Bilirubin diglucuronide synthesis by UDP-glucuronic … · 2005-04-22 · Phenobarbital Microsomes 72.5 ±2.7 5.4 ±0.5 164 75± 2.7 9±0.5 (n = 4) 33 48±4.0 36 ±6.5 Digitonin-activated

Proc. NatI. Acad. Sci. USAVol. 76, No. 4, pp. 2037-2041, April 1979Medical Sciences

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

(hepatic metabolism/coniugation/bile pigments/glycosides/UDP glucuronosyltransferase)

NORBERT BLANCKAERT, JOHN GOLLAN, AND RUDI SCHMIDDepartment of Medicine and Liver Center, University of California, San Francisco, California 94143

Contributed by Rudi Schmid, February 1, 1979

ABSTRACT Incubation of rat liver homogenate or micro-somal preparations with bilirubin or bilirubin monoglucuronidewith (BMG) resulted in formation of bilirubin diglucuronide(BDG). Both synthesis of BMG and its conversion to BDG werecritically dependent on the presence of UDP-glucuronic acid.Pretreatment of the animals with phenobarbital stimulated bothreactions. When 33 .tM bilirubin was incubated with micro-somal preparations from phenobarbital-treated rats, 80-90%of the substrate was converted to bilirubin glucuronides; thereaction products consisted of almost equal amounts of BMGand BDG. When phenobarbital pretreatment was omitted orwhen the substrate concentration was increased to 164 uM bi-lirubin, proportionally more BMG and less BDG were formed.Homogenate and microsomes from homozygous Gunn ratsneither synthesized BMG nor converted BMG to BDG. Thesefindings in vitro suggest an explanation for the observations invivo that, in conditions of excess bilirubin load or of geneticallydecreased bilirubin UDP glucuronosyltransferase (EC 2.4.1.17)activity, proportionally more BMG and less BDG are excretedin bile.

In humans and other mammals, bilirubin is excreted in bilelargely in the form of glycosidic conjugates. These are formedin the liver by esterification of one or both propionic acid sidechains of the pigment with glucuronic acid (1, 2) or, to a lesserextent, with glucose or xylose (3-5). Bilirubin diglucuronide(BDG) has been identified as the major conjugate in the bile ofadult humans, rats, dogs, and cats (1, 2, 5). Formation of BDGprobably proceeds in two enzyme-catalyzed steps-i.e., syn-thesis of bilirubin monoglucuronide (BMG) and conversion ofit to BDG (6). Whereas the enzyme involved in hepatic for-mation of BMG has been identified as a microsomal glucuro-nosyltransferase [UDP glucuronate (3-glucuronosyltransferase(acceptor-unspecific), EC 2.4.1.17] with UDP-glucuronic acidserving as the carbohydrate donor (7), the mechanism andsubcellular location of the conversion of BMG to BDG remaincontroversial. In animals that excrete predominantly BDG, livertissue preparations incubated at pH 7.4-7.8 under standardconditions with 164-300 1iM bilirubin and 2.8-5.0 mM UDP-glucuronic acid synthesize almost exclusively BMG (6, 8, 9).

This observation led to a search for a nonmicrosomal enzymesystem that converts BMG to BDG. It recently has been re-ported (10) that this reaction involves transesterification thatis catalyzed by bilirubin glucuronide glucuronosyltransferaseand does not require UDP-glucuronic acid. This enzyme,identified in rat liver plasma membrane preparations, converts2 mol of BMG to 1 mol of BDG and 1 mol of bilirubin (10). Thiscatalytic activity has been detected also in liver preparationsof homozygous Gunn rats (11) which exhibit congenital un-conjugated hyperbilirubinemia due to deficiency of hepaticbilirubin UDP-glucuronosyltransferase activity (BGTase) (12,13).

