Procyanidin dimer B2 [epicatechin-(4β-8)-epicatechin] in human plasma after the consumption of a flavanol-rich cocoa–

Abstract

Epidemiologic studies have linked flavonoid-rich foods with a reduced risk of cardiovascular mortality. Some cocoas are flavonoid-rich and contain the monomeric flavanols (-)-epicatechin and (+)-catechin and oligomeric procyanidins formed from these monomeric units. Both the monomers and the oligomers have shown potential in favorably influencing cardiovascular health in in vitro and preliminary clinical studies. Although previous investigations have shown increasing concentrations of (-)-epicatechin in human plasma after cocoa consumption, no information is available in the published literature regarding the presence of procyanidins in human plasma.
This study sought to determine whether procyanidins can be detected and quantified in human plasma after acute consumption of a flavanol-rich cocoa.
Peripheral blood was obtained from 5 healthy adult subjects before (baseline, 0 h) and 0.5, 2, and 6 h after consumption of 0.375 g cocoa/kg body wt as a beverage. Plasma samples were analyzed for monomers and procyanidins with the use of reversed-phase HPLC with coulometric electrochemical array detection and liquid chromatography-tandem mass spectrometry.
Procyanidin dimer, (-)-epicatechin, and (+)-catechin were detected in the plasma of human subjects as early as 0.5 h (16 +/- 5 nmol/L, 2.61 +/- 0.46 micro mol/L, and 0.13 +/- 0.03 micro mol/L, respectively) after acute cocoa consumption and reached maximal concentrations by 2 h (41 +/- 4 nmol/L, 5.92 +/- 0.60 micro mol/L, and 0.16 +/- 0.03 micro mol/L, respectively).
Dimeric procyanidins can be detected in human plasma as early as 30 min after the consumption of a flavanol-rich food such as cocoa.

Full text

798 Am J Clin Nutr 2002;76:798–804. Printed in USA. © 2002 American Society for Clinical Nutrition
Procyanidin dimer B2 [epicatechin-(4-8)-epicatechin] in human
plasma after the consumption of a flavanol-rich cocoa
1–3
Roberta R Holt, Sheryl A Lazarus, M Cameron Sullards, Qin Yan Zhu, Derek D Schramm, John F Hammerstone,
Cesar G Fraga, Harold H Schmitz, and Carl L Keen
ABSTRACT
Background: Epidemiologic studies have linked flavonoid-rich
foods with a reduced risk of cardiovascular mortality. Some
cocoas are flavonoid-rich and contain the monomeric flavanols
()-epicatechin and (+)-catechin and oligomeric procyanidins
formed from these monomeric units. Both the monomers and the
oligomers have shown potential in favorably influencing cardio-
vascular health in in vitro and preliminary clinical studies.
Although previous investigations have shown increasing concen-
trations of ()-epicatechin in human plasma after cocoa con-
sumption, no information is available in the published literature
regarding the presence of procyanidins in human plasma.
Objective: This study sought to determine whether procyanidins
can be detected and quantified in human plasma after acute con-
sumption of a flavanol-rich cocoa.
Design: Peripheral blood was obtained from 5 healthy adult sub-
jects before (baseline, 0 h) and 0.5, 2, and 6 h after consumption
of 0.375 g cocoa/kg body wt as a beverage. Plasma samples were
analyzed for monomers and procyanidins with the use of reversed-
phase HPLC with coulometric electrochemical array detection and
liquid chromatography–tandem mass spectrometry.
Results: Procyanidin dimer, ( )-epicatechin, and (+)-catechin
were detected in the plasma of human subjects as early as 0.5 h
(16 ± 5 nmol/L, 2.61 ± 0.46 mol/L, and 0.13 ± 0.03 mol/L,
respectively) after acute cocoa consumption and reached maximal
concentrations by 2 h (41 ± 4 nmol/L, 5.92 ± 0.60 mol/L, and
0.16 ± 0.03 mol/L, respectively).
Conclusion: Dimeric procyanidins can be detected in human
plasma as early as 30 min after the consumption of a flavanol-rich
food such as cocoa. Am J Clin Nutr 2002;76:798–804.
KEY WORDS Cocoa, chocolate, catechin, epicatechin,
procyanidin, flavonoid, reversed-phase HPLC, coulometric
electrochemical array detection
INTRODUCTION
A diet rich in fruit, nuts, and vegetables has been associated
with a reduced risk of cardiovascular disease (1, 2). In addition to
vitamins C, E, and the carotenoids, fruit, nuts, and vegetables con-
tain a complex array of phenolic compounds that may contribute
to increases in the plasma antioxidant capacity (3–5). Although
the extent to which an enhanced plasma antioxidant capacity may
contribute to improved health is a subject of debate, most investi-
gators view increases in oxidant defense as positive. Flavonoids
1
From the Departments of Nutrition (RRH, QYZ, DDS, and CLK) and
Internal Medicine (CLK), University of California, Davis; Analytic & Applied
Sciences, Mars Incorporated, Hackettstown, NJ (SAL, JFH, and HHS); the
Department of Biochemistry, Emory University, Atlanta (MCS); and Physical
Chemistry-PRALIB, School of Pharmacy and Biochemistry, University of
Buenos Aires (CGF).
