Covalently bound lipids in reconstructed human epithelia

The composition of free and covalently bound lipids in reconstructed epithelia generated with normal human keratinocytes, HaCaT cells and squamous carcinoma cells was investigated and compared with native skin. Stratum corneum isolated from native human and reconstructed epidermis was subjected to extensive extraction with chloroform-methanol mixtures followed by alkaline hydrolysis to release covalently bound lipids. High-performance thin layer chromatography was used for analysis of solvent-extractable and non-extractable lipids and gas liquid chromatography was performed to assess the fatty acid profile in extractable lipids. In both native and reconstructed tissue covalently bound lipids consisted of omega-hydroxyceramides, omega-hydroxyacids and free fatty acids. Small amounts of omega-hydroxyacids could already be detected in solvent-extractable fractions. omega-Hydroxyceramides consisted of Ceramide A, Ceramide B and a small fraction of unknown ceramides with intermediate polarity. The relative proportions of individual omega-hydroxyceramides were similar in both native and reconstructed stratum corneum. In contrast, differences were found in profiles of both solvent-extractable and non-extractable lipids isolated from epithelia reconstructed with transformed cell lines (HaCaT, SCC-12F2 and SCC-13 cells). Compared with native or reconstructed epidermis, in epithelia reconstructed with transformed cell lines the ceramide content was low, the most polar ceramides were missing and the content of free fatty acids was low. The same holds true for covalently bound lipids that were virtually absent in these epithelia. Marked similarities were demonstrated in the overall lipid composition of free and bound stratum corneum lipids in native epidermis and in epidermis reconstructed with normal human keratinocytes. The observed imbalance in fatty acid profile may account for differences in phase behaviour of stratum corneum lipids.     

Influence of UV irradiation on the composition of human stratum corneum lipids

Irradiation with suberythemal doses of either UV-A or UV-B yielded an increase in the amount of stratum corneum lipids extracted from the lumbar skin area of 20 volunteers. These lipids were quantified after separation by high-performance thin-layer chromatography. Ten subfractions in the ceramide region were separated; two of them (fractions 7a and 7b) were only detectable after UV-A or UV-B irradiation. Improvement of barrier function after UV irradiation of human skin with suberythemal doses may be related to an increase in the stratum corneum ceramides.     

Ceramide profiles of the uninvolved skin in atopic dermatitis and psoriasis are comparable to those of healthy skin

Ceramides are sphingolipids consisting of sphingoidbases, which are amide-linked to fatty acids. In the stratum corneum, they represent the major constituent of the free extractable intercellular lipids and play a significant role in maintaining and structuring the water permeability barrier of the skin. Using thin layer chromatography, which represents the method of the first choice in analyzing the stratum corneum ceramides, at least seven classes can be distinguished. Each ceramide class contains various species, which have the same head group and different chain lengths. As in many other skin disorders, atopic dermatitis and psoriasis show derangements in content and profile of the ceramides. Such derangements were reported for both the lesional involved as well as for the normal-appearing uninvolved skin. In this study, we focused on investigating the stratum corneum ceramides of the uninvolved skin in atopic dermatitis and psoriasis patients compared to healthy skin. The aim of the investigations was to explore possible significant and specific differences which can be accomplished for purposes of early diagnostics. The skin lipids were collected by means of an in vivo topical extraction procedure using an extraction mixture consisting of n-hexane and ethanol, (2:1). An automated multiple development-high performance thin layer chromatography (AMD-HPTLC) method with photodensitometric detection were applied to separate the ceramides and to estimate their contents. For studying their molecular profile within each ceramide class, a new method of normal phase HPLC with atmospheric pressure chemical ionization mass spectrometry were used. The results obtained by AMD-HPTLC exposed no significant alterations regarding the relative composition of the major stratum corneum lipids and primarily the ceramides. In addition, the mass spectrometric profiles within each ceramide class were similar in the patients and the healthy control subjects. In conclusion, this study revealed that the normal-appearing uninvolved skin of atopic dermatitis and psoriasis patients does not prove significant or specific deficiencies with respect to the free extractable major stratum corneum lipids and mainly the ceramides, when compared to healthy skin. Thus, they cannot be used for diagnostic purposes. Furthermore, our data are not consistent with the concept that impairments in the ceramide composition represent an obligate etiologic factor for both diseases.     

