RESUMO
Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease in which airway macrophages (AMs) play a key role. Itaconate has emerged as a mediator of macrophage function, but its role during fibrosis is unknown. Here, we reveal that itaconate is an endogenous antifibrotic factor in the lung. Itaconate levels are reduced in bronchoalveolar lavage, and itaconate-synthesizing cis-aconitate decarboxylase expression (ACOD1) is reduced in AMs from patients with IPF compared with controls. In the murine bleomycin model of pulmonary fibrosis, Acod1−/− mice develop persistent fibrosis, unlike wild-type (WT) littermates. Profibrotic gene expression is increased in Acod1−/− tissue-resident AMs compared with WT, and adoptive transfer of WT monocyte-recruited AMs rescued mice from disease phenotype. Culture of lung fibroblasts with itaconate decreased proliferation and wound healing capacity, and inhaled itaconate was protective in mice in vivo. Collectively, these data identify itaconate as critical for controlling the severity of lung fibrosis, and targeting this pathway may be a viable therapeutic strategy.
Assuntos
Carboxiliases/metabolismo , Fibrose Pulmonar Idiopática/imunologia , Macrófagos Alveolares/imunologia , Succinatos/metabolismo , Administração por Inalação , Transferência Adotiva/métodos , Adulto , Idoso , Animais , Bleomicina/administração & dosagem , Bleomicina/toxicidade , Líquido da Lavagem Broncoalveolar/imunologia , Broncoscopia , Estudos de Casos e Controles , Células Cultivadas , Modelos Animais de Doenças , Feminino , Fibroblastos , Voluntários Saudáveis , Humanos , Hidroliases/genética , Hidroliases/metabolismo , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/diagnóstico , Fibrose Pulmonar Idiopática/terapia , Pulmão/citologia , Pulmão/imunologia , Pulmão/patologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/transplante , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Cultura Primária de Células , Índice de Gravidade de Doença , Succinatos/administração & dosagem , Succinatos/imunologiaRESUMO
BACKGROUND: Persimmon fruits have been widely used in traditional medicine owing to their phenolic composition. This research aims to perform a rapid, detailed and affordable study of the profile of low-molecular-weight phenols from persimmon pulp. RESULTS: Two different HPLC-DAD/ESI-MS(n) analyses were performed using a routine three-dimensional ion trap mass spectrometer to analyze the ethanolic extract of persimmon pulp: (1) an untargeted data-dependent analysis to identify the majority of small phenols that included full MS and MS(2) scan events; (2) a targeted data-dependent analysis to identify polymerized phenols (dimers and formic acid adducts) through a source-induced dissociation analysis that included full MS and MS(2) scan events. Thirty-two low-molecular-weight phenols were detected, comprising gallic acid and its glycoside and acyl derivatives, glycosides of p-coumaric, vanillic and cinnamic acids and different flavone di-C-hexosides, most of them reported for the first time in persimmon. CONCLUSION: The use of a straightforward and affordable methodology of analysis led to obtain an up-to-date profiling of low-molecular-weight phenols in persimmon. The results can help future actions aimed to expand the understanding of the phenolic metabolome of persimmon cultivars.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Diospyros/química , Frutas/química , Fenóis/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Ácido Gálico/análogos & derivados , Ácido Gálico/análise , Peso Molecular , Fenóis/química , Extratos Vegetais/químicaRESUMO
An analytical methodology has been developed for extracting recurrent unidentified spectra (RUS) from large GC/MS data sets. Spectra were first extracted from original data files by the Automated Mass Spectral Deconvolution and Identification System (AMDIS; Stein, S. E. J. Am. Soc. Mass Spectrom. 1999 , 10 , 770 - 781 ) using settings designed to minimize spurious spectra, followed by searching the NIST library with all unidentified spectra. The spectra that could not be identified were then filtered to remove poorly deconvoluted data and clustered. The results were assumed to be unidentified components. This was tested by requiring each unidentified spectrum to be found in two chromatographic columns with slightly different stationary phases. This methodology has been applied to a large set of pediatric urine samples. A library of spectra and retention indices for derivatized urine components, both identified and recurrent unidentified, has been created and is available for download.
