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.
Study Type
INTERVENTIONAL
Allocation
RANDOMIZED
Purpose
TREATMENT
Masking
DOUBLE
Enrollment
24
Dark chocolate (85% cocoa) in tablet
white or milk chocolate (\<35% cocoa) in tablet
Sapienza University of Rome, Policlinico Umberto I
Rome, Italy
Soluble NOX2-derived peptides (sNOX2-dp)
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%
Time frame: 30 days
soluble P-selectin (sPs)
soluble P-selectin levels, a marker of in-vivo platelet activation, will be measured on citrated platelet poor plasma samples by enzyme immunoassay
Time frame: 30 days
soluble CD40 Ligands (sCD40L)
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
Time frame: 30 days
Hydrogen Peroxide (H2O2)
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.
Time frame: 30 days
flow-mediated dilation (FMD)
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.
Time frame: 30 days
Vitamin E (α-tocopherol, αT)
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.
Time frame: 30 days
Serum isoprostane (8-iso-PGF2a-III)
Serum isoprostane (8-iso-PGF2a-III) is measured by the enzyme immunoassay method (DRG International). The values are expressed as pmol/l.
Time frame: 30 days
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