First, gamma-glutamylcysteine is synthesized from L-glutamate and cysteine. This conversion requires the enzyme glutamate-cysteine ligase (GCL, glutamate cysteine synthase). This reaction is the rate-limiting step in glutathione synthesis.
Second, glycine is added to the C-terminal of gamma-glutamylcysteine. This condensation is catalyzed by glutathione synthetase.
While all animal cells are capable of synthesizing glutathione, glutathione synthesis in the liver has been shown to be essential. GCLC knockout mice die within a month of birth due to the absence of hepatic GSH synthesis.
The unusual gamma amide linkage in glutathione protects it from hydrolysis by peptidases.
Glutathione is the most abundant thiol in animal cells, ranging from 0.5 to 10 mM. It is present both in the cytosol and the organelles.
Glutathione exists in reduced (GSH) and oxidized (GSSG) states. The ratio of reduced glutathione to oxidized glutathione within cells is a measure of cellular oxidative stress. In healthy cells and tissue, more than 90% of the total glutathione pool is in the reduced form (GSH), with the remainder in the disulfide form (GSSG). An increased GSSG-to-GSH ratio is indicative of oxidative stress.
Aside from deactivating radicals and reactive oxidants, glutathione participates in thiol protection and redox regulation of cellular thiol proteins under oxidative stress by protein S-glutathionylation, a redox-regulated post-translational thiol modification. The general reaction involves formation of an unsymmetrical disulfide from the protectable protein (RSH) and GSH:
RSH + GSH + [O] -> GSSR + H2O
Glutathione is also employed for the detoxification of methylglyoxal and formaldehyde, toxic metabolites produced under oxidative stress. This detoxification reaction is carried out by the glyoxalase system. Glyoxalase I (EC 220.127.116.11) catalyzes the conversion of methylglyoxal and reduced glutathione to S-D-lactoyl-glutathione. Glyoxalase II (EC 18.104.22.168) catalyzes the hydrolysis of S-D-lactoyl-glutathione to glutathione and D-lactic acid.
It maintains exogenous antioxidants such as vitamins C and E in their reduced (active) states.
Systemic bioavailability of orally consumed glutathione is poor because the tripeptide, is the substrate of proteases (peptidases) of the alimentary canal, and due to the absence of a specific carrier of glutathione at the level of cell membrane.
Because direct supplementation of glutathione is not always successful, supply of the raw nutritional materials used to generate GSH, such as cysteine and glycine, may be more effective at increasing glutathione levels. Other antioxidants such as ascorbic acid (vitamin C) may also work synergistically with glutathione, preventing depletion of either. The glutathione-ascorbate cycle, which works to detoxify hydrogen peroxide (H2O2), is one very specific example of this phenomenon.
Additionally, compounds such as N-acetylcysteine (NAC) and alpha lipoic acid (ALA, not to be confused with the unrelated alpha-linolenic acid) are both capable of helping to regenerate glutathione levels. NAC in particular is commonly used to treat overdose of acetaminophen, a type of potentially fatal poisoning which is harmful in part due to severe depletion of glutathione levels. It is a precursor of cysteine.
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. About ten days are needed for the body to process vitamin D3 into calcitriol.
S-adenosylmethionine (SAMe), a cosubstrate involved in methyl group transfer, has also been shown to increase cellular glutathione content in persons suffering from a disease-related glutathione deficiency.
Low glutathione is commonly observed in wasting and negative nitrogen balance, as seen in cancer, HIV/AIDS, sepsis, trauma, burns, and athletic overtraining. Low levels are also observed in periods of starvation. These effects are hypothesized to be influenced by the higher glycolytic activity associated with cachexia, which result from reduced levels of oxidative phosphorylation.
Determination of glutathione
Ellman's reagent and monobromobimane
Reduced glutathione may be visualized using Ellman's reagent or bimane derivatives 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 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.
Using monochlorobimane, the quantification is done by confocal laser scanning microscopy after application of the dye to living cells. This quantification process relies on measuring the rates of fluorescence changes and is limited to plant cells.
