New insight into phase behavior and permeability of skin lipid models based on sphingosine and phytosphingosine ceramides.

The intercellular lipid matrix of the stratum corneum (SC), which consist mainly of ceramides (CERs), free fatty acids and cholesterol, is fundamental to the skin barrier function. These lipids assemble into two lamellar phases, known as the long and short periodicity phases (LPP and SPP respectively). The LPP is unique in the SC and is considered important for the skin barrier function. Alterations in CER composition, as well as impaired skin barrier function, are commonly observed in diseased skin, yet the understanding of this relationship remains insufficient. In this study, we have investigated the influence of non-hydroxy and α-hydroxy sphingosine-based CERs and their phytosphingosine counterparts on the permeability and lipid organization of model membranes, which were adjusted in composition to enhance formation of the LPP. The permeability was compared by diffusion studies using ethyl-p-aminobenzoate as a model drug, and the lipid organization was characterized by X-ray diffraction and infrared spectroscopy. Both the sphingosine- and phytosphingosine-based CER models formed the LPP, while the latter exhibited a longer LPP repeat distance. The ethyl-p-aminobenzoate flux across the sphingosine-based CER models was higher when compared to the phytosphingosine counterparts, contrary to the fact that the α-hydroxy phytosphingosine-based CER model had the lowest chain packing density. The unanticipated low permeability of the α-hydroxy phytosphingosine-based model is probably associated with a stronger headgroup hydrogen bonding network. Our findings indicate that the increased level of sphingosine-based CERs at the expense of phytosphingosine-based CERs, as observed in the diseased skin, may contribute to the barrier function impairment.

Interactions of dipalmitoylphosphatidylcholine with ceramide-based mixtures.

The outermost layer of the skin, the stratum corneum (SC), acts as the natural physical barrier. The SC consists of corneocytes embedded in a crystalline lipid matrix consisting of ceramides, free fatty acids and cholesterol. Although phospholipids are frequently present in topical formulations, no detailed information is reported on the interactions between phospholipids and SC lipids. The aim of this study was to examine the interactions between a model phospholipid, dipalmitoylphosphatidylcholine (DPPC) and synthetic ceramide-based mixtures (referred to as SC lipids). (Perdeuterated) DPPC was mixed with SC lipids and the lipid organization and mixing properties were examined. The studies revealed that DPPC participates in the same lattice as SC lipids thereby enhancing a hexagonal packing. Even at a high DPPC level, no phase separated pure DPPC was observed. When a DPPC containing formulation is applied to the skin surface it must partition into the SC lipid matrix prior to any mixing with the SC lipids. To mimic this, DPPC was applied on top of a SC lipid membrane. DPPC applied in a liquid crystalline state was able to mix with the SC lipids and participated in the same lattice as the SC lipids. However, when DPPC was applied in a rippled gel-state very limited partitioning of DPPC into the SC lipid matrix occurred. Thus, when applied to the skin, liquid crystalline DPPC will have very different interactions with SC lipids than DPPC in a (rippled-)gel phase.

Stratum corneum lipid matrix: Location of acyl ceramide and cholesterol in the unit cell of the long periodicity phase.

