Chronic Consumption of Flavanol-rich Cocoa Improves Endothelial Function and Decreases Vascular Cell...

Chronic Consumption of Flavanol-rich Cocoa ImprovesEndothelial Function and Decreases Vascular Cell

Adhesion Molecule in HypercholesterolemicPostmenopausal Women

Janice F. Wang-Polagruto, PhD,* Amparo C. Villablanca, MD,w John A. Polagruto, PhD,*Luke Lee, BS,* Roberta R. Holt, BS,* Heather R. Schrader, BS,* Jodi L. Ensunsa, MS,*Francene M. Steinberg, PhD, RD,* Harold H. Schmitz, PhD,z and Carl L. Keen, PhD*w

Abstract: Endothelial dysfunction characterizes many disease

states including subclinical atherosclerosis. The consumption of

flavanol-rich cocoa and cocoa-based products has been shown

to improve endothelial function in both compromised and

otherwise normal, healthy individuals when administered either

acutely or over a period of several days, or weeks. Women

experience increased risk for cardiovascular disease after

menopause, which can be associated with endothelial dysfunc-

tion. Whether a flavanol-rich cocoa-based product can improve

endothelial function in hypercholesterolemic postmenopausal

women is not known. The purpose of the present study was to

determine whether chronic dietary administration of flavanol-

rich cocoa improves endothelial function and markers of

cardiovascular health in hypercholesterolemic postmenopausal

women. Thirty-two postmenopausal hypercholesterolemic wo-

men were randomly assigned to consume a high-flavanol cocoa

beverage (high cocoa flavanols (CF)—446mg of total flavanols),

or a low-flavanol cocoa beverage (low CF—43mg of total

flavanols) for 6 weeks in a double-blind study (n=16 per

group). Endothelial function was determined by brachial artery-

reactive hyperemia. Plasma was analyzed for lipids (total

cholesterol, high-density lipoprotein cholesterol, low-density

lipoprotein cholesterol), hormones (follicle-stimulating hor-

mone), total nitrate/nitrite, activation of cellular adhesion

markers (vascular cell adhesion molecule 1, intercellular

adhesion molecule 1, E-Selectin, P-Selectin), and platelet

function and reactivity. Changes in these plasma markers were

then correlated to brachial reactivity. Brachial artery hyperemic

blood flow increased significantly by 76% (P<0.05 vs. baseline)

after the 6-week cocoa intervention in the high CF group,

compared with 32% in the low CF cocoa group (P=ns vs.

baseline). The 2.4-fold increase in hyperemic blood flow with

high CF cocoa closely correlated (r2=0.8) with a significant

decrease (11%) in plasma levels of soluble vascular cell adhesion

molecule-1. Similar responses were not observed after chronic

use of low CF. There were no significant differences between

high and low CF in other biochemical markers and parameters

measured. This study is the first to identify beneficial vascular

effects of flavanol-rich cocoa consumption in hypercholester-

olemic postmenopausal women. In addition, our results suggest

that reductions in plasma soluble vascular cell adhesion

molecule-1 after chronic consumption of a flavanol-rich cocoa

may be mechanistically linked to improved vascular reactivity.

Key Words: flavanols, epicatechin, procyanidin, brachial artery

reactivity, flow-mediated dilation, platelet function, adhesion,

cocoa, cardiovascular disease, nitric oxide

(J Cardiovasc PharmacolTM

2006;47[Suppl 2]:S177–S186)

The vascular endothelium is the primary regulator ofcardiovascular homeostasis in health and disease. It

functions to maintain vascular tone, regulate permeabil-ity, attenuate vascular inflammation, and inhibit smoothmuscle cell proliferation.1 Early atherosclerosis is char-acterized by a number of processes that include prele-sional endothelial dysfunction, endothelial activation,and monocyte recruitment to the endothelium andsubendothelial space. Endothelial dysfunction and im-pairment in endothelial-dependent vasodilation charac-terize many disease states, including cardiovasculardisease (CVD).2,3 Common contributors to endothelialdysfunction include cardiovascular risk factors such aselevated nascent and modified low-density lipoproteincholesterol (LDL-C), smoking, hypertension, diabetesmellitus,3 and the menopausal state.4 Activation of theendothelium is typically associated with increased con-centrations of soluble adhesion molecules in the blood,including intercellular adhesion molecule 1 (ICAM-1),vascular cell adhesion molecule 1 (VCAM-1), P-Selectin,and E-Selectin2 that can participate in the recruitment ofmonocytes and other blood-borne cells.Copyright r 2006 by Lippincott Williams & Wilkins

From the Departments of *Nutrition, wInternal Medicine, University ofCalifornia, Davis, CA; and zDepartment of Analytical and AppliedSciences, Mars, Incorporated, Hackettstown, NJ.

This work was supported in part by a grant from Mars, Incorporated,Hackettstown, NJ.

