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. chromatogram of brain homogenate

(ra b b i t 4) • • • • • • 122

H.p.l.c. chromatogram of heart homogenate

(ra b b i t 4) • « • • • • 124

H.p.l.c. chromatogram of kidney homogenate

( r abbit 4) • • • • • • 126

H.p.l.c. chromatogram of spleen homogenate

(ra b b i t 4) 128

H.p.l.c. chromatogram of liver homogenate

(ra b b i t 4) 130

H.p.l.c. chromatogram of small intestine homogenate (rabbit 4) 132

H.p.l.c. chromatogram of small intestine homogenate following hydrolysis (rabbit 4) ... 134

Radioenzymatic (PNMT) assay for the determination of free noradrenaline 143 -11-

5.2 D$H kinetic study with dopamine as the substrate ... 148

5.3 D£H kinetic study with dopamine 4-0-sulphate as

the s u b s t r a t e ...... 150

5.4 DgH kinetic study with dopamine 3-0-sulphate as

the substrate ...... 154

5.5 A possible mechanism for the formation of free

noradrenaline from dopamine 3-0-sulphate ...... 163

5.6 A possible mechanism for the formation of

noradrenaline 3-0-sulphate from dopamine 3-0-

sulphate ...... 164 -12-

C H A P T E R 1

INTRODUCTION

1.1 General Introduction 14

1.2 Biosynthesis of catecholamines 14

1.3 Enzymes involved in catecholamine formation 16

1.4 Storage of noradrenaline in sympathetic nerves 17

1.5 Release of catecholamines from the adrenal ... 18

1.6 Metabolic fate of noradrenaline 18

1.7 Routes of metabolism of catecholamines 19

1.8 Regulation of catecholamine release from nerve

endings 21

1.9 The dopaminergic receptor 22

1.10 Catecholamines in biological fluids 23

1.10.1 Catecholamines in urine 25

1.11 Factors affecting noradrenaline levels in plasma 25

1.11.1 Constitutional factors - age 25

1.11.2 Constitutional factors - sex 26

1.11.3 Sampling procedure 26

1.11.4 Postural changes: standing and tilt 26

1.11.5 Exercise: isometric and dynamic 27

1.12 Plasma catecholamines as an index of sympathoadrenal

a c t i v i t y 27

1.13 Pathological factors affecting catecholamine levels 28

1.14 Importance of sulphoconjugation in catecholamine

m e t a b o l i s m 30 - 13-

1.15 Sulphate transfer reaction 30

1.16 Biological functions of sulphate activation 32

1.17 Arylsulphate esters 33

1.18 Phenol sulphotransferase 34

1.18.1 Substrate spectrum 34

1.19 The availability of inorganic sulphate for sulphate

c o n j u g a t i o n in vivo 35

1.20 Sulphation of catecholamines in vivo and in vitro 36

1.21 Site of sulphonation and role of sulphation in

the CNS 42

1.22 The possible role of sulphoconjugates 45

1.23 Catecholamine sulphoconjugates in pathological

c o n d i t i o n s 48 - 14-

C h a p t e r 1

1.1 General Introduction.

The biological significance of catecholamines has been recognised for over eighty years. The history of catecholamine research is replete with discoveries that have opened broad areas of general importance in biology and medicine.

The pressor response to extracts of adrenal tissue was first noted by

Oliver & Schaefer in 1895, and shortly thereafter the active substance was identified as adrenaline by Abel & Crawford (1897). It was, however, nearly half a century later that the adrenaline-like neurotransmitter substance liberated on stimulation of sympathetic nerves was positively identified as noradrenaline following the work of von Euler (1948) and Peart (1949).

1.2 Biosynthesis of Catecholamines.

The similarities in structure of adrenaline and the essential amino acid, tyrosine, were noted soon after the structure of adrenaline had been confirmed. In 1911, Funk synthesised 3,4-dihydroxyphenylalanine

(dopa) because it was believed that this compound was an intermediate in the formation of adrenaline from tyrosine. Holtz (1939) discovered the enzyme, dopa decarboxylase, which converts the hydroxylated amino acid to dopamine (Fig. 1.1). The observation of Blaschko (1942), that dopa decarboxylase did not decarboxyl ate_N-methyldopa led him to propose that the biosynthetic pathway for catecholamines involved conversion of dopa to dopamine which was then converted to noradrenaline and subsequently

N-methylated. This was confirmed in vitro by Goodal & Kirshner (1957) and this biosynthetic pathway (Fig. 1.1) is now generally accepted as PHENYLALANINE Phenylalanine hydroxylase w (liver) TYROSINE Tyrosine hydroxylase ’f (nerve) DOPA L-Aromatic amino acid decarboxylase 'f (nerve) DOPAMINE Dopamine- Brhydroxylase u (nerve) NORADRENALINE Phenylethanolamine N-methvltransferase ,, (adrenal medulla & ADRENALINE brain>

ng. 1.1 The biosynthesis of dopamine, noradrenaline and adrenaline. (Blaschko, 1973). - 16-

the major route of formation of the three catecholamines, dopamine, noradrenaline and adrenaline.

1.3 Enzymes involved in catecholamine formation.

Catecholamine biosynthetic pathways have been demonstrated in adrenal medullary tissue, in sympathetic nerves and in the brain. The biosynthetic enzymes have also been demonstrated in these tissues, with the exception of phenylethanolamine-f[-methyltransferase (PNMT), which is only present in the adrenal medulla and brain.

Tyrosine hydroxylase is responsible for conversion of tyrosine to dopa (Fig. 1.1). It was partially purified and characterised by Nagatsu et a K (1964). Tyrosine hydroxylase is relatively specific for the substrate L-tyrosine and the hydroxylation of tyrosine is the rate limiting step in catecholamine biosynthesis (Spector et aj_., 1963;

Levitt et ^1_., 1965).

Aromatic L-amino acid decarboxylase has low substrate specificity and will decarboxylate many aromatic L-amino acids (Lovenberg et ^1_., 1962).

The product of dopa decarboxylation, dopamine, is not only a precursor for other catecholamines, but is itself a neurotransmitter

(Hornykiewicz, 1966). Dopamine B -hydroxylase is the copper containing enzyme present in neuronal granules and converts dopamine to noradrenaline (Levin et al^., 1960). Its distribution throughout the peripheral and central nervous system correlates with that of noradrenaline. This enzyme is capable of hydroxylating many phenylethyl and phenyl isopropyl amines (Crevel ing et a1_., 1962; Goldstein &

Contrera, 1962), and is of particular interest, since it is released from nerve endings together with noradrenaline on activation of sympathetic nerves. - 17-

In certain experimental studies the amounts of dopamine- 8-hydroxylase released are quantitatively related to sympathetic nerve activity.

However, there is little convincing data to show that the amounts of the enzyme which finally reach the circulation parallel overall sympathetic tone.

Phenyl ethanol amine-^[_-methyl transferase (PNMT) converts noradrenaline to adrenaline utilising a methyl group derived from

S-adenosyl methionine (Axelrod, 1962). The activity of this enzyme is at least in part modulated by corticosteroids and its activity may be increased in response to repeated stress. There is evidence for regulation of the synthesis of the catecholamines by end product inhibition (Fuller & Hunt, 1967). However, as will be discussed later there are many other mechanisms which are involved in the regulation of noradrenaline release and subsequent activation of the adrenergic receptors.

1.4 Storage of noradrenaline in sympathetic nerves.

Noradrenaline is stored in sympathetic nerve terminals and brain in a granular fraction (Wolfe e t ^ L , 1962). Ultrastructural, autoradiographic and histochemical studies have revealed the existence of dense cored vesicles containing not only noradrenaline and dopamine

- 8-hydroxylase, but also ATP (with which noradrenaline forms high molecular weight aggregates), chromagranin A, magnesium ions and cytochrome b561 (Kirshner, 1974; Winkler, 1976). Noradrenaline is bound to the granular membrane which contains an ATP-dependent active uptake process that counteracts the spontaneous loss of noradrenaline.

As a result of the granular storage, noradrenaline is protected from metabolic degradation by monoamine oxidase. However, subcellular fractionation studies also indicate the presence of some free - 18-

noradrenaline in an "extragranular" compartment. In the adrenal medulla, noradrenaline formed within medullary granules passes out into the cytosol where it is N-methylated to adrenaline. The latter is then taken up into the granules and stored. Dopamine that is taken up into the granules is converted to noradrenaline and stored as such.

Consequent upon activation of sympathetic nerves, noradrenaline and other granular contents are extruded from nerve endings by the process of exocytosis (De Potter et al_., 1969; Geffen et _al_., 1969; Gewirtz &

Kopin, 1970).

1.5 Release of catecholamines from the adrenal.

Activation of the splanchnic nerves liberates acetyl choline from nerve endings within the medulla. Acetyl choline increases the permeability of the chromaffin cells to calcium ions which are necessry for the release of the neurotransmitter (Burn & Gibbons, 1965; Boullin,

1967) and the granular contents including adrenaline are liberated again by an exocytotic process. Noradrenaline synthesis is activated almost

immediately after exocytosis, with newly synthesized noradrenaline released preferentially (Kopin, 1977).

1.6 Metabolic fate of noradrenaline.

Following release of noradrenaline from sympathetic nerve endings, the fate of the neurotransmitter is complex. It is important to understand the events that occur before any interpretation may be put upon changes in neurotransmitter levels occurring in tissues and biological fluids. Activation of vascular post-synaptic alpha^ receptors, or cardiac beta^ adrenoceptors, mediate vasoconstriction, chronotropic and inotropic responses respectively. Most of the noradrenaline released is immediately taken up into the nerve endings by - 19-

active transport (uptake 1) and restored in vesicles (Dengler et al.,

1962; Iversen, 1963; Burgen & Iversen, 1965). In the process, some noradrenaline may be metabolised by monoamine oxidase (Kopin &

Gordon, 1963). Alternatively, a fraction of the neurotransmitter may be removed by an extraneuronal active transport system (uptake 2). This process plays a less important role in removing noradrenaline from the synaptic cleft. This high capacity, low affinity system may be important for removal of catecholamines from the circulation (Iversen,

1965). Extraneuronal tissue uptake is followed by metabolism of catecholamines by fr-methylation (Kopin & Gordon, 1963).

1.7 Routes of metabolism of catecholamines.

Armstrong et a^. (1957) were the first to show that 3-methoxy-4- hydroxymandelic acid (vanillylmandelic acid, VMA) was a urinary metabolite of noradrenaline (Fig. 1.2). Degradation of catecholamines is a function of deamination (monoamine oxidase, MAO) and JT-methylation

(catechol-0-methyl transferase, COMT). MAO converts many biogenic amines including dopamine, noradrenaline, adrenaline and serotonin by oxidative deamination to the corresponding aldehydes which are then further oxidised to the corresponding acid or reduced to the glycol metabolite

(Fig. 1.2). Mitochondrial MAO is responsible for the breakdown of extragranular stores of catecholamines.

COMT is widely distributed and present in nearly all tissues. This enzyme requires magnesium ions and^S-adenosyl methionine as a methyl donor (Axelrod & Tomchick, 1958). It can _0_-methyl ate a wide variety of catechols and is found in highest levels in the liver and kidney, although it is present in almost all tissues.

The importance of COMT in terminating the effects of administered -20-

OH

Fig. 1.2 Metabolic routes of dopamine, noradrenaline and adrenaline.

= H OH OH R 1 = H H c h 3 R2 1 Dopamine Noradrenaline Adrenaline 2 3-()-methyl dopamine Normetanephr ine Metanephrine 3 Dihydroxyphenyl ethanol Dihydroxyphenyl ethylglycol 4 Dihydroxyphenyl acetic acid Dihydroxymandelic acid 5 3-methoxy-4 hydroxyphenyl 3-methoxy-4 hydroxyphenyl ethanol ethylglycol 6 Homovanillic acid 3-methoxy-4 hydroxymandelic acid

(Kopin, 1977). -21-

catecholamines was shown in both experimental animals (Axelrod et al.,

1958) and in man (La Brosse et al_., 1958): metanephrine was a major urinary metabolite of administered adrenaline. It has been shown that following administration of tritium-labelled adrenaline in rats (Kopin et jLL, 1961) and in human subjects (Kopin, 1960; La Brosse et al.,

1961), over two-thirds of the catecholamine was ^-methylated before being deaminated, and less than one fourth was deaminated directly.

However, it should be re-emphasized that metabolism of noradrenaline is not the essential mechanism by which the released catecholamine is inactivated.

In the periphery the major end product of endogenous catecholamine metabolism appears to be vanillylmandelic acid (VMA) (La Brosse et al.,

1961) whereas in brain 3'-methoxy-4'-hydroxy phenylglycol (MHPG) (Ebert

& Kopin, 1975) is predominantly formed.

The metabolites of dopamine found in urine are homovanillic acid and methoxytyramine. The metanephrines, acidic and alcohol metabolites are excreted in urine predominantly as their sulphate conjugates.

1.8 Regulation of catecholamine release from nerve endings.

Adrenergic agents interact with cell surface recognition sites

(receptors) and this interaction causes biochemical changes within the plasma membrane of the target cell. As a result, membrane-bound enzyme systems are stimulated and intracellular mediators for adrenergic processes are generated.

It is now well known that regulatory mechanisms participate in the control of noradrenaline release from nerve endings. Several receptors are present on the prejunctional neurone. Studies with various alpha (a) agonist and antagonist compounds indicate a subclass -22-

of alpha receptors (o^K stimulation of which leads to an inhibition of noradrenaline release from nerve terminals.

Presynaptic beta (B) receptors at low concentrations enhance noradrenaline release from nerve terminals. It is postulated that at low frequency stimulation when the amounts of neurotransmitter release are small, the presynaptic 3-mediated facilitation of noradrenaline release predominates, whereas with high rates of stimulation, a 2 receptor mediated inhibition of noradrenaline release predominates.

Other prejunctional receptors may also be involved in the local regulation of noradrenaline release.

Post-synaptic tissues may influence noradrenaline release by the synthesis and release of prostaglandins, which in turn inhibit exocytosis of noradrenaline danger, 1980).

1.9 The dopaminergic receptor.

Dopamine has been known for many years to be a weak a and

3 -adrenergic agonist. The first evidence that dopamine caused

vasodilatation by an unusual mechanism was reported by McDonald et al.

(1963, 1964). It was noted that this response was not blocked by

conventional a and B-adrenergic antagonists, such as phenoxybenzamine

and propranolol, and this observation suggested that dopamine was acting

on a unique vascular receptor. Vasodilatation in the renal, mesenteric

and cerebral vascular beds in response to dopamine has been subsequently

studied in detail and a potency series for drugs capable of eliciting

this response has been derived (Goldberg, 1975).

Dopamine is believed also to function as a neurotransmitter in

certain areas of the nervous system. Dopaminergic pathways connect the

substantia nigra to the caudate nucleus and putamen and degeneration of - 23-

these tracts is associated with Parkinson's disease (Iversen, 1975). A dopaminergic system was also demonstrated in the rabbit superior cervical sympathetic ganglia (Greengard, 1976). It was also shown that dopamine-sensitive adenylate cyclase existed in various areas of the brain including the basal ganglia, and a similar distribution of dopamine containing neurones has been noted (Iversen, 1975).

1.10 Catecholamines in biological fluids.

Several attempts have been made to determine the amounts of the mediator of sympathetic effector responses as an alternative method for evaluating sympathetic activity. However, progress in this field has been frustrated by the lack of suitable methods to determine the small amounts of catecholamines that are present in human plasma.

The early methods lacked sensitivity and specificity. Bioassay, although sensitive, suffered from the lack of absolute specificity for individual catecholamines. The early chemical methods which were colorimetric lacked both sensitivity and specificity. The development of fluorimetric assays was a great advance on previous methods.

However, at the lower concentrations of noradrenaline, such as those found in human plasma, errors were magnified and the reproducibility of assays was a major problem.

Engelman ^t aj_. (1968) published details of an elaborate double­ isotope-derivative radioenzymatic assay for catecholamines, which has since been modified to permit the isolation and quantitation of individual catecholamines with a remarkable degree of specificity and sensitivity (Engelman & Portnoy, 1970). Henry and co-workers (1975) reported details of another radioenzymatic procedure which was specific for noradrenaline. - 24-

The low levels of catecholamines in plasma can be explained by the rapid rate of metabolism and uptake into sympathetic nerve endings.

Adrenal medullae contribute little to the circulating pool of noradrenaline. Adrenaline is present in plasma in even smaller amounts

than noradrenaline and is derived solely from adrenal glands. Dopamine

is generally not detectable in plasma or on the border of the detection

limits of sensitive radioenzymatic assay (Da Prada & Zurcher, 1976).

According to Johnson et (1980), approximately 80% of plasma

noradrenaline, 75-80% of adrenaline and 99% of dopamine are present as

sulphoconjugates. Similar observations were made by Kuchel et al.

(1982).

The levels of catecholamines in plasma estimated by different methods

are presented in Table 1.1.

Table 1.1. Plasma catecholamines (pg/ml).

R e f e r e n c e Control subjects No. of NA concentration subjects (pg/ml ± S.D.)

Engel man et al^., 1970 (C) 240 + 62 32

Louis et ^1_., 1973 (N) 220 ± ? 14

Pedersen & Christensen, 1975 (N) 250 + ? 32

Lake et aj_., 1977 (N) 304 + 159 84

Sever et al_., 1977 (N) 403 + 184 59

Sever , 1978 (N) 372 + 171 48

C = catecholamines

N = noradrenaline

? = not given - 25-

1.10.1 Catecholamines in urine.

Free and conjugated catecholamines and their metabolites are excreted in urine. Measurement of the end products of catecholamine metabolism has been attempted by many groups but, with the exception of phaeochromocytoma, no consistent changes have been found in, for example, patients with hypertension. Although the kidney plays a part in the clearance of catecholamines and their metabolites from the circulation, the actual content of free amines and metabolites in urine is in part derived from nerve endings within renal tissue. Gross changes in sympathetic activity may be reflected by increments in the excretion of urinary catecholamines, but it would be naive to assume that in the presence of a change in catecholamine disposition in one or more tissues or organs, this would be reflected accurately by an examination of excretory products in urine.

1.11 Factors affecting noradrenaline levels in plasma.

1.11.1 Constitutional factors - Age.

It has been a matter of controversy as to whether or not plasma noradrenaline increases with age in normal subjects. The positive correlation between plasma noradrenaline and age was found by some

(Pedersen & Christensen, 1975; Ziegler et al_., 1976; Sever et al.,

1977; Franco-Morsel 1 i et al_., 1977; MiuraetaK, 1978; Kiowski et al., 1979; Bertel et al_., 1980; Esler et a^., 1981), but denied by others (de Quattro & Chan, 1972; de Champlain & Cousineau, 1977;

Brecht & Schoeppe, 1978). It has been suggested that the lack of older subjects in some earlier studies may have influenced their authors' conclusions (Sever, 1978). It is now generally believed that there is a

relationship between noradrenaline and age in normal subjects when a - 26-

broader range of individuals are examined (Goldstein, 1981). Esler et al. (1981) examined the rate at which tritiated noradrenaline spilled over into plasma and was cleared. This spillover rate remained fairly constant in subjects between the ages of 20 and 70; the increase in plasma noradrenaline that they observed with age was therefore due to the diminished clearance of noradrenaline.

1.11.2 Constitutional factors - Sex.

There are no differences in plasma noradrenaline concentration between males and females when appropriately age matched, according to several groups of investigators (Ziegler et al_., 1976; Sever et al.,

1978; Brecht & Schoeppe, 1978; Ibsen et al_., 1979), whereas others

(Jones et a[., 1978) found higher values in females.

1.11.3 Sampling procedure.

The site from which the blood is obtained is not critical (Watson et aT., 1979; Vecht et aj_., 1981). Venepuncture should not give higher levels of plasma noradrenaline than withdrawing blood from an indwelling catheter (Campese et^ a/L, 1977; Jones et aJ_., 1978; Robertson et al.,

1979).

As diurnal rhythms may affect plasma noradrenaline concentrations, experiments should be performed at similar times of the day (Sever,

1978).

1.11.4 Postural changes: Standing and Tilt.

Change of posture from recumbent to standing causes an increase in

plasma noradrenaline levels, especially in the older subjects (Sever et

al., 1977). - 27-

Gradual tilting also results in an increase of plasma noradrenaline levels (Rosenthal et al_., 1978).

1.11.5 Exercise: Isometric and Dynamic.

Mental arithmetic and isometric exercise do not always produce elevations in plasma noradrenaline levels (Floras, 1981).

Dynamic exercise such as bicycling results in an increase of plasma noradrenaline levels by 75% (Floras, 1981).

1.12 Plasma catecholamines as an index of sympathoadrenal activity.

With the advent of sophisticated radioenzymatic methods, plasma catecholamine measurements have been proposed as biochemical determinants of sympathoadrenal activity.

Because adrenaline is released directly into the intramedullary venous sinuses and thence to the systemic circulation, it may be postulated that circulating adrenaline is a better determinant of adrenal medullary activity than circulating noradrenaline is of sympathetic neuronal activity. The interpretation of the validity of circulating catecholamines as indices of sympathoadrenal function is complicated by a host of factors that include uptake into blood vessel walls, protein binding (40-60% protein bound) metabolism, including conjugation and clearance by the kidney, liver and other organs.

Factors contributing to the increase of noradrenaline and adrenaline in plasma are summarised in Table 1.2. - 28-

Table 1.2. Percentage increment in plasma noradrenaline and

adrenaline with various stimuli (Sever, 1982).

Stimuli Noradrenaline Adrenaline

Mental arithmetic 0 ?

V a l s a l v a 0 50

Isometric effort 0 - 30 60 - 70

Cold pressor response 60 - 70 100

Orthostasis 80 - 200 0

Dynamic exercise 200 - 400 200 - 300

1.13 Pathological factors affecting catecholamine levels.

There are many pathological conditions in which catecholamines and

their metabolites have a role including thyrotoxicosis, myxoedema,

congestive heart failure, post-myocardial infarction, cardiac

arrhythmias, idiopathic orthostatic hypotension, hypertension and

hepatic encephalopathy (Axelrod & Weinshilbaum, 1972).

An increase in the concentration of plasma catecholamines has

been noted in phaeochromocytoma (Engelman, 1977; Page & Copeland, 1968;

Louis et al_., 1972). The measurement of catecholamines and/or their

metabolites in urine and tissue in the above condition and in

neuroblastoma is necessary in the establishment of the diagnosis (Leung

& Griffith, 1974). In contrast, in subjects with autonomic

deficiencies, resting levels of plasma noradrenaline are low and changes

in the concentration of this neurotransmitter are not observed on - 29-

postural stimulation (Osikowska & Sever, 1976; Ziegler ^t al_., 1977).

Marked reductions in catecholamine levels are also found in patients with cervical cord transection (Mathias et aj_., 1976), hypothyroidism, diabetes with autonomic dysfunction (Christensen, 1972a,b), primary orthostatic hypotension (Ziegler et a K , 1977) and in patients treated with sympathetic blocking drugs such as clonidine (Wing et al_., 1977), ganglion blocking drugs (Louis e t ^ L , 1973, 1974) and adrenergic blocking drugs (Sever et al_., 1978).

The levels of plasma catecholamines in hypertensive subjects are the subject of much controversy. Several investigators (Engelman et a!.,

1970; Lutold et aj_., 1976) reported that total plasma catecholamines were increased in essential hypertension. However, these studies were criticised for the use of unsuitable controls (Sever, 1978). Others suggested that plasma noradrenaline concentrations were similar in normal and hypertensive subjects when adjusted for age (Lake et a!.,

1977), but were also criticised for having insufficient age-matched controls (Campese et al_., 1977). However, despite the controversial findings mentioned above, one interesting fact emerged from more recent studies, namely the normal rise in noradrenaline in plasma with age seen in normotensive subjects is not seen in hypertensives.

The results presented thus far indicate considerable differences of opinion between groups of investigators as to the significance of circulating catecholamines as an index of sympathoadrenal activity, factors affecting levels of these amines, the role that they play in essential hypertension and other pathological conditions. This is thought to be because plasma catecholamine concentrations are the tangible expression of several equilibria. They are the sum of amounts - 30-

added by the intersynaptic spillover, and amounts withdrawn by the mechanisms involved in their metabolic clearance.

1.14 Importance of sulphoconjugation in catecholamine metabolism.

It is not therefore surprising that recently several investigators

turned their attention to catecholamine metabolites that are present in

biological fluids and tissues in much higher concentrations than free

catecholamines, namely catecholamine ()-sulphate conjugates (Kuchel et.

al.. 1978; Johnson et al_., 1980; Joyce et ak, 1982). The existence

of an enzymatic pathway which is capable of sulphating the catecholamine

neurotransmitters has been known for over four decades. However, the

importance of this pathway and its overall contribution to the enzymatic

breakdown of these neurotransmitters have generally received less

attention. Scrutiny of the literature leads to the conclusion that only

deamination by monoaminooxidase (MAO) and ^-methylation by catechol

0-methyltransferase (COMT) play a predominant role in the enzymatic

inactivation of catecholamines. However, it has recently become

apparent that the enzyme, phenol sulphotransferase (PST), has a

relatively high affinity for the catecholamine neurotransitters dopamine

and noradrenaline.

1.15 Sulphate transfer reaction.

Before describing any of the specific studies regarding sulphate

conjugation of catecholamines, it is necessary to discuss the nature and

diversity of sulphation reactions that occur in mammalian systems. A

wide variety of substances, both endogenous and exogenous, are capable

of undergoing conjugation with sulphate. These include a variety of

lipids, steroids, phenolic drugs, alcohols, ascorbate and calciferol,

as well as the catecholamines and their metabolites (Roy, 1981). - 31-

The sulphate donor for all these reactions is 3'-phosphoadenosine-5'- phosphosulphate (PAPS), which is one of the two principal activated forms of sulphate. The other activated form of sulphate is 5 ‘- adenylsulphate (APS). PAPS and APS were first described by Hilz &

Lipmann (1955) and the discovery of these activated forms of sulphate followed a series of studies by various other investigators (Bernstein &

McGilvery, 1952a,b; De Meio et a K , 1953; Segal, 1955) on the sulphoconjugation of phenols in rat liver.

The reactions involved in sulphate activation are shown below (APS =

5'-adenylsulphate or adenosine-5 1-phosphosulphate: PAPS = 3'-phospho-

5'-adenylsulphate, or 3'-phosphoadenosine-5 1-phosphosulphate).

ATP sulphurylase ( ) + ATP ◄ ------5------APS + P P i G° + 11,000 cal/mol 1 S 0 4

APS ki n a s e (2) APS + ATP ◄ ------PAPS + ADP G° - 6,000 cal/mol

Pyrophosphatase n (3) PP. + h 20 < f c = ------► 2P. G° - 5,000 cal/mol l 1

2- S u m s o / + 2ATP < ----- p PAPS + ADP + 2Pi G° 0 cal/mol

PAPS + DA - P S T > PAP + DA 0- so4

The sulphate activating enzymes, ATP sulphurylase (ATP: sulphate

adenylyl transferase, E.C. 2.7.7.4) and ATP kinase (ATP:

adenylylsulphate-3'-phosphotransferase, E.C. 2.7.1.2.5) catalyse the

first two steps in the incorporation of inorganic sulphate into

biological molecules (Gregory & Lipmann, 1957; Robbins & Lipmann, 1957,

1958; Robbins, 1958).

The equilibrium of reaction (1) lies far to the left. Nevertheless,

the overall production of PAPS in vivo is promoted by the hydrolysis of - 32-

the inorganic pyrophosphate and the favourable APS-kinase reaction. ATP

2- 2- sulphurylase will accept other group VI anions (e.g. MoO^ , SeO^ )

(Wilson & Bandurski, 1958).

The final step in the series of reactions is the transfer of the sulphate group from PAPS to an appropriate acceptor, such as dopamine

(DA). In the case of the scheme shown above, the enzyme affecting this conversion is phenolsulphotransferase (PST, E.C. 2.8.2.1) (Gregory &

Lipmann, 1957). One of the limiting factors in the above process is sulphate availability (Bray et^l_, 1952) and the presence of large amounts of inorganic or organic sulphate, for example, in the form of cysteine or cystine will drive the reaction towards an increased formation of sulphate conjugate.

1.16 Biological functions of sulphate activation.

PAPS serves as a sulphate donor for the biosynthesis of all known sulphate esters, thus plays a role in sulphate ester biosynthesis analogous to that of ATP in phosphate ester biosynthesis. However, PAPS is more than just a sulphating agent.

In aerobic microorganisms, PAPS is the substrate for an NADPH-1inked reductase system, which yields sulphite, the latter is further reduced to sulphide which in turn condenses with £-acetylserine to yield cysteine. Recent evidence suggests that APS rather than PAPS may serve as the substrate for assimilatory sulphate reduction in higher plants and other oxygen involving eukaryotes (Dodgson & Rose, 1966). In dissimilatory sulphate reducers (Desulfovibrio), APS plays an additional role, that of terminal acceptor of anaerobic metabolism (Dodgson & Rose,

1970). However, there is evidence suggesting that reduction of sulphite does not occur in higher animals (Huovinen & Gustaffsson, 1962). -33-

35 The experiments showing the appearance in tissue of S-labelled

sulphur-containing amino acids following the injection of labelled

inorganic sulphate (Dziewiatkowski & Diferrante, 1957; Rambaut &

Miller, 1965) was most probably the result of activity of intestinal

microorganisms. It would appear that higher animals lost the facility

to utilise active forms of sulphate for reductive assimilatory (or

dissimilatory) purposes and the role of APS and PAPS is only associated

with the process of sulphoconjugation. Nevertheless, such animals are

ultimately dependent on the APS and PAPS-producing systems of plants and

microorganisms for their supplies of reduced organic sulphur compounds.

1.17 Aryl sulphate esters.

Phenol sulphotransferase (E.C. 2.8.2.1) catalyses the transfer of a

sulphate group from 3'-phosphoadenosine-5'-phosphosulphate to a phenolic

acceptor substrate resulting in sulphated phenol and adenosine-3'-5'-

diphosphate (Section 1.15). Phenol sulphation represents a major

mechanism for detoxication of endogenous and exogenous compounds bearing

phenolic groups (Roy & Trudinger, 1970; De Meio, 1975; Williams,

1979).

Although the existence in mammalian urine of several forms of

sulphate had been known since the early part of the nineteenth century,

the isolation of potassium phenol sulphate by Bauman (1876) from the

urine of dogs following administration of phenol was the first finding

that esterification of phenolic compounds by sulphuric acid occurs in

vivo. There is a large variety of compounds that form arylsulphates.

They include simple phenols and phenolic acids, heterocyclic phenols

(indoxyl and skatoxyl), phenolic hormones, including steroids such as

estrone and catecholamines. Conjugation with sulphate is not only -34-

restricted to catecholamines as such, but also their deaminated and/or

^-methylated metabolites, e.g. 3-methoxy-4-hydroxy-phenylglycol form sulphoconjugates (Schanberg et al_., 1968a,b).

Sulphate conjugation also represents a major pathway for metabolism of phenolic drugs such as salicylamide and a-methyldopa (Williams,

1979), L-dopa (Jenner & Rose, 1974; Tyce, 1974; Bronaugh et al.,

1975) and isoprenaline (Morgan et aj_., 1969; Conolly et al_., 1972).

1.18 Phenol sulphotransferase.

Phenol sulphotransferase (PST) has been partially purified from various sources including rat (Pennings et ^1_., 1978; Foldes & Meek,

1973) and human brain (Matlock & Jones, 1970; McEvoy & Carrol, 1971;

Hidaka & Austin, 1972), guinea pig (Banerjee & Roy, 1966, 1968) and rabbit liver (Hidaka et _a]_., 1969). Recently, Jakoby et ^1_. (1980) and

Sekura & Jakoby (1979, 1981) have reported the presence of four isoenzymes (I, II, III, IV) of phenol sulphotransferase in rat liver and the purification of three (I, II, IV) isoenzymes to homogeneity.

1.18.1 Substrate spectrum.

The four isolated forms of PST appear to be of two types.

