Simultaneous analysis of homovanillic acid, 5-hydroxyindoleacetic acid, 3-methoxy-4-hydroxy-phenylethylene glycol and vanilmandelic acid in plasma from alcoholics by high-performance liquid chromatography with electrochemical detection Critical comparison of solid-phase and liquid—liquid extraction methods

A method is described for the simultaneous determination of vanilmandelic acid, 3-methoxy-4-hydroxyphenylethylene glycol, 5-hydroxyindoleacetic acid, and homovanillic acid in a human plasma sample using reversed-phase high-performance liquid chromatography with column switching and amperometric detection. Two methods of sample preparation were tested. Liquid—liquid extraction yields better recoveries, is more selective and precise than solid-phase extraction and allows a shorter time of chromatographic analysis. Estimated plasma values of the metabolites from healthy controls are in good agreement with previously reported levels. Studies of alcoholics at the beginning of the delirium tremens provided different plasma levels of the metabolites, dependent on the different duration — and hence the severity — of the delirium.     

Amino Acids in Plasma and CSF and Monoamine Metabolites in CSF: Interrelationship in Healthy Subjects

Plasma and cerebrospinal fluid (CSF) concentrations of amino acids were measured in 65 healthy volunteers (50 men and 15 women). The CSF levels of the monoamine metabolites homovanillic acid (HVA), 3-methoxy-4-hydroxyphenylethylene glycol (MOPEG), and 5-hydroxyindoleacetic acid (5-HIAA) were also determined. Sex differences were observed in both plasma and CSF amino acid levels as well as in the relationship between these concentrations. No significant correlations were observed between the CSF levels of HVA and 5-HIAA, and the concentrations of their precursor amino acids in either plasma or CSF. The MOPEG level in CSF correlated positively with the plasma concentrations of several amino acids.     

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.     

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.     

NOREPINEPHRINE METABOLISM IN THE CENTRAL NERVOUS SYSTEM OF MAN: STUDIES USING 3-METHOXY-4-HYDROXYPHENYLETHYLENE GLYCOL LEVELS IN CEREBROSPINAL FLUID

—Levels of 3-methoxy-4-hydroxyphenylethylene glycol (MHPG), a major metabolite of norepinephrine, were measured in human CSF by gas-liquid chromatography. MHPG concentrations were similar in both ventricular and lumbar CSF samples; about 30 per cent of the MHPG from either source occurred as the sulphate conjugate. There was relatively little entry of intravenously infused [¹⁴C]MHPG into lumbar spinal fluid. Both α-methylparatyrosine, an inhibitor of tyrosine hydroxylase, and fusaric acid, an inhibitor of dopamine-β-hydroxylase, significantly diminished MHPG values. On the other hand, doses of l -DOPA or probenecid, sufficient to substantially elevate CSF levels of the dopamine metabolite, homovanillic acid, failed to alter the spinal fluid content of MHPG. CSF concentrations of MHPG in patients with Parkinson's disease or the other central nervous system disorders studied did not differ significantly from control levels. The results suggest that MHPG values in CSF may provide an index to norepinephrine metabolism in the central nervous system of man.     

MASS FRAGMENTOGRAPHIC DETERMINATION OF SOME ACIDIC AND ALCOHOLIC METABOLITES OF BIOGENIC AMINES IN THE RAT BRAIN

—A mass fragmentographic procedure is described for the simultaneous quantification of a number of deaminated metabolites derived from tyramine, octopamine, dopamine, and norepinephrine. With this method, several of the metabolites were measured in normal rat brain. The results support the central nervous system origin of tyramine, octopamine and their metabolites. The concentration of the dopamine metabolite, homovanillic acid, in the rat brain was found to be about 15% higher than that of dihydroxyphenylacetic acid. As for the metabolites of norepinephrine, vanilmandelic acid concentration was found to be about 5% that of 3-methoxy-4-hydroxyphenylglycol. The possible role of vanilmandelic acid in the CNS metabolism of norephrine is discussed.     

INHIBITION OF RAT BRAIN PHENOL SULPHOTRANSFERASE IN VITRO BY NORADRENALINE and DOPAMINE METABOLITES1

Abstract— Kinetic parameters of the sulphotransferase reaction in rat brain were investigated in vitro at pH 7.4. Evidence is presented that the enzyme phenol sulphotransferase (EC 2.8.2.1) can be assayed with 4-methylumbelliferone or 3-methoxy-4-hydroxyphenylethyleneglycol as the substrate. Both assays give identical V_max values, whereas K_m values are 0.026 mm and 0.039 mm , respectively. Normetanephrine, metanephrine and the catecholamines adrenaline and dopamine, having a positive charge on the side chain at pH 7.4, do not inhibit 4-methylumbelliferone and 3-methoxy-4-hydroxyphenylethy-leneglycol sulphotransferase at this pH. Their deaminated metabolites 3,4-dihydroxyphenylethyleneglycol, 3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, 3-methoxy-4-hydroxyphenylethylene glycol, 3-methoxy-4-hydroxyphenethanol and 3-methoxy-4-hydroxyphenylacetic acid inhibit both the enzyme activities. The type of inhibition is noncompetitive with the exception of 3-methoxy-4-hydroxy-phenylethyleneglycol, which is a competitive inhibitor of 4-methylumbelliferone sulphation. 3-Methoxy-4-hydroxy-mandelic acid does not inhibit the enzyme activities. It is concluded that the catecholamines themselves are not sulphated by rat brain in vitro at pH 7.4.     

