Health Risk BehaviorsNCT05075720
Nitrate INFORMER Meat Study
A study testing Meat with added nitrate and Meat without added nitrate in people with Health Risk Behaviors, run by Edith Cowan University at 1 site in Australia.
Trial 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… Read more about this study
- Who is it for?
- Ages 18 to 70 yearsHealthy volunteers can take part
- Conditions the study is for
- Health Risk Behaviors
- What is being tested?
- Meat with added nitrate and Meat without added nitrate
- Could I receive a placebo?
- Yes, some participants may receive a placebo
- How many people
- 25 participants
- Where
- 1 siteAustralia
- Study dates
- 27 Sep 2022to 13 Dec 2022
- Phase
- Interventional
- Are there results?
- None posted in the registry
Key requirements
Who can take part, as listed by the sponsor. Only the study team can confirm whether you are eligible, so take this list to your doctor.
- Ages 18 to 70 years
- All sexes
- Healthy volunteers accepted
- Adult, Older adult
Who may be able to join
- 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 70 years old
- healthy, ambulant, community-dwelling
- with no history of major chronic disease
Who cannot join
- 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
- systolic blood pressure > 160 mmHg
- diastolic blood pressure > 100 mmHg
- 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
- alcohol consumption > 30g/day
- who are pregnant, lactating, or wishing to become pregnant during the study
- use of antibiotics within the previous 12 weeks of the study
- regular use of mouthwash and not willing to cease mouthwash use for the duration of the study
- participation on other research studies
- major gastrointestinal tract condition e.g. Crohns disease and inflammatory bowel disease
- and inability or unwillingness to follow the study protocol.
Trial locations and contacts
1 site in 1 country.
Australia1 site
Royal Perth Hospital Research Foundation
Perth, Western Australia, 6000
About this study
Official title: Randomised Controlled Trial to Investigate N-nitrosamine Formation After Meat Intake - Meat Study of the Nitrate INFORMER Studies; Nitrate INFORMER Studies: Is Nitrosamine FORMation dEpenent on souRce
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.
Read the 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:
- A choice of 3 breakfasts: cornflakes with milk; rice krispies with milk; white bread toast with eggs.
- Lunch will be provided comprising pumpkin soup and white bread/or rice
- Dinner: a low nitrate vegetarian meal
- All water will be provided
- Participants will be asked to refrain from drinking coffee and any alcoholic beverage 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 and lunch will comprise the 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 coffee and any alcoholic beverage and do any exercise for 24 hours from the time of the first intervention.
Dietary interventions
- Meat with added nitrate: 50 g salami and 35 g ham on white bread sandwich at breakfast and lunch. This intervention will allow us to determine both endogenous formation of N-nitrosamines as well as N-nitrosamines present in the commercially prepared meat.
- Meat without added nitrate: 65 g Pork mince on white bread sandwich at breakfast and lunch. Nitrate is not an allowed additive in pork mince. This intervention will allow us to determine if there is endogenous formation of N-nitrosamines as well as N-nitrosamines present in the prepared meat due to the natural content of nitrate in meat.
- Control: low nitrate vegetable protein burger on white bread. Protein content matched to interventions 1 and 2.
Allocation 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, 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 we 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.
- Conditions
- Health Risk Behaviors
- Being tested
- Meat with added nitrate, Meat without added nitrate, Control
- Medical subject headings
- Health Risk Behaviors
What participants receive
What participants receive. In a randomised study each person is assigned to one group by chance.
Meat with added nitrateDietary supplement
The intervention comprises 50 g salami and 35 g ham on white bread sandwich at breakfast and lunch.
This intervention will allow us to determine both endogenous formation of N-nitrosamines as well as N-nitrosamines present in the commercially prepared meat.
Meat without added nitrateDietary supplement
The intervention comprises 65 g Pork mince on white bread sandwich at breakfast and lunch. Nitrate is not an allowed additive in pork mince.
This intervention will allow us to determine if there is endogenous formation of N-nitrosamines as well as N-nitrosamines present in the prepared meat due to the natural content of nitrate in meat.
