Interference of the polyphenol epicatechin with the biological chemistry of nitric oxide- and peroxynitrite-mediated reactions

Article abstract of DOI:10.1016/j.bcp.2003.11.017

The formation of reactive nitrogen species in mammalians has both beneficial and undesirable effects. Nitric oxide (NO) production in endothelial cells leads to vascular smooth muscle relaxation, but if reactive nitrogen species are generated in high amounts by cells under inflammatory conditions they are toxic. Flavonoids like (-)-epicatechin show an inverse association of their intake with diseases thought to be associated with overproduction of reactive nitrogen species. We found that the formation of cyclic GMP in cultured porcine aortic endothelial cells was not affected by up to 1mM (-)-epicatechin. Half maximal inhibition of interferon-γ/ lipopolysaccharide induced nitrite accumulation in murine macrophages required about 0.5mM of the flavonoid. In contrast, nitration of free tyrosine triggered by 0.1 and 1mM authentic peroxynitrite was inhibited by (-)-epicatechin with IC₅₀ values of 6.6 and 28.0μM, respectively. The presence of 15mM sodium bicarbonate had no significant effect. Nitration of protein-bound tyrosine in phorbol 12-myristate 13-acetate treated HL-60 cells in the presence of nitrite was inhibited by (-)-epicatechin at a similar concentration range (IC₅₀=10-100μM). Myeloperoxidase activity of phorbol 12-myristate 13-acetate stimulated HL-60 cells was inhibited by (-)-epicatechin with an IC₅₀ value of 77.4μM. Epicatechin inhibited dihydrorhodamine oxidation by 50μM authentic peroxynitrite and 1mM 3-morpholino-sydnonimine with IC₅₀ values of 11.8 and 0.63μM, respectively. Our data suggest that at up to 0.1mM (-)-epicatechin preferentially inhibits NO-related nitration and oxidation reactions without affecting NO synthesis and cyclic GMP signaling.

Full text of DOI:10.1016/j.bcp.2003.11.017

Biochemical Pharmacology 67 (2004) 1285–1295
Interference of the polyphenol epicatechin with the biological chemistry
of nitric oxide- and peroxynitrite-mediated reactions
Ru¨diger Wippel, Margit Rehn, Antonius C.F. Gorren, Kurt Schmidt, Bernd Mayer*
¨ ¨ ¨
Institut fur Pharmakologie and Toxikologie, Karl-Franzens Universitat Graz, Universitatsplatz 2, A-8010 Graz, Austria
Received 23 September 2003; accepted 13 November 2003
Abstract
The formation of reactive nitrogen species in mammalians has both beneficial and undesirable effects. Nitric oxide (NO) production in
endothelial cells leads to vascular smooth muscle relaxation, but if reactive nitrogen species are generated in high amounts by cells under
inflammatory conditions they are toxic. Flavonoids like (ꢀ)-epicatechin show an inverse association of their intake with diseases thought
to be associated with overproduction of reactive nitrogen species. We found that the formation of cyclic GMP in cultured porcine aortic
endothelial cells was not affected by up to 1 mM (ꢀ)-epicatechin. Half maximal inhibition of interferon-g/lipopolysaccharide induced
nitrite accumulation in murine macrophages required about 0.5 mM of the flavonoid. In contrast, nitration of free tyrosine triggered by 0.1
and 1 mM authentic peroxynitrite was inhibited by (ꢀ)-epicatechin with IC50 values of 6.6 and 28.0 mM, respectively. The presence of
15 mM sodium bicarbonate had no significant effect. Nitration of protein-bound tyrosine in phorbol 12-myristate 13-acetate treated HL-60
cells in the presence of nitrite was inhibited by (ꢀ)-epicatechin at a similar concentration range (ic50 ¼ 10–100 mM). Myeloperoxidase
activity of phorbol 12-myristate 13-acetate stimulated HL-60 cells was inhibited by (ꢀ)-epicatechin with an IC value of 77.4 mM.
50
Epicatechin inhibited dihydrorhodamine oxidation by 50 mM authentic peroxynitrite and 1 mM 3-morpholino-sydnonimine with IC
50
values of 11.8 and 0.63 mM, respectively. Our data suggest that at up to 0.1 mM (ꢀ)-epicatechin preferentially inhibits NO-related
nitration and oxidation reactions without affecting NO synthesis and cyclic GMP signaling.
# 2003 Elsevier Inc. All rights reserved.
Keywords: Epicatechin; Tyrosine nitration; Nitric oxide; Nitric oxide synthase; HL-60 cells; Myeloperoxidase
1. Introduction
residues is of particular interest in human pathology of
inflammation. The product 3-nitrotyrosine has been iden-
tified in numerous infectious and inflammatory diseases
[2]. The functional consequences of protein nitration have
not been fully clarified, but in some diseases 3-nitrotyr-
osine formation may be causally linked to pathogenesis
through modulation of signaling cascades [3,4], interfer-
ence with enzyme function [5,6], or structural protein
assembly [7,8].
During the last years evidence has accumulated that
inflammation is involved in many of the diseases that afflict
modern Western population, including atherosclerosis and
coronary artery disease. Inflammation is accompanied by
an increased number of cells mediating non-specific
immune response. Phagocytic white blood cells are impor-
tant in host defense and may inflict oxidative damage
during inflammatory processes [1]. Besides the oxidation
of cellular biomolecules, nitration of protein tyrosine
Two main pathways of tyrosine nitration have been
described. According to the earlier proposal, peroxynitrite,
which is formed in a fast reaction of NO with superoxide
anion radicals (O2 ꢀ), triggers nitration via nitrogen diox-
ꢁ
ꢁ
* Corresponding author. Tel.: þ43-316-380-5567;
ide radicals (NO2 ) or a CO2 adduct [9–13]. In an alter-
fax: þ43-316-380-9890.
native mechanism MPO (EC 1.11.1.7) is thought to be the
major trigger of tyrosine nitration, either via oxidation oꢁf
nitrite, the stable endproduct of NO autooxidation to NO2
by compounds I and II [14] or via reaction of nitrite with
the main MPO product, hypochlorous acid to form NO2Cl
as nitrating species [15–18]. Phagocytic white blood cell
E-mail address: mayer@kfunigraz.ac.at (B. Mayer).
