The application of dimethyldioxirane for the selective oxidation of polyfunctional steroids.

Oxidation and epoxidation reactions of a series of structurally different steroids related to methyl 5 beta-cholanoates having hydroxyl groups and/or double bonds by treatment with dimethyldioxirane (DMDO) are described. Steroidal alcohols, olefines, and unsaturated alcohols and conjugated enones with DMDO were transformed into ketones, epoxides, and epoxy-ketones, respectively, in good isolated yields. The regio- and stereoselectivities for DMDO reaction differing from those observed for organic peracids, tert-butyl hydroperoxide and alkaline hydrogen peroxide are also discussed.

Capillary gas chromatographic behaviour of tert.-hydroxylated steroids by trialkylsilylation.

Capillary gas chromatographic behaviour was studied for a variety of structurally different bile acids and sterols having one to two tert.-hydroxy groups, together with several sec.-hydroxy groups, at positions C-3, -5, -7, -12, -14, -17, -20, -24, and/or -25. The tert.-hydroxylated steroids were subjected to trimethylsilylation with hexamethyldisilazane/trimethylchlorosilane /pyridine and N,O-bis(trimethylsilyl)acetamide/N-trimethylsilylimidazole/trimethylchlorosilane, and dimethylethylsilylation with N,N,-dimethylethylsilylimidazole. The methylene unit values of the resulting trialkylsilylation products were used for determining their structures of partially and/or fully derivatised ethers. The reactivity of the trialkylsilylation of tert.-hydroxy groups was found to be significantly dependent not only on the derivatisation reagents and conditions used, but also on the position and steric factor of the tert.-hydroxy groups. The following general order of the decreasing reactivity of tert.-hydroxy groups in steroids by trialkylsilyl etherification was observed: 25>20, 24>5beta>17alpha>>14alpha.

High-performance ion-pair chromatographic behaviour of conjugated bile acids with di-n-butylamine acetate.

This paper dealt with a simple and efficient method for separating a mixture of different series of ionic, high polar, and hydrophilic conjugates of bile acids by high-performance ion-pair chromatography (HPIPC) with a new volatile ion-pair chromatographic reagent, di-n-butylamine acetate (DBAA), as a mobile phase additive. The substrates examined included eleven different classes of C-24 glycine- or taurine-amidated, 3-sulfated, 3-glucosylated, 3-N-acetylglucosaminidated, and 3-glucuronidated conjugates of cholic, chenodeoxycholic, urosodeoxycholic, and deoxycholic acids, as well as their double-conjugated forms. The anionic conjugated bile acids were chromatographed on a C18, reversed-phase ion-pair column, eluting with methanol-water (65:35, v/v) containing 5 mM of DBAA as a counter ion. Satisfactory chromatographic separation and column performance were attained by DBAA, compared with conventionally used non-volatile tetra-n-butylammonium phosphate. The present HPIPC method with DBAA provides an insight into the separation and structural elucidation of these biologically important bile acid conjugates and may be proved to be applied to HPLC-mass spectrometric analysis.

Preparation of specific antisera to 15alpha-hydroxyestrogen 15-N-acetylglucosaminides.

3-(1-Carboxypropyl) ether derivatives of 15alpha-hydroxyestradiol 15-N-acetylglucosaminide (15alpha-OHE2 15NAG) and 15alpha-hydroxyestriol (E4) 15NAG were synthesized and conjugated with bovine serum albumin. Antisera elicited in rabbits possessed high affinity and specificity for the 15alpha-hydroxyestrogen (15alpha-OHEs) 15NAG, exhibiting no significant cross-reactivity with 15alpha-OHEs and their positional isomers such as 16NAG and 17NAG. Enzyme immunoassay methods developed by using the purified antisera and horseradish peroxidase-labeled antigens were applied to the measurement of 15alpha-OHEs 15NAG and E4 15NAG in normal pregnancy urine. We demonstrated for the first time that the conjugation of N-acetylglucosamine to E4 occurs at the C-15alpha position.

