The Fecal Metabolome of Irritable Bowel Syndrome

Abstract

Irritable bowel syndrome (IBS) is increasingly common in Canada, effecting upwards of 18% of the population. The cause of functional gut disorders is not well understood, and new tools are urgently needed to help understand these complex chronic diseases and more accurately diagnose patients. Comprehensive metabolite profiling is a promising strategy to derive new insights into microbiome activity, but a clear set of guidelines for the handling and storage of human fecal specimens has yet to be thoroughly developed. The objective of this thesis is to create standardized sample handling procedures to enable reliable untargeted metabolite analysis of stool samples from IBS patients for biomarker discovery and differential diagnosis. Our results indicated that lyophilization prior to sample extraction not only increased the extraction efficiency on average by 28.5% compared to crude extraction, but also provided good long-term stability with less then 50% of metabolites showing altered responses after long-term storage while frozen up to 21 weeks. Additionally, lyophilization increased study repeatability, by simplifying the weighing and extraction process, reducing variability due to inconsistent stool water content as metabolite concentrations can be normalized to dried weight. This approach was subsequently applied in a pilot metabolomics study involving a cohort of IBS patients (n = 60) and healthy non-IBS controls (n = 20), where lyophilized stool extracts were analyzed by multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS) with stringent quality control. The study included a differential stool metabolome analysis of diarrhoea and constipation predominate IBS subgroups (IBS-D; IBS-C), while also classifying IBS patients during contrasting periods of active or dormant symptoms based on their self-reported symptom severity and Bristol stool scale scores. Untargeted and targeted metabolite profiling of stool extracts by MSI-CE-MS under full-scam data acquisition in positive and negative ion modes revealed several promising biomarkers unique to IBS subtypes and symptomology, while also identifying novel metabolic signatures underlying IBS pathophysiology. Stool metabolomic studies aim to better decipher the underlying mechanisms of debilitating digestive disorders having complex aetiologies, which may also improve diagnostic testing and therapeutic treatments optimal for individual patients.