Assay of dopamine β-hydroxylase in human serum as a modification of the assay for the enzyme in rat serum by high-performance liquid chromatography

Abstract

Dopamine h 3,4-dihydroxyphenylethylamine, ascorbate : oxygen oxidoreductase (P-hydroxylating), EC 1_14_17.1] catalyzes the P-hydrosylation of doparnine, the Iast step in the biosynthesis of norepinephrine. The enzyme is released with catecholamines into the blood stream from the peripheral sympathetic nerve endings [I J , and its activity in human serum may reflect the degree of diseases accompanying changes in the Ievel of serum DBH activity, such as hypertension and renal and neurological diseases [Z] , and has therefore drawn much attention from clinical and biomedical investigators. We reported recently a sensitive method for the assay of DBH in rat serum based on the following principle_ Octopamine formed enzymatically from the substrate tyrarnine, is separated by Dowex 5OW-X4 column chromatography and oxidized with periodate to p-hydroxybenzaldehyde, which is then converted into a fluorescent compound with 2,2’-dithiobis(laminonaphthaIene) (DT_k?S)_ The compound, after extraction with n-hexane--chloroform, is separated by normal-phase high-performance liquid chromatography (HPLC) on LiChrosorb Alox T with fluorescence detection using n-hexane-chloroform containing a small amount of acetic acid as mobile phase [3]. DBH activity in human serum is much higher than that in rat serum; and the optimal conditions of the reaction with the enzyme in human serum were found to be slightly different from those with the enzyme in rat serum. Thus, the reported DBH assay method has been applied to the assay of the enzyme in a minute of human serum with some modifications_