The biosynthesis of 23,24-dinor-5-cholene-3beta,20-diol and 23,24-dinor-5-cholene-3beta,21-diol.
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Acetates↗
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Biosynthesis of adrenal corticosteroids by the sesterterpene pathway via 23,24-dinor-4-cholen-3-one.
Rat adrenal gland preparations were incubated with 23,24-dinor-5-cholen-3 beta-ol (Guneribol), a proposed intermediate in the sesterterpene pathway for steroid biosynthesis. Steroids were isolated, purified by thin layer and high-performance liquid chromatographies and crystallized to constant specific activity. These preparations converted the substrate to 23,24-dinor-4-cholen-3-one. Radioactive 23,24-dinor-4-cholen-3-one was synthesised and incubated with further tissue preparations and shown to be converted to corticosteroids. These findings suggest that 23,24-dinor-4-cholen-3-one is an intermediate on the sesterterpene pathway for steroidogenesis.
Adrenal Cortex Hormones↗
Biosynthesis of androgens by the sesterterpene pathway via 23,24-dinor-4-cholen-3-one.
Rat testicular and adrenal gland microsomal preparations were incubated with 23,24-dinor-5-cholen-3 beta-ol (Guneribol) a proposed intermediate in the sesterterpene pathway for steroid biosynthesis. Steroids were isolated, purified by thin layer and high-performance liquid chromatography and crystallized to constant specific radioactivity. These preparations converted the substrate to 23,24-dinor-4-cholen-3-one. Radioactive 23,24-dinor-4-cholen-3-one was synthesized and incubated with further tissue preparations and shown to be converted to steroid hormones. These findings suggest that 23,24-dinor-4-cholen-3-one is an intermediate on the sesterterpene pathway for steroidogenesis.
Adrenal Glands↗
Determination of 3 beta,12 alpha-dihydroxy-5-cholen-24-oic acid and related bile acids in human serum by gas chromatography-mass spectrometry.
The glycine, taurine and sulphate conjugates of 3 beta,12 alpha-dihydroxy-5-cholen-24-oic acid were synthesized as authentic samples for the analysis of this unusual bile acid. A highly sensitive and specific quantitative assay of the bile acid and related compounds has been developed by selected-ion monitoring in gas chromatography-mass spectrometry of their methyl ester-trimethylsilyl ether derivatives, using the deuterium labelled internal standards: [2H6]3 beta,12 alpha-dihydroxy-5-cholen-24-oic acid, [2H5]3 beta-hydroxy-5-cholen-24-oic acid, [2H7]cholic acid and their sulphates. Calibration curves for these bile acids were linear over the range 0.01-100 micrograms/ml in human serum. Recoveries of the bile acids and their conjugates ranged from 95 to 103% of the added amounts of their standard samples. The unsaturated bile acid was identified in a significant amount of 25.2 micrograms/ml in serum of an infant with liver disease, and its sulphate comprised 55.1% of the amount of the bile acid.
Bile Acids and Salts↗
Chemical synthesis of the 3-sulfooxy-7-N-acetylglucosaminyl-24-amidated conjugates of 3beta,7beta-dihydroxy-5-cholen-24-oic acid, and related compounds: unusual, major metabolites of bile acid in a patient with Niemann-Pick disease type C1.
The chemical synthesis of 3beta,7beta-dihydroxy-5-cholen-24-oic acid, triply conjugated by sulfuric acid at C-3, by N-acetylglucosamine (GlcNAc) at C-7, and by glycine or taurine at C-24, is described. These are unusual, major metabolites of bile acid found to be excreted in the urine of a patient with Niemann-Pick disease type C1. Analogous double-conjugates of 3beta-hydroxy-7-oxo-5-cholen-24-oic acid were also prepared. The principal reactions involved were: (1) beta-d-N-acetylglucosaminidation at C-7 of methyl 3beta-tert-butyldimethylsilyloxy (TBDMSi)-7beta-hydroxy-5-cholen-24-oate with 2-acetamido-1alpha-chloro-1,2-dideoxy-3,4,6-tri-O-acetyl-d-glucopyranose in the presence of CdCO(3) in boiling toluene; (2) sulfation at C-3 of the resulting 3beta-TBDMSi-7beta-GlcNAc with sulfur trioxide-trimethylamine complex in pyridine; and (3) direct amidation at C-24 of the 3beta-sulfooxy-7beta-GlcNAc conjugate with glycine methyl ester hydrochloride (or taurine) using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride as a coupling agent in DMF. The structures of the multi-conjugated bile acids were characterized by liquid chromatography-mass spectrometry with an electrospray ionization probe under the positive and negative ionization modes.
Bile Acids and Salts↗
Chenodeoxycholic acid synthesis in the hamster: a metabolic pathway via 3 beta, 7 alpha-dihydroxy-5-cholen-24-oic acid.
