Gert S. Gooris

Structure of human stratum corneum as a function of temperature and hydration: A wide-angle X-ray diffraction study

Wide-angle X-ray diffraction measurements were performed as a function of temperature and hydration on human stratum corneum. Reflections located close to the beam stop (d > 1 nm) could be explained on the basis of two lamellar structures. These findings confirmed results obtained with small-angle X-ray diffraction (Bouwstra et al., J. Invest. Dermatol., 97 (1991b) 1005–1012), in which the several complicated diffraction peaks could be ascribed to two lamellar structures with repeat distances of 6.4 and 13.4 nm. In the wide-angle region of the diffraction pattern, we found two diffraction lines, that are characteristic of orthorhombic and hexagonal lateral packing of the lipids. Moreover, the strongest reflections of polycrystalline cholesterol were detected. Upon heating stratum corneum, a phase transition from an orthorhombic to a hexagonal phase in the lateral packing of the lipids at 40°C was observed. Between 60 and 75°C disordering of the lamellar structure occurred, while the hexagonal lateral packing was still present. Between 75 and 95°C, the hexagonal lateral packing disappeared. No changes were found in the reflection lines characteristic of protein. After recrystallisation of the lipids from 120 or 95°C, several higher diffraction orders of the lamellar structure with a repeat distance of 13.4 nm were found. Variation in the hydration level between 6 and 40% w/w did not lead to a shift in the position of the reflections characteristic of lateral packing. In addition, in the recent paper cited above, swelling of the bilayers was not observed, indicating that no water is absorbed between the lamellar regions of human stratum corneum.

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.

Modes of action of terpene penetration enhancers in human skin; Differential scanning calorimetry, small-angle X-ray diffraction and enhancer uptake studies

The mechanisms through which the terpenes, d-limonene, 1–8-cineole and nerolidol, increase the permeability of human stratum corneum (s.c.) and the mechanisms underlying propylene glycol (PG)/terpene synergy were investigated using differential scanning calorimetry (DSC), small-angle X-ray diffraction (SAXD) and enhancer uptake studies. DSC experiments identified two major lipid transitions at 72° and 83°C. d-Limonene reduced the temperatures of both transitions by approx. 20°C without affecting their enthalpies (ΔH). 1–8-Cineole also reduced the temperatures of both transitions by approx. 20°C but also reduced ΔH for the first major lipid transition; ΔH for the second was unaffected. d-Limonene increased the combined entropy change (ΔS) associated with both lipid transitions by 11% whereas 1–8-cineole decreased ΔS by 32%. The decrease in ΔS produced by 1–8-cineole provides evidence that this enhancer is lipid disruptive at normal skin temperature. Nerolidol reduced the transition temperatures of both major lipid transitions by approx. 4°C and also decreased their cooperativity. Reduced bilayer cooperativity indicates that this enhancer also disrupts the intercellular lipids. Lack of a clear baseline prevented accurate measurement of ΔH and ΔS values following nerolidol treatment. SAXD experiments showed that d-limonene and 1–8-cineole act to reduce the intensity of lipid based reflections. Decreases in reflection intensities may be linked to a disruption of lipid packing within the bilayers and/or to a disturbance in the stacking of the bilayers. Treatment with nerolidol did not markedly reduce the intensities of the bilayer based reflections. Uptake studies revealed that large quantities of terpenes can be accommodated by the s.c. (mean uptake of d-limonene, 1–8-cineole and nerolidol was 8.90%, 26.2% and 39.6% w/w dry s.c.). The possibility that terpene enhancers pool in the s.c. is discussed. DSC and SAXD investigations provided fragmented evidence that PG/terpene synergy may produce enhanced lipid bilayer disruption. Enhancer uptake studies showed that PG does not significantly increase terpene delivery to the s.c. above that provided by application of neat terpenes.

