Plasma 5-Hydroxyindoleacetic Acid as an Indicator of Monoamine Oxidase-A Inhibition in Rat Brain and Peripheral Tissues

We have examined the changes induced by the monoamine oxidase (MAO; EC 1.4.3.4) inhibitors tranylcypromine, clorgyline, and deprenyl on MAO activity and 5-hydroxytryptamine (serotonin, 5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) content in rat brain and blood (plasma and whole blood). The decreases of MAO-A activity observed in the liver and lungs after different doses of clorgyline or tranylcypromine correlated significantly (r > 0.80 in all cases) with the decline of plasma 5-HIAA. This was unaffected by 0.25 and 5 mg kg^?1 of deprenyl, indicating that 5-HT was deaminated exclusively in the periphery by MAO-A. It is interesting that very potent and significant correlations (r > 0.75) were found between plasma 5-HIAA and MAO-A activity, 5-HIAA and 5-HT content in brain tissue. These results suggest that plasma 5-HIAA can be used confidently as a peripheral indicator of the inhibition of MAO-A in brain. This may represent a favorable alternative to the analysis of 5-HIAA in CSF in psychiatric patients undergoing antidepressant treatment with nonspecific MAO inhibitors or with the new selective MAO-A inhibitors.     

Current concepts: II. Measurement of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in discrete brain nuclei using reverse phase liquid chromatography with electrochemical detection

A rapid and sensitive method has been outlined for the measurement of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) utilizing a weak cation-exchange resin and liquid chromatography with electrochemical detection. The sensitivity of the procedure allows measurement of the amine in punches of rat substantia nigra even after local injection of the neurotoxin 5,7-dihydroxytryptamine. Increases in 5-HT and decreases in 5-HIAA concentrations after pargyline, and selective increases in 5-HIAA concentrations after probenecid were detected in selected brain regions (nucleus accumbens, anterior striatum, substantia nigra). Thus, this procedure is sensitive enough to estimate 5-HT turnover in discrete nuclei of the rat brain.     

The effects of the anticonvulsant valproic acid on cerebral indole amine metabolism.

The effects of valproic acid (500 mg/kg, ip, 1 h prior to testing) on indole amine metabolism were studied in rats by measurement of the contents of tryptophan, 5-hydroxytryptophan (5-HTP), 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) in the cerebral hemisphere. Tryptophan and 5-HIAA levels were increased, whereas 5-HTP and 5-HT remained unchanged. Furthermore, valproic acid failed to alter the levels of 5-HTP and DOPA, 5-HT and DA, and 5-HIAA in animals pretreated, respectively, with 3-hydroxybenzyl hydrazine (a decarboxylase inhibitor), pargyline (a monoamine oxidase inhibitor), or probenecid (a compound which blocks 5-HIAA transport out of the brain and cerebrospinal fluid). These results militate against the possibility that valproic acid alters the rate of tryptophan hydroxylation or the synthesis of 5-HT. However they do support the concept that valproic acid increases brain 5-HIAA by inhibition of the transport mechanism which removes 5-HIAA from the brain.     

Simultaneous Effects of p-Chloroamphetamine, d-Fenfluramine, and Reserpine on Free and Stored 5-Hydroxytryptamine in Brain and Blood

The effects of acute treatment with p-chloramphetamine, d-fenfluramine, and reserpine on intracellular (brain tissue and whole blood) and extracellular (CSF and platelet-free plasma) compartments of 5-hydroxytryptamine (5-HT) in the brain and blood of the same rats have been examined. These treatments affected 5-HT in brain tissue and whole blood similarly (r = 0.823). Reserpine significantly reduced both intracellular pools at 2 and 24 h. p-Chloroamphetamine and d-fenfluramine were more effective on brain tissue 5-HT. The concentration of 5-HT in CSF was significantly increased by all treatments. p-Chloroamphetamine induced a dramatic 70-fold increase of CSF 5-HT, paralleling a 42% decrease in brain tissue. d-Fenfluramine significantly increased CSF 5-HT to 212% of controls and reduced whole brain 5-HT (-23%). The effects of p-chloroamphetamine and d-fenfluramine on 5-HIAA in brain, CSF, and plasma were nonsignificant. Individual values of 5-hydroxyindoleacetic acid (5-HIAA) in CSF and brain were highly correlated (r = 0.855), indicating that CSF 5-HIAA reflects well the concentration of 5-HIAA in brain tissue. Yet the intra- and extracellular concentrations of 5-HIAA were unrelated to the 5-HT changes. This indicates that CSF 5-HIAA does not reflect the active (extracellular) compartment of 5-HT in brain.     

