ABSTRACT: A method for detecting glutathione selectively in whole blood deposited on filter paper is described. GSH is fractionated from proteins, hemoglobin and other potentially interfering components and determined using a resorufin-acrylate fluorescent probe. The relative standard deviation is lower than 5% (n = 5). Recoveries of GSH from whole blood are between 94% and 108.6%.
Project description:BACKGROUND/OBJECTIVES:Glutathione (GSH) is the most abundant endogenous antioxidant and a critical regulator of oxidative stress. Maintenance of optimal tissues for GSH levels may be an important strategy for the prevention of oxidative stress-related diseases. We investigated if oral administration of liposomal GSH is effective at enhancing GSH levels in vivo. SUBJECTS/METHODS:A 1-month pilot clinical study of oral liposomal GSH administration at two doses (500 and 1000?mg of GSH per day) was conducted in healthy adults. GSH levels in whole blood, erythrocytes, plasma and peripheral blood mononuclear cells (PBMCs) were assessed in 12 subjects at the baseline and after 1, 2 and 4 weeks of GSH administration. RESULTS:GSH levels were elevated after 1 week with maximum increases of 40% in whole blood, 25% in erythrocytes, 28% in plasma and 100% in PBMCs occurring after 2 weeks (P<0.05). GSH increases were accompanied by reductions in oxidative stress biomarkers, including decreases of 35% in plasma 8-isoprostane and 20% in oxidized:reduced GSH ratios (P<0.05). Enhancements in immune function markers were observed with liposomal GSH administration including Natural killer (NK) cell cytotoxicity, which was elevated by up to 400% by 2 weeks (P<0.05), and lymphocyte proliferation, which was elevated by up to 60% after 2 weeks (P<0.05). Overall, there were no differences observed between dose groups, but statistical power was limited due to the small sample size in this study. CONCLUSIONS:Collectively, these preliminary findings support the effectiveness of daily liposomal GSH administration at elevating stores of GSH and impacting the immune function and levels of oxidative stress.
Project description:A fluorescent probe for glutathione (GSH) detection was developed. Our study indicates a possible mechanism which couples a conjugate addition and micelle-catalyzed large membered ring formation/elimination sequence. This method enables excellent selectivity towards GSH over other biological thiols such as cysteine (Cys) and homocysteine (Hcy). The proposed method is precise with a relative standard deviation (R.S.D) lower than 6% (n = 3) and has been successfully applied to determine GSH in human plasma with recoveries between 99.2% and 102.3%.
Project description:Human liver glutathione S-transferases (GSH S-transferases) were fractionated into cationic and anionic proteins. During fractionation with (NH4)2SO4 the anionic GSH S-transferases are concentrated in the 65%-saturated-(NH4)2SO4 fraction, whereas the cationic GSH S-transferases separate in the 80%-saturated-(NH4)2SO4 fraction. From the 65%-saturated-(NH4)2SO4 fraction two new anionic GSH S-transferases, omega and psi, were purified to homogeneity by using ion-exchange chromatography on DEAE-cellulose, Sephadex G-200 gel filtration, affinity chromatography on GSH bound to epoxy-activated Sepharose and isoelectric focusing. By a similar procedure, cationic GSH S-transferases were purified from the 80%-saturated-(NH4)2SO4 fraction. Isoelectric points of GSH S-transferases omega and psi are 4.6 and 5.4 respectively. GSH S-transferase omega is the major anionic GSH S-transferase of human liver, whereas GSH S-transferase psi is present only in traces. The subunit mol.wt. of GSH S-transferase omega is about 22500, whereas that of cationic GSH S-transferases is about 24500. Kinetic and structural properties as well as the amino acid composition of GSH S-transferase omega are described. The antibodies raised against cationic GSH S-transferases cross-react with GSH S-transferase omega. There are significant differences between the catalytic properties of GSH S-transferase omega and the cationic GSH S-transferases. GSH peroxidase II activity is displayed by all five cationic GSH S-transferases, whereas both anionic GSH S-transferases do not display this activity.
Project description:t-Butyl hydroperoxide and cumene hydroperoxide, both known to be substrates for glutathione peroxidase, were used to oxidize erythrocyte GSH. Addition of concentrations of hydroperoxides equimolar with respect to GSH in the erythrocytes or whole blood quantitatively oxidizes GSH in the erythrocytes with a half-time of 4.5s at 37 degrees C and about three times as long at 4 degrees C. In the presence of glucose, normal erythrocytes regenerate all the GSH in about 25min. However, glucose 6-phosphate dehydrogenase-deficient erythrocytes failed to regenerate GSH. Treatment of erythrocytes with hydroperoxides does not affect erythrocyte survival in rabbits. Oxidation of erythrocyte GSH with equimolar concentrations of hydroperoxides does not lead to formation of mixed disulphides of haemoglobin and GSH. The hydroperoxides do not affect erythrocyte glycolytic and hexose monophosphate-shunt-pathway enzymes. Previous studies on transport of GSSG from erythrocytes were confirmed by using t-butyl hydroperoxide to oxidize erythrocyte GSH.