Method for the separation of the unconjugates and conjugates of chenodeoxycholic acid and deoxycholic acid by two-dimensional reversed-phase thin layer chromatography with methyl beta-cyclodextrin.

A simple and efficient method for the separation of individual unconjugated bile acids and their glycine- and taurine-amidated, 3-sulfated, 3-glucosylated and 3-glucuronidated conjugates is described. The method involves the use of a two-dimensional (2D) reversed-phase (RP) high-performance thin-layer chromatographic (HPTLC) technique with methyl beta-cyclodextrin (Me-beta-CD). Five major unconjugated bile acids, chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), ursodeoxycholic acid and lithocholic acid, and their conjugates were examined as the solutes. A high degree of separation of individual bile acids in each homologous series was achieved on a RP-HPTLC plate by developing with aqueous methanol in the first dimension and the same solvent system containing Me-beta-CD in the second dimension. In particular, all of the six 'difficult-to-separate' pairs, unconjugated CDCA and DCA and their conjugated forms with glycine, taurine, sulfuric acid, D-glucose and D-glucuronic acid, were effectively resolved by adding Me-beta-CD in the aqueous mobile phases with the formers having larger mobilities than the latter. The application of this 2D inclusion RP-HPLC method to the separation of glycine-conjugated bile acids in human bile is also described. The present method would be useful for separating and characterizing these bile acids present in biological materials.

Structure-retention correlation of isomeric bile acids in inclusion high-performance liquid chromatography with methyl beta-cyclodextrin.

The structure-retention correlation of various C24 bile acid isomers was studied by the addition of methyl beta-cyclodextrin (Me-beta-CD) to mobile phases in reversed-phase high-performance liquid chromatography (HPLC). The compounds examined include a series of monosubstituted bile acids related to cholanoic acids differing from one another in the position and configuration of an oxygen-containing function (hydroxyl or oxo group) at the position C-3, C-6, C-7, or C-12 and the stereochemistry of the A/B-ring fusion (trans 5 alpha-H and cis 5 beta-H) in the steroid nucleus. The inclusion HPLC with Me-beta-CD was also applied to biologically important 4 beta- and 6-hydroxylated bile acids substituted by three to four hydroxyl groups in the 5 beta-steroid nucleus. These bile acid samples were converted into their fluorescence prelabeled 24-pyrenacyl ester derivatives and chromatographed on a Capcell Pak C18 column eluted with methanol-water mixtures in the presence or absence of 5 mM Me-beta-CD. The effects of Me-beta-CD on the retentions of each compound were correlated quantitatively to the decreasing rate of capacity factors and the relative strength of host-guest interactions. On the basis of the retention data, specific and nonspecific hydrogen-bonding interactions between the bile acids and the Me-beta-CD were discussed.

Comparative abilities and optimal conditions for beta-glycosidase enzymes to hydrolyse the glucuronide, glucoside, and N-acetylglucosaminide conjugates of bile acids.

Enzymatic hydrolyses were described for three variants of glycosidic conjugated bile acids with one beta-glucuronidase (Helix pomatia), three beta-glucosidase (almonds, sweet almonds, and Escherichia coli), and four beta-N-acetylglucosaminidase (jack beans, bovine kidney, human placenta, and Diplococcus pneumoniae) preparations. The substrates include the beta-glucuronide, beta-glucoside, and beta-N-acetylglucosaminide conjugates of bile acids related to hyodeoxycholic, murideoxycholic, chenodeoxycholic, and ursodeoxycholic acids possessing a sugar moiety at position C-3, C-6 or C-7. The comparative abilities and optimal conditions for the beta-glycosidases to catalyze the hydrolyses of the substrates were clarified by changing pHs and incubation times. Hydrolysis rates of the bile acid glycosides with beta-glycosidase treatments were influenced by both the source of the enzyme preparations and the conjugated position of a sugar moiety in the substrates, and the 3-glucoside and 3-N-acetylglucosaminide conjugates were usually hydrolyzed more efficiently than their corresponding 6- and 7-analogs. Escherichia coli and jack bean enzymes were chosen to hydrolyse the glucosidic and N-acetylglucosaminidic conjugated bile acids, respectively.

