The Role of the Trans Double Bond in Skin Barrier Sphingolipids

Article pubs.acs.org/Langmuir

The Role of the Trans Double Bond in Skin Barrier Sphingolipids: Permeability and Infrared Spectroscopic Study of Model Ceramide and Dihydroceramide Membranes Barbora Školová, Kateřina Jandovská, Petra Pullmannová, Ondřej Tesař, Jaroslav Roh, Alexandr Hrabálek, and Kateřina Vávrová* Skin Barrier Research Group, Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Heyrovského 1203, 50005 Hradec Králové, Czech Republic ABSTRACT: Dihydroceramides (dCer) are members of the sphingolipid family that lack the C4 trans double bond in their sphingoid backbone. In addition to being precursors of ceramides (Cer) and phytoceramides, dCer have also been found in the extracellular lipid membranes of the epidermal barrier, the stratum corneum. However, their role in barrier homeostasis is not known. We studied how the lack of the trans double bond in dCer compared to Cer influences the permeability, lipid chain order, and packing of multilamellar membranes composed of the major skin barrier lipids: (d)Cer, fatty acids, cholesterol, and cholesteryl sulfate. The permeability of the membranes with long-chain dCer was measured using various markers and was either comparable to or only slightly greater than (by up to 35%, not significant) that of the Cer membranes. The dCer were less sensitive to acyl chain shortening than Cer (the short dCer membranes were up to 6-fold less permeable that the corresponding short Cer membranes). Infrared spectroscopy showed that long dCer mixed less with fatty acids but formed more thermally stable ordered domains than Cer. The key parameter explaining the differences in permeability in the short dCer and Cer was the proportion of the orthorhombic phase. Our results suggest that the presence of the trans double bond in Cer is not crucial for the permeability of skin lipid membranes and that dCer may be underappreciated members of the stratum corneum lipid barrier that increase its heterogeneity.

■

INTRODUCTION Ceramides (Cer) belong to a large family of sphingolipids that share common sphingoid backbones, including sphingosine, dihydrosphingosine (sphinganine), phytosphingosine (4-hydroxysphinganine), and 6-hydroxysphingosine. Sphingosinebased Cer have gained much attention as cell-signaling molecules1−3 and as key constituents of the skin barrier that prevent desiccation and the entry of unwanted substances into the human body.4−8 Since the 1990s, the C4 trans double bond in the sphingosine backbone has been presumed to be essential for a variety of Cer biological functions, and the corresponding dihydroceramides (dCer) were described to lack activity. This assumption originated from studies comparing short-chain (C2 or C6) dCer and Cer (reviewed in Fabrias et al.9). Later studies showed that the long-chain dCer are not inactive, although their effects differ from those of Cer: for example, dCer inhibited the channel formation induced by Cer in mitochondrial membranes and mitigated apoptotic cell death,10 and dCer accumulation induced autophagy with no sign of cell death.11 In fact, dCer are precursors of Cer in their de novo biosynthesis from L-serine. The double bond is introduced by dCer desaturase (Des). Des1 mainly exhibits high Δ4desaturase activity, leading to Cer, whereas Des2 exhibits both Δ4-desaturase and 4-hydroxylase activities, leading to both Cer and phytoceramides (phytoCer) (Figure 1).9,12−16 © 2014 American Chemical Society

Figure 1. Structures and biosynthesis of Cer from dCer. The C4 trans double bond in Cer is introduced by the action of Des1 and Des2 on dCer. Des2 also exhibits 4-hydroxylation activity, leading to phytoCer (panel A). The structures and abbreviations of the investigated Cer and dCer are given in panel B.

In human skin, Cer together with cholesterol (Chol), free fatty acids (FFA), and small amounts of cholesterol sulfate (CholS) form multiple lamellae that fill the intercellular space Received: February 15, 2014 Revised: April 1, 2014 Published: April 29, 2014 5527

dx.doi.org/10.1021/la500622f | Langmuir 2014, 30, 5527−5535

Langmuir

Article

(24C) and short acyl chains (C2−C8) in model membranes composed of (d)Cer/Chol/FFA/CholS that mimic some of the important aspects of the skin lipid barrier. The permeability of the studied membranes was assessed using three different markers: electrical impedance, steady-state flux of theophylline (TH), and steady-state flux of indomethacin (IND). Additionally, the biophysical behavior of the membranes containing dCer was studied by attenuated total reflectance Fouriertransform infrared spectroscopy (ATR-FTIR) and compared to those with Cer.

