Quantitation of reduced and total glutathione at the femtomole level by high-performance liquid chromatography with fluorescence detection: application to red blood cells and cultured fibroblasts

A new rapid and highly sensitive HPLC method with ortho-phthalaldehyde (OPA) pre-column derivatization has been developed for determination of reduced glutathione (GSH) and total glutathione (GSHt) in human red blood cells and cultured fibroblasts. OPA derivatives are separated on a reversed-phase HPLC column with an acetonitrile-sodium acetate gradient system and detected fluorimetrically. An internal standard (glutathione ethyl ester) is added to facilitate quantitation. Total glutathione is determined after reduction of disulfide groups with dithiothreitol; the oxidized glutathione (GSSG) concentration is calculated by subtraction of the GSH level from the GSHt level. The assay shows high sensitivity (50 fmol per injection, the lowest reported), good precision (C.V. <5.0%), an analytical recovery of GSH and GSSG close to 100%, and linearity (r > 0.999). This HPLC technique is very simple and rapid. Its wide applicability and high sensitivity make it a convenient and reliable method for glutathione determination in various biological samples.     

Liver glutathione and glutathione reductase response of endotoxin-treated mice.

The decrease in the level of liver glutathione (GSH) in endotoxin-treated mice was in part due to formation of glutathione disulfide (GSSG). An electron-generating system (EGS) had no effect when incubated with soluble liver extracts from normal controls but resulted in recovery of GSH amounting to 25% in endotoxin-treated animals. Incubation in the absence of the EGS caused a decline of 16% in the GSH in extracts from normal animals compared with a 50% decrease in endotoxin-treated animals. Exclusion of nicotinamide adenine dinucleotide phosphate (NADP) from the EGS resulted in a slight decline in the GSH of the extract from the normal controls but 25% for the endotoxin-treated animals. Reduction of exogenous GSSG by the liver extracts required that exogenous NADP be added to ghe incubation mixtures.     

Glutathione and glutathione assays

Glutathione, the very important intracellular antioxidant, is present in intracelullar environment in milimolar concentrations. Glutathione is a tripeptide molecule, which plays an essential role in the antioxidant system, as well as in maintenance of the intracellular redox state. This thiol compound exists in two forms, the reduced (GSH) and the oxidized (GSSG), and the ratio of both forms is crucial for the characterization of the oxidative stress in cells. Number of analytical methods have been developed for the measurement of the glutathione. Especially, High Performance Liquid Chromatography methods (HPLC) are mostly used linked to different types of detection, including electrochemical, UV/VIS or fluorimetric detection. Another approach for glutathione assay is using the spectral methods, either fluorimetric or spectrophotometric assays. In enzymatic assay, glutathione reductase reduces GSSG with simultaneous oxidation of specific substrate, which is sequentially photometrically detected. The fluorimetric method is based on the detection of derivatized GSH molecule.     

Characterization of in vivo tissue redox status, oxygenation, and formation of reactive oxygen species in postischemic myocardium

The current study aims to characterize the alterations of in vivo tissue redox status, oxygenation, formation of reactive oxygen species (ROS), and their effects on the postischemic heart. Mouse heart was subjected to 30 min LAD occlusion, followed by 60 min reperfusion. In vivo myocardial redox status and oxygenation were measured with electron paramagnetic resonance (EPR). In vivo tissue NAD(P)H and formation of ROS were monitored with fluorometry. Tissue glutathione/glutathione disulfide (GSH/GSSG) levels were detected with high-performance liquid chromatography (HPLC). These experiments demonstrated that tissue reduction rate of nitroxide was increased 100% during ischemia and decreased 33% after reperfusion compared to the nonischemic tissue. There was an overshoot of tissue oxygenation after reperfusion. Tissue NAD(P)H levels were increased during and after ischemia. There was a burst formation of ROS at the beginning of reperfusion. Tissue GSH/GSSG level showed a 48% increase during ischemia and 29% decrease after reperfusion. In conclusion, the hypoxia during ischemia limited mitochondrial respiration and caused a shift of tissue redox status to a more reduced state. ROS generated at the beginning of reperfusion caused a shift of redox status to a more oxidized state, which may contribute to the postischemic myocardial injury.     

