Your body has a lot of Red Blood Cells (RBCs) (aka erythrocytes) (~5 billion per mL). And they’re produced at staggering rates. But they have limited lifetimes – only ~120 days in healthy people. So each day your body turns over about 5 million of them *PER SECOND!* And this turnover is responsible for the color of your bruises, your pee, and your poop. How cool is that?! My parents didn’t think it was that cool when I excitedly told them, but I find it fascinating that one molecule, heme, which we normally think about as that thing that carries oxygen in our blood, is ultimately responsible for not just the color of our blood but also the color of all those other things. It’s also responsible for the yellowness of the eyes and skin referred to as jaundice, which can occur if there’s excess turnover (hemolytic anemia) and/or problems with the liver (which is responsible for some of the breakdown).
I think it’s cool, so today I want to tell you about heme catabolism and hyperbilrubinemia. If those terms don’t sound interesting, I get into how we can use light to treat neonatal jaundice… And I also get into some less-cool but really important yet overlooked things about how a condition called G6PD deficiency, which affects over 1 in 10 Black males puts people, especially, newborns at higher risk, yet isn’t regularly tested for.
A quick breakdown of the breakdown and then the details.
Heme is the non-protein part of hemoglobin, the protein that carries oxygen throughout your blood. Heme consists of a ring-y thing called protoporphyrin bound to iron. In order to recycle heme, the iron (and CO) get removed to give you biliverdin, which gets turned into bilirubin. This is yellowy and if it builds up it can cause that yellowness called jaundice (the jaundice itself isn’t the big deal, but the bilirubin is toxic, and jaundice is a warning sign that things are wrong). To prevent bilirubin’s accumulation, it’s sent to the liver for further processing. Problem is, bilirubin isn’t water-soluble so it can’t just travel through the blood unattended. Instead, it hitches a ride on an abundant blood protein called albumin. After the drop-off, the liver converts that “indirect bilirubin” into conjugated bilirubin (aka “direct bilirubin”) by adding on (conjugating) a couple of glucuronate sugars. These sugars make it soluble without the need for a chaperone, so the conjugated bilirubin gets sent through the bile duct into the intestines. There, bacteria act on it and convert it into colorless urobilinogens, some of which get absorbed & travel to the kidneys while others remain in the intestines. The urobilinogens then oxidize to colored products including uribilin (pee color) and sercobilin (poop color).
now for the details…
One of the reasons why RBCs have limited half-lives (the time it takes for half to get degraded) is that RBCs have to deal with a lot of oxygen – I mean, carrying it’s kinda their job! Each cell in your body needs oxygen in order to produce energy and function, so RBCs contain a protein called hemoglobin that transports oxygen throughout the body, picking it up in the lungs and dropping it off where needed.
Sounds great, right? Problem is, where there’s oxygen, there’s the potential for Reactive Oxygen Species (ROS) to form. And where there’s ROS, there is the potential for “oxidative damage.”
Quick terminology note: Atoms join together to form molecules by sharing subatomic particles called electrons – each atom “owns” a certain number of them but can share, steal, and/or donate to get to their ideal number. “Oxidation” is a term we use to describe the loss of electrons, and it goes hand in hand with its counterpart, “reduction” which is the gain of electrons. The giver is called an oxidant (or oxidative agent) and the taker is the reductant (or reducing agent). You can remember this with the mnemonic OIL RIG: Oxidation is Loss, Reduction is Gain.
With ROS, you basically have these super energetic oxygen atoms that act as oxidants (things that can oxidize other things, getting reduced in the process). These oxygens desperately want an electron and they’re willing to attack proteins, lipids (fats & oils like those making up cell membranes), DNA, etc. in order to get it.
This, as you might imagine, isn’t very good for those other molecules… In fact, if enough ROS damage builds up, the very membranes of the RBCs can “fall apart” and the cell’s content can spill out (a kind of intravascular hemolysis). To prevent this from happening, your body tries to keep ROS under control and remove old RBCs under a more orderly process called extravascular hemolysis, which doesn’t involve all that potentially dangerous cell stuff from just being dumped out into your blood stream. In this controlled pathway, RBCs get swallowed up (phagocytosed) by macrophage cells that recycle their contents and handle the hazardous waste carefully. This takes place primarily in the spleen and liver, but also in bone marrow, as well as other places to lesser extent. In addition to old(senescent) RBCs, defective immature RBCs of the bone marrow also go through this turnover process.
The biggest cellular hazmat concern with regards to RBCs is the hemoglobin – that protein that holds onto oxygen. The problem isn’t the protein part (the globin), instead, it’s the heme. Heme is this ring-y thing called protoporphyrin that holds an iron atom in its center. If you want to learn more about it, I take about its synthesis (anabolism) yesterday: http://bit.ly/leadheme But today I want to focus on its breakdown (catabolism).
