Determination of dansylated monoamine and amino acid neurotransmitters and their metabolites in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry

The determination of neurotransmitters (NTs) and their metabolites facilitates better understanding of complex neurobiology in the central nervous system disorders and has expanding uses in many other fields. We present a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) method for the quantification of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), norepinephrine (NE), vanillymandelic acid (VMA), 3-methoxy-4-hydroxy phenylglycol (MHPG), 5-hydroxytryptamine (5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA), glutamate (Glu), and γ-aminobutyric acid (GABA). The NTs and their metabolites were dansylated and analyzed by an LC gradient on a C18 column on-line with a tandem mass spectrometer. This method exhibited excellent linearity for all of the analytes with regression coefficients higher than 0.99. The lower limit of quantification (LLOQ) values for DA, DOPAC, HVA, NE, VMA, MHPG, 5-HT, 5-HIAA, Glu, and GABA were 0.57, 0.37, 0.35, 0.40, 0.35, 0.91, 0.27, 0.43, 0.65, and 1.62 pmol/ml, respectively. The precision results were expressed as coefficients of variation (CVs), ranging from 1.5% to 13.6% for intraassay and from 2.9% to 13.7% for the interassay. This novel LC-ESI/MS/MS approach is precise, highly sensitive, specific, and sufficiently simple. It can provide an alternative method for the quantification of the NTs and their metabolites in human plasma.     

Simultaneous measurement of monoamines, their metabolites and 2,3- and 2,5-dihydroxybenzoates by high-performance liquid chromatography with electrochemical detection. Application to rat brain dialysates

A reversed-phase chromatographic method with electrochemical detection was developed for the simultaneous determination of 2,3- and 2,5-dihydroxybenzoates, indicators of in vivo hydroxyl free radical formation, monoamines (NE, DA, 5-HT) and their metabolites (MHPG, DOPAC, HVA, 3MT, 5-HIAA). Linearity was observed from 10 pg to 10 ng injected. Reproducibility is correct (C.V. about 9%) except for 3MT and 5-HT. The limit of detection for almost all products was about 20 pg injected on the column. An application of this method in the study of the neurotoxicity of high pressure oxygen in rat is described. The limit of quantification for all compounds was 5 ng/ml except for HVA (10 ng/ml). Some basal levels DA, 5-HT, 5-HIAA, HVA, DOPAC, 3MT, 2,5-DHBA and 2,3-DHBA in microdialysates coming from striatum of normoxic restrained rats are given.     

Ventricular Cerebrospinal Fluid Monoamine Transmitter and Metabolite Concentrations Reflect Human Brain Neurochemistry in Autopsy Cases

Concentrations of dopamine (DA), its metabolites 3-methoxytyramine and homovanillic acid (HVA), noradrenaline (NA), its metabolites normetanephrine (NM) and 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxytryptamine (5-HT, serotonin), and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were measured in 14 brain regions and in CSF from the third ventricle of 27 human autopsy cases. In addition, in six cases, lumbar CSF was obtained. Monoamine concentrations were determined by reversed-phase liquid chromatography with electrochemical detection. Ventricular/lumbar CSF ratios indicated persistence of rostrocaudal gradients for HVA and 5-HIAA post mortem. Ventricular CSF concentrations of DA and HVA correlated positively with striatal DA and HVA. CSF NA correlated positively with NA in hypothalamus, and CSF MHPG with levels of MHPG in hypothalamus, temporal cortex, and pons, whereas CSF NM concentration showed positive correlations with NM in striatum, pons, cingulate cortex, and olfactory tubercle. CSF 5-HT concentrations correlated positively with 5-HT in caudate nucleus, whereas the concentration of CSF 5-HIAA correlated to 5-HIAA levels in thalamus, hypothalamus, and the cortical areas. These data suggest a specific topographic origin for monoamine neurotransmitters and their metabolites in human ventricular CSF and support the contention that CSF measurements are useful indices of central monoaminergic activity in man.     

