Cholestane pentol

Special laboratory Cholestane pentol glucuronides (U) (m/z 627 in LSDMS) + + + + +... 

GC-MS analysis is used to confirm the identities of ions in the LSI-MS urine spectrum and show that the excretion of abnormal cholanoids is >20 times normal. In the case of 5 ff-reductase deficiency GC-MS analysis should show that S-oxo-A" bile acids account for >70% of the total urinary bile acid excretion. In the case of sterol 27-hydroxylase deficiency (CTX), GC-MS analysis should indicate that the major cholestane pentols in the urine are 3,7,12,22,25 and 3,7,12,23,25-pentols. (One patient has been described who had familial cholestatic liver disease associated with greatly increased urinary excretion of 5jff-cholestane-3a,7a,12a,24 S,25-pentol [see previous table]). Liquid secondary ion-tandem mass spectrometry (LSI-MS/ MS) is an alternative method to GC-MS and can rapidly confirm the identity of a number of diagnostic ions that are found in the LSI-MS spectrum of urine. These include sulphated and taurine-conjugated abnormal metabolites such as those observed in 3 ff-HSDH deficiency (32.1), 5)9-reductase deficiency (32.2), oxysterol 7a-hydroxylase deficiency (32.4) and peroxisomal disorders [13]. 

Bile alcohols are polyhydroxy C27 sterols that serve as intermediates in the biosjmthesis of cholic acid and chenodeoxycholic acid from cholesterol (1, 2). Recently several studies have shown that increased amounts of bile alcohols namely 27-nor-5p-cholestane-3a,7a,12a,24, 25-pentol and 5P-cholestane-3a, 7a,12a,25,26-pentol are excreted (as glucuronides in urine of patients with liver diseases such as primary biliary cirrhosis (3), liver cirrhosis (4, 5) and a-antitrypsin deficiency (6). Ichimiya et. al., described the occurrence of 5P-cholestane-3a,7a,12a,26,27-pentol (5P-cyprinol) and 5P-cholestane-3a,7a,... 

Chromatographic analysis of the polar sterol fractions of the bile and feces, in conjunction with gas-liquid chromatography (GLC)-mass spectrometry indicated two major components, 5P-cholestane-3a,7a,12a,25-tetrol and 5P-cholestane-3a,7a,12a,23,25-pentol, and a minor component, 5P-cholestane-3a, 7a,12a,24,25-pentol(18-21). Only minute amounts of 5P-cholestane-3a, 7a,12a,23R-tetrol, 5P-cholestane-3a,7a,12a,24R-tetrol, 5P-cholestane-3a,7a, I2a,24S-tetrol and 5P-cholestane-3a,7a,12a,25S,26-pentol have been detected (20-24). The presence of 5P-cholestane-3a,7a,12a,25-tetrol was positively identified by comparison with the synthesized sample prepared in our laboratory (20-22,24-27) (Fig.l). The predominent bile alcohol of the pentol fraction was 5P-cholestane-3a,7a,12a,23,25-pentol, amounting to approximately 80% by weight, while 5P-cholestane-3a,7a,12a,24,25-pentol accounted for approximately 20% of this fraction. The less abundant pentol was shown to be identical with 5P-cholestane-3a,7a,12a,24a,25-pentol, which had been prepared from 5P-cholestane-3a,7a,12a,25-tetrol (20,21). 

The biosynthetic 5p-cholestanetetrol and pentol had the same melting point, TLC and GLC properties, and infrared and mass spectra as the reference compound (19-21). In addition, the recently described two-dimensional H-NMR studies (28-30) from our and other laboratories have further elucidated their structure and stereochemistry. Fig. 2 illustrates 2D-NMR H- H COSY spectra of 5P-cholestane-3a,7a, 12a 25-tetrol (for comparison 2D-NMR H- H COSY spectra of 5P-cholestane-3a,7P, 12a,25-tetrol is also provided). The 13c-DEPT spectra of (24R) and 5P-cholestane-3a,7a,12a,24a,25-pentol and (23R)-5P-cholestane-3a,7a,12a, 24a,25-pentol has been recently described (21). 

The occurrence of bile alcohols hydroxylated at position 25 in CTX patients indicated the presence of an alternate pathway of bile acid synthesis from cholesterol (via 25-hydroxylated intermediates ). In particular, the identification of 5P-cholestane-3a,7a,12a,24a,25-pentol indicated that cholic acid arised from the cleavage of a 24,25- glycol. 

During the course of these studies, the two 5P-cholestane-3a,7a, 12a,24,25-pentols, epimeric at C-24, and 5P-cholestane-3a,7a,12a,23,25-pentol epimeric at C-23 were further resolved by analytical and preparative TLC, and were either synthesized or isolated and characterized. In addition, the absolute stereochemistry at C-23 of 5P-cholestane-3a,7a, 12a,23,25-pentol and at C-24 of 5P-cholestane-3a,7a,12a,24,25-pentol (39,40) was established by circular dichroism (CD) studies employing the method of Nakanishi (41,42). These experiments conclusively defined the chirality of these pentahydroxy bile alcohols having 1,2 and 1,3 glycol systems in the side chain. Using Eu(fod)3... 

Similarly lanthanide-induced CD and C NMR studies elucidated the absolute configuration at C-2S in Sfl-cholestane-3a,7o,12a,2S,26-pentol as 2SS (44). 

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 This pathway has been demonstrated in both rat and human liver (2,10,56). It involves the 25-hydroxylation of 5P-cholestane-3a,7a,12a-triol to give 5P-choiestane-3a,7a,12a,25- tetrol (XIV) followed by stereospecific 24S-hydroxylation to yield 5P-cholestane-3a,7a,12a,24S,25-pentol (XV, Fig. 9). The pentoi is then oxidized to 5P-choiestane-3a,7a,12a, 25-tetrahydroxy-5P-cholestan-24-one (XVI) (59,60), which is degraded by... 

