Finding
Despite this, it has emerged as a significantly important component with Sato et al.
Paper
Age-related changes to human stratum corneum lipids detected using ToF-SIMS following in vivo sampling
Abstract
This work demonstrates the ability to detect changes in both quantity and spatial distribution of human stratum corneum (SC) lipids from samples collected in vivo. The SC functions as the predominant barrier to the body, protecting against the penetration of xenobiotic substances. Changes to the SC lipid composition have been associated with barrier impairment and consequent skin disorders and it is therefore important to monitor and quantify changes to this structure. This work demonstrates the first reported use of time of flight secondary ion mass spectrometry (ToF-SIMS) to assess physiological changes to human SC as a function of depth. This technique provides exceptional sensitivity and chemical specificity, allowing analysis of single tape stripped samples taken from volunteers. Using this methodology we were able to successfully identify chemical differences in human SC resulting from both intrinsic and extrinsic (photo) aging. Samples were collected from women of two age groups (under 27 and post-menopausal) and from two body sites with varying UV exposure (inner forearm and dorsal hand) and differences were identified using multivariate data analysis. The key finding was the significant aged-related increase and change in spatial distribution of the sterol cholesterol sulfate, a membrane stabilizing lipid. Significant changes in the prevalence of both lignoceric acid (C24:0) and hexacosanoic acid (C26:0) were also observed. This work describes previously unreported age-related chemical changes to human SC, providing an insight into aging mechanisms which may improve the design of both pharmaceutical and cosmetic topical products. The stratum corneum consists of tightly stacked layers of corneocytes surrounded by an intercellular lipid-rich domain and despite being only 10-20 μm thick it provides the primary barrier to the body, preventing transepidermal water loss and the penetration of xenobiotic substances. Although previously thought to be a metabolically inert structure, the role of the SC as a natural biosensor is now known to be crucial in the defence mechanism of the skin. Examples include changes to external humidity which initiate filaggrin proteolysis and the release of preformed cytokines to regulate epidermal lipid synthesis in response to acute barrier disruption. Changes in the SC lipid composition have also been linked to various skin disorders, including atopic dermatitis, psoriasis and ichthyoses, reviewed by Harding and more recently Jungersted and Agner. Detailed chemical analysis of this tissue and the changes it undergoes in response to both internal and external factors is therefore hugely important in order to facilitate more effective skin treatments. Time of flight secondary ion mass spectrometry (ToFSIMS) is a surface analysis technique that provides high chemical sensitivity and specificity. Successful analysis of a wide range of biological compounds have been demonstrated using ToF-SIMS, including proteins and lipids from a variety of biological substrates, including bacteria, individual cells and both healthy and diseased tissue. ToF-SIMS analysis has recently been employed to monitor permeation of topically applied compounds through ex vivo skin tissue, both animal 11 and human. However, this technique has not yet been utilised to study the lipid composition of native human skin, despite offering several advantages over traditionally used chromatographic mass spectrometry methods. ToF-SIMS requires no prior sample preparation except the removal of excess moisture, which allows biological samples to be analysed in their original state without the need for a dissolution or extraction process. The technique can also simultaneously detect ions from multiple components within a sample, eliminating the need for the often complicated separation step found in chromatographic methods. However, the key advantage offered by ToF-SIMS is the ability to map the spatial distribution of detected ions within a sample, with a resolution of 1 μm achievable. The lipid composition of the SC is unique and significantly different to other biological membranes. Typical keratinocyte plasma membranes contain high levels of phospholipids yet the SC contains virtually none, as depletion of these lipids accompanies cornification. Instead this tissue consists predominantly of three main species, ceramides, cholesterol and fatty acids, in an equimolar ratio. Cholesterol sulfate is also present in small quantities, declining from 5% of the total lipid content in the underlying layers of the SC to only 1% in the most superficial layers. Despite this, it has emerged as a significantly important component with Sato et al. proposing that it plays a role in regulating desquamation, through inhibition of proteases which facilitate corneocyte loss. Elias et al. had previously demonstrated a fivefold increase in this molecule in patients with recessive