Label-free silver triangular nanoplates for spectrophotometric determination of catecholamines and their metabolites.

A novel method towards spectrophotometric determination of catecholamines and their metabolites differing in their functional groups has been developed. This method is based on a change in morphology of silver triangular nanoplates upon the action of cateсholamines and their metabolites, which is manifested by the decrease of the nanoparticle local surface plasmon resonance (LSPR) band intensity or its shift to the short-wavelength region of the spectrum. The shift value of the LSPR band or the change of its intensity increases with increasing concentration of catecholamines or their metabolites, which is proposed for their spectrophotometric determination. The limits of detection of catecholamines and their metabolites under selected conditions increase in the series homovanillic acid < vanillylmandelic acid < L-epinephrine < L-norepinephrine < dopamine and are 0.25, 1.2, 3.0, 64, and 130 µmol L-1, respectively. The selectivity of the proposed method was assessed using vanillylmandelic acid as example. It was found that the determination of vanillylmandelic acid does is not interfered in the presence of 4000-fold excess of Na+, K+, CH3COO-, and 1000-fold excess of Mg2+, Ca2+, Al3+, NO3-. The method also allows for the selective determination of vanillylmandelic acid in the presence of a 1000-fold excess of structurally related substances that do not contain either a catechol fragment or an electron donor substituent. The proposed approach was successfully applied to the determination of catecholamines in pharmaceuticals and artificial urine. Graphical abstract.

A sensitive analytical method for the measurement of neurotransmitter metabolites as potential population biomarkers in wastewater.

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.

Determination of tumour biomarkers homovanillic and vanillylmandelic acid using flow injection analysis with amperometric detection at a boron doped diamond electrode.

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.

Specific Urinary Metabolites in Malignant Melanoma.

Background and objectives: Melanin, which has a confirmed role in melanoma cell behaviour, is formed in the process of melanogenesis and is synthesized from tryptophan, L-tyrosine and their metabolites. All these metabolites are easily detectable by chromatography in urine. Materials and Methods: Urine samples of 133 individuals (82 malignant melanoma patients and 51 healthy controls) were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC). The diagnosis of malignant melanoma was confirmed histologically. Results: Chromatograms of melanoma patients showed increased levels of 5,6-dihydroxyindole-2-carboxylic acid, vanilmandelic acid, homovanilic acid, tryptophan, 5-hydroxyindole-3-acetic acid, and indoxyl sulphate compared to healthy controls. Concentration of indoxyl sulphate, homovanilic acid and tryptophan were significantly increased even in the low clinical stage 0 of the disease (indoxyl sulphate, homovanilic acid and tryptophan in patients with clinical stage 0 vs. controls expressed as medium/ interquartile range in µmol/mmol creatinine: 28.37/15.30 vs. 5.00/6.91; 47.97/33.08 vs. 7.33/21.25; and 16.38/15.98 vs. 3.46/6.22, respectively). Conclusions: HPLC detection of metabolites of L-tyrosine and tryptophan in the urine of melanoma patients may play a significant role in diagnostics as well as a therapeutic strategy of melanoma cancer.

Neuroblastoma.

Neuroblastoma is the most common extracranial solid tumour occurring in childhood and has a diverse clinical presentation and course depending on the tumour biology. Unique features of these neuroendocrine tumours are the early age of onset, the high frequency of metastatic disease at diagnosis and the tendency for spontaneous regression of tumours in infancy. The most malignant tumours have amplification of the MYCN oncogene (encoding a transcription factor), which is usually associated with poor survival, even in localized disease. Although transgenic mouse models have shown that MYCN overexpression can be a tumour-initiating factor, many other cooperating genes and tumour suppressor genes are still under investigation and might also have a role in tumour development. Segmental chromosome alterations are frequent in neuroblastoma and are associated with worse outcome. The rare familial neuroblastomas are usually associated with germline mutations in ALK, which is mutated in 10-15% of primary tumours, and provides a potential therapeutic target. Risk-stratified therapy has facilitated the reduction of therapy for children with low-risk and intermediate-risk disease. Advances in therapy for patients with high-risk disease include intensive induction chemotherapy and myeloablative chemotherapy, followed by the treatment of minimal residual disease using differentiation therapy and immunotherapy; these have improved 5-year overall survival to 50%. Currently, new approaches targeting the noradrenaline transporter, genetic pathways and the tumour microenvironment hold promise for further improvements in survival and long-term quality of life.

