I always love seeing molecular teamwork and one of the best examples of this is how your body carries oxygen throughout the body, gets rid of CO₂, and, while it’s at it, regulates your blood pH! We’re talking cooperation out the wazoo! From hemoglobin molecules cooperatively binding to oxygen, the first oxygen peer-pressuring more oxygens to latch on, to the respiratory and renal (kidney) systems having the other’s back! Plus, there’s some cool technology doctors use to monitor what’s going on. This is my lame-o intro to a post that’s a bit of a grab bag of related awesomeness! 

You know that little “finger closepin” that doctors put on someone’s finger to monitor their vital signs? It’s called a pulse ox, which is short for pulse oximeter because it measures (-meter) a person’s pulse and oxygen saturation. 100% oxygen saturation obviously doesn’t mean that your blood is all oxygen – instead it means that all of the copies of oxygen-carrying proteins in your blood, proteins called hemoglobin, are loaded up with oxygen. Knowing the oxygen saturation helps doctors understand how well someone’s body is able to maintain adequate oxygen delivery to all the tissues in need – and how this technique works is quite cool indeed! Especially since, unlike Arterial Blood Gasses (ABGs) it can work continuously and doesn’t require blood drawing. These techniques tell you slightly different things, and today I’ll give an overview of what each brings.

Here’s an overview of the techniques and then I’ll tell you more about the biochemistry and some more details on the techniques. Quick technical note: as I’ll get into more later, concentrations of gasses are often reported as “partial pressures,” which is just a measurement of how much of that gas there is, and is signified by “Pa” before the gas name. So, for example, the partial pressure of oxygen is reported as PaO₂, and uses units like kPa (kiloPascals) or mmHg (millimeters of mercury). Sometimes, partial pressures of gases in the blood are referred to as “blood gas tensions”

You can find out PaO₂ (and a couple other things) using an ABG (Arterial Blood Gas). This common hospital procedure takes a sample of blood from a person’s artery (one of the blood vessels leaving your heart to go take oxygen (O₂) to the rest of your body) and measures a few things, including the oxygen concentration (aka oxygen content). This concentration tells you about how well the lungs are doing their job, and it depends on a few things:

  • FiO₂ (Fraction of Inspired oxygen): the partial pressure of the oxygen you breathe in (how much oxygen is in the air to begin with?)
  • how well ventilation & gas exchange is working (are the lungs doing that expand-contract-expand-contract thing they need to be doing in order to pull fresh, oxygen-rich air in & push used, CO₂-rich, oxygen-poor air out?)
  • concentration of hemoglobin (how many oxygen carriers are present?)
  • affinity of hemoglobin for oxygen (how willing are those carriers to actually carry oxygen?)

Pulse oximetry (pulse ox) focuses on the hemoglobin, looking to see what percentage of the hemoglobin proteins (our oxygen carriers) are bound to oxygen (i.e. what is the oxygen saturation). And it does this “looking” non-invasively – you just have to stick that digital finger hairpin on your finger. The oxygen saturation is hopefully 95-100%. If the saturation’s lower than this, the person is said to be “hypoxic” (hypo, below). 

Now that I’ve given that brief introduction, let’s get into why we care, and then I’ll get into some more details. The”cardiopulmonary system” is your heart + lungs + blood vessels + blood, etc. working together to take oxygen that’s in the air and distribute it all around your body. Even the furthest reaches in your body – the tips of your teeny toes – need oxygen in order to function normally because, among other things, oxygen plays a crucial role in energy production https://bit.ly/cellularrespiration2 

Energy is stored in our body in the form of a molecule called ATP, which you can think of a bit like energy arcade tokens – lots of different energy sources (fats, proteins, carbs) can be broken down and their energy stored as the same molecule, ATP, which can be “spent” to drive energetically-costly reactions like making proteins and copying DNA. This is analogous to how a variety of currencies and bill/coin denominations can all be converted into various amounts of the same type of arcade token, which can be spent on any game in the arcade. https://bit.ly/atpenergymoney 

The most efficient form of ATP production is a process called oxidative phosphorylation (oxphos) followed by the electron transport chain (ETC) and oxygen is the star player in the ETC. The ETC drives ATP production by passing electrons from one molecule to another to another to another to OXYGEN! Without oxygen there to drive the process, it shuts down (your body can make some energy through anaerobic (non-oxygen-requiring) respiration, but you make a lot less ATP that way, and you also get the buildup of byproducts like lactic acid). 

