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Although extracellular vesicles (EVs) have been confirmed as promising sources of disease biomarkers, little is known about the metabolite compositions of its subpopulations and their roles in the diagnosis of pleural effusion. Here, we performed metabolomics and lipidomics analysis to investigate the metabolite characteristics of two EV subpopulations derived from pleural effusion by differential ultracentrifugation, namely large EVs (lEVs, pelleted at 20,000\u00a0\u00d7\u00a0g) and small EVs (sEVs, pelleted at 110,000\u00a0\u00d7\u00a0g), and assessed their metabolite differences between tuberculosis and malignancy. A total of 579\u00a0metabolites, including amino acids, acylcarnitines, organic acids, steroids, amides and various lipid species, were detected. The results showed that the metabolic profiles of lEVs and sEVs overlapped with and difference from each other but significantly differed from those of pleural effusion. Additionally, different type of vesicles and pleural effusion showed unique metabolic enrichments. Furthermore, lEVs displayed more significant and larger metabolic alterations between the tuberculosis and malignancy groups, and their differential metabolites were more closely related to clinical parameters than those of sEV. Finally, a panel of four biomarker candidates, including phenylalanine, leucine, phosphatidylcholine 35:0, and sphingomyelin 44:3, in pleural lEVs was defined based on the comprehensive discovery and validation workflow. This panel showed high performance for distinguishing TPE and MPE, particularly in patients with delayed or missed diagnosis, such as the area under the receiver\u2010operating characteristic curve (AUC) &gt;0.95\u00a0in both sets. We conducted comprehensive metabolic profiling analysis of EVs, and further explored the metabolic reprogramming of tuberculosis and malignancy at the level of metabolites in lEVs and sEVs, providing insight into the mechanism of pleural effusion, and identifying novel biomarkers for diagnosing TPE and MPE.<\/jats:p>","DOI":"10.1080\/20013078.2020.1790158","type":"journal-article","created":{"date-parts":[[2020,7,14]],"date-time":"2020-07-14T12:18:55Z","timestamp":1594729135000},"update-policy":"https:\/\/doi.org\/10.1080\/tandf_crossmark_01","source":"Crossref","is-referenced-by-count":72,"title":["Metabolic characteristics of large and small extracellular vesicles from pleural effusion reveal biomarker candidates for the diagnosis of tuberculosis and malignancy"],"prefix":"10.1002","volume":"9","author":[{"given":"Ping","family":"Luo","sequence":"first","affiliation":[{"name":"Center for Translational Medicine Union Hospital Tongji Medical College Huazhong University of Science and 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