Protective effects of flavanol-rich dark chocolate on endothelial function and wave reflection during acute hyperglycemia.

Abstract

Nitric oxide plays a pivotal role in regulating vascular tone. Different studies show endothelial function is impaired during hyperglycemia. Dark chocolate increases flow-mediated dilation in healthy and hypertensive subjects with and without glucose intolerance; however, the effect of pretreatment with dark chocolate on endothelial function and other vascular responses to hyperglycemia has not been examined. Therefore, we aimed to investigate the effects of flavanol-rich dark chocolate administration on (1) flow-mediated dilation and wave reflections; (2) blood pressure, endothelin-1 and oxidative stress, before and after oral glucose tolerance test (OGTT). Twelve healthy volunteers (5 males, 28.2±2.7 years) randomly received either 100 g/d dark chocolate or flavanol-free white chocolate for 3 days. After 7 days washout period, volunteers were switched to the other treatment. Flow-mediated dilation, stiffness index, reflection index, peak-to-peak time, blood pressure, endothelin-1 and 8-iso-PGF(2α) were evaluated after each treatment phase and OGTT. Compared with white chocolate, dark chocolate ingestion improved flow-mediated dilation (P=0.03), wave reflections, endothelin-1 and 8-iso-PGF(2α) (P<0.05). After white chocolate ingestion, flow-mediated dilation was reduced after OGTT from 7.88±0.68 to 6.07±0.76 (P=0.027), 6.74±0.51 (P=0.046) at 1 and 2 h after the glucose load, respectively. Similarly, after white chocolate but not after dark chocolate, wave reflections, blood pressure, and endothelin-1 and 8-iso-PGF(2α) increased after OGTT. OGTT causes acute, transient impairment of endothelial function and oxidative stress, which is attenuated by flavanol-rich dark chocolate. These results suggest cocoa flavanols may contribute to vascular health by reducing the postprandial impairment of arterial function associated with the pathogenesis of atherosclerosis.

