Physiology of bile and and its metabolism

This chapter tries to address Section N1(iii) from the 2017 CICM Primary Syllabus, which asks the exam candidates to "describe the physiology of bile and its metabolism". Question 5 from the first paper of 2016 was the only question that had ever asked about this comprehensively ("describe the composition, formation and functions of bile"). Judging from the unusually specific comments of the examiners that actually resemble a good model answer, a lot of detail about the molecular mechanisms of bile formation and recirculation was expected. It appears that a quick  revision note would benefit the 69% of candidates who did not pass that question:

To present this information in a logical structure, one might be tempted to track the progress of bile along the path of its formation, from the hepatocyte to the gallbladder; and then to explain the biological and chemical characteristics of the end product. However in doing this one must constantly refer to various molecular and ionic components of bile, potentially losing the reader's attention (as they try to figure out what those components are and what they do). Thus it turned out to make more sense to first discuss the composition of bile, and while doing so to explain where each component comes from and what its role is. Only at the end is everything put together into a sequence of simplified steps. 

For the reader unwilling to waste any minutes on unreliable non-peer-reviewed resources, the work by Nathanson & Boyer (1991), Boyer (1986), Boyer (2013), or Boyer & Soroka (2021) are among the most comprehensive, even in a field where detailed reviews are abundant. To discriminate between them according to the level of detail was difficult because they each contain a potentially suffocating amount of it; but if one can still function at that sort of depth, one might enjoy going even deeper with Bile Acids in Health and Disease (Hofmann et al, 1988).

Contents and properties of bile

It is somewhat difficult to pin down the exact composition of bile, because it varies from site to site in the biliary tree, and between patients, and between species. For example, canalicular bile is thought to be isoosmolar with plasma and usually contains essentially the same ions as plasma, with the exception perhaps of bicarbonate. Then, after it has had some time to stew in the gall bladder, being concentrated 10-fold,  it's obviously a very different substance. What follows is an effort to describe the different components of bile and their chemical properties without getting bogged down in the discussion of their obviously highly variable concentrations. 

Water: bulk fluid volume of bile

Bile is a ridiculously complex substance (like all body fluids, really) about 5% of which is a mélange of organic and inorganic solutes, and the rest is water. This water comes from the plasma, and is mainly attracted into the lumen of the bile duct by the osmotic force of the other 5%. There are supposed to be tight junctions along the entire path of bile synthesis and delivery, which means we should expect of the water will probably be coming through aquaporin channels on the surface of the hepatocytes and cholangiocytes. In reality, the tight junctions are not perfectly impermeable, and plasma water can sometimes leak through, bringing with it some impurities (for example plasma proteins and plasma ions). Still, the bottom line is that biliary volume is mainly derived from water, and the rate of its flow is related mainly to the rate of solute excretion into the bile.

Solute content of bile

Bile contains quite a wide variety of solutes, ranging from dissolved ions to large plasma proteins. To borrow from an excellent table in Boyer (2013), the major solutes in the earlier parts of the biliary tree can be summarised as follows:

Solute	Concentration
Electrolytes	Same as plasma
Bile salts	3-45 mmol/L
Bilirubin	1-2 mmol/L
Cholesterol	2.5-8.0 mmol/L
Plasma proteins	< 1g/L

A lot of other solutes are also present in bile, including immunoglobulins (IgA), heavy metals (copper, manganese, etc), nutrients (glucose), vitamins (25-OH-D, B12, folate, riboflavin), intestinal growth promoter peptides, and various weird little organic molecules like glutamic acid, glutathione, ATP ADP and AMP, glycine, aspartate, plus surely countless others. These latter small organic molecules probably don't need to occupy our attention any more than is required to discuss their role as an osmotic stimulus for the secretion of bile. Their role is played in the distal canaliculus, where bile secretion is not able to rely on the osmotic force produced by the secretion of bile acids.

Bile acids

A "bile acid" is what you call a carboxylic acid which happens to incorporate a sterol ring and which is found in bile. It's basically a steroid body with an acidic aliphatic side chain hanging off it. Sterol acids in general are a group of weak acids (you wouldn't try to dissolve a body with them) and there are a whole range of them documented in chemistry textbooks ("some 150 cholanic acids are mentioned in Elsevier's Encyclopedia of Organic Chemistry, and about 440 monocarboxylic unsubstituted hydroxyl and carbonyl substituted bile acids are cataloged by Sobotka", reported Carey & Small in 1972). Of these multitudes, the vast majority do not occur in nature, or occur only as a middle step in some synthetic pathway or another - for example, humans can only naturally synthesise two. They are the end products of cholesterol metabolism which is performed by the hepatocytes, and quite a large amount of cholesterol meets its end in this way. According to Chiang (1998), conversion to bile acids accounts for 50% of the daily cholesterol catabolism in the body. Most textbooks and papers would at this stage interrupt the flow of narrative with a massive complex biochemical pathway diagram of bile acid biosynthesis, which could not possibly be expected to have any educational value for the future intensivist, as (for one) none of the enzymes involved are amenable to their manipulation. We will omit that step here, and move on to describe the final products. 

