Gut: first published as 10.1136/gut.28.2.125 on 1 February 1987. Downloaded from
Gut, 1987, 28, 125-130
Porphyrin metabolism and haem biosynthesis in Gilbert's syndrome
K E L McCOLL, G G THOMPSON, E EL OMAR, M R MOORE, AND A GOLDBERG From the University Dept. Medicine, Gardiner Institute, Western Infirmary, Glasgow
SUMMARY Studies in 14 patients with unconjugated hyperbilirubinaemia caused by Gilbert's syndrome have revealed abnormalities of the enzymes of haem biosynthesis measured in peripheral blood cells. The activity of the penultimate enzyme of haem biosynthesis protopor- phyrinogen (PROTO) oxidase was reduced at 3-1±2.6 nmol PROTO/g protein/h (mean±ISD) compared with 8*2±5-1 in controls (p<0.005). This was associated with a compensatory increase in the activity of the initial and rate controlling enzyme of the pathway delta-aminolaevulinic acid (ALA) synthase at 866±636 nmol ALA/g/protein/h compared with 156±63 in controls (p<0.001). Unlike variegate porphyria in which there is a genetic deficiency of PROTO oxidase there was no increased excretion of porphyrins or their precursors in Gilbert's syndrome. Accentuation and subsequent correction of the unconjugated hyperbilirubinaemia with rifampicin produced reciprocal changes in PROTO oxidase activity indicating that bilirubin may be inhibiting the activity of this enzyme. http://gut.bmj.com/
Gilbert's syndrome originally described in 1901 by ferase activity.`8 Gilbert's syndrome may be a Gilbert and Lereboullet' is a benign disorder affect- heterogenous disorder.9 We wish to report a pre- ing 2-5% of the population. It is characterised by viously unrecognised biochemical abnormality in
mild, chronic, variable unconjugated hyperbili- patients with the syndrome which involves the on September 24, 2021 by guest. Protected copyright. rubinaemia in the presence of otherwise normal enzymes of haem biosynthesis and discuss its standard liver function tests and normal liver relationship to the previously reported disturbances histology. The hyperbilirubinaemia is mainly the in haem degradation. result of reduced bilirubin clearance2 though in approximately 50% of subjects with the syndrome, Methods there is also an increased rate of bilirubin formation because of slightly shortened red blood cell survival.3 PATI ENTS The cause of the impaired bilirubin clearance Eleven male and three female patients previously remains unclear. Compartmental analysis of radio- diagnosed to have Gilbert's syndrome were studied. bilirubin kinetic studies suggests a reduction in both Their mean age was 33 years (range 19-59). Total the uptake and conjugation of bilirubin,24 though bilirubin concentrations at the time studied ranged non-compartmental analytical techniques have failed from 19-54 [imol/l (normal range 5-17) with a mean to confirm the presence of an uptake defect.5 In vitro value of 31 I,mol/l. In each case the bilirubin was assays of bilirubin conjugation have consistently predominantly unconjugated and the reticulocyte shown reduced bilirubin-UDP-glucuronyl trans- count less than 2%. None of the patients was on any medication. For comparison, 20 normal subjects (15 men and five women, mean age 32 years) and nine Address for correspondence: Dr Kenneth E L McColl, University Dept. patients with variegate porphyria (eight women and Medicine, Gardiner Institute, Western Infirmary, Glasgow G11 6NT. one man, mean age 31 years) were also studied. Each Received for publication 26 June 1986. of the porphyria patients had experienced an attack 125 Gut: first published as 10.1136/gut.28.2.125 on 1 February 1987. Downloaded from
126 McColl, Thompson, El Omar, Moore, and Goldberg
