Monoconjugated Bilirubin Research Articles

The Occurrence and Significance of the Bilirubin Species, Including Delta Bilirubin, in Jaundiced Infants

An improved technique for bilirubin analysis, using high-performance liquid chromatography, has enabled us to study the occurrence and significance of four species of bilirubin (unconjugated, monoconjugated, diconjugated, and delta bilirubin) in 40 infants with jaundice of various etiologies. We found that: (a) infants with indirect hyperbilirubinemia showed greater than 90% of their total serum bilirubin as unconjugated bilirubin. The small remaining fraction consisted of conjugated bilirubin; predominantly delta bilirubin (5%); (b) infants with elevated direct serum bilirubin (greater than or equal to 2 mg/dl) showed almost twice more monoconjugated than diconjugated bilirubin fractions; (c) the standard diazo test for bilirubin analysis underestimates the direct bilirubin by as much as 34%; and (d) delta bilirubin, a tightly protein bound bilirubin, was observed in significant amounts in infants with elevated direct bilirubin. Its concentration, which ranged from 10-73% of the total bilirubin, was related to the duration rather than to the cause of the jaundice. It was also observed at birth in an infant with giant-cell hepatitis. It is concluded that the identification of more specific bilirubin species in jaundiced infants, especially in those with elevated direct serum bilirubin, will further help in the understanding and management of their disease.

Serum bilirubin subfractions in patients with alcohol abuse during detoxication.

Sera were obtained from 41 alcohol abusers consecutively admitted for detoxication. Blood samples were withdrawn on the second, fourth and seventh days of abstention. Initial bilirubin values were moderately elevated in 10 patients. Determination of the bilirubin subfractions by high performance liquid chromatography showed elevated values of unconjugated (alpha), monoconjugated (beta), diconjugated (gamma) and albumin-bound (delta) bilirubin, in 8, 15, 4 and 15 patients, respectively. During abstention, the total bilirubin value normalized rapidly, only three patients still had values above the upper reference limit after 7 days. In patients with initially elevated values of bilirubin subfractions, only a few had elevated beta and gamma levels on the seventh day, whereas delta levels decreased at a slower rate and remained virtually unchanged. On admission, 27 patients exhibited elevated levels of serum bile acids; these values decreased during abstention and after 7 days only six patients had slightly elevated values. Only five patients had initially elevated levels of alkaline phosphatase (ALP). These became normalized in all but two patients during abstention. The results suggest that mild cholestasis is common among alcohol-abusers without clinically evident liver disease and that these changes are reversible on abstention.

Thyroid hormones and the hepatic handling of bilirubin: II. Effects of hypothyroidism and hyperthyroidism on the apparent maximal biliary secretion of bilirubin in the Wistar rat

This study was undertaken in the Wistar R/A Pfd rat to investigate the effects of hypothyroidism and of hyperthyroidism on the maximal biliary excretion (Tm) of bilirubin and on the concentration and composition of bilirubin in liver and plasma at the end of a bilirubin load. Hypothyroidism caused a cholestatic condition with a 50% decrease in bile flow and in bilirubin Tm, and with an increased proportion of conjugated bilirubin in liver and plasma. This was associated with an increased ratio of bilirubin diconjugates to monoconjugates in bile, liver, and plasma, which can be ascribed to the increased hepatic conjugation activity towards bilirubin and/or to the prolonged retention of bile pigments in the hepatocytes with increased conversion of monoconjugates to diconjugates. Cholestasis induced by hypothyroidism was further characterized by a decreased biliary output of unconjugated bilirubin. The latter phenomenon might represent an indirect effect related to a decreased output of bilirubin monoconjugates with impaired hydrolysis to unconjugated bilirubin; it might also reflect the cholestatic condition with decreased excretion of the unesterified bile pigment as such. Hyperthyroidism resulted in a 1.3-1.4-fold increase in bile flow. The maximal bilirubin concentration in bile decreased 1.3-1.4-fold, so that the apparent maximal bilirubin excretion rate remained unchanged at 115 nmol.min-1.100 g-1, as observed in untreated rats. Hyperthyroidism lowered the bilirubin UDP-glucuronosyltransferase activity, produced a decreased ratio of bilirubin di- to monoconjugates in bile and plasma, and a decreased ratio of conjugated to total bile pigment concentration in liver and in plasma. Similar findings are present in the heterozygous Gunn rat strain and in patients with hepatic bilirubin UDP-glucuronosyltransferase deficiency. We therefore propose the hyperthyroid rat as an experimental animal model of Gilbert's syndrome.