Cholic acid and chenodeoxycholic acid are those products, generally referred to as "primary" bile acids. From a chemical perspective "cholic" has nothing to do with choline, an organic amino compound unhelpfully described by PubChem as "a choline that is the parent compound of the cholines class". Cholic acid and choline only have bile in common, having been first identified in bile (hence the "chole"), and are otherwise completely different molecules. 

Both cholic acid and chenodeoxycholic acid need to be conjugated before they are ready for use, because neither are especially acidic, with a pKa of around 5.0-6.5, which means that at normal physiological pH of the bile duct or recently fed duodenum their water solubility would be poor. And if their water solubility were poor, they would neither emulsify anything, nor remain in the gut reliably (i.e. they could absorb more easily because of their lipid solubility). There is in fact a constant recirculation of unconjugated bile acids which are passively reabsorbed in the bile duct, and then make their way back to the hepatocyte in the portal blood. In short, conjugation with an amino acid (taurine or glycine) is a necessary chemical modification that generally takes place in the hepatocytes before these acids are exported into the lumen of the bile duct. 

The result of this conjugation step is four possible daughter products: 

• Taurocholic acid
• Glycocholic acid
• Taurochenodeoxycholic acid
• Glycochenodeoxycholic acid

These conjugated bile acids have pKa in the range of 1-2, which means they are fully ionised and therefore totally water-soluble. As the result, they are often referred to by their conjugate base (eg. taurocholate). 

Bile acids vs. bile salts

The terms "bile acid" and "bile salt" are tossed around rather freely in the hepatobiliary literature, and nobody seems interested in disambiguating them, perhaps because that field grows thick with biochemists and most specialist authors would assume a certain background knowledge on the part of their readers. In case you're a non-specialist who happens to ask "what is a bile salt and how is it different to a bile acid", these greyhairs will scowl professorially, and possibly create a subcommittee to decide how they should discipline you.

It would appear that:

• Bile acids are insoluble fatty (sterol) acids
• Bile salts are soluble ionised fatty (sterol) acids

The same sterol acid can be a bile acid while non-ionised, and a bile salt when fully ionised (Carey, 1988). Specifically, bile salts are often considered to be the sodium and potassium salts of conjugated bile acids which are said to form when these bile acid anions associate with metal cations in solution. Conjugated bile acid anions are usually co-secreted with sodium, which means that, in effect, the hepatocyte is secreting sodium taurocholate (which is fully dissociated in solution).

It is therefore entirely accurate to refer to the biologically relevant anionic products of bile acid secretion as bile salts; and in fact some tend to view the act of conjugation as the step which converts bile acids into bile salts (as this is the point where previously poorly dissociated unconjugated and insoluble bile acids are converted into something that dissociates and produces anions). On the other hand, occasionally the authors refer to these substances as bile acids throughout their publication, as for example with the chapter by Fickert & Dawson for Sleisenger & Fordtran's Gastrointestinal and Liver Disease (p.1999). As there does not seem to be any agreement within the scientific community as to how one should use this terminology, the CICM trainee cohort is put in an awkward position, where in any given exam sitting half the answers will probably use "bile acid" and half will use "bile salt". We can only hope that the examiners will have minimal appreciation of this matter, and continue grading fairly. As for the rest of this chapter, to remain consistent with the peer-reviewed literature, the use of "bile acid" and "bile salt" will be completely random and inconsistent.

Primary  and secondary bile acids 

"It was often not appreciated bacteria in gut produce “secondary” bile acids", the examiners complained in their answer to Question 5 from the first paper of 2016. The author can virtually guarantee that the reader will forget the names of these substances as soon as they finish reading this chapter, and that the exam candidates who failed to appreciate their importance in Question 5 had still gone on to enjoy rich rewarding careers as intensivists. Still, to completely nail this part of the question, the exam candidate would have had to have namedropped some of these substances. It should be easy; there are only two.

