Dynamics of iron‐ascorbate‐induced lipid peroxidation in charged and uncharged phospholipid vesicles

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Abstract
Peroxidation of egg yolk phosphatidylcholine (egg PC) liposomes was induced by addition of ascorbic acid (AsA) and Fe(II) in the presence of a trace of autoxidized egg PC (PC−OOH), but not in the absence of PC−OOH. PC−OOH was degraded upon addition of AsA and Fe(II) but not of either one alone. The results suggest that PC−OOH is necessary to initiate lipid peroxidation by AsA/Fe(II). AsA oxidation in the bulk water phase was also associated with an increase in lipid peroxidation by AsA/Fe(II) in the presence of PC−OOH, but not in the absence of PC−OOH. Furthermore, the spin probe 12‐NS [12‐(N‐oxyl‐4,4′‐dimethyloxazolidin‐2‐yl)stearic acid], which labels the hydrophobic region of dimyristoyl phosphatidylcholine (DMPC) liposomal membranes, was degraded upon addition of AsA and Fe(II) in the presence of PC−OOH, but not in the absence of PC−OOH. These results indicate that the “induction message” that is associated with decreases of PC−OOH and AsA in the initiation step of lipid peroxidation must be transferred from the membrane surface to the inner hydrophobic membrane region. AsA in the bulk phase was oxidized faster and more extensively upon its addition together with Fe(II) to egg PC liposomes than to DMPC liposomes, though the initial content of PC−OOH in the former was 5–10 times lower than in the latter. This suggests that, in egg PC liposomes, the OOH‐groups of new PC−OOH generated in the inner membrane regions must become accessible from the surface, enabling reaction with AsA/Fe(II) which in turn would result in an extensive decrease in AsA. By contrast, in DMPC liposomes, that do not generate PC−OOH, AsA is only oxidized slightly in connection with the degradation of the PC−OOH initially present. The effect of surface charges on the membrane surface was also studied to obtain further information on the initiation step of lipid peroxidation. The rate of lipid peroxidation by AsA/Fe(II) or Fe(III) decreased in the order, egg PC liposomes ≫negatively charged egg PC liposomes containing dicetylphosphate>positively charge egg PC liposomes containing stearylamine. The rate of associated AsA oxidation was in the order, egg PC liposomes≫egg PC/stearylamine liposomes>egg PC/dicetylphosphate liposomes. However, in DMPC liposomes that do not generate PC−OOH, the rates of AsA oxidation associated with the reductive cleavage of PC−OOH by AsA/Fe(II) and coupled with the reduction of Fe(III) to Fe(II) were in the order, DMPC liposomes =DMPC/stearylamine liposomes≫DMPC/dicetylphosphate liposomes. These differences in the rates of lipid peroxidation, depending on differences in membrane charge, are discussed in relation to two properties of AsA: (i) its antioxidant property through trapping of lipid radicals and (ii) its prooxidant properties (a) by being an effective iron chelator thus altering the reactivity of iron with oxygen and peroxides and (b) by being an iron reductant and providing a source of Fe(II).