Last post for PHYL3001- for real this time! This lecture was pretty much just about two methods of measuring the traction force, as well as some clarification on mechanomemory. Since I've already discussed mechanomemory here, this will just be a very short post that explains how we measure traction force.
The two methods of measuring traction force are the micropillar/microneedle technique, as well as traction force microscopy (TFM).
Micropillar/Microneedle
Instead of creating a flat hydrogel, you can create a hydrogel with lots of little micropillars (or "microneedles"). When cells pull on the micropillars, they bend. Micropillars have a spring constant, which depends on several other factors, such as the height of the pillars and the material used. The force generated by the cell can be calculated by multiplying the spring constant of the micropillars by the displacement.
If you are using the micropillar method, care should be taken to ensure that the pillars are not too long or too far apart. If pillars are too long, then they are at risk of "lateral collapse," which basically means they can tilt even if force isn't applied to them (imagine a tall cylinder made out of jelly- a short cylinder would keep its shape but a long one would easily bend and break). If pillars are too far apart, there may be a "sagging effect" where the gel in between pillars touches the cell.
The micropillar method is cheaper than the TFM method that I'm about to introduce, and requires less specialised equipment. However, it can only measure forces in the x and y directions, and the discontinuity of the hydrogel (i.e. spaces between micropillars) means that there will also be discontinuity in your results.
Traction Force Microscopy (TFM)
To do TFM, you need a special hydrogel with fluorescent microbeads embedded. When cells pull on the gel, they pull the beads as well. When the cells are removed by using trypsin, the beads return back to their original place. The location of the beads (before and after trypsin) can be determined by using a confocal microscope, and the distance that the beads move can be measured in order to determine the amount of force.
TFM has an advantage over the micropillar method in that it can measure forces in three dimensions (x, y and z), as confocal microscopes can take pictures at different depths. Unfortunately, specialised equipment such as confocal microscopes and microbeads can be expensive, and so not every lab will be able to use this technique.
Sunday, June 4, 2017
Addiction
Last post for PHAR3303! This was probably my favourite lecture from this guy. It covers three main theories of drug addiction: negative reinforcement, positive reinforcement and incentive sensitisation.
Withdrawal-Based Negative Reinforcement
Negative reinforcement is when a behaviour is done in order to avoid a negative response. For example, you might hurry up and hand in an assignment in order to avoid a late penalty. In the case of drugs, people take drugs to avoid withdrawal effects.
What are withdrawal effects? Well, when we take drugs, there are usually compensatory effects that cause us to develop tolerance to the drugs. When we stop taking drugs, the compensatory effects continue even though the drug effects have stopped, causing withdrawal effects. Withdrawal effects are usually opposite to those produced by the drug, so if the drug caused analgesia (pain relief), you might get hyperalgesia (excessive pain) instead. No wonder withdrawal effects can be quite aversive!
There are, however, some limitations to this theory. Firstly, animals will give themselves drugs, even if they have never had the drug before (and therefore don't have any withdrawal symptoms to avoid). Secondly, some drugs (such as cocaine and amphetamine) only have minor withdrawal effects, which are not bad enough to fully explain addiction. Furthermore, locking up people until the withdrawal effects have subsided doesn't seem to cure them of addiction.
Positive Reinforcement: "Wise's Psychomotor Stimulant Theory of Addiction"
Positive reinforcement is when a behaviour is done in order to get a positive response. For example, you might work hard on an assignment because you want good marks. In the case of drugs, people take drugs because they stimulate pleasure centres in the brain. In fact, drugs may stimulate pleasure centres better than natural rewards (like food). This is known as Wise's Psychomotor Stimulant Theory of Addiction.
The "reward system" is made up of neurons in the mesolimbic dopamine system. Cell bodies of these neurons sit in the ventral tegmental area (a.k.a. A10) and project to the nucleus accumbens. Dopamine terminals can be selectively lesioned by first pretreating with desimipramine (in order to prevent lesioning of noradrenaline terminals) and then infusing 6-OHDA into the nucleus. When dopamine terminals are lesioned, or when high doses of dopamine blockers are administered, animals stop seeking cocaine and amphetamine. They would, however, keep seeking amphetamine if it was infused straight into the nucleus accumbens, which to me suggests that receptors on cell bodies in the nucleus accumbens may be responsible (though I could be wrong, I don't know much about neuroscience). Interestingly enough, lesioning dopamine terminals didn't prevent heroin-seeking, but destruction of the cell bodies of the nucleus accumbens did.
