Improved liquid chromatographic determination of dopamine-beta-hydroxylase activity in tissues and plasma.
We describe a new assay for dopamine-beta-hydroxylase (D beta H) activity in human and rat plasma and rat tissues using reversed-phase high-performance liquid chromatography with electrochemical detection. Human and rat plasma D beta H activity was measured directly, without extraction of the enzyme. The D beta H from rat tissues was extracted on Concanavalin A-Sepharose before the assay to avoid interference from the presence of tissue catecholamines. Dopamine, the natural substrate of D beta H, was utilized at optimal (enzyme-saturating) concentration. The reaction product, norepinephrine, was isolated on Dowex AG 50W-X4 (H+ form) column. An internal standard, [3H]norepinephrine, was included to correct for the loss of norepinephrine during the procedure. This method allows for the first time the determination of D beta H activity in small volumes of rat and human plasma (5-20 microliters) and tissues. The procedure can be easily set up in any laboratory equipped with a high-performance liquid chromatograph, an electrochemical detector, and a liquid scintillation counter.
D000818:Animals; D002853:Chromatography, Liquid; D004299:Dopamine beta-Hydroxylase / Q000097:blood / Q000378:metabolism*; D004837:Epinephrine / Q000097:blood; D006801:Humans; D008297:Male; D009638:Norepinephrine / Q000097:blood; D051381:Rats; D011919:Rats, Inbred Strains; D014018:Tissue Distribution
D004299:Dopamine beta-Hydroxylase; D009638:Norepinephrine; D004837:Epinephrine