ControlDietary supplement
The control comprises low nitrate vegetable protein burger on white bread. Protein content matched to interventions 1 and 2.
| Study group | Role | What they receive |
|---|---|---|
| Dietary Supplement: Meat with added nitrate | Experimental | The intervention comprises 50 g salami and 35 g ham on white bread sandwich at breakfast and lunch.
This intervention will allow us to determine both endogenous formation of N-nitrosamines as well as N-nitrosamines present in the commercially prepared meat. Receives: Dietary Supplement: Meat without added nitrate; Dietary Supplement: Control |
| Dietary Supplement: Meat without added nitrate | Experimental | The intervention comprises 65 g Pork mince on white bread sandwich at breakfast and lunch. Nitrate is not an allowed additive in pork mince.
This intervention will allow us to determine if there is endogenous formation of N-nitrosamines as well as N-nitrosamines present in the prepared meat due to the natural content of nitrate in meat. Receives: Dietary Supplement: Meat with added nitrate; Dietary Supplement: Control |
| Dietary Supplement: Control | Sham comparator | The control comprises low nitrate vegetable protein burger on white bread. Protein content matched to interventions 1 and 2. Receives: Dietary Supplement: Meat with added nitrate; Dietary Supplement: Meat without added nitrate |
What is being measured
The primary outcome is the main question the study is designed to answer.
N-nitrosamines in urine post intervention (up to 24 hours)
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. N-nitrosamines will be measured by gas chromatography mass spectrometry (GCMS).
Measured: At each clinic visit, all urine from the start of intervention up till 24 hours will be collected.
N-nitrosamines in stool samples post intervention (up to 24 hours)
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 will be measured by gas chromatography mass spectrometry (GCMS).
Measured: At each clinic visit, all stool samples from the start of intervention up till 24 hours will be collected.
Timeline
From the start of the study to its completion; the mark is today. Estimated dates are the sponsor’s plan.
- First posted13 Oct 2021
- Study start27 Sep 2022
- Primary completion13 Dec 2022
- Study completion13 Dec 2022
Resources
- Official record on ClinicalTrials.govThe registry entry this page is built from, with its full change history.
- Publications (18)Papers the sponsor has linked to this study.
- Other trials for Health Risk BehaviorsStudies of the same condition, open and under way.
- What the trial phases meanHow a treatment moves from first tests in people to approval.
More details
For doctors and researchers: how the study is designed, who runs it, and its registry IDs.
Study design
- Study type
- Interventional
- Phase
- Phase N/A
- Primary purpose
- Prevention
- Allocation
- Randomised
- Intervention model
- Crossover
- Model description
- prospective, mono-centre, randomised, controlled, crossover study
- Masking
- Single blind
- Who is masked
- Outcomes assessor
- Enrolment
- 25 participants (actual)
Sponsor and investigators
- Lead sponsor
- Edith Cowan University
- Sponsor type
- University, hospital or other
- Collaborators
- The University of Western Australia, Flinders University
- Responsible party
- Sponsor
- Investigators
- Catherine P Bondonno, PhD, RNutr., Principal investigator, Edith Cowan University
IDs, dates and publications
- ClinicalTrials.gov ID
- NCT05075720
- Sponsor's protocol ID
- 2021-02629-BONDONNO
- Status last verified
- Jan 2023
- First submitted
- 30 Aug 2021
- Last updated
- 1 Feb 2023
- Results posted
- No
- FDA-regulated drug
- No
- FDA-regulated device
- No
- Individual data sharing
- No
Publications
Lundberg JO, Weitzberg E. NO-synthase independent NO generation in mammals. Biochem Biophys Res Commun. 2010 May 21;396(1):39-45. doi: 10.1016/j.bbrc.2010.02.136.
PubMed 20494108Blekkenhorst LC, Bondonno NP, Liu AH, Ward NC, Prince RL, Lewis JR, Devine A, Croft KD, Hodgson JM, Bondonno CP. Nitrate, the oral microbiome, and cardiovascular health: a systematic literature review of human and animal studies. Am J Clin Nutr. 2018 Apr 1;107(4):504-522. doi: 10.1093/ajcn/nqx046.
