Study participants and study design
We recruited YOUNG (<35 years) and ELDERLY (50–80 year) healthy male Caucasian adult subjects without history, signs, or symptoms indicative of cardiovascular disease, including previous myocardial infarction, stroke, and peripheral artery disease or current or previous medication (Fig. 2). Participants were randomly assigned to either the CF intake group (FLAVANOL; 450 mg total flavanols two times daily) or a nutrient-matched CF-free group (CONTROL) based on a double-masked, parallel-group study design. All interventions were provided in anonymized sachets. Study participants were instructed to prepare the drinks by emptying the contents of each packet into ∼500 ml of water; drinks were prepared just prior to consumption. Drinks were consume two times per day, one beverage in the morning with breakfast and one with the evening meal. This regimen was maintained for 14 days, with compliance assessed by the collection of empty sachets on the last study day visit.
Measurements were taken fasted before (baseline) and 1 h after the first drink on day 1 and day 14. Endothelial function (primary end point) was measured as flow-mediated vasodilation (FMD). Secondary endpoints included measures of cardiac output, the physicomechanical properties of the large conduit arteries, the dilatory capacity and tone of resistance arteries, and perfusion in the microcirculation (Fig. 1). Tertiary endpoints included total plasma epicatechin metabolites. The study protocol was approved by the ethics committee of the Heinrich-Heine-University; all subjects gave written informed consent (Clinicaltrials.gov: NCT01639781).
Both interventions used a low-calorie fruit-flavored beverage mix (provided by Mars, Inc.), standardized and matched in composition. All beverage mixes were agglomerated powders, utilizing a maltodextrin base into which flavoring and sweeteners were incorporated. The beverage mixes were provided in sachets (7 g, equals one serving) labeled with an alphanumeric identifier to enable a double-masked study design.
A high flavanol cocoa extract (Cocoapro®-processed cocoa extract, Mars Inc.) was the source of flavanols in the CF-containing drink. The CF-containing drink (FLAVANOL) provided 450 mg of total cocoa flavanols per serving (Adamson et al. 1999). The total amount of CF in milligrams represents the sum of all monomeric flavanols and their oligomers (i.e., procyanidins) with a degree of polymerization up to and including 10 (i.e., DP 1–10). The predominant monomeric flavanol in this drink was (−)-epicatechin (see Table 1).
The control beverage mix did not contain any cocoa extract and thus provided 0 mg CF (CONTROL). Given the natural presence of theobromine and caffeine in cocoa extract, both theobromine and caffeine were added to the control beverage mix in order to match the composition of alkaloids in the CF-containing test product. Coloring was also added so that the 0 mg CF drink was also indistinguishable in appearance. Compositional details for the 0 mg (CONTROL) and 450 mg CF (FLAVANOL) test drinks are provided in Table 2.
Cardiac function was determined by heart rate (HR), cardiac output (CO), stroke volume (SV), and total peripheral resistance (TPR); the function of major conduit arteries, elastic aorta and muscular brachial artery, was characterized based on their physicomechanical properties and capacity to dilate (Fig. 1). Physicomechanical properties of the aorta are central blood pressure (BP), pulse wave velocity (PWV), and augmentation index (AIX); dilatory capacity of brachial artery was measured by flow-mediated and nitroglycerin-mediated vasodilation (FMD and NMD, respectively) as well as peripheral blood pressure that was determined at the upper arm and finger; the conductance function of arterioles was evaluated as resting and maximal forearm blood flow (FBF) during reactive hyperemia. Important readouts of microvascular perfusion: Cutaneous perfusion was evaluated by laser Doppler perfusion imaging (LDPI) at rest and maximal perfusion during reactive hyperemia, and blood rheology was determined as red blood cell (RBC) deformability.