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 < 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 < 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 < 0.01), serum total bilirubin (p < 0.01), and serum free hemoglobin (p < 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.

Measurement of direct bilirubin by use of bilirubin oxidase.

We developed an enzymatic method for measuring direct-reacting bilirubin (DBIL) in serum. At pH 4.5, bilirubin oxidase (BOX) oxidizes mono-conjugated bilirubin, di-conjugated bilirubin, and most of the delta-bilirubin to biliverdin. The resulting decrease in absorbance at 460 nm is linearly related to the concentration of DBIL in serum. Mean DBIL values in the 51 patients' sera examined by the BOX method and a diazo procedure (Clin Chem 1982;28:2305) were 45.4 and 42.8 mg/L, respectively. For the same samples, mean values for DBIL and conjugated bilirubin by the Kodak "Ektachem" methods were 50.2 and 24.8 mg/L, respectively. Hemoglobin, up to 1.5 g/L, does not interfere. Unconjugated bilirubin reacts negligibly. Day-to-day CVs were 2.2% and 2.4% at DBIL concentrations of 37 and 74 mg/L, respectively.

Isomers of bilirubin glucuronide in serum and bile before and after relief of common duct obstruction

Isomers of bilirubin glucuronide with the bilirubin acyl group attached to the C1-, C2-, C3- and C4-positions of the glucuronyl residue are present in bile of patients with extrahepatic cholestasis, whereas in normal bile only C1-isomers are found. In the present study, these bilirubin glucuronide isomers, and the fractions of unconjugated bilirubin, and bilirubin mono- and diconjugates were determined in serum and bile of 8 patients before and after relief of common duct obstruction by endoscopic papillotomy. Before papillotomy we found 39.6% C1-isomers (median value), 22.2% C2-isomers, 19.3% C3-isomers and 11.4% C4-isomers in the bile. The values in serum before papillotomy were comparable. Twenty-four hours after papillotomy, the level of C1-isomers in bile increased significantly to 56.3% (P less than 0.05) with a concomitant decrease of the non-C1-isomers. In contrast, in serum the isomers of bilirubin glucuronide did not change significantly at 24 h after papillotomy. Before papillotomy, the fraction of unconjugated bilirubin in bile was 3.6% of the total, with 15.8% bilirubin monoconjugates and 75.5% bilirubin disconjugates. After papillotomy, unconjugated bilirubin decreased to 1.6% (n.s.) and bilirubin monoconjugates to 11.9% (n.s.), while bilirubin diconjugates increased to 86.1% (P less than 0.05). In serum, the elevated fractions of bilirubin diconjugates and monoconjugates decreased from 38.4 to 32.2% (P less than 0.05) and from 29.6 to 23.4% (n.s.), respectively. In parallel, the fraction of unconjugated bilirubin in serum increased from 24.1 to 37.0% (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of pH on the deconjugation of conjugated bilirubin in human bile.

The enzymatic activity of bacterial beta-glucuronidase plays an essential role in the formation of calcium bilirubinate in bile. There are, however, many unsettled problems such as methodology of the assay for its enzymatic activity. In the present study (1) the azopigments from monoconjugated bilirubin (MCB) and unconjugated bilirubin (UCB) in native bile were semiquantitatively determined, (2) the deconjugation of conjugated bilirubin (CB) in bile was estimated with azopigment analysis and (3) factors affecting the deconjugation of CB in bile, especially for pH value, were investigated. CB in bile was stable at physiologic pH during 6-hr incubation at 37 degrees C, but was hydrolyzed at alkaline pH. At physiologic pH, addition of beta-glucuronidase from E. coli hydrolyzed CB in bile and increased MCB and UCB in bile. Based upon the results mentioned above, it is suggested that alkaline pH and enzymatic activity of beta-glucuronidase should cause the increase of UCB in bile. It can be said that beta-glucuronidase is essential for the formation of calcium bilirubinate gallstone at physiologic pH.

