Inaccuracies in measurement of conjugated and unconjugated bilirubin in bile with ethyl anthranilate diazo and solvent-partition methods.

A criticial evaluation was made of the ethyl anthranilate diazo and two solvent-partition methods for the determination of conjugated and unconjugated bilirubin in human and rat bile. The ethyl anthranilate diazo reagent, which reacts only with conjugated bilirubin in serum, also diazotized a variable proportion of unconjugated bilirubin in bile and thus overestimated the concentration of monoconjugates. With the Weber-Schalm and modified Folsch solvent-partition methods applied to human or rat bile, 4--9% of added 14C-labelled unconjugated bilirubin partitioned with the conjugated bilirubin in the upper phase, and 4--9% of added 14C-labelled conjugated bilirubin partitioned into the lower phase. With dog bile, the spill-over of 14C-labelled bilirubin into the lower phase was 9--11%. Analysis of azopigments from the Weber-Schalm partition confirmed that over two-thirds of the bilirubin in the lower phase represents monoconjugates, principally the less-polar monoxylosides and monoglucosides. These solvent-partition methods thus overestimate the concentration of unconjugated bilirubin in bile.     

Mass-spectrometric study of the azopigments obtained from bile pigments with diazotized ethyl anthranilate

The structures of some azopigments obtained by diazotization of conjugated and unconjugated bile pigments with diazotized ethyl anthranilate were studied by mass spectrometry. The alpha(0)-azopigments derived from rat bile and human bile were shown to be identical (t.l.c. and mass spectra) with azobilirubin derived from unconjugated bilirubin. The presence of two methyl vinyl isomers (Ia) and (Ib) in equal amounts was shown by t.l.c. and mass spectrometry. The structure of the delta-azopigment derived from rat bile was studied by two methods: (a) ammonolysis gave rise to an amide having a CH(2).CH(2).CO.NH(2) side chain as shown by its mass spectrum; (b) the mass spectrum of a trimethylsilyl derivative of the delta-azopigment methyl ester confirmed the ester to be a beta-d-monoglucuronide ester of azobilirubin I.     

Extra hepatic formation of bilirubin glucuronides in the rat

After total hepatectomy in the rat, the presence of conjugated bilirubin in the plasma has been demonstrated using an extraction technique. This is particularly striking after loading the animals with unconjugated bilirubin. The identification of bilirubin glucuronide has been achieved using thin-layer chromatography of the azopigments formed (1) with ethyl anthranilate and (2) with p-iodoaniline.     

Heterogeneity of bile pigment conjugates as revealed by chromatography of their anthranilate azopigments

1. Azopigments derived from conjugated bile pigments by coupling with the diazonium salt of ethyl anthranilate are analysed conveniently by quantitative t.l.c. or by column chromatography on CM-cellulose. 2. By chromatographic studies combined with a series of chemical tests six groups of azopigments were demonstrable in preparations from bile and from icteric urine of man. Azobilirubin and its β-d-monoglucuronide have hitherto been considered to be the only major derivatives that can be obtained from human bile pigments. In the present work, other azopigments accounted for 30–40% of the total azopigment material, and the amounts of these showed considerable variation among biological fluids. 3. The divergence of the present results from earlier work is probably related to the use of milder diazotization conditions and of chromatographic techniques with a high resolving power. 4. The thin-layer chromatographic systems developed allow rapid and quantitative analysis of azopigments derived from bile pigments.     

Heterogeneity of bile pigments formed by the breakdown of haemoglobin in the isolated male-dog kidney.

Normothermic perfused isolated male-dog kidneys formed radioactive bile pigments by the breakdown of radioactive haemoglobin prepared from [2-14C]glycin. After column chromatographic separation and preparation of dipyrrolic azopigments, 86.3 +/- 2.2% of the bile pigments seemed to be conjugated bilirubin. Thin-layer chromatographic separation of the azopigments of ethyl anthranilate revealed a good correlation between photometric scanning, radiochromatographic scanning and the radioactivity of the azopigments scraped off the thin-layer glass plates and counted in a liquid scintillation counter. Although the same heterogenity of the azopigments was observed as in dog bile, the isolated male-dog kidney formed significantly less alpha2- and significantly more gamma-fractions.     