2
Supported by grants from the National Institutes of Health (DK-35747)
and Mars Incorporated.
3
Address reprint requests to CL Keen, Department of Nutrition, University
of California, Davis, One Shields Avenue, Davis, CA 95616-8669. E-mail:
clkeen@ucdavis.edu.
Received April 13, 2001.
Accepted for publication October 18, 2001.
represent one class of phenolic compounds that has attracted con-
siderable attention. Epidemiologic studies suggest that the con-
sumption of a flavonoid-rich diet reduces an individual’s risk of
cardiovascular morbidity and mortality (6–8). Cocoa can be a rich
source of flavonoids and was historically used as a medicine to
combat inflammation, pain, and numerous other ailments (9). Like
wine and tea, cocoa flavanols consist of a complex mixture of the
monomeric (+)-catechin (catechin) and ()-epicatechin (epicate-
chin) and the oligomers of these monomeric base units known as
procyanidins (10).
In in vitro systems, extracts of cocoa flavanols and their
oligomers have the ability to inhibit LDL oxidation (11), prevent
peroxynitrite-dependent oxidation and nitration (12, 13), promote
endothelium-dependent relaxation (14), and modulate the pro-
duction of inflammatory cytokines (15, 16). The in vitro effects
observed with the monomers are often significantly different from
those observed with the oligomers, suggesting that there may be
considerable structure specificity for these compounds. Although
the potent in vitro effects of the flavanols and their oligomers have
generated considerable interest in these compounds, it has been
suggested that their in vivo effects may be minimal because of
gastric degradation (17). Thus, it is important to determine
whether the oligomers can be absorbed from the diet.
In a recent study (18) we used a sensitive and selective method
to measure plasma epicatechin concentrations after acute cocoa
consumption. In the current study we modified this method to
enable us to simultaneously measure epicatechin, catechin and
the procyanidin dimer B2 [epicatechin-(4-8)-epicatechin] in
human plasma after the acute consumption of a flavanol-rich
cocoa. Furthermore, the chemical identity of these compounds
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EPICATECHIN, CATECHIN, AND DIMERS IN PLASMA 799
was assessed by liquid chromatography–tandem mass spectrom-
etry (LC-MS/MS).
SUBJECTS AND METHODS
Subjects and clinical study design
Five nonsmoking adult volunteers (3 men and 2 women aged
23–34 y with an average body weight of 70.5 ± 4.6 kg) with no
apparent disease participated in the study. The health status of all
participants was evaluated via questionnaire, and written,
informed consent was given before participation in the study. The
study protocol was conducted, and approved, according to guide-
lines set by the Human Subjects Review Committee of the Uni-
versity of California, Davis.
Subjects were asked to abstain from alcohol, analgesics, and
flavonoid-rich foods (eg, fruit, vegetables, nuts, coffee, tea, juice,
and chocolate) on the day before the experiment. Baseline sam-
ples were drawn after a 12-h evening fast. The subjects then con-
sumed 0.375 g cocoa (Cocoapro; Mars Inc, Hackettstown, NJ)/kg
body wt in 300 mL water. One gram of the cocoa provided
12.2 mg monomers, 9.7 mg dimers, and 28.2 mg procyanidins
(trimers through decamers) (3). On average, the subjects ingested
a total dose of 26.4 g cocoa, providing 323 mg monomers and
256 mg dimers. In addition to the cocoa beverage, the subjects
were allowed to consume white bread.
Blood samples were drawn via venipuncture at baseline (0 h)
and 0.5, 2, and 6 h after cocoa consumption. Blood was collected
into two 10-mL Vacutainer tubes (Becton Dickinson, Franklin
Lakes, NJ) containing sodium heparin as an anticoagulant. Blood
was centrifuged at 1500  g at 4 C for 10 min, and the plasma
was separated and frozen at 80 C until analyzed.
Determination of plasma catechin, epicatechin, and
procyanidin dimers
Chemicals were purchased from Sigma Chemical Co (St Louis)
unless otherwise stated. Plasma samples were extracted as
described by Richelle et al (19) and Rein et al (18). The resulting
solution was filtered with a 0.22-m Ultrafree-MC low-binding
Durapore centrifugal filter (Millipore, Bedford, MA) and cen-
trifuged (10 000  g, 5 min, 4 C); 50 L of the filtered solution
was analyzed for catechin, epicatechin, and procyanidin dimers by
reversed-phase HPLC with coulometric multiple-array detection.
