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 atherogenic event owing to the diverse array of biologic effects attributed to modified LDL. Recently, we and others have demonstrated that the lipophilic nitrone spin trap alpha-phenyl-N-tert-butyl nitrone (PBN) can inhibit Cu(2+)-dependent LDL oxidation while the related, more hydrophilic analog alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone is ineffective. Because the inhibitory activity of PBN is relatively weak as compared to hydrophobic phenolic antioxidants, we have synthesized a number of cyclic analogues of PBN that encompass a wide range of hydrophobicity and examined their ability to inhibit LDL oxidation in vitro. Formation of a six-membered ring by a bond formed from one methyl of the tert-butyl group to the aromatic ring yielded MDL 101,002, which was 3- and 24-fold more active than PBN against Cu2+ and 2,2'-azobis-2-amidinopropane hydrochloride-dependent oxidation, respectively. The effect of aromatic substituents was examined and, in general, activity positively correlated with hydrophobicity, particularly with Cu2+. Electron spin resonance (ESR) spectroscopy demonstrated that the PBN adduct in oxidized LDL is composed of a mobile component (exposed to the LDL aqueous phase) and an immobilized component, localized in the lipid-protein interface or in the bulk lipid. The most active cyclic nitrones exhibited only highly immobilized adducts, suggesting they are buried within the particle. Studies with MDL 105,185 (which is a chloro-substituted 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 substituted for the gem-dimethyl methylene (MDL 102,832 and 101,694) formed hydrophobic Cu2+ complexes that were observed in the lipid fraction by ESR. This result was confirmed by fractionation of LDL oxidation reaction mixtures and spectrophotometric quantitation of associated Cu2+. The ability to bind Cu2+ was dependent upon the presence of the spirocyclohexyl ring. These data demonstrate that cyclic nitrones can inhibit LDL oxidation at exceedingly low concentrations by multiple mechanisms: 1) trapping of lipid-derived radicals, 2) trapping of apoprotein B-derived radicals, and 3) binding of Cu2+ ions. It is suggested that this new class of highly potent spin traps may be used as effective radical traps in free radical biology and medicine.
Marion Merrell Dow Research Institute, Cincinnati, Ohio 45215.
C E
CE
Thomas
D F
DF
Ohlweiler
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B
Kalyanaraman
D003300:Copper / Q000737:chemistry / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D008077:Lipoproteins, LDL / Q000097:blood / Q000187:drug effects*; D009589:Nitrogen Oxides / Q000494:pharmacology*; D010084:Oxidation-Reduction; D011485:Protein Binding; D013113:Spin Labels; D013329:Structure-Activity Relationship
D008077:Lipoproteins, LDL; D009589:Nitrogen Oxides; D013113:Spin Labels; D003300:Copper