Induction of fecal cholesterol excretion is not effective for the treatment of hyperbilirubinemia in Gunn rats.

BACKGROUND: Unconjugated hyperbilirubinemia, a feature of neonatal jaundice or Crigler-Najjar syndrome, can lead to neurotoxicity and even death. We previously demonstrated that unconjugated bilirubin (UCB) can be eliminated via transintestinal excretion in Gunn rats, a model of unconjugated hyperbilirubinemia, and that this is stimulated by enhancing fecal fatty acid excretion. Since transintestinal excretion also occurs for cholesterol (TICE), we hypothesized that increasing fecal cholesterol excretion and/or TICE could also enhance fecal UCB disposal and subsequently lower plasma UCB concentrations. METHODS: To determine whether increasing fecal cholesterol excretion could ameliorate unconjugated hyperbilirubinemia, we treated hyperbilirubinemic Gunn rats with ezetimibe (EZE), an intestinal cholesterol absorption inhibitor, and/or a liver X receptor (LXR) and farnesoid X receptor (FXR) agonist (T0901317 (T09) and obeticholic acid (OCA), respectively), known to stimulate TICE. RESULTS: We found that EZE treatment alone or in combination with T09 or OCA increased fecal cholesterol disposal but did not lower plasma UCB levels. CONCLUSIONS: These findings do not support a link between the regulation of transintestinal excretion of cholesterol and bilirubin. Furthermore, induction of fecal cholesterol excretion is not a potential therapy for unconjugated hyperbilirubinemia. IMPACT: Increasing fecal cholesterol excretion is not effective to treat unconjugated hyperbilirubinemia. This is the first time a potential relation between transintestinal excretion of cholesterol and unconjugated bilirubin is investigated. Transintestinal excretion of cholesterol and unconjugated bilirubin do not seem to be quantitatively linked. Unlike intestinal fatty acids, cholesterol cannot "capture" unconjugated bilirubin to increase its excretion. These results add to our understanding of ways to improve and factors regulating unconjugated bilirubin disposal in hyperbilirubinemic conditions.

Disruption of HNF1α binding site causes inherited severe unconjugated hyperbilirubinemia.

Crigler-Najjar syndrome presents as severe unconjugated hyperbilirubinemia and is characteristically caused by a mutation in the UGT1A1 gene, encoding the enzyme responsible for bilirubin glucuronidation. Here we present a patient with Crigler-Najjar syndrome with a completely normal UGT1A1 coding region. Instead, a homozygous 3 nucleotide insertion in the UGT1A1 promoter was identified that interrupts the HNF1α binding site. This mutation results in almost complete abolishment of UGT1A1 promoter activity and prevents the induction of UGT1A1 expression by the liver nuclear receptors CAR and PXR, explaining the lack of a phenobarbital response in this patient. Although animal studies have revealed the importance of HNF1α for normal liver function, this case provides the first clinical proof that mutations in its binding site indeed result in severe liver pathology stressing the importance of promoter sequence analysis.

Metastable and equilibrium phase diagrams of unconjugated bilirubin IXα as functions of pH in model bile systems: Implications for pigment gallstone formation.

Metastable and equilibrium phase diagrams for unconjugated bilirubin IXα (UCB) in bile are yet to be determined for understanding the physical chemistry of pigment gallstone formation. Also, UCB is a molecule of considerable biomedical importance because it is a potent antioxidant and an inhibitor of atherogenesis. We employed principally a titrimetric approach to obtain metastable and equilibrium UCB solubilities in model bile systems composed of taurine-conjugated bile salts, egg yolk lecithin (mixed long-chain phosphatidylcholines), and cholesterol as functions of total lipid concentration, biliary pH values, and CaCl2 plus NaCl concentrations. Metastable and equilibrium precipitation pH values were obtained, and average pKa values of the two carboxyl groups of UCB were calculated. Added lecithin and increased temperature decreased UCB solubility markedly, whereas increases in bile salt concentrations and molar levels of urea augmented solubility. A wide range of NaCl and cholesterol concentrations resulted in no specific effects, whereas added CaCl2 produced large decreases in UCB solubilities at alkaline pH values only. UV-visible absorption spectra were consistent with both hydrophobic and hydrophilic interactions between UCB and bile salts that were strongly influenced by pH. Reliable literature values for UCB compositions of native gallbladder biles revealed that biles from hemolytic mice and humans with black pigment gallstones are markedly supersaturated with UCB and exhibit more acidic pH values, whereas biles from nonstone control animals and patients with cholesterol gallstone are unsaturated with UCB.

