Inherited disorders of bilirubin transport and conjugation: new insights into molecular mechanisms and consequences.

Inherited disorders of bilirubin metabolism might reduce bilirubin uptake by hepatocytes, bilirubin conjugation, or secretion of bilirubin into bile. Reductions in uptake could increase levels of unconjugated or conjugated bilirubin (Rotor syndrome). Defects in bilirubin conjugation could increase levels of unconjugated bilirubin; the effects can be benign and frequent (Gilbert syndrome) or rare but severe, increasing the risk of bilirubin encephalopathy (Crigler-Najjar syndrome). Impairment of bilirubin secretion leads to accumulation of conjugated bilirubin (Dubin-Johnson syndrome). We review the genetic causes and pathophysiology of disorders of bilirubin transport and conjugation as well as clinical and therapeutic aspects. We also discuss the possible mechanisms by which hyperbilirubinemia protects against cardiovascular disease and the metabolic syndrome and the effects of specific genetic variants on drug metabolism and cancer development.

The roles of MRP2, MRP3, OATP1B1, and OATP1B3 in conjugated hyperbilirubinemia.

Increased concentrations of bilirubin glucuronides in blood plasma indicate hepatocellular dysfunction. Elucidation of the transport processes of bilirubin conjugates across the basolateral (sinusoidal) and the canalicular plasma membrane domains of hepatocytes has decisively contributed to our current understanding of the molecular basis of conjugated hyperbilirubinemia in human liver diseases. Under normal conditions, unconjugated bilirubin is taken up into hepatocytes by transporters of the organic anion-transporting polypeptide (OATP) family, followed by conjugation with glucuronic acid, and ATP-dependent transport into bile. This efflux across the canalicular membrane is mediated by multidrug resistance protein 2 (MRP2 or ABCC2), which is a 190-kDa glycoprotein transporting with high affinity and efficiency monoglucuronosyl bilirubin and bisglucuronosyl bilirubin into bile. MRP2 is hereditarily deficient in human Dubin-Johnson syndrome. Under pathophysiological conditions such as cholestatic liver injury and MRP2 inhibition, the basolateral efflux pump multidrug resistance protein 3 (MRP3 or ABCC3) is responsible for the occurrence of conjugated hyperbilirubinemia. MRP3 is a glycoprotein with a similar molecular mass as MRP2, with 48% amino acid identity, and with overlapping substrate specificity. Human MRP3 is the only basolateral efflux pump shown to transport bilirubin glucuronides. In human and rat hepatocytes, MRP3/Mrp3 is strongly upregulated under conditions of cholestasis and MRP2 deficiency. This is in line with the concept that basolateral efflux pumps of the hepatocyte compensate for impaired canalicular efflux of compounds into bile and contribute to balance the rate of uptake or synthesis of compounds in hepatocytes with the capacity for efflux into bile.

[Neonatal hyperbilirubinemia and molecular mechanisms of jaundice]. / Vrozené hyperbilirubinemie a molekulární mechanizmy zloutenky.

The introductory summarises the classical path of heme degradation and classification of jaundice. Subsequently, a description of neonatal types of jaundice is given, known as Crigler Najjar, Gilberts, DubinJohnson and Rotor syndromes, emphasising the explanation of the molecular mechanisms of these metabolic disorders. Special attention is given to a recently discovered molecular mechanism of the Rotor syndrome. The mechanism is based on the inability of the liver to retrospectively uptake the conjugated bilirubin fraction primarily excreted into the blood, not bile. A reduced ability of the liver to uptake the conjugated bilirubin contributes to the development of hyperbilirubinemia in common disorders of the liver and bile ducts and to the toxicity of xenobiotics and drugs using transport proteins for conjugated bilirubin.

Rotor Syndrome: Glucuronidated Bile Acidemia From Defective Reuptake by Hepatocytes.

