Ferrous iron mediated oxidation of arachidonic acid: studies employing nitroblue tetrazolium (NBT)

The oxidation of arachidonic acid by ferrous sulfate provides a useful model to study the role of iron in lipid oxidation reactions. We have employed nitroblue tetrazolium (NBT) in the present investigation to evaluate the mechanism of this reaction. In the presence of arachidonic acid, Fe +++, and O2, the yellow dye NBT was rapidly reduced to the blue form, NBTH2. The molar ratio of arachidonic acid to Fe++ in this rapid reaction was 1:1, showing an interaction of one fatty acid molecule per iron molecule. Approximately one molecule of NBT was reduced per four molecules of arachidonic acid and Fe++. Reduction of NBT was accompanied by oxidation of Fe++ to Fe+++, suggesting the transfer of four electrons from the Fe++ to NBT to reduce the dye. Arachidonic acid was found to be unchanged when extracted at the end of the reaction, indicating formation of a complex that could dissociate leaving intact arachidonic acid. Evidence for the presence of such a complex which slowly dissociates during the reaction was obtained after longer incubations with small amounts of arachidonic acid. NBT reduction was not inhibited by agents which hydrolyze superoxide, by catalase or by agents which trap hydroxy radicals. We, therefore, propose a model in which NBT traps a radical generated on the arachidonic acid molecule. The proposed model suggests mechanisms for other fatty acid oxidation reactions such as prostaglandin and hydroperoxy fatty acid synthesis.