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.

Truncated UDP-glucuronosyltransferase (UGT) from a Crigler-Najjar syndrome type II patient colocalizes with intact UGT in the endoplasmic reticulum.

Mutations in the gene encoding bilirubin UDP-glucuronosyltransferase (UGT1A1) are known to cause Crigler-Najjar syndrome type II (CN-II). We previously encountered a patient with a nonsense mutation (Q331X) on one allele and with no other mutations in the promoter region or other exons, and proposed that CN-II is inherited as a dominant trait due to the formation of a heterologous subunit structure comprised of the altered UGT1A1 gene product (UGT1A1-p.Q331X) and the intact UGT1A1. Here, we investigated the molecular basis of CN-II in this case by expressing UGT1A1-p.Q331X in cells. UGT1A1-p.Q331X overexpressed in Escherichia coli or mammalian cells directly bound or associated with intact UGT1A1 in vitro or in vivo, respectively. Intact UGT1A1 was observed as a dimer using atomic force microscopy. Fluorescent-tagged UGT1A1-p.Q331X and intact UGT1A1 were colocalized in 293T cells, and fluorescence recovery after photobleaching analysis showed that UGT1A1-p.Q331X was retained in the endoplasmic reticulum (ER) without rapid degradation. These findings support the idea that UGT1A1-p.Q331X and UGT1A1 form a dimer and provide an increased mechanistic understanding of CN-II.

Beyond plasma bilirubin: the effects of phototherapy and albumin on brain bilirubin levels in Gunn rats.

BACKGROUND & AIMS: Severe unconjugated hyperbilirubinemia, as occurs in Crigler-Najjar disease and neonatal jaundice, carries the risk of neurotoxicity. This neurotoxicity is related to the increased passage of free bilirubin (UCB(free)), the fraction of bilirubin that is not bound to plasma proteins, into the brain. We hypothesized that albumin treatment would lower the UCB(free) fraction, and thus decrease bilirubin accumulation in the brain. METHODS: We treated chronic (e.g., as a model for Crigler-Najjar disease) and acute hemolytic (e.g., as a model for neonatal jaundice) moderate hyperbilirubinemic Gunn rats with phototherapy, human serum albumin (HSA) or phototherapy+HSA. RESULTS: In the chronic model, adjunct HSA increased the efficacy of phototherapy; it decreased plasma UCB(free) and brain bilirubin by 88% and 67%, respectively (p<0.001). In the acute model, adjunct HSA also increased the efficacy of phototherapy; it decreased plasma UCB(free) by 76% (p<0.001) and completely prevented the hemolysis-induced deposition of bilirubin in the brain. Phototherapy alone failed to prevent the deposition of bilirubin in the brain during acute hemolytic jaundice. CONCLUSIONS: We showed that adjunct HSA treatment decreases brain bilirubin levels in phototherapy-treated Gunn rats. We hypothesize that HSA decreases these levels by lowering UCB(free) in the plasma. Our results support the feasibility of adjunct albumin treatment in patients with Crigler-Najjar disease or neonatal jaundice.

Gilbert and Crigler Najjar syndromes: an update of the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene mutation database.

