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The term glutathione is typically used as a collective term to refer to the tripeptide L-gamma-glutamyl-L-cysteinylglycine in both its reduced and dimeric forms. Monomeric glutathione is also known as reduced glutathione and its dimer is also known as oxidized glutathione, glutathione disulfide and diglutathione. In this monograph, reduced glutathione will be called glutathione— this is its common usage by biochemists—and the glutathione dimer will be referred to as glutathione disulfide.

Glutathione is widely found in all forms of life and plays an essential role in the health of organisms, particularly aerobic organisms. In animals, including humans, and in plants, glutathione is the predominant non-protein thiol and functions as a redox buffer, keeping with its own SH groups those of proteins in a reduced condition, among other antioxidant activities. Glutathione has the following structural formula:

Glutathione (GSH), whose IUPAC name is 2-amino-5-{[2-[(carboxymethyl)amino]-1-(mercaptomethyl)-2-oxoethyl]amino}-5-oxopentanoic acid, is γ-glutamylcysteinylglycine, a tripeptide. It contains an unusual peptide linkage between the amine group of cysteine and the carboxyl group of the glutamate side chain. Glutathione, an antioxidant, protects cells from toxins such as free radicals .

DESCRIPTION:

Thiol groups are kept in a reduced state within ~5 mmol in animal cells. In effect, glutathione reduces any disulfide bonds formed within cytoplasmic proteins to cysteines by acting as an electron donor. Glutathione is found almost exclusively in its reduced form, since the enzyme which reverts it from its oxidized form (GSSG), glutathione reductase, is constitutively active and inducible upon oxidative stress. In fact, the ratio of reduced to oxidized glutathione within cells is often used scientifically as a measure of cellular toxicity.

Glutathione is present in tissues in concentrations as high as one millimolar. Cysteine, the business residue of glutathione, neither has the solubility nor activity of glutathione at physiological pH. It appears that nature has built the cysteine molecule into the glutathione tripeptide to make the amino acid more soluble and allow it to have redox buffering activity in a living tissue environment. Glutathione also plays roles in catalysis, metabolism, signal transduction, gene expression and apoptosis. It is a cofactor for glutathione S-transferases, enzymes which are involved in the detoxification of xenobiotics, including carcinogenic genotoxicants, and for the glutathione peroxidases, crucial selenium-containing antioxidant enzymes (see Selenium). It is also involved in the regeneration of ascorbate from its oxidized form, dehydroascorbate (see Vitamin C). There are undoubtedly roles of glutathione that are still to be discovered.

Glutathione is present in the diet in amounts usually less than 100 milligrams daily, and it does not appear that much of the oral intake is absorbed from the intestine into the blood (see Pharmacokinetics). Glutathione is not an essential nutrient since it can be synthesized from the amino acids L-cysteine, L-glutamate and glycine. It is synthesized in two ATP-dependent steps: first, gamma-glutamylcysteine is synthesized from L-glutamate and cysteine via the enzyme gamma-glutamylcysteine synthetase—the rate limiting step— and second, glycine is added to the C-terminal of gamma-glutamylcysteine via the enzyme glutathione synthetase. The liver is the principal site of glutathione synthesis. In healthy tissue, more than 90% of the total glutathione pool is in the reduced form and less than 10% exists in the disulfide form. The enzyme glutathione disulfide reductase is the principal enzyme that maintains glutathione in its reduced form. This latter enzyme uses as its cofactor NADPH (reduced nicotinamide adenine dinucleotide phosphate). NADPH is generated by the oxidative reaction in the pentose phosphate pathway.

The consequences of a functional glutathione deficiency, which results in tissue oxidative stress, can be seen in some pathological conditions. For example, those with glucose 6-phosphate dehydrogenase deficiency produce lower amounts of NADPH and hence, lower amounts of reduced glutathione. This condition is characterized by a hemolytic anemia. Conditions causing chronic glutathione deficiency all result in hemolytic anemia, among other pathological consequences. Oxidative stress caused by glutathione deficiency results in fragile erythrocyte membranes. Malaria-causing organisms (Plasmodia species) do not like to feed on these sick erythrocytes. That is about the only good news regarding this situation. Chronic functional glutathione deficiency is also associated with immune disorders, an increased incidence of malignancies, and in the case of HIV disease, probably accelerated pathogenesis of the disease. Acute manifestations of functional glutathione deficiency can be seen in those who have taken an overdosage of acetaminophen. This results in depletion of glutathione in the hepatocytes, leading to liver failure and death, if not promptly treated.