Acute Effect of Protein or Carbohydrate Breakfasts on Human Cerebrospinal Fluid Monoamine Precursor and Metabolite Levels

Patients with normal pressure hydrocephalus who had three lumbar punctures during 1 week ingested either water, a protein breakfast, or a carbohydrate breakfast 2.5 h before each of the lumbar punctures. The CSF was analyzed for biogenic amine precursors and metabolites. The protein meal raised CSF tyrosine levels, a finding consistent with animal data, but did not alter those of tryptophan or any of the biogenic amine metabolites. The carbohydrate meal increased CSF 3-methoxy-4-hydroxyphenylethylene glycol, an unexplained finding. The carbohydrate meal did not affect CSF tryptophan, tyrosine, 5-hydroxyindoleacetic acid, or homovanillic acid. Our results support the idea that in humans protein or carbohydrate meals do not alter plasma amino acid levels sufficiently to cause appreciable changes in CNS tryptophan levels or 5-hydroxytryptamine synthesis.     

Relationship Between Serotoninergic Measures in Blood and Cerebrospinal Fluid Simultaneously Obtained in Humans

The relationships between the concentration of serotonin (5-HT) and related metabolites in human blood and CSF have been studied. Plasma tryptophan (TP), 5-HT, 5-hydroxyindoleacetic acid (5-HIAA), and indoleacetic acid (IAA), whole-blood 5-HT, and CSF TP, 5-HT, 5-HIAA, IAA, homovanillic acid, and 3-methoxy-4-hydroxyphenylethylene glycol were determined in 35 unmedicated outpatients who underwent minor surgical operations and had no history of psychiatric or neurological illnesses. Significant correlations were found between the serotoninergic parameters analyzed in blood and CSF. Plasma free 5-HT correlated significantly with CSF 5-HT (r = 0.411, p less than 0.02), and plasma 5-HIAA correlated with the CSF 5-HIAA/5-HT ratio (r = 0.508, p less than 0.004). The concentration of 5-HIAA in CSF correlated with the plasma 5-HIAA/5-HT ratio (r = 0.405, p less than 0.026) (which can be taken as an index of monoamine oxidase type A activity in peripheral tissues) and with the platelet 5-HT/plasma 5-HT ratio (r = 0.375, p less than 0.05). The concentrations of IAA in CSF and plasma were strongly correlated (r = 0.899, p less than 0.001). The significance of these results and their relationship to the use of "in vivo" measures of 5-HT and related metabolites in plasma and platelets as an index of serotoninergic function in affective disorders are discussed.     

The effect of probenecid on catecholamine metabolites in human cerebrospinal fluid analyzed by mass fragmentography.

A gas chromatography-mass fragmentography (GC-MS) method was used to measure homovanillic acid (HVA), vanillylmandelic acid (VMA) and 3-methoxy-4-hydroxyphenethylene glycol (MHPG) in lumbar cerebrospinal fluid (CSF) of 31 patients before and after treatment with probenecid. HVA values increased from 24.6 ± 2.6 S.E.M. to 210 ± 17 ng/ml. The increase in VMA was from 1.06 ± 0.23 to 2.22 ± 0.17 ng/ml and that of MHPG was from 12.2 ± 1.08 to 15.6 ± 1.27 ng/ml. All increases were significant (p = < .01). The results for MHPG and HVA are consistent with results of earlier studies using different methods. VMA concentrations increased significantly but at a rate much lower than those of HVA.     

