RESUMO
The recessive N-ethyl-N-nitrosourea-induced phenotype toku is characterized by delayed hair growth, progressive hair loss, and excessive accumulation of dermal cholesterol, triglycerides, and ceramides. The toku phenotype was attributed to a null allele of Gk5, encoding glycerol kinase 5 (GK5), a skin-specific kinase expressed predominantly in sebaceous glands. GK5 formed a complex with the sterol regulatory element-binding proteins (SREBPs) through their C-terminal regulatory domains, inhibiting SREBP processing and activation. In Gk5toku/toku mice, transcriptionally active SREBPs accumulated in the skin, but not in the liver; they were localized to the nucleus and led to elevated lipid synthesis and subsequent hair growth defects. Similar defective hair growth was observed in kinase-inactive GK5 mutant mice. Hair growth defects of homozygous toku mice were partially rescued by treatment with the HMG-CoA reductase inhibitor simvastatin. GK5 exists as part of a skin-specific regulatory mechanism for cholesterol biosynthesis, independent of cholesterol regulation elsewhere in the body.
Assuntos
Glicerol Quinase/metabolismo , Lipídeos/biossíntese , Processamento de Proteína Pós-Traducional , Pele/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Animais , Glicerol Quinase/genética , Lipídeos/genética , Camundongos , Camundongos Knockout , Domínios Proteicos , Sinvastatina/farmacologia , Proteínas de Ligação a Elemento Regulador de Esterol/genéticaRESUMO
OBJECTIVE: Bariatric surgery induces weight loss and improvement of insulin resistance; one aspect of both is an amelioration of hepatic steatosis. This study was undertaken to assess the changes in the hepatic lipidome after duodenal-jejunal bypass (DJB) surgery. METHODS: A DJB surgical model was developed and characterized in diet-induced obese mice. In comparison with sham-operated mice, an unbiased lipidomic profiling of hepatic lipids was performed together with measurements of gene expression within key pathways of hepatic lipid metabolism. RESULTS: In the liver of DJB mice, a dramatic reduction (by 77%) in hepatic triacylglycerols was observed. Global lipidomic profiling identified marked decreases of triacylglycerols comprised of medium length fatty acids and with low double bond content. Specific diacylglycerol species were also among the most dramatic decreases in hepatic lipids, whereas lysophosphatidic acids and phosphatidic acids were increased. Expression of fatty acid transporter and lipogenic genes was down-regulated. CONCLUSIONS: From in-depth analysis of hepatic lipid composition, specific lipid intermediates were identified that are preferentially changed following DJB surgery. These changes were most likely due to DJB-induced weight loss, and only further studies will be able to distinguish weight loss-dependent from weight loss-independent changes.
Assuntos
Duodeno/cirurgia , Fígado Gorduroso/metabolismo , Resistência à Insulina , Jejuno/cirurgia , Animais , Cirurgia Bariátrica , Glicemia/metabolismo , Fígado Gorduroso/cirurgia , Derivação Gástrica/métodos , Masculino , CamundongosRESUMO
Oxylipins are oxidation products of polyunsaturated fatty acids (PUFAs) that affect a broad range of physiological processes, including cell proliferation, inflammation, inflammation resolution, and vascular function. Moreover, oxylipins are readily detectable in plasma, and certain subsets of oxylipins have been detected in human atherosclerotic lesions. Taken together, we set out to produce a detailed quantitative assessment of plasma and plaque oxylipins in a widely used model of atherosclerosis, to identify potential biomarkers of disease progression. We administered regular chow or regular chow supplemented with 0.5% cholesterol (HC) to male New Zealand white rabbits for 12 weeks to induce hypercholesterolemia and atherosclerosis. Our targeted lipidomic analyses of oxylipins on plaques isolated from rabbits fed the HC diet detected 34 oxylipins, 28 of which were in compliance with our previously established quality control acceptance criteria. The arachidonic acid (AA) metabolite derived from the COX pathway, 6-keto-PGF1α was the most abundant plaque oxylipin, followed by the linoleic acid (LA) metabolites 9-HODE, 13-HODE and 9,12,13-TriHOME and the arachidonic acid (AA)-derivatives 11-HETE and 12-HETE. We additionally found that the most abundant oxylipins in plasma were three of the five most abundant oxylipins in plaque, namely 11-HETE, 13-HODE, and 9-HODE. The studies reported here make the first step towards a comprehensive characterization of oxylipins as potentially translatable biomarkers of atherosclerosis.
