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The use of a radioiodinated fatty acid analogue in the study of myocardial ischemia and infarction Hudon, Marck P.J. 1988

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The Use of a Radioiodinated Fatty Acid Analogue i n the Study of Myocardial Ischemia and Infarction By Marck P.J. Hudon B.Sc. (Zoology), University of B r i t i s h Columbia, 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department of Academic Pathology i n a f f i l i a t i o n with Department of Radiology, Div i s i o n of Nuclear Medicine and Department of Surgery, Division of Cardiovascular and Thoracic Surgery We accept this thesis as conforming to the required standard The University of B r i t i s h Columbia December, 1988 © Marck P.J. Hudon, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. 1 further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of P^-WaVc^w The University of British Columbia Vancouver, Canada D ^ e \ ft / 1 at / Q -9 -DE-6 (2/88) ABSTRACT The purpose of this study was to u t i l i z e 15-p-123I-phenylpenta-decanoic acid (123IPPA) and 15-p-12 3I-/3-methyl iodophenylpentadecanoic acid (B123IPPA) in the assessment of myocardial metabolism and perfusion following ischemia and infarction. Canine models of global ischemia, regional ischemia and infarction were used. Time-activity curve analysis was performed on the f i r s t two models using 15-p-123I-iodophenylpentadecanoic acid to determine changes in metabolic status of the myocardium. Reported are the h a l f - l i f e values + SD. B123IPPA, 201x1 and a histochemical method (tetrazolium staining) were used in the latter model to assess perfusion defects, including ischemic risk zone. Significant differences in the elimination rate (mins) of 1 2 3IPPA metabolic by-products for the early and late phases of the time-activity curves of the global ischemia model in the lateral wall (early and late phase) and apical wall (late phase) were observed with Iso-osmolar (Tyers) solution. There were no significant differences for either the Iso-osmolar + superoxide dismutase (SOD) or Iso-osmolar + allopurinol. Also there was no significant difference in th values between the three areas of the l e f t ventricle (apex, septal wall and lateral wall) within the control group. Results with the regional ischemia model indicate an increase in early phase th values representing 123IPPA washout, between control and 14 days i i post-operative animals i n both the l a t e r a l wall (22+10 to 60+107 min -p>0.05) and the apical wall (23+10 to 37±33 min - p>0.05) and a decrease i n I 2 3 I P P A washout i n the septal wall (22±14 to 14+7 min - p>0.05). An increase i n th between control and 14 days was seen i n the late phase post-operatively i n the l a t e r a l wall (34+12 to 71+50 min - p>0.05), the apex (44+8 to 115+104 min - p>0.05) and the septal wall (34+2 to 626+1325 min -p>0.05). The results of the myocardial i n f a r c t i o n model indicated a greater degree of correlation between the size of perfusion defects estimated by a histochemical method (TTZ staining) vs. B1 2 3IPPA (r = 0.65, p<0.005), than vs. 2 o i T i ( r = o.49, p<0.005). The following conclusions can be made on the basis of the results obtained. ' The u t i l i z a t i o n of iodinated free f a t t y acids within each area of the l e f t v e n t r i c l e ( l a t e r a l w a l l , apical wall and septal wall) demonstrate simi l a r rates of 1 2 3IPPA washout. The effect of reversible global ischemia on myocardial washout of 15-1 2 3IPPA may be modified by the addition of SOD or al l o p u r i n o l to the control iso-osmolar cardioplegic solution. The imaging agent 1 2 3IPPA may be a useful indicator of metabolic status of the myocardium, revealing changes i n washout rates during the period of developing regional ischemia. L a s t l y , the u t i l i z a t i o n of B1 2 3IPPA does not appear to be effective i n determining the size of perfusion defects, or i n identifying the ischemic r i s k zone because of border demarcation problems. i i i TABLE OF CONTENTS A b s t r a c t i i T a b l e o f C o n t e n t s i v L i s t o f T a b l e s i x L i s t o f F i g u r e s x i L i s t o f A p p e n d i c e s x v A b b r e v i a t i o n s and Fo rmu l ae x v i Acknow ledgments x i x I I n t r o d u c t i o n 1 I I L i t e r a t u r e Rev i ew 7 (1) M y o c a r d i a l M e t a b o l i s m 7 a) F a t t y A c i d s 7 i ) membrane t r a n s p o r t 7 i i ) a c t i v a t i o n and t r a n s f e r 8 i i i ) / 3 - o x i d a t i o n . 8 b ) G l u c o s e 10 c ) F a t t y A c i d s and Ene rgy P r o d u c t i o n 10 (2) M y o c a r d i a l I s c h e m i a and I n f a r c t i o n 11 a) C y t o s k e l e t a l A l t e r a t i o n s 11 b ) E l e c t r o p h y s i o l o g i c a l A l t e r a t i o n s 12 c ) F u n c t i o n a l A l t e r a t i o n s 13 i ) e c h o c a r d i o g r a p h y 13 i i ) hemodynamics 13 d) M e t a b o l i c A l t e r a t i o n s 13 e) E x p e r i m e n t a l Mode l s o f I s c h e m i a 15 i v (3) Radiopharmaceuticals i n the C l i n i c a l Diagnosis of Ischemia and Infarction 18 a) Technetium ("mTc) . 18 i ) pyrophosphate 18 i i ) i s o n i t r i l e s 21 b) Thallium - 201 25 i ) H i s t o r i c a l perspectives 25 i i ) Radionuclidic properties 26 i i i ) C l i n i c a l Applications and Experimental Evaluation 26 c) Iodine - 123, 131 28 i ) H i s t o r i c a l Perspectives of Iodinated Fatty Acids. 28 i i ) i23I odine ( i 2 3 i ) 31 1. Production of 1 2 3I 31 2. Fatty Acid Iodination Procedures 33 i i i ) Applications: C l i n i c a l and Experimental 39 1. Iodinated Decenoic acid .- 39 2. Iodinated Decanoic acid 42 3. Iodinated Pentadecanoic acid 46 4. Miscellaneous Fatty Acids 52 (4) Ischemia and Cardioplegia 61 I I I Purpose 64 IV Experimental Materials and Methods 65 (1) Global Ischemia 65 a) Experimental Model of Cardioplegia 65 b) Protocol 65 c) Preparation of 15-p-1 2 3I-iodophenylpentadecanoic acid ( i 2 3I P P A) 67 i ) Radipiodine 67 i i ) Production of 1 2 3IPPA 67 i i i ) Hexane Extraction 67 v iv) P u r i f i c a t i o n 68 v) Preparation of 1 2 3IPPA for Injection 68 v i ) Quality Control 68 d) Electron Microscopy 68 i ) Tissue Collection 68 i i ) Processing 69 i i i ) Sectioning 69 e) Light Microscopy 69 f) Data Acquisition 70 i ) Imaging 70 i i ) Blood-Gas Analysis 70 g) Data Analysis 71 i ) 1 2 3IPPA Image Analysis 71 i i ) S t a t i s t i c a l Analysis 71 (2) Regional Ischemia 72 a) Experimental Model of Regional Ischemia 72 b) Metabolic Assessment 73 c) Preparation of 15-p-1 2 3I-iodophenylpentadecanoic acid (1 2 3IPPA) 74 i ) Radioiodine 74 i i ) Production of *2 3IPPA 74 i i i ) Hexane Extraction 74 iv) P u r i f i c a t i o n 74 v) Preparation of 1 2 3IPPA for Injection 74 v i ) Quality Control 74 d) Data Acquisition 74 i ) Imaging 74 i i ) Blood Gas Analysis 74 v i e) Electron Microscopy 75 i ) Tissue Collection 75 i i ) Processing 75 i i i ) Sectioning 75 f) Light Microscopy 75 g) Data Analysis 75 i ) 1 2 3IPPA Image Analysis 75 i i ) S t a t i s t i c a l Analysis 75 (3) Myocardial Infarction 76 a) Experimental Model of Myocardial Infarction 76 b) Metabolic Assessments 77 c) Preparation of 15-p-Beta-Methyl-1 2 3I-iodophenylpentadecanoic acid (B1 2 3IPPA) 77 d) Data Acquisition 78 i ) Imaging 78 i i ) Histochemical Assessment 80 i i i ) Autoradiography 82 iv) Histology 84 e) Data Analysis 84 i ) B1 2 3IPPA Image Analysis 84 i i ) S t a t i s t i c a l Analysis 85 V Results 86 (1) Global Ischemia 86 a) Analysis of 1 2 3IPPA curves 86 b) Hemodynamics 93 c) Histology 93 i ) Electron Microscopy 93 i i ) Light Microscopy 95 v i i (2) Regional Ischemia 97 a) Analysis of i2 3IPPA Curves 97 b) Metabolic Assessments 102 c) Hemodynamic Assessments 102 d) Histology 106 i ) Electron Microscopy 108 i i ) Light Microscopy 108 (3) Myocardial Infarction I l l a) Metabolic Analysis I l l b) Analysis of Perfusion Defect Assessments 113 i ) B1 2 3IPPA 113 i i ) 2 o iTi 113 i i i ) Histochemical 113 c) Autoradiography - Assessment of Ischemic Risk Zone .... 120 i ) B1 2 3IPPA 120 i i ) 2o i T l 120 d) Histology - Light Microscopy 122 e) Inter-observer and Intra-observer Variance 122 VI Discussion 124 (1) Global Ischemia 124 (2) Regional Ischemia 130 (3) Myocardial Infarction 136 VII Summary and Conclusions 143 VIII Bibliography 145 IX Appendices 168 v i i i List of Tables Table 1 - Imaging properties of the ideal radiopharmaceutical 5 Table 2 - Imaging properties of 2 0 1 T 1 , 1 2 3IFFA and physical characteristics of an ideal radiopharmaceutical 6 Table 3 - Physical properties of 1 2 3I , i2&I and " i i 30 Table 4 - Washout rate (th in minutes + SD) of 1 2 3IPPA pre-operatively and post-operatively. Myocardial regions examined by time-activity (T-A) curve analysis included the apical wall, septal wall and lateral wall 89 Table 5 - Measured and calculated parameters of cardiac hemodynamic status. Parameters were assessed at 2 time intervals including pre-cardiopulmonary bypass (as a control assessment) and post-CPB (as a post-operative assessment) 92 Table 6 - 123IPPA washout for both early and late phases of the T-A curves over 14 days of developing ischemia. Areas examined were the l a t e r a l , apical and septal walls of the l e f t ventricle 99 Table 7 - Hemodynamic and Metabolic Assessments. Hemodynamic and blood analysis at various time periods during progressive coronary artery occlusion over 14 days 105 ix Table 8 - Perfusion defect size calculated as % of total slice size for each of the three methods - histochemical (TTZ staining) B123IPPA and 2 0 iTi 115 Table 9 - Autoradiographic and scintigraphic assessment of ischemic risk zone calculated as % of total slice size 121 x L i s t of Figures Figure 1 - Schematic from WHO showing causes of mortality in developed countries (1986) 2 Figure 2 (A)- Various sizes of ameroid constrictors available 17 (B)- Ameroid constrictor in place on LAD coronary artery - Heart has been excised following 14 days of gradual coronary occlusion to demonstrate co l lateral c irculat ion 17 Figure 3 - Catabolic pathways of 1 2 3 I P P A and 1 2 3 I H A 34 Figure 4 - Aromatic electrophil ic and nucleophilic substitution reaction 38 Figure 5 - Metabolic trapping scheme 55 Figure 6 (A)- 2 0 1 T h a l l i u m SPECT-scan in dog (anterior projection at 6 hours post-infarction). (B)- B 1 2 3 IPPA SPECT-scan in same dog as above (anterior projection at 6 hours post-infarction) 79 Figure 7 - Heart showing l igat ion and section cutting technique... 81 Figure 8 (A)- Autoradiographic assessment of perfusion in sl ices of myocardium from one experiment with s l ice tracings of myocardial sl ices overlaid onto the autoradiograph showing borders of the sl ices and infarcts . (B)- The clear acetate paper overlaid onto autoradiograph showing demarcation of the infarction and the ischemic r isk zone 83 x i Figure 9 - Time-Activity curves for 123IPPA washout from the myocardium (normalized to 2 min peak counts) (A) pre-operatively and (B) post-operatively, following 2 hrs of global ischemia 87 Figure 10 - Washout rate (th in min) of 123IPPA for the (A) early phase and (B) late phase of the T-A curve 88 Figure 11 - Washout rate (th in min) assessed pre-operatively for the (A) early phase; and (B) late phase of the T-A curves 91 Figure 12 - Electron microscopic evidence of ischemic changes at the ultrastructural level: (A) normal tissue (B) (i) Iso-osmolar (Tyers') solution ( i i ) 10 + SOD ( i i i ) 10 + Allopurinol 94 Figure 13 (A)- Control specimen showing macroscopic anatomy of the myocardium. (B)- Ischemic specimen showing gross changes at the cellular level: (i) Iso-osmolar (Tyers') solution ( i i ) 10 + SOD ( i i i ) 10 + Allopurinol 96 Figure 14 (A)- Regional myocardial washout rate of 1 2 3IPPA (th in min) for the early phase of the T-A curve. (B)- Regional myocardial washout rate of 1 2 3IPPA (th in min) for the late phase of the T-A curve 98 x i i Figure 15 (A)- Control scintigram i n dog injected with 15-p-1 2 3I-iodophenyl pentadecanoic acid (anterior view) with T-A curve and th values (Cl) (B)- Post-operative scintigram i n dog injected with 15-p-1 2 3I-iodophenyl pentadecanoic acid (anterior view) at 14 days with T-A curve and th values (Tl) 101 Figure 16 (A)- Lactate dehydrogenase and creatine kinase levels pre- and post-operatively. (B)- CK-MB% assessed at pre-operative and post-operative time intervals 103 Figure 17 - Lactate assessed at pre- and post-operative time intervals 104 Figure 18 (A)- Microscopic assessment of LAD coronary artery showing normal diameter with no c o n s t r i c t i o n . (B) - Assessment of LAD coronary artery i n one experiment at 14 days of c o n s t r i c t i o n . (C) - Different degree of closure i n another animal i n the study. (D) - Microscopic assessment of LAD coronary artery showing complete closures of vessel from animal which collapsed from myocardial i n f a r c t i o n . (E) - Ameroid constrictors - at 0 days and after 14 days i n s i t u 107 Figure 19 - Electron microscopic assessment of normal myocardial tissu e , (A) Dog 1; (C) Dog 2. Ul t r a s t r u c t u r a l changes of myocardial tissue showing evidence of ischemia, (B) Dog 1; (D) Dog 2 109 x i i i Figure 20 - Light microscopic changes produced by ischemia at 14 days i n two different animals (A) Dog 1, and (B) Dog 2 110 Figure 21 - Venous concentrations of lactate dehydrogenase, creatine kinase and creatine kinase-MB isoenzyme at pre- and post-operative intervals 112 Figure 22 - Staining of a myocardial i n f a r c t using a s t a i n combining nitroblue tetrazolium and triphenyl tetrazolium. The staining of formazans within viable tissue can be seen as dark tiss u e , while the infarcted region remains pale 114 Figure 23 - Relationship between estimates of i n f a r c t size by the histochemical method (TTZ staining) versus (A) B1 2 3IPPA and (B) 2 0 iTi 118 Figure 24 - Graph of B1 2 3IPPA versus 2°iTl showing the relationship between the estimations of perfusion defect size by these two imaging agents 119 Figure 25 (A)- Light microscopic assessment of normal canine myocardium. (B)- Gross c e l l u l a r changes following acute myocardial i n f a r c t i o n 123 x i v L i s t of Appendices Appendix I - Histochemical assessment (TTZ staining) of perfusion defect size using planimetric quantification 167 Appendix I I - Scintigraphic assessment of perfusion defect using B 1 2 3 I P P A and a o iTi 171 Appendix I I I - B1 23ippA/ 2 0 i x i ratios for each experiment and corresponding area-of-interest 180 Appendix IV - Infarcted tissue as percent of the entire s l i c e expressed for each of the three techniques of perfusion defect assessment, including histochemistry (TTZ staining), B1 2 3IPPA and 201TI . 184 Appendix V - Autoradiographic assessment of the ischemic r i s k zone for dogs 8 and 9 using planimetric quantitation 188 xv Abbreviations and Formulae ANOVA - Analysis of Variance gi23ippA. - Beta-methyl-iodophenylpentadecanoic acid BMIPP - Beta-methyl iodinated pentadecanoic acid CAD - Coronary Artery Disease CI - Cardiac Index CK-MB% - The % of Creatine kinase in the form of the MB iso-enzyme J1CPA - 11Carbon-palmitic acid CVD - Cardiovascular Disease Cs - Cesium CPI - "mTc-hexakis (carbomethoxyisopropyl-isonitrile) technetium (I) Cl - Chlorine DMIPP - Dimethyl iodinated pentadecanoic acid dp/dt - First Derivative of Left Ventricular Pressure DMIVN - Dimethyl-125I-18-nonadecanoic acid DCM - Dilated cardiomyopathy ECG - Electrocardiography EF - Ejection Fraction FFA - Free Fatty Acids 7 - Gamma HCM - Hypertrophic cardiomyopathy HR - Heart Rate HDA - Hexadecanoic acid HSA - Human serum albumin IPP - Iodinated phenylpentadecanoic acid 1 2 3,1 3 1I - Iodine 123IPPA - Iodinated phenylpentadecanoic acid IPI - "mTc-hexakis (isopropyl-isonitrile) technetium (I) IHD - Ischemic Heart Disease IHA - Hexadecanoic acid KI - Potassium iodide KeV - Kiloelectron volts xvi LVP - Left Ventricular Systolic Pressure LAD - Left Anterior Descending Coronary Artery LCX - Left Circumflex Coronary Artery MVO2 - Oxygen extraction MUGA - Multiuser gated angiography MIBI - "mTc-hexakis (2-methoxyisobutyl-isonitrile) technetium (I) mCi - Millicuries M.W. - Molecular Weight PCTA - Percutaneous transluminal angioplasty PP - Pyrophosphate Rf - Regional front RP-30 - "mTc-methoxy-isobutyl-isonitrile RMBF - Regional Myocardial Blood Flow SI - Stroke Index SWI - Stroke Work Index SBP - Systolic Blood Pressure SVR - Systemic Vascular Resistance SPECT - Single Photon Emission Computed Tomography Sb - Antimony T-A - Time-Activity TBI - "mTc-hexakis-(t-butyl-isonitrile) technetium (I) "mT c - Technetium 201TI - Thallium th - Half-lives Te - Tellurium TTZ - Tetrazolium TPT - Triphenyltetrazolium WHO - World Health Organization Xe - Xenon x v i i ( i ) Body BSA (m2) = KfwO2/3 Surface 104 Area wt = weight (Constant) K = 11.2 (dog) ( i i ) Cardiac CI = MCO Index BSA (L/min/m2) MCO = Mean Cardiac Output ( i i i ) S t r o k e SI = CI (1000') Index HR (mL/beat/m2) HR = Heart rate (iv) Systemic SVR = (MBP - RAP) Vascular ( MCO ) Resistance 80 (dynes . sec/cm5) MBP = Mean Blood Pressure RAP = Right A t r i a l Pressure (v) Ejection EF = SV Fraction EDV x 100 SV = Stroke volume EDV = End-diastolic volume x v i i i Acknowledgments I thank Dr. D.M. Lyster, Dr. W.R.E. Jamleson and Dr. A.K. Qayumi for their assistance and guidance in the completion of this study. I wish to express sincere thanks to Dr. David V. Godin for his guidance and support during this thesis preparation. I thank the members of my committee for their comments and suggestions. I would like to thank Mr. Tom Wells, Dr. R.R. Miller, Dr. D.A. Owen, Mrs. Maureen Day and Mrs. Eva Chung, in the Department of Anatomic Pathology and Laboratories for their assistance in many aspects of this study. I thank Diane Minshall, Bruce Gregg and Dr. Maria Kiess for their assistance and for the use of their computer f a c i l i t i e s at St. Paul's Hospital. I wish to thank Dr. M. Shulzer and Ms. Eva Germman for their assistance with the s t a t i s t i c a l analysis for the study. I thank Mr. Craig Sartori and Hayes Dougan for their guidance with iodinated free fatty acid preparation. I express my gratitude to Dr. D.A. Jackson for his assistance and time he devoted to the discussion of various aspects of this thesis. I wish to thank Miss Cathy Legg, Mr. Greg Funk, Miss Donna MacKenzie, Mr. Trevor Neufeld and Mr. Darryl Barber for their encouragement and support throughout this thesis preparation. In addition, I would like to thank Mrs. Christine Kirkham for the typing of the thesis. I dedicate this thesis to my family, especially to my father. xix TO MY FATHER AND MOTHER BROTHER AND SISTER "There is no wealth but Life". John Ruskin: Unto this Last, 1862. "You cannot find a medicine for life when once man is dead". Chrysippus: 550 B.C. x x - 1 -INTRODUCTION Myocardial ischemia continues to plague many people i n most developed countries of the world (WHO s t a t i s t i c s , 1986). Cardiovascular disease (CVD) was the primary cause of death i n Canada i n 1986 (Figure 1, WHO s t a t i s t i c s , 1986). I t has been reported that 4.3 b i l l i o n d ollars i s l o s t by the Canadian economy per year as a resul t of cardiovascular disease (B.C. Heart Foundation Fact Letter, 1987). Clearly, CVD i s a major problem that must be addressed. A major c l i n i c a l goal i n CVD i s i t s i d e n t i f i c a t i o n p r i o r to i t s presentation as myocardial i n f a r c t i o n , or i r r e v e r s i b l e myocardial damage. Currently, diagnosis of myocardial ischemia i s based on electrocardiography, elevations of s p e c i f i c serum enzymes, angiography and patient history (angina pectoris) (Braunwald, 1982). However, the diagnosis remains inconclusive with these factors, only becoming conclusive subsequent to myocardial damage. C a l i f f et a l . (1988) have developed an angina score based on the cause of angina, frequency of angina and ECG changes to grade the severity of CAD. The score also incorporates the patient's age, sex, coronary anatomy and l e f t ventricular function as independent varia b l e s . However, ca l c u l a t i o n of the angina score, which can range from 0 to >9, w i l l only i d e n t i f y those patients who may require aggressive therapy and w i l l not i d e n t i f y those patients before angina appears. Early diagnosis may prevent some of the deaths associated with myocardial ischemia. Since the diagnosis of myocardial Ischemia remains inconclusive, development of alternative diagnostic modalities, which w i l l i d e n t i f y myocardial ischemia early i n i t s pathogenesis, are needed. Nuclear medicine may serve t h i s need. However, no radiopharmaceutical exists at present which possesses a l l of the desired characteristics necessary for optimal - 2 -Figure 1 - Schematic from WHO, showing causes of death in developed countries (1986). I.H.D. - Ischemic Heart Disease C.V.D. - Cerebro-Vascular Disease Diseases of the circulatory system A - I.H.D. B - C.V.D. C - Other diseases of C S . Neoplasms Other and ill—defined causes Respiratory diseases - 3 -myocardial imaging. Thallium-201, having been considered the most suitable radiopharmaceutical for c l i n i c a l application, i s u t i l i z e d i n cardiac centres as an accepted non-invasive test to evaluate myocardial perfusion. However, 2 0 1T1 does have some drawbacks, including 7 energy, which f a l l s below the optimal range of commercially available imaging systems, a long physical h a l f - l i f e and a high t o t a l body radiation exposure to the patient (Zaret, 1977). Alternative imaging agents are presently being developed and evaluated for optimum assessment of myocardial ischemia and i n f a r c t i o n . Two such groups of compounds are radiolabeled free f a t t y acids and i s o n i t r i l e s . The use of radioactive tracers to study the cardiovascular system dates back to the 1920's (Blumgart and Weiss, 1927). The use of radioiodinated f a t t y acids as myocardial imaging agents was f i r s t developed by Evans and colleagues i n 1965. Evans u t i l i z e d 1 3 1I - o l e i c acid to demonstrate the p o s s i b i l i t y of photoscanning the hearts of mongrel dogs. The authors chose free f a t t y acids because they are an important energy substate for the myocardium (Bing et a l . . 1956). However, a problem with the 1 3 1I - l a b e l e d f a t t y acids was a 7 energy of 364 KeV f a l l i n g above the optimal range of the imaging systems of that time (decreasing diagnostic quality of the images obtained). Another problem associated with t h i s early work by Evans was the double-bond saturation technique used to iodinate the o l e i c acid. The products of this procedure exhibited a reduced myocardial extraction e f f i c i e n c y and a lower rate of blood clearance, r e l a t i v e to the non-iodinated parent compound (Poe et a l . . 1973). In order to a l l e v i a t e t h i s problem, Poe et a l . (1975) developed a radioiodine exchange-type process, y i e l d i n g a terminally-labeled product which retained the - 4 -c h a r a c t e r i s t i c s o f t h e p a r e n t compound. I n a d d r e s s i n g t h e p r o b l e m o f an i d e a l r a d i o p h a r m a c e u t i c a l s u i t a b l e f o r gamma i m a g i n g Poe e t a l . (1976b) p r o p o s e d t h e use o f i z s j b e cause o f i t s more i d e a l i m a g i n g p r o p e r t i e s ( T a b l e s 1 & 2). The t e c h n i q u e o f u s i n g t h e s e i o d i n a t e d f a t t y a c i d s i n m y o c a r d i a l i m a g i n g ha s b e en e s t a b l i s h e d by Poe and h i s c o l l e a g u e s . The a p p l i c a t i o n o f t h i s t e c h n i q u e t o d i a g n o s e m y o c a r d i a l i s c h e m i a and i n f a r c t i o n , howeve r , r ema i n s an a r e a o f a c t i v e r e s e a r c h . - 5 -Table 1. Imaging Properties of the "IDEAL" Radiopharmaceutical Gamma Energy (KeV) 100-200 Extraction E f f i c i e n c y (%) 100 Physical H a l f - L i f e (min) 30-60 B i o l o g i c a l H a l f - L i f e (min) short Total Body Exposure (mrad/Ci) low - 6 -Table 2. Various physical characteristics of 2 0 1- T h a l l i u m , 1 2 3IFFA and the ideal radiopharmaceutical 2 0 l f l IDEAL 1 2 3I-FFA Gamma Energy (KeV) 80 100-200 159 Extraction E f f i c i e n c y (%) 87 100 78 Physical H a l f - L i f e 72 hrs 30-60 min 13.3 hrs B i o l o g i c a l H a l f - L i f e 7 hrs short 0.5 hrs Total Body Exposure(mrads) 210 low 30 - 7 -I I L I T E R A T U R E REVIEW (1) MYOCARDIAL METABOLISM In view of the c l i n i c a l use of these iodinated fatty acids, a br ie f review of myocardial metabolism is necessary. Cardiac muscle has the a b i l i t y to maintain a variety of energy stores. However, i t has long been appreciated that the myocardium preferential ly u t i l i ze s free fatty acids above a l l others (Bing, 1956). (a) Fatty Acids - The metabolic process of fatty acid oxidation is well-documented and may be divided into various steps including: i) membrane transport; i i ) activation and acyl transfer to carnitine; i i i ) beta-oxidation (Idell-Wenger et a l . . 1978). i ) Free Fatty acids are removed from the blood by the myocardium. While in the blood, the fatty acids may exist in two forms: as free fatty acids bound to albumin, or as triglycerides (Lehninger, 1982). The majority of serum fatty acids are bound to albumin (Idell-Wenger et a l . . 1978 and Goodman, 1958). Following release from albumin, the transport of the FFA through the c e l l membrane occurs v ia passive diffusion (Idell-Wenger et a l . . 1978). - 8 -An equilibrium, however, i s maintained between plasma and c e l l u l a r pools by an energy-dependent process (Opie, 1968). This equilibrium-maintaining extraction process i s controlled by a number of factors, including the albumin:FFA molar r a t i o , metabolic status of the tissue, concentration of the substrate i n plasma, a v a i l a b i l i t y of alternative substrates, mechanical a c t i v i t y of the heart, rate of oxidative r e s p i r a t i o n and plasma levels of certain hormones (Neely, 1972). The other form of the f a t t y acid within the blood i s as a t r i g l y c e r i d e . The former i s composed of a glycerol backbone with three f a t t y acid molecules attached to i t . Triglycerides are the major storage form of f a t t y acids i n c e l l s , but are not normally found i n membranes (Lehninger, 1982). i i ) Following d i f f u s i o n through the c e l l membrane, FFA's are transformed into f a t t y acyl-CoA esters (Lehninger, 1982). This step occurs on the outer mitochondrial membrane and i s enzymatically catalyzed (Idell-Wenger et a l . . 1978) and allows the f a t t y acid to continue through the three-step transport process, i n which f a t t y acid moves from the outer mitochondrial membrane into the mitochondrial matrix. i i i ) There are three known pathways of f a t t y acid oxidation of which only beta-oxidation predominates i n the body (Antony and - 9 -Landau, 1968). Beta-oxidation occurs within the matrix of the mitochondrion. The sequence of beta-oxidation has been known since the early 1900's. Knoop (1904) demonstrated the successive removal of 2-carbon fragments (as acetyl-CoA) from the long chain f a t t y acid. Each successive removal of acetyl CoA results i n the loss of hydrogen atoms from the f a t t y acid (through the action of dehydrogenases), which subsequently enter the electron transport chain also located i n the mitochondria (Lehninger, 1982). The product i s the o r i g i n a l f a t t y acid less 2 carbon atoms, which can then re-enter the beta-oxidation sequence. Fatty acid oxidation i s not l i m i t e d to the mitochondria, having been shown to also occur i n peroxisomes (Christensen et a l . . 1986). These authors used hepatocytes from fasted rats to show that peroxisomal f a t t y acid oxidation plays an important role i n chain-shortening of very long chain f a t t y acids, i e . C2 2 and longer. These findings have been confirmed by Yamada et a l . (1987) who, again using isolated rat hepatocytes, showed that peroxisomal beta-oxidation i s involved i n the chain-shortening of omega-phenyl f a t t y acids. The products of the peroxisomal beta-oxidation sequence, with fewer carbon atoms, were then able to enter the mitochondria and undergo mitochondrial beta-oxidation with subsequent generation of ATP through coupled processes. Several important differences exist between peroxisomal and - 10 -mitochondrial beta-oxidation including individual enzymes, cofactor requirements, coupling to energy production and specificity for fatty acids of different chain lengths (Wanders et a l . . 1987). Much of the work reported on peroxisomal beta-oxidation confirms the presence of these organelles within the liver. However, other studies have also shown their existence in other tissues including the myocardium (Connock and Perry, 1983). Therefore, when examining beta-oxidation of free fatty acids within the myocardium, one must refer to both components, ie. a mitochondrial and a peroxisomal sequence. (b) Glucose - It is known that the myocardium preferentially utilizes fatty acids over glucose as a metabolic fuel (Bing, 1956; Evans et al.. 1962). The glycolytic cycle must be inhibited in some way for this to occur. Randle et al. (1964), Neely et a l . (1969), and Neely (1972) have demonstrated that glucose transport is inhibited by fatty acids or ketone bodies. Opie (1972) has also reported on the inhibition of glycolysis by free fatty acids. (c) Fatty acids and energy production - The end result of fatty acid oxidation is the production of acetyl-CoA units. The latter can then enter the citric acid cycle via a condensation reaction with oxaloacetate to form citrate (Lehninger, 1982). The latter continues on through the citric acid cycle, producing reducing equivalents and C02. The electrons from the reducing - 11 -equivalents are transferred to the electron transport chain within the inner mitochondrial membrane, which transports them to oxygen. The energy liberated from these electrons cascading down th i s chain i s coupled to the synthesis of ATP from ADP and phosphate i n a process c a l l e d oxidative phosphorylation. (2) MYOCARDIAL ISCHEMIA AND INFARCTION Ischemia i s defined as a deficiency of blood due to functional c o n s t r i c t i o n or actual obstruction of a blood vessel, leading to a reduction i n the delivery of oxygen to that p a r t i c u l a r tissue (Dorlands, 1988). Pathological myocardial ischemia e l i c i t s a series of progressive subcellular and c e l l u l a r changes which affect many of the c e l l ' s normal metabolic processes. I t i s a dynamic process, which can have varying e f f e c t s , including: a) c e l l u l a r alterations; b) electrophysiological alterations; c) functional alterations; d) metabolic a l t e r a t i o n s . a) C e l l u l a r Alterations - Ischemia i s a dynamic, heterogeneous process characterized by a continuum of stages sometimes leading to i r r e v e r s i b l e injury (Trump, 1982). Although the effects of ischemia on c e l l homeostasis *T"~f" are many, an increase i n cytosolic Ca i s very important. The l a t t e r - 12 -j j leads to an increase i n Ca -calmodulin complexes, which a l t e r s the c e l l ' s cytoskeleton i n a number of ways, including c e l l shape, organelle orientation and al t e r s p a r t i c l e s within the membranes (Trump, 1982). The increased number of Ca -calmodulin complexes also activates phospholipases, which leads to hydrolysis of membrane phospholipids which, i n turn, w i l l cause c e l l membrane damage and mitochondrial membrane damage (Trump, 1982). The l a t t e r may affect the transport of f a t t y acids and their release. b) Electrophysiological- Alterations - Under normal resting conditions, heart rate may be within a range of 60-80 beats/min (Guyton, 1986) . The P wave i s produced by excitation of a t r i a l muscle, with the right activated p r i o r to the l e f t atrium. The in t e r v a l between the P wave and the QRS complex represents the time taken for the action potential to tra v e l from the SAN ( s i n o a t r i a l node), through the AVN (atr i o v e n t r i c u l a r node) and the Purkinge system. The QRS complex represents the excita t i o n of the vent r i c l e s and the T wave i s the repolarization of the v e n t r i c l e s . Electrophysiological evidence of ischemia i s lacking i n s p e c i f i c i t y , i e . one cannot diagnose ischemia based on ECG evidence alone. I t has been demonstrated on many occasions that S-T segment elevation often precedes coronary artery l i g a t i o n (Opie, 1972; Coraboeuf et a l . . 1976). Sampson et  a l . (1960) have attributed this elevation i n the S-T segment to a flow of current towards the ischemic area, which i s surrounded by normal tis s u e . Therefore, the ECG w i l l be deflected i n the positive d i r e c t i o n during the S-T segment. The exact mechanism of this phenomenon i s s t i l l c ontroversial. Other changes include T-Q depression (Fozzard and Makielski, - 13 -1985), shortening of action potential duration, slowed intramyocardial conduction and dispersion of refractory currents (Corr and Sobel, 1979). The c e l l u l a r events underlying these electrophysiological changes remain o inconclusive, but these are believed to be m u l t i f a c t o r i a l . c) Functional Alterations - The functional consequences of ischemia on the myocardium have been examined extensively. Vatner (1980) demonstrated that myocardial dysfunction i s correlated with a reduction i n blood flow i n the conscious dog. Echocardiographic assessments demonstrated that indices of l e f t ventricular function, including regional wall thickening, ejection f r a c t i o n and end-systolic pressure/area r a t i o decreased (Akaishi et a l . . 1985). These authors also demonstrated a f a l l i n cardiac output, l e f t v e ntricular stroke work and ^ / ^t following severe stenosis of the myocardial vessels. The cause of th i s dysfunction continues to be a controversial issue. Some believe that the f a l l i n tissue ATP levels i s the cause (Braunwald et a l . . 1982; Swain et a l . . 1982), while others believe that dysfunction associated with global ischemia i s due to oxygen free radicals (Myers et a l . . 1986). Becker et a l . . (1986) have demonstrated that the ischemic myocardium possesses s i g n i f i c a n t functional capacity and therefore deficient levels of ATP may not be the ultimate cause of the dysfunction. Currently, many authors believe that oxygen free radicals are very important i n discerning the underlying cause of any vent r i c u l a r dysfunction associated with ischemia. d) Metabolic Alterations - Many studies have been performed examining the effects of ischemia on c e l l u l a r metabolic a c t i v i t y . The - 14 -oxidation of f a t t y acids i s one pathway which i s dramatically affected by an ischemic episode. Early work by Evans (1964) demonstrated that subsequent to an ischemic episode, f a t t y acids are diverted away from beta-oxidation and converted to the storage form of f a t t y acids ( t r i g l y c e r i d e s ) , which are then deposited i n the l i v e r and adipose t i s s u e . The effects on mitochondria and free f a t t y acid oxidation w i l l also be discussed under the radiopharmaceuticals section of t h i s t h e s i s . The metabolic consequences of ischemia result from a f a l l i n oxygen tension. Since the myocardium i s dependent upon aerobic metabolism, a f a l l i n oxygen tension w i l l give r i s e to an increase i n anaerobic g l y c o l y s i s , i n an attempt to generate high energy phosphates. Associated with t h i s s h i f t i n metabolism to the anaerobic state i s an accumulation of end-products of metabolism, such as l a c t a t e , hydrogen ion, purine bases and adenosine, leading to a f a l l i n myocardial pH. In response to a l i m i t e d supply of energy, hormones are released which stimulate cAMP synthesis within plasma membranes of adipocytes (Trump, 1984). This induces a l i p o l y t i c cascade which results i n activation of lipases which hydrolyze t r i g l y c e r i d e s to f a t t y acids and g l y c e r o l . The l a t t e r i s released into plasma for transport to the l i v e r and subsequent use i n the formation of glucose (Lehninger, 1982) . Shug et a l . (1973) reported that free f a t t y acids accumulate i n the mitochondrial f r a c t i o n of myocardial c e l l s subjected to ischemia because of a reduced u t i l i z a t i o n . Concomitant with this increased concentration of i n t r a c e l l u l a r free f a t t y acid i s an accumulation of long-chain acyl-CoA and long-chain acylcarnitine esters (Subramanian et a l . . 1987). These esters have been shown to i n h i b i t a number of enzyme systems including the mitochondrial adenine nucleotide translocator, an enzyme involved i n the - 15 -translocation of ATP and ADP across the inner membrane of the mitochondria (Shug et a l . (1975), Paulson et a l . (1984), Paulson et a l . (1986), Shug et a l . (1987)). Thus, hearts exposed to th i s increased concentration of free f a t t y acids during ischemia may show enhanced deterioration of metabolic function with subsequent lim i t e d energy production. Also affected by ischemia are the processes of l i p i d and protein metabolism (Reimer and Jennings, 1986). Thus, ischemia w i l l a l t e r many aspects of the c e l l ' s normal metabolic machinery, along with f a t t y acid metabolism. e) Experimental Models of Ischemia - There are a large number of available parameters which can be u t i l i z e d to assess myocardial ischemia. In view of the many physiological changes associated with ischemia, the proper choice of a representative system w i l l be dependent upon the purpose of the study. The experimental models used i n th i s study include: i ) Global Ischemia i n s i t u - Global ischemia involves the production of ischemia throughout the heart. Animals are placed on cardiopulmonary bypass, with oxygenator support, caval occlusion and l e f t a t r i a l venting. An aorti c cross-clamp i s positioned e f f e c t i v e l y eliminating coronary flow, producing global ischemia. Cardiac surgical procedures r e l y on th i s technique to obtain a quiescent, bloodless f i e l d i n which the surgeon can work. Many studies have been completed v a l i d a t i n g t h i s model i n the examination of the metabolic and functional consequences of temporary t o t a l ischemia (Conti et a l . . 