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The lipid regions in the outermost layer of the skin (stratum corneum) form the main barrier for diffusion of substances through the skin. In this layer the main lipid classes are ceramides, cholesterol (CHOL), and FFA. Previous studies revealed a coexistence of two crystalline lamellar phases with periodicities of approximately 13 nm (referred to as long periodicity phase) and 6 nm (short periodicity phase). Additional studies showed that lipid mixtures prepared with isolated pig ceramides (pigCER) mimic lipid phase behavior in stratum corneum closely. Because the molecular structure of pigCER differs in some important aspects from that of human ceramides (HCER), in the present study the phase behavior of mixtures prepared with HCER has been examined. Phase behavior studies of mixtures based on HCER revealed that in CHOL:HCER mixtures the long periodicity phase dominates. In the absence of HCER1 the short periodicity phase is dominant. Addition of FFA promotes the formation of the short periodicity phase and induces a transition from a hexagonal sublattice to an orthorhombic sublattice. Furthermore, the presence of FFA promotes the formation of a liquid phase. Finally, cholesterol sulfate, a minor but important lipid in the stratum corneum, reduces the amount of cholesterol that phase separates in crystalline domains. From these observations it can be concluded that the phase behavior of mixtures prepared from HCER differs in some important aspects from that prepared from pigCER. The most prevalent differences are the following: i) the addition of FFA promotes the formation of the short periodicity phase; and ii) liquid lateral packing is obviously present in CHOL:HCER:FFA mixtures. These changes in phase behavior might be due to a larger amount of linoleic acid moiety in HCER mixtures compared with that in pigCER mixtures.—Bouwstra, J. A., G. S. Gooris, F. E. R. Dubbelaar, and M. Ponec. Phase behavior of lipid mixtures based on human ceramides: coexistence of crystalline and liquid phases.

Abstract The main diffusion barrier for drugs penetrating through the skin is located in the intercellular lipid matrix in the upper layer of the skin, the stratum corneum (SC). The main lipid classes in the SC are ceramides (CER), free fatty acids (FFA) and cholesterol (CHOL). The lipids in SC are organized into two lamellar phases with periodicities of approximately 13 and 6 nm, respectively. Similar lipid organization has been found with equimolar CHOL:CER:FFA mixtures in SAXD studies performed at room temperature. However, one may conclude that the phase behavior of the mixtures is similar to that in SC only when the lipid organization of the lipid mixtures resembles that in SC over a wide temperature range. Therefore, in the present study, the organization of the lipid mixtures has been studied in a temperature range between 20° and 95°C. From these experiments it appeared that at elevated temperatures in equimolar CHOL:CER:FFA mixtures a new prominent 4.3 nm phase is formed between 35–55°C, which is absent or only weakly formed in intact human and pig SC, respectively. As it has been suggested that gradients of pH and cholesterol sulfate exist in the SC and that Ca2+ is present only in the lowest SC layers, the effect of pH, cholesterol sulfate, and Ca2+ on the lipid phase behavior has been investigated with lipid mixtures. Both an increase in pH from 5 (pH at the skin surface) to 7.4 (pH at the SC–stratum granulosum interface) and the presence of cholesterol sulfate promote the formation of the 13 nm lamellar phase. Furthermore, cholesterol sulfate reduces the amount of CHOL that is present in crystalline domains, causes a shift in the formation of the 4.3 nm phase to higher temperatures, and makes this phase less prominent at higher temperatures. The finding that Ca2+ counteracts the effects of cholesterol sulfate indicates the importance of a proper balance of minor SC components for appropriate SC lipid organization. In addition, when the findings are extrapolated to the in vivo situation, it seems that cholesterol sulfate is required to dissolve cholesterol in the lamellar phases and to stabilize SC lipid organization. Therefore, a drop in cholesterol sulfate content in the superficial layers of the SC is expected to destabilize the lipid lamellar phases, which might facilitate the desquamation process.—Bouwstra, J. A., G. S. Gooris, F. E. R. Dubbelaar, and M. Ponec. Cholesterol sulfate and calcium affect stratum corneum lipid organization over a wide temperature range.

The main barrier of the skin is formed by the lipids in the apical skin layer, the stratum corneum (SC). In SC mainly ceramides (CER), free fatty acids (FFA) and cholesterol (CHOL) are present. The CER are composed of at least six different fractions. CER 1 has an exceptional molecular structure as it contains a linoleic acid linked to a long-chain ω-hydroxy acid (C > 30). The SC lipids are organized in two lamellar phases with periodicities of approximately 6 and 13 nm, respectively. Recent studies revealed that ceramides isolated from pig SC mixed with cholesterol in confined ratios mimic stratum corneum lipid phase behavior closely (Bouwstra, J.A., et al. 1996. J. Lipid Res. 37: 999–1011). In this paper the role of CER 1 for the SC lipid lamellar organization was studied. For this purpose lipid phase behavior of mixtures of CHOL and total ceramide fraction was compared with that of mixtures of CHOL and a ceramide mixture lacking CER 1. These studies showed that in the absence of CER 1 almost no long periodicity phase was formed over a wide CHOL/CER molar ratio. A model is proposed for the molecular arrangement of the two lamellar phases. This model is based on the dominant role CER 1 plays in the formation of the long periodicity phase, electron density distribution calculations, and observations, such as i) the bimodal distribution of the fatty acid chain lengths of the ceramides, ii) the phase separation between long-chain ceramides and short-chain ceramides in a monolayer approach, and iii) the absence of swelling of the lamellae upon increasing the water content organization in SC. In this molecular model the short periodicity phase is composed of only two high electron density regions indicating the presence of only one bilayer, similar to that often found in phospholipid membranes. The molecular arrangement in the long periodicity phase is very exceptional. This phase most probably consists of two broad and one narrow low electron density regions. The two broad regions are formed by partly interdigitating ceramides with long-chain fatty acids of approximately 24–26 C atoms, while the narrow low-electron density region is formed by fully interdigitating ceramides with a short free fatty acid chain of approximately 16 to 18 C atoms.—Bouwstra, J. A., G. S. Gooris, F. E. R. Dubbelaar, A. M. Weerheim, A. P. IJzerman, and M. Ponec. Role of ceramide 1 in the molecular organization of the stratum corneum lipids. J. Lipid Res. 1998. 39: 186–196.