Full text

Page 1

Blumberg, Michele Stornello and Claudio Ferri
Davide Grassi, Giovambattista Desideri, Stefano Necozione, Fabrizio Ruggieri, Jeffrey B.
Reflection During Acute Hyperglycemia
Protective Effects of Flavanol-Rich Dark Chocolate on Endothelial Function and Wave
Print ISSN: 0194-911X. Online ISSN: 1524-4563
Copyright © 2012 American Heart Association, Inc. All rights reserved.
is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Hypertension
published online July 30, 2012;
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Protective Effects of Flavanol-Rich Dark Chocolate on
Endothelial Function and Wave Reflection During
Acute Hyperglycemia
Davide Grassi, Giovambattista Desideri, Stefano Necozione, Fabrizio Ruggieri,
Jeffrey B. Blumberg, Michele Stornello, Claudio Ferri
Abstract—Nitric oxide plays a pivotal role in regulating vascular tone. Different studies show endothelial function is impaired
during hyperglycemia. Dark chocolate increases flow-mediated dilation in healthy and hypertensive subjects with and without
glucose intolerance; however, the effect of pretreatment with dark chocolate on endothelial function and other vascular
responses to hyperglycemia has not been examined. Therefore, we aimed to investigate the effects of flavanol-rich dark
chocolate administration on (1) flow-mediated dilation and wave reflections; (2) blood pressure, endothelin-1 and oxidative
stress, before and after oral glucose tolerance test (OGTT). Twelve healthy volunteers (5 males, 28.2?2.7 years) randomly
received either 100 g/d dark chocolate or flavanol-free white chocolate for 3 days. After 7 days washout period, volunteers
were switched to the other treatment. Flow-mediated dilation, stiffness index, reflection index, peak-to–peak time, blood
pressure, endothelin-1 and 8-iso-PGF2?were evaluated after each treatment phase and OGTT. Compared with white
chocolate, dark chocolate ingestion improved flow-mediated dilation (P?0.03), wave reflections, endothelin-1 and
8-iso-PGF2?(P?0.05). After white chocolate ingestion, flow-mediated dilation was reduced after OGTT from 7.88?0.68 to
6.07?0.76 (P?0.027), 6.74?0.51 (P?0.046) at 1 and 2 h after the glucose load, respectively. Similarly, after white chocolate
but not after dark chocolate, wave reflections, blood pressure, and endothelin-1 and 8-iso-PGF2?increased after OGTT.
OGTT causes acute, transient impairment of endothelial function and oxidative stress, which is attenuated by flavanol-rich
dark chocolate. These results suggest cocoa flavanols may contribute to vascular health by reducing the postprandial
impairment of arterial function associated with the pathogenesis of atherosclerosis.
00-00.) ● Online Data Supplement
(Hypertension. 2012;60:
Key Words: flavonoids ? cocoa ? nitric oxide ? endothelial function ? wave reflections ? oxidative stress
D
(T2DM).1Atherosclerotic pathology is a common sequela to
T2DM, with macrovascular and microvascular diseases being
among the leading causes of morbidity and mortality in patients
with T2DM.2Even after adjustment for conventional risk
factors, individuals with T2DM exhibit a 2- to 4-fold increased
risk of mortality in comparison with individuals without diabe-
tes.3Thus, several investigations have been directed to the
potential adverse effects of hyperglycemia on the arterial wall.
Observational data reveal high blood glucose concentrations are
a risk factor for mortality, even in nondiabetic individuals.4
Postprandial hyperglycemia has also been implicated in the
development of cardiovascular disease. Elevated postprandial
glucose may have a direct toxic effect on the vascular endothe-
lium mediated by oxidative stress, independent of other cardio-
iets high in readily absorbable carbohydrates are asso-
ciated with an increased risk for type 2 diabetes mellitus
vascular risk factors, such as hyperlipidemia.2,5A high-
carbohydrate meal has been described to increase oxidative
stress and favor proinflammatory effects.5
Several in vivo studies have found impaired nitric oxide
(NO)-mediated endothelial function during acute episodes of
hyperglycemia.6–10NO is a key endothelium-derived relaxing
factor and plays a pivotal role in the maintenance of basal
vascular tone and vascular responses to dilative stimuli.11–13
NO acts to negate the actions of endothelium-derived contract-
ing factors such as angiotensin II and endothelin-1 (ET-1), as
well as inhibit platelet aggregability11–13and endothelial activa-
tion.11–13Therefore, reduced NO bioavailability may contribute
to the onset and progression of endothelial dysfunction, includ-
ing impaired vasodilative and increased adhesive properties,
leadingtoatherogenesis.11–13Additionally,endothelium-derived
NO may act to regulate arterial stiffness and wave reflec-
Received February 27, 2012; first decision March 14, 2012; revision accepted July 7, 2012.
From the Department of Internal Medicine and Public Health, University of L’Aquila, L’Aquila, Italy (D.G., G.D., S.N., F.R., C.F.); Antioxidants
Research Laboratory, Jean Mayer United States Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, MA
(J.B.B.); Centre for the Hypertension Study and Therapy, General Hospital Umberto I, Syracusae, Italy (M.S.).
The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.
112.193995/-/DC1.
Correspondence to Davide Grassi, University of L’Aquila Department of Internal Medicine and Public Health, Viale S Salvatore, Delta 6 Medicina,
67100 Coppito, L’Aquila, Italy. E-mail davide.grassi@cc.univaq.it
© 2012 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org DOI: 10.1161/HYPERTENSIONAHA.112.193995
1 by CLAUDIO FERRI on August 14, 2012 http://hyper.ahajournals.org/ Downloaded from

