A total of 18 non-smoking subjects, between 30 and 65 years of age and with body mass index (BMI) > 35 kg/ m2, were recruited between November 2016 and March 2017. Participants were excluded if they had medical conditions (i.e., diabetes, acute coronary syndrome or transient ischaemic attack within the past 3 months, using oral anticoagulation therapy), if they reported excessive alcohol consumption (> 14 U/ week), if they were pregnant or lactating or had dietary restrictions that could interfere with the nutritional intervention and study outcomes. Written informed consent was obtained from all participants prior to participation in the study. The study was approved by the School of Medicine, Dentistry and Biomedical Sciences Research Ethics Committee of Queen’s University Belfast and adhered to the guidelines contained within the Declaration of Helsinki.
The study was a one-week, randomized crossover trial (Figure 1). Participants were randomized either to BRJ or GLVJ by use of a block design (block size n= 4; www.randomization.com) for 1 week, with a 2-weeks washout between the two interventions.
The registration number is NCT04736784. The first trial registration date on the website is 03/02/2021. Actual study start date: 30/1/2017, Actual study completion date: 15/5/2017. The source of the registration is ClinicalTrials.gov.
The intervention was designed to give the participants equal amount of nitrate (approximately 340 mg nitrate daily) from both juices. Therefore, participants were randomized to receive 115 ml of BRJ or 250 ml of GLVJ daily for 7 days. Both juices were made in the UK. The amount of nitrate in the BRJ and in the leafy juice was calculated based on previous research (1,27,28). The leafy vegetable juice contained 30 % celery, 30% cucumber, 15 romaine, 15% spinach, 5 % kale, 2.5 % lemon and 2.5 ginger. The calculated amount of nitrate in the GLVJ (340.30 mg per 250 ml) was less than the amount of the nitrate in BRJ (735.75mg/ 250 ml). Therefore, the participants were asked to drink 250 ml of the GLVJ daily for 7 days and 115 ml BRJ daily for 7 days. Previous studies have suggested that the peak action time for dietary nitrate to reduce BP is ~2.5–3 h after ingestion (29). Therefore, participants were requested to drink both juices in the evening, 2–3 h before measuring home BP.
Participants were advised to follow their usual diet and not to make any changes to dietary or lifestyle habits and instructed that their body weight was to remain stable for the duration of the intervention. They were also advised to avoid mouth wash during the study, as antibacterial mouthwash has been shown to attenuate the rise in plasma nitrite (3).
Height was assessed at baseline using a Leicester portable height measure. Before and after each intervention period, participants were weighed using recently calibrated digital weighing scales (Seca 876, SECA, Birmingham, UK), and BP was measured.
Questionnaires to assess physical activity and education level were completed at baseline. General physical activity was assessed (i.e. over the last 4 weeks) using the general practice physical activity questionnaire (GPPAQ). The primary end- point of this study was to compare the effect of two different high nitrate juices on clinic and home- measured BP between intervention groups (over the course of the 4- week intervention period).
Blood pressure measurements
Clinic blood pressure measurements
With the patient seated comfortably for 15 minutes prior to measurement and the arm supported at the level of the heart, clinical BP was measured in triplicate by using an automated BP monitor (Omron Healthcare, The Netherlands). The final value was calculated by the average from the 2nd and 3rd readings.
Daily home blood pressure monitoring
An automated BP monitor (Omron Healthcare, The Netherlands) was provided to each participant to measure daily resting BP at home. Participants were asked to conduct duplicate measurements, in a seated position, at evening, two hours after juice consumption and before going to bed. Participants were trained on how to use the monitor, with an emphasis on the correct positioning of the monitor and arm. In addition, written instructions on measuring BP were provided. A form was provided to participants to record the BP readings (SBP and DBP) and time of each measurement. The average of the measurements was calculated; the average of the values for each week was calculated to evaluate differences in BP between the two interventions. To increase participant compliance, text messages were sent to the participants, as a reminder, daily, during the course of the study before juice drinking to drink the juice and after juice drinking to record BP values, and other messages as a reminder before each lab visit. A total of 32 ml non- fasting whole blood, collected into serum and EDTA tubes, was obtained from participants, using standard phlebotomy procedures before and after each intervention period. All participants were seated for venepuncture. Samples were centrifuged at 3000 rpm for 15 minutes at 4°C and separated appropriately for the proposed assays. Sample aliquots were stored at -80°C until analysis.
Analysis of plasma nitrate and nitrite concentrations
Plasma nitrate concentrations were quantified using a commercial kit and calibrators from R&D Systems (Minneapolis, MN, USA) according to the manufacturers’ instructions. Kits were run on a Triturus automated ELISA analyser supplied by Grifols UK Ltd. Plasma samples were filtered using Amicon Ultra 2, 10,000 MW centrifugal filter units (Millipore UK Ltd). The assay procedure measured total nitrite by converting nitrate to nitrite. Samples and standards, as well as quality controls, were prepared as per the manufacturer instructions. The plate was then incubated at room temperature for 10 minutes and samples were read at 540 nm with wavelength correction at 690 nm.
Participants’ view of the intervention juices
A series of 5- point Likert- scales were used to assess ease of consumption of the intervention juices in the following question: Based on your experience in this study, did you find the juice (either BRJ or GLVJ based on the intervention week): very easy, easy, sometimes hard sometimes easy, hard, very hard. The same question was asked to assess the acceptability, with the following response choices: very acceptable, acceptable, unsure, unacceptable, very unacceptable. In addition, participants were asked if drinking the juice for seven days is shown to help improve blood pressure: (a) I would be willing to continue drinking the juice after the study finished and (b) I would be willing to spend approximately £2.25 pound per week for BRJ or £14.00 per week for GLVJ and how much they agreed with the statements given, with the following responses: strongly agree, agree, unsure, disagree, strongly disagree.
In addition, open and closed questions were also used such as whether the participants found the study products gave any side effects and if they would be willing to continue taking the products post- intervention, if found to be beneficial to health. For example, ‘’Did you experience any side effects when drinking (juice name), during the study’’, ‘’If you found drinking (juice name) over a period of seven days hard or unacceptable, please tell us why’’.
The data were checked for normality by using the Kolmogorov- Smirnov test. Descriptive statistics were obtained for all variables. Normal variables were reported as mean (SD) and categorical variables were reported as percentages. Descriptive statistics and paired t- test were performed using SPSS v17.0 for Windows (SPSS Inc, Chicago, IL). Paired sample t- tests were performed on BP data to compare the within-group effect of each juice before and after each intervention. Hills and Armitage analysis(30) used for two- period crossover design using STATA software (version 9.0, StataCorp, TX, USA) and Excel (Windows 2013). Treatment effects (difference in mean adjusted for period) and 95% CI were calculated. P values < 0.05 (2-tailed) were considered statistically significant.