Mechanism of Bilirubin Diglucuronide Formation in Intact Rats BILIRUBIN DIGLUCURONIDE FORMATION IN VIVO NORBERT BLANCKAERT, JOHN GOLLAN, and RUDI SCHMID, Department of Medicine and Liver Center, University of California School of Medicine, San Francisco, California 94143; Laboratory of Hepatology, Department of Medical Research, University of Leuven, B-3000, Leuven, Belgium A B S T R A C T Although it is well established that bili- ['4C]monoglucuronide, no transfer or exchange of the rubin monoglucuronide is formed in the liver from bili- ['4C]glucuronosyl group between injected and en- rubin by a microsomal bilirubin uridine diphosphate dogenously produced bilirubin monoglucuronide (UDP)-glucuronosyltransferase, the subcellular site of could be detected in the excreted bilirubin diglucuron- conversion of monoglucuronide to diglucuronide and ide. Third, in homozygous Gunn rats, injected "4C- the molecular mechanism involved in diglucuronide labeled or unlabeled bilirubin mono- or digluc- synthesis have not been identified. Based on in vitro uronides were excreted in bile unchanged (except that studies, it has been proposed that two fundamentally diglucuronide was hydrolyzed to a minor degree). This different enzyme systems may be involved in di- indicates that Gunn rats, which lack bilirubin UDP- glucuronide synthesis in rat liver: (a) a microsomal glucuronosyltransferase activity, are unable to convert UDP-glucuronosyltransferase system requiring UDP- injected monoglucuronide to diglucuronide. Collec- glucuronic acid as sugar donor or (b) a transglucuroni- tively, these findings establish that a transglucuronida- dation mechanism that involves transfer of a glucurono- tion mechanism is not operational in vivo and support syl residue from one monoglucuronide molecule to the concept that bilirubin diglucuronide is formed by another, catalyzed by a liver plasma membrane en- a microsomal UDP-glucuronosyltransferase system. zyme. To clarify the mechanism by which bilirubin monoglucuronide is converted in vivo to diglucuron- INTRODUCTION ide, three different experimental approaches were used. First, normal rats were injected with either equal Bilirubin,' the principal degradation product of amounts of bilirubin-IIIa ['4C]monoglucuronide and heme in mammals (1), is excreted in bile largely in unlabeled bilirubin-XIIIa monoglucuronide, or bili- the form of polar derivatives in which one or both rubin-XIIIa ['4C]monoglucuronide and unlabeled bili- propionic acid ,8-substituents of the pigment are esteri- rubin-IIIa monoglucuronide. Analysis of radiolabeled fied with a sugar, predominantly glucuronic acid (2, 3). diglucuronide excreted in bile showed that ["4C]- This conjugation is thought to disrupt the multiple in- glucuronosyl residues were not transferred between tramolecular hydrogen bonds responsible for the non- monoglucuronide molecules. Second, in normal rats in- polar character of bilirubin and thereby renders the fused intravenously with dual-labeled [3H]bilirubin pigment excretable in bile (4). It is well established that formation of bilirubin monoglucuronide (BMG) is catalyzed by microsomal uridine diphosphate (UDP)- This work was presented in part at the 79th Annual Meeting glucuronosyltransferase [UDP-glucuronate /3-glucuron- of the American Gastroenterology Association 22-25 May osyltransferase (acceptor unspecific), EC 2.4.1.17], 1978. Gastroenterology. 74: 1166. (Abstr.) with UDP-glucuronic acid (UDPGlcUA) serving as Dr. Blanckaert is an Appointed Investigator of the Belgian National Research Council and a grantee of a North Atlantic sugar donor (5). It remained unexplained, however, Treaty Organization research fellowship and Senior Ful- bright-Hays scholarship. Address reprint requests to Dr. I Abbreviations used in this paper: bilirubin, bilirubin-IXa; Blanckaert in Califomia. BDG,bilirubin diglucuronide; BMG, bilirubin monoglucuronide; Received for publication 24 August 1979 and in revised TLC, thin-layer chromatography; UDP, uridine diphosphate; form 7 February 1980. UDPGlcUA, uridine diphosphate glucuronic acid. 1332 J. Clin. Invest. © The American Society for-Clinical Investigation, Inc. * 0021-9738/80/06/1332/11 $1.00 Volume 65 June 1980 1332-1342 whv on incubation of liver homogenate or microsomiies et al. (11) reported formation of BDG from administered frolm humans, rats, dogs, and cats with UDPGlcUA, BMG in amounts comparable to those in normal rats. the reaction product was largely BMG (6), while in The present study was undertaken to clarify the these species bile contains predominantly bilirubin mechanism of BDG formation in vivo. Experiments diglucuronide (BDG) (2, 3, 7-9). Because of this ap- were devised to examine whether BDG is synthesized parent inconsistenicy between the findings in vivo