ACTIVATION OF HUMAN POLYMORPHONUCLEAR LEUKOCYTES BY PRODUCTS DERIVED FROM THE PEROXIDATION OF HUMAN RED-BLOOD-CELL MEMBRANES

Oxidation of red blood cell (RBC) ghost preparations initiated by tert -butyl hydroperoxide (tBuOOH) was employed to explore the formation of lipid products derived from endogenous phospholipids that specificall y expressed biological activity toward the human polymorphonuclear leu kocyte (PMN). Common measure of lipid peroxidation, thiobarbituric aci d-reactive substances (TBARS) and the increased absorbance at 235 nm c onsistent with the formation of conjugated dienes, was observed follow ing a 90-min incubation of RBC ghosts with tBuOOH. Saponification of p hospholipids and separation of the resultant fatty acids by RP-HPLC pe rmitted direct mass spectrometric analysis of oxidized fatty acids. In dividual HPLC fractions were assayed for their ability to increase int racellular free calcium ion concentrations in human PMN to guide struc tural investigations. Two fractions were found to contain biologically active components, and tandem mass spectrometric analysis of the abun dant ions observed in these fractions resulted in the characterization of several oxidized polyunsaturated fatty acids derived from arachido nic and linoleic acids. The major components in these fractions includ ed 5-hydroxyeicosatetraenoic acid (5-HETE) and 5-hydroperoxyeicosatetr aenoic acid (5-HpETE). The dose-dependent increases in intracellular c alcium in the neutrophil using synthetic 5(rac)METE, Ei(rac)-HpETE, an d 5-oxo-ETE were found to have EC50's Of 250, 6, and 3 nM, respectivel y. The quantity of 5-oxygenated arachidonate components present in oxi dized RBC was consistent with the observed biological response elicite d by fractions A and B. This study suggests that 5-HETE and 5-HpETE ar e abundant products of lipid peroxidation of cellular membranes and th at these racemic products possess significant biological activity. Suc h compounds could play important roles as mediators of the cellular re sponse to toxicologic stimuli that generate free radical species.