Applying a vernix caseosa based formulation accelerates skin barrier repair by modulating lipid biosynthesis
2017; Elsevier BV; Volume: 59; Issue: 2 Linguagem: Inglês
10.1194/jlr.m079186
ISSN1539-7262
AutoresWalter Boiten, Tineke Berkers, Samira Absalah, Jeroen van Smeden, A.P.M. Lavrijsen, Joke A. Bouwstra,
Tópico(s)Therapeutic Uses of Natural Elements
ResumoRestoring the lipid homeostasis of the stratum corneum (SC) is a common strategy to enhance skin barrier function. Here, we used a ceramide containing vernix caseosa (VC)-based formulation and were able to accelerate barrier recovery in healthy volunteers. The recovery was examined over 16 days by monitoring trans-epidermal water loss (TEWL) after barrier disruption by tape-stripping. Four skin sites were used to examine the effects of both treatment and barrier recovery. After 16 days, samples were harvested at these sites to examine the SC ceramide composition and lipid organization. Changes in ceramide profiles were identified using principal component analysis. After barrier recovery, the untreated sites showed increased levels of ceramide subclass AS and ceramides with a 34 total carbon-atom chain length, while the mean ceramide chain length was reduced. These changes were diminished by treatment with the studied formulation, which concurrently increased the formulated ceramides. Correlations were observed between SC lipid composition, lipid organization, and TEWL, and changes in the ceramide subclass composition suggest changes in the ceramide biosynthesis. These results suggest that VC-based formulations enhance skin barrier recovery and are attractive candidates to treat skin disorders with impaired barrier properties. Restoring the lipid homeostasis of the stratum corneum (SC) is a common strategy to enhance skin barrier function. Here, we used a ceramide containing vernix caseosa (VC)-based formulation and were able to accelerate barrier recovery in healthy volunteers. The recovery was examined over 16 days by monitoring trans-epidermal water loss (TEWL) after barrier disruption by tape-stripping. Four skin sites were used to examine the effects of both treatment and barrier recovery. After 16 days, samples were harvested at these sites to examine the SC ceramide composition and lipid organization. Changes in ceramide profiles were identified using principal component analysis. After barrier recovery, the untreated sites showed increased levels of ceramide subclass AS and ceramides with a 34 total carbon-atom chain length, while the mean ceramide chain length was reduced. These changes were diminished by treatment with the studied formulation, which concurrently increased the formulated ceramides. Correlations were observed between SC lipid composition, lipid organization, and TEWL, and changes in the ceramide subclass composition suggest changes in the ceramide biosynthesis. These results suggest that VC-based formulations enhance skin barrier recovery and are attractive candidates to treat skin disorders with impaired barrier properties. Vital functions of the stratum corneum (SC) include preventing excessive water loss from the body and protecting the body from the hostile outside environment by acting as an inside-out and an outside-in barrier, respectively. The SC is generated by a dynamic process in which keratinocytes in the lower layer of the epidermis undergo terminal differentiation and transform into corneocytes (1.Eckhart L. Lippens S. Tschachler E. Declercq W. Cell death by cornification.Biochim. Biophys. Acta. 2013; 1833: 3471-3480Crossref PubMed Scopus (282) Google Scholar). During this differentiation process, lipids are synthesized, stored in lamellar bodies and, at the interface between the viable epidermis and SC, these lamellar bodies are secreted in the intercellular regions (2.Feingold K.R. Elias P.M. Role of lipids in the formation and maintenance of the cutaneous permeability barrier.Biochim. Biophys. Acta. 2014; 1841: 280-294Crossref PubMed Scopus (239) Google Scholar). The secreted lipids form an extracellular lipid matrix crucial for a proper skin barrier (3.van Smeden J. Janssens M. Gooris G.S. Bouwstra J.A. The important role of stratum corneum lipids for the cutaneous barrier function.Biochim. Biophys. Acta. 2014; 1841: 295-313Crossref PubMed Scopus (3) Google Scholar, 4.Sahle F.F. Gebre-Mariam T. Dobner B. Wohlrab J. Neubert R.H. Skin diseases associated with the depletion of stratum corneum lipids and stratum corneum lipid substitution therapy.Skin Pharmacol. Physiol. 