ABSTRACT
Glutathione has long been considered a key biomarker for determining the antioxidant response of the cell. Hence, it is a primary marker for reactive oxygen species studies. The method utilizes Ortho-phthalaldehyde (OPA) to quantify the cellular concentration of glutathione(s). OPA conjugates with reduced glutathione (GSH) via sulfhydryl binding to subsequently form an isoindole, resulting in a highly fluorescent conjugate. To attain an accurate result of both oxidized glutathione (GSSG) and GSH, a combination of masking agents and reducing agents, which have been implemented in this protocol, are required. Treatments may also impact cellular viability. Hence, normalization via protein assay is presented in this multiparametric assay. The assay demonstrates a pseudo-linear detection range of 0.234 - 30µM (R2=0.9932±0.007 (N=12)) specific to GSH. The proposed assay also allows for the determination of oxidized glutathione with the addition of the masking agent N-ethylmaleimide to bind reduced glutathione, and the reducing agent tris(2-carboxyethyl) phosphine is introduced to cleave the disulfide bond in GSSG to produce two molecules of GSH. The assay is used in combination with a validated bicinchoninic acid assay for protein quantification and an adenylate kinase assay for cytotoxicity assessment.
Subject(s)
Glutathione , Oxidation-Reduction , o-Phthalaldehyde , o-Phthalaldehyde/chemistry , Glutathione/analysis , Glutathione/chemistry , Glutathione/metabolism , Humans , Animals , Glutathione Disulfide/analysis , Glutathione Disulfide/metabolism , Glutathione Disulfide/chemistry , Phosphines/chemistryABSTRACT
Glutathione (GSH) is indispensable for maintaining redox homeostasis in biological fluids and serves as a key component in cellular defense mechanisms. Accurate assessment of GSH relative to its oxidized counterpart, glutathione disulfide (GSSG), is critical for the early diagnosis and understanding of conditions related to oxidative stress. Despite existing methods for their quantification, the label-free and simultaneous measurement of GSH and GSSG in biological fluid presents significant challenges. Herein, we report the use of an alpha-hederin (Ah) nanopore for the direct measurement of the GSH:GSSG ratio in simulated biological fluid, containing fetal bovine serum (FBS). This system hinges on detecting characteristic relative ion blockades (ΔI/Io) as GSH and GSSG molecules pass through the Ah nanopore under an applied electric field. The distinct current blockage signals derived from the translocation of GSH and GSSG enabled us to determine the molar ratio of GSH and its oxidized form. Notably, the interactions between the hydroxyl groups of the sugar moiety lining the nanopore's inner surface and the sulfhydryl group of GSH significantly influence the translocation dynamics, resulting in a longer translocation time for GSH compared to GSSG. The Ah nanopore technology proposed in this study offers a promising approach for real-time, single molecule-level monitoring of glutathione redox status in biological fluids, eliminating the need for labeling or extensive sample preparation.
Subject(s)
Biosensing Techniques , Glutathione Disulfide , Glutathione , Nanopores , Oxidation-Reduction , Glutathione/chemistry , Glutathione/analysis , Glutathione/blood , Biosensing Techniques/methods , Glutathione Disulfide/analysis , Glutathione Disulfide/chemistry , Glutathione Disulfide/blood , Animals , Humans , Cattle , Oxidative StressABSTRACT
A novel method is introduced for extracting and enriching Cd(II) and Pb(II) from edible oils using glutathione disulfide (GSSG) as both an extractant and a phase-separation agent. The ions in the oils were initially extracted into an aqueous solution containing GSSG. After mixing the solution with acetonitrile at the appropriate volume ratio, a new phase formed, resulting in enrichment of the analytes. The experimental conditions were optimized using response surface methodology with a central composite design. Under optimal conditions, the method offered a combined enrichment factor of >660, with combined extraction efficiencies of 84.31% and 83.35% for Cd(II) and Pb(II), respectively. Finally, the method was conjugated to capillary electrophoresis to determine Cd(II) and Pb(II) in edible oil samples, with detection limits of 0.45 and 1.24 ppb, respectively. In comparison to traditional approaches, the GSSG-based method demonstrates rapidity, efficiency, and recyclability in extracting heavy metal ions from complex matrices.
