Determination of 5-hydroxytryptophan, 5-hydroxytryptamine, and 5-hydroxyindoleacetic acid in 20 rat brain nuclei using liquid chromatography with electrochemical detection

High-performance liquid chromatography with electrochemical detection is utilized for the simultaneous determination of serotonin, its precursor 5-hydroxytryptophan, and its major metabolite 5-hydroxyindoleacetic acid in nervous tissue samples. Tissue preparation required only homogenization in acidic solution and centrifugation prior to application to the chromatograph. Detection limits in the low picogram range were obtained for those indoles separated. This assay was used in combination with a micropunch dissection technique of 20 discrete rat brain nuclei to measure serotonin, its precursor, and major metabolite. The specificity of the assay was checked with pharmacological experiments aimed to increase or decrease serotonin levels. Pargyline, a monoamine oxidase inhibitor, led to a marked increase in serotonin and a decrease of 5-hydroxyindoleacetic acid while p-chlorophenylalanine, by blocking the conversion of tryptophan to 5-hydroxytryptophan, selectively depleted 5-hydroxytryptophan, serotonin, and 5-hydroxyindoleacetic acid.     

Biogenic amines and their metabolites in Trichostrongylus colubriformis, a nematode parasite of ruminants

The following biogenic amines (BA) and BA metabolites were identified via HPLC in homogenates prepared from adults of Trichostrongylus colubriformis (Nematoda) recovered from the intestines of goats: N-acetyldopamine, DOPA, dopamine, epinephrine, epinine, 5-hydroxyindoleacetic acid, 4-hydroxy-3-methoxy-phenylglycol, 3-(p-hydroxyphenyl) proprionic acid, metanephrine, O-methyl-DOPA, 3-methoxytyramine, norepinephrine, normetanephrine, octopamine, p-hydroxymandelic acid, serotonin, synephrine, tyramine and vanillylmandelic acid. The mean concentrations of these compounds in groups of worms collected from different goats did not differ significantly with sex, but between the groups variance was high with probable components of both host and nematode origin.     

Measurement of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in cultured rat mesencephalic neurons by high-performance liquid chromatography with electrochemical detection

An HPLC method with electrochemical detection for the simultaneous measurement of serotonin (5-hydroxytryptamine) and 5-hydroxyindoleacetic acid in primary mesencephalic cell culture is described. The serotonin and 5-hydroxyindoleacetic acid cell content was measured on different days of growth in vitro; after twelve days in culture the amounts of serotonin and 5-hydroxyindoleacetic acid detected were 916.0 ± 70.2 and 215.8 ± 15.5 pg per well, respectively. The heterogeneity of neurons in our cultures and their capacity to take up serotonin were assessed by measuring the amounts of exogenous serotonin taken up in the presence of different monoamine uptake inhibitors. This method, sensitive and reliable, can represent a valid alternative to the use of labelled compounds.     

Increased central serotonergic activity associated with nocturnal anorexia induced by Walker 256 carcinoma

The role of brain serotonin levels in Walker 256 tumor induced anorexia was investigated. Total and free plasma tryptophan, regional brain serotonin and 5-hydroxyindoleacetic acid were determined at night, and their relationship to nocturnal anorexia assessed by linear regression analysis. No significant difference in tryptophan, serotonin, or 5-hydroxyindoleacetic acid levels was detected between pair fed and tumor bearing rats exhibiting a 20% reduction of nighttime food intake. Tumor bearing rats with a 40% reduction in food intake had higher nighttime plasma free tryptophan and regional 5-hydroxyindoleacetic acid levels than their pair fed malnourished controls. These results indicate that increased plasma free tryptophan and elevated serotonin metabolism may not be the initial dysfunction responsible for nocturnal anorexia. However, it may contribute to the decreasing nocturnal food intake in severely anorexic tumor rats.     

