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1.
Metabolites ; 12(8)2022 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-36005595

RESUMO

Quantitative analysis of bile acids in human feces can potentially help to better understand the influence of the gut microbiome and diet on human health. Feces is a highly heterogeneous sample matrix, mainly consisting of water and indigestible solid material (as plant fibers) that show high inter-individual variability. To compare bile acid concentrations among different individuals, a reliable normalization approach is needed. Here, we compared the impact of three normalization approaches, namely sample wet weight, dry weight, and protein concentration, on the absolute concentrations of fecal bile acids. Bile acid concentrations were determined in 70 feces samples from healthy humans. Our data show that bile acid concentrations normalized by the three different approaches are substantially different for each individual sample. Fecal bile acid concentrations normalized by wet weight show the narrowest distribution. Therefore, our analysis will provide the basis for the selection of a suitable normalization approach for the quantitative analysis of bile acids in feces.

2.
Cell Rep ; 37(1): 109797, 2021 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-34610315

RESUMO

Membrane lipids and their metabolism have key functions in neurotransmission. Here we provide a quantitative lipid inventory of mouse and rat synaptic junctions. To this end, we developed a multiomics extraction and analysis workflow to probe the interplay of proteins and lipids in synaptic signal transduction from the same sample. Based on this workflow, we generate hypotheses about novel mechanisms underlying complex changes in synaptic connectivity elicited by environmental stimuli. As a proof of principle, this approach reveals that in mice exposed to an enriched environment, reduced endocannabinoid synthesis and signaling is linked to increased surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) in a subset of Cannabinoid-receptor 1 positive synapses. This mechanism regulates synaptic strength in an input-specific manner. Thus, we establish a compartment-specific multiomics workflow that is suitable to extract information from complex lipid and protein networks involved in synaptic function and plasticity.


Assuntos
Metabolismo dos Lipídeos , Transdução de Sinais , Sinapses/metabolismo , Amidoidrolases/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Endocanabinoides/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Metabolismo dos Lipídeos/genética , Lipídeos/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monoacilglicerol Lipases/metabolismo , Proteoma/análise , Proteômica/métodos , Ratos , Ratos Wistar , Receptores de AMPA/metabolismo , Transdução de Sinais/genética , Espectrometria de Massas em Tandem
3.
Br J Pharmacol ; 178(16): 3342-3353, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33751575

RESUMO

BACKGROUND AND PURPOSE: The analysis of human faecal metabolites can provide an insight into metabolic interactions between gut microbiota and the host organism. The creation of metabolic profiles in faeces has received little attention until now, and reference values, especially in the context of dietary and therapeutic interventions, are missing. Exposure to xenobiotics significantly affects the physiology of the microbiome, and microbiota manipulation and short-chain fatty acid administration have been proposed as treatment targets for several diseases. The aim of the present study is to give concomitant concentration ranges of faecal sterol species, bile acids and short-chain fatty acids, based on a large cohort. EXPERIMENTAL APPROACH: Sterol species, bile acids and short-chain fatty acids in human faeces from 165 study participants were quantified by LC-MS/MS. For standardization, we refer all values to dry weight of faeces. Based on the individual intestinal sterol conversion, we classified participants into low and high converters according to their coprostanol/cholesterol ratio. KEY RESULTS: Low converters excrete more straight-chain fatty acids and bile acids than high converters; 5th and 95th percentile and median of bile acids and short-chain fatty acids were calculated for both groups. CONCLUSION AND IMPLICATIONS: We give concentration ranges for 16 faecal metabolites that can serve as reference values. Patient stratification into high or low sterol converter groups is associated with significant differences in faecal metabolites with biological activities. Such stratification should then allow better assessment of faecal metabolites before therapeutic interventions. LINKED ARTICLES: This article is part of a themed issue on Oxysterols, Lifelong Health and Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.16/issuetoc.