in the uppermost epidermal layer, the stratum corneum (SC). These lipids are the most important part of the skin barrier that prevents water loss from the human body.4−8 Although the presence of free dihydrosphingosine in skin was described in 1990,17 dCer were detected only recently with the improvement of mass spectrometry analytical techniques. In 2008, Masukawa et al. described dCer with non-hydroxylated (NdS) and α-hydroxylated acyls (AdS) in the human SC,18 and in 2011, Bouwstra’s group found that the unique epidermal Cer with an extremely long ω-linoleyloxyacyl (also termed acylCer) also contained the dihydrosphingosine backbone (i.e., EOdSclass Cer).19,20 Their results indicate that dCer constitute approximately 10% of the sphingolipids in the human skin barrier. The importance of Cer in the skin is supported by the observation of diminished Cer levels in prominent skin diseases, such as atopic dermatitis, ichthyosis, and psoriasis.6,8 Lower Cer levels lead to higher water loss and to the penetration of allergens and toxic substances, which trigger inflammation and disturb the homeostasis of the skin barrier. This “vicious circle” can be blocked by a topical supplementation of Cer or their less expensive analogues (pseudoCer).21−24 Although the potencies of some pseudo-Cer have been well documented, the structural requirements and mechanisms of action of exogenous Cer and their synthetic analogues are seldom defined.5 We have previously demonstrated that the acyl chain length in Cer is crucial for maintaining skin barrier properties: shortchain Cer increase the permeability of both the skin25,26 and model lipid membranes27 by forming separated domains with shorter lamellar periodicity and less orthorhombic packing than those of the native long-chain lipids. The short and long Cer also penetrate to different depths of the epidermis after their topical application to the human skin.28 Here, we focus on the C4−C5 unsaturation of Cer because the construction of this double bond is, together with the correct stereochemistry, the most difficult step in Cer chemical synthesis, which impedes Cer use in barrier repair. Our question was the following: What is the role of the C4 trans double bond of Cer in SC lipid membranes? The first possible answer to this question is that the C4 trans double bond is not necessary and that Cer could be replaced by dCer (or even more simplified analogues) in the treatment of skin diseases. This possibility is supported by the fact that dCer are physiologically present in the SC.18−20 However, a certain Cer/dCer ratio might be crucial for proper skin barrier function: Takagi et al. showed that keratinocyte-specific Arntdeficient mice died shortly after birth due to severe water loss. These mice showed similar histological characteristics with only slightly narrower spacing in the SC lipid lamellae but a significantly altered Cer/dCer ratio and decreased Des2 transcript levels.29 Another study demonstrated that in Des1−/− mice the overall Cer levels were decreased, while the dCer levels were dramatically higher.30 The surviving animals were small and had scaly skin and sparse hair, suggesting an altered epidermal function with the altered Cer/dCer ratio. For free sphingoid bases, a markedly increased sphingosine/ dihydrosphingosine ratio (from 5.5 to 15.2) was found in atopic eczema.31,32 At the molecular level, the C4 trans double bond was found to influence the biophysical properties of Cer vs dCer membranes.33,34 In this work, we aimed at comparing the permeability and biophysical behavior of a series of Cer and dCer having long

