Preparation of intervention supplements
Fruitflow® SD is commercially produced by DSM Nutritional Products, Basel, Switzerland, in powder format. The composition of Fruitflow® has been described previously [11]. Briefly, a standard 150 mg dose (as per the approved EC health claim) delivers up to 9 mg nucleoside derivatives, up to 10 mg simple phenolic conjugates (e.g., chlorogenic acid, other caffeic/phenolic acid derivatives), and up to 7 mg flavonoid derivatives, of which at least 2.4 mg are quercetin derivatives. The commercially produced Fruitflow® SD is standardized to ensure minimum quantities of these three compound groups are contained in each manufactured ingredient batch, ensuring that the bioactive components’ intake is consistent from batch to batch. In this study, the powder was administered as a single dose at concentrations of 30 mg, 75 mg, 150 mg, and 300 mg Fruitflow® SD (FF30, FF75, FF150, FF300). Tapioca starch was used as a placebo control (Con) (Essential Nutrition Ltd., Brough, UK). All supplements were encapsulated using size 00 Vegecaps (LGA, La Seyne-sur-Mer, France), and the final weight of each capsule was 600 mg (weight of Fruitflow® SD plus weight of tapioca starch filler).
Randomization and coding of supplements
Capsules were coded following a randomization protocol: Genstat (VSN International, 17th /18th edition) was used to generate 20 sets of randomly allocated treatments numbered 1–5. Each treatment set was allocated to a subject number, in numerical order, and the appropriate treatments were boxed and labelled with the subject number and visit number, e.g., S1–1, S1–2, S1–3, etc. All supplements were coded off-site at the Human Nutrition Unit of the University of Aberdeen and provided to the investigators prior to the start of recruitment. Subject numbers were then assigned to subjects in order of recruitment into the study. Supplements were identical concerning appearance and only differed in the coding of the capsules. The treatment code of the intervention supplements was blinded for subjects, investigators, and staff involved in the study’s conduct.
Recruitment and screening of subjects
Fifteen males aged 30–65 years were assessed for eligibility, of which 12 were recruited into the study. Recruitment was carried out from the local population, by poster advertisement within the Faculty of Medicine. Study numbers were estimated on the basis of the expected response to the 150 mg dosage: 15–20% ± 9–15% reduction from baseline ADP-mediated aggregation after 150 mg Fruitflow® SD. For such a response, we calculated a minimum of 10 subjects required to allow the effect to be detected (vs. placebo) with 80% power and a 95% confidence interval for the mean. Extra subjects were recruited to allow for failure to complete all interventions. Suitability for inclusion into the study was assessed using diet and lifestyle questionnaires and medical screening, during which blood pressure and platelet function were evaluated. Individuals with low haematology counts (platelet number < 170 × 109/ L; haematocrit < 40%; haemoglobin < 120 g/L), or low platelet function (as determined by response to 3 μmol/L ADP agonist) were not included into the study. Any subject habitually consuming dietary supplements (for example, fish oils, evening primrose oil) suspended these supplements for a minimum of 1 month before participating in the study.
Ethical considerations
Written informed consent was obtained from all subjects prior to participation, and all study procedures were in accordance with the Helsinki Declaration of 1975 (revised in 1983). The local ethical committee approved the study at Oslo University Hospital, Norway (Reference No.: 2015/ 396) and it was subsequently registered as ISRCTN53447583.
Study design
This was an active control equivalence study (a positive control study), following a double-blinded, randomized crossover design, in which the treatment interventions Con, FF30, FF75, and FF300 were compared to FF150 (standard dose). Subjects undertook all five interventions, with each intervention separated by a period of at least 7days. All study activities were undertaken at the Nutrition Dept. of the Faculty of Medicine, University of Oslo, Norway. Each intervention period was of 24-h duration. Subjects presented at the Nutrition Department facility, and baseline measurements, including fasted baseline blood samples (approximately 40 mL), were taken (t0). The intervention supplements, randomly assigned and blinded, were consumed in the presence of study investigators after the baseline venepuncture. Breakfast was then supplied, and subjects were free to leave the facility. After 24 h, subjects returned to the facility, and a 12-h fasted blood sample was taken for analysis (t24). Subjects were again given breakfast and were free to leave the facility, returning after a minimum of 7 days to repeat the procedure for the next interventions, as required, until all five interventions had been completed.
