Background Dietary nitrate improves exercise performance by reducing the oxygen cost of exercise, although the mechanisms responsible are not fully understood. Objectives We tested the hypothesis that nitrate and nitrite treatment would lower the oxygen cost of exercise by improving mitochondrial function and stimulating changes in the availability of metabolic fuels for energy production. Methods We treated 9-mo-old zebrafish with nitrate (sodium nitrate, 606.9 mg/L), nitrite (sodium nitrite, 19.5 mg/L), or control (no treatment) water for 21 d. We measured oxygen consumption during a 2-h, strenuous exercise test; assessed the respiration of skeletal muscle mitochondria; and performed untargeted metabolomics on treated fish, with and without exercise. Results Nitrate and nitrite treatment increased blood nitrate and nitrite levels. Nitrate treatment significantly lowered the oxygen cost of exercise, as compared with pretreatment values. In contrast, nitrite treatment significantly increased oxygen consumption with exercise. Nitrate and nitrite treatments did not change mitochondrial function measured ex vivo, but significantly increased the abundances of ATP, ADP, lactate, glycolytic intermediates (e.g., fructose 1,6-bisphosphate), tricarboxylic acid (TCA) cycle intermediates (e.g., succinate), and ketone bodies (e.g., β-hydroxybutyrate) by 1.8- to 3.8-fold, relative to controls. Exercise significantly depleted glycolytic and TCA intermediates in nitrate- and nitrite-treated fish, as compared with their rested counterparts, while exercise did not change, or increased, these metabolites in control fish. There was a significant net depletion of fatty acids, acyl carnitines, and ketone bodies in exercised, nitrite-treated fish (2- to 4-fold), while exercise increased net fatty acids and acyl carnitines in nitrate-treated fish (1.5- to 12-fold), relative to their treated and rested counterparts. Conclusions Nitrate and nitrite treatment increased the availability of metabolic fuels (ATP, glycolytic and TCA intermediates, lactate, and ketone bodies) in rested zebrafish. Nitrate treatment may improve exercise performance, in part, by stimulating the preferential use of fuels that require less oxygen for energy production.

The nitric oxide (NO) metabolites nitrite (NO2−) and nitrate (NO3−) can be quantified as an endpoint of endothelial function. We developed a LC-MS/MS method of measuring nitrite and nitrate isotopologues, which has a lower limit of quantification (LLOQ) of 1 nM. This method allows for isotopic labeling to differentiate newly formed nitrite and nitrate from nanomolar to micromolar background levels of nitrite and nitrate in biological matrices. This method utilizes 2,3-diaminonaphthalene (DAN) derivatization, which reacts with nitrite under acidic conditions to produce 2,3-naphthotriazole (NAT). NAT was chromatographically separated on a Shimadzu LC System with an Agilent Extend-C18 5 μm 2.1 × 150 mm column and detected using a multiple reaction monitoring (MRM) method on an ABSciex 3200 QTRAP mass spectrometer operated in positive mode. Mass spectrometry allows for the quantification of 14N-NAT (m/z 170.1) and 15N-NAT (m/z 171.1). Both nitrite and nitrate demonstrated a linear detector response (1 nM – 10 μM, 1 nM – 100 nM, respectively), and were unaffected by common interferences (Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), phenol red, and NADPH). This method requires minimal sample preparation, making it ideal for most biological applications. We applied this method to develop a cell culture model to study the development of nitrate tolerance in human endothelial cells (EA.hy926).

Dietary nitrate lowers blood pressure and improves athletic performance in humans, yet data supporting observations that it may increase cerebral blood flow and improve cognitive performance are mixed. We tested the hypothesis that nitrate and nitrite treatment would improve indicators of learning and cognitive performance in a zebrafish (Danio rerio) model. We utilized targeted and untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to examine the extent to which treatment resulted in changes in nitrate or nitrite concentrations in the brain and altered the brain metabolome. Fish were exposed to sodium nitrate (606.9 mg/L), sodium nitrite (19.5 mg/L), or control water for 2–4 weeks and free swim, startle response, and shuttle box assays were performed. Nitrate and nitrite treatment did not change fish weight, length, predator avoidance, or distance and velocity traveled in an unstressed environment. Nitrate- and nitrite-treated fish initially experienced more negative reinforcement and increased time to decision in the shuttle box assay, which is consistent with a decrease in associative learning or executive function however, over multiple trials, all treatment groups demonstrated behaviors associated with learning. Nitrate and nitrite treatment was associated with mild anxiogenic-like behavior but did not alter epinephrine, norepinephrine or dopamine levels. Targeted metabolomics analysis revealed no significant increase in brain nitrate or nitrite concentrations with treatment. Untargeted metabolomics analysis found 47 metabolites whose abundance was significantly altered in the brain with nitrate and nitrite treatment. Overall, the depletion in brain metabolites is plausibly associated with the regulation of neuronal activity including statistically significant reductions in the inhibitory neurotransmitter γ-aminobutyric acid (GABA; 18–19%), and its precursor, glutamine (17–22%). Nitrate treatment caused significant depletion in the brain concentration of fatty acids including linoleic acid (LA) by 50% and arachidonic acid (ARA) by 80%; nitrite treatment caused depletion of LA by ~90% and ARA by 60%, change which could alter the function of dopaminergic neurons and affect behavior. Nitrate and nitrite treatment did not adversely affect multiple parameters of zebrafish health. It is plausible that indirect NO-mediated mechanisms may be responsible for the nitrate and nitrite-mediated effects on the brain metabolome and behavior in zebrafish.

