Alteration of stratum corneum ceramide profiles in spontaneous canine model of atopic dermatitis

Ceramides (CERs) in the stratum corneum (SC) are thought to play a key role in cutaneous barrier function. It has been reported that human SC contains 11 free CER classes and that their profiles are altered in humans with atopic dermatitis (AD). Although decreased proportions of free CERs or quantities of protein-bound CERs in the SC have been reported in dogs with AD, the overall profile of CERs in the canine SC has not been fully elucidated. The aim of this study was thus to investigate the profile of free CERs in the canine SC and to identify alterations in the CER profiles in dogs with AD. Normal-phase liquid chromatography-electrospray ionization-mass spectrometry indicated 11 clusters of peaks for free CER classes, similar to those recognized in the human SC. The fractions of free SC CER in dogs with AD and in breed- and age-matched healthy dogs were quantitatively compared using high-performance thin-layer chromatography. CER[EOS], CER[EOP] and CER[NP], which are known to be decreased in the skin of humans with AD, were also decreased in the skin of dogs with AD. These findings highlight canine AD as a spontaneous animal model for investigating the disruption of CER-associated cutaneous barrier functions in the corresponding human disease.     

Topically applied fatty acids are elongated before incorporation in the stratum corneum lipid matrix in compromised skin

In several skin diseases, both the lipid composition and organization in the stratum corneum (SC) are altered which contributes to the impaired skin barrier function in patients. One of the approaches for skin barrier repair is treatment with topical formulations to normalize SC lipid composition and organization. Vernix caseosa (VC), a white cheesy cream on the skin during gestational delivery, has shown to enhance skin barrier repair. In this study, we examined how a fatty acid (FA) containing formulation mimicking the lipid composition of VC interacts with the lipid matrix in the SC. The formulation was applied on ex vivo human skin after SC removal. Subsequently, the ex vivo human skin generated SC during culture. The effect of FA containing formulations on the lipid organization and composition in the regenerated SC was analysed by Fourier transform infrared (FTIR) spectroscopy and liquid chromatography mass spectroscopy (LC/MS), respectively. FTIR results demonstrate that the FAs are intercalated in the lipid matrix of the regenerated SC and partition in the same lattice with the endogenous SC lipids, thereby enhancing the fraction of lipids forming an orthorhombic (very dense) packing in the SC. LC/MS data show that the topically applied FAs are elongated before intercalation in the lipid matrix and are thus involved in the lipid biosynthesis in the skin.     

Molecular interactions of plant oil components with stratum corneum lipids correlate with clinical measures of skin barrier function

Plant‐derived oils consisting of triglycerides and small amounts of free fatty acids (FFAs) are commonly used in skincare regimens. FFAs are known to disrupt skin barrier function. The objective of this study was to mechanistically study the effects of FFAs, triglycerides and their mixtures on skin barrier function. The effects of oleic acid (OA), glyceryl trioleate (GT) and OA/GT mixtures on skin barrier were assessed in vivo through measurement of transepidermal water loss (TEWL) and fluorescein dye penetration before and after a single application. OA's effects on stratum corneum (SC) lipid order in vivo were measured with infrared spectroscopy through application of perdeuterated OA (OA‐d₃₄). Studies of the interaction of OA and GT with skin lipids included imaging the distribution of OA‐d₃₄ and GT ex vivo with IR microspectroscopy and thermodynamic analysis of mixtures in aqueous monolayers. The oil mixtures increased both TEWL and fluorescein penetration 24 h after a single application in an OA dose‐dependent manner, with the highest increase from treatment with pure OA. OA‐d₃₄ penetrated into skin and disordered SC lipids. Furthermore, the ex vivo IR imaging studies showed that OA‐d₃₄ permeated to the dermal/epidermal junction while GT remained in the SC. The monolayer experiments showed preferential interspecies interactions between OA and SC lipids, while the mixing between GT and SC lipids was not thermodynamically preferred. The FFA component of plant oils may disrupt skin barrier function. The affinity between plant oil components and SC lipids likely determines the extent of their penetration and clinically measurable effects on skin barrier functions.     