Assuntos
Ácidos e Sais Biliares , Espectrometria de Massas em Tandem , Colesterol , Cromatografia Líquida , Ácidos Graxos Voláteis , Fezes , Humanos
4.
Anal Chem ; 92(20): 13672-13676, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-32865986

RESUMO

We introduce STAMPS, a pathway-centric web service for the development of targeted proteomics assays. STAMPS guides the user by providing several intuitive interfaces for a rapid and simplified method design. Applying our curated framework to signaling and metabolic pathways, we reduced the average assay development time by a factor of ∼150 and revealed that the insulin signaling is actively controlled by protein abundance changes in insulin-sensitive and -resistance states. Although at the current state STAMPS primarily contains mouse data, it was designed for easy extension with additional organisms.


Assuntos
Redes e Vias Metabólicas , Proteômica/métodos , Transdução de Sinais , Animais , Cromatografia Líquida de Alta Pressão , Bases de Dados de Proteínas , Insulina/metabolismo , Redes e Vias Metabólicas/genética , Camundongos , Peptídeos/análise , Transdução de Sinais/genética , Espectrometria de Massas em Tandem
5.
Anal Bioanal Chem ; 412(10): 2315-2326, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32198533

RESUMO

The intestinal microbiome plays an important role in human health and disease and fecal materials reflect the microbial activity. Thus, analysis of fecal metabolites provides insight in metabolic interactions between gut microbiota and host organism. In this work, we applied flow injection analysis coupled to Fourier transform mass spectrometry (FIA-FTMS) to identify and quantify lipid species in human fecal samples. Fecal homogenates were subjected to lipid extraction and analyzed by FIA-FTMS. The analysis of different subjects revealed a vast heterogeneity of lipid species abundance. The majority of samples displayed prominent signals of triacylglycerol (TG) and diacylglycerol (DG) species that could be verified by MS2 spectra. Therefore, we focused on the quantification of TG and DG. Method validation included limit of quantification, linearity, evaluation of matrix effects, recovery, and reproducibility. The validation experiments demonstrated the suitability of the method, with exception for approximately 10% of samples, where we observed coefficients of variation higher than 15%. Impaired reproducibility was related to sample inhomogeneity and could not be improved by additional sample preparation steps. Additionally, these experiments demonstrated that compared with aqueous samples, samples containing isopropanol showed higher amounts of DG, presumably due to lysis of bacteria and increased TG lipolysis. These effects were sample-specific and substantiate the high heterogeneity of fecal materials as well as the need for further evaluation of pre-analytic conditions. In summary, FIA-FTMS offers a fast and accurate tool to quantify DG and TG species and is suitable to provide insight into the fecal lipidome and its role in health and disease.


Assuntos
Diglicerídeos/química , Fezes/química , Análise de Injeção de Fluxo/métodos , Espectrometria de Massas/métodos , Triglicerídeos/química , Humanos , Espectrometria de Massas/instrumentação
6.
mSystems ; 4(4)2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138673

RESUMO

Long-term consumption of dietary fiber is generally considered beneficial for weight management and metabolic health, but the results of interventions vary greatly depending on the type of dietary fibers involved. This study provides a comprehensive evaluation of the effects of a specific dietary fiber consisting of a wheat-bran extract enriched in arabinoxylan-oligosaccharides (AXOS) in a human intervention trial. An integrated multi-omics analysis has been carried out to evaluate the effects of an intervention trial with an AXOS-enriched diet in overweight individuals with indices of metabolic syndrome. Microbiome analyses were performed by shotgun DNA sequencing in feces; in-depth metabolomics using nuclear magnetic resonance in fecal, urine, and plasma samples; and massive lipid profiling using mass spectrometry in fecal and serum/plasma samples. In addition to their bifidogenic effect, we observed that AXOS boost the proportion of Prevotella species. Metagenome analysis showed increases in the presence of bacterial genes involved in vitamin/cofactor production, glycan metabolism, and neurotransmitter biosynthesis as a result of AXOS intake. Furthermore, lipidomics analysis revealed reductions in plasma ceramide levels. Finally, we observed associations between Prevotella abundance and short-chain fatty acids (SCFAs) and succinate concentration in feces and identified a potential protective role of Eubacterium rectale against metabolic disease given that its abundance was positively associated with plasma phosphatidylcholine levels, thus hypothetically reducing bioavailability of choline for methylamine biosynthesis. The metagenomics, lipidomics, and metabolomics data integration indicates that sustained consumption of AXOS orchestrates a wide variety of changes in the gut microbiome and the host metabolism that collectively would impact on glucose homeostasis. (This study has been registered at ClinicalTrials.gov under identifier NCT02215343)IMPORTANCE The use of dietary fiber food supplementation as a strategy to reduce the burden of diet-related diseases is a matter of study given its cost-effectiveness and the positive results demonstrated in clinical trials. This multi-omics assessment, on different biological samples of overweight subjects with signs of metabolic syndrome, sheds light on the early and less evident effects of short-term AXOS intake on intestinal microbiota and host metabolism. We observed a deep influence of AXOS on gut microbiota beyond their recognized bifidogenic effect by boosting concomitantly a wide diversity of butyrate producers and Prevotella copri, a microbial species abundant in non-Westernized populations with traditional lifestyle and diets enriched in fresh unprocessed foods. A comprehensive evaluation of hundreds of metabolites unveiled new benefits of the AXOS intake, such as reducing the plasma ceramide levels. Globally, we observed that multiple effects of AXOS consumption seem to converge in reversing the glucose homeostasis impairment.

