Barbara Bojko (Presenter)
Nicolaus Copernicus University
Authorship: Iga Stryjak, Barbara Bojko
Department of Pharmacodynamics and Molecular Pharmacology, Faculty of Pharmacy, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun
In situ solid phase microextraction (SPME) was used for monitoring of organ function after 2, 4, 6 and 21 hours of kidney resection in rabbit model. The method offers minimum invasiveness of the procedure preventing tissue damage as well as broad coverage of small molecules, and fast and simple sampling/sample preparation protocol. Instrumental analysis was done on UPLC-Q-Exactive Focus system. The data showed significant changes in compounds associated with glutathione, amino acids and purine metabolism as a result of oxidative stress and ischemia.
Transplantation is life-saving procedure. However, despite of rapidly developing technologies there is still dramatic limitations of tools available for efficient evaluation of organs quality and function. The effective selection of grafts would enable to decrease number of rejections and increase the pool of high quality organs available for transplantation without waste of those which carry potential risk of rejection based on the visual inspection or basic-parameter assessment. In the previous work it was demonstrated that in situ solid phase microextraction (SPME) could serve as a “chemical biopsy tool” due to the low invasiveness and good coverage of low molecular weight compounds. In the current work the approach was used for monitoring of metabolic profile of kidneys in rabbit model from organ resection over the period of 21 hours storage.
The SPME probes with 7 mm length of mixed-mode coating were used for the experiment. Time of sampling/extraction was 30 min followed by desorption in 250 uL of acetonitrile:water 1:1 v/v. The extracts were analyzed on Q Exactive Focus in positive and negative ionization mode. Reversed phase chromatographic separation was performed using 3 um, 10 cm x 2.1 mm Discovery HS F5 (pentafluorophenylpropyl) column. The Compound Discoverer software v. 184.108.40.2063 was used for data processing and analysis. For the significance test the p-value of <0.05 and two-fold change were set.
The chemometric analysis showed separation of the data related to given time points. The alteration observed after two and four hours after kidneys resection were mainly associated with changes in the level of dipeptides involved in glutathione metabolism. Depletion of glutathione has been reported previously for liver preservation and reperfusion after liver transplantation as a result of oxidative stress. However, after sixth hour post-resection the most significant changes were observed in metabolism of purines and amino acids. This can be explained by progressing ischemia. Similar trend has been already reported for heart and skeletal muscles.
The obtained results showed that in situ SPME coupled to UPLC-HRMS platform could be successfully used for timecourse metabolomics studies of grafts by eliminating need for biopsy collection thus preventing organ damage. In the same time gained information reflects well status of the organ, and selected compounds after proper validation could be used as biomarkers of graft condition enabling physician making more confident and data-supported decision on its transplantation.
References & Acknowledgements:
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