Biosynthesis and Biotransformation of Bile Acids A

Introduction: Bile acids are steroidal compounds, which contain 24 carbon atoms. They can be classifi ed into two major groups: primary and secondary.The most abundant bile acids: The primary bile acids include cholic acid and chenodeoxycholic acid, while the major secondary bile acids are deoxycholic acid and litocholic acid. Bile acids are important physiological agents for intestinal absorption of nutrients and are used for biliary lipid secretion, toxic metabolites and xenobiotics. The aim of this paper is to analyze biosynthesis and biotransformation of bile acids, as preparation for practical usage in laboratory and clinical conditions. Topic: Biosynthesis and biotransformation of bile acids: The biosynthesis of bile acids is the dominant metabolic pathway for catabolism of cholesterol in humans. The classical route of biosynthesis of bile acids is embarking on the conversion of cholesterol into 7α−hydroxycholesterol using enzyme 7α−cholesterol hydroxylase (CYP7A1). This enzyme is one of the microsomal cytochrome P450 enzyme is localized exclusively in the liver. Classical road is the main road in the biosynthesis of bile acids, and its total contribution amounts to 90% for people, and 75% in mice. CYP 7A1 enzyme is considered to be sensitive to the inhibition of carbon monoxide, and the condition for the eff ect of NADPH, the oxygen, lecithin, and the NADPH-cytochrome P450 reductase. Bile acids are important signaling molecules and metabolic controls which activate the nuclear receptor and the G protein-coupled receptors (GPCR), a signaling lipid regulation of the liver, glucose and energy homeostasis. Also, bile acids maintain metabolic homeostasis. Biotransformation of bile acids: The conversion of cholesterol into bile acids just important for maintenance of cholesterol homeostasis, but also to prevent the accumulation of cholesterol, triglycerides and toxic metabolites as well as violations of the liver and other organs. Enterohepatic circulation of bile acids from the liver to the intestine and back to the liver occupies the most important role in the processes of absorption and distribution, as well as in metabolic regulation and homeostasis. Conclusions: This physiological process is complicated and regulates the membrane transport system in the liver and intestine by means of nuclear receptors. It is very dangerous fact that toxic bile acids may be causes of infl ammation, apoptosis and cell death. On the other hand activated GPCR signaling and nuclear bile acid protects against infl ammation of the liver, intestine and macrophages. Bile acid metabolism disorders cause cholestatic liver disease, dyslipidemia, fatty liver disease, cardiovascular disease and diabetes.


INTRODUCTION
All of the bile acids are characterized by the appearance of cyclopentanoperhydrophenanthrene ring.Th e primary bile acids are synthesized in the liver.Further, they can be conjugated with glycine and taurine, before secretion into the bile ducts and the digestive tract.Th e conjugation of reducing the hydrophobicity of a bile acid.Also, in this manner increases the amphiphilic nature of the bile acids and makes them less toxic.Th ese conjugates are membrane permeable and have the ability to transform a series of lamellar lipid into the mixed micelles.Th e secondary bile acids (deoxycholic acid and litocholic acid) occur 7-α dehydroxylation and deconjugation of cholic acid and deoxycholic acid , wherein is then resorbed, conjugated and excreted from the bile.Th e modifi cation of the primary bile acids in the secondary is carried out by means of anaerobic bacteria in the colon.Th e required amount of bile acids in the body are maintained by a recirculation phenomenon enterohepatic [1].Bile acids have an important role in pharmaceutical terms.It is refl ected in the emulsifi cation of fats and cholesterol in the bile and intestines and reabsorption of the fat and fat-soluble vitamins in the intestinal lumen.In this case, the bile acids, used as replacement therapy in conditions when their synthesis is insuffi cient.On the other hand, it is important to note that chenodeoxycholic and ursodeoxycholic acids used in the treatment of breaking stones in the bile [2].Th ey may also have a signifi cant antibacterial eff ect, with better results related to the cholic acid derivatives, but derivatives of deoxycholic acid.Since the antibacterial eff ect, are used for the production of new antibiotics, which are very effi cient and have low toxicity content [3].Th e fl uorescent derivatives of bile acids, aff ect the characterization of the liver and transport through the intestine, as well as to itself during the transportation of biotransformation.In this case, the bile acid containing fl uorophores attached to the side chain.Fluorophores have a signifi cant impact, since the bound bile acids, aff ect on the transport of hepatocytes and enterocytes [4].Pass through the cell membrane and aff ect on the biotransformation of hepatocytes during transport [5].In aqueous solution the bile acid aggregate and form aggregates polymolecularity, rod clusters (socalled.Micelles).Th ey may be incorporated into cholesterol, and phospholipids.Bile acids have an important role and in agriculture [1].Th ey are used as a food supplement in animals.Some of them due to increased growth, or at Volume 4 • Number 1 • January 2017 • HOPH Scheme 1. Conformational display of cholanic acid [7] Scheme 2. The structures of the most widespread of bile acids [6] prebiotic treatment may be contributory factors of increased secretion of cholesterol and bile stones creation.Th e aim of this paper is to analyze biosynthesis and biotransformation of bile acids.

