Date of Award

2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Pharmacology

First Advisor

Ian A. Blair

Abstract

Coenzyme A (CoA) and CoA thioesters are central to numerous metabolic pathways. However, the inability to synthesize isotopically labeled CoA has limited analytical methods to measure these compounds. In this thesis, we developed Stable Isotope Labeling with Essential nutrients in Cell culture (SILEC), a method to generate isotopically labeled CoA derivatives by growing cells in a labeled CoA precursor, [13C15N]-pantothenate. Labeled CoA derivatives were used as internal standards in a stable isotope dilution liquid chromatography-mass spectrometry (LC-MS) method to measure changes in short chain acyl-CoA species. This assay was also adapted to include measurement of glutathione-CoA (CoASSG), a mixed disulfide increased in mitochondrial oxidative stress. Menadione significantly increased intracellular levels of CoASSG, and decreased levels of CoASH through the formation of CoA-menadione adduct. In addition, rotenone, an organic pesticide and potent complex I inhibitor, induced a dose-dependent decrease in succinyl-CoA and increase in beta-hydroxybutyryl-CoA (BHB-CoA) in multiple human cell lines, as well as inhibited glucose-derived acetyl-CoA and succinyl-CoA biosynthesis. This SILEC assay was further adapted for use in freshly isolated human platelets to assess metabolic changes in Friedreich's Ataxia (FA), an inherited mitochondrial disease caused by mutations in the frataxin gene. FA patients were found to have significantly decreased acetyl-CoA: succinyl-CoA ratio, consistent with an in vitro siRNA knockdown model of frataxin. Finally, platelets were demonstrated to serve as a powerful ex vivo metabolic and toxicologic challenge platform evidenced by treatment with propionate, rotenone and a number of stable isotope metabolic tracers. Together, these methods provide a paradigm for the discovery of novel biomarkers for metabolic and mitochondrial disease.

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