Changes in the composition of molecular species of covalently bound and free ceramides, and their correlation with disease severity in atopic dermatitis 

Changes in the composition of molecular species of covalently bound and free ceramides, and their correlation with disease severity in atopic dermatitis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Changes in the composition of molecular species of covalently bound and free ceramides, and their correlation with disease severity in atopic dermatitis Akari Kondo, Yuko Takenaka, Anna Fujiwara, Saori Takahashi, Masami Kitade-Miyayama, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2509454/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Ceramides (Cers) are major constituents of the stratum corneum intercellular lipids, which are involved in the barrier function of the skin. Here, we examined the ratio of molecular species of Cers and their correlation with disease severity in patients with atopic dermatitis (AD). The levels of unsaturated fatty acids (USFAs) in both covalently bound and free Cers were higher in the lesional skin of patients with AD than in the non-lesional skin of patients with AD and normal skin of healthy controls. The proportion of USFAs (C30:1, C32:1, and C34:1) was considerably higher than that of other Cer molecular species in both covalently bound and free Cers in patients with AD. The proportion of USFAs in covalently bound Cers positively correlated with the levels of transepidermal water loss (TEWL) (r = 0.542) in the lesional skin of patients with AD. Additionally, the proportion of USFAs in covalently bound Cers positively correlated with thymus and activation-regulated chemokine (TARC), which is an index of disease severity, in the non-lesional (r = 0.676) and lesional (r = 0.503) skin of patients with AD. The proportion of USFA (C32:1), which was the highest in covalently bound Cers, also positively correlated with the TARC level in non-lesional (r = 0.733) and lesional (r = 0.515) skin of patients with AD. Our study is the first to show that there is an increase in USFA in not only lesional but also non-lesional skin of patients with AD. Moreover, this increase in USFA was associated with dryness and impaired barrier function and correlated with the TARC levels, a marker for the degree of type 2 inflammation. We speculate that exacerbation of type 2 inflammation may lead to abnormal epidermal lipid metabolism in the skin of patients with AD. atopic dermatitis ceramides molecular species stratum corneum thymus and activation-regulated chemokine Figures Figure 1 Figure 2 Figure 3 Introduction The skin protects against invasion by foreign particles and functions as a barrier to limit water evaporation from the body. Particularly, the stratum corneum (SC), the outermost layer of the epidermis, and sphingolipid ceramides (Cers), play an important role in the barrier function. Cers consist of fatty acids, long-chain bases, and carbon chains of different lengths [1]. The three types of fatty acids are non-OH fatty acids [N], α-OH fatty acids [A], and esterified ω-OH fatty acids [EO]. The four types of sphingoid bases are dihydrosphingosine [DS], sphingosine [S], 6-OH sphingosine [H], and phytosphingosine [P]. The combinations of these types can be classified into 12 species. Based on the length of the carbon chain, approximately 480 types of Cers have been identified in human SC [2–6]. Acylceramides, which are epidermis-specific molecular species, have three hydrophobic structures with linoleic acid and long fatty acids with carbon chains of more than 28 atoms (C28). Acylceramides are required for the skin barrier function [1]. Covalently bound Cers are formed by the hydrolysis of linoleic acids and binding of the cross-linked proteins of the cornified envelope to the ω-OH group. Acylceramides are important precursors of covalently bound Cers [1]. The corneocyte lipid envelope comprises Cers that are covalently bound to the cornified envelope. The covalently bound Cers connect lipid lamellae and keratinocytes, and they are essential for skin barrier formation [1]. The levels of covalently bound Cers decrease upon ultraviolet-B irradiation, sodium dodecyl sulfate application, and tape stripping [7]; their levels are lower in the winter than in the summer [8]. In normal skin, the ratio of saturated free fatty acids (SFAs) and unsaturated free fatty acids (USFAs) in covalently bound Cers changes with the season and age [8]. Increased USFA proportion reportedly decreased skin barrier function in dry-skinned mice [9]. The levels of covalently bound Cers decrease and the molecular species composition of Cers changes in atopic dermatitis (AD) [10]. In this condition, Cer [NH] consists of non-OH fatty acid and 6-OH sphingosine, Cer [NP] consists of non-OH fatty acid and phytosphingosine, Cer [EOS] consists of esterified ω-OH fatty acid and sphingosine, and Cer [EOH] consists of esterified ω-OH fatty acid; 6-OH sphingosine levels are low. Cer [AS] consists of α-OH fatty acid and sphingosine levels are high. Acylceramide Cer [EOS] levels are the most depleted among the Cer molecular species [11, 12]. To our knowledge, no study has assessed the correlation between molecular species and lipid composition associated with epidermal barrier function in AD. Assessment of the association between molecular species and lipid composition and AD severity in clinical practice is also required. Based on previous reports [8, 9] we speculated that AD, which occurs in men and women of all ages, decreases epidermal barrier function and increases USFA proportions, changing the molecular species composition of Cers. Here, we focused on Cer [EOS], the most important Cer molecular species for the barrier function [13] and flexibility of the SC, and a modulator of SC liquid balance [14]. Furthermore, we examined the lipid composition and molecule types of Cer in the SC and their correlation with AD severity. Methods Participants We examined Japanese patients who visited Tokyo Women’s Medical University Hospital between August 2017 and September 2019. We enrolled 38 patients with AD and 32 healthy individuals (controls) with no clinical skin rash. Informed consent was obtained from all participants according to the principles of the Declaration of Helsinki. This study was approved by the Institutional Review Board of Tokyo Women’s Medical University Hospital (No. 4430; approved on August 1, 2017) and Meiji Co., Ltd. (No. 120; approved on August 31, 2017). AD diagnosis in the patients was made clinically based on the Japanese Guidelines for Atopic Dermatitis 2020 [15]. Biophysical Measurements of SC The SC sheets were collected by stripping three times with a 30 mm × 25 mm piece of polyphenylene sulfide (PPS) tape (Teraoka Seisakusho, Tokyo, Japan) at room temperature (24°C ± 2°C and 50% ± 15% relative humidity). In 38 patients with AD, SC sheets were collected from the lesional skin of upper extremities in 26, lower extremities in 7, trunks in 2, and palm in 1, respectively. In 2 patients, SC sheets could not be collected, since they were in remission with no active lesion. In the 38 patients with AD, SC sheets were collected from the non-lesional skin of upper extremities in 24, lower extremities in 10, and face in 1, respectively. In 3 patients, SC sheets could not be collected, since they were in erythroderma. In the healthy control, SC sheets were also collected from the upper and lower extremities. The collected samples were stored at −80°C until analysis. Measurement of Transepidermal Water Loss Transepidermal water loss (TEWL) was measured using a Tewameter TM 300 (Courage + Khazaka Electronic, Cologne, Germany). Owing to limited outpatient time, measurements were made from the same area where the SC sheets were collected in eight out of 26 patients with AD and 31 healthy controls. Measurements were conducted in an air-conditioned room (26°C ± 3°C and 46% ± 18% relative humidity). Clinical Assessment Blood samples from patients with AD who gave consent were collected simultaneously with the other samples. We examined eczema area and severity index (EASI) [16], investigator's global assessment (IGA) [17], serum immunoglobulin E (IgE), thymus and activation-regulated chemokine (TARC), lactate dehydrogenase (LDH), and total eosinophil count (TEC). Extraction of Cers Cers were extracted according as previously described [8, 18]. Half of each of the three tapes collected was cut, immersed in hexane, and sonicated in ice-cold water for 30 min to remove the tape adhesive. SC cells were recovered from the extracted liquid on a piece of filter paper (Kiriyama Roto Filter paper 124; Nippon Rikagaku Kikai, Tokyo, Japan) using suction filtration. After drying, the filter paper was immersed in chloroform/methanol (2:1, v/v) and sonicated for 30 min under cool conditions. The filtrate, containing free Cers, was recovered, dried, and re-dissolved in methanol for the analysis of free Cers. The protein pellet was recovered on filter paper and dried using a centrifugal evaporator. The filter paper was incubated in 1 M KOH in 95% methanol at 60°C for 2 h to release the lipids that were covalently bound to the SC via ester-like bonds. The solution, including the filter paper, was neutralized with 1 N HCl and centrifuged at 1,830 × g for 5 min at room temperature (H-700FRS; Kokusan Co., Ltd., Saitama, Japan). The supernatant containing ω-OH Cers was recovered. The sediment was washed again with methanol. The supernatant was recovered, combined, dried, and re-dissolved in methanol for the analysis of covalently bound Cers. The pellet containing the proteins was immersed in phosphate buffered saline (pH 7.4) containing 1% sodium dodecyl sulfate and incubated at 60°C for 2 h to solubilize the protein. The protein concentration was measured using a commercial kit (Micro BCA Assay Kit; Pierce Biotechnology, Rockford, IL, USA). Analysis of Cers Covalently bound Cers and free Cers in the SC samples were identified using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) (Quattro Premier XE; Waters Corporation, Milford, MA, USA). All analyses were performed on a 2 mm × 100 mm column with a particle size of 1.7 µm (ACQUITY UPLC ® BEH C18; Waters, Milford, MA, USA). The analysis of covalently bound Cers was performed as follows. Mobile phase A consisted of 5 mM ammonium acetate in 95% methanol and mobile phase B consisted of 5 mM ammonium acetate in methanol. The initial eluent composition was 100% A, followed by an increase to 100% B for 30 min, 100% B for 2 min, and then 0% B for 3 min. The total running time was 35 min at an eluent flow of 0.4 mL/min and a column temperature of 40°C. The analytes were detected using electrospray ionization in the positive mode. Multiple-reaction monitoring was performed using characteristic fragmentation ions (m/z 676.7/264.3 for d18:1 (sphingosine) /C26:0 m/z 704.7/264.3 for d18:1/C28:0, m/z 732.7/264.3 for d18:1/C30:0, m/z 730.7/264.3 for d18:1/C30:1, m/z 760.8/264.3 for d18:1/C32:0, m/z 758.8/264.3 for d18:1/C32:1, m/z 788.8/264.3 for d18:1/C34:0, m/z 786.8/264.3 for d18:1/C34:1, and m/z 814.8/264.3 for d18:1/C36:1). The parameters for the HPLC-MS/MS analysis were as follows: capillary voltage, 3000 V; source temperature, 120°C; desolvation temperature, 400°C; desolvation gas flow, 850 L/h; cone voltage, 40 V; cone gas flow, 50 L/h; and collision energy, 30 eV. The Cer molecular species contents were determined using N-omega-hydroxytriacontanoyl-D-erythro-sphingosine (Matreya, Pleasant Gap, PA, USA) according to the d18:1/C30:0 Cers standard. The free Cers analysis was performed as follows. Mobile phase A consisted of 5 mM ammonium acetate in 95% methanol and mobile phase B consisted of 5 mM ammonium acetate in 95% isopropanol and 5% methanol. The initial eluent composition was maintained at 100% A for 5 min followed by an increase to 50% B for 20 min, 50% B for 5 min, and then 0% B for 5 min. The total running time was 35 min at an eluent flow of 0.4 mL/min and a column temperature of 50°C. The analytes were detected using electrospray ionization in the positive mode. Multiple-reaction monitoring was performed using characteristic fragmentation ions (m/z 957.0/264.3 for d18:1/C26:0/C18:2, m/z 985.0/264.3 for d18:1/C28:0/C18:2, m/z 1013.0/264.3 for d18:1/C30:0/C18:2, m/z 1011.0/264.3 for d18:1/C30:1/C18:2, m/z 1041.0/264.3 for d18:1/C32:0/C18:2, m/z 1039.0/264.3 for d18:1/C32:1/C18:2, m/z 1069.0/264.3 for d18:1/C34:0/C18:2, m/z 1067.0/264.3 for d18:1/C34:1/C18:2, and m/z 1095.0/264.3 for d18:1/C36:1/C18:2). The parameters for the HPLC-MS/MS analysis were as follows: capillary voltage, 3000 V; source temperature, 150°C; desolvation temperature, 400°C; desolvation gas flow, 850 L/h; cone voltage, 30 V; cone gas flow, 50 L/h; and collision energy, 45 eV. The Cer molecular species contents were determined using N-(30-linoleoyloxy-triacontanoyl)-sphingosine (Matreya, Pleasant Gap, PA, USA) according to the d18:1/C30:0/C18:2 Cers standard. Statistical Analysis All data are presented as mean ± standard error of the mean. Data were analyzed using Tukey–Kramer test (comparison between different participants) or paired t -test (comparison between the same participants) (SPSS ver. 25; IBM SPSS, Armonk, NY, USA). Differences between groups were considered significant at P < 0.05 and P < 0.01. Results Baseline Characteristics of Patients with AD Thirty-two healthy individuals (controls) with no clinical skin rash (6 men and 26 women) were enrolled; 38 patients with AD (24 men and 14 women) were enrolled (Table 1). There were 36 adult patients (≥15 years) and 2 adolescent patients (7). Details of the patients are shown in Figure 1. Lipid Composition in Cers Figure 2(a) shows the SFA and USFA levels in covalently bound and free Cers, and total levels. The total levels of covalently bound Cers were significantly higher in lesional skin than in non-lesional skin in patients with AD. In covalently bound Cers, the USFA levels were significantly higher in lesional skin of patients with AD than in non-lesional skin of patients with AD and normal skin in healthy controls. There was no difference in SFA levels among the three samples. The total levels of free Cers in lesional skin of patients with AD were lower than those in normal skin of healthy controls. However, this difference was not significantly different and the levels in non-lesional skin of patients with AD were significantly lower than those in normal skin of healthy controls. The levels of USFAs in free Cers were significantly higher in lesional skin of patients with AD than in non-lesional skin of patients with AD and normal skin of healthy controls. The USFA levels were higher in lesional skin of patients with AD than in normal skin of healthy controls and non-lesional skin of patients with AD in both covalently bound and free Cers. Figure 2(b) shows the ratio of USFAs to SFAs in covalently bound and free Cers. In both covalently bound and free Cers, the USFA proportion was significantly higher in lesional skin of patients with AD than in the skin of the other groups. The USFA proportion in covalently bound Cers decreased as follows: lesional skin of patients with AD > non-lesional skin of patients with AD > normal skin of healthy controls. In contrast, in both covalently bound and free Cers, the proportion of SFAs decreased as follow: normal skin of healthy controls > non-lesional skin of patients with AD > lesional skin of patients with AD. These results showed that USFA proportion was significantly higher and SFA proportion was significantly lower in lesional skin of patients with AD. We identified nine molecular species of Cer [EOS], consisting of sphingosine (d18:1) with a long-chain ω-OH fatty acid (C26:0, 28:0, 30:0, 30:1, 32:0, 32:1, 34:0, 34:1, or 36:1), in 15 of the 38 patients with AD. Figure 2(c) shows the proportion of the nine molecular species in the covalently bound and free Cers. In non-lesional skin of patients with AD and normal skin of healthy controls, SFA (C30:0) was the most common in both covalently bound and free Cers. USFA (C32:1) was the most common in covalently bound Cers in lesional skin of patients with AD and the percentage of SFA (C30:0) was the highest in free Cers. In both covalently bound and free Cers, the percentage of SFA (C30:0) decreased as follows: normal skin of healthy controls > non-lesional skin of AD > lesional skin of AD. The percentage of USFAs (C32:1 and C34:1) increased as follows: normal skin of healthy controls < non-lesional skin of AD < lesional skin of AD. The percentage of USFAs (C30:1, C32:1, and C34:1) was significantly higher in lesional skin of patients with AD than in non-lesional skin of patients with AD and normal skin of healthy controls. Correlation of USFAs in Covalently Bound Cers with Other Indices Figure 3(a) shows the correlation between USFA proportion in covalently bound Cers and TEWL in eight patients with AD. A positive correlation was found between USFA proportion in covalently bound Cers and TEWL in lesional skin of patients with AD (r = 0.542). Figure 3(b) shows the correlation between USFA proportion in covalently bound Cers and the levels of TARC. A positive correlation was found between USFA proportion in covalently bound Cers and TARC levels in non-lesional skin of 13 patients (r = 0.676) and lesional skin of 17 patients with AD (r = 0.503). Figure 3(c) shows the correlation between the USFA (C32:1) proportion and TARC level. The USFA (C32:1) proportion in covalently bound Cers, which was the highest USFA in covalently bound Cers, also positively correlated with the TARC levels in non-lesional skin of 13 patients (r = 0.733) and lesional skin of 17 patients with AD (r = 0.515). Discussion Cer [NH] and Cer [NP] levels correlate strongly with the indicator conductance, which indicates the state of dryness [19]. Cer [NH] and Cer [NP] levels show a negative correlation with TEWL, whereas Cer [NS] and Cer [AS] levels are positively correlated with TEWL [12]. The Cer [NP]/[NS] ratio is a marker for SC function, and its low values suggest a poor skin condition [20]. In AD, the average chain length of long-chain acylceramides is shortened [5, 10, 21], and the Cer [NH] and Cer [NP] levels decrease; the changes in the molecular species of Cers are related to the barrier function of SC [12, 20]. Cer [EOS], an important acylceramide for the function of the SC [14], notably decreases in both lesional and non-lesional skin of patients with AD compared with that in normal skin of healthy controls [11, 12, 21]. Additionally, Cer [EOS] is important for the maintenance of the qualitative and quantitative balance between SFAs and USFAs in the skin barrier [5, 22]. An increase in USFA levels is associated with a decrease in the epidermal barrier function of SC [8, 9]. In AD, the Cer/cholesterol ratio or the lipid/protein ratio in the skin is reduced compared with that in the normal skin of healthy controls, which causes a change in the composition of SC lipids. This effect is considered to contribute to the disruption of the skin barrier function [23–26]. Regarding fatty acid saturation of free Cers, the structure of SC lipids changes considerably, even at the ultrastructural level, when the linoleate of Cer [EOS] is replaced with oleate in the normal skin of healthy controls. Because of the increase in oleic acid, the USFA levels in Cer [EOS] are higher in the winter than in the summer [14, 27]. Regarding Cer desaturation and changes in molecular type in diet-induced dry-skin mice, the average proportion of Cers with SFAs (C30:0, C32:0, and C34:0) decreased and that of USFAs (C32:1, C34:1, and C36:1) increased among covalently bound Cers [9]. In clinical practice, TEWL is used for evaluating the barrier function of the SC and can be measured non-invasively and quantitatively. High TEWL levels have been observed [28] in AD and psoriasis, and there is no significant difference in the TEWL level between normal skin of healthy controls and non-lesional skin in AD [12]. However, TEWL does not always explain the severity and pathophysiology of AD. In contrast, the TARC levels more accurately reflect the changes in AD pathology during treatment compared to peripheral blood eosinophil counts and IgE and LDH levels [29]. In the early stage of AD, the expression of cytokines, such as interleukins IL-4, IL-5, and IL-13, increases, which is a predominant Th2 state. CCR4, a chemokine receptor, is highly specific to Th2 cells and TARC is involved in AD pathophysiology by inducing the migration of CCR4-expressing Th2 lymphocytes. The serum TARC levels in AD are higher than those in other skin diseases, reflecting clinical severity, and decrease with treatment [30, 31]. Here, we focused on Cer [EOS] in AD, which is a typical skin disease characterized by decreased epidermal barrier function. We expected changes in the lipid and molecular species composition of Cers derived from Cer [EOS] because of the decreased epidermal barrier function. We aimed to determine the relationship between Cer [EOS] composition and AD severity. We identified nine molecular species of Cer [EOS], which contained sphingosine (d18:1) with a long-chain ω-OH fatty acid (C26:0, 28:0, 30:0, 30:1, 32:0, 32:1, 34:0, 34:1, or 36:1). The USFA levels in both covalently bound Cers and free Cers increased in lesional skin of patients with AD compared with those in non-lesional skin of patients with AD and normal skin of healthy controls. Furthermore, the USFA proportion in both covalently bound and free Cers increased in the following order: normal skin of healthy controls < non-lesional skin of patients with AD < lesional skin of patients with AD. Regarding the molecular composition of Cers, the proportion of Cers with USFAs (C30:1, C32:1, and C34:1) was significantly higher in both covalently bound and free Cers in lesional skin of patients with AD. Here, a positive correlation was found between USFA proportion in covalently bound Cers and TEWL. Additionally, the USFA proportion in covalently bound Cers positively correlated with the serum TARC levels in both non-lesional and lesional skin of patients with AD. This finding suggests that a state of Th2 predominance may increase USFA proportion and molecular composition changes. SFAs are more resistant to oxidation than USFAs and are involved in membrane stability and development of a stronger lipid lamellar structure [22]. USFAs synthesized from phospholipids via phospholipase A2 promote premature lysis of corneodesmosomes in the SC [32]. When the pH of the SC increases, the catalytic activity of β-glucocerebrosidase and acidic sphingomyelinase, which are important for Cer synthesis, decrease [8, 33]. Furthermore, when the pH of the SC increases, serine protease-activated receptor 2 (PAR2) is activated, which suppresses the secretion of lamellar bodies, resulting in a decrease in skin barrier function [8, 34]. Skin pH increases in patients with AD [35]. The composition of Cer molecular