Serotonin turnover in individual brain nuclei: evaluation of three methods using liquid chromatography with electrochemical detection

Liquid chromatography with electrochemical detection and brain microdissection techniques were used to evaluate three methods of studying serotonin turnover in 10 individual brain nuclei. The increase in serotonin (5-HT) and decline in 5-hydroxyindole acetic acid (5-HIAA) after administration of the monoamine oxidase inhibitor, pargyline, as well as the accumulation of 5-hydroxytryptophan (5-HTP) after the L-amino acid decarboxylase inhibitor, m-hydroxybenzylhydrazine, were measured. Serotonin accumulation and 5-HIAA decline could be detected in the n. caudatus, globus pallidus, cortical amygdala, n. interstitialis striae terminalis, n. preopticus medialis, and n. dorsomedialis. Only serotonin accumulation could be accurately assessed in the n. ventromedialis, n. arcuatus, and median eminence. The pattern of increase of serotonin after pargyline varied in different nuclei. There was a linear increase of serotonin over 90 minutes in the caudate, globus pallidus, and ventromedial nucleus and over 60 minutes in the n. preopticus medialis, and cortical amygdala. This contrasted with a maximal increase at 30 minutes in the other nuclei. However, 5-HIAA decline tended to be greatest after 30 minutes in most nuclei. Increases in 5-HTP concentrations after decarboxylase inhibition were not reliably detected in these areas. These results indicate that two nonsteady state methods may be used to evaluate changes in serotonin turnover in selected individual, nonpooled hypothalamic and forebrain nuclei.     

Measurement of Serotonin Turnover Rate in Rat Dorsal Raphe Nucleus by In Vivo Electrochemistry

5-Hydroxytryptamine (5-HT; serotonin) turnover rate in dorsal raphe nucleus of the urethane-anesthetized rat was estimated by using the in vivo electrochemical detector to measure the decay of extraneuronal 5-hydroxyindole acetic acid (5-HIAA) after monoamine oxidase inhibition. Carbon paste electrodes were scanned by semiderivative voltammetry and revealed two peaks: one at +0.15 V and the other at +0.25 V. The higher potential peak is composed primarily of the 5-HT metabolite 5-HIAA. After administration of pargyline, 75 mg/kg i.p., this peak declined exponentially. Regression analysis of these data by an exponential decay model yielded the fractional rate constant 0.82 +/- 0.06 h-1 (mean +/- SEM). This rate constant of 5-HIAA disappearance measured by in vivo electrochemistry is identical to the rate constant found by others measuring 5-HIAA disappearance by direct tissue assay methods. In animals not treated with pargyline, tissue 5-HIAA concentrations in the dorsal raphe nucleus were measured by HPLC with electrochemical detection. The average 5-HT turnover rate calculated as the product of the fractional rate constant and steady-state tissue 5-HIAA concentration was 12.6 nmol/g/h. These results demonstrate that electrochemical detection of extraneuronal 5-HIAA combined with monoamine oxidase inhibition can be used to measure neurotransmitter turnover in vivo in a discrete brain region.     

