Excretion of administered and endogenous photobilirubins in the bile of the jaundice gunn rat.

Radiolabeled photobilirubins, prepared in vitro by anaerobic illumination of [34C]bilirubin, were injected intravenously into homozygous jaundiced Gunn rats with an external bile fistula. With the animals kept in darkness, the labeled photobilirubins were excreted rapidly in bile. Photobilirubins IA and IB were excreted primarily as unconjugated bilirubin, whereas photobilirubin II was excreted primarily as photobilirubin II and not converted into bilirubin. Bile of Gunn rats given no exogenous pigments, but undergoing phototherapy, contained a large proportion of photobilirubin II and, if collected in liquid nitrogen, traces of photobilirubins I; neither was found in bile when these rats were kept in the dark. Because there is prior evidence that these rats were kept in the dark. Because there is prior evidence that these photobilirubins are isomers of bilirubin, these experiments indicate that the major mechanism of phototherapy is photoisomerization of bilirubin. Photobilirubin II is the unidentified major photoderivative described previously, whereas formation of photobilirubins IA and IB, and their reversion to bilirubin-IXalpha, account for the remarkably increased output of the parent pigment during phototherapy.     

Photocatabolism of labeled bilirubin in the congenitally jaundiced (Gunn) rat

To elucidate the mechanism by which phototherapy reduces serum bilirubin, studies were performed on the catabolism of labeled bilirubin in homozygous jaundiced Gunn rats before, during, and after a period of exposure to 1700 foot candles of daylight fluorescent light. Following equilibration with the body pool of an intravenously administered tracer dose of (3)H- or (14)C-bilirubin, radioactive and diazo reactive compounds were excreted in the bile at a slow, steady rate and plasma specific activity declined semilogarithmically. Subsequent exposure to light caused a marked increase in the biliary excretion of radioactive and diazoreactive compounds. Fecal and urinary radioactivity increased also but remained minor fractions of the total excreted radioactivity. After extinguishing the lights, these variables reverted gradually to control values. Spectral and chromotographic analysis of the excreted pigments and their azopigments demonstrated that the increased biliary radioactivity during phototherapy consisted of two roughly equal fractions: (a) unconjugated bilirubin, excreted at rates comparable to the output of conjugated bilirubin in the bile of normal nonjaundiced rats; and (b) water-soluble bilirubin derivatives, chromatographically identical with those found in Gunn rat bile under control lighting conditions but different from the products of photodecomposition of bilirubin in vitro. In some animals, phototherapy produced little decline in plasma bilirubin despite comparable acceleration of bilirubin catabolism. This was attributed tentatively to increased synthesis of early labeled bilirubin in these animals.     

Bilirubin excretion in rats with normal and impaired bilirubin conjugation: effect of phenobarbital

The effect of phenobarbital on bilirubin excretion was studied in rats with different capacities for bilirubin conjugation. Drug treatment induced substantial increases in bilirubin UDP-glucuronyl transferase activity in the liver of both normal and heterozygous Gunn rats, but not homozygous Gunn rats in which enzyme activity is completely absent. However, enhancement of bilirubin excretion in vivo was observed only in heterozygous Gunn rats. In these animals the maximum capacity to excrete bilirubin into bile (T(max)), like the activity of the conjugating enzyme, was half normal; phenobarbital caused an increase in T(max) to levels characteristic of normal animals, with a twofold rise in the excretion of conjugated pigment. This appeared to be largely unrelated to enhancement of bile flow, and there was no stimulation of alternate pathways of bilirubin excretion.Conjugated bilirubin was consistently recovered from the plasma and urine of both untreated normal and heterozygous Gunn rats infused with unconjugated pigment. The quantities thus recovered comprised a similar fraction of the total pigment conjugated in both types of animal. Moreover, there were linear correlations between T(max) and both the rate of bile flow and the activity of the conjugating enzyme over the range of values represented by control rats of both types. These findings suggest that the process by which conjugated bilirubin is secreted into the bile is closely related to conjugation and limits the final excretory rate at different levels of pigment excretion. The phenobarbital effect uniquely observed in heterozygous Gunn rats appears to be mediated primarily by enhancement of the limited capacity for bilirubin conjugation with an associated rise in functional secretory capacity.     

