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(Hypertension. 2005;46:398.)
© 2005 American Heart Association, Inc.

Original Articles

Cocoa Reduces Blood Pressure and Insulin Resistance and Improves Endothelium-Dependent Vasodilation in Hypertensives

Davide Grassi; Stefano Necozione; Cristina Lippi; Giuseppe Croce; Letizia Valeri; Paolo Pasqualetti; Giovambattista Desideri; Jeffrey B. Blumberg; Claudio Ferri

From the Department of Internal Medicine and Public Health (D.G., S.N., C.L., G.C., L.V., P.P., G.D., C.F.), University of L’Aquila, Italy; and Jean Mayer USDA Human Nutrition Research Center on Aging (J.B.B.), Tufts University, Boston, Mass.

Correspondence to Claudio Ferri, MD, Dipartimento MISP, Università di L’Aquila, Piazzale Salvatore Tommasi 1, 67100 Coppito, L’Aquila, Italy. E-mail claudio.ferri{at}cc.univaq.it

	Abstract

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Consumption of flavanol-rich dark chocolate (DC) has been shown to decrease blood pressure (BP) and insulin resistance in healthy subjects, suggesting similar benefits in patients with essential hypertension (EH). Therefore, we tested the effect of DC on 24-hour ambulatory BP, flow-mediated dilation (FMD), and oral glucose tolerance tests (OGTTs) in patients with EH. After a 7-day chocolate-free run-in phase, 20 never-treated, grade I patients with EH (10 males; 43.7±7.8 years) were randomized to receive either 100 g per day DC (containing 88 mg flavanols) or 90 g per day flavanol-free white chocolate (WC) in an isocaloric manner for 15 days. After a second 7-day chocolate-free period, patients were crossed over to the other treatment. Noninvasive 24-hour ambulatory BP, FMD, OGTT, serum cholesterol, and markers of vascular inflammation were evaluated at the end of each treatment. The homeostasis model assessment of insulin resistance (HOMA-IR), quantitative insulin sensitivity check index (QUICKI), and insulin sensitivity index (ISI) were calculated from OGTT values. Ambulatory BP decreased after DC (24-hour systolic BP –11.9±7.7 mm Hg, P<0.0001; 24-hour diastolic BP –8.5±5.0 mm Hg, P<0.0001) but not WC. DC but not WC decreased HOMA-IR (P<0.0001), but it improved QUICKI, ISI, and FMD. DC also decreased serum LDL cholesterol (from 3.4±0.5 to 3.0±0.6 mmol/L; P<0.05). In summary, DC decreased BP and serum LDL cholesterol, improved FMD, and ameliorated insulin sensitivity in hypertensives. These results suggest that, while balancing total calorie intake, flavanols from cocoa products may provide some cardiovascular benefit if included as part of a healthy diet for patients with EH.

Key Words: endothelium • insulin • hypertension, essential

	Introduction

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Observational studies suggest dietary flavonoids decrease the risk of death from coronary heart disease,¹ cancer,¹ and stroke.² Flavonoid-rich foods include fruits and vegetables as well as tea, red wine, and chocolate.³ The high flavonoid content, particularly in flavanols (ie, catechins) and their procyanidin oligomers, of these foods may contribute to some of their putative cardiovascular benefits.^4,5 The antioxidant protection afforded by flavonoids in the vascular endothelium may reduce the risk for atherosclerosis, including their action of inhibiting the oxidative conversion of NO to peroxynitrite.⁶ Accordingly, cocoa flavonoids decreased oxidant-induced peroxynitrite production in vitro⁷ and increased NO synthase (NOS) expression and NO-dependent vasorelaxation in rabbit aortic rings.⁸ In healthy adults, drinking flavanol-rich cocoa increased NO-dependent vasorelaxation in finger arteries,⁹ and eating flavanol-rich dark chocolate (DC) improved flow-mediated dilation (FMD) in brachial arteries in association with an increase in plasma epicatechin.¹⁰

Impaired NO-dependent vasorelaxation also contributes to a dysregulation of blood pressure (BP)¹¹ and a decrement of insulin-mediated glucose uptake.¹² In contrast, increased endothelial NOS expression and NO bioavailability ameliorate endothelial dysfunction, and thereby have the potential to decrease BP, increase insulin sensitivity, and slow down atherogenetic processes. In this regard, the anthocyanin cyanidin-3-glucoside was able to increase NOS expression and NO bioavailability in vascular endothelial cells.¹³ We recently demonstrated decrements in BP and increments in insulin sensitivity in healthy volunteers after 15 days of DC intake.¹⁴ Thus, we studied patients with essential hypertension (EH) to evaluate the effects of flavanol-rich DC on 24-hour ambulatory BP monitoring (ABPM), endothelium-dependent vasorelaxation via FMD of the brachial artery, insulin sensitivity via oral glucose tolerance tests (OGTTs), and 2 serum biomarkers of vascular inflammation: high-sensitive C-reactive protein (hsCRP) and intercellular adhesion molecule-1 (ICAM-1).

	Methods

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Subject Selection
Twenty never-treated EH patients (10 males and 10 females; mean age 43.65±7.8 years) referred to our outpatient unit were recruited and tested in 2004. Entry criteria were: 25 to 60 years of age; no diabetes or impaired glucose tolerance;^15,16 systolic BP (SBP) between 140 to 159 mm Hg or diastolic BP (DBP) between 90 to 99 mm Hg; absence of macroproteinuria; body mass index between 18 and 27 kg/m² for males and 18 to 26 kg/m² for females; total serum cholesterol <6.1 mmol/L; and serum triglyceride <1.7 mmol/L. Exclusion criteria were pregnancy, concomitant diseases, and use of medications including dietary supplements. Smokers and consumers of wine or other alcoholic beverages were also excluded. Echo-Doppler examinations of limb and neck vessels excluded patients with atherosclerotic lesions. M-mode and B-mode echocardiograms excluded patients with cardiac abnormalities. The study was conducted according to the Declaration of Helsinki of the World Medical Association (Edinburgh revision, 2000).