species changes and levels of USFAs increase in the skin of patients with AD; the increase in the pH of the SC causes a decrease in Cers synthase activity, a structural change in the membrane, and PAR2 activation. We inferred that this decrease caused the suppression of lamellar body secretion, resulting in a decrease in skin barrier function. A limitation of this study is that the analysis of molecular species and lipid composition is in the preliminary stage and cannot be easily performed in daily medical care. Additionally, only Cers derived from Cer [EOS] were analyzed. Further studies are required to elucidate the pathogenic mechanisms of AD, the maintenance of external therapies and systemic therapies that will help control fluctuating molecular species or eliminate changes in lipid composition, and the maintenance of lipid composition. Dietary supplementation might also be considered for these changes. Analysis of Cer molecular species and lipid composition might help develop treatments for improving skin barrier function tailored to individual AD pathology. Here, an increase in USFA levels was observed in both lesional and non-lesional skin of patients with AD. This increase in USFA levels was associated with dryness and deterioration of barrier function and was correlated with TARC level, a marker for the degree of type 2 inflammation. These findings suggest that exacerbation of type 2 inflammation may lead to abnormal epidermal lipid metabolism in the skin of patients with AD. Declarations Compliance with Ethical Standards Competing interests: The authors declare that they have no conflict of interest. Research involving Human Participants Ethics approval: This study was approved by the Institutional Review Board of Tokyo Women’s Medical University Hospital (No. 4430; approved on August 1, 2017) and Meiji Co., Ltd. (No. 120; approved on August 31, 2017). Consent to Participate: Informed consent was obtained from all participants according to the principles of the Declaration of Helsinki. Consent to Publish: Not applicable. Acknowledgments None declared. Funding None. Author contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by [Akari Kondo], [Yuko Takenaka], [Anna Fujiwara], [Saori Takahashi],[ Masami Kitade-Miyayama], and [Masashi Morifuji]. The first draft of the manuscript was written by [Akari Kondo] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. References Ohno Y (2017) Elucidation of the synthetic mechanism of acylceramide, an essential lipid for skin barrier function (in Japanese). Yakugaku Zasshi 137: 1201–1208. https://doi.org/10.1248/yakushi.17-00126 Ishikawa J, Shimotoyodome Y, Ito S, Miyauchi Y, Fujimura T, Kitahara T, Hase T (2013) Variations in the ceramide profile in different seasons and regions of the body contribute to stratum corneum functions. Arch Dermatol Res 305: 151–162. https://doi.org/10.1007/s00403-012-1286-5 Masukawa Y, Narita H, Sato H, Naoe A, Kondo N, Sugai Y, et al (2009) Comprehensive quantification of ceramide species in human stratum corneum. J Lipid Res 50: 1708–1719. https://doi.org/10.1194/jlr.D800055-JLR200 Masukawa Y, Narita H, Shimizu E, Kondo N, Sugai Y, Oba T, et al (2008) Characterization of overall ceramide species in human stratum corneum. J Lipid Res 49: 1466–1476. https://doi.org/10.1194/jlr.M800014-JLR200 van Smeden J, Janssens M, Gooris GS, Bouwstra JA (2014) The important role of stratum corneum lipids for the cutaneous barrier function. Biochim Biophys Acta 1841: 295–313. https://doi.org/10.1016/j.bbalip.2013.11.006 Jia ZX, Zhang JL, Shen CP, Ma L (2016) Profile and quantification of human stratum corneum ceramides by normal-phase liquid chromatography coupled with dynamic multiple reaction monitoring of mass spectrometry: development of targeted lipidomic method and application to human stratum corneum of different age groups. Anal Bioanal Chem 408: 6623–6636. https://doi.org/10.1007/s00216-016-9775-6 Takagi Y, Nakagawa H, Kondo H, Takema Y, Imokawa G (2004) Decreased levels of covalently bound ceramide are associated with ultraviolet B-induced perturbation of the skin barrier. J Invest Dermatol 123: 1102–1109. https://doi.org/10.1111/j.0022-202X.2004.23491.x Fujiwara A, Morifuji M, Kitade M, Kawahata K, Fukasawa T, Yamaji T, et al (2018) Age-related and seasonal changes in covalently bound ceramide content in forearm stratum corneum of Japanese subjects: determination of molecular species of ceramides. Arch Dermatol Res 310: 729–735. https://doi.org/10.1007/s00403-018-1859-z Morifuji M, Oba C, Ichikawa S, Ito K, Kawahata K, Asami Y, et al (2015) A novel mechanism for improvement of dry skin by dietary milk phospholipids: effect on epidermal covalently bound ceramides and skin inflammation in hairless mice. J Dermatol Sci 78: 224–231. https://doi.org/10.1016/j.jdermsci.2015.02.017 Macheleidt O, Kaiser HW, Sandhoff K (2002) Deficiency of epidermal protein-bound omega-hydroxyceramides in atopic dermatitis. J Invest Dermatol 119: 166–173. https://doi.org/10.1046/j.1523-1747.2002.01833.x Imokawa G, Abe A, Jin K, Higaki Y, Kawashima M, Hidano A (1991) Decreased level of ceramides in stratum corneum of atopic dermatitis: an etiologic factor in atopic dry skin? J Invest Dermatol 96: 523–526. https://doi.org/10.1111/1523-1747.ep12470233 Ishikawa J, Narita H, Kondo N, Hotta M, Takagi Y, Masukawa Y, et al (2010) Changes in the ceramide profile of atopic dermatitis patients. J Invest Dermatol 130: 2511–2514. https://doi.org/10.1038/jid.2010.161 Conti A, Rogers J, Verdejo P, Harding CR, Rawlings AV (1996) Seasonal influences on stratum corneum ceramide 1 fatty acids and the influence of topical essential fatty acids. Int J Cosmet Sci 18: 1–12. https://doi.org/10.1111/j.1467-2494.1996.tb00131.x Oldroyd J, Critchley P, Tiddy G, Turner J, Rawlings AV (1994) Specialized role for ceramide one in the stratum corneum water barrier. J Invest Dermatol 102: 525 Katoh N, Ohya Y, Ikeda M, Ebihara T, Katayama I, Saeki H, et al (2020) Japanese guidelines for atopic dermatitis 2020. Allergol Int Jul 69: 356–369. https://doi.org/10.1016/j.alit.2020.02.006 Hanifin JM, Thurston M, Omoto M, Cherill R, Tofte SJ, Graeber M (2001) The eczema area and severity index (EASI): assessment of reliability in atopic dermatitis. EASI evaluator group. Exp Dermatol 10: 11–18. https://doi.org/10.1034/j.1600-0625.2001.100102.x Simpson E, Bissonnette R, Eichenfield LF, Guttman-Yassky E, King B, Silverberg JI et al. (2020) The validated investigator global assessment for atopic dermatitis (vIGA-AD): the development and reliability testing of a novel clinical outcome measurement instrument for the severity of atopic dermatitis. J Am Acad Dermatol 83: 839–846. https://doi.org/10.1016/j.jaad.2020.04.104 Ohno Y, Kamiyama N, Nakamichi S, Kihara A (2017) PNPLA1 is a transacylase essential for the generation of the skin barrier lipid ω-O-acylceramide. Nat Commun 8: 14610. https://doi.org/10.1038/ncomms14610 Ishikawa J, Yoshida H, Ito S, Naoe A, Fujimura T, Kitahara T, et al (2013) Dry skin in the winter is related to the ceramide profile in the stratum corneum and can be improved by treatment with a Eucalyptus extract. J Cosmet Dermatol 12: 3–11. https://doi.org/10.1111/jocd.12019 Yokose U, Ishikawa J, Morokuma Y, Naoe A, Inoue Y, Yasuda Y, et al (2020) The ceramide [NP]/[NS] ratio in the stratum corneum is a potential marker for skin properties and epidermal differentiation. BMC Dermatol 20: 6. https://doi.org/10.1186/s12895-020-00102-1 Danso M, Boiten W, van Drongelen V, Gmelig Meijling K, Gooris G, El Ghalbzouri A, et al (2017) Altered expression of epidermal lipid Bio-Synthesis enzymes in atopic dermatitis skin is accompanied by changes in stratum corneum lipid composition. J Dermatol Sci 88: 57–66. https://doi.org/10.1016/j.jdermsci.2017.05.005 Bouwstra JA, Gooris GS, Dubbelaar FE, Ponec M (2002) Phase behavior of stratum corneum lipid mixtures based on human ceramides: the role of natural and synthetic ceramide 1. J Invest Dermatol 118: 606–617. https://doi.org/10.1046/j.1523-1747.2002.01706.x Di Nardo A, Wertz P, Giannetti A, Seidenari S (1998) Ceramide and cholesterol composition of the skin of patients with atopic dermatitis. Acta Derm Venereol 78: 27–30. https://doi.org/10.1080/00015559850135788 Groen D, Poole DS, Gooris GS, Bouwstra JA (2011) Investigating the barrier function of skin lipid models with varying compositions. Eur J Pharm Biopharm 79: 334–342. https://doi.org/10.1016/j.ejpb.2011.05.007 Bouwstra JA, Gooris GS (2010) The lipid organization in human stratum corneum and model systems. Open Dermatol J 4: 10–13. https://doi.org/10.2174/1874372201004010010 Janssens M, van Smeden J, Puppels GJ, Lavrijsen AP, Caspers PJ, Bouwstra JA (2014) Lipid to protein ratio plays an important role in the skin barrier function in patients with atopic eczema. Br J Dermatol 170: 1248–1255. https://doi.org/10.1111/bjd.12908 Rogers J, Harding C, Mayo A, Banks J, Rawlings A (1996) Stratum corneum lipids: the effect of aging and the seasons. Arch Dermatol Res 288: 765–770. https://doi.org/10.1007/BF02505294 du Plessis J, Stefaniak A, Eloff F, John S, Agner T, Chou TC, et al (2013) International guidelines for the in vivo assessment of skin properties in non-clinical settings: Part 2. transepidermal water loss and skin hydration. Skin Res Technol 19: 265–278. https://doi.org/10.1111/srt.12037 Kataoka Y (2014) Thymus and activation-regulated chemokine as a clinical biomarker in atopic dermatitis. J Dermatol 41: 221–229. https://doi.org/10.1111/1346-8138.12440 Kakinuma T, Nakamura K, Wakugawa M, Mitsui H, Tada Y, Saeki H, et al (2001) Thymus and activation-regulated chemokine in atopic dermatitis: serum thymus and activation-regulated chemokine level is closely related with disease activity. J Allergy Clin Immunol 107: 535–541. https://doi.org/10.1067/mai.2001.113237 Tamaki K, Kakinuma T, Saeki H, Horikawa T, Kataoka Y, Fujisawa T, et al (2006) Serum levels of CCL17/TARC in various skin diseases. J Dermatol 33: 300–302. https://doi.org/10.1111/j.1346-8138.2006.00072.x Fluhr JW, Kao J, Jain M, Ahn SK, Feingold KR, Elias PM (2001) Generation of free fatty acids from phospholipids regulates stratum corneum acidification and integrity. J Invest Dermatol 117: 44–51. https://doi.org/10.1046/j.0022-202x.2001.01399.x Feingold KR (2009) The outer frontier: the importance of lipid metabolism in the skin. J Lipid Res 50 Supplement: S417–S422. https://doi.org/10.1194/jlr.R800039-JLR200 Hachem JP, Houben E, Crumrine D, Man MQ, Schurer N, Roelandt T, et al (2006) Serine protease signaling of epidermal permeability barrier homeostasis. J Invest Dermatol 126: 2074–2086. https://doi.org/10.1038/sj.jid.5700351 Eberlein-König B, Schäfer T, Huss-Marp J, Darsow U, Möhrenschlager M, Herbert O, et al (2000) Skin surface pH, stratum corneum hydration, trans-epidermal water loss and skin roughness related to atopic eczema and skin dryness in a population of primary school children. Acta Derm Venereol 80: 188–191. https://doi.org/10.1080/000155500750042943 Tables Table 1. Baseline characteristics of patients with atopic dermatitis. Sex, n (%) Male 24 (63.2) Female 14 (36.8) Age (years) a 30 (24.25-30) Age group, n (%) <15 years 2 (5.3) ≧ 15 years 36 (94.7) Treatment, n (%) Topical corticosteroids alone 32 (84.2) Topical tacrolimus alone 1 (2.6) Systemic corticosteroids 1 (2.6) Phototherapy 1 (2.6) Dupilumab 1 (2.6) Clinical index EASI b 18.4 IGA b 4 Laboratory parameter IgE (IU/mL) b 5330 TARC (pg/mL) b 3090 LDH (IU/mL) b 285 TEC (/μL) b 607 a Data provided as median [first quartile-second]. b Data provided as median [interquartile range]. EASI, eczema area and severity index; IGA, investigator's global assessment; IgE, immunoglobulin E; LDH, lactate dehydrogenase; TARC, thymus and activation-regulated chemokine; TEC, total eosinophil count. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2509454","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":170496214,"identity":"1e82e34b-6405-4f83-aa3f-4e3dfd2a0526","order_by":0,"name":"Akari Kondo","email":"","orcid":"","institution":"Tokyo Women's Medical University Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Akari","middleName":"","lastName":"Kondo","suffix":""},{"id":170496215,"identity":"9a09ad4d-0b54-4a97-b5ef-2c2769274382","order_by":1,"name":"Yuko Takenaka","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIiWNgGAWjYFACNhCRACKYGRgqUIWxAh5ULWcMSNXC2GaAUyUc2LMfS3xcUJMmzyCRY2zwcd6fxLUzEhg//GDgy8NpC0/aYeMZx3IMG4BaEmduM0jcdiOBWbKHga0Yt8PS26R52CoYQVoO824zyAVqYZAG+iWxAZcW/uftv3n+VdhDtMwBa2H+jVeLRNoxZt62nESQlmTeBrAWNvy23HiWLM3bl5bcxvOs2HDGMeP6bWcetln2GOD2C3t/muFnnm/Jtv3syZslPtTIGZsdTz5840fFMZwhBgdsAgkwJiPQSQbHEnAqhQP+AyjcGiK0jIJRMApGwQgBAMHfUdZcch+0AAAAAElFTkSuQmCC","orcid":"","institution":"Tokyo Women's Medical University Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yuko","middleName":"","lastName":"Takenaka","suffix":""},{"id":170496216,"identity":"2c2b5e62-8bee-425a-b1c8-53ccb621bae7","order_by":2,"name":"Anna Fujiwara","email":"","orcid":"","institution":"Meiji (Japan)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Fujiwara","suffix":""},{"id":170496218,"identity":"04effd40-120a-487b-aa31-db45401afaf9","order_by":3,"name":"Saori Takahashi","email":"","orcid":"","institution":"Meiji (Japan)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Saori","middleName":"","lastName":"Takahashi","suffix":""},{"id":170496219,"identity":"79b69d5a-dbec-44eb-95e2-ddd47faafbf2","order_by":4,"name":"Masami Kitade-Miyayama","email":"","orcid":"","institution":"Meiji (Japan)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Masami","middleName":"","lastName":"Kitade-Miyayama","suffix":""},{"id":170496221,"identity":"5133c4d1-c115-468b-9547-98176e804632","order_by":5,"name":"Masashi Morifuji","email":"","orcid":"","institution":"Meiji (Japan)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Masashi","middleName":"","lastName":"Morifuji","suffix":""},{"id":170496223,"identity":"eb2d3da0-efd0-46d5-b47f-ae54a4904030","order_by":6,"name":"Makoto Kawashima","email":"","orcid":"","institution":"Tokyo Women's Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Makoto","middleName":"","lastName":"Kawashima","suffix":""},{"id":170496225,"identity":"f71de57a-8804-4661-a62d-b199579859af","order_by":7,"name":"Naoko Ishiguro","email":"","orcid":"","institution":"Tokyo Women's Medical University Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Naoko","middleName":"","lastName":"Ishiguro","suffix":""}],"badges":[],"createdAt":"2023-01-24 08:14:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2509454/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2509454/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":32135329,"identity":"0ce42c41-aa00-4b38-a26b-48729d8a3c55","added_by":"auto","created_at":"2023-01-27 20:26:34","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":238760,"visible":true,"origin":"","legend":"\u003cp\u003eStudy flow chart and details of the study patients with AD\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2509454/v1/630235d26ced0a1ebd1dcdec.jpg"},{"id":32135330,"identity":"01e17e86-f114-4fc4-9602-8a0596435721","added_by":"auto","created_at":"2023-01-27 20:26:34","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":174728,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Total levels of covalently bound and free ceramides and the levels of unsaturated fatty acids (USFAs) and saturated fatty acid (SFAs), (b) ratio of USFAs and SFAs in covalently bound and free ceramides, and (c) ratio of nine molecular species, which consisted of USFAs (C26:0, 28:0, 30:0, 32:0, and 34:0) and SFAs (C30:1, 32:1, 34:1, and 36:1) in covalently bound and free ceramides in normal skin of healthy controls (n = 32) and non-lesional and lesional skin of patients with atopic dermatitis (AD) (n = 15). Values represent mean ± SEM. Significant differences between groups are indicated by *P \u0026lt; 0.05 and **P \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2509454/v1/bc56dbe5c368a45c5a891b7e.jpg"},{"id":32135328,"identity":"f4f8d506-b8f8-456e-b8b8-856b438e2ca3","added_by":"auto","created_at":"2023-01-27 20:26:34","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":126438,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Correlation between the proportion of unsaturated fatty acids (USFAs) in covalently bound ceramides and the levels of transepidermal water loss (TEWL) in lesional skin of patients with atopic dermatitis (AD) (n = 8 ).\u003c/p\u003e\n\u003cp\u003e(b) Correlation between the proportion of USFAs in covalently bound ceramides and the levels of activation-regulated chemokine (TARC) in the non-lesional (n = 13) and lesional (n = 17) skin of patients with atopic dermatitis (AD).\u003c/p\u003e\n\u003cp\u003e(c) Correlation between the proportion of USFA (C32:1) and the levels of thymus and activation-regulated chemokine (TARC) in the non-lesional (n = 13) and lesional (n = 17) skin of patients with atopic dermatitis (AD)\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2509454/v1/5a27a88e8e2628f26f330ce0.jpg"},{"id":32611880,"identity":"7bff34a6-f2f6-403d-bc60-90079ae6082d","added_by":"auto","created_at":"2023-02-07 20:44:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":573443,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2509454/v1/817f810c-5064-4f45-ae32-f94122f51f90.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Changes in the composition of molecular species of covalently bound and free ceramides, and their correlation with disease severity in atopic dermatitis ","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe skin protects against invasion by foreign particles and functions as a barrier to limit water evaporation from the body. Particularly, the stratum corneum (SC), the outermost layer of the epidermis, and sphingolipid ceramides (Cers), play an important role in the barrier function. Cers consist of fatty acids, long-chain bases, and carbon chains of different lengths [1]. The three types of fatty acids are non-OH fatty acids [N], \u0026alpha;-OH fatty acids [A], and esterified \u0026omega;-OH fatty acids [EO]. The four types of sphingoid bases are dihydrosphingosine [DS], sphingosine [S], 6-OH sphingosine [H], and phytosphingosine [P]. The combinations of these types can be classified into 12 species. Based on the length of the carbon chain, approximately 480 types of Cers have been identified in human SC [2\u0026ndash;6].\u003c/p\u003e\n\u003cp\u003eAcylceramides, which are epidermis-specific molecular species, have three hydrophobic structures with linoleic acid and long fatty acids with carbon chains of more than 28 atoms (C28). Acylceramides are required for the skin barrier function [1]. Covalently bound Cers are formed by the hydrolysis of linoleic acids and binding of the cross-linked proteins of the cornified envelope to the \u0026omega;-OH group. Acylceramides are important precursors of covalently bound Cers [1]. The corneocyte lipid envelope comprises Cers that are covalently bound to the cornified envelope. The covalently bound Cers connect lipid lamellae and keratinocytes, and they are essential for skin barrier formation [1].\u003c/p\u003e\n\u003cp\u003eThe levels of covalently bound Cers decrease upon ultraviolet-B irradiation, sodium dodecyl sulfate application, and tape stripping [7]; their levels are lower in the winter than in the summer [8]. In normal skin, the ratio of saturated free fatty acids (SFAs) and unsaturated free fatty acids (USFAs) in covalently bound Cers changes with the season and age [8]. Increased USFA proportion reportedly decreased skin barrier function in dry-skinned mice [9]. The levels of covalently bound Cers decrease and the molecular species composition of Cers changes in atopic dermatitis (AD) [10]. In this condition, Cer [NH] consists of non-OH fatty acid and 6-OH sphingosine, Cer [NP] consists of non-OH fatty acid and phytosphingosine, Cer [EOS] consists of esterified \u0026omega;-OH fatty acid and sphingosine, and Cer [EOH] consists of esterified \u0026omega;-OH fatty acid; 6-OH sphingosine levels are low. Cer [AS] consists of \u0026alpha;-OH fatty acid and sphingosine levels are high. Acylceramide Cer [EOS] levels are the most depleted among the Cer molecular species [11, 12].\u003c/p\u003e\n\u003cp\u003eTo our knowledge, no study has assessed the correlation between molecular species and lipid composition associated with epidermal barrier function in AD. Assessment of the association between molecular species and lipid composition and AD severity in clinical practice is also required.\u003c/p\u003e\n\u003cp\u003eBased on previous reports [8, 9] we speculated that AD, which occurs in men and women of all ages, decreases epidermal barrier function and increases USFA proportions, changing the molecular species composition of Cers. Here, we focused on Cer [EOS], the most important Cer molecular species for the barrier function [13] and flexibility of the SC, and a modulator of SC liquid balance [14]. Furthermore, we examined the lipid composition and molecule types of Cer in the SC and their correlation with AD severity.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eParticipants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe examined Japanese patients who visited Tokyo Women\u0026rsquo;s Medical University Hospital between August 2017 and September 2019. We enrolled 38 patients with AD and 32 healthy individuals (controls) with no clinical skin rash.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all participants according to the principles of the Declaration of Helsinki. This study was approved by the Institutional Review Board of Tokyo Women\u0026rsquo;s Medical University Hospital (No. 4430; approved on August 1, 2017) and Meiji Co., Ltd. (No. 120; approved on August 31, 2017).\u003c/p\u003e\n\u003cp\u003eAD diagnosis in the patients was made clinically based on the Japanese Guidelines for Atopic Dermatitis 2020 [15].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiophysical Measurements of SC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe SC sheets were collected by stripping three times with a 30 mm \u0026times; 25 mm piece of polyphenylene sulfide (PPS) tape (Teraoka Seisakusho, Tokyo, Japan) at room temperature (24\u0026deg;C \u0026plusmn; 2\u0026deg;C and 50% \u0026plusmn; 15% relative humidity).\u0026nbsp;In 38 patients with AD, SC sheets were collected from the lesional skin of upper extremities in 26, lower extremities in 7, trunks in 2, and palm in 1, respectively. In 2 patients, SC sheets could not be collected, since they were in remission with no active lesion. In\u0026nbsp;the\u0026nbsp;38 patients with AD, SC sheets were collected from the non-lesional skin of upper extremities in 24, lower extremities in 10, and face in 1, respectively.\u0026nbsp;In 3 patients, SC sheets could not be collected, since they were in erythroderma. In the healthy control, SC sheets were also collected from the upper and lower extremities.