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University Microfilms International 300 N INFORMATION TO USERS This was produced from a copy of a document sent to us for microfilming. While the most advanced technological means to photograph and reproduce this document have been used, the quality is heavily dependent upon the quality of the material submitted. The following explanation of techniques is provided to help you understand markings or notations which may appear on this reproduction. 1. The sign or "target” for pages apparently lacking from the document photographed is "Missing Page(s)”. If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting through an image and duplicating adjacent pages to assure you of complete continuity. 2. When an image on the film is obliterated with a round black mark it is an indication that the film inspector noticed either blurred copy because of movement during exposure, or duplicate copy. 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In all cases we have filmed the best available copy. University Microfilms International 300 N. ZEEB HU., ANN ARBOR, Ml 48106 8214094 Gendler, Stephen Marshall METABOLISM OF CITRATE TO OXALATE IN THE RAT AND THE ANALYSIS OF OXALATE IN URINE The Ohio State University Ph.D. 1982 University Microfilms International300 N. Zeeb Road, Ann Arbor, MI 48106 Copyright 1982 by Gendler, Stephen Marshall All Rights Reserved METABOLISM OF CITRATE TO OXALATE IN THE RAT AND THE ANALYSIS OF OXALATE IN URINE DISSERTATION Presented in Partial fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Stephen Marshall Gendler, B.S. ***** The Ohio State University 1982 Reading Committee: Approved By Howard A. I . Newman, Ph.D. John S. Rieske, Ph.D. Keith E. Richardson, Ph.D. C y/\ A d v iser Department of Physiological C hem istry ACKNOWLEDGMENTS I would like to express my deepest gratitude to my research advisor, Dr. Keith Richardson. Without his scientific and spiritual guidance, this document would have been impossible. I would like to thank the other members of the reading com m ittee, Dr. John R ieske and Dr. Howard Newman, fo r t h e i r suggestions and help in the writing of this dissertation. As my academic advisor, Dr. Newman has been especially helpful in my clinical chemistry career. I am also thankful for the technical assistance given me by Dr. Dennis Pollack, who also served as Graduate School Representative. There is one person who stands above all others in the support and advice given me. I would have quit long ago had it not been for my wife, Kathy. I will have this degree because of her love and understanding, and I thank God for her presence. VITA November 19, 19 51 ................. Born, Detroit, Michigan 197 3 ................................................... B.S., Michigan State University, East Lansing M ichigan 197 4 ................................................... Registered Medical Technologist, American Society of Clinical P athology 1982.................................................... Assistant Director, Clinical Biochemistry, Michael Reese Hospital, Chicago, Illinois PUBLICATIONS Gendler, S.M., M.P. Farinelli, K.E. Richardson, and D.W. Fry, Two enzymes with potential use in spectrophotometric oxalate determination, Clinical Chemistry, 2<>, 1032 (1981) (Abstract). FIELDS OF STUDY Major Field: Clinical Chemistry Studies in Oxalate Metabolism and Oxalate Analysis. Professor Keith E. Richarson iii TABLE OF CONTENTS Page ACKNOWLEDGMENTS............................................................................................ i i VITA.......................................................................................................................... i i i LIST OF TABLES................ v LIST OF FIGURES .............................................................................. vi INTRODUCTION..................................................................... 1 C h ap ter I . THE METABOLISM OF CITRATE TO OXALATE 4 Literature Review ............................................................ 4 H y p o th e s is.............................................................................. 20 M ethods....................................................................................... 21 Results and Discussion.............................................. 26 Conclusions............................................................................ 30 I I . A POTENTIAL ASSAY FOR OXALATE UTILIZING COUPLED ENZYMATIC REACTIONS................................. 32 Literature Review ............................................................ 32 H y p o th e sis.............................................................................. 59 M ethods...................................................................................... 60 Results and Discussion.............................................. 68 Conclusions............................................................................ 81 I I I . AN HPLC METHOD FOR URINARY OXALATE 84 Literature Review ............................................................ 84 H y p o th e sis............................................................................... 91 M ethods................................................ 92 Results and Discussion.............................................. 97 Conclusions............................................................................ 105 REFERENCES 106 LIST OF TABLES T able Page 1. Results of the Study Showing the Metabolism of Citrate to Oxalate in the Male Wistar R a t................................................................................................................. 28 2. Influence of Media on Oxalate Decarboxylase Production in Aspergillus phoenicis at 30°C... 69 3. P u r if i c a t i o n of O x alate D ecarb o x y lase........................ 73 4. Retention Time of Organic Acids of the TCA Cycle and Oxalate Metabolism .............................................. 98 v LIST OF FIGURES / Figure Page 1. Pathways of Oxalate Synthesis.......................................... 6 2. Influence of Manganese on Oxalate Decarb­ oxylase Production in Aspergillus p h o e n ic is.............................................................................................. 71 3. Influence of Growth Temperature on Oxalate Decarboxylase Production in Aspergillus p h o e n ic is................... *........................................................................ 7 2 4. Influence of pH on the Activity of Oxalate Decarboxylase and Formate Dehydrogenase.............. 7 5 5. Reaction Progression of the Coupled R e a c tio n s.............................................................................................. 80 6. Separation of Radiolabelled Acids ............................... 99 7. Chromatogram of Blank............................................................... 101 8. OD Treated Urine ............................................................................ 103 9. Chromatogram of Urine............................................................... 104 vi INTRODUCTION Problem Statement Oxalate, a normal constituent of human urine, is excreted at a rate of 20 to 40 mg per 24 hours. The source of this oxalate is dietary and endogenous synthesis. Endogenous synthesis accounts for 89 to 97 percent of the total oxalate excreted by the kidney ( Richardson and Farinelli, 1981 ). However, this excretion is exceeded in several diseases in man. Primary hyperoxaluria, a rare genetic disease, is characterized by recurrent calcium oxalate nephrolithiasis, chronic renal failure, and early death from uremia ( Williams and Smith, 1978 ). Hyper­ oxaluria is also seen in patients who have undergone ileal resection or have inflammatory ileal disease ( Earnest et a l., 1974 ). In addition, there are 40 deaths per year resulting from ethylene glycol poisoning, where formation and deposition of oxalic acid occurs in renal, cardiopulmonary and central nervous system tissues ( Berman et al., 1957 ). Oxalic acid is present in 73 percent of all kidney stones and is the major component in over 70 percent of a series of 10,000 urinary calculi analyzed from across the United States ( Herring, 1962 ). A kidney stone will develop in one to five percent of the population ( Boyce et a l . , 1956 ). It is important to understand the metabolism of oxalic acid as it is a component of many major diseases. Citrate is a intermediary metabolite present in all tissues. Citrate is reported to be metabolized to oxalate in the rat ( Hodgkinson, 1978 ). Should a metabolite
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