Endogenous Metabolite

One of the most common non-protein thiols found in living cells is L-reduced glutathione. Important biological processes in organisms include the synthesis of proteins and DNA, enzyme activity, metabolism, and cell protection, all of which depend on L-reduced glutathione. L-reduced glutathione has been found to be a sign of oxidative stress and has the ability to scavenge oxygen free radicals [1].

The ROS were decreased after GSH treatment, and the mRNA levels of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, matrix metalloproteinase (MMP)-1, MMP-3, were also significantly inhibited after GSH stimulation. However, the IL-10 levels were enhanced, and GSH increased the expression of PTEN. The GSH suppressed the activation of phosphorylated (p)-PI3K and p-AKT. The supplementation of the BSO restored the activation of PI3K/AKT pathway with a high production of ROS. The levels of TNF-α, IL-1β and IL-6 were also elevated, when the BSO was added.[2]
Conclusion: These findings suggest that GSH can act as an inflammatory suppressor by downregulating the PTEN/PI3K/AKT pathway in MH7A cells. These data indicated a novel function of GSH for improving the inflammation of RA SFs and may help to alleviate the pathological process of RA.[2]

Modified electrodes coated by adsorbed cobalt phthalocyanines are known to show substantial electrocatalytic activity for the electro-oxidation of several thiols in alkaline aqueous solution. In this context, we explore in this study the electrocatalytic activity of adsorbed cobalt phthalocyanine (CoPc) on ordinary pyrolytic graphite electrode for the oxidation of reduced L-glutathione GSH and the reduction of its disulfide GSSG at physiological pH. To do so, cyclic and rotating disk voltammetries were performed and the amperometric results show that a stable electrochemical sensing material, with good reproducibility and sensitivity (in accordance with the concentrations of GSH expected in biological media), can be easily achieved. This opens the way for the design of an electrochemical sensor able to detect these two analytes in biologically relevant experimental conditions (in terms of pH)[1].

The MH7A cells and mouse SFs were treated with indicated concentrations of GSH (100 μg/mL) with or without Escherichia coli lipopolysaccharide (LPS) (100 ng/mL), the control group was treated with equal amount of phosphate buffered saline (PBS) for 24 h at 37°C. The supernatants were used to detect the protein levels of cytokines via enzymelinked immunosorbent assay (ELISA). The cells were used to detect the messenger ribonucleic acid (mRNA) expression levels of cytokines via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and were used to measure the protein expression levels of PTEN, phosphorylated (p)-PI3K, and p-AKT via Western blotting[2].
The levels of ROS were determined using a ROS assay kit according to the manufacturer’s instructions. Cellular ROS production was measured using a 2´,7´-dichlorofluorescein diacetate (DCFDA) assay kit according to the manufacturer’s instructions. The DCFDA is a cell permeable fluorogenic dye that measures hydroxyl, peroxyl, and activity of ROS within the cell. Briefly, dilute DCFH-DA with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μmoL/L. Remove the cell culture medium and add 500 µL diluted DCFH-DA in 24-well plate. Incubate for 20 min at 37°C in a humidified atmosphere (5% CO2). Wash the cells three times with serum-free cell culture medium to fully remove the redundant DCFH-DA. Finally, capture the figures with laser confocal microscope[2].
The RNA was isolated from MH7A cells and mouse SFs using TRIzol® reagent, and RT was conducted using PrimeScript™ RT Master mix. Then, complementary deoxyribonucleic acid (cDNA) was amplified using SYBR® Premix Ex Taq™ with gene-specific primers. The RT-qPCR analyses were performed in a LightCycler® 480 II detection system under the following thermal cycler conditions: initial denaturation for 5 min at 95°C, followed by 45 cycles for 15 sec at 95°C, 15 sec at 60°C and 15 sec at 72°C, using the primers listed in Tables 1 and 2. All experiments were performed in triplicate and the comparative cycling threshold values (Ct values) were normalized to endogenous reference (GAPDH). The levels of mRNA expression were calculated using the 2-ΔΔCq method[2].

A total of 30 DBA/1J female mice were used in this study. The release of ROS in MH7A cells was examined using a ROS assay kit. The effects of GSH on the messenger ribonucleic acid (mRNA) expression and protein levels of inflammatory cytokines were determined via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) in mouse SFs and MH7A cells, respectively. The PTEN/PI3K/AKT pathway was investigated via Western blotting. The effects of buthionine-sulfoximine (BSO), as an inhibitor of GSH, on these molecules were examined.[2]

[1]. Pereira-Rodrigues N, et al. Electrocatalytic activity of cobalt phthalocyanine CoPc adsorbed on a graphite electrode for the oxidation of reduced L-glutathione (GSH) and the reduction of its disulfide (GSSG) at physiological pH. Bioelectrochemistry. 2007 Jan;70(1):147-54.
[2]. Antioxidant glutathione inhibits inflammation in synovial fibroblasts via PTEN/PI3K/AKT pathway: An in vitro study. Arch Rheumatol . 2021 Dec 24;37(2):212-222

Glutathione is a tripeptide compound consisting of glutamic acid attached via its side chain to the N-terminus of cysteinylglycine. It has a role as a skin lightening agent, a human metabolite, an Escherichia coli metabolite, a mouse metabolite, a geroprotector, an antioxidant and a cofactor. It is a tripeptide, a thiol and a L-cysteine derivative. It is a conjugate acid of a glutathionate(1-).

