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 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 , 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 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 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 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 (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 . 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 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. Protected copyright. divided into four main groups a, ,8, y, and 8, the 8 bilirubin acyl group can occur in vivo. The bile group consisting essentially of the glucuronide used by Kuenzle was obtained after cholecystectomy moiety (Fig. 1). Asazo-pigment aocanresult only from and then stored in a deep freeze so that it is likely the monoconjugates, this technique can be used to that acyl migration could also have occurred in his assess the relative amounts ofmono and diconjugates. pigments. If this is true, then his azopigments B4, From analysis of the azo-pigments formed with dog B5, and B6 might be mixtures of 2-, 3-, and 4-0 bile it has been possible to recognise more than 20 acylglucuronides instead of disaccharides as orig- diazo positive tetra-pyrrolic bile pigments (Heirwegh inally proposed; contamination could possibly et al., 1975), whose conjugating groups are linked as account for the glucose moiety (Gordon et al., esters to the aglycone bilirubin lXo. Glucuronic 1976; Compernolle et al., 1978) acid appears to be the dominant conjugating group The ease with which the transformation of the in all the animals so far tested, apart from the 1- O-acyl glucuronide occurs probably accounts chicken, (Fevery et al., 1977); in most animals the for the fact that bilirubin glucuronide has not yet monoconjugates predominate but, in man, dicon- been synthesised, except in a form which is not jugates are usually present in greater amounts. attacked by fl-glucuronidase (Thompson and Significant concentrations of xylose and glucose Hofmann, 1970). It is to be hoped that, now that conjugates are present in dog bile (Fevery et al., there is awareness of this problem of transformation, 1971; Gordon et al., 1974) and may also be found in synthesis of both the natural bilirubin monoglucu- human bile in obstructive jaundice (Fevery et al., ronide and bilirubin diglucuronide will be possible 1972). Although thin layer chromatographic systems so that a better understanding of the factors influ- have now been developed for the separation of the encing their metabolism can be attained. Gut: first published as 10.1136/gut.19.6.481 on 1 June 1978. Downloaded from 484 Barbara H. Billing

ENZYMATIC FORMATION OF BILIRUBIN The fate of the unconjugated bilirubin formed in GLUCURONIDES the trans-glucuronidation reaction is unknown; it With the identification of 'direct' bilirubin as could either mix with the pigment formed from the bilirubin glucuronide it was quickly established that, haemoproteins or free haem, or else gain direct in common with other glucuronides, the main site of access to the microsomal membrane for recon- synthesis of bilirubin monoglucuronide is in the jugation. It certainly seems to be a rather compli- with uridine diphosphate cated system, especially as bilirubin monoglucuronide as the glucuronyl donor (Schmid et is sufficiently polar to enable it to be excreted al., 1957; Lathe and Walker, 1958). Two models directly into the bile. One looks forward to further have been proposed to explain the mechanism of information on this aspect of glucuronidation. UDP-glucuronyl transferase activation. In the 'compartmental' model, it is postulated that the UDPGA is synthesised in the cytoplasm and gains Relation between haem metabolism and bilirubin access to the enzyme, which is situated in the lumen formation of the cisternae of the microsomal membrane, as the result of the action of a permease (Berry and It has long been known that the main source of Hallinan, 1976). In contrast, in the 'conformational' bilirubin is the breakdown of haemoglobin. The model (Zakim and Vessey, 1976), it is assumed that senescent red cells are removed from the circulation activation of the enzyme involves release of the either by intravascular lysis and release of free constraint of the enzyme by the phospholipids in the haemoglobin or as intact red cells, thereby involving membrane with the breakdown of the permeability two different cell populations (Bissell et al., 1972). barrier. Probably both hypotheses contain elements The intact