The lipid alterations in the stratum corneum of dogs with atopic dermatitis are alleviated by topical application of a sphingolipid-containing emulsion

Background. Atopic dermatitis (AD) results from an altered skin barrier associated with defects in the lipid composition of the skin. Dogs with AD present similar clinical symptoms to humans, and may be a useful model for investigations into AD. Aim. To analyse the changes occurring in the lipids of the stratum corneum (SC) of dogs with AE after 3 weeks of topical treatment with an emulsion containing ceramides, free fatty acids (FFAs) and cholesterol (skin lipid complex; SLC). Methods. Nonlesional SC was collected by tape stripping from control and treated areas. Free and protein‐bound lipids were purified, and the various classes were isolated by column chromatography, analysed by thin‐layer chromatography and assayed. Results. Ceramides, FFA and cholesterol were all found to be lower in the skin of untreated dogs with AD than in normal dogs, and the topical treatment resulted in significantly increased values for ceramides. Conversely, only trace amounts of glucosylceramides were present in normal SC, but a high concentration (27 μg per mg protein) was detected in canine atopic SC, which disappeared after treatment with SLC. There was a heterogeneous distribution of all of the lipids in the different layers of canine atopic SC, which was more pronounced for protein‐bound than for free lipids. Following topical treatment, the protein‐bound lipid content normalized. Conclusions. Topical treatment with SLC resulted in a significant improvement of the lipid biosynthesis of keratinocytes in atopic dogs, thereby potentially enabling the formation of a tighter epidermal barrier.     

Barrier lipid composition and response to plasma lipids: A direct comparison of mouse dorsal back and ear skin

The skin of the ear and the back are frequently selected sites in skin research using mouse models. However, distinct responses to treatment have been described between these two sites in several studies. Despite the crucial role of the stratum corneum (SC) in the skin barrier function of both dorsal back and ear skin, it remains unclear whether differences in lipid composition might underlie altered responses. Here, we compared the skin morphology and the barrier lipid composition of the ear with the back skin of wild-type mice. The ear contained more corneocyte layers in the SC and its barrier lipid composition was enriched with sphingosine ceramide subclasses, especially the short ones with a total chain length of 33-34 carbons. The free fatty acid (FFA) profile in the ear skin shifted towards shorter chains, significantly reducing the mean chain length to 23.3 vs 24.7 carbons in the back skin. In line, FFA species in the ear displayed a twofold increase in unsaturation index (P < .001). Gene expression in the ear skin revealed low expression of genes involved in lipid synthesis and uptake, indicating a reduced metabolic activity. Finally, the effects of hypercholesterolaemia on SC FFA composition was compared in ear and back skin of apolipoprotein E knockout (APOE^−/−) mice. Interestingly, the FFA profile in APOE^−/− ear skin was minimally affected, while the FFA composition in the back skin was markedly changed in response to hypercholesterolaemia. In conclusion, ear and back skin have distinct barrier lipids and respond differently to elevated plasma cholesterol.     

Distinct barrier integrity phenotypes in filaggrin-related atopic eczema following sequential tape stripping and lipid profiling

