CHEMICAL SYNTHESIS, DISPOSITION AND METABOLISM OF DOPAMINE AND NORADRENALINE SULPHATES BARBARA ALEKSANDRA OSIKOWSKA A thesis submitted for the degree of Doctor of Philosophy to the University of London December 1983 - 2- ABSTRACT The existence of an enzymatic pathway which is capable of sulphating the catecholamine neurotransmitters has been known for over four decades. The importance of this metabolic pathway and its overall contribution to the enzymatic breakdown of these neurotransmitters has generally received less attention than deamination and JKmethylation. The aim of this work was to synthesise authentic dopamine and noradrenaline ^-sulphates, for use as standards in studies on the disposition and metabolism of these important products of dopamine and noradrenaline metabolism. 1. Three products resulted from chemical sulphonation of dopamine: dopamine 3-0-sulphate, dopamine 4-0-sulphate and dopamine 6- s u l p h o n i c acid. 2. Because all three products of dopamine sulphonation are isomeric, chemically similar organic acids and could not be distinguished by analytical techiques such as elemental analysis, ultraviolet spectroscopy and infrared spectroscopy, high performance liquid chromatography was employed for the separation and purification of these products, and nuclear magnetic resonance spectroscopy was considered to be the only technique powerful enough to distinguish between these isomers. 3. Noradrenaline 3- and 4-0-sulphates were isolated from a one-step synthetic reaction. They were separated, purified and characterised using techniques applied for the synthesis and separation of dopamine ^-sulphates. - 3- 4. The disposition of dopamine 3- and 4-0-sulphates was investigated in human urine before and following L-dopa administration and in multiple urine samples from a single subject. Both dopamine O-sulphates were present in urine under physiological conditions and after L-dopa; however, dopamine 3-0-sulphate was the major urinary conjugate in both circumstances. 5. The disposition of dopamine and noradrenaline 3- and 4-0-sulphates were investigated in rabbit brain, heart, kidney, liver, spleen and small intestine. Dopamine O-sulphates were not detected in any of the tissues with the method of analysis used; both noradrenaline O^-sulphate isomers were present in the above tissues in ratios depending on the animal and tissue investigated. In tissue homogenates of one rabbit, noradrenaline 4-0-sulphate was not detected. 6. Experiments investigating the in vitro metabolism of dopamine and noradrenaline O-sulphates showed that the aforementioned compounds were substrates for dopamine 3 -hydroxylase, the enzyme responsible for the conversion of dopamine to noradrenaline. Two products were formed following incubation of dopamine 3 -hydroxylase with dopamine O-sulphates, namely free noradrenaline and noradrenaline ()-sulphates. In the presence of active dopamine 3 -hydroxylase in the reaction mixture noradrenaline O-sulphates yielded free noradrenaline. The effects of a dopamine 3 -hydroxylase inhibitor on the above reactions were also investigated. 7. Observations in both experimental animals and man suggest that there may be important interindividual variations in the extent of 4-0- sulphation of these amines, the underlying basis for which cannot at present be explained. - 4- ACKNOWLEDGEMENTS I would like to thank all those whose help in this project made its completion possible. I am grateful to Professors P.S. Sever and W.S. Peart for allowing me to carry out this work in their departments. Professor P.S. Sever and Dr. J. Idle provided help, new ideas, continuous encouragement, criticism and friendship. Dr. F Swinbourne assisted in providing facilities for nuclear magnetic resonance spectroscopy and helped in the interpretation of the spectra. I would also like to express my thanks to the following: Mr. R. Mattin for his technical assistance, Miss G. Bartlett for typing the thesis and Drs. R. Unwin and S. Thom for proof-reading. I am grateful to all my friends at St. Mary's Hospital Medical School, past and present, whose friendship and encouragement allowed the satisfactory completion of this thesis. The work was generously supported by the Wellcome Trust. I reserve final thanks for my husband and my parents, whose support and understanding given freely through the duration of this work I could never fully return. - 5- CONTENTS P a g e ABBREVIATIONS 6 LIST OF TABLES 7 LIST OF FIGURES 9 CHAPTER: INTRODUCTION ...12 2 STUDIES ON THE CHEMICAL SULPHONATION OF DOPAMINE AND NORADRENALINE ... 52 3 DISPOSITION OF DOPAMINE O-SULPHATES IN HUMAN URINE 98 4 DISPOSITION OF NORADRENALINE 3- AND 4-0- SULPHATES IN RABBIT TISSUES ... 