A sensitive radioenzymatic assay for dopamine, norepinephrine, and epinephrine in plasma and tissue

N. Ben Jonathan, J. C. Porter

Research output: Contribution to journalArticle

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Abstract

A double isotope, radioenzymatic assay for measuring dopamine, norepinephrine, and epinephrine in one sample is described. The assay procedure includes incubation, solvent extraction, and thin layer chromatography. Dopamine, norepinephrine and epinephrine were incubated with catechol O methyl transferase (COMT) and [3H]S adenosyl methionine ([3H]SAM) and were converted to the O methylated tritiated derivatives: [3H] methoxytyramine, [3H]normetanephrine, and [3H] metanephrine, respectively. After several extraction steps, the O methylated products were purified by means of two dimensional, thin layer chromatography using silica gel. The thin layer chromatographic system resulted in complete separation of the three O methylated compounds with an overlap of only 1-2%. The assay was linear from 0 to 5 ng for each catecholamine and had a sensitivity of 10-30pg. The addition of large amounts of plasma reduced the activity of COMT, but increasing the magnesium concentration in the incubation mixture and the addition of EGTA to plasma samples improved the recoveries. Each sample was corrected for losses incurred during extraction and chromatography by using [14C]methoxytyramine, [14C]normetanephrine, and [14C] metanephrine that were added at the end of incubation. Several catechol compounds known to be O methylated by COMT were examined for crossreactivity. Of the substances tested, only dihydroxyphenylalanine (DOPA) exhibited cross reactivity. However, the apparent 30% cross reactivity of DOPA with dopamine was due to the presence of decarboxylase activity in the COMT preparation. As little as 50 μl of trunk plasma from decapitated rats was sufficient for the determination of the three catecholamines.

Original languageEnglish (US)
Pages (from-to)1497-1507
Number of pages11
JournalEndocrinology
Volume98
Issue number6
StatePublished - 1976

Fingerprint

Guaiacol
Transferases
Epinephrine
Dopamine
Norepinephrine
Normetanephrine
Metanephrine
Dihydroxyphenylalanine
Thin Layer Chromatography
Catecholamines
Carboxy-Lyases
Egtazic Acid
Silica Gel
Isotopes
Methionine
Magnesium
Chromatography
3-methoxytyramine

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

A sensitive radioenzymatic assay for dopamine, norepinephrine, and epinephrine in plasma and tissue. / Ben Jonathan, N.; Porter, J. C.

In: Endocrinology, Vol. 98, No. 6, 1976, p. 1497-1507.

Research output: Contribution to journalArticle

Ben Jonathan, N. ; Porter, J. C. / A sensitive radioenzymatic assay for dopamine, norepinephrine, and epinephrine in plasma and tissue. In: Endocrinology. 1976 ; Vol. 98, No. 6. pp. 1497-1507.
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