Subject(s)
Cadmium , Electrophoresis, Capillary , Food Contamination , Glutathione Disulfide , Lead , Plant Oils , Cadmium/isolation & purification , Cadmium/chemistry , Cadmium/analysis , Electrophoresis, Capillary/methods , Lead/isolation & purification , Lead/analysis , Lead/chemistry , Plant Oils/chemistry , Plant Oils/isolation & purification , Food Contamination/analysis , Glutathione Disulfide/analysis , Glutathione Disulfide/chemistry , Glutathione Disulfide/isolation & purification , Chemical Fractionation/methodsABSTRACT
The antioxidant capacity of wine depends on its quality and aging potential. Aging on lees can improve this capacity thanks to the release of glutathione (GSH), as can the addition of yeast derivatives (YD). Therefore, the GSH potential of wine lees (WL) and YD requires investigation. We propose an optimized method to extract and quantify GSH from WL and YD. First, a method was developed to detect and quantify GSH and glutathione disulfide (GSSG) using LC-HRMS. Second, Box-Behnken response surface methodologies (RSM) were applied to both matrices. Results showed that the main parameter affecting GSH extraction efficiency was ethanol concentration. Quantitation of various samples revealed GSH concentrations of up to 900 µg/g for WL and 40 mg/g for YD. To our knowledge, the absolute quantitation of GSH/GSSG in these matrices has not been reported until now.
Subject(s)
Saccharomyces cerevisiae , Wine , Glutathione Disulfide/analysis , Wine/analysis , Glutathione/analysis , Antioxidants/analysisABSTRACT
Metabolic stress caused by a lack of glucose significantly affects the state of red blood cells, where glycolysis is the main pathway for the production of ATP. Hypoglycemia can be both physiological (occurring during fasting and heavy physical exertion) and pathological (accompanying a number of diseases, such as diabetes mellitus). In this study, we have characterized the state of isolated erythrocytes under metabolic stress caused by the absence of glucose. It was established that 24 h of incubation of the erythrocytes in a glucose-free medium to simulate blood plasma led to a two-fold decrease in the ATP level into them. The cell size, as well as intracellular sodium concentration increased. These findings could be the result of a disruption in ion transporter functioning because of a decrease in the ATP level. The calcium level remained unchanged. With a lack of glucose in the medium of isolated erythrocytes, there was no increase in ROS and a significant change in the level of nitric oxide, while the level of the main low-molecular weight thiol of cells, glutathione (GSH) decreased by almost 2 times. It was found that the metabolic stress of isolated red blood cells induced hemoglobin glutathionylation despite the absence of ROS growth. The cause was the lack of ATP, which led to a decrease in the level of GSH because of the inhibition of its synthesis and, probably, due to a decrease in the NADPH level required for glutathione (GSSG) reduction and protein deglutathionylation. Thus, erythrocyte metabolic stress induced hemoglobin glutathionylation, which is not associated with an increase in ROS. This may have an important physiological significance, since glutathionylation of hemoglobin changes its affinity for oxygen.
Subject(s)
Glutathione , Hemoglobins , Glutathione Disulfide/analysis , Glutathione Disulfide/metabolism , Reactive Oxygen Species/metabolism , Oxidation-Reduction , Glutathione/analysis , Glutathione/metabolism , Hemoglobins/analysis , Hemoglobins/metabolism , Erythrocytes/chemistry , Erythrocytes/metabolism , Oxidative Stress , Glucose/analysis , Glucose/metabolism , Adenosine TriphosphateABSTRACT
Oxidation of glutathione (GSH) to its disulfide dimer (GSSG) is the major mechanism by which cells balance reactive oxygen species (ROS) and mitigate oxidative stress. Thus, measuring the ratio of GSH/GSSG is an ideal way to assess oxidative stress within a cell. Quantitative mass spectrometry offers an ideal method to measure the GSH/GSSG ratio and can be applied to a variety of biological matrices and disease models. The following chapter details the design, optimization, and execution of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to measure the GSH/GSSG ratio.
