[Bilirubin excretion into bile. I. Effect of the medium on bilirubin secretion. Analysis of criteria for bilirubin excretion].
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Animals↗
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[Studies on bilirubin glucuronide. 2. A study on conjugated bilirubin fraction in bile and bilirubin glucuronide].
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Acute Disease↗
Anti-bilirubin monoclonal antibody. II. Enzyme-linked immunosorbent assay for bilirubin fractions by combination of two monoclonal antibodies.
Among 16 monoclonal antibodies raised against covalently coupled bilirubin-bovine serum albumin, we selected two antibodies: one (designated 24G7) reacted with unconjugated and conjugated bilirubin and the other (designated 25H17) reacted only with unconjugated bilirubin. Combination of these two antibodies enabled us to determine extremely low concentrations of unconjugated and conjugated bilirubin independently by enzyme-linked immunosorbent assay (ELISA). In the assay, samples were incubated with each anti-bilirubin IgG, and then free remaining IgG was allowed to bind to the immunotiter plates coated with bilirubin-bovine serum albumin. The bound fraction of the IgG was visualized with horseradish peroxidase-conjugated rabbit anti-mouse IgG and substrate. Bilirubin concentration was determined from the absorbance at 425 nm. In this system, we could measure 10(-7)-10(-5) M unconjugated and conjugated bilirubin in samples, which is 100-fold more sensitive than Michaëlsson's diazocoupling method. The assay results gave a good correlation coefficient (0.86) compared with those determined with high performance liquid chromatography.
Antibodies, Monoclonal↗
Serum bilirubin fractions in analbuminemic rats after bile duct ligation. Albumin requirement in the formation of covalently protein-bound bilirubin.
To clarify the role of albumin in the production of covalently protein-bound bilirubin (delta bilirubin, B delta) in the blood and the pathophysiological relevance of B delta, changes in the serum bilirubin level and histological findings in the liver, kidneys and myocardium were studied in Sprague-Dawley (SD) rats and Nagase analbuminemic rats (NAR) after bile duct ligation (BDL). In SD rats, the serum bilirubin level increased until 3 days after BDL, and was followed by the appearance of B delta. The increase in serum bilirubin was smaller in NAR, and no serum B delta was noted. Albumin administration to NAR 1 day after BDL increased serum bilirubin with the appearance of B delta. Serum bilirubin decreased in both SD rats and NAR 7 days after BDL. Marked deposition of bile pigments was noted in NAR in the renal tubular epithelium. The renal bilirubin content was decreased after albumin administration. From these results it is concluded that albumin is necessary for the production of B delta, and that renal deposition of bile pigments progresses in the absence of albumin.
Animals↗
Does bilirubin play a role in the pathogenesis of both cholesterol and pigment gallstone formation? Direct and indirect influences of bilirubin on bile lithogenicity.
Bilirubin is found in the center of cholesterol gallstones, but its pathogenic role in their formation is unknown. Bilirubin causes a disproportionate reduction of biliary lipid secretion without affecting bile salt secretion in association with a change of biliary lecithin species, which modulates the cholesterol crystallization process. Therefore, the present study investigated whether bilirubin can influence the cholesterol crystallization procedure, and the mechanism(s) of any such action. Supersaturated model bile was prepared (taurocholate/lecithin/cholesterol at 71:18:11, a total lipid concentration of 9.0 g/dl, and cholesterol saturation index of 1.8), and cholesterol crystallization was monitored over time using a spectrophotometer and video-enhanced differential contrast microscopy in the absence or presence of bilirubin (at a final concentration of 10 microM, 20 microM, 40 microM, and 100 microM). Bilirubin enhanced the onset of cholesterol crystallization by 50%, whereas the crystal growth rate and final crystal mass were reduced at a high concentration of bilirubin. Taken together, these results suggest that bilirubin influences the cholesterol crystallization process, by either a direct interaction with biliary lipids that alters metastability, an indirect alteration of the bile salt-micellar lipid holding capacity, or both. Thus, bilirubin may play a role in the pathogenesis of both cholesterol and pigment gallstones.
Bile↗
pKa and aggregation of bilirubin: titrimetric and ultracentrifugation studies on water-soluble pegylated conjugates of bilirubin and fatty acids.
