Measurement of serum bilirubin and its mono- and diconjugates: application to patients with hepatobiliary disease.

A technique has recently been described by Blanckaert and his colleagues that specifically and accurately quantifies unconjugated bilirubin, diconjugated bilirubin, and the C-8 and C-12 isomers of monoconjugated bilirubin. This technique has now been used to determine the distribution pattern of bilirubin and its ester conjugates in 91 sera from 65 patients with hepatobiliary disease, and the results were compared with two conventional diazo assays. Both diazo assays yielded values for total bilirubin concentration that were markedly and unpredictably higher than those obtained by the new technique, and the direct-reacting fraction by diazo assay showed little or no agreement with the fraction of total ester conjugates determined by the new method. Previous studies using the new method had shown that bilirubin conjugates are undetectable in sera from healthy adults or individuals with Gilbert's syndrome, but they were found in 89 of the 91 present patient sera. The fraction of total serum bilirubin represented by C-8 monoconjugates, C-12 monoconjugates, diconjugates, and total ester conjugates was higher in patients with biliary obstruction than in those with parenchymal liver disease, but extensive overlap between groups prevented determination of these conjugated species from being diagnostically useful. Overall, bilirubin ester conjugates in serum consisted of 30% C-8 monoconjugates, 37% C-12 monoconjugates, and 33% diconjugates, while urine contained predominantly diconjugates.     

Subfractionation of serum bilirubins by alkaline methanolysis and thin-layer chromatography. An aid in the differential diagnosis of icteric diseases

The determination of direct and indirect-reacting bilirubin fractions by diazo procedures does not allow a definite diagnosis of icteric diseases. Therefore, the clinical relevance of serum bilirubin subfractionation by alkaline methanolysis and subsequent thin-layer chromatography (AM-TLC) was evaluated. Esterified bilirubins could be detected and quantitated in all serum samples investigated. The ratio of serum esterified to total bilirubin was 10-28% in 60 healthy adults (mean 17 +/- 5% S.D.), 1-11% in 77 patients with Gilbert's syndrome (mean 6 +/- 2%), and 2 and 3%, respectively, in two patients with Crigler-Najjar disease type II. The difference was highly significant (p less than 0.001) and the overlap was restricted to three of 139 individuals. The ratio of esterified to total bilirubin was similar to that obtained with HPLC when corrected for with a blank run. The absolute concentration of bilirubin esters in serum from Gilbert's syndrome patients was similar to that from healthy controls, but the unconjugated pigment was increased. In patients with chronic haemolysis (n = 9) and chronic persistent hepatitis (n = 12), the hyperbilirubinaemia consisted of a proportional increase of both unconjugated and esterified bilirubin. As such, the ratio of conjugated to total bilirubin was not significantly different from control values. Patients with acute hepatitis during the first (n = 18) and third ('remission') week of the disease (n = 15), liver cirrhosis (n = 34), and extrahepatic cholestasis (n = 20) predominantly showed an increase in bilirubin conjugates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of ioglycamide on the hepatic transport of bilirubin and its mono- and diconjugates in the rat

Bilirubin seems to share the biliary excretion pathway with other organic anions, but not with bile acids. We studied the effects of the organic anion ioglycamide, an iodinated contrast agent, on bilirubin metabolism in Wistar rats. This compound does not undergo conjugation and is characterized by a maximal biliary secretory rate (Tm). The results show that in spite of producing a 3-fold increase in bile flow, ioglycamide excretion under Tm conditions decreased the output of unconjugated bilirubin and its monoconjugate by approximately 90%. Diconjugated bilirubin decreased by only 50% and became by far the predominant pigment in bile (86.5 +/- 6.0% of total pigment vs. 61.0 +/- 4.0% in basal conditions, n = 12). Unconjugated and monoconjugated bilirubins changed in parallel suggesting that the former arises from the monoconjugates. In serum, diconjugated bilirubin augmented from trace amounts to 1.15 +/- 0.17 mumole per liter. Total conjugated pigments in serum increased from 5 to 85% of total bilirubin. Bile acid output remained unchanged. Pretreatment of rats with ioglycamide altered neither the activity of bilirubin UDP-glucuronyltransferase nor the ratio of diconjugate to monoconjugate formed at both low (25 microM) and high (164 microM) bilirubin concentrations. The observed biological effects of ioglycamide were dose-dependent and fully reversible. We suggest that ioglycamide interferes with the excretion of conjugated bilirubins ("bilirubinostasis"). The monoconjugates retained in the hepatocyte might then undergo more efficient transformation to diconjugates, the latter thus becoming the most important bile pigments in serum and bile.     

