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Bile salts: structure, function, synthesis, enterohepatic circulation

Bile salts and bile acids are polar cholesterol derivatives, and represent the major route for the elimination of the steroid from the body.
They are molecules with similar but not identical structures, and diverse physical and biological characteristics.
They are synthesized in the liver, stored in the gallbladder, secreted into the duodenum, and finally, for the most part, reabsorbed in the ileum.
Because at physiological pH these molecules are present as anions, the terms bile acid and bile salts are used herein as synonyms.

Contents

Chemical structure

Bile salts have similarities and differences with cholesterol molecule.
Like the steroid, they have a nucleus composed of four fused rings: three cyclohexane rings, labeled A, B and C, and a cyclopentane ring, labeled D. This structure is the perhydrocyclopentanophenanthrene, more commonly known as steroid nucleus.

Bile Acids and Their Conjugates

In higher vertebrates, they have 24 carbon atoms, as the side chain is three carbons shorter than the original. In lower vertebrates, bile acids have 25, 26, or 27 carbon atoms. The side chain ends with a carboxyl group, ionized at pH 7, that can be linked to the amino acid glycine or taurine.
In addition to the hydroxyl group at position 3, they have hydroxyl groups at positions 7 and/or 12.
All this makes them much more polar than cholesterol.
Since A and B rings are fused in cis configuration, the planar structure of the steroid nucleus is curved, and it is possible to identify:

Cholic Acid Structure

Therefore, having both polar and nonpolar groups, they are amphiphilic molecules and excellent surfactants. However, their chemical structure makes them different from many other surfactants, often composed of a polar head region and a nonpolar tail.

Primary, conjugated and secondary bile salts

Primary bile acids are those synthesized directly from cholesterol in the hepatocytes. In humans, the most important are cholic acid and chenodeoxycholic acid, which make up 80 percent of all bile acids. Before being secreted into the biliary tree, they are almost completely conjugated, up to 98 percent, with the glycine or taurine, to form glycoconjugates and tauroconjugates, respectively. In particular, approximately 75 percent of cholic acid and chenodeoxycholic acid are conjugated with glycine, to form glycocholic acid and glycochenodeoxycholic acid, the remaining 25 percent with taurine, to form taurocholic acid and taurochenodeoxycholic.

Synthesis of Conjugated Bile Acids

Conjugated bile acids are molecules with more hydrophilic groups than unconjugated bile acids, therefore with a increased emulsifying capacity. In fact, conjugation decreases the pKa of bile acids, from about 6, a value typical of non-conjugated molecules, to about 4 for glycocholic acid, and about 2 for taurocholic acid. This makes that conjugated bile acids are ionized in a broader range of pH to form the corresponding salts.
The hydrophilicity of the common acid and bile salts decreases in the following order: glycine-conjugated < taurine-conjugated < lithocholic acid < deoxycholic acid < chenodeoxycholic acid < cholic acid < ursodeoxycholic acid.
Finally, conjugation also decreases the cytotoxicity of primary bile acids.

Secondary bile acids are formed from primary bile acids which have not been reabsorbed from the small intestine. Once they reach the colon, they can undergo several modifications by bacteria of the gut microbiota, which is part of the larger human microbiota, to form secondary bile acids, which, along with short-chain fatty acids, are two major type of bacterial metabolites produced in the gut. They make up the remaining 20 percent of the body’s bile acid pool.

Another way of categorizing bile salts is based on their conjugation with glycine and taurine and their degree of hydroxylation. On this basis, three categories are identified.

Function of bile acids

All their physiological functions are performed in the conjugated form.

lipase activity, mainly pancreatic lipase, (bile salts also play a direct role in the activation of this enzyme);

intestinal esterase activity.

Subsequently, they facilitate lipid absorption, as well as absorption of fat soluble vitamins by the intestinal mucosa, thanks to the formation of mixed micelles.
Bile acids perform a similar function in the gallbladder where, forming mixed micelles with phospholipids, they prevent the precipitation of cholesterol.
Note: as a consequence of the arrangement of polar and nonpolar groups, bile acids form micelles in aqueous solution, usually made up of less than 10 monomers, as long as their concentration is above the so-called critical micellar concentration or CMC.

