Nitrate INFORMER Meat Study
Study Details
Study Description
Brief 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 meat with and without added nitrate.
| Condition or Disease | Intervention/Treatment | Phase |
|---|---|---|
|
N/A |
Detailed Description
Study design:
A crossover study design will be used with a 1-week washout period. Each participant will complete the scheduled study visits for one of the dietary interventions which will be assigned in random order. Following the washout, the participants will then complete the scheduled study visits for the remaining intervention. Participants will be provided with a low nitrate and N-nitrosamine meal on the preceding evening of each study visit. These meals will be consistent across all study visits Participants will be asked to refrain from drinking coffee and any alcoholic beverage and do any exercise 24 hours prior to their study visit.
Dietary interventions:
Meat with added nitrate: Prosciutto/pancetta/Parma ham/salami (all derived from pork) prepared by a commercial butcher with sodium nitrate and nitrite as an additive.
Meat without added nitrate and nitrite: Pork sausages, prepared by a commercial butcher, equivalent in weight to the meat plus nitrate intervention. Nitrate is not an allowed additive in sausages.
Assessments:
Prior to the first clinic visit, each participant will complete a food frequency questionnaire (FFQ) to assess background habitual diet.
At each clinic visit, baseline samples of blood, saliva, and urine will be collected for measurement of N-nitrosamines (plasma and urine), nitrate and nitrite (plasma, saliva, and urine). An oral (tongue scraping) sample and faecal stool sample will be collected for oral and gut microbiome analysis. A small blood sample (20 mL, approximately 1.5 tablespoons) will be taken at this time. A sterile container will be provided to participants for saliva collection over a time period of 5 min.
After the intervention, a saliva sample will be taken at 30-minute intervals up to 240 minutes for measurement of nitrate and nitrite. At 60 min and 240 minutes, a blood sample will be collected for measurement of N-nitrosamines, nitrate, and nitrite. The amount of the blood sample at each time point is the same as collected at baseline. After the intervention, all urine within the first 240 minutes and then subsequent 20-hour period will be collected for measurement of N-nitrosamines, nitrate, and nitrite.
Study Design
Arms and Interventions
| Arm | Intervention/Treatment |
|---|---|
| Experimental: Meat with added nitrate Prosciutto/pancetta/Parma ham/salami (all derived from pork) prepared by a commercial butcher with sodium nitrate as an additive. |
Dietary Supplement: Meat with added nitrate
Prosciutto/pancetta/Parma ham/salami (all derived from pork) prepared by a commercial butcher with sodium nitrate and nitrite as an additive.
|
| Experimental: Meat without added nitrate Pork sausages, prepared by a commercial butcher, equivalent in weight to the meat plus nitrate intervention. Nitrate is not an allowed additive in sausages. |
Dietary Supplement: Meat without added nitrate
Pork sausages, prepared by a commercial butcher, equivalent in weight to the meat plus nitrate intervention. Nitrate is not an allowed additive in sausages.
|
Outcome Measures
Primary Outcome Measures
- N-nitrosamines in plasma pre each intervention. [At each clinic visit, baseline samples of blood will be collected.]
A blood sample will be collected into EDTA tubes by venepuncture. Immediately after collection the blood will be centrifuged at 3000 x g (5 min, 4°C), and the plasma extracted and frozen at -80°C until analysis using analysis. Level of N-nitrosamines will be measured by gas chromatography mass spectrometry (GCMS).
- N-nitrosamines in urine pre each intervention. [At each clinic visit, baseline samples of urine will be collected.]
Participants will be provided with a sterilized container and instructions to collect the first urine sample of the day which will be brought into the clinic. Urine aliquots will be frozen at -80°C until analysis. Level of N-nitrosamines will be measured by gas chromatography mass spectrometry (GCMS).
- N-nitrosamines in plasma post each intervention at 60 min [After the intervention at each clinic visit, at 60 min a blood sample will be collected.]
A blood sample will be collected into EDTA tubes by venepuncture. Immediately after collection the blood will be centrifuged at 3000 x g (5 min, 4°C), and the plasma extracted and frozen at -80°C until analysis using analysis. Level of N-nitrosamines will be measured by gas chromatography mass spectrometry (GCMS).
- N-nitrosamines in plasma post each intervention at 240 min [After the intervention at each clinic visit, at 240 min a blood sample will be collected.]
A blood sample will be collected into EDTA tubes by venepuncture. Immediately after collection the blood will be centrifuged at 3000 x g (5 min, 4°C), and the plasma extracted and frozen at -80°C until analysis using analysis. Level of N-nitrosamines will be measured by gas chromatography mass spectrometry (GCMS).