■

EXPERIMENTAL SECTION

Chemicals. Cer and dCer were purchased from Avanti Polar Lipids (Alabaster, AL) or synthesized (see below). Deuterated lignoceric acid (DFFA) was obtained from C/D/N isotopes (Pointe-Claire, Canada). All other chemicals and solvents were from Sigma-Aldrich (Schnelldorf, Germany). Water was deionized, distilled, and filtered through a Millipore Q purification system. Synthesis of N-[(2S,3R)-1,3-Dihydroxyoctadecan-2-yl]butyramide (dCer4). Butyric acid (26.1 mg, 0.269 mmol), dihydrosphingosine (81.2 mg, 0.269 mmol), and N-hydroxysuccinimide (31 mg, 0.269 mmol) were dissolved in 5 mL of dry CH2Cl2 under nitrogen and cooled to 0 °C. Next, 1-ethyl-3-(3(dimethylamino)propyl)carbodiimide (34 mg, 0.269 mmol) in dry CH2Cl2 was slowly added and then stirred for 0.5 h at 0 °C, and the reaction mixture was then allowed to warm to room temperature and stirred overnight. Next, the reaction mixture was washed with 0.1 M HCl and water. The organic phase was dried with Na2SO4 and then evaporated; the residue was purified by column chromatography on silica gel using 50:1 CHCl3/methanol (v/v) as the mobile phase (Rf = 0.36). Yield: 51%, white crystals, mp = 117−120 °C, [α] = +4.1° (c = 0.37; CHCl3). IR (ATR): ν 3279, 2917, 2850, 1644, 1549, 1467, 721 cm−1. 1H NMR (CDCl3, 300 MHz): δ 6.58−6.52 (m, 1H), 4.02−3.96 (m, 1H), 3.87−3.74 (m, 3H), 2.99−2.67 (m, 2H), 2.24 (t, J = 7.1 Hz, 2H), 1.76−1.62 (m, 2H), 1.59−1.47 (m, 2H), 1.41−1.12 (m, 26H), 0.97 (t, J = 7.3 Hz, 3H), 0.88 (t, J = 6.5 Hz, 3H) ppm. 13C NMR (CDCl3, 75 MHz): δ 173.8; 74.1; 62.4; 53.9; 38.6; 34.5; 31.9; 29.7; 29.7; 29.6; 29.6; 29.4; 26.0; 22.7; 19.2; 14.1; 13.7 ppm. MS (ESI+) m/ z 372.4 (M + H+), 394.3 (M + Na+). Preparation of Model SC Lipid Membranes. The model SC lipid membranes were prepared as equimolar mixtures of Cer (or dCer), Chol, and lignoceric acid (most abundant FFA in the SC35) with the addition of 5 wt % of CholS. The lipids were dissolved in 2:1 hexane/96% ethanol (v/v) at 4.5 mg/mL (note: the use of 96% (not absolute) ethanol is necessary to dissolve CholS). These lipid solutions (3 × 100 μL per cm2) were slowly sprayed on Nuclepore polycarbonate filters with 15 nm pores (Whatman, Kent, UK) under a stream of nitrogen using a Linomat IV (Camag, Muttenz, Switzerland) equipped with additional y-axis movement. These lipid films were heated to 90 °C, a temperature that is well above the main phase transition, equilibrated for 10 min, and then slowly (∼3 h) cooled to room temperature. Then, they were incubated at 32 °C for 24 h at 30% air humidity. The membranes for ATR-FTIR were prepared in the same way. For details, see our previous work.27 Permeability Experiments. The permeability of the model SC lipid membranes was evaluated using Franz diffusion cells with an available diffusion area of 0.5 cm2 and an acceptor volume of approximately 6.5 mL. The membranes were mounted into the diffusion cells with the lipid film facing the donor compartment. The acceptor compartment of the cell was filled with phosphate-buffered saline (PBS, containing 10 mM phosphate buffer, 137 mM NaCl and 2.7 mM KCl) at pH 7.4 with 50 mg/L gentamicin. The acceptor phase was stirred at 32 °C; the precise volume was measured for each cell and was included in the calculation. After a 1 h equilibration, the electrical impedance was measured (see below). Next, 100 μL of the donor sampleeither 5% theophylline (TH) or 2% indomethacin (IND) suspensions in 60% propylene glycol (see ref 27 for comments)was applied to the membrane. This setup ensured sink 5528

dx.doi.org/10.1021/la500622f | Langmuir 2014, 30, 5527−5535

Langmuir

Article

Figure 2. Permeabilities of the model SC membranes containing the studied dCer having various acyl chain lengths, FFA, Chol, and CholS (black bars) in comparison with the pertinent Cer-based membranes27 (white bars). Three permeability markers of the model membranes were measured: the electrical impedance (panel A) and steady-state fluxes of TH (B) and IND (C). Data are presented as the means ± SEM, n ≥ 6, ∗, statistically significant against the membrane containing the appropriate long-chain lipid (24C acyl chain, i.e., dCer24 or Cer24), +, statistically significant difference between dCer and Cer with the same chain length as indicated by the lines above the particular columns (p < 0.05). conditions for the selected drugs. Samples of the acceptor phase (300 μL) were withdrawn every 2 h over 8 h and were replaced with the same volume of PBS. During this period, a steady-state situation was reached. The samples were analyzed by HPLC (see below). The cumulative amount of the drug that penetrated across the lipid membrane was corrected for the acceptor phase replacement and was plotted against time, and the steady-state flux was calculated from the linear region of the plot. Electrical Impedance Measurement. The electrical impedance of the model SC lipid membranes was recorded using an LCR meter 4080 (Conrad Electronic, Hirschau, Germany, measuring range of 20 Ω−10 MΩ, error at kΩ values 7 cm−1 splitting between the components of the doublet contour. An interesting behavior was observed in the long-chain dCer24-based membranes. Small domains of phase-separated DFFA were formed in this membrane, as evidenced by the CD2 doublet with 2 cm−1 splitting between its components, suggesting reduced mixing of DFFA and dCer24 compared with DFFA and Cer24, where only a singlet was observed. Above 30 °C, the doublet components merged into one band, indicating well-mixed dCer24 and DFFA chains, but the components split again at 52 °C and remained as a doublet until the onset of the main phase transition at 62 °C. Thus, the pretransition rearrangement in dCer24 involves the separation of some DFFA. This behavior of dCer24 membrane might be connected with a transition between metastable and stable phases or between interdigitated phases that were shown to occur for long asymmetric Cer.44,45

