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Institut für Physiologische Chemie I, Heinrich-Heine-Universität Düsseldorf, D-40001 Düsseldorf, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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KEY WORDS: • flavonoids • inflammation • procyanidins • cocoa • peroxynitrite • antioxidants
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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). One form
of oxidative stress is that associated with enhanced production of
reactive nitrogen species, nitric oxide and peroxynitrite. This
condition, which is also called nitrosative stress, may occur in states
of inflammation, because inflammatory cells produce enhanced amounts of
nitric oxide and of superoxide, both of which react rapidly to form
peroxynitrite. While this is used by the organism to attack invading
microorganisms, there may be an overproduction of these oxidants, which
could be hazardous to surrounding healthy tissue. Indeed, as a potent
oxidizing and nitrating species, peroxynitrite leads to tissue damage
in a number of pathological conditions in humans and experimental
animals (Beckman 1996
, Beckman et al. 1990
). Therefore, there is a need for defense against
peroxynitrite. The physiological and pharmacological strategies for
protection against peroxynitrite are organized into three categories:
prevention, interception and repair
(Arteel et al. 1999
, Sies 1993
).
Flavonoids occur in different classes, including procyanidins, as
natural products in plants, and these polyphenols are ingested with the
diet (Haslam 1998
, Lazarus et al. 1999
).
Flavonoids are general free radical scavengers (Bors et al. 1994
) and chelate transition metals (Korkina and Afanas’ev 1997
, Morel et al. 1993
).
Procyanidins (e.g., epicatechin) are exceptionally efficient radical
scavengers (Bors and Michel 1999
, Kondo et al. 1999
). Possible health benefits of polyphenols include the
suppression of inflammatory cytokine production (Mao et al. 1999
, Sanbongi et al. 1997
), protection against
cardiovascular disease (Kondo et al. 1996
,
Waterhouse et al. 1996
) and anticarcinogenic effects
(Stoner and Mukhtar 1995
, Yang et al. 1998
).
Flavonoids react with nitric oxide (van Acker et al. 1995
) and superoxide (Girard et al. 1995
,
Robak and Gryglewski 1988
) and protect against
peroxynitrite oxidation and nitration reactions (Haenen et al. 1997
, Pannala et al. 1997
). Recent work with the
procyanidin (-)-epicatechin and the respective procyanidin oligomers,
ranging up to the nonamer isolated from Theobroma cacao,
showed that these compounds effectively prevent oxidation and nitration
reactions of peroxynitrite (Arteel and Sies 1999
), with
the tetrameric compound being of particular interest. It is likely that
these compounds react with nitrating and oxidizing intermediate species
formed during peroxynitrite decay and not with peroxynitrite proper,
similar to simple phenolic compounds (Ramezanian et al. 1996
). Here, we present some characteristics of the reaction of
peroxynitrite with epicatechin and the tetrameric compound.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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The tetrameric procyanidin oligomer was isolated and purified from
Cocoapro cocoa from the seeds of T. cacao
(Adamson et al. 1999
, Hammerstone et al. 1999
) and was kindly supplied by Mars (Hackettstown, NJ). Stock
solutions (10 mg/ml) of the tested procyanidin preparations were made
in methanol but were readily soluble in aqueous solutions used in the
studies. Diethylenetriamine pentaacetic acid
(DTPA),4
5,5'-dithiobis-2-nitrobenzoic acid (DTNB), (-)-epicatechin and
(-)-epigallocatechin gallate were from Sigma (Deisenhofen, Germany).
Dihydrorhodamine 123 (DHR-123) was from Molecular Probes (Eugene, OR).
MnO2 was from Fluka (Buchs, Switzerland).
2-Phenyl-1,2-benzioselenazol-3-(2H)-one (ebselen) was
kindly provided by Rhône-Poulenc-Rorer (Cologne, Germany).
Peroxynitrite was synthesized from sodium nitrite and
H2O2 using a quenched-flow reactor
(Koppenol et al. 1996
), and H2O2
was eliminated by passage of the peroxynitrite solution over
MnO2 powder. The final peroxynitrite concentration was
determined spectrophotometrically at 302 nm (
= 1700 L ·
mol-1 · cm-1).
Assay of peroxynitrite-mediated oxidation reactions.
The peroxynitrite-mediated oxidation of DHR-123 was performed as
described previously (Kooy et al. 1994
) with minor
modifications (Sies et al. 1997
). Briefly, peroxynitrite
(100 nmol/L) was added to 0.5 µmol/L DHR-123 in 0.1 mol/L phosphate
buffer, 0.1 mmol/L DTPA, pH 7.3, under intense stirring at room
temperature, and fluorescence was detected with a fluorescence
spectrophotometer LS-5 (Perkin–Elmer Cetus, Norwalk, CT) with
excitation and emission wavelengths of 500 and 536 nm, respectively.
