Dark Chocolate and Platelet Function in Humans

Study Overview
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Brief Summary
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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.

Detailed Description
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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: whether acute intake of 60 g dark chocolate enriched in flavan-3-ols, as compared with standard dark chocolate low in flavan-3-ols and white chocolate containing no flavan-3-ols, affects platelet aggregation, thromboxane A2 formation upon aggregation, in vitro bleeding time, P-selectin expression, and activation of the fibrinogen receptor; whether and how acute intake of 60 g dark chocolate enriched in flavan-3-ols, as compared with standard dark chocolate and white chocolate, affects the platelet proteome, and thereby potential new biomarkers of platelet function, as well as protein levels of anti-oxidant enzymes; identities and concentrations of flavan-3-ols and their metabolites in plasma and/ or urine 2 and 6 h after acute intake of 60 g dark chocolate enriched in flavan-3-ols, as compared with standard dark chocolate and white chocolate.

Official Title
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Acute Effects of the Consumption of Dark Chocolate Enriched in Flavan-3-ols on Platelet Function and the Platelet Proteome

Conditions
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Cardiovascular Disease

Intervention / Treatment
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* Dietary Supplement: Dark chocolate enriched in flavan-3-ols and procyanidins
* Dietary Supplement: Standard dark chocolate
* Dietary Supplement: White chocolate


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Healthy male and/or female volunteers, aged between 18 and 70 years Exclusion Criteria: Subjects are excluded if: they are taking aspirin or aspirin-containing drugs, other anti-inflammatory drugs, or any drugs or herbal medicines known to alter platelet function or the haemostatic system in general (without a minimum washout period of one month) they are taking fish oils or evening primrose oil, or fat soluble vitamin supplements within the last 4 weeks they are taking any medicine known to affect lipid and/or glucose metabolism they are taking hormone replacement therapy they have any known clinical signs of diabetes, hypertension, renal, hepatic, hematological disease, gastrointestinal disorders, endocrine disorders, coronary heart disease, infection or cancer they are suffering from alcohol or any other substance abuse or are having eating disorders they are usually consuming a vegetarian diet they have a BMI below 18 or above 35 kg/ sqm they are undertaking more than 6 hours of vigorous exercise per week they are having an abnormal menstrual cycle they are pregnant they suffer from an allergy to cocoa or any of the ingredients contained within either of the chocolate bars they have been giving a pint of blood for transfusion purposes within the last month they have a low platelet count (< 170 x 10E09/ L) they have unsuitable veins for blood sampling and/ or cannulation their hematocrit is below 40 % for males and 35 % for females their haemoglobin is below 130 g/ L for males and 115 g/ L for females they are not able to travel on their own to the Rowett Institute of Nutrition and Health, Aberdeen for each of the interventions

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 70 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Prevention
Allocation: Randomized
Intervention Model: Crossover Assignment
Masking: Single


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: 42 healthy volunteers - crossover<br>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<br>* Acute consumption (within 15 minutes) of 60 g of chocolate containing  900 mg of total flavan-3-ols and procyanidins.<br>* Other names: CocoanOX12%-containing chocolate;Dietary Supplement: Standard dark chocolate<br>* Acute consumption (within 15 minutes) of 60 g of chocolate containing  400 mg total flavan-3-ols and procyanidins.<br>Dietary Supplement: White chocolate<br>* Acute consumption (within 15 minutes) of 60 g of white chocolate containing no flavan-3-ols and procyanidins.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| 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) | Post-prandial, up to 6 hours after chocolate consumption | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change in ex vivo bleeding time using the Platelet Function Analyzer-100 (PFA-100) | Using collagen-epinephrine coated cartridges. | 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 | 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 | 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. | 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 | 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 | Post-prandial, up to 6 hours after chocolate consumption | 
| Total phenolics in urine | Using the Folin-Ciocalteu assay | Post-prandial, up to 6 hours after chocolate consumption | 
| Total catechins in urine | Using an adaption of the DMACA assay | 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. | Post-prandial, up to 6 hours after chocolate consumption | 
| Analysis of flavan-3-ol and procyanidin contents in study chocolates | Using an HPLC method | 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 | 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 | Post-prandial, just before and 6 hours after chocolate consumption | 
| Markers of oxidative stress in plasma | Plasma levels of lipid peroxides (thiobarbituric acid-reactive substances, TBARS) Activity of glutathione peroxidase (Only at t = 2 h after chocolate ingestion) | 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 | Shortly after the intervention period was finished (February 2009) | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Platelets, Platelet aggregation, Platelet activation, Platelet function, Light transmission aggregometry, Platelet Function Analyzer-100 (PFA-100), P-selectin, Fibrinogen receptor, Glycoprotein IIb-IIIa, Chocolate, Cocoa, Flavan-3-ols, Procyanidins