Dopamine Metabolites Dihydroxyphenylacetic Acid Research Articles

Simple high-performance liquid chromatographic method for the concurrent determination of the amine metabolites vanillylmandelic acid, 3-methoxy-4-hydroxyphenylglycol, 5-hydroxyindoleacetic acid, dihydroxyphenylacetic acid and homovanillic acid in urine using electrochemical detection

A simple method for the concurrent analysis of the noradrenaline metabolites vanillylmandelic acid and 3-methoxy-4-hydroxyphenylglycol, the dopamine metabolites dihydroxyphenylacetic acid and homovanillic acid, and the serotonin metabolite 5-hydroxyindoleacetic acid in human urine is described. Following organic extraction of the metabolites from acidified urine, they are separated by single-step gradient elution high-performance liquid chromatography on a reversed-phase column. Detection and quantification are achieved with an electrochemical detector using a carbon-paste electrode; samples can be injected at 40-min intervals. Optimisation of analytical parameters is described, and examples of the application of the method in the fields of clinical chemistry and clinical neuroscience are given. This provides a convenient method for the concurrent study of the metabolism of three major biogenic amines, and is readily adaptable for studies on cerebrospinal fluid and brain tissue.

A study on the acute effect of amphetamine on the urinary excretion of biogenic amines and metabolites in monkeys.

1 The effects of an acute dose (3 mg/kg) of amphetamine on the urinary excretion of phenylethylamine (PEA), p-tyramine, their metabolites, catecholamine metabolites and p-hydroxymandelic acid, a major metabolite of p-octopamine were evaluated in the monkey. Amphetamine excretion was also measured. 2 Amphetamine was slowly eliminated from the body, being found in the urine at least six days after administration. 3 Amphetamine increased the excretion of PEA and decreased that of its major metabolite, phenylacetic acid (PAA). This pattern of changes is similar to that previously found in the urine of chronic schizophrenics. 4 The excretion of the dopamine metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC) was markedly reduced, that of vanilmandelic acid (VMA) remained unchanged while 3-methoxy-4-hydroxyphenylglycol (MHPG) was increased on the day of drug administration and persisted for at least a further six days. A similar extended effect on the excretion of p-hydroxymandelic acid (it was reduced) was also observed. 5 The excretion of p-tyramine but not its metabolite, p-hydroxyphenylacetic acid, was decreased by amphetamine during treatment and returned to normal levels six days later. 6 From the results obtained, it was concluded that amphetamine effects on behaviour cannot exclusively be attributed to its influence on catecholamines and that other biogenic amines may be involved. 7 Since PEA elicits many behavioural changes similar to those seen with amphetamine, and since amphetamine increases PEA excretion, we suggest that amphetamine may exert some of its behavioural responses through the release of PEA.

Kainic acid-induced changes of serotonin and dopamine metabolism in the striatum and substantia nigra of the rat

Levels of the putative neurotransmitters serotonin (5-HT) and dopamine (DA) and their respective metabolites 5-hydroxyindoleacetic acid (5-HIAA) and dihydroxyphenylacetic acid (DOPAC) were determined in the rat striatum after unilateral intrastriatal injection of the convulsive neurotoxin kainic acid. Two days after intrastriatal kainic acid injection, levels of the 5-HT metabolite 5-HIAA were increased by about 200% in the injected striatum and by about 150% in the contralateral striatum. An elevated striatal 5-HIAA content was still detectable 10 days after the kainate lesion, but approached normal values 10 weeks after the injection of the neurotoxin. Two days after the lesion, but not at the other time intervals, a moderate increase of 5-HIAA also occurred bilaterally in other brain areas such as the substantia nigra, frontal cortex and hypothalamus. Levels of 5-HT were decreased significantly in the injected striatum 2 days after the intrastriatal application of kainic acid and increased by about 40% after 10 weeks. The 5-HT concentration in the contralateral striatum or in the three other brain areas examined was unchanged at all time intervals. Levels of the DA metabolite DOPAC and DA turnover were increased in the lesioned striatum 2 days after kainic acid injection; concomitantly the DOPAC level was increased in the substantia nigra of the contralateral side. DOPAC levels of the contralateral striatum were unchanged or slightly reduced 2 days after the injection. Ten days as well as 10 weeks after the lesion there was a slightly increased DOPAC concentration in both striata. The levels of DA were not altered at any time interval after the injection of kainic acid.

