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A clinical appraisal of propofol-mediated, antioxidant-based cardioprotection during coronary artery… Raedschelders, Koen 2011

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A CLINICAL APPRAISAL OF PROPOFOL-MEDIATED, ANTIOXIDANTBASED CARDIOPROTECTION DURING CORONARY ARTERY BYPASS GRAFTING WITH CARDIOPULMONARY BYPASS by Koen Raedschelders B.Sc., The University of Alberta, 2004  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Pharmacology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2011  © Koen Raedschelders, 2011  Abstract Coronary artery disease is the leading cause of death in North America. The invasiveness of its treatment depends on its severity; less severe disease can be treated pharmacologically or surgically without significantly different outcomes, but coronary artery bypass grafting (CABG) clearly reduces mortality among medium- and high-risk patients compared to percutaneous and non-surgical intervention. Although the majority of patients undergoing surgical revascularization emerge without severe postoperative complications, a significant portion of patients encounter a postoperative complication known as low cardiac output syndrome which can quadruple the overall mortality rate for CABG. Intraoperative ischemia reperfusion injury is a major factor in the development of low cardiac output syndrome; so effective intraoperative myocardial protection is central to reducing its incidence, and represents an opportunity to considerably improve patient outcomes. The introductory chapter of this thesis describes the origin and role of reactive oxygen species (ROS) in myocardial ischemia-reperfusion injury. In addition, it introduces key strategies targeted to reduce ROS-mediated myocardial ischemiareperfusion injury, highlighting key clinical studies that translated these strategies to reduce the severity of ischemia-reperfusion injury during CABG. The central hypothesis of the clinical project on which this thesis is based states that propofol reduces the incidence of low cardiac output syndrome subsequent to CABG with CPB by decreasing the magnitude of 15-F2t-isoprostane generation during ischemia-reperfusion. The second chapter introduces propofol, and will review previous studies that explore its cardioprotective potential.  ii  The experimental section of this thesis describes the development of a quantitative technique for propofol analysis in whole blood, and its application in a dose finding study that define the parameters for achieving experimentally relevant concentrations of propofol during cardiopulmonary bypass. These two studies were fundamental to the development of a clinical study evaluating ROS generation and the incidence low cardiac output syndrome in patients undergoing CABG surgery. Preliminary results that address the central hypothesis are subsequently presented, along with an alternative proposed mechanism for propofol-mediated cardioprotection. This thesis will conclude with a summary of findings and a description of several future studies aimed at testing, generating, and evaluating new hypotheses.  iii  Preface The author, Koen Raedschelders, performed the majority of the research and analysis leading to the results included in this dissertation, and performed the entirety of the writing. The contributions of other researchers and collaborations are summarized below: Contributions from other researchers: Chapter 3:  Yu Hui and Koen Raedschelders performed linearity, reproducibility, and precision analyses, and analyzed patient whole blood samples. Hong Zhang modified the liquid-liquid extraction to make it amenable for capillary electrophoresis separations and performed exploratory separations.  Chapter 4:  Yu Hui and Hong Zhang analyzed patient whole blood samples. Bradley Laferlita was the anesthesiology resident responsible for hemodynamic monitoring while Tao Luo was responsible for the study protocol.  Chapter 5:  David Ansley, the principal investigator, developed the study described in this chapter with input from Peter Choi.  Chapter 6:  Yu Hui performed quantitative propofol analyses, 15-F2t-isoprostane analyses, and 3-nitrotyrosine assays. NO2- assays were performed in the laboratory of Pascal Bernatchez.  Ethics approval: This study was reviewed by and received approval from the UBC Clinical Research Ethics Board (Certificate number H04-70456)  iv  Publications arising from work presented in the dissertation: 1. Hui Y, Raedschelders K, Zhang H, Ansley D, Chen D. Quantitative analysis of propofol in whole blood using capillary electrophoresis. J Chromatogr B. 2009;877:703-709. Material from this article is included in Chapter 3 2. Raedschelders K, Hui Y, Laferlita B, Luo T, Zhang H, Chen DD, Ansley DM. Target-achieved propofol concentration during on-pump cardiac surgery: a pilot dosefinding study. Can J Anaesth. Sep 2009;56(9):658-666. Material from this article is included in Chapter 4 3. Ansley DM, Raedschelders K, Chen DD, Choi PT. Rationale, design and baseline characteristics of the PRO-TECT II study: PROpofol CardioproTECTion for Type II diabetics A randomized, controlled trial of high-dose propofol versus isoflurane preconditioning in patients undergoing on-pump coronary artery bypass graft surgery. Contemp Clin Trials. Mar 29 2009;30(4):380-385. Material from this article is included in Chapter 5  v  Table of Contents Abstract ......................................................................................................................... ii Preface.......................................................................................................................... iv Table of Contents ......................................................................................................... vi List of Tables................................................................................................................. x List of Figures .............................................................................................................. xi List of Symbols and Abbreviations ........................................................................... xiii Acknowledgments ....................................................................................................... xv Dedication................................................................................................................... xvi 1.  Introduction to reactive oxygen species generation during myocardial ischemia  and reperfusion ............................................................................................................. 1 1.1. Myocardial ischemia-reperfusion injury............................................................. 2 1.2. Free radical generation during myocardial ischemia-reperfusion........................ 6 1.3. Sources of superoxide during myocardial ischemia and reperfusion................... 8 1.3.1 Mitochondrial electron transport ................................................................ 12 1.3.2 Cellular xanthine oxidase........................................................................... 16 1.3.3 The immune system................................................................................... 18 1.4. Nitric oxide generation during myocardial ischemia and reperfusion ............... 20 1.4.1 Nitric oxide synthases................................................................................ 21 1.4.2 Nitrite reduction......................................................................................... 23 1.5. Hydroxyl radical generation during myocardial ischemia and reperfusion........ 25 1.5.1 Iron-catalyzed hydroxyl formation............................................................. 26 1.5.2 Peroxynitrous acid degradation.................................................................. 27 1.6. Free radical-mediated damage during myocardial ischemia and reperfusion .... 30 1.6.1 Lipid peroxidation ..................................................................................... 32 1.6.2 Protein oxidation and nitration................................................................... 36 1.6.3 DNA damage............................................................................................. 39 1.6.4 Matrix metalloproteinases.......................................................................... 40 1.6.5 Mitochondrial permeability transition ........................................................ 41 1.7. Protective strategies during cardiac surgery ..................................................... 44 1.7.1 Ischemic preconditioning........................................................................... 45 vi  1.7.2 Volatile anesthetic preconditioning ............................................................ 48 1.7.3 Antioxidants .............................................................................................. 50 1.8. Conclusions and perspectives .......................................................................... 53 1.9. Thesis objectives and outline ........................................................................... 54 2.  Clinical-based cardioprotection beyond volatile anesthetic preconditioning .... 59 2.1. Propofol-mediated, antioxidant-based cardioprotection.................................... 61 2.2. Propofol modulation of ion fluxes.................................................................... 62 2.3. Propofol mediated reactive oxygen species scavenging.................................... 64 2.4. Precedents for propofol-mediated cardioprotection.......................................... 65  3.  Quantitative analysis of propofol in whole blood using capillary  electrophoresis............................................................................................................. 68 3.1. Introduction..................................................................................................... 68 3.2. Experimental ................................................................................................... 70 3.2.1 Apparatus .................................................................................................. 70 3.2.2 Chemicals and reagents.............................................................................. 70 3.2.3 Preparation of standard solutions ............................................................... 71 3.2.4 Patients and sampling ................................................................................ 71 3.2.5 Sample extraction ...................................................................................... 73 3.2.6 Capillary electrophoresis............................................................................ 73 3.3. Results and discussion ..................................................................................... 74 3.3.1 Specificity ................................................................................................. 74 3.3.2 Selection of buffer type and separation voltage .......................................... 78 3.3.3 Linearity.................................................................................................... 79 3.3.4 Reproducibility.......................................................................................... 80 3.3.5 Optimization of the liquid-liquid sample extraction.................................... 80 3.3.6 Optimization of the sample resuspension solution...................................... 82 3.3.7 On-line pH-difference induced focusing .................................................... 82 3.3.8 Indices of precision and recovery of whole blood analysis ......................... 84 3.3.9 Limit of detection and quantitation ............................................................ 86 3.3.10 Patient samples ........................................................................................ 87 3.4. Conclusion ...................................................................................................... 89 vii  4.  Target achieved propofol concentration during on-pump cardiac surgery: A  pilot dose finding study............................................................................................... 90 4.1. Introduction..................................................................................................... 90 4.2. Methods .......................................................................................................... 92 4.2.1 Study design .............................................................................................. 92 4.2.2 Study population........................................................................................ 93 4.2.3 Perioperative procedures............................................................................ 94 4.2.4 Anesthesia protocol ................................................................................... 94 4.2.5 Application of propofol during cardiopulmonary bypass............................ 95 4.2.6 Measurement of propofol concentration..................................................... 95 4.2.7 Hemodynamic data collection.................................................................... 95 4.2.8 Inotropic and vasoactive drug protocol ...................................................... 96 4.2.9 Sample size and statistical analysis. ........................................................... 97 4.3. Results............................................................................................................. 98 4.3.1 Patient and operative characteristics........................................................... 98 4.3.2 Propofol concentrations in whole blood ..................................................... 99 4.3.3 Intraoperative hemodynamic function...................................................... 101 4.4. Discussion ..................................................................................................... 104 5.  Rationale and design of the PROpofol CardioproTECTion for Type II Diabetics  (PRO-TECT II) Study: A randomized, controlled trial of high-dose propofol versus isoflurane preconditioning in patients undergoing on-pump cardiac surgery ....... 109 5.1. Introduction................................................................................................... 109 5.2. Materials and methods................................................................................... 111 5.2.1 Study design ............................................................................................ 111 5.2.2 Study population...................................................................................... 111 5.2.3 Randomization......................................................................................... 112 5.2.4 Study protocol ......................................................................................... 112 5.2.5 Blinding................................................................................................... 115 5.2.6 Outcome measures................................................................................... 116 5.2.7 Ethical considerations .............................................................................. 117 5.2.8 Sample size.............................................................................................. 117 viii  5.2.9 Data analysis ........................................................................................... 118 5.2.10 Funding ................................................................................................. 118 5.2.11 Trial status............................................................................................. 118 5.3. Discussion ..................................................................................................... 120 6.  Propofol does not differ from isoflurane preconditioning in terms of 15-F2t-  isoprostane generation during ischemia and reperfusion in patients undergoing coronary artery bypass grafting with cardiopulmonary bypass............................. 123 6.1. Introduction................................................................................................... 123 6.2. Methods ........................................................................................................ 125 6.2.1 Study design ............................................................................................ 125 6.2.2 Study population...................................................................................... 125 6.2.3 Perioperative procedures.......................................................................... 126 6.2.4 Blinding................................................................................................... 127 6.2.5 Measurement of propofol concentration................................................... 127 6.2.6 Coronary sinus blood sampling................................................................ 128 6.2.7 15-F2t-isoprostane analysis....................................................................... 128 6.2.8 Nitrite analysis......................................................................................... 129 6.2.9 Measurement of 3-nitrotyrosine generation during ischemia and reperfusion… ...................................................................................................... 130 6.2.10 Hemodynamic data collection ................................................................ 130 6.2.11 Sample size............................................................................................ 131 6.2.12 Statistical analysis.................................................................................. 131 6.3. Results........................................................................................................... 132 6.3.1 Patient and operative characteristics......................................................... 132 6.3.2 15-F2t-isoprostane generation during ischemia and reperfusion ................ 135 6.3.3 NO2- generation during ischemia and reperfusion..................................... 135 6.3.4 3-Nitrotyrosine generation during ischemia and reperfusion..................... 135 6.3.5 Perioperative hemodynamic endpoints..................................................... 137 6.4. Discussion ..................................................................................................... 139 7.  Summary and concluding remarks ................................................................... 146  Bibliography.............................................................................................................. 156 ix  List of Tables Table 1: General patient and operative characteristics. .................................................. 72 Table 2: Reproducibility of three clinically relevant standard propofol concentrations on the same day. ............................................................................................ 74 Table 3: Reproducibility of three clinically relevant standard propofol concentrations across three days....................................................................................... 76 Table 4: Contribution of sample solution pH to the resolution and focusing effect of the separation. ................................................................................................ 83 Table 5: Indices of precision of three independently prepared clinically relevant propofol concentrations spiked into and extracted from whole blood....................... 86 Table 6: Suggested dose regimen for attending anesthesiologists................................... 97 Table 7: Patient demographic and perioperative characteristics. .................................... 99 Table 8: Demographic details of the first fifty subjects enrolled in the Pro-TECT II study. ............................................................................................................... 119 Table 9: Patient demographic and perioperative characteristics. .................................. 134  x  List of Figures Figure 1: Myocardial ionic alterations during myocardial ischemia. ................................ 5 Figure 2: Major sources of O2• - during myocardial ischemia and reperfusion. ............... 11 Figure 3: Main hydroxyl radical producing pathways during myocardial ischemia and reperfusion................................................................................................ 29 Figure 4: Generalized example scheme of lipid peroxidation......................................... 34 Figure 5: Generalized examples of free-radical mediated amino acid modification scheme...................................................................................................... 37 Figure 6: Summary of the major processes and consequences of mitochondrial permeability transition. ............................................................................. 42 Figure 7: Flow chart describing the conceptual link between the individual chapters of this thesis.................................................................................................. 58 Figure 8: Structures of propofol and alpha-tocopherol with their similarities highlighted in bold. ..................................................................................................... 65 Figure 9: Representative electropherograms of a) standard solution of propofol and thymol, and b) whole blood containing propofol and thymol..................... 77 Figure 10: Ohm's law plot generated to determine the optimal separation potential. ...... 79 Figure 11: Concentration distribution of propofol in whole blood of 30 patients undergoing CABG with CPB. Samples were obtained 15 minutes after aortic declamping under a propofol infusion rate of 120 µg•kg-1•min-1. ... 88 Figure 12: Propofol concentrations in whole blood at reperfusion during CABG with CPB in 24 patients receiving one of three infusion rates; 50, 100, and 150 µg•kg-1•min-1. ........................................................................................ 100 Figure 13: Scatter plots of propofol concentration plotted against a) age, b) weight c) body surface area and d) crossclamp duration for 23 patients receiving 120 µg•kg-1•min-1 of propofol during CPB.................................................... 102 Figure 14: Intraoperative profiles of cardiac index, systemic vascular resistance index, and left ventricular stroke work index. .................................................... 103 Figure 15: Representative diagram of study interventions applied during coronary artery bypass graft surgery in the PRO-TECT II Study...................................... 114  xi  Figure 16: Ion chromatogram of 15-F2t-isoprostane with 15-F2t-isoprostane-D4 internal standard in patient plasma. HPLC separation on a C18 column in tandem with ion trap mass spectrometry.............................................................. 129 Figure 17: Breakdown of patient randomization and study completion........................ 133 Figure 18: Change in 15-F2t-isoprostane generation during myocardial ischemia and reperfusion a) plotted according to the anesthetic protocol that patients were randomized to, and b) plotted against the concentration of propofol achieved in whole blood at reperfusion.................................................................. 136 Figure 19: Nitrite generation changes during myocardial ischemia and reperfusion during CABG with CPB in patients randomized to propofol or isoflurane anesthesia protocols................................................................................................. 137 Figure 20: Protein-bound 3-nitrotyrosine changes in plasma during myocardial ischemia and reperfusion during CABG with CPB in patients randomized to propofol or isoflurane anesthesia protocols............................................................ 137 Figure 21: Perioperative profiles of cardiac index, systemic vascular resistance index, and left ventricular stroke work index............................................................ 138  xii  List of Symbols and Abbreviations ADP  Adenosine diphosphate  ATP  Adenosine triphosphate  BH2  Dihydrobiopterin  BH4  Tetrahydrobiopterin  CAT  Catalase  CABG  Coronary artery bypass grafting  CE  Capillary electrophoresis  CK-MB  Myocardial creatine kinase  CO2  Carbon dioxide  CPB  Cardiopulmonary bypass  DNA  Deoxyribonucleic acid  ELISA  Enzyme-linked immunosorbent assay  ET-1  Endothelin-1  FAD/FADH2  Flavin adenine dinucleotide: oxidized / reduced  FMN/FMNH•  Flavin mononucleotide oxidized /reduced semiquinone radical  GABA  Gamma-aminobutyric acid  GSH  Glutathione  GSSG  Glutathione disulfide  H2O2  Hydrogen peroxide  HbO2  Oxygenated Hemoglobin  ICU  Intensive care unit  LD  Capillary length from inlet to the detection window, in CE  LT  Total capillary length from, in CE  L-NAME  NG-nitro-Levo-arginine methyl ester; competitive NOS inhibitor  L-NMMA  NG-monomethyl-Levo-arginine; competitive NOS inhibitor  MDA  Malondialdehyde  MEKC  Micellar electrokinetic capillary chromatography  MetHb  Hemoglobin with ferric (as opposed to ferrous) iron  MMP  Matrix metalloproteinase  xiii  MPT  Mitochondrial permeability transition  NAD(P)+/NAD(P)H Nicotinamide adenine dinucleotide (phosphate): oxidized /reduced NO•  Nitric Oxide  NO2  Nitrite  NO2•-  Nitrogen dioxide radical  NOS  Nitric oxide synthase -  ONOO  Peroxynitrite  ONOOH  Peroxynitrous acid  O2  Molecular Oxygen  O2•-  Superoxide  PKC  Protein kinase C  prn  Denotes “as per routine practice”  rcf  Relative centrifugal force  ROS  Reactive Oxygen Species: Encompasses O2•-, NO•, NO2•-, ONOO-, and ONOOH  Rs  Resolution  SOD  Superoxide dismutase  TCA cycle  Tricarboxylic acid cycle  tmig  Migration time (of a given analyte)  TNFα  Tumor necrosis factor-alpha  UQ/ UQH•  Ubiquinone: oxidized quinone / reduced semiquinone  UV  Ultraviolet  XDH  Xanthine dehydrogenase  XO  Xanthine oxidase  σ  Standard deviation  µ  Mean  µepA  Electrophoretic mobility (in this case, of analyte “A”)  Ψm  Inner mitochondrial membrane potential  xiv  Acknowledgments The completion of this work would not have been possible without the guidance, support, and assistance of a host of colleagues and friends. I have the utmost respect for them. Foremost among these are my research supervisors and mentors. Dr. David Ansley and Dr. David Chen, who were generous with their time and guidance, and who provided me with the freedom to explore and the structure to succeed. I am indebted to you both. I know how fortunate I am to have Dr. David Ansley, Dr. David Chen, Dr. David Godin, Dr. Adam Frankel, and Dr. William Jia as a supervisory committee. I am in awe of your collective work and inspired by the creativity and ingenuity inherent therein. I am grateful to my student colleagues, without exception but specifically E. Jane Maxwell, Yu (Joe) Hui, and Dr. Baohua Wang, whom I have had the absolute pleasure of working with. I am equally grateful to the faculty members within the department of Chemistry and the department of Anesthesiology, Pharmacology and Therapeutics. They specifically include the aforementioned names as well as Dr. David Fedida, Dr. Michael Walker, Dr. Christopher Ahern, and Dr. Pascal Bernatchez. I am proud to know them as mentors, examples of scientific excellence, and friends. The small size of our research team belies the many people that were involved in enabling this clinical project at Vancouver General Hospital. These include the cardiac surgeons, the perfusionists, the anesthesiologists and anesthesiology technicians, the OR and intensive care unit nursing staff, and the recruiting team (Marion Eng, Rebecca Fox), and the patient volunteers. To my close family primarily, and to my close friends within and beyond science, whose creative thinking and dialogue were critical in my scientific progress, including but not limited to Robert Fougere, Alan Hokazono, Andy Jeffries, Kathy Gratton, Dr. Nizar Bahlis, Dr. Oliver Bathe, Dr. Nicole Baryla, and Dr. Nick Toltl. I am beholden to you for the patient support, advice, and friendship that you’ve extended my way. Financial support over the course of this thesis was provided by the citizens of Canada in the form of a Canada Graduate Doctoral Scholarship from the Canadian Institutes of Health Research.  xv  Dedication  “Every child knows that play is nobler than work.” –Cormac McCarthy. Blood Meridian This thesis has no dedication. That nobler pursuit is most often dedicated to Monique Raedschelders -proud, principled, and independent. A pure fire that grew until her body could no longer contain the flames, and her wild soul was set free.  xvi  1.  Introduction to reactive oxygen species generation during myocardial ischemia and reperfusion Coronary artery disease is the leading cause of death in North America1, 2, accounting  for an estimated direct and indirect cost of over $448.5 billion2, and $18.5 billion in Canada3. Less severe coronary artery disease can be treated pharmacologically or surgically without significant differences in outcomes4, 5, but coronary artery bypass grafting (CABG) clearly reduces mortality among medium- and high-risk patients, including diabetics6, 7, over non-surgical management8 and percutaneous interventions6, 9, 10  . The 515,000 annual CABG surgeries done in the United States alone testify to the  effectiveness of this technique11. Although the majority of patients undergoing surgical revascularization emerge without severe postoperative complications, a significant proportion of patients encounter a postoperative complication known as low cardiac output syndrome. Low cardiac output syndrome can quadruple the overall mortality rate for CABG from 2% to 8%12, 13. Intraoperative ischemia-reperfusion injury is a major factor in the development of low cardiac output syndrome; so effective intraoperative myocardial protection is central to reducing the incidence of this high-risk scenario. Low cardiac output syndrome affects up to 26% of diabetic patients, compared to 8% to 15% of non-diabetic patients recovering from cardiac surgery14-16. Several factors likely contribute to this discrepancy, chief among these are defective antioxidant defenses17, 18, increased oxidative stress, and impaired endogenous myocardial protective pathways identified in the diabetic heart19, 20. Diabetics are two to five times more likely to develop cardiovascular disease and therefore account for up to 30% of open heart procedures21. Diabetic patients are 1  particularly at an elevated risk of complications after CABG and accordingly have a lower 10-year survival16. Managing ischemia-reperfusion injury during to CABG represents an opportunity to considerably improve patient outcomes. 1.1. Myocardial ischemia-reperfusion injury During CABG with cardiopulmonary bypass (CPB), venous blood is redirected from the vena cava to the “heart-lung machine”, which mediates artificial gas exchange and pumps oxygenated blood back through the body via the aorta. The heart-lung machine provides the body with adequate perfusion through constant delivery of oxygenated blood downstream of the aorta, but cardiac perfusion is a direct physical consequence of cardiac systole/diastole cycling. Cardioplegia solution is administered through the coronary vasculature to electrically silence the heart during CPB, thus inhibiting excitationcontraction and reducing myocardial energy demand. Despite the administration of cardioplegia and the absence of contraction, the myocardium still consumes oxygen at a rate of 2 mL-of-O2-per-100g-per-min22. When the heart is electrically silenced, its oxygen and nutrient demands are decreased, but its blood flow is also effectively halted. The resulting ischemia can range in severity from reversible to fatal, and as the duration and severity of ischemia increases, so does the extent of myocyte injury and death23, 24. Ischemic injury is more complex than O2 and ATP depletion, and CO2 buildup; these events simply represent the first consequences of ischemia, which in turn induce a myriad of changes at the cellular, tissue, and organ levels. Over 95% of ATP production in the non-ischemic heart results from mitochondrial oxidative phosphorylation, the vast majority being fuelled by fatty acid β-oxidation25. The insufficient blood supply during ischemia causes oxidative substrates to accumulate at the expense of their reductive 2  counterparts; the myocardial NADH/NAD+ concentration ratio can increase more than 10-fold during ischemia26. In response, the heart shifts its metabolic phenotype from β-oxidation towards glycolysis. This phenotypic shift is accomplished in part by increasing the expression of the GLUT-1 and GLUT-4 transporters27-29 and increasing glycogenolysis30, and is stimulated by the increase in inorganic phosphates and decreased pH that accompany ischemia. The final metabolite of glycolysis is pyruvate, but at low oxygen tension, pyruvate is used as an electron acceptor and reduced to lactate. Non-mitochondrial ATP turnover during ischemic episodes, combined with the accumulation of lactate from glycolysis at low oxygen tension, results in ischemic intracellular acidosis31. Glycolysis is a short-term solution to the problem of ischemia because myocardial glucose stores are limited32. Aside from changing their metabolic phenotype, ischemic myocytes can re-establish their oxygen demand to meet its decreased supply by reducing contractility with a generalized downgrading of cardiac function. This process results in myocardial hibernation33. Even in the less active hibernating state, myocytes still require ATP for housekeeping processes like ion homeostasis. If oxygen supplies are depleted below the levels required for hibernation, then the myocardium will sustain prolonged dysfunction. Ion homeostasis is the major metabolic priority for the heart. This remains true during myocardial ischemia, hibernation, and stunning alike. The myocardium requires ATP to restore the limited energy reserves that are quickly exhausted during ischemia. Ion homeostasis depends primarily on key energy-dependent ion exchangers including the Na+/K+ ATPase, and Ca2+-ATPases. Secondary channels include the Na+/Ca2+ and the  3  Na+/ H+ exchanger. Proton accumulation resulting from unbalanced ATP hydrolysis drives the Na+/H+ exchanger leading to an initial intracellular Na+ overload, and an eventual intracellular Ca2+ overload (Figure 1) (Reviewed in34, 35). Myocardial acidosis reduces cardiac contractility by interfering with the normal Ca2+-troponin C interaction36, thus promoting arrhythmia. Ultimately, ischemia results in energy starved, Ca2+-loaded, acidotic myocytes. These conditions strongly contribute to ischemic arrhythmia37, contraction failure38, and cell death39.  