102 JOURNAL OF COSMETIC SCIENCE THE SKIN BARRIER: A UNIQUE INTERPLAY BETWEEN CORNEOCYTES AND LIPIDS Joke Bouwstra', Ph.D., G. Gooris' and M. Ponec s •Leiden/Amsterdam Center Jbr Drug Research, Leiden, The Netherlands 2Department of Dermatology, Leiden University Medical Center, The Netherlands Introduction: One of the most important functions of the skin is to protect the body against unwanted effects from the environment. The barrier for permeation of substances across the skin resides in the outermost layer of the skin, the stratum corneum (SC). The SC consists of dead cells, comeocytes, surrounded by hydrophobic lipid lameline. The interconnection between the lipid lamellae and corneocytes is formed by a monolayer of lipids bound to the cornified envelope. Visualisation studies revealed that the penetration route across the SC resides in the intercellular tortuous pathway between the corneocytes. However, there is at least one exception. Water can be absorbed into the corneocytes in large amounts. For all substances the SC lipids play a key role in the skin barrier function. In SC mainly hydrophobic lipids, such as ceramides (CER), long chain free fatty acids (FFA) (most abundant chain lengths C22 and C24) and cholesterol are present. Eight subclasses of ceramides have been identified in human SC. The ceramides consist of a sphingosine, phytosphingosine or a 6-OH- sphinganine base to which a nonhydroxy fatty acid or an to-hydroxy fatty acid is chemically linked. In Molecular arrangement Lateral packing Figtire 1 Left: The periodicity (d) of the 12.8 and 5.5 Long periodicity (LP) Liquid (high permeability) rllIl lamellar phases. i••ii•!l i• Right. The lateral packing is either liquid (not "•..•b•., . well defined spacings between molecules), the d Hexagonal linedlure permeaioihty) hexagonal packing (equal intermolecular • spacing) and the orthorhombic phase (two •1• .... possible spacing between the molecules) Short periocli,city (SP) . human SC two ceramides, CER1 and CER4, have been identified with a linoleic acid chemically bound to the C31/33 c0-hydroxy fatty acid. Information on the relationship between lipid organization and composition is of great importance to unravel the mechanism controlling the skin barrier function. Methods: Small angle and Wide angle X-ray diffraction: By using small and wide angle X-ray diffraction the following parameters can be determined. A. The periodicity (d) is of one of the imporantant characteristics of the lamellar phases. This is the distance over which the structure of the lamellar phase perpendicular to its basal plane is repeated, see figure 1. B. Lateral packing of SC lipids, which can be liquid, hexagonal and/or orthorhombic. With Electron diffraction is easier to distinguish between a hexagonal and orthorhombic lattice and to study lateral packing in vivo in man. Human stratum cornebro: In human SC two lamellar phases are present with a periodicity of approximately 6.4 nm and 13.4 rim, respectively (1). Since the 13 nm lamellar phase has always been present in all species studied so far, this phase is considered to be important for the skin barrier function. Increasing the hydration level from 0 to 300Vow/w does almost not lead to an increase in the periodicity. This indicates an absence of swelling of the lamellar phases. From the WAXD pattern of human SC the

2002 ANNUAL SCIENTIFIC MEETING 103 presence of an orthorhombic lateral packing has been established. Recently the lateral packing has also been studied by electron diffraction using sequential strips of SC (2). With this approach we have established that throughout the SC the lipids form an orthorhombic lattice with the exception of the most superficial layers in which occasionally hexagonal packing has been observed. CHO]L:CER:FFA mixtures: In mixtures prepared from CHOL and CER (isolated from human SC), t•vo lamellar phases are formed with periodicities of 5.4 and 12.8 nm, respectively (3). This presence of the lamellar phases is insensitive to the CHOL:CER. In the equimolar CHOL:CER:FFA mixture the periodicity of the lamellar phases increases to 5.5 and 13.0 nm, respectively, mimicking even closer the lamellar phase behaviour in intact SC. Lateral packing: In the absence of FFA, a diffraction pattern indicating a hexagonal packing is observed. Addition of FFA induces a transition from a hexagonal to an orthorhombic packing, demonstrating a crucial role of FFA for the formation of a competent barrier (3). CHOL:CER:FFA mixtures prepared in the absence of CERI: When CER1 is absent only a very small population of lipids forms the 12.8 nm lamellar phase. Molecular organisation in the repeating unit of the 12-13 nm lamellar phase: The presence of a broad- narrow-broad sequence of the electron lucent bands of the lamellae in the RuO4-fixed SC (4) and an electron density distribution profile in the repeating unit of the 12.8 nm lamellar phase demonstrate a broad-narrow-broad sequence of the hydrocarbon regions. Based on this and on the key role CER1 plays in the formation of the 12-13 nm lamellar phase we proposed a model for molecular organization of this phase presented in figure 2. In this model the lipids are organised in 3 layers: a narrow layer that is located in the center with a broad layer on both sides. CER 1 links this tri-layer unit together and consequently dictates the broad-narrow-broad sequence in the tri-layer unit (4). It has been demonstrated that also in isolated model systems and in intact SC the orthorhombic phase coexists with the fluid phase. We propose that this liquid sublattice is located in domains in the central layer of the 12-13 nm phase. In this layer the unsaturated linoleate and cholesterol are present and that this fluid phase will acts as a diffusion pathway of substances and might facilitate the deformation of the lipid lamellae in the SC. This model is referred to as the "sandwich model". Conclusions:The lipids are organized in crystalline lamellae, but most probably a liquid phase is also present. The important role of CER1 in the formation of the 13.4 nm lamellar phase and the transition from a hexagonal to an orthorhombic phase induced by FFA have been observed in mixtures prepared with isolated SC lipids as well as in intact human SC. The in vitro findings can predict in vivo situation. SANDWICH MODEL 12-13 nm periodicity Figure 2 ]•$' "l'•l•i '•"•"•'.•/• 3,avers: A "sandwich model" is propos The Ir •' • • I• •b Crystaliineiattice ' '. i-•.• characteristics are: a liquid sublattice is in the • '• . . .-- ,Fluid phase central lipid layer of this phase and a gradual -'-- •...'..'_'. Crystaiiinelattice change in lipid mobility occurs due the I I presence of less mobile long saturated Gradual change in chain Linoleate CERt hydrocarbon chains. The central lipid layer is mobility Stacking of lamellae: aiternating fluid and crystalline sublattices not a continuous fluid phase ß . . •v.. _•.• - • •(Shear stress r ::: •:..i...• . -• :.: '•: -• Sandwich model: t : .. • _, _ _ . :7'/: •:•:!:::!: • ': : -• facilitates r "::: :•' ...... -• deformation 1 Bouwstra JA, Gooris GS, van der Spek JA and Bras W. J Invest Dermatol. 1991 96: 1006-1014. 2 Pilgram GSK, Engelsma-van Pelt AM, Bouwstra JA, Koerten HK. J Invest Dermatol 1999 133:403-409 3 Bouwstra JA, Gooris GS, Dubbelaar, FER, Ponce M, J. Invest. Dermatol. 2002 118: 606-616. 4 Madison KC, Swarzendruber DC, Wertz PW, Downing DT, J. Invest Dermatol 1987 88:714-718