Characterization of Low-Temperature (i.e.,

Disorders of skin barriers: clinical implications.

Oleic acid is known to be a penetration enhancer for polar to moderately polar molecules. A mechanism related to lipid phase separation has been previously proposed by this laboratory to explain the increases in skin transport. In the studies presented here, Fourier transform infrared spectroscopy (FT-IR) was utilized to investigate whether or not oleic acid exists in a separate phase within stratum corneum (SC) lipids. Per-deuterated oleic acid was employed allowing the conformational phase behavior of the exogenously added fatty acid and the endogenous SC lipids to be monitored independently of each other. The results indicated that oleic acid exerts a significant effect on the SC lipids, lowering the lipid transition temperature (Tm) in addition to increasing the conformational freedom or flexibility of the endogenous lipid alkyl chains above their Tm. At temperatures lower than Tm, however, oleic acid did not significantly change the chain disorder of the SC lipids. Similar results were obtained with lipids isolated from the SC by chloroform:methanol extraction. Oleic acid, itself, was almost fully disordered at temperatures both above and below the endogenous lipid Tm in the intact SC and extracted lipid samples. This finding suggested that oleic acid does exist as a liquid within the SC lipids. The coexistence of fluid oleic acid and ordered SC lipids, at physiological temperatures, is consistent with the previously proposed phase-separation transport mechanism for enhanced diffusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Stratum Corneum Lipids Serve as a Bound-Water Modulator

Stratum corneum (SC) lipids are of particular importance in maintaining the permeability barrier function. Although many studies have demonstrated that UVB irradiation of mammalian skin reduces barrier function, the responsible alterations in SC lipid profiles are not known. In this study, we investigated both compositional and morphological alterations in SC lipids with the development of barrier abnormalities caused by daily UVB irradiation in hairless rat skin. The UVB irradiation of suberythemal doses (0.5 minimal erythema dose) significantly increased transepidermal water loss (TEWL) relative to nonirradiated control, indicating a diminished barrier function. Under these conditions, the total amounts of major SC lipid species (ceramides, cholesterol, free fatty acids) in UVB-irradiated SC did not differ from those in nonirradiated SC. However, electron microscopic observations revealed marked abnormalities in the intercellular domains of UVB-irradiated SC, where naturally occurring intercellular multilamellar structures were often absent and leaving the area with the appearance of an empty space. Moreover, in UVB-irradiated SC, individual corneocytes often showed small amounts of intercellular deposition product with abnormal lamellar structure, where lamellar body sphingomyelinase activity was present. These observations demonstrated a partial failure of lamellar body secretion in UVB-irradiated SC and suggested that a defect in the secretion of lamellar body-derived lipids and enzymes to SC intercellular space is, at least in part, responsible for the observed abnormal intercellular structure and barrier disruption.

Phospholipids are a major class of lipids in epidermis, where they serve as a source of free fatty acids that are important for the maintenance of epidermal permeability barrier function. The phospholipid biosynthetic enzyme, 1-acyl-sn-glycerol-3-phosphate acyltransferase (AGPAT), catalyzes the acylation of lysophosphatidic acid to form phosphatidic acid, the major precursor of all glycerolipids. We identified an expression pattern of AGPAT isoforms that is unique to epidermis, with relatively high constitutive expression of mouse AGPAT (mAGPAT) 3, 4, and 5 but low constitutive expression of mAGPAT 1 and 2. Localization studies indicate that all five isoforms of AGPAT were expressed in all nucleated layers of epidermis. Furthermore, rat AGPAT 2 and 5 mRNAs increased in parallel with both an increase in enzyme activity and permeability barrier formation late in rat epidermal development. Moreover, after two methods of acute permeability barrier disruption, mAGPAT 1, 2, and 3 mRNA levels increased rapidly and were sustained for at least 24 h. In parallel with the increase in mRNA levels, an increase in AGPAT activity also occurred. Because upregulation of mAGPAT mRNAs after tape-stripping could be partially reversed by artificial barrier restoration by occlusion, these studies suggest that an increase in the expression of AGPATs is linked to barrier requirements.

The outermost layer of the mammalian skin, the stratum corneum (SC), is known to provide an epidermal barrier against water loss as well as protection against chemical, biological, and environmental stress. SC is composed of corneocytes in a lipid matrix. Intercellular lipids play a fundamental role in the barrier function of the skin. SC lipid multilayers consist mainly of ceramides, free fatty acids, and cholesterol. For elucidating the unique nature of SC, it is of considerable interest to examine mixtures of isolated SC lipids. In this paper, we review the thermotropic phase behaviour of various binary mixtures of SC lipids [1–3].