- N-nitrosamines in urine post each intervention up to 240 min [At each clinic visit, all urine within the first 240 min will be collected.]
Participants will be provided with a sterilized container and instructions to collect all urine until 4 hours post intervention. Urine aliquots will be frozen at -80°C until analysis. Level of N-nitrosamines will be measured by gas chromatography mass spectrometry (GCMS).
- N-nitrosamines in urine post each intervention after 4 hours up till 24 hours [After each clinic visit, all urine from 4-hour to 24-hour period will be collected.]
Participants will be provided with a sterilized container and instructions to collect all urine from 4 hours until 24 hours post intervention. Urine aliquots will be frozen at -80°C until analysis. Level of N-nitrosamines will be measured by gas chromatography mass spectrometry (GCMS).
Secondary Outcome Measures
- Salivary nitrate pre each intervention [At each clinic visit, baseline saliva sample will be collected.]
Participants will be asked to expectorate saliva into a specimen container for 5 minutes while sitting quietly. Saliva aliquots will be frozen at -80°C until analysis. Concentrations of nitrate will be measured using gas chromatography mass spectrometry (GCMS).
- Salivary nitrate post each intervention at 30 min [At each clinic visit, a saliva sample will be collected at 30 min post the intervention.]
Participants will be asked to expectorate saliva into a specimen container for 5 minutes while sitting quietly. Saliva aliquots will be frozen at -80°C until analysis. Concentrations of nitrate will be measured using gas chromatography mass spectrometry (GCMS).
- Salivary nitrate post each intervention at 60 min [At each clinic visit, a saliva sample will be collected at 60 min post the intervention.]
Participants will be asked to expectorate saliva into a specimen container for 5 minutes while sitting quietly. Saliva aliquots will be frozen at -80°C until analysis. Concentrations of nitrate will be measured using gas chromatography mass spectrometry (GCMS).
- Salivary nitrate post each intervention at 90 min [At each clinic visit, a saliva sample will be collected at 90 min post the intervention.]
Participants will be asked to expectorate saliva into a specimen container for 5 minutes while sitting quietly. Saliva aliquots will be frozen at -80°C until analysis. Concentrations of nitrate will be measured using gas chromatography mass spectrometry (GCMS).
- Salivary nitrate each intervention at 120 min [At each clinic visit, a saliva sample will be collected at 120 min post the intervention.]
Participants will be asked to expectorate saliva into a specimen container for 5 minutes while sitting quietly. Saliva aliquots will be frozen at -80°C until analysis. Concentrations of nitrate will be measured using gas chromatography mass spectrometry (GCMS).
- Salivary nitrate post each intervention at 150 min [At each clinic visit, a saliva sample will be collected at 150 min post the intervention.]
Participants will be asked to expectorate saliva into a specimen container for 5 minutes while sitting quietly. Saliva aliquots will be frozen at -80°C until analysis. Concentrations of nitrate will be measured using gas chromatography mass spectrometry (GCMS).
- Salivary nitrate post each intervention at 180 min [At each clinic visit, a saliva sample will be collected at 180 min post the intervention.]
Participants will be asked to expectorate saliva into a specimen container for 5 minutes while sitting quietly. Saliva aliquots will be frozen at -80°C until analysis. Concentrations of nitrate will be measured using gas chromatography mass spectrometry (GCMS).
- Salivary nitrate post each intervention at 210 min [At each clinic visit, a saliva sample will be collected at 210 min post the intervention.]
Participants will be asked to expectorate saliva into a specimen container for 5 minutes while sitting quietly. Saliva aliquots will be frozen at -80°C until analysis. Concentrations of nitrate will be measured using gas chromatography mass spectrometry (GCMS).
- Salivary nitrate post each intervention at 240 min [At each clinic visit, a saliva sample will be collected at 240 min post the intervention.]
Participants will be asked to expectorate saliva into a specimen container for 5 minutes while sitting quietly. Saliva aliquots will be frozen at -80°C until analysis. Concentrations of nitrate will be measured using gas chromatography mass spectrometry (GCMS).
- Plasma nitrate pre each intervention [At each clinic visit, a blood sample will be collected at baseline.]
A blood sample will be collected into EDTA tubes by venepuncture. Immediately after collection the blood will be centrifuged at 3000 x g (5 min, 4°C), and the plasma extracted and frozen at -80°C until analysis. Concentrations of nitrate will be measured using gas chromatography mass spectrometry (GCMS).
- Plasma nitrate post each intervention at 60 min [At each clinic visit, a blood sample will be collected at 60 min post the intervention.]
A blood sample will be collected into EDTA tubes by venepuncture. Immediately after collection the blood will be centrifuged at 3000 x g (5 min, 4°C), and the plasma extracted and frozen at -80°C until analysis. Concentrations of nitrate will be measured using gas chromatography mass spectrometry (GCMS).
