Sweat lipid mediator profiling: a noninvasive approach for cutaneous research

Abstract

Recent advances in analytical and sweat collection techniques provide new opportunities to identify noninvasive biomarkers for the study of skin inflammation and repair. This study aims to characterize the lipid mediator profile including oxygenated lipids, endocannabinoids, and ceramides/sphingoid bases in sweat and identify differences in these profiles between sweat collected from nonlesional sites on the unflared volar forearm of subjects with and without atopic dermatitis (AD). Adapting routine procedures developed for plasma analysis, over 100 lipid mediators were profiled using LC-MS/MS and 58 lipid mediators were detected in sweat. Lipid mediator concentrations were not affected by sampling or storage conditions. Increases in concentrations of C30–C40 [NS] and [NdS] ceramides, and C18:1 sphingosine, were observed in the sweat of study participants with AD despite no differences being observed in transepidermal water loss between study groups, and this effect was strongest in men (P < 0.05, one-way ANOVA with Tukey's post hoc HSD). No differences in oxylipins and endocannabinoids were observed between study groups. Sweat mediator profiling may therefore provide a noninvasive diagnostic for AD prior to the presentation of clinical signs. Recent advances in analytical and sweat collection techniques provide new opportunities to identify noninvasive biomarkers for the study of skin inflammation and repair. This study aims to characterize the lipid mediator profile including oxygenated lipids, endocannabinoids, and ceramides/sphingoid bases in sweat and identify differences in these profiles between sweat collected from nonlesional sites on the unflared volar forearm of subjects with and without atopic dermatitis (AD). Adapting routine procedures developed for plasma analysis, over 100 lipid mediators were profiled using LC-MS/MS and 58 lipid mediators were detected in sweat. Lipid mediator concentrations were not affected by sampling or storage conditions. Increases in concentrations of C30–C40 [NS] and [NdS] ceramides, and C18:1 sphingosine, were observed in the sweat of study participants with AD despite no differences being observed in transepidermal water loss between study groups, and this effect was strongest in men (P < 0.05, one-way ANOVA with Tukey's post hoc HSD). No differences in oxylipins and endocannabinoids were observed between study groups. Sweat mediator profiling may therefore provide a noninvasive diagnostic for AD prior to the presentation of clinical signs. Blood and urine have been extensively studied in the context of biomedical research and diagnostics. However, the collection of these biofluids can be physically and/or culturally invasive, affecting subject compliance. The use of noninvasive matrices, such as hair, oral fluid, sweat, and tears, has improved subject compliance in pharmacokinetic studies (1Raju K.S. Taneja I. Singh S.P. Wahajuddin Utility of noninvasive biomatrices in pharmacokinetic studies.Biomed. Chromatogr. 2013; 27: 1354-1366Crossref PubMed Scopus (34) Google Scholar). Recently, metabolomic analyses of sweat have begun for similar reasons (2Mena-Bravo A. Luque de Castro M.D. Sweat: a sample with limited present applications and promising future in metabolomics.J. Pharm. Biomed. Anal. 2014; 90: 139-147Crossref PubMed Scopus (162) Google Scholar). Sweat is a complex fluid excreted by apocrine and eccrine glands of the skin, reported to contain small molecules, including electrolytes, urea, lactate, amino acids, metals, and xenobiotics (2Mena-Bravo A. Luque de Castro M.D. Sweat: a sample with limited present applications and promising future in metabolomics.J. Pharm. Biomed. Anal. 2014; 90: 139-147Crossref PubMed Scopus (162) Google Scholar). Sweat can be used for diagnostics when disease alters its composition, as has been demonstrated in cystic fibrosis where sweat chloride levels are elevated (3Gibson L.E. Cooke R.E. A test for concentration of electrolytes in sweat in cystic fibrosis of the pancreas utilizing pilocarpine by iontophoresis.Pediatrics. 