Formation of bilirubin monoglucuronide and diglucuronide in isolated rat hepatocytes. Effect of spironolactone.
The formation of bilirubin monoglucuronide (BMG) and diglucuronide (BDG) was studied in isolated rat hepatocytes with appropriate viability. Isolated cells were obtained from normal rats and from rats pretreated with spironolactone (SP). A fixed number of cells (4.8 X 10(6)) was incubated in a medium containing uridine diphosphoglucuronic acid (UDPGA, 3.4 mM) and bilirubin (11.3 microM, 29 microM, 50 microM and 81 microM) for different time intervals (from 0 to 25 min). The pellet of cells and the supernatant fraction were subjected to alkaline methanolysis, and the proportions of BMG and BDG were estimated by thin-layer chromatography. No conjugates were detected at time O or in the absence of UDPGA in the incubation system. BMG and BDG were detected after 2 min of incubation and then they increased up to 15 min of incubation. Both conjugates were mostly found in the supernatant fraction, and a predominance of BMG was apparent. Normal cells also synthesized increasing amounts of BMG and BDG with the increase of bilirubin substrate concentration up to 50 microM. When hepatocytes from SP-treated rats were used, a more rapid rate of glucuronidation, that was mainly produced at the expense of BMG found in the supernatant fraction, was clear. The results probably indicate that enzymic conversion of BMG to BDG may be rate limiting in isolated hepatocytes although other possible mechanisms were not excluded.
D000818:Animals; D001663:Bilirubin / Q000031:analogs & derivatives* / Q000096:biosynthesis; D066298:In Vitro Techniques; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D013148:Spironolactone / Q000494:pharmacology*
C019270:bilirubin diglucuronide; C016266:bilirubin glucuronate; D013148:Spironolactone; D001663:Bilirubin