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The Etiological Role of Carbon Monoxide in Fibrinolytic Activities

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The Etiological Role of Carbon Monoxide in Fibrinolytic Activities W< 1 74-21,978 KALMAZ, Ekrem Vahap, 1940- THE ETIOLOGICAL ROLE OF CARBON MONOXIDE IN FIBRINOLYTIC ACTIVITIES. The University of Oklahoma, Ph.D., 1974 Environmental Sciences ; University Microfilms, A XEROX Company, Ann Arbor, Michigan © 1974 EKREM VAHAP KALMAZ ALL RIGHTS RESERVED THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED. THE UNI\ŒRSITY OF OKLAHOMA GRADUATE COLLEGE THE ETIOLOGICAL ROLE OF CARBON MONOXIDE IN A DISSERTATION SUBMITTED TO THE GRADUATE FACULTY in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY BY EKREM VAHAP KALMAZ Norman, Oklahoma 1974 THE ETIOLOGICAL ROLE OF CARBON MONOXIDE IN FIBRINOLYTIC ACTIVITIES APPROVED BY DISSERTATION COMMITTEE THIS WORK IS DEDICATED TO MY PARENTS AND DR. CARL A. NAU 1X1 ACKNOWLEDGEMENTS It is of immense importance that an advisor set a fine example by judicious guidance, valued criticism and continuous encouragement for the successful completion of a graduate career for his students. To Dr. Larry W. Canter, I express my deep sense of gratitude and indebted­ ness for all of this. His guidance will have a lasting and positive effect upon me throughout my professional career. I wish to express my deep appreciation and gratitude to Dr. Carl A. Nau for his advice and support of this thesis. While working under him, I have gained much knowledge and many experiences. My gratitude to Drs. J. M. Robertson, J. W. Hampton, and R. Y. Nelson for their valuable suggestions and discussions. My gratitude to Dr. R. L. Coleman for his guidance and help throughout the course of this study. My gratitude to Dr. W. Joel for his guidance and help in histo­ logical and microscopic studies. My gratitude to Dr. R. Cain for his guidance and help in immuno­ logical studies. Gratitude is expressed to the following for volunteering their technical assistance: Mr. K. Cobb, Ms. A. Tuttle, Mr. M. W. Grimsley, Mr. J. Hall, Dr. Husseyin, Mr. D. Warner and Dr. L. Foyer. Individual consideration is extended to Dr. A. Weidenback and Ms. H. Mauldin for their help in preparation of the manuscript. iv I acknowledge the typing assistance given by Brenda Montaperto for the excellent job she did on the typing of this manuscript for me. TABLE OF CONTENTS Page LIST OF TABLES ................................................... viii LIST OF ILLUSTRATIONS = .......................................... ix Chapter I. INTRODUCTION ............................................ 1 General Description o,f Carbon Monoxide Gas and Its Relation to Human Health ....................... 3 Purpose and Scope .................................... 16 II. LITERATURE REVIEW ...................................... 18 Description of Importance of Hemoglobin Molecule in Oxygenation and Deoxygenation of Blood ......... 18 Physico-chemical Behavior of Hemoglobin .............. 18 Basis for Recommended Standard ....................... 35 Fibrinolysis and Thrombosis .......................... 38 The Present Knowledge of the Roles of Vascular System and Circulating Blood in Relation to the Fibrinolytic Enzyme System ..................... 50 III. EQUIPMENT, METHODS AND PROCEDURE ....................... 59 Material and Methods ................................. 59 Animals Used .......................................... 59 Exposure Chambers ........................... 59 Measurement of hemoglobin, carboxyhemoglobin and oxy­ hemoglobin (CO-oxymeter) ........................... 67 Spectrophotometric Determination of Carboxyhemoglobin, Oxyhemoglobin and Hemoglobin ....................... 71 Euglobulin Lysis Time (ELT) .......................... 76 Modified Method (ELT) ................................ 76 Whole Blood Clotting Time ....................... 80 Whole Blood Lysis Time ......... 81 Platelet Morphology .................................. 84 Platelet Counting ............ 85 vi Page Isolation of Transglutaminase (Factor XIII) Free of Fibrinogen for Fibrin Plate ..................... 86 Cohn Fractionation ............................... 86 Preparation of Fibrin Plate ............................. 96 Fibrin Plate Test (Hyland) ..... 98 Available Plasmin Test ............................... 99 Purification of Plasminogen for the Caseinolytic Assay ................................................. 100 Caseinolytic Assay (For Activator) ..................... 104 Standard Urokinase Curve ............................... 105 Urokinase Determination in Rabbit Urine ................ 106 Urokinase Standard Curve ............................... 110 Determination of Fibrinogen ............................ 114 Spectrophotometric Method of Determination of Fibrin­ ogen .................................................. 