Chromatography was carried out by using an HP 1100 HPLC
system with Chemstation software, equipped with a quaternary
pump, temperature-controlled autosampler, column oven, and
diode array detector (Hewlett-Packard, Wilmington, DE) in series
with a CoulArray 5600 detector (ESA, Chelmsford, MA). Separa-
tion was achieved by using a reversed-phase Alltima C
18
column
(5 m, 150 mm  4.6 mm; Alltech Associates, Deerfield, IL) with
a C
18
5-m guard column (Alltech Associates) . The mobile phase
was mixed with 2 solvents: solvent A [40% methanol, 60%
100 mmol sodium acetate/L in water (EM Sciences, Darmstadt,
Germany), pH 5.0] and solvent B (7% methanol, 93% 100 mmol
sodium acetate/L in water, pH 5.0). A gradient elution was used at
a flow rate of 1 mL/min with the initial concentration of solvent A
set at 20%, which was held until 2 min. This was followed by a lin-
ear increase to 40% solvent A by 10 min, immediately followed
with another linear increase to 85% solvent A by 13 min. The sys-
tem was held at 85% of solvent A until 17 min, at which time a lin-
ear increase to 100% solvent A was achieved by 20 min. The system
was then linearly decreased to 40% solvent A by 23 min, followed
by another linear decrease to 20% solvent A by 25 min.
Coulometric electrochemical array detection was carried out by
using the following cell settings: 50, 65, 150, 200, 250, 300, 700,
and 800 mV. The resulting chromatographs were analyzed by using
CoulArray for WINDOWS software (ESA, Chelmsford, MA). Iden-
tification of epicatechin and catechin at 150 mV was based on
coelution with authentic standards and quantified by using external
standards. Identification of the procyanidin dimer peak at 700 mV
was based on coelution with authentic standards and quantified by
using external standards extracted from cocoa (Cocoapro) (3, 20).
Confirmation of procyanidin dimers with liquid
chromatography–tandem mass spectrometry
Experiments were performed on a Sciex API 3000 triple
quadropole mass spectrometer (Perkin-Elmer, Norwalk, CT) equipped
with a turbo ionspray source. All experiments were performed in the
negative ion mode. The ionspray needle was held at 4500 V while
the inlet voltage (orifice) was kept at 65 V to minimize collisional
decomposition of molecular ions before entry into the first quadropole.
Molecular ions of dimer were identified by simple MS analy-
sis of standard solutions. Product ion spectra of these species were
acquired by using Q1 to pass the molecular ion of interest. Nitro-
gen was used to collisionally activate precursor ion decomposi-
tion in the second quadropole, which was offset from the first
quadropole by 35 eV. Subsequently, formed product ions were
then detected by scanning the third quadropole.
Quantitative MS/MS data for procyanidin dimers was acquired by
multiple reaction monitoring experiments in which the first
quadropole was set to pass a specific precursor ion mass-to-charge
ratio (m/z) and the third quadropole was set to pass a structurally dis-
tinctive product ion m/z. The dimer transitions were 577.3/407.0,
577.3/289.0, and 577.3/125.0. Each transition was optimized with
regard to both ionization and collision energy to minimize collisional
decomposition of molecular ions before entry into the first quadropole
and maximize formation of structurally distinctive product ions. The
dwell time for multiple reaction monitoring transitions of each indi-
vidual molecular species was 30 ms when both monomer and dimer
were monitored and 60 ms when only the dimer was monitored.
Procyanidin dimer was confirmed in the plasma by using
reversed-phase LC-MS/MS. A 100-L aliquot of the plasma extract
in 0.25 mol perchloric acid/L in water was diluted to a final volume
of 200  L with water:methanol (90:10 by vol). A 50-L volume
was injected onto a 150  2.1 mm Discovery C
8
column (Supelco,
Bellefonte, PA) at a flow rate of 300 L/min. Solvent A was
water:methanol (90:10 by vol) and solvent B was 100% methanol.
The column eluent was directly infused into the ion source of the
mass spectrometer, which was operated at 500  C with a 6 L/min
nebulizing nitrogen gas flow. The elution protocol consisted of col-
umn preequilibration with 100% solvent A for 3 min followed by
sample injection, 7 min at 100% solvent A, a 1-min linear gradient
to 100% solvent B, and a 7-min wash with 100% solvent B.
Statistical analyses
Data were analyzed for differences by one-way analysis of
variance or Kruskal-Wallis one-way analysis of variance on
ranks (SIGMASTAT for WINDOWS, version 2.03; SPSS, Rich-
mond, CA). When appropriate, Tukey’s or Dunn’s all-pairwise-
comparison tests were used to identify differences between base-
line and results 0.5, 2, and 6 h after cocoa consumption.
Statistical significance was set at P < 0.05.
