From derivatization to distribution : developing methods for vitamin D analysis and preliminary insights into an overlooked HDL transport
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Vitamin D is essential for numerous physiological processes, yet its transport remains incompletely understood. The classical model proposes chylomicronmediated delivery; however, current literature and this dissertation explores an overlooked high-density lipoprotein (HDL)-mediated transport pathway, given vitamin D's structural similarity to cholesterol. This research aimed to develop analytical methods to detect, and where possible quantify, cholecalciferol (D3) and its hydroxylated metabolites in human plasma and lipoprotein samples. Methodological development began by qualitatively confirming D3's derivatization with DMEQ-TAD for UV detection. While successful, this initial work showed insufficient sensitivity for physiological quantification, leading to a sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The platform integrated refined extraction protocols and optimized DMEQ-TAD derivatization, demonstrating excellent sensitivity with low limits of quantification for comprehensive vitamin D metabolite profiling. This study involved human subjects who consumed a controlled meal with a D3 dietary supplement, followed by serial blood draws for plasma analysis. Applying developed methodology, we investigated D3 and its hydroxylated metabolites within human plasma lipoprotein fractions separated via sequential ultracentrifugation. Preliminary time-course data revealed D3 increasingly detected in HDL. These findings provide preliminary, yet compelling, evidence challenging the sole reliance on the classical chylomicron pathway for Vitamin D transport, suggesting an overlooked role for HDL as an inter-organ carrier.
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Ph. D