Effect of Lettuce With Different Nitrate Contents on Blood Pressure
Condition: Dietary Modification; Blood Pressure
Study type: Interventional
Phase: Phase 1/Phase 2

Summary: 
      Leafy vegetables are a natural source of dietary nitrate, which may reduce systolic blood
      pressure (BP). However, the evidence has been obtained from studies using nitrate solutions
      (i.e., potassium or sodium nitrate) or beetroot juice supplementation which have a suitable
      placebo for the design of double-blind clinical trials. However, the design of food-based
      nutritional interventions is complicated by the fact that an appropriate placebo treatment is
      not available and, therefore, it is not possible to meet the criteria for proper double-blind
      randomised placebo-controlled intervention trials. In addition, the biological effects of
      processed products such as beetroot juice or solutions with a pharmacological grade may be
      different from that of fresh vegetables, e.g. due to conversion of nitrate to nitrite in the
      mouth during chewing.

      Objectives: to investigate whether two sets of lettuce specifically grown with different
      nitrate content but otherwise similar composition show different effects on nitrate uptake
      and bioavailability in humans. Additionally, the investigators also aim to design human
      intervention studies to investigate the effect of intake of lettuce with different nitrate
      content on vascular health.

      These objectives will be tested by growing lettuce with different fertiliser compositions
      resulting in high and low nitrate content and then investigating the bioavailability and
      short-term effect on BP in healthy young volunteers in a double-blind cross-over design.
      Eligible subjects will consume one meal each of either low or high nitrate lettuce. Urine,
      blood and saliva samples will be collected at baseline, for 6 hours after the ingestion and
      then again after 24hr. Blood Pressure BP will be measured continuously for 24 hours starting
      at baseline. The volunteers will repeat the intervention with the second treatment.
    

Detailed description: 
      Study Design and Subjects: This study is designed as a double-blind, randomized, crossover,
      intervention trial in which thirty healthy young volunteers (BMI: 20-25 kg/m2), non-smoking
      subjects (age: 18-35 years) will be recruited. In each of two intervention periods, subjects
      will be asked to eat 50g of either high nitrate lettuce or low nitrate lettuce (placebo) on a
      single occasion (the second visit) with dietary restriction for 3 days prior to the second
      visit and in the 24 hr period following after the consumption, concluded with a third visit,
      with at least 3 weeks washout period before the second 2-day intervention period. Urine,
      saliva and blood samples will be collected during a 6-hour period during the second visit.
      Urine samples will be collected during 24h from consumption until the third visit, to assess
      the pharmacokinetics and bioavailability and compliance to the interventions (details in the
      diagram of human trial).

      The primary outcomes are 24-hour BP and nitrate content in urine, plasma and saliva. Samples
      will be collected into tubes and then analysed in the laboratory for the nitrate content
      (NO3-) by gas chromatography-mass spectroscopy (GCMS). Participants will follow specific
      dietary and lifestyle instructions to minimise confounding effects.

      Participant will receive a small honorarium after the study and reasonable travel expenses
      will be reimbursed.

      Dietary Interventions: Participants will be asked to arrive fasting in the morning of the
      second (main) visit and will consume a standard meal (Chicken Hotpot from ASDA) with the same
      drink water at the evening before and the evening after the visit, as well as following a
      diet that excludes a list of specific foods containing high nitrate (rocket, spinach, other
      leafy vegetables, radish and beet root), cured meat, cured seafood and cured fish, mature
      cheese for 24hr prior to the study and fasting on the morning of the study.

      Anthropometry: This includes the measurements of body weight, height and waist circumference
      according to standardised protocols. Procedures are safe, not invasive and induce minimal
      discomfort for the participants.

      Body Composition: This includes the measurement of body fat and muscle using a non-invasive
      technique called bio impedance analysis using a leg-to-leg bioelectrical impedance device
      (TANITA 300 MA). The procedure is safe, non-invasive and induces minimal discomfort for the
      participant. The duration of the measurement is of approximately 3-5 minutes.

      24-hr BP monitoring: participants will be fitted with an automated portable device to measure
      BP over a 24-hr period. Participants will be instructed on how to safely operate the device.
      The measurement are safe and minimally invasive. BP readings will be recorded every 30
      minutes during day time and every hour during the night to minimise the potential impact on
      sleep quality.

      Urine samples: The first morning void (FMV) will be collected at home (pre supplementation)
      and after arrival full urine samples (all that the volunteer is able to produce) at set times
      before and after the supplementation (between baseline and 6hr) at NU-Food research facility.
      Subsequent collections afterwards (between 6-12hr and 12-24 hr) will take place at home in
      appropriate plastic containers and the samples will be collected at the final visit 24hr
      after the consumption. After measuring the volume of each sample, sub samples will be stored
      for analysis and for calculation of pharmacokinetics.

      Saliva samples: a small saliva sample (~2ml) will be collected in disposable plastic
      containers at baseline and at every hour for 6 hours at NU-Food research facility; and then
      after 9 and 12 hour at home, with the final sample taken after 24 hr.

      Blood Samples: Venous blood samples (LH 6 ml) will be collected at baseline, three and six
      hours post supplementation of treatments and then again after 24 hr.
    

Intervention name(s): High Nitrate Lettuce; Low Nitrate Lettuce
Intervention type(s): Dietary Supplement; Dietary Supplement
Intervention description(s): The lettuce has been grown with high concentration of nitrogen fertilizer (150ppm) to produce the high of nitrate content in the lettuce, the nitrate content of 50 grams of high nitrate lettuce was (~530 mg nitrate derived).; The lettuce has been grown with low concentration of nitrogen fertilizer (25ppm) to produce the low amount of nitrate in the lettuce, the nitrate content of 50 grams of high nitrate lettuce was (~3 mg nitrate derived).
Allocation: Randomized
Intervention model: Crossover Assignment
Masking: Quadruple (Participant, Care Provider, Investigator, Outcomes Assessor)
Primary purpose: Prevention
Keywords: Nitric oxide; Dietary intervention trail