Presentation description

Heart disease is the leading cause of premature death and disability in the US and responsible for 1 in 5 deaths. The pathophysiology of heart disease involves metabolic rewiring and oxidative stress, but gaps exist in the complete understanding of the exact mechanisms and metabolites required for metabolic changes. One such family of metabolites-NAD+ and similar cofactors-is of interest, as they are key regulators of cellular redox homeostasis and signaling molecules in transduction pathways activated in heart disease. However, quantification of the NAD cofactors in cell culture and tissue samples is challenging, owing to the reactivity and instability of their chemistry. This project sought to optimize and validate protocols for the quantitation and extraction of the NAD metabolome using liquid chromatography-mass spectroscopy, to support quantitative isotope-tracer studies to characterize NAD metabolic fluxes. Using NAD standards and mammalian HCT116 cells, extraction reagents were validated and a Z-HILIC liquid chromatography column was found to produce best results. Development of methods to quantify NAD metabolic fluxes began with deuterated serine and its folate-mediated catabolism to produce isotopically-labeled NADH for mass spectroscopy analysis. The findings of this project can be applied in future metabolomics studies in animal models and human tissue.