Simultaneous determination of reduced and oxidized glutathione in peripheral blood mononuclear cells by liquid chromatography-electrospray mass spectrometry.

We developed a sensitive and specific liquid chromatography-electrospray mass spectrometric (HPLC-ESI-MS) assay for the simultaneous determination of reduced and oxidized glutathione (GSH and GSSG) in peripheral blood mononuclear cells (PBMC). Following derivatization with N-ethylmaleimide to prevent GSH auto-oxidation, addition of thiosalicylic acid as internal standard, and protein precipitation with cold acetonitrile, the samples were injected into a diol column, eluted with acetonitrile-1% aqueous acetic acid (25:75) and detected by the ESI-MS system. The optimized method exhibited a good detection limit for both analytes (0.01 and 0.05 microM for GSH and GSSG, respectively). Good linearity was reached in the 0.01-20 microM range for GSH and 0.05-20 microM for GSSG. The mean recoveries of GSH and GSSG were 98.5-100.6% and 105.8-111.5%, respectively. The run-to-run repeatability for retention time and peak area was RSD% 0.06 and 1.75 for GSH and 0.18 and 2.50 for GSSG. The optimized method was applied to GSH and GSSG assay in PBMC analyzing 20 healthy individuals.     

Assessment of endoplasmic reticulum glutathione redox status is confounded by extensive ex vivo oxidation

Glutathione (GSH) and glutathione disulfide (GSSG) form the principal thiol redox couple in the endoplasmic reticulum (ER); however, few studies have attempted to quantify GSH redox status in this organelle. To address this gap, GSH and GSSG levels and the extent of protein glutathionylation were analyzed in rat liver microsomes. Because of the likelihood of artifactual GSH oxidation during the lengthy microsomal isolation procedure, iodoacetic acid (IAA) was used to preserve the physiological thiol redox state. Non-IAA-treated microsomes exhibited a GSH:GSSG ratio between 0.7:1 to 1.2:1 compared to IAA-treated microsomes that yielded a GSH:GSSG redox ratio between 4.7:1 and 5.5:1. The majority of artifactual oxidation occurred within the first 2 h of isolation. Thus, the ER GSH redox ratio is subject to extensive ex vivo oxidation and when controlled, the microsomal GSH redox state is significantly higher than previously believed. Moreover, in vitro studies showed that PDI reductase activity was markedly increased at this higher thiol redox ratio versus previously reported GSH:GSSG ratios for the ER. Lastly, we show by both HPLC and Western blot analysis that ER proteins are highly resistant to glutathionylation. Together, these results may necessitate a re-evaluation of GSH and its role in ER function.     