The concentration of epicatechins, epigallocatechin gallate and the
tetrameric oligomer required to inhibit DHR-123 oxidation by 50% were
determined. There was no significant interference of the test compounds
in fluorescence determination of DHR-123. The effect of vehicle
(0.3–1.5% methanol) under these conditions was negligible.
To determine the effect of peroxynitrite on glutathione, peroxynitrite
(10 µmol/L) was added to 10 µmol/L glutathione (GSH) and
different concentrations of epicatechin in 0.1 mol/L phosphate buffer,
0.1 mmol/L DTPA, pH 7.4, under intense stirring at room temperature for
30 s. GSH was determined colorimetrically by reaction with DTNB as
described previously (Ellman 1959
) (412
= 13,600 L · mol-1 · cm-1).
Peroxynitrite-mediated oxidation of benzoic acid was performed as
described previously (Sies et al. 1997
,
Szabó et al. 1997
) with modifications.
Peroxynitrite (500 µmol/L) was given by bolus addition under constant
mixing at room temperature into a mixture (2 ml) containing benzoic
acid (1 mmol/L) and DTPA (0.1 mmol/L) in 0.1 mol/L potassium phosphate
buffer (pH 7.3). The effect of epicatechin or of the tetrameric
oligomer (50 µmol/L) was determined under these conditions. Product
formation was quantified with HPLC analysis (see later).
Assay of peroxynitrite-mediated nitration of tyrosine.
Protection against peroxynitrite-mediated nitration of tyrosine was
performed as described previously (Pannala et al. 1997
)
with minor modifications. Peroxynitrite (500 µmol/L) was added via
bolus addition under constant vortexing to 100 µmol/L tyrosine in 0.1
mol/L phosphate buffer (pH 7.3) containing 0.1 mmol/L DTPA. Under these
conditions, the effects of the tested compounds (0–20 µmol/L) were
determined and quantified with HPLC (see later).
HPLC analysis.
To quantify hydroxylated benzoic acid formation by peroxynitrite,
samples (50 µl) were injected onto a C18 reversed phase column (150
x 4.6 mm; Merck, Darmstadt, Germany) with a Waters (Milford, MA)
720 WISP autosampler. Separation was performed with 50 mmol/L ammonium
acetate (pH 7.0)/methanol (68:32) at a flow rate of 1.0 ml/min. The
fluorescent product (2-hydroxybenzoic acid) formation was monitored
with a Merck-Hitachi F-1000 fluorescent detector (
ex
= 300 nm, em = 410 nm) equipped with a D-2500
Chromato-Integrator. Calibration curves with 2-hydroxybenzoic acid
were used to determine concentrations. Under these conditions, the
effects of epicatechin or of the tetrameric oligomer (50 µmol/L) were
determined relative to the effect of peroxynitrite alone ("No
addition").
To determine the effect of peroxynitrite on free tyrosine, samples (50
µL; containing 100 µmol/L 3-hydroxy-4-nitrobenzoic acid as an
internal standard) were injected onto a C18 reversed phase column (150
x 4.6 mm; Merck) with a Waters 720 WISP autosampler. Separation
was performed with a 50 mmol/L potassium phosphate buffer (pH
7.0)/acetonitrile step gradient on a Merck-Hitachi L-655A 12 HPLC
unit coupled with a Merck-Hitachi L-5000 controller unit at a flow
rate of 1.0 ml/min. The initial ratio of buffer to acetonitrile was
95:5, follow by a stepwise decrease to 50:50 at 5 min; after 13 min,
the ratio was returned to 95:5 and was maintained for an additional 13
min. Such a step gradient was necessary to achieve separation of the
compounds of interest and then to elute the flavonoids. The formation
of 3-nitrotyrosine was monitored with a Merck-Hitachi L-4200 UV/Vis
detector equipped with a D-2500 Chromato-Integrator at 430 nm,
whereas the disappearance of tyrosine was monitored at 280 nm.
Calibration curves of the ratio of peak area of 3-nitrotyrosine
standard to internal standard were used to determine concentrations
(Fig. 1
).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Table 1
summarizes the protection of epicatechin, epigallocatechin gallate and
the tetrameric compound against peroxynitrite-mediated oxidation of
DHR-123. For comparison, the results obtained with ebselen are also
shown (Table 1)
. The half-maximal inhibitory concentration of
ebselen was ~150 nmol/L, which is similar to our previous findings
(Briviba et al. 1996
, Sies et al. 1997
).
