RESUMO
We describe a sensitive high performance liquid chromatography (HPLC)-based method for the determination of cholesterol in brain tissue. The method does not require the derivatization of the analyte and uses separation and quantification by reversed-phase HPLC coupled to UV detection. Lipids were methanol/chloroform extracted following the method of Bligh and Dyer, and separated using isopropanol/acetonitrile/water (60/30/10, v/v/v) as mobile phase. We observed lineal detection in a wide range of concentrations, from 62.5 to 2000 ng/µL, and were able to detect a significant increase in the brain cholesterol levels between postnatal days 2 and 10 in C57BL6 mice. Based on our validation parameters, we consider this analytical method a useful tool to assess free cholesterol in rodent brain samples and cell cultures.
Assuntos
Encéfalo/metabolismo , Colesterol/análise , Cromatografia Líquida de Alta Pressão/métodos , Animais , Colesterol/isolamento & purificação , Cromatografia de Fase Reversa , Limite de Detecção , Extração Líquido-Líquido , Camundongos , Camundongos Endogâmicos C57BL , Espectrofotometria UltravioletaRESUMO
Nowadays, data concerning the composition of Caryodendron orinocense Karst. (Euphorbiaceae) and Bactris gasipaes Kunth (Arecaceae) seed oils are lacking. In light of this fact, in this paper fatty acids and unsaponifiable fraction composition have been determined using GC-MS, HPLC-DAD (Diode Array Detector), NMR approaches and possible future applications have been preliminary investigated through estimation of antioxidant activity, performed with DPPH test. For C. orinocense linoleic acid (85.59%) was the main component, lauric (33.29%) and myristic (27.76%) acids were instead the most abundant in B. gasipaes. C. orinocense unsaponifiable fraction (8.06%) evidenced a remarkable content of ß-sitosterol, campesterol, stigmasterol, squalene and vitamin E (816 ppm). B. gasipaes revealed instead ß-sitosterol and squalene as main constituents of unsaponifiable matter (3.01%). Antioxidant capacity evidenced the best performance of C. orinocense seed oil. These preliminary results could be interesting to suggest the improvement of the population's incomes from Amazonian basin. In particular the knowledge of chemical composition of C. orinocense and B. gasipaes oils could be helpful to divulge and valorize these autochthones plants.
Assuntos
Antioxidantes , Arecaceae/química , Euphorbiaceae/química , Ácidos Graxos/isolamento & purificação , Ácidos Graxos/farmacologia , Nozes/química , Óleos de Plantas/química , Sementes/química , Colesterol/análogos & derivados , Colesterol/análise , Colesterol/isolamento & purificação , Colesterol/farmacologia , Cromatografia Líquida de Alta Pressão , Ácidos Graxos/análise , Sequestradores de Radicais Livres , Cromatografia Gasosa-Espectrometria de Massas , Ácidos Láuricos/análise , Ácidos Láuricos/isolamento & purificação , Ácidos Láuricos/farmacologia , Ácido Linoleico/análise , Ácido Linoleico/isolamento & purificação , Ácido Linoleico/farmacologia , Espectroscopia de Ressonância Magnética , Ácido Mirístico/análise , Ácido Mirístico/isolamento & purificação , Ácido Mirístico/farmacologia , Fitosteróis/análise , Fitosteróis/isolamento & purificação , Fitosteróis/farmacologia , Óleos de Plantas/isolamento & purificação , Sitosteroides/análise , Sitosteroides/isolamento & purificação , Sitosteroides/farmacologia , Esqualeno/análise , Esqualeno/isolamento & purificação , Esqualeno/farmacologia , Estigmasterol/análise , Estigmasterol/isolamento & purificação , Estigmasterol/farmacologia , Vitamina E/análise , Vitamina E/isolamento & purificação , Vitamina E/farmacologiaRESUMO
In the present paper the assessment of a novel molecularly imprinted polymer, poly(methacrylic acid)/silica, for clean-up and selective extraction of cholesterol in milk samples is described. The relative selectivity coefficient (k) values for cholesterol/5-α-cholestane and cholesterol/7-dehydrocholesterol systems were found to be 5.08 and 6.08, respectively, thus attesting the selectivity of the MIP for cholesterol under competitive adsorption with structurally analogous steroid compounds. The milk analysis was initially based on saponification followed by liquid-liquid extraction with n-hexane. Then, the protocol of molecularly imprinted solid phase extraction (MISPE) was carried out by loading the milk hexanic extract through 200 mg of MIP or NIP (non-imprinted polymer) packed into SPE cartridges at a flow rate of 0.6 mL min(-1). The washing step was performed by using n-hexane followed by further elution with ethanol and HPLC-UV analysis at 208 nm. From the breakthrough curve the maximum adsorption capacity of the MIP towards cholesterol was found to be 29.51 mg g(-1). The precision of the MISPE protocol was assessed as intra- and inter-days yielding RSD (relative standard deviations) lower than 4.10%. Cleaner HPLC chromatograms were obtained for milk samples submitted to the MISPE protocol in comparison to the solid phase extraction using the NIP or modified octadecyl silica (C18). Recoveries varying from 96.6 up to 102.2% for milk samples spiked with cholesterol were achieved, thus ensuring the accuracy of the proposed method.
