Liquid Chromatography-Mass Spectrometry Analysis of Dysfunctional Mitochondrial Metabolism: Insights into Rotenone Toxicity and Friedreichâ's Ataxia

Mitochondrial dysfunction plays a role in a wide range of diseases resulting in an enormous public health burden. The goal of this thesis is to identify metabolic pathways that are disrupted in response to mitochondrial insults. A large proportion of this work is based on the generation of stable isotope labelled metabolites to allow for the rigorous quantification of intracellular metabolites by liquid chromatographymass spectrometry. Once developed, this methodology was employed in cell culture models initially to characterize an unidentified acyl-CoA thioester induced by propionate metabolism. This novel pathway was identified as the direct formation of 2-methyl-2-pentenoyl-CoA, and using isotopic labeling by metabolic precursors served as a critical component to this pathway elucidation. These same techniques were then applied to studying rotenone, a mitochondrial complex I inhibitor associated with Parkinsonâ??s disease. Previous work by our group has shown that rotenone inhibits components of glucose metabolism. As demonstrated in this thesis, lipid oxidation and glutamine anaplerosis serve as important compensatory mechanisms in this setting. Furthermore, chiral analysis of 2-hydroxyglutarate, a metabolite linked to glutamine metabolism, revealed stereospecific alterations in response to rotenone. These previously unknown metabolic adaptations induced by rotenone may contribute to neurological phenotypes resulting from diminished complex I activity. Finally, a collaborative effort was initiated to study metabolic defects in Friedreichâ??s ataxia, a genetic disease suspected to occur, in part, due to deficiencies in mitochondrial complex I. Utilizing isolated platelets in combination with isotopic labeling it was shown that Friedreichâ??s ataxia patients exhibit diminished glucose metabolism with a concomitant increase in lipid oxidation. Taken together these findings suggest adaptations to glucose and lipid metabolism are metabolic characteristics resulting from disrupted mitochondrial function across multiple models, and description of these disruptions gives insight into basic metabolic biotransformation, toxicology, and etiology of poorly understood diseases. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Pharmacology First Advisor Ian A. Blair