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.     

Gas chromatographic method for the simultaneous determination of dopamine and norepinephrine metabolites

A new gas chromatographic method, using only flame ionization detection which can determine nanogram quantities of homovanillic acid, 3,4-dihydroxyphenylacetic acid, 3-methoxy-4-hydroxyphenylethyleneglycol and 3,4-dihydroxyphenylethyleneglycol in the same reaction, is described. These compounds are treated with diazoethane and n-butylboronic acid. Homovanillic acid and 3,4-dihydroxyphenylacetic acid are converted to their ethyl esters while 3-methoxy-4-hydroxyphenylethyleneglycol and 3,4-dihydroxyphenylethyleneglycol from cyclic boronates and are thus assayed. This method is quantitative, highly specific and sensitive. It has been applied to the analysis of these compounds in urine.     

Differential Effect of Stress on In Vivo Dopamine Release in Striatum, Nucleus Accumbens, and Medial Frontal Cortex

Microdialysis was used to assess extracellular dopamine in striatum, nucleus accumbens, and medial frontal cortex of unanesthetized rats both under resting conditions and in response to intermittent tail-shock stress. The dopamine metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid also were measured. The resting extracellular concentration of dopamine was estimated to be approximately 10 nM in striatum, 11 nM in nucleus accumbens, and 3 nM in medial frontal cortex. In contrast, the resting extracellular levels of 3,4-dihydroxyphenylacetic acid and homovanillic acid were in the low micromolar range. Intermittent tail-shock stress increased extracellular dopamine relative to baseline by 25% in striatum, 39% in nucleus accumbens, and 95% in medial frontal cortex. 3,4-Dihydroxyphenylacetic acid and homovanillic acid also were generally increased by stress, although there was a great deal of variability in these responses. These data provide direct in vivo evidence for the global activation of dopaminergic systems by stress and support the concept that there exist regional variations in the regulation of dopamine release.     

Catechol O-methyltransferase and monoamine oxidase A genotypes,and plasma catecholamine metabolites in bipolar and schizophrenic patients

Metabolites of dopamine and norepinephrine measured in the plasma have long been associated with symptomatic severity and response to treatment in schizophrenic, bipolar and other psychiatric patients. Plasma concentrations of catecholamine metabolites are genetically regulated. The genes encoding enzymes that are involved in the synthesis and degradation of these monoamines are candidate targets for this genetic regulation. We have studied the relationship between the Val158Met polymorphism in catechol O-methyltransferase gene, variable tandem repeat polymorphisms in the monoamine oxidase A gene promoter, and plasma concentrations of 3-methoxy-4-hydroxyphenylglycol, 3,4-dihydroxyphenylacetic acid and homovanillic acid in healthy control subjects as well as in untreated schizophrenic and bipolar patients. We found that the Val158Met substitution in catechol O-methyltransferase gene influences the plasma concentrations of homovanillic and 3,4-dihydroxyphenylacetic acids. Although higher concentrations of plasma homovanillic acid were found in the high-activity ValVal genotype, this mutation did not affect the plasma concentration of 3-methoxy-4-hydroxyphenylglycol. 3,4-dihydroxyphenylacetic acid concentrations were higher in the low-activity MetMet genotype. Interestingly, plasma values 3-methoxy-4-hydroxyphenylglycol were greater in schizophrenic patients and in bipolar patients than in healthy controls. Our results are compatible with the previously reported effect of the Val158Met polymorphism on catechol O-methyltransferase enzymatic activity. Thus, our results suggest that this polymorphism, alone or associated with other polymorphisms, could have an important role in the genetic control of monoamine concentration and its metabolites.     

Free and Conjugated Amines in Human Lumbar Cerebrospinal Fluid

Significant amounts of acid-hydrolyzable conjugates of 3,4-dihydroxyphenylethylamine, norepinephrine, and 5-hydroxytryptamine were detected in lumbar CSF from 22 awake unpremedicated healthy individuals. In the CSF samples, the amounts of conjugated amines almost always exceeded the amounts of free amines, but were less than the amounts of the acid metabolites 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid.     

