Feasibility and acceptability of a dietary intervention study to reduce salt intake and increase high-nitrate vegetable consumption among middle-aged and older Malaysian adults with elevated blood pressure: a study protocol

Introduction Global population ageing is one of the key factors linked to the projected rise of dementia incidence. Hence, there is a clear need to identify strategies to overcome this expected health burden and have a meaningful impact on populations’ health worldwide. Current evidence supports the role of modifiable dietary and lifestyle risk factors in reducing the risk of dementia. In South-East Asia, changes in eating and lifestyle patterns under the influence of westernised habits have resulted in significant increases in the prevalence of metabolic, cardiovascular and neurodegenerative non-communicable diseases (NCDs). Low vegetable consumption and high sodium intake have been identified as key contributors to the increased prevalence of NCDs in these countries. Therefore, nutritional and lifestyle strategies targeting these dietary risk factors are warranted. The overall objective of this randomised feasibility trial is to demonstrate the acceptability of a dietary intervention to increase the consumption of high-nitrate green leafy vegetables and reduce salt intake over 6 months among Malaysian adults with raised blood pressure. Methods and analysis Primary outcomes focus on feasibility measures of recruitment, retention, implementation and acceptability of the intervention. Secondary outcomes will include blood pressure, cognitive function, body composition and physical function (including muscle strength and gait speed). Adherence to the dietary intervention will be assessed through collection of biological samples, 24-hour recall and Food Frequency Questionnaire. A subgroup of participants will also complete postintervention focus groups to further explore the feasibility considerations of executing a larger trial, the ability of these individuals to make dietary changes and the barriers and facilitators associated with implementing these changes. Ethics and dissemination Ethical approval has been obtained from Monash University Human Research Ethics Committee and Medical Research and Ethics Committee of Malaysia. Results of the study will be disseminated via peer-reviewed publications and presentations at national and international conferences. ISRCTN47562685; Pre-results.

I, the Submitting Author has the right to grant and does grant on behalf of all authors of the Work (as defined in the below author licence), an exclusive licence and/or a non-exclusive licence for contributions from authors who are: i) UK Crown employees; ii) where BMJ has agreed a CC-BY licence shall apply, and/or iii) in accordance with the terms applicable for US Federal Government officers or employees acting as part of their official duties; on a worldwide, perpetual, irrevocable, royalty-free basis to BMJ Publishing Group Ltd ("BMJ") its licensees and where the relevant Journal is co-owned by BMJ to the co-owners of the Journal, to publish the Work in this journal and any other BMJ products and to exploit all rights, as set out in our licence.
The Submitting Author accepts and understands that any supply made under these terms is made by BMJ to the Submitting Author unless you are acting as an employee on behalf of your employer or a postgraduate student of an affiliated institution which is paying any applicable article publishing charge ("APC") for Open Access articles. Where the Submitting Author wishes to make the Work available on an Open Access basis (and intends to pay the relevant APC), the terms of reuse of such Open Access shall be governed by a Creative Commons licence -details of these licences and which Creative Commons licence will apply to this Work are set out in our licence referred to above.
Other than as permitted in any relevant BMJ Author's Self Archiving Policies, I confirm this Work has not been accepted for publication elsewhere, is not being considered for publication elsewhere and does not duplicate material already published. I confirm all authors consent to publication of this Work and authorise the granting of this licence. have resulted in significant increases in the prevalence of metabolic, cardiovascular and neurodegenerative non-communicable diseases (NCDs). Low vegetable consumption and high sodium intake have been identified as key contributors to the increase in the prevalence of NCDs in these countries. Therefore, nutritional and lifestyle strategies targeting these dietary risk factors are warranted in these populations to prevent cardio-metabolic and neurodegenerative chronic diseases.

Methods and Analysis
The primary aim of this randomised 2*2 factorial trial is to demonstrate the feasibility, acceptability and the potential to deliver a dietary intervention to increase the consumption in high-nitrate green leafy vegetables and reduce salt intake over a 6-month period among Malaysian adults with raised blood pressure. Additional secondary outcomes will be collected including blood pressure, cognitive function, body composition, physical function and biological samples to assess adherence to the dietary intervention. A subgroup of participants will also complete post-intervention focus groups to further explore the feasibility considerations of executing this research, the ability of these individuals to make changes to their diet as well as the barriers and facilitators associated with implementing dietary change.

Ethics and Dissemination
Ethical approval has been obtained from MUHREC (Monash University Human Research Ethics Mixed-method approach to obtain key information on feasibility of study 6. Owing to the small sample size and the feasibility nature of this study, the planned comparative analysis is only exploratory, and therefore, the efficacy of the intervention between groups cannot be determined. However, the results of this study will be instrumental in the design and calculation of the sample size of a larger, follow-on efficacy trial Dementia is an incurable progressive syndrome characterised by multiple cognitive deficits and loss 3 of independence, and its pathogenesis is linked to multiple distinct neuropathological processes 4 including Alzheimer's disease (AD) and/or vascular pathology. 1 Observational evidence suggests that 5 several of the pathological processes underlying dementia might be delayed or prevented by 6 interventions focussed on dietary and behavioural changes 2 . Hence, in the absence of definite 7 pharmacological treatments, these options represent a key strategy to alleviate its individual and 8 social impact. 3 Optimal control of modifiable cardiovascular factors such as blood pressure, as well as 9 adherence to lifestyle recommendations such as diet and physical activity, have been consistently 10 associated with reduced dementia risk in observational studies. [2][3][4] The role of nutrition for the 11 prevention of cognitive decline in cognitively healthy individuals and populations with cognitive 12 impairment has been extensively investigated by testing the role that single nutrients, such as n-3 13 PUFA or B vitamins 5 as well as whole dietary patterns, such as the DASH diet 6 , the Mediterranean diet 7 14 8 and the Mediterranean-DASH diet Intervention for Neurodegeneration Delay (MIND diet). 7   and dementia by the World Health Organisation (WHO) which promotes physical activity and healthy 20 diets rich in fruits, vegetables, wholegrains and reduced salt content. 12 21 22 The prevalence of people with dementia in low and middle income countries (LMICs), particularly in 23 Asia, is rising and is estimated to increase from 60% in 2001 to 71% by 2040. 13 Malaysia is one of many 24 Asian countries experiencing rapid socio-economic and nutritional transitions 14  Intervention Study to Prevent Cognitive Impairment and Disability) trial has successfully pioneered 43 such an approach for dementia prevention. 24 25 44 45 The key nutrients associated with the beneficial effects of increased fruit and vegetables consumption 46 within these dietary patterns are difficult to disentangle, but recent analyses have identified the high 47 content of inorganic nitrate as a potential nutrient involved the improvement of vascular and 48 metabolic health. 26 Inorganic nitrate is closely linked to the metabolism of Nitric Oxide (NO) which is 49 known for its multiple effects on physiological function such as control of blood pressure, brain 50 function and immunity. 27 An increase in inorganic nitrate consumption has been associated with an 51 increase in NO production via complex mechanisms involving oral microbiota, gastric environment 52 and activation of enzymes with a capacity to reduce nitrate into nitrite first, and then finally into NO 28 . high-nitrate vegetables and increased nitrate intake with improved blood pressure, metabolic health 55 and cognitive function. 29 30 Beetroot and green leafy vegetables, such as spinach, lettuce and rocket, 56 are a key source of naturally available inorganic nitrate, containing over 250 mg of nitrate per 100 g 57 of fresh vegetable weight. 31 A systematic review and meta-analysis demonstrated that inorganic 58 nitrate and beetroot juice supplementation were associated with a significant decrease in blood 59 pressure. The pooled effect for both interventions showed a reduction in systolic blood pressure of 60 4.4 mmHg, with a more modest decrease of 1.1 mmHg in diastolic blood pressure. 32 However, the 61 results on cognition are still preliminary, with studies of short intervention durations and among small 62 samples, 27 and studies conducted in developing countries are also limited. Nevertheless, with 63 hypertension highlighted as an important modifiable risk factor for dementia, 3 12 more studies that 64 test the implementation of dietary interventions that aim to improve blood pressure are warranted.