A water-soluble conjugate (1) with intact carboxyl groups was prepared by addition of poly(ethylene glycol) thiol (MPEG-SH) regiospecifically to the exo vinyl group of bilirubin. (1)H and (13)C NMR and absorbance spectroscopy in CDCl(3) and DMSO-d(6) confirmed the assigned structure and showed that pegylation did not disrupt the hydrogen-bonded ridge-tile conformation of the pigment moiety. Aqueous solutions of 1 were optically clear, but NMR signals were seen only from the MPEG portion and none from the tetrapyrrole, consistent with dissolved assemblies containing aggregated bilirubin cores within mobile polyether chains. On alkalinization (pH >12), signals from the pigment moiety reappeared. Titrimetric measurements on 1 in water showed the pK(a)'s of the two carboxyl groups to be similar (average 6.42). Control studies with pegylated half-esters of succinic, suberic, brassylic, thapsic, and 1,20-eicosanedioic acid showed that pegylation per se has little, if any, effect on carboxyl ionization. However, aggregation increases the apparent pK(a) by approximately 1-2 units. The molecularity of bilirubin in solution was further characterized by ultracentrifugation. Over the pH range 8.5-10 in buffer, bilirubin formed multimers with aggregation numbers ranging from approximately 2-7. Bilirubin is monomeric in DMSO or CHCl(3) at approximately 2 x 10(-)(5) M, but aggregation occurred when the CHCl(3) was contaminated with trace adventitious (perhaps lipoidal) impurities. These observations show that aggregation increases the pK(a)'s of aliphatic carboxylic acids relative to their monomer values in water. They are consistent with earlier (13)C NMR-based estimates of approximately 4.2 and approximately 4.9 for the aqueous pK(a)'s of bilirubin and similar studies of bilirubin in micellar bile-salt solutions. Together with earlier work, they confirm that the pK(a)'s of bilirubin are about normal for aliphatic carboxyls and suggest that the high (>7.5) values occasionally reported, including those based on CHCl(3) partitioning, are artifacts of aggregation or technique.
Bilirubin↗
Bilirubin conjugates of human bile. Isolation of phenylazo derivatives of bile bilirubin.
A method is presented that allows the isolation of eight different phenylazo derivatives of bile bilirubin. In step I of the isolation procedure, three bilirubin fractions (bilirubin fractions 1, 2 and 3) from human hepatic bile are separated by reverse-phase partition chromatography on silicone-treated Celite with the use of a solvent system prepared from butan-1-ol and 5mm-phosphate buffer, pH6.0. Azo coupling is then performed with diazotized aniline. The three azo pigment mixtures are subjected to step II, in which the above chromatography system is used again. With each azo pigment mixture this step brings about the separation of a non-polar and a polar azo pigment fraction (azo 1A and azo 1B, azo 2A and azo 2B, and azo 3A and azo 3B from bilirubin fractions 1, 2 and 3 respectively). Approximately equal amounts of non-polar and polar pigments are obtained from bilirubin fractions 1 and 2, whereas bilirubin fraction 3 yields azo 3B almost exclusively. In step IIIA the non-polar azo pigment fractions are fractionated further by adsorption chromatography on anhydrous sodium sulphate with the use of chloroform followed by a gradient of ethyl acetate in chloroform. Three azo pigments are thus obtained from both azo 2A (azo 2A(1), azo 2A(2) and azo 2A(3)) and azo 3A (azo 3A(1), azo 3A(2) and azo 3A(3)). The 2A pigments occur in approximately the following proportions: azo 2A(1), 90%; azo 2A(2), 10%; azo 2A(3), traces. The pigments are purified by crystallization, except for the A(3) pigments, which are probably degradation products arising from the corresponding A(2) pigments. In step IIIB the polar azo pigment fractions are subjected to reverse-phase partition chromatography on silicone-treated Celite with the use of a solvent system prepared from octan-1-ol-di-isopropyl ether-ethyl acetate-methanol-0.2m-acetic acid (1:2:2:3:4, by vol.). Azo pigment fractions 2B and 3B each yield six azo pigments (azo 2B(1) to azo 2B(6) and azo 3B(1) to azo 3B(6) respectively) together with small amounts of products of hydrolysis (azo 2A(B) and azo 3A(B)). Only one azo B pigment is obtained from bilirubin fraction 1, and this azo pigment is probably of the B(2) type. The yields of the azo 3B pigments suggest that these pigments are present in approximately the following proportions: azo 3B(1), 0-0.4%; azo 3B(2), traces; azo 3B(3), traces; azo 3B(4), 10%; azo 3B(5), 50%; azo 3B(6), 40%. Azo pigments 2B(1) to 2B(6) are estimated to occur in similar proportions. Since pairs of correspondingly numbered azo pigments from bilirubin fractions 1, 2 and 3 do not separate on rechromatography together (e.g. azo 2A(1) co-chromatographs with azo 3A(1), and azo 2B(6) co-chromatographs with azo 3B(6)), it is concluded that such pigments are chemically identical. The structures of the isolated phenylazo derivatives are discussed in an accompanying paper (Kuenzle 1970c).
