Chemistry and Physics of Lipids 164 (2011) 643–653 Contents lists available at ScienceDirect Chemistry and Physics of Lipids j ourna l ho me p ag e : www.elsevier.com/locate/chemphyslip The thermotropism and prototropism of ternary mixtures of ceramide C16, cholesterol and palmitic acid. An exploratory study Sofia L. Souza a , Joana Valério a , Sérgio S. Funari b , Eurico Melo a,c,∗ a Instituto de Tecnologia Química e Biológica – UNL, Av. da República-EAN, 2780-157 Oeiras, Portugal b Hasylab, DESY, Notkestrasse 85, D-22607 Hamburg, Germany c Instituto Superior Técnico, Technical University of Lisbon, Av. Rovisco Pais, 1049-001 Lisboa, Portugal a r t i c l e i n f o Article history: Received 24 March 2011 Received in revised form 1 June 2011 Accepted 15 June 2011 Available online 22 June 2011 Keywords: Differential scanning calorimetry Fatty acids Ceramide SAXS–WAXS Stratum corneum pH a b s t r a c t 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 traf- ficking. 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 scan- ning 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 pro- tonation 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 ◦ 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%. © 2011 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Most membrane-forming lipids used by nature in the building of cellular membranes or other biological structures are fluid at the physiological temperature or, at least, the mixtures in which they are found in nature are fluid. The notable exception is the lipid matrix of the stratum corneum whose properties rely on the rigidity conferred by a particular mixture of ceramides and hydroxylated ceramides, together with cholesterol and saturated fatty acids. Ceramides by themselves form lamellar crystalline phases that melt to give lamellar liquid crystalline phases at quite high temperatures Abbreviations: C16-Cer, N-palmitoyl-d-erythro-sphingosine; Ch, cholesterol; PA, palmitic acid; SC, stratum corneum. ∗ Corresponding author at: Instituto de Tecnologia Química e Biológica, Apartado 127, P-2780 Oeiras, Portugal. Tel.: +351 214469724. E-mail address: eurico@itqb.unl.pt (E. Melo). (Shah et al., 1995; Souza et al., 2009). This happens even for rela- tively short-chain ceramides because of the strong hydrogen-bonds connecting the headgroups and the network formed between the headgroups and the adjacent water layer (Moore et al., 1997). The lipidic mixture found in the stratum corneum is, at least in part, laterally organized in a crystalline structure (White et al., 1988) that has been referred as being important to the particular prop- erties of this system (Elias, 2005; Madison, 2003). The presence of a quite large molar percentage of fatty acid (ca. 18%) and the fact that the stratum corneum interfaces a region with pH 7.4 and pH ca. 5.5, raised the interest of several researchers on how the physical–chemical characteristics of the lipid matrix is modulated by pH (Mimeault and Bonenfant, 2002; Kitson et al., 1994; Kitagawa et al., 1995; Bouwstra et al., 2000). Ceramides are also known to be involved in cell signaling (Stancevic and Kolesnick, 2010) and are a potent inducer of apo- ptosis (Hannun and Obeid, 1995). Although the mechanism is unknown, recent findings suggest that the biological functions 0009-3084/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2011.06.009