66
Similarly, these healthy dietary patterns are generally characterised by a low consumption of salt, 67 which is mostly derived as a result of consumption of foods with low salt content, as well as a 68 consumption of fresh, unprocessed food products 33 . The DASH diet has been specifically developed 69 around healthy dietary advice in order to promote consumption of a healthy dietary choices alongside 70 a strict control of sodium intake 34 . Secondary analyses derived from seminal DASH studies showed 71 additional benefits on blood pressure and cardio-metabolic health in individuals with lower salt intake 72 and adherence to a healthy dietary pattern 35 . These analyses have been extended to cognitive 73 function with promising results, 19 21 but the evidence has mostly come from studies conducted in the 74 United States and no studies to our knowledge have been conducted in LMICs. Sodium is the main 75 electrolyte contained in extracellular fluids and involved in regulation of blood pressure and cellular 76 activities. However, an excess sodium intake has been linked to extracellular volume expansion 77 resulting in increased blood pressure and renal sodium excretion. 36 Epidemiological studies have 78 repeatedly proven the association of high salt diets with impaired blood pressure control and 79 increased cardiovascular risk; 37 38 this evidence has been confirmed in several clinical trials testing the 80 effects of salt-reducing intervention and demonstrating the protective effects of salt reduction on 81 cardiovascular health. 39 40 Excessive sodium consumption is recognised as a global health issue 41 with 82 efforts aiming to reduce salt intake at the population level by 30% by 2025, and 75 countries have 83 already implemented national sodium reduction programmes to help meet this target. 42 The 84 strategies used to reduce sodium intake at the population level have used a variety of approaches, to 85 include working with industry to reformulate food products, establishing sodium content targets for 86 foods, revising food labelling schemes, taxation of high-salt foods and development of educational interventions. 43 In sum, an increased consumption of dietary nitrate and lower salt intake may 88 therefore have additional benefits on health outcomes such as blood pressure and cognition, 89 mediated by an increase in NO generation (i.e., nitrate) and control of vascular tone and cellular 90 metabolism (i.e., sodium). However, this hypothesis has never been tested in previous interventions 91 which represents the main aim of the research study proposed in this protocol.  The community will be informed about this current study before it is initiated. Participants will be 157 randomly identified by the application of the inclusion and exclusion criteria to the SEACO health 158 surveillance database. Potential participants will be approached via home visit and will be provided 159 with information about the study. They will be given an opportunity to read an information sheet, 160 containing detailed information about the study procedures and will have the opportunity to ask 161 questions. Thereafter, their interest in participating to the study will be evaluated. If they agree to 162 take part, a screening assessment will be completed. If eligible, participants will be invited to attend a 163 baseline study visit at the health facilities within the community.   Week 2 Week 4 Week 6 Week 8 Week 10 Week 12 Week Whole blood samples will be analysed to measure changes in nitrite concentrations during the 333 intervention. Samples will also be analysed to assess for biomarkers of cardio-vascular risk (C-reactive   334 protein, glycated haemoglobin, nitro-tyrosine) and direct brain measures such as plasma BDNF, 335 plasma amyloid β42 and amyloid β40. A capillary blood sample will be taken and analysed for the 336 concentration of glucose in the blood using a portable glucometer. Dried blood spot samples will be 337 analysed to measure changes in nitrate concentrations during the intervention. Blood spots will be 338 first processed using a standardised elution protocol to obtain a liquid solution of the samples and 339 then analysed for nitrate concentrations using ozone-based chemiluminescence, which is the 340 reference method for the analysis on nitrate in biological fluids. All blood sampling assessment 341 methods will be conducted at baseline and end of study visits.  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45

354
Although participants will not be involved in the recruitment and conduct of this study, participants 355 will be invited to provide feedback on their participation intervention study via focus groups 356 discussions. The focus group topic guide will also be piloted among a random sample of participants 357 prior to implementation to allow feedback, refinement and tailoring prior to implementation. Two 358 focus group discussions will be conducted within each arm of trial (total 8). Purposive sampling will be 359 used to capture broad variation in age, ethnicity, house location and for better understanding of the 360 phenomenon being studied. All focus groups discussions will be audio recorded and the topics covered

381
The main aims from the feasibility study are to show that the two interventions can be followed with 382 high adherence during the trial. Secondary aims are largely descriptive, aiming to provide bounds for 383 key parameters to inform the main trial. The recruitment and retention rate in each arm will be 384 described. It is unlikely that statistically significant differences in retention rates will be discovered, 385 but upper and lower bounds will be estimated, and reasons for groups with apparently low 386 recruitment or retention rates will be explored qualitatively. Normality of the distribution of the 387 variables and appropriate transformations (LogX, 1/X, Xn) will be performed if necessary. Summary 388 data will be expressed as mean (SD) or frequency (percent). General linear models for repeated 389 measures will be used to detect significant differences between the intervention groups with and 390 without adjustment for baseline levels. Chi square test will be used for categorical variables. An 391 interaction term (time x group) will be built to assess between-group interactions in changes in the 392 measured outcomes during the interventions. Dietary data will be analysed using the Nutritionist Pro

Instructions to authors
Complete this checklist by entering the page numbers from your manuscript where readers will find each of the items listed below.
Your article may not currently address all the items on the checklist. Please modify your text to include the missing information. If you are certain that an item does not apply, please write "n/a" and provide a short explanation.
Upload your completed checklist as an extra file when you submit to a journal.
In your methods section, say that you used the SPIRITreporting guidelines, and cite them as:

Methods and Analysis
The primary aim of this randomised feasibility trial is to demonstrate the acceptability of a dietary intervention to increase the consumption of high-nitrate green leafy vegetables and reduce salt intake over a 6-month period among Malaysian adults with raised blood pressure. Secondary outcomes will include blood pressure, cognitive function, body composition and physical function (including muscle strength and gait speed). Adherence to the dietary intervention will be assessed through collection of biological samples, 24 hour recall and food frequency questionnaire. A subgroup of participants will also complete post-intervention focus groups to further explore the feasibility considerations of executing a larger trial, the ability of these individuals to make dietary changes and the barriers and facilitators associated with implementing these changes. Owing to the small sample size and the feasibility nature of this study, the planned comparative analysis is only exploratory, and therefore, the efficacy of the intervention between groups cannot be determined. However, the results of this study will be instrumental in the design and calculation of the sample size of a larger, follow-on efficacy trial

