J Clin Pathol 1989;42:466-469

Sclerosing cholangitis with hepatic microvesicularsteatosis in cystic fibrosis and chronic pancreatitis

I BENETT, B SALH, N Y HABOUBI,* J M BRAGANZA

From the University Department of Gastroenterology, Royal Infirmary, and the *Histopathology Department,Withington Hospital, Manchester

SUMMARY The association between asymptomatic primary sclerosing cholangitis and exocrinepancreatic disease was underlined by the findings in a patient with cystic fibrosis and in another withchronic pancreatitis. In each case hepatocytes showed extensive microvesicular steatosis and studiesof drug metabolism suggested hepatic enzyme induction: biliary or serum analysis, or both, disclosedraised concentrations of a lipid-based marker of free radical oxidation. These findings suggest thattoxic metabolites of oxygen or other chemicals may have a role in the pathogenesis of the bile ductlesion.

The listed hepatobiliary abnormalities in cysticfibrosis include fatty liver in 15-30% of cases, biliarycirrhosis in up to 25% with portal hypertension insome 2%, and gallstones in 4-12% of cases.' Primarysclerosing cholangitis, as defined in standard texts, ishardly mentioned, although a report by Porta et a12 ofsix infants with cystic fibrosis showed changes that fellwithin the range of the recognised histological signs ofthe disease.3 Similar changes have been recorded inpatients with chronic pancreatitis.45 The inverseassociation of abnormal pancreatograms, or reducedexocrine secretory capacity, or both, in patients withprimary sclerosing cholangitis, has been documentedby many authors.45 The overlap between thehepatobiliary abnormalities in cystic fibrosis andchronic pancreatitis may be greater still, as illustratedby the two cases reported below.

Case reports

CYSTIC FIBROSIS AND SCLEROSING CHOLANGITISA 31 year old chartered surveyor was referred forinvestigation of steatorrhoea (640 mmol fat in threedays, normal < 60 mmol). He was neither a smokernor a drinker. Gross clubbing of fingers and toes was astriking clinical feature. A low para-aminobenzoicacid (PABA)'4C excretion index (0 34, normal > 0-76)indicated pancreatic exocrine failure; an abnormalendoscopic pancreatogram was typical of chronic

Accepted for publication 12 January 1989

pancreatitis. The raised serum alkaline phosphataseactivity (476 U/1, normal < 300 U/1) was investigatedby endoscopic retrograde cholangiography (fig I a)and liver biopsy (fig 1 b): both showed the typicalchanges of sclerosing cholangitis. Semithin sectionsstained with toluidine blue as well as electron micro-scopic examination of the liver biopsy specimenshowed microvesicular steatosis (fig I c and I d).Special stains for x,-antitrypsin, hepatitis B surfaceantigen, inspissated mucin, iron and copper bindingprotein were negative.The high concentration of sodium in sweat

stimulated by pilocarpine (121 mmol/l), azospermia,increased lung markings on chest x-ray picture and arestrictive pulmonary function test profile confirmedthe clinical suspicion of cystic fibrosis. The familyhistory showed that of the patient's three brothers,one, now aged 30, had been diagnosed as having cysticfibrosis at the age of 18. The brothers were tissuetyped: HLA A 2, 9; B 15, 16 in our patient and A 2, 28;B 15 in his affected brother.

CHRONIC PANCREATITIS AND SCLEROSINGCHOLANGITISA 34 year old despatch clerk was referred with athree year history of recurrent pancreatitis. He drank60-80 g alcohol daily between the ages of 16 and 31when he became teetotal, but the attacks ofpancreatitis continued. He had smoked 20 cigarettes aday since the age of 16. An occupational historydisclosed daily exposure to diesel exhaust fumes. Thediagnosis of chronic pancreatitis was confirmed by the

