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1.
J Lipid Res ; 61(3): 306-315, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31953305

RESUMO

The composition-function relationship of HDL particles and its effects on the mechanisms driving coronary heart disease (CHD) is poorly understood. We tested the hypothesis that the functionality of HDL particles is significantly influenced by their lipid composition. Using a novel 3D-separation method, we isolated five different-sized HDL subpopulations from CHD patients who had low preß-1 functionality (low-F) (ABCA1-dependent cholesterol-efflux normalized for preß-1 concentration) and controls who had either low-F or high preß-1 functionality (high-F). Molecular numbers of apoA-I, apoA-II, and eight major lipid classes were determined in each subpopulation by LC-MS. The average number of lipid molecules decreased from 422 in the large spherical α-1 particles to 57 in the small discoid preß-1 particles. With decreasing particle size, the relative concentration of free cholesterol (FC) decreased in α-mobility but not in preß-1 particles. Preß-1 particles contained more lipids than predicted; 30% of which were neutral lipids (cholesteryl ester and triglyceride), indicating that these particles were mainly remodeled from larger particles not newly synthesized. There were significant correlations between HDL-particle functionality and the concentrations of several lipids. Unexpectedly, the phospholipid:FC ratio was significantly correlated with large-HDL-particle functionality but not with preß-1 functionality. There was significant positive correlation between particle functionality and total lipids in high-F controls, indicating that the lipid-binding capacity of apoA-I plays a major role in the cholesterol efflux capacity of HDL particles. Functionality and lipid composition of HDL particles are significantly correlated and probably both are influenced by the lipid-binding capacity of apoA-I.

3.
N Engl J Med ; 382(8): 697-705, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31860793

RESUMO

BACKGROUND: The causative agents for the current national outbreak of electronic-cigarette, or vaping, product use-associated lung injury (EVALI) have not been established. Detection of toxicants in bronchoalveolar-lavage (BAL) fluid from patients with EVALI can provide direct information on exposure within the lung. METHODS: BAL fluids were collected from 51 patients with EVALI in 16 states and from 99 healthy participants who were part of an ongoing study of smoking involving nonsmokers, exclusive users of e-cigarettes or vaping products, and exclusive cigarette smokers that was initiated in 2015. Using the BAL fluid, we performed isotope dilution mass spectrometry to measure several priority toxicants: vitamin E acetate, plant oils, medium-chain triglyceride oil, coconut oil, petroleum distillates, and diluent terpenes. RESULTS: State and local health departments assigned EVALI case status as confirmed for 25 patients and as probable for 26 patients. Vitamin E acetate was identified in BAL fluid obtained from 48 of 51 case patients (94%) in 16 states but not in such fluid obtained from the healthy comparator group. No other priority toxicants were found in BAL fluid from the case patients or the comparator group, except for coconut oil and limonene, which were found in 1 patient each. Among the case patients for whom laboratory or epidemiologic data were available, 47 of 50 (94%) had detectable tetrahydrocannabinol (THC) or its metabolites in BAL fluid or had reported vaping THC products in the 90 days before the onset of illness. Nicotine or its metabolites were detected in 30 of 47 of the case patients (64%). CONCLUSIONS: Vitamin E acetate was associated with EVALI in a convenience sample of 51 patients in 16 states across the United States. (Funded by the National Cancer Institute and others.).


Assuntos
Lesão Pulmonar Aguda/patologia , Líquido da Lavagem Broncoalveolar/química , Sistemas Eletrônicos de Liberação de Nicotina , Vaping/efeitos adversos , Vitamina E/análise , Lesão Pulmonar Aguda/etiologia , Adolescente , Adulto , Idoso , Fumar Cigarros , Óleo de Coco/análise , Feminino , Humanos , Limoneno/análise , Masculino , Pessoa de Meia-Idade , Estados Unidos , Adulto Jovem
4.
MMWR Morb Mortal Wkly Rep ; 68(45): 1040-1041, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31725707

RESUMO

CDC, the Food and Drug Administration (FDA), state and local health departments, and multiple public health and clinical partners are investigating a national outbreak of e-cigarette, or vaping, product use-associated lung injury (EVALI). Based on data collected as of October 15, 2019, 86% of 867 EVALI patients reported using tetrahydrocannabinol (THC)-containing products in the 3 months preceding symptom onset (1). Analyses of THC-containing product samples by FDA and state public health laboratories have identified potentially harmful constituents in these products, such as vitamin E acetate, medium chain triglyceride oil (MCT oil), and other lipids (2,3) (personal communication, D.T. Heitkemper, FDA Forensic Chemistry Center, November 2019). Vitamin E acetate, in particular, might be used as an additive in the production of e-cigarette, or vaping, products; it also can be used as a thickening agent in THC products (4). Inhalation of vitamin E acetate might impair lung function (5-7).


