The main, neutral or classical pathway

In this metabolic route, the modification of the cholesterol ring precedes the cleavage of its side chain (Figure 2). The pathway starts with the hydroxylation at C7, which is catalyzed by the microsomal enzyme CYP7A1, a member of the cytochrome P450 family of proteins. CYP7A1 is exclusively expressed in hepatocytes and, owing to its critical role in this biosynthetic pathway, is the element on which most regulatory factors converge [9]. The product of the CYP7A1-mediated reaction is 7α-hydroxycholesterol, which is then converted into 7α-hydroxy-4-cholesten-3-one (7αC4) by the action of microsomal HSD3B7. Then the microsomal enzyme sterol 12α-hydroxylase (CYP8B1) can add a hydroxyl group at C12, generating a series of intermediates whose final product is CA. If 12α-hydroxylation does not occur, the final compound generated by this route is CDCA, which highlights the role of CYP8B1 in controlling the CA/CDCA ratio and justifies its tight regulation [10]. In both cases, the double bond at the C4 position is then reduced by the cytosolic enzyme AKR1D1, resulting in the presence of a hydrogen at the 5β position. The last steroid ring modification is catalyzed in the cytosol by 3α-hydroxysteroid-dehydrogenase (AKR1C4), which reduces the 3-oxo group to a 3-hydroxyl with α-stereochemistry.

Display full size

Figure 2. 

Enzymes involved in the main, neutral, or classical pathway of bile acid (BA) synthesis. Several inborn errors leading to autosomal recessive disorders have been described to affect some of these enzymes (red bars). Although all resulting diseases are infrequent, the level of incidence is indicated by the number of asterisks as low (***), very low (**), and extremely low (*). 7αC4:7α-hydroxy-4-cholesten-3-one; CA: cholic acid; CDCA: chenodeoxycholic acid; DHCA: 3α,7α-dihydroxy-5β-cholestanoic acid; THCA: 3α,7α,12α-trihydroxy-5β-cholestanoic acid

The shortening of the cholesterol side chain begins with the 26-hydroxylation due to the mitochondrial enzyme CYP27A1 [11]. Subsequent oxidation to an aldehyde and then to a carboxylate group allows the formation of the C27-BA intermediates DHCA and THCA. The hydroxylation of the pro-chiral side chain by CYP27A1 results in the exclusive formation of the 25R-stereoisomer of both compounds, which must then be activated by a thioester bond with an acetyl-CoA molecule. Both BACS and very long-chain acyl-CoA synthetase (VLCS) are involved in the generation of C27-BA-CoAs, which are taken up by the peroxisome through the ABCD3 transporter, also called PMP70. The reactions in this organelle start with the racemization of the C25 chiral center from the R- to the S-configuration by AMACR. Next, the stereospecific enzyme ACOX2 oxidizes the 25S-isomers, generating a double bond at position C24, which is then hydroxylated and subsequently oxidized to generate a 24-keto group by the action of HSD17B4 or DBP. SCPx or SCP2 accounts for the thiolytic cleavage of this ketone into propionyl-CoA and CA-CoA or CDCA-CoA. Finally, the conjugation of these BA-CoAs with glycine or taurine is carried out by BAAT, generating the four primary human BAs, i.e., taurocholic acid (TCA), GCA, taurochenodeoxycholic acid (TCDCA) and glycochenodeoxycholic acid (GCDCA).

The acidic pathway

In this metabolic pathway, which is quantitatively more important during early life and infancy, the first step is the formation of a C27-carboxylic acid, which is then followed by modifications of the steroid nucleus and further peroxisomal chain shortening to finally generate C24-BAs, preferentially CDCA [12]. The acidic pathway is initiated by CYP27A1, which carries out the hydroxylation of the C26 position generating the 25R-stereoisomer. Despite catalyzing the first reaction of the process, CYP27A1 is not its limiting enzyme. Instead, the crucial step appears to be the steroidogenic acute regulatory protein-related lipid transfer domain 1 (STARD1)-mediated transport of cholesterol to the inner mitochondrial membrane [13]. Once (25R)-3β-hydroxy-5-cholestenoic acid is obtained, its 7α-hydroxylation is carried out by CYP7B1. This constitutes a difference from the neutral pathway in which this hydroxylation is mediated by CYP7A1. Next, the same enzymes involved in the classic pathway (HSD3B7, AKR1D1, and AKR1C4) complete the ring modifications. Other oxysterols such as 24- and 25-hydroxycholesterol, highly relevant in regulating lipid metabolism, can also be incorporated into the biosynthesis of BAs at the beginning of this pathway. Both metabolites can be obtained by the action of CYP27A1. Moreover, brain CYP46A1 can also minimally contribute to this metabolic route through the synthesis and release to the blood of 24-hydroxycholesterol [14].