Assuntos
Hipercolesterolemia/sangue , Oxilipinas/sangue , Placa Aterosclerótica/sangue , Animais , Cromatografia Líquida de Alta Pressão , Ácidos Graxos Insaturados/metabolismo , Humanos , Hipercolesterolemia/metabolismo , Masculino , Espectrometria de Massas , Oxilipinas/metabolismo , Placa Aterosclerótica/metabolismo , CoelhosRESUMO
Studies in lipoprotein kinetics almost exclusively rely on steady-state approaches to modeling. Herein, we have used a non-steady-state experimental design to examine the role of cholesteryl ester transfer protein (CETP) in mediating HDL-TG flux in vivo in rhesus macaques, and therefore, we developed an alternative strategy to model the data. Two isotopomers ([(2)H11] and [(13)C18]) of oleic acid were administered (orally and intravenously, respectively) to serve as precursors for labeling TGs in apoB-containing lipoproteins. The flux of a specific TG (52:2) from these donor lipoproteins to HDL was used as the measure of CETP activity; calculations are also presented to estimate total HDL-TG flux. Based on our data, we estimate that the peak total postprandial TG flux to HDL via CETP is â¼ 13 mg · h(-1) · kg(-1) and show that this transfer was inhibited by 97% following anacetrapib treatment. Collectively, these data demonstrate that HDL TG flux can be used as a measure of CETP activity in vivo. The fact that the donor lipoproteins can be labeled in situ using well-established stable isotope tracer techniques suggests ways to measure this activity for native lipoproteins in free-living subjects under any physiological conditions.
Assuntos
Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Oxazolidinonas/farmacologia , Triglicerídeos/metabolismo , Animais , Lipoproteínas HDL/sangue , Macaca mulatta , Masculino , Modelos Biológicos , Triglicerídeos/sangueRESUMO
Glucagon and insulin have opposing action in governing glucose homeostasis. In type 2 diabetes mellitus (T2DM), plasma glucagon is characteristically elevated, contributing to increased gluconeogenesis and hyperglycemia. Therefore, glucagon receptor (GCGR) antagonism has been proposed as a pharmacologic approach to treat T2DM. In support of this concept, a potent small-molecule GCGR antagonist (GRA), MK-0893, demonstrated dose-dependent efficacy to reduce hyperglycemia, with an HbA1c reduction of 1.5% at the 80 mg dose for 12 weeks in T2DM. However, GRA treatment was associated with dose-dependent elevation of plasma LDL-cholesterol (LDL-c). The current studies investigated the cause for increased LDL-c. We report findings that link MK-0893 with increased glucagon-like peptide 2 and cholesterol absorption. There was not, however, a GRA-related modulation of cholesterol synthesis. These findings were replicated using structurally diverse GRAs. To examine potential pharmacologic mitigation, coadministration of ezetimibe (a potent inhibitor of cholesterol absorption) in mice abrogated the GRA-associated increase of LDL-c. Although the molecular mechanism is unknown, our results provide a novel finding by which glucagon and, hence, GCGR antagonism govern cholesterol metabolism.
