Evaluation of dopamine metabolism in rat striatum by a gas chromatographic technique

Dopamine metabolism in rat striatum was evaluated by gas chromatographic quantitation of 3,4-dihydroxy-phenylacetic acid (DOPAC) and homovanillic acid (HVA). The level of DOPAC (5.21 nmoles/g ± 0.40 S.E.M., N = 12) exceeded that of HVA (3.63 nmoles/g ± 0.25 S.E.M., N = 12). 2 hr following administration of probenecid (200 mg/kg i.p.) the level of striatal HVA was approximately doubled whereas the level of DOPAC was not significantly elevated. Parggline (75 mg/kg i.p.) produced a rapid depletion of DOPAC and HVA. The rate of disappearance of DOPAC (t^1/2 = 10 min) exceeded that of HVA (t^1/2 = 18 min). Rates of metabolite formation were computed assuming steady state kinetics. The rate of formation of DOPAC (20.5 nmoles/g/hr) was much greater than that of HVA (10.1 nmoles/g/hr). We conclude that DOPAC is the major dopamine metabolite in rat striatum and that its measurement may provide the best index of functional of neuronal activity in this species.     

The effect of chlorpromazine on homovanillic acid levels in cerebrospinal fluid of schizophrenic patients

A mass fragmentometric method was used for determination of homovanillic acid levels in cerebrospinal fluid of acutely admitted schizophrenic patients. Spinal puncture was performed before and 12 days following the beginning of chlorpromazine treatment (200–600 mg/day). The level of homovanillic acid in cerebrospinal fluid before treatment was significantly higher in women than in men. Chlorpromazine treatment resulted on the average in a more than 2-fold elevation of the homovanillic acid level in cerebrospinal fluid. There were marked individual differences in the effect. The results are compatible with the view that therapeutic doses of chlorpromazine accelerate brain dopamine synthesis in man.A close analysis of the relation between effects of chlorpromazine on brain dopamine synthesis and psychotic and motor behaviour in schizophrenic patients seems required.     

Plasma and cerebrospinal fluid probenecid concentrations as related to accumulation of acidic biogenic amine metabolites in man

The oral administration of probenecid (100 mg/kg/18 h) to depressed patients results in marked increases in the acidic metabolites of biogenic amines in the cerebrospinal fluid (CSF). In contrast to previous reports (where lower dose rates of probenecid were employed) the increases in 5-hydroxyindoleacetic acid and homovanillic acid were independent of plasma or CSF probenecid concentrations. Higher plasma and CSF probenecid concentrations were found in this study. The binding of probenecid to plasma proteins for each patient was also determined to examine the pharmacodynamics of probenecid distribution. The ratio of probenecid in CSF to the calculated free probenecid in plasma was higher (0.4) and more constant than previously noted. At these higher dose levels, probenecid successfully competes with the active transport process for biogenic amine acidic metabolites and also appears to block its own removal from CSF.A preliminary report of this work was presented (Fed. Proc. 32, 696 (1973).     

Mass fragmentographic analysis of homovanillic acid and its homoiso analogue in cerebrospinal fluid using the α-dideutero acid as internal standard

The preparation and use of HVA-d₂ for the mass fragmentographic determination of HVA levels in the CSF was described. Following extraction and synthesis of the methyl ester heptafluorobutyryl derivatives samples were injected into a combined gas chromatograph-mass spectrometer connected to a multiple ion detector. The two fragments set into focus were the molecular ions of authentic HVA and HVA-d₂ respectively. The experimental error was less than 4%. The recovery of small amounts of authentic HVA added to CSF was satisfactory. Multiple ion determination using two sets of fragments gave similar results demonstrating high specificity and reproducibility of the method used.The mass spectral characteristics of the methyl ester heptafluorobutyryl derivative of ISO-HVA suggested that this compound could be differentiated from HVA by mass fragmentography. Thus the base peak did not constitute the molecular ion in the spectrum of the ISO-HVA-derivative as it is for the derivative of HVA. Multiple ion analysis of CSF before and after the addition of ISO-HVA failed to supply positive evidence for the presence of ISO-HVA in CSF in neurological or schizophrenic patients.     

Alterations in cerebrospinal fluid dopamine metabolites following physostigmine infusion

The effect of IV physostigmine administration on the cerebrospinal fluid (CSF) levels of homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) in normal subjects was determined. After an adjustment for differing CSF concentrations of probenecid, physostigmine was found to elevate CSF HVA and DOPAC concentrations. The authors discuss these changes in CSF HVA and DOPAC and their possible relationship to the ability of physostigmine to decrease the symptoms of some patients with tardive dyskinesia.     