Reprints: Carl L. Keen, Department of Nutrition, University ofCalifornia, Davis, One Shields Avenue, Davis, CA 95616 (e-mail:[email protected])

J Cardiovasc PharmacolTM� Volume 47, Supplement 2, 2006 S177

Clinical studies indicate that high dietary intake ofthe antioxidant vitamins, folic acid, L-arginine, ando-3 fatty acids favorably affects vascular endothelialfunction.2,5,6 Plant-derived foods rich in polyphenoliccompounds, such as the flavonoids, have also received agreat deal of attention for their potential cardiovascularhealth benefits, and certain cocoas can be an especiallyrich source of a specific subclass of these phytochemicalsknown as flavanols. Cocoa flavanols (CFs) and flavanol-rich cocoa products have demonstrated potential, both invitro and in human studies, to modulate cardiovascularhealth in at least 4 important ways, including: improvedendothelial function, modulation of inflammation,reduced platelet reactivity, and increases in oxidativedefense. For example, the consumption of flavanol-richcocoa and cocoa-based products increases plasma anti-oxidant capacity and lag time to LDL oxidation.7–13 Inaddition, the consumption of a flavanol-rich cocoa orcocoa-based product has been shown to reduce plateletreactivity, as determined by assays measuring plateletaggregation and primary hemostasis, and markerof platelet activation.11,14–17 Consumption of flavanol-rich cocoa beverages has been shown to improveendothelial function in healthy smoking and nonsmokingsubjects.18–23 Finally, although the duration of the effectand the mechanism(s) of action are apparently quitedifferent, the effect of flavanol-rich cocoa on plateletactivation is qualitatively similar to that after aspirinadministration on an acute basis, albeit of lessermagnitude.16,24

Flavonoid-rich beverages favorably affect endothe-lial function in healthy men and women subjects, andthose with vascular compromise. For example, vascularendothelial function in subjects with established CVDimproved after the consumption of flavonoid-rich bev-erages.5,25,26 Similarly, in subjects with no history ofCVD, flow-mediated dilation (FMD) after the consump-tion of dealcoholized red wine27 and black tea28 increasedsignificantly (5.6% and 2.1%, respectively). Heiss et al22

demonstrated that flavanol-rich, but not flavanol-poor,cocoa could significantly increase both FMD and thenitric oxide (NO) pool 2 hours after ingestion of a singledose (176mg of total flavanols). They have recentlyshown that flavanol-rich cocoa (176 to 185mg/dose) canreverse endothelial dysfunction in a group of smokers,also at 2 hours postconsumption.21 Importantly, Fisheret al19 added substantially to the field by unambiguouslydemonstrating that the enhancement in peripheral vaso-dilation in otherwise normal, healthy subjects afteringestion of flavanol-rich cocoa (821mg of total flavanolsper day) was NO-dependent.

Although the literature describing flavanol-richcocoa and its ability to improve important markers ofcardiovascular health is impressive, additional questionsremain to be answered. It is unknown, for example,whether sex differences exist with respect to endothelialresponse to flavanol-rich cocoa. It is also not knownwhether premenopausal and postmenopausal womendiffer in their endothelial response to flavanol-rich cocoa,

and how this may be modulated by hyperlipidemia. Thus,in the present study, we investigate the effects of flavanol-rich cocoa consumption on markers of cardiovascularrisk and endothelial function in hypercholesterolemic,postmenopausal women, not taking hormone-replace-ment therapy (HRT) or cholesterol lowering medication.As this group represents individuals in the populationwho are at risk for subclinical atherosclerosis because oftheir age, menopausal status, and hyperlipidemia,4,29–31

we hypothesized that they would display a cardioprotec-tive response after the intake of flavanol-rich cocoa. Inaddition, because little is known regarding whether thereported vascular actions of acute flavanol-rich cocoaadministration can persist during chronic use, we alsochose to investigate the impact of chronic flavanol-richcocoa consumption by postmenopausal women.

METHODS

SubjectsThe study group consisted of 32 hypercholester-

olemic postmenopausal women. Subjects were nonsmo-kers, did not use cholesterol-lowering drugs, and did notregularly use antioxidant supplements. Participants hadno history of CVD, thyroid disorder, or diabetes mellitus.Inclusion criteria included a fasting serum total cholester-ol (TC) level >200mg/dL, postmenopausal state (definedby absence of a menstrual period for 12 consecutivemonths and follicle-stimulating hormone (FSH) level of23 to 116.3mIU/mL), body mass index<30kg/m2, andno use of HRT. Those previously on HRT were asked tostop HRT with the consent of their physician, andwashout for 6 weeks before starting the study. Studyparticipants provided written, informed consent, follow-ing protocols approved by the Institutional Review Boardat the University of California, Davis.

Study DesignThe study was a randomized, double-blind, parallel-

arm study with 2 treatment groups: a high-flavanol cocoabeverage (high CF), and a low-flavanol cocoa beverage(low CF) (n=16 women in each group). Subjects wereentered on a rolling-enrollment basis, and the study wasconducted over a total of 9 months. All subjectsparticipated in the 11-week study protocol, and madeup to 6 visits to the clinic (Fig. 1). Low CF was consumeddaily for the first 2 weeks of the study (week 0 to 2, run-in),subjects were then randomized to consume high or low CFdaily for a 6-week period (week 2 to 8, treatment period),followed by a 3-week washout period (week 8 to 11)during which time no cocoa beverage was consumed.

A 3-day diet record was kept at 4 time pointsthroughout the study, at weeks 0, 2, 8, and 11. Subjectsfollowed a low-flavonoid diet for 24 hours before eachclinic visit by avoiding all fruits and vegetables, and juicesderived from them, wine, and tea. Cocoa and otherchocolate products were avoided during the entire11-week study period. Subjects otherwise maintainedtheir regular diet.

