Determination of reduced, oxidized, and protein-bound glutathione in human plasma with precolumn derivatization with monobromobimane and liquid chromatography

This assay measures reduced (GSH), oxidized (GSSG, GSSR), and protein-bound (glutathione-protein mixed disulfides, ProSSG) glutathione in human plasma. Oxidized glutathione and ProSSG are converted to GSH in the presence of NaBH4, and, after precolumn derivatization with monobromobimane, GSH is quantitated by reversed-phase liquid chromatography and fluorescence detection. The NaBH4 concentration is optimized so that total recovery of oxidized glutathione is obtained and no interference with the formation/stability of the GSH-bimane adduct occurs. The presence of 50 microM dithioerythritol prevents reduced recovery at low concentrations of GSH, and the standard curve for GSH is linear over a wide concentration range and is super-imposed upon that obtained with GSSG. Selective determination of oxidized glutathione exploits the fact that N-ethylmaleimide (NEM) blocks free sulfhydryl groups and excess NEM is inactivated by the subsequent addition of NaBH4. To measure total glutathione including the protein-bound forms, the protein is solubilized with dimethyl sulfoxide, which is compatible with the other reagents and slightly increases the yield of the fluorescent GSH derivative. The assay is characterized by a sensitivity (less than 2 pmol) sufficiently high to detect the various forms of glutathione in plasma, by an analytical recovery of GSH and GSSG close to 100%, and by a within-day precision corresponding to a coefficient of variation of 7%. The assay was used to determine the dynamic relationships among various glutathione species in human plasma.     

Analysis of glutathione and glutathione disulfide in human saliva using hydrophilic interaction chromatography with mass spectrometry

A sensitive method for the determination of glutathione (GSH) and glutathione disulfide (GSSG) in human saliva was developed and validated. GSH was captured and stabilized by the addition of N-ethylmaleimide (NEM). Solid-phase extraction (SPE) using an Oasis MAX extraction cartridge was employed for sample preparation and analysis was performed on a Shimadzu LCMS-2010 A that was operated in the single ion monitoring mode using positive ion electrospray ionization (ESI) as the interface. The monitored ion for GSH-NEM was m/z 433 and that for GSSG was m/z 613. Chromatography was carried out on an Atlantis HILIC silica column (150 mm x 2.1 mm, 5 microm) with acetonitrile and formate buffer as the mobile phase at the flow rate of 0.2 ml/min. The calibration curve was linear over the range of 0.1-100 microM for GSH-NEM. The extraction recoveries of GSH-NEM spiked at concentrations of 25 and 50 microM were 97.1 and 104.4%, respectively. Similar results were obtained for GSSG. The newly developed hydrophilic interaction chromatography with mass spectrometry (HILIC/MS) method showed superior sensitivity for the determination of GSH and GSSG in human saliva samples.     

Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: dependence of the rate on the composition of the redox buffer

The velocity of the oxidative renaturation of reduced ribonuclease A catalyzed by protein disulfide isomerase (PDI) is strongly dependent on the composition of a glutathione/glutathione disulfide redox buffer. As with the uncatalyzed, glutathione-mediated oxidative folding of ribonuclease, the steady-state velocity of the PDI-catalyzed reaction displays a distinct optimum with respect to both the glutathione (GSH) and glutathione disulfide (GSSG) concentrations. Optimum activity is observed at [GSH] = 1.0 mM and [GSSG] = 0.2 mM. The apparent kcat at saturating RNase concentration is 0.46 +/- 0.05 mumol of RNase renatured min-1 (mumol of PDI)-1 compared to the apparent first-order rate constant for the uncatalyzed reaction of 0.02 +/- 0.01 min-1. Changes in GSH and GSSG concentration have a similar effect on the rate of both the PDI-catalyzed and uncatalyzed reactions except under the more oxidizing conditions employed, where the catalytic effectiveness of PDI is diminished. The ratio of the velocity of the catalyzed reaction to that of the uncatalyzed reaction increases as the quantity [GSH]2/[GSSG] increases and approaches a constant, limiting value at [GSH]2/[GSSG] greater than 1 mM, suggesting that a reduced, dithiol form of PDI is required for optimum activity. As long as the glutathione redox buffer is sufficiently reducing to maintain PDI in an active form [( GSH]2/[GSSG] greater than 1 mM), the rate acceleration provided by PDI is reasonably constant, although the actual rate may vary by more than an order of magnitude. PDI exhibits half of the maximum rate acceleration at a [GSH]2/[GSSG] of 0.06 +/- 0.01 mM.     

