Intercellular Lipid Structure Research Articles

Distribution of Domains Formed by Lateral Packing of Intercellular Lipid in the Stratum Corneum.

Intercellular lipids fill the interstices of corneocytes and serve a barrier function. The amount of transdermal water evaporation varies depending on the packing structure of intercellular lipids, as this structure is important for maintaining barrier efficacy. This packing structure consists of a mixture of crystals (orthorhombic and hexagonal) and liquid crystals (fluid phase), and the proportion of these phases is thought to affect barrier function. However, there have been no methods to visualize the actual distribution of the domains formed by packing structure in intercellular lipids. In this study, the planar distribution of intercellular lipid structures was determined using focal plane array (FPA)-based Fourier transform (FT) IR imaging analysis of stratum corneum cell units obtained by grid stripping. The lipid composition of ceramides was revealed by electrospray ionization tandem mass spectrometry (ESI-MS/MS)-based shotgun lipidomics. The distribution of domains formed by packing structures and the lipid composition of ceramides was compared in skin with high- or low-transepidermal water loss (TEWL). The orthorhombic proportion was lower in high-TEWL skin than in low-TEWL skin. ESI-MS/MS-based shotgun lipidomics analysis showed that the alpha-hydroxyceramide content in the low- and high-TEWL groups differed regarding the distribution of fatty acid chain lengths. The evaluation of stratum corneum cell units using FPA-based FTIR imaging is an innovative technology that can visualize the distribution of domains formed by intercellular lipid-packing structures. Increased proportions of alpha-hydroxyceramide subclasses such as alpha-hydroxy-sphingosine ceramide and alpha-hydroxy-phytosphingosine ceramide were associated with a reduced proportion of the orthorhombic packing structure domain.

Stratum Corneum Structure and Function Studied by X-ray Diffraction

X-ray diffraction is one of the powerful tools in the study of a variety of structures in the stratum corneum at the molecular level. Resolving structural modifications during functioning is an important subject for clarifying the mechanism of operating principles in the function. Here, the X-ray diffraction experimental techniques used in the structural study on the stratum corneum are widely and deeply reviewed from a perspective fundamental to the application. Three typical topics obtained from the X-ray diffraction experiments are introduced. The first subject is concerned with the disruption and the recovery of the intercellular lipid structure in the stratum corneum. The second subject is to solve the moisturizing mechanism at the molecular level and the maintenance of normal condition with moisturizer, being studied with special attention to the structure of soft keratin in the corneocytes in the stratum corneum. The third subject is the so-called 500 Da rule in the penetration of drugs or cosmetics into skin, with attention paid to the disordered intercellular lipid structure in the stratum corneum.

Amount of cholesterol in intercellular lipid models inversely correlates with hexagonal structure ratio in packing structures

The packing structure of intercellular lipids in skin forms an important biological barrier but little is known about the changes in packing structure caused by surfactant-induced lipid effluence. In this study, to examine the effect of the decrease in lipids in the lipid model (LM) on the packing structure, a single component was reduced from stratum corneum intercellular LM comprising ceramide ADS, cholesterol, and palmitic acid. The packing structure was assessed using differential scanning calorimetry, Raman spectroscopy, and powder X-ray diffraction. Surfactants were applied to LM to investigate lipid elution and changes in the packing structure. The results showed that the decrease in cholesterol caused disorder in the lipid structure and the amount of cholesterol was strongly inversely correlated with the hexagonal structure ratio (RHex/Ort) in the packing structure. Furthermore, the RHex/Ort values were highly correlated with the amount of lipids eluted by surfactants, making it possible to measure lipid elution using RHex/Ort values. The methods used in this study are a useful alternative to native intercellular lipids for elucidating mechanisms underlying skin irritation due to surfactants.

Amino Acids and Their N-Acetylated Derivatives Maintain the Skin's Barrier Function.