erythrocytes are sequestered by the of truth and may involve UDP-N-acetyl- reticuloendothelial cells in the liver or spleen while glucosamine. the circulating haemoglobin, whether haptoglobin Although bilirubin glucuronyl transferase has not bound or free, is taken up by the hepatic parenchymal been purified, it is generally accepted that there is a cells (Bissell et al., 1972). If the haptoglobin binding specific enzyme for the glucuronidation of bilirubin. capacity is exceeded, then the free haemoglobin may Bilirubin glucoside and xyloside conjugates are also be filtered by the renal glomeruli and reabsorbed formed in the endoplasmic reticulum (Fevery et al., by the epithelial cells of the proximal convoluted http://gut.bmj.com/ 1971). It has been shown in vitro that, if the constraint tubules (Bunn and Jandl, 1969); thus, in intravascular ofmicrosomal UDP glucuronyl transferase activity is haemolysis both the liver and the kidney can become released by the addition of deoxycholate or bile, important sites of bilirubin production (Pimstone et then if both UDPGA and UDP- glucose are added al., 1971). It is not known what proportion of ery- bilirubin glucosidation is increased preferentially throcyte catabolism in normal subjects involves (Bock and Remmer, 1977). It is possible that this intravascular lysis. If severe haemolysis occurs, then observation could provide an explanation for the the haem will be transported bound to a variety on September 29, 2021 by guest. Protected copyright. rise in glucose and xylose conjugate formation in of carrier proteins to form haem-haemopexin, obstructive jaundice (Fevery et al., 1972). methaemalbumin, and methaemoglobin (Muller- There is no clear evidence that the diglucuronide Erberhard and Liem, 1974). It is of interest that, is also formed in the endoplasmic reticulum and whereas oxyhaemoglobin and haem-haemopexin recently Jansen et al. (1977) presented data which enter the as an intact haem-protein indicated that the conversion of bilirubin mono- complex (Muller-Erberhard and Liem, 1974), the glucuronide to bilirubin diglucuronide may occur haem ofmethaemalbumin isseparated from itsprotein at the surface membrane of the liver cell. (2 mol moiety before its uptake by the liver (Bissell et al., bilirubin monoglucuronide -* 1 mol bilirubin di- 1972). glucuronide and 1 mol bilirubin). Support for this hypothesis comes from a recent publication by 'EARLY BILIRUBIN' Wolkoff and colleagues (1978) who found that the Studies with isotopic precursors of haem have intrahepatic pool of conjugated bilirubin was almost established that 10-20% of the circulating bilirubin exclusively bilirubin monoglucuronide, which comes from haem compounds which have a short accumulated as a ligand bound to several gluta- half life. This so-called 'early labelled bilirubin' has thione-S-transferases, including ligandin. Their two major components (Yamamoto et al., 1965), one failure to find the diglucuronide can be understood if of which is erythropoietic in origin and is probably one accepts that its formation occurs at the cana- formed in the bone marrow from reticulocytes and licular membrane in a site or configuration which normoblasts which fail to reach maturation; it is facilitates its immediate biliary excretion. significantly increased in disease associated with 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) 485 ineffective erythropoiesis (Israels, 1970). The presence been postulated that the apoprotein of this cyto- of the non-erythropoietic component can be demon- chrome, which is located in the membrane of the strated by the administration of 8-amino- endoplasmic reticulum, may serve as the binding aevulinic acid (ALA), as it labels hepatic haem protein for haem (Bissell, 1975; Schmid and almost exclusively and only penetrates erythropoietic McDonagh, 1975; O'Carra and Colleran, 1976). The cells to a limited extent (Robinson, 1972). It is NADPH and the microsomal electron transport generally considered that at least two haemoproteins system are then used to reduce the iron of the are involved, one of which is associated with cyto- bound haem, and to generate a reactive oxygen ,chrome p450, which has a half life of eight hours, radical which attacks the haem at its o-bridge to while the other unknown compound has an estimated form a-oxyhaem (Jackson, 1974). The inter- halflife ofless than one hour and could be free haem. mediates formed between oxyhaem and Recent studies (Berk et al., 1976) indicate that in are not known but the process certainly involves loss normal