Because in intact Sprague-Dawley rats or homozygous Gunnrats we have been unable to obtain evidence for formation ofBDG by transglucuronidation of BMG (14), we reevaluated themechanism by which bilirubin is conjugated by the microsomalsystem. The observation that, in conditions associated withdecreased hepatic BGTase (e.g., Gilbert syndrome, Crigler-Najjar disease) and in heterozygous Gunn rats, bile containspredominantly BMG (15-18) suggested that, in the presenceof high hepatic bilirubin concentrations relative to the conju-gating enzyme activity, the liver may preferentially form BMG.This was supported by the additional finding that BMG ex-cretion is proportionally enhanced in normal rats infused in-travenously with bilirubin (19). The standard procedure forassaying microsomal BGTase (6) utilizes bilirubin concentra-tions (164-300 ,uM) greatly in excess of those present in normalrat liver; under steady-state conditions of hepatic bilirubintransport in the rat, the bilirubin concentration in the liver isof the same order of magnitude as the plasma concentration(20), approximately 0.5 ,4M (unpublished data). It thereforeseemed possible that, under these assay conditions, the observedpreferential formation of BMG might be related to the highsubstrate concentrations used.To test this hypothesis, we examined the formation of bili-

rubin glucuronides by rat liver preparations at two differentsubstrate concentrations. In addition, we investigated the directconversion of BMG to BDG by rat liver microsomes and eval-uated the effect of pretreating the animals with phenobarbitalto enhance hepatic conjugating ability (21). We found that ratliver microsomes contain a UDP-glucuronic acid-dependentenzyme system that converts bilirubin or BMG to BDG and thatthe activity of this enzyme system is enhanced by pretreatmentof the animals with phenobarbital.

MATERIALS AND METHODSChemicals. The following chemicals were used: bilirubin

(E452 in chloroform, 61.0 X 103 liter molh'cm-') containing1% IIIa, 93% IXa, and 6% XIIIa isomers (Koch-Light Labo-ratories, Colnbrook, U. K.); UDP-glucuronic acid (ammoniumsalt) and NAD+ (Sigma); glucaro-1,4-lactone (A grade) anddigitonin (Calbiochem); ethyl anthranilate (Eastman Kodak);chloroform stabilized with 0.75% ethanol and pentan-2-onedried over CaSO4 and redistilled (Mallinckrodt); SephadexLH-20 (Pharmacia). All other reagents were of analytical re-agent grade.Animals and Preparation of Cell Fractions. Male

Sprague-Dawley rats (300-350 g; "normal rats"), in which thebilirubin glucuronides in bile were predominantly (70%) in thediconjugated form, and homozygous Gunn rats (340-350 g;"Gunn rats") were used. Sodium phenobarbital (Mallinckrodt)

Abbreviations: BMG, bilirubin monoglucuronide; BDG, bilirubin di-glucuronide; BGTase, bilirubin UDP-glucuronosyltransferase ac-

tivity.

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was administered in the drinking water (0.1%, wt/vol) for 72hr and the animals were starved for 18 hr before being killed.The abdomen was opened under ether anesthesia, the portalvein was perfused with ice-cold 1.54 mM KCl, and the liver wasrapidly removed. After excision of gross connective tissue, a 25%(wt/vol) liver homogenate was prepared in ice-cold 0.25 Msucrose, pH 7.4, containing 1 mM disodium EDTA and passedthrough a coarse gauze filter.

For preparation of microsomes, homogenate was centrifugedat 41,000 X g for 7 min at 4VC and then the supernatant wascentrifuged at 80,000 X g for 25 min. The pellet was resus-pended in the initial volume of sucrose/EDTA solution andagain centrifuged as described above. Gunn rat microsomeswere washed twice with defatted bovine serum albumin (50mg/ml; fraction V, Sigma) between the two centrifugation stepsto remove bilirubin. The final bilirubin concentration in Gunnrat microsomes (2.0 g wet weight equivalent of liver per ml) was0.9 nmol/mg of microsomal protein.The washed microsomes were dispersed and diluted in the

sucrose/EDTA solution to a volume equivalent to 0.2 or 2.0 gwet weight of liver per ml of sucrose/EDTA solution. The moredilute microsomal preparations and 25% (wt/vol) homogenateswere used for assaying BGTase (22). The more concentratedmicrosomal preparations were used in all other experimentswith microsomes and were activated by preincubation at 0°Cfor 30 min with an equal volume of digitonin solution (25mg/ml in sucrose/EDTA medium). In control experiments,homogenates or microsomal preparations were inactivated byheating to 70°C for 30 min. Protein concentrations were de-termined by the method of Lowry et al. (23) with bovine serumalbumin as standard.

