Identifications of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 23 beta-tetrol, 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 24 alpha-tetrol, and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 24 beta-tetrol in cerebrotendinous xanthomatosis

The bile alcohols present in the feces of a patient with cerebrotendinous xanthomatosis were studied. Three bile alcohols which are different from any known natural bile alcohol were isolated as minor components of the fecal bile alcohol fraction. The structures of these compounds were established as 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 23 beta-tetrol, 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 24 alpha-tetrol, and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 24 beta-tetrol by comparison with synthetic samples.     

Synthesis of 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol and 5beta-cholestane-3alpha, 7alpha, 245, 25-pentol.

This paper describes syntheses of 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol and 5beta-cholestane-3alpha, 7alpha, 12alpha, 24xi, 25-pentol which give higher yields than previously published methods. In addition, 5beta-cholestane-3alpha, 7alpha, 12alpha, 24xi, 25-pentol was synthesized by a different procedure, namely via performic acid oxidation of the correspinding unsaturated triol, which gave a lower yield but avoided the formation of 5beta-cholestane-3alpha, 7alpha, 12alpha, 25, 26-pentol, which normally tends to contaminate the final product. Structures were confirmed by gas-liquid chromatography, infrared-, proton magnetic resonance- and mass spectrometry, 5beta-Cholestane-3alpha, 7alpha, 12alpha, 25-tetrol and 5beta-cholestane-3alpha, 7alpha, 12alpha, 24xi, 25-pentol were required for in vivo and in vitro studies of the (hypothetical) 25-hydroxylation pathway of cholic acid biosynthesis.     

Identification of new bile alcohols, 5 beta-cholestane-3 alpha,7 alpha,24,26-tetrol, 5 beta-cholestane-3 alpha,7 alpha,25,26-tetrol, and 5 beta-cholestane-3 alpha,7 alpha,26,27-tetrol in human gallbladder bile

The nature of cholestanetetrols present as the glucurono-conjugates in human gallbladder bile was studied. Glucurono-conjugated bile alcohols were isolated by ion exchange chromatography and, after enzymatic hydrolysis, were fractionated by reversed phase partition chromatography to give a fraction containing tetrahydroxy bile alcohols which was analyzed by gas-liquid chromatography and mass spectrometry. Along with the three previously identified bile alcohols, 5 alpha- and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha,24-tetrols, and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha,26-tetrol, three new cholestanetetrols, possessing two hydroxyl groups in the ring system and two in the side chain, were detected in the tetrahydroxy bile alcohol fraction. These new bile alcohols were identified as 5 beta-cholestane-3 alpha, 7 alpha,24,26-tetrol, 5 beta-cholestane-3 alpha, 7 alpha,25,26-tetrol, and 5 beta-cholestane-3 alpha, 7 alpha,26,27-tetrol by direct comparison of their gas-liquid chromatographic behaviors and mass spectral data with those of authentic standards prepared from chenodeoxycholic acid by partial synthesis.     

Conversion of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 26-tetrol into 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid by rabbit liver mitochondria

Rabbit liver mitochondria in the presence of NAD+ were found to catalyze the conversion of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 26-tetrol into 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid. The peroxisomal fraction did not catalyze the reaction. Sonication of the mitochondria or dialysis overnight against a hypotonic buffer increased the rate of oxidation twofold. Most of the enzyme activity was recovered in the supernatant fraction after centrifugation at 100,000xg of sonicated mitochondria. 4-Heptylpyrazole, an inhibitor of cytosolic ethanol dehydrogenase, inhibited the mitochondrial formation of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid by 70%. Disulfiram, an inhibitor of cytosolic acetaldehyde dehydrogenase, did not inhibit the reaction. The role of the mitochondrial dehydrogenase system in bile acid biosynthesis is discussed.     

Metabolism of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 26-tetrol and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 25-tetrol into cholic acid in normal human subjects.