2015; 28: 42-55Crossref PubMed Scopus (120) Google Scholar). Three main lipid classes have been identified: cholesterol, fatty acids, and ceramides. The latter constitutes the group of SC lipids that have the most diverse chemical structure. Their building blocks are an array of sphingoid bases and acyl chains chemically linked together by an amide bond, resulting in 16 ceramide subclasses (5.t'Kindt R. Jorge L. Dumont E. Couturon P. David F. Sandra P. Sandra K. Profiling and characterizing skin ceramides using reversed-phase liquid chromatography-quadrupole time-of-flight mass spectrometry.Anal. Chem. 2012; 84: 403-411Crossref PubMed Scopus (149) Google Scholar). Both chains (the sphingoid base and acyl chain) can vary substantially in carbon chain length. The composition of the SC ceramide fraction (the ceramide profile) has been correlated with and shown to be important for lipid organization and skin barrier function (6.Mojumdar E.H. Kariman Z. van Kerckhove L. Gooris G.S. Bouwstra J.A. The role of ceramide chain length distribution on the barrier properties of the skin lipid membranes.Biochim. Biophys. Acta. 2014; 1838: 2473-2483Crossref PubMed Scopus (66) Google Scholar, 7.Stahlberg S. Lange S. Dobner B. Huster D. Probing the Role of Ceramide Headgroup Polarity in Short-Chain Model Skin Barrier Lipid Mixtures by (2)H Solid-State NMR Spectroscopy.Langmuir. 2016; 32: 2023-2031Crossref PubMed Scopus (21) Google Scholar, 8.Janssens M. van Smeden J. Gooris G.S. Bras W. Portale G. Caspers P.J. Vreeken R.J. Hankemeier T. Kezic S. Wolterbeek R. et al.Increase in short-chain ceramides correlates with an altered lipid organization and decreased barrier function in atopic eczema patients.J. Lipid Res. 2012; 53: 2755-2766Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). In the present study, the effect of a ceramide-containing formulation on skin barrier recovery, ceramide composition, and lipid organization were studied. Through the processes of cornification and desquamation, the SC is continuously rejuvenated (9.Matsui T. Amagai M. Dissecting the formation, structure and barrier function of the stratum corneum.Int. Immunol. 2015; 27: 269-280Crossref PubMed Scopus (168) Google Scholar, 10.Rawlings A.V. Molecular basis for stratum corneum maturation and moisturization.Br. J. Dermatol. 2014; 171: 19-28Crossref PubMed Scopus (49) Google Scholar) and can thereby quickly recover the barrier function after disruption (11.Tanaka M. Zhen Y.X. Tagami H. Normal recovery of the stratum corneum barrier function following damage induced by tape stripping in patients with atopic dermatitis.Br. J. Dermatol. 1997; 136: 966-967Crossref PubMed Scopus (34) Google Scholar). An impaired skin barrier function is encountered in several inflammatory skin diseases, one of which is atopic dermatitis (AD). In AD, an inadequate recovery of the impaired skin barrier perpetuates the condition. Normalization of the barrier function in AD is crucial to reduce the penetration of irritants and allergens into the skin. Application of lipophilic formulations that act as an additional barrier is a common treatment (12.Wolf R. Parish L.C. Barrier-repair prescription moisturizers: do we really need them? Facts and controversies.Clin. Dermatol. 2013; 31: 787-791Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 13.Hon K.L. Leung A.K. Barankin B. Barrier repair therapy in atopic dermatitis: an overview.Am. J. Clin. Dermatol. 2013; 14: 389-399Crossref PubMed Scopus (88) Google Scholar, 14.Corazza M. Minghetti S. Bianchi A. Virgili A. Borghi A. Barrier creams: facts and controversies.Dermatitis. 2014; 25: 327-333Crossref PubMed Scopus (16) Google Scholar, 15.van Zuuren E.J. Fedorowicz Z. Christensen R. Lavrijsen A. Arents B.W.M. Emollients and moisturisers for eczema.Cochrane Database Syst. Rev. 2017; 2: CD012119PubMed Google Scholar). Another approach aims at normalization of ceramide composition (4.Sahle F.F. Gebre-Mariam T. Dobner B. Wohlrab J. Neubert R.H. Skin diseases associated with the depletion of stratum corneum lipids and stratum corneum lipid substitution therapy.Skin Pharmacol. Physiol. 