Preparation of BMG. Glycine-HCl buffer (pH 2.7 or 2.0),citrate/phosphate buffer (pH 6.0), and triethanolamine-HClbuffer (pH 7.6) were prepared as described (22, 24). BMG wasprepared biosynthetically, purified by adsorption chroma-tography, and isolated by thin-layer chromatography (un-published data). Bile enriched with bilirubin glucuronides wascollected from Sprague-Dawley rats infused with bilirubin (5,umol/hr per 100 g body weight). Enriched bile (2 ml), washedtwice with 30 ml of n-hexane, was mixed with 750 mg of Se-phadex LH-20 and the suspension was freeze-dried. The Se-phadex LH-20 containing the adsorbed pigments was thenwashed in a column (internal diameter, 2 cm) at 4°C with 400ml of chloroform/ethanol, 1:1 (vol/vol), followed by 200 ml ofethanol. The pigments were eluted from the sorbent with eth-anol/water 1:1 (vol/vol) and the eluate was concentrated invacuo to 5 ml. An equal volume of ethanol and then 2 ml ofglycine-HCl buffer (pH 2.0) were added and the pigment wasextracted with 300 ml of ethyl acetate. The extract was con-centrated in vacuo and then applied to thin-layer chromatog-raphy plates precoated with silica gel (silica gel 60 F-254, 5765,0.25 mm, EM Laboratories, Elmsford, NY). The plates weredeveloped with chloroform/methanol/water, 10:5:1 (vol/vol),and the BMG band was immediately scraped from the plateand the pigment was eluted with methanol. These BMGpreparations contained 2-6% unconjugated bilirubin and6-16% BDG which were formed by dipyrrole exchange duringchromatography. The authentic BMG in the preparation waspredominantly of the IXa isomer type (28-29% exovinyl*isomer; 48-51% endovinyl isomer) but contained also 7-9% and13-15% of the IIIa and XIIIa isomers, respectively. Enzymatic

and chemical tests on the ethyl anthranilate azo derivatives (24,25) demonstrated that BMG had the 1-O-acyl f3-glucuronidestructure.

Incubations. BGTase in digitonin-activated homogenate(0.125 g wet weight equivalent of liver per ml of suspension)or microsomal preparations (0.1 g wet weight equivalent perml) was determined as described (22).

All other incubation experiments were performed with di-gitonin-activated homogenates (0.125 g wet weight equivalentper ml) or microsomes (1.0 g wet weight equivalent per ml);incubation mixtures were prepared at 00C in glass-stopperedcentrifuge tubes as follows. Appropriate amounts (40 or 200nmol) of bilirubin (dissolved in chloroform) or BMG (dissolvedin methanol) were delivered to the tubes and the solvent wasevaporated under a stream of N2. Bilirubin was dissolved in 200,1l of 0.1 M NaOH, and 800 Ail of a mixture containing equalvolumes of digitonin-activated enzyme preparation andtriethanolamine.HCl buffer (pH 7.6) were added, followed by20 Al of 1 M HCL. When BMG was used as substrate, the pig-ment residue was directly dissolved in 800Wl of the mixture ofdigitonin-activated enzyme preparation and triethanolam-ine-HCI buffer (pH 7.6); 60 ,ul of 125 mM MgCl2, 100 p1A of asolution containing 100 mM glucaro-1,4-lactone and 20 mMNAD+, and 40 ,l of 86 mM UDP-glucuronic acid were addedsequentially to the mixture of buffered enzyme preparation andbilirubin or BMG substrate. The mixtures were incubated for20 min under argon at 37°C in a water bath shaker. Controlincubations were performed under identical conditions butcontained 40 ,ul of disodium EDTA/sucrose medium insteadof UDP-glucuronic acid solution. When BMG was used assubstrate, additional controls were used as follows: (i) an incu-bation mixture identical to that used in the tests was analyzedimmediately before incubation; and (ii) incubations wereperformed with heat-inactivated enzyme preparations. In allinstances, tests and controls were prepared and analyzed induplicate, and the results are expressed as the mean of the twovalues.