Assuntos
Cromatografia Gasosa-Espectrometria de Massas , Bibliotecas de Moléculas Pequenas/química , Ácido Cítrico/química , Humanos , Urina/químicaRESUMO
Although chemical derivatization for signal enhancement in drug testing is most often associated with gas chromatography, it also has the potential to improve the detection of analytes poorly ionized by atmospheric pressure ionization techniques, such as electrospray ionization used in liquid chromatography-mass spectrometry. A number of acidic compounds, namely drug glucuronides (e.g. conjugates of temazepam, oxazepam, lorazepam, morphine, testosterone, epitestosterone, 5-α-dihydrotestosterone, androsterone, p-nitrophenol, and paracetamol) were successfully derivatized with tris(trimethoxyphenyl) phosphoniumpropylamine to introduce a quaternary cation functionality to the analytes. Benzodiazepine glucuronides were more specifically investigated, and following positive mode electrospray ionization mass spectrometry, average improvements to peak areas as a result of derivatization were 67-, 6-, and 7- fold for temazepam, oxazepam, and lorazepam glucuronides. Average improvements to the signal-to-noise ratios for temazepam, oxazepam, and lorazepam glucuronides were 1336-, 371- and 217-fold, respectively. The values obtained for the derivatized conjugate were also typically higher than those for the underivatized parent drug. Urine containing benzodiazepine glucuronides was also successfully derivatized. The data indicates potential for the use of charge derivatization to improve the detection of molecules with acidic functionalities by liquid chromatography-mass spectrometry (LC-MS) techniques in certain scenarios.
Assuntos
Ciências Forenses/métodos , Glucuronídeos/química , Compostos Organofosforados/química , Preparações Farmacêuticas/química , Preparações Farmacêuticas/urina , Propilaminas/química , Benzodiazepinas/química , Benzodiazepinas/metabolismo , Benzodiazepinas/urina , Cromatografia Líquida , Glucuronídeos/urina , Humanos , Preparações Farmacêuticas/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
The commercial cultivation of genetically engineered (GE) crops in Europe has met with considerable consumer resistance, which has led to vigorous safety assessments including the measurement of substantial equivalence between the GE and parent lines. This necessitates the identification and quantification of significant changes to the metabolome and proteome in the GE crop. In this study, the quantitative proteomic analysis of tomato fruit from lines that have been transformed with the carotenogenic gene phytoene synthase-1 (Psy-1), in the sense and antisense orientations, in comparison with a non-transformed, parental line is described. Multidimensional protein identification technology (MudPIT), with tandem mass spectrometry, has been used to identify proteins, while quantification has been carried out with isobaric tags for relative and absolute quantification (iTRAQ). Fruit from the GE plants showed significant alterations to their proteomes compared with the parental line, especially those from the Psy-1 sense transformants. These results demonstrate that MudPIT and iTRAQ are suitable techniques for the verification of substantial equivalence of the proteome in GE crops.
Assuntos
Alquil e Aril Transferases/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteoma/metabolismo , Solanum lycopersicum/metabolismo , Transformação Genética , Alquil e Aril Transferases/metabolismo , Frutas/genética , Frutas/metabolismo , Geranil-Geranildifosfato Geranil-Geraniltransferase , Solanum lycopersicum/enzimologia , Solanum lycopersicum/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Proteoma/genéticaRESUMO
Hepcidin is a peptide hormone that functions as a key regulator of mammalian iron metabolism. Biological levels are increased in end-stage renal disease and during inflammation but suppressed in hemochromatosis. Thus hepcidin levels have diagnostic importance. This study describes the development of an analytical method for the quantitative determination of the concentration of hepcidin in clinical samples. The fragmentation of hepcidin was investigated using triple quadrupole and linear ion trap mass spectrometers. A standard quantity of a stable isotopically labelled hepcidin internal standard was added to serum samples. Extraction was performed by protein precipitation and weak cation-exchange magnetic nanoparticles. Chromatography was carried out on sub 2 microm particle stationary phase, using ultra-high-pressure liquid chromatography and a linear ion trap for quantitation. The lower limit of quantitation was 0.4 nmol/L with less than 20% accuracy and precision. The mean hepcidin concentration in sera for controls was 4.6 +/- 2.7 nmol/L, in patients with sickle cell disease, 7.0 +/- 8.9 nmol/L; in patients with end-stage renal disease, 30.5 +/- 15.7 nmol/L; and patients with penetrant hereditary hemochromatosis, 1.4 +/- 0.8 nmol/L.