Paraquat induces alternation of the dopamine catabolic pathways and glutathione levels in the substantia nigra of mice

The herbicide paraquat (PQ) is a strong redox agent that participates in the formation of reactive oxygen species (ROS) and induces toxicity in the nigrostriatal dopaminergic system. In this study, we investigated the effect of PQ on dopamine (DA) and glutathione levels in the substantia nigra (SN) of mice. Male C57BL/6 mice (aged 7 weeks and 23–25 g) were used for this study. The mice were treated with normal saline (vehicle) and PQ (10 mg/kg, i.p.) twice weekly for three consecutive weeks. We measured changes in tyrosine hydroxylase (TH) immunoreactivity, DA and its metabolites, and glutathione (reduced and oxidized) in the SN.     

Further studies on the sequence of dopamine metabolism in the rat brain.

The time—response curve of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) concentrations in the striatum of the rat were measured during apomorphine, haloperidol, morphine, oxotremorine or reserpine treatment as well as after a brain lesion. The results showed that changes in DOPAC concentrations consistently preceded changes in HVA concentrations. It was concluded that HVA must be considered as a “second metabolite” rather than as a marker of extra-neuronal metabolism. The turnover of HVA calculated after tropolone treatment revealed that this compound can be considered as a reliable COMT inhibitor. The turnover of 3-methoxytyramine (3-MT) calculated after tropolone treatment (during MAO inhibition) appeared to be 1.9 nmol/g/h. From this relatively low value it was concluded that 3-MT cannot be considered as an alternative metabolite for DA degradation during MAO inhibition. 3-MT concentrations measured after apomorphine or γ-hydroxybutyric acid treatment also suggest that 3-MT leaves the brain to only a minor extent during MAO inhibition. The effects of reserpine on pargyline-induced 3-MT concentrations indicate that there is hardly any COMT-activity in dopaminergic terminals. Evidence was provided that, at least during reserpine or pargyline treatment, dopamine leaves the brain unmetabolized or via unknown metabolic pathways.     

Changes in striatal dopamine metabolism during precipitated morphine withdrawal

Precipitation of withdrawal in morphine tolerant/dependent rats by either naloxone or the partial agonist ZK 48491 caused a significant increase in the contration of striatal DA, which persisted for at least 1 h. During the same time the probenecid-induced accumulation of HVA and DOPAC was reduced in the striatum in relation to probenecid-treated tolerant/dependent controls. 20 min after precipitation of withdrawal by naloxone, the striatal concentration of 3-methoxytyramine was decreased by about 40%, while the activity of the DA metabolizing enzymes, MAO and COMT, remained unchanged. Naloxone-precipitated withdrawal was, further, found to delay the depletion of striatal DA caused by inhibition of synthesis 90 min after alpha-methyl-p-tyrosine treatment. All these results provide evidence for a decreased release of DA from the striatum during precipitated morphine withdrawal.     

Decrease in dopamine,its metabolites and noradrenaline in cerebrospinal fluid of schizophrenic patients after withdrawal of long-term neuroleptic treatment

Dopamine (DA), homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC), noradrenaline (NA), and 5-hydroxyindolacetic acid (5HIAA) were measured in cerebrospinal fluid (CSF) of 15 chronic schizophrenic patients before and 2 weeks after withdrawal of long-term neuroleptic treatment. Total neuroleptic-like activity in serum (NLA) was determined at the same times. Levels of DA and its metabolites (DOPAC and HVA) and NA were significantly reduced after the discontinuation of neuroleptic treatment. No change was observed in 5HIAA values. NLA was substantially reduced, but still remained detectable. The decrease in DA, DOPAC, and HVA all showed positive correlations with each other, and correlated negatively with NLA measured after 2 weeks. Our data implies that the decrease in DA turnover is the result of the discontinuance of DA receptor blockade, while the change in NA level is independent of it.This work was supported by the State Office for Technical Development, Hungary     

Effect of electroshock on dopamine metabolism in rat brain

Summary A single electroshock stimulation (EST) by means of a conventional clinical EST apparatus was administered to 40 rats. Ten minutes after the start of general convulsions the animals were sacrificed. Corpus striatum was analyzed for dopamine (DA) and homovanillic acid (HVA). A statistically not significant increase of DA and a highly significant increase of HVA was noted in the EST group compared to the control animals. The data indicate an acceleration of the DA synthesis induced by EST. The results are discussed in relation to the action of antidepressive drugs on the monoaminergic neurons and a supposed biochemical correlate of endogenous depression.     