On-line concentration and separation of cationic and anionic neurochemicals by capillary electrophoresis with UV absorption detection.

This paper presents on-line simultaneous concentration and separation of cationic and anionic neurochemicals by capillary electrophoresis (CE) with UV absorbance spectroscopy. Neurochemical stacking exploits differences in local electric field and viscosity between the sample zone and the background electrolyte (BGE). To achieve these discontinuous conditions for CE, neurochemicals were prepared in a solution containing 1mM formic acid and 20% (v/v) acetonitrile (ACN). The capillary was filled with a solution of 500 mM Tris-borate (TB) and 10% (v/v) glycerol. The buffer vial contained 500 mM TB and 0.5% (v/v) polyethylene oxide (PEO). After injecting a large sample volume, PEO enters the capillary by electro-osmotic flow (EOF). Anionic neurochemicals stacked at the sample zone and PEO-containing BGE boundary. Simultaneously, cationic neurochemicals were concentrated at the boundary between the sample zone and the glycerol-containing BGE. The concentrated cationic neurochemicals were baseline separated in the presence of glycerol, mainly due to hydrogen bonding interactions between glycerol hydroxyl groups and the neurochemical's hydroxyl and amino groups. Under optimal stacking conditions, we observed the following: (a) the maximum sample injection volume was 720 nL; (b) the limit of detection for signal-to-noise ratio of 3 ranged from 14.7 to 313.4 nM; and (c) sensitivity enhancements compared to normal injection (15 nL) ranged from 116 to 281-fold. We evaluated the proposed method by the determination of neurochemicals in urine samples.

Coupling reaction and complex formation for the spectrophotometric determination of physiologically active catecholamines in bulk, pharmaceutical preparations and urine samples of schizophrenic patients.

A simple and sensitive spectrophotometric method for the determination of three catecholamines namely dopamine hydrochloride (DO.HCl), dobutamine hydrochloride (DOB.HCl) and vanillymandelic acid (VMA), in both pure form or in their commercially available pharmaceutical formulations or urine samples of schizopherinic patients is described. The method is based on the reaction of diazotized 4-aminoantipyrine (4-AAP) with catecholamines in a basic medium (pH = 10-11) to yield pink-coloured products having absorption maxima at 500, 505 and 480 nm for DO.HCl, VMA and DOB.HCl, respectively. Before carrying out Beer's Law, different experimental conditions, such as time, temperature, sequence of addition, and pH are optimized. The coloured species obeyed Beer's Law in the range of 47.4-417.2, 59.45-445.9 and 67.57-405.4 mg/L for DO.HCl, VMA and DOB.HCl, respectively. The molar absorptivity values as obtained from Beer's Law data were found to be 2.979 x 10(4), 4.39 x 10(4) and 1.036 x 10(4) L mol(-1) cm(-1), while Sandell's sensitivity values were observed to be 3 x 10(-3), 4.4 x 10(-3) and 1.3 x 10(-3) microg cm(2-) for DO.HCl, VMA and DOB.HCl, respectively. Common excipients did not interfere with the proposed method. The results of the proposed method compare favourably with those of official methods. The proposed method offers simplicity, reliability, rapidity, and accuracy compared to the existing methods.

Application of quartz crystal nanobalance for simultaneous determination of vanillylmandelic and homovanillic acids by a net analyte signal-based method.

Homovanillic acid (HVA) and vanillylmandelic acid (VMA) were selectively determined by quartz crystal nanobalance sensor in conjunction with net analyte signal (NAS)-based method called HLA/GO. An orthogonal design was applied for the formation of calibration and prediction sets including HVA, VMA, and some common and structurally similar urine compounds. The selection of the optimal time range involved the calculation of the NAS regression plot in any considered time window for each test sample. The searching of a region with maximum linearity of NAS regression plot (minimum error indicator) and minimum of predicted error sum of squares value was carried out by applying a moving window strategy. Based on the obtained results, the differences on the adsorption profiles in the time range between 1 and 300 s were used to determine mixtures of compounds by HLA/GO method. Several figures of merit like selectivity, sensitivity, analytical sensitivity, and limit of detection were calculated for both compounds. The results showed that the method was successfully applied for the determination of VMA and HVA.

