Aims and hypotheses
It is hypothesised that the gut microbiota and their metabolites acetate and butyrate prevent the development of hypertension and that medicinal food containing prebiotic fibres and postbiotics (i.e. acetate and butyrate), such as HAMSAB, can be used to lower BP. Thus, our aim is to determine if HAMSAB, which produces high levels of SCFAs as well as leading to the release of high levels of acetate and butyrate as a result of microbial fermentation, could be used as a new strategy to lower BP.
Study design
This is a double-blinded, randomised, cross-over, placebo controlled trial as shown in Figure 1.
Recruitment summary
Recruitment is being conducted through advertisement placed around the Alfred Hospital (Melbourne, Australia) precinct as well as public places (GP offices, shopping malls/centres, noticeboards) in this locality. Previous research participants who approved to be contacted for future research were also contacted via email through a large database to be considered for the trial. Online social media platforms are also used to recruit participants (Monash FODMAP platforms, Marques Lab, Facebook, Twitter, Google ads). The trial gained exposure through an article in a tabloid based in Melbourne (The Herald Sun) as well as an article through a Monash University online news platform (Monash Lens). Recruitment started in July 2019 and is estimated to finish in June 2021.
Study population
Males and females with untreated hypertension as defined by the Australian National Heart Foundation guidelines (office BP ≥140/90 mmHg and 24h ambulatory blood pressure monitoring (ABPM) ≥130/80) are being recruited for this study. Participants with masked hypertension are included and defined as (office BP ≤140/90 mmHg but ABPM ≥130/80). Participants are also required to have a BMI of 18.5-35kg/m2. Exclusion criteria include the use of anti-hypertensive medication, office BP ≥165/100 mmHg, recent use of antibiotics (<3 months) or probiotics (<6 weeks), Type 1 or 2 diabetes, pregnancy and the presence of gastrointestinal diseases. Participants are also excluded if there are any dietary requirements (e.g., vegetarian and coeliac). Figure 2 describes the current study population at time of publication.
Sample size
In this proof-of-principle study, we estimate we will require 26 subjects to achieve 80% power with α=0.05 (calculated effect size 0.8) to determine a 7 mmHg difference in ambulatory daytime systolic BP after intake of modified HAMS for 3 weeks. The 3 weeks intervention was chosen based on previously published papers (17, 20). We will aim to recruit 31 participants per group to allow for a 20% drop out rate. We have recruited 17 participants to date, with one dropout due to BP >165/100 and requiring medication. One participant had to be excluded after completion of the trial due to antibiotic intake needed while in the second arm of the study, resulting in 16 participants that have completed the trial. Due to the SARS-COV-2 pandemic, we were forced to stop the trial, which has recommenced in 2021.
Study protocol and measurements
A diagram of the study protocol is presented in Figure 1. Participants are randomly stratified based on age, sex and BMI using REDCap (Version 9.1.0 Tennessee) to either a diet containing 40g HAMSAB or placebo maize-starch daily. Both the study participants and study coordinator are blinded to the diet containing the prebiotic supplement. The study period is 9 weeks: 3 weeks actively on either diet, with a 3-week washout period in between. Participants are provided 2 meals daily and are instructed to eat their usual foods around the trial’s food. The dietary intake of the participants is not restricted, and the foods provided to them are suggested to in place of normal foods (i.e. replacing a usual breakfast of toast with a ‘breakfast muffin’). To increase compliance, we have opted to not replace every meal. A sample 7-day meal plan that is cycled during the 3-week diet phase is shown in Table 1. Participants are provided with a list of foods containing natural resistant starches and SCFAs to avoid during the study period (e.g., green banana flour, waxy maize starch, kombucha, vinegars). Participants are provided with a food diary for the duration of the study, where they document approximate intake in quartiles (i.e. 0, ¼, ½, ¾, all). This data is then translated for nutritional analysis using FoodWorks Professional Software (Version 7.01, Xyris, Queensland).
Participants are initially screened over the phone to assess eligibility according to the inclusion and exclusion criteria. Those that meet the inclusion criteria are invited to attend a baseline assessment where signed consent is obtained. At the baseline assessment, participants spend approximately 60-90 minutes with the researcher for all BP measurements, including testing of office BP, carotid-to-femoral pulse wave velocity, as well as providing the 24h ABPM device and home monitoring device. The researcher discusses the 3-day food diary with the participants, collects the 24-hr dietary recall, and provides the faecal tubes with instructions for collection.
