Penny J. Beuning, Philip W. LeQuesne, Patricia Ann Mabrouk, Satyanarayana Reddy
Date of Award
Doctor of Philosophy
Department or Academic Unit
College of Arts and Sciences. Department of Chemistry and Chemical Biology.
Chemistry, Mass spectrometry, DNA adducts, Vitamin D
Mass spectrometry, Vitamin D, DNA adducts
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.
Caroline Ceailles Flarakos
Flarakos, Caroline Ceailles, "Applications of mass spectrometry techniques to the elucidation of novel metabolic pathways of Vitamin D and the quantification of DNA adducts" (2008). Chemistry Dissertations. Paper 1. http://hdl.handle.net/2047/d10016673
Click button above to open, or right-click to save.