Chip-based SALDI-MS for rapid determination of intracellular ratios of glutathione to glutathione disulfide

Alterations in the ratio of glutathione(GSH) to glutathione disulfide(GSSG) reveal the cell living state and are associated with a variety of diseases. In this study, an Au NPs grafted nanoporous silicon chip was used for surface assisted laser desorption ionization-mass spectrometry(SALDI-MS) detection of GSH. Due to the bond interaction between thiol of GSH and Au NPs modified on the chip surfaces, GSH could be captured from the complex cellular lysate. Meanwhile, the composite nanostructures of Au NPs grafted porous silicon surface presented good desorption/ionization efficiency for GSH detection. The GSH levels in different tumor cells were successfully detected. Chip-based SALDI-MS was optimized for quantification of intracellular GSH/GSSG ratio changing under drug stimulation in liver tumor cells, GSSG was reduced to GSH by reductant of tris(2-carboxyethyl)phosphine(TCEP) and isotope-labeling GSH was as an internal standard. It was found that the increasing concentration of drug irinotecan and hypoxia culture condition caused the rapid consumption of GSH and a decrease of GSH/GSSG ratio in liver tumor cells. The developed SALDI-MS method provided a convenient way to accurately measure and rapidly monitor cellular GSH value and the ratios of GSH/GSSG.     

A novel approach for concurrent quantitation of glutathione,glutathione disulfide,and 2‐hydroxyethylated glutathione in lungs of mice exposed to ethylene oxide,using liquid chromatography–positive electrospray tandem mass spectrometry

Glutathione (GSH), glutathione disulfide (GSSG) and 2‐hydroxyethylated glutathione (HESG) are important biomarkers for exploring the genotoxicity mechanism of ethylene oxide (EO) or ethylene in vivo. A liquid chromatography–tandem mass spectrometry method was developed for simultaneous determination of GSH, GSSG and HESG in mouse lung tissues after inhalation exposure to EO. The lower limit of quantitation for all these biomarkers was 0.002 µg/mL. The linearity of the calibration curves for all analytes was >0.998. The intra‐day assay precision relative standard deviation (RSD) values for quality control samples for all analytes were ≤12.8% with accuracy values ranging from 87.2 to 113%. The inter‐day assay precision (RSD) values for all analytes were ≤13.1% with accuracy values ranging from 86.9 to 103%. This method was applied to concurrently determine the levels of GSH, GSSG and HESG in lung samples isolated from mouse after 4‐week inhalation exposure to EO at 0, 10, 50, 100 and 200 ppm. Copyright © 2015 John Wiley & Sons, Ltd.     

Disulfide bond cleavage induced by a platinum(II) methionine complex

The cleavage of a disulfide bond and the redox equilibrium of thiol/disulfide are strongly related to the levels of glutathione (GSH)/oxidized glutathione (GSSG) or mixed disulfides in vivo. In this work, the cleavage of a disulfide bond in GSSG induced by a platinum(II) complex [Pt(Met)Cl2] (where Met = methionine) was studied and the cleavage fragments or their platinated adducts were identified by means of electrospray mass spectrometry, high-performance liquid chromatography, and ultraviolet techniques. The second-order rate constant for the reaction between [Pt(Met)Cl2] and GSSG was determined to be 0.4 M(-1) s(-1) at 310 K and pH 7.4, which is 100- and 12-fold faster than those of cisplatin and its monoaqua species, respectively. Different complexes were formed in the reaction of [Pt(Met)Cl2] with GSSG, mainly mono- and dinuclear platinum complexes with the cleavage fragments of GSSG. This study demonstrated that [Pt(Met)Cl2] can promote the cleavage of disulfide bonds. The mechanistic insight obtained from this study may provide a deeper understanding on the potential involvement of platinum complexes in the intracellular GSH/GSSG systems.     

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.     

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.     