2015; 28: 42-55Crossref PubMed Scopus (120) Google Scholar). Changes in this composition were correlated to the reduced skin barrier function in AD (8.Janssens M. van Smeden J. Gooris G.S. Bras W. Portale G. Caspers P.J. Vreeken R.J. Hankemeier T. Kezic S. Wolterbeek R. et al.Increase in short-chain ceramides correlates with an altered lipid organization and decreased barrier function in atopic eczema patients.J. Lipid Res. 2012; 53: 2755-2766Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 16.Ito S. Ishikawa J. Naoe A. Yoshida H. Hachiya A. Fujimura T. Kitahara T. Takema Y. Ceramide synthase 4 is highly expressed in involved skin of patients with atopic dermatitis.J. Eur. Acad. Dermatol. Venereol. 2017; 31:: 135-141Crossref PubMed Scopus (22) Google Scholar, 17.Ishikawa J. Narita H. Kondo N. Hotta M. Takagi Y. Masukawa Y. Kitahara T. Takema Y. Koyano S. Yamazaki S. et al.Changes in the ceramide profile of atopic dermatitis patients.J. Invest. Dermatol. 2010; 130: 2511-2514Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Previously, ceramide-containing formulations have been used to study skin barrier repair (18.Meckfessel M.H. Brandt S. The structure, function, and importance of ceramides in skin and their use as therapeutic agents in skin-care products.J. Am. Acad. Dermatol. 2014; 71: 177-184Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 19.Lynde C.W. Andriessen A. A cohort study on a ceramide-containing cleanser and moisturizer used for atopic dermatitis.Cutis. 2014; 93: 207-213PubMed Google Scholar, 20.Oh M.J. Nam J.J. Lee E.O. Kim J.W. Park C.S. A synthetic C16 omega-hydroxyphytoceramide improves skin barrier functions from diversely perturbed epidermal conditions.Arch. Dermatol. Res. 2016; 308: 563-574Crossref PubMed Scopus (9) Google Scholar, 21.Barba C. Parra J.L. Coderch L. Semenzato A. In vivo and in vitro evaluation of topical formulations containing physiological lipid mixture for replacement of skin barrier function.G. Ital. Dermatol. Venereol. 2014; 149: 347-353PubMed Google Scholar) and it was reported that the ratios between the three main SC lipids in a formulation was important for short-term recovery (22.Man MQ.M. Feingold K.R. Thornfeldt C.R. Elias P.M. Optimization of physiological lipid mixtures for barrier repair.J. Invest. Dermatol. 1996; 106: 1096-1101Abstract Full Text PDF PubMed Scopus (249) Google Scholar). Furthermore, it was suggested that long-term application could enhance barrier recovery (21.Barba C. Parra J.L. Coderch L. Semenzato A. In vivo and in vitro evaluation of topical formulations containing physiological lipid mixture for replacement of skin barrier function.G. Ital. Dermatol. Venereol. 2014; 149: 347-353PubMed Google Scholar). Nonetheless, it is disputed whether ceramides in a formulation actually normalize SC lipid composition or remain on the skin surface (23.Zhang Q. Flach C.R. Mendelsohn R. Mao G. Pappas A. Mack M.C. Walters R.M. Southall M.D. Topically applied ceramide accumulates in skin glyphs.Clin. Cosmet. Investig. Dermatol. 2015; 8: 329-337PubMed Google Scholar). It has been shown that the orally administered essential fatty acids were incorporated in the epidermis (24.Rhodes L.E. O'Farrell S. Jackson M.J. Friedmann P.S. Dietary fish-oil supplementation in humans reduces UVB-erythemal sensitivity but increases epidermal lipid peroxidation.J. Invest. Dermatol. 1994; 103: 151-154Abstract Full Text PDF PubMed Scopus (106) Google Scholar). How these formulations accelerate barrier recovery is not clearly understood. Therefore, the primary aim of this study was to examine how treatment with a formulation during barrier recovery affects SC lipid composition and subsequently lipid organization. This was done by examining the normal physiological responses in healthy volunteers. In this study, we applied a ceramide-containing formulation based on the composition of the vernix caseosa (VC). This is a natural ceramide-containing formulation that serves as a protective lipid film covering the fetus in the last trimester of pregnancy and during delivery (25.Rissmann R. Oudshoorn M.H. Zwier R. Ponec M. Bouwstra J.A. Hennink W.E. Mimicking vernix caseosa–preparation and characterization of synthetic biofilms.Int. J. Pharm. 