Page 3

tions.13,14Reversal of endothelial dysfunction has been sug-
gested to slow the rate of atherogenesis and improve the
prognosis of patients with cardiovascular disease.13–15
Consumption of dietary flavonoids, including intake from
flavonoid-rich foods such as tea, wine, and cocoa has been
linked to reduced endothelial dysfunction and increased resis-
tance of low-density lipoprotein cholesterol to oxidation.15,16
Flavonoids appear to affect endothelial function by serving as
antioxidants and as modulators of signal transduction path-
ways.15,16We observed that flavanol-rich dark chocolate (DC)
ingestion improves NO-dependent flow-mediated dilation
(FMD) and insulin sensitivity and decreases blood pressure (BP)
in healthy17and hypertensive subjects with18and without19
glucose intolerance. Similarly, in patients with T2DM, Balzer et
al20have reported that acute as well as thrice-daily consumption
for 30 days of flavanol-containing cocoa increased FMD in a
dose-dependent manner; however, the effect of pretreatment
with flavonoids on the endothelial response to hyperglycemia
hasnotyetbeentested.Therefore,wehypothesizedDCcouldbe
able to protect arterial function from a glucose load and
examined the effects of DC and flavanol-free white chocolate
(WC) administration for 3 days on endothelial function and
wave reflections, as well as BP, circulating ET-1 and total
isoprostanes (8-iso-PGF2?) before and after an oral glucose
tolerance test (OGTT) in healthy subjects.
Methods
Subjects
Twelve healthy volunteers (5 males, mean age 28.2?2.7 years) were
recruited from staff at the San Salvatore Regional Hospital and
University of L’Aquila. Individuals were excluded if they had an
acute or chronic disease, including any type of metabolic abnormal-
ity and/or major cardiovascular risk factor. Habitual smokers and use
of any prescribed medication and/or dietary supplement within 2
weeks of entering the study were excluded. To further limit potential
confounding factors, individuals were excluded if they reported daily
intense sports activities (?10 hours/wk), changes of ?10% body
weight within 6 months of entering the study, current dietary treatment
regimen, and/or participation in another clinical study within 3 months
of entering this trial. The study was approved by the responsible Ethics
Committee, and all participants gave written informed consent.
Study Design
After a 7-day, cocoa-free, run-in phase, the volunteers were randomly
assigned to receive either 100-g DC bars (Cioccolato Bonajuto, Antica
Dolceria Bonajuto) or 100-g WC bars (Milka, Kraft Foods) in an
isocaloric fashion over a period of 3 days. FMD; office systolic (SBP)
and diastolic (DBP) BP; pulse contour analysis for measuring stiffness
index (SI), reflection index (RI), and peak-to-peak time (PPT); serum
ET-1and8-iso-PGF2?wereevaluatedbeforeandafterOGTTfollowing
each intervention phase. The homeostasis model assessment of insulin
resistance, quantitative insulin sensitivity check index, and insulin
sensitivity index were calculated from OGTT results.
After evaluation of exclusion/inclusion criteria, volunteers were
carefully instructed to maintain their usual diet except to refrain from
flavonoid-rich foods and beverages (a detailed list was given to each
participant), including wine, as well as all other alcoholic beverages.
All participants were encouraged to continue with their usual
physical activity throughout the study period. The subjects were
instructed to consume their chocolate bars in the morning and not in
the afternoon or at dinner. They consumed the first dose at breakfast
on day 1 and the last dose on the day before the visit. Analysis of the
DC using high-performance liquid chromatography quantified 447
mg of epicatechin, 59 mg of catechin, and 14 mg of quercetin; the
same method showed WC contained only trace amounts of polyphe-
nols (0.04 mg of catechin). DC and WC contained similar amounts
of energy and macronutrients (online-only Data Supplement Table S1).
At the end of the first phase of intervention, patients entered a second
7-day, chocolate-free phase and then were crossed over to the other
treatment.Chocolatedosesforeachsubjectwererolledinaluminumfoil
and administered in dated, sequentially numbered, nontransparent boxes
that were not labeled regarding content. All the involved physicians and
staff were blinded to the group assignment. Patients did not receive any
information regarding the chocolate and were instructed not to disclose
their assigned intervention group to investigators until the study com-
pletion. To avoid changes in body weight during the intervention,
participants were carefully instructed on how to substitute the chocolate
bars with foods of similar energy and macronutrient composition.
Patientswerecontinuouslyandcarefullyfollowedwithtailoredpersonal
diet counseling provided throughout the study by trained dieticians and
physicians. Please see the online-only Data Supplement for the addi-
tional and detailed methods section.
Results
According to the inclusion/exclusion criteria, participants
presented with normal ranges for waist circumference, lipid
profiles, and glucose-insulin metabolism. The baseline clini-
cal characteristics of the 12 study subjects are shown in the
online-only Data Supplement Table S2. All subjects com-
pleted both parts of the study.
Endothelial Function
Compared with the WC intervention period, DC increased
baseline FMD (8.51?0.69 versus 7.88?0.68%; P?0.03)
(Figure 1, A). After the WC phase, FMD fell from 7.88?0.68
at baseline to 6.07?0.76 (P?0.027), 6.74?0.51 (P?0.046),
and 7.16?0.97% (P?0.07) at 1, 2, and 3 hours after the
glucose load, respectively (Figure 1B). In contrast, after DC
ingestion, FMD was not significantly different in response to
the OGTT (from 8.51?0.69 to 8.25?0.92, n.s.; 7.96?0.84,
n.s.; and 8.48?1.4%, n.s. at 1, 2, and 3 hours after the glucose
load, respectively (Figure 1B). Compared with WC, DC admin-
istration protected endothelial function (P?0.0007 for treat-
ment) by preventing the attenuation of FMD induced by the
glucose load (Figure 1B).
Wave Reflections
Compared with the WC phase, DC ingestion decreased baseline
SI (Figure 2A) and RI values (59.3?12.4 versus 50.4?8%;
P?0.04) and increased PPT (265.5?35.1 versus 295.6?36.2
ms; P?0.05). After WC, SI increased after the glucose load
(Figure 2B). After DC, SI increased only after 60 minutes
from glucose load. Similarly, after WC, RI increased after 30,
60, and 90 minutes, and PPT decreased after 30, 60, 90, 120,
and 180 minutes from the glucose load, while, after DC, RI
increased after 90 minutes, and PPT decreased after 60, 90,
and 180 minutes (online-only Data Supplement Figures S1A
and S1B, respectively). The DC attenuated the impairment of
these measures of wave reflection induced by acute hyper-
glycemia (SI: P?0.0089 for treatment; RI: P?0.032 for
treatment; PPT: P?0.0001 for treatment) (Figure 2 and online-
only Data Supplement Figure S1).
Blood Pressure
Baseline office SBP and DBP were not statistically different
for treatment between the DC and WC intervention phases
(SBP: 110.6?9.3 versus 111.2?6.7 mm Hg; DBP: 70.0?7.4
2 Hypertension
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Page 4