Subject(s)
Glutathione , Tandem Mass Spectrometry , Glutathione Disulfide/analysis , Chromatography, Liquid/methods , Tandem Mass Spectrometry/methods , Glutathione/metabolism , Oxidative Stress , Oxidation-ReductionABSTRACT
Polysulfide degradation in wine can result in hydrogen sulfide (H2S) release, imparting a rotten-egg smell that is detrimental to wine quality. Although the presence of wine polysulfides has been demonstrated, their biogenesis remains unclear. This study investigated the role of Saccharomyces cerevisiae in polysulfide formation during fermentation, with and without 5 mM cysteine supplementation as an H2S source. Using an established liquid chromatography-tandem mass spectrometry method, monobromobimane derivatives of hydropolysulfides, including CysSSSH, CysSSSSH and GSSSSH, and two oxidized polysulfides, GSSG and GSSSSG, were detected in yeast cells at the end of fermentation in a grape juice-like medium. Polysulfide production by four S. cerevisiae single deletion mutants (BY4743 Δcys3, Δcys4, Δmet17 and Δtum1) showed no significant differences compared to BY4743, suggesting that uncharacterized pathways maintain cellular polysulfide homeostasis. Five mM cysteine addition increased the formation of shorter sulfur chain species, including GSS-bimane and GSSG, but did not elevate levels of longer sulfur chain species. Additionally, polysulfides with even numbers of sulfur atoms tended to predominate in cellular lysates. Oxidized polysulfides and longer chain hydropolysulfides were not detected in finished wines. This evidence suggests that these polysulfides are unstable in wine-like environments or not transported extracellularly. Collectively, our data illustrate the complexity of yeast polysulfide metabolism under fermentation conditions.
Subject(s)
Vitis , Wine , Wine/analysis , Saccharomyces cerevisiae/metabolism , Vitis/metabolism , Cysteine/analysis , Glutathione Disulfide/analysis , Glutathione Disulfide/metabolism , Fermentation , Sulfur/metabolism , Dietary SupplementsABSTRACT
A new approach has been developed for the direct determination of reduced (glutathione [GSH]) and oxidized (glutathione disulfide [GSSG]) GSH in whole blood by means of capillary electrophoresis. Its features include GSH-stabilizing sample preparation, the use of an internal standard, and pH-mediated stacking. Blood stabilized with acid citrate and K3 EDTA was treated with acetonitrile with N-ethylmaleimide, and then the analytes were extracted with diethyl ether. The total analysis time was 8 min using a 50-µm (i.d.) by 32.5-cm (eff. length) silica capillary. The background electrolyte was 0.075-M citrate Na pH 5.8 with 200-µM cetyltrimethylammonium bromide and 5-µM sodium dodecyl sulfate, and the separation voltage was -14 kV. The quantification limit (S/N = 15) of the method was 1.5 µM for GSSG. The accuracy levels of GSH and GSSG analysis were 104% and 103%, respectively, and between-run precision levels were 2.6% and 3.2%, respectively. Analysis of blood samples from healthy volunteers (N = 24) showed that the levels of GSH and GSSG and the GSH/GSSG ratio in the whole blood were 1.05 ± 0.14 mM, 3.9 ± 1.25 µM, and 256 ± 94, respectively. Thus, the presented approach can be used in clinical and laboratory practice.
