Gut: first published as 10.1136/gut.19.6.481 on 1 June 1978. Downloaded from Gut, 1978, 19, 481-491 Twenty-five years of progress in bilirubin metabolism (1952-77) BARBARA H. BILLING From the Department of Medicine, Royal Free Hospital, Hampstead, London SUMMARY This review deals with the development of our understanding of the chemistry of bilirubin and its glucuronide derivatives during the years 1952-77. It examines the relation between haem metabolism and bilirubin formation and our present knowledge of hepatic transport of bilirubin. The heterogeneity of familial hyperbilirubinaemia is discussed. The progress made in our understanding of bilirubin 8) have significantly different physical properties so metabolism during the last 25 years is due to the that, unlike the naturally occurring lXo isomer, they combined activities of the biochemist, organic can be excreted directly into bile, without requiring chemist, mass spectroscopist, electron microscopist, modifications to their structure (Blanckaert et al., and cell biologist, as well as the hepatologist, surgeon, 1977). and haematologist. This multi-disciplinary approach With chemical treatment it is possible to cleave the has resulted in a better appreciation of the mech- unsymmetrical bilirubin lXoa molecule into pairs of anisms involved in the formation and excretion of dipyrrole methene units which then undergo random bilirubin. It is, therefore, now possible to describe in recombination to give mixtures of the isomers biochemical terms some of the abnormalities respon- bilirubin lllx, bilirubin 1Xoa, and bilirubin XIIlx sible for hyperbilirubinaemia and in some circum- (McDonagh and Assisi, 1972). Although bilirubin http://gut.bmj.com/ stances to introduce appropriate therapeutic measures IHix and bilirubin XIIIo are rarely found in nature, to modify the degree ofjaundice. it is important to appreciate that, under certain conditions, dipyrrole exchange occurs, as this Chemistry of bilirubin can influence the interpretation of data obtained when determining the structure of bile pigments Although bilirubin was isolated from bile by Stadler (Jansen, 1973; Heirwegh et al., 1975). as early as 1864 it was not until 1942 that Fischer When a solution of bilirubin is exposed to visible on September 29, 2021 by guest. Protected copyright. and Plieninger (1942) demonstrated that it had an light with wavelengths in the region of 430 to 470 nm open chain structure consisting of four pyrrole it undergoes photo-oxidation, due to the formation rings joined by three carbon bridges (Fig. 1). The of singlet oxygen (McDonagh, 1971); this results in sequence of the four methyl, two vinyl, and two the formation ofcolourless products whicharesoluble propionic side chains was identical with that found in water. A similar reaction is thought to occur in the in the proptoporphyrin IX ring of haem after re- skin of jaundiced infants during phototherapy. In moval of carbon at the ax methene bridge, so it was addition, experiments in the Gunn rat suggest that, designated bilirubin IXcx. It has now been established during treatment, bilirubin may be temporarily that the terminal pyrrole rings exist predominantly converted from the 'trans' (Z) to the 'cis' (E) form in the lactam form (Hutchinson et al., 1971). The (Bonnett et al., 1976), which is free of hydrogen linear structure of the molecule would favour a bonding and therefore capable of being excreted compound with polar properties but this is not the directly into the bile (Ostrow, 1971; Zenone et al. case at a physiological pH and an explanation for 1977). the lipid solubility of bilirubin has long been sought. It was first postulated by Fog and Jellum (1963) that Identification of bilirubin derivatives there was strong intramolecular hydrogen bonding and recently Bonnett et al. (1976), using x-ray 'INDIRECT' AND 'DIRECT' BILIRUBIN analysis, have demonstrated that the molecule is stabilised by six intramolecular hydrogen bonds With the use of the Van den Bergh reaction for the (Fig. 2). The other three bilirubin isomers (fi, y, and measurementof bilirubin (Vanden Bergh and Muller, 481 Gut: first published as 10.1136/gut.19.6.481 on 1 June 1978. Downloaded from 482 Barbara H. Billing C-OH *HOC I I CHI CH2 CH2 CHI . l I I CH$ CH CH5 CH2 CH1 CH5 CHOCH H H H H H H H BILIRU BIN Diazonium salt of ~0 ethyl anthranilate 0 C OH C-OH ~~~~~~~~~~~~~~~~~~~I CH2 CH2 CH2 CHi. 11I CH CH2 CH CH3 CH3 CH2 1tO H H H Vinyl isomer of azopigment A Iso-vinyl isomer azopigment A http://gut.bmj.com/ Fig. 1 Coupling ofbilirubin with the diazonium salt ofethyl anthranilate to form two isomeric azo pigments (A or a,,) Asterisk indicates site ofconjugate formation. The glucuronide conjugates of azopigment A have been designated azopigment B (or 8). obstructive jaundice gave a 'direct' reaction in the test. It was therefore concluded that the liver produced a change in the bilirubin molecule in the course of its passage through the organ into the bile on September 29, 2021 by