Chronic persistent hepatitis and unconjugated hyperbilirubinemia.

The authors studied 12 patients with chronic persistent hepatitis and persistent or intermittent mild unconjugated hyperbilirubinemia. Maximum serum total bilirubin concentration ranged from 2.1 to 3.6 mg/dl. Hemolysis was not evident. Hepatic bilirubin UDP-glucuronyltransferase activity assayed in each patient ranged from 0.16 to 0.39 U (mean +/- SEM = 0.27 +/- 0.02) compared to 0.68-1.99 (1.35 +/- 0.08) in 23 normals, 0.78-2.28 (1.41 +/- 0.05) in 53 patients with acute hepatitis, 0.34-1.74 (0.81 +/- 0.09) in 16 patients with anicteric chronic persistent hepatitis, and 0-0.62 (0.24 +/- 0.03) in 33 patients with Gilbert's syndrome. The mean UDP-glucuronyltransferase activity was significantly lower in anicteric chronic persistent hepatitis compared to normals, but higher than in Gilbert's syndrome. The incidence of unconjugated hyperbilirubinemia among first degree relatives was 0:32 in icteric chronic persistent hepatitis compared to 24:85 (28%) in Gilbert's syndrome. These results show that the likely cause for the unconjugated hyperbilirubinemia associated with chronic persistent hepatitis is an acquired depression of hepatic bilirubin UDP-glucuronyltransferase activity. The data suggest that the enzyme defect is related to chronic persistent hepatitis.     

The differential diagnosis of Crigler-Najjar disease, types 1 and 2, by bile pigment analysis

Phenobarbital response, bile pigment composition, and the fractional biliary excretion ratio of bilirubin were studied in nine children with Crigler-Najjar disease. In five children, serum bilirubin levels decreased during phenobarbital treatment by 26% or more and the pigment composition in bile changed with a decrease in the proportion of unconjugated bilirubin from 33% +/- 12% to 13% +/- 1% and an increase in monoconjugates and diconjugates from 57% +/- 14% and 10% +/- 2%, respectively, to 72% +/- 4% and 16% +/- 3%. In four children, serum bilirubin levels did not change significantly during phenobarbital treatment. In these patients, bile pigments comprised 91% +/- 10% unconjugated bilirubin, 9% +/- 11% monoconjugates, and 1% +/- 1% diconjugates. On the basis of these differences, the former group can be classified as having type 2 Crigler-Najjar disease and the latter, type 1. Bile pigment analysis in parents of patients with Crigler-Najjar disease showed an increased proportion of monoconjugates in at least one of the partners in three of four couples tested, despite normal serum bilirubin levels. Serum bilirubin levels were about the same in type 1 and 2 patients and amounted to 236 +/- 62 mumol/L and 214 +/- 82 mumol/L, respectively. In addition the fractional bilirubin excretion ratio, calculated as the ratio ([bilirubin in bile]/[bilirubin in serum])/([bile acid in bile]/[bile acid in serum]) could not differentiate between these two groups. However, there was a 10-fold and 100-fold difference of this ratio between patients with Crigler-Najjar disease and those with Gilbert's syndrome and between patients with Crigler-Najjar disease and controls. The fractional bilirubin excretion ratio proved an excellent tool to differentiate between Gilbert's syndrome and Crigler-Najjar disease, whereas Crigler-Najjar disease types 1 and 2 could be differentiated on the basis of bile pigment analysis.     