CMFDA has also been mistakenly used as a glutathione probe. Unlike monochlorobimane, whose fluorescence increases upon reacting with glutathione, the fluorescence increase of CMFDA is due to the hydrolysis of the acetate groups inside cells. Although CMFDA may react with glutathione in cells, the fluorescence increase does not reflect the reaction. Therefore, studies using CMFDA as a glutathione probe should be revisited and reinterpreted.
The major limitation of these bimane-based probes and many other reported probes is that these probes are based on irreversible chemical reactions with glutathione, which renders these probes incapable of monitoring the real-time glutathione dynamics. Recently, the first reversible reaction based fluorescent probe-ThiolQuant Green (TQG)-for glutathione was reported. ThiolQuant Green can not only perform high resolution measurements of glutathione levels in single cells using a confocal microscope, but also be applied in flow cytometry to perform bulk measurements.
The RealThiol (RT) probe is a second-generation reversible reaction-based GSH probe. A few key features of RealThiol: 1) it has a much faster forward and backward reaction kinetics compared to ThiolQuant Green, which enables real-time monitoring of GSH dynamics in live cells; 2) only micromolar to sub-micromolar RealThiol is needed for staining in cell-based experiments, which induces minimal perturbation to GSH level in cells; 3) a high-quantum-yield coumarin fluorophore was implemented so that background noise can be minimized; and 4) equilibrium constant of the reaction between RealThiol and GSH has been fine-tuned to respond to physiologically relevant concentration of GSH. RealThiol can be used to perform measurements of glutathione levels in single cells using a high-resolution confocal microscope, as well as be applied in flow cytometry to perform bulk measurements in high throughput manner.
Organelle-targeted RT probe has also been developed. A mitochondria targeted version, MitoRT, was reported and demonstrated in monitoring the dynamic of mitochondrial glutathione both on confocoal microscope and FACS based analysis.
Protein-based glutathione probes
Another approach, which allows measurement of 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)
GSSG because its very low physiological concentration is difficult to measure accurately unless the procedure is carefully executed and monitored and the occurrence of interfering compounds is properly addressed. GSSG concentration ranges from 10 to 50 ?M in all solid tissues, and from 2 to 5 ?M in blood (13-33 nmol per gram Hb). GSH-to-GSSG ratio ranges from 100 to 700.
Other biological implications
The sulfur-rich aspect of glutathione results in it forming relatively strong complexes with lead(II).
Once a tumor has been established, elevated levels of glutathione may act to protect cancerous cells by conferring resistance to chemotherapeutic drugs. The antineoplastic mustard drug canfosfamide was modelled on the structure of glutathione.
Several studies have been completed on the effectiveness of introducing inhaled glutathione to people with cystic fibrosis with mixed results.
While extracellular amyloid beta (A?) plaques, neurofibrillary tangles (NFT), inflammation in the form of reactive astrocytes and microglia, and neuronal loss are all consistent pathological features of Alzheimer's disease (AD), a mechanistic link between these factors is yet to be clarified. Although the majority of past research has focused on fibrillar A?, soluble oligomeric A? species are now considered to be of major pathological importance in AD. Upregulation of GSH may be protective against the oxidative and neurotoxic effects of oligomeric A?.[medical ]
Depletion of the closed form of GSH in the hippocampus may be a potential early diagnostic biomarker for AD. 
Glutathione is the most common agent taken by mouth in an attempt to whiten the skin. It may also be used as a cream. Whether or not it actually works is unclear as of 2019. Due to side effects that may result with intravenous use, the government of the Philippines recommends against such use.
^Pompella A, Visvikis A, Paolicchi A, De Tata V, Casini AF (October 2003). "The changing faces of glutathione, a cellular protagonist". Biochemical Pharmacology. 66 (8): 1499-503. doi:10.1016/S0006-2952(03)00504-5. PMID14555227.
^Chen Y, Yang Y, Miller ML, Shen D, Shertzer HG, Stringer KF, Wang B, Schneider SN, Nebert DW, Dalton TP (May 2007). "Hepatocyte-specific Gclc deletion leads to rapid onset of steatosis with mitochondrial injury and liver failure". Hepatology. 45 (5): 1118-28. doi:10.1002/hep.21635. PMID17464988.