Abbreviations: cGMP, cyclic GMP; HPLC, high performance liquid
chromatography; IFN, interferon; LPS, lipopolysaccharide; MPO, myelo-
peroxidase; NO, nitric oxide; NOS, NO synthase; eNOS, endothelial NO
synthase (type III); iNOS, inducible NO synthase (type II); PBS,
phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; SIN-1,
3-morpholinosydnonimine.
0006-2952/$ – see front matter # 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2003.11.017

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R. Wippel et al. / Biochemical Pharmacology 67 (2004) 1285–1295
activation can principally boost both peroxynitrite and
MPO-mediated nitration. The generation of oxidants by
neutrophils results from activation NADPH oxidase whicꢀh
to study the potential interference of (ꢀ)-epicatechin with
NO/cGMP-mediated signaling. Intracellular accumulation
of cGMP was measured upon Ca2þ stimulation to see
whether or not this compound affects any of the compo-
nents of endothelial NO/cGMP signaling; (ii) murine
macrophages were used to determine the interference of
(ꢀ)-epicatechin with INF-g/LPS-induced iNOS expression
and iNOS activity of these cells; (iii) human leukocytes,
which contain up to 5% MPO, were used to study the effect
of (ꢀ)-epicatechin on the nitration of protein-bound tyr-
osine. These cells were also used to find out whether (ꢀ)-
epicatechin is a direct inhibitor of MPO activity. In addi-
tion, the effect of (ꢀ)-epicatechin on eNOS activity and
peroxynitrite-mediated nitration and oxidation reactions
was studied.
reduces molecular oxygen to O2 ꢀ. Dismutation of O2
ꢁ
ꢁ
yields hydrogen peroxide (H2O2), but both O2 ꢀ and H2O2
are relatively non-toxic to bacteria. Activated neutrophils
also secrete MPO, which is a heme protein that localizes to
azurophilic granules and is present in these cells at up to
5% of their dry weight [1]. MPO utilizes H2O2 and chloride
as substrates to form hypochlorous acid (HOCl) according
to Eq. (1).
ꢁ
H2O2 þ Clꢀ þ Hþ ! HOCl þ H2O
(1)
Activation of phagocytic white blood cell is also associated
with the induction iNOS and sustained release of NO [19].
NO itself is not particularly toxic to cells, but may form
secondary oxidants that are responsible for cytotoxicity.
An important pathwayꢁthat enhances NO cytotoxicity is its
rapid reaction with O2 ꢀ to yield peroxynitrite (ONOOꢀ).
While peroxynitrite may serve as a beneficial molecule in
the defense against invading microorganisms, overproduc-
tion may have a wide range of pathophysiological con-
sequences, including oxidation and nitration of
biomolecules [20].
2. Material and methods
2.1. Materials
All solvents used were of HPLC grade. Dihydrorhoda-
mine 123 (DHR), 3-nitrotyrosine were from Fluka-Sigma.
Recombinant mouse IFN-g and Pronase from Strepto-
myces griseus were from Roche Diagnostics GmbH,
anti-iNOS antibody was obtained from BD Transduction
Laboratories. Penicillin/streptomycin, amphotericin B, and
fetal calf serum were from PAA Laboratories GmbH. L-
[2,3,4,5-3H]Arginine hydrochloride (57 Ci mmolꢀ1) was
from American Radiolabeled Chemicals Inc., purchased
through Humos Diagnostica GmbH. Tetrahydrobiopterin
was obtained from Dr. B. Schircks Laboratories. NADPH
was purchased from Pharma Waldhof GmbH. Recombi-
nant human eNOS (maximal enzyme activity of 100–
150 nmol of L-citrulline mgꢀ1 minꢀ1) was expressed in
the yeast Pichia pastoris as described in detail elsewhere
[32]. Centrifuge tube filters (0.22 mm cellulose acetate)
were from Szabo (Vienna, Austria). NG-Nitro-L-arginine,
LPS from Salmonella typhosa, and all other chemicals
were from Sigma.
The flavonoid (–)-epicatechin (Fig. 1), present in high
amounts in dietary sources, particularly green tea, certain
chocolates, and red wine has been shown to be a potent
scavenger of peroxynitrite [21,22]. Recently it has been
shown that (–)-epicatechin is cell permeable [23]. Sug-
gested health benefits of these polyphenolic compounds
include protection against cancer [24] and inhibition of
inflammatory cytokine production [25]. These compounds
are also reported to function as potent antioxidants due to
their reducing properties [26]. The Rotterdam study [27]
showed an inverse association of flavonoid intake with
incident of myocardial infarction. In addition, some fla-
ꢀ
ꢁ
vonoids can act as scavengers of NO [28] and O2 [29],
but the underlying mechanisms are still unclear. Further-
more, it has been proposed that flavonoids like (ꢀ)-epi-
ꢁ
catechin act as potent scavengers of NO2 ꢁ [30].
Experimental evidence for the formation of NO2 pro-
duced by the MPO/nitrite system was recently reported
[31].
2.2. Solutions
In the present study, we investigated the potential of the
flavonoid (ꢀ)-epicatechin on various reactions that involve
NO and related species. Three different cell lines were
used: (i) cultured porcine aortic endothelial cells were used
All solutions were prepared freshly each day. Water was
ultrafiltered type I (resistance ꢂ18 MO cmꢀ1) from a
Milli-Q Water System. PBS is 8 mM Na2HPO4, 1.5 mM
KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4. NO solu-
tions were prepared as described [33]. Stock solutions of
(ꢀ)-epicatechin (100 mM) in H2O/DMSO (v/v) were
further diluted with the buffer solution used in the assays
(either PBS or as indicated in the legends of the figures).
DHR was dissolved in acetonitrile to 10 mM and kept in
the dark until use. SIN-1 was dissolved to 10 mM at pH 5.0.
Alkaline solutꢀions of peroxynitrite were prepared from
acidified NO2 and H2O2 as described [34,35]. Stock
solutions were diluted with H2O to 10 mM (pHꢃ12.8)
OH
OH
O
HO
OH
OH
Fig. 1. Chemical structure of (ꢀ)-epicatechin.