Wang-Polagruto et al J Cardiovasc PharmacolTM� Volume 47, Supplement 2, 2006

S178 r 2006 Lippincott Williams & Wilkins

Cocoa BeverageSubjects were supplied with individually sealed

servings of 36 g of cocoa powder (Mars, Incorporated,Hackettstown, NJ) labeled with a 3-digit code, and askedto reconstitute it in 240mL of hot water. To assesscompliance, empty packets and any unused servings werecollected at follow-up visits and counted. The cocoapowder consisted of 18.8 g cocoa powder, 18 g sucrose,and trace amounts of carrageenan, vanillin, cinnamon,and salt (for suspension of solids and flavoring). High CFcontained an average of 12.4mg total CFs/g and 446mgof total flavanols. Low CF beverage contained an averageof 1.2mg total CFs/g and 43mg of total flavanols.

Sample CollectionFasting (12 h) blood samples were obtained from

study participants at weeks 0 (baseline), 2, 8, and 11.Subjects were instructed to refrain from the use ofnonsteroidal anti-inflammatory medications for 4 daysbefore a blood draw. To assess changes in markers ofvascular function, lipids, and platelet reactivity, bloodwas collected in evacuated tubes containing ethylenediaminetetraacetic acid, sodium heparin, or sodiumcitrate, respectively. Plasma was separated by low-speedcentrifugation (1800g for 15min at 41C) and stored at� 801C until the end of the study. Platelet reactivity wasdetermined on whole blood within 4 hours of collectionusing a Platelet Function Analyzer (PFA-100; DadeBehring International, Miami, FL). For a given assay,individual samples from all of the study time points wereanalyzed concurrently to prevent interassay variation.Blood was also collected for fasting TC, LDL (LDL-C)and high-density lipoprotein cholesterol (HDL-C),triglycerides (TG), and glucose. Lipid and chemistrypanels were performed at the UC Davis Medical Centerclinical pathology laboratory using a Synchron LX-20System (Beckman Coulter Inc, Brea, CA). LDL-C wascalculated using the Friedewald equation.32 Postmeno-pausal status of study participants was confirmed byassay of serum FSH at the UC Davis Medical Centerclinical lab using a 2-site chemiluminotic immunoassay(Bayer Diagnostics, Medfield, MA). Subject’s height was

measured once at the first visit, and at this and subsequentvisits weight, blood pressure, and heart rate wererecorded.

Plasma Nitrate and NitriteNitrate and nitrite levels were measured in both

plasma and urine samples by a modified method of theGriess reaction that converts all nitrate to nitrite usingthe bacterial enzyme nitrate reductase.33 The colori-metric reaction between nitrite, sulfanilamide andN-(1-naphthyl) ethylenediamine produces a pink/magenta azo product with a maximum absorbance at543 nm. Absorbance was read using a Multiskan Ascentmicroplate photometer (Labsystems Inc, Franklin, MA).

Biochemical Markers of Endothelial Functionand Cardiovascular Risk

To determine whether changes in brachial arteryreactivity were associated with changes in biochemicalmarkers of endothelial function and vascular inflamma-tion, biomarkers were measured on plasma samplescollected at weeks 2, 8, and 11 using standard commer-cially available enzyme immunoassay kits following themanufacturer’s instructions. Markers included thoseassociated with early monocyte binding to endotheliumand endothelial activation including: soluble intercellularadhesion molecule-1 (sICAM-1), soluble vascular celladhesion molecule-1 (sVCAM-1), soluble P-Selectin(sP-Selectin), and soluble E-Selectin (sE-Selectin).

Assay of Platelet FunctionTo determine if chronic consumption of flavanol-

rich cocoa affects platelet reactivity, platelet function wasmeasured with whole citrated blood using the PlateletFunction Analyzer (PFA-100) (Dade Behring Interna-tional, Miami, FL) as described previously.16 Care wastaken to ensure that blood collected for this assayremained undisturbed for at least 30 minutes beforeanalysis, and only blood drawn within 4 hours beforeperforming the assay was used. The PFA-100 measurescollagen-epinephrine or collagen-ADP-induced hemo-static plug formation under simulated small vessel shearconditions (5000 to 6000/s), and the time in seconds toocclude the aperture of a collagen-coated membrane(closure time). If occlusion of the aperture did not occurby 300 seconds, the test was stopped. Samples wereprocessed in duplicate.

Determination of Plasma EpicatechinHeparinized plasma samples were extracted and

analyzed for plasma epicatechin concentration. For thesestudies, an HP 1100 high-pressure liquid chromatographysystem with Chemstation software, equipped witha quaternary pump, temperature-controlled autosampler,column oven, and diode array detector (Hewlett-Packard,Wilmington, DE) in series with a CoulArray 5600detector (ESA, Chelmsford, MA) was used as previouslydescribed.34

Run-inperiod

:

Recruit

for 2

HighCF

LowCF

Study Timeline

Run-inperiod

Randomization to one of the following

groups

Treatment period

Washout

isubject

Consume Low CFcocoa

beverage

weeks

HighCF

LowCF

6 weeksdaily cocoa beverage

3 weekswashout

Week 0 2 8 11

BD BD/BR BD/BR BD

FIGURE 1. Schematic summary of study design and timelineof clinic visits and blood draws. BD, blood draw; BR, brachialartery reactivity; CF, cocoa flavanols.