For determination of the relative amounts of bilirubin, BMG,and BDG, the pigments in the incubation mixtures weresubjected to alkaline methanolysis as follows. Methanol (6 ml),about 50 mg of ascorbic acid, a trace of disodium EDTA, and6 ml of KOH/methanol (20 g/liter) were added sequentiallyto the incubation mixture, and methanolysis was allowed toproceed at 20-25°C for 60 sec. Chloroform (6 ml) and 12 mlof glycine-HCI buffer (pH 2.7) were then added to extract thepigments. Bilirubin, the four-isomeric monomethyl esters, andthe dimethyl ester in the extract were separated by thin-layerchromatography with chloroform/methanol/glacial acetic acid,97:2:1 (vol/vol), and determined spectrophotometrically. Re-covery of added bilirubin after incubation and analytical pro-cedures, determined eight times, ranged from 95 to 104%.The structure of the bilirubin glucuronides formed during

incubation was determined as follows. BMG and BDG wereextracted from the incubation mixture (24) and separated bythin-layer chromatography using chloroform/methanol/water,10:5:1 (vol/vol), as the solvent system. Individual pigment bandswere scraped from the plate and transferred to glass-stopperedtubes containing 2 ml of methanol and 1 ml of diazotized ethylanthranilate (8). After 15 min at 20-25°C, 0.5 ml of ascorbicacid solution (100 mg/ml) and 4 ml of glycine-HCl buffer (pH2.7) were added. The azopigments were extracted with 2 mlof pentan-2-one and analyzed chromatographically and by aseries of chemical and enzymatic tests for structure elucidation(24-26). The relative proportions of IIIa, IXa, and XIIIa iso-mers in BDG were determined by thin-layer chromatography(27) of the bilirubin obtained after saponification of the isolated

* For the purpose of this publication, bilirubin-IXa monoglucuronidecarrying the glucuronyl group on the dipyrrolic half of the asym-metric bilirubin-IXa molecule with the exovinyl group is denotedas the exovinyl isomer; the opposite is considered to be the endovinylisomer.

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Table 1. Formation ofBMG and BDG-f bilirubin bygT$ liver homogenate or microsomes

Enzyme preparation Incubation conditions Glucuronides formed,Pretreatment of Subcellular BGTase, nmol/ Protein, Bilirubin, % of bilirubin substrate

rats fraction 10 min/mg protein mg/ml uM BMG BDG

None Homogenate 3.5 ± 0.2 7.6 ± 0.9 164 29± 1.0 1 ± 0.8(n = 3) 33 57+ 7.6 11 ±4.2

Phenobarbital Homogenate 5.9; 6.5 9.0; 8.6 164 39; 45 2; 4(n = 2) 33 68; 70 24; 20

None Microsomes 12.5 ± 1.5 2.7 ± 0.1 164 35 i 3.1 2 ± 0.5(n = 4) 33 77 1.2 9 1.2

Phenobarbital Microsomes 72.5 ± 2.7 5.4 ± 0.5 164 75 ± 2.7 9 ± 0.5(n = 4) 33 48±4.0 36 ±6.5

Digitonin-activated homogenate or microsomes from untreated or phenobarbital-treated Sprague-Dawley rats were incubated at 370C for20 min with bilirubin (33 or 164 gM) and UDP-glucuronic acid (2.8 mM) atpH 7.6 in the presence of MgCl2 (6.15 mM), glucaro-1,4-lactone (8.2mM), and NAD+ (1.64 mM). Data are given as mean ±SEM or as individual values.

bilirubin dimethyl esters formed by alkaline methanolysis ofthe original BDG.