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

In hepatobiliary disease bilirubin becomes bound covalently to serum albumin, producing a nondissociable bile pigment-protein complex (biliprotein). To elucidate the mechanism of biliprotein formation we studied the bile pigment composition of blood from animals with experimental cholestasis and carried out comparative studies on the rate of biliprotein formation in vivo and in vitro during incubation of bilirubin glucuronides with albumin. Bile duct ligation in the rat and guinea pig led to rapid accumulation in the circulation of bilirubin, heterogeneous bilirubin esters of glucuronic acid, and a biliprotein that migrated along with albumin on high performance liquid chromatography. When the obstruction was removed, biliprotein remained longer in the circulation than did the other bile pigment species. Biliprotein and heterogeneous bilirubin esters of glucuronic acid were not formed in bile duct-ligated homozygous Gunn rats but they were formed when bilirubin glucuronides were incubated with Sprague-Dawley rat serum or human serum albumin at 37 degrees C in vitro. Bilirubin glucuronide rearrangement in vitro was accompanied by nonenzymic hydrolysis. We conclude that the formation of biliprotein in vivo is probably nonenzymic and suggest that mammalian biliprotein is formed by acyl migration of bilirubin from a bilirubin-glucuronic acid ester to a nucleophilic site on albumin.     

Rapid clearance of a structural isomer of bilirubin during phototherapy.

During phototherapy for neonatal jaundice, bilirubin is converted into a variety of photoproducts. Determination of the relative importance of these photoproducts to the elimination of bilirubin requires knowledge of their rates of excretion. We have measured the rate at which the structural isomer of bilirubin, lumirubin, disappeared from the serum of nine jaundiced premature infants after the cessation of phototherapy. In all patients studied, the decline in serum lumirubin could be approximated by a first-order rate equation with a half-life of 80 to 158 min. This rate of disappearance is much faster than that previously determined for the other major bilirubin photoproducts. In samples of bile aspirated from the duodenum of infants undergoing phototherapy, lumirubin was the principal bilirubin photoproduct found. These results indicate that formation and excretion of lumirubin is an important route for bilirubin elimination during phototherapy.     

Hepatic Conversion of Bilirubin Monoglucuronide to Diglucuronide in Uridine Diphosphate-Glucuronyl Transferase-Deficient Man and Rat by Bilirubin Glucuronoside Glucuronosyltransferase

The microsomal enzyme uridine diphosphate (UDP) glucuronate glucuronyltransferase (E.C. 2.4.1.17) catalyzes formation of bilirubin mono-glucuronide from bilirubin and UDPglucuronic acid. Bilirubin glucuronoside glucuronosyltransferase (E.C. 2.4.1.95), an enzyme concentrated in plasma membrane-enriched fractions of rat liver, converts bilirubin monoglucuronide to bilirubin diglucuronide. Bilirubin glucuronoside glucuronosyltransferase activity was studied in homogenates of liver biopsy specimens obtained from patients with the Crigler-Najjar syndrome (Type I) and in subcellular liver fractions of rats homozygous for UDP glucuronate glucuronyltransferase deficiency (Gunn strain). In patients with the Crigler-Najjar syndrome (Type I) and in Gunn rats, hepatic UDPglucuronate glucuronyltransferase activity was not measurable; however, bilirubin glucuronoside glucuronosyltransferase activity was similar to that in normal controls. The subcellular distribution of bilirubin glucuronoside glucuronosyltransferase activity in Gunn rat liver was similar to the distribution observed in normal Wistar rat liver.When bilirubin monoglucuronide was infused intravenously into Gunn rats, 29+/-5% of the conjugated bilirubin excreted in bile was bilirubin diglucuronide. After transplantation of normal Wistar rat kidney, which contained UDPglucuronate glucuronyltransferase activity, in Gunn rats, the serum bilirubin concentration decreased by 80% in 4 days. The major route of bilirubin removal was biliary excretion of conjugated bilirubin, approximately 70% of which was bilirubin diglucuronide. Although patients with the Crigler-Najjar syndrome (Type I) and Gunn rats lack UDP glucuronate glucuronyltransferase, their livers enzymatically convert bilirubin monoglucuronide to diglucuronide in vitro. Conversion in bilirubin monoglucuronide to diglucuronide was demonstrated in Gunn rats in vivo.     

