RESUMO
The sterols 7 alpha-hydroxycholest-4-en-3-one (I) and 5 alpha-cholestane-3 alpha,7 alpha-diol (II) are competitive inhibitors for rabbit hepatic microsomal preparations of steroid 12 alpha-hydroxylase with apparent Ki values of 56 and 93 microM, respectively. To ascertain the optimum structure for a substrate with maximal enzymic activity, nine sterols or steroidal acids containing the 7 alpha-hydroxy-4-en-3-one or 3 alpha,7 alpha-dihydroxy-5 alpha configuration were prepared and studied as inhibitors with enzyme preparations in the presence of NADPH, oxygen and appropriate cofactors. Although each of these compounds exhibited competitive inhibition, the best inhibitor for sterol (I) was 7 alpha,25-dihydroxycholest-4-en-3-one (IV) (Ki 36 microM). Steroidal acids (3-oxo-7 alpha-hydroxychol-4-enoic acid and 3-oxo-7 alpha-hydroxy-4-cholene-24-carboxylic acid) were poor inhibitors (Ki 1080 and 654 microM, respectively). For sterol (II) the best inhibitors were sterol (IV) (Ki 35 microM) and 5 alpha-cholestane-3 alpha,7 alpha,25-triol (VIII) (Ki 45 microM). The 12 alpha-hydroxylated products of sterols (I) and (IV) were less tightly bound to the enzyme (Ki 88 and 98 microM, respectively) in the presence of sterol (II). Allochenodeoxycholic acid (Ki 495 microM) was not a good inhibitor for sterol (II). 12 alpha-Hydroxylated products of sterols (IV) and (VIII) were isolated from larger scale incubations, separated by HPLC and identified by mass spectrometry.
Assuntos
Microssomos Hepáticos/enzimologia , Esteroide 12-alfa-Hidroxilase/antagonistas & inibidores , Esteroide Hidroxilases/antagonistas & inibidores , Esteróis/farmacologia , Animais , Cromatografia/métodos , Técnicas In Vitro , Masculino , Coelhos , Solubilidade , Esteroide 12-alfa-Hidroxilase/metabolismo , Esteróis/metabolismo , Especificidade por SubstratoRESUMO
Rabbit liver microsomal preparations fortified with 0.1 mM NADPH effectively promote hydroxylation of [3beta-3H]- or [24-14C]allochenodeoxycholic acid or [5alpha,6alpha-3H2]5alpha-cholestane-3alpha,7alpha-diol to their respective 12alpha-hydroxyl derivatives in yields of about 25 or 65% in 60 min. Minor amounts of other products are formed from the diol. The requirements for activity of rabbit liver microsomal 12alpha-hydroxylase resemble those of rat liver microsomes. Of a number of enzyme inhibitors studied only p-chloromercuribenzoate demonstrated a marked ability to inhibit the reaction with either tritiated substrate. There was no difference in the quantity of product produced from the tritiated acid or the 14C-labeled acid. No clear sex difference was found in activity of the enzyme, nor was an appreciable difference noted in activity of the enzyme between mature and immature animals.
Assuntos
Ácidos e Sais Biliares/metabolismo , Microssomos Hepáticos/metabolismo , Oxigenases de Função Mista/metabolismo , Envelhecimento , Animais , Feminino , Cinética , Masculino , NADP , Oxirredução , Coelhos , Fatores SexuaisRESUMO
[25R]3beta,7alpha-Dihydroxy-[5alpha,6alpha- -3 H2]-5alpha-cholestanoic acid was prepared, purified and 0.34 mg was administered intraperitoneally as the potassium salt to each of three adult male rats with cannulated bile ducts. Bile collected in the first 24 h, containing 97% of the administered 3-H was hydrolysed and the free bile acids were separated by acetic acid partition chromatography. Of the chromatographed tritium 58% was associated with allochenodeoxycholic acid and 14% with its 3beta-isomer; only 5% of the 3-H was found in allocholic acid and 1% with the substrate or more polar unidentified materials. Thus, this dihydroxy-5alpha-cholestanoic acid is metabolized in the rat primarily to dihydroxy-5alpha-cholanic acids, comparable to the metabolism of 3alpha, 7alpha-dihydroxy-5beta-cholestanoic acid in man.
