RESUMO
A chiral analytical method was proposed based on capillary electrophoresis with laser-induced fluorescence detection coupled with microwave-assisted derivatization for the simultaneous baseline separation and sensitive detection of four stereoisomers of 3-hydroxyaspartate. The derivatization reaction of 3-hydroxyaspartate with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole was greatly accelerated by microwave irradiation. Under the optimized conditions, the derivatization yield was increased by 20% and the derivatization time was shortened by 20 min when compared with those from conventional water bath heating. In addition, the sensitivity was improved by online sample concentration methods. The detection limit of l-threo-3-hydroxyaspartate obtained by large-volume sample stacking with polarity switching was 5.3 nmol/L, which was around 1000-fold lower than that of the capillary electrophoresis/laser-induced fluorescence without stacking. The excellent analytical performance in terms of linearity and precision was also achieved. Furthermore, the developed method was successfully applied to the determination of 3-hydroxyaspartate in the spiked urine, and satisfactory recoveries were obtained ranging from 90.5 to 107.0%.
Assuntos
Ácido Aspártico/análogos & derivados , Eletroforese Capilar/métodos , Espectrometria de Fluorescência/métodos , Ácido Aspártico/análise , Ácido Aspártico/química , Ácido Aspártico/isolamento & purificação , Fracionamento Químico , Modelos Lineares , Micro-Ondas , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeAssuntos
Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Antibacterianos/isolamento & purificação , Ácido Aspártico/análogos & derivados , Ácido Aspártico/isolamento & purificação , Ácido Aspártico/farmacologia , Aspergillus/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Micotoxinas/isolamento & purificação , Micotoxinas/farmacologia , Microbiologia do SoloRESUMO
We present sequential CE analysis of amino acids and L-asparaginase-catalyzed enzyme reaction, by combing the on-line derivatization, optically gated (OG) injection and commercial-available UV-Vis detection. Various experimental conditions for sequential OG-UV/vis CE analysis were investigated and optimized by analyzing a standard mixture of amino acids. High reproducibility of the sequential CE analysis was demonstrated with RSD values (n = 20) of 2.23, 2.57, and 0.70% for peak heights, peak areas, and migration times, respectively, and the LOD of 5.0 µM (for asparagine) and 2.0 µM (for aspartic acid) were obtained. With the application of the OG-UV/vis CE analysis, sequential online CE enzyme assay of L-asparaginase-catalyzed enzyme reaction was carried out by automatically and continuously monitoring the substrate consumption and the product formation every 12 s from the beginning to the end of the reaction. The Michaelis constants for the reaction were obtained and were found to be in good agreement with the results of traditional off-line enzyme assays. The study demonstrated the feasibility and reliability of integrating the OG injection with UV/vis detection for sequential online CE analysis, which could be of potential value for online monitoring various chemical reaction and bioprocesses.
Assuntos
Eletroforese Capilar/métodos , Espectrofotometria Ultravioleta/métodos , Asparagina/análise , Asparagina/química , Asparagina/isolamento & purificação , Ácido Aspártico/análise , Ácido Aspártico/química , Ácido Aspártico/isolamento & purificação , Limite de Detecção , Modelos Químicos , Reprodutibilidade dos TestesRESUMO
We describe an analytical methodology to obtain high sensitivity and better resolution through the study of fluorometric excitation (λex) and emission (λem) spectrum wavelengths of OPA-amino acids. The spectrum emission study revealed a maximum signal peak at 450 nm for aspartate and glutamine. For glycine, taurine, and GABA, the maximum signal peak was at 448 and for glutamate at 452 nm. The remaining amino acids analyzed showed a maximum emission around 450 nm. The best signal obtained within the spectrum excitation experiments was using 229- to 450-nm λex-λem. The drawbacks observed at these wavelengths were a baseline drift and negative peaks occurrence. Thus, the excitation wavelength of 240 nm was chosen (240- to 450-nm λex-λem) as a compromise between a very good signal response and a baseline stability to resolve the 18 amino acids studied. Furthermore, this protocol was properly validated. On the other hand, the elution gradient program used for neuroactive amino acids (aspartate, glutamate, glycine, taurine and GABA) showed separation to the baseline, in a 15-min run in all of them. Other amino acids, up to 18, also exhibited a very good separation in a 25-min run. In conclusion, we propose the use of 240- to 450-nm λex-λem wavelengths, in OPA-amino acids analysis, as the most suitable protocol to obtain the best signal response, maintaining an optimum chromatographic resolution.
