Effect of cholesterol on miscibility and phase behavior in binary mixtures with synthetic ceramide 2 and octadecanoic acid. Infrared studies.

The three main lipid components of the stratum corneum, namely ceramides, free fatty acids and cholesterol, play a fundamental role in the maintenance of the skin barrier. The current investigation is aimed toward understanding the miscibility and intermolecular interactions of these lipids. Toward this end, Fourier transform infrared spectroscopic studies of the three possible equimolar binary mixtures of cholesterol, a synthetic non-hydroxylated fatty acid N-acyl sphingosine with a C18 chain length (N-stearoylsphingosine, approximating human ceramide 2), and stearic acid were undertaken. The thermotropic responses of the methylene stretching and scissoring vibrations were used to evaluate chain conformation and packing respectively. Selective perdeuteration, of either the stearic acid or the ceramide acid chains, permitted separate and simultaneous evaluation of the conformational order and packing properties of the sphingosine chain, the amide linked fatty acid chains and/or the stearic acid chain. Whereas cholesterol mixed well with ceramide at physiological temperatures, the stearic acid was miscible with the cholesterol only at relatively high temperatures where the fatty acid is disordered. A complex interaction between stearic acid and ceramide was detected. A separate fatty acid-rich phase persisted until at least 50 degrees C, whereas at higher temperatures the components appear to be quite miscible. However, a preferential association of the fatty acid with the ceramide base chain is indicated. None of the binary systems studied exhibit miscibility and interactions resembling those in the ternary mixtures of these substances, which is widely used to model stratum corneum. The role of cholesterol in controlling the miscibility characteristics in the ternary system is evident.

Fourier transform infrared spectroscopy and differential scanning calorimetry studies of fatty acid homogeneous ceramide 2.

Ceramides provide a major component of the barrier function of skin. An understanding of barrier organization requires a detailed characterization of ceramide phase behavior and molecular interactions. Toward this end, Fourier transform infrared (FTIR) and differential scanning calorimetry (DSC) studies of ceramide 2 analogues (non-hydroxylated fatty acid N-acyl sphingosines) of specific chain lengths (C(14), C(16), C(18), C(20)) are presented. In addition, the molecular interactions of the individual chains in each molecule are elucidated through thermotropic FTIR studies of derivatives possessing perdeuterated fatty acid chains. DSC data showed a much smaller chain length variation (for the C(16), C(18), C(20) derivatives) in the main order-disorder transition temperature (approx. 93+/-1 degrees C) than is observed in the corresponding series of phosphatidylcholines, consistent with minimal ceramide hydration. The temperature dependence of the methylene stretching and scissoring modes revealed a solid-solid phase transition at 20-25 degrees C below the main order-disorder transition accompanied by chain packing alterations from orthorhombic-->hexagonal subcells. The chain packing transition was accompanied by enhanced penetration of water into the polar region. This was deduced from the temperature dependence of the amide I and II modes, which provide direct evidence for H-->D exchange. The CD(2) scissoring mode splitting of the deuterated fatty acid constituent of the C(16), C(18), C(20) chains revealed preferential segregation of microdomains (3-5 chains) of this species within the orthorhombic phase. In contrast, the sphingosine base chains appeared to be sufficiently separated so as to inhibit interchain vibrational coupling between them. FTIR spectroscopy provides a convenient means for characterizing domain formation, chain packing, and hydration sites of these phases, which are highly ordered under physiological conditions.

Insights into the molecular organization of lipids in the skin barrier from infrared spectroscopy studies of stratum corneum lipid models.

In order to gain some insight into the molecular organization of lipids in the skin barrier we used Fourier transform infrared (FTIR) spectroscopy to investigate models of the stratum corneum (SC) containing deuterated hexadecanoic acid, cholesterol, and ceramide 2 or ceramide 5. In both models there is clear evidence of separate conformationally ordered domains of ceramide and fatty acids. In addition, these chains are packed in orthorhombic subcells at physiological temperatures. The ceramide headgroup behavior indicates distinct hydrogen bonding patterns between the ceramide 2 and ceramide 5 models. In the ceramide 2 model the amide I mode is split into two components suggesting strong transverse intermolecular hydrogen bonding between headgroups. In contrast, no amide splitting is observed for ceramide 5 although the amide frequencies are indicative of strong hydrogen bonding. These observations on the molecular organization of SC lipids are discussed in terms of skin barrier function.

