This chapter tries to address Section N2(i) from the 2017 CICM Primary Syllabus, which asks the exam candidates to “describe the interpretation of laboratory assessment of liver function (Albumin, Glucose, Bilirubin, Coagulation profile, Ammonia).” Specifically, the discussion focuses on bilirubin, because it has appeared in the CICM First Part exam as Question 6(p2) from the second paper of 2008. The exam candidates were asked to "describe the physiology of bilirubin production, metabolism and clearance" for 70% of the marks. That "production, metabolism and clearance" line reads a bit pharmacological, as if the intention was for us to regard bilirubin as a drug, albeit one which has its origins in the body. This chapter is structured accordingly, where possible using the ADME framework to discuss the comings and goings of bilirubin.
- Production of bilirubin
- Bilirubin is a tetrapyrrole product of haem metabolism
- It is produced by a series of enzymatic steps which mainly take place in hepatocytes and reticuloendothelial macrophages
- Haem oxygenase converts haem into CO, iron and biliverdin
- Biliverdin reductase converts biliverdin into bilirubin
- Distribution of bilirubin
- Unconjugated bilirubin is poorly water soluble
- It circulates mainly bound to albumin (92% protein-bound)
- It can displace highly protein-bound drugs such as warfarin and frusemide from their albumin binding sites
- Metabolism of bilirubin
- Unconjugated bilirubin enters the hepatocytes by a combination of active and passive processes
- In hepatocytes, bilirubin is conjugated with glucouronide by the enzyme UDP-GT which
- Conjugated bilirubin is more water soluble, and 5-10 times less protein bound
- Clearance of bilirubin
- Conjugated bilirubin is pumped against its concentration gradient by the efflux pump MRP2, expressed on the apical (canalicular) surface of hepatocytes.
- Biliary concentration of conjugated bilirubin is 1000-2000 μmol/L
- In the intestine, conjugated bilirubin is converted into urobilinogen by intestinal bacterial enzymes, and then reduced into stercobilinogen.
- Urobilinogen and stercobilinogen then oxidise into urobilin or stercobilin, which give the urine and faeces their characteristic colours.
- Interpretation of bilirubinuria and bilirubin ratio
- Only conjugated bilirubin can be eliminated in the urine
- Thus, hyperbilirubinemia with bilirubin-stained urine is likely to be due to an abnormally large amount of conjugated bilirubin in the blood
- An abnormally large amount of conjugated bilirubin is usually due to obstructive jaundice
- A large amount of unconjugated bilirubin is likely to be due to excessive haem breakdown (eg. haemolysis) or parenchymal liver disease rather than biliary obstruction
Fevery (2008) and Ruiz et al (2021) are probably the best references for pre-exam preparation, and anything they do not cover would probably not be examinable. This is not a question-rich topic, and the CICM trainee should redirect their last-minute revision to something with higher mark density. Still, if you're just completely nuts for bile pigments, you can indulge your appalling fetish in Gray's "Bile Pigments" from the 1963 edition of Comprehensive Biochemistry.
Bilirubin is usually described in some vague alchemist-like statements or frankly pejorative terms, such as a "toxic metabolite of haem", "a dark orange-yellow pigment", or "a toxic hydrophobic waste product". To be slightly more precise, it is a linear tetrapyrrole that belongs to the family of biladienes, i.e. it is a molecule made of four pyrrole rings where some of the pyrroles are joined to each other by methene bridges, where the carbon atoms are connected by double bonds, C=C, instead of normal methylene C-C bridges (for the record there are also bilatrienes where the bridges are C≡C). This is one of those things probably best explained through pictures, and there's probably some instructional value to displaying them below, even if it is to demonstrate for a future generation of hobbyist science authors how not to present this material.
To add a veneer of legitimacy to this shoddy operation, these structures were stolen directly from PubChem, so you know they are real (even though molecular structures are probably entirely pointless, as no CICM exam candidate will ever be asked to draw or recognise them). Of the biologically interesting biladienes and bilanes, bilirubin and its family of haem metabolites are the most important to human metabolism, but there are many others in nature - for example, chlorophyll is also porphyrin (a four-pyrrole ring like haem, except instead of iron the central metal atom is magnesium) and it produces phyllobilin biladienes as the products of its catabolism. In fact a lot of these substances are pigments with light sensitive properties, and even human bilirubin - as some of you may recall from those two stressful weeks spent in the neonatal ICU - can react with UV light. But before this section dives into a swamp of algae biology, it is probably better to refocus.