• Lithocholic acid
• Deoxycholic acid

These are produced by the actions of gut bacteria. They take the bile acids, deconjugate them to liberate taurine and glycine, and then dehydroxylate the product at different points (removing hydroxy groups at C-7 and C-12) producing the bile acids mentioned above. Having resisted up until this point, the author cannot help but assault the reader with some molecular structures here, if only to show that the bile acids all look very similar:

 

So: what do they do, and what is the point of knowing about them? Well, in short, they have the same detergent activity as the primary bile acids, except they are somewhat weaker (being unconjugated bile acids), which mitigates some of their toxicity to the gut bacteria - this is most likely the evolutionary reason for this bacterial metabolism of bile. The consequences for the mammal host are numerous and related to the effect these salts have on feedback control of bile synthesis and the gut microbiome, but these are matters we do not have space for here, and the interested reader is instead referred to Ridlon et al (2016). 

One other secondary bile acid needs to be mentioned here, even though it is present in only very small quantities in the human digestive tract. Ursodeoxycholic acid, a tautomer of chenodeoxycholic acid, is also named after an animal, having been originally identified in the bile of polar bears (whereas chenodeoxycholic acid was isolated in the domestic goose, hence the "cheno"). Weirdly, though lithocholic acid was identified from the gallstones of a calf, nobody decided to call it vitulocholic or bovicholic acid. Anyway, ursodeoxycholic acid is used therapeutically to decrease the absorption of cholesterol, increase the conversion of cholesterol into bile acids, and increases the flow of bile - all by a series of fascinating but poorly understood mechanisms. The ICU trainee needs to know about this substance because it is occasionally used to help bile flow in primary biliary cirrhosis, liver allograft rejection and GVHD.

Organic macromolecules in bile

Most proteins in the bile appear to come from plasma. This should be strange, because blood is separated from bile by tight junctions, and so theoretically no blood proteins should be able to cross into the bile. Still, they seem to make it across into the bile by simple diffusion, as the concentration of these truant substances in the bile is inversely proportional to their molecular size. Additionally, some proteins in the bile (IgA) seem to be there non-accidentally, i.e. they seem to perform some useful role and there is some evidence that they are secreted actively by vesicle-mediated exocytosis. The overall list of proteins and peptides includes:

• Probably passively filtered:
  • Albumin
  • Transferrin
  • Apolipoproteins
  • Haptoglobin
  • Ceruloplasmin
• Probably intentionally secreted:
  • IgA, IgG, IgM
  • Epidermal growth factor
  • Cholecystokinin
  • Amylase
  • Lysosomal hydrolases

Without going into too much more detail, the best reference to leave here is the article by LaRusso (1984), which has the most informative name of any paper: "Proteins in bile: how they get there and what they do".

Bilirubin in bile

The discussion of bilirubin and its metabolism had attracted "additional credit" in the already dense answer to Question 5 from the first paper of 2016. Rather than describe the movements of haem and bilirubin it may just be better to mention that bile contains "bile pigment" of which conjugated bilirubin and biliverdin (oxidised bilirubin) are the main constituents. Bilirubin is actively transported out of the hepatocytes and into the canaliculi, and the bilirubin content of bile is therefore high (1000-2000 μmol/L),  usually at least ten times higher (and up to a hundred times higher) than in blood, to the extent that testing drain fluid for bilirubin can be used as a means of detecting a post-operative bile leak (Rahbari et al, 2012).

Ionic composition of bile

Bile in the early branches of the biliary tree appears to have the same sort of ionic composition as plasma, aside from having a rather large "anion gap" as the result of bile acid (salt) secretion. Even with a large number of those molecules excusing themselves from solution to chill quietly in micelles, the bilestream is full of dissolved taurocholate and glycocholate. Interestingly, the result of the lipid fraction creates a "fat trapping effect", where the total concentration of measurable taurocholate is massive (say, 300 mmol/L) but the total osmolality of the aqueous bile solution is still only isoosmolar with plasma (about 290-320 mOsm/kg). 

It would probably be rather pointless to give prescriptive concentrations of biliary electrolytes. The composition of bile is subject to wild fluctuation, depending on the rate of flow, prevailing hormonal conditions, plasma electrolyte concentration, and how long the bile has spent cooking in the gall bladder. Wheeler (1961) and Wheeler et al (1960) produced excellent work to describe the changes in the composition of bile with some of these factors, and looking at their data would discourage anybody from setting up a table of biliary electrolyte concentrations. Still, people like tables; so here's a representative one from Ho (1996), listing the typical electrolyte concentration of the bile of patients who did not form gallstones (aspirated directly from their biliary structures during a laparotomy performed for some other totally elective reason). 