Increase in short-chain ceramides correlates with an altered lipid organization and decreased barrier function in atopic eczema patients

A hallmark of atopic eczema (AE) is skin barrier dysfunction. Lipids in the stratum corneum (SC), primarily ceramides, fatty acids, and cholesterol, are crucial for the barrier function, but their role in relation to AE is indistinct. Filaggrin is an epithelial barrier protein with a central role in the pathogenesis of AE. Nevertheless, the precise causes of AE-associated barrier dysfunction are largely unknown. In this study, a comprehensive analysis of ceramide composition and lipid organization in nonlesional SC of AE patients and control subjects was performed by means of mass spectrometry, infrared spectroscopy, and X-ray diffraction. In addition, the skin barrier and clinical state of the disease were examined. The level of ceramides with an extreme short chain length is drastically increased in SC of AE patients, which leads to an aberrant lipid organization and a decreased skin barrier function. Changes in SC lipid properties correlate with disease severity but are independent of filaggrin mutations. We demonstrate for the first time that changes in ceramide chain length and lipid organization are directly correlated with the skin barrier defects in nonlesional skin of AE patients. We envisage that these insights will provide a new therapeutic entry in therapy and prevention of AE.

The lipid and protein structure of mouse stratum corneum: a wide and small angle diffraction study.

The structure of mouse stratum corneum was investigated using small and wide angle X-ray scattering. Diffraction patterns were collected as a function of temperature and hydration. The lipid lamellar structure is characterized by a repeat distance of 13.4 nm. Occasionally a second lipid lamellar phase has been found with a repeat distance of 6.1 nm. Upon hydration neither swelling of the lamellae nor lateral swelling of the lipids was found. On the basis of these facts it was concluded that the size of the crystallographic unit cell of the lipid structure is insensitive to the water content. The 13.4 nm lamellar phase disappeared upon heating to 55 degrees C. At 45 degrees C the orthorhombic lateral packing disappeared. At this temperature only an hexagonal and liquid lateral packing of the lipids was observed. The hexagonal lateral packing transformed to a liquid one between 45 degrees C and 80 degrees C. Model calculations were carried out to obtain the electron density profile of the lamellar structure. In all models three electron lucent regions were fitted between which electron dense regions are located indicating that the 13.4 nm lamellar structure consist of three bilayers.

New Aspects of the Skin Barrier Organization

In the superficial layer of the skin, the stratum corneum (SC), the lipids form two crystalline lamellar phases with periodicities of 6.4 and 13.4 nm (long-periodicity phase). The main lipid classes in SC are ceramides, free fatty acids and cholesterol. Studies with mixtures prepared with isolated ceramides revealed that cholesterol and ceramides are very important for the formation of the lamellar phases, and the presence of ceramide 1 is crucial for the formation of the long-periodicity phase. This observation and the broad-narrow-broad sequence of lipid layers in the 13.4-nm phase led us to propose a molecular model for this phase. This consists of one narrow central lipid layer with fluid domains on both sides of a broad layer with a crystalline structure. This model is referred to as ‘the sandwich model’. While the presence of free fatty acids does not substantially affect the lipid lamellar organization, it is crucial for the formation of the orthorhombic sublattice, since the addition of free fatty acids to cholesterol/ceramide mixtures results in transition from a hexagonal to a crystalline lipid phase. Studies examining lipid organization in SC derived from dry or lamellar X-linked ichthyosis skin revealed that in native tissue the role of ceramide 1 and free fatty acids is similar to that observed with mixtures prepared with isolated SC lipids. From this we conclude that the results obtained with lipid mixtures can be used to predict the SC lipid organization in native tissue.

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.

Small angle X-ray scattering: possibilities and limitations in characterization of vesicles.

The use of small angle X-ray scattering (SAXS) for characterization of lipid vesicle dispersions is described. The effect of curvature of the membrane, the presence of proteins in the core and on the surface of the membrane, variations in membrane thickness and distribution in the number of bilayers of the vesicles in the dispersion on the scattering curve is discussed. Concerning unilamellar vesicles, either the membrane curvature of vesicles smaller than 50 nm or variations in membrane thickness result in a disappearance of the first node in the scattering curve, even if the bilayer is symmetric with respect to the electron density distribution. In the case of dispersion in which unilamellar as well as multilamellar vesicles are present it is shown that a small fraction of multilamellar liposomes changes the scattering curve dramatically. Liposomes were prepared from various compositions of dipalmitoylphosphatidylcholine (DPPC) and cholesterol hemisuccinate (CHEMS) by the film method. The electron density profile of the bilayers and distribution in the number of bilayers of the liposome dispersions were determined. The average number of bilayers increased as a function of the decrease in CHEMS content. Liposomes with higher CHEMS content than 10 mol% were unilamellar. It seems that increase in charge intercalated in the bilayers resulted in unilamellar vesicles.

