University of Windsor Scholarship at UWindsor Electronic Theses and Dissertations Theses, Dissertations, and Major Papers 1-1-1970 Determination of total and conjugated glucuronic acid in serum and urine employing a modified naphthoresorcinol reagent. Angelo Mazzuchin University of Windsor Follow this and additional works at: https://scholar.uwindsor.ca/etd Recommended Citation Mazzuchin, Angelo, "Determination of total and conjugated glucuronic acid in serum and urine employing a modified naphthoresorcinol reagent." (1970). Electronic Theses and Dissertations. 6086. https://scholar.uwindsor.ca/etd/6086 This online database contains the full-text of PhD dissertations and Masters’ theses of University of Windsor students from 1954 forward. These documents are made available for personal study and research purposes only, in accordance with the Canadian Copyright Act and the Creative Commons license—CC BY-NC-ND (Attribution, Non-Commercial, No Derivative Works). 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Further reproduction prohibited without permission. / THIS DISSERTATION HAS BEEN EXAMINED AND APPROVED BY; 'A EXTERNAL EXAMINER 322757 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT A quantitative determination of free, conjugated, and total glucuronic acid in pure and biological systems (serum and urine) has been developed employing a modified naphthoresorcinol procedure. Glucose interference is eliminated selectively with glucose oxidase which permits the determination of glucuronic acid in the presence of high concentrations of glucose. Free glucuronic acid is decomposed with NaOH to a naphthoresorcinol-negative product in the determination of 0-glucuronides. The procedure utilizes a modified naphthoresorcinol reagent and a reduced volume of extracting agent. The method is six to eight times more sensitive than the previous naphthoresorcinol procedures. The reagent is stable for seven days and yields reproducible results. The method of standard addition is employed for the analysis, which results in increased accuracy at low concentrations in the determination of free and conjugated glucuronic acid. The accuracy of the method is + 5^ with a precision of + 0.35 119 per 3 ml in the optimum concentration range of 3.5 to 16.6 |ig per 3 ml. The total time required for ii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I l l a complete analysis on either serum or urine is four hours. A titrimetric determination of glucose, galactose, and xylose has been developed employing N-bromosuccinimide. The reactions are performed at room temperature for I5 minutes and the errors are less than + 6^ at the milligram level. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS Grateful acknowledgement is made to Professor R. J. Thibert, Ph.D., for his many constructive suggestions regarding the design and direction of this research project. I also wish to gratefully acknowledge Dr. R. J. Walton for his assistance in the execution of this project. I also wish to acknowledge the financial assistance provided by the Ontario Graduate Fellowships. IV Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DEDICATION To Dale, whose love and moral support inspired in me the confidence and patience required throughout graduate school. V Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS Page TITLE P A G E ................................... i ABSTRACT ........................................... ii ACKNOWLEDGEMENTS................................... iv DEDICATION................................... V TABLE OF CONTENTS ................... vi ABBREVIATIONS ..................................... ix LIST OF FI G U R E S .............................. x LIST OF TA B L E S ................................ xi Chapter I INTRODUCTION .......................... 1 II EXPERIMENTAL.......................... 12 A. Modified naphthoresorcinol procedure - Materials and M e t h o d s ....................... 12 1) Apparatus................. 12 2) Reagents ................. 12 3) Modified Naphthoresorcinol R e a g e n t ................... 13 4) study of Variables in the NR Procedure ............ 14 5) Spectral and Stability Study of MNR Product . l6 VI Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. v i l Chapter Page 6) MNR Procedure for Glucuronic Acid ...... l6 7) Stability of MNR Reagent. 17 8) Glucose Interference Study 17 9) Gluconic Acid Study .... 18 10) Determination of Glucuronic Acid in the Presence of G l u c o s e ................... 19 11) Determination of Conjugated Glucuronic Acid in the Presence of Free Glucuronic Acid...................... 19 12) Determination of Total and Conjugated Glucuronic Acid in Synthetic Mixtures by Method of Standard A d d i t i o n ................ 20 13) Glucuronide Study in Serum. 23 14) Dénaturation Studies ... 23 15) Analysis in Urine ........ 24 B. Titrimetric determination of Glucose, Galactose, and Xylose with N-bromosuccinimide - Materials and M e t h o d s ........ 25 1 ) Reagents 25 2) pH Studies .............. 26 3) Titrimetric Determination of G l u c o s e .............. 26 4) Titrimetric Determination of Galactose and Xylose . 27 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Vlll Chapter Page III RESULTS AND DISCUSSION ....... 29 A. Modified Naphthoresorcinol P r o c e d u r e .......... 29 B. Titrimetric Determination of Glucose, Galactose, and Xylose with N-Bromo succ inimide .... 71 IV SUMMARY AND CONCLUSION.. 78 BIBLIOGRAPHY.................... 80 VITA AUCTORIS ........................... 87 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABBREVIATIONS GA glucuronic acid MNR modified naphthoresorcinol NBS N-bromosuccinimide NR naphthoresorcinol Spike method of standard addition P-Gide phenolphthalein momo-jB-glucuronic acid IX Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES Figure Page 1. Absorption Spectra of Various NR Products............................. 35 2. Beer's Law Study of Various NR Procedures........................... 37 3. stability Study of MNR Reagent .... 40 4. Glucose Interference Study with Glucose Oxidase ..................... 45 5. Flow Diagram Outlining Procedure for a Single Sample ..................... 52 6. Determination of Total and Conjugated Glucuronic Acid in Synthetic Mixtures by the Method of Standard Addition . 57 7. Loss of P-Gide on Deproteinization . 59 8. Comparison of Beer's Law Curves Obtained Throughout this Study .... 70 X Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES Table Page I Study of Variables in the NR Procedure............................. 31 II Determination of D-glucuronic Acid and P-Gide in Various Mixtures .... 48 III Determination of Total and Conjugated Glucuronic Acid in Synthetic Mixtures . 53 IV Recovery Studies in Serum ............. 6l V Effect of Protein Concentration on S l o p e s ............................... 64 VI Determination of Glucuronic Acid and Glucuronide in Serum . ............ 66 VII Determination of Glucuronic Acid and Glucuronide in Urine ................. 68 VIII pH Range for Maximum -Recovery......... 75 IX Recovery Studies ........... 77 XX Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. , CHAPTER I INTRODUCTION The uronic acids are generally defined as aldose derivatives retaining the