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A liquid chromatographic assay for 5-hydroxytryptophan, serotonin and 5-hydroxyindoleacetic acid in human body fluids.

We describe the precision and accuracy of a liquid chromatographic method, which uses internal standards, 6-fluoroserotonin and 5-hydroxyindolecarboxylic acid, in quantitating serotonin and 5-hydroxyindoleacetic acid in human cerebrospinal fluid, plasma and urine. In addition, 5-hydroxytryptophan is measured in the cerebrospinal fluid. The limit of sensitivity of this method is 0.1 pmol/injection, the peak height/concentration ratio is linear in the concentration range of 0.1 pmol to 50 mumol/injection, and the coefficient of variation is of the order of 15% at the limit of sensitivity and below 10% at amounts above 0.5 pmol/injection. No endogenous monoamines or their metabolites interfere with the quantitation of the substances of interest in the body fluids studied.

5-Hydroxytryptophan↗

Cerebrospinal fluid adenosine 3',5'-monophosphate, 5-hydroxyindoleacetic acid and homovanillic acid in patients with sleep apnoea syndrome.

Adenosine 3',5'-monophosphate (cyclic AMP), 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) were determined in the cerebrospinal fluid of patients with respiratory disorder and hypersomnia and in control patients. Patients with the sleep apnoea syndrome confirmed polygraphically showed elevated levels of cyclic AMP and 5-HIAA. Cyclic AMP levels were inversely correlated with arterial Po(2), measured under resting conditions. The level of HVA also was raised, but the change was not statistically significant.

Cyclic AMP↗

3-methoxy-4-hydroxyphenylglycol, 5-hydroxyindoleacetic acid, and homovanillic acid in human cerebrospinal fluid. Storage and measurement by reversed-phase high-performance liquid chromatography and coulometric detection using 3-methoxy-4-hydroxyphenyllactic acid as an internal standard.

To simultaneously measure 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxyindoleacetic acid (5HIAA), and homovanillic acid (HVA) in human cerebrospinal fluid (CSF), we used an acetonitrile protein precipitation, reversed-phase high-performance liquid chromatography with coulometric detection, and 3-methoxy-4-hydroxyphenyllactic acid (MHPLA) as an internal standard for all three metabolites. MHPG, 5HIAA, HVA, and MHPLA were stable for one month when stored in CSF at -70 degrees C. Three determinations were made in triplicate for each of seven subjects over a 30-day storage period and the coefficients of variation within subject for these determinations ranged from 0.075 to 0.165 for MHPG, 0.045 to 0.148 for 5HIAA and 0.053 to 0.181 for HVA. Means and standard deviations of CSF concentrations were 10.7 +/- 3.0 ng/ml for MHPG, 22.4 +/- 9.9 ng/ml for 5HIAA, and 39.9 +/- 21.4 ng/ml for HVA. This method provides simple sample preparation, sensitivity, and cost advantages, as well as simultaneous extraction and quantitation of MHPG, 5HIAA, and HVA using an internal standard.

Chromatography, High Pressure Liquid↗

Effects of inhibition of serotonin synthesis on 5-hydroxyindoleacetic acid excretion, in healthy subjects.

The urinary excretion of 5-hydroxyindoleacetic acid (5-HIAA), the main metabolite of serotonin, reflects the content and turnover of gastrointestinal (GI) serotonin. Employing longitudinal measurements of 5-HIAA, the authors investigated in healthy subjects (n = 43) how manipulations of serotonin synthesis affect GI serotonin. Under conditions of serotonin-free diets, the intersubject and intrasubject variability (coefficient of variation) for 5-HIAA excretion averaged 33% and 14%, respectively. Dietary tryptophan restrictions to 50% of minimal daily requirements (which is equivalent to a 10-fold reduction in baseline tryptophan intake) decreased by half the urinary excretion of 5-HIAA, irrespective of the caloric content of diet. Restoration to the regular tryptophan intake produced a rapid normalization of the 5-HIAA excretion. Neutral amino acids are known to compete with the intestinal transport absorption mechanisms of tryptophan. Administration of neutral amino acids (1.8 g, by mouth, three times a day, before each meal) or of carbidopa (50 mg, by mouth, three times a day for 3 days) to a normal tryptophan diet failed to alter significantly the 5-HIAA excretion. Further, neutral amino acids failed to enhance the reduction in 5-HIAA produced by the low-tryptophan diet. The failure of these treatments to reduce 5-HIAA excretion could be due to large capacity transport and decarboxylation systems for tryptophan. Other possibilities are discussed. In summary, dietary tryptophan is essential for the maintenance of GI serotonin. Reductions or increases in dietary tryptophan are the easiest and most effective method to alter GI serotonin.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

CSF 5-hydroxyindoleacetic acid levels in suicidal schizophrenic patients.

