Analysis of catecholamines and related substances using porous graphitic carbon as separation media in liquid chromatography-tandem mass spectrometry

Capillary porous graphitic carbon (PGC) columns have been utilized for separation of several catecholamines and related compounds (i.e. L-tyrosine, L-DOPA, 3-O-methyl-DOPA, dopamine, 3,4-dihydroxy-phenyl-acetic acid (DOPAC), homovanillic acid, noradrenaline, vanillomandelic acid and adrenaline) on-line with electrospray ionization tandem mass spectrometry (ESI-MS/MS). The use of a mobile phase without ion-pairing agents and with high content of organic modifier facilitated the coupling to the selective and sensitive mass spectrometric detection. Minimum detectable sample concentration (MDC sample) for noradrenaline, dopamine and L-tyrosine in a standard solution was estimated to 3, 10 and 30 nM, respectively (3 S/N corresponds to MDQ for L-tyrosine of approximately 8 x 10(-14)mol). The developed strategy was applied for analysis of brain tissue, i.e. a substantia nigra (ns) sample.     

F prostaglandins function as potent olfactory stimulants that comprise the postovulatory female sex pheromone in goldfish

This study establishes that ovulated female goldfish release F type prostaglandins (PGFs) to the water where they stimulate male spawning behavior and comprise the goldfish postovulatory pheromone. We first demonstrated that ovulated and prostaglandin-injected female goldfish release immunoreactive PGFs to the water. Next, using electro-olfactogram recording (EOG), we determined that waterborne prostaglandins function as potent olfactory stimulants for mature male goldfish. Prostaglandin F2 alpha (PGF2 alpha) and its metabolite 15-keto-prostaglandin F2 alpha (15K-PGF2 alpha) were the most potent prostaglandins; the former had a detection threshold of 10(-10) M and the latter a detection threshold of 10(-12) M. Studies of prostaglandin-injected fish indicated that PGF metabolites are an important component of the pheromone. Cross-adaptation experiments using the EOG demonstrated that goldfish have separate olfactory receptor sites for PGF2 alpha and 15K-PGF2 alpha that are independent from those that detect other olfactory stimulants. Finally, we established that male goldfish exposed to low concentrations of waterborne PGFs exhibit reproductive behaviors similar to those elicited by exposure to the odor of ovulated fish. Together with our recent discovery that a steroidal maturational hormone functions as a preovulatory "priming" pheromone for goldfish, these findings suggest that hormones and their metabolites may commonly serve as reproductive pheromones in fish.     

Determination of monoamines and 10 of their metabolites in the brain and heart of the rat by high-pressure liquid chromatography with electrochemical detection

The adequate parameters for simultaneous determination of more than 10 monoamines, their precursors and metabolites (noradrenaline, 3-methoxy-4-hydroxyphenylglycol, 3,4-dihydrooxyphenylglycol++, vanylylmandelic acid, normetanephrine, adrenaline, metanephrine, dopamine, 3-methoxytytramine, 3,4-dihydroxphenylacetic acid, 3,4-dihydroxyphenylalanine, 5-hydroxytryptophane, 5-hydroxyindolacetic acid) by liquid chromatography with electro-chemical detection were suggested for the rat brain and heart. The influence of reserpine, iproniazid, and imipramine on the content of the changes of monoamines and their metabolite levels in the rat brain and heart were also investigated.     

INHIBITION OF RAT BRAIN PHENOL SULPHOTRANSFERASE IN VITRO BY NORADRENALINE and DOPAMINE METABOLITES1

Abstract— Kinetic parameters of the sulphotransferase reaction in rat brain were investigated in vitro at pH 7.4. Evidence is presented that the enzyme phenol sulphotransferase (EC 2.8.2.1) can be assayed with 4-methylumbelliferone or 3-methoxy-4-hydroxyphenylethyleneglycol as the substrate. Both assays give identical V_max values, whereas K_m values are 0.026 mm and 0.039 mm , respectively. Normetanephrine, metanephrine and the catecholamines adrenaline and dopamine, having a positive charge on the side chain at pH 7.4, do not inhibit 4-methylumbelliferone and 3-methoxy-4-hydroxyphenylethy-leneglycol sulphotransferase at this pH. Their deaminated metabolites 3,4-dihydroxyphenylethyleneglycol, 3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, 3-methoxy-4-hydroxyphenylethylene glycol, 3-methoxy-4-hydroxyphenethanol and 3-methoxy-4-hydroxyphenylacetic acid inhibit both the enzyme activities. The type of inhibition is noncompetitive with the exception of 3-methoxy-4-hydroxy-phenylethyleneglycol, which is a competitive inhibitor of 4-methylumbelliferone sulphation. 3-Methoxy-4-hydroxy-mandelic acid does not inhibit the enzyme activities. It is concluded that the catecholamines themselves are not sulphated by rat brain in vitro at pH 7.4.     