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.     

Hyperoxia caused by microdialysis perfusion decreased striatal monoamines: involvement of oxidative stress

Due to complex influence, such as utilization and permeability of arterial vessels to oxygen, there is a considerable difference of oxygen tension between extracellular fluid and perfusate usually used in microdialysis (30-60 Torr versus 145 Torr). Dialysate dopamine and monoamine metabolites-3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindoleacetic acid were measured under different kinds of oxygen tension solutions (145, 72, 48 Torr). In the acute and anesthetized group, dopamine, 3,4-dihydroxyphenylacetic acid, 5-hydroxyindoleacetic acid and homovanillic acid increased 72, 93, 86 and 65%, respectively when changing the perfusate from 145 Torr to near physiological 48 Torr, while in chronic and conscious group, carried out 72 h after surgery, these compounds showed obscure increases (only homovanillic acid produced a significant change of 14%).The different effect of perfusate oxygen tension on dialysate levels of monoamines in anesthetized and conscious rats might be caused by oxidative stress triggered by hyperoxia combined with anesthesia and surgical trauma.     

Monoamines and their metabolites in somatosensory,visual, and cingulate cortices of adult rat: Differences in content and lack of sidedness

Small areas of somatosensory, visual and cingulate cortex were microdissected and assayed for their monoamine content by high-performance liquid chromatography with electrochemical detection. No differences were found between the right and the left hemisphere for any area nor for any of the monoamines. The values averaged from left and right hemispheres for the sensory areas were significantly different from the cingulate in the content of norepinephrine, 4-hydroxy-3-methoxyphenylglycol, dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, 5-hydroxyl-tryptophan, serotonin and 5-hydroxyindole-3-acetic acid. The two sensory cortices differed in their levels of norepinephrine, dopamine, 3–4-dihydroxyphenylacetic acid and homovanillic acid. In the latter comparison, the measured amounts were higher in somatosensory than in visual cortex. This biochemical heterogeneity in monoamine distribution may reflect specific innervation patterns for these compounds in these discrete cortical areas and allows differences in content to be related to functional specialization of the cerebral cortex.     

A simple, sensitive method for measuring 3,4-dihydroxyphenylacetic acid and homovanillic acid in rat brain tissue using high-performance liquid chromatography with electrochemical detection.

A sensitive, specific, and very simple method, using high-performance liquid chromatography combined with electrochemical detection, measured 3,4-dihydroxyphenylacetic acid and homovanillic acid in small areas of rat brain. The dopamine metabolites were extracted with diethyl ether and a known amount of vanillic acid as internal standard, and were separated on a micro-particulate reverse-phase column using a sodium acetate buffer (pH 5.0) as mobile phase.     

Oxalic acid stabilizes dopamine, serotonin, and their metabolites in automated liquid chromatography with electrochemical detection

Use of antioxidative agents is required in automated LC assay of microdialysis samples, due to rapid degradation of the monoamine neurotransmitters and their metabolites. Addition of oxalic acid prevented degradation of dopamine, serotonin, 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindoleacetic acid efficiently: after a 24-h incubation at room temperature the decreases in peak heights were less than 10%. The long-term stability of the analytes, however, was still enhanced when acetic acid and -cysteine were included in the solution. Using this antioxidative solution, the monoamine neurotransmitters and their metabolites could be determined with an automated LC assay even at room temperature.     