Arylsulphotransferase I and II form one group that has an optimum with

2-naphthol at pH 6.5; each enzyme reacts with antibody prepared to the other. The second group, sulphotransferases III and IV have a more acid pH optimum for 2-naphthol, pH 5.5, and neither enzyme reacts with antibody to either sulphotransferase I or II (Sekura & Jakoby, 1981).

Although both groups are catalytically active with a large variety of simple phenols (e.g. 2-naphthol; phenol; m-chlorophenol; p-chlorophenol; p-nitrophenol; tyrosine methylester), none use hydroxysteroids, bile -35-

acids, aryl amines or either primary or secondary alcohols as substrates.

The major difference among the two groups is a capability of sulphotransferase IV in catalyzing sulphation of certain physiologically occurring catecholamines as substrates as well as tyrosine derivatives and organic hydroxyl amines. The enzyme referred to as N^hydroxy-2- acetylfluorene sulphotransferase by Wu & Straub (1976) is probably aryl sulphotransferase III or IV, or possibly a mixture of the two (Sekura &

Jakoby, 1981). Unlike enzymes I and II, sulphotransferase IV has been shown to be active in sulphating thejf; terminal tyrosine present in such peptides as the enkephalins and the heptapeptide of cholecystokinin, but not of gastrin, angiotensin I and II or the octapeptide of cholecystokinin (Sekura & Jakoby, 1981). Both groups catalyse sulphation of the thyroid hormones and their degradation products.

1.19 The availability of inorganic sulphate for sulphate conjugation

in vivo.

Much research in the 1940s and 1950s concentrated on investigations to identify the source of sulphate for sulphoconjugation reactions and, as a result, two views were proposed. Certain investigators using 35 inorganic [ S]-sulphate demonstrated a direct link between dietary sulphate and the appearance of sulphoconjugates in the urine

(Dziewiatkowski, 1949; Laidlow & Young, 1948). By contrast, others argued that although the incorporation of Na^ S0^ into phenolic sulphates was indeed possible, dietary inorganic sulphate was not used extensively for sulphoconjugation. Reduced sulphur compounds, particularly cysteine, cystine and methionine, were considered to be good if not better sources of intracellular sulphate (Binkley, 1949;

Wellers et al., 1960; Michels & Smith, 1965). -36-

Recently, a number of mechanisms have been described which may

contribute to the production of intracellular sulphate:- (1) The most

direct mechanism is the uptake of inorganic sulphate itself from the gastrointestinal tract, from the perfusing blood and from the

extracellular fluid (Curtis, 1982); (2) The activity of the

intracellular sulphatases which catalyse the hydrolysis of naturally

occurring low molecular weight esters such as steroid sulphatases,

release inorganic sulphate (Dodgson & Rose, 1980); (3) The lysosomal

degradation of sulphated biopolymers such as chondroitin sulphate,

heparin and heparin sulphate also provides sulphates (Wood et _al_., 1973;

MacNicholl et al_., 1980); (4) The sulphur present in reduced sulphur

containing compounds such as sulphides, polysulphides, thiosulphate,

sulphite and cysteine may undergo oxidation to inorganic sulphate

(Bartholomew et a!., 1980; Koj et al_., 1967). For each tissue or cell

type a relevant consideration should be, if all four mechanisms are in

operation, what is the relative contribution of each mechanism to

sulphoconjugation of endogenous and exogenous substrates. Finally, the

question may be raised whether the cell can switch from one sulphur

source to another. At present, the answers to all these problems are

not known.

1.20 Sulphation of catecholamines in vivo and in vitro.

The sulphation of catecholamines in man was first reported by Richter

(1940). This author reported that 70% of an orally administered dose of

adrenaline was present in urine as the sulphate ester. Richter

indicated that unlike phenylethylamine and most other amines,

conjugation of adrenaline predominated over oxidation by monoamine

oxidase and suggested that it was probably the main physiological -37- pathway by which this catecholamine was metabolised in the body.

In the following year, Richter & Macintosh (1941) demonstrated that adrenaline sulphate was pharmacologically inactive when tested on a number of physiological systems including the cardiovascular system, the nictitating membrane and the intestine of the cat. The original observations made by Richter were subsequently confirmed by Deichmann

(1943), Beyer & Shapiro (1945) and Haggendal (1963). This last author reported that conjugated noradrenaline and adrenaline were present in plasma at concentrations two or three times that of free noradrenaline and adrenaline. When the amounts of the free amines increased, for example, during muscular work or insulin-induced hypoglycaemia, the levels of conjugates did not markedly change.

Kahane et ^1_. (1967) demonstrated that approximately 70% of the endogenous catecholamines, noradrenaline and adrenaline, were present in urine as their sulphate conjugates.

In contrast, Clark et^ al_. (1951) isolated from urine of rabbits fed with adrenaline bitartrate, a conjugate which was identified as a glucuronide. La Brosse & Mann (1960) were the first to report that metabolites of the catecholamines also underwent extensive sulphation in humans. These authors claimed that_0_-methylated metabolites of noradrenaline and adrenaline were excreted in urine mainly as their sulphate conjugates. However, the studies involving intravenous injections of tracer doses of radiolabel led noradrenaline, showed that after 60-70 min only 14% of this amine was excreted as the sulphate ester (Goodall & Rosen, 1963). These authors also reported that the deaminated metabolites, 3,4-dihydroxyphenylglycol, 3,4-dihydroxymandelic acid and 3-methoxy-4-hydroxymandelic acid were present in urine as sulphate conjugates. Similar experiments performed with adrenaline -38-

revealed that greater than 30% of the total metabolites formed was normetanephrine sulphate (Goodall & Alton, 1965, 1968).

Although the above authors demonstrated extensive sulphate conjugation of the 0-methylated and deaminated metabolites of noradrenaline and adrenaline, little if any sulphation of the parent compound was observed.

The sulphate ester of the major central nervous system (CNS) metabolite of noradrenaline, 3-methoxy-4-hydroxyphenylethyleneglycol

(MHPG), has also been identified in human cerebral spinal fluid (CSF) and brain (Karoum et aj_., 1977).

Approximately 20% of the total MHPG in the CSF was conjugated, whereas the levels of this sulphate conjugate varied from 10 to 40% in several areas of human brain. The content of the sulphate conjugate of the other metabolites of noradrenaline and the other catecholamines has not been estimated in human CNS.

The findings presented above concentrated primarily on the conjugation reactions involving noradrenaline and adrenaline. Numerous reports similarly showed that dopamine and its metabolites are extensively conjugated in vivo in mammals.

The first to examine the metabolic fate of dopamine were Goodall &

Alton (1968). These authors found that following intravenous injection of dopamine into human subjects, only 1.5% of the dose was recovered in urine as dopamine sulphate. The sulphoconjugates of the deaminated and

^-methylated products varied between 2 and 6% of the total dose. This is contrary to the excretion patterns observed for endogenous dopamine which indicated that greater amounts of the sulphate ester are formed in vivo. -39-

It has been reported that Parkinsonian patients excreted

significantly less free dopamine than normal control subjects, but the

levels of dopamine sulphate in urine were comparable between the two

groups. The dopamine sulphoconjugate represented up to 80% of the total

dopamine excreted (Weil-Malherbe & Van Buren, 1969).

Several studies investigating the metabolism of dopamine have

concentrated on the degradative pathways of the precursor of dopamine,

L-dopa. Thus, after intravenous administration of L-dopa, 64% of the

infused dose was recovered in urine as dopamine and metabolic products

of dopamine (Goodall & Alton, 1972). Products of dopamine metabolism

consisted of the sulphoconjugates of dopamine and the free and

conjugated forms of 3-methoxytyramine, homovanillic acid (HVA),

dihydroxyphenylacetic acid (DOPAC) and several unknown metabolites.

Dopamine sulphate conjugate in urine represented 6.2% of administered L-

dopa or 80% of the total dopamine present in urine. These findings were

also confirmed by Rutledge & Hoehn (1973).

Jenner & Rose (1974) were the first to identify two different isomers

of dopamine 0-sulphate in the urine of Parkinsonian patients. They

claimed that dopamine 3-0-sulphate was the predominant conjugate. This

finding was confirmed by Bronaugh et _al_. (1975) and Arakawa et al.

(1979). In this last study dopamine 3-0-sulphate was a major dopamine

conjugate following oral administration of L-dopa to normal volunteers. 14 In contrast, when L-dopa [ C] was administered to the pigtail monkey, dopamine 4-0-sulphate was a predominant metabolite (Bronaugh et

al., 1974b). Bronaugh et a K (1975a) have also shown that the amount of

conjugate formed in vivo is dependent on the route of administration.

When L-dopa is given orally, more than 90% of the dopamine formed is

converted to the sulphate ester, whereas only 40% is conjugated after -40-

intravenous injection. The major metabolites of dopamine, DOPAC and HVA were also extensively conjugated when L-dopa was administered orally.

It was also shown that more dopamine sulphate and DOPAC were formed when tracer doses of L-dopa were administered compared to when larger, therapeutic, doses were employed (Bronaugh et ^1_., 1975).

The data presented above on dopamine metabolism after L-dopa administration, probably reflect the fate of endogenous dopamine in tissues rather than in the circulation. In support of this view is the fact that, when dopamine was injected directly into the circulation, dopamine sulphate constituted only a small percentage (1.9%) (Alton &

Goodall, 1968) of the total dose as in comparison with dopamine sulphate formation following oral administration of L-dopa (7.8%, Jenner & Rose,

1974); (6.2%, Goodall & Alton, 1972); (7.5%, Arakawa et aj_., 1979).

These results indicate that catecholamines formed from L-dopa within tissues have a greater propensity to undergo a conjugation with sulphate. However, when these amines are present in the circulation, metabolism occurs primarily by either deamination orjO-methylation.

Consistent with these findings were some of the data described above by Goodall and coworkers (Goodall & Rosen, 1963; Goodall & Alton, 1965,

1968) for noradrenaline metabolism. They reported that only a minor fraction of noradrenaline sulphate was found in urine following administration of tracer doses of this amine. In contrast, Buu & Kuchel

( 1977) and Johnson et al_. (1980) have shown that greater than 99% of circulating dopamine and 70% of circulating noradrenaline and adrenaline are present in plasma conjugated with sulphate. The above results show that endogenous catecholamines in plasma occur predominantly in the conjugated form, and exogenous administered amines undergo extensive deamination and/or J^-methylat ion. This might suggest that the formation -41-

of sulphate esters of neurotransmitters occurs prior to their release into the circulation, or alternatively that circulating catecholamine sulphates may be derived in part from the gut or liver and other tissues. Clearly, the data presented above demonstrated that catecholamines undergo extensive conjugation in vivo.

The in vitro investigations concerned with catecholamine conjugation with sulphate concentrated mainly on reactions between various substrates and crude or partially purified preparations containing PST of a variety of tissues. Thus, Jenner & Rose (1973) were the first to show that dopamine incubated with rat liver and brain preparations containing PST resulted in the formation of two dopamine conjugates, namely dopamine 3- and 4-0-sulphates. Dopamine 3-0-sulphate was found to be the main sulphate conjugate (> 99%) formed in a reaction with crude rat liver preparation.

Brain preparations incubated with dopamine resulted in the formation of nearly twice as much dopamine 3-0-sulphate in comparison with 4-0- sulphate (ratio 3- to 4-0-sulphate = 1.7). Several other groups of workers confirmed the finding that the 3-0-sulphate was the major conjugate when PST preparations were incubated with dopamine (Merits,

1976; Renskers ^t a K , 1980), adrenaline (Wong, 1978) and 3,4- dihydroxybenzoic acid (Pennings & van Kempen, 1980).

Besides the aforementioned substrates, the 3-methylated products of

COMT action also serve as substrates. In fact, 3-methoxytyramine was found to be preferentially sulphated compared to dopamine (Crooks et al., 1978). However, dopamine itself is one of the best substrates for

PST (Foldes & Meek, 1974; Hart et^l_., 1979).

The formation of a glycol sulphate conjugate from noradrenaline in brain tissue was demonstrated by Schanberg et ^1_. (1968). -42-

Further studies of the sulphotransferase system that uses catecholamine metabolites in rat brain showed that a larger number of these metabolites may act as substrates for one or more sulphotransferase(s) (Eccleston & Ritchie, 1973). It was also shown that MHPG was a better substrate for PST than DHPG (Sudgen & Eccleston,

1971; Eccleston & Ritchie, 1973). It would appear that methylation predisposed the molecule to subsequent sulphate conjugation.

It is tempting to suggest that the exclusive presence of MHPG- sulphate in brain could be the result of such a complementary or synergistic sequence of Ojmethylation and sulphate conjugation.

However, because dopamine 3-0-sulphate is the predominant sulphate isomer of dopamine, sulphate conjugation could possibly compete with

3-0-methyl ation in determining the resultant conjugated product

(Bronaugh et a K , 1975).

It has been shown that, in vitro, CL-methylation can precede

_0_-sulphation, resulting in sulphated methoxy compounds. Sulphation prevents, however, further metabolism by 0_-methylation.

On the action of another catecholamine metabolising enzyme, MAO, it is worth noting that of both dopamine sulphates neither ester could be further metabolised by MAO from human brain (Renskers et al_., 1980).

1.21 Site of sulphonation and role of sulphation in the CNS.

PST, a soluble cytoplasmic enzyme (Dodgson, 1977) is widely distributed in the mammalian body, with the highest specific activity usually being found in the jejunum, although this varies with species

(Sandler, 1981).

In earlier studies conjugation was generally thought to occur in the liver (Bernstein & McGilvery, 1952a). More recent experiments showed -43-

that the intestine of dog and monkey (Merits, 1976; Wong, 1978) were also sites for catecholamine sulphation.

Recently, PST activity has been detected in human erythrocytes and in platelets (Anderson & Weinshilboum, 1979, 1980). Other peripheral tissues in which PST activity was found, and therefore could be sites for sulphation were kidney, spleen and heart (Buu & Kuchel, 1981). Also brain has the ability to form sulphoconjugates of phenols (Hidaka &

Austin, 1972; Meek & Neff, 1972, 1973; Jenner & Rose, 1973; Pennings et _al_., 1978). Some aspects of the conjugation in vivo in the periphery of mammals have already been discussed.

An important question is whether sulphoconjugation plays an important role in the inactivation of catecholamines in the central nervous system. Several forms of PST which are capable of sulphating catecholamines have been shown in the rat brain. They have already been discussed.

Recently, a form of PST which is capable of sulphating catecholamines has been shown to be present in human brain (Roth et ^1_., 1981) and the sulphate conjugates of the catecholamines have been identified in human

CSF (Tyce et^l_., 1980). This last study demonstrated the presence of dopamine sulphate in the cerebrospinal fluid of Parkinsonian patients and suggested that this esterification reaction may also occur in brain.

Indeed, studies by Renskers et al_. (1980) revealed that both dopamine and noradrenaline undergo sulphate conjugation in subcellular preparations of human brain. The enzyme (PST) was found primarily in the soluble fraction of brain homogenates.

As a continuation of the above study, Roth et a K (1981) carried out investigations of human brain PST activity with regard to its capacity to inactivate dopamine. These authors found that PST activity could -44- account for approximately 10% of the total enzymatic activity involved in the metabolism of this catecholamine. However, the detection of sulphate esters of the catecholamines in human brain has not been reported, but the presence of sulphate esters of catecholamines in several areas of rat brain has recently been reported (Buu et a!.,

1981). Because of the very low levels of dopamine and noradrenaline sulphates found in the rat brain, these authors claimed that the presence of PST and free catecholamine substrates in most regions of rat brain in high concentrations does not necessarily lead to catecholamine sulphate formation. They also found low PST activity in the richly innervated tissues such as spleen and heart, together with very small concentrations of catecholamine sulphates in these tissues and argued against a role of sulphoconjugation as a major inactivation pathway of catecholamines (Buu & Kuchel, 1981).

It should be stressed that the form of PST in human brain which conjugates catecholamines appears to be quite a different enzyme to that found in common laboratory animals (Roth e t ^ L , 1981). These authors claimed that the affinity of human sulphotransferase for dopamine is almost 100-times greater than that of the sulphotransferase in rat, mouse, guinea pig, bovine and dog brain. The only animal examined that had a Km value for dopamine similar to that of the human transferase was the African green monkey.

The above findings suggest distinctive roles for PST and therefore for sulphoconjugation in the different animal species. It is an important observation, for it emphasizes that simple metabolic studies assessing the levels of sulphation of catecholamines, accordingly, may not reflect the extent to which these reactions occur in humans.

Based on the controversial data presented above, PST has the -45- potential to play a significant role in regulating the concentrations of the catecholamine neurotransmitters in both the CNS and the periphery.

It became possible to assess the activity of this enzyme in vivo following the discovery that human platelets are capable of sulphating catecholamines and their metabolites (Hart et al_., 1979; Weinshilboum &

Anderson, 1981; Sandler e t ^ L , 1981). If PST activity is under genetic control, then the activity measured in platelets may reflect sulphotransferase activity throughout the body.

Two forms of PST have been detected in human platelets and the properties of these enzymes appear to resemble those previously described for human brain. Of the substrates that have been tested, the catecholamines have the lowest Km values for the platelet transferases.

A recent study (Carter et al_., 1981) has demonstrated that the relative activities of the two enzymes in platelets vary in different individuals.

1.22 The possible role of sulphoconjugates.

To this point the above data demonstrated that sulphate conjugates of the catecholamines occur both in the CNS and in the periphery.

Sulphoconjugates of catecholamines are often present in tissues and biological fluids in much higher concentrations than the free amine. At least one form of sulphotransferase has a relatively high affinity for catecholamines and it is possible to measure PST activity by a relatively non-invasive procedure in man. However, it is still unclear if catecholamine sulphates are final products of these amines metabolism or if they act as intermediates in the neurotransmitter catabolic pathway.

Another important question is whether the catecholamine -46-

sulphoconjugates in biological fluids could be a better index of

sympathoadrenal activity than the free catecholamines, because of their

greater stability. Unlike free catecholamines, sulphoconjugates are

resistant to degradation by MAO (Renskers et aK, 1980) and COMT (Buu et.

_al_., 1981).

The physical and chemical properties of catecholamine sulphates

differ considerably from those of a free amine. They are resilient to

degradation by catecholamine catabolic enzymes and are capable of

forming internal salts. The absence of charge in the molecule permits

it to penetrate tissue membranes more easily (Jenner & Rose, 1973). For

example, dopamine sulphate conjugates have been suggested to be a

transport and storage form of free dopamine (Jenner & Rose, 1973) for

the above reasons. The findings of Rutledge & Hoehn (1973) that 80% of

the dopamine excreted by Parkinsonian patients receiving L-dopa therapy

is in the conjugated form, raised the possibility that sulphoconjugation

may constitute an inactivation mechanism of dopamine (Bronaugh et a!.,

1973).

Previously, the sulphate ester of adrenaline has been shown to

be devoid of any physiological actions of its parent compound (Richter &

McIntosh, 1942). However, dopamine sulphate is not necessarily inert:

it can be metabolised as shown by Merits (1976). This author claimed 14 that when trace amounts of [ C]-dopamine 3-0-sulphate were administered

to dog, rat and guinea pig, the compound was further metabolised. The 14 guinea pig [ C]-dopamine 3-0-sulphate was almost completely desulphated

and metabolised according to the pattern characteristic of orally

administered dopamine in the animal species. In the rat, about 40% of 14 the administered [ C]-dopamine 3-0-sulphate in a relatively high dose

was excreted in urine unchanged, whereas a smaller dose was totally ■47-

metabolised according to the pattern characteristic in the rat. In dog urine, however, more than 80% of the radioactive dose emerged in urine

as unchanged [^C]-dopamine 3-0-sulphate, the normal metabolism end

product of dopamine in the dog.

The above findings are not totally surprising. Dodgson & Tudball 35 (1960) showed that injection of p-nitrophenyl [ S]-sulphate into rats was followed by the appearance in urine of up to 30% of the

radioactivity of the dose as inorganic [°°S]-sulphate. Estrone [ S 3

sulphate is almost completely desulphated in vivo in the rat (Hanahan et 35 al., 1949), and man (Twombly & Levitz, 1960). Also nitrocatechol [ S]

sulphate is partly desulphated in vivo (Flynn et , 1967). In other

instances, quite a different transformation of sulphoconjugates of

various compounds can take place in vivo. Injected L-tyrosine-0_-

sulphate was rapidly and completely deaminated without a loss of the

ester sulphate group (Dodgson et a K , 1961). Similar types of

transformation have been noted with the estrogens; for example,

conversion of estrone sulphate to 15a -hydroxyestrone sulphate has been

observed in humans (Jirku et al_., 1967), also androstenediol can be

directly converted to dehydroisoandrosterone sulphate (Baulieu, 1965).

These results indicate the existence of a group of sulphates that

undergo further metabolism. However, the above findings can be

controversial. Jenner & Rose (1974) claimed that in the rat dopamine

3-0-sulphate was metabolically inert. It did not undergo desulphation

when incubated under a variety of conditions with preparations of three

arylsulphatase enzymes from rat tissues and, when administered

intraperitoneally or intravenously to rats, was rapidly excreted in the

urine unchanged. In contrast, dopamine 4-0-sulphate was more readily

desulphated in vitro than the 3-isomer, although even the most active -48-

enzyme had very low affinity for the 4-0-sulphate (Jenner & Rose, 1978).

These authors also showed that administration of dopamine 3- and 4-0- 35 C S] sulphates to rats did not result in desulphation by mammalian arylsulphatases in vivo. However, these compounds were hydrolysed by the gut flora following oral administration, and were also partially desulphated after i.p. administration as a result of biliary excretion oc into the gut lumen. Dopamine 4-0-C S] sulphate was also shown to be extensively metabolised, without desulphation, yielding two metabolites.

The major metabolite of the above compound was identified as

3,4-dihydroxyphenylacetic acid 4-0-sulphate, a probable product of monoamine oxidase action.

These collective observations suggested that there may be two types of arylsulphate ester from the point of view of physiological importance. Firstly, those that are metabolically inert in vivo and are presumably the end product, and, secondly, those that can undergo metabolic transformation in vivo and which may be therefore of some biological importance. However, there is not enough evidence to state whether catecholamine sulphates may belong to this second group, although it has been shown that dopamine 3- and 4-0-sulphates can be converted directly to free noradrenaline in vitro (Buu & Kuchel,

1979a,b).

1.23 Catecholamine sulphoconjugates in pathological conditions.

In DOCA hypertensive rats, the decrease in tissue conjugated dopamine

preceded the decrease of tissue free adrenaline and noradrenaline (Buu et aT_., 1978) implying that the measurement of dopamine sulphate reflects the early changes in the activity of the peripheral adrenergic

system better than the determination of free catecholamines. -49-

Kuchel et al_. (1978) suggested that essential hypertensive patients compared with control subjects have higher conjugated plasma dopamine, less urinary free and conjugated dopamine with blunted urinary free dopamine and sodium responsiveness to frusemide. Conjugated noradrenaline and adrenaline, mean arterial pressure and age were positively correlated. These authors also found elevated plasma and urine levels of total dopamine in patients with primary aldosteronism.

They implied that elevated conjugated dopamine appeared to reflect a compensatory activation of the dopaminergic vasodilator pathway in hypertension, and the total dopamine urinary excretion an intrinsic deficiency or compensatory increase of a dopamine-modulated natriuretic mechanism. The same authors in a different series of studies on hyperthyroid hypertension suggested that with increasing doses of exogenous adrenaline given on separate days, the concentration of free plasma adrenaline increased proportionally to an increase in conjugated adrenaline, which implied a rapid conversion of free into conjugated adrenaline (Kuchel et al_., 1982). At the same time there was an increase of platelet PST, probably through induction of this enzyme by rising concentrations of adrenaline. There were also findings that conjugation was one of the major inactivation pathways of catecholamines released from phaeochromocytoma (Kuchel et _aK, 1979). 3 3 When L-dopa [ H] and [ H]-dopamine sulphates were administered to 3 bilaterally adrenalectomised rats, they were found to generate [ H]- adrenaline in the urine and kidney tissues (Buu et a^., 1981). This observation suggested that in the rat adrenaline could be synthesised outside the adrenals and that dopamine sulphate can serve as an 3 intermediate in such synthesis. [ H]-dopamine sulphate was also 3 converted to [ H]-noradrenaline, indicating that it can be metabolised -50- in vivo and could be the source of free dopamine in urine.

Unger et a K (1979) showed that following the strong sympathoadrenergic stimulus of surgical stress in dogs, this was accompanied by an increase in blood pressure and heart rate.

Conjugated dopamine showed a twofold rise in arterial plasma, while conjugates of noradrenaline and adrenaline decreased in the circulation.

In the latest of their studies these investigators (Kuchel et al_., 1982) showed that patients with essential hypertension had increased plasma conjugated dopamine, increasing with age, lower conjugated noradrenaline and adrenaline, higher free adrenaline, lower urinary free and total dopamine, but higher noradrenaline than control subjects.

Thus, the data presented above very strongly suggested that catecholamine sulphates may have a role as intermediates in metabolism and provide a good index of sympathoadrenal activity. However, all the findings that confirmed the role of catecholamine sulphates in metabolism and as an index of sympathoadrenal activity in several pathological conditions have resulted from one group of workers only.

Data presented previously in this Chapter have indicated that there is some controversy concerning the involvement of catecholamine sulphates as intermediates in catecholamine metabolism.

In view of all the facts presented here, it is important to determine the role of catecholamine conjugates in mammalian systems. None of the studies presented in this introductory Chapter have included measurements of catecholamine sulphoconjugates in tissues and/or biological fluids by direct methods. Instead authors have relied upon hydrolysis and subsequent estimation of free amines or measurements in vitro of catecholamine sulphate formation catalysed by phenolsulphotransferase. Few investigators have stated clearly the -51- purity and identity of their synthesized catecholamine sulphates.

In this thesis special emphasis was placed on the detailed characterisation and authentication of synthesized dopamine and noradrenaline O-sulphates together with the development of direct assays to estimate these compounds in tissues and biological fluids. In addition, a study of the metabolism of dopamine and noradrenaline sulphates has been carried out which may provide the basis for further studies on the disposition of these important compounds.

There are many questions that need to be answered before the significance of sulphoconjugation as a reaction regulating catecholamine concentrations in mammalian systems can be adequately assessed. -52-

CHAPTER 2.

STUDIES ON THE CHEMICAL SULPHONATION OF DOPAMINE AND NORADRENALINE.

2.1 Introduction 55

2.2 Chemical sulphonation of dopamine ...... 60

2.2.1 Materials ...... 60

2.2.2 Methods: Chemical sulphonation of dopamine according

to published methods ...... 60

2.2.3 Variation of reaction temperature and specific

gravity of sulphuric acid ...... 61

2.2.4 High performance liquid chromatography (h.p.l.c.) 62

2.2.5 Nuclear magnetic resonance spectroscopy (n.m.r.) 62

2.2.6 Results: Sulphonation of dopamine by published

methods ...... 63

2.2.6.1 Elemental analysis ...... 63

2.2.6.2 Gibbs' reaction and hydrolysis ...... 63

2.2.6.3 H.p.l.c. and n.m.r. analysis ...... 64

2.2.6.4 Ion-exchange chromatography ...... 64

2.2.7 Effect of reaction temperature and H2SO4

sp.g. on the products of dopamine sulphonation ... 64

2.2.8 Structural determination of the reaction

products by nuclear magnetic resonance

spectroscopy (n.m.r.) ...... 66

2.2.9 Product I (Dopamine 6-sulphonic acid)

characterisation ...... 70

2.2.9.1 Elemental analysis and melting point ...... 70

2.2.9.2 Nuclear magnetic resonance spectra ...... 70

2.2.10 Product II (Dopamine-4-0-sulphate)

characterisation ...... 73 2.2.10.1 Elemental analysis and melting point

2.2.10.2 Nuclear magnetic resonance spectra

2.2.11 Product III (Dopamine 3-0-sulphate)

characterisation

2.2.11.1 Elemental analysis and melting point

2.2.11.2 Nuclear magnetic resonance spectra

2.3 Chemical sulphonation of noradrenaline ...

2.3.1 Materials

2.3.2 Methods: Reaction of noradrenaline with

H2S04 (sp*9* ]*84)

2.3.3 High performance liquid chromatography

(h.p.l.c.) ......

2.3.4 Results: High performance liquid chromatography

analysis of products formed during sulphonation

of noradrenaline by H^SO^ (sp.g. 1.84) ...

2.3.5 Hydrolysis by sulphatase and HC1

2.3.6 Reaction of noradrenaline with pyridine-sulphur

trioxide

2.3.7 Sulphonation of noradrenaline by chlorosulphonic

acid ......

2.3.8 High performance liquid chromatography analysis

of noradrenaline sulphonation products ...

2.3.9 Sulphonation of noradrenaline by chlorosulphonic

acid: effect of varying reaction time

2.3.10 Sulphonation of noradrenaline by chlorosulphonic

acid: effect of varying reaction mixture

composition 2.3.11 Preparative scale synthesis of authentic

noradrenaline 3- and 4-0-sulphates

2.3.12 Results: Product I structural determination

2.3.12.1 Product I: Elemental analysis and melting point

2.3.12.2 Nuclear magnetic resonance spectroscopy ...

2.3.13 Results: Product II structural determination

2.3.13.1 Product II: Elemental analysis and melting point

2.3.13.2 Nuclear magnetic resonance spectroscopy ...

2.4 Discussion - 55-

Chapter 2

2.1 INTRODUCTION

Conjugation of dopamine and noradrenaline with sulphate constitutes a major pathway in the metabolism of these catecholamines. Several workers have investigated the metabolic transformation of these amines

by sulphoconjugation, and demonstrated that these compounds are present

in biological fluids such as plasma and urine in much higher

concentrations than free dopamine and noradrenaline (Richter, 1940;

Richter & McIntosh, 1941; Kahane et al_., 1967; Imai et^l_., 1970;

Buu & Kuchel, 1977; Buu et aJ_., 1978).

Measurements of dopamine and noradrenaline sulphoconjugates were

performed by using indirect methods, based on hydrolysis of

sulphoconjugates and subsequent estimation of appropriate

catecholamine(s). No efforts were made to investigate the existence and

the significance of specific isomers of the aforementioned amines,

because of the lack of authenticated synthetic standards, (Unger et a!.,

1979; Johnson et al_., 1980; Kuchel et aK, 1981; Cuche et^l_., 1982;

Joyce et^ al_., 1982).

Jenner & Rose (1973) were the first to demonstrate the in vitro

conversion of dopamine to its 3- and 4-0-sulphates using

sulphotransferase preparations from rat liver and brain. A part of this

study included a one-step synthesis of the authentic metabolites from

dopamine and sulphuric acid, a method which has been used extensively

hitherto.

This latter study involved a reaction of dopamine hydrochloride with

a 10-fold molar excess of cone. H^SO^ (sp.g. 1.86) at 0°C, and gave rise

to dopamine 3-0-sulphate and dopamine-4-0-sulphate in 10 and 12% yield - 56-

respectively. The techniques of ion-exchange chromatography and differential crystallisation were used to resolve the two isomers, which were characterised by chemical procedures involving methylation and oxidative degradation to isovanillic and vanillic acids. No attempts were made to perform direct structural analysis, except for the reaction with Gibbs' reagent, which should give a positive colour reaction with the 4-0-isomer, but not with the 3-0-isomer. Subsequent workers

(Bodnaryk & Brunet, 1973) modified the reaction conditions using a 35- fold excess of cone. at -12°C with a consequent low yield (3%) of the sulphate conjugates. Again, chemical derivatisation was accomplished to characterise the sulphates, but in this case by methylation and oxidation to the corresponding aldehydes isovanillin and vanil1 in.

From this point, the literature becomes rather more obscure. Claims that dopamine 3-0-sulphate is the major urinary metabolite of orally administered dopamine in the dog (Merits, 1976), that both sulphate isomers can be quantitated in the urine by high-performance liquid chromatography (h.p.l.c.) after 1-DOPA administration to man (Arakawa et al., 1979) and that the sulphates are converted by dopamine

3-hydroxylase directly to noradrenaline (Buu & Kuchel, 1979a) cannot be evaluated, since none of these workers give any data to substantiate the nature or purity of their synthesised compounds.