Serotonin Metabolism by Monoamine Oxidase in Rat Primary Astrocyte Cultures

The oxidative deamination of serotonin (5-HT) to 5-hydroxyindoleacetic acid (5-HIAA) by rat primary astrocyte cultures was investigated in intact cells using HPLC. All detectable 5-HIAA accumulated in the extracellular medium, and its rate of production was proportional to the 5-HT concentration over the tested range of 5 x 10(-7) to 10(-4) M. At 5 x 10(-7) M 5-HT, intracellular 5-HT was detectable only in astrocytes treated with monoamine oxidase (MAO) inhibitors. These findings are consistent with the idea that 5-HT taken up into astrocytes is not stored for re-release, but is rapidly metabolized to 5-HIAA, which is then extruded from the cell. At 5 x 10(-7) M 5-HT, 5-HIAA formation in intact cells was blocked 63% by the selective high-affinity 5-HT uptake inhibitor fluoxetine. 5-HT oxidation to 5-HIAA is carried out principally by MAO-A, because clorgyline was more effective at inhibiting the production of 5-HIAA than was pargyline. Radioenzymatic determinations of MAO activity in cell homogenates supported these findings, because under these conditions clorgyline was 1,000-fold more effective than pargyline at inhibiting MAO activity toward 14C-labelled 5-HT. However, the relatively selective MAO-B substrate beta-phenylethylamine (PEA) was also oxidized, showing that these cultures also contained MAO-B activity; the Km values for MAO-A oxidation of 5-HT and MAO-B oxidation of PEA were 135 and 45 microM, and Vmax values were 88 and 91 nmol/mg of total cell protein/h, respectively. Higher concentrations of PEA (greater than 20 microM) were oxidized by both MAO-A and MAO-B isozymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of barbitone sodium and thiopental sodium on brain dopamine, noradrenaline, serotonin and 5-hydroxyindoleacetic acid content in Arvicanthis niloticus

The quantitative estimation of total dopamine (DA), noradrenaline (NE), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) content in the whole brain tissue of normal Nile grass rat, Arvicanthis niloticus, gives and average of 631 +/- 12 ng DA/g, 366 +/- 12 ng NE/g, 617 +/- 15 ng 5-HT/g and 431 +/- 10 ng 5-HIAA/g fresh brain tissue. The effect of barbitone sodium and thiopental sodium on the total DA, NE, 5-HT and 5-HIAA content in the brain tissue of the Nile grass rat, Arvicanthis niloticus, was studied. The total DA, NE, 5-HT and 5-HIAA contents were determined 5 hr after i.p. injection of different doses of barbitone sodium (20, 40 and 80 mg/ml/100 g body wt) and thiopental sodium (5, 10 and 20 mg/ml/100 g body wt). The effect of different time intervals (1, 10, 30 min, 1, 2.5, 5, 8, 16, 24 and 48 hr) on the total brain DA, NE, 5-HT and 5-HIAA content was investigated after i.p. injection of 40 mg of barbitone sodium and 10 mg of thiopental sodium/ml/100 g body wt. Both barbitone sodium and thiopental sodium caused an increase in DA, NE and 5-HT content and a decrease in 5-HIAA content in the brain tissue of Arvicanthis niloticus. The increase in the whole brain contents of DA, NE and 5-HT after the administration of barbitone sodium and thiopental sodium may be due either to inhibition of transmitter release by an action at the monoamine nerve terminal or to effects causing a decrease in nerve impulse flow. On the other hand, the decrease in 5-HIAA may be due to the decrease in the turnover of 5-HT.     

Regional Distribution of Extracellular 5-Hydroxytryptamine and 5-Hydroxyindoleacetic Acid in the Brain of Freely Moving Rats

The extracellular concentrations of 5-hydroxytryptamine (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) have been determined in six brain areas of awake rats (frontal cortex, striatum, hypothalamus, hippocampus, inferior colliculus, and raphe nuclei) using intracerebral microdialysis. The extracellular levels of 5-HT showed no significant differences among the brain regions studied. The tissue levels of 5-HT and 5-HIAA as well as the extracellular concentration of 5-HIAA were significantly higher in raphe nuclei. The regional distribution of tissue and extracellular 5-HIAA were very similar, suggesting that extracellular 5-HIAA depends mainly on the output from the intracellular compartment. On the other hand, extracellular 5-HT and tissue 5-HT showed a different distribution pattern. The tissue/extracellular ratio for 5-HT ranged from 739 in frontal cortex to 2,882 in raphe, whereas it only amounted to 1.8-3.6 for 5-HIAA. The relationship between the present results and the density of 5-HT uptake sites in these areas is discussed.     

