Effects of Daily Nitrate Consumption on Oral Bacteria Composition, Blood Pressure and Vascular Function

Study Overview
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Brief Summary
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Diseases of the heart and blood vessels, including raised blood pressure, are one of the leading causes of death worldwide. Higher intakes of dietary nitrate, found abundantly in root vegetables such as beetroot, have been shown to have health benefits including lowering blood pressure and improving the elasticity of blood vessels. Bacteria which reside in the mouth and those in the gastrointestinal tract play an important role in converting dietary nitrate to nitrite and nitric oxide (a chemical which promotes the relaxation of blood vessels). In particular, removal of oral bacteria by using antiseptic mouthwash is accompanied by an increase in blood pressure in subjects with normal blood pressure, even after consuming nitrate-rich foods. To date, very little is known about the role of these oral bacteria in the control of blood pressure, and if there are any differences in bacterial composition between individuals.

Detailed Description
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Interested volunteers will be provided with an outline of the study and asked to complete a medical and lifestyle questionnaire (in person, by email or over the phone). Potentially suitable participants will be identified and asked to attend a screening session after a 12 h overnight fast (not eating during this time and only drinking water) during which the study will be explained in more detail before a consent form is signed. Anthropometric measurements will then be taken including weight, height and blood pressure. A 9 ml blood sample will also be collected on site at the Department of Food and Nutritional Sciences (Hugh Sinclair Unit of Human Nutrition). Subjects who meet the initial inclusion criteria will be invited to a further screening session during which time a dentist will check for dental diseases (e.g. current dental cavities or periodontal infection) and saliva flow rate. Volunteers without periodontal disease and with normal saliva flow will then be invited to participate in the study. Eligible subjects will then be randomised to one of two treatments: i) up to 70 ml (the amount given will be equivalent to 3.7 mg/kg body weight) beetroot juice (James White Drinks Limited, UK) or ii) a matched volume of placebo beetroot juice (control, James White Drinks Limited, UK) to be consumed daily for 8 weeks. As drinking water is a major source of dietary nitrate, subjects will be provided with a water filter to reduce the nitrate level in their tap water for drinking and cooking during the study period. There will be a wash out period of 4 weeks between the intervention juices. In total, volunteers will be involved in the study for 20 weeks (five months). On the day before each baseline study visit (weeks 0 and 12), volunteers will be required to fast overnight after having a low nitrate evening meal and only drinking low nitrate mineral water (Buxton mineral water), which will be provided to them, during this time. They will refrain from strenuous exercise and alcohol. On the morning of the study visit, the volunteers will be asked not to brush their teeth before they come to the clinical unit for their study visit. When they arrive in the fasted state, the participants will be asked to provide a spot urine sample and a stool sample. Body weight and composition will be measured using the Tanita weighing scale, saliva sample and an oral bacteria sample will be collected by swabbing the tongue with a cotton swab. Volunteers will be asked to lie down for 20 minutes prior to the measurement of blood pressure and pulse wave analysis (PWA) using the Mobil-O-Graph device followed by the assessment of microvascular reactivity using Laser Doppler Imaging with iontophoresis. A blood sample of 20 ml (equivalent to just over 1 tablespoon) will then be taken before a light breakfast is provided. Volunteers will then be given a toothbrush and toothpaste to brush their teeth and asked to complete an online food frequency questionnaire (EatwellUK). During the four-week wash-out period between interventions, participants will be instructed to avoid any beetroot juice drinks and maintain their usual dietary and lifestyle habits.

