Beet the Cold: The Effect of Inorganic Nitrate Supplementation on Peripheral Blood Flow and Pain in Individuals With Raynaud's Phenomenon. A Pilot, Double-blind, Placebo Controlled, Randomised Crossover Trial
Phase: NA
Conditions: Raynaud Phenomenon
Sponsor: University of Portsmouth

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Study Title: Beet the cold: The effect of inorganic nitrate supplementation on peripheral 
blood flow and pain in individuals with Raynaud’s phenomenon. A pilot, double-blind, 
placebo controlled, randomised crossover trial.  
Internal Reference No: N/A 
Ethics Ref: 17/SC/0148 
Date and Version No: 15/08/2017 version 1.3       
Chief Investigator: 
Dr Anthony Shepherd  
Investigators:  
See protocol development team below  
Sponsor:  
Portsmouth Hospitals NHS Trust  
Funder (if applicable): 
Grant under review by Scleroderma and Raynaud’s UK 
Signatures:  
 
Protocol Development Team:   
Chief Investigator   
- Dr Anthony Shepherd 
(University of Portsmouth) 
Co-investigator 1  
 
- Dr Clare Eglin   
 
(University of Portsmouth) 
Co- investigator 2 
 
- Dr Joe Costello  
 
(University of Portsmouth) 
Co- investigator 3 
 
- Dr Zoe Saynor  
 
(University of Portsmouth) 
Co- investigator 4 
 
- Dr Paul Gorczynski  
(University of Portsmouth) 
Co- investigator 5 
 
- Dr Heather Massey  
(University of Portsmouth) 
Co- investigator 6 
 
- Dr Steven Young Min 
(Queen Alexandra Hospital) 
Co- investigator 7 
 
- Mrs Jan MacDonald 
(Patient and public involvement 
member)   
Co- investigator 8 
 
- Dr Mark Gilchrist  
(Royal 
Devon 
and 
Exeter                     
Hospital) 
Co- investigator 9 
 
- Dr Stephen Bailey   
(Loughborough University) 

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TABLE OF CONTENTS 
 
1. 
AMENDMENT HISTORY ................................................................................................ 5 
2. 
SYNOPSIS ..................................................................................................................... 6 
3. 
ABBREVIATIONS ........................................................................................................... 8 
4. 
BACKGROUND AND RATIONALE ................................................................................. 9 
5. 
PRELIMINARY STUDIES AND EXPERIENCE OF INVESTIGATORS .......................... 11 
6. 
AIMS AND OBJECTIVES ............................................................................................. 12 
6.1 
Primary Objective .................................................................................................. 12 
Primary objectives: Does CBJ increase peripheral blood flow, improve endothelial function 
and increase skin temperature compared to placebo in people with RP. .......................... 12 
6.2 
Secondary Objectives ............................................................................................ 12 
7. 
STUDY DESIGN ........................................................................................................... 12 
7.1 
Summary of Study Design ..................................................................................... 12 
7.2 
Primary and Secondary Endpoints/Outcome Measures ......................................... 14 
8. 
STUDY PARTICIPANTS ............................................................................................... 15 
8.1 
Study Setting ......................................................................................................... 15 
8.2 
Overall Description of Study Participants ............................................................... 15 
8.3 
Eligibility Criteria .................................................................................................... 15 
9. 
SAMPLING ................................................................................................................... 16 
10. 
STUDY PROCEDURES ............................................................................................ 16 
10.1    Recruitment ........................................................................................................... 17 
10.2   Screening and Enrolment ....................................................................................... 17 
10.3 Randomisation.......................................................................................................... 18 
10.4 Study Assessments .................................................................................................. 18 
10.5 
Discontinuation/Withdrawal of Participants from Study Treatment ......................... 20 

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10.6 
Definition of End of Study ...................................................................................... 20 
11. 
INTERVENTIONS ..................................................................................................... 20 
11.1 
Description of Study Intervention / Treatment ........................................................ 20 
11.2     Adherence to Study Treatment ............................................................................. 21 
11.3     Accountability of the Study Treatment .................................................................. 21 
11.4     Concomitant Medication / Therapies ..................................................................... 21 
12. 
ASSESSMENT OF SAFETY (if applicable) ............................................................... 22 
12.1 
Definitions .............................................................................................................. 22 
12.2 
Reporting Procedures for Serious Adverse Events ................................................ 22 
12.3 
Recording and Reporting Procedures for All Adverse Events ................................ 23 
13. 
Data Handling and Record Keeping .......................................................................... 23 
13.1 
Data Collection Forms ........................................................................................... 23 
13.2     Data Management ................................................................................................ 23 
14. 
DATA ANALYSIS ...................................................................................................... 23 
14.1      Description of Analysis Populations ..................................................................... 23 
14.2      Analysis of Endpoints .......................................................................................... 23 
14.3     Procedure for Dealing with Missing, Unused and Spurious Data .......................... 24 
14.4     Procedures for Reporting any Deviation(s) from the Original Statistical Analysis 
Plan .................................................................................................................................. 24 
14.5      Interim analysis and criteria for early study termination ........................................ 24 
15. 
ETHICS ..................................................................................................................... 24 
15.1 
Participant Confidentiality ...................................................................................... 25 
15.2     Other Ethical Considerations ................................................................................ 25 
15.3     Declaration of Helsinki .......................................................................................... 25 
15.4     ICH Guidelines for Good Clinical Practice ............................................................. 25 
16. 
PATIENT PUBLIC INVOLVEMENT (PPI) .................................................................. 26 

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16.1    Study design .......................................................................................................... 26 
16.2    Study implementation ............................................................................................ 26 
16.3    Dissemination ........................................................................................................ 26 
17. 
FINANCING AND INSURANCE ................................................................................ 27 
17.1    Research Costs ..................................................................................................... 27 
17.2    Service Support Costs ........................................................................................... 27 
No service support costs are being sort. ........................................................................... 27 
17.3    Excess Treatment Costs ........................................................................................ 27 
17.4    Study Sponsorship ................................................................................................ 28 
18. 
TIMETABLE AND ORGANISATIONAL CHART ........................................................ 28 
19. 
RESOURCES, EQUIPMENT AND PHYSICAL FACILITIES ...................................... 30 
20. 
DISSEMINATION AND OUTCOME ........................................................................... 30 
21. 
REFERENCES .......................................................................................................... 31 
22. 
APPENDIX 1     SCHEDULE OF PROCEDURES ..................................................... 36 
23. 
APPENDIX 2    STUDY FLOW CHART ..................................................................... 36 
24. 
APPENDIX 3    PARTICIPANT INFORMATION SHEET ........................................... 38 
25. 
APPENDIX 4    INFORMED CONSENT FORM ......................................................... 38 
26. 
APPENDIX 5    SAMPLE QUESTIONNAIRES .......................................................... 38 
27. 
APPENDIX 6    SAMPLE DATA COLLECTION FORMS ........................................... 38 
28. 
APPENDIX 
7 
 
 
 