4  Figure 1: i) Myocardial ionic alterations during myocardial ischemia. 1. Depolarization via Na+ influx; 2. Ca2+ influx/excitation-contraction coupling via (a) voltage gated Ca2+ influx and (b) Ryanodine receptor mediated Ca2+-induced- Ca2+-release; 3. Repolarization via K+ efflux; 4. Re-establishing electrochemical gradients via (a) Na+/ Ca2+ exchange, (b) ATP-driven Ca2+ sequestration, (c) ATP-driven Na+/K+ exchange; 5. Acidosis-induced Na+/H+ exchange. ii) Depiction of the role that altered cellular energetic plays in promoting ischemic acidosis and altered myocardial ion fluxes.  5  The principle that timely reperfusion could salvage the ischemic myocardium, and that the extent of ischemia was reflected by the extent of the myocardial apoptosis and necrosis was clearly defined in the mid 1970’s23, 24, 40, 41. Additionally, the role of calcium overload and contracture had been established by the finding that verapamil42 and propranolol43 could influence post-ischemic myocardial cell death. Nevertheless, these results contrasted with a 1973 report describing a massive protein and enzyme release from the myocardium immediately after aerobic reperfusion44. The effect of reperfusion was subsequently characterized by ionic and histological differences that were not present during ischemia45, 46. Namely, post-ischemic reperfused myocytes showed increased intracellular Na+, Cl-, and Ca2+ concentrations, as well as decreased intracellular K+ concentrations, with large contractile proteins forming distinct bands while mitochondria (and indeed cells) became swollen. Several studies in the late 1970’s and early 1980’s further described reperfusion injury from a functional perspective in animal models47, 48, and even from the perspective of CABG49. Despite these observations, the concept that a distinct injury was generated at reperfusion initially remained controversial. A major obstacle was that reperfusion injury, at that time, was a concept that lacked a mechanism. 1.2. Free radical generation during myocardial ischemia-reperfusion The concept that the reintroduction of molecular oxygen to the myocardium could induce a unique type of injury was first proposed by Hearse et al in 1973 who noticed that a large fraction of cellular enzymes were released not during hypoxia, but rather upon sudden reoxygenation44. The concept was further supported when the inclusion of exogenous glucose to anoxic perfusate to supplement the limited endogenous myocardial 6  supply was found to relieve cell injury at reperfusion50, 51. Thus, myocyte injury subsequent to post-ischemic reperfusion was paradoxically predicated on the reintroduction of oxygen to cells that are energy-starved, which clearly implicated cellular metabolism. In 1980, Guarnieri et al demonstrated that ischemia and reperfusion impaired superoxide dismutase activity and decreased cellular glutathione-to-glutathione disulfide ratio (see equations 3 and 5) while increasing lipid peroxidation, suggesting that the extent of superoxide anion radical (O2•-) produced at reperfusion exceeded the capacity of endogenous cellular antioxidant systems52. The overall results of these and other similar studies introduced the notion that metabolic reducing equivalents accumulate during ischemia, which in turn serve as substrates for oxygen-centered free radical generation at reperfusion53. Experiments using isolated heart models in the presence or absence of superoxide dismutase further implicated oxygen-derived radicals as likely mediators of reperfusion injury54-56, but due to the lack of direct evidence of production, identity, and mechanism of action, their role remained controversial. In the late 1980’s, the application of electron spin resonance and spin trapping directly implicated oxygen-derived free radicals in ischemia-reperfusion injury57-63. Pioneering work using electron spin resonance spectroscopy for the first time directly characterized the generation of oxygen-, nitrogen-, and carbon-centered free radicals during ischemia and reperfusion in an isolated rabbit heart model57. Similar results were independently produced in isolated rat model59. Although these spin trapping experiments could not definitively identify the radicals that were generated, they clearly showed that the signals for oxygen- and nitrogen-centered radicals increased marginally during ischemia at the apparent expense of the carbon-centered semiquinone radical signal57.  7  The ischemic pattern of reactive oxygen and nitrogen species (collectively ROS) generation was unchanged with hypoxic reperfusion, but all three signals spiked with oxygenated reperfusion; their peak occurring approximately 20 seconds thereafter. Arroyo et al subsequently used spin trapping to identify O2•-, hydroxyl radical (•OH), and a carbon centered radical at reperfusion, and found that their cumulative generation at reperfusion was abolished by superoxide-dismutase. Accordingly, O2•- was identified as the parent radical that servers as a precursor to the formation of both •OH and the carboncentered radical58 which the same group subsequently identified as a group of oxyradical-mediated lipid peroxidation products64. Bolli et al demonstrated that this pattern of free-radical generation extended to an intact dog model60, in which O2•- was similarly identified as the parent radical at reperfusion61. The advent of techniques capable of direct detection and identification of oxygencentered radicals in the late 1980’s was critical to establish their generation during myocardial ischemia and reperfusion. Subsequent studies, in which exogenously administered ROS at levels akin to those observed during reperfusion induced similar calcium overloading, functional depression, and metabolic changes65, 66, cemented the ROS as central mediators of this injury. 1.3. Sources of superoxide during myocardial ischemia and reperfusion O2 consists of two oxygen atoms with an electron configuration containing two unpaired electrons in separate orbitals. As such, O2 is a diradical with a high electron affinity and is itself relatively reactive. O2 is completely reduced by accepting four electrons to yield two water molecules. Alternatively, partially reduced oxygen yields highly unstable intermediates. The addition of a single electron to O2 yields O2•-, the 8  addition of another electron in an aqueous environment yields hydrogen peroxide (H2O2), which can get further reduced to the highly reactive •OH. Each of these partially reduced forms of oxygen are generated during ischemia and reperfusion, but experiments using superoxide dismutase clearly indicate that O2•- is the parent radical -without it, neither H2O2 or •OH are appreciably generated58, 61. Low levels of O2•- are generated mainly by electron leakage within the mitochondrial electron transport chains of normally functioning cells. In contrast, O2•- generation during ischemia and reperfusion occurs on a much larger scale, with multiple potential cellular sources.  9  10  Figure 2: Major sources of O2•- during myocardial ischemia and reperfusion. Top panel left: Tricarboxylic acid cycle (TCA) coupled to complete O2 reduction at complex IV with electron flux (e-) through mitochondrial electron transport complexes. H+ pumping at complex I, III, and IV enables ATP synthesis. Top panel centre: Ischemic inhibition of electron flux beyond complex III and decreased TCA cycle activity results in a modest increase in O2•- generation primarily at complex III, decreased H+ pumping, and F1FoATPase reversal. Top panel right: TCA cycle reactivation at reperfusion with sustained electron flux inhibition beyond complex III increases O2•- generation at complex III. H+ pumping at complex I and III weakly reestablishes the proton motive force and enables low-level ATP synthesis. Middle panel left: Xanthine dehydrogenase activity under normal perfusion. Mid panel centre/right: Prolonged ischemia converts xanthine dehydrogenase to xanthine oxidase, which mediated O2•- generation. 2nd centre panel right: NAD(P)H dehydrogenase in activated immune cells produces O2•- generation subsequent to cell damage. Lower panel left: Coupled constitutive nitric oxide synthase (NOS) production of NO•. Lower panel centre: Prolonged ischemia results in the oxidation of tetrahydrobiopterin (BH4), a NOS cofactor, to yield dihydrobiopterin (BH2). Lower panel centre/right: Uncoupled constitutive NOS-BH2 results in O2•- generation.  11  1.3.1 Mitochondrial electron transport The mitochondrial electron transport chain is a series of enzyme complexes that utilize the free energy released by a succession of spontaneous redox reactions to generate a proton gradient during the inner mitochondrial membrane. The high-energy intermediates that fuel this process are NADH and FADH2, which are supplied by the tricarboxylic acid cycle and ultimately lead to the reduction of molecular oxygen to water. During mitochondrial oxidative phosphorylation, the enzyme F1Fo-ATP synthase uses the potential energy stored within the mitochondrial electrochemical proton motive force to generate ATP. The generation of the mitochondrial proton motive force is entirely dependent on the rapid cycling of successive redox intermediates within the mitochondrial electron transport chain. The complexes of the electron transport chain undergo reduction and oxidation in order of increasing reduction potentials in a highly regulated and tightly coupled process. When the system is functioning normally, electron transport is initiated with the oxidation of NADH by the NADH dehydrogenase complex and virtually always results in the complete reduction of oxygen to water at the cytochrome oxidase complex. Only an estimated 1-2% of electrons passing through the mitochondrial electron transport chain under normal physiological conditions result in the production of O2•- 67, 68. The consistent leakage of electrons from the electron transport chain to produce O2•- under normal physiological conditions has fueled speculation of their role as cell signaling intermediates. To this end, Aon et al suggest that this physiological superoxide production is part of an oscillating O2•- pattern which serves a critical role to couple myocardial energy production with energy demand69. Indeed, O2•- generated under 12  normal physiological conditions regulates important cell functions including metabolism, proliferation, and apoptosis (reviewed in70). Mitochondrial O2•- generation occurs in the inner mitochondrial membrane by a nonenzymatic, single electron transfer to O2 primarily by ubisemiquinone (equation 1) in complex III and secondarily by reduced flavin mononucleotide in the NADH dehydrogenase complex (equation 2)68, 71, 72. Most cells contain enzymatic antioxidant defense mechanisms that quickly convert ROS to water. Specifically, superoxide dismutase enzymes, which contain either copper, manganese, or a nickel metal centers that can be either reduced or oxidized to convert O2•- to O2 and H2O2 in a reaction that is effectively diffusion-limited73, 74 (equation 3). H2O2 is subsequently converted to water by either catalase75 (equation 4) or by the glutathione peroxidase system76, 77 (equation 5). Variations of these endogenous antioxidant enzyme systems are expressed in both the mitochondrial matrix and the cytoplasm of their host cells in the myocardium. Equation 1:  UQH " +O2 # UQ + H + + O2•$  Equation 2:  FMNH " +O2 # FMN + H + + O2•$  ! Equation 3(a/b): !  SOD(n +1)+ + O2•" # SOD(n ) + O2  Equation 4(a/b): ! Equation 5(a/b): !  SOD(n ) + O2•" + 2H + # SOD(n +1)+ + H 2O2 CAT(Fe III ) + H 2O2 " CAT(Fe IV = O) + H 2O CAT(Fe IV = O) + H 2O2 " CAT(Fe III ) + H 2O + O2 H 2O2 + 2(GSH) "Glut.peroxidase " " ""# GSSG + 2H 2O GSSG + NADPH + H + "Glut.reductase """ "# 2(GSH) + NADP +  The idea that myocardial injury results from the ROS burst at reperfusion is ! somewhat misleading. Sub-lethal levels of ROS are clearly generated in the ischemic  myocardium57, 78, 79. ROS generated during ischemia induce peroxidation of cardiolipin, a 13  mitochondrial lipid integral to the stability and activity cytochrome oxidase dimers80, 81. Ischemic inhibition of cytochrome oxidase prevents electron flux beyond the level of cytochrome c80, 82-84, and causes redox intermediate cycling to cease. A new equilibrium establishes under these conditions in which upstream intermediates predominantly exist in their reduced form and in which partial O2 reduction to O2•- by ubisemiquinones of complex III is favored. The result is a modest increase in O2•- generation during ischemia79. Complex III of the mitochondrial electron transport chain contains two ubisemiquinone moieties; Q0 and Qi85, 86. Ischemic damage also occurs at complex III87, and this damage appears to enhance O2•- generation at the Q0 site which is oriented toward the intermembrane space where cellular and mitochondrial antioxidant defenses are not present72, 88. By contrast, O2•- generated at the Qi site, like that generated by NADH dehydrogenase, is more likely to enter the mitochondrial matrix, where it would quickly be met with mitochondrial antioxidant defenses72, 86, 88. O2•- released into the intermembrane space can diffuse into the cytosol in its neutral protonated form (HO2•) or through the voltage-dependent anion channel89. The generation of O2•- during brief ischemia is thought to play an important role in preconditioning90-92 and may also play a role in post-reperfusion energy recovery93, so its generation and release to the intermembrane space by Q0 may be important from a signaling perspective. Alternatively, cardiolipin peroxidation and cytochrome oxidase uncoupling originating during ischemia persists into reperfusion, at which time equation 1 becomes the de facto terminal redox step for damaged mitochondrial electron transport. In contrast with ischemia, tricarboxylic acid cycle intermediates and O2 tension are readily  14  replenished at reperfusion, and O2•- generated from damaged electron transport chains and subsequent H2O2 production by superoxide dismutase are free to occur on a much larger scale94. In this way, radicals generated during ischemia work in concert with the oxidative burst at reperfusion to mediate ischemia-reperfusion injury in cardiomyocytes95. O2•- generation during ischemia appears to be a self-limiting phenomenon; decreased flux through the tricarboxylic acid cycle does not lend itself to efficiently supplying single electrons to ubisemiquinone after it leaks them to O2. This reasoning extends to Zweier’s original electron spin resonance observation of a marginal increase in the alkylperoxyl centered radical signal during ischemia at the apparent expense of the carboncentered semiquinone signal57, and also extends to a subsequent study, in which ischemic alkyl-peroxyl and O2•- concentrations reach a plateau rather than increasing indefinitely with prolonged ischemia78. The energy demand of the myocytes is reflected by their large mitochondrial content96, which makes these cells especially vulnerable to electron transport chain damage and to increased mitochondrial O2•- generation during ischemia and reperfusion97, 98  . The consequences of this persistent mitochondrial damage are exemplified by the  finding that cardiac work, normalized to O2 consumption or tricarboxylic acid cycle activity, only fractionally recovers at reperfusion99. If ischemia is brief, a relatively large fraction of mitochondrial electron transport will catalyze the complete reduction of oxygen at reperfusion. If ischemia is more severe, the proportion of damaged mitochondrial electron transport proteins increases along with the amount of O2•- that is produced at early reperfusion (Figure 2).  15  1.3.2  Cellular xanthine oxidase  The mitochondrial electron transport chain is an important source of O2•- during ischemia and reperfusion, but several other sources also contribute. The xanthine dehydrogenase/xanthine oxidase enzyme system was identified early as a potential source of cellular O2•- generation100, 101. Xanthine dehydrogenase catalyzes the catabolic oxidation of hypoxanthine to xanthine and subsequently to uric acid by coupling the reaction with NAD+ reduction to yield NADH (equation 6). During ischemia, xanthine dehydrogenase can be converted to xanthine oxidase by the modification of a sulfhydryl group102 or by proteolytic cleavage103. Although xanthine oxidase is still capable of catalyzing the conversion of hypoxanthine to uric acid, it does so by coupling the reaction with the reduction of molecular oxygen104(equation 7). The cessation of oxidative phosphorylation during ischemia causes ATP degradation to successively lower energy products of purine catabolism, of which hypoxanthine is an intermediate. Thus, hypoxanthine accumulates during ischemia, and serves as a substrate for equation 7a when O2 tension is restored at reperfusion. Equation 7a also provides xanthine as a substrate for equation 7b at reperfusion. Equation 6 (a/b):  Equation 7 (a/b): !  hypoxanthine + NAD+ "XDH " "# xanthine + NADH xanthine + NAD + "XDH " "# uric _ acid + NADH XO hypoxanthine + O2 "" # xanthine + O2•$ XO xanthine + O2 "" # uric _ acid + O2•$  The contribution of xanthine oxidase as a significant source of O2•- generation during ischemia and!reperfusion remains rather controversial. The xanthine oxidase inhibitor allopurinol was shown to preserve myocardial glutathione and catalase activity and decreased infarct size in separate dog models of ischemia-reperfusion injury in the 16  1980’s105, 106. Shortly thereafter, Grum et al failed to detect any appreciable xanthine oxidase or dehydrogenase activity in the rabbit heart107. Similarly undetectable xanthine oxidase activity was reported in the human heart108, 109, and its contribution to postischemic O2•- production in the rabbit heart was discounted110. These results contradict allopurinol-mediated free-radical reduction. Xanthine oxidase is readily expressed in endothelial cells that line the myocardial vasculature111, 112, thus allopurinol may protect the myocardium by reducing post-ischemic O2•- production in endothelial cells. Alternatively, allopurinol has been shown to improve post-ischemic myocardial function independently of xanthine oxidase inhibition113-115, possibly through an intrinsic antioxidant activity116 or cellular metabolic modulation117, 118. These secondary effects are more likely explanations for allopurinol-mediated protective effects recently reported in patients undergoing percutaneous intervention following acute myocardial infarction119. Xanthine dehydrogenase activity was found to be relatively stable in human heart tissue120, and its conversion to xanthine oxidase is a relatively slow process with a halflife of 7 hours in ischemic rat tissue103. This xanthine dehydrogenase stability suggests that xanthine oxidase is unlikely to be appreciably formed during transient ischemia. By the same token, one would expect xanthine oxidase levels to increase under prolonged pathological conditions such as ischemic heart disease (Figure 2). This is in agreement with previously reported results121, and therefore provides mechanistic support to reports of allopurinol-mediated reductions in O2•- markers with improved functional recovery within the context of CABG122 and improved exercise tolerance in patients with chronic stable angina123.  17  1.3.3 The immune system The spin-trap and resonance studies that definitively identified O2•- as the parent free radical produced in the post-ischemic heart showed an important difference between experimental models: isolated heart models had maximal free-radical intensities within a minute of reperfusion before quickly dropping to near-baseline levels57-59, 62, 64, 78, 124, while similar experiments in intact animals showed that the oxidative burst was still elevated 3 hours after reperfusion60, 61. This discrepancy in the timeframe for ROS generation between isolated heart and whole animal models illustrated that whole blood either prolongs myocardial O2•- generation, or contains components that, once activated by the post-ischemic myocardium, take over O2•- production. It is in this context that the cells of the immune system are another source of post-ischemic myocardial O2•generation. The activation of the immune system at reperfusion in response to myocardial injury is a stepwise process whose magnitude depends on the extent of myocyte and endothelial cell damage. Myocardial damage incurred during ischemia and early reperfusion activates the complement system125, 126, which in turn leads to the secretion of strong chemotactic stimuli and the expression of proteins that promote infiltration of phagocytes (reviewed in127). The contribution of phagocytes to post-ischemic myocardial O2•- generation occurs primarily as a result of neutrophil infiltration127-129. These cells contain the enzyme NAD(P)H oxidase, which can use either NADH or NADPH as substrates for the single electron reduction of O2 (equation 8, Figure 2)130. Equation 8:  !  _ oxidase NAD(P)H + 2O2 "NAD(P " ")H" ""# NAD(P) + + H + + 2O2•$  18  The generation of O2•- by the immune system is less a consequence of ischemia and reperfusion than an immune response to tissue and cell damage. As such, one would expect a brief delay between the cellular post-ischemic oxidative burst and that originating from leukocytes. This delay helps to reconcile the temporal difference in postischemic O2•- generation between isolated heart and intact animal models. The contribution of O2•- generation from phagocytes during CABG with CPB is more difficult to assess. The compliment system is activated during CPB131, but also during off pump CABG132. Furthermore, while off-pump CABG is generally associated with decreased inflammation133-136, several studies indicate that the effect of the surgical procedure itself far outweighs the use of CPB as a pro-inflammatory factor during CABG137-139. Additionally, intraoperative reductions in inflammation do not necessarily reduce acute postoperative inflammation140. More specifically, the question of whether polymorphonuclear leukocytes play an active role in mediating post-ischemic myocardial injury remains controversial. Inflammatory mediators are clearly activated and recruited to the post-ischemic myocardium, but neutrophil depletion does not appear to inhibit myocardial stunning and studies of clinical anti-neutrophil therapies do not generally produce clear benefits (reviewed in141). Furthermore, superoxide dismutase scavenging of extracellular O2•during ischemia and reperfusion has long been known to be ineffective at reducing myocardial ischemia-reperfusion injury142, while intracellular superoxide dismutase is more effective143, 144. These results indicate that endogenous intracellular free-radical generation is the central mediator of injury during ischemia and early reperfusion. These results support the concept that while the immune system plays a significant role in the  19  pathophysiology of ischemia-reperfusion injury; inflammation appears to be the consequence rather than the cause of myocardial injury. 1.4. Nitric oxide generation during myocardial ischemia and reperfusion Since its identification as “endothelial derived relaxation factor”145-147, several important roles spanning a host of physiological processes have been ascribed to nitric oxide (NO•). These include vasoregulation148, neurotransmission149, immunomodulation and defence150, 151, and cell signaling152. The diverse physiological importance of NO• belies its relatively recent discovery, but underscores its acclamation in 1992 by Science Magazine as “molecule of the year”153, 154. NO• is a relatively stable free radical whose half-life of a few seconds, combined with its solubility in both aqueous and organic solutions, allow it to freely diffuse across cell membranes. It spontaneously reacts with oxygen and water to form nitrite (equation 9), and is further oxidized in the presence of oxyhemoproteins to nitrate (equation 10a/b)155. Although dietary nitrates significantly contribute to their concentration in plasma, the concentrations of nitrate and nitrite after several hours of fasting, is a good indicator of overall NO• activity156. Equation 9:  6  "1 "1  s 4NO• + O2 + 2H 2O #k=#2x10 #M## $ 4NO2" + 4H +  2RFe 2+O2 + 3NO2" + 2H + # 2Fe 3+ + 3NO3" + H 2O  Equation 10(a/b): 4RFe 2+O2 + 4NO2" + 4H + # 4Fe 3+ + 4NO3" + O2 + 2H 2O ! NO• is important in the context of myocardial ischemia-reperfusion injury primarily ! with O •- to produce the peroxynitrite anion (ONOO-)157 (equation 11), because it reacts 2  which is itself far more reactive and toxic to biomolecules than its reactants. The reaction between O2•- and NO• is diffusion limited, and since the rate of ONOO- formation 20  depends on the product of its reactant concentrations, small increases in O2•- or NO• can rapidly increase ONOO- levels. Equation 11:  9  "1 "1  #7x10 M s NO• + O2•" $k$ $ $$% ONOO"  When Zweier et al reported electron paramagnetic resonance spectra during ischemia ! they noticed that the pattern of nitrogen-centered radical generation and reperfusion,  mirrored that of the oxygen-centered radical during ischemia and early reperfusion57. Its uncertain identity was subsequently assumed to be a peroxylamine radical product of free-radical mediated peptide bond breakage78. Similarly obtained electron spin resonance spectra demonstrated nitroxyl radical adducts59. The benefit of more recent findings suggests that this signal could have indicated the generation NO• and/or nitrogen dioxide radical (NO2•), the latter being a breakdown product of peroxynitrous acid (ONOOH) (equation 17). Myocardial NO• generation during both ischemia and reperfusion is a prerequisite for both cases. There are two likely sources of myocardial NO• generation during ischemia and reperfusion; nitric oxide synthase (NOS) activity and nitrite reduction. 1.4.1 Nitric oxide synthases The enzyme-catalyzed 5-electron oxidation of L-arginine to L-citrulline is the best studied NO• producing biosynthetic pathway (equation 12). This reaction is catalyzed by one of three NOS enzyme isoforms; neuronal-, inducible-, and endothelial-NOS (nNOS, iNOS, and eNOS, respectively)158. The myocardium constitutively expresses both eNOS and nNOS159-161, and can be induced to express iNOS161, 162. Equation 12: 2LArg + 3NADPH + 3H + + 4O2 " "# 2LCit + 3NADP + + 2NO• + 4H 2O  !  21  Enzymatically active NOS exist as a homodimeric oxidoreductase associated with the cofactor tetrahydrobiopterin, and contains heme, flavin adenine dinucleotide, and flavin mononucleotide moieties. Electron flux through all NOS isoforms requires calmodulin binding, but only in constitutive NOS isoforms (nNOS and eNOS) does calmodulin binding occur with calcium dependence163. Since L-arginine is the common substrate for enzymatic NO• production, the pharmacology of several of L-arginine analogs, namely LNMMA (NG-monomethyl-L-arginine) and L-NAME (NG-nitro-L-arginine methyl ester) were studied and identified as potent NOS inhibitors164. Constitutive NOS expressed in various cell types of the myocardium are a constant source of low-level NO• generation in the normally perfused heart165. During ischemia, several factors that directly influence constitutive NOS activity are altered; intracellular calcium loading may by itself promote constitutive NOS activity, but decreased oxygen tension would conversely decrease enzyme activity. The restoration of oxygen tension at reperfusion in a calcium-loaded cell would by extension be expected to result in a sudden increase in NOS activity, assuming enzyme integrity was preserved throughout ischemia. This question was partly resolved using of electron paramagnetic resonance techniques capable of analyzing NO•, by the same group that used the same technique to definitively identify and characterize O2•- production during ischemia and reperfusion one decade earlier166. These experiments, and similar studies clearly showed that NO• is generated throughout the myocardium during ischemia, the majority of it being a product of NOS activity166-168. NO• production was subsequently also shown to be increased at reperfusion, and here too the increase was largely dependent on NOS activity169. This result is somewhat expected because endothelial cells increase NO• release in response to  22  shear stress through elevated eNOS activity (reviewed in170), and shear stress increases in the coronary vasculature as a physical consequence of reperfusion. Paradoxically, the increased NO• production at reperfusion also increases ONOO- generation (equation 11), which irreversibly damages the heme domain of eNOS and oxidizes its tetrahydrobiopterin cofactor171. Although these results clearly demonstrate that myocardial NOS actively produces NO• during ischemia and reperfusion, a parallel line of evidence shows that the enzyme is not unaffected by ischemia. The intracellular acidosis that accompanies prolonged ischemia can inhibit eNOS activity in a manner that, depending on the severity and duration of the acidosis, can be either reversible or irreversible172. Interestingly, the heme domain of nNOS isolated from brain tissue was shown to be capable of producing O2•under depleted L-arginine concentrations173, 174. Similar results were subsequently reported for eNOS175, 176 and the reductase domain of iNOS177, 178. The conditions under which constitutive NOS isoforms shift to O2•- production are now recognized to be dependent not only on the intracellular L-arginine supply, but also on tetrahydrobiopterin oxidation, which is a major consequence of prolonged ischemia179, 180. In this way, uncoupled myocardial NOS can also contribute to O2•- generation during ischemia and early reperfusion (Figure 2). 1.4.2 Nitrite reduction The evidence that intracellular acidosis denatures and uncouples NOS, combined with the decreased O2 availability as a NOS substrate appears to run counter to the observed increase in NO• generated during ischemia. Furthermore, careful inspection of electron paramagnetic resonance results indicate that NO• generation increases with prolonged 23  ischemia, and is not abolished under conditions of complete NOS inhibition166. These results indicate the activity of a NOS-independent cellular NO• source. This second source was identified as the reduction of nitrite (equation 13), which occurs physiologically in a non-enzymatic, pH-dependent fashion, when the cell exists in a sufficiently hypoxic and reduced state181. Equation 13:  2NO"2 + 4H + # 2H 2O + 2NO•  In contrast to the L-arginine-NOS pathway of NO• production, nitrite represents a ! • production during both ischemia and early reperfusion, when NOS reservoir for NO  enzymes can no longer be relied upon. Equation 13 implies that under sufficiently reductive and acidic conditions during ischemia, nitrites continuously feed chemical NO• production. This reaction appears to proceed within the cellular context of myocardial ischemia only when the pH dropped below 6181. If severe ischemic conditions can permanently denature a fraction of the myocardial NOS pool, and intracellular pH recovers sharply at reperfusion182, then nitrite must be reduced to NO• by an alternative mechanism in the early phase of reperfusion. One of these alternatives is mediated in blood under low oxygen tension by deoxygenated hemoglobin183. This mechanism is presumed to induce vasodilation in hypoxic tissues, and may therefore be more relevant in blood circulating through ischemic tissues. Because the product of NO• degradation also serves as its non-enzymatic generation, non-enzymatic nitrite reduction does not affect overall plasma nitrite and nitrate levels. A second alternative pathway is mediated by xanthine oxidase. This enzyme was described as a source of nitrite reduction under zero-flow ischemia184-188, where nitrite and O2 compete for the O2•- generating mechanism illustrated by equation 7. Xanthine  24  oxidase can also catalyze nitrite reduction under the aerobic conditions encountered at reperfusion189. NO• produced by the myocardium during ischemia and reperfusion likely represents the sum of the mechanisms described above: During ischemia, nitrite reduction –direct and/or xanthine oxidase mediated, depending on the degree of intracellular acidosis- produces NO• in conjunction with functional NOS when they encounter substrate. At reperfusion, a burst of NO• synthesis is driven by the fraction of nondenatured NOS enzymes, activated both by the shear stress that reperfusion imposes on the vascular endothelium and by intracellular Ca2+ overload with the influx of O2 substrate, complemented by nitrites that are in competition with O2 for xanthine oxidase derived electrons. 