Elucidating urinary biomarkers of exercise and bone metabolism by 1H-NMR spectroscopy and LC-MS based metabolomics

© 2012 Dr. John Robert SheedyThe resurgence in the last decade of the application of computer-assisted spectroscopic and spectrometric based chemical analysis of biological samples has spurred the evolution of the metabolomics research field. Techniques such as nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) have been applied for chemical analysis of biological samples for several decades, but recent technological advances of these analytical platforms have allowed for a more comprehensive analysis of chemically complex samples. The metabolomics research field has been previously limited by computer processing power. Nowadays, the data processing speed of modern day computers equipped with user-friendly statistical software packages has allowed for more rapid processing of the plethora of biological information that is obtained from NMR and MS analytical techniques. Organisations such as the Metabolomics Standards Initiative are working to streamline metabolomics-based methodology by international research groups, to formulate minimum reporting standards of metabolomics results, and provide transparency and accuracy in research reporting for metabolomics-style research projects. Cohesiveness in reporting of results is necessary to establish a robust peer-review process so that novel discoveries made using a metabolomics research approach can be validated and accepted by the wider scientific and medical community. The implications of peer-reviewed and accepted discoveries through metabolomics will provide new means for diagnosis of disease based on quantification of novel biomarkers, evaluating environmental effects of toxicity in many organisms, improve quality control procedures in industry, and provide a deeper level of understanding of general biology through integration of metabolomics into the central dogma of systems biology. This thesis firstly provides a workflow for novel biomarker discovery through the analysis of human urine samples, where 1H-NMR and liquid chromatography (LC)-MS have both been evaluated for their efficacy in holistic profiling of urinary metabolites applied to altered metabolic states, for the identification of biomarkers related to a detectable chemical change in the urinary metabolome. Secondly, this workflow has been specifically applied to the analysis of urine due to the effect of aerobic and anaerobic exercise and associated bone turnover (Study 1 and Study 2 respectively), to identify biomarkers as indicators of skeleto-muscular turnover due to the effect of exercise. Biomarkers of aerobic exercise were putatively identified through the analysis of pre-exercise and post-exercise urine samples from participants in the 2008 Hawaiian Ironman Competition (Study 1), and biomarkers of anaerobic exercise were identified in individuals that underwent an 18-month intervention of progressive intensity resistance exercise (Study 2). However due to the nature of the 1H-NMR and LC-MS analytical techniques used in this thesis and the sample types available for analysis, novel urinary biomarkers of bone turnover were not able to be confidently identified. Study 1 provided a validation of the proposed metabolomics workflow for biomarker discovery through a current, accepted understanding of aerobic exercise physiology, and putatively identified N,N-dimethylglycine as a potential urinary biomarker for aerobic exercise. N,N-dimethylglycine is a poorly researched metabolite in the field of aerobic exercise physiology, but some published literature suggests that N,N-dimethylglycine dietary supplementation may prevent aerobic exercise-induced lactic acidosis. Study 2 identified creatinine, choline, guanidinoacetate and hypoxanthine as biomarkers of progressive resistance (anaerobic) exercise training, indicating that a long term resistance exercise training regime promotes a favourable adaptation for the utilisation of anaerobic ATP-generating metabolic pathways, notably in the creatine/phosphocreatine ATP synthesis pathway. Identification of these novel biomarkers of exercise may provide a platform for monitoring and assessing aerobic and anaerobic exercise regimes, and be suggestive of novel dietary supplements that can be used to enhance anaerobic and aerobic exercise performance