Plasma free 5-hydroxytryptamine (5-HT): Evidence of an increase induced by LM 5008, a potent 5-HT uptake inhibitor

When 5-HT platelet uptake was inhibited in rats by single or repeated oral administration of 4-[2-(3-indolyl)ethyl]piperidine (LM 5008), 5-hydroxy-indole-acetic acid (5-HIAA) and 5-HT platelet concentration decreased. An oral administration of LM 5008 (10 mg/kg) to rats whose platelets were previously labeled with tritiated 5-HT provoked an increase in plasma free 5-HT and 5-HIAA. The maximum rise in 5-HT occured at 15 min while that of 5-HIAA appeared later (30 min). Concurrently urinary excretion of 5-HT was dramatically increased (about 5 times the control value) which indicates that 5-HT metabolism was not stimulated. According to the similarity between blood platelets and tryptaminergic neurons, plasma free 5-HT variations appeat to reflect changes of the neurotransmitter level into the synaptic cleft. Moreover, the excess of plasma free 5-HT induced by LM 5008 could improve 5-HT effects on vascular tone and pain.     

PASSAGE OF 5-HYDROXYTRYPTAMINE THROUGH THE BLOOD-BRAIN BARRIER, ITS METABOLISM IN THE BRAIN AND ELIMINATION OF 5-HYDROXYINDO-LEACETIC ACID FROM THE BRAIN TISSUE

Abstract— 5-HT was injected intravenously in rats (10 mg/kg) and a marked increase in brain 5-HT and 5-HIAA was observed. For the first 10 min after injection the penetration of 5-HT into the brain and formation of 5-HIAA is evident. After 10 min degradation of exogenous 5-HT and elimination of 5-HIAA are prominent. Metabolism of exogenous 5-HT in the brain is very fast (half-life between 5 and 10 min; completely metabolized in approximately 80 min). The importance of these results in explaining the permeability of blood-brain barrier to 5-HT is discussed. Experiments on brain slices show that 5-HT is more readily metabolized in brain tissue than eliminated into incubation medium. In contrast, 5-HIAA very easily leaves brain tissue.     

Blood 5-hydroxytryptamine, 5-hydroxyindoleacetic acid and melatonin levels in patients with either Huntington's disease or chronic brain injury

Following a study of oxidative tryptophan metabolism to kynurenines, we have now analysed the blood of patients with either Huntington's disease or traumatic brain injury for levels of 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and melatonin. There were no differences in the baseline levels of these compounds between patients and healthy controls. Tryptophan depletion did not reduce 5-HT levels in either the controls or in the patients with Huntington's disease, but it increased 5-HT levels in patients with brain injury and lowered 5-HIAA in the control and Huntington's disease groups. An oral tryptophan load did not modify 5-HT levels in the patients but increased 5-HT in control subjects. The tryptophan load restored 5-HIAA to baseline levels in controls and patients with brain injury, but not in those with Huntington's disease, in whom 5-HIAA remained significantly depressed. Melatonin levels increased on tryptophan loading in all subjects, with levels in patients with brain injury increasing significantly more than in controls. Baseline levels of neopterin and lipid peroxidation products were higher in patients than in controls. It is concluded that both groups of patients exhibit abnormalities in tryptophan metabolism, which may be related to increased inflammatory status and oxidative stress. Interactions between the kynurenine, 5-HT and melatonin pathways should be considered when interpreting changes of tryptophan metabolism.     

Effect of Tryptophan on Extracellular Concentrations of Tryptophan and 5-Hydroxyindoleacetic Acid in the Striatum and Cerebellum