Effects ofp-chlorophenylalanine on cortical monoamines and on the activity of noradrenergic neurons

The catecholamines noradrenaline, dopamine, adrenaline, the indoleamine 5-hydroxy-tryptamine (5-HT; serotonin), and some of their major metabolites were assayed, using high performance liquid chromatography (HPLC), in the neocortex of normal rats as well as in animals in which 5-HT synthesis had been inhibited withp-chlorophenylalanine. Besides important depletions in serotonin and in 5-hydroxyindole-3-acetic acid, noradrenaline levels were significantly reduced, but the content in 3-methoxy-4-hydroxyphenylglycol was increased, indicating an augmented utilization of this amine. The levels of dopamine and 3-methoxytyramine were also reduced, although homovanillic acid and 3,4-dihydroxyphenylacetic acid levels remained constant. The spontaneous unitary activity of identified noradrenergic neurons in the Locus coeruleus was increased, indicating an hyperactivity of this system. These results can be interpreted in relation to functional interactions between the catecholamines and serotonin; i.e.: a decrease in endogenous serotonin results in the loss of a negative feedback control of noradrenaline release.Special Issue dedicated to Prof. Eduardo De Robertis.     

Effects of Handling or Immobilization on Plasma Levels of 3,4-Dihydroxyphenylalanine, Catecholamines, and Metabolites in Rats

In conscious animals, handling and immobilization increase plasma levels of the catecholamines norepinephrine (NE) and epinephrine (EPI). This study examined plasma concentrations of endogenous compounds related to catecholamine synthesis and metabolism during and after exposure to these stressors in conscious rats. Plasma levels of 3,4-dihydroxyphenylalanine (DOPA), NE, EPI, and dopamine (DA), the deaminated catechol metabolites 3,4-dihydroxyphenylglycol (DHPG), and 3,4-dihydroxyphenylacetic acid (DOPAC), and their O-methylated derivatives methoxyhydroxyphenylglycol (MHPG) and homovanillic acid (HVA) were measured using liquid chromatography with electrochemical detection at 1, 3, 5, 20, 60, and 120 min of immobilization. By 1 min of immobilization, plasma NE and EPI levels had already reached peak values, and plasma levels of DOPA, DHPG, DOPAC, and MHPG were increased significantly from baseline, whereas plasma DA and HVA levels were unchanged. During the remainder of the immobilization period, the increased levels of DOPA, NE, and EPI were maintained, whereas levels of the metabolites progressively increased. In animals immobilized briefly (5 min), elevated concentrations of the metabolites persisted after release from the restraint, whereas DOPA and catecholamine levels returned to baseline. Gentle handling for 1 min also significantly increased plasma levels of DOPA, NE, EPI, and the NE metabolites DHPG and MHPG, without increasing levels of DA or HVA. The results show that in conscious rats, immobilization or even gentle handling rapidly increases plasma levels of catecholamines, the catecholamine precursor DOPA, and metabolites of NE and DA, indicating rapid increases in the synthesis, release, reuptake, and metabolism of catecholamines.     

Simultaneous analysis of human plasma catecholamines by high-performance liquid chromatography with a reversed-phase triacontylsilyl silica column

The clinical importance of simultaneous analysis of 3,4-dihydroxyphenylglycol with other human plasma catecholamines has been investigated to better understand the sympathetic nervous system. However, previous reports have had analytical difficulties with both resolution and extraction. The current study uses a reversed-phase triacontylsilyl silica (C30) column under the mobile phase condition without ion-pair reagents to separate catecholamines and their metabolites, with above 91% recoveries for intra-assay, above 85% for inter-assay, and less than 10% (n=5) coefficient of variation. Lower detection limits (S/N=4) and quantification limits (S/N=6) were 40 and 100 pg/mL for norepinephrine, 3,4-dihydroxyphenylglycol, and 3,4-dihydroxyphenylalanine, 10 and 20 pg/mL for epinephrine, 10 and 40 pg/mL for dopamine. Linear ranges were from 40 to 5000 pg/mL for norepinephrine and 3,4-dihydroxyphenylalanine, from 100 to 5000 pg/mL for 3,4-dihydroxyphenylglycol, and from 10 to 2000 pg/mL for epinephrine and dopamine. The C30 column may prove clinically useful, as it provides a convenient and simultaneous method of evaluation of human plasma catecholamines.     