Apomorphine-haloperidol interactions: Different types of antagonism in cortical and subcortical brain regions

Apomorphine, a dopamine (DA) receptor stimulant induced a dose-dependent decrease in the content of the dopamine metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the olfactory tubercle and striatum of rats, but it was ineffective in the frontal cortex. Apomorphine also antagonized the haloperidol-induced accumulation of DOPAC and HVA in the olfactory tubercle and striatum, whereas in the frontal cortex it antagonized the effects of haloperidol on DOPAC but not on HVA. The total (free and conjugated) content of DA metabolites was measured in these experiments.

Partial lesions of the dopaminergic nigrostriatal system in rat brain: biochemical characterization

Various doses of 6-hydroxydopamine injected into the rat substantia nigra produced partial, dose-dependent lesions of the dopaminergic nigrostriatal tract. The resulting reduction in striatal dopamine concentrations and tyrosine hydroxylase activities tended to be proportional, allowing these measurements to serve as indices for lesion severity in any particular animal. Lesions destroying two-thirds or more of the nigrostriatal neurons accelerated dopamine's synthesis in, and release from, surviving neurons, as indicated by increased striatal levels of the dopamine metabolites dihydroxyphenylacetic acid and homovanillic acid. Formation of these metabolites was also enhanced in dendrites of dopaminergic neurons in the substantia nigra. Supersensitivity of striatal postsynaptic receptors, as judged by induction of rotational behavior after apomorphine or l-DOPA administration, occurred when 90% or more of the nigrostriatal neurons had been destroyed. In contrast, rotational behavior could be induced by amphetamine in animals with only 50% of these neurons destroyed.

The effect of phencyclidine on dopamine synthesis and metabolism in rat striatum

Previous behavioral and neurochemical studies indicate that phenyclidine (PCP), a potent psychotomimetic agent, interacts with central dopaminergic systems. We have examined the effects of PCP on the rate of accumulation of 3,4-dihydroxyphenylalanine (DOPA) after the inhibition of L-aromatic amino acid decarboxylase and on the levels of dopamine (DA) metabolites: 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in rat striatum. PCP, in doses from 2.5 to 50 mg/kg, decreased the rate of striatal DOPA accumulation. PCP did not antagonize the increase in the rate of striatal DOPA formation caused by haloperidol, reserpine or γ-butyrolactone (GBL). When given alone, PCP decreased striatal levels of DOPAC and HVA, while it greatly potentiated the haloperidol-induced rise in striatal levels of these two metabolites. PCP is considerably less effective than d-amphetamine in promoting the release of 3H-DA from preloaded striatal slices in vitro. Our results are consistent with the interpretation that PCP potentiates the synaptic effects of endogenous DA. Its mechanism of action appears to be closely related to that of a category of drugs known as non-amphetamine stimulants, which, among others, includes methylphenidate, amfonelic acid and cocaine.

Tyrosine administration increases striatal dopamine release in rats with partial nigrostriatal lesions.