Glutathione is an orphan drug for the treatment of AIDS-associated cachexia. It is thought that this disorder is due, in part, to oxidatively-stressed and damaged enterocytes. There is some evidence that although orally administered glutathione may not be absorbed into the blood from the small intestine to any significant extent, that it may be absorbed into the enterocytes where it may help repair damaged cells. Glutathione in one form or another is the subject of some medicinal chemistry research and some clinical trials. For example, an aerosolized form of glutathione is being studied in AIDS and cystic fibrosis patients. Glutathione, the principal antioxidant of the deep lung, appears to be diminished in those with AIDS. Prodrugs of gamma-L-glutamyl-L-cysteine are being evaluated as anticataract agents.

Glutathione (reduced) is known chemically as N-(N-L-gamma-glutamyl-L-cysteinyl)glycine and is abbreviated as GSH. Its molecular formula is C10H17N3O6S and its molecular weight is 307.33 daltons. Glutathione disulfide is also known as L-gamma-glutamyl-L-cysteinyl-glycine disulfide and is abbreviated as GSSG. Its molecular formula is C20H32N6O12S2.

MECHANISM OF ACTION:

Glutathione is the principal intracellular non protein thiol and plays a major role in the maintenance of the intracellular redox state. It may be thought of as an intracellular redox buffer. Glutathione is a nucleophilic scavenger and an electron donor via the sulfhydryl group of its business residue, cysteine. Its reducing ability maintains molecules such as ascorbate and proteins in their reduced state. Glutathione is also the cofactor for the selenium-containing glutathione peroxidases (see Selenium), which are major antioxidant enzymes. These enzymes detoxify peroxides, such as hydrogen peroxide and other peroxides. Another antioxidant activity of glutathione is the maintenance of the antioxidant/reducing agent ascorbate in its reduced state. This is accomplished via glutathione-dependent dehydroascorbate reductase which is comprised of glutaredoxin and protein isomerase reductase. Glutathione may also react with the reactive nitrogen species peroxynitrite to form S-nitrosoglutathione.

Glutathione S-transferases (GSTs) consist of a family of multifunctional enzymes that metabolize a wide variety of electrophilic compounds via glutathione conjunction. GSTs are involved in the detoxification of xenobiotic compounds and in the protection against such degenerative diseases as cancer. The mechanism of these enzymes involves a nucleophilic attack by glutathione on an electrophilic substrate. The resulting glutathione conjugates that form are more soluble than the original substrates and thus more easily exported from the cell. The release of glutathione-S-conjugates from cells is an ATP-dependent process mediated by membrane glycoproteins belonging to the multidrug-resistance protein (MRP) family. Proteins of the MRP family are essential for the transport of glutathione S-conjugates into the extracellular space. They are also known as glutathionine-S-conjugate pumps.

Absorption of orally administered glutathione has been observed in some animals (mice, rats, guinea pigs). Oral glutathione has been demonstrated to reverse age-associated decline in immune responsiveness in mice. In one study, glutathione was found to enhance T-cell mediated responsiveness, including delayed-type hypersensitivity (DTH). The mechanism of this effect was ascribed to the antioxidant activity of glutathione.

Parenterally administered glutathione was found to improve sperm motility in a small human trial. Again, the effect was thought to be due to the antioxidant activity of this substance.

Noise-induced hearing loss is thought to be due to oxidative stress. Intraperitoneal administration of glutathione to guinea pigs was found to protect against noise-induced hearing loss and once more, the antioxidant activity of glutathione was thought to account for this effect.