Background Filaggrin gene (FLG) loss‐of‐function mutations have been shown to represent the strongest so far known genetic risk factor for atopic dermatitis (AD). Whereas the barrier characteristics in FLG mutation carriers under baseline conditions have been investigated, there are only limited data on the permeability barrier function in filaggrin‐AD under compromised conditions. Aim We investigated: (i) stratum corneum (SC) integrity/cohesion; (ii) barrier recovery after controlled mechanical and irritant‐induced barrier abrogation; and (iii) the lipid composition of the non‐lesional and lesional skin of AD patients harbouring the European R501X, 2282del4, 3702delG, R2447X or S3247X FLG variants. Methods Thirty‐seven AD patients (14 FLG mutation carriers and 23 non‐carriers) and 20 healthy controls participated in the study. Stratum corneum integrity/cohesion was assessed by measurement of transepidermal water loss (TEWL) and amount of removed protein following sequential tape stripping. Barrier recovery was monitored by repeated measurements of TEWL and erythema up to 96 h after barrier abrogation. Samples for lipid analysis were obtained from non‐lesional and lesional skin using the cyanoacrylate method. Results Tape stripping revealed distinct genotype‐related impairment of the SC integrity/cohesion. No differences in the rate of barrier recovery among the groups were found. The SC lipid analysis revealed significant differences regarding the percentage amount of cholesterol, ceramide/cholesterol ratio and triglycerides in the uninvolved skin as well as the amounts of free fatty acids, CER[EOH] and triglycerides in the skin lesions of the AD FLG mutation carriers. Conclusions Our results provide evidence for discernible FLG‐related barrier integrity phenotypes in atopic eczema.     

Acylceramide head group architecture affects lipid organization in synthetic ceramide mixtures

The lipid organization in the upper layer of the skin, the stratum corneum (SC), is important for the skin barrier function. This lipid organization, including the characteristic 13 nm lamellar phase, can be reproduced in vitro with mixtures based on cholesterol, free fatty acids and natural as well as synthetic ceramides (CER). In human SC, nine CER classes have been identified (CER1-CER9). Detailed studies on the effect of molecular structure of individual ceramides on the SC lipid organization are only possible with synthetic lipid mixtures, as their composition can be accurately chosen and systematically modified. In the present study, small-angle X-ray diffraction was used to examine the organization in synthetic lipid mixtures of which the synthetic ceramide fraction was prepared with sphingosine-based CER1 or phytosphingosine-based CER9. The latter acylceramide contains an additional hydroxyl group at the sphingoid backbone. The results show that a gradual increase in CER1 level consistently promotes the formation of the 13 nm lamellar phase and that partial replacement of CER1 by CER9 does not affect the phase behavior. Interestingly, complete substitution of CER1 with CER9 reduces the formation of the long periodicity phase and results in phase separation of CER9.     

The human skin barrier is organized as stacked bilayers of fully extended ceramides with cholesterol molecules associated with the ceramide sphingoid moiety

The skin barrier is fundamental to terrestrial life and its evolution; it upholds homeostasis and protects against the environment. Skin barrier capacity is controlled by lipids that fill the extracellular space of the skin's surface layer-the stratum corneum. Here we report on the determination of the molecular organization of the skin's lipid matrix in situ, in its near-native state, using a methodological approach combining very high magnification cryo-electron microscopy (EM) of vitreous skin section defocus series, molecular modeling, and EM simulation. The lipids are organized in an arrangement not previously described in a biological system-stacked bilayers of fully extended ceramides (CERs) with cholesterol molecules associated with the CER sphingoid moiety. This arrangement rationalizes the skin's low permeability toward water and toward hydrophilic and lipophilic substances, as well as the skin barrier's robustness toward hydration and dehydration, environmental temperature and pressure changes, stretching, compression, bending, and shearing.     

Permeability Barrier Abnormality of Hairless Mouse Epidermis after Topical Corticosteroid: Characterization of Stratum Corneum Lipids by Ruthenium Tetroxide Staining and High-Performance Thin-Layer Chromatography

