Sweat lipid mediator profiling: a noninvasive approach for cutaneous research
2016; Elsevier BV; Volume: 58; Issue: 1 Linguagem: Inglês
10.1194/jlr.m071738
ISSN1539-7262
AutoresKaran Agrawal, Lauren A. Hassoun, Negar Foolad, Theresa L. Pedersen, Raja K. Sivamani, John W. Newman,
Tópico(s)Biochemical Analysis and Sensing Techniques
ResumoRecent 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. Biophys. Acta. 2014; 1841: 295-313Crossref PubMed Scopus (3) Google Scholar). While lipid mediators have been studied in cutaneous research, these studies have focused on a limited number of analytical targets and/or a single class of analytes (16Kendall A.C. Nicolaou A. Bioactive lipid mediators in skin inflammation and immunity.Prog. Lipid Res. 2013; 52: 141-164Crossref PubMed Scopus (149) Google Scholar), barring the identification of mediator pathway cross-talk and limiting the scope of discovery in these studies. Understanding the actions and interactions of lipid mediators has proven useful in other contexts (14Murakami M. Lipid mediators in life science.Exp. Anim. 2011; 60: 7-20Crossref PubMed Scopus (99) Google Scholar), and doing so in a noninvasive manner in the context of skin research could enhance our understanding of cutaneous biochemistry while ensuring minimal subject discomfort. The present study aims to demonstrate the potential for sweat analysis to reflect biochemical impacts of skin diseases by examining the impact of AD on the sweat profile of >100 lipid mediators from three chemical super-classes using targeted analyses. Ultra-performance LC (UPLC)-grade methanol, acetonitrile, 2-propanol, and water used during sample preparation and chromatographic analysis were purchased from Fisher Scientific (Waltham, MA). Glacial acetic acid, formic acid, and ammonium formate used during chromatographic analysis were purchased from either Fisher Scientific or Sigma-Aldrich (St. Louis, MO). Lipid mediator standards, analytical surrogates, and internal standards were synthesized or purchased from Cayman Chemicals (Ann Arbor, MI), Avanti Polar Lipids Inc. (Alabaster, AL), or Larodan (Malmö, Sweden). A total of 26 subjects (n = 13 in each group) were recruited to participate in a case-control study to examine the effects of AD on the sweat mediator lipidome between February 2015 and February 2016. Subjects were recruited from the University of California-Davis Dermatology Clinic and the Sacramento region. Inclusion criteria for the study included a diagnosis of AD by a dermatologist or the absence of any inflammatory skin conditions for control subjects. Subjects on systemic immunosuppressive medications were excluded, and all subjects with AD were sampled while they were in the unflared state. Written informed consent was obtained from all subjects prior to participation, and all study protocols were approved by the Institutional Review Board of the University of California-Davis (Protocol #605131). Of the 26 subjects recruited, three were excluded. Two subjects with AD did not produce sweat upon stimulation, and one subject without AD had flared acne vulgaris at the time of sampling. Therefore, the study proceeded with 11 subjects with and 12 subjects without AD. Group characteristics along with sampling and storage parameters are shown in Table 1.TABLE 1Sampling and storage parameters of sweat collected from subjects with and without ADParameterAD (n = 11)Control (n = 12)Gender (male/female) (n)7/47/5Age (years)aData reported as mean ± SD.33.8 ± 12.0bThese data include one female subject with AD was an outlier with respect to age (62.2 years vs. ADn=10 = 30.9 ± 7.7 years), but not with respect to observed lipid mediators.29.3 ± 3.8Sweat collected (μl)cData reported as geometric mean [range].31.2 [7.1–73]dP < 0.05 versus control group. Significance assessed by two-tailed heteroscedastic Student's t-test.61.3 [25–100]Transepidermal water loss (g/h/m2)aData reported as mean ± SD.8.4 ± 3.97.4 ± 3.9Sampling time (n)0900–1400661400–180056Storage at −80°C (n)0–19 days7520–30 days47a Data reported as mean ± SD.b These data include one female subject with AD was an outlier with respect to age (62.2 years vs. ADn=10 = 30.9 ± 7.7 years), but not with respect to observed lipid mediators.c Data reported as geometric mean [range].d P < 0.05 versus control group. Significance assessed by two-tailed heteroscedastic Student's t-test. Open table in a new tab Subjects were asked to refrain from using any topical moisturizers or medications for at least 12 h prior to the study visit. Sweat was stimulated and collected from an approximately 7 cm2 area on the volar surfaces of the bilateral forearms at nonlesional sites present within 8 cm of the wrist using the Macroduct® sweat collector (generously donated by Wescor, Inc.) according to manufacturer's instructions (http://wescor.com/translations/Translations/M2551-7A-EN.pdf, accessed July 11, 2016). Briefly, the forearm was prepared by wiping with a 70% isopropanol swab (Covidien, Minneapolis, MN) followed by distilled water-saturated sterile cotton gauze. Sweating was then stimulated sequentially on each forearm by pilocarpine iontophoresis using manufacturer-supplied pilocarpine gel disks attached to a power source containing two 9 V batteries. Pilocarpine iontophoresis was conducted for 5 min, after which the forearm was wiped with fresh distilled water-saturated sterile cotton gauze, and sweat was collected using the Macroduct® device. An image of the Macroduct® sweat collector and the typical sweat collection site used in this study can be found in supplemental Fig. S1. Collected sweat was exuded into methanol-rinsed 2 ml amber vials with Teflon lined closures (Waters, Milford, MA) by passing air through the collection tubing using a gastight syringe (Hamilton, Reno, NV) and samples were stored at −80°C until analysis. Transepidermal water loss was measured at a nonlesional site immediately adjacent to the site of sweat collection using a Tewameter TM 300 (Courage and Khazaka Electronic GmbH, Cologne, Germany) in accordance with the manufacturer's instructions. Briefly, the Tewameter probe was placed at the sampling site and triplicate measurements of transepidermal water loss were recorded in 30 s intervals. Data were reported in grams of water lost per hour per square meter of skin (g/h/m2). Oxylipins, endocannabinoids, sphingoid bases, and ceramides were isolated from sweat by direct evaporation of the matrix. Prior to evaporation, sample volume was determined by a gastight syringe (Hamilton) and samples were enriched with 5 μl anti-oxidant solution (0.2 mg/ml solution butylated hydroxytoluene/EDTA in 1:1 methanol:water) and 5 μl of 1 μM deuterated or C17-analog analytical surrogates in methanol. A complete list of target analytes and their associated analytical surrogates are shown in supplemental Tables S1–S3. Samples were then diluted with 100 μl methanol and 10 μl of a 20% glycerol solution in methanol was added as a keeper to reduce analyte loss during evaporation. Samples were dried by vacuum evaporation and reconstituted in 35 μl of an internal standard solution containing 50 nM each of 1-cyclohexyl-3-ureido dodecanoic acid (Sigma-Aldrich) and 1-phenyl,3-ureido hexanoic acid (gift from B. D. Hammock, University of California-Davis) in 1:1 (v/v) methanol:acetonitrile prior to analysis. UPLC-MS/MS analysis was conducted using modifications of previously published protocols (17Bielawski J. Pierce J.S. Snider J. Rembiesa B. Szulc Z.M. Bielawska A. Comprehensive quantitative analysis of bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry.Methods Mol. Biol. 2009; 579: 443-467Crossref PubMed Scopus (128) Google Scholar, 18Grapov D. Adams S.H. Pedersen T.L. Garvey W.T. Newman J.W. Type 2 diabetes associated changes in the plasma non-esterified fatty acids, oxylipins and endocannabinoids.PLoS One. 2012; 7 (10.1371/journal.pone.0048852.): e48852Crossref PubMed Scopus (97) Google Scholar). Briefly, three 10 μl aliquots of the reconstituted sample were sequentially injected onto an Acquity UPLC system (Waters), with one injection per lipid mediator profile analysis. Oxylipins and endocannabinoids were separated on a 2.1 × 150 mm, 1.7 μm BEH C18 column (Waters), while ceramides/sphingoid bases were separated on a 2.1 × 100 mm, 1.7 μm BEH C8 column (Waters) using the solvent gradients described in supplemental Tables S1–S3. MS/MS was performed on an API 4000 QTrap (Sciex, Framingham, MA) with either negative mode (oxylipins) or positive mode (endocannabinoids and ceramides/sphingoid bases) electrospray ionization. Ionization voltages, MS/MS parameters, and chromatographic retention time are listed in supplemental Tables S1–S3. Analytes were quantified using internal standard methodology with five to seven point calibration curves (r ≥ 0.997). Data were processed using AB Sciex MultiQuant version 3.0.2 and all calculated lipid mediator concentrations were reported in picomoles per milliliter (i.e., nM) of collected sweat. The abbreviations used to describe the oxylipins, endocannabinoids, sphingoid bases, and ceramides quantified in this study follow standard conventions in the field and are fully described in the supplemental data. Statistical analysis generally followed previously published protocols (19Dunn T.N. Keenan A.H. Thomas A.P. Newman J.W. Adams S.H. A diet containing a nonfat dry milk matrix significantly alters systemic oxylipins and the endocannabinoid 2-arachidonoylglycerol (2-AG) in diet-induced obese mice.Nutr. Metab. (Lond.). 