DRUG 
INFORMATION 
(SUMMARY 
OF 
PRODUCT 
CHARACTERISTICS) .......................................................................................................... 38 
29. 
APPENDIX 8 MANUFACTURERS BROCHURE FOR NOVEL EQUIPMENT ............ 38 
30. 
APPENDIX 9 CONTRACTUAL AGREEMENTS WITH OUTSIDE CONSULTANTS / 
COLLABORATORS / INSTITUTIONS (E.G. INDUSTRY, CONTRACT RESEARCH 
ORGANISATIONS) .............................................................................................................. 38 
 
 

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1. 
AMENDMENT HISTORY 
Amendment 
No. 
Protocol 
Version 
No. 
Date 
issued 
Author(s) of changes 
Details of Changes 
made 
1 
1.2 
 
Dr A I Shepherd 
Added 
sodium 
nitroprusside 
to 
the 
iontophoresis 
technique.  
2 
1.3 
 
Dr A I Shepherd 
“participants who do 
not 
want 
their 
GP 
informed 
 will 
be 
provided with the GP 
letter” 

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2. 
SYNOPSIS 
Study Title 
Beet the cold: The effect of inorganic nitrate supplementation on 
peripheral blood flow and pain in individuals with Raynaud’s 
phenomenon. A pilot, double-blind, placebo controlled, randomised 
crossover trial. 
 
Internal ref. no. 
n/a 
 
Problem statement 
Raynaud’s Phenomenon (RP) can cause significant discomfort and pain 
to individuals during a vasospasm. Dietary nitrate appears to offer a 
simple, low cost means of modifying blood flow to the peripheries and, 
ultimately, reducing both the discomfort and pain experienced by 
individuals with RP. This study will also advance our understanding of 
the aetiology and pathophysiology of RP, specifically the role that the 
nitrate-nitrite-nitric oxide pathway might play in modulating RP 
symptoms. An understanding of the effects of concentrated beetroot 
juice on microvascular blood flow and pain may lead to a range of 
simple, low cost and effective therapeutic interventions to prevent and 
treat episodes of RP.  
 
Research question / 
hypothesis 
Primary objectives: Does concentrated beetroot juice (CBJ) increase 
peripheral blood flow, improve endothelial function and increase skin 
temperature compared to placebo in people with RP. Secondary 
objectives: 1) Does CBJ reduce perceived discomfort and pain and, 2) is 
our protocol acceptable to individuals with RP (i.e. cold water stimulus, 
CBJ palatability) and feasible to perform in a future multi-centre 
randomised control trial (RCT) and, 3) to establish recruitment and 
retention rates. Resulting estimated effect size in relation to their 
confidence intervals from our primary objective(s) measures will be 
utilised to identify our primary outcome measure and to power a larger, 
definitive, multi-centre randomised, control trial. 
 
Study Design 
The proposed study will be a pilot, randomised placebo-controlled, 
double-blind, crossover trial. 
 

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Study Participants 
Individuals with primary and secondary Raynaud’s phenomenon.  
 
Planned Sample Size 
Twenty-five individuals with RP would need to be recruited in order to 
estimate a moderate to large effect size in a pilot study. Therefore, we 
aim to recruit 30 individuals (at a rate of 1 per week) to account for drop 
outs. 
 
Follow-up duration 
2 weeks 
 
Planned Study 
Period 
1 year 
 
Primary Objective 
Primary objectives: Does CBJ increase peripheral blood flow, improve 
endothelial function and increase skin temperature compared to placebo 
in people with RP. 
 
Secondary 
Objectives 
Secondary objectives: 1) Does CBJ reduce perceived discomfort and 
pain and, 2) is our protocol acceptable to individuals with RP (i.e. cold 
water stimulus, CBJ palatability) and feasible to perform in a future 
multi-centre randomised control trial (RCT) and, 3) to establish 
recruitment and retention rates. Resulting estimated effect size in 
relation to their confidence intervals from our primary objective(s) 
measures will be utilised to identify our primary outcome measure and to 
power a larger, definitive, multi-centre randomised, control trial.  
 
 
Primary Endpoint 
N/A: pilot study.  
 
Secondary 
Endpoints 
N/A: pilot study. 
 
Intervention (s) 
4 weeks of CBJ supplementation. 2 with nitrate rich CBJ and 2 weeks 
with nitrate depleted CBJ.  
 

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3. 
ABBREVIATIONS 
 
Ach 
 
= Acetylcholine 
CBJ 
 
= Concentrated beetroot juice 
CRF 
 
= Clinical records folder  
eGFR   
= Estimate of glomerular filtration rate 
flux/MAP  
= Cutaneous vascular conductance 
FTE  
 
= Full time equivalent 
GTN  
= Glyceryl trinitrate 
NHS  
= National Health Service 
NO• 
 
= Nitric oxide  
PCPI   
= Patient, carer and public involvement 
RCT 
 
= Randomised control trial  
REC  
 
= Research Ethics Committee 
RP 
 
= Raynaud ’s phenomenon  
SF-36  
= Short Form Health Survey 
TBC  
 
= Total blood count 
 

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4. 
BACKGROUND AND RATIONALE 
Raynaud’s phenomenon (RP) is characterised by a recurrent transient vasospasm of the 
fingers or toes in response to a cold or stressful stimulus (Wigley 2002). Nitric oxide (NO•) is 
a known vasodilator and NO• donors, such as Glyceryl Trinitrate (GTN), improve blood flow 
in patients with RP (Anderson et al., 2002; Teh et al., 1995) and in cold sensitive individuals 
(Hope, Eglin, Golden, & Tipton, 2014) (Figure 1, see accompanying document). However, 
individuals develop a tolerance to GTN and show diminishing vasodilatory effects with 
chronic treatment (Needleman & Johnson, 1973). In addition, the deleterious side effects such 
as headaches (Hsi, Roshandel, Singh, Szombathy, & Meszaros, 2005; Wigley 2002) means 
that organic nitrates (i.e. GTN and isosorbide mononitrate) are not optimal long-term 
therapies for RP. Alternative treatments therefore, warrant further investigation. 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1. Effect of GTN on foot skin temperature of cold sensitive individuals during a 2 min 
cold challenge test. (A) individual thermogram taken after 5 min of rewarming following a 2 
min immersion in 15°C water with GTN and (B) with placebo (C) depicts mean toe 
temperature before during and after the cold challenge with placebo (closed circles) and GTN 
(open circles; n=6) (Hope et al., 2014).  
Diets rich in fruit and vegetables have been shown to be effective in reducing blood pressure 
(Appel et al., 1997; Rouse, Beilin, Armstrong, & Vandongen, 1983). In addition, they lower 
the risk of morbidity and mortality from cardiovascular disease (Bazzano et al., 2001; 
20
22
24
26
28
30
32
34
36
38
Baseline
Rewarming	Time	(min)
Placebo	/	GTN
Cold	challenge
10
5
0
**
*
Skin	Temperature	(°C)
C 
A
BC
D
A
BC
D
A
BCA 
A
BC
D
A
BCB 