R. Wippel et al. / Biochemical Pharmacology 67 (2004) 1285–1295
1287
immediately before the experiments and added to 50 mM
K2HPO4/KH2PO4 buffer, pH 7.4, to obtain final peroxyni-
trite concentrations as indicated in the text. Changes of
buffer pH were <0.1 unit.
at 378 and 5% CO2 in Dulbecco’s modified Eagle’s med-
ium supplemented with 10% (v/v) heat-inactivated fetal
calf serum, 100 unit mLꢀ1 penicillin/streptomycin,
1.25 mg mLꢀ1 amphotericin B, and 3.7 g Lꢀ1 NaHCO3.
Cells were grown to confluence (ꢃ5 Â 107 cells per dish)
and incubated for 16 hr in the presence of IFN-g (50 uni-
t mLꢀ1) and LPS (0.5 unit mLꢀ1) in fresh phenol red-free
Dulbecco’s MEM. The cells were assayed for nitrite
accumulation and iNOS expression (see below).
2.3. Culture of HL-60 Cells
HL-60 promyelocytic leukemia cells (passage 20–60)
were obtained form the American Type Culture Collection
and cultured in RPMI 1640 medium supplemented with
10% (v/v) heat-inactivated fetal calf serum, 100 unit mLꢀ1
penicillin/streptomycin, 1.25 mg mLꢀ1 amphotericin B,
and 2 g Lꢀ1 NaHCO3 in 50 mL flasks at 378 in a 5%
CO2 humidified atmosphere. Cultures were passaged
and fed two to three times per week to maintain log phase
growth. Once a week cells were centrifuged and resus-
pended in fresh RPMI. Cell density for the experiments
was 1 Â 106 cells mLꢀ1 (or as indicated in the text). Cells
2.6. Determination of nitrite accumulation
Cell culture supernatants of RAW 264.7 macrophages
(ꢃ300 mL) were centrifuged (20,000 g), and the concen-
tration of NO2ꢀ was determined photometrically with the
Griess assay [37]. Aliquots (100 mL) were mixed with
100 mL of freshly prepared Griess reagent (20 mg N-(1-
naphthyl)-ethylenediamine and 0.2 g sulfanilamide dis-
solved in 20 mL of 5% (w/v) phosphoric acid), and the
absorbance at 546 nm was measured with a microplate
reader. Calibration curves were established with NaNO2
dissolved in the cell culture medium.
were counted with
a
haemacytometer (Neubauer
improved). The cells were assayed for: formation of pro-
tein-bound 3-nitrotyrosine and MPO activity. For the
determination of protein-bound 3-nitrotyrosine the cells
wereꢀincubated for 2 hr with 200 nM PMA and 1 mM
NO2 in the presence of various concentrations of (ꢀ)-
epicatechin. MPO activity was determined after treatment
of the cells with 200 nM PMA for 1 hr as described below.
2.7. SDS–polyacrylamide gel electrophoresis and
Western blotting
RAW 264.7 macrophages were washed with PBS, har-
vested, and centrifuged for 5 min at 200 g. After resuspen-
sion cells were washed followed by sonication 3Â
approximately 10 s at 50 W. SDS–polyacrylamide gel
electrophoresis was performed using 8% polyacrylamide
gels with electrophoresis at 180 V for 45 min in a Bio-Rad
miniprotean chamber as described in [38]. The separated
proteins were transferred to nitrocellulose membranes by
electroblotting for 90 min at 240 mA, followed by immu-
nodetection with an anti-iNOS (1:2500 dilution) antibody,
using horseradish peroxidase-conjugated anti-mouse IgG.
For detection of the protein, the ECL system from Amer-
sham Pharmacia Biotech was used. Densitometric analysis
was performed using a Herolab E.A.S.Y Win32 HiRes
densitometric system additionally equipped with an UV
Transilluminator UVT-28 ME. Captured images were
analyzed using E.A.S.Y Win32 image analysis software
(from Herolab GmbH).
2.4. Culture of endothelial cells and determination of
intracellular cGMP
Porcine aortic endothelial cells were cultured as pre-
viously described [36]. Endothelial cells were isolated by
enzymatic treatment (0.1% collagenase in PBS) and cul-
tured up to three passages in Dulbecco’s modified Eagle’s
medium containing 10% fetal calf serum and 100 uni-
ts mLꢀ1 penicillin/streptomycin, 1.25 mg mLꢀ1 amphoter-
icin B. Prior to experiments, endothelial cells were
subcultured in 24-well plastic plates and grown to con-
fluence (ꢃ2 Â 105 cells per well). The culture medium was
removed, and the cells were washed once and equilibrated
in incubation buffer (isotonic 50 mM Tris buffer, pH 7.4,
containing 3 mM CaCl2, 1 mM MgCl2, 1 mM 3-isobutyl-
1-methylxanthine, and 10 mM indomethacin) in the
absence or presence various concentrations of (ꢀ)-epica-
techin, or 0.1 mM NG-nitro-L-arginine. After 15 min, Ca2þ
ionophore A23187 was added to give initial final concen-
trations of 1 mM. Reactions were terminated 4 min later by
removal of the incubation buffer and treatment for 1 hr
with 1 mL of 0.01 N HCl. Intracellular cGMP was mea-
sured in the supernatants of the lysed cells by radioimmu-
noassay.
2.8. Determination of eNOS activity
The activity of eNOS was determined as formation of L-
[2,3,4,5-3H]citrulline from L-[2,3,4,5-3H]arginine [32,39].
Incubations were for 10 min at 378 in 0.1 mL of 50 mM
triethanolamine–HCl buffer, pH 7.4, containing 0.1–0.2 mg
of eNOS, 0.1 mM L-[2,3,4,5-3H]arginine (ꢃ80,000 counts
per min), 0.5 mM CaCl2, 10 mg mLꢀ1 calmodulin, 0.2 mM
NADPH, 10 mM tetrahydrobiopterin, 5 mM FAD, 5 mM
FMN, and 0.2 mM 3-[(3-cholamidopropy-l) dimethylam-
monio]-1-propanesulfonic acid (CHAPS). L-[3H]Citrulline
2.5. Culture and activation of macrophages
RAW 264.7 macrophages (form American Type Culture
Collection) were cultured in Petri dishes (diameter 90 mm)

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R. Wippel et al. / Biochemical Pharmacology 67 (2004) 1285–1295
was separated from L-[3H]arginine by cation exchange
chromatography (Dowex 50 W, 8% cross-linkage, 200–
400 mesh size, Hþ-form, from Sigma).
determinations. After incubation, cells were centrifuged
three times at 1300 g for 5 min and resuspended in PBS.
