Clinical-Grade Patches as a Medium for Enrichment of Sweat-Extracellular Vesicles and Facilitating Their Metabolic Analysis

Abstract

Cell-secreted extracellular vesicles (EVs), carrying components such as RNA, DNA, proteins, and metabolites, serve as candidates for developing non-invasive solutions for monitoring health and disease, owing to their capacity to cross various biological barriers and to become integrated into human sweat. However, the evidence for sweat-associated EVs providing clinically relevant information to use in disease diagnostics has not been reported. Developing cost-effective, easy, and reliable methodologies to investigate EVs’ molecular load and composition in the sweat may help to validate their relevance in clinical diagnosis. We used clinical-grade dressing patches, with the aim being to accumulate, purify and characterize sweat EVs from healthy participants exposed to transient heat. The skin patch-based protocol described in this paper enables the enrichment of sweat EVs that express EV markers, such as CD63. A targeted metabolomics study of the sweat EVs identified 24 components. These are associated with amino acids, glutamate, glutathione, fatty acids, TCA, and glycolysis pathways. Furthermore, as a proof-of-concept, when comparing the metabolites’ levels in sweat EVs isolated from healthy individuals with those of participants with Type 2 diabetes following heat exposure, our findings revealed that the metabolic patterns of sweat EVs may be linked with metabolic changes. Moreover, the concentration of these metabolites may reflect correlations with blood glucose and BMI. Together our data revealed that sweat EVs can be purified using routinely used clinical patches, setting the foundations for larger-scale clinical cohort work. Furthermore, the metabolites identified in sweat EVs also offer a realistic means to identify relevant disease biomarkers. This study thus provides a proof-of-concept towards a novel methodology that will focus on the use of the sweat EVs and their metabolites as a non-invasive approach, in order to monitor wellbeing and changes in diseases.

Critical Factors in Sample Collection and Preparation for Clinical Metabolomics of Underexplored Biological Specimens

“…It is secreted by apocrine, eccrine, and sebaceous glands and plays roles in the regulation of body temperature, the protection and hydration of the skin, and body homeostatic control [ 219 ]. It is a non-invasively collectible sample composed of metabolites, proteins, nucleic acids, and small particles like extracellular vesicles [ 220 ]. This biological specimen has been used in doping control testing, the exposure evaluation of heavy metals, and the routine diagnosis of cystic fibrosis (CF) [ 221 ].…”

“…This biological specimen has been used in doping control testing, the exposure evaluation of heavy metals, and the routine diagnosis of cystic fibrosis (CF) [ 221 ]. In metabolomics studies, sweat samples can provide insights into health status and have been used to investigate autoimmune disorders [ 222 , 223 ] and T2DM [ 220 ] and for monitoring treatment efficacy for CF [ 224 ].…”

“…Sweat samples are usually extracted through protein precipitation using methanol [ 224 ], 50% acetonitrile [ 225 , 226 , 227 ], formic acid aqueous solution [ 228 , 229 ], or the mixture methanol/acetonitrile (1:1, v / v ) [ 220 ] for nonpolar and polar metabolite coverage through LC–MS. After protein removal, a second extraction with dichloromethane was performed by Delgado-Povedano and collaborators [ 230 ], in which the organic phase was derivatized to evaluate the differences in metabolic profile between fresh and dry sweat-sample collection.…”

Enrichment of sweat-derived extracellular vesicles of human and bacterial origin for biomarker identification

“…To apply these innovations to sweat EVs analysis we need a better characterization of their cargo, such as RNA and proteins. RNA and DNA composition of EV-enriched human sweat 27 , 28 , metabolomics analysis of sweat 11 , 29 - 32 and sweat EVs 33 , as well as proteomics analysis of whole sweat 3 , 4 , 9 , 11 , 34 - 36 and sweat-derived exosomes of human origin pooled from several individuals 37 have been reported. Our aim in the present study was to characterize protein cargo of EVs in individual unpooled sweat samples collected during a biking exercise.…”

Metabolic patterns of sweat-extracellular vesicles during exercise and recovery states using clinical grade patches

“…Our recent publication demonstrating, for the first time, that metabolite levels from sweat EVs may serve as a means for reflecting metabolic changes in healthy and type 2 diabetes groups after heat exposure, is highly relevant. Furthermore, with the aid of clinical patches, we described a new non-invasive methodology allowing the cost-effective enrichment and the isolation of sweat EVs from healthy and diseased participants ( Rahat et al, 2023 ).…”