Incentive Sensitisation Theory of Craving
The Incentive Sensitisation Theory of Craving was developed after findings that dopamine levels increase before a reward (like sex, food or drugs).
How did we discover this? you might ask. Well, there are several different ways of measuring dopamine levels in vivo. These include in vivo microdialysis and in vivo chronoamperometry. In in vivo microdialysis, a very small dialysis membrane is inserted into a brain region. Artificial CSF is passed through, and chemicals diffuse into this liquid. This method has very good chemical resolution (i.e. it's very accurate at saying which chemical is what) but poor temporal resolution (i.e. you only get results a little while after the action- in this case, it takes minutes). In vivo chronoamperometry involves insertion of an electrode into the brain, and an electrical signal is measured when current is passed through at the same oxidation potential of a specific chemical. Chemical resolution for this method is poor, but temporal resolution is only seconds.
In vivo microdialysis came earlier, and since its temporal resolution is so poor, it made it seem like dopamine levels went up with drug administration, supporting the positive reinforcement theory. Chronoamperometry, however, found that dopamine went up before drug administration, and down right after they get the drug. Therefore, we have to distinguish between wanting something and liking something once you have it.
It turns out that dopamine is important for wanting, but not for liking. MOR receptors (mu opioid receptors) are important for liking whereas KOR receptors are aversive. Interestingly enough, MOR receptors also increased dopamine release, whereas KOR receptors decrease it. It is also important to note that while wanting can be sensitised (i.e. the more you get, the more you want), liking is desensitised (i.e. you get sick of things over time). Also, just as an aside, you're probably wondering how they measure "liking." The answer, for now, is mainly body language: animals make different faces depending on whether they like or dislike something. Also, rats make 50 000Hz vocalisations when they like something. Fun facts!
And that concludes my last post for PHAR3303! Good luck everyone!
Withdrawal-Based Negative Reinforcement
Negative reinforcement is when a behaviour is done in order to avoid a negative response. For example, you might hurry up and hand in an assignment in order to avoid a late penalty. In the case of drugs, people take drugs to avoid withdrawal effects.
What are withdrawal effects? Well, when we take drugs, there are usually compensatory effects that cause us to develop tolerance to the drugs. When we stop taking drugs, the compensatory effects continue even though the drug effects have stopped, causing withdrawal effects. Withdrawal effects are usually opposite to those produced by the drug, so if the drug caused analgesia (pain relief), you might get hyperalgesia (excessive pain) instead. No wonder withdrawal effects can be quite aversive!
There are, however, some limitations to this theory. Firstly, animals will give themselves drugs, even if they have never had the drug before (and therefore don't have any withdrawal symptoms to avoid). Secondly, some drugs (such as cocaine and amphetamine) only have minor withdrawal effects, which are not bad enough to fully explain addiction. Furthermore, locking up people until the withdrawal effects have subsided doesn't seem to cure them of addiction.
Positive Reinforcement: "Wise's Psychomotor Stimulant Theory of Addiction"
Positive reinforcement is when a behaviour is done in order to get a positive response. For example, you might work hard on an assignment because you want good marks. In the case of drugs, people take drugs because they stimulate pleasure centres in the brain. In fact, drugs may stimulate pleasure centres better than natural rewards (like food). This is known as Wise's Psychomotor Stimulant Theory of Addiction.
