RESUMO
A surface enhanced Raman spectroscopy (SERS) substrate made of gold nanoparticles-embedded mesoporous silica (AuNPs@mesoSiO2) varying with size and gold concentrations was applied for SERS applications. In this study, the AuNPs@mesoSiO2 substrate produced notable intensity and more distinguishable peaks when compared with the spectra collected directly from an Au/Cr-coated substrate with regard to the detection of a lower concentration of chemicals or environmental contamination. Both aqueous and dried coffee rings of 5-5'-Dithio-bis (2-nitrobenzoic acid) (DTNB) solutions were examined. SERS spectra obtained from the substrate showed more fingerprint peaks with significant enhancement on the spectra signals. The correlation between the SERS signal and the DTNB concentration was found to be linear within a range of 10(-2) to 10(-12) M. SERS enhancement between 25 and 8 times greater can be achieved for DTNB detection using AuNPs@mesoSiO2 compared with the normal Raman spectra obtained from the aqueous solution and the contact line of the dried coffee ring, respectively.
Assuntos
Ácido Ditionitrobenzoico/análise , Ouro/química , Nanopartículas/química , Dióxido de Silício/química , Análise Espectral Raman/métodos , Limite de Detecção , Nanopartículas/ultraestrutura , Propriedades de SuperfícieRESUMO
INTRODUCTION: The capacity to overcome the oxidative stress imposed by phagocytes seems to be critical for Candida species to cause invasive candidiasis. METHODS: To better characterize the oxidative stress response (OSR) of 8 clinically relevant Candida sp., glutathione, a vital component of the intracellular redox balance, was measured using the 5,5'-dithiobis-(2-nitrobenzoic acid (DTNB)-glutathione disulfide (GSSG) reductase reconversion method; the total antioxidant capacity (TAC) was measured using a modified method based on the decolorization of the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic) acid radical cation (ABTS*+). Both methods were used with cellular Candida sp. extracts treated or not with hydrogen peroxide (0.5 mM). RESULTS: Oxidative stress induced by hydrogen peroxide clearly reduced intracellular glutathione levels. This depletion was stronger in Candida albicans and the levels of glutathione in untreated cells were also higher in this species. The TAC demonstrated intra-specific variation. CONCLUSIONS: Glutathione levels did not correlate with the measured TAC values, despite this being the most important non-enzymatic intracellular antioxidant molecule. The results indicate that the isolated measurement of TAC does not give a clear picture of the ability of a given Candida sp. to respond to oxidative stress.
Assuntos
Antioxidantes/farmacologia , Candida/efeitos dos fármacos , Candidíase/microbiologia , Glutationa/análise , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Candida/metabolismo , Candida/patogenicidade , Ácido Ditionitrobenzoico/análise , Oxidantes/farmacologia , Oxirredução , Reagentes de Sulfidrila/análise , VirulênciaRESUMO
INTRODUCTION: The capacity to overcome the oxidative stress imposed by phagocytes seems to be critical for Candida species to cause invasive candidiasis. METHODS: To better characterize the oxidative stress response (OSR) of 8 clinically relevant Candida sp., glutathione, a vital component of the intracellular redox balance, was measured using the 5,5'-dithiobis-(2-nitrobenzoic acid (DTNB)-glutathione disulfide (GSSG) reductase reconversion method; the total antioxidant capacity (TAC) was measured using a modified method based on the decolorization of the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic) acid radical cation (ABTS*+). Both methods were used with cellular Candida sp. extracts treated or not with hydrogen peroxide (0.5 mM). RESULTS: Oxidative stress induced by hydrogen peroxide clearly reduced intracellular glutathione levels. This depletion was stronger in Candida albicans and the levels of glutathione in untreated cells were also higher in this species. The TAC demonstrated intra-specific variation. CONCLUSIONS: Glutathione levels did not correlate with the measured TAC values, despite this being the most important non-enzymatic intracellular antioxidant molecule. The results indicate that the isolated measurement of TAC does not give a clear picture of the ability of a given Candida sp. to respond to oxidative stress.
