Proc. NatI. Acad. Sci. USA Vol. 76, No. 4, pp. 2037-2041, April 1979 Medical 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 SCHMID Department 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- Because in intact Sprague-Dawley rats or homozygous Gunn somal preparations with bilirubin or bilirubin monoglucuronide rats we have been unable to obtain evidence for formation of with (BMG) resulted in formation of bilirubin diglucuronide (BDG). Both synthesis of BMG and its conversion to BDG were BDG by transglucuronidation of BMG (14), we reevaluated the critically dependent on the presence of UDP-glucuronic acid. mechanism by which bilirubin is conjugated by the microsomal Pretreatment of the animals with phenobarbital stimulated both system. The observation that, in conditions associated with reactions. When 33 .tM bilirubin was incubated with micro- decreased hepatic BGTase (e.g., Gilbert syndrome, Crigler- somal preparations from phenobarbital-treated rats, 80-90% Najjar disease) and in heterozygous Gunn rats, bile contains of the substrate was converted to bilirubin glucuronides; the predominantly BMG (15-18) suggested that, in the presence reaction products consisted of almost equal amounts of BMG and BDG. When phenobarbital pretreatment was omitted or of high hepatic bilirubin concentrations relative to the conju- when the substrate concentration was increased to 164 uM bi- gating enzyme activity, the liver may preferentially form BMG. lirubin, proportionally more BMG and less BDG were formed. This was supported by the additional finding that BMG ex- Homogenate and microsomes from homozygous Gunn rats cretion is proportionally enhanced in normal rats infused in- neither synthesized BMG nor converted BMG to BDG. These travenously with bilirubin (19). The standard procedure for findings in vitro suggest an explanation for the observations in vivo that, in conditions of excess bilirubin load or of genetically assaying microsomal BGTase (6) utilizes bilirubin concentra- decreased bilirubin UDP glucuronosyltransferase (EC 2.4.1.17) tions (164-300 ,uM) greatly in excess of those present in normal activity, proportionally more BMG and less BDG are excreted rat liver; under steady-state conditions of hepatic bilirubin in bile. transport in the rat, the bilirubin concentration in the liver is of the same order of magnitude as the plasma concentration In humans and other mammals, bilirubin is excreted in bile (20), approximately 0.5 ,4M (unpublished data). It therefore largely in the form of glycosidic conjugates. These are formed seemed possible that, under these assay conditions, the observed in the liver by esterification of one or both propionic acid side preferential formation of BMG might be related to the high chains of the pigment with glucuronic acid (1, 2) or, to a lesser substrate concentrations used. extent, with glucose or xylose (3-5). Bilirubin diglucuronide To test this hypothesis, we examined the formation of bili- (BDG) has been identified as the major conjugate in the bile of rubin glucuronides by rat liver preparations at two different adult humans, rats, dogs, and cats (1, 2, 5). Formation of BDG probably proceeds in two enzyme-catalyzed steps-i.e., syn- substrate concentrations. In addition, we investigated the direct thesis of bilirubin monoglucuronide (BMG) and conversion of conversion of BMG to BDG by rat liver microsomes and eval- it to BDG (6). Whereas the enzyme involved in hepatic for- uated the effect of pretreating the animals with phenobarbital mation of BMG has been identified as a microsomal glucuro- to enhance hepatic conjugating ability (21). We found that rat nosyltransferase [UDP glucuronate (3-glucuronosyltransferase liver microsomes contain a UDP-glucuronic acid-dependent (acceptor-unspecific), EC 2.4.1.17] with UDP-glucuronic acid enzyme system that converts bilirubin or BMG to BDG and that serving as the carbohydrate donor (7), the mechanism and the activity of this enzyme system is enhanced by pretreatment subcellular location of the conversion of BMG to BDG remain of the animals with phenobarbital. controversial. In animals that excrete predominantly BDG, liver MATERIALS AND METHODS tissue preparations incubated at pH 7.4-7.8 under standard conditions with 164-300 1iM bilirubin and 2.8-5.0 mM UDP- Chemicals. The following chemicals were used: bilirubin glucuronic acid synthesize almost exclusively BMG (6, 8, 9). (E452 in chloroform, 61.0 X 103 liter molh'cm-') containing This observation led to a search for a nonmicrosomal enzyme 1% IIIa, 93% IXa, and 6% XIIIa isomers (Koch-Light Labo- system that converts BMG to BDG. It recently has been re- ratories, Colnbrook, U. K.); UDP-glucuronic acid (ammonium ported (10) that this reaction involves transesterification that salt) and NAD+ (Sigma); glucaro-1,4-lactone (A grade) and is catalyzed by bilirubin glucuronide glucuronosyltransferase digitonin (Calbiochem); ethyl anthranilate (Eastman Kodak); and does not require UDP-glucuronic acid. This enzyme, chloroform stabilized with 0.75% ethanol and pentan-2-one identified in rat liver plasma membrane preparations, converts dried over CaSO4 and redistilled (Mallinckrodt); Sephadex 2 mol of BMG to 1 mol of BDG and 1 mol of bilirubin (10). This LH-20 (Pharmacia). All other reagents were of analytical re- catalytic activity has been detected also in liver preparations agent grade. of homozygous Gunn rats (11) which exhibit congenital un- Animals and Preparation of Cell Fractions. Male conjugated hyperbilirubinemia due to deficiency of hepatic Sprague-Dawley rats (300-350 g; "normal rats"), in which the bilirubin UDP-glucuronosyltransferase activity (BGTase) (12, bilirubin glucuronides in bile were predominantly (70%) in the 13). diconjugated form, and homozygous Gunn rats (340-350 g; "Gunn rats") were used. Sodium phenobarbital (Mallinckrodt) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Abbreviations: BMG, bilirubin monoglucuronide; BDG, bilirubin di- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate glucuronide; BGTase, bilirubin UDP-glucuronosyltransferase ac- this fact. tivity. 2037 Downloaded by guest on September 30, 2021 2038 Medical Sciences: Blanckaert et al. Proc. Nati. Acad. Sci. USA 76 (1979) was administered in the drinking water (0.1%, wt/vol) for 72 and chemical tests on the ethyl anthranilate azo derivatives (24, hr and the animals were starved for 18 hr before being killed. 25) demonstrated that BMG had the 1-O-acyl f3-glucuronide The abdomen was opened under ether anesthesia, the portal structure. vein was perfused with ice-cold 1.54 mM KCl, and the liver was Incubations. BGTase in digitonin-activated homogenate rapidly removed. After excision of gross connective tissue, a 25% (0.125 g wet weight equivalent of liver per ml of suspension) (wt/vol) liver homogenate was prepared in ice-cold 0.25 M or microsomal preparations (0.1 g wet weight equivalent per sucrose, pH 7.4, containing 1 mM disodium EDTA and passed ml) was determined as described (22). through a coarse gauze filter. All other incubation experiments were performed with di- For preparation of microsomes, homogenate was centrifuged gitonin-activated homogenates (0.125 g wet weight equivalent at 41,000 X g for 7 min at 4VC and then the supernatant was per ml) or microsomes (1.0 g wet weight equivalent per ml); centrifuged at 80,000 X g for 25 min. The pellet was resus- incubation mixtures were prepared at 00C in glass-stoppered pended in the initial volume of sucrose/EDTA solution and centrifuge tubes as follows. Appropriate amounts (40 or 200 again centrifuged as described above. Gunn rat microsomes nmol) of bilirubin (dissolved in chloroform) or BMG (dissolved were washed twice with defatted bovine serum albumin (50 in methanol) were delivered to the tubes and the solvent was mg/ml; fraction V, Sigma) between the two centrifugation steps evaporated under a stream of N2. Bilirubin was dissolved in 200 to remove bilirubin. The final bilirubin concentration in Gunn ,1l of 0.1 M NaOH, and 800 Ail of a mixture containing equal rat microsomes (2.0 g wet weight equivalent of liver per ml) was volumes of digitonin-activated enzyme preparation and 0.9 nmol/mg of microsomal protein. triethanolamine.HCl buffer (pH 7.6) were added, followed by The washed microsomes were dispersed and diluted in the 20 Al of 1 M HCL. When BMG was used as substrate, the pig- sucrose/EDTA solution to a volume equivalent to 0.2 or 2.0 g ment residue was directly dissolved in 800Wl of the mixture of wet weight of liver per ml of sucrose/EDTA solution. The more digitonin-activated enzyme preparation and triethanolam- dilute microsomal preparations and 25% (wt/vol) homogenates ine-HCI buffer (pH 7.6); 60 ,ul of 125 mM MgCl2, 100 p1A of a were used for assaying BGTase (22). The more concentrated solution containing 100 mM glucaro-1,4-lactone and 20 mM microsomal preparations were used in all other experiments NAD+, and 40 ,l of 86 mM UDP-glucuronic acid were added with microsomes and were activated by preincubation at 0°C sequentially to the mixture of buffered enzyme preparation and for 30 min with an equal volume of digitonin solution (25 bilirubin or BMG substrate. The mixtures were incubated for mg/ml in sucrose/EDTA medium). In control experiments, 20 min under argon at 37°C in a water bath shaker. Control homogenates or microsomal preparations were inactivated by incubations were performed under identical conditions but heating to 70°C for 30 min. Protein concentrations were de- contained 40 ,ul of disodium EDTA/sucrose medium instead termined by the method of Lowry et al.