Assuntos
Peptídeos Catiônicos Antimicrobianos/sangue , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Adulto , Idoso , Anemia Falciforme/sangue , Anemia Falciforme/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacocinética , Estudos de Coortes , Estabilidade de Medicamentos , Feminino , Hemocromatose/sangue , Hemocromatose/metabolismo , Hepcidinas , Humanos , Falência Renal Crônica/sangue , Falência Renal Crônica/metabolismo , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A strategy to target functionalized quantum dot-liposome (f-QD-L) hybrid vesicles in the solid tumor tissue of tumor-bearing mice is explored. Functionalized polyethylene glycol (PEG)-lipid coated QD (f-QD) were encapsulated into the aqueous core of 100 nm cationic (DOPC:Chol: DOTAP); sterically stabilized, fluid-phase (DOPC:Chol:DSPE-PEG2000); and sterically stabilized, gel-phase (DSPC:Chol:DSPE-PEG2000) liposome vesicles. Double tracking of f-QD-L in blood was performed at different time points after intravenous administration in B16F10 melanoma tumor-bearing C57BL6 mice. Cholesteryl [-1-14C] oleate lipids probed the vesicle membrane were followed by liquid scintillation counting while QD were determined independently by elemental (Cd2+) analysis using inductively coupled plasma mass spectrometry (ICP-MS). Rapid blood clearance was observed following intravenous administration of the cationic hybrid vesicles, while incorporation of PEG at the surface of zwitterionic vesicles dramatically prolonged their blood circulation half-life after systemic administration. The "rigid" PEGylated f-QD-L (DSPC:Chol:DSPE-PEG2000) hybrid vesicles led to rapid tumor accumulation of peak values (approximately 5% of injected dose per gram tissue) of QD compared to long-circulating f-QD that accumulated in the tumor tissue at longer time points. More interestingly, this hybrid vesicle tumor retention persisted for at least 24 h. For almost all types of systems, a preferential cadmium uptake by liver and spleen was obtained. Overall, f-QD-L hybrid vesicles offer great potential for tumor imaging applications due to their rapid accumulation and prolonged retention within the tumor. Furthermore, f-QD-L offer many opportunities for the development of combinatory therapeutic and imaging (theranostic) modalities by incorporating both drug molecules and QD within the different compartments of a single vesicle.
Assuntos
Antineoplásicos/administração & dosagem , Portadores de Fármacos/farmacocinética , Lipídeos/química , Melanoma Experimental/tratamento farmacológico , Animais , Lipossomos , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas , Polietilenoglicóis , Pontos Quânticos , Distribuição TecidualRESUMO
The present work describes the pharmacokinetics of recently developed liposome-quantum dot (L-QD) hybrid vesicles in nude mice following systemic administration. Hydrophobic QD were incorporated into different bilayer compositions, and the serum stability of such hybrid vesicles was evaluated using turbidity and carboxyfluorescein release measurements. L-QD hybrid blood profile and organ biodistribution were also determined by elemental (cadmium) analysis. Following intravenous administration, different tissue biodistribution profiles and tissue affinities were observed depending on the L-QD lipid bilayer characteristics. Immediate blood clearance was observed with cationic (DOTAP/DOPE/Chol) hybrid with rapid lung accumulation, while incorporation of PEG at the surface of zwitterionic vesicles dramatically prolonged their blood circulation half-life after systemic administration. Overall, the L-QD hybrid vesicle system is considered a viable platform that allows QD delivery to different tissues through facile modulation of the hybrid vesicle characteristics. In addition, L-QD offers many opportunities for the development of combinatory therapeutic and imaging (theranostic) modalities by incorporating both drug molecules and QD within the different compartments of a single vesicle.