Electrolyte composition of bile

Electrolytes/property	Average concentration (common bile duct)	Average concentration (gall bladder)
Na	169 mmol/L	202 mmol/L
K	5.4 mmol/L	10 mmol/L
Cl	107 mmol/L	110 mmol/L
Ca	1.8 mmol/L	6.8 mmol/L
Mg	0.36 mmol/L	3.2 mmol/L
Phosphate	4.0 mmol/L	10 mmol/L
HCO3-	Variable: from 12 to 55 mmol/L
pH	7.40	6.96
Osmolality	285 mOsm/Kg	282 mOsm/kg

This is probably representative of the sort of bile an intensivist should expect from the average ICU patient (i.e. fasted). The author is well guarded against any complaints about the possible inaccuracy or poor generalizability of this table by the vast range of interpersonal variability in bile composition and by the complete clinical irrelevance of these data. Most textbooks that do list references typically reference the older of the two Boyer articles (2013) when they mention anything about the properties of bile, but their numbers all vary rather wildly (for example, here's the table from Boyer, and here's one from Guyton & Hall). 

Chemical properties of bile

Bile obviously has all sorts of chemical properties, which are extensively documented to the most minute level of detail by Carey (1985) and Small (1971). Molecular volume of bile acids in cubic angstrom, for example. As there is no reason for the ICU trainee to know these, or to even think too long about them, they are left for more adventurous readers to explore on their own. The most important properties of bile for the CICM First Part exams are:

• It is a detergent emulsifier. Coming back to the chemical characteristics of bile acids, following conjugation what we are left with is a bunch of compounds that consist of a highly lipid-soluble hydrophobic sterol structure on one end and a highly polar hydrophilic acid tail on the other. This imparts a surfactant property, which is probably the most important property of bile acids for their role in digestion.
• It is initially alkaline. The cholangiocytes alkalinise it during its transit, to the point where pH in the range of 8.0-8.5 can be recorded (but around 7.5 is more normal)
• It is acidic after spending some time in the gallbladder. Bile in the gall bladder can get to a pH as low as 5.0-6.0 (Marteau et al, 1990)
• It contains surprisingly little fat;  usually no more than 20g/L, most of which is cholesterol and phosphatidylcholine or lecithin (Carey, 1988). 

Formation of bile

Bile is created at the tiny apical membrane of the hepatocytes that forms the biliary canaliculi. Some sources report that only 1% of the total surface area of hepatocytes is dedicated to this task, others say it is 10-15%, and it is not even clear what the importance of that number really is (most likely, none). Bile then undergoes extensive modification in the bile ducts. About 90% of the bile produced during the day comes to rest in the gallbladder before being released to work its magic on some ingested fats, and while it waits the gallbladder also slowly works to modify its composition

Reclamation and biosynthesis of bile salts by hepatocytes

Bile salts returning via the portal blood are reclaimed from the basolateral membrane of the hepatocytes by various channels, some of which are sodium-dependent (for example sodium-taurocholate co-transporter) and some sodium-independent (eg. organic anion-transporting polypeptides, or OATPs). The reason these names are even mentioned here is because these transporters also facilitate things like hepatitis virus entry and the uptake of xenobiotics into the hepatocyte. Secondary bile acids absorbed from the portal blood in this fashion are reconjugated and all of them are considered part of the total biliary circulation pool, which consists of:

• 30-40% cholic acid
• 30-40% chenodeoxycholic acid
• 20-30% deoxycholic acid
• <5% lithocholic acid

This pool, in total, is about 3g, and it continues recirculating (as 95% of all the bile salts are absorbed in the terminal ileum). In addition to this, liver synthesis contributes about 200-600mg of new primary bile acids every day, and about the same amount ends up excreted in the faeces.

Secretion of bile by the hepatocytes

A biliocentric view of the universe would take the position that the liver is basically just an epithelial layer that separates blood from bile. Hepatocytes do have a basal membrane that faces the bloodstream (well, the space of Disse, to be precise) and an apical membrane that faces the biliary canaliculus, so this is actually a reasonable position to take. This means only a layer of thin (10-20 μm) hepatocytes, and the tight junctions between them, are the only things that separate blood from bile. This is actually relevant because a substantial amount of the "tightness" of those junctions is actively maintained, which means in liver injury (eg. ischaemic hepatitis) the barriers may fall and regurgitation of bile back into the bloodstream can occur. And it would do so readily, as the concentration of bile acids in the canaliculus is about 200 times higher.

Reabsorbed and synthesised bile acids traverse the hepatocyte by some unclear process, widely expected to be active and probably even related to cytoskeleton contraction, as the transit of labelled bile acids is much too fast for anything like passive diffusion. They are then secreted from the apical membrane by two main processes, one dependent on bile salts, and the other independent from them. 

