ObjectiveTo determine if orally dosed γ-glutamylcysteine (γ-GC) can increase cellular glutathione (GSH) levels above homeostasis. Many chronic and age-related disorders are associated with down-regulation, or impairment, of glutamate cysteine ligase (GCL). This suggests that γ-GC supply may become limiting for the maintenance of cellular GSH at the normal levels required to effectively protect against oxidative stress and any resulting physiological damage.MethodsGSH levels were measured in lymphocytes of healthy, non-fasting participants before and after single oral doses (2 and 4 g) of γ-GC. Blood samples were immediately processed using high speed fluorescence-activated cell sorting to isolate 106 lymphocytes that were then assayed for GSH content.ResultsA single 2 g dose of γ-GC increased lymphocyte GSH content above basal levels (53±47%, p<0.01, n=14) within 90 min of administration. A randomized dosage (2 and 4 g γ-GC) crossover design was used to explore the pharmacokinetics of this GSH increase. In general, for both dose levels (n=9), GSH increased from initial basal levels over 3 h (tmax) before reaching maximum GSH concentrations (Cmax) that were near two (2 g γ-GC) to three (4 g γ-GC) fold basal levels (0.4 nmol/106 lymphocytes). Beyond tmax, GSH levels progressively declined reaching near basal levels by 5 h. The GSH half-life was between 2 and 3 h with exposure (AUC) to increased GSH levels of 0.7 (2 g γ-GC) and 1.8 (4 g γ-GC) nmol.h/106 lymphocytes.ConclusionsOral γ-GC is a non-toxic form of cysteine that can be directly taken up by cells and transiently increase lymphocyte GSH above homeostatic levels. Our findings that γ-GC can increase GSH levels in healthy subjects suggests that it may have potential as an adjunct for treating diseases associated with chronic GSH depletion. This trial was registered at anzctr.org.au as ACTRN12612000952842.

Glutathione (GSH) is one of the most abundant thiol antioxidants in cells. Many chronic and age-related diseases are associated with a decline in cellular GSH levels or impairment in the catalytic activity of the GSH biosynthetic enzyme glutamate cysteine ligase (GCL). γ-glutamylcysteine (GGC), a precursor to glutathione (GSH), can replenish depleted GSH levels under oxidative stress conditions, by circumventing the regulation of GSH biosynthesis and providing the limiting substrate. Soluble amyloid-β (Aβ) oligomers have been shown to induce oxidative stress, synaptic dysfunction and memory deficits which have been reported in Alzheimer’s disease (AD). Calcium ions, which are increased with age and in AD, have been previously reported to enhance the formation of Aβ 40 oligomers, which have been casually associated with the pathogenesis of the underlying neurodegenerative condition. In this study, we examined the potential beneficial effects of GGC against exogenous Aβ 40 oligomers on biomarkers of apoptosis and cell death, oxidative stress, and neuroinflammation, in human astrocytes. Treatment with Aβ 40 oligomers significantly reduced the cell viability and apoptosis of astrocyte brain cultures and increased oxidative modifications of DNA, lipids, and protein, enhanced pro-inflammatory cytokine release and increased the activity of the proteolytic matrix metalloproteinase enzyme, matric metalloproteinase (MMP)-2 and reduced the activity of MMP-9 after 24 h. Co-treatment of Aβ 40 oligomers with GGC at 200 μM increased the activity of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) and led to significant increases in the levels of the total antioxidant capacity (TAC) and GSH and reduced the GSSG/GSH ratio. GGC also upregulated the level of the anti-inflammatory cytokine IL-10 and reduced the levels of the pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) and attenuated the changes in metalloproteinase activity in oligomeric Aβ 40 -treated astrocytes. Our data provides renewed insight on the beneficial effects of increased GSH levels by GGC in human astrocytes, and identifies yet another potential therapeutic strategy to attenuate the cytotoxic effects of Aβ oligomers in AD.

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Accumulating evidence has suggested the involvement of oxidative stress in the pathogenesis of Alzheimer's disease (AD). The main endogenous antioxidant, glutathione (GSH), has been shown to decline with ageing and in several age-related degenerative diseases, including AD. Potential options for replenishing GSH levels as a therapeutic target to treat these conditions include the administration of GSH itself, and low toxicity forms of the limiting amino acid for GSH synthesis; cysteine. However, passive GSH uptake is limited due to an unfavourable concentration gradient between the plasma and cytosol. Similarly, cysteine prodrugs have demonstrated limited efficacy to elevate depleted GSH levels in several in vivo and in vitro models of disease. It has been suggested that the decline in GSH levels in AD, may be associated with down regulation of GSH homeostasis rather than substrate limitation. Cellular GSH homeostasis is regulated by non-allosteric feedback inhibition exerted by GSH on glutamate cysteine ligase (GCL), which is responsible for the synthesis of the GSH precursor γ-glutamylcysteine (GGC). In conditions involving down regulated GSH homeostasis, GGC serves as a crucialrate-limiting substrate for GSH synthetase, the main enzyme responsible for condensing glycine with GGC to form the final thiol tripeptide, GSH. In this review, we focus on the therapeutic potential of GGC to elevate cellular GSH levels. We also discuss the efficacy of GGC prodrugs which would be taken up and converted by the unregulated GS to GSH, and the administration of modified GSH compounds, such as GSH esters that could potentially overcome the concentration gradient that prohibits passive GSH uptake, in AD.

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