Project description:The essential thiol antioxidant, glutathione (GSH) is recruited into the nucleus of mammalian cells early in cell proliferation, suggesting a key role of the nuclear thiol pool in cell cycle regulation. However, the functions of nuclear GSH (GSHn) and its integration with the cytoplasmic GSH (GSHc) pools in whole cell redox homeostasis and signaling are unknown. Here we show that GSH is recruited into the nucleus early in cell proliferation in Arabidopsis thaliana, confirming the requirement for localization of GSH in the nucleus as a universal feature of cell cycle regulation. GSH accumulation in the nucleus was triggered by treatments that synchronize cells at G1/S as identified by flow cytometry and marker transcripts. Significant decreases in transcripts associated with oxidative signaling and stress tolerance occurred when GSH was localized in the nucleus. Increases in GSH1 and GSH2 transcripts accompanied the large increase in total cellular GSH observed during cell proliferation, but only GSH2 was differentially expressed in cells with high GSHn relative to those with an even intracellular distribution of GSH. Of the 7 Bcl-2 associated (BAG) genes in A. thaliana, only the nuclear-localized BAG 6 was differentially expressed in cells with high GSHn compared to GSHc. We conclude that GSHn is associated with decreased oxidative signaling and stress responses and that whole cell redox homeostasis is restored as the cell cycle progresses by enhanced GSH synthesis and accumulation in the cytoplasm. Arabidopsis cells were harvested at points during cell proliferation where GSH was localized either in the nucleus (GSHn) or where GSH was distributed throughout the cytoplasm (GSHc) for RNA extraction and hybridization on Affymetrix microarrays. We selected three stages where the GSH was into the nucleus and three stages where the GSH was distributed throughout the cells.
Project description:<h4>Background</h4>In vitro and rodent studies have shown that arsenic (As) exposure can deplete glutathione (GSH) and induce oxidative stress. GSH is the primary intracellular antioxidant; it donates an electron to reactive oxygen species, thus producing glutathione disulfide (GSSG). Cysteine (Cys) and cystine (CySS) are the predominant thiol/disulfide redox couple found in human plasma. Arsenic, GSH, and Cys are linked in several ways: a) GSH is synthesized via the transsulfuration pathway, and Cys is the rate-limiting substrate; b) intermediates of the methionine cycle regulate both the transsulfuration pathway and As methylation; c) GSH serves as the electron donor for reduction of arsenate to arsenite; and d) As has a high affinity for sulfhydryl groups and therefore binds to GSH and Cys.<h4>Objectives</h4>We tested the hypothesis that As exposure is associated with decreases in GSH and Cys and increases in GSSG and CySS (i.e., a more oxidized environment).<h4>Methods</h4>For this cross-sectional study, the Folate and Oxidative Stress Study, we recruited a total of 378 participants from each of five water As concentration categories: < 10 (n = 76), 10-100 (n = 104), 101-200 (n = 86), 201-300 (n = 67), and > 300 µg/L (n = 45). Concentrations of GSH, GSSG, Cys, and CySS were measured using HPLC.<h4>Results</h4>An interquartile range (IQR) increase in water As was negatively associated with blood GSH (mean change, -25.4 µmol/L; 95% CI: -45.3, -5.31) and plasma CySS (mean change, -3.00 µmol/L; 95% CI: -4.61, -1.40). We observed similar associations with urine and blood As. There were no significant associations between As exposure and blood GSSG or plasma Cys.<h4>Conclusions</h4>The observed associations are consistent with the hypothesis that As may influence concentrations of GSH and other nonprotein sulfhydryls through binding and irreversible loss in bile and/or possibly in urine.
Project description:Mitochondrial disorders are associated with decreased energy production and redox imbalance. Glutathione plays a central role in redox signaling and protecting cells from oxidative damage. In order to understand the consequences of mitochondrial dysfunction on in vivo redox status, and to determine how this varies by mitochondrial disease subtype and clinical severity, we used a sensitive tandem mass spectrometry assay to precisely quantify whole blood reduced (GSH) and oxidized (GSSG) glutathione levels in a large cohort of mitochondrial disorder patients. Glutathione redox potential was calculated using the Nernst equation. Compared to healthy controls (n = 59), mitochondrial disease patients (n = 58) as a group showed significant redox imbalance (redox potential -251 mV ± 9.7, p<0.0001) with an increased level of oxidation by ? 9 mV compared to controls (-260 mV ± 6.4). Underlying this abnormality were significantly lower whole blood GSH levels (p = 0.0008) and GSH/GSSG ratio (p = 0.0002), and significantly higher GSSG levels (p<0.0001) in mitochondrial disease patients compared to controls. Redox potential was significantly more oxidized in all mitochondrial disease subgroups including Leigh syndrome (n = 15), electron transport chain abnormalities (n = 10), mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (n = 8), mtDNA deletion syndrome (n = 7), mtDNA depletion syndrome (n = 7), and miscellaneous other mitochondrial disorders (n = 11). Patients hospitalized in metabolic crisis (n = 7) showed the greatest degree of redox imbalance at -242 mV ± 7. Peripheral whole blood GSH and GSSG levels are promising biomarkers of mitochondrial dysfunction, and may give insights into the contribution of oxidative stress to the pathophysiology of the various mitochondrial disorders. In particular, evaluation of redox potential may be useful in monitoring of clinical status or response to redox-modulating therapies in clinical trials.