Cathepsin B responsiveness to glutathione and lipoic acid redox

Some subcomponents of cell protein degradation exhibit an unexplained reductive energy requirement; and diverse cysteine proteases are among multiple effector mechanisms requiring reduction. Present studies investigated whether cathepsin B activity is graded in response to (a) reduced glutathione (GSH) and dihydrolipoic acid (DHLA) concentrations, (b) their redox ratios, and (c) their differential potencies and efficacies. Purified bovine cathepsin B activity was assayed with carbobenzyloxy-Arg-Arg-aminomethylcoumarin by standard methods following inactivation by spontaneous air oxidation. Endogenous GSH concentration (2-3 mM) maintained 30-40% of the maximal cathepsin B reaction rate observed under dithiothreitol (5 mM). Following activation with GSH, the cathepsin B reaction rate was inhibited in proportion to nonphysiologic GSH:GSSG redox ratio above 1% oxidized (e.g., 85% inhibited at 3 mM:2 mM). Thus, cathepsin B can be redox buffered by the GSH:GSSG ratio. DHLA was identified as a potent cathepsin activator with threshold near 1 microM and 80% maximal activation near 10 microM. Conversely, oxidized lipoamide disulfide inhibited cathepsin B over 5-250 microM. DHLA at 5-50 microM superimposed severalfold additional activation upon the stable submaximal cathepsin B reaction rate maintained by endogenous GSH concentration (2-3 mM). Cell protein degradation was bioassayed by release of [3H] leucine from the biosynthetically labeled rat heart under nonrecirculating perfusion. The pro-oxidant, diamide (100 microM), reversibly inhibited 80% of basal proteolysis. Supraphysiologic extracellular DHLA (80 microM) doubled the basal rate of averaged cell protein degradation in 15 min. Thus, the cell redox system buffers an intermediate rate of protein degradation, which can be decreased by supraphysiologic exposure to diamide pro-oxidant or increased by DHLA reductant.     

Quantitation of loperamide and N-demethyl-loperamide in human plasma using electrospray ionization with selected reaction ion monitoring liquid chromatography-mass spectrometry

We report here the development and validation of an LC-MS method for quantitation of loperamide (LOP) and its N-demethyl metabolite (DMLOP) in human plasma. O-Acetyl-loperamide (A-LOP) was synthesized by us for use as an internal standard in the assay. After addition of the internal standard, the compounds of interest were extracted with methyl tert.-butylether and separated by HPLC on a C18 reversed-phase column using an acetonitrile-water gradient containing 20 mM ammonium acetate. The three compounds were well separated by HPLC and no interfering peaks were detected at the usual concentrations found in plasma. Analytes were quantitated using positive electrospray ionization in a triple quadrupole mass spectrometer operating in the MS-MS mode. Selected reaction monitoring was used to quantify LOP (m/z 477-266), DMLOP (m/z 463-->252) and A-LOP (m/z 519-->266) on ions formed by loss of the 4-(p-chlorophenyl)-4-hydroxy-piperidyl group upon low energy collision-induced dissociation. Calibration curves, which were linear over the range 1.04 to 41.7 pmol/ml (LOP) and 1.55 to 41.9 pmol/ml (DMLOP), were run contemporaneously with each batch of samples, along with low (4.2 pmol/ml), medium (16.7 pmol/ml) and high (33.4 pmol/ml) quality control samples. The lower limit of quantitation (LLQ) of LOP and DMLOP was about 0.25 pmol/ml in plasma. The extraction efficiency of LOP and DMLOP from human plasma was 72.3+/-1.50% (range: 70.7-73.7%) and 79.4+/-12.8% (64.9-88.8%), respectively. The intra- and inter-assay variability of LOP and DMLOP ranged from 2.1 to 14.5% for the low, medium and high quality control samples. The method has been used successfully to study loperamide pharmacokinetics in adult humans.     

Effects of age on levels of cysteine, glutathione and related enzyme activities in livers of mice and rats and an attempt to replenish hepatic glutathione level of mouse with cysteine derivatives

There was a large statistically significant decrease in the hepatic level of cysteine and glutathione (GSH) in 24 month-old mice compared to young mice, while, cystine and glutathione disulfide (GSSG) contents in the liver did not differ between young and old mice. Activities of cystathionine γ-lyase and β-synthase in mouse live of the 24 month-old group were significantly decreased. In rats, the hepatic levels of cysteine, cystine, GSH and GSSG exhibited no statistically significant change during aging to 30 month. As the rats matured, total hepatic activities of both cystathionine γ-lyase and β-synthase increased with maximum levels at 24 months of age and decreased to the same level found in 5 week old for the former and to 22% of that in 5 week old for the latter. Intraperitoneal administration of diethyl maleate to mice led to depletion of hepatic GSH. When N-acetylcysteine and a thiazolidine derivative were intravenously injected after diethyl maleate administration, the hepatic GSH level of mice was restored to the normal level.     