Assuntos
Bile/metabolismo , Colestanos/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Ductos Biliares , Cateterismo , Ácido Quenodesoxicólico/biossíntese , Cromatografia , Cromatografia em Camada Fina , Ratos , Fatores de TempoRESUMO
In this paper, we have constructed deletion and deletion-insertion mutations of the chicken 5-aminolevulinate synthase 5' flanking region and examined the expression of these constructs in microinjected Xenopus oocytes. Utilising this assay, we have delimited the boundary of the 5' flanking region required for expression to be 80 bp upstream from the transcription initiation site. A second major focus of this study has been to define the role of known putative cis-acting sequences in regulating 5-aminolevulinate synthase gene expression. Expression of the insertion-deletion mutants demonstrated that only a TATA box at position -28, and a single GC box at position -78 was necessary for expression of the 5-aminolevulinate synthase gene in Xenopus oocytes. This result is unusual in view of the current state of knowledge of the function of cis-acting sequence elements in transcriptional regulation.
Assuntos
5-Aminolevulinato Sintetase/genética , DNA/genética , Regulação da Expressão Gênica , Oócitos/metabolismo , Sequências Reguladoras de Ácido Nucleico , Animais , Sequência de Bases , Galinhas , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , Transcrição Gênica , Transformação Genética , Xenopus laevisRESUMO
The bile acids derived from [4-14-C]cholesterol administered intracardially to rats with cannulated bile ducts were identified and quantitated. Over a period of 28 days about 90% of the administered 14-C was found in bile of which 73% was retained in the biliary acid fraction. [7beta-3-H]cholic acid, alpha-muri[3beta-3-H]cholic acid, beta-muri[3beta-3-H]cholic acid and litho[3beta-3-H]cholic acid were prepared with specific activities of about 30 muCi/mg by reduction of appropriate ketonic precursors with NaB3H4 and were added to the biliary acid fraction. After separation and purification of the bile acids, cholic, chenodeoxycholic, alpha- and beta-muricholic acids accounted for 70, 16, 7.5 and 6.1%, respectively, of the 14-C in the biliary acid fraction. The specific activities of these isolated 14-C-labeled acids were almost identical. Lithocholic acid accounted for a maximum of 0.2% and ursodeoxycholic acid and 7-oxolithocholic acid could account for no more than 2% of the biliary 14-C. Gas-liquid chromatography on 3% OV-17 of the trimethylsilyl ether derivatives of the methyl esters of the common bile acids of rat bile results in their complete separation and provides a convenient means of estimating the relative proportions of these acids in rat bile. By this method, the relative amounts of the four major acids, cholic, chenodeoxycholic, alpha- and beta-muricholic acids were 63, 20, 8 and 6%, respectively.
Assuntos
Ácidos e Sais Biliares/metabolismo , Colesterol/metabolismo , Animais , Ácidos e Sais Biliares/urina , Ductos Biliares , Radioisótopos de Carbono , Cateterismo , Ácido Quenodesoxicólico/metabolismo , Ácidos Cólicos/análogos & derivados , Ácidos Cólicos/metabolismo , Cromatografia Gasosa , Cromatografia em Camada Fina , Ácido Desoxicólico/metabolismo , Ácido Litocólico/metabolismo , Masculino , Ratos , Fatores de TempoRESUMO
We have examined the effect of heme on the activity of native delta-aminolevulinate synthase isolated from drug-induced chick embryo liver mitochondria. The enzyme was not inhibited by concentrations of heme up to 1 mM and this finding makes it improbable that heme acts physiologically to control mitochondrial delta-aminolevulinate synthase activity.