PubMed 29635489Bondonno CP, Blekkenhorst LC, Liu AH, Bondonno NP, Ward NC, Croft KD, Hodgson JM. Vegetable-derived bioactive nitrate and cardiovascular health. Mol Aspects Med. 2018 Jun;61:83-91. doi: 10.1016/j.mam.2017.08.001. Epub 2017 Sep 7.
PubMed 28802834Spiegelhalder B, Eisenbrand G, Preussmann R. Influence of dietary nitrate on nitrite content of human saliva: possible relevance to in vivo formation of N-nitroso compounds. Food Cosmet Toxicol. 1976 Dec;14(6):545-8. doi: 10.1016/s0015-6264(76)80005-3. No abstract available.
PubMed 1017769Tannenbaum SR, Weisman M, Fett D. The effect of nitrate intake on nitrite formation in human saliva. Food Cosmet Toxicol. 1976 Dec;14(6):549-52. doi: 10.1016/s0015-6264(76)80006-5. No abstract available.
PubMed 1017770Gangolli SD, van den Brandt PA, Feron VJ, Janzowsky C, Koeman JH, Speijers GJ, Spiegelhalder B, Walker R, Wisnok JS. Nitrate, nitrite and N-nitroso compounds. Eur J Pharmacol. 1994 Nov 1;292(1):1-38. doi: 10.1016/0926-6917(94)90022-1.
PubMed 7867685Mirvish SS. Role of N-nitroso compounds (NOC) and N-nitrosation in etiology of gastric, esophageal, nasopharyngeal and bladder cancer and contribution to cancer of known exposures to NOC. Cancer Lett. 1995 Jun 29;93(1):17-48. doi: 10.1016/0304-3835(95)03786-V.
PubMed 7600541IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. IARC monographs on the evaluation of carcinogenic risks to humans. Ingested nitrate and nitrite, and cyanobacterial peptide toxins. IARC Monogr Eval Carcinog Risks Hum. 2010;94:v-vii, 1-412. No abstract available.
PubMed 21141240Hord NG, Tang Y, Bryan NS. Food sources of nitrates and nitrites: the physiologic context for potential health benefits. Am J Clin Nutr. 2009 Jul;90(1):1-10. doi: 10.3945/ajcn.2008.27131. Epub 2009 May 13.
PubMed 19439460Blekkenhorst LC, Prince RL, Ward NC, Croft KD, Lewis JR, Devine A, Shinde S, Woodman RJ, Hodgson JM, Bondonno CP. Development of a reference database for assessing dietary nitrate in vegetables. Mol Nutr Food Res. 2017 Aug;61(8). doi: 10.1002/mnfr.201600982. Epub 2017 May 3.
PubMed 28105786Bartsch H, Ohshima H, Pignatelli B. Inhibitors of endogenous nitrosation. Mechanisms and implications in human cancer prevention. Mutat Res. 1988 Dec;202(2):307-24. doi: 10.1016/0027-5107(88)90194-7.
PubMed 3057363Levallois P, Ayotte P, Van Maanen JM, Desrosiers T, Gingras S, Dallinga JW, Vermeer IT, Zee J, Poirier G. Excretion of volatile nitrosamines in a rural population in relation to food and drinking water consumption. Food Chem Toxicol. 2000 Nov;38(11):1013-9. doi: 10.1016/s0278-6915(00)00089-2.
PubMed 11038239Bartholomew B, Hill MJ. The pharmacology of dietary nitrate and the origin of urinary nitrate. Food Chem Toxicol. 1984 Oct;22(10):789-95. doi: 10.1016/0278-6915(84)90116-9.
PubMed 6541617Bondonno CP, Croft KD, Puddey IB, Considine MJ, Yang X, Ward NC, Hodgson JM. Nitrate causes a dose-dependent augmentation of nitric oxide status in healthy women. Food Funct. 2012 May;3(5):522-7. doi: 10.1039/c2fo10206d. Epub 2012 Feb 16.
PubMed 22336776Bondonno CP, Downey LA, Croft KD, Scholey A, Stough C, Yang X, Considine MJ, Ward NC, Puddey IB, Swinny E, Mubarak A, Hodgson JM. The acute effect of flavonoid-rich apples and nitrate-rich spinach on cognitive performance and mood in healthy men and women. Food Funct. 2014 May;5(5):849-58. doi: 10.1039/c3fo60590f.