Enterohepatic circulation

After fat intake, enteroendocrine cells of the duodenum secrete cholecystokinin into the blood stream. Hormone binding to receptors on smooth muscle cells of the gallbladder promotes their contraction; the hormone also causes the relaxation of the sphincter of Oddi. All this results in the secretion of the bile, and therefore of bile acids into the duodenum.
Under physiological conditions, human bile salt pool is constant, and equal to about 3-5 g. This is made possible by two processes:

Up to 95 percent of the secreted bile salts is reabsorbed from the gut, not together with the products of lipid digestion, but through a process called enterohepatic circulation.
It is an extremely efficient recycling system, which seems to occur at least two times for each meal, and includes the liver, the biliary tree, the small intestine, the colon, and the portal circulation through which reabsorbed molecules return to the liver. Such recirculation is necessary since liver’s capacity to synthesize bile acids is limited and insufficient to satisfy intestinal needs if the bile salts were excreted in the feces in high amounts.
Most of the bile salts are reabsorbed into the distal ileum, the lower part of the small intestine, by a sodium-dependent transporter within the brush border of the enterocytes, called sodium-dependent bile acid transporter or ASBT, which carries out the cotransport of a molecule of bile acid and two sodium ions.
Within the enterocyte, it is thought that bile acids are transported across the cytosol to the basolateral membrane by the ileal bile acid-binding protein or IBABP. They cross the basolateral membrane by the organic solute transporter alpha-beta or OSTalpha/OSTbeta, pass into the portal circulation, and, bound to albumin, reach the liver.
It should be noted that a small percentage of bile acids reach the liver through the hepatic artery.
A hepatic level, their extraction is very efficient, with a first-pass extraction fraction ranging from 50 to 90 percent, a percentage that depends on bile acid structure. The uptake of conjugated bile acids is mainly mediated by a Na+-dependent active transport system, that is, the sodium-dependent taurocholate cotransporting polypeptide or NTCP. However, a sodium-independent uptake can also occur, carried out by proteins of the family of organic anion transporting polypeptides or OATP, mainly OATP1B1 and OATP1B3.
The rate limiting step in the enterohepatic circulation is their canalicular secretion, largely mediated by the bile salt export pump or BSEP, in an ATP-dependent process. This pump carries monoanionic bile salts, which are the most abundant. Bile acids conjugated with glucuronic acid or sulfate, which are dianionic, are transported by different carriers, such as MRP2 and BCRP.

Note: Serum levels of bile acids vary on the basis of the rate of their reabsorption, and therefore they are higher during meals, when the enterohepatic circulation is more active.

Intestinal metabolism

Bile acids which escape ileal absorption pass into the colon where they partly undergo modifications by intestinal microbiota and are converted to secondary bile acids.
The main reactions are listed below.

Intestinal Metabolism of Bile Acids

Some of the secondary bile acids are then reabsorbed from the colon and return to the liver. In the hepatocytes, they are reconjugated, if necessary, and resecreted. Those that are not reabsorbed, are excreted in the feces.
Whereas oxidations and deconjugations are carried out by a broad spectrum of anaerobic bacteria, 7alpha-dehydroxylations is carried out by a limited number of colonic anaerobes.
7alpha-Dehydroxylations and deconjugations increase the pKa of the bile acids, and therefore their hydrophobicity, allowing a certain degree of passive absorption across the colonic wall.
The increase of hydrophobicity is also associated with an increased toxicity of these molecules. And a high concentration of secondary bile acids in the bile, blood, and feces has been associated to the pathogenesis of colon cancer.

Soluble fibers and reabsorption

The reabsorption of bile salts can be reduced by chelating action of soluble fibers, such as those found in fresh fruits, legumes, oats and oat bran, which bind them, decreasing their uptake. In turn, this increases bile acid de novo synthesis, up-regulating the expression of the 7alpha-hydroxylase and sterol 12alpha-hydroxylase, and thereby reduces hepatocyte cholesterol concentration.
The depletion of hepatic cholesterol increases the expression of the LDL receptor, and thus reduces plasma concentration of LDL cholesterol. On the other hand, it also stimulates the synthesis of HMG-CoA reductase, the key enzyme in cholesterol biosynthesis.
Note: Some anti-cholesterol drugs act by binding bile acids in the intestine, thereby preventing their reabsorption.

Synthesis

Quantitatively, bile acids are the major product of cholesterol metabolism.
As previously said, enterohepatic circulation and their de novo synthesis maintain a constant bile acid pool size. In particular, de novo synthesis allows the replacement of bile salts excreted in the faces, about 5-10 percent of the body pool, namely ~ 0.5 g/day.
Below, the synthesis of cholic acid and chenodeoxycholic acid, and their conjugation with the amino acids taurine and glycine, is described.
There are two main pathways for bile acid synthesis: the classical pathway and the alternative pathway. In addition, some other minor pathways will also be described.