Assuntos
Lipídeos/farmacocinética , Pontos Quânticos , Animais , Meia-Vida , Injeções Intravenosas , Lipídeos/administração & dosagem , Lipídeos/sangue , Lipossomos/química , Lipossomos/farmacocinética , Camundongos , Distribuição TecidualRESUMO
In vitro biosynthesis using pooled human liver microsomes was applied to help identify in vivo metabolites of ketamine by liquid chromatography (LC)-tandem mass spectrometry. Microsomal synthesis produced dehydronorketamine, seven structural isomers of hydroxynorketamine, and at least five structural isomers of hydroxyketamine. To aid identification, stable isotopes of the metabolites were also produced from tetra-deuterated isotopes of ketamine or norketamine as substrates. Five metabolites (three hydroxynorketamine and two hydroxyketamine isomers) gave chromatographically resolved components with product ion spectra indicating the presence of a phenolic group, with phenolic metabolites being further substantiated by selective liquid-liquid extraction after adjustments to the pH. Two glucuronide conjugates of hydroxynorketamine were also identified. Analysis by LC-coupled ion cyclotron resonance mass spectrometry gave unique masses in accordance with the predicted elemental composition. The metabolites, including the phenols, were subsequently confirmed to be present in urine of subjects after oral ketamine administration, as facilitated by the addition of deuterated metabolites generated from the in vitro biosynthesis. To our knowledge, phenolic metabolites of ketamine, including an intact glucuronide conjugate, are here reported for the first time. The use of biologically synthesized deuterated material as an internal chromatographic and mass spectrometric marker is a viable approach to aid in the identification of metabolites. Metabolites that have particular diagnostic value can be selected as candidates for chemical synthesis of standards.
Assuntos
Anestésicos Dissociativos/farmacocinética , Ketamina/farmacocinética , Metabolômica/métodos , Microssomos Hepáticos/enzimologia , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Administração Oral , Anestésicos Dissociativos/administração & dosagem , Anestésicos Dissociativos/química , Anestésicos Dissociativos/urina , Biotransformação , Cromatografia Líquida , Estado de Consciência/efeitos dos fármacos , Ciclotrons , Deutério , Feminino , Análise de Fourier , Glucuronídeos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Isomerismo , Ketamina/administração & dosagem , Ketamina/análogos & derivados , Ketamina/química , Ketamina/metabolismo , Ketamina/urina , Masculino , Estrutura Molecular , Fenóis/metabolismo , Reprodutibilidade dos Testes , Detecção do Abuso de SubstânciasRESUMO
Hepcidin is known to be a key systemic iron-regulatory hormone which has been demonstrated to be associated with a number of iron disorders. Hepcidin concentrations are increased in inflammation and suppressed in hemochromatosis. In view of the role of hepcidin in disease, its potential as a diagnostic tool in a clinical setting is evident. This study describes the development of a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) assay for the quantitative determination of hepcidin concentrations in clinical samples. A stable isotope labeled hepcidin was prepared as an internal standard and a standard quantity was added to urine samples. Extraction was performed with weak cation-exchange magnetic nanoparticles. The basic peptides were eluted from the magnetic nanoparticles using a matrix solution directly onto a target plate and analyzed by MALDI-TOF MS to determine the concentration of hepcidin. The assay was validated in charcoal stripped urine, and good recovery (70-80%) was obtained, as were limit of quantitation data (5 nmol/L), accuracy (RE <10%), precision (CV <21%), within -day repeatability (CV <13%) and between-day repeatability (CV <21%). Urine hepcidin levels were 10 nmol/mmol creatinine in healthy controls, with reduced levels in hereditary hemochromatosis (P < 0.000005) and elevated levels in inflammation (P < 0.0007). In summary a validated method has been developed for the determination of hepcidin concentrations in clinical samples.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Adulto , Peptídeos Catiônicos Antimicrobianos/urina , Feminino , Hepcidinas , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
The introduction of genetically modified (GM) crops into the market has raised a general alertness relating to the control and safety of foods. The applicability of protein separation hyphenated to mass spectrometry to identify the bacterial enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) protein expressed in GM crops has been previously reported [M.F. Ocana, P.D. Fraser, R.K.P. Patel, J.M. Halket, P.M. Bramley, Rapid Commun. Mass Spectrom. 21 (2007) 319.]. Herein, we investigate the suitability of two strategies that employ heavy stable isotopes, i.e. AQUA and iTRAQ, to quantify different levels of CP4 EPSPS in up to four GM preparations. Both quantification strategies showed potential to determine whether the presence of GM material is above the limits established by the European Union. The AQUA quantification procedure involved protein solubilisation/fractionation and subsequent separation using SDS-PAGE. A segment of the gel in which the protein of interest was located was excised, the stable isotope labeled peptide added at a known concentration and proteolytic digestion initiated. Following recovery of the peptides, on-line separation and detection using LC-MS was carried out. A similar approach was used for the iTRAQ workflow with the exception that proteins were digested in solution and generated tryptic peptides were chemically tagged. Both procedures demonstrated the potential for quantitative detection at 0.5% (w/w) GM soya which is a level below the current European Union's threshold for food-labelling. In this context, a comparison between the two procedures is provided within the present study.