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.

Pigment gallstone disease.

Black and brown pigment gallstones are morphologically, compositionally, and clinically distinct. Black stones form primarily in the gallbladder in sterile bile and are associated with advanced age, chronic hemolysis, alcoholism, cirrhosis, pancreatitis, and total parenteral nutrition. Brown stones form not only within the gallbladder but also within the intrahepatic and extrahepatic ducts; they are uniformly infected with enteric bacteria and are usually associated with ascending cholangitis. Brown stones are related to juxtapapillary duodenal diverticula and are the predominant type of de novo common bile duct stones. Cholecystectomy is usually curative in black pigment stone disease, whereas stones often recur after cholecystectomy for brown stone disease. The pathogenesis of black stones is probably related to nonbacterial, nonenzymatic hydrolysis of bilirubin conjugates. At the pH of bile, this results in two monohydrogenated bilirubin anions that precipitate with calcium ions. Bilirubin monoconjugates that are increased in several conditions, such as Gilbert's syndrome and chronic hemolysis, may play a pivotal role in black stone formation as a source of unconjugated monohydrogenated bilirubin and as a possible co-precipitant with calcium. The precipitation of calcium carbonate and phosphate is influenced by local gallbladder factors. Brown pigment stones are formed in bile infected with enteric bacteria that elaborate hydrolytic enzymes: beta-glucuronidase, phospholipase A, and conjugated bile acid hydrolase. The resulting anions of bilirubin and fatty acids form insoluble calcium salts. We used nb/nb mice with a chronic hemolytic anemia as a model of hemolysis-induced black stone disease. The presence of 40% bilirubin monoconjugates in mouse gallstones indicated the importance of this moiety in the pathogenesis of black stones. Other data obtained by marrow transplantation experiments in mice revealed the relative importance of genotype versus the hemolytic anemia on determinants such as biliary bile acid composition and mucin secretory glands in the mouse gallbladder neck. Additional physical chemical studies of the interaction of unconjugated bilirubin in model bile solutions will be helpful in further delineating the pathogenesis of both black and brown pigment gallstones.

Glucuronidation of bilirubin and the occurrence of pigment gallstones in patients with chronic haemolytic diseases

A group of thirty-seven patients with increased haem catabolism has been studied to gain insight in their bilirubin conjugating capacity. Bilirubin UDP-glucuronyl transferase activity (GlcATa) in the liver and bilirubin monoconjugates in bile were measured and the hepatic bilirubin clearance was calculated from the radio-chromium-survival data. In the present group, 41% of the patients clearly had a deficiency in bilirubin conjugation similar to what is classically found in Gilbert's syndrome. The association may facilitate detection of these patients as serum bilirubin levels were higher (65.8 microM +/- 19) (m +/- 1 SD) in the fifteen patients with associated Gilbert's syndrome versus thirteen having only haemolysis (43.6 microM +/- 15). A fair correlation was found between the percentage of monoconjugates in bile and the GlcATa levels in the liver as well as with the calculated hepatic bilirubin clearance, although some discrepancies exist. Using these determinations, a clearcut separation from normal values was not obtained, suggesting at least in the present group of patients that Gilbert' syndrome represents only one end of a continuum of bilirubin conjugation rates and not a separate entity. Pigment stones in the gall-bladder were documented in 51% of the patients and usually at an early age. There was no relationship towards sex, serum bilirubin, GlcATa in liver, total bilirubin or monoconjugates in bile. Age played some role as well as the type of haemolysis as all patients with congenital dyserythropoiesis (n = 4) or acquired haemolysis (n = 3) had lithiasis. Moderate chronic cholecystitis was present, whereas an accumulation of iron and bile pigment was evident in the liver.

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.