individuals 'early bilirubin' synthesis, assessed of the x-meso carbon atom as CO as well as by determining the difference between plasma oxidation at the two adjacent carbon atoms. bilirubin turnover and bilirubin derived from red Experiments with 018 labelling suggest that these blood cell degradation, constitutes approximately terminal lactam atoms are derived from different 25% of the total bilirubin turnover. There appears oxygen molecules (Brown and King, 1978) (Fig. 3). to be a constant hepatic component ofapproximately 22% so that the contribution from ineffective erythropoeisis is normally very small. HAEM o(-OXYHAEM MECHANISM OF BILIRUBIN FORMATION 0 The classical studies of Lemberg and Legge (1949) had shown that the degradation ofhaem compounds to bile pigments can be achieved in vitro by treatment M4 ,V with oxygen in the presence of a mild reducing agent such as ascorbic acid andit has beenpostulated that coupled oxidation might also occur in vivo. The observation that when the reaction is performed M' M http://gut.bmj.com/ with haemin (O'Carra and Colleran, (1969), a mixture of four biliverdin isomers is produced would argue P P P P against this hypothesis. However, if the haem is boundto asuitableapoprotein(O'Carraand Colleran, 1970), stereospecificity is conferred on the reaction and biliverdin lXox is formed. There has been much controversy as to whether haem catabolism is an V on September 29, 2021 by guest. Protected copyright. enzymatic ornon-enzymatic process(Grayetal., 1972) and it would now seem that the two mechanisms BILIVERDIN are not necessarily mutually exclusive (Schmid and M McDonagh, 1975, O'Carra and Colleran, 1976). Of the several enzyme systems which convert haem to bile pigments, it is the microsomal haem oxygenase P P Fig. 3 Pathway of haem catabolism (Brown et al., of Tenhunen et al. (1969) which has received most 1978) (M,-CH3: V,-CH= CH2:P,CH2CH2H). attention, as its specific activity is highest in the spleen, bone-marrow, and liver where haem degradation is known to occur. The microsomal The biliverdin-iron complex is subsequently haem oxygenase system has an absolute and hydrolysed and reduced to bilirubin IXox by stoichimetric requirement for NADPH and requires biliverdin reductase, which is found in most mam- 3 mol of oxygen for the conversion of methaem- maliantissues and is specific for the natural a isomer. albumin, and other haem compounds, to equimolar In birds, amphibians, and reptiles biliverdin is amounts of bilirubin lXox and CO. Haemoglobin excreted directly into bile. As biliverdin is a harmless is not a substrate in this reaction, so it has been compound, it is difficult to appreciate why in concluded that in vivo haem is separated from globin mammals the conversion to bilirubin is necessary and transferred to another protein before degra- since bilirubin is a highly toxic and insoluble com- dation occurs. There is now good evidence that pound, which requires a specific transport system in cytochrome p450 is not directly involved and it has the liver (Colleran and O'Carra, 1977). A possible Gut: first published as 10.1136/gut.19.6.481 on 1 June 1978. Downloaded from 486 486Barbara H. Billing explanation is that biliverdin does not cross the between the plasma membrane and the endoplasmic placenta and can be removed from the mammalian reticulum. Meuwissen and his colleagues (1977) foetus only iffirst converted to bilirubin (Palma et al., suggest that the binding proteins act as a 'passive 1977). rotary carrier system in an aqueous barrier' and that the metabolic rate of bilirubin is regulated by the concentration of these carriers. On the other hand, Hepatic transport of bilirubin others (Wolkoff et al., 1977) consider that ligandin acts as a determinant of net hepatic uptake of UPTAKE bilirubin by controlling its efflux from the liver into The bilirubin, which is derived from senescent the plasma. erythrocytes sequestered in phagocytic cells, is transported in the plasma tightly bound to albumin COMPARTMENTATION (see future research p. 487) while approximately 10% may be bound to red cells (Barnhart and Clarenberg, 1973). Compounds, SECRETION which compete with bilirubin for common binding Unless conjugation is defective, the rate-limiting sites on the albumin molecule, such as fatty acids and factor in the transport of bilirubin from plasma to a large number of organic anions, drugs, and anti- bile appears to be the biliary excretion of bilirubin biotics, will facilitate the diffusion of bilirubin, in the glucuronide, as the uptake process