Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

While the precise mechanisms by which neural cells die during ischemia are still being defined, key interacting components include depletion of ATP, glutamate excitotoxicity, calcium overload, and production of strong oxidants that can overwhelm the antioxidant defense system. While the increased superoxide anion produced under ischemia and reperfusion conditions can adversely affect cell function, it can also interact to produce powerful oxidizing agents, including the hydroxyl radical. The superoxide dismutase family constitutes the mechanism developed by cells to dismutate the increased superoxide anion produced during ischemia into hydrogen peroxide. It is essential that this hydrogen peroxide be removed as it can be dismutated to the hydroxyl radical as mediated by reduced transition metals such as ferrous iron. The hydroxyl radical can damage cells by causing DNA strand breaks, protein oxidation, and lipid peroxidation. The latter is a particularly damaging effect since once initiated, this starts a chain of peroxidations, ultimately resulting in the formation of lipid peroxyl radicals and lipid hydroperoxides that disturb cell membrane function. The lipid hydroperoxides in the presence of iron can be converted to alkoxy and peroxy radicals resulting in new chains of lipid peroxidation (1-8).

Juurlink (6) and Juurlink and Sweeney (9) have reviewed evidence that reduced glutathione (GSH) and glutathione peroxidase (GPX) are of key importance to peroxide scavenging in neural cells. In most tissues, about 90% of cellular GPX activity is GPX1, the classical isoform localized to the cytosol and mitochondria that can scavenge hydrogen peroxide as well as organic peroxides such as free fatty acid hydroperoxides. Other major cellular activity is accounted for by a phospholipid hydroperoxide glutathione peroxidase (GPX4) that can scavenge membrane-bound phospholipid hydroperoxides, other organic peroxides, and hydrogen peroxide (3-8, 10, 11). GPX activity is dependent upon the presence of GSH which is oxidized in the process. As the affinity of GPX for peroxides increases as a function of GSH concentration, small changes in GSH can have a large influence on the ability of the cell to scavenge peroxides (6). GSH is also important for regenerating vitamin E that is important for scavenging lipid peroxyl radicals in membranes; GSH reduces oxidized ascorbate which directly reduces the alpha-tocopherol radical. GSH may also exert direct antioxidant effects (1-8, 10).

That brain GSH and GPX activity are important determinants of tissue damage associated with stroke has been illustrated in a number of ways. Ebselen, a seleno-organic compound that inhibits lipid peroxidation through a glutathione peroxidase-like activity, was shown in a placebo-controlled, double-blind clinical trial in acute ischemic stroke to be protective if administered within 24 hours of stroke onset (12). GSH depletion by buthionine sulfoximine enhances ischemic injury in rat cerebrum (13), and increasing brain GSH by administering a GSH ester immediately after an ischemic insult offers neuroprotection (14). The administration of N-acetylcysteine, a compound that promotes GSH synthesis, is also protective in transient brain ischemia models (15).

Current approaches to enhance the ability of the brain to scavenge peroxides as a means of reducing the extent of brain damage associated with stroke should include an investigation of nutritional factors. It is hypothesized that nutritional intervention targeted to enhance brain GSH and GSH-Px may be an effective means of reducing this damage. Conversely, specific nutrient deficiencies, acting through the same mechanism, may worsen the degree of functional recovery. The elderly, the group at highest risk for stroke, are known to be vulnerable to a variety of nutritional problems resulting from factors such as anorexia associated with depression, poor dental status, drugs, chronic disease and decreased activity (16, 17).

We have addressed two questions related to this hypothesis. The first is whether brain GSH concentration is responsive to a short-term dietary deficiency of sulfur amino acids (SAA). Both the reduction of oxidized-glutathione by glutathione reductase and the de novo synthesis of GSH will determine the GSH status of tissues under conditions of oxidative stress. In the synthesis of GSH (L-gamma-glutamyl-L-cysteinylglycine), gamma-glutamylcysteine is formed in the initial rate-limiting step that is catalyzed by gamma-glutamylcysteine synthetase. In the second step, GSH synthetase catalyzes the reaction between glycine and gamma-glutamylcysteine to form GSH (18-20). Cysteine is the limiting amino acid for GSH synthesis, and the sulfur amino acid content of the diet is a major determinant of GSH concentration in tissues such as liver (19). Lung and liver GSH is decreased by fasting, low-protein diets, or diets limiting in sulfur amino acids (21-23). The response of liver GSH to dietary protein is found only in the physiological range, with no further increase with excessive dietary protein (21, 24). Much less information is available on the responsiveness of brain GSH to SAA supply although one report suggested that brain GSH was reduced with a short-term SAA deficiency (25). Increases in rat brain GSH have also been reported following administration of the cysteine prodrug, L-2-oxothiazolidine-4-carboxylate (26). The latter compound, which is converted intracellularly to cysteine by the enzyme 5-oxoprolinase, has been used as a cysteine delivery system to overcome the toxicity associated with cysteine supplementation (27).

A second question that we have begun to address is whether dietary Se regulates the expression of GPX1 in brain. The family of GPX selenoproteins incorporate Se into their active sites in the form of selenocysteine. In the majority of mammalian tissues, there is a relationship between GPX activity, dietary Se level, and susceptibility to certain types of oxidative stress (28). In rat liver, Se deficiency can result in a loss of GPX1 activity to less than 1% of control values and this is accompanied by loss of immunoreactive GPX1 protein (29). Selenoproteins are also regulated individually through changes in their mRNA levels. This is thought to allow maintenance of certain selenoproteins at the expense of others when selenium supply is inadequate for optimal expression of all selenoproteins (30). Lei et al. (31) have demonstrated in liver, heart, kidney, and lung that GPX1 activity and mRNA levels are more susceptible to selenium depletion than GPX4. The data on dietary Se and isoforms of GPX in brain are much less clear although it has been shown that brain is better able to retain Se at the expense of other tissues when Se supply is limited (28). Some have reported dietary Se restriction to decrease total GPX activity in rat brain by 25-39% (32-35). Castaņo et al. (36) have even reported decreases of 21-22% in substantia nigra and striatum after only 15 days on a low Se diet. In contrast, Prohaska and Ganther (37) and Buckman et al. (28) found no influence of dietary selenium on total GPX activity in brain. The discrepancy in results may be due to varying degrees of Se deficiency among experiments or to blood contamination of the tissue. Also, little data have been available on the response of brain GPX to supplementing dietary Se above requirement. We have therefore begun to re-evaluate the effects of both deficient and supplemental levels of dietary selenium on GPX activity in brain regions.

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