The "reward system" is made up of neurons in the mesolimbic dopamine system. Cell bodies of these neurons sit in the ventral tegmental area (a.k.a. A10) and project to the nucleus accumbens. Dopamine terminals can be selectively lesioned by first pretreating with desimipramine (in order to prevent lesioning of noradrenaline terminals) and then infusing 6-OHDA into the nucleus. When dopamine terminals are lesioned, or when high doses of dopamine blockers are administered, animals stop seeking cocaine and amphetamine. They would, however, keep seeking amphetamine if it was infused straight into the nucleus accumbens, which to me suggests that receptors on cell bodies in the nucleus accumbens may be responsible (though I could be wrong, I don't know much about neuroscience). Interestingly enough, lesioning dopamine terminals didn't prevent heroin-seeking, but destruction of the cell bodies of the nucleus accumbens did.
Incentive Sensitisation Theory of Craving
The Incentive Sensitisation Theory of Craving was developed after findings that dopamine levels increase before a reward (like sex, food or drugs).
How did we discover this? you might ask. Well, there are several different ways of measuring dopamine levels in vivo. These include in vivo microdialysis and in vivo chronoamperometry. In in vivo microdialysis, a very small dialysis membrane is inserted into a brain region. Artificial CSF is passed through, and chemicals diffuse into this liquid. This method has very good chemical resolution (i.e. it's very accurate at saying which chemical is what) but poor temporal resolution (i.e. you only get results a little while after the action- in this case, it takes minutes). In vivo chronoamperometry involves insertion of an electrode into the brain, and an electrical signal is measured when current is passed through at the same oxidation potential of a specific chemical. Chemical resolution for this method is poor, but temporal resolution is only seconds.
In vivo microdialysis came earlier, and since its temporal resolution is so poor, it made it seem like dopamine levels went up with drug administration, supporting the positive reinforcement theory. Chronoamperometry, however, found that dopamine went up before drug administration, and down right after they get the drug. Therefore, we have to distinguish between wanting something and liking something once you have it.
It turns out that dopamine is important for wanting, but not for liking. MOR receptors (mu opioid receptors) are important for liking whereas KOR receptors are aversive. Interestingly enough, MOR receptors also increased dopamine release, whereas KOR receptors decrease it. It is also important to note that while wanting can be sensitised (i.e. the more you get, the more you want), liking is desensitised (i.e. you get sick of things over time). Also, just as an aside, you're probably wondering how they measure "liking." The answer, for now, is mainly body language: animals make different faces depending on whether they like or dislike something. Also, rats make 50 000Hz vocalisations when they like something. Fun facts!
And that concludes my last post for PHAR3303! Good luck everyone!
Saturday, June 3, 2017
Biomimicry on a dish
This lecture mainly spoke about some of the newer developments in cell culture technology. Let's see what the future has to offer!
Lab on a chip
Efforts are being made to simulate organs using other materials (rather than having to operate on unfortunate animals or humans). In this lecture, we looked at the lung on a chip, though there are other organs on chips that are being developed too. The "lung on a chip" consists of a main chamber with two side chambers. The side chambers are connected to vacuums, allowing them to contract and expand, stretching the middle chamber in the process. The middle chamber is divided in half by a porous membrane with cells cultured on either side- one side, simulating the lung, is coated with alveolar epithelial cells, and the other side, simulating the blood vessel, is coated with endothelial cells. The area simulating the blood vessel is filled with a viscous fluid and the area simulating the alveoli is filled with air. Processes such as immune cell migration (through the porous membrane) have been simulated on these "lungs on a chip."
Biomechanical mimicry
In these lectures, we've been talking a lot about how mechanical stimuli (such as tissue stiffness) affect cell behaviour. Therefore, we need to try and mimic these mechanical forces in vitro. These mechanical forces can include stiffness (as already repeated ad nauseum), pattern alignment (as mentioned here, shape can affect cell differentiation) and other dynamic forces (such as cyclic stretching and relaxation, microfluidic systems etc.).
Protein patterns
ECM proteins can be "patterned" onto a hydrogel by literally stamping them on. Cells can only adhere to ECM proteins (and not directly to the hydrogel- if you don't trust me on this one, you can also guarantee specific binding by "blocking" with pluronic F127). Therefore, such patterning limits the area where cells can grow, which may affect their behaviour.
Stiffness (and stiffness patterns)
Chances are, cells in vivo are introduced to a variety of different stiffnesses. This can be explored in vitro by using a step gradient (where you have alternating lines of stiff vs. soft) or a linear gradient (gel gradually changes from soft to stiff).
