Bilirubin is a toxic product derived during the metabolism of heme. Detoxification of bilirubin appears to depend on metabolism to sugar derivatives because excretion from the body depends on addition of hydrophilic groups to the propionic acid residues of bilirubin. The predominant pathway for this process is addition of glucuronic acid residues, which allows for excretion of bilirubin into bile. Despite the importance of this pathway, there is no detailed knowledge of the processes involved, as for examining the number of enzymes required to account for glucuronidation or regulation of these enzymes. The long term objectives of this research are to determine the pathways by which bilirubin is metabolized by liver to sugar conjugates that are excreted into the bile and to deliniate the ways in which the function of these pathways is modulated. Emphasis will be placed on the pathways for synthesis of the glucuronides of bilirubin. The methodological approach is to purify the enzyme or enzymes catalyzing the synthesis of the C8 and C12 monoglucuronides and the diglucuronide of bilirubin from liver microsomes of pig and dog. Pure enzymes will be delipidated completely and then characterized kinetically after reconstitution into unilamellar bilayers of defined lipid composition. This will be done in order to study the effect of the chemical and physical properties of the phospholipid environment on the function of the pure enzymes. The kinetic properties of the glucuronidation reactions for bilirubin catalyzed by intact liver microsomes will be characterized so that results from studies of the enzyme reactions in intact, untreated microsomes can be used for guiding work on the reconstitution of function of pure enzymes. That is, we will attempt to reconstitute the activity of enzymes such that they have properties identical to those for enzymes conjugating bilirubin in intact microsomes. Since pig excretes bilirubin as the monoglucuronides whereas dog excretes bilirubin diglucuronide, purification of enzymes from both species will provide an opportunity to examine why some but not all species can synthesize the diglucuronide of bilirubin. These data in turn may provide insight into why human liver under certain abnormal conditions synthesizes the monoglucuronides whereas the diglucuronide normally is the predominant metabolite of bilirubin.