1.5. Hydroxyl radical generation during myocardial ischemia and reperfusion The ability of exogenous superoxide dismutase to reduce ischemia-reperfusion injury established O2•- as a harmful radical well before its generation could directly be measured in the myocardium. By contrast, O2•- has a relatively benign reactivity towards biomolecules insofar as free radicals are concerned190-193. Furthermore, cellular antioxidant defenses convert O2•- to O2 and H2O at rates that approach diffusion limits (equations 3, 4, 5). These contradictory findings can be reconciled by a secondary O2•reaction, capable of competing with superoxide dismutase, which becomes more favorable during ischemia and reperfusion, which yields a product with more reactivity than its reactants. The reactivity of the hydroxyl radical made it an obvious candidate as the end effecter of oxidative cellular damage, and its definitive identification in the post-ischemic reperfused myocardium cemented this notion62, 78, 194, 195. Furthermore, the finding that its 25  signal was abolished by superoxide dismutase validated the concept that O2•-, through some reaction within the cell, was a reactant in •OH generation. Two pathways have been identified by which O2•- reactions can yield •OH: iron-catalyzed reduction and ONOO/ONOOH decomposition. 1.5.1 Iron-catalyzed hydroxyl formation The ability of transition metal ions to assume multiple oxidation states underscores their central role as catalysts for a host of cellular redox reactions. One such metal ion is iron. Biologically complexed iron can stably undergo single electron redox reactions that are not only critical to mitochondrial electron transport, but to cell function in general196. This same attribute can also facilitate Fe2+-mediated •OH formation through the Fenton reaction (equation 14). In order for the Fenton reaction to proceed past one cycle, Fe3+ must be reduced back to Fe2+, and it is in this capacity (equation 15) that O2•- serves to perpetuate the Fenton reaction. The sum of the Fenton reaction and the superoxidemediated reduction of Fe3+ results in a complete redox reaction known as the HaberWeiss reaction (equation 16)197. Equation 14:  Fe 2+ + H 2O2 " Fe 3+ + •OH + OH #  Equation 15:  O•2 + Fe 3+ # O2 + Fe 2+  ! Equation 16:  #1000M s O•" + H 2O2 $k$ $$ $% O2 + •OH + OH " 2  "  "1 "1  ! Iron-catalyzed •OH generation is problematic on several fronts. First, ferric iron ! aqueous solutions at physiological pH, while ferrous iron, despite being precipitates in  soluble, reduces O2 to produce ferric iron and O2•-. Virtually all physiological iron consequently exists as heme, or protein complexes, leaving only trace amounts of non-  26  chelated iron198. This limitation can be overcome because O2•- is known to destabilize several iron-sulfur containing enzymes, liberating Fe2+ in the process to catalyze the Fenton reaction199. Even if the extent to which this happens is small, the overall HaberWeiss reaction can proceed at very low H2O2 and iron concentrations with rate constant of about 1000 M-1•s-1, provided adequate O2•- can be supplied200. Another problem with iron-catalyzed •OH generation results from its relatively low kinetic rate constant. Not only does the reaction between O2•- and superoxide dismutase have a rate constant in excess of five orders of magnitude greater than that estimated for the Haber-Weiss reaction, the high concentration of superoxide dismutase dictates that O2•- is much more likely to encounter superoxide dismutase than cellular Fe2+. Catalasemediated H2O2 oxidation is also more likely than H2O2 reduction in the Fenton reaction for similar reasons201. Despite these limitations, the finding that the iron chelator desferrioxamine can reduce ischemia-reperfusion injury202-204 (although not universally so205, 206) lends support to the notion that cellular Fe2+ can contribute to •OH generation during ischemia and reperfusion. 1.5.2 Peroxynitrous acid degradation The extent to which trace metals catalyze •OH generation is unresolved. The kinetics of the overall Haber-Weiss reaction in a superoxide dismutase - and catalase-containing environment makes it an unlikely pathway for the increases in •OH generation during myocardial ischemia and reperfusion. The alternative source of •OH must be able to sufficiently compete with the kinetics and cellular concentration of superoxide dismutase. To this end, the reaction between O2•- and NO• which yields ONOO- (equation 11)157  27  occurs spontaneously and with a rate constant that, like those of superoxide dismutase and catalase, approaches diffusion207, 208. ONOO- is relatively stable and has a 1.9 second half-life at pH 7.4, which permits diffusion over a distance of several cell diameters, presumably through anion channels209. Conversely, ONOOH is a strong oxidizing agent that is inherently unstable. ONOOH can rapidly decompose to produce NO2• and •OH radicals209 (equation 17). The pKa of the ONOO-/ONOOH acid-base pair is 6.8 at 37oC210, so ONOO- becomes fractionally more protonated with increasing intracellular acidification during ischemia. The decay of ONOOH is an attractive model for •OH generation because it does not depend on the Fenton reaction, is not inhibited by catalase activity211, and preferentially yields •OH during conditions of ischemia and reperfusion. Equation 17:  pK a = 6.8 ONOO" + H + #% % %$ ONOOH % %$ NO2• + •OH  Despite the fact that myocardial generation of O2•- and NO• are not restricted to ischemia and!reperfusion, the generation of ONOO- and •OH are not appreciable until the process has taken place78, 124, 169. The likelihood that O2•- will react with superoxide dismutase or NO• depends on their relative cellular concentrations. The concentration of superoxide dismutase in the normally functioning myocardium exceeds that of NO• by several orders of magnitude, so equation 11 is unlikely to occur and very little ONOOwill be generated. The concentration of NO• approaches and surpasses that of superoxide dismutase during reperfusion, and equation 11 becomes the dominant reaction209, 212. The finding that exogenous superoxide dismutase can decrease myocardial •OH generation further reflects the notion that a balance exists between equation 3 and equation 11 as the dominant O2•- reaction.  28  The relative contribution of ONOO-/ONOOH degradation and the Haber-Weiss reaction to•OH generation during ischemia and reperfusion remains a question of debate. The rate constant of the Haber-Weiss reaction is low in comparison to that of ONOOgeneration, but myocardial H2O2 content can dramatically increase during ischemia and reperfusion213 and both desferrioxamine and catalase can reduce post-ischemic myocardial •OH generation214. Furthermore, lipid peroxidation products have somewhat paradoxically been found to directly correlate with superoxide dismutase and H2O2 concentrations, while chelators and •OH scavengers appear break this relationship215. On the other hand, the protective effects of desferrioxamine have partly been ascribed to its ability to inhibit ONOO- -mediated oxidation reactions rather than iron chelation209, 210 and catalase activity does not necessarily reduce infarct size216, 217.  Figure 3: Main hydroxyl radical producing pathways during myocardial ischemia and reperfusion.  29  The loss of a protective effect at higher doses of superoxide dismutase218 has also been attributed to an increase in H2O2 production by superoxide dismutase, which in turn inhibits superoxide dismutase activity and drives •OH generation through the Fenton reaction219-221. In support of this notion, covalent superoxide dismutase -catalase conjugates can inhibit •OH generation and reduce ischemia-reperfusion injury at concentrations that induce toxicity with non-conjugated superoxide dismutase 222. Several mathematical models contest the notion that superoxide dismutase influences cellular H2O2 levels unless a large proportion of O2•- is eliminated from the system by a mechanism that is independent of superoxide dismutase223-225. To this end, O2•- can be rapidly eliminated by reacting with NO• during ischemia and reperfusion. Thus, O2•derived •OH radicals likely result from ONOO-/ONOOH degradation proceeding in concert with the Haber-Weiss reaction during ischemia and reperfusion (Figure 3). 1.6. Free radical-mediated damage during myocardial ischemia and reperfusion The myocardium functions as a large interconnected electrical circuit in which synchronized diastole-systole cycling and contraction rely on coordinated conductivity during its component cardiomyocytes. Impaired heart function subsequent to ischemia and reperfusion is by extension a symptom of cell damage that regionally uncouples the myocardial conductivity network. The severity of cell damage incurred, which ranges from reversible to irreversible, is proportional to the magnitude of ROS reactions within cardiomyocytes226. Radical reactions are stepwise, spontaneous, self-propagating chain reactions. Free radical propagation reactions in the context of biological injury primarily result in hydrogen abstraction from alkene groups. Since these groups occur in many, but not all 30  biomolecules, these reactions are semi-random in nature. From the perspective of the cell, the semi-random uncontrolled nature of radical reactions makes them inherently damaging. Free radicals are generally strong oxidants, but the degree to which they exert these reactions depends on their individual reactivity towards the biomolecules that they encounter. For example, the short half-life and reactivity of the •OH radical dictates that it abstracts hydrogen atoms from a wide array of biomolecules provided they are located in the cellular vicinity of the •OH radical at the time of its generation. Alternatively, the less reactive O2•- and NO• radicals can diffuse to more distal locations from their origins prior to mediating radical reactions on more specific targets. The first phase of radical reactions is an initiation that generates the original radical. In the context of ischemia-reperfusion injury, initiation reactions are those that produce O2•-, NO•, and •OH. These processes have been discussed in detail within the context of myocardial ischemia and reperfusion in sections 1.2, 1.3, 1.4, and 1.5. The second phase is a series of propagation reactions. These include substitution reactions that often involve hydrogen atom abstraction from an alkene group to produce an alkyl radical, and addition reactions exemplified by the integration of molecular oxygen to the alkyl radical to yield a peroxyl radical. The reaction is further propagated because any reaction involving a radical and a non-radical invariably yields a free-radical product. The aforementioned peroxyl radical, for example, can abstract a hydrogen atom from another alkene to yield a hydroperoxide and a new alkyl radical. The final phase of radical reactions is the termination step, which occurs when a radical undergoes a second single-electron redox reaction to produce a terminal nonradical. The products of radical reactions are non-enzymatically modified organic  31  biomolecules that are usually disruptive to the function of the larger cell structures in which they occur. Thus, lipid peroxidation products can compromise the integrity of cell membranes or of proteins embedded therein; amino acid nitration or oxidation can impair normal protein function; and DNA oxidation is mutagenic. The semi-random nature of radical reactions does not necessarily yield irreversible cell damage: if non-critical proteins are only fractionally damaged, or if the magnitude of membrane, protein, or DNA damage does not adversely affect function, then the cell will survive. In this case, normal cell processes such as phospholipid and protein turnover can remove the altered biomolecule. Alternatively, radical reactions can denature or activate critical biomolecules to initiate pro-apoptotic processes. In the most severe cases, when radical reactions compromise critical cell functions and/or membrane integrity, the cell becomes necrotic. The cellular consequence of radical reactions within the myocardium is therefore a question of scale and radical concentration, and a question of the probability that certain critical cellular components will be affected. 1.6.1 Lipid peroxidation Phospholipid bilayers form the biological membranes that encapsulate cells and their organelles. The balance between membrane integrity and fluidity depend on their constituent saturated and unsaturated fatty acid components. The alkenes in unsaturated fatty acids are susceptible to hydrogen abstraction by ROS to yield carbon-centered and peroxyl radicals210. These are the carbon- and oxygen-centered lipid radicals that were observed in early paramagnetic resonance studies and were first definitively identified as such by Arroyo et al in 198764. The propagation phase of lipid peroxidation is effectively an amplification of the initial oxygen radical mediated hydrogen abstraction that 32  continues until two radical species combine in a termination reaction. Interestingly, NO• is not reactive enough to initiate lipid peroxidation, but it can act as a chain terminator227. Peroxyl and lipid isoforms and can react within and between one another to produce a heterogeneous array of end products that encompass isoprostane-like compounds and acid-mediated fragmentation products that include malondialdehyde (MDA) (Figure 4)228-231. These altered fatty acids can adversely affect the functional integrity of lipid membranes, and are therefore quickly cleaved and released by phospholipases232.  33  Figure 4: Generalized example scheme of lipid peroxidation. X and Y represent independent chain lengths and structures. Note that hydrogen abstraction can occur with similar frequency at other regions within a given precursor structure, that additions can occur at a variety of resonance structures, and that “isoprostane like compounds” represent a host of compounds with a host of structures (loosely adapted from233 and230).  34  In vivo free radical reactions produce a host of compounds that are not otherwise catalyzed by the cell. The non-enzymatic catalyzed origin of these compounds makes them potentially useful markers of oxidative stress provided they can be accurately and quantitatively analyzed. To this end, multi-center studies of various non-invasive biomarkers of oxidative stress specifically identified two lipid-peroxidation products, MDA and isoprostanes, as indicators of increased free radical activity234-236. MDA and isoprostane levels are known to increase during myocardial ischemia and reperfusion237240  , one isoprostane isomer, 15-F2t-isoprostane, induces dose-dependent vasoconstriction  in the coronary vasculature241, 242. As such, 15-F2t-isoprostane formation may exacerbate or prolong cardiac dysfunction after ischemia and reperfusion243-245. From a clinical perspective, 15-F2t-isoprostane levels increase during coronary angioplasty and coronary artery bypass grafting procedures246-248, and it has been associated with a decline in postoperative cardiac function244, 245, 249. Unsaturated fatty acids in the mitochondrial membrane are especially vulnerable to peroxidation because of their intimate proximity to ROS generated by complex III during myocardial ischemia and reperfusion. One particular mitochondrial lipid, cardiolipin, constitutes nearly 20% of the inner membrane250, and is a key regulator of mitochondrial integrity and function251. Specifically, electron transport through cytochrome c oxidase depends on its association with cardiolipin252, 253. Cardiolipin content decreases with increased lipid peroxidation during myocardial ischemia81, 82, which appears to facilitate cytochrome c release and apoptotic signaling during reperfusion254 (reviewed in255). These alterations coincide with and result in the inhibition of cytochrome c oxidase activity256, 257, and may ultimately also inhibit post-ischemic electron flux through  35  complex I258 and complex III259. Cardiolipin depletion and cytochrome c release can be preserved if complex I of the electron transport chain is pharmacologically inhibited prior to ischemia260. These results further underscore the central role that mitochondria play in the pathological ROS generation during myocardial ischemia-reperfusion injury. 1.6.2 Protein oxidation and nitration The alkene groups of unsaturated fatty acids in cell membranes are prone to hydrogen abstraction, but ROS can induce cardiomyocyte apoptosis and necrosis even when cellular and subcellular membranes remain functionally intact. This occurs because cellular proteins can be non-enzymatically modified by ROS. Amino acid oxidation and nitration within proteins underlies several clinical ischemia-reperfusion injury pathologies. Specifically, the activation of necrosis and pro-apoptotic proteins underlies infarction261 while damaged contractile proteins impairs contraction to yield myocardial stunning262-266. Proteins are susceptible to ROS damage because the functional groups and backbone α-carbons of all amino acids are susceptible to hydrogen abstraction by •OH 267. The nature of these oxidation reactions and the products that they produce is a function of the amino acid functional group, the type of ROS, and the semi-random nature of radical reactions (Figure 5)267-271. A complete catalogue of the potential reactions would have to account for post-translationally modified amino acids, the presence or absence of adjacent functional groups, and reactions between amino acids and non-protein radicals such as lipid radicals.  36  Figure 5: Generalized examples of free-radical mediated amino acid modification scheme. Upper panel: •OH -mediated hydrogen abstraction from tertiary carbons within an amino acid or peptide. Middle panel: •OH -mediated hydrogen abstraction or ONOOH-mediated oxidation of the aromatic group of phenolic amino acid. Lower panel: OH-mediated hydrogen abstraction from a sulfhydryl or methionine amino acid. R  •  represents a generalized continued amino acid or peptide motif. 37  Free radical oxidation reactions can denature proteins by cleaving peptide bonds, by cross-linking functional groups, and by altering the hydrophobicity of amino acids on protein surfaces267, 272. However, not all of these reactions are irreversible; methionine oxidation is enzymatically reduced back to methionine in a system that may contribute to cellular antioxidant defences273, 274. Disulfide bonds can likewise be enzymatically reduced275. Conversely, H2O2 generated during ischemia by the increasing activity of superoxide dismutase can deplete the glutathione antioxidant system. In this vein, cellular and mitochondrial sulfhydryl groups are significantly oxidized after ischemia and reperfusion, while tyrosine dimerization and protein-lipid peroxide conjugation products increase276. Oxidation of regulatory sulfhydryl groups in mitochondrial complex II markedly reduces electron transport in rat hearts following ischemia and reperfusion277. The ONOO- -mediated oxidation of sulfhydryl groups proceeds with a rate constant that exceeds that of H2O2 by three orders of magnitude278, and its generation is thus capable of rapidly depleting endogenous antioxidant stores. Mass spectrometry proteomic techniques applied to myocardial biopsies enable the analysis of protein changes during ischemia and reperfusion279. The use of these techniques in isolated heart models indicates that a variety of myocardial proteins are physically and chemically modified during ischemia and early reperfusion rather than differentially expressed280. More comprehensive studies revealed that protein modifications during ischemia involve several ROS-independent and a limited number or ROS-dependent modifications, while damage during reperfusion is primarily ROSdependent and is a major cause of post-reperfusion myocardial contractile dysfunction281.  38  These findings support the notion that ischemic ROS generation depletes endogenous antioxidant defenses and prime the cell for ROS damage at reperfusion. The phenolic groups of tyrosine residues are the functional units for a wide variety of cell signaling proteins collectively called tyrosine kinases and protein tyrosine phosphatases. The integrity of these signaling proteins and the signal transduction pathways that they encompass are vulnerable to ONOO- -mediated oxidation reactions. These tyrosine oxidation reactions can yield dityrosine and 3-nitrotyrosine282-284. Accordingly, myocardial 3-nitrotyrosine and dityrosine content increases after ischemia and reperfusion124, 169, 212, 285. Proteomic analysis subsequent to ischemia and reperfusion revealed that mitochondrial proteins were particularly at risk of tyrosine nitration286, reflecting their proximity to the cellular ONOO- source287. To this end, nitration and oxidation of tyrosine and cysteine residues within mitochondrial complex II at reperfusion is one mechanism by which decreased mitochondrial respiration persists despite the recovery of myocardial oxygen tension288, 289. 1.6.3 DNA damage DNA molecules are susceptible to ROS damage in the same basic way that proteins are, namely, an initial •OH -mediated hydrogen abstraction followed by purine or pyrimidine modification and/or fragmentation290. Interestingly, MDA, an ROS-mediated lipid peroxidation product, is a carcinogen because it has the ability to form adducts with DNA bases291. In this way, ROS-mediated lipid peroxidation can both directly and indirectly damage DNA. Genomic damage can initiate apoptotic pathways and impair myocardial protein expression. The initiation of these processes can deplete myocardial energy and NAD+ stores, and can impair repletion of cellular energy stores by inhibiting 39  glycolysis and mitochondrial function292. On the other hand, apoptotic cascades are more likely to result from protein or cell membrane damage than from DNA damage, and ROS-mediated genetic mutations are not generally considered hallmarks of myocardial ischemia-reperfusion injury. 1.6.4 Matrix metalloproteinases Myocardial regions that are subject to prolonged sub-lethal ischemia undergo structural rearrangements that are characteristic of the hibernating myocardium. This myocardial remodeling proceeds in part on account of extracellular matrix metalloproteinase (MMP) activity. MMPs can be transcriptionally upregulated under conditions of chronic tissue remodeling, but they are also acutely activated inside the cell by ONOO- or H2O2 mediated proenzyme cleavage293-297. Accordingly, MMP activity can contribute to ischemia-reperfusion injury in which post-operative myocardial function is depressed despite the absence of appreciable apoptosis and necrosis. During cardiac surgery, ischemia-reperfusion injury often manifests itself by persistently reduced contractility in the absence of large-scale myocyte loss. This post-operative phenomenon is called myocardial stunning, and is a major contributing factor to post-operative low cardiac output syndrome. The impaired contractility of individual myocytes is in part the result of MMP-2-mediated degradation of intracellular proteins including myosin light chain298, α-actinin299, and troponin I300, 301. From the perspective of cardiac surgery, ischemia and reperfusion incurred during CABG with CPB activates both MMP-9 and MMP-2 inside the cell where they digest intracellular contractile target proteins. This activation inversely correlates with postoperative cardiac function302-304. By extension, ONOO- -mediated contractile dysfunction in the isolated cardiomyocyte can be decreased 40  by pharmacologically inhibiting MMP activity305. Furthermore, MMP inhibition decreases endothelial injury and improves myocardial functional recovery in an isolated heart model of ischemia-reperfusion injury300, 306. Thus, from the perspective of contractile proteins, ROS generated during ischemia and reperfusion can persistently impair myocardial function by non-enzymatic protein degradation and modification, as well as by activating specific proteolytic enzymes. 1.6.5 Mitochondrial permeability transition Large-scale mitochondrial ROS generation during ischemia and reperfusion induces a cellular process called mitochondrial permeability transition307-313(Figure 6). This process is strongly inhibited during ischemia by intracellular acidosis below pH 7, but favored by depleted energy stores313, 314 and increased calcium loading314. Accordingly, mitochondrial permeability transition takes place during the first few minutes of reperfusion when intracellular pH starts to recover315-318.  41  Figure 6: Summary of the major processes and consequences of mitochondrial permeability transition. (ROS=reactive oxygen species; Casp=Caspase; Apaf=apoptotic peptidase activation factor,Ψm=inner mitochondrial membrane potential).  42  Mitochondrial permeability transition involves the formation and opening of pores on the inner mitochondrial membrane that allow nonspecific conductance of molecules smaller that 1.5kDa in size319, 320. Mitochondrial permeability transition rapidly uncouples oxidative phosphorylation, dissipates the mitochondrial membrane potential, and further depletes cellular ATP stores. Matrix proteins, generally being too large to pass through the pores, exert a colloidosmotic pressure that causes the mitochondrial matrix to swell, thus compromising the integrity of the outer membrane. Simultaneous cardiolipin peroxidation in the inner membrane liberates cytochrome c321, 322, which can subsequently be released to the cytoplasm where it initiates strong pro-apoptotic signal cascades323. The severity of mitochondrial permeability transition directly affects the ability of the cell to maintain ATP stores. If mitochondrial permeability transition is transient and the outer membrane remains intact, then the cell can recover and survive310. Alternatively, a sufficiently energized cell will become apoptotic under sustained mitochondrial permeability transition. Lastly, sustained mitochondrial permeability transition in cells that have severely depleted ATP stores will become necrotic324, 325. Accordingly, the number of mitochondria undergoing mitochondrial permeability transition correlates with the likelihood of cardiomyocyte loss312 and the severity of mitochondrial permeability transition is inversely proportional to the recovery of contractile function in an isolated heart model326. Postconditioning, a phenomenon in which protection is afforded by staggered intermittent reperfusion, appears to extend the mitochondrial permeability transition-inhibitory features of ischemia into the early phase of reperfusion327, 328.  43  1.7. Protective strategies during cardiac surgery The prevalence of coronary artery disease, combined with the lethality of its manifestations account for a major cause of death and disability worldwide. Stable atherosclerotic plaques result in chronic ischemia, which in turn leads to myocardial stunning, hibernation and remodeling anterograde of the plaque. These changes result in heart failure, and require revascularization to restore adequate cardiac performance. More urgently, unstable atherosclerotic plaques can rupture to yield coronary artery occlusions that, unless treated with emergency revascularization, can result in fatal acute myocardial infarction (reviewed in329). Revascularization by definition exposes the heart to ischemiareperfusion injury, the magnitude of which directly impacts post-operative patient outcomes. Strategies aimed at reducing ROS-mediated damage incurred during myocardial ischemia and reperfusion are generally targeted towards the three phases of free radical reaction progression. Thus, protection may involve inhibition or quenching of O2•-, NO•, or •OH generation during ischemia and early reperfusion. Alternatively increasing the likelihood that a stabile, less reactive free radical will be generated can promote chain termination reactions. These strategies are made inherently more complex by the important physiological –and even cardioprotective role that both O2•- and NO• play as signaling molecules, and by the clinical nature in which these strategies must ultimately be employed. Indeed, several such cardioprotective strategies have been developed, but translating laboratory-based successes to the clinical setting remains elusive330, 331. Despite these difficulties, several interventions are known to reduce the severity of ischemia-reperfusion injury, and do so by altering one or more phases of free-radical  44  reactions in the myocardium. Chief among these is ischemic preconditioning, which involves a host of endogenous signaling steps that ultimately reduce ROS generation at reperfusion. Volatile anesthetics can pharmacologically activate protective pathways that may be common to ischemic preconditioning, thus potentially making preconditioning more clinically accessible. Finally, the application of compounds with antioxidant capacities to supplement endogenous antioxidant systems has long been thought to inhibit the propagation phase and promote the termination phase of radical reactions. 