Assuntos
Colesterol/análise , Cromatografia Líquida de Alta Pressão , Leite/química , Impressão Molecular , Espectrofotometria Ultravioleta , Animais , Colestanos/análise , Colestanos/isolamento & purificação , Colesterol/isolamento & purificação , Desidrocolesteróis/análise , Desidrocolesteróis/isolamento & purificação , Hexanos/química , Extração Líquido-Líquido , Ácidos Polimetacrílicos/química , Dióxido de Silício/química , Extração em Fase SólidaRESUMO
The first phytochemical study of Simira eliezeriana Peixoto (Rubiaceae) allowed the isolation and structural determination of two new diterpenes named simirane A [(5R,6R,8R,9R,10S,11S,13S)-6ß,11ß-dihydroxy-2,4(18),15-erythroxylatrien-1-one] (1) and simirane B [(5S,8R,9R,10S,11S,13S)-11ß-hydroxy-2,4(18),15-erythroxylatrien-1-one] (2), together with seven known compounds: sitosterol (3), stigmasterol (4), campesterol (5), coniferaldehyde (6), vanillin (7), pinoresinol (8) and harman (9) from the bark of the plant. The structures of the compounds were established on the basis of spectroscopic methods, including 1-D and 2-D NMR, HRESI-MS and CD analysis and comparisons with available literature data of known compounds.
Assuntos
Diterpenos/isolamento & purificação , Casca de Planta/química , Extratos Vegetais/isolamento & purificação , Rubiaceae/química , Acroleína/análogos & derivados , Acroleína/isolamento & purificação , Benzaldeídos/isolamento & purificação , Colesterol/análogos & derivados , Colesterol/isolamento & purificação , Diterpenos/análise , Diterpenos/química , Etanol , Furanos/isolamento & purificação , Harmina/análogos & derivados , Harmina/isolamento & purificação , Lignanas/isolamento & purificação , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Fitosteróis/isolamento & purificação , Extratos Vegetais/análise , Extratos Vegetais/química , Sitosteroides/isolamento & purificaçãoRESUMO
BACKGROUND: The cold and menthol receptor, TRPM8, is a non-selective cation channel expressed in a subset of peripheral neurons that is responsible for neuronal detection of environmental cold stimuli. It was previously shown that members of the transient receptor potential (TRP) family of ion channels are translocated toward the plasma membrane (PM) in response to agonist stimulation. Because the spatial and temporal dynamics of cold receptor cell-surface residence may determine neuronal activity, we hypothesized that the movement of TRPM8 to and from the PM might be a regulated process. Single particle tracking (SPT) is a useful tool for probing the organization and dynamics of protein constituents in the plasma membrane. METHODOLOGY/PRINCIPAL FINDINGS: We used SPT to study the receptor dynamics and describe membrane/near-membrane behavior of particles containing TRPM8-EGFP in transfected HEK-293T and F-11 cells. Cells were imaged using total internal reflection fluorescence (TIRF) microscopy and the 2D and 3D trajectories of TRPM8 molecules were calculated by analyzing mean-square particle displacement against time. Four characteristic types of motion were observed: stationary mode, simple Brownian diffusion, directed motion, and confined diffusion. In the absence of cold or menthol to activate the channel, most TRPM8 particles move in network covering the PM, periodically lingering for 2-8 s in confined microdomains of about 800 nm radius. Removing cholesterol with methyl-beta-cyclodextrin (MßCD) stabilizes TRPM8 motion in the PM and is correlated with larger TRPM8 current amplitude that results from an increase in the number of available channels without a change in open probability. CONCLUSIONS/SIGNIFICANCE: These results reveal a novel mechanism for regulating TRPM8 channel activity, and suggest that PM dynamics may play an important role in controlling electrical activity in cold-sensitive neurons.