Side chain oxidation and cleavage of precursors in cholic acid synthesis is thought to involve initial hydroxylation at either position 25 or 26 of the side chain. Therefore, the conversion of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 26-tetrol and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 25-tetrol into cholic acid was studied in normal subjects after single intravenous injections of these labeled alcohols. Eighty-six percent and 82% of 5 beta-cholestane, 3 alpha, 7 alpha, 12 alpha, 26-tetrol was converted into cholic acid in two subjects, respectively. However, only 14 and 16% of the injected 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 25-tetrol was converted into cholic acid in two subjects, respectively. Thus, this study indicates that 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 25-tetrol is an inefficient substrate for cholic acid biosynthesis in man and that the major route of cholic acid synthesis probably involves the 26-hydroxylated intermediate.     

Development of a 3D pharmacophore for nonspecific ligand recognition of alpha1, alpha2, alpha3, alpha5, and alpha6 containing GABA(A)/benzodiazepine receptors

Transfected cells containing GABA(A) benzodiazepine receptors (BDZRs) have been utilized to systematically determine the affinity of ligands at alpha1, alpha2, alpha3, alpha5 and alpha6 subtypes in combination with beta2 and gamma2. All but a few of the ligands thus far studied have relatively high affinities for each of these alpha subtype receptors. Thus, these ligands must contain common stereochemical properties favorable for recognition by each of the subtype combinations. In the present work, such a common three-dimensional (3D) pharmacophore for recognition of alpha1, alpha2, alpha3, alpha5 and alpha6 containing GABA(A)/BDZRs types of receptors has been developed and assessed, using as a database receptor affinities measured in transfected cells for 27 diverse compounds. The 3D-recognition pharmacophore developed consists of three proton accepting groups, a hydrophobic group, and the centroid of an aromatic ring found in a common geometric arrangement in the 19 nonselective ligands used. Three tests were made to assess this pharmacophore: (i) Four low affinity compounds were used as negative controls, (ii) Four high affinity compounds, excluded from the pharmacophore development, were used as compounds for pharmacophore validation, (iii) The 3D pharmacophore was used to search 3D databases. The results of each of these types of assessments provided robust validation of the 3D pharmacophore. This 3D pharmacophore can now be used to discover novel nonselective ligands that could be activation selective at different behavioral end points. Additionally, it may serve as a guide in the design of more selective ligands, by determining if candidate ligands proposed for synthesis conform to this pharmacophore and selecting those that do not for further experimental assessment.     

Synthesis and androgen effects of 7 alpha,17 beta-dihydroxy-5 alpha-androstan-3-one, 5 alpha-androstan-3 alpha,7 alpha,17 beta-triol and 5 alpha-androstane-3 beta,7 alpha,17 beta-triol

The steroids 7 alpha,17 beta-dihydroxy-5 alpha-androstan-3-one (7 alpha-hydroxy-Dht), 5 alpha-androstan-3 alpha,7 alpha,17 beta-triol (7 alpha-hydroxy-3 alpha-A'DIOL) and 5 alpha-androstane-3 beta,7 alpha,17 beta-triol (7 alpha-hydroxy-3 beta-A'DIOL) have been synthetized from 7 alpha,17 beta-dihydroxy-4-androsten-3-one (7 alpha-hydroxy-testosterone). The effect of administering 7 alpha-hydroxy-Dht, 7 alpha-hydroxy-3 alpha-A'DIOL or 7 alpha-hydroxy-3 beta-A'DIOL on serum levels of LH, FSH and on ventral prostate and seminal vesicle weight were investigated in gonadectomized adult male rats. Each steroid was administered for seven days in a dose of 300 micrograms per day. No suppression of serum LH or FSH levels was recorded following injections of these 7 alpha-hydroxylated steroids to castrated rats, compared to castrated control rats receiving vehicle only. Administration of 7 alpha-hydroxy-Dht or 7 alpha-hydroxy-3 alpha-A'DIOL to castrated mature rats could maintain ventral prostate and seminal vesicle weights above that of castrated control rats. Administration of 7 alpha-hydroxy-3 beta-A'DIOL to castrated mature rats resulted in ventral prostate weights slightly above castrate control levels, while seminal vesicle weight in such rats were in the same range as castrated control rats. Intraperitoneal administration of testosterone or of 5 alpha-androstane-3 beta,17 beta-diol (3 beta-A'DIOL) to castrated rats maintained activity of the androgen dependent isoenzyme of acid phosphatase in the ventral prostate; 7 alpha-hydroxy-testosterone or 7 alpha-hydroxy-3 beta-A'DIOL showed, however, no effect on this enzymic activity.     

