Naturally occurring ω-Hydroxyacids.

ω-Hydroxyacids are fatty acids bearing a hydroxyl group on the terminal carbon. They are found in mammals and higher plants and are often involved in providing a permeability barrier, the primary purpose of which is to reduce water loss. Some ω-hydroxyacid derivatives may be involved in waterproofing and signalling. The purpose of this review was to survey the known natural sources of ω-hydroxyacids. ω-Hydroxyacids are produced by two different P450-dependent mechanisms. The longer (30-34 carbons) ω-hydroxyacids are produced by chain extension from palmitic acid until the chain extends across the membrane in which the extension is taking place, and then the terminal carbon is hydroxylated. Shorter fatty acids can be hydroxylated directly to produce C16 and C18 ω-hydroxyacids found in plants and 20-eicosatetraenoic acid (20-HETE) by a different P450. The C16 and C18 ω-hydroxyacids are components of polymers in plants. The long-chain ω-hydroxyacids are found in epidermal sphingolipids, in giant-ring lactones from the sebum of members of the equidae, as a component of meibum and in carnauba wax and wool wax.

Current understanding of skin biology pertinent to skin penetration: skin biochemistry.

The purpose of this review is to summarize some of the biochemical or chemical findings that have contributed most significantly to our current understanding of the permeability barrier of the skin. This literature survey covers the period from the 1970s up to the present. This seems appropriate since earlier progress was comprehensively covered in a 1978 review by Bob Scheuplein entitled 'Permeability of the skin: a review of major concepts' and in the earlier review by Scheuplein and Blank entitled 'Permeability of the skin'. Both of these review articles are still being cited, and the earlier one has been cited more than 800 times. Overlap with material covered in these earlier publications will be minimized. The overall significance of findings from some of the most recent years may not yet be determined. The emphasis will be placed on the determination of the composition and structures of the epidermal lipids, especially those of the stratum corneum, key enzymes in the biosynthesis of these lipids and some of the physical chemical properties of these lipids as revealed by X-ray diffraction, infrared spectroscopy and other physical methods.

Sphingoid bases are taken up by Escherichia coli and Staphylococcus aureus and induce ultrastructural damage.

Sphingoid bases found in the outer layers of the skin exhibit antimicrobial activity against gram-positive and gram-negative bacteria. We investigated the uptake of several sphingoid bases by Escherichia coli and Staphylococcus aureus, and assessed subsequent ultrastructural damage. E. coli and S. aureus were incubated with D-sphingosine, dihydrosphingosine, or phytosphingosine at ten times their MIC for 0.5 and 4 h, respectively, to kill 50% of viable bacteria. Treated bacterial cells were immediately prepared for SEM, TEM, and analyzed for lipid content by QTLC. E. coli and S. aureus treated with sphingoid bases were distorted and their surfaces were concave and rugate. Significant differences were observed in the visual surface area relative to controls for both E. coli and S. aureus when treated with dihydrosphingosine and sphingosine (p < 0.0001) but not phytosphingosine. While sphingoid base-treated S. aureus exhibited disruption and loss of cell wall and membrane, E. coli cytoplasmic membranes appeared intact and the outer envelope uncompromised. Both E. coli and S. aureus cells contained unique internal inclusion bodies, likely associated with cell death. QTLC demonstrated extensive uptake of sphingoid bases by the bacteria. Hence, sphingoid bases induce both extracellular and intracellular damage and cause intracellular inclusions that may reflect lipid uptake.

The relationship of hydrogen peroxide exposure protocol to bleaching efficacy.

