This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Dandona, P. Articles by Magsino, C. H. Search for Related Content PubMed PubMed Citation Articles by Dandona, P. Articles by Magsino, C. H., Jr Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB (L)-PHENYLALANINE CARVEDILOL L-TYROSINE Medline Plus Health Information Antioxidants Related Collections Oxidant stress

(Circulation. 2000;101:122.)
© 2000 American Heart Association, Inc.

Brief Rapid Communications

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

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{at}kaleidahealth.org

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—The purpose of this study was to test whether carvedilol has an antioxidant effect in humans in vivo.

Methods and Results—We administered 3.125 mg of carvedilol twice daily to normal subjects for 1 week. ROS generation by polymorphonuclear leukocytes and mononuclear cells fell from 314±183.43 and 303±116 mV to 185±157 and 189±63 mV (P<0.025), respectively. m-Tyrosine fell from 4.24±0.99 to 4.03±0.97 ng/mL (P=0.01), and o-tyrosine fell from 4.59±1.10 to 4.24±0.99 ng/mL (P=0.004) in the absence of a change in phenylalanine concentrations.

Conclusions—We conclude that carvedilol significantly inhibits ROS generation by leukocytes and oxidative conversion of phenylalanine to m- and o-tyrosine.

Key Words: carvedilol • oxygen • leukocytes • amino acids

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
It has been suggested that carvedilol may provide greater benefit than traditional ß-blockers in chronic heart failure because of its antioxidant actions that synergize with its nonspecific ß- and -blocking effects.¹ Carvedilol has been shown to inhibit lipid peroxidation of myocardial cell membranes and thus protect endothelial, neuronal, and vascular smooth muscle cells from oxygen radical–mediated 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 antioxidant 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 polymorphonuclear leukocytes (PMNLs) and mononuclear cells (MNCs). We also measured o-tyrosine and m-tyrosine in plasma as indices of oxidative damage to phenylalanine.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
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 hundred microliters of PMNLs or MNCs (2x10⁵ 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 µL of 10 mmol/L formylmethionylleucinylphenylalanine (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 chemiluminescence by superoxide dismutase and catalase: superoxide dismutase inhibited chemiluminescence by 82% at 10 µg/mL, whereas catalase inhibited chemiluminescence by 47% at 40 µg/mL. Chemiluminescence is also inhibited by diphenyleneiodonium chloride (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 chloride at nanomolar concentrations.

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.

	Results

Top Abstract Introduction Methods Results Discussion References

 
ROS generation by PMNLs at baseline was 313.75±183.43 mV (mean±SD). After carvedilol administration, ROS generation fell to 185.00±156.98 mV. The mean fall was 43.75±15.31% (range, 20% to 65%; P=0.025) (Figure 1). The mean ROS generation by PMNLs in the control group was 544±85 mV at baseline, and it was 515±80 mV 1 week later (P=NS).

View larger version (13K): [in this window] [in a new window]   Figure 1. ROS generation by PMNLs before and after 7-day treatment with carvedilol. ROS generation–induced chemiluminescence was measured in millivolts, assayed in 200 000 MNCs in HBSS, and stimulated with FMLP.

ROS generation by MNCs at baseline was 302.50±115.70 mV (mean±SD). After carvedilol administration, ROS generation fell to 189.25±63.09 mV. The mean fall was 34.77±14.58% (range, 21% to 57%; P=0.025) (Figure 2). Mean ROS generation by MNCs in control subjects was 336±45 mV at baseline and 330±42 mV a week later (P=NS).

View larger version (13K): [in this window] [in a new window]   Figure 2. ROS generation by MNC (in millivolts).

Plasma phenylalanine concentration did not change after carvedilol. Plasma m-tyrosine concentration fell from 4.24±0.99 to 4.03±0.97 ng/mL (P=0.01). The ratio of m-tyrosine to phenylalanine changed from 0.35±0.07 to 0.33±0.07 mmol/mol phenylalanine (P=0.005).

Plasma o-tyrosine concentration fell from 4.59±1.10 to 4.24±0.90 ng/mL (P=0.004). The ratio of o-tyrosine to phenylalanine changed from 0.37±0.08 to 0.35±0.07 mmol/molphenylalanine (P=0.002) (Tables 1 and 2).

