Search

In order to make high-performance liquid chromatography (HPLC) more widely available at home and in small-scale settings, we have simplified two of its most costly modules, namely the pump and the detector. This should make the setup affordable for home or small laboratory use. A manual HPLC pump was constructed so as to fit into a caulk gun from a local hardware store enabling the generation of 100-150 bar of pressure. In order to limit the pressure drop during the running of a chromatogram, a pulse dampener was developed. We further modified the electrochemical detection (ECD) system so as to use a cheap boron-doped diamond electrode with an overlay of thin filter paper, causing an eluent flow over the electrode by wicking and gravity. Both the pump and the detector are at least ten times cheaper than conventional HPLC modules. Using a home-packed Jupiter Ⓡ Proteo reversed phase capillary column we show how this low-cost HPLC system generates well resolving chromatograms after direct injection of fresh urine. The ECD did not lose its sensitivity during regular use over more than half a year. For homovanillic acid (HVA), which is of medical interest, we measured a linear dynamic range of two orders of magnitude, a detection limit of HVA in the injected sample of 3 μM and a coefficient of variation <10%. The contribution to peak broadening by the detector was much smaller than the contributions by the injector and by the column. After consumption of table olives containing hydroxytyrosol (HT), its metabolite HVA in the corresponding urine could be measured quantitatively. An approach to quantify HT in table olives is presented, as well. This method provides a new tool for investigating physiology of oneself or of dear ones at home., Competing Interests: Declaration of Competing Interest The authors declare that JL and PvdS are board members of a non-profit Foundation for the promotion of the use of chromatography at home., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Wastewater-based epidemiology is a growing research field which provides valuable information on community drug use and chemical exposure. One parameter critical to estimations of drug use is the catchment area population. A population biomarker could be used to provide this information. This study evaluated the analytical suitability of three endogenous biomarkers of human activity: the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) which has previously been proposed and two further candidates, the catecholamine metabolites vanillylmandelic acid (VMA) and homovanillic acid (HVA). An analytical method involving derivatization was developed and validated for two candidates, 5-HIAA and HVA by liquid chromatography - mass spectrometry. The best performance was obtained for VMA as the underivatized analyte. The derivatized extracts produced a 100 times better sensitivity. The three neurotransmitter metabolites were evaluated as population biomarkers in wastewater samples. All were stable in sample, not lost upon filtration and showed stable inter-day mass loads over seven days for a metropolitan wastewater treatment plant. When applied to a small community during a festival period, mass loads of both HVA and VMA reflected the increase in the catchment population, whilst 5-HIAA proved to be more variable., Competing Interests: Declaration of Competing Interest All authors have participated in (a) conception and design, or analysis and interpretation of the data; (b) drafting the article or revising it critically for important intellectual content; and (c) approval of the final version. This manuscript has not been submitted to, nor is under review at, another journal or other publishing venue. The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript, (Copyright © 2019 Elsevier B.V. All rights reserved.)

A new method for the simultaneous determination of two tumour biomarkers, homovanillic (HVA) and vanillylmandelic acid (VMA), using flow injection analysis (FIA) with amperometric detection (AD) at a commercially available boron doped diamond electrode (BDDE) was developed. It was found that this method is suitable for the determination of HVA (in the presence of VMA) and VMA (in the presence of HVA) in optimum medium of Britton-Robinson buffer (0.04 mol L -1 , pH 3.0). Calibration dependences consist of two linear parts for both biomarkers, the first one being in the concentration range from 1 to 10 μmol L -1 and the second one from 10 to 100 μmol L -1 (with obtained LODs 0.44 μmol L -1 for HVA and 0.34 μmol L -1 for VMA, respectively). To minimize any negative effects related to the passivation of the working electrode, suitable cleaning pulses (+2.4 V for 30 s) were imposed on the working electrode after each measurement. An attempt to use FIA with multiple pulse amperometric detection to determine both analytes in one run was not successful. Changing potentials in short intervals in multiple pulse detection probably results in mutual interaction of analytes and/or products of their electrochemical oxidation, thus preventing the application of this approach., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Two novel phenanthrenoids, juncuenin H (1) and dijuncuenin B (2), together with eight known phenanthrenoids, effusol (3), dehydroeffusol (4), juncusol (5), dehydrojuncusol (6), juncuenin B (7), dehydrojuncuenin B (8), juncuenin A (9), and dehydrojuncuenin A (10), were isolated from the underground parts of Juncus setchuenensis. The structures of the compounds were determined by 1D and 2D NMR and mass spectroscopy. The anxiolytic activities of compounds 1, 6, 9, and 10 were evaluated. In order to explore the mechanisms underlying their anxiolytic activities, the levels of serotonin (5-HT), dopamine (DA), and their metabolites in the cerebral cortex and hippocampus of mice treated with compound 1 were determined by quantitative mass spectrometry. The mice treated with compound 1 had significantly lower levels of 5-HT, 3-methoxytyramine (3-MT), 5-hydroxyindole-3-acetic acid (5-HIAA), homovanillic acid (HVA), and 3, 4-dihydroxyphenylacetic acid (DOPAC) in the cerebral cortex than those of the vehicle control-treated mice. The levels of HVA and 5-HIAA in the hippocampus were also significantly lower in the mice treated with compound 1 than in the control group mice. These results suggest that the metabolic changes, reflected in the levels of DA and/or 5-HT, may contribute to the anxiolytic activity of the phenanthrenoids studied herein., (Copyright © 2019. Published by Elsevier B.V.)