The purpose of this study was to compare two in-office bleaching methods with respect to tooth color change and level of hydrogen peroxide penetration into the pulp cavity and to evaluate relationships between penetration level and color change. Eighty extracted canines were exposed to two different bleaching regimens (conventional vs sealed bleaching technique). After exposure to 38% hydrogen peroxide gel for one hour, hydrogen peroxide amount was estimated spectrophotometrically. Color change was measured per Commission Internationale de l'Eclairage methodology. Linear regression was used to evaluate factors affecting color change, including bleaching technique. The conventional and sealed bleaching groups showed no difference for any color change parameters (ΔL, Δa, Δb, ΔE); however, there was significantly greater hydrogen peroxide penetration in the conventional bleaching group (p<0.05). Linear modeling of the change in lightness (ΔL) showed that the increase in lightness tended to be greater for teeth with lower initial L* values (r=-0.32, p<0.05). After adjustment for initial L*, there was no evidence that ΔL differed with hydrogen peroxide penetration levels (p>0.05) or bleaching technique (mean group difference in ΔL=0.36; p>0.05).

Organization, barrier function and antimicrobial lipids of the oral mucosa.

As one moves from the skin across the vermilion region of the lip and into the oral cavity, the oral mucosa is encountered. The oral mucosa consists of connective tissue known as the lamina propria covered by a stratified squamous epithelium. In the regions of the hard palate and gingiva, the epithelium is keratinized like the epidermis. In the buccal region, the floor of the mouth and the underside of the tongue, the epithelium is non-keratinized. The epithelium on the dorsum of the tongue is a specialized epithelium, but can be approximated as a mosaic of keratinized and non-keratinized epithelia. The non-keratinized epithelial regions do not produce a stratum corneum. Nuclei with intact DNA are retained in the superficial cells. In all regions, the outer portions of the epithelium provide a protective permeability barrier, which varies regionally. Antimicrobial lipids at the surfaces of the oral mucosa are an integral part of innate immunity.

The emerging role of peptides and lipids as antimicrobial epidermal barriers and modulators of local inflammation.

Skin is complex and comprised of distinct layers, each layer with unique architecture and immunologic functions. Cells within these layers produce differing amounts of antimicrobial peptides and lipids (sphingoid bases and sebaceous fatty acids) that limit colonization of commensal and opportunistic microorganisms. Furthermore, antimicrobial peptides and lipids have distinct, concentration-dependent ancillary innate and adaptive immune functions. At 0.1-2.0 µM, antimicrobial peptides induce cell migration and adaptive immune responses to coadministered antigens. At 2.0-6.0 µM, they induce cell proliferation and enhance wound healing. At 6.0-12.0 µM, they can regulate chemokine and cytokine production and at their highest concentrations of 15.0-30.0 µM, antimicrobial peptides can be cytotoxic. At 1-100 nM, lipids enhance cell migration induced by chemokines, suppress apoptosis, and optimize T cell cytotoxicity, and at 0.3-1.0 µM they inhibit cell migration and attenuate chemokine and pro-inflammatory cytokine responses. Recently, many antimicrobial peptides and lipids at 0.1-2.0 µM have been found to attenuate the production of chemokines and pro-inflammatory cytokines to microbial antigens. Together, both the antimicrobial and the anti-inflammatory activities of these peptides and lipids may serve to create a strong, overlapping immunologic barrier that not only controls the concentrations of cutaneous commensal flora but also the extent to which they induce a localized inflammatory response.

Penetration pattern of rhodamine dyes into enamel and dentin: confocal laser microscopy observation.

