ELECTROSPRAY MASS-SPECTROMETRIC ANALYSIS OF 5-HYDROPEROXY AND 5-HYDROXYEICOSATETRAENOIC ACIDS GENERATED BY LIPID-PEROXIDATION OF RED-BLOOD-CELL GHOST PHOSPHOLIPIDS

Recent evidence suggests that generation of hydroxyl radicals in the p resence of lipid membranes can lead to oxidation of arachidonic acid e sterified to glycerophospholipids and the production of compounds isom eric to prostaglandins, thromboxanes, and leukotrienes. Liquid chromat ography tandem mass spectrometry and multiple reaction monitoring were employed to quantitate the production of 5-hydroxyeicosatetraenoic ac id (5-HETE), 5-hydroperoxyeicosatetraenoic acid (5-HPETE), and 5-oxo-e icosatetraenoic acid (5-oxo-ETE) in red blood cells ghosts treated wit h t-butylhydroperoxide (tBuOOH). Untreated red blood cell ghosts were found to contain low, but measurable quantities of these three 5-oxyge nated eicosanoids as phospholipid esters. Following treatment, there w as approximately a 53- and 22.5-fold increase in 5-HETE and 5-HPETE, r espectively, and an 8.5-fold increase in 5-oxo-ETE. The formation of t hese compounds was inhibited nearly 90% by the antioxidants butylated hydroxytoluene, ascorbic acid, and resveratrol providing further evide nce for free radical mediated oxidation of arachidonic acid. This anal ytical protocol provided sufficient sensitivity for detection of these compounds in studies in which previous analysis by high-pressure liqu id chromatography with UV detection failed to detect their presence. T hese results reveal that the biologically active eicosanoids 5-HETE, 5 -HPETE, and 5-oxo-ETE are formed esterified to phospholipids following exposure of cellular membranes to reactive oxygen species and free ra dicals in a model system where intracellular antioxidant mechanisms we re depleted. (C) 1998 American Society for Mass Spectrometry.