Like all the aforementioned groups, the procedure for chemical sulphonation of dopamine was at first attempted as described by Jenner &

Rose (1973). However, considerable difficulties were experienced when isolation of the two sulphoconjugates was attempted. The fact that dopamine sulphate isomers could not be isolated is not totally unexpected, because the O-sulphates are not the preferred products of -57- reaction between phenolic derivatives and SOg donors such as cone.

In such cases, sulphonation of the aromatic nucleus, ortho/para to the phenols, in such a ring system activated towards electrophilic substitution, will be expected to predominate, yielding sulphonic acids

(Norman & Taylor, 1965). These would be the isomers of the O-sulphates, indistinguishable from the latter by Gibbs' reaction, I.R. and U.V. spectroscopy and indeed elemental analysis (C, H, N and S). In view of the biochemical importance which is attached to the dopamine

O-sulphates, both as metabolic excretory products of dopamine and L-dopa and as possible intermediates in noradrenaline biosynthesis, unequivocal evidence of the chemical structure of the synthetic products is required. The original synthetic reaction (Jenner & Rose, 1973) has thus been investigated in more detail with reference to temperature of the reaction and specific gravity of sulphuric acid employed. It will be shown that products other than dopamine ^-sulphates are formed during this reaction, and their proportions depend critically upon temperature and specific gravity of the sulphuric acid. In addition, sulphonation of dopamine was performed using chlorosulphonic acid according to Rose &

Bleszyinski (personal communication) and using pyridine-sulphur trioxide

(Mitchell, 1977).

Several reports confirm that both circulating and excreted noradrenaline are found largely as its sulphate conjugate (Buu & Kuchel,

1977; Kuchel et aj_., 1978). Thus conjugation with sulphate constitutes a major inactivation pathway of noradrenaline released from phaeochromocytomas (Kuchel et al_., 1980). It has been claimed that in patients with essential hypertension, noradrenaline sulphate conjugates are positively interrelated with mean blood pressure and age (Kuchel et al., 1978). In spite of the obvious importance of the sulphation to the -58-

disposition of noradrenaline, no direct assay of these conjugates was developed, current methods relying upon deconjugation and further assay of total noradrenaline (Buu & Kuchel, 1977; Johnson et al_., 1980).

This is thought to be because the authentic noradrenaline sulphate standards have not been available. The definitive synthesis of these has been performed, but involves a complex seven-step procedure (Wang et al_., 1972).

In this Chapter, the data are presented to demonstrate that noradrenaline isomeric sulphates could be prepared in a simple one-step 1 13 reaction. The application of the techniques of h.p.l.c. and H and C nuclear magnetic spectroscopy (n.m.r.) were employed, just as in the case of dopamine sulphates, to the separation, isolation and authentication of the reaction products. The reaction between noradrenaline and chlorosulphonic acid leading to the synthesis of authentic noradrenaline 3- and 4-0-sulphates was adapted from the method described by Rose & Bleszynski (personal communication). In this latter study noradrenaline was reacted with an equimolar amount of chlorosulphonic acid in the presence of diethylaniline and CS^ at -7° until extremely viscous. Afterwards the reaction mixture was left for

16 h at 2°C. The yields of noradrenaline monosulphate and noradrenaline disulphate esters were 4.2% and 4.0% respectively. The authors did not succeed in isolating noradrenaline 3- and 4-0-sulphates. Thus, this method allows only for synthesis and separation of mono- and disulphate esters of noradrenaline, and is suitable only when radioactively labelled chlorosulphonic acid is used, otherwise detection of the products is very difficult.

The first attempt to synthesise the noradrenaline sulphate isomers was made by utilising the aforementioned method; however, instead of -59-

repeating a long procedure from the original method that lead to the purification of noradrenaline monosulphates, high performance liquid chromatography was employed for analysing and purifying the products of this reaction. Because of the unsatisfactory yield of products other than noradrenaline itself, this reaction was investigated in relation to the different reaction mixture composition and various times of the reaction. The best conditions of the reaction established during the analytical procedure were then applied for the semi-preparative scale preparations of milligram quantities of noradrenaline 3- and 4-0- sulphates.

Additionally, the sulphonation of noradrenaline was performed using sulphuric acid and pyridine-sulphur trioxide.

Preparative h.p.l.c. was employed for the purification of these compounds to > 95% purity and 250 MHz nuclear magnetic resonance spectroscopy for authentication. -60-

2.2 CHEMICAL SULPHONATION OF DOPAMINE.

2.2.1 Materials.

Chemicals were obtained from the following sources: dopamine hydrochloride (Sigma Chemical Co., London, U.K.), sulphuric acid (sp.g.

1.92, Hopkin and Williams), pyridine-sulphur trioxide (Aldrich Chemical

Co., Gillingham, U.K.), chlorosulphonic acid (BDH Chemicals, Poole,

U.K.), 2,6,dichloro-£-benzoquinone-4-chloroimine (Gibbs' reagent, BDH

Chemicals, Pool, U.K.), Dowex 50 X 8 ion exchange resin (200-400 mesh,

H+ form, BDH Chemicals Poole, U.K.), Dowex 1 X 8 (200-400 mesh, chloride form, BDH Chemicals, Poole, U.K.). Methanol was of spectroscopic grade, water was glass distilled and deionised.

2.2.2 Methods: Chemical sulphonation of dopamine according to

published methods.

The chemical sulphonation of dopamine was performed as described by

Jenner & Rose (1973). Dopamine hydrochloride (0.4 g, 2.1 mmol), which had been keDt over PJ),. in vacuo for 24h, was added to stirred cone. c b H^SO^ (sp.g. 1.86; 1.1 ml; 20.6 mmol) at 0°C. After 20 min, the mixture was poured into crushed ice-water (10 ml) and the resultant clear solution applied to a Dowex 50 X 8 ion exchange column (200-400 mesh, H+ form; 200 X 15 mm). Elution was carried out with water, the effluent monitored at 280 nm and fractions (5 ml) were collected and monitored

for pH. All fractions with pH < 3, which contained approximately 70% of

the U.V.-absorbing material, were pooled. Similarly, fractions with

pH > 3 were pooled.

According to the published method (Jenner & Rose, 1973), fractions of

pH < 3 should be discarded since they contain largely inorganic sulphate

but, because of the quantity of organic material they obviously - 6 1 -

contained from their U.V. extinction, it was decided to investigate them further. Both pH < 3 and pH > 3 bulkend eluates were treated similarly and reduced to 10 ml jn. vacuo at 30°C. Both were frozen at -20°C and stored for 24 h in a frozen state, then allowed to thaw slowly at 0°C as described (Jenner & Rose, 1973). Unlike these previously described methods, no crystals appeared in the pH > 3 fraction. However, a copious crop of crystals were obtained from the more acidic fraction.

These were washed with ethanol-ether mixture and dried in vacuo over

P^Og, giving a white crystalline product (67 mg, yield as dopamine sulphates (13.7%), which according to published methods should be the

4-0-sulphate. The mother liquor from this fraction was applied to a column of Dowex 1 X 8 (200-400 mesh, acetate form; 250 X 10 mm), to isolate the 3-0-sulphate. Each of 60 fractions (10 ml) thus collected was retained for high performance liquid chromatography (h.p.l.c.) analysis.

Sulphonation of dopamine was also performed using chlorosulphonic acid (Rose & Bleszynski, personal communication) and using pyridine- sulphur trioxide (Mitchell, 1977). Again, aliquots of diluted reaction mixture were retained for h.p.l.c. analysis.

2.2.3 Variation of reaction temperature and specific gravity of

sulphuric acid.

The effect of varying temperature (0-38°C) and H2SO4 sp.g. (1.84 -

1.92) on product formation was investigated. Reactions were carried out as described above (Section 2.2.2), except that the required sp.g. of

H2S04 was obtained by the mixing of fuming H^SO^ (sp.g. 1.92) with cone.

H2^4 ^sp,g* 1*84), using a hydrometer and density determinations by weighings to adjust the specific gravity. Each reaction was terminated by pouring the mixture into 10 vol. crushed ice-water mixture which was -62-

retained for h.p.l.c. analysis. In such a way, reactions were performed

at 0, 20 and 38°C using of 1.84, 1.86 and 1.92 sp.g.

2.2.4 High performance liquid chromatography.

For the analysis and preparative scale separations of the products of

the various sulphonation reactions, h.p.l.c. was employed. Aliquots

(100-200 pi) of diluted reaction mixture containing 5-10 mg of dopamine

related material, were injected onto Hypersil ODS (5 pm particle size)

column (250 X 8 mm, Shandon Southern Products, Wilmslow, U.K.) and

eluted with 2% aq. methanol delivered by a Waters Associates Inst.

(Hartford, U.K.) 6000A pump at 0.8 - 1.0 ml/min. The column effluent

was monitored at 284 nm using a Waters variable wavelength detector

(Model 450). Four dopamine-related peaks were observed, of variable

size depending on reaction conditions, with elution volumes of 9.0,

26.2, 29.2,128.8 ml. The last of these co-chromatographed with dopamine

under various conditions and solvent systems, including in 0.5% aq.

methanol. The first three peaks are referred to hereafter as products

I, II and III respectively.

2.2.5 Nuclear magnetic resonance spectroscopy (n.m.r.).

Products I, II and III (Section 2.2.4) were isolated after repetitive

injection and reinjection on h.p.l.c. and subjected to various analyses

including n.m.r. ^H n.m.r. spectra were acquired on samples of the

aforementioned products (8-15 mg) as solutions in d--DMS0 (product II o and III) or D£0 (product I) using a Bruker n.m.r. spectrometer operating

at 250 MHz. Where appropriate, scale expansion of the spectral region

containing the aromatic protons signals ( 6.5 - 7.5 relative to 13 tetramethylsilane) was made. Also, natural abundance C n.m.r. spectra

were recorded for product I (80 mg in D£0) using another Bruker WM 250 -63-

spectrometer. Off-resonance-decoupled techniques were used to distinguish between quaternary carbons (ring-substituted carbons) and aromatic carbons bonded to hydrogen.

2.2.6 Results: sulphonation of dopamine by published methods.

The reaction between dopamine and 10-fold excess of sulphuric acid

(sp.g. 1.86) was performed as described previously (Jenner & Rose, 1973,

Section 2.2.2). Work-up of fractions from Dowex 50 X 8 column did not yield a crystalline product from the pH > 3 fraction. Rather, the acid fraction pH < 3, which it is advised to discard, containing approximately 70% of the U.V.-absorbing material (280 nm), when frozen and thawed, gave a white crystalline product (67 mg; m.p. 244-247(d)°C).

2.2.6.1 Elemental analysis.

Dopamine monosulphate (CgH^NO^S) requires: C 41.2%

H 4.7%

N 6.1% found: C 41.1%

H 4.9%

N 6.0%

Since this isolated product has found C, H, N values very similar to that of dopamine monosulphate requirements, at first it was thought that dopamine 4-0-sulphate had been isolated, since both chemical yield and crystallisation characteristics were very similar to those reported

(Jenner & Rose, 1973).

2.2.6.2 Gibbs* reaction and hydrolysis.

This isolated material gave a positive colour reaction (blue) when exposed to 1% (w/v) Gibbs* reagent in ethanol and then to NH^ vapour. -64-

This observation, together with elemental analysis results, strongly suggested that this material was most probably dopamine 4-0-sulphate.

However, the substance was poorly labile to hydrolysis with either sulphatase (sulphatase ex Helix pomatia, Sigma Chemical Co., London,

U.K.) or 0.1 M HC1, which would not have been expected for an 0- sulphate.

2.2.6.3 H.p.l.c. and n.m.r. analysis.

Further examination of the above material by h.p.l.c. (conditions of analysis as described in Section 2.2.4) revealed that it was a composite of three substances (products I-111 vide supra). Characterisation by n.m.r. (see 3.2.9) of products I-111 which this substance contains, showed that this crystalline product contained both 3 and 4-0-sulphates and a large quantity of a sulphonic acid. Thus, one of the major products of this reaction is dopamine 6-sulphonic acid [2-(21 - aminoethyl)-4,5-dihydroxyphenyl-sulphonic acid], a hitherto undescribed compound, which is neither labile to sulphatase nor to 0.1 M HC1.

2.2.6.4 Ion-exchange chromatography.

The mother liquor from the pH < 3 fraction was chromatographed on

Dowex 1 X 8, the method described to isolate the 3-0-sulphate (Oenner &

Rose, 1973). Sixty fractions (10 ml) were collected from this column and inspected by h.p.l.c. Fractions 1-25 contained products I and fractions 26-60 contained only dopamine. The identity of product I is given later (Section 4.2.8; 4.2.9).

2.2.7 Effect of reaction temperature and H^SO^ SP«9« on the products

of dopamine sulphonation.

To check the proportion of products formed during the reaction of dopamine with sulphuric acid, various reaction conditions were employed. Table 2.1 Products of reaction of various sulphonating agents with dopamine at various temperatures.

Approximate yield of product (%)

Reagent T (°C) I (9.0) II (26.2) III (29.2) Dopamine (128.8)

H2S04 (sp.g. 1.92) 0 90 5 5 n.d.

20 100 n.d. n.d. n.d.

H2S04 (sp.g. 1 .86) 0 30 35 35 n.d.

20 95 2.5 2.5 n.d.

38 95 2.5 2.5 n.d.

H2S04 (sp.g. 1.84) 0 5 20 20 55

Chlorosulphonic acid 20 n.d. 15 15 70

Pyridine-sulphur 20 n.d. n.d. n.d. 100 trioxide

★ Elution volume (ml) in h.p."I.c. system (see text). n.d. means not detected

i CN InI -66-

Sulphonation of dopamine was performed at 0, 20, 38°C using either 1.84,

1.86 or 1.92 sp.g. F^SO^. The approximate content of products I-III and unreacted dopamine was determined in each case by h.p.l.c.

The results are presented in Table 2.1. When fuming F^SO^ (sp.g.

1.92) was reacted with dopamine at 0°C one polar product predominated on h.p.l.c. (product I). Small amounts (approximately 5%) of two chromatographically similar products were also observed (products II and

III). No unreacted dopamine was found. Sulphonation of dopamine carried out at 20°C resulted in the formation of only product I.

Reactions using 1.86 sp.g. f^SO^ yielded all three products I-111 in a temperature-dependent fashion. At 0°C, roughly equal amounts of each of these were formed (Fig. 2.2), whilst on raising the temperature to either 20 or 38°C, product I again predominated, with only traces of products II and III. With F^SO^ sp.g. 1.84 at 0°C, approximately half the dopamine remained unreacted, most of the products being II and III

(20% each), with a small amount of product I (5%). Reaction of dopamine with chlorosulphonic acid at 20°C was found to give mainly unreacted dopamine (70%) with 15% each of products II and III. No reaction could be observed between dopamine and pyridine-sulphur trioxide.

2.2.8 Structural determination of the reaction products by nuclear

magnetic resonance spectroscopy (n.m.r.).

Variable chemical conditions resulted in the formation of three products (I-1 1 1 ) in the reaction between dopamine and different sulphonation agents. Two products, dopamine 3- and 4-0-sulphates have been claimed previously by many workers (Jenner & Rose, 1973; Bodnaryk

& Brunet, 1974; Merits, 1976; Arakawa et a., 1979; Buu & Kuchel,

1979a, b). As discussed previously (Section 2.1), sulphonation of dopamine might not only result in the formation of 0-sulphates, but also -67-

Fig. 2.1 Products of chemical sulphonation of dopamine.

I - dopamine 6-sulphonic acid II - dopamine 4-0-sulphate III - dopamine 3-()-sulphate t INJECT

Fig. 2.2 Hplc chromatogram of products formed during chemical sulphonation of dopamine (for legend, see over). i CN CO i -69-

Fig. 2.2

Hplc chromatogram of products formed during chemical sulphonation of dopamine.

I dopamine 6-sulphonic acid, elution volume in 2% methanol 9 ml.

II dopamine 4-0-sulphate, elution volume in 2% methanol 26.2 ml.

III - dopamine 3-0-sulphate, elution volume in 2% methanol 29.2 ml. -70-

one or more sulphonic acids, the ratio between such products being dependent upon the reaction conditions (Norman & Taylor, 1965).

Therefore, products I, II and III were isolated (20-100 mg of each) from the various reactions (Table 2.1) for analysis by n.m.r. techniques.

Since both ^-sulphates and ring sulphonic acids would be isomers, they could only be realistically distinguished by a technique such as n.m.r. which gives discrete information about the aromatic hydrogens (protons) and carbon atoms and their microenvironment. The spectra obtained from n.m.r. show that products I, II and III are dopamine 6-sulphonic acid, dopamine 4-0-sulphate and dopamine 3-0-sulphate respectively (Fig. 2.1).

2.2.9 Product I (dopamine 6-sulphonic acid) characterisation.

2.2.9.1 Elemental analysis and melting point.

This material was isolated by preparative h.p.l.c. (elution vol: 9 ml in 2% methanol) as described previously (Fig. 2.2). Dopamine 6- sulphonic acid monohydrate CgH^NO^S.H^O requires:

38.2%

5.2%

5.6% found: 38.5%

5.5%

5.9%

This compound decomposed at 272-275 u .

2.2.9.2 Nuclear magnetic resonance spectra.

The 250 MH^ n.m.r. spectrum showed signals corresponding to the four aliphatic protons of the dopamine side-chain and two signals down field each corresponding to the aromatic proton (Fig. 2.3). Such a -71-

Fig. 2.3 250 MHz H nmr spectrum (aromatic region) of dopamine 6-sulphonic acid (product I, bottom), including expanded spectrum (top). 13 Off“resonance decoupled C nmr spectrum of dopamine 6-sulphonic acid. -73-

configuration could only arise from sulphonation of the aromatic ring, since sulphonation of either phenolic group would give signals arising from three aromatic protons (positions 2,5,6; Fig. 2.3).

Additionally, since no coupling (splitting) was detectable between these two aromatic protons, even on scale expansion (Fig. 2.3, top) the protons must be para to one another. Interestingly, the lowest frequency signal (2-proton) is broadened, presumably due to a weak through-bond coupling to the aliphatic protons. Thus, the only assignable structure to the empirical formula CgH^NOgS is that of the

6-sulphonic acid. This was confirmed by off-resonance-decoupled C n.m.r. spectroscopy, which shows four singlets corresponding to four quarternary (substituted) carbons and two doublets (up-field) corresponding to the two carbon-hydrogen bonds (Fig. 2.4). Dopamine

6-sulphonic acid [2-(2‘-aminoethyl)-4,5-dihydroxyphenylsulphonic acid] has not previously been described.

2.2.10 Product II (Dopamine 4-0-sulphate) characterisation.

2.2.10.1 Elemental analysis and melting point.

This product was isolated by preparative h.p.l.c. (elution vol. 26.2 ml in 2% aq. methanol, Fig. 2.2).

Dopamine monosulphate (CgH^NO^S) requires: C 41.2%

H 4.7%

N 6.1%

found: C 41.2%

H 4.9%

N 5.9%

Melting point 244-247°C (d). -74-

2.2.10.2 Nuclear magnetic resonance spectra.

Proton [^H] n.m.r. analysis of product II revealed that the most

likely structural assignment is that of dopamine 4-0-sulphate. The 250

MHz spectrum showed the four aliphatic protons and three aromatic signals corresponding to each of three protons. The most deshielded proton in this structure (Fig. 2.5) would be that adjacent to the OSO^-

grouping, i.e. that in the 5-position. The appearance of this signal as

a doublet with a typical ortho coupling is consistent with this

interpretation. Next up-field, a doublet with a small coupling (meta) was observed, which can be assigned to the proton in the 2-position.

Finally, a quartet was observed with both ortho and meta coupling, which

is indicative of the proton in the 6-position. Such a spectrum could not have been obtained from a ring substituted sulphonic acid, nor the

3-0-sulphate (vide infra). Thus, product II is dopamine 4-0-sulphate.

2.2.11 Product III (Dopamine 3-0-sulphate) characterisation.

2.2.11.1 Elemental analysis and melting point.

This compound was isolated by preparative h.p.l.c. (elution vol. 29.2 ml in aq. methanol, Fig. 2.2).

Dopamine monosulphate (C 8H11N05S) requires: C 41.2%

H 4.7%

N 6.1% found: C 37.8%

H 5.1%

N 6.4%

Melting point 253-255°C (d). -75-

OH

8 7

— 9~ 2'5 250 H nmr spectrum of dopamine 4-0-sulphate (product II). -76-

2.2.11.2 Nuclear magnetic resonance spectra.

n.m.r. analysis of product III showed that it is undoubtedly dopamine 3-0-sulphate (Fig. 2.6). Apart from the low frequency signals corresponding to the four aliphatic protons, again three aromatic proton resonances were observed. The highest frequency of these (down field) was a doublet of small coupling (meta) corresponding to a proton adjacent to the OSO^" group (2-position). Next was observed a quartet corresponding to the proton in the 6-position, which is coupled both ortho and meta. Finally, the doublet corresponding to the 5-proton, coupled only ortho was observed. Thus, this spectrum could not have arisen from either a sulphonic acid or dopamine 4-0-sulphate and therefore product III is dopamine 3-0-sulphate.

Inspection of Figs. 2.5 and 2.6 shows that the 3-0-sulphate is contaminated with a small quantity (approximately 5%) of the

4-0-sulphate, which is consistent with h.p.l.c. purity checks. -77-

+ CHjCHjNH j

Fig. 2.6 250 MHz nmr spectrum of dopamine 3-C>-sulphate (product III). -78-

2.3 CHEMICAL SULPHONATION OF NORADRENALINE.

2.3.1 Materials.

Chemicals were obtained from the following sources: noradrenaline

base, noradrenaline bitartrate (Sigma Chemical Co., London, U.K.) and

N^N-diethyl aniline (Aldrich Chemical Co., Gillingham, U.K.). The

following chemicals were obtained from BDH Chemicals, Poole, U.K.:

chlorosulphonic acid, potassium acetate, potassium hydroxide,

phosphorus pentoxide, Dowex 50 X 8 (200-400 mesh, H+ form), CSg*

Sulphatase type IV from Aerobacter aerogenes was purchased from Sigma

Chemical Co., London, U.K.

2.3.2 Methods: Reaction of noradrenaline with H^SO^ (sp.g. 1.84).

Five batches of the reaction mixture were set up at the same time.

In each case the reaction was performed by adding noradrenaline

bitartrate (7.2 mg, 0.23 mmol) which has been kept over P^O^ in vacuo

for 24 h to stirred cone. H^SO^ (sp.g. 1.84, 22 pi, 0.31 mmol) at 0, 20

and 37°C. The reaction was stopped by pouring the mixture into ice-

cold H^O (1 ml) at the following time intervals: 5, 10, 20 min for

samples reacting at 0°C, and after 20 min for samples reacting at 20 and

37°C. The above reaction mixtures were retained for h.p.l.c. analysis.

2.3.3 High performance liquid chromatography (h.p.l.c.).

Systems used for both analytical and preparative scale chromatography

consisted of Hypersil ODS (5 pm particle size) column (250 X 10 mm,

Shandon Southern Products Ltd.), Waters Associates pump 6000A, Waters

variable wavelength detector (model 450). Solvents used were either:

potassium acetate (0.001M; pH 4.3) for analytical scale preparations, or

H2O for preparative preparations. Solvent was usually delivered at the

speed of 2 ml min ^ and the effluent monitored at 280 nm. -79-

2.3.4 Results: High performance liquid chromatography analysis of

products formed during sulphonation of noradrenaline by — 2^ 4

(sp.g. 1.84).

Diluted reaction mixture (3-5 pi) containing approximately 15-25 pg of noradrenaline-related material was injected onto h.p.l.c. with potassium acetate (0.001 M, pH 4.3) as eluent. The results are summarised in

Table 2.2.

Table 2.2. Products of sulphonation of noradrenaline with H^SO^

at various temperatures and time intervals.

Approximate yield of product %

* ** Temp 0°C Time min (18) (23) (24) (28) (38)

0 5 46 2.0 7.0 2.0 43.0

0 10 60 2.0 2.0 10.0 26.0

0 20 80 0 2.0 5.0 15.0

20 20 nd nd nd 95.0 5.0

37 20 nd nd nd 100.0 nd

★ Elution volume (ml) in above h.p.l.c. system

Co-chromatographed with noradrenaline bitartrate

Approximate yield of product was calculated from the peak area, nd - non detected

When H2SO4 (sp.g. 1.84) reacted with noradrenaline bitartrate at 0°C for 5 min, approximately half of the noradrenaline remained unreacted with the major product eluting at 18 ml (46%) and a small amount of -80-

products eluting at 23 ml (2%), 24 ml (7%) and 28 ml (2%). This product

(18 ml elution volume) also predominated in the time-dependent fashion when reaction was carried out at 10 and 20 min. It constituted 60 and

80% of total products respectively. However, quite a different product composition was observed when the reaction was performed at 20 and 37°C

for 20 min. In this case, the only product that was formed eluted at

28 ml with no presence of other products and only 5% of unreacted noradrenaline was noted at 20°C. Since products eluting at 18 and 28 ml were the major products of the reaction between noradrenaline and ^2 ^ 4

under the described conditions, they were isolated and purified after

repetitive injection and reinjection on h.p.l.c., to obtain mg quantities of these for further analysis.

2.3.5 Hydrolysis by sulphatase and HC1.

The major products of the aforementioned reaction (18 and 28 ml

elution volume in 0.001 M CH3C00K pH 4.3) were subjected to hydrolysis

by sulphatase type IV from Aerobacter aeroqenes (0.18 units in 2 M Tris

for 2 h at 37°C) and by 0.5 M HC1 at 100°C fo 10 min. Neither of these compounds hydrolysed to give free noradrenaline under the above

conditions, which would not have been expected for an 0-sulphate.

Therefore it was thought that isolated materials are not monosulphates of noradrenaline and alternative ways of synthesizing the sulphates were

tr i ed.

2.3.6 Reaction of noradrenaline with pyridine-sulphur trioxide.

This reaction was adapted from a method described by Mitchell (1977)

used for sulphonation of tyramine. Noradrenaline bitartrate (17.2 mg,

0.56 mmol) was dissolved in DMS0 (100 pi) and pyridine-sulphur trioxide

(1.6 mg) was added to dry pyridine (70 pi) and dry triethyl amine (5 pi) -81-

and allowed to react at 22°C for 16 h. The reaction mixture was then diluted with to 10 ml. An aliquot (5 pi) containing approximately

9 pg of noradrenaline-related material was injected onto h.p.l.c.

(conditions of analysis as described in Section 2.3.3). Only one peak

(38 ml elution volume in 0.001 M CH^COOK pH 4.3) was observed, which co­ chromatographed with noradrenaline. Since no products were formed

during this reaction, it was not pursued further.

2.3.7 Sulphonation of noradrenaline by chlorosulphonic acid.

Sulphonation of noradrenaline was performed using chlorosulphonic

acid according to the method described by Rose and Bleszynski (personal

communication), whereby chlorosulphonic acid (0.33 ml, 5 mmol) and

carbon disulphide (2 ml) were mixed and cooled to -7°C on ice-NaCl

mixture, with continuous stirring, N^-diethylaniline (1.6 ml, 10 mmol)

was added dropwise over 10 min, the mixture was then cooled to -10° and

noradrenaline base (0.85 g, 5 mmol) was added. Noradrenaline which

adhered to the walls of the test tube was washed off with a further

2 ml of CSg*

The reaction mixture was stirred in an ice bath at -7°C until very

dense. It was then transferred to the water bath at 25°C and mixing

continued for another 10 min. The sample was left for 16 h at 2°C,

whereafter the layers of CS^ were poured off and the solid reaction

mixture was heated to 40°C to make it plastic and then mixed with ice-

cool water (15 ml). During the solubilization of the sulphonation

products, NHg solution was added to keep the pH 4.0 - 6.0. Aliquots of

this reaction mixture were retained for h.p.l.c. analysis. Further

purification procedures described by the above authors and consisting of

several steps including column chromatography, paper chromatography, -82-

electrophoresis, precipitation and concentration of synthetized

compounds were not applied.

2.3.8 H.p.l.c. analysis of noradrenaline sulphonation products.

An aliquot (2 pi, 100 pg) of the above reaction mixture containing

noradrenaline-related material was analysed by h.p.l.c. Three peaks

(I,II,III) which eluted at 23, 24 and 38 ml respectively were observed.

Peak III which contained 96% of the material co-chromatographed with

noradrenaline, peaks I and II contained only 2% each of the analysed material. These two peaks (I and II) were isolated from the reaction mixture by preparative h.p.l.c. Aliquots (200 pi) containing 10 mg of

material were injected and reinjected onto h.p.l.c. and eluted with h^O.

Elution volumes of product I and II in water were 23 and 24 ml

respectively. Fractions from h.p.l.c. containing products I and II were concentrated in vacuo at 37°C and subsequently subjected to hydrolysis

by Aerobacter sulphatase and 0.5 M HC1 according to the procedures described in Section 2.3.5. Both products (I and II) were hydrolysed

under the above conditions to yield free noradrenaline. This observation suggested that these products might be noradrenaline

sulphates. From the experience with the synthesis of dopamine sulphates

it is known that other products which might be formed during such a reaction i.e. sulphonic acids are resistant to hydrolysis by both sulphatase and 0.5 M HC1. These two compounds were also minor products of noradrenaline reaction with H^SO^ sp.g. 1.84 (elution volumes 23 and

24 ml, Table 2.2). However, the yield of products I and II from the

aforementioned reactions was rather small and insufficient for further analysis including elemental analysis and nuclear magnetic resonance.

Therefore, attempts were made to modify the reaction of noradrenaline -83-

with chlorosulphonic acid to improve the yield of the aforementioned products.

2.3.9 Sulphonation of noradrenaline by chlorosulphonic acid: the

effect of varying reaction time.

Reaction mixtures, each consisting of noradrenaline base (5 mg,

0.029 mmol), chlorosulphonic acid (60 pi; 0.9 mmol) and

U, N-di ethyl an i 1 ine (10 pi, 0.062 mmol), were left to react in the ice

bath (temp, of the reaction mixture 7°C) for 5, 10, 30 min, 2 h and

16 h. After the appropriate time the reaction was stopped by pouring

the mixture into H^O (2 ml). These diluted samples were kept frozen at

-20°C until analysis by h.p.l.c. The results are presented in

Table 2.3.

Table 2.3. Products of reaction of chlorosulphonic acid with

noradrenaline at various time intervals.

Approximate yield of product %

Reaction time Product l Product II Product III (23 ml) (24 ml) (38 ml)

5 min 3 38 59

10 min 4 48 48

30 min 5 50 45

2 h 5 50 45

16 h 2 2 96

* elution volume ** co-chromatographed with noradrenaline -84-

The above results indicate that products I and II are formed in a much shorter time (5 - 120 min) than the original method of Rose and

Bleszynski (16 h) suggested. Since at 30 min and 2 h the total product content was 55%, it was decided to use 30 min as the reaction time. All subsequent reactions were performed during this time. Although the total product formation was 55% after 30 min and 2 h, product II was formed in 10-fold higher proportion (50% of total product) than product I (5% of total product). To achieve a better proportion in the formation of these two products, variations in reaction mixture composition were investigated.

2.3.10 Sulphonation of noradrenaline by chlorosulphonic acid: the

effect of varying reaction mixture composition.

Reaction mixtures containing noradrenaline base (5 mg, 0.029 mmol) and variable amounts of chlorosulphonic acid and N^N-diethyl ani1ine were allowed to react for 30 min on the ice bath (reaction mixture temp.

7°C). The reaction was stopped by diluting the mixture in 1^0 (2 ml).

As before, the aliquots of these were retained for h.p.l.c. analysis.

The data are presented in Table 2.4.