Organic anion transporting polypeptide (OATP) 1B1 (gene, solute carrier organic anion transporter family member 1B1 [SLCO1B1]) and OATP1B3 (SLCO1B3) serve as transporters for hepatic uptake of important endogenous substances and several commonly prescribed drugs. Inactivation of both proteins together causes Rotor syndrome. How this OATP1B1/1B3 defect disturbs bile acid (BA) metabolism is largely unknown. In this study, we performed detailed BA analysis in 3 patients with genetically diagnosed Rotor syndrome. We found that BAs glucuronidated at the C-3 position (BA-3G) accounted for 50% or more of total BAs in these patients. In contrast but similarly to healthy controls, only trace amounts of BA-3G were detected in patients with constitutional indocyanine green excretory defect (OATP1B3 deficiency) or sodium-taurocholate cotransporting polypeptide (NTCP; gene, solute carrier family 10 member 1 [SLC10A1]) deficiency. Therefore, substantial amounts of BA-3G are synthesized in hepatocytes. The cycling pathway of BA-3G, consisting of excretion from upstream hepatocytes and uptake by downstream hepatocytes by OATP1B1/1B3 may exist to reduce the burden on upstream hepatocytes. Conclusion: Detailed BA analysis revealed glucuronidated bile acidemia in patients with Rotor syndrome. Further exploration of the physiologic role of glucuronidated BAs is necessary.

Bilirubin and biliverdin protect rodents against diabetic nephropathy by downregulating NAD(P)H oxidase.

We recently found a markedly lower prevalence of vascular complications, including kidney disease, in diabetic patients with Gilbert syndrome, a congenital form of hyperbilirubinemia, suggesting a beneficial effect of bilirubin (BIL) on diabetic nephropathy. To directly examine this, we determined whether hereditary hyperbilirubinemic Gunn j/j rats and biliverdin (BVD)-treated diabetic db/db mice were resistant to the development of renal disease. Both rodent models had less albuminuria and complete protection against the progression of mesangial expansion accompanied by normalization of transforming growth factor-ß1 and fibronectin expression. Simultaneously, there was normalization of urinary and renal oxidative stress markers, and the expression of nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase subunits in the kidney. In cultured vascular endothelial and mesangial cells, BIL and BVD significantly inhibited NADPH-dependent superoxide production, and both high glucose- and angiotensin II-induced production of reactive oxygen species. Collectively, our findings suggest that BIL and BVD may protect against diabetic nephropathy and may lead to novel antioxidant therapies for diabetic nephropathy.

Transfer of bilirubin uridine diphosphate-glucuronyltransferase to enzyme-deficient rats.

Cells from a clonal strain (MH(1)C(1)) of rat hepatoma were transplanted subcutaneously into two homozygous Gunn rats, which are jaundiced because the enzyme bilirubin uridine diphosphate-glucuronyltransferase is absent from the liver. Because of the enzyme activity present in the transplanted cells, the recipient rats developed the capacity to conjugate bilirubin and reverted in large part to a normal pattern of bilirubin excretion.

Substrate-induced conjugation of bilirubin in genetically deficient newborn rats.

Bilirubin appears to induce its own conjugation with glucuronide. Bilibrubin uridine diphosphate-glucuronyltransferase activity is relatively high at birth in heterozygous offspring of permanently jaundiced female rats. The postnatal development of the transferase is accelerated in hyperbilirubinemic heterozygotes.

[Gilbert syndrome]. / Gilbert-szindróma.

Gilbert disease is a benign disorder of the bilirubin conjugation, which affects 7-10% of the average population. The symptoms are usually only mild jaundice and the slightly elevated unconjugated bilirubin level, other laboratory tests and the liver functions are usually normal. In most cases, mutation of the UDP glucuronyltransferase gene leads to impaired bilirubin conjugation. Besides the usual laboratory methods, genetic analyses of the UDP glucuronyltransferase gene can help in the diagnosis. In 80-100% of the patients the (TA)-insertion in the promoter-region of the gene is present in homozygous - (TA) 7 /(TA) 7 - form, and leads to the decrease of the amount of functionally active enzyme. The role of missense mutations localized in the coding region has not been clarified yet, but their co-occurrence with the (TA) 7 promoter-variant might mean an explanation to the elevated bilirubin level, jaundice, and the familiar aggregation of Gilbert disease.

