body is no longer able to survive.
The crucian carp (and to a smaller extent the painted turtle) has an unusually large glycogen reserve in the liver (30% of the total liver mass). Such large stores of glycogen allow the crucian carp to survive in anoxia by glycolysis alone for a much longer period of time.
The crucian carp will typically only face anoxic conditions during the winter months, when a thick layer of ice forms over the water sources they inhabit. Because of this, the crucian carp has adapted to build up glycogen stores throughout the year so that come winter, the glycogen stores are as great as possible. To do this, during the warmer summer months, instead of using carbohydrates for energy (i.e. Glucose / glycogen) lipids are used, allowing the crucian carp to build glycogen stores.
Metabolic Depression in the Crucian Carp
Although increased glycogen stores length the period of time the crucian carp is able to survive in anoxic conditions, it is not the only factor which prolongs this survival period. Metabolic depression means slowing down the rate at which the body consumes ATP, so combining reduced ATP requirements with greater stores of glycogen to produce ATP ensures a near maximum survival time in anoxia.
To reduce metabolic rate, locomotion is reduced during anoxia this means muscles use as little energy as possible. Also, some sensory functions are suppressed, these include vision and hearing. During the winter months when anoxic conditions arise, the thick layer of ice above reduces light levels to a minimum so vision is not a necessity.
Crucian carp will also induce voluntary hypothermia during anoxia. This is actually beneficial to the carp because at lower temperatures, metabolic rate decreases. Therefore when anoxic conditions arise, the crucian carp will swim to cooler waters to reduce their metabolism.
Avoiding Acidification in the Crucian Carp
The problem with glycolysis is the production of lactate and hence lactic acid, causing a decrease in cellular pH and thus acidification. If the crucian carp is producing ATP from glycolysis alone then cellular concentrations of lactic acid would normally quickly rise and pose a major threat for the carp. However, instead of producing lactate as the end product of glycolysis, the crucian carp has adapted to produce ethanol.
By producing ethanol instead of lactate, the crucial carp benefits in a number of ways. Primarily ethanol is non-acidic and thus will not decrease intracellular pH (therefore avoiding the associated damage to the cell as a result of acidification). Ethanol is also lipid soluble meaning it will not build up in the body as it is easily excreted across the gills.
Normally, during glycolysis, pyruvate is metabolised to acetyl CoA by pyruvate dehydrogenase – producing lactate as the end product. Yet a mutation in the pyruvate dehydrogenase enzyme of the crucian carp means that instead of acetyl CoA, acetaldehyde is produced. Acetaldehyde is then metabolised by alcohol dehydrogenase to produce ethanol.
However, the brain is unable to produce ethanol in this manner, only skeletal muscle can. Any glycolysis occurring in the brain will therefore produce lactate as the end product. This lactate however, is transported via the blood, to the skeletal muscle. Here, the lactate is turned into pyruvate where glycolysis will metabolise it into ethanol, it is subsequently excreted across the gills.
Avoiding Acidification in the Painted Turtle
The painted turtle deals with anoxia in a different way to the crucian carp. Whereas the crucian carp has very large glycogen stores and avoids acidification by producing ethanol, the painted turtle favours metabolic depression. Although the crucian carp also depresses its metabolism, it is on a much smaller scale. The crucian carp is still aware and the brain functions as normal. The painted turtle on the other hand becomes almost ‘comatose’ as many more body systems are shut down.
The painted turtle must too deal with the excess of lactate produced during glycolysis to avoid acidification. The painted turtle produced lactate as the end product of glycolysis and does not have the ability to form ethanol from pyruvate. To avoid acidification, it must therefore buffer the low pH of the lactic acid.
To buffer the lactic acid, the painted turtle uses bicarbonate and carbonate. The turtle has large stores of these in its shell (which has a rich blood and nerve supply). The carbonates are able to ‘mop up’ excess H ions (which make the blood acidic) and through a number of reactions, the carbonate and H ions and turned into carbon dioxide and water.
The shell is also a rich store of calcium ions. These calcium ions are able to bind with lactate and the resulting molecule becomes stored in the shell until it is possible to excrete the lactate during aerobic respiration.
This method of avoiding acidification is arguably not as efficient as the crucian carp (producing non-acidic ethanol) but it does help the turtle to avoid acidification for a much longer period of time than would normally be possible.
Metabolic Depression in the Painted Turtle
The painted turtle is able to survive anoxia for such extended periods of time due to the amazing amount at which it is able to decrease its metabolic rate by (90-95%). All non-essential systems are shut down which causes the turtle to become ‘comatose’ as the brain becomes quiescent. Even essential systems such as protein synthesis cut their ATP demand drastically. However on system reduces its ATP demand as little as possible -the Na/ K ATPase pump.
