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.     

Rapid and reliable high-performance liquid chromatographic method for analysing human plasma serotonin, 5-hydroxyindoleacetic acid, homovanillic acid and 3,4-dihydroxyphenylacetic acid

The simultaneous measurement of homovanillic acid, 3,4-dihydroxyphenylacetic acid, serotonin and 5-hydroxyindoleacetic acid in human plasma by an ultrafiltration and microbore high-performance liquid chromatography—electrochemical detection technique is established. Conventional preparation of blood is very tedious and time-consuming, but isocratic separation of the analytes in plasma ultrafiltrates using a microbore column could be achieved within 10 min. Hence, theoretically, over 140 analyses can be performed in a working day. The detection limit (signal-to-noise ratio = 3) of this method is about 0.1–0.5 pg per injection for all analytes. The required volume of plasma samples can be less than 100 μl. Hence, blood loss is minimal, especially in repeated blood sampling. This rapid, simple and sensitive method can, therefore, be used as a routine clinical tool in the simultaneous measurement of plasma homovanillic acid, 3,4-dihydroxyphenylacetic acid, serotonin and 5-hydroxyindoleacetic acid.     

Improved detection limit for catecholamines using liquid chromatography-electrochemistry with a carbon interdigitated array microelectrode

The detection limit of catecholamines can be lowered by using a carbon-based interdigitated array (IDA) microelectrode as a detector for liquid chromatography (LC). The IDA electrode is more sensitive than conventional glassy carbon electrodes due to the high current density caused by radial diffusion at each microband, and redox cycling between two microband arrays. Since the number of redox cycles increases at lower flow-rates, the carbon IDA is particularly useful for microbore LC. In an LC system with a 1-mm microbore column and a carbon IDA electrode, the peak height of dopamine (DA) and DOPAC did not decrease with decreasing flow-rate because of this redox cycling. A low detection limit of 5 fg (32 amol) and 9.6 fg (57 amol) was obtained for DA and DOPAC due to the high current density and low background noise level (0.1 pA) at the carbon IDA electrode. The total charge generated by oxidizing DA at the anodic array was more than the value calculated by assuming that all the DA molecules were oxidized.     

Simultaneous Measurement of Extracellular Morphine and Serotonin in Brain Tissue and CSF by Microdialysis in Awake Rats

In this report, we describe an HPLC with electrochemical detection assay for the simultaneous measurement of levels of morphine, serotonin, 5-hydroxyindole-3-acetic acid, and homovanillic acid in dialysates of various brain areas and CSF in the awake rat. Morphine could be detected in the dialysates after a single intraperitoneal injection, with doses as low as 1.0 mg/kg. The time course of extracellular morphine content in the lateral hypothalamus, striatum, cerebellum, periaqueductal gray, and dorsal horn of the spinal cord and in CSF, from the ventricles and cisterna magna, was similar. We detected morphine in the first 15-min sample, and levels peaked 45-60 min after injection. Maximal dialysate levels, however, varied with the type of dialysis probe used and the area sampled. The most efficient in vivo recovery was in CSF dialysates from the cisterna magna, presumably because of minimal tissue interference with the dialysis probe. For this reason, the cisterna is an ideal region for sampling CSF. Morphine had no significant effect on the extracellular concentrations of serotonin in any of the areas studied and did not modify or only slightly increased levels of tissue metabolites; however, morphine markedly increased the CSF levels of 5-hydroxyindole-3-acetic acid and homovanillic acid. Because microdialysis in freely moving animals permits assessment of the behavioral effects of morphine while continuously monitoring the drug levels in discrete brain regions, this approach will greatly facilitate future studies of the neurochemical basis of morphine's effects in the brain.     

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.     

High-performance liquid chromatography with electrochemical detection for the determination of levodopa, catecholamines and their metabolites in rat brain dialysates

A high-performance liquid chromatographic assay with electrochemical detection is described for the simultaneous determination of levodopa, 3-O-methyldopa, dopamine, dihydroxyphenylacetic acid, homovanillic acid, 3-methoxytyramine, noradrenaline, adrenaline, 3-methoxy-4-hydroxyphenylethylene glycol and 5-hydroxyindoleacetic acid in rat brain dialysates. Samples are obtained in vivo using the microdialysis technique. Microdialysis probes are placed in the brain area to be studied and neurochemicals are collected by perfusion of the probe with modified Ringer's solution. Direct injection of the dialysates allows rapid and reliable results to be obtained.     

