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Transcript of Unconjugated Bilirubin Increased Proportion Bilirubin ... · PDF fileUnconjugated Bilirubin...

  • Unconjugated Bilirubin and an Increased Proportion ofBilirubin Monoconjugates in the Bile of Patients withGilbert's Syndrome and Crigler-Najjar Disease

    JOHAN FEVERY, NORBERT BLANCKAERT, and KAREL P. M. HEIRWEGH,Laboratory for Liver Physiopathology, Department ofMedical Research,University of Leuven, 3000 Leuven, Belgium

    ANNE-MARIE PREAUX and PIERRE BERTHELOT, Unite de Recherches U-99,Institut National de la Sante et de la Recherche Medicale, Hopital HenriMondor, 94010 Creteil, France

    A B S T R A C T Bilirubin pigments were studied in thebile of 20 normal adults, 25 patients with Gilbert'ssyndrome, 9 children with Crigler-Najjar disease, and 6patients with hemolysis, to determine how a deficiencyof hepatic bilirubin UDP-glucuronosyltransferasewould affect the end products of bilirubin biotrans-formation.

    In the bile from patients with Gilbert's syndrome, astriking increase was found in the proportion ofbilirubin monoconjugates (48.69.8% of total con-jugates) relative to that in normal bile (27.27.8%).This increase was even more pronounced in childrenwith Crigler-Najjar disease, in whom, even in the mostsevere cases, glucuronide could always be demon-strated in the bile. Furthermore, unconjugatedbilirubin-IXa was unquestionably present in the bileof these children and amounted to 30-57% of theirtotal bilirubin pigments (

  • Decreased hepatic bilirubin UDP-GTA anid de-creased hepatic clearance of labeled bilirubin canl bothbe used for diagnosis as they yield indices of theconjugation rate of bilirubin-IXa (by far the predomi-nant isomer in man). In the present work, another ap-proach has been investigated. It is indeed reasonableto assume that the relative amounts of bilirubin-IXaand of its mono- and diconjugates in bile reflect theconjugating processes operating in the hepatocyte,provided that the secreted pigments are inot signifi-cantly altered in the biliary system. For these reasons,we have analyzed the nature and amounts of coinjuga-ted and unconjugated bilirubins that are present inbile. Bile analysis was compared with the results ofhepatic bilirubin UDP-GTA. Recent developmenits inmethods for bile pigment analysis have made this ap-proach possible (19).

    METHODSBile collection and patients. Bile was obtained by duo-

    clenal intubation in 20 normal controls, 25 adults with

    Gilbert's syncdrome, 8 children with Crigler-Najjar disease,and 6 patients with hemolytic jaundice who awaitedsplenectomiiy. The cause of the hemolysis was conigenitalspherocytosis in five, and autoimmune hemolysis in thesixth patient. In one additional child with Crigler-Najjardisease (child S), the bile sample was obtained by gallbladderpunicture at time of surgery. The conitrols were informiie(dpatients examinied for psychosomatic complaints or memiibersof the laboratory. Gilbert's syndromiie was dlefinecl as chronicunconijugated hyperbilirubinemia (>1.5 mg/100 ml) in theabsence of overt hemolysis and of any other disease; thesepatients included 14 males and 11 females; their agesranged from 15 to 64 yr.The ninle children with Crigler-Najjar disease were un-

    related to each other; there were five males and four females,ranging from 1 mo to 3 yr of age; clinical data on1 sonme ofthem have been published (20, 21). The nine children withCrigler-Najjar disease (Table I) showedl serumil concenitrationisof unconjugated bilirubin ranginig from 20 to 35 mg/100 ml.During the survey period, kernicterus developed in sixchildren. In these six patients, adminiistrationi of pheno-barbital failed to lower serum bilirubin, whereas it clearlyrdecreased the hyperbilirubinemiiia in two of the remiiainingthree children (20, 21). Exact data on the effect of pheno-barbital in the third child (S) could not be obtainied. If onetakes the response to phenobarbital and the developmenit of

    TABLE ICharacteristics of the Nine Children with Crigler-Najjar Disease

    Bile analysisBiliary

    Unconjugated Responise bilirubin (a,-PIA-bilirubin Kernic- to pheno- Hepatic ao-EA" a0-EA) &-EA1

    Child in serum terus* barbital* UDP-GTAt PIA EA as-PIA (pH 2.7) (pH 2.7) (pH 2.7)

    mgIlOO ml lAglh/g mg/1O0 ml % of total azopigment

    K 25.0 - + 145 2.6 1.9 68 51 17 27B 23.5 - + 0; 0; 0; 252 2.9 1.2 73 52 21 24S** - NDtt 93.0 62.0 68 56 12 23M, 24.0 + - 0 0.9 0.4 72 60 12 17M2 2.9 1.2 73 52 21 15D, 19.6 + - ND 2.7 1.1 81 47 34 40D252.4 0.8 56 41 15 48D3 1.3 0.6 84 61 23 25E 23.1 + - ND 3.2 1.3 99 77 22 traceVI 5.2 3.0 87 69 18 3V2 24.0 + - 0 0.6 0.3 97 58 39 traceV3A 1.1 0.7 90 80 10 traceC 35.0 + - 0 4.8 1.6 86 60 26 23G, 25.0 + - ND 7.1 4.0 87 70 17 9G255 8.5 3.2 86 66 20 18GM55 5.3 3.4 83 68 15 30

    * Absence (-) or presence (+) of kernicterus or positive response to treatment.Bilirubin UDP-glucuronosyltransferase activity.

