Dietary Nitrate, Vascular Function and Inflammation

This clinical research is funded by the Austrian Science Fund (FWF, project number: KLI 858). It is based on close collaborations between the University of Vienna and the Medical University of Vienna, with the Department of Clinical Pharmacology at the Medical University of Vienna as the clinical trial center and Prof. M.D. Michael Wolzt as the trial center's coordinating physician. The sponsor is the University of Vienna, as represented by the Principal Investigator Dr. Oliver Neubauer (Research Platform Active Ageing). International collaborations include Dr. Catherine Bondonno, Prof. Hodgson (both Edith Cowan University) Dr. Jonathan Peake (Queensland University of Technology), and Prof. Richard Woodman (Flinders University) (all Australia).

Wider research context / theoretical framework:

Aging is a major risk factor for cardiovascular disease (CVD). Developing effective strategies to improve cardiovascular health with ageing is a high priority. Emerging data indicate that inorganic dietary nitrate, found in green leafy vegetables and beetroot juice (BRJ), is important for cardiovascular health (Blekkenhorst et al. 2018). These data suggest that small increases in nitrate intake among those with elevated risk for CVD would have a substantial impact, by improving the bioavailability of nitric oxide (NO) through the enterosalivary nitrate- nitrite-NO pathway (involving the oral microbiome). Results from the study group's research suggest beneficial short-term effects of nitrate-rich BRJ on blood pressure and vascular inflammation markers in healthy older adults (Raubenheimer et al. 2017).

Hypotheses / research questions / objectives:

The proposed clinical study is aimed to examine whether the daily consumption of 2×70 mL BRJ over 4 weeks translates into improved clinical outcomes in older adults with treated grade 1 hypertension. The primary hypothesis is that the increased dietary nitrate intake lowers blood pressure and improves vascular function. The secondary hypotheses are that the increased dietary nitrate intake results in changes in the oral microbiome, favorably modulates the systemic inflammatory status, and that these changes correlate with cardiovascular-related outcomes.

Approach / methods:

This randomized, placebo-controlled crossover study will involve two 4-week treatment periods, consisting of interventions with nitrate-rich BRJ and nitrate-depleted BRJ, separated by a 4-week washout period. Thirty-five men and women, between the ages of 55 and 70 years, who have been diagnosed with grade 1 hypertension will be recruited. Twenty-nine participants will be required to complete the study, based on power/sample size calculations with the forearm blood flow (FBF) response to acetylcholine and 24 h-ambulatory systolic blood pressure as the primary endpoints. Principal secondary endpoints are the number of CD11b-expressing blood granulocytes and the FBF response to glyceryltrinitrate. Among various additional secondary and exploratory endpoints, plasma nitrate and nitrite and a broad range of inflammation biomarkers will be measured. Furthermore, the oral microbiome will be analyzed.

Level of originality / innovation:

Beneficial effects of nitrate have predominantly been observed in healthy populations, but evidence of cardiovascular benefits and anti-inflammatory effects in clinical populations is limited (Raubenheimer et al. 2019). This study addresses the need to determine the efficacy of dietary nitrate to improve cardiovascular health and counteract chronic low-grade inflammation in humans at risk of CVD.

Inclusion criteria

• Ambulant male and females between 55 and 75 years of age, inclusive
• Resting systolic blood pressure between 130 and 170 mmHg, inclusive
• Written informed consent

Exclusion criteria

• Diastolic blood pressure >110 mmHg
• Type 1 or 2 diabetes mellitus or fasting glucose >7.0 mmol/L
• Any evidence of acute or chronic disease such as symptomatic cardiovascular or peripheral vascular disease, moderate or severe chronic kidney disease (estimated glomerular filtration rate (GFR) <50 ml/min)), or cancer which is not in remission
• Consumption of ≥5 serves of vegetables per day
• Consumption of a diet estimated to contain >200 mg/d of nitrate
• Being vegan or vegetarian
• BMI ≥35 kg/m2 or <18.5 kg/m2
• Use of nitric oxide donors, organic nitrites/nitrates, sildenafil and related drugs, anti-coagulation, non-steroidal anti-inflammatory or statin-related drugs
• Use of antibacterial mouthwash (volunteers willing to cease using antibacterial mouth wash for a period of 4 weeks before randomisation will be included)
• Use of antibiotics (within previous 2 months)
• A change in drug therapy likely to influence blood pressure or major secondary outcomes within the previous month, or the likelihood that drug therapy would change during the study
• Current or recent (within previous 3 months) engagement in regular structured exercise training (more than 2 hours of moderate to more intense exercise per week)
• Current or recent (within previous 6 months) loss or gain of >6% of body weight
• Current or recent (<12 months) regular smoking of >5 cigarettes/day
• Alcohol intake >140 g per week for women or >210 g per week for men and/or binge drinking behaviour
• Inability or unwillingness to follow the study protocol
• Pregnancy or lactation