The thiobarbituric acid assay reflects susceptibility to oxygen induced lipid peroxidation in vitro rather than levels of lipid hydroperoxides in vivo: a methodological approach.

Although the hypothesis of oxidative stress as a pathogenetic factor of neurodegenerative diseases became a matter of interest recently, direct evidence supporting this hypothesis is rare. The most prominent assay being currently used as an index for lipid peroxidation products in vivo is the thiobarbituric acid assay. Thiobarbituric acid reactive substances are mainly formed during the decomposition of lipid hydroperoxides in vitro. It is questionable however, that all species detectable with thiobarbituric acid are derived from in vivo preformed lipid hydroperoxides. These studies were undertaken to investigate the influence of autoxidation reactions on colour production during the acid heating stage of the assay. If driven aerobically, more than 90% of thiobarbituric acid reactive substances are newly generated in vitro during incubation at 95 degrees C for 75 min. This process can be enhanced by addition of ferric iron. Chain breaking antioxidants like butylated hydroxytoluene decrease colour formation in the absence or in the presence of iron. If driven anaerobically under argon, colour formation was only 10% of aerobically heated homogenates or lipid extracts of human brain tissue. These results may indicate that measurement of thiobarbituric acid reactive substances under the aerobic conditions described here reflects to a great extent the susceptibility of brain tissue or lipids to oxygen-induced formation of lipid hydroperoxides in vitro rather than degradation products of in vivo performed lipid hydroperoxides.