Enamel and dentin are susceptible to extrinsic and intrinsic stains. The purposes of this study were to determine the penetration pattern of Rhodamine B and dextran-conjugated Rhodamine B into the enamel and dentin as observed by confocal laser microscopy and to relate it to the penetration pattern of hydrogen peroxide commonly used as an active ingredient in tooth-whitening agents and high-molecular-weight staining molecules. Eighteen recently extracted human maxillary anterior teeth were used. Teeth were cleaned and painted with nail varnish except for the crown area above the cemento-enamel junction (CEJ). The painted teeth were then immersed in Rhodamine B and dextran-conjugated Rhodamine B (70 000 MW) for 4, 7, 10 and 15 days. Teeth were sliced to 3 mm thickness in transverse plane and mounted on a glass slide just prior to observation with confocal laser microscopy. Rhodamine B and dextran-conjugated Rhodamine B readily penetrated into the enamel and dentin when exposed for 4 and 7 days, respectively. Rhodamine B penetrated along the interprismatic spaces of the enamel into the dentin. The penetration was accentuated in sections with existing crack lines in the enamel. Rhodamine B was readily absorbed into the dentinal tubules at the dentino-enamel junction and continued to penetrate through the dentin via the dentinal tubules into the pre-dentin. Within the limitations of this study, it is concluded that Rhodamine B and dextran-conjugated Rhodamine B when applied to the external surface of the tooth readily penetrate into the enamel and dentin via the interprismatic spaces in the enamel and dentinal tubules in the dentin, suggesting that stain molecules and bleaching agents possibly exhibit similar penetration pathways.

Comparative analysis of skin surface lipids of the labia majora, inner thigh, and forearm.

The purpose of this study was to develop a procedure for the collection of skin surface corneocyte lipids from the semioccluded and intimate regions of the labia majora and inner thigh of women, to evaluate the polar and nonpolar composition, and to compare the distribution of the lipid classes relative to a collection of lipids from the forearm. The solvent system of ethanol-cyclohexane was well tolerated across all sites. While the yield of polar lipids was similar across all 3 sites, there were only marginal differences in the relative abundance of ceramides, a class of lipids closely associated with skin barrier activity. The yield of neutral lipids was significantly less for the labia majora and was associated with a reduced yield of wax esters, triglycerides and free fatty acids, likely associated with reduced sebaceous gland activity. Factors that may contribute to an inferior skin barrier activity for the labia majora are discussed and suggest a possible deficiency of ω-6 fatty acid linked to the sphingosine base of ceramide EOS.

Glycolipids in mammalian epidermis: structure and function in the water barrier.

In the epidermis of terrestrial vertebrates, lipid lamellae between the horny cells are thought to form a barrier to water loss. The lipids are extruded from living cells after assembly in lamellar granules. This assembly might be promoted by recently identified 1-(3'-O-acyl)-beta-D-glucosyl-N-(omega-hydroxyacyl)sphingosines, which have 30- and 32-carbon hydroxy acids as amides and linoleic acid esterified to glucose. Such a role for these molecules could explain the effects of essential fatty acid deficiency, in which the lamellar granules fail to assemble and the barrier to water diffusion is lost.

Human synthetic sebum formulation and stability under conditions of use and storage.

The human skin surface and hair are generally coated with a thin film of liquid phase sebaceous lipids. This surface lipid film contributes to the cosmetic properties of the skin. Synthetic sebum has been used for studies on properties of skin and hair. However, there has been no standardized formulation of synthetic sebum and many of the synthetic sebum formulations that have been used do not closely resemble actual sebum. In this study, a formulation for a standardized and inexpensive synthetic sebum is proposed, and the chemical stability of this lipid mixture is demonstrated under conditions of use and storage. The proposed synthetic sebum consists of 17% fatty acid, 44.7% triglyceride, 25% wax monoester (jojoba oil) and 12.4% squalene. This lipid mixture takes up approximately 6% of its weight in water when equilibrated in an atmosphere saturated with water vapour. It is stable on exposure to the atmosphere at 32 degrees C for at least 48 h, and it is also stable on storage at 4 or -20 degrees C, either dry or in chloroform : methanol solution for at least 6 months. This synthetic sebum could be useful in studies on cosmetic properties of the skin surface or hair, on penetration of chemicals into the skin or in development of standardized tests of laundry detergent performance.

New arrangement of proteins and lipids in the stratum corneum cornified envelope.