Topical corticosteroids (TCS) are among the most frequently used topical therapeutics. Recently, it has been shown that TCS not only has antiproliferative actions, but also inhibits the differentiation of the epidermis and finally perturbates stratum corneum (SC) barrier function. It is well established that epidermal barrier function resides within the intercellular lipids of the SC. However, to date, little is known about the effects of TCS on the structure and composition of SC lipids. We therefore used hairless mouse skin to study the sequential changes of the SC permeability barrier and their intercellular lipids by ruthenium tetroxide staining and high-performance thin-layer chromatography (HPTLC) during topical use of corticosteroids. The results demonstrated a progressive increase in transepidermal water loss accompanied by a diminution in the SC intercellular lipid lamellae, which showed a normal structure of individual lamella. Analysis of lipid composition by HPTLC after a 6-week application of TCS also showed an obvious decrease in all the main components of SC lipids, which are known to constitute the permeability barrier of the skin. In light of these results, our work provides direct morphological evidence that TCS deteriorates the permeability barrier of epidermis when applied to normal skin.     

Modulation of stratum corneum lipid composition and organization of human skin equivalents by specific medium supplements

Our in‐house human skin equivalents contain all stratum corneum (SC) barrier lipid classes, but have a reduced level of free fatty acids (FAs), of which a part is mono‐unsaturated. These differences lead to an altered SC lipid organization and thereby a reduced barrier function compared to human skin. In this study, we aimed to improve the SC FA composition and, consequently, the SC lipid organization of the Leiden epidermal model (LEM) by specific medium supplements. The standard FA mixture (consisting of palmitic, linoleic and arachidonic acids) supplemented to the medium was modified, by replacing protonated palmitic acid with deuterated palmitic acid or by the addition of deuterated arachidic acid to the mixture, to determine whether FAs are taken up from the medium and are incorporated into SC of LEM. Furthermore, supplementation of the total FA mixture or that of palmitic acid alone was increased four times to examine whether this improves the SC FA composition and lipid organization of LEM. The results demonstrate that the deuterated FAs are taken up into LEMs and are subsequently elongated and incorporated in their SC. However, a fourfold increase in palmitic acid supplementation does not change the SC FA composition or lipid organization of LEM. Increasing the concentration of the total FA mixture in the medium resulted in a decreased level of very long chain FAs and an increased level of mono‐unsaturated FAs, which lead to deteriorated SC lipid properties. These results indicate that SC lipid properties can be modulated by specific medium supplements.     

Comparison of structure and organization of cutaneous lipids in a reconstructed skin model and human skin: spectroscopic imaging and chromatographic profiling

The use of animals for scientific research is increasingly restricted by legislation, increasing the demand for human skin models. These constructs present comparable bulk lipid content to human skin. However, their permeability is significantly higher, limiting their applicability as models of barrier function, although the molecular origins of this reduced barrier function remain unclear. This study analyses the stratum corneum (SC) of one such commercially available reconstructed skin model (RSM) compared with human SC by spectroscopic imaging and chromatographic profiling. Total lipid composition was compared by chromatographic analysis (HPLC). Raman spectroscopy was used to evaluate the conformational order, lateral packing and distribution of lipids in the surface and skin/RSM sections. Although HPLC indicates that all SC lipid classes are present, significant differences are observed in ceramide profiles. Raman imaging demonstrated that the RSM lipids are distributed in a non‐continuous matrix, providing a better understanding of the limited barrier function.     

Raman spectroscopy: feasibility of in vivo survey of stratum corneum lipids, effect of natural aging

The main function of the stratum corneum (SC) is for protection against external aggression. This is described as the barrier function. It mainly depends on the presence of a lipid matrix composed of ceramides, free fatty acids, cholesterol and its derivatives in the intercellular spaces. Previous studies have reported the application of Raman spectroscopy to reveal the organization of SC lipids and the state of their barrier functions. Several spectral features are directly informative about the lateral packing and the conformational order. In this work, in vivo Raman spectroscopy is used to asses the state of the SC lipid content and thus its barrier function, directly on the skin. To study the effect of natural aging on the organization of these lipids, spectra were collected from the internal side of the forearms of twenty volunteers aged from 22 to 64. Multivariate data processing enabled separation of the in vivo spectra according to the volunteers' ages. Spectral signatures show small variations, indicating a slight change in the lateral packing of SC lipids with aging of the skin.     