1984; Rousou et a l . . 1986). This approach can also be used to assess the effectiveness of cardioplegic solutions i n myocardial protection ( C a t i n e l l a et a l . . 1984). - 16 -i i ) Regional Ischemia i n s i t u - Regional ischemia d i f f e r s from the f i r s t i n a number of ways (Schaper, 1979): (a) c o l l a t e r a l c i r c u l a t i o n ; (b) mechanical stress of one portion of myocardium (non-ischemic) compared to the ischemic portion; (c) l i p i d accumulation; (d) c e l l death accompanied by extravasation of blood c e l l s and proteins. The process of coronary artery disease involves the progressive closure of a coronary artery usually by the development of an atherosclerotic plaque. A r e l i a b l e experimental model, therefore, should produce a gradual coronary occlusion over a known time period, so that various ischemia-related parameters can be assessed. One method, developed i n the late 1950's by Litvak and Vineberg, u t i l i z e s a casein p l a s t i c , Ameroid, which has been cured i n formalin to harden i t (Figure 2A). The ameroid constrictor i s composed of the ameroid, which i s sheathed by a metal jacket. There i s a central lumen, which communicates with the outer surface by a narrow arm (Figure 2B). The constrictor can be placed onto the coronary artery by way of th i s arm. The material w i l l slowly engorge with f l u i d and progressively swell and pressure w i l l be directed towards the central lumen. Thus, the coronary artery w i l l be gradually stenosed, mimicking the progression of coronary artery disease. Vineberg and Mahonti (1960) demonstrated that this technique i s an excellent model of gradual coronary occlusion with death of the animals occurring when 50% or more of the lumen of the two l e f t coronary arteries was blocked. - 17 -Figure 2 (A) - Various sizes of ameroid constrictors available. (B) - Ameroid constrictor in place on LAD coronary artery - Heart has been excised following 14 days of gradual coronary occlusion to demonstrate collateral circulation. - 18 -i i i ) Myocardial Infarction i n s i t u - Myocardial i n f a r c t i o n i s an inevitable outcome of untreated coronary artery disease. Gradually, the le s i o n from CAD w i l l grow inwards and result i n complete blockage of that artery. The i n f a r c t i o n i n this study was produced following a thoracotomy and dissection of the coronary artery. U t i l i z i n g a dexon suture, the coronary artery was l i g a t e d , producing an acute loss of blood flow to the regional myocardial bed supplied by that artery. Myocardial i n f a r c t i o n ( i e . a heart attack) may vary i n i t s outcome. With small i n f a r c t s , the remaining viable myocardium can continue to function, such that hemodynamic status i s r e l a t i v e l y well maintained. However, i n many cases i t i s f a t a l (See Figure 1, p.2). RADIOPHARMACEUTICALS IN THE CLINICAL DIAGNOSIS OF MYOCARDIAL  INFARCTION AND ISCHEMIA. "m (a) Technetium ( Tc) "m (i ) Tc-pyrophosphate One approach to imaging myocardial i n f a r c t i o n i s the use of 9 9m i n f a r c t - a v i d agents, such as Tc pyrophosphate. "m " n L , "m -Technetium ( T c ) , as Tc stannous pyrophosphate, was f i r s t used to image acute infarcted human myocardium by Parkey and "m his coworkers i n 1974. Of 23 patients, thirteen had positive Tc scans, following planar gamma imaging - eleven of these patients were scanned 3-5 days following i n f a r c t i o n and two, 7-10 days after "m i n f a r c t i o n . Localization of i n f a r c t i o n by Tc scan and ECG correlated w e l l . These authors believed that pyrophosphate was - 19 -i n c o r p o r a t e d i n t o t h e h y d r o x y a p a t i t e c r y s t a l l i n e s t r u c t u r e f o u n d i n t h e m i t o c h o n d r i a o f i r r e v e r s i b l y d a m a g e d m y o c a r d i u m . S i n c e t h i s s t u d y , a n umber o f i n v e s t i g a t o r s h a v e a t t e m p t e d t o s i z e m y o c a r d i a l i n f a r c t i o n s u n d e r e x p e r i m e n t a l c o n d i t i o n s u s i n g 9 9 " T c PP a n d S PECT. K e y e s e t a l . ( 1 9 7 8 ) w e r e t h e f i r s t t o a t t e m p t t h i s 3 - d i m e n s i o n a l r e p r e s e n t a t i o n o f t h e m y o c a r d i u m u s i n g 15 m C i o f 9 9m T c p y r o p h o s p h a t e . I n f a r c t i o n s w e r e p r o d u c e d b y l i g a t i o n o f t h e LAD a n d L C X a r t e r i e s a n d i m a g i n g p e r f o r m e d 6 0 - 9 0 m i n u t e s l a t e r . T h e m a i n p r o b l e m s r e p o r t e d b y K e y e s a n d h i s c o - w o r k e r s r e l a t e d t o a t t e n u a t i o n a n d i m a g e r e s o l u t i o n . T h e c o r r e l a t i o n b e t w e e n t h e h i s t o c h e m i c a l i n f a r c t s i z e ( t e t r a z o l i u m s t a i n i n g ) a n d S P E C T i n f a r c t s i z e , b o t h m e a s u r e d i n g r a m s , was g o o d ( r = 0 . 8 5 ) , e v e n t h o u g h t h e l a t t e r p r o b l e m s d i d e x i s t . I n a s i m i l a r s t u d y , C a l d w e l l e t a l . ( 1 9 8 4 ) r e p o r t e d a h i g h d e g r e e o f c o r r e l a t i o n ( r = 0 . 9 2 ) b e t w e e n i n v i v o d e f e c t v o l u m e b y S P E C T a n d i n v i t r o d e f e c t v o l u m e , b y w e l l - c o u n t i n g o f m y o c a r d i a l s a m p l e s . T h e a u t h o r s d e f i n e d d e f e c t v o l u m e a s t h a t v o l u m e o f l e f t v e n t r i c l e w h i c h f e l l b e l o w 2 o r m ore s t a n d a r d d e v i a t i o n s o f c o u n t s f r o m n o r m a l m y o c a r d i u m . I f 2 0 1 T 1 was u s e d w i t h t h i s t e c h n i q u e , t h e c o r r e l a t i o n b e t w e e n t h e s e two a s s e s s m e n t s o f d e f e c t v o l u m e was r = 0 . 7 0 . A l t h o u g h c o r r e l a t i o n s w e r e d eemed a p p r o p r i a t e , m o r e a c c u r a t e b o r d e r s w e r e n e e d e d i n o r d e r t o d i f f e r e n t i a t e i n f a r c t e d t i s s u e f r o m i s c h e m i c t i s s u e . K a u l e t a l . ( 1 9 8 5 ) a d d r e s s e d t h i s i s s u e , u t i l i z i n g 9 9 m T c - l a b e l e d m i c r o s p h e r e s , S P E C T a n d m a c r o a u t o r a d i o g r a p h y . T h e s e a u t h o r s c o n c l u d e d t h a t a s s e s s m e n t s o f p e r f u s i o n d e f e c t s , i n c l u d i n g t h e a r e a a t r i s k , i s d i f f i c u l t t o d e t e r m i n e i n t h e a p i c a l r e g i o n . T h e a v e r a g e c o r r e l a t i o n o n a s l i c e - b y - s l i c e c o m p a r i s o n was r = 0.88 w i t h - 20 -the basal slices showing the highest degree of correlation and the apical slices showing the worst. It must be noted that these studies were performed in excised hearts. With the evident success of this infarct-avid agent in the experimental setting, Holman et a l . (1982) took i t to the next level -the c l i n i c a l setting. In 20 patients with acute myocardial infarction, 9 9 ™ 9 9 ™ Tc pyrophosphate and xc-labeled RBC's were used to quantitate infarct size, both by planar imaging and SPECT. Infarct size assessments were then correlated with patient prognosis. Results showed that 85% of patients with infarcts of >40g had complications during the 17.8 month follow-up period. Of the patients with infarcts of <40g, only 29% had complications. This study also indicated that two-dimensional imaging cannot be used to assess the size of perfusion defects because of problems with edge detection and boundary overlap. Anatomical location of the infarction also limits the usefulness of planar imaging with only anterior infarcts being seen. SPECT imaging proved to be more accurate with better delineation of borders and better able to detect both interior and anterior infarcts. In a similar study, Corbett et a l . (1984) assessed infarct size via planar imaging and SPECT with and without blood pool overlay to determine i t s sensitivity. Blood pool overlay refers to the activity within the chambers of the heart. It was found that SPECT with blood pool overlay was significantly better than SPECT or planar imaging, alone, at determining myocardial infarct site and size, especially in the sensitivity for detection of non-transmural infarcts. A number of other studies have validated the use of SPECT as a tool for infarct - 21 -q u a n t i f i c a t i o n - J a n s e n e t a l . ( 1 9 8 5 ) ^Ic-PP, - N o h a r a e t a l . ( 1 9 8 4 ) - 2 0 1 T 1 a n d S PECT. S o c h e r e t a l . ( 1 9 8 7 ) h a v e d e m o n s t r a t e d a n e t r e t e n t i o n o f 9 9m T c - P P a p p r o a c h i n g z e r o w i t h i n n o r m a l m y o c a r d i u m v e r s u s >30% i n " m r e p e r f u s e d m y o c a r d i u m . I t a p p e a r e d t h a t t h e e x t e n t o f T c u p t a k e was d e p e n d e n t u p o n t h e d e g r e e o f i r r e v e r s i b i l i t y o f m y o c a r d i a l damage. T h e s e a u t h o r s f u r t h e r s u g g e s t e d a n d d e m o n s t r a t e d a m o re a c c u r a t e a s s e s s m e n t o f m y o c a r d i a l t i s s u e damage u s i n g a d u a l t r a c e r t e c h n i q u e . A l t h o u g h a p p e a r i n g t o b e t h e o p t i m a l a g e n t f o r a s s e s s m e n t o f p e r f u s i o n i n c l i n i c a l n u c l e a r m e d i c i n e , b a s e d o n i t s r a d i o n u c l i d i c p r o p e r t i e s , 9 9™ T c h a s y e t t o r e p l a c e 2 0 1 T 1 i n t h e a s s e s s m e n t o f c a r d i a c 9 9m i s c h e m i a . T c - P P i s a l s o a n o n - p h y s i o l o g i c a l a g e n t , t h e r e b y l i m i t i n g i t s p o t e n t i a l . " m ( i i ) T c - I s o n i t r i l e s " m T h e m o r e o p t i m a l gamma e n e r g y ( 1 4 0 K e V ) o f T c , i t s h a l f - l i f e o f 6 h o u r s a n d i t s e a s e o f g e n e r a t i o n h a s l e d t o t h e s e a r c h f o r a n i m a g i n g a g e n t w h i c h i s t a k e n u p b y n o r m a l m y o c a r d i u m . One 9 9m p o s s i b l e T c - b a s e d m y o c a r d i a l i m a g i n g a g e n t , w h i c h h a s r e c e i v e d a g r e a t d e a l o f a t t e n t i o n r e c e n t l y , i s t h e l a b e l e d i s o n i t r i l e . I n 1 9 8 1 , 99™ D e u t s c h a n d c o w o r k e r s s c r e e n e d a l a r g e n u m b e r o f c a t i o n i c T c c o m p l e x e s f o r m y o c a r d i a l u p t a k e i n d o g s a n d r a t s . N i n e t e e n c a t i o n i c c o m p l e x e s i n v e s t i g a t e d w e r e d i v i d e d i n t o 5 g r o u p s , i n c l u d i n g t h e d i m e t h y l a r s i n e s , d i p h e n y l a r s i n o - e t h a n e s , d i p h e n y l p h o s p h i n o -e t h y l p h o s p h i n e s , m o n o t h i o g l y c e r o l s a n d d i p h e n y l p h o s p h i n o e t h y l a m i n e s . O f t h e s e g r o u p s , o n l y o n e , t h e d i m e t h y l a r s i n e s , e x h i b i t e d d e t e c t a b l e - 22 -m y o c a r d i a l u p t a k e u p o n i m a g i n g . E v e n among t h e 4 h a l o g e n s p e c i e s w i t h i n t h e g r o u p , v a r i a b i l i t y e x i s t e d i n t e r m s o f i m a g e q u a l i t y a n d b i o l o g i c a l d i s t r i b u t i o n . T h e b r o m o a n d f l u o r o c o m p l e x e s y i e l d e d t h e b e s t i m a g e s . H o w e v e r , t h e d a n g e r a n d d i f f i c u l t y o f w o r k i n g w i t h h y d r o f l u o r i c a c i d f a v o r s f u r t h e r i n v e s t i g a t i o n o f t h e f o r m e r c o m p o u n d s . D i s t r i b u t i o n s t u d i e s r e v e a l e d a n a v e r a g e h e a r t u p t a k e o f 0.022 % d o s e / g f o r d i m e t h y l a r s i n e c o m p a r e d w i t h 0 .038% d o s e / g f o r 2 0 1 T 1 . A l t h o u g h t h e u p t a k e was l o w e r , m y o c a r d i a l i m a g e s w e r e o f g o o d q u a l i t y w i t h d i m e t h y l a r s i n e . I n l a t e r s t u d i e s , t h e c h l o r o d e r i v a t i v e a p p e a r e d t o b e t h e b e s t o f t h e h a l o g e n s ( K e t r i n g e t a l . . 1 9 8 3 ) . J o n e s e t a l . ( 1 9 8 4 ) s t a t e t h a t n o n e o f t h e s e a g e n t s , t h u s f a r , h a v e s h o w n a n y s u c c e s s i n i m a g i n g human m y o c a r d i u m , a l t h o u g h a n i m a l s t u d i e s h a v e s hown t r e m e n d o u s p r o m i s e . T h e r e f o r e , t h e s e a u t h o r s 9 9 m „ d e v e l o p e d a n a l t e r n a t i v e c l a s s o f x c c o m p l e x e s - h e x a k i s ( a l k y l i s o n i t r i l e ) t e c h n e t i u m ( I ) c a t i o n s t h e d i f f e r e n c e b e i n g t h a t i n t h e l a t t e r , t h e i s o n i t r i l e l i g a n d i s m o n o d e n t a t e , w h i l e i n t h e w o r k o f D e u t s c h a n d c o l l e a g u e s i t was a b i d e n t a t e l i g a n d . B i o d i s t r i b u t i o n s t u d i e s i n m i c e r e v e a l e d m a r k e d l i v e r a n d s k e l e t a l m u s c l e u p t a k e a n d some m y o c a r d i a l u p t a k e ( 1 . 2 2 + 0 . 1 2 % o f t h e i n j e c t e d d o s e ) a t 5 m i n s . p o s t - i n j e c t i o n . T h e l i p o p h i l i c i t y was a l s o s h o wn t o a f f e c t u p t a k e w i t h i n c r e a s e d l i p o p h i l i c i t y b e i n g a s s o c i a t e d w i t h i n c r e a s e d u p t a k e . A l t h o u g h n o r m a l m y o c a r d i u m was b e i n g i m a g e d , t h e m e c h a n i s m o f t h e u p t a k e o f t h e s e a g e n t s was s t i l l u n c l e a r a n d t h e r e w e r e n o s t u d i e s i n v o l v i n g human m y o c a r d i u m . 9 9 H o l m a n e t a l . ( 1 9 8 4 ) u t i l i z e d " V c h e x a k i s ( t - b u t y l i s o n i t r i l e ) t e c h n e t i u m ( I ) ( T B I ) f o r i m a g i n g 4 n o r m a l p e r s o n s a n d 2 - 23 -p a t i e n t s w i t h CAD. T h e s t u d y d e m o n s t r a t e d v e r y g o o d q u a l i t y i m a g e s o b t a i n e d b y b o t h p l a n a r a n d t o m o g r a p h i c s c a n n i n g . T h i s a g e n t , h o w e v e r , may n o t b e s u i t a b l e f o r c l i n i c a l u s e , b e c a u s e o f v e r y h i g h u p t a k e i n t h e l u n g , w h i c h d e c r e a s e s t h e q u a l i t y o f t h e i m a g e i n t h e f i r s t h o u r p o s t - i n j e c t i o n ( G e r u n d i n i , 1 9 8 6 ) . A t t i m e s > 1 h r , l i v e r a n d s p l e e n u p t a k e o b s c u r e s t h e a p i c a l w a l l o f t h e i m a g e . A n o t h e r p r o b l e m was t h e l o w r a d i o c h e m i c a l y i e l d ( 3 0 - 6 0 % ) a s r e p o r t e d b y H o l m a n e t a l . ( 1 9 8 4 ) . I n o r d e r t o b e t t e r u n d e r s t a n d t h e u p t a k e m e c h a n i s m o f t h e s e a g e n t s , S a n d s e t a l . ( 1 9 8 6 ) e x a m i n e d t h e k i n e t i c s o f T B I a n d x c - h e x a k i s ( i s o p r o p y l i s o n i t r i l e ) t e c h n e t i u m ( I ) ( I P I ) , u s i n g n e o n a t a l r a t m y o c y t e s a n d human e r y t h r o c y t e s . I t was f o u n d t h a t t h e u p t a k e was u n a f f e c t e d b y o u a b a i n o r K C l , s o t h a t i t was p r o b a b l y u n r e l a t e d t o t h e N a + / K + A T P a s e s y s t e m . S a n d s e t a l . ( 1 9 8 6 ) s u g g e s t e d t h a t l i p o p h i l i c i t y was i n v o l v e d i n m y o c a r d i a l u p t a k e j u s t a s J o n e s e t a l . ( 1 9 8 4 ) h a d s h own p r e v i o u s l y . M a u b l a n t e t a l . ( 1 9 8 8 ) h a v e d e m o n s t r a t e d t h a t i n c u l t u r e s o f m y o c a r d i a l c e l l s o f n e w b o r n r a t s 9 9m T c - h e x a k i s - 2 - m e t h o x y i s o b u t y l i s o n i t r i l e -T e c h n e t i u m ( x c - M I B I ) shows s l o w e r u p t a k e a n d i s r e l a t i v e l y i n s e n s i t i v e t o m e t a b o l i c i n h i b i t o r s , s u c h a s c y a n i d e , i o d o a c e t a t e a n d o u a b a i n . 99™ ( u?c)MIBI u p t a k e was a l s o f o u n d t o b e i n s e n s i t i v e t o c h a n g e s i n pH. P i w n i c a - W o r m s e t a l . ( 1 9 8 8 ) h a v e f o u n d t h a t h e x a k i s 9 9 „ ( c a r b o m e t h o x y - i s o p r o p y l i s o n i t r i l e ) t e c h n e t i u m ( I ) ( T : c - C P I ) u p t a k e i s a l s o i n s e n s i t i v e t o s e v e r a l m e t a b o l i c i n h i b i t o r s , b u t was 9 9™ t e m p e r a t u r e - s e n s i t i v e . C l e a r l y , t h i s a g e n t , l i k e T c p y r o p h o s p h a t e , i s a n o n - p h y s i o l o g i c a l t r a c e r a n d t h u s w o u l d b e u s e f u l - 24 -a s a t r a c e r f o r t i s s u e p e r f u s i o n s t u d i e s b u t w o u l d y i e l d n o m e t a b o l i c i n f o r m a t i o n . I t was a l s o f o u n d t h a t e x t r a c e l l u l a r a l k a l i n i z a t i o n ( p H -9 9m 8.5) p a r t i a l l y i n h i b i t e d T ' c - C P I u p t a k e , w h i l e i n t r a c e l l u l a r pH c h a n g e s h a d n o e f f e c t . A l t h o u g h t h e u p t a k e m e c h a n i s m i s s t i l l n o t c l e a r , d a t a a r e s t i l l b e c o m i n g a v a i l a b l e w h i c h w i l l a n s w e r some o f t h e q u e s t i o n s w h i c h r e m a i n ( Z a n e l l i e t a l . . 1 9 8 8 ) . A n o t h e r i s o n i t r i l e w h i c h h a s s h o w n e x c e l l e n t m y o c a r d i a l u p t a k e a n d i m a g i n g p o t e n t i a l i s T ^ c - m e t h o x y - i s o b u t y l i s o n i t r i l e , o r RP-30 ( M a d d a h i e t a l . . 1 9 8 6 ) . B a c k g r o u n d - t o - t a r g e t r a t i o s w e r e shown t o b e much i m p r o v e d f o r RP-30 o v e r T B I . N o r m a l e n d o c a r d i a l a n d e p i c a r d i a l m o t i o n a s w e l l a s n o r m a l m y o c a r d i a l w a l l t h i c k e n i n g w e r e s e e n w i t h g a t e d p l a n a r i m a g i n g a n d R P -30. I n a r e c e n t c l i n i c a l s t u d y , T a t u m e t a l . ( 1 9 8 8 ) u t i l i z e d RP-30 a n d 2 0 ix l t o i d e n t i f y t h e p e r f u s i o n d e f e c t i n a p a t i e n t s u f f e r i n g f r o m a c u t e m y o c a r d i a l i n f a r c t i o n . N i n e d a y s p o s t - i n f a r c t i o n , t h e p a t i e n t u n d e r w e n t a s t r e s s t e s t u s i n g 2 0 1 T 1 , d e m o n s t r a t i n g a n a r e a o f d e c r e a s e d a c t i v i t y , b u t no e v i d e n c e o f i s c h e m i a . T h e f o l l o w i n g d a y , t h e p a t i e n t u n d e r w e n t a s t r e s s t e s t u s i n g RP-30. T h e r e s u l t s i n d i c a t e d a s m a l l m y o c a r d i a l i n f a r c t , s e e n b o t h o n t h e r e s t i n g a n d p o s t - e x e r c i s e s c a n s . A d e f e c t , h o w e v e r , was s e e n o n l y o n t h e p o s t - e x e r c i s e s c a n r e p r e s e n t i n g a n a r e a o f i s c h e m i a . T h e r e f o r e , t h e i s o n i t r i l e s , u n l i k e 2 0 1 T l , may p r o v i d e a means o f i d e n t i f y i n g i s c h e m i c a r e a s a r o u n d t h e b o r d e r s o f a m y o c a r d i a l i n f a r c t i o n . - 25 -( b ) T h a l l i u m - 201 ( i ) H i s t o r i c a l P e r s p e c t i v e s - T h e u t i l i z a t i o n o f r a d i o i s o t o p i c s t u d i e s o f t h e c a r d i o v a s c u l a r s y s t e m d a t e s b a c k t o t h e 1 9 2 0 ' s . B l u m g a r t a n d W e i s s , i n 1 9 2 7 , d e s c r i b e d t h e u s e o f r a d i u m C i n d e t e r m i n i n g t h e v e l o c i t y o f b l o o d f l o w t h r o u g h t h e p u l m o n a r y c i r c u l a t i o n i n man. W i t h t h e a d v e n t o f r a d i o i s o t o p e s , many saw t h e p o s s i b i l i t y o f u t i l i z i n g t h e s e a g e n t s t o t r a c e p a t h w a y s a n d l o c a l i z e v a r i o u s p h y s i o l o g i c a l e l e m e n t s , s u c h a s 4 5 K ( G r e e n b e r g e t a l . . 1 9 3 8 ) a n d 4 2 K ( J o s e p h e t a l . . 1 9 3 9 ) . T h e s e a u t h o r s u t i l i z e d t h e s e a g e n t s t o s t u d y t h e p r e d o m i n a n t p a t h w a y s o f a b s o r p t i o n a n d b i o d i s t r i b u t i o n i n v a r i o u s o r g a n s y s t e m s . L o v e e t a l . ( 1 9 5 4 ) r e p o r t e d t h a t t h e u s e o f 4 2 K may b e u n s u i t a b l e f o r s t u d y i n g m y o c a r d i a l c i r c u l a t i o n , b e c a u s e o f t h e n e e d f o r l a r g e a m o u n t s o f i n j e c t e d c a r r i e r K. I n v i e w o f t h i s , C a r r e t a l . ( 1 9 6 2 ) e x a m i n e d t h e u t i l i z a t i o n o f 8 6 R b a s a n a g e n t w h i c h w o u l d show a r e d u c e d u p t a k e i n a r e a s o f m y o c a r d i a l i n f a r c t i o n . T h u s , t h e f e a s i b i l i t y a n d p o s s i b i l i t y o f u s i n g r a d i o i s o t o p i c s t u d i e s o f t h e m y o c a r d i u m was e s t a b l i s h e d . A t t e n t i o n now t u r n e d t o t h e d e v e l o p m e n t a n d r e f i n e m e n t o f m ore s u i t a b l e t r a c e r s . I t was n o t u n t i l 1 9 7 0 , w i t h t h e r e a l i z a t i o n t h a t a v a i l a b l e p o t a s s i u m i s o t o p i c a n a l o g s h a d e m i s s i o n s w h i c h w e r e t o o h i g h , d i d K a w a n a e t a l . ( 1 9 7 0 ) s u g g e s t t h e u s e o f t h e t h a l l o u s i o n , T l + , f o r r a d i o n u c l i d e i m a g i n g . L e b o w i t z e t a l . ( 1 9 7 4 ) d e m o n s t r a t e d t h a t 2 0 1 T 1 may b e a u s e f u l a g e n t i n m y o c a r d i a l s c a n n i n g . S t u d i e s u s i n g g o a t s d e m o n s t r a t e d h i g h c o n c e n t r a t i o n s o f t h e t r a c e r w i t h i n t h e k i d n e y , l i v e r a n d h e a r t . I n many o t h e r s p e c i e s , i n c l u d i n g r a b b i t s , d o g s a n d m i c e , no - 26 -a d v e r s e r e a c t i o n s t o t h e t r a c e r w e r e f o u n d . I t s p h y s i c a l c h a r a c t e r i s t i c s a n d g o o d r e s o l u t i o n w i t h s t a n d a r d gamma i m a g i n g s y s t e m s made 2 0 1 T 1 a n a c c e p t a b l e means o f n o n - i n v a s i v e t e s t i n g o f p a t i e n t s s u s p e c t e d o f h a v i n g IHD ( B o t v i n i c k e t a l . . 1 9 8 0 ) . i i ) R a d i o n u c l i d i c P r o p e r t i e s - M u c h o f t h e w o r k o n t h e c h e m i s t r y o f t h a l l i u m l i e s i n t h e f i e l d o f o r g a n o m e t a l l i c c h e m i s t r y . T h a l l i u m i s a m e t a l l i c e l e m e n t , f o u n d i n g r o u p I I I A o f t h e p e r i o d i c t a b l e , w i t h a n a t o m i c n u m b e r o f 8 1 . I t h a s a M.W. o f 20 4 . 3 9 a n d h a s two s t a b l e v a l e n c e s +1 a n d +3 (Kemp, 1 9 8 0 ) . I t i s s i m i l a r t o p o t a s s i u m a n d c a n s u b s t i t u t e f o r K + i n t h e a c t i v a t i o n o f p y r u v a t e k i n a s e ( S t r a u s s e t a l . . 1 9 7 5 ) . T w e n t y i s o t o p i c f o r m s o f T l e x i s t , w i t h a t o m i c m a s s e s r a n g i n g f r o m 191 t o 2 1 0 . i i i ) C l i n i c a l A p p l i c a t i o n a n d E x p e r i m e n t a l E v a l u a t i o n - 2 0 1 T l g ^ , a s a r a d i o i s o t o p e , h a s a h a l f - l i f e o f 73 h r s a n d d e c a y s b y o r b i t a l e l e c t r o n c a p t u r e a n d i s u t i l i z e d a s a m y o c a r d i a l i m a g i n g a g e n t ( L e b o w i t z e t a l . . 1 9 7 5 ) . A l t h o u g h u t i l i z e d i n many c a r d i a c c e n t r e s a s t h e s t a n d a r d m y o c a r d i a l p e r f u s i o n i m a g i n g a g e n t , o v e r t h e p a s t 1 0-15 y e a r s o f c l i n i c a l u s e , 2 0 1 T 1 h a s shown some d o w n f a l l s . Z a r e t ( 1 9 7 7 ) d i s c u s s e d t h e p r o b l e m o f a th = 72 h r s , w h i c h p r e c l u d e s a n y s e q u e n t i a l i m a g i n g ( M a u b l a n t e t a l . . 1 9 8 1 ) , a n d a l s o i t s e n e r g y o f 89 K e V was n o t o p t i m a l f o r c o m m e r c i a l l y a v a i l a b l e gamma i m a g i n g s y s t e m s . A t t e n u a t i o n was a l s o f o u n d t o b e o f c o n s i d e r a b l e i m p o r t a n c e . K e y e s e t a l . ( 1 9 7 8 ) a n d B u r d i n e ( 1 9 7 9 ) b o t h r e p o r t o n p o o r r e s o l u t i o n o f m y o c a r d i a l p l a n a r i m a g e s , d u e t o a t t e n u a t i o n b y o t h e r t i s s u e s o f t h e t h o r a c o - a b d o m i n a l - 27 -c a v i t y . A n o t h e r a r e a o f c o n c e r n was t h a t 2 0 1 T 1 i s a n o n - p h y s i o l o g i c a l i m a g i n g a g e n t t h u s w o u l d y i e l d l i t t l e i n f o r m a t i o n o n t h e m e t a b o l i c s t a t u s o f t h e t i s s u e ( G e w i r t z , 1 9 7 8 ) . I n v i e w o f t h e f a c t t h a t w a s h o u t r a t e s o f 2 0 1 T 1 w i t h i n n o r m a l a n d i s c h e m i c m y o c a r d i u m w e r e u n a f f e c t e d b y r e g i o n a l m y o c a r d i a l b l o o d f l o w a n d b o t h a r e a s e x h i b i t e d s i m i l a r w a s h o u t r a t e s , 2 0 1 T 1 was n o t a b l e t o y i e l d a n y i n f o r m a t i o n w h i c h w o u l d d i s t i n g u i s h b e t w e e n t h e s e two a r e a s ( G e w i r t z , 1978 a n d L e p p o e t a l . . 1 9 8 0 ) . A n o t h e r d i s a d v a n t a g e o f 2 0 l T l was i t s l a c k o f s p e c i f i c i t y ( W a c k e r s e t a l . . 1 9 8 0 ) . T h e u s e o f 2 0 1 T 1 d i d n o t d i s t i n g u i s h b e t w e e n r e c e n t a n d o l d m y o c a r d i a l i n f a r c t i o n o r i s c h e m i c a n d n e c r o t i z e d t i s s u e . T h e u t i l i z a t i o n o f 2 0 1 T 1 t o d e t e r m i n e t h e s i z e o f m y o c a r d i a l i n f a r c t i o n c o n t i n u e s t o b e i n v e s t i g a t e d . A m a j o r p r o b l e m a s s o c i a t e d w i t h t h i s t e c h n i q u e i s r e d i s t r i b u t i o n ( B e l l e r e t a l . . 1 9 8 0 ) . R e d i s t r i b u t i o n was f i r s t r e p o r t e d b y P o h o s t e t a l . ( 1 9 7 7 ) . T h e s e a u t h o r s d e m o n s t r a t e d r e d i s t r i b u t i o n o f 2 0 1 T 1 i n t o i s c h e m i c m y o c a r d i u m d u r i n g t r a n s i e n t c o r o n a r y o c c l u s i o n i n d o g s a n d f o l l o w i n g e x e r c i s e - s t r e s s t e s t s i n man. B e l l e r e t a l . ( 1 9 8 0 ) , i n c l i n i c a l s t u d i e s , d e m o n s t r a t e d t h a t d e f e c t a r e a s ( c o l d s p o t s ) o n i n i t i a l s c a n s r e s o l v e d o v e r t i m e . T h u s , a n a p p a r e n t i n f a r c t may r e p r e s e n t a n a r e a o f i s c h e m i a , w h i c h r e s o l v e d i t s e l f o v e r t i m e , a s t h e r e s u l t o f i n c r e a s e d c o l l a t e r a l c i r c u l a t i o n a n d d e c r e a s e d m y o c a r d i a l o x y g e n demand. M a s s i e e t a l . ( 1 9 8 4 ) h a v e u t i l i z e d t h i s o b s e r v a t i o n t o i d e n t i f y a r e a s o f a c u t e m y o c a r d i a l i n f a r c t i o n , b y r e p e a t i n g t h e 2 0 1 T 1 s t u d y a f t e r 3 t o 4 h o u r s . T h e p r o b l e m , h o w e v e r , i s a s s e s s i n g t h e s i g n i f i c a n c e o f t h i s r e d i s t r i b u t i o n p h e n o m e n o n . T a t u m e t a l . ( 1 9 8 8 ) d e s c r i b e a c a s e - s t u d y - 28 -i n w h i c h t h e i n f o r m a t i o n o b t a i n e d f o l l o w i n g a 2 0 1 T 1 r e d i s t r i b u t i o n s t u d y was i n a c c u r a t e , d e f i n i n g a l e s i o n much l a r g e r t h a n t h a t d e f i n e d 99m b y ( T c ) i s o n i t r i l e . T a t u m e t a l . ( 1 9 8 8 ) a l s o r e p o r t t h a t b o t h i n v i v o a n d i n v i t r o s t u d i e s h a v e d e m o n s t r a t e d a n a c c e l e r a t e d e f f l u x o f 2 0 1 T 1 f r o m t h e i n f a r c t e d m y o c a r d i u m . C l e a r l y , p r o b l e m s a s s o c i a t e d w i t h 2 0 1 T 1 c a l l e d f o r f u r t h e r r e s e a r c h i n t o t h e d e v e l o p m e n t o f a m o r e i d e a l r a d i o p h a r m a c e u t i c a l ( T a b l e 1 ) . One o f t h e m o re p r o m i s i n g s u b s t a n c e s w h i c h p o s s e s s m o r e i d e a l c h a r a c t e r i s t i c s f o r o p t i m a l m y o c a r d i a l i m a g i n g a r e i o d i n a t e d f r e e f a t t y a c i d s . T h e u s e o f i o d i n a t e d f r e e f a t t y a c i d s , h o w e v e r , r e m a i n s i n i t s e a r l y s t a g e s o f c l i n i c a l a n d e x p e r i m e n t a l a s s e s s m e n t . ( c ) I o d i n e - 1 2 3 , 131 ( i ) H i s t o r i c a l P e r s p e c t i v e s o f I o d i n a t e d F a t t y A c i d s - T h e p r e f e r e n t i a l u t i l i z a t i o n u n d e r a e r o b i c c o n d i t i o n s o f f r e e f a t t y a c i d s b y t h e h e a r t h a s b e e n a p p r e c i a t e d s i n c e t h e 1 9 5 0 ' s ( B i n g , 1 9 5 6 ) . I n o r d e r t o a s s e s s t h e i m p o r t a n c e o f f a t t y a c i d s a s a n e n e r g y p o o l , E v a n s e t a l . ( 1 9 6 3 ) e x a m i n e d t h e e x t r a c t i o n a n d f a t e o f 1 4 C - l a b e l e d p a l m i t i c a c i d i n a n i s o l a t e d r a t h e a r t m o d e l . T h e i n a b i l i t y o f 1 4 C - l a b e l e d p a l m i t i c a c i d t o a c c u r a t e l y a s s e s s b o t h t h e m y o c a r d i a l e x t r a c t i o n a n d t r i g l y c e r i d e s t o r a g e i n v o l v e d i n f a t t y a c i d m e t a b o l i s m r e s u l t e d i n a t t e m p t s b y E v a n s a n d c o - w o r k e r s t o u t i l i z e 1 3 1 I , a g a m m a - e m i t t i n g i s o t o p e , l a b e l e d t o t h e f a t t y a c i d ( E v a n s e t a l . . 1964 a , b ) . T h i s r a d i o i s o t o p e a l l o w e d b o t h m y o c a r d i a l e x t r a c t i o n a n d i n c o r p o r a t i o n o f f a t t y a c i d s i n t o t h e t r i g l y c e r i d e f o r m t o b e - 29 -a s s e s s e d . E v a n s a n d h i s c o - a u t h o r s r e c o g n i z e d t h e d i a g n o s t i c p o t e n t i a l o f t h e s e l a b e l e d f a t t y a c i d s f o r t h e p u r p o s e o f p h o t o s c a n n i n g t h e h e a r t . P r o b l e m s r e m a i n e d w i t h t h e 1 3 1 l l a b e l , w h i c h l i m i t e d i t s u s e f u l n e s s a s a c l i n i c a l i m a g i n g a g e n t . T h e s e i n c l u d e d a l a r g e r a d i a t i o n d o s e t o t h e p a t i e n t b e c a u s e o f t h e /? e m i s s i o n s a n d a l a r g e r e s i d u a l e x p o s u r e o f a c t i v i t y a n d b e c a u s e o f i t s e x t r e m e l y l o n g h a l f - l i f e (-193 h r s - a v e r a g e l i f e 279 h r s ) ( M y e r s , 1 9 7 4 ) ( T a b l e 3 ) . T h e r e f o r e , w o r k was n e e d e d t o i d e n t i f y t h e a v a i l a b i l i t y o f s u i t a b l e r a d i o n u c l i d e s f o r i n v i v o u s e a n d r e f i n e m e n t s i n t h e l a b e l i n g p r o c e d u r e s t o o b t a i n g r e a t e r p u r i t y o f t h e f i n a l p r o d u c t s . - 30 -T a b l e 3 P h y s i c a l P r o p e r t i e s o f 1 2 3 I , 1 2 S I a n d 1 3 1 I ( M y e r s e t a l . , 1 9 7 4 ) - c l i n i c a l l y u s e d r a d i o i o d i d e s . ( R a d i o n u c l i d e T r a n s f o r m a t i o n s , I C R P P u b l i c a t i o n 38, 1 9 8 3 . e d . F.D. Sowby, Pergammon P r e s s , N.Y. , N.Y.) 123 J 1 25J 131 j H a l f - l i f e 13 h r s . 1344 h r s . 193 h r s . Modes o f d e c a y EC EC B e t a P a r t i c l e s n o n e n o n e 9 0 . 4 % 192 K e V a v e r a g e 7 e n e r g y 158 K e V n o n e ( X - r a y s 27 & 35 k e V ) 363 k e V O t h e r p h o t o n s 7 - 3 % 2 7 4 - 5 3 0 k e V n o n e 7 - 6.8% 637 k e V - 1 . 6 % 723 k e V - 31 -T h e d e c r e a s e d e n t h u s i a s m o v e r i o d i n e a s a r a d i o n u c l i d e o v e r t h e n e x t d e c a d e p r o m p t e d t h e d e v e l o p m e n t o f a l t e r n a t i v e i m a g i n g a g e n t s f o r n o n - i n v a s i v e a s s e s s m e n t o f t h e c a r d i o v a s c u l a r s y s t e m . T h e s e a g e n t s i n c l u d e d 1 2 9 C s ( Y a n o e t a l . . 1 9 7 0 ) , 4 3 K ( H u r l e y e t a l . . 1 9 7 1 ) , 1 3 N - N H 4 ( H u n t e r & M o n a h a n , 1 9 7 1 ) , a n d 8 1 R u ( M a r t i n e t a l . . 1 9 7 4 ) . Many w o r k e r s c o n t i n u e d t o u s e t h e 1 4 C l a b e l ( A n t o n y a n d L a n d a u , 1 9 6 8 ) . T h a l l i u m , a s d i s c u s s e d e a r l i e r , was a l s o b e i n g a c t i v e l y e x p l o r e d a s a n i n d i c a t o r o f m y o c a r d i a l p e r f u s i o n . I t was n o t u n t i l 1975 t h a t Poe a n d c o - w o r k e r s u t i l i z e d 1 3 1 I i n t h e t e r m i n a l p o s i t i o n o f h e x a d e c a n o i c a c i d (HDA) a n d f o u n d t h a t e x t r a c t i o n p e r c e n t a g e s c o m p a r e d f a v o u r a b l y w i t h t h e n a t u r a l l y o c c u r r i n g c o m p o u n d s (77+11% f o r HDA c o m p a r e d t o 70+7.5% f o r s t e a r i c a c i d a n d 61+7.8% f o r o l e i c a c i d s l a b e l e d w i t h X 1 C ) . i i ) 1 2 3 I o d i n e ( 1 2 3 I ) 1) P r o d u c t i o n o f 1 2 3 I . T h e r e e x i s t 29 r a d i o i s o t o p e s o f I o d i n e w h i c h c a n b e u s e d i n m e d i c i n e t o d a y ( M a n i , 1 9 8 5 ) . A l l a r e p r o d u c e d i n r e a c t o r s a n d i n v o l v e a n y o n e o f f i v e p o s s i b l e n e u t r o n - i n d u c e d r e a c t i o n s , i n c l u d i n g ( n , 7 ) , ( n , p ) , ( n , a ) , ( n , 7 ) f o l l o w e d b y d e c a y , a n d l a s t l y , ( n , f i s s i o n ) . O f t h e 29 r a d i o i s o t o p e s o f i o d i n e a v a i l a b l e , 1 2 3 I h a s t h e p h y s i c a l p r o p e r t i e s w h i c h s a t i s f i e s t h e r e q u i r e m e n t s o f a n i d e a l r a d i o p h a r m a c e u t i c a l f o r i n v i v o m e d i c a l u s e ( M y e r s e t a l . . 1 9 6 2 ) . T h e s e e a r l y c l i n i c a l s t u d i e s o n i n v i v o m e d i c a l u s e o f t h e 1 2 3 I l a b e l c e n t e r e d a r o u n d t h y r o i d u p t a k e a n d s c a n n i n g . T h e 1 2 3 l was f i r s t p r o d u c e d b y t h e S b 1 2 1 ( H e 4 , 2 n ) I 1 2 3 r e a c t i o n w i t h a 30 MeV beam. - 32 -T h i s r e s u l t e d i n c o n t a m i n a t i o n w i t h h i g h - e n e r g y 7-rays f r o m 1 2 4 I . T h e t e c h n i c a l d i f f i c u l t i e s a s s o c i a t e d w i t h t h i s m e t h o d n e c e s s i t a t e d t h e d e v e l o p m e n t o f a l t e r n a t i v e r e a c t i o n s , i n c l u d i n g 1 2 2 T e ( a , 3 n ) 1 2 3 X e , ( S o d d a n d B l u e , 1 9 6 8 ) a n d 1 2 2 T e ( 3 H e , 2 n ) 1 2 3 X e — 1 2 3 I , ( S o d d e t a l . . 1 9 6 9 ) , w h i c h p r o d u c e d c a r r i e r - f r e e i o d i d e i o n b y t h e d e c a y o f 1 2 3 X e . I n o r d e r t o o b t a i n h i g h y i e l d s o f 1 2 3 I , L a m b r e c h t a n d W o l f ( 1 9 7 2 ) e x a m i n e d t h e u s e o f t h e 1 2 2 T e ( 4 H e , 3 n ) 1 2 3 X e > 1 2 3 I g e n e r a t o r a n d f o u n d a y i e l d p u r i t y o f 9 9 . 8 % 1 2 3 I , w i t h <0.2% c o n t a m i n a t i n g 1 2 5 I . T h e p r e s e n c e o f t h i s i m p u r i t y , w h i c h i n c r e a s e d r a d i a t i o n d o s e t o t h e p a t i e n t , l e d t o t h e d e v e l o p m e n t o f a n a l t e r n a t i v e r e a c t i o n b y F u s c o e t a l . ( 1 9 7 2 ) , i n w h i c h i t was r e p o r t e d t h a t v i r t u a l l y a l l r a d i o a c t i v e c o n t a m i n a n t s w e r e e l i m i n a t e d . T h e s e a u t h o r s u t i l i z e d t h e 1 2 7 I ( p , 5 n ) 1 2 3 X e r e a c t i o n , r e s u l t i n g i n 3.0 mCi/7*A/hr. o f a c t i v i t y a n d o n l y 0.1% 1 2 5 I i m p u r i t y a n d n o 1 2 4 I . H o w e v e r , t h e p r o t o n beam e n e r g y r e q u i r e d f o r t h i s r e a c t i o n i s >50 MeV, w h i c h i s n o t r e a d i l y a v a i l a b l e f r o m m o s t c y c l o t r o n s . C u r r e n t l y , t h e r e a r e 4 m a i n t y p e s o f n u c l e a r r e a c t i o n s s u i t a b l e f o r p r e p a r i n g 1 2 3 I : t h e 1 2 7 I ( p , 5 n ) 1 2 3 X e ( E C , 0+)123I(70MeV), 1 2 4 T e ( p , 2 n ) 1 2 3 I ( 2 5 - 4 0 M e V ) a n d , l a s t l y , 1 2 4 X e ( p , 2 n ) 1 2 3 C s (/9+) 1 2 3 X e ( E C , £ + ) 1 2 3 I ( 2 5 - 4 0 M e V ) , ( M a c h u l l a e t a l . . 1 9 8 6 ) a n d a f i n a l s e q u e n c e i n v o l v i n g b o t h a d i r e c t r e a c t i o n 1 2 4X e ( p , 2 p ) >1 2 3l ( 7 5 % ) a n d a n i n d i r e c t r e a c t i o n 1 2 4 X e ( p , p n ) 1 2 3 X e > 1 2 3 I ( 2 5 % ) ( A E C L , V a n c o u v e r , B.C., C a n a d a ) . T h i s l a t t e r m e t h o d was t h e p r e p a r a t i o n o f 1 2 3 I u s e d f o r t h i s e x p e r i m e n t a l e n d e a v o u r . T h e a v a i l a b i l i t y a n d p u r i t y o f 1 2 3 I i s n e c e s s a r y a s t h e t h r u s t o f much o f t h e r e s e a r c h t o d a y i s i n t o t h e d e v e l o p m e n t o f a s i m p l e a n d q u i c k l a b e l i n g k i t , w h i c h w i l l y i e l d a p u r e p r o d u c t . - 33 -2) F a t t y A c i d I o d i n a t i o n P r o c e d u r e s . E a r l y a t t e m p t s a t i o d i n a t i o n i n v o l v e d i o d i n e m o n o c h l o r i d e a d d i t i o n t o a d o u b l e b o n d ( E v a n s e t a l . . 1 9 6 5 ) o r i o d i d e r e p l a c e m e n t a t t h e t e r m i n a l p o s i t i o n i n o m e g a - b r o m o - ( l o n g c h a i n ) - f a t t y a c i d s ( R o b i n s o n e t a l . . 