An improved synthesis of taurine- and glycine-conjugated bile acids.

A simple and efficient method for the synthesis of taurine- and glycine-conjugated bile acids is described. The condensation reaction was achieved by the simple mixing of unconjugated bile acid (1.0 eq.), taurine (2.0 eq.) (or glycinate ester), diethyl phosphorocyanidate (1.2 eq.) in the presence of triethylamine at room temperature for 30-60 min. Sample clean-up was effected by the use of a prepacked Sep-Pak C18 cartridge for reversed-phase solid extraction or by direct recrystallization, yielding the desired taurine and glycine conjugates in 89-93 and 92-96% isolated yields, respectively.

Preparation of specific antiserum to 17 alpha-estradiol 17-N-acetylglucosaminide.

A specific enzyme immunoassay (EIA) method has been developed for 17 alpha-estradiol 17-N-acetylglucosaminide as a model compound instead of 15 alpha-hydroxyestrogen 15-N-acetylglucosaminides. Two new haptens, 3-(omega-carboxyalkyl) ether derivatives of 17-alpha-estradiol 17-N-acetylglucosaminide, were synthesized and conjugated with bovine serum albumin (BSA). The EIA was newly established using specific antiserum elicited against 3-(1-carboxypropyl) ether of 17 alpha-estradiol 17-N-acetylglucosaminide (17NAG CPE)-BSA conjugate and beta-galactosidase-labeled 17NAG CPE as a labeled antigen. An appropriate dose-response curve of EIA for 17 alpha-estradiol 17-N-acetylglucosaminide was obtained in the range of 20-1000 pg/tube. The specificity of EIA proved to be satisfactory in terms of cross-reactivities to related compounds including 15 alpha-N-acetylglucosaminides. The proposed method will be applicable to the preparation of antisera for use in EIA of 15 alpha-hydroxyestrogen 15-N-acetylglucosaminides.

A novel enzyme system for the reduction of 3-oxo bile acids in human red blood cells.

7 alpha,12 alpha-Dihydroxy-3-oxo- and 3,7,12-trioxo-5 beta-cholanoic acids labeled with 18O atoms were incubated with human red blood cells, and the biotransformation products were separated and characterized by gas chromatography-mass spectrometry as the pentafluorobenzyl ester-trimethylsilyl and -dimethylethylsilyl ether derivatives with the negative ion chemical ionization mode. The reduced products, 3 beta,7 alpha,12 alpha-trihydroxy-5 beta-cholanoic acid for the former, and 3 alpha-hydroxylated dioxo bile acid together with 3 beta-hydroxylated 7,12-dioxo-5 beta-cholanoic acid for the latter, were identified as metabolites. When 3-oxo bile acid was incubated with human blood denatured at 70 degrees C for 2 min, no metabolites were formed. The enzymic reduction activity has been localized in the red blood cell fraction.

Synthesis of N-acetylcysteine conjugates of catechol estrogens.

The synthesis of N-acetylcysteine conjugates of 2-hydroxyestrone (2-OHE1) and 4-hydroxyestrone (4-OHE1) is described. The reaction of estrone 2,3-quinone with N-acetylcysteine provided 2-OHE1 and its C-4 and C-1 thioether conjugates in a ratio of 1:1, while estrone 3,4-quinone with N-acetylcysteine gave 4-OHE1 and its C-2 thioether conjugate as a sole product. Their structures were characterized by inspection of NMR spectra, chemical derivatization (methylation and acetylation), and comparison with the reactivity of 4-bromoestrone 2,3-quinone or 2-bromoestrone 3,4-quinone toward N-acetylcysteine.

Stereospecific dehydrogenation of (25R)- and (25S)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acids by acyl-CoA oxidase in rat liver light mitochondrial fraction.