Hepatic disposition and biliary excretion of bilirubin and bilirubin glucuronides in intact rats. Differential processing of pigments derived from intra- and extrahepatic sources.

Mechanisms for transport of bilirubin and its conjugates in hepatocytes have not been defined. We investigated the hepatic processing of bilirubin glucuronides and their precursors, and characterized the disposition of bile pigments arising from intraversus extrahepatic sources. Tracer doses of purified radiolabeled biliverdin, bilirubin, bilirubin monoglucuronide (BMG) or diglucuronide (BDG) were administered intravenously to intact normal or jaundiced homozygous Gunn rats. Rapid sequential analysis of radiolabeled BMG and BDG in bile revealed comparable excretion patterns following biliverdin and bilirubin injection, with BDG as the major pigment. Biliary excretion of radiolabeled conjugates from injected BMG was more rapid, with BMG predominating. Excretion of injected BDG in normal rats and BMG or BDG in Gunn rats was virtually identical to that of unaltered BMG in normal rats. Model independent analysis by deconvolution provided objective comparison of the disposition of radiolabeled pigments from the different sources. These findings indicate that bilirubin glucuronides formed in the liver from endogenous (hepatic) and exogenous (extrahepatic) sources of bilirubin follow a similar excretory pathway. BMG formed endogenously is converted preferentially to BDG, whereas circulating BMG is excreted predominantly unchanged. Exogenous conjugated bilirubins are excreted more rapidly than those generated intrahepatically, by a transcellular pathway that is largely independent of the conjugation system.     

Bilirubin Diglucuronide Formation in Intact Rats and in Isolated Gunn Rat Liver

Bilirubin diglucuronide (BDG) may be formed in vitro by microsomal UDP glucuronosyl transferase (EC 2.4.1.17)-mediated transfer of a second mole of glucuronic acid from UDP-glucuronic acid, or by dismutation of bilirubin monoglucuronide (BMG) to BDG and unconjugated bilirubin, catalyzed by an enzyme (EC 2.4.1.95) that is concentrated in plasma membrane-enriched fractions of rat liver. To evaluate the role of these two enzymatic mechanisms in vivo, [³H]bilirubin mono-[¹⁴C]glucuronide was biosynthesized, purified by thin-layer chromatography, and tracer doses were infused intravenously in homozygous Gunn (UDP glucuronyl transferase-deficient) rats or Wistar rats. Bilirubin conjugates in bile were separated by high-pressure liquid chromatography and ³H and ¹⁴C were quantitated. In Gunn rats, the ¹⁴C:³H ratio in BDG excreted in bile was twice the ratio in injected BMG. In Wistar rats the ¹⁴C:³H ratio in biliary BDG was 1.25 ± 0.06 (mean ± SEM) times the ratio in injected BMG. When double labeled BMG was injected in Wistar rats after injection of excess unlabeled unconjugated bilirubin (1.7 μmol), the ¹⁴C:³H ratio in BDG excreted in bile was identical to the ratio in injected BMG. Analysis of isomeric composition of bilirubin conjugates after alkaline hydrolysis or alkaline methanolysis indicated that the bile pigments retained the IX_α configuration during these experiments. The results indicate that both enzymatic dismutation and UDP glucuronyl transferase function in vivo in BDG formation, and that dismutation is inhibited by a high intrahepatic concentration of unconjugated bilirubin. This hypothesis was supported by infusion of [³H]bilirubin-monoglucuronide in isolated perfused homozygous Gunn rat liver after depletion of intrahepatic bilirubin by perfusion with bovine serum albumin (2.5%), and after bilirubin repletion following perfusion with 0.34 mM bilirubin. From 20 to 25% of injected radioactivity was recovered in BDG in bile in the bilirubin-depleted state; only 8-10% of radioactivity was in BDG in bile after bilirubin repletion.     