    Unconjugated azodipyrrole formed by coupling with PIA.11 Unconjugated azodipyrrole formed by coupling with diazotized EA, a system in which, at pH 2.7, only the conjugatedbilirubin reacts. Therefore the difference (ao-PIA minus ao-EA) gives an estimate of the amount of unconjugated bilirubinpresent. Glucuronidated azodipyrrole formed by coupling with diazotized EA.$* Bile obtained by gallbladder puncture at surgery.

    4 Not determined. Samples taken under phenobarbital therapy. Subscripts after the letters refer to successive bile samples from the samepatient.

    Unconijugated and Alonoconjugated Bilirubin in Humant Bile 971

  • UCBUCB J'N=~~~NN-R


    ' sN=N-R


    FIGURE 1 Schematic representation ofdiazo-cleavage ofbili-rubin, leading to dipyrrolic azopigments. Diazotized p-iodoaniline (PIA) reacts with both unconjugated (UCB) andconjugated bilirubin, whereas only the conjugates react withdiazotized ethyl anthranilate (EA) at pH 2.7. In the PIA sys-tem, one molecule of UCB leads to the formation of twomolecules of unconjugated azodipyrrole (ao). In both PIAand EA systems: one molecule of bilirubin monoglucuronideproduces one molecule of a0 and one of conjugated 8-azopig-ment; diglucuronide yields only 8-azopigment.

    kernicterus as discriminative features, then the former sixchildren would belong to the so-called group I (22). In fourof these patients, two consecutive bile samples were ob-tained, one before and one during treatment with pheno-barbital, for periods varying from 1 mo to 1 yr.

    Gallbladder bile was obtained after stimulation with intra-duodenal magnesium sulfate (15 g) or intravenous (i.v.) in-jection of cholecystokinin (1 Ivy dog U/kg; CCK-PZ, GIHUnit, Karolinska Institute, Stockholm, Sweden). In all cases,a first sample (5-15 ml) taken before gallbladder stimula-tion was discarded because gastric juice present as a contami-nant may induce artificial changes in the pattern of biliarybilirubin (23). The bile samples were analyzed either freshor after storage at -20C in the dark. Storage for at least 3wk under these conditions did not alter the bilirubin com-position, provided the samples were deep-frozen immediatelyafter collection.

    In the patients, but not in the controls, liver tissue wasobtained by needle biopsy or at the time of splenectomy inthe hemolytic patients. Specimens were assayed for bilirubinUDP-GTA immediately or after storage of the biopsy as suchat -20C. The activity remains unaltered for periods up to 6wk at least under these conditions (24, 25).Chemical methods. Bilirubin UDP-GTA was determined

    in digitonin-activated liver homogenates as described byBlack et al. (25). In two children (C and B), transferaseactivity was measured by a slight modification of thismethod yielding essentially comparable results (26). /3-glucuronidase activity was assayed with phenolphthalein asthe substrate (27).

    Bile pigments were assayed with various diazo-couplingtechniques (Fig. 1). Before the assays, bile samples from thenormal controls and adult patients were diluted 21 to 51-foldwith distilled water. To obtain sufficient sensitivity in theassay, bile of patients with Crigler-Najjar disease was dilutedonly 3 to 11-fold, except in the child S (101-fold). Conjugatedbilirubin was determined by treatment of diluted bile withdiazotized ethyl anthranilate (EA) at pH 2.7 (28, 29). Totalbile pigment was determined by coupling with diazotizedp-iodoaniline (PIA) in the presence of a reaction ac-celerator (30). To detect any acid-labile conjugating bonds,bile was treated with diazotized EA at pH 6.0 (31); thisalso promotes reaction of unconjugated bilirubin-IXa ifpresent.

    The azopigments formed were separated by thin-layerchromatography (TLC). The silica gel plates were firstdeveloped with benzene:ethyl acetate, 85:15 (vol/vol), to re-move lipids and excess of diazoreagent. This washingprocedure was followed by successive developments withchloroform:methanol:water, 65:25:3 (vol/vol/vol), for 10 cmand with chloroform:methanol, 85:15 (vol/vol), for 16 cm. Theseparated azopigments were quantitated either by densitome-try or by photometric reading of methanol eluates (19).Preparations of azodipyrrole (ao; from bilirubin-IXa), ofazodipyrrole f3-D-monoxyloside (a2), 8-D-monoglucoside(a3), and 8-D-monoglucuronide (8; from normal rat and dogbile), whose structures have all been previously established,were used as chromatographic references (19, 32-34). Theratio of the a,,-EA (pH 2.7) over total azopigment wascalculated, thus permitting the determination of the relativeamounts of mono- and diconjugated bilirubin-IXa (seebelow, Validity of methods).The spectral purity ofthe separated azopigments was moni-

    tored by comparing the "characteristic spectra," obtained inmethanol, with those of pure reference compounds (35).Azopigment a0 was identified as azodipyrrole by TLC separa-tion into a mixture of the vinyl and isovinyl isomers ofazodipyrrole, both as the free acids and as their methylesters (19). Separation of the methyl esters allows easy dif-fere