A deuterium NMR and steady-state fluorescence anisotropy study of the effects of cholesterol on the lipid membrane-disordering actions of ethanol.
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-d62) 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-d62 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 Pharmacol 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 Pharmacol 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.
Johnson
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DA
Division of Biomedical Sciences, University of California, Riverside 92521-0121.
Valenzuela
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CF
Zidovetzki
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R
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000737:chemistry; D002784:Cholesterol / Q000494:pharmacology*; D004161:Diphenylhexatriene / Q000737:chemistry; D004347:Drug Interactions; D000431:Ethanol / Q000494:pharmacology*; D005454:Fluorescence Polarization; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes; D009682:Magnetic Resonance Spectroscopy; D010743:Phospholipids / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D008051:Lipid Bilayers; D008081:Liposomes; D010743:Phospholipids; D004161:Diphenylhexatriene; D015060:1,2-Dipalmitoylphosphatidylcholine; D000431:Ethanol; D002784:Cholesterol