INTRODUÇÃO: A capacidade de suportar o estresse oxidativo imposto por fagócitos parece ser crítica para que espécies de Candida causem candidíase invasiva. MÉTODOS: Para melhor caracterizar a resposta ao estresse oxidativo (REO) de oito Candida sp. clinicamente relevantes, um componente vital do balanço redox intracelular, a glutationa, foi mensurada pelo método de reconversão DTNB-GSSG redutase e a capacidade antioxidante total (CAT) foi mensurada por um método modificado baseado na descoloração do ABTS*+. Ambos os métodos foram utilizados em extratos celulares das espécies de Candida tratadas ou não com peróxido de hidrogênio (0,5mM). RESULTADOS: O estresse oxidativo induzido pelo peróxido de hidrogênio claramente reduziu os níveis intracelulares de glutationa. Esta diminuição foi mais intensa em C. albicans e os níveis de glutationa em células não tratadas foram também maiores nesta espécie. A capacidade antioxidante total demonstrou variação intraespecífica na capacidade antioxidante. CONCLUSÕES: Os níveis de glutationa não se correlacionaram com a capacidade antioxidante total mensurada, apesar desta ser a defesa antioxidante intracelular não-enzimática mais importante. Os resultados indicam que a medição isolada da CAT não fornece um quadro claro da habilidade de certa espécie de Candida responder ao estresse oxidativo.
Assuntos
Antioxidantes/farmacologia , Candida/efeitos dos fármacos , Candidíase/microbiologia , Glutationa/análise , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Candida/metabolismo , Candida/patogenicidade , Ácido Ditionitrobenzoico/análise , Oxirredução , Oxidantes/farmacologia , Reagentes de Sulfidrila/análise , VirulênciaRESUMO
This work presents an assay for total thiols and total disulfides in biological samples via HPLC quantification of 5-thio-2-nitrobenzoic acid (TNB) derived from the reaction of thiols with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent). This method also provides simultaneous quantification of glutathione (GSH) via the measurement of the GSH-DTNB adduct (GSH-TNB). By using 326nm as the detecting wavelength, the HPLC detection limit for TNB and the GSH-TNB adduct was determined to be 15 and 7.5pmol respectively. A recovery study with OVCAR-3 cells revealed that the recovery yields for TNB in the procedures for determining non-protein thiols, protein thiols, non-protein disulfides, and protein disulfides were 99.4+/-1.2% (n=3), 98.1+/-5.0% (n=3), 95.6+/-0.9% (n=3), and 96.6+/-2.3% (n=3) respectively. The recovery yield for GSH-TNB in the procedures for determining non-protein thiols, protein thiols, non-protein disulfides, and protein disulfides was 99.0+/-0.3% (n=3), 95.1+/-4.9% (n=3), 96.8+/-0.6% (n=3), and 95.1+/-2.9% (n=3) respectively. The reproducibility, expressed as the relative standard deviation for the analyte, for TNB was determined to be 2.8% (n=6) for non-protein thiols, 3.9% (n=6) for protein thiols, 3.6% (n=6) for non-protein disulfides and 4.6% (n=6) for protein disulfides. The reproducibility for GSH-TNB was determined to be 1.6% (n=6) for non-protein thiols and 2.6% (n=6) for non-protein disulfides. By comparing the amount of GSH determined in a biological sample before NaBH(4) reduction with that after the reduction, this method can provide information associated with thiol glutathionylation which would be useful for protein glutathionylation study. This method should be applicable to cellular, subcellular, protein, or other biomatrix samples for thiol and disulfide quantification and will be a useful analytical method in the study of thiol redox state and thiol glutathionylation.
Assuntos
Dissulfetos/química , Ácido Ditionitrobenzoico/química , Compostos de Sulfidrila/química , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão/métodos , Dissulfetos/análise , Dissulfetos/metabolismo , Ácido Ditionitrobenzoico/análise , Feminino , Glutationa/química , Humanos , Estrutura Molecular , Neoplasias Ovarianas/metabolismo , Oxirredução , Reprodutibilidade dos Testes , Soluções , Espectrofotometria Ultravioleta , Compostos de Sulfidrila/análise , Compostos de Sulfidrila/metabolismo , Água/químicaRESUMO
Two novel potentiometric sensors that are highly selective to Hg2+ ions are described. These are based on the use of 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) and tricyclazole (TCZ) as neutral carriers in plasticized poly(vinyl chloride) membranes. Fast Nernstian responses are obtained for Hg2+ ions over the concentration ranges 7.0 x 10(-6) - 1.0 x 10(-2) and 7.7 x 10(-6) - 1.0 x 10(-2) mol l(-1) at pH 1.8 - 3.3 with lower detection limits of 5.0 x 10(-6) and 5.6 x 10(-6) mol l(-1) (approximately 1 microh ml(-1)) and calibration slopes of 30.0 and 29.7 mV decade(-1) with DTNB- and TCZ-based membrane sensors, respectively. Validation of the assay method reveals good performance characteristics, including long life span, good selectivity for Hg2+ ions over a wide variety of other metal ions, long term response stability, and high reproducibility. Applications for direct determination of mercury in hazardous wastes including dental amalgam, mercury bulbs, and fluorescent lamps give results with good correlation with data obtained using cold vapor atomic absorption spectrometry.