Assuntos
Colesterol/sangue , Pirazóis/farmacologia , Receptores de Glucagon/antagonistas & inibidores , beta-Alanina/análogos & derivados , Animais , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Humanos , Hipercolesterolemia/induzido quimicamente , Concentração Inibidora 50 , Absorção Intestinal , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pirazóis/efeitos adversos , beta-Alanina/efeitos adversos , beta-Alanina/farmacologiaRESUMO
The cellular, macromolecular and neutral lipid composition of the atherosclerotic plaque has been extensively characterized. However, a comprehensive lipidomic analysis of the major lipid classes within atherosclerotic lesions has not been reported. The objective of this study was to produce a detailed framework of the lipids that comprise the atherosclerotic lesion of a widely used pre-clinical model of plaque progression. Male New Zealand White rabbits were administered regular chow supplemented with 0.5% cholesterol (HC) for 12 weeks to induce hypercholesterolemia and atherosclerosis. Our lipidomic analyses of plaques isolated from rabbits fed the HC diet, using ultra-performance liquid chromatography (UPLC) and high-resolution mass spectrometry, detected most of the major lipid classes including: Cholesteryl esters, triacylglycerols, phosphatidylcholines, sphingomyelins, diacylglycerols, fatty acids, phosphatidylserines, lysophosphatidylcholines, ceramides, phosphatidylglycerols, phosphatidylinositols and phosphatidylethanolamines. Given that cholesteryl esters, triacylglycerols and phosphatidylcholines comprise greater than 75% of total plasma lipids, we directed particular attention towards the qualitative and quantitative assessment of the fatty acid composition of these lipids. We additionally found that sphingomyelins were relatively abundant lipid class within lesions, and compared the abundance of sphingomyelins to their precursor phosphatidylcholines. The studies presented here are the first approach to a comprehensive characterization of the atherosclerotic plaque lipidome.
Assuntos
Hipercolesterolemia/metabolismo , Metabolismo dos Lipídeos , Placa Aterosclerótica/metabolismo , Animais , Ceramidas/sangue , Ésteres do Colesterol/sangue , Ácidos Graxos/sangue , Glicerofosfolipídeos/sangue , Hipercolesterolemia/complicações , Masculino , Placa Aterosclerótica/etiologia , Coelhos , Esfingomielinas/sangue , Triglicerídeos/sangueRESUMO
RATIONALE: Lipids are involved in various biochemical and signaling pathways, cell structure and function, and the pathophysiology of many diseases. We took advantage of ion mobility spectrometry (IMS) in conjunction with ultra-performance liquid chromatography (UPLC) and high-resolution mass spectrometry to gain quantitative and deeper qualitative structural insight within a single experiment. METHODS: Human plasma lipid extracts were analyzed using an Acquity UPLC system coupled to a Synapt G2-HDMS mass spectrometer system. The ion mobility gas employed was helium for the helium cell (150 mL/min) and nitrogen (80 mL/min) for the T-wave drift tube. The wave height for the T-wave cell was ramped in a linear fashion between 5-40 V. The mass spectra were acquired in an electrospray positive ionization mode. RESULTS: We resolved chromatographically co-eluting lipids further by ion mobility tube drift time and then subjected them to low- and high-energy fragmentation without pre-selecting respective precursor species. The fragment ions produced in a high-energy mode were aligned with their precursor ions in a low-energy mode. By aligning intact molecular spectra and fragment spectra for these lipids at a given ion mobility drift time and chromatographic retention time, we were able to obtain much cleaner fragment ion spectra for structural elucidation. For quantitative analysis we obtained a dynamic linear range from 0.002 to 2 µg/mL with and without an additional dimension of IMS. CONCLUSIONS: The additional dimension of IMS allowed us to perform quantitative and qualitative analysis within a single experiment in a relatively high-throughput manner thus providing deeper structural insights into lipids of biological interest and resulting in an information-rich dataset.