116 5 FURTHER METABOLISM OF DOPAMINE AND NORADRENALINE SULPHATES ... 137 6 DISCUSSION AND CONCLUDING REMARKS ... 166 BIBLIOGRAPHY ... 183 RELEVANT PUBLICATIONS ...212 ABBREVIATIONS COMT catechol ()-methyl transferase DA d o p a m i n e DA 3 - 0 - S 0 4 dopamine 3-0-sulphate DA 4-0-S0. dopamine 4-0-sulphate - 4 DBH d o p a m i n e 3 -hydroxylase DHP 6 3,4-dihydroxyphenylglycol d o p a d i hydroxyphenylalan i ne DOPAC dihydroxyphenylacetic acid h . p . 1.c. high performance liquid chromatography I.R. infrared MAO monoamine oxidase MHPG 3-methoxy-4-hydroxyphenylglycol NA noradrenaline N A 3 - 0 - S 0 4 noradrenaline 3-0-sulphate N A 4 - 0 - S 0 4 noradrenaline 4-0-sulphate n.m.r. nuclear magnetic resonance PAP 3'-phosphoadenosine-5'-phosphate PAPS 3 ‘-phosphoadenosine-5'-phosphosulphate PNMT phenylethanolamine-Tf-methyltransferase PP. inorganic phosphate l PST phenolsulphotransferase S.D. standard deviation U.V. ultraviolet VMA 4-hydroxy-3-methoxymandelic acid - 7- LIST OF TABLES 1.1 Plasma catecholamines (pg/ml) ...24 1.2 Percentage increment in plasma noradrenaline and adrenaline with various stimuli ... 28 2.1 Products of reactions of various sulphonating agents with dopamine at various temperatures ... 65 2.2 Products of sulphonation of noradrenaline with H^SO^ at various temperatures and time intervals 79 2.3 Products of reaction of chlorosulphonic acid with noradrenaline at various time intervals 83 2.4 Products of reaction of chlorosulphonic acid with noradrenaline: effect of varying reaction mixture composition ... 85 3.1 Dopamine 0-sulphate content in urine under physiological conditions ... 106 3.2 Dopamine ^-sulphate isomers in urine following oral administration of L-dopa (0.5 g) ... Ill 3.3 Recovery of orally administered L-dopa (0.5 g) in 24 h urine as dopamine 3- and 4-0-sulphates ... 112 4.1 Noradrenaline 3- and 4-0-sulphates in rabbit brain tissue ... 121 4.2 Noradrenaline 3- and 4-0-sulphates in rabbit heart tissue ... 123 4.3 Noradrenaline 3- and 4-0-sulphates in rabbit kidney tissue ... 125 4.4 Noradrenaline 3- and 4-0-sulphates in rabbit spleen tissue ...127 Noradrenaline 3- and 4-0-sulphates in rabbit liver tissue 129 Noradrenaline 3- and 4-0-sulphates in rabbit small intestine tissue 131 Dopamine 4-0-sulphate incubation with D$H at different time intervals 146 Dopamine 3-0-sulphate incubation with D$H at different time intervals 147 Kinetic study of D$H with dopamine as the s u b s t r a t e 147 Kinetic study of D$H with dopamine 4-0-sulphate as the substrate 149 Concentrations of free noradrenaline formed during incubation of dopamine 4-0-sulphate with D3H in the absence or presence of fusaric acid 151 Products formed by the incubation of dopamine 4-0- sulphate with Df3H 152 Kinetic study of DBH with dopamine 3-0-sulphate as the substrate 153 Concentrations of free noradrenaline formed during incubation of dopamine 3-0-sulphate with DBH in the absence or presence of fusaric acid 155 Products formed by the incubation of dopamine 3-0- sulphate with D^H 156 Noradrenaline 4-0-sulphate incubation in the p r e s e n c e of active D$H ... ... Noradrenaline 3-0-sulphate incubation in the presence of active D&H - 9- L1ST OF FIGURES 1.1 The biosynthesis of dopamine, noradrenaline and a d r e n a l i n e 15 1.2 Metabolic routes of dopamine, noradrenaline and a d r e n a l i n e 20 2.1 Products of chemical sulphonation of dopamine 67 2.2 H.p.l.c. chromatogram of products formed during chemical sulphonation of dopamine ... 68 2.3 250 MHz *H n.m.r. spectrum (aromatic region) of dopamine 6-sulphonic acid (product I, bottom), including expanded spectrum (top) ... 71 13 2.4 Off-resonance decoupled C n.m.r. spectrum of d o p a m i n e 6-sulphonic acid 72 2.5 250 MHz n.m.r. spectrum of dopamine 4-0- sulphate (product II) 75 2.6 250 MHz *H n.m.r. spectrum of dopamine 3-0- sulphate (product III) 77 2.7 Products of chemical sulphonation of noradrenaline 88 2.8 H.p.l.c. chromatogram of products formed during chemical sulphonation of noradrenaline by chlorosulphonic acid 89 2.9 250 MHz *H n.m.r. spectrum of noradrenaline 4-0- sulphate (product I) 91 2.10 250 MHz ^H n.m.r. spectrum of noradrenaline 3-0- sulphate (product II) 93 3.1 Standard curve for the estimation of dopamine 3- and 4-0-sulphates 105 H.p.l.c. chromatogram of dopamine £-sulphate s t a n d a r d s 107 H.p.l.c. chromatogram of normal urine containing both dopamine 0- s u l p h a t e s 108 H.p.l.c. chromatogram of urine after L-dopa administration, for estimation of dopamine 4-0- sulphate content 109 H.p.l.c. chromatogram of urine p r e s e n t e d in Fig. 3.4, appropriately i iluted for estimation of 3-0-sulphate content • • • • • • 110 H.p.l.c. chromatogram of authentic noradrenaline 3- and 4-0-sulphates • • • • • • 120 H.p.l.c.