Abstract:
Polyunsaturated fatty acids (PUFAs) are essential components of biological membranes and of lipoproteins; they regulate a number of cellular and physiological processes, mostly by influencing cellular membrane lipid composition, metabolism and signal-transduction pathways, either in a tissue-specific way or by oxidation. The methylene group between two double bonds makes a fatty acid vulnerable to free radical-induced damage converting it to a polar hydroperoxide. This peroxidation is f tremendous importance for the transport of different nutrients through membranes and has a significant function as modulator of enzymes and as intermediate in biosynthetic processes. Moreover, peroxidation plays a central role in numerous pathological processes such as atherosclerosis and cancer. Two difficulties arise when separating oxidized lipids via HPLC: (1) the amphipathic nature of many membrane lipids makes peak resolution difficult; (2) most membrane lipids lack accessible spectral signals making detection difficult. Using a reverse phase HPLC system tosharpen the peak resolution, we report on an improved method for postcolumn detection of lipid hydroperoxides using diphenyl-1-pyrenylphosphine (DPPP) that no longer requires a heated reaction chamber. The reaction rate of DPPP with tent-butylhydroperoxide (tBuOOH) is measured by detecting the quantity of fluorescent DPPP-oxide, while the levels of two catalysts, pyridine and Fe+3, are adjusted.Key words: radical-mediated oxidation, lipid hydroperoxides, secondary products of lipid peroxidation, Biphenyl- l -pyrenylphosphine (DPPP), high pressure liquid chromatography (HPLC), tert butylhydroperoxide (tBu00H), pyridine, Fe+3.