High-performance liquid chromatographic separation of bilirubin conjugates

A fast sensitive method for the isolation and quantitation of biliary bile pigments by reverse-phase high-performance liquid chromatography has been developed. Nine conjugates of bilirubin as well as unconjugated bilirubin and an internal standard, unconjugated mesobilirubin IX alpha, were all separated to baseline by gradient elution. The following sequence of eluted compounds was chemically identified by separating their ethyl anthranilate derivatives by thin-layer chromatography and by their enzymatic formation with UDP-bilirubin transferase and cosubstrate: bilirubin diglucuronide, bilirubin monoglucuronide monoglucoside, bilirubin monoglucuronide monoxyloside, bilirubin monoglucuronide (C-8, C-12), bilirubin diglucoside, bilirubin monoglucoside monoxyloside, bilirubin dixyloside, bilirubin monoglucoside (C-8, C-12), and bilirubin monoxyloside. The use of the commercially available mesobilirubin IX alpha as an internal standard was found to facilitate quantitation of the bilirubin conjugates.     

Analyses of azopigments obtained from the delta fraction of bilirubin from mammalian plasma (mammalian biliprotein).

Azopigments were obtained from the delta fraction of bilirubin (mammalian biliprotein) in cholestatic sera of men, rats and guinea pigs by diazo reaction with diazotized p-iodoaniline and analysed by t.l.c. Delta bilirubin of men and rats generated both unconjugated and glucuronide-conjugated azodipyrroles, whereas that of guinea pigs, in which the predominant form of conjugated bilirubin in serum was bilirubin monoglucuronide, generated only unconjugated azodipyrrole. We further analysed the azopigments by reversed-phase h.p.l.c. to distinguish their endovinyl and exovinyl isomers. The results indicated (a) that covalent binding of bilirubin to protein occurs exclusively on the conjugated dipyrrolic (either endovinyl or exovinyl) half of the parent conjugated bilirubin, (b) that both bilirubin monoglucuronide and bilirubin diglucuronide generate delta bilirubin, the latter yielding a 'conjugated' form of delta bilirubin that preserves the glucuronic acid moiety on the dipyrrolic half not bound covalently to protein, and (c) that therefore at least four forms of delta bilirubin exist in jaundiced sera of men and rats.     

The isolation of an azobilirubin β-d-monoglucoside from dog gall-bladder bile

An ethyl anthranilate azopigment of bilirubin conjugated to beta-d-monoglucoside was isolated from dog gall-bladder bile. Glucose was cleaved from the azopigment by treatment with beta-glucosidase and beta-glucuronidase. Mild alkaline hydrolysis of the compound by sodium methoxide yielded two kinds of compounds, water-soluble and organic-soluble. The former were shown, by enzymic analysis, t.l.c., nuclear magnetic resonance, and combined g.l.c. and mass spectrometry, to contain glucose. No evidence was obtained from these data that a disaccharide was present in this fraction. The organic-soluble compounds formed during this methanolysis were shown, by t.l.c. and mass spectrometry, to be the isomeric dipyrrole azopigments of bilirubin. These findings contribute further evidence to the controversy surrounding the nature of conjugated bilirubin.     

Occurrence of ditaurobilirubin, bilirubin conjugated with two moles of taurine, in the gallbladder bile of yellowtail, Seriola quinqueradiata

An unknown bilirubin conjugate was detected by HPLC analysis in the bile of yellowtail, Seriola quinqueradiata. The retention time of this unknown conjugate was identical with that of authentic ditaurobilirubin, a bilirubin conjugated with 2 mol of taurine. The retention times of the ethyl anthranilate diazo derivatives of both bilirubin conjugates were the same. The azo derivatives of this unknown conjugate were purified by Amberlite XAD-2 treatment and preparative TLC and hydrolyzed with 6 N HCl at 105 degrees C for 12 h. The hydrolyzates obtained were analyzed with an amino acid analyzer. The ninhydrin-positive substance in the hydrolyzates was purified by column chromatography and analyzed by TLC and mass spectrometry. The results of these analyses indicated that this ninhydrin-positive substance was taurine. Therefore, this unknown bilirubin conjugate was identified as ditaurobilirubin.     