The quantitative significance of the metabolism of 3 beta, 7 alpha-dihydroxy-5-cholen-24-oic acid to chenodeoxycholic acid was evaluated in the hamster. A precursor-product relationship was established in this species by the finding that intravenous administration to an animal previously given cholesterol-4-14C caused a significant reduction in the specific activity of chenodeoxycholic acid. Administration of 12.9 mumole of the precursor was followed by a 10-fold increase in chenodeoxycholic acid excretion although the predominant excretory pathway was via biliary excretion as a monosulfate. The data indicate that synthesis of bile acid from cholesterol via the intermediate 3 beta, 7 alpha-dihydroxy-5-cholen-24-oic acid can be a quantitatively important pathway.
Animals↗
Preparation and antigenic property of methyl 3 beta-hydroxy-19-oxo-5-cholen-24-oate 19-(O-carboxymethyl) oxime-bovine serum albumin conjugate.
The preparation and antigenic property of 3 beta-hydroxy-5-cholen-24-oic acid-bovine serum albumin (BSA) conjugate in which the hapten is linked to the carrier protein through an (O-carboxymethyl) oxime bridge at the C-19 position on the steroid nucleus is described. Antibody raised against antigen in the rabbit possessed high titer and specificity to 3 beta-hydroxy-5-cholen-24-oic acid, exhibiting no significant cross-reactions with various bile acids.
Antigen-Antibody Complex↗
Preparation and comparative properties of antisera against glyco-3 beta,12 alpha-dihydroxy-5-cholen-24-oic acid linked to bovine serum albumin at the C-3, C-15 alpha, and C-24 positions.
Anti glyco-3 beta,12 alpha-dihydroxy-5-cholen-24-oic acid antisera were prepared by immunizing rabbits with hapten-bovine serum albumin (BSA) conjugates coupled at the C-3, C-15 alpha, and C-24 positions on the bile acid molecule, and their properties were investigated by heterologous combination assay using 125I-labeled tracer. The antiserum raised against the C-3 BSA conjugate showed poor titer and specificity, while the antisera from the other two conjugates showed satisfactorily high affinity constants (Ka = 5.0 x 10(8) and 7.0 x 10(8) M-1) and reasonable specificity, exhibiting negligible cross-reactivities with other major human bile acids and cholesterol. Among the unsaturated bile acids tested, high reactivity was observed with tauro-3 beta,12 alpha-dihydroxy-5-cholen-24-oic acid, which suggested that bridge phenomena were significant in this assay system.
Antibodies↗
Inhibition of androgen synthesis by 22-hydroximino-23,24-bisnor-4-cholen-3-one.
The 22-hydroximino-23,24-bisnor-4-cholen-3-one (22-oxime) was synthesized and evaluated as an inhibitor of 17 alpha-hydroxylase/C17,20-lyase in rat testicular microsomes and the 5 alpha-reducatase of human prostatic microsomes from patients with benign prostatic hypertrophy. The 22-oxime demonstrated moderate inhibition for the 17 alpha-hydroxylase (Ki 74 nM vs. Km 29 nM) with progesterone as substrate and potent inhibition (Ki 18 nM vs. Km 76 nM) for the C17,20-lyase activity with 17 alpha-hydroxyprogesterone as substrate. Further investigation of this enzyme with progesterone as substrate demonstrated the inhibition occurred mainly at the 17 alpha-hydroxylation step of the progesterone substrate. The 22-oxime also demonstrated potent and competitive inhibition of 5 alpha-reductase in human prostatic microsomes (Ki 1.4 nM vs. Km 14 nM). When adult male rats were injected subcutaneously (sc) daily with 22-oxime (50 mg/kg/day) for 21 days, the concentrations of serum and testicular testosterone were significantly reduced by 65% and 59%, respectively, in comparison to vehicle-treated controls. Furthermore, both testosterone and DHT concentrations in rat prostatic tissue were significantly decreased by 60% and 44% compared to control tissue. Serum LH concentrations were unchanged in the 22-oxime-treated group compared to the control group. This indicates that the reduction in androgen concentrations in animals treated with this compound is not due to its influence on pituitary feedback mechanisms which result in reduced LH secretion. These results suggest that 22-oxime is effective in reducing androgen synthesis through the inhibition of 17 alpha-hydroxylase, C17,20-lyase, and 5 alpha-reductase both in vitro and in vivo.
5-alpha Reductase Inhibitors↗
Effect of ionic strength on the transfer of 1-pyrenemethyl-3 beta-hydroxy-22,23-bisnor-5-cholenate between bilayer vesicles containing phosphatidylserine.
