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Current Therapeutic Research, Clinical and Experimental logoLink to Current Therapeutic Research, Clinical and Experimental
. 2008 Oct;69(5):423–439. doi: 10.1016/j.curtheres.2008.10.006

Effect of intravenous administration of antioxidants alone and in combination on myocardial reperfusion injury in an experimental pig model

Dimitrios N Nikas 1,a, Georgios Chatziathanasiou 1, Anna Kotsia 1, Nikos Papamichael 2, Christoforos Thomas 3, Michail Papafaklis 1, Katerina K Naka 1,2, Nikos Kazakos 1, Haralampos J Milionis 4, Kostas Vakalis 1, Christos S Katsouras 1,2, Vasiliki Mpoumpa 5, Theodoros Vougiouklakis 5, Lampros Michalis 1,2
PMCID: PMC3969921  PMID: 24692817

Abstract

Background: Several antioxidants have been found to have conflicting results in attenuating myocardial reperfusion injury. These studies were done primarily in experimental protocols that did not approximate clinical situations.

Objective: The aim of this study was to test the efficacy of 3 different antioxidants (ascorbic acid [AA], desferrioxamine, and N-acetylcysteine [NAC]) administered IV alone and in combination in a closed-chest pig model.

Methods: Farm-raised domestic male pigs (aged 3–5 months, weight of 30–35 kg) were assigned to 1 of 5 groups to receive treatment as follows: group A, AA 100 mg/kg; group B, desferrioxamine 60 mg/kg; group C, a loading dose of NAC 100 mg/kg for 20 minutes and a 20-mg/kg maintenance dose; group D, all 3 drugs in combination; and group E, normal saline (control group). The infusion of all drugs was started 15 minutes before and completed 5 minutes after reperfusion, except for the administration of NAC, which was terminated 60 minutes postreperfusion. Myocardial ischemia (45 minutes) and reperfusion (210 minutes) were achieved percutaneously by circumflex artery balloon occlusion. Ejection fraction, left ventricular end-diastolic pressure (LVEDP), flow in the infarcted artery, and all ventricular arrhythmias were recorded. Oxidative stress was estimated by serial measurements of thiobarbituric acid reactive substance (TBARS) concentration in coronary sinus blood. Infarct size was assessed as a percentage of the area at risk (I/R ratio) using the tetrazolium red staining method.

Results: The 25 pigs were divided into 5 groups of 5 pigs each. No significant between-group differences were found in I/R ratio or in oxidative stress (as measured by TBARS concentration). Group C developed significantly more ventricular atrhythmias than the control group (80% vs 0%, P = 0.02). No other differences among groups were found. LVEDP was significantly elevated in all treatment groups (mean LVEDP difference [SD] for group A, 6.0 [1.6] mm Hg; group B, 17.6 [1.9] mm Hg; group C, 3.6 [1.7] mm Hg; group D, 6.8 [3.2] and group E, 5.4 [3.4] mm Hg). LVEDP elevation was found to be significantly higher in group B compared with all the other groups (all, P < 0.001). No significant between-group differences were found in the other parameters measured.

Conclusion: In this experimental pig model, the antioxidants AA, desferrioxamine, and NAC administered alone or in combination did not reduce the deleterious effects of reperfusion injury and specifically the extent of myocardial necrosis.

