Nitrate INFORMER Water Study
Condition: Health Risk Behaviors
Study type: Interventional

Summary: 
      Nitrate is a controversial component of vegetables, meat, and drinking water. The now
      well-established benefits of nitrate, through the enterosalivary nitrate-nitrite-nitric oxide
      (NO) pathway, on cardiovascular risk factors and long-term cardiovascular disease risk are
      tarnished by a continuing concern about a link between nitrate ingestion and cancer. This can
      result in misguided advice to avoid consumption of high-nitrate leafy green vegetables by
      both the media and the scientific literature. A recent media headline stated, "Cancer alert
      over rocket: trendy salad leaves exceed safe levels of carcinogenic nitrates in one in every
      ten samples". One scientific review stated, "the presence of nitrate in vegetables, as in
      water and generally in other foods, is a serious threat to man's health". Controversy in the
      literature, and gaps in the knowledge are leading to confusing messages around vegetables
      that may play a critical role in cardiovascular health.

      The major dietary sources of nitrate are vegetables, meat, and drinking water. Source of
      nitrate could be a crucial factor determining whether the consumption of nitrate is linked
      with beneficial (such as improving cardiovascular health) versus harmful (N-nitrosamine
      formation) effects. For example, unlike meat and water-derived nitrate, vegetables contain
      high levels of vitamin C and/or polyphenols that may inhibit the production of
      N-nitrosamines. So far, no study has investigated the formation of N-nitrosamines after
      consumption of these different sources in humans. This study will compare N-nitrosamine
      formation after intake of water with and without added nitrate.
    

Detailed description: 
      Study design

      A crossover study design will be used with a 1-week washout period between interventions.
      Participants diet for the day before and the day of the intervention will be standardised
      (food choices on visit 1 will be matched for visit 2) as follows:

      For the day before the study visit:

        -  Breakfast: white bread toast with eggs, cheese, peanut butter or jam.

        -  Lunch will be provided comprising pumpkin soup and white bread

        -  Dinner: a low nitrate vegetarian meal

        -  All water will be provided

        -  Participants will be asked to refrain from drinking any coffee or caffeine containing
           beverages 17 and any alcoholic beverage 18 and do any exercise 24 hours prior to their
           study visit.

      For the day of the study visit:

        -  Participants will arrive at the study unit fasting

        -  Breakfast will comprise white bread toast with cheese, peanut butter or jam together
           with the first water intervention

        -  Lunch will comprise pumpkin soup and white bread together with the second water
           intervention

        -  Participants will be provided with a standardised dinner: a low nitrate vegetarian meal

        -  All water will be provided

        -  Participants will be asked to refrain from drinking any coffee or caffeine containing
           beverages 17 and any alcoholic beverage 18 and do any exercise 24 hours prior to their
           study visit.

      Interventions

      • Water with added nitrate: 137 mg sodium nitrate or 164 mg potassium nitrate (100mg of
      nitrate) in a 500 mL water bottle (200 mg/L) provided twice, one with breakfast and one with
      lunch. This is equivalent to 200mg nitrate in total.

      In Australia, guidelines published in 2011 recommend that levels less than 50 mg/L are safe
      to drink, levels of 50 - 100 mg/L are safe to drink for all adults including pregnant women
      but not infants less than 3 months of age, drinking water with nitrate concentrations greater
      than 100 mg/L is not recommended.

      • Water without added nitrate: 137 mg sodium chloride or 164 mg potassium chloride in a 500
      mL water bottle provided twice, one with breakfast and one with lunch.

      Sequence generation. The sequence of intervention allocation will be generated via block
      randomisation using computer-generated random numbers. Random block sizes of 2 and 4 will be
      used.

      Concealment mechanism. Fifty randomly generated sequences of the interventions for each of
      the study participants will be printed on separate pieces of paper and sealed in opaque
      envelopes, numbered 1-50, by a study investigator not involved in performing the
      intervention, the data collection or the data analysis.

      Implementation. Once a participant is deemed eligible and enrolled in the study, the study
      coordinator will contact the study investigator responsible for randomisation and
      intervention allocation to obtain the next available envelope and randomly generated
      intervention sequence.

      Blinding. Given the nature of the interventions, participants, and the investigators
      responsible for delivering the interventions will be unblinded throughout the trial. However,
      all researchers performing the laboratory analyses and data analyses will be blinded to the
      interventions that the participants received until after the data analysis has been
      performed.

