Oral nitrate supplementation improves cardiovascular risk markers in COPD: ON-BC, a randomised controlled trial

Background Short-term studies suggest that dietary nitrate (NO3−) supplementation may improve the cardiovascular risk profile, lowering blood pressure (BP) and enhancing endothelial function. It is not clear if these beneficial effects are sustained and whether they apply in people with COPD, who have a worse cardiovascular profile than those without COPD. Nitrate-rich beetroot juice (NR-BRJ) is a convenient dietary source of nitrate. Methods The ON-BC trial was a randomised, double-blind, placebo-controlled parallel group study in stable COPD patients with home systolic BP (SBP) measurement ≥130 mmHg. Participants were randomly allocated (1:1) using computer-generated, block randomisation to either 70 mL NR-BRJ (400 mg NO3−) (n=40) or an otherwise identical nitrate-depleted placebo juice (0 mg NO3−) (n=41), once daily for 12 weeks. The primary end-point was between-group change in home SBP measurement. Secondary outcomes included change in 6-min walk distance (6MWD) and measures of endothelial function (reactive hyperaemia index (RHI) and augmentation index normalised to a heart rate of 75 beats·min−1 (AIx75)) using an EndoPAT device. Plasma nitrate and platelet function were also measured. Results Compared with placebo, active treatment lowered SBP (Hodges–Lehmann treatment effect −4.5 (95% CI −5.9– −3.0) mmHg), and improved 6MWD (30.0 (95% CI 15.7–44.2) m; p<0.001), RHI (0.34 (95% CI 0.03–0.63); p=0.03) and AIx75 (−7.61% (95% CI −14.3– −0.95%); p=0.026). Conclusions In people with COPD, prolonged dietary nitrate supplementation in the form of beetroot juice produces a sustained reduction in BP, associated with an improvement in endothelial function and exercise capacity.


GLOSSARY OF ABBREVIATIONS
-AECOPD in the preceding month -Significant comorbidity limiting exercise tolerance -Significant comorbidity limiting life expectancy -Significant renal impairment (estimated glomerular filtration rate (eGFR) <30 ml.min 1 ) -Use of >3 blood pressure lowering medications -Change in medication in the previous month -Oral nitrate medication -Current (in the last month) use of Beet Shots.

TREATMENT
Active: 70mls Beet It Stamina shot from James White Ltd (6.5mmol Nitrate) Placebo: 70mls matched placebo shot with nitrate removed.

months
The ON-BC study Protocol V2 24 th June 2020 Page 7 of 28

Clinic BP Endothelial function
Blood tests (U&E, BNP, platelet function, nitrate levels) Oral samples for microbiome 6 minute walk test (final test)

Lay Summary
Background: Dietary nitrate supplementation, in the form of beetroot juice, has a number of potentially advantageous effects in COPD.These include improving the response to pulmonary rehabilitation programme, making muscle contraction more efficient so it uses less oxygen, and improving how far people with low oxygen levels because of their lung disease can walk.Although COPD is a lung disease, people with the condition are at a higher risk of heart disease and stroke.There is also some evidence that beetroot juice can reduce blood pressure, but studies so far have been short term.A nutritional treatment that could produce a lasting reduction in blood pressure would be appealing, especially if it also improves people's ability to exercise.

Aims:
The purpose of this study is to investigate the prolonged treatment effects of daily beetroot juice on blood pressure in people with COPD.We will also look at how far people can walk.We will make measurements of how well blood vessels function and take blood samples to look at the mechanisms involved including how "sticky" platelets are.These are the cells in the blood that cause it to clot.
Study Design: 72 patients with COPD will be enrolled in the study.Half will drink a 70ml beetroot juice "shot" each morning for three months.This contains 6.5mmol nitrate, the active ingredient.The other half will take identical juice drink which has had the nitrate removed.Which group participants are in will be decided at random by a computer.The main outcome will be blood pressure measured by participants at home for 4 days at the beginning and end of the study.In addition we will measure how far people can walk, how well blood vessels work using a device that measures blood flow, and blood tests looking at nitrate levels and platelet function.We will also collect mouth swabs to look at bacteria in the mouth to see if that changes with treatment.The study is funded by The Saudi Cultural Centre.
Expected outcome: If the study is positive this will help in the development of beetroot juice as a therapy for people with COPD and other long term conditions.