Subject(s)
Ether , Glutathione , Acetonitriles , Cetrimonium , Citrates , Edetic Acid , Electrophoresis, Capillary/methods , Ethylmaleimide , Glutathione/analysis , Glutathione Disulfide/analysis , Humans , Hydrogen-Ion Concentration , Silicon Dioxide , Sodium Dodecyl SulfateABSTRACT
A fast, sensitive and reproducible method using LC-MS/MS for simultaneous quantification of glutathione (GSH), glutathione disulfide (GSSG) and glutathione-S-sulfonate (GSSO3H) was developed, optimised and applied in analysis of grape juice and wine samples. The results show that only GSH (10-60 mg·L-1) and GSSG (2-11 mg·L-1) are found in grape juice when SO2 is not added. GSSO3H was detected in must samples treated with SO2 but only at a low concentration (<1 mg L-1). In the wine samples, the dominant form of glutathione was GSSO3H (5-11 mg L-1), followed by GSH (0-5 mg L-1) and GSSG (0-6 mg L-1), underscoring the importance of GSSO3H quantification. GSSO3H formation in wine was correlated with the total SO2 level in the wine. We believe this is the first report on GSSO3H quantification in wine.
Subject(s)
Vitis , Wine , Chromatography, Liquid , Glutathione/analysis , Glutathione Disulfide/analysis , Tandem Mass Spectrometry , Wine/analysisABSTRACT
Evaluation of the compounds and metabolites, and studying their side effects in the workplace is essential. This study was designed to evaluate the exposure of dry cleaning workers to perchloroethylene (PEC), and its liver and kidney damage, and oxidative stress in B-lymphocytes isolated from the workers. Blood samples were evaluated for liver (alanine transaminase [ALT] and aspartate transaminase [AST]) and kidney (BUN and creatinine) markers. For measurement of PEC, exhaled, personal, and ambient air samples were collected and analyzed gas chromatography (GC-FID) through the NIOSH 1003 and 3704 methods. Also, the parameters of oxidative stress including the level of reactive oxygen species (ROS), glutathione (GSH), oxidized glutathione (GSSG), and lipid peroxidation (LPO) in B-lymphocytes were evaluated. The results showed that the levels of liver enzymes ALT and AST in dry cleaning workers are higher than in the control group. The personal exposure levels and exhaled air concentration of PEC in dry cleaning workers were above the recommended national occupational exposure limits (OELs) and the biological exposure index (BEI). The levels of ROS, LPO, and GSSG in B-lymphocytes from the dry cleaning workers are higher than the control group, and the levels of GSH in dry cleaning workers are lower. The results suggested that exposure of dry cleaning workers to PEC could be associated with liver damage and oxidative damage in B-lymphocytes.
Subject(s)
Air Pollutants, Occupational , Laundering , Tetrachloroethylene , Air Pollutants, Occupational/analysis , Air Pollutants, Occupational/toxicity , Environmental Monitoring/methods , Glutathione Disulfide/analysis , Humans , Laundering/methods , Lymphocytes , Oxidative Stress , Reactive Oxygen Species , Tetrachloroethylene/analysis , Tetrachloroethylene/toxicityABSTRACT
Glutathione and its disulfide were determined in a single run using liquid chromatography with on-line post-column derivatization and fluorimetric detection (340 nm/425 nm). The analytes were separated using a reversed-phase column capable of operating at 100% aqueous mobile phase and detected following direct on-line reaction with o-phthalaldehyde (7.5 mmol L-1) in highly basic medium (0.37 mol L-1 NaOH). The instrumental and chemical variables were carefully investigated towards high sensitivity and throughput, while special attention was paid to validating potential matrix effects. Glutathione and its disulfide could be selectively determined with respective LODs of 0.10 and 0.30 µmol L-1 in the absence of matrix effect (<6%). The endogenous content of the analytes was accurately determined in various food samples with recoveries ranging between 80 and 120% in all cases. The proposed method is reliable and promising as a generic analytical tool for the convenient estimation of the redox status of glutathione in various food matrices.