The hydrocarbon-chain packing structure of intercellular lipids in the stratum corneum (SC) is critical to the skin's barrier function. We previously found that formation of V-shaped ceramide reduces the barrier function of skin. There are few agents, apart from ceramides and fatty acids that can improve the orthorhombic packing (Orth) ratio of the intercellular lipid packing structure. In this study, we investigated agents that directly increase the Orth ratio. We selected an intercellular lipid model consisting of ceramide, cholesterol, and palmitic acid and performed differential scanning calorimetry. We focused on natural moisturizing factor components in the SC, and therefore investigated amino acids and their derivatives. The results of our intercellular lipid model-based study indicate that N-acetyl-L-hydroxyproline (AHYP), remarkably, maintains the lamellar structure. We verified the effect of AHYP on the lamellar structure and hydrocarbon chain packing structure of intercellular lipids using time-resolved X-ray diffraction measurements of human SC. We also determined the direct physicochemical effects of AHYP on the Orth ratio of the hydrocarbon-chain packing structure. Hence, the results of our human SC study suggest that AHYP preserves skin barrier function by maintaining the hydrocarbon-chain packing structure of intercellular lipids via electrostatic repulsion. These findings will facilitate the development of skincare formulation that can maintain the skin's barrier function.

Association of dry skin with intercellular lipid composition of stratum corneum after erlotinib administration.

Erlotinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, causes skin disorders such as dry skin, which impairs the skin barrier function. Stratum corneum (SC) lipids play an important role in skin barrier function; therefore, this study aimed to investigate the relationship between erlotinib-related dry skin and changes in the intercellular lipid composition and structure of the SC. Overall, 21 patients with non-small lung cancer were enrolled in this study. All patients received 150mg/day erlotinib orally. A SC sample of each patient was collected from the inner forearm using the tape stripping method on days 0, 7, 14, 28, and 56 after erlotinib administration. The intercellular lipid components of ceramide (CER), free fatty acid (FFA), and cholesterol sulfate (CS) in samples extracted from the tape were analyzed using liquid chromatography/time-of-flight/mass spectrometry. SC samples from six healthy subjects were collected as controls on days 0, 28 and 56 and analyzed similarly. Although total CER and FFA levels were not changed after erlotinib administration, the levels of CER subclasses [AP] and [AH] and hydroxy FFA, which are structural components of CER subclass [A], decreased. In contrast, the CS levels increased after erlotinib administration. Moreover, higher CS levels in the SC correlated with the clinical condition of dry skin. No changes were observed in the SC lipid composition in healthy subjects. Erlotinib-related dry skin was associated with a higher CS level in the SC.

Increased 1-Deoxysphingolipids and Skin Barrier Dysfunction in the Skin of X-ray or Ultraviolet B Irradiation and Atopic Dermatitis Lesion Could Be Prevented by Moisturizer with Physiological Lipid Mixture.

BackgroundSkin diseases characterized by epithelial barrier dysfunction show altered sphingolipid metabolism, which results in changes in the stratum corneum intercellular lipid components and structure. Under pathological conditions, 1-deoxysphingolipids form as atypical sphingolipids from de novo sphingolipid biosynthesis.ObjectiveThis study investigated the potential role of 1-deoxysphingolipids in skin barrier dysfunction secondary to X-ray and ultraviolet B (UVB) irradiation in vitro and in vivo. It was also evaluated changes in the expression of 1-deoxysphingolipids in lesional human skin of atopic dermatitis.MethodsIn this study, the changes in these 1-deoxysphingolipids levels of skin and serum samples were investigated in skin barrier dysfunction associated with X-ray and UVB irradiation in vitro and in vivo.ResultsIncreased 1-deoxysphingolipids were observed in cultured normal human epidermal keratinocytes after X-ray irradiation. X-ray or UVB irradiation increased the production of 1-deoxysphingosine in a reconstituted 3-dimensional (3D) skin model. Interestingly, treatment with a physiological lipid mixture (multi-lamellar emulsion contained pseudoceramide), which can strengthen the epidermal permeability barrier function, resulted in decreased 1-deoxysphingosine formation in a reconstituted 3D skin model. Further investigation using a hairless mouse model showed similar preventive effects of physiological lipid mixture against 1-deoxysphingosine formation after X-ray irradiation. An increased level of 1-dexoysphingosine in the stratum corneum was also observed in lesional skin of atopic dermatitis.Conclusion1-deoxysphingosine might be a novel biomarker of skin barrier dysfunction and a physiological lipid mixture treatment could prevent 1-deoxysphingosine production and consequent skin barrier dysfunction.

