PT  - JOURNAL ARTICLE
AU  - Charles P. Hinzman
AU  - Meth Jayatilake
AU  - Sunil Bansal
AU  - Brian L. Fish
AU  - Yaoxiang Li
AU  - Yubo Zhang
AU  - Shivani Bansal
AU  - Michael Girgis
AU  - Anton Iliuk
AU  - Xiao Xu
AU  - Jose A. Fernandez
AU  - John H. Griffin
AU  - Elizabeth A Ballew
AU  - Keith Unger
AU  - Marjan Boerma
AU  - Meetha Medhora
AU  - Amrita K. Cheema
TI  - An optimized method for the isolation of urinary extracellular vesicles for molecular phenotyping: detection of biomarkers for radiation exposure
AID  - 10.1101/2022.01.28.477909
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.01.28.477909
4099  - http://biorxiv.org/content/early/2022/01/28/2022.01.28.477909.short
4100  - http://biorxiv.org/content/early/2022/01/28/2022.01.28.477909.full
AB  - Background 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 analysis 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.Methods 3 EV isolation methods were compared: 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.Results 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. We have provided standard operating procedures for implementation of these methods in other laboratories.Conclusions We 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 to develop future methods to monitor response to radiotherapy and can be extended to an array of molecular phenotyping studies aimed at characterizing EV cargo. Competing Interest StatementThe authors have declared no competing interest.