The extracellular lipid matrix in the skin's outermost layer, the stratum corneum, is crucial for the skin barrier. The matrix is composed of ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs) and involves two lamellar phases: the short periodicity phase (SPP) and the long periodicity phase (LPP). To understand the skin barrier thoroughly, information about the molecular arrangement in the unit cell of these lamellar phases is paramount. Previously we examined the molecular arrangement in the unit cell of the SPP. Furthermore X-ray and neutron diffraction revealed a trilayer arrangement of lipids within the unit cell of the LPP [D. Groen et al., Biophysical Journal, 97, 2242-2249, 2009]. In the present study, we used neutron diffraction to obtain more details about the location of lipid (sub)classes in the unit cell of the LPP. The diffraction pattern revealed at least 8 diffraction orders of the LPP with a repeating unit of 129.6±0.5Å. To determine the location of lipid sub(classes) in the unit cell, samples were examined with either only protiated lipids or selectively deuterated lipids. The diffraction data obtained by means of D2O/H2O contrast variation together with a gradual replacement of one particular CER, the acyl CER, by its partly deuterated counterpart, were used to construct the scattering length density profiles. The acyl chain of the acyl CER subclass is located at a position of ~21.4±0.2Å from the unit cell centre of the LPP. The position and orientation of CHOL in the LPP unit cell were determined using tail and head-group deuterated forms of the sterol. CHOL is located with its head-group positioned ~26±0.2Å from the unit cell centre. This allows the formation of a hydrogen bond with the ester group of the acyl CER located in close proximity. Based on the positions of the deuterated moieties of the acyl CER, CHOL and the previously determined location of two other lipid subclasses [E.H. Mojumdar et al., Biophysical Journal, 108, 2670-2679, 2015], a molecular model is proposed for the unit cell of the LPP. In this model CHOL is located in the two outer layers of the LPP, while CER EOS is linking the two outer layers with the central lipid layers. Finally the two other lipid subclasses are predominantly located in the central layer of the LPP.

Phase behavior of skin lipid mixtures: the effect of cholesterol on lipid organization.

The lipid matrix in the stratum corneum (SC), the upper layer of the skin, plays a critical role in the skin barrier. The matrix consists of ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs). In human SC, these lipids form two coexisting crystalline lamellar phases with periodicities of approximately 6 and 13 nm. In the studies reported here, we investigated the effect of CHOL on lipid organization in each of these lamellar phases separately. For this purpose, we used lipid model mixtures. Our studies revealed that CHOL is imperative for the formation of each of the lamellar phases. At low CHOL levels, the formation of the lamellar phases was dramatically changed: a minimum 0.2 CHOL level in the CER/CHOL/FFA (1 : 0.2 : 1) mixture is required for the formation of each of the lamellar phases. Furthermore, CHOL enhances the formation of the highly dense orthorhombic lateral packing. The gradual increment of CHOL increases the fraction of lipids forming the very dense orthorhombic lateral packing. Therefore, these studies demonstrate that CHOL is an indispensable component of the SC lipid matrix and is of fundamental importance for appropriate dense lipid organization and thus important for the skin barrier function.

The role of ceramide chain length distribution on the barrier properties of the skin lipid membranes.

The skin barrier function is provided by the stratum corneum (SC). The lipids in the SC are composed of three lipid classes: ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs) which form two crystalline lamellar structures. In the present study, we investigate the effect of CER chain length distribution on the barrier properties of model lipid membranes mimicking the lipid composition and organization of SC. The membranes were prepared with either isolated pig CERs (PCERs) or synthetic CERs. While PCERs have a wide chain length distribution, the synthetic CERs are quite uniform in chain length. The barrier properties were examined by means of permeation studies using hydrocortisone as a model drug. Our studies revealed a reduced barrier in lipid membranes prepared with PCERs compared to synthetic CERs. Additional studies revealed that a wider chain length distribution of PCERs results in an enhanced hexagonal packing and increased conformational disordering of the lipid tails compared to synthetic CERs, while the lamellar phases did not change. This demonstrates that the chain length distribution affects the lipid barrier by reducing the lipid ordering and density within the lipid lamellae. In subsequent studies, the effect of increased levels of FFAs or CERs with a long acyl chain in the PCERs membranes was also studied. These changes in lipid composition enhanced the level of orthorhombic packing, reduced the conformational disordering and increased the barrier of the lipid membranes. In conclusion, the CER chain length distribution is an important key factor for maintaining a proper barrier.

The important role of stratum corneum lipids for the cutaneous barrier function.

