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Xerox University Microfilms 300 North Zeab Road Ann Arbor, Michigan 4S106 75-26,557 CHERNQFF, Harvey Norman, 1936- THE EFFECT OF PHENYLKETONURIA ON OXALATE BIOSYNTHESIS. The Ohio State University, Ph.D., 1975 Chemistry, biological Xerox University Microfilmst Ann Arbor. Michigan 48106 THE EFFECT OF PHENYLKETONURIA ON OXALATE BIOSYNTHESIS Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University By Harvey Norman Chemoff, B.Sc., M.Sc. * * * * The Ohio State University 1975 Reading Committee; Approved By G. F. Grannis, Ph.D. H.-D. Grueraer, M.D. K. E. Richardson, Ph.D. 1 0 1 A/T- - f\\ \ } d L & i d L J. S. Rieske, Ph.D. /Adviser H. W. Sprecher, Ph.D. Department of Physiological Chemistry ACKNOWLEDGEMENTS To Jane, whose support and encouragement throughout graduate school were invaluable, and without whose scientific contribution this project would not have been completed. She made the long hours and short nights seem worthwhile. Special thanks to Dr. Keith Richardson, who made the project possible and whose support will always be remembered and appreciated. Thanks to Drs. Sprecher and Gruemer, who contributed much of their time and scientific expertise to this project. Thanks also to Drs. Rieske and Grannis for their contribution to this project. A spe cial thanks to Dr. Boutwell for his personal encouragement and interest in this training and to the Center for Disease Control for their support throughout. ii VITA May 3, 1936 ...........................B o m - Douglas, Georgia 1958 ................................. B.S., The University of Georgia, Athen s, Georgia 1958 - 1960 ........................ Managed Family Store, Rainbows Department Store, Broxton, Georgia 1960 - 1961 ......................... .. U. S . Army Active Duty. Then in U. S. Army Reserves at Fort McPherson, Atlanta, Georgia 1962 - 1963 ...... ........ Analytical Chemist, Food and Drug Administration, Atlanta, Georgia 1963 — 1967 .................. Research Associate, Biochemistry Department, The University of . Georgia, Athens, Georgia 1967 ..................................M.S., The University of Georgia, Athens, Georgia 1967 - 1972 .................... ..... Supervisory Chemist, Clinical Chemistry Unit, Center for Disease Control, Atlanta, Georgia PUBLICATIONS "A Stable Liquid Urine Preparation", Clin. Chem., 17, 634 (1971). "Effect of Phenylketonuria on Oxalate Synthesis", Clin. Chem., in press. iii FIELDS OF STUDY Major Field: Clinical Chemistry (Pathology) Studies in: Pathology, Professor H.-D. Gruemer Biochemistry, Professor Keith E. Richardson iv TABLE OF CONTENTS ACKNOWLEDGEMENTS.................................................. 11 VITA.............................................................. Ill LIST OF TABLES.................................................... vl LIST OF FIGURES. '................. vii ABBREVIATIONS........... viii Chapters I STATEMENT OF THE PROBLEM..................................... 1 II LITERATURE REVIEW................................... .'....... 3 III MATERIALS AND METHODS........... 29 Phenyllactate................... 30 O-hydroxyphenylacetate • • • • 31 Oxalate..... 31 Glycolic Acid. ........................... 31 Glyoxylic Acid......... 32 IV RESULTS..................................................... 34 V DISCUSSION.................................................. 46 VI SUMMARY..................................................... 52 BIBLIOGRAPHY............................... 53 v LIST OF TABLES Table Page 1. Urinary Excretion of Creatinine, Oxalate, Glyoxylate, Glycolate, Phenyllactate and O-Hydroxyphenyl- acetate...•..•................... ................ .. 35 2. Correlation Coefficients for 24 Hr. Excretion of Oxalate, Oxalate Precursors and Phenylketonuric Metabolites with Creatinine. .................. 