of porphyria in the past but were all in clinical bilirubin did not interfere with the fluorometric remission when studied. The diagnosis of variegate measurement of protoporphyrin and thus affect the porphyria had been confirmed previously by measurement of PROTO oxidase activity. In place of markedly increased faecal porphyrin excretion, con- the washed leucocytes, bilirubin IXai was dissolved in sisting predominantly of protoporphyrin, together PROTO oxidase buffer (pH 9.2) in varying concen- with increased urinary excretion of ALA and trations up to 300 [tmol/l, and shown to have no porphobilinogen. effect on the measurement of protoporphyrin in the In six of the subjects with Gilbert's syndrome, the final incubation solution. Studies were also per- effect of rifampicin on the serum bilirubin concentra- formed to ensure that concentrations of rifampicin up tion and activities of leucocyte ALA synthase and to 20 ,ug/ml did not interfere with the measurement PROTO oxidase was studied. It was administered in of bilirubin or protoporphyrin. single daily doses of 600 mg at 23 00 hours and blood samples for bilirubin and rifampicin concentrations STATISTICAL ANALYSIS and enzyme activities obtained nine hours after the Statistical comparison of the values in the three second, seventh and 10th doses. In one subject it was different groups of subjects studied was done using not possible to obtain the two later samples. the non-parametric Mann Whitney U test. The The activities of the mitochondrial enzymes of the Spearman rank correlation was used to assess any haem pathway ALA synthase, coproporphyrinogen relationship between bilirubin concentration and oxidase, PROTO oxidase and ferrochelatase were enzyme activities. The study was approved by the measured in leucocytes and the cytosolic enzymes, Hospital Ethical Committee. ALA dehydratase, porphobilinogen deaminase and uroporphyrinogen decarboxylase in erythrocytes as Results previously described.'0 The PROTO oxidase assay involved the spectrofluorimetric measurement of the The activities of the enzymes of haem biosynthesis ill rate of protoporphyrin formation using protopor- the three groups of subjects studied are shown in phyrinogen as substrate by a modification of the Table 1. In the subjects with Gilbert's syndrome the method of Brenner and Bloomer." In this the mean ALA synthase activity was increased at 866 reaction was run at pH 9.2 and without the addition (range 360-2310) nmol ALA/g protein/h compared of sodium EDTA. The serum total bilirubin concen- with 156 (range 60-352) in the normal subjects http://gut.bmj.com/ tration was determined by the Michaelsson diazo (p
Table 1 Activities ofthe enzymes ofhaem biosynthesis in subjects with Gilbert's syndrome, patients with variegateporphyria andnormal controls
ALA synthase ALA PBGdeaminase Uropor- Copropor- PROTOoxidase Ferro- (nmolALAIg dehydratase (nmol UROI phyrinogen phyrinogen (nmolPROTO/g chelatase protlh) ([nolALA/ IRBClh) decarboxylase oxidase (nmol protlh) (pmol IRBC1min) (nmol COPROI PROTOIg meschaem/ IRBClh) protlh) gprotlh) Normal(n=20) 156±63 30-8±87 34-7±6-0 50±25 141±45 8-2±5-1 3 8±2-2 Gilbert'ssyndrome 866±636t 28-1±12-8 36-8±4-7 36±24 194 5±72 3.1+2.6* 4-0±2-2 (n= 14) Variegate 1178±655t 25-6±7-4 28-7±6-2 59-6±39 204+119 2.6+2.4* 2-12±1-8 porphyria (n=9)
Results expressed as mean± ISD. *p Porphyrin metabolism and haem biosynthesis in Gilbert'ssyndrome 127 2500- 241 0 -c 0 0 2000- * ; 18 0 '._ -c 7612- E 0 -p 1500- 0 4) 0 .. 0 "V 0 -J 00 0 .0 0 0 00 0 0 100- 0 0- 0 0 0 g. 00 Normals Gilbert's Variegate 0 syndrome porphyria 500 0 Fig. 2 Activity ofPROTO oxidase inperipheral leucocytes in Gilbert's syndrome, variegateporphyria and normal 0 0 controls. 0;.i00Its the second dose the mean total bilirubin concentra- tion was 67 Fmol/l (range 35-140) compared with the _ , . .