Cholic acid precursors

Ox bile, which contains cholic acid as its principal constituent, provided one of the earliest mammalian sources of steroid raw materials for the commercial manufacture of the androgens. In nature, cholesterol itself is the mammalian precursor of the androgens, the biosynthesis passing through progesterone (XII). 

The insertion of hydroxyl groups into the 23- or 24-position of 5P-cholestane-3a,7a,12a,25-tetrol was found to be stereospecific. Although all these compounds were potential precursors of bile acid, studies in vivo and in vitro experiments using [3P- H] and (24- C) 5P-cholestane-3a,7a,12a,25-tetrol (46) (Figs.6, 7), (24- C) 5p-cholestane-3a,7a,12a,24R,25-pentol and (24- C) 5P-cholestane-3a,7a,12a,24S,25-pentol demonstrated the existence of a new 25-hydroxylation pathway for the transformation of cholesterol to cholic acid in these patients (2,10). The reaction sequence involved the stereospecific formation of a 24S-hydroxy pentol, 5P-cholestane-3a,7a,12a,24S,25-pentol, 3a7a,12a,25-tetrahydroxy-5P-cholestan-24-one and did not involve SP-cholestanoic acids as intermediates (Fig. 8). The two bile pentols, SP-cholestane-3a,7a,12a,24R, 25-pentol and 5P-cholestane-3a,7a,12a,23R,25-... 

Hvdroxvlation pathway An alternative explanation for the bile acid synthetic defect in CTX has been proposed by Oftebro and colleagues which starts via 26-hydroxylation of 5P-cholestane-3a,7a,12a-triol (IX, Fig. lOa and 10b). In this pathway the mitochondrial fraction of both human and rat liver contains a 26-hydroxylase enzyme (63) which can convert 5P-cholestane-3a,7a,12a-triol (IX ) to 5P-cholestane-3a,7a,12a,26-tetrol (XI) (Fig. 10a and 10b ). This tetrol is oxidized to 3a,7a,12a-trihydroxy-5P-cholestan-26-oic acid (THCA, XII) by liver cytosol (2,64). Further hydroxylation at C-24 forms varanic acid (XIV) and its side chain is shortened with oxidation at C-24 to yield cholic acid (X,Fig. 10 a). These investigators demonstrated diminished mitochondrial 26-hydroxylation of 5p-cholestane-3a,7a,12a-triol and 5P-cholestane-3a,7a-diol, possible precursors for cholic acid and chenodeoxycholec acid in CTX liver. As a consequence, neither 26-hydroxylated intermediates can be formed so that total primary bile acid synthesis would be diminished. Accordingly, the accumulation of 5P-cholestane-3a,7a,12a,25-tetrol arises from 25-hydroxylation of 5P-cholestane-3a,7a,12a-triol by the alternative microsomal 25-hydroxylation mechanism. 

In summary, these studies demonstrated that in CTX the impaired synthesis of bile acids is due to a defect in the biosynthetic pathway involving the oxidation of the cholesterol side-chain. As a consequence of the inefficient side-chain oxidation, increased 23, 24 and 25-hydroxylation of bile acid precursors occurs with the consequent marked increase in bile alcohol glucuronides secretions in bile, urine, plasma and feces (free bile alcohols). These compounds were isolated, synthesized and fully characterized by various spectroscopic methods. In addition, their absolute stereochemistiy determined by Lanthanide-Induced Circular Dichroism (CD) and Sharpless Asymmetric Dihydroxylation studies. Further studies demonstrated that (CTX) patients transform cholesterol into bile acids predominantly via the 25-hydroxylation pathway. This pathway involves the 25-hydroxylation of 5P-cholestane-3a,7a, 12a-triol to give 5P-cholestane-5P-cholestane-3a,7a,12a,25- tetrol followed by stereospecific 24S-hydroxylation to yield 5P-cholestane-3a,7a,12a,24S,25-pentol which in turn was converted to cholic acid. 

Bile Salts Enable the Digestion of Lipids Cholesterol is the precursor of both steroids and bile salts and is an integral component of cell membranes. It is eliminated from the body via conversion to bile salts and direct secretion into the bile. In fact, the word cholesterol (from the Greek chole (bile) and stereos (solid)) was used originally to describe the material of which gallstones are made. In the process of degradation, it is converted to the primary bile acids cholic acid and chenodeoxycholic acid in approximately equal amounts. The salts of these acids are excreted in bile. They perform two important functions in the digestive tract ... 

Other clinical signs consist of progressive neurologic dysfunction, cataracts, and premature atherosclerosis (SI). The disease is inherited as an autosomal recessive trait, but is usually only detected in adults when cholesterol and cholestanol have accumulated over many years (S2). Biochemical features of the disease include striking elevations in tissue levels of cholesterol and cholestanol and the presence of unusual bile acids, termed bile alcohols, in bile. These bile alcohols are mainly 5 -cholestane-3a,7a,12a,24S, 25-pentol, Sp-diolestane-3a,7a,12a,23 ,25-pentol and 5P-du)lestane-3a,7a,12a,25-tetrol (S2). As chenodeoxycholic acid is deficient in the bile of patients with CTX, it was postulated that early bile salt precursors are diverted into the cholic acid pathway and 12a-hydroxy bile alcohols with an intact side chain accumulate because of impaired cleavage of the cholesterol side chain and decreased bile acid production (S2). HMG-CoA reductase and cholesterol 7a-hydroxylase activity are elevated in subjects with CTX (N4, N5), so that sufficient 7a-hydroxycholesterol should be available for bile acid synthesis. 