INTRODUCTION 1.1 BACKGROUND
COPD is a common condition responsible for considerable morbidity and mortality.There are 1.3 million people with a diagnosis of COPD in the UK and existing treatments are relatively ineffective.The presence of multiple medical problems in COPD has become the norm rather than the exception, and 50% of patients have three or more other long term conditions 1 .We have shown that a novel approach, dietary nitrate supplementation in the form of beetroot juice, has a number of potentially advantageous effects in COPD.These include enhancing the effect of pulmonary rehabilitation on exercise capacity (the ON-EPIC trial), reducing the oxygen cost of exercise in people with less severe COPD 2 and improving exercise capacity in hypoxic COPD patients (EDEN-OX trial).
At least 40% of COPD patients have concomitant cardiovascular disease 1 .Cardiovascular disease is the cause of death in about one third of individuals with COPD 3,4 .Although smoking and social deprivation are common risk factors for both conditions, COPD does appear to be an independent risk factor for cardiovascular disease 5 .Outcomes in patients with COPD are worse after acute coronary episodes than conventional scoring systems (the GRACE score) would anticipate, again suggesting that COPD should be treated as a risk marker, like diabetes. 6Dietary nitrate supplementation has been shown to reduce blood pressure both in normotensive 7 and hypertensive 8 subjects, though studies to date have been of relatively short durationthe longest for 5 weeks (reviewed here 9,10 ).There are also data linking NO with the development of metabolic syndrome -a clinical and biochemical expression of insulin resistance, representing a clustering of central obesity, hypertension, hyperglycemia, and dyslipidemia. 11,12etroot juice (BRJ) drinks are commercially available, marketed for their potential health benefits and widely used by sports people.The Beet It Stamina shot from James White Ltd is 70mls and contains 6.5mmol nitrate.A matched placebo beetroot beverage is available with identical appearance and taste, but which has been passed through an ion exchange column to remove the active nitrate component.

INTRODUCTION TO NITRATE METABOLISM
Nitric oxide (NO) is an important physiological mediator in the body.NO is endogenously produced via the action of the NO synthase (NOS) family of enzymes acting on the amino acid L-arginine, including endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS).These enzymes catalyse the conversion of arginine to NO and L-citrulline in a reaction dependent on the presence of molecular oxygen and several cofactors 13 .Endothelial NOS (eNOS) is itself activated by shear stress on the endothelial membrane, leading to NO production and vasodilatation 14,15 .
In addition to the action of the NOS enzyme family, NO is produced via the reduction of exogenous dietary nitrate (NO 3 -) in an oxygen independent manner (Figure 1).Nitrate can be found at high levels in leafy greens and beetroot, with vegetables providing 60-80% of our daily dietary intake 16 .Ingested inorganic nitrate passes readily into the circulation, with the majority being renally excreted 17,18 .Approximately 25% of ingested nitrate is actively taken up by the salivary glands via the enterosalivary circulation 18 , where concentrations may be over 10-fold greater than those measured in the plasma.It is then excreted in saliva and reduced from inert nitrate to bioactive nitrite (NO 2 -) via nitrate reductases produced by oral commensal facultative anaerobic bacteria 19,20 .Salivary nitrite may be absorbed as saliva is swallowed and enter the circulation directly as nitrite 19 , or be reduced to nitrogen oxides such as NO in the acidic environment of the stomach 21-23 and then absorbed.Thus the supplementation of the diet with nitrate provides a means of increasing circulating plasma nitrite levels which are an indicator of bioavailable NO.The importance of commensal bacteria in facilitating the metabolism of nitrate can be demonstrated by the prevention of the normal rise in plasma nitrite levels after an oral nitrate bolus following the administration of an antibacterial mouthwash 24 .
The reduction of plasma nitrite to NO is a process enhanced in hypoxic and acidic conditions as may occur in tissues during exercise 25,26 .This is an important process as under such conditions hypoxia may be limiting NO generation via the oxygen-dependent NOS pathway 27 .Lundberg et al. 19 Thus there are two common nitrate-nitrite-NO pathways, via the NOS family of enzymes and via reduction of dietary nitrate.In fact where endogenous NO production is limited via the knock-out of the eNOS gene, the supplementation of nitrite 28 or nitrate 29 enhances plasma nitrite and nitrate levels towards those seen in normal subjects.This would suggest there is an important interplay between the endogenous and exogenous sources of NO production.Although health concerns have been raised regarding supplementation with pure sodium nitrate or nitrite due to increased generation of N-nitrosamines with increased risk of gastric cancer, the generation of nitric oxides from nitrite is thought to be an important physiological pathway in humans.In fact it has been proposed that nitrite is an important source of reactive nitrogen intermediates including NO that possess important antimicrobial activity, gastric protective effects and control of vascular tone particularly in ischaemic tissues 22 .In addition nitrate-rich whole vegetables or juices also contain antioxidants and polyphenols that may reduce the formation of harmful nitrogenous compounds 30,31 .Thus the general consensus is that nitrate supplementation from vegetable sources is likely to be an important component of a healthy diet.