Bilirubin originates from the metabolism of haem. Under normal circumstances, nobody ever ingests any meaningful quantity of bilirubin, nor has it infused into themselves intentionally, so basically all the bilirubin in the body is homegrown. There is really not enough bilirubin in anybody's diet to make much of a difference to the total daily bilirubin turnover (250-350mg, or about 5000 μmol per day), even if one's diet consists entirely of animal livers. Still, it must be said that bilirubin (especially unconjugated bilirubin) is well absorbed in the intestine, which means that theoretically one would be able to consume an oral dose of it with reasonably good bioavailability. This is not something the reader needs to seriously contemplate, as most bilirubin in the normal human comes from haem liberated in the process of red cell phagocytosis (~85%) as well as cytochrome and myoglobin catabolism (~15%), of which some may be dietary.
The structure and function of haem are discussed elsewhere, as it was felt that this sort of thing belonged wherever the carriage of oxygen in the blood was being explained. Here, it will suffice to say that haem is a complex of protoporphyrin IX and iron, i.e. a thing where four pyrrole molecules form a ring surrounding an iron atom and then bully it into flip-flopping between oxidation states (ferric, Fe3+, and ferrous, Fe2+).
This makes haem both a desirable source of iron and a dangerously reactive redox agent. In short, you need to clean up your haem to reclaim all that iron and to protect yourself from oxidative damage. The mammal way of dealing with it is through a series of steps that converts it into progressively less and less toxic intermediaries, of which bilirubin is only the most diagnostically interesting one.
The origin of the haem is mainly from haemoglobin, which is retrieved from senescent red cells by the macrophages of the reticuloendothelial system. There is a nonzero amount of circulating free haemoglobin and free haem, but these are trivial quantities in comparison to what is engulfed each day by the macrophages. An additional source of haem is a constant turnover of haem-containing enzymes (type b cytochrome enzymes, myoglobin, etc) which also contributes to the liberation of some haem - typically textbooks tend to put the ratio as 80:20, where 20% of haem originates from these non-red-cell sources. Additionally, the digestive tract of carnivores contributes a variable amount where haem from animal tissue (mainly myoglobin, unless) is absorbed as whole protoporphyrin complexes and degraded by the microsomal enzymes of enterocytes.
At this point the attentive reader may scoff at the inefficiency of absorbing perfectly good bovine haem, degrading it into iron and various tetrapyrroles, juggling them for a bit, and then reconstituting them into haem again. Surely it would be more efficient to just export raw haem into the bloodstream, for cells to do whatever they please with? Because how toxic could it possibly be?
Reader, the answer is "very". Very toxic. Smith & McCulloh (2017) and Chiabrando et al (2014) discuss this in elaborate detail. To restate the main points of their article, the horrors of intravascular haem excess are well represented by the clinical course of people who experience massive haemolysis, for example those with severe malaria or those with sickle cell disease. The authors report free haem levels as high as 3g/L. The multi-system damage it does is partly due to the direct oxidation of lipids and proteins, partly due to the sensitisation of vascular endothelium to oxidative stress, and partly due to its tendency to activate neutrophils and macrophages (Smith & McCulloh, 2017). It gets into your cell membrane, incorporated itself into the lipid bilayer, and then starts catalysing the formation of lipid peroxide, making the membrane totally permeable and basically killing the cell. The net result is a profoundly inflammatory state with widespread organ damage.
In short, there are sound reasons for why haem must be dismantled for transport and mammals have all sorts of mechanisms to minimise the oxidative stress related to the necessary evil of having to handle this molecule. Among these, one is the pathway of degradation that leads to bilirubin, which starts with haem oxygenase.
It being toxic even if accumulating intracellularly in relatively small concentrations, haem must be converted to something less terrible almost as soon as it becomes available. This is the task performed by haem oxygenase, an enzyme that seems to be one of the fundamental elements of life, like Na+/K+ ATPase and DNA (for example, it is shared by both animals and plants). Haem oxygenase takes haem and cleaves one of the methene bridges, unrolling the ring molecule, liberating the iron ion, and producing a carbon monoxide molecule in the process (one of the few human biological reactions that produces carbon monoxide).