Assuntos
Técnicas de Química Analítica/métodos , Íons , Mercúrio/análise , Polímeros/química , Espectrofotometria Atômica/métodos , Calibragem , Ligas Dentárias , Ácido Ditionitrobenzoico/análise , Resíduos Perigosos , Concentração de Íons de Hidrogênio , Ionóforos/análise , Modelos Químicos , Cloreto de Polivinila/química , Potenciometria , Espectrofotometria Atômica/instrumentação , Tiazóis/análiseRESUMO
Shelf-stable milk could benefit from sensory quality improvement. Current methods of heating cause flavor and nutrient degradation through exposure to overheated thermal exchange surfaces. Rapid heating with microwaves followed by sudden cooling could reduce or eliminate this problem. The objectives for this study were focused on designing and implementing continuous microwave thermal processing of skim fluid milks (white and chocolate) to compare sensory, microbiological, and biochemical parameters with conventionally prepared, indirect UHT milks. All test products were aseptically packaged and stored at ambient temperature for 12 mo. Every 3 mo, samples were taken for microbiological testing, reactive sulfhydryl determinations, active enzyme analysis, instrumental viscosity readings, color measurements, and descriptive sensory evaluation. Microbiological plate counts were negative on all milks at each time point. Enzymatic assays showed that plasmin was inactivated by both heat treatments. 5,5'-dithio-bis(2-nitrobenzoic acid) analysis, a measure of reactive sulfhydryl (-SH-) groups, showed that the initial thiol content was not significantly different between the microwave-processed and UHT-treated milks. However, both heating methods resulted in an increased thiol level compared with conventionally pasteurized milk samples due to the higher temperatures attained. Sulfhydryl oxidase, a milk enzyme that catalyzes disulfide bond formation using a variety of protein substrates, retained activity following microwave processing, and decreased during storage. Viscosity values were essentially equivalent in microwave- and UHT-heated white skim milks. Sensory analyses established that UHT-treated milks were visibly darker, and exhibited higher caramelized and stale/fatty flavors with increased astringency compared with the microwave samples. Sweet aromatic flavor and sweet taste decreased during storage in both UHT and microwave milk products, whereas stale/fatty flavors increased over time. Sensory effects were more apparent in white milks than in chocolate varieties. These studies suggest that microwave technology may provide a useful alternative processing method for delivery of aseptic milk products that retain a long shelf life.
Assuntos
Manipulação de Alimentos/métodos , Temperatura Alta , Micro-Ondas , Leite/química , Leite/microbiologia , Sensação , Animais , Cor , Ácido Ditionitrobenzoico/análise , Gorduras/análise , Conservação de Alimentos , Oxirredutases/metabolismo , Compostos de Sulfidrila/análise , Paladar , Fatores de Tempo , ViscosidadeRESUMO
Metallothionein-3/growth inhibitory factor is a brain-specific member of the metallothionein gene family, and impairs the survival and neurite formation of cultured neurons. Metallothionein-3 can bind heavy metals such as Zn, Cu, or Cd almost in the same way as other metallothionein family. However, its biological function as growth inhibitory factor apparently distinguishes metallothionein-3 from other metallothioneins. To better understanding of the relationship between the growth inhibitory activity of metallothionein-3 and metals bound to metallothionein-3, the metal-binding ability of metallothionein-3 was analyzed in comparison with those of metallothionein-1 and -2. The metal-binding ability of metallothionein-3 was evaluated by pH titration and 5-5' dithiobis (2-nitrobenzoic) acid (DTNB) analysis as compared with those of the other metallothioneins. The affinity of metal ions for metallothionein-3 was indicated as follows, Cu>Cd>Zn, same as metallothionein-1 and -2. However, the affinity of metallothionein-3 to Cu was much higher than that of metallothionein-1 and -2. The strong affinity to Cu of metallothionein-3 might be related to its growth inhibitory activity.
