Bile Acids and Alcohols

1.  Structures and Occurrence

The bile acids (mainly C24 but also C27) are the end products of cholesterol catabolism in animals, the main functions of which 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. Very many different bile acids and alcohols occur in nature, presumably because multiple biochemical 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. For example, nitrogen-free cholic acid was isolated as long ago as 1838, but the correct structure was not elucidated until 1932. 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.

In 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 be 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, deoxycholic and cholic acids, 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 and cholic acids, so experimental data from laboratory animals cannot always be extrapolated to humans (only hamster bile is similar).

Structural formulae for typical members of the bile acids and alcohols

The three classes of bile acids and alcohols can be considered in terms of a ‘default’ structure of nuclear hydroxylation in each class. This has 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 structure, either on the nucleus or the side chain or on both.

In this way, 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. For example, 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 only found in bears, while 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)).

Biosynthesis of bile acids - conformational changes

A further complication is that much of the bile acids are secreted into bile in the form of conjugates with the amino acids taurine and to a lesser extent glycine (taurine tends to predominate in carnivores and glycine in herbivores). Bile alcohols are conjugated with sulfate. These 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.

 

 taurocholic acid formula

2.  Biosynthesis 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 classical or neutral’ pathway to the biosynthesis of the ‘root’ bile acid, chenodeoxycholic acid, involves first the synthesis of 7α-hydroxy-cholesterol, as described in our web page on oxysterols, in the endoplasmic reticulum. 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, before it is cleaved in the peroxisomes by the same enzyme that produces 27-hydroxycholesterol, i.e. sterol-27-hydroxylase (CYP27) (see also our web page on oxidized sterols). Ultimately, before secretion into bile, a high proportion of the bile acids are converted in the peroxisomes to conjugates by N-acylamidation with the amino acids taurine, a sulfonic acid-containing compound derived from cysteine (see our web page on sulfonolipids), and glycine.

Biosynthesis of bile acids

Deoxycholic acid synthesis 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.

An alternate ('acidic') 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. Oxysterol 7α-hydroxylases are the key enzymes in this second pathway, illustrated for 27-hydroxycholesterol.

Biosynthesis of bile acids by acidic pathway

While the biosynthetic process begins in the endoplasmic reticulum, the intermediates must be transferred to the cytoplasm, where some of the biosynthetic enzymes are situated, and thence to mitochondria and finally to the peroxisomes, all by appropriate transport mechanisms, which are not fully understood. Finally, conjugation of the bile acids with amino acids is effected by a bile acid:CoA synthase and a bile acid:amino acid transferase. Fully formed bile acids are transported efficiently across the sinusoidal membrane in the liver by the Na+ taurocholate co-transporting polypeptide with help from a family of polypeptides that function in the transport of organic anions.

An alternate 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.

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. Thence in response to gut hormones 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 acidBile acid and cholesterol homeostasis are maintained by an enterohepatic circulation system. As part of this process, microflora in the intestines deconjugate the bile acids and they can modify the steroidal structures to produce some ‘secondary’ bile acids, for example by removing the 7-hydroxyl group to produce 7-deoxy bile acids (e.g. lithocholic and deoxycholic acids). Further modification of secondary bile acids by sulfation and/or glucuronidation can also occur.

Regulation of bile acid synthesis involves complex processes, which are linked to the metabolism of cholesterol 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.

 

3.  The Functions of Bile Acids

As discussed briefly above and in our web page on triacylglycerol metabolism, the main 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.

Bile acids are stored in the gallbladder and are cycled between the intestines and liver via the enterohepatic circulation. The nature of the conjugates requires membrane transporters for cellular uptake and secretion, but once their main task is completed approximately 95% of the nonconjugated bile acids are reabsorbed passively throughout the small and large intestines. The conjugated bile acids require an apical sodium-dependent bile acid transporter. A further transporter molecule enables bile acids to exit 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 4 to 12 times each day. The average pool of bile acids is roughly 2 g, and because of recycling, hepatic secretion into the duodenum is about 12 g/day. A small proportion avoids hepatic extraction and enters the general circulation.

