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Sterols:   5. Bile Acids and Alcohols



The bile acids (mainly C24 but also C27) are the end products of cholesterol catabolism in animals, and their best-known functions are to act as powerful detergents or emulsifying agents in the intestines to aid the digestion and absorption of fatty acids, monoacylglycerols and other fatty products and to prevent the precipitation of cholesterol in bile. In addition, it is now recognized that they are involved in the regulation of multiple biological reactions, especially in liver cells but also in extrahepatic tissues via interactions with receptors of various kinds. Very many different bile acids and alcohols occur in nature, often dependent on the animal species, presumably because multiple enzymatic pathways have evolved to convert cholesterol into these highly water-soluble, amphipathic molecules.

The nomenclature is complex for historical reasons; many were given trivial names in the 19th century long before their structures were determined, and for example, nitrogen-free cholic acid was isolated as long ago as 1838. Although Heinrich Wieland obtained the 1927 Nobel Prize in Chemistry for his work on bile acids, the correct structure of the steroid nucleus was not determined until 1932 by the X-ray diffraction studies of Desmond Bernal, incidentally the first application of this technique to a biological problem.


1.  Structures and Occurrence

The bile acids and alcohols, sometimes termed 'cholanoids' or 'cholestanoids', are usually subdivided into three main classification groups, i.e. C27 bile alcohols, C27 bile acids and C24 bile acids. The C27 bile alcohols and acids contain the C8 side chain of cholesterol, while the C24 bile acids have a truncated C5 side chain.

cholic acidIn mammals, C24 bile acids predominate and they are major components of bile amounting to about 12% of the total (with roughly 4% phospholipids and 1% cholesterol). In non-mammalian vertebrates, such as fish and reptiles, bile alcohols (non-acidic) are formed, while invertebrates do not produce bile acids or alcohols. During vertebrate evolution, there appears to have been a pattern of progressive molecular development from C27 alcohols to C27 acids to C24 acids. The main components in human bile are the C24 compounds chenodeoxycholic (45%), deoxycholic and cholic acids (31%), with hydroxyl groups of the 3α,7α-, 3α,12α- and 3α,7α,12α-configurations, respectively. In contrast, bile in mice contains mainly the more hydrophilic muricholic (hydroxylated at the 6β position) and cholic acids, so experimental data from laboratory animals cannot always be extrapolated to humans (only hamster bile is similar to that of humans). The trivial names in common use are derived from cholic acid, the first to be characterized, with an element added that is derived from either the chemical structure or the primary animal source.

Structural formulae for typical bile acids and alcohols

The three classes of bile acids and alcohols can be considered in terms of ‘default’ structures of nuclear hydroxylation, which have hydroxyl groups at C-3 (epimerized from the 3β-hydroxyl group of cholesterol) and at C-7, together with either a primary alcohol or a carboxyl group at the terminal carbon atom of the side chain. Further substituents can then be added to the default structures, either on the nucleus or the side chain or on both.

Bile alcohols and acids exhibit great structural diversity among animal species. For example, structural variation occurs in the stereochemistry of the A/B ring juncture in the steroid nucleus, in the sites of hydroxyl or keto groups, and in the orientation of hydroxyl groups, i.e. whether they are α or β to the ring. The length of the side chain can vary, while hydroxyl groups of variable orientation and double bonds may be present or absent. In addition, the stereochemistry of the C-25 carbon atom and the site of the carboxyl group can differ. At the last count, 25 different bile alcohols with three to six hydroxyl and/or keto groups are known, together with 45 C27 and 40 C24 bile acids, and novel structures continue to be reported. The differing structures can be interpreted in terms of evolutionary relationships between species and families. For example, a 7β-hydroxyl group is found in ursodeoxycholic acid primarily in bears, i.e. it is an epimer of chenodeoxycholic acid with very different physicochemical and biological properties. Hyocholic acid with 3α,6α,7α-hydroxyls is a major component of pig bile.

A change to a cis-fused configuration at the A/B ring junction as illustrated accentuates the change in polarity of the molecule, creating hydrophilic (α) and hydrophobic (β) faces. (allo-bile acids in lower vertebrates are flat because of an A/B trans-fusion (5α-stereochemistry)). Bile acids are thus amphipathic molecules with powerful detergent properties.

