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.

Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver.

Bilirubin, a breakdown product of heme, is normally glucuronidated and excreted by the liver into bile. Failure of this system can lead to a buildup of conjugated bilirubin in the blood, resulting in jaundice. The mechanistic basis of bilirubin excretion and hyperbilirubinemia syndromes is largely understood, but that of Rotor syndrome, an autosomal recessive disorder characterized by conjugated hyperbilirubinemia, coproporphyrinuria, and near-absent hepatic uptake of anionic diagnostics, has remained enigmatic. Here, we analyzed 8 Rotor-syndrome families and found that Rotor syndrome was linked to mutations predicted to cause complete and simultaneous deficiencies of the organic anion transporting polypeptides OATP1B1 and OATP1B3. These important detoxification-limiting proteins mediate uptake and clearance of countless drugs and drug conjugates across the sinusoidal hepatocyte membrane. OATP1B1 polymorphisms have previously been linked to drug hypersensitivities. Using mice deficient in Oatp1a/1b and in the multispecific sinusoidal export pump Abcc3, we found that Abcc3 secretes bilirubin conjugates into the blood, while Oatp1a/1b transporters mediate their hepatic reuptake. Transgenic expression of human OATP1B1 or OATP1B3 restored the function of this detoxification-enhancing liver-blood shuttle in Oatp1a/1b-deficient mice. Within liver lobules, this shuttle may allow flexible transfer of bilirubin conjugates (and probably also drug conjugates) formed in upstream hepatocytes to downstream hepatocytes, thereby preventing local saturation of further detoxification processes and hepatocyte toxic injury. Thus, disruption of hepatic reuptake of bilirubin glucuronide due to coexisting OATP1B1 and OATP1B3 deficiencies explains Rotor-type hyperbilirubinemia. Moreover, OATP1B1 and OATP1B3 null mutations may confer substantial drug toxicity risks.

[Hereditary pigmentary hepatoses (functional hyperbilirubinemias)].

Reviewed in this paper are bilirubin metabolism, definition and classification of hereditary pigmentary hepatoses, their pathogenesis and forms of inheritance, clinical picture, laboratory, instrumental and differential diagnosis, morphological changes in the hepatic tissue, and therapeutic strategies.

The patient presenting with isolated hyperbilirubinemia.

Hyperbilirubinemia is a common laboratory finding in clinical practice, being found in several haematological and liver diseases as well as in familial conditions (5-10% in Western countries). Although most of the familial forms of hyperbilirubinemia are classically viewed as benign conditions, they have gained an increased interest in the last few years since recent data have indicated that subjects with an impaired bilirubin metabolism may have an increased susceptibility to drug toxicity. The authors briefly review the main steps of bilirubin metabolism, with a special emphasis on the emerging concepts on the molecular mechanisms of regulation by nuclear receptors (NRs) and genetic factors. Then the different forms of isolated hyperbilirubinemia occurring in both adults and paediatrics are systematically analysed, and a new categorisation is also proposed in light of the recent advances in bilirubin research. Finally, a diagnostic algorithm is discussed, along with a correct approach to its management, in order to avoid unnecessary medical investigations.

Genes for jaundice.

The inheritable causes of jaundice comprise a large group of conditions of varying frequency, from Gilbert's syndrome which is relatively common, to the very rare Crigle-Najjar syndrome. Although these conditions have been well characterized clinically and in some cases biochemically, the underlying molecular defects were unknown because of a lack of knowledge about the process of bile secretion by hepatocytes. The recent cloning of several transporters for bile acids and other organic anions has enabled a greater understanding of this process and allowed correlation of the malfunction of these genes with specific disease processes. This new knowledge will provide for precision in diagnosis, allow antenatal testing and provide opportunities for gene therapy for some of the more serious disorders.

Intestinal excretion of unconjugated bilirubin in man and rats with inherited unconjugated hyperbilirubinemia.

