Nitrate INFORMER Meat Study

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.

Study Overview

• Status: Not yet recruiting
• Conditions: Health Risk Behaviors
• Intervention / Treatment: Dietary supplement: Meat with added nitrate; Dietary supplement: Meat without added nitrate

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 Type: Interventional
• Enrollment (Anticipated): 25
• Phase: Not Applicable

Contacts and Locations

• Study Contact: Name: Catherine P Bondonno, PhD, RNutr.; Phone Number: +61 8 6304 4601; Email: c.bondonno@ecu.edu.au
• Study Contact Backup: Name: Alex H Liu, PhD; Email: haoci.liu@ecu.edu.au

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years to 55 years (Adult)
• Accepts Healthy Volunteers: No
• Genders Eligible for Study: All

Description

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
• systolic blood pressure > 160 mmHg
• diastolic blood pressure > 100 mmHg
• any major illness such as cancer, psychiatric illness, diagnosed diabetes
• 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.

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Prevention
• Allocation: Randomized
• Interventional Model: Crossover Assignment
• Masking: Single

Number of Arms

2

Arms and Interventions

Participant Group / 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.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
N-nitrosamines in plasma pre each 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 using analysis. Level of N-nitrosamines will be measured by gas chromatography mass spectrometry (GCMS).	At each clinic visit, baseline samples of blood will be collected.
N-nitrosamines in urine pre each intervention.	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).	At each clinic visit, baseline samples of urine will be collected.
N-nitrosamines in plasma post each intervention at 60 min	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).	After the intervention at each clinic visit, at 60 min a blood sample will be collected.
N-nitrosamines in plasma post each intervention at 240 min	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).	After the intervention at each clinic visit, at 240 min a blood sample will be collected.
N-nitrosamines in urine post each intervention up to 240 min	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).	At each clinic visit, all urine within the first 240 min will be collected.
N-nitrosamines in urine post each intervention after 4 hours up till 24 hours	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).	After each clinic visit, all urine from 4-hour to 24-hour period will be collected.

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Salivary nitrate pre each 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).	At each clinic visit, baseline saliva sample will be collected.
Salivary nitrate post each intervention at 30 min	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).	At each clinic visit, a saliva sample will be collected at 30 min post the intervention.
Salivary nitrate post each intervention at 60 min	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).	At each clinic visit, a saliva sample will be collected at 60 min post the intervention.
Salivary nitrate post each intervention at 90 min	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).	At each clinic visit, a saliva sample will be collected at 90 min post the intervention.
Salivary nitrate each intervention at 120 min	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).	At each clinic visit, a saliva sample will be collected at 120 min post the intervention.
Salivary nitrate post each intervention at 150 min	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).	At each clinic visit, a saliva sample will be collected at 150 min post the intervention.
Salivary nitrate post each intervention at 180 min	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).	At each clinic visit, a saliva sample will be collected at 180 min post the intervention.
Salivary nitrate post each intervention at 210 min	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).	At each clinic visit, a saliva sample will be collected at 210 min post the intervention.
Salivary nitrate post each intervention at 240 min	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).	At each clinic visit, a saliva sample will be collected at 240 min post the intervention.
Plasma nitrate pre each 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).	At each clinic visit, a blood sample will be collected at baseline.
Plasma nitrate post each intervention at 60 min	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).	At each clinic visit, a blood sample will be collected at 60 min post the intervention.
Plasma nitrate post each intervention at 240 min	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).	At each clinic visit, a blood sample will be collected at 240 min post the intervention.
Urinary nitrate pre each intervention	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).	At each clinic visit, baseline samples of urine will be collected.
Urinary nitrate post each intervention up to 240 min	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).	At each clinic visit, all urine within the first 240 min will be collected.
Urinary nitrate post each intervention after 4 hours up till 24 hours	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).	After each clinic visit, all urine from 4-hour to 24-hour period will be collected.
Salivary nitrite pre each 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).	At each clinic visit, baseline saliva sample will be collected.
Salivary nitrite post each intervention at 30 min	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).	At each clinic visit, a saliva sample will be collected at 30 min post the intervention.
Salivary nitrite post each intervention at 60 min	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).	At each clinic visit, a saliva sample will be collected at 60 min post the intervention.
Salivary nitrite post each intervention at 90 min	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).	At each clinic visit, a saliva sample will be collected at 90 min post the intervention.
Salivary nitrite each intervention at 120 min	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).	At each clinic visit, a saliva sample will be collected at 120 min post the intervention.
Salivary nitrite post each intervention at 150 min	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).	At each clinic visit, a saliva sample will be collected at 150 min post the intervention.
Salivary nitrite post each intervention at 180 min	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).	At each clinic visit, a saliva sample will be collected at 180 min post the intervention.
Salivary nitrite post each intervention at 210 min	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).	At each clinic visit, a saliva sample will be collected at 210 min post the intervention.
Salivary nitrite post each intervention at 240 min	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).	At each clinic visit, a saliva sample will be collected at 240 min post the intervention.
Plasma nitrite pre each 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).	At each clinic visit, a blood sample will be collected at baseline.
Plasma nitrite post each intervention at 60 min	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).	At each clinic visit, a blood sample will be collected at 60 min post the intervention.
Plasma nitrite post each intervention at 240 min	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).	At each clinic visit, a blood sample will be collected at 240 min post the intervention.
Urinary nitrite pre each intervention	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).	At each clinic visit, baseline samples of urine will be collected.
Urinary nitrite post each intervention up to 240 min	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).	At each clinic visit, all urine within the first 240 min will be collected.
Urinary nitrite post each intervention after 4 hours up till 24 hours	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).	After each clinic visit, all urine from 4-hour to 24-hour period will be collected.
Oral microbiome	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.	Oral bacteria will be collected at baseline at each clinic visit.
Gut Microbiome	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.	A stool sample will be collected at baseline at each clinic visit.