Like I said, the globin part is no problem – like all proteins, it can just get chopped up by the proteasome so its building blocks (amino acids) can be recycled. But the heme presents a problem for 2 reasons. First is the iron. As I will get back to later, that iron has the potential to generate ROS, so as soon as it gets released another protein, such as ferritin, grabs it up so it doesn’t hang out free in the cell. You release the iron (and a molecule of carbon monoxide (CO) when you oxidize the heme. That’s done with the help of an enzyme (reaction mediator) called heme oxidase which oxidizes heme and breaks open the ring to give you biliverdin. This biliverdin is green and is responsible for that color in bruising and it also makes bird poop green in case you were wondering.
Our poop isn’t green because we convert that biliverdin to bilirubin. This is done with the help of biliverdin reductase. Our problem now is that bilirubin isn’t water soluble. Instead it’s lipophilic (it loves lipids). So if you tried to get it to travel through the bloodstream by itself it would just try to slip through all the lipidy membranes. So, in order to take it to the liver for the next processing steps, the bilirubin hitches a ride on an abundant blood protein called albumin. note: Albumin serves as a carrier for a lot of molecules, so certain molecules can compete and contribute to jaundice-related problems as we’ll see later.
Bound to albumin, the bilirubin (at this point referred to as unconjugated bilirubin aka indirect bilirubin) travels through the bloodstream to the liver, where hepatocytes (liver cells) take it (but not the albumin) in. And then they use an enzyme called glucuronosyltransferase (GT) to add (aka conjugate) 2 (sometimes just one) glucuronate sugars to it. note: This enzyme transfers those sugars in a pass off from a phosphate-primed version of those sugars, uridine-diphosphoglucuronate, so you might see this written as UDP-GT.
The main benefit of this conjugation is that now you have a nice, hydrophilic (water-loved) molecule that makes it water-soluble, so it can go with the bile into the intestines un-babysat. And another benefit of that hydrophilicity is that it, along with the added bulk, prevents the bilirubin from getting passively diffused through the intestinal membranes (since those membranes are all lipid-y, the conjugated bilirubin can’t just slip through anymore.
So, now our (conjugated, aka “direct”) bilirubin is stuck in the intestines. And, you know what else in our our intestines? Bacteria. Lots and lots of bacteria. Don’t freak out, most of them are really nice and friendly and do things like break down compounds we can’t (though they sometimes release gas in the process…). Anyways, there are bacteria that will take that (conjugated) bilirubin, eat off the sugars (un-conjugate it) and reduce it to a variety of colorless molecules referred to as urobilinogens.
Some of those urobilinogens get absorbed by the intestines into the bloodstream and end up going through our kidneys and getting peed out. Others stay in the intestines. Either way, they can get oxidized to form colored products. A lot of the urobilinogen that gets excreted in the feces is oxidized to form sercobilin, which makes poop brown. Some of the urobilinogen that goes the kidney route gets oxidized to urobilin (which is that characteristic yellow).
Note that what we’re excreting is the breakdown products of bilirubin, not bilirubin itself. Under certain conditions, bilirubin can get into the urine, a condition known as bilirubinuria. This bilirubin is conjugated (aka direct). Unconjugated (aka indirect) bilirubin isn’t water-soluble so can’t get peed out. This might seem like some weird arcane point to bring up, but it actually comes up frequently in medicine. And can help piece together medical mysteries – for example, if someone comes in with jaundice and doctors find bilirubin in the urine, that indicates a “direct” jaundice. Which might indicate a post-liver problem like a blocked duct.
Conjugated (direct) bilirubin can’t get into the pee, but it can build up in the bloodstream, and so can the unconjugated (indirect). The elevation of (either kind of) bilirubin levels in the blood is called hyperbilirubinemia.
So, even without looking to pee, doctors can get an idea of what’s going on by doing a blood test for bilirubin, which typically measures total & direct bilirubin (from which you can calculate the indirect). So, what’s with the “direct” and “indirect” terminology? It might seem kinda backwards because you’d think that direct would be the plain ole bilirubin – no conjugation. BUT it’s not.
- conjugated bilirubin = direct bilirubin
- unconjugated bilirubin = indirect bilirubin
And the reason for this is that the terminology has to do with the test for it. The test works by using a dye that will react with dissolved bilirubin. So, with the conjugated (and thus soluble) form, you can just add the dye and see it directly. But, the unconjugated form is NOT water-soluble, so you have to add methanol to get it to dissolve and thus react with the dye. That gives you the total, and then you subtract the direct from the total to get the unconjugated. Thus, “indirect”
Normally, doctors will measure both the total and the direct bilirubin because they can give hints as to where a problem might lie.
