Pathways and Inborn Errors of Bile Acid Synthesis

Bile acids are synthesized from cholesterol through 17 different enzymes located in different intracellular compartments of hepatocytes. Defects have been identified in the genes encoding the enzymes involved in the bile acid synthesis pathways and nine different diseases have been identified so far. In this review, four different biosynthetic pathways of bile acids together with disorders of bile acid synthesis are described. In inborn errors of bile acid synthesis clinical findings can range from liver failure to cirrhosis in infancy or progressive neuropathy in adolescence / adulthood. Laboratory analysis of urine profiling of bile acids is important in early diagnosis and


INTRODUCTION
Bile acids are amphipathic molecules consisting of a 24-carbon steroid core and a carboxyl side chain [1,2]. Beside their role in the cholesterol catabolism, their amphipathic natures make them highly effective detergents in the digestion and absorption of dietary lipids and lipid-soluble vitamins in the small intestine [3]. Primary bile acids are synthesized by multistep enzymatic reactions involving 17 enzymes found in different intracellular compartments (endoplasmic reticulum, mitochondria, peroxisome, cytosol) of the hepatocytes [4]. Primary bile acids are then conjugated with glycine or taurine prior to secretion into bile [5]. Conjugation increases the water solubility of bile acids and reduces the passive diffusion during passage through the small intestine [6,7]. Conjugated bile acids are actively absorbed in the terminal ileum by specific receptors and enter to the enterohepatic circulation where they return to the liver for further modification [3]. The synthesis of bile acid is tightly controlled to keep the bile acid concentrations at constant levels, since the accumulation of bile acids in liver and other tissues can be highly toxic [8]. In this review, four different biosynthetic pathways of bile acids together with the disorders of bile acid synthesis are described with the recent studies in the literature.

Chemical Structure and Properties of Bile Acids
Bile acids are amphipathic molecules. Hydroxyl groups are found on only one side of the molecule and give the molecule hydrophilic character. The other face does not contain hydroxyl groups and it is hydrophobic. Due to this amphipathic nature, increased bile acid concentrations decreases the surface tension. When a certain amount of bile acid is present in the water, the hydrophobic groups are clustered together and the hydrophilic groups are positioned towards the aqueous environment. Above a critical bile acid concentration, micelles are formed. This critical bile acid concentration is called critical micelle concentration (CMC). Surface tension remains constant above CMC, but increasing bile acid concentration increases the micelle concentration [9]. The hepatocytes synthesize two primary bile acids: Cholic acid (CA; 3α, 7α, 12α-trihydroxy-5β-cholanoic acid) and chenodeoxycholic acid (CDCA; 3α, 7α-dihydroxy-5β-cholanoic acid). They are often present as N-aminoacyl conjugates. All of the primary bile acids are α-hydroxylated. This polar hydrophilic group is on one side of the molecule that gives characteristic surface-active properties to bile acids. Deoxycholic acid (DCA; 3α, 12α-dihydroxy-5β-cholanoic acid) and lithocholic acid (LCA; 3α-monohydroxy-5β-cholanoic acid) are secondary bile acids, and formed by bacterial 7-dehydroxylation of CA and CDCA. Five percentage of total amount of bile acid is in the form of ursodeoxycholic acid (UDCA; 3α, 7β-dihydroxy-5β-cholanoic acid) which is formed in the liver and intestines [10,11].

Enterohepatic Circulation
The enterohepatic circulation of bile acids between the liver and intestine was first described by Borelli in 1681 [12,13]. Approximately 95% of the bile acids in the pool are recovered by the enterohepatic circulation. It also serves as a feedback mechanism in which cholesterol 7α-hydroxylase (CYP7A1) gene transcription and bile acid synthesis are inhibited to maintain bile acid homeostasis in humans [14]. The enterohepatic circulation of bile acids mainly consists of biosynthesis, secretion and reabsorption. After their synthesis from cholesterol by a series of enzymatic reactions in the liver, most of the bile acids stored in the gall bladder. By the stimulation of food intake, bile acids are released into the intestines through the bile duct and metabolized to secondary bile acids by intestinal bacterial flora [15]. Unconjugated bile acids are removed by passive diffusion in the jejunum and colon, while the conjugated bile acids are taken up into enterocytes by the apical sodium-dependent bile acid carrier (ASBT). In total, 95% of bile acids are reabsorbed by enterocytes and the remaining is excreted through the feces. This amount (5%) is compensated by de novo bile acid synthesis in the hepatocytes [16].