Assuntos
Líquido da Lavagem Broncoalveolar/química , Surtos de Doenças , Lesão Pulmonar/epidemiologia , Vaping/efeitos adversos , Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estados Unidos/epidemiologia , Adulto Jovem
5.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1118-1119: 137-147, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-31035135

RESUMO

Progress toward better diagnosis and treatment of lipid metabolism-related diseases requires high throughput approaches for multiplexed quantitative analysis of structurally diverse lipids, including phospholipids (PLs). This work demonstrates a simplified "one-pot" phospholipid extraction protocol, as an alternative to conventional liquid-liquid extraction. Performed in a 96-well format, the extraction was coupled with high throughput UPLC and multiplexed tandem mass spectrometry (MS/MS) detection, allowing non-targeted quantification of phosphatidylcholines (PC), sphingomyelins (SM), lysophosphatidylcholines (LPC), phosphatidylethanolamines (PE), and phosphatidylinositols (PI). Using 50 µL aliquots of serum samples from 110 individuals, lipoproteins were fractionated by size, and analyzed for phospholipids and non-polar lipids including free cholesterol (FC), cholesteryl esters (CEs) and triglycerides (TGs). Analysis of serum samples with wide range of Total-TG levels showed significant differences in PL composition. The correlations of molar ratios in lipoprotein size fractions, SM/PL with FC/PL, PE/PL with TG/CE, and PE/PL with PI/PL, demonstrate the applicability of the method for quantitative composition analysis of high, low and very-low density lipoproteins (HDL, LDL and VLDL), and characterization of lipid metabolism related disease states.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Dislipidemias/sangue , Dislipidemias/diagnóstico , Fosfolipídeos/sangue , Espectrometria de Massas em Tandem/métodos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Fosfolipídeos/isolamento & purificação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
6.
Anal Bioanal Chem ; 411(12): 2493-2509, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30911800

RESUMO

Inhalation of Bacillus anthracis spores can cause a rapidly progressing fatal infection. B. anthracis secretes three protein toxins: lethal factor (LF), edema factor (EF), and protective antigen (PA). EF and LF may circulate as free or PA-bound forms. Both free EF (EF) and PA-bound-EF (ETx) have adenylyl cyclase activity converting ATP to cAMP. We developed an adenylyl cyclase activity-based method for detecting and quantifying total EF (EF+ETx) in plasma. The three-step method includes magnetic immunocapture with monoclonal antibodies, reaction with ATP generating cAMP, and quantification of cAMP by isotope-dilution HPLC-MS/MS. Total EF was quantified from 5PL regression of cAMP vs ETx concentration. The detection limit was 20 fg/mL (225 zeptomoles/mL for the 89 kDa protein). Relative standard deviations for controls with 0.3, 6.0, and 90 pg/mL were 11.7-16.6% with 91.2-99.5% accuracy. The method demonstrated 100% specificity in 238 human serum/plasma samples collected from unexposed healthy individuals, and 100% sensitivity in samples from 3 human and 5 rhesus macaques with inhalation anthrax. Analysis of EF in the rhesus macaques showed that it was detected earlier post-exposure than B. anthracis by culture and PCR. Similar to LF, the kinetics of EF over the course of infection were triphasic, with an initial rise (phase-1), decline (phase-2), and final rapid rise (phase-3). EF levels were ~ 2-4 orders of magnitude lower than LF during phase-1 and phase-2 and only ~ 6-fold lower at death/euthanasia. Analysis of EF improves early diagnosis and adds to our understanding of anthrax toxemia throughout infection. The LF/EF ratio may also indicate the stage of infection and need for advanced treatments.


