Lipid peroxidation of rat liver microsomal fractions was monitored by its low-level cheniiluniinescence in preparations from controls and vitamin-E-deficient animals. Measurements were made (a) of the duration of the lag phase T~ after initiation with NADPH/iron-ADP and (b) of the slope of the chemiluminescence increase.In microsomes with normal vitamin E (a-tocopherol) level the lag phase T,, was substantially increased by ascorbate; in contrast, even an enhanced peroxidation was observed with ascorbate in vitamin-E-deficient microsomes. Therefore, the ascorbate-mediated protection of microsomal membranes against lipid peroxidation is dependent on vitamin E in the membrane. In vitamin E deficiency the pro-oxidant effect of ascorbate was abolished when glutathione (GSH) was present.Likewise, GSH does not prolong the lag phase zo in vitamin E deficiency. However, GSH (but not cysteine) exerts an antioxidant effect both in controls and in vitamin E deficiency by decreasing the slope of the chemiluminescence increase during lipid peroxidation. The involvement of GSH in an enzyme-dependent mechanism is suggested.Vitamin E is regarded as the major lipid-soluble antioxidant, preventing oxidative attack of membrane lipids and other membrane-associated compounds (see [ 11 for review). The initiating process by a primary radical R' leads to lipid radicals L', which then add oxygen in a diffusion-limited reaction. The resulting lipid peroxyl radicals continue a chain process.(1) (2) Chain-breaking antioxidants shorten the length of the chain reaction by trapping the peroxyl radical LOO'. Vitamin E has been shown to be a very effective scavenger of these radicals [2, 31, whereas it does not react at comparable rates with carbon-centered radicals [4].Ascorbate [5 -71 and glutathione (GSH) [8 -151 have been described to be involved in several types of protective mechanism. In ESR studies regeneration of vitamin E from the cr-chromanoxyl radical by GSH and vitamin C was detected [lo, 161. In experiments investigating the effect of vitamin E and vitamin C on methyl linoleate peroxidation, vitamin E was kept in the reduced state as long as there was vitamin C present [17]. In addition to this synergistic effect of vitamin C with vitamin E, a direct chain-breaking activity of vitamin C was also reported [17]. In human blood plasma the oxidation of protein sulfhydryl groups is one of the first events when peroxyl radicals are generated [I 81, thus a chain-breaking

The reaction of superoxide with reduced glutathione (GSH) was studied with two 0;-producing systems : xanthine oxidase using xanthine or acetaldehyde as substrates and, secondly, quinol autoxidation.The capability of GSH to quench superoxide radicals was detected by lowered 0;-mediated cytochrome c3+ reduction. The formation of the oxidation products, glutathione disulfide (GSSG) and glutathione sulfonate (the latter at levels of about 6 -15 % compared to GSSG), was dependent on the 0; production and was inhibited by superoxide dismutase. The presence of GSH together with an 06-producing system led to an extra uptake of oxygen, which was also depressed by superoxide dismutase. The observed O2 uptake was accounted for by the formation of GSSG and GSO; from GSH; the data are in accordance with a mechanism involving thiyl radicals.Low-level chemiluminescence measurement indicated the formation of excited oxygen species. The intensity of photoemission was dependent on the GSH concentration and on the 0 6 production rate. Chemiluminescence was inhibited by superoxide dismutase and also by glutathione peroxidase, but not by catalase or OH. quenchers. Spectral analysis and the effects of 1,4-diazabicyclo[2.2.2]octane and sodium azide indicated the contribution of singlet molecular oxygen to the light emission. It is suggested that singlet oxygen results from an intermediate oxygen addition product such as a glutathione peroxysulphenyl radical.The superoxide anion radical 0; (and its protonated form HO;) is a radical of biological importance known to be generated in a large number of enzymatic and non-enzymatic reactions. Though cellular defense mechanisms against cell toxicity of 0 6 or HOi and further products of electron transfer to oxygen (H202, OH') are effective, these may be overwhelmed in oxidative stress with excessive formation of 0;. As a consequence, pronounced effects on the cellular glutathione (GSH) status have been described [l -31. The loss of GSH is explainable by GSH-peroxidase-mediated reduction of H20z, the latter being the product of HOi dismutation. However, the possibility that GSH also reacts directly with 0; or HO; may be considered. McNeil et al.[4] described the inhibition effect of GSH on superoxide-dependent epinephrine oxidation and on the oxidation of dianisidine and suggested that GSH may play a major role in controlling 0; concentrations in the cell. Moreover, in recent work [3] we described the effect of lowered GSH content on menadione-induced chemiluminescence of perfused rat liver; the light emission was lowered when the GSH level was decreased, thus suggesting that GSH could play a role in the generation of light-emitting species such as singlet molecular oxygen. Similar effects were found with microsomes during redox cycling of the menadione-GSH conjugate [3].Abbreviations. DABCO, 1,4-diazabicyclo[2.2.2]octane; GSH, glutathione, reduced form; GSSG, glutathione disulfide; menadione-GSH conjugate, 2-methyl-3-glutathionyl-1 ,4-naphthoquinone ; HPLC, high-pressure liquid chromatograph...

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