guest. Protected copyright. ducts. Whether this involved alterations in the chemical structure of the molecule or was associated with differences in protein binding was not established until two bile pigment fractions were obtained free of protein and other biliary constituents from BILIRUBIN IX d obstructive jaundice serum (Cole and Lathe, 1953). Fig. 2 Structure of bilirubin (Bonnett et al., 1976) rings A nd B lie in one plane and rings Cand D in another. BILIRUBIN GLUCURONIDES Dotted lines indicate six intramolecular hydrogen bonds (two sets, each involving a carboxylic group, Using reverse phase chromatography it was shown pyrrole-amino hydrogen, and a terminal lactam system.) that 'direct' bilirubin was a mixture of two pigments, arbitrarily called pigments I and II (Cole et al., 1954). Identification of these pigments was made possible 1916) in plasma it became evident that there were at using the stable azo pigments formed in the van den least two different types of bilirubin. The bilirubin in Bergh reaction as the result of an electrophilic ion the plasma of patients with haemolytic disease attacking the bilirubin at either side of the methylene required the presence of alcohol to give a positive bridge so that two dipyrroles are produced, which reaction with diazotised sulphanilic acid and was react covalently with the diazonium salt. It was considered to give an 'indirect' reaction. In contrast, shown that, whereas 'indirect' bilirubin gave rise to bile and plasma or urine from a patient with two molecules of azo-pyrrole A, the major 'direct Gut: first published as 10.1136/gut.19.6.481 on 1 June 1978. Downloaded from Twenty-five years ofprogress in bilirubin metabolism (1952-77) 483 reacting' pigment of bile (pigment II) formed two tetra pyrroles (Noir, 1976; Heirwegh et al., 1975; molecules of a more polar azo-pyrrole B. Purification Gordon et al., 1976) into mono and diconjugates of azo-pyrrole B by chromatography (Billing et al., most structural information has had to be derived 1957) revealed that it was the glucuronide of azo- from the analysis of the dipyrrolic azo-pigments, as pyrrole A. As the result of this observation and these are more stable and therefore more suitable for similar findings by other investigators (Talefant, such analytical techniques as mass spectrometry. 1956; Schmid, 1957) it was concluded that bilirubin It has been difficult to reconcile the findings of the was converted by the liver to a diglucuronide above investigators with those of Kuenzle (1970) ester. who, using reverse phase chromatography on As pigment I yielded equimolar amounts of azo- silicone treated columns for the isolation of azo- pyrroles A and B, it was postulated (Billing et al., pigments from 15 litres of human bile and very 1957) that bilirubin monoglucuronide was also precise analytical techniques, was unable to detect formed by the liver. Its existence as a chemical the presence of bilirubin glucuronide. Instead, entity was questioned for some years, as it was Kuenzle proposed that the major bilirubin con- somewhat unstable and, moreover, a complex of jugates in man were acidic disaccharides. New bilirubin and bilirubin diglucuronide would also light has, however, recently been shed on this prob- yield equimolar amounts of azo pigments A and B in lem by the observation that in aqueous solution the diazo reaction. The matter was finally resolved above pH7 bilirubin lXcs 1-0-acyl glucuronide when it was shown that two mono-amidederivatives of rapidly isomerises to non-C-1 glucuronides by bilirubin (Jansen and Billing, 1971) could be formed sequential migration of the bilirubin acyl group from from bile, which would be possible only if two position 1 to positions 2, 3, and 4 of the sugar isomers of bilirubin monoglucuronide were moiety (Compernolle et al., 1978). These non-C present. -1 glucuronides are not attacked by /-glucuronidase. When normal bile from man and rats is collected at OTHER BILIRUBIN CONJUGATES OOC and then immediately analysed it has been found that bilirubin lXcx mono- and diglucuronides With the development of a thin layer chromato- consist exclusively of 1-0-acyl isomers, and that 8 a graphic procedure with high resolving power for the azo-dipyrrole is the major azopigment formed http://gut.bmj.com/ analysis of azo pyrrole derivatives, Heirwegh and on diazotisation. If, however, the bile is incubated associates were able to show that the bile pigment at 370C or is obtained from rats or patients with pattern was considerably more complex (Heirwegh cholestasis (Fevery et al., 1972), then relatively large et al., 1970) than had previously been appreciated amounts of fi and y azopigments, which are and that there were significant species differences derived from 2-, 3-, and 4-0-acyl glucuronides (Fevery et al., 1977). With their techniques the (Blanckaert et al., 1978), are detected, thereby azo-pigments formed in a 'direct' reaction can be indicating that sequential migration of the on September 29, 2021 by guest.