1959; 23: 545-549Crossref PubMed Google Scholar). Sweat has also been used in forensic settings as a test matrix, as most illicit drugs are excreted in sweat following administration by a variety of routes (4De Giovanni N. Fucci N. The current status of sweat testing for drugs of abuse: a review.Curr. Med. Chem. 2013; 20: 545-561PubMed Google Scholar). Despite the potential diagnostic uses, there are few studies examining the metabolic profile of sweat due to a lack of uniform and reproducible sweat collection protocols that generate sweat in sufficient quantities for analytical sample processing (2Mena-Bravo A. Luque de Castro M.D. Sweat: a sample with limited present applications and promising future in metabolomics.J. Pharm. Biomed. Anal. 2014; 90: 139-147Crossref PubMed Scopus (162) Google Scholar, 5Kutyshenko V.P. Molchanov M. Beskaravayny P. Uversky V.N. Timchenko M.A. Analyzing and mapping sweat metabolomics by high-resolution NMR spectroscopy.PLoS One. 2011; 6: e28824Crossref PubMed Scopus (65) Google Scholar). Reported sweat collection devices include simple occlusive or nonocclusive absorbent bandages applied to a body surface, bags that can encase a limb, and the Macroduct® sweat collector (Wescor Inc., Logan, UT). In each case, sweat collection is preceded by stimulation of sweating by either physiological (exercise or thermal induction) or pharmacological (pilocarpine iontophoresis) means (2Mena-Bravo A. Luque de Castro M.D. Sweat: a sample with limited present applications and promising future in metabolomics.J. Pharm. Biomed. Anal. 2014; 90: 139-147Crossref PubMed Scopus (162) Google Scholar). Of these, the Macroduct® sweat collector, which uses a concave disk and plastic capillary tubing to immediately collect and isolate sweat excreted to the skin surface following stimulation by pilocarpine, is perhaps the most suitable for metabolomics analysis because sweat collection is relatively fast (∼30 min per sample) and the design of the device prevents hydromeiosis and/or encapsulation of the skin surface (2Mena-Bravo A. Luque de Castro M.D. Sweat: a sample with limited present applications and promising future in metabolomics.J. Pharm. Biomed. Anal. 2014; 90: 139-147Crossref PubMed Scopus (162) Google Scholar, 6Ely M.R. Ely B.R. Chinevere T.D. Lacher C.P. Lukaski H.C. Cheuvront S.N. Evaluation of the megaduct sweat collector for mineral analysis.Physiol. Meas. 2012; 33: 385-394Crossref PubMed Scopus (19) Google Scholar). While several proteomic analyses of sweat have been reported (7Raiszadeh M.M. Ross M.M. Russo P.S. Schaepper M.A. Zhou W. Deng J. Ng D. Dickson A. Dickson C. Strom M. et al.Proteomic analysis of eccrine sweat: implications for the discovery of schizophrenia biomarker proteins.J. Proteome Res. 2012; 11: 2127-2139Crossref PubMed Scopus (98) Google Scholar, 8Csősz É. Emri G. Kalló G. Tsaprailis G. Tőzsér J. Highly abundant defense proteins in human sweat as revealed by targeted proteomics and label-free quantification mass spectrometry.J. Eur. Acad. Dermatol. Venereol. 2015; 29: 2024-2031Crossref PubMed Scopus (41) Google Scholar), only five attempts at metabolomic analyses of sweat have been described. High-resolution nuclear magnetic resonance spectroscopy of sweat suggested the presence of several amino acids and lipid-associated groups (5Kutyshenko V.P. Molchanov M. Beskaravayny P. Uversky V.N. Timchenko M.A. Analyzing and mapping sweat metabolomics by high-resolution NMR spectroscopy.PLoS One. 2011; 6: e28824Crossref PubMed Scopus (65) Google Scholar, 9Harker M. Coulson H. Fairweather I. Taylor D. Daykin C.A. Study of metabolite composition of eccrine sweat from healthy male and female human subjects by 1H NMR spectroscopy.Metabolomics. 2006; 2: 105-112Crossref Scopus (39) Google Scholar). More recent MS-based approaches confirmed the presence of amino acid- and lipid-derived molecules, while additionally showing the presence of carbohydrates and organic acids in sweat (10Calderón-Santiago M. Priego-Capote F. Jurado-Gámez B. Luque de Castro M.D. Optimization study for metabolomics analysis of human sweat by liquid chromatography-tandem mass spectrometry in high resolution mode.J. Chromatogr. A. 2014; 1333: 70-78Crossref PubMed Scopus (58) Google Scholar, 11Delgado-Povedano M.M. Calderón-Santiago M. Priego-Capote F. Luque de Castro M.D. Development of a method for enhancing