PubMed 24676365
Study at a glance
From the registry record, last updated by the sponsor on 1 Feb 2023. Recruitment status is as the sponsor reported it: confirm with the study team.
- Official title
- Randomised Controlled Trial to Investigate N-nitrosamine Formation After Meat Intake - Meat Study of the Nitrate INFORMER Studies; Nitrate INFORMER Studies: Is Nitrosamine FORMation dEpenent on souRce
- ClinicalTrials.gov ID
- NCT05075720
- Acronym / sponsor ID
- 2021-02629-BONDONNO
- Conditions
- Health Risk Behaviors
- Study type
- Interventional
- Recruitment status
- Completed
- Enrolment
- 25 participants (actual)
- Lead sponsor
- Edith Cowan UniversityUniversity, hospital or other
- Collaborators
- The University of Western Australia, Flinders University
- Study start
- 27 Sep 2022 (actual)
- Primary completion
- 13 Dec 2022 (actual)
- Study completion
- 13 Dec 2022 (actual)
- First posted
- 13 Oct 2021 (actual)
- Results
- Not posted in the source record
Clinical summary
The research question as the sponsor registered it. This describes what the study plans to test, not what it found.
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.
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:
- A choice of 3 breakfasts: cornflakes with milk; rice krispies with milk; white bread toast with eggs.
- Lunch will be provided comprising pumpkin soup and white bread/or rice
- Dinner: a low nitrate vegetarian meal
- All water will be provided
- Participants will be asked to refrain from drinking coffee and any alcoholic beverage 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 and lunch will comprise the 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 coffee and any alcoholic beverage and do any exercise for 24 hours from the time of the first intervention.
Dietary interventions
- Meat with added nitrate: 50 g salami and 35 g ham on white bread sandwich at breakfast and lunch. This intervention will allow us to determine both endogenous formation of N-nitrosamines as well as N-nitrosamines present in the commercially prepared meat.
- Meat without added nitrate: 65 g Pork mince on white bread sandwich at breakfast and lunch. Nitrate is not an allowed additive in pork mince. This intervention will allow us to determine if there is endogenous formation of N-nitrosamines as well as N-nitrosamines present in the prepared meat due to the natural content of nitrate in meat.
- Control: low nitrate vegetable protein burger on white bread. Protein content matched to interventions 1 and 2.
Allocation 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, 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 we 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.
Study design
- Primary purpose
- Prevention
- Allocation
- Randomised
- Intervention model
- Crossover
- Masking
- Single blind (outcomes assessor)
- Number of arms
- 3
- Model description
- prospective, mono-centre, randomised, controlled, crossover study
| Arm | Type | Receives |
|---|---|---|
| Dietary Supplement: Meat with added nitrate | Experimental | Dietary Supplement: Meat without added nitrate, Dietary Supplement: ControlThe intervention comprises 50 g salami and 35 g ham on white bread sandwich at breakfast and lunch. This intervention will allow us to determine both endogenous formation of N-nitrosamines as well as N-nitrosamines present in the commercially prepared meat. |
| Dietary Supplement: Meat without added nitrate | Experimental | Dietary Supplement: Meat with added nitrate, Dietary Supplement: ControlThe intervention comprises 65 g Pork mince on white bread sandwich at breakfast and lunch. Nitrate is not an allowed additive in pork mince. This intervention will allow us to determine if there is endogenous formation of N-nitrosamines as well as N-nitrosamines present in the prepared meat due to the natural content of nitrate in meat. |
| Dietary Supplement: Control | Sham comparator | Dietary Supplement: Meat with added nitrate, Dietary Supplement: Meat without added nitrateThe control comprises low nitrate vegetable protein burger on white bread. Protein content matched to interventions 1 and 2. |
Who may qualify
- Ages
- 18 to 70 years
- Sex
- All
- Healthy volunteers
- Accepted
- Age groups
- Adult, Older adult
Key inclusion criteria (4 listed)
- 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 70 years old
- healthy, ambulant, community-dwelling
- with no history of major chronic disease
Key exclusion criteria (14 listed)
- 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
- systolic blood pressure > 160 mmHg
- diastolic blood pressure > 100 mmHg
Eligibility is decided by the study team after screening; this overview does not establish that a patient qualifies. Full inclusion and exclusion criteria.