versus 70.8?5.8 mm Hg, respectively). After the WC but not
DC intake, DBP increased (from 70.0?7.4 at baseline to
76.4?7.1 mm Hg; P?0.0079) after 30 minutes, and SBP
increased (from 110.6?9.3 at baseline to 118?6.7 mm Hg
after 30 minutes, P?0.0001 to 116.3?7.6 mm Hg after 60
minutes, P?0.0002 and to 114.5?6.2 mm Hg after 90
minutes, P?0.0191 from the glucose load. DC ingestion
prevented the increase in BP (SBP: ?0.0001 and DBP:
P?0.019 for treatment) induced by the glucose load.
Endothelin-1
Baseline serum ET-1 level was higher after the WC compared
with the DC intervention phase (Figure 3A). After the WC
phase, ET-1 increased from 5.9?1.3 at baseline to 6.4?1.7
(P?0.049), 6.5?1.4 (P?0.011), 6.9?1.5 (P?0.0001), 7.2?1.5
(P?0.0001), and 7.3?1.5 pg/mL (P?0.0001) at 30, 60, 90, 120,
and 180 minutes after the glucose load, respectively. In contrast,
DC ingestion prevented the increase in ET-1 (P?0.0023 for
treatment) induced by the glucose load (Figure 3B).
Total Isoprostanes
Baseline concentrations of serum 8-iso-PGF2?were higher
after the WC compared with the DC intervention phase
(233.9?34.2 versus 208.1?22.8 pg/L; P?0.04). After the
WC phase, 8-iso-PGF2?increased from 233.9?34.2 at base-
line to 269.6?42.7 (P?0.047), 285.3?46.0 (P?0.0139) at
30 and 60 minutes after the glucose load, respectively. In
contrast, DC ingestion prevented the increase of this bio-
marker of lipid peroxidation (P?0.0008 for treatment) in-
duced by the OGTT.
Insulin Resistance and ?-Cell Function
Although a trend toward improvement was noted after DC
but not WC ingestion, no significant differences were ob-
served regarding glucose and insulin responses during the
OGTT, homeostasis model assessment of insulin resistance,
quantitative insulin sensitivity check index, and homeostatic
model assessment %? (data not shown).
*
10
8
6
Flow-mediated dilation (%)
After white After dark
chocolate chocolate
*
*
Baseline 60’ 120’ 180’
Time
after glucose load and 3d WC
after glucose load and 3d DC
baseline
A B
Flow-mediated dilation (%)
0
-5
-10
-15
-20
-25
Figure 1. Effect of 3 days of white choco-
late (WC) and dark chocolate (DC) admin-
istration on baseline flow-mediated dila-
tion (FMD) (A) and during OGTT (B). Data
are presented as mean? SD in (A) and as
% difference respect to baseline levels in
(B). Vertical lines indicate SD and aster-
isks (*) indicate significant differences with
respect to the WC phase (A) and baseline
(square) values (B). Differences are con-
sidered significant when P?0.05.
0
2
4
6
8
10
12
14
16
18
20
2
4
6
8
10
AB
*
Baseline 30’ 60’ 90’ 120’ 180’
Time
after glucose load and 3d WC
after glucose load and 3d DC
baseline
*
*
*
*
*
Stiffness Index (m/s)
After white After dark
chocolate chocolate
Stiffness Index (%)
Figure 2. Effect of 3 days of white choco-
late (WC) and dark chocolate (DC) admin-
istration on baseline SI (A) and after
OGTT (B). Data are presented as
mean?standard deviation (SD) in (A) and
as % difference with respect to baseline
levels in (B). Vertical lines indicate SD,
and asterisks (*) indicate significant differ-
ences with respect to the WC phase (A)
and baseline (square) values (B). Differ-
ences are considered significant when
P?0.05.
Grassi et alArterial Protection by Dark Chocolate
3
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Discussion
Our results show that, in healthy subjects, short-term treat-
ment with flavanol-rich DC, compared with a dietary control
of flavanol-free WC, was able to increase endothelium-
dependent vasodilation and reduce wave reflections, lipid
peroxidation, and ET-1 levels, also protecting from acute
vascular alterations induced by a glucose load. Although we
did not examine the mechanism(s) of action underlying this
effect of DC, we might postulate that the cocoa flavanols in
DC blunted the production or action of reactive oxygen
species (ROS) induced by the transient hyperglycemia and
subsequently decreased bioavailability of NO. ROS rapidly
combines with NO to decrease its bioavailability and impair
normal endothelial function by disrupting the balance be-
tween vasodilating and vasoconstricting factors.6,12,15Fur-
ther, the measurement of 8-iso-PGF2?, formed nonenzymati-
cally through free radical catalyzed attack on esterified
arachidonate, provides a reliable tool for identifying populations
with enhanced rates of lipid peroxidation. Indeed, enhanced forma-
tion of 8-iso-PGF2?has been reported in association with several
cardiovascular risk factors. Accordingly, it has been suggested that
8-iso-PGF2?may transduce oxidant stress-dependent platelet acti-
vation and endothelial dysfunction.21Data suggest the relationship
between flavonoids and other dietary antioxidants, oxidative stress,