An assay to measure the simultaneous uptake of [3H]dopamine and [14C]serotonin by the human platelet reveals an imipramine-insensitive [3H]dopamine uptake mechanism.

To determine whether a specific dopamine uptake mechanism is present on the human platelet the simultaneous uptake of [3H]dopamine and [14C]serotonin by platelets was measured. Utilising a dual radiolabel uptake technique, platelets have been shown to take up serotonin more rapidly and to a greater extent than they take up dopamine. Furthermore, at high concentrations serotonin was able to reduce dopamine uptake by platelets by 60% whereas dopamine had no effect on serotonin uptake. Similarly, imipramine and reserpine reduced (97% and 74% respectively) serotonin uptake by platelets in a dose-dependent manner, but did not affect the uptake of dopamine. Our data show that platelets take up dopamine by a mechanism independent of the imipramine-sensitive serotonin uptake mechanism. Furthermore, the increased capacity of platelets to store serotonin is because serotonin, unlike dopamine, is transported into the dense granules of the platelet.     

Simultaneous determination of norepinephrine,serotonin, and 5-hydroxyindole-3-acetic acid in microdialysis samples from rat brain by microbore column liquid chromatography with fluorescence detection following derivatization with benzylamine

A microbore column liquid chromatographic method for the simultaneous determination of norepinephrine (NE), serotonin (5-HT), and 5-hydroxyindole-3-acetic acid (5HIAA) in microdialysis samples from rat brain is described. The method is based on precolumn derivatization of NE, 5HT, and 5HIAA with benzylamine in the presence of potassium hexacyanoferrate(III) resulting in the corresponding highly fluorescent and stable benzoxazole derivatives. A 15-microl sample was mixed with 15 microl derivatization reagent solution containing 0.3M 3-cyclohexylaminopropanesulfonic acid buffer (pH 12.0), 0.5M benzylamine, 10mM potassium hexacyanoferrate(III), and methanol (1/1/1/12, v/v/v/v). The derivatization was carried out at 50 degrees C for 20 min. The benzylamine derivatives of NE, 5HT, and 5HIAA were separated on a reversed-phase column (100 x 1.0mm i.d., packed with C18 silica, 5 microm) within 30 min. The mobile phase consisted of 15 mM acetate buffer (pH 5.0) and acetonitrile (31%, v/v); the flow rate was 50 microl/min. The detection limits (signal-to-noise ratio of 3) for NE, 5HT, and 5HIAA in the injection volume of 20 microl were 90, 210, and 260 amol, respectively. Microdialysis samples were collected in 7.5-min intervals from the probes implanted in the hippocampus and prefrontal cortex of awake rats. The basal levels of NE, 5HT, and 5HIAA in the dialysates from the hippocampus were 4.2+/-0.5, 4.9+/-0.6, and 934.1 +/- 63.4 fmol/20 microl, and those from the prefrontal cortex were 6.0+/-1.2,5.51.3, and 669.1 +/- 96.0 fmol/20 microl (mean +/- SE, n=25), respectively. The NE and 5HT levels were altered by perfusion of high-potassium or low-calcium solution and following antidepressant drugs imipramine and desipramine. It is concluded that the new fluorescence derivatization method in combination with microbore column liquid chromatography allows the simultaneous determination of NE, 5HT, and 5HIAA in the microdialysis samples at higher sensitivity, providing easier maintenance in routine use than that achieved by high-performance liquid chromatographic methods with electrochemical detection.     

Syntheses of novel diphenyl piperazine derivatives and their activities as inhibitors of dopamine uptake in the central nervous system

A new series of diphenyl piperazine derivatives containing the phenyl substituted aminopropanol moiety, which were modified at sites between the diphenyl and piperazine moieties, was prepared and evaluated for dopamine transporter binding affinity with [(3)H]GBR12935 in rat striatal membranes. These synthesized compounds showed apparent dopamine transporter binding affinities (IC(50)<30 nM) and some of them were approximately equivalent in activity to GBR12909 known as a potent dopamine uptake inhibitor, showing the activities with IC(50) values of nanomolar range. Among them, 1-[4,4-bis(4-fluorophenyl)butyl]-4-[2-hydroxy-3-(phenylamino)propyl]piperazine 2 was evaluated for extracellular dopamine levels in rat striatum using in vivo brain microdialysis. The intraperitoneal administration of 2 (0.01, 0.03, or 0.1 mmol/kg) induced dose-dependent increases of dopamine levels in rat striatal dialysates. The maximum increases in dopamine levels induced by 2 were greater than those by GBR12909. The pharmacological data of these novel diphenyl piperazine derivatives show that the compounds have potent dopamine uptake inhibitory activities in the central nervous system.     