The K+-induced increases in noradrenaline and dopamine release are accompanied by reductions in the release of their intraneuronal metabolites from the rat anterior hypothalamus

Summary The novel technique of microdialysis has been used to examine the basal and K⁺-induced release of catecholamines and metabolites from the anterior hypothalamus of the urethane-anaesthetized rat in vivo. A high pressure liquid chromatographic assay was developed to simultaneously measure endogenous noradrenaline, dopamine and their intraneuronal metabolites 3,4-dihydroxyphenylglycol (DOPEG) and 3,4-dihydroxyphenylacetic acid (DOPAC) respectively, in each 60 l dialysate sample. The effect of replacing Ca²⁺ in the perfusion fluid with a low concentration of Cd²⁺, which blocks Ca²⁺ effects, was also studied. Increasing the K⁺ concentration in the perfusion fluid elicited a concentration-dependent increase in noradrenaline and dopamine release. In contrast, there were marked reductions in DOPEG and DOPAC which were not concentration-dependent. In the Ca²⁺-depleted conditions, the K⁺-induced increase in amine release was significantly attenuated, but the reductions in the metabolites were not affected. We suggest that the mechanisms contributing to the observed reductions in the metabolites may be inhibition of neuronal reuptake, an increase in neuronal efflux, an enhancement of vesicular uptake and a decrease in vesicular efflux. Send offprint requests to E. Badoer at the above address     

The dynamics of dopamine metabolism in various regions of rat brain

Dopamine metabolism was studied in various regions of rat brain by following the decline of 3,4-dihydroxyphenylacetic acid (DOPAC) and 3-methoxy-4-hydroxyphenylacetic acid (HVA) from brain after treatment with pargyline, or from the accumulation of the acids after treatment with probenecid. The decline of DOPAC and HVA after pargyline treatment appeared exponential in all regions of brain studied with half-lives of about 13 min for HVA and 6.5 min for DOPAC. DOPAC was the major metabolite of dopamine, with various brain regions producing between 2–5 times more DOPAC than HVA. HVA accumulated after treatment with probenecid but the accumulation in 1 h did not account for all of the HVA apparently eliminated from brain. DOPAC accumulated in some regions of brain (medulla, hypothalamus and midbrain) and not in others (cerebellum, cortex, striatum and hippocampus) after probenecid treatment. We conclude that dopamine metabolism is not uniform in brain and that the accumulation of DOPAC and HVA in brain after probenecid treatment only accounts for a minor fraction of the dopamine formed in brain.     

Effect of ECT and imipramine treatment on the concentration of 5-hydroxyindoleacetic acid (5HIAA) and homovanillic acid (HVA) in the cerebrospinal fluid of depressed patients

The influence of probenecid administration on 5HIAA and HVA concentrations in the CSF of depressed patients, was studied before and after treatment with imipramine or ECT.The average increase of the two metabolites in the CSF after probenecid was similar in the untreated depressed patients and in the same patients improved after both imipramine or ECT treatment.The treatment determined a significant increase in the CSF concentration of the acid metabolites also before the probenecid administration.     

Cerebrospinal fluid 5-hydroxyindoleacetic acid (5HIAA) and homovanillic acid (HVA) following probenecid in unipolar depressives treated with amitriptyline

Lumbar cerebrospinal fluid 5-HIAA, HVA, and the ratio 5-HIAA/HVA were measured followed probenecid administration in eleven patints with unipolar depression before and during treatment with amitriptyline (AMI). Control values were obtained from a group of inmate volunteers. Prior to treatment CSF 5HIAA formation in the depressives was not different from controls. During treatment with AMI, CSF 5-HIAA formation decreased. One patient with psychotic symptoms prior to AMI and two patients who developed psychotic reactions on AMI showed relatively low CSF 5HIAA formation prior to antidepressant therapy. Compared to controls CSF HVA values were higher in the depressives prior to AMI therapy.     

Effects of tiapride on homovanillic acid levels in human cerebrospinal fluid drawn at pneumoencephalography

The effect of tiapride on HVA and 5-HIAA levels in the CSF drawn at pneumoencephalography (PEG) was studied. Five consecutive 5 ml fractions of CSF were drawn from control and tiapride-treated subjects. In both groups, a linear increase in HVA concentrations was found between the first and subsequent fractions. On the contrary, no significant difference in 5-HIAA concentrations was found in sequential CSF samples. Tiapride increased the mean HVA concentrations and caused a steeper caudocranial gradient of this metabolite but failed to modify 5-HIAA concentrations. The results suggest that tiapride blocks dopamine (DA) receptors and increases DA synthesis.     

