Glutathione (overview)

GSH presents mainly inside the cells

While GSH can be found in plasma in low concentration, most of GSH is located inside the cells (including Red Blood Cells):

Intracellular glutathione concentrations range between 0.5 and 10 mM whereas extracellular glutathione concentrations are significantly lower, with estimated values in the micromolar range. (R1)

Cells must make own Glutathione

Extracellular supply of GSH is limited by low concentration and by mandatory degradation of GSH on the cell’s membrane

TBD

Limiting factors of GSH synthesis

Availability of Cysteine

It’s widely believed that availability of L-Cysteine is a limiting factor for GSH synthesis:

The limiting reagent in the synthesis of glutathione is cysteine, which is synthesized from homocysteine via the transsulfuration pathway. In liver, ∼50% of the cysteine in glutathione is derived from homocysteine via the transsulfuration pathway. The importance of this pathway in glutathione-based redox homeostasis is further supported by a decrease in the steady-state glutathione concentration in liver (to 63%) and brain (to 71%) in transgenic mice with homozygous disruption of the cystathionine β-synthase gene. (R2)

Availability of Glycine

While cysteine availability is regarded as the main limiting factor for GSH synthesis, a number of studies have shown that glycine is also important, and its level can be lower than needed, therefore limiting GSH synthesis.

These data indicate that a major contributor to glutathione deficiency and associated oxidative stress in aging humans is a diminished rate of glutathione synthesis, which in turn is due to low availability of its precursor amino acids. Replenishing the supply of cysteine and glycine by oral supplementation is effective at restoring glutathione concentrations and reducing oxidative stress to levels observed in young healthy humans. (R3)

Neurons and astrocytes can make cysteine

Contrary to a common belief that brain cells lack transsulfuration capability, they were shown to be able to make glutathione from methionine:

In this study, we incubated mouse and human neurons and astrocytes and murine brain slices in medium with [³⁵S]methionine and detected radiolabel incorporation into glutathione. This label transfer was sensitive to inhibition of γ-cystathionase. In adult brain slices, ∼40% of the glutathione was depleted within 10h following γ-cystathionase inhibition. In cultured human astrocytes, flux through the transsulfuration pathway increased under oxidative stress conditions, and blockade of this pathway led to reduced cell viability under oxidizing conditions. This study establishes the presence of an intact transsulfuration pathway and demonstrates its contribution to glutathione-dependent redox-buffering capacity under ex vivo conditions in brain cells and slices. (R2)

GSH is a limiting factor for remethylation in neurons

Handling of oxidative stress in neurons is different from other tissues:

In most tissues SAM is utilized to methylate oxidized cobalamin, in conjunction with electron donation by methionine synthase reductase, thereby restoring methylcobalamin and allowing resumption of activity. This mode of reactivation is required approximately every 100–1,000 turnovers, even under strictly anaerobic laboratory conditions. Under physiological conditions, oxidation of cobalamin is undoubtedly much more common, illustrating how vitamin B12 serves as a sensor of redox status. During oxidative stress, cobalamin is more frequently oxidized and more HCY is diverted toward cysteine and GSH synthesis. (R4)

Neuronal cells have a uniquely different strategy for reactivating methionine synthase, which is tightly dependent upon GSH status. Whereas SAM-dependent methylation of oxidized cobalamin occurs in most cell types, in neuronal cells oxidized cobalamin dissociates from the enzyme and is replaced by methylcobalamin, allowing reactivation. However, methylcobalamin synthesis proceeds through an intermediate step that requires GSH, so methionine synthase will remain inactive longer when GSH levels are below normal. (R4)

Open questions

1. What happens to oxidized cobalamin after it dissociates from the MS enzyme in neurons?

Cystine deficiency causes export of GSH

Cystine deprivation induced GSH efflux and extracellular degradation, which aimed to restore cellular cysteine.

Inhibition of γ-glutamyl transpeptidase (GGT) impaired the ability of GSH or cell-permeable GSH to restore mTORC1 signaling and the ISR, suggesting that the capacity of GSH to release cysteine, but not GSH per se, regulated the signaling networks. (R5)

Detoxifying pathway of GSH

A major function of GSH is detoxification of xenobiotics and/or their metabolites. These compounds are electrophiles or electron-loving substances (Fig. 2, indicated as X) and form conjugates with GSH either spontaneously or enzymatically in reactions catalyzed by GSH-S-transferase (R).

The conjugates formed are usually excreted from the cell or into bile as in the case of hepatocytes. GSH conjugates can undergo GGT-mediated cleavage of the γ-glutamyl moiety, leaving a cysteinyl-glycine conjugate.

The cysteinyl-glycine bond is then cleaved by dipeptidase, resulting in a cysteinyl conjugate.

This is followed by N-acetylation of the cysteine conjugate, forming a mercapturic acid (Fig. 2). The metabolism of GSH conjugates to mercapturic acid begins either in the biliary tree, intestine or kidney, but the formation of the N-acetylcysteine conjugate usually occurs in the kidney.

In addition to exogenous compounds, many endogenously formed compounds also follow similar metabolic pathways. Although the majority of the conjugation reactions to GSH result in detoxification of the compound, occasionally the product itself is highly reactive. GSH conjugation irreversibly consumes intracellular GSH. (R6)

Steps

1. metabolite binds to GSH by Glutathione-S-Transferase
2. The conjugate is exported from the cell
3. Gamma-glutamyl is cleaved from the conjugate by GGT (extracellular). The result is Cysteine-Glycine-metabolite conjugate (Cys-Gly-X).
4. Cys-Gly-X is processed by Dipeptidase, releasing glycine and Cys-X conjugate.
5. Cys-X is acetylated by N-acetylase, producing N-Acetyl-Cysteine-X conjugate (a mercapturic acid). This happens in intestines, kidneys or in biliary tree usually.

GSH as a cysteine storage

Storage of cysteine is one of the most important functions of GSH because cysteine is extremely unstable extracellularly and rapidly auto-oxidizes to cystine, in a process that produces potentially toxic oxygen free radicals (Meister, 1988).

Meister first described the γ-glutamyl cycle in the early 1970’s, which allows GSH to serve as a continuous source of cysteine (R6)