Assuntos
Lipídeos/sangue , Lipídeos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Calibragem , Humanos , Íons/químicaRESUMO
We have previously reported on a liquid chromatography-mass spectrometry method to determine the disposition of [(13)C18]-oleic acid following intravenous and oral administration in vivo. This approach has enabled us to study a variety of aspects of lipid metabolism including a quantitative assessment of triglyceride synthesis. Here we present a more rigorous evaluation of the constraints imposed upon the analytical method in order to generate accurate data using this stable-isotope tracer approach along with more detail on relevant analytical figures of merit including limits of quantitation, precision, and accuracy. The use of mass isotopomer distribution analysis (MIDA) to quantify plasma triglyceride synthesis is specifically highlighted, and a re-evaluation of the underlying mathematics has enabled us to present a simplified series of equations. The derivation of this MIDA model and the significance of all underlying assumptions are explored in detail, and examples are given of how it can successfully be applied to detect differences in plasma triglyceride synthesis in lean and high-fat diet fed mouse models. More work is necessary to evaluate the applicability of this approach to triglyceride stores with slower rates of turnover such as in adipose or muscle tissue; however, the present report provides investigators with the tools necessary to conduct such studies.
Assuntos
Espectrometria de Massas/métodos , Ácido Oleico/análise , Triglicerídeos/biossíntese , Triglicerídeos/sangue , Animais , Isótopos de Carbono , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/sangue , Obesidade/diagnóstico , Ácido Oleico/administração & dosagemRESUMO
Apolipoprotein B100 (apoB100) and apolipoprotein A1 (apoA1) are the primary protein components of low density lipoprotein (LDL) and high density lipoprotein (HDL) particles, respectively, and plasma levels of these proteins are associated with risks of cardiovascular disease. Existing apoB100 quantitation methods for animal models have been limited to affinity capture techniques such as enzyme-linked immunosorbent assay (ELISA) and Western blot which require specialized reagents for each species and in many cases are not readily available. Here we demonstrate a single translatable ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) assay that is fast and robust and can be used to measure apolipoprotein concentrations in plasma for six species. When possible, peptide sequences that are conserved across species were identified for this assay. The sample preparation is limited and can be carried out in 96-well microtiter plates and thus allows for multiplexed preparation of samples for analysis of large numbers of samples in a short time frame when combined with UPLC/MS/MS. Separation and quantitation of the tryptic peptides is carried out at 700 µL/min using a 1.7 µm core shell C18 column (2.1 × 50 mm). The chromatography is designed for the analysis of over 100 samples per day, and the UPLC run is less than 10 min. This assay is capable of supporting cardiovascular research by providing a single assay to measure critical biomarkers across multiple species without the need for antibodies, and does so in a high-throughput manner.
Assuntos
Apolipoproteína B-100/sangue , Apolipoproteína B-48/sangue , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Animais , Apolipoproteína A-I/sangue , Apolipoproteína B-100/genética , Apolipoproteína B-48/genética , Doenças Cardiovasculares/sangue , Simulação por Computador , Cricetinae , Cães , Técnicas de Silenciamento de Genes , Humanos , Modelos Lineares , Macaca mulatta , Camundongos , Fragmentos de Peptídeos/análise , RNA Interferente Pequeno/genética , Ratos , Especificidade da Espécie , Tripsina/química , Tripsina/metabolismoRESUMO
To assess cardiovascular risk in both clinical and basic research settings, it is imperative to be able to accurately measure plasma lipid levels. Here, methods commonly used to measure lipoproteins and lipids: ultracentrifugation (UC), fast protein liquid chromatography (FPLC), Roche auto-analyzer, and enzymatic assays were tested and compared. Plasma samples from 20 healthy humans and 22 cynomolgus monkeys were analyzed for their total cholesterol (TC), cholesterol in low density lipoproteins (LDL) and high density lipoproteins (HDL), and triglycerides (TG). Major lipid classes from UC and FPLC separated lipoprotein fractions from human plasma were further characterized by liquid chromatography-mass spectrometry analysis. All the tested methods showed acceptable performance with Roche analyzer among the best in approximate dilution linearity and recovery for most lipids as well as in repeatability between measurements of the same samples. TC, LDL, HDL, and TG values measured in human vs. monkey were-183.9 ± 35.5 (mean ± SD) vs. 105.6 ± 24.6 mg/dl, 106.0 ± 30.1 vs. 42.8 ± 13.0 mg/dl, 50.0 ± 11.4 vs. 53.4 ± 14.8 mg/dl, and 107.6 ± 50.7 vs. 58.0 ± 52.3 mg/dl. While no single method was uniformly the best, we recommend the Roche analyzer for routine measurements. UC or FPLC separation is needed for further functional characterization for specific lipid fraction. We have shown athero-protective profile in cynomolgus monkey compared with humans.