Then the cell pellets were resuspended in 250 mL 0.1 M
K2HPO4/KH2PO4 buffer, pH 7.4, giving protein concen-
trations of 10–20 mg mLꢀ1 according to the Bradford
method with bovine serum albumin as a standard [42].
Protein was precipitated with 0.8 mL acetone (cooled to
ꢀ208), the samples were thoroughly vortexed, kept on ice
for 10 min and centrifuged (8000 g, 5 min), followed by
resuspension of the precipitate in 250 mL of 0.1 M phos-
phate buffer, pH 7.4, and sonication for 2Â approxi-
mately 10 s at 50 W. This procedure was repeated
three times to remove non-protein material and nitrite.
The final suspension (in 250 mL buffer) was incubated for
15–18 hr at 378 with 2 mg pronase and 0.5 mM CaCl2.
Samples (ꢃ300 mL) were centrifuged (20,000 g) and an
equal volume of 3 M potassium phosphate buffer, pH 9.6,
was added to the supernatant, followed by the addition of
acetic anhydride (30 mL). After 15 min of incubation at
ambient temperature, ethyl acetate (1 mL) and formic
acid (125 mL) were added. The samples were vortexed for
30 s and then centrifuged at 20,000 g for 1 min. The ethyl
acetate phase was concentrated to dryness under a stream
of N2 at 458. For deacetylation of the phenolic acetate
group, samples were resuspended in 60 mL 1 N NaOH.
After 30 min of incubation at 378, 120 mL of 1 M
K2HPO4/KH2PO4 buffer (pH 6.5) was added, followed
by addition of 10 mL 0.1 M sodium dithionite to reduce
the nitro substituent to the corresponding amine. The
samples were incubated for 10 min at ambient tempera-
ture, acidified by the addition of concentrated HCl
(20 mL) and centrifuged at 20,000 g for 10 min in cen-
trifuge tube filters. One hundred and forty microliters
samples (cooled to 48 by a Varian ProStar 420 autosam-
pler) were injected onto a 250 mm  4 mm C18 reversed
phase column equipped with a 4 mm  4 mm C18 guard
column (LiChrospher 100 RP-18, 5 mm particle size)
which was held at 228 by Varian column oven ProStar
510. Elution was with 10 mM H3PO4, containing 2% (v/
v) methanol (solvent A) and 50% (v/v) methanol/50%
(v/v) 10 mM H3PO4 (solvent B) at 0.7 mL minꢀ1 with a
gradient program (0–18 min 100% solvent A, 18–24 min
linear ramp to 100% solvent B, 24–39 min 100% solvent
B, 39–43 min linear ramp to 100% solvent A and 43–
75 min 100% solvent A). N-Acetyl 3-aminotyrosine was
detected electrochemically with an ESA Coulochem II
detector equipped with a 5011 analytical cell. The poten-
tials of the two electrodes were adjusted to ꢀ70 and
þ70 mV, respectively. Standards of N-acetyl 3-aminotyr-
osine were prepared as described [41], and calibration
curves were recorded daily (5–500 nM). Tyrosine was
detected as N-acetyltyrosine (prepared as N-acetyl 3-
aminotyrosine) at 280 nm (standards 50–1000 mM) with
a Varian ProStar 310 UV-Vis detector. Blank values of
non-stimulated cells were subtracted for every experi-
ment.
2.9. Determination of MPO activity in HL-60 cells
The activity of MPO was determined as formation of
hypochlorous acid by taurine chloramine assay as
described [40]. HL-60 cells (1 Â 106 cells mLꢀ1) were
stimulated with PMA (200 nM final) and incubated at
378 in 0.5 mL of 10 mM Na2HPO4/KH2PO4 buffer, pH
7.4, containing 1 mg mLꢀ1 of glucose, 138 mM NaCl,
10 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, 20 mM taurine,
and varying concentrations of (ꢀ)-epicatechin as indi-
cated. Reactions were terminated after 60 min with
20 mg mLꢀ1 catalase and putting the reaction mixture on
melting ice for 10 min. Cells were pelleted at 10,000 g for
2 min. The concentration of accumulated taurine chlora-
mine, the reaction product of taurine and hypochlorous
acid, present in the supernatants was determined as oxida-
tion of 5-thio-2-nitrobenzoic acid (100 mM final) to 5,50-
dithiobis (2-nitrobenzoic acid) by monitoring the decrease
in 5-thio-2-nitrobenzoic acid absorbance at 412 nm [40].
Blank values were measured separately for every (ꢀ)-
epicatechin concentration.
2.10. Tyrosine nitration and HPLC analysis of
3-nitrotyrosine in buffer
To study tyrosine nitration by authentic peroxynitrite,
alkaline stock solutions of peroxynitrite (final concentration
as indicatedinthetext)wereaddeddropwiseundervigorous
vortexing to tyrosine (1 mM) in 50 mM K2HPO4/KH2PO4
buffer, pH 7.4, containing various (ꢀ)-epicatechin concen-
trations, followed by incubation for 1 hr. Samples were kept
on ice until HPLC analysis (typically 1–2 hr). Samples
(50 mL) were injected onto a 250 mm  4 mm C18 reversed
phase column equipped with a 4 mm  4 mm C18 guard
column (LiChrospher 100 RP-18, 5 mm particle size,
Merck). Elution was with 50 mM KH2PO4/H3PO4 buffer
(pH 3) containing 10% (v/v) methanol at a flow rate of
1.0 mL minꢀ1. 3-Nitrotyrosine was detected by its absor-
bance at 274 nm with a Merck Hitachi L-4250 UV-Vis
detector. Identification and quantification of 3-nitrotyrosine
was based on calibration curves established with the
authentic compound (1–100 mM) under the same condi-
tions.
2.11. Determination of protein-bound 3-nitrotyrosine
and tyrosine in cell lysates
Protein-bound 3-nitrotyrosine was determined by
HPLC and electrochemical detection after derivatization
to N-acetyl 3-aminotyrosine as described [41], but with a
few modifications. HL-60 cells from two flasks (corre-
sponding to 100 mL cell suspension) were used for single

R. Wippel et al. / Biochemical Pharmacology 67 (2004) 1285–1295
1289
2.12. Determination of DHR oxidation
tions of VCl3 and the Griess reagent as described [46]. The
nitrite and total NOx concentrations in samples were sub-
tracted to give the nitrate concentrations. The amount of
NO2ꢀ present in stock solutions of conventionally prepared
peroxynitrite was subtracted from the measured values
[34].