Assuntos
Canais de Cátion TRPM/metabolismo , Linhagem Celular , Colesterol/química , Colesterol/isolamento & purificação , Humanos , Microscopia de Fluorescência , beta-Ciclodextrinas/químicaRESUMO
7-Hydroperoxycholesterol is considered to be an intermediate compound of the cholesterol oxidation path as the first product formed when cholesterol is oxidized by triplet oxygen. However, there is a limitation on cholesterol mechanism studies because of the lack of 7-hydroperoxycholesterol analytical standard due to its low stability. To verify the formation of hydroperoxides in cholesterol model systems heated at 140, 180, and 220 °C, 7α-hydroperoxycholesterol was synthesized by cholesterol photooxidation followed by rearrangement at room temperature in chloroform. Its structure was confirmed on the basis of 13C NMR and mass spectra obtained by APCI-LC-MS. The synthesized compound was also used as standard for the quantification of 7-hydroperoxycholesterol as the sum of 7α- and 7ß-hydroperoxycholesterol. The results demonstrated that 7-hydroperoxycholesterol is the first compound formed when the temperature is lower (140 °C). However, the concentration of the 7-hydroperoxycholesterol depends on the temperature and time of exposure: the higher the time, the higher the amount of 7-hydroperoxycholesterol at lower temperatures, and the lower the time, the lower the amount of 7-hydroperoxycholesterol at higher temperatures (180 and 220 °C). By the formation of 7-hydroperoxycholesterol, the known cholesterol oxidation mechanism in three phases (initiation, propagation, and termination) could be confirmed; once at lower temperatures, the stage of cholesterol oxidation is at initiation, at which hydroperoxide formation predominates.
Assuntos
Colesterol/análogos & derivados , Temperatura Alta , Modelos Químicos , Colesterol/análise , Colesterol/síntese química , Colesterol/química , Colesterol/isolamento & purificação , Cinética , Processos FotoquímicosRESUMO
The analytical conditions for the extraction of cholesterol and cholesterol oxides in chicken meat were optimized by means of response surface methodology. The separation and identification were performed by normal phase HPLC using UV and refractive index (RI) detectors, and the confirmation of the 11 cholesterol oxides identities in the samples was verified by HPLC-APCI-MS. The developed methodology showed good analytical performance, presenting recovery levels from 84 to 103% and detection limits varying from 0.01 to 0.06 microg/g for UV detection and from 1.98 to 2.12 microg/g for RI detection. The present study demonstrated the presence of 22 R-hydroxycholesterol, 24 S-hydroxycholesterol, and 22 S-hydroxycholesterol for the first time in chicken meat.
Assuntos
Galinhas , Colesterol/isolamento & purificação , Carne/análise , Óxidos/isolamento & purificação , Animais , Cromatografia Líquida de Alta Pressão/métodos , Temperatura Alta , Espectrometria de Massas/métodos , Reprodutibilidade dos Testes , SaponinasRESUMO
A new method was developed for the simultaneous determination of cholesterol and its oxidation products in eggs, using HPLC with UV and refractive index (RI) detectors, and HPLC interfaced with atmospheric pressure chemical ionization coupled to MS (HPLC-APCI-MS). The best conditions for direct saponification of the sample and extraction of the non-saponifiable material were defined using complete factorial designs with central points. The method showed accuracy and precision with a detection limit between 0.002 and 0.079 microg/g. The oxides cholest-5-ene-3beta,20alpha-diol and cholest-5-ene-3beta,25-diol identified by HPLC-UV-RI were not confirmed by HPLC-APCI-MS.