The results shown in Table 2.4 show that proportions of chlorosulphonic acid and N^-diethylanil ine used in relation to one another and to the amount of noradrenaline present in the reaction mixture play an important role in product formation. Thus, high concentration of chlorosulphonic acid (1 mmol, 34-fold excess in relation to noradrenaline base) and low concentration of N^-di ethyl - aniline (0.0186 mmol) resulted in 77% of unreacted noradrenaline and only 23% of products I and II. Similar observations could be made when a low concentration of chlorosulphonic acid (0.45 mmol, 15.5-fold Table 2.4 Products of reaction of chlorosulphonic acid with noradrenaline: the effect of varying

reaction mixture composition.

Approximate yield of product %

Chlorosulphonic fM-di ethyl aniline noradrenaline base Product*I Product II Product*III acid, mmol mmol mmol (23 ml) (24 ml) (38 ml)

1.00 0.0186 0.029 3.0 20 77

0.30 0.0186 0.029 15.0 51 34

0.97 0.031 0.029 5.0 35 60

0.90 0.062 0.029 5.0 50 45

0.75 0.124 0.029 4.0 41 55

0.60 0.186 0.029 5.0 41 54

0.45 0.248 0.029 3.0 25 72

Elution volume

Co-chromatographed with noradrenaline base

i Ln0 0 i - 8 6 -

excess in relation to noradrenaline used) and a high concentration of

N^N-diethyl aniline (0.248 mmol, 8.5-fold excess in relation to noradrenaline) was used, again unreacted noradrenaline constituted over

70% of total product, the rest being products I and II (28%). With decreasing concentrations of chlorosulphonic acid in relation to noradrenaline (33-, 31-, 25.8-, 20.6-fold excess) and increasing concentrations of N^N-diethylaniline (1.1-, 2.1-, 4.3-, 6.4-fold excess in relation to the substrate) products I and II constituted 40, 55, 45,

46% respectively, the rest being unreacted noradrenaline. The best results (66% formation of product I and II) were noted when 0.3 mmol of chlorosulphonic acid (10.3-fold excess in relation to the substrate) and

0.0186 mmol of J^,N-diethyl aniline (0.64 ratio with noradrenaline) were reacted with 0.029 mmol of noradrenaline base. This reaction yielded also the highest formation of product I (15%) in comparison with various others (Table 2.4). The above reaction mixture was then scaled up to obtain milligram quantities of products I and II.

2.3.11 Preparative scale synthesis of authentic noradrenaline 3- and

4-0-sulphates.

Noradrenaline (2.5 g, 14.7 mmol) was reacted with chlorosulphonic acid (10 ml, 151.5 mmol) containing jM -d i ethyl anil ine (1.5 ml,

9.3 mmol) for 30 min at 7°C. The reaction was stopped by pouring the reaction mixture into water (10 ml), BaCO^ was added until pH 5 was reached, BaSO^ formed during this procedure was removed by centrifugation. The supernatant was applied to Dowex 50 X column (580 X

16 mm, 200-400 mesh, H+ form) and eluted with water. Fractions (6-8 ml each) were collected and examined by h.p.l.c. and for pH. Fractions 11-28

(A) (elution volume 192 ml) pH > 5.0 contained a mixture of products I -87-

and II (h.p.l.c. elution volume in H^O or 0.001 M CH^COOK pH 4.3; 23 and 24 ml). Fraction A was then kept for further purification by h.p.1 .c.

Fractions 50-200 (B) (elution volume 1120 ml) pH > 5.0 contained mainly product I (h.p.l.c. elution volume 23 ml). All the other fractions (29-49) contained a small proportion of the aforementioned products and were discarded. Fractions A and B were concentrated in vacuo at 37°C to 10 ml. Preparative h.p.l.c. (eluent H^O) was employed to isolate and purify products I and II.

Portions containing 1-2 mg of noradrenaline material were injected and reinjected onto h.p.l.c., fractions containing product I and product

II were collected. Fractions from h.p.l.c. containing product I

(elution volume 23 ml) were concentrated in vacuo at 37°C to approximately 0.5 ml, then ethanol (5 ml) and ether (50 ml) were added.

The white precipitate that appeared was separated from the supernatant by centrifugation and dried in N2 stream and afterwards in the dessicator over Po0c and K0H. The same procedure was repeated for fractions containing product II (elution volume 24 ml in H^O). Products of chemical sulphonation of noradrenaline are shown in Fig. 2.7.

2.3.12 Results: Product I structural determination.

2.3.12.1 Product I: Elemental analysis and melting point.

This material was isolated by preparative h.p.l.c. (elution volume

23 ml, single peak, Fig. 2.8); yield 47 mg of white powder.

Elemental analysis:

CoH NO S (noradrenaline monosulphate) o i l 0 requires: C 38.6%

H 4.4%

N 5.6% Fig. 2.7 Products of chemical sulphonation of noradrenaline.

I noradrenaline 4-0-sulphate II noradrenaline 3-0-sulphate -89-

r t INJECT

Fig. 2.8 Hplc chromatogram of products formed during chemical sulphonation of noradrenaline by chlorosulphonic acid.

I noradrenaline 4-0-sulphate II - noradrenaline 3-()-5ulphate III - unreacted noradrenaline -90-

found: C 38.6%

H 4.4%

N 5.5%

Melting point 161-162°C.

2.3.12.2 Nuclear magnetic resonance spectroscopy.

Product I was also subjected to analysis by nuclear resonance.

n.m.r. spectra were acquired on samples of the above product (5-10 mg)

as solutions in d^-DMSO using a Bruker n.m.r. spectrometer operating at

250 MHz. The spectrum of product I is shown in Fig. 2. 9.

n.m.r. analysis of product I revealed that the most likely

structural assignment is that of noradrenaline-4-0-sulphate. The 250

MHz spectrum showed three aromatic signals corresponding to each of

three protons. The most deshielded proton in this structure (Fig. 2.9)

would be that adjacent to the OSO^" grouping, i.e. that in the 5-

position. The appearance of this signal as a doublet with a typical

ortho-coupling is consistent with this interpretation (doublet 7.15;

J ). Next upfield a doublet with coupling meta was observed (6.87, J_), o --- m which can be assigned to the proton in position 2. Finally a quartet

was observed with both ortho and meta coupling which is indicative of

the proton in the 6-position (6.77, J ). a,m Such a spectrum could not have been obtained from noradrenaline

3-0-sulphate nor free noradrenaline. Aliphatic protons gave a complex

signal identical to noradrenaline showing absence of side-chain

sulphation ( 6 ppm are relative to TMS).

2.3.13 Results: Product II structural determination.

2.3.13.1 Product II: Elemental analysis and melting point.

This compound was isolated by preparative h.p.l.c. (elution volume -91-

o3so 3

Fig. 2.9 250 MHz nmr spectrum of noradrenaline 4-0-sulphate (product I).

6.77 (J ), 6.87 (J ), 7.15 (J ). a,m m a -92-

24 ml, Fig. 2.8) as described in Section 2.3.11; yield 63 mg of white powder. H.p.l.c analysis showed contamination of this material with product I (1 .6%).

Elemental analysis:

CgHnNOgS (noradrenaline monosulphate) requires: C 38.6%

H 4.4%

N 5.6% found: C 39.1%

H 4.5%

N 5.7%

Melting point 164°C.

2.3.13.2 Nuclear magnetic resonance spectroscopy.

n.m.r. spectra were acquired on samples of product II (5-10 mg) as solutions in dc-DMS0 using a Bruker spectrometer operating at 250 MHz. o The spectrum of product II is shown in Fig. 2.10.

n.m.r. analysis of product II showed that it is undoubtedly noradrenaline 3-0-sulphate. Again, three aromatic resonances were observed. The highest frequency was a doublet of a small coupling meta

(6.83, JCT) corresponding to the proton in the 2-position. Next was observed a quartet corresponding to the proton in the 6-position which

is coupled both ortho and meta (6.97, J ). Finally, the doublet ------a ,m corresponding to the proton 5, coupled only ortho (7.20 Jm ) was observed. Again, aliphatic protons gave a signal identical to noradrenaline and no side-chain sulphation was observed. Thus this spectrum could have arisen only from noradrenaline 3-0-sulphate.

(6 ppm are relative to TMS). -93-

Fig. 2.10 250 MHz nmr spectrum of noradrenaline 3-()-sulphate (product II).

6.83 (J ), 6.97 (J J , 7.20 (J ). a a,m m -94-

2.4 DISCUSSION.

In this study, the reactions leading to the synthesis and characterisation of dopamine and noradrenaline 3- and 4-0-sulphates were investigated with reference to different sulphating agents and conditions of the reactions.

For the synthesis of dopamine sulphate isomers, the original reaction described by Jenner & Rose (1973) was investigated in more detail with reference to temperature and specific gravity of the sulphuric acid employed. This study has shown that the published synthesis of dopamine 3- and 4-0-sulphates does not yield these two products solely, rather a mixture of dopamine 6-sulphonic acid together with the isomeric sulphates and some unreacted dopamine. This observation strongly suggests that subsequent workers (Merits, 1976; Arakawa et al_., 1979;

Buu & Kuchel, 1979a, b) who followed the original method (Jenner & Rose,

1973) could not have isolated the pure sulphates. Nevertheless, claims of biological significance, either as metabolic excretory products or intermediates in noradrenaline biosynthesis, have been made for these

O-sulphates. Of particular interest is the notion that noradrenaline can be formed from both dopamine_0-sulphates in a single step reaction by dopamine $-hydroxylase (Buu & Kuchel, 1979a, b).

In this study even the Michaelis-Menten kinetics of the reaction were described. This would seem an unwarranted extrapolation without a chemical foundation, since in this chapter it is clearly shown that small changes in sp.g. of sulphuric acid or in reaction temperature can give rise to the presence of either dopamine 6-sulphonic acid or unreacted dopamine in the product. Because all the three products of dopamine sulphonation are isomeric chemically-similar organic acids, they will not be distinguished by elemental analysis, ultra-violet, -95-

infra-red spectroscopy or by Gibbs' reaction, particularly when they are difficult to obtain in a pure form. It is considered that n.m.r. was the only technique powerful enough to distinguish these isomers and h.p.l.c. the most efficient method of separation and purification.

Because of the formation of dopamine 6-sulphonic acid, the use of h.p.l.c. is strongly recommended both to monitor the purity of the synthesised dopamine sulphates and to separate milligram quantities of these for biochemical experiments. The products of chemical sulphonation of dopamine, i.e. 6-sulphonic acid, 4-£-sulphate and 3-0- sulphate could be identified from their retention volumes (2% aq. methanol) on reverse phase h.p.l.c. (5 pm Hypersil ODS) relative to the retention volumes of dopamine itself. Relative retention volumes calculated from the data presented in Table 2.1 are 0.07, 0.20 and 0.23 respectively for the above derivatives.

Dopamine, the third endogenous catecholamine, has been recognised as a neurotransmitter in the CNS for over 20 years (Hornykiewicz, 1972).

Recently it has assumed greater importance due to the recognition of its physiological effects mediated through specific receptors in the peripheral nervous system (Goldberg, 1972; Thorner, 1975).

Furthermore, the endogenous precursor of dopamine, L-dopa, is widely used in the treatment of Parkinsonism (Anden et al_., 1970; Rutledge &

Hoehn, 1973). Obviously, the metabolic transformation of the neurohormone and its precursor may be important factors in the physiological regulation of dopaminergic effects. If conjugation with sulphate is a major component of dopamine metabolism as suggested

(Jenner & Rose, 1973; Jenner & Rose, 1974; Merits, 1976; Arakawa et al., 1979), it is important to quantitate the sulphates using authenticated synthetic standards. -96-

Similar methodology to that of dopamine sulphate synthesis was applied to the synthesis and characterisation of noradrenaline 3 and

4-0-sulphates. However, noradrenaline being a more polar compound than dopamine reacted differently with the sulphating agent used for dopamine sulphate synthesis, i.e. sulphuric acid. Thus, the reaction between noradrenaline and sulphuric acid resulted in the formation of five products:- unreacted noradrenaline, two noradrenaline sulphates, which were only minor products of this reaction, yielding 9% and 4% of the total product; and two remaining major products were unidentified

(Table 2.2). The latter were isolated, but since they proved resilient to hydrolysis by Aerobacter sulphatase and 0.5 M HC1, they were thought to be noradrenaline sulphonic acids, and were not analysed further.

Pyridine-sulphur trioxide, as in the case of dopamine, proved to be too mild a sulphonating agent and did not give rise to noradrenaline sulphates. The most successful reaction leading to the formation of noradrenaline sulphates was that adapted from the method of Rose and

Bleszynski (personal communication). However, it had to be modified to a large extent to improve the yield of products of interest. The changes made in the conditions of this sulphonation involved the use of chlorosulphonic acid in 10-fold molar excess in relation to noradrenaline not only as a sulphonating agent, but also a solvent.

Therefore, CS^ used in the original method was omitted from the reaction, N,Nhdiethyl aniline content was reduced by 70% and the reaction time was shortened from 16 h to 30 min.

Under the above conditions, noradrenaline monosulphates constituted

66% of the total products, whereas in the original reaction the total yield of these was 8%. Further purification was also simplified. The original method included column chromatography on Amber!ite IR 120 (H+ ), -97-

precipitation of excess inorganic sulphate, concentration in vacuo of fractions from the Amberlite column followed by further purification on another Amberlite column, and finally chromatography on Ecteola- cellulose column and on paper. It is not surprising that such an elaborate procedure resulted in great losses of products and did not lead to the separation of the isomers. To achieve better recovery for noradrenaline 3 and 4-0-sulphates the isolation and purification was limited to chromatography of the reaction mixture on Dowex 50 X 8 (H+ ) columns and further purifications of fractions that contained the mixture of products on preparative h.p.l.c. to > 95% purity. It is possible that noradrenaline 3 and 4-0-sulphates could be identified from their retention volumes (H^O, 0.001 M CH^COOK pH 4.3) on reverse phase h.p.l.c. (5 pm Hypersil ODS) relative to the retention volumes of noradrenaline itself. Relative elution volumes calculated from the data in Table 2.4 are 0.60 and 0.63 respectively for the 4- and 3-0-sulphate derivative.

Since h.p.l.c. is now widely available, noradrenaline and dopamine sulphates can be easily separated and purified. In theory, material isolated from a single preparative h.p.l.c. run whether it be from biological/chemical origin should be sufficient for definitive structural assignment by modern 250 or 300 MHz n.m.r. techniques in the small scale isolation and characterisation of, for example, noradrenaline and dopamine sulphates. The above techniques combined together present the best opportunity for obtaining pure, characterised synthetic sulphate conjugates of catecholamines and related compounds.

Additionally, the study presented in this chapter provides the basis for reappraisal of the biological importance of dopamine sulphates and also provides the basis for the development of a direct assay for noradrenaline sulphates. -98-

CHAPTER 3.

DISPOSITION OF DOPAMINE O-SULPHATES IN HUMAN URINE.

3.1 Introduction ...... 99

3.2 Materials ...... 101

3.3 Methods: Preparation of urine samples ...... 101

3.3.1 High performance liquid chromatography assay for

the estimation of dopamine 3- and 4-0-sulphates in

urine ...... 102

3.3.2 The estimation of dopamine ^-sulphates in urine

before and after L-dopa administration and in

multiple samples from a single subject ...... 103

3.4 Results: Recovery of dopamine O-sulphates in

purified urine samples ...... 104

3.4.1 Estimation of dopamine ^-sulphates by high

performance liquid chromatographic method

with electrochemical detection ...... 104

3.4.2 Disposition of dopamine O-sulphates in urine

before and after L-dopa administration and

in the multiple samples from a single subject ... 106

3.5 Discussion ...... 113 3.1 INTRODUCTION

Several workers have studied the disposition of dopamine sulphate isomers in human urine following L-dopa administration (Jenner & Rose,

1974; Bronaugh et _al_., 1975; Arakawa et aj_., 1979). Jenner & Rose

(1974) were first to develop a method to estimate dopamine 3- and

4-0-sulphates in human urine. The assay they used was time consuming and tedious. It involved ion-exchange chromatography of large volumes of urine, subsequent hydrolysis of fractions containing the appropriate dopamine isomer and finally estimation of free dopamine by the trihydroxyindole procedure. This method required the addition of labelled dopamine O-sulphate isomers to locate the appropriate isomer and to calculate recoveries from the ion-exchange column and also the efficiency of the hydrolysis procedure. Bronaugh et al_. (1975) were first to develop a method to estimate dopamine O-sulphate conjugates by high performance liquid chromatography (h.p.l.c.). The sample preparation procedure they used required more than 12 h. The sensitivity of the detection was low (pg level) and the separation of dopamine 3- and 4-0-sulphates by h.p.l.c. seemed incomplete even with a

2-m column of Zipax SAX (Du Pont).

Arakawa et a]_. (1979) developed a simpler and more rapid method for the measurement of dopamine sulphate conjugates in urine. This method involved purification of urine on small cation and anion exchange resin columns, and the quantitation of the conjugates by using silica gel h.p.l.c. with a spectrophotometric detector. The sensitivity of this method was approximately 50 mg. None of the aforementioned groups of workers attempted to measure the dopamine sulphate conjugates in urine under physiological conditions, because the above methods did not show high enough sensitivity. Buu & Kuchel (1977) developed an indirect -1 0 0 -

method for the estimation of urinary dopamine sulphates. These workers hydrolysed dopamine sulphates in urine, then estimated free and total dopamine and the difference in free and total constituted conjugated dopamine. Although this method was sensitive (ng level), it did not allow for estimation of the two isomers separately, and it is not known

if complete hydrolysis of dopamine conjugates occurred.

In this Chapter a simple, rapid and sensitive method for the measurement of dopamine 3- and 4-0-sulphates in urine is described.

This method utilizes reverse phase h.p.l.c. with electrochemical

detection and could be used for the estimation of dopamine sulphate

isomers in urine at physiological levels as well as in urine after

L-dopa treatment. Furthermore, data are presented on the disposition of

dopamine sulphate isomers in urine before and after L-dopa

administration and in multiple urine samples from a single subject. -1 0 1 -

3.2 MATERIALS

Dopamine 3- and 4-0-sulphates and [^C]-dopamine 3- and 4-0-sulphates 5 S (sp. activity 8.4 X 10 cpm/mg and 4.7 X 10 cpm/mg for 3- and 4-0- isomers respectively) were prepared as described in Chapter 2. The remaining chemicals were obtained from the following sources: 14 3,4-dihydroxyphenyl(2'- C)ethylamine hydrochloride (sp. activity

61 mCi/pmol) (The Radiochemical Centre, Amersham, U.K.); Dowex 1 X 8

(200-400 mesh, chloride form, BDH Chemicals, Poole, U.K.); ammonium acetate and ascorbic acid (BDH Chemicals, Poole, U.K.); acetic acid

(Fisons Scientific, Loughborough, U.K.); tetrabutylammonium hydroxide

(40%) and EDTA (Sigma Chemical Co. Ltd., London, U.K.); ammonium-backed cellulose plates (Merck, Darmstadt, W. Germany, DC-Alutolien Cellulose

^254)’ butan"l"0^ was h.p.l.c. grade (Fisons Scientific,

Loughborough, U.K.).

3.3 Methods: Preparation of urine samples.

An aliquot (1 ml) of 24 h urine containing ascorbic acid and EDTA

(0.1 mg of each/1 ml) was applied to a Dowex 1 X 8 (200-400 mesh, acetate form, 10 mm X 8 mm) column. The column was washed with ice-cold

H^O (10 ml) and the elution was carried out with cold acetic acid

(0.2 M, 10 ml). The acetic acid eluate from an ion-exchange column was freeze-dried on a Modulyo (Edwards High Vacuum, Crawley, U.K.) and afterwards reconstituted in methanol (0.4 ml). Methanol was evaporated under ^ stream to approximately 0.05 ml and applied to aluminium-backed cellulose strips (20 X 5 cm). The plates were developed overnight

(15 h) in the following solvent system: butan-l-ol :acetic a c i d ^ O

(12:3:5 v/v). To locate compounds of interest on the tic plate, authentic dopamine 3- and 4-0-sulphates (10 pg) were applied to a strip -102

alongside the samples. Tic plates were dried at room temperature and dopamine O-sulphates were located by spraying the strip containing authentic compounds with ninhydrin (0.25%) in acetone and then heating for 5 min (100°C) (dopamine sulphates showed as a purple band). Strips

(0.5 X 5 cm) with comparable Rf values to authentic dopamine O-sulphates were cut out, and compounds of interest eluted from cellulose with acetic acid (0.2 M, 100 pi). Before application onto h.p.l.c., the samples were centrifuged at 10,000 g for 2 min in an Eppendorf 5412 centrifuge (Anderman, East Molesey, U.K.). The standard samples contained H^O (1 ml, containing 0.1 mg ascorbic acid and EDTA) and authentic dopamine 3- and 4-0-sulphates (in concentration 200-1000 ng/ml; 0.8 - 4 nmol/1 ml). They were taken through the same procedure as urine samples and were used to calculate recovery of compounds of interest in the urine samples. To calculate recovery after each step of 14 the purification procedure, standards containing [ C] dopamine £- 2 sulphates were used (5 pg; sp. activity 42 X 10 cpm/5 pg, 3-0-isomer or 2 sp. activity 23 X 10 cpm/5 pg, 4-0-isomer).

3.3.1 High performance liquid chromatography assay for the estimation

of dopamine isomeric sulphates in urine.

An aliquot (1-6 pi) of acetic acid eluate (from T.L.C.) was injected onto the h.p.l.c. system that consisted of: solvent delivery system 6000

A (Waters Associates Inst. Ltd., Hartford, U.K.) operating at 0.8 ml/min, U6K injector (Waters Associates), pre-column (Hypersil-ODS, 5 pm,

25 X 5 mm, Anachem Ltd., Luton, U.K.), column Ultrasphere IP (250 X 46 mm, Anachem Ltd.), detector cell (TL-5, Bioanalytical Systems Inc., West

Lafayette) with glassy carbon electrode and Ag/AgCl reference electrode.

The potential between electrodes (+ 0.75 V) was kept constant and the current flow measured by an electronic controller unit (LC-4, -103-

Bioanalytical Systems Inc.). The chromatograms were recorded on a Data

Module (Waters Associates).

The mobile phase consisted of ammonium acetate (0.08 M), tetrabutyl-

ammonium hydroxide (ion pairing reagent, 0.015 M), EDTA (0.0001 M),

methanol (5%, v/v) and was adjusted to pH 5.8. Before use, the mobile

phase was filtered through 0.45 p acetate filters (Millipore HAWP) and

de-gassed.

Dopamine 3- and 4-0-sulphates in the urine samples were identified by

their retention time which corresponded with the authentic standards.

The content of dopamine 3- and 4-0-sulphates in urine was determined

by reference to standard samples of the two sulphates taken through the

same procedure. Several standard concentrations were used and a

standard curve was constructed (peak height v. amount injected).

External standards were run at regular intervals to determine the

sensitivity of the detector. Retention times for dopamine ^-sulphates

varied slightly (10%) from assay to assay as freshly prepared solvent

was used for each assay.

3.3.2 The estimation of dopamine 0-sulphates in urine before and

after L-dopa administration and in the multiple samples from

a single subject.

Subjects were 4 healthy men (24 to 39 years, mean age 30.5 years).

Urine (24 h) samples were collected, and the volume measured. Then

L-dopa (0.5 g, Levodopa, Hoffman-La-Roche) was administered orally.

Following the administration of L-dopa, urine was collected for a

further 24 h. An aliquot of urine from each collection was kept at

-20°C until assayed.

In a further study, urine (24 h) from a single subject (male, 30

years) was collected over a period of 12 days (12 collections); an -104-

aliquot from each collection was kept at -20° until assayed.

Results are expressed as mean ± standard deviation (S.D.).

3.4 Results: Recovery of dopamine O-sulphates in purified urine

samples. (Dowex 1 X 8 step and TLC step). 14 This study was performed as described in Section 3.3. [ C]Dopamine

O-sulphates applied to Dowex 1 X 8 column were recovered in

86.8 ± 4.1% (n = 6) in acetic acid (10 ml).

The pH of the mixture applied to the Dowex 1 X 8 column had no effect on recovery from the samples. Samples of urine adjusted to various pH (2.5, 4.5, 5.5, 6.5, 7.5, 8.0) were tested and showed very similar recoveries (80.8%, 85.6%, 90%, 84.1%, 90.8%, 89.8% respectively for each pH). Therefore urine samples were applied to Dowex 1 X 8 ion exchange column without being adjusted to any particular pH.

An eluate from Dowex 1 X 8 column, prepared as described previously, was applied onto TLC. Recovery was calculated by counting an eluate from TLC (0.2 M acetic acid, 100 pi) in PPO monophase (New England

Nuclear, 10 ml) for 10 min (6880 Liquid Scintillation, Merck III, Searle

Analytic).

From TLC, 63.5 ± 4 . 4 % (n = 6) of dopamine sulphates were recovered.

This value represents also the overall recovery of dopamine ^-sulphates after all the steps of the purification procedure.

3.4.1 Estimation of dopamine isomeric sulphates by h.p.l.c. with

electrochemical detection.

Retention times on h.p.l.c. for authentic dopamine 4-() and 3-0- sulphates under conditions described previously were 13.1 and 13.9 min

(± 10%) respectively. Inter-assay variation as calculated for recovery -105-

Fig. 3.1 Standard curve for estimation of dopamine 3- and 4-0-sulphates. -106-

of a standard (10 ng) was 14% for dopamine 4-0-sulphate and 8.0% for noradrenaline 3-0-sulphate (n = 4). The standard curve for dopamine 3- and 4-0-sulphates is shown in Figure 3.1.

The amounts of dopamine ^-sulphates in urine were corrected for injection volume since in some assays, e.g. those following L-dopa administration, samples were diluted by a factor of 5 or 10 prior to injection onto the h.p.l.c.. The detection limit of this assay was approximately 3 ng. Chromatograms from h.p.l.c. are presented in

Figures 3.2, 3.3, 3.4 and 3.5.

Retention times for dopamine 0-sulphates in samples after L-dopa were slightly shorter because of greater concentration injected.

3.4.2 Disposition of dopamine 0-sulphates in urine before and after

L-dopa administration and in the multiple samples from a single

subject.

The samples were collected and purified as described previously. The data obtained for control subjects (before L-dopa administration,

"normal" urine) are presented in Table 3.1.

Table 3.1. Dopamine 0-sulphate isomers content in urine under

physiological conditions.

Subject pmol/24 h % excreted as DA 3 0-S0^

R.M. 5.04 100

P.S. 6.06 100

R.U. 1.98 100 ★ A.M. 5.10 83 -107-

c c

Fig. 3.2 Hplc chromatogram of dopamine ^-sulphate standards (prepared as described in Section 3.3). -108-

Fig. 3.3 Hplc chromatogram of normal urine containing both dopamine ^-sulphates. -109-

Fig. 3.4 Hplc chromatogram of urine after L-dopa administration, for estimation of dopamine 4-0-sulphate content. -1 1 0 -

Fig. 3.5 Hplc chromatogram of urine presented in Fig. 3.4, appropriately diluted for estimation of dopamine 3-0-sulphate content. -1 1 1 -

* represents total dopamine £-sulphate content (dopamine 4-0-sulphate

constituted 0.86 pmol/24 h, 3-0-isomer 4.24 pmol/24 h).

Mean value for four subjects 4.5 ± 1.6 pmol/24 h.

The data presented above showed that only in one subject dopamine

4-0-sulphate was detected and constituted only 17% of total dopamine

(^-sulphates excreted. The ratio of 3-0 to 4-0 calculated for this subject was 4.9. The mean value of total dopamine 0-sulphates excreted showed quite large individual variation (4.5 ± 1.6 pmol). The analysis of samples following the oral administration of L-dopa confirmed the observation that dopamine 3-0-sulphate was the major urinary conjugate of dopamine. The data are presented in Table 3.2.

Table 3.2. Dopamine 0-sulphate isomers in urine following oral

administration of L-dopa (0.5 g).

Subject DA 4-0-S04 DA 3-0-SO Total excreted % of dose recovered as pmol/24 h pmol/24 h pmol/24 h total DA ()-S04

R.M. 49.5 245.7 295.2 11.6

P.S. 19.5 270.9 290.4 11.4

R.U. 63.2 187.6 250.7 9.9

A.M. 34.4 129.9 164.3 6.4

The results presented in Table 3.2 showed that following oral administration of L-dopa all subjects excreted in urine both dopamine-0- sulphate isomers. During 24 h, 9.8 ± 2.4% of orally administered L-dopa

(0.5 g) was excreted as total dopamine-O-sulphates. This value for the -1 1 2 -

recovery of L-dopa as dopamine sulphate conjugates was higher than that found by Jenner & Rose (1974) in the urine of Parkinsonian patients

(7.8%) and Arakawa et^l- (1979) in the urine of normal subjects after the oral administration of L-dopa (0.5 g) and a 6 h urine collection

(7.5%). The higher recovery for dopamine-0-sulphates found in this study in comparison with the results obtained by others, could be explained by the higher sensitivity of the assay procedure used.

The data for recovery of individual dopamine ^-sulphate isomers are presented in Table 3.3.

Table 3.3. Recovery of orally administered L--dopa (0.5 g) in 24 h urine

as dopamine 3- and 4-0-sulphates.

Subject % of dose recovered as % of dose recovered as

dopamine 3-£-sulphate dopamine 4-0-sulphate

R.M. 9.6 1.9

P.S 10.6 0.8

R.U. 7.4 2.5

A.M. 5.1 1.3

During 24 h, 8.2 ± 2.4% of orally administered L-dopa was recovered in urine as dopamine 3-0-sulphate and 1.6 + 0.74% as dopamine 4-0-sulphate. These results showed quite large individual variations, which is in agreement with results published by Jenner &

Rose (1974), but not with Arakawa et al_. (1979). Therefore, it can be concluded that after oral administration of L-dopa, dopamine 3-0- -113-

sulphate is the major conjugate found in the urine, 83 ± 7.7% (Table

3.2) and dopamine 4-£-sulphate constituted 17 + 7.9% of total dopamine sulphoconjugates found in the urine.

In multiple urine samples (12) from a single individual, a considerable variation existed in the excretion of total dopamine

^-sulphates (range: 1.66 - 5.39 pmol/24 h., mean 3.15 ± 1.07 pmol).

Dopamine 4-0-sulphate was identified in four out of twelve samples.

This isomer constituted 39.6 ± 18.5% of total dopamine 0-sulphate excretion. Dopamine 3-0-sulphate was found in all urine samples and constituted from 100 to 60.4 + 18.7% of total dopamine 0-sulphates.

These results indicated that greater differences existed in the excretion of dopamine 4-0-sulphate than dopamine 3-0-sulphate. Similar observations were made when urine samples from subjects before and following L-dopa administration were analysed.

3.5 DISCUSSION.

In this Chapter a new method for the estimation of urinary dopamine

3- and 4-0-sulphates is described. High resolution and sensitive detection (3 ng) of the two dopamine 0-sulphates was achieved by the use of reverse phase high performance liquid chromatography with electrochemical detection. The high sensitivity of this procedure allowed for the estimation of dopamine 0-sulphates in "normal" urine.

The direct assay for the estimation of the above sulphoconjugates in urine at physiological levels has not been described previously, as published methods relied on hydrolysis of dopamine 0-sulphates and further estimation of total dopamine (Buu & Kuchel, 1977; Imai et a!.,

1970). Published direct methods for the estimation of dopamine -114-

sulphates in urine lacked sensitivity and could only be used when the above compounds were estimated in the urine of Parkinsonian patients on high doses of L-dopa, or in subjects after oral L-dopa administration

(Jenner & Rose, 1974; Bronaugh et aT., 1975; Arakawa et a h , 1979).

These were referred to in the Introduction to this Chapter.

The method described in this Chapter requires a two step purification procedure before dopamine sulphates could be estimated by high performance liquid chromatography. Step one involves chromatography of urine on a small Dowex 1 X 8 column, which was necessary to separate dopamine conjugates from less polar compounds, i.e. free catecholamines.

A further purification procedure involved thin layer chromatography.