Liver repopulation with bone marrow derived cells improves the metabolic disorder in the Gunn rat.

BACKGROUND: Reversible ischaemia/reperfusion (I/R) liver injury has been used to induce engraftment and hepatic parenchymal differentiation of exogenous beta2-microglubulin(-)/Thy1(+) bone marrow derived cells. AIM: To test the ability of this method of hepatic parenchymal repopulation, theoretically applicable to clinical practice, to correct the metabolic disorder in a rat model of congenital hyperbilirubinaemia. METHODS AND RESULTS: Analysis by confocal laser microscopy of fluorescence labelled cells and by immunohistochemistry for beta2-microglubulin, 72 hours after intraportal delivery, showed engraftment of infused cells in liver parenchyma of rats with I/R, but not in control animals with non-injured liver. Transplantation of bone marrow derived cells obtained from GFP-transgenic rats into Lewis rats resulted in the presence of up to 20% of GFP positive hepatocytes in I/R liver lobes after one month. The repopulation rate was proportional to the number of transplanted cells. Infusion of GFP negative bone marrow derived cells into GFP positive transgenic rats resulted in the appearance of GFP negative hepatocytes, suggesting that the main mechanism underlying parenchymal repopulation was differentiation rather than cell fusion. Transplantation of wild type bone marrow derived cells into hyperbilirubinaemic Gunn rats with deficient bilirubin conjugation after I/R damage resulted in 30% decrease in serum bilirubin, the appearance of bilirubin conjugates in bile, and the expression of normal UDP-glucuronyltransferase enzyme evaluated by polymerase chain reaction. CONCLUSIONS: I/R injury induced hepatic parenchymal engraftment and differentiation into hepatocyte-like cells of bone marrow derived cells. Transplantation of bone marrow derived cells from non-affected animals resulted in the partial correction of hyperbilirubinaemia in the Gunn rat.

Hepatic excretion of circulating bilirubin photoproducts in the Gunn rat.

To investigate the origin and metabolism of the intermediates that occur in blood during phototherapy of neonatal jaundice, serum from irradiated homozygous Gunn rats was injected intravenously into other homozygous Gunn rats fitted with bile fistulas, and the excretion of pigment in the bile of the recipient rats was studied. In some experiments the donor rats were labeled with [14C]bilirubin; in others the recipient rats were labeled. Injection of donor serum from irradiated rats caused a transient burst of pigment excretion in the bile of the recipient rats. However, simultaneous bursts of pigment and 14C excretion were observed only when the donor rat was labeled and the recipient rat was not, and not when the donor rat was unlabeled and the recipient rat was labeled. In addition, there was simultaneous transient enhanced excretion of pigment and label when labeled recipient rats were exposed briefly to blue light. We conclude that (a) the phototherapy intermediates previously detected spectroscopically in serum are formed from bilirubin and are excreted in bile independently of bilirubin; (b) the enhanced excretion of pigment in bile during phototherapy is not caused by complex formation between bilirubin and photoproducts, or by liver damage produced by photoproducts or light.

Effect of sodium phenobarbital on bilirubin metabolism in an infant with congenital, nonhemolytic, unconjugated hyperbilirubinemia, and kernicterus.