is normally not unbound form, into the liver and other tissues by saturated. Secretion of the conjugated pigments non-ionic diffusion (Brodersen, 1974). The adminis- probably involves a carrier-mediated active transport tration of drugs such as sulphonamides and sal- system which is shared by many other organic anions. icylates to neonates may therefore increase the risk Maximal bilirubin excretion can be enhanced by of kernicterus, as their blood-brain barrier is more taurocholate in common with other substances permeable to 'unbound' bilirubin than that of the excreted in bile at high concentrations relative adult (Diamond and Schmid, 1966). to plasma (Goresky et al., 1974). A high proportion The process of hepatic uptake follows detachment of the conjugated bilirubin formed is incorporated from albumin at the sinusoidal plasma membrane; into mixed micelles (Goresky et al., 1974; Goresky, it appears to be carrier-mediated and is shared by a 1975; Scharschmidt and Schmid, 1977). Goresky variety of organic anions, excluding bile acids has postulated that the bile salts form a 'miceller http://gut.bmj.com/ (Goresky, 1975; Scharschmidt et al., 1975; sink' in the bile canaliculus which removes the Paumgartner and Reichen, 1976). The fluxofbilirubin conjugated bilirubin from the non-miceller phase so across the plasma membrane is bidirectional and that a net concentration gradient is formed which in congenital non-haemolytic jaundice the reflux enhances the flux of pigment from the liver cell into of the bilirubin taken up by the hepatocyte into the the bile. The role of the Golgi apparatus and micro- plasma may be significantly increased (Billing et filaments in the intrahepatic transport of conjugated al., 1964; Berk et al., 1970). It has been well bilirubin remains to be defined. on September 29, 2021 by guest. Protected copyright. established that there are several non-specific binding proteins (aryl-glutathione transferases) ELIMINATION OF BILE PIGMENTS in the hepatic cytosol which bind bilirubin Conjugated bilirubin is not significantly reabsorbed reversibly-for example, Y and Z protein (Levi in the intestine. In the terminal ileum and large et al., 1969). There has been considerable debate as intestine it is hydrolysed by bacterial ,B glucuronidase to whether these proteins, and in particular Y and reduced to a complex mixture of , protein (ligandin), play an essential role in the uptake their structure depending on the gut flora (Watson, and transport of bilirubin or merely serve to 1969). They are then excreted in the faeces as increase the solubility of the pigment in the cell so oxidised urobilins, the measurement of which does that it can be stored in a non-toxic form (Fleischner not give a valid assessment of bilirubin production and Aras, 1976). The major problem has been that (Bloomer et al., 1970). A small fraction of the in vitro, plasma albumin appears to have a greater urobilinogens is reabsorbed and then excreted in the affinity for 'unbound' bilirubin than purified ligandin, bile; in the presence of excess bilirubin production which may be due to technical artefacts. However, or renal excretion of may if the cytosol is submitted to moving boundary sedi- be observed (Bourke et al., 1965). mentation then the high bilirubin binding affinity of Only in cholestasis, when the pigment accumulat- the ligandin can be maintained (Meuwissen et al., ing in plasma is almost entirely bilirubin glucuronide, 1977). Using data obtained with this technique it are bile pigments excreted in the urine. The 'unbound' has been claimed that the binding proteins are conjugated bilirubin is freely filtered by the glomeruli concerned with the facilitated diffusion of bilirubin and then largely reabsorbed in the proximal tubules 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) 487

(Gollan et al., 1978). Studies in an isolated kidney baum and colleagues (1976) in a patient with preparation support the view that, in contrast to the Gilbert's syndrome it has been postulated that two liver, conjugated bilirubin and bile acids are independent hepatic bilirubin pools are involved; reabsorbed by a similar mechanism (Barnes et al., on the basis of their size and kinetics these have been 1967) so that bilirubin glucuronide may inhibit the tentatively assigned to cytochrome P450, and reabsorption of bile acids. cytochrome b5 together with other mitochondrial cytochromes. As the haem of haemoproteins is bound to the microsomal membrane it could be Some areas for future research in bilirubin metabolism cleaved