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Zeeb Road, Ann Arbor, Michigan 48106 8618867 Wiener, Harvey Lewis ETHYLENE- AND DIETHYLENE GLYCOL METABOLISM, TOXICITY AND TREATMENT The Ohio State University Ph.D. 1986 University Microfilms International300 N. Zeeb Road, Ann Arbor, Ml 48106 ETHYLENE- AND DIETHYLENE GLYCOL METABOLISM, TOXICITY AND TREATMENT DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio S ta te U n iv ersity By Harvey Lewis Wiener, B. S. ***** The Ohio State University 1986 Dissertation Committee: Approved by K. E. Richardson A. J. Merola J . S. Rieske H. W. Sprecher Department of Physiological Chemistry To Cathy ACKNOWLEDGMENTS I wish to thank my adviser, Dr. Keith E. Richardson, for his never ending guidance, support, and encouragement throughout my tenure at Ohio State. I also wish to thank the members of my dissertation committee, Drs. A. John Merola, John S. Rieske, and Howard W. Sprecher for their many helpful conversations. Special thanks to Drs. Rhobert W. Evans and Michael M. Milks for their useful suggestions. I thank Dr. C. Ryzewski for generously providing chromatographic equipment and Dr. A. Schumann for his gift of (2-hydroxyethoxy)acetic acid. To the donors to The Ohio State University Development Fund, I offer my sincere thanks for partially supporting this work through the Graduate Student Alumni Research Award. To my classmates Drs. Jenifer A. Lindsey and Royal D. Saunders, and my colleagues from Dr. Richardson's laboratory I extend my sincere appreciation for their never ending friendship. I wish to thank my parents, Mr. and Mrs. Bernard Wiener, and my family for their never ending thoughts, prayers, and encouragement. Finally, I offer my most sincere thanks to Cathy D. Mahle for making it seem all worthwhile. VITA November 30/ 1959 ................................. Born - New York, New York 1 9 8 1 .................................................................... B. S., Fordham University, Bronx, New York 1981 .................................................................... Teaching Assistant, Department of Chemistry, Fordham University, Bronx, New York 1981-1986 ......................................................... Graduate Teaching Associate and Graduate Research Associate, Department of Physiological Chemistry, The Ohio S tate University, Columbus, Ohio 1983-1984 ......................................................... Graduate Teaching Associate, Division of Medical Education, The Ohio State University College of Medicine, Columbus, Ohio HONORS New York State Regents Scholarship, 1977-1981 Dean's List, Fordham University, Bronx, New York, 1980-1981 American Institute of Chemists Student Award, 1981 Graduate Student Alumni Research Award, The Ohio State U n iv e rsity , Columbus, Ohio, 1984 iv SOCIETIES American Association for the Advancement of Science American Chemical Society American Institute of Chemists PRESENTATIONS Competition between guests for sites in the host tetrakis(4-methylpyridineJnickel(II) isothiocyanate, H. L. Wiener and N. O. Smith, at the American Chemical Society New York Section Twenty-Ninth Annual Regional Undergraduate Research Symposium, 1981. PUBLICATIONS Concepts of Basic Biochemistry: Part A, Introduction to Metabolism, Harvey L. Wiener, Copyright by The Ohio State University College of Medicine, Division of Computing Services for Medical Education and Research, Columbus, Ohio, 1984. Concepts of Basic Biochemistry: Part C, Biochemical Equilibria and Energetics, Harvey L. Wiener, Copyright by The Ohio State University College of Medicine, Division of Computing Services for Medical Education and Research, Columbus, Ohio, 1984. FIELDS OF STUDY Major Field: Physiological Chemistry v TABLE OF CONTENTS DEDICATION................................................................................................................. i i ACKNOWLEDGMENTS ...................................................................................................... i i i VITA .................................................................................................................................. iv LIST OF TABLES ................................................................... ix LIST OF FIGURES............................................................................................ xi LIST OF ABBREVIATIONS ............................ xiv INTRODUCTION ........................................................................................................... 1 CHAPTER PAGE I. STUDIES ON ETHYLENE GLYCOL METABOLISM, TOXICITY, AND TREATMENT ................................................... 3 Literature Review ......................................................... 3 Metabolism of Ethylene Glycol, Glycolaldehyde, Glycolate, Glyoxylate and O xalate .......................................................... 6 Toxic M ediator .................................................... 28 Pathobiochemical Sequence of Events in Acute Ethylene Glycol Ingestion . 30 Three Clinical Stages of Ethylene Glycol Toxicity ......................................................... 35 Current Methods Used in Treating Ethylene Glycol Toxicity .................................. 39 Enzyme Therapy .............................................................. 42 Materials and Methods ............................................. 47 DCIP Assay of Glycolic Acid Oxidase . 47 Glyoxylate Phenylhydrazone Assay of Glycolic Acid Oxidase ........................ 48 Protein Determination .............................................. 49 Electrophoresis ......................................................... 51 Visible Spectroscopy ............................................. 51 DEAE-Cellulose Column P rep aratio n . 51 H ydroxylapatite ColumnPrep aratio n . 54 vi Sephadex G-25 M Column P rep aratio n . 55 Purification of Glycolic Acid O x i d a s e ......................................................................... 56 Determination of Glyoxylate ....................... 59 Oxidation of Glycolic Acid to Glyoxylic Acid in vitro in Whole Rat Blood by Pig Liver Glycolic Acid Oxidase . ............................ 60 Oxidation of [ 4C]-Glycolic Acid to [C]-Glyoxylic Acid in vitro in Whole Rat Blood by Pig Liver Glycolic Acid Oxidase ........................................................... 63 Care of Laboratory Animals .................... 64 Determination of Counting Efficiency . 64 Determination of Repiratory [C]-Carbon Dioxide ......... 65 Determination of Urinary Radiolabeled M etabolites .............................................................. 67 In vivo Studies Using Pig Liver Glycolic Acid O xidase .................................. 68 Results and Discussion .............................................. 71 I I . STUDIES ON DIETHYLENE GLYCOL METABOLISM, TOXICITY, AND TREATMENT ........................................................ 100 Literature Review .............................................................. 100 Metabolism of Diethylene Glycol by Man and Experimental Anim als ................. 103 Metabolism of Diethylene Glycol by B a c t e r i a ....................................................................... 105 Metabolism of Structurally Related Compounds; Diethyl Ether ....................................... 107 Metabolism of Structurally Related Compounds; 2-Ethoxyethanol ................................. 108 Metabolism of Structurally Related Compounds; 1,4-Dioxane ........................................ 109 C lin ic a l Signs and Symptoms of Diethylene Glycol Poisoning ............................ 110