and in vivo in normal rats by transglucuronidation of BMG, in vitro, it has been postulated that conversion of B.MG and whether homozygous Gunn rats can convert ad- to BDG is catalyzed by a separate enzyme that differs ministered BMIG to BDG. fromii bilirubin UDP-glucuronosyltransferase. Recenitly, it has been reported that in rat liver, synthesis of BDG Experimiental approach f'rom BMG is catalyzed by BMIG glucuronosyltrans- f'erase, an enzyme which is distinct from microsomiial In intact normal rats we carried out two different UDP-glucuronosyltransferase in its reaction mechanismii types of' experiments designed to detect transfer of and subcellular location (10). This enzyme is believed glucuronosyl residues betweeni BMG molecules (trans- to convert BMG to BDG by transf'er of' a glucuronisyl glucuronidationi). The first approach (series I) was group from one BMG molecule to aniother, whereby based on the dissymmiletry of bilirubin, wlhich is due to 1 mol of bilirubini aind 1 mol of BDG are f'ormed f'rom the different sequence of' the mnethyl and vinyl 8-sub- 2 mol of BMG. This transglucuroniidlationi reaction does stitueints oIn the outer pyrrolinoine rings. The molecule not require UDPGlcUA as sugar dconor, aind the enzymile thus contaiins two dissimilar (lipyrivlm-ietheines, which involved exhibits highest specific activity in the plasma we shall designate as A (with endovinyl side chain) and membrane fraction. The reported reactioni rate of'BMG B (with exovinyl side chaini), respectively. For con- glucturonosyltransf'erase in rat aind humain liver f:ar venience the comiiplete molecule is denoted as A-B (Fig. exceeds that of' microsomal bilirubin UDP-glucuronio- 1). Bilirubin monoglucuronide theref'ore exists in syltransf'erase. An unimilpaired rate of' conversion of' two isomeric f'orms that diff'er by the attachment of BMG to BDG in vitro hals been observed in liver prep- the sugar mnoiety to either the A or the B dipyrmylmethene.2 arations f'rom patients with Crigler-Naijjir disease type 1 Both isomers are formed when liver homnogenate or and f'rom homozygous Gunn rats (11), 1)oth of whiclh microsomiies are inculbated with bilinibin and UDPGlcUA lack detectable hepatic bilirubin UDP-glucturonosvl- (17, 19), anl 1)oth have been idlentified in rat bile (8, 9). tranisf'erase activity. For unknown reasons, the moinoglucuronide con-jugated Several well-established observations in humansi anid in A uisually predominiiates (9, 17, 19). rats in vivo have indlicated that, in the presence of' If'BDG were f'ormed by a tranisglucuronidatioin mechi- either a high hepatic bilirubin conicentration or reduce(d anisin, 14C-labeled glucuroniosyl groups f'or exacmple, mnicrosomial bilirtubin UDP-glucturoniosyltransferase ac- attached exclusively to dipvrrvlmlethene A of' injected tivity, the liver preferenitially formns BMG (9, 12-16). BMG would be tranisf'erredl to both the A and B halves Theref'ore, we examinied the microsomn-al UDP-gltuc- of' other BCIG molecules. However, since the two iso- tironosyltranisf'erase system of'rat liver to see whether, inieric bilirubiin miioinoglucuroinides have Inot beenI undi(ler the standard assay conditionis, the pref'erential isolated or syinthesized individually, BMG with a radio- formncationi of BMGI is related to the uniiphysiologicallv labeled conijugatinig group attaclhed exclusively to ei- high bilirubfin substrate coincenitrationis used. 'We fotund ther the A or B dipvrrylmethene is not available. We that wheni the pigmnenit substrate conicentrationi in the tlheref'ore used as mnodel comiipounds the radiolabeled incubation Imlediumiil was substantially (lecreased, liver monoglucuroinides of' the symmiiiietrical bilirubin-IIIa mierosomiies formncapproximately e(lqtal amiiounts of'B\IG aind -XI11a (Fig. 1). Sinice each of'these molecules conl- anll BDG firom bilirtubin andl readily convert addled taiins either two B or two A dlipvrrylmnethenes, the ra- B.MG to BDG (17). Both reactions are critically depend- dliolabele(l glucuronosyl group is attached solely to B enit on UDPGlcUA, as neither liver hlomogenate nor in the Illa anid to A in the XIIIa isomiier. Physiologi- mnicrosomiies conivert bilirubin or BNIG to BDG in the cally, the bilirubin-IIIa anid -XIIIa isomers and their absence of'addledl UDPGlcUA. Mloreover, synthesis in monioglucuroniides behave simiiilatrly to the correspond- vitro of' BDG fromn BMG was found to be deficienit ini ing IXa isomiiers (Restults). homnozygouis Gunnli rats. These findings in vitro stug- Rats providedl witlh an externlcal bile fistultla were ini- gestedl that formation of both B.NIG aindl BDG is catalyzedl jecte(l intravenoisvly with at miiixture of' equial aImlounlts by a microsomiial U DP-glucuroniosyltransferase systemii, of' bilirtubin-XII11a