Impaired glutathione metabolism in hemolytic anemia

During the delivery of oxygen by erythrocytes, highly reactive oxygen species such as superoxide anion arise. The presence of reactive species damages the cell constituents. Glutathione (GSH) functions to repair cells when they are attacked by oxidative stress. GSH is synthesized in erythrocytes and glutathione disulfide (GSSG) is transported outside the cells to maintain a high GSH/GSSG ratio. The redox cycle of GSH by glutathione reductase and glutathione peroxidase is closely related to G6PD. Hereditary enzyme deficiency related to GSH metabolism, with hemolytic anemia has been reported. G6PD deficiency causes hemolytic anemia due to insufficiency of the redox cycle of GSH. Deficiency of GSH synthesizing enzymes or glutathione reductase also causes hemolysis. Pyrimidine 5'-nucleotidase deficiency causes hemolytic anemia even when there is a high concentration of GSH. Accumulation of nucleotides in red cells causes inhibition of G6PD activity.     

Impaired synthesis contributes to diabetes-induced decrease in liver glutathione

Diabetes-induced glutathione (GSH) decrease is usually ascribed to GSH oxidation. Here we investigate, in streptozotocin-treated rats, if impairment of GSH synthesis contributes to GSH decrease in diabetic liver, and if antioxidant treatments can provide protection. Diabetic rats were divided into 3 groups: untreated diabetic rats (UD); N-acetyl-cysteine (NAC)-treated diabetic rats; taurine (TAU)-treated diabetic rats; a group of non-streptozotocin-treated rats was used as control (CTR). All rats were sacrificed at 40 weeks of age. Diabetes induced hepatic glutathione decrease, but oxidized glutathione (GSSG) did not increase significantly. Accumulations of cysteine and cysteinyl-glycine in UD suggest respectively decreased glutathione synthesis and increased loss through the plasma membrane with subsequent degradation. Decreased expression of γ-glutamyl-cysteine synthetase in UD is consistent with repressed GSH synthesis. Moreover, diabetes caused increase of GSSG/GSH ratio and induction of heme oxygenase-1, both signs of oxidative stress. Supplementation with NAC or TAU resulted in amelioration of glutathione levels, probably depending on antioxidant activity, more efficient glutathione synthesis and decreased GSH loss and degradation. In conclusion, impaired synthesis and increased loss and degradation of GSH appear to contribute to a decrease in GSH levels in diabetic liver. NAC and TAU are able to partially protect from oxidative stress and GSH decrease, while enhancing GSH synthesis and restricting GSH loss.     

Quantitative analysis of intracellular glutathione levels in murine bone marrow cells by using reverse-phase high-performance liquid chromatography

Intracellular reduced glutathione (GSH) and oxidized glutathione (GSSG) amounts in murine bone marrow cells were determined by using a simple and rapid quantitative analysis that used reverse-phase high-performance liquid chromatography. Freshly prepared murine bone marrow cells lysed with water were applied to C18 double columns, were eluted with 0.1% trifluoroacetic acid-H2O at 35EC and were detected by ultraviolet absorption (220 nm). GSH and GSSG peaks with retention times of 9.2 and 27.5 min, respectively, were clear and were free of interference from the peaks of contaminated molecules in the cells. The results of this study indicate that our simple method to determine intracellular GSH and GSSG amounts can be used for a small number of cells (>3 x 10(6) cells).     