Key Words: myocardial infarction, reperfusion injury, antioxidants, combination

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References

  • 1.Kloner RA, Arimie RB, Kay GL. Evidence for stunned myocardium in humans: A 2001 update. Coron Artery Dis. 2001;12:349–356. doi: 10.1097/00019501-200108000-00003. [DOI] [PubMed] [Google Scholar]
  • 2.Coronel R. Heterogeneity in extracellular potassium concentration during early myocardial ischaemia and reperfusion: Implications for arrhythmogenesis. Cardiovasc Res. 1994;28:770–777. doi: 10.1093/cvr/28.6.770. [DOI] [PubMed] [Google Scholar]
  • 3.VanBenthuysen KM, McMurtry IF, Horwitz LD. Reperfusion after acute coronary occlusion in dogs impairs endothelium-dependent relaxation to acetylcholine and augments contractile reactivity in vitro. J Clin Invest. 1987;79:265–274. doi: 10.1172/JCI112793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Lesnefsky EJ, Dauber IM, Horwitz LD. Myocardial sulfhydryl pool alterations occur during reperfusion after brief and prolonged myocardial ischemia in vivo. Circ Res. 1991;68:605–613. doi: 10.1161/01.res.68.2.605. [DOI] [PubMed] [Google Scholar]
  • 5.Bolli R, Jeroudi MO, Patel BS. Direct evidence that oxygen-derived free radicals contribute to postischemic myocardial dysfunction in the intact dog. Proc Natl Acad Sci U S A. 1989;86:4695–4699. doi: 10.1073/pnas.86.12.4695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Ambrosio G, Flaherty JT, Duilio C. Oxygen radicals generated at reflow induce peroxidation of membrane lipids in reperfused hearts. J Clin Invest. 1991;87:2056–2066. doi: 10.1172/JCI115236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Dhalla NS, Elmoselhi AB, Hata T, Makino N. Status of myocardial antioxidants in ischemia-reperfusion injury. Cardiovasc Res. 2000;47:446–456. doi: 10.1016/s0008-6363(00)00078-x. [DOI] [PubMed] [Google Scholar]
  • 8.Moens AL, Claeys MJ, Timmermans JP, Vrints CJ. Myocardial ischemia/reperfusion-injury, a clinical view on a complex pathophysiological process. Int J Cardiol. 2005;100:179–190. doi: 10.1016/j.ijcard.2004.04.013. [DOI] [PubMed] [Google Scholar]
  • 9.Collis CS, Davies MJ, Rice-Evans C. Comparison of N-methyl hexanoylhydroxamic acid, a novel antioxidant, with desferrioxamine and N-acetyl cysteine agains reperfusion-induced dysfunctions in isolated rat heart. J Cardiovasc Pharmacol. 1993;22:336–342. doi: 10.1097/00005344-199308000-00025. [DOI] [PubMed] [Google Scholar]
  • 10.Kupatt C, Hinkel R, Horstkotte J. Selective retroinfusion of GSH end cariporide attenuates myocardial ischemia-reperfusion injury in a preclinical pig model. Cardiovasc Res. 2004;61:530–537. doi: 10.1016/j.cardiores.2003.11.012. [DOI] [PubMed] [Google Scholar]
  • 11.Maxwell SR, Lip GY. Reperfusion injury: A review of the pathophysiology, clinical manifestations and therapeutic options. Int J Cardiol. 1997;58:95–117. doi: 10.1016/s0167-5273(96)02854-9. [DOI] [PubMed] [Google Scholar]
  • 12.Wang QD, Pernow J, Sjöquist PO, Rydén L. Pharmacological possibilities for protection against myocardial reperfusion injury. Cardiovasc Res. 2002;55:25–37. doi: 10.1016/s0008-6363(02)00261-4. [DOI] [PubMed] [Google Scholar]
  • 13.Gao F, Yao CL, Gao E. Enhancement of glutathione cardioprotection by ascorbic acid in myocardial reperfusion injury. J Pharmacol Exp Ther. 2002;301:543–550. doi: 10.1124/jpet.301.2.543. [DOI] [PubMed] [Google Scholar]
  • 14.Bolli R, Becker L, Gross G. Myocardial protection at a crossroads: The need for translation into clinical therapy. Circ Res. 2004;95:125–134. doi: 10.1161/01.RES.0000137171.97172.d7. [DOI] [PubMed] [Google Scholar]