Partial, unilateral nigrostriatal lesions of varying severity were produced in rats by injecting graded doses of 6-hydroxydopamine into the substantia nigra. Formation of the dopamine metabolites dihydroxyphenylacetic acid and homovanillic acid in each surviving nigrostriatal neuron (estimated by the ratios of dihydroxyphenylacetic acid to dopamine and homovanillic acid to dopamine in the striatum) increased significantly when dopamine concentrations in striata containing lesions had been reduced to 25% or less of control values, but remained unchanged in rats with less severe lesions. These findings suggest that, in rats with severe damage of nigrostriatal dopaminergic neurons, surviving neurons increase their firing rates and accelerate dopamine synthesis and release. In rats that had lesions and enhanced striatal dopamine release, but not in rats with lesser lesions (i.e., which reduced ipsilateral dopamine concentrations by less than 75%), administration of tyrosine (250 mg/kg) caused further significant increases in formation of dihydroxyphenylacetic acid and homovanillic acid. These findings provide further evidence that tyrosine availability can enhance dopamine synthesis in and release from nigrostriatal neurons if the firing rates of these neurons are accelerated.

Subcellular distribution of dopamine metabolites and their elimination from the rat brain

A compartmentation of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) was found in the rat brain when its synthesis was inhibited by the monoamine oxidase inhibitor pargyline. Homovanillic acid (HVA) did not show such compartmentation after pargyline administration, but similar compartmentation was observed for HVA after treatment with tropolone which is an inhibitor of catechol- o-methyl transferase. The most rapid HVA compartment was eliminated after inhibition of the active transport with probenecid. No anatomical substrate could be found for these compartments. A relatively larger portion of DOPAC than of HVA was found to be associated with the crude mitochondrial/synaptosomal fraction when the subcellular distribution of these two compounds was investigated. Intraventricular injection of DOPAC in conjunction with either probenecid or tropolone resulted in increased DOPAC levels compared to intraventricular DOPAC alone, but no potentiating effect was observed when probenecid and tropolone were given in combination. It is concluded that the compartmentation of DOPAC is related to the HVA/DOPAC ratio in the various brain regions and the different metabolic pathways involved. The compartmentation of HVA shows that the acid is transported between different structures prior to removal from the brain. These results indicate that great care must be taken in estimating dopamine metabolic turnover on the basis of the elimination of metabolites subsequent to the inhibition of dopamine synthesis.

Effects of (8β)-8-[(Methylthio)methyl]-6-propylergoline on dopaminergic function and brain dopamine turnover in rats

(8β)-8-[(Methylthio)methyl]-6-propylergoline induced contralateral turning in rats with nigrostriatal lesions, lowered serum prolactin in reserpinized rats, and caused stereotyped hyperactivity. In addition to these functional effects typical of dopaminergic agonists, (8β)-8-[(methylthio)methyl]-6-propylergoline decreased dopamine turnover in rat brain. Decreased turnover was indicated by a diminished depletion of dopamine content after inhibition of its synthesis by α-methyltyrosine and by a decreased steady state concentration of the dopamine metabolite, 3, 4-dihydroxyphenylacetic acid (DOPAC). DOPAC concentration in whole brain was decreased after doses of (8β)-8-[(methylthio)methyl]-6-propylergoline as low as 0.003 mg/kg, and the lowering of DOPAC persisted for up to 16 hrs. after a 0.3 mg/kg dose. (8β)-8-[(Methylthio)-methyl]-6-propylergoline had less effect than a structurally-related compound, lergotrile, on 3-methoxy-4-hydroxy-phenyl-ethyleneglycol sulfate levels in whole brain and did not affect 5-hydroxy-indoleacetic acid levels over a dose range from .01–10 mg/kg. The behavioral and neuroendocrine effects of this new ergoline compound and its reduction of dopamine turnover in rat brain indicate that it is a potent dopamine receptor agonist in vivo .

Alterations in brain dopamine and serotonin metabolism during the development of tolerance to human beta-endorphin in rats.