Diagnosis of EH
Grade I EH was diagnosed according to European Societies of Hypertension and Cardiology criteria.¹⁷ For this purpose, before enrollment into the study, BP and heart rate were measured after 10 minutes in a seated position in a comfortable room. SBP/DBP for inclusion in the protocol were 140/90 and <160/100 mm Hg on 4 visits performed at 1-week intervals. During each visit, BP was measured with a standard mercury sphygmomanometer and a stethoscope 4x at 2-min intervals. The first BP reading was discarded and the average of the last 3 measurements recorded. On each occasion, BP was recorded by the same physician who was unaware of the study design, objectives, and results (ie, was not a member of the research team). Secondary hypertension was excluded by clinical examination and appropriate tests.

Normotensive Control Group
Fifteen control subjects (7 males; mean age 33.9±7.6 years) who had participated in a related previous study¹⁴ were recruited from the medical staff to serve as a normotensive reference group. General office BP and OGTT data from these subjects have been published recently.¹⁴ These subjects had histories of normal SBP/DBP but were assessed in the outpatient unit on 3 occasions at 1-week intervals to confirm their values as <130/85 mm Hg. Remaining entry criteria for this group were the same as those for the EH patients.

Experimental Protocol
After evaluation of exclusion/inclusion criteria, EH subjects and controls were carefully instructed to maintain their usual diet but asked 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 asked to continue their usual physical activity throughout the study period and were found to comply with this instruction based on self-reports in daily physical activity diaries. Similar to the trial described by Taubert et al¹⁸ on elderly patients with isolated systolic hypertension, our patients and reference group entered a 7-day run-in phase, which excluded all cocoa foods. At the end of the run-in period, both groups were assigned randomly to receive daily either 100-g DC bars (Ritter Sport Halbbitter; Alfred Ritter GmbH & Co.) which, by our determination, contained 21.91 mg catechin, 65.97 mg epicatechin, 0.59 mg quercetin, 0.03 mg kaempferol, and 0.31 mg isorhamnetin, or 90-g flavanol-free white chocolate (WC) bars absent of any flavonoids (Milka; Kraft Foods) over a period of 15 days. DC and WC bars contained 480 kcal energy and similar amounts of cocoa butter, macronutrients, fiber, electrolytes, and vitamins.¹⁸ At the end of the first phase of intervention, patients and controls entered a second 7-day chocolate-free phase. After this period, all participants were crossed over to the other treatment (Figure 1).¹⁸ To avoid changes in body weight during the intervention, subjects were instructed how to substitute chocolate bars for foods of similar energy and macronutrient composition. The diet during the study was assessed by a diary of daily food intake and by daily measurement of body weight.¹⁸

View larger version (20K): [in this window] [in a new window]   Figure 1. Study design. After a cocoa-free run-in phase of 7 days, 20 never-treated essential hypertensives (10 males, 10 females, mean age 43.65±7.8 years) were randomly assigned to receive either 100-g DC bars containing 88 mg of flavanols or 90-g flavanol-free WC bars for 15 days. Successively, subjects entered a further cocoa-free washout phase of 7 days and then were crossed over to the other treatment. OGTTs were performed at the end of each period to calculate HOMA-IR, QUICKI, and ISI. ABPM, office BP, FMD, and hematochemical checks were performed at the same study phases. An identical protocol was used for evaluating controls.

Assessment of Insulin Sensitivity
After the run-in phase and after both intervention phases, OGTTs using 75 g of D-glucose were performed according to standard procedures^19,20 after an overnight fast and 12 hours from the last chocolate intake. Plasma glucose and insulin were assessed at baseline (0 minutes) and 30, 60, 90, 120, and 180 minutes after the 75-g glucose load. OGTT results were used for the homeostasis model assessment of insulin resistance (HOMA-IR),^19–21 the quantitative insulin sensitivity check index (QUICKI),²⁰ and the insulin sensitivity index (ISI).²²

Hematochemical Assessment
A routine hematochemical assessment with serum electrolytes and lipid profile, including total cholesterol, HDL and LDL cholesterol, and triglyceride, was conducted at the same time periods as that for assessing insulin sensitivity.

Endothelial Function
FMD of the brachial artery was assessed after a 15-minute rest period during scheduled visits (ie, after fasting [12 hours from the last chocolate ingestion]). FMD was always determined by the same physician, who was blinded to the study design and objectives (ie, was not a member of the research team), according to Ghiadoni et al.²³ Briefly, a B-mode scan of the right brachial artery was obtained in longitudinal section between 5 and 10 cm above the elbow using a 7.0-MHz linear array transducer and a standard MEGAS-platelet glycoprotein system (ESAOTE Biomedica) as described previously.^24,25 The transducer was held at the same point throughout the scan by a stereotactic clamp. End-diastolic frames (ECG-triggered) were acquired every second on a personal computer with the use of a commercial software program (MovieBox-Studio version 9; Pinnacle Systems GmbH). Arterial flow velocity was obtained by a pulsed Doppler signal at 70° to the vessel with the range gate (1.5 mm) at the center of the artery. A cuff was placed around the forearm just below the elbow. After 1-minute acquisition to measure basal diameter, the cuff was inflated for 5 minutes at 250 mm Hg and then deflated to induce reactive hyperemia. Endothelium-dependent vasodilation was considered the maximal dilation of the brachial artery induced by increased flow.^23–25 Endothelium-independent dilation was obtained by 25-µg sublingual glyceryl trinitrate.²³

BP Monitoring
Before and after each study period, 24-hour ABPM was recorded by a noninvasive oscillometric device (Medical 90207-30; Spacelabs, Inc.). BP was recorded at 15-minute intervals (daytime 6 AM to 10 PM) or 20-minute intervals (nighttime 10 PM to 6 AM). The mean 24-hour, daytime, and nighttime BP were calculated for statistical evaluation. Before starting with ABPM, sitting BP was also measured and recorded by standard mercury sphygmomanometer and stethoscope, as described above.

