RESUMO
For evaluation of the cancer risk by the tryptophan pyrolysates a sensitive enzyme immunoassay for 3-amino-1-methyl-5H-pyrido-[4,3-b]indole (Trp-P-2), a mutagenic and carcinogenic substance, found in foods and biological fluids has been developed. The dose-response curve obtained was suitable for the determination of Trp-P-2 in the range of 20-800 pg. The immunoassay system was characterized to be satisfactorily specific as judged by cross-reactivity to gamma-carboline derivatives and their precursors, and comparison of immunoreactive Trp-P-2 levels in the basic and neutral fraction derived from pyrolyzed amino acids and related compounds.
Assuntos
Carbolinas/análise , Carcinógenos/análise , Mutagênicos/análise , beta-Galactosidase/análise , Temperatura Alta , Técnicas Imunoenzimáticas , Indicadores e Reagentes , Estrutura Molecular , Alcatrões , TriptofanoRESUMO
A direct radioimmunoassay method using highly specific antisera without prior deconjugation has been developed for determination of estradiol 17-glucuronide and estriol 16-glucuronide in human plasma. Antisera were elicited in the rabbit by immunization with antigens in which the steroid haptens are linked to a carrier protein through the C-2 or C-4 position. After treatment with Rivanol the protein of albumin-free antiserum was covalently bound to a p-arylamine glass bead support through the cross-linkage with glutaraldehyde. A simple and reliable assay method employing the antibody-glass preparation was established and applied to measurement of estrogen ring D glucuronide concentration in peripheral plasma throughout normal pregnancy.
Assuntos
Estradiol/sangue , Estriol/sangue , Glucuronatos/sangue , Gravidez , Animais , Feminino , Humanos , Soros Imunes , Coelhos , RadioimunoensaioRESUMO
A radioimmunoassay method using highly specific antiserum without prior deconjugation has been developed for determination of estrone sulfate in human plasma. Antiserum was elicited in the rabbit by immunization with antigen in which the sulfated steroid hapten is linked to a carrier protein through the C-6 position. After treatment with Rivanol, the protein of albumin-free antiserum was covalently bound to a p-arylamine glass bead support through the cross-linkage with glutaraldehyde. A simple and reliable method for measurement of estrone sulfate which involves elimination of endogenous interferences by use of Sep-pak C18 followed by radioimmunoassay employing the antibody-glass preparation, was established and applied to peripheral plasma throughout normal pregnancy.
Assuntos
Estrogênios Conjugados (USP)/sangue , Estrona/análogos & derivados , Animais , Reações Cruzadas , Estrona/sangue , Feminino , Humanos , Masculino , Gravidez , Coelhos , Radioimunoensaio/métodosRESUMO
A method for simultaneous determination of major bile acids in human bile without prior hydrolysis is described. The unconjugated, glycine- and taurine-conjugated bile acids are separated into groups by ion-exchange chromatography on a newly developed lipophilic gel, piperidinohydroxypropyl Sephadex LH-20 (PHP-LH-20). Subsequently, resolution of each group into cholate, ursodeoxycholate, chenodeoxycholate, deoxycholate and lithocholate is attained into two stages by high-performance liquid chromatography on a micron-Bondapak C18 column. First, 0.3% ammonium carbonate/acetonitrile (9 : 4, v/v) is used for separation of the latter three as a mobile phase. Cholate and ursodeoxycholate are separated in 0.3% ammonium carbonate/acetonitrile (11 : 4, v/v). The present method is applicable to quantitation of free and conjugated bile acids in human bile with satisfactory accuracy and precision.
Assuntos
Ácidos e Sais Biliares/análise , Bile/análise , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Estudos de Avaliação como Assunto , Humanos , MétodosRESUMO
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.
Assuntos
Ácidos e Sais Biliares/química , Butilaminas/química , Cromatografia Líquida de Alta Pressão/métodos , Íons , Espectrometria de MassasRESUMO
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.
Assuntos
Cromatografia Gasosa/métodos , Ácidos Siálicos/química , Esteroides/química , Hidroxilação , Estrutura MolecularRESUMO
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.
Assuntos
Ácidos e Sais Biliares/química , Cromatografia Gasosa/métodos , Glucosídeos/isolamento & purificação , Glucuronatos/isolamento & purificação , Cromatografia Gasosa/instrumentação , Ionização de ChamaRESUMO
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.
Assuntos
Ácido Quenodesoxicólico/isolamento & purificação , Ácido Desoxicólico/isolamento & purificação , beta-Ciclodextrinas , Bile/química , Ácido Quenodesoxicólico/química , Cromatografia em Camada Fina , Ciclodextrinas , Ácido Desoxicólico/química , Humanos , Indicadores e Reagentes , SolventesRESUMO
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.
