Letters Section Editor: Stephen J. Lurie,
MD, PhD, Senior Editor.
To the Editor: In vitro studies have suggested
that flavonoids may have specific vascular effects, but their mechanism of
action has not been clarified.1 A subclass
of flavonoids—flavan-3-ols and their oligomers (procyanidins)—are
constituents of cocoa beans, which can be detected in human plasma after ingestion
of cocoa.2 In turn, plant extracts rich
in flavan-3-ols can increase the activity of nitric oxide synthase (NOS) in
endothelial cells.3 Nitric oxide is an essential
signaling molecule in vascular physiology. Nitric oxide bioactivity can be
preserved in human plasma in a circulating pool via increases in a number
of nitrosated compounds.4,5 Thus,
it is possible that cocoa rich in flavan-3-ols may lead to improved endothelium-dependent
dilation via an increase of nitric oxide bioactivity.
However, commercially available cocoa drinks contain only small amounts
of flavan-3-ols due to roasting and alkalization of cocoa beans, which are
known to degrade flavan-3-ols. We tested the hypothesis that ingestion of
flavan-3-ol rich cocoa can increase the circulating pool of nitric oxide in
human plasma, thus increasing endothelium-dependent dilation.
Participants were 26 outpatients with at least 1 cardiovascular risk
factor, including history of coronary artery disease, hypertension, hyperlipidemia,
diabetes, or current tobacco use. Individuals were excluded if they had C-reactive
protein levels greater than 0.5 mg/dL, atrial fibrillation, acute coronary
syndrome, or New York Heart Association class III or IV heart failure. Individuals
were studied in the morning after a 12-hour fasting period.
In an initial study involving the first 6 participants, we assessed
the time course of flavan-3-ol effects on flow-mediated dilation (FMD). This
was measured at 0, 2, 4, and 6 hours after ingestion of 100 mL of cocoa drink
containing 176 mg of flavan-3-ols (70 mg of epicatechin plus catechin, 106
mg of procyanidins [The Positive Food Co, Wokingham, England]) (n = 6) or
control (100 mL cocoa drink with <10 mg of flavan-3-ols [Dovedrink, Mars
Inc, Hackettstown, NJ] or water) (n = 3).
We then used these results to guide the timing of a double-blind crossover
study. Twenty participants received 100 mL of cocoa drinks with high or low
levels of flavan-3-ols, in random order, on 2 consecutive days. The sum of
nitrosylated and nitrosated species (collectively referred to as RNO) was
measured by reductive chemiluminescence assay 2 hours after ingestion on both
days.4 Nitrate and nitrite levels were measured
as previously described.6 Endothelium-dependent
dilation was assessed by measuring FMD of the brachial artery. In addition,
we measured a number of other vascular parameters that would not be expected
to change as a result of flavan-3-ol, including blood pressure, heart rate,
and plasma levels of nitrite and nitrate. Similarly, we measured endothelium-independent
dilation of the brachial artery following sublingual application of 400 µg
of glyceroltrinitrate, diameter of the brachial artery, and forearm blood-flow
at rest and during reactive hyperemia, as assessed by venous occlusion plethysmography.
(Technical details are available from the authors.)
All variables except endothelium-independent dilation were measured
both before and after ingestion of the cocoa. Endothelium-independent dilation
was measured only after ingestion of each drink, as nitroglycerine could have
interfered with measurement of the other variables. Differences were assessed
by paired t tests, with P values
for multiple comparisons adjusted by the Bonferroni criterion. Our study was
approved by the ethics board of the Medical Faculty of the Heinrich Heine-University,
and all participants gave written informed consent.
The sample had a mean (SD) age of 41 (14) years and body mass index
(calculated as weight in kilograms divided by the square of height in meters)
of 25 (4). Sixty-two percent were male, 19% had coronary heart disease, 27%
had arterial hypertension, 31% had hyperlipidemia, 8% had diabetes mellitus,
and 77% were smokers.
In the initial study (Figure 1),
ingestion of 100 mL of cocoa drink rich in flavan-3-ols increased FMD maximally
at 2 hours, whereas the cocoa drink low in flavan-3-ols did not affect this
parameter. There was no significant difference in FMD in the 3 individuals
receiving either water or the cocoa drink low in flavan-3-ols.
In the crossover study (Figure 2),
ingestion of cocoa rich in flavan-3-ols increased plasma levels of RNO from
22 to 36 nmol/L and FMD from 3.4% to 6.3% (P<.001
for both comparisons). Changes in plasma levels of RNO and in FMD were correlated
(r = 0.42; P = .02). As
expected, there were no significant differences in other vascular measures
before vs after ingestion of the cocoa drink rich in flavan-3-ol (diameter
of the brachial artery, 4.54 vs 4.53 mm; forearm blood-flow at rest and during
reactive hyperemia, 1.8 vs 2.1 and 11.4 vs 11.6 mL/min per 100 mL of tissue,
respectively; mean blood pressure, 91 vs 90 mm Hg; heart rate, 66 vs 62 bpm;
levels of plasma nitrite [128 vs 119 nmol/L] and nitrate [32 vs 28.5 µmol/L]).
Flow-mediated dilation, levels of RNO, and all other vascular variables were
unchanged before and 2 hours after ingestion of cocoa drink low in flavan-3-ols.
Endothelium-independent dilation of the brachial artery was not significantly
different after ingestion of either cocoa drink (12.3% vs 12.9%; P = .82).
We found that a single dose of a cocoa drink rich in flavan-3-ols transiently
increased nitric oxide bioactivity in human plasma and significantly reversed
endothelial dysfunction. The correlation between FMD and levels of RNO suggests
that flavan-3-ols induce arterial dilation via their effects on nitric oxide
availability, a conclusion that is supported by the negative results for the
other vascular variables. The long-term clinical effect of flavan-3-ols, however,
remains to be established.
Funding/Support: This work was supported by
grants from the Deutsche Forschungsgemeinschaft (SFB 575 and 612), Biomedizinisches
Forschungszentrum of the University of Düsseldorf, and Mars, Inc (Slough,
England). Dr Sies is a Fellow of the National Foundation for Cancer Research.
Acknowledgment: We gratefully acknowledge the
technical assistance of S. Matern and G. Doemer. We thank Drs J. Wills, H.
Schmitz, and J. Hammerstone, of Mars Inc, for supplying and analyzing the
Heiss C, Dejam A, Kleinbongard P, Schewe T, Sies H, Kelm M. Vascular Effects of Cocoa Rich in Flavan-3-ols. JAMA. 2003;290(8):1030-1031. doi:10.1001/jama.290.8.1030