Neuroblastoma is a pediatric neuroblastic tumor arising in the sympathetic nervous crest cells. A high grade of Neuroblastoma is characterized by a high urinary excretion of homovanillic acid and dopamine. In this work l-leucine modified Sol-Gel-Carbon electrode was used for a sensitive voltammetric determination of homovanillic acid and dopamine in urine. The electrochemical response characteristics were investigated by cyclic and differential pulse voltammetry; the modified electrode has shown an increase in the effective area of up to 40%, a well-separated oxidation peaks and an excellent electrocatalytic activity. High sensitivity and selectivity in the linear range of 0,4-100μML -1 of homovanillic acid and 10-120μML -1 of dopamine were also obtained. Moreover, a sub-micromolar limit of detection of 0.1μM for homovanillic acid and 1.0μM for the dopamine was achieved. Indeed, high reproducibility with simple preparation and regeneration of the electrode surface made this electrode very suitable for the determination of homovanillic acid and dopamine in pharmaceutical and clinical preparations. The mechanism of homovanillic acid and the electrochemical oxidation at l-leucine modified Sol-Gel-Carbon electrode is described out the B3P86/6-31+G(d,p) level of theory as implemented in Gaussian software., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Different grades of genuine and counterfeit Fraxinus excelsior exudates, marketed as natural sweeteners or mild laxatives, were evaluated for their proximate composition and for saccharidic, organic acids, lipidic and phenolic profile by means of GC-MS and (1)H NMR. Genuine samples contained mannitol (39-48 g/100 g, according to the grade), fructose (9-16 g/100 g), glucose (2-3.7 g/100 g), sorbitol (0,5-0,6 g/100 g), galactose (0.02-0.74 g/100 g), oligosaccharides as mannotriose (13-22 g/100 g) and stachyose (1-11 g/100 g), and traces of myo-inositol, mannose, sucrose. On the contrary, counterfeit samples contained mostly mannitol and sorbitol, with traces of fructose, glucose and mannose. Differences in ash, total polyphenolic content and fatty acid composition allowed a quick identification of counterfeit products, confirmed by a distinct mono-, oligosaccharidic and phenolic pattern. Elenolic acid (63-1628 mg/kg), tyrosol (15-774 mg/kg), homovanillic acid (2,39-52.8 mg/Kg), dopaol (0.8-63 mg/kg), pinoresinol (4.2-18.5 mg/kg) and fraxetin (0.25-11.64 mg/kg), albeit showing a wide concentration range, were the most abundant substances detected in the phenolic fraction of Fraxinus manna, while esculetin, p-hydroxybenzoic acid, 4-hydroxyphenacetic acid, 3,4 hydroxybenzoic acid, hydroxy-pinoresinol, medioresinol and siringaresinol were present in low amounts. The polyphenolic profile may be used as a marker for authentication and should be considered in the evaluation of nutritional and health properties ascribed to Fraxinus manna., (© 2013.)

An extensive literature has documented adverse effects on mental health in anabolic androgenic steroids (AAS) abusers. Depression seems a common adverse reaction in AAS abusers. Recently it has been reported that in a rat model of AAS abuse stanozolol induces behavioural and biochemical changes related to the pathophysiology of major depressive disorder. In the present study, we used the model of AAS abuse to examine possible changes in the monoaminergic system, a neurobiological substrate of depression, in different brain areas of stanozolol-treated animals. Wistar rats received repeated injections of stanozolol (5mg/kg, s.c.), or vehicle (propylene glycol, 1ml/kg) once daily for 4weeks. Twenty-four hours after last injection, changes of dopamine (DA) and relative metabolite levels, homovanilic acid (HVA) and 3,4-dihydroxy phenylacetic acid (DOPAC), serotonin (5-HT) and its metabolite levels, 5-hydroxy indolacetic acid (5-HIAA), and noradrenaline (NA) amount were investigated in prefrontal cortex (PFC), nucleus accumbens (NAC), striatum (STR) and hippocampus (HIPP). The analysis of data showed that after chronic stanozolol, DA levels were increased in the HIPP and decreased in the PFC. No significant changes were observed in the STR or in the NAC. 5-HT and 5-HIAA levels were decreased in all brain areas investigated after stanozolol exposure; however, the 5-HIAA/5-HT ratio was not altered. Taken together, our data indicate that chronic use of stanozolol significantly affects brain monoamines leading to neurochemical modifications possibly involved in depression and stress-related states., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Electrochemical detection is often used to detect catecholamines and indolamines in brain samples that have been separated by conventional reverse-phase high performance liquid chromatography (HPLC). This paper presents the transfer of an existing chromatographic method for the determination of monoamines in brain tissues using 5 μm granulometry HPLC columns to columns with a particle diameter less than 3 μm. Several parameters (repeatability, linearity, accuracy, limit of detection, and stability of samples) for this new ultrafast high performance liquid chromatography (UHPLC) method were examined after optimization of the analytical conditions. The separation of seven compounds, noradrenaline, dopamine and three of its metabolites, dihydroxyphenylacetic acid, homovanillic acid, and 3-methoxytyramine, and serotonin and its metabolite, 5-hydroxyindole-3-acetic acid was analyzed using this UHPLC-electrochemical detection method. The final method, which was applied to brain tissue extracts from mice, rats, and cats, decreased analysis time by a factor of 4 compared to HPLC, while guaranteeing good analytical performance., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A sensitive assay method was developed for a parallel, rapid and precise determination of dopamine and its metabolites, homovanillic acid, 3-methoxytyramine and 3,4-dihydroxyphenylacetic acid, from brain microdialysates. The method consisted of a pre-treatment step, freeze-drying (lyophilization), to concentrate dopamine and its metabolites from the microdialysates, and a detection step using liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In particular, the reaction monitoring mode was selected for its extremely high degree of selectivity and the stable-isotope-dilution assay for its high precision of quantification. The developed method was characterized by the following parameters: the precision of the developed method was determined as ≥88.6% for dopamine, ≥89.9% for homovanillic acid, ≥86.1% for 3-methoxytyramine and ≥88.1% for 3,4-dihydroxyphenylacetic acid; the mean accuracy was determined as ≥88.2% for dopamine, ≥88.3% for homovanillic acid, ≥85.9% for 3-methoxytyramine and ≥88.6% for 3,4-dihydroxyphenylacetic acid. The developed method was compared to (1) other combinations of pre-treatment methods (solid phase extraction and nitrogen stripping) with LC-MS and (2) another detection method, liquid chromatography, with electrochemical detection. The novel developed method using combination of lyophilization with LC-ESI-MS/MS was tested on real samples obtained from the nucleus accumbens of rat pups after an acute methamphetamine administration. It was proven that the developed assay could be applied to both a simultaneous analysis of all four substrates (dopamine, homovanillic acid, 3-methoxytyramine and 3,4-dihydroxyphenylacetic acid) in microdialysis samples acquired from the rat brain and the monitoring of their slight concentration changes on a picogram level over time following methamphetamine stimulus., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Biogenic amines (norepinephrine, dopamine, homovanillic acid, serotonin and 5-hyroxyindole acetic acid) were measured by HPLC method in adult F1 generation rats' brain regions (brainstem, hypothalamus, hippocampus, striatum and frontal cortex), whose mothers (P generation) were treated with vitamin A or vitamin D neonatally (hormonal imprinting). Many significant differences were found, related to the maternally untreated controls. In the earlier studied P generation females, vitamin A consistently influenced the serotonerg system (5HIAA), while vitamin D the dopaminerg system (DA or HVA). Vitamin A imprinting always resulted in reduced, while that by vitamin D always in increased tissue levels. In the present case (directly untreated F1 generation) the transgenerational effect was not unidirectional, however biogenic amine tissue levels were strongly disturbed and brain-area dependent. The results call attention to the transgenerational effect of hormonal imprinting in the case of receptor level acting vitamins which are frequently used in the most imprinting-sensitive period (perinatally) of human life and suggests that caution is warranted.