Assuntos
Antraz/patologia , Antígenos de Bactérias/sangue , Bacillus anthracis/patogenicidade , Toxinas Bacterianas/sangue , Cromatografia Líquida de Alta Pressão/métodos , Infecções Respiratórias/patologia , Espectrometria de Massas em Tandem/métodos , Toxemia/patologia , Trifosfato de Adenosina/metabolismo , Animais , Antraz/sangue , Estudos de Casos e Controles , AMP Cíclico/biossíntese , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Humanos , Limite de Detecção , Macaca mulatta , Reação em Cadeia da Polimerase , Infecções Respiratórias/sangue , Toxemia/sangue , Toxemia/microbiologia
7.
Methods Mol Biol ; 1871: 295-311, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30276747

RESUMO

Protein digestion coupled to liquid chromatography and tandem mass spectrometry (LC-MS/MS) detection enables multiplexed quantification of proteins in complex biological matrices. However, the reproducibility of enzymatic digestion of proteins to produce proteotypic target peptides is a major limiting factor of assay precision. Online digestion using immobilized trypsin addresses this problem through precise control of digestion conditions and time. Because online digestion is typically for a short time, the potential for peptide degradation, a major source of measurement bias, is significantly reduced. Online proteolysis requires minimal sample preparation and is easily coupled to LC-MS/MS systems, further reducing potential method variability. We describe herein a method optimized for the multiplexed quantification of several apolipoproteins in human serum using on-column digestion. We highlight key features of the method that enhance assay accuracy and precision. These include the use of value-assigned serum as calibrators and stable isotope-labeled (SIL) peptide analogs as internal standards. We also comment on practical aspects of column switching valve design, instrument maintenance, tandem mass spectrometry data acquisition, and data processing.


Assuntos
Cromatografia Líquida , Proteínas , Espectrometria de Massas em Tandem , Tripsina , Apolipoproteínas/análise , Apolipoproteínas/química , Apolipoproteínas/metabolismo , Proteínas Sanguíneas/química , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida/métodos , Análise de Dados , Enzimas Imobilizadas , Humanos , Peptídeos/química , Proteínas/análise , Proteínas/química , Proteólise , Software , Espectrometria de Massas em Tandem/métodos , Tripsina/química
8.
Diabetes ; 67(12): 2494-2506, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30213825

RESUMO

In clinical trials, inhibition of cholesteryl ester transfer protein (CETP) raises HDL cholesterol levels but does not robustly improve cardiovascular outcomes. Approximately two-thirds of trial participants are obese. Lower plasma CETP activity is associated with increased cardiovascular risk in human studies, and protective aspects of CETP have been observed in mice fed a high-fat diet (HFD) with regard to metabolic outcomes. To define whether CETP inhibition has different effects depending on the presence of obesity, we performed short-term anacetrapib treatment in chow- and HFD-fed CETP transgenic mice. Anacetrapib raised HDL cholesterol and improved aspects of HDL functionality, including reverse cholesterol transport, and HDL's antioxidative capacity in HFD-fed mice was better than in chow-fed mice. Anacetrapib worsened the anti-inflammatory capacity of HDL in HFD-fed mice. The HDL proteome was markedly different with anacetrapib treatment in HFD- versus chow-fed mice. Despite benefits on HDL, anacetrapib led to liver triglyceride accumulation and insulin resistance in HFD-fed mice. Overall, our results support a physiologic importance of CETP in protecting from fatty liver and demonstrate context selectivity of CETP inhibition that might be important in obese subjects.


Assuntos
Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , Proteínas de Transferência de Ésteres de Colesterol/genética , HDL-Colesterol/sangue , Dieta Hiperlipídica , Fígado Gorduroso/genética , Resistência à Insulina/fisiologia , Animais , Anticolesterolemiantes/farmacologia , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Fígado Gorduroso/metabolismo , Camundongos , Camundongos Transgênicos , Oxazolidinonas/farmacologia
9.
Clin Chem ; 64(10): 1485-1495, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30087138