I've already mentioned a step gradient hydrogel (zebraxis) here. Step gradient hydrogels, like zebraxis, can be created by using a photomask during UV polymerisation of the hydrogel. A more recent invention is a glass slide of sorts that comes in two halves. Each half has "teeth" that mesh with the "teeth" of the other half. A different hydrogel can be created on each half, and the two halves can be interlocked or moved apart at will. This might allow us to view the effect of direct contact between cells of two different stiffnesses, or paracrine signalling between cells of the different stiffnesses. Yet another invention is that of "digital stiffness writing," which uses a special heat-sensitive gel filled with gold nanorods. When a laser hits the gold nanorods, they vibrate, producing heat. This heat then causes further polymerisation (and an increase in stiffness) in the area touched by the laser.
Formation of linear hydrogels has traditionally used a gradient photomask. Unfortunately, the gradient photomask method has poor reproducibility, and can be very expensive. Therefore, our lecturer came up with an easier way to make a gradient hydrogel. I'm not 100% certain on the method (I did my research placement with him, but I'll admit right now that my participation in that placement has been very casual at best), but I'm fairly sure that it entails making a wedge-shaped hydrogel in a square-shaped well and then pouring another hydrogel mix on top.
Dynamic systems
Moving ahead, we are looking at new ways to culture cells that take a range of other factors into account. For example, the beating bioreactor uses a magnet that turns off and on in order to relax and stretch the gel, respectively. This simulates other tissues that stretch and relax periodically (such as the heart).
And I think that's it...? Or not- there's an extra mechanobiology lecture on LMS, which I should watch at some point. So maybe this isn't the last PHYL3001 post after all...!
Lab on a chip
Efforts are being made to simulate organs using other materials (rather than having to operate on unfortunate animals or humans). In this lecture, we looked at the lung on a chip, though there are other organs on chips that are being developed too. The "lung on a chip" consists of a main chamber with two side chambers. The side chambers are connected to vacuums, allowing them to contract and expand, stretching the middle chamber in the process. The middle chamber is divided in half by a porous membrane with cells cultured on either side- one side, simulating the lung, is coated with alveolar epithelial cells, and the other side, simulating the blood vessel, is coated with endothelial cells. The area simulating the blood vessel is filled with a viscous fluid and the area simulating the alveoli is filled with air. Processes such as immune cell migration (through the porous membrane) have been simulated on these "lungs on a chip."
Biomechanical mimicry
In these lectures, we've been talking a lot about how mechanical stimuli (such as tissue stiffness) affect cell behaviour. Therefore, we need to try and mimic these mechanical forces in vitro. These mechanical forces can include stiffness (as already repeated ad nauseum), pattern alignment (as mentioned here, shape can affect cell differentiation) and other dynamic forces (such as cyclic stretching and relaxation, microfluidic systems etc.).
Protein patterns
ECM proteins can be "patterned" onto a hydrogel by literally stamping them on. Cells can only adhere to ECM proteins (and not directly to the hydrogel- if you don't trust me on this one, you can also guarantee specific binding by "blocking" with pluronic F127). Therefore, such patterning limits the area where cells can grow, which may affect their behaviour.
Stiffness (and stiffness patterns)
Chances are, cells in vivo are introduced to a variety of different stiffnesses. This can be explored in vitro by using a step gradient (where you have alternating lines of stiff vs. soft) or a linear gradient (gel gradually changes from soft to stiff).
I've already mentioned a step gradient hydrogel (zebraxis) here. Step gradient hydrogels, like zebraxis, can be created by using a photomask during UV polymerisation of the hydrogel. A more recent invention is a glass slide of sorts that comes in two halves. Each half has "teeth" that mesh with the "teeth" of the other half. A different hydrogel can be created on each half, and the two halves can be interlocked or moved apart at will. This might allow us to view the effect of direct contact between cells of two different stiffnesses, or paracrine signalling between cells of the different stiffnesses. Yet another invention is that of "digital stiffness writing," which uses a special heat-sensitive gel filled with gold nanorods. When a laser hits the gold nanorods, they vibrate, producing heat. This heat then causes further polymerisation (and an increase in stiffness) in the area touched by the laser.