This reaction is the rate-limiting step in the metabolism of haem, and theoretically could take place in any human tissues, as haem oxygenase is a truly ubiquitous microsomal enzyme, found in the endoplasmic reticulum of almost every cell. Practically speaking, its activity is greatest in the tissues which see the greatest amount of haem, i.e. the liver, spleen, intestine and bone marrow. The cells which express the most of this enzyme depend on where the haem load is greatest, as haem oxygenase is induceable - for example, where free haemoglobin release from haemolysis is the main reason for the haem excess, the kidneys become the most important site of expression. Under normal conditions, enterocytes take care of dietary haem, and reticuloendothelial cells such as Kupffer cells and splenic reticuloendothelial macrophages are responsible for degrading the haem that comes from red cells (Pimstone et al, 1971).
The linear tetrapyrrole product of haem oxygenase is biliverdin, which is a downright pleasant chemical compared to haem. This substance is a green pigment that ends up released into the cytosol of whatever cells happen to be metabolising haem. It is a relatively benign antioxidant, and many species of mammals actually stop here and excrete biliverdin as their main biliary pigment. It's beautiful, as these lovely images by Katie Pulsipher demonstrate:
It is in fact a common source of vivid blue or green colouration in the animal kingdom, and some animals are naturally saturated with this pigment (for example the Indonesian skink Prasinohaema prehensicauda, who appear to have put all their attribute points into biliverdin). In humans the plasma biliverdin levels are virtually zero because it hardly has time to make its way out of the cells, as cytosolic biliverdin reductase catalyses the conversion of biliverdin into unconjugated bilirubin.
Even though it might sound as if the cytosolic conversion of biliverdin into bilirubin is a completely cosmetic step, there does appear to be some advantages to doing this. Apart from being green on the inside, biliverdin reductase knockout mice are more susceptible to oxidative stress because bilirubin happens to be a better antioxidant than biliverdin. This idea is supported by the finding that high-normal bilirubin levels are associated with a lower risk of atherosclerotic cardiovascular disease, and the observation that infants use bilirubin as a dominant antioxidant mechanism (before their other antioxidant systems mature). The antioxidant effect is apparently related to the ability of bilirubin to react with reactive oxygen species and to turn back into biliverdin, thereby acting as a scavenger of superoxide. Not everybody has been able to demonstrate that this reverse conversion is responsible for the antioxidant activity - for example, the experiments by Jansen & Daiber (2012) did not detect this reaction - which means the CICM trainee should probably not mention it in any exam answers. It would probably just be safer to report that bilirubin is an antioxidant. Biliverdin can also act as an antioxidant, but is about three times weaker, which means there is still some merit in converting it to bilirubin.
Birubin which is produced by the reduction of biliverdin is an orange-brown pigment which differs from biliverdin only by one hydrogen atom and a double bond. To be perfectly specific, the reactions described above produce bilirubin IX-α, where the Roman numerals and Greek letters refer to the positions of the methyl and vinyl groups according to the accepted nomenclature of tetrapyrroles. The reader is invited to scroll past this explanatory image from Defreese et al (1984), left here in honour of the fine tradition of torturing exam candidates with irrelevant molecular structures.
As you can see, to make bilirubin, the central methylene bridge of biliverdin is reduced by biliverdin reductase. The resulting bilirubin IX-α is often referred to as "unconjugated" bilirubin, as the next step in its metabolism is typically conjugation.
Unconjugated bilirubin has some major structural defects. For one, it is a an inherently unstable molecule. As already mentioned above, it can be easily oxidised back into biliverdin, or into some disorganised pile of dipyrroles. It can also undergo isomerisation under the effects of electromagnetic radiation, for example being turned into a series of polar isomers under the effect of UV light. Even in the absence of light it undergoes slow isomerisation, and even solid pure bilirubin in refrigerated storage (4°C) has a lifespan of only about six months before a substantial fraction of it has degraded, becoming unusable for laboratory purposes.