A new arrangement of proteins and lipids of stratum corneum (SC) cornified envelope (CE) is proposed. The chemical analysis of CE revealed the presence of free fatty acids (FFA), ceramides (Cer), and important percentages of glutamic acid/glutamine (Glx) and serine (Ser) residues. The molecular structure of these components suggests the existence of covalent links not only between Cer and Glx but also between FFA and Ser. The protein distribution of extracellular surface of CE, i.e., the proteins that could be involved in the bonds with lipids, was studied using post- and pre-embedding immunolabeling electron microscopy. Some loricrin (protein rich in Ser) was detected in the outermost part of the CE protein layer. The external arrangement of some domains of this protein may give rise to form linkages with FFA, yielding further insight into the CE arrangement in which Cer-Glx bonds and FFA-Ser bonds would be involved. Although the importance of fatty acids in the cohesion and barrier function of SC has been widely demonstrated, their role could be associated not only to the presence of these lipids in the intercellular lamellae but also in the CE, in the same way that Cer.

Characterization of rabbit ear skin as a skin model for in vitro transdermal permeation experiments: histology, lipid composition and permeability.

AIM: The aim of this work was to characterize rabbit ear skin in view of its use in transdermal permeation experiments. METHOD: The characterization included histological analysis of the tissue, qualitative and quantitative analysis of stratum corneum (SC) lipids, differential scanning calorimetry and permeation experiments (caffeine, nicotinamide, progesterone). As a reference, pig ear skin was used. RESULTS: The results obtained show that rabbit ear skin has a similar SC thickness compared to pig skin although the viable epidermis has a different structure. The lipid composition of rabbit SC was similar to pig SC but was characterized by a lower content of ceramides and a higher content of cholesterol esters and triglycerides. In terms of permeability, rabbit ear skin was 4-7 times less permeable to hydrophilic compounds, probably because of the higher lipophilicity of its SC. The permeability to progesterone was comparable between isolated pig epidermis and rabbit ear skin. CONCLUSION: Overall, the results obtained in this work support the usefulness of rabbit ear skin as barrier for skin penetration studies, for both lipophilic and hydrophilic permeants.

A new covalently bound ceramide from human stratum corneum -omega-hydroxyacylphytosphingosine.

The outermost layer of the skin, the stratum corneum, consists of non-viable keratin-filled cells, or corneocytes, embedded in a matrix of lipids. The boundary of the cells consists of cross-linked proteins with covalently bound lipids on the outer surface. The spaces between cells are filled with a mixture of ceramides, cholesterol and fatty acids. The stratum corneum provides a protective barrier against water loss through the skin and limits the penetration of potentially harmful substances from the environment. Among the covalently bound lipids on the corneocyte surface are omega-hydroxyacylsphingosine and omega-hydroxyacyl-6-hydroxysphingosine. The previously suspected presence of omega-hydroxyacylphytosphingosine is confirmed in this report through its specific isolation and characterization based on chromatographic behaviour and proton magnetic resonance spectral data.

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-accelerated phospholipid metabolism by 5,8,11,14-eicosatetraynoic acid.

The tumor-promoting agent, 12-O-tetradecanoylphorbol-13-acetate (TPA), accelerates choline phospholipid synthesis in bovine lymphocytes by an oxygen-dependent mechanism. This action is prevented by high concentrations of the cyclooxygenase inhibitor, indomethacin (1 to 3 mM), suggesting a possible involvement of lipid oxidation in the response. The acetylenic analog of arachidonic acid, 5,8,11,14-eicosatetraynoic acid (ETYA), at concentrations in the 10 to 50 microM range also prevents the acceleration of the incorporation of [methyl-3H]choline hydrochloride into choline phospholipids, and it appears more selective in its action. This antagonistic effect of ETYA, an agent which inhibits both the cyclooxygenase and lipoxygenase routes of arachidonic acid oxidation, can be precluded by arachidonic acid but not by other unsaturated or saturated fatty acids. If ETYA is added to lymphocyte cultures after 1 hr of TPA treatment, the established rate of [methyl-3H]choline hydrochloride incorporation is unaffected, but further acceleration is blocked. The inhibition by ETYA cannot be counteracted by any of the prostaglandins, Types A, B, D, E, or F alpha, or by the prostacyclin compounds, PGI1 and 6,9-thia-PGI2. The thromboxane pathway also appears not to be involved since 9,11-azoprosta-5,13-dienoic acid (Azo I), a selective inhibitor of thromboxane synthetase, does not affect the TPA response. These results suggest that TPS may activate the lipoxygenase rather than the cyclooxygenase pathway for lipid oxidation and that an arachidonic acid hydroperoxide or a subsequent metabolite plays a key role in the stimulation of choline phospholipid synthesis by the tumor-promoting phorbol ester, TPA.