and converted to conjugated bilirubin in situ and then transported into the bile by the tubular Considerable progress has been made in the last 25 system of the endoplasmic reticulum without entering years in our understanding of bilirubin metabolism the cytosol. A third very rapidly turning over bili- at a molecular level. An enzymatic mechanism for rubin pool has also been postulated which probably the formation of biliverdin has been described, but represents pigment derived from newly formed haem to what extent it plays a physiological role has still in transit from its site of synthesis in the mito- to be assessed. It is not known whether all haem chondria. The pigment from this pool is not excreted proteins are catabolised in the same way or, indeed, into bile but first refluxes so that it mixes with the whether a proportion of the haem liberated from plasma bilirubin pool. The bilirubin formed when senescent red cells gets reutilised. There is indirect biliverdin is administered intravenously appears to evidence that bilirubin may not necessarily be the reflux in a similar way (Gollan et al., 1977). It only end product of haem catabolism (Landaw et al., therefore seems possible that elimination of bilirubin 1970). Also, in the Gunn rat and patients with the from the plasma compartment occurs by hepatic Crigler-Najjar syndrome, who have an absolute channels which do not communicate with those deficiency of bilirubin glucuronyl transferase activity, responsible for endogenously formed pigment. it is obvious that there must be an alternative route Experimental studies are now required to provide for the disposal of bilirubin which does not involve direct support for these hypotheses relating to glucuronidation (Schmid and Hammaker, 1963). compartmentalisation in the liver. The role of intracellular compartmentation in the The exact role of the plasma membranes and http://gut.bmj.com/ regulation of haem and bilirubin metabolism is of ligandin in the uptake of both unconjugated and increasing interest to many investigators. It is conjugated bilirubin still requires to be defined so known that the sinusoidal cells are able to sequester that a better understanding of what is meant by and degrade intact erythrocytes (Bissell et al., 1972) the term 'hepatic clearance' can be obtained. There but their functional relationship with the paren- is also uncertainty about the mechanism and site of chymal cells is uncertain. Moreover, there is good concentration of bilirubin before its excretion in bile evidence that the parenchymal cells are not homo- and further information is needed about the factors geneous and may undergo differentiation on a controlling the formation of bilirubin diglucuronide. on September 29, 2021 by guest. Protected copyright. lobular basis so that the centrilobular and periportal Although it has been known for 20 years that cells may have different functions (Drochmans et al., bilirubin is mainly excreted as mono- and diglucuro- 1975; Jones et al., 1976). According to the lobular nides, a precise and quantitative method for their gradient hypothesis of Goresky (1975), there is a determination has not been devised. The most concentration gradient for bile acids from the portal promising approach would appear to be that of high to the central zones in each lobule. This would pressure liquid chromatography but the instability suggest that the bilirubin excretion related to bile of the pigments may present technical problems. acid dependent flow is greatest in the centrilobular Meanwhile, separation of the pigments by thin zone. layer chromatography has to be used for qualitative Comparisons of bilirubin production obtained by purposes and one of the many variations of the Van measuring the rate of disappearance from the circu- den Bergh reaction for the semi-quantitative measure- lation of a tracer dose of radioactive bilirubin with ment of unconjugated and conjugated bilirubin. CO production have shown that plasma bilirubin turnover is less than total bilirubin production Familial hyperbilirubinaemia (Landaw et al., 1970). This observation, together with studies by Jones and collegues (1972) on the It has become increasingly apparent that jaundice is measurement of 'early bilirubin' production in man, rarely associated with a single abnormality in suggested that a fraction of the bilirubin which bilirubin metabolism even in familial hyperbili- entered the bile directly was non-haemoglobin in rubinaemia. Nevertheless, there is usually a domi- origin. From the computer-derived data