That cellular use of oxygen for making energy is called “cellular respiration” but, outside of biochemistry, the term “respiration” is more  commonly used to refer to pulmonary respiration. Pulmonary refers to lungs and this type of respiration is where your lungs expand to suck air in and then contract to push used air out. Lungs are often depicted as big sack-like things, but within those “sacks” are branching tubes leading to lots of tiny grape-like ends called alveoli, which are surrounded by tiny blood vessels (capillaries) where gas exchange can occur. The sucked-in air contains oxygen and a protein called hemoglobin in the blood in the capillaries surrounding the alveoli grabs onto that oxygen and whisks it off. To help give it the energy it needs to get to your toes, the next step is your heart, which, through its pumping, gives that oxygenated blood a push out.

But how does hemoglobin know where on its route to give oxygen out? A lot more details on hemoglobin yesterday, but it’s basically just seriously amazing… http://bit.ly/bohreffect And the amazingness (like many amazing things) all comes from biochemistry, which many an amazing thing does bring!

The hemoglobin protein is a “tetramer” –  it’s made up of 4 protein chains working together to form a functional protein. But this “globin” is not functional unless it has help from a cofactor (bound, non-protein molecule) called heme. The globin protein holds onto heme (one per chain, so 4 total per hemoglobin) and heme holds onto iron and they all work together to hold onto oxygen. And they *really* work together – hemoglobin’s oxygen-binding is a great example of “cooperativity” – when none of the sites are bound to oxygen, hemoglobin isn’t that eager to grab oxygen. But when one of the 4 sites binds, it causes the protein to shape shift a little so that it’s easier for the other 3 sites to bind. And, when one of the sites lets go, the other sites are quick to follow. 

You can think of it kinda like peer pressure on the molecular scale, and it leads to a sort of all or nothing approach, which you can visualize with a hemoglobin-oxygen dissociation curve plotting hemoglobin saturation (y-axis) against oxygen concentration. As the available oxygen levels increase, it becomes more likely that one site will bump into an oxygen molecule and bind, leading to the shape shift which makes it easier for the others to follow suit. So you see an S-like curve. 

Assuming that all is normal, in your lungs, there’s a lot of oxygen, so the hemoglobin “all” binds – but in your toes, there’s less oxygen because you’re further from the source and some of it’s been delivered along the way. Since there’s less oxygen, the hemoglobin is more likely to dump some out, especially thanks to something called the Bohr effect, which we discussed in detail yesterday, and I’ll get into more in a sec, but here’s the gist: making energy doesn’t just use oxygen (O₂), it also produces carbon dioxide (CO₂). When that CO₂ dissolves, it produces carbonic acid, which makes the blood more acidic (more protons, H⁺, and thus lower pH) and lowers hemoglobin’s affinity for oxygen, making it more willing to dump it out. 

Normal room air contains ~21% oxygen (in geek speak it has an FiO₂ of 0.21), but If someone is having oxygenation problems, doctors often give them “extra” oxygen to try to get their hemoglobin to the right side of the curve. This works well if you’re on the left side of the curve, where there’s still a lot of hemoglobin open, but not enough oxygen’s getting there, but as you can see, if you’re already on the right side of the curve, and the hemoglobin’s full, that oxygen won’t help much. And it also won’t help if the lungs can’t suck in that oxygen-rich air (that is, if there’s a problem with ventilation). Instead, the lungs might need help expand-contract-expand-contract-ing, which is where mechanical ventilators can help. “Gentler” methods of providing pressure like CPAP & BiPAP, which work via face masks instead of tubes down in your lungs, can help the air get down into those alveoli. 