Concentrations of 5-hydroxyindoleacetic acid (5-HIAA) in the lumbar CSF were measured in a group of suicidal schizophrenic patients and in a matched group of nonsuicidal schizophrenic patients. The suicidal group had a significantly lower level. This finding is consistent with reports of low levels of CSF 5-HIAA in suicidal patients with other psychiatric diagnoses and suggests that low CSF 5-HIAA may be a generalized marker of aggressive behavior against the self and others.

Adult↗

Serotonin and 5-hydroxyindoleacetic acid in brains of suicide victims. Comparison in chronic schizophrenic patients with suicide as cause of death.

Serotonin (5-HT) and 5-hydroxyindoleacetic acid concentrations were determined in various brain areas of nonschizophrenic suicide victims, chronic schizophrenic patients with or without suicide as the cause of death, and normal control subjects without psychiatric or neurologic disorders. Serotonin concentrations in the basal ganglia were significantly elevated in suicide victims and chronic schizophrenic patients, as were 5-hydroxyindoleacetic acid concentrations in the occipital cortex. These differences were not specific to either patient group and may have been caused by neuroleptic or antidepressant treatment. A decreased 5-HT concentration was found in the hypothalamus of nonschizophrenic suicide victims. Among the chronic schizophrenic patients, there was no significant difference in the hypothalamic 5-HT content between the suicide victims and others, indicating that low 5-HT levels in the hypothalamus are not characteristic of schizophrenic patients who died of suicide.

Adolescent↗

Effects of African trypanosomiasis on brain levels of dopamine, serotonin, 5-hydroxyindoleacetic acid, and homovanillic acid in the rabbit.

Serotonin (5-HT) levels fell by 21% in the mid-brain-thalamus-hypothalamus (MTH) region of the rabbit brain after chronic infection with the protozoan Trypanosoma brucei gambiense. 5-HT did not decrease significantly in the caudate/putamen (CP) or the pons/medulla (PM) region. 5-Hydroxyindoleacetic acid (5-HIAA) levels were unchanged in the MTH and caudate/putamen (CP) but increased by 17% in the pons/medulla (PM) after infection. Dopamine (DA) levels rose by 19% and homovanillic acid (HVA) by 33% in the PM during infection. DA and HVA tended to be lower in the CP of infected rabbits, but the apparent decreases were not statistically significant. DA and HVA levels in the MTH were also unchanged by infection. These neurochemical changes may be involved in the behavioral symptoms that frequently accompany this disease in man and cattle.

Animals↗

Motor activity increases tryptophan, 5-hydroxyindoleacetic acid, and homovanillic acid in ventricular cerebrospinal fluid of the conscious rat.

An investigation was made into the effects of running (1 h at 20 m/min) on central serotonergic and dopaminergic metabolism in trained rats. Methodology involved continuous withdrawal of cerebrospinal fluid (CSF) from the third ventricle of conscious rats and measurements of tryptophan (TRP), 5-hydroxyindoleacetic acid (5-HIAA), and homovanillic acid (HVA) levels during a 2 h post-exercise period. All three compounds were increased during the hour following exercise and returned to their basal values within an hour later. CSF flow rate was stable when metabolite levels were elevated. Brain determinations indicated that CSF metabolite variations only qualitatively paralleled brain changes. Indeed, post-exercise TRP, 5-HIAA, and HVA levels were increased to a greater extent in brain when compared to CSF. It is suggested that increased serotonergic and dopaminergic metabolism, caused by motor activity, may be involved in the behavioral effects of exercise.

Animals↗

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 microliters 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.

Animals↗

Plasma 5-hydroxyindoleacetic acid as an endogenous index of renal plasma flow.

5-Hydroxyindoleacetic acid (5-HIAA), the major serotonin metabolite, was measured in human plasma and urine using liquid chromatography and electrochemical detection. The renal extraction of 5-HIAA was 78%, a value compatible with an almost complete extraction during a single passage of the blood through the kidney. In addition, plasma levels of 5-HIAA measured in patients with a wide range of renal function was inversely correlated (r = 0.85) with the clearance of para-aminohippuric acid (PAH). The results indicate that 5-HIAA determinations can be used as an alternative procedure to the PAH clearance method for the estimation of renal plasma flow in clinical practice.