The Yamasaki pathway

This route shares with the acidic pathway the initial formation of (25R)-3β-hydroxy-5-cholestenoic acid through CYP27A1. Subsequently, the shortening of the side chain is carried out at the peroxisome to obtain 3β-hydroxy-5-cholenoic acid, whose steroidal nucleus is then modified to finally generate C24-BAs, mainly CDCA. Although the contribution of this route to the biosynthesis of BAs is unknown, the presence of 3β-hydroxylated intermediates in amniotic fluid suggests its importance during intrauterine life [15].

The 25-hydroxylation pathway

This alternative route of BA synthesis, in which the side chain shortening is independent of peroxisomal β-oxidation, begins with the 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol by the action of the microsomal enzyme CYP3A4 [16]. The product of this reaction, 5β-cholestane-3α,7α,12α,25-tetrol is then hydroxylated to 5β-cholestane-3α,7α,12α,24S,25-pentol [17]. This hydroxylation is likely catalyzed by CYP27A1, because patients with cerebrotendinous xanthomatosis (CTX), who lack active CYP27A1, accumulate cholestanetetrol when the direct precursor is administered [18, 19]. Finally, conversion to CA would be carried out rapidly through a 24-keto intermediate by one or more cytosolic enzymes with stereospecific selectivity for the 24S isomer [18].

Additional conjugation reactions

Besides the amidation with taurine or glycine, under certain circumstances mature C24-BAs can be additionally conjugated with other polar polyatomic moieties, which results in compounds with enhanced water-solubility, reduced cytotoxicity, and facilitated renal elimination. More than 70% of BAs in urine are conjugated at 3α-position with a sulfate group, being this reaction carried out by sulfotransferase family 2A member 1 (SULT2A1) [20]. This biotransformation is particularly relevant in the case of lithocholic acid (LCA), a very hydrophobic and toxic BA, which is sulfated in high proportion, thus increasing its water solubility and facilitating its renal and fecal excretion [21]. Glucuronidation is mediated by uridine-diphospho (UDP) glucuronosyltransferase family 1 member A3 (UGT1A3), as well as other enzymes of the UGT family. This conjugation reaction can be performed at different sites of the molecule, depending on the involved isoenzyme, e.g., UGT1A3 mainly generates glucuronide-BAs at C24 position [22]. These conjugated BA species are a minority in healthy individuals but can become quantitatively relevant in patients with cholestasis [20, 23].

CYP7A1

Deficient CYP7A1 activity was described in three siblings with statin-resistant hypercholesterolemia. Interestingly, two of these patients were male and had hypertriglyceridemia and a history of biliary lithiasis. One presented an accumulation of cholesterol in the liver biopsy, along with decreased fecal excretion of BAs and an increased proportion of CDCA in feces. All three individuals were homozygous for a mutation in the CYP7A1 gene, leading to a frameshift (L413fsX414) that markedly alters the active site of the enzyme and subsequent results is the lack of function [24]. CYP7A1 deficiency does not lead to acute liver disease, as the reduction in BA synthesis by the neutral pathway can be compensated by an increment of the alternative acidic route. However, pathological repercussions appear in adulthood. As its inheritance seems co-dominant, individuals with this defect in heterozygosis also have high cholesterol levels, early gallstones formation, and even premature coronary and peripheral vascular disease [24]. Mice lacking CYP7A1 display reduced BA synthesis, while cholesterol serum levels and mortality differ between models probably due to their different genetic backgrounds [25–28].