Simultaneous determination of the monoamine neurotransmitters and glucose in rat brain by microdialysis sampling coupled with liquid chromatography-dual electrochemical detector

A new type of chemically modified electrode based ring-disk electrode as the dual electrochemical detector (DECD) for high-performance liquid chromatography (HPLC) to simultaneously determine the monoamine neurotransmitters and glucose is described. The ring electrode was modified with an ion-exchange polymer-overoxidized polypyrrole (OPPy) and the disk electrode was modified with nano Au colloid and glucose oxidase (GOD). The electrochemical behaviors of dopamine (DA) and ascorbic acid (AA) at the OPPy chemically modified electrode (CME) were investigated by differential pulse voltammetry (DPV). It was found that the CME could permeate dopamine cations and repelled the ascorbate anions, which could be used to determine the monoamine neurotransmitters and avoid the interference of AA. The electrochemical behavior of glucose at the Nafion/GOD-Au colloid/GC CME was investigated by amperometry and flow injection analysis (FIA). It was found that the sensitivity of the CME increased apparently in determination of glucose. In order to obtain better separation and current responses of the analytes in HPLC-DECD, several operational parameters have been investigated. Under the optimum conditions, the method showed good stability and reproducibility. The application of this method coupled with microdialysis sampling for in vivo simultaneous determination of monoamine neurotransmitters and glucose in rat brain was satisfactory.     

Simultaneous detection of indoleamines and dopamine in rat dorsal raphe nuclei using specific antibodies

Using a monoclonal antibody against dopamine and a rabbit antiserum against serotonin, 5-methoxytryptamine or tryptamine, we were able to achieve the simultaneous localization of two amines in glutaraldehyde-fixed sections of rat dorsal raphe nuclei. In this staining procedure, the first antigen was localized using 3,3'-diaminobenzidine (DAB), while the second antigen was stained using the 1-naphthol basic dye (2-NBD) method. The two antigens were localized in different cells or structures. No overlap of the staining was observed, thus indicating that dopamine is not localized with serotonin, 5-methoxytryptamine or tryptamine.     

Microbore high-performance liquid chromatographic method for measuring acetylcholine in microdialysis samples: optimizing performance of platinum electrodes

A microbore high-performance liquid chromatographic method with electrochemical detection was applied to the measurement of acetylcholine in microdialysis samples. There was an excellent linear relationship (r=0.99998) between the concentration of acetylcholine injected onto the column and the peak height (0.05–10 pmol/5 μl). During the validation of this method, we noticed that the peak height for acetylcholine decreased over time, coupled with the appearance of a brown coating on the surface of the platinum electrode. Repeated measurement of acetylcholine standards which had been stored at 4°C and −20°C before and after cleaning the platinum electrode with ethanol or methanol indicated that the decrease in the peak height of acetylcholine is caused by a decrease in sensitivity of the electrode itself. Results with a second microbore high-performance liquid chromatographic system confirmed these findings. On the basis of these results, we recommend that the platinum electrode is cleaned periodically with ethanol or methanol, and that quantitation is regularly calibrated with external acetylcholine.     

Simple and rapid determination of serotonin and catecholamines in biological tissue using high-performance liquid chromatography with electrochemical detection

Using the CNS of Lymnaea stagnalis a method is described for the rapid analysis of neurotransmitters and their metabolites using high performance liquid chromatography coupled with electrochemical detection. Tissue samples were homogenised in ice-cold 0.1 M perchloric acid and centrifuged. Using a C(18) microbore column the mobile phase was maintained at a flow rate of 100 microl/min and consisted of sodium citrate buffer (pH 3.2)-acetonitrile (82.5:17.5, v/v) with 2 mM decane-sulfonic acid sodium salt. The potential was set at +750 mV versus Ag|AgCl reference electrode at a sensitivity of 50 nA full scale deflection. The detection limit for serotonin was 11.86 ng ml(-1) for a 5 microl injection. Preparation of tissue samples in mobile phase reduced the response to dopamine and serotonin compared with perchloric acid. In addition it was found that the storage of tissue samples at -20 degrees C caused losses of dopamine and serotonin. As a result of optimising the sample preparation and mobile phase the total time of analysis was substantially reduced resulting in a sample preparation and assay time of 15-20 min.     

