light and electron microscopic of the liver in case of …phyria (epp) first described by magnus,...

J. clin. Path., 1974, 27, 698-709

A light and electron microscopic study of the liverin case of erythrohepatic protoporphyria andin griseofulvin-induced porphyria in miceA. MATILLA AND ELIZABETH A. MOLLAND

From the Institute ofPathology, The London Hospital

syNopsis The light- and electron-microscopic appearances of a liver biopsy from a case of erythro-hepatic protoporphyria are compared with the appearances of the liver in griseofulvin-inducedporphyria in mice. Crystals are present in hepatocytes and in the bile secretory pathways in eachcase. The nature and significance of these is discussed.

The porphyrias may be classified as 'hepatic' or'erythropoietic' according to whether the liver orbone marrow is the principal site of disturbed por-phyrin metabolism (Schmid, Schwartz, and Watson,1954). Usually included in the erythropoietic groupis the inherited disorder erythropoietic protopor-phyria (EPP) first described by Magnus, Jarrett,Prankerd, and Rimington (1961). It presents withphotosensitivity and is characterized by an excessof protoporphyrin in the circulating erythrocytesand in the stools. The site of the principal lesion isnot yet known but present evidence suggests theexistence of a hepatic as well as an erythropoieticlesion. Following reports of intrahepatic deposits ofprotoporphyrin in erythropoietic protoporphyriaby Cripps and Scheuer (1965), cases of cirrhosis andliver cell failure have been described (Barnes,Hurworth, and Millar, 1968; Donaldson, McCall,Magnus, Simpson, Caldwell, and Hargreaves, 1971;Schmidt and Stich, 1971; Thompson, Molland,Nicholson, and Gray, 1973), and the term 'erythro-hepatic protoporphyria' has been suggested(Donaldson et al, 1971) as a more comprehensivedescription of the disease.A disturbance of porphyrin metabolism charac-

terized by biochemical changes similar to those ofhuman erythropoietic protoporphyria and the depo-sition of protoporphyrin in liver cells follows theadministration of griseofulvin to mice (De Matteisand Rimington, 1963; Weston-Hurst and Paget1963), and 3 :5-dicarbethoxy 1 :4-dihydrocollidine(DDC) to guinea pigs, star and to weanling pigs(Granick and Urata, 1963; Magnus, Roe, andReceived for publication 30 May 1974.

Bhutani, 1969; Roe, Krook, and Wilkie, 1970). Wehave studied the light microscopic and ultrastruc-tural appearances of a liver biopsy from a patientwith erythropoietic protoporphyria who had ab-normal liver function tests and have compared themwith the morphological changes in the livers ofmice during the development of griseofulvin-inducedporphyria.

Materials and Methods

CASE REPORTA 30-year-old women had a history of photosensi-tivity for nine months. She had been taking the pill(Gynovlar) regularly for the previous 18 months.

Laboratory investigationsHaemoglobin 12-4 g/lOOml; ESR 41 mm in thefirst hour; alkaline phosphatase, 16-0 King-Arm-strong units/100 ml; serum glutamic oxaloacetictransaminase. 50 I units/100 ml (normal upper limit40 I units/100 ml); bilirubin 0-8 mg/100 ml.The bromsulphthalein/retention test showed 15%

retention at 45 minutes (normal: less than 5%).

Red cell porphyrinsFirst specimen: protoporphyrin 2195 ,uLgf100 ml,coproporphyrin 5 ,ug/100 ml; Second specimen:protoporphyrin 232 ,ug/100 ml, coproporphyrin3.4 ,ug/100 ml (normal: protoporphyrin 3-5-30 ,ug/100 ml, coproporphyrin 0-44-1 ,ug/100 ml).

Faecal porphyrinsFirst specimen: protoporphyrin 1578 ,ug/g of dry

698

copyright. on January 29, 2020 by guest. P

rotected byhttp://jcp.bm

j.com/

J Clin P

athol: first published as 10.1136/jcp.27.9.698 on 1 Septem

ber 1974. Dow

nloaded from

http://jcp.bmj.com/

A light and electron microscopic study of the liver

weight stool, coproporphyrin 51 ,g/g; second speci-men: protoporphyrin 567 ,ug/g, coproporphyrin31 ,ug/g (normal: protoporphyrin 0-113 ,ug/g,coproporphyrin 0-50 ,ug/g). 'X' porphyrins: normal.

