Postgrad Med J: first published as 10.1136/pgmj.39.450.176 on 1 April 1963. Downloaded from POSTGRAD. MED. J. (1963), 39, 176

BILIRUBIN METABOLISM BARBARA H. BILLING, Ph.D. Department of Medicine, Royal Free Hospital, London, W.C.I

DURING the last ten years considerable advances have been made in our knowledge of the metabo- lism of bilirubin and thus in our understanding of the basic defects underlying jaundice, partic- ularly in the newborn and in some types of familial hyperbilirubinaemia. These advances are due mainly to the characterization of 'direct' bilirubin as an ester glucuronide of bilirubin. S~. -006 Many of the classical concepts of bilirubin meta- I:/ i\ -°I bolism can therefore now be explained in chemical 0a2 4 -004 terms. ~0.I ' x.' · - 0.02 An exciting new development has been the 4. production of radioactive bilirubin (Ostrow, Ham- 20 40 60 60 100 120 140 160 1) maker and Schmid, I96I; Grodsky, Carbone, Time (days) Fanska and Peng, 1962); this tool has enabled the FIG. I.-15N contents of haem in samples (full line) and existence of an of stercobilin (dotted line) at various times after entero-hepatic circulation for bili- administration of glycine (I2 g.) containing rubin to be demonstrated and will doubtless help 31.65% excess 15N to a normal subject (Gray andcopyright. unravel many ofthe unsolved problems of bilirubin others, I950). metabolism. Formation of bilirubin subject the first peak is responsible for approxi- Most of the circulating bilirubin results from mately Io% of the pigment excreted but in the catabolism of haemoglobin in the reticulo- diseases such as pernicious anemia, congenital endothelial system, particularly in the bone porphyria and thalassaemia 40-80% of the bile marrow, spleen and liver. The exact pathway of pigment excreted may be due to this fraction. haemoglobin breakdown first to biliverdin and It was originally thought that this 'early sterco- http://pmj.bmj.com/ then, after reduction, to bilirubin is still not bilin' arose by direct synthesis from simple clear. It is known that the administration of both precursors such as aminolaevulic acid or porpho- hamatin and protoporphyrin results in the bilinogen. It is now considered, however, to be formation of bilirubin but there is no conclusive mainly erythrohaemopoietic in origin since this evidence that either of these compounds is early incorporation of labelled glycine into sterco- involved in the normal catabolism of haemoglobin. bilin is increased when erythrohaemopoiesis is In vitro studies have that stimulated. This suggested haemoglobin erythrohaemopoietic stercobilin on October 2, 2021 by guest. Protected may be degraded to bilirubin via choleglobin but may be derived from three sources, namely, the its formation has not been demonstrated in vivo. immature red cells in the bone marrow (which are The studies of Gray, Neuberger and Sneath degrading haemoglobin as well as synthesising it), (I950), together with those of other investigators, from haem formed in excess of globin or from have established that not all the bile pigment haem formed by the destruction of newly formed formed results from the breakdown of the red red cells as soon as they reach the peripheral cells at the end of their life span. They used circulation. A very small amount of bilirubin could faecal stercobilin as a measure of bile pigment theoretically come from haem pigments such as production and after the administration of 15N myoglobin or the cytochromes but there is no glycine determined its specific activity at suitable information regarding the role played by these time intervals (Fig. i). The 15N stercobilin time- proteins in bile pigment formation. The possibility curve had two peaks; the first occurred immediately of an hepatic origin of the early labelled sterco- following the glycine feeding while the second had bilin has been suggested by the work of Watson- a maximum about the I3oth day when the labelled James and Abbott (I961) who found an increase in red cells were breaking down. In the normal a patient with aplastic anaemia. Postgrad Med J: first published as 10.1136/pgmj.39.450.176 on 1 April 1963. Downloaded from April 1963 BILLING: Bilirubin Metabolism I77 Bilirubin is a lipid-soluble, non-polar pigment BILE PIGMENTS AZO PIGMENTS which gives an 'indirect' reaction in the presence of alcohol in the van den Bergh test. Prior to excretion in the bile, it is converted into a pigment I-- ILIRUBINb which is water-soluble at a physiological pH and gives a 'direct' van den Bergh reaction with diazo- PIGMENT I * n tised sulphanilic acid. Using chromotographic techniques Cole and Lathe (1953) were able to show that the properties of 'direct' bilirubin were not due to a different type of protein attachment but to an alteration in the chemical structure of the pH 6 pH pH pigment. By means of partition chromatography CHCtl3-CCt -CH3, OH n-BUTANOL n-BUTANOL (Cole, Lathe and Billing, 1954) (Fig. 2) or paper FIG. 2.-Behaviour of bile pigments and their diazo- chromatography (Giovanneti, Maggiore and products on reverse phase partition chromatograms. Vivaldi, I96I) it is possible to demonstrate that (Nosslin, I960). 