Abstract
We describe a new assay for dopamine-β-hydroxylase (DβH) activity in human and rat plasma and rat tissues using reversed-phase high-performance liquid chromatography with electrochemical detection. Human and rat plasma DβH activity was measured directly, without extraction of the enzyme. The DβH 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 DβH, 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 DβH activity in small volumes of rat and human plasma (5–20 μl) 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.
Published Version
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