Evaluation of Adding Nitrate Into Foods for Regulating Nitric Oxide Bioavailability in Healthy Individuals

Whether adding nitrate to commonly eaten foods can improve nitric oxide levels in the body and help lower blood pressure in healthy volunteers will be investigated.

Design: An open-label, randomised, crossover trial. Target population: Thirty healthy participants (15 males and 15 females) will be recruited.

Experimental intervention: Equal numbers of healthy male and female volunteers, aged 18-60 years, will be recruited. Following recruitment, participants will attend the clinical centre on seven occasions. At baseline, clinic blood pressure will be measured, and plasma, urine, and saliva samples will be collected. Participants will then receive one of the three nitrate-fortified food products according to the randomisation plan.

Clinic blood pressure will be measured at 1, 2, 3, and 4 hours post-supplementation. Blood samples will be collected at 0.5, 1, 1.5, 2, 2.5, 3, and 4 hours; urine at 4 hours; and saliva at 3 and 4 hours. At 24 hours post-supplementation, participants will return to the clinical centre for repeat blood pressure measurement and collection of blood, urine, and saliva samples. All human biological samples will be stored for subsequent determination of nitrate and nitrite concentrations.

If participants receive nitrate-fortified biscuits during one visit, they will be given either nitrate-fortified porridge or a cereal bar at the following visits, according to the randomisation plan. All participants will eventually receive all three nitrate-fortified food products, each providing approximately 4 mmol nitrate, an amount within the Acceptable Daily Intake (ADI) range.

The study will be conducted at the Clinical Research Centre at the William Harvey Research Institute, Queen Mary University of London.

Analysis: A two-way repeated measures ANOVA will be used to analyse the differences in [NO3-], [NO2-] in plasma, urine, and saliva, plasma [cGMP] across time and between different NO3--fortified foodstuffs (cereal bar, porridge, and biscuits) and clinic BP. Matching the objectives of comparing the changes in measures following the ingestion of the three food products, the description of the analysis will initially focus on the difference between the three food products and so post-hoc tests (accounting for multiple comparisons) will initially focus on comparing measures at each timepoint.

The acceptability of the three different NO3--fortified food products will be analysed using Friedman tests. As this is a crossover study, checks will be conducted for period effect and carry-over effect as part of the statistical analysis plan.

Inclusion criteria

• Healthy volunteer.
• Aged ≥18 years and ≤ 60 years.
• Willing to provide informed consent.
• Able to understand and comply with protocol requirements, instructions, and stated restrictions.

Exclusion criteria

A volunteer will not be eligible for inclusion in this study if any of the following criteria are met:

• Unwilling to provide consent.
• People with chronic health conditions requiring medication.
• Pregnant females, or those with a possibility of being pregnant.
• History of hypertension and /or diabetes.
• History of any serious illnesses, including recent infections or trauma.
• History of symptomatic coronary artery disease, stroke, or other known atherosclerotic diseases.
• People who will commence or who are likely to commence treatment with non-steroidal anti-inflammatory drugs (NSAIDs) other than aspirin, from screening until study completion.
• Self-declared alcohol or drug abuse within the past 6 months.
• Three-month prior history of regular alcohol consumption exceeding an average weekly intake of > 28 units (or an average daily intake of greater than 3 units) for males, or an average weekly intake of > 21 units (or an average daily intake of greater than 2 units) for females. One unit is equivalent to a half pint (284mL) of beer/lager; 25mL of spirits, or 125mL of wine.
• Taking systemic medication (other than the oral contraceptive pill).
• Recent (within 2 weeks) self-reported use of mouthwash or tongue scrapers.
• Recent (within 2 weeks) or current antibiotic use.
• Recent (within 1 week) use of NO3- or NO2- supplements.
• History, or recent treatment of (within the last 3 months) for any oral condition (excluding caries), including gingivitis, periodontitis and halitosis.
• History of, or recent treatment for, any blood-borne infectious disease such Hepatitis B or C virus, or HIV.
• Current smokers (including vaping) or have smoked within the last 6 months.
• Diagnosis of rheumatoid arthritis, connective tissue disorders, and other conditions known to be associated with chronic inflammation (e.g., Inflammatory Bowel Disease).
• People who have donated more than 500mL of blood within 56 days prior to the study commencement.
• Known allergy to celery, gluten, crustaceans, eggs, lupin, milk, mustard, peanuts, sesame, soybeans, tree nuts, oats, palm oil, sugar, cranberries, sunflower oil, invert syrup, sodium bicarbonate.