      Participant timeline. Prior to the first clinic visit, each participant will complete a food
      frequency questionnaire (FFQ) to assess background habitual diet.

      At each clinic visit (Figure 1), a baseline urine and faecal stool sample will be collected
      for measurement of N-nitrosamines. Prior to the intervention participants will be instructed
      to void their bladder into baseline urine collection container. After the first intervention
      all urine for the subsequent 24-hour period will be collected for measurement of
      N-nitrosamines, nitrate, and nitrite. A faecal stool will be collected for measurement of
      N-nitrosamines, nitrate, and nitrite.

      Assessments

      Urine and faecal stool collection. A urine sample will be collected at baseline, and from
      intervention up until 24 hours post-intervention. For the baseline sample, participants will
      be provided with a sterilized container and instructions to discard the first urine sample of
      the day and then collect all urine until the intervention which will be brought into the
      clinic. Participants will be instructed to drink 250 ml water on waking. For the 24-hour
      urine samples, participants will be provided with sterilized containers and instructions to
      collect all urine until 24 hours post intervention. Urine aliquots will be frozen at -80°C
      until analysis. A stool sample will be collected at baseline and for the 24 hour period post
      the first intervention. Participants will be provided with instructions and a stool sample
      collection pack (collection bags, cable ties, large zip lock bags, freezer ice blocks and a
      designated cooler bag for transport). Collected stool samples will be weighed and frozen at
      -80°C until analysis.

      N-nitrosamines. Nitrosamines in the interventions, urine and stool samples will be measured
      by gas chromatography mass spectrometry (GCMS). Volatile nitrosamines will be extracted with
      dichloromethane and are well separated by gas chromatography. Identification will be by
      retention time and unique molecular ions for each of five common nitrosamines. Quantitation
      will be made by using deuterium labelled internal standards, N-nirtrosodimethylamine-D6;
      N-nitrosodiethylamine D10; N-nitrosomorpholine D8 (Cambridge Isotopes) and
      N-nitrosopiperidine D10 (Toronto Research Chemicals). The most commonly detected nitrosamines
      are NDMA and N-Nitrosopiperidine (NPIP). Control experiments in which water stored in the
      same containers as those used for sample collection and storage will be performed to test for
      any leaching of volatile nitrosamines.

      Sample size and power

      Sample size is based on a crossover design and the primary outcome of N-nitrosamines,
      specifically N-Nitrosodimethylamine (NDMA) in urine. At α=0.05, 2 measures per subject
      (baseline and post) and a correlation of ρ=0.6 between measures, 25 participants will provide
      80% power to detect a 0.6 SD increase in NDMA concentration. To allow for a 10% withdrawal
      rate, we plan to recruit 25 participants into the study. A rolling recruitment of
      participants will be performed until the sample size is reached.

      Statistical methods

      Statistical analyses will be performed using IBM SPSS Statistics for Windows, version 25
      (IBM) and STATA/IC 17.0 (StataCorp LLC). Descriptive statistics of normally distributed
      continuous variables will be expressed as mean (± standard deviation, SD), non-normally
      distributed continuous variables as median (interquartile range, IQR) and categorical
      variables as number (proportion, %). Data will be assessed for outliers and normality prior
      to analysis. Non-normally distributed data will be log transformed if necessary. Treatment
      effects for outcomes will be obtained using linear mixed models including baseline
      measurements, treatment order, period, and time (as a categorical variable) as fixed effects.
      We will also include a treatment X period interaction term to assess for possible
      treatment-period interactions and will separately assess for carryover effects with dummy
      variables to indicate the previous treatment. Treatment effects for outcomes with a single
      post-intervention measurement will be obtained using linear mixed models including baseline
      measurements, treatment order, and period as predictors. The subject ID number will be
      included as a random intercept in each model. An overall 2-sided type-1 error rate of P<0.05
      will be used to assess statistical significance for all hypothesis testing.
    

Intervention name(s): Water with added nitrate; Water without added nitrate
Intervention type(s): Dietary Supplement; Dietary Supplement
Intervention description(s): Water with added nitrate; Water without added nitrate
Allocation: Randomized
Intervention model: Crossover Assignment
Intervention model description: prospective, mono-centre, randomised, controlled, crossover study
Masking: Single (Outcomes Assessor)
Primary purpose: Prevention