Corticotropin-releasing factor (CRF) plays an important role in stress responses and is mediated through two subtypes of receptors, CRF receptor type 1 (CRFR1) and CRF receptor type 2 (CRFR2). Each CRF receptor might have a different function through several neurotransmitter systems; however, the mechanism remains unclear. To clarify the role of each receptor in dopamine (DA) metabolism, we measured the change of extracellular concentrations of DA and the metabolites in the ventromedial hypothalamus (VMH) that played important roles in the stress response of freely moving female rats in response to the direct administration of comparative CRFR1 selective agonist, CRF, or CRFR2 selective agonist, Urocortin II (Ucn II), into the brain region. Administration of 10 microg CRF increased extracellular concentrations of DA compared with 2 microg CRF immediately after injection, and this effect was not observed after 60 min of 10 microg CRF injection. On the other hand, this change did not always occur after Ucn II administration. These results suggest that the activation of CRFR1, but not CRFR2, modulates the release of DA in VMH.

Administration of green tea or caffeine was shown previously to inhibit ultraviolet B light-induced carcinogenesis in SKH-1 mice, and this effect was associated with a reduction in dermal fat. In the present study, oral administration of 0.6% green tea (6 mg tea solids/ml) or 0.04% caffeine (0.4 mg/ml; equivalent to the amount of caffeine in 0.6% green tea) as the sole source of drinking fluid to SKH-1 mice for 15 weeks increased total 24 hr locomotor activity by 47 and 24%, respectively (p<0.0001). Oral administration of 0.6% decaffeinated green tea (6 mg tea solids/ml) for 15 weeks increased locomotor activity by 9% (p<0.05). The small increase in locomotor activity observed in mice treated with decaffeinated green tea may have resulted from the small amounts of caffeine still remaining in decaffeinated green tea solutions (0.047 mg/ml). The stimulatory effects of orally administered green tea and caffeine on locomotor activity were paralleled by a 38 and 23% increase, respectively, in the dermal muscle layer thickness. In addition, treatment of the mice with 0.6% green tea or 0.04% caffeine for 15 weeks decreased the weight of the parametrial fat pad by 29 and 43%, respectively, and the thickness of the dermal fat layer was decreased by 51 and 47%, respectively. These results indicate that oral administration of green tea or caffeine to SKH-1 mice increases locomotor activity and muscle mass and decreases fat stores. The stimulatory effect of green tea and caffeine administration on locomotor activity described here may contribute to the effects of green tea and caffeine to decrease fat stores and to inhibit carcinogenesis induced by UVB in SKH-1 mice.

We established a sensitive and simultaneous determination method of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) using HPLC-fluorescence detection. This method adopted the column-switching system, which included an on-line extraction of carboxylic acids by a strong anion-exchange column followed by separation on an ODS column, coulometric oxidation, fluorogenic reaction with ethylenediamine, and fluorescence detection. The detection limits were 50 and 100 fmol/injection for DOPAC and HVA, respectively (a signal-to-noise ratio of 3). The method was applicable to 50 microl of rat kidney microdialysate with a sufficient accuracy and precision. The concentrations of DOPAC and HVA in rat kidney microdialysate were 131+/-29 and 404+/-44 nM, respectively (n=5). This is the first report of DOPAC and HVA quantified in rat kidney microdialysate.

