Glutathione Monoethyl Ester Research Articles

Electron Paramagnetic Resonance Studies on Nitroxide Radical 2,2,5,5-Tetramethyl-4-piperidin-1-oxyl (TEMPO) Redox Reactions in Human Skin

Electron paramagnetic resonance (EPR) is currently being explored for the study of living biological systems. Among biophysical and biochemical applications, the study of nitroxide radical interactions with tissue antioxidants and oxidants is of growing interest. Skin is a target organ of the EPR methodology and is frequently exposed to oxidative stress. We investigated the piperidine-type nitroxide 2,2,5,5-tetramethyl-4-piperidin-1-oxyl (TEMPO) because it is skin permeable and readily accepts electrons in biological systems. TEMPO is readily scavenged on the surface of cultured human skin. Pretreatment of skin cultures with butylhydroperoxide, which decreases intracellular ascorbate and glutathione, causes inhibition of nitroxide scavenging. Exposure of skin cultures to dehydroascorbate, which is internalized and converted to ascorbate, leads to stimulation of nitroxide scavenging. In human keratinocytes and fibroblasts, the TEMPO radical is reversibly reduced to the hydroxylamine depending on the oxygen concentration and the availability of intracellular glutathione and ascorbate. Cell exposure to the glutathione synthetase inhibitor buthionine–sulfoximine depleted intracellular glutathione and inhibited nitroxide reduction; exposure to dehydroascorbate or glutathione–monoethylester increased intracellular ascorbate or glutathione concentration and stimulated nitroxide reduction. Quantitative considerations indicate that the major reduction site of TEMPO in skin and skin cells is the cytosol ascorbate/glutathione redox cycle. We suggest that analysis of TEMPO radical scavenging by the EPR technique is a convenient method for measuring skin ascorbate and thioldependent antioxidant activity in vitro and in vivo. Copyright © 1997 Elsevier Science Inc.

Thiol regulation of endotoxin-induced release of tumour necrosis factor alpha from isolated rat Kupffer cells.

Proinflammatory cytokines released by hepatic macrophages (Kupffer cells) have a central role in the pathogenesis of liver injury and the cardiovascular abnormalities of sepsis. Because cytokine release is controlled primarily at the level of gene expression, intracellular signalling mechanisms that control the transcription of cytokine genes are critical links to organ injury. Oxidant stress up-regulates and antioxidants down-regulate the pleiotropic transcription factor NF-kappa B, a DNA-binding protein that induces the expression of cytokines and vascular adhesion molecules. Thiol-bearing molecules are also important inhibitors of NF-kappa B activation, but whether this inhibition represents an antioxidant effect is unknown. This study was undertaken to determine whether important endogenous and pharmacological thiols modulate the activation of NF-kappa B and the release of tumour necrosis factor alpha (TNF-alpha) from Kupffer cells and to ascertain whether these effects are mediated through glutathione. Exposure of rat Kupffer cells to a physiologically relevant concentration of lipopolysaccharide (10 ng/ml) activated NF-kappa B within 1 h and induced the release of TNF-alpha over 5 h. Cellular glutathione content remained unchanged after lipopolysaccharide exposure, but both glutathione monoethyl ester and N-acetyl-L-cysteine increased cellular glutathione levels, blocked NF-kappa B activation and inhibited the release of TNF-alpha. Inhibition of glutathione synthesis prevented the NAC-induced increase in Kupffer cell glutathione, yet it did not prevent the inhibition of TNF-alpha release by NAC. Thus the inhibition of NF-kappa B activation by pharmacological thiols such as NAC might reflect a more general role of the intracellular thiol redox status in NF-kappa B regulation rather than the antioxidant properties of these agents.

Generation of DNA base modification following treatment of cultured murine keratinocytes with benzoyl peroxide.