From a stereochemical point of view, the dehydrogenation mechanism of the biotransformation of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) into (24E)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-enoic acid (delta 24-THCA) has been studied with capillary gas chromatography (GC)/negative ion chemical ionization (NICI)-mass spectrometry. After incubation of (24R,25R)- or (24S,25S)-[24,25-2H2]THCA, synthesized from (24E)-delta 24-THCA by a deuterated diimide reduction, with a rat liver light mitochondrial fraction, 5 beta-cholestanoic acids were extracted and derivatized into a pentafluorobenzyl (PFB) ester-dimethylethylsilyl (DMES) ether. Subsequent resolution into THCA and delta 24-THCA was attained by GC on a cross-linked 5% phenylmethyl silicone fused-silica capillary column monitored with a corresponding characteristic carboxylate anion [M-PFB]- in the NICI mode. The stereospecific elimination of a pro-R hydrogen at C-24 in both (25R)- and (25S)-THCA indicated syn-elimination for the former, whereas anti-elimination for the latter was observed.

Potential bile acid metabolites. 23. Syntheses of 3-glucosides of nonamidated and glycine- and taurine-amidated bile acids.

The 3-glucosides of nonamidated lithocholic, chenodeoxycholic, ursodeoxycholic, deoxycholic, and cholic acids, and their double conjugate forms with glycine and taurine were synthesized. The key reactions used were 1) beta-D- glucosidation at C-3 by the Koenigs-Knorr condensation reaction of 3 alpha-hydroxylated bile acid methyl (or p-nitrophenyl) esters with 1 alpha-bromo-1-deoxy-2, 3, 4, 6-tetra-O-acetyl-D-glucopyranose in the presence of cadmium carbonate in refluxing benzene; 2) indirect and direct amidations at C-24 by the activated p-nitrophenyl ester and by the diethylphosphorylcyanide methods, respectively, using glycinate ester and taurine as coupling agents; and 3) simultaneous alkaline hydrolysis of the hydroxyl-protecting and ester groups in both the sugar and aglycone moieties.

Synthesis of 15 alpha-hydroxyestrogen 15-N-acetylglucosaminides.

The synthesis of 15-N-acetylglucosaminides of 15 alpha-hydroxyesterone, 15 alpha-hydroxyestradiol, and 15 alpha-hydroxyestriol (estetrol) is described. The latter two were prepared by condensation of 2-acetamido-1 alpha-chloro-1,2-dideoxy-3,4,6-trio-O-acetyl-D-glucopyranose with appropriately protected 15 alpha-hydroxyestrogens by the Koenigs-Knorr reaction employing cadmium carbonate as a catalyst. Subsequent removal of protecting groups with methanolic potassium hydroxide provided the desired conjugates. 15 alpha-Hydroxyestrone 15-N-acetylglucosaminide was synthesized from the corresponding 15 alpha-hydroxyestradiol derivative by Jones oxidation followed by brief alkaline hydrolysis. These conjugates underwent enzymatic hydrolysis with beta-N-acetylglucosaminidase from Jack beans to produce 15 alpha-hydroxyestrogens.

Gas chromatographic separation of bile acid 3-glucosides and 3-glucuronides without prior deconjugation on a stainless-steel capillary column.

A method for the gas chromatographic (GC) separation of the 3-glucoside and 3-glucuronide conjugates of bile acids without the necessity for a hydrolytic step is described. The bile acid glycosides were derivatized to their complete methyl ester trimethylsilyl (Me-TMS) or methyl ester dimethylethylsilyl (Me-DMES) ether derivatives, which in turn were chromatographed on an inert and thermostable stainless-steel capillary column, Ultra ALLOY-1 (HT), coated with a thin film (0.15 micron) of chemically bonded and cross-linked dimethylsiloxane. They exhibited a single peak of the theoretical shape without any accompanying peaks due to thermal decomposition, even at oven temperatures of 320-330 degrees C. Excellent GC separation of isomeric bile acid glycosides was achieved by the combined use of suitable derivatives and column. This method, which does not need the prior deconjugation of the glycosidic moiety, could be usefully applied to biosynthetic and metabolic studies of bile acids in biological materials.

Analysis of conjugated bile acids in human biological fluids. Synthesis of hyodeoxycholic acid 3- and 6-glycosides and related compounds.