Th e most abundant bile acids
Bile acids are an integral part of the bile of animals and humans.All the bile acids of higher vertebrates can be considered derivatives of cholanic acids, which is the steroid system to 24 carbon atoms.Th e diversity of a bile acid is a result of the presence of a variable number of hydroxyl groups in their molecules.In nature, the most widespread are cholic and deoxycholic acids.Th ere are two isomeric of cholanic acid [2,6].

Biosynthesis and biotransformation of bile ACIDS
Th e biosynthesis of bile acids is the dominant metabolic pathway for catabolism of cholesterol in humans.It is important to note that the transformation of cholesterol into a bile acids occurs in complex biochemical pathways, which include the eff ect of diff erent enzymes [7].Many of these enzymes are predominantly expressed in the liver, and are localized in a number of diff erent sub-cellular compartments.It is most oft en in the human liver in the adult is converted to about 500 mg of cholesterol to bile acids [8,9].When it comes to the biosynthesis of bile acids, it involves modifi cation of the ring of cholesterol, then oxidation, shortening the side chain and eventually carried bile acid conjugation with amino acids.Consider the classical route of biosynthesis of bile acids, which is embarking on the conversion of cholesterol into 7α− hydroxycholesterol using enzyme 7α−cholesterol hydroxylase (CYP7A1).Th is enzyme is one of the microsomal cytochrome P450 enzyme is localized exclusively in the liver [10].

Biosynthesis of bill acids
Classical road is the main road in the biosynthesis of bile acids, and its total contribution amounts to 90% for men, and 75% in mice.CYP 7A1 enzyme is considered to be sensitive to the inhibition of carbon monoxide, and the condition for the eff ect of NADPH, the oxygen, lecithin, and the NADPH-cytochrome P450 reductase.It is believed that this enzyme is very important for the reason, which suppresses the backfl ow of hydrophobic bile acids returning to the liver via the portal circulation [11].By the enzyme cholesterol 7α−hydroxylase, cholesterol is converted to a 7α−hydroxycholesterol, which is in turn under the action of the enzyme 3β−hydroxy Δ 5 −C 27 −steroid oxidoreductases converted to 4-cholesten-7α-ol-3-one [11].Th e hydroxyl group of cholesterol is in the C 3 β−orientation and must be epimerize to the α-orientation during the synthesis.Th e epimerization is catalysed by HSD3B7 (3βhydroxy-Δ 5 -C 27 -steroid oxidoreductase) [11].Th at the reaction is essential for the biosynthesis and functions of bile acids, it is proved by the presence of the hidden mutations at HSD3B7.Children who have these hidden mutations develop progressive liver disease, which is characterized as cholestatic jaundice.Further in the biosynthetic pathway of enzymes 12α sterol-hydroxylase ( acid, which is a precursor for the synthesis of chenodeoxycholic acid [12].However, little is known about the rest of the enzymes, which is involved in the conversion of metabolites in chenodeoxycholic acid [14].Th e recent observation that can be formed a cholic acid and chenodeoxycholic acid when cholesterol 7α−hydroxylase is inhibited in rat hepatocytes, provides suggestive evidence that hydroxylated intermediates in 12α−position products cholic acid via the acid pathway [14].Has been shown to modify the bile acid signaling pathways in the cell, including calcium mobilization, cyclic AMP synthesis and protein kinase C activation [5].Bile acids are activated protein kinase C/Janus N-terminal kinase pathway.Stimulate secretion of pro-infl ammatory cytokines, tumor necrosis factor (TNF) and interleukin 1 (IL-1) from Kupff er cells (macrophages resistant hepatocytes), that are acti-472 Volume 4 • Number 1 • January 2017 • HOPH Scheme 3. Schematic view of primary bile acids biosynthesis pathway [17,18] vated TNF receptor signaling protein and mitogen-activated kinase (MAPK)/JNK [16,17].