View this table: [in this window] [in a new window]   Table 1. Plasma m-Tyrosine and o-Tyrosine Concentrations (ng/mL) and Phenylalanine Concentrations (µg/mL) Before and After 7 Days of Treatment With 6.25 mg Carvedilol

View this table: [in this window] [in a new window]   Table 2. Plasma m-Tyrosine/Phenylalanine and o-Tyrosine/Phenylalanine Ratios Before and After 7 Days of Treatment With 6.25 mg Carvedilol (mmol/mol Phenylalanine)

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
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 markedly 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 concentrations fall without a change in phenylalanine concentrations. Because o- and m-tyrosine are formed by ROS attack on phenylalanine, 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 physiological functions. It is possible that apoprotein and lipoprotein molecules 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 congestive heart failure by reducing morbidity and mortality and the rate of hospital admissions.¹ Although the reduction in sudden death in such patients may be a function of the antiarrhythmic effects of carvedilol, the reduction of deterioration of congestive heart failure may be due to its antioxidant effects, possibly through the protection of the myocardium from ROS damage.

The mechanism underlying this inhibitory effect of carvedilol on ROS generation is not clear from our data. Carvedilol has been shown to possess antioxidant properties in various animal models.^{9 10} The experimental data have been focused on carvedilol as a chemical antioxidant. There is only 1 report based on a human study, which demonstrates that the ex vivo oxidizability of LDL prepared from sera of patients treated with carvedilol is significantly diminished.¹¹ Our assay system determines actual ROS generation by leukocytes, thus focusing on the biological antioxidant property of carvedilol. It is possible that carvedilol exerts its antioxidant effect by both chemical and biological mechanisms.

In conclusion, we have demonstrated that carvedilol inhibits ROS generation by PMNLs and MNCs significantly, even after a short-term treatment at a relatively small dose. This reduction in 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.

Received June 22, 1999; revision received October 20, 1999; accepted November 2, 1999.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Packer M, Bristow MR, Cohn JN, Colucci WS, Fowler MB, Gilbert EM, Shusterman NH (for US Carvedilol Heart Failure Study Group). The effect of carvedilol on morbidity and mortality in patients with chronic heart failure. N Engl J Med. 1996;334:1349–1355.[Abstract/Free Full Text]
   
2. Feuerstein G, Shusterman NH, Ruffolo RR Jr. Carvedilol update IV: prevention of oxidative stress, cardiac remodeling and progression of congestive cardiac failure. Drugs Today. 1997;33:453–473.
   
3. Aruoma OI. Scavenging of hypochlorous acid by carvedilol and ebselen in vitro. Gen Pharmacol. 1997;28:269–272.[Medline] [Order article via Infotrieve]
   
4. Maggi E, Marchesi E, Covini D, Negro C, Perani G, Giorgio B. Protective effect of carvedilol, a vasodilating ß-blocker, against low density lipoprotein oxidation in essential hypertension. J Cardiovasc Pharmacol. 1996;27:532–538.[Medline] [Order article via Infotrieve]
   
5. Ferrante A, Thong YH. Optimal conditions for simultaneous purification of mononuclear and polymorphonuclear leucocytes from human blood by the Hypaque-Ficoll technique. J Immunol Methods. 1980;36:109–114.[Medline] [Order article via Infotrieve]
   
6. Dandona P, Thusu K, Hafeez R, Abdel-Rahman E, Chaudhuri A. Effect of hydrocortisone on oxygen free radical generation by mononuclear cells. Metabolism. 1998;47:788–791.[Medline] [Order article via Infotrieve]
   
7. Tosi MF, Hamedani A. A rapid specific assay for superoxide release from phagocytes in small volumes of whole blood. Am J Clin Pathol. 1992;97:566–573.[Medline] [Order article via Infotrieve]
   
8. Hancock JT, Jones OT. The inhibition of dephenylene iodonium and its analogues of superoxide generation by macrophages. Biochem J. 1987;242:103–107.[Medline] [Order article via Infotrieve]
   
9. Ishimitsu S, Fujimoto S, Ohara A. Determination of m-tyrosine and o-tyrosine in human serum by high performance liquid chromatography with fluorometric detection. J Chromatogr. 1986;378:222–225.[Medline] [Order article via Infotrieve]
   
10. Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med. 1999;340:115–126.[Free Full Text]
    
11. Baynes JW, Thorpe SR. Role of oxidative stress in diabetic complications: a new perspective on an old paradigm. Diabetes. 1999;48:1–9.[Abstract]
    
12. Dandona P, Thusu K, Cook S, Snyder B, Makowski J, Armstrong D, Nicotera T. Oxidative damage to DNA in diabetes mellitus. Lancet. 1996;347:444–445.[Medline] [Order article via Infotrieve]
    
13. Devaraj S, Li D, Jialal I. The effects of alpha tocopherol supplementation on monocyte function. J Clin Invest. 1996;98:756–763.[Medline] [Order article via Infotrieve]
    

This article has been cited by other articles:

				T. M. Paravicini and R. M. Touyz NADPH Oxidases, Reactive Oxygen Species, and Hypertension: Clinical implications and therapeutic possibilities Diabetes Care, February 1, 2008; 31(Supplement_2): S170 - S180. [Abstract] [Full Text] [PDF]

				N. Kalay, E. Basar, I. Ozdogru, O. Er, Y. Cetinkaya, A. Dogan, T. Inanc, A. Oguzhan, N. K. Eryol, R. Topsakal, et al. Protective Effects of Carvedilol Against Anthracycline-Induced Cardiomyopathy J. Am. Coll. Cardiol., November 8, 2006; (2006) j.jacc.2006.07.052v1. [Abstract] [Full Text] [PDF]

				B. J. Biesiadecki and R. J. Solaro When Hearts Fail So Does Skeletal Muscle: Breaking a Vicious Cycle Circ. Res., June 23, 2006; 98(12): 1456 - 1458. [Full Text] [PDF]

				S. Dhindsa, D. Tripathy, N. Sanalkumar, S. Ravishankar, H. Ghanim, A. Aljada, and P. Dandona Free Fatty Acid-Induced Insulin Resistance in the Obese Is Not Prevented by Rosiglitazone Treatment J. Clin. Endocrinol. Metab., September 1, 2005; 90(9): 5058 - 5063. [Abstract] [Full Text] [PDF]

				J. Lee, M. Lee, J.-U. Kim, K. I. Song, Y. S. Choi, and S.-S. Cheong Carvedilol Reduces Plasma 8-Hydroxy-2'-Deoxyguanosine in Mild to Moderate Hypertension: A Pilot Study Hypertension, May 1, 2005; 45(5): 986 - 990. [Abstract] [Full Text] [PDF]

				A. Aljada, P. Mohanty, H. Ghanim, T. Abdo, D. Tripathy, A. Chaudhuri, and P. Dandona Increase in intranuclear nuclear factor {kappa}B and decrease in inhibitor {kappa}B in mononuclear cells after a mixed meal: evidence for a proinflammatory effect Am. J. Clinical Nutrition, April 1, 2004; 79(4): 682 - 690. [Abstract] [Full Text] [PDF]

				Z. Yuan, K. Shioji, Y. Kihara, H. Takenaka, Y. Onozawa, and C. Kishimoto Cardioprotective effects of carvedilol on acute autoimmune myocarditis: anti-inflammatory effects associated with antioxidant property Am J Physiol Heart Circ Physiol, January 1, 2004; 286(1): H83 - H90. [Abstract] [Full Text] [PDF]

				M. Drent, A. P. Gorgels, and A. Bast Cardiac Failure Associated With G6PD Deficiency Circ. Res., October 17, 2003; 93 (8): e75 - e75. [Full Text] [PDF]

				P. Dandona, V. Kumar, A. Aljada, H. Ghanim, T. Syed, D. Hofmayer, P. Mohanty, D. Tripathy, and R. Garg Angiotensin II Receptor Blocker Valsartan Suppresses Reactive Oxygen Species Generation in Leukocytes, Nuclear Factor-{kappa}B, in Mononuclear Cells of Normal Subjects: Evidence of an Antiinflammatory Action J. Clin. Endocrinol. Metab., September 1, 2003; 88(9): 4496 - 4501. [Abstract] [Full Text] [PDF]

				S.-P. Yang, L.-J. Ho, Y.-L. Lin, S.-M. Cheng, T.-P. Tsao, D.-M. Chang, Y.-L. Hsu, C.-Y. Shih, T.-Y. Juan, and J.-H. Lai Carvedilol, a new antioxidative {beta}-blocker, blocks in vitro human peripheral blood T cell activation by downregulating NF-{kappa}B activity Cardiovasc Res, September 1, 2003; 59(3): 776 - 787. [Abstract] [Full Text] [PDF]

				P. Dandona, A. Aljada, A. Chaudhuri, and A. Bandyopadhyay The Potential Influence of Inflammation and Insulin Resistance on the Pathogenesis and Treatment of Atherosclerosis-Related Complications in Type 2 Diabetes J. Clin. Endocrinol. Metab., June 1, 2003; 88(6): 2422 - 2429. [Full Text] [PDF]

				A. Avogaro, E. Pagnin, and L. Calo Monocyte NADPH Oxidase Subunit p22phox and Inducible Hemeoxygenase-1 Gene Expressions Are Increased in Type II Diabetic Patients: Relationship with Oxidative Stress J. Clin. Endocrinol. Metab., April 1, 2003; 88(4): 1753 - 1759. [Abstract] [Full Text] [PDF]