Official Title
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Investigation of the Effects of Daily Nitrate Consumption on Oral Bacteria Composition, Blood Pressure and Vascular Functio

Conditions
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No Dental Disease, Non-smoking

Intervention / Treatment
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* Other: Nitrate rich beetroot juice
* Other: Placebo beetroot juice


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Male and female; A signed consent form; Blood pressure <140/90 mm Hg; BMI range 18.5 - 39.9 kg/m2; Age 18-64 years; Non-smoking; Not taking blood pressure lowering medication or using medications known to influence vascular function such as a glyceryl trinitrate (GTN) spray; No recent (within 3 months) or current use of antibiotics. Exclusion criteria are: Diagnosed with a chronic illness; Anaemia defined as a haemoglobin < 115 g/l for women and <130 g/l for men; Individuals with food allergies or allergies to medicated mouthwash or ingredients in the oral products; Requirements to take long-term medication active on the oral cavity including prebiotics and probiotics or have taken antibiotics within the last 3 months; Gingivitis diagnosis, or periodontal disease or chronic oral complaints or existing oral pathology. Less than four natural (enamel) buccal surfaces of upper molars available; Presence of fixed or removable oral appliances (e.g., dentures, orthodontic wires); xerostomia (a persistent dry mouth) diagnosis; Current smoker; Already participating in a dietary intervention study or clinical trial; Excessive alcohol consumption (> 14 units/wk); Females who are pregnant or lactating; Individuals not willing to give up using mouthwash or change their dental hygiene routine. Regular consumption or use of xylitol-based products such as toothpaste and chewing gum.

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 64 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Treatment
Allocation: Randomized
Intervention Model: Crossover Assignment
Masking: Double


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Active Comparator: Nitrate-rich Beetroot juice<br>Nitrate-rich beetroot juice equivalent to 3.7 mg nitrate per kg body weight daily for 8 weeks. | Other: Nitrate rich beetroot juice<br>* James White Drinks Limited beetroot juice (0.4-0.45 g nitrate/70 ml drink).<br>|
| Placebo Comparator: Placebo beetroot juice<br>Placebo beetroot juice (equivalent volume to the amount of nitrate-rich beetroot juice consumed) daily for 8 weeks. | Other: Placebo beetroot juice<br>* James White Drinks Limited Placebo beetroot juice (0 nitrate/70 ml drink).<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change in blood pressure measure | Systolic blood pressure, diastolic blood pressure and pulse pressure | Before and after each 8 week intervention | 
| Change in oral bacteria composition | Oral bacteria composition determined using next generation sequencing. | Before and after each 8 week intervention | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change in vascular reactivity | Laser Doppler Imaging with iontophoresis. | Before and after each 8 week intervention | 
| Change in Nox concentrations in serum, urine and saliva | Nitrate and nitrite concentrations will be determined using high performance liquid chromatography in plasma, urine and saliva. Nitric oxide will be calculated as the sum of nitrate and nitrite concentrations. Creatinine will be measured in the urine samples to normalise the nitrate and nitrite concentrations in urine for hydration status. | Before and after each 8 week intervention | 
| Change in gut bacteria composition | Stool sample will be analysed to determine composition using next generation sequencing will be used to find the differences between treatments. | Before and after each 8 week intervention | 
| Change in fasting lipid profile | Total cholesterol, HDL-C, and triacylglycerol will be measured using a clinical chemistry analyser. LDL-C will be calculated using the Friedewald formula. | Before and after each 8 week intervention | 
| Change in C-reactive protein | C-reactive protein will be measured using a clinical chemistry analyser | Before and after each 8 week intervention | 
| Change in endothelial function | Pulse wave analysis will be measured using Mobil-O-Graph | Before and after each 8 week intervention | 
| Change in body weight | Body weight will be measured using a Tanita scale | Before and after each 8 week intervention | 
| Height measurement | Height will be measured using a stadiometer | Before the intervention | 
| Change in body mass index | Body mass index will be calculated from the body weight and height measurement | Before and after each 8 week intervention | 
| Change in insulin | Insulin levels will be measured by ELISA | Before and after each 8 week intervention | 
| Change in glucose | Glucose levels will be measured using a clinical chemistry analyser | Before and after each 8 week intervention | 
| Change in insulin sensitivity | Glucose and insulin measurements will be used to calculate the homeostatic model assessment of insulin resistance (HOMA-IR) | Before and after each 8 week intervention |