Once oxidized, glutathione can be reduced back by glutathione reductase, using NADPH as an electron donor. The ratio of reduced glutathione to oxidized glutathione within cells is often used as a measure of cellular toxicity.
Glutathione is not an essential nutrient, since it can be synthesized in the body from the amino acids L-cysteine, L-glutamic acid, and glycine. The sulfhydryl (thiol) group (SH) of cysteine serves as a proton donor and is responsible for the biological activity of glutathione. Cysteine is the rate-limiting factor in cellular glutathione synthesis, since this amino acid is relatively rare in foodstuffs.
First, gamma-glutamylcysteine is synthesized from L-glutamate and cysteine via the enzyme gamma-glutamylcysteine synthetase (glutamate cysteine ligase, GCL). This reaction is the rate-limiting step in glutathione synthesis.
Animal glutamate cysteine ligase (GCL) is a heterodimeric enzyme composed of a catalytic (GCLC) and modulatory (GCLM) subunit. GCLC constitutes all the enzymatic activity, whereas GCLM increases the catalytic efficiency of GCLC. Mice lacking GCLC (i.e., lacking all de novo GSH synthesis) die before birth. Mice lacking GCLM demonstrate no outward phenotype, but exhibit marked decrease in GSH and increased sensitivity to toxic insults.
While all cells in the human body are capable of synthesizing glutathione, liver glutathione synthesis has been shown to be essential. Mice with genetically-induced loss of GCLC (i.e., GSH synthesis) only in the liver die within 1 month of birth.
The plant glutamate cysteine ligase (GCL) is a redox-sensitive homodimeric enzyme, conserved in the plant kingdom. In an oxidizing environment, intermolecular disulfide bridges are formed and the enzyme switches to the dimeric active state. The midpoint potential of the critical cysteine pair is -318 mV. In addition to the redox-dependent control is the plant GCL enzyme feedback inhibited by GSH. GCL is exclusively located in plastids, and glutathione synthetase is dual-targeted to plastids and cytosol, thus are GSH and gamma-glutamylcysteine exported from the plastids. Both glutathione biosynthesis enzymes are essential in plants; knock-outs of GCL and GS are lethal to embryo and seedling.
Glutathione exists in both reduced (GSH) and oxidized (GSSG) states. In the reduced state, the thiol group of cysteine is able to donate a reducing equivalent (H++ e−) to other unstable molecules, such as reactive oxygen species. In donating an electron, glutathione itself becomes reactive, but readily reacts with another reactive glutathione to form glutathione disulfide (GSSG). Such a reaction is probable due to the relatively high concentration of glutathione in cells (up to 5 mM in the liver).
GSH can be regenerated from GSSG by the enzyme glutathione reductase (GSR): NADPH reduces FAD present in GSR to produce a transient FADH-anion. This anion then quickly breaks a disulfide bond (Cys58 - Cys63) and leads to Cys63's nucleophilically attacking the nearest sulfide unit in the GSSG molecule (promoted by His467), which creates a mixed disulfide bond (GS-Cys58) and a GS-anion. His467 of GSR then protonates the GS-anion to form the first GSH. Next, Cys63 nucleophilically attacks the sulfide of Cys58, releasing a GS-anion, which, in turn, picks up a solvent proton and is released from the enzyme, thereby creating the second GSH. So, for every GSSG and NADPH, two reduced GSH molecules are gained, which can again act as antioxidants scavenging reactive oxygen species in the cell.
In healthy cells and tissue, more than 90% of the total glutathione pool is in the reduced form (GSH) and less than 10% exists in the disulfide form (GSSG). An increased GSSG-to-GSH ratio is considered indicative of oxidative stress.
Glutathione has multiple functions:
It is the major endogenous antioxidant produced by the cells, participating directly in the neutralization of free radicals and reactive oxygen compounds, as well as maintaining exogenous antioxidants such as vitamins C and E in their reduced (active) forms.
Regulation of the nitric oxide cycle, which is critical for life but can be problematic if unregulated
It is used in metabolic and biochemical reactions such as DNA synthesis and repair, protein synthesis, prostaglandin synthesis, amino acid transport, and enzyme activation. Thus, every system in the body can be affected by the state of the glutathione system, especially the immune system, the nervous system, the gastrointestinal system and the lungs.
