Evaluation of the Equivalence of Different Intakes of Fruitflow to Affect Platelet Aggregation and Thrombin Generating Capacity in a Randomized Double-Blind Pilot Study in Male Subjects | BMC Nutrition


Preparation of additional intervention

Fruitflow® SD is produced commercially by DSM Nutritional Products, Basel, Switzerland, in powder form. The composition of Fruitflow® has been described previously [11]. In short, a standard dose of 150 mg (in accordance with the EC approved health claim) provides up to 9 mg of nucleoside derivatives, up to 10 mg of 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 that minimum amounts of these three groups of compounds are contained in each batch of ingredients manufactured, ensuring that the consumption of bioactive components 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 of 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 (Fruitflow® SD weight plus loading weight d. ‘tapioca starch).

Randomization and coding of supplements

The capsules were coded according to a randomization protocol: Genstat (VSN International, 17th / 18th edition) was used to generate 20 sets of treatments randomly assigned and numbered 1 to 5. Each treatment set was assigned a number. subject number, in numerical order, and the appropriate treatments have been boxed and labeled with the subject number and visit number, for example, S1–1, S1–2, S1–3, etc. All supplements were coded offsite at the University of Aberdeen Human Nutrition Unit and provided to investigators prior to the start of recruitment. Subject numbers were then assigned to the subjects in the order of enrollment into the study. The supplements were identical in appearance and differed only in the coding of the capsules. The treatment supplement intervention code was blinded to the subjects, investigators, and staff involved in the conduct of the study.

Recruitment and selection of subjects

Fifteen men aged 30 to 65 were assessed for eligibility, of whom 12 were recruited into the study. Recruitment was done from the local population, by poster advertising within the Faculty of Medicine. The number of studies was estimated based on the expected response at the 150 mg dose: 15 to 20% ± 9 to 15% reduction from baseline ADP-mediated aggregation after 150 mg of Fruitflow® SD. For such a response, we calculated a minimum of 10 subjects necessary to allow detection of the effect (vs placebo) with a power of 80% and a confidence interval of 95% for the mean. Additional subjects were recruited to account for the failure of all interventions. Suitability for inclusion in the study was assessed using diet and lifestyle questionnaires and medical screening, during which blood pressure and platelet function were assessed. Individuals with low blood counts (platelet count 9/ THE ; hematocrit

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 1983). The local ethics committee approved the study at University Hospital in Oslo, Norway (reference number: 2015/396) and it was subsequently registered under number ISRCTN53447583.

Study design

This was an active control equivalence study (a positive control study), following a randomized, double-blind, crossover design, in which the treatment interventions Con, FF30, FF75 and FF300 were compared to FF150 ( standard dose). Subjects undertook all five interventions, each intervention being separated by a period of at least 7 days. All study activities were undertaken at the Department of Nutrition, Faculty of Medicine, University of Oslo, Norway. Each intervention period lasted 24 hours. Subjects presented to the nutrition service facility and baseline measurements, including fasting baseline blood samples (approximately 40 ml), were taken (t0). The randomly assigned, blinded intervention supplements were consumed in the presence of study investigators after baseline venipuncture. Breakfast was then served and subjects were free to leave the establishment. After 24 h, subjects returned to the facility and a 12 h fasting blood sample was collected for analysis (t24). Subjects again ate breakfast and were free to leave the facility, then return after a minimum of 7 days to repeat the procedure for subsequent procedures, as needed, until all five procedures were completed. .

Study measures

Platelet aggregation test

For measurement of platelet aggregation at baseline (t0) and after surgery (t24), blood was mixed with 3.8% trisodium citrate (9: 1 (v / v), blood / citrate). Blood collection using the Monovettes system (Sarstedt, UK), preparation of platelet rich plasma (PRP) and light transmission aggregometry using an AggRam aggregometer (Helena Biosciences , Sunderland, United Kingdom) were carried out as described above. [11, 12, 15]. Platelet aggregation in 200 L of adjusted PRP was initiated by the addition of 20 L of ADP (Helena Laboratories, Beaumont, TX) at concentrations ranging from 1 to 8 μmol / L of ADP. Based on the pre-intervention baseline measurements, a concentration of ADP was preselected for each individual, so that an optimal response could be recorded for each. All measurements were performed in duplicate, within 2 hours of blood collection. The effects on platelet aggregation observed after the intervention were expressed as the percentage change in the area under the aggregation curve (% AUC) after the intervention compared to baseline.

Thrombin Generating Capacity (TGC) Assay

The thrombin generating capacity (TGC) was measured at baseline (t0) and after the procedure (t24) in citrated platelet poor plasma (PPP), which was then processed to ensure that only the microparticles were remained in the plasma (no platelets or large fragments of platelets). In this way, the TGC related to the plasma microparticle load was measured. 25 L aliquots were stored frozen at -80 ° C for up to 1 month prior to analysis. Analyzes were performed using the Technothrombin® Thrombin Generation Assay (Diapharma Group Inc., West Chester, Ohio, US), using the protocol specified by the manufacturer but with some modifications to sample preparation: platelet poor plasma was generated directly by double centrifugation. from citrated whole blood, and not sequentially after generation of platelet-rich plasma. The final tissue factor (TF) concentrations used were 1 pmol / L. Plasma without microparticles and plasma rich in microparticles (Diapharma Group Inc., West Chester, Ohio, USA) were used as negative and positive controls.

Additional measures

After each blood sample, a hematology analyzer (Hemocytometer, Horiba ABX micros60, Montpellier, France) was used to monitor the hematological parameters. Baseline plasma C-reactive protein (CRP) concentration was measured in EDTA-anticoagulated blood using a semi-quantitative latex agglutination test (Dade Behring, Milton Keynes, UK ), which allowed the sample’s CRP status to be classified as ‘normal’ or ‘high’ (> 6 ng / ml CRP). Data from samples with high CRP were discarded.

statistical analyzes

Data are presented as the mean ± standard deviation (SD). Data from procedures where there was evidence of platelet pre-activation due to venipuncture or where elevated CRP was recorded were removed from the pool. A preliminary assessment of the data distribution was made by inspecting the histograms, and data points classified as outliers were removed. Changes from baseline (t0) within the study population were analyzed using a mixed model following the maximum residual likelihood (REML) approach. Initially, the random effect terms were subject / (visit x time point), while the fixed effect terms were (order + treatment) x time point. Significance was tested with the Wald statistic. As no significant order x treatment interaction was observed, the model was simplified (without the order term, treatment and visit are equivalent). Random effects then became the subject / (treatment x time point), and fixed effects were treatment x time point. The Con, FF30, FF75 and FF300 treatments were compared to FF150, to determine equivalence or non-equivalence. Due to the small sample size, no post-hoc comparison was performed. Statistical analyzes were performed using Genstat (VSN International, 17th / 18th ed.), And the differences were considered significant at P

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