The glucuronide, glucoside and N-acetylglucosaminide conjugates of hyodeoxycholic acid were synthesized. In addition, murideoxycholic acid 3-glycosides and some of their C-5 epimeric analogs were also prepared. The principal reactions used are 1) the Koenigs-Knorr condensation reaction of 3-oxo-6 alpha-hydroxy and 6-oxo-3 alpha-hydroxy esters with an appropriate alpha-acetohalosugar catalyzed by cadmium carbonate in benzene under reflux, 2) reduction of the resulting bile acid glycoside methyl ester-acetates with tert-butylamine-borane complex, and 3) subsequent hydrolysis with aqueous lithium hydroxide.

Immunoaffinity extraction of 4-hydroxy-2-(4-methylphenyl)benzothiazole and its metabolites for determination by gas chromatography-mass spectrometry.

Immunoaffinity extraction of 4-hydroxy-2-(4-methylphenyl)benzothiazole and its metabolites, together with the corresponding meta-isomers has been achieved by the use of an antibody raised against an immunogen, an O-carboxymethyloxime-bovine serum albumin conjugate of 4-hydroxy-2-(4-formylphenyl)benzothiazole. The antibody produced exhibited a broad spectrum of affinity, not only for metabolites oxidized at the 4-methyl group of the benzene moiety but also for the corresponding meta-isomers. Up to 4 micrograms in total of these benzothiazoles could be extracted on the immunoaffinity adsorbent and recovered almost quantitatively by elution with 90% methanol. The resulting chromatogram was free from any interference. The eluted compounds were derivatized by conversion to their methyl esters and/or trimethylsilyl ethers, and subsequently separated into individual benzothiazoles by means of gas chromatography-mass spectrometry. The derivatized compounds were monitored using a characteristic ion, [M-CH3]+., and the limit of detection was 10 fmole. The peak height ratio of each metabolite to its corresponding meta-isomer internal standard was plotted against the concentration of the former and good linearity was observed over the range 0.2-5 ng/ml.

Novel immunoaffinity extraction for liquid chromatographic determination of major metabolites of 4-acetoxy-2-(4-methylphenyl)benzothiazole in plasma.

Group extraction of the metabolites of 4-acetoxy-2-(4-methylphenyl)benzothiazole has been achieved through the use of an immunoaffinity adsorbent. The antisera elicited from an immunogen, 4-hydroxy-2-(4-formylphenyl)benzothiazole O-carboxymethyloxime-bovine serum albumin conjugate, were characterized to have a broad affinity spectrum for major metabolites oxidized at the 4-methyl group of the benzene moiety. One millilitre of the immunoaffinity adsorbent prepared by immobilization of antibodies (12.5 mg ml-1) was capable of retaining up to 4 micrograms of benzothiazoles. The adsorbates were recovered quantitatively by elution with 90% (v/v) methanol without any interfering peaks on the high-performance liquid chromatogram. The peak-height ratio of each metabolite to an internal standard was plotted against the concentration of the former substance; good linearity was observed in the range of 10-500 ng ml-1.

Enzymatic O-methylation of catechol estrogens in red blood cells: differences in animal species and strains.

Enzymatic O-methylation of catechol estrogens in red blood cells has been investigated with respect to species difference. In the presence of S-adenosylmethionine, 2- or 4-hydroxyestradiol (2-OHE2 or 4-OHE2) was incubated with blood lysate obtained from rats (five strains), guinea pigs, mice, rabbits, dogs, monkeys, and humans, respectively. The yielded guaiacols and unchanged substrate were determined by gas chromatography/mass spectrometry in a selected ion monitoring mode employing the corresponding 2H4-labeled compounds as internal standards. The total amounts of guaiacols formed from 2-OHE2 and 4-OHE2 were different, being the highest (79.6% and 38.1%) in monkeys and the lowest (5.1% and 1.9%) in humans. The ratios of isomeric guaiacols formed from 4-OHE2 (4Me/3Me) were 7.6-71, while those from 2-OHE2 (2Me/3Me) were 1.4-3.2. Thus, marked differences in O-methylation of catechol estrogens were observed among animal species, but no significant strain difference was detected in rats.