Screening tests on faecal porphyrins from severalrelatives were normal. Biopsy of skin from the handshowed PAS-positive hyaline material around thepapillary vessels, characteristic of erythropoieticprotoporphyria.

Skin tests with narrow waveband artificial ultra-violet light (Magnus, Porter, McCree, Moreland,and Wright, 1959) showed abnormal reactions at360, 380, 400, and 500 mn. Ultraviolet microscopyof peripheral blood films revealed occasional fluores-cent red cells.

Liver biopsyThis was performed because of the abnormal liverfunction tests.The biopsy measured 1X7 cm in length. A portion

699

was postfixed in 1 % osmium tetroxide and embeddedin araldite. Ultra-thin sections were stained byuranyl acetate and lead citrate, and examined in theAEI EM6B electron microscope. As a reference forthe ultrastructural studies 1-2 p, sections were stainedwith toluidine blue. The remainder of the materialwas fixed in 4% formal saline, paraffin embedded,and 5 ,u sections were stained with haematoxylin andeosin, Gordon and Sweet's reticulin, Perls' Prussianblue, Fouchet, periodic acid-Schiff, Verhoeff-vanGieson, and Sudan Black. Unstained sections wereexamined under ultraviolet light, using a mercuryvapour light source and a Schott BG 12 primaryfilter with a barrier cut off at 500 m,u.

ANIMAL STUDIESTwenty male mice weighing about 25 g at the begin-ning of the experiment were given a standard pow-dered diet of Thompson's mixture and water adlibitum. After one week griseofulvin was added as a

,.gm~~~~~

Fig la Humanliver: granules in

cytoplasm, thrombiin canaliculiin Kupifer cells(A). H& E x 140.

Figib Viewed5- 5C i11 I under polarized

light: birefringent* 1 granules andMaltese crosses.

'f.. 1'1.0

lWi



j.com/

J Clin P


ber 1974. Dow

nloaded from

http://jcp.bmj.com/

A. Matilla and Elizabeth A. Molland

25% mixture by weight to the diet of 10 of theanimals. Thereafter they were sacrificed at inter-vals, beginning at 24 hours, and the experiment wascompleted five weeks later. Frozen sections of eachliver were examined immediately under ultravioletlight. A portion of each was fixed in 2% glutaralde-hyde, postfixed in osmium tetroxide, and processedas above for ultrastructural examination. The re-mainder was fixed in 4% formal saline, and treatedin the same way as the human material for lightmicroscopic examination.

Results

LIVER BIOPSY IN HUMAN ERYTHROHEPATICPROTOPORPHYRIA

Light microscopyThe architecture of the liver was normal. Deep red-brown granules were present within some of thehepatocytes, chiefly in the periportal areas and to alesser extent around central veins. Groups of cellsshowed swelling with vacuolation of their cyto-plasm. Kupffer cells were prominent, due to largeamounts of dark brown pigment in their cytoplasm.Some had formed multinucleate cells. Many canali-culi and bile ducts were occluded by dark brownthrombi (fig la). Pigment-containing macrophagesand scanty lymphocytes were also observed in the

portal tracts. Occasional inclusions were also ob-served in Kupffer cells and macrophages. There wasno bile duct proliferation.Viewed between crossed polars with a first order

quartz red compensator in position, weakly posi-tively birefringent pigment was seen within hepato-cytes, Kupffer cells, and macrophages (Bloomer,Davidson, and Klatskin, 1973). 'Maltese cross'figures were seen in some of the thrombi, and in thelarger granules in the hepatocytes (fig lb).

Fouchet's stain demonstrated bile in varyingamounts in most of the inclusions. The pigment wasPAS-positive, negative for iron, and Sudan Blacknegative.Examination of the pigment using ultraviolet

light revealed that it emitted dull red fluorescencewhich was strongest in the pigment masses in theKupffer cells and macrophages. Thrombi did notexhibit fluorescence at their centres, but had a rimof red fluorescence. There was no diffuse cytoplas-mic fluorescence, but some of the hepatocyte gran-ules exhibited weak fluorescence.

