Lipid peroxidation products inhibit Glutathione based protection of Oxymyoglobin; a Model system

This study was performed as an in vitro model system to investigate the antioxidant role of glutathione in protecting oxymyoglobin from oxidation in competition with its role in detoxifying intracellular toxins such as 4-Hydroxynonenal (HNE). Briefly, the model consisted of a control solution (0.15mM oxymyoglobin solution), a positive control (0.2mM reduced glutathione (GSH) and glutathione peroxidase activity) and test samples (added HNE at 0.1, 0.2 and 0.4, 0.6 and 0.8mM). All samples were incubated for 300 minutes at pH 5.6 and 25C. Rapid oxidation of oxymyoglobin occurred in the control solution, while in the positive control, strong antioxidant activity was observed over the entire incubation period. Antioxidant activity was observed up to 240 mins in the presence of 0.1mM HNE, while in the presence of 0.2-0.6mM HNE antioxidant activity was observed for no more than 60 mins before the GSH antioxidant activity was apparently inactivated. A complete loss was observed at HNE concentrations of 0.8mM and above. At molar ratios of HNE to GSH greater than 1:1 the antioxidant activity of GSH seems to be inhibited. The level of HNE in meat are likely to exceed this ratio, thus it seems doubtful that GSH would be an important antioxidant in post mortem muscle. Introduction Browning of meat during retail display costs the US meat industry $1 billion each year (Williams et al., 1992). Browning of meat is caused by the oxidation of the red pigment, oxymyoglobin, to metmyoglobin. This brown metmyoglobin form is visually undesirable and consumers will quickly discriminate based on visual appearance. This oxidation can be slowed by antioxidants, such as glutathione (Tang et al., 2003), which occur naturally in muscle tissue. Glutathione is an intracellular tripeptide with a free sulfhydryl group and is involved in the detoxification of hydrogen peroxide and other organic peroxides, catalysed by the enzyme glutathione peroxidase (Stryer, 1975). Hydrogen peroxides are potent oxidants of lipids and proteins such as myoglobin (Morey et al., 1973), glutathione plays an antioxidant role by removing these cytotoxic species. Glutathione also plays an important role in detoxification of intracellular toxins by forming conjugates with its free sulfhydryl group. 4-hydroxynonenal (HNE) is a cytotoxic product of lipid peroxidation derived from omega -6polyunsaturated fatty acids such as arachidonic and linoleic acid (Esterbauer et al., 1991). HNE is known to covalently bind to oxymyoglobin, readily forming metmyoglobin (Faustman et al., 1999), however this can be prevented by glutathione which forms conjugates with HNE (Alin et al., 1985; Esterbauer et al., 1991). Thus accelerated formation of HNE will rapidly deplete intracellular glutathione concentrations (Siems & Grune, 2003). Therefore the interaction of glutathione with cellular toxins such as HNE, is likely to impact on its antioxidant activities. This study tested the hypothesis that glutathione will not protect oxymyoglobin from oxidation in the presence of HNE. Materials and methods This experiment was performed as an in-vitro model system. The model consisted of 0.15mM oxymyoglobin (prepared from commercial metmyoglobin), 0.4mM EDTA, 1 unit glutathione peroxidase, 0.2mM reduced glutathione (GSH) and 0.1, 0.2, 0.4, 0.6 and 0.8mM HNE. Controls contained ethanol (in the same amount used to deliver HNE) and oxymyoglobin. The model had a total volume of 1ml and was buffered to pH 5.6 (sodium citrate), to represent the conditions of post-mortem muscle. HNE and GSH were pre-incubated at 25C for 90 minutes before oxymyoglobin was added, after which the rate of formation of metmyoglobin was monitored spectrophotometrically over 5 hours at 25C. The percentage metmyoglobin was calculated using the revised krzywicki method (Tang et al., 2004). GSH was replaced by 0.2mM N-