Mathematical-model-guided development of full-thickness epidermal equivalent

Epidermal equivalents prepared with passaged keratinocytes are typically 10–20 μm thick, whereas intact human epidermis is up to 100 μm thick. Our established mathematical model of epidermal homeostasis predicted that the undulatory pattern of the papillary layer beneath the epidermis is a key determinant of epidermal thickness. Here, we tested this prediction by seeding human keratinocytes on polyester textiles with various fiber-structural patterns in culture dishes exposed to air, aiming to develop a more physiologically realistic epidermal model using passaged keratinocytes. Textile substrate with fiber thickness and inter-fiber distance matching the computer predictions afforded a three-dimensional epidermal-equivalent model with thick stratum corneum and intercellular lamellar lipid structure. The basal layer structure was similar to that of human papillary layer. Cells located around the textile fibers were proliferating, as indicated by BrdU and YAP (Yes-associated protein) staining and expression of melanoma-associated chondroitin sulfate proteoglycan. Filaggrin, loricrin, claudin 1 and ZO-1 were all appropriately expressed. Silencing of transcriptional coactivator YAP with siRNA disturbed construction of the three-dimensional structure. Measurement of trans-epidermal water loss (TEWL) indicated that the model has excellent barrier function. Our results support the idea that mathematical modeling of complex biological processes can have predictive ability and practical value.

Human skin barrier structure and function analyzed by cryo-EM and molecular dynamics simulation.

In the present study we have analyzed the molecular structure and function of the human skin's permeability barrier using molecular dynamics simulation validated against cryo-electron microscopy data from near native skin. The skin's barrier capacity is located to an intercellular lipid structure embedding the cells of the superficial most layer of skin - the stratum corneum. According to the splayed bilayer model (Iwai et al., 2012) the lipid structure is organized as stacked bilayers of ceramides in a splayed chain conformation with cholesterol associated with the ceramide sphingoid moiety and free fatty acids associated with the ceramide fatty acid moiety. However, knowledge about the lipid structure's detailed molecular organization, and the roles of its different lipid constituents, remains circumstantial. Starting from a molecular dynamics model based on the splayed bilayer model, we have, by stepwise structural and compositional modifications, arrived at a thermodynamically stable molecular dynamics model expressing simulated electron microscopy patterns matching original cryo-electron microscopy patterns from skin extremely closely. Strikingly, the closer the individual molecular dynamics models' lipid composition was to that reported in human stratum corneum, the better was the match between the models' simulated electron microscopy patterns and the original cryo-electron microscopy patterns. Moreover, the closest-matching model's calculated water permeability and thermotropic behaviour were found compatible with that of human skin. The new model may facilitate more advanced physics-based skin permeability predictions of drugs and toxicants. The proposed procedure for molecular dynamics based analysis of cellular cryo-electron microscopy data might be applied to other biomolecular systems.

Disruption of human stratum corneum lipid structure by sodium dodecyl sulphate.

Surfactants are major ingredients of body soaps and cleansers, and such harsh ones have been demonstrated to damage the skin. Stratum corneum (SC), the outermost barrier of the skin layer, is rich in intercellular lipids. This lipid structure can be disrupted by surfactants, impairing the barrier function of the skin. Thus, we investigated the surfactant-induced disruption of the intercellular lipid structure of human SC at the molecular level using synchrotron X-ray diffraction. SC samples from the breast of female Caucasians were treated with sodium dodecyl sulphate (SDS) and analysed by small-angle and wide-angle X-ray diffraction. We found that an aqueous SDS solution affected the long lamellar structure, which became disorganized. The final disordered lipid state was reached through two or more types of structural change. We propose that the disordered lipid state results from incorporation of SDS into the long lamellar structure. In contrast, the lattice constants in the short lamellar and the hydrocarbon-chain packing structures remained almost unchanged after SDS treatment. We conclude that the disruption of the long lamellar structure plays a key role in the damage to the SC caused by detergents. To our knowledge, this is the first report to clarify the details of the disorganization of the intercellular lipid structure upon surfactant application. The knowledge obtained herein may allow the development of skin restoration methods and cleanser products that do not affect skin barrier functions.

Molecular Interaction between Intercellular Lipids in the Stratum Corneum and l-Menthol, as Analyzed by Synchrotron X-Ray Diffraction.