metabolomics coverage of human sweat by gas chromatography-mass spectrometry in high resolution mode.Anal. Chim. Acta. 2016; 905: 115-125Crossref PubMed Scopus (37) Google Scholar). MS-based metabolomics of sweat was also used to develop a screening tool for lung cancer (12Calderón-Santiago M. Priego-Capote F. Turck N. Robin X. Jurado-Gámez B. Sanchez J.C. Luque de Castro M.D. Human sweat metabolomics for lung cancer screening.Anal. Bioanal. Chem. 2015; 407: 5381-5392Crossref PubMed Scopus (79) Google Scholar). Despite the potential utility of sweat in diagnostic settings, it has rarely been used in the context of cutaneous research. To the best of our knowledge, only a single study exists that examines the composition of sweat in the context of cutaneous disease, a study that demonstrated no difference in the inflammation regulating lipid prostaglandin (PG)E2 levels in subjects with atopic dermatitis (AD), psoriasis, or hyperhidrosis, relative to healthy controls (13Förström L. Goldyne M.E. Winkelmann R.K. Prostaglandin activity in human eccrine sweat.Prostaglandins. 1974; 7: 459-464Crossref PubMed Scopus (18) Google Scholar). Bioactive lipids come in many forms and regulate a variety of processes, including inflammation, cell growth and differentiation, and vascular homeostasis (14Murakami M. Lipid mediators in life science.Exp. Anim. 2011; 60: 7-20Crossref PubMed Scopus (99) Google Scholar). These mediators, which include oxygenated lipids (“oxylipins”), endocannabinoids, and ceramides, are generally thought to be produced locally via biosynthetic pathways in response to extracellular stimuli and function similarly to local hormones or autacoids (14Murakami M. Lipid mediators in life science.Exp. Anim. 2011; 60: 7-20Crossref PubMed Scopus (99) Google Scholar). Additionally, ceramides play an important structural role in the epidermal barrier (15van Smeden J. Janssens M. Gooris G.S. Bouwstra J.A. The important role of stratum corneum lipids for the cutaneous barrier function.Biochim. Acta. 2014; PubMed Scopus Google Scholar). While lipid mediators have been studied in cutaneous these studies have on a limited of analytical and/or a single of A. Bioactive lipid mediators in skin inflammation and Lipid Res. 2013; PubMed Scopus Google Scholar), the of mediator and the of discovery in these the and of lipid mediators has in (14Murakami M. Lipid mediators in life science.Exp. Anim. 2011; 60: 7-20Crossref PubMed Scopus (99) Google Scholar), and in a noninvasive in the context of skin research our of cutaneous while subject The present study aims to the potential for sweat analysis to of skin by examining the of AD on the sweat profile of lipid mediators from using targeted and water used sample and analysis were from and used analysis were from either or Lipid mediator analytical and were or from or A of subjects in each were to in a study to the of AD on the sweat mediator between and were from the of and the for the study a of AD by a or the of skin for on were and subjects with AD were while were in the unflared was from subjects prior to and study protocols were by the of the of Of the subjects were subjects with AD not sweat and subject without AD at the of the study with subjects with and subjects without with sampling and storage are in and storage of sweat collected from subjects with and without reported as include female subject with AD was an with to not with to observed lipid collected reported as < by water loss reported as at reported as These include female subject with AD was an with to not with to observed lipid reported as < by in a new were to from using or for at prior to the study Sweat was and collected from an on the volar of the at nonlesional sites present of the using the Macroduct® sweat collector by to the forearm was by with a by was on each forearm by pilocarpine using pilocarpine to a was for which the forearm was with and sweat was collected using the Macroduct® of the Macroduct® sweat collector and the sweat collection used in this study can be in sweat was with by the collection tubing using a and were at water loss was at a nonlesional immediately to the of sweat collection using a and in with the the was at the sampling and of transepidermal water loss were in were reported