Pathways of Bile Acid Synthesis
The primary bile acids are synthesized from cholesterol with a complex series of reactions involving 17 different enzymes found in endoplasmic reticulum, mitochondria, cytoplasm and peroxisomes [17]. Recent studies have identified four major biosynthetic pathways of bile acids, named the Classic / Neutral pathway, the Alternative / Acidic pathway, the Yamasaki pathway and the 25-hydroxylation pathway [18,19] (Figure 1). Cholesterol is a C27-sterol and has a double bond at position C5 (cholest-5-en-3β-ol). Cholesterol is converted into primary bile acids CA (3α, 7α, 12α-trihydroxy-5β-cholanoic acid) and CDCA (3α, 7α-dihydroxy-5β-cholanoic acid). One hydroxyl group (at C7α position) and two hydroxyl groups (at C7α and C12α positions) are added for the synthesis of CDCA and CA, respectively. The double bond at the 5-position is reduced; the 3β-hydroxyl group is converted to a 3α-hydroxyl group. The aliphatic side chain is oxidized to a carboxyl group and the structure is three carbon atoms shortened resulting in C24cholanoic acid formation [19].

1.Classical / Neutral Pathway:
The classical or neutral pathway is the most important biosynthetic pathway responsible for the production of 90% of the total amount of bile acids [20]. It is named as neutral pathway because the intermediate metabolites are neutral sterols. This pathway is only in the liver and synthesizes two primary bile acids, CA (trihydroxy-bile acid with hydroxyl groups at C3, C7 and C12) and CDCA (dihydroxy-bile acid with hydroxyl groups at C3 and C7) [21,22]. CA and CDCA are synthesized at almost equal amounts in this pathway [23,24]. The classical pathway of bile acid synthesis begins with the rate-limiting enzyme cholesterol 7α-hydroxylase (CYP7A1). The reaction of CYP7A1 produces 7α-hydroxycholesterol which is converted to 7α-hydroxy-4-cholesten-3-one. 7α-hydroxy-4cholesten-3-one is a common precursor of CA and CDCA which are synthesized in the subsequent reaction steps. It is also called C4 and currently used as a biomarker in determining the rate of bile acid synthesis. CA is formed from C4 by the action of sterol 12α-hydroxylase (CYP8B1), whereas CDCA is formed in the absence of CYP8B1. The ratio of 12α-hydroxylated bile acids (CA and DCA) to 12α-non-hydroxylated bile acids (CDCA and LCA) in the bile acid pool is determined by the CYP8B1. Subsequently, steroid side chain oxidation reactions take place by mitochondrial enzyme sterol 27-hydroxylase (CYP27A1), and followed by the removal of the propionyl group for the formation of C24 bile acids with peroxisomal β-oxidation [14].

3.Yamasaki Pathway:
C24 bile acids and 3β-hydroxy-5-cholestenoic acid in the Yamasaki pathway are formed in a similar way as in the alternative / acidic way. After this step, the hydroxylation reaction carried out by the enzyme 7α-hydroxylase in humans produces 3β, 7α-dihydroxy-5-cholenoic acid. It is the CDCA precursor, and at the last step of the reactions CDCA occurs [33]. CDCA is thought to be the main product of the Yamasaki pathway in humans. The exact contribution of the Yamasaki pathway to the bile acid pool in humans has not been fully established. However, the presence of monohydroxy bile acids in fetal bile and the relatively high levels of these bile acids in meconium and amniotic fluid suggest that this pathway is at least important during development [34].

Bile Acid Conjugation
Primary bile acids synthesized by the enzymatic reactions in the liver, are conjugated with glycine and taurine with amidation reactions at carboxyl groups. The conjugation reaction is mediated by two enzymes: Bile acid coenzyme A synthase (BACS) and bile acid CoA-amino acid N-acyltransferase (BAAT) [12]. (Figure 1)   Conjugation with amino acids significantly increases the polarity of the molecule [21]. Unconjugated bile acids have about 5 pKa. Conjugation reduces pKa of bile acids, increases water solubility, and reduces their ability to cross lipid membranes [10].

