<clinical_study>
  <!-- This xml conforms to an XML Schema at:
    https://clinicaltrials.gov/ct2/html/images/info/public.xsd -->
  <required_header>
    <download_date>ClinicalTrials.gov processed this data on September 20, 2023</download_date>
    <link_text>Link to the current ClinicalTrials.gov record.</link_text>
    <url>https://clinicaltrials.gov/ct2/show/NCT03288623</url>
  </required_header>
  <id_info>
    <org_study_id>CE 4662</org_study_id>
    <nct_id>NCT03288623</nct_id>
  </id_info>
  <brief_title>The Effects of Dark Chocolate Implementation in Elite Athletes</brief_title>
  <official_title>The Effects of Dark Chocolate Implementation in the Reduction of Oxidative Stress and Improvement of Vascular Function and Physical Performance in Elite Athletes</official_title>
  <sponsors>
    <lead_sponsor>
      <agency>University of Roma La Sapienza</agency>
      <agency_class>Other</agency_class>
    </lead_sponsor>
  </sponsors>
  <source>University of Roma La Sapienza</source>
  <oversight_info>
    <has_dmc>Yes</has_dmc>
    <is_fda_regulated_drug>No</is_fda_regulated_drug>
    <is_fda_regulated_device>No</is_fda_regulated_device>
  </oversight_info>
  <brief_summary>
    <textblock>
      Dark chocolate (DC) is rich in epicatechin which augments nitric oxide (NO) production&#xD;
      through endothelium-dependent influences. The increased bioavailability and activity of NO&#xD;
      have been demonstrated to statistically increase flow-mediated dilation in healthy subjects&#xD;
      and in hypertensive patients. DC supplementation has been hailed for its positive effects on&#xD;
      cardiovascular health and it has been proposed as a booster of physical performance in&#xD;
      athletes, however the mechanisms by which DC improves oxidative stress, vascular function and&#xD;
      athletic performance are not fully understood. The investigators designed a human study&#xD;
      assessing how DC improves NO bioavailability and activity in elite athletes. Twenty-four&#xD;
      elite soccer players (aged 18-35 years old, all males) are divided in 2 groups and randomly&#xD;
      assigned to receive DC (85% cocoa), 40g per day or white/milk chocolate (&lt;35% cocoa) for 30&#xD;
      days. The primary outcome measure is the evaluation of Soluble NOX2-derived peptide&#xD;
      (sNOX2-dp), a direct marker of NADPH oxidase activation. The secondary outcome measures are&#xD;
      other markers of oxidative stress, as the soluble P-selectin (sPs), Vitamin E, soluble CD40&#xD;
      Ligand (sCD40L), a marker of in vivo platelet activation and flow-mediated dilation assessed&#xD;
      by vascular ultrasound. All parameters are assessed at baseline and after 30 days in both&#xD;
      groups.&#xD;
    </textblock>
  </brief_summary>
  <overall_status>Completed</overall_status>
  <start_date type="Actual">September 25, 2017</start_date>
  <completion_date type="Actual">November 1, 2018</completion_date>
  <primary_completion_date type="Actual">March 31, 2018</primary_completion_date>
  <phase>N/A</phase>
  <study_type>Interventional</study_type>
  <has_expanded_access>No</has_expanded_access>
  <study_design_info>
    <allocation>Randomized</allocation>
    <intervention_model>Parallel Assignment</intervention_model>
    <primary_purpose>Treatment</primary_purpose>
    <masking>Double (Investigator, Outcomes Assessor)</masking>
  </study_design_info>
  <primary_outcome>
    <measure>Soluble NOX2-derived peptides (sNOX2-dp)</measure>
    <time_frame>30 days</time_frame>
    <description>a marker of nicotinamide adenine dinucleotide phosphateoxidase activation, was detected in serum by ELISA. The peptide was recognised by the speciﬁc monoclonal antibody against the amino-acidic sequence (224-268) of the extra membrane portion of NOX2. Values were expressed as pg/mL; intra-assay and inter-assay coefﬁcient of variation is 5%</description>
  </primary_outcome>
  <secondary_outcome>
    <measure>soluble P-selectin (sPs)</measure>
    <time_frame>30 days</time_frame>
    <description>soluble P-selectin levels, a marker of in-vivo platelet activation, will be measured on citrated platelet poor plasma samples by enzyme immunoassay</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>soluble CD40 Ligands (sCD40L)</measure>
    <time_frame>30 days</time_frame>
    <description>soluble CD40 Ligand, a marker of in vivo platelet activation, will be measured using a commercially available method by Luminex Technology according to the manufacturer's instructions (Milliplex Map Kit Millipore, Darmstadt, Germany) in platelet components</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Hydrogen Peroxide (H2O2)</measure>
