Relationships Between Nitric Oxide Congeners in Human Biological Samples and Physiological Responses to Dietary Nitrate Supplementation

Abstract

Plasma nitrite concentration (plasma [NO2-]) is the most oft-used biomarker of nitric oxide (NO) bioavailability, representing both the activity of the nitric oxide synthase (NOS) pathway and the potential for NO production via the nitrate (NO3-) reduction pathway. However, the correlations between increased plasma [NO2-] and changes in physiological responses following dietary NO3- ingestion have been inconsistent in the literature, suggesting that [NO2-] alone, measured in a single blood compartment, may not fully reflect changes in NO bioavailability. In addition to NO2-, S-nitrosothiols (RSNOs) serve as a storage form of NO due to its long half-life in biological samples and chemical stability. Recent research also claim that, compared to blood, skeletal muscle plays a more central role in NO3- production, storage, and metabolism. Therefore, it remains unknown whether other NO congeners, such as S-nitrosothiols, in different biological samples, may be more effective in mediating the physiological responses to dietary NO3- supplementation, such as reductions in blood pressure (BP), effects on mitochondrial respiration, and improvements in muscle contractility. The purpose of this thesis was to investigate the relationships between various NO congeners across different blood compartments and in skeletal muscle and the physiological responses to NO3- supplementation. Based on the identification of the most effective NO congener, NO3- supplementation regimens were developed with a focus on optimising duration and dose. Healthy individuals participated in the investigations presented in this thesis. Significant correlations were observed between peak increases in red blood cell (RBC) [RSNOs], rather than plasma [NO2-], and corresponding reductions in systolic BP (SBP) (rs=-0.68, P<0.01), diastolic BP (DBP) (rs=-0.59, P<0.01) and mean arterial pressure (MAP) (rs=-0.64, P<0.01) at 1, 2, 3, 4 and 24 h post-bolus ingestion of 12.8 mmol NO3- in Experimental Chapter 1. Similarly, in Experimental Chapter 2, increased whole blood (WB) [RSNOs], rather than plasma [NO2-], was significantly correlated with decreased DBP (r=-0.68, P=0.02) at 2.5 h post-ingestion of 12.8 mmol NO3-. Two weeks of NO3- supplementation (12.8 mmol NO3-.day-1) did not result in greater increases in [RSNOs] in WB and RBC or larger reductions in resting BP compared to a single bolus ingestion of NO3-. While reductions in BP and increases in blood [RSNOs] were not influenced by supplementation duration, BP reductions occurred only following ingestion of 19.2 mmol NO3-, and not at lower doses (6.4 mmol, and 12.8 mmol NO3-). Additionally, RBC and WB [RSNOs] peaked after 19.2 mmol NO3- ingestion in Experimental Chapter 4. Mitochondrial respiration function and efficiency, as well as time to exhaustion during high-intensity exercise, were not improved after either acute or two weeks of NO3- supplementation, and changes in mitochondrial respiration did not correlate with changes in muscle [NO3-] or [NO2-]. Different concentrations of sodium nitrite (NaNO2) (0 µM, 1.5 µM and 3.0 µM) were administered to permeabilized muscle fibres to exam the dose-response effects on mitochondrial respiration in Experimental Chapter 3. A significant increase in the leak control ratio, indicating reduced mitochondrial respiration efficiency, was observed following administration of the highest dose (3.0 µM) of NaNO2. In summary, this thesis found no beneficial effects of NO3-/NO2- supplementation on mitochondrial respiration function and efficiency in permeabilized muscle fibres in young, healthy adults. Experimental Chapter 2 also revealed significant correlations between changes in RBC [RSNOs], rather than plasma [NO2-], and changes in the absolute rate of muscle torque development (RTD) at 0-30 ms, 30-50 ms, and 0-50 ms (rs=0.62-0.63, P=0.04) following bolus NO3- ingestion, whereas increased skeletal muscle [NO3-] was correlated with enhanced RTD at 0-30 ms (r= 0.64, P=0.03) after 2 weeks of NO3- supplementation. Similarly, although two weeks of NO3- supplementation did not yield further increases in muscle [NO3-] compared to acute ingestion, muscle [NO3-] increased in a dose-dependent manner up to 19.2 mmol NO3-, as shown in Experimental Chapter 4. A novel finding of this thesis is that NO3- ingestion affects muscle contractile torque and velocity in opposite ways, depending on the dose: higher doses (12.8 mmol and 19.2 mmol NO3-) were required to enhance contractile torque (increasing by 6-7%), whereas a lower dose (6.4 mmol NO3-) was sufficient to improve contractile velocity (increasing by ~18%). Notably, significant correlations were found between changes in RBC [RSNOs] and changes in absolute RTD at 0-50 ms (rs=-0.70, P=0.02) and 0-100 ms (rs=-0.84, P<0.01) following the ingestion of 6.4 mmol NO3-. The findings of this thesis indicate that [RSNOs] in blood, as well as [NO3-] in skeletal muscle, may be more relevant NO congeners than plasma [NO2-] in reflecting, and perhaps mediating, the lowering of BP and the enhancements in muscle contractile function following NO3- supplementation. No beneficial effects of dietary NO3- supplementation on mitochondrial respiration were found, but positive and dose-dependent effects on muscle contractile torque and velocity were reported. This thesis also highlights that NO3- supplementation dose, rather than duration, plays a more critical role in regulating NO metabolism and its associated physiological responses in healthy adults.