Benzoyl peroxide (BzPO) is a free radical generating compound that acts as a tumor promoter and progressor in mouse skin. BzPO is cleaved in the presence of copper to produce benzoyloxyl and phenyl radicals. Treatment of mutation reporter plasmids with BzPO and copper yields predominantly single-strand breaks and G-->T transversion mutations. To explore the role of base modifications in the possible mammalian mutagenicity of BzPO the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) within the DNA of cultured murine keratinocytes was investigated. Treatment with 10 microM BzPO produced a maximum 3-fold increase in levels of 8-OHdG versus vehicle controls within 1-2 h, with significant levels of 8-OHdG persisting 6 h after initial exposure to BzPO. Pretreatment with the copper chelator bathocuproine disulfonic acid reduced the levels of 8-OHdG generated by BzPO to near background. However, treatment with the iron chelator desferal did not. The stable metabolic product of BzPO benzoic acid was ineffective in producing 8-OHdG. Depletion of cellular glutathione with L-buthionine-(S,R)-sulfoximine increased the amount of BzPO-generated 8-OHdG, while supplementation with glutathione monoethyl ester reduced the number of 8-OHdG molecules formed. Collectively, these results suggest that BzPO at non-cytotoxic concentrations undergoes copper-dependent activation to a reactive product to generate 8-OHdG within cultured murine keratinocytes.

Mechanism of defect in mitochondrial transport of GSH in ethanol-FED (ETOH) rats: membrane lipid composition and effects of S-adenosyl-L-methionine (SAM): [formula omitted] Liver Unit, Hospital Clinic i Provincial, Barcelona, Spain, and ∗GI and Liver Diseases, USC School of Medicine, La., CA

Methylmercury (MeHg) exposure at high concentrations poses significant neurotoxic threat to humans worldwide. The present study investigated the mechanisms of glutathione-mediated attenuation of MeHg neurotoxicity in primary cortical culture. MeHg (5 μM) caused depletion of mono- and disulfide glutathione in neuronal, glial and mixed cultures. Supplementation with exogenous glutathione, specifically glutathione monoethyl ester (GSHME) protected against the MeHg induced neuronal death. MeHg caused increased reactive oxygen species (ROS) formation measured by dichlorodihydrofluorescein (DCF) fluorescence with an early increase at 30 min and a late increase at 6 h. This oxidative stress was prevented by the presence of either GSHME or the free radical scavenger, trolox. While trolox was capable of quenching the ROS, it showed no neuroprotection. Exposure to MeHg at subtoxic concentrations (3 μM) caused an increase in system xc− mediated 14C-cystine uptake that was blocked by the protein synthesis inhibitor, cycloheximide (CHX). Interestingly, blockade of the early ROS burst prevented the functional upregulation of system xc−. Inhibition of multidrug resistance protein-1 (MRP1) potentiated MeHg neurotoxicity and increased cellular MeHg. Taken together, these data suggest glutathione offers neuroprotection against MeHg toxicity in a manner dependent on MRP1-mediated efflux.

Decreased Levels of Total and Reduced Glutathione in CD4+ Lymphocytes in Common Variable Immunodeficiency Are Associated With Activation of the Tumor Necrosis Factor System: Possible Immunopathogenic Role of Oxidative Stress

We have previously shown chronic immune activation and enhanced generation of reactive oxygen species in common variable immunodeficiency (CVI). In the present study, we examined levels of glutathione, the dominant intracellular thiol, that play an important protective role against oxidative and inflammatory stress in plasma and in monocytes and lymphocyte subsets in 20 CVI patients and in 16 healthy controls. CD4+ lymphocytes from CVI patients had significantly lower levels of both total and reduced glutathione as well as a lower ratio of reduced to total glutathione compared with healthy controls. This decrease in glutathione levels in CD4+ lymphocytes was most pronounced in the CD45RA+ subset. Plasma levels of total glutathione were also significantly decreased in CVI. In contrast, monocytes from CVI patients exhibited increased levels of both total and reduced glutathione compared with blood donor monocytes. CVI patients had significantly raised serum levels of tumor necrosis factor alpha (TNF alpha) and TNF alpha concentration was strongly associated with glutathione depletion in CD4+ lymphocytes. Furthermore, the lowest levels of both total and reduced glutathione were found in a subgroup of CVI patients characterized by persistent immune activation in vivo, decreased numbers of CD4+ lymphocytes in peripheral blood, and splenomegaly. Finally, supplementation of cell cultures with glutathione-monoethyl ester did significantly enhance interleukin-2 production from peripheral blood mononuclear cells in CVI patients. These glutathione abnormalities in CVI indicate increased oxidative stress, particularly in CD4+ lymphocytes, and intracellular depletion of reduced glutathione of the demonstrated magnitude may have profound implications for CD4+ lymphocyte function and the immunodeficiency in CVI.

