Characterization of neutrophil-dependent initiation of lipid peroxidation of endogenous plasma lipids.

Neutrophils (1 × 10⁶/mL) isolated from healthy subjects (PMN) were incubated at 37°C in HBSS supplemented with DTPA (100 μM, pH 7.0) and fresh human plasma (50%, vol/vol). Cells were activated by the addition of phorbol myristate acetate (200 nM) and then incubated for 2 hours (Complete System). The contents of 9-H(P)ODE and 9-H(P)ETE formed within endogenous plasma lipids were then determined by LC/ESI/MS/MS as described in “Materials and methods.” Additions or deletions to the Complete System were as indicated. Final concentrations of additions to the Complete System were 30 nM human MPO, 1 mM NaN₃, 300 nM catalase, 300 nM heat-inactivated catalase, 100 μM methionine, 100 μM ascorbate, and 10 μg/mL SOD. Data represent the mean ± SD of 3 independent experiments.