So how do doctors now if a patient’s oxygen’s too low? One noninvasive way is with a pulse oximeter and how it works is pretty cool. Pulse ox’s can tell based on how well specific wavelengths of light can pass through your finger. Light can be thought of as little packets of energy called photons traveling in waves. Different wavelengths correspond to photons with different energies and we perceive them as different colors. White light has all the colors of the rainbow and things look colored if they “steal” (absorb) some of those visible wavelengths, leaving us to see just the leftovers, which can be transmitted (let through) or reflected (bounced back at us). The molecular makeup of things determines which wavelengths those things can absorb, and different things have different molecular makeups, so they absorb different wavelengths and look different colors. http://bit.ly/lightleafcolor 

Visible light, light our eyes have detectors for, is only a small slice of the ElectroMagnetic Radiation (EMR) spectrum. There’s also a lot of types of light we can’t detect with our eyes, including higher-energy, shorter wavelength, ultraviolet (UV) light and lower-energy, longer-wavelength infrared (IR) light. Molecules can still sometimes absorb these, but we need special detectors to tell that this invisible light has been absorbed. 

Your blood “picks up” oxygen in your lungs – that oxygen binds to the hemoglobin protein in the blood to form a complex called oxyhemoglobin. Importantly for our body, this binding isn’t permanent (that would defeat the purpose of carrying the oxygen from the lungs where it’s aplenty to the toe where it’s harder to go). Importantly for our pulse ox, in the bound state, hemoglobin absorbs a slightly different wavelength of light than when there’s no oxygen bound. So, by shining light through and seeing how much makes it through, the pulse ox can calculate what proportion of the hemoglobin molecules are bound to oxygen. 

You might have heard that venous blood (the blood returning to the heart) is blue because it doesn’t have oxygen. This is a myth. There are a couple reasons your veins look bluish – one is that blue light doesn’t penetrate skin as far as red light. So the blue light is more easily reflected back at us than red light. And the second reason is that, while venous blood may not be blue, it *is* less red – it’s darker than the oxygen-rich blood in the arteries leaving your heart to deliver oxygen to the body. And this is because it’s that oxyhemoglobin state that’s bright red. You still have a lot of this in your veins, just not *as much*. Here’s a cool explanation: https://bit.ly/3mWM8GX 

So you’re not looking for red vs. blue here, but you can use 2 different wavelengths – one the unbound hemoglobin likes and one the oxyhemoglobin likes. And then you look at the ratio of the 2 to calculate the oxygen saturation. You can’t just shine a single wavelength – that wouldn’t tell you much because the absorption will also depend on things like how big your finger is, how thick your skin is, etc. 

So you have 2 lights:

  1. a red light (wavelength 660 nm), which gets absorbed by the unoxygenated form (since it gets absorbed, it gets “hidden” and it does NOT look red. The oxygenated form does NOT absorb this wavelength so it DOES look red). 
  2. an infrared light (wavelength 940 nm) – the oxyhemoglobin likes this one, and absorbs it better than the deoxyhemoglobin does. 

The lights switch on-off-on-off-on-off. One goes on, then goes off and the other goes on. Then they both go off. 

1

2

12

And they do this over and over, ~30 times per second. They’re not the only thing “cycling” – the amount of arterial blood is changing with each pulse of the heart. And this offers a cool opportunity – by subtracting the minimum transmitted (not absorbed) light from the maximum, you can account for other tissues that might be interfering but aren’t fluctuating like that (it lets you subtract out your “background noise”).

A pulse ox can tell you if the oxygen saturation is too low, but it doesn’t tell you about how the body is responding. To get some hints, doctors can use an Arterial Blood Gas (ABG). An ABG doesn’t just measure the oxygen (the PaO₂, which in case you’re wondering is usually 75-100 mmHg). It also measures the CO₂ and the pH to report on acid-base balance. pH is a measure of how many free protons (H⁺) there are in a solution. But it’s an inverse log, so the higher the concentration of protons (more acidic), the lower the pH and vice versa – the lower the concentration of protons (less acidic/more basic) the higher the pH. The log part just makes it so that the numbers we’re dealing with are smaller.