Repeated intracisternal injections of human beta-endorphin lead to development of tolerance with respect to the catalepsy, analgesia, and hypothermia which are seen following a single injection. The initial injection of beta-endorphin results in increases in the dopamine metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in neostriatum, as well as increases in the serotonin metabolite, 5-hydroxyindoleacetic acid (5-HIAA), in hypothalamus and brainstem and a decrease in 5-HIAA in hippocampus. In the present study, we report changes in metabolism of dopamine and serotonin in specific brain areas during the development of tolerance to beta-endorphin. Thus, the development of tolerance to beta-endorphin with respect to catalepsy, analgesia, and hypothermia may be mediated by development of tolerance to the effects of beta-endorphin on brain dopamine and serotonin release.

β-endorphin-induced increase in striatal dopamine turnover

Human β-endorphin administered intracisternally in a dose of 15 μg per rat increased striatal concentrations of the dopamine metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) as well as producing catalepsy. These effects were inhibited by naloxone. Pargyline-induced decreases in striatal DOPAC and HVA were greater in endorphin-treated than in saline-treated animals, supporting the concept that β-endorphin increases striatal dopamine turnover. β-endorphin increased the rate of decline in striatal dopamine concentration following synthesis inhibition with α-methyltyrosine, further suggesting that endorphin increases striatal dopamine turnover. β-endorphin and probenecid interacted competitively to decrease the effects of each other to increase striatal HVA. Naloxone prevented the effect of endorphin to decrease the HVA response to probenecid. Thus, probenecid cannot be used to assess the effects of endorphin on striatal dopamine turnover. If β-endorphin acts presynaptically to decrease dopamine release in striatum, the increases in striatal DOPAC and HVA probably represent a compensatory attempt to increase dopamine synthesis. Although turnover of dopamine to its metabolites is increased, dopamine release may be suppressed by β-endorphin.

Effect of probenecid on endogenous and exogenous 3,4-dihydroxy-phenylacetic acid and homovanillic acid in the rat brain

The two dopamine metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were measured in corpus striatum and hippocampus in rats after an intracerebroventricular injection of DOPAC or HVA. Probenecid 200 mg/kg i.p. increased the concentration of HVa by the same factor in both control animals and animals treated with HVA. Probenecid had no effect on the DOPAC concentration in control animals or in animals treated with DOPAC. After DOPAC intraventricularly some increase in the HVA concentration could be seen. Pargyline 75 mg/kg was given i.m. and the decline of HVA and DOPAC in corpus striatum was compared in animals with or without probenecid treatment (200 mg/kg i.p.). In the control animals DOPAC declined more rapidly than did HVA. In the probenecid-treated animals there was no change in the DOPAC curve while the HVA showed a slower elimination but still with an exponential curve. The conclusion is drawn that there is an active mechanism for the excretion of DOPAC which is not identical with the excretion mechanism fo HVA. A considerable part of the dopamine is directly deaminated to DOPAC in vivo and some of this DOPAC is probably O-methylated to HVA.

Dopamine metabolism in the tuberculum olfactorium

The occurrence of the major dopamine metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the tuberculum olfactorium of the rat was demonstrated by gas chromatography. The ratio of DOPAC to HVA in the tuberculum olfactorium was greater than the ratio of these metabolites in the striatum. The effect of pargyline and probenecid on dopamine metabolite levels was similar for both the tuberculum olfactorium and striatum.

Effect of morphine on the cerebral contents of metabolites of dopamine in normal and tolerant mice: its possible relation to analgesic action

1. The administration of an analgesic dose (10 mg/kg, s.c.) of morphine increased the concentrations of the dopamine metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the brains of normal mice, and the time course of the change in the DOPAC concentration corresponded approximately to that of morphine analgesia. The increase in the concentration of the DOPAC induced by morphine (20 mg/kg, s.c.) was completely suppressed by nalorphine (2 mg/kg, s.c.) given 5 min after the morphine administration.2. In morphine-tolerant mice the concentrations of DOPAC and HVA in the brain did not differ from those observed in normal mice, and the increase in the concentrations of DOPAC and HVA in brain after the acute administration of morphine no longer occurred.3. Nalorphine (2 mg/kg) given alone did not cause any change in brain DOPAC and HVA concentrations in normal mice.4. The morphine-induced increase in DOPAC and HVA concentrations in the brain are discussed in the light of the hypothesis that dopamine might participate not only in the extrapyramidal motor system but also in the sensory mechanisms of the brain.