Mínimas alterações hormonais em paciente com grande feocromocitoma / Slight hormonal alterations in a patient with a large pheochromocytoma

Relatamos caso clínico no qual o paciente apresentou sintomas sugestivos de feocromocitoma, grande tumor (maior que 50 g) e mínima alteração laboratorial, exemplificando uma armadilha diagnóstica. Um homem de 31 anos apresentou dois episódios de abdômen agudo, sendo o último acompanhado por cefaléia, hipertensão arterial, rubor facial, sudorese e palidez cutânea. Em outra internação, o paciente apresentava hipertensão arterial sustentada e arritmia cardíaca. Em relação aos testes laboratoriais, apenas o ácido vanil-mandélico foi levemente alterado. Cintilografia com MIBG foi realizada e sugeriu a presença de grande massa adrenal compatível com feocromocitoma. Uma amostra histopatológica da peça foi obtida após cirurgia e confirmou esta hipótese. Esse caso sugere que em pacientes que possuem sintomas sugestivos de feocromocitoma, mesmo com valores normais de catecolaminas plasmáticas e metanefrinas urinárias, devemos considerar as possibilidades de um grande tumor metabolizando catecolaminas em seu interior ou que sofreu necrose hemorrágica.

Development of a gas chromatography silicon-based microsystem in clinical diagnostics.

The accurate determination of biological parameters by means of rapid, on-line measurements at low-concentrations is an important task within the fields of pharmaceutical screening and medical diagnostic. Nevertheless, in biological samples, the analytes of interest are present as minor components in complex mixtures and with interfering species. Biosensors are the best candidates for these applications providing a direct solution to this need of accuracy, but their intrinsic selectivity often excludes all the other components in the sample. A separation step introduced prior to the sensing component could allow both the increase of selectivity with respect the interfering species and the identification of a large spectrum of molecular components in the sample. This work reports the development of a silicon-based integrated separation microsystem for gas chromatography aimed to biomedical applications, with particular emphasis to monitor the homovanillic acid (HVA) and vanillylmandelic acid (VMA) ratios in mass population screening for neuroblastoma diagnosis and prognosis. The miniaturised system consists of two main modules: (i) a metal oxide semiconductor detector and (ii) a micromachined separation capillary column. As first step, the metal oxide semiconductor capability to detect HVA and VMA has been demonstrated. Then, a technology for a silicon separation capillary microcolumn including the on-chip gas sensor housing has been proposed and a first prototype has been developed. The proposed microsystem is an analytical device with biosensing capabilities for diagnostic and biomedical applications, which yield an electronic signal proportional to the concentration of a specific analyte or group of analytes.

Comparative values of catecholamines and metabolites for the diagnosis of neuroblastoma.

UNLABELLED: In order to diagnose neuroblastomas, we assayed the three adrenal hormones and five of their metabolites by high-performance liquid chromatography followed by electrochemical detection in urine samples of 395 children with tumours of unknown nature (including 29 neuroblastomas). The analytes (expressed as analyte/creatinine ratios) performances were determined by calculating the related sensitivity and specificity and receiver operating characteristics curves within the different age groups. Normetanephrine (NME), vanillylmandelic and homovanillic acids (VMA, HVA) were the best analytes. Calculated optimal thresholds (best specificity/sensitivity couples) of these analytes minimised the number of false-positive diagnosis. CONCLUSION: combined determination of normetanephrine with vanillylmandelic acid (0-1 year) or homovanillic acid (1-5 years and 5-10 years) further enhanced the diagnostic power up to 100% sensitivity and specificity of the testing depending on the age group. Plotting individual levels (normetanephrine versus vanillylmandelic acid or homovanillic acid) allowed a rapid visual analysis that would have missed only one small low grade non-secreting tumour.

Voltammetric sensor for vanillylmandelic acid based on molecularly imprinted polymer-modified electrodes.

Despite the increasing number of applications of molecularly imprinted polymers (MIPs) in analytical chemistry, the construction of a biomimetic voltammetric sensor remains still challenging. This work investigates the development of a voltammetric sensor for vanillylmandelic acid (VMA) based on acrylic MIP-modified electrodes. Thin layers of MIPs for VMA have been prepared by spin coating the surface of a glassy carbon electrode with the monomers mixture (template, methacrylic acid, a cross-linking agent and solvent), followed by in situ photopolymerisation. After extraction of the template molecule, the peak current recorded with the imprinted sensor after rebinding was linear with VMA concentration in the range 19-350 microg ml(-1), whereas the response of the control electrode is independent of incubation concentration, and was about one-tenth of the value recorded with the imprinted sensor at the maximum concentration tested. Under the conditions used, the sensor is able to differentiate between VMA and other closely structural-related compounds, such as 3-methoxy-4-hydroxyphenylethylene glycol (not detected), or 3,4- and 2,5-dihydroxyphenilacetic acids, which are adsorbed on the bare electrode surface but not at the polymer layer. Homovanillic acid was detected with the imprinted sensors after incubation, indicating that the presence of both methoxy and carboxylic groups in the same position as in VMA is necessary for effective binding in the imprinted sites. Nevertheless, both species can be differentiated by the oxidation potential. It can be concluded that MIP-based voltammetric electrodes are very promising analytical tool for the development of highly selective analytical sensors.