1 9 7 4 ) . I t b e c a m e a p p a r e n t f r o m t h e s e s t u d i e s t h a t t e r m i n a l l y i o d i n a t e d f a t t y a c i d a n a l o g s r e s u l t e d i n h i g h e r l e v e l s o f a c t i v i t y w i t h i n t h e t i s s u e w hen c o m p a r e d t o i m a g e s o b t a i n e d f r o m u n s a t u r a t e d f a t t y a c i d s . T h e r e a s o n f o r s u c h a d i s c r e p a n c y was d e t e r m i n e d b y Poe e t a l . ( 1 9 7 5 ) a n d r e c o n f i r m e d b y M a c h u l l a e t a l . ( 1 9 8 0 ) . I t was e s t a b l i s h e d t h a t m y o c a r d i a l e x t r a c t i o n o f t e r m i n a l l y - i o d i n a t e d f a t t y a c i d s was 4 5 % h i g h e r t h a n t h a t o f f a t t y a c i d s l a b e l e d b y i o d i n a t i o n a c r o s s t h e d o u b l e b o n d . T h u s , a l a r g e r a mount o f t h e f r e e f a t t y a c i d was b e i n g r e m o v e d f r o m t h e c i r c u l a t i o n f a c i l i t a t i n g gamma s c i n t i g r a p h y . A l t h o u g h i m a g e i n t e n s i t y was i n c r e a s e d b y l a b e l i n g t h e f a t t y a c i d a t t h e t e r m i n a l p o s i t i o n , R o b i n s o n a n d L e e ( 1 9 7 4 ) h a d c o n c e r n s r e g a r d i n g t h e p o s s i b i l i t y t h a t r e c i r c u l a t i o n o f f r e e i o d i d e may b e l i m i t i n g t h e a c c u r a c y o f m y o c a r d i a l i m a g e s . M a c h u l l a e t a l . ( 1 9 7 8 ) , d e m o n s t r a t e d t h a t t h e h i g h e s t u p t a k e o f omega f a t t y a c i d o c c u r r e d when c a r b o n c h a i n l e n g t h was 17 c a r b o n u n i t s . T h e y a l s o n o t e d a v e r y h i g h l e v e l o f b a c k g r o u n d r a d i o a c t i v i t y , d u e t o f r e e i o d i d e r e c i r c u l a t i n g w i t h i n t h e b l o o d s t r e a m f o l l o w i n g b e t a - o x i d a t i o n o f t h e f a t t y a c i d s ( F i g u r e 3 ) . I n o r d e r t o e l i m i n a t e t h i s b a c k g r o u n d p r o b l e m , t h e p r e p a r a t i o n o f i o d i n a t e d - 1 2 3 I - p h e n y l p e n t a d e c a n o i c a c i d ( I P P A ) f o r i n v i v o m e t a b o l i c s t u d i e s was f i r s t d e s c r i b e d b y M a c h u l l a e t a l . ( 1 9 8 0 ) a n d M a c h u l l a a n d D u t s c h k a ( 1 9 8 1 b ) . I n t h e i r p r e p a r a t i o n o f I P P A , t h e a u t h o r s u t i l i z e d n u c l e o p h i l i c a r o m a t i c s u b s t i t u t i o n o f i o d i n e - 34 -F i g u r e 3 - C a t a b o l i c p a t h w a y s o f 1 2 3 I P P A a n d 1 2 3 I H A . - 34a -123 co— I-Heptadecanoic A c i d 1 23 co- (p- l - P h e n y l ) - P e n t a d e c a n o i c A c i d 1 23i C O O H 123 C O O H I3-Oxidation A c e t y l - C o A 1 23, / \ / C O O H 1 23i A c e t y l - C o A C O O H - 35 -o n t o t h e p h e n y l r i n g . T h e r e s u l t was t h e p r o d u c t i o n o f 70% p a r a s u b s t i t u e n t a n d 30% o r t h o s u b s t i t u e n t i n a r a d i o c h e m i c a l y i e l d o f 7 0%. H i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y was t h e n u s e d t o s e p a r a t e a n d p u r i f y e a c h o f t h e i s o m e r i c f o r m s o f t h e l a b e l e d f a t t y a c i d . T h e p a r a - I P P A was t h e n d i s s o l v e d i n 4% HSA f o r a d m i n i s t r a t i o n i n t o t h e t e s t a n i m a l s . M a c h u l l a c h o s e t h e p a r a i s o m e r b e c a u s e o f i t s g r e a t e r u p t a k e b y t h e m y o c a r d i u m . T h i s p r e p a r a t i o n m e t h o d was f o u n d t o s u f f e r f r o m a n u m b e r o f p r o b l e m s , i n c l u d i n g t h e f o r m a t i o n o f a n i s o m e r i c m i x t u r e ( 7 0 % p a r a , 30% o r t h o ) , t h e r e q u i r e m e n t o f c o m p l i c a t e d HPLC p r o c e d u r e s t o i s o l a t e t h e p a r a f o r m , a n d , l a s t l y , t h e l o s s o f 29% o f 1 2 3 I l a b e l a t t h e e n d o f t h e p r e p a r a t i o n . I n a n a t t e m p t t o a l l e v i a t e t h e s e p r o b l e m s , a s s o c i a t e d w i t h t h e n u c l e o p h i l i c a r o m a t i c s u b s t i t u t i o n m e t h o d , E i s e n h u t ( 1 9 8 2 ) d e v i s e d a f a s t a n d s i m p l e s y n t h e s i s o f p - 1 2 3 I P P A f r o m p u r e , u n l a b e l e d p - I P P A b y i o d i n e - i s o t o p e e x c h a n g e . T h e p r o c e d u r e i n v o l v e d t h e p r e p a r a t i o n o f i s o m e r i c 1 5 - i o d o p h e n y l - p e n t a d e c a n o i c a c i d , w i t h s u b s e q u e n t i s o l a t i o n o f t h e p a r a d e r i v a t i v e , u s i n g r e c r y s t a l l i z a t i o n f r o m h e x a n e . T h e p r o d u c t was t h e n r a d i o i o d i n a t e d b y i o d i n e - i s o t o p e e x c h a n g e . T h e r e a c t i o n c o n d i t i o n s i n c l u d e d a t e m p e r a t u r e o f 1 7 0 ° C f o r 30 m i n u t e s , y i e l d i n g 95% p a r a i s o m e r . S t u d i e s o f t h e r e a c t i o n a t v a r i o u s t e m p e r a t u r e s d e m o n s t r a t e d h i g h e s t r a d i o c h e m i c a l y i e l d a t 1 7 0 ° C , w i t h d e c r e a s i n g y i e l d s a t 1 4 0 ° C a n d 1 0 2 ° C ( 9 5 % , 78% a n d 50% y i e l d s , r e s p e c t i v e l y ) . T h e a u t h o r s t h e n e x a m i n e d s c i n t i g r a m s o f r a b b i t h e a r t , l i v e r a n d b a c k g r o u n d a r e a s o f i n t e r e s t , n o t i n g d i f f e r e n c e s b e t w e e n 1 2 3 I H A a n d 1 2 3 I P P A . T h e l a t t e r d e m o n s t r a t e d i n c r e a s e d h e a r t u p t a k e a n d r e d u c e d l i v e r u p t a k e a n d b a c k g r o u n d a c t i v i t y c o m p a r e d t o 1 2 3 I H A . - 36 -H o w e v e r , t h i s p r e p a r a t i v e m e t h o d r e s u l t s i n a r e l a t i v e l y l o w s p e c i f i c a c t i v i t y . T h e r e f o r e , a l t e r n a t i v e means o f p r e p a r a t i o n w e r e d e v e l o p e d , i n c l u d i n g t r i a z e n e d e c o m p o s i t i o n (Goodman e t a l . . 1 9 8 2 ) , d e s t a n n y l a t i o n ( F r a n k e e t a l . . 1 9 8 3 ) , b o r o n r e p l a c e m e n t ( K a b a l k a e t a l . . 1 9 8 1 ) , a n d t h a l l i u m r e p l a c e m e n t ( K u l k a r n i a n d P a r k e y , 1 9 8 2 ) . T h e l a t t e r r e p r e s e n t s a n i m p o r t a n t c o m p o n e n t o f t h e i o d i n a t i o n p r o c e d u r e s u s e d f o r t h e p r e s e n t s t u d y . I n o r d e r t o a c q u i r e f a s t e r a n d m o r e e f f i c i e n t l a b e l i n g t e c h n i q u e s , t h a l l a t i o n was i n t r o d u c e d a s a c o m p o n e n t o f a r o m a t i c e l e c t r o p h i l i c s u b s t i t u t i o n i n 1934 ( G i l m a n a n d A b b o t t , 1 9 3 4 ) . T h e s e e a r l y r e a c t i o n s , h o w e v e r , w e r e l i m i t e d i n u s e b y t h e p o o r e l e c t r o p h i l i c i t y o f t h e r e a g e n t u s e d . I n 1 9 6 9 ( a ) , M c K i l l o p a n d c o - w o r k e r s d e v e l o p e d a n a l t e r n a t i v e r e a g e n t f o r t h e t h a l l a t i o n o f a r o m a t i c c o m p o u n d s w h i c h a l s o i n d u c e d some i s o m e r i c s p e c i f i c i t y i n t o t h e p r o c e d u r e . T h e p r e f e r e n c e f o r t h e p a r a p o s i t i o n o n t h e p h e n y l r i n g b y i o d i n e was b e l i e v e d t o b e d u e t o t h e s t e r i c b u l k o f t h e t h a l l i u m m o l e c u l e . T h e s e t h a l l a t e d a r o m a t i c c o m p o u n d s w e r e f o u n d t o b e v e r y e f f e c t i v e i n t e r m e d i a t e s f o r t h e f o r m a t i o n o f s u b s t i t u t e d a r o m a t i c c o m p o u n d s t h r o u g h t h e c l e a v a g e o f t h e C - T l b o n d . M c K i l l o p e t a l . ( 1 9 6 9 b ) l a t e r u s e d t h i s r e a g e n t t o d e v e l o p a h i g h - y i e l d , o n e - s t e p s y n t h e s i s o f a r y l i o d i d e s . T h e s e a u t h o r s f o u n d t h a t t r e a t i n g t h e a r y l t h a l l i u m i n t e r m e d i a t e s w i t h a q u e o u s K I r e s u l t e d i n t h e f o r m a t i o n o f t h e c o r r e s p o n d i n g a r o m a t i c i o d i d e s . T h e r e a c t i o n was r e p o r t e d t o b e c o m p l e t e i n a f e w m i n u t e s a t r o o m t e m p e r a t u r e . T h u s , a n e x t r e m e l y s i m p l e s y n t h e s i s o f a r o m a t i c i o d i d e s was d e v e l o p e d . - 37 -T h e r e a c t i o n s e q u e n c e s h own i n F i g u r e 4 d e s c r i b e s t h e p r e p a r a t i o n o f 1 5 - p - 1 2 3 I - i o d o p h e n y l p e n t a d e c a n o i c a c i d u t i l i z e d i n t h i s s t u d y ( D o u g a n e t a l . . 1 9 8 6 a , b ) . T h e o r i e n t a t i o n o f t h e i n c o m i n g T l e l e c t r o p h i l e c o u l d b e i n f l u e n c e d b y t h e n a t u r e o f t h e a r o m a t i c n u c l e a r s u b s t i t u e n t s ( M c K i l l o p e t a l . . 1 9 7 1 ) a n d b y t h e t e m p e r a t u r e o f t h e r e a c t i o n m i x t u r e ( E i s e n h u t , 1 9 8 2 ) . T h e l a t t e r a u t h o r s a l s o d e m o n s t r a t e d t h a t t h e c o n c e n t r a t i o n o f b a s e c o u l d a l t e r t h e r a d i o i o d i n a t i o n y i e l d , w h i c h was m a x i m a l w i t h 0.1 M NaOH. M a c h u l l a e t a l . ( 1 9 8 1 a , b ) u s e d e l e c t r o p h i l i c a r o m a t i c s u b s t i t u t i o n t o g e n e r a t e I P P A w i t h i s o m e r i c y i e l d s o f 70% p a r a a n d 30% o r t h o . K u l k a r n i a n d P a r k e y ( 1 9 8 3 ) f o u n d t h a t c o m b i n i n g t h a l l a t i o n w i t h i o d i n a t i o n r e s u l t e d i n y i e l d s o f 99% p a r a a n d 1% o r t h o . T h e y r e a s o n e d t h a t t h e s t e r i c h i n d r a n c e a s s o c i a t e d w i t h t h a l l a t i o n s e v e r e l y l i m i t s o r t h o s u b s t i t u t i o n , w h i l e p r o m o t i n g p a r a s u b s t i t u t i o n . R a d i o c h e m i c a l y i e l d s w e r e s t i l l l o w , h o w e v e r , w i t h v a l u e s b e t w e e n 50 a n d 7 0 % . T h e r e f o r e , e f f o r t s c o n t i n u e t o d e v e l o p a l a b e l i n g m e t h o d w h i c h i s m ore e f f i c i e n t , a l l o w i n g g r e a t e r r a d i o c h e m i c a l y i e l d s . - 38 -F i g u r e 4 - A r o m a t i c e l e c t r o p h i l i c a n d n u c l e o p h i l i c s u b s t i t u t i o n r e a c t i o n . - 38a -( C H 2 ) — C + Tl ( C F 3 C \ 14 \ _ . . 3 \ Q 3 Electrophillc Aromatic Substitution ( C F , c 2 V W 3 Nucleaphilic Aromatic Substitution O H room temperature darkness 60 mins 0 T I - C 3 K ( C H 2 ) 1 4 - < Kl(aq) 60 mins 68 -60°C Na 1 2 3 l in 0.1 M NaOH 123, _// V(CH 2) U-C^ O O H - 39 -i i i ) A p p l i c a t i o n s - C l i n i c a l a n d E x p e r i m e n t a l a ) I o d i n a t e d h e x a d e c e n o i c a c i d A s m e n t i o n e d e a r l i e r , Poe a n d c o w o r k e r s ( 1 9 7 5 ) r e - e m p h a s i z e d t h e p o t e n t i a l u s e f u l n e s s o f i o d i n a t e d f a t t y a c i d s f o r d i a g n o s t i c i m a g i n g . T h e s e a u t h o r s made a c o m p a r a t i v e e v a l u a t i o n o f l o n g - c h a i n f a t t y a c i d s l a b e l e d b y X 1C i n t h e c a r b o x y l p o s i t i o n , 1 3 1l a d d i t i o n b y a d o u b l e b o n d s a t u r a t i o n t e c h n i q u e a n d , l a s t l y , 1 3 1I s u b s t i t u t i o n a t t h e t e r m i n a l p o s i t i o n . C a n i n e h e a r t s w e r e u t i l i z e d t o e x a m i n e t h e e x t r a c t i o n e f f i c i e n c i e s o f e a c h o f t h e s e p r o d u c t s . T h e 1 X C c o m p o u n d s w e r e s t e a r i c a c i d a n d o l e i c a c i d . T h e f a t t y a c i d s l a b e l e d b y t h e d o u b l e b o n d s a t u r a t i o n t e c h n i q u e i n c l u d e d o l e i c , l i n o l e i c a n d l i n o l e n i c a c i d s . T h e t e r m i n a l l y l a b e l e d a c i d was h e x a d e c e n o i c a c i d . D i r e c t c o r o n a r y a r t e r y i n j e c t i o n o f t h e f a t t y a c i d s r e s u l t e d i n t h e g e n e r a t i o n o f t i m e - a c t i v i t y c u r v e s u s i n g a s o d i u m - i o d i d e gamma s c i n t i l l a t i o n d e t e c t o r a n d f l a t - h e a d c o l l i m a t o r . B i p h a s i c m y o c a r d i a l w a s h o u t c u r v e s i n d i c a t e d t h a t t e r m i n a l i o d i n a t i o n y i e l d e d a n e n d - p r o d u c t w h i c h was e x t r a c t e d b y t h e m y o c a r d i u m a t a n e f f i c i e n c y s i m i l a r t o t h a t o f t h e p a r e n t c o m p o u n d . T h e s e a u t h o r s a l s o c o n c l u d e d t h a t t h e i o d i n e - l a b e l p r o t r u d i n g f r o m t h e d o u b l e b o n d s t e r i c a l l y a l t e r s t h e n o r m a l m e t a b o l i c c a s c a d e a s s o c i a t e d w i t h f r e e f a t t y a c i d o x i d a t i o n , s u c h t h a t e x t r a c t i o n e f f i c i e n c y was much r e d u c e d (77 ± 11% f o r IHA c o m p a r e d t o 33 + 5.2% f o r i o d i n a t e d s a t u r a t e d o l e i c a c i d ) . I n v i e w o f t h e o p t i m a l p h y s i c a l c h a r a c t e r i s t i c s o f t h e 1 2 3 I l a b e l , P o e e t a l . ( 1 9 7 7 ) u n d e r t o o k a c l i n i c a l s t u d y u t i l i z i n g 1 6 . 1 2 3 i - i o d o - 9 - h e x a d e c e n o i c a c i d ( I H A ) . T w e n t y - o n e p a t i e n t s w i t h a mean a g e o f 60, u n d e r g o i n g c a r d i a c c a t h e t e r i z a t i o n , w e r e i m a g e d a n d - 4 0 -w a s h o u t r a t e s c a l c u l a t e d . A n u mber o f v i e w s , i n c l u d i n g l e f t a n d r i g h t a n t e r i o r o b l i q u e , a n t e r i o r a n d l a t e r a l , w e r e a s s e s s e d . T h e s t u d y d e m o n s t r a t e d r e d u c e d IHA u p t a k e i n v a r i o u s r e g i o n s o f t h e m y o c a r d i u m i n a s y m p t o m a t i c a n g i n a p e c t o r i s p a t i e n t s a t r e s t . D e f i n i t e d e f i c i e n c i e s i n t r a c e r u p t a k e w e r e s e e n i n 4 a n g i n a p e c t o r i s p a t i e n t s who h a d n o i n f a r c t i o n o r a n y r e g i o n a l w a l l a b n o r m a l i t i e s . R o b i n s o n a n d L e e ( 1 9 7 5 ) r e p o r t e d o n t h e h i g h e x t r a c t i o n e f f i c i e n c y o f 1 6 - i o d o - 9 - h e x a d e c e n o i c a c i d ( - 7 0 % ) a n d b e l i e v e d i t s m e t a b o l i c b e h a v i o u r r e f l e c t e d t h a t o f a t r u e a n a l o g o f a p h y s i o l o g i c , u n s a t u r a t e d f a t t y a c i d . B o t h e x p e r i m e n t a l a n d c l i n i c a l s t u d i e s d e m o n s t r a t e d t h e f e a s i b i l i t y o f m y o c a r d i a l i m a g i n g w i t h 1 2 3 I H A ( P o e e t a l . . 1 9 7 6 a a n d Poe e t a l . . 1 9 7 7 ) . V a n d e r W a l l e t a l . ( 1 9 8 0 ) c o m p a r e d t h e i m a g i n g p r o p e r t i e s o f 1 2 3 I - 1 6 - i o d o - 9 - h e x a d e c e n o i c a c i d a n d t h a l l i u m - 2 0 1 . T h e s e a u t h o r s s t u d i e d t w e l v e p a t i e n t s w i t h d o c u m e n t e d c o r o n a r y a r t e r y d i s e a s e - 8 w i t h a c u t e MI a n d 4 w i t h u n s t a b l e a n g i n a . T h e r e s u l t s r e v e a l e d s i m i l a r t r a c e r d i s t r i b u t i o n b e t w e e n 2 0 1 T 1 a n d 1 2 3 I H A , w i t h t h e l a t t e r d e m o n s t r a t i n g i n c r e a s e d h e p a t i c a c t i v i t y . V a n d e r W a l l e t a l . ( 1 9 8 1 ) s t u d i e d t h e v a l u e o f 1 3 1 I H A i n d e t e r m i n i n g r e g i o n a l m y o c a r d i a l m e t a b o l i s m i n a c a n i n e m o d e l . T h e y f o u n d t h a t th v a l u e s w e r e l o n g e r i n i s c h e m i c s e g m e n t s a s c o m p a r e d t o n o r m a l c o n t r o l v a l u e s . I t h a s a l s o b e e n f o u n d t h a t m y o c a r d i a l u p t a k e o f t h i s a g e n t a n d 2 0 1 T 1 s h o w e d a g o o d c o r r e l a t i o n ( W e s t e r a e t a l . . 1 9 8 0 ) . I n v i e w o f t h e m o r e o p t i m a l p h y s i c a l c h a r a c t e r i s t i c s o f t h e 1 2 3 I l a b e l , D e m a i s o n e t a l . ( 1 9 8 4 ) u t i l i z e d t h i s same f a t t y a c i d w i t h a 1 2 3 I t a g f o r i n t r a c o r o n a r y i n j e c t i o n i n a n i s o l a t e d r a t h e a r t p e r f u s e d w i t h K r e b ' s s o l u t i o n . T h e s e a u t h o r s d e m o n s t r a t e d c h a n g e s i n f a t t y a c i d m e t a b o l i s m i n d u c e d b y t h e p r e s e n c e o r a b s e n c e o f g l u c o s e . - 41 -Some y e a r s e a r l i e r , i n 1978, M a c h u l l a e t a l . d e m o n s t r a t e d t h a t t h e c h a i n l e n g t h a n d s i t e o f l a b e l i n g o f a f a t t y a c i d w o u l d a f f e c t i t s u p t a k e b y t h e m y o c a r d i u m . T h e s e a u t h o r s f o u n d t h a t m y o c a r d i a l e x t r a c t i o n o f o m e g a - h a l o g e n a t e d f a t t y a c i d s w i t h a c h a i n l e n g t h o f 15 c a r b o n a t o m s o r h i g h e r o c c u r r e d m o re r e a d i l y t h a n t h a t o f a l p h a - h a l o g e n a t e d f a t t y a c i d s o f s i m i l a r l e n g t h . T h e a u t h o r s p e r f o r m e d c o m p a r a t i v e k i n e t i c s t u d i e s o f a c c u m u l a t i o n a n d w a s h o u t f r o m t h e m y o c a r d i u m o f m i c e d e m o n s t r a t i n g a h i g h b a c k g r o u n d l e v e l o f r a d i o a c t i v i t y d u e t o f r e e i o d i d e r e l e a s e . T h e e f f e c t s o f f a t t y a c i d c h a i n l e n g t h o n m y o c a r d i a l e x t r a c t i o n was e x a m i n e d b y O t t o e t a l . ( 1 9 8 1 ) . E x p e r i m e n t s u s i n g s i x o m e g a - h a l o g e n a t e d f a t t y a c i d s ( n = 10, 12, 15, 18, 21, 2 6 ) , l a b e l e d w i t h 1 2 3 I , s h o w e d t h a t c h a i n l e n g t h i n f l u e n c e s b o t h u p t a k e a n d r e s i d e n c e t i m e w i t h i n r a t m y o c a r d i u m . C h a i n l e n g t h s o f n < 15 w e r e f o u n d s u i t a b l e f o r s t u d y o f b e t a - o x i d a t i o n , w h i l e n > 19 w e r e w e l l - s u i t e d f o r m y o c a r d i a l p e r f u s i o n i m a g i n g b e c a u s e o f l o n g e r r e s i d e n c e t i m e s w i t h i n t h e m y o c a r d i u m . C u r r e n t l y , n o n a d e c e n o i c f a t t y a c i d s a r e b e i n g i n v e s t i g a t e d a s p o t e n t i a l p e r f u s i o n i m a g i n g a g e n t s . A m b r o s e e t a l . ( 1 9 8 7 b , c ) h a v e e x a m i n e d t h e e f f e c t s o f m e t h y l - b r a n c h i n g o n m y o c a r d i a l r e t e n t i o n o f t e r m i n a l l y i o d o v i n y l s u b s t i t u t e d f a t t y a c i d s a n d h a v e s hown t h a t s i g n i f i c a n t d i f f e r e n c e s e x i s t i n t h e m e t a b o l i c b e h a v i o r o f t h e s e a g e n t s . Goodman e t a l . ( 1 9 8 7 ) h a v e d e m o n s t r a t e d g r e a t e r h e a r t u p t a k e , s l o w e r m y o c a r d i a l r e l e a s e a n d h i g h e r h e a r t : b l o o d r a t i o s f o r ( E ) - 1 9 - [ 1 2 5 I ] i o d o - 3 , 3 - d i m e t h y l - 1 8 - n o n a d e c e n o i c a c i d ( D M I V N ) . T h e l a t t e r h a s b e e n u s e d b y Som e t a l . ( 1 9 8 8 ) t o e x a m i n e t h e e f f e c t s o f h y p e r t e n s i o n a n d v e r a p a m i l o n f a t t y a c i d k i n e t i c s i n r a t s . - 42 -b ) I o d i n a t e d h e x a / h e p t a d e c a n o i c a c i d T h e i n f l u e n c e o f c h a i n l e n g t h p r o m p t e d e x a m i n a t i o n i n t o t h e p o s s i b l e u s e o f l o n g e r c h a i n f a t t y a c i d s ( n > 1 5 ) , s u c h a s h e x a / h e p t a d e c a n o i c a c i d . V a n d e r W a l l e t a l . ( 1 9 8 1 ) e x a m i n e d t h e m y o c a r d i a l d i s t r i b u t i o n o f 1 2 6 I - h e x a d e c e n o i c a c i d a n d 1 3 ^ - h e x a d e c a n o i c a c i d . I t was r e p o r t e d t h a t m y o c a r d i a l u p t a k e o f h e x a d e c e n o i c a c i d was 40% l o w e r t h a n h e x a d e c a n o i c a c i d . T h u s , t h e l a t t e r may h a v e g r e a t e r s c i n t i g r a p h i c v a l u e . T h e p r o b l e m o f h i g h b a c k g r o u n d a c t i v i t y was a d d r e s s e d b y F r e u n d l i e b ( 1 9 7 8 ) , w h o s e m e t h o d was u t i l i z e d b y V y s k a ( 1 9 7 9 a , b ) a t t h e I n s t i t u t e o f N u c l e a r M e d i c i n e , N u c l e a r R e s e a r c h C e n t e r , i n t h e F e d e r a l R e p u b l i c o f G e r m a n y . T h e s e a u t h o r s a s s e s s e d m y o c a r d i a l f a t t y a c i d m e t a b o l i s m u s i n g o m e g a - h a l o g e n a t e d - ( 1 2 3 I ) - h e p t a - d e c a n o i c a c i d i n 20 n o r m a l p e r s o n s , 35 c o r o n a r y h e a r t d i s e a s e p a t i e n t s a n d 3 p a t i e n t s w i t h o l d m y o c a r d i a l i n f a r c t i o n s . T h e b a c k g r o u n d s u b t r a c t i o n t e c h n i q u e o f i n j e c t i n g a 300/*Ci b o l u s o f N a 1 2 3 I 20 m i n u t e s f o l l o w i n g t h e r o u t i n e s c a n was e m p l o y e d . T h e a c t i v i t y r e l e a s e d b y t h e m y o c a r d i u m was d e t e r m i n e d f r o m t h e b a c k g r o u n d - c o r r e c t e d t i m e - a c t i v i t y c u r v e s . I n f a r c t e d z o n e s w e r e c l e a r l y v i s i b l e a s c o l d s p o t s a n d i s c h e m i c r e g i o n s i d e n t i f i e d a s h a v i n g p r o l o n g e d th v a l u e s . T h e a u t h o r s c o n c l u d e d t h a t HDA was a n e x c e l l e n t r a d i o p h a r m a c e u t i c a l c a p a b l e o f n o n - i n v a s i v e l y d i f f e r e n t i a t i n g n o r m a l f r o m p a t h o l o g i c a l m y o c a r d i u m . Many c l i n i c a l s t u d i e s f o l l o w e d t h i s w o r k i n a n a t t e m p t t o i d e n t i f y f a t t y a c i d ( i o d i n a t e d h e x a d e c a n o i c a c i d ) m e t a b o l i c a b e r r a t i o n s i n v a r i o u s c a r d i a c a b n o r m a l i t i e s . F r e u n d l i e b e t a l . ( 1 9 8 0 ) e x a m i n e d th v a l u e s i n n o r m a l v o l u n t e e r s , p a t i e n t s s u f f e r i n g f r o m CAD a n d , l a s t l y , - 43 -p a t i e n t s w i t h c o n g e s t i v e h e a r t f a i l u r e . T h e s e a u t h o r s w e r e a b l e t o i d e n t i f y a r e a s o f i n f a r c t i o n a n d i s c h e m i a b y a r e d u c t i o n i n t r a c e r u p t a k e . E x a m i n a t i o n o f th v a l u e s , h o w e v e r , r e v e a l e d n o s i g n i f i c a n t d i f f e r e n c e s b e t w e e n t h e s e t h r e e g r o u p s . S i m i l a r f i n d i n g s w e r e r e p o r t e d b y D u d c z a k e t a l . ( 1 9 8 2 a ) . V a n d e r W a l l e t a l . ( 1 9 8 1 a ) a s s e s s e d t h e t h v a l u e s o f 30 p a t i e n t s who h a d s u f f e r e d a m y o c a r d i a l i n f a r c t i o n o f l e s s t h a n o n e w e e k ' s d u r a t i o n . W a s h o u t r a t e s o b t a i n e d f r o m m o n o - e x p o n e n t i a l t i m e - a c t i v i t y c u r v e s o f i n f a r c t e d r e g i o n s r e v e a l e d s i g n i f i c a n t l y l o w e r v a l u e s c o m p a r e d t o n o n - i n f a r c t e d a r e a s ( 1 6 . 8 + 3.5 c o m p a r e d w i t h 34.8 + 7.7 m i n , r e s p e c t i v e l y ) . T h e s e a u t h o r s a t t r i b u t e d t h i s p h e n o m e n o n t o r a p i d i n f l u x a n d o u t f l o w o f t i s s u e f l u i d s w i t h i n i n f l a m e d m y o c a r d i u m a n d / o r a n i n c r e a s e i n c o n t r a c t i l i t y o f t h e s u b e p i c a r d i u m t o c o m p e n s a t e f o r n o n - f u n c t i o n a l s u b e n d o c a r d i u m , t h u s i n c r e a s i n g o x y g e n r e q u i r e m e n t s a n d u t i l i z a t i o n o f F F A b y t h e s u b e p i c a r d i u m . T h e p r o l o n g e d w a s h o u t r a t e s a s s o c i a t e d w i t h r e v e r s i b l e i s c h e m i a w e r e e x p l a i n e d b y a s h i f t i n m e t a b o l i c s u b s t r a t e f r o m f a t t y a c i d s t o g l u c o s e i n 30 p a t i e n t s s u f f e r i n g f r o m s t a b l e a n g i n a p e c t o r i s ( V a n d e r W a l l e t a l . . 1 9 8 1 b ) . V a n d e r W a l l e t a l . ( 1 9 8 3 ) e x a m i n e d t h e w a s h o u t r a t e s o f 1 2 3 I - h e x a d e c a n o i c a c i d i n p a t i e n t s w i t h u n s t a b l e a n g i n a p e c t o r i s a n d f o u n d s i m i l a r r e s u l t s . W a s h o u t r a t e s i n t h e i s c h e m i c s e g m e n t s ( p e r f u s i o n d e f e c t s ) w e r e s i g n i f i c a n t l y p r o l o n g e d when c o m p a r e d t o n o r m a l l y p e r f u s e d a r e a s ( 4 5 . 4 + 4 . 8 c o m p a r e d w i t h 29.1 + 3.6 m i n , r e s p e c t i v e l y ) . T h e p r o l o n g e d w a s h o u t r a t e s w e r e r e p o r t e d t o b e due t o m y o c a r d i a l i s c h e m i a , w h i c h d i m i n i s h e s f a t t y a c i d u p t a k e , r e d u c e s b e t a - o x i d a t i o n a n d i n c r e a s e s t r i g l y c e r i d e f o r m a t i o n . F r e u n d l i e b e t a l . ( 1 9 8 2 ) e x a m i n e d m y o c a r d i a l w a s h o u t r a t e s o f 1 2 3 I - h e x a d e c a n o i c a c i d i n - 4 4 -p a t i e n t s b e f o r e a n d a f t e r c a r d i a c s u r g e r y f o r c o r o n a r y a r t e r y g r a f t i n g . R e s u l t s w e r e v a r i a b l e , i n t h a t , a f t e r r e v a s c u l a r i z a t i o n , th v a l u e s i n c r e a s e d , d e c r e a s e d o r w e r e u n c h a n g e d . I n a s i m i l a r s t u d y , F r i d r i c h e t a l . ( 1 9 8 6 ) e x a m i n e d t h e t r a c e r k i n e t i c s o f 1 2 3 I - h e p t a - d e c a n o i c a c i d i n p a t i e n t s f o l l o w i n g a o r t o c o r o n a r y b y p a s s g r a f t i n g c o m p a r e d t o n o n - g r a f t e d p a t i e n t s . T h e s e a u t h o r s a l s o a s s e s s e d v e n t r i c u l a r f u n c t i o n a n d c o r r e l a t e d i t w i t h m y o c a r d i a l m e t a b o l i s m o f HDA. I t was f o u n d t h a t n o r m a l f u n c t i o n ( E F > 50% a t r e s t ) was i n d i c a t i v e o f n o r m a l m e t a b o l i s m . V i s s e r e t a l . ( 1 9 8 5 ) u t i l i z e d i o d o - h e p t a - d e c a n o i c a c i d t o a s s e s s m y o c a r d i a l m e t a b o l i s m i n p a t i e n t s f o l l o w i n g s u c c e s s f u l t h r o m b o l y s i s f o r m y o c a r d i a l i n f a r c t i o n . B e c k u r t s e t a l . ( 1 9 8 5 ) u t i l i z e d t h i s a g e n t t o e x a m i n e t h e k i n e t i c s o f f a t t y a c i d m e t a b o l i s m i n n o r m a l a n d d i a b e t i c r a t s . K u i k k a e t a l . ( 1 9 8 8 ) a s s e s s e d HDA k i n e t i c s i n p a t i e n t s w i t h n o n - i n s u l i n - d e p e n d e n t d i a b e t e s . V i l l a v e c c h i a e t a l . ( 1 9 8 5 ) e x a m i n e d w a s h o u t o f 1 2 3 I - h e p t a - d e c a n o i c a c i d i n n o r m a l s u b j e c t s a n d p r o p o s e d i t s u s e f u l n e s s f o r m o n i t o r i n g m y o c a r d i a l m e t a b o l i s m f o l l o w i n g t h e a d m i n i s t r a t i o n o f d r u g s w h i c h may c a u s e m y o c a r d i a l t o x i c i t y . S p e c u l a t i o n a s t o t h e v a l i d i t y o f m e a s u r e d m y o c a r d i a l e l i m i n a t i o n r a t e ( m i n s ) o f h e p t a - d e c a n o i c a c i d w e r e r a i s e d b y V i s s e r e t a l . ( 1 9 8 5 ) . T o t h i s p o i n t , many i n v e s t i g a t o r s b e l i e v e d t h a t w a s h o u t r a t e s r e f l e c t e d t h e p r o c e s s o f b e t a - o x i d a t i o n . V i s s e r a n d h i s c o l l e a g u e s , h o w e v e r , d e m o n s t r a t e d t h a t t h e w a s h o u t r a t e s o f t h e f a t t y a c i d may n o t b e r e p r e s e n t i n g b e t a - o x i d a t i o n , s i n c e f i v e m i n u t e s a f t e r I V i n j e c t i o n , 69% o f t h e r a d i o a c t i v i t y was p r e s e n t a s f r e e i o d i n e , 24% a s l i p i d a n d 7% a s u n c h a n g e d f a t t y a c i d . T h u s , w a s h o u t r a t e s may r e p r e s e n t w a s h o u t - 45 -o f f r e e i o d i d e . V i s s e r e t a l . ( 1 9 8 6 ) , h o w e v e r , d e m o n s t r a t e d t h a t d i s t i n c t p a t t e r n s o f d i s t r i b u t i o n a n d e l i m i n a t i o n r a t e ( m i n s ) o f t h i s t r a c e r may a l l o w i t s u s e i n t h e s t u d y o f m y o c a r d i a l m e t a b o l i s m f o l l o w i n g v a r i o u s m e t a b o l i c i n t e r v e n t i o n s . L u t h y e t a l . ( 1 9 8 8 ) h a v e d e m o n s t r a t e d t h a t d e - i o d i n a t i o n o f HDA i s r e p r e s e n t a t i v e o f o x i d a t i v e f a t t y a c i d m e t a b o l i s m . T h e v a l i d i t y o f m y o c a r d i a l w a s h o u t r a t e s o f 1 2 3 l - h e p t a - d e c a n o i c a c i d , a n d w h a t i t r e p r e s e n t s , h o w e v e r , c o n t i n u e s t o b e q u e s t i o n e d . S c h o n e t a l . ( 1 9 8 6 ) b e l i e v e d t h a t t h e h a l f - t i m e o f HDA was n o t a q u a n t i t a t i v e a s s e s s m e n t o f b e t a - o x i d a t i o n , s i n c e t h e k i n e t i c s d i f f e r e d f r o m t h o s e o f 1 1 C P A . A l s o , HDA w a s h o u t was p r o l o n g e d w i t h i n c r e a s e d MV0 2, w h i c h i s o p p o s i t e t o w h a t s h o u l d a c t u a l l y o c c u r . T h e r a t i o o f t h e s i z e ( y - i n t e r c e p t o n T - A c u r v e ) o f t h e e a r l y p h a s e o v e r t h e s i z e o f t h e l a t e p h a s e may b e a b e t t e r i n d i c a t o r o f m y o c a r d i a l m e t a b o l i s m o f HDA ( S c h o n e t a l . . 1 9 8 6 ) . T h e s e a u t h o r s d e m o n s t r a t e d a d i r e c t c o r r e l a t i o n b e t w e e n t h i s r a t i o a n d MV0 2. S t o d d a r t e t a l . ( 1 9 8 7 ) d e m o n s t r a t e d t h e v a r i a b i l i t y o f th v a l u e s i n p a t i e n t s s u f f e r i n g f r o m a c u t e m y o c a r d i a l i n f a r c t i o n , who h a d u n d e r g o n e a n HDA s c a n a n d a MUGA s c a n ( M u l t i u s e r - g a t e d - a n g i o g r a p h i c - E F ) w i t h i n 6 d a y s o f i n f a r c t i o n . T h e a u t h o r s c o n c l u d e d t h a t i m p r o v e m e n t s i n i m a g i n g a n d b a c k g r o u n d s u b t r a c t i o n t e c h n i q u e s a r e n e e d e d b e f o r e t h i s i n f o r m a t i o n b e c o m e s u s e f u l . S i m i l a r c o n c l u s i o n s w e r e r e p o r t e d b y R a i l t o n e t a l . ( 1 9 8 7 ) a s a t e s t f o r CAD a n d b y G a v i n e t a l . ( 1 9 8 7 ) a s a n a s s e s s m e n t o f p e r c u t a n e o u s t r a n s l u m i n a l a n g i o p l a s t y . - 46 -c ) I o d i n a t e d P h e n y l p e n t a d e c a n o i c a c i d T h e p r o b l e m o f r a p i d m e t a b o l i c w a s h o u t o f s t r a i g h t c h a i n f a t t y a c i d i m a g i n g a g e n t s was a d d r e s s e d b y M a c h u l l a e t a l . ( 1 9 8 0 ) . T h e a u t h o r s p r o p o s e d a n a l t e r a t i o n i n t h e f a t t y a c i d c a r r i e r , s u c h t h a t h i g h i o d i n e b a c k g r o u n d a c t i v i t y w o u l d b e m i n i m i z e d . M a c h u l l a a n d c o w o r k e r s c h o s e o m e g a - p - 1 2 3 I - p h e n y l p e n t a d e c a n o i c a c i d ( 1 2 3 I P P A ) a s t h e i m a g i n g a g e n t b a s e d o n w o r k b y K n o o p ( 1 9 0 4 ) a n d D a k i n ( 1 9 0 9 ) . T h e r e s u l t o f a d d i n g a p h e n y l g r o u p , t o w h i c h t h e i o d i n e c a n b i n d , was a s u b s t a n t i a l d e c r e a s e i n f r e e r a d i o i o d i d e r e l e a s e . I n m i c e , i t was d e m o n s t r a t e d t h a t 4% o f i n j e c t e d r a d i o a c t i v i t y a c c u m u l a t e d i n t i s s u e w i t h a h e a r t / b l o o d r a t i o o f 20. I n r a b b i t s , t h e i m a g e / b a c k g r o u n d r a t i o was 2 a t 2 0 - 3 0 m i n p o s t - i n j e c t i o n . E i s e n h u t ( 1 9 8 2 ) d e m o n s t r a t e d h i g h e r u p t a k e a n d l o w e r b a c k g r o u n d a c t i v i t y w i t h 1 2 3 I P P A i n t h e r a b b i t h e a r t , w h e n c o m p a r e d t o i o d o - h e x a d e c a n o i c a c i d . T h e c a t a b o l i c e n d p r o d u c t o f 1 2 3 I P P A m e t a b o l i s m was i d e n t i f i e d a s i o d o b e n z o i c a c i d , w h i c h was k nown t o b e e x c r e t e d l i k e h i p p u r i c a c i d . T h i s e f f e c t i v e l y e l i m i n a t e d a n y r e s i d u a l 1 2 3 I f r o m t h e c i r c u l a t i o n a n d l i m i t e d t h e b a c k g r o u n d a c t i v i t y . R e s k e e t a l . ( 1 9 8 4 b , c ) a l s o e x a m i n e d t h e d i f f e r e n c e s b e t w e e n 1 2 3 I P P A a n d t h e p h y s i o l o g i c a l a g e n t 1 4 C - p a l m i t i c a c i d . I n a L a n g e n d o r f f r a t h e a r t m o d e l , t h e a u t h o r s m e a s u r e d r a t e s o f p r o d u c t i o n o f 1 4 C 0 2 a n d 1 2 3 I - b e n z o i c a c i d . T h e c o r r e l a t i o n c o e f f i c i e n t b e t w e e n t h e two b y - p r o d u c t s was r = 0.87. S i m i l a r r e s u l t s w e r e r e p o r t e d b y E c k h a r d t e t a l . ( 1 9 8 4 ) . T h e r e f o r e , a l t h o u g h 1 2 3 I P P A i s n o t a p h y s i o l o g i c a l a g e n t , i t d o e s a p p e a r t o a d e q u a t e l y r e f l e c t t h e m e t a b o l i s m o f f a t t y a c i d s . - 47 -I n o r d e r t o e x a m i n e t h e p h a r m a c o k i n e t i c s o f 1 2 3 I P P A , D a u s e t a l . ( 1 9 8 1 ) u t i l i z e d mouse m y o c a r d i u m a n d d e m o n s t r a t e d e f f e c t i v e e x t r a c t i o n . T h e a u t h o r s a l s o s h o w e d t h a t a p o r t i o n o f t h e f a t t y a c i d was i n c o r p o r a t e d i n t o t h e t r i g l y c e r i d e p o o l . R e s k e e t a l . ( 1 9 8 2 a ) e x a m i n e d m y o c a r d i a l u p t a k e a n d w a s h o u t o f 1 2 3 I P P A f r o m r a t m y o c a r d i u m . A m a x i m a l u p t a k e o f 17% d o s e / g t i s s u e o c c u r r e d a t 2 m i n u t e s p o s t - i n j e c t i o n . T h e a b s o l u t e u p t a k e o f b o t h i o d o - h e x a d e c a n o i c a c i d a n d 1 2 3 I P P A was s i m i l a r ; h o w e v e r , t h e r e l a t i v e u p t a k e w i t h i n i s c h e m i c m y o c a r d i a l s e g m e n t s was g r e a t e r f o r t h e s t r a i g h t c h a i n a n a l o g u e t h a n f o r t h e p h e n y l a t e d d e r i v a t i v e ( W e s t e r a e t a l . . 1 9 8 3 ) . T h e a u t h o r s b e l i e v e d t h a t two u p t a k e m e c h a n i s m s e x i s t e d - p a s s i v e d i f f u s i o n a n d a c a r r i e r - m e d i a t e d d i f f u s i o n . T h e y h y p o t h e s i z e d t h a t t h e l a t t e r b e c o m e s l e s s a c t i v e a s i s c h e m i a o c c u r s . T h e m a x i m a l u p t a k e b y t h e m y o c a r d i u m r e p o r t e d b y R e s k e e t a l . ( 1 9 8 2 a ) was f o l l o w e d b y a two c o m p o n e n t w a s h o u t c u r v e . M o s t o f t h e 1 2 3 I P P A was p r e s e n t a s m y o c a r d i a l l i p i d s w i t h <1% d o s e / g p r e s e n t a s f r e e 1 2 3 I P P A . T h i s f i n d i n g was a l s o r e p o r t e d b y F u c h s e t a l . ( 1 9 8 2 ) . T h e m a j o r c a t a b o l i c p r o d u c t f o u n d i n t h e o u t f l o w p e r f u s a t e o f t h e L a n g e n d o r f f p r e p a r a t i o n was 1 2 3 I - b e n z o i c a c i d ( E r c a n e t a l . . 1 9 8 3 ) . S c h m i t z e t a l . ( 1 9 8 4 ) u t i l i z e d a g a s - l i q u i d c h r o m a t o g r a p h i c - m a s s s p e c t r o m e t r i c a n a l y s i s m e t h o d t o c h a r a c t e r i z e t h e m e t a b o l i t e s f o r m e d i n t h e l i p i d e x t r a c t o f t h e p e r f u s a t e a s i t was r e m o v e d f r o m t h e p e r f u s e d r a t h e a r t . T h r e e m a j o r m e t a b o l i t e s w e r e i d e n t i f i e d , i n c l u d i n g o m e g a - p - i o d o - p h e n y l p r o p i o n i c a c i d , o m e g a - p - i o d o - p h e n y l p r o p e n o i c a c i d a n d , l a s t l y , p - i o d o b e n z o i c a c i d . - 48 -T i m e - a c t i v i t y c u r v e s , d e r i v e d f r o m t h e w a s h o u t r a t e s o f 1 2 3 I P P A , f o l l o w i n g i n t r a c o r o n a r y a d m i n i s t r a t i o n i n p a t i e n t s w i t h v a l v u l a r h e a r t d i s e a s e u s i n g p l a n a r gamma i m a g i n g , e x h i b i t e d a v a s c u l a r s p i k e a t 0-10 s e c o n d s , w h i c h was f o l l o w e d b y a m u l t i c o m p o n e n t w a s h o u t ( R e s k e e t a l . . 1 9 8 2 c , d ) . T h e c u r v e c o u l d b e d i v i d e d i n t o 3 s l o p e s e x h i b i t i n g th v a l u e s o f 0 . 2 4 - 0 . 5 m i n , 6-10 m i n a n d 4 0 - 6 0 m i n . S i m i l a r f i n d i n g s w e r e r e p o r t e d b y R e s k e e t a l . ( 1 9 8 4 a ) i n w h i c h p a t i e n t s w i t h CAD w e r e s t u d i e d . A n a l y s i s o f b l o o d r a d i o a c t i v i t y a t 1-5 m i n u t e s p o s t - i n j e c t i o n r e v e a l e d t h a t 5 0 - 8 0 % o f t h e a c t i v i t y was i n t h e f o r m o f 1 2 3 I - h i p p u r i c a c i d a n d 1 2 3 I - b e n z o i c a c i d . R e s k e e t a l . ( 1 9 8 2 a , d ) a l s o r e p o r t e d t h a t 2 5 - 3 0 % o f 1 2 3 I P P A was e x t r a c t e d b y n o r m a l l y p e r f u s e d m y o c a r d i u m a t e v e r y b l o o d p a s s a g e . K u l k a r n i e t a l . ( 1 9 8 5 ) o b t a i n e d c l e a r m y o c a r d i a l i m a g e s f r o m 4 t o 40 m i n f o l l o w i n g t h e i n j e c t i o n o f 1 2 3 I P P A a t r e s t a n d a f t e r p e a k e x e r c i s e i n n o r m a l v o l u n t e e r s w i t h n o a d v e r s e e f f e c t s . F o l l o w i n g t h i s w o r k , many s t u d i e s w e r e p e r f o r m e d , e x a m i n i n g t h e p o t e n t i a l o f 1 2 3 I P P A i n a n a l y z i n g c h a n g e s i n f a t t y a c i d w a s h o u t i n v a r i o u s p a t h o l o g i c a l s t a t e s . I n p a t i e n t s s u f f e r i n g f r o m CAD, D u d c z a k e t a l . ( 1 9 8 2 b ) a s s e s s e d m y o c a r d i a l w a s h o u t r a t e s o f 1 2 3 I - H D A a n d 1 2 3 I P P A . T h e y f o u n d t h a t b o t h a g e n t s e x h i b i t e d b i e x p o n e n t i a l c u r v e s a n d t h a t h a l o t h a n e a n e s t h e s i a p r o l o n g e d th v a l u e s . I n a l a t e r s t u d y , D u d c z a k e t a l . ( 1 9 8 3 a ) f o u n d th v a l u e s o f 69.3 m i n , w h i c h w e r e i n a c c o r d a n c e w i t h t h e v a l u e s r e p o r t e d f o r p a t i e n t s w i t h CAD f r o m h i s e a r l i e r w o r k . T h e p a r a i s o m e r was a l s o s h o wn t o h a v e a d e c r e a s e d m y o c a r d i a l / b a c k g r o u n d r a t i o r e l a t i v e t o t h e o r t h o i s o m e r ( 1 . 