RESULTSConversion of Bilirubin to BMG and BDG. Incubation of

bilirubin at the standard concentration (164 ,uM) with liverhomogenate or microsomal preparations, from either untreatedor phenobarbital-treated Sprague-Dawley rats, yielded BMGalmost exclusively or largely (Tables 1 and 2). Analysis of theBMG formed indicated that it contained more endovinyl thanexovinyl bilirubin IXa monoglucuronide (Table 2), suggestingthat esterification with glucuronic acid preferentially occurson the dipyrrolic half of the bilirubin molecule that carries theendovinyl side chain.When the bilirubin substrate concentration was decreased

to 33 ,M, the amount of BDG synthesized was increased rela-tive to that of BMG, but the absolute amount of BDG producedwas comparable to that obtained with the higher substrateconcentration (Tables 1 and 2). With 33 ,M bilirubin, micro-

somes from phenobarbital-treated rats formed almost equalamounts of BMG and BDG. Under these incubation conditionsthe BMG synthesized contained also predominantly the IXaendovinyl isomer. The isomeric composition (IIIa, IXa, XIIIa)of the BDG formed was similar to that of the bilirubin sub-strate.

As reported (21), pretreatment of rats with phenobarbitalsubstantially enhanced hepatic BGTase (Table 1); this increasewas seen with homogenate or microsomes. In the absence ofUDP-glucuronic acid, formation of BMG or BDG could not be

demonstrated. Both homogenate and microsomal preparationsfrom homozygous Gunn rats failed to exhibit detectable enzymeactivity.

Conversion of BMG to BDG. To determine whether con-version of BMG to BDG by the microsomal system requiresUDP-glucuronic acid, BMG in concentrations ranging from 33

to 173 1AM was substituted for bilirubin as substrate. Pretreat-ment with phenobarbital substantially enhanced the conversionof BMG to BDG, with both homogenate and microsomalpreparations (Table 3). With microsomes from phenobarbi-tal-treated rats, 40-43% of the BMG was converted to BDG(Tables 2 and 3). No conversion of BMG to BDG was observedwhen UDP-glucuronic acid was omitted from the incubationmixture or when heat-inactivated microsomes were used; lessthan 4% of BMG substrate was hydrolyzed in these control ex-

periments. The ratio of exovinyl to endovinyl IXa isomers ofBMG at the end of the incubations was similar to that of theini; ial BMG used as substrate (Table 2), indicating that bothBNV- isomers had served as substrates for BDG synthesis. Aswith bilirubin as substrate, higher BMG concentrations resultedin a proportionally smaller fraction of the BMG being convertedto BDG (Table 3). No conversion of BMG to BDG was observedwith microsomal preparations of homozygous Gunn rats.

Structural Analysis of BMG and BDG Formed by RatLiver Microsomes. The isomeric composition (I1a, IXa,XIIIa) of BDG formed from bilirubin or BMG was similar tothat of the substrate used (Table 2), which excludes the possi-bility of significant dipyrrole exchange during the incubationand isolation procedures. The following findings obtained by

Table 2. Isomeric composition of bilirubin glucuronides formed by incubation of bilirubin or BMG with rat liver microsomes

Isomeric composition of synthesized glucuronidesGlucuronides formed, BMG

% of substrate XIa IXa BDGSubstrate BMG BDG Ila endovinyl exovinyl XIIIa IIIa IXa XIIIa

Bilirubin, 164,uM 73 2.3 9 + 1.3 3 0.6 69 0.7 22 + 0.3 6 0.3 ND ND NDBilirubin, 33,uM 49 5.5 42 ± 4.7 3 0.7 65 0.9 26 + 0.3 6 0.3 3 0.6 91 1.2 6± 0.6BMG,33,uM 40+2.5 9±0.3 47±0.6 30+0.3 14±0.3 9±0.3 77±0.7 14±0.3