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Joshipura et al., 1999; Joshipura et al., 2001) and are thought to be beneficial to 
cardiovascular health due to their vasodilatory effects (M Gilchrist, Winyard, & Benjamin, 
2010). As diet exhibits such tremendous intra- and inter-individual variation, elucidating 
which components of such a diet are responsible for this effect is difficult. There is a growing 
weight of evidence from both human and animal studies that nitrate and nitrite derived from 
the diet can serve as a source for nitric oxide (NO; please see below), particularly where it is 
deficient (V. Kapil, Weitzberg, Lundberg, & Ahluwalia).  Indeed, the greatest protective 
effect on cardiovascular disease is to be found in those diets with the greatest consumption of 
green leafy and or cruciferous vegetables (Joshipura et al., 1999; Joshipura et al., 2001) which 
typically have high nitrate content.   
NO is produced in the body in two ways.  The first requires the availability the amino acid L-
arginine, molecular oxygen, and families of enzymes, the nitric oxide synthases ((NOS); 
(Moncada & Higgs, 1993; Moncada, Palmer, & Higgs, 1989)); that is the NOS pathway. The 
second pathway is independent of the NOS pathway and involves the stepwise enzymatic and 
chemical reduction of inorganic nitrate to nitrite (Benjamin et al., 1994; Björne et al., 2004; 
Lundberg, Weitzberg, & Gladwin, 2008; Zweier, Wang, Samouilov, & Kuppusamy, 1995).  
A major source of nitrite in humans is the reduction of dietary nitrate by facultative anaerobic 
bacteria in the mouth (Duncan et al., 1995). The remaining nitrite is then absorbed into the 
circulation where it acts as a storage pool for subsequent NO• production, which is expedited 
in hypoxaemia (Cosby et al., 2003). 
Localised hypoxemia such as that observed in the digital vasculature of individuals with RP 
(Wigley 2002) is a potential therapeutic target for dietary nitrate supplementation. In contrast 
to organic nitrates (GTN), inorganic nitrate (in the form of beetroot juice) does not cause the 
same negative side effects (Anthony I. Shepherd et al., 2015) or demonstrate tachyphylaxis 
(Vanhatalo et al., 2010) whilst it does notably improve skin blood flow (Levitt, Keen, & 
Wong, 2015), microvascular function (Hobbs et al., 2013; Vikas Kapil, Khambata, 
Robertson, Caulfield, & Ahluwalia, 2015) and lower blood pressure (BP) in healthy 
individuals and chronic conditions such as hypertension (Vikas Kapil et al., 2015), peripheral 
arterial disease, (Kenjale et al., 2011) heart failure (Zamani et al., 2015) and chronic 
obstructive pulmonary disease (Berry et al., 2014). Thus concentrated beetroot juice (CBJ) 

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may offer an inexpensive, safe and potentially effective intervention to improve the pain and 
reduced peripheral blood flow characterising individuals with RP.  
RP can cause significant discomfort and pain to individuals during a vasospasm. Dietary 
nitrate appears to offer a simple, low cost means of modifying blood flow to the peripheries 
and, ultimately, reducing both the discomfort and pain experienced by individuals with RP. 
This study will also advance our understanding of the aetiology and pathophysiology of RP, 
specifically the role that the nitrate-nitrite-nitric oxide pathway might play in modulating RP 
symptoms. An understanding of the effects of CBJ on microvascular blood flow and pain may 
lead to a range of simple, low cost and effective therapeutic interventions to prevent and treat 
episodes of RP.  
The proposed study will be a pilot, randomised placebo-controlled, double-blind, crossover 
trial. The effect of acute (1 day) and chronic (2 weeks) CBJ supplementation on 
microvascular function (skin blood flow and speed of rewarming of the hand and foot) in 
people with RP will be compared to a placebo (CBJ with the nitrates removed). A summary of 
the main risks of this study include; a well-recognised side effect of beetroot consumption, is 
that it may turn urine and faeces red.  This is not harmful and will pass rapidly after 
discontinuation of the juice.  Subjects will be told of this during the consent visit. Intravenous 
access can cause discomfort associated with insertion of the needle. We will take a blood 
sample on three occasions. Some discomfort can be caused during iontophoresis (an itchy 
feeling) and some pain may be felt when immersing the hand and foot in to 15°C water for 2 
minutes. Participants will be made aware that they can withdraw at any stage in accordance 
with GCP and informed consent.  
5. 
PRELIMINARY STUDIES AND EXPERIENCE OF INVESTIGATORS 
Dr’s Shepherd, Gilchrist and Bailey are leaders in the field of nitrate supplementation 
(particularly beetroot) and have over 70 peer reviewed publications between them in the field. 
The Department of Sport and Exercise Science (DSES) at the University of Portsmouth (UoP) 
has a world leading environmental group. Dr’s Eglin and Costello from this group are leaders 
in this field and will be helping with controlling environmental conditions and the specific 
tests for this study (i.e. iontophoresis and cold sensitivity tests). Dr Gorczynski has extensive 
experience of running focus groups and performing qualitative research in patient groups. 

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Dr’s Young Min and Gilchrist are clinicians with extensive experience of running clinical 
trials. See reference list for publications and attached CV’s. 
6. 
AIMS AND OBJECTIVES 
The overall aim is to establish if this protocol is acceptable and feasible to perform whilst 
establishing effect sizes for our primary and secondary outcomes to power a larger definitive 
trial.  
6.1 
Primary Objective 
Primary objectives: Does CBJ increase peripheral blood flow, improve endothelial function 
and increase skin temperature compared to placebo in people with RP. 
6.2 
Secondary Objectives 
Secondary objectives: 1) Does CBJ reduce perceived discomfort and pain; 2) is our protocol 
acceptable to individuals with RP (i.e. cold water stimulus, CBJ palatability) and feasible to 
perform in a future multi-centre randomised control trial (RCT); and 3) to establish 
recruitment and retention rates. Resulting estimated effect sizes in relation to their confidence 
intervals from our primary objective(s) measures will be utilised to identify our primary 
outcome measure and to power a larger, definitive, multi-centre randomised, control trial.  
7. 
STUDY DESIGN 
7.1 
Summary of Study Design 
The proposed study will be a pilot, randomised placebo-controlled, double-blind, crossover 
trial. The effect of acute and chronic CBJ supplementation on microvascular function in 
people with RP will be compared to a placebo (CBJ with the nitrates removed). The placebo 
is the same in colour, odour, texture and taste as CBJ (Mark Gilchrist et al., 2014) but does 
not cause an increase in plasma nitrate or nitrite (Shepherd et al., 2016). The volunteers will 
be given a full briefing about the study and given the participant information sheet before we 
seek their written consent (see below). The individual’s severity of RP symptoms will be 
established according to the International Consensus Criteria for Diagnosing RP (Maverakis 
et al., 2014).  