- Plasma nitrate post each intervention at 240 min [At each clinic visit, a blood sample will be collected at 240 min post the intervention.]
A blood sample will be collected into EDTA tubes by venepuncture. Immediately after collection the blood will be centrifuged at 3000 x g (5 min, 4°C), and the plasma extracted and frozen at -80°C until analysis. Concentrations of nitrate will be measured using gas chromatography mass spectrometry (GCMS).
- Urinary nitrate pre each intervention [At each clinic visit, baseline samples of urine will be collected.]
Participants will be provided with a sterilized container and instructions to collect the first urine sample of the day which will be brought into the clinic. Urine aliquots will be frozen at -80°C until analysis. Concentrations of nitrate will be measured using gas chromatography mass spectrometry (GCMS).
- Urinary nitrate post each intervention up to 240 min [At each clinic visit, all urine within the first 240 min will be collected.]
Participants will be provided with a sterilized container and instructions to collect all urine until 4 hours post intervention. Urine aliquots will be frozen at -80°C until analysis. Concentrations of nitrate will be measured using gas chromatography mass spectrometry (GCMS).
- Urinary nitrate post each intervention after 4 hours up till 24 hours [After each clinic visit, all urine from 4-hour to 24-hour period will be collected.]
Participants will be provided with a sterilized container and instructions to collect all urine from 4 hours until 24 hours post intervention. Urine aliquots will be frozen at -80°C until analysis. Concentrations of nitrate will be measured using gas chromatography mass spectrometry (GCMS).
- Salivary nitrite pre each intervention [At each clinic visit, baseline saliva sample will be collected.]
Participants will be asked to expectorate saliva into a specimen container for 5 minutes while sitting quietly. Saliva aliquots will be frozen at -80°C until analysis. Concentrations of nitrite will be measured using gas chromatography mass spectrometry (GCMS).
- Salivary nitrite post each intervention at 30 min [At each clinic visit, a saliva sample will be collected at 30 min post the intervention.]
Participants will be asked to expectorate saliva into a specimen container for 5 minutes while sitting quietly. Saliva aliquots will be frozen at -80°C until analysis. Concentrations of nitrite will be measured using gas chromatography mass spectrometry (GCMS).
- Salivary nitrite post each intervention at 60 min [At each clinic visit, a saliva sample will be collected at 60 min post the intervention.]
Participants will be asked to expectorate saliva into a specimen container for 5 minutes while sitting quietly. Saliva aliquots will be frozen at -80°C until analysis. Concentrations of nitrite will be measured using gas chromatography mass spectrometry (GCMS).
- Salivary nitrite post each intervention at 90 min [At each clinic visit, a saliva sample will be collected at 90 min post the intervention.]
Participants will be asked to expectorate saliva into a specimen container for 5 minutes while sitting quietly. Saliva aliquots will be frozen at -80°C until analysis. Concentrations of nitrite will be measured using gas chromatography mass spectrometry (GCMS).
- Salivary nitrite each intervention at 120 min [At each clinic visit, a saliva sample will be collected at 120 min post the intervention.]
Participants will be asked to expectorate saliva into a specimen container for 5 minutes while sitting quietly. Saliva aliquots will be frozen at -80°C until analysis. Concentrations of nitrite will be measured using gas chromatography mass spectrometry (GCMS).
- Salivary nitrite post each intervention at 150 min [At each clinic visit, a saliva sample will be collected at 150 min post the intervention.]
Participants will be asked to expectorate saliva into a specimen container for 5 minutes while sitting quietly. Saliva aliquots will be frozen at -80°C until analysis. Concentrations of nitrite will be measured using gas chromatography mass spectrometry (GCMS).
- Salivary nitrite post each intervention at 180 min [At each clinic visit, a saliva sample will be collected at 180 min post the intervention.]
Participants will be asked to expectorate saliva into a specimen container for 5 minutes while sitting quietly. Saliva aliquots will be frozen at -80°C until analysis. Concentrations of nitrite will be measured using gas chromatography mass spectrometry (GCMS).
- Salivary nitrite post each intervention at 210 min [At each clinic visit, a saliva sample will be collected at 210 min post the intervention.]
Participants will be asked to expectorate saliva into a specimen container for 5 minutes while sitting quietly. Saliva aliquots will be frozen at -80°C until analysis. Concentrations of nitrite will be measured using gas chromatography mass spectrometry (GCMS).
- Salivary nitrite post each intervention at 240 min [At each clinic visit, a saliva sample will be collected at 240 min post the intervention.]
