Bilirubin UDP-glucuronosyltransferase Activity Research Articles

Isolated Silymarin Flavonoids Increase Systemic and Hepatic Bilirubin Concentrations and Lower Lipoperoxidation in Mice.

Bilirubin is considered to be one of the most potent endogenous antioxidants in humans. Its serum concentrations are predominantly affected by the activity of hepatic bilirubin UDP-glucuronosyl transferase (UGT1A1). Our objective was to analyze the potential bilirubin-modulating effects of natural polyphenols from milk thistle (Silybum marianum), a hepatoprotective herb. Human hepatoblastoma HepG2 cells were exposed to major polyphenolic compounds isolated from milk thistle. Based on in vitro studies, 2,3-dehydrosilybins A and B were selected as the most efficient compounds and applied either intraperitoneally or orally for seven days to C57BL/6 mice. After, UGT1A1 mRNA expression, serum, intrahepatic bilirubin concentrations, and lipoperoxidation in the liver tissue were analyzed. All natural polyphenols used increased intracellular concentration of bilirubin in HepG2 cells to a similar extent as atazanavir, a known bilirubinemia-enhancing agent. Intraperitoneal application of 2,3-dehydrosilybins A and B (the most efficient flavonoids from in vitro studies) to mice (50 mg/kg) led to a significant downregulation of UGT1A1 mRNA expression (46 ± 3% of controls, p < 0.005) in the liver and also to a significant increase of the intracellular bilirubin concentration (0.98 ± 0.03vs.1.21 ± 0.02 nmol/mg, p < 0.05). Simultaneously, a significant decrease of lipoperoxidation (61 ± 2% of controls, p < 0.005) was detected in the liver tissue of treated animals, and similar results were also observed after oral treatment. Importantly, both application routes also led to a significant elevation of serum bilirubin concentrations (125 ± 3% and 160 ± 22% of the controls after intraperitoneal and oral administration, respectively, p < 0.005 in both cases). In conclusion, polyphenolic compounds contained in silymarin, in particular 2,3-dehydrosilybins A and B, affect hepatic and serum bilirubin concentrations, as well as lipoperoxidation in the liver. This phenomenon might contribute to the hepatoprotective effects of silymarin.

Combined UGT1A1 and UGT1A7 variant alleles are associated with increased risk of Gilbert’s syndrome in Taiwanese adults

Gilbert's syndrome (GS) is caused by a reduction in the activity of hepatic bilirubin UDP-glucuronosyltransferase (UGT). This reduction is associated with UGT1A1*28 and UGT1A1*6 polymorphisms. Recent research also showed that carriage of UGT1A1*6 allele were significantly related with UGT1A7*3. Polymerase chain reaction-restriction fragment length polymorphism were utilized to determine UGT1A7 and UGT1A1 genes for 207 patients with GS and 207 gender/age-matched healthy controls. For the 207 healthy controls, linkage disequilibrium was observed between -57UGT1A7 and 622UGT1A7 loci (D' = 1.00 and r(2) = 1.00), -57UGT1A7 and 211UGT1A1 loci (D' = 0.72 and r(2) = 0.36), respectively. A dose-response effect for number of at-risk allele of UGT1A1 and risk for GS was noted (odds ratio (OR) = 8.19 for heterozygous UGT1A1*28 genotype; OR = 124.96 for homozygous UGT1A1*28 genotype; and p for trend <0.05). Patients with combined genotypes carrying UGT1A7 variant alleles and UGT1A1 variant alleles (including UGT1A1*28 and UGT1A1*6) are associated with increased risk of GS (OR = 13.96 for patients with combined genotype carrying at least one variant allele of UGT1A1 and UGT1A7). In conclusion, the -57UGT1A7 (T>G) is highly associated with UGT1A7*3 and moderately associated with 211UGT1A1 (G>A). Certain UGT1A1/UGT1A7 combined genotypes are risk factors of GS.