As we know, if the Na/ K ATPase pump stops functioning, then the transmembrane gradient breaks down due to the leakage of Na & K ions across the membrane. This allows calcium ions to enter the cell and cause fatal destruction if normoxic conditions are not resumed. However, the painted turtle is not able to maintain 100% efficiency of this pump during anoxia due to the large demand of ATP it requires. Its efficiency is essential cut down to 25%.
To combat the reduction in the ATPase pump’s efficiency a phenomenon known as ‘Channel Arrest’ occurs. This allows the retention of the transmembrane gradient. Normally Na & K are able to leak into and out of a cell via transmembrane ion channels, down their respective concentration gradients. However during channel arrest these ion channels are forced to close greatly decreasing the amount of ion leakage into and out of the cell. This essentially counteracts the 4-fold reduction in the ATPase pump’s efficiency and allows for the preservation of the transmembrane gradient – thus preventing cell damage.
Another phenomenon of the painted turtle which allows for further depression of the metabolic rate is ‘Spike arrest’. This inactivates Na & K ion channels normally associated with producing action potentials. However as these channels are inactivated the action potential threshold becomes greatly elevated – thus forcing the brain into a quiescent state and preventing responses to stimuli.
The Role of Neurotransmitters and Metabolites
Certain molecules are involved with mediating the transition to an anoxic state. The main ones are the neurotransmitters; glutamate and GABA as well as the metabolite adenosine.
Adenosine release is associated with low levels of oxygen. When low blood oxygen is detected, adenosine is released shortly after. The immediate result is an increase of cerebral blood flow. This decreases soon after in the painted turtle as the metabolic rate is lowered – however as the crucian carp remains ‘active’ during anoxia, cerebral blood flow does not reduce.
Adenosine is basically responsible for ensuring ATP production is able to meet demand as it is responsible for a number of factors contributing to the metabolic depression associated with anoxia. It blocks the production of glutamate (an excitatory neurotransmitter) and has been found to suppress protein synthesis and down-regulate NDMA receptor activity.
The NDMA receptor is highly permeable to Ca2+ ions, which are responsible for cell damage in anoxic conditions of unspecialized organisms. Normally in anoxic conditions excessive glutamate over-stimulates this receptor and causes a massive influx of calcium ions into the cell. Therefore the ability of adenosine to down regulate this receptor is highly beneficial during anoxia.
GABA and glutamate are highly related neurotransmitters; GABA is an inhibitory neurotransmitter and glutamate an excitatory neurotransmitter. During anoxia, we see a steady rise in GABA and a steady decline in glutamate. An initial steep rise of GABA in the painted turtle is due to increased levels of adenosine (released at low blood oxygen levels). Because GABA is an inhibitory neurotransmitter it is able to effectively reduce the amount of ATP the body requires, as the painted turtle reduces its metabolism by 90-95% we see very large amounts of GABA released. Release of GABA is seen in the crucian carp too, but as the carp remains active, GABA levels observed are much more modest.
Conclusion
Effectively we see two different strategies for combating anoxic conditions. The painted turtle, although it has larger glycogen stores than other animals, chooses to reduce its metabolism by such a large amount that it becomes almost comatose. This is achieved by Spike arrest, channel arrest, nearly a total suppression of nervous activity and a huge increase in extracellular inhibitory neurotransmitter, GABA. The limiting factor of how long the painted turtle can remain in anoxia is therefore the build-up of lactate. Although it is steadily removed and stored in the shell, it eventually builds up enough to reduce cellular pH to fatal levels if normoxic conditions are not returned.
The crucian carp on the other hand has glycogen stores so large it is able to survive on glycolysis alone at a metabolic rate not too far below normal. In terms of metabolic depression the crucian carp only moderately suppresses nervous activity, coupled with moderate release of GABA and suppression of locomotor activity (as well as voluntary hypothermia to decrease metabolic rate further). The key strategy for survival is the production of the non-acidic glycolytic end product – ethanol, thus preventing cellular pH from falling to fatal levels. Because of this, the limiting factor of anoxia survival in crucian carp is therefore the amount of glycogen stored in the liver.
Summary
| Crucian Carp | Painted Turtle | |
| Activity level during anoxia | Slightly reduced | Comatose, quiescent brain |
| Production of ATP from glycolysis | Great | Above average |
| Glycogen storage | Great | Above average |
| End product of glycolysis | Ethanol | Lactate (Lactic acid) |
| Blood pH and cellular pH | Stable (ethanol is non-acidic) | Slowly rises when uptake of lactate decreases |
| Overall metabolic depression | 70% | 90-95% |
| ATP levels and membrane potential of the brain | Maintained at normoxic levels – Brain function is preserved | Maintained at normoxic levels – Brain function is preserved |
| Suppression of neuronal activity | Above average | Great |
| Channel & Spike Arrest | No | Yes |
References: Voelkel, S (2010), “Surviving Anoxia”, BIOL 445, University of Liverpool, Unpublished.
