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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
Source: PubMed

ABSTRACT

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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    • "Reduced (GSH) and oxidized (GSSG) content were spectrophotometrically determined by monitoring the formation of TNB for 10 min at 415 nm[31]. Briefly, 20 µL of cell extract was added to a reaction mixture (0.84 mM DTNB, 0.28 mM NADPH, 5 mM EDTA, in 100 mM phosphate buffer pH 7.5. "
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    ABSTRACT: Hyperglycemia has been implicated in the development of endothelial dysfunction through heightened ROS production. Since nitrones reverse endothelial nitric oxide synthase (eNOS) dysfunction, increase antioxidant enzyme activity, and suppress pro-apoptotic signaling pathway and mitochondrial dysfunction from ROS-induced toxicity, the objective of this study was to determine whether nitrone spin traps DMPO, PBN and PBN-LA were effective at duplicating these effects and improving glucose uptake in an in vitro model of hyperglycemia-induced dysfunction using bovine aortic endothelial cells (BAEC). BAEC were cultured in DMEM medium with low (5.5 mM glucose, LG) or high glucose (50 mM, HG) for 14 days to model in vivo hyperglycemia as experienced in humans with metabolic disease. Improvements in cell viability, intracellular oxidative stress, NO and tetrahydrobiopterin levels, mitochondrial membrane potential, glucose transport, and activity of antioxidant enzymes were measured from single treatment of BAEC cells with nitrones for 24 h after hyperglycemia. Chronic hyperglycemia significantly increased intracellular ROS by 50%, decreased cell viability by 25%, reduced NO bioavailability by 50%, and decreased BH4 levels by 15% thereby decreasing NO production. Intracellular glucose transport and superoxide dismutase (SOD) activity were also decreased by 50% and 25% respectively. Nitrone (PBN and DMPO, 50 μM) treatment of BAEC cells grown in hyperglycemic conditions resulted in the normalization of outcome measures except for SOD and catalase activities. Our findings demonstrate that the nitrones reverse the deleterious effects of hyperglycemia in BAEC cells. We believe that in vivo testing of these nitrone compounds in models of cardiometabolic disease is warranted.
    Full-text · Article · Jan 2016 · Biochemical pharmacology
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    • "Reduced glutathione (GSH) cytosolic content was determined according toRahman et al. (2006), using reduced glutathione as standard. Absorbance was read at 412 nm and GSH expressed in μmol per g of FW. "
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    ABSTRACT: Ocean acidification processes are major threats to marine calcifying organisms, mostly affecting biomineralization related processes. Abiotic stressors acting on marine systems do not act alone, rather in a combination of multiple stressors, especially in coastal habitats such as estuaries, where anthropogenic and environmental pressures are high. Arsenic (As) is a widely distributed contaminant worldwide and its toxicity has been studied on a variety of organisms. However, the effect of low pH on the toxicity of As on marine organisms is unknown. Here, we studied the combined effects of ocean acidification and As exposure on two closely related oyster species (Crassostrea angulata and Crassostrea gigas), by use of a biochemical approach. Oxidative stress related parameters were studied along with the assessment of biomineralization enzymes activity after 28 days of exposure. Results showed that both species were sensitive to all tested conditions (low pH, As and pH + As), showing enhancement of antioxidant and biotransformation defenses and impairment of biomineralization processes. Glutathione S-transferases (GSTs) activity were significantly higher in oysters exposed to As, showing activation of detoxification mechanisms, and a lower GSTs activity was observed in low pH + As condition, indicating an impact on the oysters capacity to detoxify As in a low pH scenario. Carbonic anhydrase (CA) activity was significantly lower in all tested conditions, showing to be affected by both As and low pH, whereas the combined effect of low pH + As was not different from the effect of low pH alone. Multivariate analysis of biochemical data allowed for the comparison of both species performance, showing a clear distinction of response in both species. C. gigas presented overall higher enzymatic activity (GSTs; superoxide dismutase; catalase; CA and acid phosphatase) and higher cytosolic GSH content in As exposed oysters than C. angulata. Results obtained indicate a higher tolerance capacity of the Pacific oyster C. gigas towards the tested conditions.
    Full-text · Article · Jan 2016 · Science of The Total Environment
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    • "The GSH recycling assay was performed according to a protocol by Rahman et al. (2006): The protein precipitation of subcellular fractions was carried out with 5 % mphosphoric acid. After 10 min of incubation at 4 °C, the samples were centrifuged at 12,000 g and 4 °C for 10 min. "
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    ABSTRACT: Aging is characterized by a progressive decrease of cellular functions, because cells gradually lose their capacity to respond to injury. Increased oxidative stress is considered to be one of the major contributors to age-related changes in all organs including the liver. Our study has focused on elucidating whether important antioxidative enzymes, the mTOR pathway, and MAPKs exhibit age-dependent changes in the liver of rats during aging. We found an age-dependent increase of GSH in the cytosol and mitochondria. The aged liver showed an increased SOD enzyme activity, while the CAT enzyme activity decreased. HO-1 and NOS-2 gene expression was lower in adult rats, but up-regulated in aged rats. Western blot analysis revealed that SOD1, SOD2, GPx, GR, γ-GCL, and GSS were age-dependent up-regulated, while CAT remained constant. We also demonstrated that the phosphorylation of Akt, JNK, p38, and TSC2Ser1254 decreased while ERK1/2 and TSC2Thr1462 increased agedependently. Furthermore, our data show that the mTOR pathway seems to be activated in livers of aged rats, and hence stimulating cell proliferation/regeneration, as confirmed by an age-dependent increase of PCNA and p-eIF4ESer209 protein expression. Our data may help to explain the fact that liver cells only proliferate in cases of necessity, like injury and damage. In summary, we have demonstrated that, age-dependent changes of the antioxidant system and stress-related signaling pathways occur in the livers of rats, which may help to better understand organ aging. © 2015, Leibniz Research Centre for Working Environment and Human Factors. All rights reserved.
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