[Biochemical diagnosis of pheochromocytoma and neuroblastomas]. / Diagnostic biochinique du phéochromocytome et du neuroblastome.

Pheochromocytoma and neuroblastoma are distinct tumours, but their biological diagnosis is based on secretion increase of one or several catecholamines. Assays have to be very sensible and specific for an early diagnosis. 24 hours urinary catecholamines and metabolites are currently measured, but technical improvements permit plasma metanephrine assay, an excellent indicator of pheochromocytoma. HPLC coupled to electrochemical detection represents the most efficient methodology. After a review of urinary and plasma assay methods, the authors show usual values of catecholamines, metanephrines, HVA and VMA, according to ages, and give examples of results encountered in classical or not tumours and in falsely positive cases. Urinary metanephrine assay is the most sensible and specific in biological diagnosis of pheochromocytoma, while catecholamines and VMA assays lack of sensibility. Results have to be given by 24 hours and by creatinine ratio. Metanephrine assay can be performed also in plasma and exhibits the same interest. However, in urine as in plasma, in case of renal failure, results cannot be interpreted. Neuroblastoma biological diagnosis is based classically on HVA, VMA, and dopamine assays, nowadays only in 24 hours urine (or in urinary micturition for screening), and results are also expressed as creatinine ratio. But even if several assays are advisable, 5% of the neuroblastoma cases do not produce increased catecholamine values. In some cases, metanephrine assay could be of interest. After the age of 12 months, clinical expression of neuroblastoma is dramatic in 70% of cases. So, a biological screening has been experimented in several countries including France. A French translation of the consensus conference report (1998) is appended, which shows the complexity of neuroblastoma screening. Now, there is no evidence that early tumour detection by screening lessens the mortality rate, but a weak benefit is not excluded.

Evaluation of catecholamine metabolites, mIBG scan, and bone marrow cytology as response markers in stage 4 neuroblastoma.

BACKGROUND: The early biological response has been proved an excellent predictor in acute lymphoblastic leukemia and nephroblastoma. We asked whether catecholamine metabolites, mIBG scan, and bone marrow evaluation might be relevant response markers in disseminated neuroblastoma. PROCEDURE: Three hundred sixty-seven unselected stage 4 neuroblastoma patients treated according the German cooperative trial NB90 were entered into the study. Catecholamine plasma and urine levels were centrally determined by gas chromatography/ mass spectrometry. Bone marrow cytology and mIBG scans were evaluated by local investigators. RESULTS: At diagnosis, mIBG scan was positive in 306 patients (92%), borderline in seven patients (2%), and negative in 19 patients (6%). Bone marrow aspirates were cytologically positive in 292 patients (84%) and negative in 57 patients (16%). Plasma catecholamine levels were elevated in 79% (206 of 260 patients.), urinary levels in 91% (307 of 338 patients). The outcome of patients with normalized mIBG scan after four courses of chemotherapy [5 year EFS (event free survival) 0.22 +/- 0.07] was not superior to the outcome of patients with still abnormal uptake (5 year EFS 0.30 +/- 0.05). The event free survival of patients with still positive bone marrow aspirates after four courses (0.16 +/- 0.06) was inferior to the EFS of patients with negative bone marrow aspirates (0.26 +/- 0.04, P = 0.0054). Urinary catecholamine normalization after four cycles of chemotherapy (5 year EFS 0.35 +/- 0.06 versus 0.26 +/- 0.10) had no influence on outcome, whereas plasma catecholamine normalization after the first (5 year EFS 0.40 +/- 0.09 versus 0.14 +/- 0.07, P= 0.0364) or the fourth cycle (5 year EFS 0.35 +/- 0.06 versus 0.26 +/- 0.10, P = 0.0242) indicated a better outcome. CONCLUSIONS: These data show that serial plasma catecholamine levels and bone marrow aspirates in the course of the disease are useful tools in predicting outcome.