Assuntos
Escherichia coli/genética , Ligantes , Metalotioneína/genética , Metais Pesados/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Técnicas de Cultura de Células , Ácido Ditionitrobenzoico/análise , Estabilidade Enzimática , Escherichia coli/química , Escherichia coli/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Metalotioneína/isolamento & purificação , Metalotioneína/metabolismo , Metais Pesados/química , Metais Pesados/farmacologia , Dados de Sequência Molecular , Plasmídeos/genética , Plasmídeos/metabolismo , Mutação Puntual , Reação em Cadeia da Polimerase/métodos , Coelhos , Análise de Sequência/métodosRESUMO
A new sensitive high performance liquid chromatographic method for the determination of L-cysteine in an enzymatic reaction mixture using ultra violet spectrometric detection was developed. The sample reacted with 5,5'-dithio-bis-nitrobenzoic acid (DTNB) and was analyzed on a Shimadzu VP-ODS column at room temperature, using gradient elution with detection at 330 nm. The L-cysteine chromatographic peak was determined in comparison with derivatives of 2-mercapto ethanol and dithiothreitol. The linear range was 5-950 micromol/L. The recoveries were 99.7%-100.5% and the relative standard deviations (RSDs) were less than 1.3%. The detection limit was 0.8 micromol/L. The method is simple and accurate.
Assuntos
Cromatografia Líquida de Alta Pressão , Cisteína/análise , Cromatografia Líquida de Alta Pressão/métodos , Cisteína/química , Ácido Ditionitrobenzoico/análise , Ditiotreitol/análise , Mercaptoetanol/análise , Espectroscopia Fotoeletrônica/métodosRESUMO
Enteropeptidase (synonym:enterokinase, EC 3.4.21.9) is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)(4)-Lys. The DNA sequence encoding the light chain (catalytic subunit) of human enteropeptidase (GenBank Accession No. U09860) was synthesized from 26 oligonucleotides by polymerase chain reaction and cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin immediately after the DNA sequence encoding enteropeptidase recognition site. The fusion protein thioredoxin/human enteropeptidase light chain was expressed in Escherichia coli BL21(DE3) strain in both soluble and insoluble forms. The soluble recombinant fusion protein failed to undergo autocatalytic cleavage and activation; however, autocatalytic cleavage and activation of recombinant human enteropeptidase light chain (L-HEP) were achieved by solubilization and renaturation of the fusion protein from inclusion bodies and the active L-HEP was purified on agarose-linked soybean trypsin inhibitor. The purified L-HEP cleaved the synthetic peptide substrate Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide with kinetic parameters K(m)=0.16 mM and k(cat)=115 s(-1) and small ester Z-Lys-SBzl with K(m)=140 microM, k(cat)=133 s(-1). L-HEP associated with soybean trypsin inhibitor slowly and small ester Z-Lys-SBzl cleavage was inhibited with K(i)(*)=2.3 nM. L-HEP digested thioredoxin/human epidermal growth factor fusion protein five times faster than equal activity units of bovine recombinant light chain (EKMax, Invitrogen) at the same conditions.