Ichimiya, H., Yanagisawa, J. and Nakayama, F. (1984). Significance of bile alcohols in urine of a patient with cholestasis identification of Sp-cholestane-3a,7a,12a,26,27-pentol (5p-bufoI) and 5P-cholestane-3a,7a, 12a,26-tetrol (27-deoxy-5P-cyprinol). Chem. Pham. Bull. 32 2874-2877. 

Dayal, B., Salen, G., Padia, P., Shefer, S., Tint, G. S., Williams, T. H., Toome, V., and Sasso,.G. (1992). Stereoselective synthesis of (24R,24S) 5P-Cholestane-3a,7a,12a, 24,25-pentol and (25R,25S ) SP-cholestane-3a, 7a,12a,25, 26-pentol using a modified osmium catalyzed Sharpless Asymmetric Dihydroxylation Process. Chem. Phys. Lipids, 61 271-281... 

Cholic acid has been converted into 5/S-cholestane-3a,7a,12a,25-tetrol and the 3a,7a,12a,24,25-pentols. Extension of the side-chain involved conversion of the acid via the diazo-ketone (361) into a homocholanic ester derivative (362). Grignard reaction then gave the 25-hydroxy-cholestane (363). Dehydration followed by hydroxylation (OSO4) afforded the 24,25-diols with a little of the 25,26-diol. ... 

The 22-isomeric 5)8-cholestane-3a,7a,12a,22,25-pentols (365) have been synthesized from cholic acid after a preliminary oxidative degradation to the dinor-22-aldehyde (364), which was followed by a Grignard step with 3-methyl-3-(tetrahydropyran-2 -yloxyl)-butynylmagnesium bromide, and hydrogenation. 

S,25-pentoI (XII, Fig. 4). The pentol is then oxidized to 3a,7a,12a,25-tetrahydroxy-SP-cholestan-24-one (XIII) (S26), which is degraded by cytosolic enzymes to cholic acid (X) and acetone (Fig. 4). 

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. 

Cholestane-3a,7a,12a-triol is efficiently 25-hydroxylated in the microsomal fraction of hver from both rat and man [40,41]. Shefer et al. [184] and Salen et al. [185] have shown that 5/8-cholestane-3a,7a,12a,25-tetrol is converted to S S-choles-tane-3a,7a,12a,24a,25-pentol, 5i8-cholestane-3a,7a,12a,24j8,25-pentol, 5j8-choles-tane-3 ,7a,12a,23,25-pentol and 5j8-cholestane-3 ,7a,12a,25,26-pentol in the presence of microsomes fortified with NADPH. In the presence of NAD", 5j8-choles-tane-3a,7a,12a,24, 25-pentol, but not the other 5j8-cholestanepentols formed, is efficiently converted to chohc add by soluble enzymes (Fig. 13). The latter conversion must be assumed to involve formation of acetone. These experiments demonstrate the existence of a new pathway for side-chain degradation in chohc acid synthesis which does not involve hydroxylation at C-26 or the participation of mitochondria. The relative importance of this pathway is a matter of controversy. Salen et al. suggested that this may be the major pathway for biosynthesis of chohc acid in man [185]. The finding that 5i8-cholestane-3a,7a,12a,25-tetrol is converted into chohc add in vivo in rat and man considerably less efficiently than 5j8-choles-tane-3a,7a,12a,26-tetrol does not support this contention [40,186]. 

This rare inherited hpid storage disease is characterized by xanthomas, progressive neurological dysfunction, cataracts and the development of xanthomatous lesions in the brain and lung. In contrast to other diseases with tendon xanthomatosis, plasma cholesterol levels are remarkably low. Large deposits of cholesterol and cholestanol are present in most tissues, and the concentration of cholestanol is 10-100 times higher than normal. Salen and collaborators have made extensive and elegant studies on the various metabolic aspects of this disease [184,185,187-192]. They have conclusively shown that there is a subnormal synthesis of bile acids and that the metabolic defect is an impaired oxidation of the cholesterol side chain. The synthesis of chenodeoxycholic acid is reduced more than that of cholic acid. These patients excrete considerable amounts of bile alcohol in bile and faeces. The bile alcohols have been identified as 5)S-cholestane-3a,7a,12a,25-tetrol, 5 8-cholestane-3a,7a,12a,24,25-pentol and 5/8-cholestane-3 ,7a,12a,23,25-pentol. Two different explanations for the accumulation of these bile alcohols have been presented. 

Arapaimol-A 5/S-Cholestane-2/8,3 ,7 , 12 a,26-pentol Arapaima gigas-. some frogs... 

Chimaerol 5a-Cholestane-3a,7a,12a,24,26-pentol White sucker and related fishes lungfish... 

Bufol 5/8-Cholestane-3a,7a,l 2 a,25,26-pentol Toads of genus Bufo some frogs... 

Cyprinol 5/8-Cholestane-3o,7a,l 2 a,26,27-pentol Several bony fishes some frogs... 

A principal bile salt of Arapaima gigas, the very large South American fresh-water teleost of the family Osteoglossidae, is a sulfate ester of arapaimol-A [8]. The bile also contains a sulfate ester of a second bile alcohol, arapaimol-B [8], Spectral analysis indicated that arapaimol-A and -B are 5y3-cholestane-2/3,3a,7a,12 ,26-pentol and 5 -cholestane-2j8,3a,7a,12a,26,27-hexol, respectively [8], Arapaimol-A also occurs in the bile of some species of frogs [9]. 

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