Decreased noradrenaline (norepinephrine) synthesis in familial dysautonomia.

Noradrenaline synthesis and metabolism of dopamine was evaluated in three patients with familial dysautonomia and compared with that of six normal subjects. Each patient and subject was infused with 104.8 muCi of dopamine-2-(14)C dissolved in 1000 ml of physiological saline. The urine was collected during the infusion period and at intervals thereafter. Using a specially designed flow monitor system, the various biosynthetic and metabolic products of dopamine were separated, identified, and their radioactivity measured. The results indicate that in familial dysautonomia the synthesis of noradrenaline is significantly decreased; this is reflected by a decrease in recovery of radioactive noradrenaline as well as various metabolic products of noradrenaline, i.e. 3-methoxy-4-hydroxymandelic acid (MOMA), normetadrenaline, and normetadrenaline conjugate. Concomitant with this decrease in noradrenaline synthesis, there was a shift towards dopamine metabolism as reflected by an increase in the recovery of primary and secondary dopamine metabolites; 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxy-4-hydroxyphenylacetic acid (HVA), 3-methoxytyrosine, and respective conjugates, etc. Whereas all dysautonomic patients showed the same general metabolic pattern as was expected, they varied in degree.

Acid metabolites of monoamines in avian brain; effects of probenecid and reserpine.

1. The concentration of the dopamine (DA) metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the anterior part of the nucleus basalis of pigeon brain was found to be 0.17 +/- 0.01 mug/g, which is about one-fifth of the concentration of homovanillic acid (HVA) in this region. In the chicken, the concentration of HVA in the (entire) nucleus basalis was 0.06 +/- 0.006 mug/g, lower than in any other species examined, and giving a ratio of DA to HVA of about 50. The concentration of DOPAC in the 8 day old chick was 0.053 +/- 0.002 mug/g.2. Probenecid, 200 mg/kg intramuscularly, doubled the content of DOPAC in the nucleus basalis of the pigeon and increased the concentration of HVA in both the pigeon and the chicken by a factor of 4 to 5. These findings demonstrate the existence, in avian brain, of an active transport mechanism for the removal of acidic substances and explain the low concentrations of the acids found in bird brain.3. A method is described for the estimation of 5-hydroxytryptamine (5-HT) and 5-hydroxyindolylacetic acid (5-HIAA) in the same tissue sample. Probenecid caused an increase in the 5-HIAA content but produced no change in the 5-HT content of the nucleus basalis of pigeon brain.4. Reserpine caused a fall in the content of acidic DA metabolites in the nucleus basalis of the pigeon. The effect was more pronounced after raising the concentration of these acids with probenecid.5. Treatment of pigeons with pargyline (100 mg/kg 17 hr before decapitation) did not significantly increase the DA content of the nucleus basalis, but it prevented to some extent the loss in DA caused by reserpine.6. Pigeons and chickens were sedated by probenecid. The deepest sedation occurred at about the same time as the greatest increase in the acidic amine metabolites in the brain.7. Intracisternal injection of HVA in the pigeon and intravenous injection of large amounts of HVA, DOPAC, 5-HIAA or 3,4-dimethoxyphenylacetic acid into newly hatched chicks did not produce any sedation or other effects on behaviour. In contrast, injection of sodium gamma-hydroxybutyrate caused paralysis followed by prostration and eye closure.8. Estimation of the concentration of HVA in the brain of the young chick after intravenous injection of the acid (100 mg/kg) showed that the concentration was of the same order of magnitude as it is in animals given probenecid; this suggests that the sedation which follows probenecid is not related to the accumulation of acidic amine metabolites.