Defects of Bile Acid Synthesis
Inborn errors of bile acid synthesis (IEBAS) are rare genetic disorders of liver metabolism that cause chronic liver disease and fat-soluble vitamin deficiencies in childhood [35,36]. These defects are characterized by the inability to produce normal bile acids. There is accumulation of unusual bile acid and bile acid intermediates due to enzyme defects in the bile acid biosynthetic pathways from cholesterol [37,38]. Although 17 enzymes are present in the bile acid biosynthetic pathway, only 9 types of IEBAS have been identified to date [39] (Table 1).
Long-term jaundice in the neonatal period is one of the main findings of IEBAS. In cases of obstructive jaundice with normal levels of total cholesterol and gamma-glutamyl transpeptidase (GGT); hepatic dysfunction or cirrhosis with an unknown cause; family history of hepatic dysfunction; no improvement with the treatments after a diagnosis of neonatal hepatitis; clinical symptoms similar to tyrosinemia; no itching despite cholestasis; recurrent diarrhea or steatorrhea; symptoms of fat-soluble vitamin deficiency (intracranial hemorrhage, rickets, hypocalcemic seizures, etc.), IEBAS should be considered. To date, 9 types of IEBAS have been discovered. Urine bile acid profiling is an important tool in the diagnosis of IEBAS (Table 2).

2.Δ4-3-Oxosteroid-5β-reductase deficiency:
This deficiency in bile acid synthesis is due to a defect in the aldo-ketoreductase family 1 member D1 (AKR1D1) gene, which encodes the key enzyme ∆4-oxosteroid 5β-reductase [47]. This enzyme catalyzes the reduction of Δ4-3-ketosteroid AB to form the cis ring structure AB [48]. The deficiency of this cytosolic enzyme results in misreduction of the double bond between the C-4 and C-5 of the sterol A-ring, which is the basic step in major bile acid synthesis. Thus, there is a misconversion of the 3-oxo intermediates to the corresponding 3α-hydroxyl products. This deficiency results in significantly reduced primary bile acid synthesis and deposition of 3-oxo-Δ4 and allo (5α-H) -bile acids [32,49]. Accumulated unsaturated C27-3-oxo-∆4 steroids are converted to the corresponding C24bile acids and can be detected in both blood and urine [19]. The main findings in 5β-reductase deficiency are normal or slightly elevated TBA and GGT in blood; high levels of conjugated bilirubin and alanine aminotransferase (ALT), and steatorrhea.

4.Cholesterol 7α-hydroxylase deficiency:
Cholesterol 7α-hydroxylase is found in the endoplasmic reticulum and expressed only in the liver. It is the rate-limiting step of bile acid synthesis [21]. It is located in the classical pathway, the main bile acid biosynthetic pathway in adults. Since the alternative pathway can still produce bile acids via oxysterol 7α-hydroxylase, CYP7A1 defect does not cause deficiency in bile acids [53].  [54]. It is caused by mutations in the CYP27A1 gene encoding sterol 27-hydroxylase. The defect blocks the initial modification of the cholesterol side chain, which leads to a reduction in the synthesis of primary bile acids together with the production and excretion of bile alcohols [55]. As a result of CYP27A1 deficiency, 5α-cholestanol (a 5α-dihydro cholesterol derivative) is accumulated in the tissues. Intense accumulation of 5α-cholestanol in the brain results in severe neurological dysfunction [56]. The increase in 5α-cholestan-3β-ol in the nervous system and high levels of this sterol in blood are unique features of CTX patients [57,58].

7.
Bile acid-CoA: amino acid N-acyltransferase deficiency: Conjugation of CA and CDCA with an amide bond to C-24 of an amino acid (taurine or glycine) is the final step in primary bile acid synthesis [61]. BAAT deficiency is caused by mutations in the BAAT gene encoding the liver-specific bile acid-CoA: amino acid N-acyltransferase (a peroxisomal enzyme), which converts CoA esters of bile acids into taurine or glycine conjugates [38].
8.Bile acid-CoA ligase deficiency: The final step in bile acid synthesis involves conjugation with amino acid glycine and taurine [62]. The two enzymes catalyze reactions that lead to amidation of bile acids. First, a CoA thioester is formed by the rate-limiting enzyme bile acid-CoA ligase, followed by the combination of glycine or taurine in a reaction catalyzed by a cytosolic BAAT [52]. BACS is the first enzyme required for amidation of primary bile acids and converts C24-bile acids into their corresponding bile acyl-CoAs [63]. CoA is then replaced by BAAT with taurine or glycine. BACS is a liver-specific enzyme found in the endoplasmic reticulum and is required for the conjugation of bile acids deconjugated by enterohepatic circulating intestinal bacteria [64]. Due to a homozygous mutation in SLC27A5, only two siblings were identified with BACS deficiency [65].

CONCLUSION
In IEBAS clinical findings can range from liver failure to cirrhosis in infancy or progressive neuropathy in adolescence or adulthood. Early diagnosis is important for early treatment and crucial to prevent fatal outcomes in which urine bile acid profiling is an important tool [38,39].

CONFLICT of INTEREST
There is no conflict of interest.