Biomarkers of Vascular Inflammation
Before and after each study phase, serum was also collected from all subjects for the determination of hsCRP (CRPLX Roche Diagnostics GmbH) and ICAM-1 (R & D Systems Inc.) by commercially available enzyme-linked immunonephelometric and immunoassay kits^26,27 according to manufacturer instructions.

Chocolate Flavonoid Content
Fats were extracted from the DC and WC bars with hexane and the defatted material dissolved in an extraction buffer of acetone:water:acetic acid (70.0:29.5:0.5), centrifuged, and filtered. The extract was dried, dissolved in metaphosphoric acid, and injected into high-performance liquid chromatography (ESA CoulArray) for flavonoid determination according to Milbury.²⁸

Statistical Analysis
Continuous normally distributed data are expressed as mean±SD. Differences in BP and metabolic indices between hypertensives and normotensives were analyzed by paired Student’s t test. Within each treatment group, changes in insulin sensitivity indices, BP, hsCRP, and ICAM-1 from baseline were analyzed by 1-factor ANOVA. For multiple comparisons, data were analyzed with a 2-factor repeated-measures ANOVA with time and treatment as the 2 factors. Post hoc comparisons were performed by Tukey’s honestly significant difference (HSD) test. Statistical analyses and power calculations were performed with SAS (2000; SAS Institute Inc.).

	Results

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Baseline characteristics of the study participants are provided in Table 1. According to entry criteria, no subjects had abnormal glucose metabolism. In hypertensives, baseline HOMA-IR, QUICKI, and ISI were similar between those randomized to WC and DC interventions (HOMA-IR 2.8±1.6 versus 2.8±1.5, P=0.92; QUICKI 0.3±0.03 versus 0.3±0.03, P=0.91; ISI 3.9±3.1 versus 3.9±3.0, P=0.99, respectively). Baseline SBP/DBP were also similar between the randomized groups (142.3±4.4 versus 142.3±4.3 mm Hg, P=0.97; 91.6±2.3 versus 90.8±3.2 mm Hg, P=0.36, respectively).¹⁴ Compared with controls, hypertensives had higher BP (P<0.0001) and HOMA-IR (P=0.005) and lower QUICKI (P=0.0007) and ISI (P=0.01).

View this table: [in this window] [in a new window]   TABLE 1. General Characteristics of Essential Hypertensives (n=20; 10 Males, 10 Females; Age 43.6± 7.8 Years) at Baseline and After 15 Days of DC or WC Consumption

Compared with baseline, DC consumption by hypertensives lowered HOMA-IR (F=16.57; P<0.0001 by 1-factor ANOVA; Figure 2A) and raised QUICKI (F=29.37; P<0.0001; Figure 2B), whereas WC was ineffective (Figure 2C and 2D). Similarly, compared with baseline, ISI was higher after DC than WC ingestion (F=39.62; P<0.0001 by ANOVA; Figure 2E and 2F). Improvement in glucose and insulin responses during OGTTs was observed after DC but not WC (P<0.05). With DC intervention, significant effects were noted in glucose responses for treatment (F=32.17; P<0.0001), time (F=72.48; P<0.0001), and treatment–time interactions (F=2.89; P=0.0003) with 2-factor repeated-measures ANOVA. Fasting glucose levels decreased after DC (from 4.7±0.5 to 4.4±0.4 mmol/L; P<0.0001). No significant variations were observed with WC intervention in glucose responses and fasting glucose levels (from 4.7±0.4 to 4.7±0.3 mmol/L; NS). Fasting insulin levels decreased after DC (from 13.1±6.7 to 9.3±4.4 mUI/mL; P<0.0001). Consistent relationships were noted after DC ingestion in insulin responses, with significant effects of treatment (F=33.55; P<0.0001), time (F=19.80; P<0.0001), and treatment–time interactions (F=4.47; P<0.0001) by 2-factor repeated-measures ANOVA. No significant variations were observed after WC for insulin responses and fasting insulin (from 13.1±6.4 to 13.1±6.5 mUI/mL; NS).

View larger version (25K): [in this window] [in a new window]   Figure 2. Effects of DC (A, B, and E) and WC (C, D, and F) on HOMA-IR, QUICKI, and ISI in 20 never-treated essential hypertensive patients. In all panels, laterally to individual values, corresponding symbols indicate means, and vertical lines indicate SDs. Asterisks indicate significant differences between DC vs WC and baseline values as evaluated by Tukey’s HSD test (P<0.05) when a 1-factor repeated-measures ANOVA showed a significant interaction for treatment.

DC reduced SBP (–11.0±6.3 mm Hg; F=55.39; P<0.0001 versus baseline) and DBP in hypertensives (–6.2±4.2 mm Hg; F=17.35; P<0.0001 versus baseline) with results tested by 1-factor repeated-measures ANOVA (Figure 3A and 3B). SBP (–0.5±1.6 mm Hg; NS) and DBP (–0.3±3.1 mm Hg; NS) remained unchanged after WC (Figure 3C and 3D). ABPM results confirmed significant reductions after DC (24-hour SBP –11.9±7.7 mm Hg, F=33.78, P<0.0001; 24-hour DBP –8.5±5.0 mm Hg, F=38.80, P<0.0001 versus baseline) but not WC ingestion (24-hour SBP –0.9±2.7 mm Hg; 24-hour DBP –0.1±2.5 mm Hg; NS; Table 2).