Assuntos
5-Aminolevulinato Sintetase/metabolismo , Heme/farmacologia , Mitocôndrias Hepáticas/enzimologia , Animais , Embrião de Galinha , Ativação Enzimática , Sulfato de Magnésio/farmacologia , Cloreto de Sódio/farmacologiaRESUMO
Exposure of animals to foreign chemicals results in the induction of many enzymes. The mitochondrial enzyme 5-aminolaevulinate synthase (ALV-S) is induced to supply haem for cytochrome P-450 (P-450) enzymes, the key proteins in drug detoxification. The drugs phenobarbital and 2-allyl-2-isopropylacetamide (AIA), although structurally different, elevate levels of ALV-S and a specific class of P-450s, the phenobarbital-inducible P-450s. Synthesis of ALV-S is negatively controlled by the end-product haem and it is proposed that drugs induce P-450 apoprotein which sequesters haem. Studies at the mRNA level show that ALV-S and P-450 are drug induced in a highly tissue-specific fashion and that haem represses mRNA levels in all but erythroid tissues. In liver, drugs activate ALV-S gene transcription and haem represses, but this mechanism does not operate in erythroid cells. Studies on the drug-induction mechanism of phenobarbital-inducible P-450s in chick embryo liver show that increased mRNA levels result from P-450 gene activation together with a significant post-transcriptional mechanism.
Assuntos
5-Aminolevulinato Sintetase/biossíntese , Regulação da Expressão Gênica , Fenobarbital/farmacocinética , Alilisopropilacetamida/farmacocinética , Sequência de Aminoácidos , Animais , Embrião de Galinha , Sistema Enzimático do Citocromo P-450/metabolismo , Ativação Enzimática , Heme/metabolismo , Humanos , Inativação Metabólica , Fígado/enzimologia , Dados de Sequência Molecular , RNA Mensageiro , Ratos , Transcrição Gênica , Ativação TranscricionalRESUMO
The rat-fetal-resorption test currently is used to assess the biological activity of vitamin E compounds. Previous studies in humans, however, suggest that rat assays underestimate the potency of free tocopherol relative to the acetate ester form and of RRR-alpha-tocopheryl acetate relative to all-rac-alpha-tocopheryl acetate. Therefore, we studied the serum concentrations of alpha-tocopherol after ingestion of RRR-alpha-tocopheryl acetate, all-rac-alpha-tocopheryl acetate, RRR-alpha-tocopherol, RRR-alpha-tocopheryl succinate, and RRR-alpha-tocopheryl acetate plus apple pectin by each of 20 adult human subjects. Measurements were made at 0, 8, 24, and 48 h after ingestion of 800 IU of the various preparations. The results at 24 h were representative of the differences observed. The mean increase in concentration of alpha-tocopherols (mg/g lipid) in 24 h was 71.2% after RRR-alpha-tocopherol, 63.3% after RRR-alpha-tocopherol acetate, and 41.2% after RRR-alpha-tocopherol succinate. Animal assay data do not correlate with data from studies of absorption and retention in serum of alpha-tocopherols ingested by humans.
Assuntos
Vitamina E/sangue , alfa-Tocoferol/análogos & derivados , Adulto , Feminino , Humanos , Absorção Intestinal , Lipídeos/sangue , Masculino , Pessoa de Meia-Idade , Estereoisomerismo , Tocoferóis , Vitamina E/análogos & derivados , Vitamina E/metabolismoRESUMO
Dihydrotestosterone glucuronide (DHTG), a series of 5 alpha-bile acids, or allo-bile acids (3 alpha-hydroxy-5 alpha-cholanic acid, 3-keto-5 alpha-cholanic acid and 3 beta-hydroxy-5 alpha-cholanic acid) and their normal bile acid analogues (3 alpha-hydroxy-5 beta-cholanic acid or lithocholate, 3-keto-5 beta-cholanic acid and 3 beta-hydroxy-5 beta-cholanic acid) were administered intravenously to female rats in order to determine their effects on bile flow. All agents caused a rapid and profound inhibition of bile flow which was dose-dependent. The logarithm of the dose vs the cholestatic response curve for DHTG, the allo-bile acids and lithocholate were all parallel. DHTG was the most potent congener and was two times more potent than 3-keto-5 alpha-cholanic acid and 5 times more potent than lithocholate. These data indicate that the glucuronic acid moiety and the trans configuration of the A and B rings of the steroid nucleus confer the greatest cholestatic potency.
