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 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 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 (GSH), with the remainder in the disulfide form (GSSG).
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 188.8.131.52) catalyzes the conversion of methylglyoxal and reduced glutathione to S-D-lactoyl-glutathione. Glyoxalase II (EC 184.108.40.206) 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.
In another study, researchers reported long term glutathione supplementation offers protection from oxidative damage. In this study 500 mg oral GSH supplementation not only increased erythrocytic GSH but also decreased 8-OHdG significantly within three months in elderly (age above 55 years) diabetic individuals
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 (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.
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).
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 (13–33 nmol per gram Hb). 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 (e.g. more oxidized in the ER, more reduced in the mitochondrial matrix), 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 appeared regularly in peer-reviewed medical journals. 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.
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.
Several studies have been completed on the effectiveness of introducing inhaled glutathione to people with cystic fibrosis with mixed results.
Although extracellular amyloid beta (Aβ) plaques, neurofibrillary tangles (NFT), inflammation in the form of reactive astrocytes and microglia, and neuronal loss are consistent pathological features of Alzheimer's disease (AD), a mechanistic link between these factors is not clear. 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.[medical citation needed]
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.
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