Changes in the glutathione level induced by transplasma membrane electron transport in maize (Zea mays L.)

Summary We investigated changes of thiols (GSH, GSSG, and cysteine) induced by transplasma membrane electron transport after addition of artificial electron acceptors and the influence of the thiol level on redox activity. GSH, GSSG, and cysteine content of maize (Zea mays L. cv. Golden Bantam) roots and coleoptile segments was determined by high performance liquid chromatography with a fluorescence detector. GSSG increased after treatment with 0.8 mM diamide, an SH-group oxidizer. GSH level of roots increased after treatment with diamide, while GSH levels of coleoptiles decreased. Incubation of roots with the GSH biosynthesis inhibitor buthionine-D,L-sulfoximine for 6 days lowered the glutathione level up to 80%. However, the GSH/GSSG ratio of maize roots remained constant after treatment with both effectors. The GSH/GSSG ratio and the glutathione level were changed by addition of artificial electron acceptors like hexacyanoferrate (III) or hexabromoiridate (IV), which do not permeate the plasma membrane. Hexacyanoferrate (III) reduction was inhibited up to 25% after the cellular glutathione level was lowered by treatment with diamide or buthionine-D,L-sulfoximine. Proton secretion induced by reduction of the electron acceptors was not affected by both modulators. The change in glutathione level is different for roots and coleoptiles. Our data are discussed with regard to the role of GSH in electron donation for a plasma membrane bound electron transport system.Abbreviations Buthionine-D,L-sulfoximine s-n-butyl-homocysteine sulfoximine - cys cysteine - diamide 1,1-azobis (N,N-dimethyl-formamide) - DTE dithioerythritol - EDTA ethylenediaminetetraacetic acid - GSH reduced glutathione - GSSG oxidizied glutathione, glutathione disulfide - HBI IV hexabromoiridate (IV) (K₂[IrBr₆]) - HCF III hexacyanoferrate (III) (K₃[Fe(CN)₆] - NEM N-ethylmaleimide - PM plasma membrane - Tris Tris(hydroxymethyl)aminomethane     

Iac operon operator DNA: isolation and trimming for NMR spectroscopy

A method for measurement of both glutathione (GSH) and glutathione disulfide (GSSG) in biological samples has been developed by using an isotachophoretic analyzer. The determination of the amount of GSH was carried out by measuring a zone length of GSH in isotachophoresis. The method gave recoveries of 92 to 106% for GSH and was quite specific for GSH. The measurement of GSSG levels was carried out by measuring differences in the length of mixed zones containing GSSG determined before and after reduction of GSSG by treatment with dithiothreitol or glutathione reductase. The method gave recoveries of 80 to 103% for GSSG. The results determined by using this method for GSH and GSSG levels in rat tissues agreed well with earlier reports.     

Turnover and functions of glutathione studied with isolated hepatic and renal cells

Suspensions of freshly isolated rat hepatocytes and renal tubular cells contain high levels of reduced glutathione (GSH), which exhibits half-lives of 3-5 and 0.7-1 h, respectively. In both cells types the availability of intracellular cysteine is rate limiting for GSH biosynthesis. In hepatocytes, methionine is actively converted to cysteine via the cystathionine pathway, and hepatic glutathione biosynthesis is stimulated by the presence of methionine in the medium. In contrast, extracellular cystine can support renal glutathione synthesis; several disulfides, including cystine, are rapidly taken up by renal cells (but not by hepatocytes) and are reduced to the corresponding thiols via a GSH-linked reaction sequence catalyzed by thiol transferase and glutathione reductase (NAD(P)H). During incubation, hepatocytes release both GSH and glutathione disulfide (GSSG) into the medium; the rate of GSSG efflux is markedly enhanced during hydroperoxide metabolism by glutathione peroxidase. This may lead to GSH depletion and cell injury; the latter seems to be initiated by a perturbation of cellular calcium homeostasis occurring in the glutathione-depleted state. In contrast to hepatocytes, renal cells metabolize extracellular glutathione and glutathione S-conjugates formed during drug biotransformation to the component amino acids and N-acetyl-cysteine S-conjugates, respectively. In addition, renal cells contain a thiol oxidase acting on extracellular GSH and several other thiols. In conclusion, our findings with isolated cells mimic the physiological situation characterized by hepatic synthesis and renal degradation of plasma glutathione and glutathione S-conjugates, and elucidate some of the underlying biochemical mechanisms.     