J Cardiovasc PharmacolTM� Volume 47, Supplement 2, 2006 Chronic Consumption of Flavanol-rich Cocoa

r 2006 Lippincott Williams & Wilkins S179

Brachial Artery ReactivityFMD of the brachial artery was measured at the

beginning and end of the 6-week cocoa intervention ineach study subject. Changes in vessel diameter afterreactive hyperemia (endothelium-dependent vasodila-tion), and after sublingual nitroglycerin (endothelium-independent vasodilation) were measured according topreviously described methods.35 Brachial artery ultra-sound scans were performed after an overnight fast withthe subjects at rest after lying in the supine position for atleast 10 minutes. Studies were performed using a PhillipsHDI 3500 ultrasound machine and a high-resolution(7.5MHz) linear array transducer by 2 experiencedvascular ultrasound technicians. The variability in mea-sures between the 2 technicians was <5%.

The right brachial artery was imaged above (3 to7 cm) the antecubital fossa and arterial bifurcation with avascular probe positioned at an angle of 60 degrees. First,baseline brachial artery diameter and flow were measured.Next, a blood pressure cuff was inflated to suprasystolicpressure (at least 30mm Hg higher than the subject’ssystolic blood pressure) and the vessel compressed for5 minutes. The cuff was deflated and brachial arterydiameter and flow were measured within 15 seconds afterdecompression during reactive hyperemia to yield thepostocclusion data. A second baseline brachial arterydiameter and flow were measured after 10 minutes todetermine that the vessel returned to baseline. Then,nitroglycerin was administered sublingually as a tablet(0.4mg) and the postnitroglycerin brachial artery flowvelocity and diameter were determined after 4 minutes.

All subjects participated in the vascular functionprotocol. The following parameters were measuredduring each ultrasound session at baseline, postocclusion,at a second baseline, and after nitroglycerin administra-tion: systolic peak flow velocity (cm/s), vessel diameter(mm), mean flow velocity (cm/s), and flow volume bydiameter (cm/s). FMD was calculated as the percentchange in vessel diameter postocclusion compared withthe first baseline. Hyperemic flow was defined asblood flow in mL/min during reactive hyperemia andcalculated as postocclusion systolic peak flow velocity�(p)� (radius of brachial artery)2 and reported as mL/min.Change in peak blood flow was calculated as the percentchange in hyperemic flow postocclusion compared withthe baseline value. To calculate endothelium-independentvasodilation, nitroglycerin-mediated dilation was deter-mined using the postnitroglycerin arterial diameter valuecompared with the second baseline.

Statistical AnalysisData are presented as the mean±SEM. Statistical

analysis was performed using SigmaStat for Windowsversion 2.03 (SPSS Inc) and SAS for Windows version 8.1(SAS Institute, Cary, NC). Data were analyzed by 2-wayrepeated measures analysis of variance (ANOVA). Gen-eral linear models were used to examine differences inpostintervention values compared with baseline values.Logarithmic transformations were performed on all

variables. Differences were considered significant atPr0.05. All parameters were correlated using thePearson product correlation.

RESULTS

Subject CharacteristicsBaseline subject characteristics were similar between

the 2 study groups (high and low CF), and there were nostatistically significant differences between groups in anyof the demographic variables we determined. Mean age ofthe subjects in the high and low CF groups was 57.7±2.2and 55.4±1.7 years, respectively. The high and low CFgroups had an average body mass index of 24.9±1.0 and25.3±0.8 kg/m2, respectively. There were no differencesin FSH levels between the high and low CF groups(90.7±6.6, 81±7.4, respectively), and all women wereconfirmed to be postmenopausal (FSH levels 23 to116.3mIU/mL). Three women in the high CF group,and 2 women in the low CF group were on hormone-HRT before the study, and with the consent of theirphysician, stopped using HRT 6 weeks before enteringthe study. Subjects that were subsequently determined tohave abnormal vascular responses, defined by paradox-ical vasoconstrictor responses to vessel occlusion or nochange in vascular reactivity in response to vesselocclusion (a total of 15 women), were excluded fromsubsequent statistical analysis as these findings suggestedunderlying vascular dysfunction and possible subclinicalvascular disease. This heterogeneous subset includes:5 subjects (3 high CF and 2 low CF) that were unableto provide a FMD measurement at week 8, 4 subjects (1high CF and 3 low CF) that had a negative FMD percentchange at week 2, 1 high CF subject with no percentchange in FMD at week 2, and 5 subjects (2 high CF and3 low CF) with negative FMD percent changes at week 8.Thus, data from a total of 9 subjects (56% of women) inthe high CF group and 8 subjects (50% of women) in thelow CF groups were used for the lipid, biochemical, andvascular function analyses.

Dietary AnalysisIndividual food intake records from weeks 0, 2, 8,

and 11 were analyzed using Nutritionist V (First DataBank, San Bruno, CA), and data from 3-day diet recordsat each time point averaged. As shown in Table 1, dietaryintake of energy, macronutrients, fats, vitamins, andother substances did not change significantly over the6-week treatment period in either cocoa treatment studygroup. In addition, no significant differences wereobserved in any of the dietary parameters between the2 cocoa treatment groups.

Hemodynamic ParametersThere were no significant changes observed in

systolic or diastolic blood pressure in the high CF groupover the 6-week dietary intervention period, Table 2. Incontrast, in the low CP group, both systolic and diastolicblood pressure decreased significantly by 9.3% and 6.5%,



respectively, after the 6-week dietary intervention period(P<0.05). In addition, diastolic blood pressure values atweek 2 were also significantly (P<0.01) different in thehigh and low CF groups. There were no significantchanges in heart rate in either group after the cocoabeverage interventions.