Figure 1. Nitrate-nitrite-NO pathway in humans. Plasma nitrate originates from the oxidation of endogenously produced NO and dietary sources. Nitrite reduction to NO is enhanced in acidic (H + ), hypoxic and reducing (e.g. vitamin C) conditions. Other proteins and enzymes may promote this step including Cytochrome P-450 (Cyt P-450), Xanthine Oxidase (XO) and Deoxyhaemoglobin (DeoxyHb). Taken from
Abnormalities in NO synthesis occur in ageing and may be linked to poor exercise tolerance seen in old age.Older subjects show reduced plasma nitrite production in response to exercise than younger individuals 32 .As nitrite is the main oxidation product of NO this is reflective of reduced eNOS activity during exertion, and as such can be abolished by the systemic administration of the NOS inhibitor L-NMMA (L-NG-monomethylarginine). This reduced NOS activity is accompanied by impaired endothelial vasodilator function in response to exercise in older persons as assessed by brachial artery flow mediated dilation studies 32 .

Impact of nitrate supplementation on exercise in COPD.
NO is key regulator of skeletal muscle blood flow, contractility, glucose and calcium haemostasis and mitochondrial function 33 .The landmark study of nitrate supplementation by Larsen et al (2007)  34 found that, in healthy individuals, acute nitrate supplementation reduced the oxygen cost of submaximal exercise.A finding confirmed in subsequent healthy subject trials 35 .
Patients with chronic obstructive pulmonary disease (COPD) have symptoms of exercise limitation that affect their activities of daily living.Exercise training through pulmonary rehabilitation (PR) is a well-established and effective therapy to ameliorate this 36 .Various research groups have investigated the role of dietary nitrate supplementation in COPD.We found that dietary nitrate supplementation reduced the oxygen cost of sub-maximal exercise assessed via cycle ergometry albeit without increasing exercise capacity in patients with COPD 2 .Berry et al also assessed the effect of dietary nitrate supplementation on submaximal exercise via cycle ergometry and found an increase in exercise capacity 37 .Kerley et al also found an increase in exercise capacity as assessed by an incremental shuttle walk test 38 .In another study, a week of BRJ 140mls BD lowered DBP but did not improve 6MWD in COPD, not using oxygen, mean FEV 1 44.7±15.1. 39 the ON-EPIC trial (presented ERS 2018) we randomised patients participating in pulmonary rehabilitation to take 140mls of BRJ or matched placebo before each exercise session.At the end of the program the improvement in exercise capacity in the active group, assessed using the incremental shuttle walk test (ISWT), was double that seen in the placebo arm: nitrate group +60.3 m (-110.0,180.0) vs. placebo +30.0 m (-70.0,290.0), p = 0.011.Furthermore, in a cross over study in hypoxic patients (EDEN-OX) (presented ERS 2018), we found that 140mls beetroot supplementation increased endurance shuttle walk time substantially compared to placebo, with an estimated treatment effect of 62sec (95%CI 33, 106) p<0.0001.
There is therefore a compelling evidence base to support pursuing the hypothesis that oral nitrate supplementation with BRJ may improve exercise performance in COPD.