Assuntos
Ácido Aspártico/isolamento & purificação , Ácido Glutâmico/isolamento & purificação , Glutamina/isolamento & purificação , Neurotransmissores/isolamento & purificação , Taurina/isolamento & purificação , Ácido gama-Aminobutírico/isolamento & purificação , o-Ftalaldeído/química , Animais , Ácido Aspártico/química , Cerebelo/química , Córtex Cerebral/química , Cromatografia Líquida de Alta Pressão , Ácido Glutâmico/química , Glutamina/química , Masculino , Neurotransmissores/química , Ratos , Ratos Sprague-Dawley , Taurina/química , Ácido gama-Aminobutírico/químicaRESUMO
A novel chiral stationary phase consisting of an amidopropyltrimethylammonium chloride divinylbenzene (APAT-DVB) polymer containing the chiral selector heptakis(2,3-di-O-acetyl-6-O-sulfo)-ß-cyclodextrin (HSßCD) has been employed in porous layer open tubular (PLOT) capillary column format with various conditions evaluated to optimize the polymerization and chiral selector immobilization. Scanning electron microscopy demonstrated a near homogenous longitudinal open path in the column with a polymer film of uniform thickness. IR spectroscopy characterized the functional groups of the polymer and energy-dispersive X-ray spectroscopy provided further evidence of the successful polymer modification with HSßCD. Optimum electrochromatographic separation conditions were elaborated with respect to organic solvent content and pH of the background electrolyte. Colum-to-column and long-term reproducibility was excellent. The effectiveness of the new capillary column was demonstrated with the successful separation of d-and l-aspartic acid, d- and l-tyrosine and d-and l-lysine.
Assuntos
Ciclodextrinas/química , Resinas Acrílicas/química , Ácido Aspártico/isolamento & purificação , Eletrocromatografia Capilar/métodos , Concentração de Íons de Hidrogênio , Lisina/isolamento & purificação , Microscopia Eletrônica de Varredura , Polimerização , Porosidade , Reprodutibilidade dos Testes , Estereoisomerismo , Propriedades de Superfície , Tirosina/isolamento & purificaçãoRESUMO
The bulk of indole-3-acetic acid (IAA) in plants is found in the form of conjugated molecules, yet past research on identifying these compounds has largely relied on methods that were both laborious and inefficient. Using recent advances in analytical instrumentation, we have developed a simple yet powerful liquid chromatography-mass spectrometry (LC-MS)-based method for the facile characterization of the small IAA conjugate profile of plants. The method uses the well-known quinolinium ion (m/z 130.0651) generated in MS processes as a signature with high mass accuracy that can be used to screen plant extracts for indolic compounds, including IAA conjugates. We reinvestigated Glycine max (soybean) for its indoles and found indole-3-acetyl-trytophan (IA-Trp) in addition to the already known indole-3-acetyl-aspartic acid (IA-Asp) and indole-3-acetyl-glutamic acid (IA-Glu) conjugates. Surprisingly, several organic acid conjugates of tryptophan were also discovered, many of which have not been reported in planta before. These compounds may have important physiological roles in tryptophan metabolism, which in turn can affect human nutrition. We also demonstrated the general applicability of this method by identifying indolic compounds in different plant tissues of diverse phylogenetic origins. It involves minimal sample preparation but can work in conjunction with sample enrichment techniques. This method enables quick screening of IAA conjugates in both previously characterized as well as uncharacterized species, and facilitates the identification of indolic compounds in general.
