Clinical trial NCT01099150: Dark Chocolate and Platelet Function in Humans
Official title: Acute Effects of the Consumption of Dark Chocolate Enriched in Flavan-3-ols on Platelet Function and the Platelet Proteome
Study type: INTERVENTIONAL
Overall status: COMPLETED
Phase: NA
Conditions: Cardiovascular Disease
Keywords: 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
Lead sponsor: University of Aberdeen
Sponsor class: OTHER
Collaborators: name: Rural and Environment Research and Analysis Directorate (RERAD, UK)
name: Biotechnology and Biological Sciences Research Council
name: Natraceutical Industrial S.L.U., Valencia, Spain
First posted: 2010-04-06
Enrollment: 42
Sex: ALL
Minimum age (years): 18
Maximum age (years): 70
Standard ages: ADULT
OLDER_ADULT
Accepts healthy volunteers: yes
Allocation: RANDOMIZED
Intervention model: CROSSOVER
Primary purpose: PREVENTION

Brief summary: 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: 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:

1. 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;
2. 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;
3. 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.

Eligibility criteria: 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

Primary outcomes:
measure: Change in light transmission aggregometry of platelet-rich plasma
timeFrame: Post-prandial, up to 6 hours after chocolate consumption
Secondary outcomes:
measure: Change in ex vivo bleeding time using the Platelet Function Analyzer-100 (PFA-100)
timeFrame: Post-prandial, up to 6 hours after chocolate consumption
measure: Change in P-selectin expression and activation of the fibrinogen receptor by flow cytometry
timeFrame: Post-prandial, up to 6 hours after chocolate consumption
measure: Levels of flavan-3-ols and their metabolites in plasma and urine
timeFrame: Post-prandial, up to 6 hours after chocolate consumption
measure: Changes in the platelet proteome
timeFrame: Post-prandial, 2 hours after chocolate ingestion
measure: Changes in thromboxane A2 production induced by ADP and TRAP
timeFrame: Post-prandial, up to 6 hours after chocolate consumption
measure: Levels of prostacyclin and/ or leukotrienes in plasma
timeFrame: Post-prandial, up to 6 hours after chocolate consumption
measure: Total phenolics in urine
timeFrame: Post-prandial, up to 6 hours after chocolate consumption
measure: Total catechins in urine
timeFrame: Post-prandial, up to 6 hours after chocolate consumption
measure: Urinary creatinine
timeFrame: Post-prandial, up to 6 hours after chocolate consumption
measure: Analysis of flavan-3-ol and procyanidin contents in study chocolates
timeFrame: At the beginning (April 2009) and end (October 2009) of the intervention period
measure: Non-targeted 1H-NMR of plasma and urine samples
timeFrame: Post-prandial, up to 6 hours after chocolate consumption
measure: Non-targeted LC-MS of urine samples
timeFrame: Post-prandial, just before and 6 hours after chocolate consumption
measure: Markers of oxidative stress in plasma
timeFrame: Post-prandial, up to 6 hours after chocolate consumption
measure: Fatty acid analysis of study chocolates
timeFrame: Shortly after the intervention period was finished (February 2009)
Other outcomes:
Locations:
city: Aberdeen
country: United Kingdom
facility: University of Aberdeen Rowett Institute of Nutrition and Health
state: Aberdeenshire
Study officials:
affiliation: University of Aberdeen Rowett Institute of Nutrition and Health
name: Baukje de Roos, MSc PhD
role: PRINCIPAL_INVESTIGATOR
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