in of water per per of skin endocannabinoids, and ceramides were from sweat by of the to sample was by a and were with in and of or analytical in A of and analytical are in were with 100 and of a in was as a to loss were by and in of an each of and from B. D. of in prior to analysis was using of protocols J. J. B. A. analysis of by liquid chromatography-tandem mass PubMed Scopus Google Scholar, D. in the plasma acids, oxylipins and One. 2012; PubMed Scopus Google Scholar). of the sample were an with per lipid mediator profile and endocannabinoids were on a while ceramides/sphingoid bases were on a 100 using the in was on an with either or and ceramides/sphingoid and are in were using with five to were using and lipid mediator concentrations were reported in per of collected sweat. The used to the endocannabinoids, and ceramides in this study in the and are in the analysis generally protocols A a alters oxylipins and the in 2014; 11: PubMed Scopus Google Scholar). lipid mediator were by analysis using disease and/or subject of or storage as the analyses were in the for using a D. a to analysis in 2012; PubMed Scopus (34) Google Scholar). to were such that with of were from were for using the test for testing Google Scholar), and were by a analysis with C. W. between and for 2006; PubMed Google Scholar). Additionally, and were not collected for male with and without To the use of these and in this was as the of the in each AD The were not used for the of to the procedures of and analysis of Scholar), was using the with and were by using the and means were using J. B. metabolomics Res. 2015; PubMed Scopus Google Scholar). subjects with and without AD by differences were by discovery one-way ANOVA with Tukey's post hoc differences in sampling or storage were for AD groups using with discovery Additionally, subjects with and subjects without AD were and sweat lipid mediator concentrations from the and volar were by discovery ANOVA with Tukey's post hoc A of 58 lipid mediators were in the sweat of subjects with and without including endocannabinoids, [NS] or [NdS] ceramides, and A of lipid mediators for and detected with concentrations in eccrine sweat can be in of of lipid mediators can be in in the of and and demonstrated with the present at concentrations the However, as the of was in the analytical with these this was and on each be While female subject with AD was the in the study or this not the lipid in subjects with AD (P < concentrations of ceramides, [NS] and and [NdS] C18:1 and the subjects with and without AD and in ceramides, and C18:1 as the The also a with mediator profiles in men in and where men with and without AD are the relative differences in ceramides and between subjects with and without when by this men with AD have to ceramides and with men without with AD only have to ceramides and with without water an of skin barrier was not between subjects with and without AD sweat lipid mediator of men and with and without The showing AD and The showing in are by the in with in the were by using a analysis with by a and are elevated in subjects with of lipid mediators in male subjects with AD relative to the subject without In AD a to in ceramides and C18:1 of lipid mediators in female subjects with AD relative to the subject without In AD to concentrations of ceramides and C18:1 of lipid mediator can be in the Lipid mediators between subjects with and without AD ceramides and were by the sampling sampling or storage used in this In no differences were observed each subject subjects with and without when the were by of collection or of storage at or or or volar (P and storage a few of the lipid mediators in subjects without AD these were discovery for physiological role in is as is its composition, water and However, sweat also a of small that can be by disease and to a and noninvasive Lipid a variety of that have important in inflammation and cell and many of these are produced by and in the This study an of lipid mediators in eccrine sweat. The only was to a single lipid mediator in sweat collected from the forearm following thermal stimulation of sweating (13Förström L. Goldyne M.E. Winkelmann R.K. Prostaglandin activity in human eccrine sweat.Prostaglandins. 