The physical barrier of the skin is located in its outermost layer – the stratum corneum (SC). SC consists of cornified cells embedded in the highly organized extracellular lipid matrix containing mainly ceramides (Cer), free fatty acids (FFA), and cholesterol (Chol). The skin lipids behaviour is studied in models of various complexity. We have studied the simple model based on Cer with C24 acyl chain (type NS or NH), FFA (C16 – C24 chains), and Chol at the equimolar ratio and a complex model prepared from isolated and purified human skin lipids (but still missing cornified cells). The models were prepared in the form of thin layers applied onto supporting wafers. They were annealed at the temperature above the melting point at low or high humidity. The regular structure was evaluated by the following methods:
1. X-ray diffraction measured with an X’pert PRO θ-θ powder diffractometer with the Bragg-Brentano geometry (PANalytical B.V., Netherlands)
2. Neutron diffraction using a cold-neutron three-axis spectrometer MIRA located at the neutron source in Heinz Maier-Leibnitz Zentrum (Garching, Germany) (a simple model)
3. Neutron diffraction using a D16 small-q diffractometer (Institut Laue-Langevin, Grenoble, France) (the human skin lipids)
The simple model based on Cer C24/FFA/Chol showed immense structural variability dependent on the annealing conditions. Bragg peaks at small angles provided a repeat distance (d) of 5.3 nm (Fig. 1A a), which is known from the literature [1], but also the repeat distances of 10.6 nm (Fig.1A b), 15.9 nm (Fig.1A c), and 21.2 nm (Fig. 1A d) were found [2]. All the peaks belong to the one-dimensional lamellar structures with an orthorhombic lipid chain sublattice (peaks at wide angles). The separated Chol (*) was also found. The repeat distance of 10.6 nm is double the theoretical maximal length of a two-chain Cer molecule in the splayed-chain conformation. The composition of the simple model with d = 10.6 nm was adjusted for the neutron diffraction experiment at different contrast conditions (D2O/H2O ratio). The relative neutron scattering length density ρ(z) was calculated according to the equation (1):
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(1) |
Fh is the scattering form factor for the order h and z is the distance from the lamellar centre. The phase angles for a centrosymmetric structure attain the values of +1 or -1. We calculated 57 ρ(z) profiles at 100% D2O based on different combinations of the phase angles in two subsequent steps. The most plausible ρ(z) profiles (Fig. 1B) were selected to be consistent with the molecular composition of the sample and to provide feasible water distribution profile. The plausible ρ(z) profiles rely on a splayed-chain conformation of Cer [3] leading to the asymmetric distribution of hydrocarbon chains in leaflets. The isolated human skin lipids formed a lamellar phase with a longer d of 13.4 nm due to the presence of ultra-long skin Cer. This long periodicity phase (LPP) [1] has a feature common with the structure with d = 10.6 nm – low intensity of the 1st order peak. The human skin lipids were measured by neutron diffraction at different contrast conditions (D2O/H2O ratio). The ρ(z) profile of LPP based on one combination of the phase angles for 6 Fh is shown in Fig. 1C. We suppose that the molecular arrangement of the LPP is also based on the splayed-chain conformation of Cer. The ρ(z) profiles of LPP need to be corroborated with electron density profiles and other experiment to verify the plausibility of our model.
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The study was supported by the Czech Science Foundation (19-09135J and 19-09600S), Charles University (SVV 260 547), EFSA-CDN (No. CZ.02.1.01/0.0/0.0/16_019/0000841) co-funded by ERDF, Heinz Maier-Leibnitz Zentrum, and Institut Laue-Langevin.