氧化型谷胱甘肽对还原型谷胱甘肽清除自由基的协同作用

利用分光光度法和基质辅助飞行质谱法研究了谷胱甘肽对1,1-二苯基-2-苦肼基(DPPH)自由基的清除作用.通过比较不同浓度和不同配比的还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)以及Na2SeO3混合溶液的自由基清除率,发现GSH/GSSG的配比对自由基清除率有明显影响.当GSH/GSSG的配比大于50∶ 1时,自由基清除率比同浓度的GSH大,且自由基清除率随GSH和GSSG的绝对浓度的增加而明显增加,说明适量的GSSG可协同催化GSH清除自由基过程.质谱测定结果表明: 此协同作用与GSSG 参与自由基清除过程中的自由基反应有关.Na2SeO3对GSH的清除自由基的影响主要是通过与GSH反应生成GSSG来调控GSH/GSSG配比的结果.通过测定和分析一定配比的GSH+GSSG混合溶液与DPPH作用前后的质谱图,提出了少量的GSSG共存下,GSH催化清除DPPH自由基的作用机理.     

Study of Oxidation of Glutathione Treated with Hypochlorous Acid by Capillary Electrophoresis

Glutathione (GSH) protects human and animal's cells by the exchange with reversible oxidized glutathione (GSSG) when cells meet with oxidants, such as hydrogen peroxide (H2O2) and lipid peroxide. But when GSH meets with hypochlorous acid (HOCl), via the myeloperoxidase-catalysed oxidation of chloride by H2O2, the amount of GSSG formed does not account for all the GSH lost, which is quite different from that we has investigated in the reaction of GSH with H2O21. Prutz proposed the fo…     

Selective detection of the reduced form of glutathione (GSH) over the oxidized (GSSG) form using a combination of glutathione reductase and a Tb(III)-cyclen maleimide based lanthanide luminescent 'switch on' assay

The synthesis of a novel Tb(III) luminescent probe for the detection of thiols is presented. The probe 1.Tb, possessing a maleimide moiety, as its sulfhydryl acceptor, was poorly emitting in aqueous pH 7 solution in the absence of a thiol. However, upon addition of thiols such as glutathione (GSH), large enhancements were observed, particularly within the physiological pH range. In contrast no enhancements were observed in the presence of the oxidized form of glutathione (GSSG), except in the presence of the enzyme glutathione reductase and NADPH which enabled 1.Tb to be used to observe the enzymatic reduction of GSSG to GSH in real time.     

Ultrarapid capillary electrophoresis method for the determination of reduced and oxidized glutathione in red blood cells

We describe a very rapid high-performance capillary electrophoresis method for the separation and quantification of reduced (GSH) and oxidized (GSSG) glutathione in red blood cells. Two procedures for sample preparation have been compared, Microcon-10 membrane filtration and acid precipitation. The separation is obtained in an uncoated capillary using a high ionic strength borate buffer at pH 7.8. The intra-assay coefficients of variation (CVs%) are 1.53 and 1.66 for GSH and GSSG, respectively. The run is shorter than 90 s and the migration time is highly reproducible both for GSH (CV% 0.22) and GSSG (CV% 0.17). When the filtration step is used only GSH is found, whereas both GSH and GSSG are detectable after acid precipitation, suggesting that GSSG revealed after acid treatment may be an artefact due to GSH oxidation. Because of its good analytical performance this method could be used for routine red blood cell glutathione measurement in healthy or pathological conditions.     