Interventions
| Intervention | Type | Arms |
|---|---|---|
| Meat with added nitrateThe intervention comprises 50 g salami and 35 g ham on white bread sandwich at breakfast and lunch. This intervention will allow us to determine both endogenous formation of N-nitrosamines as well as N-nitrosamines present in the commercially prepared meat. | Dietary supplement | Dietary Supplement: Control, Dietary Supplement: Meat without added nitrate |
| Meat without added nitrateThe intervention comprises 65 g Pork mince on white bread sandwich at breakfast and lunch. Nitrate is not an allowed additive in pork mince. This intervention will allow us to determine if there is endogenous formation of N-nitrosamines as well as N-nitrosamines present in the prepared meat due to the natural content of nitrate in meat. | Dietary supplement | Dietary Supplement: Control, Dietary Supplement: Meat with added nitrate |
| ControlThe control comprises low nitrate vegetable protein burger on white bread. Protein content matched to interventions 1 and 2. | Dietary supplement | Dietary Supplement: Meat with added nitrate, Dietary Supplement: Meat without added nitrate |
Primary outcome
N-nitrosamines in urine post intervention (up to 24 hours)
Time frame: At each clinic visit, all urine from the start of intervention up till 24 hours will be collected.
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. N-nitrosamines will be measured by gas chromatography mass spectrometry (GCMS).
N-nitrosamines in stool samples post intervention (up to 24 hours)
Time frame: At each clinic visit, all stool samples from the start of intervention up till 24 hours will be collected.
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 will be measured by gas chromatography mass spectrometry (GCMS).
Results and safety
The study is completed, but results are not available in the source record. That does not mean the treatment did or did not work.
Research links
- Official record on ClinicalTrials.govLast updated by the sponsor 1 Feb 2023.
- Results publication: PubMed 20494108Lundberg JO, Weitzberg E. NO-synthase independent NO generation in mammals. Biochem Biophys Res Commun. 2010 May 21;396(1):39-45. doi: 10.1016/j.bbrc.2010.02.136.
- Results publication: PubMed 29635489Blekkenhorst LC, Bondonno NP, Liu AH, Ward NC, Prince RL, Lewis JR, Devine A, Croft KD, Hodgson JM, Bondonno CP. Nitrate, the oral microbiome, and cardiovascular health: a systematic literature review of human and animal studies. Am J Clin Nutr. 2018 Apr 1;107(4):504-522. doi: 10.1093/ajcn/nqx046.
- Results publication: PubMed 28802834Bondonno CP, Blekkenhorst LC, Liu AH, Bondonno NP, Ward NC, Croft KD, Hodgson JM. Vegetable-derived bioactive nitrate and cardiovascular health. Mol Aspects Med. 2018 Jun;61:83-91. doi: 10.1016/j.mam.2017.08.001. Epub 2017 Sep 7.
- Results publication: PubMed 1017769Spiegelhalder B, Eisenbrand G, Preussmann R. Influence of dietary nitrate on nitrite content of human saliva: possible relevance to in vivo formation of N-nitroso compounds. Food Cosmet Toxicol. 1976 Dec;14(6):545-8. doi: 10.1016/s0015-6264(76)80005-3. No abstract available.
- Results publication: PubMed 1017770Tannenbaum SR, Weisman M, Fett D. The effect of nitrate intake on nitrite formation in human saliva. Food Cosmet Toxicol. 1976 Dec;14(6):549-52. doi: 10.1016/s0015-6264(76)80006-5. No abstract available.
- Results publication: PubMed 7867685Gangolli SD, van den Brandt PA, Feron VJ, Janzowsky C, Koeman JH, Speijers GJ, Spiegelhalder B, Walker R, Wisnok JS. Nitrate, nitrite and N-nitroso compounds. Eur J Pharmacol. 1994 Nov 1;292(1):1-38. doi: 10.1016/0926-6917(94)90022-1.