Immunoaffinity purification of GABAA receptor alpha-subunit iso-oligomers. Demonstration of receptor populations containing alpha 1 alpha 2, alpha 1 alpha 3, and alpha 2 alpha 3 subunit pairs.

Novel methods for the isolation of gamma-aminobutyric acidA (GABAA) receptor alpha subunit iso-oligomers have been developed. Thus, populations of GABAA receptors containing the GABAA receptor alpha 1 subunit, the alpha 2 subunit, and the alpha 3 subunit have been purified from sodium deoxycholate extracts of bovine cerebral cortex with the retention of specific [3H]flunitrazepam-binding activity by anti-alpha 1 324-341, anti-Cys alpha 2 414-424, or anti-Cys alpha 3 454-467 antibody affinity chromatography, respectively. The relative abundance of the different specificity alpha subunits in these preparations was compared with benzodiazepine affinity chromatography-purified GABAA receptors by immunoblotting. In each case, it was found that although the immunoreactivity with the specific alpha subunit antibody that was used for purification was enriched in immunoaffinity-purified receptors, reactivity with the other alpha subunit specificity antibodies, together with anti-gamma 2 1-14 Cys immunoreactivity was found. Immunoprecipitation of GABAA receptors purified by anti-alpha 1 324-341 antibody affinity chromatography by all three anti-alpha subunit antibodies employed, together with the use of anti-alpha 1 324-341 and anti-Cys alpha 2 414-424 antibody affinity columns in series, further substantiated the partial co-purification of the different polypeptides. These results demonstrate the copurification of the gamma 2 subunit with each population of alpha 1, alpha 2, alpha 3 subunit-enriched GABAA receptors. They also show the existence of minor populations of GABAA receptors that contain alpha 1 alpha 2, alpha 1 alpha 3, and alpha 2 alpha 3 subunit pairs within single oligomers.     

Identification of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25,26-hexol in human urine

A new bile alcohol, 5 beta-cholestanehexol, was identified in the urine of healthy humans as the glucuronide. The bile alcohol glucuronide fraction was isolated by an ion exchange chromatography on piperidinohydroxypropyl Sephadex LH-20. After enzymatic hydrolysis, the bile alcohols were converted into trimethylsilyl ether derivatives and analyzed by a combination of gas-liquid chromatography and mass spectrometry. The major bile alcohol was 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentol. As minor constituents the following C26 and C27 bile alcohols were identified: 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25,26-hexol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,26-pentol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol. In addition to these bile alcohols, a new bile alcohol was identified as a sixth component of the urinary bile alcohols. The structure was assigned as (24S)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25,26-hexol by the direct comparison of mass spectral data and chromatographic properties with synthetic standard. The average daily excretion of the new bile alcohol was 28.6 micrograms and 3.0% of the total bile alcohols. The presence of 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentol and 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25,26-hexol suggests that 26-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentol is most likely for the biosynthesis of this new bile alcohol.     

Identification of pentahydroxy bile alcohols in cerebrotendinous xanthomatosis: characterization of 5beta-cholestane-3alpha, 7alpha, 12alpha, 24xi, 25-pentol and 5beta-cholestane-3alpha, 7alpha, 12alpha, 23xi, 25-pentol.