INTRODUCTION
Dementia is an incurable progressive syndrome characterised by multiple cognitive deficits and loss of independence, and its pathogenesis is linked to multiple distinct neuropathological processes including Alzheimer's disease (AD) and/or vascular pathology. 1 Observational evidence suggests that several of the pathological processes underlying dementia might be delayed or prevented by interventions focussed on dietary and behavioural changes 2 . Hence, in the absence of definite pharmacological treatments, these options represent a key strategy to alleviate its individual and social impact. 3 Optimal control of modifiable cardiovascular factors such as blood pressure, as well as adherence to lifestyle recommendations such as a healthy diet and physical activity, have been consistently associated with reduced dementia risk in observational studies. [2][3][4] The role of nutrition for the prevention of cognitive decline in cognitively healthy individuals and populations with cognitive impairment has been extensively investigated by testing the role that single nutrients, such as n-3 PUFA or B vitamins 5 as well as whole dietary patterns, such as the DASH diet 6 and the Mediterranean diet 7 8 . There are a small number of RCTs available to support the effect of diet on cognitive function in Mild Cognitive Impairment 9 and among those at risk of cognitive decline or cognitively healthy populations 10 ; however the trials have a small sample size and overall modest quality.
Malaysia is one of many Asian countries experiencing rapid socio-economic and nutritional transitions 11 . Changes in individual eating and lifestyle patterns under the influence of westernised as a result, significant beneficial effects for a specific intervention were often not seen in the metaanalysis. 10 Therefore, there is a need for further well-designed nutritional intervention studies to test the efficacy of interventions for the early prevention of cognitive decline and dementia.
Recent analyses have identified inorganic nitrate as a potential nutrient involved the improvement of vascular and metabolic health. 13 Beetroot and green leafy vegetables, such as spinach, lettuce and rocket, are a key source of naturally available inorganic nitrate, containing over 250 mg of nitrate per 100 g of fresh vegetable weight. 14 Inorganic nitrate is closely linked to the metabolism of Nitric Oxide (NO) which is known for its multiple effects on physiological function such as control of blood pressure, brain function and immunity. 15 An increase in inorganic nitrate consumption has been associated with an increase in NO production via complex mechanisms involving oral microbiota, gastric environment and activation of enzymes 16  Results showed that a consumption of approximately 30 mg/day higher nitrate intake from vegetables was associated with a 21% lower risk of ASVD mortality. This nitrate amount equates to approximately 10-30 g/day of nitrate-rich vegetables. 19 Furthermore, a systematic review and meta-analysis demonstrated that inorganic nitrate and beetroot juice supplementation were associated with a significant decrease in blood pressure. The pooled effect for both interventions showed a reduction in systolic blood pressure of 4.4 mmHg, with a more modest decrease of 1.1 mmHg in diastolic blood pressure. 20 However, the impact on cognition is still unknown with studies of short intervention durations and among small samples, and limited studies conducted in developing countries. 14 17 Nevertheless, with hypertension highlighted as an important modifiable risk factor for dementia, 3 21  Sodium is the main electrolyte contained in extracellular fluids and involved in regulation of blood pressure and cellular activities. However, an excess sodium intake has been linked to extracellular volume expansion resulting in increased blood pressure and renal sodium excretion. 22 The DASH diet has been specifically developed to promote consumption of healthy dietary choices alongside strictly controlled sodium intake. 23 Secondary analyses derived from seminal DASH studies showed additional benefits on blood pressure and cardio-metabolic health in individuals with lower salt intake and adherence to a healthy dietary pattern 24 . These analyses have been extended to cognitive function with promising results, 25 26 but the evidence has mostly come from studies conducted in the United States and no studies to our knowledge have been conducted in Low and Middle Income Countries (LMICs). Epidemiological studies have repeatedly proven the association of high salt diets with impaired blood pressure control and increased cardiovascular risk; 27 28 this evidence has been confirmed in several clinical trials testing the effects of salt-reducing interventions and demonstrating the protective effects of salt reduction on cardiovascular health. 29 30 A prospective study looked at the relationship between a reduction in salt intake with blood pressure, mortality from stroke and ischaemic heart disease (IHD). 31 A decrease in salt intake by 1.4 g/day, as measured by 24 hour urinary sodium, contributed to a reduction in stroke and IHD mortality and in addition, among individuals who were not on antihypertensive medication, there was significant a fall in systolic blood pressure of 2.7±0.34mmHg. 31 Excessive sodium consumption is recognised as a global health issue 32 with efforts aiming to reduce salt intake at the population level by 30% by 2025. Seventy-five countries have already implemented national sodium reduction programmes to help meet this target. 33 The strategies to reduce sodium intake at the population level have used a variety of approaches, including working with industry to reformulate food products, establishing sodium content targets for foods, revising food labelling schemes, taxation of high-salt foods and development of educational interventions. 34 In sum, an increased consumption of dietary nitrate and lower salt intake may have additional benefits on health outcomes such as blood pressure and cognition, mediated by an increase in NO generation (i.e., nitrate) and control of vascular tone and cellular metabolism (i.e., sodium). However, this hypothesis has never been tested in previous interventions which represents the main aim of the feasibility study proposed in this protocol. The Global Health Dementia Prevention and Enhanced Care (DePEC) project is a National Institute for Health Research (NIHR) funded research study with a key

Purpose and Aims
The overall objective of the DePEC-Nutrition feasibility study is to demonstrate the acceptability of a dietary intervention to increase the consumption in high-nitrate green leafy vegetables and reduce salt intake over a 6-month period among Malaysian adults with raised blood pressure. The objectives focus on the core areas of a trial that need to work for the study to succeed procedurally, such as recruitment ability and participant retention, data collection procedures and assessment methods used, potential to deliver the dietary intervention and resource requirement. Information on the effect size of the intervention on cognition and blood pressure will also be determined, which will be instrumental in the design and calculation of the sample size of a follow-on efficacy trial.

Study design
The DePEC-Nutrition feasibility study is a 6 month randomised 2*2 factorial trial including four parallel arms: (1) control, (2)  be screened in detail, and then will be informed of the study and invited for a screening visit as outlined in the study procedures.

Recruitment
The community will be informed about this study via a local Community Engagement Committee (CEC) who meet regularly to distribute information on projects planned within SEACO. Participants will be randomly identified by the application of the inclusion and exclusion criteria to the SEACO health surveillance database. Potential participants will be approached via a home visit and will be provided with information about the study. They will be given an opportunity to read detailed information about the study procedures and can ask questions. Thereafter, their interest of participating in the study will be evaluated. If they agree to take part, a screening assessment will be completed. If eligible, participants will be invited to attend a baseline study visit at the health facilities within the community.