Electron microscopy of hepatocytesThe nuclei, endoplasmic reticulum and Golgiapparatus were normal. The mitochondria showedslight variation in size and measured up to 2-3 u inlength: some had lost some of their cristae and many

Fig 2 Human liver:membrane-boundvacuoles, containinglipid droplets andglycogen: crystalaggregates in cytoplasm(arrowed). Pb citrateand uranyl acetatex 10 000.

700

lk MOow

IA



j.com/

J Clin P


ber 1974. Dow

nloaded from

http://jcp.bmj.com/


Fig 3 Human liver:sublaminatedstructure of crystalsurrounded by a unitmembrane. Pb citrateand uranyl acetatex 160 000.

Fig 4 Human liver:crystals lying free incytoplasm, possiblyrelated tomitochondrion. Pbcitrate and uranylacetate x 42 000.(Inset x 126 000.)

701

.4k

41



j.com/

J Clin P


ber 1974. Dow

nloaded from

http://jcp.bmj.com/


Fig 5 Human liver: dilated canaliculus, containingthrombus ofcrystals and granular material. Pb citrateand uranyl acetate x 24 000.

contained electron-dense granules 40-60 nm dia-meter. Lipid droplets were present, some withinmembrane-bound, low-density structures also con-taining lamellar structures and particles of glycogen(fig 2). Densely osmiophilic vacuolated bodies, somehaving a myelin substructure, were also seen.Crystals, usually forming aggregates, were presentin the cytoplasm (fig 2). Each crystal had a denseborder 3-8 nm in thickness, and a less dense centrecomprising two or more layers 3 nm thick. They hadadiameter of 30 to 120 nm, and a length of 0-5 to1-5 p,. Some were enclosed by a unit membrane con-tinuous with the endoplasmic reticulum (fig 3);others appeared in close relationship to the outermembrane of mitochondria (fig 4); some were withinthe periphery of lipid droplets.The canaliculi showed variable dilatation, blunt-

ing, and loss of villi, and the formation of micro-villous blebs. Granular material and lamellar bodieswere present in the lumen. Many canaliculi wereoccluded by thrombi, measuring up to 14,u diameter,and containing granular material and crystals whichhad the same appearance as those seen in the cyto-plasm (fig 5).

<7t51 ~~~~Fig6 Hunmanliver: part of

3tes> Kupffer cell withinwhich is a thrombuscontaining bile (A);vacuoles containinglipid and crystals

. (arrowed), andi* l ~~whorls ofcrystals

bounded by a unitmembrane (B). Pb:fB=w^- citrate and uranyl

_ acetate x 16 000.Inset: crystals(arrowed) withinunit membranex 50 000.

702



j.com/

J Clin P


ber 1974. Dow

nloaded from

http://jcp.bmj.com/


Fig 7a Fig 7bFig 7a Mouse liver: portal tract with thrombi in bile ducts, bile duct proliferation, pigment (arrowed) in adjacentKupffer cells. H & E x 140.Fig 7b Viewed under polarized light: birefringent granules and Maltese crosses.

In the cytoplasm of the Kupffer cells there werenumerous phagolysosomes, containing lipid andcrystals at the periphery; whorls of crystals con-tained within a unit membrane were also present(fig 6). Some cells contained inclusions similar tothe canalicular thrombi.No portal tracts were present in the material.

MOUSE LIVER AFTER ADMINISTRATION OFGRISEOFULVIN

Light microscopyForty-eight hours after starting griseofulvin treat-ment there was a marked increase in mitotic activityof hepatocytes. Small dark brown granules alsoappeared and were initially situated on the canali-cular side of the hepatocyte. Kupffer cells werenormal.At 96 hours there were a few foci of eosinophilic

necrosis, associated with polymorphonuclear leuco-

cytes. Proliferation of bile ductules had occurred,and some extended into the parenchyma at the edgeof the limiting plate.At seven days dark brown thrombi appeared in the

canaliculi; they were also present in ductules andbile ducts in the portal tracts, where they wereassociated with a mild infiltration of lymphocytes(fig 7a).