    <time_frame>30 days</time_frame>
    <description>Hydrogen peroxide (H2O2) is produced by inflammatory and vascular cells. Extracellular H2O2 is detected using incubation with Amplex Red (10 μM) and horseradish peroxidase (0.2 U/mL) for 60 min at 37 °C in Krebs-Ringer's phosphate glucose buffer (in mM: 145 NaCl, 5.7 sodium phosphate, 4.86 KCl, 0.54 CaCl2, 1.22 MgSO4, and 5.5 glucose) protected from light. Fluorescence is detected at 590 nm using an excitation of 530 nm every 5 min for 60 min. H2O2 is expressed as fluorescence per minute.</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>flow-mediated dilation (FMD)</measure>
    <time_frame>30 days</time_frame>
    <description>Evaluation of brachial artery FMD was performed at 60 seconds (FMD60s) and 120 seconds (FMD120s) postischemic stress by vascular ultrasound. Early FMD was defined as peak FMD60s and delayed FMD as peak FMD120s.</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Vitamin E (α-tocopherol, αT)</measure>
    <time_frame>30 days</time_frame>
    <description>Serum samples will be supplemented with tocopheryl acetate (internal standard), deproteinized by the addition of ethanol, and extracted with hexane. Phase separation is achieved by centrifugation. The collected upper phase is evaporated and analysed using an Agilent 1200 Infinity series high-performance liquid chromatography system equipped with an Eclipse Plus C18 column (4.6 × 100 mm). Serum levels will be given as the ratio (μmol/mmol) between serum α-tocopherol concentration (μmol/L) and serum total cholesterol concentration (mmol/L), which better express the circulating levels of vitamin E.</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Serum isoprostane (8-iso-PGF2a-III)</measure>
    <time_frame>30 days</time_frame>
    <description>Serum isoprostane (8-iso-PGF2a-III) is measured by the enzyme immunoassay method (DRG International). The values are expressed as pmol/l.</description>
  </secondary_outcome>
  <number_of_arms>2</number_of_arms>
  <enrollment type="Actual">24</enrollment>
  <condition>Oxidative Stress</condition>
  <condition>Athletes Heart</condition>
  <condition>Physical Activity</condition>
  <arm_group>
    <arm_group_label>Dark Chocolate</arm_group_label>
    <arm_group_type>Experimental</arm_group_type>
    <description>Dark chocolate (85% cocoa) 40 mg per day for 30 days</description>
  </arm_group>
  <arm_group>
    <arm_group_label>White/Milk Chocolate</arm_group_label>
    <arm_group_type>Placebo Comparator</arm_group_type>
    <description>White chocolate or Milk chocolate administration in tablet (&lt;35% cocoa) per day for 30 days</description>
  </arm_group>
  <intervention>
    <intervention_type>Dietary Supplement</intervention_type>
    <intervention_name>Dark Chocolate (85% cocoa)</intervention_name>
    <description>Dark chocolate (85% cocoa) in tablet</description>
    <arm_group_label>Dark Chocolate</arm_group_label>
  </intervention>
  <intervention>
    <intervention_type>Dietary Supplement</intervention_type>
    <intervention_name>White/Milk chocolate (&lt;35% cocoa)</intervention_name>
    <description>white or milk chocolate (&lt;35% cocoa) in tablet</description>
    <arm_group_label>White/Milk Chocolate</arm_group_label>
  </intervention>
  <eligibility>
    <criteria>
      <textblock>
        Inclusion Criteria:&#xD;
&#xD;
          -  Elite male athletes volunteers, aged between 18 and 35 years&#xD;
&#xD;
        Exclusion Criteria:&#xD;
&#xD;
          -  they suffer from an allergy to cocoa or any of the ingredients contained within either&#xD;
             of the chocolate bars&#xD;
&#xD;
          -  they have a low platelet count (&lt; 170 x 10E09/ L)&#xD;
&#xD;
          -  they are taking aspirin or aspirin-containing drugs, other anti-inflammatory drugs, or&#xD;
             any drugs or herbal medicines known to alter platelet function or the haemostatic&#xD;
             system in general (without a minimum washout period of one month)&#xD;
&#xD;
          -  they are taking fish oils or evening primrose oil, or fat soluble vitamin supplements&#xD;
             within the last 4 weeks&#xD;
&#xD;
          -  they have unsuitable veins for blood sampling and/ or cannulation&#xD;
&#xD;
          -  they have a BMI below 18 or above 35 kg/ sqm&#xD;
&#xD;
          -  they are taking any medicine known to affect lipid and/or glucose metabolism&#xD;
&#xD;
          -  they are suffering from alcohol or any other substance abuse or are having eating&#xD;