Dithiophosphate-Induced Redox Conversions of Reduced and Oxidized Glutathione

Phosphorus species are potent modulators of physicochemical and bioactive properties of peptide compounds. O,O-diorganyl dithiophoshoric acids (DTP) form bioactive salts with nitrogen-containing biomolecules; however, their potential as a peptide modifier is poorly known. We synthesized amphiphilic ammonium salts of O,O-dimenthyl DTP with glutathione, a vital tripeptide with antioxidant, protective and regulatory functions. DTP moiety imparted radical scavenging activity to oxidized glutathione (GSSG), modulated the activity of reduced glutathione (GSH) and profoundly improved adsorption and electrooxidation of both glutathione salts on graphene oxide modified electrode. According to NMR spectroscopy and GC–MS, the dithiophosphates persisted against immediate dissociation in an aqueous solution accompanied by hydrolysis of DTP moiety into phosphoric acid, menthol and hydrogen sulfide as well as in situ thiol-disulfide conversions in peptide moieties due to the oxidation of GSH and reduction of GSSG. The thiol content available in dissolved GSH dithiophosphate was more stable during air oxidation compared with free GSH. GSH and the dithiophosphates, unlike DTP, caused a thiol-dependent reduction of MTS tetrazolium salt. The results for the first time suggest O,O-dimenthyl DTP as a redox modifier for glutathione, which releases hydrogen sulfide and induces biorelevant redox conversions of thiol/disulfide groups.     

The Antioxidant Effect of Curcumin and Rutin on Oxidative Stress Biomarkers in Experimentally Induced Periodontitis in Hyperglycemic Wistar Rats

Background: There is a growing interest in the correlation between antioxidants and periodontal disease. In this study, we aimed to investigate the effect of oxidative stress and the impact of two antioxidants, curcumin and rutin, respectively, in the etiopathology of experimentally induced periodontitis in diabetic rats. Methods: Fifty Wistar albino rats were randomly divided into five groups and were induced with diabetes mellitus and periodontitis: (1) (CONTROL)—control group, (2) (DPP)—experimentally induced diabetes mellitus and periodontitis, (3) (DPC)—experimentally induced diabetes mellitus and periodontitis treated with curcumin (C), (4) (DPR)—experimentally induced diabetes mellitus and periodontitis treated with rutin (R) and (5) (DPCR)—experimentally induced diabetes mellitus and periodontitis treated with C and R. We evaluated malondialdehyde (MDA) as a biomarker of oxidative stress and reduced glutathione (GSH), oxidized glutathione (GSSG), GSH/GSSG and catalase (CAT) as biomarkers of the antioxidant capacity in blood harvested from the animals we tested. The MDA levels and CAT activities were also evaluated in the gingival tissue. Results: The control group effect was statistically significantly different from any other groups, regardless of whether or not the treatment was applied. There was also a significant difference between the untreated group and the three treatment groups for variables MDA, GSH, GSSG, GSH/GSSG and CAT. There was no significant difference in the mean effect for the MDA, GSH, GSSG, GSH/GSSG and CAT variables in the treated groups of rats with curcumin, rutin and the combination of curcumin and rutin. Conclusions: The oral administration of curcumin and rutin, single or combined, could reduce the oxidative stress and enhance the antioxidant status in hyperglycemic periodontitis rats.     

Accurate quantification of the redox-sensitive GSH/GSSG ratios in the yeast Pichia pastoris by HILIC–MS/MS

A novel method for the simultaneous quantification of both glutathione (GSH) and its oxidized form glutathione disulfide (GSSG) by hydrophilic interaction chromatography–MS/MS has been developed and is critically discussed. Internal standardization based on isotopically labeled standards for both analytes is an absolute prerequisite for accurate quantification of this redox pair. Hence, a highly efficient and selective miniaturized procedure for the synthesis of isotopically labeled GSSG from commercially available glutathione-(glycine-¹³C₂,¹⁵N) was established using H₂O₂ as oxidant and NaI as catalyst. Moreover, a tool is presented to monitor and hence uncover artifactual GSSG formation due to oxidation of GSH during sample preparation, which is the main source of systematic error in GSSG analysis. For this purpose, we propose to monitor the oxidation product formed by reaction of naturally occurring GSH with the isotopically labeled GSH used as internal standard. For the determination of GSH/GSSG ratios in yeast, different extraction methods based on (1) hot extraction with aqueous, acidic, or organic solvents, (2) mechanical cell lysis, and (3) extraction at subambient temperature were investigated in terms of recovery, extraction efficiency, and artifactual formation of GSSG. Total combined uncertainties of as low as 25–30 % (coverage factor?=?2) for the determination of GSH/GSSG ratios without derivatization were made possible by the addition of the internal standards early in the analytical procedure (before extraction) and immediate analysis of the analytes.     