Assuntos
Ácidos e Sais Biliares/farmacologia , Bile/metabolismo , Di-Hidrotestosterona/análogos & derivados , Ácido Litocólico/farmacologia , Animais , Di-Hidrotestosterona/farmacologia , Relação Dose-Resposta a Droga , Feminino , Estrutura Molecular , Ratos , Ratos Endogâmicos , Taxa Secretória/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
1,4-Dien-3-ones of various bile acids (IIa-d), their methyl esters (IIe-h), and their formylated derivatives (IIi-k) were synthesized and their reduction investigated by both catalytic and chemical methods as an alternative route to the synthesis of allo bile acids. Lithium-ammonia reduction proved to be the better method for the reduction of these 1,4-dien-3-ones producing the 3-keto- and 3 beta-hydroxy-allo bile acids (Vb-d) and (VIb-d) in 66-72% yields.
Assuntos
Ácidos e Sais Biliares/síntese química , Colenos , Amônia , Fenômenos Químicos , Química , Hidrogenação , Lítio , OxirreduçãoRESUMO
To aid in the identification of trihydroxy acidic metabolite(s) derived from beta-sitosterol, 3alpha,7alpha,12alpha-trihydroxy-24-nor-5beta-cholan-23-oic acid was prepared and its methyl ester was treated with Raney nickel in boiling p-cymene to provide methyl 3-oxo-7alpha,12alpha-dihydroxy-24-nor-5alpha-cholanate, 3-oxo-7alpha,12alpha-dihydroxy-24-nor-5beta-cholante and 3-oxo-7alpha,12alpha-dihydroxy-24-norchol-4-enoate. The latter compound was synthesized from the 3-oxo-5beta-derivative with SeO2 to provide a product with identical properties. Catalytic reduction of either saturated 3-oxo-derivative provided the appropriate 3,7,12-triols isomeric at C-3. Results from gas liquid and partition chromatography, mass spectrometry, and otherr physical properties of the acids, their methyl esters and other derivatives are compatible with the assigned structures.
Assuntos
Ácidos Cólicos/síntese química , Fenômenos Químicos , Química , Métodos , EstereoisomerismoRESUMO
To provide 7-oxocholesterol derivatives in yields superior to those obtained by chemical oxidation, the preparation of steroidal allylic or benzylic ketones was studied. Air-induced oxidation was investigated with a highly selective low energy UV lamp in the presence of mercuric bromide. With this procedure cholesterol acetate, 5-cholestene-3 beta, 27-diol diacetate, 24(R)-24-methyl-5-cholesten-2 beta-yl acetate, 24(R)-24-ethyl-5-cholesten-28-yl acetate and 24(R)-24-ethyl-(22E)-cholesta-5,22-dien-3 beta-yl acetate were oxidized to the allylic keto-derivative in good yields; estradiol-17 beta diacetate was similarly converted to the 6-oxo-product in improved yield. This method can be very useful in the synthesis of 7-oxocholesteryl acetate and its analogs and 6-oxo-estratrienes.