'direct' bilirubin consists of two components (pig- ments I and II). The most polar pigment (pigment II) is the main pigment present in bile and together with an intermediate pigment (pigment I) is found COOR, COOR, in the icteric urine and sera of patients with I I obstructive jaundice, hepatitis and cirrhosis. The CH, CH2 extreme instability of these pigments has so far I I prevented their isolation in a pure form. The Me V Me CH, CH2 M Me V structure of pigment II has therefore been established by characterizing the azo-pigment formed in the van den this Bergh reaction; HNJHAHN H N pigment (azo-pigment B) is considerably more H H H H copyright. polar than azo-pigment A and can be separated FIG. 3.-Structure of bile pigments (bilirubin, RI and from it and other contaminants in diazotised bile R2 = H; Pigment I, R1 = glucuronyl and R2 = H; by partition chromatography (Fig. 2), counter- pigment II, RI and R2 = glucuronyl; Me = methyl current distribution (Billing, Cole and Lathe, and V = vinyl). 1957) and paper chromatography (Schmid, I957). The application of the latter two techniques has shown azo-pigment B to be the ester glucuronide curonide, and further investigations are now of azo-pigment A. Since two molecules of azo- needed to determine the factors involved in its. pigment A are known to be formed from one formation. http://pmj.bmj.com/ molecule of bilirubin it has been concluded that It is generally accepted that over 80% of the pigment II behaves similarly and is therefore an bilirubin in bile is normally present as the glucuro-- ester diglucuronide of bilirubin, the glucuronic nide. Examination of chromatograms and the acid radicals being linked to the carboxyl groups observation that mild alkali treatment does not of the proprionic substituents of the molecule as convert all the 'direct' bilirubin in icteric serum indicated in Fig. 3. A similar conclusion was or bile into bilirubin indicate that other con- reached by Talafant (1956) as the result of experi- jugates may be present. Isselbacher and McCarthy on October 2, 2021 by guest. Protected ments involving the electrophoresis of bile. (I959) by means of 35SO4 tracer studies have- The structure of pigment I has been a more demonstrated the presence of sulphate conjugates controversial matter. Since both azo-pigment A and of bilirubin in the bile of rats, cats and humans. azo-pigment B are formed from pigment I in the These findings have been confirmed in the rat by van den Bergh reaction it seemed likely that the Schoenfield, Bollman and Hoffman (1962) who pigment is the monoglucuronide of bilirubin. demonstrated that the sulphate conjugate of However, if pigment I is eluted from a chromato- bilirubin is associated with pigment II but not gram and then rechromatographed it has been with pigment I. Gregory and Watson (I962) observed that both bilirubin and pigment II have not, however, been able to demonstrate the appear on a second chromatogram (Billing, Cole presence of bilirubin sulphate in the bile of dogs or and Lathe, 1957; Nosslin, 1960). This observation human subjects and question the assumption of together with other studies (Gregory, 1962; Isselbacher and McCarthy that alkali-stable Weber, Schalm and Witmans, I963) gives support 'direct' bilirubin is identical with bilirubin to the hypothesis that pigment I is a labile equimo- sulphate. At present there is no evidence that lecular complex of bilirubin and bilirubin diglu- defective conjugation of bilirubin as a glucuronide Postgrad Med J: first published as 10.1136/pgmj.39.450.176 on 1 April 1963. Downloaded from 178 POSTGRADUATE MEDICAL JOURNAL April I963 in man can be compensated for by an appreciable ATP GLUCOSE increase in the excretion of bilirubin as a sulphate or some other conjugate. Hexokinast Biosynthesis of Bilirubin Glucuronide ADP J GLUCOSE-6-PHOSPHATE It has been demonstrated that uridine diphosphate glucuronic acid (UDPGA), and not Phosphoglucomutase glucuronic acid, is the glucuronyl donor necessary for the formation of bilirubin glucuronide (Fig. 4). The conjugating enzyme is a glucuronyl trans- GLUCOSE-I-PHOSPHATE \ UTP ferase (UDP-trans-glucuronylase) which is located YV ADP in the microsomes; the evidence regarding the PP-Uridyl Tronsferase specificity of this enzyme is equivocal. Both the DPN+ NPP kidney and gastrointestinal tract as well as the URIDINE DIPHOSPHATE liver contain an enzyme system capable of con- GLUCOSE jugating bilirubin. In the intact adult animal no UDPG Dehydrogenase satisfactory evidence has, however, been presented / JRIDINE DIPHOSPHATE BILIRUBIN to indicate that conjugation of bilirubin takes GLUCURONIC ACID place in any organ other than the liver. Hepa- DPNH V I tectomy in experimental animals results in the Glucuronyl Transfcrase gradual accumulation of both bilirubin and a " TP pigment which gives a 'direct' van den Bergh BILIRUBIN GLUCURONIDE \UDP reaction and behaves chromatographically like FIG. 4.-A possible mechanism for the conjugation of pigment I. This pigment has been identified in bilirubin with glucuronic acid (Billing and Lathe, the dog as bilirubin monoglucuronide (Schoen- I958). field, Grindley, Foulk and Bollman, 1961) but the

