Organization of skin stratum corneum extracellular lamellae: diffraction evidence for asymmetric distribution of cholesterol

Abstract

Lipid suspensions containing 2:1:1 skin ceramides:palmitic acid:cholesterol, similar to the lipid composition found in the extracellular matrix of skin stratum corneum, were analyzed by X-ray diffraction methods. These suspensions gave a sharp wide-angle reflection at 4.1 A, indicating tight hydrocarbon chain packing that would function as a water barrier, and low-angle lamellar diffraction with a repeat period near 130 A, similar to that previously recorded from intact stratum corneum. The lamellar repeat increased from 121 A at pH 6 to 133 A at pH 8.5, allowing phase angles of the lamellar data to be obtained by a sampling theorem "swelling" analysis. Electron density profiles showed that each repeating unit contained two asymmetric bilayers, with a fluid space on one side of the bilayer that increased with increasing pH, due to electrostatic repulsion between bilayers because of ionization of the palmitic acid. Profiles obtained from lamellae with cholesterol sulfate partially substituted for cholesterol showed large density increases on that same side of the bilayer, indicating that cholesterol is asymmetrically distributed in each bilayer. A molecular model was developed postulating that this asymmetry is due to the exclusion of cholesterol from lipid monolayers containing the ester-linked unsaturated (linoleic) hydrocarbon chain of skin ceramide 1. This model can explain the altered organization of extracellular lamellae in epidermal cysts (P. W. Wertz, D. C. Swartzendruber, K. C. Madison, D. T. Downing. 1987. J. Invest. Dermatol. 89:419-425) where the ester-linked chains have a higher percentage of saturated fatty acids than found in normal epidermis.

FIGURE 1

Molecular structures of the major porcine skin ceramides. Ceramides 4 and 5 differ only in the chain-length of the α-hydroxy acid moiety (longer in ceramide 4). (Reproduced with permission from McIntosh et al., 1996, copyright American Chemical Society.)

FIGURE 2

Low-angle X-ray diffraction pattern for a suspension of 2:1:1 skin ceramides:palmitic acid:cholesterol in buffer at pH 7. Three diffraction rings are visible—the first three orders of a 126-Å lamellar repeat period.

FIGURE 3

Structure amplitudes plotted versus reciprocal spacing for six different suspensions of 2:1:1 skin ceramide:palmitic acid:cholesterol, with repeat periods ranging from d = 121 Å to d = 133 Å. The solid, dotted, small dashed, and large dashed lines are absolute values of continuous transforms for one particular data set (d = 132 Å) with the phase combinations indicated in the figure.

FIGURE 4

Structure factors for 2:1:1 skin ceramide:palmitic acid:cholesterol (solid circles) and 2:1:0.5:0.5 skin ceramide:palmitic acid:cholesterol:cholesterol sulfate (open circles). The solid line is the continuous Fourier transform for 2:1:1 skin ceramide:palmitic acid:cholesterol calculated using the sampling theorem for the d = 132 Å data set.

FIGURE 5

Electron density profiles for 2:1:1 skin ceramide:palmitic acid:cholesterol suspensions at pH 6.0 (solid line) and pH 8.5 (dotted line).

FIGURE 6

Electron density profiles for 2:1:1 skin ceramide:palmitic acid:cholesterol (solid line) and 2:1:0.5:0.5 skin ceramide:palmitic acid:cholesterol:cholesterol sulfate (dotted line).