MSACL 2019 EU Abstract

Solid Phase Microextraction (SPME) in Kidney Examination – LC-MS/MS-based Identification of Potentially Significant Metabolites in Graft Quality Assessment

Iga Stryjak(1), Natalia Warmuzińska(1), Kamil Łuczykowski(1), Matyas Hama(2), Markus Selzner(2,3), Barbara Bojko(1)
(1)Department of Pharmacodynamics and Molecular Pharmacology, Faculty of Pharmacy, Nicolaus Copernicus University in Toruń, Collegium Medicum in Bydgoszcz, Poland; (2)Multi Organ Transplant Program, Department of Surgery, Toronto General Hospital, University Health Network,Toronto Canada; (3)Department of Medicine, Toronto General Hospital Toronto, Toronto, Canada

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Iga Stryjak (Presenter) Nicolaus Copernicus University in Toruń >> POSTER (PDF)

Presenter Bio: In 2017, I completed my Master’s degree in pharmacy at Nicolaus Copernicus University in Toruń, Collegium Medicum in Bydgoszcz. My master thesis was entitled „In situ solid phase microextraction (SPME) in kidney examination – step towards a better diagnostic in transplantation”. During my studies I belong to the Mass Spectrometry and Microextraction Methods Student Scientific Association at the Department of Pharmacodynamics and Molecular Pharmacology and did several internships including a six-month traineeship at the pharmacy.

After graduation, I began PhD studies and started work in Dr. Barbara Bojko’s group at Faculty of Farmacy, Nicolaus Copernicus University in Toruń, Collegium Medicum in Bydgoszcz. My current research is the continuation of kidney metabolic profile analysis in the peritransplant period with direct in vivo and in situ SPME application and LC-MS(/MS) instrumental analysis. The main goal of the project is to monitor changes in kidney metabolomic profile at the time of preservation, correlate them with graft outcome and identify metabolites responsible for those changes as potential biomarkers in molecular diagnostics during transplantation. The study is carried out on human kidneys in the cooperation with Department of Transplantation and General Surgery in University Hospital No. 1 in Bydgoszcz and on experimental porcine model in the cooperation with Toronto General Hospital. Suggested method may be in the future considered as a low-invasive complementary solution or even an alternative for invasive biopsy.
In addition, as a part of my work, I am engaged in other projects of laboratory animals’ metabolomic tissue analyses with the use SPME-LC-MS platform.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

Introduction:Transplantation is the treatment associated with increased survival rate and greater quality of patient’s life when compared to conventional dialysis. Even nowadays transplantology suffers from the lack of reliable methods of organ quality assessment. The standard protocols are limited to macroscopic appearance inspection or invasive tissue biopsy which do not provide a comprehensive information about the graft. Kidney is the organ largely associated with metabolic processes, thus measurements of metabolites concentrations may permit determining potential organ quality biomarkers and predicting the graft outcome. Hence, there is a need for new diagnostic solution allowing on site graft monitoring and quick decision-making processes during the surgery.
The goal of the project is to identify metabolites associated with changes occurring in transplanted kidneys during preservation with the use of in vivo and in situ low-invasive solid phase microextraction followed by LC-MS/MS instrumental analysis.
Methods:The study was performed on kidneys harvested from two types of porcine model donors: heart beating donor (HBD) and donor after cardiac death (DCD). Sample collection was performed according to the SPME method with the use of probes coated with 7 mm mixed-mode extraction phase. Sampling was conducted directly from the graft tissue: in vivo before transplantation, in situ after 1h, 3h, 5h, 7h of perfusion, in vivo 3 and 7 days after revascularization in the recipient, and additionally for DCD after 45 min and 2h of warm ischemia time. The untargeted metabolomic analysis was done with the use of liquid chromatography (RPC, HILIC) coupled with high resolution mass spectrometry (Q-Exactive Focus) in both positive and negative ionization modes. The putative identification of detected metabolites was done by comparison of accurate masses with metabolomic databases. In order to confirm identities of metabolites selected as significant ones in organ quality assessment, their retention times and fragmentation pattern were compared with chromatograms and MS/MS spectra of authentic standards.
Results:Monitoring the metabolomic profiles of kidneys allowed to observe biochemical changes occurring in the organ during preservation as well as biochemical differences between HBD and DCD types of donor. Alterations are mostly related to concentrations of metabolites which might be the part of ischemia/reperfusion injury mechanism due to their involvment in i.e. oxidative stress, ischemia and energy metabolism pathways.
Conclusions:SPME is a low-invasive method for direct in vivo kidney extraction without removing any tissue from the graft which makes the method an alternative for biopsy. The small size probe and minimal invasiveness of the approach permits for repeatable samplings from the same organ. Identified metabolites could be considered for further analyses towards decreased organ quality or progressing graft dysfunction biomarker validation.