Experimental and Clinical Studies on Catecholamine Metabolism

Report I. Estimation of Homovanillic Acid and Vanillylmandelic Acid in Urine

Abstract

Report I.Estimation of Homovanillic Acid and Vanillylmandelic Acid in Urine
Homovanillic acid is the end product of dopamine and vanillylmandelic acid is that of noradrenaline and adrenaline. The major portion of these metabolites is excreted in the urine of normal subjects and the estimation of these is important in studying of the catecholamine metabolism. Especially, the simultaneous measurement of urinary homovanillic acid and vanillylmandelic acid excretion is a useful diagnostic procedure for neuro-genic tumors.
For the estimation of homovanillic acid and vanillylmandelic acid, urinary phenolic acids were separated on the column chromatography of Amberlite CG-50 resin by the elution with 0.1 N acetic acid and the fractions of these two acids were obtained.
Contaminations of these eluates were examined by the two dimensional paper chromatography. The homovanillic acid elute was contaminated with p-hydroxyphenylacetic acid, and the vanillylmandelic acid elute with p-hydroxymandelic acid. For the elimination of contaminating substances, homovanillic acid and vanillylmandelic acid were converted into 3-4-dihydroxyphenylacetic acid and 3-4-dihydroxymandelic acid, respectively, by the method of demethylation as described by Ruthven and associates. These catechol acids were absorbed on alumina, removed with acid and measured colorimetrically.
In normal adults, the mean excretion of homovanillic acid was 4.93 ± 1.89 μg/mg creatinine and that of vanillylmandelic acid was 4.66 ± 1.62 μg/mg creatinine.
It was found that this method for homovanillic acid and vanillylmandelic acid estimation was excellent in specificity and convenient in simultaneous estimation of these metabolites in the same urine samples.
Report II. Catecholamine Metabolism in Neuroblastoma
Catecholamine metabolism was studied in eight patients with neuroblastoma. The following results were obtained.
1) The urinary excretion of homovanillic acid ranged from 296 to 22 μg/mg creatinine and that of vanillylmandelic acid ranged from 1175 to 7 μg/mg creatinine. The marked elevation in the urinary excretion of these two acids was found in all cases.
2) The urinary excretions of dopamine and noradrenaline ranged from 10.552 to 0.987 μg/mg creatinine and 8.380 to 0.052 μg/mg creatinine, respectively, but those of adrenaline were within normal range or in trace.
3) The tumor tissues and the metastatic liver tissues in four cases contained dopamine and noradrenaline in a high concentration but adrenaline could not be found.
4) The activities of catechol-O-methyltransferase, a metabolizing enzyme of catecholamines and phenylethanolamine-N-methyltransferase, a synthesizing enzyme of adrenaline from noradrenaline, were assayed in two cases. The catechol-O-methyltransferase activity was detected in the tumor, but the phenylethanolamine-N-methyltransferase activity was not detected.
The presence of dopamine and noradrenaline in the tumors, the increased urinary excretions of dopamine, noradrenaline, homovanillic acid and vanillylmandelic acid, and the presence of the catechol-O-methyltransferase activity in the tumor provide direct evidence of the existence of the dopa → dopamine → noradrenaline pathway in neuroblastoma.
These observations strongly suggested that catecholamines, especialy dopamine and noradrenaline, which were synthesized in the tumor, might be metabolized into 3-methoxy-4-hydroxyphenyl compounds in it, at least by the mechanism of O-methylation. This possible mechanism might be supported by the fact that cystathionine is usually found in the urine of the patients with neuroblastoma because of abundant formations of s-adeno-sylmethionine from methionine and ATP for the O-methylation.