IN-VITRO GLUCURONIDATION OF 7-HYDROXYCOUMARIN AND DETERMINATION OF 7-HYDROXYCOUMARIN AND 7-HYDROXYCOUMARIN GLUCURONIDE BY CAPILLARY ELECTROPHORESIS

A method was developed for the determination of the in vitro productio n of 7-hydroxycoumarin glucuronide and beta-glucuronidase activity in rabbit tissue homogenates (liver, kidney, heart, lung, spleen, large i ntestine and fat). Separation of 7-hydroxycoumarin (7-HC) and 7-hydrox ycoumarin glucuronide (7-HCG) by capillary electrophoresis was carried out on a 27 cm untreated fused silica capillary using a 100 mM phosph ate buffer, pH 7.0 at 17.5 kV, with detection at 320 nm. 7-HC and 7-HC G were separated within 4 min and could be determined in the same run. Rabbit tissues, containing uridine diphosphate (UDP) glucuronyl trans ferase (UDPGT), were homogenised in Tris-HCl, pH 7.4, and used for the production of 7-HCG by the reaction of 7-HC and UDP-glucuronic acid ( UDPGA). The conversion of 7-HC to 7-HCG is catalysed by UDPGT and the reverse reaction by beta-glucuronidase. A sample of the reaction mixtu re was removed and added to acetonitrile, centrifuged and analysed by capillary electrophoresis. For the reverse reaction (beta-glucuronidas e reaction), the rabbit tissue samples were homogenised in 100 mM acet ate buffer, pH 4.3. To this 7-HCG was added and its metabolism to 7-HC and the decrease in 7-HCG content was determined after stopping the r eaction with a beta-glucuronidase inhibitor, protein precipitation and centrifugation. beta-Glucuronidase activity was observed in all tissu e types, but not all tissues displayed UDPGT activity. The highest UDP GT activity was detected in the liver, followed by the kidney. The lim it of detection was 1 mu g/ml for 7-HC, and 2 mu g/ml for the glucuron ide, with a linear detection range for both analytes from 0-100 mu g/m l.