Adult↗

Excessive mortality in young free-ranging male nonhuman primates with low cerebrospinal fluid 5-hydroxyindoleacetic acid concentrations.

BACKGROUND: The purpose of this study was to assess the impact of central serotonin turnover rate on survival to adulthood among nonhuman primates living in a large, free-ranging colony. METHODS: The rate of mortality was ascertained over a 4-year period after obtaining blood and cisternal cerebrospinal fluid (CSF) samples from 49 free-ranging, 2-year-old prepubertal male rhesus monkeys. Cerebrospinal fluid was assayed for 5-hydroxyindoleacetic acid (5-HIAA), norepinephrine, 3-methoxy-4-hydroxyphenylgycol, and homovanillic acid. Blood plasma was assayed for adrenocorticotropic hormone, cortisol, and testosterone. Following the sampling of body fluids, records of scars and wounds and aggressive encounters were used to rank the subjects from low to high in aggressiveness. Direct observations of aggressive behavior were collected from 27 of the subjects over a 3-month period. RESULTS: Four years later, 6 of the 49 subjects were known to be dead and an additional 5 had been missing for more than 2 years and were presumed dead. The CSF 5-HIAA concentrations were predictive of which subjects died, with 46% of the subjects with low CSF 5-HIAA concentrations dead or presumed dead. None of the subjects from the highest CSF 5-HIAA concentration quartile were dead or missing. Indeed, 91% of the dead subjects came from the 2 lowest quartiles of CSF 5-HIAA concentrations. Direct observations of aggressive behavior showed that dead or missing subjects had initiated escalated aggression, a measure of unrestrained aggression that has a high probability of trauma or injury, at a higher rate than subjects that were known to be alive. The cause of death could be ascertained for 6 of the 11 missing subjects. The 4 subjects that were known to die as a consequence of aggressive encounters came from the lowest quartile of CSF 5-HIAA concentrations and had been rated as more aggressive during their initial capture. Subjects captured more than once possessed lower CSF 5-HIAA concentrations, were rated as more aggressive, and were more likely to suffer early death than those captured only once. CONCLUSION: These findings suggest that low CSF 5-HIAA concentrations quantified early in life is a powerful biological predictor of future excessive aggression, risk taking, and premature death among nonhuman primate males.

Aggression↗

Effect of tryptophan administration on tryptophan, 5-hydroxyindoleacetic acid and indoleacetic acid in human lumbar and cisternal cerebrospinal fluid.

Tryptophan 5-hydroxyindoleacetic acid and indoleacetic acid were measured in cerebrospinal fluid taken during pneumoencephalography from patients, some of whom took a 3 g or 6 g tryptophan load at various times before. Measurements were made on both lumbar and cisternal cerebrospinal fluid and the results showed similarities between indoleamine metabolism in human brain and spinal cord. Our data suggested that (1) the blood-brain barrier active transport system for tryptophan is not far from saturation with tryptophan and the rate-limiting enzyme in 5-hydroxytryptamine (5HT) synthesis, tryptophan hydroxylase, is about half saturated. Therefore, both 3 g and 6 g tryptophan loads produced the same maximum rise in 5HT synthesis of just under 100%, (2) tryptamine differs from 5HT in two respects. It is more sensitive to changes in tryptophan availability than 5HT and the 6 g load increased brain tryptamine metabolism more than the 3 g load; also some of the tryptamine in brain is derived from peripheral sources and diffuses from blood to brain, (3) although the brain tryptamine content is much lower than that of 5HT, its rate of metabolism as indicated by CSF metabolite levels is not. In controls the rate of tryptamine metabolism is 15% of the rate of 5HT metabolism and this can increase to 40% after a 6 g tryptophan load.

Adolescent↗

Diurnal rhythm in extracellular levels of 5-hydroxyindoleacetic acid in the medial prefrontal cortex of freely moving rats: an in vivo microdialysis study.

The cerebral monoaminergic nervous system was monitored for its diurnal changes to find whether these can be used as neurochemical indices for emotional states. Freely moving rats that were not anesthetized were used: by employing an in vivo microdialysis technique, the levels of extracellular monoamine metabolites were measured. Under an alternating 12 h light and dark (LD) cycle and subsequent constant light (LL) condition, the extracellular 5-hydroxyindoleacetic acid (5-HIAA) level was significantly higher in the dark phase than in the light phase in the medial prefrontal cortex (mPFC). However, the 5-HIAA levels in the light and dark phases in the striatum or hippocampus did not significantly differ. The homovanillic acid (HVA) level was significantly higher in the dark phase than in the light phase in the mPFC and striatum. These findings indicate that the extracellular 5-HIAA level in the mPFC has site-specific circadian rhythmicity. Furthermore, considering the relationship among diurnal rhythm, emotional disturbance and the mPFC, the site-specific diurnal rhythmicity of 5-HIAA in the mPFC may be a useful index in neurochemical studies on emotional states, such as seen in affective disorders and psychiatric diseases.