5 0 v s . 1.96, r e s p e c t i v e l y ) . M a c h u l l a e t a l . ( 1 9 8 6 ) , h o w e v e r , r e p o r t e d - 49 -d e c r e a s e d m y o c a r d i a l u p t a k e f o r t h e o r t h o i s o m e r . K a i s e r e t a l . ( 1 9 8 8 ) h a v e d e m o n s t r a t e d t h a t o - 1 2 3 I P P A i s r e t a i n e d i n human m y o c a r d i u m w i t h l o n g r e t e n t i o n t i m e s r e l a t i v e t o p - 1 2 3 I P P A . A n u m b e r o f o t h e r s t u d i e s h a v e b e e n p e r f o r m e d e x a m i n i n g f a t t y a c i d w a s h o u t r a t e s i n p a t i e n t s w i t h CAD, i n c l u d i n g V y s k a e t a l . ( 1 9 8 7 ) , D a v i e s e t a l . ( 1 9 8 7 ) a n d R e s k e e t a l . ( 1 9 8 7 ) . P a t i e n t s s u f f e r i n g f r o m c a r d i o m y o p a t h y h a v e a l s o b e e n a s s e s s e d u s i n g t h i s t y p e o f a n a l y s i s . D u d c z a k e t a l . ( 1 9 8 2 b ) d e m o n s t r a t e d e x t r e m e l y p r o l o n g e d th v a l u e s i n s u c h p a t i e n t s s h o w i n g b i e x p o n e n t i a l l a b e l e d - f a t t y a c i d w a s h o u t c u r v e s . T h e s e c a r d i o m y o p a t h i c p a t i e n t s e x h i b i t e d g r o s s , i r r e g u l a r a c t i v i t y , w i t h f a t t y a c i d w a s h o u t r a t e s o f 4 3 - 1 5 0 m i n ( D u d c z a k e t a l . . 1 9 8 3 b ) . K n a p p e t a l . ( 1 9 8 7 ) e x a m i n e d f a t t y a c i d m y o c a r d i a l e x t r a c t i o n i n c a r d i o m y o p a t h y p a t i e n t s a n d 4 n o r m a l s u b j e c t s . T h e c a r d i o m y o p a t h y p a t i e n t s w e r e s u b d i v i d e d i n t o d i l a t e d c a r d i o m y o p a t h y (DCM) ( n = 9) a n d h y p e r t r o p h i c c a r d i o m y o p a t h y (HCM) ( n = 1 1 ) . W h i l e u n d e r g o i n g f a s t i n g e r g o m e t r y , p a t i e n t s w e r e i n j e c t e d w i t h 1 2 3 I P P A I V . T h e p a t i e n t s s u f f e r i n g f r o m DCM h a d e x t r a c t i o n f r a c t i o n s ( E F ) 10% b e l o w n o r m a l a n d i n t h e HCM g r o u p , E F v a l u e s w e r e 50% b e l o w n o r m a l i n t h e s e p t u m a n d 12% b e l o w n o r m a l i n t h e p o s t e r o l a t e r a l w a l l . A n i m a l s t u d i e s a s s e s s i n g m y o c a r d i a l w a s h o u t r a t e s o f 1 2 3 I P P A h a v e p r o d u c e d s i m i l a r f i n d i n g s a s d e s c r i b e d a b o v e . D u d c z a k e t a l . ( 1 9 8 2 b ) f o u n d b i e x p o n e n t i a l w a s h o u t c u r v e s f o r 1 2 3 I - H D A a n d 1 2 3 I P P A i n c a l v e s a n d p r o l o n g e d w a s h o u t r a t e s f o l l o w i n g c o r o n a r y a r t e r y o c c l u s i o n . R e s k e e t a l . ( 1 9 8 8 ) e x a m i n e d 1 2 3 I P P A u p t a k e i n a c o r o n a r y o c c l u s i o n ( 4 0 m i n ) / r e p e r f u s i o n ( 3 0 m i n ) L a n g e n d o r f f s w i n e h e a r t m o d e l . C a r d i a c 1 2 3 I P P A u p t a k e was f o u n d t o b e p r e s e r v e d i n v i a b l e t i s s u e - 50 -f o l l o w i n g r e v e r s i b l e i s c h e m i a . C l e a r l y , 1 2 3 I P P A shows p o t e n t i a l a s a m y o c a r d i a l i m a g i n g a g e n t c a p a b l e o f a s s e s s i n g m e t a b o l i s m a s w e l l a s f l o w . S i n c e p e r f u s i o n d i r e c t l y i n f l u e n c e s 1 2 3 I P P A a v a i l a b i l i t y , t h e r e l a t i o n s h i p b e t w e e n m y o c a r d i a l b l o o d f l o w a n d u p t a k e o f 1 2 3 I P P A m u s t b e a d d r e s s e d ( R e s k e e t a l . . 1 9 8 4 d ) . R e g i o n a l m y o c a r d i a l b l o o d f l o w (RMBF) was f o u n d t o b e 9 0 - 1 2 0 m l / m i n / 1 0 0 g i n c o n t r o l a n i m a l s w i t h a t o t a l c a r d i a c u p t a k e o f 1 2 3 I P P A o f 4 . 5 - 6 . 0 % o f t h e i n j e c t e d d o s e . I n o r d e r t o d e t e r m i n e t h e r e l a t i o n s h i p b e t w e e n t h e two, c o r r e l a t i o n a n a l y s i s was p e r f o r m e d . I t r e v e a l e d a c l o s e c o r r e l a t i o n ( r = 0 . 9 4 ) b e t w e e n RMBF a n d 1 2 3 I P P A u p t a k e u n d e r c o n t r o l c o n d i t i o n s a n d a s w e l l a s u n d e r c o n d i t i o n s o f a c u t e i s c h e m i a w h e r e RMBF = 2 0 - 5 0 m l / m i n / l O O g . A t i n c r e a s e d f l o w r a t e s , s u c h a s t h o s e s e e n w i t h p a c i n g , t h e s e a u t h o r s f o u n d o n l y a m o d e r a t e i n c r e a s e o f 1 2 3 I P P A u p t a k e . I t was a l s o n o t e d t h a t a p l a t e a u o r u p p e r l i m i t e x i s t e d a t 1 5 0 - 1 7 0 m l / m i n / l O O g a n d t h a t e x c e e d i n g t h i s l e v e l r e s u l t e d i n a f a l l i n 1 2 3 I P P A u p t a k e . S c h o e n e t a l . ( 1 9 8 4 ) r e p o r t e d s i m i l a r f i n d i n g s w i t h l i m i t e d u p t a k e a t h i g h e r f l o w r a t e s . C a l d w e l l e t a l . ( 1 9 8 7 ) d e t e r m i n e d w h e t h e r 1 2 3 I P P A u p t a k e a t h i g h f l o w r a t e s c o u l d r e f l e c t a c c u r a t e l y RMBF. T h e a u t h o r s c o m p a r e d 1 2 3 I P P A u p t a k e a n d m i c r o s p h e r e d e p o s i t i o n d u r i n g t r e a d m i l l e x e r c i s e i n 6 c h r o n i c a l l y i n s t r u m e n t e d d o g s . W h i l e t h e a n i m a l s w e r e r u n n i n g , t h e c o r o n a r y a r t e r y was o c c l u d e d a n d t h e a g e n t s i n j e c t e d i n t o t h e l e f t a t r i u m . I t was f o u n d t h a t 1 2 3 I P P A c o u l d b e u s e d t o a c c u r a t e l y a s s e s s RMBF. A n i n t e r e s t i n g f i n d i n g w i t h m a j o r i m p l i c a t i o n s i s t h e i n h i b i t i o n o f 1 2 3 I P P A o r f a t t y a c i d m e t a b o l i s m b y l a c t a t e ( E c k a r d t e t a l . . 1 9 8 4 ) . - 51 -R e s k e e t a l . ( 1 9 8 6 ) a l s o r e p o r t e d t h a t l a c t a t e i n h i g h c o n c e n t r a t i o n s d e c r e a s e d f a t t y a c i d o x i d a t i o n i n c a n i n e h e a r t s a n d L a n g e n d o r f f r a t h e a r t s . D u w e l e t a l . ( 1 9 8 7 ) e x a m i n e d t h e e f f e c t s o f s o d i u m l a c t a t e i n f u s i o n b e f o r e s c i n t i g r a p h y i n p a t i e n t s s u f f e r i n g f r o m CAD. S i m i l a r th p a t t e r n s w e r e shown f o r t h e l a c t a t e i n t e r v e n t i o n g r o u p a n d i s c h e m i a g r o u p , i e . i n h i b i t i o n o f f a t t y a c i d o x i d a t i o n . D u w e l e t a l . ( 1 9 8 8 ) c o n t i n u e d t h i s w o r k a n d f o u n d t h a t n o t o n l y d o e s l a c t a t e i n h i b i t F F A o x i d a t i o n u n d e r i s c h e m i c c o n d i t i o n s , b u t i t a l s o d o e s s o u n d e r n o r m a l c o n d i t i o n s . T h i s c o u l d h a v e p a r t i c u l a r l y p r o f o u n d e f f e c t s d u r i n g a n a e r o b i c s t a t e s o r i s c h e m i c c o n d i t i o n s . I n v i e w o f i t s a p p a r e n t s u c c e s s i n t h e a s s e s s m e n t o f m y o c a r d i a l m e t a b o l i s m , some i n v e s t i g a t o r s h a v e a t t e m p t e d t o a s s e s s c a r d i a c g e o m e t r y u s i n g 1 2 3 I P P A a n d SPECT i m a g i n g . R e s k e e t a l . ( 1 9 8 2 d ) o b t a i n e d q u a l i t y i m a g e s , v i s u a l i z i n g m y o c a r d i a l i n f a r c t s a s a r e a s o f r e d u c e d 1 2 3 I P P A u p t a k e . E x a m i n a t i o n o f k i n e t i c s r e v e a l e d m a r k e d p r o l o n g a t i o n o f th v a l u e s w i t h i n i n f a r c t e d d o g s a s c o m p a r e d t o c o n t r o l a n i m a l s ( 2 0 2 + 65 c o m p a r e d t o 41.5 + 7.1 m i n , r e s p e c t i v e l y ) . No a t t e m p t was made i n t h i s s t u d y t o q u a n t i t a t e t h e m y o c a r d i a l i n f a r c t i o n i n t e r m s o f t i s s u e v o l u m e s . R e l l a s e t a l . ( 1 9 8 3 ) c o n d u c t e d a s i m i l a r e x p e r i m e n t i n w h i c h u p t a k e a n d w a s h o u t o f 1 2 3 I P P A u n d e r c o n t r o l c o n d i t i o n s a n d a f t e r p e r m a n e n t a n d t e m p o r a r y c o r o n a r y a r t e r y o c c l u s i o n i n d o g s w e r e m e a s u r e d . I n f a r c t e d m y o c a r d i a l s e g m e n t s w i t h f i x e d LAD c l o s u r e e x h i b i t e d r e d u c e d u p t a k e . I n d o g s w i t h t e m p o r a r y o c c l u s i o n f o l l o w e d b y r e p e r f u s i o n , m y o c a r d i a l u p t a k e was o n l y s l i g h t l y d e c r e a s e d c o m p a r e d t o n o r m a l m y o c a r d i a l s e g m e n t s . T h e z o n e s o f i n f a r c t i o n e s t i m a t e d b y - 52 -e x a m i n i n g 1 2 3 I P P A k i n e t i c s c o r r e l a t e d c l o s e l y w i t h z o n e s o f i n f a r c t i o n d e m a r c a t e d b y t e t r a z o l i u m s t a i n i n g . . I t was n o t e d , h o w e v e r , t h a t t h e r e was a r a p i d i n i t i a l w a s h o u t f r o m t h e m y o c a r d i u m w h i c h was p r o b a b l y due t o b e t a o x i d a t i o n . K i e s s e t a l . ( 1 9 8 7 ) a l s o c o u l d d i f f e r e n t i a t e b e t w e e n a r e a s o f i n f a r c t i o n a n d o f s t u n n e d , b u t v i a b l e , m y o c a r d i u m b y 1 2 3 I P P A w a s h o u t r a t e s . S c h w a i g e r e t a l . ( 1 9 8 7 ) e x a m i n e d t h e m e t a b o l i c c o n s e q u e n c e s o f s h o r t p e r i o d s ( 3 0 m i n ) o f m y o c a r d i a l i s c h e m i a . T h e o u t c o m e was a n i n c r e a s e i n t h e r e l a t i v e u p t a k e o f 1 8 F - d e o x y g l u c o s e , e v e n t h o u g h n o r m a l m y o c a r d i a l b l o o d f l o w was r e i n s t i t u t e d . P i p p i n e t a l . ( 1 9 8 7 ) d e m o n s t r a t e d t h a t , i n p a t i e n t s w i t h CAD, 123JPPA is more s e n s i t i v e t h a n t h a l l i u m i n t h e d e t e c t i o n o f r e v e r s i b l e a b n o r m a l i t i e s , b u t was e q u i v a l e n t t o t h a l l i u m f o r d e t e c t i o n o f c o r o n a r y a r t e r y s t e n o s i s . d ) M i s c e l l a n e o u s F a t t y A c i d s S t r a i g h t - c h a i n f a t t y a c i d s a n d t h e i o d o p h e n y l f a t t y a c i d s h a v e b e e n s hown t o s u f f e r f r o m r a p i d i n i t i a l w a s h o u t , w h i c h d e c r e a s e s i m a g e q u a l i t y o v e r t i m e . T h u s , t h e b e n e f i t s o f i n c r e a s i n g m y o c a r d i a l r e s i d e n c e t i m e c a n b e a p p r e c i a t e d when a n a t t e m p t i s made t o a s s e s s p e r f u s i o n d e f e c t s , i n p a r t i c u l a r , m y o c a r d i a l i n f a r c t i o n . S u c h a n a s s e s s m e n t may h a v e i m p o r t a n t c l i n i c a l s i g n i f i c a n c e a n d i n f l u e n c e p a t i e n t management a n d t h e r a p e u t i c i n t e r v e n t i o n s . T h e u t i l i z a t i o n o f r a d i o n u c l i d e s t o a s s e s s m y o c a r d i a l p e r f u s i o n d e f e c t s h a s b e e n a t t e m p t e d b y many g r o u p s . C o n v e n t i o n a l i m a g i n g s y s t e m s ( i e . p l a n a r i m a g i n g ) h a v e a t t e m p t e d t o c o m p r e s s t h e 3 - d i m e n s i o n a l i m a g e o f t h e h e a r t o n t o a 2 - d i m e n s i o n a l p l a n e . K e y e s e t a l . ( 1 9 7 8 ) b e l i e v e d t h a t t o a c c u r a t e l y - 53 -q u a n t i f y t h e s e p e r f u s i o n d e f e c t s , a m e t h o d was r e q u i r e d w h i c h w o u l d d i s p l a y 3 - d i m e n s i o n a l i n f o r m a t i o n . R e s u l t s f r o m t h e i r w o r k d e m o n s t r a t e d t h e l o c a t i o n o f t h e m y o c a r d i a l i n f a r c t i o n i n a l l t h r e e p l a n e s ( x , y a n d z ) . T h e u t i l i z a t i o n o f SPECT o f f e r e d d i s t i n c t a d v a n t a g e s o v e r c o n v e n t i o n a l p l a n a r i m a g i n g when a t t e m p t i n g t o q u a n t i t a t e p e r f u s i o n d e f e c t s ( C a l d w e l l e t a l . . 1 9 8 4 ) . One m e t h o d o f r a d i o p h a r m a c e u t i c a l d e v e l o p m e n t , ( p r o p o s e d 34 y e a r s a g o i n 1 9 5 4 ) , w h i c h w o u l d a i d i n p e r f u s i o n i m a g i n g , was m e t a b o l i c t r a p p i n g , ( G a l l a g h e r e t a l . . 1 9 7 8 ) . B y s u b s t i t u t i n g a f l u o r i n e a t o m f o r a h y d r o x y l g r o u p a t c a r b o n 2 o f d e o x y g l u c o s e , G a l l a g h e r s h o w e d a r e d u c t i o n i n t i s s u e w a s h o u t r a t e s . One a t t e m p t a t m e t a b o l i c t r a p p i n g was p e r f o r m e d b y Goodman e t a l . ( 1 9 8 2 ) . T h e i n t r o d u c t i o n o f a t e l l u r i u m h e t e r o a t o m o n t o c a r b o n 6 o f 1 5 - p - i o d o p h e n y l p e n t a d e c a n o i c a c i d b l o c k e d b e t a - o x i d a t i o n a n d i n c r e a s e d t h e m y o c a r d i a l r e s i d e n c e t i m e . T h i s a g e n t a l s o d e m o n s t r a t e d r a p i d m y o c a r d i a l u p t a k e a n d l o w i n v i v o d e i o d i n a t i o n . A s i m i l a r s t u d y u s i n g a new t e l l u r i u m - i o d i n a t e d - p h e n y l p e n t a d e c a n o i c a c i d was p e r f o r m e d s h o w i n g m i n i m a l d e i o d i n a t i o n a n d p r o l o n g e d r e t e n t i o n t i m e s i n d o g s a n d c a t s (Goodman e t a l . . 1 9 8 5 ) . H o w e v e r , p r o b l e m s w i t h t o x i c i t y a n d s t a b i l i t y o f t h e t e l l u r i u m h e t e r o a t o m i n t h e p r e s e n c e o f o x y g e n h a s d r a m a t i c a l l y l i m i t e d i t s c l i n i c a l u s e f u l n e s s ( M a c h u l l a e t a l . . 1 9 8 6 ) . E f f o r t s a r e c o n t i n u i n g t o w a r d s t h e d e v e l o p m e n t o f a t e l l u r i u m f a t t y a c i d a n a l o g u e f o r p e r f u s i o n s t u d i e s ( S r i v a s t a v a e t a l . . 1 9 8 7 ) . A l t e r n a t i v e l y , L i v n i e t a l . ( 1 9 8 2 ) h y p o t h e s i z e d t h a t a l a b e l e d s t r a i g h t - c h a i n f a t t y a c i d t h a t i s p a r t i a l l y m e t a b o l i z e d a n d t r a p p e d w i t h i n t h e m y o c a r d i u m b y i n t r o d u c i n g a m e t h y l g r o u p a t t h e b e t a c a r b o n - 54 -may o f f e r a n i d e a l p e r f u s i o n i m a g i n g a g e n t . I n t h e i r s c h e m e , b e t a - m e t h y l [ 1 1 C ] - h e p t a d e c a n o i c a c i d was u t i l i z e d w i t h t h e b e t a - o x i d a t i o n s e q u e n c e b e i n g h a l t e d p r i o r t o t h e f o r m a t i o n o f t h e c o r r e s p o n d i n g b e t a - k e t o - a c y l CoA ( F i g u r e 5 ) . - 55 -F i g u r e 5 - M e t a b o l i c t r a p p i n g s c h e m e - 55a -S t r a i g h t - C h a i n Fatty A c i d Beta-Methyl F a t t y A c i d RCH2CH2C00H RCH2CH2C0SCoA Dehydrogenase RCH = CHCOSCoA Hydratase RCH — CH,C0SCoA ) 2 OH Dehydrogenase RCCHoC0SCoA II 2 0 HSCoA RCOSCoA + CH3COSC0A RCHCH2C00H CH-RCHCH-COSCoA I 2 CH3 Dehydrogena se RC = CHCOSCoA I CH3 Hydratase OH RCCH2COSCoA CH. Dehydrogenase^ - 56 -T h e r a d i o a c t i v i t y o f t h i s a g e n t i n b o t h r a t s a n d d o g s s h o w e d v e r y l i t t l e d e c r e a s e o v e r t i m e ( 2 . 3 % d o s e / g t i s s u e a t 5 m i n u t e s t o 2.7% d o s e / g t i s s u e a t 60 m i n u t e s ) . T h u s , t h e f a t t y a c i d was e x t r a c t e d b u t m e t a b o l i s m h a l t e d w i t h t h e r a d i o a c t i v e l a b e l r e m a i n i n g w i t h i n t h e m y o c a r d i u m . T h e m e c h a n i s m o f m y o c a r d i a l r e t e n t i o n was a l s o s u g g e s t e d t o b e s t e r i c a n d / o r c h e m i c a l i n h i b i t i o n f r o m t h e m e t h y l g r o u p (Goodman e t a l . . 1984 a n d L i v n i e t a l . . 1 9 8 5 ) . A b e n d s c h e i n e t a l . ( 1 9 8 4 ) a l s o u t i l i z e d 1 1C - b e t a - m e t h y l - h e p t a d e c a n o i c a c i d i n o p e n - c h e s t d o g s f o l l o w i n g L C X m a r g i n a l b r a n c h o c c l u s i o n a n d r e p e r f u s i o n . E x t r a c t i o n f r a c t i o n s w e r e c a l c u l a t e d (25 + 5% i n n o r m a l a n d 45 + 15% i n i s c h e m i c z o n e s ) a n d i t was n o t e d t h a t 5% o f t h e e x t r a c t e d a g e n t was o x i d i z e d i n n o r m a l z o n e s , w h i l e <1% was o x i d i z e d w i t h i n i s c h e m i c z o n e s . B l o c k i n g t h e b e t a - o x i d a t i o n s e q u e n c e r e s u l t s i n m i n o r v e n t r i c u l a r d y s f u n c t i o n , i n c l u d i n g d e c r e a s e d L V S P a n d % s y s t o l i c w a l l t h i c k e n i n g ( W i j n s e t a l . . 1 9 8 5 ) . M e t a b o l i c a l t e r a t i o n s a l s o e n s u e d , i n c l u d i n g d e c r e a s e d MV0 2, a 60% d e c r e a s e i n F F A u t i l i z a t i o n a n d a n 18% i n c r e a s e i n g l u c o s e a n d l a c t a t e u s a g e ( W i j n s e t a l . . 1 9 8 5 ) . T h e l a t t e r w o u l d i n c r e a s e t h e m y o c a r d i a l r e t e n t i o n o f f a t t y a c i d s a n d i n c r e a s e t h e i r t i m e s p e n t w i t h i n t h e m y o c a r d i u m , t h e r e b y i m p r o v i n g i m a g e q u a l i t y when s c a n n i n g . S i n c e Goodman e t a l . ( 1 9 8 2 ) f i r s t s u g g e s t e d t h e p o t e n t i a l v a l u e o f p h e n y l p e n t a d e c a n o i c a c i d w i t h b e t a - m e t h y l a t i o n , some i n v e s t i g a t o r s h a v e e x t e n d e d t h i s w o r k . M i l l e r e t a l . ( 1 9 8 5 ) e x a m i n e d t h e m e t a b o l i c a c t i v i t y i n a n o r m a l z o n e ( p o s t e r i o r w a l l ) a n d b o r d e r a n d c e n t r a l i s c h e m i c z o n e s o f c a n i n e m y o c a r d i u m f o l l o w i n g LAD o c c l u s i o n / r e p e r f u s i o n a n d s c a n n i n g o f c l o s e d ' c h e s t d o g s . T h e m e t a b o l i c b e h a v i o u r o f n o r m a l , - 57 -b o r d e r i s c h e m i c a n d c e n t r a l i s c h e m i c ( p a r t i a l l y i n f a r c t e d ) z o n e s d i f f e r e d . T h e l a t t e r two a r e a s w e r e s i g n i f i c a n t l y d i f f e r e n t f r o m n o r m a l z o n e s , w i t h th v a l u e s w i t h i n t h e i s c h e m i c z o n e s b e i n g s i g n i f i c a n t l y p r o l o n g e d r e l a t i v e t o t h e c o n t r o l z o n e s ( 1 1 5 . 6 + 29 c o m p a r e d t o 86.5 + 3 m i n , r e s p e c t i v e l y ) . K n a p p e t a l . ( 1 9 8 6 a , b ) r e p o r t t h a t m e t h y l s u b s t i t u t i o n c o n s i d e r a b l y i n c r e a s e d t h e m y o c a r d i a l h a l f - l i v e s i n f a s t e d r a t s : i o d o p h e n y l p e n t a d e c a n o i c a c i d ( I P P ) , 5-10 m i n ; b e t a - m e t h y l - i o d o p h e n y l p e n t a d e c a n o i c a c i d ( B M I P P ) , 30-45 m i n ; d i m e t h y l - i o d o p h e n y l p e n t a d e c a n o i c a c i d ( D M I P P ) , 6-7 h r s . De G r a d o - e t a l . ( 1 9 8 7 ) , i n a w o r k i n g i s o l a t e d r a t h e a r t m o d e l , d e m o n s t r a t e d h i g h e r m y o c a r d i a l r e t e n t i o n t i m e s f o r b e t a - m e t h y l - I P P A c o m p a r e d t o I P P A . I n o r d e r t o f u r t h e r u n d e r s t a n d t h e s e d i f f e r e n c e s i n m y o c a r d i a l r e t e n t i o n , A m b r o s e e t a l . ( 1 9 8 7 a ) e x a m i n e d s u b c e l l u l a r d i s t r i b u t i o n p a t t e r n s o f t h e s e a g e n t s . T h e d i m e t h y l a n a l o g u e was f o u n d p r e d o m i n a n t l y i n t h e m i t o c h o n d r i a a n d m i c r o s o m e s ( 3 4 % a n d 3 8 % ) , w h i l e I P P s h o w e d l i t t l e a c c u m u l a t i o n w i t h i n t h e s e o r g a n e l l e s ( 1 8 % a n d 1 5 % ) . I n a n e a r l i e r s t u d y , O t t o e t a l . ( 1 9 8 5 a ) r e p o r t e d a n a c c u m u l a t i o n o f 13.4 + 1.7% a n d 8.3 + 1.1% o f I P P w i t h i n m i t o c h o n d r i a a n d m i c r o s o m e s , r e s p e c t i v e l y . T h e b e t a - m e t h y l a n a l o g u e , h o w e v e r , s h o w e d 13.7 ± 0.3% a n d 3.7 ± 0.2% a c c u m u l a t i o n , r e s p e c t i v e l y , w i t h r e t e n t i o n o f a c t i v i t y o v e r t i m e . D u d c z a k e t a l . ( 1 9 8 6 ) a s s e s s e d t h e m e t a b o l i c b e h a v i o r o f B 1 2 3 I P P A i n p a t i e n t s s u f f e r i n g f r o m CAD a n d a c u t e MI. F r o m p l a s m a a n d u r i n e s a m p l e s , b e n z o i c a n d h i p p u r i c a c i d s w e r e i d e n t i f i e d a s t h e m a j o r m e t a b o l i c b y p r o d u c t s u s i n g T L C . I n o r d e r t o d e t e r m i n e t h e c l i n i c a l u s e f u l n e s s o f s u c h a n a g e n t , i n v e s t i g a t o r s h a v e c o m p a r e d t h e c u r r e n t l y u s e d p e r f u s i o n i m a g i n g a g e n t , - 58 -2 0 1 T 1 w i t h t h e b e t a - m e t h y l - f a t t y a c i d . O k a n o e t a l . ( 1 9 8 7 ) m e a s u r e d u p t a k e a n d r e l e a s e o f B 1 2 5 I P P A , 1 2 5 I P P A a n d 2 0 1 T 1 i n c u l t u r e d m y o c y t e s f r o m mouse e m b r y o . T h e s e v a l u e s w e r e c o m p a r e d t o i n t r a c e l l u l a r A T P c o n t e n t a f t e r m e t a b o l i c i n h i b i t i o n b y c y a n i d e (CN) o r 2 - d e o x y g l u c o s e ( 2 - D G ) . T h e l a t t e r d e c r e a s e d B 1 2 5 I P P A u p t a k e a l t h o u g h i t d i d n o t a f f e c t i t s r e l e a s e . CN h a d no e f f e c t o n u p t a k e o r r e l e a s e o f B 1 2 5 I P P A . T h e c o r r e l a t i o n o f B 1 2 S I P P A w i t h i n t r a c e l l u l a r [ A T P ] was h i g h w i t h r = 0.89 ( p < 0 . 0 5 ) . T h e c o r r e l a t i o n o f 2 o i T 1 t o [ A T P ] , h o w e v e r , was p o o r w i t h a n r = 0.53 ( p N S ) . T h u s , f a t t y a c i d s o f f e r t h e d i s t i n c t a d v a n t a g e o f b e i n g a n i n d i c a t o r o f b o t h m y o c a r d i a l m e t a b o l i s m a n d p e r f u s i o n . I n c a n i n e s t u d i e s e x a m i n i n g t h e d i f f e r e n c e s o f b e t a - m e t h y l f a t t y a c i d s a n d 2 0 1 T 1 , K a i r e n t o e t a l . ( 1 9 8 8 ) r e p o r t e d t h a t b o t h a g e n t s a r e s u i t a b l e f o r d e t e c t i n g i n f a r c t i o n ( s e e n a s d e c r e a s e d a c t i v i t y w i t h i n t h e i n f a r c t e d t i s s u e ) . T h e 2 0 1 T 1 c o n s i s t e n t l y o v e r e s t i m a t e d t h e s i z e o f t h e p e r f u s i o n d e f e c t . R e d i s t r i b u t i o n was q u o t e d a s a n o t h e r i n a c c u r a c y w i t h t h e u s e o f 2 0 1 T 1 . F i s c h m a n n e t a l . ( 1 9 8 8 ) e x a m i n e d t h e p o s s i b l e b e n e f i t s o f c o m b i n e d f a t t y a c i d a n d t h a l l i u m i m a g i n g 7 d a y s a f t e r a c u t e l i g a t i o n o f LAD i n d o g s . T h e s e a u t h o r s f o u n d t h a t i n z o n e s o f d e c r e a s e d p e r f u s i o n , f a t t y a c i d a c c u m u l a t i o n o c c u r r e d i n 7 o f 7 a n i m a l s . T h e r e f o r e , i t a p p e a r s t h a t w i t h i n a r e a s o f d e c r e a s e d RMBF, f a t t y a c i d s c o n t i n u e t o b e t a k e n up b y t h e m y o c a r d i u m . A l t h o u g h a n i m a l s t u d i e s seem t o i n d i c a t e t h e u s e f u l n e s s o f c o m b i n i n g f a t t y a c i d w i t h t h a l l i u m f o r i m a g i n g o f m y o c a r d i a l i n f a r c t i o n , c l i n i c a l s t u d i e s w e r e n e e d e d t o o p t i m i s e i t s u s e f u l n e s s . - 59 -P a t i e n t s s u f f e r i n g f r o m s e v e r e m y o c a r d i a l i s c h e m i a ( 1 2 p t s - u n s t a b l e a n g i n a ( n = 2 ) a n d a c u t e MI, who u n d e r w e n t t h r o m b o l y s i s w i t h t r a n s l u m i n a l p e r c u t a n e o u s a n g i o p l a s t y ( n = 1 0 ) ) w e r e i m a g e d u s i n g t h a l l i u m a n d f a t t y a c i d ( S t r a u s s e t a l . . 1 9 8 7 ) . F r o m t h e s e s t u d i e s , v a r i a b i l i t y i n u p t a k e was n o t e d b a s e d o n t h e d e g r e e o f c o r o n a r y s t e n o s i s . T h r e e p a t t e r n s e m e r g e d : F F A u p t a k e » T 1 i n s e v e r e s t e n o s i s w i t h some f l o w o r g o o d c o l l a t e r a l c i r c u l a t i o n ( n = 6 ) ; F F A = T l i n t o t a l l y o c c l u d e d o r s e v e r e s t e n o s i s w i t h m i n i m a l f l o w ; a n d F F A < T l i n r e p e r f u s e d m y o c a r d i u m s u p p l i e d b y v e s s e l s w i t h m a r k e d r e s i d u a l s t e n o s i s b u t w i t h n o r m a l 2 0 1 T 1 d i s t r i b u t i o n a t r e s t ( n = 4 ) . T h u s , b o t h F F A a n d T l u s e d i n c o m b i n a t i o n may i n d e e d s u p p l y more i n f o r m a t i o n t h a n i f t h e a g e n t s a r e u s e d s i n g l y . W i t h t h e d i s c o v e r y o f m e t a b o l i c d e g r a d a t i o n o f m o n o - m e t h y l a t e d f a t t y a c i d s , i t was p o s t u l a t e d t h a t d i m e t h y l a t e d a n a l o g u e s may b e b e t t e r a b l e t o r e m a i n w i t h i n t h e m y o c a r d i u m ( M a c h u l l a e t a l . . 1 9 8 6 ; K n a p p e t a l . . 1 9 8 6 b ) . M y o c a r d i a l r e t e n t i o n t i m e s w e r e r e p o r t e d f o r n o n - m e t h y l a t e d , m o n o m e t h y l a t e d a n d d i m e t h y l a t e d - I P P A a n a l o g u e s , w i t h t h e l a t t e r s h o w i n g t h e l o n g e s t t i m e s w i t h i n t h e m y o c a r d i u m o f r a t s . H i g h h e a r t / b l o o d r a t i o s w e r e r e p o r t e d f o r a l l t h r e e a g e n t s ; h o w e v e r , t h e 4,4-DMIPP was shown t o h a v e t h e h i g h e s t h e a r t u p t a k e a n d b e s t h e a r t / b l o o d r a t i o . T h e s e f i n d i n g s v a r y , h o w e v e r , s i n c e 3,3-DMIPP h a s a l s o b e e n s hown t o h a v e t h e l o n g e s t r e t e n t i o n t i m e i n r a t s . I n v e s t i g a t i o n s c o n t i n u e i n a n a t t e m p t t o i d e n t i f y t h e i d e a l i m a g i n g a g e n t . J o n e s e t a l . ( 1 9 8 8 ) i n j e c t e d 1 - [ 1 X C ] - 3 , 3 - d i m e t h y l -h e p t a d e c a n o i c a c i d i n f a s t e d r a t s . T h e a g e n t s h o w e d m o d e r a t e u p t a k e w i t h c o n s i d e r a b l e w a s h o u t o f a c t i v i t y f r o m t h e m y o c a r d i u m o v e r t h e - 60 -i n i t i a l 30 m i n u t e p o s t - i n j e c t i o n p e r i o d . I n a c a n i n e s t u d y , u p t a k e was a g a i n l o w w i t h m a j o r l o c a l i z a t i o n w i t h i n t h e l u n g . T h e s e d a t a s u g g e s t t h a t d i m e t h y l s u b s t i t u t i o n a t t h e b e t a - p o s i t i o n may b e i n a d e q u a t e , due t o d e f i c i e n c i e s i n m y o c a r d i a l u p t a k e a n d r e t e n t i o n . D e m a i s o n e t a l . ( 1 9 8 8 ) , u s i n g 1 6 - ( 1 2 3 I ) - i o d o - 3 , 3 - m e t h y l h e x a d e c a n o i c a c i d i n j e c t e d d i r e c t l y i n t o t h e c o r o n a r y a r t e r y o f i s o l a t e d r a t h e a r t s , d e m o n s t r a t e d t h a t m o n o - b e t a , d i - a l p h a a n d d i - b e t a - m e t h y l a t e d i o d i n a t e d f a t t y a c i d s a r e s u i t a b l e a g e n t s f o r s t u d y i n g c e l l u l a r u p t a k e b e c a u s e o f t h e i r r e l a t i v e l y l o w o x i d a t i o n r a t e s . O t h e r p e r f u s i o n i m a g i n g a g e n t s b e i n g i n v e s t i g a t e d a r e t h e i o d o - v i n y l l o n g c h a i n f a t t y a c i d s , ( A m b r o s e e t  a l . . 1 9 8 7 b , c ; Som e t a l . . 1 9 8 8 ) . H o w e v e r , t h e r e i s c u r r e n t l y a l a c k o f a n i d e a l i m a g i n g a g e n t . - 61 -( 4 ) I S C H E M I A AND C A RDIOPLEGIA I s c h e m i a - i n d u c e d c h a n g e s h a v e b e e n i m p l i c a t e d i n t h e p o s t - o p e r a t i v e v e n t r i c u l a r d y s f u n c t i o n a s s o c i a t e d w i t h c o r o n a r y a r t e r y d i s e a s e (CAD) ( J a m i e s o n e t a l . . 1 9 8 3 ) . T h e s u b c e l l u l a r a n d c e l l u l a r c h a n g e s a s s o c i a t e d w i t h i s c h e m i a a r e d i v e r s e , a f f e c t i n g t r a n s m e m b r a n e i o n b a l a n c e , m i t o c h o n d r i a l a c t i v i t y , A T P p r o d u c t i o n a n d o x i d a t i v e m e t a b o l i s m ( T r u m p , 1 9 8 2 ) . I n o r d e r t o m i t i g a t e t h e s e d i v e r s e c h a n g e s , c a r d i o p l e g i c t e c h n i q u e s r e l y o n c o n s e r v a t i o n o f h i g h e n e r g y s t o r e s ( r a p i d d i a s t o l i c a r r e s t ) , s l o w i n g o f m e t a b o l i c r a t e ( h y p o t h e r m i a ) a n d c o m b a t t i n g t h e d e l e t e r i o u s s u b c e l l u l a r a n d c e l l u l a r c o n s e q u e n c e s o f i s c h e m i a t h r o u g h t h e u s e o f p r o t e c t i v e a d d i t i v e s . T h e r e a r e two b a s i c c a r d i o p l e g i c f o r m u l a t i o n s : 1) t h o s e w h i c h m i m i c e x t r a c e l l u l a r i o n c o n c e n t r a t i o n s ; a n d 2) t h o s e w i t h i n t r a c e l l u l a r i o n c o n c e n t r a t i o n s ( H e a r s e e t a l . . 1 9 8 6 ) . T h e f o r m e r c a n b e e i t h e r c r y s t a l l o i d p o t a s s i u m s o l u t i o n o r b l o o d - b a s e d . M o s t e x t r a c e l l u l a r s o l u t i o n s c o n t a i n N a + a n d C a + + . T h e i n t r a c e l l u l a r s o l u t i o n s c o n t a i n n o N a + o r C a ^ . E a c h t y p e o f s o l u t i o n h a s a d v a n t a g e s a n d d i s a d v a n t a g e s w h i c h m u s t b e c o n s i d e r e d when a c h o i c e b e t w e e n t h e m i s made. E x p e r i e n c e w i t h t h e s o l u t i o n b y t h e s u r g e o n w i l l u s u a l l y d e t e r m i n e t h e c h o i c e o f w h i c h s o l u t i o n i s u s e d . A n i m p o r t a n t a s p e c t o f m y o c a r d i a l p r o t e c t i o n w h i c h m u s t b e a d d r e s s e d i s r e p e r f u s i o n i n j u r y . Two c r i t i c a l f a c t o r s i n v o l v e d i n t h e l a t t e r p h e n o m e n o n i s t h e a c c u m u l a t i o n o f C a a n d t h e g e n e r a t i o n o f o x y g e n f r e e r a d i c a l s ( S h e n a n d J e n n i n g s , 1972; a n d G u a r n i e r i , 1 9 8 0 ) . S h e n a n d J e n n i n g s h a v e r e p o r t e d o n t h e e x p l o s i v e u p t a k e o f C a , o n r e p e r f u s i o n f o l l o w i n g 40 - 62 -m i n s . o f i s c h e m i a , w i t h a n a l m o s t 1 0 - f o l d i n c r e a s e a b o v e n o r m a l t i s s u e C a + + l e v e l s . I n d e e d , many r e s e a r c h e r s h a v e a t t e m p t e d t o c o n t r o l t h i s l a r g e i n f l u x o f C a i n t o t h e t i s s u e s f o l l o w i n g r e p e r f u s i o n b y t h e a d d i t i o n o f c a l c i u m e n t r y b l o c k e r s , s u c h a s v e r a p a m i l , n i f e d i p i n e o r d i l t i a z e m , a n d h a v e a c h i e v e d some d e g r e e o f s u c c e s s . T h e s e a g e n t s b l o c k c a l c i u m r e c e p t o r s i t e s , t h u s l i m i t i n g t h e amount o f t h i s i o n w h i c h e n t e r s t h e c e l l s . A l t h o u g h a p p e a r i n g b e n e f i c i a l , a l t e r i n g C a m o v e m e n t s w i t h i n t h e t i s s u e may a l s o b e d a n g e r o u s , s i n c e C a + + i o n s m e d i a t e membrane e x c i t a t i o n a n d c o n t r a c t i o n . T h e r e f o r e , c a l c i u m e n t r y b l o c k e r s a r e n o t w i t h o u t r i s k s . G u a r n i e r i ( 1 9 8 0 ) p r o p o s e d t h a t t h e e t i o l o g y o f r e p e r f u s i o n i n j u r y was r e l a t e d t o r e - o x y g e n a t i o n o f t h e m y o c a r d i u m . I t i s now b e l i e v e d t h a t m y o c a r d i a l damage a s s o c i a t e d w i t h r e p e r f u s i o n i n j u r y i s p r o d u c e d b y c y t o t o x i c m e t a b o l i t e s o f o x y g e n , w h i c h i n c l u d e s u p e r o x i d e a n i o n , h y d r o g e n p e r o x i d e a n d f r e e h y d r o x y r a d i c a l ( S u b r a m a n i a n e t a l . . 1 9 8 7 ) . T h e e f f e c t s o f t h e s e o x y g e n f r e e r a d i c a l s a r e w i d e s p r e a d . T h e y c a u s e a l t e r a t i o n s i n membrane p h o s p h o l i p i d s b y p e r o x i d a t i o n , c a u s i n g a n i n c r e a s e d r e l e a s e o f m a l o n d i a l d e h y d e ( S u b r a m a n i a n , e t a l . . 1 9 8 7 ) , m y o c a r d i a l edema b e c a u s e o f e n h a n c e d membrane p e r m e a b i l i t y ( G u a r n i e r i , e t a l . . 1 9 8 0 ) , d e c r e a s e d m i t o c h o n d r i a l a c t i v i t y a n d i n c r e a s e d c e l l u l a r p e r m e a b i l i t y ( F o x , e t a l . . 1 9 8 5 ) . C a r d i o p l e g i a p r o v i d e s a v e h i c l e t h r o u g h w h i c h p r o t e c t i v e a g e n t s , w h i c h c a n c o u n t e r a c t some o f t h e s e c h a n g e s , c a n b e a d m i n i s t e r e d a n d a s s e s s e d . T h e s o l u t i o n u t i l i z e d i n t h i s s t u d y was i s o - o s m o l a r T y e r s s o l u t i o n , w h i c h was f i r s t p r e p a r e d b y T y e r s e t a l . ( 1 9 7 4 ) . I n 10 c a n i n e e x p e r i m e n t s , 60 m i n u t e s o f h y p o t h e r m i c i s c h e m i c a r r e s t u s i n g c o r o n a r y p e r f u s i o n w i t h i s o - o s m o l a r - 63 -s o l u t i o n r e s u l t e d i n n o r m a l f u n c t i o n a n d o n l y m i l d p e r i v a s c u l a r edema o n h i s t o p a t h o l o g i c a l a s s e s s m e n t s . T h e s o l u t i o n was f i r s t u s e d c l i n i c a l l y b y T y e r s e t a l . ( 1 9 7 7 ) a n d s h o w e d o n l y m o d e r a t e p r o t e c t i o n . O f r e c e n t i n t e r e s t i s t h e i n c l u s i o n o f e n z y m e s t o s c a v e n g e o r b l o c k c y t o t o x i c o x y g e n m e t a b o l i t e s ( M c C o r d , 1 9 8 5 ) . S t u d i e s h a v e b e e n p e r f o r m e d u t i l i z i n g a l l o p u r i n o l ( w h i c h b l o c k s t h e g e n e r a t i o n o f h y d r o x y l r a d i c a l s a s a r e s u l t o f i n h i b i t i o n o f x a n t h i n e o x i d a s e ) , s u p e r o x i d e d i s m u t a s e ( w h i c h b r e a k s down s u p e r o x i d e a n i o n ) a n d c a t a l a s e ( w h i c h r e d u c e s H 2 0 2 ) i n a n a t t e m p t t o m i n i m i z e t h e damage i n d u c e d b y o x y g e n f r e e r a d i c a l s ( A m b r o s i o e t a l . . ( 1 9 8 7 ) , Das e t a l . . ( 1 9 8 7 a , b ) , G o d i n e t a l . . ( 1 9 8 6 ) , M v e r s e t a l . . ( 1 9 8 6 ) ) . A g r e a t d e a l o f l i t e r a t u r e i s c u r r e n t l y a v a i l a b l e w h i c h e x a m i n e s t h e f u n c t i o n a l c o n s e q u e n c e s o f g l o b a l i s c h e m i a . A n u m b e r o f s t u d i e s a r e a v a i l a b l e w h i c h e x a m i n e t h e f a t e o f f r e e f a t t y a c i d s , t h e p r i m a r y e n e r g y - g e n e r a t i n g s u b s t a t e s o f t h e m y o c a r d i u m , f o l l o w i n g g l o b a l i s c h e m i a , a n d t h e c a r d i o p r o t e c t i v e b e n e f i t s o f L - c a r n i t i n e d e r i v a t i v e s ( P a u l s o n e t a l . ( 1 9 8 6 ) , R e g i t z e t _ a l . ( 1 9 8 7 ) . F e r r a r i e t a l . ( 1 9 8 8 ) ) . H o w e v e r , t h e p r e c i s e m e c h a n i s m o f t h e s e c a r d i o p r o t e c t i v e e f f e c t s a r e s t i l l u n k nown. T h e f i e l d o f c a r d i o p l e g i a i s a d y n a m i c o n e w i t h many c o n t r o v e r s i e s s t i l l b e i n g d e b a t e d . T h e s e i n c l u d e t h e b e n e f i t s o f o x y g e n a t e d v s . n o n - o x y g e n a t e d s o l u t i o n s ( T a b a y a s h i e t a l . . ( 1 9 8 8 ) , de W i t e t a l . . ( 1 9 8 8 ) , L e d i n g h a m e t a l . . ( 1 9 8 8 ) ) , b l o o d v s . c r y s t a l l o i d s o l u t i o n s ( K h u r i e t a l . . 1 9 8 8 ) , t h e p r o v i s i o n o f v a r i o u s a d d i t i v e s a n d t h e i r p o t e n t i a l b e n e f i t s ( G r e e n f i e l d e t a l . . 1 9 8 8 ) , a n d a n u m b e r o f p r o c e d u r a l q u e s t i o n s , s u c h a s r a t e a n d m e t h o d o f d e l i v e r y o f s o l u t i o n , a s w e l l a s t h e o p t i m a l t e m p e r a t u r e ( R o u s o u e t a l . . 