1974; 7: 459-464Crossref PubMed Scopus (18) Google Scholar). despite differences in sweat and analysis between and (13Förström L. Goldyne M.E. Winkelmann R.K. Prostaglandin activity in human eccrine sweat.Prostaglandins. 1974; 7: 459-464Crossref PubMed Scopus (18) Google and the present the concentrations of in the sweat of healthy and were not (P between the While it be to that sweat lipid mediator concentrations are by thermal pilocarpine sweat these are To the potential of a skin on sweat lipid mediator composition, and men and with either unflared AD or healthy with AD elevated concentrations of [NS] and [NdS] ceramides and C18:1 in sweat. Increases in and ceramides have been reported in the stratum corneum of subjects with AD and have been with an of the epidermal barrier J. H. N. M. et in the profile of atopic dermatitis Dermatol. PubMed Scopus Google Scholar, M. Smeden J. Gooris G.S. W. G. et in ceramides with an lipid and barrier function in atopic Lipid Res. 2012; PubMed Scopus Google Scholar). More in C18:1 have also been to be with in in an AD N. H. et profiles and in atopic Dermatol. 2013; PubMed Scopus Google Scholar). The observed in C18:1 in the sweat of subjects with AD to the reported in C18:1 in the of subjects with AD J. M. M. G. levels of sphingosine, a may be with of the stratum corneum from with atopic dermatitis to by Dermatol. PubMed Scopus Google Scholar). However, the of C18:1 activity of Dermatol. PubMed Scopus Google Scholar), these in subjects with AD are with to epidermal by such as J. M. M. G. levels of sphingosine, a may be with of the stratum corneum from with atopic dermatitis to by Dermatol. PubMed Scopus Google Scholar). it is to that the elevated sweat C18:1 levels may a to this to the skin The in [NS] ceramides to be at with the generally reported in skin ceramides with AD G. of in the barrier function of the stratum implications for the of atopic Dermatol. Res. 2014; Google Scholar). However, ceramides as a are complex molecules, of reported and present in in the skin, as in Of these and ceramides and are to and the epidermal barrier and of skin ceramides (15van Smeden J. Janssens M. Gooris G.S. Bouwstra J.A. The important role of stratum corneum lipids for the cutaneous barrier function.Biochim. Acta. 2014; PubMed Scopus Google Scholar). of of these in the stratum corneum of subjects with with a epidermal barrier J. H. N. M. et in the profile of atopic dermatitis Dermatol. PubMed Scopus Google Scholar, M. Smeden J. Gooris G.S. W. G. et in ceramides with an lipid and barrier function in atopic Lipid Res. 2012; PubMed Scopus Google Scholar). Of the [NS] and [NdS] ceramides of skin ceramides and to have to (15van Smeden J. Janssens M. Gooris G.S. Bouwstra J.A. The important role of stratum corneum lipids for the cutaneous barrier function.Biochim. Acta. 2014; PubMed Scopus Google Scholar, role in cell and in human Dermatol. Res. PubMed Scopus Google Scholar). concentrations of [NS] and [NdS] ceramides have been to in the stratum corneum of subjects with while concentrations of [NS] and [NdS] ceramides have been to in the stratum corneum of subjects with AD J. H. N. M. et in the profile of atopic dermatitis Dermatol. PubMed Scopus Google Scholar). In in [NS] and [NdS] ceramides have also been with an epidermal and lipid in the stratum corneum due to these has been as the for epidermal barrier L. Gooris G.S. Bouwstra J.A. The role of on the barrier of the skin lipid Acta. 2014; PubMed Scopus Google Scholar). has been reported that subjects with AD have of J. A. H. A. is in skin of with atopic Eur. Acad. Dermatol. Venereol. 2016; of 2016; Scholar). is for the of a to a to [NdS] or ceramides that are to [NS] or ceramides and its are acids A. and its regulate of J. PubMed Scopus Google Scholar). of the of are for the of and activity has been M. in the Acta. 2014; PubMed Scopus Google Scholar). that C18:1 is the most abundant in human skin M.E. of human skin include and Dermatol. PubMed Scopus (39) Google Scholar), the of on of its [NdS] ceramides, which be to [NS] have reported in [NS] ceramides and and [NdS] ceramides in the sweat of subjects with it that our are with the reported in activity in subjects with in is in subjects with no of levels being affected by AD G. of in the barrier