Lung antioxidant enzymes, peroxidation, glutathione system and oxygen consumption in catalase inactivated young and old Rana perezi frogs

In the lung of Rana perezi no differences as a function of age have been found for any of the five major antioxidant enzymes, reduced (GSH), oxidized (GSSG) or glutathione ratio (GSSG/GSH), oxygen consumption (VO2) and for in vivo or in vitro stimulated tissue peroxidation. This frog shows a moderate rate of oxygen consumption and a life span substantially longer than that of rats and mice. Chronic (2.5 months) catalase depletion in the lung did not affect survival or any additional antioxidant enzyme, GSH, GSSG or in vivo and in vitro lung peroxidation in any age group. Only the GSSG/GSH ratio and the VO2 were elevated in catalase depleted old but not young frogs. After comparison of these results with those obtained in other animal species by other authors we suggest the possibility that decreases in antioxidant capacity in old age be restricted to species with high basal metabolic rates. Nevertheless, scavenging of oxygen radicals can not be 100% effective in any species. Thus, aging can still be due to the continuous presence of small concentrations of O2 radicals in the tissues throughout the life span in animals with either high or low metabolic rates.     

Hepatic efflux and renal extraction of plasma glutathione: Marked differences between healthy subjects and the rat

Summary Hepatic efflux of glutathione accounts for almost 85% of the plasma level in the rat. However, the expected high concentration in the hepatic vein in man has not been demonstrated as yet. Our findings in ten patients without liver dysfunction reveal that substantial translocation of glutathione from the liver to the plasma does not occur. The removal rate of circulating glutathione by the kidney is far lower in man than in the rat (49.5% vs 84.6%). Moreover, the inferior vena cava in man contains more circulating glutathione than the artery, which is not consistent with the results obtained in the rat. Furthermore, the plasma concentration in man is about one tenth of that in the animals. These results clearly indicate that species-specific differences in the overall biosynthesis and metabolism of the tripeptide occur, resulting in marked variations in its plasma concentration.Abbreviations GSH reduced glutathione - GSSG oxidized glutathione - GSH+GSSG glutathione — the sum of reduced and oxidized glutathione - Na₄EDTA ethylenediamintetraacetic acid as its tetra-sodium salt - HCl hydrochloric acid - 2-VP 2-vinylpyridine - -GT -glutamyltranspeptidase - AT 125 [S, 5S]--amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid     

Oxidation of glutathione by the myeloperoxidase system

Oxidation of glutathione (GSH) by the myeloperoxidase (MPO) system was studied. The combination of MPO, H2O2, and a halide ion oxidized GSH. This occurred at a H2O2 concentration too low to oxidize GSH by itself. The MPO-mediated oxidation of GSH required the simultaneous presence of MPO, H2O2, and a halide ion. The system had an acid pH optimum of pH 5.5-6.0. Iodide was more effective than bromide which in turn was more effective than chloride. The oxidative product was shown to be GSSG, since it could be reduced back to GSH by glutathione reductase and NADPH. The MPO-mediated oxidation of GSH may be one mechanism by which this system damages microorganisms.     

Comparison between the effects of aging and hyperoxia on glutathione redox state and protein mixed disulfides in Drosophila melanogaster

The main purpose of this study was to determine whether experimental enhancement of oxidative stress by exposure to hyperoxia is an appropriate model for the acceleration of the normal aging process or for establishing a causal association between oxidative stress and aging. Insect tissues are directly exposed to ambient air via the tracheolar invaginations and are thus highly susceptible to oxidative stress under hyperoxic conditions. Amounts of glutathione (GSH), glutathione disulfide (GSSG) and protein mixed disulfides (PrSSG) were compared under normoxic and 100% ambient oxygen in males of two different strains of Drosophila melanogaster (Oregon R (WT) and y w strains). The reason for using two different strains was to preclude the effects of genetic background and to determine whether variations in longevity of the two strains are associated with resistance to oxidative stress. Amounts of GSSG and PrSSG increased, whereas GSH:GSSG ratios declined as a function of age in both strains. Under hyperoxia, y w flies did not exhibit an increase in GSSG amount or a decline in GSH:GSSG ratio, whereas WT flies showed a decline in GSH:GSSG ratio only during the later part of hyperoxic exposure. In neither strain there was a progressive increase in PrSSG amount under hyperoxia. Results indicate that hyperoxia (100% oxygen) neither reproduces nor accelerates the pattern of alterations in glutathione redox state and PrSSG content observed during aging under normoxic conditions, although some other indicators of oxidative stress may be affected.     

