Dark Chocolate and Platelet Function in Humans

April 15, 2012 updated by: University of Aberdeen

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

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.

Study Overview

Status

Completed

Conditions

Intervention / Treatment

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.

Study Type

Interventional

Enrollment (Actual)

42

Phase

  • Not Applicable

Contacts and Locations

This section provides the contact details for those conducting the study, and information on where this study is being conducted.

Study Locations

  • United Kingdom
    • Aberdeenshire
      • Aberdeen, Aberdeenshire, United Kingdom, AB21 9SB
        • University of Aberdeen Rowett Institute of Nutrition and Health

Participation Criteria

Researchers look for people who fit a certain description, called eligibility criteria. Some examples of these criteria are a person's general health condition or prior treatments.

Eligibility Criteria

Ages Eligible for Study

18 years to 70 years (Adult, Older Adult)

Accepts Healthy Volunteers

No

Genders Eligible for Study

All

Description

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

Study Plan

This section provides details of the study plan, including how the study is designed and what the study is measuring.

How is the study designed?

Design Details

  • Primary Purpose: Prevention
  • Allocation: Randomized
  • Interventional Model: Crossover Assignment
  • Masking: Single

Arms and Interventions

Participant Group / Arm
Intervention / Treatment
Experimental: 42 healthy volunteers - crossover

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
Acute consumption (within 15 minutes) of 60 g of chocolate containing ~900 mg of total flavan-3-ols and procyanidins.
Other Names:
  • CocoanOX12%-containing chocolate
Dietary supplement: Standard dark chocolate
Acute consumption (within 15 minutes) of 60 g of chocolate containing ~400 mg total flavan-3-ols and procyanidins.
Dietary supplement: White chocolate
Acute consumption (within 15 minutes) of 60 g of white chocolate containing no flavan-3-ols and procyanidins.

What is the study measuring?

Primary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Change in light transmission aggregometry of platelet-rich plasma
Time Frame: Post-prandial, up to 6 hours after chocolate consumption
  • 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)
Time Frame: Post-prandial, up to 6 hours after chocolate consumption
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
Time Frame: Post-prandial, up to 6 hours after chocolate consumption
  • 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
Time Frame: Post-prandial, up to 6 hours after chocolate consumption
  • 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
Time Frame: Post-prandial, 2 hours after chocolate ingestion
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
Time Frame: Post-prandial, up to 6 hours after chocolate consumption
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
Time Frame: Post-prandial, up to 6 hours after chocolate consumption
Using high performance liquid chromatography (HPLC) and/ or immunoassays
Post-prandial, up to 6 hours after chocolate consumption
Total phenolics in urine
Time Frame: Post-prandial, up to 6 hours after chocolate consumption
Using the Folin-Ciocalteu assay
Post-prandial, up to 6 hours after chocolate consumption
Total catechins in urine
Time Frame: Post-prandial, up to 6 hours after chocolate consumption
Using an adaption of the DMACA assay
Post-prandial, up to 6 hours after chocolate consumption
Urinary creatinine
Time Frame: Post-prandial, up to 6 hours after chocolate consumption

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
Time Frame: At the beginning (April 2009) and end (October 2009) of the intervention period
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
Time Frame: Post-prandial, up to 6 hours after chocolate consumption
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
Time Frame: Post-prandial, just before and 6 hours after chocolate consumption
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
Time Frame: Post-prandial, up to 6 hours after chocolate consumption
  1. Plasma levels of lipid peroxides (thiobarbituric acid-reactive substances, TBARS)
  2. 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
Time Frame: Shortly after the intervention period was finished (February 2009)
Using the fatty acid methyl ester (FAME) analysis and a gas chromatographic approach
Shortly after the intervention period was finished (February 2009)

Collaborators and Investigators

This is where you will find people and organizations involved with this study.

Sponsor

University of Aberdeen

Collaborators

Biotechnology and Biological Sciences Research Council
Rural and Environment Research and Analysis Directorate (RERAD, UK)
Natraceutical Industrial S.L.U., Valencia, Spain

Investigators

  • Principal Investigator: Baukje de Roos, MSc PhD, University of Aberdeen Rowett Institute of Nutrition and Health

Publications and helpful links

The person responsible for entering information about the study voluntarily provides these publications. These may be about anything related to the study.

General Publications

Helpful Links

Study record dates

These dates track the progress of study record and summary results submissions to ClinicalTrials.gov. Study records and reported results are reviewed by the National Library of Medicine (NLM) to make sure they meet specific quality control standards before being posted on the public website.

Study Major Dates

Study Start

March 1, 2009

Primary Completion (Actual)

November 1, 2009

Study Completion (Actual)

May 1, 2011

Study Registration Dates

First Submitted

March 25, 2010

First Submitted That Met QC Criteria

April 5, 2010

First Posted (Estimate)

April 6, 2010

Study Record Updates

Last Update Posted (Estimate)

April 17, 2012

Last Update Submitted That Met QC Criteria

April 15, 2012

Last Verified

April 1, 2012

More Information

Terms related to this study

Keywords

Additional Relevant MeSH Terms

Other Study ID Numbers

  • 600
  • 09/002 (Other Identifier: Rowett Human Studies Ethical Review Panel)

Drug and device information, study documents

Studies a U.S. FDA-regulated drug product

No

Studies a U.S. FDA-regulated device product

No

product manufactured in and exported from the U.S.

No

This information was retrieved directly from the website clinicaltrials.gov without any changes. If you have any requests to change, remove or update your study details, please contact register@clinicaltrials.gov. As soon as a change is implemented on clinicaltrials.gov, this will be updated automatically on our website as well.

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