Digitonin-activated microsomes from phenobarbital-treated Sprague-Dawley rats were incubated with bilirubin orBMG and UDP-glucuronicacid. Protein concentration in the incubation mixtures ranged from 4.4 to 6.3 mg/ml. After alkaline methanolysis, bilirubin and mono- and dimethylesters derived from the synthesized bilirubin glucuronides were extracted and isolated by thin-layer chromatography. The four isomeric bilirubinmonomethyl esters were separated chromatographically and quantitated by spectrophotometry. The three isomeric bilirubin .dimethyl esters,which move as a single band on chromatography, were eluted from the silica gel with methanol and saponified, and the bilirubin MIIa, IXa, andXIIIa isomers were identified by thin-layer chromatography and determined spectrophotometrically. All data are mean ±SEM from threeexperiments. ND, not determined.

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Table 3. Formation of BDG from BMG by homogenate or microsomes from rat liver

Enzyme preparation Incubation conditions BDG formed,Rat Subcellular BGTase, nmol/ Protein, BMG, % of BMG

Strain n Pretreatment fraction 10 min/mg protein mg/ml gM substrate

Normal 1 None Homogenate 3.5 11.4 35 11Normal 1 None Microsomes 14.4 3.2 34 12Normal 1 Phenobarbital Homogenate 7.5 11.0 36 22Normal 4 Phenobarbital Microsomes 26.9 ± 2.2 6.6 ± 0.6 33-37 43 i 3.1Normal 2 Phenobarbital Microsomes 24.0; 29.0 7.8; 5.3 70; 69 26; 33Normal 1 Phenobarbital Microsomes 24.0 7.8 173 16Gunn 2 Phenobarbital Microsomes 0; 0 7.8; 8.2 35; 35 0; 0

Digitonirnactivated homogenate or microsomes from untreated or phenobarbital-treated Sprague-Dawley or homozygous Gunn rats wereincubated at 370C for 20 min with BMG (33-173 ,uM) and UDP-glucuronic acid (3.4 mM) at pH 7.6, in the presence of MgCl2 (7.5 mM), glu-caro-1,4-lactone (10 mM), and NAD+ (2.0 mM). The BMG concentrations given have been corrected for contamination of the substrate withbilirubin and BDG. Data are given as individual values or mean ±SEM.

analysis of the azo derivatives of synthesized bilirubin conju-gates indicated that the reaction products had the 1-O-acylf3-D-glucuronide structure. (i) Methylation and subsequentacetylation of conjugated azo derivatives (azopigment S) at eachstep resulted in formation of a pigment with chromatographicbehavior identical to that of the corresponding derivative ofauthentic azodipyrrole 1-O-acyl f-D-glucuronide. (ii) Incu-bation of the conjugated azopigment with ,B-glucuronidase ledto virtually complete (94-97%) hydrolysis; this reaction wasinhibited by glucaro-1,4-lactone. (iii) The carbohydrate residueliberated by ammonolysis corresponded chromatographicallyto D-glucuronic acid. (iv) After ammonolysis or alkaline etha-nolysis, the carboxyl amide or ethyl ester derivatives of theexovinyl and endovinyl isomers of the azopigments were ob-tained on chromatographic analysis, indicating that the esterlinkage in the parent glucuronides was at the propionic acid sidechain.

DISCUSS-IONThe present findings demonstrate that, when incubated withbilirubin and UDP-glucuronic acid, rat liver homogenate ormicrosomal preparations form both BMG and BDG (Table 1).Under all experimental conditions used, production of BMGexceeded that of BDG and formation of both BMG and BDGwas stimulated by pretreatment of the rats with phenobarbital(Tables 1 and 3). Characterization of the reaction productsshowed that the conjugated pigments had the 1-O-acylf-D-glucuronide structure, similar to those formed in the intactanimal. The bilirubin IXa monoglucuronide synthesized invitro consisted of a mixture of the exovinyl and endovinyl iso-mers, with the latter isomer predominating (Table 2). It is un-known whether the preferred formation of the endovinyl iso-mer is due to the asymmetry of bilirubin IXa or reflects thepresence of two separate glucuronosyltransferases. Kineticanalysis of the first step in the conjugation of bilirubin IXatherefore requires individual determination of the two isomericreaction products. For the biosynthesis of BDG, both IXamonoglucuronide isomers were used as substrates in proportionto their availability (Table 2).

