This study evaluates the potential anti-inflammatory effects of inorganic dietary nitrate in a model of acute inflammation relevant to cardiovascular disease (CVD).

Green leafy vegetables contain large amounts of inorganic nitrate, and research suggests that this nitrate has beneficial effects on the heart and blood vessels. The Ahluwalia Group have shown anti-inflammatory benefits of inorganic nitrate in pre-clinical models of CVD, early mechanistic studies in healthy volunteers, and in patients with hypertension, hypercholesterolaemia and those suffering acute heart attacks that translate to cardiovascular benefits. Understanding the mechanism of how this is achieved may open new therapeutic options in CVD.

The Investigators therefore wish to explore whether inorganic nitrate might alter inflammatory responses using a blister-model of acute skin inflammation. This study is a randomised control trial with parallel limbs where half of patients receive nitrate-rich beetroot juice, and the other half a nitrate-deplete placebo beetroot juice.

Nitric oxide (NO) is an important substance produced continuously by all blood vessels. It is thought to maintain health, in part, by preventing and suppressing inflammatory responses. NO is normally generated at the endothelium by a group of important enzymes called nitric oxide synthases. However, in chronic inflammatory states such as those seen in cardiovascular disease (CVD), the endothelial isoform of the enzyme becomes dysfunctional and produces less NO, and the NO that is produced is scavenged by the products of oxidative stress.

Giving NO back to the blood vessel to mediate its beneficial effects is not straightforward. However, the Ahluwalia Group have shown that dietary inorganic nitrate, both as a capsule and a dietary intervention with nitrate-rich beetroot juice, has a number of beneficial effects in CVD states. This beneficial effect is mediated through it's endogenous conversion from nitrate to nitrite in the mouth, and then from nitrite to NO via nitrite reductases within the blood vessel. The Investigators have shown benefit of inorganic nitrate in pre-clinical models of CVD, early mechanistic studies in healthy volunteers, and anti-inflammatory benefits in patients with hypertension, hypercholesterolaemia and those suffering acute heart attacks.

The Investigators wish to prospectively investigate the anti-inflammatory effects of inorganic nitrate using a cantharidin-induced blister model of acute inflammation. This allows investigation of the innate immune system's response to an acute insult and characterise the inflammatory and resolution phases. Cantharidin is commonly used as a vesicant in the treatment of plantar verrucae and molluscum contagiosum, and experimentally to study the pharmacokinetics of drugs within the interstitial space.

This will be a double-blind placebo-controlled parallel limb study supplementing 24 healthy volunteers with dietary-rich beetroot juice versus nitrate-deplete placebo juice, to investigate blister formation, and inflammatory cell recruitment and activation.

Inclusion Criteria:

• Healthy volunteers
• Caucasian
• Willing to provide informed consent

Exclusion Criteria:

• Healthy subjects unwilling to consent
• Non-caucasian volunteers
• History of any serious illnesses, including recent infections or trauma
• Subjects taking systemic medication (other than the oral contraceptive pill)
• Subjects with self-reported use of mouthwash or tongue scrapers
• Subjects with recent or current antibiotic use
• Subjects with a history, or recent treatment of (within last 3 months) any oral condition (excluding caries), including gingivitis, periodontitis and halitosis
• Subjects with a history of skin conditions
• Subjects with and history of allergic reaction to any topical application
• Subjects with any history of a bloodborne infectious disease such Hepatitis B or C virus, or HIV

We have excluded non-caucasian volunteers due to a small incidence of prolonged skin hyperpigmentation in non-caucasians in previous studies.

We have excluded subjects using mouthwash/tongue scrapers and oral conditions as we know that the nitrate ingested from the diet (i.e. beetroot juice) is converted to nitrite by the commensal bacteria in the back of the mouth. Subjects who have oral conditions or use mouthwash/tongue scrapers would have differences in oral bacterial populations, both in number and species. Therefore, by excluding these subjects, we will try and keep this variable as similar as possible in both groups.

N/A - no plan to make IPD available to others

All members of the study team and volunteer participants will be blinded to treatment versus placebo arm