Assuntos
3-Fosfoshikimato 1-Carboxiviniltransferase/análise , Glycine max/enzimologia , Marcação por Isótopo/métodos , Plantas Geneticamente Modificadas/enzimologia , Sementes/enzimologia , Sementes/genética , Glycine max/genéticaRESUMO
Hepcidin is a peptide hormone that functions as a key regulator of mammalian iron metabolism. Serum and urine levels are increased in inflammation and suppressed in hemochromatosis, and they may have diagnostic importance. This study describes the development and validation of an analytical method for the quantitative determination of the concentration of hepcidin in clinical samples. A stable, isotopically labeled internal standard, [15N,13C2]Gly12,20-hepcidin, was synthesized and a standard quantity was added to urine samples. Extraction was performed using weak cation exchange magnetic nanoparticles. An ion trap mass spectrometer was used to quantify hepcidin in the samples. The hepcidin assay was validated, and good recovery of hepcidin was obtained. The assay is accurate and precise. Urinary hepcidin levels of 3 to 9 nmol/mmol creatinine(-1) were found in healthy controls, with reduced levels in hemochromatosis (P<0.00006) and elevated levels in inflammation (P<0.00035). In sickle cell disease, a wide range was found, with the mean value not differing significantly from controls (P<0.26). In summary, a validated method has been developed for the quantitation of hepcidin using a stable, isotopically labeled internal standard and applied to determine the concentrations of hepcidin in the low nanomolar range in urine samples from patients and controls.
Assuntos
Antibacterianos/urina , Peptídeos Catiônicos Antimicrobianos/urina , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Adulto , Calibragem , Feminino , Hepcidinas , Humanos , Marcação por Isótopo , Masculino , Pessoa de Meia-IdadeRESUMO
Salicylic acid (SA), which is central to defense mechanisms in plants and the principal metabolite of aspirin, occurs naturally in man with higher levels of SA and its urinary metabolite salicyluric acid (SU) in vegetarians overlapping with levels in patients on low-dose aspirin regimens. SA is widely distributed in animal blood. Fasting for major colorectal surgery did not cause disappearance of SA from plasma, even in patients following total proctocolectomy. A (13)C(6) benzoic acid load ingested by six volunteers led, between 8 and 16 h, to a median 33.9% labeling of urinary salicyluric acid. The overall contribution of benzoic acid (and its salts) to the turnover of circulating SA thus requires further assessment. However, that SA appears to be, at least partially, an endogenous compound should lead to reassessment of its role in human (and animal) pathophysiology.
Assuntos
Ácido Benzoico/metabolismo , Jejum/sangue , Jejum/urina , Ácido Salicílico/sangue , Ácido Salicílico/urina , Adulto , Animais , Aspirina , Ácido Benzoico/administração & dosagem , Feminino , Hipuratos/sangue , Hipuratos/urina , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Current analytical methods used for screening drugs and their metabolites in biological samples from victims of drug-facilitated sexual assault (DFSA) or other vulnerable groups can lack sufficient sensitivity. The application of liquid chromatography, employing small particle sizes, with tandem mass spectrometry (MS/MS) is likely to offer the sensitivity required for detecting candidate drugs and/or their metabolites in urine, as demonstrated here for ketamine. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) was performed following extraction of urine (4 mL) using mixed-mode (cation and C8) solid-phase cartridges. Only 20 microL of the 250 microL extract was injected, leaving sufficient volume for other assays important in DFSA cases. Three ion transitions were chosen for confirmatory purposes. As ketamine and norketamine (including their stable isotopes) are available as reference standards, the assay was additionally validated for quantification purposes to study elimination of the drug and primary metabolite following a small oral dose of ketamine (50 mg) in 6 volunteers. Dehydronorketamine, a secondary metabolite, was also analyzed qualitatively to determine whether monitoring could improve retrospective detection of administration. The detection limit for ketamine and norketamine was 0.03 ng/mL and 0.05 ng/mL, respectively, and these compounds could be confirmed in urine for up to 5 and 6 days, respectively. Dehydronorketamine was confirmed up to 10 days, providing a very broad window of detection.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Ketamina/metabolismo , Ketamina/urina , Espectrometria de Massas em Tandem/métodos , Adulto , Feminino , Humanos , Ketamina/análogos & derivados , MasculinoRESUMO
