Analysis of free and protein-bound ceramides by tape stripping of stratum corneum from dogs

The free and protein-bound ceramides of dog stratum corneum (SC) were analyzed by thin-layer chromatography after tape stripping of the abdomen of five dogs. The sphingoid bases were identified by gas–liquid chromatography as sphingosine, phytosphingosine, and 6-hydroxysphingosine. Electrospray ionization-ion trap mass spectrometry was used to characterize the protein-bound ceramides containing sphingosine and omega-hydroxy long-chain fatty acids. Although the molecular species were the same ones in all dogs, wide quantitative variations in the patterns of SC ceramides were observed in different breeds of dogs. The free ceramide concentration changed with the depth of SC, with a higher concentration in the deep layers, whereas the concentration of protein-bound ceramides remained constant. These results show that canine SC is close to that of humans with respect to ceramides.     

Atopic dermatitis is associated with a decrement in health-related quality of life

BACKGROUND: Although atopic dermatitis is a chronic skin disease that can have a major impact on a patient's life, the burden of illness associated with this condition has not been well characterized. OBJECTIVE: To determine the health-related quality of life (HRQL) of patients with atopic dermatitis by disease severity and to compare it with that of the general public and of patients suffering from other chronic illnesses or skin disorders. METHODS: Two hundred and thirty-nine atopic dermatitis patients aged 4-70 years completed the Medical Outcomes Study Short Form-36 Health Survey (SF-36) and the Dermatology Life Quality Index (DLQI) or the Children's Dermatology Life Quality Index. These HRQL scores were compared by self-reported patient disease severity ratings. Health-related quality of life scores were compared with those of the general population and those of patients with other chronic conditions (clinical depression, hypertension, type 2 diabetes) or skin disease (psoriasis). Dermatology Life Quality Index scores were also compared with those of other skin diseases (such as psoriasis, Darier's disease, and Hailey-Hailey disease). RESULTS: Patients with atopic dermatitis had inferior scores on the SF-36 vitality, social functioning, and mental health subscales compared with individuals in the general population. In seven of eight subscales, individuals reporting more severe disease had inferior DLQI and SF-36 scores. Patients with atopic dermatitis had inferior mental health scores compared with those with diabetes or hypertension, and inferior social functioning scores compared with patients with hypertension. When compared with a psoriasis cohort, patients with atopic dermatitis had inferior scores in the role-physical, vitality, social functioning, role-emotional, and mental health SF-36 domains. Patients with atopic dermatitis had similar DLQI scores to patients with other chronic dermatologic diseases. CONCLUSIONS: These results demonstrate that atopic dermatitis has an impact on HRQL, particularly in social functioning and psychological wellbeing. Patient-assessed severity of atopic dermatitis correlates with HRQL decrements, indicating greater HRQL impact with greater disease severity. Atopic dermatitis has as large an impact on HRQL as several chronic conditions and other dermatologic conditions.     

Water permeation of reaggregated stratum corneum with model lipids

Corneocytes were prepared from stratum corneum after extraction of the lipids and then were reaggregated with model lipids to form a membrane. The transport of water through the membrane was found to be similar to earlier published values for reaggregated stratum corneum formed with the indigenous lipids. Similar values were also obtained when only partially saponified free fatty acids were present as lipids. These results support an earlier hypothesis, that the lipid barrier to water penetration of the stratum corneum is determined by the structural organization of the lipids, not by the exact chemical structure of individual species.     

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.     

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.     

Aberrant lipid organization in stratum corneum of patients with atopic dermatitis and lamellar ichthyosis

There are several skin diseases in which the lipid composition in the intercellular matrix of the stratum corneum is different from that of healthy human skin. It has been shown that patients suffering from atopic dermatitis have a reduced ceramide content in the stratum corneum, whereas in the stratum corneum of lamellar ichthyosis patients, the amount of free fatty acids is decreased and the ceramide profile is altered. Both patient groups also show elevated levels of transepidermal water loss indicative of an impaired barrier function. As ceramides and free fatty acids are essential for a proper barrier function, we hypothesized that changes in the composition of these lipids would be reflected in the lipid organization in stratum corneum of atopic dermatitis and lamellar ichthyosis patients. We investigated the lateral lipid packing using electron diffraction and the lamellar organization using freeze fracture electron microscopy. In atopic dermatitis stratum corneum, we found that, in comparison with healthy stratum corneum, the presence of the hexagonal lattice (gel phase) is increased with respect to the orthorhombic packing (crystalline phase). In lamellar ichthyosis stratum corneum, the hexagonal packing was predominantly present, whereas the orthorhombic packing was observed only occasionally. This is in good agreement with studies on stratum corneum lipid models that show that the presence of long-chain free fatty acids is involved in the formation of the orthorhombic packing. The results of this study also suggest that the ceramide composition is important for the lateral lipid packing. Finally, using freeze fracture electron microscopy, changes in the lamellar organization in stratum corneum of both patient groups could be observed.     