Patients with Crigler-Najjar syndrome and Gunn rats cannot form bilirubin glucuronides owing to a lack of bilirubin UDP-glucuronosyltransferase activity. Because increased serum and tissue bilirubin levels remain constant, an alternative excretory route has to substitute for this deficiency. Gunn rats excrete in bile only 2-13% of the bilirubins eliminated in Wistar rats. In contrast, the biliary excretion rate of urobilinogen in Gunn and Wistar rats is comparable. The sum of bilirubins and urobilinogen excreted in the bile of Gunn rats amounts to 10-30% of pigments excreted in Wistar rats. Despite this low biliary excretion, the intestinal content and fecal excretion of bile pigments in Gunn and Wistar rats were similar. These data support an extrabiliary entrance of unconjugated bilirubin into the intestine. Additional proof for this was found in that the intestinal lumen of Gunn rats still contains a high amount of bilirubins and urobilinogen after 3 d of external biliary drainage. A similar procedure in Wistar rats resulted in the complete disappearance of bile pigments from the intestine. The direct transmural transport of bilirubin from blood to all parts of the intestinal lumen was demonstrated by injecting 14C-bilirubin i.v. into Gunn rats with isolated parts of small and large intestine. In Crigler-Najjar and Gilbert's syndrome patients, the biliary excretion of bile pigments has previously been shown to be strongly reduced. Their stools, however, contained approximately the same amount of bile pigments as in normal subjects. Although only traces of unconjugated bilirubin were detected in the stool of normal persons (4 +/- 3% of total bile pigments), higher amounts were found in patients with Crigler-Najjar disease (20 +/- 12&). These results suggest a direct intestinal permeation of unconjugated bilirubin in severe unconjugated hyperbilirubinemia both in man and rats.

Effects of luminal nutrients and small bowel transplants on congenital indirect hyperbilirubinemia.

The Gunn rat is an excellent model of Crigler-Najjar syndrome, type 1. In previous studies we demonstrated that heterotopic 15-20-cm jejunal transplants from Wistar rats lowered serum bilirubin levels by 40%, and the reduction was transient (6 weeks). In contrast, orthotopic transplants decreased bilirubin levels by 60% and the effect persisted throughout the 8-week study. This study was initiated to identify the luminal substance(s) which are responsible for the persistent bilirubin-lowering effect of jejunal transplants. Thirty-one Wistar to Gunn 15-20-cm jejunal transplants were randomized to receive daily Thiry-Vella graft irrigation with 5 ml of normal saline (n = 8); bile salts (cholate + deoxycholate, 40 mg/ml, n = 5; fats (Microlipid, 20 mg/ml, n = 5); proteins (Casec caseinate, 40 mg/ml, n = 5); and sugars (Moducal + Polycose, 40 mg/ml, n = 8). Bilirubin levels were measured spectrophotometrically at weekly intervals. At 4 and 8 weeks, enzyme-induced bilirubin conjugation activity was measured using added known amounts of added bilirubin. Irrigation of the transplants with saline, protein, and sugar resulted in moderate (40%) lowering of serum total and indirect bilirubin levels. Fat was significantly more effective, lowering mean total bilirubin levels from 9.6 +/- 0.4 to 1.6 +/- 0.2 mg/dl at 6 weeks. After this time, bilirubin levels increased slightly. Bile salts were slightly less effective, lowering bilirubin levels at 6 weeks by only 75%. However, this effect persisted and at 8 weeks levels averaged 2.4 +/- 0.2 mg/dl. Conjugating enzyme activity in the transplants increased from 1.4 +/- 0.3 to 2.5 +/- 0.5 mg bilirubin conjugated/mg tissue/hr. Luminal fats and bile salts appear to augment enzyme-induced bilirubin conjugation in heterotopic jejunal transplant recipients.

Hepatic transport of serum bilirubin, bromsulfophthalein, and indocyanine green in patients with congenital non-hemolytic hyperbilirubinemia and patients with constitutional indocyanine green excretory defect.

We carried out a retrospective study of 71 patients with congenital non-hemolytic hyperbilirubinemia who had been treated at our institution over the 25 years from 1965 to 1990. Twenty patients had Gilbert's syndrome, 1 had Crigler-Najjar syndrome, 1 had new type unconjugated hyperbilirubinemia, 21 had Dubin-Johnson syndrome, and 28 had Rotor's syndrome. We also reviewed 20 patients with constitutional indocyanine green (ICG) excretory defect. The study focused on the hepatic transport of serum bilirubin, bromsulfophthalein (BSP), and ICG. In Dubin-Johnson syndrome, a defect appeared in late-stage transport, while uptake and storage capacity were normal. In Rotor's syndrome, defects were found in the early stage, and storage capacity was reduced, while excretion into bile was slightly suppressed. A secondary rise in serum ICG was seen in 5 of the 10 patients with Dubin-Johnson syndrome. The transport defect in Gilbert's syndrome was unclear. It could not be considered to be homogeneous, but it may exist at multiple sites, from the conjugation with serum proteins to excretion into bile. Following phenobarbital administration, the ICG secondary rise in the 5 patients with Dubin-Johnson syndrome disappeared, and ICG was rapidly cleared from blood. However, in patients with Dubin-Johnson syndrome, BSP clearance in serum did not show any change before and after phenobarbital administration. ICG excretion in patients with constitutional ICG excretory defect was due only to the impairment o ICG transport, and the defect was suggested to be hepatic uptake. These results indicate that studies of the hepatic transport of bilirubin, BSP, and ICG are useful for determining the etiological factors involved in congenital hyperbilirubinemia and constitutional ICG excretory defect.