Assuntos
Domínio Catalítico , Enteropeptidase/biossíntese , Escherichia coli/genética , Lisina/análogos & derivados , Proteínas Recombinantes/biossíntese , Animais , Catálise , Domínio Catalítico/genética , Bovinos , Cromatografia de Afinidade/métodos , Clonagem Molecular , DNA Complementar/genética , Ácido Ditionitrobenzoico/análise , Ácido Ditionitrobenzoico/química , Eletroforese em Gel de Poliacrilamida , Enteropeptidase/genética , Enteropeptidase/isolamento & purificação , Inibidores Enzimáticos , Fator de Crescimento Epidérmico/metabolismo , Escherichia coli/metabolismo , Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Humanos , Hidrólise , Corpos de Inclusão/química , Interleucina-13/metabolismo , Isopropiltiogalactosídeo/farmacologia , Cinética , Lisina/metabolismo , Oligopeptídeos/metabolismo , Reação em Cadeia da Polimerase , Dobramento de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Especificidade por Substrato , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Inibidores da Tripsina/química , Inibidores da Tripsina/farmacologiaRESUMO
Objetivo: Optimación de un programa regional de cribado con alfafetoproteína sérica materna con objeto de que la prevalencia de los defectos de cierre del tubo neural en el Principado de Asturias sea próxima a cero.Sujetos y métodos: Las participantes han sido gestantes pertenecientes a las diferentes áreas de salud del Principado de Asturias independientemente de su procedencia, sanidad pública o privada. Se estudiaron a 63.163 embarazadas en 11 años. Todas las técnicas bioquímicas se realizaron en el Laboratorio de Bioquímica del Hospital San Agustín.Resultados: Se ha conseguido optimizar el programa de cribado con alfafetoproteína en suero materno con porcentajes muy bajos de falsos positivos y amniocentesis realizadas, el 1,9 y el 0,5 por ciento, respectivamente. Se ha observado que las anencefalias presentan valores de alfafetoproteína en suero materno significativamente más elevados que las espinas bífidas abiertas, y se ha conseguido disminuir la prevalencia de este tipo de malformaciones en el Principado de Asturias de una media de 1,3 a prácticamente cero.Conclusiones: La puesta en funcionamiento de un programa de estas características requiere un equipo multidisciplinario para que los resultados obtenidos sean satisfactorios. Después de varios años de desarrollo los resultados obtenidos han convencido a los obstetras y médicos generalistas de la utilidad de incorporar dicho programa al protocolo del embarazo. Este programa ha servido, además, para obtener un conocimiento exhaustivo de este tipo de malformaciones en el Principado de Asturias. (AU)
Assuntos
Adulto , Gravidez , Feminino , Humanos , Defeitos do Tubo Neural/diagnóstico , Defeitos do Tubo Neural/sangue , alfa-Fetoproteínas/análise , alfa-Fetoproteínas , Ácido Ditionitrobenzoico/análise , Eletroforese/métodos , Biomarcadores/análise , Isoenzimas/análise , Programas de Rastreamento , Valor Preditivo dos Testes , Valor Preditivo dos TestesRESUMO
En este trabajo se pretende estudiar y evaluar las principales fuentes de variación que pueden provocar variabilidad en la determinación de colinesterasa en sangre entera. Para este fin se analizaron alícuotas de sangre entera de perro bajo distintas condiciones analíticas. Se observó un aumento de la actividad colinesterasa conforme se incrementó la temperatura de reacción. La mayor actividad colinesterasa se encontró en un intervalo de pH entre 8.0 y 8.5; sin embargo, una fuerte elevación de la hidrólisis no enzimática del sustrato tiene lugar cuando el pH excede de 7.5, lo que haría recomendable el empleo de pH 7.5. Aunque los valores más elevados de actividad se alcanzaron con una concentración del sustrato acetiltiocolina iodada de 5x10-3 M y de butiriltiocolina iodada de 10x10-3 M, se describió un fuerte aumento de la hidrólisis no enzimática de los sustratos a estas concentraciones, por lo que se prefirió emplear una concentración de ambos sustratos de 1x10-3 M. El cromóforo ácido 5,5-ditiobis-2-nitrobenzoico, y los sustratos acetiltiocolina iodada y butiriltiocolina iodada, permanecieron estables al menos durante tres meses si eran conservados a una temperatura de -20ºC. Los datos aportados permitirán contribuir a un mejor conocimiento de las fuentes de variación que afectan a la determinación de colinesterasa en sangre entera, y a la estandarización de las condiciones analíticas utilizadas (AU)
Assuntos