7.
J Steroid Biochem Mol Biol ; 190: 115-125, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30940596

RESUMO

Serum concentrations of lathosterol, the plant sterols campesterol and sitosterol and the cholesterol metabolite 5α-cholestanol are widely used as surrogate markers of cholesterol synthesis and absorption, respectively. Increasing numbers of laboratories utilize a broad spectrum of well-established and recently developed methods for the determination of cholesterol and non-cholesterol sterols (NCS). In order to evaluate the quality of these measurements and to identify possible sources of analytical errors our group initiated the first international survey for cholesterol and NCS. The cholesterol and NCS survey was structured as a two-part survey which took place in the years 2013 and 2014. The first survey part was designed as descriptive, providing information about the variation of reported results from different laboratories. A set of two lyophilized pooled sera (A and B) was sent to twenty laboratories specialized in chromatographic lipid analysis. The different sterols were quantified either by gas chromatography-flame ionization detection, gas chromatography- or liquid chromatography-mass selective detection. The participants were requested to determine cholesterol and NCS concentrations in the provided samples as part of their normal laboratory routine. The second part was designed as interventional survey. Twenty-two laboratories agreed to participate and received again two different lyophilized pooled sera (C and D). In contrast to the first international survey, each participant received standard stock solutions with defined concentrations of cholesterol and NCS. The participants were requested to use diluted calibration solutions from the provided standard stock solutions for quantification of cholesterol and NCS. In both surveys, each laboratory used its own internal standard (5α-cholestane, epicoprostanol or deuterium labelled sterols). Main outcome of the survey was, that unacceptably high interlaboratory variations for cholesterol and NCS concentrations are reported, even when the individual laboratories used the same calibration material. We discuss different sources of errors and recommend all laboratories analysing cholesterol and NCS to participate in regular quality control programs.


Assuntos
Colesterol/sangue , Fitosteróis/sangue , Colestanol/sangue , Colesterol/análogos & derivados , Cromatografia Gasosa/métodos , Cromatografia Líquida/métodos , Humanos , Sitosteroides/sangue , Inquéritos e Questionários
8.
Biomolecules ; 9(4)2019 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-30925749

RESUMO

Short chain fatty acids (SCFAs) are generated by the degradation and fermentation of complex carbohydrates, (i.e., dietary fiber) by the gut microbiota relevant for microbe⁻host communication. Here, we present a method for the quantification of SCFAs in fecal samples by liquid chromatography tandem mass spectrometry (LC-MS/MS) upon derivatization to 3-nitrophenylhydrazones (3NPH). The method includes acetate, propionate, butyrate, and isobutyrate with a run time of 4 min. The reproducible (coefficients of variation (CV) below 10%) quantification of SCFAs in human fecal samples was achieved by the application of stable isotope labelled internal standards. The specificity was demonstrated by the introduction of a quantifier and qualifier ions. The method was applied to investigate the pre-analytic stability of SCFAs in human feces. Concentrations of SCFA may change substantially within hours; the degree and kinetics of these changes revealed huge differences between the donors. The fecal SCFA level could be preserved by the addition of organic solvents like isopropanol. An analysis of the colon content of mice either treated with antibiotics or fed with a diet containing a non-degradable and -fermentable fiber source showed decreased SCFA concentrations. In summary, this fast and reproducible method for the quantification of SCFA in fecal samples provides a valuable tool for both basic research and large-scale studies.