Assuntos
Compostos de Epóxi/química , Esteroides/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , OxirreduçãoRESUMO
16 alpha-Hydroxyestrone, a product of estrogen 16 alpha-hydroxylation in humans that is suspected to be implicated in cell transformation, has been found to form stable adducts with nuclear components. The stable covalent adduct formed from 16 alpha-hydroxyestrone with 2-methoxyethylamine via the Heyns rearrangement of the alpha-hydroxyimine was identified as 3-hydroxy-17 beta-(2-methoxyethylamino)estra-1,3,5(10)-trien-16-one. Since the same product was obtained from 16 beta-hydroxyestrone with the amine, the alpha-hydroxyenamine is the most likely intermediate of the Heyns rearrangement. The adduct was fairly stable at 37 C in phosphate buffer (pH 7.4)/methanol (1:1 v/v), while the adduct formed from 16-oxoestradiol was disrupted reversely and completely within 6 hours. The evidence suggests that N-(3-hydroxy-16-oxoestra-1,3,5(10-trien-17 beta-yl)amine is the partial structure of the stable adducts formed from D-ring alpha-ketol estrogens with proteins.
Assuntos
Hidroxiestronas/química , Aminas , Estrogênios , Esteroide 16-alfa-HidroxilaseRESUMO
The ring A monoglucuronides and monosulfates of 2-hydroxyestriol were synthesized from 2-hydroxyestriol 16,17-diacetate by means of the Koenigs-Knorr reaction with methyl alpha-acetobromoglucuronate and sulfation with sulfur trioxide-pyridine complex, respectively. The conjugated positions of these compounds were definitely established by conversion to 2-hydroxyestriol monomethyl ethers by methylation, then enzymatic hydrolysis. The ring D monoglucuronides and monosulfates of 2-hydroxyestriol were also prepared from 2-hydroxyestriol 2,3-dibenzyl ether by glucuronidation and sulfation in a similar fashion followed by debenzylation, respectively. The positions of conjugation were established on the basis of their 1H-nuclear magnetic resonance spectral data.
Assuntos
Animais , Estriol/síntese química , Glucuronatos/síntese química , Glucuronidase/metabolismo , Caracois Helix/enzimologia , Sulfatases/metabolismo , Ácidos Sulfúricos/síntese químicaRESUMO
Four possible monoglucuronides of estetrol (estra-1,3,5(10)-triene-3,15 alpha, 16 alpha, 17 beta-tetraol) have been prepared from appropriately protected estetrol by the Koenigs-Knorr reaction employing cadmium carbonate as a catalyst. Condensation of methyl acetobromoglucuronate with estetrol 15,16,17-triacetate provided the 3-glucuronide acetate-methyl ester in a satisfactory yield. Introduction of the glucuronyl residue into C-17 was similarly attained by the use of estetrol 3-benzoate 15,16-acetonide. When estetrol 3,17-diacetate and acetobromosugar were stirred in anhydrous toluene in the presence of cadmium salt, the reaction occurred at C-16 and C-15 yielding two isomeric monoglucuronide derivatives in a ratio of ca. 5 to 2. Removal of the protecting groups in the four glucuronide acetate-methyl esters gave the desired estetrol glucuronides, respectively. These synthetic substrates underwent readily enzymatic hydrolysis with beef-liver beta-glucuronidase to afford estetrol.
Assuntos
Estetrol/síntese química , Estriol/análogos & derivados , Animais , Cádmio , Bovinos , Fenômenos Químicos , Química , Glucuronatos/síntese química , Glucuronidase , Hidrólise , Técnicas In Vitro , Fígado/enzimologia , Espectroscopia de Ressonância Magnética , MétodosRESUMO
3-N-Acetylglucosaminides of unconjugated, glycine- and taurine-conjugated bile acids have been synthesized. Bile acids appropriately protected were condensed with acetochloroglucosamine through the 3 alpha-hydroxyl group by means of the Koenigs-Knorr reaction using cadmium carbonate as a catalyst. Subsequent borohydride reduction and/or alkaline hydrolysis provided desired 3-N-acetylglucosaminides of unconjugated bile acids. Glycine-conjugates were obtained from N-acetylglucosaminides of unconjugated bile acids and ethyl glycinate by the carbodiimide method. The preparation of N-acetylglucosaminides of taurine-conjugates was attained by the Koenigs-Knorr reaction of bile acid p-nitrophenyl esters followed by condensation with taurine. 7-N-Acetylglucosaminides of ursodeoxycholates were prepared in a similar fashion. The convenient synthesis of 3-N-acetylglucosaminides of unconjugated bile acids is also described.
Assuntos
Acetilglucosamina/análogos & derivados , Ácidos e Sais Biliares/química , Acetilglucosamina/síntese química , Boroidretos , Cádmio , Carbonatos , Catálise , Glicina/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Taurina/metabolismo , Ácido Ursodesoxicólico/químicaRESUMO
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.