BACKGROUND: The leaves of the khat plant (Catha edulis) are chewed for their pleasurable effects. Chewing releases cathinone which may decrease appetite through an unknown mechanism. Levels of the peptide ghrelin increase with hunger and decrease immediately post-prandially, while peptide YY is released following a meal. We hypothesised that the anorexigenic effects of khat may be mediated through changes in these hormones. MATERIALS AND METHODS: Six habitual khat chewers attended on two separate occasions. For a period of 3h they chewed either khat leaves or lettuce. Blood pressure (BP) and pulse rate (PR) were monitored throughout, as were subjective assessments of hunger and fullness. Plasma samples were analysed for cathinone, ghrelin and PYY levels. RESULTS: Chewing khat significantly decreased subjective feelings of hunger and increased fullness (p<0.05) but had no effect on ghrelin and PYY levels. Khat led to an increase in cathinone levels as well as an increase in BP and PR. Cathinone levels correlated positively with fullness and pulse rate and negatively with hunger. CONCLUSIONS: Chewing khat decreases subjective feelings of hunger and increases systemic sympathetic tone, but has no effect on ghrelin and PYY levels. We conclude that the anorexigenic effect of khat may be secondary to central mechanisms mediated via cathinone.
Assuntos
Depressores do Apetite/farmacologia , Apetite/efeitos dos fármacos , Catha/química , Grelina/metabolismo , Peptídeo YY/metabolismo , Extratos Vegetais/farmacologia , Adulto , Alcaloides/sangue , Apetite/fisiologia , Área Sob a Curva , Pressão Sanguínea/efeitos dos fármacos , Estudos Cross-Over , Frequência Cardíaca/efeitos dos fármacos , Humanos , Masculino , Mastigação/fisiologia , Pessoa de Meia-Idade , Folhas de Planta/química , Período Pós-PrandialRESUMO
In tomato (Solanum lycopersicum), phytoene synthase-1 (PSY-1) is the key biosynthetic enzyme responsible for the synthesis of fruit carotenoids. To further our understanding of carotenoid formation in tomato fruit, we characterized the effect of constitutive expression of an additional tomato Psy-1 gene product. A quantitative data set defining levels of carotenoid/isoprenoid gene expression, enzyme activities, and metabolites was generated from fruit that showed the greatest perturbation in carotenoid content. Transcriptional upregulation, resulting in increased enzyme activities and metabolites, occurred only in the case of Psy-1, Psy-2, and lycopene cyclase B. For reactions involving 1-deoxy-d-xylulose5-phosphate synthase, geranylgeranyl diphosphate synthase, phytoene desaturase, zeta-carotene desaturase, carotene isomerase, and lycopene beta-cyclase, there were no correlations between gene expression, enzyme activities, and metabolites. Perturbations in carotenoid composition were associated with changes in plastid type and with chromoplast-like structures arising prematurely during fruit development. The levels of >120 known metabolites were determined. Comparison with the wild type illustrated that key metabolites (sucrose, glucose/fructose, and Glu) and sectors of intermediary metabolism (e.g., tricarboxylic [corrected] acid cycle intermediates and fatty acids) in the Psy-1 transgenic mature green fruit resembled changes in metabolism associated with fruit ripening. General fruit developmental and ripening properties, such as ethylene production and fruit firmness, were unaffected. Therefore, it appears that the changes to pigmentation, plastid type, and metabolism associated with Psy-1 overexpression are not connected with the ripening process.
