Journal of Cutaneous Pathology 1981: 8: 404-410

Melanin pigmentation of the skin inprimary biliary cirrhosis

PETER R . MILLS, CHRISTINE J. SKERROW and RONA M . MACKIE

Gastroenterology Unit, Royal Infirmary and University Department of Dermatology, WesternInfirmary, Glasgow, Seotland

A histologieal and ultrastruetural study has demonstrated that eutaneous pigmen-tation in primary biliary eirrhosis (PBC) is due to the presence of increased amountsof melanin, widely dispersed throughout both epidermis and dermis. No deposits ofstainable iron were observed. Compared with skin from matched sites from eontrolpatients with alcoholic cirrhosis and no pigmentation, the melanocyte: keratinoeyteratio was not signifieantly higher in PBC. However, in PBC, melanosomes persistedto unusually high levels in the epidermis and were paekaged in larger mem-brane-bound elusters than was the ease in the eontrols.Whether exeess melanin results from increased melanogenesis or defective melanindegradation remains unelear, although there is some evidenee favouring the lattermechanism. No hormonal (P-MSH and ACTH) or ehemieal (bile salt irritation)stimuli to inerease melanogenesis were demonstrated.

Accepted for publication May 2, 1981 .

Primary biliary cirrhosis (PBC) is a slowlyprogressive form of chronic liver disease ofuncertain aetiology which predominantly af-fects middle-aged women. Patients usuallypresent with pruritus with referal initially to adermatologist and it is only later that jaundicedevelops due to progressive intrahepaticcholestasis. One of the striking physical signsof the condition is excessive skin pigmentationwhich initially involves exposed areas butgradually becomes generalised. Skin pigmen-tation in PBC is common, may be an earlypresenting feature and can become clinicallyobtrusive (Schaffner 1975). The cause of thisimpressive clinical sign, which rarely occurs in

other forms of chronic liver disease and is nota feature of secondary biliary cirrhosis, hasnever been investigated. Whilst themechanisms influencing cutaneous pigmenta-tion are generally not well understood (Mac-Kie 1978) pigmentation in chronic renal fail-ure has recently been shown to be due to in-creased cutaneous melanin production as-sociated with elevated plasma immunoreac-tive P-melanocyte-stimulating hormone(P-MSH) levels (Smith et al. 1975). Wetherefore undertook a histological study of thecause of excess skin pigmentation in PBC andexamined plasma P-MSH and ACTH levels.

0303-6987/81/060404-07 $02.50/0 © 1981 Munksgaard, Copenhagen

PIGMENTATION IN CIRRHOSIS 405

Material and methods ; .] . :L .Clinical assessment

Sixteen Caucasian patients with histologieally-proven chronic liver disease underwent a skinbiopsy. (See Table 1). There were nine pa-tients with PBC and associated abnormal skinpigmentation. The control group consisted ofsix patients with alcoholic cirrhosis, two withand four without excessive skin pigmentationand one patient with primary haemochroma-tosis and pigmentation. All the patients hadcirrhosis on liver biopsy, with the exception ofpatient 9 who showed the early histologicalchanges of Stage II PBC. A clinieal record ofthe degree of pigmentation was made as fol-lows: 1- normal; 2 - mild pigmentation, justvisible; 3 - moderate pigmentation, obvious,usually patchy; 4 - marked pigmentation,confluent, negroid. Serum bilirubin was re-corded as an indication of the clinieal stage ofPBC (Shapiro et al. 1979).

] ^Skin biopsies ;. .

Skin biopsies were taken from the flank areaand divided into portions for use in the fol-lowing studies, whieh were carried out withoutknowledge of the patients' diagnosis.a) Light microscopy

Tissue was fixed in buffered formalin, em-bedded in paraffin and stained withhaematoxylin and eosin, and with Perl'sstain for iron. Frozen tissue was used fordetection of DOPA-oxidase (tyrosinase)in order to visualise melanoeytes and as-sess melanocyte: keratinocyte ratios in thebasal layer of the epidermis.

b) ImmunofluorescenceSkin was snap-frozen in liquid nitrogenand examined for the presenee of the eon-jugated primary bile salts; cholate andchenodeoxyeholate, using antibodyobtained from immunised rabbits (Baqir etal. 1979).

e) Electron microscopyPrimary fixation was in 2.5% bufferedglutaraldehyde, followed by 1% osmiumtetroxide. After embedding in Epon 812,thin seetions were cut and stained withuranyl acetate and lead citrate. This mater-ial was used to assess basal melanoeyte:keratinocyte ratios; the overall number ofmelanosomes in keratinoeytes (expressedas a range 0 to 3, normal 1); the degree ofdermal pigmentary incontinence (range 0to 3, normal 0); the persistence of mela-nosomes in various epidertnal layers(B-basal only, normal; S-spinous; G-granular; H-horny) and the presence, inkeratinoeytes, of giant eompound mela-nosomes containing from 20-40 individualmelanosomes (range 0 to 3, normal 0).