Assuntos
Ácidos Indolacéticos/química , Indóis/química , Plantas/química , Aminoácidos/química , Aminoácidos/isolamento & purificação , Ácido Aspártico/química , Ácido Aspártico/isolamento & purificação , Cromatografia Líquida , Cocos/química , Flores/química , Ginkgo biloba/química , Ácidos Indolacéticos/isolamento & purificação , Indóis/isolamento & purificação , Solanum lycopersicum/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Folhas de Planta/química , Triptofano/químicaRESUMO
The emergence and spread of carbapenem-resistant Gram-negative pathogens is a global public health problem. The acquisition of metallo-ß-lactamases (MBLs) such as NDM-1 is a principle contributor to the emergence of carbapenem-resistant Gram-negative pathogens that threatens the use of penicillin, cephalosporin and carbapenem antibiotics to treat infections. To date, a clinical inhibitor of MBLs that could reverse resistance and re-sensitize resistant Gram-negative pathogens to carbapenems has not been found. Here we have identified a fungal natural product, aspergillomarasmine A (AMA), that is a rapid and potent inhibitor of the NDM-1 enzyme and another clinically relevant MBL, VIM-2. AMA also fully restored the activity of meropenem against Enterobacteriaceae, Acinetobacter spp. and Pseudomonas spp. possessing either VIM or NDM-type alleles. In mice infected with NDM-1-expressing Klebsiella pneumoniae, AMA efficiently restored meropenem activity, demonstrating that a combination of AMA and a carbapenem antibiotic has therapeutic potential to address the clinical challenge of MBL-positive carbapenem-resistant Gram-negative pathogens.
Assuntos
Ácido Aspártico/análogos & derivados , Carbapenêmicos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Tienamicinas/farmacologia , Resistência beta-Lactâmica/efeitos dos fármacos , Inibidores de beta-Lactamases , Animais , Antibacterianos/farmacologia , Ácido Aspártico/isolamento & purificação , Ácido Aspártico/farmacologia , Aspergillus/química , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Feminino , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/genética , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , Meropeném , Camundongos , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
Amino acids (AAs) derived from hydrolysis of protein rest streams are interesting feedstocks for the chemical industry due to their functionality. However, separation of AAs is required before they can be used for further applications. Electrodialysis may be applied to separate AAs, but its efficiency is limited when separating AAs with similar isoelectric points. To aid the separation, specific conversion of an AA to a useful product with different charge behavior to the remaining compounds is desired. Here the separation of L-aspartic acid (Asp) and L-glutamic acid (Glu) was studied. L-Glutamate α-decarboxylase (GAD, Type I, EC 4.1.1.15) was applied to specifically convert Glu into γ-aminobutyric acid (GABA). GABA has a different charge behavior from Asp therefore allowing a potential separation by electrodialysis. Competitive inhibition and reduced operational stability caused by Asp could be eliminated by maintaining a sufficiently high concentration of Glu. Immobilization of GAD does not reduce the enzyme's initial activity. However, the operational stability was slightly reduced. An initial study on the reaction operating in a continuous mode was performed using a column reactor packed with immobilized GAD. As the reaction mixture was only passed once through the reactor, the conversion of Glu was lower than expected. To complete the conversion of Glu, the stream containing Asp and unreacted Glu might be recirculated back to the reactor after GABA has been removed. Overall, the reaction by GAD is specific to Glu and can be applied to aid the electrodialysis separation of Asp and Glu.
