An interventional study of Dark chocolate enriched in flavan-3-ols and procyanidins and Standard dark chocolate in Cardiovascular Disease, sponsored by University of Aberdeen. Completed at 1 site in United Kingdom. Open to participants aged 18 Years to 70 Years, including healthy volunteers. Per ClinicalTrials.gov, last updated 2012-04-17.
Sponsored by University of Aberdeen · Not applicable, Interventional, and Prevention
Cardiovascular disease is a major cause of mortality worldwide and responsible for one out of three global deaths. A main characteristic of cardiovascular disease is impaired blood flow and formation of blood clots. Platelets are clot-forming cells responsible for the prevention of bleeding. However, in disease conditions they may be overly activated, promoting blood clots and blockage of blood vessels.
Consumption of diets rich in fruits and vegetables decreases mortality from cardiovascular disease through a number of mechanisms, including the prevention of platelet clotting and aggregation. There is some evidence suggesting that platelet aggregation may be modulated through a group of compounds known as flavan-3-ols, which are found in various foods, and especially in cocoa. However, the mechanisms by which those compounds affect platelet function are not yet fully understood. We designed a human study assessing the mechanisms by which flavan-3-ols from cocoa beneficially affect platelet function and the platelet proteome.
Cardiovascular disease (CVD) is a primary cause of premature deaths worldwide, with incidence rates in the United Kingdom, particularly in Scotland, being amongst the highest worldwide. Thus identification of dietary components that most effectively prevent CVD is potentially of wide public health benefit.
Consumption of diets rich in plant-based products protects against the development of CVD. Such effects have been ascribed in part to polyphenols, which are non-nutritive but, potentially bioactive secondary metabolites ubiquitous found in fruits, vegetables, herbs, spices, teas and wines. The beneficial effects of polyphenols on CVD is believed to be mediated, at least in part, though improving platelet function. At least 10 human intervention studies found a consistent and robust beneficial effect of cocoa products on platelet function, but unfortunately all of these studies used only one or two methods to assess platelet function, therefore only getting limited insights into the complex physiological behavior of platelets. In addition, none of these studies assessed potential mechanisms by which flavan-3-ols may inhibit platelet function. Schramm et al. have shown that consumption of chocolate rich in flavan-3-ols and their oligomers (procyanidins) lead to increased production of prostacyclin, a strong platelet inhibitor. This finding has also been observed when aortic endothelial cells are treated with procyanidins in vitro. Thus the stimulation of prostacyclin production in endothelial cells may reflect one pathway by which flavan-3-ols indirectly inhibit platelet activation. Many other potential mechanisms are discussed in the literature but so far the evidence for such mechanisms is limited or non-existing.
In this study we assess effects of consumption of chocolate enriched in flavan-3-ols on platelet function by measuring not only platelet aggregation, but also in vitro coagulation and platelet activation in healthy humans. In addition, we examine the effects of consumption of flavan-3-ols on the regulation of the platelet proteome to elucidate pathways by which these bioactive cocoa compounds affect platelet function.
HYPOTHESIS
Acute consumption of a moderate amount of dark chocolate enriched in flavan-3-ols results in decreased platelet activation and aggregation by decreasing the levels of thromboxane A2 produced by endothelial cells.
OBJECTIVES
The main objective of the proposed study is to determine whether consumption of 60 g dark chocolate enriched in flavan-3-ols results in decreased platelet activation and aggregation by decreasing levels of thromboxane A2, as well as assessing what other mechanisms could be involved.
The specific objectives of the proposed study are to determine:
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Exclusion Criteria:
Subjects are excluded if:
Acute consumption of three interventions (60 g dark chocolate enriched in flavan-3-ols, 60 g standard dark chocolate, or 60 g white chocolate) on three separate days (at least 2 weeks apart) in random order. Post-prandial measurements at t = 0 h, t = 2 h and t = 6 h.