Bile salt-dependent bile secretion

Of the total bile flow over a 24-hour period, about 35% will be "bile salt dependent". Logically, the greatest amount of bile salt content is in the portal blood, as bile salts are constantly reabsorbed in the intestine, and so the periportal hepatocytes tend to secrete bile in mainly this bile salt-dependent fashion.

In wordy form:

• Hepatocytes transport bile acids from the portal blood by making use of a Na+/K+ ATPase generated sodium gradient. The sodium-gradient-powered transporters responsible for this process are saturable and specific for each bile acid
• These bile salts are then transported into the biliary canaliculus
• In the canaliculus, they act as the main osmotic diver for water secretion via the aquaporins which sit in the apical membranes of hepatocytes
• At the same time the hepatocytes are busily secreting cholesterol and phosphatidylcholine  into the lumen
• As the lipid and sterol content of the bile increases, micelles tend to form, and bile salts are incorporated into these micelles
• Incorporation of bile salts into micelles withdraws bile salts from the solution, which means the concentration of  "free" bile salts in the lumen drops, making room for more secretion (otherwise the concentration gradient would be too steep and the transporters would probably not cope).

Bile salt-independent bile secretion 

As the result of the activity of periportal hepatocytes, the bile salt content of the blood at the end of the sinusoid is low, and bile-salt-dependent transport would not work particularly well. The cells in these distal regions of the lobule must secrete bile in a way that does not rely on a bile salt concentration gradient. Fortunately there's plenty of other stuff to secrete, for example:

• Glutathione
• Glutathione conjugates (with various random organic molecules)
• Sulfated secondary bile acids and their derivatives
• Conjugated bilirubin (with glucouronide)
• Organic cations (like drugs and xenobiotics)
• Weird trace elements and heavy metals (manganese, zinc, etc)
• Bicarbonate

All of these substances are expelled from the apical membrane of the hepatocyte by the action of several relatively nonspecific transport systems, such as the multidrug resistance-associated protein 2 (MRP2).

Modification of bile by cholangiocytes

Cholangiocytes receive an early beta version of bile, unready for the public. The role of cholangiocytes is to significantly alkalinise this product, and to reabsorb anything worth keeping  (for example, if some sort of valuable nutrient molecule has escaped into the bile, the cholangiocytes will often be able to reclaim it by endocytosis). As they work to change the ionic composition of the bilestream, they also increase its osmolality, and water is secreted into the lumen of the bile duct. By this activity it is said they contribute 40% of the total bile volume, and apparently this happens mostly in the large columnar cholangiocytes in the big ducts, rather than the little cuboidal cells that line the terminal cholangioles. When Rous & McMaster (1921) isolated the bile ducts of cats and dogs from the remainder of the biliary tree, they found they continued to issue "a clear sterile watery fluid, slightly alkaline to litmus, and with a specific gravity of 1.011", entirely the product of cholangiocytes.  

Changes to the composition of bile in the gallbladder

From the tables detailing the composition of bile listed above, it is easy to see that the gallbladder removes a lot of the water and electrolyte content of bile, making it more concentrated and acidic. The details of these processes are thankfully already well represented by Turumin et al (2013), which means we do not need to revisit them here. The main objective of this process is to make sure that the concentration of bile salts in the stored bile is maximised, so that it has the greatest effect on dietary fats when it is unleashed upon them.

Function of bile

The CICM model answer to Question 5 from the first paper of 2016 posits that "the major role of bile is in lipid, cholesterol and lipid soluble vitamin absorption with a minor role in excretion of bile pigments". This oversimplification represents the absolute maximum that could be expected from a trainee answering that question. Bile actually has a large range of functions, which have already been discussed in a roundabout way in various other sections of this website. To summarise, with links to specific pages:

• Digestive functions of bile:
  • Emulsification of dietary fat, to aid its transport in water
  • Misellisation, to increase the surface area for lipases to work on
  • Exclusion of other adsorbents off the surface of micelles
  • Solubilisation of fatty acids to help them absorb more effectively
  • All of these also benefit the absorption of fat-soluble micronutrients, eg. vitamins
• Excretory role of bile:
  • Major excretory route for potentially harmful exogenous lipophilic substances
  • Excretory route for bilirubin 
  • Major route for elimination of cholesterol
• Immune function of bile:
  • IgA and IgG are secreted into bile, which helps neutralise any organisms which might have refluxed up into the biliary tree
• Growth stimulus for enterocytes
  • Bile contains various growth promoters and signalling molecules which act as trophic stimuli for enterocytes

.