The influence of ionic strength or the concentration of K+ ([K+]) of the aqueous phase on the spontaneous transfer of cholesterol between negatively charged bilayer vesicles composed of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylserine (DPPS) (1:1, mole:mole) was studied using a pyrene-labelled cholesterol analogue, 1-pyrenemethyl-3 beta-hydroxy-22,23-bisnor-5-cholenate (PMC), as the probe. The decrease in PMC excimer fluorescence was best fitted to a bi-exponential function. Increasing [K+] from 0.1 M to 0.3 M had little effect on the shorter half-time (1.4 +/- 0.2 min) but increased the longer half-time from 16.3 +/- 1.9 min to 26.7 +/- 2.1 min. Fluorescence quenching and titration of an interface-located fluorophore, 1-anilinonaphthalene-8-sulfonic acid (ANS) revealed an increase in interfacial hydrophobicity upon increasing in ionic strength. The physical state of the acyl chains was not affected by ionic strength as indicated by a constant PMC excimer:monomer fluorescence intensity ratio. However, an increase in enthalpy change of the lipid phase transition from 15.7 kJ/mol ([K+] = 0.1 M) to 21.3 kJ/mol ([K+] = 0.3 M), together with a slight increase in the transition temperature, implies that interactions between adjacent molecules in the charged lipid bilayer vesicles became stronger at higher ionic strength. Our results suggest that the van der Waals attraction between PMC and phospholipid molecules could be affected by conformation changes in the charged head group region brought about by changes of ionic strength in the aqueous phase, with consequent effects on the desorption of cholesterol from the bilayer surface.
1,2-Dipalmitoylphosphatidylcholine↗
A new approach to the synthesis of 3 beta, 23-diacetoxy-24-nor-5-cholene from 3 beta, 21-diacetoxy-5-pregnen-20-one.
Synthesis of 3 beta, 23-diacetoxy-24-nor-5-cholene in six steps from 3 beta, 21-diacetoxy-5-pregnen-20-one has been achieved by a new approach. The method would allow to label the side chain of a bile acid at carbon-atom 21.
17-alpha-Hydroxypregnenolone↗
A convenient synthesis of 3 beta,12 alpha-, 3 beta,7 alpha-, and 3 beta,7 beta-dihydroxy-5-cholen-24-oic acids: unusual bile acids in human biological fluids.
The unusual bile acids 3 beta,12 alpha- (V), 3 beta,7 alpha- (XIIIa), and 3 beta,7 beta- (XIIIb) dihydroxy-5-cholen-24-oic acids were synthesized conveniently from the 3-oxo derivatives of deoxycholic (I) and lithocholic (VI) acids, respectively, to provide authentic samples for the gas chromatography-mass spectrometric determination of these bile acids in the abnormal metabolism of bile acids.
Bile Acids and Salts↗
Nuclear magnetic resonance spectroscopy of 3 beta,7 beta-dihydroxy-5-cholen-24-oic acid multi-conjugates: unusual bile acid metabolites in human urine.
Complete 1H and 13C resonance assignments were made for a new type of 3 beta,7 beta-dihydroxy-5-cholen-24-oic acid doubly conjugated with sulfuric acid at C-3 and N-acetylglucosamine at C-7 and its glycine- and taurine-amidated triple-conjugates by the combined use of several homonuclear and heteronuclear shift-correlated 2D NMR techniques. The effects of sulfation at C-3, N-acetylglucosaminidation at C-7, and aminoacyl amidation at C-24 on the 1H and 13C chemical shifts and signal multiplicity were clarified. The shielding data serving to characterize each of the bile acid multi-conjugates are also discussed.
Bile Acids and Salts↗
Determination of 3 beta-hydroxy-5-cholen-24-oic acid and its sulfate in human serum by gas chromatography-mass spectrometry.
The glycine conjugate of 3 beta-hydroxy-5-cholen-24-oic acid and its sulfate labeled with deuterium at the C-2, -4, and -23 positions were synthesized. A highly sensitive and specific quantitative assay of the bile acid has been developed by selected ion monitoring in gas chromatography-mass spectrometry of the methyl ester trimethylsilyl ether derivatives using the deuterium labeled conjugates as internal standards. Calibration curves for the bile acid and its sulfate exhibited a linear relationship over the range of 0.01-100 micrograms/ml in human serum.
Bile Acids and Salts↗
Inhibition of steroidogenesis in cultured Leydig tumor cells by 22-amino-23,24-bisnor-5-cholen-3 beta-O1 and (20R) 20-phenyl-5-pregnene-3 beta,20-diol.