Relationship Between Plasma and Cerebrospinal Fluid Norepinephrine and Dopamine Metabolites in a Nonhuman Primate

Major and minor pathways of metabolism in the mammalian CNS result in the formation of 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) and normetanephrine (NMN) from norepinephrine (NE), and homovanillic acid (HVA) and 3-methoxytyramine (3-MT) from dopamine (DA), respectively. The correlational relationships between HVA and 3-MT and between MHPG and NMN in primate CSF and plasma have not been described. These relationships may help to elucidate the usefulness of CSF and plasma metabolites as indices of CNS NE and DA activity. In addition, because NMN is unlikely to cross the blood-brain barrier. CSF NMN concentrations would not be confounded by contributions from plasma, which is a major issue with CSF MHPG. We have obtained repeated samples of plasma and CSF from drug-naive male squirrel monkeys and have measured the concentrations of MHPG, HVA, NMN, and 3-MT to define their correlational relationships. For the NE metabolites, significant correlations were obtained for CSF MHPG and NMN (r = 0.806, p less than 0.001), plasma MHPG and CSF NMN (r = 0.753, p less than 0.001), and plasma and CSF MHPG (r = 0.776, p less than 0.001). These results suggest that CSF and plasma MHPG and CSF NMN may reflect gross changes in whole brain steady-state noradrenergic metabolism. Only a single significant relationship was demonstrated for the DA metabolites, with CSF 3-MT correlating with plasma HVA (r = 0.301, p less than 0.025). The results for the DA metabolites probably reflect regional differences in steady-state brain dopaminergic metabolism.     

Catecholamines increase in the urine of non-segmental vitiligo especially during its active phase

Neural factors appear to play a major role in the pathogenesis of vitiligo. To investigate the possible correlation between vitiligo and peripheral monoaminergic system activity, we used high-pressure liquid chromatography and electrochemical detector methods to evaluate the basal urine excretion values of catecholamines [norepinephrine (NE), epinephrine and dopamine (DA)], their relative metabolites [3-methoxy-4-hydroxyphenylglycol (MHPG), normetanephrine (NMN), metanephrine (MN), vanilmandelic acid (VMA) and homovanillic acid], as well as 5-hydroxyindoleacetic acid (5-HIAA), in 35 healthy subjects and in 70 patients, suffering from non-segmental vitiligo at different stages of the disease. Levels of NE, DA, NMN, MN, MHPG, VMA and 5-HIAA were found to be significantly higher in patients than in controls. The patients with progressive vitiligo (n = 56) presented increased urinary excretion values for all parameters (in particular, NE levels) than other patients. Interestingly, in patients at its more recent vitiligo onset (<1 yr), NE values were different to those of subjects affected from 1 to 5 yr and from 6 to 10 yr. This result was confirmed by the significant negative relationship detected between NE excretion values and disease duration. In both vitiligo and control groups, significant correlations were found between monoamines as well as between these monoamines and their metabolites. The increase in catecholamine turnover, mainly occurring at the onset of the disease, is probably due to the stress associated with the appearance of lesions. Moreover, considering that these compounds readily produce toxic free-radicals and that vitiliginous subjects have a defective free radical defence mechanism, they may also contribute to the disappearance of melanocytes in the early phases of vitiligo.     

Effect of ventral noradrenergic bundle lesions on concentrations of monoamine neurotransmitters and metabolites in several discrete areas of the rat brain

1. The effects of radiofrequency lesions of the ventral noradrenergic bundle (VNB) on monoamine and metabolite concentrations in several discrete areas of the rat hypothalamus were examined. Monoamines and metabolites were analyzed utilizing high-performance liquid chromatography coupled with electrochemical detection. 2. VNB lesions decreased the concentrations of norepinephrine (NE) and 3-methoxy-4-hydroxyphenylethylene glycol in all areas examined except in the ventromedial nucleus (VMN). Dopamine and 3,4-dihydroxyphenylacetic acid concentrations were selectively decreased in the dorsomedial nucleus (DMN) and also slightly decreased in the medial forebrain bundle following VNB lesions. Serotonin and 5-hydroxyindole-3-acetic acid concentrations were not altered by VNB lesion in any area examined. 3. The results indicate that the NE innervation to the hypothalamus is extensive and that NE in the VMN may not be derived from the VNB. The source of the DA innervation to the DMN may be located in or pass through the area affected by the VNB lesion.     

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.     

Effect of age on cisternal cerebrospinal fluid concentrations of monoamine metabolites in nonhuman primates

There are conflicting reports of the effects of aging on human neurotransmitter systems as estimated by monoamine metabolite concentrations in cerebrospinal fluid (CSF). These discrepancies may be due to sampling site, age or sex of the subjects or other variables that affect CSF metabolite determinations. Cisternal CSF concentrations of homovanillic acid (HVA), 3-methoxy-4-hydroxyphenyl-ethylene glycol (MHPG) and 5-hydroxyindoleacetic acid (5-HIAA), major metabolites of dopamine, norepinephrine and serotonin, respectively, were measured in rhesus monkeys (Macaca mulatta) of two age groups. Concentrations of HVA and MHPG were significantly lower in the older group of monkeys, whereas no changes in 5-HIAA were found. This supports the hypothesis that brain catecholamine concentrations decline with age.     

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.     