Glutathione protection against gentamicin ototoxicity depends on nutritional status

This study demonstrates that gentamicin ototoxicity depends on dietary factors and correlates with tissue glutathione levels. After 15 days of gentamicin injections (100 mg/kg/day s.c.) guinea pigs on a regular protein diet (18.5% protein) had an average hearing loss of 9 dB at 3 kHz, 31 dB at 8 kHz and 42 dB at 18 kHz. Guinea pigs on a 7% protein diet showed an increased hearing loss of 52 dB at 3 kHz, 63 dB at 8 kHz and 74 dB at 18 kHz. Supplementing the low protein diet with either essential or sulfur-containing amino acids did not protect against gentamicin ototoxicity. Glutathione levels in the cochlear sensory epithelium were decreased in animals on a low protein diet and could be restored to normal by oral administration of glutathione monoethyl ester (1.2 g/kg/day) in combination with vitamin C (100 mg/kg/day). Glutathione supplementation significantly reduced the magnitude of hearing loss in the low protein diet group at all frequencies (43 dB reduction at 3 kHz, 27 dB reduction at 8 kHz and 21 dB reduction at 18 kHz). In animals on a full protein diet, dietary glutathione neither increased cochlear glutathione levels nor attenuated hearing loss. Serum gentamicin levels did not differ between animals on the various diets with or without glutathione supplement. These results suggest that gentamicin toxicity and detoxifying mechanisms are affected by the metabolic state of the animal and the glutathione content of the tissue. Thus, compounds that could potentially protect against gentamicin ototoxicity may be more correctly assessed in animal models of deficient nutritional states in which endogenous detoxifying mechanisms are compromised. This animal model might also be more realistically related to the clinical situation of a critically ill patient receiving gentamicin treatment.

Suppression of nitric oxide toxicity in islet cells by α-tocopherol

We show here that preincubation of pancreatic islet cells with α-tocopherol significantly improves their resistance to toxic doses of nitric oxide (NO). No protection was afforded by other antioxidants such as vitamin C or glutathione-monoethyl ester. The pathway of NO induced islet cell death involves DNA damage and excessive activation of poly(ADP-ribose)polymerase leading to irreversible depletion of intracellular NAD +. α-Tocopherol was found to interfere at early steps of this pathway, by preventing the occurrence of DNA strand breaks. This indicates that α-tocopherol directly interacts with NO or its reactive intermediates. We conclude that α-tocopherol is not only part of the cellular defence system against oxygen radicals but also protects eukaryotic cells from NO toxicity.

Comparison of the delivery of reduced glutathione into P388D 1 cells by reduced glutathione and its mono- and diethyl ester derivatives

The effect of reduced glutathione, reduced glutathione monoethyl ester and reduced glutathione cliethyl ester on the cellular concentration of reduced glutathione and cysteine in P388D 1 macrophages in vitro, and the cellular and extracellular de-esterification of reduced glutathione esters, was investigated. At 1 mM reduced glutathione derivative, only reduced glutathione diester markedly increased the cellular concentration of reduced glutathione. There was little delivery of reduced glutathione monoethyl ester into the cells. Reduced glutathione, and monoethyl and diethyl ester derivatives all increased the cellular concentration of cysteine; reduced glutathione diethyl ester also increased the cellular concentration of γ-glutamylcysteine. Reduced glutathione esters were de-esterified intracellularly where the diester was rapidly converted to the monoester. The diester was also converted to the monoester extracellularly by interaction with cell surface esterases and by a much slower spontaneous hydrolysis. This indicates that the diester of reduced glutathione was a much more effective vehicle for delivery of reduced glutathione into cells than the monoester. Reduced glutathione diester also increased the cellular concentrations of cysteine and γ-glutamylcysteine, suggesting that de novo synthesis of reduced glutathione was also stimulated.