Lithocholate-3-O-glucuronide-induced cholestasis. A study with congenital hyperbilirubinemic rats and effects of ursodeoxycholate conjugates.

The mechanism of lithocholate-3-O-glucuronide-induced cholestasis is unknown. In this study, we investigated the cholestatic effects of this agent in a congenital hyperbilirubinemic rat, EHBR. We also studied the effects of ursodeoxycholate-3-O-glucuronide and tauroursodeoxycholate on lithocholate-3-O-glucuronide-induced cholestasis in rats. Lithocholate-3-O-glucuronide, administered at the rate of 0.1 mumol/min/100 g for 40 min, a cholestatic dose in control rats, failed to cause cholestasis in EHBR, and biliary lithocholate-3-O-glucuronide excretion was delayed. Biliary concentrations of this agent did not correlate with the severity of cholestasis. Both tauroursodeoxycholate and ursodeoxycholate-3-O-glucuronide, infused at the rate of 0.2 mumol/min/100 g for 120 min, completely inhibited cholestasis induced by lithocholate-3-O-glucuronide administered at the rate of 0.1 mumol/min/100 g for 40 min. Only tauroursodeoxycholate enhanced biliary lithocholate-3-O-glucuronide excretion. These findings indicate that lithocholate-3-O-glucuronide-induced cholestasis is induced by damage at the level of the bile canalicular membrane. Ursodeoxycholate-3-O-glucuronide inhibits this cholestasis, possibly by inhibiting the access of lithocholate-3-O-glucuronide to the bile canalicular membrane.

The uncoupling of biliary lipid from bile acid secretion by organic anions in the rat.

A number of organic anions has been shown to inhibit biliary phospholipid and cholesterol secretion without affecting bile acid secretion. However, the mechanism of this uncoupling phenomenon is still unclear. This study shows a comparison of the effects of ampicillin (18 mumol/100g body wt), sulfated taurolithocholic acid (0.2 and 1.0 mumol/100 g body wt), and indocyanine green (0.6 mumol/100 g body wt) in control and Groningen Yellow-Wistar rats with chronic (8 days) biliary drainage. Groningen Yellow rats have a hereditary defect in hepatobiliary transport of various organic anions. Bile secretion, but not hepatic uptake, of the three organic anions was strongly impaired in Groningen Yellow rats compared with controls. Ampicillin and sulfated taurolithocholic acid caused a strong uncoupling in control rats but had no effect or a much smaller effect on lipid secretion in Groningen Yellow rats. Indocyanine green did not affect lipid secretion, in either control or in Groningen Yellow rats. Gel-filtration chromatography of bile showed a specific coelution of ampicillin and sulfated taurolithocholic acid with the bile acid fraction, whereas indocyanine green coeluted with the phospholipid/cholesterol fraction. This study concludes that the uncoupling by ampicillin and sulfated taurolithocholic acid occurs after their secretion into bile and is caused by interaction of these compounds with bile acids. It is hypothesized that this interaction inhibits the capacity of bile acids to induce secretion of phospholipids and cholesterol into the bile.

Defective ATP-dependent bile canalicular transport of organic anions in mutant (TR-) rats with conjugated hyperbilirubinemia.