■

DISCUSSION Sphingolipids, particularly Cer, are essential compounds for regulating skin barrier homeostasis. Their hydrogenated counterparts, dCer, have only recently been identified in the SC. dCer constitute approximately 10% of human SC sphingolipids,46 but whether dCer fulfill some special function in the skin or their presence in SC is just a consequence of incomplete desaturation/hydroxylation of dCer into Cer or phytoCer is unknown. Because a previous study suggested that a specific dCer/Cer ratio is important for skin barrier homeostasis,29 we aimed at studying the biophysical differences between Cer and dCer in the skin lipid membrane environment. In particular, we focused on how the lack of the C4 trans double bond in dCer compared to Cer influences the permeability, lipid chain order, and lipid packing of model membranes containing the major SC lipids. We used model multilayered lipid membranes composed of Cer (or dCer), FFA, Chol, and a small amount of CholS. Our membrane models mimic several but not all aspects of the skin barrier: they lack corneocytes; therefore, the permeation pathway is not tortuous, the lipid composition is simpler, and the long periodicity lamellar phase is not present (for a detailed comparison with the SC and discussion, see de Jager et al.47). Nevertheless, our previous study on short-chain Cer showed correlation between the permeabilities of this simple membrane model and those of skin. The advantage of such a simplified four-component model is that we can control and easily vary its composition, which is necessary for structure−activity studies of Cer. First, we compared dCer and Cer with long C24 acyl chains, i.e., the lipids that are found in human skin,18−20 also referred to as CerNdS and CerNS according to the Motta nomenclature.48 We did not find any significant changes in membrane permeability upon the exchange of Cer by dCer, although the dCer24 membranes provided slightly but 5532

dx.doi.org/10.1021/la500622f | Langmuir 2014, 30, 5527−5535

Langmuir

Article

minor alterations in their composition by allowing various compensatory mechanisms.

previous observations that short-chain dCer do not mimic the effects of short Cer.9 The biophysical parameter that explains this difference best is the proportion of the orthorhombic phase; this phase is similar among the dCer membranes in contrast to the Cer ones, where it decreases with chain length and has minima at the Cer with C4−6 acyl.27 This observation might be connected to the higher flexibility of the C4−C5 single bond in dCer compared with the trans double bond in Cer, which affects the molecular shape, hydrogen bonding pattern34 and, in the case of dihydrosphingomyelin, interactions with Chol.51 In particular, the higher permeability of the Cer4− 6 membranes can be explained by that their short acyl chains wobble between the hydrophobic and hydrophilic region of the membranes, preventing tight lipid packing in their vicinity.54 The higher flexibility of the single bond in dCer may lead to a more favorable molecular shape. This lower sensitivity of dCer to the acyl chain shortening might be an advantage in case of the replacement of the long sphingolipids by their shorter counterparts due to, for example, altered activity of Cer synthases or elongases.46,52,53 When considering the possibility of using dCer or pseudoCer without the 4,5 unsaturation as skin barrier repair agents, our results on membrane permeability suggest that the C4 trans double bond in Cer may be substituted by easier-to-synthesize molecular features during the rational design of such compounds. However, this topic seems to be controversial because dCer (palmitoyldihydrosphinganine) was used as a competitive sphingomyelinase inhibitor that slowed barrier repair after acute insult by acetone lipid extraction in mice.55 Nevertheless, certain levels of dCer are physiologically present in the SC. Moreover, L’Oreal’s Ceramide R, which is basically a stereochemically undefined dCer (oleyldihydrosphingosine or 2-oleamidooctadecane-1,3-diol), is widely used as a skin care product, and many other pseudoCer also lack the double bond.56 This topic definitely requires further clarification, including the question of how deep into the skin topically applied Cer or pseudoCer really penetrate during lipid supplementation and to what extent they interfere with sphingolipid metabolism in keratinocytes. A widely accepted paradigm based on a study that used fluorescent short-chain Cer is that topically applied Cer traverse the SC into the nucleated epidermis, are uptaken by cells, reprocessed, and are delivered into the SC via lamellar granules.57,58 However, we showed that long-chain fluorescent Cer do not penetrate into the living epidermis; instead, they stay in the SC.28 This finding may also apply to topical dCer simply because of their lipophilicity. In addition, dCer do not even induce transbilayer (flip-flop) lipid motion in liposomes, in contrast to Cer.59 In conclusion, the permeability differences of the membranes with the long-chain dCer24 and Cer24 were rather subtle, suggesting that the C4 double bond in Cer (essential e.g., for Cer cell signaling) is not crucial for the permeability of SC model lipid membranes provided that the proper lipid chain length is maintained. Although dCer24 mixed less with FFA and these membranes contained fewer orthorhombically packed lipids, these unfavorable features were partly balanced by the stronger cohesive forces and lower sensitivity to acyl chain length variation of dCer compared with Cer. We speculate that the presence of dCer in the skin barrier is not a result of an inability to completely desaturate them during keratinization but of a deliberate process leading to increased heterogeneity in SC Cer classes. Such a heterogeneous mixture of lipids may be more resistant to environmental stressors or

