Isolation and Lipid Biochemical Characterization of Stratum Corneum Membrane Complexes: Implications for the Cutaneous Permeability Barrier

Abstract

Despite substantial histochemical and ultrastructural evidence suggesting that mammalian stratum corneum is a heterogenous tissue composed of lipid-depleted corneocytes embedded in a neutral lipid-enriched matrix, controversy remains about the distribution of lipids in this layer. In this study we attempted to resolve this issue by cell fractionation and lipid biochemistry of isolated stratum corneum membrane complexes. Intact homogeneous sheets of stratum corneum were obtained from neonatal mice by treatment first with the staphylococcal epidermolytic toxin and then by removal of residual granular cells with trypsin. The resultant sheets were pooled, pulverized at −196°C and homogenized at 20–30,000 psi in a Stansted cell disrupter. Treatment with 0.005% Subtilisin (protease-type VIII) in Tris-HCl buffer, pH 8.8, for 60min at 25°C stripped keratin filaments from beneath the plasma membrane. The resulting homogeneous population of apposed membrane complexes revealed trapped intercellular lipid bilayers when it was examined ultrastructurally. Whereas whole stratum corneum sheets contained 10% lipid/total lipid + protein weight, purified membrane complexes contained about 50% lipid, a 5-fold concentration of lipid in membrane fractions over whole cells. From available quantitative data stratum corneum cytoplasmic domains contained less than 3% lipid, while membrane complexes accounted for over 80% of the total stratum corneum lipid. Moreover, freeze-fracture replicas of isolated membrane complexes revealed intercellular membrane bilayers that were: ( a ) identical to those encountered in whole stratum corneum and ( b ) removed by lipid solvent extraction. Finally, thin-layer chromatography of lipids extracted from membrane complexes revealed a similar spectrum and weight distribution of lipids as encountered in extracts from whole sheets. These results provide direct evidence that stratum corneum cell membrane regions are preferentially enriched in lipids over whole tissue, confirming earlier morphological studies that lipids are progressively segregated in membrane domains during cornification. This heterogeneous structural/chemical organization has profound implications for skin barrier function, stratum corneum cohesion, and for avenues of percutaneous transport.