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Reactive oxygen species, probably hydroxyl radicals (OH.), have been suggested to be generated during arachidonic acid (AA) metabolism and, once released, these species can modify the rate and extent of various reactions involved in AA metabolism. We have studied this phenomenon in washed human platelets. OH. generation was quantitated using 14C-benzoic acid as a specific trap in a continuous ionization chamber system. Resting platelets did not produce any detectable signal, whereas addition of AA resulted in gradual OH. production with peak values detected at approximately 20 min. Similar studies conducted under nitrogen or after boiling the platelets almost abolished OH. generation. Aspirin had no significant effect, whereas 5,8,11,14-eicosatetraynoic acid decreased the signal by greater than 90%, thus suggesting that OH. is produced primarily through the lipoxygenase pathway. Superoxide dismutase (SOD) and catalase had no effect and, as expected, phenol and mannitol decreased OH. production considerably, by greater than 50% and 90%, respectively. Azide and cyanide also reduced the OH. generation by about two thirds. We conclude that OH. is generated during AA metabolism by human platelets. It is primarily produced via the lipoxygenase pathway and may require a heme-dependent peroxidase. This highly reactive oxidant may play an important role in normal and abnormal hemostasis.

Separation of all major cyclooxygenase and lipoxygenase metabolites of arachidonic acid were obtained by high performance liquid chromatography (HPLC). A C18 reverse-phase column was used in ion suppression mode to separate underivatized metabolites of arachidonic acid isolated from human and rabbit platelets. The metabolites were monitored by measuring radioactivity or ultraviolet light absorption at 192 nm (absorption by double bonds). Comparisons of TLC and HPLC separations demonstrated that the HPLC separation of metabolites of [1-14C]arachidonic acid was quantitative. HPLC also resolved several metabolites that were not detected by scanning of TLC separations.