This paper describes studies dealing with the nature of the C27 pentahydroxy bile alcohols present in the bile and feces of two patients with cerebrotendinous xanthomatosis (CTX). The presence of a bile alcohol having the structure 5beta-cholestane-3alpha,7alpha,12alpha,24alpha,25-pentol was confirmed by separation of the two 24-hydroxy epimers of 5beta-cholestane-3alpha,7alpha,12alpha,24,25-pentol and characterization of the dpimers by gas-liquid chromatography and infrared and mass spectrometry. Tentative assignment of the 24alpha and 24beta configuration was made on the basis of molecular rotation differences. A second major bile alcohol excreted by the CTX subjects was 5beta-cholestane-3alpha,7alpha,12alpha,23xi,25-pentol. Its structure was determined by infrared spectrometry, proton magnetic resonance spectrometry, and mass spectrometry because a reference compound was not available.     

Identification of 3 alpha, 6 alpha, 7 alpha, 12 alpha-tetrahydroxy-5 beta-cholestanoic acid in Zellweger's syndrome

In order to confirm the occurrence of 3 alpha, 6 alpha, 7 alpha, 12 alpha-tetrahydroxy-5 beta-cholestanoic acid in Zellweger's syndrome, the nature of tetrahydroxycholestanoic acids present in a patient with this disease was studied. Urinary bile acids were extracted with a Sep-pak C18 cartridge and methylated after alkaline hydrolysis. The methyl esters were purified by silica gel column chromatography, and the methyl tetrahydroxycholestanoate fraction was analyzed by gas liquid chromatography-mass spectrometry. Along with already known side chain hydroxylated derivatives of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid, 3 alpha, 7 alpha, 12 alpha, 24- and 3 alpha, 7 alpha, 12 alpha, 26-tetrahydroxy-5 beta-cholestanoic acids, three nuclear hydroxylated derivatives of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid were found. One of them was identified as 3 alpha, 6 alpha, 7 alpha, 12 alpha-tetrahydroxy-5 beta-cholestanoic acid by direct comparison with the authentic standard which was chemically synthesized from 3 alpha, 6 alpha, 7 alpha, 12 alpha-tetrahydroxy-5 beta-cholanoic acid by side chain elongation.     

Radioimmunoassay of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol 3-glucuronide in serum of patients with cerebrotendinous xanthomatosis.

A rapid, convenient, and specific radioimmunoassay for 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol has been developed. Specific antiserum was obtained from rabbits immunized by the bile alcohol-bovine serum albumin conjugate, which was coupled by an (O-carboxymethyl)oxime bridge at the C-3 position. The assay produces values for serum concentrations of bile alcohol glucuronides in patients with cerebrotendinous xanthomatosis.     

Synthesis of 3 alpha,6 beta,7 alpha,12 beta- and 3 alpha,6 beta,7 beta,12 beta-tetrahydroxy-5 beta-cholanoic acids.

Chemical synthesis of 3 alpha,6 beta,7 alpha,12 beta- and 3 alpha,6 beta,7 beta,12 beta-tetrahydroxy-5 beta-cholan-24-oic acids is described. 3 alpha,12 beta-Dihydroxy-5 beta-chol-6-en-24-oic acid used as the starting material in the synthesis was prepared via oxidation of 3 alpha,12 alpha-dihydroxy-5 beta-chol-6-en-24-oic acid 3-hemisuccinate at C-12 followed by reduction with potassium/tertiary amyl alcohol. alpha-Epoxidation of the ester diacetate of 3 alpha,12 beta-dihydroxy-5 beta-chol-6-en-24-oic acid with m-chloroperbenzoic acid followed by cleavage of the epoxide with acetic acid and alkaline hydrolysis yielded 3 alpha,6 beta,7 alpha,12 beta-tetrahydroxy-5 beta-cholan-24-oic acid (overall yield 25%). N-Methylmorpholine-N-oxide-catalyzed osmium tetroxide oxidation of the ester diacetate of 3 alpha,12 beta-dihydroxy-5 beta-chol-6-en-24-oic acid followed by alkaline hydrolysis yielded 3 alpha,6 beta,7 beta,12 beta-tetrahydroxy-5 beta-cholan-24-oic acid (overall yield 33%). The structures of the synthesized bile acids were confirmed from their proto nuclear magnetic resonance and mass spectral fragmentation patterns.     

New bile alcohols--synthesis of 5beta-cholestane-3alpha, 7alpha, 25-triol and 5beta-cholestane-3alpha, 7alpha, 25-24 (14C)-triol.