View larger version (30K): [in this window] [in a new window]   Figure 3. Effects of DC (A and B) or WC (C and D) on sitting SBP and DBP in 20 never-treated essential hypertensives. In both panels, laterally to individual values, corresponding symbols indicate means, and vertical lines indicate SDs. Asterisks indicate significant differences between DC vs WC and baseline values as evaluated by Tukey’s HSD test (P<0.05) when a 1-factor repeated-measures ANOVA showed a significant interaction for treatment.

View this table: [in this window] [in a new window]   TABLE 2. 24-Hour ABPM Data at Baseline and After 15 Days of DC or WC Ingestion in 20 Essential Hypertensives

Evaluating ABPM results for daytime and nighttime revealed a significant effect of treatment (P<0.0001) and time (P<0.0001) but not treatment–time interactions (P=0.9901 and P=0.5, respectively) by 2-factor repeated-measures ANOVA. DC reduced SBP (daytime –12.0±7.3 mm Hg, P<0.05; nighttime –11.9±11.6 mm Hg, P<0.05 versus baseline) and DBP (daytime –7.8±5.2 mm Hg, P<0.05; nighttime –10.2±6.3 mm Hg, P<0.05 versus baseline), whereas WC did not (SBP daytime –0.7±3.6 mm Hg, SBP nighttime –1.4±2.9 mm Hg, NS; DBP daytime –0.2±2.9 mm Hg, DBP nighttime –0±2.8 mm Hg, NS; Table 2).

Responses of SBP and DBP (daytime and nighttime) to DC were similar in controls, with a significant effect of treatment (P<0.0001) and time (P<0.0001) by 2-factor repeated-measures ANOVA. In contrast, a significant treatment–time interaction (P=0.001) was found for SBP but not for DBP (P=0.33). DC intake reduced SBP (daytime –6.3±5.5 mm Hg, P<0.05; nighttime –5.3±5.4 mm Hg, P<0.05 versus baseline) and DBP (daytime –4.2±4.5 mm Hg, P<0.05; nighttime –3.1±3.8 mm Hg, P<0.05 versus baseline), whereas WC affected neither SBP (daytime –0.5±3.8 mm Hg; nighttime –0.7±3.5 mm Hg; NS) nor DBP (daytime –0.3±2.2 mm Hg; nighttime –0.9±2.0 mm Hg; NS; Table 3). As in hypertensives, DC reduced 24-hour SBP (–5.9±5.4 mm Hg; F=15.21; P<0.0001 versus baseline) and 24-hour DBP (–4.1±4.1 mm Hg; F=11.59; P<0.0001 versus baseline), whereas WC did not (24-hour SBP –0.5±3.7 mm Hg; 24-hour DBP –0.6±2.1 mm Hg; NS) when examined by 1-factor repeated-measures ANOVA (Table 3).

View this table: [in this window] [in a new window]   TABLE 3. 24-Hour ABPM Data Obtained in 15 Control Subjects at Baseline and After DC or WC for 15 Days

Baseline FMD was impaired in hypertensives compared with controls (7.4±1.4% versus 9.9±0.9%, respectively; P<0.0001) and increased in hypertensives almost to normal values after DC (8.9±1.4%; F=13.25; P<0.0001) but not WC (7.5±1.3%; Figure 4A and 4B). FMD also increased after DC (11.8±1.3%; F=19.86; P<0.0001 versus baseline) but not WC (10.1±0.9%; NS) in controls (Figure 4C and 4D).

View larger version (25K): [in this window] [in a new window]   Figure 4. Effects of DC and WC on brachial artery measurements by FMD in 20 never-treated essential hypertensives (A and B, respectively) and 15 controls (C and D, respectively). In both panels, laterally to individual values, corresponding symbols indicate means, and vertical lines indicate SDs. Asterisks indicate significant differences between DC vs WC and baseline values as evaluated by Tukey’s HSD test (P<0.05) when a 1-factor repeated-measures ANOVA showed a significant interaction for treatment.

Baseline glyceryl trinitrate–induced dilation did not differ between hypertensives (8.6±1.4%) and controls (9.0±1.2%) and remained unchanged after DC (hypertensives 8.5±1.2%, NS; normotensives 9.2±1.3%, NS) and WC (hypertensives 8.7±1.7%, NS; normotensives 9.1±1.7%, NS).

In hypertensives, serum total cholesterol (F=7.37; P=0.0003; P<0.05 versus WC and baseline) and LDL cholesterol (F=2.94; P=0.04) but not triglycerides or HDL cholesterol levels decreased after DC but not WC (Table 1). Other variables including serum hsCRP and ICAM-1 levels remained unchanged after DC in hypertensives (hsCRP from 0.37±0.39 to 0.33±0.39 mg/dL, NS; ICAM-1 from 144.9±6.5 to 144.2±6 µg/L, NS) and in normotensives (hsCRP from 0.26±0.2 to 0.26±0.2 mg/dL, NS; ICAM-1 from 140.8±5.5 to 140.5±5 µg/L, NS) as well as after WC in hypertensives (hsCRP from 0.35±0.31 to 0.36±0.31 mg/dL, NS; ICAM-1 from 144.9±6.2 to 144.8±6.3 µg/L, NS) and in normotensives (hsCRP from 0.3±0.24 to 0.3±0.22 mg/dL, NS; ICAM-1 from 140.8±5.4 to 140.6±5.5 µg/L, NS).