2009; 372: 59-65Crossref PubMed Scopus (11) Google Scholar). Previously, it was shown that VC-like formulations were able to significantly accelerate barrier recovery in mice (26.Oudshoorn M.H. Rissmann R. van der Coelen D. Hennink W.E. Ponec M. Bouwstra J.A. Effect of synthetic vernix biofilms on barrier recovery of damaged mouse skin.Exp. Dermatol. 2009; 18: 695-703Crossref PubMed Scopus (12) Google Scholar, 27.Oudshoorn M.H. Rissmann R. van der Coelen D. Hennink W.E. Ponec M. Bouwstra J.A. Development of a murine model to evaluate the effect of vernix caseosa on skin barrier recovery.Exp. Dermatol. 2009; 18: 178-184Crossref PubMed Scopus (32) Google Scholar). This vernix formulation has the advantage that ceramides can be formulated into it. The formulation is semi-occlusive (28.Visscher M.O. Barai N. LaRuffa A.A. Pickens W.L. Narendran V. Hoath S.B. Epidermal barrier treatments based on vernix caseosa.Skin Pharmacol. Physiol. 2011; 24: 322-329Crossref PubMed Scopus (24) Google Scholar). To examine the effect of the formulation on barrier recovery, we used barrier disruption by tape-stripping, as this is an effective way to remove the barrier and induce barrier recovery in healthy skin (27.Oudshoorn M.H. Rissmann R. van der Coelen D. Hennink W.E. Ponec M. Bouwstra J.A. Development of a murine model to evaluate the effect of vernix caseosa on skin barrier recovery.Exp. Dermatol. 2009; 18: 178-184Crossref PubMed Scopus (32) Google Scholar, 29.Sextius P. Marionnet C. Bon F.X. de La Chapelle A.L. Tacheau C. Lahfa M. Mauviel A. Bernard B.A. Leclaire J. Bernerd F. et al.Large scale study of epidermal recovery after stratum corneum removal: dynamics of genomic response.Exp. Dermatol. 2010; 19: 259-268Crossref PubMed Scopus (15) Google Scholar, 30.Bandier J. Carlsen B.C. Rasmussen M.A. Petersen L.J. Johansen J.D. Skin reaction and regeneration after single sodium lauryl sulfate exposure stratified by filaggrin genotype and atopic dermatitis phenotype.Br. J. Dermatol. 2015; 172: 1519-1529Crossref PubMed Scopus (23) Google Scholar, 31.O'Connor R.J. Ogle J. Odio M. Induction of epidermal damage by tape stripping to evaluate skin mildness of cleansing regimens for the premature epidermal barrier.Int. J. Dermatol. 2016; 55: 21-27Crossref PubMed Scopus (4) Google Scholar, 32.Breternitz M. Flach M. Prassler J. Elsner P. Fluhr J.W. Acute barrier disruption by adhesive tapes is influenced by pressure, time and anatomical location: integrity and cohesion assessed by sequential tape stripping. A randomized, controlled study.Br. J. Dermatol. 2007; 156: 231-240Crossref PubMed Scopus (120) Google Scholar). Here, it is shown that treatment with the formulation resulted in accelerated barrier recovery of tape-stripped skin compared with untreated tape-stripped skin. In regenerated untreated SC, an altered ceramide composition was observed. These alterations correlated to a decrease in skin barrier function. When applying the ceramide-containing formulation, the ceramide composition was modulated toward a ceramide composition more closely mimicking that of control skin. Combined, these results indicate that barrier disruption induces changes in lipid processing during recovery, related to a decreased barrier function and that these changes in lipid processing can be modulated by application of the VC formulation. The following solvents were used for lipid extraction and analysis: Millipore water (18.2 mΩ), HPLC-grade methanol and chloroform from Lab-Scan (Gliwice, Poland), UPLC-grade isopropanol and ethanol from Biosolve (Valkenswaard, The Netherlands), and HPLC-grade heptane from Actu-All (Oss, The Netherlands). The synthetic ceramides listed in supplemental Table S1 were used as calibrators for the LC/MS analysis. Trypsin and trypsin inhibitor for SC isolation were purchased from Sigma-Aldrich (Zwijndrecht, The Netherlands). The clinical study was approved by the Leiden University Medical Center Ethical Committee and performed according to the Declaration of Helsinki. All volunteers signed an informed consent. Eight female and 7 male Caucasian volunteers, aged 18 to 29 years (mean age 23 years), were recruited and checked by a dermatologist. Volunteers