The urinary concentrating defect in the Gunn strain of rat. Role of bilirubin.

The role of high serum and tissue levels of unconjegated bilirubin in the pathogenesis of the impaired urinary concentrating ability was investigated in homozygous (jj) Gunn rats with the congenital absence of hepatic glucuronyl transferase. Continuous phototherapy with blue fluorescent lights at a wave length of 460 nm or oral cholestyramine feeding or both reduced serum levels of unconjugated hilirubin to levels consistently below 3.0 mg/100 ml for several weeks in both weanling and adult jj Gunn rats. The renal concentrating defect was already present in weanling jj Gunn rats by 21 days of age. In treated weanling jj animals, maximum concentrating ability and the concentration of urea and nonurea solutes in the papilla and medulla, determined after 24 h of fluid deprivation, were normal when compared to unaffected heterozygous (Jj) littermates. Solute-free water reabsorption which is reduced in jaundiced jj Gunn rats was restored to normal in treated weanling jj rats. The tissue concentration of unconjugated bilirubin was reduced throughout the papilla and inner and outer medulla in the treated jj rats in comparison with untreated jj littermates. The defect in urinary concentrating ability was only partially reversible and sometimes irreversible in adult jj rats, probably because of permanent renal parenchymal damage occurring secondary to massive crystalline deposits in the papilla and medulla. It is concluded that unconjugated bilirubin is directly involved in the pathogenesis of the concentrating defect in jaundiced jj Gunn rats.     

Nonviral gene transfer into liver and muscle for treatment of hyperbilirubinemia in the gunn rat

We evaluated naked plasmid DNA (pDNA)-mediated expression of human hepatic bilirubin UDP-glucuronosyltransferase (hUGT1A1) in skeletal muscle to correct hyperbilirubinemia in the UGT1A1-deficient Gunn rat, an animal model of Crigler-Najjar syndrome type I (CN-I). After delivery of pDNA encoding hUGT1A1 via hepatic vein or femoral artery, in vitro bilirubin glucuronidation activity was detectable in Gunn rat liver and muscle extracts. Expression of hUGT1A1 in Gunn rat liver or muscle resulted in excretion of bilirubin glucuronides in bile. Total biliary bilirubin concentrations increased from a pretreatment average of 10.5 +/- 2.1 microM to 29.2 +/- 4.2 microM after gene transfer into the liver, and to 28.6 +/- 3.8 microM after gene transfer into muscle. Total serum bilirubin decreased by up to 31.2 +/- 6.9 and 29.2 +/- 3.7% and remained significantly lower for at least 1 and 2 weeks, respectively. Tissue damage associated with the procedure was minimal and reversible. Our results demonstrate that muscle can be genetically modified to glucuronidate bilirubin, leading to elimination in bile. A 30% decrease in serum bilirubin, if sustained, would provide meaningful clinical benefit for CN-I patients. However, to be clinically useful, this method needs further optimization and stable gene expression must be achieved.     