Biosynthesis of bile acids - conformational changes

taurocholic acid formulaA further complication is that much of the bile acids are secreted into bile in the form of conjugates with the carboxyl group and the amino acids taurine and to a lesser extent glycine. Taurine conjugation is the rule in fish, amphibians, reptiles and birds, and it also occurs to some extent in mammals; C27 bile acids are conjugated exclusively with taurine. Glycine conjugation occurs in herbivores and primates, but other forms of conjugation can occur to a lesser extent, e.g. with glucose or by sulfation, or with N‑acetylglucosamine at C‑7. As bile alcohols are conjugated with sulfate, they have a pKa similar to that of taurine conjugates. Although the amount of sulfated bile acids in serum and bile tends to be small, they constitute a high proportion of those in urine.

The physical properties of bile acid conjugates are obviously the key to their function. The additions increase substantially the acidity of the molecules and their solubility in water. At the physiological pH values in the intestines, the bile conjugates ionize and exist in salt form. In this conjugated state, the molecules cannot enter the epithelial cells of the biliary tract and small intestines. Their amphipathic nature enables them to form mixed micelles with phosphatidylcholine in solution, and these micelles are relatively stable in the presence of calcium ions.


2.  Biosynthesis and Metabolism of Bile Acids

Although there are a number of different biosynthetic routes to bile acids from cholesterol, there are four main steps, and the liver is the only organ concerned in the production of the ‘primary’ bile acids. In fact, there are at least 16 enzymes that catalyse up to 17 reactions to convert insoluble cholesterol into a highly soluble conjugated bile salt. What has been termed the ‘classical or neutral’ pathways to the biosynthesis of the ‘root’ bile acid, chenodeoxycholic acid, is believed to be the main mechanism in humans under normal physiological conditions.

In brief, the first step is rate limiting and involves the synthesis of 7α-hydroxy-cholesterol by a cholesterol 7α-hydroxylase (CYP7A1) in the endoplasmic reticulum, as described in our web page on oxysterols. In the next step, epimerization of the 3β-hydroxyl group is effected by a specific oxidoreductase, before the double bond in position 5 is hydrogenated by one of two reductases. The side-chain is oxidized in the mitochondria and the resulting 3α,7α-dihydroxy-5β-cholestanoic acid is converted to the CoA ester in the endoplasmic reticulum for transport into the peroxisomes where the side-chain is cleaved by the same enzyme that produces 27-hydroxycholesterol, i.e. sterol-27-hydroxylase (CYP27) (see also our web page on oxidized sterols), to remove the three terminal carbons and eventually produce chenodeoxycholic acid. Finally, before secretion into bile, a high proportion of the bile acids are converted in the peroxisomes to conjugates with the amino acids taurine, a sulfonic acid-containing compound derived from cysteine (see our web page on sulfonolipids), and glycine by N-acylamidation by means of a bile acid:CoA synthase and a bile acid:amino acid transferase.

Biosynthesis of bile acids

Synthesis of deoxycholic and cholic acids occurs by a similar route, except that a sterol 12α-hydroxylase (another of the cytochrome P450 family) introduces a 12-hydroxyl group into the steroidal side-chain. The eventual result is that cholic and chenodeoxycholic acids are produced in approximately equal amounts.

An alternative minor pathway for the synthesis of bile acids is now known to exist that utilizes other oxysterols as the precursors. It is often termed the 'acidic pathway' as acidic intermediates are formed when the oxidation of the side-chain of cholesterol precedes the modification of the steroid ring. Oxysterol 7α-hydroxylases are the key enzymes in this second pathway, illustrated for 27-hydroxycholesterol. In this instance, bile acid synthesis is initiated in the inner membrane of mitochondria by sterol 27-hydroxylase, but the rate limiting step may be cholesterol transport into the mitochondria. The process continues in the endoplasmic reticulum and the cytoplasm, to produce chenodeoxycholic acid, and finally the conjugated forms are produced in the peroxisomes. At each step, specific transport mechanisms are required, which are not fully understood. This biosynthetic pathway is believed to produce less than 10% of the bile acids in humans.