Brachial Artery ReactivityThere were no differences in resting or postnitro-

glycerin arterial diameter at the beginning and end of theintervention in either of the CF dietary groups. Asexpected, hyperemic diameter and postnitroglycerindiameter increased from baseline in both CF beveragestudy groups. After 6-week CF dietary intervention,FMD increased 2% in the high CF group, and decreased1.5% in the low CF group. The increase in FMD in thehigh CF displayed a strong trend toward statisticalsignificance (P=0.0585). Furthermore, in the high CFbeverage group hyperemic blood flow increased by 76%

compared with baseline (P=0.002), consistent with asignificant vasodilatory effect. Compared with baseline,hyperemic blood flow increased by 32% in the low CFgroup; however, this change did not reach statisticalsignificance (Fig. 2).

Adhesion MarkersLevels of biochemical markers of endothelial func-

tion and adhesion molecules during the 6 weeks on thecocoa treatments are reported in Table 3. After 6-weekintervention, sVCAM-1 decreased by 10.7% in the highCF group (P=0.009), suggesting a significant attenua-tion of activation of this marker of vascular health inresponse to daily flavanol-rich cocoa intake. In contrast,sVCAM-1 increased in the low CF group. The differentialresponse between high and low CF on sVCAM-1 washighly statistically significant (P=0.010). Because con-siderable attention has focused on soluble adhesionmolecules as early biomarkers of alterations in vascular

TABLE 1. Dietary Nutrient Intake for Subjects in Each Treatment Group During the 6-week Cocoa Beverage Intervention Period

High CF Cocoa Group Low CF Cocoa Group

Week 2 Week 8 Week 2 Week 8

Energy (MJ) 4.6±0.4 3.7±0.3 3.6±0.2 3.2±0.3Protein (g) 67.5±5.9 67.7±4.8 60.8±3.9 63.5±6.3CHO (g) 199.3±21.2 174±17.3 195.8±17.3 172.7±16.4Fat (g) 59.9±7.8 56±7.5 50.2±4.1 41.2±4.1Sat fat (g) 19.3±2.5 17.6±3.1 15.1±1.7 11.8±1.6MUFA (g) 19.2±3.4 16.9±2.7 17.1±2.7 12.6±1.4PUFA (g) 10.1±1.8 11±1.2 8.6±1 9.2±1.2Cholesterol (mg) 217.7±26.7 163±18.4 175.6±25.2 191.8±37.2Vitamin C (mg) 108.2±15.7 93.1±15.2 104.5±14.3 121.1±23.2Vitamin E (IU) 9.1±1.7 11.9±3.2 8.9±1.5 10.4±1.8Dietary fiber (g) 16.8±2.1 16.1±3.2 15.9±1.2 15.3±1.3Caffeine (mg) 81.1±21.9 73±14 78.1±21.7 77.1±17.8Alcohol (g) 5.4±1.4 12.5±5.3 7.5±2.5 5.5±2.4Fruit (servings/d) 1.6±0.6 1.4±0.3 1.5±0.4 1.5±0.4

(Values are mean±SEM); n=16 in the high CF group, n=16 in the low CF group.Values reflect average daily intakes over each 3-day food record period, at weeks 2 and 8.CF indicates cocoa flavanols; CHO, carbohydrates; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; MJ, mega joules; IU, international units.There were no significant differences for any variable by RM ANOVA between time points (2 and 8wk) or between treatment groups (high and low CF).

TABLE 2. Brachial Artery Reactivity and Hemodynamic Parameters



Systolic blood pressure (mm Hg)* 125±6 128±7 140±10 127±8*Diastolic blood pressure (mm Hg)w 64±3 63±3 76±2w 71±3*Heart rate (bpm) 68±3 69±4 73±6 63±6Resting arterial vessel diameter (mm) 3.3±0.1 3.2±0.1 3.3±0.2 3.3±0.2Hyperemic arterial diameter (mm) 3.7±0.1 3.7±0.1 3.7±0.1 3.7±0.1Post nitroglycerin arterial diameter (mm) 4.1±0.1 3.9±0.1 4.0±0.2 3.9±0.1Flow-mediated dilation (% change) 12.1±1.9 14.1±3.9 12.5±1.4 11±2

(Values are mean±SEM); n=9 in the high CP group, n=8 in the low CP group.CF indicates cocoa flavanols.*Systolic and diastolic blood pressure decreased significantly (P<0.05) with low CF during the 6-week intervention.wDiastolic blood pressure values at week 2 were significantly (P<0.01) different in the high and low CF groups by RM ANOVA.There were no significant differences for any other variable by RM ANOVA between time points (2 and 8wk) or between treatment groups (high and low CF).



function, we investigated whether in the high CF studygroup there was a relationship between the decrease insVCAM-1 levels and the increase in hyperemic flow. Thisanalysis demonstrated these parameters to be tightlycorrelated (r=0.814, P=0.014) in a unique way as therewere no significant changes in sP-selectin, sE-selectin, orsICAM-1 over the 6-week study period for either cocoatreatment group. In addition, there were no significantdifferences in these parameters, or total nitrates andnitrites, between the 2 cocoa treatment groups.