Office BP is measured under resting conditions (>5 minutes sitting), with the researcher not in the room and an average of three measurements is taken using an automated digital BP monitor (Omron Healthcare, Japan, HEM-907). During the office baseline BP measurements participants are seated with their back supported, arms and legs uncrossed, and not speaking. Participants are also instructed on how to correctly measure their BP at home according to the Australian Heart Foundation guidelines, and they are provided with a calibrated and research approved BP monitor (Omron Healthcare, Japan, HEM-7121) for the duration of the study for home BP measurements. At home, participants take two readings at the same time each day under rested conditions whilst on either diet which they document in a BP diary. Participants are also instructed to document any factors that they believe may affect their BP in this diary (e.g. medication changes, illnesses, severe stress).
Measurements of carotid-femoral pulse wave velocity and pulse wave analysis are performed in duplicate using the Sphygmocor XCEL device (AtCor Medical, Sydney, NSW). Participants are seated and rested for the brachial measurements of pulse wave analysis and in the supine position for the pulse wave velocity measurement. Mobil-O-Graph (I.E.M Industrielle Entwicklung Medizintechnik GmbH) BP monitoring devices are used for the 24h ABPM measurements. This takes BP measurements every 15 minutes during the day time and 30 minutes during the night time period. Night and day time periods are confirmed by a diary completed by the subject. Once the ABPM monitor has been returned to the researcher and hypertension has been confirmed, participants are provided with study food.
Three-day food diaries and 24 hour food recalls are used to understand habitual dietary intake amongst the study participants at each visit. Three-day food diaries are to be completed over two week days and one weekend day. This dietary information is recorded in FoodWorks Professional Software (Version 7.01, Xyris, Queensland) to calculate the average energy and macronutrient intake for each participant. Quantification of resistant starch is calculated from the Monash University FODMAP database and resistant starch report (21). Participants are also instructed to follow the Australian Government’s ‘Eat for Health’ guidelines on alcohol, which limits alcohol to no more than two standard drinks on any day, and no more than four standard drinks on any occasion.
BMI and waist-to-hip ratio are measured at each visit. Patients are instructed to not plan any intended weight loss or weight gain strategies during the duration of the study, due to the effect of weight changes on BP. Participants also record the amount and types of physical activity they have participated in, during the week prior to each visit.
A fasting blood sample is taken on each visit and all testing is performed at the site hospital (The Alfred Hospital, Melbourne). Standard biochemical testing is performed including fasting blood glucose and fasting lipids and blood electrolytes and liver enzyme tests to determine renal and liver function.
Plasma and faecal SCFAs will be measured at the end of the study using gas chromatography in triplicates, as previously described (22). Faecal DNA preserved in DNA/RNA Shield and stored at -80oC will be extracted using the QIAamp PowerFecal DNA kit (Qiagen). The V4 region of the bacterial 16S rRNA will be amplified by PCR using 20 ng of DNA, Platinum Hot Start PCR master mix (ThermoFisher Scientific), 515F and 926R primers (Bioneer) and methods previously described (23) in a Veriti Thermal Cycler (ThermoFisher Scientific). Two hundred and forty ng of the product will be pooled, cleaned and then sequenced in a Illumina MiSeq sequencer (300 bp paired-end reads, with minimum 100,000 reads per sample). 16S data will be analysed using the QIIME2 workflow (24). We will analyse faecal samples collected from before, during (7 days) and after the interventions in terms of alpha diversity (i.e., number of bacteria and distribution within samples), beta diversity (i.e., types of bacteria and prevalence between samples) and taxonomic changes according to recent guidelines (25) (Figure 3). This will inform further metagenomics analyses.
Data analysis
We will perform linear mixed model analyses for repeated measures with between and within subjects to analyse the phenotype such as BP and pulse wave velocity using SPSS (version 25), where fixed effects include time (baseline, after first arm, before second arm, after second arm) and treatment group (placebo versus HAMSAB) (26, 27). Gut microbiome will be analysed using QIIME2 (24) and other in-house bioinformatics pipelines (such as (20).