You might be wondering why you need to measure both CO₂ and pH, since I just told you that CO₂ can dissolve and decrease the pH. So let’s get into a little more detail. When CO₂ is made, often as a byproduct of carb break-down, it doesn’t just wait around doing nothing – a protein enzyme (reaction mediator/speed-upper) called carbonic anhydrase takes it, and water (another byproduct of respiration) and combines them to make carbonic acid, H₂CO₃. 

sidenote: carbonic anhydrase is a beast! It’s mind-blowing-ly fast, combining CO₂ with water to make carbonic acid – or doing the reverse – 1 MILLION TIMES PER SECOND.  The reaction can happen on its own at a moderate pace, but not fast enough to keep up with the rates our body needs it to in order to, for example, make CO₂ to exhale as HCO₃⁻ races through your lungs. more here: https://www.ncbi.nlm.nih.gov/books/NBK22599/ 

H₂CO₃ is an acid because it can donate a proton, i.e. it can “dissociate” into its “conjugate base” bicarbonate, HCO₃⁻. That’s called a conjugate base because, having lost a proton, it can now act as a base, which is something that then accept a proton. So, the process is reversible 

H₂CO₃ ⇌ H⁺ + HCO₃⁻ 

That protonation/deprotonation doesn’t require any enzymatic help or anything, so how does it determine which direction to go? Concentrate on the concentrations! At the extreme, if you have no H₂CO₃, you can only go left, but if you have a lot of H₂CO₃, the reaction is driven to the right. And what determines how much H₂CO₃ there is? In part, the CO₂ it’s made from! (produced during cellular respiration) 

CO₂ + H₂O ⇌ H₂CO₃ 

So overall you have 

CO₂ + H₂O ⇌ H₂CO₃ ⇌ H⁺ + HCO₃⁻  (acidic conditions that favor oxygen dumping)

So, the more cellular respiration (energy-making), the more CO₂ & H₂O. And the more of those, the more H₂CO₃ you make. And the more H₂CO₃ there is, the more protons you produce. And the more protons there are, the more acidic the solution (lower the pH). 

Thing is, your body  doesn’t want the pH to go too low (a condition called acidosis, so it has great “buffering” capacity (that equation’s reversible remember, so if you have a lot of bicarbonate ions (HCO₃⁻ ), the reaction will get get driven back to the left, sopping up excess protons and raising pH. more on pH buffers: http://bit.ly/phbuffers 

Your kidneys normally excrete the sodium bicarbonate (HCO₃⁻) that’s made during the acid-making, in your pee, but the kidneys can also conserve, thus boosting this base to neutralize the acid. But if they keep “too much,” the pH can get too high, a condition called alkalemia (normal pH is ~7.4). Typically, acidemia is below 7.35 and alkalemia is above 7.45.

Basically, your respiratory system can control pCO₂ (by adjusting ventilation rates, such as making you breathe faster to get rid of excess CO₂) and your renal system (kidneys) can control HCO₃⁻ levels (and to some extent H⁺ levels by excreting excess acid). So, pH abnormalities can be caused by problems with either or both systems. Ideally, the two can work in concert to keep things normal, and if there’s a problem with one system, the other might be able to partially compensate. 

The pCO₂ (which should be 35-45 mmHg) can be used to calculate what you’d expect the pH to be and then you can compare it to the actual pH to figure out the “base excess” (BE), but it’s usefulness is controversial, so I’m not gonna go into more detail. If you want to learn more about these things: https://www.ncbi.nlm.nih.gov/books/NBK536919/ 

There are exceptions to everything because the human body is full of surprises and tricks up its sleeves, but If the pH is low AND the pCO₂ is high, that points to respiratory problems (although the pH might be normal due to the body compensating appropriately). But if the pH is low and the pCO₂ is also low, or at least not high, that points to the problem being “metabolic” (metabolism referring to molecular making and breaking – so basically some biochemical pathway is acting up, and it’s likely not the cardiopulmonary system’s fault). 

When researching this, I found a helpful mnemonic: ROME (Respiratory Opposite, Metabolic Equal). This says, if the pH and pCO₂ are off in opposite directions (e.g. one’s too high and the other is too low) the problem is likely respiratory and if the pH and pCO₂ are off in the same direction (both too high or both too low) the problem is likely metabolic. 