Acetates↗
Interaction of bilirubin with human erythrocyte membranes. Bilirubin binding to neuraminidase- and phospholipase-treated membranes.
Saturable bilirubin binding to human erythrocyte membranes was measured before and after digestion with neuraminidase and phospholipases. Neuraminidase-treated erythrocyte membranes did not show any change in their binding properties, indicating that gangliosides could be excluded as candidates for saturable bilirubin-binding sites on erythrocyte membranes. Although bilirubin-binding properties of the membranes did not change after phospholipase D digestion, either, phospholipase C treatment greatly enhanced bilirubin binding. Thus it is suggested that a negatively charged phosphoric acid moiety of phospholipids on the membrane surface may play a role to prevent a large amount of bilirubin from binding to the membranes. Further saturable bilirubin binding to inside-out sealed erythrocyte membrane vesicles showed values comparable with those of the right-side-out sealed membranes, suggesting that the bilirubin-binding sites may be distributed on both outer and inner surfaces of the membranes, or may exist in the membranes where bilirubin may be accessible from either side.
Bilirubin↗
Bilirubin, reserve albumin for binding of bilirubin and pH in plasma during phototherapy (ordinary and double light) of term newborn infants.
Forty-five term newborn infants with uncomplicated hyperbilirubinaemia were treated continuously with phototherapy for 24 hours. Twenty-eight infants received double light treatment and 17 infants ordinary phototherapy. During both treatments a significant decrease in the serum unconjugated bilirubin concentration, a significant increase in the serum reserve albumin concentration for binding of bilirubin determined by the [14C] MADDS method, and a significant decrease in the index of serum bilirubin toxicity occurred. The changes in these parameters were significantly greater during the double light treatment than during the ordinary phototherapy. During the treatment the fall in index was constant. No significant change in plasma pH was seen. Thus, the study gives further evidence that the risk of bilirubin encephalopathy is reduced by phototherapy and that double light treatment is in the respect superior to ordinary phototherapy. Prior to phototherapy the molar ratio in serum of unconjugated bilirubin plus reserved albumin for binding of bilirubin to albumin was only 0.60, on average, and during the treatment the increase in the serum reserve albumin concentration was less than the decrease in the serum bilirubin concentration. This can be explained either by the presence in infant serum of an unknown ligand interfering competitively or allosterically in the binding of MADDS and bilirubin to albumin, or by the existence of a foetal albumin with a lower affinity for MADDS than adult albumin.
Bilirubin↗
Bilirubin as an anti-oxidant for surgical stress: a preliminary report of bilirubin oxidative metabolites.
BACKGROUND: Bilirubin has been recognized as an anti-oxidant. The purpose of this study was to examine whether bilirubin would act as an antioxidant for surgical stress in humans. MATERIALS AND METHODS: Serum bilirubin and urinary bilirubin oxidative metabolites (BOM) were measured in 96 patients who underwent surgery. The antioxidant activity of bilirubin was assessed using BOM measured by enzyme-linked immunosorbent assay with an anti-bilirubin monoclonal antibody. RESULTS: Serum bilirubin levels increased after surgery in all 96 patients (p < 0.01), but did not correlate with operation time or blood loss (p = 0.53 and p = 0.28, respectively). BOM increased only in patients with major surgeries (p = 0.048). Significant correlations between BOM and operation time and blood loss were found (p < 0.01). CONCLUSIONS: Bilirubin appears to act as an antioxidant for invasive surgery in humans. Urinary BOM could be a reliable marker for the degree of surgical stress.
Adolescent↗
Relationship of unbound bilirubin concentration to reserve albumin-binding concentration for bilirubin in human neonatal plasma.
We examined the relationship of plasma unbound bilirubin concentration and reserve albumin-binding concentration for bilirubin in a sample of 545 neonates. Plasma unbound bilirubin concentration and total bilirubin-binding concentration were determined with the peroxidase assay. Contrary to published reports, we found that plasma unbound bilirubin concentration and plasma reserve albumin for bilirubin-binding concentration are highly correlated (r = -0.706; p less than 0.001) and that the relationship between these two parameters is dependent upon the total bilirubin-binding concentration. That these measured parameters correlate in the same manner as predicted for free bilirubin and free albumin by the law of mass action, suggests that these measurements may be meaningful.
Bilirubin↗
Hepatic disposition and biliary excretion of bilirubin and bilirubin glucuronides in intact rats. Differential processing of pigments derived from intra- and extrahepatic sources.