RESUMO

BACKGROUND: Despite the usefulness of standard lipid parameters for cardiovascular disease risk assessment, undiagnosed residual risk remains high. Advanced lipoprotein testing (ALT) was developed to provide physicians with more predictive diagnostic tools. ALT methods separate and/or measure lipoproteins according to different parameters such as size, density, charge, or content, and equivalence of results across methods has not been demonstrated. METHODS: Through a split-sample study, 25 clinical specimens (CSs) were assayed in 10 laboratories before and after freezing using the major ALT methods for non-HDL particles (non-HDL-P) or apolipoprotein B-100 (apoB-100) measurements with the intent to assess their comparability in the current state of the art. RESULTS: The overall relative standard deviation (CV) of non-HDL-P and apoB-100 concentrations measured by electrospray differential mobility analysis, nuclear magnetic resonance, immunonephelometry, LC-MS/MS, and vertical autoprofile in the 25 frozen CSs was 14.1%. Within-method comparability was heterogeneous, and CV among 4 different LC-MS/MS methods was 11.4% for apoB-100. No significant effect of freezing and thawing was observed. CONCLUSIONS: This study demonstrates that ALT methods do not yet provide equivalent results for the measurement of non-HDL-P and apoB-100. The better agreement between methods harmonized to the WHO/IFCC reference material suggests that standardizing ALT methods by use of a common commutable calibrator will improve cross-platform comparability. This study provides further evidence that LC-MS/MS is the most suitable candidate reference measurement procedure to standardize apoB-100 measurement, as it would provide results with SI traceability. The absence of freezing and thawing effect suggests that frozen serum pools could be used as secondary reference materials.


Assuntos
Apolipoproteína B-100/sangue , Doenças Cardiovasculares/sangue , Técnicas de Laboratório Clínico , Lipoproteínas LDL/sangue , Lipoproteínas VLDL/sangue , Calibragem , Técnicas de Laboratório Clínico/instrumentação , Técnicas de Laboratório Clínico/métodos , Técnicas de Laboratório Clínico/normas , Humanos , Padrões de Referência , Sensibilidade e Especificidade , Manejo de Espécimes
10.
PLoS One ; 13(4): e0194797, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29634782

RESUMO

Lipoproteins are complex molecular assemblies that are key participants in the intricate cascade of extracellular lipid metabolism with important consequences in the formation of atherosclerotic lesions and the development of cardiovascular disease. Multiplexed mass spectrometry (MS) techniques have substantially improved the ability to characterize the composition of lipoproteins. However, these advanced MS techniques are limited by traditional pre-analytical fractionation techniques that compromise the structural integrity of lipoprotein particles during separation from serum or plasma. In this work, we applied a highly effective and gentle hydrodynamic size based fractionation technique, asymmetric flow field-flow fractionation (AF4), and integrated it into a comprehensive tandem mass spectrometry based workflow that was used for the measurement of apolipoproteins (apos A-I, A-II, A-IV, B, C-I, C-II, C-III and E), free cholesterol (FC), cholesterol esters (CE), triglycerides (TG), and phospholipids (PL) (phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and lysophosphatidylcholine (LPC)). Hydrodynamic size in each of 40 size fractions separated by AF4 was measured by dynamic light scattering. Measuring all major lipids and apolipoproteins in each size fraction and in the whole serum, using total of 0.1 ml, allowed the volumetric calculation of lipoprotein particle numbers and expression of composition in molar analyte per particle number ratios. Measurements in 110 serum samples showed substantive differences between size fractions of HDL and LDL. Lipoprotein composition within size fractions was expressed in molar ratios of analytes (A-I/A-II, C-II/C-I, C-II/C-III. E/C-III, FC/PL, SM/PL, PE/PL, and PI/PL), showing differences in sample categories with combinations of normal and high levels of Total-C and/or Total-TG. The agreement with previous studies indirectly validates the AF4-LC-MS/MS approach and demonstrates the potential of this workflow for characterization of lipoprotein composition in clinical studies using small volumes of archived frozen samples.


Assuntos
Apolipoproteínas/sangue , Cromatografia Líquida/métodos , Fracionamento por Campo e Fluxo/métodos , Lipídeos/sangue , Lipoproteínas/sangue , Espectrometria de Massas em Tandem/métodos , Apolipoproteína A-I/metabolismo , Apolipoproteína B-100/metabolismo , Análise Química do Sangue/métodos , Calibragem , Colesterol/química , Humanos , Luz , Modelos Estatísticos , Tamanho da Partícula , Controle de Qualidade , Espalhamento de Radiação , Fluxo de Trabalho
11.
J Lipid Res ; 58(12): 2275-2288, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28986437

RESUMO

As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950-Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.