Effects of K-stimulation and precursor loading on the in vivo release of dopamine, serotonin and their metabolites in the nucleus accumbens of the rat

The efflux of endogenous 3,4-dihydroxyphenylethylamine (DA) 5-hydroxytryptamine (5-HT), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in the nucleus accumbens of the anesthetized rat was studied using a push-pull cannula. Local perfusion for 10 minutes with 35 mM K⁺ significantly (P<0.01) increased the release of DA and 5-HT, but not their metabolites, from their respective control levels of 0.95 and 0.04 pmol/15 min to 2.5 and 0.23 pmol/15 min. Exposure to 35 mM K⁺ a second and third time resulted in a decrement in the amount of stimulated release for both DA and 5-HT. This decrease was prevented by local perfusion for 10 minutes with 50 uM L-tyrosine and -tryptophan starting 30 minutes before each episode of depolarization. The baseline amounts of DOPAC, HVA and 5-HIAA observed in the perfusates were several fold higher than the basal levels found for 5-HT and Da. In the absence of precursors, the efflux of DOPAC, HVA and 5-HIAA decreased approximately 60, 40 and 25%, respectively, from the first to the last baseline fraction collected. Addition of precursors prevented the decrease for DOPAC and 5-HIAA but not for HVA. The data indicated that (a) the release of DA and 5-HT, along with their metabolites, could be simultaneously measured with the present procedure, and (b) when using the push-pull cannula, local perfusion with precursors may be necessary following periods of sustained and/or repeated stimulation in order to replenish the monoamine transmitter pools.     

Neuropeptide Y perfused in the preoptic area of rats shifts extracellular efflux of dopamine,norepinephrine and serotonin during hypothermia and feeding

This study examined the localized action of neuropeptide Y (NPY) on monoamine transmitter activity in the hypothalamus of the unrestrained rat as this peptide induced hypothermia, spontaneous feeding or both responses simultaneously. A guide tube was implanted in the anterior hypothalamic pre-optic area (AH/POA) of Sprague-Dawley rats. Then either control CSF vehicle or NPY in a dose of either 100 ng/μl or 250 ng/μl was perfused by push-pull cannulae in this structure in the fully sated, normothermic rat. Successive perfusions were carried out at a rate of 20 μl/min for 6.0 min with an interval of 6.0 min elapsing between each. Samples of perfusate were assayed by HPLC for their levels of dopamine (DA), norepinephrine (NE), serotonin (5-HT) and their respective metabolites. Whereas control CSF was without effect on body temperature (T_b) or feeding, repeated perfusions of NPY over 3.0 hr caused dose—dependent eating from 4 to 39 g of food, hypothermia of 0.9 to 2.3°C or both responses concurrently. As the rats consumed 11–39 g of food, the efflux of NE, MHPG, DOPAC and 5-HT was enhanced significantly, whereas during the fall in T_b the efflux of NE, DOPAC and 5-HIAA from the AH/POA increased. When the T_b of the rat declined simultaneously with eating behavior, the levels in perfusate of DOPAC and HVA increased significantly while MHPG declined. During perfusion of the AH/POA with NPY the turnover of NE declined while DA and 5-HT turnover increased during hypothermia alone or when accompanied by feeding. These results demonstrate that the sustained elevation in NPY within the AH/POA causes a selective alteration in the activity of the neurotransmitters implicated in thermoregulation, satiety and hunger. These findings suggest that both DA and NE comprise intermediary factors facilitating the action of NPY on neurons involved in thermoregulatory and ingestive processes. The local activity of NPY on hypothalamic neurons apparently shifts the functional balance of serotonergic and catecholaminergic neurons now thought to play a primary role in the control of energy metabolism and caloric intake.     