High-performance liquid chromatographic analysis of monoamines and some of theirγ-glutamyl conjugates produced by the brain and other tissues ofHelix aspersa (gastropoda)

1. Earlier reports from this and other laboratories have indicated that wide variations exist in estimates of the concentrations of norepinephrine in the brain and heart of the snail Helix aspersa. This is a report on investigations of norepinephrine concentrations in Helix aspersa tissues using high-performance liquid chromatography with electrochemical detection. In addition, the effects of treatment with some amino acid precursors or enzyme inhibitors on the concentrations of norepinephrine, dopamine, 5-hydroxytryptamine, and some of their metabolites were investigated. 2. The levels of norepinephrine in the brain were low (46 ng/g) in comparison to dopamine (2.1) micrograms/g) and 5-hydroxytryptamine (2.6 micrograms/g). Epinephrine was not observed in either snail heart of snail nervous tissue. 3. Administration of L-3,4-dihydroxyphenylalanine resulted in elevated snail brain dopamine, while 3,4-dihydroxyphenylserine treatment increased norepinephrine. Treatment with blockers of tyrosine hydroxylase and aromatic-L-amino acid decarboxylase reduced dopamine concentrations without affecting 5-hydroxytryptamine. 4. The dopamine metabolite 3,4-dihydroxyphenylacetic acid was observed only after administration of L-3,4-dihydroxyphenylalanine or dopamine and then only in very small amounts. At no time was the dopamine metabolite homovanillic acid or the 5-hydroxytryptamine metabolite 5-hydroxyindoleacetic acid observed in brain, heart, or whole-body extracts of the snail. 5. Incubation of nervous tissue with either dopamine or 5-hydroxytryptamine resulted in the production of electrochemically active metabolites which were identified by oxidation characteristics and cochromatography with synthesized standards as the gamma-glutamyl conjugates of the amines. Treatment of snails with 5-hydroxytryptamine or dopamine also resulted in the production of gamma-glutamyl conjugates. 6. The present experiments show that great care must be exercised when measuring monoamines and their metabolites in gastropod tissues by high-performance liquid chromatography with electrochemical detection.     

Support for a physiological role of endogenous catecholamines in the stimulation of bovine luteal progesterone production

To determine if catecholamines were present in bovine luteal tissue, corpora lutea (CL) were obtained during the mid-luteal phase (Days 10-12) and the concentration of dopamine (DA) and norepinephrine (NE) was determined by high-performance liquid chromatography. Both DA and NE were detected in luteal tissue at mean concentrations of 41.9 +/- 5.73 and 10.2 +/- 2.51 ng/g for DA and NE, respectively. These concentrations represented a luteal content of 306.6 +/- 66.88 ng/CL for DA and 70.5 +/- 16.88 ng/CL for NE. In vitro, DA at concentrations of 1.0 mM to 0.01 mM stimulated the production of progesterone (P4, p less than 0.05). The response to DA was inhibited by propranolol (a beta-adrenergic receptor antagonist, p less than 0.05) but not by phentolamine, phenoxybenzamine (alpha-adrenergic receptor antagonists), or haloperidol (a DA receptor antagonist, p greater than 0.05). Neither L-tyrosine nor L-dopa altered P4 production (p greater than 0.05). Inhibition of DA beta-hydroxylase, the enzyme that catalyzes the conversion of DA to NE by FLA-63 blocked the DA-induced increases in luteal P4 production (p less than 0.05). These results demonstrate the existence of DA and NE in bovine luteal tissue and indicate that exogenous DA can be converted to NE in luteal tissue. The results support a physiological role for catecholamines in the stimulation of bovine luteal function.     

THE METABOLISM OF TRITIATED DOPAMINE IN REGIONS OF THE RAT BRAIN IN VIVO—II

Abstract— The levels of tritiated catecholamines and metabolites were measured in regions of the rat brain at intervals after the intraventricular injection of [³H]dopamine, [³H]nor-adrenaline and [³H]normetanephrine. The disappearance of catecholamines and appearance of metabolites with time and the regional turnover rates of these amines indicate that the major pathway of the metabolism of noradrenaline and dopamine actively released from physiological storage sites is to the neutral alcoholic metabolites. The acid metabolites, homovanillic acid and 3,4-dihydroxyphenylacetic acid appear to be only minor products of normal dopamine metabolism in rat brain regions including the striate, but are the main end products of the metabolism of excess exogenous dopamine.
The active metabolism of stored noradrenaline to alcohol metabolites is also indicated by the increase in neutral alcohol metabolites accompanying the increased noradrenaline turnover when rats were subjected to electroshock stress. Therefore in the rat brain, neutral alcohol metabolites of dopamine and noradrenaline have great significance in the study of physiological catecholamine turnover in any region.     