Increased production, hepatic conjugation, and biliary secretion of bilirubin in the rat after chronic ethanol consumption

Disturbances of bilirubin metabolism such as jaundice or pigment gallstone formation, or both, occur in alcoholic cirrhosis of the liver. We have studied the influence of chronic ethanol consumption on bilirubin metabolism as well as on biliary calcium and bile acids in 16 pair-fed male rats. The animals received nutritionally adequate liquid diets containing 36% of total calories either as ethanol or isocaloric carbohydrates for 4 wk. Bile flow was significantly enhanced after chronic ethanol feeding (p less than 0.05 after 90-min bile collection) and was found to be mainly bile acid-independent. The biliary output and concentration of bilirubin monoconjugates, bilirubin diconjugates, and total calcium was significantly increased (p less than 0.01) in alcohol-fed rats compared with controls. This was not the case for unconjugated bilirubin and for the calcium/bile acid ratio. Hepatic bilirubin uridine-5'-diphosphate-glucuronosyltransferase activity (p less than 0.01), serum total bilirubin (p less than 0.01), and serum free hemoglobin (p less than 0.001) were significantly increased after ethanol consumption. These data provide evidence for enhanced bilirubin production, probably due to hemolysis, after alcohol ingestion. The enhanced bile production is associated with an increased hepatic conjugation and subsequent biliary secretion of bilirubin conjugates. In advanced alcoholic liver disease, these compensatory mechanisms may fail and contribute to the development of jaundice.     

Hereditary chronic conjugated hyperbilirubinemia in mutant rats caused by defective hepatic anion transport

A mutant rat strain is described with autosomal recessive conjugated hyperbilirubinemia. Transport of conjugated bilirubin and tetrabromosulfophthalein from liver to bile is severely impaired whereas uptake of organic anions from plasma to liver is normal. During the first 10 days of life, serum bilirubin levels are 147 +/- 11 mumoles per liter with 68.7% diconjugates and 27.9% monoconjugates. In adult rats, serum bilirubin is 33 +/- 8 mumoles per liter with 81.8% diconjugates and 12.1% monoconjugates vs. 0.3 +/- 0.1 mumole per liter unconjugated bilirubin in normal adult rats. Bile acid metabolism is only mildly affected. In young rats, serum bile acid levels are normal. In adult rats, bile acid levels are elevated to 49 +/- 11 mumoles per liter vs. 10 +/- 6 mumoles per liter in normal rats. The bile flow in mutant rats is reduced to about 50%. This might be caused by a reduction of the bile acid-independent bile fraction. Liver marker enzyme activities in mutant rat serum are normal. Liver morphology is also normal. Total urinary coproporphyrin excretion is not elevated but urinary coproporphyrin isomer I excretion is increased, a pattern like that in Dubin-Johnson syndrome in humans. However, unlike Dubin-Johnson syndrome, the mutant rats do not have the characteristic black hepatic pigment. These rats provide a unique model to study mechanisms of bile formation and cholestasis.     

Monoconjugated bilirubin is a major component of hemolysis-induced gallstones in mice