1 9 8 8 ) . - 64 -I I I PURPOSE A s h a s b e e n i l l u s t r a t e d , t h e p r e v a l e n c e o f c a r d i o v a s c u l a r d i s e a s e i n t o d a y ' s s o c i e t y n e c e s s i t a t e s r e s e a r c h i n t o t h e r e d u c t i o n o f m o r t a l i t y r a t e s a n d t h e i m p r o v e m e n t o f a s s e s s m e n t t e c h n i q u e s . B o t h d i a g n o s t i c t e s t s a n d e v a l u a t i o n o f c u r r e n t t e c h n i q u e s a r e r e q u i r e d . C u r r e n t d i a g n o s t i c t e s t s , a i m e d a t a s s e s s i n g m y o c a r d i a l i s c h e m i a a n d i n f a r c t i o n i n c l i n i c a l n u c l e a r m e d i c i n e , l a c k a n i d e a l r a d i o p h a r m a c e u t i c a l . I o d i n a t e d ( 1 2 3 I ) f r e e f a t t y a c i d s may s a t i s f y t h e o p t i m a l r e q u i r e m e n t s f o r t h e i d e a l i m a g i n g a g e n t f o r m y o c a r d i a l i s c h e m i a a n d i n f a r c t i o n ( b o t h a s a d i a g n o s t i c t o o l a n d m e t h o d o f e v a l u a t i o n ) : T h e r e f o r e , t h e o b j e c t i v e s o f t h i s t h e s i s a r e t h r e e - f o l d : 1) t o e v a l u a t e t h e e f f e c t o f r e v e r s i b l e g l o b a l i s c h e m i a i n a c a n i n e m o d e l o n m y o c a r d i a l m e t a b o l i s m o f 1 5 - p - ( 1 2 3 I ) - i o d o p h e n y l -p e n t a d e c a n o i c a c i d ( 1 2 3 I P P A ) ; 2) t o a s s e s s t h e l o n g i t u d i n a l e f f e c t o f p r o g r e s s i v e r e g i o n a l i s c h e m i a o n m y o c a r d i a l m e t a b o l i s m o f 1 2 3 I P P A i n a c a n i n e m o d e l ; a n d 3) t o u t i l i z e 2 0 1 T h a l l i u m a n d B e t a - m e t h y l - 1 2 3 I P P A ( B 1 2 3 I P P A ) f o r a s s e s s i n g p e r f u s i o n d e f e c t s a n d t o d e t e r m i n e t h e i r a b i l i t y t o i d e n t i f y t h e i s c h e m i c r i s k z o n e i n a c a n i n e m o d e l . - 65 -I V E XPERIMENTAL MATERIALS AND METHODS ( 1 ) G l o b a l I s c h e m i a ( a ) E x p e r i m e n t a l M o d e l o f C a r d i o p l e g i a A d u l t d o g s ( 1 7 - 2 8 k g ) w e r e f a s t e d o v e r n i g h t a n d a n a e s t h e t i z e d w i t h s o d i u m p e n t o b a r b i t a l ( 2 5 mg/kg) I V . S u p p l e m e n t a l s o d i u m p e n t o b a r b i t a l was g i v e n a s n e c e s s a r y t h r o u g h o u t t h e p r o c e d u r e . T h e d o g s w e r e i n t u b a t e d a n d m a i n t a i n e d o n a p r e s s u r e - l i m i t e d v e n t i l a t o r ( M a r k - 7 R e s p i r a t o r - B i r d C o r p o r a t i o n ) . A s t a n d a r d m e d i a n s t e r n o t o m y was p e r f o r m e d . A c a t h e t e r was p l a c e d i n t o t h e r i g h t i n t e r n a l mammary a r t e r y t o m o n i t o r b l o o d p r e s s u r e . P o l y e t h y l e n e c a t h e t e r s w e r e i n s e r t e d i n t o t h e l e f t a t r i u m a n d r i g h t a t r i u m t o m o n i t o r a t r i a l p r e s s u r e s . A 7 - F r e n c h Swan-Ganz t h e r m o d i l u t i o n c a t h e t e r was i n s e r t e d i n t o t h e p u l m o n a r y a r t e r y . T h e c a t h e t e r s w e r e a t t a c h e d t o s t a n d a r d t r a n s d u c e r s t o m o n i t o r p r e s s u r e . T h e ECG, h e a r t r a t e a n d p r e s s u r e s w e r e d i s p l a y e d o n a t w e l v e - c h a n n e l o s c i l l o s c o p e w i t h c o n c u r r e n t d i g i t a l r e a d o u t a n d r e c o r d i n g c a p a b i l i t i e s ( S E L a b o r a t o r i e s , E n g l a n d ; J o h n F o r t i n M f g . L t d . , C a n a d a ) . R e c t a l a n d m y o c a r d i a l t e m p e r a t u r e s w e r e m o n i t o r e d c o n t i n u o u s l y . T h e a n i m a l s w e r e p l a c e d o n c a r d i o p u l m o n a r y b y p a s s ( C P B ) , w i t h o x y g e n a t o r s u p p o r t , c a v a l o c c l u s i o n a n d l e f t a t r i a l v e n t i n g . T h e d o g s r e c e i v e d 0.5 mg/kg h e p a r i n p r i o r t o i n i t i a t i o n o f CPB. T h e CPB pump was p r i m e d w i t h d o n o r d o g b l o o d . ( b ) P r o t o c o l F o l l o w i n g c a r d i a c c a n n u l a t i o n , c o n t r o l m e a s u r e m e n t s w e r e r e c o r d e d . T h e s e i n c l u d e d h e a r t r a t e , a r t e r i a l b l o o d p r e s s u r e , p u l m o n a r y - 66 -a r t e r y p r e s s u r e a n d l e f t a n d r i g h t a t r i a l p r e s s u r e s . C a r d i a c o u t p u t s w e r e m e a s u r e d u s i n g a c a r d i a c o u t p u t c o m p u t e r a n d c o l d s a l i n e i n j e c t i o n s i n t o t h e r i g h t a t r i u m . T h e h e a r t was s u b j e c t e d t o two h o u r s o f i s c h e m i c a r r e s t a t a m y o c a r d i a l t e m p e r a t u r e o f 1 5 - 1 9 ° C . C a r d i o p l e g i c s o l u t i o n was a d m i n i s t e r e d i n t e r m i t t e n t l y t o m a i n t a i n m y o c a r d i a l t e m p e r a t u r e a t t h e d e s i r e d r a n g e . A c i d - b a s e s t a t u s was a s s e s s e d b y p e r i o d i c b l o o d - g a s a n a l y s i s . S y s t e m i c t e m p e r a t u r e was m a i n t a i n e d a t 3 0 ° C b y t h e CPB u n i t . T h r e e g r o u p s o f e x p e r i m e n t s w e r e p e r f o r m e d . C a r d i o p l e g i a was i n i t i a t e d + +2 +2 w i t h i s o - o s m o l a r s o l u t i o n ( 3 1 0 mOsm/L, K 25 meq/L, Mg 3 meq/L, C a 2 meq/L a n d N a + 144 m e q / L ) . I n G r o u p A ( n = 5) i s o - o s m o l a r s o l u t i o n was u t i l i z e d w i t h o u t a d d i t i o n s . I n G r o u p B ( n = 7) , s u p e r o x i d e d i s m u t a s e (SOD -C a r l s b e r g B i o t e c h n o l o g y , C o p e n h a g e n , D e n m a r k ) was a d m i n i s t e r e d a t 6.5 mg/kg. d i s s o l v e d i n s a l i n e . One t h i r d o f t h e t o t a l d o s e ( 1 1 0 - 1 8 2 mg) was a d d e d t o t h e f i n a l d e l i v e r y o f c a r d i o p l e g i a a n d t h e r e m a i n i n g d o s e a d m i n i s t e r e d s y s t e m i c a l l y I V d u r i n g t h e f i r s t f i v e m i n u t e s o f r e p e r f u s i o n . I n G r o u p C ( n = 7 ) a l l o p u r i n o l ( W e l l c o m e F o u n d a t i o n L t d . , L o n d o n , E n g l a n d ) was a d m i n i s t e r e d a t 40 mg/kg w i t h t h e m a j o r i t y ( 9 5 % o f t o t a l d o s e ) b e i n g a d d e d s y s t e m i c a l l y I V a n d t h e r e m a i n d e r ( 5 % o f t o t a l d o s e ) t o t h e f i n a l i n f u s i o n o f c a r d i o p l e g i c s o l u t i o n . C a r d i o p l e g i c s o l u t i o n t e m p e r a t u r e was m a i n t a i n e d a t 8 - 1 0 ° C a n d i n f u s e d t h r o u g h a n a o r t i c c a n n u l a c o n n e c t e d t o a CPB c i r c u i t pump. T h e f i n a l i n f u s i o n o f c a r d i o p l e g i a was a d m i n i s t e r e d t w e n t y m i n u t e s p r i o r t o r e m o v a l o f t h e a o r t i c c r o s s - c l a m p a n d r e p e r f u s i o n . S y s t e m i c r e w a r m i n g was a l s o i n i t i a t e d a t t h e f i n a l i n f u s i o n o f c a r d i o p l e g i c s o l u t i o n . F o l l o w i n g r e m o v a l o f t h e a o r t i c c r o s s - c l a m p a n d r e l e a s e o f c a v a l o c c l u s i o n , t h e h e a r t was d e f i b r i l l a t e d i f n e c e s s a r y . T h r o u g h o u t t h e - 67 -c r o s s - c l a m p p e r i o d , a o r t i c p r e s s u r e was m a i n t a i n e d a b o v e 60 mmHg t h r o u g h t h e i n f u s i o n o f c a r d i o p l e g i c s o l u t i o n a t a f l o w r a t e o f l O O m L / k g / m i n . P e r i o d i c b l o o d - g a s a n a l y s i s was p e r f o r m e d d u r i n g t h e p o s t - C P B p e r i o d a n d s o d i u m b i c a r b o n a t e was a d m i n i s t e r e d , i f r e q u i r e d . H e m o d y n a m i c a s s e s s m e n t s w e r e p e r f o r m e d t h i r t y m i n u t e s a f t e r w e a n i n g f r o m CPB. ( c ) P r e p a r a t i o n o f 1 5 - p - 1 2 3 I - i o d o p h e n y l p e n t a d e c a n o i c a c i d  ( 1 2 3 I P P A ) ( i ) R a d i o i o d i n e . r R a d i o i o d i n e ( 1 2 3 I ) N a I was o b t a i n e d t h r o u g h t h e 1 2 4 X e ( p , 2 p ) 1 2 3 I (26MeV) d i r e c t r e a c t i o n a n d 1 2 4 X e ( p , p n ) 1 2 3 X e * 2 3 l ( 26MeV) i n d i r e c t r e a c t i o n ( A t o m i c E n e r g y o f C a n a d a L t d . , V a n c o u v e r , B . C . ) w i t h a r a d i o n u c l i d e p u r i t y g r e a t e r t h a n 9 9 . 9 % a t c a l i b r a t i o n t i m e . ( i i ) P r o d u c t i o n 1 2 3 I P P A - 1 2 3 I P P A was p r o d u c e d b y a m e t h o d s i m i l a r t o t h a t o f K u l k a r n i a n d P a r k e y ( 1 9 8 2 ) . T h a l l a t i o n was c a r r i e d o u t i n a 1.0 m l . r e a c t i v i a l u s i n g 0.75 mg. PPA a n d 3.0 mg. t h a l l i u m t r i s t r i f l u o r o a c e t a t e [ T 1 ( T F A ) 3 ] d i s s o l v e d i n 225 /*L t r i f l u o r o a c e t i c a c i d ( C h i e n e t a l . . 1 9 8 3 ) . T h e m i x t u r e was a l l o w e d t o p r o c e e d a t r o o m t e m p e r a t u r e f o r o n e h o u r . T o t h e i n s i t u p a r a t h a l l i u m c o m p l e x was a d d e d ( 1 2 3 I ) N a I ( l O m C i ) a n d 3/iL K I s o l u t i o n (6 mg/ml i n d i s t i l l e d H 2 0 ) . T h i s was a l l o w e d t o r e a c t f o r o n e h o u r a t 5 8 - 6 0 ° C a n d r e s u l t e d i n i o d o - d e t h a l l a t i o n f o r m i n g 1 2 3 I P P A . ( i i i ) H e x a n e E x t r a c t i o n - T h e 1 2 3 I P P A was e x t r a c t e d f r o m t h e r e a c t i o n m i x t u r e i n t o 1 m l . o f h e x a n e a n d w a s h e d s e r i a l l y t h r e e m o r e t i m e s ( l m l h e x a n e / e x t r a c t i o n ) . T h e a c t i v i t y was m e a s u r e d i n a w e l l - c o u n t e r t h r o u g h o u t t h e p r o c e d u r e w i t h t h e m a j o r i t y ( > 80%) o c c u r r i n g i n t h e i n i t i a l two v a c u t a i n e r s . - 68 -( i v ) P u r i f i c a t i o n - P u r i f i c a t i o n o f t h e 1 2 3I P P A was c o m p l e t e d u s i n g a s m a l l S i 0 2 c o l u m n ( W a t e r s S i 0 2 S e p - P a k P I N 5 1 9 0 0 ) a n d e l u t i o n w i t h 2.5% m e t h a n o l i n m e t h y l e n e c h l o r i d e . T h e 1 2 3I P P A f r a c t i o n s w e r e c o l l e c t e d a n d t h e s o l v e n t e v a p o r a t e d . ( v ) P r e p a r a t i o n o f 1 2 3I P P A f o r I n j e c t i o n - F o l l o w i n g e v a p o r a t i o n o f t h e s o l v e n t u s i n g a n i t r o g e n s t r e a m , t h e 1 2 3I P P A was d i s s o l v e d i n 2 0 0 / i l 99% e t h a n o l a n d s u s p e n d e d w i t h s o n i c a t i o n i n 3 m i s . 6.0% human s e r u m a l b u m i n . T h e p r o d u c t was f i l t e r e d u s i n g a 0.22/im m i l l i p o r e f i l t e r t o r e m o v e a n y c o n t a m i n a n t p a r t i c u l a t e m a t t e r p r i o r t o i n j e c t i o n . ( v i ) Q u a l i t y C o n t r o l - T h i n l a y e r c h r o m a t o g r a p h y was c a r r i e d o u t o n s i l i c a g e l p l a t e s ( B a k e r f l e x 1 B 2 - F ) w i t h 2.5% m e t h a n o l i n m e t h y l e n e c h l o r i d e a s t h e s o l v e n t . T h e o r i g i n was s p o t t e d w i t h 1 0 - 2 0 / i l s m e t h a n o l . T e n nls o f I P P A ( q u a l i t y c o n t r o l ) was t h e n a p p l i e d t o t h e o r i g i n a n d s u b s e q u e n t l y t h e s h e e t s w e r e d e v e l o p e d . T h e v a l u e f o r 1 2 3 I P P A i s 0.6 w i t h t h e i o d i d e r e m a i n i n g a t t h e o r i g i n a n d a n y i m p u r i t y t r a v e l l i n g w i t h t h e s o l v e n t f r o n t . Two i m p u r i t i e s w h i c h w e r e e n c o u n t e r e d i n c l u d e d b e n z o i c a c i d ( a t s o l v e n t f r o n t ) a n d f r e e 1 2 3 I ( R ^ = 0 . 0 ) . ( d ) E l e c t r o n M i c r o s c o p y ( i ) T i s s u e C o l l e c t i o n - P u n c h b i o p s i e s o f t h e l e f t v e n t r i c l e w e r e t a k e n f o r e l e c t r o n m i c r o s c o p i c e x a m i n a t i o n p r i o r t o s a c r i f i c e , p r o v i d i n g u l t r a s t r u c t u r a l e v i d e n c e o f a n y i s c h e m i c c h a n g e s . T h e s a m p l e s w e r e i m m e d i a t e l y p l a c e d i n c o l d g l u t a r a l d e h y d e a n d r e f r i g e r a t e d f o r 1 2 - 1 5 h r s ( o v e r n i g h t ) . T h e g l u t a r a l d e h y d e - f i x e d s a m p l e s w e r e t h e n t r a n s f e r r e d t o a 0.1M c a c o d y l a t e b u f f e r s o l u t i o n , i n w h i c h t h e s a m p l e c a n b e s t o r e d f o r s e v e r a l y e a r s . - 69 -( i i ) P r o c e s s i n g - F o l l o w i n g t h e c o m p l e t i o n o f t h e s e r i e s , g l u t a r a l d e h y d e - f i x e d s p e c i m e n s w e r e c u t i n t o 1-2 mm c u b e s , w i t h a n u n u s e d r a z o r b l a d e . T h e s e c u b e s w e r e f i x e d i n 1% o s m i c a c i d f o r 14 h o u r s a t 4 ° C . T h e s a m p l e s w e r e t h e n w a s h e d 3 t i m e s w i t h d i s t i l l e d w a t e r a t 5 m i n u t e i n t e r v a l s . T h e s a m p l e was t h e n s t a i n e d w i t h 5% a q u e o u s u r a n y l a c e t a t e f o r 15 m i n u t e s a t 3 7 ° C , f o l l o w e d b y r i n s i n g w i t h d i s t i l l e d w a t e r a n d d e h y d r a t i o n i n i n c r e a s i n g c o n c e n t r a t i o n s o f a l c o h o l a n d f i n a l l y p r o p y l e n e o x i d e . T h e f i n a l m i x t u r e f o r d e h y d r a t i o n was 50% p r o p y l e n e o x i d e a n d 50% e f f a p o x y m i x t u r e , i n w h i c h t h e s a m p l e was i n c u b a t e d , c l o s e d t o a i r , f o r 3h h o u r s a n d t h e n e x p o s e d o v e r n i g h t t o a i r . T h e s a m p l e s w e r e t h e n e m b e d d e d i n f r e s h 100% e f f a p o x y m i x t u r e i n Beem c a p s u l e s . T h e Beem c a p s u l e s w e r e i n c u b a t e d o v e r n i g h t a t 3 7 ° C a n d a t 6 0 ° C f o r 48 h o u r s . ( i i i ) S e c t i o n i n g - U s i n g a m i c r o t o m e , 1 /im t h i c k s e c t i o n s w e r e c u t f r o m t h e Beem c a p s u l e a n d s t a i n e d w i t h 1% t o l u i d i n e b l u e i n 1% b o r a x f o r 30 s e c o n d s o n a h o t p l a t e . T h e s e s e c t i o n s w e r e p l a c e d o n s l i d e s a n d e x a m i n e d u n d e r l i g h t m i c r o s c o p y . A s u i t a b l e s e c t i o n was c h o s e n a n d t h a t b l o c k was t h e n c u t i n t o t h i n s e c t i o n s ( 6 0 - 1 0 0 nm) u s i n g a n u l t r a m i c r o t o m e . T h e s e s e c t i o n s w e r e p l a c e d o n g r i d s a n d s t a i n e d w i t h R e y n o l d s l e a d c i t r a t e f o r 8 m i n u t e s i n a c o v e r e d P e t r i d i s h c o n t a i n i n g NaOH p e l l e t s . T h e f i n a l s e c t i o n s w e r e t h e n e x a m i n e d u n d e r t h e e l e c t r o n m i c r o s c o p e . ( e ) L i g h t M i c r o s c o p y F o l l o w i n g s a c r i f i c e , t h e h e a r t s w e r e e x c i s e d a n d s a m p l e s o f t h e r i g h t v e n t r i c l e , s e p t u m a n d l e f t v e n t r i c l e t a k e n f o r l i g h t m i c r o s c o p i c e x a m i n a t i o n . S m a l l s e c t i o n s o f t i s s u e w e r e c u t w i t h a s c a l p e l a n d t h e n - 70 -p l a c e d i n t o t i s s u e s a m p l e b o x e s a n d p a s s e d t h r o u g h a H i s t o m a t i c T i s s u e P r o c e s s o r . T h i s m a c h i n e a u t o m a t i c a l l y p a s s e s t h e s a m p l e t h r o u g h a s e r i e s o f d e h y d r a t i o n s t e p s i n p r o g r e s s i v e l y i n c r e a s i n g c o n c e n t r a t i o n s o f a l c o h o l , f o l l o w e d b y c l e a r i n g i n x y l o l a n d f i n a l l y i m p r e g n a t i o n a n d e m b e d d i n g i n wax. T h e b l o c k s w e r e t h e n c u t i n t o t h i c k s e c t i o n s u s i n g a m i c r o t o m e a n d p l a c e d o n t o a s l i d e . T h e s l i d e s w e r e t h e n s t a i n e d w i t h h e m a t o x y l i n a n d e o s i n , w h i c h s t a i n e d n u c l e i b l u e b e c a u s e o f t h e i r b a s o p h i l i c n a t u r e a n d c y t o p l a s m a n d c o n n e c t i v e t i s s u e s h a d e s o f p i n k . ( f ) D a t a A c q u i s i t i o n ( i ) I m a g i n g - T h e a n i m a l s w e r e f a s t e d o v e r n i g h t a n d a n a e s t h e t i z e d w i t h s o d i u m p e n t o b a r b i t a l ( 2 5 m g / k g ) . S u p p l e m e n t a l s o d i u m p e n t o b a r b i t a l was g i v e n i f n e c e s s a r y . P l a n a r gamma i m a g i n g was p e r f o r m e d w i t h a P i c k e r I n t e r n a t i o n a l D a t a M o + m - M o b i l e c a m e r a ( f i t t e d w i t h a l o w e n e r g y a l l - p u r p o s e c o l l i m a t o r ) a n d a m o b i l e c o m p u t e r t e r m i n a l (ADAC-Cam I I ) . T h e a n t e r i o r v i e w o f t h e d o g was i m a g e d f o l l o w i n g t h e i n j e c t i o n o f 3-5 m C i o f 1 2 3 I P P A . T h e s c a n s w e r e p e r f o r m e d o n e w e ek p r e - o p e r a t i v e l y ( c o n t r o l ) a n d two a n d o n e - h a l f h o u r s p o s t - o p e r a t i v e l y . I m a g e s w e r e c o l l e c t e d o n a 128 x 128 x 8 m a t r i x o v e r a 30 m i n u t e p e r i o d , c o m m e n c i n g i m m e d i a t e l y a f t e r i n j e c t i o n . F r a m e s w e r e c o l l e c t e d e v e r y f i v e s e c o n d s o v e r t h e f i r s t 2 m i n u t e s a n d e v e r y 1 m i n u t e f r o m 2 t o 30 m i n u t e s . ( i i ) B l o o d - G a s A n a l y s i s - B l o o d g a s e s w e r e a s s e s s e d u s i n g a C o r n i n g 175 A u t o m a t i c p H / B l o o d Gas S y s t e m ( C o r n i n g M e d i c a l , M e d f i e l d , MA). B l o o d s a m p l e s w e r e i n j e c t e d i n t o t h e s y s t e m t h r o u g h t h e i n t a k e p o r t . T h e s y s t e m m e a s u r e d pH, p C 0 2 , p 0 2 a n d b a r o m e t r i c p r e s s u r e d i r e c t l y . T h e - 71 -s y s t e m a l s o c a l c u l a t e d b i c a r b o n a t e , b a s e e x c e s s , o x y g e n c o n t e n t a n d o x y g e n s a t u r a t i o n b a s e d o n t h e m e a s u r e d v a l u e s l i s t e d p r e v i o u s l y . A p r i n t e r , w h i c h i s i n c o r p o r a t e d i n t o t h e m a c h i n e , r e c o r d e d t h e s a m p l e d a t a . ( g ) D a t a A n a l y s i s ( i ) Image A n a l y s i s - Image a n a l y s i s was p e r f o r m e d o n a n ADAC L a b o r a t o r i e s C o m p u t e r (Cam I I ) . T h e f i v e s e c o n d f r a m e s w e r e summed i n t o o n e m i n u t e i m a g e s . A r e a s o f i n t e r e s t w e r e c r e a t e d a r o u n d t h e s u p e r i o r v e n a c a v a ( b a c k g r o u n d ) , l a t e r a l w a l l , a p i c a l w a l l , s e p t a l w a l l , a n d t h e t o t a l h e a r t . T h e s e c o m p r e s s e d d a t a w e r e u t i l i z e d f o r n u m e r i c a l d a t a m a n i p u l a t i o n s . T h e d a t a w e r e n o r m a l i z e d t o p e a k c o u n t s a n d t h e b a c k g r o u n d ( S V C ) was s u b t r a c t e d . T h e s e n u m e r i c a l d a t a w e r e t h e n t r a n s f e r r e d t o a n IBM-PC. T i m e - a c t i v i t y c u r v e s w e r e g e n e r a t e d u s i n g a p r o g r a m s u p p l i e d b y RS/1 ( R e s e a r c h S y s t e m / 1 : BBN S o f t w a r e P r o d u c t s C o r p . , C a m b r i d g e , MA). T h i s same p r o g r a m was u t i l i z e d t o a n a l y z e t h e c u r v e s u s i n g a b i e x p o n e n t i a l l e a s t s q u a r e s f i t w i t h c u r v e p e e l i n g , r e s u l t i n g i n a b e s t f i t l i n e a n d e q u a t i o n f o r e a c h c u r v e . 1 2 3 I P P A h a l f - l i v e s (th) w e r e c a l c u l a t e d f r o m t h e s l o p e s o f t h e l i n e s . ( i i ) S t a t i s t i c a l A n a l y s i s - H e m o d y n a m i c a s s e s s m e n t s a r e r e p o r t e d a s mean + SD a n d p a i r e d S t u d e n t T - T e s t s w e r e u s e d t o t e s t f o r a n y s i g n i f i c a n t d i f f e r e n c e s b e t w e e n c o n t r o l a n d p o s t - o p e r a t i v e v a l u e s . 1 2 3 I P P A h a l f - l i v e s (th) a r e r e p o r t e d a s mean + SD a n d a W i l k - S h a p i r o t e s t f o r n o r m a l i t y was p e r f o r m e d o n t h e p e r c e n t c h a n g e b e t w e e n c o n t r o l a n d p o s t - o p e r a t i v e v a l u e s . C h a n g e s i n th b e t w e e n g r o u p s w e r e t h e n s u b j e c t e d t o a n F - t e s t f o r t e s t i n g t h e h o m o g e n e i t y o f t h e v a r i a n c e . I f t h e d a t a w e r e 72 -h o m o g e n o u s , a p o o l e d v a r i a n c e e s t i m a t e T - t e s t was a p p l i e d . I f t h e d a t a w e r e n o t h o m o g e n o u s , a s e p a r a t e v a r i a n c e e s t i m a t e T - t e s t was a p p l i e d . T e s t i n g t h e d a t a w i t h a M a n n - W h i t n e y t e s t p r o d u c e d s i m i l a r s t a t i s t i c a l r e s u l t s . C h a n g e s b e t w e e n c o n t r o l a n d p o s t - o p e r a t i v e v a l u e s o f th w i t h i n g r o u p s w e r e a s s e s s e d u s i n g a S t u d e n t P a i r e d T - t e s t . C o n t r o l m e a s u r e m e n t s o f th w e r e p o o l e d f o r e a c h i n d i v i d u a l a r e a ( l a t e r a l w a l l , a p i c a l w a l l , s e p t a l w a l l ) a n d a n y d i f f e r e n c e s w e r e a n a l y z e d f o r b y ANOVA. ( 2 ) R e g i o n a l I s c h e m i a ( a ) E x p e r i m e n t a l M o d e l o f R e g i o n a l I s c h e m i a T h e s t u d y g r o u p c o n s i s t e d o f t w e l v e c a n i n e e x p e r i m e n t s . A d u l t d o g s ( 1 7 - 3 3 k g ) w e r e f a s t e d o v e r n i g h t a n d a n a e s t h e t i z e d w i t h s o d i u m p e n t o b a r b i t a l ( 2 5 mg/kg) I V . S u p p l e m e n t a l s o d i u m p e n t o b a r b i t a l was g i v e n a s n e c e s s a r y . T h e d o g s w e r e i n t u b a t e d a n d m a i n t a i n e d o n a p r e s s u r e - l i m i t e d v e n t i l a t o r ( M a r k - 7 R e s p i r a t o r - B i r d C o r p o r a t i o n ) . T h e h e a r t was e x p o s e d v i a a f o u r t h i n t e r c o s t a l s p a c e t h o r a c o t o m y . T h e ECG a n d h e a r t r a t e w e r e d i s p l a y e d o n a 1 2 - c h a n n e l o s c i l l o s c o p e w i t h c o n c u r r e n t d i g i t a l r e a d o u t a n d r e c o r d i n g c a p a b i l i t i e s ( S E L a b o r a t o r i e s , E n g l a n d ; J o h n F o r t i n M f g . , L t d . , C a n a d a ) . A p p r o x i m a t e l y 1.0-2.0 cm o f t h e l e f t a n t e r i o r d e s c e n d i n g c o r o n a r y a r t e r y was d i s s e c t e d o u t d i s t a l t o t h e b i f u r c a t i o n w i t h t h e l e f t c i r c u m f l e x c o r o n a r y a r t e r y . V e s s i l o o p s w e r e u t i l i z e d t o e x t e n d t h e a r t e r y a n d a n a m e r o i d c o n s t r i c t o r ( T h r e e P o i n t s P r o d u c t s , M o n t r e a l , Q u e b e c , C a n a d a ) o f a p p r o p r i a t e s i z e was p o s i t i o n e d . A t r i a l p a c i n g w i r e s w e r e i m p l a n t e d a n d d i r e c t e d t o a s u b c u t a n e o u s p o c k e t l y i n g a t t h e b a s e o f t h e n e c k o f t h e a n i m a l . T h e - 73 -p e r i c a r d i u m was c l o s e d f o l l o w i n g 30 m i n u t e s o r when t h e a n i m a l was s t a b i l i z e d . T h e i n c i s i o n was t h e n c l o s e d a n d t h e a n i m a l s r e t u r n e d t o t h e h o l d i n g f a c i l i t i e s . T h e a n i m a l s w e r e g i v e n D e r a p e n , a p e n i c i l l i n , IM, o n c e a d a y f o r 3 d a y s p o s t - o p e r a t i v e l y . T h e a n i m a l s w e r e s a c r i f i c e d two w e e k s p o s t - o p e r a t i v e l y , i m m e d i a t e l y a f t e r t h e f i n a l s c a n . ( b ) M e t a b o l i c A s s e s s m e n t M e t a b o l i c b l o o d a n a l y s i s was p e r f o r m e d o n v e n o u s b l o o d s a m p l e s a c q u i r e d p r e - o p e r a t i v e l y a n d 24 h r s , 5 d a y s , 7 d a y s a n d 14 d a y s p o s t - o p e r a t i v e l y . S a m p l e s w e r e a p p o r t i o n e d i n t o a p p r o p r i a t e v i a l s f o r a n a l y s i s . A D u p o n t A u t o m a t i c C l i n i c a l A n a l y z e r (ACA) ( D u p o n t Co., C l i n i c a l S y s t e m s D i v i s i o n , W i l m i n g d o n , DE) was u s e d t o q u a n t i t a t e l a c t a t e l e v e l s . L a c t a t e d e h y d r o g e n a s e a n d c r e a t i n e k i n a s e a c t i v i t y i n b l o o d s a m p l e s w e r e a n a l y z e d o n a C o b a s B i o s p e c t r o p h o t o m e t r i c A n a l y z e r . E a c h enzyme h a s a s p e c i f i c t e s t p a c k , i e . l a c t a t e - L A p a c k , LDH - B e c k m a n D r i - S T A T L D L R e a g e n t p a c k a n d CK - B e c k m a n D r i - S T A T o p t i m i z e d CK-NAC R e a g e n t p a c k . T o d e t e r m i n e l a c t a t e l e v e l s , t h e a p p r o p r i a t e a n a l y t i c a l t e s t p a c k a n d s a m p l e a r e l o a d e d i n t o t h e ACA. T h i s m a c h i n e a u t o m a t i c a l l y a d v a n c e s t h e p a c k t h r o u g h t h e t e s t p r o c e d u r e a n d p r i n t s o u t t h e r e s u l t s . L a c t a t e d e h y d r o g e n a s e a n d CK d e t e r m i n a t i o n s r e q u i r e a m i n o r amount o f o p e r a t o r p r e p a r a t i o n . U s i n g t h e a p p r o p r i a t e p a c k s , t h e r e a g e n t s m u s t b e p r e p a r e d . T h e s a m p l e t r a y c a n t h e n b e l o a d e d a p p r o p r i a t e l y . T h e a p p r o p r i a t e t e s t k e y i s s e l e c t e d a n d t h e p a r a m e t e r l i s t w i l l b e p r i n t e d o u t . - 74 -( c ) P r e p a r a t i o n o f 1 5 - p - 1 2 3 I - i o d o p h e n y l p e n t a d e c a n o i c a c i d  ( 1 2 3 I P P A ) ( i ) R a d i o i o d i n e - M e t h o d t h e same a s i n G l o b a l I s c h e m i a s e c t i o n . ( i i ) P r o d u c t i o n 1 2 3 I P P A - M e t h o d t h e same a s i n G l o b a l I s c h e m i a s e c t i o n . ( i i i ) H e x a n e E x t r a c t i o n - M e t h o d t h e same a s i n G l o b a l I s c h e m i a s e c t i o n . ( i v ) P u r i f i c a t i o n - M e t h o d t h e same a s i n G l o b a l I s c h e m i a s e c t i o n . ( v ) P r e p a r a t i o n o f 1 2 3 I P P A f o r I n j e c t i o n - M e t h o d t h e same a s i n G l o b a l I s c h e m i a s e c t i o n . ( v i ) Q u a l i t y C o n t r o l - M e t h o d t h e same a s i n G l o b a l I s c h e m i a s e c t i o n . ( d ) D a t a A c q u i s i t i o n ( i ) I m a g i n g - I m a g i n g p r o t o c o l t h e same a s i n G l o b a l I s c h e m i a S e c t i o n . T h e s c a n s , h o w e v e r , w e r e p e r f o r m e d o n e week p r e - o p e r a t i v e l y a n d a t 6 h r s . , 5 d a y s , 7 d a y s a n d 14 d a y s p o s t - o p e r a t i v e l y . T h e l a t t e r two t i m e a s s e s s m e n t s w e r e p e r f o r m e d w i t h a t r i a l p a c i n g . T h e p a c i n g w i r e s w e r e e x p o s e d f r o m t h e s u b c u t a n e o u s p o c k e t . A M e d t r o n i c p a c i n g u n i t ( M e d t r o n i c M e d i c a l I n c . , L o s A n g e l e s , C a ) was u s e d . H e a r t s w e r e p a c e d a t a r a t e o f 185 b e a t s / m i n . ( i i ) B l o o d - G a s A n a l y s i s - B l o o d g a s a n a l y s i s was p e r f o r m e d o n a B e c k m a n m o d e l 175 A u t o m a t i c p H / B l o o d Gas S y s t e m ( C o r n i n g M e d i c a l , M e d f i e l d , MA) a s d e s c r i b e d i n G l o b a l I s c h e m i a M o d e l e a r l i e r . - 75 -( e ) E l e c t r o n M i c r o s c o p y ( i ) T i s s u e C o l l e c t i o n - P u n c h b i o p s i e s o f t h e r e g i o n a l m y o c a r d i a l b e d w e r e t a k e n p r i o r t o s a c r i f i c e f o r e l e c t r o n m i c r o s c o p i c e x a m i n a t i o n , p r o v i d i n g u l t r a s t r u c t u r a l e v i d e n c e o f a n y i s c h e m i c c h a n g e s . A b i o p s y was t a k e n f r o m t h e n o n - i s c h e m i c a r e a p r o x i m a l t o t h e a m e r o i d c o n s t r i c t o r , a s a c o n t r o l s p e c i m e n , a n d a s p e c i m e n a l s o t a k e n f r o m t h e i s c h e m i c a r e a o f t h e l e f t v e n t r i c l e d i s t a l t o t h e a m e r o i d c o n s t r i c t o r . R e m a i n i n g p r o c e d u r e s w e r e i d e n t i c a l t o t h o s e d e s c r i b e d i n t h e G l o b a l I s c h e m i a s e c t i o n . ( i i ) P r o c e s s i n g - A s r e p o r t e d i n G l o b a l I s c h e m i a s e c t i o n . ( i i i ) S e c t i o n i n g - A s r e p o r t e d i n G l o b a l I s c h e m i a s e c t i o n . ( f ) L i g h t M i c r o s c o p y F o l l o w i n g s a c r i f i c e , t h e h e a r t s w e r e e x c i s e d a n d s a m p l e s o f t h e r e g i o n a l m y o c a r d i a l b e d t a k e n w i t h i n t h e i s c h e m i c a r e a a n d t h e n o n - i s c h e m i c a r e a . M e t h o d s t h e same a s t h a t r e p o r t e d i n G l o b a l I s c h e m i a s e c t i o n . ( g ) D a t a A n a l y s i s ( i ) 1 2 3 I P P A Image A n a l y s i s - s e e s e c t i o n E ( i ) o f G l o b a l I s c h e m i a M o d e l . ( i i ) S t a t i s t i c a l A n a l y s i s - S t a t i s t i c a l a n a l y s i s o f t h e m e t a b o l i c d a t a a n d a s s e s s m e n t o f 1 2 3 I P P A w a s h o u t r a t e s w e r e p e r f o r m e d - 76 -u s i n g a m u l t i v a r i a t e r e p e a t e d m e a s u r e s d e s i g n t o d e s i g n a t e d i f f e r e n c e s b e t w e e n mean v a l u e s o f t h e p r e - o p e r a t i v e a n d p o s t - o p e r a t i v e t i m e i n t e r v a l s . D a t a w e r e e x p r e s s e d a s mean + SD. L i n e a r r e g r e s s i o n a n a l y s i s was p e r f o r m e d t o e x a m i n e t h e c o r r e l a t i o n b e t w e e n a n y o f t h e m e t a b o l i c p a r a m e t e r s a n d 1 2 3 I P P A w a s h o u t w i t h t h e f u n c t i o n a l d a t a o v e r t h e p r o g r e s s i o n o f i s c h e m i a . ( 3 ) M y o c a r d i a l I n f a r c t i o n M o d e l ( a ) E x p e r i m e n t a l R e p r e s e n t a t i o n o f M y o c a r d i a l I n f a r c t i o n N i n e a d u l t d o g s ( 1 5 - 2 3 k g ) w e r e f a s t e d o v e r n i g h t a n d a n a e s t h e t i z e d w i t h s o d i u m p e n t o b a r b i t a l ( 2 5 mg/kg) I V . S u p p l e m e n t a l s o d i u m p e n t o b a r b i t a l was g i v e n a s n e c e s s a r y . T h e d o g s w e r e i n t u b a t e d a n d m a i n t a i n e d o n a p r e s s u r e - l i m i t e d v e n t i l a t o r ( M a r k - 7 R e s p i r a t o r - B i r d C o r p o r a t i o n ) . T h e e l e c t r o c a r d i o g r a p h was d i s p l a y e d o n a t w e l v e - c h a n n e l o s c i l l o s c o p e w i t h c o n c u r r e n t d i g i t a l r e a d o u t a n d r e c o r d i n g c a p a b i l i t i e s ( S E L a b o r a t o r i e s , E n g l a n d ; J o h n F o r t i n M f g . , L t d . , C a n a d a ) . T h e h e a r t was e x p o s e d v i a a f o u r t h i n t e r c o s t a l s p a c e t h o r a c o t o m y . T h e f e m o r a l a r t e r y was d i s s e c t e d c l e a r a n d c a n n u l a t e d t o a l l o w m o n i t o r i n g o f a r t e r i a l p r e s s u r e t h r o u g h o u t t h e p r o c e d u r e . T h e f e m o r a l v e i n was c a n n u l a t e d t o o b t a i n b l o o d s a m p l e s a t v a r i o u s i n t e r v a l s d u r i n g t h e p r o c e d u r e . T h e l e f t a n t e r i o r d e s c e n d i n g c o r o n a r y a r t e r y was d i s s e c t e d c l e a r a t a p o i n t a p p r o x i m a t e l y 3 cm f r o m t h e b i f u r c a t i o n o f t h e LAD f r o m t h e m a i n l e f t c o r o n a r y a r t e r y . P r i o r t o l i g a t i o n o f t h e LAD, t h e d o g s w e r e p l a c e d o n 100% o x y g e n f o r 15 m i n u t e s , g i v e n 50 mg. X y l o c a i n e , I V , 0.5 gm C a l c i u m g l u c o n a t e , I V a n d h a m p u l e o f s o d i u m b i c a r b o n a t e , I V . T h e LAD was l i g a t e d c o m p l e t e l y u s i n g a 2-0 d e x o n s u t u r e a n d t h e d o g s a l l o w e d t o s t a b i l i z e f o r 30 m i n u t e s - 77 -p r i o r t o c l o s i n g t h e i n c i s i o n . T h e d o g s w e r e t h e n w e a n e d o f f t h e r e s p i r a t o r . ( b ) M e t a b o l i c A s s e s s m e n t s M e t a b o l i c a s s e s s m e n t s o f v e n o u s b l o o d s a m p l e s i n c l u d e d l a c t a t e d e h y d r o g e n a s e , c r e a t i n e k i n a s e a n d CK-MB i s o e n z y m e d e t e r m i n a t i o n s . B l o o d was w i t h d r a w n p r e - o p e r a t i v e l y , p r e - i n f a r c t i o n , H - h o u r p o s t - i n f a r c t i o n , 2 h o u r s p o s t - i n f a r c t i o n a n d i m m e d i a t e l y p r i o r t o s a c r i f i c e . D e t e r m i n a t i o n s w e r e made a s d e s c r i b e d i n s e c t i o n ( B ) o f R e g i o n a l I s c h e m i a . ( c ) P r e p a r a t i o n o f B e t a - M e t h v l - 1 5 - p - 1 2 3 I - i o d o p h e n y l p e n t a d e c a n o i c  A c i d ( B 1 2 3 I P P A ) ( i ) R a d i o i o d i n e - i d e n t i c a l t o s e c t i o n C ( i ) o f G l o b a l I s c h e m i a . ( i i ) P r o d u c t i o n o f B 1 2 3 I P P A - i d e n t i c a l t o s e c t i o n C ( i i ) o f G l o b a l i s c h e m i a , e x c e p t f o r s u b s t i t u t i o n o f b e t a - m e t h y l - P P A f o r PPA. ( i i i ) H e x a n e E x t r a c t i o n - i d e n t i c a l t o s e c t i o n C ( i i i ) o f G l o b a l I s c h e m i a . ( i v ) P u r i f i c a t i o n - i d e n t i c a l t o s e c t i o n C ( i v ) o f G l o b a l I s c h e m i a . ( v ) P r e p a r a t i o n o f B 1 2 3 I P P A f o r I n j e c t i o n - i d e n t i c a l t o s e c t i o n C ( v ) o f G l o b a l I s c h e m i a . ( v i ) Q u a l i t y C o n t r o l - i d e n t i c a l t o s e c t i o n C ( v i ) o f G l o b a l I s c h e m i a . - 78 -( d ) D a t a A c q u i s i t i o n ( i ) I m a g i n g - A t 6 h o u r s p o s t - i n f a r c t i o n , s i n g l e - p h o t o n e m i s s i o n c o m p u t e r i z e d t o m o g r a p h y ( S P E C T ) was p e r f o r m e d u s i n g a S i e m a n s R o t a C a m e r a ( S i e m a n s G a m m a S o n i c s I n c . , Des P l a i n e s , I l l i n o i s ) . I n 7 o f 9 d o g s , i n s i t u s c a n n i n g was commenced i m m e d i a t e l y f o l l o w i n g t h e i n j e c t i o n o f 2 m C i o f 2 0 1 T h a l l i u m - c h l o r i d e ( D u p o n t ) . T h e d u a l c a m e r a h e a d s r o t a t e d a t 6 ° i n c r e m e n t s t a k i n g 30 s e r i a l i m a g e s o v e r a t o t a l s c a n n i n g t i m e o f 20 m i n u t e s . I m m e d i a t e l y f o l l o w i n g t h e 2 o iT 1 s c a n > 3 . 5 m C i o f B 1 2 3 I P P A was i n j e c t e d a n d a s i m i l a r s c a n p e r f o r m e d ( F i g u r e 6 ) . I m a g e s w e r e c o l l e c t e d o n a 64 x 64 x 8 m a t r i x a n d r e c o r d e d f o r 40 s e c o n d s a t e a c h a n g l e . I n t h e f i n a l two a n i m a l s , e a c h d o g was i m a g e d w i t h o n l y o n e o f t h e r a d i o n u c l i d e s a n d s i m i l a r a n a l y s i s c a r r i e d o u t . T h u s , i m a g e s w e r e o b t a i n e d f o r 2 0 1 T 1 a l o n e , a s w e l l a s B 1 2 3 I P P A a l o n e . T h e a n i m a l s w e r e s a c r i f i c e d f o l l o w i n g t h e i m a g i n g p r o c e d u r e . - 79 -2 0 1 T h a l l i u m S P E C T - s c a n i n d o g ( a n t e r i o r p r o j e c t i o n a t 6 h o u r s p o s t - i n f a r c t i o n ) , s h o w i n g t h e v e n t r i c u l a r c a v i t y a s a b l a c k h o l e s u r r o u n d e d b y a r i n g o f a c t i v i t y . T h e i n f a r c t a p p e a r s a s a n a r e a o f d e c r e a s e d a c t i v i t y w i t h i n t h e r i n g . B 1 2 3 I P P A S P E C T - s c a n i n same d o g a s a b o v e ( a n t e r i o r p r o j e c t i o n a t 6 h o u r s p o s t - i n f a r c t i o n ) , s h o w i n g t h e i n f a r c t ( I N F ) , v e n t r i c u l a r c a v i t y ( V C ) , n o r m a l m y o c a r d i u m (N) a n d t h e i s c h e m i c r i s k z o n e s ( I R Z ) . - 80 -( i i ) H i s t o c h e m i c a l A s s e s s m e n t - I m m e d i a t e l y a f t e r t h e a n i m a l s w e r e s a c r i f i c e d , t h e h e a r t s w e r e e x c i s e d , w e i g h e d a n d s l i c e d t r a n s v e r s e l y ( t o t h e l e v e l o f t h e m i t r a l v a l v e ) i n t o 10 mm. s e c t i o n s , u s i n g a n e l e c t r i c m e a t s l i c e r . T h e n u mber o f s l i c e s v a r i e d f r o m A t o 9 ( F i g u r e 7 ) . E a c h o f t h e s l i c e s was w e i g h e d a n d p l a c e d i n t o a b a t h c o n t a i n i n g 0.2 M p h o s p h a t e b u f f e r ( p H 7 . 4 ) , 0.4 % w t / v o l t r i p h e n y l t e t r a z o l i u m , a n d 0 . 0 4 % w t / v o l n i t r o b l u e t e t r a z o l i u m . T h e s e l a t t e r two a g e n t s w e r e o b t a i n e d f r o m S i g m a C h e m i c a l s I n c . , ( S t . L o u i s , MO). T h e s l i c e s r e m a i n e d i n t h e b a t h f o r 20 m i n u t e s a t 3 7 ° C . T h i s t e c h n i q u e a l l o w e d f o r v i s u a l i z a t i o n o f i n f a r c t e d m y o c a r d i u m ( H e a r s e , 1976, C a l d w e l l e t a l . . 