The Azerbaijani scrub: a simple method to harvest large amounts of stratum corneum

BACKGROUND: The study of skin barrier constituents may require collection of much stratum corneum. Existing methods are inadequate and/or difficult. METHODS: A simple and safe method to harvest stratum corneum from glabrous human skin derives from the bathing practices of people in Azerbaijan. The method requires water immersion of the subject for 30 min immediately followed by vigorous scrubbing of skin with a moist rough crepe mitten. RESULTS: This scrubbing method causes the separation of large amounts of stratum corneum which is easily harvested. CONCLUSION: The method facilitates study of stratum corneum components, including intercellular lipids.     

Electron diffraction provides new information on human stratum corneum lipid organization studied in relation to depth and temperature.

The outermost layer of mammalian skin, the stratum corneum, provides the body with a barrier against transepidermal water loss and penetration of agents from outside. The lipid-rich extracellular matrix surrounding the corneocytes in the stratum corneum is mainly responsible for this barrier function. In this study (cryo-) electron diffraction was applied to obtain information about the local lateral lipid organization in the extracellular matrix in relation to depth in human stratum corneum. For this purpose, stratum corneum grid-strips were prepared from native skin in vivo and ex vivo. It was found that the lipid packing in samples prepared at room temperature is predominantly orthorhombic. In samples prepared at 32 degrees C the presence of a hexagonal packing is more pronounced in the outer layers of the stratum corneum. Gradually increasing the specimen temperature from 30 to 40 degrees C induced a further transition from an orthorhombic to a hexagonal sublattice. At 90 degrees C all lipids were present in a fluid phase. These results are in good agreement with previously reported wide angle X-ray diffraction and Fourier transformed infrared spectroscopy studies. We conclude that the lipids in human stratum corneum are highly ordered throughout the stratum corneum and that electron diffraction allows monitoring of the local lipid organization, which contributes to the understanding of stratum corneum barrier function.     

Quantitative Analysis of Stratum Corneum Lipids in Xerosis and Asteatotic Eczema

Sphingolipids, a major constituent of intercellular lipids, are an important determinant for both water-holding and permeability barrier function in the stratum corneum. To assess the pathogenic role of sphingolipids in the stratum corneum of dry skin disorders such as xerosis and asteatotic eczema in leg skin, ceramides were quantified by thin layer chromatography after n-hexane/ethanol extraction of resin-stripped stratum corneum and evaluated as μg/mg stratum corneum. In healthy leg skin (n=49), there was age-related decline in the total ceramide, whereas xerosis (n=25) and asteatotic eczema (n=16) suffering significantly reduced water-holding properties, exhibited no definite decrease, rather slight increase in ceramide quantity with the same composition of each individual ceramide as compared to healthy age-matched controls. These data indicate that the seemingly elevated level of ceramide is an artificial effect due to inflammatory processes which result from susceptibility to dryness. Analysis of sebum-derived lipids present in the stratum corneum revealed that there was a significant decline in free fatty acids in xerosis and asteatotic eczema as compared to age-matched healthy controls, and a similar decline in triglycerides in the above three groups when compared to younger controls. Although the observed decrease in the stratum corneum lipids may well explain the high incidence of winter dry skin in older people, the progression toward asteatotic eczema can not be accompanied solely by a decrease in ceramide quantity, suggesting that the evolution of xerotic skin is associated with other moisturizing factors and/or environmental stimuli.     

Decreased level of ceramides in stratum corneum of atopic dermatitis: an etiologic factor in atopic dry skin?

Stratum corneum lipids are an important determinant for both water-retention function and permeability-barrier function in the stratum corneum. However, their major constituent, ceramides, have not been analyzed in detail in skin diseases such as atopic dermatitis that show defective water-retention and permeability-barrier function. In an attempt to assess the quantity of ceramides per unit mass of the stratum corneum in atopic dermatitis, stratum corneum sheet was removed from the forearm skin by stripping with cyanoacrylate resin and placed in hexane/ethanol extraction to yield stratum corneum lipids. The stratum corneum was dispersed by solubilization of cyanoacrylate resin with dimethylformamide, and after membrane filtration, the weight of the stratum corneum mass was measured. The ceramides were quantified by thin-layer chromatography and evaluated as microgram/mg stratum corneum. In the forearm skin of healthy individuals (n = 65), the total ceramide content significantly declined with increasing age. In atopic dermatitis (n = 32-35), there was a marked reduction in the amount of ceramides in the lesional forearm skin compared with those of healthy individuals of the same age. Interestingly, the non-lesional skin also exhibited a similar and significant decrease of ceramides. Among six ceramide fractions, ceramide 1 was most significantly reduced in both lesional and non-lesional skin. These findings suggest that an insufficiency of ceramides in the stratum corneum is an etiologic factor in atopic dry skin.     