Assuntos
Benchmarking , Ensaio de Proficiência Laboratorial/estatística & dados numéricos , Lipídeos/sangue , Humanos , Cooperação Internacional , Metabolismo dos Lipídeos/fisiologia , Lipídeos/normas , Variações Dependentes do Observador , Padrões de Referência , Reprodutibilidade dos Testes
12.
J Am Soc Mass Spectrom ; 28(11): 2319-2329, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28801822

RESUMO

We demonstrate the application of in-source nitrogen collision-induced dissociation (CID) that eliminates the need for ester hydrolysis before simultaneous analysis of esterified cholesterol (EC) and triglycerides (TG) along with free cholesterol (FC) from human serum, using normal phase liquid chromatography (LC) coupled to atmospheric pressure chemical ionization (APCI) tandem mass spectrometry (MS/MS). The analysis requires only 50 µL of 1:100 dilute serum with a high-throughput, precipitation/evaporation/extraction protocol in one pot. Known representative mixtures of EC and TG species were used as calibrators with stable isotope labeled analogs as internal standards. The APCI MS source was operated with nitrogen source gas. Reproducible in-source CID was achieved with the use of optimal cone voltage (declustering potential), generating FC, EC, and TG lipid class-specific precursor fragment ions for multiple reaction monitoring (MRM). Using a representative mixture of purified FC, CE, and TG species as calibrators, the method accuracy was assessed with analysis of five inter-laboratory standardization materials, showing -10% bias for Total-C and -3% for Total-TG. Repeated duplicate analysis of a quality control pool showed intra-day and inter-day variation of 5% and 5.8% for FC, 5.2% and 8.5% for Total-C, and 4.1% and 7.7% for Total-TG. The applicability of the method was demonstrated on 32 serum samples and corresponding lipoprotein sub-fractions collected from normolipidemic, hypercholesterolemic, hypertriglyceridemic, and hyperlipidemic donors. The results show that in-source CID coupled with isotope dilution UHPLC-MS/MS is a viable high precision approach for translational research studies where samples are substantially diluted or the amounts of archived samples are limited. Graphical Abstract ᅟ.


Assuntos
Colesterol/sangue , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas em Tandem/métodos , Triglicerídeos/sangue , Ésteres do Colesterol/sangue , Humanos , Hidrólise , Reprodutibilidade dos Testes
13.
Proteomics Clin Appl ; 11(7-8)2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28296203

RESUMO

PURPOSE: Apolipoprotein A-I (ApoA-I) and apolipoprotein B-100 (ApoB-100) are amphipathic proteins that are strong predictors of cardiovascular disease risk. The traceable calibration of apolipoprotein assays is a persistent challenge, especially for ApoB-100, which cannot be solubilized in purified form. EXPERIMENTAL DESIGN: A simultaneous quantitation method for ApoA-I and ApoB-100 was developed using tryptic digestion without predigestion reduction and alkylation, followed by LC separation coupled with isotope dilution MS analysis. The accuracy of the method was assured by selecting structurally exposed signature peptides, optimal choice of detergent, protein:enzyme ratio, and incubation time. Peptide calibrators were value assigned by isobaric tagging isotope dilution MS amino acid analysis. RESULTS: The method reproducibility was validated in technical repeats of three serum samples, giving 2-3% intraday CVs (N = 5) and <7% interday CVs (N = 21). The repeated analysis of interlaboratory harmonization standards showed -1% difference for ApoA-I and -12% for ApoB-100 relative to the assigned value. The applicability of the method was demonstrated by repeated analysis of 24 patient samples with a wide range of total cholesterol and triglyceride levels. CONCLUSIONS AND CLINICAL RELEVANCE: The method is applicable for simultaneous analysis of ApoA-I and ApoB-100 in patient samples, and for characterization of serum pool calibrators for other analytical platforms.