Study measurements
Platelet aggregation assay
For measurement of platelet aggregation at baseline (t0) and post-intervention (t24), blood was mixed with 3·8% trisodium citrate (9:1 (v/v), blood/citrate). Blood collection using the Monovettes system (Sarstedt, UK), platelet-rich plasma (PRP) preparation, and light-transmission aggregometry using an AggRam aggregometer (Helena Biosciences, Sunderland, UK) were carried out as described by us previously [11, 12, 15]. Platelet aggregation in 200 μL adjusted PRP was initiated by the addition of 20 μL of ADP (Helena Laboratories, Beaumont, TX) at concentrations ranging from 1 to 8 μmol/L ADP. Based on the pre-intervention baseline measurements, an ADP concentration was pre-selected for each individual, such that an optimal response could be recorded for each. All measurements were carried out in duplicate, within 2 h of blood sampling. Effects on platelet aggregation observed post-intervention were expressed as the percentage change in the area under the aggregation curve (%AUC) post-intervention compared with baseline values.
Thrombin generation capacity (TGC) assay
Thrombin generation capacity (TGC) was measured at baseline (t0) and post-intervention (t24) in citrated platelet-poor plasma (PPP), which was further treated to ensure only microparticles remained in the plasma (no platelets or large platelet fragments). In this way, the TGC related to plasma microparticle load was measured. Aliquots of 25 μL were stored frozen at − 80 °C for up to 1 month before analysis. Analyses were carried out using the Technothrombin® Thrombin Generation Assay (Diapharma Group Inc., West Chester, Ohio, US), using the protocol specified by the manufacturer but with some modification to sample preparation: platelet-poor plasma was generated by double centrifugation directly from citrated whole blood, not sequentially after generation of platelet-rich plasma. The final concentrations of tissue factor (TF) used were 1 pmol/L. Microparticle-free plasma and microparticle-high plasma (Diapharma Group Inc., West Chester, Ohio, US) were used as negative and positive controls.
Supplementary measurements
After each blood withdrawal, a haematology analyzer (Hemocytometer, Horiba ABX micros60, Montpellier, France) was used to monitor haematological parameters. Baseline plasma C-reactive protein (CRP) concentration was measured in EDTA-anticoagulated blood using a semi-quantitative latex agglutination assay (Dade Behring, Milton Keynes, UK), which allowed classification of sample CRP status as either ‘normal’ or ‘elevated’ (> 6 ng/ml CRP). Data from samples with elevated CRP was discarded.
Statistical analysis
Data are presented as mean ± standard deviation (SD). Data from interventions where evidence of platelet pre-activation due to venepuncture existed or where elevated CRP was recorded were removed from the set. Preliminary assessment of the data distribution was carried out by inspecting histograms, and data points classified as outliers were removed. Changes from baseline (t0) within the study population were analysed using a mixed model following the residual maximum likelihood (REML) approach. Initially, random effect terms were subject / (visit x timepoint), while fixed effect terms were (order + treatment) x timepoint. Significance was tested with the Wald statistic. As no significant order x treatment interactions were observed, the model was simplified (without the order term, treatment and visit are equivalents). Random effects then became subject / (treatment x timepoint), and fixed effects were treatment x timepoint. Treatments Con, FF30, FF75 and FF300 were compared to FF150, to determine equivalence or non-equivalence. Due to the small sample size, no post-hoc comparisons were made. Statistical analyses were carried out using Genstat (VSN International, 17th / 18th edition), and differences were considered significant at P < 0.05.