Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications across clinical research, including monitoring radiation exposure. A key limitation to their implementation is minimal standardization in EV isolation and analytical methods. Further, most urinary EV isolation protocols necessitate large volumes of sample. This study aimed to compare and optimize isolation and analytical methods for EVs from small volumes of urine.
Researchers at the Georgetown University Medical Center compared 3 EV isolation methods: ultracentrifugation, magnetic bead-based, and size-exclusion chromatography from 0.5 mL or 1 mL of rat and human urine. EV yield and mass spectrometry signals (Q-ToF and Triple Quad) were evaluated from each method. Metabolomic profiling was performed on EVs isolated from the urine of rats exposed to ionizing radiation 1-, 14-, 30- or 90-days post-exposure, and human urine from patients receiving thoracic radiotherapy for the treatment of lung cancer pre- and post-treatment.
Size-exclusion chromatography is the preferred method for EV isolation from 0.5 mL of urine. Mass spectrometry-based metabolomic analyses of EV cargo identified biochemical changes induced by radiation, including altered nucleotide, folate, and lipid metabolism. The researchers have provided standard operating procedures for implementation of these methods in other laboratories.
Extracellular vesicle (EV) isolation method comparison workflow
EVs were isolated from two different initial volumes of urine (0.5 mL or 1 mL) from rats exposed to either 0 Gy (sham) or 13 Gy, X-irradiation, using 3 independent methods; ultracentrifugation with filtration (UC), size-exclusion chromatography (SEC) or a proprietary magnetic bead-based method (MBB). EV isolates were then characterized using cryogenic electron microscopy, nanoparticle tracking analysis and immunoblot array. Finally, the biochemical content of EVs was evaluated using untargeted quadrupole time of flight (QToF) mass spectrometry for both overall detection signals and potential contaminants
These researchers demonstrate that EVs can be isolated from small volumes of urine and analytically investigated for their biochemical contents to detect radiation induced metabolomic changes. These findings lay a groundwork for future development of methods to monitor response to radiotherapy and can be extended to an array of molecular phenotyping studies aimed at characterizing EV cargo.