Glutathione: Systemic Protectant Against Oxidative and Free Radical Damage Dedicated to the memory of Professor Daniel Mazia, my PhD mentor and a pioneer in cell biology

The tripeptide thiol glutathione (GSH) has facile electron-donating capacity, linked to its sulfhydryl (-SH) group. Glutathione is an important water-phase antioxidant and essential cofactor for antioxidant enzymes; it provides protection also for the mitochondria against endogenous oxygen radicals. Its high electron-donating capacity combined with its high intracellular concentration endows GSH with great reducing power, which is used to regulate a complex thiol-exchange system (-SH <---> S-S-). This functions at all levels of cell activity, from the relatively simple (circulating cysteine/-SH thiols, ascorbate, other small molecules) to the most complex (cellular -SH proteins). Glutathione is homeostatically controlled, both inside the cell and outside. Enzyme systems synthesize it, utilize it, and regenerate it as per the gamma-glutamyl cycle. Glutathione is most concentrated in the liver (10 mM), where the "P450 Phase II" enzymes require it to convert fat-soluble substances into water-soluble GSH conjugates, in order to facilitate their excretion. While providing GSH for their specific needs, the liver parenchymal cells export GSH to the outside, where it serves as systemic source of -SH/reducing power. GSH depletion leads to cell death, and has been documented in many degenerative conditions. Mitochondrial GSH depletion may be the ultimate factor determining vulnerability to oxidant attack. Oral ascorbate helps conserve GSH; cysteine is not a safe oral supplement, and of all the oral GSH precursors probably the least flawed and most cost-effective is NAC (Nacetylcysteine). (Alt Med Rev 1997; 2(3):155-176) Glutathione (g-glutamylcysteinylglycine, GSH) is a sulfhydryl (-SH) antioxidant, antitoxin, and enzyme cofactor. Glutathione is ubiquitous in animals, plants, and microorganisms, and being water soluble is found mainly in the cell cytosol and other aqueous phases of the living system. Glutathione often attains millimolar levels inside cells, which makes it one of the most highly concentrated intracellular antioxidants. Glutathione exists in two forms (Fig. 1): The antioxidant "reduced glutathione" tripeptide is conventionally called glutathione and abbreviated GSH; the oxidized form is a sulfur-sulfur linked compound, known as glutathione disulfide or GSSG. The GSSG/GSH ratio may be a sensitive indicator of oxidative stress. GSH has potent electron-donating capacity, as indicated by the high negative redox potential of the GSH/GSSH "redox couple" (E'0 =-0.33v). Its high redox potential renders GSH both a potent antioxidant per se and a convenient cofactor for enzymatic reactions that require readily available electron pairs, the so-called "reducing equivalents." Lewin6 articulated how a substance with great readiness to donate electrons, when present at high concentrations, has greatly enhanced effectiveness as a reductant. This is reducing power, and is most expressed by GSH where its concentrations are highest (as in the liver). The reducing power of GSH is a measure of its freeradical scavenging, electron-donating, and sulfhydryl-donating capacity. Reducing power is also the key to the multiple actions of GSH at the molecular, cellular, and tissue levels, and to its effectiveness as a systemic antitoxin. The reduced glutathione molecule consists of three amino acids glutamic acid, cysteine, and glycine covalently joined end-to-end (Fig. 1). The sulfhydryl (-SH) group, which gives the molecule its electron-donating character, comes from the cysteine residue. Glutathione is present inside cells mainly in its reduced (electron-rich, antioxidant) GSH form. In the healthy cell GSSG, the oxidized (electron-poor) form, rarely exceeds 10 percent of total cell glutathione.1 Intracellular GSH status appears to be a sensitive indicator of the cell's overall health, and of its ability to resist toxic challenge. Experimental GSH depletion can trigger suicide of the cell by a process known as apoptosis. The peer-reviewed literature on glutathione is too extensive to be adequately discussed in a single review. This review summarizes the salient features of GSH as antioxidant and systemic protectant, examines instances of GSH abnormalities linked to tissue and organ system breakdown, and explores the possibilities for GSH replacement therapy to benefit degenerative conditions. Glutathione Biosynthesis, Metabolism, and Utilization The metabolism of GSH has been worked out to an extent that cannot be fully detailed herein; publications by the late Alton Meister and his colleagues provide greater detail. Glutathione status is homeostatically controlled, being continually self-adjusting with respect to the balance between GSH synthesis (by GSH synthetase enzymes), its recycling from GSSG (by GSH reductase), and its utilization (by peroxidases, transferases, transhydrogenases, and transpeptidases). The overall