No significant changes from baseline were observed in 24-hour urinary NaCl excretion in hypertensives after either DC (from 128.3±36.8 to 123.1±31.8 mmol/24 h; NS) or WC (from 126.4±29.6 to 121.1±26.7 mmol/24 h; NS). Similar findings were observed in normotensives with DC (from 114.2±27.9 to 117±22.6 mmol/24 h; NS) and WC (from 116.5±25.1 to 112.6±18.9 mmol/24 h; NS). Age, gender, and other variables did not influence the effect of chocolate on HOMA-IR, QUICKI, ISI, ABPM, and FMD values in either group.

	Discussion

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The current study shows that consumption of flavanol-rich DC decreased daytime and nighttime BP, reduced insulin resistance, and improved NO-dependent vasorelaxation. These outcomes may be closely interrelated because impairment of NO-dependent vasorelaxation appears to contribute substantially to EH,¹¹ a disease often associated with insulin resistance and metabolic syndrome.^29,30 It is important to note that hypertension, and its association with atherogenesis,³¹ inflammation,³¹ platelet activation,³² and oxidative stress,³³ has been considered the most important cardiovascular risk factor worldwide.³⁴

Our results in EH patients and normotensives support studies indicating flavanol intake can reduce BP in healthy, young individuals¹⁴ as well as geriatric patients with isolated systolic hypertension.¹⁸ However, 24-hour BP reduction induced by DC in control subjects found here and also reported by Grassi et al¹⁴ are in contrast to related interventions conducted by Fisher et al⁹ and Engler et al.¹⁰ These investigators did not observe any BP change in their healthy volunteers drinking a flavanol-rich chocolate beverage for 1 or 4 days⁹ or eating DC bars for 2 weeks.¹⁰ The absence of a reduction in BP in these 2 studies may be attributable to their shorter duration, use of different chocolate products, demographics or health status of the subjects, or other differences between the studies. Nevertheless, it is worthwhile to note that Engler et al¹⁰ found a slight reduction in office SBP (–1.0±1.4 mm Hg; NS) in their trial. Importantly, and in contrast with these studies, we used ABPM, a more robust determination of treatment-induced BP changes than office BP measurements.³⁵

Consistent with our results, Fisher et al⁹ and Engler et al¹⁰ observed an increase in FMD with their chocolate interventions. Similarly, Heiss et al³⁶ found consumption of flavanol-rich chocolate increased FMD in patients with coronary artery disease, severe hypertension, or diabetes. In these patients, chocolate significantly increased plasma nitrite and nitrate, an integrated biomarker of NO and NO metabolism.³⁷ Thus, despite the potential of flavanols to induce prostacyclin production,³⁸ their capability to improve FMD may result from modulating NO status, potentially via increasing NOS³⁹ or inhibiting the NO conversion to peroxynitrite.^6,14 In vitro, flavanols have been reported to increase NOS expression³⁹ and activity^8,39 and prevent NO–superoxide anion reactions to form peroxynitrite.⁷ In humans, drinking tea, a flavonoid-rich beverage, has been shown to improve FMD in the brachial arteries of hypertensives and normotensives with coronary artery disease.³⁸ Similarly, flavonoid-rich purple grape juice has been reported to increase FMD and decrease oxidative stress in patients with coronary artery disease.⁴⁰

Insulin sensitivity is partly dependent on insulin-mediated NO release.⁴¹ Thus, flavanols and other dietary antioxidants may decrease insulin resistance by ameliorating NO bioavailability. Consistent with this hypothesis, intravenous infusion of ascorbic acid to glucose intolerant subjects and smokers improved FMD and insulin sensitivity while reducing plasma thiobarbituric acid–reactive substances, an index of lipid peroxidation.⁴² Similarly, Hirashima et al⁴³ reported that in patients with vasospastic angina, a condition associated with reductions in insulin sensitivity⁴⁴ and NO status,⁴⁵ ascorbic acid infusion normalized FMD and reduced insulin resistance.

CRP, an acute phase protein, is regulated by inflammatory cytokines and oxidants and decreases in response to therapies with agents possessing antioxidant properties.²⁷ However, flavanol-rich DC did not affect circulating hsCRP or ICAM-1 in our subjects. Similarly, Mathur et al,⁴⁶ although demonstrating the antioxidant action of cocoa on the susceptibility of LDL to oxidation in healthy adults, found no effect of cocoa on hsCRP. Also, using a less sensitive agglutination technique for the determination of CRP, Davidsson et al⁴⁷ found no change in plasma CRP in children provided iron-fortified chocolate drinks.⁴⁷ Nonetheless, it is possible that the absence of an effect of flavanols on hsCRP or ICAM-1 may reflect an inadequate study dose or duration.

At variance with previous findings we obtained on normotensives,¹⁴ a significant reduction of serum total and LDL cholesterol levels was observed in hypertensives after DC ingestion. A few studies have evaluated the effects of flavonoids contained in tea and cocoa on serum cholesterol.⁴⁸ Mursu et al⁴⁹ found that ingestion of DC bars for 15 days increased HDL cholesterol in healthy subjects without variations in total and LDL cholesterol. Recently, Fraga et al⁵⁰ showed significant decrements in serum total cholesterol (–11%) and LDL cholesterol levels (–15%) after short-term consumption of flavanol-rich chocolate in young subjects. Also, Davies et al⁵¹ found significant decrements in serum total cholesterol (–6.5%) and LDL cholesterol (–11.1%), with unchanged HDL cholesterol levels after 3 weeks of black tea in hypercholesterolemic adults. Similarly, experimental data from rats showed a specific hypocholesterolemic effect of catechins.⁵² Further, chocolate also contains linoleic and oleic acids, 2 fatty acids known to modulate cholesterol metabolism.⁵³ Therefore, although a study in smokers⁵⁴ and an early study in healthy subjects⁵⁵ showed no changes in serum total cholesterol and cholesterol fractions after black^54,55 or green tea,⁵⁴ our results are consistent with previous reports showing positive effects of flavonoids on serum lipid profiles.