- Results publication: PubMed 7600541Mirvish SS. Role of N-nitroso compounds (NOC) and N-nitrosation in etiology of gastric, esophageal, nasopharyngeal and bladder cancer and contribution to cancer of known exposures to NOC. Cancer Lett. 1995 Jun 29;93(1):17-48. doi: 10.1016/0304-3835(95)03786-V.
- Results publication: PubMed 21141240IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. IARC monographs on the evaluation of carcinogenic risks to humans. Ingested nitrate and nitrite, and cyanobacterial peptide toxins. IARC Monogr Eval Carcinog Risks Hum. 2010;94:v-vii, 1-412. No abstract available.
- Results publication: PubMed 19439460Hord NG, Tang Y, Bryan NS. Food sources of nitrates and nitrites: the physiologic context for potential health benefits. Am J Clin Nutr. 2009 Jul;90(1):1-10. doi: 10.3945/ajcn.2008.27131. Epub 2009 May 13.
- Results publication: PubMed 28105786Blekkenhorst LC, Prince RL, Ward NC, Croft KD, Lewis JR, Devine A, Shinde S, Woodman RJ, Hodgson JM, Bondonno CP. Development of a reference database for assessing dietary nitrate in vegetables. Mol Nutr Food Res. 2017 Aug;61(8). doi: 10.1002/mnfr.201600982. Epub 2017 May 3.
- Results publication: PubMed 3057363Bartsch H, Ohshima H, Pignatelli B. Inhibitors of endogenous nitrosation. Mechanisms and implications in human cancer prevention. Mutat Res. 1988 Dec;202(2):307-24. doi: 10.1016/0027-5107(88)90194-7.
- Results publication: PubMed 11038239Levallois P, Ayotte P, Van Maanen JM, Desrosiers T, Gingras S, Dallinga JW, Vermeer IT, Zee J, Poirier G. Excretion of volatile nitrosamines in a rural population in relation to food and drinking water consumption. Food Chem Toxicol. 2000 Nov;38(11):1013-9. doi: 10.1016/s0278-6915(00)00089-2.
- Individual participant dataSharing planned: No
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Referral and contact
AzTrials does not enrol participants or take referrals. Contact the study team or a site listed below; they decide who can take part. This study is not recruiting at the moment.
Catherine P Bondonno, PhD, RNutr. principal investigator
Edith Cowan University
1 site in 1 country: see every site and its status.
Questions about this trial
What is this clinical trial studying?
A study testing Meat with added nitrate and Meat without added nitrate in people with Health Risk Behaviors, run by Edith Cowan University at 1 site in Australia.
The full registered description is in the About this study section and the official record.
Who may be eligible to take part?
The sponsor lists 4 inclusion and 14 exclusion criteria. Eligibility is decided by the study team after screening, so read the full criteria and contact a listed site.
Is this clinical trial currently recruiting?
The record says: finished, as of the sponsor's last update on 1 Feb 2023. Status can change between updates, so confirm with the study contact before making plans.
Where is the study taking place?
The record lists 1 site in Australia. Whether a site is taking part right now should be confirmed with that site. See every site.
What are the possible benefits and risks?
They depend on the study and the treatment, and no study can promise a benefit. Some participants in this study may receive a placebo. The study team explains the known risks during informed consent, before anyone joins; the record itself does not list them.
Are results available for this study?
The study is completed, but results are not available in the source record. That does not mean the treatment did or did not work.
How can a doctor refer a patient or contact the research team?
The record lists no contact. The official record names the sponsor, who can be asked directly. AzTrials does not enrol participants or take referrals.
What does the primary outcome measure?
N-nitrosamines in urine post intervention (up to 24 hours) (time frame: At each clinic visit, all urine from the start of intervention up till 24 hours will be collected.). The study lists 2 primary outcomes in all. It is what the study was designed to measure, not a finding.
Where can I read the original study information?
On the ClinicalTrials.gov record for NCT05075720, which also lists 18 linked publications.
Does a listing mean the treatment is approved or proven to work?
No. A listing means the study is registered. It does not show that the treatment is safe, works, or is approved by a regulator.
How often is this information updated?
AzTrials copies changed records from ClinicalTrials.gov every night and reloads the whole registry every week. The sponsor last updated this record on 1 Feb 2023.