A new high-temperature transition of crystalline cholesterol in mixtures with phosphatidylserine

Phosphatidylserine and cholesterol are two major components of the cytoplas mic leaflet of the plasma membrane. The arrangement of cholesterol is marke dly affected by the presence of phosphaticlylserine in model membranes. At relatively low mol fractions of cholesterol in phosphaticlylserine, compare d with other phospholipids, cholesterol crystallites are formed that exhibi t both thermotropic phase transitions as well as diffraction of x-rays. In the present study we have observed and characterized a novel thermotropic t ransition occurring in mixtures of phosphatidylserine and cholesterol. This new transition is observed at 96 degreesC by differential scanning calorim etry (DSC), using a heating scan rate of 2 degreesC/min. Observation of the transition requires that the hydrated lipid mixture be incubated for sever al days, depending on the temperature of incubation. The rate of formation of the material exhibiting a transition at 96 degreesC is more rapid at hig her incubation temperatures. At 37 degreesC the half-time of conversion is similar to7 days. Concomitant with the appearance of the 96 degreesC peak t he previously known transitions of cholesterol, occurring at -38 degreesC a nd 75 degreesC on heating scans of freshly prepared suspensions, disappear. These two transitions correspond to the polymorphic transition of anhydrou s cholesterol and to the dehydration of cholesterol monohydrate, respective ly. The loss of the 75 degreesC peak takes a longer time than that of the 3 8 degreesC peak, indicating that anhydrous cholesterol first gets hydrated to the monohydrate form exhibiting a transition at 75 degreesC and subseque ntly is converted by additional time of incubation to an altered form of th e monohydrate, showing a phase transition at 96 degreesC. After several wee ks of incubation at 37 degreesC, only the form with a phase transition at 9 6 degreesC remains. If such a sample undergoes several successive heating a nd cooling cycles, the 96 degreesC peak disappears and the 38 degreesC tran sition reappears on heating. For samples of 1-paimitoyl-2-oleoyl phosphatic lylserine or of 1-stearoyl-2-oleoyl phosphaticlylserine having mol fraction s of cholesterol between 0.4 and 0.7, the 38 degreesC transition that reapp ears after the melting of the 96 degreesC component generally has the same enthalpy as do freshly prepared samples. This demonstrates that, at least f or these samples, the amount of anhydrous cholesterol crystallites formed i s indeed a property of the lipid mixture. We have also examined variations in the method of preparation of the sample and find similar behavior in all cases, although there are quantitative differences. The 96 degreesC transi tion is partially reversible on cooling and reheating. This transition is a lso scan rate dependent, indicating that it is, at least in part, kinetical ly determined. The enthalpy of the 96 degreesC transition, after incubation of the sample for 3 weeks at 37 degreesC is dependent on the ratio of chol esterol to 1-palmitoyl-2-oleoyl phosphaticlylserine or to 1-stearoyl-2oleoy l phosphatidyl seri ne, with the enthalpy per mole cholesterol increasing b etween cholesterol mol fractions of 0.2 and 0.5. Dimyristoyl phosphatidylse rine at a 1:1 molar ratio with cholesterol, after incubation at 37 degreesC , exhibits a transition at 95 degreesC that reverses on cooling at 44 degre esC, instead of 60 degreesC, as observed with either 1-palmitoyl-2-oleoyl p hosphaticlylserine or 1-stearoyl-2-oleoyl phosphaticlylserine. These findings along with the essential absence of the 96 degreesC transiti on in pure cholesterol or in cholesterol/phosphatidylcholine mixtures, indi cates that the phospholipid affects the characteristics of the transition, and therefore the cholesterol crystallites must be in direct contact with t he phospholipid and are not simply in the form of pure crystals of choleste rol. These observations are particularly important in view of recent observ ations of the presence of cholesterol crystals in biological systems.