l-Menthol increases drug partitioning on the surface of skin, diffusion of drugs in the skin, and lipid fluidity in the stratum corneum and alters the rigidly arranged lipid structure of intercellular lipids. However, l-menthol is a solid at room temperature, and it is difficult to determine the effects of l-menthol alone. In this study, we vaporized l-menthol in order to avoid the effects of solvents. The vaporized l-menthol was applied to the stratum corneum or lipid models comprising composed of ceramides (CER) [EOS], the longest lipid acyl chain of the ceramides in the stratum corneum lipids that is associated with the barrier function of the skin; CER [NS], the shorter lipid acyl chain of the ceramides, and the most components in the stratum corneum of the intercellular lipids that is associated with water retention in the intercellular lipid structure of the stratum corneum; cholesterol; and palmitic acid. Synchrotron X-ray diffraction, differential scanning calorimetry, and attenuated total reflection Fourier transform infrared spectroscopy analyses revealed that the lipid models were composed of hexagonal packing and orthorhombic packing structures of different lamellar periods. Taken together, our results revealed that l-menthol strongly affected the lipid model composed of CER [EOS]. Therefore, l-menthol facilitated the permeation of drugs through the skin by liquid crystallization of the longer lamellar structure. Importantly, these simple lipid models are useful for investigating microstructure of the intercellular lipids in the stratum corneum.

Influence of Characteristics of Oily Vehicle on Skin Penetration of Ufenamate.

Skin penetration amounts of a highly lipophilic drug, ufenamate, prepared in four oily vehicles, including white petrolatum (WP), liquid paraffin (LP), isopropyl myristate (IPM), and isocetyl stearate (ICS), were compared. Ufenamate was mixed in each vehicle at 5% and applied at a rate of 2 mg/cm2 to intact, stripped, and delipidized Yucatan micropig skin. The amounts of ufenamate and IPM in the stratum corneum (SC), epidermis, and dermis were determined. The skin penetration amounts of ufenamate from liquid oils were significantly higher than those from WP; the amounts of ufenamate were in the order WP<LP≤ICS<IPM, which was the same as that of the vehicle viscosities. The IPM skin penetration amount was approximately 20 times that of ufenamate. The skin penetration amounts of ufenamate from the liquid vehicles decreased after application to delipidized skin and were not significantly different among the four vehicles. The skin penetration amounts of the vehicle oils were significant and might disrupt intercellular lipid structures, especially in the strips 1-6 of the SC. In the deeper SC, there was no effect of the vehicle or skin condition. Thus, ufenamate mixed in liquid vehicles was found to be an effective dosage form.

Lipid organization and stratum corneum thickness determined in vivo in human skin analyzing lipid–keratin peak (2820–3030 cm−1) using confocal Raman microscopy

While the intercellular lipid structure of the stratum corneum (SC) plays an important role in the skin barrier function, the depth‐dependent profile of the intercellular lipids contributes decisively to deepen the understanding of the skin barrier function, drug penetration, development of skin diseases and their medication. The depth‐dependent profile of the lipids' chemico‐physical properties, such as the solid–fluid phase transition and the order–disorder transition, can exclusively be measured in human skin in vivo by means of confocal Raman microscopy. In the present paper, the lipid–keratin peak (2820–3030 cm−1) was investigated. The lipid‐related Raman peaks centered at 2850 cm−1 and 2880 cm−1 were deconvoluted using Gaussian functions and investigated for their depth‐dependent shape and positional changes. Different fitting procedures show that even an additional Gaussian function cannot be used to fully characterize the lipid's polymethylene chains around 2880 cm−1, which justifies the introduction of the sharpness of the peak centered near 2880 cm−1. The results show that the 2880 cm−1 peak sharpness might be used for determining the SC thickness. The concentration of the lipids with long‐chain carbon backbone (free fatty acids and ceramides) semi‐quantitatively decreases from 10 µm to 20 µm (SC thickness is 19.8 µm). The maximum position and broadness of the Gaussian peak centered at 2850 cm−1 show that near the surface and in the deeper layers of the SC, the state of the lipids is more fluid and disordered compared to the medium layers of the SC. Copyright © 2016 John Wiley &amp; Sons, Ltd.

Effect of Nerolidol and/or Levulinic Acid on the Thermotropic Behavior of Lipid Lamellar Structures in the Stratum Corneum.

Permeation enhancers are required to deliver drugs through the skin efficiently and maintain effective blood concentrations. Studies of the barrier function of the stratum corneum using l-menthol, a monocyclic monoterpene widely used in medicines and foods, have revealed an interaction between characteristic intercellular lipid structures in the stratum corneum and permeation enhancers. The variety of permeation enhancers that can be used to contribute to transdermal delivery systems beyond l-menthol is increasing. In this study, we focused on nerolidol and levulinic acid and investigated their influence on stratum corneum lipid structures. Nerolidol, a sesquiterpene, has been reported to enhance the permeation of various drugs. Levulinic acid is reported to enhance the permeability of buprenorphine and is used as a component of the buprenorphine® patch. Synchrotron X-ray diffraction and attenuated total reflectance Fourier transform IR spectroscopy measurements revealed that nerolidol disturbs the rigidly arranged lipid structure and increases lipid fluidity. Levulinic acid had a smaller effect on stratum corneum lipid structures, but did increase lipid fluidity when co-administered with nerolidol or heat. We found that nerolidol has an effect on stratum corneum lipids similar to that of l-menthol, and levulinic acid had an effect similar to that of oleic acid.