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.     

A possible function of structural lipids in the water-holding properties of the stratum corneum

In order to clarify the possible role of lipids in the water-holding property of stratum corneum, the forearm skin of 6 healthy male volunteers was treated with acetone/ether (1/1) for 1, 5, 10, and 20 min. A prolonged treatment period of 5-20 min produced a chapped and scaly appearance of the stratum corneum without any inflammatory reactions. Under these conditions, there was a marked decrease in the water-holding capacity of the stratum corneum accompanied by a considerable and selective loss of intercellular lipids such as cholesterol, cholesterol esters, and phospholipids. These impairments persisted until day 4 after treatment. Electron microscopic observation of the altered stratum corneum revealed that naturally occurring intercellular materials were absent, leaving the area with the appearance of a vacant space. These findings suggest an additional and essential role of the specific structural lipids for the water-holding properties of the stratum corneum.     

In vitro and in vivo antistaphylococcal activity of human stratum corneum lipids

Despite the assumption that sebum-derived fatty acids are responsible for cutaneous antimicrobial defense, no studies have assessed the contribution of epidermis-derived lipids. Herein, we tested the antistaphylococcal effects of human stratum corneum lipids, enriched in endogenous, keratinocyte-derived species obtained by lipid extraction and thin-layer chromatography, for antimicrobial activity in both in vitro and in vivo systems. Whereas the most potent species in vitro were the free fatty acids, polar lipids and glycosphingolipids also demonstrated antistaphylococcal activity in vitro, while other neutral lipids displayed virtually none, results that were confirmed with authentic standards in vitro. In a pilot study on delipidized forearm test sites in human volunteers, naturally occurring free fatty acids, polar lipids, and glycosphingolipids exhibited significantly more antistaphylococcal activity than other stratum corneum lipids or vehicle controls. Finally, biopsy specimens of incubated skin sites demonstrated penetration of staphylococci through lipid-enriched intercellular domains. These results provide the first evidence that endogenous, epidermis-derived skin lipids may contribute to cutaneous antimicrobial resistance.     

The non‐homogenous distribution and aggregation of carotenoids in the stratum corneum correlates with the organization of intercellular lipids in vivo

The human stratum corneum (SC) contains an abundant amount of carotenoid antioxidants, quenching free radicals and thereby protecting the skin. For the precise measurements of the depth‐dependent carotenoid concentration, confocal Raman microscopy is a suitable method. The quantitative concentration can be determined by the carotenoid‐related peak intensity of a Gaussian function approached at ≈1524 cm^?1 using non‐linear regression. Results show that the carotenoid concentration is higher at the superficial layers of the SC then decreases to a minimum at 20% SC depth and increases again towards the bottom of the SC. In the present work, two carotenoid penetration pathways into the SC are postulated. The first pathway is from the stratum granulosum to the bottom of the SC, while in the second pathway, the carotenoids are delivered to the skin surface by sweat and/or sebum secretion and penetrate from outside. The carotenoids are aggregated at the superficial layers, which are shown by high correlation between the aggregation states of carotenoids and the lateral organization of lipids. At the 30%‐40% SC depths, the ordered and dense lipid molecules intensify the lipid‐carotenoid interactions and weaken the carotenoid‐carotenoid interaction and thus exhibit the disaggregation of carotenoids. At 90%‐100% SC depths, the carotenoid‐lipid interaction is weakened and the carotenoids have a tendency to be aggregated. Thus, the molecular structural correlation of carotenoid and SC lipid might be reserved in the intercellular space of the SC and also serves as the skeleton of the intercellular lipids.     

Biophysical and morphological changes in the stratum corneum lipids induced by UVB irradiation