Detection of oxidized and reduced glutathione with a recycling postcolumn reaction

A rapid, sensitive, and selective method for the quantitation of both oxidized (GSSG) and reduced (GSH) glutathione in biological materials is described. Oxidized and reduced glutathione are resolved by anion-exchange high-performance liquid chromatography and detected with an in-line, recycling postcolumn reaction. The recycling reaction specifically amplifies the response to oxidized and reduced glutathione 20-100 times over that obtained with a stoichiometric reaction, permitting the detection of 2 pmol glutathione. Oxidized and reduced glutathione levels were measured in rat liver and in dog heart mitochondria. Special precautions are necessary to avoid artifacts which lead to either underestimation or overestimation of GSSG levels. GSH/GSSG ratios of approximately 100-300 were observed in samples prepared from rapidly frozen rat liver. Somewhat higher GSH/GSSG ratios were observed in isolated dog heart mitochondria.     

OXIDIZED AND REDUCED GLUTATHIONE IN THE RAT BRAIN UNDER NORMOXIC AND HYPOXIC CONDITIONS

In order to test the proposition that hypoxia leads to a change in the concentration ratio of reduced (GSH) and oxidized (GSSG) glutathione in the brain, enzymatic, fluorometric assays were worked out for measuring GSH and GSSG. In lightly anaesthetized and immobilized rats. GSH concentrations in the cerebral cortex and the cerebellum were close to 2 μmol.g^-1 while a slightly lower concentration (approx 1.4μmol.g^-1) was found in the brain stem. In order to avoid artefactual oxidation of GSH during sample preparation for GSSG determination the tissue was extracted with trichloroacetic acid, following alkylation of SH groups with N-ethylmaleimide. With these precautions GSSG concentrations were approx 0.7% of the corresponding GSH concentrations. However. the results indicated that the true GSSG concentrations may be even lower. During hypoxia there was neither a decrease in GSH nor an increase in GSSG concentrations in cortical tissue or cisternal CSF.     

Determination of reduced and oxidized glutathione in erythrocytes by high-performance liquid chromatography with ultraviolet absorbance detection

A method is described for simultaneous quantitation of reduced (GSH) and oxidized (GSSG) glutathione in erythrocytes by HPLC. They were determined by standard addition method. Blood samples were collected in tubes containing 1,10-phenanthroline. The separated erythrocytes were hemolyzed with water containing standard. After deproteinization, GSH and GSSG were converted to N-(2,4-dinitrophenyl) derivatives and analyzed by HPLC with UV detection. The coefficients of variation of GSH and GSSG on replicate assays were 6% and 8%, respectively. The stabilities of GSH and GSSG and of the derivatives were also examined. The present method appears to be satisfactory for determination of these physiological concentrations in erythrocytes.     

Characterization of Glutathione Uptake in Broad Bean Leaf Protoplasts

Transport of reduced glutathione (GSH) and oxidized glutathione (GSSG) was studied with broad bean (Vicia faba L.) leaf tissues and protoplasts. Protoplasts and leaf discs took up GSSG at a rate about twice the uptake rate of GSH. Detailed studies with protoplasts indicated that GSH and GSSG uptake exhibited the same sensitivity to the external pH and to various chemical reagents. GSH uptake was inhibited by GSSG and glutathione conjugates. GSSG uptake was inhibited by GSH and GS conjugates, and the uptake of metolachlor-GS was inhibited by GSSG. Various amino acids (L-glutamic acid, L-glutamine, L-cysteine, L-glycine, L-methionine) and peptides (glycine-glycine, glycine-glycine-glycine) affected neither the transport of GSH nor GSSG. Uptake kinetics indicate that GSH is taken up by a single saturable transporter, with an apparent Km of 0.4 mM, whereas GSSG uptake exhibits two saturable phases, with an apparent Km of 7 [mu]M and 3.7 mM. It is concluded that the plasma membrane of leaf cells contains a specific transport system for glutathione, which takes up GSSG and GS conjugates preferentially over GSH. Proton flux measurements and electrophysiological measurements indicate that GSH and GSSG are taken up with proton symport. However, a detailed analysis of these measurements suggests that the ion movements induced by GSSG differ from those induced by GSH.     