of Kirshen- nant defect which is responsible for the patient Gut: first published as 10.1136/gut.19.6.481 on 1 June 1978. Downloaded from 488 Barbara H. Billing becoming icteric. ation is enzyme replacement; this has been attempted The capacity of the liver to eliminate bilirubin in Gunn rats with the transplantation of is greatly in excess of normal total haem catab- (Matas et al., 1976) and tissue grafting (Foliot et olism, so that haemolysis rarely produces gross al., 1975) but would present considerable problems jaundice unless there are concomitant defects in in man. biliary and urinary excretion (Fulop et al., 1971). The hyperbilirubinaemia of the Dubin-Johnson Mild defects in glucuronyl transferase activity have and Rotor syndromes is essentially of the conjugated been documented in patients with chronic haemolytic type. Excretory defects for several organic anions, disease (Auclair et al., 1976) but it is unlikely that but not for bile acids, have been demonstrated and this would be sufficient to cause jaundice if it were for some time it was accepted that the two syndromes not for the marked increase in bilirubin production. were variants of the same condition (Aras, 1961), Similarly, the shortened erythrocyte life span seen the only significant difference being that, in the in 50% of patients with Gilbert's syndrome (Powell Dubin-Johnson syndrome, the liver appeared to be et al., 1967; Berk and Blaschke, 1972) would not be black due to retention of a melanin-like substance. sufficient to cause jaundice if it were not for a Recent studies have, however, revealed further significant reduction in their ability to synthesise differences. The abnormality causing BSP retention bilirubin monoglucuronide. in the appears to be related to a Analysis of the bile of patients with Gilbert's defect in hepatic uptake rather than secretion as syndrome and the Crigler-Najjar syndrome has originally demonstrated in the Dubin-Johnson shown that, unlike that found in the normal syndrome (Abe and Okuda, 1975). Both syndromes subject, bilirubin monoglucuronide is the dominant have abnormalities in urinary coproporphyrin pigment (Fevery et al., 1977; Gordon et al., 1977), excretion (Wolkoff et al., 1976); in the Dubin- which suggests that there may be a second enzyme Johnson syndrome the totalcoproporphyrin excretion defect relating to the conversion of monoglucuronide is slightly reduced or normal, 90%Y being in the to diglucuronide. The rate of disappearance of a form of isomer I, whereas in the Rotor syndrome the tracer dose of radioactive bilirubin from the plasma total coproporphyrin is greater than normal but the is always significantly reduced in patients with percentage of coproporphyrin I is only about 60%

unconjugated hyperbilirubinaemia (Berk et al., 1970; of the total. It is not yet understood whether there is http://gut.bmj.com/ Black et al., 1974) and compartmental analysis has any relation between canalicular secretion and por- indicated that this is due to a defect in hepatic phyrin metabolism or if they are just two indepen- clearance. As the concentration of ligandin in these dent abnormalities in these syndromes. patients appears to be normal, the possibility exists that there are also abnormalities at the level of the The period under review coincides with that spent by plasma membrane. Further evidence of the hetero- the author as a colleague of Sheila Sherlock working of this condition is apparent from the obser- geneity on bilirubin metabolism; her constant encouragement on September 29, 2021 by guest. Protected copyright. vation that a small proportion of patients have a and support is gratefully acknowledged. mild impairment in their ability to eliminate a standard dose of BSP (Berk et al., 1972). A reduced clearance of tolbutamide (Carulli et al., 1976), which References does not undergo glucuronidation, has also been re- ported. The multiplicity of these abnormalities Abe, H., and Okuda, K. (1975). Biliary excretion of con- jugated sulfobromophthalein (BSP) in constitutional would argue against the suggestion that Gilbert's conjugated hyperbilirubinaemias. Digestion, 13, 272-283. syndrome is not a real disease (Bailey et al., 1977) Arias, I. M. (1961). Studies of chronic familial non-hemolytic and that the hyperbilirubinaemia merely constitutes jaundice with conjugated bilirubin in the serum with and the upper end of the normal range. without an unidentified pigment in the liver cells. American a Journal of Medicine, 31, 510-518. 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