| Summary: | This dissertation demonstrates the widespread application of mass spectrometry to the qualitative and quantitative analysis of small molecules. Specifically, this manuscript describes the implementation of GC-MS techniques towards the elucidation of novel metabolic pathways of vitamin D and the development and validation of an LC-MS/MS analytical assay for the quantification of DNA adducts. Chapter 1 provides an introduction to mass spectrometry with an overview of the major
methodologies utilized to address our research goals. Chapter 2 highlights the superseding role of mass spectrometry in the structural characterization and quantification of vitamin D, its metabolites and other emerging analogs. After a review of the vitamin D biochemistry and the development of synthetic analogs, an overview of the current techniques for the detection and characterization of vitamin D compounds is given, with specific emphasis on the contribution made by mass
spectrometry. Chapter 3 describes the elucidation of a novel metabolic pathway of vitamin D and its effect on further metabolism of the hormone. Specifically, we demonstrated that vitamin D can be metabolized into its C-3 epimer by inversion of stereochemistry around C-3 of the A-ring. In the second part of Chapter 3, we investigated the effect of the C-3 epimerization conversion on the metabolism of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), biologically active form of vitamin D3.
Through a comparative study between 1α,25(OH)2D3 and its C-3 epimer, we confirmed that both compounds undergo the same C-24 oxidation pathway, a well described metabolic pathway leading to the catabolic inactivation of vitamin D compounds. However, the C-3 epimerization was found to slow down and partially block the metabolism of 1α,25(OH)2-3epi-D3 through the C-24 oxidation pathway, resulting in the accumulation of stable intermediary metabolites. These observations provided further
evidence for the metabolic stability of 1α,25(OH)2-3epi-D3, proposed as one of the possible mechanisms responsible for its unique biological actions. Chapter 4 focuses on the metabolism of 20-epi-vitamin D analogs and reports the elucidation of a novel pathway, namely C-1 esterification with fatty acids. Over the past decade, 20-epi analogs, in which the methyl group at C-20 is in its unnatural orientation, have been of particular interest because they have been shown to decrease cell
proliferation and promote cell differentiation with a potency significantly greater than 1α,25(OH)2D3. In order to understand the mechanisms responsible for this enhanced potency, we investigated the metabolism of such analogs. Selected 20-epi-vitamin D analogs were reported to be metabolized via C-1 esterification with fatty acids. HPLC, GC-MS, ESI-MS and 1H-NMR were used in a complementary fashion in order to elucidate this novel metabolic pathway. Chapter 5 describes the development
and validation of a highly sensitive LC-MS/MS assay for the quantification of DNA adducts derived from benzo[a]pyrene diol epoxide (B[a]PDE), a carcinogenic and mutagenic metabolite of benzo[a]pyrene (B[a]P). A brief introduction to DNA adducts is provided, with specific focus on polycyclic aromatic hydrocarbons (PAHs). The synthesis and characterization of B[a]PDE-deoxyguanosine (B[a]PDE-dG) reference standards and B[a]PDE-[15N5]-dG internal standard constituted the first step in the
method development and is described in detail. The development and validation of the LC-MS/MS quantitative method is then reported. In the last section of Chapter 5, the validated LC-MS/MS assay is applied to the quantification of B[a]PDEdeoxyguanosine adducts formed in human lymphoblastoid TK6 cells treated with B[a]PDE. We investigated the relationship between DNA adduct formation, toxicity, and gene expression and observed a positive dose-response correlation, providing further
evidence for the potential involvement of DNA adducts in carcinogenesis. Chapter 6 presents suggested future directions in the areas of vitamin D and DNA adducts research based on the results presented in this dissertation.
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