Beetroot juicevascular impact in COPD
Several studies support a short term effect of nitrate supplementation in the form of beetroot juice on blood pressure in COPD.We have demonstrated that nitrate supplementation lowers the oxygen cost of exercise in COPD 40 .In that single dosing study diastolic blood pressure (DBP) fell by 7 ± 8 mmHg.Another study in COPD patients, using 3 days dosing with twice daily 4.8 mmol nitrate 70 mL beetroot juice, found a 10mmHg reduction systolic blood pressure (SBP) 41 .Kerley et al. used acute doses of 12.9 mmol and SBP, DBP and mean arterial pressure (MAP) decreased by −12 ± 19; −1.6 ± 16; −5 ± 4 mmHg respectively 42 .Berry et al., used a single dose of 7.58 mmol and found SDP was 8mmHg lower and DBP 3mmHg lower. 37A week of Beetroot 140mls BD lowered DBP by a mean 4.6mmHg 39 .One study used 6.77 mmol twice a day for 2.5 days and found no difference in blood pressure. 43OPD also involves damage to the pulmonary vascular bed which can progress to pulmonary hypertension 44 .There is intriguing data, in idiopathic pulmonary hypertension, to show a link between low circulating nitrate levels and both the extent of pulmonary hypertension and its progression and mortality. 45he ON-BC study Protocol V2 24 th June 2020 Page 12 of 28 An outstanding question is whether dietary nitrate supplementation will cause a sustained fall in blood pressure associated with a reduction/reversal in end organ damage (arterial stiffness).

The oral microbiome and nitrate metabolism
The community of microorganisms that inhabit the human body are referred to as the human microbiome.It consists mainly of bacteria, fungi and viruses and has been associated with both health and disease.Between individuals there is a common catalogue of microorganisms, but which any one person has and the relative abundances of each of these is unique to them 46,47 .In processes mediated by bacteria, such as nitrate reduction, this uniqueness is a potential source of variation as different microbial communities are not always functionally identical. 48Previous investigators have noted a change in the composition of the oral microbiome in response to nitrate supplementation in a modest cohort, with an increase in the genera Neisseria and Rothia 49 .Smoking has been demonstrated to have an impact on the oral microbiome with selective depletion of certain organisms including Neisseria. 50There is little data on the composition of the COPD oral microbiome, as usually the lung microbiome is studied in these individuals, so which organisms are responsible for the conversion of nitrate and how they vary between subjects will be determined here.If the oral microbiome is indeed a source of variation in response to nitrate supplementation then it might be possible to identify a nitrate reducing strain that could be used as a probiotic to normalise the nitrate-nitrite-NO functional pathway prior to supplementation with nitrate.
Factors potentially influencing the oral microbiota in COPD and thus nitrate/nitrite metabolism include smoking history, poor dentition, inhaled medication, gastric acid suppressing medication and gastro-oesophageal reflux.Advances in understanding of the oral microbiota have the potential to support novel probiotic approaches to improve nitrate metabolism in COPD patients.

Flow mediated dilatation to assess endothelial function
Flow mediated dilatation is non-invasive method of assessing endothelial function and assessing atherosclerosis in patients who are at high risk.NO is a known activator of guanylate cyclase which synthesises cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (cGTP) leading to the relaxation of vascular smooth muscle.Thus not surprisingly the administration of NO 3 -has been correlated with reduced systolic and diastolic blood pressure 51,52 and potential cardioprotective effects.
Endothelial function can be assessed using the Endopat device which measures fingertip vascular response to brachial artery occlusion and then release after a period without blood flow.

RATIONALE FOR CURRENT STUDY
Dietary nitrate supplementation, in the form of beetroot juice, has a number of potentially advantageous effects in COPD.These include improving the response to pulmonary rehabilitation programme, making muscle contraction more efficient so it uses less oxygen, and improving how far people with low oxygen levels because of their lung disease can walk.
Although COPD is a lung disease, people with the condition are also at a higher risk of heart disease and stroke.There is also some evidence that beetroot juice can reduce blood pressure, but studies so far have been short term.A nutritional treatment that could produce a lasting reduction in blood pressure would be appealing, especially if it also improves people's ability to exercise.
Thus the PICOT research question is whether in people with stable COPD, oral dietary nitrate supplementation in the form of daily 70ml beetroot shot compared to a placebo drink of nitrate-depleted beetroot juice, reduces blood pressure over a three month period.
Additional outcomes will be effect on six minute walk distance, endothelial function, platelet aggregation, blood nitrate levels, plasma concentration of arginine/asymmetric dimethylarginine (ADMA) levels), and Fractional exhaled NO (FeNO).
We will also perform oral microbiome sampling.