1.7.1 Ischemic preconditioning The roots of preconditioning stems from a study by Murry et al, who coined the term and were first to report that infarct size subsequent to a 40-minute circumflex occlusion was significantly smaller in canine hearts that had been preconditioned with four transient five-minute circumflex occlusions separated by five minutes of reperfusion, than in canine hearts in which the 40-minute occlusion was not preceded by a preconditioning stimulus332. In a related paper, the same group demonstrated that four successive tenminute coronary occlusions did not deplete myocardial adenine nucleotide pools beyond the first occlusion, and that the initial depletion was less severe than during a single, continuous 40-minute occlusion333. Seven years earlier, Verdouw et al, investigating if pigs could serve as their own controls to study myocardial ischemia, noticed that the first ischemic episode induced a quantitatively different metabolic profile than the second ischemic episode334. These authors speculated that the metabolomic difference resulted from myocardial glycogen and high-energy phosphate depletion, and/or necrosis, but failed to recognize that the first ischemic episode may have been inducing a cardioprotective state in anticipation of the second. In 1993, it was discovered that 45  ischemic preconditioning also induced longer-term changes that were protective between 12-72 h after the initial ischemic episode335, 336. These changes were found to be mediated, at least in part, by increasing the translation of stress proteins including HSP72 and HSP60335. In the clinical setting, Kloner et al observed that patients presenting with acute myocardial infarctions who had any previous history of angina were less likely to suffer from severe congestive heart failure, in hospital death, shock, or a combination thereof relative to those patients with no previous history of angina337. Ischemic preconditioning is mechanistically complex and not completely understood. The end effectors of the early preconditioning involve post-translational differences to the myocardial proteome, while late preconditioning recruits transcriptional changes. Preconditioning in general is a complex mix of cell signaling and channel activation: the phenomenon appears to be predicated on the indirect activation of several key prosurvival pathways including the phosphatidylinositol-3-kinase-Akt and extracellular signal regulated kinase pathways, which in turn converge on key end-effectors338. Chief among these are the ATP-sensitive potassium (KATP) channel338, 339. KATP channels are found on the membranes of mitochondria and sarcolemma, and become activated by low ATP concentrations. Mitochondrial KATP channels play an important physiological role in the maintenance of mitochondrial matrix volume during oxidative phosphorylation by regulating osmotic gradients. Preconditioning activates sarcolemmal KATP channels, allowing a net inward potassium current to the cytoplasm. This inward potassium current stabilizes the cell membrane potential, thereby decreasing Ca2+ influx340. In turn, decreased Ca2+ loading is thought to reduce both contracture and the severity of the ROS burst. The activation of mitochondrial KATP channels also increases O2•- generation at  46  complex I of the electron transport chain, which presumably initiates protective signaling cascades prior to ischemia and reperfusion341. Paradoxically, both O2•- and NO• signaling play critical roles in ischemic preconditioning92, 342-344 but neither ONOO- nor •OH appear to be appreciably formed345, 346  . Regardless of the role that ROS play in preconditioning signaling, ischemic  preconditioning ultimately reduces the magnitude injurious ROS activity at reperfusion347. Several possibilities could explain this apparent paradox. First, the ischemic episodes that induce a preconditioning effect are relatively short, so the magnitude with which NO• is generated during ischemic preconditioning may be insufficient to effectively compete with superoxide dismutase-mediated O2•- dismutation. Thus ONOO- generation would be averted. Second, O2•- and NO• generation may be temporally and/or spatially separated during ischemic preconditioning. Either of these possibilities would ultimately reduce the magnitude of ONOO- generation during the signaling phase of this phenomenon. In support of this, NO• generation during ischemia reduces the magnitude of ROS generation during both ischemia and reperfusion348, and ischemic preconditioning in turn relies on moderate O2•- increases in the absence of large scale NO• generation, which does not effectively lend itself to ONOO- formation and thus reduces the likelihood of intracellular MMP activation349. Ultimately, preconditioning reduces intracellular 3-nitrotyrosine staining and maintains electron transport chain coupling as evidenced by conserved NADH dehydrogenase and cytochrome oxidase activity subsequent to ischemia and reperfusion346. From a clinical perspective, ischemic preconditioning is restricted to those contexts in which prolonged ischemia can be anticipated. To this end, a host of studies have  47  investigated various clinical outcomes subsequent to ischemic preconditioning protocols within the context of CABG with CPB (reviewed in350, 351). Notably, analysis of excised biopsies revealed that preconditioning can preserve myocardial ATP stores during CABG352, 353. A systematic review and meta-analysis of clinical ischemic preconditioning studies in human subjects identified clear reductions in postoperative ventricular arrhythmias, inotropic requirements, and intensive care unit stay among patients receiving ischemic preconditioning protocols351. Ischemic preconditioning within the context of cardiac surgery necessitates the application of an invasive treatment to the heart in the form of repetitive aortic clamping and declamping, which has the potential to destabilize plaques and may increase the likelihood of thromboembolism, among other risks. Some of these risks may be reduced with other conditioning strategies such as ischemic postconditioning and remote ischemic conditioning. The impact of postconditioning and remote preconditioning on ROS formation in first few minutes of reperfusion are not as clear as that of ischemic preconditioning, and these conditioning strategies are extensively reviewed elsewhere354, 355  . In the absence of clear evidence from a uniform large clinical trial speaking to its  benefits with respect to overall post-operative mortality, clinicians will continue to be reluctant in adopting these strategies in the operative setting. 1.7.2 Volatile anesthetic preconditioning In 1983, Davis et al reported that halothane anesthesia was capable of reducing infarct size in dogs undergoing coronary artery occlusion356. Six years later, Davis and Sidi reported that isoflurane-anesthetized dogs had a decreased myocardial infarct size and reduced necrosis and O2 consumption after left anterior descending coronary artery 48  occlusion, compared to control dogs357. Still, it was not until 1997 that the capacity of anesthetics to induce a preconditioning-like effect was realized358, 359. A variety of anesthetics appear to induce a preconditioning-like effect in a variety of animal and experimental models of ischemia-reperfusion injury (reviewed in360-362). Like ischemic preconditioning, volatile anesthetics indirectly increase the open probability of KATP channels359, 363, 364. By contrast, volatile anesthetics appear to induce different gene and protein expression patterns than ischemic preconditioning365-368. Whether these latter changes can be sufficiently induced and functional at reperfusion within the operative timeline is questionable. Volatile anesthetic preconditioning is easily translated to the operative setting. To this end, several studies have been conducted in which volatile anesthetics were compared either with one another and/or with intravenous anesthetic, showing improvements in biochemical markers of myocardial damage, hemodynamic function, and hospital stay369377  . Unfortunately, two notable multi-center, randomized trials failed to replicate  decreases in troponin I with a sevoflurane preconditioning protocol378, 379, while a third study found a similar lack of troponin I reduction with an isoflurane protocol380. To this end, the preconditioning protocol itself appears to influence its successful activation374, and it has been argued that patient and operative factors may play an important role in whether or not volatile anesthetics confer additional benefits during cardiac surgery381. Of equal interest, a recent meta-analysis comparing sevoflurane to propofol anesthesia found that patients receiving sevoflurane generally had higher post-bypass cardiac indices, decreased serum troponin I, shorter intensive care unit and hospital stays, and lower incidence of myocardial ischemia382. Notably, this meta analysis failed to  49  detect any significant differences in postoperative ventilation time, inotropic support, mortality, myocardial infarction, and atrial fibrillation382. These latter results are in agreement with a previous retrospective review comparing sevoflurane to propofol anesthesia in 10,535 patients undergoing cardiac surgery. This study failed to detect any significant differences in 30 day mortality, post-operative myocardial infarction and arrhythmia383. In a similar vein, a recent study comparing sevoflurane, isoflurane and propofol during CABG found that patients receiving volatile anesthesia had improved troponin I profiles while those receiving propofol had less lipid peroxidation, improved glutathione peroxidase activity, and decreased NO• production384. A more comprehensive study comparing isoflurane and propofol anesthesia failed to find any significant differences in postoperative troponin-I, hospital or intensive care unit stays, or in hospital-, 30-day- and 1-year cardiac morbidity and mortality385. 1.7.3 Antioxidants Chemical antioxidants can be characterized as compounds that inhibit the propagation step of free radical reactions. The antioxidant capacity of a compound depends on two critical factors: its solubility in the medium in which radical reactions are occurring and its ability to stabilize an unpaired electron. Antioxidant inhibition of the radical propagation phase necessarily involves a chemical reaction, which necessitates its interaction with a given reactive radical. Thus, if the radical reaction to be inhibited involves lipid peroxidation, it would be desirable if the antioxidant in question were lipophillic. The propagation phase is stalled when a relatively stable radical with lower reactivity is produced. The stability of a radical depends in part on the area across which the lone electron can be resonance-stabilized. Accordingly, large conjugated networks 50  can resonance-stabilize lone electrons throughout their conjugated polyene or aromatic Pi orbitals, and tertiary radicals are inherently more stable than secondary-, and in turn primary radicals386. β-Carotene, for example, is an organic compound containing a network of 22 conjugated carbon atoms387. Its antioxidant capacity is derived from its ability to stabilize an unpaired electron across a large conjugation network of 11 unsaturated bonds containing eight tertiary carbons subsequent to hydrogen abstraction388. Although the resonance stabilization of an unpaired electron is not a termination reaction, more stable radicals are more likely to react with more reactive radicals than with less reactive non-radicals. In the case of the β-carotene radical, reaction with a second peroxyl radical can yield a host of non-radical products, each corresponding to a possible resonance structure389. Antioxidant supplementation is a logical adjuvant therapy during CABG, because the operative procedure itself is associated with a depletion of intrinsic antioxidant capacity390. Accordingly, several clinical studies have measured surrogate markers of oxidative stress and preliminary outcomes when various antioxidants have been supplied during CABG procedures. The first of these antioxidants is N-acetylcysteine, which reacts with •OH and to a lesser extent H2O2, and is a physiological precursor to glutathione391. The administration of N-acetylcysteine appears to effectively decrease MDA levels, but this decrease is not associated with improved troponin-I or myocardial creatine kinase (CK-MB) profiles392-395, with few exceptions396. Furthermore, while one comprehensive randomized, double-blind, placebo-controlled clinical trial found that Nacetylcysteine administered during CPB decreased 3-nitrotyrosine and 15-F2t-isoprostane  51  staining –albeit in the absence of hemodynamic and clinical outcome differences397, a second similarly designed trial failed to detect any differences in a host of clinical and biochemical outcomes, notably including death, myocardial infarction, length of hospitalization, troponin-T, and CK-MB among patients that were prophylactically treated with N-acetylcysteine prior to CABG398. The second compound supplemented as an antioxidant during CABG is α-tocopherol, or vitamin E, which scavenges peroxyl radicals in biological lipid membranes399, 400. Similar to N-acetylcysteine, α-tocopherol appears capable of reducing markers of oxidative stress, but the overall clinical benefit appears to be limited401-403. Interestingly, the intracoronary administration of α-tocopherol was found to improve post-reperfusion troponin-I and CK-MB profiles relative to control404. These results suggest that both Nacetylcysteine and α-tocopherol must be applied intraoperatively to reduce ROS damage during ischemia-reperfusion injury, but the overall benefit in terms of clinical outcomes may be limited. Finally, the intravenous anesthetic propofol (2,6-diisopropylphenol) has an intrinsic antioxidant capacity owing to its di-substituted phenolic structure. Like N-acetylcysteine, propofol is capable of scavenging •OH405, 406, but not necessarily O2•- 407. Propofol completely inhibits in vitro lipid peroxidation of a linoleic acid emulsion at a concentration beyond 140 µM, which, although exceeding clinically relevant concentrations by an order of magnitude, is more potent than α-tocopherol405. Importantly, phenolic antioxidants can structurally incorporate a nitroso- group in the presence of NO•, which decreases their antioxidant capacities408. Nevertheless, the concentration range of 10 µM and below of propofol can inhibit in vitro lipid  52  peroxidation of artificial membrane and whole blood by 50%406, 409. Clinically relevant infusion rates can achieve propofol concentrations that enhance erythrocyte antioxidant capacity of during CABG with CPB410, 411. Propofol has also been shown to scavenge ONOO- in cultured endothelial cells and astrocytes, in a reaction that appears to occur to a greater degree than to tyrosine nitration412, 413. Several studies have demonstrated that propofol anesthesia during CABG is associated with a decrease in lipid peroxidation414-417. Despite these results, it remains unclear whether propofol or volatile anesthetics produce significantly different outcomes subsequent to cardiac surgery. On one hand, several aforementioned studies indicate that volatile anesthetic preconditioning is associated with improved functional and troponin I profiles compared to propofol373, 375-377. On the other hand, other aforementioned studies fail to detect significant differences in several patient outcomes including mortality and morbidity382, 383, 385. It may ultimately remain difficult to assess clinical differences in the absence of standard volatile anesthetic preconditioning protocols and measures of achieved propofol concentrations in whole blood. 1.8. Conclusions and perspectives Myocardial ischemia and reperfusion is a major source of cardiomyocyte injury within the context of a host of clinical pathologies. These pathologies include cardiac arrest, acute myocardial infarction, and low cardiac output syndrome after cardiac surgery. The financial and societal relevance of this type of injury is underscored by the increasing prevalence of heart disease. Any improvement in the clinical outcomes associated with these pathologies must be predicated on reducing the magnitude of  53  ischemia-reperfusion injury, which by extension requires an understanding of the central role that oxygen and nitrogen radicals play in its pathogenesis. There are several potential sources of free radicals during tissue ischemia and reperfusion, but mitochondria appear to be especially important in the context of the myocardium. Several factors underscore this reasoning: 1. The constant energy demand of the myocardium dictates that cardiac myocytes contain large amounts of mitochondria96. 2. Mitochondria are an important source of O2•-, •OH, and NO• generation during ischemia and reperfusion418-421. 3. Mitochondria contain all basic types of biomolecules that would be susceptible to free radical reactions in the cell as a whole. Furthermore, the mitochondrial membranes contain high levels of unsaturated fatty acids that are susceptible to peroxidation reactions422. 4. Mitochondria are key regulators of ROS mediated cell death423. Although xanthine oxidase and inflammatory mediators likely contribute to the myocardial pool of O2•-, especially in the diseased heart, and •OH can mediate its toxicity throughout the cell, cardiac mitochondria likely represent the most important source and site of myocardial ROS generation during myocardial ischemia and reperfusion. As such, mitochondria serve as a useful microcosm of the cardiomyocytes in which they reside; their protection from ROS-mediated damage during ischemia and reperfusion may pay dividends beyond even their myocyte hosts. 1.9. Thesis objectives and outline The unsaturated fatty acids that comprise cellular and subcellular lipid membranes are susceptible to ROS-mediated peroxidation reactions. These self-propagating reactions are initiated by •OH, and yield a heterogeneous array of products that encompass acidcatalyzed fragmentation products including MDA, as well as a host of isoprostane 54  isomers. Lipid peroxidation products can adversely affect the functional integrity of lipid membranes, and are therefore quickly cleaved and released by membrane phospholipases232. One isoprostane isomer, 15-F2t-isoprostane, induces dose-dependent vasoconstriction in the coronary vasculature with sub-µM potency241-243. Levels of 15-F2t-isoprostane increase during coronary angioplasty and CABG procedures246-248, it has been associated with a decline in postoperative cardiac function244, 249, and its formation may exacerbate or prolong cardiac dysfunction after ischemia and reperfusion243-245. Intraoperative ischemia-reperfusion injury does not universally translate to persistent or significant decreases in postoperative clinical outcomes, so the ability to detect additional clinical outcome advantages is often limited to large, multi-centered, and adequately powered trials. By contrast, effective lipid soluble antioxidant therapies should reduce 15-F2tisoprostane generation in a patient cohort at large. In light of previous results that point toward a propofol-mediated cardioprotective effect, and given the duality of 15-F2t-isoprostane as an indicator of ROS-mediated lipid peroxidation and a mediator of myocardial ischemia-reperfusion injury, this thesis is set around the following central hypothesis: Propofol reduces the incidence of low cardiac output syndrome subsequent to CABG with CPB by decreasing the magnitude of 15-F2t-isoprostane generation during myocardial ischemia and reperfusion. Implicit in this hypothesis is that propofol’s antioxidant capacity can decrease •OH mediated, self-propagated lipid peroxidation. Of equal importance, •OH generation can be decreased by a reduction in ONOO- formation. This reduction in ONOO- formation 55  can in turn result from a decrease in the generation of its reactants, namely NO• and O2•-. Accordingly, reducing NO• generation or temporally separating NO• and O2•- generation could influence the magnitude of •OH, and subsequently 15-F2t-isoprostane generation during ischemia and reperfusion. Accordingly, this thesis aims to address the following research questions: 1.  Does propofol reduce the magnitude of •OH-initiated, self-propagating lipid peroxidation reactions over isoflurane preconditioning as evidenced by 15-F2tisoprostane generation during ischemia and reperfusion within the context of CABG with CPB?  2.  Is NO• generation during ischemia and reperfusion differentially affected by propofol or isoflurane preconditioning during CABG with CPB?  3.  Does propofol differ from isoflurane preconditioning in terms of ONOOgeneration, during myocardial ischemia and reperfusion injury as evidenced by plasma 3-nitrotyrosine levels within the context of CABG with CPB?  The central hypothesis also postulates that the conditions that yield propofol-based cardioprotection in laboratory models of ischemia-reperfusion injury translate to the clinical setting, and that previous results indicative of the inability of isofluranepreconditioning to decrease •OH generation are robust. Consequently, several clinicalbased cardioprotective strategies will briefly be reviewed in Chapter 2, followed by a discussion of the pharmacology of propofol, its antioxidant capacity from the perspective of relevant laboratory-based studies, and the precedents that exist in the literature in support of its use as a potential cardioprotectant.  56  The flow chart in Figure 7 is a general outline of this thesis. The development of a clinical study capable of addressing the central hypothesis underscores the objectives of this thesis. This clinical study required a maneuver that could reliably and predictably deliver a concentration of propofol to the heart at reperfusion that corresponds to concentrations that tend to be associated with laboratory-based cardioprotection. Accordingly, Chapter 3 describes the development of a capillary electrophoresis-based technique that enables rapid and quantitative analysis of propofol in whole blood from patients undergoing CABG with CPB. The analytical technique described in Chapter 3 was used within the context of a dose finding study. The results from this dose-finding study, which is described in Chapter 4, helped inform the clinical parameters that translate laboratory-based conditions for propofol-based cardioprotection to a clinical study. Chapter 5 describes the rationale and design of this larger clinical study, within which the research questions and central hypothesis can be addressed. Finally, preliminary results that address the research questions are presented in Chapter 6. This thesis concludes with a summary of findings, with future directions for exploration, and with a prospective outlook of the research.  57  Figure 7: Flow chart describing the conceptual link between the individual chapters of this thesis.  58  2.  Clinical-based cardioprotection beyond volatile anesthetic preconditioning Patients presenting for cardiac surgery are a heterogeneous clinical population. This  heterogeneity may be reflected by one patient receiving a slight but significant outcome benefit from a given treatment or procedure, to the detriment or indifference of another. For example, less severe coronary artery disease can be treated pharmacologically or surgically without significant differences in outcomes4, 5, but medium- and higher-risk patients with more severe disease, and those with co-morbidities clearly benefit from reduced mortality when treated with CABG compared to non-surgical management8 and percutaneous interventions6, 9, 10. A particularly significant and prevalent co-morbidity is diabetes mellitus. Mortality from cardiovascular pathologies is doubled among patients with diabetes, and these patents, who are two to five times more likely to develop cardiovascular disease and have smaller vessel diameter, often have multi-vessel disease and lower left ventricular ejection fraction at a significantly greater frequency than their non-diabetic counterparts424. Unsurprisingly, diabetics represent 30% of patients presenting for cardiac surgery21. Although patients with diabetes mellitus may benefit from CABG over percutaneous intervention6, 7, their condition is associated with an increased rate of early and late adverse postoperative events following cardiac surgery, including increased perioperative morbidity and mortality, reduced long term survival, and recurrence of angina12, 13, 16, 425-429. In the early postoperative period, low cardiac output syndrome affects up to 26% of diabetic patients, compared to 8% to 15% of non-diabetic patients recovering from cardiac surgery14-16. This syndrome is defined by persistent hypotension (a systolic blood  59  pressure less than 90 mmHg) and/or low cardiac index (less than 2.2 L⋅min⋅m2) despite hemodynamic optimization, and can quadruple the overall mortality rate for CABG surgery from 2% to 8%12, 13. Intraoperative ischemia-reperfusion injury is a major factor in the development of low cardiac output syndrome, so its disproportionate frequency among patients with diabetes reflects either their increased susceptibility to ischemiareperfusion injury and/or their decreased susceptibility to intraoperative cardioprotection. Isoflurane preconditioning mimics ischemic preconditioning with respect to KATP channel activation339, 359, 363, 379, 430. Both isoflurane and ischemic preconditioning recruit Akt signaling and Bcl-2431-433, but they appear to diverge in their ability to decrease •OH generation at reperfusion –ischemic preconditioning being capable of doing so, but not isoflurane345, 434, 435. This divergence may reflect the finding that isoflurane only marginally improves outcomes subsequent to CABG380. Alternatively, Akt signaling and Bcl-2 activation may sufficiently induce a protective phenotype in a subset of patients so that the additional benefit of decreased •OH generation is not necessary for cardioprotection. Experimental models of diabetes suggest that signal transduction pathways required for ischemic or anesthetic preconditioning are corrupted19, 436. Hyperglycemia-induced oxidative stress can suppress myocardial KATP channel activity437 and inhibit Akt signaling20, 438-441. These differences are illustrated clinically by different gene expression profiles between the diabetic and non-diabetic myocardium subsequent to CPB442. Concerning the ROS-induced end-effectors of ischemia-reperfusion injury, experimental studies indicate that mitochondrial permeability transition may be enhanced by diabetes438, 443-445, while MMP expression and activation is increased by  60  hyperlipidemia446 and diabetes447, 448. These results underscore and reflect in the notion that volatile anesthetic preconditioning is compromised in the diabetic myocardium436, 449, and that therapeutic approaches that demonstrated clinical effective cardioprotection in these patients remain largely elusive449. 2.1. Propofol-mediated, antioxidant-based cardioprotection A recent meta analysis of observational studies found that the integrity of endogenous antioxidant defenses inversely correlated with clinical outcomes in patients with coronary artery disease450. These data suggest that patients with coronary artery disease have insufficient or depleted endogenous defenses to ROS, which would make them vulnerable to the ROS that are generated during myocardial ischemia-reperfusion injury. Antioxidants have the potential to benefit patients during cardiac surgery by intercepting and neutralizing ROS generated during ischemia and reperfusion. In doing so, antioxidants could inhibit ROS-activated apoptotic processes such as mitochondrial permeability transition, and decrease the activation of MMPs, while the integrity of cellular and sub-cellular membranes and membrane-protein complexes. Although this concept tends to bear fruit in experimental models of ischemia and reperfusion, clinical evidence is more ambiguous (reviewed in451). Effective antioxidant therapy represents an attractive opportunity because it is mechanistically orthogonal to isoflurane-based preconditioning and accordingly circumvents signaling pathways that may be compromised in the diabetic heart. Clinical studies of adjuvant antioxidant therapy indeed tend to show decreases in surrogate markers of oxidative stress, but benefits in terms of patient outcomes or markers of myocardial damage are less evident. One likely reason behind the marginal clinical 61  benefits is that the majority of patients who undergo cardiac surgery emerge without major intraoperative myocardial injury that translates into prolonged or significantly depressed clinical outcomes, so the detection of any additional clinical outcome advantages necessitates much larger studies of adequate power. The contrasting results between laboratory models and clinical studies may also indicate that antioxidant therapies have not been effectively translated. Specifically, antioxidants are likely compartmentalized, eliminated, or otherwise diluted in vivo, so that their myocardial concentrations no longer reflect experimental ones. To this end, intracoronary administration of α-tocopherol was found to improve post-reperfusion troponin-I and CK-MB profiles relative to control404 and its intraoperative administration marginally improved myocardial function402. Conversely, its prophylactic application has no bearing on markers of myocardial injury in the early postoperative period403. Similarly, N-acetylcysteine administration during CPB or as a cardioplegia solution supplement is capable of reducing myocardial oxidative stress392, 394, 395, 397, at times with decreased markers of myocardial injury396, while prophylactic N-acetylcysteine administration again has no bearing on any marker of postoperative myocardial damage or functional outcome393, 398. 2.2. Propofol modulation of ion fluxes Propofol is an intravenous anesthetic whose lipophilicity accounts for its rapid distribution half-life (2-8 minutes), and whose high rate of clearance, which exceeds hepatic blood flow, accounts for a relatively quick post-operative arousal time452-456. The anesthetic activity of propofol ultimately stems from its ability induce membrane hyperpolarization. The mechanism by which this is accomplished is derived from 62  propofol’s ability to bind and modulate a host of receptors across a small concentration range, these include glycine receptors457, voltage dependent Na+ channels458, and chiefly GABAA receptors (reviewed in459). Interestingly, propofol positively modulates GABAA activity in two separate and independent ways: first, propofol increases the open probability of the channel in the presence of GABA by binding the GABAA receptor independently of its subunit composition457, 460; second, propofol directly activates GABAA by binding near the extracellular site of a transmembrane motif on the β-subunit of this receptor460-465. The whole blood concentration of propofol at which half of patients are awake is 1.07 µg/mL, while the average surgical concentration varies from 2.97 to 4.05 µg/mL, depending on the procedure466. Propofol induces significant effects on ionic gradients in the myocardium within this concentration range. Specifically, propofol directly reduces the open probability of L-type Ca2+ channels467, 468 and decreases inward Ca2+ current starting at concentrations of 4.5 µg/mL469, 470. Interestingly, propofol appears to offset these actions by increasing myofilament sensitivity to calcium471, 472, thus minimizing any effect on cardiac contractility. Furthermore, its negative inotropic action is not apparent at clinically relevant concentrations472. Propofol causes a hyperpolarizing shift with decreased peak sodium current amplitudes, slowed rates of recovery from inactivation through reduced single Na+ channel open duration, and dose-dependently blocked whole cell Na+ currents in rat ventricular myocytes458. Although this inward Na+ current-reducing effect has not directly been implicated in cardioprotection, it likely contributes to propofol’s overall ability to reduce intracellular Ca2+ concentrations, as would its ability to induce PKC-  63  dependent Na+/Ca2+ exchanger reversal and its preservation of Na+/H+ exchanger activity473-476. 2.3. Propofol mediated reactive oxygen species scavenging Propofol is a phenol that is substituted at the 2- and 6- position with isopropyl groups. This structure resembles that of tocopherols (Figure 8), and is thought to contribute to propofol’s antioxidant activity, which mirrors that of α-tocopherol405, 477. Propofol’s capacity to scavenge •OH radicals in vitro is generally acknowledged405, 406, and although there is some question whether this capacity extends to O2•- 407, it clearly decreases MDA, a marker of lipid peroxidation405-408. This capacity is illustrated by the finding that 140 µM of propofol completely inhibits in vitro lipid peroxidation of a linoleic acid emulsion. Although this concentration exceeds clinical relevance by more than an order of magnitude, the effect was more potent than that of α-tocopherol405. At the more relevant concentration range of 10 µM, propofol can inhibit in vitro lipid peroxidation of artificial membrane and in whole blood by 50%406, 409. Experimentally, propofol dose-dependently reduces MDA production in red blood cells409, 410, 478 cultured cardiomyocytes479, and isolated heart models of ischemiareperfusion injury480-483. These isolated heart studies, and similar ones that did not measure MDA levels484-486, report reductions in measures of ischemia-reperfusion injury with improved functional endpoints. One such study reported that propofol during ischemia and reperfusion can reduce mitochondrial lipid peroxidation and H2O2 production in association with preserved mitochondrial respiration and cardiolipin content487. This study extrapolated that 5 µg/mL of propofol in patient blood would be expected to reduce the severity of myocardial ischemia-reperfusion injury. 