Subject(s)
Chromatography, Liquid/methods , Food Analysis/methods , Glutathione Disulfide/analysis , Glutathione/analysis , Chromatography, High Pressure Liquid/methods , Limit of Detection , Oxidation-Reduction , Vegetables/chemistry , Wine/analysis , o-Phthalaldehyde/chemistryABSTRACT
Accumulation, contents of protein, non-enzymatic antioxidant glutathione (GSH and GSSG), lipid peroxidation product (melondialdehyde-MDA) and organic acids (fumarate, succinate, malate and citrate), and activities of neurological (acetylcholinesterase-AChE), detoxification (glutathione S-transferase-GST) and metabolic (lactate dehydrogenase-LDH, aspartate transaminase-AST and alanine transaminase-ALT) enzymes were recorded in the hatchlings of Cyprinus carpio, Ctenopharyngodon idella, Labeo rohita and Cirrhinus mrigala after 7 and 14 days exposure and 10 days post exposure (recovery period) to sublethal concentrations (0.005, 0.01, 0.02 and 0.05 mg/L) of triclosan, a highly toxic and persistent biocide used in personal care products. Accumulation was maximum between 7-14 days at 0.01 mg/L for C. carpio and L. rohita but at 0.005 mg/L for C. idella and C. mrigala. No triclosan was observed at 0.005 mg/L in C. carpio and C. mrigala after recovery. Significant decline in protein, glutathione and acetylcholinesterase but increase in glutathione S-transferase, lactate dehydrogenase, aspartate transaminase, alanine transaminase, melondialdehyde and organic acids over control during exposure continued till the end of recovery period. Integrated biomarker response (IBR) analysis depicted higher star plot area for glutathione and glutathione S-transferase during initial 7 days of exposure, thereafter, during 7-14 days of exposure and the recovery period, higher star plot area was observed for acetylcholinesterase, aspartate transaminase, alanine transaminase and organic acids. Higher star plot area was observed for protein in all the species throughout the study. The study shows that L. rohita is most sensitive and glutathione, acetylcholinesterase, aspartate transaminase and alanine transaminase are the biomarkers for the toxicity of sublethal concentrations of TCS.
Subject(s)
Anti-Infective Agents, Local/toxicity , Biomarkers/analysis , Carps/growth & development , Oxidants/toxicity , Triclosan/toxicity , Water Pollutants, Chemical/toxicity , Animals , Anti-Infective Agents, Local/administration & dosage , Anti-Infective Agents, Local/pharmacokinetics , Carps/metabolism , Citric Acid/analysis , Cosmetics/chemistry , Dicarboxylic Acids/analysis , Dose-Response Relationship, Drug , Enzymes/analysis , Glutathione/analysis , Glutathione Disulfide/analysis , Malondialdehyde/analysis , Oxidants/administration & dosage , Oxidants/pharmacokinetics , Proteins/analysis , Species Specificity , Triclosan/administration & dosage , Triclosan/pharmacokinetics , Water Pollutants, Chemical/administration & dosage , Water Pollutants, Chemical/pharmacokineticsABSTRACT
The present paper is aimed at providing an overview of the recent advances in the electrochemical sensing of glutathione (GSH), an important electrochemically and biologically active molecule, for the period 2012-2018. Herein, the analytical performances of newly developed electrochemical methods, procedures and protocols for GSH sensing are comprehensively and critically discussed with respect to the type of method, electrodes used (new electrode modifications, advanced materials and formats), sample matrices, and basic validation parameters obtained (limit of detection, linear dynamic range, precision, selectivity/evaluation of interferences). This paper considers electrochemical methods used alone as well as the hyphenated methods with electrochemical detection (ECD), such as HPLC-ECD or CE-ECD. The practical applicability of the platforms developed for GSH detection and quantification is mostly focused on pharmaceutical and biomedical analysis. The most significant electrochemical approaches for GSH detection in multicomponent analyte samples and multicomponent matrices and for real-time in vivo GSH analysis are highlighted. The great variability in the electrochemical techniques, electrode approaches, and obtainable performance parameters, discussed in this review, brought new insights not only on current GSH and glutathione disulfide (GSSG) determinations, but, along with this, on the advances in electrochemical analysis from a more general point of view.