Eligibility criteria
To be eligible for inclusion in this study, participants must report as: Have systolic blood pressure more than or equal to 160 mmHg and / or diastolic blood pressure more than or equal to 100 mmHg 4. Adhere to any therapeutic diet such as weight loss treatments or a gluten-free diet  Data collection will be undertaken directly on an electronic hand held device (Samsung Galaxy Tab 3v -Survey CTO). Questionnaire items will be read out-loud by the data collector and participant responses will be recorded directly onto the electronic tablet. The flow of participants through the DePEC-Nutrition feasibility study is illustrated in Figure 1. At the screening visit, written informed consent will be first obtained, after which participants will complete a screening questionnaire to collect information on demographics, co-morbidities, medication history, cognitive function (MMSE), body weight, height and resting blood pressure. If eligible, participants will undergo baseline assessments, consisting of two parts; a home visit (part 1), followed by an appointment at KK, Segamat (part 2). Baseline assessments will include: obtaining respondent's signed informed consent for study participation, eligibility assessment (medical and medication history), dietary assessment (24-hour recall and food frequency questionnaire (FFQ)), cognitive assessment, collection of biological samples (blood, dried blood spots, 24-urine and spot urine samples, saliva samples, and salivary strips), measurements of hand grip and gait speed, physical activity (IPAQ) and depression (GDS).
An interim two-month visit will take place for those in the intervention groups only, to provide reinforcement messages relating to the allocated dietary intervention and with a view to enhancing compliance with the intervention. In addition, a brief adherence questionnaire will be administered, body weight and resting blood pressure will be measured and salivary strip and spot urine samples will be collected. The same measurements and further dietary reinforcement messages will be conducted at an interim four-month visit for the participants allocated to the intervention arms.
Participants in the control arm will only have one interim visit at month four and will have their weight and blood pressure measured. The end of study visit (at KK, Segamat) will include the conduction of the measurements collected at baseline, including cognitive assessment, dietary assessment, and collection of biological samples (blood, dried blood spots, 24-urine and spot urine samples, saliva samples, salivary strips). In addition, an exit group session (focus group evaluation) and a selfadministered feedback questionnaire will be scheduled to evaluate the adherence to the interventions and obtain detailed feedback on the overall study protocol. Finally, the participants will be offered a token of appreciation to acknowledge their participation in the study and to compensate for travel expenses. This is usually to the value of RM 25.

Randomisation and blinding
The SEACO health round database will serve as the sampling frame, from which eligible participants are identified. Block randomisation will be carried out to assign eligible participants into 10 one of the four arms. Block sizes of four participants will be used in the randomisation and will be generated by a member of the research team not involved in the data collection. The interviewers will be blinded to the participant assignment to intervention arms. Participant blinding to the intervention allocation will not be possible due to the nature of the dietary intervention study.

Intervention and control arms
This dietary and behavioural intervention will focus on key components that are related to changing local dietary patterns influenced by nutritional transition trends in Malaysia. It aims to target two key components that have protective (dietary nitrate) and negative (salt intake) effects on cardiovascular and cognitive health. The intervention will target individuals with a higher CVD risk to adopt a tailored strategy to enhance daily consumption of green leafy vegetables and / or reduce salt intake.
Nutritional counselling sessions with a medical doctor will be conducted at baseline to provide sustainable and effective strategies to improve dietary habits as well as monitor and address key barriers to dietary change. These sessions will be delivered in a group setting using PowerPoint slides and practical activities to educate the group on the allocated dietary intervention. All intervention groups will receive written information focussed on health benefits, sources, recommended intakes and practical tips related to one of the following dietary interventions; (1) to increase dietary nitrate (2) to reduce salt intake (3) to increase dietary nitrate and to reduce salt intake. Those who are randomised to the salt intervention (either alone or combined) will receive a measuring spoon as an additional strategy to support individuals to understand portion size and measure salt intake, as successfully tested by an intervention study in China. 36

Outcome measures
Outcome assessments will be conducted at baseline, month four and month six for all groups, with an additional assessment visit at month two for those in the intervention groups. Assessments at baseline and month 6 will be conducted in clinic / at home, with remaining assessments completed in the participants own home. The same instruments will be used for measurements at each time point.
Assessors are trained prior to the assessments to follow a standardised protocol. A timeline of the study procedures can be found in Table 1.  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59   Week 2 Week 4 Week 6 Week 8 Week 10 Week 12 Week The primary outcome measures are related to feasibility and will be collected via questionnaire, review of the study procedures and by review of data collector field notes. Patient feedback, acceptance and adherence issues will be additionally assessed through qualitative focus groups conducted post-intervention. The specific feasibility considerations that will be assessed in the study have been outlined in Table 2. The CONSORT 2010 statement 39 will be used to guide the reporting of the feasibility results.

Blood pressure
Three consecutive measurements of resting blood pressure readings will be recorded in a sitting position using a calibrated OMRON monitor. The median measure will be used to estimate the blood pressure. Blood pressure will be measured at all outcome assessment visits.

Anthropometry and body composition
Body weight will be recorded in kilograms using calibrated, electronic scales to the nearest 0.1kg where possible. Weight will be measured at all outcome assessment visits. Height will be recorded in metres using a stadiometer and measured to the nearest 0.01m where possible. The recordings for weight and height will be subsequently used to calculate BMI by dividing weight (kg) by height (m 2 ).
BMI will be calculated at baseline and end of study visits. Body composition will be measured by bioelectrical impedance. A Tanita Body Composition analyser will be used to measure body fat percentage and classification, segmental subcutaneous fat and skeletal muscle percentage (whole body, trunk, legs and arms), resting metabolism, visceral fat level and classification and body age. Body composition will be measured at baseline and end of study visits.

Physical assessments
The following physical assessments will be conducted at baseline and end of study visits. (1) Muscle strength will be measured by a hand grip-strength dynamometer. Using their right arm, the participant will be asked to make three measurements and an average calculated. The same process will be repeated for the left arm. (2) A 4 meter walk test will be done to assess the gait speed. Two trials will be conducted and the average measure of time in minutes will be used to assess the gait speed. (3) In the Timed Up and Go (TUG) test, the time taken for participants to rise from an armless chair (46 cm height), to walk 3 metres, turn, walk back and sit down will be measured. The TUG test will be performed twice consecutively, and the average of the two scores will be used. (4) The Geriatric Depression Scale (GDS) (short form) will be used to assess depressive symptoms 40 -scores range from zero to 15, with a score of zero to five indicating a normal score and a score greater than five  41 will be used to assess physical activity.

Dietary Assessment
Conventional approaches to assessing dietary intake are associated with measurement error. There is a growing consensus that combining the use of self-report instruments (like FFQ and 24 hour recall) together with biomarker analyses, could increase the accuracy of individual intake estimates, especially for episodically consumed foods. 42 43 Thus, dietary assessment data will be collected by two methods: 24 hour recall and FFQ. Participants will complete a one day 24 hour recall facilitated by a trained data collector. The FFQ is a validated questionnaire used in previous dietary research in Malaysia. 44 Information will be entered into Nutrition Pro Software (v7.5) and daily intake will be calculated.

Biological Sample Collection
The following bio-specimen will be collected in this study:

Capillary blood
All samples will be used only for the purpose of the study. The venous blood will first be centrifuged, separated into aliquots and then stored at -20°C at the KK Segamat and then transported to Monash University Sunway campus for storage. Other samples will be stored at -20°C storage. The freezer room for the stored samples will be under lock and key with restricted access.