Assuntos
Colesterol/análogos & derivados , Colesterol/química , Cromatografia Líquida de Alta Pressão/métodos , Ovos/análise , Ionização do Ar , Pressão Atmosférica , Colesterol/análise , Colesterol/isolamento & purificação , Colesterol/metabolismo , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida , Desmosterol/análise , Desmosterol/isolamento & purificação , Análise de Alimentos , Hidroxicolesteróis/análise , Hidroxicolesteróis/isolamento & purificação , Cetocolesteróis/análise , Cetocolesteróis/isolamento & purificação , Espectrometria de Massas/métodos , Oxirredução , Sensibilidade e Especificidade , Espectrofotometria Ultravioleta , Raios UltravioletaRESUMO
Analysis of the sterol fraction obtained from the Colombian Caribbean sponge Topsentia ophiraphidites revealed that this sponge is a rich source of C30 and C31 sterols. Among them, a new C31 sterol, named ophirasterol, was isolated, and its structure was established as (22E,24R)-24-(1-buten-2-yl)cholesta-5,22-dien-3beta-ol (1) by spectral means and comparison with synthetic C-24 epimers with known configuration. Other isolated C30 and C31 sterols were the known 24-ethyl-24-methyl-22-dehydrocholesterol (2), 24-isopropyl-22-dehydrocholesterol (3), 24-isopropylcholesterol (4), 24-ethyl-24-methylcholesterol (5), 24-isopropenyl-25-methyl-22-dehydrocholesterol (6) and 24-isopropenyl-25-methylcholesterol (7), and 24-isopropenyl-22-dehydrocholesterol (8).
Assuntos
Poríferos/química , Esteróis/química , Animais , Colesterol/análogos & derivados , Colesterol/química , Colesterol/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Estereoisomerismo , Esteróis/isolamento & purificaçãoRESUMO
El objetivo de éste estudio fue optimizar la determinación de ácido siálico en LDL aislada por precipitación selectiva y establecer relaciones entre componentes que puedan ser indicadores de aterogenicidad. Se empleó polivinilsulfato disuelto en polietilenglicol como reactivo precipitante, se ajustaron las condiciones de los lavados para garantizar la ausencia de otras proteínas del suero y se solubilizó la LDL en 5 por ciento de NaCI. Se determinó apoB, colesterol, proteínas y se optimizó la cuantificación de ácido siálico según el método de Warren modificado por Sobenin y col. Se realizó la evaluación del método analítico adaptado en cuanto a precisión intra- e inter-ensayos (CV 8 y 9 por ciento respectivamente), linealidad (hasta 7 nmol de ácido siálico/tubo); efecto de los lavados; especificidad; ausencia de interferencia de blancos de reactivo precipitante; ensayo de recuperación (entre 88 y 120 por ciento). Los resultados obtenidos sobre 30 muestras de personas normolipémicas de ambos sexos (edades entre 30 y 65 años) expresados como media ñ SEM fueron: colesterol 3,8 ñ 0,2 mmol/l, ácido siálico 34,7 ñ 2,7 Amol/l, ApoB 1,27 ñ 0,09 Amol/l, proteínas 1,57 ñ 0,08 g/l. Indices de composición: ácido siálico/colesterol: 9,24 ñ 0,48 mmol/mol, ácido siálico/apoB 34,4 ñ 3 mol/mol y ácido siálico/proteína 39,6 ñ 1,31 Amol/g. Son necesarios estudios clínicos que permitan evaluar los alcances de los índices de composición propuestos como posibles indicadores de aterogenicidad (AU)
Assuntos
Humanos , Masculino , Feminino , Lipoproteínas LDL/isolamento & purificação , LDL-Colesterol/isolamento & purificação , Ácido N-Acetilneuramínico/isolamento & purificação , Técnicas de Laboratório Clínico , Arteriosclerose/diagnóstico , Lipoproteínas LDL/diagnóstico , LDL-Colesterol/diagnóstico , Ácido N-Acetilneuramínico/diagnóstico , Apolipoproteínas B/isolamento & purificação , Apolipoproteínas B/diagnóstico , Colesterol/isolamento & purificação , Precipitação Química , Fatores de RiscoRESUMO
A previously isolated trihydroxy-gorgosterol derivative (2), a new gorgosterol (3), and the known sterol cholestane-3beta,5alpha, 6beta-triol are reported from the Caribbean octocoral Eunicea laciniata collected off northeast Venezuela. Using high-resolution 2D NMR techniques, the total structural assignment and detailed stereochemistry of 2 is presented for the first time and the full structure of 3 advanced.