Liquid chromatography–electrospray tandem mass spectrometry of acidic monoamine metabolites

A new method based on liquid chromatography–tandem mass spectrometry has been developed for the determination of monoamine metabolites, i.e., homovanillic acid (HVA), vanilmandelic acid (VMA), 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) in human urine. Analytes were separated on a C₁₆ amide (5 cm, 5 μm) column and ionized by negative ion electrospray. Operating in the selected-reaction monitoring mode, linearity was established over three-orders of magnitude and limits of detection were in the range 30–70 μg/l. Precision calculated as RSD was within 0.8–5.2% for all intra- and inter-day determinations. The method was applied to the quantitative analysis of monoamine metabolites in 700 urine samples from occupationally (adults) and environmentally (both children and adults) exposed people living in areas with different soil contamination from lead. The urinary excretion of monoamine metabolites was significantly higher (P<0.001) in the subgroup of children living in polluted areas as compared to the control group (HVA, 6.03 vs. 4.57 mg/g creatinine; VMA, 5.33 vs. 4.37 mg/g creatinine; 5-HIAA 3.24 vs. 2.45 mg/g creatinine). In adults belonging to both groups of subjects occupationally and environmentally exposed, no differences were detected in the urinary concentration of monoamine metabolites. However, adults showed lower values of HVA (2.57 mg/g creatinine), VMA (2.17 mg/g creatinine) and 5-HIAA (2.09 mg/g creatinine) as compared to children groups.     

Phenol sulphotransferase: purification and characterization of the rat kidney and stomach enzymes

3'-Phosphoadenosine-5'-phosphosulphate-dependent enzymes that catalyse sulphation of p-nitrophenol have been purified from rat kidney and stomach mucosa by affinity chromatography on the p-hydroxyphenylacetic acid-agarose conjugate, by chromatography on DEAE-cellulose and Sephadex G-100. The phenol sulphotransferase (PST) from rat kidney had Mr of 69 000 and that of the stomach enzyme was 32 000. With p-nitrophenol as the sulphate acceptor, the pH optima were 6.4 for the stomach PST and 5.4 and 6.6 for the kidney enzyme. Both enzymes were inhibited by 2,6-dichloro-4-nitrophenol and phenylglyoxal, an arginine specific modifying reagent. Both enzymes readily sulphated p-nitrophenol, 2-naphthol, 1-naphthol and salicylamide and did not act on biogenic amines (e.g. epinephrine, norepinephrine, dopamine, serotonin), acid metabolites of catecholamines (e.g. 3,4-dihydroxyphenylacetic acid, homovanillic acid), and O-methylated metabolites of catecholamines. Only the stomach enzyme sulphated such catecholamine metabolites as homovanillic alcohol and 3-methoxy-4-hydroxyphenylglycol. In contrast to the brain enzyme, but similarly to the liver enzyme, the kidney and stomach phenol sulphotransferases appear to sulphate exogenous phenolic substrates in preference to potential endogenous substrates.     

Catecholamines and vitiligo.

The levels of some catecholamine metabolites, namely homovanillic acid (HVA), vanilmandelic acid (VMA), 3-methoxytyramine (MT), normetanephrine (NMN), metanephrine (MN), 3,4-dihydroxy mandelic acid (DOMAC), and 3,4-dihydroxy phenylacetic acid (DOPAC), have been evaluated in the 24 hr urines of 150 patients affected with different types of vitiligo and in 50 healthy age-matched individuals. The patients were divided into three groups according to the different phases of the disease. The first group included subjects affected either with the early active phase or with progressive increase in both number and/or the size of previous lesions. The second group included patients in whom no new lesions had appeared for between 4-8 months. In the third group the white areas had been stable for 1-5 years. The first and second groups showed values of HVA and VMA from 4 to 10 times and from 1/2 to 3 times higher respectively than those of controls, while no significant differences were found between the third group and controls. Our results clearly show that a significant increase of urinary levels of HVA and VMA, deriving respectively from dopamine and from norepinephrine and epinephrine characterizes the onset and the progressive active phases of vitiligo, irrespective of the type of distribution. The increased release of catecholamines from the autonomic nerve endings in the microenvironment of melanocytes in the affected skin areas might be involved in the etiopathogenesis of vitiligo through two main mechanisms: (1) a direct cytotoxic action of catecholamines and/or their o-diphenol catabolites; (2) an indirect action.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of creatinine, vanilmandelic acid, homovanillic acid and uric acid in urine by micellar electrokinetic chromatography

A simultaneous determination of vanilmandelic acid, homovanillic acid, creatinine and uric acid using capillary electrophoresis was investigated. The optimum conditions of buffer concentration, pH and surfactant concentration were studied, and high resolution was obtained using a 30 mM phosphate buffer (pH 7.0) containing 150 mM sodium dodecyl sulfate. The detection was by UV absorbance at 245 nm and the column was a fused-silica capillary of 67 cm×75 μm I.D.. The determination of these metabolites in human urine was completed within 15 min without any interferences.