Phase separation in ceramide[NP] containing lipid model membranes: neutron diffraction and solid-state NMR

Abstract

The stratum corneum is the outermost layer of the skin and protects the organism against external influences as well as water loss. It consists of corneocytes embedded in a mixture of ceramides, fatty acids, and cholesterol in a molar ratio of roughly 1 : 1 : 1. The unique structural and compositional arrangement of these stratum corneum lipids is responsible for the skin barrier properties. Many studies investigated the organization of these barrier lipids and, in particular, the exact conformation of ceramides. However, so far no consensus has been reached. In this study, we investigate a model system comprised of N-(non-hydroxy-tetracosanoyl)-phytosphingosine/cholesterol/tetracosanoic acid (CER[NP]-C24/CHOL/TA) at a 1 : 1 : 1 molar ratio using neutron diffraction and H solid-state NMR spectroscopy at temperatures from 25 °C to 80 °C. Deuterated variants of all three lipid components of the model system were used to enable their separate investigation in the NMR spectra and quantification of the amount of molecules in each phase. Neutron scattering experiments show the coexistence of two lipid phases at low temperatures with repeat spacings of 54.2 Å and 43.0 Å at a physiological skin temperature of 32 °C. They appear to be indistinguishable in theH NMR spectra as both phases are crystalline and ceramide molecules do not rotate around their long axis on a microsecond timescale. The evolution of these phases upon heating is followed and with increasing temperature fluid and even isotropically mobile molecules are observed. A model of the organization of the lamellar phases is proposed in which the thicker phase consists of CER[NP]-C24 in a hairpin conformation mixed with CHOL and TA, while the phase with a repeat spacing of 43.0 Å contains CER[NP]-C24 in a V-shape conformation.

V-Shaped Molecular Configuration of Wax Esters of Jojoba Oil in a Langmuir Film Model

“…This indicates that both chains are being progressively orientated towards a more vertical disposition as they are compressed. However, S CD values would be more consistent with a "V-shape" 63 rather than a hairpin configuration as proposed by Adam 13 Moreover, this slight ordering tendency would explain neither the apparent dipole moment drop between 100 and 80 Å 2 observed in Fig.2 nor the slight reorientation of dipoles suggested by the analyses done in the previous section. This strengthens the possibility that reorientation of a population of water molecules is taking place as suggested above.…”

Stratum corneum lipid matrix with unusual packing: A molecular dynamics study

“…Magnus Lundborg and collaborators published recently two extensive works in which they also developed an LM model based on Iwai work, although the final lipid composition and configuration were changed [56,57]. Their model also presented stability with the splayed CER and some tilt in the zone where the FFA are located, remarks that are also presented in one recent work of neutron diffraction of skin lipids [58]. These results agree with the performed tests in our work, however, the PMF profiles in the Lundborg's work were different, which could result from more precise calculations, since the model was not coarse-grained, or due to less compactness in the LM model.…”

Quantification of the amount of mobile components in intact stratum corneum with natural-abundance13C solid-state NMR

“…In addition, as also discussed in that paper, there is a risk of incomplete lipid extraction as well as preferential extraction of more fluid lipids. There have been several 2 H NMR studies employing deuterated lipids to quantify the amount of fluid lipids in SC model lipid mixtures, [34][35][36][37][38][39] but not in intact SC. The amount of the mobile lipids obtained for studies of model mixtures naturally varies with the chosen lipid compositions.…”