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Participants and Recruitment: Twenty-five individuals with RP would need to be recruited in 
order to estimate a moderate to large effect size in a pilot study (Johanson & Brooks, 2009). 
Therefore, we aim to recruit 30 individuals (at a rate of 1 per week) with RP to account for a 
15% drop-out rate (typically seen in our clinical trials). Participants will be recruited from a 
database of known individuals with RP who attend outpatient clinics at the Queen Alexandra 
Hospital (Portsmouth) and via posters and word of mouth within the University of 
Portsmouth. The participants will attend the lab on five occasions during which cold 
sensitivity and endothelial function (iontophoresis) will be assessed (Spinnaker Building, 
University of Portsmouth).   
Consent and Screening: Visit 1 will act as a screening, consent and baseline assessment (3.5-
4 hours).  
Participants will be given the opportunity to ask any questions they may have after reading 
the participant information sheet.  A standard medical history and clinical examination will be 
undertaken by a research nurse or member of the research team, including for example height, 
mass, electrocardiogram, ankle-brachial pressure index and blood sample collection for lipids 
and biochemical markers, urea and electrolytes, and liver function tests. Seated resting blood 
pressure measurements will be performed to calculate systolic, diastolic blood pressure and 
cutaneous vascular conductance (flux/MAP). The participants will then undertake an 
endothelial function (iontophoresis) test in an ambient temperature of 23°C followed by a 
cold challenge in an ambient temperature of 30°C (both described below) as baseline 
measures. Quality of life will be examined through the provision of the Medical Outcomes 
Study 36-item Short Form Health Survey (SF-36). |Following this visit, a letter will be sent to 
their General Practitioner (GP), confirming their participation within the trial. Participants who 
do not want their GP informed will be provided with the GP letter.     
Randomisation: During the first visit the participants will be randomised by a nurse to begin 
either the CBJ or placebo arm of the study. A computer programme will be utilised to 
randomly allocate to treatment order, whilst stratifying for disease type and gender. 
Participants will be provided with instructions and their first acute dose to take away with 
them.  

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Visit 2 and 4 (acute supplementation visits ~ 1.5-2 hours). One and a half hours before visits 
2 and 4, participants will be instructed to ingest an acute dose of 140 ml of either CBJ 
(delivers ~ 12.4 mmol·L1 of inorganic nitrate) or placebo (~ 0.002 mmol·L1 Beet It, Heartbeet 
Ltd.). The primary objectives (peripheral blood flow, endothelial function and skin 
temperature; see below for details) will be assessed ~ 1.5 hours after arrival in the research 
facility. The timing of the supplement is key to ensure plasma / saliva nitrate and nitrite are 
elevated throughout the testing period (Webb et al., 2008). On arrival at the laboratory, the 
participants will have their BP measured and a ~ 20 ml venous blood sample taken. The 
participants will then undertake an endothelial function (iontophoresis) test in an ambient 
temperature of 23°C followed by a cold challenge in an ambient temperature of 30°C (both 
described below). The SF-36 will also be measured here. Visits 3 and 4 (crossover period) 
will be separated by at least 7 days to allow washout (Wylie et al., 2013) and all visits will be 
conducted at the same time of day in a counter-balanced order at a time suitable for the 
participants. 
Visit 3 and 5 (chronic supplementation visits ~ 1.5-2 hours). Visits 3 and 5 will be identical in 
nature to visit 2 and 4, however, following chronic supplementation of 70 ml·day-1 of either 
CBJ or placebo for 14 days (Vanhatalo et al., 2010; Wylie et al., 2013). With consent, 
participants will be reminded to take the juice via text message or voice mails. Please see the 
accompanying documents for the participant flow through the study. 
The participants diet will be standardised prior to testing and they will be asked to abstain 
from using antibacterial mouth wash for 7 days prior to each test and throughout the period of 
testing as this has been shown to reduce the concentration of oral microflora that are 
responsible for the reduction of nitrate to nitrite (Govoni, Jansson, Weitzberg, & Lundberg, 
2008). Participants will also be asked to refrain from using other medicines during the testing 
period that may affect blood flow or the release of nitrite from the CBJ with such as organic 
nitrates, proton pump inhibitors, ranitidine and nicorandil. 
7.2 
Primary and Secondary Endpoints/Outcome Measures 
This is a pilot study and as such, primary outcome measures are N/A. See above for our 
primary and secondary objectives. These will be used to inform what our primary outcomes 
will be in a future trial.  

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8. 
STUDY PARTICIPANTS 
8.1 
Study Setting 
Firstly, the Extreme Environments Laboratory at the University of Portsmouth has a 30 year 
history of conducting innovative human research trials. The laboratory contains some of the 
world’s leading facilities for conducting environmental research including three bespoke 
environmental chambers, with temperature, humidity, solar, wind and hypoxia control, and a 
research group involving over 20 academics and researchers. Two of the applicants (Drs 
Eglin and Costello) have extensive experience of conducting examinations of cold sensitivity 
and endothelial function in both healthy and clinical populations (Eglin, Golden, & Tipton, 
2013; Hope et al., 2014; Maley, House, Tipton, & Eglin, 2015). Dr. Saynor also has 
significant experience working with patient groups, including several rheumatic conditions 
and in assessing endothelial function etc. in current research. Presently, the team are 
completing similar research on individuals with cold sensitivity and the proposed 
methodology, including infrastructure, equipment and expertise, is therefore feasible and 
requires no additional training or time to develop. 
8.2 
Overall Description of Study Participants 
Participants with a clinical diagnosis of Primary or Secondary Raynaud’s Phenomenon.  
8.3 
Eligibility Criteria 
Inclusion Criteria 
The participants must meet ALL of the following criteria to be considered eligible for the 
study: 
 
Male or Female, aged 18 years or above. 
 
Diagnosed with Raynaud’s Phenomenon.  
 
Participant is willing and able to give informed consent for participation in the study. 
 
Exclusion Criteria 
The participant may not enter the study if ANY of the following apply: 
 Patients with significant renal impairment (eGFR<30),  

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 Uncontrolled hypertension,  
 Taking regular organic nitrates, nicorandil, or thiazolidinidiones,  
 or any medication which may interfere with data interpretation or safety,  
 who have had a myocardial infarction or cerebro−vascular event,  
 who smoke,  
 or any other serious medical condition which would interfere with data interpretation or 
safety will be excluded from participation. 
9. 
SAMPLING 
Twenty-five individuals with RP would need to be recruited in order to estimate a moderate 
to large effect size in a pilot study (Johanson & Brooks, 2009). Therefore, we aim to recruit 
30 individuals (at a rate of 1 per week) with RP to account for a 15% drop-out rate (typically 
seen in our clinical trials). We will utilise an opportunistic sampling process, from both 
clinics and posters and word of mouth.  
10. 
STUDY PROCEDURES 
Endothelial function test: Following a 30 minute acclimatisation period, acetylcholine (ACh) 
and sodium nitroprusside will be delivered transdermally using iontophoresis to three sites, 
the middle phalanx of the middle finger of the left hand, the dorsal aspect of the left foot and 
the volar aspect of the left forearm. Cutaneous vascular conductance and skin temperature 
will be measured to determine the dose response to ACh at each site separately (Maley et al., 
2015).  
Cold challenge test: Following a 15 min acclimatisation period, participants will undertake 
light exercise on a cycle ergometer for 12 min (maximum of 50 W, to elevate their core 
temperature by approximately 0.3°C). Prior exercise has been found to increase the 
reproducibility of the test (Eglin et al. 2005) by removing the effect of varying central 
vasoconstrictor tone. Participants will then undertake a standardised cold challenge test by 
immersing their hand and foot separately into 15°C water for 2 minutes followed by 
spontaneous rewarming for 10 minutes (Eglin et al., 2013; Hope et al., 2014). Skin 