A new HPLC-based method for the quantitative analysis of inner stratum corneum lipids with special reference to the free fatty acid fraction

Abstract The inner stratum corneum is likely to represent the location of the intact skin barrier, unperturbed by degradation processes. In our studies of the physical skin barrier a new high-performance liquid chromatography (HPLC)-based method was developed for the quantitative analysis of lipids of the inner stratum corneum. All main lipid classes were separated and quantitated by HPLC/light scattering detection (LSD) and the free fatty acid fraction was further analysed by gas-liquid chromatography (GLC). Mass spectrometry (MS) was used for peak identification and flame ionization detection (FID) for quantitation. Special attention was paid to the free fatty acid fraction since unsaturated free fatty acids may exert a key function in the regulation of the skin barrier properties by shifting the physical equilibrium of the multilamellar lipid bilayer system towards a noncrystalline state. Our results indicated that the endogenous free fatty acid fraction of the stratum corneum barrier lipids in essence exclusively consisted of saturated long-chain free fatty acids. This fraction was characterized as a very stable population (low interindividual peak variation) dominated by saturated lignoceric acid (C24:0, 39 molar%) and hexacosanoic acid (C26:0, 23 molar%). In addition, trace amounts of very long-chain (C32-C36) saturated and monounsaturated free fatty acids were detected in human forearm inner stratum corneum. Our analysis method gives highly accurate and precise quantitative information on the relative composition of all major lipid species present in the skin barrier. Such data will eventually permit skin barrier model systems to be created which will allow a more detailed analysis of the physical nature of the human skin barrier. Received: 12 March 1998     

Lipid composition of outer stratum corneum in hereditary palmoplantar keratodermas

BACKGROUND: The analysis of lipid composition of the outer stratum corneum is a promising approach to study the pathophysiology of inherited disorders of keratinization. OBJECTIVE: The purpose of the study was the search for biochemical alterations of stratum corneum lipids in hereditary palmoplantar keratoderma (PPK). METHODS: Using high-performance thin-layer chromatography, we performed an analysis of all major stratum corneum lipid classes in scales of 29 patients with 8 different types of hereditary PPK. RESULTS: In comparison to the controls, slight differences in the lipid pattern were found in all keratodermas. Reduced amounts of total ceramides and increased levels of free fatty acids were noted in nearly all types. CONCLUSIONS: The study indicates that the abnormal composition of stratum corneum lipids in PPK is probably not caused by genetic defects of the epidermal lipid metabolism, but it appears to represent an epiphenomenon of a disturbed cornification.     

Analysis of the non-ionic detergent-soluble lipids of human stratum corneum

Mechanical stimulation of the skin surface in the presence of non-ionic detergent (Triton X-100)-containing buffers has been used to prepare isolated corneocytes and non-ionic detergent-soluble (NIDS) material from human stratum corneum. Analysis of the major lipid classes present in the corneocytes and NIDS material has been performed by thin layer chromatography with flame ionization detection and quantitation based on the introduction of an internal standard. Results indicate that the NIDS lipid represents a disproportionately large amount of the total stratum corneum lipid and that it differs in composition from the corneocyte lipid, there being a greater proportion of the cholesteryl ester fraction in the corneocytes. Both these findings support the 'brick wall' model of the stratum corneum and provide further evidence that the NIDS material is intercellular in nature.     