The role of bilirubin conjugates in the formation of pigment gallstones is not known. In this study, we completely solubilized and then analyzed by high-performance liquid chromatography specimens of black pigment gallstones from eight nb/nb mice with hereditary hemolytic anemia. Each dried gallstone specimen of about 200 micrograms was dissolved in 5 ml of dimethyl sulfoxide/0.15 M HCI/50 mM disodium-EDTA (8:1:1 by volume) at room temperature. Stone dissolution was complete by 30 min as monitored by the A456 and direct observation, and no oxidative products of bilirubin were observed in the visible spectrum, 350 to 750 nm. By high-performance liquid chromatography, the intact tetrapyrroles were separated as diconjugated and monoconjugated bilirubins; unconjugated bilirubin was resolved as XIII, IX and III alpha-isomers. The isocratic solvent system used was 0.1 M di-n-dodecylamine acetate/0.1 M di-n-octylamine acetate (4:1, v/v) in methanol, pH 7.4, at a flow of 1 ml per min. Diconjugated bilirubin accounted for 6.0 +/- 2.4 molar % (mean +/- S.E.), monoconjugated bilirubin for 37.4 +/- 8.4% and unconjugated bilirubin for 56.3 +/- 8.9% of the solubilized pigments. The IX alpha-isomer represented 96 +/- 1.9% of the unconjugated bilirubin. The presence of bilirubin conjugates in gallstones was confirmed by ethylanthranilate diazotization: the conjugated azodipyrrole in stone had the same retention time as that of conjugated azodipyrrole from rat and mouse bile. A majority of the bilirubin conjugates was sensitive to beta-glucuronidase of liver origin, indicating that the C-1 glucuronide ester was present.(ABSTRACT TRUNCATED AT 250 WORDS)     

An unusual case of Crigler-Najjar disease in the adult. Classification into types I and II revisited

We present the case of a 25-year-old man with Crigler-Najjar disease who had since birth a marked unconjugated hyperbilirubinemia without bilirubin overproduction, without any neurological involvement and in whom phenobarbital administration failed to produce any effect. Analysis of his biliary bile pigments on two occasions showed (i) a decrease excretion of bilirubin, as indirectly suggested by a high ratio of biliary bile acids over total bilirubin; (ii) an increase in unconjugated bilirubin IX alpha quantitated by thin-layer chromatography (TLC) following alkaline methanolysis and by direct extraction and TLC of the tetrapyrroles; (iii) a high proportion of bilirubin monoconjugates whereas the excretion of diconjugates was very low. Classification of the present patient into Crigler-Najjar disease type I or II was not possible. The most striking and practical difference among the various cases of Crigler-Najjar disease remains the response to phenobarbital. Among cases of Crigler-Najjar disease which respond to enzyme induction and Gilbert's syndrome, the continuous spectrum suggests a common defect.     

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.     

Delayed gastric emptying in subjects with Gilbert's syndrome

BACKGROUND/AIMS: Recently, it has been proposed that decreased intestinal motility in fasting-induced hyperbilirubinemic rats probably results in an increase in the enterohepatic cycling of unconjugated bilirubin. We investigated the association among gastric emptying, intestinal transit time, and serum unconjugated levels in subjects with Gilbert's syndrome. METHODOLOGY: Ten subjects with Gilbert's syndrome were included in this study according to the following criteria: fasting hyperbilirubinemia; no hemolysis or gastrointestinal disorders and free of any medication. Five normal, healthy volunteers acted as controls. Gastric emptying and intestinal transit time were evaluated after overnight fasting by administration of a standard meal mixed with 1-2ci of 99Tc-labeled diethylene-triamine-pentacetic acid. Serum unconjugated bilirubin levels were determined by high-performance liquid chromatography. RESULTS: The gastric emptying in Gilbert's syndrome subjects was 134.1 +/- 38.9 and 90.9 +/- 6.5 min in controls, P < 0.03. It was a tendency to have a shorter intestinal transit time in subjects with Gilbert's syndrome, 138.3 +/- 59.0, than in control subjects, 183.8 +/- 11.3 min. Serum unconjugated bilirubin levels (mg/dL) were 2.6 +/- 1.04 and 0.95 +/- 0.34, P < 0.01. CONCLUSIONS: Gastric emptying is delayed significantly in Gilbert's syndrome, and intestinal transit time differences between Gilbert's syndrome subjects and controls were not significantly different.     