In designing the present experiments, three different mo-lecular mechanisms for the conversion of BMG to BDG wereconsidered. The first was dipyrrole exchange between indi-vidual BMG molecules resulting in formation of equimolaramounts of BDG and bilirubin, which would be associated witha relative increase in bilirubin IIIa and XIIIa isomers (27).Dipyrrole exchange was ruled out by the finding that the isomercomposition of the BDG synthesized from bilirubin or BMGwas similar to that of the substrate (Table 2). A second mecha-nism considered was enzymatic transesterification of BMG,

which would yield equimolar amounts of BDG and bilirubinwithout requiring UDP-glucuronic acid (10). Such a transes-terification mechanism is inconsistent with our finding thatconversion of BMG to BDG is critically dependent on thepresence of UDP-glucuronic acid. On the other hand, thepresent findings suggest a third mechanism-namely, forma-tion of BDG by transfer of a glucuronyl residue from UDP-glucuronic acid to BMG, catalyzed by a microsomal UDP-glucuronosyltransferase.The capacity of this microsomal UDP-glucuronosyltrans-

ferase system for BDG formation is considerably lower than thatfor BMG synthesis. This is reflected by the observation that, permg of microsomal protein, the absolute quantity of BDG syn-thesized from bilirubin or BMG changed little when the sub-strate concentration was increased above 33 ,uM (Tables 1 and3). Consequently, BMG formation by microsomal preparationsgreatly exceeds that of BDG when high bilirubin substrateconcentrations are used, such as those used in the standardprocedure for assaying BGTase (Table 1). On the other hand,almost equal proportions of BMG and BDG were synthesizedby the microsomes when the substrate concentration was low-ered to 33 ,gM (Table 1), which still is far in excess of the bili-rubin concentration in normal rat liver in vivo. Similar findingshave been reported for conjugation of bilirubin with xylose inrat liver preparations (6).

In homozygous Gunn rats which lack microsomal BGTase(12, 13) and hence are unable to form BMG, no conversion ofBMG to BDG was detectable in liver homogenate or micro-somal preparations (Table 3). This supports the concept thata microsomal UDP-glucuronosyltransferase system catalyzesthis conversion; it remains to be determined whether the sameor separate microsomal UDP-glucuronosyltransferases are in-volved in the two conjugation steps. These conclusions areconsistent with previous observations in intact Gunn rats in vivo,which indicated that, although these hyperbilirubinemic ani-mals can excrete injected BMG and BDG, they cannot syn-thesize either (14, 28).

In the present experiments, as in most previous work (6),microsomal preparations were "activated" with digitonin toincrease enzyme activity. Because it is unknown whether nativeor activated enzyme preparations more closely reflect bilirubinconjugation in the intact liver cell, unqualified extrapolationof the present findings in vitro to the physiological mechanismof hepatic bilirubin conjugation in vivo is not warranted.Nonetheless, the present observations appear to offer a plausibleexplanation for the reported patterns of conjugated bilirubinexcretion in the intact organism. Thus, in the presence ofphysiological rates of heme turnover, resulting in low hepaticbilirubin concentrations, bile contains predominantly BDG as

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expected (1, 17). On the other hand, when the bilirubin loadis increased experimentally (19) or when hepatic bilirubinUDP-glucuronosyltransferase is decreased genetically as inCrigler-Najjar disease or Gilbert syndrome (15-17), BMG ex-cretion is proportionally increased, as would be predicted onthe basis of the present findings in vitro.We are indebted to Gail MacNeil for expert editorial assistance. This

work was supported in part by National Institutes of Health GrantsAM-21899, AM-11275, and P50 AM-18520 and by the Walter C. PewFund for Gastrointestinal Research. N.B. is an "Appointed Researcher"of the Belgian National Research Council and recipient of a NorthAtlantic Treaty Organization Research Fellowship and Senior Ful-bright-Hays Scholarship.