■

AUTHOR INFORMATION

Corresponding Author

*Tel +420 495 067 497; Fax +420 495 067 166; e-mail [email protected] (K.V.). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS

This work was cofinanced by the European Social Fund and the state budget of the Czech Republic (project no. CZ.1.07/ 2.3.00/30.0061), Czech Science Foundation (13-23891S), and Charles University in Prague (SVV 267 001, GAUK 652412).

■

ABBREVIATIONS dCer, dihydroceramide(s); Cer, ceramide(s); Chol, cholesterol; CholS, cholesterol sulfate; Des, dihydroceramide desaturase; DFFA, perdeuterated free fatty acid; FFA, free fatty acid(s); IND, indomethacin; phytoCer, phytoceramides; SC, stratum corneum; TH, theophylline.

■

REFERENCES

(1) van Blitterswijk, W. J.; van der Luit, A. H.; Veldman, R. J.; Verheij, M.; Borst, J. Ceramide: second messenger or modulator of membrane structure and dynamics? Biochem. J. 2003, 369 (Pt 2), 199. (2) Ogretmen, B.; Hannun, Y. A. Biologically active sphingolipids in cancer pathogenesis and treatment. Nat. Rev. Cancer 2004, 4 (8), 604. (3) Morales, A.; Fernandez-Checa, J. C. Pharmacological modulation of sphingolipids and role in disease and cancer cell biology. Mini Rev. Med. Chem. 2007, 7 (4), 371. (4) Mizutani, Y.; Mitsutake, S.; Tsuji, K.; Kihara, A.; Igarashi, Y. Ceramide biosynthesis in keratinocyte and its role in skin function. Biochimie 2009, 91 (6), 784. (5) Novotny, J.; Hrabalek, A.; Vavrova, K. Synthesis and structureactivity relationships of skin ceramides. Curr. Med. Chem. 2010, 17 (21), 2301. (6) Proksch, E.; Brandner, J. M.; Jensen, J. M. The skin: an indispensable barrier. Exp. Dermatol. 2008, 17 (12), 1063. (7) Feingold, K. R. The outer frontier: the importance of lipid metabolism in the skin. J. Lipid Res. 2009, 50 (Suppl.), S417. (8) Holleran, W. M.; Takagi, Y.; Uchida, Y. Epidermal sphingolipids: metabolism, function, and roles in skin disorders. FEBS Lett. 2006, 580 (23), 5456. (9) Fabrias, G.; Munoz-Olaya, J.; Cingolani, F.; Signorelli, P.; Casas, J.; Gagliostro, V.; Ghidoni, R. Dihydroceramide desaturase and dihydrosphingolipids: debutant players in the sphingolipid arena. Prog. Lipid Res. 2012, 51 (2), 82. (10) Stiban, J.; Fistere, D.; Colombini, M. Dihydroceramide hinders ceramide channel formation: Implications on apoptosis. Apoptosis 2006, 11 (5), 773. (11) Signorelli, P.; Munoz-Olaya, J. M.; Gagliostro, V.; Casas, J.; Ghidoni, R.; Fabrias, G. Dihydroceramide intracellular increase in response to resveratrol treatment mediates autophagy in gastric cancer cells. Cancer Lett. 2009, 282 (2), 238. (12) Ternes, P.; Franke, S.; Zahringer, U.; Sperling, P.; Heinz, E. Identification and characterization of a sphingolipid delta 4-desaturase family. J. Biol. Chem. 2002, 277 (28), 25512. (13) Michel, C.; van Echten-Deckert, G.; Rother, J.; Sandhoff, K.; Wang, E.; Merrill, A. H., Jr. Characterization of ceramide synthesis. A dihydroceramide desaturase introduces the 4,5-trans-double bond of sphingosine at the level of dihydroceramide. J. Biol. Chem. 1997, 272 (36), 22432.

