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J Lipid Res. 1996 May;37(5):999-1011.

Phase behavior of isolated skin lipids.

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Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden University, The Netherlands.


Ceramides were isolated from the pig stratum corneum (SC) and mixed in varying molar ratios with either cholesterol or with cholesterol and free fatty acids. The phase behavior of the mixtures was studied by small-(SAXD) and wide-angle (WAXD) X-ray diffraction. Ceramides alone did not exhibit a long range ordering. Upon addition of cholesterol to ceramides, lamellar phases were formed and a hexagonal lateral packing was detected similar to that seen in intact SC. At a cholesterol/ceramide molar ratio of 0.1, only one reflection at 5.9 nm was observed. At a cholesterol/ceramide molar ratio of 0.2, three reflections corresponding to 12.3, 5.56, and 4.26 nm appeared. The reflections were based on two phases. Increasing the cholesterol/ceramide ratio to 0.4, the peak positions were slightly shifted. The diffraction pattern revealed the presence of two lamellar phases with periodicities of 12.2 and 5.2 nm, respectively. The positions of the peaks remained unchanged when the cholesterol/ceramide ratio was increased up to 1.0. At a cholesterol/ceramide molar ratio of 2.0, the intensity of various peaks based on the 12.2 nm phase decreased in intensity. The phase behavior of the cholesterol/ceramide mixtures in a ratio between 0.4 and 1.0 was very similar to that found in intact pig SC in which two lamellar phases with periodicities of 6.0 and 13.2 nm are present. Our data further indicate that the formation of the 5.2 nm lamellar phase requires a higher cholesterol content than the formation of the 12.2 nm lamellar phase. Furthermore, when the relative amount of cholesterol is very high, the 5.2 nm phase is the most pronounced one. Addition of free fatty acids increased the solubility of cholesterol, indicating the role free fatty acids may play for the skin barrier function. The phase behavior of cholesterol/ceramide/fatty acid mixtures was found to be dependent on the chain length of fatty acids used. Namely, addition of short-chain free fatty acids (C14-C18) did not change the periodicity of the 12.2 and 5.2 nm phases, but induced the formation of an additional 4.2 nm phase. In the presence of long-chain free fatty acids (C16-C26), the periodicity of the lamellar phases was slightly increased (to 13.0 and 5.3 nm, respectively) but no additional 4.2 nm phase was formed. These results indicate that the lipid phase behavior of the cholesterol/ceramide/free fatty acid mixtures closely mimics that of the intact stratum corneum only in the presence of long-chain free fatty acids.

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