Oxidation of DHR by 50 mM authentic peroxynitrite
and was monitored at 500 nm with a Hewlett-Packard
8452A Diode Array spectrophotometer at ambient tem-
perature in a total volume of 0.2 mL of a 50 mM K2HPO4/
KH2PO4 buffer, pH 7.4 containing 0.1 mM DHR and
0.1 mM of the metal chelator diethylenetriamine pentaa-
cetic acid, as described [43,44].
2.15. Statistics and data evaluation
With 1 mM SIN-1 as the source of peroxynitrite, the
oxidation of DHR was monitored for 10 min in the pre-
sence of various concentrations of (ꢀ)-epicatechin. Aero-
bic decay of SIN-1 is a two-step reaction with intermediate
formation of SIN-1A, which reacts with O2 to yield SIN-
Unless otherwise indicated, data are given as
means ꢄ SE of three independent experiments. The con-
centration producing 50% inhibition of the maximal
response (IC ) was calculated from individual concentra-
50
tion–response curves which were calculated by non-linear
regression analysis. The computations were performed
with an IBM-compatible personal computer using Sigma-
Plot 2002 for Windows Version 8.0.
ꢀ
ꢁ
1C, O2
and NO. Consequently, the time course of
formation of SIN-1C (and of NO/O2 ꢀ) exhibits a lag
phase of several minutes. Since subsequent formation of
peroxynitrite and reaction with DHR are much faster, DHR
oxidation will follow the same time course as SIN-1C
formation, exhibiting a similar lag phase. To take this into
account, we fitted the time course of DHR oxidation to an
equation (Eq. (2)) describing the time course of formation
of SIN-1C, using previously measured rate constants for
SIN-1A and SIN-1C formation [45].
ꢁ
3. Results
3.1. Interference of (ꢀ)-epicatechin with NO/cGMP-
mediated signaling, eNOS activity, iNOS expression
and accumulation of nitrite
A500 nm ¼ að1 þ 0:939 eꢀ0:064t ꢀ 1:939 eꢀ0:031tÞ þ b
(2)
Interference of (ꢀ)-epicatechin with NO/cGMP-
mediated signaling was studied in cultured porcine aortic
endothelial cells. As shown in Fig. 2A, (ꢀ)-epicatechin had
no effect on cGMP accumulation under basal conditions.
Stimulation of the cells with Ca2þ ionophore A23187 led
to a pronounced accumulation of intracellular cGMP
(51:5 ꢄ 4:9 pmol per 106 cells) which was completely
inhibited by NG-nitro-L-arginine (0.1 mM). This rise in
cGMP was not affected by up to 1 mM (ꢀ)-epicatechin,
indicating that the polyphenol does neither interfere with
Ca2þ-triggered endothelial NO synthesis nor with the
activation of soluble guanylyl cyclase by NO. Under
standard assay conditions (ꢀ)-epicatechin was a very poor
The fitting parameters a and b represent the observed
absorbance change and the initial absorbance, respectively.
The obtained values of a, which are proportional to the
effective concentration of the oxidant reacting with DHR,
were replotted against the concentration of (ꢀ)-epicatechin
as apparent relative reactivities, with the value in the
absence of (ꢀ)-epicatechin taken as 100%, and fitted to
the Hill equation.
2.13. Decomposition of peroxynitrite
Peroxynitrite decomposition was studied by stopped-
flow absorbance spectroscopy at 378 (Bio-Sequential SX-
17MV stopped-flow ASVD spectrofluorimeter, Applied
Photophysics, Leatherhead, UK) with the wavelength set
at 302 nm (e ¼ 1670 Mꢀ1 cmꢀ1) according to a published
procedure [34]. Reservoir 1 contained 0.2 mM peroxyni-
trite in 0.01 M NaOH, and reservoir 2 contained the
varying concentrations of (ꢀ)-epicatechin (0–10 mM) in
1 M K2HPO4/KH2PO4 buffer, pH 7.4.
inhibitor of purified eNOS with an IC value of about
50
0.5 mM (Fig. 2B). Activation of RAW 264.7 macrophages
with IFN-g/LPS led to iNOS expression apparent as for-
mation of ꢃ40 mM nitrite. Addition of 1 mM (ꢀ)-epica-
techin decreased iNOS expression to ꢃ40% of control
(data not shown). As shown in Fig. 2C, presence of up to
100 mM (ꢀ)-epicatechin did not affect nitrite accumulation
in the supernatant of induced RAW cells (ic50 > 1 mM).
