Abstract
(1) Relative contribution of phenylalanine and tyrosine as precursors of catecholamine in man
Tracer amounts of L-phenylalanine-U-14C (7.7 mμ Ci) and L-tyrosine 3, 5-3H (91 mμ Ci) were given (i. v.) simultaneously to a patient of neuroblastoma (5 yrs) and his urine was collected for 48 hours after the injection. DOPA, VMA, and HVA were purified chromatographically from the urine and the 3H/14C ratios in the metabolites were analyzed. In the DOPA fraction, the 14C content was in a range of marginal detection and the 3H/14C ratios in VMA and HVA were 61 and 64, respectively, while the expected ratio was 5.07 on the assumption of an instantaneous conversion of phenylalanine to tyrosine after the injection and of possible loss of 3H from tyrosine during its hydroxylation. Essentially identical results were obtained from anotherneuroblastoma patient (4 yrs) The results suggest that phenylalanine in vivo is not utilized, to a significant extent, as a substrate for tyrosine hydroxylase in sympathetic tissues.
(2) Identification and quantification of 3-methoxy-4-hydroxyphenylglycol (MHPG) in human urine
Contrary to the generally accepted reports that sulfate is only a urinary conjugate of MHPG, a glucuronide of MHPG was isolated from urine of a ganglioneuroma patient (58 yrs) in a relatively large quantity. Purification and analysis of the glucuronide from the urine after simultaneous administration of 14C-norepinephrine and 3H-MHPG to the patient revealed that the glucuronide is a new metabolite of norepinephrine. Relative amounts of free and conjugated MHPG in various human urine specimens (e. g. pheochromocytoma and neuroblastoma) were also estimated.
(3) Selective O-methylation of physiological catechols by catechol-O-methyl-transferase (COMT) In vitro, O-methylation of physiological substrates of COMT, such as (nor) epinephrine, dopamine, 3, 4-dihydroxyphenylacetic acid, 3, 4-dihydroxymandelic acid., 4-dihydroxyphenethanol, 3, 4-dihydroxyphenylglycol, 3, 4-dihydroxybenzoic acid, N-acetyldopamine, N-acetylnorepinephrine and DOPA, gave mixtures of meta- and para-O-methyl derivatives. The extent of para-methylation relative to meta-methylation was low with substrates containing an ionized moiety in the side chain, i. e., amino acid, acids, and amines, and increased as the polar (hydrophilic) character of the side chain is decreased. With a non-polar substituent, such as in 4-ethylcatechol, the metal/para ratio was around unity. The results were interpreted as suggesting the presence of a hydrophobic region in the vicinity of the catechol-binding site of COMT which migrates against binding of polar substrates in the orientation necessary for Para-methylation, while non-polar substrates would appear to bind in a random fashion resulting in formation of nearly equal amounts of meta- and para-O-methylated products. No difference in the metal/para ratios was observed with COMT of liver and brain and no change in the ratios during more than 400 fold purification of liver COMT, which suggest that only one enzyme is involved in both meta-and para-O-methylation.