Assuntos
Eritrócitos/enzimologia , Estrogênios de Catecol/sangue , Adulto , Animais , Cães , Estradiol/análogos & derivados , Estradiol/metabolismo , Cobaias , Humanos , Macaca fascicularis , Metilação , Camundongos , Camundongos Endogâmicos ICR , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos SHR , Ratos Sprague-Dawley , Ratos Wistar , S-Adenosilmetionina/metabolismo , Especificidade da EspécieRESUMO
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.
Assuntos
Acetilcisteína/síntese química , Estrogênios de Catecol/química , Hidroxiestronas/química , Acetilcisteína/química , Espectroscopia de Ressonância Magnética , Estrutura MolecularRESUMO
The 2 beta-hydroxylated derivatives of lithocholic, chenodeoxycholic, deoxycholic, and cholic acids were synthesized from the respective parent bile acids by established procedures. The principal reactions involved were (1) bromination of 3-oxo formylated bile acids in N,N-dimethylformamide, (2) rearrangement and substitution of the resulting 4 beta-bromo-3-oxo derivatives to the 2 beta-acetoxy-3-oxo compounds with potassium acetate, and (3) reduction to the 2 beta-acetoxy-3 alpha-hydroxy compounds with tert-butylamine-borane complex. As for the prepared 2 beta-hydroxylated bile acids with a diequatorial trans-glycol structure, proton and carbon-13 nuclear magnetic resonance spectroscopic and gas-liquid chromatographic/mass spectrometric properties are discussed.
Assuntos
Ácidos Graxos/síntese química , Acetatos , Ácido Acético , Fenômenos Químicos , Química , Ácido Quenodesoxicólico/análogos & derivados , Ácido Cólico , Ácidos Cólicos/química , Ácido Desoxicólico/análogos & derivados , Dimetilformamida , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Hidroxilação , Ácido Litocólico/análogos & derivados , Espectroscopia de Ressonância MagnéticaRESUMO
Anti-testosterone and anti-5 alpha-dihydrotestosterone antisera have been produced by pretreatment of mice with a cross-reacting steroid coupled to a copolymer of D-glutamic acid and D-lysine to inhibit production of cross-reacting antibodies before immunization with the specific steroid conjugated to a protein. 15 beta-Carboxyethylmercapto derivatives were used as haptens. The cross-reactivities of the anti-testosterone and anti-5 alpha-dihydrotestosterone antisera with 5 alpha-dihydrotestosterone and with testosterone were 0.49 and 13.5%, respectively.
Assuntos
Di-Hidrotestosterona/imunologia , Soros Imunes , Testosterona/imunologia , Animais , Fenômenos Químicos , Química , Reações Cruzadas , Glutamatos , Lisina , Camundongos , PolímerosRESUMO
An improved procedure for the syntheses of stereoisomeric 3,6-dihydroxy- and 6-hydroxy-5 alpha-cholanoic acids (and their methyl esters) is described. The principal reactions employed are those reported in the preceding paper of this series, with the commercially available hyodeoxycholic acid as starting material. The final step in the procedure is the reduction of the key 5 alpha C-6 ketones with either the stereoselective equatorial reagent, Li/NH3/MeOH, or the axial reagent, Zn(BH4)2. The results of analysis of the prepared 6-monohydroxylated and 3,6-dihydroxylated stereoisomers by thin-layer chromatographic, high performance liquid chromatographic and gas-liquid chromatographic mobilities, and 1H and 13C nuclear magnetic resonance spectra are discussed along with the data for the corresponding compounds in the 5 beta-series.
Assuntos
Ácidos e Sais Biliares/metabolismo , Ácido Desoxicólico/análogos & derivados , Ácido Desoxicólico/síntese química , Cromatografia Gasosa , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Ácido Desoxicólico/química , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Oxirredução , EstereoisomerismoRESUMO
New synthetic routes to the four possible stereoisomeric 3 alpha,6,7,12 alpha-tetrahydroxy-5 beta-cholanoic acids (and their methyl esters), one of which (3 alpha,6 alpha 7 beta,12 alpha) is new, and some related compounds are described. In addition, the 5 alpha-epimer of the new acid was obtained. The final products were obtained in high purity for use as reference compounds in the analysis of bile acids in human biologic samples. The results of analysis of the prepared stereoisomers by proton and carbon 13 nuclear magnetic resonance spectroscopies are briefly discussed along with the thin-layer and gas-liquid chromatographic properties.
Assuntos
Ácidos e Sais Biliares/metabolismo , Ácidos Cólicos/síntese química , Fenômenos Químicos , Química , Ácidos Cólicos/química , Cromatografia Gasosa , Cromatografia em Camada Fina , Espectroscopia de Ressonância Magnética , Estrutura Molecular , EstereoisomerismoRESUMO
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.