Nitric oxide (NO) is a signaling molecule that regulates blood pressure and vascular tone. Humans produce NO by endothelial nitric oxide synthase (eNOS), which is impaired in patients with cardiovascular disease leading to increased blood pressure, endothelial dysfunction, and an increased risk of adverse cardiovascular outcomes. Maintaining optimal levels of NO is critical for human health. Inorganic nitrate (NO3-) is reduced to NO and can be an alternate pathway to restore NO production. Diets high in nitrate reduce blood pressure and improve exercise performance in humans. In patients with advanced cardiovascular disease, organic nitrates such as nitroglycerin (glyceryl trinitrate, GTN) release NO and are essential for managing symptoms. This dissertation emphasizes mass spectrometry techniques to elucidate novel mechanisms for the effects of both organic and inorganic nitrate on cardiovascular health and exercise performance.Pharmaceutical Nitrate. Patients develop tolerance to GTN after several weeks of continuous use, limiting the potential for long-term therapy. The cause of nitrate tolerance is relatively unknown. I developed a cell culture model of nitrate tolerance that utilizes stable isotopes to measure metabolism of 15N3-GTN into 15N-nitrite. I performed global metabolomics to identify the mechanism of GTN-induced nitrate tolerance and to elucidate the protective role of vitamin C. Metabolomics demonstrated that GTN impaired purine metabolism and depleted intracellular ATP and GTP. GTN inactivated the enzyme xanthine oxidase (XO), an enzyme that is critical for the metabolism of GTN into NO. Vitamin C prevented inactivation of XO, resulting in increased NO production from GTN. Vitamin C supplementation should be further investigated as a simple and inexpensive strategy to prevent nitrate tolerance.Dietary Nitrate. Inorganic nitrate improves exercise performance by reducing the oxygen cost of exercise. Although previous research has suggested that nitrate improves mitochondrial efficiency and stimulates mitochondrial biogenesis, we have an incomplete understanding of the mechanism. In a zebrafish (Danio rerio) model, nitrate reduced the oxygen cost of exercise during a vigorous 2-hour exercise test. Although there were no significant differences in mitochondrial function between control and nitrate-treated zebrafish, nitrate treatment increased resting ADP, ATP, and cyclic AMP levels. Metabolomics analysis of whole fish illustrated that nitrate stimulated glycolysis and ketogenesis. I conclude that nitrate reduces the oxygen cost of exercise by favoring glycolysis for energy production, thereby producing more ATP while consuming less oxygen. Nitrate-stimulated alteration of metabolic fuels for energy production is a novel mechanism for the improvement in exercise performance.Summary. This dissertation is a significant contribution to scientific knowledge because of the development of a novel assay to measure 15N-labeled nitrate and nitrite, which I have applied to study the metabolism of pharmaceutical and dietary nitrates. Using metabolomics, I elucidated the novel mechanism that vitamin C prevents nitrate tolerance by protecting XO from inactivation. Furthermore, I demonstrated that nitrate alters the utilization of metabolic fuels, leading to reduced oxygen consumption during exercise.
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