If the ratio of indirect to direct is elevated (that is, you have more unconjugated bilirubin than you should) this suggests that there’s an elevated turnover of RBCs that the liver can’t keep up with. This can happen with hemolytic anemia, such as that which occurs as a complication of G6PD deficiency as I will get into in a minute.
If you have the opposite scenario – If the ratio of direct to indirect is elevated (that is, you have more conjugated bilirubin than you should) that indicates that the problem is after the liver step (the liver’s done its job already) and suggests there might be a blockage of the biliary duct. You often also get brownish urine because you get excess conjugated bilirubin in the urine. (the unconjugated stuff is attached to albumin so it can’t get filtered into the urine and thus you can’t pee it out).
And what if the ratio is unchanged? You just have more? In this “mixed” situation where you have a lot of both, the problem is likely something with the liver itself.
As for how the test works, it involves “diazo reagents.” “Diazo” means that these molecules have an end with 2 N’s linked through a triple bond. These groups are highly reactive under certain conditions and can attack the central carbon bridge of bilirubin, splitting it and producing 2 azodipyrroles. (a pyrrole is one of those ring things within bilirubin and now instead of the 4 (tetra) you have in bilirubin (a tetrapyrrole) you have 2 (di)). This is called the “Van den Bergh reaction” Those azobilirubins are purply, and you can measure purpleness using a spectrophotometer, which measures light absorption at chosen wavelengths.
So you measure the direct (what you get without adding methanol) and then the total (what you get after add methanol) and then you subtract direct from total to get indirect. Hence the kinda weird terminology.
In addition to revealing underlying problems, it’s important to catch hyperbilirubinemia because bilirubin is toxic to tissues. And, to make things worse, if unattended (not bound to albumin) it can cross the blood-brain barrier and cause a life-threatening “brain jaundice” called kernicterus.
Newborns are at especially high risk of this for a couple reasons. One is that they have a lot of RBCs to get rid of. In utero they express a different form of the globin part of hemoglobin – a different protein chain. So, they make a different form of hemoglobin, called fetal hemoglobin. It has a higher affinity for oxygen, which helps them take some out of the mother’s blood even though that blood isn’t very oxygen rich. Once they’re out in the real world, though, they don’t need that extra affinity, so they start making adult hemoglobin and have to ditch all that fetal stuff.
So they have a lot of RBCs to turn over and, at the same time, especially with preterm infants, the liver hasn’t started making enough glucuronosyltransferase (GT) yet. In utero, the mom’s liver disposes of the fetus’ heme. But after birth, the baby’s all on its own for that and it has to kick into gear and make its own liver enzymes. And it might not be able to keep up well with the demand.
Further complicating matters, breast milk is great for a lot of things, but it contains long fatty acid chains that can compete with the bilirubin for albumin binding sites. Which can leave more free bilirubin in the blood (the kind that can get into the brain).
Thankfully, if caught early, there are effective, non-invasive treatments for neonatal jaundice. The main one is phototherapy. The reason heme & some of its breakdown products have different colors has to do with their ability to absorb certain wavelengths of light. Different wavelengths of light correspond to different colors. White light has them all – the full rainbow – and if something absorbs a certain color it “removes it” from the rainbow of white light so that thing looks a different color. What wavelengths a molecule does or doesn’t absorb has to do with the molecular makeup and if you want to know more, check out this post: http://bit.ly/lightleafcolor
Light is “just” little packets of energy traveling in waves (with different wavelengths corresponding to different energies). So when you’re talking about absorbing light you’re really talking about absorbing energy. And when molecules absorb energy, they can sometimes shift around their bonds a bit to give you “isomers.” And those isomers can behave differently. So when you shine blue-green wavelength light (460-490 nm) on unconjugated bilirubin, which is not water soluble, it converts into isomers that *are* water-soluble and thus can be peed out.
Having to recycle RBCs to get rid of fetal hemoglobin is only a problem you have as a neonate, but there are other reasons you might have to turn these cells over at high rates. And one of these reasons is if you have poorly-controlled ROS – those Reactive Oxidative Species we want to avoid because they can damage the cells and lead to hemolysis.
Your RBCs do their best to control ROS in a couple ways. One is by preventing its formation through the “babysitting” of ROS generators. For example, metal ions (charged forms of metal atoms) like iron have the potential to generate ROS, so your body tries to make sure all metal ions are held by proteins in their least dangerous form. For example, that iron that gets released when you break open the heme doesn’t just float around. Instead, it gets bound by proteins – including one called ferritin which is a storage protein for iron. Ferritin makes sure it stays in its oxidized, ferric, form, Fe³⁺, and not its reduced, ferrous, form, Fe²⁺. This is important because, in something called the Fenton reaction, Fe²⁺ (but *not* Fe³⁺) can react with hydrogen peroxide to generate a hydroxy radical, which is a super ROS-y ROS that has an unpaired electron and an oxygen that’s desperate to pair it.