\u0026nbsp;The collected samples were stored at \u0026minus;80\u0026deg;C until analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of Transepidermal Water Loss\u003c/strong\u003eTransepidermal water loss (TEWL) was measured using a Tewameter TM 300 (Courage + Khazaka Electronic, Cologne, Germany). Owing to limited outpatient time, measurements were made from the same area where the SC sheets were collected in eight out of 26 patients with AD and 31 healthy controls. Measurements were conducted in an air-conditioned room (26\u0026deg;C \u0026plusmn; 3\u0026deg;C and 46% \u0026plusmn; 18% relative humidity).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood samples from patients with AD who gave consent were collected simultaneously with the other samples.\u0026nbsp;We\u0026nbsp;\u003cspan lang=\"\"\u003eexamined\u0026nbsp;\u003c/span\u003eeczema area and severity index (EASI) [16], investigator\u0026apos;s global assessment (IGA) [17], serum immunoglobulin E (IgE), thymus and activation-regulated chemokine (TARC), lactate dehydrogenase (LDH), and total eosinophil count (TEC).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtraction of Cers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCers were extracted according as previously described [8, 18]. Half of each of the three tapes collected was cut, immersed in hexane, and sonicated in ice-cold water for 30 min to remove the tape adhesive. SC cells were recovered from the extracted liquid on a piece of filter paper (Kiriyama Roto Filter paper 124; Nippon Rikagaku Kikai, Tokyo, Japan) using suction filtration. After drying, the filter paper was immersed in chloroform/methanol (2:1, v/v) and sonicated for 30 min under cool conditions. The filtrate, containing free Cers, was recovered, dried, and re-dissolved in methanol for the analysis of free Cers. The protein pellet was recovered on filter paper and dried using a centrifugal evaporator. The filter paper was incubated in 1 M KOH in 95% methanol at 60\u0026deg;C for 2 h to release the lipids that were covalently bound to the SC via ester-like bonds. The solution, including the filter paper, was neutralized with 1 N HCl and centrifuged at 1,830 \u003cem\u003e\u0026times;\u003c/em\u003e \u003cem\u003eg\u003c/em\u003e for 5 min at room temperature (H-700FRS; Kokusan Co., Ltd., Saitama, Japan). The supernatant containing \u0026omega;-OH Cers was recovered. The sediment was washed again with methanol. The supernatant was recovered, combined, dried, and re-dissolved in methanol for the analysis of covalently bound Cers. The pellet containing the proteins was immersed in phosphate buffered saline (pH 7.4) containing 1% sodium dodecyl sulfate and incubated at 60\u0026deg;C for 2 h to solubilize the protein. The protein concentration was measured using a commercial kit (Micro BCA Assay Kit; Pierce Biotechnology, Rockford, IL, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of Cers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCovalently bound Cers and free Cers in the SC samples were identified using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) (Quattro Premier XE; Waters Corporation, Milford, MA, USA). All analyses were performed on a 2 mm \u0026times; 100 mm column with a particle size of 1.7 \u0026micro;m (ACQUITY UPLC\u003csup\u003e\u0026reg;\u003c/sup\u003e BEH C18; Waters, Milford, MA, USA).\u003c/p\u003e\n\u003cp\u003eThe analysis of covalently bound Cers was performed as follows. Mobile phase A consisted of 5 mM ammonium acetate in 95% methanol and mobile phase B consisted of 5 mM ammonium acetate in methanol. The initial eluent composition was 100% A, followed by an increase to 100% B for 30 min, 100% B for 2 min, and then 0% B for 3 min. The total running time was 35 min at an eluent flow of 0.4 mL/min and a column temperature of 40\u0026deg;C. The analytes were detected using electrospray ionization in the positive mode. Multiple-reaction monitoring was performed using characteristic fragmentation ions (m/z 676.7/264.3 for d18:1 (sphingosine) /C26:0 m/z 704.7/264.3 for d18:1/C28:0, m/z 732.7/264.3 for d18:1/C30:0, m/z 730.7/264.3 for d18:1/C30:1, m/z 760.8/264.3 for d18:1/C32:0, m/z 758.8/264.3 for d18:1/C32:1, m/z 788.8/264.3 for d18:1/C34:0, m/z 786.8/264.3 for d18:1/C34:1, and m/z 814.8/264.3 for d18:1/C36:1). The parameters for the HPLC-MS/MS analysis were as follows: capillary voltage, 3000 V; source temperature, 120\u0026deg;C; desolvation temperature, 400\u0026deg;C; desolvation gas flow, 850 L/h; cone voltage, 40 V; cone gas flow, 50 L/h; and collision energy, 30 eV. The Cer molecular species contents were determined using N-omega-hydroxytriacontanoyl-D-erythro-sphingosine (Matreya, Pleasant Gap, PA, USA) according to the d18:1/C30:0 Cers standard.\u003c/p\u003e\n\u003cp\u003eThe free Cers analysis was performed as follows. Mobile phase A consisted of 5 mM ammonium acetate in 95% methanol and mobile phase B consisted of 5 mM ammonium acetate in 95% isopropanol and 5% methanol. The initial eluent composition was maintained at 100% A for 5 min followed by an increase to 50% B for 20 min, 50% B for 5 min, and then 0% B for 5 min. The total running time was 35 min at an eluent flow of 0.4 mL/min and a column temperature of 50\u0026deg;C. The analytes were detected using electrospray ionization in the positive mode. Multiple-reaction monitoring was performed using characteristic fragmentation ions (m/z 957.0/264.3 for d18:1/C26:0/C18:2, m/z 985.0/264.3 for d18:1/C28:0/C18:2, m/z 1013.0/264.3 for d18:1/C30:0/C18:2, m/z 1011.0/264.3 for d18:1/C30:1/C18:2, m/z 1041.0/264.3 for d18:1/C32:0/C18:2, m/z 1039.0/264.3 for d18:1/C32:1/C18:2, m/z 1069.0/264.3 for d18:1/C34:0/C18:2, m/z 1067.0/264.3 for d18:1/C34:1/C18:2, and m/z 1095.0/264.3 for d18:1/C36:1/C18:2). The parameters for the HPLC-MS/MS analysis were as follows: capillary voltage, 3000 V; source temperature, 150\u0026deg;C; desolvation temperature, 400\u0026deg;C; desolvation gas flow, 850 L/h; cone voltage, 30 V; cone gas flow, 50 L/h; and collision energy, 45 eV. The Cer molecular species contents were determined using N-(30-linoleoyloxy-triacontanoyl)-sphingosine (Matreya, Pleasant Gap, PA, USA) according to the d18:1/C30:0/C18:2 Cers standard.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are presented as mean \u0026plusmn; standard error of the mean. Data were analyzed using Tukey\u0026ndash;Kramer test (comparison between different participants) or paired \u003cem\u003et\u003c/em\u003e-test (comparison between the same participants) (SPSS ver. 25; IBM SPSS, Armonk, NY, USA). Differences between groups were considered significant at P \u0026lt; 0.05 and P \u0026lt; 0.01.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eBaseline Characteristics of Patients with AD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThirty-two healthy individuals (controls) with no clinical skin rash (6 men and 26 women) were enrolled; 38 patients with AD (24 men and 14 women) were enrolled (Table 1). There were 36 adult patients (\u0026ge;15\u0026thinsp;years) and 2 adolescent patients (\u0026lt;15\u0026thinsp;years). Prior treatment for AD; EASI; IGA level; and serum IgE, TARC, LDH, and TEC levels are shown in Table 1. Most patients had more than moderate AD (EASI\u003csup\u003e\u0026nbsp;\u003c/sup\u003e[6] \u0026gt;7). Details of the patients are shown in Figure 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLipid Composition in Cers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 2(a) shows the SFA and USFA levels in covalently bound and free Cers, and total levels. The total levels of covalently bound Cers were significantly higher in lesional skin than in non-lesional skin in patients with AD. In covalently bound Cers, the USFA levels were significantly higher in lesional skin of patients with AD than in non-lesional skin of patients with AD and normal skin in healthy controls. There was no difference in SFA levels among the three samples. The total levels of free Cers in lesional skin of patients with AD were lower than those in normal skin of healthy controls. However, this difference was not significantly different and the levels in non-lesional skin of patients with AD were significantly lower than those in normal skin of healthy controls. The levels of USFAs in free Cers were significantly higher in lesional skin of patients with AD than in non-lesional skin of patients with AD and normal skin of healthy controls. The USFA levels were higher in lesional skin of patients with AD than in normal skin of healthy controls and non-lesional skin of patients with AD in both covalently bound and free Cers.\u003c/p\u003e\n\u003cp\u003eFigure 2(b) shows the ratio of USFAs to SFAs in covalently bound and free Cers. In both covalently bound and free Cers, the USFA proportion was significantly higher in lesional skin of patients with AD than in the skin of the other groups. The USFA proportion in covalently bound Cers decreased as follows: lesional skin of patients with AD \u0026gt; non-lesional skin of patients with AD \u0026gt; normal skin of healthy controls. In contrast, in both covalently bound and free Cers, the proportion of SFAs decreased as follow: normal skin of healthy controls \u0026gt; non-lesional skin of patients with AD \u0026gt; lesional skin of patients with AD. These results showed that USFA proportion was significantly higher and SFA proportion was significantly lower in lesional skin of patients with AD.\u003c/p\u003e\n\u003cp\u003eWe identified nine molecular species of Cer [EOS], consisting of sphingosine (d18:1) with a long-chain \u0026omega;-OH fatty acid (C26:0, 28:0, 30:0, 30:1, 32:0, 32:1, 34:0, 34:1, or 36:1), in 15 of the 38 patients with AD.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFigure 2(c) shows the proportion of the nine molecular species in the covalently bound and free Cers. In non-lesional skin of patients with AD and normal skin of healthy controls, SFA (C30:0) was the most common in both covalently bound and free Cers. USFA (C32:1) was the most common in covalently bound Cers in lesional skin of patients with AD and the percentage of SFA (C30:0) was the highest in free Cers. In both covalently bound and free Cers, the percentage of SFA (C30:0) decreased as follows: normal skin of healthy controls \u0026gt; non-lesional skin of AD \u0026gt; lesional skin of AD. The percentage of USFAs (C32:1 and C34:1) increased as follows: normal skin of healthy controls \u0026lt; non-lesional skin of AD \u0026lt; lesional skin of AD. The percentage of USFAs (C30:1, C32:1, and C34:1) was significantly higher in lesional skin of patients with AD than in non-lesional skin of patients with AD and normal skin of healthy controls.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrelation of USFAs in Covalently Bound Cers with Other Indices\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 3(a) shows the correlation between USFA proportion in covalently bound Cers and TEWL in eight patients with AD. A positive correlation was found between USFA proportion in covalently bound Cers and TEWL in lesional skin of patients with AD (r = 0.542).\u003c/p\u003e\n\u003cp\u003eFigure 3(b) shows the correlation between USFA proportion in covalently bound Cers and the levels of TARC. A positive correlation was found between USFA proportion in covalently bound Cers and TARC levels in non-lesional skin of 13 patients (r = 0.676) and lesional skin of 17 patients with AD (r = 0.503).