Ratios of biliary glutathione disulfide (GSSG) to glutathione (GSH): a potential index to screen drug-induced hepatic oxidative stress in rats and mice

Hepatotoxicity of drug candidates is one of the major concerns in drug screening in early drug discovery. Detection of hepatic oxidative stress can be an early indicator of hepatotoxicity and benefits drug selection. The glutathione (GSH) and glutathione disulfide (GSSG) pair, as one of the major intracellular redox regulating couples, plays an important role in protecting cells from oxidative stress that is caused by imbalance between prooxidants and antioxidants. The quantitative determination of the GSSG/GSH ratios and the concentrations of GSH and GSSG have been used to indicate oxidative stress in cells and tissues. In this study, we tested the possibility of using the biliary GSSG/GSH ratios as a biomarker to reflect hepatic oxidative stress and drug toxicity. Four compounds that are known to alter GSH and GSSG levels were tested in this study. Diquat (diquat dibromide monohydrate) and acetaminophen were administered to rats. Paraquat and tert-butyl hydroperoxide were administered to mice to induce changes of biliary GSH and GSSG. The biliary GSH and GSSG were quantified using calibration curves prepared with artificial bile to account for any bile matrix effect in the LC–MS analysis and to avoid the interference of endogenous GSH and GSSG. With four examples (in rats and mice) of drug-induced changes in the kinetics of the biliary GSSG/GSH ratios, this study showed the potential for developing an exposure response index based on biliary GSSG/GSH ratios for predicting hepatic oxidative stress.     

Determination of glutathione disulfide levels in biological samples using thiol-disulfide exchanging agent, dithiothreitol

A reverse-phase HPLC method incorporating dithiothreitol (DTT) reduction for quantitative determination of oxidized glutathione (GSSG) in biological samples is described here. This method is based on our previous enzymatic reduction technique that uses N-1-(pyrenyl) maleimide (NPM) as a derivatizing agent. In our earlier method, glutathione disulfide (GSSG) was measured by first reducing it to GSH with glutathione reductase (GR) in the presence of NADPH. However, this is a very costly and time-consuming technique. The method described here employs a common and inexpensive thiol-disulfide exchanging agent, DTT, for reduction of GSSG to GSH, followed by derivatization with NPM. The calibration curves are linear over a concentration range of 25-1250 nm (r(2) > 0.995). The coefficients of variations for intra-run precision and inter-run precision range from 0.49 to 5.10% with an accuracy range of 1.78-6.15%. The percentage of relative recovery ranges from 97.3 to 103.2%. This new method provides a simple, efficient, and cost-effective way of determining glutathione disulfide levels with a 2.5 nm limit of detection per 5 microL injection volume.     

Simultaneous analysis of oxidized and reduced glutathione in cell extracts by capillary zone electrophoresis

Glutathione (GSH) and glutathione disulfide (GSSG) levels in cells constitute a thiol redox system. They can be used as an indicator of oxidative stress of the cell. In this study, a capillary zone electrophoresis (CZE) method is described that enables quantitation of GSH and GSSG from cellular extracts. The CZE buffer used was 20 mM ammonium acetate containing 5% (v/v) acetic acid at pH 3.1 in conjunction with a polybrene coated capillary operated in reverse polarity mode. Effects of different acids used to prepare cell samples were investigated on CZE performance. The acids include meta phosphoric acid (MPA), trichloroacetic acid (TCA), phosphoric acid (PA) and sulfosalicylic acid (SSA) and are used to stabilize GSH and GSSG before performing CZE analysis. The method features a limit of detection of 4 microM and a limit of quantitation of 12 microM for both GSSG and GSH and recoveries of 94% for GSH and 100% for GSSG. Quantitative analysis of GSSG and GSH in HaCaT cell extracts (5% SSA, w/v) was performed with this method and changes in the ratio of GSH to GSSG in N-ethylmaleimide treated cell sample was observed by comparing with control cell samples.     

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.     

Scanning electrochemical microscopy studies of glutathione-modified surfaces. An erasable and sensitive-to-reactive oxygen species surface

A surface sensitive to reactive oxygen species (ROS) was prepared by reduction of a diazonium salt on glassy carbon electrode followed by the chemical coupling of glutathione (GSH) playing the role of an antioxidant species. The presence of active GSH was characterized through spectroscopic studies and electrochemical analysis after labeling of the -SH group with ferrocene moieties. The specific reactivity of GSH vs ROS was evaluated with scanning electrochemical microscopy (SECM) using the reduction of O(2) to superoxide, O(2)(?-), near the GSH-modified surface. Approach curves show a considerable decrease of the blocking properties of the layer due to reaction of the immobilized GSH with O(2)(?-) and the passage of GSH to the glutathione disulfide (GSSG). The initial surface could be regenerated several times with no significant variations of its antioxidant capacity by simply using the biological system glutathione reductase (GR)/NADPH that reduces GSSG back to GSH. SECM imaging shows also the possibility of writing local and erasable micropatterns on the GSH surface by production of O(2)(?-) at the tip probe electrode.     