				E. Azeka, J. A. Franchini Ramires, C. Valler, and E. Alcides Bocchi Delisting of infants and children from the heart transplantation waiting list after carvedilol treatment J. Am. Coll. Cardiol., December 4, 2002; 40(11): 2034 - 2038. [Abstract] [Full Text] [PDF]

				F.D.R. Hobbs, J.E. Kenkre, A.K. Roalfe, R.C. Davis, R. Hare, and M.K. Davies Impact of heart failure and left ventricular systolic dysfunction on quality of life. A cross-sectional study comparing common chronic cardiac and medical disorders and a representative adult population Eur. Heart J., December 1, 2002; 23(23): 1867 - 1876. [Abstract] [Full Text] [PDF]

				K. Nakamura, K. Kusano, Y. Nakamura, M. Kakishita, K. Ohta, S. Nagase, M. Yamamoto, K. Miyaji, H. Saito, H. Morita, et al. Carvedilol Decreases Elevated Oxidative Stress in Human Failing Myocardium Circulation, June 18, 2002; 105(24): 2867 - 2871. [Abstract] [Full Text] [PDF]

				P. Mohanty, H. Ghanim, W. Hamouda, A. Aljada, R. Garg, and P. Dandona Both lipid and protein intakes stimulate increased generation of reactive oxygen species by polymorphonuclear leukocytes and mononuclear cells Am. J. Clinical Nutrition, April 1, 2002; 75(4): 767 - 772. [Abstract] [Full Text] [PDF]

				S. Engelhardt, Y. Grimmer, G.-H. Fan, and M. J. Lohse Constitutive Activity of the Human beta 1-Adrenergic Receptor in beta 1-Receptor Transgenic Mice Mol. Pharmacol., October 1, 2001; 60(4): 712 - 717. [Abstract] [Full Text] [PDF]

				A. Aljada, H. Ghanim, J. Friedman, R. Garg, P. Mohanty, and P. Dandona Troglitazone Reduces the Expression of PPAR{{gamma}} While Stimulating That of PPAR{{alpha}} in Mononuclear Cells in Obese Subjects J. Clin. Endocrinol. Metab., July 1, 2001; 86(7): 3130 - 3133. [Abstract] [Full Text] [PDF]

				A. Aljada, R. Garg, H. Ghanim, P. Mohanty, W. Hamouda, E. Assian, and P. Dandona Nuclear Factor-{{kappa}}B Suppressive and Inhibitor-{{kappa}}B Stimulatory Effects of Troglitazone in Obese Patients with Type 2 Diabetes: Evidence of an Antiinflammatory Action? J. Clin. Endocrinol. Metab., July 1, 2001; 86(7): 3250 - 3256. [Abstract] [Full Text] [PDF]

				M. Packer, A. J.S. Coats, M. B. Fowler, H. A. Katus, H. Krum, P. Mohacsi, J. L. Rouleau, M. Tendera, A. Castaigne, E. B. Roecker, et al. Effect of Carvedilol on Survival in Severe Chronic Heart Failure N. Engl. J. Med., May 31, 2001; 344(22): 1651 - 1658. [Abstract] [Full Text] [PDF]

				P. Dandona, P. Mohanty, H. Ghanim, A. Aljada, R. Browne, W. Hamouda, A. Prabhala, A. Afzal, and R. Garg The Suppressive Effect of Dietary Restriction and Weight Loss in the Obese on the Generation of Reactive Oxygen Species by Leukocytes, Lipid Peroxidation, and Protein Carbonylation J. Clin. Endocrinol. Metab., January 1, 2001; 86(1): 355 - 362. [Abstract] [Full Text]

				L. Rossig, J. Haendeler, Z. Mallat, B. Hugel, J.-M. Freyssinet, A. Tedgui, S. Dimmeler, and A. M. Zeiher Congestive heart failure induces endothelial cell apoptosis: protective role of carvedilol J. Am. Coll. Cardiol., December 1, 2000; 36(7): 2081 - 2089. [Abstract] [Full Text] [PDF]

				R. Garg, Y. Kumbkarni, A. Aljada, P. Mohanty, H. Ghanim, W. Hamouda, and P. Dandona Troglitazone Reduces Reactive Oxygen Species Generation by Leukocytes and Lipid Peroxidation and Improves Flow-Mediated Vasodilatation in Obese Subjects Hypertension, September 1, 2000; 36(3): 430 - 435. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Dandona, P. Articles by Magsino, C. H. Search for Related Content PubMed PubMed Citation Articles by Dandona, P. Articles by Magsino, C. H., Jr Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB (L)-PHENYLALANINE CARVEDILOL L-TYROSINE Medline Plus Health Information Antioxidants Related Collections Oxidant stress