Adrenergic hyperactivity and metanephrine excess in the nucleus accumbens after prefrontocortical dopamine depletion.

Selective dopamine depletion within the medial prefrontal cortex in rats is known to enhance dopamine and norepinephrine levels in the nucleus accumbens and to induce characteristic behavioral disturbances. The present study was designed to determine levels of adrenaline, apart from dopamine and norepinephrine, and metabolites in the nucleus accumbens after prefrontocortical dopamine depletion. Prefrontocortical dopamine depletion was carried out by injecting 6-hydroxydopamine, and it was validated through: the emergence of behavioral disturbances such as amphetamine-induced stereotypies, spontaneous motor hyperactivity, and enhanced "anxiety-like" responses and through postmortem quantification of catecholamine levels by using high-performance liquid chromatography. The findings indicated that lesioned rats exhibited more oral stereotypies after amphetamine, were hyperlocomotive, and showed more pronounced anxiety-like behaviors than controls. Following prefrontocortical dopamine depletion, postmortem concentrations of dopamine and norepinephrine, along with the metabolites 3,4-dihydroxyphenylacetic acid and vanillylmandelic acid, were reliably enhanced in the nucleus accumbens as expected, and dopamine turnover was decreased. Furthermore the nucleus accumbens contained higher levels of adrenaline and its transmethylated metabolite metanephrine. To sum up, prefrontocortical dopamine depletion induces motor and emotional disturbances in rats and alters the neurochemical profile of the nucleus accumbens, not only inducing dopaminergic and noradrenergic hyperactivity but also leading to adrenaline and metanephrine excess.

A 5-year (1990-1994) neuroblastoma screening feasibility study in France. Methodology and preliminary observations.

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.

The influence of total sleep deprivation on urinary excretion of catecholamine metabolites in major depression.

To elucidate the influence of total sleep deprivation (TSD) on catecholaminergic neurotransmission, which is assumed to be disturbed in depression, 9 depressive patients collected consecutive 24-h urine samples prior to (baseline), during (TSD) and following total sleep deprivation (post-TSD). Urine samples were analysed for total MHPG (3-methoxy-4-hydroxyphenylglycol), conjugates of MHPG (glucuronide and sulfate), excretion of HVA (homovanillic acid) and VMA (3-methoxy-4-hydroxymandelic acid). TSD increased the urinary excretion of MHPG-sulfate as a marker of the central norepinephrine metabolism and the excretion rates of VMA and HVA as indices of the peripheral catecholamine metabolism. Patients with higher VMA values prior to TSD reacted worse, and the VMA increase due to TSD was positively correlated with the response. The results demonstrate that TSD, besides acting as a stimulus on the peripheral sympathetic nervous system, influences central nervous noradrenergic neurotransmission, as reflected by the increase of MHPG-sulfate.

Effects of N-ethyl,N-nitrosourea on monoamine concentrations and metabolizing enzymes in mouse brain regions.

N-ethyl,N-nitrosourea is a well known alkylating agent and produces central nervous system-specific tumors in several laboratory animal species. In the present study, young male CD-1 mice were treated by i.p. injections of 0, 2, 8, or 32 mg/kg body weight N-ethyl,N-nitrosourea, twice a week for 3 weeks. Endogenous levels of brain monoamine neurotransmitters and their selected metabolites; norepinephrine (NE), dopamine (DA), 5-hydroxytryptamine (5-HT), vanillylmandelic acid (VMA), dihydroxyphenyl acetic acid (dopac), homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA), and dihydroxyphenylalanine (dopa) were measured using HPLC with electrochemical detection. N-ethyl,N-nitrosourea treatment caused an increase of NE and 5-HT in the hypothalamus and striatum. Increased levels of 5-HIAA were noticed in the same brain regions. Elevated levels of NE were also observed in the cerebral cortex, medulla oblongata and the cerebellum. The major metabolite of NE, VMA, was decreased in several brain regions to non-detectable levels. Histopathological examination of brain tissue did not reveal any pathologic lesions. The increases in brain amines were associated with increased activity of tryptophan hydroxylase in the hypothalamus and corpus striatum. Dopa-decarboxylase was elevated in the cerebral cortex at a low dose of N-ethyl,N-nitrosourea only, whereas the monoamine oxidase activity was unaltered. Results indicated that N-ethyl,N-nitrosourea exposure may cause an elevation of steady state levels of various biogenic amines in brain areas and these changes to some extent are consistent with the altered activity of metabolizing enzymes.