Subject(s)
Electrochemical Techniques , Glutathione/analysis , Animals , Glutathione Disulfide/analysis , Humans , Molecular StructureABSTRACT
Nitroxyl (HNO), the one-electron-reduction product of NO has recently been revealed to have potentially beneficial pharmacological properties in cardiovascular health as a result of interactions with specific thiols such as glutathione (GSH). To disentangle the complicated inter-relationship between HNO and GSH in the signal transduction and oxidative pathways, we designed and synthesized a dual-site fluorescent probe NCF to indicate cellular HNO and GSH-GSSG balance. The sensitive and selective detection of HNO was achieved by incorporating an organophosphine group to naphthaldehyde-TCF. Then the resulted fluorescent product is able to monitor the conversion of GSH and GSSG reversibly. Additionally, outstanding biocompatibility make it capable of monitoring intracellular HNO and consequently GSH-GSSG oscillationsin living cells. We anticipate that NCF will be a unique molecular tool to investigate the interplaying roles of HNO and GSH.
Subject(s)
Fluorescent Dyes/chemistry , Glutathione Disulfide/analysis , Glutathione/analysis , Nitrogen Oxides/analysis , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/toxicity , Glutathione/metabolism , Glutathione Disulfide/metabolism , Hep G2 Cells , Humans , Limit of Detection , Microscopy, Confocal/methods , Microscopy, Fluorescence/methods , Models, Chemical , Naphthols/chemical synthesis , Naphthols/chemistry , Naphthols/toxicity , Nitrogen Oxides/metabolism , Phosphines/chemical synthesis , Phosphines/chemistry , Phosphines/toxicityABSTRACT
Identifying and mapping the wide range of sulfur species within complex matrices presents a challenge for understanding the distribution of these important biomolecules within environmental and biological systems. Here, we present a coupled micro X-ray fluorescence (µXRF) and X-ray absorption near-edge structure (XANES) spectroscopy method for determining the presence of specific sulfur species in coral tissues and skeletons at high spatial resolution. By using multiple energy stacks and principal component analysis of a large spectral database, we were able to more accurately identify sulfur species components and distinguish different species and distributions of sulfur formerly unresolved by previous studies. Specifically, coral tissues were dominated by more reduced sulfur species, such as glutathione disulfide, cysteine, and sulfoxide, as well as organic sulfate as represented by chondroitin sulfate. Sulfoxide distributions were visually correlated with the presence of zooxanthellae endosymbionts. Coral skeletons were composed primarily of carbonate-associated sulfate (CAS) along with minor contributions from organic sulfate and a separate inorganic sulfate likely in the form of adsorbed sulfate. This coupled XRF-XANES approach allows for a more accurate and informative view of sulfur within biological systems in situ and holds great promise for pairing with other techniques to allow for a more encompassing understanding of elemental distributions within the environment.
Subject(s)
Anthozoa/chemistry , Cysteine/analysis , Glutathione Disulfide/analysis , Sulfates/analysis , Animals , Chondroitin Sulfates/analysis , Spectrometry, X-Ray Emission , X-Ray Absorption SpectroscopyABSTRACT
Glutathione is a ubiquitous antioxidant that protects cells against reactive oxygen species and other chemical stressors. Despite its functional importance, the impact of genetics on the glutathione system has yet to be fully appreciated. Here, we investigated the heritability of glutathione levels and redox status in a disease-relevant condition: advanced age. We assembled a panel of 18-21-month-old mice representing 19 inbred strains and quantified the levels of reduced and oxidized glutathione, and their sums and ratios, in liver, kidney, heart, pancreas, cerebral cortex, and striatum. Heritability values were calculated for each phenotype and the results varied by tissue of origin. Cardiac glutathione phenotypes exhibited the highest heritabilities (G2 = 0.44-0.67), while striatal glutathione was least heritable (G2 = 0.11-0.29). Statistical relationships between tissues were evaluated, and the emergence of significant correlations suggested that despite tissue-specific heritabilities, at least some shared regulatory mechanisms may exist. Overall, these data highlight another mechanism by which genetic background determines antioxidant protection and stress resistance.