Percutaneous absorption of sunscreens through micro-yucatan pig skin in vitro.

PURPOSE: The objectives of this study were to develop an in vitro model for studying sunscreen permeation in skin, and evaluate the influence of formulation differences. METHODS: The sunscreens studied were two of the most widely used agents, octyl methoxycinnamate (OMC) and benzophenone-3. Preparations containing radiolabeled actives were applied to micro-Yucatan pig skin dermatomed to a thickness of 250-300 microm as a finite dose in a flow-through diffusion system. At the end of each experiment the amounts removed by washing, retained inside stratum corneum (SC) and penetrated into receptor and viable skin were determined. RESULTS: The two sunscreens reached a peak level in SC within an hour. Benzophenone-3 penetrated skin to a greater extent than OMC. The opposite was true when comparisons of SC retention were made. The ratio of retained to penetrated amount of sunscreens from a hydroalcoholic formulation at the end of 10 hours was higher when the sunscreens were present together than alone. CONCLUSIONS: Despite the highly lipophilic nature of sunscreens, particularly OMC, SC is the rate limiting skin layer for penetration. Penetration and SC retention were formulation dependent. The ratio of SC content to the amount penetrated is a useful tool for evaluating sunscreen permeation.

Role of ceramides 2 and 5 in the structure of the stratum corneum lipid barrier.

We utilized Fourier transform infrared (FTIR) spectroscopy to investigate headgroup and chain interactions in model SC lipid barriers containing equimolar amounts of deuterated hexadecanoic acid, cholesterol, and ceramide 2 (non-hydroxy sphingosine) or ceramide 5 (alpha-hydroxy sphingosine). In the ceramide 2 model the thermotropic response of the CD _ 2 and CH _ 2 stretching modes indicates that hexadecanoic acid begins to disorder at 42 degrees C while ceramide 2 remains ordered until 52 degrees C. Additionally, splitting of the CD _ 2 bending and CH _ 2 rocking modes provides evidence for separate orthorhombic hexadecanoic acid and ceramide domains. The ceramide amide I mode (1650 cm ; -1) is split into two components indicating strong intermolecular hydrogen bonding between headgroups. In the ceramide 5 model, the CH _ 2 and CD _ 2 stretching frequencies again reveal highly conformationally ordered ceramide 5 and hexadecanoic acid chains. Splitting of both the CD _ 2 bending and CH _ 2 rocking modes is observed. However, the CH _ 2 rocking frequencies indicate distorted packing of the ceramide. The collapse of these highly ordered phases, and the onset of conformational disorder, occurs at 50 degrees C for both ceramide 5 and hexadecanoic acid. The amide I and II frequencies of ceramide 5 indicate strong H-bonding, although neither mode is split. Our results demonstrate that model SC lipid systems have quite different physical properties depending on whether they contain ceramide 2 or 5. From this we infer that ceramide 2 and 5 make distinct contributions to the structural biophysics of the SC lipid barrier. Our observation of ordered lipid domains is also consistent with the recently proposed domain mosaic model of the skin barrier.

Lipid domains and orthorhombic phases in model stratum corneum: evidence from Fourier transform infrared spectroscopy studies.

A three component model for the lipid barrier of the stratum corneum (SC) consisting of ceramide III, cholesterol, and perdeuterated palmitic acid, has been characterized by Fourier transform infrared spectroscopy. At physiological temperature the CD2 scissoring mode of the palmitic acid methylenes, and the CH2 rocking mode of the ceramide methylenes, are each split into two components. This indicates that both components exist in separate, conformationally ordered phases, probably with orthorhombic perpendicular subcells. The magnitude of the splitting indicates that the domains are at least 100 chains in size. The thermotropic behavior of the CD2 stretching vibrations demonstrates that conformational disordering of the palmitic acid commences at 42 degrees C with a transition midpoint of 50 degrees C. The CH2 stretching frequency indicates the ceramide chains remain ordered until 50 degrees C then disorder with a midpoint of 67 degrees C. The results provide a molecular characterization for the complex low temperature (10-40 degrees C) dynamic behavior suggested by recent 2H NMR experiments.