Collaborators and Investigators

• Sponsor: Edith Cowan University
• Collaborators: Flinders University; The University of Western Australia
• Investigators: Principal Investigator: Catherine P Bondonno, PhD, RNutr., Edith Cowan University

Publications and helpful links

General Publications

• Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJ, Holmes SP. DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods. 2016 Jul;13(7):581-3. doi: 10.1038/nmeth.3869. Epub 2016 May 23.
• 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. Review.
• Blekkenhorst 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.
• Bondonno 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. Review.
• Spiegelhalder 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.
• Tannenbaum 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.
• Gangolli 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.
• Mirvish 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. Erratum In: Cancer Lett 1995 Nov 6;97(2):271.
• IARC 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.
• Hord 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.
• Blekkenhorst 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.
• Bartsch 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.
• Levallois 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.
• Bartholomew 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.
• Bondonno 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.
• Bondonno 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.
• Cole JR, Wang Q, Fish JA, Chai B, McGarrell DM, Sun Y, Brown CT, Porras-Alfaro A, Kuske CR, Tiedje JM. Ribosomal Database Project: data and tools for high throughput rRNA analysis. Nucleic Acids Res. 2014 Jan;42(Database issue):D633-42. doi: 10.1093/nar/gkt1244. Epub 2013 Nov 27.
• Parks DH, Chuvochina M, Waite DW, Rinke C, Skarshewski A, Chaumeil PA, Hugenholtz P. A standardized bacterial taxonomy based on genome phylogeny substantially revises the tree of life. Nat Biotechnol. 2018 Nov;36(10):996-1004. doi: 10.1038/nbt.4229. Epub 2018 Aug 27.

Study record dates

Study Major Dates

• Study Start (Anticipated): May 11, 2022
• Primary Completion (Anticipated): December 16, 2022
• Study Completion (Anticipated): December 16, 2022

Study Registration Dates

• First Submitted: August 30, 2021
• First Submitted That Met QC Criteria: September 29, 2021
• First Posted (Actual): October 13, 2021

Study Record Updates

• Last Update Posted (Actual): April 1, 2022
• Last Update Submitted That Met QC Criteria: March 30, 2022
• Last Verified: March 1, 2022

More Information

Terms related to this study

• Other Study ID Numbers: 2021-02629-BONDONNO

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: No

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No