Although epicatechin was not as effective as ebselen, epigallocatechin
gallate was more effective. Of particular interest was the tetrameric
compound with a half-maximal inhibitory concentration of <100
nmol/L.
|
Table 1
summarizes the protection of the tested compounds against
nitration of free tyrosine by peroxynitrite (see Figs. 2
,
3
). When peroxynitrite (500 µmol/L) was added to
tyrosine-containing buffer, a peak that absorbs at 430 nm appeared
(Fig. 2B
) with a concomitant decrease in tyrosine (Fig. 3B
); this effect was blunted with low micromolar
concentrations of epicatechin or epigallocatechin gallate (Table 1)
,
leading to protection of tyrosine (Fig. 3C
). Similar to
protection against DHR-123 oxidation, the tetrameric compound was more
effective on a molar basis (Table 1
, Fig. 3D
). It is of
interest that the peak corresponding to procyanidin parent compound
(e.g., Fig. 3E
) disappeared on the addition of
peroxynitrite, leading to the formation of new product peaks (Fig. 3F
). These data suggest that the procyanidins are modified
during reaction with peroxynitrite, leading to new products that appear
to be more water soluble.
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); therefore, the results of the
DHR-123 oxidation assay could suggest a competition-type reaction.
However, simpler phenolic compounds tend to react via a first-order
pathway (Ramezanian et al. 1996
), dependent only on
peroxynitrite concentrations. To further determine the mechanism of
protection of the tested compounds against peroxynitrite, we
investigated whether epicatechin can protect against the oxidation of
GSH, another second-order reaction (Radi et al. 1991
). Peroxynitrite (10 µmol/L), added via bolus addition to
GSH (10 µmol/L), caused a 40% oxidation of this thiol compound, as
assayed by the DTNB-reactive content (Table 2
). Molar ratios of epicatechin as high as 20-fold had no protective
effect on GSH oxidation. Indeed, lower concentrations tended to
increase GSH oxidation (Table 2)
. The lack of competitive
protection against GSH oxidation by peroxynitrite with procyanidin
oligomers suggest that these compounds do not react directly with
peroxynitrite, similar to simpler phenolics (Ramezanian et al. 1996
). Conversely, epicatechin and the tetrameric compound were
both effective at protecting against hydroxylation of benzoic acid by
peroxynitrite (Table 3
), also a first-order reaction (Ramezanian et al. 1996
), with the tetrameric compound again more effective than
epicatechin (Table 3)
. Furthermore, although epicatechin did not
enhance the decay rate of peroxynitrite, an increase in absorbance at
430 nm was observed that occurred with a time frame longer than
peroxynitrite decay (data not shown).
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The role of dietary polyphenols in health and disease has received
recent attention (Rice-Evans and Packer 1998
,
Ursini et al. 1999
). These compounds have been shown to
inhibit nitration (Pannala et al. 1997
) and oxidation
reactions (Haenen et al. 1997
), as well as DNA damage
and strand breakage (Fiala et al. 1996
, Ohshima et al. 1998
), caused by peroxynitrite. Previous studies have
shown that polyphenolic compounds may be both oxidized and nitrated by
the addition of peroxynitrite (Kerry and Rice-Evans 1998
, Pannala et al. 1998
), similar to simpler
phenolics (Ramezanian et al. 1996
). It has been
previously suggested that polyphenolic compounds scavenge peroxynitrite
(Haenen et al. 1997
), suggesting a second-order type
of reaction, dependent on concentration of both procyanidin and
peroxynitrite. However, the results of this current study, coupled with
previous work (Arteel and Sies 1999
), suggest that
procyanidins do not directly react with peroxynitrite but most likely
with reactive oxidizing/nitrating intermediates. Currently, it is not
known whether the product or products formed from this reaction can be
recycled in vivo (i.e., to maintain a catalytic cycle of defense) or
whether this reaction is a one-time event. However, because the
daily dietary intake of procyanidins is quite high, it may be
unnecessary for these compounds to be recycled. Furthermore,
destruction/modification of procyanidins during the reaction with
peroxynitrite may protect more important sites from damage (e.g.,
tyrosine residues on proteins). Although epicatechin from chocolate was
found to reach a concentration of 0.7 µmol/L in plasma after the
intake of 80 g of black chocolate (Richelle et al. 1999
), it is not yet known how well cocoa procyanidin oligomers
are absorbed into the bloodstream. Recent work (Spencer et al. 1999
) using the isolated rat intestine showed that for certain
flavonoids, glucuronidation and possibly other metabolism may occur at
the level of the intestinal mucosa. Future research will involve
further investigation of some of these issues.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 This study was supported by Deutsche Forschungsgemeinschaft (SFB 503, Project B1) and by the National Foundation for Cancer Research (Bethesda, MD). G.E.A. was a Research Fellow of the Alexander von Humboldt Foundation (Bonn, Germany).
3 To whom reprint requests should be addressed.
4 Abbreviations used: DTPA, diethylenetriamine pentaacetic acid; DHR-123, dihydrorhodamine 123; DTNB, 5,5'-dithiobis-2-nitrobenzoic acid; GSH, glutathione.
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