64  Figure 8: Structures of propofol (left) and alpha-tocopherol (left) with their similarities highlighted in bold.  2.4. Precedents for propofol-mediated cardioprotection In 2005, Lim et al reported a swine model of cardioplegic arrest with CPB, which serves as a clinically relevant model of CABG with CPB. This study compared pigs receiving propofol during CPB with those receiving sodium thiopentone-diazepamfentanyl intravenous anesthesia throughout. Pigs receiving propofol had reduced troponin I levels at reperfusion, reduced hemodynamic dysfunction after CPB, and reduced ischemia-reperfusion injury as evidenced by preserved myocardial tissue levels of adenine nucleotides, lactate, and amino acids. The estimated a propofol concentration in blood at 3.7 µg/mL488. Although Lim et al did not measure markers of lipid peroxidation, several other clinical studies do. Specifically, propofol’s ability to enhance erythrocyte antioxidant capacity appears to extend to CABG with CPB, provided sufficient drug concentrations 65  are achieved in blood410, 489. Similarly, several studies have demonstrated that propofol anesthesia during CABG is associated with decreased lipid peroxidation414-417, 490. A critical difference between experimental models of myocardial ischemia-reperfusion injury and the clinical setting is that increased lipid peroxidation may in part reflect an inflammatory response to intraoperative tissue damage. To this end, propofol has been shown to decrease systemic inflammation and lipid peroxidation during and after aortic crossclamp application in pigs subject to myocardial ischemia-reperfusion injury491, 492. Likewise, propofol reduces iNOS activity and inflammation during general surgery493 and improves pulmonary inflammation subsequent to CPB in patients494. Unfortunately these results can neither attribute the decreased lipid peroxidation to reduced ROS generation in the myocardium nor to a reduction in the inflammatory response. In a similar vein, one clinical study found that patients receiving an average of 7 µg/mL of propofol had decreased indices of post-ischemic inflammation and significantly lower levels of MDA in coronary sinus blood immediately after crossclamp removal, but no significant differences in post-operative MDA generation in systemic blood416. A subsequent study from the same group reported similar findings with respect to MDA417. Another critical difference between experimental models of myocardial ischemiareperfusion injury and its clinical manifestation is that volatile anesthetics likely influence myocardial tolerance to ischemia. As such, clinical studies, in which several choices for the primary anesthetic have demonstrated some laboratory based benefit, almost certainly lack a clear control group. In this light, it remains unsurprisingly unclear whether propofol can significantly improve outcomes over volatile anesthetics during cardiac surgery. On one hand, several studies indicate that volatile anesthetic  66  preconditioning is associated with improved functional and troponin I profiles compared to propofol373, 375-377. On the other hand, other studies fail to detect significant differences in several patient outcomes including mortality and morbidity382, 383, 385. A meta-analysis comparing sevoflurane to propofol anesthesia found that patients receiving sevoflurane generally had higher post-bypass cardiac indices, decreased serum troponin I, shorter intensive care unit and hospital stays, and lower incidence of myocardial ischemia, but failed to detect any significant differences in postoperative ventilation time, inotropic support, mortality, myocardial infarction, and atrial fibrillation382. These latter results are in agreement with another retrospective review comparing sevoflurane to propofol anesthesia in 10,535 patients undergoing cardiac surgery, which failed to detect significant differences in 30-day mortality, post-operative myocardial infarction and arrhythmia. This second study suggested that propofol and sevoflurane likely conferred mechanistically distinct cardioprotective effects383. In a similar vein, a recent study comparing sevoflurane, isoflurane and propofol during CABG found that patients receiving volatile anesthesia had improved troponin I profiles while those receiving propofol had less lipid peroxidation, improved glutathione peroxidase activity, and decreased NO• production384. Finally, a more comprehensive study comparing isoflurane and propofol anesthesia failed to find any significant differences in postoperative troponin-I, hospital or intensive care unit stays, or in hospital-, 30-day- and 1-year cardiac morbidity and mortality385.  67  3.  Quantitative analysis of propofol in whole blood using capillary electrophoresis  3.1. Introduction Propofol is a frequently used intravenous anesthetic for the introduction and maintenance of anesthesia. Our research group is interested in the potential cardioprotective effects of propofol during CPB. Target controlled infusion devices, which predict whole blood propofol concentrations based on mathematical algorithms that link patient characteristics and pharmacokinetics with infusion rates, are currently in use. Unfortunately, there are often discrepancies between predicted and achieved concentrations. Furthermore, target controlled infusion devices are not universally approved for clinical use. In order to overcome these limitations, techniques and devices capable of determining actual drug concentrations in whole blood are required. In order to maximize their clinical usefulness, analytical devices should be simple, highly automated, fast, accurate, and precise. Several methods for the quantitative determination of propofol in biological samples have been reported, examples include high performance liquid chromatography-UV spectrophotometry495-498, gas chromatography with flame ionization detection498, 499, and chromatography techniques coupled with mass spectrometry detectors 497-502. While these techniques offer accurate and precise quantitative results, they have qualities that do not necessarily translate well to the clinical setting. Specifically, mass spectrometry techniques that measure propofol concentrations in expired breath are rendered void by the use of CPB. Mass spectrometry detectors usually require front-end separation techniques when analyzing biological samples that originate from complex matrices such  68  as blood. Liquid chromatography-based techniques can produce efficient and fast separations, but they often require longer column regeneration steps between runs that involve larger organic solvent volumes. A final impediment to the routine use of many modern separation techniques for target-achieved type analysis in the clinical context is the physical size of commercially available mass spectrometry, liquid chromatography, and capillary electrophoresis (CE) instruments. CE is one of the most powerful tools for chemical separation. The advantages of high resolution, short analysis time, low cost, and small buffer and solvent volume requirements make CE an attractive technique to analyze complex matrices503, 504. Although the size of commercially available CE instruments precludes their routine presence in a clinical setting, the much smaller microfluidic devices have the potential to meet this need. The conceptual similarities between CE and electrophoretic-based microfluidic separations represents a strong opportunity to foster the development of target-achieved drug delivery strategies and devices, in which drug dosing is dictated by actual concentrations achieved in whole blood, that are better suited to routine use in clinical settings. The aim of the present study was to develop and validate a CE-based method capable of quantitative propofol analysis in whole blood. Propofol has an estimated theoretical triglyceride-water partition coefficient of λtr/w = 4715505, it strongly binds to plasma proteins and cellular blood constituents, and it has a mean free fraction in plasma of 13%506. Capillary electrophoresis with micellar additives, also called micellar electrokinetic chromatography (MEKC), first introduced by Terabe et al. 507, has been widely used for the analysis of biological samples508, particularly where neutral and  69  hydrophobic compounds are concerned. We have developed and validated a fast and highly selective MEKC-based method for the quantitative analysis of propofol in whole blood using a commercially available CE system. This method was used to determine the concentration of propofol in whole blood samples obtained from patients undergoing CABG with CPB. 3.2. Experimental 3.2.1 Apparatus All experiments were carried out on a Beckman Coulter P/ACE MDQ System (Beckman Coulter Inc., Fullerton, CA, USA) with a UV absorption detector. The detection wavelength that was used for the analysis of propofol was 200 nm. An uncoated fused-silica capillary (50 cm total length, 40 cm length to detector, 50 µm inner diameter, 360 µm outer diameter) (Polymicro Technologies, Phoenix, AZ, USA) was used throughout. A separation temperature of 25°C was maintained for all CE experiments. 3.2.2 Chemicals and reagents Borax and sodium dodecyl sulfate were purchased from Sigma-Aldrich (Oakville, ON, Canada). Separation buffer consisted of an aqueous solution containing 50 mM sodium dodecyl sulfate and 15 mM borax. This separation buffer was sonicated and filtered through a 0.45 µm membrane filter. Tetramethylammonium hydroxide (25% in methanol) was purchased from Alfa Aesar (Ward Hill, MA, USA) and diluted before use with HPLC grade 2-propanol (Fisher Scientific, Ottawa, ON, Canada) (3:37 v/v). Cyclohexane, acetonitrile, and methanol were purchased from Fisher Chemicals (Fisher Scientific, Ottawa, ON, Canada) Deionized water was obtained using a NANOpure 70  Infinity Reagent Grade Water System (Apple Scientific Inc., Chesterland, OH, USA). All solutions were filtered through 0.45 µm membrane filters (Toyo Roshi Kaisha, Ltd., Tokyo, Japan) prior to use. Propofol (2,6-diisopropylphenol), 97% purity, was purchased from Sigma-Aldrich. Thymol, 99% purity, was purchased from Acros Organics (Morris Plains, NJ, USA) and used as internal standard. Durapore centrifugal filters with 0.1 µm pores for the liquid-liquid extracted solutions, were purchased from Millipore, Inc. (Bedford, MA, USA). 3.2.3 Preparation of standard solutions Initial stock solutions of propofol and thymol were prepared separately in 50% acetonitrile-50% deionized water. These solutions consisted of 800 µg/mL propofol and 100 µg/mL of thymol. Propofol was subsequently serially diluted with 6% acetonitrile to the following desired concentrations: 16, 8, 4, 2, 1, 0.5 µg/mL. Two additional concentrations (0.3, 0.1 µg/mL) were prepared from 1 µg/mL. These solutions were supplemented with thymol stock solution for a final internal standard concentration of 7 µg/mL. 3.2.4 Patients and sampling This investigation conforms to the principles outlined in the Declaration of Helsinki. Following institutional approval and written informed patient consent, 30 patients scheduled for primary CABG surgery were enrolled in an ongoing parallel study investigating the short-term application of propofol during CPB. All patients received a 1.0 mg/kg bolus dose of propofol at heparinization (approximately 10 minutes prior to aortic crossclamp placement) followed by an infusion of 120 µg•kg-1•min-1 for the  71  duration of CPB. A whole blood sample of 5 mL was withdrawn from the central venous line 15 minutes after aortic declamping using a vacutainer containing heparin (Becton Dickinson, NJ), and stored in 1.25 mL aliquots at -80oC for subsequent CE analysis. Patient and operative characteristics are listed in Table 1. Blank venous blood, which did not contain prpofol, was sampled from the central venous line intraoperatively in the interim time between induction of anesthesia and the administration of propofol. This blood was sampled and stored as described for central venous samples. Perioperative patient care was administered according to the routine clinical practice at Vancouver General Hospital.  Table 1: General patient and operative characteristics. Mean  Standard Deviation  Age (years)  63  9  Weight (kg)  90  14  Height (cm)  174  7  Body Surface Area (m2)  2.05  0.17  Aortic Crossclamp Time (min)  96.6  23.0  Cardiopulmonary bypass time (min)  125.4  35.8  Gender Distribution (f/m)  1/29  72  3.2.5 Sample extraction A liquid-liquid extraction procedure described by Plummer509 is frequently used for the analysis of propofol in blood samples. We have modified this procedure to suit the CE analysis. In brief, 30 µL of thymol internal standard stock solution (100 µg/mL), 200 µL of deionized water, and 25 µL of 1 M NaH2PO4 (pH 4.2) were added to a 400 µL aliquot of thawed whole blood. The sample mixture was then vortexed for 1 min, after which 650 µL of cyclohexane was added, followed by at least 2 minutes of inverting and vortexing. Organic and aqueous layers were separated by centrifugation (1200 rcf for 1 minute at 4oC). A 500 µL aliquot of the cyclohexane layer was transferred through a 0.1 µm centrifugal filter into a clean tube containing 10 µL of diluted Tetramethylammonium hydroxide solution (2% v/v in propanol). The solvent was evaporated to dryness under vacuum centrifuge (down to 0.6 torr) and the residue was re-dissolved in 80 µL of 6% acetonitrile in water. 3.2.6 Capillary electrophoresis Prior to daily use, the capillary was conditioned with 0.1 M NaOH, methanol, purified water, and separation buffer in successive rinses of 20 minutes each. Individual CE runs were preceded by four successive 3 minute rinses using 0.1 M NaOH, methanol, purified water, and separation buffer. A pressure difference of 20 psi was used in all rinse procedures. Upon filling the capillary with separation buffer, conventional hydrodynamic sample injection was performed at 0.5 psi (3447 Pa) for 5 seconds. A separation voltage of 25 kV under normal polarity was applied continuously for 12 minutes. Propofol and thymol were detected with UV absorbance at λ=200nm. 73  3.3. Results and discussion Propofol is a highly lipophillic compound505 with a pKa of 11. We developed a MEKC-based separation to overcome the low aqueous solubility of the drug in its neutral form in solutions with pH < 11. Specific aspects of the method development process for the quantitative analysis of propofol in whole blood are described below. 3.3.1 Specificity In order to identify the thymol and propofol peaks, and to match them with specific migration times and apparent electrophoretic mobilities, we added either propofol or thymol reference materials into the blank- and blank whole blood matrices. The peak identities were further confirmed during the construction of the calibration curve, in which a series of standard solutions were prepared with increasing propofol concentrations but one consistent thymol concentration. Accordingly, the propofol peak could be distinguished from the thymol peak because the former had an incrementally increasing peak area. In CE analysis, apparent electrophoretic mobility (µepA) is more representative of a given analyte than migration time because it normalizes the rate of migration with respect to both the medium and the electric field strength, and is independent of electroosmotic flow. Accordingly, our group always converts migration times to electrophoretic mobility when conducting CE. The apparent electrophoretic mobilities of thymol and propofol always have coefficients of variation below 0.7 (Table 2, Table 3, Table 5). Based on specific apparent electrophoretic mobility, we can assign thymol and propofol peaks in different samples without ambiguity. In a similar vein, there appears to  74  be a difference between the migration times of propofol and thymol from Figure 9a to Figure 9b. These electropherograms were produced with two separately prepared capillaries. This difference may manifest itself in the electroosmotic flow, and therefore alter the migration times. Calculating apparent electrophoretic mobilities reveals the magnitude of the actual difference (Figure 9a: µepT=-22.08 cm2·kV-1· min-1, µepP=-23.08 cm2·kV-1·min-1; Figure 9b: µepT=-22.19 cm2·kV-1· min-1, µepP=-23.22 cm2·kV-1· min-1).  Table 2: Reproducibility of three clinically relevant standard propofol concentrations on the same day. Run 1  Run 2  Run 3  µ  Areacorr P/T µ epA T 2 µg/mL P T tmig P  0.149 -21.99 -23.01 6.70 7.33  0.138 -22.07 -23.08 6.71 7.34  0.148 -22.10 -23.11 6.73 7.36  0.145 -22.05 -23.07 6.72 7.34  0.006 0.06 0.06 0.02 0.02  4.3 0.3 0.3 0.3 0.2  Areacorr P/T µ epA T  0.301 -21.86  0.316 -21.89  0.298 -21.95  0.305 -21.90  0.010 0.04  3.2 0.2  P  -22.87  -22.88  -22.94  -22.90  0.04  0.2  T P  6.77 7.40  6.89 7.53  6.93 7.58  6.86 7.50  0.08 0.09  1.2 1.22  0.603  0.621  0.587  0.604  0.017  2.7  T P T  -21.89 -22.91 6.93  -21.93 -22.94 6.78  -22.01 -23.03 7.00  -21.95 -22.96 6.90  0.06 0.27 0.11  0.3 0.3 1.6  P  7.59  7.40  7.68  7.56  0.14  1.9  4 µg/mL  tmig  Areacorr P/T 8 µg/mL  µ epA tmig  σ  % RSD  P= Propofol; T= Thymol; Areacorr= corrected area; µepA= apparent electrophoretic mobility (cm2·kV-1· min-1); tmig= migration time (min); µ = mean; σ = standard deviation; % RSD = relative standard deviation expressed as a percentage.  75  Table 3: Reproducibility of three clinically relevant standard propofol concentrations across three days. Day 1  Day 2  Day 3  µ  σ  % RSD  2 µg/mL  Areacorr P/T T µ epA P T tmig P  0.145 -22.05 -23.07 6.72 7.34  0.145 -22.11 -23.11 6.70 7.32  0.145 -21.82 -22.84 6.61 7.22  0.145 -22.01 -23.02 6.68 7.29  0.004 0.16 0.16 0.06 0.07  3.1 0.7 0.7 0.9 0.9  4 µg/mL  Areacorr P/T T µ epA P T tmig P  0.305 -21.90 -22.90 6.86 7.50  0.287 -22.03 -23.03 6.76 7.38  0.301 -21.89 -22.93 6.58 7.20  0.300 -21.96 -22.97 6.73 7.36  0.011 0.07 0.06 0.14 0.15  3.5 0.3 0.3 2.1 2.1  Areacorr P/T T µ epA P  0.604 -21.95 -22.96  0.605 -22.11 -23.09  0.626 -21.98 -23.01  0.609 -21.99 -22.99  0.015 0.08 0.07  2.5 0.4 0.3  8 µg/mL  T 6.90 6.77 6.60 6.76 0.15 2.2 P 7.56 7.38 7.21 7.38 0.17 2.3 A P= Propofol; T= Thymol; Areacorr= corrected area; µep = apparent electrophoretic tmig  mobility (cm2·kV-1·min-1); tmig= migration time (min); µ = mean; σ = standard deviation; % RSD = relative standard deviation expressed as a percentage.  The specificity of the method was also assessed by its ability to separate propofol and thymol from other nonspecific blood components, and to resolve the peaks from one another. In brief, the optimized method produced sharp, gaussian peaks for both propofol and thymol with baseline resolution (Rs ≥ 2.6) at concentrations of 2, 4, and 8 µg/mL in both standard and sample extracted from whole blood matrices. Additionally, blank blood samples did not produce any detectable signals that interfered with the propofol and thymol peaks. Figure 9 shows the representative electropherograms for propofol and thymol from a) 6% acetonitrile and b) whole blood matrices.  76  Figure 9: Representative electropherograms of a) standard solution of propofol and thymol, and b) whole blood containing propofol and thymol. Run Conditions: Sample injection: 0.5psi for 5 seconds. Run Buffer: 50 mM sodium dodecyl sulfate, 15 mM borax Separation: 25 kV normal polarity over 12 minutes across an uncoated capillary (ID=50 µm; LT=50 cm; LD=40 cm). Detection: UV absorbance at λ=200 nm.  77  3.3.2 Selection of buffer type and separation voltage The solubility of propofol is proportional to the concentrations of sodium dodecyl sulfate in the buffer, particularly because propofol is exists almost entirely in its protonated and neutral in this buffer (pH=8.5). This limitation could be overcome by the addition of organic solvent in the running buffer, but this significantly compromised the consistency of run-times. The varying migration times reflect the difficulty of accurately controlling and reproducing the organic content in the buffer. The increased ionic strength of higher borax concentrations in the separation buffer translated into longer migration times. Alternatively, a lower ionic strength separation buffer resulted in broader peaks, lower resolution, and an inferior limit of detection. The composition of the separation buffer represents a compromise between the solubility and stacking efficiency of the analyte, and the running time. A 15 mM borax buffer (60 mM borate) with 50 mM sodium dodecyl sulfate was chosen as optimum condition based on the solubility of propofol, as well as the reproducibility and length of the runs. The separation voltage of 25 kV across a 50 cm capillary of 50 µm inner diameter was chosen based on the highest potential difference within the linear region in the Ohm’s law plot (Figure 10). The optimized separation conditions produce propofol and thymol peaks in less than 8 minutes (thymol tmig: 6.72±0.02 min; µepT: -22.05±0.06 cm2·kV-1·min-1; propofol tmig: 7.34±0.02 min; µepP: -23.07±0.06 cm2·kV-1· min-1. Values in mean ± standard deviation).  78  Figure 10: Ohm's law plot generated to determine the optimal separation potential. (Capillary: LT =50 cm, 50 µm inner diameter, Buffer: 15 mM borax buffer with 50 mM sodium dodecyl sulfate).  3.3.3 Linearity The clinically relevant concentration range of propofol was estimated between 1.5 and 10 µg/mL(whole blood)410, 454. The linearity of the current method was assessed using the corrected area of standard propofol solutions between 0.1 and 16 µg/mL relative to that of a fixed concentration of 7 µg/mL thymol. This ratio was plotted against the propofol concentration. This curve has a goodness of fit of r2 = 0.9995 throughout the tested range, with the equation: y = 0.0740x + 0.0019 (slope: 0.00064, 95% CI slope: 0.0725 to 0.0760; y-intercept: 0.0039, 95% CI: -0.0075 to 0.0112, n=15 concentrations).  79  3.3.4 Reproducibility Reproducibility was assessed by injecting 3 clinically relevant concentrations of propofol (2, 4, and 8 µg/mL) dissolved in 6% acetonitrile, and injected 3 times on the same day and on 3 successive days. The electrophoretic mobilities of propofol and thymol were calculated to assess the precision, selectivity, and specificity of the separation, while the corrected area ratio of the propofol and thymol peaks was used to measure the quantitative precision. Table 2 and Table 3 summarize the results of the reproducibility assays for propofol at 2, 4 and 8 µg/ml on the same day and on 3 successive days, respectively. The apparent electrophoretic mobilities of thymol and propofol had acceptable consistency within a given day and throughout a three-day interval for each of the tested concentrations (% RSD ≤ 0.7). The consistency of the corrected area ratio was also acceptable on the same day and over the three days across the three tested concentrations (% RSD ≤ 4.3). 3.3.5 Optimization of the liquid-liquid sample extraction The procedure used to extract propofol from whole blood was adapted from the liquid-liquid extraction first described by Plummer509. We have modified this method to suit the requirement for CE analysis. The first modification was to run the liquid-liquid extracted organic layer through a syringe filter of 0.1 µm pore diameter in order to prevent the capillary from becoming plugged and to reduce the variation in the migration time. Additionally, rather than using a stream of N2(g) to dry the extracted organic layer, we were able to achieve better precision using a vacuum centrifuge at 0.6 torr. We found  80  that the dry pellet could be stably stored under N2(g) at -80oC until subsequent resuspension and analysis with minimal degradation (data not shown). In order to control the volatility and reduce the loss of propofol during the drying process, tetramethylammonium hydroxide needs to be added to the organic layer prior to drying and resuspension. In order to optimize the tetramethylammonium hydroxide content for subsequent CE analysis, we added 5, 15, 25, 35, and 45 µL of 1% tetramethylammonium hydroxide (in methanol) to the organic layer prior to drying and resuspension. The best electropherograms, in terms of resolution, peak height, and baseline stability, were obtained with 15 or 25 µL of 1% tetramethylammonium hydroxide. We subsequently made all of our tetramethylammonium hydroxide dilutions at 2% v/v in propanol because of its superior solubility with cyclohexane compared to methanol. Indeed, we found this change to yield better run-to-run reproducibility. The pH of the resulting resuspended sample solutions was consistently between 11 and 12. We chose to use 400 µL blood and 650 µL cyclohexane to constitute the extraction system which satisfied the need for both accuracy and operability. 500 µL of organic phase out of 650 µL was then aspired to preclude aqueous contamination. The smaller total volume of this extraction system (<1.5 mL) than that of the original design is not only more convenient from a clinical perspective, but also in the laboratory because common and disposable lab supplies and equipment such as eppendorf tubes, spin filters and microcentrifuges can be used. The precision and accuracy of the separation and recovery requires that errors in all upstream steps be minimized. Pipetting errors, weighing errors, and inconsistencies in the extraction steps tend to propagate throughout subsequent steps. This is especially true when smaller starting volumes of blood are used,  81  as with our method, because the relative error is larger. Weighing small quantities of solid should be avoided because they tend to yield relatively large sampling errors. 3.3.6 Optimization of the sample resuspension solution We investigated the effects of the organic content and the ionic strength in the resuspension solution. Pellets were resuspended in aqueous solutions containing 6%, 30%, or 60% acetonitrile. 6% acetonitrile was a practical organic content from an analytical perspective because it was capable of keeping the analyte homogeneously dissolved without negatively affecting the peak shape. Higher organic contents tended to produce poor peak shapes. In order to optimize the ionic strength of the resuspension solution, we resuspended pellets in three buffers of increasing ionic strength. Solutions containing 6% acetonitrile with 0%, 1%, or 10% running buffer were studied. We found that the lowest ionic strength resuspension solution (6% acetonitrile with 0% running buffer) yielded the best resolution and peak shapes. We found that the main determinant for the pH of the extracted and prepared sample ready to be injected into the capillary was the amount of tetramethylammonium hydroxide that was added to the organic phase prior to drying under vacuum centrifuge, as opposed to the amount of borax buffer contained within the resuspension solution. These modifications enabled us to quantitatively analyze propofol from a 400 µL starting volume of whole blood. 3.3.7 On-line pH-difference induced focusing The resuspended solutions consistently had a pH of between 11 and 12, which is similar to the pKa of both propofol (pKa = 11) and thymol (pKa = 10.5). Accordingly, propofol and thymol are likely fractionally ionized in the injected sample, establishing  82  equilibria between their respective anionic and neutral species. The analytes in the sample plug could experience an effect similar to pH junction velocity-difference induced focusing510. In order to verify this effect we separated propofol and thymol in two distinct sample solutions: the first sample consisted of propofol and thymol resuspended in an aqueous solution containing 6% acetonitrile adjusted to a final pH of 12 with tetramethylammonium hydroxide. The second sample consisted of an equal concentration of propofol and thymol as condition 1, resuspended in 6% acetonitrile aqueous solution, with a final pH of 6. Table 4 describes the peak width, migration time, velocity, and end length of the propofol and thymol peaks derived from these samples, as well as the resolution between them. These results, which show improved peak width, length, and resolution, suggest that our method induces an online focusing effect. We were able to achieve sufficient sensitivity for routine clinically relevant propofol concentrations, but this focusing effect could be further investigated to increase the sensitivity if needed. Table 4: Contribution of sample solution pH to the resolution and focusing effect of the separation. Sample Solution pH 12 Thymol Propofol  Sample Solution pH 6 Thymol Propofol  Peak Width (min)  0.13  0.12  0.27  0.26  Migration Time (min)  6.70  7.30  6.75  7.38  Analyte Velocity (mm/min)  59.70  54.79  59.26  54.24  Peak Length (mm)  7.76  6.58  16.00  14.10  Resolution  4.80  2.36  83  3.3.8 Indices of precision and recovery of whole blood analysis In order to assess the precision of the propofol analysis from whole blood, we spiked propofol into blank whole blood to obtain one of three clinically relevant final concentrations (2, 4, and 8 µg/mL). Thymol was subsequently added to a final concentration of 7 µg/mL. We performed the extraction, and analyzed the sample using our optimized CE method. Each concentration was independently prepared and analyzed in triplicate. The percent-recovery was calculated using the standard curve to convert the propofol/thymol (P/T) corrected area ratio of each sample to its corresponding propofol concentration, and expressing this value as a percentage of the actual spiked propofol concentration (equation 18). Equation 18:  R=  (P /T " bstd .curve )mstd .curve #100 [Pspike ]  P/T denotes the corrected area ratio of propofol to thymol; bstd.curve and mstd.curve  ! denote the y-intercept and slope of the standard curve; and [Pspike] denotes the concentration of propofol supplemented into the blank blood sample. Table 5 shows indices of precision for propofol at 2, 4 or 8 µg/ml. The electrophoretic mobilities of propofol and thymol were consistent for each of the tested propofol concentrations (% RSD ≤ 0.2). The migration times of extracted samples were similar to those achieved using standard prepared solutions containing propofol and thymol, suggesting that the extraction and residual whole blood components did not significantly alter the separation process. Migration times of propofol and thymol peaks derived from extracted samples all have % RSD ≤ 0.1, indicating that the sample  84  preparation step consistently produces samples of similar composition that do not negatively influence the regeneration of the capillary after each separation process. The consistency of the P/T corrected area ratio and the percent recovery reflect the cumulative error of the operator, the extraction, and the instrument. The coefficients of variation associated with these values are therefore expected to be elevated. Accordingly, the P/T corrected area ratio derived from propofol in whole blood has % RSD values at or below 5.2%, while the percentage recovery has % RSD values at or below 5.1%. We report percent recovery values that consistently exceed 100% over the concentration range (2, 4, or 8 µg/mL) we examined (120.3%, 123.8% or 120.8%). The distribution and partition coefficients (log D at pH of 3-7, and log P, at 25oC) were calculated using Advanced Chemistry Development software (V8.14 for solaris ACD/Labs). Thymol has log D and log P values of 3.28, while those of propofol are 4.16. Accordingly, the efficiency with which propofol is extracted into cyclohexane is greater than that of thymol, and the P/T signal ratio subsequent to the extraction is therefore elevated. Since this elevated P/T signal ratio is used to derive the numerator in equation 18, the percent recovery can be expected to exceed 100%.  85  Table 5: Indices of precision of three independently prepared clinically relevant propofol concentrations spiked into and extracted from whole blood.  2 µg/mL  4 µg/mL  Areacorr P/T  Prep 1 0.182  Prep 2 Prep 3 0.181 0.166  µ 0.176  σ 0.009  % RSD 5.2  % R P/T µ epA T P tmig T P  124.0 -22.02 -23.01 6.72 7.33  123.8 -21.97 -22.95 6.72 7.33  113.2 -21.99 -22.98 6.70 7.31  120.3 -21.99 -22.98 6.71 7.32  6.2 0.03 0.03 0.01 0.01  5.1 0.1 0.1 0.1 0.1  Areacorr P/T % R P/T µ epA T P  0.359 121.6 -22.07 -23.06  0.356 120.4 -22.04 -23.02  0.382 129.4 -21.98 -22.97  0.366 123.8 -22.03 -23.02  0.015 4.9 0.05 0.05  4.0 3.9 0.2 0.2  6.72 7.32  6.73 7.34  6.73 7.34  6.73 7.33  0.01 0.01  0.1 0.1  0.717 121.0 -22.04 -23.03 6.70 7.30  0.737 124.3 -22.03 -23.01 6.69 7.29  0.694 117.0 -22.05 -23.05 6.70 7.31  0.716 120.8 -22.04 -23.03 6.70 7.30  0.022 3.7 0.01 0.02 0.01 0.01  3.0 3.0 0.05 0.07 0.1 0.1  tmig  8 µg/mL  T P  Areacorr P/T % R P/T µ epA T P T tmig P  P= Propofol; T= Thymol; Areacorr= corrected area; %R= % recovery; µepA= apparent electrophoretic mobility (cm2·kV-1·min-1); tmig= migration time (min); µ= mean; σ= standard deviation % RSD = relative standard deviation expressed as a percentage. Prep= sample preparation  3.3.9 Limit of detection and quantitation The limit of detection of a standard solution of propofol using our method was determined using the signal-to-noise ratio of 3 relative to the signal of the standardized thymol concentration (7 µg/mL). The slope from the standard curve was used to translate this P/T ratio to the corresponding propofol concentration. The limit of quantitation was determined similarly, using a signal-to-noise ratio of 10. The current method has a limit  86  of detection of 0.07 µg/mL and a limit of quantitation of 0.24 µg/mL. The above values are determined using thymol prepared in a neat solution, and therefore describe the limits of detection and quantitation for the instrument. A second series of calculations were done using electropherograms derived from whole blood samples. The signal-to-noise ratio from these electropherograms includes the contribution of the extraction and the original whole blood matrix, and therefore more closely reflects the conditions for which the method was intended. The limit of detection and limit of quantitation values were determined as described above, but included a correction for the recovery (see section 3.8). The resulting limit of detection and limit of quantitation values from electropherograms of whole blood samples were 0.07 µg/mL and 0.23 µg/mL, respectively. The similarity between the noise from neat and extracted injections testifies to the effectiveness of the modified sample extraction procedure. Although these values are higher than what has been achieved with some other methods, we found that the sensitivity is sufficient for clinically relevant propofol concentrations with acceptable % RSD. 3.3.10 Patient samples We used our method to analyze the concentration of propofol in the blood of 30 patients receiving propofol as the primary anesthetic during CPB. Whole blood samples were drawn 15 minutes after aortic declamping. Figure 11 shows the concentration distribution of these 30 patients. The average concentration of propofol was 5.36 µg/mL (95% CI 4.48 to 6.24 µg/mL). This concentration of propofol reflects the infusion rate of 120 µg•kg-1•min-1. The distribution in Figure 11 shows that propofol infusions normalized to patient weight results in a considerable amount of variation in the 87  concentration achieved in whole blood. The magnitude of this distribution is in agreement with previous reports454, 456, 502 and likely reflects both population variance and altered pharmacokinetics during CPB. Whatever the reason, the data show the need for a reliable method to monitor the actual whole blood concentrations achieved in patients during surgery. Such techniques will improve patient safety by reducing the likelihood of complications related to elevated concentrations, including the rare but fatal “propofol infusion syndrome”511.  