In adult humans, roughly 0.5 g 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.

In addition to their function in the absorption of dietary lipids and in cholesterol homeostasis, bile acids act as signalling molecules. They activate a specific G-protein-coupled receptor (TGR5), and they also interact with a receptor in the nucleus (FXRα), which regulates the expression of many genes involved in sterol, triacylglycerol and carbohydrate metabolism. These two receptors have selective affinities for different bile acids and exhibit different patterns of expression corresponding to different signalling functions in tissues. Via various signalling pathways, bile acids regulate their own biosynthesis and enterohepatic circulation, and 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. 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 the specific receptors.

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 metabolism of bile acids is associated with a number of disease states. At high concentrations, they are toxic and their presence is relevant to the pathogenesis of certain liver diseases and colon cancer. In contrast, hydrophilic ursodeoxycholic acid and its taurine conjugate are used therapeutically for cholesterol gallstone dissolution. 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 deconjugated 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.

 

Recommended Reading

  • Agellon, L.B. Metabolism and function of bile acids. In: Biochemistry of Lipids, Lipoproteins and Membranes (5th Edition). pp. 423-440 (Vance, D.E. and Vance, J. (editors), Elsevier, Amsterdam) (2008).
  • Amaral, J.D., Viana, R.J.S., Ramalho, R.M., Steer, C.J. and Rodrigues, C.M.P. Bile acids: regulation of apoptosis by ursodeoxycholic acid. J. Lipid Res., 50, 1721-1734 (2009) (DOI: 10.1194/jlr.R900011-JLR200).
  • Baptissart, M., Vega, A., Maqdasy, S., Caira, F., Baron, S., Lobaccaro, J.M.A. and Volle, D.H. Bile acids: From digestion to cancers. Biochimie, 95, 504-517 (2013) (DOI: 10.1016/j.biochi.2012.06.022).
  • Chiang, J.Y.K. Regulation of bile acid synthesis. www.bioscience.org.
  • Ferdinandusse, S., Denis, S., Faust, P.L. and Wanders, R.J.A. Bile acids: the role of peroxisomes. J. Lipid Res., 50, 2139-2147 (2009) (DOI: 10.1194/jlr.R900009-JLR200).
  • Griffiths, W.J. and Sjövall, S. Bile acids: analysis in biological fluids and tissues. J. Lipid Res., 51, 23-41 (2010) (DOI: 10.1194/jlr.R001941-JLR200).
  • Hofmann, A.F. and Hagey, L.R. Bile acids: chemistry, pathochemistry, biology, pathobiology, and therapeutics. Cell. Mol. Life Sci., 65, 2461-2483 (2008) (DOI: 10.1007/s00018-008-7568-6).
  • Hofmann, A.F., Hagey, L.R. and Krasowski, M.D. Bile salts of vertebrates: structural variation and possible evolutionary significance. J. Lipid Res., 51, 226-246 (2010) (DOI: 10.1194/jlr.R000042).
  • Hylemon, P.B., Zhou, H., Pandak, W.M., Ren, S., Gil, G. and Dent, P. Bile acids as regulatory molecules. J. Lipid Res., 50, 1509-1520 (2009) (DOI: 10.1194/jlr.R900007-JLR200).
  • Lefebvre, P., Cariou, B., Lien, F., Kuipers, F. and Staels, B. Role of bile acids and bile acid receptors in metabolic regulation. Physiol. Rev., 89, 147-191 (2009) (DOI: 10.1152/physrev.00010.2008).
  • Russell, D.W. The enzymes, regulation, and genetics of bile acid synthesis. Annu. Rev. Biochem., 72, 137-174 (2003) (DOI: 10.1146/annurev.biochem.72.121801.161712).
  • Zwicker, B.L. and Agellon, L.B. Transport and biological activities of bile acids. Int. J. Biochem. Cell Biol., 45, 1389-1398 (2013) (DOI: 10.1016/j.biocel.2013.04.012).

 

Updated August 26, 2013

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