Effects of Chlorpromazine on Bilirubin Metabolism and Biliary Secretion in the Rat

Chlorpromazine (CPZ) is a long recognized cause of jaundice, but the mechanism by which this agent produces icteric liver disease is not well defined. This study examined some effects of CPZ administration on bilirubin metabolism and biliary secretion in the rat. Acute CPZ treatment (25 mg per kg intravenously) produced hemolysis and resulted in increased bilirubin output and stimulation of hepatic heme synthesis. Both unconjugated bilirubin and a gamma-azopigment found primarily in obstructed rat bile were seen. Acute infusion of hemoglobin solutions to simulate the hemolysis produced by acute CPZ treatment resulted in increased bilirubin output and increased hepatic heme synthesis, but no alterations in the normal conjugation pattern of bilirubin in bile were seen. Chronic CPZ treatment for 4 and 8 days (25 mg per kg intraperitoneally) increased bilirubin output without elevating plasma hemoglobin levels and increased the fraction of monoconjugated bilirubin in bile; the 8-day chronic treatment regimen also caused unconjugated bilirubin to appear in bile. No change in liver weight, bile salt, phospholipid, or cholesterol output was seen with chronic CPZ therapy. Changes in bilirubin production and conjugation caused by CPZ may be a contributing factor in this agent's hepatotoxicity.

Glucuronidation of bilirubin and the occurrence of pigment gallstones in patients with chronic haemolytic diseases

Abstract. A group of thirty-seven patients with increased haem catabolism has been studied to gain insight in their bilirubin conjugating capacity. Bilirubin UDP-glucuronyl transferase activity (GlcATa) in the liver and bilirubin monoconjugates in bile were measured and the hepatic bilirubin clearance was calculated from the radio-chromium-survival data. In the present group, 41% of the patients clearly had a deficiency in bilirubin conjugation similar to what is classically found in Gilbert's syndrome. The association may facilitate detection of these patients as serum bilirubin levels were higher (65-8 μM ± 19) (m ± 1 SD) in the fifteen patients with associated Gilbert's syndrome versus thirteen having only haemolysis (43-6 μ± 15). A fair correlation was found between the percentage of monoconjugates in bile and the GlcATa levels in the liver as well as with the calculated hepatic bilirubin clearance, although some discrepancies exist. Using these determinations, a clearcut separation from normal values was not obtained, suggesting at least in the present group of patients that Gilbert's syndrome represents only one end of a continuum of bilirubin conjugation rates and not a separate entity.

Isolation and properties of conjugated bilirubin from bile.

1. A simple, rapid solvent partition method is described for isolation of conjugated bilirubin, free of unconjugated bilirubin, bile salts, phospholipids and cholesterol, from rat bile. Yields are 40-58%. The product is a phosphate-buffered solution containing approx. 0.4mg of bilirubin/ml, principally as mono- and di-glucuronide conjugates. The method may be modified for isolation of conjugates from human bile with 15-22% yield, and for preparation of unconjugated bilirubin from rat or human bile with yields of 55-62%. 2. The conjugated pigment has red-brown fluorescence and an absorption maximum at 450nm with in(mM) 59.8cm(-1). Diazotization by the Malloy-Evelyn method gives a direct Van den Bergh reaction (in water) 12% greater than the total reaction (in methanol), with in(total) 28.4x10(3)lmol(-1)cm(-1) at 550nm. After desalting by elution from Sephadex LH-20 in 50% (v/v) ethanol, the product gave water-soluble mustard-yellow crystalline needles. Such desalted conjugates were precipitated by Pb(2+) but not by Ba(2+), Ca(2+) or Zn(2+). 3. At pH7.0 and 37 degrees C the conjugated bilirubin was oxidized at a rate of 1%/h without hydrolysis, whereas 84% was hydrolysed by beta-glucuronidase or aqueous alkali. 4. Mono- and di-glucuronides were separated by elution from Sephadex LH-20 in 95% (v/v) ethanol or by extraction with chloroform at pH3.2-3.4. The monoconjugated bilirubin did not become labelled during incubation with unconjugated [(14)C]bilirubin, and chromatographed as a single spot without dissociating into unconjugated bilirubin and diglucuronide as would be expected of a complex. 5. After intravenous injection of mono- or di-conjugated [(14)C]bilirubin into normal or Gunn rats, 79-91% was excreted in bile and 2-7% in urine over 2h. In these experiments injected diglucuronide was not hydrolysed whereas 30-41% of injected monoglucuronide was converted into diglucuronide by the normal but not by the Gunn rats. The evidence favours the existence of a true bilirubin mono-glucuronide that is not a complex.