Figure 11: Concentration distribution of propofol in whole blood of 30 patients undergoing CABG with CPB. Samples were obtained 15 minutes after aortic declamping under a propofol infusion rate of 120 µg•kg-1•min-1. Run Conditions: Sample injection: 0.5 psi for 5 seconds. Run Buffer: 50 mM sodium dodecyl sulphate, 15 mM borax Separation: 25kV normal polarity over 12 minutes across an uncoated capillary (ID=50 µm; LT=50 cm; LD=40 cm). Detection: UV absorbance at λ=200 nm.  88  3.4. Conclusion We describe a MEKC-based method capable of quantitative propofol analysis in whole blood over a clinically relevant concentration range with acceptable precision. This method is capable of producing sharp, baseline-resolved analyte peaks in less than 8 minutes from 400 µL of whole blood. More importantly, the method is robust and accurate enough to provide reliable propofol concentrations from patient samples collected in clinic settings. The procedures described in this work that were aimed at improving the reproducibility and robustness should be applicable to method development for other pharmaceutical analysis. Target achieved type techniques will require analytical methods with a high degree of automation, fast run times, and small and simple instrumentation. We recognize that the current method does not meet all of these criteria, especially insofar as the sample preparation step is concerned. However, this accurate and reliable method will provide a foundation for the development of other types or devices that more closely meets this clinical demand.  89  4.  Target achieved propofol concentration during on-pump cardiac surgery: A pilot dose finding study  4.1. Introduction Ischemia-reperfusion injury during CABG is a source of intraoperative cardiac injury512. Therapeutic pharmacologic options during surgery to preserve the viability of ischemic myocardium include volatile anesthetic pre- and post-conditioning and antioxidant therapies376, 513-515. Unfortunately, the clinical reproducibility and effectiveness of volatile anesthetic preconditioning has recently come into question378, 380. Anesthetic preconditioning has not translated easily to the clinical scenario and is not universally effective. Patient factors including diabetic status436, 449 and aortic crossclamp intervals exceeding 30 to 40 minutes516 could mitigate the effects of the preconditioning stimulus. Research into alternative approaches of cardioprotection is required. Conditions at reperfusion significantly contribute to tissue injury and repair517. The antioxidant409 and cell signaling properties518, 519 of propofol, as well as its ability to inhibit mitochondrial permeability transition520, 521 are well suited to reduce reperfusion injury. Unfortunately, clinical anesthetic conditioning studies have demonstrated that target controlled infusion devices, which predict whole blood propofol concentrations based on mathematical algorithms that link patient characteristics and pharmacokinetics with infusion rates, set to a target propofol concentration from 1 to 4 µg/mL failed to protect against myocardial injury375, 376. Based on work from our lab and others using both simulated models of ischemia-reperfusion injury and studies in patients481, 482, 484, 485,  90  488, 522-524  , we postulate that propofol confers cardioprotection when a target range of 4.5  µg/ml to 8.9 µg/ml (25 to 50 µM) is achieved. There have been limited in vivo studies evaluating the effect of increased propofol dosing to achieve the therapeutic concentration range defined in vitro. Recently, a clinically relevant swine model of normothermic blood cardioplegic arrest with CPB demonstrated that a 1 mg/kg bolus followed by a 100 µg•kg-1•min-1 continuous propofol infusion was cardioprotective without negative hemodynamic consequences488. The authors estimated whole blood propofol concentration of 3.7 µg/mL based on other clinical studies with similar operative procedures456, 525, 526. We previously found that a 2 to 2.5 mg/kg bolus of propofol followed by an infusion of 200 µg•kg-1•min-1 produces drug concentrations associated with increased antioxidant capacity (8.2+/- 2.1 µg/mL), but showed signs of intraoperative cardiac depression compared to conventional propofol or isoflurane anesthesia maintenance410. More recently, increasing propofol anesthetic maintenance from 60 to 120 µg•kg-1•min-1 intraoperatively was associated with a reduction in biomarkers of cardiac injury and oxidative stress, although the range of values was consistent with those expected during cardiac surgery414. Clinically relevant differences in hemodynamics and left ventricular function were not detected in this study and drug concentrations were not measured. Further systematic investigations are needed to evaluate the role of propofol in cardiac surgery. It is unknown if experimental cardioprotective propofol concentrations can routinely be achieved at reperfusion during CABG with CPB using short-term continuous infusion, or if such concentrations are associated with an increased risk of cardiac instability upon  91  emergence from CPB. To address this question, we conducted a pilot dose finding study and developed a predictive mathematical modeling for optimal dosing in patients. In this pilot study, we hypothesize that a whole blood propofol concentration of 5 µg/mL can be achieved clinically with continuous drug delivery during CABG with CPB. We focused our treatment interval to the CPB interval of CABG, and measured the resulting propofol concentrations in whole blood 15 minutes after reperfusion. We also sought to identify any evidence of clinically significant cardiac depression upon separation from bypass, and measured intraoperative hemodynamic performance using cardiac index, systemic vascular resistance index, and left ventricular stroke work index. 4.2. Methods 4.2.1 Study design We report on two successive studies, with the aim of establishing a clinical anesthetic maneuver that reliably yields a target whole blood propofol concentration of 5 µg/mL. The first study (Study 1) was an open label pilot dose finding study in 24 patients who received one of three propofol doses by continuous infusion during CPB. Propofol concentrations were mathematically described as a function of the infusion rate with an empirical line of best fit, constructed using nonlinear regression followed by Akaike’s Information Criteria comparison. The pharmacokinetics of propofol are most accurately described by a three compartment model 454. True pharmacokinetic steady state for propofol under a 3-compartment model would require in excess of 3 days for non-obese patients, and beyond 10 days for obese patients455. Given these timescales, it is unreasonable to anticipate steady state conditions for propofol during cardiac surgery –let 92  alone during the ischemic or reperfusion phase. For these reasons, the mathematical function describing the relationship between the infusion rate and the propofol concentration achieved at reperfusion was not modeled on pharmacokinetic or physiological principles. We used this mathematical function solely to determine the infusion rate predicted to yield our target propofol concentration. The infusion rate derived from Study 1 was employed in a subsequent and ongoing randomized controlled trial, entitled PRO-TECT II (www.clinicaltrials.gov NCT00734383, see Chapter 5)527. We planned an analysis of propofol concentrations at the midpoint of the PRO-TECT II trial (n=72) in those patients randomized to the propofol treatment arm (n=30). The purpose of this interim analysis (Study 2) was to assess the reliability with which our clinical maneuver achieves our target propofol concentration at reperfusion. Both studies focus on propofol concentrations in whole blood sampled 15 minutes after reperfusion in vacutainer tubes containing EDTA as the anticoagulant (Becton Dickinson, NJ). This operative timeframe precludes pharmacokinetic analysis. Hemodynamic measures of cardiac index, systemic vascular resistance index, and left ventricular stroke work index were also recorded prior to CPB, upon separation from CPB, and prior to patient transfer to the intensive care unit in both part 1 and in part 2. 4.2.2 Study population This investigation conforms to the principles outlined in the Declaration of Helsinki. Following institutional approval and informed patient consent, we enrolled hemodynamically stable patients scheduled for revascularization of 3 or more coronary  93  vessels where a minimum continuous aortic crossclamp time of 60 minutes was anticipated. We excluded patients who: 1) were less than 18 or greater than 80 years of age; 2) refused consent; 3) had co-existing valvular heart disease; 4) had an acute or evolving myocardial infarction; 5) had a history of hypersensitivity to propofol or any formulation component. 4.2.3 Perioperative procedures Perioperative monitoring (arterial, central, and pulmonary catheterization), surgical, and cardioplegia techniques (warm, intermittent, antegrade delivery of blood:crystalloid (8:1 ratio)) were standardized. Tranexamic acid 0.05 mg/kg then 0.10 µg•kg-1•min-1 was the antifibrinolytic therapy of choice. CPB was conducted at 34-37°C. Intraoperative hematocrit was maintained at 0.25 to 0.27 during CPB and facilitated by retrograde autologous prime procedure528. 4.2.4 Anesthesia protocol Anesthesia was standardized to induction with fentanyl 10-15 µg/kg, midazolam 2-4 mg and sodium thiopental as required for loss of consciousness. Muscle relaxation and tracheal intubation were achieved with rocuronium 0.1 mg/kg. Anesthesia was maintained with isoflurane (0.5-2%, end tidal), except during CPB when propofol was administered. Post-CPB anesthesia was as per clinical practice of the attending anesthesiologist.  94  4.2.5 Application of propofol during cardiopulmonary bypass Delivery of isoflurane was discontinued approximately ten minutes prior to aortic crossclamp. Propofol was then applied as a 1.0 mg/kg bolus followed by a continuous infusion of 50 (n=8), 100 (n=9) or 150 µg•kg-1•min-1 (n=7) in Part One, or 120 µg•kg1  •min-1 (n=30) in Part Two, until 15 minutes after release of the aortic crossclamp  (reperfusion). 4.2.6 Measurement of propofol concentration Four milliliters of whole blood was sampled from the central venous line 15 minutes after reperfusion to accommodate the surgeon after crossclamp removal. Whole blood was sampled using vacutainer tubes containing EDTA as the anticoagulant (Becton Dickinson, NJ), then stored as ~1.25 mL aliquots and stored at -80°C for subsequent quantitative propofol analysis by capillary electrophoresis529 (see Chapter 3). 4.2.7 Hemodynamic data collection Intraoperative central venous pressure and mean pulmonary catheter wedge pressure were maintained to within ±20% of baseline values by volume transfusion from the CPB reservoir. Transesophageal echocardiography was employed during the perioperative period to facilitate volume loading, and to rule out cardiac tamponade, pneumo- or hemothorax as possible causes of cardiac depression. Intraoperative cardiac function (cardiac index, systemic vascular resistance index, left ventricular stroke work index) was measured and derived at three timepoints: pre-CPB, post-CPB emergence, and just prior to admission to the intensive care unit (pre-ICU).  95  4.2.8 Inotropic and vasoactive drug protocol Intraoperative hemodynamic management included use of phenylephrine (1-2 µg/kg prn) for blood pressure below 85 systolic or mean arterial pressures below 50 mmHg. Arterial blood pressure greater than 140 mmHg systolic or mean arterial pressures above 80 mmHg was treated by deepening anesthesia by using fentanyl (1-2 µg/kg) followed by the vasodilator of choice prn at the discretion of the attending anesthesiologist. If preCPB heart rate was above 85 beats per minute, and if the attending anesthesiologist felt that adequate anesthesia and analgesia had been achieved, patients were treated with metoprolol intravenous prn. Systolic blood pressure less than 90 mmHg in the presence or absence of a cardiac index below 2.2 L•min-1•m-2, despite a pulmonary capillary wedge pressure range of 12 to15 mmHg at the time of separation from CPB, was treated with dopamine or dobutamine (> 4 µg•kg-1•min-1), epinephrine or norepinephrine (> 0.04 µg•kg-1•min-1), alone or in combination with milrinone (0.25 to 0.75 µg•kg-1•min-1) at the discretion of the attending anesthesiologist. Inotropic support exceeding 30 minutes in duration under the conditions described in Table 6 was considered clinically significant.  96  Table 6: Suggested dose regimen for attending anesthesiologists. Inotrope  Starting Dose  Dose Range  epinephrine  0.5 to 2 µg/min  0-8 µg/min  milrinone  0.125-0.25 µg•kg-1•min-1  0-0.75 µg•kg-1•min-1  dobutamine  3.5-7.5 µg•kg-1•min-1  0-10 µg•kg-1•min-1  dobutamine  1.5 to 3.5 µg•kg-1•min-1  0-10 µg•kg-1•min-1  2-4 µg/min  0-8 µg/min  SVR<600 norepinephrine SVR>1200 milrinone  0.125-0.75 µg•kg-1•min-1  mPAP>25 NTG and/or milrinone 0.125-0.75 µg•kg-1•min-1 SVR: systemic vascular resistance, mPAP: mean pulmonary arterial pressure, NTG: Nitroglycerine.  4.2.9 Sample size and statistical analysis. Based on two of our previous studies409, 410, we anticipated a one-tailed difference in whole blood propofol concentrations of 2.2 µg/mL between doses in study 1, with a standard deviation of 1.4 µg/mL. The type 1 error rate was set at α=0.05 and the power at 0.9. Accordingly, we determined a minimum sample size of seven patients per group. At the midpoint of PRO-TECT II, 72 patients had been randomized, 30 to the propofol treatment arm. This comprises the sample available for analysis for Study 2; no formal sample size calculation was performed.  97  All data are reported and presented as the mean and standard deviation except for predicted values and constant of proportionality, which are described using 95% confidence intervals. Hemodynamic parameters from Part 1 were analyzed using a twoway repeated-measures ANOVA. Bonferroni/Dunn post-tests for pair-wise comparisons of averages for doses across time were performed when the variance of the dose-time interaction reached a significance level of p<0.05. Hemodynamic parameters from Part 2 are presented descriptively. All analyses were performed using GraphPad Prism 4.0c software. 4.3. Results 4.3.1 Patient and operative characteristics Patient and operative characteristics according to experimental group are described in Table 7. Insufficient anesthesia, as evidenced clinically by elevated mean arterial pressure (exceeding 80 mmHg) and low mixed venous oxygenation (less than 65%) on CPB, was suspected by the attending anesthesiologist in three patients who had received a propofol infusion of 50 µg•kg-1•min-1 during CPB. These patients received supplemental isoflurane. Two patients receiving a propofol infusion of 150-, and one receiving a propofol infusion of 100 µg•kg-1•min-1, were described as clinically unstable at separation from CPB. They required two or more inotropes, alone or in combination with norepinephrine, for hemodynamic stabilization prior to intensive care unit transfer.  98  Table 7: Patient demographic and perioperative characteristics. Experimental group 50 µg•kg-1•min-1  100 µg•kg-1•min-1  150 µg•kg-1•min-1  120 µg•kg-1•min-1  n  8  9  7  30  Age (yr)  59±7  70±4  62±10  63±8  Weight (kg)  71.0±17.0  76.9±12.9  77.8±16.4  89.4±13.9  Height (cm)  162.5±8.5  165.7±7.3  166.4±9.8  173.1±7.2  BSA (m2)  1.78±0.23  1.88±0.19  1.86±0.24  2.03±0.17  Gender (m:f)  4:4  7:2  4:3  29:1  LVEF (%)  50±17  45±13  51±9  50±14  ACC (min)  65±29  101±39  73±20  89±28  CPB (min)  89±35  140±59  140±59  119±35  Data are expressed as mean± standard deviation or patient numbers. BSA: body surface area, LVEF: left ventricular ejection fraction, ACC: aortic cross-clamp interval, CPB: cardiopulmonary bypass interval.  4.3.2 Propofol concentrations in whole blood Part One The whole blood concentrations of propofol in patients treated with infusion rates of 50, 100, and 150 µg•kg-1•min-1 were 2.10 (1.20), 2.96 (1.87) and 14.28 (4.79) µg/mL, respectively (Figure 12). The empirical line of best fit for the relationship between propofol concentrations and infusion rates was determined using non-linear curve fitting. According to an Akaike’s Information Criteria comparison, an exponential growth nonlinear model was preferred over the alternative power series model. Equation 18 describes 99  the model mathematically, in which the y-variable represents the achieved whole blood concentration, the x represents the infusion rate, and a (0.215; 95%CI=-0.088 to 0.519) and K (0.0279; 95%CI=0.0181 to 0.0376) are constants of proportionality. The line had a coefficient of determination of r2=0.781, and predicted that 113 µg•kg-1•min-1 was required to achieve a mean concentration of 5 µg/mL. Equation 18:  y = a " e Kx  !  Figure 12: Propofol concentrations in whole blood at reperfusion during CABG with CPB in 24 patients receiving one of three infusion rates; 50, 100, and 150 µg•kg-1•min-1. The solid line represents the empirical line of best fit (r2=0.781); the dotted line represents its 95% confidence interval. All concentrations were determined using capillary electrophoresis from 400 µL of whole blood, sampled 15 minutes postreperfusion during CPB.  100  Part Two There were 4 study protocol violations; two patients received Propofol 2.0 mg/kg bolus (propofol concentrations: 10.6, 11.0 µg/ml); two additional patients received no loading dose (propofol concentrations: 2.6, 2.6 µg/ml). Three cases had operative aortic crossclamp intervals below 60 minutes (propofol concentrations: 2.4, 4.0, 6.5 µg/ml), thus intraoperatively violating study inclusion criteria. These seven patients were excluded from subsequent analysis. The whole blood propofol concentration from the remaining 23 patients was 5.39±1.45 µg/mL, with a range of 2.60 to 7.54 µg/mL. The 25%, 50%, and 75% quartiles were 4.36 µg/mL, 5.63 µg/mL, and 6.34 µg/mL, respectively. Propofol levels were 4.45 µg/mL (25 µM) or higher in 18/23 patients (78%), and above 5 µg/mL 15/23 patients (65%). Propofol concentrations showed no apparent correlation with patient age, weight, body surface area, or aortic crossclamp duration in this series (Figure 13). 4.3.3 Intraoperative hemodynamic function Figure 14 depicts intraoperative profiles for cardiac index, systemic vascular resistance index and left ventricular stroke work index in patients receiving propofol infusions of 50, 100, 150 µg•kg-1•min-1 along side those from patients receiving infusions of 120 µg•kg-1•min-1. We did not detect significant differences in the dose-time interaction for any hemodynamic parameters (Figure 14a,c,e). The hemodynamic profiles described in Figure 14 were similar for patients in Study 1 and Study 2.  101  Figure 13: Scatter plots of propofol concentration plotted against a) age, b) weight c) body surface area and d) crossclamp duration for 23 patients receiving 120 µg•kg-1•min-1 of propofol during CPB.  102  Figure 14: Intraoperative profiles of cardiac index (a,b), systemic vascular resistance index (c,d), and left ventricular stroke work index (e,f). Left panels (a,c,e) show profiles from patients receiving one of three propofol infusion rates (50, 100, and 150 µg•kg1  •min-1; n=24) during CPB. Right panels (b,d,f) show profiles from patients randomized  to receive 120 µg•kg-1•min-1 of propofol (n=23) during cardiopulmonary bypass. The horizontal axis is given in terms of operative timepoints: prior to CPB (pre-CPB), on separation from CPB (post-CPB), and immediately prior to transfer to the intensive care unit (pre-ICU). Data for each treatment group are presented as the mean and standard deviation.  103  4.4. Discussion The current study describes conditions under which laboratory-based propofol mediated cardioprotection was translated to an experimental clinical maneuver. In order to minimize alterations to the operative procedure, and to facilitate clinical investigation, the method employed a loading bolus followed by constant infusion focused to the CPB interval. The primary research question relates to whether cardioprotective concentrations could be reliably achieved in vivo without undue risk of cardiac depression. The principle findings of this study are: 1) the constant infusion rate predicted to achieve a mean propofol concentration of 5 µg/mL in whole blood was 113 µg•kg-1•min-1; 2) the propofol concentration achieved with the nearest practical rate of 120 µg•kg-1•min-1, was 5.39 (1.45 µg/mL), with quartiles of 25% = 4.36 µg/mL; 50% = 5.63 µg/mL; and 75% = 6.34 µg/mL. Our model predicted a concentration of 6.10 (± 1.76) µg/ml at this infusion rate; 3) patient age, weight, body surface area, or aortic crossclamp duration were not found to influence propofol concentration at reperfusion; 4) There was no evidence of depressed left ventricular function at emergence from CPB in patients receiving 120 µg•kg-1•min-1 propofol infusions during CPB. The dosing groups in Part One of our study were partly modeled after a pharmacokinetic study, conducted in a nonsurgical setting, by Gepts et al530. The mathematical model we used represents an empirical means to fit our data in order to predict the infusion rate most likely to produce a given propofol concentration under similar operative and anesthetic conditions. As a result, a 1 mg/kg propofol bolus followed by a 120 µg•kg-1•min-1 continuous infusion was chosen for our PRO-TECT II  104  protocol. Given its primary importance to tissue injury and repair, our sampling coincides with the early stage of reperfusion. The method of drug application in our study produced a wide range of blood concentrations for a given infusion rate. This variability appears to be in line with that of several other studies where propofol concentrations were measured under similar operative conditions452, 456, 531-533. It is clear that steady state conditions were not achieved, but it is also clear that steady state conditions for propofol cannot reasonably be anticipated within the context of cardiac surgery. We suggest that a significant reduction in the variance of propofol concentrations in the absence of steady state conditions will require monitoring of the concentration achieved during the course of surgery. By extension, drug level monitoring may be required to appropriately evaluate the role of propofol in cardioprotection, and its absence in experimental clinical studies makes interpretation of findings difficult. We are satisfied that the experimental clinical maneuver derived in this study is capable of producing a propofol concentration associated with laboratory based cardioprotection. Indeed, the effect of increased propofol dosing to achieve the therapeutic concentration range associated with laboratory based propofol mediated cardioprotection (25-50 µM) was achieved clinically in 78% of patients in our study. The highest level we measured was approximately 7.5 µg/ml (45 µM) as seen in 17% of cases. This concentration is clinically and experimentally relevant, given these levels have been previously associated with the range expected to inhibit both lipid peroxidation487 and mitochondrial permeability transition521. The absence of high drug levels among patients in study 2 suggests that a large dose of propofol, applied during 105  CPB, has no detrimental effect on early post-bypass functional recovery relative to lower infusion rates. This contrasts with reports where total intravenous anesthesia with propofol and remifentanil was used for cardiac surgery375. We did not observe a decrease in cardiac index, consistent with our definition of cardiac depression, upon emergence from CPB across dosing groups. This pattern was associated with a decrease in systemic vascular resistance index and the maintenance of left ventricular stroke work index (Figure 14). By extension, elevated doses of propofol during cardioplegic arrest do not appear to increase the risk of cardiac instability on emergence from CPB. The benefit of this method with respect to clinical outcomes and cardioprotection cannot be extrapolated in the current study, and remains to be determined. Patient characteristics of age, disease state, and weight have been identified as significant covariates that influence propofol pharmacokinetics454, 530. Their effect on data spread effects is likely to be amplified in non-steady state conditions. We did not find any systematic influence of these parameters on propofol concentrations at reperfusion (Figure 13), suggesting that non-steady state conditions and variability in total infused drug volume have a larger influence. We used capillary electrophoresis to quantitatively analyze propofol in whole blood529 (see Chapter 3). The separation is completed in less than 8 minutes, but the length of the preparative step still precludes its use for point of care target-achieved type dosing. Quantitative analysis that provides target-achieved drug infusion would likely facilitate perioperative care of high-risk patients. Technologies that enable target-  106  achieved dosing could then be adopted for routine use in studies designed to determine clinical outcomes. There are limitations to the present study. Propofol concentrations were only measured in central venous blood collected at one time point, which limits any pharmacokinetic interpretations of the data. Central venous sampling was used for quantitative propofol analysis. Site-effect studies have confirmed that venous sampling is equally representative of arterial drug concentrations provided the infusion interval prior to sampling is longer than 20 minutes453, 454, 534-537. Secondly, the mathematical model described in this study is inherently susceptible to changes in the anesthetic maneuver, and is incapable of predicting propofol concentrations in routine clinical practice, or beyond the infusion rates used in our study. In the absence of controls that omit propofol anesthesia, we are unable to attribute either the magnitude or the pattern of hemodynamic changes to the administration of propofol. The volume of propofol delivered in our study prior to sampling is entirely dependent on patient weight and crossclamp interval. Crossclamp intervals are neither consistent between surgical cases nor sufficient to establish near-steady state pharmacokinetic conditions455, 530. For these reasons, our line of best fit has no pharmacokinetic basis, its constants are not known to represent any physiological parameters, and there is no known basis for the apparent log-linear relationship between the infusion rate and the concentration that equation 18 suggests. The current study focused on the intraoperative interval. Any patterns of hemodynamic performance are not known to extend to the postoperative period. Finally, our hemodynamic findings are not known to apply to patients with severe ventricular  107  dysfunction and profoundly low cardiac output, or to patients treated with drugs used to treat low cardiac output, such as milrinone. The current study introduces an experimental clinical maneuver focused to the CPB interval, capable of yielding an elevated propofol concentration at reperfusion. In summary, the administration of a 1 mg/kg bolus dose of propofol followed by a continuous infusion of 120 µg•kg-1•min-1 during CPB produced relevant cardioprotective drug concentrations in whole blood at reperfusion. These concentrations were associated with an increase in cardiac index at emergence from CPB, in the absence of additional inotropic support. The achieved drug concentrations have previously been associated with enhanced red cell and tissue antioxidant capacity in vitro and in vivo406, 409, 489, reduced dysfunction subsequent to experimental ischemia-reperfusion injury488, and reduced endothelial and cardiomyoblast apoptosis519, 538. Failure to prevent cardiac injury with conventional propofol doses could be explained by inadequate concentrations and timing of administration. It remains to be determined if achieving a target concentration of 5 µg/ml will improve clinical outcomes (morbidity and mortality) in high-risk patient populations undergoing cardiac surgery.  108  5.  Rationale and design of the PROpofol CardioproTECTion for Type II Diabetics (PRO-TECT II) Study: A randomized, controlled trial of high-dose propofol versus isoflurane preconditioning in patients undergoing on-pump cardiac surgery  5.1. Introduction Diabetic patients are up to five times more likely to develop cardiovascular disease16. Unsurprisingly, these patients account for nearly a third of CABG surgeries21. Following cardiac surgery, individuals with diabetes suffer higher rates of perioperative morbidity and mortality, recurrence of angina and lower long-term survival rates compared to patients without diabetes12-14, 427, 428. In particular, diabetic patients are at elevated risk for low cardiac output syndrome14, 15  , defined as persistent hypotension (systolic blood pressure < 90 mmHg) and/or low  cardiac output (cardiac index < 2.2 L•min-1•m-2) despite hemodynamic optimization. Prolonged use of high doses of inotropes, vasopressors, and/or intra-aortic balloon counterpulsation are required. The causes of low cardiac output syndrome may include the injury that follows ischemia and reperfusion of the heart and inadequate revascularization. The diabetic heart is more sensitive to this form of injury due to defective antioxidant defenses17, 18, increased oxidative stress, and impaired endogenous myocardial protective pathways19, 20. If inadequately treated, low cardiac output syndrome can quadruple the overall mortality rate for CABG surgery427. Major efforts have focused on increasing the myocardial tolerance to ischemia (preconditioning) via physical (intervals of ischemia) or pharmacological (volatile  109  anesthetics) means332, 516, 539-541. The myocardial mitochondrial ATP-regulated KATP channel is essential for protection by preconditioning338, 339. Unfortunately in diabetes, signal transduction pathways required for ischemic or anesthetic preconditioning are corrupted19, 20 and sulphonylurea oral hypoglycemic agents can block KATP channel opening359. Preconditioning is insufficient to prevent injury in the context of prolonged ischemic intervals (greater than 25 to 30 min)516. Such circumstances require a different therapeutic approach. Elevated oxidative stress occurs during hyperglycemia and during myocardial ischemia and reperfusion. Oxidative stress promotes the conversion of NO• to ONOOand stimulates tumor necrosis factor-α (TNFα)542, which in turn inhibits cardioprotective endothelial NOS (eNOS)543, 544 and enhances endothelin-1 (ET-1) formation545. These factors cause cardiac dysfunction. Effective antioxidant intervention during ischemia and reperfusion appears important for preserving myocardial function. Thus, rather than increasing the myocardial tolerance to ischemia, we have focused on alleviating oxidantmediated post-ischemic injury by increasing antioxidant defenses (cardioprotection). Our previous work suggests that propofol, an intravenous anesthetic with antioxidant potential, may confer cardioprotection410, 414, 482, 483. Although conventional low doses of propofol have not been clinically effective in reducing postoperative cardiac injury or improving cardiac function, in vitro dose-finding and animal based studies from our laboratory suggest that high doses of propofol can reach a therapeutic concentration (≥10 to 25 µmol/L) needed for cardioprotection483, 519, 546. We have translated our approach of applying high-dose propofol in the laboratory into a safe experimental maneuver during cardiac surgery.  110  This paper describes the design of the PROpofol CardioproTECTtion for Type II Diabetics (PRO-TECT II) Study, a Phase II randomized controlled trial designed to explore the relationships of biomarkers of oxidative or nitrosative stress in diabetes, determine the effect of high dose propofol cardioprotection to counteract these effects in patients undergoing elective primary CABG with CPB, and provide feasibility and sample size data needed to conduct Phase III RCTs. 5.2. Materials and methods 5.2.1 Study design The PRO-TECT II Study is a Phase II RCT comparing high-dose propofol cardioprotection versus isoflurane preconditioning in diabetic and nondiabetic patients at risk of an adverse perioperative cardiac event who are undergoing CABG surgery requiring extracorporeal circulation. Participants, health care providers, investigators, data collectors, and laboratory staff are blinded to whether patients receive propofol or isoflurane. 