Influence of the sebaceous gland density on the stratum corneum lipidome

www.nature.com/scientificreports OPEN Received: 20 March 2018 Accepted: 13 July 2018 Published: xx xx xxxx Influence of the sebaceous gland density on the stratum corneum lipidome Matteo Ludovici1, Nina Kozul1,2, Stefano Materazzi2, Roberta Risoluti2, Mauro Picardo1 & Emanuela Camera 1 The skin surface lipids (SSL) result from the blending of sebaceous and epidermal lipids, which derive from the sebaceous gland (SG) secretion and the permeability barrier of the stratum corneum (SC), respectively. In humans, the composition of the SSL is distinctive of the anatomical distribution of the SG. Thus, the abundance of sebum biomarkers is consistent with the density of the SG. Limited evidence on the influence that the SG exerts on the SC lipidome is available. We explored the differential amounts of sebaceous and epidermal lipids in areas at different SG density with lipidomics approaches. SC was sampled with adhesive patches from forearm, chest, and forehead of 10 healthy adults (8F, 2M) after mechanical removal of sebum with absorbing paper. Lipid extracts of SC were analysed by HPLC/(-)ESI-TOF-MS. In the untargeted approach, the naïve molecular features extraction algorithm was used to extract meaningful entities. Aligned and normalized data were evaluated by univariate and multivariate statistics. Quantitative analysis of free fatty acids (FFA) and cholesterol sulfate (CHS) was performed by targeted HPLC/(-)ESI-TOF-MS, whereas cholesterol and squalene were quantified by GC-MS. Untargeted approaches demonstrated that the relative abundance of numerous lipid species was distinctive of SC depending upon the different SG density. The discriminating species included FFA, CHS, and ceramides. Targeted analyses confirmed that sebaceous FFA and epidermal FFA were increased and decreased, respectively, in areas at high SG density. CHS and squalene, which are biomarkers of epidermal and sebaceous lipid matrices, respectively, were both significantly higher in areas at elevated SG density. Overall, results indicated that the SG secretion intervenes in shaping the lipid composition of the epidermal permeability barrier. Skin is a tissue exceptionally enriched in lipids. The involvement of skin lipids in the maintenance of skin and entire body homeostasis is well documented1,2. Additionally, disturbance of the skin lipid arrangement is linked to several skin diseases such as acne, atopic dermatitis, and psoriasis, to mention the main ones3–6. The skin surface lipids (SSL) result from the mixture of lipids deriving from the two main cutaneous lipid sources. Sebum, an amorphous lipid matrix secreted by the sebaceous gland (SG), and epidermal lipids in the stratum corneum (SC), represent the main lipid compartments at the skin surface7. SC covers the entire body surface, even though features of the SC such as thickness and hydration change considerably from site to site. The density of the SG is particularly high on the scalp and foreheads, where it reaches 400–900 glands/cm2. In contrast, limbs show a considerably lower SG density8. Abundance of sebum is consistent with the SG density although sebometry and follicle number are not linearly correlated9,10. Sebum and SC have a remarkably diverse and unique lipid composition. Sebum is an oily to waxy mixture composed by 30–60% triglycerides (TG), 20–30% wax esters (WE), 10–30% FFA, and 10–20% squalene, as weight/weight percent (w/w %)11. Squalene is specific to human sebum where it is present at exceptionally high concentrations compared to other body lipid compartments. Minor components of sebum are cholesterol that together with sterols esters accounts for the 2–4%, and diglycerides (1–2%)10,12. The epidermal lipid compartment localized in the SC contains approximately 50% ceramides, 15% FFA, and 25% cholesterol and cholesterol sulfate (CHS)6,13,14. The latter one is present at a fairly high concentration in the SC wherein it regulates cohesion and desquamation. Extrusion of CHS and amounts of FFA take part to the regulation of skin pH15. Altogether, lipids account for the 10% of the 1 Laboratory of Cutaneous Physiopathology, San Gallicano Dermatological Institute IRCCS, Rome, Italy. 2Department of Chemistry, University of Rome “Sapienza”, Rome, Italy. Matteo Ludovici and Nina Kozul contributed equally to this work. Correspondence and requests for materials should be addressed to E.C. (email: ) SCIENtIfIC Reports | (2018) 8:11500 | DOI:10.1038/s41598-018-29742-7 1 www.nature.com/scientificreports/ SC weight. In the SC, the lipid and protein matrices are tightly intertwined to form the skin permeability barrier (SPB)16. Formation of a competent SPB is a complex process wherein several lipid biosynthetic pathways concur in the build-up of the lipid barrier. The epidermal sphingolipid metabolism leads to the biosynthesis of broad spectra of ceramides that are key regulators of the epidermal differentiation and inflammation17. The ceramide families found in the SC present very long chain FA, normally longer than 20 carbons, bound to the sphingoid bases. Moreover, ceramides bearing hydroxylated FA are partly transformed into acylceramides upon binding to long chain FA18,19. Little is known about the influence of the SG product on the epidermal lipid arrangement. It has been suggested that sebum secreted by the SG contributes to the ‘self-sterilizing’ properties of human skin20. Sapienic acid (FA C16:1n-10) is a sebum specific FA and the most active antimicrobial skin surface lipid21. Moreover, sebaceous FFA participate to the net value of superficial pH22. Thus, abundance and quality of sebum might influence the biochemical environment and the resident flora on the skin surface. Moreover, sebum can be regarded as a surface active biofluid that could impact physical properties of the SC. The diversity of skin lipids together with the different sources of their production pose a challenge for their qualitative and quantitative assessments. Lipidomics has gained importance in several fields of bioscience and has enhanced its potential in deciphering the complexity of the lipid composition in different biological matrices23. Conditions of suitable lipidomics approaches depend on the lipid source and the specific problem of interest. Lipidomics methods include well established separation and detection techniques such as thin layer chromatography (TLC), liquid chromatography (LC), gas chromatography (GC), mass spectrometry (MS)24,25. Approaches to investigate the two major skin lipid sources have been designed for both sebum and epidermal lipids12,18,26–35. A shotgun MS-based lipidomic approach has been presented that addressed the comprehensive identification and profiling of ceramide classes, TG, and cholesterol esters (CE) in human SC36. Intense research devoted to the characterization of sebaceous lipids by GC-MS dates back to the last century. More recent GC-MS methods have widened the lipid class coverage compared to the traditional (...truncated)