function of the stratum implications for the of atopic Dermatol. Res. 2014; Google Scholar). is that [NS] levels as a to a epidermal research be to this or for the of in discovery of AD on sweat concentrations was studies examining [NS] ceramides in AD not differences in [NS] concentrations J. H. N. M. et in the profile of atopic dermatitis Dermatol. PubMed Scopus Google Scholar, M. Smeden J. Gooris G.S. W. G. et in ceramides with an lipid and barrier function in atopic Lipid Res. 2012; PubMed Scopus Google Scholar). While differences in skin ceramides, with men skin have been reported by A. D. M. of epidermal lipids of the healthy human affecting the design of a PubMed Scopus Google Scholar), have no differences in either or when men and M. J. J. I. M. M. H. and in stratum corneum Dermatol. Res. PubMed Scopus Google Scholar, J. and barrier function in healthy Venereol. 90: PubMed Scopus Google Scholar). However, while the of was in the of was between with AD in the and concentration in the sweat, that these in [NS] and [NdS] ceramides and C18:1 may be to the of To the best of our knowledge, no have been between levels of or and However, in cutaneous concentrations have been reported in A. The of acids in PubMed Scopus Google and in response to L.E. M. M. A. The response in human skin is by profiles that may its and J. 23: PubMed Scopus Google Scholar), and skin has been observed in our The role of in cutaneous disease it is that as a may cutaneous inflammation L.E. M. M. A. The response in human skin is by profiles that may its and J. 23: PubMed Scopus Google Scholar). the has been demonstrated to be a in A. of of proteins and by the acids and Med. 2014; PubMed Scopus Google Scholar), and to the of disease, an atopic similar to by inflammation and S.P. The 2013; PubMed Scopus Google Scholar). it is that and cutaneous inflammation and and testing of AD skin may be to an between these lipid mediators and lipid mediators with AD include and the or of which have been in cutaneous inflammation and D. A. of cell mediator new in 20: PubMed Scopus Google Scholar, G. of inflammation by PubMed Scopus Google Scholar, A. G. I. D. A. P. and prostaglandin the of and and by human 2006; PubMed Scopus Google Scholar). While and were in the sweat of subjects with and without concentration differences were not observed between the groups (P was not that the study subjects with AD were in an unflared and were from nonlesional skin, these the of and with the of its therefore to be between cutaneous lipid mediator and observed in sweat in the context of with to ceramides and is that these differences in ceramides and bases with epidermal barrier were observed in nonlesional skin from subjects with unflared that an epidermal barrier in AD is with or nonlesional skin to M. M. M. G. of epidermal barrier function by spectrometry in to its in the of atopic PubMed Scopus Google Scholar). the sweat mediator to a in subjects with AD and may provide an for diagnostics. This potential utility studies to the and of the observed by the of skin such as or on the sweat mediator may also be of to collect sweat from and nonlesional skin of subjects with AD in the in to the of epidermal barrier and/or inflammation on the sweat mediator While the of lipid mediators in sweat and plasma are is a of and the of the sweat are of the endocannabinoids, and G. J. C. L. regulate growth and of human eccrine sweat Dermatol. 2012; PubMed Scopus (19) Google Scholar). small xenobiotics are to in sweat by either or from Sweat testing for and Anal. PubMed Scopus Google Scholar), and the may be for lipid the of mediators in the sweat be important for the of the and this studies in the this study a of the sweat mediator the presence of endocannabinoids, [NS] and [NdS] ceramides, and bases in eccrine sweat collected from the volar Additionally, this study an and noninvasive tool that be used to cutaneous research. studies the of sweat to on the of skin inflammation, the of sweat and the in sweat composition with collection from as to the surface of this and to cutaneous research. The the and by M. I. J. W. and B. sample and analysis and the by M. subject The also the of the Macroduct® sweat collection by Inc., an with atopic dermatitis prostaglandin analysis