Assuntos
Ácidos Graxos Voláteis/análise , Animais , Cromatografia Líquida , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Controle de Qualidade , Espectrometria de Massas em Tandem
9.
Biochimie ; 153: 26-32, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30063945

RESUMO

Increasing numbers of laboratories develop new methods based on gas-liquid and high-performance liquid chromatography to determine serum concentrations of oxygenated cholesterol metabolites such as 7α-, 24(S)-, and 27-hydroxycholesterol. We initiated a first international descriptive oxycholesterol (OCS) survey in 2013 and a second interventional survey 2014 in order to compare levels of OCS reported by different laboratories and to define possible sources of analytical errors. In 2013 a set of two lyophilized serum pools (A and B) was sent to nine laboratories in different countries for OCS measurement utilizing their own standard stock solutions. In 2014 eleven laboratories were requested to determine OCS concentrations in lyophilized pooled sera (C and D) utilizing the same provided standard stock solutions of OCS. The participating laboratories submitted results obtained after capillary gas-liquid chromatography-mass selective detection with either epicoprostanol or deuterium labelled sterols as internal standards and high-performance liquid chromatography with mass selective detection and deuterated OCS as internal standard. Each participant received a clear overview of the results in form of Youden-Plots and basic statistical evaluation in its used unit. The coefficients of variation of the concentrations obtained by all laboratories using their individual methods were 58.5-73.3% (survey 1), 56.8-60.3% (survey 2); 36.2-35.8% (survey 1), 56.6-59.8, (survey 2); 61.1-197.7% (survey 1), 47.2-74.2% (survey 2) for 24(S)-, 27-, and 7α-hydroxycholesterol, respectively. We are surprised by the very great differences between the laboratories, even under conditions when the same standards were used. The values of OCS's must be evaluated in relation to the analytical technique used, the efficiency of the ample separation and the nature of the internal standard used. Quantification of the calibration solution and inappropriate internal standards could be identified as major causes for the high variance in the reported results from the different laboratories. A harmonisation of analytical standard methods is highly needed.


Assuntos
Colesterol/análise , Cromatografia Gasosa/métodos , Cromatografia Líquida/métodos , Colesterol/normas , Humanos , Padrões de Referência , Inquéritos e Questionários
10.
Anal Chem ; 90(14): 8487-8494, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-29920210

RESUMO

There has been an increasing interest during recent years in the role of the gut microbiome on health and disease. Therefore, metabolites in human feces related to microbial activity are attractive surrogate marker to track changes of microbiota induced by diet or disease. Such markers include 5α/ß-stanols as microbiome-derived metabolites of sterols. Currently, reliable, robust, and fast methods to quantify fecal sterols and their related metabolites are missing. We developed a liquid chromatography-high-resolution mass spectrometry (LC-MS/HRMS) method for the quantification of sterols and their 5α/ß-stanols in human fecal samples. Fecal sterols were extracted and derivatized to N, N-dimethylglycine esters. The method includes cholesterol, coprostanol, cholestanol and sitosterol, 5α/ß-sitostanol, campesterol and 5α/ß-campestanol. Application of a biphenyl column permits separation of isomeric 5α- and 5ß-stanols. Sterols are detected in parallel reaction monitoring (PRM) mode and stanols in full scan mode. HRMS allows differentiation of isobaric ß-stanols and the [M + 2] isotope peak of the coeluting sterol. Performance characteristics meet the criteria recommended by Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines. Analysis of fecal samples from healthy volunteers revealed high interindividual variability of sterol and stanol fractions. Interestingly, cholesterol and sitosterol showed similar fractions of mainly 5ß-stanols. In contrast, campesterol is substantially converted to 5α-campestanol and might be a poorer substrate for bacterial metabolism. Robust and fast quantification of fecal sterols and their related stanols by LC-MS/HRMS offers great potential to find novel microbiome-related biomarker in large-scale studies.


