Determination of primary and secondary lipid peroxidation products: Plasma lipid hydroperoxides and thiobarbituric acid reactive substances

Summary

Lipid peroxidation is a complex process whereby unsaturated lipid undergoes reaction with molecular oxygen to yield lipid hydroperoxides. In most situations involving biological samples the lipid hydroperoxides are degraded to a variety of products including alkanals, alkenals, hydroxyalkenals, ketones, alkanes. Although attack by singlet oxygen on unsaturated lipid has been shown to give hydroperoxides by a nonradical process, the vast majority of situations involving lipid peroxidation proceeds through a free radicalmediated chain reaction initiated by the abstraction of a hydrogen atom from the unsaturated lipid by a reactive free radical, followed by a complex sequence of propagation reactions. The involvement of free oxygen radicals in the pathology, of certain diseases explains the growing interest in the assay of lipid peroxides. Assay of polyunsaturated fatty acid degradation products is currently performed by measuring the so-called thiobarbituric acid-reactive substances, of which malondialdehyde is the best known. Because this assay is controversial, a second index of free radical attack would be useful to confirm the peroxidative process.

In this paper, we describe the determination of plasma lipid hydroperoxides and thiobarbituric acid reactive substances (TBARS). We proposed an improved enzymatic technique for assay of lipid hydroperoxides in biological fluids. The technique previously described by Heath and Tappel cannot be used in biological determinations. In fact, the presence of endogenous enzymes such as glutathione peroxidase and glutathione reductase in the sample interferes with the reaction and makes the results unreliable. Elimination of these endogenous enzymes by deproteinization before assaying for lipid hydroperoxides in the plasma gives simple, reliable, and reproducible measurements. The determination of TBARS is a widely used method for investigating overall lipid peroxidation. The TBARS assay is accomplished by mixing the sample with a TBA reagent in acid medium and placing in a boiling water bath. After extracting the TBARS by organic solvent, their optical density or fluorescence intensity is measured. TBARS assay detects both preexisting malondialdehyde (MDA) plus whatever substances give rise to MDA during the assay. Lipid hydroperoxides can decompose during heating in the presence of acid and metals and give rise to MDA and other aldehydes capable of interacting with TBA during the assay. Many researchers use this assay in their laboratories but the procedures used vary; the variability could arise from differences in sample volume, acid type, pH of medium, heating duration, blank undertaking and detection conditions. These variations render impossible the comparison of results between laboratories. The use of an assay kit for plasma TBARS assay would enable the method to be standardized. The results reported here indicate that the MDA-kit manufactured by SOBIODA (GRENOBLE, France) complies with criteria of good analytical practices. However, we concluded that no single method sufficiently meets analytical standards in all application to make it the choice (let alone universal) one. We thus emphasize the need to integrate different analytical approaches in the assessment of oxidant stress in vivo.