Formation of linear hydrogels has traditionally used a gradient photomask. Unfortunately, the gradient photomask method has poor reproducibility, and can be very expensive. Therefore, our lecturer came up with an easier way to make a gradient hydrogel. I'm not 100% certain on the method (I did my research placement with him, but I'll admit right now that my participation in that placement has been very casual at best), but I'm fairly sure that it entails making a wedge-shaped hydrogel in a square-shaped well and then pouring another hydrogel mix on top.
Dynamic systems
Moving ahead, we are looking at new ways to culture cells that take a range of other factors into account. For example, the beating bioreactor uses a magnet that turns off and on in order to relax and stretch the gel, respectively. This simulates other tissues that stretch and relax periodically (such as the heart).
And I think that's it...? Or not- there's an extra mechanobiology lecture on LMS, which I should watch at some point. So maybe this isn't the last PHYL3001 post after all...!
Hypoxia
Last post for PHYL3002!
Define hypoxia, hypoxemia and cyanosis.
Hypoxia can be divided into peripheral and central hypoxia. Peripheral hypoxia only affects some tissues, whereas central hypoxia affects the whole body. Peripheral hypoxia can be ischaemic (due to poor perfusion of tissue, as might happen if you tie a tourniquet too tightly), histotoxic (due to mitochondrial failure, as happens in cyanide poisoning) or in heavy exercise (oxygen supplies are depleted, causing muscle to switch to anaerobic metabolism).
Explain why there is no hypoxemia in anaemic hypoxia
Anaemia can cause hypoxia without causing hypoxemia. In anaemia, there are fewer red blood cells, and therefore less haemoglobin for oxygen to bind to. Therefore, the total oxygen concentration (oxygen dissolved in arterial blood + oxygen bound to haemoglobin) is less overall, resulting in hypoxia. Anaemia, however, does not cause oxygen dissolved in arterial blood to decrease, and therefore does not cause hypoxemia.
Identify the five causes of hypoxemia
Explain the changes in gases cause by the five types of hypoxemia
The five causes of hypoxemia are as follows:
Low PiO2
As mentioned, this can occur at high altitudes. It can also occur due to occupational hazards (e.g. a nitrogen gas leak that displaces oxygen).
Hypoventilation
Hypoventilation = ventilation insufficient to meet respiratory demands. This can have several causes, including but not limited to the following:
Calculate the AaDO2 from blood gases
Since hypoventilation is the only cause of hypoxemia that causes an increase in PaCO2, it can be distinguished by using the alveolar gas equation. If you don't remember from 2nd year, the alveolar gas equation is as follows:
PAO2 = PiO2 - (PACO2/RQ)
where PAO2 is alveolar partial pressure of O2, PiO2 is inspired partial pressure of O2, PACO2 is alveolar partial pressure of CO2 and RQ is respiratory quotient, which in turn is V'CO2/V'O2 (i.e. moles of CO2 produced per moles of O2 consumed). RQ is 1 for a pure carbohydrate diet, around 0.7 for a pure fat diet, and around 0.8 for a normal Western diet.
Since we can't actually measure alveolar gases directly, we assume that they are similar to arterial gases (which they are if gas exchange is normal). Therefore, we can substitute PAO2 for PaO2 and PACO2 for PaCO2 in the above equation. PiO2 is something we can easily measure, and for RQ we can estimate 0.8 (I don't know if there are better ways to measure it though). By substituting our measured PaCO2 into the equation, we can find a theoretical PaO2, which we can then compare to the real PaO2 to find the AaDO2 (Alveolar-arterial difference in oxygen). If AaDO2 is less than 15mmHg, then hypoventilation is the cause of the hypoxemia.