Participants will be asked to expectorate saliva into a specimen container for 5 minutes while sitting quietly. Saliva aliquots will be frozen at -80°C until analysis. Concentrations of nitrite will be measured using gas chromatography mass spectrometry (GCMS).
- Plasma nitrite pre each intervention [At each clinic visit, a blood sample will be collected at baseline.]
A blood sample will be collected into EDTA tubes by venepuncture. Immediately after collection the blood will be centrifuged at 3000 x g (5 min, 4°C), and the plasma extracted and frozen at -80°C until analysis. Concentrations of nitrite will be measured using gas chromatography mass spectrometry (GCMS).
- Plasma nitrite post each intervention at 60 min [At each clinic visit, a blood sample will be collected at 60 min post the intervention.]
A blood sample will be collected into EDTA tubes by venepuncture. Immediately after collection the blood will be centrifuged at 3000 x g (5 min, 4°C), and the plasma extracted and frozen at -80°C until analysis. Concentrations of nitrite will be measured using gas chromatography mass spectrometry (GCMS).
- Plasma nitrite post each intervention at 240 min [At each clinic visit, a blood sample will be collected at 240 min post the intervention.]
A blood sample will be collected into EDTA tubes by venepuncture. Immediately after collection the blood will be centrifuged at 3000 x g (5 min, 4°C), and the plasma extracted and frozen at -80°C until analysis. Concentrations of nitrite will be measured using gas chromatography mass spectrometry (GCMS).
- Urinary nitrite pre each intervention [At each clinic visit, baseline samples of urine will be collected.]
Participants will be provided with a sterilized container and instructions to collect the first urine sample of the day which will be brought into the clinic. Urine aliquots will be frozen at -80°C until analysis. Concentrations of nitrite will be measured using gas chromatography mass spectrometry (GCMS).
- Urinary nitrite post each intervention up to 240 min [At each clinic visit, all urine within the first 240 min will be collected.]
Participants will be provided with a sterilized container and instructions to collect all urine until 4 hours post intervention. Urine aliquots will be frozen at -80°C until analysis. Concentrations of nitrite will be measured using gas chromatography mass spectrometry (GCMS).
- Urinary nitrite post each intervention after 4 hours up till 24 hours [After each clinic visit, all urine from 4-hour to 24-hour period will be collected.]
Participants will be provided with a sterilized container and instructions to collect all urine from 4 hours until 24 hours post intervention. Urine aliquots will be frozen at -80°C until analysis. Concentrations of nitrite will be measured using gas chromatography mass spectrometry (GCMS).
- Oral microbiome [Oral bacteria will be collected at baseline at each clinic visit.]
Oral bacteria will be collected from the posterior dorsal surface of the tongue using a sterile stainless-steel metal tongue cleaner. Participants will be instructed to scrape the tongue cleaner over the tongue until a coating is visible on the tongue cleaner (approximately 5 times). A sterile collection swab will be used to remove this coating which will subsequently be frozen at -80°C for later microbiome compositional analysis.
- Gut Microbiome [A stool sample will be collected at baseline at each clinic visit.]
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 for later microbiome compositional analysis.
Eligibility Criteria
Criteria
Inclusion Criteria:
The recruitment will be as inclusive as possible so that the results are relevant to much of the general population. Twenty-five men and women will be recruited from the Perth general population according to the following criteria:
-
aged between 18 to 55 years old
-
healthy, ambulant, community-dwelling
-
with no history of major chronic disease
Exclusion Criteria:
Individuals volunteering to participate in the study will be excluded according to the following criteria:
-
current or recent (<12 months) smoking
-
body mass index (BMI) <18 or > 35 kg/m2
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systolic blood pressure > 160 mmHg
-
diastolic blood pressure > 100 mmHg
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any major illness such as cancer, psychiatric illness, diagnosed diabetes
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use of any of the following medications: statins, antihypertensives, nitric oxide donors, antithrombotic medication, anti-coagulant medication, anti-arrhythmic drugs, beta-blockers, regular aspirin use, regular proton pump inhibitor use
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alcohol consumption > 30g/day
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who are pregnant, lactating, or wishing to become pregnant during the study
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use of antibiotics within the previous 12 weeks of the study
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regular use of mouthwash and not willing to cease mouthwash use for the duration of the study
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participation on other research studies
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major gastrointestinal tract condition e.g. Crohns disease and inflammatory bowel disease
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and inability or unwillingness to follow the study protocol.
Contacts and Locations
Locations
No locations specified.Sponsors and Collaborators
- Edith Cowan University
- The University of Western Australia
- Flinders University
Investigators
- Principal Investigator: Catherine P Bondonno, PhD, RNutr., Edith Cowan University
Study Documents (Full-Text)
None provided.More Information
Publications
None provided.- 2021-02629-BONDONNO