Assuntos
Apolipoproteína A-I/química , Apolipoproteína B-100/química , Análise Química do Sangue/métodos , Espectrometria de Massas , Fragmentos de Peptídeos/sangue , Proteólise , Tripsina/metabolismo , Sequência de Aminoácidos , Apolipoproteína A-I/metabolismo , Apolipoproteína B-100/metabolismo , Calibragem , Cromatografia Líquida , Humanos , Isótopos/química , Modelos Lineares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Reprodutibilidade dos Testes
14.
J Proteomics ; 150: 258-267, 2017 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-27667389

RESUMO

Apolipoproteins measured in plasma or serum are potential biomarkers for assessing metabolic irregularities that are associated with the development of cardiovascular disease (CVD). LC-MS/MS allows quantitative measurement of multiple apolipoproteins in the same sample run. However, the accuracy and precision of the LC-MS/MS measurement depends on the reproducibility of the enzymatic protein digestion step. With the application of an immobilized enzyme reactor (IMER), the reproducibility of the trypsin digestion can be controlled with high precision via flow rate, column volume and temperature. In this report, we demonstrate the application of an integrated IMER-LC-MS/MS platform for the simultaneous quantitative analysis of eight apolipoproteins. Using a dilution series of a characterized serum pool as calibrator, the method was validated by repeated analysis of pooled sera and individual serum samples with a wide range of lipid profiles, all showing intra-assay CV<4.4% and inter-assay CV<8%. In addition, the method was compared with traditional homogeneous digestion coupled LC-MS/MS for the quantification of apoA-I and apoB-100. Applied in large scale human population studies, this method can serve the translation of a wider panel of apolipoprotein biomarkers from research to clinical application. SIGNIFICANCE: Currently, the translation of apolipoprotein biomarkers to clinical application is impaired because of the high cost of large cohort studies using traditional single-analyte immunoassays. The application of on-line tryptic digestion coupled with LC-MS/MS analysis is an effective way to address this problem. In this work we demonstrate a high throughput, multiplexed, automated proteomics workflow for the simultaneous analysis of multiple proteins.


Assuntos
Apolipoproteínas/análise , Proteólise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Tripsina/metabolismo , Apolipoproteínas/metabolismo , Biomarcadores/sangue , Análise Química do Sangue/métodos , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/metabolismo , Cromatografia Líquida , Estudos de Viabilidade , Humanos , Reprodutibilidade dos Testes
15.
J Lipid Res ; 57(4): 674-86, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26908829

RESUMO

HDL cholesterol (HDL-C) efflux function may be a more robust biomarker of coronary artery disease risk than HDL-C. To study HDL function, apoB-containing lipoproteins are precipitated from serum. Whether apoB precipitation affects HDL subspecies composition and function has not been thoroughly investigated. We studied the effects of four common apoB precipitation methods [polyethylene glycol (PEG), dextran sulfate/magnesium chloride (MgCl2), heparin sodium/manganese chloride (MnCl2), and LipoSep immunoprecipitation (IP)] on HDL subspecies composition, apolipoproteins, and function (cholesterol efflux and reduction of LDL oxidation). PEG dramatically shifted the size distribution of HDL and apolipoproteins (assessed by two independent methods), while leaving substantial amounts of reagent in the sample. PEG also changed the distribution of cholesterol efflux and LDL oxidation across size fractions, but not overall efflux across the HDL range. Dextran sulfate/MgCl2, heparin sodium/MnCl2, and LipoSep IP did not change the size distribution of HDL subspecies, but altered the quantity of a subset of apolipoproteins. Thus, each of the apoB precipitation methods affected HDL composition and/or size distribution. We conclude that careful evaluation is needed when selecting apoB depletion methods for existing and future bioassays of HDL function.


Assuntos
Apolipoproteínas B/deficiência , Apolipoproteínas B/isolamento & purificação , Precipitação Química , Lipoproteínas LDL/metabolismo , Adulto , Apolipoproteína A-I/metabolismo , Apolipoproteínas B/metabolismo , Transporte Biológico/efeitos dos fármacos , Precipitação Química/efeitos dos fármacos , Cloretos/farmacologia , HDL-Colesterol/química , HDL-Colesterol/metabolismo , Sulfato de Dextrana/farmacologia , Feminino , Heparina/farmacologia , Humanos , Lipoproteínas LDL/química , Compostos de Manganês/farmacologia , Oxirredução/efeitos dos fármacos , Tamanho da Partícula , Polietilenoglicóis/farmacologia
16.
Toxicon ; 95: 72-83, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25576235