Protective effect of glutathione (GSH) over glutathione monoethyl-ester (GSH-E) on cold preservation of isolated rat liver cells

Hepatocyte suspensions provide a rapid method to determine how hypothermic storage affects liver cell metabolism and viability. We investigated whether reduced Glutathione (GSH) inclusion into a modified University of Wisconsin (UW) solution, has a protective effect over Glutathione derivatives, such as Glutathione-monoethylester (GSH-E), when suspensions of hepatocytes are cold stored for several days. Isolated rat liver cells were cold preserved 96 h in UW, UW plus 3 mM GSH and UR' plus 3 mM GSHE. During the cold storage, not significant changes in cell viability were observed, but the total Glutathione content was higher in systems with extracellular GSII over those with GSH-E or without. After cold storage, the liver cells were gently resuspended in Krebs-Henseleit—1% Albumin and used for 120 min of normothermic (37°C incubation. We evaluate the functional response of the cells measuring the exclusion of Trypan Blue (TBE). This response was clearly different in preserved cells in presence of GSH. These results indicate a protective role of extracellular Glutathione, due to an accumulation of it, rather than the derivative, for hepatic cell during the cold storage in UW solutions. And also, it is possible to extend experiments with hepatocytes from a single cell isolation over 4 or more consecutive days

Effect of Oral Glutathione Monoethyl Ester and Glutathione on Circulating and Hepatic Sulfhydrils in the Rat

Glutathione (GSH) plays an important role in the detoxification of reactive metabolites of oxygen and xenobiotics and as a source of cysteine. Since several clinical situations characterized by low circulating and intracellular GSH have been identified, there is a growing interest in pharmacological interventions to correct a deranged sulfhydril status. Therefore, the systemic bioavailability of orally administered GSH and glutathione monoethyl ester (GSHE) was examined in the rat. Following the intraduodenal administration of 0.5 mmol/kg of GSH and GSHE there was no significant increase in the concentrations of cysteine and GSH in plasma, but hepatic cysteine and GSH increased significantly, albeit transiently. Five mmol/kg of GSH and GSHE significantly increased circulating and hepatic cysteine and GSH. Following the administration of 0.5 and 5 mmol/kg of GSHE low concentrations of the ester were found in plasma and the liver, indicating that GSHE is not readily absorbed from the gastrointestinal tract, although it is not a substrate for gamma-glutamyl-transferase. GSHE resulted in a delayed release of cysteine and GSH compared to GSH, such that the concentrations of GSH and cysteine in liver and plasma were significantly higher 2 h after administration of GSHE than after GSH. The data indicate that the bioavailability of GSH and GSHE is low in the rat. Orally administered GSH and GSHE do not affect the circulating concentrations of GSH and cysteine unless very high doses are administered, but increase hepatic cysteine and GSH at lower doses because of the efficient extraction by the liver of cysteine originating from the breakdown of GSH and GSHE in the gut.

Status of Glutathione during Oxidant-Induced Oxidative Stress in the Preimplantation Mouse Embryo1

Experiments were conducted to elucidate the status of glutathione present in the oxidized (GSSG), reduced (GSH), and protein-mixed disulfide (GSSprotein) forms in preimplantation mouse embryos during development and after treatment with tertiary-butyl hydroperoxide (tBH) to cause oxidative stress. Glutathione was measured at picomolar levels by fluorimetric HPLC after derivatization of extracted embryonic samples with dansyl chloride. GSH content decreased approximately 10-fold from that in the unfertilized oocyte to 0.12 pmol/blastocyst, representing an estimated change in concentration from 7 to 0.7 mM. GSH levels were lower in embryos cultured in vitro than in embryos that developed in vivo. Addition of GSH to the culture medium improved in vitro development of mouse embryos, but surprisingly the addition of glutathione monoethyl ester did not. Addition of low levels of the oxidant tBH (13.2 microM) to culture medium decreased the percentage of two-cell and blastocyst stage embryos that exhibited further development. After 15-min exposure to 13.2 microM tBH, GSH levels were markedly decreased in the two-cell stage embryo (75%), but only slightly decreased (25%) in the blastocyst. The loss of GSH was accounted for by increases in GSSG and GSSprotein, indicating that the embryo was undergoing oxidative stress. These data indicate that preimplantation embryos are very sensitive to conditions that can cause oxidative stress and show also that their glutathione status changes dramatically during development.