1 9 8 4 ) . - 81 -F i g u r e 7 - H e a r t s h o w i n g l i g a t i o n a n d s e c t i o n c u t t i n g t e c h n i q u e . - 81a -- 82 -( i i i ) A u t o r a d i o g r a p h y - I n t h e f i n a l two e x p e r i m e n t s , i n w h i c h i m a g i n g was c o m p l e t e d w i t h 201^ 1 a l o n e a n d B 1 2 3 I P P A a l o n e , i s c h e m i c r i s k z o n e s w e r e d e t e r m i n e d b y a m a c r o a u t o r a d i o g r a p h i c t e c h n i q u e . T h e s t a i n e d , i n t a c t s l i c e s w e r e p l a c e d b e t w e e n two a c r y l i c s h e e t s a n d t h e p o s i t i o n m a r k e d . T h e s l i c e s w e r e f r o z e n a t - 7 0 ° C f o r 20 m i n u t e s . A s h e e t o f X - r a y f i l m was t h e n i n t e r p o s e d b e t w e e n o n e a c r y l i c s h e e t a n d t h e s l i c e s . T h e w h o l e a p p a r a t u s was p l a c e d i n a l i g h t - p r o o f b o x a n d s t o r e d a t - 2 0 ° C f o r 24 h o u r s . T h e f i l m s w e r e r e m o v e d a t t h e e n d o f t h i s t i m e p e r i o d a n d d e v e l o p e d u s i n g a n a u t o m a t i c p r o c e s s o r . A s h e e t o f c l e a r a c e t a t e p a p e r was p l a c e d o v e r t h e m a r k e d s l i c e s a n d t h e i n d i v i d u a l s l i c e s d r a w n m a n u a l l y i n c l u d i n g t h e i n f a r c t b o r d e r s . T h e p e r f u s e d a r e a s o n t h e m a c r o a u t o r a d i o g r a p h s a p p e a r e d a s r a d i o d e n s e a r e a s , w h i l e u n p e r f u s e d r e g i o n s a p p e a r e d o p a q u e . T h e c l e a r a c e t a t e t r a c i n g s w e r e s u p e r i m p o s e d o n t o t h e m a c r o a u t o r a d i o g r a p h s a n d i s c h e m i c r i s k z o n e s s e e n , i e . t h a t a r e a o f u n d e r p e r f u s i o n b e t w e e n t h e i n f a r c t b o r d e r a n d t h e r a d i o d e n s e a r e a s ( F i g u r e 8 ) . B o t h t h e a c e t a t e t r a c i n g s a n d m a c r o a u t o r a d i o g r a p h s w e r e q u a n t i t a t e d u s i n g p l a n i m e t r y . P l a n i m e t r i c q u a n t i f i c a t i o n was c o m p l e t e d b y o n e o b s e r v e r t w i c e w i t h a t h r e e m o n t h i n t e r v a l b e t w e e n a n d b y a s e c o n d o b s e r v e r o n c e . - 83 -T h e f i n d i n g s o f t h e a u t o r a d i o g r a p h i c t e c h n i q u e u t i l i z e d i n t h e p r e s e n t s t u d y a r e shown. P e r f u s i o n i n t h e s l i c e s o f h e a r t f r o m o n e e x p e r i m e n t w i t h t h e s l i c e t r a c i n g s o v e r l a i d o n t o t h e a u t o r a d i o g r a p h , s h o w i n g b o r d e r s o f t h e s l i c e s a n d i n f a r c t s , i s s e e n . A c l o s e r v i e w o f t h e c l e a r a c e t a t e p a p e r o v e r l a i d o n t o o n e s l i c e o f t h e a u t o r a d i o g r a p h s h o w i n g d e m a r c a t i o n o f t h e i n f a r c t i o n a n d t h e i s c h e m i c r i s k z o n e . - 84 -( i v ) H i s t o l o g y - F o l l o w i n g m a c r o a u t o r a d i o g r a p h y , t h e s l i c e s w e r e r e m o v e d f r o m t h e a p p a r a t u s a n d i n f a r c t e d t i s s u e c a r e f u l l y d i s s e c t e d o u t a n d w e i g h e d . S a m p l e s w e r e t h e n d i s s e c t e d f r o m t h e s t a i n e d a r e a ( v i a b l e t i s s u e ) a n d i n f a r c t e d r e g i o n a n d p l a c e d i n t o f o r m a l i n f o r s u b s e q u e n t h i s t o l o g i c a l s t a i n i n g a n d e x a m i n a t i o n . T h e t i s s u e s w e r e t h e n p l a c e d i n t o t i s s u e s a m p l e b o x e s a n d p a s s e d t h r o u g h a H i s t o m a t i c T i s s u e P r o c e s s o r . T h i s m a c h i n e a u t o m a t i c a l l y p a s s e s t h e s a m p l e t h r o u g h a s e r i e s o f d e h y d r a t i o n s t e p s i n p r o g r e s s i v e l y i n c r e a s i n g c o n c e n t r a t i o n s o f a l c o h o l , f o l l o w e d b y c l e a r i n g i n x y l o l a n d f i n a l l y i m p r e g n a t i o n a n d e m b e d d i n g i n wax. T h e b l o c k s w e r e t h e n c u t i n t o t h i c k s e c t i o n s u s i n g a m i c r o t o m e a n d p l a c e d o n t o a s l i d e . T h e s l i d e s w e r e t h e n s t a i n e d w i t h h e m a t o x y l i n a n d e o s i n , w h i c h s t a i n e d n u c l e i b l u e b e c a u s e o f t h e i r b a s o p h i l i c n a t u r e a n d c y t o p l a s m a n d c o n n e c t i v e t i s s u e s h a d e s o f p i n k . ( e ) D a t a A n a l y s i s ( i ) B 1 2 3 I P P A Image a n a l y s i s - Image a n a l y s i s was p e r f o r m e d o n a n ADAC S y s t e m s L a b o r a t o r i e s c o m p u t e r (Cam I I ) . S t a n d a r d a n d o b l i q u e r e c o n s t r u c t i o n was c o m p l e t e d b y f i l t e r e d b a c k p r o j e c t i o n . T h i r t y c o m p u t e r - r e c o n s t r u c t e d s l i c e s w e r e o b t a i n e d b e g i n n i n g f r o m t h e a p i c a l r e g i o n a n d t r a v e l l i n g t o w a r d s t h e b a s e . E a c h s i n g l e s h o r t - a x i s i m a g e r e p r e s e n t e d a t h i c k n e s s o f 0.53 cm i n r e a l s p a c e . T h e r e f o r e , 2 s h o r t - a x i s i m a g e s c o r r e s p o n d e d t o a s i n g l e 1 cm m y o c a r d i a l s l i c e . T o t a l h e a r t l e n g t h was c h e c k e d f o r a c c u r a c y b e t w e e n t h e e x c i s e d h e a r t a n d t h e c o m p u t e r - c a l c u l a t e d l e n g t h . I m a g e s w e r e a n a l y z e d q u a n t i t a t i v e l y f r o m a p e x t o b a s e e n d i n g a t t h e l e v e l o f t h e m i t r a l v a l v e . Q u a n t i f i c a t i o n was c o m p l e t e d b y c a l c u l a t i n g t h e - 85 -t o t a l n u m b e r o f p i x e l s w i t h i n e a c h a r e a o f i n t e r e s t , i . e . t h e s l i c e , t h e i n f a r c t , t h e v e n t r i c u l a r c a v i t y a n d t h e i s c h e m i c r i s k z o n e . T h e n , b a s e d o n k n own p i x e l n u m b e r s , p e r c e n t a g e s c o u l d b e c a l c u l a t e d . A s was t h e c a s e f o r p l a n i m e t r i c q u a n t i f i c a t i o n , c o m p u t e r q u a n t i f i c a t i o n was c o m p l e t e d b y o n e o b s e r v e r t w i c e w i t h a 3 m o n t h p e r i o d b e t w e e n a n d b y a s e c o n d o b s e r v e r o n c e . ( i i ) S t a t i s t i c a l A n a l y s i s - I n t e r - o b s e r v e r a n d i n t r a - o b s e r v e r d i f f e r e n c e s w e r e c a l c u l a t e d f o r t h e c o m p u t e r - g e n e r a t e d a n d h i s t o c h e m i c a l o b s e r v a t i o n s u s i n g t h e UBC S t a t i s t i c a l P a c k a g e , G e n l i n a n d p e r f o r m i n g a n ANOVA ( K a u l e t a l . . 1 9 8 5 ) . D a t a w e r e e x p r e s s e d a s a mean +SD. L i n e a r r e g r e s s i o n a n a l y s i s a s w e l l a s t h e c o r r e l a t i o n c o e f f i c i e n t w e r e u s e d t o d e t e r m i n e t h e m o re a c c u r a t e m e t h o d o f a s s e s s i n g p e r f u s i o n d e f e c t s i n c l u d i n g t h e i s c h e m i c r i s k z o n e . - 86 -V R E S U L T S ( 1 ) G l o b a l I s c h e m i a ( a ) A n a l y s i s o f T i m e - A c t i v i t y C u r v e s T a b l e 4 r e p o r t s t h e mean v a l u e s o f t h e e l i m i n a t i o n r a t e (th i n m i n s ) o f 12 3 J P P A i n t h e l a t e r a l w a l l , a p i c a l w a l l a n d s e p t a l w a l l o f t h e l e f t v e n t r i c l e f o r t h e e a r l y a n d l a t e p h a s e s o f t h e T - A c u r v e . T h e c u r v e i s b i - e x p o n e n t i a l a n d i t i s h y p o t h e s i z e d t h a t t h e e a r l y p h a s e ( f i r s t p o r t i o n o f t h e c u r v e ) r e p r e s e n t s b e t a - o x i d a t i o n , w h i l e t h e s e c o n d c o m p o n e n t ( o r l a t e p h a s e ) r e p r e s e n t s s t o r a g e o f t h e f a t t y a c i d s a s t r i g l y c e r i d e s . A n o r m a l i z e d c u r v e , a s s h o wn b y F i g u r e 9, was u t i l i z e d t o c a l c u l a t e t h e th v a l u e s l i s t e d i n T a b l e 4 a n d i s g r a p h i c a l l y r e p r e s e n t e d i n F i g u r e 10. C u r v e s w e r e n o r m a l i z e d t o p e a k c o u n t s a t t h e 2 m i n t i m e i n t e r v a l . E l i m i n a t i o n r a t e ( m i n s ) o f 1 2 3 I P P A i s shown b y F i g u r e 10 a n d T a b l e 4 ( e a r l y p h a s e a n d l a t e p h a s e ) . A n i n c r e a s e i n th was o b s e r v e d p o s t - o p e r a t i v e l y i n t h e l a t e r a l w a l l o n l y f o r g r o u p A ( F i g u r e 1 0 ( A ) ) . T h e s e p t a l w a l l a n d t h e a p i c a l w a l l f o r g r o u p A s h o w e d no s i g n i f i c a n t c h a n g e p o s t - o p e r a t i v e l y . G r o u p B s h o w e d a d e c r e a s i n g t r e n d i n t h e l a t e r a l w a l l a n d s e p t a l w a l l , w i t h n o c h a n g e i n t h e a p i c a l w a l l . T h e f i n a l g r o u p , C, i l l u s t r a t e s a d e c r e a s i n g t r e n d i n th v a l u e s f o r t h e l a t e r a l w a l l a n d a p i c a l w a l l , w i t h l i t t l e c h a n g e i n t h e s e p t a l w a l l . F i g u r e 1 0 ( B ) a n d T a b l e 4 i l l u s t r a t e t h e e f f e c t o f v a r i o u s c a r d i o p l e g i c t e c h n i q u e s o n t h e l a t e p h a s e o f 1 2 3 I P P A w a s h o u t . T h e th i s i n c r e a s e d f o r G r o u p A i n a l l t h r e e a r e a s o f t h e l e f t v e n t r i c l e i e . t h e l a t e r a l w a l l , s e p t a l w a l l a n d a p i c a l w a l l ( F i g u r e 1 0 ( B ) ) . I n c r e a s i n g t r e n d s a r e shown f o r g r o u p B i n t h e l a t e r a l w a l l , s e p t a l w a l l a n d t h e a p i c a l w a l l . I n g r o u p C, a s l i g h t d e c r e a s e i n t h e l a t e r a l w a l l a n d i n c r e a s e s i n t h e a p i c a l w a l l a n d s e p t a l w a l l w e r e s e e n . - 87 -T i m e - A c t i v i t y c u r v e s f o r 1 2 3 I P P A w a s h o u t f r o m t h e m y o c a r d i u m ( n o r m a l i z e d t o 2 m i n p e a k c o u n t s ) ( A ) p r e - o p e r a t i v e l y a n d ( B ) p o s t - o p e r a t i v e l y , f o l l o w i n g 2 h r s o f g l o b a l i s c h e m i a . -87a-0 10 20 30 Time (min) - 88 -F i g u r e 10 - W a s h o u t r a t e ( t 4 i n m i n ) o f 1 2 3 I P P A f o r t h e ( A ) e a r l y p h a s e a n d ( B ) l a t e p h a s e o f t h e T - A c u r v e . - 88a -( A) Lateral Wall 4°r-( p=0.018) .9 30 ^ 20 10 0 (A) EARLY PHASE (E) Apical Wall ±11 p<0.05 pNS pNS Iso-osmolar 10+ 10+ (10) SOD ALLO ±24 20 10 0 P N S pNS Iso-osmolar 10+ 10+ (10) SOD ALLO (C) Septal Wall 40 r = 20 0 ±33 I pNS pNS pNS Iso-osmolar 10+ 10 + (10) SOD ALLO • Control = Ischemia (A) Lateral Wall 400 -_ 300 c '6 200 100 0 p=0.051 pNS pNS Iso-osmolar 10+ io+ (10) SOD ALLO (B) LATE PHASE (B) Apical Wall 300 200 -p<0.05 pNS (p=0.034) Iso-osmolar 10+ (10) SOD pNS IO+ A L L O (C) Septal Wall 600 400 m 2 0 0 = ±1162 JL H i pNS pNS pNS Iso-osmolar 10+ 10+ (10) SOD ALLO Control Ischemia - 89 -T a b l e 4 W a s h o u t r a t e ( t h i n m i n s + SD) o f 1 5 - p - i o d o p h e n y l P e n t a d e c a n o i c A c i d a t c o n t r o l a n d p o s t - o p e r a t i v e t i m e i n t e r v a l s e x a m i n e d b y T - A c u r v e a n a l y s i s . M y o c a r d i a l r e g i o n s i n c l u d e d t h e l a t e r a l w a l l , a p i c a l w a l l a n d s e p t a l w a l l . G r o u p A - T y e r s ' I s o - o s m o l a r S o l u t i o n ( 1 0 ) G r o u p B - 10 + SOD G r o u p C - 10 + A l l o p u r i n o l G r o u p A G r o u p B G r o u p C C o n t r o l P o s t - o p e r a t i v e C o n t r o l P o s t - o p e r a t i v e C o n t r o l P o s t - o p e r a t i v e EARLY PHASE L a t e r a l W a l l 1 1 ± 6 2 4 + 1 0 ( p < 0 . 0 5 ) 20+17 1 4 ± 1 2 1 9 ± 2 4 1 3 ± 9 A p e x 13+10 1 6 ± 1 5 14+ 7 1 4 ± 7 14+ 6 1 1 ± 5 S e p t a l W a l l 1 6 ± 1 1 16+13 24+34 17+ 9 1 3 ± 1 4 1 5 ± 2 3 L A T E PHASE L a t e r a l W a l l 5 6 ± 6 0 4 1 6 ± 2 3 8 ( p = 0 . 0 5 ) 1 1 1 ± 1 0 1 2 4 0 ± 270 1 4 6 ± 2 1 5 1 3 2 ± 8 5 A p e x 3 9 ± 1 3 8 9 ± 2 6 ( p < 0 . 0 5 ) 2 1 7 ± 3 3 6 2 8 1 ± 344 1 0 8 ± 62 1 2 2 ± 8 0 S e p t a l W a l l 72+96 1 0 9 ± 46 194+272 6 9 9 ± 1 1 6 2 6 9 ± 16 1 1 6 ± 9 5 - 90 -F i g u r e 1 1 ( A ) shows t h e mean c o n t r o l w a s h o u t r a t e ( t ^ f i n m i n ) f o r t h e e a r l y p h a s e i n t h e l a t e r a l w a l l , a p e x a n d s e p t a l w a l l o f t h e l e f t v e n t r i c l e . F i g u r e 11(B) d e p i c t s t h e s i t u a t i o n f o u n d f o r t h e l a t e p h a s e o f t h e I P P A m e t a b o l i c t i m e - a c t i v i t y c u r v e . - 91 -W a s h o u t r a t e (th i n m i n ) a s s e s s e d p r e - o p e r a t i v e l y f o r t h e ( A ) e a r l y p h a s e a n d ( B ) l a t e p h a s e o f t h e T - A c u r v e s . - 91a -40 30 20 10 0 400 300 200 h 100 Early Phase Lateral wall Apex Septal wall Control (n l^^  0 Late Phase - 92 -T a b l e 5 M e a s u r e d a n d c a l c u l a t e d p a r a m e t e r s o f c a r d i a c h e m o d y n a m i c s t a t u s = C a r d i a c i n d e x , S I = S t r o k e i n d e x , SWT = S t r o k e w o r k i n d e x , SVR = S y s t e m i c v a s c u l a r r e s i s t a n c e , SBP = S y s t o l i c b l o o d p r e s s u r e , E F = E j e c t i o n f r a c t i o n ) . P a r a m e t e r s w e r e a s s e s s e d a t 2 - t i m e i n t e r v a l s , i n c l u d i n g " p r e " - c a r d i o p u l m o n a r y b y p a s s ( a s a c o n t r o l a s s e s s m e n t ) a n d " p o s t " - c a r d i o p u l m o n a r y b y p a s s ( a s a p o s t - o p e r a t i v e a s s e s s m e n t ) . T i m e C I S I SWI SVR HR SBP E F G r o u p A P r e - C B P 1 9 ± 0 84 1 5 ± 1 2 0 ± 1 5 1 5 3 ± 713 1 2 6 ± 5 121+11 7 3 ± 5 P o s t - C B P 2 6+0 3 22+3 22+3 3079+ 582 ( p < 0 . 0 5 ) 125+4 104+6 7 1 ± 8 G r o u p B P r e - C B P 2 6 ± 0 3 1 9 ± 2 2 4 ± 3 3 9 0 6 ± 394 1 3 5 ± 4 1 2 7 ± 8 8 0 ± 2 P o s t - C B P 3 0 ± 0 4 1 9 ± 2 2 3 ± 3 3 5 1 7 ± 584 1 5 2 ± 7 ( p < 0 . 0 5 ) 131+9 7 7 ± 5 G r o u p C P r e - C B P 2 5 ± 0 3 1 8 ± 2 2 5 ± 3 4 5 7 9 ± 605 1 4 3 ± 7 143+9 7 8 ± 4 P o s t - C B P 2 6 ± 0 2 16+1 21+3 4 0 2 3 ± 511 1 5 8 ± 7 141+7 7 9 ± 4 - 93 -( b ) H e m o d y n a m i c s C a r d i a c i n d e x ( C I ) was u n c h a n g e d i n g r o u p s A, B a n d C ( T a b l e 5 ) . S t r o k e I n d e x ( S I ) a l s o r e m a i n e d u n c h a n g e d i n a l l t h r e e g r o u p s . T h e r e was no c h a n g e i n l e f t v e n t r i c u l a r s t r o k e w o r k i n d e x (SWI) i n g r o u p s A, B a n d C. E j e c t i o n f r a c t i o n ( E F ) was u n c h a n g e d i n g r o u p A, g r o u p B a n d g r o u p C. S y s t e m i c v a s c u l a r r e s i s t a n c e ( S V R ) d e c r e a s e d i n g r o u p A, b u t r e m a i n e d u n c h a n g e d i n g r o u p s B a n d C. H e a r t r a t e (HR) was u n c h a n g e d i n g r o u p A a n d g r o u p C, b u t i n c r e a s e d i n g r o u p B. S y s t o l i c b l o o d p r e s s u r e ( S B P ) was u n c h a n g e d i n a l l t h r e e g r o u p s . ( c ) H i s t o l o g y ( i ) E l e c t r o n M i c r o s c o p y - U l t r a s t r u c t u r a l c h a n g e s a s s o c i a t e d w i t h e a c h o f t h e c a r d i o p l e g i c s o l u t i o n s a r e d e m o n s t r a t e d i n F i g u r e 12. A l l t h r e e g r o u p s show m i n i m a l u l t r a s t r u c t u r a l c h a n g e s i n m y o f i b r i l l a r i n t e g r i t y , m i t o c h o n d r i a l s w e l l i n g , i n t e r s t i t i a l a n d i n t r a c e l l u l a r edema, v a c u o l a t i o n o f t h e s a r c o p l a s m i c r e t i c u l u m a n d d i s r u p t i o n o f t h e m i t o c h o n d r i a l c r i s t a e . - 94 -E l e c t r o n m i c r o s c o p i c e v i d e n c e o f i s c h e m i c c h a n g e a t t h e u l t r a s t r u c t u r a l l e v e l s . ( A ) n o r m a l t i s s u e ( X 8 5 5 0 ) . ( B ) ( i ) I s o - o s m o l a r ( T v e r ' s ) s o l u t i o n ( X 8 5 5 0 ) ( i i ) 10 + SOD (X 8 5 5 0 ) . ( i i i ) 10 + A l l o p u r i n o l ( X 8 5 5 0 ) . MY = m y o f i b r i l P L s p l a s m a membrane Z s Z - l i n e G = g l y c o g e n M = m i t o c h o n d r i a - 94a -- 94b -- 94c -- 95 -( i i ) L i g h t M i c r o s c o p y - G r o s s c e l l u l a r c h a n g e s f o u n d i n a l l t h r e e s p e c i m e n s i n c l u d e edema a n d c e l l u l a r i n f i l t r a t i o n ( F i g u r e 1 3 ) . No d i s t i n c t i o n c o u l d b e made b e t w e e n t h e t h r e e c a r d i o p l e g i c s o l u t i o n s b a s e d o n g r o s s c e l l u l a r a n a t o m y . - 96 -C o n t r o l s p e c i m e n s h o w i n g m a c r o s c o p i c a n a t o m y o f t h e m y o c a r d i u m (X 6 3 ) . I s c h e m i c s p e c i m e n s h o w i n g g r o s s c h a n g e s a t t h e c e l l u l a r l e v e l . ( i ) I s o - o s m o l a r ( T y e r ' s ) s o l u t i o n (X 9 6 ) . ( i i ) 10 + SOD ( X 9 6 ) . ( i i i ) 10 + A l l o p u r i n o l ( X 9 6 ) . (1 B ( i i i - 97 -( 2 ) R e g i o n a l I s c h e m i a T h e p l a c e m e n t o f a n a m e r o i d c o n s t r i c t o r o n t h e l e f t a n t e r i o r d e s c e n d i n g c o r o n a r y a r t e r y was c o m p l e t e d o n 12 d o g s . S e v e n d o g s , w h i c h c o m p r i s e d t h e s t u d y g r o u p , s u r v i v e d t o 14 d a y s . T h e r e m a i n i n g a n i m a l s d i d n o t s u r v i v e t o t h e c o m p l e t i o n o f t h e e x p e r i m e n t b e c a u s e o f i n f a r c t i o n a n d t h u s w e r e e x c l u d e d f r o m t h e s t u d y . ( a ) A n a l y s i s o f 1 2 3 I P P A c u r v e s R e g i o n a l m y o c a r d i a l w a s h o u t o f 1 2 3 I P P A ( T a b l e 6 a n d F i g u r e 1 4 ( A ) ) f o r t h e e a r l y p h a s e o f t h e t i m e - a c t i v i t y c u r v e s s h o w e d no s i g n i f i c a n t c h a n g e s ; h o w e v e r , t r e n d s i n d i c a t e d d e c r e a s e d w a s h o u t i n t h e i s c h e m i c s e g m e n t s ( l a t e r a l w a l l a n d a p i c a l w a l l ) a s shown b y i n c r e a s e d t^i v a l u e s . S i m i l a r l y i n c r e a s e d th v a l u e s w e r e o b s e r v e d f o r t h e l a t e p h a s e i n t h e i s c h e m i c s e g m e n t s a s w e l l ( F i g u r e 1 4 ( B ) ) . T h e n o n - i s c h e m i c s e g m e n t ( s e p t a l w a l l ) s h o w e d no s t a t i s t i c a l l y s i g n i f i c a n t c h a n g e i n th f o r t h e e a r l y p h a s e ( F i g u r e 1 4 ( A ) ) . T h e l a t e p h a s e s h o w e d a n i n c r e a s e w h i c h was n o t s t a t i s t i c a l l y s i g n i f i c a n t ( F i g u r e 1 4 ( B ) ) . R e s u l t s o b t a i n e d w i t h o n e s t u d y a n i m a l l e d t o t h e l a r g e mean a n d s t a n d a r d d e v i a t i o n o b s e r v e d a n d i f t h i s a n i m a l was e x c l u d e d , t h e v a l u e was 85+40-p>0.05. T h e a n i m a l s h o w e d a l a r g e th v a l u e i n d i c a t i n g l i t t l e w a s h o u t o f t h e f a t t y a c i d . - 98 -R e g i o n a l m y o c a r d i a l w a s h o u t r a t e o f 1 2 3 I P P A (th i n m i n ) f o r t h e e a r l y p h a s e o f t h e T - A c u r v e . R e g i o n a l m y o c a r d i a l w a s h o u t r a t e (th i n m i n ) o f 1 2 3 I P P A f o r t h e l a t e p h a s e o f t h e T - A c u r v e . - 98a -IPPA ANAYLS IS Control Immediate 6 Days 7 Days 14 Days TIME *p<o.05 c o m p a r e d to control - 99 -T a b l e 6 1 2 3 I P P A w a s h o u t f o r b o t h t h e e a r l y a n d l a t e p h a s e s o f t h e T - A c u r v e s o v e r 14 d a y s o f d e v e l o p i n g i s c h e m i a . A r e a s e x a m i n e d w e r e t h e l a t e r a l , a p i c a l a n d s e p t a l w a l l o f t h e l e f t v e n t r i c l e . * l a r g e s t a n d a r d d e v i a t i o n i s d u e t o o n e d o g . CONTROL 6 HRS POST-O P E R A T I V E L Y 5 DAYS 7 DAYS 14 DAYS E a r l v P h a s e i i n m i n ) L a t e r a l W a l l 2 2 ± 1 0 34+14 13+11 2 1 ± 1 3 6 0 ± 1 0 7 A p e x 2 3 ± 1 0 2 9 ± 7 24+15 2 1 ± 1 2 3 7 ± 33 S e p t a l W a l l 2 2 ± 1 4 2 7 ± 8 22+14 19+12 14+ 7 L a t e P h a s e ( t h i n m i n ) * L a t e r a l W a l l 34+12 45+12 114+9 ( p < 0 . 0 5 ) 7 1 ± 50 ( p < 0 . 0 5 ) A p e x 44+ 8 47+23 6 9 ± 4 9 1 1 5 ± 104. S e p t a l W a l l 34+ 2 3 8 ± 2 8 62+63 1 0 7 ± 1 4 6 6 2 6 + 1 3 2 5 * - 100 -C o n t r o l a n d p o s t - o p e r a t i v e s c i n t i g r a m s a t 14 d a y s w i t h a s s o c i a t e d t i m e - a c t i v i t y c u r v e s a n d v a l u e s a r e s h o wn i n F i g u r e s 1 5 ( A ) a n d ( B ) . - 101 -F i g u r e 15 ( A ) - C o n t r o l s c i n t i g r a m i n d o g i n j e c t e d w i t h 1 5 - p - 1 2 3 I - i o d o p h e n y l p e n t a d e c a n o i c a c i d ( a n t e r i o r v i e w ) w i t h T - A c u r v e a n d th v a l u e s . ( C l ) . ( B ) - P o s t - o p e r a t i v e s c i n t i g r a m i n d o g i n j e c t e d w i t h 1 5 - p - 1 2 3 I - i o d o p h e n y l p e n t a d e c a n o i c a c i d ( a n t e r i o r v i e w ) a t 14 d a y s w i t h T - A c u r v e a n d th v a l u e s . ( T l ) . - 101a -- 102 -( b ) M e t a b o l i c A s s e s s m e n t s M e t a b o l i c d a t a ( T a b l e 7) s h o w e d no s t a t i s t i c a l l y s i g n i f i c a n t c h a n g e s i n a n y o f t h e p a r a m e t e r s e x c e p t f o r CK v a l u e s , i n w h i c h t h e i m m e d i a t e p o s t - o p e r a t i v e v a l u e o f 6333.4+1728 was s i g n i f i c a n t l y i n c r e a s e d ( p < 0 . 0 0 2 ) , b u t r e t u r n e d t o c o n t r o l l e v e l s a t 5 d a y s ( T a b l e 7 a n d F i g u r e 1 6 ( A ) ) . L a c t a t e d e h y d r o g e n a s e l e v e l s r e m a i n e d u n c h a n g e d t h r o u g h o u t t h e 14 d a y s , ( T a b l e 7 a n d F i g u r e 1 6 ( A ) ) . L e v e l s o f t h e i s o e n z y m e CK-MB r o s e s i g n i f i c a n t l y i m m e d i a t e l y f o l l o w i n g s u r g e r y a n d w e r e n o r m a l a t 14 d a y s p o s t - o p e r a t i v e l y ( F i g u r e 1 6 ( B ) ) . L a c t a t e l e v e l s s h o w e d n o s i g n i f i c a n t c h a n g e s t h r o u g h o u t t h e e x p e r i m e n t ( T a b l e 7 a n d F i g u r e 1 7 ) . ( c ) H e m o d y n a m i c A s s e s s m e n t s ( T a b l e 7) T h e r e was n o s i g n i f i c a n t c h a n g e i n h e a r t r a t e t h r o u g h o u t t h e e x p e r i m e n t . P a c i n g r e s u l t e d i n a s t a t i s t i c a l l y s i g n i f i c a n t i n c r e a s e i n h e a r t r a t e ( p < 0 . 0 0 1 ) , d u r i n g t h e a s s e s s m e n t o f m y o c a r d i a l m e t a b o l i s m u s i n g 1 2 3 I P P A a t b o t h s e v e n d a y s a n d 14 d a y s p o s t - o p e r a t i v e l y . - 103 -L a c t a t e d e h y d r o g e n a s e a n d CK l e v e l s p r e - a n d p o p e r a t l v e l y . CK-MB% a s s e s s e d a t c o n t r o l a n d p o s t - o p e r a t i v e i n t e r v a l s . - 103a -E n z y m a t i c A n a l y s i s A U/L thousands 8 r C K - M B % Control Immediate 5 Days Time (days) 7 Days 14 Days - 104 -F i g u r e 17 - L a c t a t e a s s e s s e d a t p r e - a n d p o s t - o p e r a t i v e t i m e i n t e r v a l s . - 104a -Metabolic Analysis Lactate meq/l Contro l Immediate 5 Days Time(days) BBi L a c t a t e - 105 -T a b l e 7. H e m o d y n a m i c a n d b l o o d a n a l y s e s a t v a r i o u s t i m e p e r i o d s d u r i n g g r a d u a l c o r o n a r y o c c l u s i o n o v e r a 14 d a y p e r i o d . * p a c e d t o a r a t e o f 185 b e a t s / m i n u t e . V A R I A B L E CONTROL IMMEDIATE 5 DAYS 7 DAYS ( p a c e d HR) 14 DAYS ( p a c e d HR) P u l s e 1 2 0 ± 3 6 1 3 5 ± 1 9 1 2 7 ± 2 9 1 2 3 ± 2 2 1 3 6 ± 2 4 b e a t s / m i n ) * ( 1 8 5 ) ( p < 0 . 0 0 1 ) * ( 1 8 5 ) ( p < 0 . 0 0 1 ) pH 7 . 3 6 ± 0 . 0 4 7.34+0.03 7 . 3 8 ± 0 . 0 2 7 . 3 8 ± 0 . 0 3 7 . 3 6 ± 0 . 0 4 PCO2 mmHg 4 3 ± 7 4 1 ± 6 4 2 ± 3 4 1 . 4 3 ± 2 . 5 41+4 p 0 2 mmHg 5 1 ± 1 4 3 7 ± 9 43+14 3 7 . 4 3 ± 7 . 9 54+11 H C 0 3 mmol/1 2 4 ± 1 . 8 2 2 ± 1 . 5 25.1+1.8 2 4 . 2 9 ± 0 . 9 5 2 3 . 0 0 ± 2 . 5 L a c t a t e meq/1 1 . 2 0 ± 0 . 7 4 1 . 2 3 ± 0 . 5 4 0 . 9 8 ± 0 . 2 7 0 . 8 7 ± 0 . 2 2 1 . 2 3 ± 0 . 5 7 LD U / l 1 2 5 ± 1 0 3 1 6 0 ± 5 6 2 1 2 ± 1 6 7 175+114 2 1 3 ± 1 4 6 CK U / l 2 0 6 ± 2 0 4 6 3 3 3 ± 1 7 2 8 ( p < 0 . 0 0 2 ) 220+79 1 8 3 ± 6 6 3 2 1 ± 3 3 2 - 106 -( d ) H i s t o l o g y P o s t - m o r t e m e x a m i n a t i o n o f t h e l e f t a n t e r i o r d e s c e n d i n g c o r o n a r y a r t e r y r e v e a l e d p a t e n c y i n a l l s e v e n a n i m a l s o f t h e s t u d y g r o u p ( F i g u r e 1 8 ( B a n d C ) ) . A m e r o i d c o n s t r i c t i o n , h o w e v e r , d i d r e s u l t i n s e v e r e c l o s u r e a t t h e 14 d a y i n t e r v a l ( F i g u r e 1 8 ( E ) ) . A l l f i v e a n i m a l s w h i c h h a d d i e d s h o w e d e v i d e n c e o f m y o c a r d i a l i n f a r c t i o n a s a s s e s s e d b y t e t r a z o l i u m s t a i n i n g a n d c o m p l e t e c l o s u r e o f t h e a m e r o i d c o n s t r i c t o r ( F i g u r e 1 8 ( D ) ) . F i g u r e 1 8 ( A ) shows a n o r m a l c o r o n a r y a r t e r y w i t h n o r m a l p a t e n c y . - 107 -M i c r o s c o p i c a s s e s s m e n t o f LAD c o r o n a r y a r t e r y s h o w i n g n o r m a l d i a m e t e r w i t h n o c o n s t r i c t i o n (X 9 6 ) . A s s e s s m e n t o f LAD c o r o n a r y a r t e r y i n o n e e x p e r i m e n t a t 14 d a y s o f c o n s t r i c t i o n ( X 9 6 ) . D i f f e r e n t d e g r e e o f c l o s u r e i n a n o t h e r a n i m a l i n t h e s t u d y (X 9 6 ) . M i c r o s c o p i c a s s e s s m e n t o f LAD c o r o n a r y a r t e r y s h o w i n g c o m p l e t e c l o s u r e s o f v e s s e l f r o m a n i m a l w h i c h c o l l a p s e d f r o m m y o c a r d i a l i n f a r c t i o n ( X 9 6 ) . A m e r o i d c o n s t r i c t o r s - a t 0 d a y s a n d a f t e r 14 d a y s i n s i t u . - 108 -( i ) E l e c t r o n m i c r o s c o p i c s t u d i e s o f t h e r e g i o n a l m y o c a r d i a l b e d s h o w e d i s c h e m i c c h a n g e s a t 14 d a y s p o s t - o p e r a t i v e l y , ( F i g u r e s 1 9 ( B ) a n d ( D ) ) . G r o s s c h a n g e s i n c l u d e d edema a n d m y o f i l a m e n t c l u m p i n g . M i t o c h o n d r i a a p p e a r e d g r o s s l y s w o l l e n a n d i r r e g u l a r i n s h a p e , w i t h t h i c k e n e d c r i s t a e . I t m u s t a l s o b e n o t e d t h a t t h e i s c h e m i a r a n g e d f r o m m i l d ( F i g u r e 1 9 ( B ) ) t o s e v e r e ( F i g u r e 1 9 ( D ) ) w i t h i n t h i s s t u d y . N o r m a l m y o c a r d i a l u l t r a s t r u c t u r e i s s e e n i n F i g u r e 1 9 ( A a n d C ) . ( i i ) L i g h t m i c r o s c o p y r e v e a l e d e v i d e n c e o f i s c h e m i a a s s h o wn i n F i g u r e 20. Damage was r e s t r i c t e d t o g r o s s c h a n g e s , s u c h a s edema. - 109 -E l e c t r o n m i c r o s c o p i c a s s e s s m e n t o f n o r m a l m y o c a r d i a l t i s s u e , ( A ) Dog 1, ( C ) D o g 2 ( X 8 5 5 0 ) . U l t r a s t r u c t u r a l c h a n g e s o f m y o c a r d i a l t i s s u e s h o w i n g e v i d e n c e o f i s c h e m i a , ( B ) Dog 1, (D) D o g 2 ( X 8 5 5 0 ) . - 109a -- 110 -L i g h t m i c r o s c o p i c c h a n g e s p r o d u c e d b y i s c h e m i a a t 14 d a y s i n two d i f f e r e n t a n i m a l s ( A ) D o g 1, a n d ( B ) D o g 2 (X 2 6 5 ) . » 110a -- I l l -( 3 ) M y o c a r d i a l I n f a r c t i o n M o d e l ( a ) M e t a b o l i c A s s e s s m e n t s V e n o u s c o n c e n t r a t i o n s o f l a c t a t e d e h y d r o g e n a s e ( L D H ) , c r e a t i n e k i n a s e ( C K ) a n d c r e a t i n e - k i n a s e - M B i s o e n z y m e (CK-MB) a r e d e p i c t e d i n F i g u r e 2 1 . LDH i n c r e a s e d f r o m a c o n t r o l v a l u e o f 74.56+25.30 U / L t o a p r e - s a c r i f i c e v a l u e o f 5 4 8 . 6 0 + 6 8 4 . 2 0 U / L ( p < 0 . 0 5 ) . CK v a l u e s i n c r e a s e d f r o m 1 60.33+46.44 U / L t o 5030.60+2232 U / L ( p < 0 . 0 5 ) . T h e c o n t r o l a n d p r e - s a c r i f i c e v a l u e s f o r CK-MB w e r e 3 1 8 . 5 0 ± 1 5 1 . 1 U / L a n d 8 2 9 . 8 7 ± 8 1 . 3 8 U/L, r e s p e c t i v e l y ( p < 0 . 0 5 ) . - 112 -V e n o u s c o n c e n t r a t i o n s o f l a c t a t e d e h y d r o g e n a s e , c r e a t i n e k i n a s e a n d c r e a t i n e k i n a s e - M B i s o e n z y m e p r e - a n d p o s t - o p e r a t i v e i n t e r v a l s . - 112a -Venous LDH,CK,CK-MB LDH=laciate dehydrogenase CK=creatine kinase CK-MB=creatine kinase 'MB' isoenzyme Control Pre-ligation 30 ' post- inf Time ±2232^ £±4820 i 20 ' post-inf 6 hr post-inf (pre-sacrifice) - 113 -( b ) A n a l y s i s o f P e r f u s i o n D e f e c t A s s e s s m e n t s F i g u r e 22 shows t h e c l e a r d e m a r c a t i o n o f t h e p e r f u s i o n d e f e c t w i t h T T Z s t a i n i n g . T a b l e 8 l i s t s t h e p l a n i m e t r i c i n f a r c t q u a n t i f i c a t i o n f o r e a c h o f t h e t h r e e m e t h o d s c a l c u l a t e d a s % o f t o t a l s l i c e s i z e . A l s o l i s t e d a r e t h e r a t i o s o f B 1 2 3 I P P A t o 2 0 1 T l f o r e a c h p e r f u s i o n d e f e c t a s s e s s m e n t . ( M e a n r a t i o = 1 . 0 1 ± 1 . 2 5 ) . - 114 -S t a i n i n g o f a m y o c a r d i a l i n f a r c t i o n u s i n g a s t a i n c o m b i n i n g n i t r o b l u e t e t r a z o l i u m a n d t r i p h e n y l t e t r a z o l i u m . T h e s t a i n i n g o f f o r m a z a n s w i t h i n v i a b l e t i s s u e c a n b e s e e n a s d a r k t i s s u e , w h i l e t h e i n f a r c t e d r e g i o n r e m a i n s p a l e . - 115 -T a b l e 8 - P e r f u s i o n d e f e c t s i z e c a l c u l a t e d a s % o f t o t a l s l i c e s i z e f o r e a c h o f t h e t h r e e m e t h o d s - h i s t o c h e m i c a l ( T T Z s t a i n i n g ) , B 1 2 3 I P P A a n d 2 0 1 T 1 . Do g S l i c e ( H i s t o c h e m i c a l ) ( B 1 2 3 I P P A ) ( 2 0 1 T 1 ) R a t i o s M e a n o f 2 . M e a n o f 2 Mean o f 2 r B i 2 3 I P P A l O b s e r v e r ( % ) O b s e r v e r ( % ) O b s e r v e r ( % ) [ 2 0 1 T 1 j 1 1 27 4 30 8 19 4 1.59 1 2 32 6 15 9 10 8 1.47 1 3 28 8 7 0 10 2 0.69 1 4 21 2 11 2 23 8 0.47 1 5 23 9 15 2 34 3 0.44 1 6 0 12 3 40 1 0.31 2 1 9 6 45 8 49 5 0.93 2 2 10 4 30 7 49 1 0.63 2 3 9 1 12 0 23 8 0.51 2 4 4 2 9 9 31 6 0.31 2 5 9 3 20 0 5 3 3.77 2 6 14 3 8 2 0 0 0.0 3 1 42 1 38 1 28 9 1.32 3 2 32 7 31 4 25 4 1.22 3 3 38 5 17 8 21 1 0.84 3 4 36 7 7 1 . 24 9 0.29 3 5 27 3 9 1 17 0 0.54 3 6 19 8 5 1 20 1 0.25 4 1 2 3 2 8 0 0.0 4 2 0 0 0 0.0 4 3 0 0 0 0.0 4 4 3 9 0 4 5 0.0 4 5 7 4 0 23 2 0.0 4 6 0 0 37 2 0.0 5 1 29 7 12 8 34 2 0.37 5 2 19 8 13 0 28 5 0.46 5 3 21 6 13 9 10 4 1.34 5 4 22 5 32 .8 17 0 1.93 5 5 7 9 39 3 14 4 2.73 6 1 24 7 31 5 24 4 1.29 6 2 20 4 23 4 25 8 0.91 6 3 27 2 10 5 11 4 0.92 6 4 13 5 22 .9 0 0 0.0 6 5 14 1 31 3 6 7 4.67 - 116 -T a b l e 8 ( c o n t i n u e d ) D o g S l i c e ( H i s t o c h e m i c a l ) ( B 1 2 3 I P P A ) ( 2 0 1 T 1 ) R a t i o s M e a n o f 2 M e an o f 2 M e a n o f 2 ( B1 2 3 I P P A l O b s e r v e r ( % ) O b s e r v e r ( % ) O b s e r v e r ( % ) [2 0 1 T 1 j 7 1 41 3 27 4 33.6 0.82 7 2 23 1 11 4 21.5 0.53 7 3 15 5 4 6 3.6 1.28 7 4 11 7 17 9 3.2 5.59 mean r a t i o • • 1 . 0 1 ± 1 . 2 5 8 1 48 5 34.6 8 2 38 0 50.2 8 3 47 2 36.5 8 4 40 6 39.1 8 5 38 1 37.4 8 6 4 1 7 32.5 8 7 4 1 3 5.8 9 1 20 3 24 8 9 2 16 9 10 1 9 3 10 4 0 0 9 4 0 25 5 9 5 0 16 9 9 6 0 7 6 \ - 117 -T h e c o r r e l a t i o n b e t w e e n e a c h o f t h e m e t h o d s i s d e p i c t e d b y F i g u r e 23. T h e a c t u a l i n f a r c t s i z e , a s a s s e s s e d b y t e t r a z o l i u m s t a i n i n g , i s c o m p a r e d t o B 1 2 3 I P P A ( r = 0 . 6 5 ) ( p < 0 . 0 0 5 ) a n d 2 0 1 T 1 ( r = 0 . 4 9 ) ( p < 0 . 0 0 5 ) . F i g u r e 24 d e m o n s t r a t e s t h e r e l a t i o n s h i p b e t w e e n B 1 2 3 I P P A a n d ? 0 1 T 1 ( r = 0 . 5 4 p < 0 . 0 0 5 ) . T h e o v e r a l l r a t i o f o r B 1 2 3 I P P A / 2 0 X T 1 i s 1.01 ± 1.25. - 118 -R e l a t i o n s h i p b e t w e e n e s t i m a t e s o f i n f a r c t s i z e b y t h e h i s t o c h e m i c a l m e t h o d ( T T Z s t a i n i n g ) v s . ( A ) B 1 2 3 I P P A a n d ( B ) 2 0 1 T 1 - 118a -% Inf - Histochemistry (TTZ) - 119 -F i g u r e 24 - G r a p h o f B 1 2 3 I P P A v s . 2 0 1T1 s h o w i n g t h e r e l a t i o n s h i p b e t w e e n t h e e s t i m a t i o n o f p e r f u s i o n d e f e c t s i z e b y t h e s e two i m a g i n g a g e n t s . - 119a -- 120 -( c ) A u t o r a d i o g r a p h i c a s s e s s m e n t o f I R Z T a b l e 9 l i s t s t h e p l a n i m e t r i c q u a n t i t a t i o n o f t h e i s c h e m i c r i s k z o n e a s w e l l a s t h e i s c h e m i c r i s k z o n e a s s e s s e d b y B 1 2 3 I P P A a n d 2 0 1 T 1 . - 121 -T a b l e 9 - A u t o r a d i o g r a p h i c a n d s c i n t i g r a p h i c a s s e s s m e n t o f i s c h e m i c r i s k z o n e c a l c u l a t e d a s % o f t o t a l s l i c e s i z e . D o g - s l i c e A u t o r a d i o g r a p h y B1 2 3 I P P A 201x1 8-1 0.0 _ 24.9 8-2 67.5 - 16.5 8-3 69.7 - 0.0 8-4 56.3 - 0.0 8-5 60.4 - 0.0 8-6 4 8 . 3 - 0.0 8-7 4 9 . 1 - 0.0 9-1 0.0 9.5 9-2 0.0 0.0 -9-3 0.0 18.7 -9-4 0.0 0.0 -9-5 0.0 19.2 -9-6 0.0 23.2 -- 122 -( d ) H i s t o l o g y L i g h t m i c r o s c o p y was u s e d t o e x a m i n e g r o s s c e l l u l a r c h a n g e s o f t e n a s s o c i a t e d w i t h m y o c a r d i a l i n f a r c t i o n ( F i g u r e s 2 5 ( A ) n o r m a l a n d ( B ) i n f a r c t e d t i s s u e ) . T h e m o s t e v i d e n t f i n d i n g i s t i s s u e edema. ( e ) I n t e r - o b s e r v e r a n d I n t r a - o b s e r v e r V a r i a n c e T h e r e was m a r g i n a l i n t e r - o b s e r v e r v a r i a n c e e x p r e s s e d b y ANOVA a n d D u n c a n m u l t i p l e r a n g e t e s t s . A n a l y s i s s h o w e d two h o m o g e n o u s s u b s e t s i n w h i c h p>0.05. I n t r a - o b s e r v e r v a r i a n c e was l a r g e d e m o n s t r a t i n g s i g n i f i c a n t d i f f e r e n c e s a t p<0.05. - 123 -L i g h t m i c r o s c o p i c a s s e s s m e n t o f n o r m a l c a n i n e m y o c a r d i u m (X 6 3 ) . G r o s s c e l l u l a r c h a n g e s f o l l o w i n g a c u t e m y o c a r d i a l i n f a r c t i o n ( X 6 3 ) . - 123a -124 -VI DISCUSSION ( a ) G l o b a l I s c h e m i a T h e h e m o d y n a m i c r e s u l t s o f t h i s s t u d y i n d i c a t e l i t t l e p r o t e c t i o n a f f o r d e d b y t h e a d d i t i o n o f a l l o p u r i n o l o r SOD t o t h e c a r d i o p l e g i c s o l u t i o n . R e s u l t s i n d i c a t e t h a t g r o u p A ( i s o - o s m o l a r c a r d i o p l e g i c s o l u t i o n ) i s s i m i l a r t o g r o u p B ( 1 0 + SOD) a n d g r o u p C ( 1 0 + a l l o p u r i n o l ) . M y e r s e t  a l . ( 1 9 8 6 ) , u s i n g a m o d e l o f g l o b a l i s c h e m i a i n i s o l a t e d r a b b i t h e a r t s , r e p o r t e d m a r g i n a l f u n c t i o n a l p r o t e c t i o n a f f o r d e d b y a l l o p u r i n o l a d d e d t o t h e c a r d i o p l e g i c s o l u t i o n a n d no p r o t e c t i o n a f f o r d e d b y SOD i n c l u d e d i n t h e c a r d i o p l e g i c s o l u t i o n . A n u mber o f s t u d i e s h a v e b e e n p e r f o r m e d ( S t e w a r t e t a l . . 1 9 8 5 ; a n d G o d i n e t a l . . 1 9 8 6 ) i n w h i c h p r e t r e a t m e n t o f t h e a n i m a l s w i t h a l l o p u r i n o l d e m o n s t r a t e d p r e s e r v a t i o n o f l e f t v e n t r i c u l a r f u n c t i o n a s a s s e s s e d b y h e m o d y n a m i c m e a s u r e m e n t s a n d u l t r a s t r u c t u r a l p r e s e r v a t i o n , r e s p e c t i v e l y . T h u s , t h e i n e f f e c t i v e n e s s o f a l l o p u r i n o l a n d SOD i n p r e s e r v i n g l e f t v e n t r i c u l a r f u n c t i o n i n t h i s s t u d y may b e a r e f l e c t i o n o f t h e f a i l u r e t o p r e t r e a t t h e a n i m a l s . A l l h e m o d y n a m i c m e a s u r e m e n t s w e r e made o n l y 30 m i n u t e s p o s t - o p e r a t i v e l y . I t i s i m p o r t a n t t o n o t e t h a t l e f t v e n t r i c u l a r f u n c t i o n s h o r t l y f o l l o w i n g CPB may c o n s i s t e n t l y b e d e p r e s s e d ( S w a n s o n e t a l . . 1 9 8 3 ) . I n o r d e r t o b e t t e r a s s e s s t h e e f f e c t s o f t h e i n t e r v e n t i o n s , m e a s u r e m e n t s s h o u l d h a v e b e e n r e p e a t e d a f t e r s e v e r a l h o u r s o r i d e a l l y 24-48 h o u r s p o s t - r e p e r f u s i o n . T h e r e p o r t o f f a v o r a b l e p r e s e r v a t i o n o f m i t o c h o n d r i a l u l t r a s t r u c t u r e a n d f u n c t i o n b y G o d i n e t a l . ( 1 9 8 6 ) , u s i n g a l l o p u r i n o l p r e t r e a t m e n t , a s w e l l a s t h e r e p o r t e d p r o t e c t i o n o f m i t o c h o n d r i a l o x i d a t i v e p h o s p h o r y l a t i o n u s i n g - 125 -SOD a n d c a t a l a s e i n t h e c a r d i o p l e g i a s o l u t i o n b y S h l a f e r e t a l . ( 1 9 8 2 ) , s u g g e s t s c o n t i n u e d f a t t y a c i d m e t a b o l i s m . T h e a s s e s s m e n t o f f a t t y a c i d m e t a b o l i s m b y r a d i o l a b e l e d f a t t y a c i d s may p r o v i d e a u s e f u l t o o l f o r i n d i c a t i n g m i t o c h o n d r i a l i n t e g r i t y a n d d e s e r v e s f u t u r e c o n s i d e r a t i o n a s s u c h . T h e u t i l i z a t i o n o f h y p o t h e r m i c c a r d i o p l e g i c a r r e s t h a s b e c o m e i n c r e a s i n g l y p o p u l a r o v e r t h e p a s t 25 y e a r s ( R o b e r t s , 1 9 8 7 ) . M e t a b o l i s m o f 1 2 3 I P P A i n t h e l e f t v e n t r i c u l a r w a l l s o f h e a r t s s u b j e c t e d t o r e v e r s i b l e g l o b a l i s c h e m i a i s shown i n T a b l e 4 a n d F i g u r e 10. E x a m i n a t i o n o f t r e n d s i n d i c a t e s t h a t t h e i s o - o s m o l a r s o l u t i o n was l e s s e f f e c t i v e i n m a i n t a i n i n g m y o c a r d i a l f a t t y a c i d m e t a b o l i s m , c o m p a r e d t o t h e s o l u t i o n s s u p p l e m e n t e d w i t h SOD a n d a l l o p u r i n o l . H e m o d y n a m i c a l l y , a l l g r o u p s s h o w e d s i m i l a r r e s u l t s . I n a s t u d y e x a m i n i n g h e m o d y n a m i c s a n d m e t a b o l i s m a t s i m i l a r t i m e s , F r i d r i c h e t a l . ( 1 9 8 6 ) d e m o n s t r a t e d t h a t n o r m a l h e m o d y n a m i c s a r e i n d i c a t i v e o f n o r m a l m e t a b o l i s m . I n t h i s s t u d y , h e m o d y n a m i c s w e r e a s s e s s e d 30 m i n u t e s a f t e r w e a n i n g f r o m CPB, c o m p a r e d w i t h m e t a b o l i s m w h i c h was a s s e s s e d 3 h o u r s p o s t CPB. T h i s c o u l d h a v e a b e a r i n g o n o u r r e s u l t s . D u r i n g t h e i s c h e m i c p e r i o d , a n a e r o b i c g l y c o l y s i s i s t h e d o m i n a n t m e c h a n i s m o f e n e r g y p r o d u c t i o n ( C o n t i a n d K a o , 1 9 8 3 ) . H o w e v e r , f o l l o w i n g r e p e r f u s i o n , t h e r e may e x i s t a m i x e d p o p u l a t i o n o f c e l l s , some f u n c t i o n i n g a e r o b i c a l l y , w h i l e t h e m a j o r i t y m a i n t a i n a n a n a e r o b i c s t a t e , a s d e m o n s t r a t e d b y O p i e e t a l . ( 1 9 7 3 ) . T h e s e a u t h o r s d e m o n s t r a t e d i n c r e a s e d u t i l i z a t i o n o f g l u c o s e w i t h i n t h e i s c h e m i c z o n e , f o l l o w i n g c o r o n a r y a r t e r y l i g a t i o n o v e r a two h o u r i n t e r v a l . We f o u n d a t r e n d o f i n c r e a s i n g f u n c t i o n a l i n d i c e s d u r i n g t h e i n i t i a l p o s t - b y p a s s p e r i o d f o r t h e i s o - o s m o l a r g r o u p ( T a b l e 5) a l t h o u g h o n l y SVR was s i g n i f i c a n t a t p<0.05. I t m u s t b e n o t e d , h o w e v e r , t h a t s y s t e m i c v a s c u l a r r e s i s t a n c e was - 126 -d e c r e a s e d t o a g r e a t e r e x t e n t i n g r o u p A, c o m p a r e d t o g r o u p s B a n d C ( T a b l e 5 ) . T h u s , t h e t r e n d t o w a r d s i m p r o v e d f u n c t i o n o f g r o u p A c o u l d s i m p l y r e f l e c t t h i s f a l l i n SVR. I n d e e d , e j e c t i o n f r a c t i o n r e m a i n e d r e l a t i v e l y u n c h a n g e d i n t h e T y e r s ' g r o u p ( 7 3 + 5 t o 71 + 8 ) ( p > 0 . 