Correlation between high-performance liquid chromatography and automated fluorimetric methods for the determination of dopamine, 3, 4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindoleacetic acid in nervous tissue and cerebrospinal fluid

The correlation between automated fluorimetric methods and high-performance liquid chromatography is described for the determination of homovanillic acid and 5-hydroxyindoleacetic acid in cerebrospinal fluid, and for dopamine, 3, 4-dihydroxyphenylacetic acid and homovanillic acid in striata of rat brain. The automated fluorimetric methods for 3, 4-dihydroxyphenylacetic acid and homovanillic acid showed a good correlation with the high-performance liquid chromatographic methodology. The fluorimetric determination for dopamine was somewhat less reliable than the high-performance liquid chromatographic assay. The fluorimetric assay for 5-hydroxyindoleacetic acid correlated poorly with the chromatographic method.     

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.     

Reverse-phase high-performance liquid chromatographic separation and electrochemical detection of norepinephrine,dopamine, serotonin,and related major metabolites

A convenient method for the determination of biogenic amines and their metabolites in small samples of brain tissue weighing from 0.5 to 5 mg, based on reverse-phase chromatography, is described. The biogenic amines, norepinephrine, dopamine, and serotonin, and the metabolites normetanephrine and 3-methoxytyramine were separated by ion-pair chromatography on a μBondapak phenyl column with an aqueous eluant, while the metabolites 3,4-dihydroxyphenylacetic acid, homovanillic acid, 3-methoxy-4-hydroxyphenylglycol, and 5-hydroxyindoleacetic acid were separated on a μBondapak C18 column with a methanol aqueous mobile phase. The sensitivity of the method is in the picogram range, from 20 to 100 pg, depending on the substances analyzed.     

Analysis of monoamines and their metabolites by high performance liquid chromatography with coulometric electrochemical detection

High performance liquid chromatography with coulometric electrochemical detection has been used to achieve simultaneous determination of norepinephrine, epinephrine, 5-hydroxytryptophan, normetanephrine, dopamine, metanephrine, 3,4-dihydroxyphenylacetic acid, N-acetyldopamine, tyramine, tryptophan, 5-hydroxyindoleacetic acid, 5-hydroxytryptamine, N-acetyl-5-hydroxytryptamine, homovanillic acid, tyrosine, p-octopamine, N-acetyl-p-octopamine, and p-synephrine. The procedure has been applied to study monoamine degradation in the insect brain and to demonstrate that N-acetylation rather than oxidative deamination is the primary route of monoamine catabolism in insects.     

Effects of Graded Levels of Tissue Oxygen Pressure on Dopamine Metabolism in the Striatum of Newborn Piglets

Abstract: The effect of graded levels of tissue hypoxia on the extracellular levels of dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid has been monitored in vivo by microdialysis. Reproducible levels of decreased oxygen in the brain were obtained by increasing the rate of ventilation from the control value of 25/min to as high as 95/min. With increasing ventilatory rate, the oxygen pressure in the cortex decreased from ∼40 torr to 16 torr. As the oxygen pressure decreased stepwise from 40 to 27, 22, and 16 torr, the dopamine levels in the extracellular medium rose by 70, 90, and 150%, respectively, returning to baseline within a few minutes of return to control ventilation rates. Levels of the catabolic products 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid decreased with decreasing tissue oxygen. Unlike the dopamine levels, these catabolite levels continued to decrease through 30 min of recovery (to 50% of control), returning to baseline only after recovery periods of 1–2 h. These data suggest that hypoxia induces long-term alterations in the neurotransmitter turnover. The marked effects of mild tissue hypoxia (decrease of oxygen from 40 torr to 26 torr) on both the extracellular dopamine concentration and dopamine metabolism indicate that the metabolic consequences of decreased tissue oxygen pressure extend to higher values than generally appreciated.     

Determination of Acidic Metabolites of Biogenic Amines in Human Aqueous Humour by Gas Chromatography-Negative Ion Chemical Ionisation Mass Spectrometry

The concentrations of acidic metabolites derived from the biogenic amines o-, m-, and p-tyramines (o-, m-, and p-hydroxyphenylacetic acids), p-octopamine/p-synephrine (p-hydroxymandelic acid), and dopamine (homovanillic acid and 3,4-dihydroxyphenylacetic acid) were measured in human aqueous humour obtained from patients undergoing elective surgery for cataract removal or for trabeculectomy as a treatment for chronic open-angle glaucoma. There were no clear differences in the pattern of metabolism of neurotransmitters between the two groups. An unexpected finding was that the o-tyramine metabolite, o-hydroxyphenylacetic acid, was present in aqueous humour.     

