Determination by HPLC of the UDP-Glucuronyl Transferase Activity For 7-Hydroxycoumarin in Rabbit Tissue Samples

Abstract

HPLC was used to determine the uridine diphosphate (UDP) glucuronyl transferase activity in rabbit tissues. The crude tissue samples prepared were from the liver, kidney, bladder, large intestine, lung, spleen, heart and fat. A metabolic reaction mixture was prepared that included the rabbit tissue sample, 7-hydroxycoumarin and UDP-glucuronic acid (UDPGA). The method was used to determine the level of production of 7-hydroxycoumarin-glucuronide from 7-hydroxycoumarin. Seperation of the analyte was carried out under gradient elution by reverse phase chromatography on a C18 column, with UV detection at 320 nm. It was possible to determine 7-hydroxycoumarin-glucuronide (7-OHCG) produced over time as well as the decrease in 7-hydroxycoumarin concentration as the reaction progressed. The rate of the reaction was calculated from a plot of the concentration of 7-OHCG produced versus time for each organ, and the rate calculated from the slope of the linear part of the curve. The liver showed the highest activity with a rate of production of 7-OHCG of 2.3 nmol per min per milligram of protein. The kidney showed an activity of 0.22 nmol of 7-OHCG produced per minute per milligram of protein, with the bladder and large intestine showing activities of 0.14 nmol and 7.8 pmol of 7-OHCG produced per minute per milligram of protein. The method used required minimal sample clean up and was reliable and accurate.