Effects of acute 17alpha-methyltestosterone,acute 17beta-estradiol,and chronic 17alpha-methyltestosterone on dopamine,norepinephrine and serotonin levels in the pituitary,hypothalamus and telencephalon of rainbow trout (Oncorhynchus mykiss)

This study investigated: (a) the effects of acute 17alpha-methyltestosterone (MT) or 17beta-estradiol (E(2)) administration on norepinephrine (NE), dopamine (DA), serotonin (5-HT), 3,4, dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA) contents in the hypothalamus, telencephalon and pituitary of previtellogenic female rainbow trout Oncorhynchus mykiss, and (b) the effects of chronic MT administration on the levels of these neurotransmitters in these brain regions in immature male rainbow trout. The acute administration of MT induced a significant decrease in pituitary levels of DOPAC as well as in the DOPAC/DA ratio. On the other hand, the acute administration of E(2) induced an increase in pituitary 5-HT levels as well as a decrease in the 5-HIAA/5-HT ratio. In a second experiment, 20 mg MT per kilogram body weight was implanted for 10, 20 or 40 days into sexually immature male rainbow trout. Implanted rainbow trout showed increased testosterone and decreased E(2) levels. In the pituitary, MT induced long-term decreases in NE, DA, DOPAC and 5-HT levels, as well as in the DOPAC/DA ratio. Hypothalamic and telencephalic DA, NE and 5-HT levels were not affected by MT implantation. However, 5-HIAA levels and the 5-HIAA/5-HT ratio were reduced by MT implantation in both brain regions. These results show that chronic treatment with MT exerts both long-term and region-specific effects on NE, DA, and 5-HT contents and metabolism, and thus that this androgen could inhibit pituitary catecholamine and 5-HT synthesis. A possible role for testosterone in the control of pituitary dopaminergic activity and gonadotropin II release is also discussed.     

Increased Cerebrospinal Fluid Dopamine and 3,4-Dihydroxyphenylacetic Acid Levels in Huntington''s Disease: Evidence for an Overactive Dopaminergic Brain Transmission

Levels of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), noradrenaline (NA), 3-methoxy-4-hydroxyphenylglycol (MHPG), and 5-hydroxyindoleacetic acid (5-HIAA) in the CSF of patients with Huntington's disease (HD) were measured by HPLC. CSF DA, DOPAC, and MHPG levels were found to be increased in HD patients. Levels of HVA, 5-HIAA, and NA in the CSF of HD patients did not differ from those of controls. Changes in CSF DA and DOPAC levels were consistent with previous findings of increased DA tissue content in some brain areas of patients with HD. These results suggest that CSF DOPAC levels could be a more reliable index of overactive dopaminergic brain systems in HD than CSF HVA levels.     

Nicotine-Induced Changes in Neurotransmitter Levels in Brain Areas Associated with Cognitive Function

Nicotine, one of the most widespread drugs of abuse, has long been shown to impact areas of the brain involved in addiction and reward. Recent research, however, has begun to explore the positive effects that nicotine may have on learning and memory. The mechanisms by which nicotine interacts with areas of cognitive function are relatively unknown. Therefore, this paper is part of an ongoing study to evaluate regional effects of nicotine enhancement of cognitive function. Nicotine-induced changes in the levels of three neurotransmitters, dopamine (DA), serotonin (5-HT), norepinepherine (NE), their metabolites, homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA), and their precursor, L-DOPA, were evaluated in the ventral and dorsal hippocampus (VH and DH), prefrontal and medial temporal cortex (PFC and MTC), and the ventral tegmental area (VTA) using in vivo microdialysis in awake, freely moving, male Sprague-Dawley rats. The animals were treated with acute nicotine (0.5 mg/kg, s.c.) halfway through the 300-min experimental period. The reuptake blockers, desipramine (100 microM) and fluoxetine (30 microM), were given to increase the levels of NE and 5-HT so that they could be detected. Overall, a nicotine-induced DA increase was found in some areas, and this increase was potentiated by desipramine and fluoxetine. The two DA metabolites, HVA and DOPAC, increased in all the areas throughout the experiments, both with and without the inhibitors, indicating a rapid metabolism of the released DA. The increase in these metabolites was greater than the increase in DA. 5-HT was increased in the DH, MTC, and VTA in the presence of fluoxetine; its metabolite, 5-HIAA, was increased in the presence and absence of fluoxetine. Except in the VTA, NE levels increased to a similar extent with desipramine and fluoxetine. Overall, nicotine appeared to increase the release and turnover of these three neurotransmitters, which was indicated by significant increases in their metabolites. Furthermore, DA, and especially HVA and DOPAC, increased for the 150 min following nicotine administration; 5-HT and NE changes were shorter in duration. As gas chromatography experiments showed that nicotine levels in the brain decreased by 75% after 150 min, this may indicate that DA is more susceptible to lower levels of nicotine than 5-HT or NE. In conclusion, acute nicotine administration caused alterations in the levels of DA, 5-HT, and NE, and in the metabolism of DA and 5-HT, in brain areas that are involved in cognitive processes.     

