X-ray Diffraction Analysis of Isolated Skin Lipids: Reconstitution of Intercellular Lipid Domains

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Abstract
Low- and wide-angle X-ray diffraction were used to determine the structural organization of lipids isolated from the stratum corneum extracellular matrix that forms the major water permeability barrier in mammalian epidermis. Hydrated pig skin ceramides gave a single low-angle reflection of about 62 Å and a wide-angle reflection at 4.15 Å. The addition of either cholesterol or fatty acid, the other major lipid components of the skin stratum corneum extracellular matrix, modified this diffraction pattern, depending on the lipid mole ratios. In the absence of water, lipid mixtures exhibited lipid phase separation, as shown by low- and wide-angle reflections typical of a separate cholesterol phase. However, a hydrated 2:1:1 mole ratio of ceramide:cholesterol:palmitic acid (similar to that found in stratum corneum) produced a diffraction pattern with a single sharp wide-angle reflection at 4.10 Å and low-angle reflections which indexed as the first eight orders of a single repeat period of 130 Å. The repeat period and intensity distribution of the low-angle data were similar to those found in intact stratum corneum [White et al. (1988) Biochemistry 27, 3725−3732; Bouwstra et al. (1994) Biochim. Biophys. Acta 1212, 183−192]. Higher concentrations of cholesterol or palmitic acid resulted in lipid phase separations. The 130 Å repeat period decreased only about 3 Å as water was removed by incubation in low-relative humidity atmospheres. The 130 Å repeat period depended on the presence of a particular ceramide, N-(ω-acyloxy)acylsphingosine, which is found only in the epidermis. In contrast, 2:1:1 mixtures of brain ceramide:cholesterol:palmitic acid gave reflections of 56 and 34 Å. These results indicate that a structure with dimensions similar to those of the lamellar repeating unit found in skin stratum corneum does not depend on the presence of protein but does depend on the presence of specific skin ceramides and appropriate concentrations of cholesterol and fatty acid.