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temperature (from a thermal imaging camera), peripheral (skin) blood flow (laser Doppler) 
thermal sensation, comfort and pain will be measured throughout the test.  
Quality of life: 
Quality of life will be examined through the provision of the Medical Outcomes Study 36-
item Short Form Health Survey (SF-36) (Ware Jr & Sherbourne, 1992). The SF-36 measures 
eight aspects of quality of life: physical functioning, bodily pain, role limitations due to 
physical health problems, role limitations due to personal or emotional problems, emotional 
well-being, social functioning, energy/fatigue, and general health perceptions. The SF-36 also 
provides a single item score that indicates a perceived change in health. The SF-36 has been 
shown to be reliable with Cronbach alpha scores ranging from .80 to .92 for various aspects 
of quality of life (Ruta, Abdalla, Garratt, Coutts, & Russell, 1994). This will be measured at 
each experimental visit post CBJ or placebo consumption.   
Qualitative analysis (acceptability):  
Semi-structured interviews will be used to examine the acceptability of CBJ and placebo 
supplementation. Specifically, interviews will explore research methods / testing procedures 
and the participant experience of consuming beetroot juice regularly. Approximately 15-20 
semi-structured interviews will be necessary to reach a point of data saturation where no new 
information is provided. Interviews will be conducted by a researcher with experience (co-
applicant) in qualitative research methods and methodologies. Interviews will be transcribed 
verbatim and analysed thematically (Braun & Clarke, 2006).   
10.1    Recruitment 
Participants will be recruited from a database of known individuals with RP who attend 
outpatient clinics at the Queen Alexandra Hospital (Portsmouth) and via posters and word of 
mouth within the University of Portsmouth. If this study is funded, a research nurse will 
utilised to find potential participants.  
10.2   Screening and Enrolment 
Consent and Screening: Visit 1 will act as a screening, consent and baseline assessment (3.5-
4 hours).  

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Participants will be given the opportunity to ask any questions they may have after reading 
the participant information sheet.  A standard medical history and clinical examination will be 
undertaken by a research nurse or member of the research team, including for example height, 
mass, electrocardiogram, ankle-brachial pressure index and blood sample collection for lipids 
and biochemical markers, urea and electrolytes, and liver function tests. Seated resting blood 
pressure measurements will be performed to calculate systolic, diastolic blood pressure and 
cutaneous vascular conductance (flux/MAP). If this study is funded, a research nurse will 
utilised to find and screen potential participants.  
10.3 Randomisation 
During the first visit the participants will be randomised by a nurse to begin either the CBJ or 
placebo arm of the study. A computer programme will be utilised to randomly allocate to 
treatment order, whilst stratifying for disease type and gender. Participants will be provided 
with instructions and their first acute dose to take away with them. 
10.4 Study Assessments 
Endothelial function test: Following a 30 minute acclimatisation period, acetylcholine (ACh) 
and sodium nitroprusside will be delivered transdermally using iontophoresis to three sites, 
the middle phalanx of the middle finger of the left hand, the dorsal aspect of the left foot and 
the volar aspect of the left forearm. Cutaneous vascular conductance and skin temperature 
will be measured to determine the dose response to ACh at each site separately (Maley et al., 
2015).  
Cold challenge test: Following a 15 min acclimatisation period, participants will undertake 
light exercise on a cycle ergometer for 12 min (maximum of 50 W, to elevate their core 
temperature by approximately 0.3°C). Prior exercise has been found to increase the 
reproducibility of the test (Eglin et al. 2005) by removing the effect of varying central 
vasoconstrictor tone. Participants will then undertake a standardised cold challenge test by 
immersing their hand and foot separately into 15°C water for 2 minutes followed by 
spontaneous rewarming for 10 minutes (Eglin et al., 2013; Hope et al., 2014). Skin 
temperature (from a thermal imaging camera), peripheral (skin) blood flow (laser Doppler) 
thermal sensation, comfort and pain will be measured throughout the test.  

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Secondary Objectives 
Quality of life: 
Quality of life will be examined through the provision of the Medical Outcomes Study 36-
item Short Form Health Survey (SF-36) (Ware Jr & Sherbourne, 1992). The SF-36 measures 
eight aspects of quality of life: physical functioning, bodily pain, role limitations due to 
physical health problems, role limitations due to personal or emotional problems, emotional 
well-being, social functioning, energy/fatigue, and general health perceptions. The SF-36 also 
provides a single item score that indicates a perceived change in health. The SF-36 has been 
shown to be reliable with Cronbach alpha scores ranging from .80 to .92 for various aspects 
of quality of life (Ruta et al., 1994). This will be measured at each experimental visit post 
CBJ or placebo consumption.   
Qualitative analysis (acceptability):  
Semi-structured interviews will be used to examine the acceptability of CBJ and placebo 
supplementation. Specifically, interviews will explore research methods / testing procedures 
and the participant experience of consuming beetroot juice regularly. Approximately 15-20 
semi-structured interviews will be necessary to reach a point of data saturation where no new 
information is provided. Interviews will be conducted by a researcher with experience (co-
applicant) in qualitative research methods and methodologies. Interviews will be transcribed 
verbatim and analysed thematically (Braun & Clarke, 2006).   
Biochemical analysis: 
Blood / saliva samples will be taken and assessed prior to testing on each study visit. Blood 
samples for plasma nitrate/nitrite will be taken in chilled Lithium Heparin tubes and EDTA 
tubes for assessment of oxidative stress markers. The blood / saliva needs to be placed in a 
chilled (4oC) centrifuge and spun at 4000g for 10 minutes immediately on collection. Once 
spun the plasma or saliva will be pipetted into aliquots. The samples will be placed in a -20oC 
freezer prior to transfer to a -80oC freezer for storage at the end of each day.  
Deproteinised samples will be analysed for nitrite and nitrate concentration using a Sievers nitric 
oxide analyser (Sievers NOA 280i, Analytix Ltd, Durham, UK) and a modification of our 
previous chemiluminescence technique. Elisa’s will be utilised using commercially available 