Microbore liquid chromatography with dual electrochemical detection for the determination of serotonin and 5-hydroxyindoleacetic acid in rat brain dialysates

A microbore liquid chromatographic assay with dual electrochemical detection is described for the determination of serotonin and its metabolite 5-hydroxyindoleacetic acid in rat brain dialysates. The concentration of serotonin in these samples is usually in the low nanomolar range (fmol per 20 μl range). To optimize separation and detection, several adaptations were made to the system with respect to the injection valve, flow-rate of the pump, connections between injector, column and detector, and cell volume of the detector. These aspects are discussed, as well as the procedure developed for optimal peak identification of serotonin and correct estimation of 5-hydroxyindoleacetic acid. The assay allows the measurement of basal serotonin release without the use of a re-uptake inhibitor added to the perfusion fluid.     

Changes in serotonin metabolism during food satiation in the mink and its effect on predatory aggression

Effect of single and protracted alimentary satiation on predatory aggression and content of serotonin and its metabolite 5-hydroxyindoleacetic acid in the amygdalar complex and hypothalamus was studied in mink--a representative of predators. A single alimentary satiation was not accompanied by any marked changes in serotonin metabolism and predatory behaviour. A long-term alimentary satiation significantly heightened the content of the 5-hydroxyindoleacetic acid in the lateral hypothalamus and amygdala without any changes in serotonin level, testifying to a high synthesis of serotonin with its simultaneous intensive destruction. Long-term satiation also greatly increased the latencies of aggression and killing the victim. It is suggested that serotonin is one of endogenous factors controlling predatory behaviour in predators, and this control is realized in interrelation with feeding behaviour.     

Attenuation and recovery of evoked overflow of striatal serotonin in rats treated with neurotoxic doses of methamphetamine

Repeated administration of methamphetamine to animals can lead to long-lasting decreases in striatal monoamine content. In the present study, the effects of neurotoxic doses of methamphetamine on basal and evoked overflow of striatal serotonin and of its primary metabolite 5-hydroxyindoleacetic acid were examined in awake rats using in vivo microdialysis. Male Fischer-344 rats were administered methamphetamine (5 mg/kg, s.c.) or saline four times in 1 day at 2-h intervals. Microdialysis studies were carried out 1 week, 1 month, and 6 months later. At 1 week posttreatment there were significant decreases in potassium- and amphetamine-evoked overflow of serotonin in the striatum of the methamphetamine-treated animals. Basal extracellular levels of 5-hydroxyindoleacetic acid but not of serotonin were also decreased. Evoked overflow of serotonin recovered by 1 month, and extracellular levels of 5-hydroxyindoleacetic acid had recovered by 6 months. Tissue levels of serotonin and 5-hydroxyindoleacetic acid were decreased at 1 week posttreatment but back to control levels by 1 month after treatment. These results indicate that presynaptic serotonergic functioning is attenuated in the striatum of rats treated 1 week earlier with neurotoxic doses of methamphetamine. However, in the model used, the changes are transient, and recovery can occur within 1-6 months posttreatment.     

Simultaneous Determination of Serotonin, 5-Hydroxyindoleacetic Acid, 3,4-Dihydroxyphenylacetic Acid and Homovanillic Acid by High Performance Liquid Chromatography with Electrochemical Detection

A rapid and highly sensitive procedure for simultaneous determination of serotonin, 5-hydroxyindoleacetic acid, 3,4-dihydroxyphenylacetic acid and homovanillic acid is described. After precipitation of proteins with perchloric acid the samples are applied directly to a high performance liquid chromatograph, with electrochemical detection. As little as 20 pg of serotonin, 5-hydroxyindoleacetic acid, and 3,4-dihydroxyphenylacetic acid and 200 pg of homovanillic acid can be detected. One chromatographic run requires less than 10 min.     