Assuntos
Ácido Aspártico/química , Ácido Glutâmico/química , Ácido gama-Aminobutírico/química , Ácido Aspártico/isolamento & purificação , Biomassa , Enzimas Imobilizadas/química , Escherichia coli/enzimologia , Glutamato Descarboxilase/química , Ácido Glutâmico/isolamento & purificaçãoRESUMO
A novel stationary phase was synthesized for chiral ligand-exchange chromatography via atom transfer radical polymerization (ATRP). Glycidyl methacrylate (GMA) was grafted onto the surface of the silica by ATRP using bromoisobutyryl bromide as an initiator, and the organic metal compound formed in the CuCl/2,2'-bipyridine(Bpy) system as a catalyst at room temperature. The chiral stationary phase was then synthesized by grafting L-phenylalanine on the surface of the silica. The stationary phase was characterized by means of elementary analysis and evaluated in detail to determine its separability. The amount of L-phenylalanine on the surface of silica was calculated to be 4.32 mg/m2. The results showed that the good enantioseparations of some DL-amino acids were obtained using ligand-exchange chromatography on the synthesized chiral stationary phase (50 degrees C) with 0.05 mol/L KH2PO4 and 0.1 mmol/L Cu(Ac)2 solution (pH 4.5) as the mobile phase at a flow rate of 1.0 mL/min and a wavelength of 223 nm. The influences of the mobile phase pH, concentration of Cu (II), and temperature of column on the resolution of DL-amino acids by ligand-exchange chromatography were investigated. The results showed that these conditions could affect the resolution of racemates. Compared with the column prepared by radical method using L-phenylalanine directly bonded onto the surface of the silica, the synthesized stationary phase showed a better separation ability, and the DL-aspartic acids and DL-asparagines could be separated at baseline.
Assuntos
Cromatografia por Troca Iônica/instrumentação , Resinas de Troca Iônica/síntese química , Fenilalanina/química , Asparagina/química , Asparagina/isolamento & purificação , Ácido Aspártico/química , Ácido Aspártico/isolamento & purificação , Cromatografia por Troca Iônica/métodos , Resinas de Troca Iônica/química , Ligantes , Polimerização , EstereoisomerismoRESUMO
A micro-free flow electrophoresis (µFFE) analytical system with voltage applied in two dimensions was proposed. Both fluid transport and separation were driven electrokinetically. The Alpha-Imager was applied as an in-situ detector, which could observe, scan and analyze the photometric state of the whole separating area. The mass-transfer process and the range of voltages applied on the chip were simulated and calculated by MATLAB software. Then, the chip design with a separating chamber, which was 12 mm in length, 5 mm in width and 20 µm in depth, was presented. Under the CZE-CZE mode, the operational conditions, such as the EOF, the pH of the buffer and the ratio of the voltage applied in two dimensions, were optimized. Mixed amino acids, including FITC-labeled L-lysine, FITC-labeled L-phenylalanine and FITC-labeled L-aspartic, were successfully separated on the chip when the borate buffer contained 3% glycerol, with pH as 11 and the ratio of field strength in two-dimension was 1:7. The resolution could achieve 2.1 and 1.9, respectively.
Assuntos
Ácido Aspártico/isolamento & purificação , Lisina/isolamento & purificação , Fenilalanina/isolamento & purificação , Técnicas Eletroquímicas , Eletroforese/instrumentação , Análise de Injeção de Fluxo/instrumentação , Concentração de Íons de Hidrogênio , Dispositivos Lab-On-A-Chip , SoftwareRESUMO
We describe the stacking and separation of d- and l-aspartic acid (Asp) by capillary electrophoresis (CE) with light-emitting diode-induced fluorescence detection (LEDIF). In the presence of cyanide, d- and l-Asp were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) to form fluorescent derivatives prior to CE-LEDIF. The separation of NDA-derivatized d- and l-Asp was accomplished using a discontinuous system - buffer vials contained a solution of 0.6% poly(ethylene oxide) (PEO), 150 mM sodium dodecyl sulfate (SDS), and 60mM hydroxypropyl-ß-cyclodextrin (Hp-ß-CD), while a capillary was filled with a solution of 150 mM SDS and 60mM Hp-ß-CD. The role of PEO, Hp-ß-CD, and SDS is to act as a concentrating media, as a chiral selector, and as a pseudostationary phase, respectively. This discontinuous system could be employed for the stacking of 600 nL of NDA-derivatized d- and l-Asp without the loss of chiral resolution. The stacking mechanism is mainly based on the difference in viscosity between sample zone and PEO as well as SDS sweeping. The limits of detection at signal-to-noise of 3 for d- and l-Asp were down to 2.4 and 2.5 × 10(-10)M, respectively. Compared to normal sample injection volume (25 nL), this stacking approach provided a 100- and 110-fold improvement in the sensitivity of d- and l-Asp, respectively. This method was further applied for determining d- and l-Asp in cerebrospinal fluid, soymilk, and beer.