Sodium phenobarbital and various hormones, compounds capable of hepatic enzyme induction, were given to an infant boy with congenital, nonhemolytic, unconjugated, hyperbilirubinemia and severe kernicterus for prolonged periods between the ages of 2 and 25 months to determine their effect on serum bilirubin concentrations. Phenobarbital, 5 mg/day orally, on two occasions decreased serum bilirubin concentrations approximately threefold over a period of 30 days. Withdrawal of phenobarbital after the first study resulted in a gradual (30 days) return of serum bilirubin to pretreatment levels. The lower serum bilirubin concentrations observed when phenobarbital therapy was reinstituted were maintained for 61 days on 2.5 mg/kg per day of the drug. Orally administered L-triiodothyronine, 0.05-0.1 mg/day for 71 days, intramuscular human growth hormone, 1 mg/day for 21 days, and testosterone propionate, 0.1 mg/day for 9 days, did not decrease serum bilirubin levels below lowest control values of 18 mg/100 ml.Bilirubin-(3)H was administered twice before and once with bilirubin-(14)C during phenobarbital therapy to study the kinetics of bilirubin metabolism. Results of the first and second control studies and of the bilirubin-(3)H and bilirubin-(14)C phenobarbital studies, respectively, were as follows: total body bilirubin pools, 200, 184, 73, and 72 mg; half-lives, 111, 84, 37, and 39 hr; and turnover, 30, 37, 33, and 31 mg/day. The data show that the approximate threefold decrease in serum bilirubin concentration and total body pool resulted from a comparable decrease in bilirubin half-life without a significant change in turnover. In vitro histological (electron microscopy) and enzymological studies of liver obtained by surgical biopsies before and during phenobaribtal administration showed that both the hepatocyte content of agranular endoplasmic reticulum (AER) and the ability of liver homogenate to conjugate p-nitrophenol were significantly increased during phenobarbital treatment. The observations suggest that phenobarbital affects bilirubin metabolism by the induction of an enzyme(s) with a slow rate(s) of degradation (or rapid rate of degradation with limited capacity).

Hepatobiliary transport of glutathione and glutathione conjugate in rats with hereditary hyperbilirubinemia.

TR- mutant rats have an autosomal recessive mutation that is expressed as a severely impaired hepatobiliary secretion of organic anions like bilirubin-(di)glucuronide and dibromosulphthalein (DBSP). In this paper, the hepatobiliary transport of glutathione and a glutathione conjugate was studied in normal Wistar rats and TR- rats. It was shown that glutathione is virtually absent from the bile of TR- rats. In the isolated, perfused liver the secretion of glutathione and the glutathione conjugate, dinitrophenyl-glutathione (GS-DNP), from hepatocyte to bile is severely impaired, whereas the sinusoidal secretion from liver to blood is not affected. The secretion of GS-DNP was also studied in isolated hepatocytes. The secretion of GS-DNP from cells isolated from TR- rat liver was significantly slower than from normal hepatocytes. Efflux of GS-DNP was a saturable process with respect to intracellular GS-DNP concentration: Vmax and Km for efflux from TR- cells was 498 nmol/min.g dry wt and 3.3 mM, respectively, as compared with 1514 nmol/min.g dry wt and 0.92 mM in normal hepatocytes. These results suggest that the canalicular transport system for glutathione and glutathione conjugates is severely impaired in TR- rats, whereas sinusoidal efflux is unaffected. Because the defect also comes to expression in isolated hepatocytes, efflux of GS-DNP from normal hepatocytes must predominantly be mediated by the canalicular transport mechanism, which is deficient in TR- rats.

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.

Unconjugated bilirubin and an increased proportion of bilirubin monoconjugates in the bile of patients with Gilbert's syndrome and Crigler-Najjar disease.

Bilirubin pigments were studied in the bile of 20 normal adults, 25 patients with Gilbert's syndrome, 9 children with Crigler-Najjar disease, and 6 patients with hemolysis, to determine how a deficiency of hepatic bilirubin UDP-glucuronosyltransferase would affect the end products of bilirubin biotransformation. In the bile from patients with Gilbert's syndrome, a striking increase was found in the proportion of bilirubin monoconjugates (48.6+/-9.8% of total conjugates) relative to that in normal bile (27.2+/-7.8%). This increase was even more pronounced in children with Crigler-Najjar disease, in whom, even in the most severe cases, glucuronide could always be demonstrated in the bile. Furthermore, unconjugated bilirubin-IXalpha was unquestionably present in the bile of these children and amounted to 30-57% of their total bilirubin pigments (<1% in the controls). It was not possible to predict from the biliary bilirubin composition whether a child would respond to phenobarbital therapy or not. Bile composition was normal in patients with hemolysis, except when there was associated deficiency of hepatic glucuronosyltransferase. Therefore, the observed alterations were not a simple consequence of unconjugated hyperbilirubinemia. The present findings suggest that Crigler-Najjar disease represents a more pronounced expression than Gilbert's syndrome of a common biochemical defect. Hepatic bilirubin UDP-glucuronosyltransferase deficiency leads to decreased formation of diconjugates with an ensuing increase in the proportion of bilirubin monoconjugates in bile; in the most severe cases, an elevated content of biliary unconjugated bilirubin is also found.