^ abcGuoyao Wu, Yun-Zhong Fang, Sheng Yang, Joanne R. Lupton, Nancy D. Turner (2004). "Glutathione Metabolism and its Implications for Health". Journal of Nutrition. 134 (3): 489-492. doi:10.1093/jn/134.3.489. PMID14988435.CS1 maint: multiple names: authors list (link)
^Halprin KM, Ohkawara A (1967). "The measurement of glutathione in human epidermis using glutathione reductase". The Journal of Investigative Dermatology. 48 (2): 149-52. doi:10.1038/jid.1967.24. PMID6020678.
^Couto N, Malys N, Gaskell SJ, Barber J (June 2013). "Partition and turnover of glutathione reductase from Saccharomyces cerevisiae: a proteomic approach". Journal of Proteome Research. 12 (6): 2885-94. doi:10.1021/pr4001948. PMID23631642.
^Steullet P, Neijt HC, Cuénod M, Do KQ (February 2006). "Synaptic plasticity impairment and hypofunction of NMDA receptors induced by glutathione deficit: relevance to schizophrenia". Neuroscience. 137 (3): 807-19. doi:10.1016/j.neuroscience.2005.10.014. PMID16330153.
^ abVarga V, Jenei Z, Janáky R, Saransaari P, Oja SS (September 1997). "Glutathione is an endogenous ligand of rat brain N-methyl-D-aspartate (NMDA) and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors". Neurochemical Research. 22 (9): 1165-71. doi:10.1023/A:1027377605054. PMID9251108.
^Zhang J, Zhou X, Wu W, Wang J, Xie H, Wu Z (2017). "Regeneration of glutathione by ?-lipoic acid via Nrf2/ARE signaling pathway alleviates cadmium-induced HepG2 cell toxicity". Environ Toxicol Pharmacol. 51: 30-37. doi:10.1016/j.etap.2017.02.022. PMID28262510.
^Garcion E, Wion-Barbot N, Montero-Menei CN, Berger F, Wion D (April 2002). "New clues about vitamin D functions in the nervous system". Trends in Endocrinology and Metabolism. 13 (3): 100-5. doi:10.1016/S1043-2760(01)00547-1. PMID11893522.
^van Groningen L, Opdenoordt S, van Sorge A, Telting D, Giesen A, de Boer H (April 2010). "Cholecalciferol loading dose guideline for vitamin D-deficient adults". European Journal of Endocrinology. 162 (4): 805-11. doi:10.1530/EJE-09-0932. PMID20139241.
^Lieber CS (November 2002). "S-adenosyl-L-methionine: its role in the treatment of liver disorders". The American Journal of Clinical Nutrition. 76 (5): 1183S-7S. doi:10.1093/ajcn/76/5.1183S (inactive 30 July 2019). PMID12418503.
^Vendemiale G, Altomare E, Trizio T, Le Grazie C, Di Padova C, Salerno MT, Carrieri V, Albano O (May 1989). "Effects of oral S-adenosyl-L-methionine on hepatic glutathione in patients with liver disease". Scandinavian Journal of Gastroenterology. 24 (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 (September 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. 29 (5): 597-604. doi:10.1093/oxfordjournals.alcalc.a045589. PMID7811344.
^Sebastià J, Cristòfol R, Martín M, Rodríguez-Farré E, Sanfeliu C (January 2003). "Evaluation of fluorescent dyes for measuring intracellular glutathione content in primary cultures of human neurons and neuroblastoma SH-SY5Y". Cytometry. Part A. 51 (1): 16-25. doi:10.1002/cyto.a.10003. PMID12500301.
^Meyer AJ, Brach T, Marty L, Kreye S, Rouhier N, Jacquot JP, Hell R (December 2007). "Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the redox potential of the cellular glutathione redox buffer". The Plant Journal. 52 (5): 973-86. doi:10.1111/j.1365-313X.2007.03280.x. PMID17892447.