Influence of Complete and Pronounced Incomplete Cerebral Ischemia and Subsequent Recirculation on Cortical Concentrations of Oxidized and Reduced Glutathione in the Rat

Abstract: The influence of complete and pronounced incomplete cerebral ischemia on cortical concentrations of reduced (GSH) and oxidized (GSSG) glutathione was studied in lightly anaesthetized (70% N₂ O) rats. GSH was extracted with HCl-methanol-perchloric acid and GSSG with trichloroacetic acid in the presence of N-ethylmaleimide and measured fluorometrically, giving normal concentrations in cortical tissue of about 2 and 0.01 μmol.g^?1 respectively. Reversible complete ischemia was induced by increasing the intracranial pressure to above the systolic blood pressure by infusing mock CSF into the cisterna magna. Reversible pronounced incomplete ischemia was induced by bilateral carotid artery clamping combined with hypovolemic hypotension. Whether complete or incomplete, a 30-min ischemic period caused a similar decrease in cortical GSH concentration (to about 90% of control) without any concomitant accumulation of GSSG in the tissue (or in CSF). Prolongation of the ischemic period (complete ischemia) to maximally 120 min caused an almost linear decrease of the tissue glutathione concentration to 45% of the preischemic value. During subsequent recirculation following a 30 min period of either complete or pronounced incomplete ischemia, there was a further decrease in cortical GSH concentrations without a reciprocal increase in GSSG concentrations. Lipid peroxidation (verified by determination of malondialdehyde production) induced in brain cortical tissue in vitro caused oxidation of tissue GSH with accumulation of GSSG. As the observed decrease in GSH during brain ischemia in vivo was not accompanied by any reciprocal increase in GSSG the results fail to support the hypothesis that peroxidative damage occurs during or following brain ischemia. The finding of an unchanged GSSG concentration does, however, not exclude the possibility of an increased turnover rate in the glutathione reductase reaction. It is concluded that the observed decrease in tissue GSH concentration mainly reflects a decrease in the glutathione pool size, due to an imbalance between breakdown and synthesis secondary to tissue energy failure.     

Alkaline hydrolysis of N-ethylmaleimide allows a rapid assay of glutathione disulfide in biological samples

The estimation of glutathione disulfide (GSSG) is based on the NADPH-dependent glutathione reductase reaction. A new method has been developed to eliminate the inactivating effect of N-ethylmaleimide (NEM), added to prevent glutathione oxidation, on glutathione reductase. This method takes advantage of instability of NEM in alkaline solutions. The product of NEM hydrolysis, N-ethylmaleamic acid, obtained under accurate pH-controlled conditions, is compatible with a good activity of glutathione reductase which allows total recovery and measurement of GSSG. The method, applied to estimation of GSSG content in human erythrocytes and rat liver, gives results in optimum agreement with values reported in literature. Because of its simple performance and rapidity, the procedure can be considered an improved method in removing NEM and is particularly advantageous when a large number of biological samples must be treated and estimated.     

Glutathione production in copper-deficient isolated rat hepatocytes.