A depth-dependent profile of the lipid conformation and lateral packing order of the stratum corneum in vivo measured using Raman microscopy.

The intercellular lipid structure of the stratum corneum (SC) plays a key role in skin barrier function. A depth profile of the intercellular lipid conformation and the lipid lateral packing order were measured in vivo in the human SC using confocal Raman microscopy. The depth profiles of the 2880 cm(-1)/2850 cm(-1) peak ratio intensity, which represent the C-H stretching and lateral packing order of lipids, and the 1080 cm(-1)/(1130 cm(-1) + 1060 cm(-1)) peak ratio, which represents the C-C skeleton vibration and trans-gauche conformation order of lipids, were investigated. The influence of keratin on the lipid peaks at 2850 cm(-1) and 2880 cm(-1) was excluded by the developed mathematical algorithm. The results show that the trans-conformation and lateral packing order of the intercellular lipids reach their maximum value in the SC at 20-40% of its depth and then decrease towards the stratum granulosum. These results show that at a depth of 20-40% (normally corresponding to a depth of 4-8 μm) the SC exhibits the most ordered lipids and therefore the highest skin barrier function. The lateral packing of lipids is more disordered on the surface and in the deeper parts of the SC, which may be associated with a reduced skin barrier function.

Understanding age-induced alterations to the biomechanical barrier function of human stratum corneum

BackgroundThe appearance and function of human skin are dramatically altered with aging, resulting in higher rates of severe xerosis and other skin complaints. The outermost layer of the epidermis, the stratum corneum (SC), is responsible for the biomechanical barrier function of skin and is also adversely transformed with age. With age the keratin filaments within the corneocytes are prone to crosslinking, the amount of intercellular lipids decreases resulting in fewer lipid bilayers, and the rate of corneocyte turnover decreases. ObjectivesThe effect of these structural changes on the mechanical properties of the SC has not been determined. Here we determine how several aspects of the SC’s mechanical properties are dramatically degraded with age. MethodsWe performed a range of biomechanical experiments, including micro-tension, bulge, double cantilever beam, and substrate curvature testing on abdominal stratum corneum from cadaveric female donors ranging in age from 29 to 93 years old. ResultsWe found that the SC stiffens with age, indicating that the keratin fibers stiffen, similarly to collagen fibers in the dermis. The cellular cohesion also increases with age, a result of the altered intercellular lipid structure. The kinetics of water movement through the SC is also decreased. ConclusionsOur results indicate that the combination of structural and mechanical property changes that occur with age are quite significant and may contribute to the prevalence of skin disorders among the elderly.

Essential oils and their constituents as skin penetration enhancer for transdermal drug delivery: a review.

In this paper, we focused on essential oils and their constituents as skin penetration enhancers for transdermal drug delivery, mechanism of their action as well as their possible toxicity. Essential oils and their volatile constituents can penetrate through the skin as well as enhance penetration of different drug from topical formulation into the lower skin layers using different mechanisms of action based on (1) disintegration of the highly ordered intercellular lipid structure between corneocytes in stratum corneum, (2) interaction with intercellular domain of protein, which induces their conformational modification, (3) increase the partitioning of a drug. After application to the skin, essential oils and their components are rapidly metabolized, not accumulated in the organism and fast excreted what strongly suggest that they can be successfully use as safe penetration enhancers. Essential oils and their constituents may be preferred over the traditionally used synthetics materials as safe and suitable permeation enhancers to promote the percutaneous absorption of hydrophilic and lipophilic drugs from topical formulation into the lower skin layers.

