The use of capillary electrophoresis for studying the in vitro glucuronidation of 7-hydroxycoumarin.
An assay utilizing CE, has been developed for studying the in vitro metabolism of 7-hydroxycoumarin to 7-hydroxycoumarin-glucuronide. A reaction mixture containing a crude preparation of bovine uridine diphosphate (UDP) glucuronyl transferase (UDPGT) and the substrates uridine diphosphate glucuronic acid (UDPGA) and 7-hydroxycoumarin was prepared. Aliquots were removed from the reaction mixture at specific time intervals and analyzed on a 57-cm untreated silica capillary for the presence of 7-hydroxycoumarin-glucuronide. Samples were electrophoresed in a 100 mM phosphate/11 mM deoxycholic acid (sodium salt)/acetonitrile electrolyte buffer, pH 7.0, at 30 kV, with detection at 320 nm. The method allowed the determination of 7-hydroxycoumarin-glucuronide without sample cleanup, with a limit of detection of 2 micrograms/mL, and a linear detection range of 0 microgram/mL to 100 micrograms/mL. The 7-hydroxycoumarin-glucuronide, concentrations were calculated from calibration curves of standards prepared in enzyme solution. The initial rate of production of 7-hydroxycoumarin-glucuronide, in the first 70 min, was 3.1 +/- 0.13 nmol/mL/min/mg protein. The method was fast and reliable with percentage relative standard deviations for concentration of 7-hydroxycoumarin-glucuronide, over time, of less than 10%.
Bogan
D P
DP
School of Biological Sciences, Dublin City University, Ireland.
Killard
A J
AJ
O'Kennedy
R
R
D000818:Animals; D002417:Cattle; D019075:Electrophoresis, Capillary; D014453:Glucuronosyltransferase / Q000737:chemistry; D014468:Umbelliferones / Q000032:analysis* / Q000737:chemistry; D014535:Uridine Diphosphate Glucuronic Acid
D014468:Umbelliferones; D014535:Uridine Diphosphate Glucuronic Acid; C031477:7-hydroxycoumarin; C039928:7-hydroxycoumarin glucuronide; D014453:Glucuronosyltransferase