site of its formation under these abnormal condi- copyright. tions has not been established. for bilirubin in man has been confirmed using The ability of the foetal and newborn liver to both bilirubin labelled with 14C (Lester and form bilirubin glucuronide is markedly reduced, Schmid, I962) and bilirubin labelled with 15N due mainly to a deficiency of glucuronyl transferase (Gilbertsen, Bossenmaier and Cardinal, 1962). activity rather than UDPGA. In the rat foetus The significance of the enterohepatic circulation the glucuronyl trransferase activity of the gastric in normal man remains to be investigated. It may mucosa is twice that of the adult liver (Stevenson provide one of the reasons for the lack of corre- and Dutton, I962). The possibility that the lation between the amount of pigment known to gastric mucosa might be an important means be formed from the breakdown of circulating red http://pmj.bmj.com/ of removing bilirubin in the fcetus has cells and that found as stercobilin in the faeces. In been considered but seems unlikely in the patients with a compensated hyperbilirubinaemia light of recent studies using 14C bilirubin in due to a deficiency of glucuronyl transferase the guinea-pig foetus. (Schenker, Dawber and (Crigler-Najjar syndrome) and a homozygous ;Schmid, 1962) and the monkey fcetus (Lester, strain of rats (Gunn rats) with the same deficiency, Behrman and Lucey, I962), which showed that Schmid and Hammaker (I962) have found that fcetal bilirubin can be after bilirubin can be efficiently removed, unconjugated both absorbed by on October 2, 2021 by guest. Protected crossing the placenta, in the maternal bile. the intestine and transferred from the plasma pool directly across the mucosa into the intestinal Enterohepatic Circulation of Bilirubin lumen. In the Gunn rats they were able to prevent The experiments of Lester, Ostrow and its reabsorption by feeding cholestyramine resin, Schmid (I961) in which 14C bilirubin was adminis- which has a high affinity for bilirubin, and in this tered intraduodenally to rats with bile fistulae have way significantly reduced the serum bilirubin clearly demonstrated that in the rat, bilirubin is levels to 30 to 45% of the control values (Lester, absorbed from the intestine and then reappears Hammaker and Schmid, I962). The value of in the bile. The absorption of unconjugated cholestyramine treatment in reducing jaundice in bilirubin appeared to be more rapid and quanti- man remains to be assessed. tativelygreater than that of conjugated bilirubin. It seems likely that conjugated bilirubin is first Conversion of Bilirubin to Urobilinogen and hydrolyzed to bilirubin and then absorbed as free Mesobilifuscin bilirubin. It has been well established that bilirubin, The existence of an enterohepatic circulation especially if in the conjugated form, is reduced Postgrad Med J: first published as 10.1136/pgmj.39.450.176 on 1 April 1963. Downloaded from April x963 BILLING: Bilirubin Metabolism I79 by the bacterial flora in the intestine and colon to dialysis have been for the most part unsuccessful. the urobilinogen group of pigments, which give If however albumin is added to the dialysis the well-known Erlich's aldehyde reaction. These fluid then it is possible for considerable quantities urobilinogens are readily dehydrogenated to give of bilirubin to be removed by intermittent urobilin IXa, stercobilin and d-urobilin, the peritoneal dialysis (Grollman and Odell, I962). relative amounts formed depending on the Conjugated bilirubin is also bound to the mobility of the intestinal contents and the rate of plasma albumin but the type of pigment-protein- filling of the colon, as well as the efficiency of the complex formed is probably different (Klatskin bacterial flora. Watson and Weimer (1959) were and Bungards, I956). It may also form a complex unable to correlate the composition of the faecal with phospholipids which can be extracted by urobilin with the patient's condition and while shaking with ether (Charbonnier and Poungouras, urobilin IXa was the dominant pigment in some 1959; Lucassen, I96I). This 'ether-soluble subjects, stercobilin predominated in others. The bilirubin' extraction test has been used to administration of broad-spectrum antibiotics may differentiate between malignant and non- result in bilirubin rather than stercobilin being maligant biliary obstruction but according to excreted in the faeces. Mertens and Croal (I960) is probably merely a The presence of an enterohepatic circulation measure of the degree of obstruction, as indicated for urobilinogen was first postulated by McMaster by the high phospholipid level. and Elman (I927). They assumed that in liver Small doses of bilirubin are quickly cleared disease re-excretion of urobilinogen into the bile from the plasma by the liver and then after was impaired and that instead, the pigment passed storage and concentration are excreted into the to the kidney and was excreted in the urine. bile at a slower rate. In the normal subject serum Urobilinoid pigments are, however, not normally levels greater than i mg/ioo ml are rarely encoun- found in bile except in the presence of infection; tered and the bilirubin is almost entirely in the if McMaster and Elman's hypothesis is correct non-conjugated form. With raised levels of this means that the pigment would have to be serum bile pigments, bilirubin will be stored in reconverted to bilirubin in the liver before it most of the tissues in the body, including thecopyright. could be excreted in the bile which, for bio- adipose tissue. It has been suggested that the chemical and structural reasons, seems unlikely. staining of the tissues of the skin and muscle may A critical re-examination of this problem with be associated with their extravascular albumin labelled stercobilin of high specific activity is content (Billing and Lathe, 1958). The differences required to establish whether urobilinogen, like in skin colour observed in infants with hemolytic bilirubin, after absorption from the intestine is jaundice and adults with obstructive jaundice re-excreted in the bile. Recent experiments by could be due to differences in tissue binding of Kahan, and Varro have shown that bilirubin and bilirubin but these