5533

dx.doi.org/10.1021/la500622f | Langmuir 2014, 30, 5527−5535

Langmuir

Article

(14) Geeraert, L.; Mannaerts, G. P.; van Veldhoven, P. P. Conversion of dihydroceramide into ceramide: involvement of a desaturase. Biochem. J. 1997, 327 (Pt 1), 125. (15) Schulze, H.; Michel, C.; van Echten-Deckert, G. Dihydroceramide desaturase. Methods Enzymol. 2000, 311, 22. (16) Mikami, T.; Kashiwagi, M.; Tsuchihashi, K.; Akino, T.; Gasa, S. Substrate specificity and some other enzymatic properties of dihydroceramide desaturase (ceramide synthase) in fetal rat skin. J. Biochem. 1998, 123 (5), 906. (17) Wertz, P. W.; Downing, D. T. Free sphingosine in human epidermis. J. Invest. Dermatol. 1990, 94 (2), 159. (18) Masukawa, Y.; Narita, H.; Shimizu, E.; Kondo, N.; Sugai, Y.; Oba, T.; Homma, R.; Ishikawa, J.; Takagi, Y.; Kitahara, T.; Takema, Y.; Kita, K. Characterization of overall ceramide species in human stratum corneum. J. Lipid Res. 2008, 49 (7), 1466. (19) van Smeden, J.; Hoppel, L.; van der Heijden, R.; Hankemeier, T.; Vreeken, R. J.; Bouwstra, J. A. LC/MS analysis of stratum corneum lipids: ceramide profiling and discovery. J. Lipid Res. 2011, 52 (6), 1211. (20) Janssens, M.; van Smeden, J.; Gooris, G. S.; Bras, W.; Portale, G.; Caspers, P. J.; Vreeken, R. J.; Kezic, S.; Lavrijsen, A. P.; Bouwstra, J. A. Lamellar lipid organization and ceramide composition in the stratum corneum of patients with atopic eczema. J. Invest. Dermatol. 2011, 131 (10), 2136. (21) Elias, P. M. An appropriate response to the black-box warning: corrective, barrier repair therapy in atopic dermatitis. Clin. Med. Dermatol. 2009, 2, 1. (22) Vavrova, K.; Hrabalek, A.; Mac-Mary, S.; Humbert, P.; Muret, P. Ceramide analogue 14S24 selectively recovers perturbed human skin barrier. Br. J. Dermatol. 2007, 157 (4), 704. (23) Chamlin, S. L.; Kao, J.; Frieden, I. J.; Sheu, M. Y.; Fowler, A. J.; Fluhr, J. W.; Williams, M. L.; Elias, P. M. Ceramide-dominant barrier repair lipids alleviate childhood atopic dermatitis: changes in barrier function provide a sensitive indicator of disease activity. J. Am. Acad. Dermatol. 2002, 47 (2), 198. (24) Man, M. M.; Feingold, K. R.; Thornfeldt, C. R.; Elias, P. M. Optimization of physiological lipid mixtures for barrier repair. J. Invest. Dermatol. 1996, 106 (5), 1096. (25) Janusova, B.; Zbytovska, J.; Lorenc, P.; Vavrysova, H.; Palat, K.; Hrabalek, A.; Vavrova, K. Effect of ceramide acyl chain length on skin permeability and thermotropic phase behavior of model stratum corneum lipid membranes. Biochim. Biophys. Acta 2011, 1811 (3), 129. (26) Novotny, J.; Janusova, B.; Novotny, M.; Hrabalek, A.; Vavrova, K. Short-chain ceramides decrease skin barrier properties. Skin Pharmacol. Physiol. 2009, 22 (1), 22. (27) Skolova, B.; Janusova, B.; Zbytovska, J.; Gooris, G. S.; Bouwstra, J. A.; Slepicka, P.; Berka, P.; Roh, J.; Palat, K.; Hrabalek, A.; Vavrova, K. Ceramides in the skin lipid membranes: Length matters. Langmuir 2013, 29 (50), 15624. (28) Novotny, J.; Pospechova, K.; Hrabalek, A.; Cap, R.; Vavrova, K. Synthesis of fluorescent C24-ceramide: evidence for acyl chain length dependent differences in penetration of exogenous NBD-ceramides into human skin. Bioorg. Med. Chem. Lett. 2009, 19 (24), 6975. (29) Takagi, S.; Tojo, H.; Tomita, S.; Sano, S.; Itami, S.; Hara, M.; Inoue, S.; Horie, K.; Kondoh, G.; Hosokawa, K.; Gonzalez, F. J.; Takeda, J. Alteration of the 4-sphingenine scaffolds of ceramides in keratinocyte-specific Arnt-deficient mice affects skin barrier function. J. Clin. Invest. 2003, 112 (9), 1372. (30) Holland, W. L.; Brozinick, J. T.; Wang, L. P.; Hawkins, E. D.; Sargent, K. M.; Liu, Y.; Narra, K.; Hoehn, K. L.; Knotts, T. A.; Siesky, A.; Nelson, D. H.; Karathanasis, S. K.; Fontenot, G. K.; Birnbaum, M. J.; Summers, S. A. Inhibition of ceramide synthesis ameliorates glucocorticoid-, saturated-fat-, and obesity-induced insulin resistance. Cell. Metab. 2007, 5 (3), 167. (31) Aburai, K.; Yoshino, S.; Sakai, K.; Sakai, H.; Abe, M.; Loiseau, N.; Holleran, W.; Uchida, Y.; Sakamoto, K. Physicochemical analysis of liposome membranes consisting of model lipids in the stratum corneum. J. Oleo. Sci. 2011, 60 (4), 197.