^Maulucci G, Labate V, Mele M, Panieri E, Arcovito G, Galeotti T, Østergaard H, Winther JR, De Spirito M, Pani G (October 2008). "High-resolution imaging of redox signaling in live cells through an oxidation-sensitive yellow fluorescent protein". Science Signaling. 1 (43): pl3. doi:10.1126/scisignal.143pl3. PMID18957692.
^Giustarini D, Dalle-Donne I, Milzani A, Fanti P, Rossi R (September 2013). "Analysis of GSH and GSSG after derivatization with N-ethylmaleimide". Nature Protocols. 8 (9): 1660-9. doi:10.1038/nprot.2013.095. PMID23928499.
^Farkas E, Buglyó P (2017). "Chapter 8. Lead(II) Complexes of Amino Acids, Peptides, and Other Related Ligands of Biological Interest". In Astrid S, Helmut S, Sigel RK (eds.). Lead: Its Effects on Environment and Health. Metal Ions in Life Sciences. 17. de Gruyter. pp. 201-240. doi:10.1515/9783110434330-008. ISBN9783110434330. PMID28731301.
^Balendiran GK, Dabur R, Fraser D (2004). "The role of glutathione in cancer". Cell Biochemistry and Function. 22 (6): 343-52. doi:10.1002/cbf.1149. PMID15386533.
^Visca A, Bishop CT, Hilton SC, Hudson VM. "Improvement in clinical markers in CF patients using a reduced glutathione regimen: an uncontrolled, observational study. J Cyst Fibros 2008
^Bishop C, Hudson VM, Hilton SC, Wilde C (January 2005). "A pilot study of the effect of inhaled buffered reduced glutathione on the clinical status of patients with cystic fibrosis". Chest. 127 (1): 308-17. doi:10.1378/chest.127.1.308. PMID15653998.
^Mandal PK, Tripathi M, Sugunan S (January 2012). "Brain oxidative stress: detection and mapping of anti-oxidant marker 'Glutathione' in different brain regions of healthy male/female, MCI and Alzheimer patients using non-invasive magnetic resonance spectroscopy". Biochemical and Biophysical Research Communications. 417 (1): 43-48. doi:10.1016/j.bbrc.2011.11.047. PMID22120629.
^Mandal PK, Saharan, S, Tripathi M, Murari G (October 2015). "Brain Glutathione Levels - A Novel Biomarker for Mild Cognitive Impairment and Alzheimer's Disease". Biological Psychiatry. 78 (10): 702-710. doi:10.1016/j.biopsych.2015.04.005. PMID26003861.
^Vallverdú-Queralt A, Verbaere A, Meudec E, Cheynier V, Sommerer N (January 2015). "Straightforward method to quantify GSH, GSSG, GRP, and hydroxycinnamic acids in wines by UPLC-MRM-MS". Journal of Agricultural and Food Chemistry. 63 (1): 142-9. doi:10.1021/jf504383g. PMID25457918.
^ abcMalathi, M; Thappa, DM (2013). "Systemic skin whitening/lightening agents: what is the evidence?". Indian Journal of Dermatology, Venereology and Leprology. 79 (6): 842-6. doi:10.4103/0378-6323.120752. PMID24177629.
^Dilokthornsakul, W; Dhippayom, T; Dilokthornsakul, P (June 2019). "The clinical effect of glutathione on skin color and other related skin conditions: A systematic review". Journal of Cosmetic Dermatology. 18 (3): 728-737. doi:10.1111/jocd.12910. PMID30895708.
^Sonthalia, Sidharth; Daulatabad, Deepashree; Sarkar, Rashmi (2016). "Glutathione as a skin whitening agent: Facts, myths, evidence and controversies". Indian J. Dermatol. Venereol. Leprol. 82 (3): 262-72. doi:10.4103/0378-6323.179088. PMID27088927.
Drevet JR (May 2006). "The antioxidant glutathione peroxidase family and spermatozoa: a complex story". Molecular and Cellular Endocrinology. 250 (1-2): 70-9. doi:10.1016/j.mce.2005.12.027. PMID16427183.