Csernay (I962) conjugated http://pmj.bmj.com/ the reabsorption of stercobilin from the isolated matters need to be investigated further. small intestine of the dog is dependent on the Both unconjugated and conjugated bilirubin formation of a mucoprotein complex and suggest may be found in the lymph, ascitic fluid, pleural that the kidney may play some part in the con- fluid and cerebrospinal fluid of the jaundiced version of urobilin to its respective urobilinogen subject. No correlation has been found between prior to excretion in the urine. the bile pigment levels in the CSF and those in It has been suggested that the discrepancies the plasma. observed between urobilinogen excretion and Bilirubin cannot usually be detected in the on October 2, 2021 by guest. Protected haemoglobin catabolism might be accounted for urine of normal subjects, or patients with an by the conversion of bilirubin or urobilinogen unconjugated hyperbilirubinamia. In icteric to dipyrrylmethenes such as mesobilifuscin. urine both pigments I and II but not bilirubin are Studies by Gilbertsen and Watson (I962) sup- found. The level of conjugated bilirubin in the port the view, however, that the naturally urine does not correlate well with that in the occurring fecal dipyrrylmethene pigments are plasma and depends on the state of the disease anabolic rather than catabolic in origin. process. In viral hepatitis, for example, bile pigments may appear in the urine even before the Bile Pigments in Plasma, Urine and Body patient becomes clinically icteric but during Fluids convalescence no pigment will be found in the At the serum bilirubin levels encountered in urine although the serum level may be as high as icteric plasma, regardless of the cause of the 6-8 mg/ioo ml. Lack of pure specimens of jaundice, virtually all the pigment is bound to conjugated bilirubin and suitable techniques for albumin (Ostrow and Schmid, I962). It is for the quantitative determination of bile pigments this reason that attempts to reduce jaundice by in urine has prevented satisfactory renal clearance Postgrad Med J: first published as 10.1136/pgmj.39.450.176 on 1 April 1963. Downloaded from i8o POSTGRADUATE MEDICAL JOURNAL April 1963 studies being carried out. It would appear that in the dog the pigments are excreted by the renal tubules, but there is no reliable evidence in man on this matter. Toxicity ofBilirubin and Kernicterus The yellow pigment that accumulates in the brain in kernicterus has been identified as uncon- jugated bilirubin. The toxic effects of bilirubin have therefore been studied and it has been shown that bilirubin has an inhibitory action on the oxygen consumption of brain tissue, particularly in the newborn. It has also been demonstrated s s that bilirubin is an inhibitor of haem synthesis and S / NL U S O I D that in rat liver and brain mitochondria it FIG. 5.-Jaundice could theoretically result from (I) an uncouples oxidative phosphorylation. According increased load of bilirubin; (2) defective uptake to Ernster (I96I) bilirubin behaves like a detergent and transport within the liver cell; (3) defective in its action on mitochondria and conjugation in the hepatic microsomes; (4) defec- causes swelling tive canalicular excretion or a mechanical block in as well as inducing ATPase activity. With high the bile duct system. (S. Sherlock (1962), Brit. concentrations of bilirubin the mitochondria are med. J., i, I950). destroyed, and an enzyme may be released which will oxidise bilirubin to biliverdin. It is possible that such a reaction may be responsible for the presence of small amounts of biliverdin in the (2) Defective uptake and transport within the serum of with advanced liver disease. liver cell. patients Defective of bilirubin in the The newborn animal is more to the (3) conjugation susceptible hepatic microsomes. copyright. toxic action of large doses of bilirubin than the (4) Defective canalicular excretion or a adult animal (Rozdilsky, I96I). In some animals mechanical block in the bile duct. staining of the nervous tissue is only observed if Although, in most instances, jaundice is not due there is previous brain damage by severe hypo- to a single causative factor this may be the case in glycaemia, anoxia or traumatic hsemorrhage. congenital hyperbilirubinaemia. Detailed studies Whether previous damage as well as high con- of bile pigment metabolism in these disorders may, centrations of bilirubin are necessary for the therefore, give some insight into the pathways of development of kernicterus in man has not been normal metabolism.

