Biosynthesis of cholic acid
Bile acid synthesis from cholesterol can occur via two pathways, one initiated by sterol 27-hydroxylase activity or one initiated by that of cholesterol 7 alpha-hydroxylase. In contrast to cholesterol 7 alpha-hydroxylase, which is found in the liver, sterol 27-hydroxylase is a widely distributed mitochondrial enzyme with high activity in vascular endothelial cells. Although both pathways lead to the production of chenodeoxycholic and cholic acids, the key step, 7 alpha-hydroxylation, is governed by two different enzymes. Both 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid, the metabolites of cholesterol occurring via sterol 27-hydroxylase activity, normally circulate in plasma. After their uptake by the liver they are metabolized mostly to chenodeoxycholic acid, which down-regulates the activity of cholesterol 7 alpha-hydroxylase, the rate-limiting step for the production of bile acids in the liver. Because of this relationship and also in view of the accelerated atherosclerosis and cholesterol deposition in tissues that occur as a consequence of genetically determined sterol 27-hydroxylase deficiency and of the potent biologic effect of 27-hydroxycholesterol in cell culture, it is proposed that this metabolic pathway serves a regulatory function. The pathway beginning with cholesterol 7 alpha-hydroxylation is modulated by genetic, hormonal, and probably dietary factors, and becomes most prominent with the interruption of the enterohepatic circulation of bile acids.
The most abundant bile acids in human bile are chenodeoxycholic acid (45%) and cholic acid (31%). These are referred to as the primary bile acids. Before the primary bile acids are secreted into the canalicular lumen they are conjugated via an amide bond at the terminal carboxyl group with either of the amino acids glycine or taurine. These conjugation reactions yield glycoconjugates and tauroconjugates, respectively. This conjugation process increases the amphipathic nature of the bile acids making them more easily secretable as well as less cytotoxic. The conjugated bile acids are the major solutes in human bile.
Structure of the conjugated cholic acids
The synthesis and excretion of bile acids comprise the major pathway of cholesterol catabolism in mammals. Synthesis provides a direct means of converting cholesterol, which is both hydrophobic and insoluble, into a water-soluble and readily excreted molecule, the bile acid. The biosynthetic steps that accomplish this transformation also confer detergent properties to the bile acid, which are exploited by the body to facilitate the secretion of cholesterol from the liver. This role in the elimination of cholesterol is counterbalanced by the ability of bile acids to solubilize dietary cholesterol and essential nutrients and to promote their delivery to the liver. The synthesis of a full complement of bile acids requires 17 enzymes. The expression of selected enzymes in the pathway is tightly regulated by nuclear hormone receptors and other transcription factors, which ensure a constant supply of bile acids in an ever changing metabolic environment. Inherited mutations that impair bile acid synthesis cause a spectrum of human disease; this ranges from liver failure in early childhood to progressive neuropathy in adults
The rates of excretion and specific activities of total bile acids, cholic acid, chenodeoxycholic acid, and cholesterol excreted in the bile were measured during a diurnal cycle in bile fistula rats fed [3H]cholesterol for 4 to 5 weeks. One [3H]-cholesterol-fed rat received a constant infusion of [2-14C]-mevalonic acid and another received a constant infusion of 7α-hydroxy[4-14C]cholesterol during collection of the bile.
The rates of excretion of the four biliary constituents measured exhibited diurnal rhythm, with a maximum during the night and a minimum at noon. The specific activities of total 3H-labeled bile acids and of [3H]cholic acid decreased during the first half of the dark period, to reach a minimum coinciding with the maximum rate of excretion, and then increased during the second half of the dark period. The specific activities of [3H]chenodeoxycholate and [3H]cholesterol did not change appreciably during the diurnal cycle. During [2-14C]mevalonate infusion the specific activity of [14C]cholate varied diurnally in parallel with the change in specific activity of [3H]cholate, but that of [14C]chenodeoxycholate did not change during the diurnal cycle. During 7α-hydroxy[4-14C]cholesterol infusion the specific activity of [14C]cholate varied diurnally in parallel with that of [3H]cholate. The specific activity of [14C]chenodeoxycholate fell markedly during the night and was higher than that of [14C]-cholate throughout the diurnal cycle.
These observations indicate that newly synthesized hepatic cholesterol is the preferred substrate for the formation of cholic acid, but they raise the possibility that some chenodeoxycholic acid is formed from a pool of cholesterol other than that from which cholic acid is formed.
The major pathway for the synthesis of the bile acids is initiated via hydroxylation of cholesterol at the 7 position via the action of cholesterol 7α-hydroxylase (CYP7A1) which is an ER localized enzyme. CYP7A1 is a member of the cytochrome P450 family of metabolic enzymes. This pathway is depicted in highly abbreviated fashion in the Figure below. The pathway initiated by CYP7A1 is referred to as the "classic" or "neutral" pathway of bile acid synthesis. There is an alternative pathway that involves hydroxylation of cholesterol at the 27 position by the mitochondrial enzyme sterol 27-hydroxylase (CYP27A1). This alternative pathway is referred to as the "acidic" pathway of bile acid synthesis. Although in rodents the acidic pathway can account for up to 25% of total bile acid synthesis, in humans it accounts for no more than 6%. The bile acid intermediates generated via the action of CYP27A1 are subsequently hydroxylated on the 7 position by oxysterol 7α-hydroxylase (CYP7B1). Although the acidic pathway is not a major route for human bile acid synthesis it is an important one as demonstrated by the phenotype presenting in a newborn harboring a mutation in the CYP7B1 gene. This infant presented with severe cholestasis (blockage in bile flow from liver) with cirrhosis and liver dysfunction.