2014; 11: 24Crossref PubMed Scopus (6) Google Scholar). All lipid mediator data were assessed by partial least squares discriminant analysis (PLS-DA) using disease state and/or subject gender, hand sampled, time of collection, or storage time as the classifier. All statistical analyses were performed in the R statistical environment (R Foundation for Statistical Computing, Vienna, Austria) using imDEV v1.42, a Microsoft Excel add-in (Microsoft Corporation, Redmond, WA) (20Grapov D. Newman J.W. imDEV: a graphical user interface to R multivariate analysis tools in Microsoft Excel.Bioinformatics. 2012; 28: 2288-2290Crossref PubMed Scopus (34) Google Scholar). Prior to PLS-DA, data were curated such that analytes with <70% completeness of data were removed from consideration. Curated data were screened for outliers using the Grubb's test (21Grubbs F.E. Sample criteria for testing outlying observations.Ann. Math. Statist. 1950; 21: 27-58Crossref Google Scholar), and missing data were imputed by a two-component probabilistic principle components analysis with univariate scaling (22Wang C. Wang W. Links between PPCA and subspace methods for complete Gaussian density estimation.IEEE Trans. Neural Netw. 2006; 17: 789-792Crossref PubMed Google Scholar). Additionally, ceramide and sphingoid base data were not collected for four male subjects, two with and two without AD. To allow the unbiased use of these subjects' oxylipin and endocannabinoid data in PLS-DA analyses, this missing data was interpolated as the gender-independent mean of the remaining subject's data in each AD group. The interpolated ceramide results were not used for the mean comparisons described below. Following normalization of data according to the procedures of Box and Cox (23Box G.E.P. Cox D.R. An analysis of transformations (with discussion).J. Roy. Statist. Soc. Ser.B. 1964; 26: 211-252Google Scholar), PLS-DA was conducted using the orthogonal scores algorithm with univariate scaling and leave-one-out cross-validation. Variables were clustered by Spearman correlation coefficients using the Minkowski distance and Ward agglomeration. All means comparisons were performed using MetaboAnalyst 3.0 (24Xia J. Sinelnikov I.V. Han B. Wishart D.S. MetaboAnalyst 3.0–making metabolomics more meaningful.Nucleic Acids Res. 2015; 43: W251-W257Crossref PubMed Scopus (2131) Google Scholar). For subjects with and without AD classified by gender, mean differences were tested by false discovery rate-corrected one-way ANOVA with Tukey's post hoc HSD. Mean differences in sampling or storage were tested for AD groups separately using Student's t-test with false discovery rate correction. Additionally, three subjects with and three subjects without AD were randomly selected and mean sweat lipid mediator concentrations from the right and left volar forearms were compared by false discovery rate-corrected repeated measures ANOVA with Tukey's post hoc HSD. A total of 58 lipid mediators were quantified in the sweat of subjects with and without AD, including 33 oxylipins, 3 nitrolipids, 13 endocannabinoids, 7 [NS] or [NdS] ceramides, and 2 sphingoid bases. A complete list of lipid mediators screened for and detected along with their associated concentrations in eccrine sweat can be found in supplemental Table S4. Representative chromatograms of all three classes of lipid mediators can be found in supplemental Figs. S2–S4. As seen in supplemental Fig. S3, the monoacylglycerol derivatives of arachidonic, linoleic, and oleic acid (i.e., 2-AG/1-AG, 2-LG/1-LG, and 2-OG/1-OG, respectively) all demonstrated isomerization, with the 1-isomer present at higher concentrations than the 2-isomer. However, as the same degree of isomerization was noted in the analytical surrogate associated with these targets (d5-2-AG), this isomerization was considered acceptable, and quantitative data on each isomer could still be obtained. While one female subject with AD was substantially older than the other women in the study (62.2 years vs. ADn=10 = 30.9 ± 7.7 years or ADfemales = 34.1 ± 10.6 years), this extreme age did not influence the measured lipid mediators. As shown in Fig. 1, subjects with AD had increased (P < 0.05) concentrations of C14–C24 ceramides, i.e., C32–C42 [NS] ceramides; C18 and C24 dihydroceramides, i.e., C36 and C42 [NdS] ceramides; C18:1 sphingosine; 10-nitrooleate; and the arachidonate-derived alcohol 8-HETE. PLS-DA discriminated subjects with and without AD (Fig. 2A), and identified increases in ceramides, dihydroceramides, and C18:1 sphingosine as the dominant discriminating factors (Fig. 2B). The PLS-DA also highlighted a gender-dependent effect, with mediator profiles increasing more in men than in women, and where men with and without AD are better separated than women (Fig. 2A). Comparing the relative fold differences in ceramides and sphingosine between subjects with and without AD, when separated by gender, highlights this observation (Fig. 2C, D). Specifically, men with AD have ∼2- to 