REGIONAL DISTRIBUTION OF MONOAMINES AND THEIR METABOLITES IN THE HUMAN BRAIN

Abstract— Noradrenaline (NA), dopamine (DA). 5-hydroxytryptamine (5-HT), 4-hydroxy, 3-methoxy-phenylethylene glycol (MHPG), homovanillic acid (HVA), 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindolylacetic acid (5-HIAA) were measured in twenty areas of post-mortem brain from ten psychiatrically and neurologically normal patients. There was a marked difference, which did not appear to be related to sex, medication, cause of death or time between death and dissection, in amine and metabolite concentrations between brains. In the cortex, 5-HT, MHPG, HVA. DOPAC and S-HIAA were approximately even in their distribution; NA and DA could not be detected. In sub-cortical areas there were clear differences in the distribution of the three amines accompanied by less marked differences in the distribution of their respective metabolites.     

INHIBITION BY APOMORPHINE OF DOPAMINE DEAMINATION IN THE RAT BRAIN

Abstract— Apomorphine (A) inhibited dopamine deamination by rat brain mitochondria, but did not influence catechol- O -methyltransferase (COMT) activity by brain homogenates. The administration of apomorphine (10mg/kg i.p.) to normal rats increased brain dopamine (DA) by 34 per cent and decreased homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) by 60 per cent. In rats treated with reserpine 15 min prior to A, the latter prevented the rise of cerebral HVA and DOPAC and the depletion of DA produced by the former. Finally, A decreased the L-DOPA-induced accumulation of HVA and DOPAC in the rat basal ganglia. These results indicate that A inhibits DA deamination by monoamine oxidase.
This inhibition seems to be specific since apomorphine did not influence 5-HIAA levels in normal rats and prevented neither central 5-HT depletion nor 5-HIAA rise induced by reserpine.     

Asymmetric metabolism of hypothalamic catecholamines alternates with side of ovulation in a lizard (Anolis carolinensis).

We determined levels of monoamines and their metabolites in 2 hypothalami dissected from the right and left hemibrains of 15 females during the right-left alternating ovulatory cycle of Anolis carolinensis. Tissue contents of the following were measured using HPLC and electrochemical (coulometric) detection: dopamine (DA) and its metabolite 2,4-dihydroxyphenylacetic acid (DOPAC), norepinephrine (NE) and its metabolites 3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxyphenylglycol (DHPG), and serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA). An asymmetry ratio (AR) was determined by subtracting hypothalamic content (pM/mg) on the larger ovary (LO) side from that on the smaller ovary (SO) side, divided by the sum of the 2 sides (AR = SO - LO/SO+LO). The Ar of MHPG and DHPG both decreased as the largest follicle in the LO grew during the cycle, from greater than 0 (content higher on the SO side) at the beginning of the cycle to less than 0 (content higher on the LO side). The average content of MHPG in the 2 sides significantly increased during the cycle. There were no significant asymmetric changes in hypothalamic DA or DOPAC. The average content of DA increased during the cycle, whereas the content of DOPAC, as well as DOPAC/DA, did not change. The average content of 5-HT increased, and the average metabolite ratio of 5-HIAA/5-HT decreased during the cycle without significant asymmetries. The metabolite ratios of NE and DA, but not 5-HT, were asymmetric on the same side in a given female.(ABSTRACT TRUNCATED AT 250 WORDS)     