Results of various in vivo experiments with labelled bile acid precursors in patients with CTX have been published [185,190,195]. All these experiments show that there is a defect in the oxidation of the steroid side chain in the biosynthesis of cholic acid but are not fully conclusive with respect to the site of defect. Bjorkhem et al. administered a mixture of [ H]7a,26-dihydroxy-4-cholesten-3-one and [ " C]7a-hy-droxy-4-cholesten-3-one to a patient with CTX [195]. The ratio between and C in the cholic acid and the chenodeoxycholic acid isolated was 40 and 60 times higher, respectively, than normal. Similar results were obtained after simultaneous administration of H-labelled 5)3-cholestane-3a,7a,26-triol and 4- C-labelled 5j8-cholestane-3a,7a-diol. The results of these experiments are in consonance with the contention that the basic defect in CTX is the lack of the 26-hydroxylase, but do not per se completely exclude other defects in the oxidation of the side chain. 

Microsomal 12a-hydroxylation is the only unique step in the formation of chohc acid and is likely to be of regulatory importance for the ratio between newly synthesized cholic and chenodeoxycholic acid. Introduction of a 26-hydroxyl group seems to prevent subsequent 12a-hydroxylation in rat liver and the 26-hydroxylase could thus also have a regulatory role. It is possible that there are different precursor pools for the synthesis of cholic acid and chenodeoxycholic acid in rats. If so, the relative size of the two pools could be of importance for the relative rate of formation of the two bile acids. 

In 1963, Carey and Haslewood isolated trace amounts of (25/ )-3a,7 ,12a-trihy-droxy-5 8-cholestan-26-oic acid from human fistula bile [96]. The stereochemistry at C-25 of this bile acid was recently confirmed by direct comparison with reference compounds of known absolute configuration [97], This trihydroxy-5j8-cholestanoic acid also occurs in baboon bile [98]. Hanson and Williams found the corresponding dihydroxy bile acid, 3 ,7a-dihydroxy-5 S-cholestan-26-oic acid, in human bile [99]. The occurrence of these higher bile acids, quantitatively of minor importance, is of interest because they are biosynthetic precursors of two primary bile acids of mammalian species, cholic acid and chenodeoxycholic acid, respectively (Chapter 9). 

Danielsson and Kazuno have shown that 5j8-ranol is an efficient precursor of cholic acid in bile fistula rats while 26-deoxy-5)S-ranol is not [134]. The mechanism of the side-chain degradation of 5 -ranol is not known but probably an oxidation of the 26-hydroxyl group to a carboxyl group followed by a /S-oxidation. 

In the 5a series allochenodeoxycholate is 12a-hydroxylated to allocholate in the bile fistula rat [131] or with a hepatic microsomal preparation from rat, rabbit or human liver fortified with NADPH [152,153]. Kallner [133] noted that small amounts of more polar derivatives were present in rat bile, with largely unchanged allochenodeoxycholate. AUohyocholate was identified as a minor metabolite [154]. With rabbit liver microsomal preparations, allochenodeoxycholate is a competitive inhibitor for 12a-hydroxylation of 7a-hydroxy-cholest-4-en-3-one and 5a-choles-tane-3a,7a-diol, precursor of cholic and allocholic acids, respectively [155]. Allo-cholic acid has also been characterized as a metabolite of 3 8,7a-dihydroxy-5-cholenic acid after intraperitoneal injection into carp [156]. 

Steroids contain a four-ring structure called the steroid nucleus (Fig. 5.23). Cholesterol is the steroid precursor in human cells from which all of the steroid hormones are synthesized by modifications to the ring or C20 side chain. Although cholesterol is not very water soluble, it is converted to amphipathic water-soluble bile salts such as cholic acid. Bile salts line the surfaces of lipid droplets called micelles in the lumen of the intestine, where they keep the droplets emulsified in the aqueous environment. 

Chenodeoxycholic acid is converted into a-muricholic acid (3a,6i8,7a-trihydroxy-5jS-cholanoic acid) and j8-muricholic acid (3a,6)J,7/J-trihydroxy-5j8-cholanoic acid) in the mouse and the rat and probably also in man (68, 102, Chapter 11 in this volume). a-Muricholic acid is a precursor of jS-muri-cholic acid in a reaction involving the intermediary formation of the 7-oxo compound (Chapter 11 in this volume). In the rat, /8-muricholic acid has been shown to be formed also from 3a,7j8-dihydroxy-5i8-cholanoic acid, which is a minor metabolite of chenodeoxycholic acid, and from 3a,6)9-dihydroxy-5)5-cholanoic acid, which is a metabolite of lithocholic acid (Chapter 11 in this volume). The microsomal 6i8-hydroxylase system in rat liver catalyzing the conversion of (tauro)chenodeoxycholic ac d into (tauro)a-muricholic acid has been studied by Hsia and collaborators (103-105), who... 

Fig. 1. The two 24-carbon primary bile acids in man are I, 3a,7a,12a-trihydroxy-5/5-cho]anoic acid (cholic acid), and II, 3a,7a-dihydroxy-5iS-cholanoic acid (chenodeoxycholic acid). Their immediate precursors are, respectively. III, 3a,7a,12a-trihydroxy-5/3-cholestanoic acid, and IV, 3a,7a-dihydroxy-5jS-cholestanoic acid.

The synthesis of cholic acid from cholesterol in rodents proceeds via 7a-OH-cholesterol, leading to a trihydroxy derivative which finally loses a side chain, resulting in the formation of cholic acid (1). The synthesis appears to proceed similarly in the human liver (12). Trihydroxycoprostanic acid, which is a precursor of cholic acid and which is formed from cholesterol... 

---