Plasma LipidsThe mean starting serum lipid concentrations for

the high and low CF groups, respectively, were: 235±8versus 240±8mg/dL (TC), 149±5 versus 150±9mg/dL (LDL), 69.7±4.6 versus 68.1±6.1mg/dL (HDL-C),3.5±0.2 versus 3.9±0.3 (TC:HDL ratio), and 79.7±7.8versus 111.1±21.1mg/dL (TGs). Although TC, LDL-C,and TG did not change within either treatment groupafter 6-week intervention, there was a differential effectof cocoa dose on HDL-C. HDL-C levels increased by6.6% with high CF, yet decreased by 9.6% in the lowCF group. This differential response on HDL-C wasstatistically significant, P<0.05. However, changes in

HDL-C did not correlate with changes in hyperemicflow or any of the other cardiovascular health markersstudied (data not shown). Measurement of blood lipidconcentrations at week 11 indicated no significantchanges to TC, LDL, the TC:HDL ratio, and TG.The significant difference in the concentration of serumHDL-C between the groups remained at week 11.

Platelet Function and Flavanol ConsumptionAfter 6 weeks of the daily cocoa beverage interven-

tion, there was a 13% increase in ADP/collagen-inducedPFA-100 closure time in the high CF compared with thelow CF group that approached statistical significance(P=0.079). Baseline values for ADP/collagen-inducedplatelet reactivity in the high and low CF groups were84.5±2.2 and 96.6±8.9 seconds, respectively. Therewere no significant changes in epinephrine/collagen-induced platelet reactivity in either CF treatment groupafter 6-week dietary intervention. Baseline values forepinephrine/collagen-induced platelet reactivity were169.1±17.5 and 173.2±22.9 seconds for the high andlow CF groups, respectively.

DISCUSSIONThe objective of this dietary intervention study was

to determine whether chronic (6 weeks) consumption offlavanol-rich cocoa attenuated cardiovascular risk mar-kers and improved vascular function, as measured byFMD and reactive hyperemia of the brachial artery, inhypercholesterolemic postmenopausal women. Our find-ings extend prior observations of improved markersof cardiovascular health resulting from consumptionof flavanol-rich cocoa either acutely or over a period ofseveral days. In addition, a vasculoprotective effect forchronic flavanol-rich cocoa was demonstrated in hyper-cholesterolemic postmenopausal women, a populationnot previously studied with regard to effects of flavanol-rich cocoa consumption. Our findings can be summarizedas follows: (1) chronic consumption of high CF, but notlow CF, improves FMD and significantly increaseshyperemic brachial artery blood flow; (2) the improve-ment in endothelial function with high CF seems to bemechanistically linked to reductions in sVCAM-1; and

Change in Hyperemic Flow after the 6-week cocoabeverage intervention

0

20

40

60

80

100

120

140

160

Low CF High CF

Wk 2 Wk 8

*

% C

han

ge

in h

yper

emic

flo

w

FIGURE 2. Change in hyperemic blood flow from week 2 to 8for the high and low CF study groups. The increase wassignificantly different in the high CF cocoa beverage group,P = 0.002 [all pairwise multiple comparison procedures (Tukeytest)].

TABLE 3. Biochemical Markers of Endothelial Function



Nitrates and nitrites (mmol/L) 16.1±3.9 16.5±4.5 21.7±5.4 20.5±4.9sP-Selectin (ng/mL) 28.3±3.2 32.2±4.5 31.4±6 32.3±6.3sE-Selectin (ng/mL) 45.3±6.3 42.3±5.6 45±6.1 47.1±7.4sVCAM-1 (ng/mL) 631.5±49.3 553.9±35.5*w 398.6±48.7 433.8±65.8sICAM-1 (ng/mL) 183.1±10.8 182.9±14.5 165.7±16.8 162.3±17.6

(Values are mean±SEM); n=9 in the high CF group, n=8 in the low CF group.CF indicates cocoa flavanols; sVCAM-1, vascular cell adhesion molecule 1; sICAM-1, intercellular adhesion molecule 1.*(sVCAM-1 significantly different after the 6-week intervention in the high CF group (P=0.009, RM ANOVA).wChange in sVCAM-1 in the high CF group significantly different from the change in the low CF group (P=0.010, t test).



(3) chronic consumption of high CF but not low CF leadsto a significant increase in HDL cholesterol.

The endothelium is important for maintainingnormal vascular function and preventing early events inatherogenesis,3,36,37 and endothelial dysfunction canprecede appreciable atherosclerotic lesion development.Noninvasive imaging with Doppler ultrasound 38 hasrevealed changes in endothelium-dependent FMD inpatients with risk factors for vascular disease. Improve-ment in FMD has been previously demonstrated with anumber of dietary interventions, including soy protein39,and cocoa.18,21–23 To our knowledge, this study is the firstto examine the effect of chronic consumption of aflavanol-rich food on endothelial function and vascularbiomarkers in mildly hypercholesterolemic postmenopau-sal women. Our study used a number of unique featurescompared with prior work. Previous studies of flavanol-rich cocoa have used male subjects, or mixed studygroups of both males and females. In addition, althoughthe menopausal status of women has not been previouslynoted, on the basis of the age of subjects it seems thatprior investigations have not included postmenopausalwomen.18–23 Postmenopausal women are at a greater riskfor CVD because of their age and menopausal status, andas our findings demonstrate, this is a group of individualsthat can exhibit early impairment of vascular function.Therefore, the present study focused on postmenopausalwomen to determine whether flavanol-rich cocoa couldinfluence risk markers associated with CVD. Further-more, our study population was mildly hypercholester-olemic, permitting us to ascertain whether flavanol-richcocoa can favorably impact lipid parameters. Lastly, wealso measured changes in vascular reactivity in responseto chronic cocoa consumption (6 weeks of daily flavanol-rich cocoa consumption) to compare the vascularresponses to the acute or shorter duration administrationof flavanol-rich cocoa used in prior studies.18–23 Becausethe effect of chronic consumption of flavanol-rich cocoahas not been well studied and there are numerousexamples of acute drug effects differing from chronicresponses, we sought to provide longer duration con-sumption in our study design.

