This pre-post trial was conducted at the Laboratory of Clinical and Experimental Pathophysiology, CLINEX, located at Pedro Ernesto University Hospital, of Rio de Janeiro State University. Written informed consent was obtained from all the enrolled patients. The study protocol was approved by the Human Ethics Committee of Pedro Ernesto University Hospital. The procedures followed in this study were in accordance with institutional guidelines.
Potential participants were recruited at the Department of Plastic Surgery, among the candidates for lipoplasty. Candidates for the study, underwent eligibility screening by registered dietitians. Subjects were screened according to the following criteria: age between 18 and 60 years, BMI from 25.0 to 34.9
and diagnosis of stage 1 hypertension (without previous antihypertensive treatment) [26
]. The exclusion criteria were current use of antioxidant and dietary supplements; use of any medication known to interfere in body weight, blood pressure, and metabolic profile; recent changes (within previous 6 months) in dietary intake, body weight (>3
kg), and intensity or frequency of physical exercise. Individuals with eating disorders, major depression, or a medical history of drug addiction were excluded. Those with any metabolic disease, such as diabetes mellitus or hypothyroidism or chronic diseases severely affecting the cardiovascular, gastrointestinal, and renal systems were also excluded. Pregnant or lactating women were not allowed into the study.
Of the 550 individuals initially screened, 28 entered the run-in period. During the 2-week run-in period all cocoa foods were excluded, and potential participants were submitted to clinical, dietary, anthropometric, and biochemical evaluation. Six subjects failed to complete the run-in period, four individuals because their levels of blood pressure were not in the range of stage 1 hypertension and two because of lost of interest. So after completing the run-in period, 22 participants were included in the study and 20 completed the follow-up period (4 weeks), being included in the final analysis (). The noncompleters left the study because of scheduling conflicts.
During the intervention phase (4 weeks), participants were submitted to clinical and nutritional assessment at baseline (week 0) and weeks 1, 2, 3, and 4. All participants were instructed to consume 50
g of chocolate 70% cocoa/day (containing 2135
mg polyphenols) for 4 weeks (). They received instructions to consume 25
g in the morning and 25
g in the afternoon. At baseline and at every week participants received the amount of chocolate sufficient for 7 days of the study. The development and reinforcement of strategies for continued success were made at the same time points.
Table 1 Nutrient composition of the dark chocolate used in the study (50g).
Body weight, waist circumference, hip circumference were measured at baseline and at weeks 1, 2, 3, and 4. At baseline and at week 4 participants were also submitted to ambulatory blood pressure monitoring (ABPM) and evaluation of endothelial function; body composition and fasting plasma levels of circulating insulin, glucose, leptin, lipid profile (triglycerides, total cholesterol, high-density lipoprotein (HDL) cholesterol, and low density lipoprotein (LDL) cholesterol), biomarkers of inflammation (C-reactive protein, interleucina-6, and tumor necrosis factor-α), biomarker of oxidative stress (oxidized LDL), and biomarkers of endothelial dysfunction (intracelluar adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin).
The dietary assessments during the run-in period used a 3-day food record, covering 2 weekdays, and 1 weekend day, to estimate current energy and nutrients consumption. To avoid weight gain during the study period, patients were instructed to reduce their habitual energy intake proportionally to the energy supplied by the chocolate (280
Kcal/day; mainly from lipid-rich foods). During the intervention phase, the food records were reviewed and clarified in an interview with a nutritionist every week to assess dietary adherence. Nutrient analysis of the 3-day food record was performed using the software NutWin (São Paulo Federal University, UNIFESP, São Paulo, Brazil). Reported total energy, percent of energy from protein, fat and carbohydrate, and fiber content at the run-in phase were similar to that of the last week of the study. Subjects were carefully instructed to refrain from flavonoid-rich foods and beverages, including tea and wine; a list of these foods and beverages was given to each participant. All participants were encouraged to continue their usual physical activity throughout the study period.
Height, weight, and waist and hip circumferences were measured from 08:00 to 10:00
h after a 12 h fast. Height was measured using a stadiometer accurate to ±0.5
cm, and weight was obtained with a calibrated scale, accurate to ±0.1
kg (Filizola S.A., São Paulo, SP, Brazil), with participants wearing light clothing and no shoes. BMI was calculated using the standard equation (kilograms per meters squared). Waist circumference was measured in the standing position, midway between the lower margin of the last rib and the iliac crest. The measurements were taken at midexhalation. Hip circumference was measured at the widest point of the hip/buttocks area with the measuring tape parallel to the floor. Waist-to-hip ratio was determined by dividing waist circumference by hip circumference. Anthropometric measurements were taken twice, and mean values were used in all analyses. Percentage of body fat was estimated by electrical bioimpedance using a Biodynamics BIA-450 body fat analyzer (Biodynamics Corp., Seattle, WA, USA).