Dietary Supplement: Dark chocolate enriched in flavan-3-ols and procyanidins · Dietary Supplement: Standard dark chocolate · Dietary Supplement: White chocolate
Acute consumption (within 15 minutes) of 60 g of chocolate containing \~900 mg of total flavan-3-ols and procyanidins.
Also known as: CocoanOX12%-containing chocolate
Acute consumption (within 15 minutes) of 60 g of chocolate containing \~400 mg total flavan-3-ols and procyanidins.
Acute consumption (within 15 minutes) of 60 g of white chocolate containing no flavan-3-ols and procyanidins.
Change in light transmission aggregometry of platelet-rich plasma
* Using a Helena Platelet Aggregation Chromogenic Kinetics System-4 (PACKS-4) light transmission aggregometer * Induced by adenosine diphosphate (ADP) and thrombin receptor-activating peptide (TRAP)
Time frame: Post-prandial, up to 6 hours after chocolate consumption
Change in ex vivo bleeding time using the Platelet Function Analyzer-100 (PFA-100)
Using collagen-epinephrine coated cartridges.
Time frame: Post-prandial, up to 6 hours after chocolate consumption
Change in P-selectin expression and activation of the fibrinogen receptor by flow cytometry
* P-selectin expression as early marker for platelet activation * Activated fibrinogen receptor as late marker for platelet activation * Induced by ADP and TRAP * Using BD FACSArray Bioanalyzer
Time frame: Post-prandial, up to 6 hours after chocolate consumption
Levels of flavan-3-ols and their metabolites in plasma and urine
* Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) * Enzyme-hydrolysed for total flavan-3-ols ((-)-epicatechin equivalents) * Non-Hydrolysed for metabolic profile
Time frame: Post-prandial, up to 6 hours after chocolate consumption
Changes in the platelet proteome
Using 2D-gel electrophoresis and LC-MS/MS identification of proteins.
Time frame: Post-prandial, 2 hours after chocolate ingestion
Changes in thromboxane A2 production induced by ADP and TRAP
Using enzyme-linked immunosorbent assay (ELISA) in plasma after platelet aggregation
Time frame: Post-prandial, up to 6 hours after chocolate consumption
Levels of prostacyclin and/ or leukotrienes in plasma
Using high performance liquid chromatography (HPLC) and/ or immunoassays
Time frame: Post-prandial, up to 6 hours after chocolate consumption
Total phenolics in urine
Using the Folin-Ciocalteu assay
Time frame: Post-prandial, up to 6 hours after chocolate consumption
Total catechins in urine
Using an adaption of the DMACA assay
Time frame: Post-prandial, up to 6 hours after chocolate consumption
Urinary creatinine
Using a Thermo KONELAB 30 selective chemistry analyser (Thermo Scientific, Hertfordshire, UK) and its respective kit To be used for normalisation of urinary flavan-3-ols and total phenolics from spot urine samples.
Time frame: Post-prandial, up to 6 hours after chocolate consumption
Analysis of flavan-3-ol and procyanidin contents in study chocolates
Using an HPLC method
Time frame: At the beginning (April 2009) and end (October 2009) of the intervention period
Non-targeted 1H-NMR of plasma and urine samples
To establish a metabolic profile - markers of intake and potential effects on host metabolism
Time frame: Post-prandial, up to 6 hours after chocolate consumption
Non-targeted LC-MS of urine samples
To establish a metabolic profile - markers of intake and potential effects on host metabolism
Time frame: Post-prandial, just before and 6 hours after chocolate consumption
Markers of oxidative stress in plasma
1. Plasma levels of lipid peroxides (thiobarbituric acid-reactive substances, TBARS) 2. Activity of glutathione peroxidase (Only at t = 2 h after chocolate ingestion)
Time frame: Post-prandial, up to 6 hours after chocolate consumption
Fatty acid analysis of study chocolates
Using the fatty acid methyl ester (FAME) analysis and a gas chromatographic approach
Time frame: Shortly after the intervention period was finished (February 2009)
This study is completed, as verified in Apr 2012. You cannot join it, but the record below documents what was studied.
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University of Aberdeen