BACKGROUND: UV irradiation induces a variety of responses in the epidermis, including sunburn cell formation, epidermal hyperplasia, and epidermal permeability barrier disruption. OBJECTIVE: The aim of present study was to assess the effects of UVB irradiation in the intercellular lipids in murine stratum corneum. METHODS: Adult hairless mice were exposed to a single UVB dose (0.15 J/cm(2)), the Fourier transform infrared (FT-IR) spectroscopic study was performed to investigate the effect on the biophysical changes in the stratum corneum lipids, barrier function was monitored by transepidermal water loss (TEWL) measurement, and the morphological alterations of stratum corneum was examined by electron microscopy using ruthenium tetroxide postfixation. RESULTS: The FT-IR spectroscopic study revealed that there was the shift to higher wavenumbers of the symmetric and asymmetric stretching peaks near 2850 and 2920 cm(-1) respectively at days 3-4 after a single UVB irradiation, reflecting to the increase in motional freedom of lipids hydrocarbon chains, call as disordering of lipids. Moreover, A single UVB irradiation also caused a significant increase in TEWL, the increase in TEWL began after 2 days and peaked at day 4. Electron microscopic observations revealed that marked morphological abnormalities in the intercellular domains, including abnormal profile of lamellar granules and its contents at the interface between stratum corneum and stratum granulosum and the persistence of the nuclei in the stratum corneum. Moreover, the separated fragmentary lipid lamellae, excessive numbers of lamellae in stacks, both the elongated and enlarged lacuna as well as the extracellular whorls were present within the widen space of the stratum corneum. CONCLUSION: The both of biophysical and morphological changes of the stratum corneum lipids may reflect to the mechanisms of perturbation of the epidermal permeability barrier induced by UVB irradiation.     

Detection and distribution of endogenous steroids in human stratum corneum

ObjectivesThe objective of the study was to investigate the presence and distribution of endogenous steroids in human stratum corneum (SC) with respect to sex, age, anatomical site, and depth into SC, using a noninvasive sampling technique and a sensitive analytic method for quantitation.Materials and methodsCorneocytes in the SC samples removed by sequential tape stripping from the forearm, forehead, and back sites were processed and analyzed using a validated liquid chromatography–tandem mass spectrometry method for the quantitation of hydrocortisone, cortisone, and testosterone.ResultsIn the 32 volunteers surveyed, testosterone was only detected at the forearm site in a single volunteer. Both hydrocortisone and cortisone were detected in 5–7 individuals out of 16 from both the age 20–35 years and age 50–65 years groups. Maximal amounts of hydrocortisone and cortisone found in SC of forehead, forearm, and back were 0.37 ng/cm², 0.96 ng/cm², and 0.49 ng/cm²; and 0.20 ng/cm², 0.12 ng/cm², and 0.06 ng/cm², respectively, and were either higher than or comparable to those reported in human hair in terms of concentration by SC weight. In the population with either hydrocortisone or cortisone detected, no significant differences relating to sex, age groups, and anatomical sites were observed for the amount of hydrocortisone and cortisone in the SC. However, significantly higher amount of cortisone was found in the surface layers of SC than deeper layers in the age 50–65 years group.ConclusionThe results demonstrate that, with the achievable sensitivity of current analytical technology, physiological concentrations of endogenous steroids, such as hydrocortisone and cortisone, can be found in the SC of some individuals.     

Image analysis of the distribution of turnover rate in the stratum corneum

Background/aims: A common method to evaluate turnover rate in the stratum corneum is to measure the change in fluorescence intensity with time after dyeing the stratum corneum with fluorescent pigments. If these changes in fluorescence over time are carefully observed, the rate of decline in fluorescence intensity differs among different small areas on the skin surface. A possible relationship between these differences and dry skin has been reported. The purpose of this research was to develop a method for analyzing turnover rate in the stratum corneum in each small area on the surface of the skin as well as to investigate the variations in the inconsistencies of turnover rate. Methods: The stratum corneum at six body regions (forehead, cheek, forearm, opisthenar, back and lower leg) was dyed with dansyl chloride (DC), and the change in fluorescence intensity over time was imaged with a highly sensitive television camera through special filters. Then, the fluorescent distribution in the images was analyzed to measure the change in fluorescence intensity with time among the small areas. Also, the decline in fluorescence intensity observed was categorized using specific characteristics into six different types. Results: By attaching a filter to an ultraviolet (UV) light source in order to transmit light at the excitation wavelength and a filter to the camera lens to transmit light at the wavelength of DC fluorescence, we could image the low intensity fluorescent light from the DC without interference from the UV light exciting the DC. The characteristics of the variation in the decline in fluorescence intensity were categorized into six patterns. Type I: pattern showing a uniform decline in fluorescence intensity. Type II: pattern showing sporadic areas where fluorescence intensity declines quickly. Type III: pattern showing relatively large areas where fluorescence intensity declines slowly. Type IV: pattern showing sporadic areas of fluorescence intensity, matched with locations of keratotic plugs. Type V: pattern showing sporadic fluorescent areas, not matched with locations of keratotic plugs. Type VI: pattern showing a partial, drastic decline in fluorescence intensity occurring on inflamed skin after sunburn. Conclusions: By analyzing the image generated from a highly sensitive television camera equipped with special filters, we could measure turnover rate of the stratum corneum at any small area. The variations in Types IV and V were believed to be derived from keratotic plugs and closed comedo. Except for Type VI, observed on significant skin inflammation, Type II and Type III were believed to be the patterns that reflected variations in turnover rate in stratum corneum itself.