Stratum corneum lipid profile and maturation pattern of corneocytes in the outermost layer of fresh scars: the presence of immature corneocytes plays a much more important role in the barrier dysfunction than do changes in intercellular lipids

BACKGROUND: The functional characteristics of the stratum corneum (SC) of fresh scars as well as keloids and hypertrophic scars are characterized by elevated transepidermal water loss (TEWL) and increased SC hydration. OBJECTIVES: To study the composition of the intercellular lipids and maturation properties of the cornified envelope (CE) of the SC, as these are the most important components for the SC barrier function in fresh scars. METHODS: SC lipids were extracted from the donor site for split-thickness skin grafting soon after re-epithelialization using a cup method, and were analysed with high-performance thin-layer chromatography. CEs, which were prepared from superficial layers of the SC, were double stained with Nile red and anti-involucrin. RESULTS: We found a significant decrease in the proportion of ceramide (CER) in the SC lipids of fresh scars. We also observed changes in the SC CER profile that consisted of an increase in CER 4 and CER 7 and a decrease in CER 3, without any significant change in the proportion of CER 1. These changes were insufficient to explain the remarkably high TEWL recorded in the early stage of fresh scars. In contrast, with double staining of CE with Nile red and anti-involucrin, we detected the presence of numerous immature and less hydrophobic corneocytes in the outermost layer of the SC of fresh scars. Scanning electron microscopy of such corneocytes revealed numerous fine wrinkles on their enlarged surface area. Most of all, we found a closely similar, time-dependent, exponential decrease in the ratio of immature corneocytes with a poorly hydrophobic CE and in the recorded TEWL values in fresh scars. There was a highly significant positive correlation between the proportion of immature corneocytes in the outermost layer of the SC and TEWL values. CONCLUSIONS: These results suggest that the SC barrier dysfunction of the fresh scars is attributable to the presence of immature corneocytes with a less hydrophobic CE, rather than to the changes in SC lipid composition.     

An ex vivo human skin model for studying skin barrier repair

In the studies described in this study, we introduce a novel ex vivo human skin barrier repair model. To develop this, we removed the upper layer of the skin, the stratum corneum (SC) by a reproducible cyanoacrylate stripping technique. After stripping the explants, they were cultured in vitro to allow the regeneration of the SC. We selected two culture temperatures 32°C and 37°C and a period of either 4 or 8 days. After 8 days of culture, the explant generated SC at a similar thickness compared to native human SC. At 37°C, the early and late epidermal differentiation programmes were executed comparably to native human skin with the exception of the barrier protein involucrin. At 32°C, early differentiation was delayed, but the terminal differentiation proteins were expressed as in stripped explants cultured at 37°C. Regarding the barrier properties, the SC lateral lipid organization was mainly hexagonal in the regenerated SC, whereas the lipids in native human SC adopt a more dense orthorhombic organization. In addition, the ceramide levels were higher in the cultured explants at 32°C and 37°C than in native human SC. In conclusion, we selected the stripped ex vivo skin model cultured at 37°C as a candidate model to study skin barrier repair because epidermal and SC characteristics mimic more closely the native human skin than the ex vivo skin model cultured at 32°C. Potentially, this model can be used for testing formulations for skin barrier repair.     

Surfactants have multi-fold effects on skin barrier function

Background

The stratum corneum (SC) is responsible for the barrier properties of the skin and the role of intercorneocyte skin lipids, particularly their structural organization, in controlling SC permeability is acknowledged. Upon contacting the skin, surfactants interact with the SC components leading to barrier damage.

Objective

To improve knowledge of the effect of several classes of surfactant on skin barrier function at three different levels.

Methods

The influence of treatments of human skin explants with six non-ionic and four ionic surfactant solutions on the physicochemical properties of skin was investigated. Skin surface wettability and polarity were assessed through contact angle measurements. Infrared spectroscopy allowed monitoring the SC lipid organization. The lipid extraction potency of surfactants was evaluated thanks to HPLC-ELSD assays.

