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1. Bilirubin UDP-glucuronyltransferase activity and its dependence on substrate concentrations in rat liver, renal cortex and intestinal mucosa microsomes were studied. 2. Bilirubin monoglucuronide synthesis from unconjugated bilirubin was a higher capacity, lower affinity step in comparison with bilirubin diglucuronide formation in the three tissues tested. 3. Bilirubin glucuronide formation in liver microsomes showed a higher capacity but a lower affinity than extrahepatic ones. Renal cortex and intestinal mucosa exhibited similar kinetics parameters. 4. In vitro bilirubin glucuronidation in renal cortex and intestinal mucosa was quantitatively important as compared with the hepatic one.

Bile flow and biliary excretion of sulfobromophthalein (BSP) was examined in isolated perfused livers from normal and spironolactone(SP)-treated rats. BSP biliary excretion contributed to the bile production in both groups. Moreover SP increased BSP biliary excretion but transfer of dye from plasma into liver was not affected.

In vitro and in vivo experimental models were designed for the study of the effect of spironolactone (SP) on bilirubin metabolism in rat liver and small intestinal mucosa. In vitro studies included uptake of bilirubin by liver slices and intestinal sheets, determination of glucuronyltransferase activity in mucosal homogenates, and the handling of bilirubin by the isolated perfused liver after bilirubin overload. In vitro studies were carried out to measure the plasma disappearance rate of bilirubin and to determine the extent of bilirubin conjugation and biliary excretion of the pigment infused intravenously. The results obtained suggested that the mechanisms involved in the uptake of bilirubin by tissues were not influenced by SP pretreatment. Glucuronyltransferase activity in the small intestinal mucosa was significantly induced by SP, as previously observed in rat liver. Isolated perfused livers from SP-treated rats, as well as treated living rats, exhibited a greater than normal capacity for bilirubin excretion into bile at the expense of bilirubin diglucuronide. Conjugated bilirubin in the small intestinal mucosa of rats infused with unconjugated pigment was also increased after SP pretreatment. The results favoured the conclusion that SP is an inducer of bilirubin conjugation in the livers as well as in extrahepatic tissues, such as the small intestinal mucosa.

The formation of bilirubin glucuronides was studied using liver microsomes from normal and spironolactone (SP)-treated rats. Increasing amounts of uridine diphosphoglucuronic acid (UDPGA) were added to the incubation mixture to investigate the influence of this nucleotide on bilirubin glucuronidation. The results demonstrated that microsomal preparations from SP-treated rats had a greater capability for bilirubin glucuronidation than normal microsomes which was mostly produced at the expense of bilirubin diglucuronide (BDG) formation. On the other hand, the increase in UDPGA concentration in the incubation mixture also produced an increase of BDG formation which was more important than that of monoglucuronide. These results support the hypothesis of different subunits in the glucuronyl-transferase moiety with different substrate specificities. In addition, the inducer effect of SP on bilirubin glucuronidation is in agreement to the results obtained in living rats which were described previously.

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.

Breakdown of UDPGA incorporated in a mixture containing rat liver or small intestinal mucosa preparations was investigated in the absence of exogenous sugar acceptor. A rapid UDPGA breakdown was seen with intestinal mucosa preparations in such a way that UDPGA decreased to about 1% or less of the initial value after 15 min of incubation. It was estimated that 60 to 70% of total products derived from UDPGA at the end of the incubation with intestinal tissue was in the form of uridine (90%) and uracil (10%). The addition of UDPG, ATP or UDP-N-acetylglucosamine protected UDPGA against degradation by tissue. The results indicated that care must be taken during the incubation of exogenous UDPGA for glucuronyltransferase assay in the intestinal mucosa due to the predominance in this tissue of enzyme systems involved in UDPGA breakdown.