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assays for markers of oxidative stress such as glutathione and nitrotyrosine. We will also 
measure plasma for the quantification of endothelin 1.  
10.5 
Discontinuation/Withdrawal of Participants from Study Treatment 
Participants may be withdrawn from treatment, or from the study as a whole; if they ask to 
withdraw, if participant safety is in question or if the study is stopped early.   
10.6 
Definition of End of Study  
The end of study will be the date of the last visit of the last participant.  
11. 
INTERVENTIONS 
11.1 
Description of Study Intervention / Treatment 
Visit 2 and 4 (acute supplementation visits ~ 3 hours). One and a half hours before visits 2 
and 4, participants will be instructed to ingest an acute dose of 140 ml of either CBJ (delivers 
~ 12.4 mmol·L1 of inorganic nitrate) or placebo (~ 0.002 mmol·L1 Beet It, Heartbeet Ltd.). 
The primary objectives (peripheral blood flow, endothelial function and skin temperature; see 
below for details) will be assessed ~ 1.5 hours after arrival in the research facility. The timing 
of the supplement is key to ensure plasma nitrate and nitrite are elevated throughout the 
testing period (Webb et al., 2008). On arrival at the laboratory, the participants will have their 
BP measured and a ~ 20 ml venous blood sample taken for determination of plasma [nitrate], 
[nitrite] and nitrotyrosine and protein carbonyl concentrations (which are clinically relevant 
markers of oxidative stress (Frijhoff et al., 2015)). The participants will then undertake an 
endothelial function (iontophoresis) test in an ambient temperature of 23°C followed by a 
cold challenge in an ambient temperature of 30°C (both described below). Visits 3 and 4 
(crossover period) will be separated by at least 7 days to allow washout (Wylie et al., 2013) 
and all visits will be conducted at the same time of day in a counter-balanced order at a time 
suitable for the participants. 
Visit 3 and 5 (chronic supplementation visits ~ 3 hours). Visits 3 and 5 will be identical in 
nature to visit 2 and 4, however, following chronic supplementation of 70 ml·day-1 of either 
CBJ or placebo for 14 days (Vanhatalo et al., 2010; Wylie et al., 2013). With consent, 

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participants will be reminded to take the juice via text message or voice mails. Please see the 
accompanying documents for the participant flow through the study. 
The participants diet will be standardised prior to testing and they will be asked to abstain 
from using antibacterial mouth wash for 7 days prior to each test and during the testing phase 
as this has been shown to reduce the concentration of oral microflora that are responsible for 
the reduction of nitrate to nitrite (Govoni et al., 2008). Participants will also be asked to 
refrain from using other medicines during the testing period that may affect blood flow or the 
release of nitrite from the CBJ with such as organic nitrates, proton pump inhibitors, 
ranitidine and nicorandil. 
11.2     Adherence to Study Treatment 
Venous blood samples will be taken on all visits. Deproteinised samples will be analysed for 
nitrate and nitrite concentration using a Sievers nitric oxide analyser (Sievers NOA 280i, 
Analytix Ltd, Durham, UK) with our established technique (S. Bailey, J  et al., 2010; S. 
Bailey, J et al., 2009; S. J. Bailey, Wilkerson, DiMenna, & Jones, 2009; M Gilchrist et al., 
2013; Mark Gilchrist et al., 2014; Anthony I Shepherd et al., 2015; Anthony I. Shepherd et 
al., 2015; Shepherd et al., 2016). This will essentially provide strong evidence of adherence to 
the supplement.  
11.3     Accountability of the Study Treatment 
This study is double-blind in nature. The research nurse (if funded) or technicians from the 
department of Sport and Exercise Science at the University of Portsmouth will dispense the 
CBJ. At each time the CBJ is dispensed, the juice will be checked for treatment arm and 
double signed by 2 trained staff members.  
11.4     Concomitant Medication / Therapies 
Participant involvement is anticipated to be ~6 weeks.  Medication will be recorded on 
enrolment and updated throughout their time on supplementation.  

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12. 
ASSESSMENT OF SAFETY (IF APPLICABLE) 
12.1 
Definitions  
A serious adverse event is any untoward medical occurrence that: 
 Results in death, 
 Is life-threatening, 
NOTE: The term "life-threatening" in the definition of "serious" refers to an event in which 
the participant 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 inpatient hospitalisation or prolongation of existing hospitalisation, 
 Results in persistent or significant disability/incapacity, or 
 Is a congenital anomaly/birth defect. 
 Other important medical events* 
  *Other events that may not result in death, are not life threatening, or do not require 
hospitalisation, may be considered a serious adverse event when, based upon appropriate 
medical judgement, the event may jeopardise the participant and may require medical or surgical 
intervention to prevent one of the outcomes listed above. 
12.2 
Reporting Procedures for Serious Adverse Events 
A serious adverse event (SAE) occurring to participant should be reported to the REC that 
gave a favourable opinion of the study where in the opinion of the Chief Investigator the 
event was: ‘related’ – that is, it resulted from administration of any of the research 
procedures; and ‘unexpected’ – that is, the type of event 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 report of serious 
adverse event form (see NRES website).  

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12.3 
Recording and Reporting Procedures for All Adverse Events 
All adverse events will be recorded in participant file notes. Distinction between SAE and AE 
will be made by an independent medical officer. All SAE will be reported to the sponsor and 
the REC regardless of if it is related or not.   
13. 
DATA HANDLING AND RECORD KEEPING 
13.1 
Data Collection Forms 
Visit 1 will act as a screening, consent and baseline assessment (4 hours). All measures for this visit 
will be stored in the clinical records folder (CRF). Separate forms will be prepared for; 
anthropometrics, medical history, screening (i.e. blood pressures etc) and blood markers. On 
subsequent visits data forms will be used to collect temperatures of the labs and the rewarming of the 
hands and feet and also for the iontophoresis test. All paper copies will be stored in the CRF’s.  
13.2     Data Management 
All study data will be entered on paper copies and stored in participant CRF’s. All data will be 
double data entered into excel / access. Macro’s will be used to check for anomalies and 
corrected. The participants will be identified by a study specific participants number and/or 
code in any database.  The name and any other identifying detail will NOT be included in any 
study data electronic file. The chief investigator (Dr Ant Shepherd) is responsible for database 
maintenance and management.  
14. 
DATA ANALYSIS 
14.1      Description of Analysis Populations  
This is a pilot study. All participants who complete all 5 visits will be entered into the analysis 
as per protocol.  
14.2      Analysis of Endpoints  
All data will be tested for normality. Where data are not normally distributed a nonparametric 
test will be performed. Data will be presented as means ± SD. Statistical analyses will be 
performed on SPSS software version 22.0 (Chicago, IL). Statistical difference will be 
accepted when P < 0.05. Statistical differences will be assessed by repeated-measures 

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ANOVAs. Where baseline differences are present, analyses of covariance (ANCOVAs) will 
be used with baseline as a covariate.  
14.3     Procedure for Dealing with Missing, Unused and Spurious Data  
Where statistic differences are present, post hoc (Bonferroni-corrected) analysis will be 
performed. Outliers will remain within the data. Within the data analysis software missing 
data will be coded 9999 and the data point missed during analysis.   
14.4     Procedures for Reporting any Deviation(s) from the Original Statistical Analysis Plan 
For this pilot a SAP is not required (excluding the above).  
14.5      Interim analysis and criteria for early study termination  
Given the small samples size and time frame, no interim analysis will be performed. Early 
termination will only occur if the safety of participants is in question.  
15. 
ETHICS 
NHS Local Research Ethics Committee (REC) approval will be obtained prior to commencement of 
the study. The REC, local NHS, research and development department and all site specific forms 
and patient identification centre forms will be forwarded to SRUK prior to recruitment of 
participants. Written informed consent will be obtained from all participants. Insurance 
indemnity will be provided by the UoP for this study.  
Every effort has been made to keep the risks and discomforts to a minimum but there are 
some risks associated to taking part. Not all volunteers will experience any or all of the risks 
stated below, but participants will be made aware of them. 
The main burden to participants is a significant lifestyle alteration because of a change in 
dietary practices for two, two week periods. This will involve drinking 70ml of either nitrate 
rich beetroot juice or nitrate depleted beetroot juice on a daily basis for two, two week 
periods.   
We will also be asking people with RP to put their hand and foot into 15°C water as part of 
the cold sensitivity test. This may cause some discomfort associated with RP. We will be 
measuring the rewarming and temperature of the hands and feet. Participants will be 
reminded that they are free to withdraw at any time point. 