Using the cholesterol side-chain cleavage enzyme purified from bovine adrenals, we have previously shown that 22-amino-23,24-bisnor-5-cholen-3-beta-ol (22-ABC) and (20R) 20-phenyl-5-pregnene-3 beta, 20 diol (20-PPD) are potent competitive inhibitors of this enzyme. The studies presented herein were designed to characterize the effects of these new inhibitors on steroid production by intact cells. Using cultured Leydig tumor cells, we compared the effects of 22-ABC, 20-PPD, and aminoglutethimide (a well-known inhibitor of the cholesterol side-chain cleavage enzyme) on hormone-stimulated steroidogenesis. Our results show that these compounds inhibit steroid production in a dose-dependent manner and that 20-PPD and 22-ABC are about 100 and 4 times more active, respectively, than aminoglutethimide. In these cells, the inhibitory effects of the new compounds seem to be localized exclusively at the conversion of cholesterol to pregnenolone.
Aminoglutethimide↗
Transfer of cholesterol and a fluorescent cholesterol analog, 3'-pyrenylmethyl-23,24-dinor-5-cholen-22-oate-3 beta-ol, between human plasma high density lipoproteins.
A fluorescent cholesterol analog, 3'-pyrenylmethyl-23,24-dinor-5-cholen-22-oate-3 beta-ol (PMCA), has been synthesized as a spectroscopic probe of cholesterol function. The substrate activity of PMCA, about two-thirds that of cholesterol, with lecithin:cholesterol acyltransferase indicates that PMCA is a reasonable cholesterol analog and that the orientation of the substituted sterol in the phospholipid interface is similar to that of cholesterol. The fluorescence properties of PMCA are similar to those of other pyrene-containing compounds that exhibit concentration-dependent excimer fluorescence. The rate of transfer of [3H]PMCA between HDL is about six times faster than cholesterol. These results indicate that the analog will be useful in studies of cholesterol function.
Cholesterol↗
Nuclear magnetic resonance identification of the taurine conjugate of 3 alpha,6 beta,7 beta-trihydroxy-5 beta,22-cholen-24-oic acid (tauro-delta 22-beta-muricholate) in the serum of female rats treated with alpha-naphthylisothiocyanate.
Recently developed 1H nuclear magnetic resonance methods, including 2-dimensional, 1H-detected-[13C] shift correlation spectroscopy and 1-dimensional HOHAHA spectroscopy at 500 MHz have been used to identify the major bile acid in the serum of rats treated with alpha-naphthylisothiocyanate as the taurine conjugate of 3 alpha,6 beta,7 beta-trihydroxy-5 beta,22-cholen- 24-oic acid (tauro-delta 22-beta-muricholate), a derivative of beta-muricholate having an unsaturated bond in the acyclic side chain. Complete stereospecific assignments of the 1H and protonated 13C spectra of the title compound and beta-muricholate are reported. The assignments were based entirely on 1H-1H and 1H-13C scalar connectivities and were made using approximately 0.5-2.0 mg of material. It is suggested that these new methods will be of general value for identifying the structure and assigning the spectra of other scarce steroid-like molecules.
1-Naphthylisothiocyanate↗
Taurine conjugate of 3 alpha,6 beta,7 beta-trihydroxy-5 beta,22-cholen-24-oic acid (tauro-delta 22-beta-muricholate): the major bile acid in the serum of female rats treated with alpha-naphthylisothiocyanate and its secretion by liver slices.
The taurine conjugate of 3 alpha,6 beta,7 beta-trihydroxy-5 beta,22-cholen-24-oic acid (tauro-delta 22-beta-muricholate) has been identified in the serum of female rats treated with alpha-naphthylisothiocyanate. Using a high performance liquid chromatographic/enzymatic method for measurement of bile acids, tauro-delta 22-beta-muricholate was the predominant bile acid in the serum of female Fischer 344 rats treated for 3 days with alpha-naphthylisothiocyanate. Other significant changes in concentrations of serum bile acids included increases in tauro-alpha-muricholate, tauro-beta-muricholate, taurocholate, taurochenodeoxycholate, and several unknown bile acids. The formation of tauro-delta 22-beta-muricholate was examined in vitro using liver slices from control rats and rats treated with alpha-naphthylisothiocyanate. Slices were incubated for 7 h in William's E medium containing no bile acids or 25 mumol/l of one of the following: beta-muricholate, tauro-beta-muricholate, or cholate. Tauro-delta 22-beta-muricholate was secreted by slices from control and treated rats and the rate was increased significantly by the addition of beta-muricholate (2.9-5.6-fold) but not tauro-beta-muricholate or cholate to the medium. Tauro-delta-beta-muricholate was formed by liver slices from endogenous precursors and from exogenous beta-muricholate. Pretreatment of rats with alpha-naphthylisothiocyanate did not alter total secretion rates but those of some important individual bile acids were affected. Because of the increased secretion of tauro-delta-beta-muricholate by liver slices with the addition of beta-muricholate to the medium, the liver may be the primary site of formation for this unsaturated bile acid.
1-Naphthylisothiocyanate↗