Yukihiko Adachi

Analysis of genes for bilirubin UDP-glucuronosyltransferase in Gilbert's syndrome

Gilberts and Crigler-Najjar syndromes are characterised by unconjugated hyperbilirubinaemia due to complete and partial absence of bilirubin UDP-glucuronosyltransferase (UGT). Nucleotide sequences of the genes for bilirubin UGT were analysed in six patients with Gilberts syndrome. All patients had a missense mutation caused by a single nucleotide substitution and the mutations were heterozygous. In addition, relatives of patients with Crigler-Najjar syndrome types I and II, and of those with Gilberts syndrome were analysed. All ten relatives with mild hyperbilirubinaemia were heterozygotes with respect to each defective allele. These results suggest that Gilberts syndrome is inherited as a dominant trait.

Long-term effects of intrasplenically transplanted adult hepatocytes and fetal liver in hyperbilirubinemic Gunn rats

We performed adult hepatocyte transplantation (HCTx) and fetal liver transplantation (FLTx) into the spleens of hyperbilirubinemic Gunn rats in congenic combination and we compared the long-term effects of these procedures for as long as 12 months. Proliferative activity of intrasplenic hepatocytes was evaluated using antiproliferating cell nuclear antigen (PCNA) immunohistochemical staining. The serum total bilirubin levels (T. Bil) significantly decreased from 7.16±0.25 mg/dl to 4.38±0.60 mg/dl 2 months after HCTx and gradually decreased thereafter until 12 months after transplantation (3.23±0.37 mg/dl, P<0.05 vs preoperative value). The T. Bil change after FLTx was similar to that of HCTx: 7.22±0.24 mg/dl before FLTx, and 4.92±0.24 and 3.06±0.47 mg/dl, 2 and 12 months after FLTx (P<0.05), respectively. Bilirubin glucuronides, which were not detectable in the bile from untreated Gunn rats, appeared in considerable amounts 4 months after HCTx and FLTx (27.5% and 36.0% of total bile, respectively). PCNA labeling indices of intrasplenic hepatocytes (4.9%±0.9% and 3.7%±0.7%, 6 months after HCTx and FLTx, respectively) were slightly higher than those of normal hepatocytes (1.0%±0.1%) in the host liver. In conclusion, both adult and fetal rat hepatocytes transplanted into the spleen in congenic combination functioned for at least a year in terms of bilirubin glucuronidation. The spleen is considered to be one of the optimal grafting sites for hepatocytes, with nearly lifelong significant function and proliferative activity.

Hepatic bilirubin-conjugating enzymes of man in the normal state and in liver disease.

SummaryBilirubin UDP-glucuronyl transferase (BGT), bilirubin UDP-glucosyl transferase (BGLT) and bilirubin UDP-xylosyl transferase (BXT) activities were measured in wedge-biopsied liver specimens obtained from patients with various liver diseases, and compared with those in controls with normal liver histology. BGT was measured alone using needle biopsy liver specimens from the patients with Gilbert’s syndrome (15 patients), Rotor s syndrome (one) and posthepatitic hyperbilirubinemia (3). BGT was decreased to about 30% of controls in Gilbert’s syndrome, but showed no change in posthepatitic hyperbilirubinemia and Rotor’s syndrome. About 90% decrease in BGT, BGLT and BXT were observed in Crigler-Najjar syndrome type II (3 patients). In patients with cholelithiasis and chronic hepatitis, statistically significant changes of these three enzymes were not observed, except the statistically significant increase in BGT activity in chronic hepatitis. Slight increases in BGT and BXT activities were observed in anicteric cases with cholelithiasis.The ratio of BGT, BGLT and BXT activities in controls was 1:0.50:0.98 (expressed as “per mg protein”). Slight differences existed between the ratios of BGT, BGLT and BXT in various liver diseases, and this may suggest the separate identities of BGLT and BXT from BGT.Determination of bilirubin-conjugation is essential in the diagnosis of Gilbert’s syndrome and Crigler-Najjar syndrome type II, but shows no specific change in the other chronic liver diseases.

Mechanism of feminization in male patients with non-alcoholic liver cirrhosis: role of sex hormone-binding globulin.

SummaryWe measured serum sex hormone-binding globulin (SHBG) using a radioimmunoassay developed by us, testosterone (T), estradiol (E2), free T and free E2 in 50 male patients with non-alcoholic liver cirrhosis (compensated: 30, decompensated; 20) and age-matched healthy male subjects. SHBG was significantly increased in patients with liver cirrhosis compared with healthy subjects. The high serum SHBG level in male compensated cirrhotic patients tended to decrease with progression to the decompensated state. Serum cholinesterase showed a positive correlation with SHBG in liver cirrhosis. Serum free T and the T/ SHBG ratio decreased, while serum E2, free E2, and the E2/T and the free E2/free T ratios increased in liver cirrhosis, resulting in estrogen predominance and feminization of male patients. These changes were more marked in decompensated than compensated liver cirrhosis. An increased free E2/free T ratio was observed in patients with gynecomastia, palmar erythema or vascular spider. The T/SHBG ratio showed a positive correlation with serum free T, suggesting that it can be used as a free T index in liver cirrhosis. From these observations, it is suggested that serum SHBG plays an important role, by regulating the serum free T level in the occurrence of feminization in male patients with non-alcoholic liver cirrhosis.