and hyperglycemia may result from reduced NO degradation by
ROS,6,9,15,22increased endothelial NO synthase activity,15,20,23
and/or decreased ET-1.15,24,25
Impaired endothelium-dependent vasodilation and, by exten-
sion, decreased bioavailability of NO has been demonstrated in
animalmodelsofdiabetesmellitusandinpatientswithbothtype
1 and type 2 diabetes.6,26Augmented generation of ROS as a
cause of this endothelial dysfunction has been implicated by
studies in healthy humans6,9and in patients with type 1 and type
2 diabetes in whom infusions of the antioxidant vitamin C
restored endothelium-dependent vasodilation toward nor-
mal.9,27,28One feature common to the in vitro, animal, and
human studies of diabetes is elevated glucose status. Indeed,
hyperglycemia per se has been observed to negatively affect
endothelium-dependent vasodilation in human arteries,10in-
crease pulse wave reflections, and decrease aortic distensibility
in healthy male volunteers.29Modest increases in postprandial
glucose can acutely lower FMD in healthy subjects as well as
those with impaired glucose tolerance and T2DM, though the
latter groups show greater reductions.7,8Consistent with our
results, the inverse association between FMD and plasma glu-
cose found in these studies suggests a continuous relationship
between increasing glucose concentrations and decreasing en-
dothelial function.
In vitro studies indicate that the exposure of endothelial
cells to elevated glucose concentrations leads to the genera-
tion of ROS, including superoxide anion, which can inacti-
vate NO or otherwise contribute to endothelial cell injury.30
Similarly, patients with T2DM have increased concentrations
of biomarkers of lipid peroxidation and reduced levels of
endogenous antioxidants compared with healthy controls.9It
has also been shown that an acute oral glucose load can
increase biomarkers of oxidative stress in healthy subjects.8
Concordantly, a recent study31reported that oxidative stress
may be an early event in a metabolic cascade elicited by a
high-glycemic index diet, ultimately increasing the risk for
cardiovascular disease and diabetes. Our results suggest that
hyperglycemia induced by an oral glucose load may increase
oxidative stress, produce a transient loss of NO bioavailabil-
ity, and an increase in ET-1, with a consequent impairment in
vascular tone. Importantly, these data also suggest that cocoa
flavanols may interfere with this chain of events.
This positive effect by cocoa flavanols might have impor-
tant implications, as data on endothelial dysfunction obtained
from coronary and upper arm circulation predicts coronary
and cerebrovascular events.12Endothelium-derived NO and
ET-1 also appear to play an important role in the regulation of
arterial stiffness and wave reflections.12–14Arterial stiffness
and wave reflections have also been shown to have an indepen-
dent predictive value for cardiovascular morbidity and all-cause
mortality and coronary events and strokes in patients with
uncomplicated essential hypertension.32Flavanols and related
polyphenolic antioxidants may protect against hyperglycemia by
decreasing the formation of ROS and thereby increasing the
0
2
4
6
8
10
0
5
10
15
20
25
*
*
*
A
B
after glucose load and 3d WC
after glucose load and 3d DC
baseline
Baseline 30’ 60’ 90’ 120’ 180’
Time
*
Endothelin-1 (pg/mL)
Endothelin-1 (%)
After white After dark
chocolate chocolate
Figure 3. Effect of 3 days of white choc-
olate (WC) and dark chocolate (DC)
administration on baseline ET-1 levels
(A) and during OGTT (B). Data are pre-
sented as mean?standard deviation (SD)
in (A) and as % difference with respect
to baseline levels in (B). Vertical lines
indicate SD, and asterisks (*) indicate
significant differences with respect to the
WC phase (A) and baseline (square) val-
ues (B). Differences are considered sig-
nificant when P?0.05.
4Hypertension
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bioavailability of NO. For example, Schroeter et al23reported
that flavanol-rich cocoa improved FMD in conduit arteries and
in microcirculation strongly correlated with the kinetics of
increased NO species and (?)-epicatechin and its metabolites in
plasma. They also found the actions of the cocoa flavanols were
closely mimicked by pure (?)-epicatechin and abolished by the
NO synthase inhibitor, L-NG-mono-methyl-arginine. Our data
are concordant with our previous work17–19and other human
studies showing a positive effect of flavanol-rich chocolate or
cocoa on FMD.15,20,33,34Similarly, Vlachopoulos et al35re-
portedthat100-g,flavonoid-richDCacutelyincreasedFMDand
decreased wave reflections, whereas it did not affect aortic