Serum binding and biliary excretion of bilirubin after bilirubin loading in Nagase analbuminemic rats and heterozygous (Jj) Gunn rats

To elucidate the effects of albumin on the handling of serum bilirubin, hepatic metabolism and biliary excretion of bilirubin were examined during intravenous bilirubin infusion in Sprague-Dawley (SD) rats, Gunn (heterozygous, Jj) rats, and Nagase analbuminemic rats (NARs). Serum bilirubin was primarily bound to a protein fraction with a molecular weight of about 600 x 10(3) or more in NARs. About 39.2% +/- 12.5% of the serum bilirubin during infusion of bilirubin was bound to the same fraction in Gunn rats. Bilirubin was substantially taken up into the liver and excreted into the bile in NARs, suggesting the role of a high molecular protein, probably a lipoprotein, in its blood transport and the hepatic uptake process. In NARs, biliary bilirubin secretion reached the peak between 20 and 40 minutes after the initiation of bilirubin loading and decreased thereafter, whereas it continued to increase in SD rats and in NARs to which albumin was administered 20 minutes after the start of bilirubin loading. Biliary bilirubin fractions before bilirubin loading were similar in SD rats and NARs, whereas an increase in bilirubin monoglucuronide (BMG) and a decrease in bilirubin diglucuronide (BDG) were observed in Gunn rats. After the initiation of bilirubin loading, a decrease in biliary BDG and an increase in BMG and unconjugated bilirubin were observed in all groups of rats.     

Uridine Diphosphate-Glucuronic Acid-independent Conversion of Bilirubin Monoglucuronides to Diglucuronide in Presence of Plasma Membranes from Rat Liver is Nonenzymic

Two routes have been proposed for conversion of bilirubin monoglucuronide to the diglucuronide: glucuronyl transfer (a) from UDP-glucuronic acid to bilirubin monoglucuronide, catalyzed by a microsomal UDP-glucuronyltransferase, and (b) from one molecule of bilirubin monoglucuronide to another (transglucuronidation), catalyzed by an enzyme present in liver plasma membranes. The evidence regarding the role of the latter enzyme for in vivo formation of bilirubin diglucuronide is conflicting. We therefore decided to reexamine the transglucuronidation reaction in plasma membranes and to study the conversion of bilirubin monoglucuronide to diglucuronide in vivo. Purified bilirubin monoglucuronide was incubated with homogenates and plasma membrane-enriched fractions from liver of Wistar and Gunn rats. Stoichiometric formation of bilirubin and bilirubin diglucuronide out of 2 mol of bilirubin monoglucuronide was paralleled by an increase of the IIIα- and XIIIα-isomers of the bilirubin aglycone, thus showing that dipyrrole exchange, not transglucuronidation, is the underlying mechanism. Complete inhibition by ascorbic acid probably reflects intermediate formation of free radicals of dipyrrolic moieties. The reaction was nonenzymic because it proceeded independently of the protein concentration and heat denaturation of the plasma membranes did not result in decreased conversion rates. Collectively, these findings show spontaneous, nonenzymic dipyrrole exchange when bilirubin monoglucuronide is incubated in the presence of rat liver plasma membranes. Because bilirubin glucuronides present in biological fluids contain exclusively the bilirubin-IXα aglycone, formation of the diglucuronide from the monoglucuronide by dipyrrole exchange does not occur in vivo. Rapid excretion of unchanged bilirubin monoglucuronide in Gunn rat bile after injection of the pigment provides confirmatory evidence for the absence of a UDP-glucuronic acid-independent process.     