Characterization of the major diazo-positive pigments in bile of homozygous Gunn rats

Bilinoid pigments in bile of homozygous Gunn rats (jj) were analysed either after formation of dipyrrolic ethyl anthranilate azo derivatives or as the unmodified parent tetrapyrroles. 1. T.l.c. of the azo derivatives revealed seven major unconjugated components which were structurally characterized by chemical tests, spectrophotometry and mass spectrometry. In addition, two minor components were identified as azodipyrrole (A+B)-glucoside and azodipyrrole (A+B)-β-d-glucuronide. 2. Extraction and t.l.c. of the tetrapyrrolic pigments showed 13 major yellow diazo-positive bands. Four of them, accounting for 59% of total diazo-positive material, were identified as unconjugated bilirubin-IXα, -IXβ, -IXγ and -IXδ. A fifth band (16%) was characterized as a mixture of two isomeric monohydroxyl derivatives and another band (8%) as a dihydroxyl derivative of bilirubin-IXα. 3. Although unconjugated bilirubin-IXα constitutes one-third of total diazo-positive material in bile of our strain of Gunn rats, the daily amount excreted represented only about 3–4% of daily bilirubin production. 4. Phototherapy caused a 2.2-fold increase in the biliary output of diazo-positive bilinoids, but did not affect markedly their composition. However, an additional diazo-negative pigment, accounting for one-third of total yellow colour, was observed but was not identified. Mass-spectral data on two dipyrrolic azopigments have been deposited as Supplementary Publication SUP 50076 (3 pages) with the British Library Lending Division, Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1977) 161, 1.     

Rearranged glucuronic acid conjugates of bilirubin-IX alpha in post-obstructive bile. Structure elucidation of azopigments beta and gamma as ethyl anthranilate N-glycosides derived from 2-, 3- and 4-o-acyl glucuronides

Azopigment analysis was performed on conjugates of bilirubin-IXalpha in bile of man and rats obtained after obstruction of the bile duct or in bile incubated under N2. The azopigments beta and gamma, formed by applying a pH 2.7 diazonium reagent containing an excess of ethyl anthranilate, correspond to rearranged ethyl athranilate N-glucuronides having the azodipyrrole acyl group on positions 2, 3 and 4 of the sugar. These assignments were verified, first by conversion of the structurally known 2-, 3- and 4-O-acyl glucuronide azopigments, unsubstituted at C-1, into ethyl anthranilate N-glucuronide reference compounds, and second, by mass spectrometry of trimethylsilyl ether methyl ester derivatives of unknown and reference compounds. The C-1 ethyl anthranilate group of the N-glucuronides triggers characteristics fragmentation reactions of the carbohydrate moiety revealing the position of the azodipyrrole O-acyl group.     

Bilirubin conjugates in bile of man, rat and dog. Semi-quantitative analysis of bile composition by thin-layer chromatography.

1. Conjugated bile pigments, separated in two fractions by semi-quantitative t.l.c. performed on silicic acid with phenol/water as the developing solvent, were treated with diazotized ethyl anthranilate. Resulting dipyrrylazo derivatives were analysed by quantitative t.l.c. 2. The tentative structure elucidation of tetrapyrrolic bilirubin conjugates and semi-quantitative evaluation of rat bile, post-obstructive human bile and dog bile composition is presented. 3. Homogeneous and mixed hexuronic acid diesters of bilirubin containing glucuronic acid constitute 51% of the total conjugates in normal rat bile, 45% of those in human post-obstructive bile and 38% of those in obstructed rat biles. 4. Monoconjugated bilirubin amounts to 33% of total conjugated bile pigments in normal rat bile, and 17 and 14% in post-obstructive hepatic human bile and gall-bladder bile of dog respectively. After loading with unconjugated bilirubin a greater amount of monoconjugates (56%) occur in the rat bile, whereas bilirubin diglucuronide excretion is decreased (34%). 5. In gall-bladder bile of normal dog, 40% of glucose-containing diconjugates, 32% of homogeneous and/or mixed hexuronic acid (mainly glucuronic acid) diesters of bilirubin and 14% of xylose-containing diconjugates are estimated. 6. Increased amounts of bilirubin conjugates, including some with unidentified uronic acid groups, were observed in cholestatic rat biles and quantities of conjugates with glucuronic acid were decreased.     