Etiology and Incidence of Unconjugated Hyperbilirubinemia after Orthotopic Liver Transplantation

Obfectives: Gilbert's syndrome, or slow bilirubin glucuronidation phenotype, is a common cause of benign hyperbilirubinemia in the general population. There have heen only two previously reported cases of Gilbert's syndrome occurring in patients after liver transplantation. This study was conducted to determine the frequency of Gilbert's syndrome in liver transplant recipients. Methods: The charts of all patients followed by the Mayo Liver Transplant Clinic for 1 yr or more after transplantation, as of June 1992, were reviewed to identify all patients with a consistent pattern of unconjugated hyperbiliruhinemia greater than two times the upper limits of normal and with a normal conjugated bilirubin level. These patients were further evaluated to exclude all other causes of hyperbilirubinemia, including biliary obstruction, rejection, viral infection, cholestatic liver disease, and hemolysis. Resutts: Five of 229 patients (2.2%) had a consistent pattern of unconjugated hyperbiliruhinemia. Only three patients (1.3%) had no other identifiable cause of hyperbilirubinemia. Conclusions: This study was performed to determine the incidence of unconjugated hyperbilirubinemia and particularly to determine the incidence of Gilbert's disease in liver transplant recipients. The apparently low frequency of Gilbert's after liver transplantation may reflect the masking of the diagnosis by other transplant-associated pathology or donor selection bias because of unexplained hyperbilirubinemia. Post-transplant patients who fit the Gilbert's syndrome profile of unconjugated hyperbilirubinemia should have a postprandial bilirubin drawn as a first step. The awareness of this syndrome may avoid a costly and invasive evaluation in the liver transplant recipient.     

Thyroid hormones and the hepatic handling of bilirubin. I. Effects of hypothyroidism and hyperthyroidism on the hepatic transport of bilirubin mono- and diconjugates in the Wistar rat

The effects of thyroidectomy and of thyroid hormone administration on the hepatic transport of endogenous bilirubin were investigated in the Wistar R/APfd rat. Hypothyroidism resulted in an enhanced hepatic bilirubin UDP-glucuronosyltransferase activity and in a decreased p-nitrophenol transferase activity. It caused a cholestatic condition with a 50% decrease in bile flow and bile salt excretion, and an increased proportion of conjugated bilirubin in serum. The biliary output of unconjugated and monoconjugated bilirubins decreased in parallel by about 65%, whereas the excretion rate of the diconjugate dropped by only 47%, resulting in an increased di- to monoconjugate ratio in bile. Hyperthyroidism was characterized by a decreased bilirubin and an increased p-nitrophenol transferase activity, and by an augmented bilirubin output in bile. The output of unconjugated and monoconjugated bilirubins increased in parallel by about 50 or 100%, whereas the excretion of the diconjugate increased by only 20 to 50%, depending on the dose of thyroxine administered; this resulted in a decreased di- to monoconjugate ratio in bile. A linear positive relationship was found between bilirubin UDP-glucuronosyltransferase activity and the ratio of bilirubin di- to monoconjugates present in bile or formed by in vitro incubation of liver homogenates at low concentration of bilirubin (10 to 15 microM), indicating that bile pigment composition is mainly determined by the conjugation activity in the liver. The inverse relationship observed between hepatic beta-glucuronidase activity and the ratio of di- to monoconjugates in bile warrants further investigation to analyze whether this enzyme activity also plays a possible role in the changes in bile pigment composition in hypo- and hyperthyroid rats.     

Serum bilirubins in hepatobiliary disease: comparison with other liver function tests and changes in the postobstructive period