stiffness. In contrast with our results, plasma oxidant status did
not change after DC. Moreover, differently from our previous
findings,17–19no variation in BP was observed between the
active and control treatments. This incongruence might be
explained, considering that the current study design involved a
small number of subjects (according to our primary outcome:
FMD) and the very short intervention phases (acute administra-
tion). Indeed, concordantly with all our findings, a very recent
meta-analysis reported that, although FMD was improved after
chronic and acute flavonoid-rich DC intakes, significant reduc-
tions in DBP and mean arterial pressure were observed after
chronic but not after acute intake of DC.36
Inherent limitations of our study design include the small
number of subjects, short duration, and the inability to blind
the participants to the test and control chocolate interventions.
Our isocaloric protocol can also be considered a limitation in
that it does not necessarily reflect usual patterns of chocolate
consumption. Indeed, in the current study, we provided ?25%
of total daily calories by chocolate. Nevertheless, chocolate
perfectly respects the daily recommended balance of macronu-
trient intake (protein: 10% to 35%; fat: 20% to 35%; carbohy-
drates: 45% to 65%), without negatively affecting the caloric
quality of nutrients. Further, considering our previous findings,
we aimed to test effects from commercial chocolate while
preserving high flavonoids intake. Starting from this, future
long-term studies should test and confirm the beneficial effects
of DC at lower doses with reduced caloric burden but preserving
the original high content of flavonoids, particularly, including
DC in the usual dietary patterns of the population.
Our choice of interventions, however, did permit us to
compare flavonoid-rich versus flavonoid-free chocolate and,
simultaneously, cocoa-rich versus cocoa-free chocolate. This
is of interest, as additional mechanisms of action of cocoa
flavanol appear to exist (eg, via modulation of the renin-
angiotensin system),37and other cocoa constituents such as
theobromine and caffeine may also contribute partly to the
observed changes in FMD and wave reflections. One study of
theobromine from dark chocolate, however, found no hemo-
dynamic or electrophysiological effects in young adults38;
another found the caffeine and theobromine content of flavanol-
poor chocolate were similar to that of flavanol-rich chocolate,
although only dark chocolate affected vascular function and NO
bioavailability,39and van den Bogaard et al40reported natural-
dose theobromine cocoa did not significantly change either
24-hour ambulatory or central SBP compared with placebo.
Perspectives
We observed for the first time that consumption of flavanol- and
cocoa-rich DC increases FMD together with a reduction of wave
reflections and ET-1. The DC intervention attenuated the endo-
thelial dysfunction, oxidative stress, and wave reflections in-
duced by a glucose load in healthy subjects with normal glucose
tolerance. Although our study design was robust, it was also
short-term, involved a small number of subjects, and employed
an isocaloric protocol. Importantly, caution is always warranted
beforerecommendingtheadditionofanycalorie-richfoodtothe
diet. Our results suggest that postprandial hyperglycemia may
contribute substantially to atherosclerotic disease and that cocoa
and flavanols might reduce the impact of this relationship. As
worldwide cocoa and chocolate are daily consumed by the
general population, our observations may have an impact on
human health with important clinical consequences.
Acknowledgments
We kindly thank Franco Ruta, Chairman, Antica Dolceria Bonajuto
of Modica, for the donation of the dark chocolate bars.
Disclosures
None.
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Novelty and Significance
What Is New?
● Flavonoid-rich dark chocolate can positively affect arterial function,
preventing the vascular alterations induced by a glucose load in healthy
subjects.
What Is Relevant?
● Prolonged and repeated postprandial hyperglycemia may contribute
substantially to atherosclerotic disease, and cocoa and flavanols may
reduce the impact of this relationship.
Summary
Consumption of flavanol- and cocoa-rich dark chocolate may
improve arterial function and offer protection from vascular
damage induced by a glucose load in healthy subjects. As
worldwide cocoa and chocolate are daily consumed by the general
population, our observations may have an impact on human
health, with important clinical consequences.
6Hypertension
September 2012
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ONLINE SUPPLEMENT