After 6-week high CF dietary intervention, weobserved a significant increase in hyperemic blood flow,an important marker of vasodilation, and a 2% increasein FMD. Although the change in FMD approachedstatistical significance it did not reach it. However, theincrease approximated the 3% increase in FMD notedby Heiss et al22 after acute (2 h) consumption of aflavanol-rich cocoa drink and a 1.3% increase after darkchocolate consumption,18 and a 2.1% increase in FMDafter black tea consumption as reported by Hodgsonet al28 It is important to note that the FMD measure-ments in these studies were all taken within hours afterconsumption of the flavonoid-rich food. Short-termflavanol-rich cocoa supplementation studies suggest thatplasma epicatechin partially contributes to an improvedFMD by increasing NO availability, a response thatpeaks B2 hours after cocoa consumption.11,19,21,40–42

However, we were unable to detect significant levels ofnonmethylated plasma epicatechin after the overnight fastthat preceded the FMD measurements. If the positiveeffects of cocoa supplementation are transient in nature,dissipating within 1 to 2 hours after stopping cocoaconsumption, this could in part explain our results.However, little is known about the long-term effects offlavanol consumption, and as recently observed forquercetin,43 it may be possible for flavanols to accumulatewithin tissues over a more prolonged period of time, toproduce a biologic effect. Therefore, it is important to notethat all subjects were given the low-flavanol cocoa for a2-week run in period before the beginning of the trial.This ‘‘low-flavanol’’ cocoa was not completely devoid inepicatechin or catechin content, but rather contained43mg of total flavanols. A 2-week supplementation studyproviding 46mg/day of epicatechin in dark chocolateproduced significant improvements in FMD 2 hours afterthe last chocolate dose.18 Recently, Heiss et al,21 reportedimprovements in FMD response with a drink containingB70mg of epicatechin and catechin. In addition, we havereported reduced platelet reactivity with as little as 220mgof total flavanols (in a small handful of chocolate chips).11

Epidemiology studies also indicate a 25% lowered risk inischemic heart disease individuals that consume 50mg ofcatechin in the diet per day.44 Thus, it is possible that the2-week run-in period with the low flavanol cocoa beverageimproved the FMD baseline to such an extent that theaddition of the high flavonoid drink did not providestatistically significant improvements. The above issueshould be addressed in future studies.

To determine the potential mechanism underlyingthe increase in hyperemic flow observed with high CF inour study, we investigated a number of importantvariables previously demonstrated to affect vascularfunction. For this analysis we considered NO, serumlipids, platelet aggregation, and vascular cell adhesionmarkers. Our findings with each of these parameters arediscussed below.

We first considered the potential role of NO. It hasbeen suggested that changes in NO signaling afterconsumption of flavanol-rich foods may be responsiblefor their vasodilatory effects. Indeed, the administrationof the NO synthase inhibitor, L-NAME, completelyprevented the dilatory effects induced by cocoa consump-tion in studies by Fisher et al.19 Although we observed nodifferences in plasma total nitrite and nitrate afterconsumption of the high or low CF cocoa beverage for6 weeks, this does not preclude the possible involvementof early effects because of an increase in NO availability.As total plasma nitrate and nitrite was measured after anovernight fast, we may have failed to detect any short-term transient effects of cocoa that could have occurredshortly after the consumption of the cocoa beverages.

Higher plasma HDL-C is atheroprotective,45 and itis associated with improved endothelial function inhypercholesterolemic patients.46 In addition, elevationof HDL-C can restore endothelial function in patientswith low HDL-C.47 We did not observe a significant



difference in HDL-C during the 6-week dietary cocoaintervention in either flavanol-rich cocoa treatmentgroup. However, compared with baseline values, theincrease in HDL-C observed with high CF was signifi-cantly different from the decrease in HDL-C observed inthe low CF group. The change in HDL-C with flavanol-rich cocoa dose observed in the current study may beimportant, particularly given the fact that a similarobservation was made by Wan et al.9 These resultssuggest that an improved lipid biomarker profile could bemechanistically linked to the observed improved vascularresponsiveness after chronic high-dose cocoa in post-menopausal women.