The biliary excretion of 25-hydroxy-[3H] vitamin D3 in the Gunn rat

1. The biliary excretion of 25-hydroxy-[3H]-vitamin D3 (25-(OH)-[3H]D3)-derived materials after the intravenous administration of 25-(OH)-[3H]D3 (1.25 nmol/100 g body weight) was studied during a period of 3 h in homozygous (icteric) and heterozygous (anicteric) Gunn rats. 2. The heterozygous rats excreted significantly more 25-(OH)-[3H]D3-derived materials than the homozygous Gunn rats (7.2 +/- 1.5% vs 3.1 +/- 0.5% of the administered dose, P < 0.025). 3. The lower biliary excretion of 25-(OH)-[3H]-D3-derived material in homozygous compared with heterozygous Gunn rats was not due to differences in the plasma 25-(OH)-[3H]D3 concentrations nor was it due to differences in the uptake of 25-(OH)-[3H]D3 by the liver since similar liver/plasma concentration ratios were observed in both groups of animals; it seemed, however, to be due to differences in the biliary transfer of 25-(OH)-[3H]-D3, as indicated by a lower bile/liver concentration ratio in homozygous than in heterozygous rats (1.39 +/- 0.23 vs 0.49 +/- 0.06, P < 0.05). 4. Moreover, samples of bile obtained from homozygous rats contained no beta-glucuronidase-sensitive 25-(OH)-[3H]D3-derived materials but contained significantly more intact 25-(OH)-[3H]-D3 than samples obtained from heterozygous Gunn rats (P < 0.05). After hydrolysis of bile obtained from homozygous and heterozygous Gunn rats with the enzyme beta-glucuronidase, the total amount of intact 25-(OH)-[3H]D3 recovered was found to be similar in the two groups of rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bilirubin conjugates in bile of man and rat in the normal state and in liver disease

Conjugates of bilirubin were studied in normal bile of man and rat, and in bile of liver patients. In general human bile was obtained by duodenal intubation. In addition T-tube bile was examined in patients operated on for mechanical obstruction. The bile pigment compositions of duodenal and T-tube bile were similar in two patients where comparison was possible. Obstruction of the bile duct in rats was used as an animal model for obstructive jaundice.Diazotized ethyl anthranilate was used for determination of total conjugated bile pigment and for thin-layer chromatography (t.l.c.) analysis of the derived azopigments. The available t.l.c. procedures are versatile and allow rapid and quantitative analysis. A variety of conjugated azopigments can be distinguished.With chloroform, negligible amounts of unconjugated bilirubin are extracted from bile of man. Therefore, the percentage of monoconjugated bile pigments present in the initial bile sample can be calculated from the percentage of azodipyrrole found after diazotization.Normal bile from man and rat yields similar azopigment patterns. The dominant component is azopigment-delta (azodipyrrole beta-D-monoglucuronoside). Small amounts of azopigments with complex conjugating structures (gamma-azopigments) are present in both cases. Human bile further yields small amounts of azopigments containing xylose or glucose (called azopigments-alpha(2) and -alpha(3), respectively). Monoconjugated bilirubin (estimated from the percentage of azodipyrrole) amounts of 22% of total bile pigments in human bile and to 39% in murine bile. In both, the bulk of bile pigment is bilirubin diglucuronoside.From bile of patients with acquired liver diseases a new azopigment group (beta-azopigment) was derived. The gamma-azopigment group was increased; the delta-azopigment group (containing azodipyrrole beta-D-monoglucuronoside) was decreased. No differentiation was possible between intra- and extrahepatic cholestasis. The percentage of beta-azopigment showed a positive correlation with serum bilirubin concentration (r = 0.6).Recovery of the diseases was accompanied by normalization of the azopigment patterns.In rats, hydrostatic or mechanical obstruction induced increases in beta- and gamma-azopigments and a decrease in delta-azopigment similar to the changes observed in bile of liver patients. Complete normalization was obtained 6 hr after relieving the hydrostatic obstruction (duration 15-21 hr). In contrast, with man after surgery for extrahepatic obstruction, T-tube bile was not normalized when the T-tube was withdrawn (10 days after operation).Hydrostatic obstruction in rats provides an easy model when postobstructive bile pigment composition and parameters have to be investigated.The present investigations stress the importance of the physiopathological state when studying bilirubin conjugation. Hindrance to bile secretion induced heterogeneity of bilirubin conjugates and stimulated the formation of complex structures.     