Maybe this will make more sense with an example! Let's say that we have a patient with a PaO2 of 68mmHg and a PaCO2 of 50mmHg. To test whether or not they are hypoventilating, we can substitute the PaCO2 into the alveolar gas equation, find the theoretical PaO2 and compare it with the real PaO2. This gives us the following:
PAO2 = PiO2 - (PACO2/RQ)
PaO2 (theoretical) = 0.21*(760-47) - (50/0.8)
PaO2 (theoretical) = 149.73 - 62.5
PaO2 (theoretical) = 87.23mmHg
AaDO2 = PaO2 (theoretical) - PaO2 (actual)
AaDO2 = 87.23 - 68
AaDO2 = 19.23mmHg
In this case, AaDO2 is more than 15mmHg, so hypoventilation is not the cause of this patient's hypoxemia. In this case, we would have to do extra tests- a 100% oxygen test to rule out shunt and a DLCO test to rule out diffusion capacity issues. If both of those tests come back negative, then the patient has V'/Q' mismatch. Note that hypoxemia due to shunts or diffusion capacity issues are quite rare.
Define hypoxia, hypoxemia and cyanosis.
- Hypoxia: Low oxygen content.
- Hypoxemia: Low partial pressure of oxygen in the arteries.
- Cyanosis: Discolouration of the skin due to unsaturated haemoglobin.
Hypoxia can be divided into peripheral and central hypoxia. Peripheral hypoxia only affects some tissues, whereas central hypoxia affects the whole body. Peripheral hypoxia can be ischaemic (due to poor perfusion of tissue, as might happen if you tie a tourniquet too tightly), histotoxic (due to mitochondrial failure, as happens in cyanide poisoning) or in heavy exercise (oxygen supplies are depleted, causing muscle to switch to anaerobic metabolism).
Explain why there is no hypoxemia in anaemic hypoxia
Anaemia can cause hypoxia without causing hypoxemia. In anaemia, there are fewer red blood cells, and therefore less haemoglobin for oxygen to bind to. Therefore, the total oxygen concentration (oxygen dissolved in arterial blood + oxygen bound to haemoglobin) is less overall, resulting in hypoxia. Anaemia, however, does not cause oxygen dissolved in arterial blood to decrease, and therefore does not cause hypoxemia.
Identify the five causes of hypoxemia
Explain the changes in gases cause by the five types of hypoxemia
The five causes of hypoxemia are as follows:
- Low PiO2 (partial pressure of inspired oxygen, such as in high altitudes)
- Hypoventilation
- Diffusion limitation (i.e. issues in gases diffusing across the alveolar wall)
- R-L shunt (i.e. mixing of deoxygenated and oxygenated blood, as occurs in several congenital heart defects)
- V'/Q' mismatch (the most common cause of hypoxemia)
Low PiO2
As mentioned, this can occur at high altitudes. It can also occur due to occupational hazards (e.g. a nitrogen gas leak that displaces oxygen).
Hypoventilation
Hypoventilation = ventilation insufficient to meet respiratory demands. This can have several causes, including but not limited to the following:
- Asphyxiation- no ventilation due to a physical obstruction (strangling etc.)
- Failure of respiratory drive- can be due to CNS damage or certain drugs
- Failure of respiratory muscle- can be due to a neuromuscular disease such as muscular dystrophy
- Failure of lung ventilation- due to a restrictive or obstructive lung disease
- Drowning- as I will explain...
The effect of drowning depends on whether you are drowning in salt or fresh water. (Okay, the overall result- death- is still the same, but the path to death is a bit different.) Also most patients who die by drowning actually die by laryngospasm rather than by the water itself, but let's ignore that for now.
If you are drowning in fresh water, your blood will have a higher osmolarity than the fluid in the alveoli, so water is drawn into the blood. This causes cells to swell and then lyse. Lysed cells release a lot of potassium, so the potassium concentration of the blood goes up (which can lead to arrhythmias, heart failure etc.). Concentrations of other solutes, such as sodium, chlorine and proteins, will decrease as they have become diluted by the extra water.
If you are drowning in salt water, your blood will have a lower osmolarity than the fluid in the alveoli, so even more water is drawn into the alveoli. Therefore, salt water drowning is harder to save someone from than fresh water drowning. Since fluid is being lost from the blood, the blood is more concentrated, and so concentrations of sodium, chloride, magnesium, proteins etc. all increase.