After 10 days pigment accumulated in Kupffercells and multinucleate cells were present. Therewas considerable variation in the amount of pigmentformed in each liver. Granules of pigment wereidentified in vessels in the portal tracts of livers con-taining abundant pigment. Extensive foci of necrosisappeared. Groups of hepatocytes showed vacuola-tion of cytoplasm with the nucleus initially remainingintact; subsequently the cells dissolved and pigmentwas liberated, which was associated with a mildpolymorphonuclear cell infiltration.Viewed between crossed polars with a first order

703

1- ..:.K.-- -9,..-IA*

I4:4



j.com/

J Clin P


ber 1974. Dow

nloaded from

http://jcp.bmj.com/


quartz red compensator in position weakly posi-tively birefringent crystals appeared in the pigmentgranules of the hepatocytes at 48 hours. The bileduct and canalicular thrombi showed a 'Maltesecross' birefringence, and smaller 'Maltese crosses'were seen in the hepatocytes (fig 7b).

Examination using ultraviolet light showed adiffuse but rapidly fading red autofluorescence, firstvisible at 48 hours. The centres of the thrombi didnot fluoresce but persistent fluorescence occurredaround the periphery. There was also persistentfluorescence in the larger hepatocyte granules andKupffer cell pigment. A proportion of red cells,especially in the central veins, showed moderatefluorescence. The gallbladder mucosa was alsofluorescent.

All other tissues examined, including the spleenand bone marrow, showed no abnormalities.

Electron microscopy of hepatocytesChanges were first observed in the mitochondria at48 hours. There was marked size variation, with an18 % increase in average length, and the largestmeasured 4 ,u iln length. Swelling, separation ofcristae, bizarre shapes, and segmented mitochon-dria were seen (fig 8). Many appeared to have under-gone degeneration and were converted to osmio-philic ring-like bodies with a myelin substructure.Later. the mitochondria again became more uni-

form and rounded and peroxisomes containingparacrystalline inclusions appeared (fig 9). Occa-sional electron-dense granules were seen in themitochondrial matrix, but these were less numerousthan those in the human liver. There was some dila-tation of the cisternae of the rough endoplasmicreticulum in the early stages; the Golgi apparatusappeared normal and glycogen was present in nor-mal amounts. Osmiophilic vacuolated bodies wereseen in the later stages.

Crystals lying free in the cytoplasm were firstobserved at 72 hours. Subsequently they appearedas small aggregates, up to 2 p, diameter, many ofwhich were enclosed by a unit membrane continuouswith the endoplasmic reticulum and were concen-trated in the region adjacent to the canaliculus(fig 10). They were similar to those found in thehuman material but were smaller with an overallwidth of 6 to 40 nm and length of up to 05,u (fig 1 1).There was a dense border and a centre comprisingup to 10 or more layers 3 nm thick.

After eight days occasional canaliculi were dilatedand lamellar material appeared in the lumen. There-after the dilatation became more extensive, with lossof villi and microvillous bleb formation (fig 9).Some of the canaliculi appeared lined by lamellarbodies composed of either degenerate villi or bile.Aggregates of crystals appeared in the canaliculiduring the first week; subsequently thrombi com-

Fig 8 Mouse liver:elongated mito-chondria, showingbizarre shapes andsegmentation. Pbcitrate and uranylacetate x 30 000.

704



j.com/

J Clin P


ber 1974. Dow

nloaded from

http://jcp.bmj.com/


Fig 9 Mouse liver:dilated canaliculuswith partial loss ofvilli. Paracrystallineinclusions in adjacentperoxisomes(arrowed). Pb citrateand uranyl acetatex 14 500.

Fig 10 Mouse liver:crystal aggregateswithin endoplasmicreticulum adjacentto canaliculus;microvillous blebformation. Pb citrateand uranyl acetatex 20 000.

705

I lw. 'V.,A 14)" li4-,z--,,,2.11.."...,Ot:",._-..v-".,-.



j.com/

J Clin P


ber 1974. Dow

nloaded from

http://jcp.bmj.com/


Fig 11 Mouse liver:crystals lying free incytoplasm. Pb citrateand uranyl acetatex 136 000.

Fig 12 Mouse liver:thrombus in can-aliculus, containingcrystals and granularmaterial. Lamellarbodies at theperiphery. Pb citrateand uranyl acetatex 24 000.(Inset: aggregate ofcrystals in canaliculusx 22 000.)

706



j.com/

J Clin P


ber 1974. Dow

nloaded from

http://jcp.bmj.com/


..-.