A model for fatal halothane hepatitis in the guinea pig.

In the guinea pig, depleting hepatic glutathione before inhaling subanesthetic 0.1% halothane increases covalent binding of halothane biotransformation intermediates to hepatic protein and potentiates resultant liver injury. Because inhalation of a higher concentration of halothane is known to produce greater levels of covalent binding than with subanesthetic halothane, this study was undertaken with 0.25-1.0% halothane concentrations to further examine glutathione depletion as an etiology for halothane hepatitis. Male Hartley guinea pigs were injected intraperitoneally with either vehicle control solution (Veh) or 1.6 g/kg buthionine sulfoximine (BSO), to decrease hepatic glutathione by > 80%, 24 h before a 4-h exposure to 0.25%, 0.5%, or (v/v) halothane with 40% O2. Some BSO-pretreated animals also received 2.0 g/kg glutathione monoethyl ester (GEE), intraperitoneally, 2 h before inhaling halothane to replenish hepatic glutathione. Glutathione-depleted animals developed significantly worse hepatic injury with each halothane concentration. One-third to one-half of BSO+halothane-treated animals developed fatal submassive to massive hepatic necrosis. Covalent binding of halothane intermediates to hepatic protein increased by 45% in BSO + 1.0% halothane-treated guinea pigs. Administration of GEE to BSO-pretreated animals before 1.0% halothane decreased binding to protein and blunted development of liver necrosis. Following Veh + 1.0% halothane, hepatic glutathione was found to be decreased by 60%. Glutathione would appear to help protect hepatocytes to some degree from covalent binding by reactive halothane biotransformation intermediates. These studies present the first animal model to produce fatal halothane-induced hepatic necrosis.

Glutathione monoethyl ester (GSHE) is a slow release form of extracellular glutathione (GSH)

Glutathione monoethyl ester (GSHE) is a slow release form of extracellular glutathione (GSH)

Glutathione monoethyl ester (GSHE) protects against ischemia-reperfusion injury of the liver

Glutathione monoethyl ester (GSHE) protects against ischemia-reperfusion injury of the liver

Transport of glutathione diethyl ester into human cells.

Glutathione monoesters in which the carboxyl group of the glycine residue is esterified were previously found, in contrast to glutathione itself, to be effectively transported into various types of cells and to be converted intracellularly into glutathione. Glutathione monoesters are thus useful for prevention of oxidative stress, certain toxicities, and for treatment of glutathione deficiency. Glutathione diethyl ester is rapidly split to the glutathione monoethyl ester by mouse plasma glutathione diester alpha-esterase activity. Thus, as expected, glutathione mono- and diesters have similar effects on cellular glutathione levels in mice. However, human plasma lacks glutathione diester alpha-esterase; thus, it became of interest to compare the transport properties of glutathione mono- and diesters in human cells. We found that human cells (erythrocytes, peripheral blood mononuclear cells, fibroblasts, ovarian tumor cells, and purified T cells) transport glutathione diethyl ester much more effectively than the corresponding monoethyl (glycyl) ester. Human cells rapidly convert glutathione diethyl ester to the monoester, whose intracellular levels rise to levels that are significantly higher than levels found after application of the monoester to the cells. High levels of the monoester provide the cells with a means of producing glutathione over a period of time. We conclude that glutathione diethyl ester is highly effective as a delivery agent for glutathione monoester, and thus for glutathione, in human cells and therefore could serve to decrease oxidative stress and toxicity. Hamster (and certain other animals) also lack plasma glutathione diester alpha-esterase and therefore would be suitable animal models. Previously reported toxicity of certain glutathione ester preparations appears to reflect the presence of impurities rather than effects of the esters.