Animals↗

Thiamine deficiency and cerebrospinal fluid 5-hydroxyindoleacetic acid: a preliminary study.

In three out of five patients with low cerebrospinal fluid thiamine concentrations, the 5-hydroxyindoleacetic acid (5HIAA) values also were low. All patients received thiamine replacement therapy; they underwent a second lumbar puncture after 13, 6, 7, 5 and 45 days of treatment. In all patients blood and cerebrospinal fluid thiamine values rose after treatment. In those patients with initially low CSF 5HIAA, thiamine treatment increased 5HIAA markedly.

Adult↗

Increase of the serotonin metabolite, 5-hydroxyindoleacetic acid, in cerebro-spinal fluid and spinal cord of bovines affected with the paraplegic syndrome.

The paraplegic syndrome of bovines is a condition characterized by impairment of locomotion, hypoalgesia and finally death within 72 h. The pathogenesis of the syndrome has not been established. In the present work we determined the levels of monoamines and their metabolites in cerebro-spinal fluid and spinal cord of affected animals in order to investigate the functional state of these neurotransmitters. The content of the main metabolite of serotonin, 5-hydroxyindoleacetic acid, was elevated in the cerebro-spinal fluid and in the gray matter of the spinal cord of paraplegic bovines. Serotonin content in the spinal cord did not differ with respect to control animals, but was decreased in the cerebro-spinal fluid of affected animals. Modifications in the noradrenergic system were also observed, but were less consistent, for which reason further studies are needed. These observations indicate an increase in the turnover rate of serotonin in the paraplegic syndrome. The meaning of the described alterations is unknown at the moment.

Animals↗

Rat tumor response to the vascular-disrupting agent 5,6-dimethylxanthenone-4-acetic acid as measured by dynamic contrast-enhanced magnetic resonance imaging, plasma 5-hydroxyindoleacetic acid levels, and tumor necrosis.

The dose-dependent effects of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) on rat GH3 prolactinomas were investigated in vivo. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was used to assess tumor blood flow/permeability pretreatment and 24 hours posttreatment with 0, 100, 200, or 350 mg/kg DMXAA. DCE-MRI data were analyzed using K(trans) and the integrated area under the gadolinium time curve (IAUGC) as response biomarkers. High-performance liquid chromatography (HPLC) was used to determine the plasma concentration of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) following treatment to provide an index of increased vessel permeability and vascular damage. Finally, tumor necrosis was assessed by grading hematoxylin and eosin-stained sections cut from the same tumors investigated by MRI. Both tumor K(trans) and IAUGC were significantly reduced 24 hours posttreatment with 350 mg/kg DMXAA only, with no evidence of dose response. HPLC demonstrated a significant increase in plasma 5-HIAA 24 hours posttreatment with 200 and 350 mg/kg DMXAA. Histologic analysis revealed some evidence of tumor necrosis following treatment with 100 or 200 mg/kg DMXAA, reaching significance with 350 mg/kg DMXAA. The absence of any reduction in K(trans) or IAUGC following treatment with 200 mg/kg, despite a significant increase in 5-HIAA, raises concerns about the utility of established DCE-MRI biomarkers to assess tumor response to DMXAA.

Angiogenesis Inhibitors↗

Effects of MPP+ on the release of serotonin and 5-hydroxyindoleacetic acid from rat striatum in vivo.

A procedure utilizing brain dialysis was employed to investigate the effects of 1-methyl-4-phenylpyridinium ion (MPP+), the major metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), on the in vivo release of 5-hydroxytryptamine (5-HT) from the rat striatum. MPP+ (10(-5)-10(-2) M) was administered through the microdialysis probe and dialysates were collected and assayed for monoamines and their metabolites by a high performance liquid chromatography coupled with an electrochemical detector (HPLC-EC). In addition to a dopamine (DA) releasing action, MPP+ caused a cumulatively dose-dependent increase in the release of 5-HT while concomitantly producing a decrease in the efflux of 5-hydroxyindoleacetic acid (5-HIAA). This MPP+-induced increase in the 5-HT release, may play an important role in the acute 5-HT syndrome seen in animals after MPTP.

1-Methyl-4-phenylpyridinium↗