Abstract/Summary

Epidemiological studies have demonstrated an inverse association between high vegetable consumption and lower blood pressure (BP), an important modifiable cardiovascular disease (CVD) risk marker. The high nitrate content of root (e.g. beetroot) and green leafy vegetables is a key dietary component related to the beneficial effects on vascular function and BP. However, the role of commensal oral bacteria which convert dietary nitrate to nitrite and potent vasodilator nitric oxide, in BP regulation and maintenance of vascular tone is unclear. A literature review (Chapter 2) was performed to determine the association between the oral microbiome and real time measures of vascular function. Elimination of oral bacteria with antiseptic mouthwash was reported to reduce conversion of inorganic and supplemental nitrate to nitrite and be associated (in some studies) with an increase in BP. This review highlighted the need for further research to focus on the effects of dietary nitrate on oral bacteria composition and CVD risk markers, and to determine underlying mechanisms. The relationship between dietary nitrate consumption from different sources (vegetable, processed meat and water) with BP and other CVD risk factors was investigated in the UK NDNS cross-sectional cohort (Chapter 3). This data analysis included 3407 adults aged 19-64 years old. A comprehensive database was developed to accurately estimate the nitrate and nitrite levels in vegetables, processed meats and drinking water. Across increasing quartiles of dietary nitrate intake from vegetables and drinking water, BP and glycated haemoglobin were significantly lower in Q3 (95-130 mg nitrate/day) than Q1 (3-65 mg nitrate/day). In addition, glucose and high sensitivity C-reactive protein concentrations were lower in Q4 compared to Q1. In contrast, there were no differences in CVD risk markers across quartiles of nitrate or nitrite from processed meats or dietary nitrite from vegetables and drinking water. Analysis of urinary nitrate and nitrite concentrations in n=1340 NDNS participants revealed a significant but weak correlation with total dietary nitrate intake (r=0.121, p<0001). In contrast to the dietary data, there were no significant difference in BP across increasing quartiles of urinary nitrate concentration which suggests that urinary nitrate may not be an ideal biomarker of intake (Chapter 4). To address the role of the oral microbiome in contributing to the beneficial effects of dietary nitrate on BP and vascular function, two human studies were performed. In REBOC1 (Chapter 5), a three-arm sequential pilot study in n=20 healthy adults, the salivary nitrite concentration was found to be significantly higher after rinsing the mouth with beetroot juice (1.09±0.34 µM) compared with the use of an antibacterial mouthwash (to eliminate oral bacteria) prior to mouth rinsing with beetroot juice (0.021±0.01 µM; p ˂ 0.001). Within the oral cavity, the posterior tongue was identified as the main site of nitrate reduction, with beetroot juice promoting an increase in the abundance of Neisseria. In a follow-up study incorporating a double-blind randomised cross-over design, the chronic effects of dietary nitrate intake on BP and other CVD risk markers were related to the changes in oral and gut microbiota compositions (Chapter 6). Consumption of nitrate-rich beetroot juice (3.7 mg/kg body weight) for 8 weeks reduced systolic and diastolic BP by 4.1 and 2.8 mmHg, respectively and increased endothelial independent microvascular reactivity by 4% compared with a control beetroot juice (n=19 healthy participants). These findings were associated with an increase in the nitrate-reducing capacity and abundance of Neisseria in the oral cavity and reduction in Clostridium in the gut microbiota after chronic dietary nitrate intake. Future research should address the impact of inorganic nitrate as a potential dietary strategy to improve cardiovascular health, particularly in those individuals with raised BP, and to determine the utility of urinary nitrate as a biomarker of intake.