Critical assessment of lifelong phenotype correction in hyperbilirubinemic Gunn rats after retroviral mediated gene transfer

Among inherited diseases of the liver, Crigler-Najjar type 1 disease (CN-1), which results from complete deficiency in bilirubin UDP-glucuronosyltransferase activity (B-UGT1), is an attractive target for gene therapy studies. Hyperbilirubinemic Gunn rats, a model of CN-1, were injected at 2 days of age with lentiviral or oncoretroviral vectors encoding the human B-UGT1. After injection, bilirubinemia was normalized for up to 95 weeks. Bilirubin conjugates were present in the bile, demonstrating liver transduction. PCR and enzyme activity analysis confirmed gene and phenotype correction in liver. We observed that when using a strong viral promoter, a complete correction was achieved with less than 5% of B-UGT1 copy per haploid genome and after a reconstitution of 12% B-UGT1 normal activity. Liver histology remained normal throughout the experiment and tissue distribution analysis revealed preferential hepatocyte transduction after systemic delivery. Finally, no adverse immune response occurred even after induction of nonspecific liver inflammation, suggesting immune ignorance to the therapeutic protein. Our present results document the lifelong safety of gene therapy for CN-1 with retroviral vectors. They offer a better delineation of liver gene correction level required to achieve complete correction of bilirubinemia and pave the way for future clinical application of gene therapy for inherited liver disorders.

Adeno-associated Virus Vector Serotypes Mediate Sustained Correction of Bilirubin UDP Glucuronosyltransferase Deficiency in Rats

Crigler–Najjar (CN) patients have no bilirubin UDP glucuronosyltransferase (UGT1A1) activity and suffer brain damage because of bilirubin toxicity. Vectors based on adeno-associated virus (AAV) serotype 2 transduce liver cells with relatively low efficiency. Recently, AAV serotypes 1, 6, and 8 have been shown to be more efficient for liver cell transduction. We compared AAV serotypes 1, 2, 6, and 8 for correction of UGT1A1 deficiency in the Gunn rat model of CN disease. Adult Gunn rats were injected with CMV-UGT1A1 AAV vectors. Serum bilirubin was decreased over the first year by 64% for AAV1, 16% for AAV2, 25% for AAV6, and 35% for AAV8. Antibodies to UGT1A1 were detected after injection of all AAV serotypes. An AAV1 UGT1A1 vector with the liver-specific albumin promoter corrected serum bilirubin levels but did not induce UGT1A1 antibodies. Two years after injection of AAV vectors all animals had large lipid deposits in the liver. These lipid deposits were not seen in age-matched control animals. AAV1 vectors are promising candidates for CN gene therapy because they can mediate a reduction in serum bilirubin levels in Gunn rats that would be therapeutic in humans.

Hepatocyte transplantation

Hepatocyte transplantation

Evidence for phosphorylation requirement for human bilirubin UDP-glucuronosyltransferase (UGT1A1) activity

Our discovery of rapid down-regulation of human bilirubin UDP-glucuronosyltransferase (UGT) in colon cell lines that was transient and irreversible following curcumin- and calphostin-C-treatment, respectively, suggested phosphorylation event(s) were involved in activity. Likewise, bilirubin-UGT1A1 expressed in COS-1 cells was inhibited by curcumin and calphostin-C. Because calphostin-C is a highly specific protein kinase C (PKC) inhibitor, we examined and found 4 to 5 predicted PKC phosphorylation sites in 11 UGTs examined. UGT1A1 incorporated [ 33 P] orthophosphate, which was inhibited by calphostin-C. Also triple mutant, T75A/T112A/S435G-UGT1A1, at predicted PKC sites failed to incorporate [ 33 P] orthophosphate. Individual or double mutants exhibited dominant-negative, additive, or no effect, while the triple mutant retained 10–15% activity towards bilirubin and two xenobiotics. Compared to wild-type, S435G and T112A/S435G shifted pH-optimum for eugenol, but not for bilirubin or anthraflavic acid, toward alkaline and acid conditions, respectively. This represents the first evidence that a UGT isozyme requires phosphorylation for activity.

Effects on extrahepatic UDP-glucuronosyltransferases in hypophysectomized rat.