\u003c/p\u003e\n\u003cp\u003eFigure 3(c) shows the correlation between the USFA (C32:1) proportion and TARC level. The USFA (C32:1) proportion in covalently bound Cers, which was the highest USFA in covalently bound Cers, also positively correlated with the TARC levels in non-lesional skin of 13 patients (r = 0.733) and lesional skin of 17 patients with AD (r = 0.515).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCer [NH] and Cer [NP] levels correlate strongly with the indicator conductance, which indicates the state of dryness [19]. Cer [NH] and Cer [NP] levels show a negative correlation with TEWL, whereas Cer [NS] and Cer [AS] levels are positively correlated with TEWL [12]. The Cer [NP]/[NS] ratio is a marker for SC function, and its low values suggest a poor skin condition [20]. In AD, the average chain length of long-chain acylceramides is shortened [5, 10, 21], and the Cer [NH] and Cer [NP] levels decrease; the changes in the molecular species of Cers are related to the barrier function of SC [12, 20]. Cer [EOS], an important acylceramide for the function of the SC [14], notably decreases in both lesional and non-lesional skin of patients with AD compared with that in normal skin of healthy controls [11, 12, 21]. Additionally, Cer [EOS] is important for the maintenance of the qualitative and quantitative balance between SFAs and USFAs in the skin barrier [5, 22]. An increase in USFA levels is associated with a decrease in the epidermal barrier function of SC [8, 9].\u003csup\u003e\u0026nbsp;\u003c/sup\u003eIn AD, the Cer/cholesterol ratio or the lipid/protein ratio in the skin is reduced compared with that in the normal skin of healthy controls, which causes a change in the composition of SC lipids. This effect is considered to contribute to the disruption of the skin barrier function [23\u0026ndash;26]. Regarding fatty acid saturation of free Cers, the structure of SC lipids changes considerably, even at the ultrastructural level, when the linoleate of Cer [EOS] is replaced with oleate in the normal skin of healthy controls. Because of the increase in oleic acid, the USFA levels in Cer [EOS] are higher in the winter than in the summer [14, 27]. Regarding Cer desaturation and changes in molecular type in diet-induced dry-skin mice, the average proportion of Cers with SFAs (C30:0, C32:0, and C34:0) decreased and that of USFAs (C32:1, C34:1, and C36:1) increased among covalently bound Cers [9].\u003c/p\u003e\n\u003cp\u003eIn clinical practice, TEWL is used for evaluating the barrier function of the SC and can be measured non-invasively and quantitatively. High TEWL levels have been observed [28] in AD and psoriasis, and there is no significant difference in the TEWL level between normal skin of healthy controls and non-lesional skin in AD [12]. However, TEWL does not always explain the severity and pathophysiology of AD. In contrast, the TARC levels more accurately reflect the changes in AD pathology during treatment compared to peripheral blood eosinophil counts and IgE and LDH levels [29]. In the early stage of AD, the expression of cytokines, such as interleukins IL-4, IL-5, and IL-13, increases, which is a predominant Th2 state. CCR4, a chemokine receptor, is highly specific to Th2 cells and TARC is involved in AD pathophysiology by inducing the migration of CCR4-expressing Th2 lymphocytes. The serum TARC levels in AD are higher than those in other skin diseases, reflecting clinical severity, and decrease with treatment\u003csup\u003e\u0026nbsp;\u003c/sup\u003e[30, 31].\u003c/p\u003e\n\u003cp\u003eHere, we focused on Cer [EOS] in AD, which is a typical skin disease characterized by decreased epidermal barrier function. We expected changes in the lipid and molecular species composition of Cers derived from Cer [EOS] because of the decreased epidermal barrier function. We aimed to determine the relationship between Cer [EOS] composition and AD severity. We identified nine molecular species of Cer [EOS], which contained sphingosine (d18:1) with a long-chain \u0026omega;-OH fatty acid (C26:0, 28:0, 30:0, 30:1, 32:0, 32:1, 34:0, 34:1, or 36:1). The USFA levels in both covalently bound Cers and free Cers increased in lesional skin of patients with AD compared with those in non-lesional skin of patients with AD and normal skin of healthy controls. Furthermore, the USFA proportion in both covalently bound and free Cers increased in the following order: normal skin of healthy controls \u0026lt; non-lesional skin of patients with AD \u0026lt; lesional skin of patients with AD. Regarding the molecular composition of Cers, the proportion of Cers with USFAs (C30:1, C32:1, and C34:1) was significantly higher in both covalently bound and free Cers in lesional skin of patients with AD. Here, a positive correlation was found between USFA proportion in covalently bound Cers and TEWL. Additionally, the USFA proportion in covalently bound Cers positively correlated with the serum TARC levels in both non-lesional and lesional skin of patients with AD. This finding suggests that a state of Th2 predominance may increase USFA proportion and molecular composition changes.\u003c/p\u003e\n\u003cp\u003eSFAs are more resistant to oxidation than USFAs and are involved in membrane stability and development of a stronger lipid lamellar structure [22]. USFAs synthesized from phospholipids via phospholipase A2 promote premature lysis of corneodesmosomes in the SC [32]. When the pH of the SC increases, the catalytic activity of \u0026beta;-glucocerebrosidase and acidic sphingomyelinase, which are important for Cer synthesis, decrease [8, 33]. Furthermore, when the pH of the SC increases, serine protease-activated receptor 2 (PAR2) is activated, which suppresses the secretion of lamellar bodies, resulting in a decrease in skin barrier function [8, 34].\u003c/p\u003e\n\u003cp\u003eSkin pH increases in patients with AD [35]. The composition of Cer molecular species changes and levels of USFAs increase in the skin of patients with AD; the increase in the pH of the SC causes a decrease in Cers synthase activity, a structural change in the membrane, and PAR2 activation. We inferred that this decrease caused the suppression of lamellar body secretion, resulting in a decrease in skin barrier function.\u003c/p\u003e\n\u003cp\u003eA limitation of this study is that the analysis of molecular species and lipid composition is in the preliminary stage and cannot be easily performed in daily medical care. Additionally, only Cers derived from Cer [EOS] were analyzed. Further studies are required to elucidate the pathogenic mechanisms of AD, the maintenance of external therapies and systemic therapies that will help control fluctuating molecular species or eliminate changes in lipid composition, and the maintenance of lipid composition. Dietary supplementation might also be considered for these changes. Analysis of Cer molecular species and lipid composition might help develop treatments for improving skin barrier function tailored to individual AD pathology.\u003c/p\u003e\n\u003cp\u003eHere, an increase in USFA levels was observed in both lesional and non-lesional skin of patients with AD. This increase in USFA levels was associated with dryness and deterioration of barrier function and was correlated with TARC level, a marker for the degree of type 2 inflammation. These findings suggest that exacerbation of type 2 inflammation may lead to abnormal epidermal lipid metabolism in the skin of patients with AD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompeting interests: The authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResearch involving Human Participants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval:\u0026nbsp;This study was approved by the Institutional Review Board of Tokyo Women\u0026rsquo;s Medical University Hospital (No. 4430; approved on August 1, 2017) and Meiji Co., Ltd. (No. 120; approved on August 31, 2017).\u003c/p\u003e\n\u003cp\u003eConsent to Participate:\u0026nbsp;Informed consent was obtained from all participants according to the principles of the Declaration of Helsinki.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsent to Publish: Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone declared.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by [Akari Kondo], [Yuko Takenaka], [Anna Fujiwara], [Saori Takahashi],[ Masami Kitade-Miyayama], and [Masashi Morifuji]. The first draft of the manuscript was written by [Akari Kondo] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eOhno Y (2017) Elucidation of the synthetic mechanism of acylceramide, an essential lipid for skin barrier function (in Japanese). Yakugaku Zasshi 137: 1201\u0026ndash;1208. \u003cspan lang=\"\"\u003ehttps://doi.org/10.1248/yakushi.17-00126\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eIshikawa J, Shimotoyodome Y, Ito S, Miyauchi Y, Fujimura T, Kitahara T, Hase T (2013) Variations in the ceramide profile in different seasons and regions of the body contribute to stratum corneum functions. Arch Dermatol Res 305: 151\u0026ndash;162.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1007/s00403-012-1286-5\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eMasukawa Y, Narita H, Sato H, Naoe A, Kondo N, Sugai Y, et al (2009) Comprehensive quantification of ceramide species in human stratum corneum. J Lipid Res 50: 1708\u0026ndash;1719.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1194/jlr.D800055-JLR200\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eMasukawa Y, Narita H, Shimizu E, Kondo N, Sugai Y, Oba T, et al (2008) Characterization of overall ceramide species in human stratum corneum. J Lipid Res 49: 1466\u0026ndash;1476.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1194/jlr.M800014-JLR200\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003evan Smeden J, Janssens M, Gooris GS, Bouwstra JA (2014) The important role of stratum corneum lipids for the cutaneous barrier function. Biochim Biophys Acta 1841: 295\u0026ndash;313.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1016/j.bbalip.2013.11.006\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eJia ZX, Zhang JL, Shen CP, Ma L (2016) Profile and quantification of human stratum corneum ceramides by normal-phase liquid chromatography coupled with dynamic multiple reaction monitoring of mass spectrometry: development of targeted lipidomic method and application to human stratum corneum of different age groups. Anal Bioanal Chem 408: 6623\u0026ndash;6636.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1007/s00216-016-9775-6\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eTakagi Y, Nakagawa H, Kondo H, Takema Y, Imokawa G (2004) Decreased levels of covalently bound ceramide are associated with ultraviolet B-induced perturbation of the skin barrier. J Invest Dermatol 123: 1102\u0026ndash;1109.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1111/j.0022-202X.2004.23491.x\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eFujiwara A, Morifuji M, Kitade M, Kawahata K, Fukasawa T, Yamaji T, et al (2018) Age-related and seasonal changes in covalently bound ceramide content in forearm stratum corneum of Japanese subjects: determination of molecular species of ceramides. Arch Dermatol Res 310: 729\u0026ndash;735.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1007/s00403-018-1859-z\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eMorifuji M, Oba C, Ichikawa S, Ito K, Kawahata K, Asami Y, et al (2015) A novel mechanism for improvement of dry skin by dietary milk phospholipids: effect on epidermal covalently bound ceramides and skin inflammation in hairless mice. J Dermatol Sci 78: 224\u0026ndash;231.