Brachial artery FMD and nitroglycerin-mediated vasodilation (NMD) were measured by ultrasound (Vivid I, GE) in combination with an automated analysis system (Brachial Analyzer, MIA, Iowa City) as described (Heiss et al. 2010a). Brachial artery (BA) FMD was measured by ultrasound (10-MHz transducer; Vivid I, GE) in combination with an automated analysis system (Brachial Analyzer, MIA, Iowa City, IO) in a 21 °C-temperature-controlled room after 15 min of supine rest. Diameter and Doppler-flow velocity were measured at baseline and immediately after cuff deflation, at 20, 40, 60, and 80 s, and maximal diameter was used to calculate FMD. At the end of each study day, nitroglycerin-mediated vasodilation (NMD) was measured at 4 min after 400 μg sublingual nitroglycerin (Nitrolingual, Pohl).
The Task Force Monitor (CN-Systems, Graz, Austria) was used for continuous beat-to-beat assessment of cardiovascular variables, including stroke volume, BP, heart rate, and total peripheral resistance by impedance cardiography, which included ECG, phonocardiography, Finapres (Finapres-Medical-Systems, Amsterdam, Netherlands), and BP monitoring system (Dynamap, Tampa, USA).
Office blood pressure was measured three times after 10 min of rest using an automated clinical digital sphygmomanometer (Dynamap, Tampa, FL, USA) with appropriately sized cuff placed around the upper arm at heart level. Furthermore, we determined 24-h ambulatory BP measurements on the day before day 1 of the study and during the last 24 h of the study on day 13 until the clinical visit on day 14. Values are expressed as day, night, and total average. Furthermore, we determined BP at the fingertip using a tonometric device (Finapres Medical Systems, Amsterdam, Netherlands). Values were taken over 5 min and results represent average of measurements taken. Central BP was derived from peripheral pulse wave analysis obtained with applanation tonometry using a proprietary transfer function (SphygmoCor®, AtCor medical, Australia).
Pulse wave analysis
Central blood pressure parameters including augmentation index (AIX) were measured by applanation tonometry using the SphygmoCor® system. Via a transfer function, the pressure waveform of the ascending aorta was synthesized. PWV was determined from measurements taken at the carotid and femoral artery as described (Van Bortel et al. 2012).
Forearm blood flow
Forearm blood flow (FBF) was measured by mercury-in-rubber strain gauge plethysmography (Periquant 833, Gutman, Eurasburg, Germany) according to standard techniques at rest and during reactive hyperemia secondary to 3 min of forearm ischemia.
Assessment of perfusion in cutaneous microcirculation using laser doppler perfusion imaging
Perfusion of the cutaneous microcirculation of the forearm skin was measured at rest and during reactive hyperemia using a scanning laser Doppler perfusion imager (PeriScan PIM III, Perimed, Sweden) as described (Keymel et al. 2010). The parameter obtained is a global circulatory index expressed in arbitrary units (au) integrating the multidirectional average velocities in skin resistance arteries (lumen <100 m). After 15-min rest, the laser beam was positioned 15 cm above the forearm scanning a field of 200 mm2 on the volar site of the forearm. Microvascular reactivity was assessed during postocclusive reactive hyperemia. Following the baseline perfusion (1 min; 20 images), a blood pressure cuff located at the proximal forearm was inflated to suprasystolic pressure. After the cuff release, the microvascular response on reactive hyperemia was recorded. Data acquisition and analysis were performed by LDPIWin Software (Perimed, Sweden). Maximum perfusion, amplitude of perfusion (maximum perfusion − baseline perfusion), ratio (maximum perfusion / baseline perfusion), percentage increase ((maximum perfusion − baseline perfusion) / baseline perfusion × 100), time to peak response, and area under curve were evaluated without subtracting biological zero from the data (Keymel et al. 2010).