0 5 ) , i n d i c a t i n g n o c h a n g e i n f u n c t i o n ( T a b l e 5 ) . G r o u p s B a n d C show n o s i g n i f i c a n t c h a n g e i n f u n c t i o n p o s t - o p e r a t i v e l y . T h e d r a m a t i c f a l l i n SVR s e e n i n g r o u p A i s i n t e r e s t i n g . A l t h o u g h we w e r e n o t e x a m i n i n g t h e c o n s e q u e n c e s o f g l o b a l i s c h e m i a o n SVR, t h i s r e s u l t i s n o t e w o r t h y . T h e f a l l i n SVR w i l l i n f l u e n c e a f t e r l o a d a n d t h u s w i l l i n d i r e c t l y a f f e c t v a r i a b l e s d e p e n d e n t u p o n t h e l a t t e r , s u c h a s c a r d i a c i n d e x a n d s t r o k e i n d e x . T h e f a l l i n r e s i s t a n c e may l e a d t o a n i n c r e a s e d c a r d i a c o u t p u t , w h i c h w i l l i n t u r n i n c r e a s e c a r d i a c i n d e x ( n o t i n g t h e f o r m u l a f o r C I ) . S i m i l a r l y , a r i s e i n C I w i l l a f f e c t S I , s i n c e t h e l a t t e r i s d e p e n d e n t u p o n t h e f o r m e r . T h e q u e s t i o n o f why t h e SVR d e c r e a s e d s o d r a m a t i c a l l y i n g r o u p A i s unknown. E x a m i n i n g t h e f o r m u l a , o n e n o t i c e s t h a t many v a r i a b l e s may i n f l u e n c e i t s v a l u e . A l t h o u g h t h e e x a c t c a u s e i s unknown, o u r m a i n c o n c e r n was n o t w i t h t h e c o n s e q u e n c e s o f g l o b a l i s c h e m i a o n SVR, b u t o n m e t a b o l i s m . S i m i l a r l y , t h e i n c r e a s e i n HR f o r g r o u p B c a n n o t b e a c c o u n t e d f o r , b u t i n v i e w o f t h e l a r g e n u mber o f v a r i a b l e s w h i c h w i l l a f f e c t i t t h e a n s w e r may b e v e r y c o m p l e x . T h e m e t a b o l i c a s s e s s m e n t was p e r f o r m e d t h r e e h o u r s f o l l o w i n g w e a n i n g f r o m b y p a s s . A s a l r e a d y m e n t i o n e d , r e p e r f u s i o n , a s w e l l a s i s c h e m i a , may l e a d t o a m i x e d p o p u l a t i o n o f c e l l s , i e . v a r i a b l e d e g r e e s o f i s c h e m i c e f f e c t s . T h e l a t e r a l w a l l p l a y s a m a j o r r o l e i n l e f t v e n t r i c u l a r f u n c t i o n a n d s h o w e d s i g n i f i c a n t l y i n c r e a s e d th v a l u e s [ 1 1 + 6 t o 2 4 + 1 0 ( p < 0 . 0 5 ) a n d 56 ± 60 t o 417 ± 238 (p = 0 . 0 5 1 ) ] f o r t h e e a r l y a n d l a t e p h a s e s , - 127 -r e s p e c t i v e l y , ( T a b l e 4 ) i n t h e i s o - o s m o l a r g r o u p . T h i s i m p l i e s i m p a i r m e n t o f f a t t y a c i d o x i d a t i o n . A l t h o u g h e j e c t i o n f r a c t i o n a p p e a r e d t o b e u n c h a n g e d i m m e d i a t e l y f o l l o w i n g b y p a s s , a s s e s s m e n t o f f u n c t i o n a n d m e t a b o l i s m s e v e r a l ( i d e a l l y 2 4 - 4 8 ) h o u r s a f t e r b y p a s s may h a v e p r o v i d e d a more m e a n i n g f u l c o m p a r i s o n . T h e a b i l i t y o f SOD a n d a l l o p u r i n o l t o a b o l i s h t h i s i s c h e m i a - r e l a t e d s h i f t i n t h e b e t a - o x i d a t i o n p h a s e o f m e t a b o l i s m i s q u e s t i o n a b l e . I t i s known t h a t g l o b a l i s c h e m i a c a u s e s a s h i f t f r o m a e r o b i c t o a n a e r o b i c m e t a b o l i s m ( O p i e , 1 9 7 6 a , b ) , a s e v i d e n c e d b y a d e c r e a s e i n o x i d a t i v e p h o s p h o r y l a t i o n , t h u s d e c r e a s i n g f a t t y a c i d m e t a b o l i s m , s i n c e b o t h p r o c e s s e s a r e c o u p l e d . F a t t y a c i d m e t a b o l i s m may a l s o b e a f f e c t e d b y o x y g e n - d e r i v e d f r e e - r a d i c a l - i n d u c e d h y d r o l y s i s o f membranes a n d m i t o c h o n d r i a l membrane damage ( T r u m p , 1 9 8 2 ) . T h e s e e v e n t s may l e a d t o i m p a i r m e n t o f f a t t y a c i d m e t a b o l i s m a s r e f l e c t e d i n d e c r e a s e d m y o c a r d i a l w a s h o u t r a t e s o f t h e f a t t y a c i d . T h e r e s u l t s o f t h i s s t u d y d e m o n s t r a t e t h a t o n l y t h e l a t e r a l w a l l i n g r o u p A ( T y e r s i s o - o s m o l a r s o l u t i o n ) i s s i g n i f i c a n t l y a l t e r e d , c o m p a r e d t o t h e two o t h e r c a r d i o p l e g i c f o r m u l a t i o n s , a s a s s e s s e d b y t h e e a r l y p h a s e o f 1 2 3 I P P A w a s h o u t ( F i g u r e 1 0 ) . T h u s , i t w o u l d a p p e a r t h a t t h e c a r d i o p l e g i c s o l u t i o n s ( g r o u p s B a n d C ) , a i m e d a t p r e v e n t i n g o x y g e n c y t o t o x i c i t y , may h a v e p r o v e n b e n e f i c i a l f o r m a i n t e n a n c e o f m y o c a r d i a l f a t t y a c i d o x i d a t i o n . H o w e v e r , t h e l a r g e s t a n d a r d d e v i a t i o n s o b t a i n e d i n t h i s s t u d y may h a v e m a s k e d a n y c h a n g e s i n th i n t h e o t h e r s o l u t i o n s . T h e r e f o r e t h e f a c t t h a t o n l y t h e i s o - o s m o l a r g r o u p s h o w e d a s i g n i f i c a n t i n c r e a s e o f th f o r t h e l a t e r a l w a l l may o r may n o t s i g n i f y p o o r m y o c a r d i a l p r e s e r v a t i o n . T h e r e m a i n i n g g r o u p s may o r may n o t p r o t e c t t h e m y o c a r d i u m w e l l ; h o w e v e r , a g a i n t h e SD v a l u e s a r e l a r g e a n d may mask s i g n i f i c a n t v a l u e s . - 128 -V a r i a b i l i t y o f t h e r e s u l t s o f t h i s s t u d y may b e e x p l a i n e d b y w o r k c o m p l e t e d b y F o x e t a l . ( 1 9 8 5 ) a n d L e r c h ( 1 9 8 6 ) . T h e s e a u t h o r s r e p o r t e d t h a t w a s h o u t r a t e s o f t h o s e f a t t y a c i d s w h i c h b a c k - d i f f u s e o r a r e n o t m e t a b o l i z e d c o u l d a f f e c t t h e w a s h o u t c u r v e s a n d , t h u s , a c c o u n t f o r t h e v a r i a b i l i t y i n t h v a l u e s . T h e y a l s o f o u n d t h a t s u p p l y o f t h e t r a c e r t o t h e m y o c a r d i u m a n d w a s h o u t o f t h e t r a c e r b y t h e c o r o n a r y c i r c u l a t i o n may i n f l u e n c e t h e r e s u l t s . T h e s e f a c t o r s w e r e n o t t a k e n i n t o a c c o u n t i n t h i s s t u d y . L a t e p h a s e p h a r m a c o k i n e t i c s r e f l e c t e i t h e r t h e i n c o r p o r a t i o n o f f a t t y a c i d s a s t r i g l y c e r i d e s a n d p h o s p h o l i p i d s ( V a n d e r W a l l , 1 9 8 5 ) o r t h e s t o r a g e a n d d e c a y o f t h e i s o t o p e ( V i s s e r e t a l . . 1 9 8 6 ) . T h e i m p o r t a n c e o f t h i s e n e r g y s t o r e i s r e l a t i v e l y s e c o n d a r y t o t h e c l i n i c a l l y m o r e r e l e v a n t b e t a - o x i d a t i o n p h a s e . C o n t r o l m y o c a r d i a l w a s h o u t r a t e s ( t h ) a r e g r e a t e r i n t h e l a t e p h a s e , r e l a t i v e t o t h e e a r l y p h a s e , i n d i c a t i n g a s l o w e r u t i l i z a t i o n o f t h i s t r i g l y c e r i d e a n d p h o s p h o l i p i d p o o l . V a n d e r W a l l e t a l . ( 1 9 8 1 ) r e p o r t t h v a l u e s i n e x c e s s o f 100 m i n i n t h i s p h a s e . V a r i a b i l i t y i n o u r r e s u l t s may r e f l e c t a s c a n n i n g p e r i o d o f o n l y 30 m i n u t e s , w h i c h w o u l d a c c o u n t f o r o n l y t h e b e g i n n i n g o f t h i s l a t e p h a s e . Many o f t h e s t u d i e s e x a m i n i n g m y o c a r d i a l m e t a b o l i s m now e x t e n d t h e s c a n n i n g t i m e t o 6 0 - 9 0 m i n u t e s ( S c h o n e t a l . . 1 9 8 6 ) . A l l w a l l s d e m o n s t r a t e d a n i n c r e a s e i n t h f o r t h e l a t e p h a s e , b e t w e e n c o n t r o l a n d p o s t - o p e r a t i v e a s s e s s m e n t s . H o w e v e r , o n l y v a l u e s f o r t h e l a t e r a l w a l l a n d a p i c a l w a l l f o r t h e i s o - o s m o l a r g r o u p w e r e s t a t i s t i c a l l y s i g n i f i c a n t l y i n c r e a s e d ( p < 0 . 0 5 ) ( F i g u r e 1 0 ) . T h e s e p r o l o n g e d t h v a l u e s , f o l l o w i n g t h e i s c h e m i c i n t e r v a l , may i m p l y i m p a i r m e n t i n t h e r e l e a s e o f t r i g l y c e r i d e s a n d p h o s p h o l i p i d s f r o m t h i s s e c o n d a r y e n e r g y p o o l , o r c o u l d r e f l e c t a n i n c r e a s e i n t h e e s t e r i f i c a t i o n o f i n t r a c e l l u l a r - 129 -f r e e f a t t y a c i d s , t h u s r e d u c i n g f a t t y a c i d a v a i l a b i l i t y f o r b e t a - o x i d a t i o n , a s p o s t u l a t e d b y O p i e e t a l . ( 1 9 7 3 ) . T h e s t a n d a r d i s o - o s m o l a r s o l u t i o n a n d t h e c a r d i o p l e g i c s o l u t i o n s s u p p l e m e n t e d w i t h SOD a n d a l l o p u r i n o l s h o w e d s i m i l a r e f f e c t s o n t h e l a t e p h a s e o f 1 2 3 I P P A m e t a b o l i s m , t h u s i m p l y i n g t h a t a l l t h r e e s o l u t i o n s r e s u l t i n a n i n c r e a s e i n t h e c o n t e n t o f t h i s l i p i d p o o l a n d , b y i m p l i c a t i o n , p o o r p r o t e c t i o n a s a s s e s s e d b y c o m p a r i n g p r e - o p e r a t i v e t o p o s t - o p e r a t i v e th v a l u e s . T e o h e t a l . ( 1 9 8 8 ) h a v e d e m o n s t r a t e d t h a t o x i d a t i o n o f f a t t y a c i d s a n d g l u c o s e b y t h e m y o c a r d i u m i s i m p a i r e d a f t e r c a r d i o p l e g i c a r r e s t f o r c o r o n a r y b y p a s s g r a f t i n g . N i n e t e e n p a t i e n t s w e r e u s e d t o e x a m i n e t h e e f f e c t o f a r t e r i a l l a c t a t e c o n c e n t r a t i o n s o n t h e m e t a b o l i s m o f f a t t y a c i d s a n d a l s o o n v e n t r i c u l a r f u n c t i o n s i n c e l a c t a t e c a n b e u s e d a s a n e n e r g y s u b s t i t u t e . P a t i e n t c r i t e r i a w e r e : s t a b l e e x e r t i o n a l a n g i n a p e c t o r i s , d o u b l e - o r t r i p l e - v e s s e l CAD, a n d L V E F>30%. I n r a n d o m i z e d g r o u p s , p a t i e n t s w i t h i n t h e l o w l a c t a t e c o n c e n t r a t i o n (0 mmol/L) g r o u p r e c e i v e d a n i n t r a v e n o u s i n f u s i o n o f 150 m l s / h r o f n o r m a l s a l i n e p e r i o p e r a t i v e l y , w h i l e t h e p a t i e n t s who w e r e i n t h e h i g h l a c t a t e g r o u p r e c e i v e d 150 m l s / h r o f L a c t a t e d R i n g e r s s o l u t i o n ( 2 4 . 3 mmol/L l a c t a t e ) . L a c t a t e 1 4 C was u s e d t o a s s e s s m y o c a r d i a l l a c t a t e m e t a b o l i s m . T h e g r o u p w h i c h r e c e i v e d t h e p e r i o p e r a t i v e i n f u s i o n w i t h h i g h l a c t a t e s h o w e d i m p r o v e d f u n c t i o n a l r e c o v e r y c o m p a r e d t o t h e g r o u p i n f u s e d w i t h l o w l a c t a t e l e v e l s w h i c h may s u p p o r t t h e h y p o t h e s i s t h a t l a c t a t e may b e t h e p r e f e r r e d s u b s t r a t e a f t e r c a r d i o p l e g i c a r r e s t . T h i s s h i f t away f r o m f r e e f a t t y a c i d m e t a b o l i s m , f o l l o w i n g c a r d i o p l e g i c a r r e s t , may a l s o e x p l a i n t h e i n c r e a s e s i n th s e e n i n t h i s s t u d y . L a c t a t e l e v e l s w e r e n o t m e a s u r e d i n t h e s t u d y , s o c o n f i r m a t i o n o f l a c t a t e m e t a b o l i s m b e f o r e a n d a f t e r c a r d i o p l e g i c a r r e s t was n o t p o s s i b l e . - 130 -R e g i o n a l h e t e r o g e n e i t y o f t h e v a r i o u s a r e a s o f t h e h e a r t was a l s o a d d r e s s e d i n t h i s s t u d y ( F i g u r e 1 1 ) . T h e r e s u l t s d e m o n s t r a t e no s i g n i f i c a n t d i f f e r e n c e i n n o r m a l c a n i n e m y o c a r d i a l w a s h o u t r a t e s o f t h e v a r i o u s a r e a s o f t h e l e f t v e n t r i c l e a l t h o u g h , a g a i n , l a r g e s t a n d a r d d e v i a t i o n s w e r e n o t e d . T h i s v a l i d a t e s t h e u s e o f c o n t r o l a r e a s a n d e x p e r i m e n t a l a r e a s w i t h i n a s i n g l e h e a r t , s i n c e t h e a r e a s d e m o n s t r a t e s i m i l a r w a s h o u t r a t e s . ( b ) R e g i o n a l I s c h e m i a M o d e l T h e l a t e r a l w a l l a n d a p i c a l w a l l w i t h i n t h e l e f t v e n t r i c l e s h o w e d s i g n i f i c a n t i n c r e a s e s i n th v a l u e s a f t e r 14 d a y s o f g r a d u a l c o r o n a r y o c c l u s i o n , s u g g e s t i n g i m p a i r m e n t o f o x i d a t i v e f r e e f a t t y a c i d m e t a b o l i s m ( T a b l e 6 ) . T h e s e r e s u l t s c o n c u r w i t h e x p e r i m e n t a l a n d c l i n i c a l s t u d i e s , w h i c h h a v e d e m o n s t r a t e d d e c r e a s e d f a t t y a c i d w a s h o u t r a t e s w i t h i n i s c h e m i c m y o c a r d i u m ( R e s k e e t a l . . 1982 a,b; D u d c z a k e t a l . . 1 9 8 2 a , b ; V a n d e r W a l l e t a l . . 1 9 8 3 ) . I s c h e m i a was c o n f i r m e d b y e l e c t r o n m i c r o s c o p i c e x a m i n a t i o n o f a c o n t r o l s p e c i m e n a n d a n i s c h e m i c a r e a w i t h i n t h e r e g i o n a l m y o c a r d i a l b e d a f t e r 14 d a y s o f g r a d u a l c o r o n a r y o c c l u s i o n ( F i g u r e 1 9 ) . U l t r a s t r u c t u r a l i s c h e m i c c h a n g e s a r e s i m i l a r t o t h o s e r e p o r t e d b y R e i m e r a n d J e n n i n g s ( 1 9 8 7 ) , i e . edema, m y o f i l a m e n t c l u m p i n g , m i t o c h o n d r i a l s w e l l i n g w i t h i r r e g u l a r i t i e s i n t h e i r s h a p e a n d c r i s t a e t h i c k e n i n g . V a r i a b i l i t y b e t w e e n a n i m a l s , a s t o e x t e n t o f i s c h e m i a , c o u l d b e o b s e r v e d . T h e f l o c c u l e n t d e n s i t i e s , o r a m o r p h o u s s p e c k s , f o u n d w i t h i n t h e m i t o c h o n d r i a a r e r e p r e s e n t a t i v e o f a c e r t a i n d e g r e e o f i s c h e m i a . H o w e v e r , n o t a l l a n i m a l s m a n i f e s t e d t h i s c h a n g e ; t h u s , c l e a r l y some d i s c r e p a n c y e x i s t s a s t o t h e l e v e l o r d e g r e e o f i s c h e m i c i n j u r y b e t w e e n a n i m a l s . - 131 -A s t u d y b y R a i l t o n e t a l . ( 1 9 8 7 ) s u g g e s t s t h a t i m a g i n g w i t h i o d i n a t e d a l i p h a t i c f a t t y a c i d s ( e . g . 1 2 3 I - h e p t a d e c a n o i c a c i d ) may n o t b e o f s u f f i c i e n t s e n s i t i v i t y o r s p e c i f i c i t y t o b e u s e d f o r r o u t i n e c l i n i c a l a s s e s s m e n t o f c a r d i a c p a t i e n t s . T h e s e a u t h o r s r e p o r t e d t h a t 40% o f p a t i e n t s w i t h a n g i n a p e c t o r i s a n d a p o s i t i v e e x e r c i s e e l e c t r o c a r d i o g r a m c o u l d n o t b e r e l i a b l y d i a g n o s e d u s i n g t h i s i m a g i n g a g e n t . H o w e v e r , t h e t e r m i n a l l y - l a b e l e d 1 7 - c a r b o n s t r a i g h t - c h a i n f a t t y a c i d u s e d ( 1 2 3 I - H D A ) r e q u i r e d b a c k g r o u n d s u b t r a c t i o n a n d t h i s p r o c e d u r e may h a v e d e c r e a s e d i t s s e n s i t i v i t y . R e c e n t l y , s t u d i e s h a v e q u e s t i o n e d t h e i n t e r p r e t a t i o n o f m y o c a r d i a l w a s h o u t r a t e s i n t e r m s o f t h e m e c h a n i s m o f w a s h o u t . I t i s r e p o r t e d t h a t t h e e a r l y e l i m i n a t i o n p h a s e o f t h e T - A c u r v e , a s s u m e d t o r e p r e s e n t b e t a - o x i d a t i o n , i s a c t u a l l y a r e f l e c t i o n o f t h e d i f f u s i o n o f t h e f r e e i o d i n e f r o m t h e m y o c y t e i n t o t h e b l o o d s t r e a m ( V i s s e r e t a l . . 1 9 8 5 ) . T h e s e a u t h o r s s u g g e s t t h e d e c r e a s e i n m y o c a r d i a l w a s h o u t r a t e s d e m o n s t r a t e d u n d e r i s c h e m i c c o n d i t i o n s may b e t h e r e s u l t o f a d e c r e a s e d w a s h o u t o f r a d i o i o d i d e . H o w e v e r , r a d i o i o d i n a t e d h e p t a d e c a n o i c a c i d , s u b s e q u e n t t o c a t a b o l i s m , r e l e a s e s f r e e r a d i o i o d i d e , w h i c h i s f r e e t o r e c i r c u l a t e . M a c h u l l a e t a l . ( 1 9 8 0 ) i n o r d e r t o p r e v e n t t h e r e l e a s e o f r a d i o i o d i d e f o l l o w i n g i n j e c t i o n o f a l a b e l e d f a t t y a c i d , u s e d r a d i o i o d i n a t e d o m e g a - p h e n y l p e n t a d e c a n o i c a c i d i n a m u r i n e m o d e l . R e s k e e t a l . ( 1 9 8 5 a , c ) e x a m i n e d t h e k i n e t i c s o f t i s s u e c o n c e n t r a t i o n s o f l a b e l e d c a t a b o l i t e s o f 1 2 3 I P P A a n d f o u n d a r a p i d o x i d a t i o n o f I P P A a n d s u b s e q u e n t w a s h o u t o f 1 2 3 I P P A c a t a b o l i t e s f r o m t h e m y o c a r d i u m . T h e r e f o r e , t h e th v a l u e s s h o u l d n o t b e i n f l u e n c e d b y r e c i r c u l a t i n g i o d i n e s i n c e t h e i o d i d e w i l l b e r e m o v e d f r o m t h e b o d y s t i l l a t t a c h e d t o t h e p h e n y l r i n g . T h e w a s h o u t o f r a d i o l a b e l e d f a t t y a c i d s w i l l r e q u i r e f u r t h e r i n v e s t i g a t i o n b e f o r e i t b e c o m e s c o m p l e t e l y u n d e r s t o o d . - 132 -T h e p h e n y l a t e d d e r i v a t i v e o f p e n t a d e c a n o i c a c i d u s e d i n t h i s s t u d y r e s u l t s i n t h e r e l e a s e o f 1 2 3 I - b e n z o i c a c i d i n t o t h e p e r i p h e r a l c i r c u l a t i o n , w h e r e i t c a n b e f u r t h e r m e t a b o l i z e d b y t h e l i v e r a n d k i d n e y s t o 1 2 3 I - h i p p u r i c a c i d a n d f i n a l l y e x c r e t e d i n t h e u r i n e ( M a c h u l l a e t a l . . 1980 a n d R e s k e e t a l . . 1 9 8 2 a , d ) . T h e m y o c a r d i a l i m a g e s o b t a i n e d a l l o w e d c l e a r d e m a r c a t i o n o f l e f t v e n t r i c u l a r w a l l s a s r e p o r t e d b y R e s k e e t a l . ( 1 9 8 2 a , d ) . T h e p r o l o n g e d th v a l u e s i n b o t h e a r l y a n d l a t e p h a s e s o f t h e t i m e - a c t i v i t y c u r v e s ( T a b l e 6 a n d F i g u r e 14) o f t h e i s c h e m i c s e g m e n t s a t 14 d a y s may b e a t t r i b u t e d t o a r e d u c t i o n i n o x y g e n s u p p l y i n d u c e d b y a d e c r e a s e i n c o r o n a r y d i a m e t e r b y way o f t h e a m e r o i d c o n s t r i c t o r . A t r i a l p a c i n g i n d u c e d a n a d d i t i o n a l s t r e s s b y i n c r e a s i n g o x y g e n demand, t h u s r e m o v i n g a n y b e n e f i t o f c o m p e n s a t o r y f l o w v i a t h e d e v e l o p m e n t o f c o l l a t e r a l c i r c u l a t i o n w i t h i n t h e i s c h e m i c b e d ( B r a z i e r e t a l . . 1975; S c h e l l e t a l . . 1 9 7 9 ; H o r n b y e t a l . . 1 9 7 6 ) . T h e r e s u l t s o b t a i n e d i n t h i s s t u d y a r e i n a g r e e m e n t w i t h t h o s e o f V a n d e r W a l l e t a l . ( 1 9 8 1 ) , i n w h i c h a g r o u p o f p a t i e n t s w i t h c o r o n a r y a r t e r y d i s e a s e w e r e e x e r c i s e d a n d s c a n n e d w i t h HDA. T h e i s c h e m i c r e g i o n s s h o w e d s i g n i f i c a n t l y i n c r e a s e d th v a l u e s , w h i l e t h e c o n t r o l g r o u p ( n o r m a l v o l u n t e e r s ) , a t r e s t , h a d n o r m a l w a s h o u t r a t e s . I n t h e p r e s e n t s t u d y , a t r i a l p a c i n g r e s u l t e d i n s i g n i f i c a n t l y i n c r e a s e d h e a r t r a t e s a n d t h e i s c h e m i c r e g i o n s s h o w e d i n c r e a s e d th v a l u e s . H a n s e n e t a l . ( 1 9 8 8 ) d e m o n s t r a t e d t h a t e x e r c i s e - i n d u c e d m y o c a r d i a l i s c h e m i a r e s u l t s i n a l t e r e d 1 2 3 I P P A u p t a k e , r e d u c e d w a s h o u t , o r b o t h . T h e s e r e s u l t s a r e c o n s i s t e n t w i t h t h e l o n g i t u d i n a l s t u d y p e r f o r m e d h e r e a n d a l s o w i t h a r e p o r t p u b l i s h e d b y K e n n e d y e t a l . ( 1 9 8 6 ) . T h e l a t t e r d e m o n s t r a t e d d r a m a t i c a l l y r e d u c e d 1 2 3 I P P A w a s h o u t i n p a t i e n t s w i t h s e v e r e CHD c o m p a r e d t o n o r m a l v o l u n t e e r s . - 133 -S c h o n e t a l . ( 1 9 8 6 ) , r e p o r t s t h a t t h e s o - c a l l e d b e t a - o x i d a t i o n p h a s e i s i n f l u e n c e d b y i n c r e a s i n g MV0 2 a n d a u g m e n t e d c a r d i a c w o r k . I f t h e s e two f a c t o r s w e r e t o i n f l u e n c e w a s h o u t r a t e s a s e x p e c t e d , a n i n c r e a s e i n f a t t y a c i d w a s h o u t s h o u l d b e s e e n . T h e i n c r e a s e d a v a i l a b i l i t y o f o x y g e n a n d i n c r e a s e d w o r k l o a d s h o u l d r e s u l t i n a n i n c r e a s e d u t i l i z a t i o n o f t h e f a t t y a c i d , t h u s i n c r e a s i n g w a s h o u t r a t e s . H o w e v e r , t h e e f f e c t s a r e s u c h t h a t m y o c a r d i a l w a s h o u t r a t e s d e c r e a s e . T h i s w o u l d s u g g e s t t h a t th o f t h i s e a r l y p h a s e i s n o t a r e f l e c t i o n o f f a t t y a c i d m e t a b o l i s m . H o w e v e r , s i n c e HDA a n d 1 2 3 I P P A o x i d a t i o n r e s u l t i n d i f f e r e n t c a t a b o l i c e n d - p r o d u c t s , s t u d i e s c o m p a r i n g t h e e f f e c t o f MV0 2 a n d c a r d i a c w o r k o n 1 2 3 I P P A m e t a b o l i s m a r e n e e d e d t o d i s t i n g u i s h a n y d i f f e r e n c e s f r o m t h e p h y s i o l o g i c a l l y o c c u r r i n g p a l m i t i c a c i d l a b e l e d w i t h 1 1 C . S c h o n h a s s t a t e d t h a t t h e s i z e s ( i e . t h e Y - i n t e r c e p t o f t h e T - A c u r v e ) o f t h e e a r l y a n d l a t e p h a s e s a r e v a l i d m e a s u r e s o f f a t t y a c i d m e t a b o l i s m a n d may p r o v i d e a more v a l i d c o m p a r i s o n . T h e d e b a t e , h o w e v e r , c e n t r e s a r o u n d w h a t t h e s e s o - c a l l e d e l i m i n a t i o n p h a s e s r e p r e s e n t . T h e i n f l u e n c e o f i s c h e m i a , b y way o f r e g i o n a l m y o c a r d i a l b l o o d f l o w a n d v a r i o u s i n t e r v e n t i o n s , o n t h e o x i d a t i o n o f 1 2 3 I P P A was a l s o e x a m i n e d b y R e s k e e t a l . ( 1 9 8 6 ) . T h e s e a u t h o r s d e m o n s t r a t e d t h a t i n c r e a s i n g c o n c e n t r a t i o n s o f l a c t a t e d e c r e a s e d f a t t y a c i d w a s h o u t , r e s u l t i n g i n s i g n i f i c a n t l y i n c r e a s e d th v a l u e s . S i m i l a r f i n d i n g s w e r e r e p o r t e d b y D u w e l e t a l . ( 1 9 8 8 ) i n w h i c h f a s t e d p a t i e n t s w e r e l o a d e d w i t h s o d i u m l a c t a t e (3mM/kg) p r i o r t o 1 2 3 I - H D A s c i n t i g r a p h y . T h e h a l f - l i f e v a l u e s i n c r e a s e d a n d u p t a k e l e v e l s d e c r e a s e d f o l l o w i n g t h e l a c t a t e i n t e r v e n t i o n . B o t h l a c t a t e a n d e x e r c i s e d e m o n s t r a t e d s i m i l a r s c i n t i g r a p h i c f i n d i n g s s u g g e s t i n g t h a t l a c t a t e w i l l i n h i b i t F F A o x i d a t i o n u n d e r n o r m a l , a s w e l l a s i s c h e m i c , - 134 -c o n d i t i o n s ( D u w e l e t a l . . 1 9 8 8 ) . P e r f u s i o n was a l s o i d e n t i f i e d a s a m a j o r d e t e r m i n a n t o f c a r d i a c 1 2 3 I P P A u p t a k e . T h e c h a n g e s i n th v a l u e s r e p o r t e d i n t h i s s t u d y may b e t h e r e s u l t o f a r e d u c t i o n i n b l o o d f l o w , t h u s r e d u c i n g o x y g e n s u p p l y , b u t n o t a n i n c r e a s e i n l a c t a t e , a n e n d - p r o d u c t o f i s c h e m i c m e t a b o l i s m . T h e l a t t e r was shown n o t t o s i g n i f i c a n t l y i n c r e a s e i n t h i s s t u d y ( T a b l e 7 ) . A n u m b e r o f c o n c e r n s a r e a p p a r e n t i n t h i s s t u d y . T h e d e g r e e o f c l o s u r e a n d r a t e o f s w e l l i n g ( F i g u r e 18) o f t h e a m e r o i d c o n s t r i c t o r c o u l d n o t b e c o n t r o l l e d , l e a d i n g t o v a r i a b l e d e g r e e s o f i s c h e m i a i n e x p e r i m e n t a l a n i m a l s a s m e n t i o n e d e a r l i e r . T h e e l e c t r o n m i c r o g r a p h s s h o w e d v a r i a b l e d e g r e e s o f i s c h e m i c damage ( F i g u r e 1 9 ) a n d t h i s d i s c r e p a n c y i n t h e e x t e n t o f i s c h e m i a may a c c o u n t f o r t h e v a r i a b i l i t y s e e n i n th v a l u e s . T h e l a r g e s t a n d a r d d e v i a t i o n s a s s o c i a t e d w i t h a s s e s s m e n t o f th may a l s o b e due t o t h e way i n w h i c h a n i m a l s i n t h e s t u d y w e r e g r o u p e d . W a s h o u t r a t e s (th) w e r e g r o u p e d a c c o r d i n g t o t h e t i m e o f a s s e s s m e n t . H o w e v e r , a s m e n t i o n e d e a r l i e r , r a t e o f s w e l l i n g c o u l d n o t b e c o n t r o l l e d . T h e r e f o r e , t h e r a d i u s o f t h e c e n t r a l l u m e n o f o n e c o n s t r i c t o r a t d a y f i v e , f o r e x a m p l e , c o u l d h a v e b e e n r e d u c e d b y 50%, w h i l e i n a n o t h e r c o n s t r i c t o r , t h e r e d u c t i o n i n r a d i u s c o u l d h a v e b e e n 8 0 % . H o w e v e r , b o t h th a s s e s s m e n t s w e r e p e r f o r m e d o n t h e same d a y a n d t h u s w e r e p l a c e d i n t o t h e same g r o u p . I f t h e a s s e s s m e n t s o f th w e r e c a t e g o r i z e d a s t o d e g r e e o f i s c h e m i a a n d n o t t i m e , t h e s t a n d a r d d e v i a t i o n s may h a v e b e e n r e d u c e d . T h a t i s , i f we w e r e a b l e t o i n j e c t m i c r o s p h e r e s , a s d i s c u s s e d e a r l i e r , a n d q u a n t i t a t e b l o o d f l o w r e d u c t i o n , c a t e g o r i e s b a s e d o n d e g r e e o f i s c h e m i a m i g h t h a v e b e e n r e c o r d e d . T h u s , a l l th v a l u e s a t 50% r e d u c t i o n i n b l o o d f l o w , f o r e x a m p l e , w o u l d h a v e b e e n a s s e s s m e n t 1, 60% a s s e s s m e n t 2, a n d s o o n . T h i s m i g h t a l l e v i a t e some o f t h e s t a t i s t i c a l - 135 -p r o b l e m s we e x p e r i e n c e d ( l a r g e s t a n d a r d d e v i a t i o n s ) i n t h e a n a l y s i s o f t h e d a t a . T h e a s s e s s m e n t o f r e g i o n a l m y o c a r d i a l b l o o d f l o w b y a m i c r o s p h e r e t e c h n i q u e was p r o p o s e d . H o w e v e r , i n j e c t i n g t h e s e m i c r o s p h e r e s a t e a c h t i m e i n t e r v a l w o u l d n e c e s s i t a t e a n a t r i a l c a n n u l a b e m a i n t a i n e d f o r 14 d a y s . T h e p r o b l e m s a s s o c i a t e d w i t h t h i s r e s u l t e d i n i t s e x c l u s i o n f r o m t h e s t u d y . We do r e a l i z e t h a t t h i s d i f f i c u l t y m u s t b e o v e r c o m e i f t h i s r e s e a r c h i s t o move f o r w a r d . F i v e o f t w e l v e a n i m a l s d i e d a s a r e s u l t o f c o m p l e t e c l o s u r e o f t h e c o n s t r i c t o r , c a u s i n g m y o c a r d i a l i n f a r c t i o n p r i o r t o t h e f i n a l 14 d a y s a s s e s s m e n t p e r i o d , s h o w i n g t h e v a r i a b i l i t y o f t h i s t e c h n i q u e . T h e d i f f e r e n t r e s p o n s e s o f t h e s e p t a l w a l l a n d t h e r e m a i n i n g two w a l l s ( T a b l e 6, F i g u r e 1 4 ) may b e e x p l a i n e d b y t h e c o r o n a r y a n a t o m y o f t h e d o g . D o n a l d a n d E s s e x ( 1 9 5 4 ) r e p o r t t h a t t h e s e p t a l b r a n c h o f t h e LAD s u p p l i e s 70 t o 75% o f t h e i n t e r v e n t r i c u l a r s e p t u m . T h e s e p t a l b r a n c h ( r a m u s s e p t a l i s ) , h o w e v e r , may o r i g i n a t e i n a n umber o f d i f f e r e n t l o c a t i o n s ( M i l l e r , 1 9 6 4 ) . T h i s c o u l d e x p l a i n t h e d i f f e r e n c e b e t w e e n t h e s e p t u m a n d o t h e r two w a l l s , s i n c e t h e a m e r o i d c o n s t r i c t o r may h a v e b e e n p l a c e d d i s t a l l y o r p r o x i m a l l y t o t h e r a m u s s e p t a l i s . T h u s , t h i s a r e a o f t h e v e n t r i c l e may o r may n o t h a v e b e e n a f f e c t e d b y t h e r e d u c t i o n i n b l o o d f l o w t h r o u g h t h e LAD d i s t a l t o t h e c o n s t r i c t o r . F l u o r o s c o p y s t u d i e s o f t h e c o r o n a r y a n a t o m y m i g h t h a v e a l l e v i a t e d t h i s p r o b l e m . S c h e l b e r t ( 1 9 8 6 ) s t a t e s t h a t a n umber o f f a c t o r s w i l l i n f l u e n c e t h e s l o p e o f t h e t i s s u e w a s h o u t c u r v e . T h e s e i n c l u d e t h e v o l u m e o f d i s t r i b u t i o n o f 1 2 3 I P P A i n t i s s u e , b a c k d i f f u s i o n , r e c i r c u l a t i o n a n d f l o w - d e p e n d e n t r e m o v a l o f 1 2 3 I P P A a n d i t s m e t a b o l i c b y - p r o d u c t s . A l t h o u g h t h e s e f a c t o r s w e r e n o t c o n s i d e r e d i n t h i s s t u d y , t h e y h a v e b e e n a d d r e s s e d i n t h e l i t e r a t u r e . R e s k e e t a l . ( 1 9 8 4 d ) c a l c u l a t e d t h a t 4.5 - 6.0% o f t h e i n j e c t e d - 136 -d o s e o f 1 2 3 I P P A was t a k e n up b y t h e m y o c a r d i u m . I n 1 9 8 5 ( a ) , R e s k e a n d c o w o r k e r s d e m o n s t r a t e d t h a t a number o f o t h e r t i s s u e s e x t r a c t e d 1 2 3 I P P A , b u t a t s i g n i f i c a n t l y s l o w e r r a t e s . T h e s e t i s s u e s i n c l u d e d l u n g , l i v e r , k i d n e y , s p l e e n a n d s k e l e t a l m u s c l e . T h e r e f o r e , a l t h o u g h t h e m a j o r i t y o f t h e f a t t y a c i d m e t a b o l i s m may o c c u r e l s e w h e r e i n t h e b o d y , t h e e f f e c t o f r e c i r c u l a t i o n i s m i n i m a l d u e t o t h e f a c t t h a t t h e 1 2 3 1 - b e n z o i c a c i d p r o d u c e d i s r e m o v e d f r o m t h e c i r c u l a t i o n b y t h e k i d n e y s a n d l i v e r a n d s u b s e q u e n t l y e l i m i n a t e d f r o m t h e b o d y i n t h e u r i n e . T h e u s e o f t h e p h e n y l a t e d d e r i v a t i v e t h u s a v o i d e d t h e p r o b l e m o f r e c i r c u l a t i o n ( M a c h u l l a e t  a l . . 1 9 8 0 ) . S i n c e o c c l u s i o n o f t h e c o r o n a r y a r t e r y was i n c o m p l e t e a n d e x t e n s i v e c o l l a t e r a l c i r c u l a t i o n was p r e s e n t , r e m o v a l o f m e t a b o l i c b y - p r o d u c t s c o u l d b e a s s u m e d t o h a v e c o n t i n u e d ( L e r c h , 1 9 8 6 ) . T h e f i n a l p a r a m e t e r n o t a d d r e s s e d was b a c k - d i f f u s i o n o f u n m e t a b o l i z e d 1 2 3 I P P A ( F o x e t a l . . 1 9 8 5 ) . T h i s may a l s o h a v e c o n t r i b u t e d t o v a r i a b i l i t y i n th v a l u e s . I n s p i t e o f t h e s e s h o r t c o m i n g s , t h e r e s u l t s o f t h i s s t u d y i n d i c a t e t h a t 1 5 - p - 1 2 3 I - i o d o p h e n y l - p e n t a d e c a n o i c a c i d may b e u t i l i z e d t o e x a m i n e d i s t u r b a n c e s i n r e g i o n a l m y o c a r d i a l m e t a b o l i s m c a u s e d b y i s c h e m i a . ( c ) M y o c a r d i a l I n f a r c t i o n M o d e l R a d i o n u c l i d e t e c h n i q u e s a i m e d a t q u a n t i f y i n g m y o c a r d i a l i n f a r c t i o n s t i l l l a c k a n i d e a l r a d i o p h a r m a c e u t i c a l . I n t h i s e x p e r i m e n t a l e n d e a v o u r , t h e u t i l i z a t i o n o f a b e t a - m e t h y l a t e d f a t t y a c i d , c h o s e n f o r i t s r e d u c e d m e t a b o l i c r a t e a n d i n c r e a s e d i n t r a m y o c a r d i a l r e s i d e n c e t i m e ( L i v i n i e t a l . . 1 9 8 2 ) , h a s s h own l i t t l e e f f e c t i v e n e s s f o r a s s e s s i n g t h e s i z e o f m y o c a r d i a l p e r f u s i o n d e f e c t s . I n c o m p a r i s o n t o a h i s t o c h e m i c a l a s s e s s m e n t o f p e r f u s i o n d e f e c t s i z e , o u r r e s u l t s show a l o w d e g r e e o f c o r r e l a t i o n f o r t h e p e r f u s i o n - 137 -d e f e c t s e s t i m a t e d b y t h e i o d i n a t e d f a t t y a c i d a n a l o g u e ( r = 0 . 6 5 - p < 0 . 0 0 5 ) a n d b y 20171 ( r = 0 . 4 9 - p < 0 . 0 0 5 ) . B o t h a g e n t s s u f f e r f r o m b o r d e r d e m a r c a t i o n p r o b l e m s w h i c h w i l l g r e a t l y l i m i t t h e i r u s e f u l n e s s i n s u c h a m a n n e r . S i g n i f i c a n t p o s t - i n f a r c t i o n i n c r e a s e s o f c r e a t i n e k i n a s e a n d CK-MB e n z y m e s i n t h i s s t u d y c o n f i r m e d t h e p r e s e n c e o f m y o c a r d i a l i n f a r c t i o n ( F i g u r e 2 1 ) . T h e i n c r e a s e i n CK a n d CK-MB e n z y m e s f o l l o w i n g m y o c a r d i a l i n f a r c t i o n h a s b e e n known s i n c e t h e 1 9 6 0 ' s ( D r e y f u s e t a l . . 1 9 6 0 ) a n d h a s b e e n c o n f i r m e d m ore r e c e n t l y b y S y l v e n ( 1 9 8 0 ) a n d H e y n d r i c k e t a l . ( 1 9 8 6 ) . A l t h o u g h t h e c o r r e l a t i o n b e t w e e n h i s t o c h e m i c a l a s s e s s m e n t o f i n f a r c t s i z e a n d t h e a s s e s s m e n t b y B 1 2 3 I P P A was h i g h e r , r e l a t i v e t o t h a t o f 2 0 1 T 1 , t h e v a l u e s w e r e s t i l l l o w , c o m p a r e d t o t h o s e r e p o r t e d i n t h e l i t e r a t u r e f o r t h e s c i n t i g r a p h i c a s s e s s m e n t o f i n f a r c t s i z e ( K a u l e t a l . . 1985; a n d W o l f e e t a l . . 1 9 8 5 ) . T h e s e l o w c o r r e l a t i o n v a l u e s may b e e x p l a i n e d b y a d e c r e a s e i n t r a c e r a v a i l a b i l i t y b e c a u s e o f m e t a b o l i c d e g r a d a t i o n a s s u g g e s t e d b y D u d c z a k e t a l . ( 1 9 8 6 ) , a s w e l l a s A m b r o s e e t a l . ( 1 9 8 7 ) . T h e s e a u t h o r s r e p o r t e d d e c r e a s e d m y o c a r d i a l r e t e n t i o n t i m e s w i t h i n t h e m y o c a r d i u m o f a b e t a - m e t h y l a t e d a g e n t . D u d c z a k e t a l . ( 1 9 8 6 ) g e n e r a t e d mono- a n d b i - e x p o n e n t i a l w a s h o u t c u r v e s i n 19 p a t i e n t s u s i n g a b e t a - m e t h y l a t e d f a t t y a c i d . M y o c a r d i a l w a s h o u t was b i - e x p o n e n t i a l i n 11 o f 19 p a t i e n t s w i t h a mean th = 13.8 m i n f o r t h e e a r l y p h a s e a n d mean th = 187 m i n f o r t h e l a t e p h a s e . I n 8 o f 19 p a t i e n t s , w a s h o u t was m o n o - e x p o n e n t i a l w i t h th = 218 m i n . S i m i l a r l y , A m b r o s e e t a l . ( 1 9 8 7 a ) r e p o r t e d t h a t f o l l o w i n g I V a d m i n i s t r a t i o n o f b e t a - m e t h y l - i o d o p h e n y l - p e n t a d e c a n o i c a c i d i n r a t s , t h e r e w e r e d i f f e r e n c e s i n h e a r t r e t e n t i o n t i m e s a n d s u b c e l l u l a r d i s t r i b u t i o n . S u b c e l l u l a r d i s t r i b u t i o n was a l s o e x a m i n e d b y O t t o e t a l . ( 1 9 8 5 a ) . T h e s e a u t h o r s m e a s u r e d w a s h o u t t i m e s f o r i o d o p h e n y l - p e n t a d e c a n o i c - 138 -a c i d ( I P P ) , b e t a - m e t h y l - I P P a n d b e t a - d i - m e t h y l - I P P a n d f o u n d s i g n i f i c a n t i n c r e a s e s i n w a s h o u t t i m e s f o r t h e l a t t e r two a g e n t s c o m p a r e d t o I P P , 5-10 m i n , 30-45 m i n , a n d 6-7 h o u r s , r e s p e c t i v e l y . T h e m e c h a n i s m b y w h i c h c a t a b o l i s m o f t h e m o n o - m e t h y l - s u b s t i t u t e d a g e n t o c c u r s i s a c o m p l e x p a t h w a y i n v o l v i n g a n u mber o f a d d i t i o n a l s t e p s p r i o r t o e n t r y i n t o t h e b e t a - o x i d a t i o n p a t h w a y ( D u d c z a k e t a l . . 