Carvedilol Inhibits Reactive Oxygen Species Generation by

Leukocytes and Oxidative Damage to Amino Acids

Paresh Dandona, MD; Rajaram Karne, MBBS; Husam Ghanim, BS; Wael Hamouda, MS;
Ahmad Aljada, PhD; Cesar H. Magsino, Jr, MD

BackgroundThe purpose of this study was to test whether carvedilol has an antioxidant effect in humans in vivo.
Methods and ResultsWe administered 3.125 mg of carvedilol twice daily to normal subjects for 1 week. ROS generation
by polymorphonuclear leukocytes and mononuclear cells fell from 3146183.43 and 3036116 mV to 1856157 and
189663 mV (P,0.025), respectively. m-Tyrosine fell from 4.2460.99 to 4.0360.97 ng/mL (P50.01), and o-tyrosine
fell from 4.5961.10 to 4.2460.99 ng/mL (P50.004) in the absence of a change in phenylalanine concentrations.
ConclusionsWe conclude that carvedilol significantly inhibits ROS generation by leukocytes and oxidative conversion
of phenylalanine to m- and o-tyrosine. (Circulation. 2000;101:122-124.)
Key Words: carvedilol n oxygen n leukocytes n amino acids
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I t has been suggested that carvedilol may provide greater

benefit than traditional b-blockers in chronic heart failure
because of its antioxidant actions that synergize with its
nonspecific b- and a-blocking effects.1 Carvedilol has been
shown to inhibit lipid peroxidation of myocardial cell mem-
branes and thus protect endothelial, neuronal, and vascular
smooth muscle cells from oxygen radicalmediated injury.2
Carvedilol has also been shown to scavenge peroxy and
hypochlorous radicals in chemical systems in vitro.3 In the
only study in humans, carvedilol was shown to have antiox-
idant actions in patients treated with moderate doses of 25
mg/d as assessed by suppression of ex vivo LDL oxidation
and reduction of anti oxidized LDL antibodies in vivo.4
Ortho-tyrosine (o-tyrosine) and meta-tyrosine (m-tyrosine)
have recently been shown to be useful markers of oxidative
damage to phenylalanine, because they are formed after
reactive oxygen species (ROS) attack on phenylalanine.
Thus, their concentration is considered to be an index of
oxidative damage to amino acids and proteins.
We undertook this study to investigate the effect of
carvedilol administration on ROS generation by polymorpho-
nuclear leukocytes (PMNLs) and mononuclear cells (MNCs).
We also measured o-tyrosine and m-tyrosine in plasma as
indices of oxidative damage to phenylalanine.
Methods
Eight normal subjects 26 to 33 years old volunteered for the study.
The study was approved by the Institutional Review Board of the
State University of New York at Buffalo. Written, informed consent
was obtained from each subject. None of the subjects were on any
medications, including NSAIDS, vitamin E, or other antioxidants.
Fasting blood samples were collected at baseline in tubes with
EDTA as an anticoagulant. The subjects were given 3.125 mg of
carvedilol PO twice a day for 7 days. On day 8, another fasting blood
sample was collected as above. ROS generation by PMNLs and
MNCs and levels of o-tyrosine and m-tyrosine in plasma were
measured at baseline and on day 8.
A group of 6 control subjects, not given any drugs, also had 2
fasting blood samples taken 1 week apart without any drug
intervention.
Preparation of PMNLs and MNCs
PMNLs and MNCs were prepared, washed, and suspended in HBSS
as previously described.5
Measurement of ROS Generation
Respiratory burst activity of PMNLs and MNCs was measured by
detection of superoxide radical via chemiluminescence.6 Five hun-
dred microliters of PMNLs or MNCs (23105 cells) was delivered
into a Lumiaggregometer (Chronolog) plastic flat-bottom cuvette to
which a spin bar was added. Fifteen microliters of 10 mmol/L
luminol was then added, followed by 1 mL of 10 mmol/L formyl-
methionylleucinylphenylalanine (FMLP). Chemiluminescence was
recorded for 15 minutes (a protracted record after 15 minutes did not
alter the relative amounts of chemiluminescence produced by various
cell samples). Our method, developed independently, is similar to
that published by Tosi and Hamedani.7 The interassay coefficient of
variation (CV) for this assay is 6%. We have further established that
in our assay system, there is a dose-dependent inhibition of chemi-
luminescence by superoxide dismutase and catalase: superoxide
dismutase inhibited chemiluminescence by 82% at 10 mg/mL,
whereas catalase inhibited chemiluminescence by 47% at 40 mg/mL.
Chemiluminescence is also inhibited by diphenyleneiodonium chlo-
ride (data not shown), a specific inhibitor of NADPH oxidase, the
enzyme responsible for the production of superoxide radicals.8 Our
assay system is exquisitely sensitive to diphenyleneiodonium chlo-
ride at nanomolar concentrations.

