Bilirubin diglucuronide synthesis by a UDP-glucuronic acid-dependent enzyme system in rat liver microsomes.

Abstract

Incubation of rat liver homogenate or microsomal preparations with bilirubin or bilirubin monoglucuronide with (BMG) In humans and other mammals, bilirubin is excreted in bile largely in the form of glycosidic conjugates. These are formed in the liver by esterification of one or both propionic acid side chains of the pigment with glucuronic acid (1, 2) or, to a lesser extent, with glucose or xylose (3-5). Bilirubin diglucuronide (BDG) has been identified as the major conjugate in the bile of adult humans, rats, dogs, and cats (1, 2, 5). Formation of BDG probably proceeds in two enzyme-catalyzed steps-i.e., synthesis of bilirubin monoglucuronide (BMG) and conversion of it to BDG (6). Whereas the enzyme involved in hepatic formation of BMG has been identified as a microsomal glucuronosyltransferase [UDP glucuronate (3-glucuronosyltransferase (acceptor-unspecific), EC 2.4.1.17] with UDP-glucuronic acid serving as the carbohydrate donor (7), the mechanism and subcellular location of the conversion of BMG to BDG remain controversial. In animals that excrete predominantly BDG, liver tissue preparations incubated at pH 7.4-7.8 under standard conditions with 164-300 1iM bilirubin and 2.8-5.0 mM UDPglucuronic acid synthesize almost exclusively BMG (6,8,9).This observation led to a search for a nonmicrosomal enzyme system that converts BMG to BDG. It recently has been reported (10) that this reaction involves transesterification that is catalyzed by bilirubin glucuronide glucuronosyltransferase and does not require UDP-glucuronic acid. This enzyme, identified in rat liver plasma membrane preparations, converts 2 mol of BMG to 1 mol of BDG and 1 mol of bilirubin (10). This catalytic activity has been detected also in liver preparations of homozygous Gunn rats (11) which exhibit congenital unconjugated hyperbilirubinemia due to deficiency of hepatic bilirubin UDP-glucuronosyltransferase activity (BGTase) (12, 13).Because in intact Sprague-Dawley rats or homozygous Gunn rats we have been unable to obtain evidence for formation of BDG by transglucuronidation of BMG (14), we reevaluated the mechanism by which bilirubin is conjugated by the microsomal system. The observation that, in conditions associated with decreased hepatic BGTase (e.g., Gilbert syndrome, CriglerNajjar disease) and in heterozygous Gunn rats, bile contains predominantly BMG (15-18) suggested that, in the presence of high hepatic bilirubin concentrations relative to the conjugating enzyme activity, the liver may preferentially form BMG. This was supported by the additional finding that BMG excretion is proportionally enhanced in normal rats infused intravenously with bilirubin (19). The standard procedure for assaying microsomal BGTase (6) utilizes bilirubin concentrations (164-300 ,uM) greatly in excess of those present in normal rat liver; under steady-state conditions of hepatic bilirubin transport in the rat, the bilirubin concentration in the liver is of the same order of magnitude as the plasma concentration (20), approximately 0.5 ,4M (unpubl...