Simultaneous detection of indoleamines and dopamine in rat dorsal raphe nuclei using specific antibodies

Summary Using a monoclonal antibody against dopamine and a rabbit antiserum against serotonin, 5-methoxytryptamine or tryptamine, we were able to achieve the simultaneous localization of two amines in glutaraldehyde-fixed sections of rat dorsal raphe nuclei. In this staining procedure, the first antigen was localized using 3,3-diaminobenzidine (DAB), while the second antigen was stained using the 1-naphtol basic dye (2-NBD) method. The two antigens were localized in different cells or structures. No overlap of the staining was observed, thus indicating that dopamine is not localized with serotonin, 5-methoxytryptamine or tryptamine.     

Enhancement of Dopamine Release In Vivo from the Rat Striatum by Dialytic Perfusion of 6R-l-erythro-5,6,7,8-Tetrahydrobiopterin

We have previously reported that intracerebroventricular administration of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (6R-BH4), a cofactor for tyrosine hydroxylase, enhances biosynthesis of 3,4-dihydroxyphenylethylamine (dopamine) in the rat brain. In the present study, we have more precisely examined the effects of 6R-BH4 on dopamine release in vivo from the rat striatum using brain microdialysis. The amount of dopamine collected in striatal dialysates was determined using HPLC with electrochemical detection after purification with an alumina batch method. When the striatum was dialyzed with Ringer solution containing various concentrations of 6R-BH4 (0.25, 0.5, and 1.0 mM), dopamine levels in striatal dialysates increased in a concentration-dependent manner. Biopterin had little effect on dopamine levels in dialysates. The 6R-BH4-induced increase in dopamine levels in dialysates was abolished after pretreatment with tetrodotoxin (50 microM) added to the perfusion fluid, but after pretreatment with nomifensine (100 mg/kg, intraperitoneal injection), an inhibitor of dopamine uptake mechanism, a larger increase was observed. After inhibition of tyrosine hydroxylase by pretreatment with alpha-methyl-p-tyrosine (250 mg/kg, intraperitoneal injection), most of the increase persisted. These results suggest that 6R-BH4 has a dopamine-releasing action, which is not dependent on biosynthesis of dopamine.     

Effects of Hypoxia on the Activity of the Dopaminergic Neuron System in the Rat Striatum as Studied by In Vivo Brain Microdialysis

The purpose of the present study is to clarify the effects of hypoxia on the activity of the dopaminergic neurons in the brain and its mechanism of action. For this purpose, the effects of hypoxia on the extracellular levels of 3,4-dihy-droxyphenylethylamine (dopamine) were examined in the rat Striatum using in vivo brain microdialysis in the presence or absence of pretreatment with either tetrodotoxin (a blocker of voltage-dependent sodium channels) or nomifensine (a blocker of dopamine reuptake). Exposure to various degrees of hypoxia (15, 10, and 8% O₂ in N₂) increased dopamine levels in striatal dialysates to 200, 400, and 1,100%, respectively, of the control value. On reoxygenation, dopamine levels in the dialysates rapidly returned to the control level. Reexposure to hypoxia increased the dopamine levels to the same extent as during the first exposure. After addition of tetrodotoxin (40 mUM) to the perfusion fluid or pretreatment with nomifensine (100 mg/kg, i.p.), exposure to hypoxia no longer increased the dopamine levels. These results suggest that although hypoxia induces an increase in the extracellular dopamine levels (hence, an apparent increase in the activity of the dopaminergic neurons), this increase is not the result of an increase in dopamine release itself, but rather the result of inhibition of the dopamine reuptake mechanism.     