5beta-Cholestane-3alpha, 7alpha, 25-triol and 5beta-cholestane-3alpha, 7alpha, 25-24(14-C)-triol were synthesized from 3alpha, 7alpha-dihydroxy-5beta-cholanoic acid (chenodeoxycholic acid). Chenodeoxycholic acid was converted to the diformoxy derivative (II) using formic acid. Reaction of II with thionyl chloride yielded the acid chloride which was treated with diazomethane (CH-2-N-2 or 14-CH-2-N-2) to produce 3alpha, 7alpha-diformoxy-24-oxo-25-diazo-25-homocholane (III, A or B). 25-Homochenodeoxycholic acid (IV, A or B) was formed from III by means of the Wolff rearrangement of the Arndt-Eistert synthesis. The methyl ester of V (A or B) was treated with methyl magnesium iodidi in ether to provide the desired triol, VI (A and B). The triol was identified by mass spectrometry and elemental analysis and was characterized by thin-layer and gas-liquid chromatography. The 3alpha, 7alpha, 25-triol is of possible significance as an intermediate in the pathway of bile acid formation from cholesterol.     

Synthesis of C-22, C-23-3H-labeled 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestane

This report describes an efficient synthesis of C-22, C-23-(3)H-labeled 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestane. - Somanathan, R., and S. Krisans. Synthesis of C-22, C-23-(3)H-labeled 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestane.     

Similar enzymes, different structures: phthalate dioxygenase is an alpha3alpha3 stacked hexamer, not an alpha3beta3 trimer like "normal" Rieske oxygenases

Phthalate dioxygenase (PDO) is a member of a class of bacterial oxygenases that contain both Rieske [2Fe-2S] and Fe(II) mononuclear centers. Recent crystal structures of several Rieske dioxygenases showed that they exist as alpha(3)beta(3) multimers with subunits arranged head-to-tail in alpha and beta stacked planar rings. The structure of PDO, which consists of only alpha-subunits, remains to be solved. Although similar to other Rieske dioxygenases in many aspects, PDO was shown to differ in the mechanism of catalysis. Gel filtration and analytical centrifugation experiments, supplemented with mass spectrometric analysis (both ESI-MS and ESI-GEMMA), in this work showed a hexameric arrangement of subunits in the PDO multimer. Our proposed model for the subunit arrangement in PDO postulates two alpha(3) planar rings one on top the other, similar to the alpha(3)beta(3) arrangement in other Rieske dioxygenases. Unlike other Rieske dioxygenases, this arrangement brings two Rieske and two mononuclear centers, all on separate subunits, into proximity, allowing their cooperation for catalysis. Potential reasons necessitating this unusual structural arrangement are discussed.     

Nicotine binding to native and substituted peptides comprising residues 188-207 of nicotinic acetylcholine receptor alpha1, alpha2, alpha3, alpha4, alpha5, and alpha7 subunits

Structural determinants of L-[(3)H]nicotine binding to synthetic peptides comprising residues 188-207 of nicotinic acetylcholine receptor alpha subunits were invesitigated by equilibrium binding analysis. Two binding components were detected, one of low affinity (K(d) approximately 1.5 microM) that did not differ significantly among peptides and another of high affinity. The high affinity binding component was higher for the neuronal peptides (K(d) = 14-23 nM) than the muscle alpha1 peptides (K(d) = 52 nM). The following nonconservative substitutions in the alpha4 peptide resulted in a significant decrease in nicotine affinity for the peptide: Y190A, Y190D, C192G, E195A, E195-, P199A, P199-, and Y203A. Substitution of alpha4P199 with a leucine which is present in the alpha1 sequence decreased the affinity of the alpha4 peptide for nicotine and substitution of alpha1L199 with a proline (alpha4) or a glutamine (alpha3) increased the affinity of the alpha1 peptide. It is concluded that aromatic residues contribute to the binding site for nicotine on the alpha4 subunit and that the residue present at position 199 partly determines differences in nicotine affinity for different alpha subunits.