It has a vital function in iron metabolism. Yeast cells depleted of or containing toxic levels of GSH show an intense iron starvation-like response and impairment of the activity of extra-mitochondrial ISC enzymes, followed by death.
In the case of N-acetyl-p-benzoquinone imine (NAPQI), the reactive cytochrome P450-reactive metabolite formed by paracetamol (or acetaminophen as it is known in the US), which becomes toxic when GSH is depleted by an overdose of acetaminophen, glutathione is an essential antidote to overdose. Glutathione conjugates to NAPQI and helps to detoxify it. In this capacity, it protects cellular protein thiol groups, which would otherwise become covalently modified; when all GSH has been spent, NAPQI begins to react with the cellular proteins, killing the cells in the process. The preferred treatment for an overdose of this painkiller is the administration (usually in atomized form) of N-acetyl-L-cysteine (often as a preparation called Mucomyst), which is processed by cells to L-cysteine and used in the de novo synthesis of GSH.
This detoxification reaction is carried out by the glyoxalase system. Glyoxalase I (EC 184.108.40.206) catalyzes the conversion of methylglyoxal and reduced glutathione to S-D-lactoyl-glutathione. Glyoxalase II (EC 220.127.116.11) catalyzes the hydrolysis of S-D-lactoyl-glutathione to glutathione and D-lactic acid.
Glutathione has recently been used as an inhibitor of melanin in the cosmetics industry. In countries like Japan and the Philippines, this product is sold as a skin whitening soap. Glutathione competitively inhibits melanin synthesis in the reaction of tyrosinase and L-DOPA by interrupting L-DOPA's ability to bind to tyrosinase during melanin synthesis. The inhibition of melanin synthesis was reversed by increasing the concentration of L-DOPA, but not by increasing tyrosinase. Although the synthesized melanin was aggregated within one hour, the aggregation was inhibited by the addition of glutathione. These results indicate that glutathione inhibits the synthesis and agglutination of melanin by interrupting the function of L-DOPA."
Richie et al. published a long-term, randomized, double-blinded, placebo-controlled study that shows for the first time, daily consumption of glutathione is effective at increasing glutathione blood levels. 
In a previous study of acute oral administration, seven healthy subjects were given a very large dose (3 grams) of oral glutathione, Witschi and coworkers found "it is not possible to increase circulating glutathione to a clinically beneficial extent by the oral administration of a single dose of 3 g of glutathione." Plasma levels of glutathione, cysteine, and glutamate were tested in the plasma after 270 minutes and three out of four of the subjects did have an increase or rise in their glutathione levels. However, it is possible to increase and maintain appropriate glutathione levels by increasing the daily consumption of glutathione rich foods and/or supplements.[non-primary source needed]
Calcitriol (1,25-dihydroxyvitamin D3), the active metabolite of vitamin D3, after being synthesized from calcifediol in the kidney, increases glutathione levels in the brain and appears to be a catalyst for glutathione production. Calcitriol was found to increase GSH levels in rat astrocyte primary cultures on average by 42%, increasing protein concentrations from 29 nmol/mg to 41 nmol/mg, 24 and 48 hours after administration; this effect was reduced to 11%, relative to the control, 96 hours after administration. It takes about ten days for the body to process vitamin D3 into calcitriol.
Zinc has been shown to have some influence over de novo glutathione synthesis. Furthermore, magnesium has significant influence on glutathione synthesis, it appears to be an essential cofactor.
Low glutathione is commonly observed in wasting and negative nitrogen balance, as seen in cancer, HIV/AIDS, sepsis, trauma, burns and athletic overtraining.
Once a tumor has been established, elevated levels of glutathione may act to protect cancerous cells by conferring resistance to chemotherapeutic drugs.