Determination of cellular redox status by stable isotope dilution liquid chromatography/mass spectrometry analysis of glutathione and glutathione disulfide

Oxidation of glutathione (GSH) to glutathione disulfide (GSSG) occurs during cellular oxidative stress. The redox potential of the 2GSH/GSSG couple, which is determined by the Nernst equation, provides a means to assess cellular redox status. It is difficult to accurately quantify GSH and GSSG due to the ease with which GSH is oxidized to GSSG during sample preparation. To overcome this problem, a stable isotope dilution liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) method has been developed using 4-fluoro-7-sulfamoylbenzofurazan (ABD-F) derivatization. ABD-F derivatization of the GSH thiol group was rapid, quantitative, and occurred at room temperature. The LC/MRM-MS method, which requires no sample clean-up, was validated within the calibration ranges of 5 to 400 nmol/mL in cell lysates for GSH and 0.5 to 40 nmol/mL in cell lysates for GSSG. Calibration curves prepared by adding known concentrations of GSH and GSSG to cell lysates were parallel to the standard curve prepared in buffers. GSH and GSSG concentrations were determined in two monocyte/macrophage RAW 267.4 cell lines with or without 15-LOX-1 expression (R15LO and RMock cells, respectively) after treatment with the bifunctional electrophile 4-oxo-2(E)-nonenal (ONE). R15LO cells synthesized much higher concentrations of the lipid hydroperoxide, 15(S)-hydroperoxyeicosatetraenoic acid (15-HPETE), which undergoes homolytic decomposition to ONE. GSH was depleted by ONE treatment in both RMock and R15LO cells, leading to significant increases in their redox potentials. However, R15LO cells had higher GSH concentrations (most likely through increased GSH biosynthesis) and had increased resistance to ONE-mediated GSH depletion than RMock cells. Consequently, R15LO cells had lower reduction potentials at all concentrations of ONE. GSSG concentrations were higher in R15LO cells after ONE treatment when compared with the ONE-treated RMock cells. This suggests that increased expression of 15(S)-HPETE modulates the activity of cellular GSH reductases or the transporters involved in removal of GSSG.     

A needle-and-thread approach to bilayer transport: permeation of a molecular umbrella-oligonucleotide conjugate across a phospholipid membrane

A di-walled molecular umbrella, composed of two choloyl groups, one spermidine moiety, and a 5-thiol(2-nitrobenzoyl) "handle", was covalently attached to a 16-mer oligonucleotide (S-dT16) through a disulfide bond. Incubation of this conjugate (1) with vesicles made from 1-palmitoyl-2-oleyol-sn-glycero-3-phosphocholine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (95/5, mol/mol) (200 nm diameter, extrusion) that contained entrapped glutathione (GSH) at 37 degrees C resulted in the liberation of the oligonucleotide and the umbrella-bound 5-mercapto(2-nitrobenzoyl) moiety (USH) via thiolate-disulfide interchange. The appearance of USH, together with the absence of leakage of entrapped GSH and a vesicular capture of the oligonucleotide that matches the extent of USH formation, provides compelling evidence for delivery of S-dT16 into the aqueous compartment of these vesicles. In a sense, the molecular umbrella functions like a "needle" in providing a pathway for the oligonucleotide (the "thread") to cross the membrane.     

Balancing conformational and oxidative kinetic traps during the folding of bovine pancreatic trypsin inhibitor (BPTI) with glutathione and glutathione disulfide

The oxidative folding of bovine pancreatic trypsin inhibitor (BPTI) has served as a paradigm for the folding of disulfide-containing proteins from their reduced form, as well as for protein folding in general. Many extracellular proteins and most pharmaceutically important proteins contain disulfide bonds. Under traditional conditions, 0.125 mM glutathione disulfide (GSSG) and no glutathione (GSH), the folding pathway of BPTI proceeds through a nonproductive route via N* (a two disulfide intermediate), or a productive route via N' (and other two disulfide intermediates which are in rapid equilibrium with N'). Both routes have the rearrangement of disulfide bonds as their rate-determining steps. However, the effects of the composition of the redox buffer, GSSG and GSH, on folding has not been extensively investigated. Interestingly, BPTI folds more efficiently in the presence of 5 mM GSSG and 5 mM GSH than it does under traditional conditions. These conditions, which are similar to those found in vivo, result in a doubly mixed disulfide between N' and glutathione, which acts as an oxidative kinetic trap as it has no free thiols. However, with 5 mM GSSG and 5 mM GSH the formation of the double mixed disulfide is compensated for by N* being less kinetically stable and the more rapid conversion of the singly mixed disulfides between N' and glutathione to native protein (N). Thus a major rate-determining step becomes the direct conversion of a singly mixed disulfide to N, a growth-type pathway. Balancing the formation of N* and its stability versus the formation of the doubly mixed disulfide and its stability results in more efficient folding. Such balancing acts may prove to be general for other disulfide-containing proteins.     