Assuntos
Técnicas de Química Analítica , Lipídeos/sangue , Lipoproteínas/sangue , Adulto , Animais , Autoanálise , Biomarcadores/sangue , Colesterol/sangue , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Cromatografia Líquida , Feminino , Humanos , Macaca fascicularis , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Triglicerídeos/sangue , UltracentrifugaçãoRESUMO
Electrophilic reactive metabolite screening by liquid chromatography/mass spectrometry (LC/MS) is commonly performed during drug discovery and early-stage drug development. Accurate mass spectrometry has excellent utility in this application, but sophisticated data processing strategies are essential to extract useful information. Herein, a unified approach to glutathione (GSH) trapped reactive metabolite screening with high-resolution LC/TOF MS(E) analysis and drug-conjugate-specific in silico data processing was applied to rapid analysis of test compounds without the need for stable- or radio-isotope-labeled trapping agents. Accurate mass defect filtering (MDF) with a C-heteroatom dealkylation algorithm dynamic with mass range was compared to linear MDF and shown to minimize false positive results. MS(E) data-filtering, time-alignment and data mining post-acquisition enabled detection of 53 GSH conjugates overall formed from 5 drugs. Automated comparison of sample and control data in conjunction with the mass defect filter enabled detection of several conjugates that were not evident with mass defect filtering alone. High- and low-energy MS(E) data were time-aligned to generate in silico product ion spectra which were successfully applied to structural elucidation of detected GSH conjugates. Pseudo neutral loss and precursor ion chromatograms derived post-acquisition demonstrated 50.9% potential coverage, at best, of the detected conjugates by any individual precursor or neutral loss scan type. In contrast with commonly applied neutral loss and precursor-based techniques, the unified method has the advantage of applicability across different classes of GSH conjugates. The unified method was also successfully applied to cyanide trapping analysis and has potential for application to alternate trapping agents.
Assuntos
Cromatografia Líquida/métodos , Mineração de Dados/métodos , Descoberta de Drogas/métodos , Espectrometria de Massas/métodos , Algoritmos , Alquilação , Biologia Computacional , Simulação por Computador , Glutationa/química , Glutationa/metabolismo , Humanos , Microssomos Hepáticos/metabolismo , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismoRESUMO
Lecithin:cholesterol acyltransferase (LCAT) is the key circulating enzyme responsible for high-density lipoprotein (HDL) cholesterol esterification, HDL maturation, and potentially reverse cholesterol transport. To further explore LCAT's mechanism of action on lipoprotein metabolism, we employed adeno-associated viral vector (AAV) serotype 8 to achieve long-term (32-week) high level expression of human LCAT in hCETP;Ldlr(+/-) mice, and characterized the lipid profiles in detail. The mice had a marked increase in HDL cholesterol, HDL particle size, and significant reduction in low-density lipoprotein (LDL) cholesterol, plasma triglycerides, and plasma apoB. Plasma LCAT activity significantly increased with humanized substrate specificity. HDL cholesteryl esters increased in a fashion that fits human LCAT specificity. HDL phosphatidylcholines trended toward decrease, with no change observed for HDL lysophosphatidylcholines. Triglycerides reduction appeared to reside in all lipoprotein particles (very low-density lipoprotein (VLDL), LDL, and HDL), with HDL triglycerides composition highly reflective of VLDL, suggesting that changes in HDL triglycerides were primarily driven by the altered triglycerides metabolism in VLDL. In summary, in this human-like model for lipoprotein metabolism, AAV8-mediated overexpression of human LCAT resulted in profound changes in plasma lipid profiles. Detailed lipid analyses in the lipoprotein particles suggest that LCAT's beneficial effect on lipid metabolism includes not only enhanced HDL cholesterol esterification but also improved metabolism of apoB-containing particles and triglycerides. Our findings thus shed new light on LCAT's mechanism of action and lend support to its therapeutic potential in treating dyslipidemia.