Norepinephrine,dopamine, and 5-HT release from perfused hypothalamus of the rat during feeding induced by neuropeptide Y

In the unrestrained rat, the hyperphagic-like ingestion of food evoked by the sustained elevation of neuropeptide-Y (NPY) in the hypothalamus was correlated with the release and turnover of monoaminergic transmitters in this structure. A single guide tube was implanted stereotaxically in the perifornical region of the hypothalamus for localized push-pull perfusion of an artificial CSF vehicle or NPY_1–36 in a concentration of 10, 50, or 100 ng/1.0 l. After the rat was fully satiated, a site reactive to NPY was perfused repeatedly at a rate of 20 l/min for 6.0 min with an interval of 6.0–12 min elapsing between each perfusion. Samples of perfusate were analyzed by HPLC with coulometric detection for DA, HVA, DOPAC, NE, MHPG, 5-HT, and 5-HIAA. Although control perfusions were without effect on feeding or monoamine activity, NPY evoked mean cumulative intakes of food of 14±2.4, 25.6±3.0 and 26.5±3.2 g in response to 10, 50, or 100 ng/l concentrations of NPY, respectively, over the 4.0–5.0 hr test interval. HPLC analyses showed that during feeding the release of both NE and DA was enhanced significantly. The turnover of both catecholamines likewise increased significantly as reflected by the elevated levels of MHPG, DOPAC and HVA. However, neither the basal efflux of 5-HT nor its turnover, as reflected by the output of 5-HIAA, was affected during feeding induced by NPY perfused in the hypothalamus. These results suggest that a sustained elevation of NPY in the hypothalamus causes a perturbation in the basal activity of NE and DA which are both implicated in the neuronal mechanism regulating normal eating behavior. Thus, these catecholamine neurotransmitters are envisaged to comprise an intermediary step in the functional role played by NPY in the hypothalamus in integrating the control of energy metabolism and caloric intake.     

Altered dopamine turnover in murine hypothalamus after low-dose continuous oral administration of aluminum.

Aluminum, a known neurotoxic substance, has been suggested as a possible contributing factor in the pathogenesis of Alzheimer's disease. Ground-water pollution by aluminum has been recently reported. In the current study groups of 5 male BALB/c mice were administered aluminum ammonium sulfate in drinking water ad libitum at 0, 5, 25, and 125 mg/L aluminum for 4 weeks. At the termination of aluminum exposure, their brains were removed and dissected into cerebrum, cerebellum, medulla oblongata, midbrain, corpus striatum, and hypothalamus. The concentration of norepinephrine (NE), dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA), were determined in each brain area. DA, DOPAC, and HVA levels were lower in the hypothalamus of aluminum-treated mice, most notably in the low-dose group, as compared with control. No marked alterations in NE, 5-HT, and 5-HIAA levels were detected in any brain region. Changes in the concentration of DA and its metabolites measured in the hypothalamus suggest an inhibition of DA synthesis by aluminum.     