114 Assay of Fibrinogen in Plasma .......... I............ 118 Modified Micro-Kieldahl Method for Fibrinogen Deter­ mination ............................................ 119 Determination of Fibrinogen Split Products (FSP) ...... 123 Preparation of Fibrinogen Coated Erythrocytes ......... 125 Determination of ag-Macroglobulin ...................... 131 Alpha-1-Antitrypsin Test ............................... 134 Histological Studies .................................... 136 Azocarmin G Solution .................................... 139 Analine-Alcohol Solution ............................... 139 Pilot Study ........................................... 141 IV. DISCUSSION AND SUMMARY .................................... 157 Summary and Conclusions ................................ 169 BIBLIOGRAPHY ........................................................ 173 vii LIST OF TABLES Table Page 1. Hemoglobin, Carboxyhemoglobin and Oxyhemoglobin Mean Values of Percent Saturation Per Week in 12 Exposed Rabbits' Blood .......................... 72 2. Hemoglobin, Carboxyhemoglobin and Oxyhemoglobin Mean Values of Percent Saturation Per Week in 6 Control Rabbits' Blood .......................................... 73 3. ELT in Normal-Control Rabbits' Blood ..................... 79 4. Variation of the Lysis Area Induced by Dialyzed Undiluted Normal Rabbit Urine on Fibrin Plate .................... Ill viii LIST OF ILLUSTRATIONS Figure Page 1. Schematic Pathway of Fibrinolysis ....................... 42 2. Sketch Indicating Principles of Operation of the Exposure Chambers ...................................... 61 3. Profiles of Mean Values of Carboxyhemoglobin, Hemoglobin and Oxygen (test groups) .............................. 65 Control group .......................................... 66 4. Sketch Indicating Principle of Operation of CO-oximeter.. 69 5. Hemoglobin Spectra ....................................... 70 6. Standard Graph for Hemoglobin and Oxyhemoglobin Deter­ mination ............................................... 75 7. Profiles of ELT in Exposed and Non-exposed Rabbit Blood.. 78 8. Whole Blood Clot Time .................................... 83 9. Platelet Quantity Profile ................................ 88 10. Chromatography of Fibrinogen ............................. 93 11. Immunoelectrophoresis of Purified Fibrinogen ............ 95 12. Pattern of Affinity Chromatography of Plasminogen .... 103 13. Urokinase Standard Curve by Caseinolytic Assay .......... 107 14. Example of Lysis Area Produced on Unheated Fibrin Plates. 109 15. Urokinase Standard Curve ................................. 113 16. Plasminogen Caseinolytic Activity ........................ 115 17. Kjeldahl Distillation Apparatus .......................... 121 18. Fibrinogen Concentration in Plasma of Exposed and Non­ exposed Rabbit Plasma .................................. 124 ix Figure Page 19. Typical Result with Microtitre Plate ........ 129 20. The FSP Concentrations in Exposed and Non-exposed Rabbit Blood .................................. 133 21. Determination of a^AT by Immune Precipitate'............. 135 22. Iramuno-Plate (Reference Rabbit Serum Curve) for a„ Globuline ......................................7 ....... 138 23. Microscopic Changes in Myocardial and Arterial Tissues .. 143 24. Comparative Distribution Patterns of 0., Hb, COHb in Rabbit Blood That Was Exposed To 300 ppm CO ....... 147 25. Whole Blood Lysis Time .................... 149 26. Fibrin Plate (Hyland) .................................... 151 27. Changes of Major Parameters of Rabbit Blood That Was Exposed to 300 ppm CO With and Without E A C A .......... 153 28. Immunoelectrophoresis of Urokinase from Urine ........... 155 THE ETIOLOGICAL ROLE OF CARBON MONOXIDE IN FIBRINOLYTIC ACTIVITIES CHAPTER I INTRODUCTION Carbon monoxide (CO), an odorless, tasteless, colorless, non­ irritating gas, is the most abundant and widely distributed air pollu­ tant found in the lower atmosphere (Goldsmith, et al., 1968). It is an active reducing agent for chemicals at elevated temperatures, but it is principally encountered as a by-product of incomplete combustion of carbonaceous material. Carbon monoxide as a component of the ambient air, has existed ever since the geologic ages prior to Pleistocene (Encyclopaedia Bri­ tannica, 1969). As a gas CO was discovered by Priestly in 1799 (Bour, et al., 1967) and the composition of CO was first established by Cle­ ment and Desormes in 1801. The Greeks knew of the effects of CO, and the Romans used it for suicides and punishment of criminals (Cooper, 1966). Tliere was relatively high concentrations of atmospheric CO from natural sources such as vegetative and animal growth as well as volca­ nic activities before man learned to make fire. At present, emission 1 2 of CO gas generally exceeds that of all other gaseous pollutants com­ bined. especially in urban
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