The influence of systemic hypoxia and reoxygenation on the glutathione redox system of brain, liver, lung and plasma in newborn rats.

The concentrations of reduced (GSH) and oxidized glutathione (glutathione disulfide, GSSG) in lung, liver, brain and plasma of newborn rats were investigated under the condition of reversible hypoxia. Brain and lung of newborn rats seem to be susceptible to reversible hypoxia. We found an increase in GSSG concentration after hypoxia in these organs. This alteration of the GSH-GSSG redox system was reversible within 2 hours of reoxygenation. A second increase in cerebral GSSG concentration after 4 hours of reoxygenation was connected with fasting during the experiment. In the liver we found a hypoxia dependent decrease in the GSH level, followed by a decrease in GSSG concentration. The increased GSSG concentrations in lung and brain are accompanied by an enhancement of plasma GSSG concentration.     

Altered glutathione redox state in schizophrenia

Altered antioxidant status has been reported in schizophrenia. The glutathione (GSH) redox system is important for reducing oxidative stress. GSH, a radical scavenger, is converted to oxidized glutathione (GSSG) through glutathione peroxidase (GPx), and converted back to GSH by glutathione reductase (GR). Measurements of GSH, GSSG and its related enzymatic reactions are thus important for evaluating the redox and antioxidant status. In the present study, levels of GSH, GSSG, GPx and GR were assessed in the caudate region of postmortem brains from schizophrenic patients and control subjects (with and without other psychiatric disorders). Significantly lower levels of GSH, GPx, and GR were found in schizophrenic group than in control groups without any psychiatric disorders. Concomitantly, a decreased GSH:GSSG ratio was also found in schizophrenic group. Moreover, both GSSG and GR levels were significantly and inversely correlated to age of schizophrenic patients, but not control subjects. No significant differences were found in any GSH redox measures between control subjects and individuals with other types of psychiatric disorders. There were, however, positive correlations between GSH and GPx, GSH and GR, as well as GPx and GR levels in control subjects without psychiatric disorders. These positive correlations suggest a dynamic state is kept in check during the redox coupling under normal conditions. By contrast, lack of such correlations in schizophrenia point to a disturbance of redox coupling mechanisms in the antioxidant defense system, possibly resulting from a decreased level of GSH as well as age-related decreases of GSSG and GR activities.     

Liquid chromatography-electrospray ionization mass spectrometric analysis of corticosterone in rat plasma using selected ion monitoring.

A simple and fast yet highly sensitive and specific method based on HPLC coupled to electrospray ionization mass spectrometry has been developed for the quantitation of corticosterone in rat plasma. After extraction of rat plasma (100 microl) with diethyl ether using 5-pregnen-3beta-ol-20-one-16alpha-carbonitrile (Sigma) as internal standard, HPLC was performed on a short C8 column (Zorbax-Eclipse, 50x4.6 mm I.D.) using a steep methanol-water gradient (methanol 54% to 90% in 6 min). Detection was performed on a single quadruple mass spectrometer in selected ion monitoring mode (m/z 369 for corticosterone and 364 for the internal standard). The detection limit of the assay was 9 fmol (3 pg) of corticosterone on column. In vitro data were subjected to curve fitting (cubic, r2=0.9999). Recovery of corticosterone after extraction ranged from 81 to 93%. The relative standard deviations for intra- and inter-assay precision ranged from 0.8 to 3.6% and 5.2 to 12.9%, respectively. Corticosterone did not undergo any appreciable degradation when stored in plasma at -20 degrees C for 2 months. The assay is routinely used in our laboratory to examine corticosterone levels as a marker of stress in rats and may also be used for the determination of 18-hydroxy-11-deoxycorticosterone.