The effects of L-tryptophan (50 mg/kg i.p.) on extracellular concentrations of tryptophan and the 5-hydroxytryptamine (5-HT) metabolite 5-hydroxyindoleacetic acid (5-HIAA) were determined in the rat striatum and cerebellum, regions with rich and poor 5-HT innervation, respectively. Determinations were on perfusates from dialysis probes in the brains of conscious, freely moving rats. The pharmacokinetic profiles of dialysate tryptophan after tryptophan load (peak concentration, time to peak concentration, area under curve, and half-life) in the two regions did not differ significantly. The dialysate 5-HIAA concentration in the striatum rose two- to threefold after the administration of tryptophan. Therefore, as 5-HIAA was undetectable in the cerebellum either before or after the administration of tryptophan, the increase of 5-HIAA in the striatum is unlikely to depend appreciably on its production within the cerebral vasculature or outside the brain or on its entering the striatum through a blood-brain barrier damaged by placement of the dialysis probe. Overall, the findings strengthen previous evidence that extracellular 5-HIAA concentrations determined by cerebral dialysis are a valid measure of the metabolism of 5-HT of brain neuronal origin.     

Turnover of Dopamine and Serotonin and Their Metabolites in the Striatum of Aged Rats

Turnover of dopamine (DA), serotonin [5-hydroxytryptamine (5-HT)], and their metabolites has been measured in adult and aged rats. Turnover rates of 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxy-3-indoleacetic acid (5-HIAA) have been assayed from the disappearance rates after blocking by pargyline inhibition of monoamine oxidase (MAO) and from the accumulation rates by probenecid inhibition of the probenecid-sensitive transport system. DA and 5-HT turnover rates have been measured as accumulation rates of 3,4-dihydroxyphenylalanine and 5-hydroxytryptophan, respectively, after central decarboxylase inhibition by 3-hydroxybenzylhydrazine (NSD-1015) and as accumulation rates of DA and 5-HT after pargyline inhibition of MAO. The DA turnover rate after NSD-1015 was 23.9% lower in aged rats than in adults, whereas after pargyline there was no significant difference between the two age groups. The HVA fractional rate constant and turnover after pargyline were lower in aged rats than in adults, and HVA turnover after probenecid was higher in aged rats than in adults. The DOPAC-HVA pathway seems to be reinforced at the expense of DOPAC conjugation. In aged and adult rats whose 5-HT steady-state levels were not statistically different, the 5-HT turnover rate after pargyline and NSD-1015 treatment was lower in aged rats than in adults. An increase of 5-HIAA levels after pargyline and probenecid treatment in aged rats could be due to the handling stress.     

Myocardial interstitial serotonin and its major metabolite, 5-hydroxyindole acetic acid levels determined by microdialysis technique in rat heart

Aims

The aim of this study was to elucidate myocardial interstitial serotonin (5-HT) kinetics in the heart, including 5-HT reuptake and enzymatic degradation to 5-hydroxyindole acetic acid (5-HIAA) via monoamine oxidase (MAO).

Main methods

Using microdialysis technique in anesthetized rats, we simultaneously monitored myocardial interstitial levels of 5-HT and its major metabolite, 5-HIAA, in the left ventricle and examined the effects of local administration of a MAO inhibitor, pargyline, or a 5-HT uptake inhibitor, fluoxetine.

Key findings

Pargyline increased dialysate 5-HT concentration from 1.8 ± 0.3 at baseline to 3.9 ± 0.5 nM but decreased dialysate 5-HIAA concentration from 20.7 ± 1.0 at baseline to 15.8 ± 1.4 nM at 60–80 min of administration. Fluoxetine increased dialysate 5-HT concentration from 1.9 ± 0.4 at baseline to 6.5 ± 0.9 nM at 60–80 min of administration, but did not change dialysate 5-HIAA concentration. Local administration of ADP (100 mM) increased dialysate 5-HT and 5-HIAA concentrations. Pargyline did not affect ADP-induced increase in dialysate 5-HT concentration but suppressed ADP-induced increase in dialysate 5-HIAA concentration during 60 min of ADP administration. Fluoxetine increased dialysate 5-HT concentration at 40–60 min of ADP administration, but did not affect ADP-induced increase in dialysate 5-HIAA concentration.

Significance

Simultaneous monitoring of myocardial interstitial 5-HT and 5-HIAA levels provides valuable information on 5-HT kinetics including reuptake and enzymatic degradation by MAO, which play a role in the regulation of myocardial interstitial 5-HT levels at baseline and when 5-HT levels are elevated.     