Quantitation of oxidized and reduced glutathione in plasma by micellar electrokinetic capillary electrophoresis.

A method for the separation of reduced (GSH) and oxidized (GSSG) glutathione was optimized in terms of buffer concentration, sodium dodecyl sulfate concentration, buffer pH, detection wavelength, run voltage and injection volume. The method demonstrated good linearity (r2 > 0.999) and reproducibility (internal standard corrected peak area RSD < 2.3%) in the range of interest (16-81 microM GSH and 8-40 microM GSSG). A detection limit of less than 1 microM GSH and GSSG was obtained using a high sensitivity flow cell. When the optimized method was applied to plasma samples, concentrations of 1.6 microM GSH and 0.8 microM GSSG were easily detected without the need for derivatization. The on-capillary detection was calculated to be 38.6 fmol of GSH and 18.3 fmol of GSSG.     

Rapid determination of reduced and oxidized glutathione levels using a new thiol-masking reagent and the enzymatic recycling method: application to the rat liver and bile samples

A microtiter plate assay for quantitation of reduced (GSH) and oxidized (GSSG) glutathione in the rat liver tissue and bile is described. The assay is based on the established enzymatic recycling method and a new thiol-masking reagent, 1-methyl-4-vinyl-pyridinium trifluoromethane sulfonate (M4VP). Samples were first processed by homogenization with (liver) or addition of (bile) sulfosalicylic acid. The total glutathione and GSSG were then determined before and after rapid (≤2 min) and efficient (100%) masking of the GSH content of the samples with M4VP followed by the enzymatic recycling assay. The percentages of error and coefficient of variation of the assay were within the accepted guidelines, indicating the accuracy and precision of the assay in the range of 6.25–100 pmol GSH per microplate well and 2.17–140 pmol GSSG per well, with lower limit of quantitation of 6.25 and 2.17 pmol per well for GSH and GSSG, respectively. Furthermore, the recoveries of added GSH or GSSG from the liver and bile samples were accurate and precise. The assay was applied to measurement of GSH, GSSG, and GSH:GSSG ratio in the liver and serially collected bile samples in sham-operated and ischemic rat livers, demonstrating a depletion of glutathione and a decrease in the GSH:GSSG ratio as a result of ischemia. The developed assay is rapid, sensitive, accurate, and precise and is suitable for studies of the redox status of liver under physiologic and pathophysiologic conditions.     

Disposable Amperometric Sensors for Thiols with Special Reference to Glutathione

The antioxidant ‘reduced glutathione’ tripeptide is conventionally called glutathione (GSH). The oxidized form is a sulfur‐sulfur linked compound, known as glutathione disulfide (GSSG). Glutathione is an essential cofactor for antioxidant enzymes; it provides protection also for the mitochondria against endogenous oxygen radicals. The ratio of these two forms can act as a marker for oxidative stress. The majority of the methods available for estimation of both the forms of glutathione are based on colorimetric and electrochemical assays. In this study, electrochemical sensors were developed for the estimation of both GSH and GSSG. Two different types of transducers were used: i) screen‐printed three‐electrode disposable sensor (SPE) containing carbon working electrode, carbon counter electrode and silver/silver chloride reference electrode; ii) three‐electrode disposable system (CDE) consisting of three copper electrodes. 5,5′‐dithiobis(2‐nitrobenzoic acid) (DTNB) was used as detector element for estimation of total reduced thiol content. The enzyme glutathione reductase along with a co‐enzyme reduced nicotinamide adenine dinucleotide phosphate was used to estimate GSSG. By combining the two methods GSH can also be estimated. The detector elements were immobilized on the working electrodes of the sensors by bulk polymerization of acrylamide. The responses were observed amperometrically. The detection limit for thiol (GSH) was less than 0.6 ppm when DTNB was used, whereas for GSSG it was less than 0.1 ppm.     