Assuntos
Ácidos e Sais Biliares/síntese química , Esteroides , Compostos Alílicos , Compostos de Benzil , Indicadores e Reagentes , Cinética , Oxirredução , Fotoquímica , Raios UltravioletaRESUMO
Interest in the structural requirements of a sterol or bile acid for maximal activity by an hepatic microsomal steroid 12 alpha-hydroxylase prompted the preparation of 5 alpha-cholestane-3 alpha, 7 alpha, 25-triol and 5 alpha-analogs of 3 alpha, 7 alpha-dihydroxy-5 beta-cholane-24-carboxylic acid. Methyl 3 alpha, 7 alpha-dihydroxy-5 beta-cholane-24-carboxylate derived from methyl chenodeoxycholate via the Arndt-Eistert reaction was allomerized with Raney nickel in boiling p-cymene to provide a number of product of which methyl 3, 7-dioxo-5 beta- and 5 alpha-cholane-24-carboxylates, methyl 3-oxo-7 alpha-hydroxy-5 beta-and 5 alpha-cholane-24-carboxylates, were identified. Reduction with K-Selectride of methyl 3-oxo-7 alpha-hydroxy-5 beta-cholane-24-carboxylate, provided a high yield of methyl 3 alpha, 7 alpha-dihydroxy-5 alpha-cholane-24-carboxylate. Treatment of this ester with an excess of methyl magnesium iodide afforded 5 alpha-cholestane-3 alpha, 7 alpha, 25-triol. The products were characterized by thin-layer and gas liquid chromatography, proton resonance, infrared and mass spectrometry.
Assuntos
Ácidos e Sais Biliares/síntese química , Colestanóis/síntese química , Cromatografia Líquida de Alta Pressão , Ésteres , Indicadores e Reagentes , Espectrometria de MassasRESUMO
Syntheses of the E and Z isomers of methyl 3 alpha-,3 alpha,7 alpha-,3 alpha,12 alpha-, and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-en-26-oates are reported. Mass spectral studies show fragmentation patterns in support of assignment as the E or Z isomers, especially in differences in loss of the side chain. Chromatographic procedures, primarily gas chromatography and high-performance liquid chromatography, support these assignments. The E isomer predominates in either of two methods of synthesis.
Assuntos
Ácidos e Sais Biliares/química , Colanos/química , Colestenos/síntese química , Espectrometria de Massas , Estrutura Molecular , EstereoisomerismoRESUMO
Analogs of 7 alpha-hydroxy-4-cholesten-3-one were prepared to ascertain structural features necessary for maximal activity of hepatic microsomal 12 alpha-steroid hydroxylase. Methyl 3 alpha,7 alpha-dihydroxy-5 beta-cholane-24-carboxylate derived from chenodeoxycholic acid was oxidized at C-3 with silver carbonate/Celite. The product was hydrolyzed and dehydrogenated with SeO2 to provide 3-oxo-7 alpha-hydroxy-4-cholene-24-carboxylic acid. 5 beta-Cholestane-3 alpha,7 alpha,25-triol and 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol were similarly oxidized at C-3 and dehydrogenated to provide 7 alpha,25-dihydroxy-4-cholesten-3-one and 7 alpha,12 alpha,25-trihydroxy-4-cholesten-3-one, respectively. The products were characterized by thin-layer and gas chromatography, ultraviolet, infrared, proton resonance and mass spectrometry.
Assuntos
Colestenos/síntese química , Colestenonas/síntese química , Microssomos Hepáticos/enzimologia , Esteroide 12-alfa-Hidroxilase/metabolismo , Esteroide Hidroxilases/metabolismo , Animais , Fenômenos Químicos , Química , Técnicas In Vitro , Especificidade por SubstratoRESUMO
In mass spectrometry, sterols and bile acids from fragment ions characteristic of certain steroid structures. After separation of derivatized sterols or bile acids by the gas chromatograph and fragmentation in the mass spectrometer, data collected by the computer can be collated to provide a reconstructed gas chromatogram and a series of fragment ion current chromatograms in which the relative abundances of characteristic fragment ions are plotted vs time or scan number. Intensities of these fragment ions will be greatest and hence coincide with peak elution of unidentified sterols or bile acids. The use of known amounts of labeled appropriate sterols of bile acids permits quantitation of the identified sterol or bile acid.