This step was essential to separate dopamine O-sulphates from other equally polar conjugates that eluted together with the above compounds from Dowex 1 X 8 column. Other methods were tried, such as chromatography on Amberlite columns. This was found unsatisfactory as dopamine sulphates were obscured by other polar constituents of urine.

When chromatograms from "normal" urine and those after oral administration of L-dopa were compared, it could be seen that on the chromatogram of "normal" urine several peaks of shorter retention time than dopamine-O-sulphates were present and at least one peak that showed longer retention time. On the chromatogram of urine after L-dopa only dopamine O-sulphate peaks were observed, because the amounts of these in the urine were so high that all other compounds present in the urine extract were insignificant in the chromatograms. Thus, it was much easier to estimate dopamine ^-sulphate conjugates in urine after L-dopa administration, as the concentrations of these were much higher than when physiological levels were recorded. The results obtained from urine samples of four subjects before L-dopa administration and from -115-

multiple samples of one subject confirmed previous observations (Jenner

& Rose, 1974; Bronaugh et a]_., 1975; Arakawa ^t al_., 1979) that dopamine 3-0-sulphate is the major urinary conjugate constituting

60-100% of total dopamine sulphoconjugates. In urine samples after

L-dopa administration, the 3-£-isomer constituted 83 ± 7.7% of total dopamine sulphoconjugates. The 4-0-sulphate conjugate was present in all urine samples after L-dopa administration, but only in 25% of the samples when measured under physiological conditions. The above results suggested that sulphoconjugation of dopamine might compete with 3-0- methyl ation, since dopamine 3-0-sulphate was a major urinary isomer.

The great variability in excretion of dopamine 4-0-sulphate implied that this isomer might be metabolised further before the excretion. Jenner &

Rose (1978) suggested that dopamine 4-0-sulphate was generally more readily desulphated that the 3-0-isomer. The lower levels of dopamine

4-0-sulphate found in urine as compared with its 3-0-isomer also might be explained by the finding that this isomer was extensively metabolised, without desulphation, yielding two metabolites (Jenner &

Rose, 1978). On the basis of the above data, it is difficult to provide a clear explanation for the differences existing in the excretion pattern of the two dopamine ^-sulphate isomers. However, since the method for the estimation of these compounds at physiological levels is now available, this study provides the basis for further experiments concerning the disposition and metabolism in vivo of dopamine 3- and

4-0-sulphates. -116-

CHAPTER 4.

DISPOSITION OF NORADRENALINE 3- AND 4-0-SULPHATES IN RABBIT TISSUES.

4.1 Introduction ...... 117

4.2 Materials ...... 118

4.3 Methods: Preparation of tissue homogenates ... 118

4.3.1 Tissue homogenates:- Preparation for analysis

by high performance liquid chromatography ... 119

4.3.2 High performance liquid chromatography with

electrochemical detection ...... 119

4.4 Results: Disposition of noradrenaline

sulphates in the brain 121

4.4.1 Disposition of noradrenaline sulphates

the heart 123

4.4.2 Disposition of noradrenaline sulphates in

the kidney 125

4.4.3 Disposition of noradrenaline sulphates

the spleen 127

4.4.4 Disposition of noradrenaline sulphates ii

the liver 129

4.4.5 Disposition of noradrenaline sulphates in

the small intestine 131

4.4.6 Products of hydrolysis of tissue homogenates 133

4.5 Discussion 135 -117-

4.1 INTRODUCTION

Several workers have studied the sulphoconjugation of catecholamines and demonstrated that this process occurs both in the central nervous system (Jenner & Rose, 1973; Renskers et _al_., 1980; Rein ^t ^1_., 1981;

Buu et aj_., 1981) and in the periphery (Jenner & Rose, 1973; Merits,

1975; Bronaugh et al_., 1975; Wong, 1978) of mammals. Recently it has been demonstrated that human platelets are capable of sulphating catecholamines (Hart et^l_., 1979; Weinshilboum & Anderson, 1981;

Sandler et al_., 1981).

Most investigators have studied sulphoconjugation of catecholamines following administration of catecholamines orally or parenterally, or by measuring phenolsulphotransferase (PST) activity in different tissues using catecholamines as substrates. However, there are only isolated reports of the investigation of the formation of and disposition of 3 and 4-0-sulphoconjugates of catecholamines.

The relative amounts of the 3- and 4-0-dopamine sulphate conjugates formed have been studied with dopamine as the acceptor by Jenner & Rose

(1973). They found dopamine 3-0-sulphate as the main product (> 99%) formed in incubations with crude rat liver preparations, whereas brain preparations formed the 3-0 and 4-0-sulphates in a molar ratio of 1.7.

Renskers et al_. (1980) found in their study on 3-0- and 4-0-sulphation of dopamine by human brain PST a ratio of 4.1.

Merits (1976) showed in vitro that the small intestine of the dog was the site of dopamine sulphate formation, whereas the small intestines of guinea pig and rat apparently lacked this ability. This author also claimed that dopamine 3-0-sulphate was a primary metabolite after the 14 oral administration of dopamine to the dog. In contrast, when C-dopa was administered to the pigtail monkey, dopamine 4-0-sulphate was the -118-

primary conjugate (Bronaugh et ak, 1974b). Except for this last observation, the above data suggest that the 3-0-sulphate isomer is the preferential catecholamine sulphate formed. This would be the expected finding since phenolsulphotransferase (PST) preferentially attacks the hydroxyl group at the 3-position (Wong, 1982), producing 3-0-sulphates with dopamine, adrenaline (Wong, 1978) and 3,4 dihydroxybenzoic acid

(Pennings & van Kempen, 1980).

In order to further investigate the distribution of catecholamine sulphate isomers in different tissues, a direct method for their estimation was developed. However, the presence of dopamine 0-sulphates was not detected in any of the tissues. The amounts of the noradrenaline ^-sulphate isomers were measured in brain, heart, liver, kidney, spleen and small intestine by a direct method utilizing high performance liquid chromatography.

4.2 MATERIALS

Noradrenaline 3 and 4-0-sulphates were prepared as described in

Chapter 2. Potassium chloride was obtained from BDH Chemicals, Poole,

U.K.

4.3 METHODS

Male rabbits (Charing Cross Half Lops) were killed by nembutal injection (rabbit 1) or by cervical dislocation (rabbits 2,3,4). The tissues were excised immediately and kept at -20°C until homogenised.

Homogenates of tissues (10%) were prepared in KC1 (1.19%, containing -119-

0.1 mg of ascorbic acid and 0.1 mg of EDTA per ml) by using a Citenco

homogeniser with Jencons teflon pestle for 1 min. Homogenates were

centrifuged for 10 min (10,000 g, 4°C) in an MSE High Speed 18

centrifuge. The supernatant was separated from the pellet and sonicated

for 1 min (MSE 150 W Ultrasonic Disintegrator), and kept at -20°C until

assayed.

4.3.1 Tissue homogenates: Preparation for analysis by high performance

1iqu id chromatography.

Tissue homogenates were thawed and an aliquot (1 ml) was applied to a

Dowex 1 X 8 (200-400 mesh, acetate form, 10 mm X 8 mm) column. The columns were washed with ice-cold H£0 (8-10 ml) and compounds of

interest eluted with cold acetic acid (0.2 M, 10 ml). The eluates were freeze-dried in a Modulyo Edwards High Vacuum and afterwards were reconstituted in acetic acid (0.2 M, 250-400 pi), centifuged at 10,000 g

in an Eppendorf 5412 centrifuge and kept for analysis on h.p.l.c..

Authentic noradrenaline 3- and 4-0-sulphates (500 ng each) were added to

KC1 (1.19%, 1 ml containing 0.1 mg of ascorbic acid and 0.1 mg of EDTA perml) and taken through the procedure described above. The recovery of the above compounds was 80-90% (see Chapter 3) and total recovery after both steps of purification was 75-85%.

4.3.2 High performance liquid chromatography with electrochemical

detection.

The system used for high performance liquid chromatography (h.p.l.c.) and the conditions for the analysis were as described in Chapter 3

(Section 3.3.1).

The noradrenaline 3- and 4-0-sulphates in tissue homogenates were -1 2 0 -

Fig. 4.1 Hplc chromatogram of authentic noradrenaline 3- and 4-0-sulphate standards (prepared as described in Section 4.3.1). -1 2 1 -

identified by their retention time which corresponded with authentic

standards. The content of noradrenaline 3- and 4-0-sulphates in tissue

homogenates was determined by measurement of the peak height ratios on

the chromatogram in comparison with standards. Tissue homogenates from

the same animal, together with noradrenaline sulphate standards, were

always analysed in the same assay. The retention time for the

noradrenaline sulphates varied slightly from assay to assay (10%) as

freshly prepared solvent was used for each assay.

4.4 Results: Disposition of noradrenaline sulphates in the brain.

The content of noradrenaline 3- and 4-£-sulphates was estimated in four animals. Examples of standard sulphates and those chromatograms obtained from various tissues are given in Figures 4.1 - 4.7. In one rabbit (No. 3, Table 4.1) no noradrenaline 4-0-sulphate was found. In the remaining animals the levels of this isomer varied from 2.0 - 5.3 ng/mg of tissue (Table 4.1). Noradrenaline 3-0-sulphate levels varied from 1.4 - 6.1 ng/mg of tissue.

Table 4.1. Noradrenaline 3- and 4-0-sulphates in rabbit brain tissue.

Rabbit No. NA 3-0-S04 NA 4-0-S0.

ng/mg tissue ng/mg tissue

1 5.7 2.0

2 1.4 2.3

3 6.1 n.d.

4 4.4 5.3

n.d. = not detected -1 2 2 -

INJECT

4 -2 HPlc chromatogram of brain homogenate (rabbit 4). -123-

Thus in this small study of four animal brains, the relative proportions of the two sulphate conjugates varied considerably. In two animals the predominant sulphate was found to be noradrenaline 3-O-SO^ and in the remaining two animals the converse was observed.

4.4.1 Disposition of noradrenaline sulphates in the heart.

This study was performed on hearts excised from rabbits 2, 3 and 4.

In one heart homogenate (rabbit 3), no noradrenaline 4-0-sulphate was found (Table 4.2). The ratio of 3-0:4-0 isomers in the other two

(2 and 4) showed values of 0.65 and 0.43 respectively. In heart homogenate from rabbit 3 the highest level of noradrenaline 3-0-sulphate was noted (Fig. 4.2).

Table 4.2 Noradrenaline 3- and 4-0-sulphates in rabbit heart tissue.

Rabbit No. NA 3-0-S04 NA 4-O-SO.

ng/mg tissue ng/mg tissue

2 1.5 2.3

3 8.7 n.d.

4 1.2 2.8

n.d. = not detected

Just as in the case of the brain homogenates, rabbits 2 and 4 showed the higher concentrations of noradrenaline 4-0-sulphate than of the 3-0- isomer.

The heart tissue homogenate chromatogram from rabbit 4 is presented in Figure 4.3. -124-

c c •H •H E E

00 o

Fig. 4.3 Hplc chromatogram of heart homogenate (rabbit 4). -125-

4.4.2 Disposition of noradrenaline sulphates in the kidney.

Noradrenaline 3- and 4-0-sulphates were estimated in kidney homogenates from four rabbits. The data are presented in Table 4.3.

Table 4.3. Noradrenaline 3- and 4-0-sulphates in rabbit kidney tissue.

Rabbit No. NA 3-0-S04 NA 4-0-S04

ng/mg tissue ng/mg tissue

1 4.9 3.2

2 1.4 1.8

3 11.0 n.d.

4 3.7 4.8

n.d. = not detected

Again, rabbit 3 apparently fails to produce noradrenaline 4-0- sulphate and also the highest levels of noradrenaline 3-0-sulphate was found in this animal.

In the remaining animals approximately similar quantities of the two sulphates were identified. -126-

Fig. 4.4 Hplc chromatogram of kidney homogenate (rabbit 4). -127-

4.4.3 Disposition of noradrenaline sulphates in the spleen.

Estimations of noradrenaline 3- and 4-0-sulphates were performed in the tissues excised from rabbits 2, 3 and 4. Noradrenaline 4-0-sulphate conjugate was not detected in two animals (Table 4.4). Spleen tissue homogenate from rabbit 4, however, showed predominantly 4-0-sulphate.

Table 4.4. Noradrenaline 3- and 4-0-sulphates in rabbit spleen tissue.

Rabbit No. NA 3-O-SO. NA 4-0-S04

ng/mg tissue ng/mg tissue

2 1.5 n.d.

3 4.3 n.d.

4 1.2 4.6

n.d. = not detected -128-

c •HE

GO

INJECT

Fig. 4.5 Hplc chromatogram of spleen homogenate (rabbit 4). -129-

4.4.4 Disposition of noradrenaline sulphates in the liver.

This study was performed on the livers excised from four rabbits as described previously. Again, noradrenaline 4-0-sulphate was not detected in tissue homogenate from rabbit 3. In tissues from the other rabbits widely varying amounts of sulphates were detected, but on the whole more 4-0-sulphate than 3-0-sulphate was found. The data are presented in Table 4.5.

Table 4.5. Noradrenaline 3- and 4-0-sulphates in rabbit liver tissue.

Rabbit No. NA 3-O-SO. NA 4-0-S04

ng/mg tissue ng/mg tissue

1 17.6 21.6 2 3.2 7.5

3 6.0 n.d. 4 8.0 40.0

n.d. = not detected -130-

t INJECT

Fig. 4.6 Hplc chromatogram of liver homogenate (rabbit 4). -131-

4.4.5 Disposition of noradrenaline sulphates in the small intestine.

Estimations of noradrenaline 3- and 4-0-sulphates were performed in

homogenates of small intestine from four rabbits. The results are

presented in Table 4.6.

Table 4.6. Noradrenaline 3- and 4-0-sulphates in rabbit small

intestine.

Rabbit No. NA 3-0-S0 NA 4-0-S0. 4 - 4 ng/mg tissue ng/mg tissue

1 5.3 8.0

2 24.0 1.9

3 23.0 n.d.

4 1.9 8.7

n.d. = not detected

The highest quantities of noradrenaline 3-0-sulphate were found in this tissue. In similarity with previous studies in these animals, rabbit 3 failed to produce noradrenaline 4-0-sulphate. Noradrenaline 3-

O-sulphate was the predominant isomer in two animals, but in the remaining two homogenates, the 4-0-sulphate isomer was found in greater

amounts. -132-

Fig. 4.7 Hplc chromatogram of small intestine homogenate (rabbit 4). -133-

4.4.6 Hydrolysis of tissue homogenates.

This experiment was performed on all tissues from rabbit 4.

Homogenates of tissues were purified on a Dowex 1 X 8 column as described previously. The eluates were freeze-dried and afterwards reconstituted in 400 pi acetic acid (0.2 M). HC1 (1 M) was then added to adjust the pH of the solution to pH 1.0, acid samples were left for 15 min at 100°C. An aliquot of this solution (2-2 pi) was injected onto h.p.l.c. After hydrolysis, all samples showed a different chromatographic profile compared with that obtained before hydrolysis

(Fig. 4.8). The disappearance of peaks at 8.4 and 9.1 min was noted, together with the appearance of much larger peaks that eluted earlier

(5.5 - 7.4 min) and also the appearance of a peak at 11 min. Since the same chromatographic profile was obtained following hydrolysis of all tissues, the chromatogram presented is that from small intestine homogenate of rabbit 4.

The formation of free noradrenaline from noradrenaline sulphate conjugates was confirmed in all tissues from rabbit 1 by radioenzymatic assay (Chapter 5) of eluates from Dowex 1 x 8 before and after hydrolysis of tissue homogenates (6.5 ng; 7.6 ng; 37.6 ng; 12.1 ng per mg of brain, kidney, liver and small intestine respectively). -134-

c c •HE E •O «r> rx

Fig. 4.8 Hplc chromatogram of small intestine homogenate following hydrolysis (rabbit 4). -135-

4.5 DISCUSSION

A new direct, sensitive assay for the estimation of noradrenaline

(^-sulphates in various tissues was developed. Certain difficulties were experienced while developing this assay. It proved impossible to find

an appropriate internal standard which would not interfere with the elution volume of compounds of interest. Ideally, such a standard should be an exogenous compound with properties similar to catecholamine

O-sulphates. For example, isoprenaline O-sulphate theoretically would be ideal, but this substance was not available. The recoveries of noradrenaline O-sulphates in tissues were therefore calculated on the basis of recoveries for standard samples that were taken through the same procedure as the tissues. Inter-assay coefficients of variation were less than 15% (Chapter 3). Also this assay showed directly which noradrenaline isomers were present in the various tissues, and in what proportions. All previous studies investigating the ratios of the 3- and 4-()-sulphate isomers formed in a particular tissue were based on measurement of products formed during reactions involving incubation of various subtrates (acceptors) with crude or partially purified tissue preparations containing PST (Jenner & Rose, 1973; Wong, 1978; Renskers et^^l_., 1980; Pennings & van Kempen, 1980). These studies indicated that the 3-0-sulphate conjugate was the major product formed. This has been previous discussed (see Introduction to this Chapter). However, it is not known to what extent the action of aryl sulphatase affected those results, especially as it has been shown that catecholamine 4-0- sulphates were hydrolysed at higher rates than 3-0-sulphates by rat liver aryl sulphatase (Jenner & Rose, 1978).

The results presented in this Chapter did not confirm previous findings. In tissues investigated in three out of four animals, both -136-

noradrenaline ^-sulphates were found. The fact that noradrenaline

4-0-sulphate was not detected in tissues from rabbit 3 is very

interesting. This finding suggested that there might be two separate

arylsulphotransferases which catalyse the synthesis of noradrenaline 3- and 4-0-sulphates. It is possible that rabbit 3 lacked the enzyme which catalyses the synthesis of noradrenaline 4-0-sulphate, and as a possible consequence more 3-0-sulphate was synthesised in almost all tissues in

this animal. If indeed two separate enzymes catalyse the synthesis of the two sulphate isomers, in other species, e.g. man, this finding may have important implications especially for the metabolism of drugs such

as L-dopa, methyldopa and isoprenaline if similar interindividual differences in sulphoconjugation occurred. The remaining rabbits showed

large individual variations in proportion of the two noradrenaline

(^-sulphates in various tissues. However, apart from the brain and kidney homogenates of rabbit 1 and small intestine homogenates of rabbit 2, all the other tissue homogenates in which both sulphate

isomers were present seem to contain more noradrenaline 4-0-sulphate than 3-0-sulphate.

It should be added that with the assay described in this Chapter, it proved impossible to detect any dopamine 0-sulphates in the tissues

investigated. This finding is not totally unexpected as it has been claimed that dopamine ^-sulphates are present in brain in very low concentrations (Buu et aj_., 1981), and that sulphoconjugation is a means of rapidly transporting free dopamine from the brain to the periphery

(Buu et aT_., 1981; Sharpless et al_., 1981). However, even in the peripheral tissues dopamine ^-sulphates were not detectable; most probably they were present in concentrations below the detection limit of the assay (2 ng). -137-

CHAPTER 5 .

FURTHER METABOLISM OF DOPAMINE AND NORADRENALINE SULPHATES

5.1 Introduction • • • 139

5.2 Materials • • • 141

5.3 Methods: Incubation of dopamine and dopamine

O-sulphates with dopamine-B-hydroxylase (DBH) • • • 141

5.3.1 Incubation of dopamine O-sulphates with DBH

in the presence of fusaric acid • • • 142

5.3.2 Kinetic study of DBH • • • 142

5.3.2.1 Linearity of DBH reaction • • • 142

5.3.2.2 The kinetic study of DBH with dopamine,

dopamine 3 and 4-0-sulphates ... • • • 144

5.3.3 Determination of free noradrenaline • • •144

5.3.4 Determination of conjugated noradrenaline • • • 145

5.3.5 Detection of sulphatase • • • 146

5.4.1 Results: Linearity of DBH reaction • • • 146

5.4.2 Kinetic study of DBH with dopamine as

substrate • • • 147

5.4.3 Product of dopamine 4-0-sulphate incubation

with D$H • • • 149

5.4.4 Inhibition of DBH activity by fusaric acid

with dopamine 4-0-sulphate as substrate • • • 151

5.4.5 Sulphatase activity • • • 152 -138-

5.4.6 Noradrenaline sulphate formation: Second

product of the reaction of dopamine

4-0-sulphate with DBH .. 152

5.5 Product of dopamine 3-0-sulphate incubation

with DBH .. 153

5.5.1 Inhibition of DBH activity by fusaric acid

with dopamine 3-0-sulphate as substrate .. 155

5.5.2 Sulphatase activity .. 156

5.5.3 Noradrenaline sulphate formation: Second

product of the reaction of dopamine

3-0-sulphate with DBH .. 156

5.6 Incubation of noradrenaline sulphates

with DBH .. 157

5.6.1 Incubation of noradrenaline 4-0-sulphate

with DBH .. 158

5.6.2 Incubation of noradrenaline 3-0-sulphate

with D^H .. 158

5.6.3 Sulphatase activity .. 159

5.7 Discussion .. 160 -139-

5.1 INTRODUCTION

Dopamine and noradrenaline readily undergo sulphoconjugation in mammalian systems, and it is known that sulphoconjugates of these amines

are present in plasma, tissue and urine in much higher cncentrations than the free dopamine and noradrenaline (Haggendal, 1963; Kahane et

al., 1967; Buu&Kuchel, 1977; B u u ^ t ^ K , 1978). However, the

biological role of these sulphate esters remains unclear. Of particular

interest are dopamine 3- and 4-()-sulphates, since free dopamine is

usually undetectable (De Champlain et aj_., 1976) or below detection

limits of present methods of analysis in plasma (Weise & Kopin, 1976)

and in urine (Buu & Kuchel, 1977).

Jenner & Rose (1973) observed that rat primarily forms dopamine 3-0-

sulphate, this isomer also predominates in the urine of Parkinsonian

patients treated with L-dopa (Jenner & Rose, 1974; Bronaugh et al., 14 1974a, 1975). In contrast, when C-L-dopa was administered to the pigtail monkey, dopamine 4-0-sulphate was the primary metabolite

(Bronaugh et aj_., 1974b). Since dopamine ethereal sulphates possess an

ability to form internal salts which may have a greater ability for penetrating biological membranes, they were thought to represent a transport form of dopamine (Jenner & Rose, 1973). Alternatively, ethereal sulphates of catecholamines were considered to be the end products of these amines' metabolism destined for excretion, since

sulphation has been generally believed to be detoxification mechanism

(Jenner & Rose, 1974; Beyer & Shapiro, 1945). Other workers (Merits,

1976) suggested that dopamine 3-0^-sulphate was an intermediate rather than the end product of dopamine metabolism. He reported that, depending on the dose injected, dopamine 3-0-sulphate was rapidly metabolised in rats and dogs. However, it is not known whether the -140- metabolism of dopamine 3-0-sulphate occurs before or after its

hydrolysis to free dopamine, and whether dopamine sulphate is directly

involved as an intermediate in the metabolism of catecholamines. The notion that a sulphate conjugate could serve as a substrate for an enzyme is well known. Thus, conversion of estrone sulphate to

15 a-hydroxyestrone sulphate has been observed in humans (Jirku et al.,

1967). Also androstenediol sulphate could be directly converted to dehydroisoandrosterone sulphate (Baulieu et a/L, 1965), showing that

17 a-hydroxydehydrogenase can have a sulphate as a substrate. Buu &

Kuchel (1979a, b) demonstrated in vitro conversion of dopamine 3 and

4-0-sulphate directly to free noradrenaline by dopamine 3-hydroxylase,

implicating both dopamine sulphates as intermediates in catecholamine metabolism. The authors postulated that the direct conversion of

dopamine 3-0-sulphate to free noradrenaline, if found to occur in vivo, may serve as an alternative pathway for the biosynthesis of noradrenaline in peripheral tissues, when the small (if present) and

short lived supply of free dopamine is exhausted. It therefore was of

interest to determine whether the conversion of dopamine sulphates to

free noradrenaline is the only transformation of these compounds

catalysed by D3H or whether these compounds could also serve as

precursors of, for example, noradrenaline sulphates. -141-

5.2 MATERIALS

Dopamine and noradrenaline sulphate isomers were prepared as described in Chapter 2. The remaining chemicals were purchased from the following sources: sodium acetate, sodium fumarate, ascorbic acid (BDH

Chemicals, Poole, U.K.),_Njethylmaleimide, pargeline, catalase

(E.C.1.11.1.6; specific activity 17,000 units/mg), fusaric acid, p-nitrocatechol sulphate and dopamine 6-hydroxylase (E.C.1.14.17.1; specific activity 2.7-5 units/mg), aryl sulphatase from Aerobacter aerogenes (E.C.3.1.6.1; specific activity 4.8 units/mg) from Sigma

Chemical Co., London, U.K.). Noradrenaline bitartrate, adrenaline bitartrate, trizma base, glutathione, tungstophosphoric acid, sodium 3 phosphate (Sigma Chemical Co., London, U.K.), [H]-S-adenos.yl-methionine

(SAM, New England Nuclear, Southampton, U.K.). DEPH (Di-(2-ethylhexyl) phosphoric acid; BDH Chemicals, Poole, U.K.); PPO and P0P0P (Fisons

Scientific, Loughborough, U.K.).

5.3 METHODS: Incubation of dopamine and dopamine 0-sulphates with D6H.

The procedure for hydroxylation of dopamine and its sulphate isomers by D6H was based on the method of Nagatsu and Udenfriend (1972). The standard incubation mixture (total volume 200 pi) contained: sodium acetate buffer (1 M, pH 5.0; 40 pi); sodium fumarate (0.2 M; 10 pi),

N-ethylmaleimide (0.2 M; 30 pi); pargeline (0.02 M; 10 pi), freshly prepared ascorbic acid (0.2 M; 10 pi); catalase (170 units; 10 pi; dopamine HC1 (0.21 - 1.71 mM) or dopamine sulphate isomers in concentrations ranging from 0.1 to 0.85 mM as substrates. _3 The amounts of D6H used varied from 16 X 10 units (kinetic _3 studies), to 21 X 10 units (incubation of dopamine 3- and 4-0- sulphates with fusaric acid). Before use, DBH was dialysed against

100 volumes of 0.001 M phosphate buffer, pH 6.5, for 12 h. After -142- dialysis, the protein content of the enzyme was estimated (Lowry, 1951).

The volume of the reaction mixture was adjusted to 200 pi with water.

The reaction mixture containing all constituents except for the

substrate was incubated for 10 min in a water bath at 37°C. The subtrate was then added and samples were incubated for a further 20 min. Control experiments included: a) samples containing reaction mixture and

substrate, but no enzyme, b) samples containing reaction mixture,

substrate and boiled enzyme (10 min; 100°C), and c) samples containing reaction mixture and active enzyme, but no substrate. For each

concentration of substrate used, the appropriate control sample

(containing the same concentration of substrate) was set up. After the

incubation the reaction was terminated by placing reaction tubes on ice

and by adding: a) 1 M Tris buffer (containing 2% EDTA, pH 8.6, 0.5 ml), alumina (50 mg) and H20 to 2 ml, when noradrenaline assay was to be

performed; b) 0.2 M tris buffer (pH 7,4; 2 ml) and Aerobacter sulphatase

(0.31 units), when noradrenaline sulphate assay was to be performed.

5.3.1 Incubation of dopamine 0-sulphates with DgH in the presence of

fusaric acid.

Dopamine 3 and 4-0-sulphates (0.85 mM) were incubated in the presence of four different concentrations of fusaric acid (0.01; 0.1; 1.0;

5.0 mM), a non-competitive inhibitor of DBH (Nagatsu et al_., 1970) under conditions described in Section 5.3.

5.3.2 Kinetic study of Db H.

5.3.2.1 Linearity of Db H reaction.

The linearity of the reaction between DBH and dopamine 3- and 4-0- sulphates was investigated by incubating different concentrations of

isomeric sulphates (2.7 - 21.4 pM for dopamine 4-0-sulphate and 10.7 - -143-

PLASMA CATECHOLAMINES

ALUMINA

ELUTE BOVINE NORADRENALINE ADRENAL MEDULLAE PNMT

^ADRENALINE

| ALUMINA

—ELUTE

— PRECIPITATE EXCESS LABEL — SOLVENT EXTRACTION

— SCINTILLATION COUNTING

HENRY et0111975)

Fig. 5.1 Radioenzymatic (PNMT) assay for the determination of free noradrenaline. -144-

42.8 pM for dopamine 3-0-sulphate) at the following time intervals:

10, 20, 30, 40 min.

5.3.2.2 The kinetic study of QBH with dopamine, dopamine 3-and 4-0-

sulphates.

The kinetics of D$H activity with dopamine isomeric sulphates and free dopamine were determined essentially as described in Section 5.3 with concentrations of dopamine-O-sulphates from 0.106 to 0.854 mM and concentrations of free dopamine (base) ranging from 0.16 to 1.3 mM.

Incubation time was 20 min.

5.3.3 Determination of free noradrenaline.

Free noradrenaline was measured with an assay based on the radioenzymatic technique of Henry et al_. (1975). The principal of this method is the conversion of noradrenaline to tritiated adrenaline by partially purified bovine adrenal phenyl ethanol amine_N-methyl transferase (PNMT) (EC 2.1.1.28) and tritiated_S-adenosyl-methionine

(SAM), and subsequent scintillation counting of radioactive adrenaline

(Fig. 5.1).

Samples that had been previously incubated with DBH were placed on

ice and Tris buffer (1M containing 2% EDTA; pH 8.6; 0.5 ml), alumina

(50 mg) and water (to 2 ml) were added. Samples were then rotated for

15 min in order to adsorb free noradrenaline onto alumina. Afterwards the samples were centrifuged for 5 min (5°C, 1000 g) and washed with water (3 ml X 3). Noradrenaline was eluted with hydrochloric acid (0.2

M; 200 pi), aliquots (5-100 pi) of HC1 were adjusted to volume 200 pi with water and subsequently incubated for 60 min with freshly prepared reaction mixture containing in 50 pi: 2 M Tris HC1 containing 5% EDTA and glutathione, 1.55 mg/ml, (pH 9.2) (38 pi), ^[H]-^-adenosyl methionine (SAM) (2 pCi, 2 pi), and purified bovine adrenal -145-

phenylethanol amine-41-methyl transferase (PNMT) (protein concentration

5 mg/ml; 10 pi), which was prepared according to the method of Axelrod

(1962). 3 [H]-Adrenaline formed during this reaction was separated from the mixture by adsorption onto alumina (100 mg), subsequent elution by perchloric acid (1 ml) and extraction into toluene: DEHP (99% v/v,

10 ml) at pH 7.3 (adjusted by 1 ml of 0.5 M Na^ P0^). The toluene layer

(9 ml) was transferred to plastic scintillation vials containing

Liquifluor scintillant (400 pi) (New England Nuclear). The samples were then counted for 10 min (6880 Liquid Scintillation Mark III, Searle

Analytic).

A standard curve was constructed from the counts per minute for the blanks (samples without substrate but with all the other constituents of the reaction mixture) and samples that contained a known amount of noradrenaline (0.025 pM - 1.5 pM). Samples that contained a known amount of noradrenaline and blanks were incubated first of all with DBH reaction mixture, and then with PNMT. The noradrenaline concentration for each samples was then read from the standard curve. All samples were analysed in duplicate.

5.3.4 Determination of conjugated noradrenaline.

An aliquot of the D£H and dopamine sulphates reaction mixture was analysed directly for free noradrenaline, while another aliquot of the same reaction mixture was first subjected to hydrolysis by sulphatase from Aerobacter aerogenes (0.31 units) in Tris. HC1 buffer (0.2 M, pH

7.4; 2 ml) at 37°C for 2 h. Afterwards the samples were analysed as described in the previous section to give the total noradrenaline. The difference between the total noradrenaline and the free noradrenaline constituted the value for noradrenaline sulphate. -146-

5.3.5 Detection of sulphatase.

In order to determine whether sulphatase activity was present in the system, p-nitrocatechol sulphate (5.5 pM) was incubated in the same incubation medium in the place of dopamine-£-sulphate. At the end of the incubation period (20 min) NaOH (0.5 M; 0.5 ml) was added and the resulting mixture was measured at 525 nm for nitrocatechol anion (Burns

& Wynn, 1975) using a Unicam SP 1800 spectrophotometer. In control experiments (reference samples) p-nitrocatechol sulphate was added after the incubation was terminated.