Following the action of HSD3B7 the bile acid intermediates can proceed via two pathways whose end products are chenodeoxycholic acid (CDCA) and cholic acid (CA). The distribution of these two bile acids is determined by the activity of sterol 12α-hydroxylase (CYP8B1). The intermediates of the HSD3B7 reaction that are acted on by CYP8B1 become CA and those that escape the action of the enzyme will become CDCA. Therefore, the activity of the CYP8B1 gene will determine the ratio of CA to CDCA. As discussed below the CYP8B1 gene is subject to regulation by bile acids themselves via their ability to regulate the action of the nuclear receptor FXR.
Synthesis of the 2 primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA). The reaction catalyzed by the 7α-hydroxylase (CYP7A1) is the rate limiting step in bile acid synthesis. Expression of CYP7A1 occurs only in the liver. Conversion of 7α-hydroxycholesterol to the bile acids requires several steps not shown in detail in this image. Only the relevant co-factors needed for the synthesis steps are shown. Sterol 12α-hydroxylase (CYB8B1) controls the synthesis of cholic acid and as such is under tight transcriptional control
Bile salts are secreted from hepatocytes, into the bile canaliculi, via the action of the bile salt export protein (BSEP; ATP-binding cassette B11, ABCB11). Transport of phospholipids into the canaliculi requires the transporter ABCB4. ABCB4 is also known as multi-drug resistance protein 3 (MDR3, a member of the P-glycoprotein family of transporters). Some free cholesterol is also transported out of hepatocytes into the canaliculi via the action of the obligate heterodimeric transporter ABCG5/ABCG8. The transport of cholesterol via this complex also requires ABCB4. The ABCB4 requirement is consistent with the known actions of phospholipids in the bile canaliculi functioning as a sink to accept cholesterol tranported out by ABCG5/ABCG8. Each of these hepatic lipid transporters is critical for normal hepato-biliary function since mutations in any of the genes encoding the transporters have been shown to be associated with cholestatic liver diseases. These transport defects result in the accumulation of toxic levels of bile salts within the hepatocytes resulting in liver failure.
Once bile salts are secreted into the duodenum and carry out their emulsification role, around 95% are reabsorbed into the distal ileum. Bile salt reabsorption occurs via the apical sodium-dependent bile transporter (ASBT) present in the brush border membrane of the enterocyte. Ileal bile acid-binding protein (IBABP; also known as fatty acid-binding protein subclass 6: FABP6) is thought to be involved in the transport of bile salts across the enterocyte cytosol to the basolateral membrane. Once bile salts reach the basolateral membrane they are transported (effluxed) into the blood by the heterodimeric transporter OSTα/OSTβ (organic solute transporters). A small percentage of the bile salts are not reabsorbed and undergo deconjugation by intestinal microbiota before either being absorbed or converted into secondary bile acids. Anaerobic bacteria present in the colon modify the primary bile acids converting them to the secondary bile acids, identified as deoxycholate and ursodeoxycholate (DCA and UDCA, from cholate) and lithocholate (LCA, from chenodeoxycholate). These secondary bile acids are either passively absorbed from the colon or they are excreted in the feces. The absorbed primary and secondary bile acids and salts are transported back to the liver where most, but not all, are actively transported into hepatocytes by sodium sodium (Na+)-taurocholate cotransporting polypeptide (NTCP/SLC10A1) and organic anion transporters (OATP) such as OAT1B2) that mediate the uptake of bile salts and bile acids, respectively. Once in the liver the bile acids are reconjugated and then re-secreted together with newly synthesized bile salts. This overall process constitutes one cycle of what is called the enterohepatic circulation. When LCA is returned to the liver it undergoes a sulfation reaction and is subsequently excreted in the feces. The bile acid pool contains about 2–4 gm of bile acids and this pool is recycled via the enterohepatic circulation on the order of six to ten times each day. Of the total bile salt pool, around 0.2–0.6 gm are excreted in the feces each day. This lost fraction of bile salts is replenished via de novo hepatic bile acid synthesis from cholesterol.
Structure of a liver lobule. Lobules in the liver represent histologically and functionally distinct domains within the liver. They are not to be confused with the anatomical lobes of the liver which are defined as the right and left lobes and the median and quadrate lobes. Lobules are histologically defined as classical, portal, and acinus lobules. Lobules contain hepatocytes, and are vascularized by the hepatic portal vein, the hepatic artery, and the central vein. In addition, the bile cannaliculi run through the lobules allowing hepatic products such as bile acids to be delivered to the bile ducts and ultimately to the gallbladder. Kuppfer cells are liver resident macrophages