Dietary copper deficiency has been shown to reduce copper-dependent superoxide dismutase (SOD) activity and to increase lipid peroxidation in rats. Circulating reduced glutathione (GSH) concentrations are elevated in copper-deficient (CuD) rats, which suggests an increased GSH synthesis or decreased degradation, perhaps as an adaptation to the oxidative stress of copper deficiency. GSH synthesis was examined in isolated hepatocytes from CuD rats. Isolated hepatocytes were prepared by collagenase perfusion and incubated in Krebs-Henseleit bicarbonate buffer, pH 7.4, 10 mM glucose, 2.5 mM Ca2+ in the presence and absence of 1.0 mM buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis. Cell viability was assessed by trypan blue exclusion. GSH and oxidized glutathione (GSSG) were measured by the glutathione reductase recycling assay. Copper deficiency depressed hepatocyte Cu by greater than 90% and increased intracellular GSH by 41-117% over the 3-h incubation, with a two- to threefold increase in the rate of intracellular GSH synthesis. Intracellular GSSG values were minimally influenced by CuD, with a constant mol% GSSG. Extracellular total glutathione (GSH + 2GSSG) synthesis was increased by approximately 33%. Both intracellular GSH and extracellular total glutathione synthesis were inhibited by BSO. The pattern of food consumption in CuD rats, meal fed versus ad libitum fed, had no effect on glutathione synthesis. The results indicate an increased hepatic GSH synthesis as a response to dietary copper deficiency and suggest an interrelationship between the essential nutrients involved in oxyradical metabolism.     

The interaction of reduced glutathione with active oxygen species generated by xanthine-oxidase-catalyzed metabolism of xanthine

The interaction of reduced glutathione (GSH) with active oxygen species generated during xanthine-oxidase-catalyzed metabolism of xanthine was investigated. The only GSH-derived product detected in this system was oxidized glutathione (GSSG). Catalase inhibited the oxidation of GSH to GSSG by more than 80%, whereas superoxide dismutase exerted a smaller but significant inhibition of GSSG formation. Hydroxyl radical (OH) scavengers or desferrioxamine (1 mM) had no effect on GSSG formation. Using EPR spectroscopy and the spin trap 5,5-dimethylpyrroline-N-oxide (DMPO), the production of superoxide was observed by the detection of a DMPO-OOH radical adduct. This spectrum was altered by the inclusion of GSH (5 - 20 mM) in the reaction mixture, indicating the generation of a different radical species consistent with DMPO-glutathionyl radical adduct generation.     

Determining glutathione and glutathione disulfide using the fluorescence probe o-phthalaldehyde

Because of the importance of glutathione (GSH) and glutathione disulfide (GSSG) in cellular signal transduction, gene regulation, redox regulation, and biochemical homeostasis, accurate determination of cellular glutathione levels is critical. Several procedures have been developed, but many suffer from overestimating GSSG or from cellular substances interfering or competing with GSH determination. Assays based on HPLC, with enzymatic reduction of GSSG by glutathione reductase and NADPH, appear to be valid but are limited in sample throughput and availability of equipment. The fluorescence probe o-phthalaldehyde (OPA, phthalic dicarboxaldehyde) reacts with GSH and has a high quantum yield, yet its use has been limited due to unidentified interfering and fluorescence-quenching substances in liver. This paper describes assay conditions under which these limitations are avoided. By using a phosphate-buffered assay at lower pH, interference with nonspecific reactants is minimal. Since enzymatic reduction is not possible due to the reaction of OPA with NAD(P)H and other stronger reducing agents, leading to an overestimation of GSSG levels, dithionite was used to reduce GSSG. High sample throughput combined with sensitive (20-pmol limit of detection) and accurate determination of GSH and GSSG using OPA is achievable with any monochromatographic spectrofluorometer. Sample preparation and storage conditions are described that return the same levels of GSH and GSSG for at least 4 weeks.     

Glutathione disulfide reduction in tumor mitochondria after t-butyl hydroperoxide treatment.

Treatment of isolated mitochondria from rat hepatoma tumor cells (AS-30D) with the oxidant, t-butyl hydroperoxide (tBuOOH, 1 or 5 mumol/ml) resulted in the oxidation of glutathione (GSH to GSSG) and the formation of protein-glutathione mixed disulfides (ProSSG). The GSSG was retained inside of the hepatoma mitochondria. In the presence of ADP+succinate (5 or 10 mM), or ketoglutarate (10 mM) or malate (5 mM), the GSSG was reduced to GSH, but the amount of ProSSG stayed constant. With saline or ADP+glutamate (10 mM)/malate (0.1 mm) no reduction of GSSG to GSH occurred. The presence of antimycin (5 micrograms/ml) with ADP+succinate inhibited reduction. At a concentration of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, 0.5 mM) which inhibited a major portion of the glutathione reductase activity, the reduction of GSSG to replenish GSH was also inhibited. NADPH may play a critical role as well, for the addition of 2.4 mM NADPH to permeabilized hepatoma mitochondria fostered the reduction of GSSG after tBuOOH treatment. Therefore, hepatoma mitochondria possess a glutathione reductase-dependent system to reduce GSSG to GSH. The reaction only occurs with actively respiring mitochondria.     