Characterization of dansylated glutathione, glutathione disulfide, cysteine and cystine by narrow bore liquid chromatography/electrospray ionization mass spectrometry

A method using reversed phase high performance liquid chromatography/electrospray ionization-mass spectrometry (RP-LC/ESI-MS) has been developed to confirm the identity of dansylated derivatives of cysteine (C) and glutathione (GSH), and their respective dimers, cystine (CSSC) and glutathione disulfide (GSSG). Cysteine, GSH, CSSC and GSSG are present at low concentrations in rainbow trout (Oncorhynchus mykiss) liver cells. Initially, hepatic cells were sampled from a suspension culture and disrupted upon addition of 10% perchloric acid. The reduced thiols present in the cell extracts were acetylated to prevent dimerization and then the C and GSH species were derivatized with dansyl chloride for fluorescence detection. An LC system using a weak anion exchange column (AE) with fluorescence detection (FLD) was used for sensitive routine analysis; however, it produced peaks of unknown origin in addition to the expected analytes. Analytes were then separated on a C18 RP-LC system using a water/acetonitrile gradient with 0.2% formic acid, and detected using LC/ESI-MS at 3.5 KV which produced an intense ion with a minimum limit of detection of less than 0.5 pmole injected (>10:1 signal-to-noise (S/N). Subsequently, fractions of effluent from the AE-LC/FLD system were analyzed by LC/ESI-MS to confirm the presence of the target analytes in routine cell extracts. Monodansylated GSSG was identified as a product that could possibly affect the quantification of GSH and GSSG.     

谷胱甘肽键合柱对变性核糖核酸酶的复性研究

将氧化还原型谷胱甘肽(GSH/GSSG)共价键合到色谱固定相上, 实现了对变性核糖核酸酶(RNase)的复性. 实验发现, 谷胱甘肽键合柱具有典型的弱阳离子交换性质, 在离子交换(IEC)模式下能够对4种标准蛋白进行基线分离, 且具有较高的柱效. 当蛋白浓度为5 mg/mL, 流速为0.2 mL/min时, 在流动相中不加GSH/GSSG的条件下, GSH/GSSG柱对变性核糖核酸酶的活性回收率可达(39.5±3.8)%, 而普通IEC柱对变性核糖核酸酶的活性回收率几乎为0, 说明其对变性蛋白二硫键的正确对接具有明显的促进作用; 在收集液中加入GSH/GSSG后, 其活性回收率可达到(81.5±4.3)%. 本文结果对蛋白折叠液相色谱法的发展及降低蛋白复性成本具有一定的应用价值.     

Sulodexide Increases Glutathione Synthesis and Causes Pro-Reducing Shift in Glutathione-Redox State in HUVECs Exposed to Oxygen–Glucose Deprivation: Implication for Protection of Endothelium against Ischemic Injury

Sulodexide (SDX), a purified glycosaminoglycan mixture used to treat vascular diseases, has been reported to exert endothelial protective effects against ischemic injury. However, the mechanisms underlying these effects remain to be fully elucidated. The emerging evidence indicated that a relatively high intracellular concentration of reduced glutathione (GSH) and a maintenance of the redox environment participate in the endothelial cell survival during ischemia. Therefore, the aim of the present study was to examine the hypothesis that SDX alleviates oxygen–glucose deprivation (OGD)-induced human umbilical endothelial cells’ (HUVECs) injury, which serves as the in vitro model of ischemia, by affecting the redox state of the GSH: glutathione disulfide (GSSG) pool. The cellular GSH, GSSG and total glutathione (tGSH) concentrations were measured by colorimetric method and the redox potential (ΔEh) of the GSSG/2GSH couple was calculated, using the Nernst equation. Furthermore, the levels of the glutamate–cysteine ligase catalytic subunit (GCLc) and the glutathione synthetase (GSS) proteins, a key enzyme for de novo GSH synthesis, were determined using enzyme-linked immunoassay (ELISA). We demonstrated that the SDX treatment in OGD conditions significantly elevated the intracellular GSH, enhanced the GSH:GSSG ratio, shifting the redox potential to a more pro-reducing status. Furthermore, SDX increased the levels of both GCLc and GSS. The results show that SDX protects the human endothelial cells against ischemic stress by affecting the GSH levels and cellular redox state. These changes suggest that the reduction in the ischemia-induced vascular endothelial cell injury through repressing apoptosis and oxidative stress associated with SDX treatment may be due to an increase in GSH synthesis and modulation of the GSH redox system.     

