Kazuhiro Maruta

Fluorescence assay for dopamine β-hydroxylase activity in human serum by high-performance liquid chromatography

Abstract As a model for the assay of enzyme activity by high-performance liquid chromatography, a simple, sensitive and specific fluorescence assay for dopamine β-hydroxylase (DBH) activity was devised using o -phthalaldehyde as reagent. Tyramine was used as a substrate and was incubated under optimal conditions. Endogenous inhibitors in the enzyme preparation were completely inactivated by addition of N -ethylmaleimide and a small amount of Cu 2+ . After isolating the amines on a small Dowex 50 column, the eluate was analyzed fluorometrically by high-performance liquid chromatography. The product, octopamine, was eluted prior to and completely separated from tyramine, and the height of the fluorescence peak gave the amount of enzymatically formed octopamine. DBH activity in serum obtained from the umbilical vein was much lower than the activity in adult human serum. Normal values of serum DBH activity from 153 subjects (1–74 years) showed a mean and standard deviation of 42.5 ± 30.9 and 34.3 ± 18.2 μmol/min/liter of serum at 37°C, before and after computation by the method of Hoffmann. Male serum had a slightly higher activity than female serum. Serum DBH activity rapidly increased during the first 10 years after birth, gradually increased up to an optimum at 40–50 years, and then decreased.

Stress-induced changes in in vivo and in vitro dopamine-β-hydroxylase activity in spontaneously hypertensive rats

Abstract The in vivo dopamine-β-hydroxylase (DBH) activity in peripheral tissues of the whole animal and the in vitro DBH activity in serum, adrenal glands, peripheral sympathetically innervated tissue, and brain were examined in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) at 12 weeks of age before and after daily immobilization stress. In vitro DBH activity did not differ significantly between SHR and WKY before stress, but in vivo DBH activity in SHR was significantly lower than that in WKY. After repeated immobilization stress, in vivo DBH activity was significantly elevated only in SHR but not in WKY. In vitro DBH activity in serum, adrenal gland, vas deferens, and brain was also significantly increased in SHR, whereas that in WKY was increased only in adrenal glands. These results suggest that SHR at 12 weeks of age, when hypertension is established, may have even lower sympathetic nerve activity than WKY, and that after repeated stress the response of the sympatho-adrenal system is higher in SHR than in WKY.