Assuntos
Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Dependovirus/genética , Dislipidemias/terapia , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos , Lipídeos/sangue , Fosfatidilcolina-Esterol O-Aciltransferase/genética , Receptores de LDL/deficiência , Animais , Proteínas de Transferência de Ésteres de Colesterol/genética , Modelos Animais de Doenças , Dislipidemias/enzimologia , Dislipidemias/genética , Humanos , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Tamanho da Partícula , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Receptores de LDL/genética , Fatores de TempoRESUMO
The purpose of this study was to evaluate the use of high resolution LC-MS together with metabolomics and D(4)-cholic acid (D(4)-CA) as a metabolic tracer to measure the metabolism and reconjugation of bile acids (BAs) in vitro and in vivo. Metabolic tracers are very important because they allow for the direct detection (substrate-to-product) of small and significant biological perturbations that may not be apparent when monitoring "static" endogenous levels of particular metabolites. Slc27a5, also known as fatty acid transport protein 5 (FATP5), is the hepatic BA-CoA ligase involved in reconjugating BAs during enterohepatic BA recycling. Using Slc27a5-cKD mice, silencing of â¼90% gene expression was achieved followed by reduction in the reconjugation of D(4)-CA to D(4)-taurocholic acid (D(4)-TCA), as well as other conjugated BA metabolites in plasma (p = 0.0031). The method described allowed a rapid measure of many D(4) and endogenous BA. Analysis of bile resulted in the detection of 39 BA metabolites from a 13 min analytical run. Finally, the utilization of a novel high resolution mass spectrometry method in combination with metabolomics and a stable isotope metabolic tracer allowed for the detection of targeted and untargeted BAs following silencing of the Slc27a5 gene in primary hepatocytes and in mice.
Assuntos
Ácidos e Sais Biliares/metabolismo , Cromatografia Líquida/métodos , Proteínas de Transporte de Ácido Graxo/metabolismo , Fígado/patologia , Espectrometria de Massas/métodos , Metabolômica/métodos , Animais , Inativação Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
A novel method to differentiate diet-induced alterations in plasma lipid phenotypes "static (concentration of lipids) and kinetic (endogenous production, e.g., denovo lipogenesis)" was employed. C57Bl6 mice were randomized into 2 groups and fed either a high-carbohydrate, low-fat (HC) or a carbohydrate-free, high-fat diet (HF) diet for 13 days; D(2)O was administered via intraperitoneal injection and then adding D(2)O to the drinking water for 96 h. Principal component analysis (PCA) revealed differences in the plasma lipid content, for example, triglycerides (TG) 50:2, 50:3, and 52:2 were up-regulated in mice fed the HC diet, whereas TG 52:4, 52:1, 54:5, 54:3, 54:4, and 54:2 were higher in animals fed the HF diet. However, although the fractional contribution of synthesis was ~10-fold lower in HF vs HC fed mice, changes in TG concentration were not entirely mediated by altered de novo lipogenesis. In addition, the ability to couple isotope labeling measurements with PCA analyses revealed cases where there were no differences in the concentration of a compound but its source was substantially altered. In summary, this strategy determined (i) the presence/absence of differences in concentration and (ii) the contribution of different pathways and synthesis that could affect lipid biology in a mouse model respectively.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Metabolismo dos Lipídeos/fisiologia , Espectrometria de Massas/métodos , Metabolômica/métodos , Animais , Aterosclerose/metabolismo , Medição da Troca de Deutério , Dieta , Carboidratos da Dieta/administração & dosagem , Carboidratos da Dieta/farmacologia , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/farmacologia , Dislipidemias/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/análise , Lipídeos/química , Fígado/química , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Análise de Componente PrincipalRESUMO