5.2.2 Study population Adult patients undergoing cardiac surgery at the Vancouver General Hospital are eligible if they are 18-80 years of age, are undergoing primary CABG surgery requiring CPB, require revascularization of three or more coronary vessels with an anticipated aortic cross clamp time of ≥ 60 minutes, and have a preoperative systolic blood pressure > 90 mmHg in the absence of inotropic or mechanical support. Patients are ineligible if they have co-existing valvular heart disease, an acute or evolving myocardial infarction, or a history of hypersensitivity to propofol or any formulation component; or are taking 111  non-steroidal anti-inflammatory drugs, vitamin C, or vitamin E within five days of surgery. 5.2.3 Randomization Subjects are randomly allocated to either the propofol group or the isoflurane group after written informed consent. The allocation process uses a computer-generated random number table, with random permuted blocks of four or six, stratified by diabetic status and left ventricular ejection fraction as diabetes mellitus and decreased left ventricular function may affect the incidence rate of low cardiac output syndrome. For diabetic status, the two strata are no diabetes mellitus, defined as no history or diagnosis of diabetes mellitus, and Type II diabetes mellitus, defined as an established history and diagnosis of adult-onset diabetes mellitus treated with oral hypoglycemic agents (regardless of insulin use). For left ventricular ejection fraction, the two strata are normal, defined as a preoperative ejection fraction of at least 45% on angiography, and low, defined as a preoperative ejection fraction of less than 45% on angiography. The randomization scheme is unavailable to individuals involved in the recruitment, data collection, or management of the subjects. 5.2.4 Study protocol Standardized anesthetic techniques are used at the Vancouver General Hospital. Intraarterial blood pressure monitoring, central venous and pulmonary artery catheterization, and transesophageal echocardiography are used in addition to routine monitors. Subjects will undergo intravenous (IV) anesthetic induction with fentanyl 10-15 µg/kg, midazolam 0.15-0.25 mg/kg, and sodium thiopental 1-2 mg/kg followed by muscle relaxation using  112  rocuronium 1-2 mg/kg to facilitate tracheal intubation. Prior to CPB, anesthesia will be maintained with isoflurane 0.5 to 1.5% (end tidal). Subjects will receive phenylephrine (1-2 µg/kg), increased anesthetic depth, fentanyl (1 to 2 µg/kg), or vasodilator therapy (e.g., nitroglycerin 0.125 to 0.25 µg•kg-1•min-1) to maintain their systolic and mean arterial blood pressures between 85 to 140 mmHg and 50 to 80 mmHg respectively. Subjects will receive metoprolol if their pre-bypass heart rates exceed 85 bpm. Tranexamic acid (0.05 mg/kg then 0.01 mg•kg-1•hr-1) is the antifibrinolytic of choice, to reduce the risk of bleeding. Following a median sternotomy, the left and right internal mammary and radial arteries will be dissected for grafts depending on the location of the coronary artery disease. Subjects will receive intermittent, antegrade blood cardioplegia during continuous aortic cross clamping. The temperature of the cardioplegia will be left to surgical preference. CPB will be conducted at 34-37oC. Intraoperative hematocrit will be maintained between 0.25 and 0.27 during CPB. For subjects allocated to propofol cardioprotection, isoflurane will be discontinued ten minutes before CPB. At this time, subjects will receive propofol 1 mg/kg intravenous as a bolus followed by an intravenous infusion at 120 µg•kg-1•min-1 until 15 minutes after release of the aortic cross clamp (reperfusion). For subjects allocated to isoflurane preconditioning, subjects will receive a preconditioning dose of isoflurane 2.5% (endtidal) for ten minutes before CPB, followed by isoflurane 0.5-1.5% (end-tidal) during and after CPB, without administration of propofol. Figure 15 outlines the study interventions.  113  Figure 15: Representative diagram of study interventions applied during coronary artery bypass graft surgery in the PRO-TECT II Study. ET: end tidal, IV: intravenous, CPB: cardiopulmonary bypass.  At the end of surgery, all subjects will transfer to the cardiac surgery intensive care unit for postoperative management. Subjects will be mechanically ventilated initially after surgery until they meet criteria for weaning and tracheal extubation. Subjects will receive inotropic support, based on standard guidelines, at the time of separation from CPB and at any time in the first 24 h after surgery when the systolic blood pressure is <90 mmHg and/or a cardiac index is <2.1 L•min-1•m-2 despite pulmonary capillary wedge pressure and/or central venous pressure being 12–15 mmHg. The use of dopamine or dobutamine >4 µg•kg-1•min-1, epinephrine or norepinephrine >0.04 µg•kg-1•min-1 or milrinone 125 µg•kg-1•min-1, alone or in combination, for greater than 30 min will be considered clinically significant. Subjects will receive a continuous intravenous infusion  114  of insulin as needed to maintain glucose levels from 8 to 12 mmol/L during and after surgery. For postoperative analgesia, subjects will receive intravenous opioids in the first 24 to 72 h postoperatively, as required. After 24 h, once oral or nasogastric intake is tolerated, subjects will switch from intravenous opioids to oral hydromorphone, oxycodone, or codeine with acetaminophen. To reduce the risk of graft occlusion, aspirin is started one day after surgery. As the antiplatelet effect of nonsteroidal anti-inflammatory drugs could confound the results of our primary outcome (see below), this class of drugs will not be prescribed for adjunctive analgesia in this study. 5.2.5 Blinding An anesthesiologist (staff or fellow), who will be uninvolved with the clinical care of the subject, will receive the allocation by telephone from the recruiting nurse, and will initiate the intervention to maintain blinding of the subject, attending anesthesiologist, surgeon, nursing staff, and investigators. Ten minutes before CPB, the same individual will place opaque drapes over the anesthetic vaporizers and discontinue the anesthetic concentration readout on the anesthetic machine and the CPB machine to avoid unmasking of the allocation. During CPB, the unblinded study anesthesiologist, instead of the attending anesthesiologist, will direct the unblinded perfusionist in the administration of the anesthetic from the CPB machine. The drapes will be removed after separation from CPB. Patients in the isoflurane group received a mock intralipid infusion, delivered to an empty plastic bag that remained hidden from view, to mimic the propofol infusion.  115  Unblinding rules apply if a serious adverse event (anaphylactoid or anaphylactic reaction) occurs during the course of the study. The Principle Investigator will report the occurrence of this serious adverse event to the institutional Ethics Committee and the Health Protection Branch of Health Canada. 5.2.6 Outcome measures The primary outcome will be the myocardial-derived plasma free 15-F2t-isoprostane level, which will be the calculated difference between the measured coronary sinus plasma level of 15-F2t-isoprostane from coronary sinus blood sampled at 5 min after release of the aortic cross clamp. Secondary biochemical outcomes include plasma total antioxidant concentration, systemic and coronary sinus levels of troponin I, ET-1, TNFα, and 3-nitrotyrosine as evidence of ONOO- formation in blood; gene and protein expression of inducible NOS (iNOS) and eNOS, protein expression of Akt and its activation, and evidence of superoxide formation in atrial tissue. The clinical outcome is the incidence rate of low cardiac output syndrome during the first 6 h after surgery. Low cardiac output syndrome is defined as a systolic blood pressure less than 90 mmHg and/or cardiac index less than or equal to 2.1 L•min-1•min-2 despite central venous or pulmonary artery occlusion pressure and/or central venous pressure of 12 to 15 mmHg, systemic vascular resistance 800 to 1200 dyne•s/cm5, and a spontaneous or atrioventricular-paced heart rate greater than 80 beats per min. Low cardiac output syndrome will be adjudicated in a blinded fashion. Since the adequacy of revascularization could impact our primary and secondary outcomes, the vessel diameters, vessel quality and adequacy of revascularization will be documented. We will confirm adequate filling volume using transesophageal echocardiography to continuously 116  assess left ventricular function during the intraoperative period and to rule out other causes of postoperative cardiac depression (e.g., cardiac tamponade, pneumo-, hemothorax). The incidence rate of inotropic support or intra-aortic balloon counterpulsation required to treat low cardiac output syndrome for more than 30 min duration, the intensive care unit length-of-stay, and the hospital length-of-stay will be recorded. 5.2.7 Ethical considerations This investigation conforms to the principles outlined in the Declaration of Helsinki. This study was approved by the University of British Columbia Clinical Research Ethics Board. The PRO-TECT II study is registered at www.clinicaltrials.gov (NCT00734383). 5.2.8 Sample size Based on our previous work in humans undergoing CABG surgery and CPB using isoflurane anesthesia, plasma free 15-F2t-isoprostane levels at ten minutes reperfusion was 160.7±120.4 pg/mL (mean ± standard deviation). In our previous rat studies, plasma free 15-F2t-isoprostane levels decreased 70% during reperfusion (relative difference)483. We anticipate a 25% decrease in humans. Based on a type I error rate of 0.05, a power of 0.80, as well as an anticipated relative difference of 25% between groups with an estimated plasma free 15-F2t-isoprostane level to be 200 pg/ml at 5 minutes reperfusion in the isoflurane group, the required sample size would be 36 subjects per strata per group; therefore, 144 subjects will be required.  117  5.2.9 Data analysis We will use the intention-to-treat principle for all our analyses. We will describe normally distributed continuous data and skewed continuous data using the mean with its standard deviation, and medians and ranges respectively and categorical data using counts, proportions, and percentages and their 95% confidence intervals. Comparisons of levels of biochemical factors will use two-way ANOVA with a Bonferroni correction for between-group comparisons and one-way repeated measure ANOVA and Tukey's multiple comparison test for within-group comparisons. The correlations between 3nitrotyrosine, 15-F2t-isoprostane, ET-1 and cardiac index and/or left ventricular ejection fraction will be evaluated by the Pearson test. All statistical analysis will be performed using GraphPad Prism (GraphPad Software Inc., La Jolla, CA, USA). 5.2.10 Funding This project is funded with peer-reviewed grants, including the International Anesthesia Research Society Clinical Scholar Award, the Canadian Institutes of Health Research grant MOP 82757, the Canadian Anesthesiologists' Society Dr. Earl Wynands Research Award in Cardiovascular Anesthesia, the Natural Sciences and Engineering Research Council of Canada and the Vancouver Coastal Health Research Institute. 5.2.11 Trial status The PRO-TECT II Study is currently enrolling patients at a single centre in Canada. Table 8 summarizes pre- and intraoperative data describing our first 50 patients.  118  Table 8: Demographic details of the first fifty subjects enrolled in the Pro-TECT II study. Characteristic Gender, male / female Age, yr (µ±σ)  n = 50 48/2 64 ± 9  Weight, kg (µ±σ)  80.6 ± 24.7  Height, cm (µ±σ)  160.8 ± 43.3  Body surface area, m2 (µ±σ)  1.86 ± 0.51  Left ventricular ejection fraction, % (µ±σ)  52.0 ± 12.4  Left ventricular ejection fraction > 45%, n (%)  72 (%)  Left ventricular ejection fraction 25-45%, n (%)  28 (%)  Cardiac risk factors Coronary artery disease History of myocardial infarction, n (%)  40 (%)  Left main stenosis > 50%, n (%)  38 (%)  Hypertension, n (%)  66 (%)  Diabetes, n (%)  44 (%)  Current smoker, n (%)  25 (%)  Preoperative medication use, n (%) β-blocker  84 (%)  Calcium-channel blocker  20 (%)  Angiotensin-converting enzyme inhibitor  62 (%)  Digoxin  4 (%)  Statin  76 (%)  Oral hypoglycemic agent  20 (%)  µ = mean; σ = standard deviation.  119  5.3. Discussion Diabetic patients are at elevated risk for low cardiac output syndrome. This high risk scenario adversely affects up to 26% of diabetic patients recovering from cardiac surgery, and low cardiac output syndrome can quadruple the overall mortality rate subsequent to CABG surgery from 2% to 8%14, 15. Aggressive hemodynamic treatments are required, but can prove inadequate. Therefore, an urgent need exists for effective forms of preemptive cardioprotection. The PRO-TECT II Study represents a novel therapeutic approach for the prevention of myocardial ischemia–reperfusion injury in patients undergoing cardiac surgery with CPB. It is the first randomized controlled trial examining two distinct intraoperative cardioprotective strategies: the effect of intravenous anesthesia applied in an elevated dose, compared to a standardized volatile anesthetic preconditioning protocol. Our research into the therapeutic potential of propofol, an intravenous anesthetic with antioxidant properties, could have a significant impact on outcome in this select population. Given that signaling pathways for endogenous myocardial protection are corrupt and oxidant stress is elevated in diabetes17, 18, 436, we will determine the effectiveness of propofol to counter these effects, reduce injury, and preserve postoperative myocardial function. The dosing, delivery, and timing of propofol administration in our study is distinct from earlier clinical preconditioning studies conducted under conditions of hypothermic crystalloid cardioplegic arrest and CPB375, 376. Our experimental intervention is designed to combine continuous drug delivery via the systemic circulation during perfusion, and intermittent delivery via blood cardioplegia during global myocardial ischemia, at 120  normothermia. We believe that unconventional large dosing of propofol during the critical interval of ischemia and early reperfusion is a requirement to achieve drug concentrations associated with protective benefit (>25 µM) during early reperfusion under conditions of CPB. Our study design is strengthened by the simulated propofol infusion in the volatile preconditioning experimental group to enhance study blinding. The PRO-TECT II Study is not powered for clinical outcomes. We have employed stratified randomization to secure equal distribution of patients based on their diabetic status and preoperative ventricular function. It will therefore provide the prerequisite knowledge for a future randomized clinical trial powered to detect clinical outcomes in diabetic patients presenting for coronary revascularization. In addition to providing information on cardiac injury (enzymatic myocardial infarction, incidence of low cardiac output syndrome) and the 15-F2t-isoprostaneendothelin-1/eNOS relation to cardiac dysfunction, gene and protein expression of the Akt/eNOS cardioprotective pathway in response to propofol cardioprotection or volatile anesthetic preconditioning are of specific interest to our group. The gene and protein expression profiles that may influence cell survival versus cell death may enable the prediction of a given patient phenotype that is at increased risk of perioperative myocardial injury. Quantitative analysis of bioamines in coronary sinus blood may provide mechanistic insight to activity resulting from gene or protein activation realized in tissue. Determination of intravenous propofol levels achieved under conditions of CPB will establish the drug dosing protocols required to provide reliable delivery of cardioprotective concentrations of propofol for use in expanded clinical trials.  121  In conjunction with the study described above, we plan to investigate the possible role for therapeutic combination of drugs for decreasing risks of cardiac events. In clinical preconditioning studies, the effects of volatile anesthetic preconditioning have been examined with no consideration for the effects of drug combinations, including insulin, statins and β-blockers. We should be able to provide important information on a therapeutic alternative to volatile anesthetic preconditioning for diabetics. The range of data from PRO-TECT II will allow for comprehensive assessment of potential benefits and optimal therapeutic combinations for this select population.  122  6.  Propofol does not differ from isoflurane preconditioning in terms of 15-F2tisoprostane generation during ischemia and reperfusion in patients undergoing coronary artery bypass grafting with cardiopulmonary bypass  6.1. Introduction Coronary artery disease remains a leading cause of death in North America1, 2, and accounts for a significant and increasing economic burden estimated in the billions of dollars2, 3. CABG with CPB is a surgical revascularization strategy that uses healthy vessels to bypass diseased coronary arteries. CABG with CPB reduces mortality among medium- and high-risk patients6, 8-10, 547. Despite its effectiveness, this procedure is associated with an important source of intraoperative myocardial ischemia-reperfusion injury512, 517. A central hallmark of ischemia-reperfusion injury is the increased generation of ROS, which begins during ischemia but culminates with a large-scale burst at reperfusion. Several studies indicate that ischemia and reperfusion directly result in increased O2•and NO• generation, which subsequently reacts with NO• to produce ONOO-124, 157, 209, 212. ONOO- is readily protonated to its conjugate acid, ONOOH, under intracellular acidosis that develop during ischemia210. In turn, ONOOH rapidly degrades into •OH and NO2• radicals209, both of which readily initiate free radical reactions with biomolecules. Although myocardial generation of O2•- and NO• are not restricted to ischemia and reperfusion, the generation of ONOO- and •OH are not appreciable in its absence78, 124, 169. The unsaturated fatty acids that comprise cellular and subcellular lipid membranes are particularly susceptible to self-propagating peroxidation reactions initiated by •OH. Lipid  123  peroxidation products can adversely affect the functional integrity of lipid membranes, and are therefore quickly cleaved and released by membrane phospholipases232. One such product, 15-F2t-isoprostane, has potent dose-dependent vasoconstrictor activity in vivo241243  and may exacerbate intraoperative ischemia-reperfusion injury243-245, 249. Antioxidant-based therapies have been shown to reduce markers of lipid  peroxidation, but the overall benefit in terms of clinical and functional outcomes remains unclear392-398, 401-403. The ability to effectively translate these therapies appears to require that the antioxidants need to be delivered with sufficient concentrations to the myocardium at reperfusion. The phenolic structure of propofol confers this intravenous anesthetic with an antioxidant capacity405, 406. Several studies indicate that this antioxidant capacity may translate to a reduction in biomarkers of ROS-mediated lipid peroxidation in various in vivo and in vitro models of ischemia-reperfusion injury412-417, 480-483  . Relatively few reports describe the antioxidant capacity of propofol from the  perspective of 15-F2t-isoprostane and increased ONOO- biosynthesis during ischemia and reperfusion during CPB, and even fewer measure and report the concentration of propofol that was achieved in whole blood at reperfusion. In this study, we sought to determine if a propofol infusion during CPB with bloodcardioplegia, designed to achieve drug concentrations associated with cardioprotection in experimental models, could reduce the generation of 15-F2t-isoprostane and ONOOrelative to a standardized isoflurane preconditioning and maintenance protocol (see Chapter 5). The primary outcome of this study was the measurement of 15-F2tisoprostane, which was used as an indicator of ROS-mediated lipid peroxidation. Secondary outcomes included plasma nitrite (NO2-) and serum protein-bound 3-  124  nitrotyrosine as markers of NO• and ONOO- generation, respectively. We measured intraoperative and early postoperative hemodynamic performance using cardiac index, systemic vascular resistance index, and left ventricular stroke work index in order to identify any evidence of clinically significant cardiac depression upon separation from CPB. 6.2. Methods 6.2.1 Study design This study is a component of an ongoing Phase II randomized controlled trial, entitled PRO-TECT II (www.clinicaltrials.gov NCT00734383, see Chapter 5)527, comparing high-dose propofol cardioprotection versus isoflurane preconditioning in diabetic and nondiabetic patients at risk of an adverse perioperative cardiac event who are undergoing CABG surgery requiring CPB at Vancouver General Hospital. Participants, health care providers, investigator, and data collectors were blinded to patient randomization until completion of all pertinent analyses. 6.2.2 Study population This investigation conforms to the principles outlined in the Declaration of Helsinki. Following institutional approval and informed patient consent, we enrolled hemodynamically stable patients scheduled for revascularization of 3 or more coronary vessels where a minimum continuous aortic crossclamp time of 60 min was anticipated. We excluded patients who: 1) were less than 18 or greater than 80 years of age; 2) refused consent; 3) had coexisting valvular heart disease; 4) had an acute or evolving myocardial infarction; 5) had a history of hypersensitivity to propofol or any formulation component. 125  6.2.3 Perioperative procedures Standardized anesthetic techniques are used at the Vancouver General Hospital. Intraarterial blood pressure monitoring, central venous and pulmonary artery catheterization, and transesophageal echocardiography are used in addition to routine monitors. Subjects underwent intravenous anesthetic induction with fentanyl 10-15 µg/kg, midazolam 0.150.25 mg/kg, and sodium thiopental 1-2 mg/kg followed by muscle relaxation using rocuronium 1-2 mg/kg to facilitate tracheal intubation. All patients received isoflurane 0.5 to 1.5% (end tidal) for the maintenance of anesthesia prior to CPB. Subjects received phenylephrine (1-2 µg/kg), fentanyl (1 to 2 µg/kg), or vasodilator therapy (e.g., nitroglycerin 0.125 to 0.25 µg•kg-1•min-1) to maintain their systolic and mean arterial blood pressures between 85 to 140 mmHg and 50 to 80 mmHg respectively. Subjects received metoprolol if their pre-bypass heart rate exceeded 85 bpm. Tranexamic acid (0.05 mg/kg then 0.01 mg•kg-1•hr-1) was used as the antifibrinolytic of choice to reduce the risk of bleeding. Following a median sternotomy, the left and right internal mammary and radial arteries were dissected for grafts depending on the location of the coronary artery disease. Subjects received intermittent, antegrade blood cardioplegia during continuous aortic cross clamping. CPB was conducted between 34-37oC. Intraoperative hematocrit was maintained between 0.25 and 0.27 during CPB. The study protocol was initiated approximately ten minutes prior to CPB at heparinization. At this time, patients randomized to the propofol arm received an initial 1 mg/kg intravenous bolus of propofol followed by a 120 µg•kg-1•min-1 intravenous infusion for the duration of CPB. Patients randomized to the isoflurane arm received a  126  standardized preconditioning dose of isoflurane (2.5% end-tidal for ten minutes before CPB), followed by isoflurane maintenance (0.5-1.0% end-tidal) throughout CPB, without administration of propofol (Figure 15). Weaning from CPB and post-CPB anesthesia was accomplished according to the routine clinical practice of the attending anesthesiologist, who was blinded to the treatment arm during CPB. 6.2.4 Blinding An anesthesiologist (staff or fellow), who was not involved with the clinical care of the subject, received the patient allocation by telephone and initiated the intervention to maintain blinding of the patient, attending anesthesiologist, surgical staff, nursing staff, and investigators. Ten minutes before CPB, this same individual placed an opaque drape over the anesthetic vaporizers and discontinued the anesthetic concentration readouts on the relevant instruments to avoid unmasking of the allocation. This unblinded study anesthesiologist directed the unblinded perfusionist in anesthetic administration through the CPB machine. The drapes were removed after separation from CPB. Patients in the isoflurane group received a mock intralipid infusion, delivered to an empty plastic bag that remained hidden from view, to mimic the propofol infusion. 6.2.5 Measurement of propofol concentration Four milliliters of whole blood was sampled from the central venous line 15 minutes after reperfusion using vacutainer tubes containing EDTA as the anticoagulant (Becton Dickinson, NJ), and subsequently stored at -80°C for subsequent quantitative propofol analysis by capillary electrophoresis529 (see Chapter 3).  127  6.2.6 Coronary sinus blood sampling Coronary sinus blood was sampled immediately prior to the initiation of CPB and 5 minutes after crossclamp removal using vacutainer tubes containing EDTA as the anticoagulant (Becton Dickinson, NJ). Samples were centrifuged (10 minutes at 10,000 rcf) and plasma was separated and stored at -80◦C in 1.25ml aliquots. Samples destined for 15-F2tisoprostane were frozen in the presence of 0.005% of butylated hydroxytoluene. 6.2.7 15-F2t-isoprostane analysis 15-F2t-isoprostane was quantitatively analyzed using immunoaffinity purification followed by liquid chromatography-mass spectrometry analysis. In brief, 500 µL of plasma was spiked with 15-F2t-isoprostane-D4 internal standard and applied to an 8isoprostane affinity column (Cayman Chemical, Ann Arbor, MI, USA) as previously described548. The eluent was dried in a vacuum centrifuge, and resuspended in water containing 2% Acetonitrile and 0.01% NH4OH for subsequent LC-MS analysis. Liquid chromatography was performed on a C18 column (Phenomenex) using a 12 minute gradient, from 5% to 20% B, with the following mobile phase composition: A) 0.01% NH4OH in water. B) 0.01% NH4OH in Acetonitrile. Quantitative analysis was accomplished using an ion trap mass spectrometer (Bruckner Daltonics), comparing the ratio of the intensity of the signal at m/z=353.1 and m/z=357.1. Figure 16 represents a typical ion chromatogram of findings in human plasma from our lab.  128  Figure 16: Ion chromatogram of 15-F2t-isoprostane with 15-F2t-isoprostane-D4 internal standard in patient plasma. HPLC separation on a C18 column in tandem with ion trap mass spectrometry.  6.2.8 Nitrite analysis The concentration of NO2- in coronary sinus plasma was measured as a stable surrogate marker of NO• generation. Plasma sample were deproteinized as follows: Plasma (100 µl) was supplemented with 200 µl of ethanol, vortexed, and centrifuged (13000 rcf for 5 min). The supernatant was recovered, dried using a vacuum centrifuge, and resuspended in 100 µl of ultrapure water (Cayman Chemical, MI). Samples containing NO2- were refluxed in glacial acetic acid containing sodium iodide to quantitatively reduce NO2- to NO•, which was subsequently reacted with ozone and analyzed by chemiluminescence detection using an NO• analyzer (Sievers)549.  129  6.2.9 Measurement of 3-nitrotyrosine generation during ischemia and reperfusion 3-Nitrotyrosine-modified serum protein was detected in coronary sinus plasma as a stable surrogate marker of ONOO- generation using a competitive ELISA assay (Upstate-Milipore, MA). In brief, a 3-nitrotyrosine-bovine serum albumin standard was used as the immobilized antigen, which competes with 3-nitrotyrosine-modified serum proteins for the primary antinitrotyrosine IgG antibody. A second anti-rabbit antibody conjugated to horseradish peroxidase subsequently binds any bound primary antibody, whose chemiluminescence signal inversely correlates with the concentration of 3-nitrotyrosine present in plasma samples. Quantitative results are derived through the construction of a standard curve with serially diluted 3-nitrotyrosine-bovine serum albumin standards550. 6.2.10 Hemodynamic data collection Intraoperative central venous pressure and mean pulmonary catheter wedge pressure were maintained to within ±20% of baseline values by volume transfusion from the CPB reservoir. Transesophageal echocardiography was employed during the perioperative period to facilitate volume loading, and to rule out cardiac tamponade and pneumo- or hemothorax as possible causes of cardiac depression. Intraoperative cardiac function (cardiac index, systemic vascular resistance index, left ventricular stroke work index) was measured and derived at five timepoints: pre-CPB, post-CPB emergence, on admission to the intensive care unit (o/aICU), and 2 and 4 hours post admission to the intensive care unit (2h ICU and 4h ICU, respectively).  130  6.2.11 Sample size The power analysis for the PRO-TECT II study as a whole was based on the variation from 15-F2t-isoprostane results that were generated using ELISA analysis483, 551 (Chapter 5). A subsequent validation study indicated that ELISA-based 15-F2t-isoprostane analyses are invalid substitutions for mass-spectrometry based analyses552. Accordingly, liquid chromatography-mass spectrometry was adopted for the quantitative analysis of 15-F2tisoprostane, including an interim analysis to approximate the variance of 15-F2tisoprostane levels among patients prior to the initiation of CPB (prior to any experimental intervention). These results, derived from the revised methodology, were used verify the suitability of the power analysis using equation 19. The type 1 error rate and power were maintained at α=0.05 (zα=1.65) and 0.8 (β=0.2; zβ=0.842), respectively, while the 15-F2tisoprostane levels at baseline were 152 ± 109 pg/mL (µ±σ) with the relative difference anticipated to attain clinical relevance set at 25%.  Equation 19:  n=  2" 2 (z# + z$ ) 2 %µ  2  Accordingly, the required sample size is calculated at 52 patients per group for a total ! of 104 patients. In anticipation of study deviations, analysis of these data would commence when 110 patients had been randomized. 6.2.12 Statistical analysis All data are reported and presented as the mean with its standard deviation except for predicted values and the constants of proportionality, which are described using 95% confidence intervals. Post-reperfusion concentration values are multiplied by the 131  fractional difference in patient hematocrit in order to correct for hemodilution. Hemodynamic parameters were analyzed using two-way repeated-measures ANOVA. Bonferroni/Dunn post-tests for pair-wise comparisons of averages for doses across time were performed when the variance of the dose-time interaction reached a significance level of p≤0.05. Comparisons between anesthetic treatments were considered significant when p≤0.05. The directionality (increase or decrease) of an effect was considered significant when the 95% confidence interval of its mean failed to extend across zero. All analyses were performed using GraphPad Prism 4.0c software. 6.3. Results 6.3.1 Patient and operative characteristics A total of 112 patients were enrolled for the current study, and allocated to either the propofol or the isoflurane arm using a computer-generated random number table within the context of the blocked randomization scheme of the PRO-TECT-II study527 (see Chapter 5). Fifteen of the 112 enrolled patients were randomized but not studied for various operative and administrative decisions made after randomization: six patients required off-pump CABG due to coexisting aortic disease, one patient required concomitant valvular surgery, one patient required a MAZE procedure, the study could not reasonably be accommodated by operating room staff in the case of three patients, and four patients were recruited immediately prior to a temporary moratorium of clinical research activities to facilitate an institutional review of an unrelated study. Of the remaining 97 patients, 46 were randomized to the propofol arm, while 51 were  132  randomized to the isoflurane arm (Figure 17). The demographic and perioperative characteristics for these patients are described in Table 9.  Figure 17: Breakdown of patient randomization and study completion. (OR denotes operating room).  133  Table 9: Patient demographic and perioperative characteristics. Experimental group Propofol  Isoflurane  n  46  51  Age (yr)  64±9  65±8  Weight (kg)  87.8±14  83.4±17  Height (cm)  171±12  171±9  BSA (m2)  2.00±0.20  1.95±0.23  Gender (m:f)  42:4  42:9  LVEF (%)  50±15  49±12  Diabetic (n)  27  28  No. of grafts (n)  4±1  4±1  ACC (min)  86±27  83±29  CPB (min)  112±36  112±45  Data are expressed as mean ± standard deviation or patient numbers. BSA: body surface area, LVEF: left ventricular ejection fraction, ACC: aortic cross-clamp interval, CBP: cardiopulmonary bypass interval  134  6.3.2 15-F2t-isoprostane generation during ischemia and reperfusion 15-F2t-isoprostane increased significantly during ischemia and reperfusion among patients receiving propofol (99% CI: 0.24 to 0.41 logarithmic fold change units) and among patients receiving isoflurane (99% CI: 0.19 to 0.40 logarithmic fold change units). No significant difference was found between the groups (Figure 18a). The generation of 15-F2t-isoprostane during ischemia and reperfusion was plotted against the propofol concentration achieved in whole coronary sinus blood at reperfusion (Figure 18b). The slope of the correlation analysis line does not significantly deviate from zero (p=0.9953; 95% CI: -0.0257 to 0.0259; r2=8.3x10-7). 