PROTECTIVE EFFECTS OF FLAVANOL-RICH DARK CHOCOLATE ON
ENDOTHELIAL-FUNCTION AND WAVE REFLECTION DURING ACUTE
HYPERGLYCEMIA




Davide Grassi1,4, MD, PhD, Giovambattista Desideri1, MD, Stefano Necozione1, MD, Fabrizio
Ruggieri1, BSc, Jeffrey B. Blumberg2, PhD, Michele Stornello3, MD and Claudio Ferri1,MD

Short title: Arterial protection by dark chocolate

1Department of Internal Medicine and Public Health, University of L’Aquila, L’Aquila, Italy (DG,
GD, SN, FR, MS, CF); 2Antioxidants Research Laboratory, Jean Mayer USDA Human Nutrition
Research Center on Aging, Tufts University, Boston, MA USA (JBB). 3Centre for the Hypertension
Study and Therapy, General Hospital Umberto I, Syracusae, Italy.




4 Corresponding author: Davide Grassi, MD, University of L’Aquila Department of Internal
Medicine and Public Health Viale S. Salvatore, Delta 6 Medicina, 67100 Coppito, L’Aquila , Italy
Phone: +39 0862 434749; fax: +39 0862 434749; e-mail: davide.grassi@cc.univaq.it
Total Word count : 5568


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EXPANDED?METHODS?SECTION?


Endothelial function. In a fasting state, FMD of the brachial artery was measured before and
after 1, 2, and 3 h of the glucose load during each scheduled visit. FMD was always determined by
the same physician, who was blinded to the study design and objectives, according to the guidelines
of the American College of Cardiology (1). 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 (VIVIDe, General Electric) as described previously (2). The transducer was held at the
same point throughout the scan by a stereotactic clamp. A cuff was placed around the forearm just
below the elbow. After a 1 min acquisition to measure basal diameter, the cuff was inflated for 5
min at 250 mmHg, and then deflated to induce reactive hyperemia. Measurements were obtained by
a system for real-time evaluation of the brachial artery diameter with automatic mathematical
contour tracking operator locating and tracking the edges (3, 4). Endothelium-dependent
vasodilation was considered the maximal dilation of the brachial artery induced by increased flow
(1, 2). Reproducibility in repeated measures with the standard technique presented coefficients of
variation of 23% in previous measurements with the old technique (3, 4).

Wave reflection. Before and after 30, 60, 90, 120, and 180 min of the glucose load, the RI
(height of the second peak/height of the first peak in the digital volume pulse [DVP]), SI (subject
height/time delay between direct and reflected waves in the DVP), and the PPT (time delay between
the SBP and DBP peaks or, in the absence of a second peak, the point of inflection) were evaluated
by a validated finger photoplethysmographic device (Micro Medical, Gillingham, Kent, UK) (5).