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Other less burdensome tests will be performed such as:  
Blood pressure; participants will feel a cuff squeeze their arm and this can be uncomfortable 
for a few moments. Blood samples: Intravenous access can cause discomfort associated with 
insertion of the needle. We will take a blood sample on four occasions. Iontophoresis may 
cause some local discomfort/itching sensation during the delivery of acetylcholine and 
sodium nitroprusside, however this is transient occurring only when the current is applied (8 x 
20 s). 
A well-recognised side effect of beetroot consumption is that it may turn urine and faeces red. 
This is not harmful and will pass rapidly after discontinuation of the juice. Subjects will be 
told of this during the consent visit. Subjects will also be asked to refrain from using anti-
bacterial mouthwashes for a week before drinking the juice and during the intervention itself. 
This is critical as it interferes with the biological pathway in which we believe this 
intervention works.  
15.1 
Participant Confidentiality 
The study staff will ensure that the participants’ anonymity is maintained.  The participants 
will be identified only by initials and a participants ID number on the CRF and any electronic 
database. All documents will be stored securely and only accessible by study staff and 
authorised personnel. The study will comply with the Data Protection Act which requires data 
to be anonymised as soon as it is practical to do so.   
15.2     Other Ethical Considerations 
N/A 
15.3     Declaration of Helsinki 
Refer to declaration and that the study protocol will be carried out in accordance with it. 
15.4     ICH Guidelines for Good Clinical Practice 
All staff will be GCP trained and will be monitored by the sponsor to ensure adherence to 
GCP. 

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16. 
PATIENT PUBLIC INVOLVEMENT (PPI) 
16.1    Study design 
Evidence of patient, carer and public involvement (PCPI):  
The chief investigator has a strong track record of PCPI work having been involved in PCPI 
award winning projects recently from the UK Stroke Forum (October 2016) for another 
acceptability and feasibility trial. We have a PCPI co-applicant who has be instrumental in the 
design and development of this pilot RCT and will continue to be involved in the trial 
steering committee, reporting, dissemination and future NIHR funding bids. Other individuals 
with RP have been consulted and have stated that they liked the juice and the quantity should 
not be an issue. Some concerns over the testing schedule (5 visits of 3 hours) for working 
aged individuals were raised. In response we have made weekend testing available to be as 
inclusive as possible. The lay summary and patient facing documentation has been reviewed 
by our PCPI co-applicant.     
16.2    Study implementation 
This is a pilot study and as such we do not have a TSC or DMC. Issues will be managed by 
the senior members of the research team. We will endeavor to involve the PCPI member 
where possible on decisions regarding the study and data.  
16.3    Dissemination 
Our PCPI member will help disseminate findings through local groups, local/national media, 
and co-authoring the paper. Our PCPI involvement has developed the protocol to this stage 
and will continue to play a large role in this project and the larger definitive trial to come.  
 
 
 
 
 
 
 
 

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17. 
FINANCING AND INSURANCE 
17.1    Research Costs 
These are the costs that have been requested from Scleroderma & Raynaud’s UK. If the study 
is not funded by SRUK, the UoP has allocated funding for this study (excluding staff costs).  
Table 1. Breakdown of budget costs.  
Costs 
  
Details  
Total  
Staff1,2 
Researcher 
(grade 
31)1 
0.5 FTE for 1 year 
21,889 
Nurse (grade 6)2 
0.2 FTE for 1 year 
7,817 
Staff advertisement 
Online advert (researcher only) 
400  
Participant transport3 
Maximum 
£10 
per 
visit 
Total 5 visits 
1500 
Screening bloods4 
£15 per participant 
Lipids, TBC, urea eGRF etc 
450 
Visit bloods5 
Nitrate and Nitrite 
£3 a sample, pre each visit in 
duplicate 
900 
Oxidative stress 
Nitrothyrosine, 4 kits  
Protein carbonyl’s, 3 kits  
  
918 
807 
 
Beetroot juice6 
Placebo  
17, 70 ml drinks @ £5 x 30 
participants 
2550 
CBJ  
17, 70 ml drinks @ £1 x 30 
participants 
510 
  
  
                                                     
Total 
£37,741 
Acronyms; FTE (Full time equivalent), TBC (total blood count), eGFR (estimate of glomerular 
filtration rate), and CBJ (concentrated beetroot juice).  
17.2    Service Support Costs 
No service support costs are being sort.  
17.3    Excess Treatment Costs 
No excess treatment costs are being sort.   

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17.4    Study Sponsorship 
This study is being sponsored by the UoP. 
18. 
TIMETABLE AND ORGANISATIONAL CHART 
See corresponding numbers below in Gantt.  
- 
1; Setting up collaborations with interested parties and PPI 
- 
2; Literature search and designing data collection tools 
- 
3; Piloting of data collection tool and subsequent review 
- 
4; R+D / ethics milestones 
- 
5; Data collection period – first inclusions 
- 
6; Data collection period – last inclusions 
- 
7; Data collection period – last follow-up visits 
- 
8; Data entry 
- 
9; Analysis 
- 
10; Writing up reports / publications 
- 
11; Dissemination 
 
 
 
 
 
 

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1 
  
  
  
2   
  
  
3 
  
  
  
  
  
  
4 
  
  
5 
  
6 
7 
8 
  
Jun-17 
Jul-17 
Aug-17 
Sep-17 
Oct-17 
Nov-17 
Dec-17 
Jan-18 
Feb-18 
Mar-18 
Apr-18 
May-18 
Jun-18 
5 
  
6 
  
7   
  
  
  
  
  
  
  
  
  
  
  
8 
  
9 
  
Jul-18 
Aug-18 
Sep-18 
Oct-18 
Nov-18 
Dec-18 
Jan-19 
Feb-19 
Mar-19 
Apr-19 
May-19 
Jun-19 
Jul-19 
9   
10   
11   
  
  
Aug-19 
Sep-19 
Oct-19 

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19. 
RESOURCES, EQUIPMENT AND PHYSICAL FACILITIES 
In addition to the departmental support, and in order to facilitate the completion of the work, 
Dr Shepherd will have full access to all of the necessary world-leading extreme environment 
facilities that are housed within the department estate given that they are pivotal to the 
success of the project. Specifically, this will enable temperature control within the facilities to 
ensure reproducibility. In addition, other equipment, including a thermal imaging camera, 
laser Doppler system and an iontophoresis kit will be made available. Finally, the department 
will ensure that technical assistance is provided to enable the set-up of the laboratories and 
testing rooms, and support with randomisation and beetroot juice dispensing to maintain the 
double-blind nature of this study.  
20. 
DISSEMINATION AND OUTCOME 
RP can cause significant discomfort and pain to individuals during a vasospasm. Dietary 
nitrate appears to offer a simple, low cost means of modifying blood flow to the peripheries 
and, ultimately, reducing both the discomfort and pain experienced by individuals with RP. 
This study will also advance our understanding of the aetiology and pathophysiology of RP, 
specifically the role that the nitrate-nitrite-nitric oxide pathway might play in modulating RP 
symptoms. An understanding of the effects of concentrated beetroot juice on microvascular 
blood flow and pain may lead to a range of simple, low cost and effective therapeutic 
interventions to prevent and treat episodes of RP.  
 
