MOISTURIZING FUNCTION OF LANOLIN 185 Uranyl acetate 0.5 g Osmium tetroxide 1.0 g Glutaraldehyde stock solution (50%) 6.0 ml Results. Figure 2 is a section of the outer layers of stratum corneum showing an excess of fanolin (grey) remaining on the surface (bottom of picture) after 16 hours, and also some fanolin (similarly grey) that has penetrated into intercellular spaces down to the third or fourth corneocyte. A dark line following the edge of some corneocytes suggests a particularly high concentration of lanolin (or at least lead marker) at those points, as though lanolin had become incorporated in the trilaminar outer membranes of corneo- cytes that in places had become detached from the cell. Most other intercellular lipids appear to have been lost during specimen processing. The surface lanolin is seen to contain some very electron-opaque particles, which are believed to be, in part, a residual impurity of unreacted lead nitrate in the doped lanolin. An examination of a thin film of this lanolin by electron microscopy showed similar small, very dense clumps. Figure 3 is of another skin section at greater magnification and shows even more clearly an accumulation of lanolin (and/or lead marker) in laminar structures. The largest such feature is particularly ionteresting since is shows a distinct triple layer consistinog of a central band about 200 A thick sandwiched between two narrower (80 to 100 A) bands that are more electron-opaque. This structure is in accordance with the findings of Swanbeck (9), who proposed the existence of fibrous protein layers sur- rounded by lipid bilayers, which, in our own case, have apparently been infiltrated by I #L. ??LAN. 16H. i 9!6552 88.ekU XI5K 5•0n• Figure 2. Lanolin on skin surface (bottom of picture) showing penetration into intercellular spaces and concentration at cell surfaces.

186 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Figure 3. Concentration of lanolin/marker in trilaminar membrane of cell envelope. lanolin and/or marker. The dimensions we see certainly agree well with Swanbeck's measurements. This trilaminar structure with a central protein band has been disputed, but lipid layers with a spacing of 50 to 80 fix were reported by Friberg et al. (10), who also showed that interaction of other lipids with the bilayers could affect the spacing. Other work reported by Ward and du Reau (l 1) also showed an alteration in spacing by the solubilization of oleyl alcohol into the bilayers and consequent increase in water capacity. It is possible in our case, therefore, that incorporation of applied substance into the lamellae has altered the spacing of lipid layers from that reported by Friberg et al. Trilaminar structures can also be detected in Figure 4, and here too, as in Figure 3, some of them have become detached from the cell surface. Trilaminar membranes have been previously reported by Odland and Holbrook (12) and Elias (13), and are now an accepted structural feature. In our case it appears that although most of the natural intercellular lipids have been lost during preparation of the specimens (except perhaps in Figure 10), the lamellae covalently bound to the corneocyte surfaces appear to have survived in places, although some have been detached from the cell surface. In an attempt to overcome this loss of lipids, further samples were prepared using a freeze- substitution technique that has been reported to fix and retain better, at low temper- ature, the natural lipids. This does not appear to have been so in our case, however, with most intercellular spaces still showing absence of natural lipids. Nevertheless, some interesting features are visible. Figure 5 shows that lanolin (grey) has penetrated here into the intercellular spaces down as far as the stratum granulosum. Such stained material was not visible in the untreated