To clarify the neurochemical backing of aurothioglucose (ATG)-induced obesity in mice, we investigated lesion sites, hypothalamic neurotransmitters and c-Fos-like immunoreactivity (Fos-IR). At day 2 after ATG, tissue loss or cells death was observed in several parts of the ventral area of the ventromedial hypothalamic nucleus (VMH), and the dorsal area of arcuate nucleus and in the nucleus of the solitary tract (NTS). However, the greater part of the VMH was retained. Body weight began to increase in week 1. Hypothalamic serotonin (5-HT) and the metabolites were increased at day 2. The contents of acetylcholine, norepinephrine and dopamine in the hypothalamus showed no significant change. In week 1, the area shown tissue loss was compacted and plugged up. In the control group, most obvious c-Fos-like immunoreactive region was paraventricular nucleus (PVN). At day 2, Fos-IR was observed around destroyed regions in the hypothalamus and NTS, but few Fos-IR was found in the other regions including PVN. The Fos-IR around destroyed regions diminished after week 1. In week 3, Fos-IR in the PVN increased. These results suggest that the development of ATG-induced obesity cannot be attributed to solely VMH destruction. The restoration processes of the neuronal dysfunction involving PVN seem to play an important role in the development of obesity. NTS lesion and 5-HT system might contribute to decrease in food intake for several days after ATG.

The anterior piriform cortex (APC) is essential for the anorectic reactions to an amino acid-imbalanced diet, and it also responds to repletion of the limiting amino acid. In the present study, we examine the dynamic changes of the interstitial dopamine metabolites in the APC following feeding of either an amino acid-corrected or -imbalanced diet. Microdialysates, collected from the APC, were analyzed using HPLC with electrochemical detection. The concentrations were 19.7 +/- 4.8 microg/L for 3, 4-dyhydroxyphenylacetic acid and 25.1 +/- 4.4 microg/L for homovanillic acid, respectively, in the baseline dialysates. After diet treatments, no significant changes occurred in 3, 4-dyhydroxyphenylacetic acid in the corrected (n = 7) or imbalanced (n = 9) groups vs. the basal group (n = 7). However, after feeding the threonine-corrected diet, the concentration of homovanillic acid was significantly less (P < 0.01) than after the basal and imbalanced diets. The homovanillic acid level in the corrected group was already significantly lower than in the basal group by 20 min (P < 0.05), and reached its lowest level at 70 min (P < 0.05). The concentrations of homovanillic acid in the corrected group remained at this low level until the end of the experiment. The present results introduce the idea that the dopaminergic system is involved in the feeding responses to essential amino acid repletion.

Repeated daily intraperitoneal (i.p.) administrations of cadmium (CdCl2, 1 mg/kg per day for 5 days) increased striatal dopamine (DA) release (180% of controls) and turnover (150% of controls) in 13-day-old rats. Cd treatment also increased striatal metallothionein (MT) content (161%), Cd (127%) and lipid peroxidation (LPO, 190%). In addition, Cd treatment decreased striatal tyrosine hydroxylase (TH) activity (-28%), and such an effect may result from D-2 receptor blockade as a consequence of excessive dopamine release, since sulpiride (a specific D-2 receptor antagonist) administration to Cd-treated rats abolished the effect of Cd on TH. No effect was observed on striatal monoamine oxidase (MAO) activity. Dexamethasone (Dx) treatment increased striatal MT content and caused no effect on either DA release or turnover. However, Dx administration prevented the effects caused by Cd, including the increased DA release and enhanced striatal lipid peroxidation. These results indicate that toxic effects on the brain are to be expected as a result of Cd exposure and that Dx administration can attenuate them.

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.

An isocratic liquid chromatographic method with electrochemical detection for the determination of L-3,4-dihydroxyphenylalanine, dopamine, norepinephrine, epinephrine, serotonin, and their major metabolites, 3,4-dihydroxyphenylacetic acid, 4-hydroxy-3-methoxyphenylacetic acid and 5-hydroxyindole-3-acetic acid in chicken brain tissue is described. Chickens were killed at different ages, the brains were quickly frozen and 300-microm cryostat sections were made. From these sections, two to six tissue micropunches (1 mm in diameter) were punched out from 20 different areas of the hypothalamus and homogenated in 100 microl 0.1 M perchloric acid which included 0.01% cysteine as antioxidant. Fifty-microl supernatants were injected directly onto the LC system, separated on a 3-microm Phase II ODS column (100 x 3.2 mm I.D.) and detected by an electrochemical detector at a potential of +0.75 V. Standard curves, recoveries, analytical precision and detection limits were investigated for each monoamine neurotransmitter and its metabolites. The method was applied to study the influence of food restriction on the concentration of monoamine neurotransmitters in different brain areas, known to be involved in feeding and reproductive behaviour of female broiler chickens. Over 1000 micropunched tissue samples from ad libitum fed and food-restricted female broiler chickens were analyzed. Our results provide a possible role for catecholamines and indolamines in the altered feeding and reproductive behaviour of the broiler chicken.