Measurement of RBC deformability
RBC deformability was measured by a laser-assisted optical rotational cell analyzer (LORCA, R&R Mechatronics, Hoorn, Netherlands) according to the manufacturers’ instructions as described (Keymel et al. 2011). After a 15-min rest in a supine position, blood was drawn from the cubital vein, collected in a heparinized tube (10 IE/ml, Liquemin 5000 IE, Roche, Grenzach-Wyhlen, Germany), and RBC deformability was measured by the laser-assisted optical rotational cell analyzer (LORCA, R&R Mechatronics, Hoorn, Netherlands) according to the manufactures’ instructions as described previously. Briefly, 20 μl of whole blood was diluted 200 times in high-viscosity L–polyvinylpyrrollidone. One milliliters of the suspension was transferred into the LORCA device and automatically subjected to varying degrees of shear stress at 0.3 to 10 Pa by stepwise increases of the rotation speed (Keymel et al. 2011).
Analysis of flavanols and their metabolites in plasma
(−)-Epicatechin and its related metabolites where analyzed in plasma by HPLC-FLD/UV and electrochemical detection using authentic standards provided by Mars Inc., as previously described (Ottaviani et al. 2012). Prior to analysis, plasma samples (0.5 ml) were defrosted on ice and then subjected to β-glucuronidase and sulfatase treatment (2000 units/ml; 40 min; 37 °C). Then, samples were mixed with 2 ml of acidified ice-cold methanol (0.5 % acetic acid in methanol, v/v) containing 3′-O-ethyl-(−)-epicatechin (500 nM) as a recovery standard. Samples were centrifuged at 17,000×g for 15 min at 4 °C, and the supernatant was collected. The pellet was extracted again with 2 ml of acidified ice-cold methanol (0.5 % acetic acid in methanol, v/v) containing 3′-O-ethyl-(−)-epicatechin (500 nM), and then with 1 ml of 50 % methanol acidified with 0.5 % acetic acid and containing 3′-O-ethyl-(−)-epicatechin (500 nM). Combined supernatants underwent concentration (to approximately 50 μl) using a Speedvac system (Thermo Fisher Scientific Inc., Basingstoke, UK) and were mixed with resorcinol (300 pmol) and catechol (300 pmol) prior to analysis by HPLC. Flavanol monomers and O-methylated metabolites were analyzed using a Hewlett-Packard 1200 series HPLC (Hewlett-Packard, Palo alto, CA, USA) equipped with diode array and fluorescent detection. Samples (50 μl) were injected onto a reversed-phase Phenomenex Luna C18(2) column (4.6 × 150 mm) with 3-μm particle size. The mobile phase consisted of (A) HPLC grade water, (B) 200 mM sodium acetate, pH 4.4/Methanol (84/16), and (C) acetonitrile, and the following gradient protocol was run: 0 min, 75 % A, 25 % B; 5 min, 75 % A, 25 % B; 20 min, 65 % A, 25 % B; 28 min, 63 % A, 25 % B; 34 min, 55 % A; 25 % B; 41 min, 45 % A, 25 % B; 45 min, 25 % B, 75 % C; 55 min, 25 % B, 75 % C; 56.1 min 75 % A, 25 % B; and 60 min, 75 % A, 25 % B. The flow rate was 0.8 ml/min. Detection of flavanols and their metabolites was performed using a fluorescent detector with excitation wavelength of 276 nm and emission wavelength of 316 nm. The concentration of each compound was determined using an external calibration curve produced with the use of authentic standards.
Results are expressed as mean ± standard error of the mean (SEM). Baseline data represent data of first visit (day 1, 0 h). The primary test for an effect was a three-way repeated mixed model measurements ANOVA (one within-subject factor: time [day 1, 0 h / day 1,1 h / day 14, 0 h / day 14, 1 h]; two between-subject factors: age (YOUNG/ELDERLY) and intervention (CONTROL/FLAVANOL). ANOVA and confidence intervals for pairwise comparisons were computed with SPSS 20 (IBM). Correlations were Pearson’s r. Values of p less than 0.05 were regarded as statistically significant.