Methods to determine glutathione
Reduced glutathione may be visualized using Ellman's reagent or bimane derivates such as monobromobimane. The monobromobimane method is more sensitive. In this procedure, cells are lysed and thiols extracted using a HClbuffer. The thiols are then reduced with dithiothreitol (DTT) and labelled by monobromobimane. Monobromobimane becomes fluorescent after binding to GSH. The thiols are then separated by HPLC and the fluorescence quantified with a fluorescence detector. Bimane may also be used to quantify glutathione in vivo. The quantification is done by confocal laser scanning microscopy after application of the dye to living cells. Another approach, which allows to measure the glutathione redox potential at a high spatial and temporal resolution in living cells is based on redox imaging using the redox-sensitive green fluorescent protein (roGFP) or redox sensitive yellow fluorescent protein (rxYFP)
^ abCouto, Narciso; Malys, Naglis; Gaskell, Simon; Barber, Jill (2013). "Partition and Turnover of Glutathione Reductase from Saccharomyces cerevisiae: a Proteomic Approach". Journal of Proteome Research12 (6): 2885–94. doi:10.1021/pr4001948. PMID23631642.
^Pastore, Anna; Piemonte, Fiorella; Locatelli, Mattia; Lo Russo, Anna Lo; Gaeta, Laura Maria; Tozzi, Giulia; Federici, Giorgio (2003). "Determination of blood total, reduced, and oxidized glutathione in pediatric subjects". Clinical Chemistry47 (8): 1467–9. PMID11468240.
^White, C. C.; Viernes, H.; Krejsa, C. M.; Botta, D.; Kavanagh, T. J. (2003). "Fluorescence-based microtiter plate assay for glutamate–cysteine ligase activity". Analytical Biochemistry318 (2): 175–180. doi:10.1016/S0003-2697(03)00143-X. PMID12814619.edit
^Dalton, T; Dieter, MZ; Yang, Y; Shertzer, HG; Nebert, DW (2000). "Knockout of the Mouse Glutamate Cysteine Ligase Catalytic Subunit (Gclc) Gene: Embryonic Lethal When Homozygous, and Proposed Model for Moderate Glutathione Deficiency When Heterozygous". Biochemical and Biophysical Research Communications279 (2): 324–9. doi:10.1006/bbrc.2000.3930. PMID11118286.
^Yang, Y.; Dieter, MZ; Chen, Y; Shertzer, HG; Nebert, DW; Dalton, TP (2002). "Initial characterization of the glutamate-cysteine ligase modifier subunit Gclm(-/-) knockout mouse. Novel model system for a severely compromised oxidative stress response". Journal of Biological Chemistry277 (51): 49446–52. doi:10.1074/jbc.M209372200. PMID12384496.
^McConnachie, L. A.; Mohar, I.; Hudson, F. N.; Ware, C. B.; Ladiges, W. C.; Fernandez, C.; Chatterton-Kirchmeier, S.; White, C. C.; Pierce, R. H.; Kavanagh, T. J. (2007). "Glutamate Cysteine Ligase Modifier Subunit Deficiency and Gender as Determinants of Acetaminophen-Induced Hepatotoxicity in Mice". Toxicological Sciences99 (2): 628–36. doi:10.1093/toxsci/kfm165. PMID17584759.
^Chen, Ying; Yang, Yi; Miller, Marian L.; Shen, Dongxiao; Shertzer, Howard G.; Stringer, Keith F.; Wang, Bin; Schneider, Scott N.; Nebert, Daniel W.; Dalton, Timothy P. (2007). "Hepatocyte-specificGclcdeletion leads to rapid onset of steatosis with mitochondrial injury and liver failure". Hepatology45 (5): 1118–28. doi:10.1002/hep.21635. PMID17464988.
^Hothorn, M.; Wachter, A; Gromes, R; Stuwe, T; Rausch, T; Scheffzek, K (2006). "Structural Basis for the Redox Control of Plant Glutamate Cysteine Ligase". Journal of Biological Chemistry281 (37): 27557–65. doi:10.1074/jbc.M602770200. PMID16766527.
^Wachter, Andreas; Wolf, Sebastian; Steininger, Heike; Bogs, Jochen; Rausch, Thomas (2004). "Differential targeting of GSH1 and GSH2 is achieved by multiple transcription initiation: implications for the compartmentation of glutathione biosynthesis in the Brassicaceae". The Plant Journal41 (1): 15–30. doi:10.1111/j.1365-313X.2004.02269.x. PMID15610346.