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800 HOLT ET AL
FIGURE 1. Coulometric multiple-array chromatograms. A: 10 nmol (on-column) catechin standard [peak 1, 12.6 min; molecular weight (MW) = 289]
and 10 nmol epicatechin standard (peak 2, 16.9 min; MW = 289). B: 12.5 nmol procyanidin dimer B2 standard [epicatechin-(4-8)-epicatechin; peak 3,
14.7 min; MW = 578] and 12.5 nmol dimer B5 standard [epicatechin-(4-6)-epicatechin; peak 4, 20.3 min; MW = 578]. C: an extracted plasma sample
from one subject 2 h after cocoa consumption. The HPLC conditions are as described in the text.
RESULTS
Analytic determination of epicatechin, catechin, and
procyanidin dimers
The sensitivity and selectivity of coulometric electrochemical
array detection allowed us to measure not only epicatechin but
also catechin and the procyanidin dimers B2 and B5. A multiple-
array chromatograph for standards of catechin and epicatechin
[peaks 1 (12.6 min) and 2 (16.9 min), respectively] is depicted in
Figure 1A. Two procyanidin dimer peaks were found in the stan-
dard that was purified from cocoa; we previously characterized
these 2 peaks by nuclear magnetic resonance (21) as dimers B2
and B5 [peak 3 (14.7 min) and peak 4 (20.3 min), respectively;
Figure 1B]. Both epicatechin and catechin exhibit peak oxidation
responses at 150 mV followed by a second peak oxidation
response at 700 mV. The sample preparation and chromatography
did not fully resolve desired analytes from other coeluting plasma
components. However, the detection system selectively detected
catechin and epicatechin at the lower (150 mV) potential. A sim-
ilar strategy was used with the procyanidin dimers; however, to
achieve greater sensitivity, 700 mV was chosen as the analytic
potential. By choosing these 2 analytic potentials we were able to
measure plasma epicatechin and catechin and to simultaneously
detect low concentrations of dimer. A typical chromatograph at
150 and 700 mV for a 2-h plasma sample is depicted in Figure 1C.
The coulometric detection response was linear for on-column
standard curves with the use of the purified dimer B2 extract
between 290 and 29 ng. When plasma was spiked with purified
extracts of dimer, the recovery of B2 dimer was 103%. As previ-
ously reported, the recovery for catechin and epicatechin was
between 70% and 90% (18). The extraction procedure did not
yield additional dimer from plasma spiked with epicatechin and
catechin. The relative SDs for unspiked samples of 7 consecutive
analyses of dimer B2, catechin, and epicatechin were 12%, 7%,
and 4%, respectively. The limits of detection for dimer B2 and
dimer B5 on-column were 290 and 723 pg, respectively. These
detection limits allow for the measurement of plasma concentra-
tions of dimer B2 and B5 as low as 10 and 25 nmol/L, respectively.
Mass spectral identification of procyanidin dimers in plasma
To enhance the MS signal response, chromatography was not
used to resolve procyanidin dimers B2 and B5 in the purified stan-
dard. The molecular ions of the procyanidin dimers were identi-
fied by simple MS analysis of a standard solution (m/z: 577).
Because of background chemical noise or other intrinsic interfer-
ences that may generate ions having the same molecular ion (m/z),
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EPICATECHIN, CATECHIN, AND DIMERS IN PLASMA 801
FIGURE 2. The structure of procyanidin dimer B2 [epicatechin-
(4-8)-epicatechin] with major fragmentations and the relative abundance
of product ion spectrum determined with tandem mass spectrometry. m/z,
mass-to-charge ratio; (M-H)

, molecular ion in negative mode.
FIGURE 3. Results of liquid chromatography–tandem mass spec-
trometry of the dimer standard, containing both dimer B2 and B5, spiked
into control plasma at 9.86 nmol/L (A) and dimers in an extracted plasma
sample (unspiked) from one subject 2 h after consumption of 0.375 g
cocoa/kg body wt (B). The data for both panels A and B represent the
mass–to–product ion pairs (577.3/407.0, 577.3/289.0, and 577.3/125.0)
produced from both dimer B2 and dimer B5. A plasma dimer B2 concen-
tration of 42.5 nmol/L was quantified in the sample represented in panel
B with the use of multiple-array coulometric detection.
we used MS/MS to further identify the ion (m/z: 577) as dimer.
MS/MS is used to fragment the ion of interest. Once fragmented,
the MS/MS product ion spectra of the dimer standard showed
3 structurally distinctive ion products of 125, 289, and 407 m/z
(Figure 2). It is important to note that when ionized, the 425-m/z
fragment represented in Figure 2 will lose a molecule of water,
producing the product ion of 407 m/z (Figure 2). The major prod-
uct ions that are produced from the dimer standard indicate that
fragmentation occurs at the linkage (m/z: 289) of the oligomer as
well as opening of the C rings (m/z: 125 and 407). The fragmen-
tation pattern of dimer B2 is represented in Figure 2. Although its
fragmentation pattern is not shown, the fragmentation dimer of B5
also involves simple opening of the C rings and linkage cleavage,
producing product ions similar to dimer B2.