1. Gordon, E. R., Goresky, C. A., Chan, T.-H. & Perlin, A. S. (1976)Biochem. J. 155,477-486.

2. Compernolle, F., Van Hees, G. P., Blanckaert, N. & Heirwegh,K. P. M. (1978) Biochem. J. 171, 185-201.

3. Compernolle, F., Van Hees, G. P., Fevery, J. & Heirwegh, K. P.M. (1971) Biochem. J. 125, 811-819.

4. Gordon, E. R., Chan, T.-H., Samodai, K. & Goresky, C. A. (1977)Biochem. J. 167, 1-8.

5. Fevery, J., Van De Vijver, M., Michiels, R. & Heirwegh, K. P. M.(1977) Biochem. J. 164, 737-746.

6. Heirwegh, K. P. M., Meuwissen, J. A. T. P. & Fevery, J. (1973)Adv. Clin. Chem. 16,239-289.

7. Dutton, G. J. (1966) in Glucuronic Acid, Free and Combined,ed. Dutton, G. J. (Academic, New York), pp. 186-299.

8. Van Roy, F. P. & Heirwegh, K. P. M. (1968) Biochem. J. 107,507-518.

9. Jansen, P. L. M. (1974) Biochim. Biophys. Acta 338, 179-182.10. Jansen, P. L. M., Chowdhury, J. R., Fischberg, E. B. & Arias, I.

M. (1977) J. Biol. Chem. 252, 2710-2716.

11. Chowdhury, J. R., Jansen, P. L. M., Fischberg, E. B., Daniller,A. & Arias, I. M. (1978) J. Clin. Invest. 62, 191-196.

12. Arias, I. M. & London, I. M. (1957) Science 126, 563-564.13. Schmid, R., Axelrod, J., Hammaker, L. & Swarm, R. L. (1958)

J. Clin. Invest. 37, 1123-1130.14. Blanckaert, N., Gollan, J. L. & Schmid, R. (1978) Gastroenter-

ology 74, 1166 (abstr.).15. Gollan, J. L., Huang, S. N., Billing, B. & Sherlock, S. (1975)

Gastroenterology 68, 1543-1555.16. Gordon, E. R., Shaffer, E. A. & Sass-Kortsak, A. (1976) Gastro-

enterology 70, 761-765.17. Fevery, J., Blanckaert, N., Heirwegh, K. P. M. Preaux, A.-M. &

Berthelot, P. (1977) J. Clin. Invest. 60,970-979.18. Van Steenbergen, W. & Fevery, J. (1978) Gastroenterology 74,

1107 (abstr.).19. Noir, B. A. (1976) Biochem. J. 155,365-373.20. Hammaker, L. & Schmid, R. (1967) Gastroenterology 53, 31-

37.21. Robinson, S. H., Yannoni, C. & Nagasawa, S. (1971) J. Clin. In-

vest. 50, 2605-2613.22. Heirwegh, K. P. M., Van De Vijver, M. & Fevery, J. (1972) Bio-

chem. J. 129,605-618.23. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.

(1951) J. Biol. Chem. 193,265-275.24. Blanckaert, N., Fevery, J., Heirwegh, K. P. M. & Compernolle,

F. (1977) Biochem. J. 164, 237-249.25. Blanckaert, N., Compernolle, F., Leroy, P., Van Houtte, R., Fe-

very, J. & Heirwegh, K. P. M. (1978) Biochem. J. 171, 203-214.

26. Blanckaert, N., Heirwegh, K. P. M. & Compernolle, F. (1976)Biochem. J. 155, 405-417.

27. McDonagh, A. F. & Assisi, F. (1971) FEBS Lett. 18, 315-317.28. Ostrow, J. D. & Murphy, N. H. (1970) Biochem. J. 120, 311-

327.

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