Thermotropic phase behavior of milk sphingomyelin and role of cholesterol in the formation of the liquid ordered phase examined using SR-XRD and DSC.

Sphingomyelin (SM) and cholesterol are major lipid components of biological membranes involved in the formation of ordered domains. In this study, we investigated the biophysical properties of milk-SM bilayers and determined the effect of cholesterol. The thermotropic phase behaviours of milk-SM and milk-SM/cholesterol mixtures were characterized using differential scanning calorimetry (DSC) and high flux synchrotron radiation X-ray diffraction (SR-XRD). The melting phase transition temperature determined for fully hydrated milk-SM bilayers was Tm  = 34.3 ± 0.1 °C. The thermotropic phase behavior of milk-SM is complex, reflecting the mixture of different molecular species. Structural reorganisations successively occurring on heating of fully hydrated milk-SM bilayers were interpreted as follows: i) melting of C16:0-SM, ii) conversion of long and saturated SM species from fully interdigitated (Lβ1 ) to mixed interdigitated (Lβ2 ) lamellar structures evidencing gel phase polymorphism and then iii) transition to the fluid liquid-crystal Lα phase. We demonstrated that cholesterol modulates the physical properties of milk-SM bilayers and that building up of the lamellar liquid-ordered Lo phase is completed for 33 mol% of cholesterol. The ordering effect of cholesterol on milk-SM bilayers and the temperature-independent behavior of the Lo phase formed by milk-SM/cholesterol complexes were characterized. The findings of this work will contribute in a better understanding of the biological functions exerted by the milk-SM as a function of its phase state and interactions with cholesterol (e.g. hypocholesterolemic effect).