Blood pressure measurements. During each visit, BP was measured before and after 30, 60,
90, 120, and 180 min of the glucose load with volunteers in a supine position in a comfortable room.
BP was assessed in quadruplicate at 2 min intervals with an oscillometric device (Omron 705 CP,
Omron). 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.

Biomarkers of endothelial damage and oxidative stress. Before and after 30, 60, 90, 120,
and 180 min of the glucose load in each study phase, serum was also collected from all subjects for
the determination of ET-1 and total 8-iso-PGF2α by commercially available enzyme-linked
immunoassay kits according to manufacturer instructions (R & D Systems Inc. and Assay Design
Inc., Ann Arbor, MI). Serum aliquots were stored at -80ºC in polypropylene tubes immediately after
centrifugation (15 min at 4ºC at 3000 rpm) of blood samples and subsequently used to assess
circulating ET-1 and total 8-iso-PGF2α.

Assessment of insulin sensitivity and β-cell function. An OGTT was performed in each
subject after both intervention phases after a 10-14 h overnight fast and 12 h from the last
chocolate ingestion. Plasma glucose and insulin were assessed at baseline and then 30, 60, 90, 120,
and 180 min after the glucose load. OGTT results were utilized for the HOMA-IR (6, 7), QUICKI
(6), and ISI as described by Matsuda and DeFronzo (6) and the index of insulin secretion HOMA
derived β-cell function (HOMA-%β) (7).

Statistical analysis. Data are expressed as mean ± standard deviation (SD). Baseline
differences between the two intervention phase groups were analyzed by paired t test. Data during
the OGTT were analyzed using Proc Mixed Procedure with subjects treated as a random factor and
treatment and sequence as fixed factors. LSMEANS was used for comparing multiple group means
with Tukey’s honestly significant difference (HSD) test. Statistical analyses were conducted on
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changes from the respective baseline values and not on direct comparison of absolute values on WC
and DC during the oral glucose tolerance test. Therefore, results during OGTT were statistically
adjusted for respective baseline values. Statistical power was based on results obtained in similar
studies with healthy individuals who consumed flavonoid-rich DC and WC bars (3). In that study
the mean FMD increased by 1.9% at a SD of 1.3% after cocoa polyphenol consumption. On the
basis of these data the same improvement in FMD could be detected in 12 individuals with a high
power (0.9 at alpha = 0.05). Differences were considered significant when p <0.05. Statistical
analyses and power calculation were performed with SAS (version 9.1.3, 2004; SAS Institute Inc.,
Cary, NC).

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Table S1. Energy , nutrient and specific component intake with flavanol-rich dark chocolate (DC)
(100 g) and flavanol-free white chocolate (WC) (100 g).
Parameter
Energy (Kcal)
Cocoa (%)
Carbohydrates (g)
Protein (g)
Total fat (g)
Sodium (mg)
Potassium (mg)
Magnesium (mg)
Catechin (mg)
Epicatechin (mg)
Quercetin (mg)
Theobromine (g)
Caffeine (mg)
*BLD, below the limit of detection


Table S2. General characteristics of the study population (mean ±SD)
Characteristic
Number of subjects
Age
BMI (Kg/m2)
Body Weight (Kg)
Waist circumference (cm)
LDL-cholesterol (mg/dl)
HDL-cholesterol (mg/dl)
Triglycerides (mg/dl)
Plasma glucose (mg/dl)
Plasma insulin (µU/ml)


?

















DC
510
80
58.6
10.4
26
17
240
7.8
59
447
14
1.2
108.8
WC
536
0
63.2
4.6
29.6
64.3
287
25
0.04
BLD*
BLD
BLD
BLD
Value
12/ 5M
28.2±2.7 y
23.2±4.2
64.5±10.7
82.7±5.8
97.8±16.7
57.1±13.8
59.3±24.3
74.7±7.7
7.5±4.5
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?
*
*
*
*
*
A) B)
*
*
*
*
*
*
Figure S1. Effect of 3 d WC (white) and DC (black) administration after OGTT on reflection index (RI) (Panel A) and peak to peak
time (PPT) (Panel B) values. Data are presented as % difference respect to baseline levels. Asterisks (*) indicate significant
differences with respect the baseline (square) values. Differences are considered significant when p <0.05.
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