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1016/j.jdermsci.2015.02.017\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eMacheleidt O, Kaiser HW, Sandhoff K (2002) Deficiency of epidermal protein-bound omega-hydroxyceramides in atopic dermatitis. J Invest Dermatol 119: 166\u0026ndash;173.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1046/j.1523-1747.2002.01833.x\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eImokawa G, Abe A, Jin K, Higaki Y, Kawashima M, Hidano A (1991) Decreased level of ceramides in stratum corneum of atopic dermatitis: an etiologic factor in atopic dry skin? J Invest Dermatol 96: 523\u0026ndash;526.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1111/1523-1747.ep12470233\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eIshikawa J, Narita H, Kondo N, Hotta M, Takagi Y, Masukawa Y, et al (2010) Changes in the ceramide profile of atopic dermatitis patients. J Invest Dermatol 130: 2511\u0026ndash;2514.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1038/jid.2010.161\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eConti A, Rogers J, Verdejo P, Harding CR, Rawlings AV (1996) Seasonal influences on stratum corneum ceramide 1 fatty acids and the influence of topical essential fatty acids. Int J Cosmet Sci 18: 1\u0026ndash;12.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1111/j.1467-2494.1996.tb00131.x\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eOldroyd J, Critchley P, Tiddy G, Turner J, Rawlings AV (1994) Specialized role for ceramide one in the stratum corneum water barrier. J Invest Dermatol 102: 525\u003c/li\u003e\n \u003cli\u003eKatoh N, Ohya Y, Ikeda M, Ebihara T, Katayama I, Saeki H, et al (2020) Japanese guidelines for atopic dermatitis 2020. Allergol Int Jul 69: 356\u0026ndash;369.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1016/j.alit.2020.02.006\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eHanifin JM, Thurston M, Omoto M, Cherill R, Tofte SJ, Graeber M (2001) The eczema area and severity index (EASI): assessment of reliability in atopic dermatitis. EASI evaluator group. Exp Dermatol 10: 11\u0026ndash;18.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1034/j.1600-0625.2001.100102.x\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eSimpson\u0026nbsp;E, Bissonnette R, Eichenfield LF, Guttman-Yassky E, King B, Silverberg JI et al.\u0026nbsp;(2020) The validated investigator global assessment for atopic dermatitis (vIGA-AD): the development and reliability testing of a novel clinical outcome measurement instrument for the severity of atopic dermatitis. J Am Acad Dermatol 83: 839\u0026ndash;846.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1016/j.jaad.2020.04.104\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eOhno Y, Kamiyama N, Nakamichi S, Kihara A (2017) PNPLA1 is a transacylase essential for the generation of the skin barrier lipid \u0026omega;-O-acylceramide. Nat Commun 8: 14610.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1038/ncomms14610\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eIshikawa J, Yoshida H, Ito S, Naoe A, Fujimura T, Kitahara T, et al (2013) Dry skin in the winter is related to the ceramide profile in the stratum corneum and can be improved by treatment with a Eucalyptus extract. J Cosmet Dermatol 12: 3\u0026ndash;11.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1111/jocd.12019\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eYokose U, Ishikawa J, Morokuma Y, Naoe A, Inoue Y, Yasuda Y, et al (2020) The ceramide [NP]/[NS] ratio in the stratum corneum is a potential marker for skin properties and epidermal differentiation. BMC Dermatol 20: 6.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1186/s12895-020-00102-1\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eDanso M, Boiten W, van Drongelen V, Gmelig Meijling K, Gooris G, El Ghalbzouri A, et al (2017) Altered expression of epidermal lipid Bio-Synthesis enzymes in atopic dermatitis skin is accompanied by changes in stratum corneum lipid composition. J Dermatol Sci 88: 57\u0026ndash;66.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1016/j.jdermsci.2017.05.005\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eBouwstra JA, Gooris GS, Dubbelaar FE, Ponec M (2002) Phase behavior of stratum corneum lipid mixtures based on human ceramides: the role of natural and synthetic ceramide 1. J Invest Dermatol 118: 606\u0026ndash;617.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1046/j.1523-1747.2002.01706.x\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eDi Nardo A, Wertz P, Giannetti A, Seidenari S (1998) Ceramide and cholesterol composition of the skin of patients with atopic dermatitis. Acta Derm Venereol 78: 27\u0026ndash;30.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1080/00015559850135788\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eGroen D, Poole DS, Gooris GS, Bouwstra JA (2011) Investigating the barrier function of skin lipid models with varying compositions. Eur J Pharm Biopharm 79: 334\u0026ndash;342.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1016/j.ejpb.2011.05.007\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eBouwstra JA, Gooris GS (2010) The lipid organization in human stratum corneum and model systems. Open Dermatol J 4: 10\u0026ndash;13.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.2174/1874372201004010010\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eJanssens M, van Smeden J, Puppels GJ, Lavrijsen AP, Caspers PJ, Bouwstra JA (2014) Lipid to protein ratio plays an important role in the skin barrier function in patients with atopic eczema. Br J Dermatol 170: 1248\u0026ndash;1255.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1111/bjd.12908\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eRogers J, Harding C, Mayo A, Banks J, Rawlings A (1996) Stratum corneum lipids: the effect of aging and the seasons. Arch Dermatol Res 288: 765\u0026ndash;770.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1007/BF02505294\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003edu Plessis J, Stefaniak A, Eloff F, John S, Agner T, Chou TC, et al (2013) International guidelines for the in vivo assessment of skin properties in non-clinical settings: Part 2. transepidermal water loss and skin hydration. Skin Res Technol 19: 265\u0026ndash;278.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1111/srt.12037\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eKataoka Y (2014) Thymus and activation-regulated chemokine as a clinical biomarker in atopic dermatitis. J Dermatol 41: 221\u0026ndash;229.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1111/1346-8138.12440\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eKakinuma T, Nakamura K, Wakugawa M, Mitsui H, Tada Y, Saeki H, et al (2001) Thymus and activation-regulated chemokine in atopic dermatitis: serum thymus and activation-regulated chemokine level is closely related with disease activity. J Allergy Clin Immunol 107: 535\u0026ndash;541.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1067/mai.2001.113237\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eTamaki K, Kakinuma T, Saeki H, Horikawa T, Kataoka Y, Fujisawa T, et al (2006) Serum levels of CCL17/TARC in various skin diseases. J Dermatol 33: 300\u0026ndash;302.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1111/j.1346-8138.2006.00072.x\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eFluhr JW, Kao J, Jain M, Ahn SK, Feingold KR, Elias PM (2001) Generation of free fatty acids from phospholipids regulates stratum corneum acidification and integrity. J Invest Dermatol 117: 44\u0026ndash;51.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1046/j.0022-202x.2001.01399.x\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eFeingold KR (2009) The outer frontier: the importance of lipid metabolism in the skin. J Lipid Res 50 Supplement: S417\u0026ndash;S422.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1194/jlr.R800039-JLR200\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eHachem JP, Houben E, Crumrine D, Man MQ, Schurer N, Roelandt T, et al (2006) Serine protease signaling of epidermal permeability barrier homeostasis. J Invest Dermatol 126: 2074\u0026ndash;2086.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1038/sj.jid.5700351\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eEberlein-K\u0026ouml;nig B, Sch\u0026auml;fer T, Huss-Marp J, Darsow U, M\u0026ouml;hrenschlager M, Herbert O, et al (2000) Skin surface pH, stratum corneum hydration, trans-epidermal water loss and skin roughness related to atopic eczema and skin dryness in a population of primary school children. Acta Derm Venereol 80: 188\u0026ndash;191.\u0026nbsp;\u003cspan lang=\"\"\u003ehttps://doi.org/10.1080/000155500750042943\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1.\u0026nbsp;\u003cspan\u003eBaseline characteristics of patients with atopic dermatitis.\u003c/span\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"586\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.47008547008547%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003eSex, n (%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.7008547008547%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.623931623931623%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.794871794871796%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.128205128205128%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.282051282051283%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.47008547008547%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.7008547008547%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003eMale\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.623931623931623%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.794871794871796%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.128205128205128%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003e24 (63.2)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.282051282051283%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.47008547008547%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.7008547008547%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003eFemale\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.623931623931623%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.794871794871796%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.128205128205128%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003e14 (36.8)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.282051282051283%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.47008547008547%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003eAge (years)\u003csup\u003ea\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.7008547008547%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.623931623931623%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.794871794871796%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"36.41025641025641%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003e30 (24.25-30)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.47008547008547%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003eAge group, n (%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.7008547008547%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.623931623931623%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.794871794871796%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.128205128205128%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.282051282051283%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.47008547008547%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.7008547008547%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003e\u0026lt;15 years\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.623931623931623%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.794871794871796%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.128205128205128%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003e2 (5.3)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.282051282051283%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.47008547008547%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.7008547008547%\"\u003e\n \u003cp\u003e≧\u003cspan lang=\"\"\u003e15 years\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.623931623931623%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.794871794871796%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.128205128205128%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003e36 (94.7)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.282051282051283%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.47008547008547%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003eTreatment, n (%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.7008547008547%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.623931623931623%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.794871794871796%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.128205128205128%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.282051282051283%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.426621160409557%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" width=\"38.225255972696246%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003eTopical corticosteroids alone\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.085324232081913%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003e32 (84.2)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.262798634812286%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.426621160409557%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" width=\"38.225255972696246%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003eTopical tacrolimus alone\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.085324232081913%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003e1 (2.6)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.262798634812286%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.426621160409557%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" width=\"38.225255972696246%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003eSystemic corticosteroids\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.085324232081913%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003e1 (2.6)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.262798634812286%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.47008547008547%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"26.324786324786324%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003ePhototherapy\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.794871794871796%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.128205128205128%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003e1 (2.6)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.282051282051283%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.47008547008547%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.7008547008547%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003eDupilumab\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.623931623931623%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.794871794871796%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.128205128205128%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003e1 (2.6)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.282051282051283%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.47008547008547%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003eClinical index\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.7008547008547%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.623931623931623%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.794871794871796%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.128205128205128%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.282051282051283%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.47008547008547%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.7008547008547%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003eEASI\u003csup\u003eb\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.623931623931623%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.794871794871796%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.128205128205128%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003e18.4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.282051282051283%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.47008547008547%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.7008547008547%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003eIGA\u003csup\u003eb\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.623931623931623%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.794871794871796%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.128205128205128%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003e4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.282051282051283%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.47008547008547%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003eLaboratory parameter\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.7008547008547%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.623931623931623%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.794871794871796%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.128205128205128%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.282051282051283%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.47008547008547%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"26.324786324786324%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003eIgE (IU/mL)\u003csup\u003eb\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.794871794871796%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.128205128205128%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003e5330\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.282051282051283%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.47008547008547%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"26.324786324786324%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003eTARC (pg/mL)\u003csup\u003eb\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.794871794871796%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.128205128205128%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003e3090\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.282051282051283%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.47008547008547%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"26.324786324786324%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003eLDH (IU/mL)\u003csup\u003eb\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.794871794871796%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.128205128205128%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003e285\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.282051282051283%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.47008547008547%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"26.324786324786324%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003eTEC (/\u0026mu;L)\u003csup\u003eb\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.794871794871796%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.128205128205128%\"\u003e\n \u003cp\u003e\u003cspan lang=\"\"\u003e607\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.282051282051283%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003ea Data provided as median [first quartile-second].\u003c/p\u003e\n\u003cp\u003eb Data provided as median [interquartile range].\u003c/p\u003e\n\u003cp\u003eEASI, eczema area and severity index; IGA, investigator\u0026apos;s global assessment; IgE, immunoglobulin E; LDH, lactate dehydrogenase; TARC, thymus and activation-regulated chemokine; TEC, total eosinophil count.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"atopic dermatitis, ceramides, molecular species, stratum corneum, thymus and activation-regulated chemokine","lastPublishedDoi":"10.21203/rs.3.rs-2509454/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2509454/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Ceramides (Cers) are major constituents of the stratum corneum intercellular lipids, which are involved in the barrier function of the skin. Here, we examined the ratio of molecular species of Cers and their correlation with disease severity in patients with atopic dermatitis (AD). The levels of unsaturated fatty acids (USFAs) in both covalently bound and free Cers were higher in the lesional skin of patients with AD than in the non-lesional skin of patients with AD and normal skin of healthy controls. The proportion of USFAs (C30:1, C32:1, and C34:1) was considerably higher than that of other Cer molecular species in both covalently bound and free Cers in patients with AD. The proportion of USFAs in covalently bound Cers positively correlated with the levels of transepidermal water loss (TEWL) (r = 0.542) in the lesional skin of patients with AD. Additionally, the proportion of USFAs in covalently bound Cers positively correlated with thymus and activation-regulated chemokine (TARC), which is an index of disease severity, in the non-lesional (r = 0.676) and lesional (r = 0.503) skin of patients with AD. The proportion of USFA (C32:1), which was the highest in covalently bound Cers, also positively correlated with the TARC level in non-lesional (r = 0.733) and lesional (r = 0.515) skin of patients with AD. Our study is the first to show that there is an increase in USFA in not only lesional but also non-lesional skin of patients with AD. Moreover, this increase in USFA was associated with dryness and impaired barrier function and correlated with the TARC levels, a marker for the degree of type 2 inflammation. We speculate that exacerbation of type 2 inflammation may lead to abnormal epidermal lipid metabolism in the skin of patients with AD.","manuscriptTitle":"Changes in the composition of molecular species of covalently bound and free ceramides, and their correlation with disease severity in atopic dermatitis ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-01-27 20:26:29","doi":"10.21203/rs.3.rs-2509454/v1","editorialEvents":[{"type":"communityComments","content":1}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fa5a46f1-7280-4076-852b-013c1505cbd3","owner":[],"postedDate":"January 27th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-02-07T20:44:17+00:00","versionOfRecord":[],"versionCreatedAt":"2023-01-27 20:26:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2509454","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2509454","identity":"rs-2509454","version":["v1"]},"buildId":"ApUGefWb6u5IBVtyqm6d5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}