1 9 8 6 ) . S c i n t i g r a p h i c i m a g e s o f s u f f i c i e n t q u a l i t y , h o w e v e r , c o u l d s t i l l b e o b t a i n e d up t o 120 m i n u t e s a f t e r i n j e c t i o n o f t h e b e t a - m e t h y l - I P P , a s r e p o r t e d b y D u d c z a k e t a l . ( 1 9 8 6 ) . A l t h o u g h some m e t a b o l i c d e g r a d a t i o n o f o u r f a t t y a c i d may h a v e t a k e n p l a c e , o u r s c a n t i m e o f 20 m i n w o u l d b e a s s o c i a t e d w i t h m i n i m a l l o s s o f t r a c e r f r o m t h e m y o c a r d i u m . O f g r e a t e r c o n c e r n w i t h t h i s s t u d y , a n d many o t h e r s t u d i e s i n t h i s f i e l d , i s b o r d e r d e m a r c a t i o n o n t h e t o m o g r a p h i c i m a g e s , e s p e c i a l l y a t t h e a p i c a l r e g i o n , f o l l o w i n g LAD o c c l u s i o n . C a l d w e l l e t a l . ( 1 9 8 4 ) r e p o r t s t h a t , i n m y o c a r d i a l i n f a r c t s a t t h e a p i c a l r e g i o n , i t was n e c e s s a r y t o o c c a s i o n a l l y e s t i m a t e t h e l o c a t i o n o f t h e a p e x f r o m a s u b j e c t i v e v i s u a l i m p r e s s i o n . S u c h a n a p p r o a c h a p p e a r s t o b e i n a d e q u a t e , i n v i e w o f o u r i n t r a - o b s e r v e r d i f f e r e n c e s ( T a b l e 8 ) . C a l d w e l l e t a l . ( 1 9 8 4 ) a l s o s t a t e d t h a t , i f t h e d e f e c t s w e r e a s s e s s e d a s v o l u m e s , t h e t o m o g r a p h i c e s t i m a t e s w o u l d b e h i g h e r t h a n a c t u a l v a l u e s . T h i s was a t t r i b u t e d t o b l o o d p o o l a c t i v i t y w i t h i n t h e v e n t r i c u l a r c a v i t y . I n o r d e r t o e x c l u d e t h i s p r o b l e m f r o m o u r s t u d y , b l o o d p o o l - e n d o c a r d i a l b o u n d a r i e s o n t h e t o m o g r a m w e r e d e f i n e d a n d s u b s e q u e n t l y t h e c e n t r a l c a v i t y s u b t r a c t e d f r o m e a c h s l i c e i m a g e . T h i s was d i f f i c u l t s i n c e e n d o c a r d i a l , a s w e l l a s e p i c a r d i a l b o r d e r s , w e r e n o t a s w e l l - d e f i n e d a s may h a v e b e e n p o s s i b l e b e c a u s e o f p r o b l e m s w i t h r e s o l u t i o n . T h i s p r o b l e m w i t h b o r d e r - 139 -c l a r i t y may h a v e c o n t r i b u t e d t o e r r o r w i t h i n t h e s t u d y a n d i n f l u e n c e d o u r i n t e r p r e t a t i o n o f i m a g e g e o m e t r y . I n v i e w o f t h e h e t e r o g e n o u s g e o m e t r y o f t e n a s s o c i a t e d w i t h m y o c a r d i a l i n f a r c t i o n , d i f f i c u l t i e s i n p l a c i n g a c c u r a t e b o r d e r s a r o u n d s u c h a n a r e a do e x i s t . I n o r d e r t o c o m p e n s a t e f o r t h i s , P r i g e n t e t a l . ( 1 9 8 7 ) u t i l i z e d maximum c o u n t c i r c u m f e r e n t i a l p r o f i l e a n a l y s i s o f 2 0 i ~ i S PECT i m a g e s t o q u a n t i f y i n f a r c t s i z e u s i n g a n u m b e r o f d e r i v e d a l g o r i t h m s . T h e s e a u t h o r s d e m o n s t r a t e d h i g h l i n e a r c o r r e l a t i o n v a l u e s w i t h t h e s e m e t h o d s , w h i c h w e r e i n d e p e n d e n t o f e d g e d e t e c t i o n . R e f i n e m e n t s i n v o l u m e d e t e r m i n a t i o n b y SPECT i n d i c a t e s t h a t t h i s t e c h n i q u e may b e a n a c c u r a t e d i a g n o s t i c t o o l ( H a l a m a a n d H e n k i n , 1986; M o r t e l m a n s e t  a l . . 1 9 8 6 ) . H a l a m a a n d H e n k i n ( 1 9 8 6 ) r e p o r t e d o n t h e u s e o f e d g e e n h a n c e m e n t a n d v o l u m e p r o j e c t i o n t o e s t i m a t e v o l u m e s b y S P E C T w i t h g o o d c o r r e l a t i o n . M o r t e l m a n s e t a l . ( 1 9 8 6 ) h a v e d e s c r i b e d a new t h r e s h o l d i n g m e t h o d , w h i c h i n c r e a s e s t h e a c c u r a c y o f e d g e d e t e c t i o n a n d t h u s i n c r e a s e s t h e a c c u r a c y o f v o l u m e d e t e r m i n a t i o n b y S PECT. T h e s e t e c h n i q u e s r e q u i r e d c o m p u t e r p r o g r a m s w h i c h w e r e e i t h e r t o o e x p e n s i v e , o r u n a v a i l a b l e , s o t h a t t h e o n l y m e t h o d a v a i l a b l e t o u s , i e . a s u b j e c t i v e a s s e s s m e n t w i t h a r e a s - o f - i n t e r e s t d r a w n m a n u a l l y , was u t i l i z e d i n t h i s s t u d y . A l t h o u g h S P E C T a p p e a r s t o o f f e r many a d v a n t a g e s , t h e r e r e m a i n some t e c h n i c a l p r o b l e m s w h i c h r e q u i r e f u r t h e r i n v e s t i g a t i o n b e f o r e t h i s c a n b e c o m e a f u l l y a c c e p t a b l e means o f q u a n t i t a t i v e l y a s s e s s i n g p e r f u s i o n d e f e c t s . A n o t h e r p o s s i b l e s o u r c e o f e r r o r may h a v e b e e n t h e u t i l i z a t i o n o f f i l t e r s i n t h e r e c o n s t r u c t i o n p r o c e s s ( J a s z c z a k , 1 9 8 8 ) . A l t h o u g h b e n e f i c i a l f o r i m a g e q u a l i t y , t h e m a s k i n g o u t o f a n y l e v e l s o f a c t i v i t y may l i m i t t h e u s e f u l n e s s o f t h i s t e c h n i q u e . T h e i m a g e s i n t h i s s t u d y w e r e r e c o n s t r u c t e d f r o m a 64 x 64 m a t r i x . I t was f e l t , a t t h e t i m e , t h a t t h i s - 140 -was a p p r o p r i a t e ; h o w e v e r , a 128 x 128 m a t r i x may h a v e i m p r o v e d i m a g e q u a l i t y . T h u s , i n a p p r o p r i a t e m a t r i x s i z e may h a v e c o n t r i b u t e d t o t h e e r r o r i n t h i s p o r t i o n o f t h e s t u d y . C l e a r l y , t h e r e a r e a n umber o f t e c h n i c a l f a c t o r s a s s o c i a t e d w i t h SPECT, w h i c h w e r e n o t a d d r e s s e d i n t h i s s t u d y a n d t h u s may h a v e c o n t r i b u t e d t o t h e l o w c o r r e l a t i o n v a l u e s . H o w e v e r , i t m u s t b e k e p t i n m i n d t h a t when d e a l i n g w i t h mm. o r cm. p r e c i s i o n , a n y s m a l l v a r i a t i o n s i n i m a g e f u z z i n e s s c o u l d b e t r a n s m i t t e d i n t o v e r y s u b s t a n t i a l e r r o r s . I t i s c l e a r t h a t t h e m e t h o d u t i l i z e d t o a s s e s s t h e s i z e o f p e r f u s i o n d e f e c t s i n t h i s s t u d y was i n a p p r o p r i a t e . A s m e n t i o n e d e a r l i e r , t h e u t i l i z a t i o n o f b o t h i m a g i n g a g e n t s t o g e t h e r may a l l o w f o r a b e t t e r a s s e s s m e n t o f t h e i s c h e m i c r i s k z o n e a n d g i v e some i n d i c a t i o n o f i r r e v e r s i b l y i n j u r e d m y o c a r d i u m . S t r a u s s e t a l . ( 1 9 8 7 ) d e m o n s t r a t e d v a r i a b l e u p t a k e p a t t e r n s when u t i l i z i n g 201x1 a n d b e t a - m e t h y l - p - 1 2 3 I - i o d o p h e n y l p e n t a d e c a n o i c a c i d . I t was f o u n d t h a t i n a r e a s s u p p l i e d b y v e s s e l s w h i c h w e r e t o t a l l y o c c l u d e d o r s e v e r e l y s t e n o s e d , F F A u p t a k e was e q u a l t o 201x1 u p t a k e . I n a r e a s i n w h i c h t h e v e s s e l was s e v e r e l y s t e n o s e d , b u t h a d some f l o w o r g o o d c o l l a t e r a l s , F F A u p t a k e was g r e a t e r t h a n t h a t o f 2 0 1 T 1 . I n z o n e s o f r e p e r f u s e d m y o c a r d i u m , F F A u p t a k e was l e s s t h a n 2 0 1 T 1 . T h e r e f o r e , i t i s c l e a r t h a t t h e u t i l i z a t i o n o f b o t h a g e n t s may d i f f e r e n t i a t e b e t w e e n v a r i o u s z o n e s o f i s c h e m i a w i t h i n t h e m y o c a r d i u m . F i s c h m a n n e t a l . ( 1 9 8 8 ) a l s o e x a m i n e d t h e r o l e o f c o m b i n e d f a t t y a c i d a n d t h a l l i u m i m a g i n g i n t h e a s s e s s m e n t o f m y o c a r d i a l i s c h e m i a a n d i n f a r c t i o n . F o l l o w i n g a c u t e LAD o c c l u s i o n i n m o n g r e l d o g s , b o t h 2 ° I T 1 a n d 1 2 3 I - p a r a - i o d o p h e n y l - 9 - m e t h y l h e p t a d e c a n o i c a c i d w e r e i n j e c t e d a n d s c a n s p e r f o r m e d . T h e a u t h o r s o b s e r v e d a r e l a t i v e e x c e s s o f t h e f a t t y a c i d i n t h e - 141 -i n f a r c t e d a r e a i n 7 o f 7 d o g s . T h e r e f o r e , i t a p p e a r s t h a t t h e i n f a r c t e d a r e a h a d t a k e n u p t h e f a t t y a c i d , p r e s u m a b l y v i a c o l l a t e r a l c i r c u l a t i o n . T h u s , b o t h 20171 a n d f a t t y a c i d w o u l d p r o v i d e i n f o r m a t i o n a s t o t h e e x t e n t o f t h e i n f a r c t i o n a n d t h e d e g r e e o f i s c h e m i a . I n t h i s s t u d y , h o w e v e r , B1 2 3 I P P A/2 O I T ; L r a t i o s i n d i c a t e d s i m i l a r e s t i m a t i o n s o f p e r f u s i o n d e f e c t s i z e (mean r a t i o 1 .01+1.25), i n d i c a t i n g n o a d v a n t a g e o f u s i n g o n e o v e r t h e o t h e r o r o f u s i n g b o t h t o g e t h e r . K a i r e n t o e t a l . ( 1 9 8 8 ) e v a l u a t e d 2 o i T i a n d i2 3 i . p . i 4 . i 0 d 0 p h e n y l -B e t a - m e t h y l - t e t r a d e c a n o i c a c i d w i t h SPECT i n a c a n i n e m o d e l o f m y o c a r d i a l i n f a r c t i o n . R e s u l t s i n d i c a t e d t h a t b o t h a g e n t s s h o w e d d e c r e a s e d a c t i v i t y w i t h i n a r e a s o f i n f a r c t i o n . I t was a l s o shown t h a t 20171 o v e r e s t i m a t e d t h e s i z e o f t h e d a m a g e d m y o c a r d i u m r e l a t i v e t o t h e f a t t y a c i d i m a g i n g a g e n t . W i t h i n t h e z o n e o f i n f a r c t i o n , h o w e v e r , i n v i e w o f t h e w e l l - d e v e l o p e d c o l l a t e r a l c i r c u l a t i o n i n t h e d o g , v a r i o u s d e g r e e s o f i s c h e m i a may e x i s t . T h u s , f a t t y a c i d u p t a k e w i t h i n t h e s e c e l l s may a l s o v a r y , t h e r e b y i n f l u e n c i n g o u r SPECT i m a g e . T h i s was a l s o s h o wn t o b e t h e c a s e i n K a i r e n t o ' s w o r k . T h e r e f o r e , t h i s t y p e o f r e s i d u a l a c t i v i t y w i t h i n t h e i n f a r c t e d a r e a c o u l d l e a d t o i m a g e f u z z i n e s s a n d t h u s g i v e r i s e t o p o s s i b l e e r r o r . I n a n i n t e r e s t i n g s t u d y b y M a u b l a n t e t a l . ( 1 9 8 8 ) , b e a t i n g v e n t r i c u l a r c e l l s f r o m 3 d a y o l d n e w b o r n r a t s w e r e c u l t u r e d a n d i n c u b a t e d w i t h 37 kBq ( 1 / i C i ) o f 2 0 1 T 1 . T h e e f f e c t o f e x t r a c e l l u l a r pH o n i n t r a c e l l u l a r [20171] W a s i n v e s t i g a t e d . A t 20 m i n , i n t r a c e l l u l a r [20171] w a s h i g h e r a t pH 8 t h a n a t p H 6, i n f e r r i n g some a l t e r a t i o n i n t h e r e m o v a l o f 20171 f r o m t h e c e l l a s pH i n c r e a s e s . T h i s may i n d i c a t e t h a t t h e r e may b e l e s s 20171 a c c u m u l a t i o n w i t h i n c e l l s d u e t o a c i d o s i s , w h i c h i s i n d i c a t i v e o f a d e c r e a s e d pH. - 142 -O u r d a t a do n o t s u p p o r t t h e c h o i c e o f t h e b e t a - m e t h y l - F F A o v e r 20171 a s a m a r k e r o f t i s s u e p e r f u s i o n . A l t h o u g h p h y s i c a l c h a r a c t e r i s t i c s w o u l d i n d i c a t e i t s s u p e r i o r i t y , F F A i m a g i n g s t i l l r e q u i r e s some f u r t h e r t e c h n i c a l a n d d e v e l o p m e n t a l r e f i n e m e n t b e f o r e a c c u r a t e q u a n t i t a t i v e a s s e s s m e n t s o f m y o c a r d i a l p e r f u s i o n d e f e c t s c a n b e made. - 143 -V I I SUMMARY AND CONCLUSIONS T h e p u r p o s e o f t h i s s t u d y was t o u t i l i z e 1 5 - p - 1 2 3 I - p h e n y l -p e n t a d e c a n o i c a c i d a n d 1 5 - p - b e t a - m e t h y l - i o d o ( 1 2 3 I ) p h e n y l p e n t a d e c a n o i c a c i d i n t h e a s s e s s m e n t o f m y o c a r d i a l m e t a b o l i s m a n d p e r f u s i o n f o l l o w i n g i s c h e m i a a n d i n f a r c t i o n . We c a n c o n c l u d e f r o m t h i s s t u d y : ( 1 ) I n a m o d e l o f g l o b a l i s c h e m i a , we f o u n d t h a t m y o c a r d i a l w a s h o u t o f 1 5 - 1 2 3 I P P A may b e m o d i f i e d b y t h e a d d i t i o n o f SOD o r a l l o p u r i n o l t o t h e c o n t r o l c a r d i o p l e g i c s o l u t i o n ( T y e r s i s o - o s m o l a r s o l u t i o n ) . I s o - o s m o l a r s o l u t i o n d o e s n o t a p p e a r a s e f f e c t i v e i n m a i n t a i n i n g F F A w a s h o u t a s do s o l u t i o n s s u p p l e m e n t e d w i t h SOD o r a l l o p u r i n o l . I t i s i m p o r t a n t t o m e n t i o n t h a t t h e d o s i n g r e g i m e a n d t i m e o f a d m i n i s t r a t i o n may m o d i f y t h e e x t e n t o f p r o t e c t i o n b y t h e s e two a g e n t s a g a i n s t o x y g e n c y t o t o x i c i t y . ( 2 ) I n a m o d e l o f r e g i o n a l i s c h e m i a , 1 2 3 I P P A may b e a u s e f u l i n d i c a t o r o f m e t a b o l i c s t a t u s o f t h e m y o c a r d i u m . D u r i n g t h e p e r i o d o f d e v e l o p i n g i s c h e m i a , b i o c h e m i c a l d e r a n g e m e n t s i n m e t a b o l i c p a t h w a y s may b e o c c u r r i n g p r i o r t o a n y f u n c t i o n a l i m p a i r m e n t . S i n c e t h e p r i m a r y e n e r g y s u b s t r a t e s f o r t h e m y o c a r d i u m a r e f r e e f a t t y a c i d s , e a r l y i s c h e m i c i n j u r y may b e m a n i f e s t e d a s a l t e r e d w a s h o u t r a t e s ( u t i l i z a t i o n ) o f t h e s e f u e l s . C h a n g e s i n r e g i o n a l w a s h o u t r a t e s o f 1 5 - 1 2 3 I P P A w e r e d e m o n s t r a t e d a t 14 d a y s o f s l o w l y d e v e l o p i n g r e g i o n a l i s c h e m i a . ( 3 ) T h e e f f e c t i v e n e s s o f t h e b e t a - m e t h y l f a t t y a c i d a n a l o g u e i n d e t e r m i n i n g t h e s i z e o f p e r f u s i o n d e f e c t s i s m i n i m a l . P r o b l e m s r e m a i n i n i m a g e c l a r i t y a n d b o r d e r d e m a r c a t i o n . - 144 -( 4 ) T h e u s e o f i o d i n a t e d f r e e f a t t y a c i d s a s a n o n - i n v a s i v e , i n v i v o d i a g n o s t i c t o o l may r e p r e s e n t a means o f e a r l y a s s e s s m e n t o f m y o c a r d i a l i s c h e m i a . I t may a l s o p r o v i d e a means o f a s s e s s i n g t h e e f f e c t i v e n e s s o f v a r i o u s c a r d i o p l e g i c t e c h n i q u e s i n o r d e r t o a c h i e v e o p t i m a l m y o c a r d i a l p r e s e r v a t i o n . 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Cardiovasc Dis 20(2):81-94, 1977. - 168 -IX APPENDICES A p p e n d i x I - H i s t o c h e m i c a l a s s e s s m e n t ( T T Z s t a i n i n g ) o f p e r f u s i o n d e f e c t s i z e u s i n g p l a n i m e t r i c q u a n t i f i c a t i o n . 0 D o g - S l i c e 1 t h i c k ( c m ) T o t a l S l i c e 2 v o l ( c m 3 ) - l s t o b s e r v e r T o t a l S l i c e 3 v o l ( c m 3 ) - 2 n d o b s e r v a t i o n s b y 1 s t o b s e r v e r T o t a l S l i c e 4 v o l ( c m 3 ) - 2 n d o b s e r v e r 1 1-1 1 0 2 60 2 95 3 20 2 1-2 1 0 7 00 7 25 7 05 3 1-3 1 2 12 80 13 50 13 50 4 1-4 1 0 20 80 21 76 21 12 5 1-5 1 0 18 56 18 24 18 32 6 1-6 1 2 28 90 28 70 28 80 7 2-1 1 0 5 08 5 12 5 56 8 2-2 1 0 12 12 12 30 12 96 9 2-3 1 0 10 25 10 10 10 35 10 2-4 1 0 23 12 23 36 23 4 4 11 2-5 1 0 16 80 17 44 16 88 12 2-6 1 0 22 82 22 54 22 61 13 3-1 1 0 1 80 1 68 1 80 14 3-2 1 0 8 65 8 60 8 90 15 3-3 1 0 16 75 16 95 16 80 16 3-4 1 0 28 63 28 49 28 63 17 3-5 1 2 33 80 33 60 34 00 18 3-6 1 0 32 40 32 56 32 48 19 4-1 1 0 5 45 4 90 5 10 20 4-2 0 3 5 28 4 75 5 55 21 4-3 1 0 8 47 8 33 8 82 22 4-4 1 0 19 74 19 39 20 72 23 4-5 1 0 16 80 16 40 16 88 24 4-6 1 0 17 52 17 76 18 08 25 5-1 1 0 9 54 9 12 9 12 26 5-2 1 0 11 69 12 39 12 25 27 5-3 1 0 21 77 21 77 23 34 28 5-4 1 0 24 50 23 59 23 80 29 5-5 1 0 23 04 25 56 23 22 30 6-1 1 0 16 32 16 96 16 72 31 6-2 1 0 19 60 19 52 19 84 32 6-3 1 0 22 24 22 24 22 88 33 6-4 1 0 25 76 23 80 24 22 34 6-5 1 0 25 20 25 12 25 76 35 7-1 1 0 18 88 19 60 19 76 36 7-2 1 0 36 24 39 04 36 80 37 7-3 1 2 32 10 31 40 31 40 38 7-4 1 0 25 84 25 52 24 24 39 8-1 1 0 4 00 4 10 3 45 40 8-2 1 0 9 84 10 38 9 90 c o n t i n u e d - 169 -A p p e n d i x I ( c o n t i n u e d ) 0 D o g - S l i c e 1 t h i c k ( c m ) T o t a l S l i c e 2 v o l ( c m 3 ) - 1 s t o b s e r v e r T o t a l S l i c e 3 v o l ( c m 3 ) - 2 n d o b s e r v a t i o n s b y 1 s t o b s e r v e r T o t a l S l i c e 4 v o l ( c m 3 ) - 2 n d o b s e r v e r 41 8-3 1 0 15 .84 15 24 16 08 42 8-4 1 0 17 .52 18 12 18 60 43 8-5 1 0 13 .35 14 30 13 25 4 4 8-6 1 0 18 .06 18 00 17 70 45 8-7 1 0 17 .70 19 02 15 78 46 9-1 1 0 4 .45 4 95 3 95 47 9-2 1 0 6 .40 7 85 7 10 48 9-3 1 0 11 .90 12 50 12 30 49 9-4 1 0 16 .14 18 06 16 80 50 9-5 1 0 9 .80 11 25 10 10 51 9-6 1 0 13 .44 14 94 13 08 - 170 -Appendix I (continued) 0 Dog-Slice 5 inf-vol(cm3)-1st 6 inf-vol(cm3)-2nd 7 inf-vol(cm3)-2nd observer observations by observer 1st observer 1 1-1 0 95 0 75 0.65 2 1-2 2 40 2 35 2.20 3 1-3 3 60 3 90 4.00 4 1-4 4 96 4 16 4.40 5 1-5 4 32 4 48 4.40 6 1-6 0 00 0 00 0.00 7 2-1 0 76 0 28 0.48 8 2-2 1 62 0 66 1.62 9 2-3 0 95 1 00 0.85 10 2-4 0 64 1 28 1.04 11 2-5 1 36 1 92 1.52 12 2-6 3 22 5 60 0.91 13 3-1 0 69 0 78 0.75 14 3-2 2 65 3 00 2.90 15 3-3 6 45 6 45 6.55 16 3-4 10 43 10 43 10.64 17 3-5 9 50 9 10 9.10 18 3-6 5 68 7 04 6.64 19 4-1 0 00 0 25 0.10 20 4-2 0 00 0 00 0.00 21 4-3 0 00 0 00 0.00 22 4-4 0 70 0 70 0.98 23 4-5 0 48 2 64 0.56 24 4-6 0 00 0 00 0.00 25 5-1 2 94 2 88 2.46 26 5-2 2 17 2 66 2.38 27 5-3 5 60 4 41 4.41 28 5-4 5 25 5 53 5.39 29 5-5 1 56 2 22 1.92 30 6-1 3 68 4 48 4.24 31 6-2 3 52 4 56 4.00 32 6-3 5 60 6 72 6.00 33 6-4 3 22 3 29 3.50 34 6-5 3 52 4 00 3.20 35 7-1 7 60 8 48 8.00 36 7-2 7 52 10 32 8.24 37 7-3 5 10 4 80 4.90 38 7-4 2 48 3 12 3.28 39 8-1 1 50 2 65 1.50 40 8-2 4 74 2 94 3.72 Continued - 171 -A p p e n d i x I ( c o n t i n u e d ) 0 D o g - S l i c e 5 i n f - v o l ( c m 3 ) - 1 s t 6 i n f - v o l ( c m 3 ) - 2 n d 7 i n f - v o l ( c m 3 ) - 2 n d o b s e r v e r o b s e r v a t i o n s b y 1 s t o b s e r v e r o b s e r v e r 41 8-3 7.08 7.62 7.56 42 8-4 5.58 9.06 7.44 43 8-5 5.15 5.55 4 . 9 0 4 4 8-6 7.50 8.34 6.60 45 8-7 7.92 7.20 6.54 46 9-1 0.60 1.35 0.80 47 9-2 1.25 1.35 1.00 48 9-3 0.95 1.60 1.30 49 9-4 0.00 0.00 0.00 50 9-5 0.00 0.00 0.00 51 9-6 0.00 0.00 0.00 - 172 A p p e n d i x II - S c i n t i g r a p h i c a s s e s s m e n t o f p e r f u s i o n d e f e c t u s i n g B123IPPA a n d 2 0 1-rl Bi2 3 i F F A ( c o l s 1-3) 2oi.xi ( c o l s 4-6) D- S-•A 1 1 s t 2 2 n d 3 2 n d 4 1 s t 5 2 n d 6 2 n d o b s e r v e r o b s e r v a t i o n s o b s e r v e r o b s e r v e r o b s e r v a t i o n s o b s e r v e r b y 1 s t b y 1 s t o b s e r v e r o b s e r v e r 1- 1 s 54.25 63 .19 149 .19 49 81 62 .31 161 . 50 V 17 44 19.44 i 1 8 .00 63 .19 10 38 12 81 30.94 r 33 69 1- 2 s 84.19 139 .81 140 00 106 56 94 31 149 .19 V 21 .31 14 81 31 .63 i 1 9 . 75 33 .69 25 88 19 06 r 29 82 29 ,44 24.56 1- 3 s 1 0 8 . 5 0 130 .63 163 56 111 75 129 44 171 . 50 V 24 .13 15 56 16 00 25 .63 i 3 2 .69 34 .00 38 44 32 13 r 15 .13 43 63 29 .38 1- 4 s 1 1 8 . 8 1 109 94 187 69 171 06 178 31 160.31 V 17 .69 15 81 25 19 23 .25 i 1 5.13 14 00 47 75 52 25 r 34 00 1- 5 s 2 0 5 . 6 9 116 .94 152 31 198 50 185 75 188.19 V 20 81 16 06 24 38 25.19 i 6 8 . 0 0 67 63 60 25 r 19 06 20 00 1- 6 s 2 2 2 . 5 6 137 .69 168 88 216 25 227 63 185.63 V 22 88 15 81 27 19 19 .00 i 7 2 .56 69 89 68 38 r 14 19 38 94 1- 7 s 218.81 180 75 152 63 187 44 170 00 203 . 75 V 33 31 20 75 38 88 25 .00 i 5 1 . 5 0 48 25 4 4 50 r 71 31 1- 8 s 252.06 149 69 146 38 219 75 1 8 6 . 1 V 25 69 14 94 27.38 i 7 3 .13 72 94 60 00 1- 9 r s 109 06 151 38 197.81 V 26 31 13 19 32.88 i 22 00 117 19 r 27 44 61 .75 c o n t i n u e d i - 173 -A p p e n d i x I I ( c o n t i n u e d ) Bi 2 3I P P A (c o l s i.2) 20 1. •Tl ( c o l s 4 - 6 ) D- S -A 1 1 s t 2 2 n d 3 2 n d 4 1 s t 5 2 n d 6 2 n d o b s e r v e r o b s e r v a t i o n s o b s e r v e r o b s e r v e r o b s e r v a t i o n s o b s e r v e r b y 1 s t b y 1 s t o b s e r v e r o b s e r v e r 2- 1 s 53.81 45 75 26 88 82 00 94 88 143.88 V 15 75 18.06 i 2 3 .56 32 62 54 31 40 38 50.00 r 52.81 32 62 79 88 26 44 26.05 2- 2 s 93.38 84 44 79 56 106 69 125 31 133.56 V 13 81 7 63 13 00 22 31 24.19 i 4 7 . 8 1 30 31 39 25 45 44 42 44 36.31 r 37.38 20 69 20 69 27 44 31 59 2- 3 s 101.88 86 19 128 13 125 19 128 94 139.75 V 10.31 16 94 12 63 16 19 20 56 21.31 i 6.75 15 19 21 19 38 19 134 75 40.00 r 7 6.69 30 75 61 75 75 4 4 19 56 2- 4 s 112.69 103 50 138 38 132 63 116 81 144.81 V 11.88 15 88 12 63 19 38 22 31 13.94 i 3.12 20 56 22 31 41 94 32 94 r 67.88 15 69 34 06 72 81 77.44 2- 5 s 113.56 112 25 144 63 122 31 115 00 136.13 V 10.13 20 38 16 00 13 94 16 31 13.31 i 30 69 17 06 19 44 r 23.56 55 63 91 25 10.94 2- 6 s 114.31 106 88 137 94 126 25 115 06 127.06 V 8.38 17 69 12 00 14 31 15 38 14.38 i 1 81 35 38 20.38 r 2 3.69 24 94 26 06 2- 7 s 1 2 2 . 9 4 96 00 136 19 99 38 117 00 153.00 V 9.69 13 25 12 44 10 75 14 52 14.38 i 0 75 30 15 36.00 r 53 44 2- 8 s 107 69 130 69 120.38 V 18 44 11 38 13.63 i 20 06 33.69 r 27 75 3- 1 s 4 7 .38 75 26 58 19 62 19 118 44 184.13 V 5 62 17 19 19.69 i 1 3 .25 36 19 25 13 14 06 51 50 32.63 r 34.13 16 06 27 06 76.56 3- 2 s 5 4 . 1 9 90 62 201 56 59 81 162 56 190.94 V 9 44 16 88 25 19 32.13 i 1 0 . 5 0 37 94 65 50 16 81 31 75 61.88 r 27.38 17 19 39 44 14 50 50 56 18.63 C o n t i n u e d - 174 -A p p e n d i x I I ( c o n t i n u e d ) B 1 2 3 T p p A ( c o i s 1_3) 2 0 1 - T 1 ( c o l s 4 - 6 ) D- S -A 1 1 s t 2 2 n d 3 2 n d 4 1 s t 5 2 n d 6 2 n d o b s e r v e r o b s e r v a t i o n s o b s e r v e r o b s e r v e r o b s e r v a t i o n s o b s e r v e r b y 1 s t b y 1 s t o b s e r v e r o b s e r v e r 3- 3 s 64 .00 98 50 106 25 9 2 . 0 0 125 31 191.31 V 17 19 13 50 20 38 19.94 i 16 .44 25 56 39 13 35.25 43 94 71.63 r 29 50 31 12 25 88 2 8 . 7 5 18 00 33.31 3- 4 s 116 .63 114 94 210 08 1 4 1 . 4 4 165 44 206.13 V 11 .56 14 06 29 19 17.63 27 75 85.63 i 22 4 4 29 38 82 50 5 7 . 9 4 19 38 r 54 69 35 00 35 13 3 4 . 4 4 31 94 30.88 3- 5 s 171 .38 118 75 168 31 1 5 7 . 6 9 191 44 191.44 V 13 .31 17 00 18 13 1 7 . 6 9 29 69 24.13 i 39 19 30 00 41 31 2 6 .38 12 31 23.44 r 54 31 35 00 31 69 63.06 48 00 44.13 3- 6 s 135 19 126 19 170 38 1 8 9 . 2 9 120 38 172.31 V 11 .81 19 88 12 88 24.88 24 75 17.13 i 37 .50 1 9 . 4 4 33 19 r 37 81 33 63 26 00 79.63 48 00 75.44 3- 7 s 167 00 121 31 145 88 1 8 7 . 6 9 134 74 131.19 V 16 .81 25 56 17 38 21.06 27 13 21.69 i 1 3 . 31 36 31 r 75 69 20 63 7 3 . 7 5 12 31 3- 8 s 109 88 158 00 125 75 149.19 V 16 38 9 69 21 19 20.50 i 22 69 r 31 94 19.88 4- 1 s 49 88 56 31 92 38 55.25 52 00 52.88 V 8 94 14 19 i 9 19 r 11 94 1 1 . 8 1 4- 2 s 32 44 77 14 91 38 59.19 65 31 63.06 V 14 81 10 31 i r 1 2 . 6 3 4- 3 s 76 63 77 69 95 06 83.19 72 31 63.75 V 8 56 17 00 11 75 4 63 i r 14 13 8 19 C o n t i n u e d . - 175 -A p p e n d i x I I ( c o n t i n u e d ) B i 2 3 i p p A ( c o i s i _ 3 ) 2 ° i - T l ( c o l s 4 - 6 ) D- S -A 1 1 s t 2 2 n d 3 2 n d 4 1 s t 5 2 n d 6 2 n d o b s e r v e r o b s e r v a t i o n s o b s e r v e r o b s e r v e r o b s e r v a t i o n s o b s e r v e r b y 1 s t b y 1 s t o b s e r v e r o b s e r v e r 4- 4 s 86.31 73 25 102 19 81 .44 92 25 71.81 V 1 0 . 4 4 16 44 11 81 5 .69 7 13 1.44 i r 25 .88 14 .75 4- 5 s 1 1 7 . 1 9 96 56 112 56 101 .88 100 00 102.94 V 14.81 16 13 10 54 6 .69 9 86 5.13 I r 22 .81 4- 6 s 101.38 107 13 90 62 117 .19 102 38 94.06 V 13.88 5 88 7 .06 7 .81 11 06 8.12 i r 1 6 .44 19 38 61 .63 4- 7 s 115.13 51 38 71 56 124 88 98 673 106.44 V 1 1 . 4 4 5 94 5 88 9 .44 15 31 10.06 i 23 25 r 3 2.50 31 .06 44.94 4- 8 s 42 06 46 69 98 69 110.56 V •1 6 19 7 44 11 13 10.00 J_ r 51.63 4- 9 s 45 06 54 25 94 81 145.50 V 12 94 13.31 i 34.94 r 41.88 5- 1 s 50.19 84 56 151 63 30 88 124 88 151.13 V 16 88 14 30 18 00 21.69 i 1 1 .25 13 81 6 00 37 75 59.81 r 38.38 39 88 7 94 23 69 5- 2 s 51.81 117 31 177 63 49 06 181 63 166.00 V 5.62 24 56 15 94 25 50 19.63 i 1 5 .13 11 81 8 19 55 00 71,63 r 3 3 . 5 0 51 38 13 88 5- 3 s 4 0 . 6 9 130 44 177 81 49 50 186 38 157.81 V 3.25 25 75 13 78 24 13 21.94 i 9.06 11 88 9 75 43 38 62.38 r 73 44 18 31 C o n t i n u e d - 176 -A p p e n d i x I I ( c o n t i n u e d ) B i 2 3 I P P A ( c o i s ! _ 3 2 ° i - T l ( c o l s 4 - 6 ) D- S -A 1 1 s t 2 2 n d 3 2 n d 4 1 s t 5 2 n d 6 2 n d o b s e r v e r o b s e r v a t i o n s o b s e r v e r o b s e r v e r o b s e r v a t i o n s o b s e r v e r b y 1 s t b y 1 s t o b s e r v e r o b s e r v e r 5- 3 s 7 6 . 9 4 113.31 151 63 59.37 165 4 4 151.25 V 6.31 24.19 18 00 17 63 19.19 i 2 5 . 9 4 1 2 . 4 4 38 88 51.25 r 1 2.50 33.38 25.88 5- 5 s 89.81 120.63 155 88 1 1 1 . 1 3 128 63 137.94 V 7.81 24. 44 19 50 1 3 . 3 1 17 19 17.63 i 1 5 . 81 18.88 38 88 r 22.13 19. 25 20 25 32.69 23 81 41.69 5- 6 s 1 1 8 . 2 5 126. 44 149 13 72.19 132 50 152.00 V 13.69 25. 69 13 50 16 75 15.31 i 1 9 .25 47 31 2 6 . 5 0 26 19 r 35.13 21 . 69 31 56 2 2 . 3 1 39.88 5- 7 s 138. 81 135 50 122 00 190.56 V 19. 44 13 75 17 25 17.69 i 22. 38 85 25 30 63 r 4 5 . 69 54.06 5- 8 s 94. 88 166 56 142 94 190.56 V 15. 75 22 88 12 25 17.69 i 16. 31 26 56 39 94 r 18. 13 55 19 54.06 5- 9 s 75. 63 135 50 146 56 169.50 V 12. 31 13 75 12 63 19.75 i 24. 94 85 25 31 69 r 40.94 6- 1 s 30.19 100. 75 179 56 4 3 . 2 5 140 19 173.88 V 24. 31 22 63 29 19 36.44 i 9.13 28. 12 65 75 58 88 45.63 r 8.81 12. 38 24 25 62 31 76.25 6- 2 s 61.38 1 0 1 . 56 125 69 1 4 0 . 1 9 141 38 169.69 V 5.13 18. 88 16 25 1 1 . 5 0 29 06 13.94 i 1 9 .25 15. 13 33 13 18.19 32 63 49.00 r 15.13 25. 44 4 4 50 49.50 6- 3 s 1 1 0 . 3 1 73. 38 136 81 1 0 2 . 8 1 174 75 165.74 V 10.69 13. 75 12 94 13.13 34 56 18.81 i 3 1.25 15. 13 33 31 1 7 . 1 9 43 00 35.94 r 1 4.31 22 81 17.88 C o n t i n u e d - 177 -A p p e n d i x I I ( c o n t i n u e d ) B i 2 3 i p P A ( c o l s i _ 3 2 0 1 - T 1 ( c o l s 4 - 6 ) D- S -A 1 1 s t 2 2 n d 3 2 n d 4 1 s t 5 2 n d 6 2 n d o b s e r v e r o b s e r v a t i o n s o b s e r v e r o b s e r v e r o b s e r v a t i o n s o b s e r v e r b y 1 s t b y 1 s t o b s e r v e r o b s e r v e r 6- 4 s 1 1 0 . 4 4 86 00 133 50 1 2 9 . 8 8 162 81 159.19 V 9.37 17 88 8 63 1 5 . 0 0 23 13 14.25 i 1 7 . 44 13 63 38 00 43.06 r 19.81 20 56 6- 5 s 1 1 1 . 6 9 100 06 114 38 1 1 4 .63 152 00 155.19 V 7.13 18 44 8 50 1 4 . 8 1 28 38 16.06 i 1 1 .25 34 88 r 16.56 33.00 6- 6 s 118.56 113 63 128 63 1 1 3 . 3 1 127 31 121.50 V 9.88 14 38 9 31 15.19 18 63 16.25 i r 27 81 16 38 6- 7 s 124.06 113 69 143 00 1 1 3 . 9 4 114 63 150.88 V 9.81 14 81 9 37 14.31 14 81 11.38 i 16 94 21 75 r 29 94 23 38 6- 8 s 119 06 128 44 129 88 140.38 V 14 88 9 25 15 00 14.94 i 40 00 36 88 r 25 19 6- 9 s 117 50 147 25 102 81 136.63 V 17 44 12 75 13 13 13.25 i 4 4 00 56 88 17 19 r 21 56 30 19 15.50 7- 1 s 4 3 . 3 8 80 50 153 56 4 5 . 1 3 130 88 187.06 V 13 88 21 38 22 00 30.50 i 1 8.25 62 81 35 75 127.81 r 27 56 39 44 42 69 7- 2 s 1 03.81 100 50 136 69 36.13 189 69 185.25 V 16.75 23 56 19 94 36 00 27.88 i 1 8 . 44 21 56 7.50 4 4 13 93.50 r 1 5 . 9 4 32 38 28 93 15.75 7- 3 s 1 2 1 . 5 0 111 19 156 19 1 1 1 . 1 9 194 13 188.38 V 22.63 22 63 12 25 13.88 25 63 18.69 i 1 8.81 23 00 11.13 39 69 39.63 r 22.13 31 25 35.75 C o n t i n u e d - 178 -A p p e n d i x I I ( c o n t i n u e d ) B 1 2 3 T p p A ( c o i s i _ 3 2 o i . T l ( c o l s 4 - 6 ) D -S -A 1 1 s t 2 2 n d 3 2 n d 4 1 s t 5 2 n d 6 2 n d o b s e r v e r o b s e r v a t i o n s o b s e r v e r o b s e r v e r o b s e r v a t i o n s o b s e r v e r b y 1 s t b y 1 s t o b s e r v e r o b s e r v e r 7 -4 s 149.13 114 50 149 .31 1 3 6 . 3 8 183 06 167.44 V 17.56 25 38 19 .31 15.38 29 06 22.44 i 1 3 .25 25. 50 34.13 r 17.13 7 -5 s 1 5 1 . 2 5 131 56 147 .44 141.38 154 94 212.13 V 16.06 27 44 17 .44 12.88 26 06 12.06 i 2 3 . 94 14. 31 r 31.88 7 -6 s 1 5 7 . 1 9 130 19 175 .19 1 6 6.13 141 38 216.63 V 22.25 30 00 21 25 18.81 12 88 16.44 i 8.19 14.69 r 30 .44 7 -7 s 149 13 196 38 136 38 144.69 V 17 56 21 13 15 38 12.19 i 13 25 37 .94 10 13 r 17 13 7 -8 s 121 50 142 88 1 1 1 . 19 159.75 V 22 63 21 13 13. 88 14.31 i 18 81 27 .44 11 13 r 22 13 30 88 8 -1 s 4 3 . 5 0 1 5 1 . 81 192.06 V 29 13 27.56 i 70. 56 81.63 r 32. 50 38.88 8 -2 s 72.38 1 5 1 . 81 142.75 V 29 13 14.13 i 5 6 . 5 7 62 88 63.44 r 28. 69 8 -3 s 1 1 8 . 6 9 172 56 116.94 V 2 0 . 8 1 27 75 15.81 i 6 9 . 5 6 76. 88 34.63 r 2 4.75 19. 19 C o n t i n u e d - 179 -A p p e n d i x I I ( c o n t i n u e d ) B 1 2 3 T p p A ( c o i s i _ 3 a o i . T l ( c o l s 4 - 6 ) D- S -A 1 1 s t 2 2 n d 3 2nd 4 L s t 5 2 n d 6 2 n d o b s e r v e r o b s e r v a t i o n s o b s e r v e r o b s e r v e r o b s e r v a t i o n s o b s e r v e r b y 1 s t b y l s t o b s e r v e r o b s e r v e r 8- 4 s 151 .81 131 00 155.00 V 29 .13 18 75 16.00 i 70 .56 19 63 49.31 r 32 .50 23 26 11.25 8- 5 s 151 .81 100 13 133.88 V 29 .13 13 25 12.50 i 62 .88 19 75 r 28 .69 55.88 8- 6 s 172 .56 131 44 183.00 V 27 .75 13 19 11.81 i 76 .88 27 63 44.88 8- 7 r S 131 .00 159 25 146.31 V 18 .75 16 81 12.44 i 19 .63 49 50 33.44 r 23 .26 8- 8 s 149 69 137.69 V 19 31 10.56 i 54 00 24.63 9- 1 r s 59 .63 91 06 219 .31 V 21 06 19 .25 i 17 .75 26 44 81 .50 r 17 .13 28 06 9- 2 s 100 .56 98 38 115 .81 V 13 .00 19 88 12 .94 i 26 .56 r 19 .69 35 62 28 .75 9- 3 s 134 .31 127 81 128 .81 V 18 .81 18 00 12 .81 i r 28 .19 50 50 9- 4 s 132 .69 124 75 129 .88 V 10 .06 24 44 17 .25 i r 27 .75 37 00 C o n t i n u e d - 180 -A p p e n d i x I I ( c o n t i n u e d ) B 1 2 3 I P P A ( c o l s 1-3 2 0 1 - T 1 ( c o l s 4 - 6 ) D- S -A 1 1 s t 2 2 n d 3 2 n d 4 1 s t 5 2 n d 6 2nd o b s e r v e r o b s e r v a t i o n s o b s e r v e r o b s e r v e r o b s e r v a t i o n s o b s e r v e r b y 1 s t b y 1 s t o b s e r v e r o b s e r v e r 9- 5 s 1 1 5 . 7 5 114 06 127 13 V 14.38 15 69 15 38 i r 4 1 . 5 0 20 00 9- 6 s 134.88 122 25 124 88 V i 1 2 .88 16 13 14 13 r 34 44 41 50 9- 7 s 98 31 118 19 V 8 75 14 00 i 30 88 42 06 9- 8 r S 100 75 121 44 V 10 94 12 13 i 30 94 4 7 , 4 4 9- 9 r s 99 69 98 00 V 9 44 10 69 i 33 06 44 25 r 28 88 - 181 -A p p e n d i x III - B 1 2 3 I P P A / 2 0 1 T 1 r a t i o s f o r e a c h e x p e r i m e n t a n d c o r r e s p o n d i n g a r e a - o f - i n t e r e s t D-S -A 7 1 s t 8 2 n d O b s e r v e r O b s e r v e r F A / T L F A / T L 1-1 s 0 49 0 98 V 0 91 i 3 19 2 66 r 1-2 s 1 09 1 01 V 0 97 0 65 i 2 32 r 1 35 1-3 s 0 86 0 87 V 0 04 0 64 i 1 73 r 1 42 1-4 s 0 75 0 93 V 0 74 0 75 i r 1-5 s 0 89 0 74 V 0 77 0 59 i r 1-6 s 0 96 0 88 V 0 73 0 82 i r 1-7 s 1 16 0 92 V 1 22 0 63 i r 1-8 s 1 06 0 82 V 0 77 0 59 i r 1-9 s 0 72 0 85 V 0 85 0 48 i 0 41 r 0 23 D-S -A 7 1 s t 8 2 n d O b s e r v e r O b s e r v e r F A / T L F A / T L 2-1 s 0 48 0 19 V i 0 81 r 1 23 2-2 s 0 67 0 60 V 0 62 0 32 i 0 71 1 08 r 0 66 2-3 s 0 67 0 92 V 0 82 0 59 i 0 11 0 53 r 1 57 2-4 s 0 89 0 96 V 0 71 0 91 i 0 62 r 0 91 2-5 s 0 98 1 06 V 1 24 1 20 i 1 58 r 5 09 2-6 s 0 93 1 09 V 1 15 0 83 i r 2-7 s 0 82 V 0 91 i r 2-8 s V i r 3-1 s 0 61 0 32 V 0 29 i 0 70 0 77 r 0 21 - 182 -A p p e n d i x I I I ( c o n t i n u e d ) D-S -A 7 l s t 8 2 n d O b s e r v e r O b s e r v e r F A / T L F A / T L 3-2 s 0 .56 1 06 V 0 37 0 53 i 1 19 1 06 r 0 34 2 12 3-3 s 0 79 0 56 V 0 84 0 68 i 0 58 0 55 r 1 73 0 78 3-4 s 0 69 1 01 V 0 51 0 34 i 1 52 r 1 10 1 38 3-5 s 0 62 0 88 V 0 57 0 75 i 2 44 1 76 r 0 73 0 71 3-6 s 1 05 0 99 V 0 80 0 75 i r 0 70 0 34 3-7 s 0 90 1 11 V 0 94 0 80 i r 1 68 3-8 s 0 87 1 06 V 0 77 0 47 i r 4-1 s 1 08 1 74 V i r 4-2 s 1 18 1 45 V i r 4-3 s 1 07 1 49 V 3 67 i r D-S -A 7 l s t 8 2 n d O b s e r v e r O b s e r v e r F A / T L F A / T L 4-4 s 0 79 1 42 V 2 31 8 20 i r 4-5 s 0 97 1 09 V 1 63 2 04 i r 4-6 s 1 05 0 96 V 0 53 0 87 i r 4-7 s 0 52 0 67 V 0 39 0 58 i r 4-8 s 0 43 0 42 V 0 56 0 74 i r 4-9 s 0 48 0 37 V i r 5-1 s 0 68 1 00 V 0 94 0 67 i 0 37 r 1 68 5-2 s 0 65 1 07 V 0 96 0 81 i 0 21 r 5-3 s 0 70 1 13 V 1 07 0 63 i 0 27 r 5-4 s 0 68 1 00 V 1 37 0 94 i r - 183 -A p p e n d i x I I I ( c o n t i n u e d ) D-S -A 7 1 s t 8 2 n d O b s e r v e r O b s e r v e r F A / T L F A / T L 5-5 s 0 94 1 13 V 1 42 1 10 i r 0 81 0 49 5-6 s 0 95 0 98 V 1 53 0 88 i r 0 79 5-7 s 1 14 0 71 V 1 13 0 78 i 0 73 r 5-8 s 0 66 0 87 V 1 29 1 29 i 0 41 r 1 02 5-9 s 0 52 0 80 V 0 97 0 70 i 0 79 r 6-1 s 0 72 1 03 V 0 83 0 62 i 0 48 1 44 r 0 20 0 32 6-2 s 0 72 0 74 V 0 65 1 17 i 0 46 0 68 r 0 57 6-3 s 0 42 0 82 V 0 40 0 69 i 0 35 0 93 r 6-4 s 0 53 0 84 V 0 77 0 61 i 0 36 r D-S -A 7 1 s t 8 2 n d O b s e r v e r O b s e r v e r F A / T L F A / T L 6-5 s 0 66 0 74 V 0 65 0 53 i r 6-6 s 0 89 1 06 V 0 77 0 57 i r 6-7 s 0 99 0 95 V 1 00 0 82 i r 6-8 s 0 92 0 91 V 0 99 0 62 i r 6-9 s 1 14 1 08 V 1 32 0 96 i 2 56 r 1 95 7-1 s 1 14 0 82 V 1 20 0 70 i 1 56 0 49 r 7-2 s 0 62 0 74 V 0 63 0 72 i r 0 65 7-3 s 0 53 0 83 V 0 65 0 66 i 0 49 r 1 12 7-4 s 0 57 0 89 V 0 88 0 86 i 0 58 r A p p e n d i x I I I ( c o n t i n u e d ) D-S -A 7 1 s t 8 2 n d O b s e r v e r O b s e r v e r F A / T L F A / T L 7-5 s 0 85 0 70 V 1 05 1 45 i r 7-6 s 0 92 0 81 V 2 33 1 29 i r 7-7 s 1 09 1 36 V 1 14 1 73 i 1 31 r 7-8 s 1 09 0 89 V 1 63 1 48 i 1 69 r 8-1 s V i r 8-2 s V i r 8-3 s V i r 8-4 s V i r 8-5 s V i r - 185 -Appendix IV - Infarcted tissue as percent of entire sl i c e expressed for each o f the three techniques of perfusion defect assessment, including histochemistry (TTZ staining), B123IPPA and 2 0 1T1. 1 2 % Inf 3 4 % Inf 2nd % Inf B123IPPA Dog-Slice 1st observations 2nd 1st observer by 1st observer observer observer 1-1 36 54 25 42 20 31 33.24 1-2 34 29 32 41 31 21 23.46 1-3 28 13 28 89 29 63 21.04 1-4 23 85 19 12 20 83 33.06 1-5 23 28 24 56 24 02 32.60 1-6 0 00 0 00 0 00 26.47 2-1 14 96 5 47 8 63 43.78 2-2 13 37 5 37 12 50 51.20 2-3 9 27 9 90 8 21 7.37 2-4 2 77 5 48 4 44 3.10 2-5 8 11 11 01 9 00 0.00 2-6 14 11 24 84 4 02 0.00 3-1 38 33 46 43 41 67 27.96 3-2 30 64 34 88 32 58 19.38 3-3 38 51 38 05 38 99 25.69 3-4 36 43 36 61 37 16 21.36 3-5 28 11 27 08 26 76 27.25 3-6 17 53 21 62 20 44 0.00 4-1 0 00 5 10 1 96 0.00 4-2 0 00 0 00 0 00 0.00 4-3 0 00 0 00 0 00 0.00 4-4 3 55 3 61 4 73 0.00 4-5 2 86 16 10 3 32 0.00 4-6 0 00 0 00 0 00 0.00 5-1 30 82 31 58 26 97 22.42 5-2 18 56 21 47 19 43 32.76 5-3 25 72 20 26 18 90 24.19 5-4 21 43 23 44 22 65 36.73 5-5 6 77 8 68 8 27 19.28 6-1 22 55 26 42 25 36 30.24 6-2 17 96 23 36 20 16 34.24 6-3 25 18 30 22 26 22 31.37 6-4 12 50 13 82 14 45 17.26 6-5 13 97 15 92 12 42 10.76 7-1 40 25 43 27 40 49 42.06 7-2 20 75 26 43 22 39 21.12 7-3 15 89 15 28 15 61 13.91 7-4 9 60 12 23 13 53 17.71 Continued - 186 -A p p e n d i x I V ( c o n t i n u e d ) D o g - S l i c e 1 % I n f l s t o b s e r v e r 2 % I n f 2 n d o b s e r v a t i o n s b y l s t o b s e r v e r 3 % I n f 2 n d o b s e r v e r 4 B1 2 3I PpA l s t o b s e r v e r 8-1 37 50 64 .63 43 .48 8-2 48 17 28 .32 37 .58 8-3 4 4 70 50 .00 47 .01 8-4 31 85 50 .00 40 .00 8-5 38 58 38 .81 36 .98 8-6 41 53 46 .33 37 .27 8-7 4 4 75 37 .85 41 .44 9-1 13 48 27 .27 20 .25 29.76 9-2 19 53 17 .20 14 .08 30.35 9-3 7 98 12 .80 10 .57 0.00 9-4 0 00 0 .00 0 .00 0.00 9-5 0 00 0 .00 0 .00 0.00 9-6 0 00 0 .00 0 .00 0.00 - 187 A p p e n d i x I V ( c o n t i n u e d ) D o g - S l i c e 5 B ^ S I P P A 6 B1 2 3I P P A 7 T L 8 T L 2 n d 9 T L o b s e r v a t i o n 2 n d l s t o b s e r v a t i o n 2 n d b y l s t o b s e r v e r o b s e r v e r b y l s t o b s e r v e r o b s e r v e r o b s e r v e r 1-1 18 5 40 6 20 84 16 0 21.4 1-2 24 2 0 0 24 28 8 0 0.0 1-3 0 0 0 0 30 48 0 0 0.0 1-4 0 0 0 0 34 07 37 4 0.0 1-5 12 9 0 0 32 31 47 8 22.8 1-6 0 0 70 5 26 59 45 9 4 8 . 3 2-1 54 1 39 6 66 23 45 5 36.7 2-2 22 8 18 0 48 49 82 7 16.1 2-3 17 0 0 0 34 96 27 6 8.7 2-4 11 7 0 0 37 03 29 4 28.4 2-5 35 1 24 8 15 74 0 0 0.0 2-6 24 5 0 0 1 62 0 0 0.0 3-1 48 2 38 2 22 61 34 9 29.2 3-2 30 2 49 5 28 10 26 1 22.1 3-3 14 4 13 4 38 31 17 5 7.3 3-4 0 0 0 0 46 80 27 8 0.0 3-5 0 0 0 0 15 05 30 2 5.1 3-6 0 0 15 4 7 99 34 2 18.2 4-1 8 4 0 0 0 0 0 0 0.0 4-2 0 0 0 0 0 0 0 0 0.0 4-3 0 0 0 0 0 0 0 0 0.0 4-4 0 0 0 0 0 0 13 4 0.0 4-5 0 0 0 0 0 0 54 5 15.0 4-6 0 0 0 0 0 0 59 4 52.3 5-1 16 0 0 0 19 44 35 3 47.7 5-2 6 1 0 0 16 69 26 5 42.4 5-3 0 0 17 4 19 70 11 5 0.0 5-4 19 5 42 1 20 95 30 0 0.0 5-5 38 8 59 8 19 30 23 9 0.0 6-1 27 2 37 1 0 0 41 0 32.3 6-2 22 5 13 4 21 3 28 9 27.1 6-3 0 0 0 0 19 17 15 0 0.0 6-4 28 1 23 2 0 0 0 0 0.0 6-5 36 8 46 2 0 0 20 21 0.0 C o n t i n u e d . . - 188 -A p p e n d i x I V ( c o n t i n u e d ) D o g - S l i c e 5 B i 2 3 I P P A 6 B1 2 3 I P P A 7 T L 8 T L 2 n d 9 T L o b s e r v a t i o n 2 n d 1 s t o b s e r v a t i o n 2 n d b y 1 s t o b s e r v e r o b s e r v e r b y 1 s t o b s e r v e r o b s e r v e r o b s e r v e r 7-1 62 63 0 00 0 00 45 21 23.36 7-2 0 00 0 00 20 75 32 83 23.54 7-3 26 93 0 00 5 10 26 02 0.00 7-4 0 00 0 00 0 00 16 56 0.00 8-1 0 .0 54 4 4 9 . 5 8-2 78 12 37 5 35.0 8-3 71 07 21 1 15.3 8-4 57 53 34 8 25.0 8-5 51 26 41 4 19.4 8-6 53 09 31 3 13.0 8-7 17 49 0 0 0.0 9-1 17 8 26 9 9-2 0 0 0 0 9-3 0 0 0 0 9-4 39 5 41 9 9-5 20 0 30 7 9-6 0 0 22 8 - 189 -A p p e n d i x V - A u t o r a d i o g r a p h i c a s se s smen t o f t h e i s c h e m i c r i s k zone f o r dogs 8 and 9 u s i n g p l a n i m e t r i c q u a n t i t a t i o n . D o g - S l i c e 1 s l i c e v o l ( c m 3 ) - l s t 2 s l i c e v o l ( c m 3 ) - 2nd 3 s l i c e v o l ( c m 3 ) - 2 n d o b s e r v e r o b s e r v a t i o n s by l s t o b s e r v e r o b s e r v e r 8 -1 3 55 3 95 3 45 8-2 10 44 10 14 9 48 8-3 15 36 15 66 15 84 8-4 16 44 17 04 17 64 8-5 11 80 13 25 13 35 8-6 16 80 17 64 17 40 8-7 22 14 19 80 19 38 9 -1 3 75 3 65 3 50 9-2 7 40 7 15 7 00 9-3 11 35 11 45 11 50 9-4 17 34 17 28 16 92 9-5 10 10 10 10 10 00 9-6 13 26 13 32 13 38 D o g - S l i c e 4 IRZ (cm3) - l s t 5 IRZ (cm3) - 2nd 6 IRZ (cm3) - 2nd o b s e r v e r o b s e r v a t i o n s by l s t o b s e r v e r o b s e r v e r 8 -1 3 55 3 95 3 45 8-2 6 54 6 84 6 60 8-3 10 74 10 92 10 44 8 -4 8 82 9 60 9 78 8-5 8 30 8 00 7 45 8-6 8 10 8 52 8 16 8-7 9 53 9 71 8 97 9 -1 0 00 0 00 0 00 9-2 0 00 0 00 0 00 9 -3 0 00 0 00 0 00 9 -4 0 00 0 00 0 00 9 -5 0 00 0 00 0 00 9-6 0 00 0 00 0 00 

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