Phase equilibria of cholesterol/dipalmitoylphosphatidylcholine mixtures: deuterium nuclear magnetic resonance and differential scanning calorimetry

Abstract

Deuterium nuclear magnetic resonance spectroscopy and differential scanning calorimetry are used to map the phase boundaries of mixtures of cholesterol and chain-perdeuteriated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine at concentrations from 0 to 25 mol % cholesterol. Three distinct phases can be identified: the L.alpha. or liquid-crystalline phase, the gel phase, and a high cholesterol concentration phase, which we call the .beta. phase. The liquid-crystalline phase is characterized by highly flexible phospholipid chains with rapid axially symmetric reorientation; the gel phase has much more rigid lipid chains, and the motions are no longer axially symmetric on the 2H NMR time scale; the .beta. phase is characterized by highly ordered (rigid) chains and rapid axially symmetric reorientation. In addition, we identify three regions of two-phase coexistence. The first of these is a narrow L.alpha./gel-phase coexistence region lying between 0 and about 6 mol % cholesterol at temperatures just below the chain-melting transition of the pure phospholipid/water dispersions, at 37.75.degree. C. The dramatic changes in the 2H NMR line shape which occur on passing through the phase transition are used to map out the boundaries of this narrow two-phase region. The boundaries of the second two-phase region are determined by 2H NMR difference spectroscopy, one boundary lying near 7.5 mol % cholesterol and running from 37 down to at least 30.degree. C; the other boundary lies near 22 mol % cholesterol and covers the same temperature range. Within this region, the gel and .beta. phases coexist. As the temperature is lowered below about 30.degree. C, the phospholipid motions reach the intermediate time scale regime of 2H NMR so that spectral subtractions become difficult and unreliable. The third two-phase region lies above 37.degree. C, beginning at a eutectic point somewhere between 7.5 and 10 mol % cholesterol and ending at about 20 mol %. In this region, the L.alpha. and .beta. phases are in equilibrium. The boundaries for this region are inferred from differential scanning calorimetry traces, for the boundary between the L.alpha.- and the two-phase region, and from a dramatic sharpening of the NMR peaks on crossing the boundary between the two-phase region and the .beta.-phase region. In this region, the technique of difference spectroscopy fails, presumably because the diffusion rate in both the L.alpha.- and .beta.-phase domains is so rapid that phospholipid molecules echange rapidly between domains on the experimental time scale.