Alterations in the composition of human stratum corneum lipids induced by inflammation

Changes in the composition of human stratum corneum lipids (SCL) were studied at various times after an induced inflammatory reaction. Three kinds of injury were inflicted: 3 MED of ultraviolet radiation, 3 MED of solar radiation or 30 strippings of the horny layer with plastic tape. The SCL were evaluated qualitatively and quantitatively at short intervals. Marked changes in the SCL were detected between the 9th and the 11th day. With all 3 insults, the SCL increased quantitatively and showed a more polar pattern, i.e. a decrease in neutral fractions and an increase in the polar ones. These changes were synchronous with the marked shedding which occurs between the 9th and the 11th day. These findings have implications for the role of lipids in stratum corneum cohesiveness.     

Two ceramide subfractions detectable in Cer(AS) position by HPTLC in skin surface lipids of non-lesional skin of atopic eczema

The non-involved skin of atopic eczema (NEAE) is characterized by severe dryness and an impaired barrier function of the stratum corneum as indicated by an increased transepidermal water loss. Previous studies have demonstrated that this barrier impairment coincides with marked alterations in the amount and composition of stratum corneum ceramides. The aim of this study was to identify specific alterations in NEAE that may be used in the diagnosis of the atopic eczema. Using a classical procedure for high performance thin layer chromatography we could confirm earlier results: apart from Cer(EOH), which contains omega-hydroxy fatty acid (O) ester-linked to linoleic acid (E) and amide-linked to 6-hydroxy-4-sphingenine (H), the quantities of all ceramide fractions were significantly decreased. Furthermore, Cer(EOH)/Certotal was significantly increased, whereas the percentage of Cer(EOS), which contains sphingosine (S), and Cer(NP), which contains non-hydroxy fatty acid (N) amide-linked to phytosphingosine (P), were significantly decreased. Using a modified procedure for high performance thin layer chromatography we could demonstrate the formation of a double peak in the position of Cer(AS), which contains alpha-hydroxy fatty acid (A), in lipids of NEAE. The subfractions of the double peak comprised 15% and 12% of Certotal. MALDITOF mass spectrometry suggested that the double peak was formed by a homologous series of mono-hydroxylated and mono-unsaturated ceramides of different chain length, e.g., Cer(AS) subfractions containing either (C16,18) or (C22,24,26) alpha-hydroxy fatty acids. In contrast, in normal skin a single peak in Cer(AS) position, which comprised 22% of Certotal, was mainly formed by the long chain subfraction. In some cases this single peak displayed a small shoulder at its right flank, but never showed a clear peak separation when developed with NEAE samples. Furthermore, even in senile xerosis, or in either non-involved skin of psoriasis or seborrhoic eczema, only a single peak occurred in Cer(AS) position. Accordingly, the double peak might be specific for NEAE and turn out to be a marker for atopic eczema.     

Ethnicity and stratum corneum ceramides

Background The barrier function of the skin is dependent on an optimal composition of the stratum corneum lipids, exemplified by the altered lipid profile in patients with atopic eczema (AE). Differences in the global prevalence of AE point to the environment as an important factor in AE. Studies on filaggrin point to a genetic aspect in AE. The influence of environment and genes needs to be explored. Objectives To investigate possible differences in stratum corneum lipids between different healthy ethnicities living in the same environment. Methods Healthy participants without any major skin diseases were enrolled in the study. Twenty‐five participants of Asian origin (Asians), 18 of African origin (Africans) and 28 of Danish origin (white‐skinned), all students at universities in the Copenhagen area of Denmark, had the ceramide profile of their stratum corneum examined using the cyanoacrylate method and analysed using high‐performance thin layer chromatography. Results For the ceramide/cholesterol ratio we found statistically significant differences between groups, with Asians having the highest ratio (P < 0·001 as compared with both white‐skinned individuals and Africans), white‐skinned individuals having intermediate values, and Africans having the lowest values. No statistically significant differences were found between any of the ceramide subgroups. Conclusions We found different ceramide/cholesterol ratios in comparable groups of different ethnicity, pointing to unknown genetic differences.     