Voltammetric Investigation of Zinc Release from Metallothioneins Modulated by the Glutathione Redox Couple and Separated with a Porous Membrane

Glutathione (GSH), in addition to serving as a redox buffer in cellular environment, has been suggested as a modulator in metal regulation and homeostasis by metallothioneins (MTs). The interactions of MTs with both GSH and its oxidized form GSSG have been shown to govern the direction of metal transfer. Common methods for the determination of zinc release from MTs modulated by GSH/GSSG either involve radioactive species or enzymes or are labor‐intensive. In this study, upon separation of Zn²⁺ from the reaction mixture of MTs and GSH with a centrifugal filter membrane, differential pulse voltammetry (DPV) was used for the Zn²⁺ quantification. The same approach is extended to the studies of metal transfer between Zn₇MT with a GSH/GSSG mixture and that between Zn₇MT with GSSG. The concomitant conversion between the free thiol and disulfide bonds was confirmed with UV‐vis spectrophotometry. The results demonstrate that GSSG, GSH, and the GSH/GSSG mixture all modulate zinc release from Zn₇MT. The percentage of zinc release increases in the order of GSH, GSSG, and the GSH/GSSG mixture. The new approach is demonstrated to be well suited for investigation of redox regulation of MT and its reaction with zinc‐containing enzymes.     

Simultaneous quantification of reduced and oxidized glutathione in plasma using a two-dimensional chromatographic system with parallel porous graphitized carbon columns coupled with fluorescence and coulometric electrochemical detection

A method for the simultaneous quantification of reduced and oxidized glutathione in human plasma employing a two-dimensional chromatographic system with parallel porous graphitized carbon (PGC) columns coupled with fluorescence (FLD) and coulometric electrochemical detection (ED) has been developed. Post-sampling oxidation of reduced glutathione (GSH) was prevented by derivatizing the -SH group with monobromobimane (MBB) and the glutathione-bimane adduct (GSMB) was detected by FLD. Oxidized glutathione (GSSG) was detected by ED optimized to give lowest possible limits of detection (LOD). The method is fully validated and is currently used for determination of GSH, GSSG and its redox potential in different clinical studies.     

Determination of glutathione in spruce needles by liquid chromatography/tandem mass spectrometry

For the determination of glutathione (GSH) and its oxidized form (GSSG) in spruce needles their electrospray mass and MS/MS spectra were recorded with an ion trap mass spectrometer (ITMS, LCQ, Finnigan) and a triple stage quadrupole mass spectrometer (TSQ, Quattro II, Micromass). A study of the stability of GSH in aqueous solutions shows the presence of dimeric and trimeric forms of GSH, as well as GSSG, GSH-sulfonate and GSH-sulfinic acid. The same components were also found in extracts of spruce needles. We developed an assay which is suitable for monitoring low concentrations of GSH and similar compounds in plant tissues, employing the sensitivity and specificity of LC/MS/MS. Preliminary results on the mass spectrometric determination of GSH in spruce needles are given.     

Simultaneous determination of reduced and oxidized glutathione in tissues by a novel liquid chromatography-mass spectrometry method: application in an inhalation study of Cd nanoparticles

The paper presents the development of an advanced extraction and fast analytical LC MS/MS method for simultaneous analyses of reduced and oxidized glutathione (GSH and GSSG, respectively) in different animal tissues. The simultaneous determination of GSH and GSSG is crucial because the amount and ratio of both GSH and GSSG may be altered in response to oxidative stress, an important mechanism of toxicity. The method uses the derivatization of free thiol groups in GSH. Its performance was demonstrated for less explored tissues (lung, brain, and liver) in mouse. The combined extraction and analytical method has very low variability and good reproducibility, maximum coefficients of variance for within-run and between-run analyses under 8 %, and low limits of quantification; for GSH and GSSG, these were 0.2 nM (0.06 ng/mL) and 10 nM (6 ng/mL), respectively. The performance of the method was further demonstrated in a model experiment addressing changes in GSH and GSSG concentrations in lung of mice exposed to CdO nanoparticles during acute 72 h and chronic 13-week exposures. Inhalation exposure led to increased GSH concentrations in lung. GSSG levels were in general not affected; nonsignificant suppression occurred only after the longer 13-week period of exposure. The developed method for the sensitive detection of both GSH and GSSG in very low tissue mass enables these parameters to be studied in cases where only a little sample is available, i.e. in small organisms or in small amounts of tissue.     