The skin protects the body from unwanted influences from the environment as well as excessive water loss. The barrier function of the skin is located in the stratum corneum (SC). The SC consists of corneocytes embedded in a lipid matrix. This lipid matrix is crucial for the lipid skin barrier function. This paper provides an overview of the reported SC lipid composition and organization mainly focusing on healthy and diseased human skin. In addition, an overview is provided on the data describing the relation between lipid modulations and the impaired skin barrier function. Finally, the use of in vitro lipid models for a better understanding of the relation between the lipid composition, lipid organization and skin lipid barrier is discussed. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.

Localization of cholesterol and fatty acid in a model lipid membrane: a neutron diffraction approach.

The intercellular lipid matrix of the skin's stratum corneum serves to protect the body against desiccation and simultaneously limits the passage of drugs and other xenobiotics into the body. The matrix is made up of ceramides, free fatty acids, and cholesterol, which are organized as two coexisting crystalline lamellar phases. In studies reported here, we sought to use the technique of neutron diffraction, together with the device of isotopic (H/D) substitution, to determine the molecular architecture of the lamellar phase having a repeat distance of 53.9 ± 0.3 Å. Using hydrogenous samples as well as samples incorporating perdeuterated (C24:0) fatty acids and selectively deuterated cholesterol, the diffraction data obtained were used to construct neutron scattering length density profiles. By this means, the locations within the unit cell were determined for the cholesterol and fatty acids. The cholesterol headgroup was found to lie slightly inward from the unit cell boundary and the tail of the molecule located 6.2 ± 0.2 Å from the unit cell center. The fatty acid headgroups were located at the unit cell boundary with their acyl chains straddling the unit cell center. Based on these results, a molecular model is proposed for the arrangement of the lipids within the unit cell.

Disposition of ceramide in model lipid membranes determined by neutron diffraction.

The lipid matrix present in the uppermost layer of the skin, the stratum corneum, plays a crucial role in the skin barrier function. The lipids are organized into two lamellar phases. To gain more insight into the molecular organization of one of these lamellar phases, we performed neutron diffraction studies. In the diffraction pattern, five diffraction orders were observed attributed to a lamellar phase with a repeat distance of 5.4 nm. Using contrast variation, the scattering length density profile could be calculated showing a typical bilayer arrangement. To obtain information on the arrangement of ceramides in the unit cell, a mixture that included a partly deuterated ceramide was also examined. The scattering length density profile of the 5.4-nm phase containing this deuterated ceramide demonstrated a symmetric arrangement of the ceramides with interdigitating acyl chains in the center of the unit cell.

New insights into the stratum corneum lipid organization by X-ray diffraction analysis.

The characteristic 13-nm lamellar phase that is formed by lipids in the outermost layer of the skin, the stratum corneum (SC), is very important for the barrier function of the skin. To gain more insight into the molecular organization of this lamellar phase, we performed small-angle x-ray diffraction (SAXD) using various lipid mixtures mimicking the lipid composition in SC. In the SAXD pattern of each mixture, at least seven diffraction orders were observed, attributed to the lamellar phase with a repeat distance ranging from 12.1 to 13.8 nm. Using the sampling method based on the variation in repeat distance, we selected phase angles for the first six diffraction orders. Using these phase angles for the lamellar phase, a high-resolution electron density distribution could be calculated. Subsequently, from SAXD patterns of isolated SC, the electron density distribution of the lamellar phase was also calculated and appeared to be very similar to that in the lipid mixtures. This demonstrates that the lipid mixtures serve as an excellent model for the lipid organization in SC, not only with respect to the repeat distance, but also in terms of the electron density distribution within the unit cell.

Infrared spectroscopy studies of mixtures prepared with synthetic ceramides varying in head group architecture: coexistence of liquid and crystalline phases.

