Dietary Nitrate Intake in Vegetarians and Omnivores

Primary outcomes

1. Dietary nitrate intake

   Time frame: 7 days

   Macro- and micronutrient intake of seven-day food diaries were analysed using a nutritional analysis software programme (Microdiet, Downlee Systems, Chapel-en-le-Frith, UK). Nitrate content of vegetables was mainly obtained from the European Food Safety Authority and additional data for spinach and lettuce from the Food Standards Agency. Nitrate and total polyphenol figures were uploaded to the Microdiet database prior to analysis.

2. Concentration of nitrate and nitrite in plasma and saliva

   Time frame: 7 days

   Whole blood was collected into lithium-heparin tubes (BD Vacutainer®, Becton Dickinson, Plymouth, UK) and rapidly centrifuged at 4,000 rpm and 4°C for 10 minutes. The plasma was then separated, frozen at -80°C until further analyses of nitrate and nitrite were performed. Non-stimulated saliva (3 milliliters) was also collected into a sterile falcon tube and rapidly frozen at -80°C. Then, nitrate and nitrite were measured in plasma and saliva using ozone-based chemiluminescence.

3. Change in blood pressure

   Time frame: 7 days

   Systolic, diastolic and mean arterial blood pressure was measured following British Hypertension Guidelines. Three successive supine readings were taken using an oscillometric device (Connex ProBP 3400 Digital Blood Pressure Device, Welch Allyn UK Ltd.) with a one minute rest between readings. The second and third readings were averaged to determine mean clinic blood pressure.

4. Change in oral nitrate-reducing capacity

   Time frame: 7 days

   Nitrate-reducing capacity of oral bacteria was analysed to compare vegetarians and omnivores and treatments (placebo mouthwash vs antibacterial mouthwash). Participants were instructed to hold 10 milliliters of water containing sodium nitrate (80 μicromols) in their mouth for 5 minutes. The mouth rinse was collected into a Falcon sterile tube and centrifuged (4,500 rpm, 4°C) for 10 minutes. The supernatant was collected and stored at -80°C before measuring absolute nitrite concentration.

5. Change in Resting Energy Expenditure

   Time frame: 7 days

   Resting Energy Expenditure was analysed using indirect calorimetry after using the placebo mouthwash and antibacterial mouthwash

6. Change oral microbiome

   Time frame: 7 days

   bundance of oral bacteria was analysed using 16S ribosomal ribonucleic acid (rRNA) after using the placebo mouthwash and antibacterial mouthwash.

   Salivary DNA was extracted using a DNA kit (QIAamp® DNeasy Blood & Tissue Kit). Polymerase chain reaction (PCR) amplification of the 16S rRNA V1-2 region was carried out using universal 16S primers 27 F (5'-AGA GTT TGA TCM TGG CTC AG-3') and 338 R (5'-GCW GCC WCC CGT AGG WGT-3'). Single band PCR products were purified and quantified with Qubit 2.0 Fluorometer (Invitrogen, CA, USA). Sequencing was performed on an Ion Torrent Personal Genome Machine.

7. Change in blood biomarkers

   Time frame: 7 days

   Blood markers were analyzed to assess differences between vegetarians and omnivores to control for diabetes and dyslipidaemia and to analyse the effect of antibacterial mouthwash.

   Whole blood glucose and lactate was measured using a biochemistry analyser (YSI 2300 Stat Plus, YSI Life Sciences, USA). Total cholesterol, triglycerides, high density lipoproteins (HDL) and low density lipoproteins (LDL) were analysed with enzymatic methods using the Roche 702 spectrophotometric module of a Cobas 8000 analyser (Roche Diagnostics Ltd, UK)