STUDY OBJECTIVES
The purpose of this study is to investigate the effects of prolonged administration of beetroot (BR) juice versus placebo beverage ingestion on cardiovascular risk markers in COPD patients.
The following hypotheses are to be tested: 1) BRJ will reduce BP 2) BRJ will increase exercise capacity (6MWD) 3) BRJ will improve health related quality of life (CAT score) 4) BRJ will improve endothelial function -assessed by Endopat score 5) BRJ will improve markers of platelet activation (platelet-monocyte aggregates) 6) BRJ will alter ratio of plasma concentration of arginine/asymmetric dimethylarginine (ADMA) 7) BRJ will reduce serum BNP levels.8) BRJ will increase fractional exhaled NO (FeNO) 9) BRJ will increase blood nitrate and nitrite levels 10) BRJ will alter the oral microbiome 11) BRJ will be an acceptable treatment (>80% participants comply with treatment defined as >80% doses consumed)

STUDY DESIGN
The study is a double-blind parallel group placebo-controlled trial to investigate the impact of dietary nitrate supplementation on BP.During the 3 month active treatment period 72 participants with stable COPD will consume 70mls beetroot juice each morning.This will either be active or placebo with the nitrate removed.

STUDY OUTCOME MEASURES
Primary endpoint Change in home monitored BP .

Secondary endpoints i)
Exercise capacity (6 minute walk test distance) ii) Health related quality of life (HRQoL) Blood Brain Natriuretic Peptide levels (BNP) iii) Markers of platelet activation (platelet-monocyte aggregates) iv) Plasma concentration of arginine/asymmetric dimethylarginine (ADMA) levels) v) Fractional exhaled NO (FeNO) vi) Blood nitrate and nitrite levels vii) Oral microbiome sampling viii) Acceptability of treatment (>80% participants comply with treatment defined as >80% doses consumed)

PRE-RANDOMISATION EVALUATIONS
Patients will have a clinical diagnosis of COPD based on symptoms and spirometry.
We will identify COPD patients in four ways: (i) opportunistically as they come into contact with the primary and secondary care teamscommunity clinics, secondary care clinics, 6 month post pulmonary rehabilitation reviews (ii) using existing research/audit registers of patients including those who have completed pulmonary rehabilitation (iii) by advertisement (local newspapers and through the BLF network of Breathe Easy Groups (iv) using the NIHR UK Clinical Trials Gateway to contact suitable patients who have consented to be contacted about research.

INITIAL ASSESSMENT FOR ELIGIBILITY
Where face to face visits are not possible, individuals who agree to be contacted will be asked to read the patient information sheet and then an initial telephone or video call will be made to go through eligibility criteria.If they agree to take part they will be asked to sign two copies of the consent form and return one.
They will then be sent a home BP monitor by courier.

WITHDRAWAL CRITERIA
No early stopping criteria are anticipated.
Data from patients who wish to be withdrawn from the study will be retained and used for analysis unless they request otherwise.

RANDOMISATION OR REGISTRATION PRACTICALITIES
Patients will be asked to provide written informed consent.Once baseline measures have been completed participants will be randomised 1:1 using an online system (from SealedEnvelope TM ).They will receive a code and be given the matched beetroot shots (active or placebo) to take.

UNBLINDING
No circumstances where unblinding would be needed are anticipated.

TREATMENT ARMS
Active: 70mls Beet It Stamina shot from James White Ltd (6.5mmol Nitrate) taken orally each morning.Placebo: 70mls Beet It Stamina placebo shot from James White Ltd (placebo with nitrate removed) taken orally each morning.

INTERACTION WITH OTHER DRUGS
No drug interactions are known.

DISPENSING AND ACCOUNTABILITY
Beetroot shots will be labelled with a code label linked to allocation i.e. active or placebo.They will be stored securely at room temperature and either given or sent to study participants by the trial team after randomization has occurred.