(32) Loiseau, N.; Moradian, S.; Elias, P. M.; Holleran, W.; Uchida, Y. Ceramide metabolites in epidermal permeability barrier function and atopic dermatitis. J. Invest. Dermatol. 2009, 129 (1s), S68. (33) Brockman, H. L.; Momsen, M. M.; Brown, R. E.; He, L.; Chun, J.; Byun, H. S.; Bittman, R. The 4,5-double bond of ceramide regulates its dipole potential, elastic properties, and packing behavior. Biophys. J. 2004, 87 (3), 1722. (34) Li, L.; Tang, X.; Taylor, K. G.; DuPre, D. B.; Yappert, M. C. Conformational characterization of ceramides by nuclear magnetic resonance spectroscopy. Biophys. J. 2002, 82 (4), 2067. (35) Norlen, L.; Nicander, I.; Lundsjo, A.; Cronholm, T.; Forslind, B. A new HPLC-based method for the quantitative analysis of inner stratum corneum lipids with special reference to the free fatty acid fraction. Arch. Dermatol. Res. 1998, 290 (9), 508. (36) Fasano, W. J.; Hinderliter, P. M. The Tinsley LCR Databridge Model 6401 and electrical impedance measurements to evaluate skin integrity in vitro. Toxicol. In Vitro 2004, 18 (5), 725. (37) Mitragotri, S. Modeling skin permeability to hydrophilic and hydrophobic solutes based on four permeation pathways. J. Controlled Release 2003, 86 (1), 69. (38) Mendelsohn, R.; Moore, D. J. Vibrational spectroscopic studies of lipid domains in biomembranes and model systems. Chem. Phys. Lipids 1998, 96 (1−2), 141. (39) Snyder, R. G.; Schachtschneider, J. H. Vibrational analysis of the n-paraffinsI: Assignments of infrared bands in the spectra of C3H8 through n-C19H40. Spectrochim. Acta 1963, 19 (1), 85. (40) Gay, C. L.; Guy, R. H.; Golden, G. M.; Mak, V. H.; Francoeur, M. L. Characterization of low-temperature (i.e., < 65° C) lipid transitions in human stratum corneum. J. Invest. Dermatol. 1994, 103 (2), 233. (41) Damien, F.; Boncheva, M. The extent of orthorhombic lipid phases in the stratum corneum determines the barrier efficiency of human skin in vivo. J. Invest. Dermatol. 2010, 130 (2), 611. (42) Arseneault, M.; Lafleur, M. Cholesterol sulfate and Ca2+ modulate the mixing properties of lipids in stratum corneum model mixtures. Biophys. J. 2007, 92 (1), 99. (43) Pare, C.; Lafleur, M. Formation of liquid ordered lamellar phases in the palmitic acid/cholesterol system. Langmuir 2001, 17 (18), 5587. (44) Pinto, S. N.; Silva, L. C.; de Almeida, R. F. M.; Prieto, M. Membrane domain formation, interdigitation, and morphological alterations induced by the very long chain asymmetric C24:1 ceramide. Biophys. J. 2008, 95 (6), 2867. (45) Pinto, S. N.; Silva, L. C.; Futerman, A. H.; Prieto, M. Effect of ceramide structure on membrane biophysical properties: The role of acyl chain length and unsaturation. Biochim. Biophys. Acta, Biomembr. 2011, 1808 (11), 2753. (46) Janssens, M.; van Smeden, J.; Gooris, G. S.; Bras, W.; Portale, G.; Caspers, P. J.; Vreeken, R. J.; Hankemeier, T.; Kezic, S.; Wolterbeek, R.; Lavrijsen, A. P.; Bouwstra, J. A. Increase in shortchain ceramides correlates with an altered lipid organization and decreased barrier function in atopic eczema patients. J. Lipid Res. 2012, 53 (12), 2755. (47) de Jager, M.; Groenink, W.; Bielsa i Guivernau, R.; Andersson, E.; Angelova, N.; Ponec, M.; Bouwstra, J. A novel in vitro percutaneous penetration model: evaluation of barrier properties with p-aminobenzoic acid and two of its derivatives. Pharm. Res. 2006, 23 (5), 951. (48) Motta, S.; Monti, M.; Sesana, S.; Caputo, R.; Carelli, S.; Ghidoni, R. Ceramide composition of the psoriatic scale. Biochim. Biophys. Acta 1993, 1182 (2), 147. (49) Kumagai, K.; Yasuda, S.; Okemoto, K.; Nishijima, M.; Kobayashi, S.; Hanada, K. CERT mediates intermembrane transfer of various molecular species of ceramides. J. Biol. Chem. 2005, 280 (8), 6488. (50) Rerek, M. E.; Chen, H. C.; Markovic, B.; Van Wyck, D.; Garidel, P.; Mendelsohn, R.; Moore, D. J. Phytosphingosine and sphingosine ceramide headgroup hydrogen bonding: Structural insights through 5534