The effects of hypophysectomy on hepatic and extrahepatic UDP-glucuronosyltransferase activities in adult male rats were observed. UDP-glucuronosyltransferase activities toward 1-naphthol decreased to 20-30% of control in the liver, kidney, lung, and testis. The mRNA of UGT1A6, which is an isoform contributing to the glucuronidation of various phenolic xenobiotics such as 1-naphthol, were decreased drastically in the liver, kidney, and testis by hypophysectomy. However, while bilirubin UDP-glucuronosyltransferase activity in the liver intensified, there was only a slight increase in the activity in the kidney and no alteration in the lung. The mRNA of UGT1A1, which is an isoform contributing to the glucuronidation of bilirubin, increased significantly in the liver and slightly in the kidney after hypophysectomy. These inductions and reductions in enzymatic activities and mRNA levels in each tissue were restored to control levels by intermittent injections of rat growth hormone. Interestingly, while hepatic UGT activity toward bisphenol A remained constant in hypophysectomized rats, the testicular UGT activity declined to 10-15% of control but returned to normal levels following growth hormone treatment, suggesting that an unknown UGT isoform (s) mediates bisphenol A glucuronidation in the testis. These results indicate that the expression of extrahepatic UGT is isoform-specific and regulated differentially in tissues by the pituitary gland.

Mechanism of indinavir-induced hyperbilirubinemia.

Indinavir is a viral protease inhibitor used for the treatment of HIV infection. Unconjugated hyperbilirubinemia develops in up to 25% of patients receiving indinavir, prompting drug discontinuation and further clinical evaluation in some instances. We postulated that this side-effect is due to indinavir-mediated impairment of bilirubin UDP-glucuronosyltransferase (UGT) activity and would be most pronounced in individuals with reduced hepatic enzyme levels, as occurs in approximately 10% of the population manifesting Gilbert's syndrome. This hypothesis was tested in vitro, in the Gunn rat model of UGT deficiency, and in HIV-infected patients with and without the Gilbert's polymorphism. Indinavir was found to competitively inhibit UGT enzymatic activity (K(I) = 183 microM) while concomitantly inducing hepatic bilirubin UGT mRNA and protein expression. Although oral indinavir increased plasma bilirubin levels in wild-type and heterozygous Gunn rats, the mean rise was significantly greater in the latter group of animals. Similarly, serum bilirubin increased by a mean of 0.34 mg/dl in indinavir-treated HIV patients lacking the Gilbert's polymorphism versus 1.45 mg/dl in those who were either heterozygous or homozygous for the mutant allele. Whereas saquinavir also competitively inhibits UGT activity, this drug has not been associated with hyperbilirubinemia, most likely because of the higher K(I) (360 microM) and substantially lower therapeutic levels as compared with indinavir. Taken together, these findings indicate that elevations in serum-unconjugated bilirubin associated with indinavir treatment result from direct inhibition of bilirubin-conjugating activity.

Expression of bilirubin UDP-glucuronosyltransferase (bUGT) throughout fetal development: intrasplenic transplantation into Gunn rats to correct enzymatic deficiency.

The aim of this work was to determine the pattern of expression of hepatic bilirubin UDP-glucuronosyltransferase throughout fetal development in rats, with the purpose of using fetal hepatocytes at the most appropiate stage of development for transplantation into Gunn rats lacking bilirubin UDP-glucuronosyltransferase activity and then assessing the therapeutic capacity of the implants. The results show that at day 13 of gestational life there is already bilirubin UDP-glucuronosyltransferase gene expression. Twenty-one-day fetal hepatocyte transplantation was also performed into the spleens of hyperbilirubinemic Gunn rats, when alpha-fetoprotein mRNA is still detectable. At 15, 30, and 90 days after transplantation, a mild decrease in total bilirubin serum levels was observed. An increase in bile conjugated bilirubin also was observed at 30 and 90 days. These data suggest the favorable evolution of transplanted cells and show its feasibility for therapy.

Molecular basis of bilirubin UDP-glucuronosyltransferase induction in spontaneously diabetic rats, acetone-treated rats and starved rats.

The co-ordinated induction of several hepatic drug-metabolizing enzymes is a common feature in the regulation of drug biotransformation under normal and pathological conditions. In the present study the activity and expression of bilirubin UDP-glucuronosyltransferase (UGT1A1) were investigated in livers of BioBreeding/Worcester diabetic, fasted and acetone-treated rats. Bilirubin glucuronidation was stimulated by all three treatments; this was correlated with an increase in the UGT1A1 protein concentration in hepatic microsomes. Transcriptional induction of UGT1A1 was also observed in diabetes and starvation but not with acetone treatment, which apparently caused translational stabilization of the enzyme protein. The hormonal/metabolic alterations in diabetes and starvation might be a model for postnatal development. The sudden interruption of maternal glucose supply signals the enhanced expression of UGT1A1, giving a novel explanation for the physiological induction of bilirubin glucuronidation in newborn infants.