So what are these metabolic causes? According to StatPearls, https://www.ncbi.nlm.nih.gov/books/NBK536919/ 

  • causes of metabolic acidosis include: “diabetic ketoacidosis, septic shock, renal failure, drug or toxin ingestion, and gastrointestinal or renal HCO3 loss” and
    • we talked about ketoacidosis a bit in our discussion of glycogenic vs ketogenic amino acids. Ketone bodies are a normal way to get energy from fats (not water-soluble) through your blood. When ketone bodies are produced faster than they can be consumed you get ketosis – which is ketone overload. Some ketone bodies have carboxylic acid groups so they mess up the pH of the blood. Diabetics are at risk for diabetic ketoacidosis when their insulin levels are too low – basically their cells aren’t taking in and using sugar, and insulin’s “opposite” hormone-wise, glucagon, goes unchecked, “yelling” at cells to burn fat. So they do –  they turn to breaking down fat and protein leading to high levels of ketones being made. 
  • causes of metabolic alkalosis include “kidney disease, electrolyte imbalances, prolonged vomiting, hypovolemia, diuretic use, and hypokalemia”
    • hypovolemia is low (hypo-) blood volume & hypokalemia is low blood potassium – it might seem weird that potassium would be involved, but when there isn’t enough potassium in the blood, protons get driven into cells, lowering the blood pH through a number of mechanisms. this a really good video explaining the kidney stuff https://youtu.be/5Dpj4UFKwzw 

Here’s a helpful book chapter I found: https://bit.ly/3x4hGzq  

Be careful not to confuse oxygen concentration (aka oxygen content) with oxygen saturation. The total oxygen concentration tells you how much oxygen is in the blood, without taking into account how much hemoglobin is present, so you could potentially have “extra” hemoglobin working at reduced capacity and still have a “normal” oxygen concentration. The oxygen saturation tells you about how “at capacity” the hemoglobin is – so, in that above scenario where you had “extra” hemoglobin working at reduced capacity, that would show up as a lower oxygen saturation. 

But – what if you had low hemoglobin working normally? You wouldn’t see that in the oxygen saturation, but you would see that in the oxygen concentration. 

So both have value, and some blood gas analyzers (the machines that measure the oxygen, carbon dioxide, and pH for the ABG) also measure oxygen saturation directly in a similar manner to the pulse oximeter – but other ABGs report calculated oxygen saturation based on the assumption that hemoglobin content is normal. Oxygen content (concentration) is often expressed in mL O₂/100mL of blood or O₂/L of blood, with the maximum (the carrying capacity) being about 20mL oxygen/100mL blood. But most of that oxygen is bound to hemoglobin. In the arterial blood of a healthy individual, about 96-98% of hemoglobin’s oxygen-binding sites are oxygen-bound. https://bit.ly/2zdmEkh 

I’ve been talking about arterial blood because the situation in your veins, the blood vessels returning all the “used” blood to your heart, is a different story. There, because of reasons including less oxygen (lower partial pressures) and lower pH (greater acidity), a lower proportion of hemoglobin is oxygen-bound. It varies, but is typically about 75% https://bit.ly/2zdmEkh – so there’s still quite a bit which is good if you need to hold your breath or something, or your muscles are working overtime!

as promised, a note on partial pressures: In a gas, the molecules are so far apart that they don’t interact with one another and they don’t take up much space or anything, so different gases act basically the same. The ideal gas law says that, when it comes to pressure, it’s the # of gas particles, not their identity, that matters. Dalton tells us we that if we have a mixture of gases, we can add together the pressure that would be generated by each gas separately and that would tell us the total pressure. And we can go the other way too – if we know what proportion of a mixture is a certain gas we can calculate what the pressure would be if we removed all of the other gases in there – and we call that the partial pressure.⠀http://bit.ly/solutionconcentrations 

Law of Partial Pressures: Ptotal = P1 + P2 + P3 ….. ⠀

For example, if we had a gas mixture that was 1/2 A & 1/2 B, 1/2 of the gas molecules would be A and half would be B. And 1/2 of the total pressure would come from A & half from B – each would have a partial pressure of 1/2 the total pressure (e.g. if the total pressure was 1 atm, the partial pressure of each would be 0.5 atm). What if you added an equal amount of C? Now, proportion-wise, you’d have 1/3 A, 1/3 B, and 1/3 C. But the partial pressures would each still be 0.5 atm (but your total pressure would be 1.5 atm). Hope that was helpful and didn’t just confuse you more!

Standard warning – I am NOT a doctor – not even a nerd one (PhD) YET. So hopefully I got it all right and your real doctors know this technical stuff better than I do! 

more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0

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