^Pasternak, Maciej; Lim, Benson; Wirtz, Markus; Hell, RüDiger; Cobbett, Christopher S.; Meyer, Andreas J. (2007). "Restricting glutathione biosynthesis to the cytosol is sufficient for normal plant development". The Plant Journal53 (6): 999–1012. doi:10.1111/j.1365-313X.2007.03389.x. PMID18088327.
^Scholz RW. Graham KS. Gumpricht E. Reddy CC. Mechanism of interaction of vitamin E and glutathione in the protection against membrane lipid peroxidation. Ann NY Acad Sci 1989:570:514-7. Hughes RE. Reduction of dehydroascorbic acid by animal tissues. Nature 1964:203:1068-9.
^Parisy, Vincent; Poinssot, Benoit; Owsianowski, Lucas; Buchala, Antony; Glazebrook, Jane; Mauch, Felix (2006). "Identification of PAD2 as a γ-glutamylcysteine synthetase highlights the importance of glutathione in disease resistance of Arabidopsis". The Plant Journal49 (1): 159–72. doi:10.1111/j.1365-313X.2006.02938.x. PMID17144898.
^Rouhier, Nicolas; Lemaire, StéPhane D.; Jacquot, Jean-Pierre (2008). "The Role of Glutathione in Photosynthetic Organisms: Emerging Functions for Glutaredoxins and Glutathionylation". Annual Review of Plant Biology59: 143–66. doi:10.1146/annurev.arplant.59.032607.092811. PMID18444899.
^Richie Jr, J.P.; Nichenametla, S.; Neidig, W.; Haley, JS; Schell, T.D.; Muscat, J.E. (2014). "Randomized controlled trial of oral glutathione supplementation on body stores of glutathione". European Journal of Nutrition. doi:10.1007/s00394-014-0706-z. PMID24791752.
^Witschi, A.; Reddy, S.; Stofer, B.; Lauterburg, B. H. (1992). "The systemic availability of oral glutathione". European Journal of Clinical Pharmacology43 (6): 667–9. doi:10.1007/BF02284971. PMID1362956.
^Lands, L. C.; Grey, V. L.; Smountas, A. A. (1999). "Effect of supplementation with a cysteine donor on muscular performance". Journal of applied physiology (Bethesda, Md. : 1985)87 (4): 1381–1385. PMID10517767. edit
^Garcion, E; Wion-Barbot, N; Montero-Menei, C; Berger, F; Wion, D (2002). "New clues about vitamin D functions in the nervous system". Trends in Endocrinology and Metabolism13 (3): 100–5. doi:10.1016/S1043-2760(01)00547-1. PMID11893522.
^Garcion, E.; Sindji, L.; Leblondel, G.; Brachet, P.; Darcy, F. (2002). "1,25-Dihydroxyvitamin D3 Regulates the Synthesis of γ-Glutamyl Transpeptidase and Glutathione Levels in Rat Primary Astrocytes". Journal of Neurochemistry73 (2): 859–866. doi:10.1046/j.1471-4159.1999.0730859.x. PMID10428085.edit
^Van Groningen, L.; Opdenoordt, S.; Van Sorge, A.; Telting, D.; Giesen, A.; De Boer, H. (2010). "Cholecalciferol loading dose guideline for vitamin D-deficient adults". European Journal of Endocrinology162 (4): 805–811. doi:10.1530/EJE-09-0932. PMID20139241.edit
^Lieber, Charles S. (2002). "S-adenosyl-L-methionine: its role in the treatment of liver disorders". The American journal of clinical nutrition76 (5): 1183S–7S. PMID12418503.
^Vendemiale, G.; Altomare, E.; Trizio, T.; Le Grazie, C.; Di Padova, C.; Salerno, M. T.; Carrieri, V.; Albano, O. (1989). "Effects of Oral S-Adenosyl-l-Methionine on Hepatic Glutathione in Patients with Liver Disease". Scandinavian Journal of Gastroenterology24 (4): 407–15. doi:10.3109/00365528909093067. PMID2781235.
^Loguercio, C; Nardi, G; Argenzio, F; Aurilio, C; Petrone, E; Grella, A; Del Vecchio Blanco, C; Coltorti, M (1994). "Effect of S-adenosyl-L-methionine administration on red blood cell cysteine and glutathione levels in alcoholic patients with and without liver disease". Alcohol and alcoholism (Oxford, Oxfordshire)29 (5): 597–604. PMID7811344.