~~~~~~Vregt prerifampicin value of 32 ,umol/l (range 19-50). http://gut.bmj.com/ Normals Gilbert s Variegate syndrome porphyria This was associated in each subject with further activity a further Fig. 1 Activity ofALA synthase inperipheral leucocytes in depression of PROTO oxidase and Gilbert's syndrome, variegateporphyria and normal increase in ALA synthase activity (Fig. 3). The mean controls. PROTO oxidase activity fell to being 1.7 nmol PROTO/g protein/h (range 0.3-5.2) compared with gate porphyria had increased faecal excretion of the pretreatment value of 2-9 (range 1-7.8) and mean protoporphyrin and coproporphyrin and in five there ALA synthase activity rose to 897 nmol ALA/g on September 24, 2021 by guest. Protected copyright. was also increased excretion of ALA and porphobi- protein/h (range 337-1500) compared with the pre- linogen in the urine (Table 2). The erythrocyte treatment value of 535 (range 287-1000). With con- protoporphyrin concentration was normal in both tinued rifampicin therapy the bilirubin concentration conditions. fell and by the 10th day of treatment four of the five Rifampicin treatment in each of the six patients subjects monitored for this length of time had values studied with Gilbert's syndrome initially increased within our normal range. Likewise with continued the total bilirubin concentration because of a rise in dosing the plasma rifampicin level (mean± 1 SD) fell, the unconjugated fraction (Fig. 3). Nine hours after being 6.54±3.1 ,ug/ml, 1*74±1.5 pg/ml, and Table 2 Excretion ofporphyrins andporphyrin precursors and erythrocyteprotoporphyrin concentration in Gilbert's syndrome and variegate porphyria Erythrocyte Urine Faecal protoporphyrin (nmol/l) ALA PBG Uroporphyrin Coproporphyrin Coproporphyrin Protoporphyrin (Wnoll24h) (nmoll24h) (nmol/24h) (nmol/24h) (nmollg dry wt) (nmollg dry wt) Normal range 0-900 0-40 0-16 0-49 0-430 0-76 0-200 Gilbert'ssyndrome 620 11-9 4 2 71 30 112 mean (range) (474-976) (1-24) (0-11) (0-4) (33-147) (22-37) (79-139) Variegate porphyria 698 45 44 72- 420 558 1243 mean (range) (370-1085) (1-82) (1-62) (8-141) (44-938) (66-1400) (355-2536) Gut: first published as 10.1136/gut.28.2.125 on 1 February 1987. Downloaded from 128 McColl, Thompson, El Omar, Moore, and Goldberg 150 nmol ALA/g protein/h compared with their pretreat- ment value of 580. 1.5 120 E Discussion - 90 Our observations indicate that in addition to dis- 0 turbed bilirubin metabolism subjects with Gilbert's 60- syndrome also have abnormalities of haem bio- I- synthesis characterised by increased activity of the initial enzyme of the pathway ALA synthase and 30 0 reduced activity of the penultimate enzyme PROTO oxidase. Delta-aminolaevulinic acid synthase is the 0 1 1 rate controlling enzyme of haem biosynthesis and under negative feedback control by haem. In the -- 8 acute porphyrias in which there are genetic defic- iencies of individual enzymes in the pathway - for .C example, PROTO oxidase in variegate porphyria, c there is a compensatory increase in ALA synthase activity. In Gilbert's syndrome the increased ALA synthase activity can be explained by the reduced PROTO oxidase activity. CL CL -6 2 The biphasic effect of rifampicin on the uncon- E jugated bilirubin concentration allowed the relation- C_ ship of the abnormal haem biosynthesis and hyper- bilirubinaemia to be examined. The initial rise in the unconjugated bilirubin concentration on starting rifampicin, which is most marked nine hours after c each dose, is because of competition for hepatic uptake, and the subsequent normalisation of the http://gut.bmj.com/ -X0 bilirubin level, and fall in the serum drug level, *_ because of the induction of this process.'617 The reciprocal changes in PROTO oxidase activity -6 suggest that the enzyme is being inhibited by uncon- jugated bilirubin. The preliminary studies excluded E the possibility that the decreased PROTO oxidase c activity was artefactual because of bilirubin or rifam- on September 24, 2021 by guest. Protected copyright. picin interfering with the measurement of its activity and the increased ALA synthase activity confirms an in vivo block in haem biosynthesis. The changes in U') ALA synthase activity with rifampicin therapy are -X£ 0 1- . also consistent with unconjugated bilirubin impeding _J x~ 1 Before +2 +7 +10 the rate of haem synthesis. The initial increase in Time after commencing rifampicin (days) ALA synthase activity on rifampicin, however, may Fig. 3 Effectofrifampicin 600 mglday on the serum total also be explained by the haemoprotein inducing bilirubin concentration and the activities ofPROTO oxidase effects of the drug.'8 andALA synthase inperipheral leucocytes in six subjects Bilirubin IXai is structurally very similar to proto- with Gilbert's syndrome. porphyrinogen, the normal substrate of PROTO oxidase, and may be acting as a competitive inhibitor. 