Phase behavior of lipid mixtures based on human ceramides: coexistence of crystalline and liquid phases.

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.

Phase behaviour of skin barrier model membranes at pH 7.4.

The main function of the skin is to protect the body against exogenous substances. The skin barrier is located in the outermost layer of the skin, the stratum corneum (SC). This layer consists of keratin enriched cells embedded in lipid lamellae that form the main barrier for diffusion of substances through the skin. The main lipid classes in this barrier are ceramides, cholesterol and free fatty acids. Cholesterol sulfate and calcium are also present in SC. Furthermore it has been suggested that a pH gradient exists. In a previous paper the effect of cholesterol sulfate and calcium on the lipid phase behaviour of mixtures prepared from cholesterol, ceramides and free fatty acids at pH 5 was reported (approximate pH at the skin surface). In the present study the phase behaviour of mixtures prepared from cholesterol, ceramides and free fatty acids prepared at pH 7.4 (the pH of viable cells) has been examined between 25 and 95 degrees C. Our studies reveal that a reversed hexagonal phase has been formed at elevated temperatures. Addition of calcium inhibits the formation of the reversed hexagonal phase, while cholesterol sulfate promotes the presence of the reversed hexagonal phase at increased temperatures. From our results we can conclude that the lipid mixtures prepared at pH 5 resemble more closely the lipid phase behaviour in intact SC than the lipid mixtures prepared at pH 7.4.

The lipid organisation in the skin barrier.

The main function of the skin is to protect the body against exogenous substances. The skin barrier is located in the outermost layer of the skin, the stratum corneum. This layer consists of keratin enriched cells embedded in lipid lamellae. These lamellae form the main barrier for diffusion of substances through the skin. In diseased skin the barrier function is often impaired. For a full understanding of the properties of the human skin barrier, insight in the stratum corneum lipid organisation is of great importance. In this paper a short description of the lipid organisation in normal human stratum corneum will be given, after which the role the main lipid classes play in the stratum corneum lipid organisation will be described. In addition the effect of cholesterol sulfate and calcium on the lipid organisation will be discussed. Finally a new model, the "sandwich model", will be proposed that describe the localisation of the fluid phases in the stratum corneum.

Cholesterol sulfate and calcium affect stratum corneum lipid organization over a wide temperature range.

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 degrees and 95 degrees 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 degrees 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 Ca(2+) is present only in the lowest SC layers, the effect of pH, cholesterol sulfate, and Ca(2+) 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 Ca(2+) 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.

The role of ceramide composition in the lipid organisation of the skin barrier.

The lipid lamellae in the stratum corneum (SC) play a key role in the barrier function of the skin. The major lipids are ceramides (CER), cholesterol (CHOL) and free fatty acids (FFA). In pig SC at least six subclasses of ceramides (referred to as CER 1, 2-6) are present. Recently it was shown that in mixtures of isolated pig SC ceramides (referred to as CER(1-6)) and CHOL two lamellar phases are formed, which mimic SC lipid organisation very closely [J.A. Bouwstra et al., 1996, J. Lipid Res. 37, 999-1011] [1]. Since the CER composition in SC originating from different sources/donors often varies, information on the effect of variations in CER composition on the SC lipid organisation is important. The results of the present study with mixtures of CHOL including two different CER mixtures that lack CER 6 (CER(1-5) mixtures) revealed that at an equimolar molar ratio their lipid organisation was similar to that of the equimolar CHOL:CER(1-6) and CHOL:CER(1,2) mixtures, described previously. These observations suggest that at an equimolar CHOL:CER ratio the lipid organisation is remarkably insensitive toward a change in the CER composition. Similar observations have been made with equimolar CHOL:CER:FFA mixtures. The situation is different when the CHOL:CER molar ratio varies. While in the CHOL:CER(1-6) mixture the lamellar organisation hardly changed with varying molar ratio from 0.4 to 2, the lamellar organisation in the CHOL:CER(1-5) mixtures appeared to be more sensitive to a change in the relative CHOL content, especially concerning the changes in the periodicities of the lamellar phases. In summary, these findings clearly indicate that at an equimolar CHOL:CER molar ratio the lamellar organisation is least sensitive to a variation in CER composition, while at a reduced CHOL:CER molar ratio the CER composition plays a more prominent role in the lamellar phases. This observation may have an implication for the in vivo situation when both the CER composition and the CHOL:CER molar ratio change simultaneously.

pH, cholesterol sulfate, and fatty acids affect the stratum corneum lipid organization.

Lipid mixtures prepared from cholesterol (CHOL), isolated ceramides (CER), and free fatty acids can serve as attractive tools to study the role various stratum corneum (SC) lipids or microenvironmental conditions play in the SC lipid organization, as the phase behavior in these mixtures and in SC are similar: two lamellar phases with periodicities of approximately 6 and 13 nm are present. Because pH and cholesterol sulfate (CSO4) gradients exist in SC and may affect the local SC lipid organization, the effects of pH and CSO4 on lipid phase behavior was examined. X-ray diffraction studies with CHOL:CER mixtures revealed that the lamellar ordering at pH 5 and 7.4 were similar: both the short and the long periodicity phases were present. Upon addition of free fatty acids the phase behavior became pH dependent; the long periodicity phase being more dominant at pH 7.4 than at pH 5. Similar observations have been made upon addition of CSO4. Furthermore, only in the presence of CSO4 did phase-separated CHOL disappear, indicating that CHOL completely dissolves in the lamellar phases. A major phase change from an hexagonal to an orthorhombic lateral packing has been observed in the presence of free fatty acids. Furthermore, in the presence of CSO4 next to orthorhombic also liquid lateral packing could be detected. In contrast to lamellar ordering, changes in pH did not affect the lateral packing in any of the lipid mixtures studied.

Role of ceramide 1 in the molecular organization of the stratum corneum lipids.

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 omega-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.