  • 15.Rose RC, Bode AM. Biology of free radical scavengers: An evaluation of ascorbate. Faseb J. 1993;7:1135–1142. [PubMed] [Google Scholar]
  • 16.Mãrtensson J, Meister A. Glutathione deficiency decreases tissue ascorbate levels in newborn rats: Ascorbate spares glutathione and protects. Proc Natl Acad Sci U S A. 1991;88:4656–4660. doi: 10.1073/pnas.88.11.4656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Chevion M, Jiang Y, Har-El R. Copper and iron are mobilized following myocardial ischemia: Possible predictive criteria for tissue injury. Proc Natl Acad Sci U S A. 1993;90:1102–1106. doi: 10.1073/pnas.90.3.1102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Zweier JL. Measurement of superoxide-derived free radicals in the reperfused heart. Evidence for a free radical mechanism of reperfusion injury. J Biol Chem. 1988;263:1353–1357. [PubMed] [Google Scholar]
  • 19.Williams RE, Zweier JL, Flaherty JT. Treatment with deferoxamine during ischemia improves functional and metabolic recovery and reduces reperfusion-induced oxygen radical generation in rabbit hearts. Circulation. 1991;83:1006–1014. doi: 10.1161/01.cir.83.3.1006. [DOI] [PubMed] [Google Scholar]
  • 20.Mao GD, Thomas PD, Lopaschuk GD, Poznansky MJ. Superoxide dismutase (SOD)-catalase conjugates. Role of hydrogen peroxide and the Fenton reaction in SOD toxicity. J Biol Chem. 1993;268:416–420. [PubMed] [Google Scholar]
  • 21.Miller DM, Buettner GR, Aust SD. Transition metals as catalysts of ‘autoxidation’ reactions. Free Radic Biol Med. 1990;8:95–108. doi: 10.1016/0891-5849(90)90148-c. [DOI] [PubMed] [Google Scholar]
  • 22.Hiraishi H, Terano A, Razandi M. Reactive oxygen metabolite-induced toxicity to cultured bovine endothelial cells: Status of cellular iron in mediating injury. J Cell Physiol. 1994;160:132–134. doi: 10.1002/jcp.1041600116. [DOI] [PubMed] [Google Scholar]
  • 23.Aruoma OI, Halliwell B, Hoey BM, Butler J. The antioxidant action of N-acetylcysteine: Its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid. Free Radic Biol Med. 1989;6:593–597. doi: 10.1016/0891-5849(89)90066-x. [DOI] [PubMed] [Google Scholar]
  • 24.Sochman J, Kolc J, Vrana M, Fabián J. Cardioprotective effects of N-acetylcysteine: The reduction in the extent of infarction and occurrence of reperfusion arrhythmias in the dog. Int J Cardiol. 1990;28:191–196. doi: 10.1016/0167-5273(90)90060-i. [DOI] [PubMed] [Google Scholar]
  • 25.National Academy of Science Guide for the care and use of laboratory animals. http://www.csupomona.edu/research/acuc/docs/GuidetoUseCareLab%20Animals.pdf Accessed July 15, 2008.
  • 26.Higgs ZC, MacAfee DA, Braithwaite BD, Maxwell-Armstrong CA. The Seldinger technique: 50 Years on. Lancet. 2005;366:1407–1409. doi: 10.1016/S0140-6736(05)66878-X. [DOI] [PubMed] [Google Scholar]
  • 27.Forman MB, Puett DW, Cates CU. Glutathione redox pathway and reperfusion injury. Effect of N-acetylcysteine on infarct size and ventricular function. Circulation. 1988;78:202–213. doi: 10.1161/01.cir.78.1.202. [DOI] [PubMed] [Google Scholar]
  • 28.Fischer UM, Cox CS, Jr, Allen SJ. The antioxidant N-acetylcysteine preserves myocardial function and diminishes oxidative stress after cardioplegic arrest. J Thorac Cardiovasc Surg. 2003;126:1483–1488. doi: 10.1016/s0022-5223(03)00792-x. [DOI] [PubMed] [Google Scholar]
  • 29.TIMI Study Group The Thrombolysis in Myocardial Infarction (TIMI) trial. Phase I findings. N Engl J Med. 1985;312:932–936. doi: 10.1056/NEJM198504043121437. [DOI] [PubMed] [Google Scholar]