Presynaptic Serotonergic Markers in Community-Acquired Cases of Alzheimer's Disease: Correlations with Depression and Neuroleptic Medication

Abstract: Presynaptic serotonergic markers, serotonin uptake sites, and concentrations of serotonin and 5-hydroxyindoleacetic acid were studied in the frontal and temporal cortex of 20 community-acquired cases of Alzheimer's disease and 16 controls matched for age, sex, postmortem delay, and storage. Clinical assessments, including behavioural symptoms, of the Alzheimer patients were made at 4-month intervals during life. There was a significant reduction in the number of serotonin uptake sites in Alzheimer cases in temporal but not frontal cortex. There was no significant alteration in the concentrations of serotonin or 5-hydroxyindoleacetic acid in either region. Alzheimer patients who had persistent depressive symptoms during life had significantly fewer serotonin uptake sites in both cortical areas compared with Alzheimer patients without these symptoms. In addition, Alzheimer patients who were receiving chronic neuroleptic medication had significantly lower concentrations of serotonin in frontal cortex and 5-hydroxyindoleacetic acid in temporal cortex than those patients not receiving such treatment. These data suggest previous studies that reported uniform serotonergic dysfunction may have been subject to unintentional selection of behaviourally disturbed Alzheimer cases or those receiving chronic neuroleptic medication. This study also provides a basis for the treatment of behaviourally disturbed Alzheimer patients with serotonomimetics.     

Neurochemical effects of cyclopiazonic acid in chickens

Chickens dosed (per os) with cyclopiazonic acid (CPA) at 0.5, 5.0, and 10 mg/kg body weight showed significant (P less than or equal to 0.05) increases in brain dopamine and serotonin concentrations 96 hr after dosing. The increases coincide with significant decreases in homovanillic acid and subtle increases of dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid concentrations. The elevated dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid concentrations may be related to the elevated concentrations of dopamine and serotonin, respectively. The observed changes in neurotransmitter/metabolite concentrations 96 hr after dosing parallel elimination of CPA from the birds skeletal muscles; however, they do not correlate with the significant weight losses in these birds at 48 and 96 hr after dosing. The brain weights of the treated birds were statistically insignificant from their respective controls, although increases in brain weight-body weight ratio within treatments and with time correlated with CPA toxicity. No significant changes were observed in dopamine, dihydroxyphenylacetic acid, homovanillic acid, serotonin, and 5-hydroxyindoleacetic acid concentrations among the treatments at 3, 24, and/or 48 hr after dosing.     

Effect of dietary tryptophan on plasma and brain tryptophan, brain serotonin, and brain 5-hydroxyindoleacetic acid in rainbow trout

In order to determine the effect of dietary tryptophan level on plasma and brain tryptophan, brain serotonin, and brain 5-hydroxyindoleacetic acid levels, juvenile rainbow trout (Salmo gairdneri) were raised for 16 weeks on semipurified diets containing 0.06%, 0.16%, 0.21%, 0.26%, 0.39%, or 0.59% tryptophan. After 14 weeks, feed intake was depressed in fish fed the diets containing 0.06% or 0.16% tryptophan. No further differences in feed intake were noted between the remaining treatments. In addition, body weight was lower in fish fed diets containing 0.06%, 0.16%, or 0.21% tryptophan compared with fish fed higher levels. After 16 weeks of feeding the test diets, plasma tryptophan levels were found to be directly related to dietary tryptophan levels. Similarly, increased dietary levels of tryptophan resulted in increased brain levels of tryptophan, serotonin, and 5-hydroxyindoleacetic acid. These results demonstrate that in rainbow trout, as in mammals, altered dietary levels of tryptophan result in alterations in plasma and brain tryptophan, brain serotonin, and brain 5-hydroxyindoleacetic acid.     