Mechanisms for transport of bilirubin and its conjugates in hepatocytes have not been defined. We investigated the hepatic processing of bilirubin glucuronides and their precursors, and characterized the disposition of bile pigments arising from intraversus extrahepatic sources. Tracer doses of purified radiolabeled biliverdin, bilirubin, bilirubin monoglucuronide (BMG) or diglucuronide (BDG) were administered intravenously to intact normal or jaundiced homozygous Gunn rats. Rapid sequential analysis of radiolabeled BMG and BDG in bile revealed comparable excretion patterns following biliverdin and bilirubin injection, with BDG as the major pigment. Biliary excretion of radiolabeled conjugates from injected BMG was more rapid, with BMG predominating. Excretion of injected BDG in normal rats and BMG or BDG in Gunn rats was virtually identical to that of unaltered BMG in normal rats. Model independent analysis by deconvolution provided objective comparison of the disposition of radiolabeled pigments from the different sources. These findings indicate that bilirubin glucuronides formed in the liver from endogenous (hepatic) and exogenous (extrahepatic) sources of bilirubin follow a similar excretory pathway. BMG formed endogenously is converted preferentially to BDG, whereas circulating BMG is excreted predominantly unchanged. Exogenous conjugated bilirubins are excreted more rapidly than those generated intrahepatically, by a transcellular pathway that is largely independent of the conjugation system.
Animals↗
Influence of glucuronosyl bilirubin and (EZ)-cyclobilirubin on determination of serum unbound bilirubin by UB-analyser.
BACKGROUND: In the enzyme reaction for the determination of the unbound (free) bilirubin concentration by glucose oxidase and peroxidase, materials with low affinity for serum protein are reactive. The influence of these materials on the determination of serum unbound bilirubin was investigated. METHODS Serum samples from patients with neonatal hyperbilirubinaemia were analysed by high-performance liquid chromatography for total glucuronosyl bilirubin concentration (TGC) and (EZ)-cyclobilirubin concentration [(EZ)-C]. Based on these measurements, the samples were classified into three groups: group I [13 samples, TGC <2 micromol/L and (EZ)-C < 2.5 micromol/L]; group II [four samples, TGC < 2 micromol/L and (EZ)-C > or = 2.5 micromol/L]; and group III (five samples, TGC > or = 2 micromol/L). The concentrations of total bilirubin and unbound bilirubin were measured in these same samples with a UB-analyser. When the absorbance at 460nm was monitored, the decrease in absorbance was non-linear (concave curve). The degree of concavity was estimated (D15 value) as the deviation from linearity at 15s. RESULTS: The D15 value was significantly higher in groups II and III than in group I. D15 value correlated significantly with TGC, (EZ)-C and unbound bilirubin concentration, and the unbound bilirubin concentration correlated significantly with TGC and (EZ)-C. CONCLUSION: These results indicated that determination of serum unbound bilirubin concentration using the UB-analyser could be positively skewed by high concentrations of TGC and (EZ)-C.
Artifacts↗
Enzymatic removal of bilirubin toxicity by bilirubin oxidase in vitro and excretion of degradation products in vivo.
The toxic effects of the degradation products of bilirubin that were formed by reaction with bilirubin oxidase were investigated with the C 1300 mouse neuroblastoma cell line by examining the following parameters: growth inhibition, morphologic characteristics, membrane transport, DNA synthesis, and protein synthesis. The addition of bilirubin to the cells resulted in definite cytotoxic effects on all of these parameters in a dose-dependent fashion; the addition of bilirubin oxidase reversed the toxic effects on the C 1300 cells in vitro. Furthermore, we found that most of these enzymatic degradation products of bilirubin were excreted by the kidney into the urine in a few hours after intravenous injection of the degradation products; in contrast, no intact bilirubin was excreted. Thus, these findings suggest that hyperbilirubinemia in newborn infants (kernicterus) may be prevented by administering polyethylene glycol-conjugated bilirubin oxidase, with a longer plasma half-life which has been reported previously to oxidize bilirubin to its nontoxic components in the bloodstream.
Animals↗
Determination of the sum of bilirubin sugar conjugates in plasma by bilirubin oxidase.