UGT1A1 enzyme defects are responsible of both Gilbert syndrome (GS) and Crigler-Najjar syndrome (CNS). GS depends on a variant TATAA element (which contains two extra TA nucleotides as compared to the wild type genotype) in the UGT1A1 gene promoter resulting in a reduced gene expression. On the contrary, CNS forms are classified in two types depending on serum total bilirubin concentrations (STBC): the more severe (CNS-I) is characterized by high levels of STBC (342-684µmol/L), due to total deficiency of the UGT1A1 enzyme, while the milder one, namely CNS-II, is characterized by partial UGT1A1 deficiency with STBC ranging from 103 to 342µmol/L. GS and CNS are caused by genetic lesions involving a complex locus encoding the UGT1A1 gene. The present report provides an update of all reported UGT1A1 gene mutations associated to GS and CNS.

[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.

Towards liver-directed gene therapy for Crigler-Najjar syndrome.

Crigler-Najjar (CN) syndrome is a recessive inherited disorder caused by deficiency of uridine diphospho-glucuronosyl transferase 1A1. This hepatic enzyme catalyzes the glucuronidation of bilirubin, an essential step in excretion into bile of this neurotoxic compound. As a result, CN patients suffer from severe unconjugated hyperbilirubinemia and are at risk of bilirubin encephalopathy. Over the last decades ex vivo and in vivo gene therapy using viral and non-viral vectors has been used to correct hyperbilirubinemia in the relevant animal model for CN syndrome, the Gunn rat. Several of these approaches did result in long-term correction of serum bilirubin levels in this animal model. However, none have been translated into a clinical trial. In this review we will recapitulate the strategies used and discuss their suitability for clinical application in the near future. We will also address specific safety measures in the gene therapy protocol needed to prevent adverse effects such as bilirubin toxicity. Since CN seems an ideal model for other monogenetic inherited metabolic liver disorders, development of liver-directed gene-therapy has relevance beyond this rare disease.

Small animal models of hepatocyte transplantation.

In this chapter, we describe techniques used to determine the efficiency of hepatocyte transplantation in animal models of liver disease. We have included the Gunn rat as a model of an inherited liver disease without hepatocyte damage and Abcb4 knockout mice as a model for an inherited liver disease with hepatocyte damage. Immunodeficient mice are included as an animal model for human hepatocyte transplantation.We describe problems that can be encountered in the maintenance and breeding of Gunn rats and immunodeficient Rag2/gamma common knockout mice. Protocols for the collection of bile in rats and mice are described, and we have also detailed the detection of green fluorescent protein (GFP)-labelled human hepatocytes in immunodeficient mice in this chapter.

Ultrasound-guided in Utero Transplantation of Placental Stem Cells into the Liver of Crigler-Najjar Syndrome Model Rat.

BACKGROUND: Advances in prenatal screening and early diagnosis of genetic disease will potentially allow for preemptive treatment of anticipated postnatal disease by in utero cell transplantation (IUCT). This strategy carries potential benefits over postnatal treatment, which might allow for improved engraftment and function of the transplanted cells. Congenital metabolic disorders may be an ideal target for this type of therapy, as in most cases, they require replacement of a single deficient hepatic enzyme, and multiple small-animal models exist for preclinical testing. METHODS: The Gunn rat, a Crigler-Najjar syndrome model animal lacking UDP-glucuronosyltransferase (UGT1A1), was used as recipient. Human amniotic epithelial cells (hAECs), which possess hepatic differentiation potential, were transplanted into the midgestation fetal Gunn rat liver via ultrasound-guided IUCT. The impact of IUCT on live birth and postnatal survival was evaluated. Human cell engraftment was immunohistochemically analyzed on postnatal day 21. RESULTS: Ultrasound-guided IUCT was conducted in rat fetuses on embryonic day 16. Following IUCT, the antihuman mitochondria-positive cells were detected in the liver of recipient rats at postnatal day 21. CONCLUSIONS: Here, we have introduced ultrasound-guided IUCT of hAEC using a small-animal model of a congenital metabolic disorder without immunosuppression. The immunological advantage of IUCT was demonstrated with xenogeneic IUCT. This procedure is suitable to conduct preclinical studies for exploring the feasibility and efficacy of ultrasound-guided transuterine cell injection using rodent disease models.