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A tripeptide with many roles in cells. It conjugates to drugs to make them more soluble for excretion, is a cofactor for some enzymes, is involved in protein disulfide bond rearrangement and reduces peroxides. Glutathione is a metabolite found in or produced by Escherichia coli (strain K12, MG1655). Glutathione is a natural product found in Zea mays, Drosophila melanogaster, and other organisms with data available.

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Glutathione is a tripeptide comprised of three amino acids (cysteine, glutamic acid, and glycine) present in most mammalian tissue. Glutathione acts as an antioxidant, a free radical scavenger and a detoxifying agent. Glutathione is also important as a cofactor for the enzyme glutathione peroxidase, in the uptake of amino acids, and in the synthesis of leukotrienes. As a substrate for glutathione S-transferase, this agent reacts with a number of harmful chemical species, such as halides, epoxides and free radicals, to form harmless inactive products. In erythrocytes, these reactions prevent oxidative damage through the reduction of methemoglobin and peroxides. Glutathione is also involved in the formation and maintenance of disulfide bonds in proteins and in the transport of amino acids across cell membranes.

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Glutathione is a compound synthesized from cysteine, perhaps the most important member of the body's toxic waste disposal team. Like cysteine, glutathione contains the crucial thiol (-SH) group that makes it an effective antioxidant. There are virtually no living organisms on this planet-animal or plant whose cells don't contain some glutathione. Scientists have speculated that glutathione was essential to the very development of life on earth. Glutathione has many roles; in none does it act alone. It is a coenzyme in various enzymatic reactions. The most important of these are redox reactions, in which the thiol grouping on the cysteine portion of cell membranes protects against peroxidation; and conjugation reactions, in which glutathione (especially in the liver) binds with toxic chemicals in order to detoxify them. Glutathione is also important in red and white blood cell formation and throughout the immune system. glutathione's clinical uses include the prevention of oxygen toxicity in hyperbaric oxygen therapy, treatment of lead and other heavy metal poisoning, lowering of the toxicity of chemotherapy and radiation in cancer treatments, and reversal of cataracts. 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 (EC 4.4.1.5) catalyzes the conversion of methylglyoxal and reduced glutathione to S-D-Lactoyl-glutathione. Glyoxalase II (EC 3.1.2.6) catalyzes the hydrolysis of S-D-Lactoyl-glutathione to glutathione and D-lactate. GSH is known as a substrate in both conjugation reactions and reduction reactions, catalyzed by glutathione S-transferase enzymes in cytosol, microsomes, and mitochondria. However, it is also capable of participating in non-enzymatic conjugation with some chemicals, as in the case of 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 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-acetylcysteine, which is used by cells to replace spent GSSG and renew the usable GSH pool. It conjugates to drugs to make them more soluble for excretion, is a cofactor for some enzymes, is involved in protein disulfide bond rearrangement and reduces peroxides.

C10H17N3O6S

307.32

307.0838

C, 39.08; H, 5.58; N, 13.67; O, 31.24; S, 10.43

70-18-8

L-Glutathione reduced-13C2,15N;815610-65-2; 20167-21-9 (sodium); 34212-83-4 (disodium); 70-18-8 (free acid)

124886

Typically exists as white to off-white solids at room temperature

1.4±0.1 g/cm3

754.5±60.0 °C at 760 mmHg

182-192ºC

410.1±32.9 °C

0.0±5.5 mmHg at 25°C

1.572

-0.87

197.62

S([H])C([H])([H])[C@@]([H])(C(N([H])C([H])([H])C(=O)O[H])=O)N([H])C(C([H])([H])C([H])([H])[C@@]([H])(C(=O)O[H])N([H])[H])=O

RWSXRVCMGQZWBV-WDSKDSINSA-N

InChI=1S/C10H17N3O6S/c11-5(10(18)19)1-2-7(14)13-6(4-20)9(17)12-3-8(15)16/h5-6,20H,1-4,11H2,(H,12,17)(H,13,14)(H,15,16)(H,18,19)/t5-,6-/m0/s1

(2S)-2-amino-5-[[(2R)-1-(carboxymethylamino)-1-oxo-3-sulfanylpropan-2-yl]amino]-5-oxopentanoic acid

GSH; NSC-400639; NSC400639; Glutatiol; NSC 400639; L-Glutathione; Glutathione;glutathione; 70-18-8; L-Glutathione; Glutathion; L-Glutathione reduced; Isethion; Tathion; Glutathione-SH;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.  (3). This product is not stable in solution, please use freshly prepared working solution for optimal results.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~62.5 mg/mL (~203.37 mM)

Solubility in Formulation 1: 100 mg/mL (325.39 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

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 (Please use freshly prepared in vivo formulations for optimal results.)