BACKGROUND: A reliable indicator of cholestasis is the presence of abnormal concentrations of bilirubin mono- and diglucuronide [conjugated bilirubin (CB)] in blood. A routine assay of CB is available only to those who possess a certain type of clinical analyzer. We describe a two-point manual method for CB that could be adapted as a rate assay to automated clinical analyzers. METHODS: The measurement of CB is based on its oxidation to biliverdin by bilirubin oxidase. The resulting decrease in absorbance at 460 nm is proportional to the CB concentration. The assay is calibrated with solutions of ditaurobilirubin in human serum. RESULTS: Under the conditions of the assay (0.1 mol/L glycine buffer, pH 10.0; reaction time, 2 min), only 5% of unconjugated bilirubin is oxidized and delta-bilirubin is not oxidized at all. Results obtained with the bilirubin oxidase method agreed well with those obtained by HPLC. The long-term CVs at CB concentrations of 6 and 63.4 mg/L were 20% and 2.6%, respectively. The reference values, established by analyzing 51 plasma specimens from healthy adults, were 0.0-1.2 mg/L, with a mean value of 0.2 mg/L. CONCLUSIONS: The proposed method for CB has good analytical specificity and obviates the requirement for HPLC or a dry chemistry analyzer. The measurement of CB in blood is superior to the measurement of direct bilirubin because an abnormal concentration of direct bilirubin does not necessarily indicate the presence of cholestasis.
Bilirubin↗
Glucagon enhances bile flow, bilirubin uridine diphosphate-glucuronyltransferase activity and biliary bilirubin monoconjugate excretion in the rat.
Intravenous infusion of glucagon (100 micrograms/hr/100 g body weight) in rats produces a 20 to 35% increase in bile flow and enhances the activity of hepatic bilirubin uridine diphosphate-glucuronyltransferase to 132% after a 90 min infusion. When a bilirubin load is given to produce a constant and apparently maximal biliary bilirubin excretion rate (or transport maximum) the administration of glucagon increased the bilirubin transport maximum. The excretion rate of bilirubin monoglucuronides was more enhanced than that of diglucuronide. The enhanced rate of glucuronidation, assayed in vitro, correlated with the augmented biliary output and inversely with the plasma unconjugated bilirubin levels. It is concluded that glucagon, at the dosage used, leads to a higher formation rate of bilirubin monoconjugates and that the choleresis, also induced by the hormone, enhances the biliary secretion of the monoconjugates formed. The enhanced conjugation results in a decreased plasma concentration of unconjugated bile pigment and the associated choleresis leads to a decreased di- to monoconjugate ratio, opposite to what has been observed during bilirubinostasis and cholestasis. The secretory efficacy, as assessed from the bile-to-plasma concentration ratio, is enhanced for all bilirubin pigments after glucagon administration.
Animals↗
The hematofluorometer: a rapid assay for bilirubin and bilirubin binding.
Serum bilirubin measurement is extremely important to clinicians caring for newborn infants. These infants are at risk for kernicterus (bilirubin staining of brain matter) due to their immature clearance mechanism and increased bilirubin load. The hematofluorometer is a newly available device that uses the fluorescent properties of bilirubin to measure 1) albumin-bound bilirubin, 2) reserve binding capacity, and 3) total whole blood bilirubin. Since it is an easy and rapid assay, it provides the clinician with immediate information regarding the infant's level of unbound bilirubin. This information permits rapid assessment of the patient's risk for bilirubin encephalopathy. Clinicians and researchers could readily use this device in several clinical situations.
Bilirubin↗
Membrane lipid composition and vesicle size modulate bilirubin intermembrane transfer. Evidence for membrane-directed trafficking of bilirubin in the hepatocyte.
To characterize the mechanisms underlying intracellular bilirubin transport, stopped-flow fluorometry was utilized to study the effects of membrane vesicle size and lipid composition on the kinetics of unconjugated bilirubin movement between model and native hepatocyte membranes. Bilirubin transfer rates declined asymptotically with increasing donor vesicle diameter, due primarily to a 1.4 kcal.mol-1 decrease in the entropy of activation for the larger vesicles. The incorporation of phosphatidylethanolamine and phosphatidylserine significantly enhanced the dissociation of bilirubin from phosphatidylcholine vesicles. Cholesterol induced a biphasic effect on the transfer rate constant; an initial decrease in rate from 248 to 217 s-1 associated with cholesterol:phospholipid ratios up to 20% was followed by a dramatic rise to 312 s-1 as the cholesterol concentration was increased to 70 mol %. The bilirubin dissociation rate from isolated rat liver endoplasmic reticulum (9.1 s-1) was significantly slower than for both basolateral and canalicular plasma membranes, which exhibited rate constants of 11.7 and 25.8 s-1, respectively. Collectively, these data suggest that the cholesterol: phospholipid ratio is the principal determinant of bilirubin dissociation from membranes. We postulate that the inherent cellular membrane cholesterol gradient in the hepatocyte creates a directed flux of bilirubin from the plasma membrane to teh endoplasmic reticulum and represents a potential driving force for intracellular bilirubin transport.
Animals↗