Results

One anionic and one cationic surfactant increased the skin polarity by removing the sebaceous and epidermal lipids and by disturbing the organization of the lipid matrix. Another cationic surfactant displayed a detergency effect without disturbing the skin barrier. Several non-ionic surfactants disturbed the lipid matrix organization and modified the skin wettability without any extraction of the skin lipids. Finally two non-ionic surfactants did not show any effect on the investigated parameters or on the skin barrier.

Conclusions

The polarity, the organization of the lipid matrix and the lipid composition of the skin allowed describing finely how surfactants can interact with the skin and disturb the skin barrier function.

     

Knock‐down of filaggrin does not affect lipid organization and composition in stratum corneum of reconstructed human skin equivalents

Human skin mainly functions as an effective barrier against unwanted environmental influences. The barrier function strongly relies on the outermost layer of the skin, the stratum corneum (SC), which is composed of corneocytes embedded in an extracellular lipid matrix. The importance of a proper barrier function is shown in various skin disorders such as atopic dermatitis (AD), a complex human skin disorder strongly associated with filaggrin (FLG) null mutations, but their role in barrier function is yet unclear. To study the role of FLG in SC barrier properties in terms of SC lipid organization and lipid composition, we generated an N/TERT‐based 3D‐skin equivalent (NSE) after knock‐down of FLG with shRNA. In these NSEs, we examined epidermal morphogenesis by evaluating the expression of differentiation markers keratin 10, FLG, loricrin and the proliferation marker ki67. Furthermore, the SC was extensively analysed for lipid organization, lipid composition and SC permeability. Our results demonstrate that FLG knock‐down (FLG‐KD) did not affect epidermal morphogenesis, SC lipid organization, lipid composition and SC permeability for a lipophilic compound in NSEs. Therefore, our findings indicate that FLG‐KD alone does not necessarily affect the functionality of a proper barrier function.     

Deficiency of epidermal protein-bound omega-hydroxyceramides in atopic dermatitis

Atopic dermatitis is a common skin disease of unknown etiology with an impaired permeability barrier function. To learn more about the molecular pathology in lesional skin, we analyzed levels of free extractable as well as protein-bound barrier lipids in the epidermis of atopic dermatitis subjects. The amount of protein-bound omega-hydroxyceramides in healthy epidermis comprised 46-53 wt% of total protein-bound lipids, whereas this percentage was decreased to 23-28 wt% in nonlesional areas and even down to 10-25 wt% in affected atopic skin areas of the subjects. Furthermore, the partial amount of free extractable very long chain fatty acids with more than 24 carbon atoms was reduced in affected regions down to 25 wt% and in nonlesional regions of the atopic dermatitis subjects down to 40 wt% compared to healthy controls. This "hydrocarbon chain length deficiency" regarding the barrier lipids in atopic skin was supported by metabolic labeling studies with [14C]-serine in cultured epidermis. The biosynthesis of free glucosylceramides and free ceramides was remarkably decreased in affected skin areas of the atopic subjects compared to healthy control subjects. Especially affected were the de novo syntheses of ceramide 4 (i.e., ceramide EOH, consisting of a very long chain N-acyl omega-hydroxy fatty acid esterified with linoleic acid and 6-hydroxysphingosine as sphingoid base) and ceramide 3 (ceramide NP, consisting of a nonhydroxy N-acyl fatty acid and phytosphingosine). In conclusion, this study revealed that the lesional epidermis in atopic dermatitis has considerable deficiencies within main barrier lipid components, which may contribute to the severely damaged permeability barrier.     