DEFINITIONS
Adverse Event (AE): any untoward medical occurrence in a patient or clinical study subject.Serious Adverse Event (SAE): any untoward and unexpected medical occurrence or effect that: • Results in death • Is life-threatening -refers to an event in which the subject was at risk of death at the time of the event; it does not refer to an event which hypothetically might have caused death if it were more severe • Requires hospitalisation, or prolongation of existing inpatients' hospitalisation • Results in persistent or significant disability or incapacity • Is a congenital anomaly or birth defect Medical judgement should be exercised in deciding whether an AE is serious in other situations.Important AEs that are not immediately life-threatening or do not result in death or hospitalisation but may jeopardise the subject or may require intervention to prevent one of the other outcomes listed in the definition above, should also be considered serious.

REPORTING PROCEDURES
All adverse events should be reported.Depending on the nature of the event the reporting procedures below should be followed.Any questions concerning adverse event reporting should be directed to the Chief Investigator in the first instance.

Non serious AEs
All such events, whether expected or not, should be recorded.

Serious AEs
An SAE form should be completed and faxed to the Chief Investigator within 24 hours.However, relapse and death due to <condition>, and hospitalisations for elective treatment of a pre-existing condition do not need reporting as SAEs.
All SAEs should be reported to the <name of REC> where in the opinion of the Chief Investigator, the event was: • 'related', ie resulted from the administration of any of the research procedures; and • 'unexpected', ie an event that is not listed in the protocol as an expected occurrence Reports of related and unexpected SAEs should be submitted within 15 days of the Chief Investigator becoming aware of the event, using the NRES SAE form for non-IMP studies.Office blood pressure: [used for eligibility assessment] this will be performed, in accordance with the British Hypertension Society's standard operating procedure, using an automated cuff device after subject has been sat quietly for 5 minutes prior to measurement.The subject's arm will be supported.
Lung function: Spirometry will be performed according to ATS/ERS guidelines, taking the best of at least 3 efforts performed seated.
ECG: a twelve lead ECG will be performed after 5 minutes rest on a couch.Rhythm, heart rate, axis and QTc interval will be recorded.
FeNO: Exhaled nitric oxide (FeNO): We will use the NIOX VERO® device.The measurement of FeNO requires the participant to exhale slowly into a mouthpiece.The exhaled NO value is measured in parts per billion (PPB).
Anthropometric measurements: subjects' height (cm) will be measured without shoes with a wall mounted measure, and we will measure their weight (kg) using standardised scales.