Unconjugated bilirubin has extremely poor water solubility, with a pKa of somewhere in the range of 6.5-6.8 (Lightner et al, 1996). At physiological pH, according to Fevery (2008), about 83% of bilirubin is present in a non-ionised form. You wouldn't expect that from looking at all the polar groups on the bilirubin molecule, but no - annoyingly, it forms internal hydrogen bonds between all of its own polar groups. Here's a diagram from Wang et al (2006) to demonstrate:
Anyway: not water soluble. And for this reason it circulates mainly bound to albumin (92% albumin-bound, if one needed to quote a precise number). It is generally said that each albumin molecule binds 2 bilirubin molecules. As albumin binding sites are finite, bilirubin molecules are in competition with other substances, for example drugs that are highly protein-bound. Maruyama et al (1984) list a whole mass of 1980s drugs that fit this description, many of which are still in use (eg. warfarin, frusemide, aspirin, ibuprofen). This protein binding has two aspects of clinical significance:
Owing to these biding phenomena, under normal conditions (ie. where the level is relatively normal) bilirubin is basically confined to the circulating blood volume, with a small amount leaking into the extracellular fluid (where albumin also hangs out). When Dekker et al (2018) infused some bilirubin into healthy volunteers, they found the volume of distribution was about 9.9L, or about 0.14L/kg.
So: by this stage in the metabolic pathway, you've got some unconjugated bilirubin circulating around bound to albumin. The next stage is uptake into hepatocytes, conjugation and elimination. Interestingly not all of these steps are well understood, and therefore they make a small target for the CICM examiners, making it unlikely that anything more than "uptake and conjugation" would be expected from a written answer.
Uptake of the albumin-bound bilirubin into hepatocytes seems to be some combination of active and passive processes, but nobody seems to know exactly how it is captured and internalised. Most authors try to casually gloss over this step so they can quickly move on to the much better-understood process of conjugation ("thought to be mediated by carrier proteins", quip UpToDate offhandedly). Bilirubin is an organic molecule and an anion, so it is plausible that it is a substrate for all the organic anion transporters on the hepatocyte basal membrane (OAT1, OAT2, OAT3, OATP, and so forth). Kamisako et al discussed this possibility in 2000. It is equally plausible that the free fraction of bilirubin (being highly lipid soluble) would just make its way into the hepatocytes by passive diffusion. In short nobody knows how the uptake process works, and because there are no documented inherited disorders of hepatocyte bilirubin uptake, nobody is looking because the clinical relevance is low.
Having gained access to the hepatocyte, bilirubin becomes an intracellular toxin, as its concentration in the cytosol ends up being quite high. Free bilirubin in such high concentrations (more than 15 µmol/L of free bilirubin, corresponding to about 150 µmol/L of plasma bilirubin) would have some impressive cytotoxic and prooxidant effects. To avoid this, it needs to be safely sequestered away though protein binding. According to Ostorow et al (1994), most of the intracellular bilirubin ends up being bound to glutathione-S-transferase A (ligandin) and to fatty acid-binding proteins (which are a group of ubiquitous intracellular lipid chaperones). Thankfully this does not need to continue for long, as intracellular bilirubin in hepatocytes is quickly nerfed by conjugation to glucuronide.
Within the hepatocyte, bilirubin is conjugated with glucouronide to form a chemically stable water-soluble complex. This reaction is performed by uridine-diphosphoglucuronic glucuronosyltransferase, understandably abbreviated as UDP-GT. Glucouronic acid is the main conjugate, but apparently glucose and xylose are also valid partners for bilirubin. These sugar moieties are attached to the propionic acid side-chains, breaking the intramolecular hydrogen bonds and "unrolling" the bilirubin molecule, making it polar. In fact two sugar moieties can be bound to one bilirubin molecule, i.e. there is the possibility of mono- or di-glucouronides. Wang et al (2006) explain this process in a lot more detail, which is completely unnecessary for the CICM exam candidate. All they would ever be expected to know is some of the properties of conjugated bilirubin:
Hanging a couple of sugar molecules on a bilirubin structure makes it straighter and more polar, which alters its chemical properties significantly. Conjugated bilirubin is highly water-soluble, less protein bound (5-10 times less affinity for albumin) and apparently less cytotoxic than unconjugated bilirubin. When encountered in massive concentrations, it appears to be almost harmless, leading to nothing more dramatic than the greenish discolouration of teeth in neonates. The improved water solubility also confers one other advantageous characteristic, which is the decreased ability to cross cell membranes (thus, less likely to reabsorb once it is excreted in the bile). In this way, bilirubin is converted into something that, once eliminated, stays eliminated.