These data were used to determine the transitions used in the
multiple reaction monitoring experiments to identify procyanidin
dimers in the plasma. Thus, the dimers are detected by pairing a
specific precursor ion (m/z: 577.3) with a structurally distinct
product ion (either m/z 407.0, 289.0, or 125.0). The LC-MS/MS
results of the purified procyanidin dimer standard are shown in
Figure 3A, and the procyanidin dimers in the plasma extract of
one subject 2 h after consumption of the cocoa beverage are rep-
resented in Figure 3B.
Plasma monomer and dimer B2 concentrations
An unknown peak with a retention time of 14.7 min was
detected in the plasma extract 0.5, 2, and 6 h after consumption of
0.375 g cocoa/kg body wt. We later confirmed by coelution with
the purified dimer standards that this unknown peak in the plasma
extract corresponded with the first peak observed in the purified
dimer standard (retention time: 14.7 min). As stated above, we
characterized the dimer peak at this retention time as dimer B2.
Representative chromatographs at 700 mV from one sub-
ject are shown in Figure 4. These chromatographs show the
presence of the dimer B2 peak in the plasma at 0.5, 2, and
6 h. Dimer B5 (retention time: 20.3 min) was not detected in
the plasma by coulometric detection at any time point in any
of the subjects. The plasma concentrations of the dimer B2
were significantly (P < 0.05) elevated at 0.5, 2, and 6 h rel-
ative to baseline (16 ± 5, 41 ± 4, and 15 ± 2 nmol/L, respec-
tively) (Figure 5).
Thirty minutes after the subjects consumed 0.375 g cocoa/kg
body wt, plasma epicatechin and catechin concentrations
increased significantly (P < 0.05) above baseline (0.08 ± 0.46
compared with 2.61 ± 0.46 mol/L and 0.00 compared with
0.13 ± 0.03 mo/L, respectively). Plasma concentrations of
epicatechin and catechin continued to increase, with values
at 2 h being 5.92 ± 0.60 and 0.16 ± 0.03 mol/L, respec-
tively. Plasma concentrations of epicatechin and catechin
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802 HOLT ET AL
FIGURE 4. The detection of dimer B2 in one subject 0, 0.5, 2, and 6 h after consumption of 0.375 g cocoa/kg body wt. At baseline (0 h), dimer B2
was undetectable, increasing in concentration to 17.5 nmol/L at 0.5 h, to 51.5 nmol/L at 2 h, and to 21.5 nmol/L 6 h after cocoa consumption. The limit
of detection for dimer B2 was 10 nmol/L. The treatment of samples and HPLC conditions are as described in the text.
FIGURE 5. Mean (± SEM) plasma dimer B2, catechin, and epicate-
chin concentrations 0, 0.5, 2, and 6 h after consumption of 0.375 g cocoa/kg
body wt (n = 5).
*
Significantly different from baseline (0 h), P < 0.05.
were lower 6 h after cocoa consumption: 1.05 ± 0.01 and
0.02 ± 0.002 mol/L, respectively (Figure 5).
DISCUSSION
The flavanols and the related oligomers of this basic subunit,
known as the procyanidins, can be found in a variety of foods,
including apples, wine, tea, peanuts, almonds, and cocoa (3, 10).
Although many papers have determined the time course of
monomer absorption into plasma (19, 22–26), there is limited
information concerning the metabolism of the procyanidins. Some
studies have used radiolabeled techniques to indicate that extracts
containing procyanidins are bioavailable (27, 28). However, the
question remains whether the procyanidins are depolymerized or
remain intact before absorption. Deprez et al (29) reported that
procyanidins, with an average polymerization of 6, could be
degraded after 48 h incubation with human colonic microflora into
low-molecular-weight aromatic acids. As for the smaller
oligomers, it was reported that radiolabeled monomers, dimers,
and trimers can be transported across an in vitro cell layer of
Caco-2 cells, whereas the larger oligomers adhere to the cell sur-
face (30). Complementing the above, using procyanidins isolated
from cocoa, Spencer et al (31) perfused a 50-mol/L dimer solu-
tion for 90 min through the small intestine of rats. Small amounts
(< 1%) of the procyanidin dimers B2 and B5 were shown to pass
through the rat enterocytes, in vitro, with 95.8% of the total pass-
ing flavanols being unconjugated epicatechin. These data support
the concept that the dimers can be absorbed in vivo, albeit to a
lesser extent than the monomer subunits of the depolymerized
dimers. In the current study, we identified and quantified dimer
B2 using coulometric multiple-array detection in the plasma after
the subjects consumed a flavanol-rich cocoa beverage. We con-
firmed the presence of the procyanidin dimers in extracted plasma
samples by using LC-MS/MS. The use of LC-MS/MS enabled us
to confirm that unpolymerized dimers were present in the plasma
samples by locking onto ions that had an m/z of 577 and were
associated with structure-dependent product (dimer) ion fragments
that are unique to this procyanidin oligomer.