Biochemical and molecular aspects of genetic disorders of bilirubin metabolism.

Bilirubin, the oxidative product of heme in mammals, is excreted into the bile after its esterification with glucuronic acid to polar mono- and diconjugated derivatives. The accumulation of unconjugated and conjugated bilirubin in the serum is caused by several types of hereditary disorder. The Crigler-Najjar syndrome is caused by a defect in the gene which encodes bilirubin UDP-glucuronosyltransferase (UGT), whereas the Dubin-Johnson syndrome is characterized by a defect in the gene which encodes the canalicular bilirubin conjugate export pump of hepatocytes. Animal models such as the unconjugated hyperbilirubinemic Gunn rat, the conjugated hyperbilirubinemic GY/TR-, and the Eisai hyperbilirubinemic rat, have contributed to the understanding of the molecular basis of hyperbilirubinemia in humans. Elucidation of both the structure of the UGT1 gene complex, and the Mrp2 (cMoat) gene which encodes the canalicular conjugate export pump, has led to a greater understanding of the genetic basis of hyperbilirubinemia.

Effect of changes in dietary components on the serum bilirubin in Gilbert's syndrome.

Patients with Gilbert's syndrome were placed on low calorie diets and isocaloric diets sequentially severely reduced in carbohydrate, protein, or fat content. Significant increases in the serum bilirubin concentration occurred after the low calorie diet, but not after the isocaloric nutrient-depleted diets. Thus caloric deprivation per se and not changes in dietary components is responsible for diet-induced hyperbilirubinemia.

Gilbert's syndrome and drug metabolism.

Gilbert's syndrome is an inherited disorder which is characterised by unconjugated hyperbilirubinaemia. In patients with Gilbert's syndrome, both bilirubin clearance and in vitro hepatic microsomal uridine diphosphoglucuronyl transferase (UDPGT) activity are reduced. In addition, there is evidence suggesting impaired hepatic uptake of bilirubin in Gilbert's syndrome. Glucuronidation of a number of substrates appears to be impaired in Gilbert's syndrome, but the significance of the reported changes in oxidation and acetylation are less clear.

Hepatic oxidative drug metabolism and the microsomal milieu in a rat model of congenital hyperbilirubinemia.

The aims of this study were to evaluate the hypothesis that impaired glucuronidation of bilirubin and possibly of drug oxidation in the liver of homozygous (jj) Gunn rats may be due to an altered microsomal milieu. Accordingly, we investigated and compared in vivo and in vitro demethylation of aminopyrine, hepatic cytochrome P-450 levels, microsomal lipid composition, and microsomal membrane fluidity in icteric, homozygous (jj) Gunn rats and in their anicteric heterozygous (jJ) littermates. In both males and females, [14C]aminopyrine demethylation in vivo, using the 14CO2 breath test, was unimpaired in the icteric animals. Likewise, cytochrome P-450 levels in the icteric and nonicteric groups were similar, and aminopyrine kinetics in vitro in the females were comparable in icteric and nonicteric littermates. The main lipid classes were also similar in the homozygous and heterozygous female Gunn rats, whereas only minor changes were seen in the phospholipid fatty acyl composition with a small, but significant, increase in the unsaturated index in the icteric group. Despite this, there was no apparent effect on hepatic microsomal membrane fluidity as measured by the order parameter of I[12,3] and the rotational correlation time of I[1,14] in either female or male sets of homozygous and heterozygous Gunn rats. Our data, therefore, do not support an alteration of composition or fluidity of the microsomal milieu as a mechanism of impaired bilirubin glucuronidation and possibly of oxidation in these animals. They also absolve long-term unconjugated hyperbilirubinemia as a mechanism of hepatic microsomal dysfunction. Our study, therefore, indirectly suggests that abnormal glucuronidation of bilirubin and some other aglycones in homozygous Gunn rats is due to genetic abnormalities involving the enzyme(s) itself.