  • 30.Khalil PN, Siebeck M, Huss R. Histochemical assessment of early myocardial infarction using 2,3,5-triphenyltetrazolium chloride in blood-perfused porcine hearts. J Pharmacol Toxicol Methods. 2006;54:307–312. doi: 10.1016/j.vascn.2006.02.010. [DOI] [PubMed] [Google Scholar]
  • 31.McMurray J, Chopra M, Abdullah I. Evidence for oxidative stress in unstable angina. Br Heart J. 1992;68:454–457. doi: 10.1136/hrt.68.11.454. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Paraskevaidis IA, Iliodromitis EK, Vlahakos D. Deferoxamine infusion during coronary artery bypass grafting ameliorates lipid peroxidation and protects the myocardium against reperfusion injury: Immediate and long-term significance. Eur Heart J. 2005;26:263–270. doi: 10.1093/eurheartj/ehi028. [DOI] [PubMed] [Google Scholar]
  • 33.Kumari R, Manchanda SC, Maulik SK. Effect of pre- and posttreatment of losartan in feline model of myocardial ischemic-reperfusion injury. Methods Find Exp Clin Pharmacol. 2004;26:39–45. doi: 10.1358/mf.2004.26.1.793471. [DOI] [PubMed] [Google Scholar]
  • 34.Wasowicz W, Nève J, Peretz A. Optimized steps in fluorometric determination of thiobarbituric acid-reactive substances in serum: Importance of extraction pH and influence of sample preservation and storage. Clin Chem. 1993;39:2522–2526. [PubMed] [Google Scholar]
  • 35.Larson MG. Analysis of variance. Circulation. 2008;117:115–121. doi: 10.1161/CIRCULATIONAHA.107.654335. [DOI] [PubMed] [Google Scholar]
  • 36.Kobayashi S, Tadokoro H, Wakida Y. Coronary venous retroinfusion of deferoxamine reduces infarct size in pigs. J Am Coll Cardiol. 1991;18:621–627. doi: 10.1016/0735-1097(91)90622-g. [DOI] [PubMed] [Google Scholar]
  • 37.Mickle DA, Li RK, Weisel RD. Myocardial salvage with trolox and ascorbic acid for an acute evolving infarction. Ann Thorac Surg. 1989;47:553–557. doi: 10.1016/0003-4975(89)90431-1. [DOI] [PubMed] [Google Scholar]
  • 38.Nishinaka Y, Sugiyama S, Yokota M. The effects of a high dose of ascorbate on ischemia-reperfusion-induced mitochondrial dysfunction in canine hearts. Heart Vessels. 1992;7:18–23. doi: 10.1007/BF01745863. [DOI] [PubMed] [Google Scholar]
  • 39.Bellows SD, Hale SL, Simkhovich BZ. Do antioxidant vitamins reduce infarct size following acute myocardial ischemia/reperfusionqqq. Cardiovasc Drugs Ther. 1995;9:117–123. doi: 10.1007/BF00877751. [DOI] [PubMed] [Google Scholar]
  • 40.Buettner GR. Ascorbate autoxidation in the presence of iron and copper chelates. Free Radic Res Commun. 1986;1:349–353. doi: 10.3109/10715768609051638. [DOI] [PubMed] [Google Scholar]
  • 41.Vera JC, Rivas CI, Fischbarg J, Golde DW. Mammalian facilitative hexose transporters mediate the transport of dehydroascorbic acid. Nature. 1993;364:79–82. doi: 10.1038/364079a0. [DOI] [PubMed] [Google Scholar]
  • 42.Reddy BR, Kloner RA, Przyklenk K. Early treatment with deferoxamine limits myocardial ischemic/reperfusion injury. Free Radic Biol Med. 1989;7:45–52. doi: 10.1016/0891-5849(89)90099-3. [DOI] [PubMed] [Google Scholar]
  • 43.Drossos G, Lazou A, Panagopoulos P, Westaby S. Deferoxamine cardioplegia reduces superoxide radical production in human myocardium. Ann Thorac Surg. 1995;59:169–172. doi: 10.1016/0003-4975(94)00726-N. [DOI] [PubMed] [Google Scholar]
  • 44.Borg DC, Schaich KM. Prooxidant action of desferrioxamine: Fenton-like production of hydroxyl radicals by reduced ferrioxamine. J Free Radic Biol Med. 1986;2:237–243. doi: 10.1016/s0748-5514(86)80004-6. [DOI] [PubMed] [Google Scholar]