X-linked ichthyosis. This hereditary condition results from a deficiency in steroid sulfatase and causes extreme scaling of the skin and both Elias et al. and Sato et al. have induced scaling of murine skin through topical cholesterol sulfate application. It is also known that this sterol is found in keratinizing membranes at much higher levels than in mucosal membranes, further supporting the suggestion that it is involved the regulation of desquamation. Research into aging skin is becoming increasingly popular, a consequence of both an aging population and an increased awareness of UV damage. The aging process in skin occurs through two pathways, intrinsic and extrinsic. Intrinsic aging is a consequence of the degenerative process the body undergoes over time, whereas extrinsic aging becomes apparent in people from a much younger age due to exposure to environmental factors, such as cigarette smoke, pollution and UV radiation. Prolonged exposure to UV radiation in particular can result in phenotypic changes, an effect termed photo damage. The majority of research surrounding aging skin is focused on changes to physical parameters, such as transepidermal water loss, surface pH or skin hydration. However, information regarding age-related changes to the SC lipid composition is less documented and different groups have reported variable effects. Both Jungersted et al. and Imokawa et al. used the cyanoacrylate method to remove human SC and conducted analysis using thin layer chromatography. Yet while Jungersted et al. noted no significant change in any of the individual ceramide classes, Imokawa et al. reported a significant decrease in total ceramide content with age. Rogers et al. also used thin layer chromatography to report an agerelated decrease in all three major lipid classes, but this analysis was conducted using pooled tape stripped samples. Denda et al. used solvent extraction coupled with thin layer chromatography to reveal both an increase and decrease in individual ceramide classes, but only in female volunteers. There is also limited information regarding the effect of environmental exposure on SC lipids. Bak et al. demonstrated a significant decrease in murine SC ceramide content following long term UV exposure and Boireau-Adamezyk et al. reported a decrease in cholesterol levels when comparing an exposed arm site to a protected one. Despite its proposed importance in desquamation, only one group to date have focused on a change in cholesterol sulfate levels with age. Haratake et al. conducted thin layer chromatographic analysis of extracted lipids from murine SC to demonstrate that cholesterol sulfate and cholesterol levels were increased and decreased respectively in aged mice. Despite the presence of a lipid gradient within the SC there is limited research assessing both lipid composition in general and age-related changes as a function of SC depth. Typical SC sampling methods, such as surface extraction and cyanoacrylate resin, sample the SC as a whole and until recently instrument sensitivity required sequential tape strips to be pooled. Both Egawa et al. and Boireau-Adamezyk et al. have recently demonstrated the ability to record measurements at varying depths within the SC in vivo using Raman spectroscopy, at depths of 2 and 4 μm apart respectively. However, this technique cannot provide the chemical specificity that is offered by mass spectrometry techniques. This study describes the first reported application of ToFSIMS to conduct chemical analysis of native human SC from individual tape stripped samples collected in vivo. In addition to exceptional sensitivity and chemical specificity, ToF-SIMS also allows the spatial distribution of compounds to be mapped. Therefore, by combining ToF-SIMS with tape stripping both the composition and distribution of lipids can be monitored as a function of SC depth. EXPERIMENTAL SECTION Sample Population. The sample population consisted of healthy female volunteers with a Fitzpatrick skin type II/III. In order to observe differences due to intrinsic aging, samples were taken from 9 females under 27 years old and 9 postmenopausal females (typically > 60 years of age). In order to observe differences caused by extrinsic aging, samples were collected from both the flexor forearm (low UV exposure) and dorsal hand (high UV exposure) for each volunteer. For the purpose of this study the following abbreviations will be used: post-menopausal (old or O), under 27 year old (young or Y), arm (A) and hand (H). All volunteers participated after providing consent and confirmed they were free from skin conditions, medication and had not recently used any form of artificial tanning methods. Sample Collection. Tape stripping experiments were performed using D-Squame® skin sampling discs (CuDerm, Dallas, Tx, USA). The circular discs had a diameter of 22 mm and used a fully cured medical grade synthetic poly-acrylate ester adhesive. The disc was applied to the skin, a constant pressure applied using a pressure disc applicato
Authors
Daniel J. Johnson, D. Scurr, Nichola J. Starr
Journal
Journal name not available for this finding