HSD3B7

Deficiency in HSD3B7 activity [Online Mendelian Inheritance in Man (OMIM): 607765] is the most frequent genetic defect affecting BA synthesis. In an American study from 1987 to 2016, HSD3B7 deficiency was found in 34% out of 297 patients with inherited errors of BA metabolism included in that cohort [29]. The inheritance of this disorder, characterized by a diverse spectrum of mutations in the HSD3B7 gene, is autosomal recessive [30]. In these patients, there are low levels of mature BAs due to their inability to reduce the double bond in the Δ5 position of the cholesterol ring, preventing the epimerization of the hydroxyl in 3β and causing the accumulation of unsaturated 3β,7α-dihydroxy-5-cholenoic acid and 3β,7α,12α-trihydroxy-5-cholenoic acid, which are usually sulfated in the C3 position. It has been proven that 3β-hydroxy-5-cholenoic acids fail to act as agonists of the main nuclear receptors involved in BA homeostasis farnesoid X receptor (FXR), pregnane X receptor (PXR) and constitutive androstane receptor (CAR), resulting in their impairment to exert hepatoprotective actions and thus explaining the mechanism for progressive cholestatic liver disease [31]. Sulfation of these toxic Δ5-intermediates will favor their elimination, as is the case for LCA [21]. Serum aminotransferases [alanine aminotransferase (ALT), aspartate aminotransferase (AST)] are elevated, while gamma-glutamyl transpeptidase (GGT) levels are normal. Clinically, HSD3B7 deficiency is characterized by neonatal cholestasis with jaundice, impaired absorption of lipids and fat-soluble vitamins, and steatorrhea, which progresses to more severe liver alterations, such as cirrhosis and hepatomegaly, with or without splenomegaly [1, 32]. However, in some cases, the clinical presentation is atypical [33] or even asymptomatic [34]. HSD3B7 deficiency in mice leads to cholesterol malabsorption and a very high mortality rate [35]. Early treatment with CA or CDCA is effective in normalizing liver function in these patients [33, 36]. Accurate determination of 3β-hydroxy-Δ5-BA sulfates is critical for both diagnosis and dose titration and monitoring response to therapy with primary BAs [4, 5].

AKR1D1

Deficiency in AKR1D1 activity (OMIM: 235555) is the second most frequent disorder of BA metabolism, accounting for 20% of cases in the American cohort mentioned above [29]. Patients with this disorder show similar symptomatology to those with HSD3B7 deficiency. Still, in this case, there is an accumulation of 4-cholenoic BAs, which may have a 3-oxo group, along with the presence of 5α-BAs (flat “allo” BAs) [37]. Liver function tests show elevated serum aminotransferases and GGT. The typical clinical presentation resembles neonatal hepatitis, with rapid progression of liver disease, leading to cirrhosis and liver failure in infancy. In contrast to patients, mice with AKR1D1 deficiency do not show overt signs of cholestasis, hepatic inflammation, or liver damage [38]. Due to the reduced synthesis of primary BAs, treatment based on BA replacement therapy could be beneficial for these patients [37]. However, conversely to HSD3B7 deficiency, treatment of pronounced AKR1D1 deficiency with CDCA worsen cholestasis and liver function tests. This may be due to the enhanced generation in these patients of 3-oxo-4-cholenoic acids which cannot be sulfated at the C3 position, and which inhibit the secretion of exogenously administered CDCA [3]. Relief of cholestasis and improvement of liver function in these patients is achieved by administration of the less toxic CA in combination with CDCA [39]. Different autosomal recessive mutations in the AKR1D1 gene, both in homozygosis and heterozygosis, have been associated with this disorder. Patients with a longer prothrombin time have a worse prognosis and a poorer response to treatment with BAs [40]. Importantly, secondary AKR1D1 deficiency may occur due to several liver diseases, highlighting the need for genotyping AKR1D1 deficient patients to elucidate the actual etiology of the disease [41].

CYP7B1

This enzyme is specific to the alternative acidic pathway. The lack of CYP7B1 enzymatic activity (OMIM: 613812) is one of the rarest defects in BA synthesis, as it has been reported in few children [29]. However, a recent study on Taiwanese infants suggested that the incidence of this disorder may be higher than believed due to its misdiagnosis in children [42]. The accumulation of Δ5-monohydroxylated and saturated 3β-monohydroxylated BAs leads to severe neonatal liver disorders such as progressive cholestasis, fibrosis, and cirrhosis with fatal consequences [43]. The serious clinical consequences of CYP7B1 deficiency highlight the importance of the alternative acidic pathway in early life in humans. Conversely, in Cyp7b1^−/− mice, BA metabolism as well as liver function are normal, and the loss of this enzyme is compensated by increases in the synthesis of BAs by other pathways [44]. Along with 3β-hydroxy-Δ5-cholenoic acid, which makes up 90% of total plasma BAs, various oxysterols such as 25-hydroxycholesterol and 27-hydroxycholesterol accumulate and help to differentiate this disorder from AKR1D1 deficiency [45]. Serum aminotransferases are markedly elevated, while GGT levels are normal. Unlike other defects in enzymes of BA synthesis involved in the biotransformation of the steroid ring, CYP7B1 deficiency is a particularly severe condition that does not respond to CA. As therapeutic options, liver transplantation [46] or CDCA treatment [47] has been effective in some patients.