In conclusion, our findings support a potentially beneficial action of chocolate flavanols on BP, vasorelaxation, and insulin sensitivity in essential hypertensives and suggest directions for further research in this area.⁵⁶ Interestingly, consumers of chocolate and other candies appear to have a lower mortality rate compared those who do not eat candy.⁵⁷ Nonetheless, it is important to note that the DC used in this and related studies differ markedly from the majority of commercially available cocoa or chocolate confectionery with very low flavanol content.⁵⁶ Further, caution is always warranted when considering dietary recommendations for foods high in fat and calories, especially for cardiovascular disease.

Received March 15, 2005; first decision April 6, 2005; accepted June 13, 2005.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Hertog MG, Kromhout D, Aravanis C, Blackburn H, Buzina R, Fidanza F, Giampaoli S, Jansen A, Menotti A, Nedeljkovic S, Pekkarinen M, Simic BS, Toshima H, Feskens EJ, Hollman PC, Katan MB. Flavonoid intake and long-term risk of coronary heart disease and cancer in the Seven Countries Study. Arch Intern Med. 1995; 155: 381–386.[Abstract]
2. Sirving OK, Hertog MG, Feskens EJM, Kromhout D. Dietary flavonoids, antioxidants vitamins and incidence of stroke. Arch Intern Med. 1996; 154: 637–642.
3. Hammerstone JF, Lazarus SA, Schmitz HH. Procyanidin content and variation in some commonly consumed foods. J Nutr. 2000; 130: 2086S–2092S.[Abstract/Free Full Text]
4. Lee KW, Kim YJ, Lee HJ, Lee CY. Cocoa has more phenolic phytochemicals and a higher antioxidant capacity than tea and red wine. J Agric Food Chem. 2003; 51: 7292–7295.[CrossRef][Medline] [Order article via Infotrieve]
5. Ross JA, Kasum CM. Dietary flavonoids: bioavailability, metabolic effects, and safety. Annu Rev Nutr. 2002; 22: 19–34.[CrossRef][Medline] [Order article via Infotrieve]
6. Pryor WA, Squadrito GL. The chemistry of peroxynitrite: a product from the reaction of nitric oxide with superoxide. Am J Physiol. 1995; 268: L699–L722.[Medline] [Order article via Infotrieve]
7. Arteel GE, Schroeder P, Sies H. Reactions of peroxynitrite with cocoa procyanidin oligomers. J Nutr. 2000; 130: 2100S–2104S.[Abstract/Free Full Text]
8. Karim M, McCormick K, Kappagoda CT. Effects of cocoa extracts on endothelium-dependent relaxation. J Nutr. 2000; 130: 2105S–2108S.[Abstract/Free Full Text]
9. Fisher ND, Hughes M, Gerhard-Herman M, Hollenberg NK. Flavanol-rich cocoa induces nitric-oxide-dependent vasodilation in healthy humans. J Hypertens. 2003; 21: 2281–2286.[CrossRef][Medline] [Order article via Infotrieve]
10. Engler MB, Engler MM, Chen CY, Malloy MJ, Browne A, Chiu EY, Kwak HK, Milbury P, Paul SM, Blumberg J, Mietus-Snyder ML. Flavonoid-rich dark chocolate improves endothelial function and increases plasma epicatechin concentrations in healthy adults. J Am Coll Nutr. 2004; 3: 197–204.
11. Panza JA, Casino PR, Kilcoyne CM, Quyyumi AA. Role of endothelium-derived nitric oxide in the abnormal endothelium-dependent vascular relaxation of patients with essential hypertension. Circulation. 1993; 87: 1468–1474.[Abstract/Free Full Text]
12. Clark MG, Wallis MG, Barrett EJ, Vincent MA, Richards SM, Clerk LH, Rattigan S. Blood flow and muscle metabolism: a focus on insulin action. Am J Physiol Endocrinol Metab. 2003; 284: E241–E258.[Abstract/Free Full Text]
13. Xu JW, Ikeda K, Yamori Y. Upregulation of endothelial nitric oxide synthase by cyanidin-3-glucoside, a typical anthocyanin pigment. Hypertension. 2004; 44: 217–222.[Abstract/Free Full Text]
14. Grassi D, Lippi C, Necozione S, Desideri G, Ferri C. Short-term administration of dark chocolate is followed by a significant increase in insulin sensitivity and a decrease in blood pressure in healthy persons. Am J Clin Nutr. 2005; 8: 611–614.
15. World Health Organization. Diabetes Mellitus: Report of a WHO Study Group. Geneva, Switzerland: WHO; 1985.
16. American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care. 2004; 27: S5–S10.[CrossRef][Medline] [Order article via Infotrieve]
17. European Society of Hypertension. European Society of Cardiology guidelines for the management of arterial hypertension. J Hypertens. 2003; 21: 1011–1053.[CrossRef][Medline] [Order article via Infotrieve]
18. Taubert D, Berkels R, Roesen R, Klaus W. Chocolate and blood pressure in elderly individuals with isolated systolic hypertension. J Am Med Assoc. 2003; 290: 1029–1030.[Free Full Text]
19. Katz A, Nambi SS, Mather K, Baron AD, Sullivan G, Quon MJ. Quantitative insulin sensitivity check index: a simple, accurate method for assessing insulin sensitivity in Humans. J Clin Endocrinol Metab. 2000; 85: 2402–2410.[Abstract/Free Full Text]