Consistent with the in vitro findings of Spencer et al (31), we
detected relatively modest concentrations of dimer B2 in the
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EPICATECHIN, CATECHIN, AND DIMERS IN PLASMA 803
plasma relative to the monomers. In the current study, a mean
plasma dimer B2 concentration of 41 nmol/L was observed 2 h
after the consumption of 256 mg dimers in the cocoa; the dimer
represented < 1% of the circulating nonmethylated flavanols com-
pared with an average plasma epicatechin concentration of
5.92 mol/L at the same time point. Interestingly, although the
measured ratio of epicatechin to catechin was 1:1 in the cocoa
preparation, epicatechin was the predominant plasma flavanol,
with plasma catechin concentrations being only 3% of the plasma
epicatechin concentration. A similar observation was previously
made by Rein et al (18). Complementing these findings, epicate-
chin was reported to be the primary bioavailable form of the pro-
cyanidin dimers B2 and B5 in an isolated intestine model (31).
The combination of these results suggests that dimers and other
procyanidin oligomers may be degraded in the gut into epicate-
chin monomers that are then adsorbed. On the basis of this con-
cept, only a limited amount of oligomer is absorbed. These obser-
vations provide an explanation for the high ratio of epicatechin to
catechin observed in the current study. Further research on this issue
is warranted. It will be interesting to characterize the effects of cat-
echin-containing dimers such as B1 [epicatechin-(4-8)-catechin]
and B3 [catechin-(4-8)-catechin], which are predominant in red
wine and grape juice, on ratios of plasma epicatechin to catechin.
Information on the above will provide insight as to whether the
observed high ratio of epicatechin to catechin in the current study
is due to the absorption of the monomers after the degradation of
the oligomers in the gut or whether the ratio represents a prefer-
ential absorption of epicatechin.
With respect to the physiologic relevance of the in vivo plasma
concentrations observed in this study, in vitro studies have
reported an IC
50
(concentration that yields 50% inhibition,
expressed as mol monomer) of a cocoa extract of dimer B2 as
5 mol/L for an azo compound–dependent liposome oxidation
and 2 mol/L for an iron- and ascorbate-dependent liposome oxi-
dation (32). A dimer extract increased the lag time of conjugated
diene formation by 47% from control and at a concentration of
290 nmol/L (32). These data support the concept that low nanomo-
lar concentrations of dimers may have significant in vivo effects.
We previously reported increases in plasma antioxidant capac-
ity and reductions in lipid oxidation products with concurrent
increases of plasma epicatechin concentrations (33). Dose-
response increases in plasma epicatechin after the feeding of var-
ious amounts of cocoa flavonoids have also been reported by
Richelle et al (19). There was considerable disparity between the
reported plasma epicatechin concentrations in the current study
and in that by Richelle et al. In a recent report by Baba et al (26),
the consumption of cocoa flavonoids was associated with a rise in
plasma epicatechin concentrations to concentrations similar to
those in the current study.
In the current study, we pretreated the plasma samples with a
solution of -glucuronidase (EC 3.2.1.31) and arylsulfatase
(EC 3.1.6.1). With this approach we measured nonmethylated
(free, glucuronide, sulfide, and glucuronide-sulfide) plasma con-
jugates of epicatechin, catechin, and dimer B2; the individual con-
jugates were not analyzed. Consistent with our findings, Baba et al
(26) reported nonmethylated plasma epicatechin concentrations
of 3.46 mol/L in healthy subjects 2 h after they consumed a
cocoa drink that provided 220 mg epicatechin. Baba et al (26)
reported a concentration of 1.46 mol methylated epicatechin/L in
the plasma at the 2-h time point; free epicatechin represented 4.5%
of the total of methylated and nonmethylated epicatechin.
Only a limited amount of studies have examined the biological
effects of the conjugates. Harada et al (34) reported that glucuronide
conjugates of epicatechin and catechin have similar superoxide anion
scavenging activities compared with the aglycones; however,
reduced activities were measured with the methylglucuronide con-
jugates of both catechin and epicatechin. Similarly, Da Silva et al
(35) reported that rat plasma containing predominantly nonmethy-
lated conjugates was more effective in inhibiting radical-generated
hydroperoxides than was plasma containing primarily methylated
conjugates. Finally, Manach et al (36) observed that quercetin glu-
curonides and sulfides increased the inhibition of copper-induced
LDL oxidation, however, to a lesser extent than that of aglycone. The
abovementioned studies suggest that the nonmethylated conjugates
possess, although possibly to a lesser extent, the biological effects
that are observed in vitro with free epicatechin, catechin, and dimer.