Glutathione-Mediated Regulation of ATP Sulfurylase Activity,SO42- Uptake,and Oxidative Stress Response in Intact Canola Roots

The dual role of glutathione as a transducer of S status (A.G. Lappartient and B. Touraine [1996] Plant Physiol 111: 147-157) and as an antioxidant was examined by comparing the effects of S deprivation, glutathione feeding, and H2O2 (oxidative stress) on SO42- uptake and ATP sulfurylase activity in roots of intact canola (Brassica napus L.). ATP sulfurylase activity increased and SO42- uptake rate severely decreased in roots exposed to 10 mM H2O2, whereas both increased in S-starved plants. In split-root experiments, an oxidative stress response was induced in roots remote from H2O2 exposure, as revealed by changes in the reduced glutathione (GSH) level and the GSH/oxidized glutathione (GSSG) ratio, but there was only a small decrease in SO42- uptake rate and no effect on ATP sulfurylase activity. Feeding plants with GSH increased GSH, but did not affect the GSH/GSSG ratio, and both ATP sulfurylase activity and SO42- uptake were inhibited. The responses of the H2O2-scavenging enzymes ascorbate peroxidase and glutathione reductase to S starvation, GSH treatment, and H2O2 treatment were not to glutathione-mediated S demand regulatory process. We conclude that the regulation of ATP sulfurylase activity and SO42- uptake by S demand is related to GSH rather than to the GSH/GSSG ratio, and is distinct from the oxidative stress response.     

Vibrational spectroscopic study of glutathione complexation in aqueous solutions

A spectroscopic study of glutathione (GSH) and glutathione disulfide (GSSG) has been performed using Fourier-transformed infrared absorption and Raman scattering in order to pinpoint the sites of complexation of these two species with water and particularly with H2O2. Molecules of GSH and GSSG were studied in KBr pellets, and in aqueous solutions of H2O, D2O, and H2O with H2O2 (1 mol L(-1)) to characterize the specific influence of the solvent molecules. A time-resolved Raman study was performed for GSH/H2O2, in aqueous solution at 1:1 molar ratio in order to observe the formation of GSSG and to discuss the mechanism of this redox reaction.     

Chromatographic and mass spectrometric analysis of glutathione in biological samples

Biological thiol compounds are classified into high-molecular-mass protein thiols and low-molecular-mass free thiols. Endogenous low-molecular-mass thiol compounds, namely, reduced glutathione (GSH) and its corresponding disulfide, glutathione disulfide (GSSG), are very important molecules that participate in a variety of physiological and pathological processes. GSH plays an essential role in protecting cells from oxidative and nitrosative stress and GSSG can be converted into the reduced form by action of glutathione reductase. Measurement of GSH and GSSG is a useful indicator of oxidative stress and disease risk. Many publications have reported successful determination of GSH and GSSG in biological samples. In this article, we review newly developed techniques, such as liquid chromatography coupled with mass spectrometry and tandem mass spectrometry, for identifying GSH bound to proteins, or for localizing GSH in bound or free forms at specific sites in organs and in cellular locations.     

Liquid chromatography-tandem mass spectrometry assay of reduced and oxidized glutathione and main precursors in mice liver

A liquid chromatography/tandem mass spectrometry assay of glutathione (GSH), glutathione disulfide (GSSG) and of precursors (gamma-glutamyl-cysteine, cysteinyl-glycine, cysteine, cystine, homocysteine and homocystine) was developed to study glutathione synthesis in mice liver. After iodoacetic acid derivatization, the analytes were analyzed using reversed-phase gradient HPLC and detected using multiple reaction monitoring. Linear calibrations were performed over the concentrations range of 100-10,000 ng/mL for the thiol-containing precursors and extended up to 100,000 ng/mL for GSH and GSSG. The method was validated for each compound with inter-day accuracy below 11.9% and with precision below 15%. The method showed low limits of quantitation of 100 ng/mL for each thiol-containing compound and GSSG and of 200 ng/mL for other disulfides.