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The use of gas chromatography-mass spectrometry to measure the relative pool size and 13C-enrichment of a broad range of intracellular metabolites to monitor changes in the metabolome of mammalian cells is described. The typical workflow for metabolomics and 13C-tracer investigations is discussed and each step optimized to increase the breadth and intensity of detected metabolites, reproducibility of measured metabolite concentrations, sample throughput, and automation of mass isotopomer and multivariate statistical analyses of the data. The protocols developed and optimized in this dissertation allow for the combination of complementary metabolomic and pathway activity data to provide a more comprehensive analysis of the system's metabolic phenotype under the condition(s) being investigated. The utility of the presented protocols were then evaluated by studying the metabolic profiles of two separate cultured human cell lines under various microenvironmental conditions. The first demonstration of the utility of our protocols for combining gas chromatography-mass spectrometry-based metabolomics and 13C-glucose tracer studies was used to correlate shifts in the central carbon metabolism of human embryonic kidney 293 cells with gold nanoparticle exposure. Prior to our studies, little was known about the metabolic perturbations associated with gold nanoparticle uptake. We discovered that gold nanoparticles modulate lipid metabolism in human embryonic kidney 293 cells while having no apparent effect on the glycolytic flux into the pentose phosphate pathway or the tricarboxylic acid cycle. We discuss the significance of these findings and propose possible reasons for the observed metabolic changes. In the second application of our methodologies, we monitored glucose and mitochondrial metabolism of human embryonic stem cells under ambient and reduced oxygen availability. We demonstrated a decoupling of glycolysis and the TCA without any change in the rate of lactate synthesis under reduced oxygen tension. Our results also suggest that alanine and glutaminolysis potentially play significant roles in the adaptation of human embryonic stem cells to reduced oxygen availability. As in the first demonstration, we discuss the biological relevancy of these findings and propose reasons for the observed metabolic changes.
A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Includes bibliographical references.
Timothy Logan, Professor Directing Dissertation; P. Bryant Chase, University Representative; Brian Miller, Committee Member; Michael Roper, Committee Member.
Florida State University
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