Quantitative Systems Pharmacology Model of hUGT1A1-modRNA Encoding for the UGT1A1 Enzyme to Treat Crigler-Najjar Syndrome Type 1.

Crigler-Najjar syndrome type 1 (CN1) is an autosomal recessive disease caused by a marked decrease in uridine-diphosphate-glucuronosyltransferase (UGT1A1) enzyme activity. Delivery of hUGT1A1-modRNA (a modified messenger RNA encoding for UGT1A1) as a lipid nanoparticle is anticipated to restore hepatic expression of UGT1A1, allowing normal glucuronidation and clearance of bilirubin in patients. To support translation from preclinical to clinical studies, and first-in-human studies, a quantitative systems pharmacology (QSP) model was developed. The QSP model was calibrated to plasma and liver mRNA, and total serum bilirubin in Gunn rats, an animal model of CN1. This QSP model adequately captured the observed plasma and liver biomarker behavior across a range of doses and dose regimens in Gunn rats. First-in-human dose projections made using the translated model indicated that 0.5 mg/kg Q4W dose should provide a clinically meaningful and sustained reduction of >5 mg/dL in total bilirubin levels.

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.

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.

Genetic predisposition to the metabolism of irinotecan (CPT-11). Role of uridine diphosphate glucuronosyltransferase isoform 1A1 in the glucuronidation of its active metabolite (SN-38) in human liver microsomes.

Irinotecan (CPT-11) is a promising antitumor agent, recently approved for use in patients with metastatic colorectal cancer. Its active metabolite, SN-38, is glucuronidated by hepatic uridine diphosphate glucuronosyltransferases (UGTs). The major dose-limiting toxicity of irinotecan therapy is diarrhea, which is believed to be secondary to the biliary excretion of SN-38, the extent of which is determined by SN-38 glucuronidation. The purpose of this study was to identify the specific isoform of UGT involved in SN-38 glucuronidation. In vitro glucuronidation of SN-38 was screened in hepatic microsomes from normal rats (n = 4), normal humans (n = 25), Gunn rats (n = 3), and patients (n = 4) with Crigler-Najjar type I (CN-I) syndrome. A wide intersubject variability in in vitro SN-38 glucuronide formation rates was found in humans. Gunn rats and CN-I patients lacked SN-38 glucuronidating activity, indicating the role of UGT1 isoform in SN-38 glucuronidation. A significant correlation was observed between SN-38 and bilirubin glucuronidation (r = 0.89; P = 0.001), whereas there was a poor relationship between para-nitrophenol and SN-38 glucuronidation (r = 0.08; P = 0.703). Intact SN-38 glucuronidation was observed only in HK293 cells transfected with the UGT1A1 isozyme. These results demonstrate that UGT1A1 is the isoform responsible for SN-38 glucuronidation. These findings indicate a genetic predisposition to the metabolism of irinotecan, suggesting that patients with low UGT1A1 activity, such as those with Gilbert's syndrome, may be at an increased risk for irinotecan toxicity.

N-Glucuronidation of the antiepileptic drug retigabine: results from studies with human volunteers, heterologously expressed human UGTs, human liver, kidney, and liver microsomal membranes of Crigler-Najjar type II.