Next, we considered the potential role of solubleadhesion molecules. The expression, principally onendothelial cells, of molecules such as VCAM-1, ICAM-1, and E-selectin mediates leukocyte recruitment to sitesof vascular inflammation and is an early event inatherogenesis.48,49 In animal models, the expression ofVCAM-1 and ICAM-1 localizes to areas predisposedto the formation of atherosclerotic plaque.48,50 Thus,considerable attention has focused on soluble adhesionmolecules as early biomarkers of alterations in vascularfunction, as they are indirect measures of vascularinflammation and endothelial cell activation. In animalmodels of atherosclerosis, for example, the use ofantibodies to adhesion markers has been shown to reducevarious CVD parameters.51 Evidence that the concentra-tion of soluble adhesion markers (eg, sVCAM-1 andsICAM-1) in plasma are predictive of cardiovascular riskhas led to the suggestion that they could be targets fortherapeutic intervention.51 Thus, identifying a foodproduct, or compounds contained in food (such asflavanols), as potential modulators of adhesion moleculeexpression is of considerable interest. We observed thatsubjects who consumed the high CF had significantlylower levels of sVCAM-1 after 6-week intervention,compared with those consuming the low CF beverage.Furthermore, the increase in hyperemic flow stronglycorrelated with the decrease in sVCAM-1 in this samegroup of postmenopausal women. This suggests that thechronic use of high flavanol cocoa in postmenopausalwomen can improve endothelial function; on the basis ofour findings, this may involve a VCAM-1-dependentmechanism.

It is not yet clear which specific components in theflavanol-rich cocoa provided to subjects in our studycontributed to the observed improved vascular reactivityresponse and concomitant reduction in VCAM-1. Similarto the cocoa used by Fisher et al,19 ingredients in both thehigh and low CF used in this investigation were similar incomposition, except for the amount of total flavanols.However, given that epicatechin and certain B-typedimer(s), and their related metabolites are the presumedpredominant flavanols in human plasma after cocoaconsumption,34,52 they are candidates of potential interestfor the effects of high CF observed in this and otherstudies. These flavonoids are reported to inhibit activa-tion of the oxidative-stress sensitive nuclear transcription

factor, nuclear factor kappa-B (NF-kB). It is pertinent tonote that in so doing flavanols can inhibit the upregula-tion of VCAM-1 endothelial expression in vitro.53,54

As the transcription factor NF-kB is a known promoterof VCAM-1 expression,39 it can be speculated thatreductions in NF-kB activation in cells of the vascularendothelium provide a mechanistic explanation for aflavanol-induced decrease in sVCAM-1. Future studiesare needed to investigate the downstream signaling eventsassociated with of flavanol-rich cocoa-induced decreasesin VCAM-1.

Certain flavonoids may be partly responsible for adecreased tendency for platelet activation and aggrega-tion, important mechanisms involved in the progressionand pathogenesis of CVD. We have also previouslydemonstrated in humans that an acute dose of a flavanol-rich cocoa product inhibited platelet activation andfunction as evidenced by changes in epinephrine/col-lagen-induced platelet reactivity.16 We have also pre-viously demonstrated that an acute dose of flavanol-richcocoa inhibits platelet activation and function over6 hours in healthy humans.15 The mechanism, in part, isvia suppression of ADP-stimulated or epinephrine-stimulated platelet activation and platelet microparticleformation, leading us to conclude that flavanol-rich cocoaconsumption can have an acute effect on primaryhemostasis that is similar to aspirin. However, we havenot yet studied the effects of chronic consumption offlavanol-rich cocoa, nor have we studied this in post-menopausal women. Therefore, the focus of the platelet-related protocols of this study was to investigate thelonger-term effect of high and low CF on plateletreactivity in this group. We observed a trend toward anincrease in ADP-stimulated platelet aperture closure timeafter consumption of the high CF product. Our findingsare in agreement with other investigators who demon-strated that flavanol-rich cocoa supplementation for 28days significantly increased plasma epicatechin andcatechin concentrations and significantly decreased plate-let function.55

The consumption of a heart-healthy diet as anadjunct to therapeutic lifestyle changes is widely recom-mended for cardiovascular risk reduction.29 Although thetotal number of subjects included in our statisticalanalyses was constrained by the fact that a significantnumber of subjects exhibited abnormal baseline vascularfunction our findings suggest that in hypercholesterolemicpostmenopausal women daily consumption of flavanol-rich cocoa can significantly improve vascular endothelialfunction. In addition, the vascular improvements aretightly correlated to a reduction in sVCAM-1 andassociated with a increase in HDL-C, all knownpredictors of CVD risk. The results from this presentstudy indicate that the chronic consumption of flavanol-rich cocoa can be beneficial for hypercholesterolemicpostmenopausal women, extending the growing body ofevidence that flavanol-rich diets provide significantcardiovascular protection. Additional studies investigat-ing the actions of other dietary flavonoids having putative



cardiovascular benefits in postmenopausal women couldtherefore be of interest.

ACKNOWLEDGMENTSThe authors thank Francine Nardinelli, Deborah

Finley, and Holly Beals for providing technical assistancewith the ultrasound exams. They also thank Neil Willits forproviding the statistical analysis for this study. J.F.W.P.,H.H.S., and C.L.K. contributed to the concept of the study.J.F.W.P., A.C.V., J.A.P., H.H.S., and C.L.K. contributedto the design of the study. J.F.W.P., J.A.P., L.L., R.R.H.,H.S., J.L.E., and F.M.S. contributed to the conduct of thestudy. J.F.W.P., A.C.V., J.A.P., and C.L.K. contributed tothe interpretation of the data. Sherri Lazarus assisted theinvestigators in obtaining the cocoa product.

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Chronic Consumption of Flavanol-rich Cocoa Improves Endothelial Function and Decreases Vascular Cell...

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