A. MECHANISM OF ACTION:

1. Glutathione (GSH) is a cysteine-containing tripeptide found in all eukaryotic cells. Intracellular GSH is synthesized within the cytosol from the amino acids glutamate, cysteine, and glycine by a two step enzymatic process utilizing ATP as the energy source. Ninety percent of the GSH that is synthesized within the cell is stored in the cytosol; a small amount (10%) is stored in mitochondria.

2. GSH plays an important role in the regulation of many enzymatic reactions and is the most important scavenger molecule, participating in several detoxification reactions. Its cellular functions include amino acid transport, acting as a cofactor in various enzymatic reactions, and maintenance of sulfhydryl redox status. GSH represents a defense against electrophilic xenobiotics and intracellular oxidants (ie, free radicals).

3. All eukaryotic cells are capable of synthesizing GSH, and the liver is the major site of glutathione synthesis in humans and animals. Once synthesized by hepatic cells, GSH is either translocated to plasma or excreted in the bile. The bulk of plasma GSH is in the reduced form (85%), while the reminder is oxidized (15%). GSH is cleared in the kidney through direct glomerular filtration and a nonfiltration mechanism using the gamma-glutamyl transpeptidation reaction.

4. GSH deficiency may be inherited or acquired. Hereditary deficiency is rare and due to a deficiency in an enzyme required to synthesize GSH in reduced form. In acquired deficiency, it is not known whether reduction in GSH is an effect or cause of the disease process (Meister, 1991; White et al, 1994).

B. DEFICIENCY STATES:

1. GSH is present in the epithelial lining fluid (ELF) of the normal respiratory tract and participates in the prevention of parenchymal oxidant injury. A deficiency of GSH in ELF has been reported in idiopathic pulmonary fibrosis and in adult respiratory distress syndrome (ARDS), diseases in which reactive oxygen species could have a pathogenic role (White et al, 1994). GSH aerosol was reported to restore respiratory epithelial surface (RES) oxidant-antioxidant balance in cystic fibrosis (Roum et al, 1999) and in chronic disease of the upper respiratory tract (Testa et al, 1995).

2. Adequate GSH levels seem to be necessary for both T and B lymphocyte function and immune function in general, and may affect HIV replication on a molecular level (Staal et al, 1992; White et al, 1994).

3. Studies performed in patients with cirrhosis showed that hepatocyte GSH levels are low, which could exacerbate hepatic injury. Ethanol-exposed hepatocytes showed increased susceptibility to oxidant injury and were protected by preincubation with a GSH ester (White et al, 1994).

4. In animals and humans, exogenous administration of high-dose reduced GSH has been reported to protect against cisplatin nephrotoxicity and from oxazaphosphorine (cyclophosphamide, ifosfamide) urotoxicity, without interfering with antitumor efficacy (Meister, 1991; Aebi et al, 1991; White et al, 1994; Links & Lewis, 1999).

5. Intracellular reduced GSH has a role in protection of endothelial cells against oxygen-free radicals, and may prevent endothelial dysfunction in arteries exposed to oxidative stress (Meister, 1991).

6. Although extracellular GSH is not effectively transported into cells, exogenous addition of GSH results in a substantial increase in intracellular and extracellular cysteine, an antioxidant, in cultured endothelial cells and in humans.

Fig. 1.   Structures of the inhibitors.

Derivaties of R-Hep (A), Glu-Oct-Hep (B) and GSH (C) are shown. * Indicates chiral atoms of which both configurations were used as a mixture. Thus two stereoisomers are present in the compounds in (A) and four in the compound in (B).

Role of glutathione and catalase in H2O2 detoxification in LPS-activated hepatic endothelial and Kupffer cells:

In the first series of experiments we investigated the effect of LPS in vivo on the GSH levels in hepatic cells using the bimane method. The accompanying level of cellular H2O2 was also determined in parallel cell incubations (Fig. 1). Basal glutathione concentration was about threefold greater in endothelial than in Kupffer cells from control rats. LPS in vivo caused a two- to threefold increase in glutathione content in Kupffer cells, whereas it had no statistically significant increase in hepatic endothelial cells. The inset in Fig. 1 shows that the same changes were observed when a conventional assay was used for the determination of cellular glutathione (45). With use of the conventional assay we found that 96-98% of total glutathione was in the reduced form in nonstimulated cells. Furthermore, the baseline concentrations of glutathione were similar in endothelial and parenchymal cells (data not shown). Basal cellular H2O2 concentrations were also elevated in sinusoidal cells after LPS treatment. The increase was more marked in activated Kupffer cells than in endothelial cells (Fig. 1B).