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Sclerosing cholangitis with hepatic microvesicular steatosis in cystic fibrosis and chronic pancreatitis_~~~~~~~~~~~~~~~~~~~~~~~~, C;.;4,#o. i

~~~~~~~~~~~~~~~~~~~~~~~~'.q.1 sa0t.

Fig I Case 1: cysticfibrosis. (a) Endoscopic cholangiogram showing beading in many intrahepatic bile ducts: theintrapancreatic bile duct was not constricted. (b) Portal tract showing almost complete obliteration of the bile duct byextensive periductalfibrosis. (Haematoxylin picro sirius red 0.) (c) Extensive variable sizefat droplets in hepatocytes.(Toluidine blue-stained semithin section.) (d) Electron microscopy of ultrathin section showing variable sizedfat droplets.

findings ofpancreatic calculi on plain abdominal x-raypicture, with grossly abnormal pancreatogram: pan-creatic exocrine function was moderately impaired(PABA/'4C index 0 64). The marginally raised serumalkaline phosphatase activity (350 U/l) was ascribed tochanges which fell within the range of features ofprimary sclerosing cholangitis on retrograde cholan-giography and percutaneous liver biopsy (fig 2): thebiopsy specimen also showed micro/macrovesicularsteatosis in hepatocytes and excessive amounts oflipofuscin, considering the patient's age. His tissuetype was HLA A3; B22, 40.

Indirect evidence of induction of the hepatic drug

metabolising enzymes glutathione S-transferase B,cytochromes P450, and glucuronyl transferase is affor-ded by an increase in the early phase disappearanceconstant after an intravenous injection of sulpho-bromophthalein (BSP ki), the clearance of theo-phylline, and the concentration of D-glucaric acid inurine, respectiyely.*" Reference ranges for these testsand for the serum and biliary concentrations ofa lipid-based marker of free radical oxidation-the 9, 11isomer (9, 1 ILA') of linoelic acid (9, 12 LA'2'3)-aregiven in the table, along with published data in groupsof patients with cystic fibrosis and chronic pan-creatitis, and the values in the subjects of this report.

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Fig 2 Case 2: chronic pancreatitis. Portion ofa portal tractshowing loose periductalfibrosis and mild inflammation.(Haematoxylin and eosin.)

Discussion

The finding of asymptomatic sclerosing cholangitis inthe patient with cystic fibrosis extends the acceptedareas of overlap between this congenital disease andacquired chronic pancreatitis, both alcoholic and non-alcoholic."' Increased osmolality of sweat, pulmonaryrestrictive defects, attacks of pancreatitis, ductalprotein plugs, pancreatic calculi, and tubular com-plexes representing dedifferentiated acini progressingto acinar effacement, are features that are shared to avarying extent between cystic fibrosis and chronicpancreatitis. Biliary cirrhosis is a recognised complica-tion of each condition but primary sclerosing cholan-gitis is not. Furthermore, the coexistence of primarysclerosing cholangitis with microvesicular fat inhepatocytes, as in both patients in this report, has notbeen recorded in any other disease. Their concurrence

Benett, Salh, Haboubi, Braganzasuggests that both lesions are produced by the samemechanism. We propose that this involves excessivefree radical activity through induction of drugmetabolising enzymes and other potential routes.'5

Primary sclerosing cholangitis preferentially affectssmall and medium sized bile ducts: the lesion has beenproduced inadvertently by infusing 5-fluorodeoxy-uridine into the hepatic artery in patients with livermetastases,'6 or experimentally by infusing a formal-dehyde solution into the bile ducts of rats.'7 Possessionof the autoimmune haplotype HLA A, B5 DR3 renderspatients with ulcerative colitis susceptible to primarysclerosing cholangitis."' These observations suggestthat an abnormal (in amount, type, or both) biliaryconstituent triggers the inflammatory process, theperpetuation of which is facilitated by, but not depen-dent on, an HLA related display of class II antigens onbiliary epithelium.'9 Microvesicular steatosis hashitherto been described in different and distinctivesettings, including acute fatty liver of pregnancy,Reye's syndrome, and on exposure to certain drugsand chemicals (sodium valproate, paracetamol, halo-genated hydrocarbons, alcohol). It is suspected thatthis change, which represents disrupted microtubulartransport in hepatocytes, is caused by toxic metabo-lites-whether oxygen free radicals, or reactive drug orchemical intermediates generated via the cytochromesP450.'5We have previously noted the coexistence of

primary sclerosing cholangitis with microvesicularsteatosis or excess lipofuscin, or both, in patients withchronic pancreatitis and have proposed that thelesions are caused by excessive hepatic free radicalactivity and the discharge into bile of reactive drug orchemical intermediates along with stable but poten-tially toxic products of free radical oxidation of lipidand protein.45 This hypothesis took into account thecapacity of certain products to change the structure of

Table Data on three in vivo tests ofhepatic enzyme induction and a lipid based marker offree radical activity

References onchronic

References on pancreatitisReference cysticfibrosis groupranges group data Case I data Case 2

Induction markers:BSP ki 94-15-86 No information in adults7 20 6 23Theophylline clearance (mg/h/kg) 15-66' T 8'0 95 9 189

50-97945-8 1'o

Urinary D-glucaric acid (mmol/mol creatinine) 0-41-482" t I° 5 50 t " 6-72