established. Neither is it known why some parts http://pmj.bmj.com/ of the brain (e.g. the corpora striata, the thal- i. Increased load of bilirubin amus and the hippocampus) tend to become Hcemolytic disease stained in the presence of high levels of plasma An excessive release of haemoglobin from the bilirubin while others do not. The part played red cells may result in the normal daily production by the blood-brain barrier has also still to be of 300 mg of bilirubin being increased as much as clarified. There is suggestive evidence that in six-fold. The capacity of the liver to excrete the newborn the permeability is increased so that bilirubin in excess of that is, however, greatly on October 2, 2021 by guest. Protected bilirubin passes more readily into the CSF and the normally required so that in haemolytic disease brain. the plasma bile pigment concentration rarely rises Conjugated bilirubin, on the other hand, above 5 mg/ioo ml. The main pigment in the appears to be non-toxic. In the adult with severe plasma is unconjugated bilirubin so that bilirubin- jaundice these pigments are predominant and uria is not usually observed. Small amounts of only on rare occasions are high levels of uncon- conjugated bilirubin may be detected in the jugated bilirubin encountered. This is probably plasma but these will not exceed 15% of the total the main reason for the apparent absence of serum bilirubin concentration unless hepato- kernicterus in the adult. cellular damage has also occurred (Tisdale, JAUNDICE Klatskin and Kinsella, 1959). On theoretical grounds increased concentrations Alternative pathways of bilirubin metabolism of bilirubin in the plasma may result from one or An overproduction of bilirubin can arise from more of the following causes (Fig. 5 ). sources other than the breakdown of mature red (I) An increased load of bilirubin. cells. Israels, Suderman and Ritzman (1959) April I963 BILLING: Bilirubin Metabolism I8I Postgrad Med J: first published as 10.1136/pgmj.39.450.176 on 1 April 1963. Downloaded from recently suggested that such a mechanism might level greater than 8 mg/ioo ml it seems likely that be responsible for the unconjugated hyper- the jaundice can be partly explained by an enzyme bilirubinamia they observed in four cases of defect the extent of which appears to be related jaundice, who had raised values of faecal and to the degree ofjaundice. urine urobilinogen excretion, in the presence of a normoblastic hyperplasia of the marrow. 3. Defects in bilirubin conjugation Determinations of the relative specific activities Jaundice in the Newborn of hsem and stercobilin following the intravenous Hepatic immaturity. In the newborn infant, administration of 50 microcuries of z-14C-glycine unconjugated bilirubin will accumulate in the to one of these patients, who had an average plasma until the necessary enzyme systems have daily excretion of 945 mg stercobilin, indicated developed to enable conjugation and excretion of that at least 82% of this pigment was derived from the pigment to take place. It has been claimed sources other than the circulating red cells. Israels that over 40% of newborn infants develop and Zipursky (I962) consider that this excess clinical jaundice of varying degrees of severity formation of bilirubin probably occurs in the (Claireaux I960). This is most likely to occur in bone marrow, either as a result of haem catabolism premature infants who attain a higher degree of or by a more direct anabolic pathway. They have jaundice and remain jaundiced for a longer time called this 'shunt hyperbilirubinaemia' and consider than those with normal birth weights. It is only that it represents a marked exaggeration of a on rare occasions that the plasma bilirubin normal pathway which is responsible for approxi- concentration exceeds 20 mg/ioo ml, and, mately 11% of bile pigment production. according to current practice, the need for an Excessive formation of bile pigments in the exchange transfusion has to be considered. This presence of minimal peripheral haemolysis has involves the removal of bilirubin not only from also been cited by Arias (g962a) and Robinson, the plasma but also from the tissues. The admini- Vanier, Desforges and Schmid (I962) as a cause stration of albumin prior to an exchange trans- of chronic jaundice. The latter authors in their fusion has therefore been suggested (Odell, 1959), studies of the kinetics of bile pigment formation in order to obtain maximum transference of bili- copyright. in a patient with thalassaemia minor, in whom rubin into the vascular compartment. Although the there was marked evidence of 'ineffective erythro- procedure has still to receive clinical evaluation, poiesis', were able to account for the pattern of results obtained from experimental work with stercobilin labelling by the greatly increased puppies (Waters, I96I) would support the use of turnover of haemoglobin in the bone marrow. albumin as a therapeutic agent and encourage the use of whole blood rather than packed cells. 2. Defects in hepatic uptake of bilirubin Preliminary reports proposing that glucuronic Almost nothing is known of the mechanism acid might be useful in preventing kernicterus whereby bilirubin is transferred from the plasma has not been substantiated: on theoretical grounds http://pmj.bmj.com/ via the sinusoids into the liver cell where it is this is not surprising since it is UDPGA and not actively transported to the microsomes for con- glucuronic acid which is the glucuronyl donor for jugation. A defect at some stage in this transport the synthesis of bilirubin glucuronide. mechanism has been shown to occur in patients It has been known for some time that bilirubin with 'Gilbert's Disease'. These comprise a hetero- is readily destroyed by light and Cremer, Perryman geneous group of benign disorders characterized and Richards (1958) and Ferreira, Cardim and a mild of Mellone have claimed by unconjugated hyperbilirubinaemia (1960) that exposure of on October 2, 2021 by guest. Protected 1-4 mg/ioo ml (Billing and Williams, I962). newborn infants to artificial light is a useful Hepatic