Fig. 1.   Effect of lipopolysaccharide (LPS) in vivo on reduced glutathione (GSH) content in Kupffer and sinusoidal endothelial cells. Eighteen hours after injection of LPS (hatched bars) or saline (open bars) changes in glutathione or H2O2 contents were determined using monochlorobimane (MCLB) and 2',7'-dichlorofluorescein diacetate (DCF) fluorescence probes as described in MATERIALS AND METHODS. Bars indicate means ± SE; n = 6-8 independent cell preparations in each group. * Statistically significant difference compared with cells from saline-injected animals. Inset: total glutathione determined by a conventional assay method.

In the next series of experiments we tested the effect of H2O2 administration in vitro on the cellular contents of GSH and H2O2. Figure 2 depicts the time-dependent changes in GSH content in response to 0.2 mM H2O2 in Kupffer and endothelial cells. H2O2 administration caused a transient drop in GSH levels, which was more pronounced in cells from saline- than from LPS-treated animals. GSH content returned to normal levels faster in cells exposed to LPS in vivo than in cells from control rats (Fig. 2). Because cellular GSH reached the lowest concentration ~10 min after H2O2 challenge, GSH determinations were performed 10 min after H2O2 administration in subsequent experiments.

Fig. 2. Time dependence of H2O2 effects in vitro on GSH in resting and LPS-activated hepatic cells. GSH content of endothelial (EC) or Kupffer cells (KC) from LPS- or saline-injected animals was determined at varying incubation times after 0.2 mM H2O2 challenge. Baseline GSH level in absence of exogenous H2O2 was considered as 100%. One typical experiment of many independent determinations is shown.

Figure 3 compares the response of cellular GSH and H2O2 to exogenously administered H2O2 in resting and LPS-activated Kupffer cells. H2O2 challenge resulted in a concentration-dependent decrease in GSH (Fig. 3A) in both resting and activated cells. However, in LPS-treated cells, GSH levels remained markedly higher even in the presence of 1 mM extracellular H2O2 compared with nonstimulated cells in the absence of H2O2. As expected, the dose-dependent decrease in cellular GSH was accompanied by a dose-dependent increase in cellular H2O2 in these cells (Fig. 3B).

Fig. 3.   Dose dependence of H2O2 effects in vitro on cellular GSH and H2O2 concentrations in resting and LPS-exposed Kupffer cells: effect of inhibition of catalase. Cells were incubated in the presence of increasing concentration of H2O2, and cellular MCLB and DCF fluorescence were determined as described in MATERIALS AND METHODS. A and B: results from 5 independent experiments; means ± SE. C and D: percent comparison of 3-amino-1,2,4-triazole (ATZ) effects determined in the absence or presence of 0.2 mM H2O2 in resting or LPS-activated Kupffer cells; means ± SE, n = 7. * and # Statistically significant difference compared with vehicle and 0.2 mM H2O2-treated cells, respectively.

To assess the contribution of catalase in the maintenance of cellular GSH and H2O2 detoxification, cells were treated with aminotriazol, a specific catalase inhibitor, before the in vitro challenges (Fig. 3). In the absence of H2O2, aminotriazol treatment caused a 30% decrease in cellular GSH in resting Kupffer cells, whereas it had no effect on LPS-treated cells (Fig. 3C). Combined treatment by aminotriazol and H2O2 caused a more pronounced decrease in GSH in resting than in LPS-treated Kupffer cells. (Fig. 3C).

After these treatments the changes in cellular contents of H2O2 and glutathione showed a proportional and inverse relationship (Fig. 3, B and D). After aminotriazol treatment the percent increase in H2O2 levels was less marked in LPS-stimulated than in resting Kupffer cells (Fig. 3, B and D).