Assuntos
Ácido Aspártico/isolamento & purificação , Cromatografia Capilar Eletrocinética Micelar/métodos , Animais , Ácido Aspártico/líquido cefalorraquidiano , Ácido Aspártico/química , Cerveja/análise , Fluorescência , Corantes Fluorescentes/química , Limite de Detecção , Polietilenoglicóis/química , Dodecilsulfato de Sódio/química , Leite de Soja/química , Estereoisomerismo , alfa-Ciclodextrinas/químicaRESUMO
Sodium cholate (SC), beta-CD, hydroxypropyl (HP)-beta-CD, HSA, and the dual mixtures of them were evaluated for the analysis of aspartic acid (Asp) and glutamic acid (Glu) enantiomers fluorescently tagged with 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein (DTAF) by CE with LIF detection. Among the investigated chiral selectors and the dual selector systems, the dual selector systems of HSA and SC resulted to be the most useful chiral selectors allowing relatively high chiral resolution. Several experimental parameters such as chiral reagent type and concentration, buffer concentration, and pH, type and concentration of organic modifier were studied in order to find the optimum conditions for the chiral resolution of the two derivatized amino acids in their enantiomers. The effect of different variables that affect derivatization (time, temperature, pH, and DTAF concentration) was studied. Under optimum conditions, the analytes were separated in a short 10.5 min analysis time, and the RSDs for migration time and peak area were less than 0.12 and 2.8%, respectively. The method was applied for the analysis of compound amino acids injection without interference from other amino acids in the sample matrices observed.
Assuntos
Ácido Aspártico/isolamento & purificação , Fluorescência , Ácido Glutâmico/isolamento & purificação , Lasers , Albumina Sérica/química , Colato de Sódio/química , Eletroforese Capilar , Humanos , EstereoisomerismoRESUMO
OBJECTIVE: To study on the comprehensive utilization value from fresh juice of Panax quinquefolium L. METHODS: The total saponins of Panax quinquefoliuml L. 9 kinds of trace elements were determined in fresh juice with ICP-AES, as well as 16 kinds of ammonia acids. RESULTS: The physiologically active. Panax quinquenoside was affluent in fresh juice. There were many kinds of trace elements and ammomnia acid in fresh juice. CONCLUSION: Fresh juice of Panax quinquefolium L. is a kind of valuable resource.