Health status questionnaires:
The COPD assessment (CAT) score will be administered to evaluate quality of lifethis 8 item symptom score has been shown to be responsive both to exacerbations and to pulmonary rehabilitation. 63,64Each item is scored 0-5 giving a score from 0 to 40 with 40 being the worst possible health status.
Blood sampling: Blood samples (a total of 20mls) will be drawn at baseline and again after 12 wks of treatment.
-Analysis of nitrite and nitrate: at visit 1 and 3 we will collect venous blood sample (3 mL) from subjects who for assessment of nitrite and nitrate levels using a chemoluminescence technique 53 .One of the research team will withdraw the blood samples through a 21-gauge butterfly needle inserted into an antecubital vein of the subjects.Blood samples will be collected in the morning between 8 and 11 am.
-Then, we will rapidly separate the plasma from red cells using a bench-top centrifuge for 3 min at 5,000 rpm.Plasma (1 mL) free of hemolysis is transferred into dark colored microtubes that also contain 6.5 mM N-ethylmaleimide (NEM) (100 mM; 6.5 µL) and 0.1 mM diethylenetriaminepentaacetic acid (DTPA) (10 mM; 10 µL).NEM and DTPA prevent the destruction of plasma S-nitrosothiols to NO 2 − .The samples are immediately frozen on dry ice and stored at −80 °C.In the lab, samples nitrite and nitrate will be first reduced to NO, which was then quantified using a NO analyser (NOA 280, Sievers).To determine total nitrite and nitrate concentrations, collectively termed 'NOx', samples will be added to 0.1 Mol/L vanadium (III) chloride in 1 M hydrochloric acid refluxing at 90°C under nitrogen.Nitrite concentrations will be then determined by addition of samples to 1.5 % potassium iodide in glacial acetic acid under nitrogen at room temperature.Finally, concentrations of nitrate will be calculated by subtraction of nitrite from NOx values 54 .Empty tubes will also be collected in each batch to allow for background nitrate levels to be measured.
-Analysis of brain natriuretic peptide (BNP): is a hormone secreted predominantly by the myocytes of the left ventricle in response to elevated wall stress.It has been shown to be a reliable diagnostic and prognostic tool in the management of congestive heart failure 55 .Recent studies show that BNP acts on the endothelium, for example, in mediating vasodilation through nitric oxide (NO), 56 possibly by influencing the activity of endothelial and inducible NO synthases (eNOS and iNOS, respectively), providing a link between natriuretic peptides and endothelial function.
-Analysis of platelet aggregation: we will study platelet function in trial participants to see whether nitrate supplementation attenuates ex vivo platelet aggregation in response to collagen and ADP.Blood will be drawn through a butterfly needle inserted into an antecubital vein of the subjects.Blood samples will be collected in the morning between 8 and 11 am and stored for analysis by PFA-100 in the lab.The PFA-100® (Dade Behring, Düdingen, Switzerland) is composed of a microprocessorcontrolled device and single-use test cartridges 57 .The test cartridges consist of a sample reservoir, a capillary and a membrane coated with 2 mg equine type I collagen and either 10 mg epinephrine bitartrate (EPI cartridge) or 50 mg adenosine 5'diphosphate (ADP cartridge).Blood is pipetted into the reservoir and aspirated through a capillary with a diameter of 200 mm with constant negative pressure resulting in high shear forces (5000-6000 s-1).The capillary ends in a membrane aperture with a diameter of 150 mm.Platelets adhere at the aperture where they are activated by the collagen and then aggregate.The two agonists epinephrine and ADP enhance aggregation.Finally, a platelet plug occludes the aperture and blood flow stops.The time measured in seconds from the beginning of the test until formation of an occluding platelet plug is called closure time (CT).If an occluding platelet plug does not form after 300 s, the analysis is stopped 58 .
-Analysis of plasma Arginine/Asymmetric dimethylarginine (ADMA): studying biochemical variables involved in the NO system such as Arginine and ADMA help us understand underling mechanisms of the effect of BRJ.ADMA is the endogenous inhibitor of the nitric oxide synthase (NOS) system.Arginine (Arg) is the substrate of NOS competing with ADMA.A low Arg/ADMA ratio is associated with the presence of vascular disease or endothelial dysfunction 59 .5 mL venous blood taken into tubes by venipuncture through a butterfly needle.To obtain the serum, a member of the research team will collect the samples and centrifuged at 3500 rpm for 10 minutes.
Then, samples will be stored at -80C till the analyses.Serum arginine and ADMA levels will be measured by HPLC (High-Performance Liquid Chromatography) using commercial kits (EUREKA srl-Laboratory Division, Chiaravalle, Italy, cat no: Z58010) with a programmable fluorescence detector.Results will be given in lmol/L 60 .
The 6-minute walk test distance (6MWD): we will be perform the 6MWD in an enclosed flat corridor, 30-m in length, following the American Thoracic Society guidelines 61 .Patients will be instructed to cover the longest distance possible in 6 minutes, with or without pause.
During the test, standardized encouragement will be given to patients.The test will be performed twice and the greater distance will be recorded for pre-and post-intervention.
Participants will wear the McRoberts MoveTest device during the test.
Oxygen saturation (SpO2) and heart rate will be monitored throughout the test using a pulse oximeter.During the test, a decrease in oxygen saturation level of equal to or less than 88% will be accepted as desaturation.The following parameters, including heart rate and oxygen saturation levels before and after the 6-minute walking test, the distance patients walk over 6 minutes, and the lowest and highest oxygen saturation levels during the test will be recorded during 6-minute walk distance test.
Analysis of oral Microbiota: Mouth swab will be taken and evaluated using 16S rRNA gene sequencing of total extracted saliva DNA on an Illumina MiSeq and analysed using an established pipeline in the section of Genomic Medicine.Comparison of microbial communities over time within patients and correlation with nitrate/nitrite levels across patients will allow identification of candidate organisms responsible for denitrification of nitrate to nitrite and NO.In addition, RT qPCR of functional genes (e.g.nirK, nirS and narG) in total extracted saliva RNA will demonstrate the expression of relevant microbial denitrification genes in response to nitrate.
Home self-activated BP monitoring: Participants will be given an Omron device to take home.They will be shown how to use it to measure their blood pressure and instructed to sit quietly for 5 minutes before they do so.They will be asked to take two readings (a minute apart) in the morning, the afternoon and the evening.They will be asked to do this for 4 days.The first day of readings will be discarded and the average of readings on days 2 to 4 taken.
In the event of a failure to do this at visit two they will be asked to repeat this before randomisation.In the event of a failure to do this before visit 3, the "clinic" blood pressure measure will be used for the primary endpoint.