Biosynthesis of bile acids by acidic pathway

A further pathway for the synthesis of bile acids is now known to exist that utilizes other oxysterols as the precursors. Production of these is catalysed by sterol 27-, 25- and 24-sterol hydroxylases, for example (see our web page on oxysterols). Most of the 24-hydroxycholesterol originates in the brain, but further conversion to bile acids takes place in the liver. The cholesterol 7α-hydroxylase is again a key enzyme in the process.

Regulation of bile acid synthesis involves complex processes, which are linked to the metabolism of cholesterol, retinoids and fatty acids. However, the main control is exerted via the rate-limiting enzyme cholesterol 7α-hydroxylase, the activity of which can be modified by a number of different pathways, but especially by the action of bile acids and cholesterol on gene transcription via specific receptors (see below).

Enterohepatic circulation and metabolism: Bile acids are stored in the gallbladder and are cycled between the intestines and liver via the enterohepatic circulation. Conjugated bile acids are secreted into the canalicular space between hepatocytes bound to a specific binding protein, and they cross the canalicular membrane in an ATP-dependent fashion by a bile salt export pump to enter the bile in the gall bladder, where the concentration of bile acids in bile is 100 to 1000 times higher than that in the hepatocytes; this transport against the concentration gradient controls the overall rate of bile acid production. Thence in response to the gut hormone cholecystokinin, they pass through bile ducts into the duodenum of the small intestine, where they assist the emulsification and absorption of the partially hydrolysed lipids from the diet (see our web page on triacylglycerol metabolism).

Formula of lithocholic acidMicroflora in the intestines de-conjugate a proportion of the bile acids by means of a bile salt hydrolase, which acts upon a wide range of bile acid conjugates including the six main components of human bile, and further microbial enzymes can modify the steroidal structures by dehydroxylation, oxidation of hydroxyls to oxo groups and epimerization to generate 'secondary' bile acids. For example, they act upon the deconjugated bile acids to produce lithocholic from chenodeoxycholic acid and deoxycholic from cholic acid by removing the 7-hydroxyl group (i.e. resulting in 7-deoxy bile acids). These secondary bile acids are cytotoxic, and they can be detoxified in humans by oxido-reduction, epimerization and side-chain desaturation to produce hyocholic and ursodeoxycholic acids in addition to by sulfation and/or glucuronidation.

The nature of the conjugates requires membrane transporters for cellular uptake and secretion, but once their main task is completed 90-95% of the non-conjugated bile acids are reabsorbed passively throughout the small and large intestines. The more abundant conjugated bile acids require an apical sodium-dependent bile acid transporter in the terminal ileum to cross the brush border membrane, before they are assisted across the enterocyte by the cytosolic ileal bile acid binding protein. A further transporter molecule enables bile acids to exit the basolateral membrane of the enterocyte, before they are returned to the liver bound to albumin in the portal blood stream, where they are absorbed by the sodium/taurocholate co-transporting polypeptide to complete the cycle. They are then re-secreted into bile together with newly synthesised bile acids to continue the process. In humans, a conjugated bile salt may complete this cycle from two to six times each day. The average pool of bile acids is roughly 2 g, and because of recycling, hepatic secretion into the duodenum is about 12g/day. A small proportion avoids hepatic extraction and enters the general circulation. Similarly, only a little of the secondary bile acids is absorbed into tissues, but these can accumulate slowly as the human liver is unable to convert deoxycholic acid back to cholic acid.

In adult humans, roughly 0.5g of cholesterol is utilized for bile acid production each day. It has become evident that the 5% of bile acids that is lost into the faeces represents an important element of the turnover of cholesterol. Indeed, this is the major pathway for the removal of cholesterol from the body, and it is important for the maintenance of cholesterol homeostasis both from quantitative and regulatory standpoints.


3.  The Functions of Bile Acids

As discussed briefly above and in more detail in our web page on triacylglycerol metabolism, a major function of bile acids is to act as powerful detergents or emulsifying agents in the intestines to aid the digestion and absorption of fatty acids, monoacylglycerols, fat-soluble vitamins and other fatty products. They stimulate lipolysis by facilitating the binding of pancreatic lipase with its co-lipase. In addition, they may control the growth of bacteria in the small intestine.

