title: Analysis of the glucuronidation of 7-hydroxycoumarin by HPLC.
abstract: The in-vitro metabolism of 7-hydroxycoumarin to 7-hydroxycoumarin-glucuronide was investigated in bovine liver homogenate. A metabolic reaction mixture was prepared that included a crude preparation of uridine diphosphate (UDP) glucuronyl transferase, 7-hydroxycoumarin and UDP-glucuronic acid. A HPLC method was developed to separate coumarin, 7-hydroxycoumarin, 7-hydroxycoumarin-glucuronide and an internal standard, 4-hydroxycoumarin. Samples were separated by reverse-phase HPLC, on a C18 column, with a 1 ml min-1 gradient elution with UV detection at 320 nm. The limit of quantification of the method, for 7-hydroxycoumarin-glucuronide, was 1.47 microM, and the linear range was from 0-295.7 microM. Concentrations of 7-hydroxycoumarin-glucuronide produced were calculated from a plot of 7-hydroxycoumarin-glucuronide concentration versus the mean absorbance ratio (n = 4) (7-hydroxycoumarin-glucuronide absorbance/4-hydroxycoumarin absorbance). It was possible to monitor the decrease in the 7-hydroxycoumarin content as it was metabolised as well as the increase in 7-hydroxycoumarin-glucuronide as it was produced enzymatically. The identity of the compound produced was confirmed by photodiode array spectral analysis. A plot of time versus 7-hydroxycoumarin-glucuronide produced indicates that the metabolism is linear for the first 90 min and reached a plateau at 150 min. The rate of reaction in the first 90 min was 2.96 +/- 0.06 (RSD 1.7%, n = 3) nmol of 7-hydroxycoumarin-glucuronide produced per minute per milligram of protein. After 150 min 0.34 +/- 0.005 mM (RSD 1.4%) 7-hydroxycoumarin-glucuronide was produced, from 0.77 mM 7-hydroxycoumarin introduced into the reaction mixture and 58.0% +/- 5.3% (or 0.44 +/- 0.02 mM) of the 7-hydroxycoumarin remained. These results show that it is possible to monitor the production of the phase II metabolite of coumarin with minimal sample clean-up and without the need for deconjugation of the glucuronide moiety. The method was very reliable and applicable for the direct determination of 7-hydroxycoumarin-glucuronide in an in-vitro metabolic assay.
authors.affiliation: School of Biological Sciences, Dublin City University, Glasnevin, Ireland.
authors.forename: A J
authors.initials: AJ
authors.lastname: Killard
authors.forename: R
authors.initials: R
authors.lastname: O'Kennedy
authors.forename: D P
authors.initials: DP
authors.lastname: Bogan
mesh_terms: D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003374:Coumarins / Q000302:isolation & purification / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000378:metabolism; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism*; D014468:Umbelliferones / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D003374:Coumarins; D014468:Umbelliferones; C031477:7-hydroxycoumarin; C039928:7-hydroxycoumarin glucuronide; C030123:coumarin; D014453:Glucuronosyltransferase