The same series of experiments was also performed on hepatic endothelial cells (Fig. 4). Addition of H2O2 in vitro resulted in a dose-dependent decrease in cellular GSH with an inverse increase in cellular H2O2. The relative decrease in intracellular GSH or increase in H2O2, however, was very similar in LPS-exposed and nonstimulated endothelial cells (Fig. 4). Aminotriazol, in the absence of exogenously administered H2O2, did not markedly alter cellular GSH or H2O2 (Fig. 4C). Furthermore, combined aminotriazol and H2O2 treatments caused similar percent changes in cellular GSH and H2O2 in endothelial cells from LPS- or saline-injected rats (Fig. 4, C and D).

Fig. 4.   Dose dependence of H2O2 effects in vitro on cellular GSH and H2O2 concentrations in resting and LPS-exposed endothelial cells: effect of inhibition of catalase. Endothelial cells were isolated and tested under same conditions as described in legend to Fig. 3. A and B: means ± SE, n = 5. C and D: means ± SE, n = 7. * and # Statistically significant differences compared with vehicle and 0.2 mM H2O2-treated cells, respectively.

Figure 5 shows that the specific activity of catalase was at least an order of magnitude higher in parenchymal than in nonparenchymal cells (please note difference in scale of the ordinate in Fig. 5A). Basal catalase activity in endothelial cells was approximately one-half of that in Kupffer cells. LPS in vivo decreased catalase activity in Kupffer cells by ~45% (Fig. 5A), whereas it had no effect on catalase in endothelial cells. LPS also decreased catalase activity in parenchymal cells. The activity of glutathione reductase was very similar in parenchymal, Kupffer, and endothelial cells. LPS treatment did not alter glutathione reductase activity in these cells (Fig. 5B).

Fig. 5. Effect of LPS in vivo on activity of catalase and glutathione reductase in Kupffer and sinusoidal endothelial cells. For comparison, findings on parenchymal cells are also shown (notice difference in scale of ordinate for catalase between parenchymal and nonparenchymal cells). Enzyme activities were determined as described in MATERIALS AND METHODS. Bars indicate means ± SE; n = 4 independent cell preparations in each group. * Statistically significant difference compared with cells from saline-injected animals.

Pathology:

Excess glutamate at synapses, which may be released in conditions such as traumatic brain injury, can prevent the uptake of cysteine, a necessary building block of glutathione. Without the protection from oxidative injury afforded by glutathione, cells may be damaged or killed.

RESEARCH SUMMARY:

The use of glutathione in cancer treatment has been two-fold. It has been investigated as an antitumor agent in its own right and as a chemoprotectant used to diminish the toxicities of some cancer drugs. In one animal study, glutathione produced significant regression of aflatoxin-induced liver cancers and significantly enhanced survival. All rats exposed to aflatoxin but not given glutathione died within 24 months of exposure to the carcinogen, but 81% of the glutathione-treated animals were still alive at the end of the 24 months. The researchers concluded that the glutathione-effect noted in this study "strongly suggests that this antioxidant merits further investigation as a potential antitumor agent in humans."

Human cancer studies, so far, have utilized glutathione in a secondary role—principally to protect against the toxicity of cisplatin. Its role in this regard has been found effective in several studies wherein it has been demonstrated to diminish cisplatin-induced nephrotoxicity and neurotoxicity.

Early research indicates that exogenous glutathione may significantly inhibit platelet aggregation and improve other hemostatic and hemorheological factors in atherosclerotic patients. In other preliminary clinical work, glutathione has been found to help preserve renal function in patients who had coronary artery bypass operations.

A glutathione aerosol preparation has been helpful in reversing the oxidant-antioxidant imbalance in idiopathic pulmonary fibrosis, and it has helped suppress lung epithelial surface inflammatory cell-derived oxidants in patients with cystic fibrosis. Similar aerosol treatment has been given to HIV patients to augment deficient glutathione levels of the lower respiratory tract with the idea of improving host defense in these immuno-compromised individuals. More research is needed.