Assuntos
Ácidos e Sais Biliares/análise , Esteróis/análise , Colestanol/análise , Computadores , Cromatografia Gasosa-Espectrometria de Massas/métodos , Indicadores e Reagentes , Compostos de TrimetilsililRESUMO
Allo bile acids and their taurine- and glycine-conjugates were synthesized by modification of existing procedures. Their chromatographic and spectral properties were investigated and compared with the 5ß-analogs. Some of the naturally occurring 5α- and 5ß-conjugates were separable by high performance liquid chromatography via straight-phase (Corasil II column) or reverse-phase systems (µBondapak/C18 column), though unsuccessful with thin layer or gas liquid chromatography. Differences between the C-5 epimers in the infrared and proton magnetic resonance spectra could be attributed to the configurations at the A/B ring junction and the configuration of the 3-hydroxyl group. The calcium salts of the allo compounds were generally less soluble in water than the 5ß-isomers.
RESUMO
Several high pressure liquid chromatographic methods for the separation of conjugated and free bile acids are presented. A mixture of synthetic conjugated bile acids has been separated by reverse-phase systems consisting of either a Waters Associated' "fatty-acid analysis" or a muBondapak/C18 column eluted with a mixture of 2-propanol/potassium phosphage buffer (pH 2.5 or 7.0). The major conjugated bile acids present in the gallbladder bile of obese subjects have been analyzed each in less than 30 min and quantitated with a U.V. detector set at 193 nm. Some of the 5 alpha- and 5 beta-isomers of conjugated bile salts could be resolved in straight-phase systems on Corasil II or muPorasil columns. Mass spectra of the conjugated bile acids obtained by electron impact were characteristics of the type of amino acid attached to the side chain, and the number of hydroxyl substituents on the nucleus. Most of the isomers could readily be differentiated by the relative intensities of the fragment ions.
Assuntos
Ácidos e Sais Biliares/análise , Aminoácidos/análise , Cromatografia Líquida de Alta Pressão/métodos , Ácidos Graxos/análise , Vesícula Biliar/análise , Humanos , Isomerismo , Espectrometria de MassasRESUMO
Significant correlations have been reported by epidemiologists between the mortality from colorectal cancer in various populations and the consumption of meat or lipids by these populations. These have directed considerable attention to possible relationships between diet and the occurrence of this neoplasm. We have carried out studies of the composition of bile from rats as influenced by diets of varying lipid content. Two cannulas were surgically implanted to form an externalized bile duct through which bile was drained from the common duct and returned to the duodenum. Small aliquots were analyzed for total bile acids by enzymatic assay and for individual bile acids by high-pressure liquid chromatography, gas chromatography and gas chromatography-mass spectometry. Animals consuming diets highest in lipid content provided bile with the greatest amounts of bile acids. The primary bile acids, taurocholic, taurochenodeoxychilic, and tauro alpha- and beta-muricholic acids made up more than 99% of the 3 alph-hydroxy bile acids and were found in approximate molar ratio of 2:1:1. Either complete drainage of bile without return to the duodenum, or biliary tract obstruction had pronounced influence on the rate of secretion of bile and its composition.
Assuntos
Ácidos e Sais Biliares/metabolismo , Neoplasias do Colo/etiologia , Neoplasias Retais/etiologia , Ração Animal/análise , Animais , Bile/análise , Bile/metabolismo , Ácidos e Sais Biliares/análise , Neoplasias do Colo/metabolismo , Dieta , Gorduras na Dieta/efeitos adversos , Gorduras na Dieta/análise , Gorduras na Dieta/metabolismo , Humanos , Ratos , Neoplasias Retais/metabolismoRESUMO
Mesenteric fat tissue obtained at autopsy from 6 patients with mesenteric panniculitis (MP) were found to contain significant amounts of cholesteryl esters (CE). In addition, samples from 3 of these cases were found to contain 0.5-1.3% free cholesterol, 0.9-1.9% free fatty acids (FFA), 0.6-2.5% l-alkyl glyceryl ether diesters and small amounts of squalene. Two of these tissues also contained alk-l-enyl glyceryl ether diesters. The fatty acid compositions of the CE, FFA, triacylglycerides and glyceryl ether diesters (GEDE) were determined and oleic acid (18:1) was found to be the major fatty acid. The alkyl group composition of the GEDE consisted essentially of 16:0 and 18:0 and 18:1 carbon atoms in both types of ethers.