Assuntos
Fezes/química , Microbioma Gastrointestinal , Esteróis/análise , Espectrometria de Massas em Tandem/métodos , Colesterol/análogos & derivados , Colesterol/análise , Cromatografia Líquida/economia , Cromatografia Líquida/métodos , Fezes/microbiologia , Humanos , Limite de Detecção , Fitosteróis/análise , Sitosteroides/análise , Espectrometria de Massas em Tandem/economia
11.
J Steroid Biochem Mol Biol ; 169: 145-151, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-27132025

RESUMO

The most common phytosterols in the human diet are sitosterol and campesterol, which originate exclusively from plant derived food. These phytosterols are taken up by NPC1L1 transport from the intestine into the enterocytes together with cholesterol and other xenosterols. Phytosterols are selectively pumped back from the enterocytes into the intestinal lumen and on the liver site from hepatocytes into bile by heterodimeric ABCG5/G8 transporters. Like cholesterol, both phytosterols are prone to ring and side chain oxidation. It could be shown that oxyphytosterols, found in atherosclerotic tissue, are most likely of in situ oxidation (Schött et al.; Biochem. Biophys. Res. Commun. 2014 Apr 11;446(3):805-10). However, up to now, the entire mechanism of phytosterol oxidation is not clearly understood. Here, we provide further information about the oxidation of sitosterol and the transport of its oxidation products out of tissue. Our survey includes data of 104 severe aortic stenosis patients that underwent an elective aortic valve cusp replacement. We studied their phytosterol concentrations, as well as absolute and substrate corrected oxyphytosterol levels in plasma and valve cusp tissue. In addition, we also examined phytosterol and oxyphytosterol concentrations in plasma and tissues (from brain and liver) of 10 male ApoE knockout mice. The ratio of 7-oxygenated-sitosterol-to-sitosterol exceeds the ratio for 7-oxygenated-campesterol-to-campesterol in plasma and tissue of both humans and mice. This finding indicates that sitosterol is oxidized to a higher amount than campesterol and that a selective oxidative mechanism might exist which can differentiate between certain phytosterols. Secondly, the concentrations of oxyphytosterols found in plasma and tissue support the idea that oxysitosterols are preferably transported out of individual tissues. Selective oxidation of sitosterol and preferred transport of sitosterol oxidation products out of tissue seem to be a metabolic pathway of forced sitosterol clearance from tissue compartments.


Assuntos
Estenose da Valva Aórtica/sangue , Oxigênio/química , Sitosteroides/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Estenose da Valva Aórtica/patologia , Apolipoproteínas E/genética , Transporte Biológico , Encéfalo/metabolismo , Colesterol/metabolismo , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Próteses Valvulares Cardíacas , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Oxirredução , Oxisteróis/metabolismo , Fitosteróis/sangue , Fitosteróis/metabolismo
12.
Steroids ; 99(Pt B): 272-80, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25814070

RESUMO

The aim of the study was to evaluate the relationship between phytosterols, oxyphytosterols, and other markers of cholesterol metabolism and concomitant coronary artery disease (CAD) in patients with severe aortic stenosis who were scheduled for elective aortic valve replacement. Markers of cholesterol metabolism (plant sterols and cholestanol as markers of cholesterol absorption and lathosterol as an indicator of cholesterol synthesis) and oxyphytosterols were determined in plasma and aortic valve tissue from 104 consecutive patients with severe aortic stenosis (n=68 statin treatment; n=36 no statin treatment) using gas chromatography-flame ionization and mass spectrometry. The extent of CAD was determined by coronary angiography prior to aortic valve replacement. Patients treated with statins were characterized by lower plasma cholesterol, cholestanol, and lathosterol concentrations. However, statin treatment did not affect the sterol concentrations in cardiovascular tissue. The ratio of campesterol-to-cholesterol was increased by 0.46±0.34µg/mg (26.0%) in plasma of patients with CAD. The absolute values for the cholesterol absorption markers sitosterol and campesterol were increased by 18.18±11.59ng/mg (38.8%) and 11.40±8.69ng/mg (30.4%) in the tissues from patients with documented CAD compared to those without concomitant CAD. Campesterol oxides were increased by 0.06±0.02ng/mg (17.1%) in the aortic valve cusps and oxidized sitosterol-to-cholesterol ratios were up-regulated by 0.35±0.2ng/mg (22.7%) in the plasma of patients with CAD. Of note, neither cholestanol nor the ratio of cholestanol-to-cholesterol was associated with CAD. Patients with concomitant CAD are characterized by increased deposition of plant sterols, but not cholestanol in aortic valve tissue. Moreover, patients with concomitant CAD were characterized by increased oxyphytosterol concentrations in plasma and aortic valve cusps.