The barrier function of the skin is provided by the stratum corneum (SC), the outermost layer of the skin.Ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs) are present in SC and form highly ordered crystalline lipid lamellae. These lamellae are crucial for a proper skin barrier function. In the present study,Fourier transform infrared spectroscopy was used to examine the lipid organization of mixtures prepared from synthetic CERs with CHOL and FFAs. The conformational ordering and lateral packing of these mixtures showed great similarities to the lipid organization in SC and lipid mixtures prepared with native CERs.Therefore, mixtures with synthetic CERs serve as an excellent tool for studying the effect of molecular architecture of CER subclasses on the lipid phase behavior. In SC the number of OH-groups in the head groups of CER subclasses varies. Furthermore, acylCERs with a linoleic acid chemically bound to a long acyl chain are also identified. The present study revealed that CER head group architecture affects the lateral packing and conformational ordering of the CER:CHOL:FFA mixtures. Furthermore, while the majority of the lipids form a crystalline packing, the linoleate moiety of the acylCERs participates in a "pseudo fluid" phase.

Interaction of lipophilic moisturizers on stratum corneum lipid domains in vitro and in vivo.

Dry skin symptoms such as scaling and itching are often treated with lipophilic moisturizers. The aim of this study was to investigate the effect of lipophilic moisturizers on the stratum corneum (SC) ultra-structure and lipid organization. Lipophilic moisturizers were applied on the forearms of 4 healthy volunteers for 3 h. Subsequently, the application sites were tape stripped, and selected tape strips prepared for Freeze Fracture Electron Microscopy (FFEM), a method to visualize the SC intercellular lipid parallel to the skin surface. To investigate the effect of lipid moisturizers on the lipid lamellae, isolated SC was pretreated with the lipophilic moisturizers for 24 h prior to performing small angle X-ray diffraction (SAXD) measurements. Additionally, the lipid organization of mixtures prepared with ceramides, cholesterol, free fatty acids and lipophilic moisturizer in a 2:1:1:1 molar ratio were studied using SAXD. The FFEM data (in vivo) as well as the SAXD data (in vitro) show that the lipophilic moisturizers do not change the lipid lamellar organization in the SC. Addition of 20% m/m lipophilic moisturizer to the ceramide:cholesterol:free fatty acids mixture did not inhibit the formation of the long periodicity phase, the characteristic lamellar phase in the SC, even though there was clear evidence that two of the three moisturizers were at least partially incorporated in the long periodicity phase. Concluding, all findings suggest that the lipophilic moisturizers investigated in this study do not drastically change the lamellar organization of the SC intracellular lipid matrix, but that the moisturizers form separate domains in the SC, as was visualized by FFEM.

Infrared spectroscopic study of stratum corneum model membranes prepared from human ceramides, cholesterol, and fatty acids.

The outermost layer of the skin, the stratum corneum, consists of corneocytes surrounded by lipid domains. The main lipid classes in stratum corneum are cholesterol, ceramides (CER), and free fatty acids forming two crystalline lamellar phases. However, only limited information is available on whether the various lipid classes participate in the same crystalline lattices or if separate domains are formed within the lipid lamellae. In this article infrared spectroscopic studies are reported of hydrated mixtures prepared from cholesterol, human CER, and free fatty acids. Evaluation of the methylene stretching vibrations revealed a conformational disordering starting at approximately 60 degrees C for all mixtures. Examination of the rotational ordering (scissoring and rocking vibrations) of mixtures prepared from equimolar cholesterol and CER with a variation in the level of free fatty acids showed that at lower free fatty acid content orthorhombic and hexagonal domains coexist in the lipid lamellae. Increasing the fatty acid level to an equimolar cholesterol/CER/fatty acid mixture reveals the dominant presence of an orthorhombic lattice, confirming x-ray diffraction studies. Replacing the protonated free fatty acid chains by their perdeuterated counterparts demonstrates that free fatty acids and CER participate in the same orthorhombic lattice up to a level of slightly less than 1:1:0.75 cholesterol/CER/free fatty acids molar ratio but that free fatty acids also form separate domains within the lipid lamellae at equimolar ratios at room temperature. However, no evidence for this has been observed at 32 degrees C. Extrapolating these findings to the situation in stratum corneum led us conclude that in stratum corneum, fatty acids and CER participate in the orthorhombic lattice at 32 degrees C, the skin temperature.

The phase behaviour of skin lipid mixtures based on synthetic ceramides.

