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Information to Users INFORMATION TO USERS While the most advanced technology has been used to photograph and reproduce this manuscript, the quality of the reproduction is heavily dependent upon the quality of the material submitted. For example: • Manuscript pages may have indistinct print. In such cases, the best available copy has been filmed. • Manuscripts may not always be complete. In such cases, a note will indicate that it is not possible to obtain missing pages. • Copyrighted material may have been removed from the manuscript. In such cases, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, and charts) are photographed by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each oversize page is also filmed as one exposure and is available, for an additional charge, as a standard 35mm slide or as a 17”x 23” black and white photographic print. Most photographs reproduce acceptably on positive microfilm or microfiche but lack the clarity on xerographic copies made from the microfilm. For an additional charge, 35mm slides of 6”x 9” black and white photographic prints are available for any photographs or illustrations that cannot be reproduced satisfactorily by xerography. 8710037 Poikey, Leonard Andrew THE USE OF IMMOBILIZED OXALATE OXIDASE IN AN ANALYTICAL ASSAY FOR URINARY OXALATE AND IN AN EXTRACORPOREAL SHUNT TREATMENT FOR HYPEROXALURIA The Ohio State University Ph.D. 1987 University Microfilms I nternsition300 el N. Zeeb Road, Ann Arbor, Ml 48106 PLEASE NOTE: In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark V 1. Glossy photographs or pages. 2. Colored illustrations, paper or print______ 3. Photographs with dark background _____ 4. Illustrations are poor copy_______ 5. Pages with black marks, not original copy. 6. Print shows through as there is text on both sides of page. 7. Indistinct, broken or small print on several pages________ 8. Print exceeds margin requirements______ 9. Tightly bound copy with print lost in spine________ 10. Computer printout pages with indistinct print. 11. P ag e(s) _ lacking when material received, and not available from school or author. 12. P ag e(s)_____ seem to be missing in numbering only as text follows. 13. Two pages numbered . Text follows. 14. Curling and wrinkied p a g e s_ 15. Dissertation contains pages with print at a slant, filmed as received 16. O ther _______________ University Microfilms International THE USE OF IMMOBILIZED OXALATE OXIDASE IN AN ANALYTICAL ASSAY FOR URINARY OXALATE AND IN AN EXTRACORPOREAL SHUNT TREATMENT FOR HYPEROXALURIA DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree, Doctor of Philosophy, in the Graduate School of the Ohio State University By Leonard A. Poikey, M.S. ***** The Ohio State University 1987 Dissertation Committee: Approved by K. E. Richardson, Ph.D. J. S. Rieske, Ph.D. Adviser, Department of M. I. Walters, Ph.D. Physiological Chemistry To Two Women 11 ACKNOWLEDGMENTS I ara extremely grateful to my adviser. Dr. Keith E. Richardson, for his support and guidance. My goal of a doctoral degree could not have been achieved without his help and knowledge. Dr. Richardson has taught me more than solely academics. I value his friendship, and I have enjoyed our frequent talks. I wish also to thank my dissertation committee, Drs. Martha I. Walters and John S. Rieske, for their advice during the course of this work. Also, Ms. Micaela Stratton's expertise in hematology was extremely helpful. On a more personal note, a sincere thank you to my family is fitting. Their thoughts and encouragement never faltered. I am especially indebted to two women. My mother instilled in her son the belief of the value of education as a means of personal growth and reward. Her support never waned, and I am sure she is very proud of her son's accomplishment. My wife instilled in her husband the belief that he could, and would, obtain a doctoral degree. She tolerated years of frustration helping iii her husband as he pursued his goal. This work, and subsequent degree, is dedicated to these two women. IV VITA July 18, 1953 Born - Detroit, Michigan 1975 B.S., Biology University of Michigan Ann Arbor, Michigan 1977 M.S., Biology Wayne State University Detroit, Michigan 1975-1979 Medical Technician Providence Hospital Southfield, Michigan 1977-1979 Research Technician Dept, of Internal Medicine Wayne State University Harper-Grace Hospital Detroit, Michigan 1979-1981 Medical Technologist Biomedical Reference Labs Birmingham, Alabama 1981-1986 Medical Technologist Roche Laboratories Dublin, Ohio 1986-1987 General Supervisor Roche Laboratories Dublin, Ohio PUBLICATIONS Lott, J.A. and Poikey, L.A. (1984) pH and blood gas determinations: An update on correction factors. Journal of Medical Technology, 1_ (2): 101-106. FIELDS OF STUDY Major Field; Clinical Chemistry Studies in: Enzymology Enzyme Immobilization Analytical Method Development Adviser : Keith E. Richardson, Ph.D. VI TABLE OF CONTENTS page DEDICATION............................................. ii ACKNOWLEDGMENTS......................................iii VITA..................................................... V LIST OF TABLES......................................viii LIST OF FIGURES........................................ x LIST OF ABBREVIATIONS............................. xiii INTRODUCTION............................................1 CHAPTER I. THE IMMOBILIZATION OF OXALATE OXIDASE AND ITS USE IN AN ANALYTICAL ASSAY........... 4 Literature Review................................4 Materials and Methods........... 24 Results and Discussion........................ 52 CHAPTER II. IMMOBILIZED ENZYME USE IN AN EXTRACORPOREAL SHUNT IN THE RAT..............82 Literature Review........... 82 Materials and Methods........ 94 Results and Discussion.......... 112 FINAL SUMMARY........................................ 147 REFERENCES............................................150 Vll LIST OF TABLES Table page 1. Immobilization of oxalate oxidase............ 53 2. Inhibitors/Activators effects................. 66 3. Stabilizer effects....... ....... 67 A. Ascorbate oxidase use to remove interference.................................... 73 5. Sodium nitrite use to remove interference..75 6 . Assay recovery with ascorbate oxidase....... 76 7. Assay recovery with sodium nitrite........... 76 8 . Assay precision with ascorbate oxidase...... 79 9. Assay precision with sodium nitrite..........80 10. Method comparison.......... 81 11. CPG-enzyme effects on cellular components in vitro........................... 119 12. CPG-enzyme effects on blood chemistry in vitro...........................120 13. Filter material effect on cellular components in vitro........................... 121 14. Filter material effect on cellular components in vitro (% change)..............122 viii 15. Antiplatelet drug effects In vitro........ 127 16. Antlplatelet drug effects In vitro (% change)..................... 128 17. Anticoagulant effect in vitro............. ..131 18. Anticoagulant effect In vitro (% change)..132 19. Acid citrate effect on cellular components In vitro........................... 134 20. Acid citrate effect on cellular components In vivo............................ 136 21. Acid citrate (IX) effects on blood chemistry in vivo.......................... .137 22. Acid citrate (2X) effects on blood chemistry in vivo....... 139 Ix LIST OF FIGURES Figure page 1. Oxalate synthesis in man........................ 5 2. Isolation and purification of oxalate oxidase....... .2 5 3. CPG-oxidase via carbodiimide linkage.........31 4 . CPG-oxidase via carbonyl linkage............. 33 5. CPG-oxidase via azide linkage.................34 6 . AminoethyIcellulose coupled oxidase..........36 7. Polyacrylamide coupled oxidase............... 38 8 . Carbony1-CPG-oxidase activity as a function of pH..................................55 9. Carbodiimide-CPG-oxidase activity as a function of pH ..................................57 10. Carbony1-CPG-oxidase activity as a function of temperature....................... 58 11. Carbodiimide-CPG-oxidase activity as a function of temperature....................... 59 12. Stability at -2Q°C of the carbonyl-CPG- oxidase.......................................... 60 13. Stability at +4°C of the carbonyl-CPG- oxidase.......................................... 62 14. Stability at +25°C of the carbony1-CPG- oxidase .......................................... 63 15. Composite stability of the carbony1-CPG- oxidase .......................................... 64 16. Reaction progress as a function of time....69 17. Standard curve with ascorbate oxidase....... 70 18. Standard curve with sodium nitrite ...... 71 19. Quench curve for the LS6800 System...........97 20. Extracorporeal shunt and COg-trapping apparatus....................................... 108 21. Carbonyl-CPG-decarboxylase activity as a function of pH ...............................113 22. Carbonyl-CPG-decarboxylase activity as a function of temperature.................... 114 23. Carbony1-CPG-decarboxylase stability....... 116 24. Carbony1-CPG-oxidase activity in vivo...... 141 25. Plasma content effect on the carbonyl- CPG-oxidase (CO2 assay)...................... 142 26. Plasma content effect on the carbonyl- CPG-oxidase (H2O 2 assay)..................... 143 XI 27, Carbony1-CPG-decarboxylase activity in vivo........................................
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