were excluded if they had dermatological disorders or a history of dermatological disorders, suffered from chronic inflammatory diseases, used systemic drug therapies, had abundant hair or unnatural abnormalities on the ventral forearms, had a history of drug abuse, or if they were pregnant. All enrolled volunteers completed the clinical study. Figure 1A depicts the timeline of the study. Before the study commenced, volunteers had a 1 week washout period in which no soaps or cosmetics were applied on the ventral forearms. At time point (td0) a site of 3.5 by 2.5 cm of SC was removed at both ventral forearms by consecutive tape-stripping using DSquame tape (Cuderm, Dallas). The tape-stripping continued until the site appeared shiny (33.Pinkus H. Examination of the epidermis by the strip method of removing horny layers. I. Observations on thickness of the horny layer, and on mitotic activity after stripping.J. Invest. Dermatol. 1951; 16: 383-386Abstract Full Text PDF PubMed Scopus (220) Google Scholar) and a trans-epidermal water loss (TEWL) of > 60 g/m2/h was reached (these sites are henceforth referred to as stripped). TEWL was measured using AquaFlux AF200 (BIOX, London, UK). Thereafter, two fingertip units of a VC-like formulation (composition is shown in supplemental Table S2) were applied twice daily for 14 days on one ventral forearm (referred to as treated), followed by a 2 day washout period. Treatment allocation to either left or right arm was alternated between volunteers. Figure 1B depicts the four sites investigated in this study: control (no treatment, no tape-stripping), treated (treated with formulation), stripped (tape-stripped at tD0), and stripped+treated (tape-stripped at TD0 and treated with formulation). At the final time point (tD16) 21 tape strips, using polyphenylene sulfide tape (Nichiban, Tokyo, Japan), were harvested at all four sites. The amount of SC on each tape strip was determined by measuring the tape strips with a SquameScan (Heiland Electronic, Wetzlar, Germany). Tape-strip zero was discarded. After every other tape strip, an infrared spectrum of the tape-stripped skin region was made (section 2.5). After tape-stripping, a 4 mm biopsy of both recovered sites was collected, near the site where Nichiban tape strips were harvested, to perform small angle X-ray diffraction (SAXD; see the section by this name below). During the study, TEWL of all sites was monitored at distinct time points; measurements were performed before formulation application. To determine barrier recovery of the stripped sites, TEWL was monitored. At each time point depicted in Fig. 1C, TEWL was measured at three different sections of each site (supplemental Figure S1). The barrier recovery percentage was calculated by defining the average of three TEWL measurements at the nonstripped sites as 100% barrier and TEWL at the stripped sites directly after tape-stripping as 0% barrier. The following equation was used (26.Oudshoorn M.H. Rissmann R. van der Coelen D. Hennink W.E. Ponec M. Bouwstra J.A. Effect of synthetic vernix biofilms on barrier recovery of damaged mouse skin.Exp. Dermatol. 2009; 18: 695-703Crossref PubMed Scopus (12) Google Scholar): For each volunteer, several values were calculated for both the treated and untreated stripped sites: i) the recovery percentage of the site at three sections at each time-point; ii) the mean of the three previous values; iii) the area under the barrier recovery curve (AUC) of the means over time, using a point-to-point linear fit; iv) using the same fit, the time it took to reach designated recovery percentages between 5 and 100%, with increments of 5%; and v) the difference in time between the stripped and stripped+treated sites at each of the previously mentioned recovery percentage. Recovery percentages >90% were excluded, as only 5 volunteers reached these values at both stripped sites, biasing these data. Tape-stripped SC can be used to examine the lipid content by mass spectrometry (34.Boiten W. Absalah S. Vreeken R. Bouwstra J. van Smeden J. Quantitative analysis of ceramides using a novel lipidomics approach with three dimensional response modelling.Biochim. Biophys. Acta. 2016; 1861: 1652-1661Crossref PubMed Scopus (39) Google Scholar, 35.Masukawa