Effect of domoic acid on metabolism of 5-hydroxytryptamine in rat brain

Domoic acid (Dom) is a neurotoxic secondary amino acid that interacts with the glutamate receptors, producing neurological problems. In the present work, we study the effects of Dom on the levels of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in discrete rat brain regions. The effects of Dom on the brain metabolism of serotonin are also discussed in this paper. Dom stimulates the rat brain serotoninergic system, increasing differentially the synthesis and the catabolism of 5-HT and the elimination of 5-HIAA.     

Effect of immobilization stress on serotonin content and turnover in regions of the rat brain.

Sprague-Dawley rats were stressed by immobilization from 30 to 300 minutes and the effects on serotonin (5-HT) and 5-hydroxy-indoleacetic acid (5-HIAA) content were determined in the cerebral cortex, diencephalon, striatum, hippocampus and the brain stem. In a subsequent study 5-HT turnover rate in these brain areas was estimated by measuring 5-HIAA accumulation 0, 30, 60 and 90 minutes after probenecid. The content of 5-HIAA and the turnover rate of 5-HT were significantly increased in the cerebral cortex shortly after the onset of immobilization. The content of 5-HIAA in the brainstem was increased by immobilization although 5-HT turnover rate was not increased. Short term increases in 5-HIAA content were observed in the striatum and hippocampus. However, no significant changes in 5-HT turnover rate were observed in either of these 2 brain areas. Immobilization did not affect 5-HIAA content or 5-HT turnover in the diencephalon. The sensitivity of the serotonergic system in the cerebral cortex to immobilization stress suggests that this brain region could be used in future studies of the interrelationships between stress and the brain serotonergic system.     

Effects of acute and chronic administrations of phencyclidine on the levels of serotonin and 5-hydroxyindoleacetic acid in discrete brain areas of mouse

The effects of phencyclidine (PCP) on the levels of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in discrete brain areas of mouse were investigated. Following a single administration, PCP significantly increased at 60 min the level of 5-HT but not 5-HIAA in the cortex. However, acute administration of PCP induced no changes of 5-HT and 5-HIAA levels in other brain areas investigated. On the other hand, chronic treatment of PCP produced a significant increase the striatal 5-HT and 5-HIAA levels by about 30% and 20%, respectively. These increased levels were gradually returned to the control levels, and there was no difference of these levels between the control group and the 48 hr withdrawal group. The changes of 5-HT level in the hypothalamus were similar to those in the striatum. These results suggest that the pharmacological actions of PCP and tolerance development to PCP may be related to the functional changes of serotonergic neuronal activity.     

Effect of cold exposure on brain 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in some vertebrate species

1. The changes in 5-HT and 5-HIAA levels were studied in the brain regions of Gerbillus pyramidum, Streptopelia senegalensis aegyptiaca and Agama stellio following exposure to cold. 2. In general, the 5-HT levels increased in the Gerbillus brain parts and decreased in those of Streptopelia. 3. Cold exposure in the Agama brain regions caused a transient decrease in the 5-HT levels of the cerebral hemispheres, midbrain and pons plus medulla after 6 hr and a general increase after 12, 24 and 48 hr. 4. It is concluded that cold exposure may be associated with increased activity of 5-HT ergic neurons and the rate of turnover of 5-HT to 5-HIAA.     

Contents of serotonin and 5-hydroxyindoleacetic acid in the rabbit brain after long-term administration of lithium oxybutyrate

Serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) levels in rat brain were analysed 24 hours after 7-, 15-, 29- days lithium hydroxybutyrate (LH) injections (10 mg/kg daily). After 7 days the drug reduced 5-HT in hypothalamus and 5-HIAA in the mid brain by 35%. After 15 days LH decreased 5-HT in striatum, hypothalamus by 32 and 17% and 5-HIAA in thalamus, hypothalamus by 28 and 44% respectively. After 29 days LH diminished 5-HT in striatum, hippocampus, amygdala by 24, 29 and 32% and 5-HIAA--in hypothalamus by 42%. The role of adaptative changes and stabilization processes in the central serotoninergic system in mechanism of LH psychotropic effects is discussed.