Dark Chocolate and Platelet Function in Humans

This study has been completed.
Sponsor:
Collaborators:
Rural and Environment Research and Analysis Directorate (RERAD, UK)
Biotechnology and Biological Sciences Research Council
Natraceutical Industrial S.L.U., Valencia, Spain
Information provided by (Responsible Party):
University of Aberdeen
ClinicalTrials.gov Identifier:
NCT01099150
First received: March 25, 2010
Last updated: April 15, 2012
Last verified: April 2012
  Purpose

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.


Condition Intervention
Cardiovascular Disease
Dietary Supplement: Dark chocolate enriched in flavan-3-ols and procyanidins
Dietary Supplement: Standard dark chocolate
Dietary Supplement: White chocolate

Study Type: Interventional
Study Design: Allocation: Randomized
Endpoint Classification: Efficacy Study
Intervention Model: Crossover Assignment
Masking: Single Blind (Investigator)
Primary Purpose: Prevention
Official Title: Acute Effects of the Consumption of Dark Chocolate Enriched in Flavan-3-ols on Platelet Function and the Platelet Proteome

Further study details as provided by University of Aberdeen:

Primary Outcome Measures:
  • Change in light transmission aggregometry of platelet-rich plasma [ Time Frame: Post-prandial, up to 6 hours after chocolate consumption ] [ Designated as safety issue: No ]
    • 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)


Secondary Outcome Measures:
  • 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 ] [ Designated as safety issue: No ]
    Using collagen-epinephrine coated cartridges.

  • 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 ] [ Designated as safety issue: No ]
    • 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

  • Levels of flavan-3-ols and their metabolites in plasma and urine [ Time Frame: Post-prandial, up to 6 hours after chocolate consumption ] [ Designated as safety issue: No ]
    • Using liquid chromatography-tandem mass spectrometry (LC-MS/MS)
    • Enzyme-hydrolysed for total flavan-3-ols ((-)-epicatechin equivalents)
    • Non-Hydrolysed for metabolic profile

  • Changes in the platelet proteome [ Time Frame: Post-prandial, 2 hours after chocolate ingestion ] [ Designated as safety issue: No ]
    Using 2D-gel electrophoresis and LC-MS/MS identification of proteins.

  • Changes in thromboxane A2 production induced by ADP and TRAP [ Time Frame: Post-prandial, up to 6 hours after chocolate consumption ] [ Designated as safety issue: No ]
    Using enzyme-linked immunosorbent assay (ELISA) in plasma after platelet aggregation

  • Levels of prostacyclin and/ or leukotrienes in plasma [ Time Frame: Post-prandial, up to 6 hours after chocolate consumption ] [ Designated as safety issue: No ]
    Using high performance liquid chromatography (HPLC) and/ or immunoassays

  • Total phenolics in urine [ Time Frame: Post-prandial, up to 6 hours after chocolate consumption ] [ Designated as safety issue: No ]
    Using the Folin-Ciocalteu assay

  • Total catechins in urine [ Time Frame: Post-prandial, up to 6 hours after chocolate consumption ] [ Designated as safety issue: No ]
    Using an adaption of the DMACA assay

  • Urinary creatinine [ Time Frame: Post-prandial, up to 6 hours after chocolate consumption ] [ Designated as safety issue: No ]

    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.


  • 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 ] [ Designated as safety issue: No ]
    Using an HPLC method

  • Non-targeted 1H-NMR of plasma and urine samples [ Time Frame: Post-prandial, up to 6 hours after chocolate consumption ] [ Designated as safety issue: No ]
    To establish a metabolic profile - markers of intake and potential effects on host metabolism

  • Non-targeted LC-MS of urine samples [ Time Frame: Post-prandial, just before and 6 hours after chocolate consumption ] [ Designated as safety issue: No ]
    To establish a metabolic profile - markers of intake and potential effects on host metabolism

  • Markers of oxidative stress in plasma [ Time Frame: Post-prandial, up to 6 hours after chocolate consumption ] [ Designated as safety issue: No ]
    1. Plasma levels of lipid peroxides (thiobarbituric acid-reactive substances, TBARS)
    2. Activity of glutathione peroxidase (Only at t = 2 h after chocolate ingestion)

  • Fatty acid analysis of study chocolates [ Time Frame: Shortly after the intervention period was finished (February 2009) ] [ Designated as safety issue: No ]
    Using the fatty acid methyl ester (FAME) analysis and a gas chromatographic approach


Enrollment: 42
Study Start Date: March 2009
Study Completion Date: May 2011
Primary Completion Date: November 2009 (Final data collection date for primary outcome measure)
Arms Assigned Interventions
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 Name: 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.

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

Ages Eligible for Study:   18 Years to 70 Years
Genders Eligible for Study:   Both
Accepts Healthy Volunteers:   Yes
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
  Contacts and Locations
Choosing to participate in a study is an important personal decision. Talk with your doctor and family members or friends about deciding to join a study. To learn more about this study, you or your doctor may contact the study research staff using the Contacts provided below. For general information, see Learn About Clinical Studies.

Please refer to this study by its ClinicalTrials.gov identifier: NCT01099150

Locations
United Kingdom
University of Aberdeen Rowett Institute of Nutrition and Health
Aberdeen, Aberdeenshire, United Kingdom, AB21 9SB
Sponsors and Collaborators
University of Aberdeen
Rural and Environment Research and Analysis Directorate (RERAD, UK)
Biotechnology and Biological Sciences Research Council
Natraceutical Industrial S.L.U., Valencia, Spain
Investigators
Principal Investigator: Baukje de Roos, MSc PhD University of Aberdeen Rowett Institute of Nutrition and Health
  More Information

Additional Information:
Publications:
Natella F, Nardini M, Virgili F, Scaccini C. Role of dietary polyphenols in the platelet aggregation network - A review of the in vitro studies. Curr Top Nutraceut Res 4(1): 1-22, 2006.

Responsible Party: University of Aberdeen
ClinicalTrials.gov Identifier: NCT01099150     History of Changes
Other Study ID Numbers: 600, 09/002
Study First Received: March 25, 2010
Last Updated: April 15, 2012
Health Authority: United Kingdom: Research Ethics Committee

Keywords provided by University of Aberdeen:
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

Additional relevant MeSH terms:
Cardiovascular Diseases
Procyanidin
Proanthocyanidin
Antioxidants
Molecular Mechanisms of Pharmacological Action
Pharmacologic Actions
Protective Agents
Physiological Effects of Drugs
Antiprotozoal Agents
Antiparasitic Agents
Anti-Infective Agents
Therapeutic Uses

ClinicalTrials.gov processed this record on July 29, 2014