Increased susceptibility of stored erythrocytes to anti-band 3 IgG autoantibody binding

When human blood was stored in a citrate-phosphate-dextrose (CPD) solution at 4°C, the susceptibility of the erythrocytes to binding of autologous IgG increased. The autologous IgG binding was partially inhibited by purified Band 3 glycoprotein and its oligosaccharides. The susceptibility of the erythrocytes to binding of 125I-labeled anti-band 3 IgG autoantibody similarly increased. The results indicate that the anti-band 3 binding sites composed of Band 3 oligosaccharides were generated on the cell surface. The rate of the increase in the susceptibility of the stored cells to the antibody binding was lowered when blood was stored in a CPD solution containing L-ascorbic acid or erythorbic acid, suggesting involvement of an oxidative mechanism in the generation of the binding sites. The cytoplasmic glutathione level of erythrocytes gradually decreased during the blood storage. Storing blood in a CPD solution containing glutathione monoethylester or glutathione monoisopropylester resulted in partial prevention of the decrease in cytoplasmic glutathione level and of the increase in the IgG-binding ability of the cells. Similar preventive effect of glutathione monoethylester was observed in the binding of 125I-labeled anti-band 3 autoantibody to the stored erythrocytes. Thus, the increase in the susceptibility of the stored erythrocytes to anti-band 3 binding may be caused, at least partially, by an oxidative stress resulting in a decreased cytoplasmic glutathione level.

Cardiac cell toxicity induced by 4-hydroperoxycyclophosphamide is modulated by glutathione.

Cardiac myocytes were exposed to 4-hydroperoxycyclophosphamide (4-HC, an activated derivative of cyclophosphamide) to assess whether early ionic events are associated with the dose limiting toxicity of this chemotherapeutic agent. Primary cultures of embryonic chick cardiac myocytes were grown to confluency and then exposed to a medium containing 4-HC. Cellular sodium, potassium, and calcium contents were measured by atomic absorption spectrophotometry and related to protein and ATP content. Pretreatment of the cultured heart cells with glutathione depleting or enhancing agents provided the basis for evaluating the involvement of glutathione in the 4-HC-induced cytotoxicity. Administration of 150 microM 4-HC to cardiac myocytes resulted in increases in cellular sodium and calcium contents and decreases in potassium, ATP, and protein contents. Pretreatment of cardiac myocytes with L-buthionine-SR-sulphoximine, a specific inhibitor of gamma-glutamylcysteine synthetase, depleted cellular glutathione to 12% of control and significantly reduced the minimum concentration of 4-HC causing cytotoxic changes. Conversely, elevation of cellular thiol content by the pretreatment of cardiac myocytes with glutathione monoethyl ester (but not glutathione) provided protection against 4-HC induced cytotoxicity. Cellular glutahione concentration can markedly influence the 4-HC induced changes in cellular ion content and ATP, which are early indicators of 4-HC induced cytotoxicity.

Oxygen-induced lung damage. Relationship to lung mitochondrial glutathione levels.

Several reports suggest there is a relationship between lung glutathione (GSH) levels and susceptibility to oxygen-induced lung damage. However, studies of other organs and cells indicate that a better relationship may exist between mitochondrial GSH levels and oxidant damage. We determined whether there is a similar relationship in the lung using a well-characterized mouse model and a series of interventions that alter lung GSH levels and susceptibility to oxygen-induced lung damage. Mice were fasted or given buthionine sulfoximine (BSO, 20 mM), which reduce total lung GSH levels and increase susceptibility to oxygen-induced lung damage. Mice were also given glutathione monoethyl ester (GSH-ME) intraperitoneally (5 or 10 mM/kg/day for 2 days) or intratracheally (0.2 mM once) in an attempt to increase lung GSH levels. Fasting for up to 3 days and the administration of BSO for 7 to 10 days decreased total lung GSH levels (p < 0.001 for both) but not lung mitochondrial GSH levels. Intraperitoneal administration of GSH-ME increased mitochondrial GSH levels (p < 0.001 in both fed and fasted mice), but it had little effect on total lung GSH levels and no effect on susceptibility to oxygen-induced lung damage. Exposure to 100% oxygen increased mitochondrial GSH levels in both the fed and fasted mice to nearly the same extent (p < 0.001 for both). However, the fasted mice had lower total lung GSH levels compared with the fed mice (p < 0.05) and increased susceptibility to 100% oxygen.(ABSTRACT TRUNCATED AT 250 WORDS)

Parenteral glutathione monoester enhances tissue antioxidant stores.

