Sensitive and specific radioenzymatic assay for norepinephrine, epinephrine and dopamine based on the thin-layer chromatographic separation of their Dns-O-methyl derivatives.
A radioenzymatic assay is described in which norepinephrine, epinephrine and dopamine are converted to their tritiated 3-O-methyl derivatives by reaction with S-[methyl-3H]adenosyl-L-methionine in the presence of catechol-O-methyltransferase. The methylated compounds are then reacted with Dns chloride, and the Dns derivatives are extracted into ethyl acetate, isolated by thin-layer chromatography and quantified by liquid scintillation spectrometry. The assay displays a high degree of specificity for each compound, due in large part to the chromatographic properties of the Dns derivatives. It is capable of measuring 2 pg of each catecholamine, and is linear to at least 5 ng. Approximately 50 samples can be assayed in 1.5 days.
D000818:Animals; D001923:Brain Chemistry; D002394:Catechol O-Methyltransferase; D002855:Chromatography, Thin Layer; D003619:Dansyl Compounds; D004298:Dopamine / Q000302:isolation & purification*; D004837:Epinephrine / Q000302:isolation & purification*; D008099:Liver / Q000201:enzymology; D008297:Male; D008745:Methylation; D009638:Norepinephrine / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains
D003619:Dansyl Compounds; D002394:Catechol O-Methyltransferase; D004298:Dopamine; D009638:Norepinephrine; D004837:Epinephrine