Assuntos
Aminoácidos/análise , Frutas/química , Ginsenosídeos/análise , Panax/química , Plantas Medicinais/química , Oligoelementos/análise , Aminoácidos/isolamento & purificação , Ácido Aspártico/análise , Ácido Aspártico/isolamento & purificação , Ginsenosídeos/isolamento & purificação , Ácido Glutâmico/análise , Ácido Glutâmico/isolamento & purificação , Ferro/análise , Ferro/isolamento & purificação , Manganês/análise , Manganês/isolamento & purificação , Oligoelementos/isolamento & purificaçãoRESUMO
A simple, sensitive and low-cost method was developed for the determination of aspartate (Asp) and glutamate (Glu) in rabbit retina. Polymer monolith microextraction (PMME) using a poly(acrylamide-vinylpyridine-N,N'-methylene bisacrylamide) (AA-VP-Bis) monolithic column was combined with derivatization of Asp and Glu using 8-phenyl-(4-oxy-acetic acid N-hydroxysuccinimide ester)-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (TMPAB-OSu), and this was used to analyze the derivatives of Asp and Glu by high-performance liquid chromatography (HPLC) with fluorescence detection. The conditions for the derivatization and the subsequent extraction of Asp and Glu derivatives were optimized. The enrichment factors for the derivatives of Asp and Glu were found to be 14.1 and 14.7, respectively, by PMME. The limits of detection of Asp and Glu were 0.14 and 0.53 nmol/L, respectively. The precision and recovery were evaluated with spiked retina. The inter- and intraday relative standard deviations were less than 10%. The proposed method was successfully applied to the determination of Asp and Glu levels in rabbit retina samples with different stages of intraocular hypertension.
Assuntos
Ácido Aspártico/análise , Ácido Aspártico/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Ácido Glutâmico/análise , Ácido Glutâmico/isolamento & purificação , Polímeros/química , Retina/química , Animais , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Calibragem , Cromatografia Líquida de Alta Pressão/instrumentação , Fluorescência , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Concentração de Íons de Hidrogênio , Hipertensão/metabolismo , Estrutura Molecular , Coelhos , Retina/metabolismoRESUMO
A new sensitive assay for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in biofluids was developed, based on the separation and detection of alanine, glutamate, and aspartate using capillary electrophoresis (CE) with electrochemiluminescence (ECL) detection. The three amino acids were separated in 5 mM phosphate of pH 2.1 as background electrolyte, and detected on a 500 microm platinum disk electrode at 1.2V (versus Ag/AgCl) in the presence of 10 mM tris(2,2'-bipyridyl)ruthenium(II) dissolved in 80 mM phosphate of pH 10.5. A mass detection limit of 37.3 fmol (or 81.5 fmol) for glutamate, corresponding to the product in the enzyme reaction catalyzed by 1.24 x 10(-9)U AST (or 2.72 x 10(-9)U ALT) in a 30 min reaction period, was achieved. This assay was applied to investigate the cytotoxicity effect of ethanol on HepG2 cells and differentiating nonalcoholic steatohepatitis (NASH) from alcoholic liver disease, indicating that the technique is promising for the application in the cell biological and clinical fields.
Assuntos
Alanina Transaminase/sangue , Alanina Transaminase/metabolismo , Aspartato Aminotransferases/sangue , Aspartato Aminotransferases/metabolismo , Líquidos Corporais/enzimologia , Medições Luminescentes/métodos , Alanina/isolamento & purificação , Ácido Aspártico/isolamento & purificação , Diagnóstico Diferencial , Eletroforese Capilar , Etanol , Feminino , Ácido Glutâmico/análise , Ácido Glutâmico/isolamento & purificação , Hepatite/sangue , Hepatite/diagnóstico , Humanos , Concentração de Íons de Hidrogênio , Hepatopatias Alcoólicas/sangue , Hepatopatias Alcoólicas/diagnóstico , Masculino , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Deprotonated peptides containing C-terminal glutamic acid, aspartic acid, or serine residues were studied by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer with ion production by electrospray ionization (ESI). Additional studies were performed by post source decay (PSD) in a matrix-assisted laser desorption ionization/time-of-flight (MALDI/TOF) mass spectrometer. This work included both model peptides synthesized in our laboratory and bioactive peptides with more complex sequences. During SORI-CID and PSD, [M - H]- and [M - 2H]2- underwent an unusual cleavage corresponding to the elimination of the C-terminal residue. Two mechanisms are proposed to occur. They involve nucleophilic attack on the carbonyl carbon of the adjacent residue by either the carboxylate group of the C-terminus or the side chain carboxylate group of C-terminal glutamic acid and aspartic acid residues. To confirm the proposed mechanisms, AAAAAD was labelled by 18O specifically on the side chain of the aspartic acid residue. For peptides that contain multiple C-terminal glutamic acid residues, each of these residues can be sequentially eliminated from the deprotonated ions; a driving force may be the formation of a very stable pyroglutamatic acid neutral. For peptides with multiple aspartic acid residues at the C-terminus, aspartic acid residue loss is not sequential. For peptides with multiple serine residues at the C-terminus, C-terminal residue loss is sequential; however, abundant loss of other neutral molecules also occurs. In addition, the presence of basic residues (arginine or lysine) in the sequence has no effect on C-terminal residue elimination in the negative ion mode.