As well as their function in the absorption of dietary lipids and in cholesterol homeostasis, bile acids act as signalling molecules. They function as nutrient signalling hormones by activating several receptors in the nucleus (farnesoid X (FXRα), pregnane X (PXR) and Vitamin D receptors (VDR)) together with G-protein coupled receptors in the plasma membrane such as the transmembrane G-protein coupled receptor 5 (TGR5), sphingosine-1 phosphate receptor 2 and muscarinic receptor. Thereby, they regulate the expression of many genes involved in sterol, triacylglycerol and carbohydrate metabolism. These receptors have selective affinities for different bile acids, for example chenodeoxycholic acid is the most potent stimulator of FXRα, and they exhibit different patterns of expression corresponding to different signalling functions in tissues.

Scottish thistleActing via the FXR receptor and various signalling pathways, bile acids exert a negative feedback regulation on their own synthesis and enterohepatic circulation, mainly through inhibition of both the activity and expression of the key enzyme CYP7A1. In collaboration with insulin, they have an influence on the metabolism of lipids and of glucose. For example, they are involved in the regulation of triacylglycerol biosynthesis and the production of very-low-density lipoproteins (VLDL) in the liver, thereby lowering plasma triacylglycerol levels. There are suggestions that modification of bile acid metabolism may be a useful pharmacological approach to the treatment of the metabolic syndrome and type 2 diabetes. The FXR receptor also has a major influence on cholesterol metabolism and thence on atherosclerosis, and pharmacological intervention with this receptor may prove to be a useful therapeutic approach to liver and metabolic diseases.

In addition, bile acids are intimately involved in the processes of apoptosis and cell survival, and they influence calcium mobilization, cyclic AMP synthesis and protein kinase C activation via their interactions with receptors. The pregnane X receptor functions as a xenobiotic sensor and is activated by high levels of bile acids to regulate bile acid synthesis possibly to prevent liver damage. Similarly, activation of the vitamin D receptor in the intestines induces the cytochrome P450 enzyme CYP3A4 that detoxifies excess bile acids. TGR5 is expressed in many different tissues in addition to the intestines, and the discovery that it was activated by bile acids lead to the realization that these had multiple effects on signalling processes throughout the body, for example by stimulating the production of cAMP and phosphorylation of key enzymes relevant to glucose homeostasis and immune cell regulation. Conjugated bile acids activate the sphingosine 1 phosphate receptor-2, which in turn leads to the activation of various kinases that impact upon the regulation of glucose metabolism.

Most of the enzymes involved in bile acid synthesis play multiple roles in intermediary metabolism. For example, some are involved in the production of oxysterols, others act on intermediates in hormone biosynthesis, some metabolize very-long-chain fatty acids, such as dietary pristanic acid, and another is utilized in vitamin D synthesis.

Inefficient biosynthesis and metabolism of bile acids can cause health problems from the neonatal period to adulthood with diverse clinical symptoms ranging from cholestatic liver disease to neuropsychiatric symptoms and spastic paraplegias. At high concentrations, they are toxic and their presence is relevant to the pathogenesis of cancer of the biliary tract and colon in addition to liver diseases. In contrast, hydrophilic ursodeoxycholic acid (and its taurine conjugate) are used therapeutically for cholesterol gallstone dissolution and in the treatment of primary biliary cirrhosis by stimulating bile flow from the liver. Also, this bile acid has inhibitory effects upon apoptosis and is being study for potential beneficial effects in a number of disease states where apoptosis is deregulated, including neurological disorders such as Alzheimer's, Parkinson's and Huntington's diseases.


4.  Analysis

For many years, gas chromatography linked to mass spectrometry has been the method of choice for the analysis of de-conjugated bile acids, and it is still invaluable because of the structural information that can be obtained by electron-impact ionization. However, high-performance liquid chromatography linked to mass spectrometry with electrospray ionization now affords much greater sensitivity when this is required, and this technique is suitable for the analysis of both conjugated and non-conjugated bile acids.


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Lipid listings Credits/disclaimer Updated: August 3rd, 2017 Author: William W. Christie LipidWeb icon