A method for the determination of the hepatic enzyme activity catalyzing bile acid acyl glucuronide formation by high-performance liquid chromatography with pulsed amperometric detection
✍ Scribed by Shigeo Ikegawa; Junji Oohashi; Naoaki Murao; Junichi Goto
- Publisher
- John Wiley and Sons
- Year
- 2000
- Tongue
- English
- Weight
- 95 KB
- Volume
- 14
- Category
- Article
- ISSN
- 0269-3879
No coin nor oath required. For personal study only.
✦ Synopsis
A method for the determination of the activity of hepatic glucuronyltransferase catalyzing formation of bile acid 24-glucuronides using high-performance liquid chromatography (HPLC) with pulsed amperometric detection (PAD) has been developed. Bile acid 24-glucuronides were simultaneously separated on a semimicrobore column, Capcell Pak C18UG120, using 20 mM ammonium phosphate (pH 6.0)-acetonitrile (27:10 and 16:10) as the mobile phase in the stepwise gradient elution mode. A 1 M potassium hydroxide solution for the hydrolysis of the 24-glucuronides, which liberates the corresponding bile acids and glucuronic acid, was mixed with the mobile phase in a post-column mode, and the resulting eluant was heated at 90 degrees C, the 24-glucuronides being monitored using a pulsed amperometric detector; the limit of detection was 10 ng. The proposed method was applied to the determination of the hepatic enzyme activity catalyzing bile acid 24-glucuronide formation and the result exhibited the efficient 24-glucuronide formation of the monohydroxylated bile acid, lithocholic acid.