Gilbert syndrome accelerates development of neonatal jaundice

Objective: Gilbert Syndrome (GS), associated with unconjugated hyperbilirubinemia and decreased bilirubin UDP-glucuronosyltransferase activity, is usually diagnosed after puberty. The role of GS in neonatal jaundice is unknown. This study tested the hypothesis that a recently identified molecular marker for GS (a TA insertion in the promoter of UGT1A, the gene encoding bilirubin UDP-glucuronosyltransferase) is associated with neonatal jaundice. Study design: Transcutaneous jaundice index was measured shortly after birth and daily for the first week of life in 151 healthy infants. Genomic DNA was isolated from blood or buccal brushings, and the UGT1A promoter was amplified by the polymerase chain reaction to yield 90 (A[TA] 6TAA, normal) or 92 (A[TA] 7TAA, GS) base pair products. Statistical analysis used Kruskal-Wallis, Wilcoxon, and Fisher's exact tests. Results: Nineteen (13%) subjects were homozygous for the A(TA) 7TAA polymorphism associated with GS. The A(TA) 7TAA homozygotes had a greater increase in jaundice index during the first 2 days of life than heterozygotes or A(TA) 6TAA homozygotes. Conclusion: Although peak jaundice levels did not differ among groups, newborn infants with the molecular marker for GS have an accelerated increase in neonatal jaundice during the first 2 days of life. (J Pediatr 1998;132:656-60.)

Genetic defects of the UDP-glucuronosyltransferase-1 (UGT1) gene that cause familial non-haemolytic unconjugated hyperbilirubinaemias

Congenital familial non-haemolytic hyperbilirubinaemias are potentially lethal syndromes caused by genetic lesions that reduce or abolish hepatic bilirubin UDP-glucuronosyltransferase activity. Here we describe genetic defects that occur in the UGT1 gene complex that cause three non-haemolytic unconjugated hyperbilirubinaemia syndromes. The most severe syndrome, termed Crigler-Najjar syndrome type I, is mainly associated with mutations in exons 2 to 5 that affect all UGT1 enzymes and many of the mutations result in termination codons and frameshifts. Crigler-Najjar type II syndrome which is treatable with phenobarbital therapy is associated with less dramatic missense mutations or heterozygous expression of mutant and normal alleles. Gilbert's syndrome, the most prevalent (2–19% in population studies) and mildest of the three syndromes is principally caused by a TA insertion at the TATA promoter region upstream of the UGT1A1 exon. Current methods used for the diagnosis and treatment of these diseases are discussed.

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.

Genetic defects at the UGT1 locus associated with Crigler-Najjar type I disease, including a prenatal diagnosis

Characterization of the UGT1 gene complex locus encoding both multiple bilirubin and phenol UDP-glucuronosyltransferases (transferases) has been critical in identifying mutations in the bilirubin isoforms. This study utilizes this information to identify the bases of deficient bilirubin UDP-glucuronosyltransferase activity encoded by the UGT1A gene for the major bilirubin isozyme, HUG-Br1, in 3 Crigler-Najjar type I individuals and the genotype of an at-risk unborn sibling of one patient. A homozygous and heterozygous two-base mutation (CCC to CGT) created the HUG-Br1P387R mutant of the major bilirubin transferase in 2 different Crigler-Najjar type I patients, B.G. and G.D., respectively. Both parents of B.G. and his unborn sibling, J.G., were determined to be carriers of the P387R mutation. G.D. also contains the CAA to TAA nonsense mutation (G1n357st). Y.A. has a homozygous CT deletion in codons 40/41. The HUG-Br1P387R mutant protein was totally inactive at the major pH optimum (6.4), but retained 26% normal activity at the minor pH optimum (7.6), which was 5.4% of the combined activities measured at the two pH values.