20. Bonora E, Kiechi S, Willeit J, Oberhollenzer F, Egger G, Targher G, Alberiche M, Bonadonna RC, Muggeo M. Prevalence of insulin resistance in metabolic disorders. The Bruneck Study. Diabetes. 1998; 47: 1643–1649.[Abstract]
21. Mannucci E, Bardini G, Ognibene A, Rotella CM. Comparison between 2 insulin sensitivity indexes in obese patients. Diabetes Care. 2000; 23: 1042–1043.[Medline] [Order article via Infotrieve]
22. Matsuda M, DeFronzo RA. Insulin sensitivity indices obtained from oral glucose tolerance testing. Comparison with the euglycemic insulin clamp. Diabetes Care. 1999; 22: 1462–1470.[Abstract/Free Full Text]
23. Ghiadoni L, Magagna A, Versari D, Kardasz I, Huang Y, Taddei S, Salvetti A. Different effect of antihypertensive drugs on conduit artery endothelial function. Hypertension. 2003; 41: 1281–1286.[Abstract/Free Full Text]
24. Corretti MC, Anderson TJ, Benjamin EJ, Celermajer D, Charbonneau F, Creager MA, Deanfield J, Drexler H, Gerhard-Herman M, Herrington D, Vallance P, Vita J, Vogel R; International Brachial Artery Reactivity Task Force. Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force. J Am Coll Cardiol. 2002; 39: 257–265.[Abstract/Free Full Text]
25. Beux F, Carmassi S, Salvetti MV, Ghiadoni L, Huang Y, Taddei S, Salvetti A. Automatic evaluation of arterial diameter variation from vascular echographic images. Ultrasound Med Biol. 2001; 27: 1621–1629.[CrossRef][Medline] [Order article via Infotrieve]
26. Willerson JT, Ridker PM Inflammation as a cardiovascular risk factor. Circulation. 2004; 109: II-2–II-10.[Medline] [Order article via Infotrieve]
27. Desideri G, Croce G, Tucci M, Passacquale G, Broccoletti S, Valeri L, Santucci A, Ferri C. Effects of bezafibrate and simvastatin on endothelial activation and lipid peroxidation in hypercholesterolemia: evidence of different vascular protection by different lipid-lowering treatments. J Clin Endocrinol Metab. 2003; 88: 5341–5347.[Abstract/Free Full Text]
28. Milbury PE. Analysis of complex mixtures of flavonoids and polyphenols by high-performance liquid chromatography with coulometric array detection. Anal Biochem. 2000; 279: 164–169.[CrossRef][Medline] [Order article via Infotrieve]
29. Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults. Executive summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults (ATP III). J Am Med Assoc. 2001; 285: 2486–2497.[Free Full Text]
30. Ferri C, Bellini C, Desideri G, De Mattia G, Santucci A. Relationship between insulin resistance and nonmodulating hypertension. Linkage of metabolic abnormalities and cardiovascular risk. Diabetes. 1999; 48: 1623–1630.[Abstract]
31. Virdis A, Schiffrin EL. Vascular inflammation: a role in vascular disease in hypertension? Curr Opin Neprol Hypertens. 2003; 12: 181–187.
32. Nadar SK, Blann AD, Lip GY. Plasma and platelet-derived vascular endothelial growth factor and angiopoietin-1 in hypertension: effects of antihypertensive therapy. J Intern Med. 2004; 256: 331–337.[CrossRef][Medline] [Order article via Infotrieve]
33. Fortuno A, Olivan S, Beloqui O, San Jose G, Moreno MU, Diez J, Zalba G. Association of increased phagocytic NADPH oxidase-dependent superoxide production with diminished nitric oxide generation in essential hypertension. J Hypertens. 2004; 22: 2169–2175.[CrossRef][Medline] [Order article via Infotrieve]
34. Yusuf S, Hawken S, Ounpuu S, Dans T, Avezum A, Lanas F, McQueen M, Budaj A, Pais P, Varigos J, Lisheng L; INTERHEART Study Investigators. Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART Study): case-control study. Lancet. 2004; 364: 937–952.[CrossRef][Medline] [Order article via Infotrieve]
35. Clement DL, De Buyzere ML, De Bacquer DA, de Leeuw PW, Duprez DA, Fagard RH, Gheeraert PJ, Missault LH, Braun JJ, Six RO, Van Der Niepen P, O’Brien E; Office Versus Ambulatory Pressure Study Investigators. Prognostic value of ambulatory blood-pressure recordings in patients with treated hypertension. N Engl J Med. 2003; 348: 2407–2415.[Abstract/Free Full Text]
36. Heiss C, Dejan A, Kleinborgard P, Schewe T, Stes H, Kelm M. Vascular effects of cocoa rich in flavan-3-ols. J Am Med Assoc. 2003; 290: 1030–1031.[Free Full Text]
37. Feelisch M, Rassaf T, Mnaimneh S, Singh N, Bryan NS, Jourd’Heuil D, Kelm M. Concomitant S-, N-, and heme-nitros(yl)ation in biological tissues and fluids: implications for the fate of NO in vivo. FASEB J. 2002; 16: 1775–1778.[Abstract/Free Full Text]
38. Duffy SJ, Keaney JF Jr, Holbrook M, Gokce N, Swerdloff PL, Frei B, Vita JA. Short- and long-term black tea consumption reverses endothelial dysfunction in patients with coronary artery disease. Circulation. 2001; 104: 151–156.[Abstract/Free Full Text]