Neurotensin perfused in hypothalamus of sated or fasted rat: HPLC analysis of release of DA, NE and 5-HT and their metabolites

This investigation was undertaken in the unrestrained rat to determine the localized effect of neurotensin (NT) on the profile of release and turnover of norepinephrine (NE), dopamine (DA) and serotonin (5-HT) within the hypothalamus. Following stereotaxic implantation of a permanent guide tube, artificial CSF was perfused in the hypothalamus of the freely moving animal by means of push-pull cannulae at a rate of 20 μl/min and for an interval of 5.0 min. After three 5.0 min control samples were collected, NT in a concentration of 0.1 μg/μl was perfused followed by additional CSF controls. Assay by HPLC-EC of each perfusate showed that when the rat was sated, NT evoked a significant increase in the release of DA and DOPAC from the hypothalamus as well as augmented NE turnover, as reflected by a significant efflux in MHPG. However, when the rat was fasted for 22 hr, the perfusion of NT reduced DA and DOPAC concentrations in the diencephalic perfusate significantly as well as levels of both MHPG and VMA. Under both sated and fasted conditions, NT failed to produce notable changes in the release of 5-HT or its metabolism to 5-HIAA. These findings thus reveal a functional interaction between NT and both of the catecholamine neurotransmitters within hypothalamic neurons, which is clearly dependent upon the nutritional status of the animal.     

Brain Dialysis: In Vivo Metabolism of Dopamine and Serotonin by Monoamine Oxidase A but Not B in the Striatum of Unrestrained Rats

A dialysis cannula was implanted into rat striatum while the animals were anesthetized, and the area was perfused with Ringer solution while the animals were unanesthetized after at least 3 days following surgery. Concentrations of the metabolites of 3,4-dihydroxyphenylethylamine (DA) and 5-hydroxytryptamine (5-HT) in the perfusate were determined by HPLC with electrochemical detection. Levels of the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the perfusate significantly decreased after pargyline administration (50 mg/kg i.p.), which may inhibit not only monoamine oxidase (MAO)-B but also MAO-A in these high doses. The level of the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA) also decreased after pargyline treatment, although change in the relative level of 5-HIAA was less than that of DOPAC or HVA. To clarify the mechanisms for the metabolism of monoamines in rat striatum, highly specific MAO-A and -B inhibitors were used in the following experiments. Treatment with l-deprenyl (10 mg/kg), a specific inhibitor for MAO-B, did not cause any statistically significant change in DOPAC, HVA, and 5-HIAA levels. No significant change was found in rat striatal homogenates at 2 h after the same treatment with l-deprenyl. In contrast, low-dose treatment (1 mg/kg) with clorgyline, a specific inhibitor for MAO-A, caused a significant decrease in levels of these three metabolites in both the perfusates and tissue homogenates. In addition to the above three metabolites, the level of 3-methoxytyramine, which is an indicator of the amount of DA released, greatly increased after treatment with a low dose (1 mg/kg) of clorgyline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stress-Related Changes in Cerebral Catecholamine and Indoleamine Metabolism: Lack of Effect of Adrenalectomy and Corticosterone

The concentrations of catecholamine and indoleamine metabolites were measured in intact and adrenalectomized mice to determine whether adrenal hormones mediate or modulate the stress-induced responses. Thirty minutes of footshock resulted in significant increases of the ratios of the dopamine (DA) catabolite, dihydroxyphenylacetic acid (DOPAC), to DA in prefrontal cortex, nucleus accumbens, striatum, hypothalamus, and brainstem, and of homovanillic (HVA)/DA ratios in nucleus accumbens, striatum, amygdala, and hypothalamus. Ratios of 3-methoxy-4-hydroxyphenylethyleneglycol to norepinephrine (NE) were also increased in prefrontal cortex, nucleus accumbens, septum, amygdala, hypothalamus, hippocampus, and brainstem. The concentration of NE was decreased in amygdala. 5-Hydroxyindoleacetic acid (5-HIAA)/5-hydroxytryptamine (5-HT, serotonin) ratios and free tryptophan were also increased in every brain region. Very similar data were obtained from mice restrained for 30 min. Adrenalectomy resulted in increased HVA/DA ratios in prefrontal cortex and striatum, and 5-HIAA/5-HT in septum. The stress-related changes were largely similar in adrenalectomized mice. Significant interactions between adrenalectomy and footshock treatment occurred in prefrontal cortical DOPAC/DA and hypothalamic NE which was depleted only in adrenalectomized mice, suggesting tendencies for these measures to be more responsive in adrenalectomized mice. Corticosterone administration (0.5-2.0 mg/kg s.c.) which resulted in plasma concentrations in the physiological range did not alter the concentrations of the cerebral metabolites measured in any region. We conclude that adrenal hormones do not mediate cerebral catecholamine or indoleamine metabolism in stress, although adrenalectomy may affect HVA and 5-HIAA metabolism, and there was a tendency for catecholamines to be more sensitive to stress in adrenalectomized animals.     