Genetic defects at the UGT1 locus associated with Crigler‐Najjar type I disease, including a prenatal diagnosis

Characterization of the UGT1 gene complex locus encoding both multiple bilirubin and phenol UDP-glucuronosyltransferases (transferases) has been critical in identifying mutations in the bilirubin isoforms. This study utilizes this information to identify the bases of deficient bilirubin UDP-glucuronosyltransferase activity encoded by the UGT1A gene for the major bilirubin isozyme, HUG-Br1, in 3 Crigler-Najjar type I individuals and the genotype of an at-risk unborn sibling of one patient. A homozygous and heterozygous two-base mutation (CCC to CGT) created the HUG-Br1P387R mutant of the major bilirubin transferase in 2 different Crigler-Najjar type I patients, B.G. and G.D., respectively. Both parents of B.G. and his unborn sibling, J.G., were determined to be carriers of the P387R mutation. G.D. also contains the CAA to TAA nonsense mutation (G1n357st). Y.A. has a homozygous CT deletion in codons 40/41. The HUG-Br1P387R mutant protein was totally inactive at the major pH optimum (6.4), but retained 26% normal activity at the minor pH optimum (7.6), which was 5.4% of the combined activities measured at the two pH values. Am. J. Med. Genet. 68:173–178, 1997 © 1997 Wiley-Liss, Inc.

Genetic background of constitutional unconjugated hyperbilirubinemia

Crigler-Najjar syndrome (types I and II) and Gilbert's syndrome are familial disorders associated with severe to mild unconjugated hyperbilirubinemia. In these conditions, the activity of bilirubin UDP-glucuronosyltransferase (UGT1 ∗1), which is located in the hepatocyte endoplasmic reticulum, is defective, and severely and moderately decreased, respectively. UGT1 ∗1 is derived from one of the UGT1 genes. It has a promoter containing a TATA box and consists of exon 1A (which is one of the individual first exons) and common exons 2–5. UGT1 ∗1 mRNA is formed by differential splicing of these exons. In recent years, gene analysis of these syndromes has been carried out, and genetic abnormalities have been clarified. Homozygous nonsense mutations, mis-sense mutations, and other relevant mutations of exons 1A-5 have been reported in almost all of the patients with Crigler-Najjar syndrome type I, while mainly homozygous mis-sense mutations of exons 1A, 2, and 5 have been reported in type II patients. Almost all patients with this syndrome (types I and II) show autosomal recessive inheritance. On the other hand, some patients with Gilbert's syndrome show heterozygous mis-sense mutations in exons 1A, 4, and 5, while others show homozygous 2-base pair-insertion mutation (TA) into the TATA box in the promoter region [A(TA) 7TAA; normal: A(TA) 6TAA]. The pattern of inheritance can be autosomal recessive or autosomal dominant. It has also been clarified that enzyme activity is lowered to about 30% (rather than 50%) by heterozygous mutations of the coding region, because of the occurrence of dominant negative mutation based on subunit-structure of the enzyme.

GILBERT SYNDROME IS ASSOCIATED WITH NEONATAL JAUNDICE. • 1809

A molecular marker for Gilbert Syndrome (GS) was recently identified (Bosma et al, NEJM 1995;333:1171-5). GS is associated with hyperbilirubinemia and decreased bilirubin UDP-glucuronosyltransferase activity (B-UGT) in adults. However, GS is usually diagnosed after puberty and the role of GS in the neonatal period is unknown. The current study tested the hypothesis that this marker (an extra TA in the TATAA box of the B-UGT gene, UGTIA, promoter) is associated with neonatal jaundice.

Bilirubin glucuronidation by intact Gunn rat fibroblasts expressing bilirubin UDP-glucuronosyltransferase

