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In all cases we have filmed the best available copy. University Microfilms International 300 N. ZEEB RD., ANN ARBOR, Ml 48106 8121836 N g , K w o kei Ja c o b GAS CHROMATOGRAPHIC-MASS SPECTROMETRIC PROFILING OF ORGANIC ACIDS OF HUMAN AMNIOTIC FLUID The Ohio Stale University PH.D. 1981 University Microfilms Internationai300 N. Zeeb Road, Ann Arbor, M I 48106 Copyright 1981 by Ng, Kwokei Jacob All Rights Reserved GAS CHROMATOGRAPHIC— MASS SPECTROMETRIC PROFILING OF ORGANIC ACIDS OF HUMAN AMNIOTIC FLUID DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University by Kwokei Jacob Ng •k ' k ' k & ' k The Ohio State University 1981 Approved by Reading Committee: Brian D. Andresen , ; ■ / // / Joseph R. Bianchine /r"~ A 1'- A I Richard H. Fertel Advisor Philip B. Hollander Department of Pharmacology College of Medicine ACKNOWLEDGMENTS Since the beginning of this project, the constant support, enthusiasm, advice and assistance in many ways from Dr. Bianchine and Dr. Andresen have enable me to accomplish the present work. It is also a great blessing and pleasure to have the opportunity to serve apprenticeship with Dr. Andresen. He has spent many long late hours teaching me patiently the fine arts of gas chromatography and mass spectrometry, showing me how to apply the scientific method for clinical assays. The design of the helium preheater would not have been completed without his expertise. I would like to thank members of the Reading Committee and Graduate School Representative, Dr. Burkman for their time in reading my dissertation and suggestions. I would also like to thank Drs. Zuspan, 0'Shaunghessy, lams, Rayburn, Freeman and Stemple for their enthusiasm and supply of amniotic fluid samples and other biological specimens. Special thanks are due to Dr. Fertel who has been very helpful in many ways. I would also like to thank Dr. Tejwani, Dr. Tjoe and Dr. Gerber for their interest in my research work. Mrs. Judy Lubbers has been very kind to share with me her capillary columns and experiences in handling them. The friendship of fellow graduate students is a cherisable memory. Lastly, but not the least, the love and care of my family and my wife, Shirley, have made the present effort the more worthwhile. ii VITA May 1, 1943......................... Born - Canton, China 1963................................ Northcote Teachers' Training College, Hong Kong 1963-1968.......................... School teacher, St. Louis School Hong Kong 1969-1973.......................... B.S., Department of Chemistry, California State University, Northridge 1973-1976........................... M.S., Departments of Biochemistry & Pharmacognosy, Columbus, Ohio State University 1977............................... Research Assistant I,II Department of Radiology, Columbus, Ohio 1978-1979........................... Research Associate, Department of Radiology, Columbus, Ohio State University 1980-1981........................... Ph. D., Department of Pharmacology, Columbus, Ohio State University PUBLICATIONS "Longevity, Stability and DNA Repair" by Hart, R.W., D'Ambrosio, S.M., Ng, K.J., and Modak, S.P. Mechanism of Ageing and Development, 9, 203 (1979). "Helium Preheater for the Open-split Interface" by B.D. Andresen, K.J. Ng, J. Wu and J.R. Bianchine. Biomedical Mass Spectrometry (in print) FIELDS OF STUDY Major Field: Pharmacology Others: Biochemistry. Drs. Serif, Barber, Gross, Behrman Ives, Royer Radioisotope Methodology. Drs. Malspeis and Feller Organic Chemistry. Drs. Newman, Paquette, Swenton Carbohydrate Chemistry. Dr. Horton Natural Product Isolation Techniques. Dr. Doskotch Medical Physiology. Drs. Grossie, Lipsky, Weiss, Dujardin Biochemical Methods. Drs. Ives, Behrman, Gross Methods in Organic Chemistry. Dr. Ouellette Gas Chromatography and Mass Spectrometry. Drs. Andresen, Hammar, Wong TABLE OF CONTENTS Page ACKNOWLEDGMENTS................... ii VITA........................................................ iii LIST OF TABLES.............................................. vii LIST OF FIGURES............................................. viii I. INTRODUCTION......................................... 1 A. Introduction..................................... 1 B. Funtions of Amniotic fluid....................... 2 C. The origin of amniotic fluid.............. 2 D. Amniocentesis................................... 5 E. Prenatal diagnosis by amniocentesis.............. 8 F. Components of amniotic fluids.................... 16 1. Cellular..................................... 16 2. Macromolecules............................... 16 3. Organic acids........................... 21 4. Organic bases................................ 44 5. Hormones..................................... 46 6. Drugs........................................ 48 G. GC-MS Methodology................................ 50 H. Objectives of present investigation.............. 56 II. Materials and Methods A. Biological samples............................... 57 v Page B. Extraction of amniotic f l u i d . ..... 58 C. Derivatization of acidic fractions...................... 62 D. Gas chromatography-mass spectrometry analysis of samples................................................. 65 III. Results A. Stabilization of baseline with open-split interface heater............................ 70 B. Cleanliness of instruments and purity of reagents........ 74 C. Capillary GC-MS profiles of acidic fractions of 1. amniotic fluid of early pregnancy................... 82 2. amniotic fluid of late pregnancy.................... 86 3. amniotic fluid of pregnancy with open tubular neural defects...................................... 88 4. amniotic fluid of pregnancies with Rh sensitization.. 89 5. amniotic fluid of pregnancy from smoking mother 92 6. amniotic fluid of pregnancy with Delalutin treatment.. 94 7. amniotic fluid of pregnancy with hydrops............ 96 8. amniotic fluid of diabetic pregnancy................ 97 9. amniotic fluid from pregnancy of Down syndrome and gastrointestinal block............................ 99 10. amniotic fluid from drug addicted mother............. 100 D. Capillary GC-MS profile of acidic fraction of serum...... 101 E. Capillary GC-MS profile of acidic fraction of ovarian cyst fluid........................................ 103 F. Comparison of organic acids identified in this study with those identified by GC-MS profiling of amniotic fluid in the literature.......................................... 105 IV. Conclusions................................................. 109 V. Bibliography...................... 115 VI. Appendices Appendix A. Compilation of capillary GC-MS profiles of basic fractions of amniotic fluid................... 124 Appendix B. Compilation of mass spectra................ 139 vi LIST OF TABLES Examples of genetic disorders and the corresponding diagnostic tests...................................... 9 Proteins, peptides and enzymes in amniotic fluid...... 20 Lists of organic acids in amniotic fluid identified by GC-MS profiling in literature...................... 30 Steroidal hormones and metabolites in amniotic fluid.. 46 Non-steroidal hormones in amniotic fluid............. 47 Drugs and exogenous chemicals detected in amniotic fluid.................. ................ .............. 48 List of capillary GC-MS analyzed samples and patient informations.......................................... 80 List of identified compounds with the corresponding peak numbers.......................................... 81 Comparison of organic acids identified in the literature with those of current