Mercury is a neurotoxic agent that produces different effects on the brain. In the present work, the effects of chronic doses of methyl mercury (MeHg) were studied on the dopaminergic system of the rat striatum, using microdialysis coupled to high-performance liquid chromatography in order to quantify the in vivo release of dopamine (DA) and its acidic metabolites DOPAC and HVA. The administration of an equivalent total dose of 6 mg/kg of MeHg induced significant increases in the striatal release of DA and/or its acidic metabolites, independently of the pattern of administration. These effects are discussed on the base of the release and the metabolization of DA. In conclusion, the effect of MeHg administered under these experimental conditions on the in vivo release of DA and its metabolites seems to have a dose-dependent component and seems to be an accumulative process., (Copyright 1997 Academic Press.)

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 L-cysteine and ascorbic acid. Using this assay, the neurotransmitters and their metabolites could easily be determined in microdialysates obtained from different rat brain areas.

Introduction: Following the pioneering Japanese experience, several European and North American groups implemented pilot studies on screening infants for neuroblastoma, considering the possibility of demonstrating a decrease in mortality rate. In France, a 5-year (1990-1994) feasibility study on neuroblastoma screening at the age of 4 months was initiated in the Rhône district (1.5 M inhabitants, 26,000 births per year)., Methods: Vanillylmandelic (VMA) and homovanillic (HVA) acids were measured by HPLC, and creatinine (Cr) by the Jaffé's kinetic method on Technicon RA-XT. VMA and HVA were expressed as microgram/mg of Cr. The method was assessed with both a daily intra-laboratory control and a sample of urine obtained from a national quality control organism., Results and Discussion: The overall participation rate for the 5-year period was 82.2%. Out of 105,293 infants tested from 128,126 births, 12 NB cases were discovered with screening (screened cases) and 1 case was discovered with a late performed test (at 13 months of age). Six neuroblastomas were found clinically before the age of 4 m. Two cases were missed because the parents did not perform the test. Three children with normal tests at screening were false-negative cases: two of them were found secreting at diagnosis, while one remained non-secretory at diagnosis and later on. Otherwise, thirty-five false-positive tests were found. Biochemical observations are discussed. It is too early to reach clinical conclusions from this screening program on neuroblastomas as it is presently being followed up.

An extract of Ginkgo biloba leaves (EGb) was administered by gastric probe to Wistar female rats, and urine and faeces samples were collected for 5 days and whole blood samples were withdrawn every 30 min for 6 h. After purification with SPE C18 cartridges, the samples were analysed by reversed-phase LC-diode array detection (LC-DAD) for residual flavonoid glycosides, aglycones and metabolites. No glycosides or aglycones were detected in urine, faeces or blood and extensive degradation of EGb flavonoids within 24 h was detected. Among the seven different phenylalkyl acids detected by LC-DAD, 3,4-dihydroxyphenylacetic acid (I), hippuric acid (II), 3-hydroxyphenylacetic acid (III), homovanillic acid (IV) and benzoic acid (VII) were directly confirmed by on-line mass spectrometry using an electrospray interface (ES-MS). Peaks V and VI needed to be collected and separately examined and they were found to be 3-(4-hydrophenyl)propionic acid and 3-(3-hydrophenyl)propionic acid, respectively. As further evidence, the identity of metabolites I, II, III, IV, V and VII was confirmed by co-chromatography with authentic standards.

The dopamine metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), was found to be decreased in the anterior cingulate cortex of individuals with schizophrenia compared with normal controls. The finding does not appear to be solely related to the presence of antipsychotic medications, age, postmortem interval, or freezer time. No changes in norepinephrine and its metabolites were found.

A microbore high-performance liquid chromatographic method with electrochemical detection was developed for the measurement of small quantities of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, in microdialysis samples from rat striatum. The limit of detection of dopamine was in the low nmol/l range (10 fmol/40 microliters dialysate). Standard mixtures of dopamine and its metabolites were stored in tubes made of polypropylene, soda-lime glass or borosilicate glass. Dopamine and homovanillic acid were stable in all three types of container for 24 h providing they were kept at an acidic pH. However, 3,4-dihydroxyphenylacetic acid was only stable in tubes made of polypropylene or borosilicate glass. Basal levels of dopamine in the dialysate obtained from rats which had been chronically implanted with a microdialysis probe in the anterior striatum were stable for one to three days following probe implantation. However, the levels of 3,4-dihydroxyphenylacetic acid and homovanillic acid in the dialysate decreased rapidly with time after the first day. This indicates that the synthesizing capacity of the neurons is compromised by the microdialysis probe after day one, and that microdialysis experiments in freely moving animals should be confined to the day immediately following probe implantation.

A growing body of evidence suggests that an interference with dopamine (DA) transmission disrupts maternal behavior in the rat. The present brain microdialysis study was therefore conducted to investigate whether infants can modulate ventral striatal DA release in mother rats. There was a significant rise in the extracellular concentrations DA, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) in the ventral striatum when mothers were reunited with their litters following separation overnight. Nursing was the predominant behavior during this phase of the experiment. More active behaviors were elicited by soiling pups with flowerpot earth, and this was accompanied by further increases in DA, DOPAC, HVA, and 5-HIAA. It is suggested that pup-induced stimulation of ventral striatal DA release facilitates parental responses such as pup retrieval.

