MULTIPLE MECHANISMS FOR INHIBITION OF LOW-DENSITY-LIPOPROTEIN OXIDATION BY NOVEL CYCLIC NITRONE SPIN TRAPS

Oxidation of low density Lipoproteins (LDL) may be a critical atheroge nic event owing to the diverse array of biologic effects attributed to modified LDL, Recently, we and others have demonstrated that the lipo philic nitrone spin trap alpha-phenyl-N-tert-butyl nitrone (PBN) can i nhibit Cu2+-dependent LDL oxidation while the related, more hydrophili c analog alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone is ineffective . Because the inhibitory activity of PBN is relatively weak as compare d to hydrophobic phenolic antioxidants, we have synthesized a number o f cyclic analogues of PBN that encompass a wide range of hydrophobicit y and examined their ability to inhibit LDL oxidation in vitro. Format ion of a six-membered ring by a bond formed from one methyl of the ter t-butyl group to the aromatic ring yielded MDL 101,002, which was 3- a nd 24-fold more active than PBN against Cu2+ and 2,2'-azobis-2-amidino propane hydrochloride-dependent oxidation, respectively. The effect of aromatic substituents was examined and, in general, activity positive ly correlated with hydrophobicity, particularly with Cu2+. Electron sp in resonance (ESR) spectroscopy demonstrated that the PBN adduct in ox idized LDL is composed of a mobile component (exposed to the LDL aqueo us phase) and an immobilized component, localized in the lipid-protein interface or in the bulk lipid. The most active cyclic nitrones exhib ited only highly immobilized adducts, suggesting they are buried withi n the particle. Studies with MDL 105,185 (which is a chloro-substitute d nitrone containing a seven-membered ring rather than six-membered as for MDL 101,002) demonstrated radical trapping in both the lipid and apoprotein fractions. Compounds in which a spirocyclohexyl ring was su bstituted for the gem-dimethyl methylene (MDL 102,832 and 101,694) for med hydrophobic Cu2+ complexes that were observed in the lipid fractio n by ESR. This result was confirmed by fractionation of LDL oxidation reaction mixtures and spectrophotometric quantitation of associated Cu 2+. Th, ability to bind Cu2+ was dependent upon the presence of the sp irocyclohexyl ring. These data demonstrate that cyclic nitrones can in hibit LDL oxidation at exceedingly low concentrations by multiple mech anisms: 1) trapping of lipid-derived radicals, 2) trapping of apoprote in B-derived radicals, and 3) binding of Cu2+ ions. It is suggested th at this new class of highly potent spin traps may be used as effective radical traps in free radical biology and medicine.