Is an orthorhombic lateral packing and a proper lamellar organization important for the skin barrier function

The lipid organization in the stratum corneum (SC), plays an important role in the barrier function of the skin. SC lipids form two lamellar phases with a predominantly orthorhombic packing. In previous publications a lipid model was presented, referred to as the stratum corneum substitute (SCS), that closely mimics the SC lipid organization and barrier function. Therefore, the SCS serves as a unique tool to relate lipid organization with barrier function. In the present study we examined the effect of the orthorhombic to hexagonal phase transition on the barrier function of human SC and SCS. In addition, the SCS was modified by changing the free fatty acid composition, resulting in a hexagonal packing and perturbed lamellar organization. By measuring the permeability to benzoic acid as function of temperature, Arrhenius plots were constructed from which activation energies were calculated. The results suggest that the change from orthorhombic to hexagonal packing in human SC and SCS, does not have an effect on the permeability. However, the modified SCS revealed an increased permeability to benzoic acid, which we related to its perturbed lamellar organization. Thus, a proper lamellar organization is more crucial for a competent barrier function than the presence of an orthorhombic lateral packing.

The role of ceramides 1 and 2 in the stratum corneum lipid organisation.

A mixture of ceramide 1 and ceramide 2 (CER(1 + 2)) was isolated from pig stratum corneum and mixed in various molar ratios with cholesterol (CHOL) or with CHOL and palmitic acid (PA). The mixtures were hydrated in a buffer solution of pH 5.0 and their phase behaviour was studied by wide- and small-angle X-ray diffraction. The small-angle diffraction curve of the CHOL/CER(1 + 2) mixture at a molar ratio of 0.4 revealed the presence of only one peak at a spacing of 6.7 nm. Increasing the amount of CHOL to a molar ratio of 0.6 was accompanied by a shift of this peak to a smaller spacing (5.7 nm) and the appearance of two weak peaks at 11.8 and 4.1 nm spacings. Increasing the CHOL content to an equimolar ratio resulted in the appearance of two lamellar phases with periodicities of 5.5 and 12 nm, respectively. In a CHOL/CER(1 + 2) mixture at a molar ratio of 2 the periodicities of the two phases were 5.6 and 12 nm, respectively. From these observations it was concluded that the CHOL/CER(1 + 2) mixtures exerted similar phase behaviour, as reported earlier for intact SC (Bouwstra et al. (1995) J. Lipid Res. 36, 496-504) and for mixtures (Bouwstra et al. (1996) J. Lipid Res., in press) prepared from CHOL and total ceramide fraction (CER) isolated from pig stratum corneum. However, in the CHOL/CER mixtures a lower relative amount of CHOL was required to acquire these lamellar phases, indicating that at low CHOL contents, CER 3, 4, 5 and 6 play a crucial role in the formation of the lamellar phases. Furthermore, the solubility of CHOL in the mixtures increased in the presence of CER 1, suggesting its important role for the barrier function of the skin. When palmitic acid (PA) was included, the phase behaviour of the CHOL/CER(1 + 2)/PA mixture was more complex. Next to two lamellar phases, an additional phase with a spacing of 3.77 nm was observed, never seen in intact stratum corneum. In the absence of CHOL, the wide-angle diffraction pattern of the CER(1 + 2) revealed one sharp reflection at 0.456 nm and two diffuse reflections at 0.430, 0.417 nm and 0.395 nm, indicating the presence of a crystalline sublattice. In an equimolar mixture of CHOL/CER(1 + 2) no sharp 0.456 nm reflection was observed indicating a more disordered packing. Furthermore, phase separation of CHOL occurred, this conclusion is based on the presence of reflections corresponding to polycrystalline cholesterol monohydrate. These findings indicate that the lateral packing of mixtures of CHOL/CER(1 + 2) is more complex than that of the CHOL/CER mixtures that reveals a hexagonal lateral packing.