The selective, reversible monoamine oxidase (MAO) A inhibitor brofaromine inhibits serotonin (5-HT) uptake in animal models in vitro and in vivo. We investigated whether such an effect can be demonstrated at clinical doses in humans by treating three groups of six volunteers with either placebo, 15 mg phenelzine three times a day, or 75 mg brofaromine twice a day in a 2-week experiment. As an indirect, although relevant parameter, binding of 3H-paroxetine to the 5-HT uptake sites on blood platelets was assessed. Moreover, whole-blood 5-HT as a measure of platelet 5-HT, and serum homovanillic acid (HVA) to tentatively estimate MAO inhibition, were determined. Brofaromine reduced 3H-paroxetine binding to platelets compared with placebo by 20%-25% throughout the treatment period, significance being reached on the last treatment day. In contrast, phenelzine tended to increase 3H-paroxetine binding. Both drugs increased whole-blood 5-HT to approximately 140%-150%. Brofaromine moderately and on some days significantly decreased serum HVA, whereas phenelzine only tended to do so. Our results suggest that brofaromine at the clinically used dosage of 150 mg/day does indeed inhibit 5-HT uptake, as evidenced by measurements of 3H-paroxetine binding to platelets.

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.

To clarify the involvement of the central nervous system in responses of organisms to noise and whole-body vibration, the activity of dopamine (DA) neuron systems was estimated by examining DA turnover rates in various discrete regions of the brains of rats exposed to noise (broad band, 102 dB) or whole-body vibration (20 Hz, 4G) for 90 min. Plasma corticosterone level (COR) was determined simultaneously as an index of stress-induced autonomic-nervous and endocrine functions. Noise and whole-body vibration increased both COR and DA turnover rates (shown by an increase of homovanillic acid (HVA) and/or HVA/DA ratio) in the frontal cortex (FC) and the nucleus accumbens (NAc). Only noise increased the DA turnover rate in the amygdala (AMY). Furthermore, strong positive correlations of the HVA/DA ratios in the FC and the NAc with COR were observed in rats exposed to noise or vibration. These results suggest that the responses of organisms to noise and whole-body vibration may be critically mediated by cerebral DA systems, in particular by the mesocortical DA system, indicating that change of DA in the AMY can be considered a specific response to noise.

Gas chromatography-high-resolution selected-ion monitoring mass spectrometry was used to analyze catecholamine metabolites in rat brain microdialysate. Dialysate samples were collected in vials containing stable isotope analogues of homovanillic acid (HVA), 3-methoxy-4-hydroxyphenylglycol (MHPG) and 5-hydroxyindoleacetic acid (5HIAA) and analyzed as their trimethylsilyl derivatives. The metabolite levels were monitored at 20-min intervals throughout the time course of the experiment, beginning immediately after surgery and implantation of the dialysis probe and ending 4 h after amphetamine treatment. The levels of HVA were observed to decrease after amphetamine treatment, while those of MHPG and 5HIAA did not change significantly.

Detection and identification modes for the rapid, selective, highly sensitive and simultaneous determination of catecholamines, indoleamines and related metabolites by high-performance liquid chromatography (HPLC) with series of coulometric working electrodes (CWE) were investigated. Five detection modes were examined: (1) oxidative single mode using a single CWE, (2) oxidative screen mode using a series of two CWE, (3) redox mode using a series of two CWE, (4) redox-reductive screen mode using a series of three CWE and (5) redox-reductive screen mode using a series of four CWE. For the highly sensitive detection of catechol compounds, oxidative single, redox and redox-reductive screen modes were suitable. Oxidative single and oxidative screen modes were better than the other modes for indole and o-methylated catechol compounds. For the selective detection of these compounds, however, the redox-reductive screen mode was best. The specific ratio obtained in HPLC with the redox or redox-reductive screen mode is useful as an index for identification purposes. These findings suggest that HPLC with the redox-reductive screen mode of detection is applicable to neuroscience studies.

A technique is presented for the separation and detection in amniotic fluid of various substances associated with catecholamine metabolism. Monoamines and their metabolites were separated using reversed-phase ion-pair high-performance liquid chromatography. Detection and quantification were performed electrochemically. The retention times of 28 standards associated with the monoamines and their precursors and metabolites were evaluated with 14 different eluents. On the basis of the retention times of each standard and the modification of the retention times of the various peaks detected in amniotic fluid, the following substances were identified in this biological fluid: 4-hydroxy-3-methoxyphenylacetic acid, 5-hydroxyindoleacetic acid, 3,4-dihydroxyphenylacetic acid, 4-hydroxy-3-methoxyphenylglycol, epinephrine, 4-hydroxy-3-methoxymandelic acid, octopamine, tyrosine and tryptophan.

Following 10 daily pairings of multiple conditioned stimuli with injection of cocaine (15 mg/kg), the presentation of the stimuli alone elicited behaviors in rats similar to those induced by cocaine. The behaviors included increased duration or frequency of rearing, sniffing, head bobbing, and horizontal locomotor activity (crossing). The level of the conditioned response for several of these behaviors approximated that induced by the drug itself. The conditioned drug effect showed decay over 15 days but little extinction during 4 daily trials. Brain concentrations of the dopamine metabolites, homovanillic acid and dihydroxyphenylacetic acid, were similar in the conditioned and pseudoconditioned control groups in both the caudate and mesolimbic areas. The behavioral results demonstrate that, in a classical conditioning paradigm, previously neutral stimuli can elicit behaviors similar to those induced by cocaine and that certain conditioned responses show time related decline. This agrees with the reported conditioning of amphetamine's behavioral effects but differs in terms of the action on brain dopamine turnover.