5.4.1 Results: Linearity of DBH reaction.

This study was performed as described in Section 3.2.1. The data for dopamine 4-0-sulphate are presented in Table 5.1.

Table 5.1. Dopamine 4-0-sulphate incubation with DBH at different

time intervals.

2 Substrate Product formation pmol X 10

cone. pM 10 min 20 min 30 min 40 min

2.7 1.2 1.8 2.5 4.1

5.4 2.4 3.2 4.8 5.4

10.7 4.0 9.0 10.3 15.3

21.4 8.6 17.2 21.3 29.9

These results suggest that the reaction between dopamine 4-0-sulphate

and DBH remains linear up to 40 min. A similar study was performed with

dopamine 3-0-sulphate. -147-

Table 5.2 Dopamine 3-0-sulphate incubation with DgH at different

time intervals. ro —* X o Substrate Product formation pmol 1

cone. pM 10 min 20 min 40 mi

10.7 4.8 8.3 12.7

21.4 8.3 16.6 24.8

42.8 17.7 31.6 48.0

Just as in the case of dopamine 4-0-sulphate, the reaction between

dopamine 3-0-sulphate and D3H remained linear up to 40 min. It was

decided to use 20 min incubation time for the kinetic studies of D$H and

all other subsequent studies.

5.4.2 Kinetic study of Db H with dopamine as the substrate.

This study was performed as described in the previous sections.

Results for product formation are expressed in pmol of free noradrenaline formed per minute per milligram of enzyme protein, and are presented in

Table 5.3.

Table 5.3 Kinetic study of Db H with dopamine as the substrate.

Substrate cone. pM [S] Product formation [V] pmol min"^ mg”^

163 219.9

326 299.8

653 425.1

1307 458.7 -148-

1 1 —- Immol IS]

Fig. 5.2 D$H kinetic study with dopamine as the substrate.

Km = 0.26 (+ 0.01 S.D.) nM Vmax. = 0.57 (+ 0.03 S.D.) mmol min ^ mg ^ -149-

These results presented in a Lineweaver-Burk plot (Fig. 5.2) showed a

Km of 0.26 (± 0.01 S.D.) mM and a Vmax of 0.57 (± 0.03 S.D.) mmol min“* mg-1.

5.4.3 Product of dopamine 4-0-sulphate incubation with Db H. Kinetic

study of Db H with the above sulphate as a substrate.

Incubation of dopamine 4-0-sulphate with bovine dopamine-B- hydroxylase lead to the formation of free noradrenaline (Table 5.4).

Substrate was checked for contamination with noradrenaline and dopamine by n.m.r. and h.p.l.c. (see Chapter 2) and was found to be free from any contamination with free catecholamines. Thus, the results presented in

Table 5.4 show that dopamine 4-0-sulphate is a substrate for dopamine-

3-hydroxylase and follows Michaelis-Menten kinetics.

Table 5.4 Kinetic study of DbH with dopamine 4-0-sulphate as the

substrate.

Substrate cone. pM [S] Product formation [V] pmol min"* mg“*

106 7.35

213 12.85

427 23.65

641 28.22

854 33.69

The resulting Linweaver-Burk plot (Fig. 5.3) revealed an apparent

Km of 0.83 (± 0.1 S.D.) mM and a Vmax of 0.07 (± 0.02 S.D.) mmol min"* mg -1 .

Comparison of Km (0.83 mM) and Vmax (0.07 mmol min"* mg"*) values obtained for dopamine 4-0-sulphate with those obtained for free dopamine

(Km = 0.26 mM) (Vmax = 0.57 mmol min"1 mg“*) shows lower affinity of DBH -150-

Fig. 5.3 DgH kinetic study with dopamine 4-£-sulphate as the substrate.

Km = 0.83 (+ 0.1 S.D.) rrtol Vmax = 0.07 (+ 0.02 S.D.) mmol min ^ mg ^ -151- for dopamine 4-0-sulphate than for free dopamine. Velocity (V) of free dopamine hydroxylation is higher than that of the dopamine 4-£-sulphate reaction. The lower Km value for free dopamine than for dopamine 4-0- sulphate indicates lower substrate requirements for achievement of half maximum velocity.

5.4.4 Inhibition of DBH activity by fusaric acid with dopamine 4-0-

sulphate as the substrate.

In order to determine whether the formation of noradrenaline was indeed catalysed by dopamine B-hydroxylase, dopamine 4-0-sulphate was incubated in the presence of various concentrations of fusaric acid.

The results (Table 5.5) showed that fusaric acid at a concentration of

0.01 mM inhibited the formation of free noradrenaline by 85%, at the concentration of 0.1 mM by 96%, while at 1.0 mM by 99% and at 5.0 mM noradrenaline formation was completed abolished.

Table 5.5 Concentrations of free noradrenaline formed during

incubation of dopamine 4-0-sulphate (0.85 mM) with Db H

in the absence or presence of fusaric acid.

Fusaric acid cone. mM Free noradrenaline formation pM

0 5.61 0.01 0.85 0.10 0.20 1.00 0.07 5.00 <0.01 -152-

5.4.5 Sulphatase activity.

To determine whether noradrenaline formed was due to free dopamine liberated from dopamine 4-0-sulphate by a sulphatase was investigated by measuring the sulphatase activity in the incubation medium using p-nitrocatechol sulphate (Section 5.3.5). The results showed that no sulphatase activity was detectable. The detection limit of this assay is approximately 1 pM.

5.4.6 Noradrenaline sulphate formation: second product of the reaction

of dopamine 4-0-sulphate and DBH.

This study was performed to explore the possibility of direct conversion of dopamine 4-:0jSulphate to corresponding noradrenaline sulphate. It was found that noradrenaline sulphate was indeed formed when dopamine 4-0-sulphate was used as a substrate for the reaction catalysed by DgH. The results are presented in Table 5.6.

Table 5.6 Products formed by the incubation of dopamine 4-0-sulphate

with dopamine-3-hydroxylase.

Substrate Free noradrenaline Conjugated noradrenaline concentration formation formation , . pM pmol min -1 mg-1 pmol min -1 mg -1

21 1.25 1.30

53 3.40 2.70

210 15.80 8.90

420 20.70 13.10

The above data suggest that at the lower concentration of the substrate (21; 53 pM) the ratio of free and conjugated noradrenaline -153- formation is almost the same (free NA/conjugated NA = 1.10 + 0.17), whereas at the higher substrate concentration (210, 420 pM) there is more free noradrenaline formed than conjugated (ratio free Na/conjugated

NA = 1.7 ± 0.1).

5.5 Product of dopamine 3-0-sulphate incubation with DgH. Kinetic

study of Db H with the above sulphate as a substrate.

This study has shown that incubation of dopamine 3-0-sulphate with

D$H resulted, just as in the case of dopamine 4-0-sulphate, in the

formation of free noradrenaline (Table 5.7). This substrate was also

free of any contamination with free noradrenaline or dopamine

(Chapter 2).

Table 5.7 Kinetic study of Db H with dopamine 3-0-sulphate as a

substrate.

Substrate cone. M [S] Product formation [V] pmol min"* mg *

106 0.79

213 1.51

427 2.84

641 3.98

854 5.29

The above results expressed as a Linweaver-Burk plot (Fig. 5.4) showed an apparent Km of 2.5 (+ 0.5 S.D.) mM and a Vmax of 0.022

(± 0.003 S.D.) mmol min”* mg”*. These data showed that dopamine 3-0-

sulphate was not as good a substrate for DgH (Km 2.5 mM) when compared with dopamine 4-0-sulphate (Km 0.83 mM) and the least reactive of all - 154-

Fig. 5.4 D$H kinetic study with dopamine 3-0-sulphate as the substrate.

Km = 2.5 (+ 0.5 S.D.) mM Vmax = 0.022 (+ 0.003 S.D.) mmol min ^ mg ^ -155-

three substrates (dopamine, dopamine 4-0-sulphate, dopamine 3-0-

sulphate) investigated. This substrate had a Vmax of 0.022 mmol min”*

mg”*, three times lower than dopamine 4-0-sulphate (Vmax 0.07 mmol min”*

mg"*) and 26 times lower than dopamine itself (Vmax 0.57 mmol min"* mg”^ ).

Therefore, it can be concluded that when formation of free

noradrenaline is involved, dopamine 4-0-sulphate is a better substrate

for dopamine 6-hydroxylase than dopamine 3-0-sulphate.

5.5.1 Inhibition of Db H activity by fusaric acid with dopamine 3-0-

sulphate as the substrate.

This study was performed to determine if the conversion of dopamine

3-0-sulphate was catalysed by D3H. Fusaric acid (5-butylpicolinic acid) is a potent inhibitor of dopamine f3-hydroxylase and the inhibition of

the enzyme by fusaric acid is non-competitive. Dopamine 3-0-sulphate

was incubated in the presence of different concentrations of fusaric

acid. The results (Table 5.8) indicated that fusaric acid at a

concentration of 0.01 mM inhibited noradrenaline formation by 70%, at

0.1 mM by 86%, and at concentrations of 1.0 and 5.0 mM, noradrenaline

formation was completely inhibited.

Table 5.8 Concentrations of free noradrenaline formed during

incubation of dopamine 3-0-sulphate (0.85 mM) with D8H in

the absence or presence of fusaric acid.

Fusaric acid cone. mM Free noradrenaline formation pM

0 0.57 0.01 0.17

0.1 0.08

1.0 <0.01

5.0 <0.01 -156-

5.5.2 Sulphatase activity.

This experiment was performed as described in Section 5.3.5. No

sulphatase activity was detectable (Section 5.4.5).

5.5.3 Noradrenaline sulphate formation: second product of the reaction

of dopamine 3-0-sulphate and DBH.

This study has shown that noradrenaline sulphate was also formed when

dopamine 3-0-sulphate was incubated with dopamine 3-hydroxylase. Just

as in the case of dopamine 4-0-sulphate there were two products arising

when dopamine 3-0-sulphate was used as a substrate. The data are

presented in Table 5.9.

Table 5.9 Products formed by the incubation of dopamine 3-0-sulphate

with Pr H.

Substrate Free noradrenaline Conjugated noradrenaline concentration formation formation i . pM pmol min"^ mg“^ pmol min —1 mg -1

21 0.27 1.31

53 0.57 3.40

210 5.45 11.40

420 6.80 18.48

The above results showed that when the low concentrations of

substrate were used (21, 53 pM), the ratio of free to conjugated noradrenaline was 0.185 (+ 0.015), but when high concentrations of substrate (210, 420 pM) were investigated, the ratio increased to 0.42

(± 0.05). A similar observation was made with dopamine 4-0-sulphate as a substrate (ratio 1.1 and 1.7 for low and high concentrations -157-

respectively). Thus, when dopamine 3-0-sulphate was incubated with DftH two products resulted: free noradrenaline and noradrenaline sulphate.

Unlike the case of dopamine 4-£-sulphate there was more conjugated noradrenaline formed than free at the higher concentration of substrate

used (approximately 1.5 times more than when dopamine 4-0-sulphate was used as a substrate).

5.6 Incubation of noradrenaline sulphates with dopamine g-hydroxylase.

The data presented previously showed that two products; free

noradrenaline and noradrenaline sulphate conjugate, resulted from the

reaction catalysed by D$H. Buu & Kuchel (1979a,b) showed that dopamine

O-sulphates were substrates for reactions catalysed by D$H, however,

according to the above authors, free noradrenaline was the only product

of this reaction. Since the data presented in this Chapter indicated

that noradrenaline sulphate conjugate was also a product of this

reaction, it was decided to investigate if the formation of free

noradrenaline was perhaps a product of noradrenaline sulphate hydrolysis

in the DftH incubation medium. In order to explore this possibility,

authentic noradrenaline 3- and 4-0-sulphates were used as substrates for

this reaction. The conditions of the reactions were exactly the same as

described in Section 5.3, with noradrenaline 4-^-sulphate in

concentrations ranging from 5 to 50 pM and noradrenaline 3-0-sulphate

from 50-400 pM. The reaction was stopped after 20 min incubation at 37°C

by placing the tubes on ice and by the addition of Tris HC1 buffer (1 M;

containing 2% EDTA; pH 8.6; 0.5 ml), alumina 50 mg and H£0 to 2 ml.

Free noradrenaline was measured as described in Section 5.3.3. Again,

DBH incubation medium was checked for sulphatase activity (Section _3 5.3.5). D$H was used in a concentration of 21 X 10 units. -158-

5.6.1 Incubation of noradrenaline 4-0-sulphate with dopamine

8-hydroxylase.

Incubation of noradrenaline 4-£-sulphate in DgH incubation medium in the presence of active D8H resulted in the formation of free noradrenaline (Table 5.10). Control experiments performed in the absence of D8H or with boiled D$H (100°C, 10 min) did not show that such a conversion took place. Therefore, it can be concluded that only in the presence of active enzyme desulphation of noradrenaline 4-0-sulphate occurred. In the presence of fusaric acid (concentrations: 1.25, 2.5,

5, 10 mM) and noradrenaline 4-0-sulphate (in cone. 20 pM), dopamine

8-hydroxylase activity was not inhibited at all, and free noradrenaline was formed. This observation suggests that fusaric acid most probably inhibits only hydroxylation of the side chain of dopamine and dopamine related compounds.

Table 5.10 Noradrenaline 4-0-sulphate incubation in the presence of

active D8H.

Substrate Free noradrenaline concentration formation . . -i -i pM pmol min mg

5 2.6 10 6.1 20 10.5 50 20.0

5.6.2 Incubation of noradrenaline 3-0-sulphate with dopamine

8-hydroxylase.

This study showed very similar results to that performed with -159-

noradrenaline 4-0-sulphate as a substrate. Incubation of noradrenaline

3-0-sulphate in the presence of active [£H gave rise to free noradrenaline (Table 5.11). Again, control experiments performed in the absence of DftH and in the presence of boiled enzyme (100°C, 10 min) showed no formation of free noradrenaline. Fusaric acid in concentrations 1.25, 2.5, 5.0, 10.0 mM did not inhibit formation of free noradrenaline (substrate concentration 200 pM). However, despite the fact that noradrenaline 3-£-sulphate concentrations used were approximately 10 times higher than those used for noradrenaline 4-0- sulphate, the formation of free noradrenaline was approximately 10 times lower.

Table 5.11 Noradrenaline 3-0-sulphate incubation in the presence of

active DgH.

Substrate Free noradrenaline concentration formation i • — pM pmol min 1 mg-1

50 0.30

100 0.63

200 1.36

400 3.08

5.6.3 Sulphatase activity.

This experiment was performed as described previously (Section

5.3.5). No sulphatase activity was detected, the detection limit of this assay is approximately 1 pM. - 1 6 0 -

5.7 DISCUSSION

This study has shown that incubation of dopamine isomeric sulphates with dopamine 13-hydroxylase resulted in the formation of two products: free noradrenaline and noradrenaline sulphate conjugate. This observation is partially in agreement with results obtained by Buu &

Kuchel (1979a,b). However, these workers claimed that free noradrenaline was the only product of this reaction. The formation of free noradrenaline following incubation of dopamine O-sulphates with dopamine 3-hydroxylase could be explained by one of the following: contamination of enzyme with free dopamine, or free dopamine liberated from dopamine O-sulphate by sulphatase present in the incubation medium may have been converted to free noradrenaline. Alternatively, since noradrenaline sulphate was formed in the above reaction, it might have been hydrolysed to yield free noradrenaline. Finally, dopamine

O-sulphates could undergo a conversion to free noradrenaline and to noradrenaline sulphate. The possibility that free noradrenalinee was

synthesized from dopamine contaminating the enzyme could be eliminated, since control samples containing incubation medium and active D3H did not differ from control samples containing boiled enzyme or no enzyme.

Involvement of sulphatase was also ruled out on the basis that no

sulphatase activity was found in the incubation mixture. The detection

limit of the sulphatase assay was approximately 1 pM, which is 260 times

less than the Km of dopamine 3-hydroxylase for dopamine (Km = 0.26 mM).

Therefore, it was assumed that the formation of free noradrenaline was not caused by liberation of dopamine from dopamine O-sulphate in the

incubation medium. The next alternative that dopamine ^-sulphates are directly converted by dopamine 3-hydroxylase to corresponding noradrenaline ^-sulphates which were then hydrolysed to yield free -1 6 1 -

noradrenaline, was very interesting.

Data presented in this Chapter indicated that noradrenaline sulphates were also products of the reaction catalysed by D$H. Of interest was the finding that more noradrenaline sulphates were formed (approximately

1.5 times) when dopamine 3-0-sulphate was a substrate than when dopamine

4-()-sulphate was used. Both results obtained by Buu & Kuchel (1979a,b) and data presented here indicated that dopamine 4-0-sulphate was a better substrate for D£H (Km = 2.6 mM, Buu & Kuchel, 1979b) (Km =

0.83 mM, Section 5.4.3) than dopamine 3-0-sulphate (Km = 17 mM, Buu &

Kuchel, 1979a) (Km = 2.5 mM, Section 5.5) when free noradrenaline formation was involved. There is, therefore, an interesting relationship between the affinity of DBH for the above substrates and the formation of the two reaction products. Dopamine 4-0-sulphate was a better substrate for D$H than dopamine 3-0-sulphate when noradrenaline was formed, and a poorer substrate when noradrenaline sulphate conjugate was formed (ratio of free to conjugated noradrenaline 1.1 - 1.7). The situation was reversed with dopamine 3-0-sulphate as a substrate (ratio of free noradrenaline to conjugated noradrenaline 0.185 - 0.42). When noradrenaline sulphate isomers were reacted with DBH under the same conditions as dopamine 0-sulphates, noradrenaline 4-0-sulphate in concentrations of 5-50 pM resulted in the formation of 10 times more free noradrenaline than noradrenaline 3-0-sulphate used in much higher concentratons (50-400 pM). The above observations suggest that perhaps dopamine and noradrenaline 4-0-sulphate are less stable compounds than their isomers and could be more readily desulphated in the incubation medium. However, the 4-0-sulphates were quite stable in the incubation mixture that contained boiled DBH or no DBH. The fact that production of free noradrenaline from dopamine 3- and 4-0-sulphates -1 6 2 -

was inhibited by fusaric acid to a high degree (70-100%) seemed to indicate that direct conversion of the above sulphates to free noradrenaline was indeed catalysed by D3H. In the case of noradrenaline

0-sulphates no such inhibition could be observed. This is probably due to the fact that fusaric acid inhibits hydroxylation of the side chain of dopamine and dopamine related compounds. The finding that dopamine

^-sulphates were, and noradrenaline sulphates could be, substrates for d o p a m i n e 3-hydroxylase was not totally surprising. The above enzyme is non-specific and several phenylethyl amines as well as dopamine can serve as substrates (Levin & Kaufman, 1961; Bridgers & Kaufman, 1962;

Goldstein & Contrera, 1961).

Buu & Kuchel (1979a) proposed a possible mechanism for the formation of free noradrenaline from dopamine 3-0-sulphate (Fig. 5.5). The authors postulated that such a mechanism was compatible with that proposed for the hydroxylation of dopamine. It involved an oxidation of the enzyme complex with the formation of two ascorbic free radicals

(Step 1) followed by an interaction of the oxidized enzyme with oxygen and dopamine sulphate to yield noradrenaline quinone (Steps 3 and 4).

Noradrenaline quinone is reduced to noradrenaline (Step 5) (with or without ascorbic acid). This mechanism explains quite well the formation of free noradrenaline from dopamine 0-sulphates and even from noradrenaline ^-sulphates. However, the formation of another product of the reaction involing dopamine 0-sulphates and D 3H, namely noradrenaline sulphates, cannot be explained by this scheme. If Steps 1, 2 and 3 of this mechanism are as shown in Fig. 5.5, and in Step 4 ascorbic acid is a hydrogen donor (Fig. 5.6), dopamine sulphate could be converted to the corresponding noradrenaline sulphate.

It is not known whether the conversion of dopamine sulphates to free -163-

r *' ^ C u *2 >Cu (D 2 Ascorbic + Enz. Enz ■+■ 2[ Ascorbic] +2 H* ^ C u '2 V

f *9- 5 -5 A possible mechanism for the formation of free

noradrenaline from dopamine 3-0-sulphate.

(Buu & Kuchel, 1979a). -164-

^ C u *2 + 2lAscorbic) +2H* id 2 Ascorbic + En *v-c»«

Fig. 5.6 A possible mechanism for the formation of noradrenaline

3-0-sulphate from dopamine 3-£-sulphate. -165-

noradrenaline and to noradrenaline sulphate conjugates occurs in vivo.

If this were the case, then such a mechanism could serve as an alternative pathway for the biosynthesis of free noradrenaline in peripheral tissues, or as a pathway for the biosynthesis of both free and conjugated noradrenaline.

Another observation which could be interesting is that dopamine

4-0-sulphate (Km = 0.83 mM, Vmax = 0.07 mmol min"^ mg"^) is a more reactive compound than dopamine 3-0-sulphate (Km = 2.5 mM; Vmax =

0.022 mmol min”^ mg”^). This could explain why this particular dopamine

0-sulphate is present in smaller quantities than its isomer in human urine (Oenner & Rose, 1973; Bronaugh et ^1_., 1975; Arakawa et al.,

1979).

The finding by Unger ^t _al_. (1979) that dopamine O-sulphates are present in greater amounts than free dopamine in the noradrenaline storage sites of the dog adrenal gland suggests that the aim of sulphoconjugation may not necessarily be the production of end products to be excreted. However, more work is required to explore the possibility that catecholamine sulphoconjugates may be involved in metabolic processes as biosynthetic intermediates. -1 6 6 -

C H A P T E R 6

DISCUSSION AND CONCLUDING REMARKS

During the last 20 years there has been a remarkable increase in our

knowledge of the biochemistry and pharmacology of catecholamines. The

properties of the biosynthetic enzymes involved in the formation of

catecholamines have been investigated and the control of catecholamine

biosynthesis has been studied extensively. The mechanisms for storage

and release of catecholamines from adrenergic nerve endings and the

adrenal medulla have been elucidated and their metabolic fate and mode

of activation determined. The effects of drugs on the various

biosynthetic and metabolic enzymes, together with their effects on

storage and release of catecholamines, have also been investigated.

Physiological, pharmacological and pathological factors that influence

catecholamine concentrations in biological fluids (such as age, sex,

posture, exercise, disease and drugs) have also been subject to

extensive investigation.

In recent years attention has focussed on certain metabolites of

catecholamines that are present in tissues and biological fluids in much

higher concentrations than the free amines, namely catecholamine

O-sulphates. Interest in these compounds has increased with the

discovery of phenolsulphotransferases (PST) in human platelets (Hart

et al_., 1979). It is thought that PST activity measured in the

platelets may reflect sulphotransferase activity throughout the body.

Also the measurement of PST in platelets may provide a non-invasive

procedure for the evaluation of overall PST activity in humans.

However, progress in the field of catecholamine sulphoconjugation has

been limited by methodological difficulties which have included the lack

of authentic catecholamine ^-sulphates as standards, and the lack of — 167—

suitable assay techniques for the direct estimation of these compounds

in tissues and biological fluids.

In the present work, special emphasis has been placed on the

synthesis, characterisation and authentication of dopamine and

noradrenaline sulphates. Many difficulties were initially experienced

in the synthesis of dopamine isomeric sulphates, despite the fact that

published methods for the synthesis of these sulphates were followed

closely (Jenner & Rose, 1973). It proved impossible to obtain pure

dopamine 3- and 4-0-sulphates, as other products of the reaction were

discovered, including dopamine 6-sulphonic acid. Using high performance

liquid chromatography with ultra-violet detection, it was possible to

identify different products of the reactions between dopamine and

sulphuric acid of varying specific gravity and other sulphating agents.

It was discovered that small changes in reaction mixture composition

and in reaction conditions influenced product composition. Dopamine

6-sulphonic acid was isolated and found to possess properties very

similar to those of dopamine 3- and 4-0-sulphates. This compound has

the same molecular weight as the dopamine ^-sulphates. Thus, elemental

analysis provided only an approximation of the structure of the

synthesised products. Unreacted dopamine was also shown to be a

contaminant of the synthesized compounds.

Since separation of the products of dopamine sulphonation was found

to be unsatisfactory when using ion exchange chromatography,

semipreparative scale h.p.l.c. was employed as an alternative method.

The solvent system chosen provided good separation of the various

products of this reaction, and was easy to remove in vacuo, leaving pure

c o mpounds.

The characterisation of the synthesized products at first proved

difficult when elemental analysis, infra-red,ultra-violet and mass -1 6 8 -

spectroscopy analytical techniques were employed. Dopamine 6-sulphonic

acid showed very similar physical and chemical properties to those of

dopamine O-sulphates. Even the reaction with Gibbs' reagent produced

misleading results as sulphonic acid reacts positively with this

reagent.

Determination of the absolute structure of dopamine 3- and 4-0-

sulphate was not attempted by exhaustive methylation, Hofmann

degradation, permanganate oxidation and acid hydrolysis sequentially as

described by Jenner & Rose, (1973). Instead, nuclear magnetic resonance

spectroscopy (n.m.r.) techniques were employed. This technique proved

to be sufficiently powerful to distinguish the structures of the

different products of dopamine sulphonation and also provided

information on the purity of synthesised compounds. In practice, the

purity of compounds obtained from sulphonation of dopamine was first

confirmed on h.p.l.c. prior to n.m.r. spectroscopy.

Dopamine 6-sulphonic acid and dopamine 3- and 4-0-sulphates were

purified to > 95%. Because of the difficulties experienced in the

synthesis of dopamine O-sulphates, it is surprising how few

investigators who have prepared these sulphates for use in a variety of

experiments in vivo and in vitro actually provided an adequate

description of the methods used for authentification of these compounds.

Merits (1976) investigated the metabolism in vivo of dopamine 3-0-

sulphate after oral administration to a variety of animals, and gave no

indication of the purity and authenticity of the compound. If, in this

author's study, dopamine 3-0-sulphate was contaminated with free

unreacted dopamine, then the finding that the guinea pig metabolised

this compound in a similar way to that described for orally administered

dopamine was misleading. Buu et al_. (1982) investigated the metabolism

of dopamine ^-sulphate in the central nervous system by injecting it -16 9 -

into the lateral ventricals of rat brains. Again no description of the purity and authenticity of the sulphates was given. These authors claimed that the conjugate caused severe clonic and tonic convulsions lasting as long as 20 minutes after the injection. The conclusions drawn from this experiment, that dopamine sulphates have direct physiological effects of their own, which are different from those of free dopamine, cannot be justified. If the dopamine 0-sulphates prepared in these experiments were contaminated with, for example, dopamine 6-sulphonic acid, it is possible that the responses elicited were caused by this compound or other contaminants and not the dopamine

0-sulphates.

Because little is known about the biological role of catecholamine sulphates in mammalian systems, it is vital that the results of such studies are reviewed critically. There should be a clear indication of the purity of the synthesised sulphates when carring out in vivo experiments, otherwise misleading conclusions may be drawn which may apply to impurities in the sulphates rather than the sulphates themselves.

There is limited information available concerning the disposition and the role of noradrenaine ()-sulphates. Again, this results from the lack of available authentic standards of these compounds. The definitive synthesis of these has been performed (Wang et ^1_., 1972), but involved a seven-step procedure which was difficult and costly. In this Thesis, a one-step synthesis of noradrenaline 0-sulphates has been described.

The conditions of this synthesis differed from those for dopamine sulphates synthesis, but the methods used for the separation, purification and authentication were similar. The main problem with this synthesis was to find an appropriate sulphating agent.

Favourable conditions were achieved using chlorosulphonic acid as the sulphate donor and N^N_-diethyl anil ine as catalizing agent. -170-

Although it is known that the major metabolite of noradrenaline,

3-methoxy-4-hydroxyphenylethyleneglycol (Schanberg et al_., 1968) occurs

in the brain of several species as a sulphate conjugate, little data are

available on the disposition of noradrenaline O-sulphates, because methods for the direct estimation of these compounds in tissues and

biological fluids have not been available hitherto.

In order to understand more fully the importance of catecholamine

sulphoconjugation and to assess whether or not catecholamine O-sulphates

play an important role in the metabolism of these amines, it was

necessary to develop direct methods for studies on the disposition of

sulphoconjugates in tissues and biological fluids.

Much research on dopamine £-sulphate disposition in man has

concentrated on examining the metabolic fate of the precursor of

dopamine, L-dopa.

Dopamine 3- and 4-0-sulphates were estimated in the urine of

Parkinsonian patients or normal volunteers following the oral

administration of L-dopa by several groups of workers (Jenner & Rose,

1974; Bronaugh et a]_., 1974a,b; 1975; Tyce et al_., 1974; Arakawa et

al., 1979). Dopamine 3-0-sulphate was found to be the predominant

sulphoconjugate. However, very few investigators estimated the

quantities of dopamine 3- and 4-0-sulphates in normal urine. Buu &

Kuchel (1977) measured dopamine O-sulphates in normal urine by a method

based on acid hydrolysis of urine followed by lyophilization and

subsequent analysis for dopamine. They found dopamine O-sulphates

present in urine in concentrations varying from 60 to 200 ng/ml.

Unfortunately these authors did not state the quantities excreted per

24 h. This method would not allow for the separate estimation of

isomeric dopamine ^-sulphates and it could not be known whether complete

hydrolysis of the sulphoconjugates occurred. Furthermore, it is not -171-

known whether acid lyophilization hydrolyses the more stable glucuronide conjugates.

It is also difficult to determine the true recovery of this procedure as it is not known to what extent free dopamine liberated from the sulphoconjugate is destroyed during this procedure.

With the method described in this study, dopamine 3- and 4-0- sulphates were estimated in the urine of healthy, male volunteers. The values obtained for the excretion of both dopamine sulphate isomers were somewhat higher (range 164-295 pmol/24 h; 250-600 ng/ml) than those found by Buu & Kuchel. Only in the urine of one subject were both dopamine sulphates identified. Dopamine 3-0-sulphate was the major sulphoconjugate in the urine of this subject and the only sulphoconjugate found in the urine of the remaining subjects. In four out of twelve urine collections from a single subject, both dopamine

O-sulphates were identified and, again, dopamine 3-0-sulphate was the major sulphoconjugate.

The h.p.l.c. method developed permits the direct estimation of both dopamine O-sulphates in urine. It involves a simple preparation procedure of urine samples which does not destroy compounds of interest and assures that dopamine O-sulphates are well separated from other constituents of urine which might interfere with the analysis. It was necessary to use h.p.l.c. with electrochemical detection, as the levels in urine were too low to be measured by U.V. detection. The development of this sensitive assay provides new facilities for investigating the metabolism and disposition of these sulphates. The sensitivity of this method obviates the need for costly radiolabel led dopamine ^-sulphates for the investigations in vivo of metabolism of these compounds (Jenner

& Rose, 1974; Merits, 1976), and small changes in the concentration of -172-

dopamine 3- and 4-£-sulphates could be detected by this non-

radiochemical method.

This procedure could also be used for the identification and

quantitive estimation of the products arising from incubation of

different acceptors, such as dopamine, noradrenaline, adrenaline,

isoprenaline and 3,4-dihydroxybenzoic acid with tissue homogenates

containing phenolsulphotransferase activity.

This method can also be used when larger amounts of dopamine 0-

sulphates are excreted, for example following the oral administration of

L-dopa. Results presented here showed that following oral

administration of L-dopa both dopamine sulphates were excreted in the

urine of all subjects, dopamine 3-£-sulphate being the major conjugate

which constituted approximatey 80% of total sulphates.

Under physiological conditions, dopamine 4-0-sulphate in urine was

found in only 25% of the cases. In contrast, following L-dopa

administration all subjects excreted dopamine 4-0-sulphate as well as

the 3-0-sulphate. This observation is in agreement with the findings of

other investigators (Jenner & Rose, 1974; Bronaugh ^t ^1_., 1975;

A r a k a w a et aj_., 1979).