Absence of effect of para-chlorophenylalanine on 5-hydroxyindoleacetic acid in cerebrospinal fluid in man

The effect of para-chlorophenylalanine (PCPA) on the 5-hydroxy-indoleacetic acid (5-HIAA) content in human cerebrospinal fluid (CSF) has been studied. There was no effect on the CSF-content of 5-HIAA 12, 24 or 48 h after a single dose of PCPA 1 g p.o. Neither was there any effect after 1 g/day during 4 days. The increase of 5-HIAA after administration of probenecid was reduced during treatment with PCPA 1 g/day. This reduction, however, is not due to a diminished production of 5-HIAA by PCPA but probably caused by a pharmacological interaction between probenecid and PCPA since the concentrations of probenecid in CSF were lower during administration of PCPA than without that drug.It is concluded that PCPA in the doses used in this study gives no effects on the CSF-concentrations of 5-HIAA.     

Potassium-induced release of tritiated histamine from rat brain tissue slices.

The release of exogenous histamine was studied by superfusing brain slices following incubation with the radiolabeled amine. Histamine was released in a calcium-dependent way by 40 mM potassium. This release was high in hypothalamus and striatum and low in hippocampus and cortex. Compound 48/80, a mast cell histamine releasing agent, also induced histamine release, but only from hypothalamic tissue slices. It is suggested that the potassium-induced release of accumulated exogenous histamine is mainly from glial cells.     

Extrapyramidal reactions and amine metabolites in cerebrospinal fluid during haloperidol and clozapine treatment of schizophrenic patients

8 male schizophrenic patients participated in a double-blind, cross over study of the extrapyramidal side-effects of haloperidol and clozapine (acute dystonia, Parkinsonism and tardive dyskinesia), together with their effect on homo-vanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in the cerebrospinal fluid (CSF). Haloperidol (9 mg/day) caused Parkinsonism, reduced tardive dyskinesias and increased the HVA concentration in the CSF. Clozapine (225 mg/day) had no effect on the neurological phenomena but reduced HVA and 5-HIAA concentrations in the CSF. During the discontinuation phase following the administration of haloperidol, tardive dyskinesia occurred or was aggravated; this did not occur after administration of clozapine. Accordingly, it is suggested that clozapine does not induce dopaminergic hypersensibility and, therefore, will not induce tardive dyskinesias.Part of this work was presented at the 9th C.I.N.P. Congress, Paris, July 7–12, 1974.     

Neurochemical investigations of the interaction of N,N-dimethyltryptamine with the dopaminergic system in rat brain

N,N-dimethyltryptamine (DMT) is a psychotomimetic indolealkylamine that has been suspected of being an endogenous causal factor in the aetiology of schizophrenia. Some aspects of its interaction with the dopaminergic system of the rat brain have been studied neurochemically, and comparisons have been made with the effects of d-amphetamine and apomorphine. In contrast to these two compounds, DMT has proved to be a potent, but short-acting MAO inhibitor with a rather selective effect on MAO A (serotonin-deaminating MAO).Like classical MAO inhibitors, DMT also greatly increases the accumulation of ³H-dopamine plus ³H-3-methoxytyramine newly formed from ³H-Dopa. Amphetamine and apomorphine have no comparable effect.Striatal levels of 3,4-dihydroxyphenylacetic acid are more efficiently lowered by DMT than those of homovanillic acid. the reverse has been observed with apomorphine, whereas amphetamine increases the concentrations of homovanillic acid and decreases those of 3,4-dihydroxyphenylacetic acid.Thus, in addition to its MAO inhibitory action, DMT probably also possesses dopamine-releasing effects. Both these properties could result in an indirect dopaminomimetic activity of DMT. It may be assumed, therefore, that the psychotomimetic activity of this compound is not noly linked to its effects on cerebral serotonergic mechanisms, but to a combination of these with an indirect dopaminergic stimulant activity.     

Milacemide effects on the temporal inter-relationship of amino acids and monoamine metabolites in rat cerebrospinal fluid

The temporal inter-relationship of various amino acids and monoamine metabolites in rat cerebrospinal fluid was examined after acute administration of milacemide (100, 200 or 400 mg/kg i.p.), a glycine prodrug. Glycine concentrations rose linearly and dose dependently (20–190%) but were only significantly elevated at the higher milacemide dose (200 and 400 mg/kg). In animals given 400 mg/kg, glycine values were still significantly elevated 8 h later. A concomitant increase (20–25%) in serine and taurine and a decrease in alanine cerebrospinal fluid values were observed at the highest milacemide dose. Other amino acids were unaffected. While cerebrospinal fluid 5-hydroxyindoleacetic acid concentrations were unaffected, the dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, exhibited a linear dose-dependent reduction. However, only homovanillic acid values were significantly decreased after 400 mg/kg milacemide. Cerebrospinal fluid analysis may be useful as a first screen in ascertaining putative neurochemical changes associated with drug administration.