Heterogeneity ofBilePigmentConjugates asRevealed by Chromatography oftheir EthylAnthranilate Azopigments

1.Azopigments derived fromconjugated bilepigments bycoupling withthe diazonium salt ofethyl anthranilate areanalysed conveniently byquantitative t.l.c. orbycolumnchromatography onCM-cellulose. 2.Bychromatographic studies combined withaseries ofchemical tests sixgroups ofazopigments weredemonstrable in preparations frombile andfromicteric urine ofman. Azobilirubin anditsf-Dmonoglucuronide havehitherto beenconsidered tobetheonlymajorderivatives thatcanbeobtained fromhumanbile pigments. Inthepresent work,other azopigments accounted for30-40%ofthetotal azopigment material, andthe amountsoftheseshowedconsiderable variation amongbiological fluids. 3.The divergence ofthepresent results fromearlier workisprobably related totheuseof milder diazotization conditions andofchromatographic techniques withahigh resolvingpower. 4.Thethin-layer chromatographic systems developed allow rapid andquantitative analysis ofazopigments derived frombile pigments. Workwithbilirubin anditsconjugates isnotoriously difficult. Byusing thereverse-phase column chromatographic systems ofCole, Lathe& Billing (1954) andofBilling (1955) theso-called pigment-I fraction hasbeenfoundtochangeconsiderably upon rechromatography (Gregory, 1963;Weber, Schalm& Witmans,1963), although thisobservationcouldnotbeconfirmed by Schoenfield & Bollman(1963). Thepigmentsaresensitivetooxidationwhenilluminated. Further, different mixtures ofazopigments may result whethercoupling with p-diazobenzenesulphonic acidatacidpH proceeds inthepresenceorintheabsence ofethanol (Pittera & Cassia, 1963). Thedifficulties involved are also reflected inthecontinuing efforts toprepare pure conjugated bilirubin (Lucassen, 1961;Jacobsen, 1969;Ostrow& Murphy,1970), andintheconflicting dataabouttheexistence ofbilirubin monoglucuronide (Noir, GarayR Heirwegh, 1968;Ostrow& Murphy,1970)andofbilirubin sulphate (With, 1968). Finally, dataon thenature ofconjugated bilirubin reviewed byWith(1968) are difficult toexplain inthelight ofcurrent conceptsandsuggest a greater degree ofheterogeneity thanhitherto suspected. Inviewoftheinstability andprobable complexity ofbile pigments (Tenhunen, 1965) itseemed preferable to characterize them as theirstable azopigment derivatives. Mostfrequently a diazoreagentcontaining p-diazobenzenesulphonic acid