Ciliated olfactory receptor neurons in goldfish (Carassius auratus) partially survive nerve axotomy,rapidly regenerate and respond to amino acids

Electro-olfactogram (EOG) recordings in response to amino acid stimulation were made from both control and experimental olfactory mucosae following unilateral axotomy. The recorded EOG amplitudes, amino acid stimulus relative effectiveness and dose-response relations for control and experimental mucosae were comparable in all pre- and postoperative recordings. Semi-thin investigations of olfactory mucosae showed degeneration of olfactory receptors but indicated that intact receptors were also present. SEM of olfactory mucosae revealed that ciliated receptor cells were present in both axotomized and control sides on postoperative days, whereas microvillous receptors completely degenerated and did not regenerate until 7 weeks post axotomy. The present findings along with previous behavioral observations suggest at least three possible sources of the EOGs recorded from the experimental olfactory mucosae following olfactory nerve transection: (1) young olfactory receptor neurons whose axons had not yet reached the region of the transected olfactory nerve; (2) newly-emerged olfactory receptor neurons; and (3) olfactory receptor neurons that had not degenerated.Abbreviations EOG electro-olfactogram - SEM scanning electron microscopy (micrograph)     

Halothane attenuated haloperidol and enhanced clozapine-induced dopamine release in the rat striatum

The effect of halothane anesthesia on changes in the extracellular concentrations of dopamine (DA) and its metabolites (3-methoxytyramine (3-MT), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA)) induced by neuroleptics was studied using in vivo microdialysis techniques. Halothane attenuated haloperidol-induced dopamine release and enhanced clozapine-induced dopamine release in the rat striatum.A microdialysis probe was implanted into the right striatum of male SD rats. Rats were given saline or the same volume of 200 microg kg(-1) haloperidol (D(2) receptor antagonist), 10 mg kg(-1) sulpiride (D(2) and D(3) antagonist), or 10 mg kg(-1) clozapine (D(4) and 5-HT(2) antagonist) intraperitoneally with or without 1-h halothane anesthesia (0.5 or 1.5%). Halothane anesthesia did not change the extracellular concentration of DA, but increased the metabolite concentrations in a dose-dependent manner. The increased DA concentration induced by haloperidol was significantly attenuated by halothane anesthesia, whereas the metabolite concentrations were unaffected. Halothane had no effect on the changes in the concentrations of DA or its metabolites induced by sulpiride. The clozapine-induced increases in DA and its metabolites were enhanced by halothane anesthesia.Our results suggest that halothane anesthesia modifies the DA release modulated by antipsychotic drugs in different ways, depending on the effects of dopaminergic or serotonergic pathways.     

Catecholamine biosynthesis in vitro and in vivo in the chromaffin tissue of the Atlantic cod, Gadus morhua

1. The biosynthesis of [3H]catecholamines from [3H]L-tyrosine in the intact chromaffin tissue of cod posterior cardinal veins was studied in vitro and in vivo at 10 degrees C. 2. The tritiated products dopamine, noradrenaline and adrenaline were separated from the [3H]tyrosine by paper chromatography of tissue extracts and the radioactivity of 1 cm strips of the chromatogram was determined by liquid scintillation spectrophotometry. DOPA could never be demonstrated in the tissue extracts from any of the experiments performed. 3. The content of [3H]noradrenaline in pieces of the cardinal veins incubated in vitro was found to increase rapidly. The tissue content of dopamine and adrenaline remained at lower levels which were reached during the first few hours of the incubation. A similar pattern could be demonstrated in the chromaffin tissue in vivo after infusion of [3H]tyrosine, but the total content of the [3H]catecholamines was lower than in the in vitro experiments. 4. The results are consistent with the view that the methylation of noradrenaline is the rate-limiting step in the biosynthesis of adrenaline in cod chromaffin tissue.     