Assuntos
Estenose da Valva Aórtica/sangue , Estenose da Valva Aórtica/complicações , Vasos Sanguíneos/metabolismo , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/etiologia , Fitosteróis/metabolismo , Idoso , Valva Aórtica/efeitos dos fármacos , Valva Aórtica/metabolismo , Estenose da Valva Aórtica/tratamento farmacológico , Vasos Sanguíneos/patologia , Colestanol/sangue , Colesterol/análogos & derivados , Colesterol/sangue , Doença da Artéria Coronariana/tratamento farmacológico , Feminino , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Masculino , Oxirredução , Fitosteróis/sangue , Sitosteroides/sangue
13.
Steroids ; 99(Pt B): 178-82, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25795151

RESUMO

In this study, we compare the distribution of intraperitoneally injected sitosterol, 7ß-hydroxysitosterol or vehicle only (control) for 28days in male ApoE-/- mice. Furthermore we examine its impact on surrogate markers of cholesterol biosynthesis and sterol absorption rate in plasma, brain and liver tissues from these animals. Injection of sitosterol revealed a 32.1% (P=0.013) lower plasma total cholesterol compared with control. Cholesterol corrected plasma and absolute brain and liver levels of sitosterol are 4.1-, 1.7-, and 7.2-fold (P<0.001 for all) higher, respectively. This is in accordance with a reduced plasma campesterol to cholesterol ratio (-16.2%; P=0.018) together with a 24.1% (P=0.047) lower concentration of hepatic lathosterol. After injection of 7ß-hydroxysitosterol the concentrations of 7ß-hydroxysitosterol in plasma, brain and liver are 21.0-, 65.8- and 42.7-fold (P<0.001 for all) higher, respectively, compared with control. Injection of 7ß-hydroxysitosterol revealed significantly lower plasma cholesterol corrected cholestanol and campesterol (-44.2%; P=0.001 and -24.5; P=0.004) as well as lower absolute liver cholestanol levels (-31.9%; P<0.001) compared with control. Intraperitoneally injected sitosterol and 7ß-hydroxysitosterol differently influence cholesterol metabolism in plasma and liver. We conclude that the polar 7ß-hydroxysitosterol compound can pass the blood brain barrier with higher efficacy than its substrate, sitosterol. Though present in higher amounts in the brain, both, sitosterol and 7ß-hydroxysitosterol do not influence cholesterol metabolism in the brain as proven by our surrogate markers.


Assuntos
Apolipoproteínas E/deficiência , Barreira Hematoencefálica/metabolismo , Sitosteroides/sangue , Animais , Apolipoproteínas E/metabolismo , Transporte Biológico , Colesterol/biossíntese , Éteres/química , Injeções Intraperitoneais , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade por Substrato
14.
Steroids ; 99(Pt B): 139-50, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25701095

RESUMO

We describe the validation of a method for the analysis of oxysterols, i.e. oxycholesterols and oxyphytosterols, in human serum using gas chromatography-mass spectrometry selected ion monitoring (GC-MS-SIM). Concentrations of 7α- and 7ß-hydroxy-, and 7oxo-cholesterol, -campesterol, and -sitosterol as well as 4ß-hydroxycholesterol and side-chain oxygenated 24S-, 25-, and 27-hydroxycholesterol were determined by isotope dilution methodology. After saponification at room temperature the oxysterols were extracted, separated from their substrates, cholesterol, campesterol, and sitosterol, by solid phase extraction, and subsequently derivatised to their corresponding trimethylsilyl-ethers prior to GC-MS-SIM. In order to prevent artificial autoxidation butylated hydroxytoluene and ethylenediaminetetraacetic acid were added. The validation of the method was performed according to the International Conference on Harmonisation guidance, including limits of detection and quantification, ranges, recovery and precision. Due to improved instrumental settings and work-up procedure, limits of detection and quantification ranged between 8.0-202.0pg/mL and 28.0-674pg/mL, respectively. Recovery data in five calibration points varied between 91.9% and 116.8% and in serum samples between 93.1% and 118.1%. The mean coefficient of variation (CV) for the recovery of all compounds was <10%. Well satisfying CVs for within-day precision (2.1-10.8%) and for between-day precision (2.3-12.1%) were obtained. More than 20 samples could be processed in a single routine day and test series of about 300 samples can be realised without impairment of the validation parameters during a sequence. Comparison of oxysterol and oxyphytosterol content in serum and plasma revealed no difference. A fully validated isotope dilution methodology for the quantification of oxycholesterols and oxyphytosterols from human serum or plasma is presented.