CYP27A1

Deficient mitochondrial CYP27A1 activity (OMIM: 213700) causes CTX, a rare autosomal recessive lipid storage disease. Its clinical manifestations in affected children include neonatal cholestasis, diarrhea, cataracts, and growth retardation. The situation worsens in adults with the onset of xanthomas and neuropsychiatric symptoms such as ataxia and dementia [48]. This alteration is characterized by a deficit of BAs and a deposit of cholesterol and cholestanol in different tissues. The blockade of the neutral pathway at this level generates an accumulation of 5β-cholestane-3α,7α,12α-triol, which is diverted through the 25-hydroxylation route for its biotransformation into CA [19]. However, this alternative pathway cannot entirely compensate for the lack of CYP27A1-mediated side chain shortening for C24-BA production. Hence, bile alcohols are generated and accumulated, which is a helpful plasma biomarker for the diagnosis of this disorder [49]. On the contrary, mice lacking CYP27A1 show a milder phenotype with lower accumulation of oxysterols and BA intermediates, probably due to a more active 25-hydroxylation pathway in this species [50, 51]. Consistently, these animals do not develop metabolic and neurologic CTX-related pathological abnormalities [52, 53]. The treatment of CTX patients with CDCA restores normal C24-BA levels and decreases those of cholestanol and bile alcohols, thus preventing severe clinical symptoms if the treatment is started early enough [54].

Peroxisomal defects

Peroxisomes are involved in many cellular functions, e.g., long-chain fatty acid β-oxidation, pipecolic and phytanic acid oxidation, synthesis of plasmalogens, cholesterol, BAs, and regulation of the oxidation-reduction status. Therefore, disorders accounting for impaired peroxisome biogenesis or specific defects in any particular peroxisomal enzyme involved in these metabolic pathways lead to the accumulation of various metabolites, which may be deposited in several tissues, consequently causing pathophysiological alterations [55]. These defects affecting BA metabolism are described below.

BACS

So far, the only patients described with deficiency concerning BACS activity were two sisters carrying a homozygous mutation in the SCL27A5 gene. Although both girls had a similar abundance (85%) of unconjugated BAs in plasma and urine, only one of them developed liver disease. The reason for this discrepancy likely was that in the patient with more severe manifestations, BACS deficiency coexisted with another homozygous deleterious mutation in the ABCB11 gene, encoding the bile salt export pump (BSEP), which plays an essential role in the canalicular secretion of BAs. In contrast, her sister was heterozygous for this mutation [81]. Mice lacking BACS show a high proportion of unconjugated BAs in bile, without signs of disease. However, these animals do no gain extra weight when fed with a high-fat diet [82, 83].

BAAT

In ten pediatric patients harboring a lack of enzymatic activity in BAAT (OMIM: 619232), this deficiency caused malabsorption of fat-soluble vitamins and increased prothrombin time. The rest of the clinical manifestations showed marked interindividual variability. Thus, neonatal cholestasis ranged from mild to very severe, with liver transplantation being necessary for one of the patients who developed biliary cholangiopathy, eventually leading to cirrhosis and liver failure. Data obtained from the liver biopsy of several of these patients showed idiopathic neonatal hepatitis with ductular proliferation and fibrosis. In addition, four patients suffered from rickets because of vitamin D deficiency. As could be expected, biochemical signs included the absence of glyco- and tauroconjugated BAs in plasma, urine, and bile, where unconjugated CA was the most abundant BA [84]. BAs in the liver of BAAT-deficient mice are mostly unconjugated. As a response to facilitate their detoxification, BA polyhydroxylation is markedly enhanced. These mice also display defective body weight gain [85]. The similarity in the BA pattern of patients with BAAT or BACS deficiency determines that the only way to discriminate between both disorders is through genotyping or immunolabeling analysis of liver tissue obtained by biopsy. Therapy based on the oral administration of conjugated CA (GCA) has improved growth rate and fat-soluble vitamin absorption in patients with BA amidation defects [86].