TR- mutant Wistar rats secrete markedly fewer organic anions other than bile acids from the liver into the bile than do control rats. Fluorescence-image analysis of isolated normal and TR- hepatocyte "doublets", which retain a bile canaliculus between them, revealed that normal hepatocytes readily transport a fluorescent bile acid (fluorescein isothiocyanate glycocholate) and a nonbile acid organic anion (carboxydichlorofluorescein diacetate) into the canaliculus. Hepatocyte doublets from TR- rats also transported fluorescein isothiocyanate glycocholate normally, but transport of carboxydichlorofluorescein diacetate into the canaliculus was negligible. Vesicles derived from the canicular domain of the plasma membrane of hepatocytes (CMV) from control and TR- rats were used to characterize the transport process for 35S-labeled bromosulphthalein and 35S-labeled bromosulphthalein glutathione, which represent nonbile acid organic anions. CMV from normal rat hepatocytes had an ATP- and temperature-dependent, saturable transport process for these 35S-labeled compounds that was absent in CMV from TR- rats. CMV from TR- rats retained normal ATP-dependent transport of daunomycin, and immunologic blots with a monoclonal antibody against the multidrug resistance gene product, P-glycoprotein, revealed no difference between normal and TR-CMV. These studies reveal that the bile canaliculus in normal rats contains an ATP-dependent organic anion transport system that is functionally absent in TR- mutant rats. The defect in TR- mutant rats is phenotypically similar to that seen in mutant Corriedale sheep and in the Dubin-Johnson syndrome in man.

Dissociation between vascular and metabolic effects of nicotinic acid in Gilbert's syndrome.

The relationship between the vasodilating and the hyperbilirubinaemic effect of low and high doses (50 and 300 mg i.v.) of nicotinic acid was studied in baseline conditions and after indomethacin pretreatment in healthy controls and patients with Gilbert's syndrome (a condition characterized by fluctuating, nonhaemolytic unconjugated hyperbilirubinaemia). The hyperbilirubinaemic effect of nicotinic acid was confirmed to be more pronounced in Gilbert's syndrome patients than in controls. The magnitude of hyperbilirubinaemia in the two groups was not dependent on the dose of nicotinic acid or indomethacin pretreatment. A dose-dependent vasodilation which was inhibited by indomethacin could be demonstrated in both controls and Gilbert's syndrome subjects. Vasodilating properties of nicotinic acid were therefore found to be dissociated from the effect on bilirubin.

Cerebellar hypoplasia in the Gunn rat with hereditary hyperbilirubinemia: immunohistochemical and neurochemical studies.

Immunohistochemical reactions were conducted, using the antibodies against GFA and S-100 proteins on sections of cerebellum from the homozygous (jj) and the heterozygous (Jj) Gunn rats. Hypertrophy of the fibrous astrocytes was observed but hyperplasia of the glial cells was not. Although the molecular layer was very thin, the Bergmann fibre appeared normal. Among the free amino acids in the cerebellum from the jj rat, glutamate concentration decreased to two-thirds of the control level. The protein profile of the cerebellum from the jj rat obtained by SDS-polyacrylamide gel electrophoresis revealed that the amount of P400 protein that is characteristic of Purkinje cells decreased considerably and there were also some changes of the other unidentified proteins. By two-dimensional electrophoresis, it was observed that in the supernatant from the jj rat cerebellum one protein spot diminished and in the particulate fraction from the jj rat one spot was enormously increased. The activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) in the cerebellum from the jj rat did not differ significantly from that of the control; however, activities of choline acetyltransferase and acetylcholinesterase of the jj rat were about twice as high as those of the control. 2-Deoxyglucose incorporation was maximum in the granular layer from both the jj and the Jj rat cerebella. However, the incorporation in the jj cerebellum was not higher than in the Jj control and even lower in some parts of the jj cerebellum than in the control Jj cerebellum.

[Constitutional hyperbilirubinemia and mixed bilirubin. Clinical and functional study]. / Iperbilirubinemie costituzionali a bilirubina mista. Studio clinico e funzionale.

Three cases of non-haemolytic constitutional icterus with prevalently conjugated bilirubin have been studied: one case of Dubin-Johnson syndrome and two cases of Rotor's syndrome. Confirmation was obtained for the differences between the two syndromes from the physiopathological (by means of the BSF load test and determination of urinary excretion of porphyrin) and morphological (macroscopic, histological, ultrastructural) viewpoints. The following were carried out in all cases: a load curve with crystalline bilirubin, study of the course of the curve and application of compartmental analysis to the curve. Findings underlie the hypothesis that Rotor's syndrome contains an uptake defect and an accumulation change on the part of the liver cell of organic anions and that it is therefore possible to suggest some identity between Rotor's syndrome and the so-called "Hepatic storage disease".