Glutathione as an Antioxidant Peptide: What the Published Research Reports

Glutathione (GSH) is described in the review literature as a ubiquitous intracellular peptide whose reported functions include detoxification, antioxidant defence, maintenance of thiol status, and modulation of cell proliferation, and it is synthesised in the cytosol of mammalian cells through a tightly regulated pathway [1]. That review identifies the availability of cysteine and the activity of the rate-limiting enzyme glutamate cysteine ligase, composed of catalytic and modifier subunits, as the major determinants of synthesis, with glutathione synthase acting as the second enzyme of the pathway [1]. Oxidative stress is reported to induce the expression of glutathione synthetic enzymes through transcription factors including Nrf2/Nrf1 acting via the antioxidant response element, activator protein-1, and NF-kappaB, and dysregulated GSH synthesis is discussed in that review in the context of conditions such as diabetes mellitus, pulmonary fibrosis, cholestatic liver injury, endotoxaemia, and drug-resistant tumour cells [1]. Reviews of neurodegenerative disease have examined GSH as a measurable brain antioxidant pool in human populations. One review recapitulates literature associating increased oxidative stress in Alzheimer's disease with decreased brain GSH levels, discusses methodologies for monitoring alterations in GSH, and highlights in vivo magnetic resonance spectroscopy as an approach for assaying GSH in human subjects [2]. A separate review of Parkinson's disease frames oxidative stress as one component of a cascade that also involves mitochondrial dysfunction, excitotoxicity, nitric oxide toxicity, and inflammation, and notes the difficulty of separating oxidative damage from other events implicated in dopaminergic cell death in human disease [3]. Several cell-based studies use the glutathione system as a mechanistic readout. In differentiated human neuronal cell lines, 24-hydroxycholesterol was reported to potentiate the pro-apoptotic and pro-necrogenic effects of amyloid-beta(1-42) through enhanced NADPH oxidase-dependent reactive oxygen species generation and a consequent shift in the GSSG/GSH ratio, an effect prevented in vitro by co-incubation with quercetin or genistein [4]. In cultured rat cortical neurons, amyloid-beta(1-40) increased reactive oxygen species and lipid peroxidation and depleted reduced glutathione in association with decreased glutathione peroxidase and glutathione reductase activities; galantamine prevented these alterations in the GSH antioxidant system in that in vitro model [5]. Peptides have been studied as modulators of glutathione-linked pathways in both cells and animals. A short peptide derived from pigment epithelium-derived factor, designated 6dS, reduced sodium iodate-induced ferrous iron accumulation, lipid peroxidation, GSH depletion, and ferroptosis in ARPE-19 cells, and, applied as eye drops in a rat model of sodium iodate-induced retinal degeneration, counteracted retinal pigment epithelium cell death, lipid peroxidation, retinal atrophy, and electrophysiological dysfunction; the authors attributed the effect to induction of SLC7A11 and GPX4, since inhibitors of the PEDF receptor, SLC7A11, and GPX4 abolished it [6]. Low molecular weight whey peptides generated by simulated gastrointestinal digestion attenuated hydrogen peroxide-induced reactive oxygen species production in glial cells in vitro, increased GSH content, and prevented lipopolysaccharide-induced GSH reduction in astrocytes, alongside attenuation of TNF-alpha and IL-6 mRNA expression and nitric oxide secretion via an NF-kappaB-dependent pathway [7]. In the murine macrophage line J774, a 6-kDa thymic peptide was reported to suppress zymosan-triggered oxidative burst in a concentration-dependent manner and to increase glutathione levels together with glutathione peroxidase, glutathione reductase, and superoxide dismutase activities, while catalase activity was unaffected [8]. A dithiol-containing peptide, GM15, derived from Arthrospira platensis glutathione oxido-reductase, scavenged superoxide and hydroxyl radicals in cell-free assays, lowered intracellular oxidative stress in hydrogen peroxide-exposed leucocytes, and induced caspase-9-mediated apoptosis in oral carcinoma KB cells in vitro [9]. Glutathione also appears in the literature as a benchmark against which other antioxidant peptides are compared. A porcine plasma-derived peptide, EDEQKFWGK, showed higher hydroxyl, ABTS, and DPPH radical scavenging percentages than glutathione in vitro and increased SOD, CAT, and GSH-Px activity in an oxidatively challenged HepG2 cell model [10]. A peptide preparation from Pinctada fucata was reported to exhibit DPPH, hydroxyl, superoxide, and cellular antioxidant activities with EC50 values indicating higher in vitro activity than glutathione, together with antiproliferative activity against HepG2, Caco-2, and MCF-7 carcinoma cells [11]. A study of duck embryo eggs tracked dynamic changes in vitamins E and C, total, reduced, and oxidised glutathione, SOD, CAT, GSH-Px, and malondialdehyde across incubation in that animal system, and screened five peptides, of which DY2 showed high antioxidant activity in vitro [12]. Peptide Inhibitor of Complement C1 (PIC1), which contains two vicinal cysteine residues, displayed dose-dependent activity in total antioxidant capacity, HORAC, ORAC, and TBARS assays performed in vitro, acting by single electron transfer and hydrogen atom transfer rather than metal chelation, with activity similar to glutathione and reduced when its cysteine residues were oxidised [13]. Structure-activity reviews of food protein-derived antioxidant peptides note that low molecular weight peptide fractions are generally reported as potent in vitro antioxidants, and that sulfur-containing residues (Cys, Met), aromatic residues (Phe, Trp, Tyr), and histidine are associated with stronger measured antioxidant properties, with glutathione cited as the reference cellular antioxidant peptide [14]. In the pharmacology literature on lipoic acid, dihydrolipoic acid formed by reduction of lipoic acid is described as able to regenerate endogenous antioxidants including vitamin E, vitamin C, and glutathione in experimental systems [15]. Formulation and analytical work has addressed the handling of GSH itself. Proliposome formulations of L-glutathione prepared by a granule method with mannitol were characterised for particle size, zeta potential, and encapsulation efficiency; circular dichroism indicated that the molecular structure of GSH was retained, total antioxidant capacity was concentration-dependent and maintained after formulation, and an oral bioavailability study in rats compared the positively charged formulation with the negative formulation, a commercial capsule, and pure GSH [16]. On the detection side, a tetraphenylethylene-based fluorescent peptide probe was developed that responds selectively to Cu2+ with fluorescence recovery upon addition of GSH, with reported detection limits in buffer and demonstrated permeability and low toxicity for live cell imaging in vitro [17]. In a cross-sectional observational study of 90 adults aged 45-65 grouped by vitamin D status, serum biomarkers including amyloid-beta 42, BDNF, calcium, glutathione, and acetylcholinesterase were measured, and glutathione levels differed significantly across vitamin D status groups in that human sample [18].