The use of stable isotopically labeled substrates and analysis by mass spectrometry have provided substantial insight into rates of synthesis, disposition, and utilization of lipids in vivo. The information to be gained from such studies is of particular benefit to therapeutic research where the underlying causes of disease may be related to the production and utilization of lipids. When studying biology through the use of isotope tracers, care must be exercised in interpreting the data to ensure that any response observed can truly be interpreted as biological and not as an artifact of the experimental design or a dilutional effect on the isotope. We studied the effects of dosing route and tracer concentration on the mass isotopomer distribution profile as well as the action of selective inhibitors of microsomal tri-glyceride transfer protein (MTP) in mice and diacylglycerol acyltransferase 1 (DGAT1) in nonhuman primates, using a stable-isotopically labeled approach. Subjects were treated with inhibitor and subsequently given a dose of uniformly ¹³C-labeled oleic acid. Samples were analyzed using a rapid LC-MS technique, allowing the effects of the intervention on the assembly and disposition of triglycerides, cholesteryl esters, and phospholipids to be determined in a single 3 min run from just 10 µl of plasma.
Assuntos
Proteínas de Transporte/metabolismo , Ésteres do Colesterol/sangue , Diacilglicerol O-Aciltransferase/metabolismo , Metabolismo dos Lipídeos , Lipoproteínas/sangue , Ácido Oleico , Triglicerídeos/sangue , Animais , Proteínas de Transporte/antagonistas & inibidores , Chlorocebus aethiops , Cromatografia Líquida , Vias de Administração de Medicamentos , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Feminino , Marcação por Isótopo/métodos , Isótopos/análise , Isótopos/sangue , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Ácido Oleico/metabolismo , Ácido Oleico/farmacologiaRESUMO
An ultraperformance liquid chromatography method using normal-phase solvents, a silica column, and evaporative light-scattering detection is presented. The method is based on a quaternary gradient profile and is capable of resolving the major neutral and polar lipids present in plasma and animal tissue in under 5 min, with a total cycle time of 11 min. Limits of quantitation for 7 different lipid classes were on the order of 200 ng of material on column which enables an accurate analysis from as little as 20 µL of plasma or 50 mg of tissue for typical samples. Intraday and interday precision for the determination of the major lipid classes in human plasma ranged from 3.6 to 10.5% CV with a variability in retention time of less than 6%. The utility of the method is demonstrated through the separation and quantitation of lipids in mouse plasma, liver, and heart tissue.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Lipídeos/análise , Animais , Colesterol/análise , Colesterol/sangue , Colesterol/isolamento & purificação , Humanos , Lipídeos/sangue , Lipídeos/isolamento & purificação , Camundongos , Fosfatidilcolinas/análise , Fosfatidilcolinas/sangue , Fosfatidilcolinas/isolamento & purificação , Fosfatidiletanolaminas/análise , Fosfatidiletanolaminas/sangue , Fosfatidiletanolaminas/isolamento & purificação , Triglicerídeos/análise , Triglicerídeos/sangue , Triglicerídeos/isolamento & purificaçãoRESUMO
A fast and robust method for lipid profiling utilizing liquid chromatography coupled with mass spectrometry has been demonstrated and validated for the analysis of human plasma. This method allowed quantification and identification of lipids in human plasma using parallel alternating low energy and high energy collision spectral acquisition modes. A total of 275 [corrected] lipids were identified and quantified (as relative concentrations) in both positive and negative ion electrospray ionization mode. The method was validated with five nonendogenous lipids, and the linearity (r(2) better than 0.994) and the intraday and interday repeatability (relative standard deviation, 4-6% and 5-8%, respectively) were satisfactory. The developed lipid profiling method was successfully applied for the analysis of plasma from osteoarthritis (OA) patients. The multivariate statistical analysis by partial least-squares-discrimination analysis suggested an altered lipid metabolism associated with osteoarthritis and the release of arachidonic acid from phospholipids.