Y. Narita H. Sato H. Naoe A. Kondo N. Sugai Y. Oba T. Homma R. Ishikawa J. Takagi Y. et al.Comprehensive quantification of ceramide species in human stratum corneum.J. Lipid Res. 2009; 50: 1708-1719Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 36.van Smeden J. Boiten W.A. Hankemeier T. Rissmann R. Bouwstra J.A. Vreeken R.J. Combined LC/MS-platform for analysis of all major stratum corneum lipids, and the profiling of skin substitutes.Biochim. Biophys. Acta. 2014; 1841: 70-79Crossref PubMed Scopus (82) Google Scholar). Samples for ceramide analysis by LC/MS were prepared from tape-strips 5–8 and 17–20, harvested at the four sites on tD16. Samples prepared from tape strips 5–8 and 17–20 were analyzed separately to determine if the SC ceramide composition generated in the initial stage of the recovery process (tapes 5–8) was different from that generated in later stages of the recovery process (tapes 17–20). Tape strips were punched to an area of 2 cm2, extracted, analyzed, and quantified as described in Boiten et al. (34.Boiten W. Absalah S. Vreeken R. Bouwstra J. van Smeden J. Quantitative analysis of ceramides using a novel lipidomics approach with three dimensional response modelling.Biochim. Biophys. Acta. 2016; 1861: 1652-1661Crossref PubMed Scopus (39) Google Scholar). Briefly, a modified four-step Bligh and Dyer extraction at 40°C was performed. Extracts were analyzed using an Acquity UPLC H-class (Waters, Milford, MA) connected to an XEVO TQ-S mass spectrometer (Waters). Separation was performed on a pva-silica column (5 μm particles, 100 × 2.1 mm inner diameter; YMC, Kyoto, Japan). Using the calibrators, a response model was build and used to quantify the ceramides in the analyzed samples. All data were corrected for the cumulative SquameScan value (SQ) of the four tape strips comprising the corresponding sample. The data resulted in quantitative SC ceramide profiles for each volunteer, at all four sites, and at two depths per site. Ceramides are named according to hydrophilic head group (subclass) and chain length (e.g., NS C42, ceramide subclass NS with a total chain length of 42 carbon atoms) (37.Motta S. Monti M. Sesana S. Caputo R. Carelli S. Ghidoni R. Ceramide composition of the psoriatic scale.Biochim. Biophys. Acta. 1993; 1182: 147-151Crossref PubMed Scopus (382) Google Scholar). For an overview of subclasses and ceramide structures, see supplemental Fig. S2. Quantitative data were used to calculate several parameters. Ceramides EOS C66 and NS C40 (supplied in the formulation) were excluded from these calculations. The following parameters were calculated: i) total amount of the individual subclasses (ng/SQ); ii) total amount of ceramides with a total chain length 34 carbons (ng/SQ); iii) mean carbon chain length: and iv) percentage of EO ceramides, as ng EO/total ng ceramides; The lateral organization and conformational ordering of the SC lipids were examined by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). FTIR spectra were collected using a Varian 670-IR spectrometer (Agilent Technologies, Inc., Santa Clara, CA) equipped with a mercury-cadmium-telluride detector and an external sample compartment containing an ATR accessory (GladiATR, PIKE Technologies, Madison, WI) with a single reflection diamond. The sample compartment was constantly purged with dry air. Each spectrum was an average of 150 scans, with a spectral resolution of 2 cm−1. Conformational ordering of the lipids was determined using the center of gravity of the peak of the CH2 symmetric stretching vibrations at 90% of the peak height. The bandwidth from the second derivative of the CH2 scissoring region was selected as a measure for lateral packing of the lipids and determined as described previously (38.Boncheva M. Damien F. Normand V. Molecular organization of the lipid matrix in intact Stratum corneum using ATR-FTIR spectroscopy.Biochim. Biophys. Acta. 2008; 1778: 1344-1355Crossref PubMed Scopus (189) Google Scholar, 39.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: 611-614Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). CH2 stretching vibration peak position values <2850 cm−1 indicate an ordered phase, whereas values above 2852 cm−1 indicate a high degree of disordering, being a liquid phase. The second derivative of the CH2 scissoring region was baseline-corrected between the endpoints of the scissoring region (∼1460–1480 cm−1) and the full width at half maximum (FWHM) was calculated (39.