function tests are normal, except for an procedure for the treatment of jaundice of the impaired bilirubin tolerance, and the composition newborn. The in vitro studies of Blondheim, of the bile and the fsecal urobilinogen excretion Lathrop and Zabriskie (I962) suggest that the are also normal. Liver histology shows only light treatment results in the formation of water- minimal changes. Very occasional reports of soluble derivatives which are not bound by reduced 51Cr red blood cell survival times have albumin. Whether these derivatives have any been made in the presence of a normal reticulocyte deleterious effect on the infant needs to be count and a normal haemoglobin concentration investigated and proper control studies are (Foulk, Butt, Owen and Whitcomb, I959; Arias, necessary before this form of treatment can be 962b) but there is no evidence of overt haemolysis. generally accepted as advantageous. No satisfactory evidence has so far been obtained It is difficult to assess the extent to which which demonstrates a deficiency of glucuronyl jaundice in the newborn constitutes a pathological transferase in these patients with Gilbert's condition or may be described as 'physiological disease. In the rarer type of patient with a plasma jaundice'. In the following discussion of other q82 POSTGRADUATE MEDICAL JOURNAL April 1963Postgrad Med J: first published as 10.1136/pgmj.39.450.176 on 1 April 1963. Downloaded from factors influencing the development of jaundice in recognized. Some of the jaundiced female infants, the newborn it is necessary to remember that there although non-reactors themselves, have been may be a pre-existing defect in conjugation and found to have fathers who are' reactors. The the concentration of unconjugated bilirubin in severity of the jaundice, therefore, does not the plasma must be interpreted accordingly. appear to be solely dependent on the degree of the enzyme defect. Fessas, Doxiadis and Valaes Haemolytic disease. In hamolytic disease, whether (I962) have found that in certain families the due to ABO or Rh incompatibility, the dominant incidence ofjaundice was greater than that found in bile pigment in the plasma is bilirubin. This 786randomlyselected Greek male neonates(2.92%) tends to rise more quickly and to greater heights which suggested that there are probably additional in the premature infant, since in addition to genetic factors contributing to the development of hepatic immaturity the infant has to contend with severe jaundice. The jaundice disappears within a a greatly increased production of bilirubin due to month of birth and does not reappear unless a excessive haemolysis. The jaundice accordingly suitable challenge is presented, such as the inges- lasts longer. tion of fava beans; in this way the disease can be The plasma of infants with haemolytic disease differentiated from congenital non-spherocytic very occasionally contains conjugated bilirubin in haemolytic anernia. This condition appears to be the cord blood and in subsequent blood samples. an extremely unlikely cause of neonatal hyper- Ifthis develops within 24 hours of birth it is usually bilirubinaemia in non-Mediterranean countries in associated with severe anaemia and hepato- Europe. splenomegaly, and the presence of high values for serum aspartate transaminase suggests hepato- Vitamin K. It is generally accepted that a cellular damage. Harris, Farrell, Shorter, Banner single dose of 2 to 5 mg. of a water-soluble vitamin and Mathieson (1962) in their post mortem study K analogue is effective in preventing haemorrhagic of the liver histology of eight such infants could disease in the newborn and does not cause hyper- find no evidence of obstruction of the bile cana- bilirubinsemia. If, however, the dosage is increased liculi islands of then the likelihood of kernicterus by extramedullary haemopoiesis developing copyright. is or swollen hepatic cells, regurgitation of bile by also increased (Bound and Telfer, 1956). This damaged liver cells or inspissation of viscous bile. toxic effect is apparent in menadiol sodium The term 'inspissated bile syndrome' would diphosphate (Synkavit) vitamin K, and some of therefore appear to have no real meaning in this the other analogues, but not in menaphthone- context (Brent, 1962) and, as will be discussed dipotassium sulphate (Vikastab) (Corner, Berry later, the accumulation of conjugated bilirubin is and Neale, I960). The mechanism whereby probably due to a secretory defect. Since con- vitamin K analogues cause jaundice is not well jugated bilirubin does not appear to be toxic the understood. Vest (1958) has shown that the serum level of unconjugated bilirubin rather than erythrocytes of premature infants given vitaminhttp://pmj.bmj.com/ of total bilirubin should be the governing factor in K, in large therapeutic doses, have a shortened assessing the need for an exchange transfusion in survival and in vitro studies with neonatal erythro- these patients. cytes incubated with Synkavit have shown a rapid decrease in the reduced glutathione content, Glucose-6-phosphate-dehydrogenase deficiency. An possibly due to interference in the regeneration hereditary deficiency of the enzyme glucose-6- of TPNH (Broberger, Ernster and Zetterstr6m, phosphate in the red cell can render 1960). dehydrogenase on October 2, 2021 by guest. Protected it susceptible to haemolysis by various agents including drugs and fava beans. This genetic Non-hcemolytic Factors. Lathe and Walker defect may become apparent in the newborn (I958) have demonstrated that in the later stages reactor following the administration of sulfanila- of pregnancy the plasma of the mother contains mide, naphthalene, primaquine or nitrofurantoin. an unkown substance which will inhibit conjuga- Maternal medication with these drugs can result tion of bilirubin by rat-liver slices. Whether this in this response occurring in the infant in utero. substance normally crosses the placenta into the Recent reports from Singapore (Smith and fcetal circulation and if so what its action in vivo Vella, 1960), Greece (Doxiadis, Fessas and would be, is unkown. It