1-15±1-0 [ig/ml after the second, seventh and 10th It has been shown in vitro to inhibit the enzyme doses respectively. The normalisation ofthe bilirubin ferrochelatase which has protoporphyrin as its sub- concentration was associated with a rise in PROTO strate.'9 Unfortunately, no purified preparations of oxidase activity and fall in ALA synthase activity. In PROTO oxidase are currently available for satis- the five subjects monitored for 10 days the mean factory in vitro inhibitory studies. If hyperbili- PROTO oxidase activity rose to 4.7 nmol PROTO/g rubinaemia is responsible for the decreased PROTO protein/h compared with their pretreatment value of oxidase activity in Gilbert's syndrome, a significant 2-6 and the mean ALA synthase activity fell to 352 negative correlation of the two might have been Gut: first published as 10.1136/gut.28.2.125 on 1 February 1987. Downloaded from Porphyrin metabolism and haem biosynthesis in Gilbert'ssyndrome 129 expected. Two reasons may account for the absence 2 Berk PD, Bloomer JR, Howe RB, Berlin NI. Constitu- of this. Firstly, it is likely to be the unconjugated and tional hepatic dysfunction (Gilbert's syndrome): a new unbound fraction of bilirubin which will inhibit the definition based on kinetic studies with unconjugated enzyme and currently there is no satisfactory assay radio-bilirubin. Am J Med 1970; 49: 296-305. for accurately and 3 Powell LW, Hemingway E, Billing BH, Sherlock S. measuring this, secondly there Idiopathic unconjugated hyperbilirubinaemia (Gilbert's may not be a direct linear correlation between the syndrome): a study of 42 families. N Engl J Med 1967; concentration of bilirubin in the serum and its 277: 1108-12. concentration in the vicinity of the enzyme PROTO 4 Frezza M, Perona G, Corrocher R, Cellerino R, oxidase which is sited on the inner mitochondrial Bassetto MA, Desandre G. Bilirubin - H3 kinetic membrane. studies: pattern of normals, Gilbert's syndrome and If subjects with Gilbert's syndrome have an enzy- hemolytic state. Acta Hepato-Gastroenterol 1973; 20: matic defect similar to variegate porphyria they 363-71. might be expected to have increased porphyrin 5 Goresky CA, Gordon ER, Shaffer EA, Pare P, excretion. Carrasavas D, Aronoff A. Definition of a conjugation This does not invariably follow, however, dysfunction in Gilbert's syndrome: studies of the handl- as a proportion of patients with variegate porphyria ing of bilirubin loads and of the pattern of bilirubin have normal porphyrin excretion when asympto- conjugates secreted in bile. Clin Sci Mol Med 1978; 55: matic.20The tissue in which the enzyme abnormalities 63-71. are present is also important. In the porphyrias, the 6 Black M, Billing BH. Hepatic bilirubin- UDP- enzyme defect is thought to be present in all cells glucuronyl transferase activity in liver disease and though the porphyrin overproduction is mainly Gilbert's syndrome. N EnglJ Med 1969; 280: 1266-71. hepatic in origin.2' In Gilbert's syndrome the enzyme 7 Felsher BF, Craig JR, Carpio N. Hepatic bilirubin defect has been shown in peripheral leucocytes and glucuronidation in Gilbert's syndrome. J Lab Clin Med we have no information regarding haem biosynthesis 1973; 81: 829-37. 