The thermotropism and prototropism of ternary mixtures of ceramide C16, cholesterol and palmitic acid. An exploratory study

Mixtures of ceramides with other lipids in the presence of water are key components of the structure of the lipid matrix of the stratum corneum and are involved in lateral phase separation processes occurring in lipid membranes. Besides their structural role, ceramides are functional for cell signaling and trafficking. We elected, as our object of study, a mixture of N-hexadecanoylceroyl-D-erythro-sphyngosine. C16-Cer, with cholesterol, Ch, in a molar proportion 54:46 in excess water to which palmitic acid, PA, is added in varying amounts. The chosen C16-Cer:Ch proportion replicates the relative abundance of ceramides and cholesterol found in the stratum corneum lipid matrix. For each lipidic composition, we identify the phases in equilibrium and study the thermotropism of the system, using differential scanning calorimetry and temperature-dependent small and wide-angle X-ray powder diffraction. Since the molecular aggregation of the system and its mesoscopic properties are affected by the degree of protonation of the PA, we explore mixtures with several PA contents at two extreme pH values, 9.0 and 4.0. A specific C16-Cer:Ch:PA composition forms at pH 9.0 a lamellar crystalline aggregate, to which we attribute the stoichiometry C16-Cer(5)Ch(4)PA(2), that melts at 88-90 degrees C to give a H(II) phase. For pH values at which there is partial or total protonation of PA another L(C) C16-Cer:Ch (2:3) stoichiometric aggregate is observed, identical to that previously reported for C16-Cer:Ch mixtures (Souza et al., 2009, J. Phys. Chem. B, 113, 1367-1375), coexisting with a lamellar fluid phase. For pH 4.0 and 7.0, the existing lamellar liquid crystalline converts into a isotropic fluid phase at high temperatures. It is also found that the miscibility of PA in the C16-Cer:Ch mixture at pH 4.0 does not exceed ca. 18 mol%, but for pH 9.0 no free PA is detected at least until 60 mol%.