2.14. Determination of the decomposition products of
ꢀ
3.2. Effect of (ꢀ)-epicatechin on tyrosine nitration
ꢀ
peroxynitrite (NO2 and NO3
)
As shown in Fig. 3A, nitration of 1 mM free tyrosine
triggered by 0.1 mM authentic peroxynitrite led to the
formation of 8:3 ꢄ 0:64 mM 3-nitrotyrosine that was inhib-
Peroxynitrite (1 mM) was decomposed by incubation
for 1 hr at 378 in 0.1 M K2HPO4/KH2PO4 buffer, pH 7.4 in
the presence or absence of 1 mM (ꢀ)-epicatechin as
ited by (ꢀ)-epicatechin with an IC
value of
50
described [34]. NO2ꢀ was determined by the Griess assay
6:6 ꢄ 1:06 mM (mean value ꢄ SE; N ¼ 3). A 10-fold
higher concentration of peroxynitrite (Fig. 3B) led to
formation of 66:8 ꢄ 6:26 mM 3-nitrotyrosine that was
as described above. For determination of NO2ꢀ þ NO3
,
samples were coincubated for 45 min with saturated solu-
ꢀ

1290
R. Wippel et al. / Biochemical Pharmacology 67 (2004) 1285–1295
60
50
40
30
20
10
0
A23187
NNA
1 µM
-
-
-
+
+
-
-
-
-
-
-
-
+
-
+
-
+
-
+
-
0.1 mM
101 102 103
101 102 103
Epicatechin [µM]
-
(A)
140
60
120
100
80
60
40
20
0
40
20
0
0.1
1
10
100
1000 10000
0.1
1
10
100
1000
(B)
(C)
Epicatechin [µM]
Epicatechin [µM]
Fig. 2. Interference of (ꢀ)-epicatechin with NO/cGMP-mediated signaling, eNOS activity and nitrite formation. (A) NO/cGMP-mediated signaling: porcine
aortic endothelial cells were subcultured in 24-well plastic plates, grown to confluence (ꢃ2 Â 105 cells per well), washed and equilibrated (as described in
Section 2) in the absence or presence of various concentrations of (ꢀ)-epicatechin, or 0.1 mM NG-nitro-L-arginine. After 15 min, Ca2þ ionophore A23187
was added to give initial final concentrations of 1 mM. Reactions were terminated 4 min later by removal of the incubation buffer and treatment for 1 hr with
1 mL of 0.01 N HCl. Intracellular cGMP was measured in the supernatants of the lysed cells by radioimmunoassay. The data are mean values ꢄ SE of four
independent experiments. (B) The activity of purified eNOS was determined as formation of L-[2,3,4,5-3H]citrulline from L-[2,3,4,5-3H]arginine (as
described in Section 2). The enzyme was preincubated with (ꢀ)-epicatechin for 10 min at 378. The data are mean values of two experiments performed in
duplicate. (C) Nitrite formation by cytokine-activated murine macrophages: RAW 264.7 macrophages were grown to confluence (ꢃ5 Â 107 cells per dish)
and incubated for 16 hr in the presence of IFN-g (50 unit mLꢀ1) and LPS (0.5 mg mLꢀ1) with the indicated concentrations of (ꢀ)-epicatechin. The
supernatants of the activated cells were assayed photometrically for nitrite accumulation (as described in Section 2) as a measure of iNOS expression and
activity. The data are mean values ꢄ SE of three independent experiments performed in duplicate.
inhibited by (ꢀ)-epicatechin with an IC
value of
ingly, HL-60 cells exhibited (ꢀ)-epicatechin-insensitive
50
28:0 ꢄ 2:33 mM (mean value ꢄ SE; N ¼ 3). 3-Nitrotyro-
sine formation was increased in the presence of 15 mM
sodium bicarbonate (81:0 ꢄ 2:63 mM with 1 mM peroxy-
nitration activity of approximately 25% (Fig. 4).
3.3. Effect of (–)-epicatechin on MPO activity in
HL-60 cells
nitrite), but the IC value for (ꢀ)-epicatechin was not
50
significantly increased (ic50 ¼ 45:8 ꢄ 5:44 mM; mean
value ꢄ SE; N ¼ 3; P ¼ 0:07; Fig. 3C).
MPO activity in PMA-stimulated HL-60 cells was
value of
50
Stimulation of HL-60 cells with 200 nM PMA in the
presence of 1 mM nitrite for 2 hr led to the formation of
0:68 ꢄ 0:18 pmol mgꢀ1 protein-bound 3-nitrotyrosine
(average blank value of non-stimulated cells was
0:16 ꢄ 0:03 pmol mgꢀ1 protein-bound 3-nitrotyrosine).
HL-60 cells contained 0:21 ꢄ 0:01 mmol mgꢀ1 protein-
bound tyrosine, indicating nitration of about three out of
one million tyrosine molecules. As shown in, nitration was
inhibited by (ꢀ)-epicatechin with an IC
77:4 ꢄ 16:19 mM (mean value ꢄ SE; N ¼ 3). Total HOCl
production was 17:9 ꢄ 2:31 nmol per 106 cells. Note that
addition of 1 mM (ꢀ)-epicatechin led to complete enzyme
inhibition (Fig. 5).
3.4. Effect of (ꢀ)-epicatechin on dihydrorhodamine
(DHR)-oxidation
inhibited by (ꢀ)-epicatechin with an IC value of 10–
50
100 mM. Thus, (ꢀ)-epicatechin inhibited nitration of free
Epicatechin inhibited DHR oxidation by 50 mM authen-
tic (Fig. 6A) with an IC value of 11:8 ꢄ 1:29 mM (mean
50
and protein-bound tyrosine with similar potency. Interest-

R. Wippel et al. / Biochemical Pharmacology 67 (2004) 1285–1295
1291
10.0
7.5
5.0
2.5
0.0
1.0
0.8
0.6
0.4
0.2
(A)
0.01
0.1
1
10
100
0.0
0.1
1
10
100
1000
80
60
40
20
0
Epicatechin [µM]
Fig. 4. Effect of (ꢀ)-epicatechin on formation of protein-bound 3-
nitrotyrosine in HL-60 cells. Cells were incubated for 2 hr with 200 nM
ꢀ
PMA and 1 mM NO2 in the presence of various concentrations of (ꢀ)-
epicatechin. 3-Nitrotyrosine was determined electrochemically after HPLC
separation as N-acetyl 3-aminotyrosine as described in Section 2. The data
are the mean values ꢄ SE of three independent experiments performed in
duplicate.
3.5. Effect of (–)-epicatechin on the decomposition of
peroxynitrite
(B)
0.1
1
10
100
1000
Decomposition of peroxynitrite was monitored as
decrease in absorbance at 302 nm at 378. All traces could
be fitted to single exponentials. The observed pseudo-first
order rate constant (1:18 ꢄ 0:03 sꢀ1) was independent of
the concentration of (ꢀ)-epicatechin (0–10 mM), which
puts an upper limit of ꢃ20 Mꢀ1 sꢀ1 to the rate constant for
a reaction between peroxynitrite and (ꢀ)-epicatechin.
Thus, the potent scavenging activity of (ꢀ)-epicatechin
cannot be explained by a direct reaction with peroxynitrite.
We consistently observed that the decomposition of
peroxynitrite, which was expected to result in formation
of about 70% NO3ꢀ and 30% NO2ꢀ at pH 7.4 and 378 [34],
was not affected by 1 mM (ꢀ)-epicatechin. Decomposition
80
60
40
20
0
0.1
1
10
100
1000
(C)
Epicatechin [µM]
Fig. 3. Effect of (ꢀ)-epicatechin on formation of 3-nitrotyrosine. Nitration
of free tyrosine in the presence of various concentrations of (ꢀ)-
epicatechin triggered by (A) 0.1 mM authentic peroxynitrite, (B) 1 mM
authentic peroxynitrite, (C) 1 mM authentic peroxynitrite in the presence
of 15 mM NaHCO3. UV-Vis detection of 3-nitrotyrosine was performed as
described in Section 2. The data are the mean values ꢄ SE of three
independent experiments performed in duplicate.