Measurement of plasma unbound unconjugated bilirubin

A method is described for measuring the unconjugated fraction of the unbound bilirubin concentration in plasma by combining the peroxidase method for determining unbound bilirubin with a diazo method for measuring conjugated and unconjugated bilirubin. The accuracy of the unbound bilirubin determination is improved by decreasing sample dilution, eliminating interference by conjugated bilirubin, monitoring changes in bilirubin concentration using diazo derivatives, and correcting for rate-limiting dissociation of bilirubin from albumin. The unbound unconjugated bilirubin concentration by the combined method in plasma from 20 jaundiced newborns was significantly greater than and poorly correlated with the unbound bilirubin determined by the existing peroxidase method (r = 0.7), possibly due to differences in sample dilution between the methods. The unbound unconjugated bilirubin was an unpredictable fraction of the unbound bilirubin in plasma samples from patients with similar total bilirubin concentrations but varying levels of conjugated bilirubin. A bilirubin-binding competitor was readily detected at a sample dilution typically used for the combined test but not at the dilution used for the existing peroxidase method. The combined method is ideally suited to measuring unbound unconjugated bilirubin in jaundiced human newborns or animal models of kernicterus.     

Separation and structural analysis of vinyl- and isovinyl-azobilirubin derivatives

The coupling reaction of bilirubin with the diazonium salts of ethyl anthranilate or of aniline yields two isomeric azopigments. These can be separated by t.l.c. as their methyl esters. The mass spectra of each pair of azopigments are very similar, showing that they are isomers. Proton-magnetic-resonance spectrometric studies show that they differ in the positions of the substituents on the pyrrolenone end ring; in one compound the methyl and vinyl groups are interposed compared with the other compound. These azo compounds were used as reference standards for determination of the site of conjugation in bilirubin monoglucuronide prepared enzymically. Analysis showed that conjugation occurs at the carboxyethyl side chain of both sides of the bilirubin molecule. During the preparation of the ethyl anthranilate reference compounds a series of minor azopigments were isolated by t.l.c. Analysis of the mass spectra of many of these showed that three side reactions can occur: (1) methylation of the imide carbonyl group; (2) addition of methanol or water to the vinyl substituent; (3) transmethylation of the ethoxycarbonyl group.     

Separation by thin-layer chromatography and structure elucidation of bilirubin conjugates isolated from dog bile.

1. A system for separation of bile pigments by t.l.c. and for their structure elucidation is presented. Separated bile pigments are characterized by t.l.c. of derived dipyrrolic azopigments. 2. At the tetrapyrrolic stage hydrolysis in strongly alkaline medium followed by t.l.c. demonstrates the presence of bilirubin-IIIalpha, -IXalpha and -XIIIalpha and allows assessment of their relative amounts. 3. Most structural information is derived from analysis of dipyrrolic azopigments. Such derivatives, obtained by treatment of separated bile pigments with diazotized ethyl anthranilate, were separated and purified by t.l.c. Micro methods showed (a) the nature of the dipyrrolic aglycone, (b) the nature of the bonds connecting aglycone to a conjugating group, (c) the ratio of vinyl/isovinyl isomers present in the aglycone and, (d) the nature of the conjugating groups (by suitable derivative formation and t.l.c. with reference to known compounds). 4. In bile of normal dogs at least 20 tetrapyrrolic, diazo-positive bile pigments could be recognized. Except for two pigments the tetrapyrrolic nucleus corresponded predominantly to bilirubin-IXalpha. All conjugated pigments had their conjugating groups connected in ester linkage to the tetrapyrrolic aglycone, Apart from bilirubin-IXalpha, monoconjugates and homogeneous and mixed diconjugates of bilirubin were demonstrated; conjugating groups of major importance were xylose, glucose and glucuronic acid. 5. Bilirubin isomer determination on native bile and isolated bile pigments, and dipyrrole-exchange assays with [14C8]bilirubin indicated (a) that the conjugates pre-exist in bile, and (b) that no significant dipyrrole exchange occurs during isolation of the pigments.     

A novel method to determine total and free serum bilirubin.