Blood sampling
Whole blood samples will be analysed to measure changes in nitrite concentrations during the intervention. Samples will also be analysed to assess for biomarkers of cardio-vascular risk (C-reactive protein, glycated haemoglobin, nitro-tyrosine) and direct brain measures such as plasma BDNF, plasma amyloid β42 and amyloid β40. A capillary blood sample will be taken and analysed for the concentration of glucose in the blood using a portable glucometer. Dried blood spot samples will be analysed to measure changes in nitrate concentrations during the intervention. Blood spots will be first processed using a standardised elution protocol to obtain a liquid solution of the samples and then analysed for nitrate concentrations using ozone-based chemiluminescence, which is the reference method for the analysis on nitrate in biological fluids. All blood sampling assessment methods will be conducted at baseline and end of study visits. Venous blood samples will be performed by a qualified medical attendant or staff nurse practising at KK Segamat and dried blood spot samples will be collected by a trained data collector.

hour and spot urine sampling
Both 24 hour urine and spot urine samples will be collected. 24 hour urine collection has been associated with a having high respondent burden owing to the time consuming nature of this method, particularly in a community and LMIC setting, where 24 hour urine sampling may be logistically difficult. Spot urine sampling is potentially a more convenient and affordable alternative. However, there are still a number of questions about the reliability of spot urine collection as a means of monitoring intervention adherence. Therefore, by using these two methods of urine collection, the authors hope to explore the feasibility of implementing these collection methods within the target population. 45 Eligible participants will be provided with a kit to collect a 24 hour urine sample during their baseline home visit (part 1). This will be provided two days before their clinic visit (part 2). The kit includes: (1)

A participant information booklet with written instructions on how to collect the sample; (2) A '24
hour urine collection record' to note essential information about the urine collection; (3) Urinecollecting equipment: (a) 2.5 litre screw-capped plastic container for storing the 24 hour urine sample, (b) 1 litre plastic jug which the urine will be voided into, (c) 1 litre screw-capped plastic container either as backup container when the 2.5 litre storage container is full, or for temporal collections of urine made outside the home; (4) A permanent marker pen to note the start and finish times of urine collection in the 2.5 litre container. Trained data collectors will verbally explain the method of 24 hour urine collection to participants. The 24 hour urine collection will be initiated one day before their clinic visit and end in the morning of their appointment. Participants will also collect a spot urine sample on the morning of their clinic visit. They will be provided with a 60 ml screw-capped plastic bottle for the spot urine collection. Participants will be instructed by a trained data collector to provide a spot urine sample using the midstream clean-catch technique.
The 24 hour urine samples collected will be assessed for completeness using assessment of the duration of urine collection, the total urine volume and 24 hour urinary creatinine excretion. The urine samples will be excluded from analysis if the time of the collection falls outside the range of [22][23][24][25][26] hours, if the total 24 hour urine volume is less than 500 ml or greater than 6000 ml, and if 24 hour creatinine excretion is less than 3 mmol, or greater than 25 mmol in women, or less than 6 mmol, or greater than 30 mmol in men. Urinary sodium will be determined using the ion-selective electrode method. For nitrate, samples will be diluted 1:100 and then analysed for nitrate concentrations using ozone-based chemiluminescence, which is the reference method for the analysis on nitrate in biological fluids.

Saliva sampling
Whole saliva will be collected using the passive drool technique. 1 ml of will be collected into one collection tube. Participants will be asked to generate some saliva in their mouth and when ready, the participant should hold the adaptor with collection tube attached to their mouth and pass saliva into the tube. This process is repeated until desired amount is collected. To measure nitrate, samples will be diluted 1:100 and then analysed for nitrate concentrations using ozone-based chemiluminescence.
For the salivary strips, a dedicated app freely available to download on mobile devices will be used to provide a quantitative reading of the salivary nitrite concentrations as a surrogate marker of dietary nitrate intake.

Patient and public involvement -Post-intervention qualitative evaluation
Participants will be invited to provide feedback on their participation in the intervention study via focus groups discussions. The focus group topic guide will also be piloted among a random sample of participants prior to implementation to allow feedback, refinement and tailoring prior to implementation. Two focus group discussions will be conducted within each arm of trial (total 8).
Purposive sampling will be used to capture broad variation in age, ethnicity, house location and for better understanding of the phenomenon being studied. All focus groups discussions will be audio recorded and the topics covered will include: (1) Access to the food items -financial (affordability), framework will be used to guide the qualitative analysis. 46 47 This will allow the feedback and recommendations from participants to directly inform refinements and tailoring of the intervention for a follow-on efficacy trial as well as to understand the barriers and facilitators to dietary change among this target group. an anticipated drop-out rate ≤20%.

Statistical Analysis
As the main aims of this feasibility study relate to the feasibility, acceptability and the potential to deliver a dietary intervention, these data will be reported narratively illustrated with descriptive statistics. The CONSORT 2010 statement will be used to guide the reporting of this information. 39 Secondary aims are largely descriptive, aiming to provide bounds for key parameters to inform the main trial. Intention-to-treat analysis will be used to include all randomised participants and determine the key outcome measures. The recruitment and retention rate in each arm will be described. It is unlikely that statistically significant differences in retention rates will be discovered, but upper and lower bounds will be estimated, and reasons for groups with apparently low recruitment or retention rates will be explored qualitatively. Normality of the distribution of the variables and appropriate transformations (LogX, 1/X, Xn) will be performed if necessary. Summary data will be expressed as mean (SD) or frequency (percent). General linear models for repeated measures will be used to detect significant differences between the intervention groups with and without adjustment for baseline levels. Chi square test will be used for categorical variables. An interaction term (time x group) will be built to assess between-group interactions in changes in the measured outcomes during the interventions. Dietary data will be analysed using the Nutritionist Pro software (v7.5).

Data collection supervision and training
SEACO is an ISO certified research platform for its operations, therefore training and supervision are carried out in accordance with SEACO's standard operating procedures. All data collectors are required to be fluent in the local language (Bahasa Melayu or Malay language or Mandarin) and have at least a working knowledge of English. To ensure data quality and consistency across interviewers, the field supervisor will undertake random concurrent supervisory visits to observe practice. The data collectors will also be periodically observed by the project leader and the field manager. Based on the Clayton. Research staff have limited access to anonymised data.

Data monitoring
All expected and unexpected adverse events reported by participants will be recorded in an events register and reported to the Human Research Ethics Committee. Due to the nature of the treatment products, i.e. commercially available food products, no adverse events are expected. However, if participants feel in anyway adversely affected by any foods or the principal investigator feels an adverse event necessitates cessation, the participant would be advised not to continue and the appropriate measures will be taken (i.e. record in field notes, contact research nurse and principal investigator if deemed necessary).

Ethics and Dissemination
The  choices focused on these key elements of the diet, would be linked to greater long-term adherence to the interventions and improved cognitive function with prospective, projected reduced risk of dementia in these populations. This mixed-method feasibility study will provide key quantitative and qualitative information on the delivery of a novel dietary intervention and will aim to estimate the effect size of the single and combined interventions on cognitive function and blood pressure, which will be instrumental in the design and calculation of the sample size of a larger, follow-on efficacy trial.