High-performance liquid chromatographic determination of catecholamines and their precursor and metabolites in human urine and plasma by postcolumn derivatization involving chemical oxidation followed by fluorescence reaction.

A high-performance liquid chromatographic method for the determination of catecholamines and their precursor and metabolites [amino compounds (norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine, 3-methoxytyramine, and L-DOPA), acidic compounds (3,4-dihydroxyphenylacetic acid, vanillyl-mandelic acid, and homovanillic acid), and alcoholic compound [4-hydroxy-3-methoxyphenyl)ethylene glycol)] in human urine and plasma. Urine and plasma samples deproteinized with perchloric acid in the presence of isoproterenol and 3,4-dihydroxyphenylpropanoic acid (internal standards) are fractionated by solid-phase extraction on a strong cation-exchange resin cartridge (Toyopak IC-SP S) into two fractions (amine fraction and acid-alcohol fraction). The compounds in each fraction are separated by an ion-pair reversed-phase chromatography on a TSK gel ODS-80TM with isocratic elution and on-line derivatized by periodate oxidation followed by a fluorescence reaction using meso-1,2-diphenylethylenediamine. The detection limits (S/N = 5) vary from 0.5 to 95 pmol/ml, depending on the compounds.     

A SENSITIVE ENZYMATIC-RADIOISOTOPIC ASSAY FOR 3,4-DIHYDROXYPHENYLACETIC ACID

Abstract— An assay for 3,4-dihydroxyphenylacetic acid (DOPAC) capable of detecting as little as 1 pmole of DOPAC is described. The basis of the assay is the O -methylation of DOPAC utilizing S -[methyl-³H]adenosyl-l-methionine and a partially purified catechol- O -methyl transferase to form [methyl-³H]homovanillic acid. The [methyl-³H]homovanillic acid is purified with ion exchange resin and either solvent partitions or TLC. Utilizing this assay, the effect of either chlorpromazine or reserpine upon the content of DOPAC both in the caudate nucleus and in the substantia nigra was determined. Both of these drugs increase the level of DOPAC in the caudate nucleus but have minimal effects upon the level of DOPAC in the substantia nigra.     

Effects of Apomorphine on In Vivo Release of Dopamine and Its Metabolites in the Prefrontal Cortex and the Striatum, Studied by a Microdialysis Method

The effects of apomorphine (0.1-2.5 mg/kg) on release of endogenous dopamine and extracellular levels of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the prefrontal cortex and the striatum were examined in vivo by a microdialysis method. Apomorphine significantly reduced release of dopamine and the extracellular levels of dopamine metabolites, DOPAC and HVA, not only in the striatum, but also in the prefrontal cortex. These findings indicate that dopamine autoreceptors modulate in vivo release of dopamine in the prefrontal cortex.     

Phencyclidine (PCP) injected in the nucleus accumbens increases extracellular dopamine and serotonin as measured by microdialysis

Phencyclidine (PCP; 20 micrograms in 0.5 microliter) was tested by local brain injection for neurochemical effects in the nucleus accumbens and striatum of rats. Changes in dopamine turnover could not be detected in postmortem tissue assays. In contrast, extracellular levels of dopamine significantly increased as measured by microdialysis in freely moving animals. PCP also increased extracellular levels of serotonin and decreased 3,4-dihydroxyphenylacetic acid (DOPAC), but did not change homovanillic acid (HVA) or 5-hydroxyindoleacetic acid (5HIAA). Microdialysis suggests that PCP acts in some dopamine terminal regions to increase extracellular dopamine and serotonin.