Subject(s)
Glutathione/genetics , Glutathione/metabolism , Animals , Cerebrum/metabolism , Female , Glutathione/analysis , Glutathione Disulfide/analysis , Glutathione Disulfide/genetics , Glutathione Disulfide/metabolism , Kidney/metabolism , Liver/metabolism , Mice , Mice, Inbred Strains , Myocardium/metabolism , Organ Specificity , Pancreas/metabolism , Phenotype , Quantitative Trait, Heritable , Species SpecificityABSTRACT
PURPOSE: To investigate the effects of Murici extract on the brain excitability-dependent phenomenon known as cortical spreading depression (CSD) and on brain oxidative stress. METHODS: Adult and aged Wistar rats were supplemented with murici extract (150 mg/kg/day or 300 mg/kg/day) by gavage for fifteen days. Afterwards, the animals were submitted to a CSD electrophysiological recording and to brain oxidative stress evaluation. RESULTS: Our results showed that aging decreased CSD propagation velocity, catalase activity and glutathione/oxidized glutathione ratio (GSH/GSSG) in the brain cortex of the rats, and increased malondialdehyde (MDA) concentrations and superoxide dismutase (SOD) activity. The highest dose (300 mg/kg/day) of murici extract accelerated CSD, whereas the lowest (150mg/kg/day) decelerated, in both adult and aged animals. In contrast, aged animals supplemented with murici extract in both doses presented low MDA levels and high GSG/GSSG ratio in comparison to the control-aged animals. CONCLUSION: Murici extract supplementation seems to revert detrimental effects in aged brains and could be considered as a strategy in the treatment of pathologies related to aging and cortical spreading depression.
Subject(s)
Aging/physiology , Antioxidants/pharmacology , Cerebral Cortex/drug effects , Cortical Spreading Depression/drug effects , Malpighiaceae/chemistry , Oxidative Stress/drug effects , Age Factors , Animals , Catalase/analysis , Cerebral Cortex/metabolism , Cortical Spreading Depression/physiology , Dietary Supplements , Glutathione/analysis , Glutathione Disulfide/analysis , Lipid Peroxidation , Male , Malondialdehyde/analysis , Oxidative Stress/physiology , Rats, Wistar , Reference Values , Reproducibility of Results , Superoxide Dismutase/analysisABSTRACT
Abstract Purpose: To investigate the effects of Murici extract on the brain excitability-dependent phenomenon known as cortical spreading depression (CSD) and on brain oxidative stress. Methods: Adult and aged Wistar rats were supplemented with murici extract (150 mg/kg/day or 300 mg/kg/day) by gavage for fifteen days. Afterwards, the animals were submitted to a CSD electrophysiological recording and to brain oxidative stress evaluation. Results: Our results showed that aging decreased CSD propagation velocity, catalase activity and glutathione/oxidized glutathione ratio (GSH/GSSG) in the brain cortex of the rats, and increased malondialdehyde (MDA) concentrations and superoxide dismutase (SOD) activity. The highest dose (300 mg/kg/day) of murici extract accelerated CSD, whereas the lowest (150mg/kg/day) decelerated, in both adult and aged animals. In contrast, aged animals supplemented with murici extract in both doses presented low MDA levels and high GSG/GSSG ratio in comparison to the control-aged animals. Conclusion: Murici extract supplementation seems to revert detrimental effects in aged brains and could be considered as a strategy in the treatment of pathologies related to aging and cortical spreading depression.