             disorders&#xD;
&#xD;
          -  they have any known clinical signs of diabetes, hypertension, renal, hepatic,&#xD;
             hematological disease, gastrointestinal disorders, endocrine disorders, coronary heart&#xD;
             disease, infection or cance&#xD;
      </textblock>
    </criteria>
    <gender>Male</gender>
    <gender_based>Yes</gender_based>
    <minimum_age>18 Years</minimum_age>
    <maximum_age>35 Years</maximum_age>
    <healthy_volunteers>No</healthy_volunteers>
  </eligibility>
  <overall_official>
    <last_name>Giacomo Frati, MD</last_name>
    <role>Study Director</role>
    <affiliation>University of Roma La Sapienza</affiliation>
  </overall_official>
  <location>
    <facility>
      <name>Sapienza University of Rome, Policlinico Umberto I</name>
      <address>
        <city>Rome</city>
        <zip>00161</zip>
        <country>Italy</country>
      </address>
    </facility>
  </location>
  <location_countries>
    <country>Italy</country>
  </location_countries>
  <link>
    <url>https://www.hindawi.com/journals/omcl/2018/4061901/</url>
    <description>Related Info</description>
  </link>
  <results_reference>
    <citation>Cavarretta E, Peruzzi M, Del Vescovo R, Di Pilla F, Gobbi G, Serdoz A, Ferrara R, Schirone L, Sciarretta S, Nocella C, De Falco E, Schiavon S, Biondi-Zoccai G, Frati G, Carnevale R. Dark Chocolate Intake Positively Modulates Redox Status and Markers of Muscular Damage in Elite Football Athletes: A Randomized Controlled Study. Oxid Med Cell Longev. 2018 Nov 21;2018:4061901. doi: 10.1155/2018/4061901. eCollection 2018.</citation>
    <PMID>30584461</PMID>
  </results_reference>
  <verification_date>April 2019</verification_date>
  <study_first_submitted>September 14, 2017</study_first_submitted>
  <study_first_submitted_qc>September 18, 2017</study_first_submitted_qc>
  <study_first_posted type="Actual">September 20, 2017</study_first_posted>
  <last_update_submitted>April 15, 2019</last_update_submitted>
  <last_update_submitted_qc>April 15, 2019</last_update_submitted_qc>
  <last_update_posted type="Actual">April 17, 2019</last_update_posted>
  <responsible_party>
    <responsible_party_type>Principal Investigator</responsible_party_type>
    <investigator_affiliation>University of Roma La Sapienza</investigator_affiliation>
    <investigator_full_name>Elena Cavarretta</investigator_full_name>
    <investigator_title>MD, PhD</investigator_title>
  </responsible_party>
  <patient_data>
    <sharing_ipd>Undecided</sharing_ipd>
    <ipd_description>De-identified individual participant data for all primary and secondary outcome measures will be made available</ipd_description>
  </patient_data>
  <!-- Results have not yet been posted for this study                                          -->
</clinical_study>


download date: ClinicalTrials.gov processed this data on September 20, 2023
link text: Link to the current ClinicalTrials.gov record.
url: https://clinicaltrials.gov/ct2/show/NCT03288623
org study id: CE 4662
nct id: NCT03288623
lead sponsor: 
has dmc: Yes
is fda regulated drug: No
is fda regulated device: No
Dark chocolate (DC) is rich in epicatechin which augments nitric oxide (NO) production
      through endothelium-dependent influences. The increased bioavailability and activity of NO
      have been demonstrated to statistically increase flow-mediated dilation in healthy subjects
      and in hypertensive patients. DC supplementation has been hailed for its positive effects on
      cardiovascular health and it has been proposed as a booster of physical performance in
      athletes, however the mechanisms by which DC improves oxidative stress, vascular function and
      athletic performance are not fully understood. The investigators designed a human study
      assessing how DC improves NO bioavailability and activity in elite athletes. Twenty-four
      elite soccer players (aged 18-35 years old, all males) are divided in 2 groups and randomly
      assigned to receive DC (85% cocoa), 40g per day or white/milk chocolate (<35% cocoa) for 30
      days. The primary outcome measure is the evaluation of Soluble NOX2-derived peptide
      (sNOX2-dp), a direct marker of NADPH oxidase activation. The secondary outcome measures are
      other markers of oxidative stress, as the soluble P-selectin (sPs), Vitamin E, soluble CD40
      Ligand (sCD40L), a marker of in vivo platelet activation and flow-mediated dilation assessed
      by vascular ultrasound. All parameters are assessed at baseline and after 30 days in both
      groups.