Scheme 3. gives a schematic view of the classical and alternative pathway in the biosynthesis of bile acids.
Conjugated bile acids produce of mitochondrial reactive oxidative species, which activated epidermal growth factor receptor and RAF-1/MEK/EPC signaling pathway.Free and conjugated bile acids have the ability to bind to the ligand binding domain of FXR, which consist of a heterodimer with retinoid X receptor and are linked to the inversely repetitive AG-GTCA similar sequences with one nucleotide spacing (IR1) which is located in the promoters of target genes FXR, to stimulate the transcription of a gene.FXR plays a central role in the regulation of bile acid synthesis, extraction and transport of lipids, glucose and energy metabolism [17,18].
Conjugation increases the solubility of bile acids.Activation is carried out with ATP and CoA (CoA−derivatives, may be conjugated).Glycine forms glycocholic acid and glycotaurocholic acid (pK 4).In the bile at pH 6 they are signifi cantly ionized and are good detergents.Taurine formes taurocholic acid and taurodeoxycholic acid (pK 2).Th ey are almost completely ionized, and represent the best detergents in the bile.Th e ratio (glycine: taurine) =3:1 to 4:1.Under the action of bacterial enzymes, primary bile acids passing through the small intestine and colon are: deconjugation and dehydroxilation.In addition formed secondary bile acids: deoxycholic acid and lithocholic acid.Deoxycholic acid is reabsorbed and conjugated, and litocholic acid is partially reabsorbed, while the remainder is excreted in the feces [14, 18,19].

Biotransfoirmations of bile acids
Early experiments involving chemical transformation of bile acids, which are carried out in order to determine their structure.In the last 50 years, carried out chemical transformations of bile acids are mainly aimed at interconversion of bile acids, the synthesis of steroid hormones and the synthesis of certain vitamins.Cholic acid is an important starting material for the synthesis of corticosteroids.It is also a necessary precursor of chenodeoxycholic acid, which is widely applied in the treatment of gallstones (Scheme 5) [20].
Regardless of whether the C 7 and C 12 hydroxyl groups replaced by hydrogen cholic acid, certain artifi cial problems are inevitable.Th ey include the following steps: -the need for a selective protection of the two hydroxyl groups (usually by acylation), and -selection of the reagent (mainly, ie.most often an oxidation) for the selective transformation of the remaining hydroxyl groups [20].
Cholic acid contains one C 3 equatorial OH group and two axial hydroxyl groups in the position of C 7 and C 12 .A equatorial hydroxyl group in the position of C 3 is subject to rapid esterifi cation than the axial hydroxyl groups in the positions of C 7 and C 12 .It has confi rmed the greater reactivity of the C 7 axial hydroxyl group compared to the axial hydroxyl group in the position of C 12 .Greater reactivity of the C 7 can not be attributed only to steric eff ects.Th ese characteristics reactivity cholic acid are experimentally confi rmed in numerous synthetic studies [20].
Borshe obtained a 3-carbethoxy derivative (cathylate) in a good yield, by the infl uence of ethyl chloroformate (ClCO 2 C 2 H 5 ) on methyl cholate in pyridine.When it is used the excess of acetyl chloride in acetic acid, the triacetyl derivative is obtained in a yield of 30-35%.Later, Fieser and associates obtained the 3-monocathylate in good yield, using piriding-dioxane as a solvent.Although, Wieland and Kapitel were synthesized the methyl 3α, 7α−diacetoxy−12α−hydroxy−5β−cholanate (compound 5) by reaction with acetic anhydride in pyridine, as well as by the infl uence of acetyl chloride in acetic acid on the methyl cholate (compound 1) [20].
In addition, they also synthesized a 3α, 7α−diacetyl derivatives, using a acetyl chloride or acetic anhydride and HCl.In the same study they were described a synthesis of 7α−acetyl derivative of cholic acid by partial acid saponifi cation of 3α, 7α−diacetyl deriva- Scheme 7. The synthesis of lithocholic acid from methyl cholate [20] diethyl ether (CH 3 ) 2 CO-(C 2 H 5 ) 2 O).In a 50% yield by partially acetylated of methyl cholate (the molar ratio of acetic anhydride: cholic acid was (4:1) in a refl ux of benzene for 2 hours (Scheme 6) [20].
