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.
Thomas
C E
CE
Marion Merrell Dow Research Institute, Cincinnati, Ohio 45215.
Ohlweiler
D F
DF
Kalyanaraman
B
B
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