Capecitabine (CAP) side effect, a prodrug of 5-fluorouracil (5-FU), is hand-foot syndrome (HFS), a localized skin disorder of the hands and feet that is believed to induce a decrease in skin barrier function. Stratum corneum (SC) lipids play an important role in skin barrier function; therefore, this study compared the SC lipid structure and composition of healthy participants with those of patients with CAP-induced HFS. Forty patients receiving a combination regimen of CAP and oxaliplatin as adjuvant chemotherapy for colorectal cancer were enrolled. All patients received 1,000 mg/m2 twice daily on days 1-14. SC samples were obtained from 11 patients with CAP-induced HFS (CSC). The SC lipid structure was analyzed using synchrotron X-ray diffraction. SC lipid components, ceramides (CERs), free fatty acids (FFAs), CAP, and its metabolites in CSC samples were analyzed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). In healthy SC (HSC), lipids formed two lamellar phases. Hexagonal and orthorhombic hydrocarbon chain packing was observed in lateral lipid organization. However, in CSC, these structures have almost disappeared. UPLC-MS/MS analysis revealed that the composition of CER and FFA differed between CSC and HSC, and that the carbon chain length of SC lipid components in CSCs was reduced compared to that in HSCs. 5-FU was detected in CSCs at 3.4 ± 1.4 ng/mg. CAP induces changes in SC lipid structure due to changes in SC lipid composition and a decrease in carbon chain length in CSCs. CAP-induced HFS is associated with 5-FU accumulation in SC.

Intercellular and covalently bound lipids within the stratum corneum (SC), the outermost layer of the epidermis, are the primary barrier to cutaneous water loss (CWL) in birds. We compared CWL and intercellular SC lipid composition in 20 species of birds from desert and mesic environments. Furthermore, we compared covalently bound lipids with CWL and intercellular lipids in the lark family (Alaudidae). We found that CWL increases in birds from more mesic environments, and this increase was related to changes in intercellular SC lipid composition. The most consistent pattern that emerged was a decrease in the relative amount of cerebrosides as CWL increased, a pattern that is counterintuitive based on studies of mammals with Gaucher disease. Although covalently bound lipids in larks did not correlate with CWL, we found that covalently bound cerebrosides correlated positively with intercellular cerebrosides and intercellular cholesterol ester, and intercellular cerebrosides correlated positively with covalently bound free fatty acids. Our results led us to propose a new model for the organization of lipids in the avian SC, in which the sugar moieties of cerebrosides lie outside of intercellular lipid layers, where they may interdigitate with adjacent intercellular cerebrosides or with covalently bound cerebrosides.

The skin barrier in healthy and diseased state

Stratum corneum lipids, particularly ceramides, are important components of the epidermal permeability barrier that are decreased in atopic dermatitis and aged skin. We investigated the effects of nicotinamide, one of the B vitamins, on biosynthesis of sphingolipids, including ceramides and other stratum corneum lipids, in cultured normal human keratinocytes, and on the epidermal permeability barrier in vivo. The rate of sphingolipid biosynthesis was measured by the incorporation of [14C]-serine into sphingolipids. When the cells were incubated with 1-30 micromol L-1 nicotinamide for 6 days, the rate of ceramide biosynthesis was increased dose-dependently by 4.1-5. 5-fold on the sixth day compared with control. Nicotinamide also increased the synthesis of glucosylceramide (7.4-fold) and sphingomyelin (3.1-fold) in the same concentration range effective for ceramide synthesis. Furthermore, the activity of serine palmitoyltransferase (SPT), the rate-limiting enzyme in sphingolipid synthesis, was increased in nicotinamide-treated cells. Nicotinamide increased the levels of human LCB1 and LCB2 mRNA, both of which encode subunits of SPT. This suggested that the increase in SPT activity was due to an increase in SPT mRNA. Nicotinamide increased not only ceramide synthesis but also free fatty acid (2.3-fold) and cholesterol synthesis (1.5-fold). Topical application of nicotinamide increased ceramide and free fatty acid levels in the stratum corneum, and decreased transepidermal water loss in dry skin. Nicotinamide improved the permeability barrier by stimulating de novo synthesis of ceramides, with upregulation of SPT and other intercellular lipids.