But, even with careful measures like that, ROS is unavoidable, especially when you have a lot of it around. So RBCs (and all cells) need molecules to intercept it, sopping up the electrons, thus acting as antioxidants, before the ROS attacks valuable things. The main antioxidant our cells rely on is this tripeptide called glutathione. It can go between reduced states in which reduced glutathione hang out separately (2 GSH) & oxidized states, where 2 glutathione link up through a disulfide bond (GSSG). ROS can do the oxidizing, but then you need a way to regenerate the reduced form. This regeneration requires a molecule called NADPH, which reduces the glutathione, becoming oxidized in the process. So, now you have NADP⁺ and you need to regenerate that! The way that it’s regenerated is through the Pentose Phosphate Pathway. More on that here: https://bit.ly/pentosephosphatepathway
But for now just now that it’s a sort of “alternative” pathway to glycolysis that’s used to breakdown glucose (blood sugar) for NADPH, RNA & DNA precursors, etc. (glycolysis is the route you take mainly when you want energy). The first step of the pathway is carried out by an enzyme called Glucose 6 Phosphate Dehydrogenase (G6PD). It takes G6P (glucose that’s been phosphorylated to trap it in the cell) & oxidizes it, reducing NADP⁺ back to NADPH.
This pathway is the main route for NADPH in all cells, but it’s the *only* route for it in RBCs. This is because those other routes involve processes that take place in mitochondria and mature RBCs don’t have mitochondria. So they’re dependent on the PPP. And thus they’re dependent on G6PD.
Now, for most people, this isn’t an issue. But, for over 400 million people around the world it can be. G6PD deficiency is one of, if not *the* most common genetic enzymatic deficiency. It involves mutations in the G6PD gene which decrease the production and/or functioning. This gene is located on the X chromosome, so it more frequently affects biological males, although biological females can be affected even if they are heterozygous (have 1 un-mutated copy and one mutated copy).
A lot of people go their whole lives without even knowing that they have the deficiency, because, although reduced, there’s usually enough G6PD to keep up. But, all it takes is 1 triggering, ROS-generating, event, and their ROS builds up to super-dangerous levels. And there’s not enough G6PD to generate enough NADPH to generate enough reduced glutathione to intercept the ROS. So the ROS attacks the proteins and cell membrane of the RBC, leading to its destruction (hemolysis). This reduces the amount of RBCs available to transport oxygen throughout the body, and thus is called hemolytic anemia. In addition to oxygen transport problems, it can cause hyperbilirubinemia because it’s more RBC turnover than the liver can handle – it can’t conjugate all the bilirubin that gets sent to it, so the unconjugated bilirubin, instead of getting conjugated, broken down, and pooped out, gets stuck in the blood serum, hanging out with albumin. This is thus a form of unconjugated hyperbilirubinemia.
People with G6PD deficiency are at risk for acute hemolytic anemia their entire life any time they encounter a trigger – these triggers include certain foods like fava beans (so this condition is sometimes referred to as “favism”) as well as certain drugs like hydroxychloroquine and sulfa drugs. Hemolytic anemia can also be provoked by infection or even certain pesticides and cosmetics.
Those risks remain their entire life, but they’re especially at risk as newborns because they have this ROS problem on top of the usual neonatal problems. People with G6PD deficiency are 2X as likely to suffer neonatal jaundice, and are sometimes at higher risk for kernicterus and poor outcomes. https://bit.ly/30r7OQM G6PD deficiency is often discovered when it’s tested for in neonates with jaundice whose parents are of African, Asian, Mediterranean, or Middle-Eastern descent. But *only* doing this targeted testing can miss a lot of people.
G6PD deficiency is *usually* not a problem. But it can be. And, many of the later in life hemolytic anemia episodes could be prevented if people knew to avoid those triggers. But most people with G6PD don’t even know they have it. Since it’s not *usually* a problem, and in my opinion, because in the US it mainly affects people of color, it is not regularly screened for as part of newborn screening (it’s only required in one state & Washington D.C.). There’s barely even any research done on it. I feel this is one more example of why it’s important that we get more people of color in positions of power within medical and scientific organizations. And if you want to learn more about it: https://bit.ly/g6pddeficiency
Note: I focused on RBCs because they’re the source of about 80% of the 4mg/kg body weight of bilirubin produced daily. https://bit.ly/2CS0ZQ8 But there are other sources of bilirubin because molecules other than hemoglobin use heme too, including myoglobin (the protein that stores oxygen in our muscles & a lot of enzymes involved in redox reactions (e.g. cytochromes, peroxidase).