Mechanism of Bilirubin Diglucuronide Formation in Intact Rats: BILIRUBIN DIGLUCURONIDE FORMATION IN VIVO

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 UDP-glucuronic acid as sugar donor or (b) a transglucuronidation mechanism that involves transfer of a 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-IIIα [¹⁴C]monoglucuronide and unlabeled bilirubin-XIIIα monoglucuronide, or bilirubin-XIIIα [¹⁴C]monoglucuronide and unlabeled bilirubin-IIIα monoglucuronide. Analysis of radiolabeled diglucuronide excreted in bile showed that [¹⁴C]glucuronosyl residues were not transferred between monoglucuronide molecules. Second, in normal rats infused intravenously with dual-labeled [³H]bilirubin [¹⁴C]monoglucuronide, no transfer or exchange of the [¹⁴C]glucuronosyl group between injected and endogenously produced bilirubin monoglucuronide could be detected in the excreted bilirubin diglucuronide. Third, in homozygous Gunn rats, injected ¹⁴C-labeled or unlabeled bilirubin mono- or diglucuronides were excreted in bile unchanged (except that diglucuronide was hydrolyzed to a minor degree). This indicates that Gunn rats, which lack bilirubin UDP-glucuronosyltransferase activity, are unable to convert injected monoglucuronide to diglucuronide. Collectively, these findings establish that a transglucuronidation mechanism is not operational in vivo and support the concept that bilirubin diglucuronide is formed by a microsomal UDP-glucuronosyltransferase system.     

Studies on the mechanism of Sn-protoporphyrin suppression of hyperbilirubinemia. Inhibition of heme oxidation and bilirubin production.

The synthetic heme analogue Sn-protoporphyrin is a potent competitive inhibitor of heme oxygenase, the rate-limiting enzyme in heme degradation to bile pigment, and can entirely suppress hyperbilirubinemia in neonatal animals and significantly reduce plasma bilirubin levels in a variety of circumstances in experimental animals and man. To further explore the mechanism by which this metalloporphyrin reduces bilirubin levels in vivo, we have examined its effects on bilirubin production in bile duct-cannulated rats, in which bilirubin derived from heme catabolism is known to be rapidly excreted in bile. The administration of Sn-protoporphyrin (10-50 mumol/kg body weight) was followed by prompt (within approximately 1 h) and sustained (up to at least 18 h) decreases in bilirubin output, to levels 25-30 percent below the levels of bilirubin output in control bile fistula animals. The metalloporphyrin had no effect on bile flow or the biliary output of bile acids. Infusions of heme, which is taken up primarily in hepatocytes, or of heat-damaged erythrocytes, which are taken up in reticuloendothelial cells, resulted in marked increases in bilirubin output in bile in control animals; these increases were completely prevented or substantially diminished by Sn-protoporphyrin administration. By contrast, the metalloporphyrin did not alter the high levels of bilirubin in plasma and bile that were achieved in separate experiments by the constant (16 h) infusion of unconjugated bilirubin to bile duct-cannulated rats. Thus, Sn-protoporphyrin exerts no major effects on the metabolic disposition of preformed bilirubin. Heme oxygenase activities were markedly decreased in microsomal preparations from liver, spleen, and kidneys in these experiments, to a degree comparable to the decreases we have observed in the intact rat. We also demonstrated that a substantial proportion (19-35%) of a dose of Sn-protoporphyrin is promptly excreted in bile and that the time course of biliary excretion of this compound more closely reflects plasma concentrations of the metalloporphyrin, which decline rapidly, rather than concentrations in liver, which are considerably more persistent. These results indicate that Sn-protoporphyrin substantially reduces the in vivo production of bilirubin from the degradation of endogenous as well as exogenous heme in the rat. Moreover, this inhibitory effect of the synthetic metalloporphyrin on bilirubin production occurs in both hepatocytes and reticuloendothelial cells, which are the major tissue sites for bilirubin formation. In other studies, we have established that heme oxygenase blockade by Sn-protoporphyrin leads to a marked and rapid excretion of heme into bile presumably because the synthetic metabolism to bile pigment and making it available for excretion via the biliary system in to the gut, These studies strongly suggest that Sn-protoporphyrin diminishes hyperbilirubinemia in animals and man by inhibiting the production of the bile pigment in vivo, and that its principal mode of action involves a potent and sustained competitive inhibition of heme oxygenase.     