Presynaptic Serotonergic Dysfunction in Patients with Alzheimer''s Disease

Indices of presynaptic serotonergic nerve endings were assayed in neocortical biopsy samples from patients with histologically verified Alzheimer's disease. The concentrations of 5-hydroxytryptamine (serotonin) and 5-hydroxyindoleacetic acid, serotonin uptake, and K+-stimulated release of endogenous serotonin were all found to be reduced below control values. Changes occurred in samples from both the frontal and temporal lobes, but they were most severe (at least a 55% reduction) in the temporal lobe. This is indicative of substantial serotonergic denervation. Values for serotonergic markers in Alzheimer's disease samples did not show correlations with rating of the severity of dementia, indices of cholinergic innervation, or senile plaque and cortical pyramidal neurone loss. However, neurofibrillary tangle count and an index of glucose oxidation (both probably reflecting pyramidal cells) correlated with the concentration of 5-hydroxyindoleacetic acid.     

THE ROLE OF BRAIN SEROTONIN IN DDT-INDUCED HYPERPYREXTA

Abstract— Administration of a single dose of p,p '-DDT to rats resulted in a marked increase in the concentration of 5-hydroxyindoleacetic acid in the brain stem. No significant change was observed in the amount of 5-hydroxytryptamine (serotonin) following treatment with this insecticide. However, when p,p' -DDT was given to pargyline-treated rats, a significantly greater increase in serotonin was observed when compared to that with the monoamine oxidase inhibitor alone. The concomitant administration of p,p' -DDT and dl - p -chlorophenylalanine not only blocked the observed rise in 5-hydroxyindoleacetic acid but also suppressed almost completely the neurotoxic symptoms (hyperpyrexia, tremors and convulsions) seen with the pesticide. In contrast, DL-6-fluorotryptophan (a more specific inhibitor of serotonin synthesis) prevented the DDT-induced rise in body temperature as well as the increase in hydroxyindoleacetic acid, but did not affect the occurrence of tremor and convulsions. Our data suggest that an increase in the turnover rate of brain serotonin may underlie the hyperpyrexia induced by p,p '-DDT.     

New antioxidant mixture for long term stability of serotonin, dopamine and their metabolites in automated microbore liquid chromatography with dual electrochemical detection

An automated microbore liquid chromatographic assay with dual electrochemical detection is described for the determination of serotonin, dopamine and their metabolites, 5-hydroxyindoleacetic acid, 3,4-dihydroxyphenylacetic acid and homovanillic acid. Due to the chemical instability of the compounds, the addition of an antioxidant is required for automated analysis over a long period of time (e.g., 20 h). Therefore, the time stability of these substances was tested with different antioxidants. The stability for serotonin and 5-hydroxyindoleacetic acid was poor in acidic medium containing Na₂EDTA but could greatly be improved by the addition of

Full-size image

-cysteine and ascorbic acid. Using this assay, the neurotransmitters and their metabolites could easily be determined in microdialysates obtained from different rat brain areas.     

Variations of brain histamine levels in germ-free and nephrectomized rats

The influence of nephrectomy on brain and peripheral tissue histamine and on brain norepinephrine, dopamine, serotonin, and 5-hydroxyindoleacetic acid was studied in germ-free and conventionally housed rats. The conventional controls had higher levels of histamine in the hypothalamus than the germ-free control animals, but no differences existed for histamine in whole brain minus the hypothalamus or in peripheral tissues. Nephrectomy increased brain histamine and 5-hydroxyindoleacetic acid levels in both germ-free and conventional rats, but had no effect on norepinephrine, dopamine or serotonin. In contrast, the histamine level in the heart of the nephrectomized germ-free animals was lower than that for germ-free controls. There were no changes in the heart or liver histamine levels of the conventional nephrectomized rats.     

Development of Monoaminergic Neurotransmitters in Fetal and Postnatal Rat Brain: Analysis by HPLC with Electrochemical Detection

The monoamines dopamine, norepinephrine, epinephrine, and serotonin and their major metabolites 3,4-dihydroxyphenylacetic acid, homovanillic acid, 3-methoxy-4-hydroxyphenylethylene glycol, and 5-hydroxyindoleacetic acid were measured in the CNS of the rat during development from fetal day 18 to young adult. The catecholamines, serotonin, and their major metabolites remained low during fetal life. Concentrations measured in total brain started to increase around birth till the end of the fourth week of life after which steady-state levels were measured. Our results suggest that although monoamine systems are already morphologically well developed during late gestational life, they probably become a significant functional system only around birth and early postnatal life.     