Inhibition of skin tumor promotion by retinoic acid and its metabolite 5,6-epoxyretinoic acid.

The ability of 5,6-epoxyretinoic acid, a biologically active metabolites of retinoic acid, to inhibit both the induction of ornithine decarboxylase (ODC) activity and skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) was evaluated. Application of 5,6-epoxyretinoic acid either concurrently with or 1 hr after each application of TPA to the initiated mouse skin inhibited the formation of skin tumors as effectively as did retinoic acid. 5,6-Dihydroretinoic acid, which is a poor substrate for epoxidation, also inhibited skin tumor promotion. 5,6-Epoxyretinoic acid, 5,6-dihydroretinoic acid, and retinoic acid were equally effective in inhibiting the induction of ODC activity by TPA. Insect juvenile hormones inhibited neither the induction of ODC activity nor skin tumor promotion by TPA. These results indicate that (a) epoxidation of retinoic acid at the 5,6-position is not a rate-limiting modification for the anti-promoting activity of retinoic acid and that (b) inhibition of the induction by TPA of mouse epidermal ODC activity may be a simple test for screening the potential prophylactic activities of new retinoids.

Beta-glucosidase activity in porcine epidermis.

One of the final steps in epidermal differentiation is the conversion of glucosylceramides to ceramides, which presumably is mediated by a beta-glucosidase activity. In the present manuscript, it is demonstrated that pig epidermis contains beta-glucosidase activity which is 3.3-times greater than alpha-glucosidase and 5-times greater than beta-galactosidase. This beta-glucosidase was found to be maximally active between pH 3.0 and essentially inactive at pH 9.0. In a standard assay, a disk of epidermis (8 mg dry weight) was submerged in 1 ml of 50 mM acetate buffer (pH 4.7) containing 150 mM NaCl and 15 mM p-nitrophenyl-beta-D-glucopyranoside at room temperature. Reaction was stopped by addition of 4 ml of 100 mM (pH 9.0) borate buffer and the supernatant was transferred to a separate tube. The nitrophenylate anion was then measured spectrophotometrically at a wavelength of 405 nm. Under these conditions, product formation was linear for at least 90 min and an apparent Km of 244 microM was estimated for the synthetic substrate. When the amount of epidermis in the assay was varied, the formation of product per unit of time remained proportional to the amount of epidermis. The level of beta-glucosidase activity was enhanced slightly by the inclusion of sodium taurocholate.

Molecular models of the intercellular lipid lamellae from epidermal stratum corneum.

The purpose of the present study was to test the hypothesis that the 13 nm trilamellar repeat units within the intercellular spaces of epidermal stratum corneum are composed of lamellae with alternating 5-3-5 nm dimensions as presented in previous models [J. Invest. Dermatol. 92 (1989) 251, P.W. Wertz, Integral lipids in hair and stratum corneum, in: P. Jolles, H. Zahn, H. Hocker (Eds.), Hair: Biology And Structure, Birkhauser Verlag, Basel, 1996, pp. 227-238, Acta Derm.-Venereol., Suppl. 208 (2000) 23]. Electron density profiles were measured from transmission electron micrographs of porcine stratum corneum prepared using ruthenium tetroxide [J. Invest. Dermatol. 92 (1989) 251]. Center-to-center distances of adjacent electron-dense bands as well as adjacent lucent bands were measured. Dense band center-to-center measurements were consistent with a 5-3-5 nm arrangement. However, lucent band center-to-center measurements revealed uniform lamellar thickness. It is suggested that linoleate chains in the central lamella reduce more ruthenium than the predominantly saturated chains in the outer lamellae and that this additional reduced ruthenium accumulates under the polar head group regions. A similar phenomenon involving the sphingosine moieties of the covalently bound omega-hydroxyceramide molecules accounts for the three-band pattern seen between the ends of adjacent corneocytes. It is concluded that the component lamellae of the several types of 13 nm trilamellar units of the stratum corneum are all of equal thickness.