In order to receive a chenodeoxycholic acid, Fisher et al also synthesized of 3α,7α−diacetyl derivative by the reaction of acetylation of methyl cholate with acetic anhydride in pyridine and pyridine-dioxane at room temperature for about 20  Dayal et al describe the reaction of esterifi cation, deacetylation and deformylation of bile acids executed under microwave conditions.Th ey used a methanesulfonic acid or p-toluenesulfonic acid in methanol, as the catalyst instead of the commonly used strong mineral acids (Th e Schemes 9 and 10).Recently exposure to microwave radiation (MW) has become very popular in terms facilitating of fast esterifi cation, hydrolysis and conjugation of bile acids.Reactions, which are carried out by standard procedures were compared , with a fi nal reactions by the infl uence of microwaves, obtained in the same purity and in the same yield , but in a much shorter period of time [20].
In .Th e reaction was conducted under the infl uence of microwaves in a period of 90s [20].
In the sixth step the 3α−hydroxy−7keto−5β−cholanate (compound 19) was subjected to the reaction of selective acetylation or formylation in the position of C3, as well as the Wolff -Kishner reduction of the keto group in position 7, to give a 3α−acetoxy (formyloxy)−5β−cholanic acid (compound 20) [20].
In later studies, Wieland and coworkers described the synthesis of which is performed in order to obtain a lithocholic acid [20].
Hydrophobic bile acids such as CDCA (chenodeoxycholic acid) is the most  [20] effi cient endogenous FXR ligand, and a hydrophilic bile acid, such as for example ursodeoxycholic acid and muricholic acid do not activate FXR [18].Bile acids bind and activate pregnane X receptor (PXR) and the vitamin D receptor (VDR) receptors.Th ese two receptors have a very important roles in the detoxifi cation of bile acids, drugs and xenobiotics [21].Deoxycholic acid actives FAS receptor and JNK induction of acide sphingomyelinsized generated ceramide in primary rat hepatocytes [16].Also, very important information is that the bile acids stimulate insulin receptor signaling.In the brown adipose tissue, bile acids have the ability to activate the receptor TGR5, Gα i protein-coupled receptor.TGR5 receptor stimulates cAMP production, which induces iodothiron diodinase (D2) and produces thyroid hormone T3, which contributes to the stimulation of energy metabolism and improvement of glucose tolerance and insulin sensitivity [10].
TGR5 receptor is not expressed in hepatocytes, and is usually localized in the sinusoidal endothelial cells.In endocrine cells, TGR5 has a role in the stimulation of glucagon, which is similar to peptide 1 and has antidiabetic activity.FXR receptor produces FGF19, which is secreted from the intestine to blood circulation in response to postprandial effl ux of bile acids to inhibit bile acid synthesis in the liver [10].CDCA and GW4064 rapidly induce FGF19 mRNA expression, FGF19 protein secretion, and tyrosine phosphorylation of FGFR4, but inhibit CYP7A1 mRNA ex-pression in primary human hepatocytes suggesting that liver-produced FGF19 is secreted from hepatocytes to activate FGFR4 signaling in hepatocytes by an autocrine or paracrine mechanism [9,13].In the intestine, bacteria overgrowth damages intestine barrier function and causes IBD, diarrhea, and impaired drug metabolism, detoxifi cation, and absorption.Bile acids control gut bacteria overgrowth and protect against infl ammation.Gut microbiota also play a role in biotransformation of bile acids and aff ected bile acid composition and metabolism via FXR and TGR5 signaling in the liver [16,17].In the liver, high levels of bile acids cause liver injury.Bile acids also have anti-infl ammatory functions by activating FXR and TGR5 signaling in hepatocytes to protect against metabolic diseases such as NAFLD, diabetes, and obesity (Scheme 16) [18−21].
Most of the litocholic acid is excreted in the feces.A small amount of litocholic acid circulates in the liver, where is conjugated on the 3-hydroxy position sulfotransferase (SUL-T2A1) and rapidly secreted into a bile [5,22].