In conclusion, although several investigators have observed
positive vascular effects in healthy adults given flavonoid-rich
foods such as whole grape juice (37–39) and cocoa (33, 40–42);
the components in these foods that are responsible for these effects
have not been definitively identified. The evidence presented here
establishes that the procyanidin oligomer dimer B2, as well as the
flavanol monomers epicatechin and catechin, can be absorbed into
the circulation. Additional work is needed to determine whether
even larger procyanidins can be absorbed. Future investigation is
also needed to determine whether the reported in vitro effects of
these procyanidin oligomers occur in vivo.
We thank Jodi L Ensunsa, Heather R Schrader, Malina Karim, and Tim J Orozco
for their assistance with this investigation
REFERENCES
1. Rimm EB, Ascherio A, Giovannucci E, Spiegelman D, Stampfer MJ,
Willett WC. Vegetable, fruit, and cereal fiber intake and risk of coro-
nary heart disease among men. JAMA 1996;275:447–51.
2. Krauss RM, Eckel RH, Howard B, et al. AHA dietary guidelines revi-
sion 2000: a statement for healthcare professionals from the nutrition
committee of the American Heart Association. Circulation 2000;102:
2284–99.
3. Adamson GE, Lazarus SA, Mitchell AE, et al. HPLC method for the
quantification of procyanidins in cocoa and chocolate samples and
correlation to total antioxidant capacity. J Agric Food Chem 1999;47:
4184–8.
4. Prior R, Cao G. Analysis of botanicals and dietary supplements for
antioxidant capacity: a review. J AOAC Int 2000;83:950–6.
5. Richelle M, Tavazzi I, Offord E. Comparison of the antioxidant activ-
ity of commonly consumed polyphenolic beverages (coffee, cocoa,
and tea) prepared per cup serving. J Agric Food Chem 2001;49:
3438–42.
6. Hertog MG, Feskens EJ, Hollman PC, Katan MB, Kromhout D.
Dietary antioxidant flavonoids and risk of coronary heart disease: the
Zutphen Elderly Study. Lancet 1993;342:1007–11.
7. Hertog MG, Kromhout D, Aravanis C, et al. Flavonoid intake and
long-term risk of coronary heart disease and cancer in the Seven
Countries Study. Arch Intern Med 1995;155:381–6.
8. Knekt P, Jarvinene R, Reunanen A, Maatela J. Flavonoid intake and
coronary mortality in Finland: a cohort study. BMJ 1996;312:478–81.
9. Dillinger TL, Barriga P, Escarcega S, Jimenez M, Lowe DS, Grivetti LE.
Food of the gods: cure for humanity? A cultural history of the medicinal
and ritual use of chocolate. J Nutr 2000;130:2057S–72S.
10. Lazarus SA, Adamson GE, Hammerstone JF, Schmitz HH. High-
performance liquid chromatography/mass spectrometry analysis of
proanthocyanidins in foods and beverages. J Agric Food Chem 1999;
47:3693–701.
by guest on June 6, 2013ajcn.nutrition.orgDownloaded from

804 HOLT ET AL
11. Pearson DA, Schmitz HH, Lazarus SA, Keen CL. Inhibition of in
vitro low-density lipoprotein oxidation by oligomeric procyanidins
present in chocolate and cocoas. In: Packer L, ed. Methods in enzy-
mology. New York: Academic Press, 2001:350–60.
12. Arteel GE, Schroeder P, Sies H. Reactions of peroxynitrite with cocoa
procyanidin oligomers. J Nutr 2000;130:2100S–4S.
13. Arteel GE, Sies H. Protection against peroxynitrite by cocoa
polyphenol oligomers. FEBS Lett 1999;462:167–70.
14. Karim M, McCormick K, Kappagoda CT. Effects of cocoa procyani-
dins on endothelium-dependent relaxation. J Nutr 2000;130:2105S–8S.
15. Mao T, Van De Water J, Keen CL, Schmitz H, Gershwin M. Cocoa
procyanidins and human cytokine transcription and secretion. J Nutr
2000;130:2093S–9S.
16. Mao T, Powell J, Van de Water J, Keen CL, Schmitz H, Gershwin M.
The influence of cocoa procyanidins on the transcription of inter-
leukin-2 in peripheral blood mononuclear cells. Int J Immunother
1999;15:23–9.
17. Spencer JP, Chaudry F, Pannala AS, Srai SK, Debnam E, Rice-Evans C.
Decomposition of cocoa procyanidins in the gastric milieu. Biochem
Biophys Res Commun 2000;272:236–41.
18. Rein D, Lotito S, Holt RR, Keen CL, Schmitz HH, Fraga CG.
Epicatechin in human plasma: In vivo determination and effect of
chocolate consumption on plasma oxidation status. J Nutr 2000;130:
2109S–14S.
19. Richelle M, Tavazzi I, Enslen M, Offord E. Plasma kinetics in man of
epicatechin from black chocolate. Eur J Clin Nutr 1999;53:22–6.
20. Hammerstone JF, Lazarus SA, Mitchell AE, Rucker R, Schmitz HH.
Identification of procyanidins in cocoa (Theobroma cacao) and
chocolate using high-performance liquid chromatography/mass spec-
trometry. J Agric Food Chem 1999;47:490–6.