Mechanistic investigation and reversible effect of 2-isopropyl-5-methylcyclohexyl heptanoate on the in vitro percutaneous absorption of indomethacin

In the present study, the enhancing effect of 2-isopropyl-5-methylcyclohexyl heptanoate (M-HEP) on the percutaneous absorption of indomethacin (IM) was evaluated by the in vitro penetration experiments using the rat abdominal skin as a barrier. Partition experiment, attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectrum and transepidermal water loss (TEWL), was employed to investigate the possible mechanisms of the action of M-HEP. Furthermore, the reversible effect of M-HEP on excised rat skin was also evaluated through in vitro permeation as a preliminary indicator of safety. The result of in vitro permeation experiment indicated that, 10% (w/w) M-HEP in combination with isopropyl palmitate (IPP) significantly increased (p < 0.05), the cumulative amount of IM in comparison with the control group (IPP only). The partition coefficient of IM between the stratum corneum (SC) and enhancer solution was also greater than that between the SC and IPP. A blue shift in the ATR-FTIR spectra of SC after treatment with M-HEP solution was observed at the CH2 band, which indicating that M-HEP disrupted the intercellular lipid structure of the SC. In addition, both M-HEP/IPP and L-menthol (MT)/IPP improved the TEWL value of rat abdominal skin. After removal of M-HEP, the skin barrier function would be restored in 8 h. In conclusion, M-HEP could reversibly enhance the percutaneous absorption of IM by increasing the partitioning of IM into the SC from enhancer solution and disturbing the organized structure of SC lipids and the reversibility of M-HEP was better than MT.

Aging enhances maceration-induced ultrastructural alteration of the epidermis and impairment of skin barrier function

Skin maceration is recognized as a risk factor for the development of certain skin lesions. In health care settings, incontinence-associated skin maceration is highly prevalent in the elderly. However, the effect of senescence on maceration has not been fully elucidated. To reveal the enhancement of the maceration-induced ultrastructural alteration and barrier function of the epidermis by aging. Skin maceration was reproduced by exposure to agarose gel in human and rat. The ultrastructural alterations in human and rat tissue were observed by transmission electron microscopy. The skin barrier function was evaluated by noninvasive methods in human, and by the transdermal penetration of small- and large-fluorescent molecules in rat. In order to reveal the effect of aging on the skin maceration, we compared these parameters between young and aged rats. In macerated skin, we observed expansion of the interstices of the stratum corneum, spinosum, and basale of the epidermis; disruption of the intercellular lipid structure in the stratum corneum; a decreased number of cell processes in the stratum spinosum and basale. The transdermal penetration test in the rat using two types of fluorescein indicated that maceration disrupted skin barrier function. Furthermore, senescence-enhanced ultrastructural and functional alterations were revealed in the rodent studies. This study demonstrates that aging enhances skin maceration. Considering that maceration is a risk factor for the skin damage, the development of technology to promote skin barrier recovery after maceration in the elderly is warranted.

Abstracts: Polyoxyethylene/polyoxypropylene dimethyl ether improves the structure of intercellular lipids in SDS‐induced dry skin

pp. 39–48 The dimethyl ether of an amphiphilic random ethylene oxide/propylene oxide copolymer (EPDME) is useful for the preparation of finely dispersed micro‐emulsions. We examined whether EPDME is effective for skin moisturization by means of electron paramagnetic resonance (EPR) studies of ex vivo specimens of stratum corneum (SC) obtained by successive stripping. The values of the order parameter S obtained by EPR measurement indicated that EPDME treatment improved sodium dodecyl sulfate (SDS)‐induced disruption of SC lipid structures. This effect appeared to be related to improved hydration of the epidermis, not occlusion by EPDME, since there was no significant change in transepidermal water loss (TEWL).

Novel method to observe subtle structural modulation of stratum corneum on applying chemical agents

In the development of functional chemicals such as percutaneous penetration enhancers and cosmetics, the structural evidence at the molecular level in stratum corneum (SC) is highly desirable. We developed a method to observe a minute structural change of intercellular lipid matrix and corneocytes on applying the chemicals to the SC using synchrotron X-ray diffraction technique. The performance of the present method was demonstrated by applying typical chemicals, chloroform/methanol mixture, hydrophilic ethanol and hydrophobic d-limonene. From the small- and wide-angle X-ray diffraction we obtained the following results: on applying chloroform/methanol mixture the intercellular lipids were extracted markedly, on applying ethanol the intercellular lipid structure was slightly disrupted, ethanol molecules were taken into the corneocytes and in addition the pools of ethanol seem to be formed in the hydrophilic region of the intercellular lipid matrix in the SC, and on applying d-limonene the repeat distance of the long lamellar structure increased by incorporating d-limonene molecules, the intercellular lipid structure was slightly disrupted, and the pools of d-limonene were formed in the hydrophobic region of the intercellular lipid matrix in the SC.