Glutathione levels in and total antioxidant capacity of Candida sp. cells exposed to oxidative stress caused by hydrogen peroxide.

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.

Glutathione levels in and total antioxidant capacity of Candida sp. cells exposed to oxidative stress caused by hydrogen peroxide / Níveis de glutationa e capacidade antioxidante total em células de Candida sp. expostas a estresse oxidativo causado por peróxido de hidrogênio

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.

Determination of thiols and disulfides via HPLC quantification of 5-thio-2-nitrobenzoic acid.

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.

[Determination of L-cysteine in enzymatic reaction mixture with pre-column derivatization by high performance liquid chromatography].

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.

5,5'-Dithiobis-(2-nitrobenzoic acid) as a probe for a non-essential cysteine residue at the medium chain acyl-coenzyme A dehydrogenase binding site of the human 'electron transferring flavoprotein' (ETF).

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.

A comparison between different methods for the determination of reduced and oxidized glutathione in mammalian tissues.

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.