Project description:A depletion of reduced glutathione (GSH) has been observed in pathological conditions and in aging. Measuring GSH in tissues using mouse models is an excellent way to assess GSH depletion and the potential therapeutic efficacy of drugs used to maintain and/or restore cellular redox potential. A high performance liquid chromatography (HPLC) method for the simultaneous determination of GSH and cysteine (Cys) in mouse organs was validated according to USA and European standards. The method was based on separation coupled with ultraviolet detection and precolumn derivatization with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). The required validation parameters, that are, selectivity, linearity, lower limit of quantification, precision, accuracy, recovery, and stability, were studied for spleen, lymph nodes, pancreas, and brain. The results showed that the lower limits of quantification were 0.313??M and 1.25??M for Cys and GSH, respectively. Intraday and interday precisions were less than 11% and 14%, respectively, for both compounds. The mean extraction recoveries of Cys and GSH from all organs were more than 93% and 86%, respectively. Moreover, the stability of both analytes during sample preparation and storage was demonstrated. The method was accurate, reliable, consistent, and reproducible and it was useful to determine Cys and GSH in the organs of different mouse strains.
Project description:A rapid, accurate and simple method was developed for the simultaneous determination of glutathione (GSH) and phytochelatins (PCs) by high-performance liquid chromatography (HPLC) with an evaporative light-scattering detector. GSH, phytochelatin 2 (PC2), PC3, PC4, PC5 and PC6 can be separated with baseline separation within 9 min using a Venusil AA column (250 mm × 4.6 mm i.d., 5 µm particle sizes). Acetonitrile and water containing 0.1% trifluoroacetic acid (0.1%) were employed as the mobile phase for the gradient elution. The drift tube temperature and flow rate of the carrier gas (N2) were 50°C and 1.5 l min-1, respectively. Under optimum conditions, good linear regression equations of six analytes were obtained with the detection limits ranging from 0.2 to 0.5 µg ml-1. The proposed method has been applied successfully for the quantification of GSH and PCs in Perilla frutescens (a cadmium hyperaccumulator) under cadmium stress. The recoveries were between 82.9% and 115.3%.
Project description:Embryonic development involves dramatic changes in cell proliferation and differentiation that must be highly coordinated and tightly regulated. Cellular redox balance is critical for cell fate decisions, but it is susceptible to disruption by endogenous and exogenous sources of oxidative stress. The most abundant endogenous nonprotein antioxidant defense molecule is the tripeptide glutathione (?-glutamylcysteinylglycine, GSH), but the ontogeny of GSH concentration and redox state during early life stages is poorly understood. Here, we describe the GSH redox dynamics during embryonic and early larval development (0-5 days postfertilization) in the zebrafish (Danio rerio), a model vertebrate embryo. We measured reduced and oxidized glutathione using HPLC and calculated the whole embryo total glutathione (GSHT) concentrations and redox potentials (Eh) over 0-120 h of zebrafish development (including mature oocytes, fertilization, midblastula transition, gastrulation, somitogenesis, pharyngula, prehatch embryos, and hatched eleutheroembryos). GSHT concentration doubled between 12h postfertilization (hpf) and hatching. The GSH Eh increased, becoming more oxidizing during the first 12h, and then oscillated around -190 mV through organogenesis, followed by a rapid change, associated with hatching, to a more negative (more reducing) Eh (-220 mV). After hatching, Eh stabilized and remained steady through 120 hpf. The dynamic changes in GSH redox status and concentration defined discrete windows of development: primary organogenesis, organ differentiation, and larval growth. We identified the set of zebrafish genes involved in the synthesis, utilization, and recycling of GSH, including several novel paralogs, and measured how expression of these genes changes during development. Ontogenic changes in the expression of GSH-related genes support the hypothesis that GSH redox state is tightly regulated early in development. This study provides a foundation for understanding the redox regulation of developmental signaling and investigating the effects of oxidative stress during embryogenesis.