Variations in lipids in different layers of porcine epidermis

Frozen cryosections, 8 microns thick, were cut parallel to the surface of porcine skin so as to provide separate samples representing various epidermal layers. These samples were dried, extracted with chloroform-methanol mixtures, and the lipids chromatographed on silica gel plates in different solvent systems. After spraying with sulfuric acid and charring, lipids were quantified using a scanning densitometer. It was thus possible to determine lipid concentrations in 12 consecutive epidermal layers, extending 96 microns into the skin. The phospholipids that were characterized all decreased in concentration toward the surface, whereas the neutral lipids and ceramides all increased. Glucosylceramide and acylglucosylceramide reached a peak concentration in the stratum granulosum and then decreased in the surface layers. Cholesterol sulfate reached a maximum concentration in the deeper stratum corneum and then abruptly decreased in the surface layer. These changes in patterns of lipid concentration are consistent with current theories regarding the formation of a water barrier in the stratum corneum that is composed mainly of neutral lipids, and with a possible function of cholesterol sulfate in cellular adhesion in the stratum corneum.     

Effects of ultraviolet A and B on the skin barrier: a functional, electron microscopic and lipid biochemical study.

To investigate the effects of ultraviolet A (UVA) and B (UVB) on the skin barrier, functional, electron microscopic and lipid biochemical studies were performed on normal and UV-irradiated skin of volunteers. Skin reactivity against primary irritants was evaluated using the alkali resistance test, the dimethylsulfoxide test and the sodium lauryl sulfate test. In all 3 irritation models, UVA- and UVB-irradiated areas were more resistant to damage than normal skin, indicating improvement of the barrier function after UV irradiation. In a second series of experiments, biopsies were taken and processed for electron microscopic evaluation of the stratum corneum. UVB significantly increased the horny cell layers; UVA did not alter the thickness of the stratum corneum. Finally, stratum corneum lipids were extracted in vivo and quantified after high-performance thin-layer chromatography. UVB and, to some extent, UVA exposure increased the amount of all stratum corneum lipids. This was also observed in all major ceramide subfractions.     

Selective recovery of deranged water-holding properties by stratum corneum lipids

Selective removal of stratum corneum lipids following applications of acetone/ether to the human forearm for extremely prolonged periods of 5-20 min induced an enduring (more than 4 days), chapped and scaly appearance of the skin which was accompanied by a significant decrease in the water-holding properties of the stratum corneum. In order to further elucidate the significance of lipids in the water-holding properties, lipids, which were extracted as sebaceous-rich lipids (SL) for the first 10-min acetone/ether treatment and as stratum corneum lipids (SCL) for the additional 30-min treatment, were topically applied daily on lipid-depleted forearm skin which had been pretreated with acetone/ether for 40 min. Two daily applications of the SCL which were solubilized in squalane containing 1% alpha-monomethyl heptadecyl glyceryl ether (GE) caused a significant increase of conductance, accompanied by a marked improvement in the level of scaling as compared with nontreatment or GE/squalane base, whereas the SL in the GE/squalane base did not exhibit any significant recovery in either conductance value or scaling. To clarify which components of the SCL are primarily responsible for the observed recovery of the water-holding properties, chromatographically separated fractions of the SCL were also topically applied in the same manner for 2 successive days. Out of the following separated fractions: cholesterol, cholesterol ester, free fatty acid, glycolipids, and ceramide, 2 daily topical applications of ceramide fraction induced a significant and the highest increase in the conductance value as compared with GE/squalane base. Furthermore, glycolipids and cholesterol fractions also exhibited a significant recovery when compared with no application at all. In contrast, free fatty acid and cholesterol ester fractions did not indicate any significant increase in the conductance value. These findings strengthen the hypothesis that structural lipids present in the intercellular spaces of the stratum corneum, especially ceramide, play a critical role in the water-holding properties of the stratum corneum.     

Lipids,proteins and corneocyte adhesion

Summary Three factors were examined for their relative contribution to corneocyte cohesion in normal adult pig ear: (1) extracellular lipids derived from membrane-coating granules (MCG); (2) corneosomes (modified stratum corneum desmosomes); and (3) corneocyte covalently bound lipid envelopes. Cohesion strength of the outer stratum corneum was measured directly by cohesometry, then altered by removing MCG lipids with solvents of varying potency. Cohesion changes were related to degree of lipid removal and ultrastructural alterations. Trypsin was also used to see if proteolysis of corneosomes promoted squame shedding. Potent solvents increased cohesion in relation to the amount of MCG lipid extracted. Tighter cohesion was due to fusion of the outer leaflets from covalently bound lipid envelopes on adjacent corneocytes. However, lipid envelopes are unlikely to mediate normal stratum corneum cohesion since MCG lipids play a significant anti-cohesive role preventing their apposition. Mild solvents partially removed MCG lipids causing a slight decrease in cohesion compared with untreated samples. This suggests a minor cohesive role for MCG lipids, consistent with maintaining their barrier function. We believe that corneosomes are the major determinant of stratum corneum cohesiveness because, in untreated skin, both cohesion and the number of corneosomes increased from the surface towards the granular layer. Furthermore, corneosome digestion with trypsin induced superficial squame shedding.     