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: 611-614Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Values of 5 spectra (tape 2–10 and tape 12–20) per site per volunteer were used for the analyses. SC isolated from biopsies harvested at stripped and stripped+treated sites were examined by SAXD. SAXD was used to determine the SC lipids lamellar organization. To isolate SC for SAXD analysis, biopsies were placed overnight in a 0.1% trypsin solution in PBS at 4°C, followed by 1 h incubation at 37°C. The SC was peeled off and washed in 0.1% trypsin inhibitor solution in PBS and twice in Millipore water. Dried SC sheets were stored under argon and over silica gel until use. All SAXD measurements were performed at the European Synchrotron Radiation Facility (Grenoble, France) at station BM26B. SAXD patterns were collected on a Pilatus 1M detector at room temperature for 5 min. The sample-to-detector distance was 2 m. Prior to data collection, the SC samples were hydrated over a 27% (w/v) NaBr solution for 24 h at room temperature. The scattering intensity I (arbitrary units) was measured as a function of the scattering vector q (in nm−1), defined as: in which Θ is the scattering angle and λ is the wavelength. From the position of the main peak (referred to as peak 2 with position q2), the spacing (d) of the lipid lamellae was calculated using d. As in SC, this peak position is determined by the structure of the two lamellar phases; a change in the spacing indicates a change in these lamellar phases (8.Janssens M. van Smeden J. Gooris G.S. Bras W. Portale G. Caspers P.J. Vreeken R.J. Hankemeier T. Kezic S. Wolterbeek R. et al.Increase in short-chain ceramides correlates with an altered lipid organization and decreased barrier function in atopic eczema patients.J. Lipid Res. 2012; 53: 2755-2766Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). All LC/MS data were processed using MassLynx and TargetLynx software (V4.1 SCN 843, Waters Inc.). FTIR data were processed and analyzed using Resolutions Pro 4.1 (Agilent Technologies, Inc.). Principal component analysis (PCA) is a commonly used analysis of lipidomics data sets (40.Checa A. Bedia C. Jaumot J. Lipidomic data analysis: tutorial, practical guidelines and applications.Anal. Chim. Acta. 2015; 885: 1-16Crossref PubMed Scopus (75) Google Scholar, 41.Vaz F.M. Pras-Raves M. Bootsma A.H. van Kampen A.H. Principles and practice of lipidomics.J. Inherit. Metab. Dis. 2015; 38: 41-52Crossref PubMed Scopus (34) Google Scholar). It is a mathematical dimensional reduction that can effectively visualize variation and the difference between samples in large data sets by reducing all variables to principal components (PCs). PCA was performed using Multibase add-in for Excel (NumericalDynamics.Com). For PCA, the quantitative data obtained from the ceramide analyses were normalized to the total amount of ceramides in the samples. The four individual sites (control, treated, stripped, and stripped+treated) of each volunteer were defined as "PCA samples". All quantified ceramides at both depths were defined as variables of that PCA sample (total of 584 variables), thereafter the mean of each ceramide/variable in all PCA samples was centered at zero (subtracting the mean). Correlation analysis and point-to-points fits were performed using GraphPad Prism (V6.05, Graphpad). Group-wise comparisons were performed using linear mixed models (LMMs) in SPSS (V24 IBM). All measurements within the same subject were treated as repeated measurement. LMMs were used because these models have the advantage that the effect size of individual variables and their interactions can be examined, and they can handle missing data (42.De Livera A.M. Zaloumis S. Simpson J.A. Models for the analysis of repeated continuous outcome measures in clinical trials.Respirology. 2014; 19: 155-161Crossref PubMed Scopus (22) Google Scholar). Supplemental Fig. S3 explains how the LMMs were used here. All interactions between fixed variables were included in t
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