is, however, of interest that Valaes, I96I) and Sardinia (Panizon, I960) have studies in infants with severe 'Transient Familial indicated that in certain parts of the world a Hyperbilirubinsemia' (Lucey and Driscoll, I96I), deficiency of glucose-6-phosphate dehydrogenase showed that the plasma of these infants and their may be an important factor in the aetiology of mothers has this inhibitory action on in vitro severe neonatal jaundice in mature infants, in conjugation of bilirubin. The relationship of the whom no blood group incompatibility has been inhibitor substance to the pathogenesis of the April I963 BILLING: Bilirubin Metabolism I83 Postgrad Med J: first published as 10.1136/pgmj.39.450.176 on 1 April 1963. Downloaded from jaundice, which subsided within the first month of hepatic glucuronyl transferase but not of of life has, however, not been established. UDPGA, which is responsible for their jaundice In a clinical trial of antibiotics in premature (Jervis, 1959; Szabo, Kovacs and Ebrey, I962). infants it was observed that the administration In all other respects liver function appears to be of sulfisoxazole reduced the concentration of normal and there is no evidence of haemolytic plasma bilirubin and at the same time increased disease. the incidence of kernicterus (Harris, Lucey and The serum bilirubin levels remain relatively con- \Iaclean, 1958). Experiments with in vitro stant in these patients in spite of a normal rate of systems and genetically jaundiced Gunn rats hamoglobin breakdown and defective pigment (Johnson, Garcia, Figueros and Sarmiento, I96I) excretion. Some alternative pathway for the have shown that organic anions such as sulphona- removal of bilirubin must therefore be present mides and salicylates compete with bilirubin for which, although less efficient than the usual albumin binding sites. An inverse relationship conjugating system since hyperbilirubinaemia exists between the blood level of the drug and occurs, is capable of maintaining a 'steady state' the plasma bilirubin. The distribution of bilirubin with respect to bilirubin. Schmid and Hammaker in the body is therefore altered and there is an (I962) have used 14C bilirubin to estimate the increase in the amount of pigment entering the rate of turnover and the magnitude of the total brain which may cause kernicterus. These miscible pool of bilirubin in a young boy with investigations emphasize the impossibility of congenital non-haemolytic jaundice. MIost of the assessing the success of a therapeutic agent in isotopic bilirubin appeared in the faeces in the reducing jaundice in the newborn merely by form of metabolites exhibiting properties different determining the serum bilirubin level; evidence from those of the known bile pigments. Further of an increased excretion of bilirubin must be work is needed to establish the site of breakdown obtained. of the bilirubir and the nature of the metabolites. Another drug known to cause jaundice is novobiocin (Cox, Foltz, Raymond and Drewyer, 4. Disturbances in hepatic excretion of 1959); in the newborn it has been reported as bilirubin causing a threefold increase in neonatal hyper- Obstructive jaundice and hepatocellular disease copyright. bilirubinaemia (Sutherland and Keller, I96I). Since over 900o of the pigments in hepatic bile In vitro studies (Hargreaves and Holton, I962) occur as pigment II it would be expected that suggest that novobiocin, which is excreted as a this would be the dominant pigment in the plasma glucuronide, acts by competing with bilirubin for of patients with extrahepatic biliary obstruction. the very limited amount of glucuronyl transferase If however the obstruction continues for some present in the newborn liver. weeks then the proportion of pigment II in the serum will decline while that of pigment I will in the Adult increase. A similar is then Jaundice pigment pattern http://pmj.bmj.com/ Crigler-Najjar Syndrome (Congenital non-hcemo- obtained to that found in the plasma of patients lyticjaundice). In very rare instances the deficiency with parenchymal liver disease due to hepatitis or of glucuronyl transferase seen in the newborn cirrhosis which has more pigment I than pigment may continue into adult life. This syndrome was II or bilirubin (Billing and Lathe, 1958). Hoffman, first described by Crigler and Najjar (1952) who Whitcomb, Butt and Bollman (1960) examined the studied the children of three related families with pigment patterns of I50 cases of jaundice and serum levels of unconjugated bilirubin ranging endeavoured to use them to differentiate between from 12-45 mg/ioo ml. Brain damage is often acute obstructive jaundice and hepatocellular on October 2, 2021 by guest. Protected caused in the neonatal period so that many of disease. There was unfortunately a significant these patients, if they survive, are to be found in overlap between the two groups which prevents mental hospitals. However, if neurological the procedure from being a useful diagnostic symptoms do not develop during early life then the tool; it is not helpful in distinguishing between prognosis is probably good (Childs and Najjar, extra and intra-hepatic cholestasis. I956; Sugar, 1961) since the toxic effects of Treatment with ACTH and corticosteroids has bilirubin are less marked in the adult. These been used for the differential diagnosis of chronic patients have an impairment in their ability to jaundice with some degree of success. In infective form glucuronides with aglycones such as hepatitis there is usually an immediate clinical salicylates, n-acetyl p-amino phenol, menthol improvement accompanied by a sharp decline in and tetrahydrocortisone (Schmid, I960). Only the plasma bilirubin level, which cannot be trace amounts of bile pigments are found in the explained in terms of increased biliary excretion bile and faecal urobilinogen excretion is low. In or a change in the renal excretion of bile pigments; vitro studies have shown that there is a deficiency the rate of red cell breakdown is also unchanged 184 POSTGRADUATE MEDICAL JOURNAL April 1963Postgrad Med J: first published as 