Pharmacological Evidence for the Existence of Multiple Functional Pools of Brain Serotonin: Analysis of Brain Perfusate From Conscious Rats

Abstract: The serotonin reuptake inhibitor fluoxetine significantly reduced levels of endogenous 5-hydroxyindoleacetic acid (5-HIAA) in brain perfusate of rats implanted with push-pull cannulas. This occurred in conjunction with its suppressant effect upon fixed-ratio operant behavior. Behavior suppressed with the serotonin agonist lysergic acid diethylamide (LSD) occurred in conjunction with a reduction of 5-HIAA only after 5-HIAA was elevated, shortly before, by 5 mg/kg of the serotonin precursor 5-hydroxytryptophan. Our data demonstrate the likely existence of multiple functional pools of serotonin in brain and support the notion that LSD preferentially affects a newly synthesized pool of this transmitter.     

The depletion of tryptophan and serotonin in the brain of developing hyperphenylalaninemic rats is abolished by the additional administration of lysine

In suckling hyperphenylalaninemic (hyper-Phe) rats, all essential amino acids including tryptophan are depleted in the blood. The inadequate supply of Trp to the developing brain leads to a decline of Trp, of serotonin, and of 5-hydroxyindoleacetic acid. The exhaustion of amino acids in both blood and brain can be restored by administration of Lys. Even though Phe is still elevated in blood and brain, Trp, serotonin and 5-hydroxyindoleacetic acid, are no longer depleted in the brain. This observation contradicts the idea that the serotonin deficit in the developing hyper-Phe brain is caused by competitive uptake inhibition of tryptophan or by the interference of Phe metabolites with the formation of serotonin. Increased accumulation of all large neutral amino acids in peripheral tissues and an impeded intestinal amino acid transport in suckling hyper-Phe rats appear to be responsible for the deficient supply of other amino acids, including Trp, to the developing brain. The availability of Lys for developing extraintestinal tissues seems to be involved in the regulation of intestinal amino acid transport.     

Differential effects of short and long term lithium on tryptophan uptake and serotonergic function in cat brain

The effcts of short and long term lithium treatment on tryptophan uptake and on tissue levels of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) were studied in twelve brain regions of the cat. Tryptophan uptake and 5-HIAA were significantly correlated in control cats. Short term treatment caused parallel increases or decreases in tryptophan uptake and 5-HIAA. Long term treatment consistently increased tryptophan uptake without corresponding changes in 5-HIAA. Relatively low cumulative doses of lithium may reduce the degree to which tryptophan uptake is a limiting factor in the the regulation of serotonin synthesis.     

Day/night differences in pineal indoles in the adult pigeon (Columba livia)

Day/night differences in concentrations of 5-hydroxy and 5-methoxy indole metabolites in the pineal gland of the pigeon are described. A simultaneous determination of 5-hydroxytryptamine (serotonin), 5-hydroxyindoleacetic acid, 5-hydroxytryptophol, N-acetyl-5-hydroxytryptamine (N-acetyl serotonin), 5-methoxyindoleacetic acid, 5-methoxytryptophol, tryptophan, indoleacetic acid and melatonin was accomplished using a recently developed procedure employing high-performance liquid chromatography with electrochemical detection. As in mammalian species, an inverse relationship was observed between N-acetylated indoles and serotonin and its acid metabolites. Melatonin and N-acetyl serotonin were increased approximately three-fold at night to concentrations of 0.730 and 1.79 ng/pineal respectively. Daytime serotonin values were 44.9 +/- 13.0 ng/pineal and decreased to 12.3 +/- 6.5 ng/pineal during the dark phase.