dx.doi.org/10.1021/la500622f | Langmuir 2014, 30, 5527−5535

Langmuir

Article

thermotropic hydrogen/deuterium exchange. J. Phys. Chem. B 2001, 105 (38), 9355. (51) Kuikka, M.; Ramstedt, B.; Ohvo-Rekila, H.; Tuuf, J.; Slotte, J. P. Membrane properties of D-erythro-N-acyl sphingomyelins and their corresponding dihydro species. Biophys. J. 2001, 80 (5), 2327. (52) Park, Y. H.; Jang, W. H.; Seo, J. A.; Park, M.; Lee, T. R.; Park, Y. H.; Kim, D. K.; Lim, K. M. Decrease of ceramides with very long-chain fatty acids and downregulation of elongases in a murine atopic dermatitis model. J. Invest. Dermatol. 2012, 132 (2), 476. (53) Sassa, T.; Ohno, Y.; Suzuki, S.; Nomura, T.; Nishioka, C.; Kashiwagi, T.; Hirayama, T.; Akiyama, M.; Taguchi, R.; Shimizu, H.; Itohara, S.; Kihara, A. Impaired epidermal permeability barrier in mice lacking elovl1, the gene responsible for very-long-chain Fatty Acid production. Mol. Cell. Biol. 2013, 33 (14), 2787. (54) Nybond, S.; Bjorkqvist, Y. J. E.; Ramstedt, B.; Slotte, J. P. Acyl chain length affects ceramide action on sterol/sphingomyelin-rich domains. Biochim. Biophys. Acta, Biomembr. 2005, 1718 (1−2), 61. (55) Schmuth, M.; Man, M. Q.; Weber, F.; Gao, W.; Feingold, K. R.; Fritsch, P.; Elias, P. M.; Holleran, W. M. Permeability barrier disorder in Niemann-Pick disease: sphingomyelin-ceramide processing required for normal barrier homeostasis. J. Invest. Dermatol. 2000, 115 (3), 459. (56) Moeller, H. In Cosmetic Lipids and the Skin Barrier; Foerster, T., Ed.; Taylor and Francis Group: New York, 2002; p 1. (57) Man, M. Q.; Feingold, K. R.; Elias, P. M. Exogenous lipids influence permeability barrier recovery in acetone-treated murine skin. Arch. Dermatol. 1993, 129 (6), 728. (58) Mao-Qiang, M.; Brown, B. E.; Wu-Pong, S.; Feingold, K. R.; Elias, P. M. Exogenous nonphysiologic vs physiologic lipids. Divergent mechanisms for correction of permeability barrier dysfunction. Arch. Dermatol. 1995, 131 (7), 809. (59) Contreras, F. X.; Basanez, G.; Alonso, A.; Herrmann, A.; Goni, F. M. Asymmetric addition of ceramides but not dihydroceramides promotes transbilayer (flip-flop) lipid motion in membranes. Biophys. J. 2005, 88 (1), 348.

5535

dx.doi.org/10.1021/la500622f | Langmuir 2014, 30, 5527−5535