The determination of total (unconjugated) and free serum bilirubin concentrations using a novel and sensitive method based on static fluorescence quenching of daneyl bovine serum albumin was developed. The method allowed the use of a sample of 5 μl or less to determine total bilirubin over a range of 10–200 μg/ml with good recovery (94.9 ± 2.2%). For the determination of total bilirubin, a denaturation medium containing 8 m urea, 10 mm dithloerythreitol, and 0.1 m Tris was employed to eliminate interference by human serum albumin itself. The method was also tested with patients' sera containing negligible conjugated bilirubin in order to compare it to a commonly used “diazo” method. The correlation between the two methods gave a practically linear relation (γ = 0.99). The effects of a number of potentially interfering substances were tested and the results showed the test was specific for bilirubin. Concentrations of free bilirubin were determined without adding a denaturation agent. The experimental values were in agreement with those calculated theoretically using the isotherm of a single binding site and an association constant of human serum albumin to bilirubin of 1.5 × 10⁸m^?1.     

Chemical synthesis of bilirubin glucuronide conjugates

In dimethylformamide, the two carboxyl groups of bilirubin react with the bifunctional coupling agent, carbonyldiimidazole, to form bilirubin diimidazole, which was isolated and crystallised. The bilirubin diimidazole, termed “activated bilirubin”, was shown to react spontaneously with primary alcohols to form diesters of bilirubin. After addition of the tetrabutyl ammonium salt of glucuronic acid, compounds with chromatographic mobilities similar to those of bilirubin mono- and diglucuronides from bile were formed.Bilirubin diglucuronides were isolated by barium precipitation followed by solvent extraction. The bilirubin diglucuronides were considered to be a mixture of α and β glucuronides esterified at positions 1, 2, 3, or 4 of glucuronic acid because the compound(s) was resistant to hydrolysis with glucuronidase and gave multiple spots by chromatography after diazotization with ethyl anthranilate. The model compounds lauryl glucuronides were synthesized similarly; the most polar product by chromatography had identical chromatographic mobility to synthetic lauryl l-d-glucuronide prepared by reductive debenzylation of lauryl (benzyl (2,3,4-tri-O-benzyl))-d-glucuronide.It is concluded that bilirubin-1-di-β-d-glucuronide can be synthesized when suitable protecting groups for the 2, 3, and 4 hydroxyl groups of glucuronic acid become available.     

Development and validation of a method for measuring the glycine and taurine conjugates of bile acids in bile by high-performance liquid chromatography

We developed and validated a simple method for measuring the individual glycine and taurine conjugates of bile acids in bile by high-performance liquid chromatography with a C₁₈ reversed-phase column using an isocratic solvent system of acidified methanol—potassium phosphate. Without preliminary derivatization or purification, complete separation of the ten major conjugated bile acids present in bile could be achieved in 65 min. Total bile acid concentrations were identical when measured enzymatically and by summing the individual bile acids determined by high-performance liquid chromatography. Bile acid composition determined by gas—liquid chromatography correlated with results by high-performance liquid chromatography. Finally, measurements of individual glycine and taurine conjugates in human bile and in mixtures of bile acid standards by high-performance liquid chromatography and thin-layer chromatography gave similar results. This high-performance liquid chromatographic system permits simultaneous quantification of total and individual bile acids and their glycine and taurine conjugates in bile.     

Differentiated quantification of human bile acids in serum by high-performance liquid chromatography-tandem mass spectrometry

Determination of quantitative changes in the pattern of serum bile acids is important for the monitoring of diseases affecting bile acid metabolism. A sensitive and specific high-performance liquid chromatography (HPLC)-MS/MS method was developed for the differentiated quantification of unconjugated as well as glycine- and taurine-conjugated cholic, chenodeoxycholic (CDCA), deoxycholic (DCA), ursodeoxycholic (UDCA) and lithocholic acid (LCA) in serum samples. After solid-phase extraction and reversed-phase HPLC separation, detection of the conjugated bile acids was performed using electrospray ionization (ESI)-MS/MS and selected reaction monitoring mode, whereas unconjugated bile acids were determined by ESI-MS and selected ion monitoring mode. The within-day and between-day coefficients of variation were below 7% for all bile acids and the recovery rates of the extraction procedure were between 84.9 and 105%. The developed method was applied to a group of 21 healthy volunteers and preliminary reference intervals in serum were established. In patients with drug-induced cholestasis, an elevation of primary bile acids has been shown.     

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 Micha?lsson's diazocoupling method. The assay results gave a good correlation coefficient (0.86) compared with those determined with high performance liquid chromatography.