Received June 22, 1999; revision received October 20, 1999; accepted November 2, 1999.
From the Division of Endocrinology, Diabetes, and Metabolism, State University of New York at Buffalo and Kaleida Health, Buffalo, NY.
Correspondence to Paresh Dandona, MD, Director, Diabetes Endocrinology Center of Western New York, Division of Endocrinology, Diabetes, and
Metabolism, State University of New York at Buffalo and Kaleida Health, 3 Gates Circle, Buffalo, NY 14209. E-mail pdandona@kaleidahealth.org
2000 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org

122
 Dandona et al Carvedilol Inhibits ROS Generation 123
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Figure 1. ROS generation by PMNLs before and after 7-day
treatment with carvedilol. ROS generationinduced chemilumi-
nescence was measured in millivolts, assayed in 200 000 MNCs
in HBSS, and stimulated with FMLP.
Assay of o-Tyrosine, m-Tyrosine,
and Phenylalanine
o-Tyrosine, m-tyrosine, and phenylalanine were measured by high
performance liquid chromatography using the technique described
by Ishimitsu et al.9
Statistical Analysis
Comparisons of the ROS generation values at baseline and on day 8
were carried out by Wilcoxon rank sum test, because the distribution of
the values was not normally distributed. The values of o-tyrosine and
m-tyrosine before and after carvedilol were compared by paired t test.
Figure 2. ROS generation by MNC (in millivolts).
Plasma phenylalanine concentration did not change after
carvedilol. Plasma m-tyrosine concentration fell from
4.2460.99 to 4.0360.97 ng/mL (P50.01). The ratio of
m-tyrosine to phenylalanine changed from 0.3560.07 to
0.3360.07 mmol/mol phenylalanine (P50.005).
Plasma o-tyrosine concentration fell from 4.5961.10 to
4.2460.90 ng/mL (P50.004). The ratio of o-tyrosine to
TABLE 1. Plasma m-Tyrosine and o-Tyrosine Concentrations
(ng/mL) and Phenylalanine Concentrations (mg/mL) Before and
After 7 Days of Treatment With 6.25 mg Carvedilol
m-Tyrosine o-Tyrosine Phenylalanine

Results Subject Baseline Day 8 Baseline Day 8 Baseline Day 8

ROS generation by PMNLs at baseline was 313.756183.43 1 4.20 4.02 4.41 4.36 11.68 11.43
mV (mean6SD). After carvedilol administration, ROS gen-
2 4.19 3.75 3.93 3.69 10.98 10.93
eration fell to 185.006156.98 mV. The mean fall was
3 3.90 3.56 5.46 4.69 10.65 10.23
43.75615.31% (range, 20% to 65%; P50.025) (Figure 1).
The mean ROS generation by PMNLs in the control group 4 6.13 5.77 6.56 5.80 12.56 12.35
was 544685 mV at baseline, and it was 515680 mV 1 week 5 4.54 4.67 4.83 4.57 10.10 10.27
later (P5NS). 6 4.65 4.55 4.87 4.59 11.22 11.56
ROS generation by MNCs at baseline was 302.506115.70 7 3.66 3.33 3.24 2.98 10.65 10.52
mV (mean6SD). After carvedilol administration, ROS genera- 8 2.63 2.60 3.40 3.27 11.35 11.56
tion fell to 189.25663.09 mV. The mean fall was Mean 4.24 4.03 4.59 4.24 11.15 11.11
34.77614.58% (range, 21% to 57%; P50.025) (Figure 2).
SD 0.99 0.97 1.10 0.90 0.75 0.75
Mean ROS generation by MNCs in control subjects was
(P50.01) (P50.04) (P50.329)
336645 mV at baseline and 330642 mV a week later (P5NS).
 124 Circulation January 18, 2000