Retigabine (D-23129), an N-2-amino-4-(4-fluorobenzylamino)phenylcarbamine acid ethyl ester, is a novel antiepileptic drug which is currently in phase II clinical development. This drug undergoes N-glucuronidation. We aimed to identify the principal enzymes involved in the N-glucuronidation pathway of retigabine and compared our findings with those obtained from human liver (a pool of 30 donors) and kidney microsomes (a pool of 3 donors) and with results from a human absorption, distribution, metabolism, and excretion study upon administration of 200 microCi of [(14)C]-D-23129. Essentially, microsomal assays with UGT1A1 produced only one of the 2 N-glucuronides, whereas UGT1A9 is capable of forming both N-glucuronides. The rates of metabolism for UGT1A9, human liver microsomes, and UGT1A1 were 200, 100, and 100 pmol N-glucuronide per minute per milligram of protein, respectively. At the 50 micromol/L uridine diphosphate glucoronic acid (UDPGA) concentration, UGT1A4 also catalyzed the N-glucuronidation of retigabine, the rates being approximately 5 and 6 pmol/(min.mg protein). With UGT1A9, the production of metabolites 1 and 2 proceeded at a K(m) of 38+/-25 and 45+/-15 micromol/L, whereas the K(m) for retigabine N-glucuronidation by human liver microsomal fractions was 145+/-39 micromol/L. Furthermore, a V(max) of 1.2+/-0.3 (nmol/[min.mg protein]) was estimated for human liver microsomes (4 individual donors). We investigated the potential for drug-drug interaction using the antiepileptic drugs valproic acid, lamotrigine, the tricyclic antidepressant imipramine, and the anesthetic propofol. These are commonly used medications and are extensively glucuronidated. No potential for drug-drug interactions was found at clinically relevant concentrations (when assayed with human liver microsomes or UGT1A9 enzyme preparations). Notably, the biosynthesis of retigabine-N-glucuronides was not inhibited in human liver microsomal assays in the presence of 330 micromol/L bilirubin, and glucuronidation of retigabine was also observed with microsomal preparations from human kidney and Crigler-Najjar type II liver. This suggests that lack of a particular UDP-glucuronosyltransferase (UGT) isoform (eg, UGT1A1 in kidney) or functional loss of an entire UGT1A gene does not completely abolish disposal of the drug. Finally, chromatographic separations of extracts from microsomal assays and human urine of volunteers receiving a single dose of (14)C-retigabine provided clear evidence for the presence of the 2 N-glucuronides known to be produced by UGT1A9. We therefore suggest N-glucuronidation of retigabine to be of importance in the metabolic clearance of this drug.

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 and bile bilirubin pigments in the differential diagnosis of Crigler-Najjar disease.

OBJECTIVE: To differentiate between Crigler-Najjar (CN) disease types 1 and 2. DESIGN: The patterns of serum bilirubins, bile pigment composition, and phenobarbital response were studied. PATIENTS: Three infants, affected by high serum unconjugated bilirubin concentrations, previously classified as type 1 CN. METHODS: Serum and bile bilirubin pigment composition, both before and after phenobarbital (PB) treatment, were determined by alkaline methanolysis and high-pressure liquid chromatography. PB was given for at least 3 weeks by oral administration (5 mg/kg bw per day). RESULTS: No diconjugated bilirubin was found either before or after PB treatment in the serum of the three studied infants. In two patients traces of monoconjugated bilirubin were detected before PB therapy, and the ratio of conjugated/total bilirubin (percent) was increased by the PB response. In the third patient, traces of monoconjugated bilirubin appeared only after PB administration. However, the serum unconjugated bilirubin concentration decreased significantly only in the second patient, following the second cycle of PB treatment, leading to the diagnosis of type 2 CN. The analysis of the methyl ester derivatives of bile pigments was also performed on bile samples obtained in two patients by Entero-Test (R) both before and after PB treatment. An absolute increment in monoesterified bilirubin concentration was found after PB administration, although the percent concentration increased in one case and decreased in the other. No diesterified bilirubin was detected in the bile samples. CONCLUSIONS: The present results show that in types 1 and 2 CN disease it is possible to detect traces of monoconjugated but not diconjugated bilirubin both in serum and in bile. Whereas PB treatment is effective in slightly increasing the serum monoconjugated bilirubin concentration even in type 1 CN disease, the diagnosis of type 1 or 2 is based on finding a substantial decrease of serum unconjugated bilirubin following PB administration.