RESUMO

The seeds of the Ricinus communis (Castor bean) plant are the source of the economically important commodity castor oil. Castor seeds also contain the proteins ricin and R. communis agglutinin (RCA), two toxic lectins that are hazardous to human health. Radial immunodiffusion (RID) and the enzyme linked immunosorbent assay (ELISA) are two antibody-based methods commonly used to quantify ricin and RCA; however, antibodies currently used in these methods cannot distinguish between ricin and RCA due to the high sequence homology of the respective proteins. In this study, a technique combining antibody-based affinity capture with liquid chromatography and multiple reaction monitoring (MRM) mass spectrometry (MS) was used to quantify the amounts of ricin and RCA independently in extracts prepared from the seeds of eighteen representative cultivars of R. communis which were propagated under identical conditions. Additionally, liquid chromatography and MRM-MS was used to determine rRNA N-glycosidase activity for each cultivar and the overall activity in these cultivars was compared to a purified ricin standard. Of the cultivars studied, the average ricin content was 9.3 mg/g seed, the average RCA content was 9.9 mg/g seed, and the enzymatic activity agreed with the activity of a purified ricin reference within 35% relative activity.


Assuntos
Semente de Rícino/química , Semente de Rícino/enzimologia , Lectinas de Plantas/química , Ricina/química , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Glicosídeo Hidrolases/metabolismo , Imunoensaio , Extratos Vegetais/química , Sementes/química , Espectrometria de Massas em Tandem
17.
Artigo em Inglês | MEDLINE | ID: mdl-22721710

RESUMO

Atrazine [6-chloro-N-ethyl-N'-(1-methylethyl)-1,3,5-triazine-2,4-diamine] is the most widely used herbicide in the United States. In recent years, there has been controversy about atrazine's potential endocrine/reproductive and neurological adverse effects in wildlife and humans. The controversy triggered several environmental and epidemiologic studies, and it generated needs for sensitive and selective analytical methods for the quantification of atrazine, atrazine metabolites, and degradation or hydrolysis products. We developed a two-dimensional high performance liquid chromatography (2D-HPLC) method with isotope dilution tandem mass spectrometry detection to measure atrazine in urine, along with 11 atrazine metabolites and hydrolysis products, including 6-chloro (Cl), 6-mercapto (Mer) and 6-hydroxy (OH) derivatives, and their desethyl, desisopropyl and diamino atrazine analogs (DEA, DIA and DAA, respectively). The 2D-HPLC system incorporated strong cation exchange and reversed phase separation modes. This versatile approach can be used for the quantitative determination of all 12 compounds in experimental animals for toxicological studies. The method requires only 10 µL of urine, and the limits of detection (LODs) range from 10 to 50 µg/L. The method can also be applied to assess atrazine exposure in occupational settings by measurement of 6-Cl and 6-Mer analogs, which requires only 100 µL of urine with LODs of 1-5 µg/L. Finally, in combination with automated off-line solid phase extraction before 2D-HPLC, the method can also be applied in non-occupational environmental exposure studies for the determination of -6-Cl and 6-Mer metabolites, using 500 µL of urine and LODs of 0.1-0.5 µg/L.


Assuntos
Atrazina/urina , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas em Tandem/métodos , Atrazina/química , Atrazina/metabolismo , Estabilidade de Medicamentos , Humanos , Limite de Detecção , Reprodutibilidade dos Testes , Extração em Fase Sólida
18.
J Virol ; 86(2): 718-25, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22072766

RESUMO

A vaginal gel containing 1% tenofovir (TFV) was found to be safe and effective in reducing HIV infection in women when used pericoitally. Because of the long intracellular half-life of TFV and high drug exposure in vaginal tissues, we hypothesized that a vaginal gel containing TFV may provide long-lasting protection. Here, we performed delayed-challenge experiments and showed that vaginal 1% TFV gel protected 4/6 macaques against vaginal simian-human immunodeficiency virus (SHIV) exposures occurring 3 days after gel application, demonstrating long-lasting protection. Despite continued gel dosing postinfection, neither breakthrough infection had evidence of drug resistance by ultrasensitive testing of SHIV in plasma and vaginal lavage. Analysis of the active intracellular tenofovir diphosphate (TFV-DP) in vaginal lymphocytes collected 4 h to 3 days after gel dosing persistently showed high TFV-DP levels (median, 1,810 fmol/10(6) cells) between 4 and 24 h that exceed the 95% inhibitory concentration (IC(95)), reflecting rapid accumulation and long persistence. In contrast to those in peripheral blood mononuclear cells (PBMCs) following oral dosing, TFV-DP levels in vaginal lymphocytes decreased approximately 7-fold by 3 days, exhibiting a much higher rate of decay. We observed a strong correlation between intracellular TFV-DP in vaginal lymphocytes, in vitro antiviral activity, and in vivo protection, suggesting that TFV-DP above the in vitro IC(95) in vaginal lymphocytes is a good predictor of high efficacy. Data from this model reveal an extended window of protection by TFV gel that supports coitus-independent use. The identification of protective TFV-DP concentrations in vaginal lymphocytes may facilitate the evaluation of improved delivery methods of topical TFV and inform clinical studies.