Instructions to authors
Complete this checklist by entering the page numbers from your manuscript where readers will find each of the items listed below.
Your article may not currently address all the items on the checklist. Please modify your text to include the missing information. If you are certain that an item does not apply, please write "n/a" and provide a short explanation.
Upload your completed checklist as an extra file when you submit to a journal.
In your methods section, say that you used the SPIRITreporting guidelines, and cite them as:  objective of this randomised feasibility trial is to demonstrate the acceptability of a dietary intervention to increase the consumption of high-nitrate green leafy vegetables and reduce salt intake over 6 months among Malaysian adults with raised blood pressure.

Methods and Analysis
Primary outcomes focus on feasibility measures of recruitment, retention, implementation and acceptability of the intervention. Secondary outcomes will include blood pressure, cognitive function, body composition and physical function (including muscle strength and gait speed). Adherence to the dietary intervention will be assessed through collection of biological samples, 24 hour recall and food frequency questionnaire. A subgroup of participants will also complete post-intervention focus groups to further explore the feasibility considerations of executing a larger trial, the ability of these individuals to make dietary changes and the barriers and facilitators associated with implementing these changes.
Malaysia is experiencing rapid socio-economic and nutritional transitions 11   Therefore, there is a need for well-designed nutritional intervention studies for the early prevention of cognitive decline and dementia.
Recent analyses have identified inorganic nitrate as a potential nutrient improving vascular and metabolic health. 13 Beetroot and green leafy vegetables, such as spinach, lettuce and rocket, are a key source of naturally available inorganic nitrate, containing over 250 mg of nitrate per 100 g of fresh vegetable weight. 14 Inorganic nitrate is closely linked to the metabolism of Nitric Oxide (NO) which is known for its multiple effects on physiological functions such blood pressure, brain function and immunity. 15 An increase in inorganic nitrate consumption has been associated with an increase in NO production via complex mechanisms involving oral microbiota, gastric environment and activation of reducing enzymes 16 . Recent epidemiological studies and clinical trials have demonstrated the association of increased nitrate intake with improved blood pressure, metabolic health and cognitive function. 17 18 A 15-year prospective cohort study investigated the association of consumption of high nitrate vegetables and risk of atherosclerotic vascular disease (ASVD) mortality. Results showed that a consumption of approximately 30 mg/day higher nitrate intake from vegetables was associated with a 21% lower risk of ASVD mortality. This nitrate amount equates to approximately 10-30 g/day of nitrate-rich vegetables. 19 However, the impact on cognition is still unknown with studies of short intervention durations and among small samples, and limited studies conducted in developing countries. 14 17 Nevertheless, with hypertension highlighted as an important modifiable risk factor for dementia, 3 20 more studies that test the implementation of dietary interventions that aim to improve blood pressure are warranted.
Sodium is the main electrolyte contained in extracellular fluids and involved in regulation of blood pressure and cellular activities. An excess sodium intake has been linked to extracellular volume expansion and increased blood pressure. 21 The DASH diet has been specifically developed to promote consumption of healthy dietary choices alongside strictly controlled sodium intake. 22 Secondary analyses derived from seminal DASH studies showed additional benefits on blood pressure and cardio- These analyses have been extended to cognitive function with promising results, 24 25 but the evidence has mostly come from studies conducted in the United States and no studies to our knowledge have been conducted in Low and Middle Income Countries (LMICs). Epidemiological studies have repeatedly proven the association of high salt diets with impaired blood pressure control and increased cardiovascular risk; 26 27 this evidence has been confirmed in several clinical trials testing the effects of salt-reducing interventions and demonstrating the protective effects of salt reduction on cardiovascular health. 28 29 A prospective study looked at the relationship between a reduction in salt intake with blood pressure, mortality from stroke and ischaemic heart disease (IHD). 30 A decrease in salt intake by 1.4 g/day, as measured by 24 hour urinary sodium, contributed to a reduction in stroke and IHD mortality, and in addition, among individuals who were not on antihypertensive medication, there was significant a fall in systolic blood pressure of 2.7±0.34mmHg. 30 Excessive sodium consumption is recognised as a global health issue 31 with efforts aiming to reduce salt intake at the population level by 30% by 2025. Seventy-five countries have already implemented national sodium reduction programmes to help meet this target. 32 In sum, an increased consumption of dietary nitrate and lower salt intake may have additional benefits on health outcomes such as blood pressure and cognition, mediated by an increase in NO generation (i.e., nitrate) and control of vascular tone and cellular metabolism (i.e., sodium). However, this hypothesis has never been tested in previous interventions which represents the main aim of the feasibility study proposed in this protocol. The Global Health Dementia Prevention and Enhanced Care (DePEC) project is a National Institute for Health Research (NIHR) funded study with a key goal to develop approaches for dementia prevention and develop more efficient post-diagnostic care in LMICs. The DePEC-Nutrition feasibility study (presented here) is one of five work streams within the overall DePEC project.

Purpose and Aims
The overall objective of the DePEC-Nutrition feasibility study is to demonstrate the acceptability of a dietary intervention to increase the consumption in high-nitrate green leafy vegetables and reduce salt intake over a 6-month period among Malaysian adults with raised blood pressure. The objectives focus on the core areas of a trial that need to work for the study to succeed procedurally, such as recruitment ability and participant retention, data collection procedures and assessment methods used, potential to deliver the dietary intervention and resource requirement. Information on the effect size of the intervention on cognition and blood pressure will also be determined, which will be instrumental in the design and calculation of the sample size of a follow-on efficacy trial.

Study design
The DePEC-Nutrition feasibility study is a 6 month randomised 2*2 factorial trial including four parallel arms: (1)   DePEC protocol R2. 02/06/2020 7 be screened in detail, and then will be informed of the study and invited for a screening visit as outlined in the study procedures.

Recruitment
The community will be informed about this study via a local Community Engagement Committee (CEC) who meet regularly to distribute information on projects planned within SEACO. Participants will be randomly identified by the application of the inclusion and exclusion criteria to the SEACO health surveillance database. Potential participants will be approached via a home visit and will be provided with information about the study. They will be given an opportunity to read detailed information about the study procedures and can ask questions. Thereafter, their interest of participating in the study will be evaluated. If they agree to take part, a screening assessment will be completed. If eligible, participants will be invited to attend a baseline study visit at the health facilities within the community.