Glutathione (GSH) is a potent endogenous antioxidant that protects major organs from oxidant injury. However, present nutrition regimens may inadequately support tissue stores of this tripeptide during critical illness. To determine whether GSH reserves can be enhanced in vivo with intravenous (IV) supplements, rats underwent central venous catheterization, were given chow and water ad libitum during a 2-day recovery period, and were then randomized to receive one of three treatments as an IV bolus: (1) dextrose, (2) glutathione (GSH), or (3) glutathione monoethyl ester. GSH monoethyl ester is transported into cells more easily than is GSH. Tissue and plasma samples were analyzed for GSH at 2 and 4 hours after drug administration. Liver, renal, and ileal mucosal GSH were significantly increased in the GSH-monoethyl ester rats compared with dextrose-treated animals. In addition, plasma GSH was dramatically increased after monoester injection. In contrast, GSH administration depressed liver GSH stores and did not significantly affect GSH concentration in the other organs analyzed. Plasma GSH concentration was elevated 2 hours after GSH administration. We conclude that: (1) the monoethyl ester of glutathione can be used in vivo to enhance tissue and plasma GSH concentration and (2) IV GSH administration does not significantly increase tissue GSH levels and may paradoxically depress hepatic GSH in normal rats. Because the malnourished and critically ill are likely to have depleted GSH stores, nutrition strategies that include the provision of GSH monoester may lend additional support to those organs that are at risk for injury from oxygen free radicals during catabolic states.

Increase in rat brain glutathione following intracerebroventricular administration of γ-glutamylcysteine

The effects of intracereboventricularly (i.c.v.) administered γ-glutamylcysteine (γ-GC) and glutathione (GSH) monoethyl ester, subcutaneously (s.c.) injected l-2-oxo-4-thiazolidinecarboxylic acid (OTC) and intraperitoneally (i.p.) administered cysteine on the concentration of GSH in rat brain were investigated. The brain content of GSH, cysteine and γ-GC was determined by HPLC with electrochemical detection (gold/mercury electrode) using N-acetylcysteine as internal standard. A dosedependent increase in the GSH concentration (145–170% of controls) was found in the substantia nigra (SN) and in the rest of the brain stem after injection of γ-GC, whereas no significant alterations in GSH were observed in the striatum and in the cerebral cortex. High levels of γ-GC could be detected in the brain tissue after the administration, and the concentration of cysteine did also increase markedly after γ-GC injection in all brain regions assessed. I.c.v. administration of l-buthionine sulfoximine (L-BSO) reduced the brain concentration of GSH by 50–70% within 24 hr. Injection of γ-GC 24 hr after L-BSO resulted in an increase in GSH up to control values within 1–3 hr in the SN and the rest of the brain stem, whereas only a slight increase in GSH was observed in the striatum and the cerebral cortex. The concentration of GSH in the striatum and SN did not change after i.p. injection of cysteine, but a slight increase in the GSH concentration in the limbic region was observed. GSH monoethyl ester (i.c.v.) and OTC (s.c.) did not produce any significant increase in the GSH concentration in the brain. When the GSH concentration had been reduced by administration of L-BSO (i.c.v.; 24 hr) subsequent injection of GSH monoethyl ester led to a slight increase in the striatal and limbic GSH levels. These data show that, of the drugs studied, γ-GC was the most effective in increasing brain GSH. It could thus serve as a valuable tool in future studies regarding metabolism and function of GSH in the brain. The observed difference in the effects of γ-GC in different brain regions indicate that the brain tissue is not homogeneous with regard to GSH synthesizing capacity.