^Micke, P.; Beeh, K. M.; Schlaak, J. F.; Buhl, R. (2001). "Oral supplementation with whey proteins increases plasma glutathione levels of HIV-infected patients". European Journal of Clinical Investigation31 (2): 171–8. doi:10.1046/j.1365-2362.2001.00781.x. PMID11168457.
^Moreno, Y. F.; Sgarbieri, VC; Da Silva, MN; Toro, AA; Vilela, MM (2006). "Features of Whey Protein Concentrate Supplementation in Children with Rapidly Progressive HIV Infection". Journal of Tropical Pediatrics52 (1): 34–8. doi:10.1093/tropej/fmi074. PMID16014759.
^Grey, V; Mohammed, SR; Smountas, AA; Bahlool, R; Lands, LC (2003). "Improved glutathione status in young adult patients with cystic fibrosis supplemented with whey protein". Journal of Cystic Fibrosis2 (4): 195–8. doi:10.1016/S1569-1993(03)00097-3. PMID15463873.
^Micke, P.; Beeh, K. M.; Buhl, R. (2002). "Effects of long-term supplementation with whey proteins on plasma glutathione levels of HIV-infected patients". European Journal of Nutrition41 (1): 12–8. doi:10.1007/s003940200001. PMID11990003.
^Ha, K. -N. (2006). "Increased Glutathione Synthesis through an ARE-Nrf2-Dependent Pathway by Zinc in the RPE: Implication for Protection against Oxidative Stress". Investigative Ophthalmology & Visual Science47 (6): 2709. doi:10.1167/iovs.05-1322.edit
^Mills, B. J.; Lindeman, R. D.; Lang, C. A. (1981). "Effect of zinc deficiency on blood glutathione levels". The Journal of nutrition111 (6): 1098–102. PMID7241230. edit
^Mills, B. J.; Lindeman, R. D.; Lang, C. A. (1986). "Magnesium deficiency inhibits biosynthesis of blood glutathione and tumor growth in the rat". Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine181 (3): 326–32. PMID3945642. edit
^Hsu, J. M.; Rubenstein, B; Paleker, A. G. (1982). "Role of magnesium in glutathione metabolism of rat erythrocytes". The Journal of nutrition112 (3): 488–96. PMID7062145. edit
^Barbagallo, M; Dominguez, L. J.; Tagliamonte, M. R.; Resnick, L. M.; Paolisso, G (1999). "Effects of glutathione on red blood cell intracellular magnesium: Relation to glucose metabolism". Hypertension34 (1): 76–82. PMID10406827. edit
^Bede; Nagy (2008). "Effects of magnesium supplementation on the glutathione redox system in atopic asthmatic children". Inflammation Research57 (6): 279–86. PMID18516713. edit
^Regan, R. F.; Guo, Y (2001). "Magnesium deprivation decreases cellular reduced glutathione and causes oxidative neuronal death in murine cortical cultures". Brain research890 (1): 177–83. PMID11164781. edit
^Meyer, Andreas J.; Brach, Thorsten; Marty, Laurent; Kreye, Susanne; Rouhier, Nicolas; Jacquot, Jean-Pierre; Hell, RüDiger (2007). "Redox-sensitive GFP inArabidopsis thalianais a quantitative biosensor for the redox potential of the cellular glutathione redox buffer". The Plant Journal52 (5): 973–86. doi:10.1111/j.1365-313X.2007.03280.x. PMID17892447.
^Maulucci, Giuseppe; Labate, Valentina; Mele, Marina; Panieri, Emiliano; Arcovito, Giuseppe; Galeotti, Tommaso; Østergaard, H; Winther, JR; De Spirito, Marco; Pani, G. (2008). "High-resolution imaging of redox signaling in live cells through an oxidation-sensitive yellow fluorescent protein". Science Signaling1 (43): pl3. doi:10.1126/scisignal.143pl3. PMID18957692.
^Influence of must composition on phenolic oxidation kinetics. Jacques Rigaud, Véronique Cheynier, Jean-Marc Souquet and Michel Moutounet, Journal of the Science of Food and Agriculture, 1991, Volume 57, Issue 1, pages 55–63, doi:10.1002/jsfa.2740570107