25
20
15
10
5
value ꢄ SE; N ¼ 3). The effect of ꢀ(ꢀ)-epicatechin on the
ꢁ
oxidation of DHR by the NO/O2 donor SIN-1 is illu-
strated in Fig. 6B. In the presence of 1 mM SIN-1, (ꢀ)-
epicatechin exhibited an IC50 value of 0:64 ꢄ 0:08 mM and
a Hill coefficient of 0:90 ꢄ 0:05. The inhibition curve
shown in Fig. 6B was obtained by taking into account
0
0.1
1
10
100
1000
the non-linear nature of NO/O2 ꢀ formation by SIN-1 (see
ꢁ
Epicatechin [µM]
Section 2). The less sophisticated procedure of plotting the
concentration of oxidized DHR obtained at the end of the
10 min incubation against the concentration of (ꢀ)-epica-
techin yielded identical results (ic50 ¼ 0:67 ꢄ 0:10 mM;
Hill coefficient ¼ 0:91 ꢄ 0:06, not shown).
Fig. 5. Effect of (ꢀ)-epicatechin on MPO activity. HL-60 were incubated
for 1 hr with 200 nM PMA in the presence of various concentrations of
(ꢀ)-epicatechin. The formed hypochlorous acid was measured with the
taurine chloramine assay as described in Section 2. The data are mean
values ꢄ SE of three independent experiments performed in duplicate.

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R. Wippel et al. / Biochemical Pharmacology 67 (2004) 1285–1295
120
100
80
inhibit the synthesis of pro-inflammatory cytokines [25].
The protective effects of these compounds may at least
partially be due to increased bioavailability and/or synth-
esis of endothelium-derived nitric oxide [52]. While (ꢀ)-
epicatechin was proposed to cause eNOS activation via
increased intracellular Ca2þ [53], related red wine poly-
phenols were reported to enhance eNOS expression in
cultured endothelial cells [54,55]. Our data provided no
evidence for increased bioavailability of endothelial NO in
the presence of (ꢀ)-epicatechin (measured as accumula-
tion of cGMP in the absence of Ca2þ stimulation; Fig. 6A),
but we cannot exclude long-term transcriptional or transla-
tional effects of the flavonoid. More importantly with
respect to the potential in vivo application of (ꢀ)-epica-
techin as radical scavenger, even very high concentrations
60
40
20
0
(A)
0.1
1
10
100
1000
100
80
(up to 1 mM) of the flavonoid did not interfere with Ca2þ
-
60
triggered NO/cGMP synthesis in endothelial cells. Since
the marginal inhibition of purified eNOS by ꢅ0.1 mM (ꢀ)-
epicatechin was not apparent in intact cells, the cellular
environment appears to provide protection against poten-
tial non-specific interaction of the flavonoid with the eNOS
protein and/or redox active cofactors of the enzyme. A
previous study reported on similarly low potency of the
related compound epigallocatechin-3-gallate to inhibit
purified nNOS activity [56]. Interestingly, this compound
was identified as relatively potent inhibitor of iNOS
mRNA expression (ic50 < 5 mM) and iNOS activity
(ic50 ꢃ 150 mM) in IFN-g/LPS-activated murine perito-
neal macrophages. Since we found that (ꢀ)-epicatechin
did not affect IFN-g/LPS-induced nitrite synthesis by
RAW 264.7 macrophages at up to 100 mM, these earlier
reports suggest a critical role of the gallate moiety [56,57].
40
20
0
0.01 0.1
1
10
100 1000
(B)
Epicatechin [µM]
Fig. 6. Effect of (ꢀ)-epicatechin on DHR oxidation. DHR oxidation was
assayed as described in Section 2 with (A) 50 mM authentic peroxynitrite
and (B) 1 mM SIN-1 (the apparent reactivities plotted along the vertical
axis were derived as detailed in Section 2). The data are mean values ꢄ SE
of three independent experiments performed in duplicate.
without and with (ꢀ)-epicatechin yielded 30:2 ꢄ 0:59%
and 30:9 ꢄ 1:47% NO2ꢀ, respectively. The total amount of
ꢀ
NO2ꢀ þ NO3
was not significantly decreased
(0:89 ꢄ 0:025 mM and 0:80 ꢄ 0:035 mM in the absence
and presence of 1 mM (ꢀ)-epicatechin, respectively; mean
values ꢄ SE; N ¼ 3; P > 0:1).