Assuntos
Biomarcadores/sangue , Cromatografia Líquida de Alta Pressão/métodos , Lipídeos/sangue , Osteoartrite/sangue , Espectrometria de Massas por Ionização por Electrospray/métodos , Feminino , Humanos , Análise dos Mínimos Quadrados , Análise Multivariada , Reprodutibilidade dos TestesRESUMO
The use of exact mass liquid chromatography/mass spectrometry (LC/MS) for drug metabolism studies has increased significantly in recent years. Firstly, exact mass measurements facilitate identification of standard biotransformations through the use of narrow window extracted ion chromatograms, which are typically highly selective relative to signals from matrix or dosing components. Secondly, novel metabolites can be characterized via elemental formula calculations and high-resolution product ion spectra. Furthermore, biological background ions can be removed by the use of mass defect filters (MDFs) which filter out ions based on the decimal component of their m/z value. Here, we describe an approach which we term 'generic dealkylation' that in association with other data interpretation tools adds significant value to the assignment process. Generic dealkylation uses a simple strategy to identify those bonds which have the potential to be cleaved by metabolism. In combination with standard phase 1 and phase 2 biotransformations, this allows creation of a chemically intelligent MDF which balances the need to remove matrix background with the requirement of avoiding filtering true metabolites. Secondly, generic dealkylation increases the hit-rate at which non-trivial (i.e. not covered by simple phase 1 oxidations or direct phase 2 conjugations) metabolites can be directly rationalized. The value of the generic dealkylation approach is illustrated by its application to determination of in vitro metabolic routes for two commercial drugs, nefazodone and indinavir.
Assuntos
Medicamentos sob Prescrição/farmacocinética , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Cromatografia Líquida de Alta Pressão , Remoção de Radical Alquila , Inativação Metabólica , Metabolômica , Microssomos Hepáticos/metabolismo , RatosRESUMO
Mass defect, neutral loss and isotope filtration techniques were applied to electrospray ionization mass spectrometry (ESI-MS) data obtained for in vivo and in vitro samples of drug metabolism studies. A combination of these post-acquisition processing techniques was shown to be more powerful than the use of one of these tools alone for the detection in complex matrices of metabolites of candidate drugs with a characteristic isotope pattern (e.g. containing bromine, chlorine, or a high proportion of radiolabeled drug ((12)C/(14)C)) or characteristic neutral losses. In combination with 'all-in-one' data acquisition this methodology is able to perform software-driven constant neutral loss scanning for an unlimited number of mass differences at any time after analysis. Highly selective MS chromatograms were obtained with excellent correlation with their corresponding radiochromatograms.
Assuntos
Isótopos de Carbono/isolamento & purificação , Fezes/química , Farmacocinética , Quinolinas/farmacocinética , Manejo de Espécimes/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Ultrafiltração/métodos , Algoritmos , Animais , Diarilquinolinas , Cães , Íons , Ratos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The application of sub-2 microm porous particle liquid chromatography (LC) operated at elevated temperatures, coupled with time-of-flight mass spectrometry (MS), to the separation and identification of metabolites of ibuprofen present in human urine following oral administrations is illustrated. The LC/MS system generated a high-resolution analytical separation that, with an analysis time of 20 min, provided a peak capacity in the order of ca. 350. Using this system a total of nine glucuronides of the drug and its metabolites were detected, including a number of isomeric acyl glucuronides of ibuprofen itself, a side-chain-oxidized carboxylic acid acyl glucuronide and a number of acyl glucuronides of various hydroxylated metabolites. The identities of the metabolites were confirmed by their accurate mass values and the presence of the common fragment ions from ibuprofen.