Intercellular lamellar lipids in plantar stratum corneum.

Plantar stratum corneum was examined by means of transmission electron microscopy after conventional osmium fixation and after fixation with ruthenium tetroxide. The latter fixative was used in order to reveal the possible existence of lamelarly ordered lipids in the intercellular space, as has previously been demonstrated for non-palmo-plantar stratum corneum. A major part of the plantar stratum corneum intercellular space was occupied by extracellular parts of desmosomes. In specimens fixed with ruthenium tetroxide the intercellular space not occupied by desmosomes was found to contain multiple alternating electron dense and electron lucid bands, suggestive of membraneous structures. This pattern appeared to be similar to that previously described for non-palmo-plantar stratum corneum. It is suggested that the intercellular lipids of palmo-plantar stratum corneum may be qualitatively similar to the intercellular lipids of non-palmo-plantar stratum corneum. The lower lipid content, expressed as weight per unit weight of tissue, in palmo-plantar stratum corneum as compared to non-palmo-plantar stratum corneum may be related to the fact that a larger portion of the intercellular space of the former tissue is occupied by desmosomes. The relatively high water permeability of palmo-plantar stratum corneum implies that desmosomes, i.e. non-lipid regions of the intercellular space, may have a high water permeability and hence could establish a hydrophilic route through the stratum corneum.     

Comparison of lipids from epidermal and palatal stratum corneum.

Stratum corneum has been isolated by tryptic digestion of porcine epidermis and palatal epithelium, and the lipid concentrations and compositions have been compared by thin-layer chromatography in conjunction with photodensitometry. Palatal stratum corneum contained 47 +/- 6 micrograms lipid/mg tissue or 115 +/- 16 micrograms lipid per cm2 of stratum corneum surface, whereas epidermal stratum corneum contained 105 +/- 17 micrograms lipid/mg tissue or 135 +/- 16 micrograms/cm2. The difference in total lipid content does not account for the tenfold higher permeability constant for the permeation of water through the former tissue compared to the latter; therefore, the difference in permeability must be based on differences in lipid composition. In this regard, palatal stratum corneum includes 12.1% phospholipids, although phospholipids were undetected in epidermal stratum corneum. Differences in the content and location of non-polar liquid-phase lipids may also be of significance for permeability. Other factors that may contribute to the greater permeability of the palatal horny layer relative to epidermal stratum corneum include generally lower proportions of cholesterol, fatty acids, and ceramides, a dramatically lower proportion of the linoleate-containing acylceramide, and a tenfold lower content of covalently bound lipids associated with the corneocyte envelope.     

Hydration characteristics of pathologic stratum corneum--evaluation of bound water

We measured in vitro the hygroscopicity and bound (non-freezing) water of various samples of pathologic horny layer obtained from the lesions of senile xerotic skin and psoriasis vulgaris and the normal horny layer from glabrous skin and plantar horny layer. The amount of water taken up by pathologic stratum corneum was much smaller than that by normal horny layer in an environment at a high relative humidity (RH). Tightly bound primary water to stratum corneum measured by Karl Fischer's method was about 5 mg/100 mg of dry stratum corneum in all the samples studied, while less tightly bound secondary water was much smaller in amount in pathologic stratum corneum than in the controls, i.e., 31.7 mg/100 mg dry scale from senile xerosis and 27.2 mg/100 mg dry psoriatic scale as compared with 38.2 mg/100 mg dry normal stratum corneum from glabrous skin and 37.3 mg/100 mg dry normal plantar stratum corneum. We believe that the low hygroscopicity of the pathologic stratum corneum is due to this smaller capacity for secondary bound water, which is responsible for the development of a dry scaly appearance even at high RH.