De Novo Synthesis of Primary Bile Acids and Their Conjugates

The classical or neutral pathway

In humans, up to 90 percent of bile salts are produced via the classical pathway, also referred to as “neutral” pathway since intermediates are neutral molecules.
It is a metabolic pathway present only in the liver, that consists of reactions catalyzed by enzymes localized in the cytosol, endoplasmic reticulum, peroxisomes, and mitochondria, and whose end products are the conjugates of cholic acid and chenodeoxycholic acid.

Cholesterol + NADPH + H+ + O2 → 7alpha-Hydroxycholesterol + NADP+ + H2O

to enter the pathway that leads to the synthesis of cholic acid, through the reaction catalyzed by 7alpha-hydroxy-4-cholesten-3-one12alpha-monooxygenase or sterol 12alpha-hydroxylase or CYP8B1 (E.C. 1.14.18.8), an enzyme localized in the endoplasmic reticulum;

to enter the pathway that leads the synthesis of chenodeoxycholic acid, through the reaction catalyzed by 3-oxo-Δ4-steroid 5beta-reductase or AKR1D1 (E.C. 1.3.1.3), a cytosolic enzyme.

It should be underlined that the activity of sterol 12alpha-hydroxylase determines the ratio of cholic acid to chenodeoxycholic acid, and, ultimately, the detergent capacity of bile acid pool. And in fact, the regulation of sterol 12alpha-hydroxylase gene transcription is one of the main regulatory step of the classical pathway.

Therefore, if 7alpha-hydroxy-4-cholesten-3-one proceeds via the reaction catalyzed by sterol 12alpha-hydroxylase, the following reactions will occur.

If 7alpha-hydroxy-4-cholesten-3-one does not proceed via the reaction catalyzed by sterol 12alpha-hydroxylase, it enters the pathway that leads to the synthesis of chenodeoxycholic acid conjugates, through the reactions described below.

Then, the conjugated bile acids glycochenodeoxycholic acid and taurochenodeoxycholic acid are formed by modifications similar to those seen for the conjugation of cholic acid, and catalyzed mostly by the same enzymes.

Note: Unconjugated bile acids formed in the intestine must reach the liver to be reconjugated.

The alternative or acidic pathway

It is prevalent in the fetus and neonate, whereas in adults it leads to the synthesis of less than 10 percent of the bile salts.
This pathway differs from the classical pathway in that:

The first step involves the conversion of cholesterol into 27-hydroxycholesterol in the reaction catalyzed by sterol 27-hydroxylase.
27-Hydroxycholesterol can follow two routes.

Route A

Route B

7alpha, 26-Dihydroxy-4-cholesten-3-one can be transformed directly to conjugates of chenodeoxycholic acid, or can be converted to 3-oxo-7alpha-hydroxy-4-colestenoic acid, and then undergo side chain modifications and other reactions that lead to the synthesis of the conjugates of chenodeoxycholic acid.

Minor pathways

There are also minor pathways that contribute to bile salt synthesis, although to a lesser extent than classical and alternative pathways.

For example:

Additionally, sterol 27-hydroxylase is expressed in various tissues, and therefore its reaction products must be transported to the liver to be converted to bile salts.

Bile salts: regulation of synthesis

Regulation of bile acid synthesis occurs via a negative feedback mechanism, particularly on the expression of cholesterol 7alpha-hydroxylase and sterol 12alpha-hydroxylase.
When an excess of bile acids, both free and conjugated, occurs, these molecules bind to the nuclear receptor farnesoid X receptor or FRX, activating it: the most efficacious bile acid is chenodeoxycholic acid, while others, such as ursodeoxycholic acid, do not activate it.
FRX induces the expression of the transcriptional repressor small heterodimer partner or SHP, which in turn interacts with other transcription factors, such as liver receptor homolog-1 or LRH-1, and hepatocyte nuclear factor-4alpha or HNF-4alpha. These transcription factors bind to a sequence in the promoter region of 7alpha-hydroxylase and 12alpha-hydroxylase genes, region called bile acid response elements or BAREs, inhibiting their transcription.
One of the reasons why bile salt synthesis is tightly regulated is because many of their metabolites are toxic.

References

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