Bile acid and steroid hormones, infl ammatory cytokines and growth factors preventing the transcription of the CYP7A1 through the 5'-upstream region of the promoter.Th e proximal promoters of CYP7A1 at rats are ligand-activated transcription factors, which play important roles in embryogenesis, development and metabolism.Human CYP7A1 promoter does not bind LXR and does not produce LXR, due to a change in the DR4 motifs in BARE-I sequences.It has been www.hophonline.orgScheme 15.The synthesis of chenodeoxycholic acid from methyl cholate [20] confi rmed that transgenic mice, carrying the human CYP7A1, do not respond to the high levels of cholesterol in the diet, are not induced by the transgene and synthesis of bile acids is not stimulated by these mice.HNF 4 transactive a CYP7A1 promoter activated with coactivator peroxisome proliferator-activated γ−receptor coactivator 1α.Mutation certain DR1 sequences drastically reduces the basal activity of CYP7A1 promoter and his answers to the inhibition of bile acid.Insulin regulates transcription factors Fox O1 is associated with insulin sequence in response to the CYP7A1 promoter in rats and contributions CYP7A1 transcription in rats [4,22].

CONCLUSION
Bile acids are synthesized via the classic pathway initiated by cholesterol 7α−hydroxylase (CYP7A 1 ), and via alternate pathways, one of which is initiated by sterol 27-hydroxylase (CYP27A 1 ).Th ese studies used mice lacking cholesterol 7α−hydroxylase (Cyp7A 1 ) to establish whether the loss of the classic pathway aff ected cholesterol homeostasis diff erently in males and females, and to determine if the rate of bile acid synthesis via alternate pathways was responsive to changes in the enterohepatic fl ux of cholesterol and bile acids.Th e acidic pathway may be quantitatively important in the synthesis of bile acids in patients with liver diseases and newborns.In mice, the majority of the bile acids is conjugated with taurin , to form taurine conjugates.Conjugation of bile acids contribute to increased ionization and solubility in physiological pH conditions, prevent precipitation of Ca 2+ , then reduce the passive absorption and resistant to cleavage using pancreatic carboxypeptidase.In the distal intestine, conjugated bile acids are fi rst deconjugated, and then bacterial 7α−dehydroxylase converted a cholic acid and chenodeoxycholic acid into a deoxycholic acid and litocholic acid.
Most litocholic acid is excreted via the feces, a small quantity is distributed in the liver and rapidly conjugated with sulfi dation process, and then excreted from the bile.As the main method for detoxifi cation of bile acids in humans servant sulphation.Bile acids such as CDCA and UDCA have been useful in the treatment of gallstones.UDCA is able to sequester cholesterol from the surface of gallstones, thereby making them smaller and easier to be excreted.Th e secondary bile acids, DCA and LCA present in the colon, have been shown to act as tumor promoters.Th e proposed mechanism may off er some insight into the benefi ts of a diet low in saturated fat and high in fi bre.UDCA and CDCA derivatives have been shown to be antiproliferative and are capable of inducing apoptosis in carcinoma cells.
In mice chenodeoxycholic acid hydroxylated to α−muricholic acid and 6β−muricholic acid, which are classifi ed in two main primary bile acids in mice.7α−hydroxy group in the chenodeoxycholic acid can be epimerized to 7β−OH group of ursodeoxycholic acid.Hydroxylation at 6α∕β or 7β−position leads to increase the solubility of bile acids, reducing their toxicity.Physiological importance of FXR receptor depends of pathways in the regulation of metabolism of bile acids.Bile acids activate FXR receptor, which plays a key role in maintaining metabolic homeostasis.Th e activated membrane G proteincoupled bile acid receptor (Gpbar-1) plays a role in the stimulation of energy metabolism.Th en, it is important to emphasize that protects the liver and intestine from infl ammation and steatosis (fatty liver) and improves insulin sensitivity.Activated GPCR, sphingosine-1-phosphate receptor 2 (S1P2) is important in lipid metabolism.Also recently discovered role of bile acids in the integrated regulation of lipids, glucose and energy metabolism.Th e serum of bile acids is higher in patients with prior gastric bypass than in overweight and severely obese patients without gastric bypass, and serum bile acids were positively correlated with serum glucagon-like peptide-1 (GLP-1).Th e mechanism underlying FXR/FGF19/FG-FR4 signaling in inhibition of CYP7A1 transcription and bile acid synthesis remains to be elucidated.