Conjugated bilirubin has two main routes of egress from the body, those being the bile and the urine.
Conjugated bilirubin is also somewhat unstable and can undergo deconjugation back to normal unconjugated bilirubin, which is technically a third possible mechanism of clearance, even though it does not eliminate any of the actual tetrapyrrole molecules from the body. Its probably not worth mentioning this in any exam answer, as the marking rubric is likely to focus on the first two mechanisms.
Once conjugated bilirubin makes its way into the lumen of the intestine, it undergoes several transformations at the hands of the gut bacteria, first becoming visually unrecognisable as colourless urobilinogen and stercobilinogen, and then oxidising into the familiar brown and yellow pigments stercobilin and urobilin.
The importance of these transformations can be safely downplayed even in this wildly excessive homage, as nobody will ever ask exam questions about it, and if they do, the bulk of the marks will be elsewhere. The CICM trainee needs to become only vaguely aware of the fact that both of these end-products are partially reabsorbed from the intestine and also eliminated in the urine, which gives urine its characteristic colour. The clinical relevance of this is that a complete biliary obstruction will prevent any such pigments from making their way into the faeces, and the patient's stool will be pale, often described as "china clay" by textbook authors. For the reader unfamiliar with pottery, which should be most of you, "china clay" is kaolin, a very white fine clay used to make high quality porcelain (and test the coagulation cascade). It is so named because it had been traditionally mined from a single hill in China (Gaoling or Kao-ling, meaning "High Hill", in Jingdezhen, Jiangxi province). With the extinction of the middle class in Western society, we should perhaps find alternative descriptions for faeces of this colour, such that modern audiences might more easily recognise them (for example KFC mashed potato or drywall patch repair mixture come to mind).
Continuing with the increasingly ill-adapted metaphor of bilirubin as a "drug" of sorts, one finally comes to consider its effects on the body, and these can be vaguely separated into "positive" and "negative". The main positive effects are antioxidant effects at normal concentrations, and the main negative effects are pro-oxidant effects at high concentrations, of which the most clinically relevant manifestation is neurotoxicity.
Antioxidant effects of bilirubin are rather potent, but their mechanism is not completely understood. Some authors claim that it can bind reactive oxygen species and re-convert into biliverdin, and biliverdin is then recycled back into bilirubin by biliverdin reductase. It appears that this is somewhat hypothetical, but the antioxidant effect remains well-recognised, whatever the mechanism. As mentioned above, this effect is sufficiently potent that the mammals have all decided that they will spend the extra time and energy on converting biliverdin into bilirubin (whereas amphibians birds and reptiles generally don't care). This antioxidant effect has multiple protective functions, and without boring the reader with a list of them, instead it might be easier to just redirect to the article by Otero et al (2009).
Pro-oxidant effects of bilirubin are only seen at high concentrations. Brites & Brito (2012) list the molecular mechanisms and their consequences, which in short are:
None of those adverse effects are really important to the clinician, as the patient will not arrive to the emergency department complaining of a swollen Golgi apparatus. The most clinically relevant upshot of all these problems is neurotoxicity, which is worth dwelling on.
To remove some ambiguity, kernicterus is the chronic neurological manifestation of the toxic hyperbilirubinemia of infancy that results in (occasionally, permanent) neurological damage. The term stems from the German kern, meaning "nucleus", and icterus, meaning "jaundice", coined by Georg Schmorl in 1904 as he was looking at yellowed nuclei in the neurons of infants who had succumbed to neonatal jaundice. The clinical features and mechanisms of kernicterus are described by Watchko & Tiribelli (2013), and here it will suffice to mention that they are mainly related to cerebellar and basal ganglia damage (eg. dystonia, choreoathetosis, hearing loss, oculomotor pareses, etc). The most important fact to retain is that these are mainly chronic findings which result from a sustained exposure to high bilirubin during infancy. The word "kernicterus" should therefore probably not be used to describe the neurological changes observed in suddenly jaundiced adults. For them, we have:
This probably occurs very rarely, and is identified even more rarely, as the bilirubin levels required to create this situation are very high, and the sort of liver failure that might cause them tends also cause the concurrent development of hepatic encephalopathy (which means you may never be able to find the bilirubin coma among all the other coma). We are talking bilirubin levels of around 500-600 μmol/L, if case reports like this one are to be believed. The condition is also known better in neonates. Features include seizures, opisthotonus, retrocollis, and abnormalities of tone (apparently it can be increased or decreased).