We describe an improved high-performance liquid chromatographic method for the determination of tyrosine, 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, tryptophan and homovanillic acid in cerebrospinal fluid and nerve tissue, using the new microbore cartridges with 5 micron average particle size. The first four substances are quantified fluorometrically and the last two electrochemically. Both detectors are connected to the same integrator through a relay which can be switched as required. Data are collected in an on-line personal computer and evaluated statistically. An improvement in the method for extraction and separation of catecholamines is also reported.

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.

The N-tetrahydrofurfuryl benzomorphan, MK-2034 is a prototype kappa agonist analgesic. Dehydrogenation and addition of a methyl group into the N-furfuryl side chain (MR-1353) results in a 2-fold decrease in analgesic potency, a 12-fold decrease in affinity for the mu receptor, a 5-fold decrease in affinity for the delta receptor and a 60-fold decrease in affinity for the kappa receptor. Both compounds possess intermediate sodium ratios for 3H-naloxone binding; however, unlike MR-2034, MR-1353 possesses a bell-shaped dose response curve for elevations in striatal dopamine metabolites. These data indicate that MR-1353 is an agonist/antagonist analgesic and suggest that the high kappa affinity of MR-2034 is responsible for the potent analgesia.

Two procedures using liquid chromatography with electrochemical detection are described for the determination of dopamine (DA) and its two acidic metabolites, homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC), in subregions of rat striatum and nucleus accumbens. A strong cation-exchange column was used for DA analysis and a C18 reversed-phase column was used for the analysis of the metabolites. Effects of pH, temperature and percentage of methanol on the retention time of HVA and DOPAC were studied. Levels of these compounds in the subregions of rat stratum and nucleus accumbens are reported.

A procedure is described for the concurrent assay of free norepinephrine, epinephrine, dopamine, vanillylmandelic acid and homovanillic acid in physiological fluids using high-performance liquid chromatography with electrochemical detection. The column packing is an octadecyl-bonded silica. A single mobile phase containing 1-octanesulphonate is used for the assay of catecholamines and for the assay of the acidic metabolites. An efficient sample preparation scheme is presented for the isolation of the catecholamines and their acidic metabolites from the same sample aliquot. Catecholamines are extracted by ion exchange on small columns and adsorption on alumina, using dihydroxybenzylamine as an internal standard. Vanillylmandelic acid and homovanillic acid are recovered from the combined loading and washing effluents of the ion-exchange column by a solvent extraction procedure. Recovery of catecholamines averages 67%. The limit of detection for individual catecholamines is ca. 30 pg. Recoveries of vanillylmandelic acid and homovanillic acid average 77% and 87%, respectively. The use of the same mobile phase for the concurrent assay of catecholamines and their acidic metabolites considerably increases the throughput of samples in the chromatographic system by eliminating the time-consuming column-equilibration periods.

The response of the plasma dopamine (DA) metabolite, homovanillic acid (HVA), to two DA agonists was investigated in the rat. Apomorphine administered i.p. (2 mg/kg) produced, within one hour, a significant decrease in plasma HVA. The response of plasma HVA to apomorphine was also investigated after pretreatment with debrisoquin, a drug which selectively blocks peripheral HVA production by inhibition of MAO. Pretreatment with debrisoquin did not significantly alter the decrement in plasma HVA produced by apomorphine indicating that a substantial portion of the plasma HVA response to apomorphine is due to the drug's action on brain. Bromocriptine (2 mg/kg) was also found to produce a significant decrease in plasma HVA. Since the response of brain HVA to DA agonists reflects the sensitivity of the DA receptor, the plasma HVA response to DA agonists might be a practical method of assessing brain DA receptor sensitivity in humans.

Acute effects of a single i.p. injection of toluene on circadian rhythms of sleep-wakefulness were investigated in rats which were chronically implanted with EEG and EMG electrodes for polygraphic recordings. The toluene injection produced an initial increase in wakefulness (W) and a subsequent increase in slow-wave sleep (SWS) during the dark period. In an attempt to clarify mechanisms of these biphasic effects of toluene on sleep-wakefulness rhythms, brain monoamines and their metabolites were determined at the times of the initial increase in W and the increased SWS. The initial increase in W was associated with an increase in cortical NA, MHPG and 5-HT together with a decrease in cortical 5-hydroxyindoleacetic acid (5-HIAA), while the increased SWS during the dark period was associated with an increase in 5-HIAA and a concomitant decrease in 3-methoxy-4-hydroxyphenylglycol (MHPG). The toluene-induced changes in sleep-wakefulness seemed to be manifested at lower blood levels of toluene than the behavioral signs of central nervous system (CNS) depression. These biphasic effects of toluene on circadian sleep-wakefulness rhythms are discussed in terms of the reciprocal interactions between central 5-HT and NA neurons.

Plasma prolactin (PRL) and homovanillic acid (HVA) levels, and urinary HVA and dopamine (DA) excretion, were measured in groups of unmedicated autistics, medicated autistics, and normal controls. No significant differences were found between unmedicated autistics and normal controls in plasma PRL and HVA levels. Excretion rates of urinary HVA and DA were also similar in the unmedicated autistic and normal subjects. Plasma PRL and HVA, as well as urinary HVA excretion, were significantly increased in the autistics on neuroleptic medication compared to the unmedicated autistics. A significant correlation (r = 0.46, p = less than 0.05) was observed between dose of neuroleptics and plasma PRL values; the correlation (r = 0.42) between neuroleptic dose and plasma HVA levels approached significance (p = 0.06). In contrast, no differences were observed in urinary DA excretion between medicated and unmedicated autistics. In general, the findings indicate that peripheral indices of dopamine functioning are normal in autistic subjects.

