162 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS plex, naturally occurring lipids, with an optimized lipid ratio, can accelerate barrier recovery in human skin, as shown above for murine skin. EFFECT OF LIPIDS ON SKIN HYDRATION Studies in hairless mice. Prior studies have demonstrated both that ceramides influence the water-holding capacity of stratum corneum (15-18) and that glucosylceramides stim- ulate epidermal proliferation (19). There is little information about the effect of other physiological lipids, applied topically, on skin hydration. We next examined the effect of the major individual stratum corneum lipids, as well as Y2, on skin hydration in acetone-treated mouse skin. As shown in Figure 3, both fatty acid (palmirate) and ceramide increase skin capacitance two hours after application. Cholesterol worsens capacitance in comparison to the vehicle. A lipid mixture consisting of cholesterol, ceramide, and palmirate (molar ratio at 1:1:3) also increases skin capacitance. However, the most dramatic increase in skin capacitance occurs following topical application of Y2 (1.6%). These results suggest that certain stratum corneum lipids, i.e., fatty acids and ceramides, improve skin hydration, and that a topical lipid mixture (Y2), enriched in all three stratum corneum lipids, produces the greatest increase in skin hydration. ._ • • • - ._ • E • '- r- [3_ 0 • --- o ._1 -- Figure 3. Effect of physiological lipids and a natural lipid mixture on skin capacitance in acetone-treated mouse skin: 40 •tl of Y2 (1.6%), cholesterol (2%), galactocerebroside II (1%), palmirate (1%), or vehicle was topically applied to acetone-treated mouse skin (about 2 x 3 cm 2 area). The lipid mixture (1.1%) contains cholesterol, galactocerebroside II, and palmitate (1:1:3 molar ratio). Skin capacitance was mea- sured before and two hours after lipid or vehicle application. The data are expressed as percentage increase after lipid or vehicle application. Results are mean -+ SEM. Significant differences are in comparison with vehicle alone.

BARRIER FUNCTION AND HYDRATION 163 Study in human volunteers. In order to explore the potential utility of physiologic lipids for improving skin hydration, we next tested whether Y2 influences stratum corneum hydration in acetone-treated vs normal human skin. As shown in Figure 4, Y2 markedly increases stratum corneum water content two hours after acetone treatment in comparison to vehicle treatment alone. Moreover, Y2 also significantly raises stratum corneum water content two hours after treatment of normal skin in comparison to vehicle treatment (Figure 5). These results demonstrate that exogenous, naturally occurring stratum corneum lipids display a moisturizating effect on both damaged and normal human skin. DISCUSSION The importance of stratum corneum lipids for barrier homeostasis is firmly established (reviewed in 1-3). Moreover, each of the three key stratum corneum lipids, i.e., cholesterol, fatty acid, and ceramides, is required for barrier homeostasis (4-6). How- ever, recent studies have shown that these lipids are required as a mixture, rather than as individual species (8). Only mixtures of these lipids, in approximately equimolar ratios, allow normal barrier recovery accompanied by formation of normal membrane bilayer structures in stratum corneum (8), and mixtures with optimized ratios of the three stratum corneum lipids further accelerate rates of barrier recovery (9, 10, 13). In contrast, incomplete mixtures result in formation of abnormal membrane bilayer struc- tures in stratum corneum and actually impede barrier recovery (8). In agreement with these prior results, the natural lipid mixture studied here, Y2, which contains a molar ratio that approximates that in optimized mixtures of physiological lipids (9,10,13), 8O a• 60 o• 4o 2o - , PO.05 ß ß Veh icle-Tre ated Y2-Treated Figure 4. Effect of natural lipid mixture on skin capacitance in acetone-treated human skin: 40 Ftl of Y2 (1.6%) or vehicle was topically applied to acetone-treated human skin (about 20 cm 2 area). Skin capacitance was measured before and two hours after Y2 or vehicle application. The data are expressed as percentage increase after Y2 or vehicle application. Results are mean + SEM. Significant difference is in comparison with vehicle alone.