Blood samples were collected after a 12 h fasting period and were stored at −80°C. Total cholesterol, HDL cholesterol, and triglyceride concentrations were assessed by using an automated analyzer. LDL cholesterol was calculated using the Friedewald formula [27
] when triglycerides did not exceed 400
mg/dL. Radioimmunoassay was used to determine plasma leptin and insulin levels (Linco Research, St Charles, MO, USA, double antibody solid-phase enzyme immunoassay). Fasting plasma glucose was determined by the use of glucose oxidase method. The insulin resistance status was assessed by the use of homeostasis model assessment of insulin resistance (HOMA-IR) index, that is, serum insulin
U/mL) × plasma glucose (mmol/L)/22.5 [28
]. The values of VCAM-1, ICAM-1 and E-Selectin were determined by immunonephelometry enzymatic immunoassay using a commercial kit of multiple dosing (LINCO Research, St Charles, MO, USA). Plasma levels of TNF-α
and IL-6 were determined by enzymatic immunometric method (TiterZyme EIA) using commercial kits (Assay Designs, Ann Arbor, MI, USA). Highly sensitive CRP (hs-CRP) was determined by turbidimetry, using commercial kit (Biosystems, Barcelona, Spain).
2.1. Endothelial Function
Endothelial function was evaluated by peripheral artery tonometry (PAT) using Endo-PAT 2000 (Itamar Medical, Caesarea, Israel), a finger plethysmographic device that allows the isolated detection of pulsatile arterial volume changes [29
]. Endo-PAT 2000 is a noninvasive technology and was approved by the Food and Drug Administration for use as a diagnostic aid in patients with signs and symptoms of ischemic heart disease [30
]. There is evidence of a significant relationship between hyperemia-induced finger pulse wave amplitude changes, defined as the PAT hyperemia ratio, and brachial artery flow-mediated dilation [31
Endo-PAT consists of two finger-mounted probes, which include a system of inflatable latex air cushions within a rigid external case. The probe design allows the application of a constant and evenly distributed near-diastolic counterpressure within the entire probe, which increases sensitivity by unloading arterial wall tension and prevents venous blood pooling to avoid venoarteriolar reflex vasoconstriction. Pulsatile volume changes of the fingertip are sensed by a pressure transducer and transferred to a personal computer where the signal is band pass filtered (0.3 to 30
Hz), amplified, displayed, and stored [29
]. The Endo-PAT studies were performed with the patient in the supine position and both hands on the same level in a comfortable, thermoneutral environment. A blood pressure cuff was placed on one upper arm (study arm), while the contralateral arm served as a control (control arm); Endo-PAT probes were placed on one finger of each hand (same finger on both hands). A continuous recording of pulsatile blood volume responses from both hands was initiated. After a 10 min equilibration period, the blood pressure cuff on the study arm was inflated to 60
mmHg above systolic pressure for 5
min. The cuff was then deflated to induce reactive hyperemia, whereas PAT recording was continued. The reactive hyperemia index (RHI) obtained with Endo-PAT is analyzed by a computer in an operator-independent manner.
2.2. Blood Pressure
During the run-in phase blood pressure was recorded by using a calibrated Dinamap 1846 Critikon automated sphygmomanometer (Critikon, Tampa, FL, USA) after a resting period of at least 10
min in the sitting position. An appropriate arm cuff was used. Arm position was adjusted so that the cuff was at the level of the right atrium. Blood pressure was measured on the nondominant arm every 3
min for 15 minutes. The first value was discarded, and the mean of the last five readings was used in the analysis. Patients were considered to have stage 1 hypertension if their systolic blood pressure levels were between 140–159
mmHg and/or diastolic blood pressure between 90–99
At baseline (week 0) and at the end of the study (week 4) blood pressure was evaluated by Ambulatory Blood Pressure Monitoring (ABPM) to improve the estimate of “true” blood pressure. Ambulatory blood pressure was recorded using the SpaceLabs 90207 oscillometric blood pressure monitors (SpaceLabs, Redmond, WA, USA) calibrated against a mercury sphygmomanometer before use on each patient. Monitors were programmed to read blood pressure and heart rate every 20
min from 6:00 to 18:00
h. and every 30
min from 18:00 to 6:00
h. Mean daytime (6:00 to 18:00
h), and nighttime (18:00 to 6:00
h) blood pressure and heart rate were calculated.
2.3. Statistical Analysis
Means ± standard errors were used to summarize continuous variables. To test the possible association of chocolate 70% cocoa intake with nutritional parameters, biochemical variables, endothelial function, and blood pressure, we compared data obtained at baseline (week 0) with data obtained at the end of the study (week 4). Paired Student's t-test was used when variables had normal distribution and Wilcoxon test was used for variables without normal distribution.
Correlation tests were conducted to determine the relationship between RHI and variables of interest. Partial correlations controlled for different confounders were also used.
On the basis of a previous study [9
], this trial was designed to have 80% power to detect a significant difference in systolic blood pressure evaluated by ABPM before and after the intake of dark chocolate. Assuming 20% dropout rate, we needed at least 12 participants in the study. GraphPad PRISM 5.0 (GraphPad Software Inc, San Diego, CA, USA) and Stata 10.0 (STATA Corp., College Station, TX, USA) were used for statistical analysis. P
< 0.05 was considered statistically significant.