The lipid lamellae present in the outermost layer of the skin, the stratum corneum (SC), form the main barrier for diffusion of molecules across the skin. The main lipid classes in SC are cholesterol (CHOL), free fatty acids (FFA) and at least nine classes of ceramides (CER), referred to as CER1 to CER9. In the present study the phase behaviour of four synthetic CER, either single or mixed with CHOL or CHOL and FFA, has been studied using small and wide angle X-ray diffraction. The lipid mixtures showed complex phase behaviour with coexistence of several phases. The results further revealed that the presence of synthetic CER1 as well as a proper composition of the other CER in the mixture were crucial for the formation of a phase with a long periodicity, characteristic for SC lipid phase behaviour. Only a mixture containing synthetic CER1 and CER3, CHOL and FFA showed similar phase behaviour to that of SC.

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.

The influence of two azones and sebaceous lipids on the lateral organization of lipids isolated from human stratum corneum.

The main problem with topical application of compounds to administer drugs to and regulate drug levels in a human body, is the barrier formed by the intercellular lipid matrix of the stratum corneum (SC). In a search for possibilities to overcome this barrier function, a good understanding of the organization and phase behavior of these lipids is required. SC lipid model studies especially provide a wealth of information with respect to the lipid organization and the importance of certain subclasses of lipids for the structure. Previously, we have shown that electron diffraction (ED) provides detailed information on the lateral lipid packing in both intact SC (G.S.K. Pilgram et al., J. Invest. Dermatol. 113 (1999) 403) and SC lipid models (G.S.K. Pilgram et al., J. Lipid Res. 39 (1998) 1669). In the present study, we used ED to examine the influence of two azones and sebaceous lipids on the lateral phase behavior of lipids isolated from human SC. We established that human SC lipids are arranged in an orthorhombic packing pattern. Upon mixing with the two enhancers the orthorhombic packing pattern was still observed; however, an additional fluid phase became more apparent. In mixtures with sebaceous lipids, the presence of the hexagonal lattice increased. These findings provide a basis for the mechanism by which these enhancers and sebaceous lipids interact with human SC lipids.

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.

Barrier characteristics of different human skin types investigated with X-ray diffraction, lipid analysis, and electron microscopy imaging.

The stratum corneum requires ceramides, cholesterol, and fatty acids to provide the cutaneous permeability barrier. The lipids are organized in intercellular membranes exhibiting short- and long-periodicity lamellar phases. In recent years, the phase behavior of barrier lipid mixtures has been studied in vitro. The relationship of human stratum corneum lipid composition to membrane organization in vivo, however, has not been clearly established. Furthermore, the special function of the different ceramide species in the stratum corneum is largely unknown. We examined lipid organization and composition of stratum corneum sheets from different subtypes of healthy human skin (normal, dry, and aged skin). Lipid organization was investigated using X-ray diffraction and demonstrated that the 4.4 nm peak attributed to the long periodicity phase was frequently missing for skin with a low Cer(EOS)/Cer(total) ratio, indicating an important part for Cer(EOS), which contains omega-hydroxy fatty acid (O) ester-linked to linoleic acid (E) and amide-linked to sphingosine (S). A deficiency in the 4. 4 nm peak was predominantly observed in young dry skin. In one case of aged skin, however, and less often in young normal skin this peak was also missing. Furthermore, the ceramide composition of samples without the 4.4 nm peak showed a deficiency of Cer(EOH), which contains 6-hydroxy-4-sphingenine (H), and an increase in Cer(NS) and Cer(AS), which contain nonhydroxy (N) or alpha-hydroxy fatty acids (A). In addition, a 3.4 nm peak attributed to crystalline cholesterol occurred in most cases of aged and dry skin, but was not observed in young normal skin. Our results do not indicate a definite pattern of correlation between lipid organization and types of human skin. They demonstrate, however, that Cer(EOS) and Cer(EOH) are key elements for the molecular organization of the long periodicity lamellar phase in the human 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.