Quantitation of 3,4-dihydroxyphenylacetaldehyde and 3, 4-dihydroxyphenylglycolaldehyde, the monoamine oxidase metabolites of dopamine and noradrenaline, in human tissues by microcolumn high-performance liquid chromatography.

We recently described the chemical synthesis of 3, 4-dihydroxyphenylacetaldehyde and 3,4-dihydroxyphenylglycolaldehyde, the monamine oxidase metabolites of dopamine and noradrenaline, respectively. We demonstrated the neurotoxicity of these compounds. Catecholamine nerve cells which synthesize these aldehydes die in degenerative brain diseases, such as Parkinson's and Alzheimer's. Here we describe a sensitive method for separating these catecholaldehydes from catecholamines and their other oxidative and methylated metabolites by microcolumn high-performance liquid chromatography with electrochemical detection. We then quantitate catecholamines and their major metabolites in human brain, plasma, and urine. The method can be used to determine the role of these catecholaldehydes in human disease.     

Analysis of Acidic Metabolites of Biogenic Amines in Bovine Retina and Vitreous and Aqueous Humour by Gas Chromatography-Negative Ion Chemical Ionisation Mass Spectrometry

Acidic metabolites of a number of biogenic amines have been identified and quantified by reaction with either acetic or propionic anhydride in the aqueous phase followed by extraction into ethyl acetate, esterification of carboxyl groups with ditrifluoromethylbenzyl bromide (DTFMBzBr), and then conversion of the remaining free hydroxyl groups to acetates. Subsequent analysis of these derivatives revealed that most (greater than 60%) of the ion current was carried by the ion resulting from the loss of DTFMBz from the molecular ion. This made the method highly specific and practical--limits of detection were established at approximately 200 pg with a potential limit of detection below the picogram level. This method establishes unequivocally that the metabolites of tyramine, dopamine, and adrenaline/noradrenaline (4-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, and dihydroxymandelic acid, respectively) are present in bovine retina and in vitreous and aqueous humour. In addition, high concentrations of the dopamine metabolite homovanillic acid were found in retina and vitreous, but not in aqueous humour. p-Hydroxymandelic acid, the acidic metabolite of p-octopamine/p-synephrine, was identified in vitreous and in aqueous humour.     

Monoamines and Their Precursors and Metabolites in the Chicken Brain, Pineal, and Retina: Regional Distribution and Day/Night Variations

Levels of norepinephrine, epinephrine, dopamine, and serotonin (5-HT) and their precursors [tyrosine, L-3,4-dihydroxyphenylalanine, tryptophan, and 5-hydroxytryptophan (5-HTP)] and metabolites [3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT), homovanillic acid, 3-methoxy-4-hydroxyphenylglycol, and 5-hydroxyindoleacetic acid (5-HIAA)] were determined concurrently in samples of chick retina, pineal gland, and nine selected areas of the brain (optic lobes, thalamus, hypothalamus, optic chiasm, pons/medulla, cerebellum, neostriatum/ectostriatum, hyperstriatum, and basal forebrain) using HPLC coupled with a coulometric electrode array detection system. The norepinephrine level was highest in the pineal gland, but it was also widely distributed throughout the chick brain, with the thalamus and hypothalamus showing substantial levels. The dopamine level was highest in the basal forebrain. The epinephrine level was highest in the hypothalamus. The thalamus and hypothalamus showed the highest levels of 5-HT. Daytime levels (1100 h) of these compounds were compared with levels in chicks killed in the middle of the dark phase (2300 h). In the brain areas examined, no day/night variations in levels of norepinephrine, epinephrine, dopamine, or 5-HT were seen, although significant nocturnal changes in levels of their metabolites were observed in some areas. Pineal levels of 5-HIAA decreased significantly at night. The retina showed significant nocturnal increases in 5-HTP, 5-HT, and 5-HIAA levels. Retinal levels of 3-MT and DOPAC were significantly decreased at night.     