10.1136/pgmj.39.450.176 on 1 April 1963. Downloaded from (Williams and Billing, 196I). In cases of intra- DUBIN-JOHNSON SYNDROME and extra-hepatic obstructive jaundice and drug 8 jaundice a favourable response is rarely seen. No satisfactory explanation has so far been put iv. Bilirubin (2mgkg) forward for the altered bile pigment metabolism e77 following steroid therapy in hepatitis. ^6cz6 Drug Jaundice 4 The first report of drug jaundice was by Hanger and Gutman (I940) in which 12 patients receiving J5 intravenous arsphenamine for the treatment of syphilis became icteric after the second or third injection. Since then an ever-increasing number of drug reactions with jaundice due to intra-hepatic U --^*^-~^~ cholestasis has been described (Popper and |5 > -Conjugated Schaffner, 1959). With many of these drugs i ilirubi n such as chlorpromazine ('Largactil'), thiouracil, para-aminosalicylic acid, chlorpropamide and I * 'Bilirubin cetyl urea, the jaundice has been attributed to a hypersensitivity reaction. i % of patients receiving 0 chlorpromazine treatment develop frank jaundice g I i together with increases in serum alkaline phos- ?0 40 60 120 IO 240 phatase and transaminases and also bromsulph- Minutes thalein retention. Out of eight patients studied by FIG. 6.-Effect of intravenous bilirubin (2 mg. kg.) on Hoffman and others (I960) four had a pigment the serum bile pigment concentration of a patient pattern similar to that seen with extra-hepatic with the Dubin-Johnson syndrome. obstruction, while the other four patients had a pattern suggestive of hepatocellular damage. copyright. Anabolic agents, such as methyl testosterone metallic poisons, naphthalene and benzene deriva- and norethandrolone ('Nilevar') with a CI7 alkyl tives. substitution in the steroid molecule, may cause jaundice to appear late in the course of therapy, Functional defects in excretion without symptoms of a hypersensitivity reaction. Defects in the excretory function of the liver Abnormalities in the microvilli lining the biliary need not necessarily be accompanied by any gross canaliculi have been reported following the of the bile ducts. The administration of these and it is histological abnormality drugs thought accumulation of conjugated as well as unconjugatedhttp://pmj.bmj.com/ that these may be responsible for the bile stasis bilirubin suggests that the difficulty lies between (Schaffner, Popper and Perez, I960). Experimental the microsomes and the bile canaliculi. It appears work with rats also supports the view that nor- to be developmental in origin but further investi- ethandrolone acts by interfering with the excretory gations of the mechanism of pigment excretion function of the liver cell. are necessary to establish whether the defect is Another group of drugs occasionally causes a a structural or metabolic one. severe of which is very type jaundice, histologically It has been observed that in some infants with and biochemically indistinguishable from viral on October 2, 2021 by guest. Protected hepatitis. This type of liver injury may follow haemolytic disease that the fall in level of plasma the administration of the mono-amine oxidase unconjugated bilirubin is preceded by a rise in inhibitors iproniazid ('Marsilid'), pheniprazine, the conjugated pigment concentration. This }-phenyl isopropyl hydrazine, isocarboxazid indicates that the conjugating enzyme system has ('Marplan') and phenalzine ('Nardil'). It has also developed satisfactorily but that the secretory been reported with iso-nicotinic acid, hydrazine, function of the liver cell is still inadequate (Harris pyrazinamide, zoxazolamine and the oral hypo- and others, 1962). This impairment in excretion glycaemic agents metahexamide and tolbutamide. may occasionally persist for several weeks and in Other drugs may cause jaundice as a result of some patients appears to have a familial incidence direct hepatocellular damage, which affects both (Jouvenceaux, Brizard, Michaud and Revol, I959; the conjugation and excretion of bilirubin. Drugs Billing, 196I). in this group include carbon tetrachloride, In some cases of chronic non-haemolytic chloroform, ethyl chloride, dichloro-diphenyl- jaundice in adolescents and adults conjugated as trichlorethane (D.D.T.) as well as muscarine, well as unconjugated bilirubin is present in the April 1963 BILLING: Bilirubin Metabolism8 Postgrad Med J: first published as 10.1136/pgmj.39.450.176 on 1 April 1963. Downloaded from plasma in spite of the absence of any histological appearance. It i's not thought that the pigment evidence of an obstructive type of jaundice /or bears any relationship to the presence or intensity hepatocellular lesion. When these patients are of the hyperbilirubinxmia (Wolf, Pizette, given an intravenous injection of bilirubin there Richman, Dreiling, Jacobs, Fernandez and Popper, is a rise in the plasma concentration of conjugated I960). The patients with the lipochrome pigment bilirubin at the time that the level of unconjugated are said to have the Dubin-Johnson syndrome bilirubin is falling, indicating a defect in hepatic (Dubin, 1958) while those without have the less excretion (Fig. 6). This excretory defect is not common Rotor syndrome (Rotor, Manahan and limited to bilirubin since there is retention of Florentin, 1948; Schiff, Billing and Oikawa, 1959). bromsulphthalein and other dyes after an intraven- An interrelationship appears to exist between the ous dose. These patients also have difficulty in two syndromes since in a particular family some excreting cholecystographic contrast media. Other affected members may have the pigment, while liver function tests are usually normal. others who are also jaundiced do not (Arias, I96I). The patients can be divided into two groups Elucidation of the pathogenesis of these genetically dependent on the presence or absence of a yellow- determined disorders should give valuable informa- brown granular pigment in the centrilobular tion about the normal mechanism of excretion by regions, which give the liver a macroscopic black the liver cell.

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