allocation: Randomized
intervention model: Parallel Assignment
primary purpose: Treatment
masking: Double (Investigator, Outcomes Assessor)
measure: Soluble NOX2-derived peptides (sNOX2-dp)
time frame: 30 days
description: a marker of nicotinamide adenine dinucleotide phosphateoxidase activation, was detected in serum by ELISA. The peptide was recognised by the speciﬁc monoclonal antibody against the amino-acidic sequence (224-268) of the extra membrane portion of NOX2. Values were expressed as pg/mL; intra-assay and inter-assay coefﬁcient of variation is 5%
measure: soluble P-selectin (sPs)
time frame: 30 days
description: soluble P-selectin levels, a marker of in-vivo platelet activation, will be measured on citrated platelet poor plasma samples by enzyme immunoassay
measure: soluble CD40 Ligands (sCD40L)
time frame: 30 days
description: soluble CD40 Ligand, a marker of in vivo platelet activation, will be measured using a commercially available method by Luminex Technology according to the manufacturer's instructions (Milliplex Map Kit Millipore, Darmstadt, Germany) in platelet components
measure: Hydrogen Peroxide (H2O2)
time frame: 30 days
description: Hydrogen peroxide (H2O2) is produced by inflammatory and vascular cells. Extracellular H2O2 is detected using incubation with Amplex Red (10 μM) and horseradish peroxidase (0.2 U/mL) for 60 min at 37 °C in Krebs-Ringer's phosphate glucose buffer (in mM: 145 NaCl, 5.7 sodium phosphate, 4.86 KCl, 0.54 CaCl2, 1.22 MgSO4, and 5.5 glucose) protected from light. Fluorescence is detected at 590 nm using an excitation of 530 nm every 5 min for 60 min. H2O2 is expressed as fluorescence per minute.
measure: flow-mediated dilation (FMD)
time frame: 30 days
description: Evaluation of brachial artery FMD was performed at 60 seconds (FMD60s) and 120 seconds (FMD120s) postischemic stress by vascular ultrasound. Early FMD was defined as peak FMD60s and delayed FMD as peak FMD120s.
measure: Vitamin E (α-tocopherol, αT)
time frame: 30 days
description: Serum samples will be supplemented with tocopheryl acetate (internal standard), deproteinized by the addition of ethanol, and extracted with hexane. Phase separation is achieved by centrifugation. The collected upper phase is evaporated and analysed using an Agilent 1200 Infinity series high-performance liquid chromatography system equipped with an Eclipse Plus C18 column (4.6 × 100 mm). Serum levels will be given as the ratio (μmol/mmol) between serum α-tocopherol concentration (μmol/L) and serum total cholesterol concentration (mmol/L), which better express the circulating levels of vitamin E.
measure: Serum isoprostane (8-iso-PGF2a-III)
time frame: 30 days
description: Serum isoprostane (8-iso-PGF2a-III) is measured by the enzyme immunoassay method (DRG International). The values are expressed as pmol/l.
arm group label: Dark Chocolate
arm group type: Experimental
description: Dark chocolate (85% cocoa) 40 mg per day for 30 days
arm group label: White/Milk Chocolate
arm group type: Placebo Comparator
description: White chocolate or Milk chocolate administration in tablet (<35% cocoa) per day for 30 days
intervention type: Dietary Supplement
intervention name: Dark Chocolate (85% cocoa)
description: Dark chocolate (85% cocoa) in tablet
arm group label: Dark Chocolate
intervention type: Dietary Supplement
intervention name: White/Milk chocolate (<35% cocoa)
description: white or milk chocolate (<35% cocoa) in tablet
arm group label: White/Milk Chocolate
criteria: 
gender: Male
gender based: Yes
minimum age: 18 Years
maximum age: 35 Years
healthy volunteers: No
last name: Giacomo Frati, MD
role: Study Director
affiliation: University of Roma La Sapienza
facility: 
country: Italy
url: https://www.hindawi.com/journals/omcl/2018/4061901/
description: Related Info
citation: Cavarretta E, Peruzzi M, Del Vescovo R, Di Pilla F, Gobbi G, Serdoz A, Ferrara R, Schirone L, Sciarretta S, Nocella C, De Falco E, Schiavon S, Biondi-Zoccai G, Frati G, Carnevale R. Dark Chocolate Intake Positively Modulates Redox Status and Markers of Muscular Damage in Elite Football Athletes: A Randomized Controlled Study. Oxid Med Cell Longev. 2018 Nov 21;2018:4061901. doi: 10.1155/2018/4061901. eCollection 2018.
PMID: 30584461
responsible party type: Principal Investigator
investigator affiliation: University of Roma La Sapienza
investigator full name: Elena Cavarretta
investigator title: MD, PhD
sharing ipd: Undecided
ipd description: De-identified individual participant data for all primary and secondary outcome measures will be made available