Visit 2
Analysis of flow mediated dilatation: This will be measured using the EndoPAT-RHI system (Itamar Medical) which is an FDA-cleared non-invasive assessment of endothelial dysfunction.Measurements will be carried out in the non-dominant arm after a 20-min rest in the supine position in a quiet, temperature-controlled room (22-24°C).The EndoPAT test measure changes in vascular tone using sensors placed on the fingertips.These changes in arterial tone are elicited by creating a down-stream hyperemic response induced following a standard 5-minute occlusion of the brachial artery using a blood pressure cuff.Measurements from the contra-lateral arm are used to control for concurrent non-endothelial dependent changes in vascular tone to derive an index of endothelial function.

LOSS TO FOLLOW-UP
Subjects lost to follow up will be excluded from analysis.

TRIAL CLOSURE
The end of the trial is defined as the last patient's last visit.

STATISTICS AND DATA ANALYSIS
The study is powered for the primary outcome measure of change in home systolic BP response at 12 wk from baseline.Data from the ON-EPIC trial showed a fall of an (mean ± SD) 5  3.7mmHg.Taking a power of 90% and significance level of 0.05, we would require 32 patients within each group to identify a 3mmHg fall with treatment.Allowing for a 10% dropout rate we will recruit 72 patients in total.
For the exploratory outcomes (blood tests, microbiome, endothelial function), will be analysed in at least 30 subjects.This is based on the number which produced a positive result in our previous trial (ON-EPIC) which investigated the effect of nitrate supplementation and pulmonary rehabilitation impact on flow mediated dilatation.Additionally, a previous study estimated of an expected increase in FMD of 25% from 5% to 6.7% after 6 weeks of beetroot juice intake in healthy volunteers, a sample size of 20 was needed to detect a difference with a power of 0.8 at significance level of α = 0.05 [assuming an SD of ultrasound FMD of 2.5] for a within-group comparison with baseline 52 .
We will analyse all data using IBM statistical package for the social sciences (SPSS) software.For each group of the study, we will summarise baseline demographic and clinical variables.
We will use ANCOVA to compare blood pressure and other continuous variables between study groups, adjusting for baseline values.We will express all data, as mean ± SEM unless otherwise stated.In all cases, P<0.05 is considered statistically significant.
Data and all appropriate documentation will be stored for a minimum of 10 years after the completion of the study, including the follow-up period.

RISK ASSESSMENT
The study involves the use of a nutritional supplement and is considered to be low risk.

MONITORING AT STUDY COORDINATION CENTRE
Data will be collected on a paper CRF and then collected into a custom Excel database and analysed using SPSS.The study will be subject to audit by the Imperial College Joint Research Office.

ETHICS APPROVAL
The Study Coordination Centre has obtained approval from the London -West London & GTAC Research Ethics Committee (REC) and Health Regulatory Authority (HRA).The study must also receive confirmation of capacity and capability from each participating NHS Trust before accepting participants into the study or any research activity is carried out.The study will be conducted in accordance with the recommendations for physicians involved in Primary endpoint Change in home monitored BP .
TITLEOral Nitrate supplementation and blood pressure in COPDa randomised clinical trial (The ON-BC study).DESIGN A double blind, placebo-controlled, parallel group study of the effect of nitrate supplementation on markers of cardiovascular risk in patients with stable COPD AIMS To establish whether, in people with stable COPD, dietary nitrate supplementation, in the form of beetroot juice administered for 3 months will: (1) reduce BP, (2) improve exercise capacity (3) improve endothelial function (4) inhibit platelet aggregation OUTCOME MEASURES ELIGIBILITY Inclusion adult patients (>21 yrs) with stable COPD GOLD I-IV established on stable pharmacotherapy for COPD -Systolic blood pressure >130 mmHg Exclusion -Unable to provide informed consent.

6 week phone call -reinforce drinking
The Chief Investigator must also notify the Sponsor of all SAEs.Local investigators should report any SAEs as required by their Local Research Ethics Committee, Sponsor and/or Research & Development Office.