Free radical oxidation products:Serum 9,11 linoleic acid/ 076- 3.32'2 T I 398 t 135009,12 linoleic acid molar ratio (%) 081- 39013Biliary 9,11 linoleic acid by duodenal drainage 1 12- 2 76'3 Duodenal aspirate too T '3 7-78

for 10 minutes after secretin (pmol) viscid for analysis

Arrows indicate significantly increased group values in published accounts.

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Sclerosing cholangitis with hepatic microvesicular steatosis in cysticfibrosis and chronic pancreatitis 469

gamma globulin (and, thereby, its immunogenecity) todisturb membrane function, and to provoke chemo-taxis and fibrosis.'2 It also considered findings of apreliminary occupational survey in patients withchronic pancreatitis" and data from pharmakokineticstudies'5 and biliary analysis.2' Case 2 in the presentreport is a typical example and, as in patients withchronic pancreatitis in general, he did not possess theautoimmune HLA haplotype.22 That haplotype is notassociated with cystic fibrosis either.23 Yet our patient(case 1) had unequivocal changes of primary scleros-ing cholangitis while hepatic oxidant stress was sug-gested by the findings of microvesicular steatosis andraised serum 9, 11 LA'. The possibility that micro-somal monoxygenases may contribute to oxidantstress in cystic fibrosis is suggested by publishedpharmakokinetic data8' and ultrastructural studiesshowing expansion or dilatation of the endoplasmicreticulum of hepatocytes24; while the possibility thatoxidant stress may underly the basic genetic defect issuggested by observations pertaining to two othertarget organs-namely, nasal epithelium2526 and theexocrine pancreas."

Finally, the two case reports indicate that bothprimary sclerosing cholangitis and microvesicularsteatosis can be completely asymptomatic. Theroutine availability of endoscopic cholangiographyhas made this point in the context of primary scleros-ing cholangitis in patients with ulcerative colitis; but itis not clear from published accounts whether micro-vesicular steatosis was looked for and excluded inthose patients.

We thank Dr P Dyer for tissue typing our patients. Weare grateful to Miss E A Wilson, Mrs J Hanbridge, andMiss M D Gamer for typing the manuscript.

References

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3 MacSween RNM, Anthony P, Scheuer P. Pathology of the liver.2nd edition. London: Churchill Livingstone, 1987.

4 Warwick F, Anderson RJL, Braganza JM. Sclerosing cholangitis-like changes in pancreatic disease. Clin Radiol 1985;36:51-6.

5 Haboubi NY, Ali HH, Braganza JM. Liver histology in patientswith pancreatitis: a clue to aetiology? Mt Sinai J Med 1986;53:380-8.

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1 1 Sandle LR, Braganza JM. An evaluation of the low-pH enzymaticassay of urinary D-glucaric acid, and its use as a marker ofenzyme induction in exocrine pancreatic disease. Clin ChimAcda 1987;162:245-56.

12 Dormandy TL. Free radical activity and diene conjugation in man.In: Poli G, Cheeseman KH, Dianzani MU, eds. Free radicals inliver injury. Oxford: IRL Press, 1985:167-74.

13 Uden S, Guyan PM, Kay P, Kay GH, Braganza JM. Heightenedfree radical activity in patients with pancreatitis. Clin Sci1988;74(suppl 18):1-32.

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16 Shea WJ, Demas BE, Goldberg HI, Hohn DC, Ferrell LD, KerlanRK. Sclerosing cholangitis associated with hepatic arterialFUDL chemotherapy: radiographic-histologic correlation. AmJ Radiol 1986;146:717-26.

17 Houry S, Languille 0, Hugier M. Experimental sclerosing cholan-gitis in rat induced by formalin injection in the biliary tract. DigDis Sci 1986;31(suppl 10):108 S.

18 Schrumpf E, Fansa 0, F0rree 0, Doblough JH, Ritland S,Thorsby E. HLA antigens and immunoregulatory T cells inulcerative colitis associated with hepatobiliary disease. Scand JGastroenterol 1982;17:187-91.

19 Chapman RW, Kelly PMA, Heryet A, Jewell DP, Fleming KA.Expression of HLA-DR antigens on bile duct epithelium inprimary sclerosing cholangitis. Gut 1988;22:422-7.

20 Braganza JM, Jolley JE, Lee WR. Occupational volatile chemicalsand pancreatitis: a link? Int J Pancreatol 1986;1:9-20.

21 Braganza JM, Wickens D, Cawood P, Dormandy TL. Lipidperoxidation (free radical oxidation) products in bile frompatients with pancreatic disease. Lancet 1983;ii:375-9.

22 Anderson RJL, Dyer P, Donnai D, Braganza JM. HLA-A and Bantigens in chronic pancreatitis. Int J Pancreatol 1988;3:83-90.

23 HennequetA, Jehanne M, HorsJ, SchmidJ, Schmid M, DaussetJ.Muscoviscidose et marquers HLA-A et B. Proceedings VllthInternational Cystic Fibrosis Congress, Paris. Paris: Jouve,1978:243-7.

24 Dominick H Ch, von Bassenwitz DB, Arends P. Ultrastructure ofthe liver in cystic fibrosis. Proceedings of VlIth InternationalCystic Fibrosis Congress, Paris. Paris: Jouve, 1978:74-7.

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26 Gavalor SM, Mezhevitch NA, Buzueva II. The ultrastucturalinvestigation of nasal mucosa glands in patients with muco-viscidosis. Proceedings of VIlth International Cystic FibrosisCongress, Paris. Paris: Jouve, 1978:127-31.

27 Wallach JD, Germaise B. Cystic fibrosis: a perinatal manifestationof selenium deficiency. In: Hemphil DD, ed. Trace substances inenvironmental health. Columbia: University of Missouri Press,1979:469-76.

Requests for reprints to: Dr J M Braganza, Departmentof Gastroenterology, Royal Infirmary, Oxford Road,Manchester Ml3 9WL, England.

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