Assuntos
Adenina/análogos & derivados , Fármacos Anti-HIV/administração & dosagem , Infecções por HIV/prevenção & controle , HIV-1/efeitos dos fármacos , Organofosfonatos/administração & dosagem , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Vagina/efeitos dos fármacos , Cremes, Espumas e Géis Vaginais/administração & dosagem , Adenina/administração & dosagem , Adenina/química , Animais , Fármacos Anti-HIV/química , Células Cultivadas , Estabilidade de Medicamentos , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/fisiologia , Meia-Vida , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Estudos Longitudinais , Organofosfonatos/química , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/fisiologia , Tenofovir , Vagina/imunologia , Vagina/virologia , Cremes, Espumas e Géis Vaginais/química
19.
J Sep Sci ; 34(24): 3606-18, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22162441

RESUMO

Three online solid phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) method examples are presented where two different types of chromatographic columns or solvent systems were coupled to meet specific analytical objectives: (i) SPE of target analytes by restricted access media from high ionic strength urine matrix was coupled with reversed phase LC-MS/MS conditions accommodating high ionization potentials of the analytes (urinary bisphenol A and other phenolic derivatives); (ii) strong cation exchange SPE of analytes of diverse polarity and pK(a) was coupled with reversed phase LC-MS/MS analysis (urinary atrazine metabolites); (iii) pre-concentration of low pg per sample analytes by weak anion exchange SPE was hyphenated with ion pair LC-MS analysis (intracellular nucleotide triphosphate analogs). With these examples we suggest a conductive generic work flow for the development of online SPE-LC-MS methods and show how advanced commercial LC devices and software allow for the design of complex yet highly versatile analytical separation systems suited to the unique physicochemical properties of the target analytes.


Assuntos
Compostos Orgânicos/análise , Extração em Fase Sólida , Cromatografia Líquida , Software , Espectrometria de Massas em Tandem
20.
Environ Sci Technol ; 45(19): 8037-45, 2011 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-21469664

RESUMO

Since 2002, practices in manufacturing polyfluoroalkyl chemicals (PFCs) in the United States have changed. Previous results from the National Health and Nutrition Examination Survey (NHANES) documented a significant decrease in serum concentrations of some PFCs during 1999-2004. To further assess concentration trends of perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA), perfluorohexane sulfonate (PFHxS), and perfluorononanoate (PFNA), we analyzed 7876 serum samples collected from a representative sample of the general U.S. population ≥12 years of age during NHANES 1999-2008. We detected PFOS, PFOA, PFNA, and PFHxS in more than 95% of participants. Concentrations differed by sex regardless of age and we observed some differences by race/ethnicity. Since 1999-2000, PFOS concentrations showed a significant downward trend, because of discontinuing industrial production of PFOS, but PFNA concentrations showed a significant upward trend. PFOA concentrations during 1999-2000 were significantly higher than during any other time period examined, but PFOA concentrations have remained essentially unchanged during 2003-2008. PFHxS concentrations showed a downward trend from 1999 to 2006, but concentrations increased during 2007-2008. Additional research is needed to identify the environmental sources contributing to human exposure to PFCs. Nonetheless, these NHANES data suggest that sociodemographic factors may influence exposure and also provide unique information on temporal trends of exposure.


Assuntos
Exposição Ambiental/estatística & dados numéricos , Fluorcarbonetos/sangue , Inquéritos Epidemiológicos/estatística & dados numéricos , Adolescente , Adulto , Distribuição por Idade , Idoso , Caprilatos/sangue , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Inquéritos Nutricionais , Ácidos Sulfônicos/sangue , Fatores de Tempo , Estados Unidos , Adulto Jovem
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