The fact that dopamine 4-0-sulphate was not detected in three out of

four normal urines could be explained by several mechanisms. It has

been claimed that more dopamine 3-0-sulphate is formed in vivo because

PST preferentially attacks the hydroxyl group of phenolic componds at

position 3 of the aromatic ring (Wong, 1982) or that dopamine

4-0-sulphate is more prone to hydrolysis in vivo by arylsulphatase than

the 3-0-isomer (Jenner & Rose, 1978) or, finally, that dopamine 4-0-

sulphate is further metabolised without being desulphated (Jenner &

Rose, 1978). -173-

The results in this study on the disposition of noradrenaline 0- sulphates in rabbit tissues suggested that both 3- and 4-0-sulphate isomers were present in various tissues in ratios depending on the tissue and animal investigated. Obviously, it is difficult to extrapolate from animal data to human. However, if similar findings were made in human tissues and the presence of 3- and 4-0-sulphates was observed, then it is possible to speculate that the catecholamine 4-0- sulphate is a less stable sulphoconjugate and further metabolised or desulphated before excretion. If this could be substantiated, then possibly the two isomeric sulphates play quite a different role in catecholamine metabolism. Catecholamine 3-0-sulphate might be the final product of metabolism whereas, catecholamine 4-0-sulphate might be an intermediary product undergoing further metabolism.

Although the analysis of homogenates was performed on tissues from a small number of animals, certain observations could still be made from the data obtained. The presence of both noradrenaline O-sulphates was detected in almost all tissues investigated, whereas dopamine (D- sulphates were not detected in any of the tissues examined. In tissue homogenates from one animal, noradrenaline 4-0-sulphate was not detected. This observation might suggest the existance of two different forms of PST that catalyse sulphoconjugation in the meta and para positions of the aromatic ring. However, before one could speculate any further, more experiments would need to be performed on larger numbers of animals.

In homogenates of rabbit liver the highest amounts of total noradrenaline sulphates were noted, whereas the greatest concentration of noradrenaline 3-0-sulphate was observed in the small intestine. The presence of noradrenaline O-sulphates was not restricted to the -174-

peripheral tissues only, they were also present in homogenates of rabbit brain.

The sulphate ester of the noradrenaline metabolite, 3-methoxy-4- hydroxyphenylethyleneglycol (MHPG) was identified in the central nervous system and cerebrospinal fluid of humans and animal species (Karoum et al., 1977). Therefore, the finding of both noradrenaline O-sulphates in rabbit brain was not surprising. Recently, Buu & Kuchel (1981) demonstrated the presence of dopamine and noradrenaline O-sulphates in the different areas of rat brain. The method used for the estimation of catecholamines in their study was based on the estimation of free catecholamines liberated f r o m s u lphoconjugates. These authors found the levels of dopamine sulphates in various areas of rat brain

(hypothalamus, striatum, hippocampus) varied from 25 to 57 ng/g of tissue and of noradrenaline O-sulphates (hypothalamus and striatum) in concentrations 20-37 ng/g of tissue.

The values obtained by the above authors are much lower than those obtained in the present study. The amounts of noradrenaline 4-()- sulphate varied from 2 to 5.3 ng/mg and noradrenaline 3-0-sulphate from

1.4 to 6.1 ng/mg of rabbit brain tissue.

This great difference in noradrenaline O-sulphate concentrations could be explained by the following: different species show different

PST activity and the availability of inorganic sulphate for sulphoconjugation reactions also varies between species. Hence the quantities of sulphoconjugates in tissues might vary between the species. The other possibility is the difference in methods used for the estimation of catecholamine sulphates. Roth & Rivett (1982) claimed that the affinity of sulphotransferase towards catecholamines varied greatly between species; for example, the affinity of human -175-

sulphotransferase for dopamine, according to these authors, was 100 times greater than that of the sulphotransferase in rat, mouse, guinea pig, ox and dog brain. It is possible that similar differences exist between commonly used laboratory animals. The other factor that could influence the quantities of catecholamine sulphoconjugates in tissues is the sulphate availability in vivo. This will be reflected in changes in the concentration of inorganic sulphate in the blood. The serum concentrations of inorganic sulphate show wide species differences. In rat and mouse, it is approximately 1 mM, rabbit and chicken have much higher concentrations, up to 2.5 mM (Mulder, 1981; Krijgsheld et a!.,

1980). The immediately available pool of inorganic sulphate varies approximately in parallel with the serum sulphate concentration.

Under normal conditions, inorganic sulphate is excreted in urine and when it is depleted in the body the excretion of sulphate is markedly decreased. The rat excretes 70 pmol/kg/h of inorganic sulphate, rabbit

25 pmol/kg/h. Taking the above observation into consideration, it seems that the rabbit has a larger pool of inorganic sulphate available for sulphoconjugation and also excretes less inorganic sulphate. Therefore, it is possible that the rabbit would have greater amounts of sulphoconjugates in various tissues than the rat.

The above considerations do not explain why dopamine O-sulphates were not detected in the rabbit tissues. The only possible explanation for this observation is that rabbit phenolsulphotransferase has a higher affinity for noradrenaline than for dopamine. It has been claimed that human brain PST affinity for endogenous catecholamines is in order dopamine < noradrenaline < adrenaline (Roth et a]_., 1981). However, the above data indicate that in the rabbit brain, noradrenaline might be the preferred substrate for PST. -176-

The investigation of dopamine and noradrenaline O-sulphate metabolism

in vitro also produced results somewhat different from the findings of

Buu & Kuchel (1979a,b) who performed similar experiments. They found

free noradrenaline to be the only product of the reaction between

dopamine O-sulphates and dopamine B-hydroxylase. In the present study

noradrenaline O-sulphates were also found to be products of this

reaction. Certain observations in both studies are similar, for

example, the above authors found dopamine 4-0-sulphate to be a better

substrate for DBH, the reaction yielded more free noradrenaline than

when the 3-0-isomer was used as a substrate, a finding which was

confirmed in this study. However, in the present study it was found

that noradrenaline 0-sulphate formation was greater when dopamine 3-0-

sulphate was used as a substrate in comparison with dopamine 4-0-

sulphate.

The presence of arylsulphatase in DBH and catalase preparations was

eliminated, however, it is very difficult to explain why noradrenaline

3- and 4-0-sulphate incubated with DBH under reaction conditions

identical to those used for dopamine 0-sulphate experiments also

resulted in the formation of free noradrenaline. Likewise, when

noradrenaline 4-0-sulphate was used as substrate, more free noradrenaline was formed than with the 3-isomer.

The possible reaction mechanisms explaining the formation of free noradrenaline and the formation of noradrenaline O-sulphates from dopamine ^-sulphates were presented in Chapter 5. Fusaric acid, a strong inhibitor of dopamine B-hydroxylase, inhibited the formation of noradrenaline from dopamine O-sulphates, however, it had no effect on the formation of free noradrenaline from noradrenaline sulphates. It is probable that this compound inhibits B-hydroxylation of the side chain -177-

of dopamine, hence it had no effect on the reaction between DBH and noradrenaline O-sulphates. However, before any firm conclusions could be drawn from the above findings, these experiments should be performed in the presence of an aryl sulphatase inhibitor. If the same products result when arylsulphatase inhibitors are added to the reaction mixture with dopamine and noradrenaline O-sulphates as substrates, this would provide unquestionable evidence that DBH has some desulphating properties, at least in vitro, in the presence of ascorbic acid and other constituents of the reaction mixture. If the finding that dopamine O-sulphates could be precursors of free noradrenaline and noradrenaline sulphates and noradrenaline O-sulphate precursors of free noradrenaline are confirmed in vivo, it would support the idea that catecholamine sulphates are convenient forms for storage of free catecholamines. However, more studies are required to confirm such speculation.

Although the exact role of catecholamine sulphoconjugates in the metabolism of these amines is still far from being fully understood, no-one could deny the importance of sulphoconjugation as a detoxication mechanism. The role of sulphoconjugation in the metabolism of dopamine following oral administration of L-dopa has already been discussed in the Introduction to this Thesis. A number of antipsychotic drugs also increase the release of dopamine in brain

(Besson et ^1_., 1973). Conjugation of dopamine might therefore be expected to assume a critically important role during chronic treatment of psychotic patients with neuroleptics. Michelot et al_. (1977) reported that cultured murine neuroblastoma cells from a strain deficient in monoamine oxidase activity, when incubated with dopamine, produced about equal amounts of 3-methoxytyramine and dopamine sulphate, the respective products of the methylation and conjugation. When the -178-

methyl ation pathway was inhibited, however, conjugation became the

predominant metabolic reaction.

Under physiological conditions, PST activity can account for

approximately 10% of the total enzymatic activity involved in the destruction of dopamine (Roth et a]_., 1981). However, even if

sulphoconjugation is normally a minor pathway of, for example, dopamine

inactivation, conjugation could assume an important role in clinical

conditions or animal studies where other pathways are functioning

abnormally or are pharmacologically blocked.

It has been postulated that in tyramine-sensitive migraine a defect

exists in sulphoconjugation of this compound (Youdim jet ^1_., 1971).

Kuchel et ^1_. (1981) claimed that a similar defect in sulphoconjugation

existed in a form of essential hypertension in which very low or

undetectable concentrations of conjugated catecholamines were found in

plasma. This was associated with an increased free catecholamine

concentration in plasma. They suggested that the increased free

catecholamine concentration reflected an excessive response to stress

and glucagon, and the conclusion was made that a defect in

sulphoconjugation left more free catecholamines (especially

noradrenaline and adrenaline) in the free state so that the adrenergic

receptors were more exposed to the actions of the catecholamines. As a

result, the symptoms and signs of sympathetic hyperactivity were

produced.

There are major controversies concerning the levels of circulating

catecholamines in hypertension. Many factors are involved in this

condition and only few investigators have found consistently increased

levels of catecholamines in hypertension (see Introduction).

Therefore, the idea postulated by Kuchel et _al_., (1981), although -179-

interesting, should be substantiated by more experimental evidence.

Despite the progress that has been made in the field of sulphoconjugation in recent years there are still many questions that need to be answered before this process is significantly assessed and the importance of this pathway in the metabolism of catechol and phenolic endogenous and exogenous compounds is fully understood.

The present study provides a good basis for further investigations of catecholamine sulphoconjugation. The next step would be the synthesis and characterisation of adrenaline and isoprenaline sulphates. With methodology described in this work for the synthesis, purification and authentication of dopamine and noradrenaline O-sulphates, the synthesis of adrenaline and isoprenaline sulphates should be straightforward.

Adrenaline O-sulphate synthesis would be an important step for the

investigation of the role of catecholamine sulphates in overall metabolism of this amine. This is of particular importance since circulating adrenaline is a far better candidate for a circulating hormone than noradrenaline or dopamine. Adrenaline O-sulphates could be used as standards for measurement of these in tissues and biological fluid. However, the work on sulphoconjugation should not be restricted to investigations of this process only in relation to endogenous c o mpounds.

Many drugs that have catechol type structure such as isoprenaline, dobutamine, terbutaline, a-methyldopa and the already mentioned L-dopa are also extensively sulphoconjugated (Conolly et _al_., 1972; Campbell ejt ^1_., 1983). Isoprenaline was administered by inhalation in asthma.

Oral or sublingual routes of administration were not recommended because of unreliability and potential toxicity, and this was thought possibly to result from differences in sulphoconjugation of this compound. It is -1 8 0 -

known that in man 80% of orally administered isoprenaline is

sulphoconjugated (Conolly et a^., 1972). Interindividual variations in

the extent of this compound's sulphation may account for its

"unreliability" when administered orally. Presently, isoprenaline has

been replaced by selective agonists such as salbutamol and terbutaline. However, it would be of interest to investigate which factors account for differences in the sulphoconjugation of this drug.

Authentic isoprenaline sulphate could be used for investigations

regarding the disposition of isoprenaline and also as a possible

internal standard for h.p.l.c. assays developed for the estimation of endogenous catecholamine ^-sulphates.

a-Methyldopa is one of the most widely used drugs in the treatment

of hypertension. Sulphoconjugation of this drug is an important route

of metabolism. It has been claimed that in man variations in platelet

PST activity reflect variations in the sulphate conjugation of

a-methyldopa (Campbell et al_., 1983). If PST activity in man is under

genetic control (Roth & Rivett, 1982), then great differences in metabolism of this and other drugs might be expected and be causally

related to the variations in drug responsiveness and toxicity.

It should be pointed out that not only are phenols and catechols

extensively sulphoconjugated, but also another very important group of

compounds undergoing sulphoconjugation are the steroids.

Sulphation is a common conjugate reaction of steroid hormones and

numerous steroid sulphates have been detected not only in faeces, urine

and bile, but also in blood and tissue extracts (Mulder, 1982). Often,

these sulphoconjugates assume a role as intermediates in steroid metabolism. In testis, for instance, four different steroid sulphates

are present in appreciable amounts; these most likely play a role as -1 8 1 -

intermediate in biosynthesis of testosterone (Vihko & Ruokonen, 1982).

Changes in the hormonal status of the organism will be followed by changes in the relative amounts of the various steroid sulphates found in tissues and body fluids. Thus, pregnancy results in profound changes in the steroid pattern, one of the main causes being the rapid growth of a new, very active, steroid metabolising organ, the fetal-placental unit

(Mulder, 1982). Obviously, sulphation is also a pathway for the removal of the biologically active form of steroids. This requires highly specific sulphatases and sulphotransferases that selectively hydrolyse or conjugate those steroids that are needed in the particular cell.

Studies on the physiologically important steroid sulphotransferases in specific cell types led to the discovery of a role of estrogen sulphotransferase in the regulation of the oestrus cycle (Brooks et a!.,

1982).

A critically regulated interplay between various metabolic routes

(among them sulphation) and specific estrogen receptors may play a role in many tissues and it is possible that such an integration is deregulated in, for example, breast tumours (Brooks et al_., 1982), as intact human breast cancer cells are incapable of sulphating oestrogens.

The above observations are only examples of possible implications of sulphoconjugation on metabolism and mode of action of many compounds, both endogenous and exogenous. They indicate that any variation in the capacity of an organism to carry out a particular conjugation reaction is to be expected to have marked consequences for the overall biological activity and disposition of compounds metabolised by that route. This may account for some species differences in drug disposition and genetically determined variations of conjugation within the same species. -1 8 2 -

The advancement in our knowledge of the importance of the sulphoconjugation pathway might, in the near future, provide many answers to the exact involvement of this process in detoxication and further metabolism of many endogenous and exogenous compounds. -183-

BIBLIOGRAPHY

Abel, J.J. & Crawford, A.C. (1897). On the blood pressure raising

constituent of the suprarenal capsule. Bull. Johns Hopkins Hosp.

8, 151-157.

Alton, H. & Goodall, M. (1968). Metabolic products of adrenaline

(epinephrine) during constant rate intravenous infusion in the human.

Biochem. Pharmac. J_7, 2163-2169.

Anden, N.E., Carlsson, A., Kerstell, J., Magnusson, J., Olsson, R.,

Roos, B.E., Steen, B., Steg, G., Svanborg, A., Thieme, G. and

Weridinius, B. (1970). Oral L-dopa treatment of Parkinsonism. Acta

Med. Scand. J87, 247-255.

Anderson, R.J. & WeinshiIboum, R.M. (1979). Phenolsulfotransferase

enzyme activity and endogenous inhibitors in the human erythrocyte.

J. Lab. Clin. Med. 94, 158-171.

Anderson, R.J. & Weinshi1 bourn, R.M. (1980). Phenolsulphotransferase

in human tissue: Radiochemical enzymatic assay and biochemical

properties. Clin. Chim. Acta. 103, 79-90.

Arakawa, Y., Imai, K. & Tamura, I. (1979). High-performance liquid

chromatographic determination of dopamine sulfoconjugates in urine

after L-dopa administration. J. Chromatogr. 162, 311-318.

Armstrong, M.D., McMillan, A. & Shaw, K.N.F. (1957). 3-Methoxy-4-

hydroxy-d-mandelic acid, a urinary metabolite of norepinephrine.

Biochim. Biophys. Acta 25, 422-429.

Axelrod, J. (1962). Purification and properties of phenylethanolamine-

N-methyltransferase. J. Biol. Chem. 237, 1657-1660. -184-

Axel rod, J., Inscoe, J.K., Senoh, S. & Witkop, B. (1958).

^-Methylation, the principal pathway for the metabolism of epinephrine

and norepinephrine in the rat. Biochim. Biophys. Acta. 27, 210-211.

Axelrod, J. & Tomchick, R. (1958). Enzymatic 0-methylation of

norepinephrine and other catechols. J. Biol. Chem. 233, 702-705.

Axelrod, J. & Weinshilboum, R. (1972). Catecholamines. N. Engl. J.

Med. 287, 237-242.

Banerjee, R.K. & Roy, A.B. (1966). The sulfotransferases of guinea pig

liver. Mol. Pharmacol. 2 , 56-66.

Banerjee, R.K. & Roy, A.B. (1968). Kinetic studies of phenol

sulfotransferase reaction. Biochim. Biophys. Acta. 151, 573-587.

Bartholomew, T.C., Powell, G.M., Dodgson, K.S. & Curtis, C.G. (1980).

Oxidation of sodium sulphide by rat liver, lungs and kidney. Biochem.

Pharmacol. 29, 2431-2437.

Baulieu, E.E., Corpechot, C., Dray, F., Emiliozzi, R., Lebeau, M.C.,

Mauvais Jarvis, P. & Robel, P. (1965). An adrenal-secreted

"Androgen": Dehydroisoandrosterone sulfate. Its metabolism and a

tentative generalisation on the metabolism of other steroid conjugates

in man. Recent Prog. Horm. Res. 21_, 411-494.

Baumann, E. (1876). Uber gepaarte Schwefelsauren in Organismus

Pflugers. Arch. Gen. Pysiol. Menschen. Thiere. 13, 285-308.

Bernstein, S. & McGilvery, R.W. (1952a). The enzymatic conjugation of

m-Aminophenol. J. Biol. Chem. 198, 195-203. -185-

Bernstein, S. & McGilvery, R.M. (1952b). Substrate activation in the

synthesis of phenyl sulfate. J. Biol. Chem. 199, 745-748.

Bertel, 0., Buhler, F.R., Kiowski, W. & Lutold, B.E. (1980). Decreased

beta-adrenoreceptor responsiveness as related to age, blood pressure

and plasma catecholamines in patients with essential hypertension.

Hypertension, 2, 130-138.

Besson, M.J., Cheramy, A., Glowinski, J. & Gauchy, C. (1973). In 3 vivo continuous release of H-DA from the cat caudate nucleus.

Effects of methyl-p-tyrosine and of transection of the nigro

neostriatal pathway. Naunyn Schmiedebergs Arch Pharmacol. 278,

101-105.

Beyer, K.H. & Shapiro, S.H. (1945). The excretion of conjugated

epinephrine related compounds. Am. J. Physiol. 144, 321-330.

Binkley, F. (1949). The source of sulfate in the formation of ethereal

sulfates. J. Biol. Chem. 178, 821-826.

Blaschko, H. (1942). The activity of l(-)dopa decarboxylase.

J. Physiol. 101, 337-349.

Blaschko, H. (1973). Catecholamine biosynthesis. Br. Med. Bull. 29,

105-109.

Bodnaryk, R.P. & Brunet, P.C.J. (1974). 3-j)_-Hydrosulphato-4-

hydroxyphenethylamine (Dopamine 3-JT-sulphate), a metabolite involved

in the sclerotization of insect cuticle. Biochem. J. 138, 463-469.

Bouillin, D.J. (1967). The action of extracellular cations on the

release of the sympathetic transmitter from peripheral nerves.

0. Physiol. 189, 85-89. -1 8 6 -

Bray, M.G., Humphries, B.G., Thorpe, W.V., White, K. & Wood, P.B.

(1952). Kinetic studies of the metabolism of foreign compounds.

4. The conjugation of phenols with sulphuric acids. Biochem. J.

52, 419-423.

Brecht, M.M. & Schoeppe, W. (1978). Relation of plasma noradrenaline

to blood pressure, age, sex and sodium balance in patients with stable

essential hypertension and in normotensive subjects. Clin. Sci. Mol.

Med. 55, 81s-83s.

Bridgers, W. & Kaufman, S. (1962). The enzymatic conversion of epinine

to epinephrine. 0. Biol. Chem. 237, 526-528.

Bronaugh, R.L., Hattox, S., Hoehn, M.M., Murphy, R.C. & Rutledge, C.O.

(1974a). Urinary excretion of dopamine sulfate isomers in

Parkinsonian patients. Trans. Amer. Soc. Neurochem. 5^, 82.

Bronaugh, R.L., Hattox, S.E., Hoehn, M.M., Murphy, R.C. & Rutledge, C.O.

(1975). The separation and identification of dopamine 3-_0_-sulphate

and dopamine 4-0_-sulphate in urine of Parkinsonian patients.

J. Pharmacol. Exp. Ther. 195, 441-452.

Bronaugh, R.L., McMurtry, R.J., Hoehn, M.M. & Rutledge, C.O. (1975).

Conjugation of L-dopa and its metabolites after oral and intravenous

administration to Parkinsonian patients. Biochem. Pharmacol. 24,

1317-1320.

Bronaugh, R.L., Wenger, G.R., Garver, D.L. & Rutledge, C.O. (1974b).

Metabolism of L-dopa in the pigtail monkey. Effect of a dopa

decarboxylase inhibitor. Pharmacologist 236. -187-

Brooks, S.C., Pack, B.A., Rozhin, J. & Christensen, C. (1982).

In situ control of estrogen activity by sulfurylation in uterus and

other target tissues. In: Sulfate Metabolism and Sulfate

Conjugation. Eds. Mulder, J., Caldwell, J., van Kempen, G.M.J.,

Vonk, R.J. London: Taylor & Francis, pp. 173-180.

Burgen, A.S.V. & Iversen, L.L. (1965). The inhibition of noradrenaline

uptake by sympathomimetic amines in the rat isolated heart. Br. J.

Pharmacol. Chemother. 25, 34-49.

Burn, J.H. & Gibbons, W.R. (1965). The release of noradrenaline from

sympathetic nerve fibres in relation to calcium concentration.

J. Physiol. J81_, 214-223.

Burns, G.R. & Wynn, C.H. (1975). Studies on the arylsulphatase and

phenol sulphotransferase activities of Aspergillus oryzae.

Biochem. J. 1 4 9 , 697-705.

Buu, N.T. & Kuchel, 0. (1977). A new method for the hydrolysis of

conjugated catecholamines. J. Lab. Clin. Med. 90, 680-685.

Buu, N.T. & Kuchel, 0. (1979a). The direct conversion of dopamine-3-CT-

sulphate to norepinephrine by dopamine 3 -hydroxylase. Life Sci. 24,

783-790.

Buu, N.T. & Kuchel, 0. (1979b). Dopamine-4-_0_-sulfate: a possible

precursor of free norepinephrine. Can. J. Biochem. 57_, 1159-1162.

Buu, N.T. & Kuchel, 0. (1981). Phenolsulphotransferase activity and

levels of catecholamine sulfates in rat brain tissue. In:

Phenolsulftransferase In Mental Health Research, ed. Sandler, M. &

Usdin, E. London: Macmillan, pp. 145-157. -1 8 8 -

Buu, N.T., Duhaime, J., Savard, C., Truong, L. & Kuchel, 0. (1981).

Presence of conjugated catecholamines in rat brain: a new method of

analysis of catecholamine sulfates. J. Neurochem. 36 (2), 769-772.

Buu, N.T., Nair, 6., Kuchel, 0. & Genest, J. (1981). The extra-adrenal

synthesis of epinephrine in rats. J. Lab. Clin. Med. 98, 527-535.

Buu, N.T., Unger, Th. & Kuchel, 0. (1978). Conjugated catecholamines in

DOCA-saline hypertensive rats. Clin. Res. ^ 6, 361A.

Campbell, N.R.C., Dunnette, J.H., van Loon, J., Mwaluko, G. &

Weinshilboum, R.M. (1983). Catechol-Ojmethyltransferase, phenol

sulphotransferase and methyldopa metabolism in man. In: 2nd World

Conference on Clinical Pharmacology and Therapeutics, Washington D.C.

p. 628.

Campese, V., Meyrs, M.R. & De Quattro, V. (1977). Plasma catecholamines

and neurogenic hypertension. N. Engl. J. Med. 297, 53.

Carter, S.M., Glover, V., Sandler, M., Gillman, P.K. & Bridges, P.K.

(1981). Human platelet phenolsulphotransferase: separate control of

the two forms and activity range in depressive illness. Clin. Chim.

Acta. 117, 333-344.

Christensen, N.H. (1972a). Increased levels of plasma noradrenaline in

hypothyroidism. J. Clin. Endocrinol. 35^, 359-363.

Christensen, N.H. (1972b). Plasma catecholamines in long term diabetes

with and without neuropathy in hypophysectomized subjects. J. Clin.

Invest. 51, 779-787. -189-

Clark, W.G., Arawie, R.J., Pogrund, R.S. & Geissman, T.A. (1951).

Conjugation of epinephrine in vivo. J. Pharm. Exp. Ther. 101, 6-7.

Conolly, M.E., Davies, D.S., Dollery, C.T., Morgan, C.D., Paterson, J.W.

& Sandler, M. (1972). Metabolism of isoprenaline in dog and man.

Br. J. P h a r m a c o l . 46, 458-472.

Creveling, C.R., Daly, J.W., Witkop, B. & Udenfriend, S. (1962).

Substrates and inhibitors of dopamine 3-oxidase. Biochim. Biophys.

Acta. 64, 125-134.

Crooks, P.A., Breakefield, X.O., Sulens, C.H., Castiglione, C.M. &

Coward, J.K. (1978). Extensive conjugation of dopamine

(3,4-dihydroxyphenethylamine) metabolites in cultured human skin

fibroblasts and rat hepatoma cells. Biochem. J. 176, 187-196.

Cuche, J.L., Prinseau, J., Ruget, G., Seitz, F., Tual, J.L.,

Baglin, A., Guedan, J. & Fritel, D. (1982). Plasma free and

sulfoconjugated catecholamines in healthy man. Eur. Health J. _3

(Suppl. C), 3-8.

Curtis, C.G. (1982). The origins of intracellular sulphate for

conjugation reactions. In: Sulphate Metabolism and Sulphate

Conjugation, eds. Mulder, G.J., Caldwell, J., Van Kempen, G.M.J.

& Vonk, R.J. London: Taylor & Francis, pp. 67-71.

Da Prada, M. & Zurcher, G. (1976). Simultaneous radioenzymatic

determination of plasma and tissue adrenaline, noradrenaline and

dopamine within the femtomole range. Life Sci. Jj), 1161-1174.

De Champlain, J. & Cousineau, D. (1977). Lack of correlation between

age and circulating catecholamines in normotensive and hypertensive

patients. N. Engl. J. Med. 297, 672. “ 190 -

Deichman, W.B. (1943). Excretion of organic sulfates following

administration of adrenalin. Proc. Soc. Exp. Biol. Med. 54, 335-336.

De Meio, R.H. (1975). Sulfate activation and transfer. In: Metabolism

of Sulfur Compounds, ed. Greenberg, D.M. London: Academic Press,

pp. 287-358.

De Meio, R.H., Wizerkaniuk, M. & Fabiani, E. (1953). Role of

adenosinetriphosphate in the enzymatic synthesis of phenyl sulfate.

J. Biol. Chem. 203, 257-263.

Dengler, H.J., Michaelson, J.A., Spiegel, H.E. & Titus, E. (1962).

The uptake of labelled norepinephrine by isolated brain and other

tissues of the cat. Int. J. Neuropharm. 2» 23-38.

de Potter, W.P., Schaepdryver, A.F., Moerman, E.J. & Smith, A.D. (1969).

Evidence for release of vesicle-proteins together with noradrenaline

upon stimulation of the splenic nerve. J. Physiol. 204, 102-104.

de Quattro, V. & Chan, S. (1972). Raised plasma catecholamines in some

patients with primary hypertension. Lancet, j_, 806-809.

Dodgson, K.S. (1977). Conjugation with sulphate. In: Drug Metabolism

from Microbe to Man. eds. Parke, D.V.W. & Smith, R.L. London: Taylor

& Francis, pp. 91-104.

Dodgson, K.S. & Rose, F.A. (1966). Some aspects of the biochemistry

of sulphur compounds. Nutr. Abstr. Rev. 36, 327-342.

Dodgson, K.S. & Rose, F.A. (1970). Sulphoconjugation and

sulfhydrolysis. In: Metabolic Conjugation and Metabolic Hydrolysis,

Vol. 1. ed. Fishman, W.H. London: Academic Press, pp. 239-325. -191-

Dodgson, K.S. & Rose, F.A. (1980). Observations on the biological roles

of sulphatases. In: Sulphur in Biology, Ciba Foundation Symposium.

Exerpta Medica, 72, 163-176.

Dodgson, K.S., Powell, G.M., Rose, F.A. & Tudball, N. (1961). oc Observations on the metaboism of Tyrosine 0 [ S]-sulphate in the rat.

Biochem. J. 79, 209-213.

Dodgson, K.S. & Tudball, N. (1960). The metabolic fate of the ester

35 sulphate group of potassium p-nitrophenyl [ S] sulphate. Biochem. J.

74, 154-159.

Dziewiatkowski, D.D. (1949). On the utilization of exogenous sulfate

sulfur by the rat in the formation of ethereal sulfates as indicated

by the use of sodium sulfate labelled with radioactive sulfur.

J. Biol. Chem. 178, 389-393.

Dziewiatkowski, D.D. & Di Ferrante, N. (1957). Association of sulfate- 35 S with serum proteins in the rat. J. Biol. Chem. 227, 347-356.

Ebert, M. & Kopin, I.J. (1975). Differential labelling of origins of

14 urinary catecholamine metabolites by dopamine- C. In: Transactions

of the Association of American Physicians, 88, 256-264.

Eccleston, D. & Ritchie, J.M. (1973). Sulphate ester formation from

catecholamine metabolites and pyrogallol in rat brain in vivo.

J. Neurochem. 21, 635-646.

Engelman, K. (1977). Phaeochromocytoma. Clinics in Endocrinology

and Metabolism. 6, 769-797. -192-

Engelman, K., Portnoy, B. & Lovenberg, W. (1968). A sensitive and

specific double-isotope derivative method for the determination of

catecholamines in biological specimens. Am. J. Med. Sci. 255,

259-268.

Engelman, K., Portnoy, B. & Sjoerdsma, A. (1970). Plasma catecholamine

concentrations in patients with hypertension. Circ. Res. 26/27

(Suppl. 1), I-141-I-145.

Esler, M., Skews, H., Leonard, P., Jackman, G., Bobik, A. & Korner, P.

(1981). Age dependence of noradrenaline kinetics in normal subjects.

Clin. Sci. 60, 217-219.

Floras, J.S. (1981). Studies on neuronal regulation of blood pressure

in hypertension. Doctor of Philosophy Thesis, University of Oxford.

Flynn, T.G., Dodgson, K.S., Powell, G.M. & Rose, F.A. (1967). The

35 metaboism of dipotassium 2-hydroxy-5-nitrophenyl [ S] sulphate, a

substrate for lysosomal arylsulphatases A & B. Biochem. J. 105,

1003-1 0 1 0 .

Foldes, A. & Meek, J.L. (1973). Rat brain phenolsulfotransferase -

partial purification and some properties. Biochim. Biophys. Acta.

327, 365-373.

Foldes, A. & Meek, O.L. (1974). Occurrence and localisation of brain

phenol sulphotransferase. J. Neurochem. 23, 303-307.

Franco-Morselli, R., Elghozi, J.L., Joly, E., Di Guilio, S. & Meyer, P.

(1977). Increased plasma adrenaline concentrations in benign

essential hypertension. Br. Med. J. 2 , 1251-1254. -193-

Fuller, R.W. & Hunt, J.M. (1967). Inhibition of phenethanolamine-N^

methyl transferase by its product, epinephrine. Life Sci. S9

1107- 1 1 1 2 .

