Bilirubin diglucuronide, the major pigment in human bile is formed in two steps. Bilirubin is converted to bilirubin monoglucuronide by transfer of the glucuronosyl moiety of uridine diphosphoglucuronic acid catalyzed by the microsomal enzyme, uridine diphosphoglucuronate glucuronosyl transferase (UDP glucuronyl transferase, EC 2.4.1.17). Bilirubin monoglucuronide is converted to bilirubin diglucuronide in u i t m by two enzymatic mechanisms: (a) UDP glucuronyl transferasemediated transfer of a second mole of glucuronic acid form UDP-glucuronic acid to bilirubin monoglucuronide; (b) dismutation of 2 moles of bilirubin monoglucuronide to 1 mole of bilirubin diglucuronide and 1 mole of unconjugated bilirubin, catalyzed by bilirubin monoglucuronide dismutase (bilirubin glucuronoside glucuronosyl transferase EC 2.4.1.95). Assay methods for the three enzymatic mechanisms in human liver homogenate by high-pressure liquid chromatography analysis of underivatized bilirubin tetrapyrroles have been developed. UDP glucuronyl transferase was activated in five human liver homogenates with digitonin, Triton X-100, or UDP-N-acetylglucosamine. Greatest activation was observed with Triton X-100. The pH optimum for conversion of bilirubin to bilirubin monoglucuronide was 7.4, and UDP glucuronyl transferase activity was 625 f 51 nmoles per 20 min per gm liver. At high initial bilirubin concentrations (342 p M ) , the product of UDP glucuronyl transferase assay with bilirubin as substrate was predominantly bilirubin monoglucuronide. At lower initial bilirubin concentrations (6.5 to 34 p M ) , up to 15% bilirubin diglucuronide was formed. Glucuronyl transferase-mediated UDP glucuronic acid-dependent conversion of bilirubin monoglucuronide to diglucuronide was assayed using UDP-'4-C-glucuronic acid. The pH optimum was 7.4, and the rate was 21 f 7 nmoles per gm liver per 20 min. The rate of bilirubin diglucuronide formation by enzymatic dismutation of bilirubin monoglucuronide was 470 f 112 nmoles per g m liver per min. The pH optimum was 6.6. The products of enzymatic dismutation were of the IXa configuration.
Uridine diphosphoglucuronate glucuronosyl transferase (UDP glucuronyl transferase, EC 2.4.1.17) activity is concentrated in liver microsomes in rats, man, and other species, and catalyzes the transfer of the glucuronosyl moiety of UDP glucuronate to many aglycones forming acyl, phenolic, thiol, or N-glucuronides (1-5). UDP glucuronyl transferase is essential for the conjugation and maximal biliary excretion of bilirubin in man and rats. Homozygous Gunn rats and patients with Crigler-Najjar syndrome (Type I) lack UDP glucuronyl transferase activity toward bilirubin, excrete only small amounts of