Animais , Cães , Ácido Ditionitrobenzoico/análise , Colinesterases/análise , Colinesterases/sangue , Acetiltiocolina/análise , Butiriltiocolina/análise , Butirilcolinesterase/análise , Concentração de Íons de Hidrogênio , Ativação Enzimática , Compostos Organofosforados/análise , Carbamatos/análise , Carbamatos/toxicidadeRESUMO
Se pretende evaluar al cromóforo 2,2´-ditiodipirina (2-PDS) para la determinación de la actividad colinesterasa en sangre entera de perro, y compararlo con el empleo del ácido 5,5´-ditiobis-2-nitrobenzoico (DTNB), cromóforo utilizado tradicionalmente en el método de ELLMAN. Ambos cromóforos proporcionaron buena precisión y repetibilidad a los ensayos (coeficientes de variación intra y entredeterminaciones > 8 por ciento). Sin embargo, el 2-PDS permitió detectar niveles más elevados de actividad colinesterasa. Asimismo, la utilización de una dilución más concentrada de sangre (1:20) únicamente fue posible con el empleo de 2-PDS. Los resultados obtenidos podrían explicarse por la capacidad del 2-PDS de evitar la interferencia de la hemoglobina a 340 nm de longitud de onda. Ambos cromóforos dieron lugar a la aparición de un nivel similar de actividad no enzimática. Tras la exposición "in vivo" e "in vitro" al insecticida organofosforado coumaphos, tanto el 2-PDS como el DTNB permitieron detectar un similar grado de inhibición en la actividad colinesterasa en la dilución 1:50 de sangre entera de perro, existiendo buena correlación entre los resultados obtenidos con ambos cromóforos (coeficientes de correlación > 0.90). La dilución 1:20, analizada con 2-PDS, presentó unos resultados similares de inhibición a los obtenidos con la dilución 1:50. De este modo, los resultadsoo obtenidos apoyan la utilización del 2-PDS como cromóforo para la determinación de la actividad colonesterasa en muestras de sangre entera de perro. (AU)
Assuntos
Animais , Cães , Colinesterases/análise , Colinesterases , Ácido Ditionitrobenzoico/análise , Ácido Ditionitrobenzoico , Coleta de Amostras Sanguíneas/veterinária , Espectrofotometria , Compostos Organofosforados/análise , Compostos Organofosforados/efeitos adversos , Inseticidas Organofosforados/efeitos adversosRESUMO
Human 'electron transferring flavoprotein' (ETF) was inactivated by the thiol-specific reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). The kinetic profile showed the reaction followed pseudo-first-order kinetics during the initial phase of inactivation. Monitoring the release of 5-thio-2-nitrobenzoate (TNB) showed that modification of 1 cysteine residue was responsible for the loss of activity. The inactivation of ETF by DTNB could be reversed upon incubation with thiol-containing reagents. The loss of activity was prevented by the inclusion of medium chain acyl-CoA dehydrogenase (MCAD) and octanoyl-CoA. Cyanolysis of the DTNB modified-ETF with KCN led to the release of TNB accompanied presumably by the formation of the thio-cyano enzyme and with almost full recovery of activity. Conservation studies and the lack of 100% inactivation, however, suggested that this cysteine residue is not essential for the interaction with MCAD.
Assuntos
Acil-CoA Desidrogenases/química , Acil-CoA Desidrogenases/metabolismo , Cisteína/química , Ácido Ditionitrobenzoico/análise , Flavoproteínas/metabolismo , Acil-CoA Desidrogenase , Sítios de Ligação , Biomarcadores/análise , Transporte de Elétrons , Flavoproteínas Transferidoras de Elétrons , Flavoproteínas/antagonistas & inibidores , Humanos , Cinética , Sondas Moleculares , Ligação Proteica , Reagentes de Sulfidrila/farmacocinéticaRESUMO
In this study, three rapid assay techniques for the determination of glutathione, one enzymatic, one fluorometric and one newly patented colorimetric method, were compared by measuring reduced (GSH) and oxidized (GSSG) glutathione in guinea-pig heart and liver. The HPLC technique was used as a standard, since it is considered the most reliable assay method. In heart, all methods measured the same levels of GSH (about 1 mumole/g wet tissue), whereas in liver the fluorometric assay gave GSH levels about half as high as those measured by the other methods (about 3 vs. 7 mumoles/g wet tissue). Conversely, the fluorometric assay grossly overestimated GSSG concentration (by 5 to 8 times) in both heart and liver. These results confirm previous doubts about the use of the fluorometric technique for GSSG determination in mammalian tissues and also raise some questions about its use for the measurement of GSH in liver. In this tissue, the GSH concentration determined by the fluorometric method was shown to be inversely correlated with the size of the sample, suggesting the presence of some quenching material.