Hypoventilation decreases oxygen concentrations and increases carbon dioxide concentrations. (This is in contrast to V'/Q' mismatch, diffusion limitation and R-L shunt, all of which cause hypoxia with little to no hypercapnia.) Therefore, hypoventilation can be distinguished from V'/Q' mismatch by use of the alveolar gas equation, but I will explain that later.
Diffusion limitation
Diffusion limitation essentially occurs to problems with gases diffusing (due to a thickened alveolar membrane etc.). This can be detected by measuring the diffusion capacity of carbon monoxide, as mentioned here. Diffusion limitation causes hypoxia with little to no hypercapnia.
R-L shunt
In a R-L shunt, there is mixture of deoxygenated and oxygenated blood. This occurs when venous blood does not get exchanged with air. There are two types of shunts: anatomical shunts, where blood fails to pass through alveoli, and physiological shunts, where air fails to get to alveoli. A normal healthy lung will have around 2% shunt, half of which is accounted for by bronchial circulation. When there is too much shunt, however, this can be problematic.
Shunt can be diagnosed by giving the patient 100% oxygen. Since 100% oxygen causes PAO2 (remember, capital A = alveolar) to increase to around 650mmHg, a normal healthy person will also have a massive increase in PaO2 (lowercase a = arteriolar). When there is a shunt involved, there is no improvement in PaO2 as the shunted blood is not exposed to the high PAO2. R-L shunt causes hypoxia with little to no hypercapnia.
V'/Q' mismatch
See earlier post: Gas Exchange and V'/Q' Ratio. Note that this is the most common cause of hypoxemia. It causes hypoxia with little to no hypercapnia.
Calculate the AaDO2 from blood gases
Since hypoventilation is the only cause of hypoxemia that causes an increase in PaCO2, it can be distinguished by using the alveolar gas equation. If you don't remember from 2nd year, the alveolar gas equation is as follows:
PAO2 = PiO2 - (PACO2/RQ)
where PAO2 is alveolar partial pressure of O2, PiO2 is inspired partial pressure of O2, PACO2 is alveolar partial pressure of CO2 and RQ is respiratory quotient, which in turn is V'CO2/V'O2 (i.e. moles of CO2 produced per moles of O2 consumed). RQ is 1 for a pure carbohydrate diet, around 0.7 for a pure fat diet, and around 0.8 for a normal Western diet.
Since we can't actually measure alveolar gases directly, we assume that they are similar to arterial gases (which they are if gas exchange is normal). Therefore, we can substitute PAO2 for PaO2 and PACO2 for PaCO2 in the above equation. PiO2 is something we can easily measure, and for RQ we can estimate 0.8 (I don't know if there are better ways to measure it though). By substituting our measured PaCO2 into the equation, we can find a theoretical PaO2, which we can then compare to the real PaO2 to find the AaDO2 (Alveolar-arterial difference in oxygen). If AaDO2 is less than 15mmHg, then hypoventilation is the cause of the hypoxemia.
Maybe this will make more sense with an example! Let's say that we have a patient with a PaO2 of 68mmHg and a PaCO2 of 50mmHg. To test whether or not they are hypoventilating, we can substitute the PaCO2 into the alveolar gas equation, find the theoretical PaO2 and compare it with the real PaO2. This gives us the following:
PAO2 = PiO2 - (PACO2/RQ)
PaO2 (theoretical) = 0.21*(760-47) - (50/0.8)
PaO2 (theoretical) = 149.73 - 62.5
PaO2 (theoretical) = 87.23mmHg
AaDO2 = PaO2 (theoretical) - PaO2 (actual)
AaDO2 = 87.23 - 68
AaDO2 = 19.23mmHg
In this case, AaDO2 is more than 15mmHg, so hypoventilation is not the cause of this patient's hypoxemia. In this case, we would have to do extra tests- a 100% oxygen test to rule out shunt and a DLCO test to rule out diffusion capacity issues. If both of those tests come back negative, then the patient has V'/Q' mismatch. Note that hypoxemia due to shunts or diffusion capacity issues are quite rare.
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