Fig 13 Mouse liver:crystals within hetero-phagocytic vacuoles inKupffer cell. Pb citrateandwranyl acetatex 24 000

posed of crystals and granular material were present(fig 12) and their appearance varied according to therelative proportions of bile and crystalline material.Similar thrombi were seen in the bile ducts and duc-tules.

In the later stages crystals appeared in Kupffercells usually within heterophagocytic vacuoles wherethey formed randomly arranged aggregates some ofwhich were associated with lipid (fig 13). Some cry-stals were bounded by a unit membrane but theextensive whorls seen in the human Kupffer cellswere not observed. Occasional inclusions, resem-bling canalicular thrombi with a high concentrationof crystals, were present.

In well defined areas hepatocytes had undergonenecrosis and polymorphonuclear leucocytes werepresent. Aggregates of crystals, some bounded by aunit membrane, were present in these areas.Macrophages in the portal tracts contained large

numbers of heterophagocytic vacuoles enclosinglipid and crystals.

SPECTROSCOPY OF PIGMENT IN HUMAN ANDMOUSE LIVERThe emission spectrum of pigment in the human andanimal livers was measured using a Zeiss pupillaryspectroscope and found to be 630 m,u, and this was

identical to that of a standard solution of proto-porphyrin.

Discussion

The administration of griseofulvin to mice leads toan early increase of liver porphyrins, includingprotoporphyrin (Janousek, 1970). Other changesinclude an increase in the excretion of faecal proto-porphyrin and coproporphyrin followed by anincrease in erythrocyte protoporphyrin concentra-tion (De Matteis and Rimington, 1963). The effectof griseofulvin is due to inhibition of mitochondrialchelatase which leads to diminished incorporationof protoporphyrin into haem, lowering the level ofthe latter which normally acts as a negative feedbackon porphyrin synthesis. There is therefore a rise inthe ALA synthetase level (Nakao, Wada, Takaku,Sassa, Yano, and Urata, 1967), with an increase inall porphyrin intermediates and accumulation ofprotoporphyrin behind the block (fig 14).ALAsynthetase is located in themitochondria,and

an increase in mitochondrial diameters from 25 to50% has been described in the livers of DDC.treated guinea-pigs (Granick and Urata, 1963). Thepresent study confirms that the earliest changes occur

707



j.com/

J Clin P


ber 1974. Dow

nloaded from

http://jcp.bmj.com/


Succi

G6

Negative Feedback

5-ALA Synthetase MitochondrialChelatase

inyl CoA+ - o 5-ALA 3- Various porphyrins go Protoporphyrin / Haem

tycine.. ueo,~~~~~~~~~~~~~~~1

Bilirubin,other degradation products

--Site of action of griseofulvin

in the mitochondria, with an 18 increase in averagelength.

Protoporphyrin diffuses from its site of formationat the mitochondrion throughout the hepatocytecytoplasm and accounts for the rapidly fadingfluorescence seen throughout the cytoplasm of thecells in the mouse livers. Our spectroscopic studieshave confirmed that the crystals in the human andanimal livers contain protoporphyrin. The factorsdetermining their formation, however, remain un-known. The laminated structure with a dense outerborder suggests incorporation of an additionalcomponent such as a lipid or protein. The earlyappearance of aggregates within membranes in themouse liver suggests that the endoplasmic reticulummay contribute to their formation. Their locationadjacent to the canaliculus and association with bilein the canaliculus suggest either that protoporphyrinmay be handled by the cell in a similar manner tobilirubin or that it has an affinity for a constituentof bile. The increased concentration could lead tofurther crystallization and to the formation of throm-bi, causing intrahepatic obstruction. The crystals mayalso cause direct damage to the canalicular lining.The appearance of the necrosis in the mouse

liver is not specific, but is not that of intrahepaticobstruction. Moreover, it occurs at a stage beforesignificant numbers of thrombi are present. Perhapsa high concentration of protoporphyrin is toxic tothe hepatocyte and a direct effect of griseofulvincannot be excluded. There was no evidence of bac-erial or viral infection.The abnormalities present in the human liver

closely resemble those seen in the later stages in themouse liver. The early mitochondrial changes inthe animal liver were not observed in the human,

Fig 14 Inhibition of liver haem synthesis by griseofulvin.

whilst the absence of necrosis, and more extensivecrystals within the Kupffer cell endoplasmic reti-culum in the latter might suggest a lower concentra-tion of protoporphyrin, accumulating over a longperiod of time.

