The antioxidant effect of spermine NONOate in human low-density lipoprotein

The nitric oxide (*NO) donor N-[4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl]-1,3- propanediamine, also referred to as Spermine NONOate (SNN), inhibited the copper(II) sulfate-initiated oxidative modification of human low-density lipoprotein (LDL) as measured by the formation of thiobarbituric acid reactive substances, conjugated diene formation, and changes in electrophoretic mobility. The presence of the nitronyl nitroxide 1-oxy-2-[p-(trimethylammoniumyl)phenyl]-4,4,5,5- tetramethylimadazoline 3-oxide, a scavenger of *NO, antagonized the inhibitory activity of SNN. The inhibition of copper-dependent LDL oxidation had a nonlinear dependence on SNN concentration. Low concentrations of SNN ( < 4 microM) were only poorly effective at inhibiting LDL oxidation; however, a dramatic enhancement of inhibition occurred above 4 microM SNN. This behavior was qualitatively different from that of butylated hydroxytoluene, a phenolic chain-breaking antioxidant, which exhibited an approximately linear concentration dependence in this system. Addition of 13[S-(E,Z)]-hydroperoxy-9,11-octadecadienoic acid, a lipid hydroperoxide, to LDL diminished the antioxidant effect of 4 and 8 microM SNN, but not that of 12 microM SNN. SNN inhibited the depletion of alpha-tocopherol during both copper-dependent and 2,2'-azobis(2-amidinopropane)-dependent oxidation of LDL. We propose that a direct reaction is occurring between NO and the lipid peroxyl radical, forming a lipid-nitroso adduct. Formation of this product would not only remove the lipid peroxyl radical, thus preventing chain propagation, but would also prohibit the regeneration of lipid hydroperoxide, thereby stopping further transition metal ion-dependent initiation. The difference in the kinetic behavior between SNN and conventional antioxidants can be explained by this effect.