A deuterium NMR and steady-state fluorescence anisotropy study of the effects of cholesterol on the lipid membrane-disordering actions of ethanol

Abstract

We examined the effects of cholesterol on the membrane-disordering action of ethanol by using deuterium nuclear magnetic resonance ( 2H-NMR) and fluorescence spectroscopy. Specifically, the effects of ethanol were measured on the 2H-NMR spectra of di (perdeuteropalmitoyl)phosphatidylcholine (DPPC-d 62) and on the steady-state emission anisotropy of diphenylhexatriene (DPH) incorporated into hydrated egg phosphatidylcholine (eggPC)/cholesterol dispersions. Analysis of the 2H-NMR spectra of DPPC-d 62 incorporated into eggPC liposomes showed that the addition of cholesterol up to 30 mol% enhanced the ability of ethanol to disorder methylene groups all along the phospholipid acyl chains. This effect was somewhat greater toward the terminal methyl groups. However, above 30 mol% cholesterol, the bilayer-disordering action of ethanol on both the upper and lower portions of the acyl chains decreased to an apparent constant change up to the highest cholesterol content examined (50 mol%). Analysis of the fluorescence anisotropy of DPH, on the other hand, suggested that cholesterol attenuated the ability of ethanol to disorder the bilayers, which is in agreement with a previous EPR study [Chin and Goldstein, Mol Pharmacot 19: 425–431,1981]. Re-analysis of our previous fluorescence anisotropy results with DPH incorporated into dispersions of brain-lipid extracts as a percent change [Johnson et al., Mol Phannacol 15: 739–746, 1979] indicated that the chemical composition of the lipid bilayers also affects the apparent ability of cholesterol to modulate the membrane-disordering action of ethanol, because the addition of cholesterol to brain-lipid extracts had no significant effect on the membrane-disordering action of ethanol. Given the greater likelihood that the 2H-NMR probes accurately monitor bulk phospholipid properties, some caution is required in the analysis of the membrane-disordering actions of drugs using EPR and fluorescence spectroscopy.