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New analyser for the determination of urinary vanillylmandelic acid, homovanillic acid and creatinine.

We have developed a novel analyser for the determination of vanillylmandelic acid, homovanillic acid and creatinine in urine by high-performance liquid chromatography using three different types of column, cation-exchange, anion-exchange and reversed-phase and the column-switching technique. In this procedure, 10 microliters of intact urine were directly injected into the cation-exchange column, and the pass-through fraction, containing vanillylmandelic acid and homovanillic acid was transferred to the anion-exchange column by column switching. The fraction partially purified from endogenous urinary impurities on the anion-exchange column was transferred to the reversed-phase column. Vanillylmandelic acid and homovanillic acid, separated by the solvent-switching technique, were detected fluorimetrically (excitation at 280 nm, emission at 320 nm). Then, creatinine eluted from the cation-exchange column is spectrophotometrically detected (254 nm). Therefore the successive simultaneous analysis of the three could be performed in a 15-min cycle; the within-assay coefficients of variation for normal and patients' urines were less than 1.9%, less than 3.3% and less than 3.0% for vanillylmandelic acid, homovanillic acid and creatinine, respectively; the recoveries averaged 100, 103 and 100%, respectively, for supplemented urines.

Anions

Improved colorimetry of urinary 3-methoxy-4-hydroxyphenylacetic acid (homovanillic acid).

Homovanillic acid is an important metabolite of dopamine, and a high proportion of patients with neuroblastoma excrete increased amounts of it in their urine. When this diagnosis is suspected, both homovanillic acid and vanilmandelic acid should be measured, because such a combined measurement reportedly leads to detection of 95% of cases. Although rapid and reliable chemical methods are available for vanilmandelic acid, the same is not true for homovanillic acid. We report here a colorimetric method for homovanillic acid that reasonably fills this void and that can be used in most clinical laboratories. In addition, we present normal values determined for children of various age groups.

Adolescent

The estimation of 3,4-dihydroxyphenylacetic acid, homovanillic acid and homo-isovanillic acid in nervous tissue by gas-liquid chromatography and electron capture detection.

1 A gas chromatographic method using electron capture detection is described for the estimation of three acidic metabolites of dopamine, 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid, HVA), 3,4-dihydroxyphenylacetic acid (DOPAC) and 3-hydroxy-4-methoxyphenylacetic acid (homo-isovanillic acid, iso-HVA). The method is based on the formation of the trifluoroacetyl-hexafluoroisopropyl derivatives of the three acids. 2 The method has been applied to the estimation of DOPAC, HVA and iso-HVA in tissues from the central and peripheral nervous systems.

3,4-Dihydroxyphenylacetic Acid

Simultaneous liquid chromatographic determination of vanillylmandelic acid, homovanillic acid, and 5-hydroxy-3-indoleacetic acid in urine, using isocratic elution and electrochemical detection.

A method for the simultaneous measurement of vanillylmandelic acid, homovanillic acid and 5-hydroxyindoleacetic acid in urine is described. Based on reversed-phase liquid chromatography with electrochemical detection, the procedure employs isocratic elution, thus making it suitable for use in the less well-equipped clinical or research laboratory. A simple extraction of the acids from acidified urine into ethyl acetate, is followed by evaporating to dryness a portion of the organic layer, and redissolving the residue in chromatographic mobile phase. Up to 20 samples can be analysed in a single working day. The method is validated and the results obtained are compared with reference methods. The cause of contamination of the glassy carbon surface of the working electrode is investigated, and a simple electrochemical pretreatment is described that overcomes this problem. Finally, the extra clinical information that can be derived from multi-metabolite assays is considered.

Biogenic Amines

Effects of halothane anaesthesia on extracellular levels of dopamine, dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindolacetic acid in rat striatum: a microdialysis study.

The effects of halothane anaesthesia on striatal extracellular levels of dopamine, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindolacetic acid (5HIAA) were investigated in microdialysis experiments. Induction of anaesthesia was accompanied by a rapid increase in dopamine levels and a slower increase in DOPAC and HVA. 5HIAA was not affected. The reduction of dopamine levels induced by apomorphine 0.05 mg/kg appeared with a shorter latency in conscious rats than in anaesthetised rats but the maximum decrease was unaffected by anaesthesia. The decreases in dopamine and DOPAC induced by alpha-methyl-p-tyrosine 50 mg/kg were affected in opposite directions by halothane: the dopamine reduction was more pronounced while the DOPAC reduction was less pronounced in anaesthetized than in conscious animals. In no case was a qualitative shift in the response observed. It is concluded that halothane may influence the levels of dopamine as well as the response to dopaminergic drugs.

3,4-Dihydroxyphenylacetic Acid

Simultaneous liquid-chromatographic determination of urinary vanillylmandelic acid, homovanillic acid, and 5-hydroxyindoleacetic acid.

We describe a liquid-chromatographic method for quantifying, simultaneously by a single procedure, vanillylmandelic acid (VMA), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) in urine. After solvent extraction of acidified urine, the analytes were chromatographed on a C8 column, with use of a mobile phase of phosphate buffer (20 mmol/L, pH 4.0) and methanol with a variable gradient elution, and detected fluorometrically. We report the analytical recovery, sensitivity, precision, working linear range, and potential for interference from similar molecules or drugs. The results of such tests demonstrate that the proposed method is sensitive and reproducible. It is, furthermore, easy to perform, and thus is suitable for use in the clinical laboratory.