Two pathways for biliary copper excretion in the rat. The role of glutathione.

To evaluate the role of glutathione in biliary copper excretion, we studied this process in control Wistar rats and in mutant Wistar rats (GY rats), in which the secretion of glutathione into bile is deficient. For comparison, biliary zinc excretion was determined simultaneously. In spite of the markedly reduced bile flow (-45%) in GY rats, biliary output rates of endogenous copper were virtually identical in GY and control rats. In contrast, zinc output was drastically reduced in GY rats compared to controls (-80%). Biliary excretion patterns after intravenous administration of copper, in doses ranging from 65 to 2265 nmol/100 g/body wt, showed a distinct rapid and slow phase in control rats. In GY rats, on the other hand, the rapid phase in copper excretion was absent but the slow phase appeared to be unaffected. Pretreatment of rats with diethylmaleate to deplete hepatic and biliary glutathione abolished the rapid phase of copper excretion in control rats, while the slow phase remained unaffected. No significant effect of diethylmaleate on the hepatic handling of exogenous copper was observed in GY rats. The maximal capacity of the slow copper excretion pathway was 40-45 nmol/hr/100 g body wt, both in control and GY rats; the capacity of rapid excretion pathway depended on the administered copper load. Intravenous injection of copper induced the biliary excretion of a substantial amount of zinc in control rats, but not in GY rats. These results indicate the existence of at least two distinct biliary excretory pathways for copper in the rat, i.e. a slow and a rapid pathway, with a glutathione dependency of the latter only. The basal excretion of (endogenous) copper, in contrast to that of zinc, can proceed independently of glutathione excretion. However, glutathione appears to be involved in the rapid secretion of excess copper.

Umbilical vein and placental vessels from newborns with hereditary haemorrhagic telangiectasia type 1 genotype are normal despite reduced expression of endoglin.

Hereditary haemorrhagic telangiectasia, HHT, is an autosomal dominant disorder that affects approximately 1 in 8000 people. HHT1 is associated with mutations in the ENG (Endoglin) gene and with haploinsufficiency. The disorder is characterized by focally dilated vessels, which can lead to arteriovenous malformations and serious complications even in young children. In the current study, umbilical cord and placenta samples from newborns with ENG mutations were analyzed to estimate the level of corresponding protein and look for potential vascular dysplasia. We confirmed, using metabolic labelling and flow cytometry, that endoglin levels were significantly reduced to median values of 47 per cent (range 32-56 per cent) and 58 per cent (46-90 per cent), respectively, in human umbilical vein endothelial cells derived from newborns with ENG mutations (HHT1 group; n=18) relative to samples from newborns shown not to have the familial mutation (non-HHT group). We also quantified the relative expression of endoglin by estimating the endoglin/PECAM-1 staining ratio in tissue sections. We observed significantly lower values in the HHT1 group, compared to the non-HHT group for the umbilical vein (n=9; median 0.6 vs 0.9; ranges 0.2-1.0 and 0.5-1.5) and for placental stem villus vessels (n=9 and 10; median 0.42 vs 0.93; ranges 0.24-0.58 and 0.56-1.18). No differences in the estimated umbilical vein cross-sectional area and in the proportion of vessels present in placental villi were observed in sections from the HHT1 group relative to the non-HHT group. Thus, blood vessels from HHT1 individuals are maintained intact in the umbilical vein and placenta during pregnancy and delivery, despite a significant reduction in endoglin expression.