Assuntos
Cromatografia Gasosa-Espectrometria de Massas/métodos , Fitosteróis/sangue , Calibragem , Colesterol/análogos & derivados , Colesterol/sangue , Humanos , Hidrólise , Técnicas de Diluição do Indicador , Isótopos , Limite de Detecção , Padrões de Referência , Reprodutibilidade dos Testes , Soro/química , Sitosteroides/sangue , Soluções , Solventes , Fatores de Tempo
15.
Biochem Biophys Res Commun ; 446(3): 805-10, 2014 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-24631689

RESUMO

Phytosterols such as campesterol and sitosterol are susceptible to oxidation by reactive oxygen species. We hypothesize that the plant sterols (PS) campesterol and sitosterol and their 7-oxygenated metabolites (POPs) correlate within and between human plasma and aortic valve cusps tissues. Plasma and tissue concentrations of PS and POPs were analyzed by gas chromatography-mass spectrometry-selected ion monitoring. Prior to analysis valve cusps tissue was mechanically separated from the calcified parts. PS and POP levels per dry cusps tissue weight were significantly higher compared with the concentrations in the calcified part. Against our hypothesis we found that despite the fact that there is a high correlation between plant sterols in and between plasma and valves cusps tissue, as well as a high correlation between plant sterols and oxyphytosterols and oxyphytosterols themselves within the valve cusps tissue, there was hardly any correlation in the amount of oxyphytosterols in plasma and between plasma and valves. Because plasma samples are easily accessible for large scale population based studies, we have to understand in more detail what the analysis of POPs implies in terms of CVD risk for the future.


Assuntos
Estenose da Valva Aórtica/sangue , Valva Aórtica/metabolismo , Fitosteróis/sangue , Fitosteróis/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Valva Aórtica/cirurgia , Colesterol/análogos & derivados , Colesterol/sangue , Colesterol/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sitosteroides/sangue , Sitosteroides/metabolismo
16.
J Lipid Res ; 55(3): 583-91, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24334219

RESUMO

Cholesterol is an important lipid of mammalian cells and plays a fundamental role in many biological processes. Its concentration in the various cellular membranes differs and is tightly regulated. Here, we present a novel alkyne cholesterol analog suitable for tracing both cholesterol metabolism and localization. This probe can be detected by click chemistry employing various reporter azides. Alkyne cholesterol is accepted by cellular enzymes from different biological species (Brevibacterium, yeast, rat, human) and these enzymes include cholesterol oxidases, hydroxylases, and acyl transferases that generate the expected metabolites in in vitro and in vivo assays. Using fluorescence microscopy, we studied the distribution of cholesterol at subcellular resolution, detecting the lipid in the Golgi and at the plasma membrane, but also in the endoplasmic reticulum and mitochondria. In summary, alkyne cholesterol represents a versatile, sensitive, and easy-to-use tool for tracking cellular cholesterol metabolism and localization as it allows for manifold detection methods including mass spectrometry, thin-layer chromatography/fluorography, and fluorescence microscopy.


Assuntos
Alcinos/química , Rastreamento de Células/métodos , Colesterol/química , Colesterol/metabolismo , 5-Aminolevulinato Sintetase/genética , Aciltransferases/metabolismo , Alcinos/metabolismo , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Colesterol/análogos & derivados , Ésteres do Colesterol/metabolismo , Colesterol Oxidase/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Humanos , Cinética , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Estrutura Molecular , Mutação , Ratos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Esteróis/metabolismo
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