Pravastatin transport across the hepatocyte canalicular membrane requires both ATP and a transmembrane pH gradient

Hepatic excretion of non‐bile acid organic anions is reported to be ATP‐dependent and a defect of this transport has been reported in congenitally jaundiced rats, animal models of human Dubin‐Johnson syndrome. To investigate the effect of the transmembrane pH gradient on hepatocyte canalicular membrane transport of ATP‐dependent organic anions, uptake of pravastatin, a 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) reductase‐inhibiting organic anion, by hepatocyte canalicular membrane vesicles was observed in the presence or absence of transmembrane pH gradients. Uptake was assessed by a rapid filtration technique. ATP‐dependent pravastatin uptake was stimulated in the presence of a transmembrane pH gradient (in > out) in Sprague‐Dawley (SD) rats. Uptake was dependent on both pravastatin and ATP concentrations and showed saturation kinetics. After intravenous injection of [14C]‐pravastatin (0.3 μmol), 81% of the dose was excreted in the bile within 35 min in SD rats, whereas only 20% was excreted in the bile in Eisai hyperbilirubinuria rats. ATP and the pH gradient also co‐stimulated the uptake of pravastatin in Eisai hyperbilirubinuria rats, although the Km was much higher and Vmax was much lower than corresponding values in SD rats. This coincided well with the marked reduction in in vivo biliary excretion of pravastatin in jaundiced rats.

Induction of rat liver bilirubin-conjugating enzymes and glutathione S-transferase by rifampicin.

SummaryAfter oral administration of rifampicin and 25-desacetylrifampicin, which is a major metabolite of rifampicin in man but not in rat, to male Wister rats for 7 days, hepatic microsomal cytochrome P450, cytochrome b5, and activities of aniline hydroxylase, aminopyrine demethylase, bilirubin-conjugating enzymes and supernatant glutathione S-transferase were measured. Rifampicin induced bilirubin UDPglucuronyltransferase, bilirubin UDP-glucosyltransferase, bilirubin UDP-xylosyltransferase and glutathione S-transferase activities, but did not induce mixed function oxidase activities. No inductive effect of desacetylrifampicin on any enzymes was observed. Serum bilirubin increased till the third day, and decreased after 7 days of rifampicin treatment. Plasma clearances of indocyanine green and sulfobromophthalein showed a marked delay after 1 day and 7 days of rifampicin treatment. Induction of bilirubin-conjugating enzymes and glutathione S-transferase by rifampicin in rats was different from that in humans, in which selective induction of mixed function oxidase is reported to occur. This species difference does not seem to be derived from the species difference of rifampicin metabolism, because no effect of desacetylrifampicin was observed. These results suggested that in rats rifampicin directly inhibits the hepatic excretion of bilirubin, whereas it enhances bilirubin conjugation due to enzyme induction.

Genetic background of constitutional unconjugated hyperbilirubinemia

Abstract Crigler-Najjar syndrome (types I and II) and Gilberts 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 Gilberts 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.

Human serum bilirubin fractionation in various hepatobiliary diseases by the newly developed high performance liquid chromatography

SummarySerum Bilirubin was fractionated by newly developed reversed phase high performance liquid chromatography (HPLC) into 5 fractions: δ (δ-Bilirubin,Bδ),γ (bilirubin diglucuronide, BDG), β (Bilirubin monoglucuronide, BMG), β′ ((Z, E,) and/or (E, Z)-bilirubin IXα) and α ((Z, Z)-bilirubinIXα). Sera of healthy subjects and of patients with unconjugated hyperbilirubinemia showed predominantly a fraction with a small amount of β′ fraction. Trace amounts of δ fraction were detected in a few cases. The results of fractionation of serum bilirubin in 159 patients with various hepatobiliary diseases suggested that the ratios B<5/(Bδ+BDG+BMG) and BMG/Bδ can be useful parameters to follow patients with jaundice, compared with the reported Bδ/total bilirubin which did not always reflect the jaundice stage, especially in cases with low serum bilirubin levels.

Analysis of bilirubin conjugates in human bile by column liquid chromatography—Changes in their composition in hepatobiliary diseases

Bilirubin in human bile was analysed by high performance liquid chromatography (HPLC), and seven peaks were identifiable. There was no difference in the proportion of bilirubin between the B bile and C bile from healthy humans obtained by the Meltzer‐Lyon method. HPLC coupled with nuclear magnetic resonance spectroscopy on bilirubin after diazotization permitted differentiation between endovinyl and exovinyl conjugates of bilirubin.

Enteric coated polymyxin B in the treatment of hyperammonemia and endotoxemia in liver cirrhosis.

SummaryEffects of enteric coated polymyxin B capsules on hyperammonemia and endotoxemia in liver cirrhosis were investigated. Six million units of polymyxin B were orally administered daily to 21 patients with liver cirrhosis and 3 patients with hepatomacum liver cirrhosis, whose plasma ammonia was higher than normal limit and/or whose plasma endotoxin was positive, for 5–32 days, and serum polymyxin B concentration (in 5 cases), changes of plasma ammonia level (in 19 cases) and plasma endotoxin (in all cases) were observed. Serum polymyxin B concentration was below the detectable limit (0.5 unit/ml) in all cases observed. In the patients with liver cirrhosis, plasma endotoxin and ammonia levels decreased rapidly after polymyxin B treatment, and the decreases in endotoxin levels were kept throughout the treatment. Twelve patients with liver cirrhosis (10 among them were treated with lactulose) were served as controls. All patients who were treated with lactulose alone showed rapid decrease in plasma ammonia, but the decrease in endotoxin in these patients was slower than that in those treated with polymyxin B. From these results, oral administration of polymyxin B is concluded to be useful in the treatment of hyperammonemia and endotoxemia in liver cirrhosis, as a poorly absorbed antibiotic and as an antiendotoxin agent.