Assuntos
Carotenoides/metabolismo , Frutas/metabolismo , Plastídeos/metabolismo , Solanum lycopersicum/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Frutas/genética , Frutas/crescimento & desenvolvimento , Cromatografia Gasosa-Espectrometria de Massas , Regulação da Expressão Gênica de Plantas , Geranil-Geranildifosfato Geranil-Geraniltransferase , Germinação/genética , Liases Intramoleculares/genética , Liases Intramoleculares/metabolismo , Solanum lycopersicum/genética , Solanum lycopersicum/crescimento & desenvolvimento , Modelos Biológicos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Plastídeos/genética , Reação em Cadeia da Polimerase , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , beta Caroteno/metabolismoRESUMO
The potential of protein fractionation hyphenated to mass spectrometry (MS) to detect and characterize the transgenic protein present in Roundup Ready soya and maize has been investigated. Genetically modified (GM) soya and maize contain the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Agrobacterium tumefaciens CP4, which confers resistance to the herbicide glyphosate. The GM soya and maize proteomes were fractionated by gel filtration, anion-exchange chromatography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) prior to MS. This facilitated detection of a tryptic peptide map of CP4 EPSPS by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and nanoelectrospray ionization quadrupole time-of-flight (nanoESI-QTOF) MS. Subsequently, sequence information from the CP4 EPSPS tryptic peptides was obtained by nanoESI-QTOF MS/MS. The identification was accomplished in 0.9% GM soya seeds, which is the current EU threshold for food-labeling requirements.
Assuntos
3-Fosfoshikimato 1-Carboxiviniltransferase/química , 3-Fosfoshikimato 1-Carboxiviniltransferase/genética , Glycine max/fisiologia , Mapeamento de Peptídeos/métodos , Plantas Geneticamente Modificadas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Zea mays/fisiologia , 3-Fosfoshikimato 1-Carboxiviniltransferase/análise , Sequência de Aminoácidos , Análise de Alimentos/métodos , Rotulagem de Alimentos/métodos , Marcadores Genéticos/genética , Dados de Sequência Molecular , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
This is the first of two reviews devoted to derivatization approaches for "soft" ionization mass spectrometry (FAB, MALDI, ESI, APCI) and deals, in particular, with small molecules. The principles of the main "soft" ionization mass spectrometric methods as well as the reasons for derivatizing small molecules are briefly described. Derivatization methods for modification of amines, carboxylic acids, amino acids, alcohols, carbonyl compounds, monosaccharides, thiols, unsaturated and aromatic compounds etc. to improve their ionizability and to enhance structure information content are discussed. The use of "fixed"-charge bearing derivatization reagents is especially emphasized. Chemical aspects of derivatization and "soft" ionization mass spectrometric properties of derivatives are considered.
Assuntos
Espectrometria de Massas/métodos , Aminas/química , Aminoácidos/química , Anisóis/química , Derivados de Benzeno/química , Brometos/química , Carboidratos/química , Indicadores e Reagentes/química , Isocianatos/química , Fenóis/química , Fosfinas/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas de Bombardeamento Rápido de Átomos/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massa de Íon Secundário/métodos , Compostos de Sulfidrila/química , Espectrometria de Massas em Tandem/métodosRESUMO
The review describes on-line derivatization/degradation methods employed in mass spectrometry to solve some structural and analytical problems. Advantages and applications of various positions of reaction systems connected mainly to a mass spectrometer or a gas chromatograph/mass spectrometer are considered. Among these are reaction systems connected directly to the mass spectrometer (reaction mass spectrometry, pyrolysis-mass spectrometry or direct pyrolysis-mass spectrometry); flash-heaters as reactors in gas chromatography/mass spectrometry (GC/MS); in-line chemical reactors located before the chromatographic column [pre-column derivatization/degradation with the use of catalytic reactions, pyrolysis (pyrolysis-GC/MS), degradation in elemental analyzers-isotope ratio mass pectrometry (EA-IRMS)]; on-column derivatization and deuteration; reactor located between the chromatographic column and a mass spectrometer [post-column catalytic derivatization, gas chromatograph-combustion-isotope ratio mass spectrometer (GC-c-IRMS)]. Post-column derivatization in high performance liquid chromatography/mass spectro-metry is briefly mentioned. Application of such on-line methodology to structure elucidation of low molecular mass compounds and polymers, to the determination of isotope ratios of the most common elements, to the investigation of catalytic reactions is discussed..
RESUMO
The review describes chemical transformations of multifunctional compounds (amino acids and peptides, amino alcohols, amino thiols, hydroxy acids, oxo acids, oxo alcohols, compounds containing simultaneously three or more different groups etc.) by using step-wise or one-step modification or protection of functional groups. Some chemical aspects of mixed derivatization performed for improving the physical-chemical properties and mass spectral characteristics are discussed. Application of mixed derivatization to qualitative and quantitative analysis of various multifunctional compounds mainly in biological fluids and other matrices by gas chromatography/mass spectrometry in electron ionization, chemical ionization, negative-ion chemical ionization and selected ion monitoring modes is considered.