Assuntos
Colestanóis/isolamento & purificação , Colesterol/análogos & derivados , Colesterol/isolamento & purificação , Cnidários/química , Animais , Colestanóis/química , Colestanóis/farmacologia , Colesterol/química , Estrutura Molecular , Análise EspectralRESUMO
High cholesterol saturated lipids ingestion has been linked to the increment of coronary diseases, particularly atherosclerosis. In this study, samples of viscera and chicken meat, as well as manufactured chicken products are characterized from the point of view of their sterol content, specially cholesterol, with the purpose to determine their nutritional quality and to contribute with the development of Venezuelan food composition tables. Gas-liquid chromatography was the method chosen for the separation and quantification of cholesterol and fitosterols eventually present. The method involves lipids extraction, direct saponification, extraction of the unsaponifiable matter and its injection in the gas chromatograph. The average cholesterol values in mg/100 g. wet sample were: 31.13 (manufactured chicken breast); 57,35 (ham like type of product made with chicken); 69.02 (chicken sausages); 60.46 (chicken "bologna").
Assuntos
Galinhas , Colesterol/isolamento & purificação , Produtos da Carne/análise , Carne/análise , Animais , VenezuelaRESUMO
The effect of denervation on the lipid metabolism and on the activity of (Na+ - K+)ATPase isoforms from the membrane fraction P3, which corresponds to the innervated electrocyte membrane, was evaluated. On a discontinuous sucrose gradient, normal P3 membranes exhibit a bimodal ("a" and "b bands) distribution of the (Na+ - K+)ATPase activity, which upon denervation changes to an unimodal ("c" band) distribution. Using these fractions, which have a higher (Na+ - K+)ATPase activity, we characterized the lipids at the hydrophobic protein surface boundary, (i.e., the bulk lipids that surround the protein). The results confirm that these lipids consist of phospholipids and cholesterol. The quantitative composition of the phospholipids is similar for both isoform fractions obtained from the discontinuous gradient of normal membranes, with phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine representing about 90% of the total phospholipids. Sphingomyelin, phosphatidylinositol, diphosphatidylglycerol and phosphatidic acid were in the minority. However, in the single band obtained after denervation, the three major phospholipid components decreased to 70% of the total, and a significant increase in the other phospholipids and in cholesterol was observed. The high cholesterol content of the denervated fraction may confer membrane stabilization, as it is likely to cause a decrease in the membrane fluidity and consequently in the enzyme activity.
Assuntos
Denervação , Órgão Elétrico/metabolismo , Lipídeos de Membrana/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Acetilcolinesterase/isolamento & purificação , Acetilcolinesterase/metabolismo , Animais , Membrana Celular/metabolismo , Colesterol/isolamento & purificação , Colesterol/metabolismo , Órgão Elétrico/inervação , Electrophorus , Lipídeos de Membrana/isolamento & purificação , Fosfolipídeos/isolamento & purificação , Fosfolipídeos/metabolismo , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/isolamento & purificaçãoRESUMO
Antes y a los 2 y 6 meses después de practicada la vasectomía se evaluaron las alteraciones que pudieran presentar las glándulas del aparato genital masculino en su fisiología y capacidad secretora, mediante el análisis químico de los constituyentes del plasma seminal (colesterol libre, colesterol esterificado y fosfolípidos). El resultado de este análisis químico, indica que la relación de la concentración del colesterol libre con respecto al colesterol esterificado, permanece constante a los seis meses de practicada la vasectomía, no así la concentración de fosfolípidos, la cual disminuye a partir de los dos meses de realizado este procedimiento quirúrgico. Estos hallazgos indican de una manera indirecta las alteraciones en la capacidad secretora que pudieran tener las glándulas genitales debido a este método de anticoncepción masculina.