The lipid and keratin structure of pig stratum corneum has been elucidated by small- and wide-angle X-ray diffraction. The measurements were carried out as a function of hydration and temperature. In addition, the stratum corneum was measured after recrystallization of the lipids at various temperatures. The results led us to conclude that the intercellular lipids in the stratum corneum are organized in at least two different lamellar structures with repeat distances of 6 and 13.2 nm. There is an indication for the presence of a third phase with a periodicity of 9 nm. The wide-angle pattern revealed a hexagonal (0.414 nm spacing) and liquid lateral packing (approximately 0.46 nm spacing). The 0.414 nm reflection started to decrease in intensity between 60 and 66 degrees C and disappeared between 72 and 95 degrees C. Furthermore, crystalline cholesterol has been indicated by both, wide- and small-angle X-ray diffraction, while the reflections of alpha-keratin were observed in the wide-angle X-ray diffraction pattern.

that could enable an epidermal targeting effect."

In order to clarify the mechanisms underlying the surfactant-induced dehydration of the stratum corneum (SC), we first examined the constitutive water-holding function of the SC by removing intercellular lipids as well as hygroscopic materials from intact human SC. The dehydration of the SC can be easily induced by depletion of intercellular lipids, but was not affected by an additional water treatment, which releases a large amount of hygroscopic materials including amino acids. Consistent with this, the application of isolated intercellular lipids or their subfractions to the lipid-depleted and dehydrated SC rescues the decreased water content as well as marked scaling, although glycerine did not have such an effect. Parallel differential scanning calorimetry analysis revealed that lipid depletion causes the SC to reduce the nonfreezable bound water content and that application of isolated intercellular lipids recovered the bound water content up to the control level. On the other hand, the additional release of hygroscopic materials from lipid-depleted SC did not affect the bound water content. Based upon the above constitutive water-holding mechanism in the SC, we assessed the physicochemical mechanisms involved in surfactant-induced dehydration of the SC, leading to tight sensation and skin roughness. Similar to treatment with acetone/ether, treatment with surfactant releases a significant amount of intercellular lipids, the intensity of which varies among various surfactants but correlates well with the intensity of the induced dehydration of the SC leading to the tight skin sensation and skin roughness. Recovery or inhibition experiments revealed that the dehydration of the SC induced by surfactant treatment is significantly attenuated by the application of isolated intercellular lipids or by the addition of monoglyceride (MG) during the washing process, which results in a significant reduction in skin roughness. The latter study showed that whereas the release of sebum components and amino acids during surfactant treatment is not affected by the addition of MG, the removal of ceramides is significantly attenuated by the addition of MG, which suggests an essential role for ceramide depletion in the induction of the surfactant-induced dehydration of the SC. In conclusion, the above findings collectively suggest that the ceramide content in the SC is strongly associated with the regulation of SC hydration and that its deficiency due to surfactant treatment is essentially responsible for the surfactant-induced dehydration of the SC. This hypothesis is further strengthened by the fact that synthetic pseudoceramides are remarkably effective in preventing or abolishing the dehydration of the SC which is an intrinsic factor for inducing the tight sensation and roughness of the skin and which provides a pathological basis for atopic dry skin.

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.

This work demonstrates the ability to detect changes in both quantity and spatial distribution of human stratum corneum (SC) lipids from samples collected in vivo. The SC functions as the predominant barrier to the body, protecting against the penetration of xenobiotic substances. Changes to the SC lipid composition have been associated with barrier impairment and consequent skin disorders, and it is therefore important to monitor and quantify changes to this structure. This work demonstrates the first reported use of time-of-flight secondary ion mass spectrometry (ToF-SIMS) to assess physiological changes to human SC as a function of depth. This technique provides exceptional sensitivity and chemical specificity, allowing analysis of single tape stripped samples taken from volunteers. Using this methodology we were able to successfully identify chemical differences in human SC resulting from both intrinsic and extrinsic (photo) aging. Samples were collected from women of two age groups (under 27 and postmenopausal) and from two body sites with varying UV exposure (inner forearm and dorsal hand), and differences were identified using multivariate data analysis. The key finding was the significant aged-related increase and change in spatial distribution of the sterol cholesterol sulfate, a membrane stabilizing lipid. Significant changes in the prevalence of both lignoceric acid (C24:0) and hexacosanoic acid (C26:0) were also observed. This work describes previously unreported age-related chemical changes to human SC, providing an insight into aging mechanisms which may improve the design of both pharmaceutical and cosmetic topical products.