It has been suggested that both erythropoieticand liver porphyrin metabolism is disturbed in thegriseofulvin-induced disease in mice (De Matteisand Rimington, 1963). Nakao et al (1967) found noevidence of raised ALA synthetase levels or inhibi-tion of mitochondrial chelatase in the erythropoieticspleen in this model and suggested that the increasederythrocyte protoporphyrin is of hepatic origin.

Isotope studies provide conflicting evidence aboutthe principal site of disturbed porphyrin metabolismin the human disease (Gray, Kulczcka, Nicholson,Magnus, and Rimington, 1964; Scholnick, Marver,and Schmid, 1969; Schwartz, Johnson, Stephenson,Anderson, Edmundson, and Fusaro, 1970). In theirclinical study of two cases, Cripps and MacEachern(1971) suggested that both liver and bone marrowwere affected. The erythrocyte protoporphyrinlevels in our case were lower than those in the lattercases, whilst our stool protoporphyrin levels werehigher. This may indicate a principal hepatic lesionin our case.Using a chick liver embryo cell culture, Granick

(1963) demonstrated the porphyria-inducing effectof griseofulvin and sex steroids. Although oestrogenichormones do not cause increased porphyrin excre-tion in normal individuals it has been reported thatthey may precipitate or aggravate acute intermittentporphyria and porphyria cutanea tarda with eleva-tion of liver ALA synthetase (Zimmerman, McMillin,and Watson, 1966). An effect of these hormones inprotoporphyria seems not to have been recorded.

708

Haemoproteinsr-%,-



j.com/

J Clin P


ber 1974. Dow

nloaded from

http://jcp.bmj.com/

Separation of abnormal cells in the peripheral blood by means of the zonal centrifuge

However, the pattern of porphyrin excretion andaccumulation depends on the relative amounts ofALA synthetase and protoporphyrin utilization.Thus low doses of DDC produces porphyria in ratswhich is similar to human protoporphyria, whilsthigher doses of DDC induce a condition similar toporphyria cutanea tarda (Magnus et al, 1969).The morphological similarities between the animal

human livers suggest a primary hepatic lesion ineach case, but they do not provide information aboutthe mechanisms leading to protoporphyrin accumu-lation. It is reasonable to suggest, however, that theremay be cases within the group of erythrohepaticprotoporphyrias where the principal lesion is inthe liver. In the case reported here the pill may haveelevated the liver ALA synthetase level sufficientlyto cause accumulation of protoporphyrin in a pre-viously abnormal but compensated system.

We wish to thank Dr P. R. Montgomery and Dr C.Ramsay for clinical information and material; Dr J.Landells for the liver biopsy; Mr Ken Swettenhamand Mr Alan Gray for technical assistance; ProfessorI. A. Magnus, Dr F. De Matteis, and Professor I.Doniach for help and advice.

References

Barnes, H. D., Hurworth, E., and Millar, J. H. D. (1968). Erythro-poietic porphyrin hepatitis. J. clin. Path., 21, 157-159.

Bloomer, J. R., Davidson, D. L., and Klatskin, G. (1973). Birefringentpigment is a diagnostic hepatic lesion in protoporphyria.Gastroenterology, 65, A-5/529.

Cripps, D. J., and MacEachern, W. N. (1971). Hepatic and erythro-poietic protoporphyria. Arch. Path., 91, 497-505.

Cripps, D. J., and Scheuer, P. J. (1965). Hepatobiliary changes inerythropoietic protoporphyria. Arch. Path., 80, 500-508.

De Matteis, F., and Rimington, C. (1963). Disturbance of porphyrinmetabolism caused by griseofulvin in mice. Brit. J. Derm., 75,91-104.

Donaldson, E.M., McCall, A. J., Magnus, I. A., Simpson, J. R.,Caldwell,R. A., and Hargreaves, T. (1971). Erythropoietic protopor-phyria: two deaths from hepatic cirrhosis. Brit. J. Derm.. 84,14-24.

