Glutathione
Glutathione is an antioxidant in plants, animals, fungi, and some bacteria and archaea. Glutathione is capable of preventing damage to important cellular components caused by reactive oxygen species such as free radicals, peroxides, lipid peroxides, and heavy metals. It is a tripeptide with a gamma peptide linkage between the carboxyl group of the glutamate side chain and cysteine. The carboxyl group of the cysteine residue is attached by normal peptide linkage to glycine.
Biosynthesis and occurrence
Glutathione biosynthesis involves two adenosine triphosphate-dependent steps:- First, gamma-glutamylcysteine is synthesized from L-glutamate and cysteine. This conversion requires the enzyme glutamate–cysteine ligase. 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.
The unusual gamma amide linkage in glutathione protects it from hydrolysis by peptidases.
Occurrence
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.Humans synthesize glutathione, but a few eukaryotes do not, including Fabaceae, Entamoeba, and Giardia. The only archaea that make glutathione are halobacteria. Some bacteria, such as cyanobacteria and proteobacteria, can biosynthesize glutathione.
Biochemical function
Glutathione exists in reduced and oxidized states. The ratio of reduced glutathione to oxidized glutathione within cells is a measure of cellular oxidative stress where increased GSSG-to-GSH ratio is indicative of greater oxidative stress. In healthy cells and tissue, more than 90% of the total glutathione pool is in the reduced form, with the remainder in the disulfide form.In the reduced state, the thiol group of cysteinyl residue is a source of one reducing equivalent. Glutathione disulfide is thereby generated. The oxidized state is converted to the reduced state by NADPH. This conversion is catalyzed by glutathione reductase:
Roles
Antioxidant
GSH protects cells by neutralising reactive oxygen species. This conversion is illustrated by the reduction of peroxides:and with free radicals:
Regulation
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 and GSH: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 catalyzes the conversion of methylglyoxal and reduced glutathione to S-D-lactoyl-glutathione. Glyoxalase II 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 states.
Metabolism
Among the many metabolic processes in which it participates, glutathione is required for the biosynthesis of leukotrienes and prostaglandins. It plays a role in the storage of cysteine. Glutathione enhances the function of citrulline as part of the nitric oxide cycle. It is a cofactor and acts on glutathione peroxidase.Conjugation
Glutathione facilitates metabolism of xenobiotics. Glutathione S-transferase enzymes catalyze its conjugation to lipophilic xenobiotics, facilitating their excretion or further metabolism. The conjugation process is illustrated by the metabolism of N-acetyl-p-benzoquinone imine. NAPQI is a reactive metabolite formed by the action of cytochrome P450 on paracetamol. Glutathione conjugates to NAPQI, and the resulting ensemble is excreted.Potential neurotransmitters
Glutathione, along with oxidized glutathione and S-nitrosoglutathione, bind to the glutamate recognition site of the NMDA and AMPA receptors. GSH and GSSG may be neuromodulators. At millimolar concentrations, GSH and GSSG may also modulate the redox state of the NMDA receptor complex. Glutathione binds and activate ionotropic receptors, potentially making it a neurotransmitter.GSH activates the purinergic P2X7 receptor from Müller glia, inducing acute calcium transient signals and GABA release from both retinal neurons and glial cells.
In plants
In plants, glutathione is involved in stress management. It is a component of the glutathione-ascorbate cycle, a system that reduces poisonous hydrogen peroxide. It is the precursor of phytochelatins, glutathione oligomers that chelate heavy metals such as cadmium. Glutathione is required for efficient defence against plant pathogens such as Pseudomonas syringae and Phytophthora brassicae. Adenylyl-sulfate reductase, an enzyme of the sulfur assimilation pathway, uses glutathione as an electron donor. Other enzymes using glutathione as a substrate are glutaredoxins. These small oxidoreductases are involved in flower development, salicylic acid, and plant defence signalling.Bioavailability and supplementation
Systemic bioavailability of orally consumed glutathione is poor because the tripeptide is the substrate of proteases 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 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 may also work synergistically with glutathione, preventing depletion of either. The glutathione-ascorbate cycle, which works to detoxify hydrogen peroxide, is one very specific example of this phenomenon.
The most effective way to increase cellular glutathione is oral supplementation with gamma-glutamylcysteine.
Additionally, compounds such as N-acetylcysteine and alpha lipoic 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, 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, 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 HCl buffer. 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.Monochlorobimane
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.
ThiolQuant Green
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 -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.RealThiol
The RealThiol 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 or redox-sensitive yellow fluorescent protein.Because its very low physiological concentration, GSSG is difficult to measure accurately. GSSG concentration ranges from 10 to 50 μM in all solid tissues, and from 2 to 5 μM in blood. GSH-to-GSSG ratio of whole cell extracts is estimated from 100 to 700. Those ratios represent a mixture from the glutathione pools of different redox states from different subcellular compartments, however. In vivo GSH-to-GSSG ratios can be measured with subcellular accuracy using fluorescent protein-based redox sensors, which have revealed ratios from 50,000 to 500,000 in the cytosol, which implies that GSSG concentration is maintained in the pM range.
Glutathione and human diseases
Comprehensive reviews on the significance of glutathione in human disease have been published on a regular basis in peer reviewed medical journals. Indisputable cause and effect links between GSH metabolism and diseases, such as diabetes, cystic fibrosis, cancer, neurodegenerative diseases, HIV and aging have been demonstrated. A variety of explanations as to why the depletion of GSH is linked to oxidative stress in these disease states have been proposed.Cancer
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 modeled on the structure of glutathione.Cystic fibrosis
Several studies have been completed on the effectiveness of introducing inhaled glutathione to people with cystic fibrosis with mixed results.Alzheimer's disease
While extracellular amyloid beta plaques, neurofibrillary tangles, inflammation in the form of reactive astrocytes and microglia, and neuronal loss are all consistent pathological features of Alzheimer's disease, a mechanistic link between these factors is yet to be clarified. Although the majority of past research has focused on fibrillar Aβ, soluble oligomeric Aβ 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 Aβ.Depletion of the closed form of GSH in the hippocampus may be a potential early diagnostic biomarker for AD.