Long-Term Ethanol Alcoholization Decreases Levodopa- Induced Aggressive Behavior in Rats

One of the old but still unsolved problems in psychiatry is the treatment of patients with a dual diagnosis of schizophrenia and alcoholism. Numerous clinical studies failed to explain unambiguously the mechanisms of formation of these comorbid disorders. Experimental modeling of dual disorders was carried out using a model of schizophrenia based on the impaired development and dopamine hypothesis of schizophrenia. To model comorbid experimental schizophrenia and alcohol abuse, the dopamine precursors, Levodopa/Carbidopa (LC), were used in combination with 15% ethanol. The aim of this study was to investigate social behavior of rats receiving LC and exposed to intermittent semi-compulsory alcoholization. After three months of the experiment, there were significantly more interactions (including aggressive–attacks, fights, bites) and submissive postures in the group of rats that received LC. Combined LC/ethanol administration did not alter rat social behavior in contrast to ethanol administered alone.     

Effect of ibogaine on serotonergic and dopaminergic interactions in striatum from mice and rats

The effect of ibogaine (Endabuse, NIH 10567) on serotonin uptake and release, and on serotonergic modulation of dopamine release, was measured in striatal tissue from rats and mice. Two hours after treatment in vivo with ibogaine (40 mg/kg i.p.), the uptake of labeled [³H]serotonin and [³H]dopamine uptake in striatal tissue was similar in the ibogaine-treated animal to that in the control. The 5HT_1B agonist CGS-12066A (10^–5 M) had no effect on stimulation-evoked tritium release from mouse or rat striatal tissue preloaded with [³H]serotonin; however, it elevated tritium efflux from striatal tissue preloaded with [³H]dopamine. This increase was not seen in mice treated with ibogaine 2 or 18 hours previously, or in rats treated 2 hours before. Dopamine autoreceptor responses were not affected by ibogaine pretreatment in either mouse or rat striatal tissue; sulpiride increased stimulation-evoked release of tritium from tissue preloaded with [³H]dopamine. The long-lasting effect of ibogaine on serotonergic functioning, in particular, its blocking of the 5HT_1B agonist-mediated increase in dopamine efflux, may have significance in the mediation of its anti-addictive properties.Special issue dedicated to Dr. Sidney Ochs.     

In vivo electrochemical studies of rat striatal dopamine and serotonin release after morphine

The effect of the reference opiate, morphine (d-morphine-sulfate), on endogenously released striatal dopamine and serotonin was studied in male, adult, anesthetized Sprague-Dawley rats. The intraperitoneal administration of morphine produced a biphasic effect on striatal dopamine release. A significant increase in the dopamine signal was seen in the first hour after drug administration; a significant decrease in the dopamine signal was seen in the second and third hour after drug administration. On the other hand, the effect of morphine on striatal serotonin release was monophasic. Morphine significantly increased serotonin release from rat striatum. The effect lasted three hours after morphine administration, i.e., the effect persisted significantly throughout the study. These data show a simultaneous opiate-dopaminergic and opiate-serotonergic interaction in rat striatum. These data further extend studies which have suggested that the pharmacological mechanism of action of morphine may have its etiology in the concurrent modulation of more than one neurotransmitter.     

Quantitative proteome analysis using differential stable isotopic labeling and microbore LC-MALDI MS and MS/MS

We demonstrate an approach for global quantitative analysis of protein mixtures using differential stable isotopic labeling of the enzyme-digested peptides combined with microbore liquid chromatography (LC) matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). Microbore LC provides higher sample loading, compared to capillary LC, which facilitates the quantification of low abundance proteins in protein mixtures. In this work, microbore LC is combined with MALDI MS via a heated droplet interface. The compatibilities of two global peptide labeling methods (i.e., esterification to carboxylic groups and dimethylation to amine groups of peptides) with this LC-MALDI technique are evaluated. Using a quadrupole-time-of-flight mass spectrometer, MALDI spectra of the peptides in individual sample spots are obtained to determine the abundance ratio among pairs of differential isotopically labeled peptides. MS/MS spectra are subsequently obtained from the peptide pairs showing significant abundance differences to determine the sequences of selected peptides for protein identification. The peptide sequences determined from MS/MS database search are confirmed by using the overlaid fragment ion spectra generated from a pair of differentially labeled peptides. The effectiveness of this microbore LC-MALDI approach is demonstrated in the quantification and identification of peptides from a mixture of standard proteins as well as E. coli whole cell extract of known relative concentrations. It is shown that this approach provides a facile and economical means of comparing relative protein abundances from two proteome samples.