谷胱甘肽的化学与医疗作用

谷胱甘肽 （glutathione,GSH）在体内以氧化型 （GSSG）和还原型 （GSH）2种形式存在。GSH分子中的巯基容易失氢氧化,因而可以消除体内的活性氧,具有保护酶和蛋白质免受氧化,保护脏器免受损伤的功效。人工合成的GSH已广泛用于临床。     

Validation of a simplified procedure for convenient and rapid quantification of reduced and oxidized glutathione in human plasma by liquid chromatography tandem mass spectrometry analysis

Endogenous glutathione (GSH) and glutathione disulfide (GSSG) status is highly sensitive to oxidative conditions and have broad application as a surrogate indicator of redox status in vivo. Established methods for GSH and GSSG quantification in whole blood display limited utility in human plasma, where GSH and GSSG levels are ~3–4 orders of magnitude below those observed in whole blood. This study presents simplified sample processing and analytical LC–MS/MS approaches exhibiting the sensitivity and accuracy required to measure GSH and GSSG concentrations in human plasma samples, which after 5-fold dilution to suppress matrix interferences range from 200 to 500 nm (GSH) and 5–30 nm (GSSG). The utility of the methods reported herein is demonstrated by assay performance and validation parameters which indicate good sensitivity [lower limits of quantitation of 4.99 nm (GSH) and 3.65 nm (GSSG), and high assay precision (intra-assay CVs 3.6 and 1.9%, and inter-assay CVs of 7.0 and 2.8% for GSH and GSSG, respectively). These methods also exhibited exceptional recovery of analyte-spiked plasma samples (98.0 ± 7.64% for GSH and 98.5 ± 12.7% for GSSG). Good sample stability at −80°C was evident for GSH for up to 55 weeks and GSSG for up to 46 weeks, with average CVs <15 and <10%, respectively.     

Speciation of Aluminum(III) Complexes with Oxidized Glutathione in Acidic Aqueous Solutions

The structural speciation aspects, including the binding sites, species, complexation abilities and effects of the oxidized glutathione (GSSG) with aluminum(III) in aqueous solutions, have been studied by means of many analytical techniques: pH-potentiometry (25 degrees C, 0.1 M KCl and 37 degrees C, 0.15 M NaCl medium) was used to characterize the stoichiometry and stability of the species formed in the interactions of the Al(III) ion and the peptide GSSG, while multinuclear ((1)H, (13)C, (27)Al) nuclear magnetic resonance (NMR) and electrospray mass spectroscopy (ESI-MS) were applied to characterize the binding sites and species of the metal ion in the complexes. Two-dimensional ((1)H, (1)H-NOESY) was also employed to reveal the difference in the conformational behavior of the peptide and its complexes. The following results were obtained: (1) Aluminum(III) can coordinate with the important biomolecule GSSG through the following binding sites: glycyl and glutamyl carboxyl groups to form various mononuclear 1:1 (AlLH(4), AlLH(3), AlLH(2), AlLH, AlL, AlLH(-1), AlLH(-2)) and several binuclear 2:1 (Al(2)LH(4), Al(2)LH(2), Al(2)L) species (where H(6)L(2+) denotes the totally protonated oxidized glutathione) in acidic aqueous solutions. (2) It indicates that the COO(-) groups at low level of preorganization in such small peptide are not sufficient to keep the Al(III) ion in solution and to prevent the precipitation of Al(OH)(3) in the physiological pH range. (3) It also suggests that the occurrence of an Al-linked complexation, the conformation of the peptide GSSG in aqueous solutions appeared to change a little, relative to the initial structure.