Results (estimates of effect sizes and confidence intervals) from this pilot study will be 
utilised to define our primary outcome and to power a larger, definitive multi-centre 
randomised control trial with our co-applicants and collaborators around Europe. Once our 
final report has been submitted to Scleroderma and Raynaud’s UK, we will apply for NIHR 
funding via a researcher-led Research for Patient Benefit (RfPB) or an Efficacy and 
Mechanism Evaluation (EME) bid (depending on the effect size and sample size) and or a 
Horizon 2020 collaboration bid. The results will be disseminated through publication in peer-
reviewed scientific and clinical journals and via presentations, local, national and 
international meetings. A summary of the results will be sent to all the participants. Finally, 
an open day and presentation will be carried out where the participants will be informed of 

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the study findings and have the opportunity to ask any pertinent questions and/or provide 
feedback. 
21. 
REFERENCES 
Anderson, M., Moore, T., Hollis, S., Jayson, M., King, T., & Herrick, A. (2002). Digital 
vascular response to topical glyceryl trinitrate, as measured by laser Doppler imaging, in 
primary Raynaud's phenomenon and systemic sclerosis. Rheumatology, 41(3), 324-328.  
Appel, L., J, Moore, T., J, Obarzenek, E., Vollmer, W., M, Svetkey, L., P, Sacks, F., M, . . . 
Karanja, N. (1997). A clinical trial of the effects of dietary patterns on blood pressure. The 
New England Journal of Medicine, 336(16), 1117-1124.  
Bailey, S., J , Fulford, J., Vanhatalo, A., Winyard, P., Blackwell, J., R, DiMenna, F., J, . . . 
Jones, A., M. (2010). Dietary nitrate supplementation enhances muscle contractile efficiency 
during knee-extensor exercise in humans. Journal of Applied Physiology, 109, 135-148.  
Bailey, S., J, Winyard, P., Vanhatalo, A., Blackwell, J., R, DiMenna, F., J, Wilkerson, D., P, . 
. . Jones, A., M. (2009). Dietary nitrate supplementation reduces the O2 cost of low-intensity 
exercise and enhances tolerance to high-intensity exercise in humans. Journal of Applied 
Physiology, 107, 1144-1155.  
Bailey, S. J., Wilkerson, D. P., DiMenna, F. J., & Jones, A. M. (2009). Influence of repeated 
sprint training on pulmonary O2 uptake and muscle deoxygenation kinetics in humans. 
Journal of Applied Physiology, 106(6), 1875-1887.  
Bazzano, L., A, He, J., Ogden, L., G, Loria, C., M, Vupputuri, S., Myers, L., & Whelton, P., 
K. (2001). Fruit and vegetable intake and risk of cardiovascular disease in US adults: the first 
National Health and Nutrition Examination Survey Epidemiologic Follow-up Study1-3. 
American Society for Clinical Nutrition 76, 93-99.  
Benjamin, N., O'Driscoll, F., Dougall, H., Duncan, C., Smith, S., Golden, M., & McKenzie, 
H. (1994). Stomach NO synthesis. Nature, 368, 502.  
Berry, M. J., Justus, N. W., Hauser, J. I., Case, A. H., Helms, C. C., Basu, S., . . . Miller, G. 
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blood 
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Björne, H., Petersson, J., Phillipson, M., Weitzberg, E., Holm, L., & Lundberg, J. O. (2004). 
Nitrite in saliva increases gastric mucosal blood flow and mucus thickness. The Journal of 
Clinical Investigation, 113(113 (1)), 106-114.  
Braun, V., & Clarke, V. (2006). Using thematic analysis in psychology. Qualitative research 
in psychology, 3(2), 77-101.  
Cosby, K., Partovi, K., Crawford, J., H, Patel, R., P, Reiter, C., D, Martyr, S., . . . Gladwin, 
M., T. (2003). Nitrite reduction to nitric oxide by deoxyhemoglobin vasodiltaes the human 
circulation Nature medicine, 9(12), 1498-1505.  
Duncan, C., Dougall, H., Johnston, P., Green, S., Brogan, R., Smith, L., . . . Benjamin, N. 
(1995). Chemical generation of nitric oxide in the mouth from the entrosalivery circulation of 
dietary nitrate. Nature medicine, 1, 546-551.  
Eglin, C. M., Golden, F. S., & Tipton, M. J. (2013). Cold sensitivity test for individuals with 
non-freezing cold injury: the effect of prior exercise. Extreme Physiology & Medicine, 2(1), 1.  
Frijhoff, J., Winyard, P. G., Zarkovic, N., Davies, S. S., Stocker, R., Cheng, D., . . . Ghezzi, P. 
(2015). Clinical Relevance of Biomarkers of Oxidative Stress. Antioxid Redox Signal, 23(14), 
1144-1170. doi: 10.1089/ars.2015.6317 
Gilchrist, M., Winyard, P., & Benjamin, N. (2010). Dietary Nitrate - Good or bad? Nitric 
Oxide, 22, 104-109.  
Gilchrist, M., Winyard, P. G., Aizawa, K., Anning, C., Shore, A., & Benjamin, N. (2013). 
Effect of dietary nitrate on blood pressure, endothelial function, and insulin sensitivity in type 
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diabetes. 
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Medicine, 
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Gilchrist, M., Winyard, P. G., Fulford, J., Anning, C., Shore, A., & Benjamin, N. (2014). 
Dietary nitrate supplementation improves reaction time in type 2 diabetes: Development and 
application of a novel nitrate-depleted beetroot juice placebo. Nitric Oxide.  
Govoni, M., Jansson, E. Å., Weitzberg, E., & Lundberg, J. O. (2008). The increase in plasma 
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22. 
APPENDIX 1    SCHEDULE OF PROCEDURES 
See Gantt chart in section 19.  
23. 
APPENDIX 2    STUDY FLOW CHART 
 

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24. 
APPENDIX 3    PARTICIPANT INFORMATION SHEET 
See attachment (booklet format)  
25. 
APPENDIX 4    INFORMED CONSENT FORM 
26. 
APPENDIX 5    SAMPLE QUESTIONNAIRES 
27. 
APPENDIX 6    SAMPLE DATA COLLECTION FORMS 
28. 
APPENDIX 
7 
 
  DRUG 
INFORMATION 
(SUMMARY 
OF 
PRODUCT 
CHARACTERISTICS)  
29. 
APPENDIX 8 MANUFACTURERS BROCHURE FOR NOVEL EQUIPMENT  
30. 
APPENDIX 9 CONTRACTUAL AGREEMENTS WITH OUTSIDE CONSULTANTS / 
COLLABORATORS / INSTITUTIONS (E.G. INDUSTRY, CONTRACT RESEARCH 
ORGANISATIONS)