Eligibility criteria
To be eligible for inclusion in this study, participants must report as:

Study procedure
Data collection will be undertaken directly on an electronic hand held device (Samsung Galaxy Tab 3v -Survey CTO). Questionnaire items will be read out-loud by the data collector and participant responses will be recorded directly onto the electronic tablet. The flow of participants through the DePEC-Nutrition feasibility study is illustrated in Figure 1. At the screening visit, written informed consent will be first obtained, after which participants will complete a screening questionnaire to collect information on demographics, co-morbidities, medication history, cognitive function (MMSE), body weight, height and resting blood pressure. If eligible, participants will undergo baseline assessments, consisting of two parts; a home visit (part 1), followed by an appointment at KK, Segamat (part 2). Baseline assessments will include: obtaining respondent's signed informed consent for study participation, eligibility assessment (medical and medication history), dietary assessment (24-hour recall and food frequency questionnaire (FFQ)), cognitive assessment, collection of biological samples (blood, dried blood spots, 24-urine and spot urine samples, saliva samples, and salivary strips), measurements of hand grip and gait speed, physical activity (IPAQ) and depression (GDS). An interim two-month visit will take place for those in the intervention groups only, to provide reinforcement messages relating to the allocated dietary intervention and with a view to enhancing compliance with the intervention. In addition, a brief adherence questionnaire will be administered, body weight and resting blood pressure will be measured and salivary strip and spot urine samples will be collected. The same measurements and further dietary reinforcement messages will be conducted at an interim four-month visit for the participants allocated to the intervention arms.
Participants in the control arm will only have one interim visit at month four and will have their weight and blood pressure measured. The end of study visit (at KK, Segamat) will include the conduction of the measurements collected at baseline, including cognitive assessment, dietary assessment, and collection of biological samples (blood, dried blood spots, 24-urine and spot urine samples, saliva samples, salivary strips). In addition, an exit group session (focus group evaluation) and a selfadministered feedback questionnaire will be scheduled to evaluate the adherence to the interventions and obtain detailed feedback on the overall study protocol. Finally, the participants will be offered a token of appreciation to acknowledge their participation in the study and to compensate for travel expenses. This is usually to the value of RM 25.

Randomisation and blinding
The SEACO health round database will serve as the sampling frame, from which eligible participants are identified. Block randomisation will be carried out to assign eligible participants into one of the four arms using R software (randomizeR) 34 . Block sizes of four participants will be used in the randomisation and will be generated by a member of the research team not involved in the data collection. The interviewers will be blinded to the participant assignment to intervention arms.
Participant blinding to the intervention allocation will not be possible due to the nature of the dietary intervention study. Matching of characteristics for group allocation will not be used in this study. The distribution of characteristics, such as age or gender, will be part of the feasibility assessment to gain information on the characteristics of the population willing to participate without imposing any restrictions on the intention to participate in the study. This information will be used to evaluate whether specific randomisation approaches will need to be used in future studies to ensure even distribution of key characteristics between groups.

Intervention and control arms
This dietary and behavioural intervention will focus on key components that are related to changing local dietary patterns influenced by nutritional transition trends in Malaysia. It aims to target two key components that have protective (dietary nitrate) and negative (salt intake) effects on cardiovascular and cognitive health. The intervention will target individuals with a higher CVD risk to adopt a tailored strategy to enhance daily consumption of green leafy vegetables and / or reduce salt intake.
Nutritional counselling sessions with a medical doctor will be conducted at baseline to provide sustainable and effective strategies to improve dietary habits as well as monitor and address key barriers to dietary change. These sessions will be delivered in a group setting using PowerPoint slides and practical activities to educate the group on the allocated dietary intervention. All intervention groups will receive written information focussed on health benefits, sources, recommended intakes and practical tips related to one of the following dietary interventions; (1) to increase dietary nitrate (2) to reduce salt intake (3) to increase dietary nitrate and to reduce salt intake. Those who are randomised to the salt intervention (either alone or combined) will receive a measuring spoon as an additional strategy to support individuals to understand portion size and measure salt intake, as successfully tested by an intervention study in China. 35

Outcome measures
Outcome assessments will be conducted at baseline, month four and month six for all groups, with an additional assessment visit at month two for those in the intervention groups. Assessments at baseline and month 6 will be conducted in clinic / at home, with remaining assessments completed in the participants own home. The same instruments will be used for measurements at each time point.
Assessors are trained prior to the assessments to follow a standardised protocol. A timeline of the study procedures can be found in Table 1.  Week 2 Week 4 Week 6 Week 8 Week 10 Week 12 Week The primary outcome measures are related to feasibility and will be collected via questionnaire, review of the study procedures and by review of data collector field notes. Patient feedback, acceptance and adherence issues will be additionally assessed through qualitative focus groups conducted post-intervention. The specific feasibility considerations that will be assessed in the study have been outlined in Table 2. The CONSORT 2010 statement 38 will be used to guide the reporting of the feasibility results.

Blood pressure
Three consecutive measurements of resting blood pressure readings will be recorded in a sitting position using a calibrated OMRON automated monitor (OMRON HEM 907, OMRON Healthcare, Milton Keynes United Kingdom). The median measure will be used to estimate the blood pressure.
Blood pressure will be measured at all outcome assessment visits.

Anthropometry and body composition
Body weight will be recorded in kilograms using calibrated, electronic scales to the nearest 0.1kg where possible. Weight will be measured at all outcome assessment visits. Height will be recorded in metres using a stadiometer and measured to the nearest 0.01m where possible. The recordings for weight and height will be subsequently used to calculate BMI by dividing weight (kg) by height (m 2 ).
BMI will be calculated at baseline and end of study visits. Body composition will be measured by bioelectrical impedance. A Tanita Body Composition analyser will be used to measure body fat percentage and classification, segmental subcutaneous fat and skeletal muscle percentage (whole body, trunk, legs and arms), resting metabolism, visceral fat level and classification and body age. Body composition will be measured at baseline and end of study visits.

Physical assessments
The following physical assessments will be conducted at baseline and end of study visits. (1) Muscle strength will be measured by a hand grip-strength dynamometer. Using their right arm, the participant will be asked to make three measurements and an average calculated. The same process will be repeated for the left arm. (2) A 4 meter walk test will be done to assess the gait speed. Two trials will be conducted and the average measure of time in minutes will be used to assess the gait speed. (3) In the Timed Up and Go (TUG) test, the time taken for participants to rise from an armless chair (46 cm height), to walk 3 metres, turn, walk back and sit down will be measured. The TUG test will be performed twice consecutively, and the average of the two scores will be used. (4) The Geriatric Depression Scale (GDS) (short form) will be used to assess depressive symptoms 39 -scores range from zero to 15, with a score of zero to five indicating a normal score and a score greater than five  40 will be used to assess physical activity.

Dietary Assessment
Conventional approaches to assessing dietary intake are associated with measurement error. There is a growing consensus that combining the use of self-report instruments (like FFQ and 24 hour recall) together with biomarker analyses, could increase the accuracy of individual intake estimates, especially for episodically consumed foods. 41 42 Thus, dietary assessment data will be collected by two methods: 24 hour recall and FFQ. Participants will complete a one day 24 hour recall facilitated by a trained data collector. The FFQ is a validated questionnaire used in previous dietary research in Malaysia. 43 Information will be entered into Nutrition Pro Software (v7.5) and daily intake will be calculated.

Biological Sample Collection
The following bio-specimen will be collected in this study:

Capillary blood
All samples will be used only for the purpose of the study. The venous blood will first be centrifuged, separated into aliquots and then stored at -20°C at the KK Segamat and then transported to Monash University Sunway campus for storage. Other samples will be stored at -20°C storage. The freezer room for the stored samples will be under lock and key with restricted access.