Rapid, concurrent analysis of dopamine, 5-hydroxytryptamine, their precursors and metabolites utilizing high performance liquid chromatography with electrochemical detection: analysis of brain tissue and cerebrospinal fluid

Assays capable of measuring picomole quantities of dopamine (DA), 5-hydroxytryptamine (5-HT), several of their precursors and metabolites concurrently within 25 minutes were developed utilizing high performance liquid chromatography with electrochemical detection (LCEC). Several parameters of the LCEC were altered in order to separate the compounds while maintaining a short assay time. The final LCEC systems demonstrated biological utility in that the DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and the 5-HT metabolite 5-hydroxy-3-indoleacetic acid (5-HIAA) were detected in rat cerebrospinal fluid; in addition to these compounds, DA and 5-HT were measurable in the striatum, hypothalamus and median eminence of the rat brain. Pargyline decreased the concentrations of DOPAC, HVA and 5-HIAA and increased the 5-HT concentration in all three brain regions, and increased the DA concentration in the striatum. Probenecid increased all three acid metabolite concentrations in the hypothalamus and median eminence, while only the HVA and 5-HIAA concentrations were increased in the striatum. The DA and 5-HT concentrations were unaltered. The LCEC methods described in this paper should be useful in elucidating the mechanisms and roles of 5-HT and DA neurons in experimental paradigms of biological interest.     

Variance in the production of homovanillic acid and 3-methoxy-4-hydroxyphenethyleneglycol by the awake primate brain

Previous experimental results, using a new technique whereby the production rates of the neurotransmitter metabolites homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenethyleneglycol (MHPG) by the awake primate brain are determined, have shown a wide variance in metabolite production among both animal and human subjects. These data suggested that either individual subjects differ in the activity of brain dopamine (DA) or norepinephrine (NE) neurons and/or that the activities of these neurons fluctuate over time. For these reasons a series of experiments were performed in which measures of HVA and MHPG production were obtained at three time points in the same animal (monkeys) over a three hour period. It was found that the group mean values for the production of HVA and MHPG by brain were similar for each of the three time points. However, it was also found that marked variations in HVA and MHPG production occur within a single animal over a three hour period. The coefficients of variation for individual animals for HVA ranged from 9.3 to 31.9% and for MHPG from 10.1 to 62.3%. These variations were not correlated with grossly observable changes in behavioral states. Using an analysis of variance it was found that the variance in MHPG production was significantly greater than that for HVA (F = 6.2, p < 0.05) suggesting that brain NE systems are more liable and/or show greater change than do brain DA systems. These data are interpreted as indicating that in the awake, resting primate brain fluctuations in the activities of DA and NE neurons occur, i.e. there is not a steady, invariant production of metabolites but rather they are produced in pulses of varying lengths. This interpretation of the data is generally consistent with electrophysiological studies which indicate that catecholamine neurons fire in bursts which are then followed by silent periods. Finally, in terms of practical application of the V-A difference technique, these data indicate that replicable group mean estimates of brain HVA and MHPG production can be obtained by averaging values from a single time point whereas accurate information about an individual animal will require multiple samplings.Recent reports from this laboratory have described a method whereby a direct measure of the rates of production of neurotransmitter metabolites such as homovanillic acid (HVA), 3-methoxy-4-hydroxyphenethyleneglycol (MHPG), and 5-hydroxyindoleacetic acid (5-HIAA) by the awake primate brain can be determined (1, 2, 3, 4). Since the quantities of HVA, MHPG, and probably 5-HIAA in the brain vary as a function of the activity of dopamine (DA), norepinephrine (NE), and serotonin (5-HT) neurons (1, 5, 6, 7, 8), it is likely that these measures of neurotransmitter metabolite production reflect the functional state of brain DA, NE, and 5-HT neuronal systems. The experimental results thus far obtained with this technique have shown a wide variance in the rates of neurotransmitter metabolite production across both animal and human subjects even though the subjects were not in clearly different behavioral or emotional states (1, 2, 4, 9). These data suggested that either individual subjects differ markedly in the activities of brain DA, NE, and 5-HT neurotransmitter systems and/or that the activity of these systems fluctuates markedly over time. For these reasons, experiments were undertaken in which repeated measures of HVA and MHPG production by brain within the same animal were determined over a three hour period. The results of these experiments, which are reported here, indicate that there are marked changes in brain metabolite production which occur within animals. The implications of these findings for our understanding of the functioning of brain neurotransmitter systems and for the practical applications of this technique are discussed.     