8 Blanckaert N, Schmid R. Physiology and pathophysio- in the liver. If the impaired haem biosynthesis in logy of bilirubin metabolism. In: Zamin D, Boyer TD, Gilbert's syndrome is the result of the unconjugated eds. Hepatology. Philadelphia: WB Saunders, 1982: hyperbilirubinaemia and Gilbert's syndrome is 246-96. because of impaired hepatic uptake of unconjugated 9 Berk PD. Pathophysiology, diagnosis and treatment of bilirubin, then intrahepatic haem biosynthesis would the hereditary hyperbilirubinaemias. In: Williams R, Maddrey WC, eds. Gastroenterology 4. London: be spared and there would be no increased porphyrin http://gut.bmj.com/ excretion because of hepatic overproduction. Butterworths, 1984: 1-51. Abnormalities of porphyrin excretion do occur in 10 Moore MR, Thompson GG, Goldberg A, Ippen H, the Dubin-Johnson syndrome and Rotor's Seubert A, Seubert S. The biosynthesis of haem in syndrome congenital (erythropoietic) porphyria. Int J Biochem in which the hereditary defects in hepatic bilirubin 1978; 9: 933-8. handling involve hepatocellular excretion rather than 11 Brenner DA, Bloomer JR. A fluorimetric assay for uptake or conjugation of the pigment,' I and also in measurement of protoporphyrinogen oxidase activity obstructive jaundice.24 In 1981, Evans et all reported in mammalian tissue. Clin Chim Acta 1980; 100: a patient with conjugated hyperbilirubinaemia 259-66. on September 24, 2021 by guest. Protected copyright. resembling that seen in Rotor's syndrome, who also 12 Michaelsson M, Nosslin B, Sjolin S. Plasma bilirubin had unexplained increased faecal porphyrin excre- determinations in the newborn infant. Pediatrics 1965; tion typical of variegate porphyria. Though some of 35: 925-31. these porphyrin abnormalities associated with conju- 13 Seubert A, Seubert S. High performance liquid chroma- tographic analysis of porphyrins and their isomers with gated hyperbilirubinaemia can be explained by radial compression columns. Anal Biochem 1982; 124: impaired hepatic porphyrin excretion, abnormal 303-7. hepatic porphyrin synthesis caused by increased 14 Moore MR. Laboratory Investigation of disturbances hepatocellular bilirubin concentrations may also be of porphyrin metabolism. Association of Clinical important. Pathologists. Broadsheet 109. London: BMA, 1983. 15 McConnell JB, Smith H, Davis M, Williams R. Plasma rifampicin assay by an improyed solvent extraction This work was supported by a biomedical research technique. BrJ Clin Pharmacol 1979; 8: 506-7. grant from the Scottish Home and Health Depart- 16 Acocella G. Clinical pharmacokinetics of rifampicin. ment. Clin Pharmacokinet 1978; 3: 108-27. 17 Frezza M, Pozzato G, Quaronto C, Tiribelli C, Piccinini C. Abnormality in the hepatic transport of rifamycin - References SV in Gilbert's syndrome. Ital J Gastroenterol 1980; 12: 1-5. 1 Gilbert A, Lereboullet P. La Cholemie simple familale. 18 McColl KEL, El Omar E, Macphee GG, et al. Effects of Sem Med 1901; 21: 241-5. rifampicin on haem biosynthesis and hepatic mono- Gut: first published as 10.1136/gut.28.2.125 on 1 February 1987. Downloaded from 130 McColl, Thompson, El Omar, Moore, and Goldberg xygenase activity in healthy subjects. Br J Clin isomers in Dubin-Johnson syndrome. Gastroenterology Pharmacol. (In press.) 1976; 70:1117-20. 19 Labbe RF, Zaske MR, Aldrich RA. Bilirubin inhibition 23 Shimizu Y, Naruto H, Ida S, Konakura M. Urinary of heme biosynthesis. Science 1959; 129: 1741-2. coproporphyrin isomers in Rotor's syndrome. A study 20 Mustajoki P. Variegate porphyria. Q J Med 1980; 44: in eight families. Hepatology 1981; 1: 173-8. 191-203. 24 Rover CM. Urinary porphyrin pattern in liver damage. 21 Marver HS, Schmid R. In: Stanbury, Wyngaarden, Fd Chem Toxic 1984; 22: 241-3. Frederickson, eds. The metabolic basis of inherited 25 Evans J, Lefkowitch J, Lim CK, Billing B. Faecal disease. New York: McGraw-Hill, 1972: 851-5. porphyrin abnormalities in a patient with features of 22 Kondo, T, Kuchiba K, Shimizu Y. Coproporphyrin Rotor's Syndrome. Gastroenterology 1981; 81: 1125-30. http://gut.bmj.com/ on September 24, 2021 by guest. Protected copyright.