6.3.3 NO2- generation during ischemia and reperfusion NO2- increased significantly during ischemia and reperfusion among patients receiving propofol (99% CI: 0.087 to 0.16 logarithmic fold change units) and among patients receiving isoflurane (99% CI: 0.117 to 0.214 logarithmic fold change units) (Figure 19). This increase is greater among patients receiving isoflurane, but not significantly so (p=0.07). 6.3.4 3-Nitrotyrosine generation during ischemia and reperfusion 3-Nitrotyrosine increased significantly during ischemia and reperfusion among patients receiving propofol (95% CI: 0.09 to 0.34 logarithmic fold change units), but we did not detect a significant increase among patients receiving isoflurane (95% CI: -0.19 to 0.07 logarithmic fold change units) (Figure 20). This difference in 3-nitrotyrosine generation between anesthetic treatments was found to be statistically significant (p=0.0029, 95% CI of the difference: 0.10 to 0.46 logarithmic fold change units). 135  Figure 18: Change in 15-F2t-isoprostane generation during myocardial ischemia and reperfusion a) plotted according to the anesthetic protocol that patients were randomized to, and b) plotted against the concentration of propofol achieved in whole blood at reperfusion.  136  Figure 19: Nitrite generation changes during myocardial ischemia and reperfusion during CABG with CPB in patients randomized to propofol or isoflurane anesthesia protocols.  Figure 20: Protein-bound 3-nitrotyrosine changes in plasma during myocardial ischemia and reperfusion during CABG with CPB in patients randomized to propofol or isoflurane anesthesia protocols. 6.3.5 Perioperative hemodynamic endpoints Perioperative hemodynamic parameters of cardiac index, systemic vascular resistance index, and left ventricular stroke work index were not significantly different between study groups (Figure 21).  137  Figure 21: Perioperative profiles of cardiac index (upper panel), systemic vascular resistance index (middle panel), and left ventricular stroke work index (lower panel). Data for each treatment group are presented as the mean and standard deviation. The horizontal axis represents the operative timeframe, so the intervals between markings do not necessarily correspond to equivalent time intervals.  138  6.4. Discussion The primary finding of this study is that the magnitude of 15-F2t-isoprostane generation during ischemia and reperfusion increased to a similar extent in patients randomized to receive either propofol or isoflurane anesthesia during CPB. This suggests that neither treatment can quench ROS formation or activity at reperfusion. The secondary findings demonstrate that the level of protein-bound 3-nitrotyrosine in plasma increased during ischemia and reperfusion among patients receiving propofol, but not among those receiving isoflurane. This difference was statistically significant, but was not reflected by the pattern of NO2- generation during the same interval. A hallmark of post-ischemic reperfusion is the transient large-scale generation of ROS originating within the cells of the reperfused tissue. This phenomenon manifests itself in the current study as the significant generation of 15-F2t-isoprostane in coronary sinus plasma (Figure 18), and stands in agreement with several other studies that clearly indicate ROS generation during myocardial reperfusion239, 246, 553. The current study differs in that we compare 15-F2t-isoprostane generation during ischemia and reperfusion within the context of CABG with CPB, where patients receive one of two potentially cardioprotective anesthesia protocols. In the first group, patients received propofol during CPB according to an experimental maneuver capable of producing drug concentrations in whole blood that reflect conditions associated with propofol mediated cardioprotection in the laboratory setting411 (see Chapter 4). In the second group, patients received isoflurane according to a standardized volatile anesthetic mediated preconditioning protocol. Although •OH cannot be enzymatically degraded, and despite its much higher reactivity and cytotoxicity than either O2•- or ONOO-193, it can be quenched by propofol407. If  139  propofol were capable of quenching ROS-mediated lipid peroxidation in the myocardium at reperfusion during CABG, we would expect the achieved propofol concentration in central venous blood at reperfusion to inversely correlate with the magnitude of 15-F2tisoprostane generation during ischemia and reperfusion. We did not detect any such correlation (Figure 18). Previous reports have demonstrated that isoflurane preconditioning is similarly incapable of reduce the magnitude of •OH generation at reperfusion434, 435. Accordingly, our finding that 15-F2t-isoprostane was generated to a similar extent in both experimental groups suggests that neither protocol can significantly reduce or quench •OH -mediated lipid peroxidation reactions at reperfusion. This interpretation contrasts with findings from several groups, including our own414, that demonstrate clear reductions in MDA generation during ischemia and reperfusion in the presence of propofol416, 417, 554, 555. Furthermore, our finding that plasma-bound 3nitrotyrosine only increased among patients receiving propofol (Figure 18) was surprising considering the similar extent to which NO2- and 15-F2t-isoprostane increased in both anesthetic groups (Figure 18, Figure 19). O2•- is rapidly converted to O2 and H2O2 in a reaction catalyzed by the widely expressed enzyme superoxide dismutase. H2O2 is then further converted to H2O and O2 by catalase and by the glutathione antioxidant systems. The increased myocardial O2•generation at reperfusion coincides with an increase in NO•, thus favoring a reaction that yields ONOO- over dismutation157. This phenomenon has been documented during ischemia and reperfusion within the context of CPB249. NO• is generated by at least two distinct pathways during myocardial ischemia and reperfusion; NOS catalyzed and acid mediated NO2- reduction (equation 12, equation 13)  140  -only the former would contribute to the overall myocardial NO2- pool at reperfusion. A significant fraction of NO• can be assumed to react with O2•- at reperfusion, which would subtract from the overall myocardial NO2- pool at reperfusion. Thus, it is reasonable to assume that the current NO2- results somewhat underestimate the true increase in NO• generation during ischemia and reperfusion. NO• spontaneously reacts with oxygen and water to form nitrite (equation 9), and is further oxidized in the presence of oxyhemoproteins to nitrate (equation 10a/b)155. Although dietary nitrates significantly contribute to their concentration in plasma, the concentrations of nitrate and nitrite after several hours of fasting, is a good indicator of overall NO• activity156. In this study, plasma concentrations of nitrite were analyzed and used as an indicator of NOS-catalyzed NO• generation. For the reasons stated above, nitrites likely underestimate the true increase in NO• generation during ischemia and reperfusion. An additional potential limitation to this approach stems from the finding that oxygenated hemoglobin can promote the conversion of NO• directly to nitrate with a rate constant that effectively renders the reaction diffusion limited for free oxygenated hemoglobin (equation 20)556. Although this reaction is somewhat inhibited by the scarcity of free oxygenated hemoglobin, NO• is can reach its stable nitrate end product by bypassing the nitrite intermediate through this pathway556. To this end, approximately 95% of nitrites are oxidized in whole blood to nitrate after 1 hour557. Equation 20  7  "1 "1  M s NO• + HbO2 #k=#6"8x10 ## # #$ MetHb + NO3"  In the present study, venous blood was sampled from the coronary sinus both before ! bypass, and 5 minutes after crossclamp removal. Blood samples were cardiopulmonary  processed within ten minutes of sampling, and the plasma was immediately frozen at -  141  80oC. Thus, nitrite oxidation is minimized due to the inherently lower oxygenated hemoglobin content of venous blood, and due to the relatively short timeframe between sampling, red blood cell removal, and sample storage. As an alternative, deoxygenated hemoglobin can act as reductase for nitrite to yield NO• under low oxygen tension or in the case of acidosis183, 558. Furthermore, nitrate concentrations far exceed those of nitrite, so nitrate analysis or total nitrite and nitrate analysis can therefore obscure any changes in NO• production that could be reflected by nitrite559. Accordingly, Lauer et al found that nitrite analyzed from venous blood reflected acute changes in regional eNOS activity, while neither nitrate nor the combined analysis of nitrate and nitrite were capable of revealing these changes560. For these reasons, we used plasma nitrite analyzed from coronary sinus samples as a marker of acute NO• production. Since the rate of ONOO- formation depends on the product of O2•- and NO• concentrations, we anticipated that the magnitude of NO2- generation would be echoed by the generation of 3-nitrotyrosine. Our data did not demonstrate any such correlation or pattern (Figure 19, Figure 20). One possible explanation for these results is that plasma protein-bound 3-nitrotyrosine may not uniformly represent cellular ONOO- generation. ONOO- is relatively stable at alkaline pH, but ONOOH is a strong oxidizing agent that itself can rapidly decompose to produce NO2• and •OH radicals209. ONOO- has a pKa of 6.8 at 37oC278, but has a 1.9-s half-life at pH 7.4 that permits diffusion over several cell diameters209, 561. Intracellular pH decreases during myocardial ischemia within the context of CABG with CPB. The extent of this acidosis is proportional to crossclamp time and reaches a lower plateau of pH 6562. The extent of intracellular acidosis during  142  ischemia dictates the relative directionality of equation 13, which in turn determines the half-life and diffusion of ONOO-. Thus, intracellular acidosis is proportional to intracellular ONOOH activity and decomposition, which mediate nitration of tyrosine residues on intracellular proteins. These intracellular protein-bound 3-nitrotyrosine residues are only be detectable in plasma after their release from the confines of the cell. Alternatively, reduced intracellular acidosis would stabilize ONOO-, facilitating its diffusion out of the cell and increasing the likelihood that it decomposes in plasma. Interestingly, intracellular myocardial acidosis during CABG with CPB is associated with an increased need for inotropic support, impaired cardiac function, and poorer long-term post-operative outcomes563, 564. Furthermore, patients with diabetes are at a greater risk for intraoperative myocardial acidosis565, and pH recovery after reperfusion is delayed by hyperlipidemia182. Propofol preserves sodium-hydrogen exchanger activity in neurons566 and cardiomyocytes471 and appears to facilitate metabolic recovery during ischemiareperfusion injury481, 567. The overall effect may account for previous findings that propofol can reduce the extent of intracellular acidosis during ischemia and can increase the rate at which intracellular pH recovers471, 566, 568. Within the context of the current study, our observation of increased plasma protein-bound 3-nitrotyrosine generation among patients receiving propofol despite similar NO2- and 15-F2t-isoprostane generation to patients receiving isoflurane may not indicate any difference in ONOO- generation, but rather a proportional difference in intracellular ONOO- degradation. In support of this notion, intracellular 3-nitrotyrosine formation is an important step in the development of post-ischemic mitochondrial dysfunction288, 289 and myocyte apoptosis569. Furthermore,  143  propofol can protect cultured endothelial cells from apoptosis induced by 3-morpholinosydnonimine derived-ONOO- generation413. Concomitant intracellular staining for 3nitrotyrosine would be a useful adjunct experiment to further clarify whether propofol and isoflurane induce differences in intracellular 3-nitrotyrosine generation. Decreased intracellular acidosis may also account for conflicting previous accounts of decreased MDA among patients receiving propofol. The first steps of free-radical mediated lipid peroxidation are similar for both 15-F2t-isoprostane and MDA generation. However, MDA cleavage from lipid peroxyl intermediates is an acid catalyzed reaction whose efficiency can reasonably be expected to inversely correlate with pH. It is possible that propofol does not reduce MDA levels by reducing lipid peroxidation, but rather by decreasing the acid catalyzed cleavage of MDA from its lipid peroxide precursor, which in turn becomes reduced to more stable lipid peroxidation end products. In contrast to 3nitrotyrosine bound to intracellular proteins, phospholipid peroxidation products are rapidly cleaved by phospholipases and released into the plasma to facilitate their detection, thus accounting for the similar 15-F2t-isoprostane generation in plasma from patients in the current study. To this end, Corcoran et al reported significantlt decreased MDA generation in post-reperfusion coronary sinus plasma among patients receiving propofol during CABG, but were unable to find a similar reduction in post-operative urinary 15-F2t-isoprostane generation, and attributed the reduction in MDA generation to propofol’s ability to reduce post-ischemic inflammation416, 417. A concomitant analysis of MDA would shed further light on the discord between previous MDA results and the current 15-F2t-isoprostane findings.  144  In order to identify any evidence of clinically significant cardiac depression during the intraoperative and early postoperative period, we recorded hemodynamic performance using cardiac index, systemic vascular resistance index, and left ventricular stroke work index. We did not detect any differences between anesthetic groups for any of these parameters (Figure 21). Intraoperative pharmacologic management of the patients by the blinded attending anesthesiologist is designed to achieve a target hemodynamic performance, and this targeted approach likely accounts for the hemodynamic similarity between treatment groups. Differences in inotrope use required to achieve target hemodynamic endpoints could reflect differences in intraoperative injury. Inotrope use and patient outcomes in the early post-operative window (specifically low cardiac output syndrome) are recorded within the context of the PRO-TECT-II study in its entirety. At the time of this manuscript preparation, the investigators, participants, and hospital staff remain blinded with respect to these parameters. Consequently, this study is incapable of providing insight into whether the biochemical phenotype reported on in this manuscript alters the likelihood of improved outcomes.  145  7.  Summary and concluding remarks Coronary artery disease results from the buildup of atherosclerotic plaques in the  lumen of coronary arteries. Such plaques, which occlude the coronary arteries and impair myocardial perfusion, lead to angina, heart failure, arrhythmia, and infarction570. Several risk factors are associated with coronary artery disease, including hypertension, diabetes mellitus, dyslipidemia, excess weight and obesity, inactivity, smoking, genetics, and stress571. The majority of these risk factors are increasingly common worldwide, and coronary artery disease unsurprisingly remains the leading cause of North American death, representing its largest health-related economic burden2, 572. Less severe coronary artery disease can be treated pharmacologically or surgically without significant differences in outcomes4, 5, but coronary artery bypass grafting clearly reduces mortality among medium- and high-risk patients, including diabetics6, 7, over non-surgical management8 and percutaneous interventions6, 9, 10. Although the majority of patients undergoing surgical revascularization emerge without severe postoperative complications, a significant proportion of patients develop postoperative low cardiac output syndrome which can quadruple the overall mortality rate for CABG from 2% to 8%12, 13. Low cardiac output syndrome disproportionately affects diabetic patients14 who are up to five times more likely to develop coronary artery disease16 and who account for nearly one third of CABG procedures21. Myocardial ischemia-reperfusion injury is a major source of cardiomyocyte damage, which manifests itself in a host of clinical pathologies that include cardiac arrest, acute myocardial infarction, and postoperative low cardiac output syndrome. Accordingly, any improvement in clinical outcomes associated with these pathologies must be predicated 146  on reducing the magnitude of ischemia-reperfusion injury, which by extension requires an understanding of the central role that oxygen radicals play in its development. The research presented in this thesis described the development of a capillary electrophoresis-based technique capable of quantitative analysis of propofol in whole blood. This technique was then applied within the context of a pilot dose finding study that helped dictate the clinical maneuver most likely to deliver relevant propofol concentrations to the heart at reperfusion. An appropriate study was designed to test the following central hypothesis: Propofol reduces the incidence of low cardiac output syndrome subsequent to CABG with CPB by decreasing the magnitude of 15-F2t-isoprostane generation during myocardial ischemia and reperfusion. The preliminary findings contained within this thesis indicate that propofol does not significantly reduce 15-F2t-isoprostane generation during myocardial ischemiareperfusion compared to a standardized isoflurane preconditioning protocol. This result suggests that neither propofol nor isoflurane can significantly quench •OH formation or activity at reperfusion. Indeed, our finding that the magnitude of 15-F2t-isoprostane generation was not inversely proportional to propofol concentration suggests that the antioxidant capacity of this drug is incapable of reducing arachidonic acid peroxidation in the myocardium during CABG with CPB. The results of this thesis further demonstrated that 3-nitrotyrosine levels increased in coronary sinus plasma among patients randomized to the propofol treatment arm, but not among those patients randomized to the isoflurane arm. Since 15-F2t-isoprostane is indicative of •OH generation –and O2•- by extension, and since 3-nitrotyrosine is 147  indicative of ONOOH generation, one would expect the pattern of nitrite generation to reflect that of 3-nitrotyrosine in accordance with equation 11 of this thesis. Paradoxically, nitrite generation increased to a similar extent in both groups –with a non-significant tendency towards increased generation among patients randomized to the isoflurane treatment arm. Previous studies have demonstrated that propofol can reduce the extent of intracellular acidosis during ischemia and can increase the rate at which intracellular pH recovers471, 566, 568. We propose that a similar effect underscores the results in our study. Deprotonated ONOO- has a half-life of approximately 1.9 second at physiological pH, which permits diffusion down its concentration gradient over several cell diameters209, 561. If previous reports indicative of reduced intracellular acidosis extend to patients randomized to receive propofol during CABG with CPB, it is conceivable that the current 3-nitrotyrosine results do not represent an increase in ONOO- generation by the myocardium, but rather an increase in its release from the myocardium. Similarly, intracellular acidosis is expected to be more severe among patients randomized to the isoflurane arm, in whom ONOO- is fractionally more protonated, and in whom tyrosine residues of intracellular myocardial proteins would undergo reciprocally greater nitration reactions than in patients randomized to receive propofol. The proposed interpretation lends itself mechanistically to reports that correlate myocardial acidosis with impaired outcomes in the postoperative period. Specifically, patients with diabetes are at a greater risk for intraoperative myocardial acidosis565, and pH recovery at reperfusion is delayed by hyperlipidemia182. These patients are also at a greater risk for developing postoperative low cardiac output syndrome.  148  Future studies should performed using atrial tissue biopsies to determine the extent of intracellular 3-nitrotyrosine generation. These immunohistochemical or western blot based results should be analyzed in conjunction with 3-nitrotyrosine results derived from coronary sinus plasma samples. This combined analysis would provide more definitive mechanistic insights to ROS-mediated myocardial ischemia-reperfusion injury. The current findings conflict with previous results that demonstrate propofolmediated MDA reductions, and propofol-mediated reductions in cardiolipin depletion. Mechanisms other than reduced •OH-mediated lipid peroxidation may be responsible for these previous findings. First, cardiolipin depletion appears to be initiated during ischemia as a result of mitochondrial ROS generation, and propofol can potentially preserve cardiolipin content by signaling mechanisms that are orthogonal to its antioxidant effect. Second, MDA reductions in whole animal models or clinical studies may reflect reduced post-ischemic inflammation rather than a decrease in •OH-initiated peroxidation reactions endogenous to the myocardium. Alternatively, several reports indicate that propofol may reduce intracellular acidosis during ischemia and promote pH recovery at reperfusion. It is conceivable that these altered conditions could differentially affect MDA and 15-F2t-isoprostane generation. Future analysis of MDA in coronary sinus blood from this patient cohort may further clarify these conflicting results. The investigators, participants, and hospital staff were blinded with respect to clinical outcomes at the time of this manuscript preparation, so the central hypothesis of the clinical study that encompasses this thesis cannot be completely addressed herein. Specifically, the incidence of low cardiac output syndrome for each treatment arm remain to be analyzed. Nevertheless, the data herein do not support the hypothesis from the  149  perspective of 15-F2t-isoprostane. In more general terms, it remains unclear whether propofol can significantly improve outcomes over volatile anesthetics among patients undergoing cardiac surgery382-385, and it seems increasingly likely that propofol and volatile anesthetics confer mechanistically distinct cardioprotective effects. The data contained in this thesis suggests that in the case of propofol, applied during CPB to a target concentration of 5 µg/ml in whole blood, this mechanism extends beyond an increased antioxidant capacity. The research presented in this thesis describes the foundational work performed to design a clinical study. Accordingly, Chapter 3 and 4 represent the individual components that made up the groundwork for the PRO-TECT II study, which is presented in Chapter 5, while Chapter 6 provides preliminary results that address specific research questions within the context of a clinical hypothesis. From a global perspective, this thesis can serve as a general illustration of the way in which laboratory-based conditions can be translated to a clinical investigation, and may accordingly be of use to other research programs with a similar goal. The more specific strength of the data generated within the PRO-TECT II trial resides in its head-to-head comparison of two potentially cardioprotective anesthetic maneuvers, initiated immediately prior to myocardial ischemia and extending past reperfusion. In one arm, patients were randomized to receive a standardized isoflurane preconditioning protocol. In the other arm, patients were randomized to receive propofol according to a maneuver that was specifically designed to achieve whole blood concentrations that have been associated with a protective effect in the laboratory.  150  The PRO-TECT II, described in Chapter 5, study is a single-center trial. Intraoperative and in-hospital patient mortality and morbidity are recorded within the context of this clinical study. Unfortunately, long-term patient outcomes beyond discharge from the hospital are not recorded. Given the relatively size of the PRO-TECT II study, the limited post-operative timeframe in which patients are followed, and the low incidence of in-hospital adverse events, it is not likely that any additional mortality benefit associated with one treatment arm over another will manifest itself in a statistically significant difference. The heterogeneity of cardiac surgeons and patients who participated in the study is likely to further contribute to the variance in patient and biomarker outcomes, and thereby provides an additional hurdle. The results of this study nevertheless provide insight into the role that anesthetics play over the course of operative myocardial ischemia and reperfusion. Additionally, the results presented in this thesis and in the PRO-TECT II study may find further value within the context of a future meta-analysis or review. Chapter 3 described the development of an electrophoresis-based technique for the quantitative analysis of propofol. This method was capable of provided a resolved and quantifiable propofol peak in less than 8 minutes. The speed of this separation is sufficient to provide feedback to clinicians, allowing them to adjust dosing in order to achieve a target concentration within the operative timeframe. Unfortunately, the sample preparation, which was not automated and which requires more than an hour and a half, precludes such intraoperative feedback. In the current study, propofol concentrations were analyzed retrospectively, but sample preparation remains a particularly significant bottleneck to intraoperative quantitative analytical feedback within and beyond the  151  studies presented in this thesis. The low sample volume requirement of CE-based separations may herald improved sample preparation efficiency and, with technological advances, may facilitate the development of automated sample preparation. Of equal importance, the CE-based method described in this thesis is mechanistic similar to microfluidic-based electrophoretic separations. Microfluidic devices have the potential to further decrease separation speed and to provide clinicians with an analytical device that is small and fast enough for routine intraoperative use. Chapter 4 described a pilot dose finding study whose results helped inform the development of a clinical maneuver capable of reliably achieving propofol concentrations in whole blood associated with whole blood cardioprotection. The results of Chapter 4 clearly demonstrate the large variance of propofol concentrations that are achieved in whole blood for any given infusion rate in patients presenting for cardiac surgery. This variance, which likely stems from a combination of patient heterogeneity and the nonsteady state pharmacokinetics that arise with propofol infusion times encountered during CABG with CPB, underscores the importance of devices capable of providing quantitative feedback to clinicians. The pharmacology of cardioprotection is an important field of research both because it sheds light on the pathogenesis of ischemia-reperfusion injury and because it may reveal more targeted strategies to improve patient outcomes. The finding in Chapter 6 that patients randomized to the propofol and isoflurane arms of the study were equivalent in terms of 15-F2t-isoprostane generation during ischemia and reperfusion implies that neither can reduce ROS generation over this operative interval. By extension, if volatile anesthetic preconditioning translates from laboratory-based protection to the clinic, then  152  the lack of clear differences in patient outcomes from meta-analyses comparing volatile preconditioning with propofol anesthesia implies that both anesthetic regimens have their own distinct cardioprotective mechanism. The mechanistic nature behind cardioprotection, whether induced by volatile anesthetics, propofol, or ischemic preconditioning, remains speculative. In the case of volatile anesthetics and ischemic preconditioning, mitochondrial KATP channel activation appears to be an important intermediate step, but this does not appear to be so for propofol. The results presented in this thesis further suggest that this mechanism extends beyond a propofol-mediated increase in tissue antioxidant capacity. Volatile anesthetic and ischemic preconditioning reduce infarct size in laboratorybased models of ischemia-reperfusion injury. This reduced infarct size is a reflection of a decrease in cardiomyocyte death. The cell-sparing effect of preconditioning is undoubtedly desirable, but cell death and infarct size may be less relevant in cardiac surgery than in the laboratory. Conversely, reversible contractile dysfunction, or myocardial stunning, which manifests itself in low cardiac output syndrome during the early post-operative period, may ultimately be a more relevant consequence of ischemia and reperfusion within the context of CABG with CPB. Accordingly, the most relevant type of cardioprotective strategies may turn out to target the preservation of contractile protein integrity and function, as well as the maintenance of high-energy phosphates in cardiomyocytes. The activation of intracellular proteolytic MMP enzymes subsequent to ischemia and reperfusion results in the targeted degradation of contractile proteins within cardiomyocytes. Ischemic preconditioning has been associated with reduced MMP  153  activity349, 573, but this association has not yet been clearly established for volatile anesthetics. It is tempting to speculate that the infarct-reducing effect of preconditioning may be robust but less relevant to cardiac surgery, while preservation of contractile function, which may be more important in terms of patient outcomes, may not be induced by these agents. Further research is required to clarify the association between volatile anesthetics and MMP activity. Modulation of the phosphotidylinositol-3-kinsase/Akt pathway has recently been associated with inhibited MMP-2 activity during ischemic preconditioning574. This result may reflect the finding that MMP-2 activity can be inhibited by phosphorylation575. Propofol has recently been shown to increase Akt activity in cultured cells538, and this may represent a mechanistic point of convergence between the potential cardioprotective effect of propofol and its ability to modulate cell signaling. Future research is required to determine whether Akt activity definitively results in intracellular MMP phosphorylation and inactivation, and whether propofol can significantly induce this effect. A second alternative stems from propofol’s ability to pharmacologically inhibit mitochondrial permeability transition. In doing so, propofol can maintain mitochondrial integrity and function subsequent to ischemia and reperfusion, and may thus preserve myocardial ATP synthesis and contractility. Studies that determine post-ischemic cardiac work normalized to tricarboxylic acid cycle activity or O2 consumption, in the presence or absence of propofol, are warranted. Such studies should additionally investigate the influence of propofol on post-ischemic levels of high-energy phosphates in the myocardium. Combined, these studies could help tease out whether propofol can preserve  154  contractility by preserving mitochondrial integrity and function, or whether it does so by preserving the integrity of the myocardial contractile machinery. Lastly, as stated in the discussion of Chapter 6, it is conceivable that propofol’s protective effect is derived, in part, from its ability to reduce the extent of myocardial acidosis during ischema, and to accelerate pH recovery upon reperfusion. In this way, propofol may facilitate ONOO- diffusion down its concentration gradient and out of the cell, while pharmacologically maintaining mitochondrial permeability transition inhibition, despite pH recovery. Future studies into the effect of propofol on intracellular pH and the effect of sustained acidosis on contractile function during ischemia and reperfusion would further clarify the role of propofol in the context of myocardial ischemia-reperfusion injury. Mechanistically distinct cardioprotective strategies may not translate to improved outcomes for that majority of patients who emerge from cardiac surgery without any significant postoperative complications; but they have the potential to improve outcomes for those patients in whom intraoperative myocardial ischemia-reperfusion injury manifests itself in postoperative cardiac dysfunction. 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