The acute effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium ion (MPP+) on mouse locomotor activity and striatal dopamine (DA) and 5-hydroxytryptamine (5-HT) levels were investigated. A single dose of either MPTP (10-30 mg/kg, i.p.) or MPP+ (5-20 ug/mouse, i.c.v.) decreased locomotor activity 10-40 min after injection: this locomotor effect was significantly suppressed by either pretreatment with nomifensine or 1-deprenyl alone, or by the combination of desmethylimipramine and 6-hydroxydopamine. Pretreatment with clorgyline did not suppress this behavior and a single dose of haloperidol enhanced the effect. The striatal levels of DA, 3-methoxytyramine and 5-HT increased in parallel with the decrease in locomotor activity caused by MPTP or MPP+. In contrast, levels of 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindoleacetic acid were decreased by injection of either MPTP or MPP+. Possible mechanism(s) of the behavioral and biochemical changes caused by the acute actions of MPTP and MPP+ with respect to their neurotoxic effects on the nigrostriatal DA system are discussed.

The effects of acute and chronic administration of haloperidol on homovanillic acid (HVA) in plasma and the brain were examined in the rat. Acute haloperidol treatment (1 mg/kg) resulted in highly significant elevations in HVA within 30 min and produced a maximal increase of HVA in 3-6 hr in both plasma and the whole brain. The response of brain HVA to increasing doses (0.05-30 mg/kg) of haloperidol showed an inverted U pattern. Plasma HVA showed a very flat response to lower doses (less than or equal to 5 mg/kg) of haloperidol and a dramatically elevated one to higher doses (greater than or equal to 10 mg/kg). Haloperidol produced a parallel increase in plasma and brain HVA at lower doses (less than or equal to 2 mg/kg) only. After chronic administration of haloperidol for up to 28 days, the response of HVA in plasma correlated mainly with, but tolerated later than, those in the whole brain and the olfactory tubercle.

The administration of MPTP to man and monkey has been shown to cause a neurotoxic effect on the nigrostriatal dopamine system. MPTP was injected in C57-BL black mice, 36 mg per kg for 7 days, which resulted in permanent reduction of dopamine and serotonin levels in the striatum. In the mice pretreated with PLG, although the striatal dopamine level was also reduced, mean dopamine and serotonin levels were significantly higher than in mice given MPTP alone. It is concluded that PLG could protect at least partially the neurotoxic effect of MPTP.

A sensitive and simple method for the simultaneous quantitation of homovanillic acid and vanilmandelic acid in small samples (0.1-1.0 ml) of urine, serum, plasma and cerebrospinal fluid is described. The stable dimethylthiophosphinyl methyl ester derivatives are detected specifically by mass fragmentography using the respective deuterated compounds as internal standards. Gas chromatographic separation is performed on a fused-silica DB-1 capillary column combined with a cold injection system for large sample volumes. Linear response curves and a detection limit of 1 ng/ml are obtained. The method has been applied to the localization of pheochromocytoma by selective determination of venous vanilmandelic acid.

Infusion of MPTP (0.2-0.8 mg/kg) into the right internal carotid artery of monkeys produces toxin-induced injury to the right nigrostriatal pathway with sparing of other dopaminergic neurones on the infused side and with negligible or little injury to the opposite, untreated side. There are contralateral limb dystonic postures, rigidity, and bradykinesia, but the animals are able to eat and maintain health without drug treatment. Spontaneous motor activity is attended by circling towards the injured side, whereas treatment with L-DOPA/-carbidopa or apomorphine stimulates circling towards the intact side. Dopamine and dopamine metabolite levels are normal in the left caudate and putamen, but markedly depressed on the right (MPTP-treated) side. This animal hemiparkinsonian model will be useful in studies of volitional movement control, drug treatments of Parkinson's disease, and functional efficacy of brain tissue implants.

We have recently found that, besides an increase in aggression, isolation increases social interaction in NIH Swiss mice. In the present study the effect of isolation in other behavioral paradigms and their relation to brain monoamine concentrations were investigated. Mice, isolated for 0-20 days, were tested in the holeboard test of exploration and locomotor activity, the plus-maze test of anxiety, and Porsolt's swim test of behavioral 'despair.' Isolation reduced exploratory head-dipping, increased locomotor activity and increased the preference of mice for the open arms of the plus-maze. The immobility time in the swim test was shortened in mice isolated for 2 or 5 days, suggesting an improved ability to cope with stressful situations. Monoamine assays failed to show significant changes in the noradrenaline, dopamine, HVA, 5-HT or 5-HIAA contents in the amygdala, hypothalamus, hippocampus or brain stem. The results indicate that isolation of NIH Swiss mice for less than three weeks induces several behavioral changes, but is not particularly stressful.

Central catecholamine concentrations were determined in autopsy samples from older schizophrenic and control subjects for both the hypothalamus and the nucleus accumbens. The results of these analyses and demographic variables were regressed on antemortem measures of cognitive function and mood state. In the hypothalamus, there are significant direct relationships of homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) with depressed mood, as measured by an adaptation of the Hamilton Rating Scale for depression. In the nucleus accumbens, dopamine (DA) and MHPG had significant inverse relationships with antemortem cognitive function, as measured by an adaptation of the Mini Mental State Exam. Results in this sample indicate that after controlling for age, the catecholamine concentrations accounted for approximately 50% of the variance in the antemortem measures of mood or cognition, depending on the loci measured.