6-fold higher ceramides and sphingosine compared with men without AD, whereas women with AD only have ∼1- to 1.5-fold higher ceramides and sphingosine compared with women without AD. Transepidermal water loss, an indicator of skin barrier status, was not different between subjects with and without AD (Table 1).Fig. 2Eccrine sweat lipid mediator discrimination of men and women with and without AD. A: The PLS-DA scores plot showing AD group and gender discrimination. B: The PLS-DA loadings plot showing variable weight in discrimination. Point sizes are defined by the variable importance in projection (VIP) scores, with VIPs >1 considered significant factors in the discriminant model. Variables were grouped by their correlation (Spearman's ρ) using a hierarchical cluster analysis with cluster identified by a unique color. Ceramides and sphingosine are elevated in subjects with AD. C: Fold-difference comparisons of lipid mediators increased in male subjects with AD relative to the average subject without AD. In men, AD caused a 2- to 6-fold elevation in ceramides and C18:1 sphingosine. D: Fold-difference comparisons of lipid mediators increased in female subjects with AD relative to the average subject without AD. In women, AD caused 1- to 1.5-fold higher concentrations of ceramides and C18:1 sphingosine. Descriptions of lipid mediator abbreviations can be found in the supplemental data.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Lipid mediators discriminating between subjects with and without AD (i.e., ceramides and sphingoid bases) were unaffected by the sampling time, sampling site, or storage conditions used in this study. In particular, no differences were observed within each subject group (i.e., subjects with and without AD) when the data were grouped by time of collection (0900–1400 or 1400–1800), duration of storage at −80°C (0–19 days or 20–30 days), or arm sampled (right or left volar forearm) (P > 0.05) (supplemental Figs. S5–S7). Sampling and storage time impacted a few of the identified lipid mediators in subjects without AD (supplemental Table S5); however, these changes were excluded after false discovery rate correction for multiple comparisons (q = 0.05). Sweat's physiological role in heat dissipation is well-known, as is its basic chemical composition, i.e., water and salt. However, sweat also contains a wide array of other small molecules that can be influenced by disease states and environmental exposures, thus offering access to a potentially valuable and noninvasive biological sample. Lipid biochemistry produces a wide variety of bioactive molecules that have important regulatory roles in inflammation and cell growth, among other things, and many of these are produced by and found in the skin. This study represents an extensive characterization of bioactive lipid mediators in eccrine sweat. The only previous attempt was to quantify a single lipid mediator (PGE2) 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). Interestingly, despite differences in sweat initiation, collection, and analysis between Forstrom, Goldyne, and Winkelman (13Förström L. Goldyne M.E. Winkelmann R.K. Prostaglandin activity in human eccrine sweat.Prostaglandins. 1974; 7: 459-464Crossref PubMed Scopus (18) Google Scholar) and the present study, the concentrations of PGE2 in the sweat of healthy individuals (1.77 ± 1.22 nM and 2.19 ± 3.02 nM, respectively) were not different (P = 0.7) between the two studies. While it would be premature to suppose that sweat lipid mediator concentrations are unaffected by thermal versus pilocarpine iontophoretic sweat stimulation, these results are intriguing. To explore the potential impact of a common skin disorder on sweat lipid mediator composition, we enrolled and evaluated men and women with either unflared AD or healthy skin. Subjects with AD showed elevated concentrations of [NS] and [NdS] ceramides and C18:1 sphingosine in their sweat. Increases in short-chain (30–40 total carbons) [NS], [AS], [NH], and [AH] ceramides have been previously reported in the stratum corneum of subjects with AD and have been correlated with an impairment of the epidermal barrier (25Ishikawa J. Narita H. Kondo N. Hotta M. Takagi Y. Masukawa Y. Kitahara T. Takema Y. Koyano S. Yamazaki S. et al.Changes in the ceramide profile of atopic dermatitis patients.J. Invest. Dermatol. 2010; 130: 2511-2514Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 26Janssens M. van Smeden J. Gooris G.S. Bras W. Portale G. Caspers P.J. Vreeken R.J. Hankemeier T. Kezic S. Wolterbeek R. et al.Increase in short-chain ceramides correlates with an altered lipid organization and decreased barrier function in atopic eczema patients.J. Lipid Res. 2012; 53: 2755-2766Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). More recently, increases in C18:1 sphingo
Referência(s)