Blood sampling
Whole blood samples will be analysed to measure changes in nitrite concentrations during the intervention. Samples will also be analysed to assess for biomarkers of cardio-vascular risk (C-reactive protein, glycated haemoglobin, nitro-tyrosine) and direct brain measures such as plasma BDNF, plasma amyloid β42 and amyloid β40. A capillary blood sample will be taken and analysed for the concentration of glucose in the blood using a portable glucometer. Dried blood spot samples will be analysed to measure changes in nitrate concentrations during the intervention. Blood spots will be first processed using a standardised elution protocol to obtain a liquid solution of the samples and  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y then analysed for nitrate concentrations using ozone-based chemiluminescence, which is the reference method for the analysis on nitrate in biological fluids. All blood sampling assessment methods will be conducted at baseline and end of study visits. Venous blood samples will be performed by a qualified medical attendant or staff nurse practising at KK Segamat and dried blood spot samples will be collected by a trained data collector.

hour and spot urine sampling
Both 24 hour urine and spot urine samples will be collected. 24 hour urine collection has been associated with a having high respondent burden owing to the time consuming nature of this method, particularly in a community and LMIC setting, where 24 hour urine sampling may be logistically difficult. Spot urine sampling is potentially a more convenient and affordable alternative. However, there are still a number of questions about the reliability of spot urine collection as a means of monitoring intervention adherence. Therefore, by using these two methods of urine collection, the authors hope to explore the feasibility of implementing these collection methods within the target population. 44 Eligible participants will be provided with a kit to collect a 24 hour urine sample during their baseline home visit (part 1). This will be provided two days before their clinic visit (part 2). The kit includes: (1) A participant information booklet with written instructions on how to collect the sample; (2) A '24 hour urine collection record' to note essential information about the urine collection; (3) Urinecollecting equipment: (a) 2.5 litre screw-capped plastic container for storing the 24 hour urine sample, (b) 1 litre plastic jug which the urine will be voided into, (c) 1 litre screw-capped plastic container either as backup container when the 2.5 litre storage container is full, or for temporal collections of urine made outside the home; (4) A permanent marker pen to note the start and finish times of urine collection in the 2.5 litre container. Trained data collectors will verbally explain the method of 24 hour urine collection to participants. The 24 hour urine collection will be initiated one day before their clinic visit and end in the morning of their appointment. Participants will also collect a spot urine sample on the morning of their clinic visit. They will be provided with a 60 ml screw-capped plastic bottle for the spot urine collection. Participants will be instructed by a trained data collector to provide a spot urine sample using the midstream clean-catch technique.
The 24 hour urine samples collected will be assessed for completeness using assessment of the duration of urine collection, the total urine volume and 24 hour urinary creatinine excretion. The urine samples will be excluded from analysis if the time of the collection falls outside the range of [22][23][24][25][26] hours, if the total 24 hour urine volume is less than 500 ml or greater than 6000 ml, and if 24 hour creatinine excretion is less than 3 mmol, or greater than 25 mmol in women, or less than 6 mmol, or  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y greater than 30 mmol in men. Urinary sodium will be determined using the ion-selective electrode method. For nitrate, samples will be diluted 1:100 and then analysed for nitrate concentrations using ozone-based chemiluminescence, which is the reference method for the analysis on nitrate in biological fluids.

Saliva sampling
Whole saliva will be collected using the passive drool technique. 1 ml of will be collected into one collection tube. Participants will be asked to generate some saliva in their mouth and when ready, the participant should hold the adaptor with collection tube attached to their mouth and pass saliva into the tube. This process is repeated until desired amount is collected. To measure nitrate, samples will be diluted 1:100 and then analysed for nitrate concentrations using ozone-based chemiluminescence.
For the salivary strips, a dedicated app freely available to download on mobile devices will be used to provide a quantitative reading of the salivary nitrite concentrations as a surrogate marker of dietary nitrate intake.

Patient and public involvement -Post-intervention qualitative evaluation
Participants will be invited to provide feedback on their participation in the intervention study via focus groups discussions. The focus group topic guide will also be piloted among a random sample of participants prior to implementation to allow feedback, refinement and tailoring prior to implementation. Two focus group discussions will be conducted within each arm of trial (total 8).
Purposive sampling will be used to capture broad variation in age, ethnicity, house location and for better understanding of the phenomenon being studied. All focus groups discussions will be audio recorded and the topics covered will include: (1) Access to the food items -financial (affordability), framework will be used to guide the qualitative analysis. 45 46 This will allow the feedback and recommendations from participants to directly inform refinements and tailoring of the intervention for a follow-on efficacy trial as well as to understand the barriers and facilitators to dietary change among this target group.  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  an anticipated drop-out rate ≤20%.

Statistical Analysis
As the main aims of this feasibility study relate to the feasibility, acceptability and the potential to deliver a dietary intervention, these data will be reported narratively illustrated with descriptive statistics. The CONSORT 2010 statement will be used to guide the reporting of this information. 38 Secondary aims are largely descriptive, aiming to provide bounds for key parameters to inform the main trial. Intention-to-treat analysis will be used to include all randomised participants and determine the key outcome measures. The recruitment and retention rate in each arm will be described. It is unlikely that statistically significant differences in retention rates will be discovered, but upper and lower bounds will be estimated, and reasons for groups with apparently low recruitment or retention rates will be explored qualitatively. Normality of the distribution of the variables and appropriate transformations (LogX, 1/X, Xn) will be performed if necessary. Summary data will be expressed as mean (SD) or frequency (percent). General linear models for repeated measures will be used to detect significant differences between the intervention groups with and without adjustment for baseline levels. Chi square test will be used for categorical variables. An interaction term (time x group) will be built to assess between-group interactions in changes in the measured outcomes during the interventions. Dietary data will be analysed using the Nutritionist Pro software (v7.5).

Data collection supervision and training
SEACO is an ISO certified research platform for its operations, therefore training and supervision are carried out in accordance with SEACO's standard operating procedures. All data collectors are required to be fluent in the local language (Bahasa Melayu or Malay language or Mandarin) and have at least a working knowledge of English. To ensure data quality and consistency across interviewers, the field supervisor will undertake random concurrent supervisory visits to observe practice. The data collectors will also be periodically observed by the project leader and the field manager. Based on the  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y scoring completed during these observation sessions, onsite training or re-training will be carried out as appropriate. Data collection is undertaken on Samsung Galaxy tablets. The data are encrypted on the tablet to ensure they are secure and inaccessible in the unlikely event that they are mislaid by data collectors. Transfer of data from the tablet to the remote server is done on a weekly basis. The data are also encrypted on the server and backed up by servers at Monash Sunway Campus and at Monash Clayton. Research staff have limited access to anonymised data.

Data monitoring
All expected and unexpected adverse events reported by participants will be recorded in an events register and reported to the Human Research Ethics Committee. Due to the nature of the treatment products, i.e. commercially available food products, no adverse events are expected. However, if participants feel in anyway adversely affected by any foods or the principal investigator feels an adverse event necessitates cessation, the participant would be advised not to continue and the appropriate measures will be taken (i.e. record in field notes, contact research nurse and principal investigator if deemed necessary).

Instructions to authors
Complete this checklist by entering the page numbers from your manuscript where readers will find each of the items listed below.
Your article may not currently address all the items on the checklist. Please modify your text to include the missing information. If you are certain that an item does not apply, please write "n/a" and provide a short explanation.
Upload your completed checklist as an extra file when you submit to a journal.