TABLE 2. Plasma m-Tyrosine/Phenylalanine and rate of hospital admissions.1 Although the reduction in sudden
o-Tyrosine/Phenylalanine Ratios Before and After 7 Days of death in such patients may be a function of the antiarrhythmic
Treatment With 6.25 mg Carvedilol (mmol/mol Phenylalanine) effects of carvedilol, the reduction of deterioration of congestive
m-Tyrosine/ o-Tyrosine/ heart failure may be due to its antioxidant effects, possibly
Phenylalanine Phenylalanine through the protection of the myocardium from ROS damage.
The mechanism underlying this inhibitory effect of carve-
Subject Baseline Day 8 Baseline Day 8
dilol on ROS generation is not clear from our data. Carvedilol
1 0.33 0.32 0.34 0.35 has been shown to possess antioxidant properties in various
2 0.35 0.31 0.33 0.31 animal models.9,10 The experimental data have been focused
3 0.33 0.32 0.47 .042 on carvedilol as a chemical antioxidant. There is only 1 report
4 0.45 0.43 0.48 0.43 based on a human study, which demonstrates that the ex vivo
5 0.31 0.29 0.28 0.26
oxidizability of LDL prepared from sera of patients treated
with carvedilol is significantly diminished.11 Our assay sys-
6 0.38 0.36 0.40 0.36
tem determines actual ROS generation by leukocytes, thus
7 0.41 0.42 0.44 0.41
focusing on the biological antioxidant property of carvedilol.
8 0.21 0.21 0.27 0.26 It is possible that carvedilol exerts its antioxidant effect by
Mean 0.35 0.33 0.37 0.35 both chemical and biological mechanisms.
SD 0.07 0.07 0.08 0.07 In conclusion, we have demonstrated that carvedilol inhibits
(P50.005) (P50.002) ROS generation by PMNLs and MNCs significantly, even after a
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short-term treatment at a relatively small dose. This reduction in

phenylalanine changed from 0.3760.08 to 0.3560.07 mmol/mol
phenylalanine (P50.002) (Tables 1 and 2).
Discussion
Our data demonstrate clearly that ROS generation by both PMNLs
and MNCs is significantly diminished by the administration of
carvedilol at a small dose (6.25 mg/d) to normal subjects. One week
of carvedilol treatment resulted in a 44% reduction in ROS
generation by PMNLs and a 35% reduction in that by MNCs. In
contrast, ROS generation by leukocytes in normal subjects not
given any drugs did not change significantly over a period of 1
week. Whether other cells in the body also respond to carvedilol by
reducing ROS generation is not clear from our study. If this effect
of carvedilol is indeed extended to other cells and tissues, the total
oxidative load of individuals on carvedilol should diminish mark-
edly after treatment with this drug. This would be reflected in the
reduction of oxidative damage to the body as measured by such
indices as o- and m-tyrosines. Oxidative damage may be an
important mechanism underlying several pathophysiological states,
eg, atherosclerosis due to oxidative modification of LDL10; diabetic
complications due to oxidative damage of lipids, proteins,11 and
DNA12; aging due to oxidative damage of proteins; and myocardial
damage/loss through oxidative injury.
Our data also demonstrate that o-tyrosine and m-tyrosine con-
centrations fall without a change in phenylalanine concentrations.
Because o- and m-tyrosine are formed by ROS attack on phenyl-
alanine, our data indicate that ROS-induced oxidative damage to
amino acids and proteins falls in association with the decline in ROS
generation by PMNLs and MNCs. This has implications for cellular
and extracellular proteins, including enzymes, and their physiolog-
ical functions. It is possible that apoprotein and lipoprotein mole-
cules may also be involved in ROS-induced damage.
The magnitude of ROS inhibition by MNCs and PMNLs (35%
and 44%) was comparable to that observed after administration of
vitamin E (1200 IU/d) for 8 weeks, a reduction of '50% in
superoxide radical generation and H2O2 production by monocytes.13
Carvedilol has been shown to improve outcomes in conges-
tive heart failure by reducing morbidity and mortality and the
ROS generation probably contributes to the diminished conversion
of phenylalanine to o- and m-tyrosine and to the previously
described antioxidant effects and related clinical benefits.
Acknowledgments
The authors thank Dr Rajesh Garg, DM, for his critical comments in
relation to this work and Pamela Maher for preparation of
the manuscript.
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 Carvedilol Inhibits Reactive Oxygen Species Generation by Leukocytes and Oxidative
Damage to Amino Acids
Paresh Dandona, Rajaram Karne, Husam Ghanim, Wael Hamouda, Ahmad Aljada and Cesar H.
Magsino, Jr

Circulation. 2000;101:122-124
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doi: 10.1161/01.CIR.101.2.122
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright 2000 American Heart Association, Inc. All rights reserved.
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