Adrenomedullary Secretion of DOPA, Catecholamines, Catechol Metabolites, and Neuropeptides

Abstract: Catecholamines and their metabolites have been proposed as markers of sympathetic nervous system stimulation. However, the adrenal medulla is a rich source of catecholamines and catecholamine metabolites and may play a significant role in plasma levels of these compounds. In addition to adrenal catecholamine metabolite efflux, the role of the catecholamine precursor 3,4-dihydroxyphenylalanine (DOPA) has not been fully evaluated. The simultaneous effluxes of catecholamines, metabolites, DOPA, and neuropeptides were measured in perfusates from isolated dog adrenals. The relative abundance of compounds detected consistently during unstimulated conditions was epinephrine ≫ norepinephrine > 3,4-dihydroxyphenylglycol > metanephrine > normetanephrine > dopamine > 3,4-dihydroxyphenylacetic acid > 3-methoxy-4-hydroxyphenylglycol ≥ DOPA ≫ [Met]enkephalin ≫ neuropeptide Y. Effluxes of analytes were not affected by cocaine and the ratios of catecholamines to metabolites increased dramatically with carbachol stimulation, consistent with negligible reuptake into adrenal cells. Thus, most of the 3,4-dihydroxyphenylglycol is expected to be derived from epinephrine and norepinephrine subsequent to translocation from chromaffin vesicles into the cytosol. The efflux of DOPA increased dramatically during stimulation with 30 µ M carbachol in a calcium-dependent manner. Efflux of DOPA during the initial stabilization period of the perfusion preparation declined exponentially, in parallel with the effluxes of the catecholamines and neuropeptides but not with metabolites. Evoked release of DOPA was Ca²⁺-dependent. These data suggest that DOPA can be stored and released exocytotically from chromaffin granules.     

Distribution of catecholamines,serotonin, and their major metabolites in the rat cingulate,piriform-entorhinal,somatosensory, and visual cortex: A biochemical survey using high-performance liquid chromatography

The catecholamines noradrenline (NA), dopamine (DA), adrenaline (AD), the indoleamine 5-hydroxytryptamine (5-HT; serotonin), as well as some of their major metabolites were assayed by high-performance liquid chromatography (HPLC) with electrochemical detection, in four well-defined areas of the rat cerebral cortex: anterior cingulate (CIN;Cg1 and Cg3), piriform and entorhinal (PiEn), hind-limb primary somatosensory (SSC;HL) and primary visual (VIS; Oc1M and Oc1B). The concentrations of NA and that of its main metabolite 3-methoxy-4-hydroxyphenylglycol were highest in PiEn, had intermediate values in CIN and were lowest for SSC and VIS cortices. The DA levels were also highest in PiEn, intermediate in CIN, while the lowest values were in SSC and VIS cortices. The different DA/NA ratios support the hypothesis that they are indeed independent neurotransmitters. In addition, the levels of 3,4-dihydroxyphenylacetic acid, homovanillic acid and 3-methoxytyramine paralleled the distribution of DA, thus confirming the presence of release sites, even in regions in which the low levels of this catecholamine could be interpreted simply as the precursor of NA. Traces of AD were detected in all the regions examined. The 5-HT contents, as well as that of its precursor 5-hydroxy-I-tryptophan and that of its metabolite 5-hydroxyindole-3-acetic acid were also found to be non-homogenous, with the highest levels measured in the PiEn and CIN regions.     

A sensitive method for the determination of plasma catecholamines using liquid chromatography with electrochemical detection

Simple and sensitive methods for the determination of plasma catecholamines are of great interest since the level of catecholamines in plasma reflects the activity of the sympatho-adrenal system. In the present work a previously described procedure based on high pressure liquid chromatography with electrochemical detection has been adapted for assay of plasma catecholamines. This method permits simultaneous detection of noradrenaline, adrenaline and dopamine in concentrations down to 0.1 nmol/1 in less than one ml plasma.     

Specific [3H]SCH23390 Binding to Dopamine D1 Receptors in Cerebral Cortex and Neostriatum: Evidence for Heterogeneities in Affinity States and Cortical Distribution

The tritiated antagonist SCH23390 was used to identify dopamine D1 receptors in the cerebral cortex and neostriatum. The kinetic properties of binding were investigated in parallel experiments with membrane preparations from both tissues. The densities of receptors (Bmax) and the dissociation constants (KD) were determined from saturation curves, and the specificity of binding verified in competition experiments using agonists and antagonists. The cortical D1 receptor displays the same pharmacological selectivity (including stereospecificity) and kinetic properties as the neostriatal D1 receptor. From both the dissociation kinetics by dilution and the competition curves, it could be established that there is an heterogeneity of binding probably due to high- and low-affinity states. Endogenous dopamine, 4-hydroxy-3-methoxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, and 3-methoxytyramine contents, as well as D1 receptor distribution, were measured for the neostriatum and four localized cortical areas: anterior cingulate, primary somatosensory, primary visual, and piriform-entorhinal. For the regions examined, the distribution of D1 receptors is heterogeneous, but correlates very well (r greater than 0.98) with the endogenous levels of dopamine and its major metabolites.