Free sphingosines in porcine epidermis.

Sphingosines and phytosphingosines serve as intermediates in the synthesis of ceramides and glucosylceramides, which are prominent components of mammalian epidermis. In the present study, we have investigated the possibility that free sphingoid bases also may be present in epidermal tissue. Samples of pig epidermis were trypsinized to separate the stratum corneum from the unkeratinized portion of the epidermis. After drying, the lipids were extracted and analyzed by thin-layer chromatography using ninhydrin to detect free amino groups. Both the stratum corneum and the unkeratinized epidermal material contained a ninhydrin-positive material with the same mobility as the sphingosine standard. Quantitation of the chromatograms by photodensitometry indicated that free sphingosine bases account for 0.44% by weight of the total stratum corneum lipid and 0.09% of the lipid in the viable portion of the epidermis. To further identify this material, it was treated with 1-fluoro-2,4-dinitrobenzene, which resulted in the production of an intensely yellow N-2,4-dinitrophenyl derivative with the same mobility as N-2,4-dinitrophenylsphingosine on thin-layer chromatography. Oxidation of the isolated dinitrophenyl derivative with lead tetraacetate produced a mixture of aldehydes which were analyzed by gas-liquid chromatography. This analysis indicates that the free sphingoid bases from the stratum corneum consist of a mixture of mainly 16- through 20-carbon sphingenines and sphinganines, the most abundant components being d17:0, d17:1, d18:1 and d20:1. The production of these free sphingosine bases may be significant in regulating epidermal differentiation.

Linoleate content of epidermal acylglucosylceramide in newborn, growing and mature mice.

Epidermis was collected from newborn, growing and adult mice. Acylglucosylceramide, a structurally unique O-acylsphingolipid, was isolated from each sample, and the ester-linked fatty acids were analyzed by capillary column gas-liquid chromatography. The esterified acids of acylglucosylceramide from newborn mice contained 12% linoleate. The linoleate content of the acylsphingolipid increased rapidly, doubling within 4 days and reaching an adult level of 45% within 2 months. The increase in the linoleate content of the epidermal lipid was accompanied by decreases in 16-carbon monoenoic fatty acids and saturated fatty acids ranging from 14 up to and including 24 carbons in length. These results indicate several potential problems for experimentation involving neonatal skin. Also, the possibility that neonatal mouse epidermis may provide a useful model system for studies on the relationship between linoleic acid and epidermal cell proliferation is also raised.

Covalently bound omega-hydroxyacylsphingosine in the stratum corneum.

Pig epidermis was heat separated, and the stratum corneum was isolated after trypsinization. Exhaustive extraction of the stratum corneum fraction with chloroform/methanol mixtures yielded 14.7% lipid on a dry weight basis. After mild saponification of the extracted residue, additional lipid could be extracted which accounted for 2.1% of the stratum corneum weight. This bound lipid proved to consist mainly (91.9%) of N-(omega-hydroxyacyl)sphingosines in which the amide-linked omega-hydroxyacids were 28 to 34 carbon atoms in length. The release of this lipid by mild alkaline hydrolysis indicates that it is bound through an ester linkage. Half of the hydroxyceramide molecules reacted in situ with acidic acetone, suggesting that half of these molecules are attached to the stratum corneum through the omega-hydroxyl function, while the other half may be linked through one of the hydroxyl groups of the sphingosine.