Granick, S. (1966). The induction in vitro of the synthesis of 8-amino-levulinic acid synthetase in chemical porphyria: a response

to certain drugs, sex hormones, and foreign chemicals. J. blolChem., 241, 1359-1375.

Granick, S., and Urata, G. (1963). Increase in activity of 8-amino-levulinic acid synthetase in liver mitochondria induced byfeeding of 3, 5- dicarbethoxy-1, 4-dihydrocollidine. J. biol.Chem., 238, 821-827.

Gray, C. H., Kulczycka, A., Nicholson, D. C., Magnus, I. A., andRimington, C. (1964). Isotope studies on a case of erythro-poietic protoporphyria. Clin. Sci., 26, 7-15.

Janousek, V. (1970). The early changes in experimental porphyria ofmice induced by griseofulvin. Trans. St. John's Hosp. Derm.Soc., 56, 14-18.

Magnus, I.A., Jarrett, A., Prankerd, T. A. J., and Rimington, C. (1961).Erythropoietic protoporphyria: a new porphyria syndromewith solar urticaria due to protoporphyrinaemia. Lancet, 2,448-451.

Magnus, I. A., Porter, A. D., McCree, K. J., Moreland, J. D., andWright, W. D. (1959). A monochromater: an apparatus for theinvestigation of the responses of the skin to ultra-violet, visibleand near infra-red radiation. Brit. J. Derm., 71, 261-266.

Magnus, 1. A., Roe, D. A., and Bhutani, L. K. (1969). Factors affectingthe induction of porphyria in the laboratory rat: biochemicaland photobiological studies using diethyl 1, 4-dihydro-2,4, 6-trimethylpyridine-3, 5-dicarboxylate (DDC) as a porphyro-genic agent. J. invest. Derm., 53, 400-413.

Nakao, K., Wada, O., Takaku, F., Sassa, S., Yano, Y., and Urata, G.(1967). The origin of the increased protoporphyria in erythro-cytes of mice with experimentally induced porphyria. J. Lab.clin. Med., 70, 923-932.

Porter, F S., and Lowe, Betty A. (1963). Congenitalery thropoieticprotoporphyria. I. Case reports, clinical studies and porphyrinanalysis in two brothers. Blood, 22, 521-531.

Roe, D. A., Krook, L., and Wilkie, B. N. (1970). Hepatic proto-porphyria in weanling pigs. J. invest. Derm., 54, 53-64.

Schmid, R., Schwartz, S., and Watson, C. J. (1954). Porphyrin con-tent ofbone marrow and liver in the various forms of porphyria.Arch. intern. Med., 93, 167-190.

Schmidt, D., and Stich, W. (1971). Erythropoetische Protopor-phyrie mit Porphyrinurie. Blut, 22, 202-210.

Scholnick, P., Marver, H., and Schmid, R. (1969). Erythropoieticprotoporphyria: a misnomer. Clin. Res., 17, 278.

Schwartz, S., Johnson, J. A., Stephenson, B. D., Anderson, A. S.,Edmondson, P. R., and Fusaro, R. M. (1970). Erythropoieticdefects in protoporphyria: a reevaluation ofearly peak labelling.J. Lab. clin. Med., 76, 893-894.

Thompson, R. P. H., Molland, E. A., Nicholson, D. C., and Gray,C. H. (1973). 'Erythropoietic' protoporphyria and cirrhosis insisters. Gut, 14, 934.

Waterfield, M .D., Favero, A. del, and Palfrey, A. J. (1969). Electron-microscopic changes in the livers of mice treated with the por-phyrinogenic agent 1 ,4-dihydro-3, 5-dicarbethoxycollidine. J.Path., 98, 151-154.

Weston-Hurst, E., and Paget, G. E. (1963). Protoporphyrin, cirrhosisand hepatomata in the livers of mice given Griseofulvin. Brit. J.Derm., 75, 105-112.

Zimmerman, T. S., McMillin, J. M., and Watson, C. J. (1966). Onsetof manifestations of hepatic porphyria in relation to the in-fluence of female sex hormones. Arch. intern. Med., 118, 229-240.

2

709



j.com/

J Clin P


ber 1974. Dow

nloaded from

http://jcp.bmj.com/

light and electron microscopic of the liver in case of …phyria (epp) first described by magnus,...

Documents