  • 45.Gao WD, Atar D, Backx PH, Marban E. Relationship between intracellular calcium and contractile force in stunned myocardium. Direct evidence for decreased myofilament Cat+ responsiveness and altered diastolic function in intact ventricular muscle. Circ Res. 1995;76:1036–1048. doi: 10.1161/01.res.76.6.1036. [DOI] [PubMed] [Google Scholar]
  • 46.Tang LD, Sun JZ, Wu K. Beneficial effects of N-acetylcysteine and cysteine in stunned myocardium in perfused rat heart. Br J Pharmacol. 1991;102:601–606. doi: 10.1111/j.1476-5381.1991.tb12219.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Calvillo L, Masson S, Salio M. In vivo cardioprotection by N-acetylcysteine and isosorbide 5-mononitrate in a rat model of ischemia-reperfusion. Cardiovasc Drugs Ther. 2003;17:199–208. doi: 10.1023/a:1026182404805. [DOI] [PubMed] [Google Scholar]
  • 48.Qiu Y, Bernier M, Hearse DJ. The influence of N-acetylcysteine on cardiac function and rhythm disorders during ischemia and reperfusion. Cardioscience. 1990;1:65–74. [PubMed] [Google Scholar]
  • 49.Rice-Evans C, Brockdorfer KR. Free radicals, lipoproteins and cardiovascular dysfunction. Mol Aspects Med. 1992;13:1–111. doi: 10.1016/0098-2997(92)90002-h. [DOI] [PubMed] [Google Scholar]
  • 50.Karwatowska-Prokopczuk E, Czarnowska E, Prokopczuk A. Combined therapy with dimethylthiourea, diltiazem and amiloride/dimethylamiloride in the ischemic/reperfused heart. Cardiovasc Res. 1995;30:70–78. [PubMed] [Google Scholar]
  • 51.Bolli R, Patel BS, Zhu WX. The iron chelator desferrioxamine attenuates postischemic ventricular dysfunction. Am J Physiol. 1987;253:H1372–H1380. doi: 10.1152/ajpheart.1987.253.6.H1372. [DOI] [PubMed] [Google Scholar]
  • 52.Dingchao H, Zhiduan Q, Liye H, Xiaodong F. The protective effects of high-dose ascorbic acid on myocardium against reperfusion injury during and after cardiopulmonary bypass. Thorac Cardiovasc Surg. 1994;42:276–278. doi: 10.1055/s-2007-1016504. [DOI] [PubMed] [Google Scholar]
  • 53.Rinne T, Mutschler E, Wimmer-Greinecker G. Vitamins C and E protect isolated cardio- myocytes against oxidative damage. Int J Cardiol. 2000;75:275–281. doi: 10.1016/s0167-5273(00)00353-3. [DOI] [PubMed] [Google Scholar]
  • 54.Laskowski H, Minczykowski A, Wysocki H. Mortality and clinical course of patients with acute myocardial infarction treated with streptokinase and antioxidants: Mannitol and ascorbic acid. Int J Cardiol. 1995;48:235–237. doi: 10.1016/0167-5273(94)02252-e. [DOI] [PubMed] [Google Scholar]
  • 55.Ambrosio G, Zweier JL, Jacobus WE. Improvement of postischemic myocardial function and metabolism induced by administration of deferoxamine at the time of reflow: The role of iron in the pathogenesis of reperfusion injury. Circulation. 1987;76:906–915. doi: 10.1161/01.cir.76.4.906. [DOI] [PubMed] [Google Scholar]
  • 56.Lesnefsky EJ, Repine JE, Horwitz LD. Deferoxamine pretreatment reduces canine infarct size and oxidative injury. J Pharmacol Exp Ther. 1990;253:1103–1109. [PubMed] [Google Scholar]
  • 57.Tossios P, Bloch W, Huebner A. N-acetylcysteine prevents reactive oxygen species-mediated myocardial stress in patients undergoing cardiac surgery: Results of a randomized, double-blind, placebo-controlled clinical trial. J Thorac Cardiovasc Surg. 2003;126:1513–1520. doi: 10.1016/s0022-5223(03)00968-1. [DOI] [PubMed] [Google Scholar]
  • 58.American Veterinary Medical Association (AVMA) Guidelines on Euthanasia. 2007. http://www.avma.org/issues/animal_welfare/euthanasia.pdf Accessed July 15, 2008. [Google Scholar]

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