New insights into ultrastructure, lipid composition and organization of vernix caseosa

The upper layer of the epidermis, the stratum corneum (SC), is very important for skin barrier function. During the last trimester of gestation, the SC of the fetus is protected by a cheesy, white biofilm called vernix caseosa (VC). VC consists of water-containing corneocytes embedded in a lipid matrix and the basic structure shows certain similarities with the SC. This study aimed to characterize VC, with the main focus on an integral analysis of free and (to the corneocytes) bound lipids, on the lipid organization, and on ultrastructure. Free lipids of VC show a wide distribution in polarity; nonpolar lipids such as sterol esters and triglycerides predominate, having a chain length of up to 32 carbon atoms. The profile of fatty acids, omega-hydroxyacids and omega-hydroxyceramides - representing the bound lipids of VC - shows high similarity to that of SC. Morphological studies revealed the presence of highly hydrated corneocytes embedded in lipids, the latter being occasionally accumulated as lipid pools. Freeze fracture electron microscopy showed smooth surfaces of corneocytes and a heterogeneous appearance of intercellular lipids. The results suggest a lower degree of ordering of VC lipids as compared to the SC. A small-angle X-ray diffraction study showed similar results.     

Barrier function in reconstructed epidermis and its resemblance to native human skin

One of the prerequisites for the use of human skin equivalents for scientific and screening purposes is that their barrier function is similar to that of native skin. Using human epidermis reconstructed on de-epidermized dermis we demonstrated that the formation of the stratum corneum (SC) barrier in vitro proceeds similarly as in vivo as judged from the extensive production of lamellar bodies, their complete extrusion at the stratum granulosum/SC interface, and the formation of multiple broad lamellar structures in the intercorneocyte space. The presence of well-ordered lipid lamellar phases was confirmed by small-angle X-ray diffraction. Although the long periodicity lamellar phase was present in both the native and the reconstructed epidermis, the short periodicity lamellar phase was present only in native tissue. In addition, the SC lipids predominantly formed the hexagonal sublattice. Analysis of lipid composition revealed that all SC lipids are synthesized in vitro. Differences in SC lipid organization in reconstructed epidermis may be ascribed to the differences in fatty acid content and profile indicating that further improvement in culture conditions is required for generation of in vitro reconstructed epidermis with stratum barrier properties of the native tissue.     

Determination of stratum corneum lipid profile by tape stripping in combination with high-performance thin-layer chromatography

Abstract Intercellular lipids in the stratum corneum (SC) are responsible for the barrier function of mammalian skin. The main components of the SC lipids are ceramides, cholesterol, and free fatty acids, as established by thin-layer chromatographic analysis of lipids extracted from the human and mammalian SC. Up to now, for lipid analysis the extracts of the entire SC has been used and information on whether the lipid composition changes with the depth in the SC is scarce. Tape stripping is a technique which removes corneocyte layers step by step with an adhesive film. The use of this technique for lipid analysis was hampered by the contamination of lipid extracts with compounds co-extracted from the tape with organic solvents used for the extraction of SC lipids. The aim of the present study was to establish a suitable analytical method for the determination of the local SC lipid composition. For this purpose, the SC samples were collected by sequential stripping with Leukoplex tape in five healthy volunteers. The lipids were extracted with ethyl acetate:methanol mixture (20:80) and separated by means of HPTLC. The results of this study revealed that the free fatty acid level is highest and the cholesterol and ceramide levels lowest in the uppermost SC layers (about 4 strippings). The levels remained unchanged in the underlying SC layers. In these layers, the ceramide level was about 60 wt% and the free fatty acid and cholesterol levels were about 20 wt% each. Ceramides could be separated into seven different fractions and the relative amounts of individual ceramide fractions did not significantly change with the SC depth. Cholesterol sulfate levels were about 5% of total cholesterol and did not change with the SC depth, except for the for the first strip where the level was about 1%. The method developed makes it possible to study the differences in the SC lipid profile in healthy and diseased human skin with relation to the SC lipid organization and to the skin barrier function in vivo. Received: 2 August 2000 / Revised: 20 December 2000 / Accepted: 26 January 2001