4.2. Effect of (ꢀ)-epicatechin on nitration of
free tyrosine by peroxynitrite
Protein tyrosine nitration, caused by peroxynitrite and/or
MPO-catalyzed oxidation of nitrite, may have deleterious
effects on cellular function and viability due to alterations
of protein structure and function, like disassembly of
structural proteins, inhibition of tyrosine phosphorylation
and decreased catalytic activity of enzymes. It has been
shown previously that (ꢀ)-epicatechin inhibits peroxyni-
trite-triggered nitration of free tyrosine and protein-bound
tyrosine [22,58]. In accordance with earlier observations
[22], we found that the potency of the flavonoid to inhibit
nitration decreased with increasing concentrations of per-
4. Discussion
4.1. Interference of (ꢀ)-epicatechin with
NO/cGMP-mediated signaling and NOS activity
Inflammatory diseases are associated with sustained
ꢀ
ꢁ
synthesis and release of free radicals, in particular O2
,
hydroxyl radicals, hydrogen peroxide, and NO [47]. Sec-
ondary reactions of these molecules yieldꢁhighly reactive
intermediates, such as peroxynitrite, NO2 and carbonate
anion radical, which cause deleterious protein and DNA
modifications [48]. Therefore, free radical scavengers are
potentially useful drugs to prevent inflammatory tissue
injury associated with oxidative stress [49]. However, some
of these compounds exhibit manifold biological activities
limiting their clinical application [50,51]. Epicatechin and
related polyphenolic flavonoids of plant origin were shown
to reduce the risk for myocardial infarction [27] and to
oxynitrite. The IC values of 6.6 and 28 mM at 0.1 and
50
1.0 mM peroxynitrite correspond to IC /[peroxynitrite]
50
ratios of 0.07 and 0.03, respectively, indicating that one
equivalent of (ꢀ)-epicatechin prevents nitration by 15–30
equivalents of peroxynitrite. Accordingly, the effect of
(ꢀ)-epicatechin is apparently not due to stoichiometric
reaction with peroxynitrite but may result from scavenging
of a reactive decomposition product. This conclusion is
supported by the virtually identical potency of the flavo-

R. Wippel et al. / Biochemical Pharmacology 67 (2004) 1285–1295
1293
noid in the presence of millimolar concentrations of bicar-
bonate and our observation that the compound did not
affect the kinetics and the concentration of end products of
peroxynitrite decomposition. There is general agreement
that formation of 3-nitrotyrosine results from reaction of
4.4. Effect of (ꢀ)-epicatechin on peroxynitrite-mediated
oxidation reactions
In accordance to previous reports [70,71], we found
that (ꢀ)-epicatechin inhibited oxidation of DHR by
ꢀ
ꢁ
ꢁ
tyrosyl radical with NO2 , with the tyrosyl radical ꢁorigi-
authentic peroxynitrite (50 mM) and the NO/O2 donor
SIN-1 (IC50 values of 12 and 0.6 mM, respectively). Since
peroxynitrite is the primary oxidant formed from SIN-1
decomposition [44], the higher potency of (ꢀ)-epicate-
chin towards SIN-1-mediated oxidation appears to be a
consequence of the low steady-state concentrations of
nating from oxidative attack of tyrosine by NO2 and
carbonate anion radical in the absence and presence of
bicarbonate, respectively [59,60]. A recent pulse radiolysis
ꢁ
study reported that (ꢀ)-epicatechin scavenged NO2 and
carbonate radicals with second-order rate constants of
9 Â 107 Mꢀ1 sꢀ1 and 5:6 Â 108 Mꢀ1 sꢀ1 [61], indicating
that inhibition of tyrosine nitration is due to quenching of
these radicals. The similar potency of (ꢀ)-epicatechin to
prevent nitration and dimerization of tyrosine suggests
that the polyphenol may also react with tyrosyl radicals
[62].
peroxynitrite rather than scavenging of O2 ꢀ. This con-
ꢁ
clusion is supported by a recent report showinꢀg that the
ꢁ
pyrogallol moiety is essential for efficient O2 scaven-
ging by flavonoids [72] and our findings that 1 mM (ꢀ)-
epicatechin (unlike superoxide dismutase) did not cause
detectable formation of free NO from SIN-1 (data not
shown). It is unclear how (ꢀ)-epicatechin inhibits DHR
oxidation by peroxynitrite. There is general agreement
that DHR becomes oxidized by a direct reaction with
peroxynitrite anion [44], which does not react with (ꢀ)-
epicatechin. However, the precise mechanism of DHR
oxidation is not completely understood. Based on the
unexpected observation that the peroxynitrite-triggered
reaction is inhibited by NO, it has been proposed that
one-electron oxidation of DHR may generate a radical
intermediate which could be quenched by radical–radical
addition of NO prior to loss of a second electron yielding
rhodamine [43]. Even though attempts to isolate this
putative nitroso-DHR adduct have proven unsuccessful
[44], one-electron oxidation may be a crucial step of
DHR oxidation, and (ꢀ)-epicatechin could inhibit the
reaction by scavenging a DHR radical intermediate.
In summary, our data identify (ꢀ)-epicatechin as selec-
tive inhibitor of NO-related oxidation and nitration reac-
tions that are thought to mediate the detrimental outcome
of NO overproduction in inflammatory diseases. Together
with the reported increased expression and/or activity of
eNOS upon long-term exposure to (ꢀ)-epicatechin and
related polyphenols, our results provide a molecular basis
for the well documented protective cardiovascular effects
of red wine and green tea.
4.3. Effect of (ꢀ)-epicatechin on nitration of
protein-bound tyrosine in HL-60 cells
At submicromolar steady-state concentrations, peroxy-
nitrite does not cause considerable nitration of free or
protein-bound tyrosine due to predominant dimerization
of tyrosyl radicals to yield dityrosine [63]. Accordingly,
NO and O2 ꢀ, co-generated endogenously from physiolo-
ꢁ
gical sources (iNOS and NADPH oxidase, respectively) or
from exogenous donor systems, do not cause significant
nitration of cellular proteins [64,65], calling into question
the pathophysiological significance of nitration by perox-
ynitrite. To account for the pivotal role of MPO and other
heme peroxidases in tyrosine nitration under inflammatory
conditions [66–69], we used phorbolester-stimulated HL-
60 cells expressing large amounts of active MPO to study
the effect of (ꢀ)-epicatechin on peroxidase-catalyzed
nitration of cellular proteins. The potency of the flavonoid
to inhibit tyrosine nitration in the presence of nitrite (but in
the absence of detectable NOS activity; data not shown)
was identical to that found for nitration of free tyrosine in
buffer, strongly suggesting the involvement of the same
reactive intermediate in peroxynitrite- and MPO-triggered
nitration. Previous suggestions have invoked nitryl chlor-
ide (NO2Cl), formed by the reaction of the MPO product
ꢁ
hypochlorous acid with nitrite, or NO2 , formed by MPO-
catalyzed oxidation of nitrite, as alternative nitrating spe-
cies [66,67]. The similar sensitivity of (ꢀ)-epicatechin to
peroxynitrite- and MPO-mediated nitration favors the
Acknowledgments
¨
This work was supported by the Fonds zur Forderung der
Wissenschaftlichen Forschung in Osterreich and by a grant
ꢁ
¨
NO2 pathway, a conclusion which is also supported by
a recent paper demonstrating lack of tyrosine nitration by
nitryl chloride [18]. In contrast to peroxynitrite-triggered
nitration of free tyrosine in buffer, there was a significant
(ꢀ)-epicatechin-insensitive component of protein tyrosine
nitration in HL-60 cells. Since MPO was completely
inhibited by high concentrations of the flavonoid
(ic50 ꢃ 80 mM), the residual nitrating activity appears to
be unrelated to MPO activity.
from the Human Frontier Science Program (HFSP):
RGP0026/2001-M.
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