Funk, C. (1911). Synthesis of d-l-3:4-dihydroxyphenylalanine.

J. Chem. Soc. 99, 554-557.

Geffen, L.B., Livett, B.G. & Rush, R.A. (1969). Immunological

localization of chromogranins in sheep sympathetic neurones and their

release by nerve impulses. J. Physiol. 204, 58-59.

Gewirtz, G.P. & Kopin, I.J. (1970). Release of dopamine-B-hydroxylase

with norepinephrine by splenic nerve stimulation. Nature, 227,

406-407.

Goldberg, W.J. (1972). Cardiovascular and renal actions of dopamine:

potential clinical applications. Pharmacological Reviews, 24, 2-23.

Goldberg, L.I. (1975). The dopamine vascular receptor. Biochem.

Pharmacol. 2A_9 651-653.

Goldstein, D.S. (1981). Plasma norepinephrine in essential

hypertension: A study of the studies. Hypertension, 3, 48-52.

Goldstein, D.S. & Contrera, J.F. (1962). The substrate specificity of

phenylamine-B-hydroxylase. J. Biol. Chem. 237, 1898-1902.

Goodall, M. & Alton, H. (1965). Urinary excretion of adrenaline

metabolites in man during intervals of 2 minutes, 5 minutes and 10

minutes after intravenous injection of adrenaline. Biochem. Pharmac.

14, 1595-1604. - 194-

Goodall, M. & Alton, H. (1968). Metabolism of 3-hydroxytryptamine

(dopamine) in human subjects. Biochem. Pharmac. J_7, 905-914.

Goodall, M. & Alton, H. (1968). Preliminary identification of 3,4-

dihydroxyphenylacetic acid and 3-methoxy-4-hydroxyphenylacetic acid

in human urine as metabolites of epinephrine. Texas Rep. Biol. Med.

26, 107-115.

Goodall, M. & Alton, H. (1972). Metabolism of 3,4-Dihydroxyphenyl-

alanine (L-dopa) in human subjects. Biochem. Pharm. 21, 2401-2408.

Goodall, M. & Kirshner, N. (1957). Biosynthesis of adrenaline and

noradrenaline in vitro. J. Biol. Chem. 226, 213-221.

Goodall, M. & Rosen, L. (1963). Urinary excretion of noradrenaline and

its metabolites at ten minute intervals after intravenous injection

of d-noradrenaline-2-C. J. Clin. Invest. 42, 1578-1588.

Greengard, P. (1976). Possible role for cyclic nucleotides and

phosphorylated membrane proteins in postsynaptic actions of

neurotransmitters. Nature, 260, 101-108.

Gregory, J.0. & Lipmann, F. (1957). The transfer of sulphate among

phenolic compounds with 3',5'-diphosphoadenosine as coenzyme.

J. Biol. Chem. 229, 1081-1090.

Haggendal, G. (1963). The presence of conjugated adrenaline and

noradrenaline in human blood plasma. Acta Physiol. Scand. 59,

255-260.

Hanahan, D.G., Everett, N.B. & Davis, C.D. (1949). Fate of S^-Na

estrone sulphate in pregnant and non-pregnant rats. Arch. Biochem.

23, 501-503. - 195 -

Hart, R.F., Renskers, K.J., Nelson, E.B. & Roth, J.A. (1979).

Localization and characterisation of phenol sulphotransferase in human

platelets. Life Sci. 24, 125-130.

Henry, D.P., Starman, B.J., Johnson, D.C. & Williams, R.H. (1975).

A sensitive radioenzymatic assay for norepinephrine in tissues and

plasma. Life Sci. J 6.» 375-384.

Hidaka, H. & Austin, J. (1972). Occurrence and distribution of

serotonin-O-sulfotransferase in human brain; a new radioisotopic

assay. Biochim. Biophys. Acta. 268, 132-137.

Hidaka, H., Nagatsu, T. & Yagi, K. (1969). Formation of serotonin-0-

sulphate by sulphotransferase of rabbit liver. Biochim. Biophys.

Acta. 177, 354-357.

Hilz, M. & Lipmann, F. (1955). The enzymatic activation of sulfate.

Proc. Natl. Acad. Sci. U.S. 4^, 880-894.

Holtz, P. (1939). Dopadecarboxylase. Naturvissenschaft 27^, 724-725.

Hornykiewicz, 0. (1966). Dopamine (3-hydroxytyramine) and brain

function. Pharmacol. Res. J_8, 925-964.

Hornykiewicz, 0. (1972). Dopamine and extrapyramidal motor function

and dysfunction. Res. Publ. Assoc. Res. Nerv. Ment. Dis. 50^, 390-415.

Huovinen, J.A. & Gustaffsson, B.E. (1967). Inorganic sulphate, sulphite

and sulphide as sulphur donors in the biosynthesis of sulphur amino-

acids in germ free and conventional rats. Biochim. Biophys. Acta.

136, 441-447. - 196 -

Ibsen, H.H., Christensen, N.J., Hollnagel, M., Leth, A.,

Kappelgaard, A.M. & Giese, J. (1979). Plasma noradrenaline

concentration in hypertensive and normotensive 40 year old

individuals: relationship to plasma renin concentration. Clin Sci.

57, 181s-183s.

Imai, K., Sugiura, M. & Tamura, Z. (1970). The presence of conjugated

dopamine in normal human plasma. Chem. Pharm. Bull. J 8, 2134.

Iversen, L.L. (1963). The uptake of noradrenaline by the isolated

perfused rat heart. Br. J. Pharmacol. 523-537.

Iversen, L.L. (1965). The uptake of catecholamines at high perfusion

concentrations in the rat isolated heart: a novel catecholamine

uptake process. Br. J. Pharmacol. Chemother. £5, 18-33.

Iversen, L.L. (1975). Dopamine receptors in the brain. Science 188,

1084-1089.

Jakoby, W.B., Sekura, R.D., Lyon, E.S., Markus, C.J. & Wang, J.L.

(1980). Sulfotransferases. In: Enzymatic Basis of Detoxification,

ed. Jakoby, W.B. New York: Academic Press, Vol. 2, pp. 199-228.

Jenner, W.N. & Rose, F.A. (1973). Studies on the sulphation of

3,4-dihydroxyphenylethylamine (dopamine) and related compounds by

rat tissues. Biochem. J. 135, 109-114.

Jenner, W.N. & Rose, F.A. (1974). Dopamine 3-0-sulphate, an end product

of L-dopa metabolism in Parkinson patients. Nature, 252, 237-238.

35 Jenner, W.N. & Rose, F.A. (1978). Dopamine 3- and 4-0 S sulphates as

substrates for arylsulphatase in vitro and their metabolism by the rat

in vivo. In: Conjugation Reactions in Drug Biotransformation.

ed. Aitio, A. Amsterdam: Elsevier/North Holland, p. 501 -197-

Jirku, H., Hogsander, U. & Levitz, M. (1967). 15 a-Hydroxyestrone

"sulfate": A biliary metabolite of estrone sulfate in the non-pregnant

female. Biochim. Biophys. Acta. 137, 588-591.

Johnson, G.A., Baker, G.A. & Smith, R.T. (1980). Radioenzymatic assay

of sulphate conjugates of catecholamines and dopa in plasma. Life

Sci. 26, 1591-1598.

Jones, D.H., Hamilton, C.A. & Reid, J.L. (1978). Plasma noradrenaline,

age and blood pressure: a population study. Clin. Sci. Mol. Med. 55,

73s-75s.

Joyce, D.A., Beilin, L.J., Vandongen, R. & Davidson, L. (1982). Plasma

free and conjugated catecholamine levels during acute physiological

stress in man. Life Sci. 58, 22-27.

Kahane, L., Esser, A.H., Kline, N.S. & Vestergaard, P. (1967).

Estimation of conjugated epinephrine and norepinephrine in urine.

J. Lab. Clin. Med. 69, 1042-1050.

Karoum, F., Moyer-Schwing, J., Potkin, S.G. & Wyatt R.J. (1977).

Presence of free, sulfate and glucuronide conjugated 3-methoxy-4-

hydroxyphenylglycol (MHPG) in human brain, cerebrospinal fluid and

plasma. Brain Res. 125, 333-339.

Kiowski, W., van Brummelen, P. & Buhler, F.R. (1979). Plasma

noradrenaline correlates with alpha-adrenoreceptor-mediated

vasoconstriction and blood pressure in patients with essential

hypertension. Clin. Sci. 57, 177s-180s.

Kirshner, N. (1974). Function and organisation of chromaffin vesicle.

Life Sci. 14, 1153-1167. -198-

oc Koj, A., Frendo, J. & Janik, Z. (1967). [ S] Thiosulphate oxidation by

rat liver mitochondria in the presence of glutathione. Biochem. J.

103, 791-795.

Kopin, I.J. (1960). Technique for the study of alternate metabolic

pathways: epinephrine metabolism in man. Science, 131, 1372-1374.

Kopin, I.J. (1977). Catecholamine metabolism (and the biochemical

assessment of sympathetic activity). Clinics in Endocrinology and

Metabolism, 6^ (3), 525-549.

Kopin, I.J., Axelrod, J. & Gordon, E.K. (1961). The metabolic fate of o 14 H -epinephrine and C metanephrine in the rat. J. Biol. Chem. 236,

2109-2113.

Kopin, I.J. & Gordon, E. (1963). Metabolism of administered and drug 3 released norepinephrine 7-H in the rat. J. Pharmacol. 140, 207-216.

Krijgsheld, K.R., Scholtens, E. & Mulder, G.J. (1980). Serum

concentration of inorganic sulphate in mammals: species differences

and circadian rhythm. Comp. Biochem. Physiol. 67A, 683-686.

Kuchel, 0., Buu, N.T., Fontaine, A., Hamet, P., Beroniade, V.,

Larochelle, P. & Genest, J. (1980). Free and conjugated plasma

catecholamines in hypertensive patients with and without

phaeochromocytoma. Hypertension 2, 177-186.

Kuchel, 0., Buu, N.T., Hamet, P. & Larochelle, P. (1982). Hypertension

in hyperthyroidism: Is there an epinephrine connection? Life Sci. 30,

603-609. - 199-

Kuchel, 0., Buu, N.T., Hamet, P., Larochelle, P. & Bourque, M. (1981).

Catecholamine sulfates and platelet phenolsulphotransferase (PST)

activity in human hypertension. In: Phenolsulfotransferase in

Mental Health Research. Eds. Sandler, M. & Usdin, E. London:

Macmillan, pp. 175-185.

Kuchel, 0., Buu, N.T., Hamet, P., Larochelle, P., Bourque, M. &

Genest, J. (1981). Unconjugated hyperepinephrinemia: a hallmark of

hypertension imitating phaeochromocytoma. Hypertension _3> 129-133.

Kuchel, 0., Buu, N.T., Hamet, P., Nowaczynski, W. & Genest, J. (1979).

Free and conjugated dopamine in phaeochromocytoma, primary

aldosteronism and essential hypertension. Hypertension 1_, 267-273.

Kuchel, 0., Buu, N.T. & Serri, 0. (1982). Sulphoconjugation of

catecholamines, nutrition and hypertension. Hypertension 4, 93-98.

Kuchel, 0., Buu, N.T., Unger, Th. & Genest, J. (1978). Free and

conjugated catecholamines in human hypertension. Clin. Sci. Mol. Med.

55, 77s-80s.

La Brosse, E.H., Axelrod, J. & Kety, S.S. (1958). 0-Methyl ation, the

principal route of metabolism of epinephrine in man. Science, 128,

593-594.

La Brosse, E.H., Axelrod, J., Kopin, I.J. & Kety, S.S. (1961). 3 Metabolism of 7-H -epinephrine-D-bitartrate in normal young man.

J. Clin. Invest. 40, 253-260.

La Brosse, E.H. & Mann, J.D. (1960). Presence of metanephrine and

normetanephrine in normal human urine. Nature 185, 40. -200-

Laidlow, J.C. & Young, L. (1948). Studies on the synthesis of ethereal

sulphates in vivo. Biochem. J. 42, 1, (Proceedings).

Lake, C.R., Ziegler, M.G., Coleman, M.D. & Kopin, I.J. (1977). Age-

adjusted plasma norepinephrine levels are similar in normotensive

and hypertensive subjects. N. Engl. J. Med. 296, 208-209.

Langer, S.Z., Briley, M.S. & Raisman, R. (1980). Regulation of

neurotransmission through presynaptic receptors and other mechanisms:

possible clinical relevance and therapeutic potential. Adv. Biochem.

Psychopharmacol. 21^, 203-212.

Leung, F. & Griffiths, J. (1974). Use of plasma catecholamine values

in diagnosing phaeochromocytoma and neuroblastoma. Can. Med. Assn. J.

Ill, 1321-1322.

Levin, E.Y. & Kaufman, S. (1961). Studies on the enzyme catalysing the

conversion of 3,4-dihydroxyphenylamine to norepinephrine. J. Biol.

Chem. 236, 2043-2049.

Levin, E.Y., Levenberg, B. & Kaufman, S. (1960). The enzymatic

conversion of 3,4-dihydroxyphenylethyl amine to norepinephrine.

J. Biol. Chem. 235, 2080-2086.

Levitt, M., Spector, S., Sjoerdsma, A. & Udenfriend, S. (1965).

Elucidation of the rate-limiting step in norepinephrie biosynthesis

in the perfused guinea-pig heart. J. Pharmacol. Exp. Ther. 148, 1-8.

Louis, W.J., Doyle, A.E. & Anavekar, S. (1973). Plasma norepinephrine

levels in essential hypertension. N. Engl. J. Med. 288, 599-601. -201 -

Louis, W.J., Doyle, A.E., Anavekar, S.N., Johnston, C.I., Geffen, L.B.

& Rush, R. (1974). Plasma catecholamine, dopamine 3-hydroxylase and

renin levels in essential hypertension. Circ. Res. 34-35 (Suppl. 1),

I-57-I-63.

Lovenberg, W., Weissbach, H. & Udenfriend, S. (1962). Aromatic-L-amino

acid decarboxylase. J. Biol. Chem. 237, 89-93.

Lowry, D.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. (1951).

Protein measurement with folin phenol reagent. J. Biol. Chem. 193,

265-273.

Lutold, B.E., Buhler, F.R. & Da Prada, M. (1976). Dynamik von Plasma

Katecholaminen und 8 -adrenorezeptor-Funktionen. Schweiz, med. Wschr.

106, 1735-1738.

MacNicholl, A.D., Wustmen, F.S., Winterburn, P.J., Powell, G.M. & 3 Curtis, C.G. (1980). Degradation of [ H] chondroitin 4-sulphate and 3 re-utilization of the [ H] hexosamine component by the isolated

perfused rat liver. Biochem. J. 186, 279-286.

Mathias, C., Christensen, N., Corbett, J., Frankel, M. & Spalding, J.

(1976). Plasma catecholamines during paroxysmal neurogenic

hypertension in quadriplegic man. Circ. Res. 39, 204-208.

Mattock, P. & Jones, J.G. (1970). Partial purification and properties

of an enzyme from rat liver that catalyses the sulphation of L-Tyrosyl

derivatives. Biochem. J. 116, 797-803.

McDonald, R.H., Goldberg, L.I., McNay, J.L. & Tuttle, E.P. (1963).

Augmentation of sodium excretion and blood flow by dopamine in man.

Clin. Res. 11, 248. -202-

McDonald, R.H., Goldberg, L.I., McNay, J.L. & Tuttle, E.P. (1964).

Effect of dopamine in man: Augmentation of sodium excretion,

glomerular filtration rate and renal plasma flow. J. Clin. Invest.

43, 1116-1124.

McEvoy, F.A. & Carrol, J. (1971). Purification from rat liver of an

enzyme that catalyses the sulphurylation of phenols. Biochem. J. 123,

901-906.

Meek, J.L. & Neff, N.H. (1972). Acidic and neutral metabolites of

norepinephrine: Their metabolism and transport from brain. J. Pharm.

exp. Ther. 181, 457-462.

Meek, J.L. & Neff, N.H. (1973). Biogenic amines and their metabolites

as substrates for phenol sulphotransferase (E.C.2.8.21) of brain and

liver. J. Neurochem. 2^, 1-9.

14 Merits, J. (1976). Formation and metabolism of [C ] Dopamine 3-0_-

sulphate in dog, rat and guinea pig. Biochem. Pharmacol. 25, 829-833.

Michelot, R.J., Lesko, N., Stout, R.W., Coward, J.K. (1977). Effect of

S-adenosylhomocysteine and_S-tubercidinylhomocysteine on catecholamine

methylation in neuroblastoma cells. Mol. Pharmacol. L3, 368-373.

Michels, F.G. & Smith, J.T. (1965). A comparison of the utilization of

organic and inorganic sulfur by the rat. J. Nutr. 87, 217-220.

Mitchell, P.D. (1973). Metabolism of tyramine. 1. A comparative study

in man, rabbit and rat. 2. An investigation in migraine and

Parkinson's disease. Ph.D. Thesis, University of London. -203-

Morgan, C.D., Sandler, M., Davies, D.S., Conolly, M., Paterson, J.W. &

Dollery, C.T. (1969). The metabolic fate of DL-[7-^H] isoprenaline in

man and dog. Biochem. J. 114, 8P.

Mulder, G.J. (1982). Sulfation of steroid hormones. Introduction. In:

Sulfate Metabolism and Sulfate Conjugation. Eds. Mulder, G.J.,

Caldwell, J., van Kempen, G.M.J. & Vonk, R.J. London: Taylor &

Francis, pp. 171-172.

Mulder, G.J., Halsema, J.C., Koster, H., Meerman, J.H.N. & Pang, K.S.

(1981). Selective inhibition of sulphation in vivo. In:

Phenolsulphotransferase in Mental Health Research. Eds. Sandler, M.

& Usdin, E. London: Macmillan, pp. 86-97.

Nagatsu, T., Hidakua, H., Kuzuya, H., Takeya, K., Umezawa, H.,

Takeuchi, T. & Hirotuki, S. (1970). Inhibition of DBH by fusaric

acid (5-butylpicolinic acid) in vitro and in vivo. Biochem.

Pharmacol. J_9, 33-44.

Nagatsu, T., Levitt, M. & Udenfriend, S. (1964). Tyrosine hydroxylase -

the initial step in norepinephrine biosynthesis. J. Biol. Chem. 239,

2910-2917.

Nagatsu, T. & Udenfriend, S. (1972). Photometric assay of dopamine -3 -

hydroxylase activity in human blood. Clin. Chem. J_8 (9), 980-983.

Norman, R.O.C. & Taylor, R. (1965). Electrophilic substitution in

benzenoid compounds. Amsterdam: Elsevier, pp. 100-110.

Oliver, G. & Schaefer, E.A. (1895). The physiological effects of

extracts of the suprarenal capsules. J. Physiol. Lond. J 8, 230-276. -204-

Osikowska, B. & Sever, P.S. (1976). Alterations in plasma noradrenaline

concentration in response to postural changes in normal subjects, in

patients with autonomic failure and in essential hypertension. B.J.

Clin. Pharmacol. _3> 963-964.

Page, L.G. & Copeland, R.B. (1968). Phaeochromocytoma. Disease a

Month, 1_, 1-40.

Peart, W.S. (1949). The nature of splenic sympathin. J. Physiol. 108,

491-501.

Pedersen, E.B. & Christensen, N.J. (1975). Catecholamines in plasma

and urine in patients with essential hypertension determined by double

isotope derivative technique. Acta med. scand. 198, 373-377.

Pennings, E.J.M. & Van Kempen, G.M.J. (1980). Studies on the meta and

para O-sulphation of the catechol compound 3,4-dihydroxybenzoic acid

by rat liver sulphotransferase in vitro. Biochem. J. 191, 133-138.

Pennings, E.J.M., Vrielink, R. & Van Kempen, G.M.J. (1978). Kinetics

and mechanism of the rat brain phenol sulphotransferase reaction.

Biochem. J. 173, 299-307.

Rambaut, P.C. & Miller, S.A. (1965). Studies of sulfur amino acid

nutrition in the adult cat. Fed. Proc. 24, 373.

Rein, G., Glover, V. & Sandler, M. (1981). Phenolsulfotransferase

in human tissue: evidence for multiple forms. In:

Phenolsulphotransferase in Mental Health Research, eds. Sandler, M.

and Usdin, E. London: Macmillan, pp. 98-126.

Renskers, K.J., Feor, K.D. & Roth, J.A. (1980). Sulfation of dopamine

and other biogenic amines by human brain sulfotransferase.

J. Neurochem. 34, 1362-1368. -205-

Richter, D. (1940). The inactivation of adrenaline in vivo in man.

J. Physiol. 98, 361-374.

Richter, D. & Macintosh, F.C. (1941). Adrenaline Ester. Amer. J.

Physiol. 135, 1-5.

Robertson, 0., Johnson, G.A., Robertson, R.M., Nies, A.S., Shand, D.G.

& Oates, J.A. (1979). Comparative assessment of stimuli that release

neuronal and adrenomedullary catecholamines in man. Circ. 59,

637-643.

Robbins, P.W. (1958). Enzymatic synthesis of adenosine-5*-

phosphosulphate. J. Biol. Chem. 233, 686-690.

Robbins, P.W. & Lipmann, F. (1957). Isolation and identification of

active sulphate. J. Biol. Chem. 229, 837-851.

Robbins, P.W. & Lipmann, F. (1958). Separation of the two enzymatic

phases in active sulfate synthesis. J. Biol. Chem. 233, 681-685.

Rose, T.H. & Bleszynski, W.S. The synthesis and characterisation of

adrenaline and noradrenaline_0-sulphates. Personal Communication.

Rosenthal, T., Birch, M., Osikowska, B. & Sever, P.S. (1978). Changes

in plasma noradrenaline concentration following sympathetic

stimulation by gradual tilting. Cardiovasc. Res. 12, 144-147.

Roth, J.A. & Rivett, A.J. (1982). Does sulfate conjugation contribute

to the metabolic inactivation of catecholamines in humans? Biochem.

Pharmacol. 31, 3017-3021. -206-

Roth, J.A., Rivett, A.J. & Renskers, K. (1981). Characterisation of

human brain phenolsulphotransferase. In: Phenolsulphotransferase in

Mental Health Research, eds. Sandler, M. & Usdin, E. London:

Macmillan, pp. 74-85.

Roy, A.B. (1981). Sulfotransferases. In: Sulfation of Drugs and

Related Compounds, ed. Mulder, G.J. Boca Raton, FL: CRC Press,

p p . 83-130.

Roy, A.B. & Trudinger, P.A. (1970). The Biochemistry of Inorganic

Compounds of Sulphur. Cambridge: Cambridge University Press.

Rutledge, C.O. & Hoehn, M.M. (1973). Sulphate conjugation and L-Dopa

treatment of Parkinsonian patients. Nature 244, 447-450.

Sandler, M. (1981). Phenolsulphotransferases: General Introduction.

In: Phenolsulphotransferase in Mental Health Research, eds.

Sandler, M. & Usdin, E. London: Macmillan, pp. 1-7.

Sandler, M., Glover, V., Bonham Carter, S.M., Lockwood, J. & Rein, G.

(1981). Clinical studies on platelet phenolsulfotransferases. In:

Phenolsulfotransferases in Mental Health Research, eds.

Sandler, M. & Usdin, E. London: Macmillan, pp. 186-207.

Schanberg, S.M., Breese, G.R., Schildkraut, J.J., Gordon, E.K. &

Kopin, I.J. (1968a). 3-Methoxy-4-hydroxyphenylglycol sulfate in brain

and cerebrospinal fluid. Biochem. Pharmacol. T7, 2006-2008.

Schanberg, S.M., Schildkraut, J.J., Breese, G.R., & Kopin, I.J. (1968b). 3 Metabolism of normetanephrine-H in rat brain - identification of

conjugated 3-methoxy-4-hydroxyphenylglycol as the major metabolite.

Biochem. Pharm. 17, 247-254. -207-

Segal, H.L. (1955). Kinetic studies on the phenol-sulfate conjugating

system of rat liver. J. Biol. Chem. 213, 161-170.

Sekura, R.D. & Jakoby, W.B. (1979). Phenol sulphotransferases.

J. Biol. Chem. 254, 5658-5663.

Sekura, R.D. & Jakoby, W.B. (1981). Aryl sulfotransferase IV from

rat liver. Arch. Biochem. Biophys. 211, 352-359.

Sever, P.S. (1978). Catecholamines in essential hypertension: the

present controversy. In: Circulating Catecholamines and Blood

Pressure, eds. Birkenhager, W.H. & Falke, H.E. Utrecht: Bunge

Scientific Publication, pp. 1-10.

Sever, P.S. (1982). The sympathetic nervous system and essential

hypertension. In: Scientific Foundations of Cardiology, Eds.

Sleight, P. & Vann Jones, J. William Heinemann, pp. 147-155.

Sever, P.S., Osikowska, B., Birch, M. & Tunbridge, R.D.G. (1977).

Plasma noradrenaline in essential hypertension. Lancet, _1, 1078-1081.

Sharpless, N.S., Tyce, G.M., Thai, L.J., Waltz, J.M., Tabaddor, K. &

Wolfson, L.I. (1981). Free and conjugated dopamine in human

ventricular fluid. Brain Res. 217, 107-118.

Spector, S., Sjoerdsma, A., Zaltzman-Nirenberg, P., Levitt, M. &

14 Udenfriend, S. (1963). Norepinephrine synthesis from tyrosine-C

in isolated perfused guinea pig heart. Science 139, 1299-1301.

Sudgen, R.F. & Eccleston, D.J. (1971). Glycol sulphate ester formation

14 from [ C] noradrenaline in brain and the influence of COMT inhibitor.

J. Neuro c h e m . 18, 2461-2468. -208-

Thorner, M.O. (1975). Dopamine is an important neurotransmitter in the

autonomic nervous system. Lancet, 1_, 662-665.

Twombly, G.H. & Levitz, M. (1960). Metabolism of estrone C^-16 sulfate

in woman. Am. J. Obstet. Gynecol. 80, 889-897.

Tyce, G.M., Sharpless, N.S., Kerr, F.W.L. & Muenter, M.D. (1980).

Dopamine conjugate in cerebrospinal fluid. J. Neurochem. 34, 210-212.

Tyce, G.M., Sharpless, N.S. & Muenter, N. (1974). Free and conjugated

dopamine during Levodopa therapy. Clin. Pharmacol. Ther. JJ5, 782-788.

Linger, Th., Buu, N.T. & Kuchel, 0. (1979). Renal handling of free

and conjugated catecholamines following surgical stress in the dog.

Am. J. Physiol. 235, 542-547.

Vecht, R.J., Gordon, D. & Sever, P. (1980). Catecholamines, isometric

exercise and left ventricular function. In: Hypertension:

Mechanisms and Management. Eds. Distler, A. & Philipp, Th.

Heidelberg: Springer, pp. 55-63.

Vihko, R. & Roukonen, A. (1982). Testicular steroid sulphates. In:

Sulfate Metabolism and Sulfate Conjugation. Eds. Mulder, G.J.,

Caldwell, J., van Kempen, G.M.J. & Vonk, R.J. London: Taylor &

Francis, pp. 199-206.

von Euler, U.S. (1948). Identification of the sympathomimetic ergone

in adrenergic nerves of cattle (Sympath in-_N_) with laevo

noradrenaline. Acta physiol, scand. 16^, 63-74.

Wang, M.T., Imai, K. & Tamura, Z. (1972). Synthesis of norepinephrine-

O-sulfates. Chem. Pharm. Bull. 20, 187-189. -209-

Watson, R.D.S., Hamilton, C.A., Reid, J.L. & Littler, W.A. (1979).

Changes in plasma norepinephrine, blood pressure and heart rate during

physical activity in hypertensive man. Hypertension 1^, 341-346.

Weil-Malherbe, H. & van Buren, J.M. (1969). The excretion of dopamine

and dopamine metabolites in Parkinson's disease and the effect of diet

thereon. J. Lab. Clin. Med. 74_, 305-318.

Weinshilboum, R.M. & Anderson, R.J. (1981). Phenol sulphotransferase

in human platelet and other tissues: endogenous inhibitors, assay

conditions, tissue and substrate correlations. In:

Phenolsulfotransferase in Mental Health Research. Eds. Sandler, M. &

Usdin, E. London: Macmillan, pp. 8-28.

Weise, V.K. & Kopin, I.J. (1976). Assay of catecholamines in human

plasma: studies of a single isotope radioenzymatic procedure.

Life Sci. 19, 1673-1786.

Wellers, G., Boelle, G. & Chevan, 0. (1960). Research on the metabolism

of sulfur. IV. Partial covering of endogenous sulfur utilization by

sodium sulfate in adult white rat. J. Physiol. 52^ 903-910.

Williams, R.T. (1959). The metabolism of phenols. In: Detoxication

Mechanisms, 2nd edition. London: Chapman & Hall. pp. 278-317

Williams, R.T. (1979). The metabolism of phenols - the sulphate

conjugate. In: Detoxication Mechanisms. New York: Wiley,

pp. 279-318.

Wilson, L.G. & Bandurski, R.S. (1958). Enzymatic reactions involving

sulfate, sulfite, selenate and molybolate. J. Biol. Chem. 233,

975-981. -210-

Wing, Reid, J.L., Hamilton, C.A., Sever, P., Davies, D.S. &

Dollery, C.T. (1977). Effects of clonidine on biochemical indices

of sympathetic function and plasma renin activity in normotensive man.

Clin. Sci. Mol. Med. 53, 45-53.

Winkler, H. (1976). The composition of adrenal chromaffin granules:

an assessment of controversial results. Neuroscience 1^, 65-80.

Wolfe, D.E., Potter, L.T., Richardson, K.C. & Axelrod, J. (1962).

Localizing tritiated norepinephrine in sympathetic axones by electron

microscopy and autoradiography. Science, 138, 440.

Wong, K.P. (1978). Measurement and kinetic study of the formation of

adrenaline sulphate in vitro. Biochem. J. 174, 777-782.

Wong, K.P. (1982). Sulphate conjugate of amines and their metabolites.

In: Sulfate Metabolism and Sulfate Conjugation. Eds. Mulder, G.O.,

Caldwell, 0., van Kempen, G.H.J. & Vonk, R.J. London: Taylor &

Francis, p.85-92.

Wood, K.M., Wusteman, F.S. & Curtis, C.G. (1973). The degradation of

intravenously injected chondroitin 4-sulphate in the rat. Biochem. J.

134, 1009-1013.

Wu, S.G. & Straub, K.D. (1976). Purification and characterisation of

_N-hydroxy-2-acetylaminofluorene sulfotransferase from rat liver.

J. Biol. Chem. 251, 6529-6536.

Youdim, M.B.H., Carter, S.B., Sandler, M., Hanington, E. & Wilkinson, M.

(1971). Conjugation defect in tyramine-sensitive migraine. Nature,

230, 127-128. -211-

Ziegler, M.1 1., Lake, C.R. & Kopin, I.J. (1976). Plasma noradrenaline

increases with age. Nature 261, 333-335.

Ziegler, M. 1., Lake, C.R. & Kopin, I.J. (1977). The sympathetic nervous

system defect in primary orthostatic hypotension. N. Engl. J. Med.

296, 293-297. -212-

RELEVANT PUBLICATIONS

The following publications appeared whilst the work described in this

Thesis was being undertaken:

J.R. Idle, B.A. Osikowska, P.S. Sever and F.J. Swinbourne.

Pitfalls in the synthesis and authentication of dopamine O-sulphates.

British Journal of Pharmacology, 74(4), 837P, 1981.

B.A. Osikowska, J.R. Idle, F.J. Swinbourne and P.S. Sever.

Unequivocal synthesis and characterisation of dopamine 3- and 4-0-

sulpha t e s .

Biochemical Pharmaology, 31 (13), 2279-2284, 1982.

J.R. Idle, R. Mattin, B.A. Osikowska, P.S. Sever and F.J. Swinbourne.

Application of HPLC and NMR to the one-step synthesis of authentic

noradrenaline 3- and 4-0-sulphates.

British Journal of Pharmacology, 77, 418P, 1982.