Mechanism of Bilirubin Diglucuronide Formation in Intact Rats

Abstract

A B S T R A C T Although it is well established that bilirubin monoglucuronide is formed in the liver from bilirubin by a microsomal bilirubin uridine diphosphate (UDP)-glucuronosyltransferase, the subcellular site of conversion of monoglucuronide to diglucuronide and the molecular mechanism involved in diglucuronide synthesis have not been identified. Based on in vitro studies, it has been proposed that two fundamentally different enzyme systems may be involved in diglucuronide synthesis in rat liver: (a) a microsomal UDP-glucuronosyltransferase system requiring UDPglucuronic acid as sugar donor or (b) a transglucuronidation mechanism that involves transfer ofa glucuronosyl residue from one monoglucuronide molecule to another, catalyzed by a liver plasma membrane enzyme. To clarify the mechanism by which bilirubin monoglucuronide is converted in vivo to diglucuronide, three different experimental approaches were used. First, normal rats were injected with either equal amounts of bilirubin-

Origin of mammalian biliprotein and rearrangement of bilirubin glucuronides in vivo in the rat.

“…(Fig. 1) and patients (33) with cholestatic jaundice and for the apparent deconjugation of bilirubin glucuronides detected in experimental studies (32, 34,35).…”

Bilirubin kinetics in intact rats and isolated perfused liver. Evidence for hepatic deconjugation of bilirubin glucuronides.

“…Third, estimation of the apparent volume of distribution of the plasma conjugated bilirubin compartment (P,) yielded an improbably high value of 166 ml (Table II). Fourth, bilirubin glucuronides added to plasma in vitro do not undergo hydrolysis (31). Thus, to resolve this apparent discrepancy in the model, a location for bilirubin deconjugation was sought which is adjacent to the plasma compartment permitting rapid equilibration with it.…”

“…It was first suggested by the observation in Gunn rats that intravenous injection of normal rat bile containing conjugated bilirubin led to an increase in the plasma unconjugated pigment level (54,55). More recently, it was found that infusion of BDG into Gunn rats resulted in biliary excretion ofabout 6% BMG (31). Moreover, in normal rats infused with BMG dual-labeled in the glucuronosyl and tetrapyrrole moieties, recovery in bile of the label contained in the glucuronosyl group consistently was less than that in the tetrapyrrole moiety (31).…”

“…BilirubinIXa 1-0-acyl ,8-D-mono-[U-_4C]glucuronide (BMG) was prepared by incubating bilirubin-IXa and UDP-[U-_4C]glucuronic acid (231 mCi/mmol, New England Nuclear) with digitonin-activated liver microsomes from rats pretreated with phenobarbital (administered for 7 d in drinking water, 0.1% wt/vol). Details of the incubation, extraction, and chromatographic isolation of BMG have been described (30,31). The pigment was quantitated spectrophotometrically assuming E450 60 x 103 liter-mol-' * cm-' for BMG in methanol.…”

The photoinduced isomerization of bilirubin in cationic detergent solutions

“…However, the route by which the monoglucuronide is transformed into the diglucuronide has been the subject of recent controversy. One proposal (Blanckaert et al, 1979) suggests that the diglucuronide is formed in a sequential process involving the enzymic transfer of a glucuronosyl residue from UDP-glucuronic acid to the free propionic acid group of bilirubin monoglucuronide. An alternative proposal (Jansen et al, 1977;Chowdhury et al, 1979) suggests that an enzyme-catalysed disproportionation of bilirubin monoglucuronide occurs and that two molecules of the monoglucuronide give rise to one molecule of unconjugated bilirubin and one molecule of the diglucuronide.…”