Unconjugated bilirubin and its mono- and diester conjugates were measured by alkaline methanolysis and normal-phase high-performance liquid chromatography (AMHPLC) in 195 serum specimens obtained from 63 patients with various hepatobiliary disorders and from 47 healthy adult controls. With this assay, esterified bilirubins were undetectable in the controls, and detection of esterified pigment in a sample was interpreted as an abnormal result. Using this criterion, the AMHPLC result in the clinical anicteric patients (n = 39) was more frequently abnormal (87%) than the corresponding value of fasting serum bile acids (48%), SGPT (52%), total bilirubins (55%), alkaline phosphatase (71%) or gamma-glutamyl transpeptidase (71%). The cumulative frequency of abnormality of these tests was comparable to that of an abnormal AMHPLC result alone. All icteric patients had detectable esterified bilirubins as well as an increased alkaline phosphatase level, while a normal result was found for serum bile acids in 34%, for SGPT in 29% and for gamma-glutamyl transpeptidase in 11%, respectively. In most hyperbilirubinemic patients, total serum bilirubin levels, as determined by a conventional diazo method, exceeded the value obtained by AMHPLC. This discrepancy, which appears to reflect the presence of bilirubin covalently bound to serum protein, was particularly pronounced following desobstructive intervention in patients with obstructive jaundice, in whom the decline of serum bilirubins showed a fast and a slow disappearance component. The latter portion seemed to correspond with slow plasma clearance of bilirubin covalently linked to serum albumin, disappearing at a rate comparable to that of the albumin moiety.     

Unconjugated hyperbilirubinemia in nonalcoholic steatohepatitis--is it Gilbert's syndrome?

BACKGROUND: Patients with nonalcoholic steatohepatitis (NASH) have normal liver function tests except for raised transaminases until they have progressed to cirrhosis of liver. The objective of this study was to evaluate patients of NASH for the presence of hyperbilirubinemia at presentation. METHOD: Sixty-seven patients of NASH were studied for the presence of hyperbilirubinemia at presentation. All patients were worked up for the presence of cirrhosis and hemolytic work up and fasting test were done in those found with unconjugated hyperbilirubinemia. RESULTS: Five out of 67 patients (7.5%) of NASH were found to have unconjugated hyperbilirubinemia. Though the fasting test was not positive, they all had a negative hemolytic workup and none of them had underlying cirrhosis. Clinical characteristics of patients with unconjugated hyperbilirubinemia were similar to those with normal serum bilirubin levels. CONCLUSION: Unconjugated hyperbilirubinemia in patients with NASH may suggest an associated Gilbert's syndrome.     

A case of Gilbert's syndrome combined with macroamylasemia

A 30-year-old Japanese male, who had no remarkable family history, visited our hospital with a complaint of abdominal pain, and unconjugated hyperbilirubinemia and hyperamylasemia were observed. He showed negative hemolysis tests, positive nicotinic acid test, low hepatic bilirubin UDP-glucuronyltransferase activity, decreased bilirubin diglucuronide and increased bilirubin monoglucuronide in bile, and a decrease in serum bilirubin after phenobarbital administration. He also showed high serum amylase level, low urine amylase level, and low amylase-creatinine clearance ratio. Gel filtration of serum with Sephadex G-200 revealed the existence of macroamylase. Countercurrent immunoelectrophoresis proved binding of serum amylase to lambda type IgA. From these results, the case was diagnosed as Gilbert's syndrome combined with macroamylasemia.     

Effects of uridine diphosphate glucuronosyltransferase activity on the maximal secretion rate of bilirubin conjugates in the rat