21. Zhu QY, Holt RR, Lazarus SA, et al. Stability of the flavan-3-ols epi-
catechin and catechin and related dimeric procyanidins derived from
cocoa. J Agric Food Chem 2002;50:1700–5.
22. Bell JR, Donovan JL, Wong R, et al. (+)-Catechin in human plasma
after ingestion of a single serving of reconstituted red wine. Am J
Clin Nutr 2000;71:103–8.
23. Lee M-J, Wang Z-Y, Li H, et al. Analysis of plasma and urinary tea
polyphenols in human subjects. Cancer Epidemiol Biomarkers Prev
1995;4:393–9.
24. Piskula MK, Terao J. Accumulation of ()-epicatechin metabolites
in rat plasma after oral administration and distribution of conjugation
enzymes in rat tissues. J Nutr 1998;128:1172–8.
25. Chen L, Lee M-J, Yang CS. Absorption, distribution, and elimination
of tea polyphenols in rats. Drug Metab Dispos 1997;25:1045–50.
26. Baba S, Osakabe N, Yasuda A, et al. Bioavailability of ()-epicatechin
upon intake of chocolate and cocoa in human volunteers. Free Radic
Res 2000;33:635–41.
27. Laparra J, Michaud J, Masquelier J. Etude pharmacocinetique des
oligomeres flavanoliques. (Pharmacokinetic study of flavonic
oligomers.) Plant Med Phytother 1977;11:133–42 (in French).
28. Jimenez-Ramsey LM, Rogler JC, Housley TL, Butler LG, Elkin RG.
Absorption and distribution of
14
C-labeled condensed tannins and
related sorghum phenolics in chickens. J Agric Food Chem 1994;
42:963–7.
29. Deprez S, Brezillon C, Rabot S, et al. Polymeric proanthocyanidins
are catabolized by human colonic microflora into low-molecular-weight
phenolic acids. J Nutr 2000;130:2733–8.
30. Santos-Buelga C, Scalbert A. Proanthocyanidins and tannin-like
compounds—nature, occurrence, dietary intake and effects on nutri-
tion and health. J Sci Food Agric 2000;80:1094–117.
31. Spencer JP, Schroeter H, Shenoy B, Srai SK, Debnam E, Rice-Evans C.
Epicatechin is the primary bioavailable form of the procyanidin dimers
B2 and B5 after transfer across the small intestine. Biochem Biophys
Res Commun 2001;285:588–93.
32. Lotito SB, Actis-Goretta L, Renart L, et al. Influence of oligomer
chain length on the antioxidant activity of procyanidins. Biochem
Biophys Res Commun 2000;276:945–51.
33. Wang JF, Schramm DD, Holt RR, et al. A dose-response effect from
chocolate consumption on plasma epicatechin and oxidative damage.
J Nutr 2000;130:2115S–9S.
34. Harada M, Kan Y, Naoki H, et al. Identification of the major
antioxidative metabolites in biological fluids of the rat with
ingested (+)-catechin and ()-epicatechin. Biosci Biotechnol
Biochem 1999;63:973–7.
35. Da Silva EL, Piskula MK, Terao J. Enhancement of antioxidative
ability of rat plasma by oral administration of ()-epicatechin. Free
Radic Biol Med 1998;24:1209–16.
36. Manach C, Morand C, Crespy V, et al. Quercetin is recovered in
human plasma as conjugated derivatives which retain antioxidant
properties. FEBS Lett 1998;426:331–6.
37. Keevil JG, Osman HE, Reed JD, Folts JD. Grape juice, but not orange
juice or grapefruit juice, inhibits human platelet aggregation. J Nutr
2000;130:53–6.
38. Stein JH, Keevil JG, Wiebe DA, Aeschlimann S, Folts JD. Purple
grape juice improves endothelial function and reduces the suscepti-
bility of LDL cholesterol to oxidation in patients with coronary artery
disease. Circulation 1999;100:1050–5.
39. Freedman JE, Parker CI, Li L, et al. Select flavonoids and whole juice
from purple grapes inhibit platelet function and enhance nitric oxide
release. Circulation 2001;103:2792–8.
40. Osakabe N, Baba S, Yasuda A, et al. Daily cocoa intake reduces
the susceptibility of low-density lipoprotein to oxidation as
demonstrated in healthy human volunteers. Free Radic Res 2001;
34:93–9.
41. Rein D, Paglieroni, Teresa G, et al. Cocoa inhibits platelet activation
and function. Am J Clin Nutr 2000;72:30–5.
42. Schramm DD, Wang JF, Holt RR, et al. Chocolate procyanidins
decrease the leukotriene-prostacyclin ratio in humans and human aor-
tic endothelial cells. Am J Clin Nutr 2001;73:36–40.
by guest on June 6, 2013ajcn.nutrition.orgDownloaded from