Rahman I, Kode A, Biswas SKAssay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nat Protoc 1: 3159-3165
Department of Environmental Medicine, Lung Biology and Disease Program, University of Rochester Medical Center, Rochester, New York, USA.Nature Protocol (Impact Factor: 9.67). 02/2006; 1(6):3159-65. DOI: 10.1038/nprot.2006.378
The spectrophotometric/microplate reader assay method for glutathione (GSH) involves oxidation of GSH by the sulfhydryl reagent 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) to form the yellow derivative 5'-thio-2-nitrobenzoic acid (TNB), measurable at 412 nm. The glutathione disulfide (GSSG) formed can be recycled to GSH by glutathione reductase in the presence of NADPH. The assay is composed of two parts: the preparation of cell cytosolic/tissue extracts and the detection of total glutathione (GSH and GSSG). The method is simple, convenient, sensitive and accurate. The lowest detection for GSH and GSSG is 0.103 nM in a 96-well plate. This method is rapid and the whole procedure takes no longer than 15 min including reagent preparation. The method can assay GSH in whole blood, plasma, serum, lung lavage fluid, cerebrospinal fluid, urine, tissues and cell extracts and can be extended for drug discovery/pharmacology and toxicology protocols to study the effects of drugs and toxic compounds on glutathione metabolism.
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- "HPLC comprises high sensitivity but due to the long duration of sample preparation and elution, there is a risk of oxidation of GSH to GSSG (Rahman et al., 2006). Therefore, determination of GSH was based on the colorimetric method via Ellman's reagent (5,5 ′ -dithio- (bis-2-nitrobenzoic) acid, DTNB, Sigma–Aldrich) based on Tietze (1969). "
ABSTRACT: In the following work a high cell density fed-batch process with Saccharomyces cerevisiae coupled with a high efficient incorporation of cysteine for glutathione (GSH) overproduction was developed. Therefore, a feeding strategy based on the respiratory quotient (RQ) was applied to ensure high biomass (96.1g/l). Furthermore, the optimal cysteine concentration and time of cysteine addition were investigated. Low concentrations of cysteine at late fermentation phases resulted in relatively high incorporation yields of about 0.40mol/mol and maintained the physiology of cultivated yeast. By changing the cysteine feeding from standard single shot to continuous addition, an often observed cell specific toxicity, triggered by high cysteine concentrations, could be prevented and the cysteine incorporation yield (0.54 ± 0.01mol/mol) and GSH content (1650.7 ± 42.8mg/l; 1.76 ± 0.08%) were maximized, respectively. The developed process was transferred from laboratory into pilot plant scale. Further, the reduced cell specific toxicity enabled the development of a repeated fed-batch procedure with a suitable performance concerning cysteine incorporation yield (0.40 ± 0.1mol/mol), biomass (84.2 ± 1.2g/l) and GSH content (1304.7 ± 61.4mg/l).Journal of Biotechnology 10/2015; 216. DOI:10.1016/j.jbiotec.2015.10.016 · 2.87 Impact Factor
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- "Reduced (GSH) and oxidized (GSSG) glutathione contents were determined according to Rahman et al. (2006). Absorbance of GSH and GSSG "
ABSTRACT: Ocean acidification and increasing discharges of pharmaceutical contaminants into aquatic systems are among key and/or emerging drivers of environmental change affecting marine ecosystems. A growing body of evidence demonstrates that ocean acidification can have direct and indirect impacts on marine organisms although combined effects with other stressors, namely with pharmaceuticals, have received very little attention to date. The present study aimed to evaluate the impacts of the pharmaceutical drug Carbamazepine and pH 7.1, acting alone and in combination, on the clamScrobicularia plana. For this, a long-termexposure (28 days)was conducted and a set of oxidative stress markers was investigated. The results obtained showed that S. plana was able to develop mechanisms to prevent oxidative damage when under low pH for a long period, presenting higher survival when exposed to this stressor compared to CBZ or the combination of CBZ with pH 7.1. Furthermore, the toxicity of CBZ on S. plana was synergistically increased under ocean acidification conditions (CBZ + pH 7.1): specimens survival was reduced and oxidative stress was enhanced when compared to single exposures. These findings add to the growing body of evidence that ocean acidification will act to increase the toxicity of CBZ to marine organisms,which has clear implications for coastal benthic ecosystems suffering chronic pollution from pharmaceutical drugs.Science of The Total Environment 10/2015; 541:977-985. DOI:10.1016/j.scitotenv.2015.09.138 · 4.10 Impact Factor
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- "Reduced (GSH) and oxidized (GSSG) glutathione content was determined according to Rahman et al. (2007), using reduced and oxidized glutathione standards (0–60 μmol/L). Absorbance was measured at 412 nm. "
ABSTRACT: Marine organisms are constantly exposed to multiple stressors creating a range of associated environmental and ecotoxicological risks. Several stressors have been identified as key drivers of environmental change that may significantly influence marine near-shore systems. These include increased frequency and duration of extreme rainy events and drought periods, arising from climate change, and the constant discharge of contaminants into aquatic systems. A growing body of evidence demonstrates that climate change can have direct and indirect impacts on marine organisms although the combined effects with other stressors, namely with metals and metalloids, have received very little attention to date. The present study evaluated the biochemical alterations induced in the clam Ruditapes philippinarum, also known as Manila clam, when simultaneously exposed (96h) to different arsenic concentrations (0, 4 and 17mg/L) and a range of salinities (14, 21, 28, 35 and 42g/L). Results obtained revealed that, when acting alone, both stressors induced oxidative stress in clams, with higher LPO levels and lower GSTs activity induced by As contamination, and a stronger inhibition of the antioxidant defenses induced by salinity increase. Furthermore, when exposed to the combination of both stressors, clams experienced stronger biochemical alterations, presenting higher LPO increases and greater decreases of antioxidant enzymes, especially noticed at higher salinities. The present findings may indicate that climate change, including predicted drought periods that will increase salinities in aquatic systems, will seriously affect the clam R. philippinarum, especially those inhabiting contaminated ecosystems.Science of The Total Environment 10/2015; 541:1106-1114. DOI:10.1016/j.scitotenv.2015.09.149 · 4.10 Impact Factor
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