39 3. Urinary Oxalate, Glyoxylate, Glycolate, Phenyllactate and O-Hydroxyphenylacetate Excreted Per Gram Creatinine...................................... 41 4. Correlation Coefficients for Oxalate Metabolites (Acid/g. Creatinine) and Phenylketonuric Metabolites (Acid/g. Creatinine)................................. 43 vi LIST OF FIGURES Figure No. Title Page 1. Metabolic Pathways of Oxalate Biosynthesis............. 12 2. Probable Pathway of L-Ascorbic Acid Metabolism to Form Oxalic Acid..... 14 3. Pathways of Serine Metabolism In Mammalian Systems 17 4. Pathways of Glyoxylate Metabolism................ 21 5. Disorders of Oxalate Metabolism in Man........ 23 6.- Linear Regression of Phenyllactate and Oxalate....... 44 7. Linear Regression of Phenyllactate and Glycolate 45 vii ABBREVIATIONS EDTA: Ethylenediaminetetraacetic acid Na: Sodium K: Potassium Li: Lithium Carbon 2 and Carbon 3 C2"C3: Cl40„: Carbon 14 (radioactive) and carbon dioxide 3-p-glycerate: 3-phosphoglycerate 2-P-glycerate: 2-phosphoglycerate 3-carbon: Beta carbon FH4: Tetrahydrofolate 5 10 N , N -methylene FH^: N**, methylene tetrahydrofolate Oxygen ° 2* Hydrogen peroxide H 2 ° 2 : NH3: Ammonia H20: Water a-keto: Alpha-keto a-keto-y-hydroxyglutamate Alpha-keto-gamma—hydroxyglutamate a-hydroxy-3-keto: Alpha-hydroxy-b e ta-keto NADH2: Nicotinamide adenine dinudeotlde, reduced Vitamin (Thiamine) Br V Vitamin Bg (Pyridoxine) viii • LDH: Lactic dehydrogenase NADPH: Nicotinamide adenine dinucleotide phosphate, reduced MgO: Magnesium oxide PLA: Phenyllactic acid OHPAA: O-hydroxyphenylacetic acid FeCl^: Ferric chloride viv CHAPTER I STATEMENT OF THE PROBLEM Kidney stones are a common problem in man. Since about 70 percent of these stones contain oxalate in quantities that cannot be accounted for by dietary oxalate absorption, the endogenous synthesis of oxalate is an Important factor in kidney stone formation. Consequently, inhibit ing the formation of oxalate should be an effective means of treating ■ patients having these kidney stones. Therapeutic measures have been directed primarily to decreasing oxalate synthesis. An early approach to this problem was to reduce the available glycine by restricting protein intake. Since glycine is a precursor of oxalate, reduction of this amino acid was should decrease the amount of oxalate formed. Another approach was used in patients with primary hyperoxaluria. This disease was observed to be associated with a vitamin Bg deficiency. Since this vitamin is a cofactor in the transamination of glyoxylate, an oxalate precursor, to glycine, administration of vitamin Bg should enhance the conversion of glyoxylate to glycine and reduce the oxidation of glyoxylate to oxalate. A third attempt has been directed at inhibiting the enzyme that oxidizes glycolaldehyde to the oxalate precursor, glycolic acid. Calcium carbamide has been used to depress oxalate synthesis by inhibiting aldehyde dehydrogenase. Many other approaches to kidney stone therapy 1 have been attempted but all of the present therapeutic approaches are inadequate. The present research is designed to investigate a new and promising approach to kidney stone therapy. This work is directed at inhibiting the major enzyme in oxalate synthesis, glycolic acid oxidase. DL- phenyllactate has been shown to inhibit this enzyme in "in vitro" and "in vivo" studies with rats. Oxalate synthesis was dramatically reduced in these rat systems. Since rat liver glycolic acid oxidase and human liver glycolic acid oxidase are very similar, DL-phenyllactate should decrease oxalate synthesis in man. Patients with phenylketonuria are known to produce high levels, of L-phenyllactate and should also have a decreased level of oxalate. In addition, these patients should have elevated levels of oxalate precursors