Crigler-Najjar (CN) disease is an inherited disorder of bilirubin metabolism. The disease is caused by a deficiency of the hepatic enzyme bilirubin UDP-glucuronosyltransferase (B-UGT). Patients with CN disease have high serum levels of the toxic compound, unconjugated bilirubin. The only defect in bilirubin metabolism of CN patients is the absence of B-UGT activity. The transplantation of cells able to glucuronidate bilirubin should therefore lower serum bilirubin levels. The Gunn rat is the animal model of CN disease. Primary Gunn rat fibroblasts (GURF) were transduced with a recombinant retrovirus, capable of transferring B-UGT cDNA. A cell line was obtained expressing B-UGT at a level comparable to hepatocytes. Bilirubin added to the culture medium of these cells was glucuronidated and excreted. The B-UGT activities of transduced GURF and freshly isolated Wistar hepatocytes were compared at different bilirubin concentrations. The specific B-UGT activities of these two cell types were comparable when physiological bilirubin concentrations (5-10 microM) were present in the culture media. At higher bilirubin concentrations (20-80 microM) the hepatocytes were more active than the transduced GURF. We conclude that with the addition of only one enzyme (B-UGT) fibroblasts can perform the complete set of reactions necessary for bilirubin glucuronidation. The difference in B-UGT activity between transduced GURF and hepatocytes at 20-80 microM bilirubin can be explained by lower UDP-glucuronic acid and glutathione S-transferase levels in GURF. Our findings also indicate that these cells could be used to develop extrahepatic gene therapy for CN disease.

Enhancement of intestinal bilirubin UDP-glucuronosyltransferase activity by modification of microsomal lipid composition

Microsomal membranes from rat small intestine exhibit a higher cholesterol/phospholipid ratio and a lower phosphatidylcholine/sphingomyelin ratio than those of the liver, which could negatively influence membrane-bound enzymes like bilirubin UDP-glucuronosyltransferase. To study the effect of in vitro modifications in the lipid composition of intestinal microsomes on bilirubin glucuronidating activity, two strategies were employed. On one hand, microsomal lipids were modified in order to mimic those of the liver tissue; on the other hand, cholesterol content of microsomal membranes was increased or decreased with respect to the normal value. Lipid changes were carried out by both an enzyme-mediated and a detergent-mediated procedure. Irrespective of the methodology employed, when a depletion in the cholesterol content was produced, enzyme activity increased about 40%, and when lipid composition approached that of the liver tissue, which not only decreased cholesterol but also modified phospholipid classes, enzyme activity increased about 80%. Both lipid modifications produced a ‘fluidification’ of microsomal membranes measured by fluorescence anisotropy of 1,6-diphenylhexatriene, being the effect of the approach to the liver higher than that of the decrease of cholesterol. In turn, the enrichment in cholesterol of microsomal membranes led to a decrease of enzyme activity of about 20% and to a ‘rigidization’ of the membranes. The present findings suggest that in rat intestine, bilirubin glucuronidation is strongly influenced by microsomal lipids. In particular, there seems to be an inverse association between enzyme activity and the cholesterol content of membrane.

THE physiology of jaundice: Molecular and functional characterization of the Crigler-Najjar syndromes

Crigler-Najjar (CN) disease is classified into two subtypes, type I and II. The molecular basis for the difference between these types is not well understood. Several mutations in the bilirubin UDP-glucuronosyltransferase (B-UGT) gene of six CN type I and two CN type II patients were identified. Recombinant cDNAs containing these mutations were expressed in COS cells. B-UGT activity was measured using HPLC and the amount of expressed protein was quantitated using a sandwich ELISA. This enabled us to determine the specific activities of the expressed enzymes. All type I patients examined had mutations in the B-UGT1 gene that lead to completely inactive enzymes. The mutations in the B-UGT1 gene of patients with CN type II only partially inactivated the enzyme. At saturating concentrations of bilirubin (75 μM) CN type II patient A had 4.4 ± 2% residual activity and CN type II patient B had 38 ± 2% residual activity. Kinetic constants for the glucuronidation of bilirubin were determined. The affinities for bilirubin of B-UGT1 expressed in COS cells and B-UGT from human liver microsomes were similar with K m of 5.1 ± 0.9 μM and 7.9 ± 5.3 μM, respectively. B-UGT1 from patient B had a tenfold decreased affinity for bilirubin, K m = 56 ± 23 μM. At physiological concentrations of bilirubin both type II patients will have a strongly reduced conjugation capacity, whereas type I patients have no B-UGT activity. We conclude that CN type I is caused by a complete absence of functional B-UGT and that in CN type II B-UGT activity is reduced. (J. Clin. Invest. 1994. 94:2385-2391.) Key words: hereditary diseases · site directed mutagenesis · enzyme-linked immunosorbent assay · enzyme kinetics · enzyme activity. (Reproduced from The Journal of Clinical Investigation, 1994, vol. 94, pp. 2385-2391 by copyright permission of The Society for Clinical Investigation.