Assuntos
Aminoácidos/isolamento & purificação , Ácido Aspártico/isolamento & purificação , Ácido Glutâmico/isolamento & purificação , Peptídeos/química , Serina/isolamento & purificação , Sequência de Aminoácidos , Ácido Aspártico/química , Ciclotrons , Ácido Glutâmico/química , Espectrometria de Massas , Isótopos de Oxigênio , Prótons , Serina/químicaAssuntos
Acetiltransferases/química , Acetiltransferases/metabolismo , Ácido Aspártico/análogos & derivados , Acetiltransferases/genética , Amidoidrolases/genética , Amidoidrolases/metabolismo , Animais , Ácido Aspártico/isolamento & purificação , Ácido Aspártico/metabolismo , Estabilidade Enzimática , Humanos , Mitocôndrias/metabolismo , Peso Molecular , Doenças do Sistema Nervoso/enzimologia , Neurônios/metabolismoRESUMO
Six chiral selectors of S-(-)-t-Leu-cyclopropylamide, S-(-)-t-Leu-cyclopentylamide, S-(-)-t-Leu-cyclohexylamide, S-(-)-t-Leu-cycloheptylamide, S-(-)-t-Leu-cyclooctylamide, S-(-)-t-Leu-cyclododecylamide have been prepared and anchored individually through amide bonding to a polydimethylsiloxane functionalized with 2,2,2-trifluoroethyl ester groups by way of nucleophilic displacement reaction. The resulting chiral polysiloxanes have been provided as stationary phases for the separation of amino acid enantiomers by capillary GC. Amino acids were derivatized into N(O)-trifluoroacetyl isopropyl esters. Especially, polydimethylsiloxane anchored with S-(-)-t-Leu-cyclooctylamide was found to be efficient for the separation of aspartic acid (Asp) enantiomers. The method was applied to the estimation of ages from the extent of Asp racemization in human dentines.
Assuntos
Aminoácidos/química , Aminoácidos/isolamento & purificação , Cromatografia Gasosa/métodos , Adulto , Idoso , Envelhecimento/metabolismo , Ácido Aspártico/química , Ácido Aspártico/isolamento & purificação , Dentina/química , Dimetilpolisiloxanos , Humanos , Silicones , EstereoisomerismoRESUMO
INTRODUCTION: The major excitatory neurotransmitters in the mammalian central nervous system are glutamate and aspartate. We developed a rapid and efficient method for the extraction and measurement of these amino acids in Planaria--a valuable model for mammalian processes because of their simple, centralized nervous system and similar neurotransmitter systems. METHOD: The method utilized buffer extraction (perchloric acid containing 0.025% of L-cystine and Na2EDTA), simple derivatization, high-pressure liquid chromatography (HPLC), and fluorescence detection. RESULTS: The mean+/-S.E.M. amounts of glutamate and aspartate were 322.6+/-43.6 and 188.6+/-27.6 pmol/mg-planarian, respectively. DISCUSSION: The method provides the ability to investigate changes in glutamate and aspartate in response to drug administration or withdrawal.