Subject(s)
Animals , Male , Aging/physiology , Cerebral Cortex/drug effects , Oxidative Stress/drug effects , Malpighiaceae/chemistry , Antioxidants/pharmacology , Reference Values , Cortical Spreading Depression/drug effects , Cortical Spreading Depression/physiology , Superoxide Dismutase/analysis , Lipid Peroxidation , Catalase/analysis , Cerebral Cortex/metabolism , Reproducibility of Results , Age Factors , Rats, Wistar , Oxidative Stress/physiology , Glutathione Disulfide/analysis , Dietary Supplements , Glutathione/analysis , Malondialdehyde/analysisABSTRACT
Glutathione (GSH) forms conjugates with polyamines in prokaryotes and eukaryotes. There is also evidence suggesting cross-talk between GSH and polyamines to regulate cellular homeostasis and function, particularly under the conditions of oxidative stress. Because of its versatile roles in cell metabolism and function, a number of high performance liquid chromatography (HPLC) methods have been developed for glutathione analysis. Here, we describe our rapid and sensitive method for the analysis of GSH and the oxidized form of glutathione (GS-SG) in animal tissues and cells by HPLC involving pre-column derivatization with o-phthalaldehyde (OPA). OPA reacts very rapidly (within 1 min) with S-carboxymethyl-glutathione at room temperatures (e.g., 20-25 °C) in an autosampler, and their derivatives are immediately injected into the HPLC column without any need for extraction. This method requires two simple steps (a total of 15 min) before samples are loaded into the autosampler: (a) the conversion of GS-SG into GSH by 2-mercaptoethanol; and (b) the oxidation of GSH by iodoacetic acid to yield S-carboxymethyl-glutathione. The autosampler is programmed to mix S-carboxymethyl-glutathione with OPA for 1 min to generate a highly fluorescent derivative for HPLC separation and detection (excitation wavelength 340 nm and emission wavelength 450 nm). The detection limit for GSH and GS-SG is 15 pmol/ml or 375 fmol/injection. The total time for chromatographic separation (including column regeneration) is 16 min for each sample. Our routine HPLC technique is applicable for analyses of cysteine and cystine, as well as polyamines and GSH-polyamine conjugates in biological samples.
Subject(s)
Chromatography, High Pressure Liquid , Glutathione/analysis , Glutathione/chemistry , o-Phthalaldehyde/chemistry , Animals , Cysteine/chemistry , Glutathione Disulfide/analysis , Glutathione Disulfide/chemistry , Humans , Plant Extracts/analysis , Plant Extracts/chemistry , Polyamines/chemistryABSTRACT
Oxidative stress and transcriptional pathways of nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear factor kappa-B (NF-κB) are critically involved in the etiopathology of amebic liver abscess (ALA). In this work, we studied the relationship between the adrenergic nervous system and ALA in the hamster. ALA was visible at 12 h of infection. While 6-hydroxidopamine (6-OHDA) decreased infection, propranolol (ß-adrenergic blocker) treatment was associated with less extensive liver damage, and phentolamine treatment (α-adrenergic blocker) significantly reduced ALA compared to 6-OHDA and propranolol. Serum enzymatic activities of alanine aminotransferase (ALT) and γ-glutamyl transpeptidase (γ-GTP) were increased at 12 h post-infection. Chemical denervation and α and ß-adrenergic blockers decreased ALT to normal levels, while 6-OHDA and propranolol showed a trend to decrease γ-GTP but phentolamine significantly reduced γ-GTP. Amebic infection increased oxidized glutathione (GSSG) and decreased both reduced glutathione (GSH) and the GSH/GSSG ratio. Propranolol and 6-OHDA showed a tendency to decrease GSSG. However, GSH, GSSG and GSH/GSSG returned to normal levels with phentolamine. Furthermore, amebic infection increased pNF-κB and interleukin-1ß (IL-1ß), and showed a tendency to decrease hemoxigenase-1 (HO-1), but not Nrf2. Chemical denervation showed a trend to decrease pNF-κB and IL-1ß, and neither Nrf2 nor HO-1 increased significantly. In addition, NF-κB and IL-1ß were attenuated by propranolol and phentolamine treatments, although phentolamine showed significant overexpression of Nrf2 and HO-1. This suggests that the adrenergic system may be involved in oxidative stress and in modulation of the Nrf2 and NF-κB pathways during ALA development.