picture of GSH metabolism is summarized by way of the gamma-glutamyl cycle in Fig. 2. Glutathione synthesis occurs within cells in two closely linked, enzymatically controlled reactions that utilize ATP and draw on nonessential amino acids as substrates. First, cysteine and glutamate are combined (by the enzyme gamma-glutamyl cysteinyl synthetase, see Reaction 1 in Fig. 2), with availability of cysteine usually being the rate-limiting factor. Cysteine is generated from the essential amino acid methionine, from the degradation of dietary protein, or from turnover of endogenous proteins. The buildup of GSH acts to feedback-inhibit this enzyme, thereby helping to ensure homeostatic control over GSH synthesis. The second GSH synthesis reaction combines gamma-glutamylcysteine with glycine to generate GSH (catalyzed by GSH synthetase, Reaction 2 in Fig. 2). Excessive accumulation of gamma-glutamylcysteine in the absence of its conversion to GSH can lead to its conversion to 5-oxoproline by the enzyme gamma-glutamyl cyclotransferase (Reaction 4). Buildup of 5-oxoproline can have adverse consequences due to metabolic acidosis. The GSH pool is drawn on for 3 major applications: (a) as cofactor for the GSG-Stransferases in the detoxicative pathways (Reaction 7 in Fig. 2); (b) as substrate for the gamma-glutamyl transpeptidases, enzymes which are located on the outer cell surface and which transfer the glutamine moiety from GSH to other amino acids for subsequent uptake into the cell (Reaction 3); and (c) for direct free-radical scavenging and as an antioxidant enzyme cofactor (Reaction 9). The GSH transferases are a large group of isozymes that conjugate GSH with fatsoluble substances as the major feature of liver detoxification. For further details of the gamma-glutamyl cycle, the reader is referred to Meister and Anderson. The oxidation-reduction pathways of GSH are summarized in Fig. 3. Glutathione is an essential cofactor for antioxidant enzymes, namely the GSH peroxidases (both Sedependent and non-Se-dependent forms exist) and the more recently described phospholipid hydroperoxide GSH peroxidases. The GSH peroxidases serve to detoxify peroxides (hydrogen peroxide, other peroxides) in the water-phase, by reacting them with GSH; the latter enzymes use GSH to detoxify peroxides generated in the cell membranes and other lipophilic cell phases. This is one instance of the water-soluble GSH providing electrons to help reduce oxidized biomolecules located away from the water phase. Enzymes collectively known as GSH transhydrogenases use GSH as a cofactor to reconvert dehydroascorbate to ascorbate, ribo-nucleotides to deoxyribonucleotides, and for a variety of -S-S<--> -SH interconversions (Fig. 3). After GSH has been oxidized to GSSG, the recycling of GSSG to GSH is accomplished mainly by the enzyme glutathione reductase. This enzyme uses as its source of electrons the coenzyme NADPH (nicotinamide adenine dinucleotide phosphate, reduced). Therefore NADPH, coming mainly from the pentose phosphate shunt, is the predominant source of GSH reducing power. Cathcart used this to explain why subjects unable to make adequate NADPH may be at increased risk of oxidative damage from GSH insufficiency. Through its significant reducing power, GSH also makes major contributions to the recycling of other antioxidants that have become oxidized. This could be the basis by which GSH helps to conserve lipid-phase anti-oxidants such as alpha-tocopherol (vitamin E), and perhaps also the carotenoids. Meister and his group used buthionine sulfoximine (BSO) to inhibit GSH synthesis in rodents, and concluded from their findings that GSH almost certainly plays such a role in vivo. The liver seems to have two pools of GSH; one has a fast turnover (half-life of 2-4 hours), while the other is avidly retained with a half-life of about 30 hours. The first corresponds to cytosolic GSH, the second mainly to mitochondrial GSH which is known to be more tightly held. Though this pool represents a minor portion of the total GSH, the mitochondria are normally under high oxidative stress and thus conserve their GSH. With regard to the essentiality of GSH for the survival of the whole organism, substantial information is available from studies on hereditary GSH depletion in the human, and from experimental depletion and repletion of GSH in animal models and cell cultures. Inherited deficiency of the enzyme gamma-glutamyl cysteine synthetase, the first of the two enzymes necessary for GSH synthesis, has been described in two human siblings. They exhibited generalized GSH deficiency, hemolytic anemia, spinocerebellar degeneration, peripheral neuropathy, myopathy, and aminoaciduria, and severe neurological complications as they moved into their fourth decade of life. Their red cell GSH was less than 3% of normal, their muscle GSH less than 25%, and their white cell GSH less than 50% normal. One of them may have been hypersensitive to antibiotics, having developed psychosis after a single dose of sulfonamide for a urinary tract