Glyceryl trinitrate (GTN) has found widespread use for the treatment of angina pectoris, a pathological condition manifested by chest pain resulting from insufficient blood supply to the heart. Metabolic conversion of GTN, a nitric oxide (NO) pro-drug, into NO induces vasodilation and improves blood flow. Patients develop tolerance to GTN after several weeks of continuous use, limiting the potential for long-term therapy. The mechanistic cause of nitrate tolerance is relatively unknown. We developed a cell culture model of nitrate tolerance that utilizes stable isotopes to measure metabolism of 15N3-GTN into 15N-nitrite. We performed global metabolomics to identify the mechanism of GTN-induced nitrate tolerance and to elucidate the protective role of vitamin C (ascorbic acid). Metabolomics analyses revealed that GTN impaired purine metabolism and depleted intracellular ATP and GTP. GTN inactivated xanthine oxidase (XO), an enzyme that is critical for the metabolic bioactivation of GTN into NO. Ascorbic acid prevented inactivation of XO, resulting in increased NO production from GTN. Our studies suggest that ascorbic acid has the ability to prevent nitrate tolerance by protecting XO, but not aldehyde dehydrogenase (another GTN bioactivating enzyme), from GTN-induced inactivation. Our findings provide a mechanistic explanation for the previously observed beneficial effects of ascorbic acid in nitrate therapy.

Humans concentrate nitrate from dietary or endogenous sources in the salivary glands, which is then reduced to nitrite, swallowed, and absorbed. Circulating nitrite acts as a reservoir for nitric oxide (NO) with its reduction to NO potentiated in acidic or hypoxic areas, such as contracting skeletal muscle. NO is an important signaling molecule with a short half‐life that regulates cardiovascular function, cellular energetics, and neurotransmission among other things. In humans, consumption of supplemental nitrate reduces blood pressure and decreases the oxygen cost of exercise, but we do not have a complete understanding of how dietary nitrate or nitrite is metabolized particularly in the presence of exercise. We used a stable isotope‐assisted metabolomics approach to assess nitrate and nitrite metabolism with and without exercise, and tested the hypothesis that nitrate and nitrite exposure reduces the cost of exercise and improves cognitive function in a zebrafish model.Adult zebrafish were exposed to either sodium nitrate (606.9 mg/L), sodium nitrite (19.5 mg/L), or control water for 21 days (n=60–66). A subset of the fish were switched to 100% 15N‐nitrite or 15N‐nitrate for the final 3 days of the exposure. Tissue nitrite and nitrate were quantified using diaminonaphthalene (DAN) derivatization and subsequently analyzed on a 3200 ABSciex QTRAP. Nitrite and nitrate exposure increased tissue nitrite (230% and 340%, respectively) and tissue nitrate (150% and 250%, respectively), and significant increases in nitrate and nitrite in blood were also observed. Nitrate exposure significantly decreased oxygen consumption during exercise in the AutoResp exercise assay. Nitrite treatment increased oxygen consumption with exercise but pathological examination indicated nitrite exposure caused mild gill damage. Metabolomics showed that up to 90% of the tissue nitrite and nitrate can be derived from exogenous sources. The data supports existing data that a feedback mechanism that inhibits endogenous NO production when high levels of nitrite or nitrate are consumed. Nitrite was depleted during exercise in all treatment groups, indicating potential increased utilization of nitrite as a substrate for NO production in hypoxia.In a free swim test nitrate and nitrite treatment significantly increased the duration of time spent in the bottom of the tank which is a possible indicator of anxiety. Nitrate treated fish also had a significant decline in movement following a startle but this was not detected in nitrite treated fish. In a negative conditioning‐learning assay nitrate treatment significantly impaired decision making time, and caused a 3‐fold increase in the period of time the fish experienced a mild shock. Nitrite treated caused a 2‐fold increase in the period of shock at the beginning of the assay, but the fish were able to learn with time. Taken together the behavioral assays demonstrate that both nitrate and nitrate treatments altered cognitive function in zebrafish and metabolomics on brains is being undertaken to understand the mechanisms contributing to these phenotypes.Support or Funding InformationOregon Agricultural Experiment Station, Endowment for the Celia Strickland and G. Kenneth Austin III Endowed Professor of Public Health