RT Journal Article
SR Electronic
T1 An optimized method for the isolation of urinary extracellular vesicles for molecular phenotyping: detection of biomarkers for radiation exposure
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.01.28.477909
DO 10.1101/2022.01.28.477909
A1 Charles P. Hinzman
A1 Meth Jayatilake
A1 Sunil Bansal
A1 Brian L. Fish
A1 Yaoxiang Li
A1 Yubo Zhang
A1 Shivani Bansal
A1 Michael Girgis
A1 Anton Iliuk
A1 Xiao Xu
A1 Jose A. Fernandez
A1 John H. Griffin
A1 Elizabeth A Ballew
A1 Keith Unger
A1 Marjan Boerma
A1 Meetha Medhora
A1 Amrita K. Cheema
YR 2022
UL http://biorxiv.org/content/early/2022/01/28/2022.01.28.477909.abstract
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.