Assay of quantitative determination of glutathione disulfide levels using enzymatic recycling methods
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.
- SourceAvailable from: Rosa Freitas
[Show abstract] [Hide abstract]
- "Reduced (GSH) and oxidized (GSSG) glutathione content were determined according to Rahman et al. (2007). For GSH the absorbance was read immediately at 412 nm. "
ABSTRACT: In the present study, the bivalve Scrobicularia plana, collected from two contrasting areas (pristine location and mercury contaminated area), was selected to assess the biochemical alterations imposed by pH decrease, carbamazepine (an antiepileptic) and the combined effect of both stressors. The effects on oxidative stress related biomarkers after 96 h exposure revealed that pH decrease and carbamazepine induced alterations on clams, with greater impacts on individuals from the contaminated area which presented higher mortality, higher lipid peroxidation and higher glutathione S-transferase activity. These results emphasize the risk of extrapolating results from one area to another, since the same species inhabiting different areas may be affected differently when exposed to the same stressors. Furthermore, the results obtained showed that, when combined, the impact of pH decrease and carbamazepine was lower than each stressor acting alone, which could be related to the defence mechanism of valves closure when bivalves are under higher stressful conditions. Copyright © 2015 Elsevier Ltd. All rights reserved.Environmental Pollution 07/2015; 202. DOI:10.1016/j.envpol.2015.03.023 · 3.90 Impact Factor
[Show abstract] [Hide abstract]
- "The colorimetric method described by Okawa et al. (1979) was used. Oxidized glutathione content (GSSG) was determined in liver and small intestine samples following the method of Rahman et al. (2006) and reduced glutathione (GSH) was measured following the glutathione-S-transferase assay described by Brigelius et al. (1983). Samples for glutathione analyses were obtained in the presence of N-ethylmaleimide and deproteinized (1:9, by vol.) using trichloroacetic acid (15% final concentration) as recommended by Asensi et al. (1994). "
ABSTRACT: Oral intake of Microcystin-LR (MC-LR) is the principal route of exposure to this toxin, with prolonged exposure leading to liver damage of unspecific symptomatology. The aim of the present paper was therefore to investigate the liver and intestine damage generated by prolonged oral exposure to low MC-LR doses (50 and 100 μg MC-LR/kg body weight, administrated every 48 hs during a month) in a murine model. We found alterations in TBARS, SOD activity and glutathione content in liver and intestine of mice exposed to both doses of MC-LR. Furthermore, the presence of MC-LR was detected in both organs. We also found hepatic steatosis (3.6 ± 0.6 % and 15.3 ± 1.6 %) and a decrease in intraepithelial lymphocytes (28.7 ± 5.0% and 44.2 ± 8.7%) in intestine of 50- and 100- μg MC-LR/kg treated animals, respectively. This result could have important implications for mucosal immunity, since intraepithelial lymphocytes are the principal effectors of this system. Our results indicate that prolonged oral exposure at 50 μg MC-LR/kg every 48 h generates significant damage not only in liver but also in intestine. This finding calls for a re-appraisal of the currently accepted NOAEL (No Observed Adverse Effect Level), 40 μg MC-LR/kg body weight, used to derive the guideline value for MC-LR in drinking water. Copyright © 2015. Published by Elsevier Ltd.Toxicon 07/2015; DOI:10.1016/j.toxicon.2015.07.011 · 2.58 Impact Factor
[Show abstract] [Hide abstract]
- "Reduced ( GSH ) and oxidized ( GSSG ) content were determined according to Rahman et al . ( 2007 ) , using reduced and oxidized glutathione as standard ( 0e60 mmol / L ) . Absorbance was measured at 412 nm and the results were expressed as mmol per g of FW ."
ABSTRACT: The present study reports metal and arsenic contamination in sediments, as well as element accumulation and partitioning in native (Ruditapes decussatus and Venerupis corrugata) and introduced (Ruditapes philippinarum) clam species living in sympatry at the Óbidos lagoon (Portugal). The biochemical performance and the human health risks derived from the consumption of these species are also discussed. The results obtained showed that R. decussatus was the most abundant species in all the sampling sites, revealing that the introduced clam has not yet supplanted the native species. The concentration of elements was higher in areas with higher Total Organic Matter (TOM) and fines content, being Chromium (Cr), Copper (Cu) and Lead (Pb) the most abundant metals. Clams from these areas showed the highest concentration of elements but the lowest bioaccumulation levels. Furthermore, except for As, higher concentration of elements was found in clams insoluble fraction, the less toxic fraction to the organisms. Due to the low contamination levels and because elements, except As, were mainly allocated to the insoluble fraction, clams presented similar biochemical parameters among distinct areas, with no significant oxidative stress induced. Furthermore, clams from the Óbidos lagoon represent a low health risk to human consumption since, except for As, their contamination levels were below the maximum permissible limits defined by international organizations. Copyright © 2015 Elsevier Ltd. All rights reserved.Marine Environmental Research 06/2015; 109. DOI:10.1016/j.marenvres.2015.06.005 · 2.33 Impact Factor