Gunn rats: a reproducible experimental model to compare the different methods of measurements of bilirubin serum concentration and to evaluate the risk of bilirubin encephalopathy

Three groups of Gunn rats were studied: group 1 was perfused with bilirubin solution alone, group 2 was perfused with bilirubin and albumin solutions simultaneously, group 3 was perfused with bilirubin solution for 30 min then bilirubin and albumin solutions for the following 10 min. Our results indicate that (1) Gunn rats are a reliable experimental model to study the risk of bilirubin encephalopathy, (2) unbound bilirubin can enter the brain when albumin binding capacity is reduced, (3) and bilirubin binding capacity of serum for unbound unconjugated serum bilirubin is a better criterion than total serum bilirubin and erythrocyte bilirubin to evaluate the risk of kernicterus. This model could also be used to study variations of permeability of the blood-brain-barrier and influences of drugs on bilirubin metabolism.     

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

Most previous compartmental models describing bilirubin transport and metabolism in the liver have been validated solely by analysis of the plasma disappearance of radiolabeled bilirubin in human subjects. We now have determined the transport kinetics of a bilirubin tracer pulse by analysis of plasma, liver, and bile radioactivity data from 30 intact rats. Plasma [3H]bilirubin disappearance was best described by the sum of three exponentials, and a six-compartment model, derived by simulation analysis, was necessary and adequate to describe all experimental data. Examination of the injected radiolabeled bilirubin by extraction with hexadecyltrimethylammonium bromide and thin-layer chromatography revealed that 6.6% (mean) of the original pigment had been degraded to labeled nonbilirubin derivatives during preparation of the tracer dose. This material exhibited a significantly longer half-life (mean 50.6 min) of the plasma terminal exponential than that of authentic radiobilirubin (20.6 min). In isolated perfused rat liver, the kinetics of [3H]bilirubin in perfusate and bile readily fitted the proposed model. Compatibility of the model with the data obtained, both in the isolated liver and in vivo, required that a fraction of bilirubin conjugated in the liver be deconjugated and returned to the plasma. Deconjugation of bilirubin glucuronides was evaluated directly by infusion of bilirubin monoglucuronides, containing 14C in the glucuronosyl group, into rats with an external bile fistula. Since metabolic degradation of hydrolyzed 14C-labeled glucuronic acid yields 14CO2, this was measured in expired air. Whereas 86% of the administered labeled pigment was recovered in bile, 7% of the label appeared in 14CO2. These findings directly validate a portion of the proposed kinetic model and suggest that hepatic deconjugation of a small fraction of bilirubin glucuronides is a physiological event. Deconjugation may also account, at least in part, for the presence of increased concentrations of unconjugated bilirubin in the plasma of patients with cholestasis.     

Metabolism of heme and bilirubin in rat and human small intestinal mucosa.

Formation of heme, bilirubin, and bilirubin conjugates has been examined in mucosal cells isolated from the rat upper small intestine. Intact, viable cells were prepared by enzymatic dissociation using a combined vascular and luminal perfusion and incubated with an isotopically labeled precursor, delta-amino-[2,3-3H]levulinic acid. Labeled heme and bile pigment were formed with kinetics similar to those exhibited by hepatocytes. Moreover, the newly formed bilirubin was converted rapidly to both mono- and diglucuronide conjugates. In addition, cell-free extracts of small intestinal mucosa from rats or humans exhibited a bilirubin-UDP-glucuronyl transferase activity that was qualitatively similar to that present in liver. The data suggest that the small intestinal mucosa normally contributes to bilirubin metabolism.