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.
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.
Ikegawa
S
S
Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Sendai, Japan.
Oohashi
J
J
Murao
N
N
Goto
J
J
D000097:Acetonitriles; D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism*; D002635:Chenodeoxycholic Acid / Q000378:metabolism; D019826:Cholic Acid / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003840:Deoxycholic Acid / Q000378:metabolism; D020719:Glucuronides / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006878:Hydroxides; D008095:Lithocholic Acid / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D010710:Phosphates; D017680:Potassium Compounds; D051381:Rats; D017208:Rats, Wistar; D014580:Ursodeoxycholic Acid / Q000378:metabolism
D000097:Acetonitriles; D001647:Bile Acids and Salts; D020719:Glucuronides; D006878:Hydroxides; D010710:Phosphates; D017680:Potassium Compounds; C024788:ammonium phosphate; D003840:Deoxycholic Acid; D002635:Chenodeoxycholic Acid; D008095:Lithocholic Acid; D014580:Ursodeoxycholic Acid; D014453:Glucuronosyltransferase; D019826:Cholic Acid; C029943:potassium hydroxide; C032159:acetonitrile