Chromatography, High Pressure Liquid

Comparative study of urinary excretion rates of para-hydroxy-mandelic acid, homovanillic acid, vanylmandelic acid in cirrhotic patients with and without encephalopathy.

A comparative study of urinary excretion of octopamine, dopamine, and noradrenaline catabolic products, respectively parahydroxymandelic acid (PHMA) homovanillic acid (HVA) and vanylmandelic acid (VMA) was carried out in 27 cirrhotic patients with (11) and without (16) porto-systemic encephalopathy (P.S.E). PHMA, HVA and VMA were significantly higher in patients with PSE, and there was a positive correlation between PHMA and HVA, and between PHMA and VMA. Higher excretion of PHMA in patients with PSE strongly suggests an increased metabolism of octopamine. HVA and VMA increased excretion, and the positive correlations could be explained by the depletion of stored dopamine and noradrenaline. These observations support the hypothesis that octopamine acts as a false neurotransmitter, and the resulting depletion of dopamine and noradrenaline could explain the neuropsychic phenomena of PSE, and the awakening effect of L. Dopa treatment.

Aged

Vanillylmandelic acid, homovanillic acid, and catecholamines in urine of infants with neuroblastoma 6- to 11-month-old.

The amounts of vanillylmandelic acid (VMA), homovanillic acid (HVA), and catecholamines (adrenaline, noradrenaline, and dopamine) in 24-hr urine specimens of 10 infants with neuroblastoma detected through mass screening in Sapporo City (patient group) were compared with those of 24 healthy infants (control group). The ages of the infants of both groups ranged from 6 to 11 months, which agrees with the age when mass screening is performed. For VMA and HVA, the mean value plus two or three times the standard deviation of the control group clearly separated the two groups in the units of mg/day, microgram/mg of creatinine, mg/kg/day, and mg/m2/day. The cut off values of 24 micrograms/mg of creatinine for VMA and 25 micrograms/mg of creatinine for HVA are now being used by us for assaying 24-hr urine specimens. No significant difference was found between the groups in levels of catecholamines, but their data were thought to be useful as normal values, since few published data regarding their normal values are available.

Catecholamines

Neurophysiological measures related to levels of 5-hydroxyindoleacetic acid, homovanillic acid and tryptophan in cerebrospinal fluid of psychiatric patients.

In 26 patients with psychiatric disorders, EEG, and visual averaged evoked responses (VAER) were recorded. In the analysis of the EEG, the mean integrated amplitude (MIA), its within patient variance (WPV), and the coefficient of variation CV=(square root of WPV/MIA) X 100 were calculated. Patients were grouped as augmenters on reducers according to their tendency in the VAER recording to augment or to reduce the intensity of incoming signals with increasing stimulus intensity. The content of monoamine metabolites, 5-hydroxyindoleacetic acid, 5-HIAA, and homovanillic acid (HVA), and --for 17 patients-- tryptophan, in the cerebrospinal fluid was determined. In the analysis of the results, WPV in the alpha-band was found to be significantly correlated to HVA (r=0.45, p less than 0.02) and to 5-HIAA (r=0.34, p less than 0.05). In a multiple regression analysis, the correlation between WPV and HVA was found to be the most important. CV was found to be signfiicantly correlated to tryptophan (r=0.68, p less than 0.01). In the analysis of the results of the VAER, reducers were found to have higher levels of 5-HIAA and tryptophan and significantly higher values of HVA. The results are discussed in relation to earlier neurophysiological and biochemical theories about psychiatric patients.

Adult

Concentration of 5-hydroxyindoleacetic acid, homovanillic acid, and tryptophan in the cerebrospinal fluid of depressed patients before and after ECT.

Cerebrospinal fluid (CSF) levels of 5-hydroxyindoleacetic acid (5HIAA), tryptophan (TRYP), and homovanillic acid (HVA), were determined prior to electroconvulsive therapy (ECT) and after an average course of 6.7 ECT in six endogenous depressed patients. Depression rating scale (DRS) scores were also obtained by a "blind" research psychiatrist before and after ECT at the time of each lumbar puncture. ECT markedly reduced DRS scores but did not significantly alter CSF levels of 5HIAA, TRYP, or HVA. We found no correlation between ECT-induced DRS score reductions and changes in any of the CSF constituents studied, or between the absolute DRS score and the corresponding CSF concentration of any of the compounds. These data are consistent with those previously reported for ECT and do not suggest that ECT alters cerebral amine metabolism in depressed patients. Neither do they provide any evidence for direct amine mediation of the depression-relieving effects of ECT in man, nor for any relation between severity of depressive illness and CSF concentrations of 5HIAA, TRYP, or HVA.

Adult

Mass fragmentographic determination of vanilmandelic acid, homovanillic acid and isohomovanillic acid in human body fluids.

Vanilmandelic, homovanillic and isohomovanillic acids in body fluids were efficiently isolated by liquid chromatography on an Amberlite XAD-4 column and by organic extraction in a special apparatus. The purified metabolites were converted into their trifluoroacetylhexafluroisopropanol esters and analyzed by mass fragmentography. The working curves of the metabolites were linear from 0.5 to 5 ng injected. The minimum detectable concentrations of all the metabolites were 2 ng/ml for plasma and cerebrospinal fluid, and 120 ng/ml for urine. The metabolite concentrations in plasma, cerebrospinal fluid and urine of normal persons and patients were determined.

Adolescent