Endocrine assessment '''

Blood samples were taken into heparinisedtubes between 09.00 and 10.00 hours, from16 patients with PBC (including the 9 above)and 19 normal laboratory staff. The plasmawas separated within 30 minutes and stored at-20°C for up to 1 month before radioim-munoassay for P-MSH and ACTH accordingto the method of Gray & Ratcliffe (1979).

Results : : : ;

The elinical, histological and ultrastructuralfindings are summarised in Table 1.

a) Histology : 'Skin pigment in the PBC patients wasshown to be due to exeess melanin with nodeposits of stainable iron observed. In thecontrol group, stainable iron was noted inpatient 10 with alcoholic cirrhosis and inpatient 16 with haemoehromatosis. Basalmelanocyte: keratinoeyte ratios, obtainedfrom DOPA-oxidase staining and from

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PIGMENTATtON IN CIRRHOSIS 407

Fig. IA. Epidermis of PBC patient 9, showing large numbers of melanosomes. Note persistence of pigmentin suprabasal eells. x 2400. . • - . .

^ #

Fig. IB. Giant compound melanosome in a basalkeratinocyte (patient 9) X 8250.

Fig. IC. Phagocytosed melanosomes in the dermis(patient 7) x 2100. ; . ,

low-power electronmicrographs, werehighly variable in both PBC and controlgroups, with no sigtiifieant differetice be-tween the groups (Table I). There was alsono correlation between the degree of elini-cal pigmentation and melanocyte:keratinoeyte ratios in either group.

b) Electron microscopyElectron microseopy showed that melano-eytes were ultrastrueturally normal in allpatients, containing few melanosomes andan occasional premelanosome. Keratino-cytes (Fig. IA), on the other hand, eon-tained grossly inereased numbers of

408 MILLS ET AL.

melanosomes in six out of eight PBC pa-tients studied and two out of the six eon-trols (Table 1). In most cases this eorre-lated with the elinical assessment of pig-mentation. Substantial numbers ofmelanosomes were observed in suprabasalkeratinoeytes in seven PBC patients andpatient 10 with aleoholic cirrhosis andpigmentation (Table 1). The presence ofunusually large eompound melanosomescontaining up to 40 individual melano-somes was noted in the same seven PBCpatients and also in patient 10. Dermalmelanosomes were observed in six out ofeight PBC patients studied eompared withtwo out of six controls. These melano-somes were present in all dermal eell types(e.g. fibroblasts, lymphocytes, mac-rophages) and in dermal appendages(nerves, ducts, vessels). (Fig. IB, IC).

c) ImmunofluorescenceImmunofluorescence studies using rabbitbile salt antibody revealed no evidence ofeonjugated primary bile salt deposition, indermis or epidermis, in any patient.

d) Radioimmunoassay of ^-MSH and ACTHThere was no significant difference inplasma P-MSH and ACTH levels betweenPBC patients and the controls. Meanplasma P-MSH values were 26.4ng/l(range: undeteeted = (ud.) - 58) for PBCpatients and 28.0 ng/1 (ud. - 86) for thecontrols. Mean ACTH values were 33.9ng/1 (ud. - 65) for PBC patients comparedwith 32.0 ng/1 (ud. - 80) for the controls.

Discussion

Increased skin pigmentation in PBC is there-fore due to the presenee of excess melaninwith a reasonable correlation between thenumber of melanosomes found in epidermalkeratinoeytes and the elinical grading of thedegree of pigmentation. There was no eon-

eomitant iron deposition in the skin of PBCpatients but this was observed in patients withalcoholic cirrhosis and haemoehromatosis.The reason for exeess melanisation eould beincreased produetion by epidermal melano-cytes, rapid transfer of melanosomes to sur-rounding keratinoeytes, a delay in melaninloss or a combination of these effeets.

The excess melanin in the skin of PBC pa-tients is not explained by an increasedmelanocyte population (Table 1). Ultra-structural observations revealed no evidence ofan increase in pre-melanosome formation ormelanosome produetion, though this possi-bility eannot be excluded. We found no hor-monal stimulus to exeess melanin produetionby P-MSH or ACTH and are in agreementwith Smith et al 1978 who suggest that theliver is probably not the site of degradation ofthese hormones. Skin irritation by bile saltshas been postulated as the eause for pruritus(Kirby, Heaton & Burton 1974) and eutane-ous pigmentation (Burton & Kirby 1975) inPBC. It was suggested that bile salts mighthave a non-speeifie eyfotoxie effeet in the skineausing release of proteolytie enzymes whichmay in turn activate epidermal tyrosinase.However, there is a poor eorrelation betweencutaneous bile salt concentrations andpruritus in patients with liver disease (James& Wongpaitoon 1980, Laneet editorial 1980)and in this study we have been unable to de-monstrate primary bile salt deposition in theskin as a stimulus for excess melanin produe-tion.

