Date of Award

2023

Document Type

Thesis

Publisher

Edith Cowan University

Degree Name

Master of Science by Research

School

School of Science

First Supervisor

Hayley Abbiss

Second Supervisor

David Broadhurst

Abstract

This study included a series of experiments focussed on comparing extraction protocols for metabolomics-based analysis of Lewis rat polycystic kidney (LPK) tissue samples and healthy Lewis rat tissue samples. Firstly, the optimal sample loading (mass of tissue) was investigated. Based on previous studies, the appropriate mass to test was approximately 5 mg. Three different volumes of tissue extract equivalent to 1, 2.5 and 5mg of pooled healthy kidney tissue and pooled polycystic kidney tissue were trialled to see how they differed in terms of the number of reproducible, uniquely identified peaks captured after metabolite extraction, derivatisation and gas chromatography-mass spectrometry analysis. Of the masses tested, the 2.5 mg samples gave the greatest number of reproducible, uniquely identified peaks and showed good internal standard reproducibility. Following this, different variables were changed to further investigate the optimal metabolite extraction conditions. Five different methods were compared including different solvent combinations and number of extraction steps used in the extraction procedure. After trialling these different methods, it was found that a single step method using pure methanol yielded more reproducible peaks than the other methods tested. Using the optimised tissue mass and extraction method, 240 metabolites were found to be significantly altered (False discovery rate (FDR) corrected p-value < 0.05) in the polycystic kidney tissue (LPK; n=10) compared to the healthy control (Lewis; n=11). These findings will further the current understanding of polycystic kidney disease pathophysiology.

DOI

10.25958/t86j-ap81

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