It is generally accepted that biliary secretion is rate-limiting for the plasma-to-bile transport of a saturating load of bilirubin. Previous studies from this laboratory have suggested a relationship between the hepatic bilirubin uridine diphosphate glucuronosyltransferase activity and the apparent maximal rate of bilirubin secretion (excretory transport maximum). The present study was undertaken to further investigate this relationship in rats with a wide range of transferase levels and to analyze the effects of transferase activity on the formation of bilirubin monoglucuronides and diglucuronides in vivo and in vitro. Animals with moderately decreased enzyme activity still showed a near-normal excretory transport maximum but decreased fractional amounts of conjugated bilirubins in plasma and liver and a decreased ratio of bilirubins diconjugates to monoconjugates in bile, liver, and plasma. A more pronounced decrease in the level of transferase activity yielded lower excretory transport maximum values as well. Enhanced transferase activities were obtained by pretreatment with a series of enzyme-inducing agents, usually at dosages not influencing bile flow. An enhanced transferase activity produced significant increases in bilirubin excretory transport maximum, decreased concentrations of both unconjugated and conjugated bilirubins in plasma, and an increased ratio of diconjugates to monoconjugates in bile and in liver. In all animals, the ratio of disconjugates to monoconjugates observed in bile was correlated with the ratio found after incubation of liver homogenates in vitro at low concentrations of bilirubin substrate. Overall, the relationship between bilirubin excretory transport maximum and uridine diphosphate glucuronosyltransferase activity can best be described by a hyperbolic curve. At "saturation" of the excretory transport maximum values, no increase of conjugates in liver or in plasma could be observed, suggesting that not the biliary secretion, per se, but rather the conjugation rate in vivo is rate-limiting. The latter seems lower than the in vitro assayed enzyme activity. The present investigations point to an equilibration of bilirubin pigments between plasma, liver, and bile. The in vivo conjugation rate can be estimated from the in vitro assay (but not at high enzyme activities) and determines the maximal biliary secretion rate, the ratio of diconjugates to monoconjugates in the three compartments studied, and the relative amount of conjugates in plasma. Our studies suggest that the conjugation rate in vivo rather than the biliary secretion step is the major determinant of the maximal bilirubin secretion rate.     

Successful treatment of severe unconjugated hyperbilirubinemia via induction of UGT1A1 by rifampicin

We report two patients with uncommon Gilbert's syndrome with severe unconjugated hyperbilirubinemia which was reduced from 200 to 60-90 micromol/L by long-term administration of rifampicin. Hepatic induction of bilirubin-glucuronosyltransferase was suggested by increased relative amounts of conjugated serum bilirubin. This molecular mechanism was confirmed in primary cultures of human hepatocytes.     

Studies on nicotinic acid interaction with bilirubin metabolism

The mechanism by which intravenous administration of nicotinic acid (NA) increases serum unconjugated bilirubin in patients with the Gilbert's syndrome has been investigated. Studies using the technique of percutaneous transhepatic catheterization of the splenic vein and coil planet centrifuge suggested that following intravenous injection of NA some of the circulating erythrocytes were rendered osmotically fragile and trapped by the spleen and that unconjugated bilirubin increased in the splenic vein blood. In patients with liver cirrhosis, the increments of unconjugated bilirubin were closely correlated with the weights of the spleens removed for the management of varices. In rats, intravenous NA injection enhanced heme oxygenase activities in the spleen, but not uridine-5-diphosphate (UDP)-glucuronyltransferase activity in the liver. These results are consistent with the hypothesis that NA-induced unconjugated hyperbilirubinemia is a result of complex reactions which include increased erythrocyte fragility, increased splenic heme oxygenase activity, and increased formation of bilirubin in the spleen.     

Increase in the relative amount of bilirubin diconjugates in rat bile and serum under infusion of phthaleins and indocyanine green

The effect of organic anions on bilirubin metabolism and excretion was investigated in rats. Biliary excretion of bromosulfophthalein, bromcresol green and indocyanine green led to a significant decrease in excretion of bilirubin pigments and to an increase of their concentration in serum. This was concomitant with a marked increase in the ratio of bilirubin diconjugates to monoconjugates in bile and serum. These changes were unrelated to either bile flow, biliary lipid output, or hepatic activity of bilirubin UDP-glucuronyl transferase. Following bolus injection of [14C]bilirubin, peak excretory rate of radioactivity was markedly delayed in rats infused with bromosulfophthalein, as compared to controls. It is concluded that administration of the organic anions increased the ratio of bilirubin diconjugates to monoconjugates in bile and serum, by slowing down the intrahepatic transit of bilirubin pigments. This, in turn, allowed more efficient enzyme-catalyzed formation of bilirubin diconjugates from the intermediate bilirubin monoconjugates.