39. Wallerath T, Li H, Godtel-Ambrust U, Schwarz PM, Forstermann U. A blend of polyphenolic compounds explains the stimulatory effect of red wine on human endothelial NO synthase. Nitric Oxide. 2005; 12: 97–104.[CrossRef][Medline] [Order article via Infotrieve]
40. Stein JH, Keevil JG, Wiebe DA, Aeschlimann S, Folts JD. Purple grape juice improves endothelial function and reduces the susceptibility of LDL cholesterol to oxidation in patients with coronary artery disease. Circulation. 1999; 100: 1050–1055.[Abstract/Free Full Text]
41. Zeng G, Nystrom FH, Ravichandran LV, Cong LN, Kirby M, Quon MJ. Roles for insulin receptor, PI3-kinase, and Akt in insulin-signaling pathways related to production of nitric oxide in human vascular endothelial cells. Circulation. 2000; 101: 1539–1545.[Abstract/Free Full Text]
42. Hirai N, Kawano H, Hirashima O, Motoyama T, Moriyama Y, Sakamoto T, Kugiyama K, Ogawa H, Nakao K, Yasue H. Insulin resistance and endothelial dysfunction in smokers: effects of vitamin C. Am J Physiol Heart Circ Physiol. 2000; 279: H1172–H1178.[Abstract/Free Full Text]
43. Hirashima O, Kawano H, Motoyama T, Hirai N, Ohgushi M, Kugiyama K, Ogawa H, Yasue H. Improvement of endothelial function and insulin sensitivity with vitamin C in patients with coronary spastic angina. J Am Coll Card. 2000; 35: 1860–1866.[Abstract/Free Full Text]
44. Gaspardone A, Ferri C, Crea F, Versaci F, Tomai F, Santucci A, Chiariello L, Gioffre PA. Enhanced activity of sodium-lithium countertransport in patients with cardiac syndrome X: a potential link between cardiac and metabolic syndrome X. J Am Coll Cardiol. 1998; 32: 2031–2034.[Abstract/Free Full Text]
45. Desideri G, Gaspardone A, Gentile M, Santucci A, Ferri C. Endothelial activation in patients with cardiac syndrome X. Circulation. 2000; 102: 2359–2364.[Abstract/Free Full Text]
46. Mathur S, Devaraj S, Grundy SM, Jialal I. Cocoa products decrease low density lipoprotein oxidative susceptibility but do not affect biomarkers of inflammation in humans. J Nutr. 2002; 132: 3663–3667.[Abstract/Free Full Text]
47. Davidsson L, Walczyk T, Morris A, Hurrell RF. Influence of ascorbic acid on iron absorption from an iron-fortified, chocolate-flavored mild drink in Jamaican children. Am J Clin Nutr. 1998; 68: 873–877.[Abstract]
48. Mennen LI, Sapinho D, de Bree A, Arnault N, Bertrais S, Galan P, Hercberg S. Consumption of foods rich in flavonoids is related to a decreased cardiovascular risk in apparently healthy French women. J Nutr. 2004; 134: 923–926.[Abstract/Free Full Text]
49. Mursu J, Voutilainen S, Nurmi T, Rissanen TH, Virtanen JK, Kaikkonen J, Nyyssonen K, Salonen JT. Dark chocolate consumption increases HDL cholesterol concentration and chocolate fatty acids may inhibit lipid peroxidation in healthy humans. Free Radic Biol Med. 2004; 37: 1351–1359.[CrossRef][Medline] [Order article via Infotrieve]
50. Fraga CG, Actis-Goretta L, Ottaviani JI, Carrasquedo F, Lotito SB, Lazarus S, Schmitz HH, Keen CL. Regular consumption of a flavanol-rich chocolate can improve oxidant stress in young soccer players. Clin Dev Immunol. 2005; 12: 11–17.[CrossRef][Medline] [Order article via Infotrieve]
51. Davies MJ, Judd JT, Baer DJ, Clevidence BA, Paul DR, Edwards AJ, Wiseman SA, Muesing RA, Chen SC. Black tea consumption reduces total and LDL cholesterol in mildly hypercholesterolemic adults. J Nutr. 2003; 133: 3298S–3302S.[Abstract/Free Full Text]
52. Ikeda I, Imasato Y, Sasaki E, Nakayama M, Nagao H, Takeo T, Yayabe F, Sugano M. Tea catechins decrease micellar solubility and intestinal absorption of cholesterol in rats. Biochem Biophys Acta. 1992; 1127: 141–146.[Medline] [Order article via Infotrieve]
53. Ascherio A, Rimm EB, Giovannucci EL, Spiegelman D, Stampfer M, Willett WC. Dietary fat and risk of coronary heart disease in men: cohort follow up study in the United States. BMJ. 1996; 313: 84–90.[Abstract/Free Full Text]
54. Princen HM, vanDuyvenvoorde W, Buytenhek R, Blonk C, Tijburg LB, Langius JA, Meinders AE, Pijl H. No effect of consumption of green and black tea on plasma lipid and antioxidant levels and on LDL oxidation in smokers. Arterioscler Thromb Vasc Biol. 1998; 18: 833–841.[Abstract/Free Full Text]
55. Aro A, Kostiainen E, Huttunen JK, Seppala E, Vapaatalo H. Effects of coffee and tea on lipoproteins and prostanoids. Atherosclerosis. 1985; 57: 123–128.[CrossRef][Medline] [Order article via Infotrieve]
56. Ferri C, Grassi G. Mediterranean diet, cocoa and cardiovascular disease: a sweeter life, a longer life, or both? J Hypertens. 2003; 21: 2231–2234.[Medline] [Order article via Infotrieve]
57. Lee IM, Paffembarger RS. Life is sweet: candy consumption and longevity. BMJ. 1998; 317: 1683–1684.[Free Full Text]

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