In normal tissue, melanosomes are de-graded in the suprabasal layers of the epider-mis by a mechanism whieh is thought to in-volve fusion of eompound melanosomes withlysosomes, and hydrolysis by lysosomal en-zymes (Olson, Nordquist & Everett 1970). InPBC patienfs, the presenee of eonsiderablenumbers of melanosomes in suprabasal layersof epidermis, together with unusually largeeompound melanosomes, suggests that

PIGMENTATION IN CIRRHOSIS 409

lysosomal melanosome degradation might bedefective. Alternatively, normal breakdownmechanisms may simply be inadequate to dealwith pigment over-produetion. However, bothsuprabasal and giant compound melanosomesare observed in PBC patients 1 and 4 in whomclinical pigmentation is slight and the totalamount of epidermal melanosomes, judgedultrastructurally, is within the normal range.With one exception in each group, both thesefeatures of abnormal persistence and packag-ing of melanosomes are found in all the PBCpatients and in none of the controls.

The combination of increased numbers ofmelanosomes and their presence in more sup-erficial layers of epidermis would both tend toinerease the pigmented appearance of the skinbut the packaging of melanosomes in largeclusters would have the opposite effect (Szaboet al. 1969). This may explain why PBC pa-tient 9, with very large numbers of melano-somes which are, however, all in giant clus-ters, had only a slight degree of clinical pig-mentation.

In conclusion, we have shown that cutane-ous pigmentation in PBC is due to excessmelanin which is widely dispersed throughoutthe epidermis, often accumulating into giantcompound melanosomes and frequentlyspilling over into the dermis. Similar changeswere seen in one patient with alcoholic cir-rhosis and skin pigmentation. Whether the ex-cess melanin results from increased produc-tion by melanocytes or defeetive lysosomaldegradation remains unresolved. We havebeen unable to demonstrate any stimulus toincreased melanin synthesis or ultrastructuralevidence of increased melanocyte activity.The evidence would perhaps tend to favourdefective degradation as the most likely causeof melanin accumulation. Histological studieson skin from patients with P-MSH stimulatedexcess melanin synthesis might help to furtherour knowledge.

Acknowledgment

We should like to thank Professor Ian Bouchier forkindly supplying rabbit antibody to primary bilesalts and Dr C. E. Gray for the hormone assays. DrG. Watkinson and Dr R. I. Russell kindly allowedus to study patients under their care.

ReferencesBaqir, Y. A., Murison, J., Ross, P. E. & Bouehier, I.

A. D. (1979) Radioimmunoassay of primary bilesalts in serum. Journal of Clinical Pathology 32,560-564.

Burton, J. L. & Kirby, J. (1975) Pigmentation andbihary eirrhosis. Lancet 1, 458.

Gray, C. E. & Ratcliffe, J. G. (f979) Clinicalevaluation of a radioimmunoassay for (i-MSH-related peptides (lipotrophins) in human plasma.Clinical Endoerinology 10, 163-172.

James, O. & Wongpaitoon, V. (1980) Cutaneousinterstitial fluid conjugated primary bile acid andalbumin concentrations in liver disease with orwithout pruritus. In: VI International bile acidmeeting. Falk Symposium No 29: Bile acid andlipids, p. 160-161, Freiburg (abstract).

Kirby, J., Heaton, K. W. & Burton, J. L. (1974)Pruritic effect of bile salts. British Medical Jour-nal 4, 693-695.

Lancet editorial: Itch (1980) Lancet 2, 568-569.MacKie, R. M. (1978) Disorders of cutaneous pig-

mentation. British Journal of Hospital Medicine20, 48-60.

Olson, R. L., Nordquist, J. & Everett, M. A. (1970)The role of epidermal lysosomes in melaninphysiology. British Journal of Dermatology 83,189-199.

Schaffner, F. (1975) Primary biliary cirrhosis. In:Cirrhosis Ed. H. Popper. Clinics in Gastroen-terology, Vol. 4, p. 351-366, W. B. Saunders Co.Ltd. London.

Shapiro, J. M., Smith, H. & Schaffner, F. (1979)Serum bilirubin: a prognostic factor in primarybiliary eirrhosis. Gut 20, 137-140.

Smith, A. G., Shuster, S., Camaish, J. S., Plummer,JM. A., Thody, A. J. Alvarex-Ude, F. & Kerr, D.N. S. (1975) Plasma immunoreactive (3-melano-cyte-stimulating hormone and skin pigmentationin chronie renal failure. British Medical Journal1, 658-659.

410 MILLS ETAL.

Smith, A. G., Shuster, S., Bomlord, A. & Williams,R. (1978) Plasma immunoreaetive p-melano-cyte-stimulating hormone in chronic liver diseaseand fulminant hepatie failure. Journal of lnves-tigative Dermatology 70, 326-327.

Szabo, G., Gerald, A. B., Pathak, M. A. & Fitzpat-rick, T. B. (1969) Racial differences in the fate ofmelanosomes in human epidermis. Nature 222,1081-1082. _,, .-._,,.- . .

Address:Dr P. R. MillsGastroenterology UnitRoyal InfirmaryCastle StreetGlasgow G4 OSF ' •Scotland. ••

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