Glutathione has also been shown to enhance insulin secretion in elderly subjects with impaired glucose tolerance. There are some further preliminary indications that glutathione might be helpful in some with diabetes, but more research is needed before any meaningful conclusions can be made.

In a double-blind, placebo-controlled study, injected glutathione demonstrated a significant positive effects on sperm motility and morphology in infertile men. And, finally, in another study that needs followup, glutathione exhibited significant in vitro inhibition of herpes simplex virus type 1 replication. It appears that the mechanism of this effect is due to glutathione's redox-modulating active. Some viral infections, including HIV infection, result in oxidative stress which may be a major mechanism of their pathogenesis, Modulating oxidative stress could be an antiviral maneuver.

Reduced Glutathione is an ubiquitous antioxidant involved in many cellular functions such as detoxification, amino acid transport, production of coenzymes and recycling of vitamins E and C. By serving as a critical nucleophilic scavenger, glutathione blocks free radical damage to all types of tissues. Glutathione is the most abundant intracellular thiol (sulfur-containing compound) and low molecular weight tripeptide found in living cells. Thiols such as glutathione, alpha lipoic acid and N-Acetyl -L-Cysteine (NAC) are powerful sulfur-bearing antioxidants. Glutathione can be found in fruits, vegetables and meats. The body also has the ability to produce glutathione in the liver. Glutathione is involved in many functions of the body. Proper amounts are crucial for maintaining good health although a deficiency may be noticed with age. Glutathione is involved in DNA creation and repair, the transport of amino acids, the metabolism of toxins and carcinogens, immune system function, the prevention of cell damage and more. Glutathione also demonstrates antioxidant activity, detoxification attributes, and immunomodulatory activity. Glutathione is connected to healthy well-being and healthy levels should be maintained.

Glutathione has recorded a positive interaction with cisplatin. When the two are used in conjunction, glutathione may help reduce the strength of cisplatin-induced nephrotoxicity and neurotocoicity without disrupting the efficiency of the drug. However, when glutathione is combined with either acetaminophen or alcohol, the drugs will decrease the therapuetic effects of the glutathione.

There have been no reported adverse effects when taking glutathione. For the most part, when recommended dosages were followed, glutathione was well tolerated.

Glutathione participates in leukotriene synthesis and is a cofactor for the enzyme glutathione peroxidase. It is also important as a hydrophilic molecule that is added to lipophilic toxins and waste in the liver during biotransformation before they can become part of the bile. Glutathione is also needed for the detoxification of methylglyoxal, a toxin produced as a by-product of metabolism. This detoxification reaction is carried out by the glyoxalase system. Glyoxalase I catalyzes the conversion of methylglyoxal and reduced glutathione to S-D-Lactoyl-glutathione. Glyoxalase II catalyzes the conversion of S-D-Lactoyl Glutathione to Reduced Glutathione and D-lactate.

GSH is known as a cofactor in both conjugation reactions and reduction reactions, catalyzed by glutathione S-transferase enzymes in cytosol, microsomes, and mitochondria. However, it is capable of participating in non-enzymatic conjugation with some chemicals, as it is hypothesized to do to a significant extent with n-acetyl-p-benzoquinone imine (NAPQI), the reactive cytochrome P450-reactive metabolite formed by acetaminophen, that becomes toxic when GSH is depleted by an overdose of acetaminophen. Glutathione in this capacity binds to NAPQI as a suicide substrate and in the process detoxifies it, taking the place of cellular protein thiol groups which would otherwise be toxically adducted; when all GSH has been spent, NAPQI begins to react with the cellular proteins, killing the cells in the process. The preferred medical treatment for an overdose of this nature, whose efficacy has been consistently supported in literature, is the administration (usually in atomized form) of N-acetylcysteine, which is used by cells to replace spent GSSG and allow a usable GSH pool.

Phorone efficiently reacts to the GSH thiol groups which makes phorone a GSH depletor. It is used to study the effects of GSH as a hydrogen peroxide scavenger in asthma [2]. The health food industry has embraced glutathione as a very efficient antioxidant against a host of diseases.