Type

Journal Article

Authors

D P Bogan
R O'Kennedy
A J Killard

Subjects

Biochemistry

Topics
liver glucuronic acid phase ii animals 7 hydroxycoumarin coumarin umbelliferones chemistry 7 hydroxycoumarin glucuronide coumarins isolation purification cattle chromatography high pressure liquid uridine diphosphate glucuronyl transferase hplc glucuronosyltransferase metabolism

Analysis of the glucuronidation of 7-hydroxycoumarin by HPLC. (1996)

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.
Collections Ireland -> Dublin City University -> Publication Type = Article
Ireland -> Dublin City University -> Status = Published
Ireland -> Dublin City University -> DCU Faculties and Centres = DCU Faculties and Schools: Faculty of Science and Health
Ireland -> Dublin City University -> DCU Faculties and Centres = DCU Faculties and Schools: Faculty of Science and Health: School of Chemical Sciences
Ireland -> Dublin City University -> Subject = Physical Sciences
Ireland -> Dublin City University -> DCU Faculties and Centres = Research Initiatives and Centres: National Centre for Sensor Research (NCSR)
Ireland -> Dublin City University -> DCU Faculties and Centres = Research Initiatives and Centres
Ireland -> Dublin City University -> Subject = Physical Sciences: Chemistry
Ireland -> Dublin City University -> DCU Faculties and Centres = DCU Faculties and Schools
Ireland -> Dublin City University -> PubMed
Ireland -> Dublin City University -> DCU Faculties and Centres = DCU Faculties and Schools: Faculty of Science and Health: School of Biotechnology

Full list of authors on original publication

D P Bogan, R O'Kennedy, A J Killard

Experts in our system

1
R O'Kennedy
Dublin City University
Total Publications: 197
 
2
Anthony J. Killard
Dublin City University
Total Publications: 73