Brain amines and neocortical EEG in young and aged rats

1. Frontal and parieto-occipital electroencephalography (EEG) of young (4 months-old) and aged (17 and 22 months-old) Wistar rats were analyzed, both during movement and during waking immobility. 2. The levels of monoamines, serotonin and their metabolites were measured from the frontal cortex, parieto-occipital cortex, hippocampus, brainstem and midbrain. 3. In aged rats, as compared to young rats, the most apparent changes of the quantitative EEG spectrum were the decreased amplitude of alpha (5-10 Hz) and beta (10-20 Hz) frequency bands in the frontal and parieto-occipital cortices during both movement and waking immobility behavior (p less than 0.05). 4. The levels of dopamine (DA), homovanillinic acid (HVA), serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) or the ratios of 5-HT/5-HIAA and DA/HVA did not differ between young and aged rats in any brain region studied, with the exceptions of brainstem DA and parieto-occipital 5-HIAA, which were elevated in aged rats (p less than 0.05). 5. In the frontal cortex, hippocampus and midbrain, noradrenaline (NA) levels of aged rats were slightly increased as compared to young rats (p less than 0.05). 6. NA levels of the parieto-occipital cortex and brainstem did not change during aging. 7. Furthermore, there were no clear correlations between the decreased amplitude of the quantitative EEG spectrum and monoamine or serotonin concentrations, or the ratios of 5-HT/5-HIAA and DA/HVA in the cerebral cortex of aging Wistar rat.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of tyrosine, tryptophan and their metabolic derivatives by liquid chromatography-electrochemical detection: application to post mortem samples from patients with Parkinson's and Alzheimer's disease

A procedure is described for the rapid determination of the major indoles and catechols. Analysis with picogram detection limits was done by high-pressure liquid chromatography on a C18 reverse-phase column using electrochemical detection (LCEC). This method provides a comprehensive list of compounds which can be simultaneously determined in brain samples and for which there is no necessity of derivatization or pre-column purification. The regional distribution of 9 neurochemicals from rat brain and the levels of 10 neurochemicals from human brain are presented. DOPA, TYR, NE, MHPG, DOPAC, 5-HIAA, TRP, DA, HVA, 3-MT and 5-HT were detected in the caudate nucleus and putamen. The levels of neurochemicals from the caudate and putamen of a demented patient with Parkinson's disease were variably decreased; catechol and indole losses were greatest in the putamen. The levels of neurochemicals in the caudate and putamen of patients with Alzheimer's disease (SDAT) were also variably decreased; loss of NE was seen only in putamen and losses of DA, HVA and 5-HT were uniform across both caudate and putamen. The CSF of SDAT patients showed changes in NE only.