Question 6(p2) from the second paper of 2008 also asked about "the changes in blood and urine of the products of bilirubin metabolism with intra and post hepatobiliary disease". The pass rate of 60% suggests that many of the of the candidates were able to parse the terms "intra-hepatobiliary" and "post-hepatobiliary" which do not occur in any other literature and which sound made-up. Presumably what they meant by this (judging by all the talk of blood and urine) was something about the difference between the biochemical and clinical presentation of patients with haemolysis or parenchymal liver disease, as opposed to patients with predominantly obstructive biliary causes.
It is often said that the patient with severe liver disease and hyperbilirubinemia will have predominantly unconjugated hyperbilirubinemia, as the sick hepatocytes lose their ability to conjugate it. The resulting bilirubin excess is all unconjugated, and therefore poorly water soluble. From this it follows that one should not expect to see bilirubin discolouration in the urine. The patient will be a fluorescent yellow colour, but their urine will look normal. In this fashion, you should be able to tell the difference between patients with obstructive jaundice (who will have predominantly conjugated hyperbilirubinemia) and patients with haemolysis (who have In reality, there are very few circumstances where the entire mass of bilirubin will be entirely unconjugated, as even a very damaged liver typically retains some ability to metabolise it, albeit slowly. You would really need to have some sort of global and total impairment of bilirubin conjugation, such as Crigler-Najjar syndrome.
Under normal circumstances, the blood should contain very little conjugated bilirubin (no more than 5-20 μmol/L) which means that the majority of it should be unconjugated and albumin bound. The ratio of conjugated and unconjugated bilirubin could therefore be informative: you should be able to discriminate between obstructive jaundice haemolysis, because in obstructive jaundice the bilirubin will be mainly conjugated, whereas in haemolysis it will be mainly unconjugated. Again, this is an excellent theory, but in reality most patients with haemolysis will usually have a relatively normal liver, capable of conjugating bilirubin, and their conjugated/unconjugated ratio will remain relatively stable.
In case the reader finds tables are better at presenting this information, the following tabular format can be offered:
Unconjugated bilirubin (no bilirubinuria)
Conjugated bilirubin (with bilirubinuria)
There are very few scenarios where bilirubin metabolism might be something you need to actively interfere with as a clinician. The only one that comes to mind is bilirubin CNS toxicity where haemolysis or immature conjugation enzymes are increasing the amount of systemic bilirubin to the point where you would need to intervene. Still, present an intensivist with a metabolic pathway, and they will work out twenty ways to mess with it. Here are a few of the more accepted ones:
Haem metabolism can be interrupted by administering tin-mesoporphyrin (SnMP), a haem impostor with a tin atom in the centre instead of iron. It acts as a competitive inhibitor of haem oxygenase and therefore prevents the first rate-limiting step of bilirubin metabolism. One might think that having all this unmetabolised haem around would be harmful, but no - apparently apart from increased photosensitivity this intervention was reasonably well tolerated (though one must add that the inhibition of all haem oxygenase is not the objective here, as you really just want to slow the rate of bilirubin formation to the point where phototherapy can control it). Which brings us to:
The abovementioned hydrogen bonds which form between the polar groups of a bilirubin molecule are susceptible to light. Specifically, under the effects of UV light (wavelengths of around 460-490 nm), these bonds tend to break, forcing a molecular configuration change on the molecule, which makes it more water-soluble (as now all the polar groups are sticking out in all directions). The photoisomers of bilirubin formed in this manner are therefore more water soluble, and this is the basis of the phototherapy used to treat neonatal jaundice and Crigler-Najjar syndrome (Itoh & Onishi, 1985).
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