Cyclic voltammetric study of the redox system of glutathione using the disulfide bond reductant tris(2-carboxyethyl)phosphine

The stabilization of the reduction state of proteins and peptides is very important for the monitoring of protein-protein, protein-DNA and protein-xenobiotic interactions. The reductive state of protein or peptide is characterized by the reactive sulfhydryl group. Glutathione in the reduced (GSH) and oxidized (GSSG) forms was studied by cyclic voltammetry. Tris(2-carboxyethyl)phosphine (TCEP) as the disulfide bond reductant and/or hydrogen peroxide as the sulfhydryl group oxidant were used. Cyclic voltammetry measurements, following the redox state of glutathione, were performed on a hanging mercury drop electrode (HMDE) in borate buffer (pH 9.2). It was shown that in aqueous solutions TCEP was able to reduce disulfide groups smoothly and quantitatively. The TCEP response at -0.25 V vs. Ag/AgCl/3 M KCl did not disturb the signals of the thiol/disulfide redox couple. The origin of cathodic and anodic signals of GSH (at -0.44 and -0.37 V) and GSSG (at -0.69 and -0.40 V) glutathione forms is discussed. It was shown that the application of TCEP to the conservation of sulfhydryl groups in peptides and proteins can be useful instrument for the study of peptides and proteins redox behavior.     

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.     

Determination of glutathione in blood via capillary electrophoresis with pH-mediated stacking

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 K₃EDTA 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.     

Simultaneous measurement of reactive oxygen species and reduced glutathione using capillary electrophoresis and laser-induced fluorescence detection in cultured cell lines.

A capillary zone electrophoretic (CZE) method coupled with laser-induced fluorescence (LIF) was developed for the simultaneous determination of two important intracellular parameters related to oxidative stress (i.e. reactive oxygen species, ROS, and reduced glutathione, GSH). This rapid and sensitive method was applied to the study of oxidative stress in cultured V79 fibroblast cells. The fluorogenic reagents selected were: (i) dihydrorhodamine-123 (DHR-123) which is converted intracellularly by ROS to the fluorescent rhodamine-123 dye (Rh-123), and (ii) naphthalene-2,3-dicarboxaldehyde (NDA), which reacts quickly with GSH in cell extracts to produce a fluorescent adduct. Separation of Rh-123, GSH-NDA and gamma-glutamylcysteine-NDA adducts was performed using an uncoated fused-silica capillary and a 100 mM borate buffer, pH 9.2, at 20 degrees C and at an applied voltage of 25 kV; LIF detection was operated using an argon laser. The cell line was also tested for its ability to alleviate oxidative stress induced by tert-butylhydroperoxide (t-BuOOH). Exposure to t-BuOOH (up to 3 mm for 2 h) did not affect the intracellular ROS and GSH concentrations. At higher (4-10 mM) t-BuOOH concentrations, an inverse relationship between the concentrations of ROS and GSH was obtained, showing that the present method can readily evaluate the gradual consumption of the primary cellular scavenger of ROS which occurs simultaneously with the increase of oxidative insult.     

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 colorimetric determination of reduced and oxidized glutathione using an end point coupled enzymatic assay

A simple and rapid colorimetric coupled enzymatic assay for the determination of glutathione is described. The proposed method is based on the specific reaction catalyzed by γ-glutamyltransferase, which transfers the γ-glutamyl moiety from glutahione to an acceptor, with the formation of the γ-glutamyl derivative of the acceptor and cysteinylglycine. The latter dipeptide is a substrate of leucyl aminopeptidase, which hydrolyzes cysteinylglycine to glycine and cysteine that can be easily measured spectrophotometrically. The proposed method was used to measure the content of glutathione in acid extracts of bovine lens, to follow the NADPH-dependent reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH) catalyzed by the enzyme glutathione reductase and to determine the glutathione content in human astrocytoma ADF cells subjected to oxidative stress. The results obtained showed that the method can be suitably used for the determination of GSH and GSSG in different biological samples and to monitor tissue or cell redox status under different conditions. It is also applicable for following reactions involving GSH and/or GSSG.

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.