Intramitochondrial crystalline inclusions were found in hepatocytes of a patient with porphyria cutanea tarda (PCT). They were composed of parallel filamentous structures which measured approximately 12 nm in diameter. Each filament was separated from an adjacent filament by a space measuring approximately 5 nm. The mitochondria containing such inclusions were usually elongated and enlarged. It seemed likely that these changes are not particular in PCT, but indicate one reversible pathological finding in the liver

Horie, Yutaka

Inoue, Takao

Kashiwagi, Ryota

Kawasaki, Hironaka

Maeda, Naoto

Okamoto, Kinya

Osatake, Hitoshi

Yonago Acta Medica

Tottori University Research Result Repository

135Yonago Acta medica 1999;42:135–140Crystalline Inclusions in Hepatocyte Mitochondria of a Patient withPorphyria Cutanea TardaRyota Kashiwagi, Kinya Okamoto, Naoto Maeda, Yutaka Horie, HironakaKawasaki, Hitoshi Osatake* and Takao Inoué*Second Department of Internal Medicine and *Second Department of Anatomy, Faculty ofMedicine, Tottori University, Yonago 683-0826, JapanIntramitochondrial crystalline inclusions were found in hepatocytes of a patient withporphyria cutanea tarda (PCT).  They were composed of parallel filamentous structureswhich measured approximately 12 nm in diameter.  Each filament was separated froman adjacent filament by a space measuring approximately 5 nm.  The mitochondriacontaining such inclusions were usually elongated and enlarged.  It seemed likely thatthese changes are not particular in PCT, but indicate one reversible pathological findingin the liver.Key words:  intramitochondrial crystalline inclusions; liver; porphyria cutanea tarda; transmission electronmicroscopyHepatic intramitochondrial inclusions, referred toas crystalline (Svoboda and Manning, 1964), crys-talloid (Haust, 1968), paracrystalline (Ruffoloand Covington, 1967), or filamentous inclusion(Mugnaini, 1964), have been thus far reported.The ultrastructure of the inclusion is character-ized by the presence of closely packed filamen-tous structures in the enlarged mitochondria.The inclusions were found in human liverin various conditions such as Weil’s disease(Sandborn et al., 1966),  chronic viral hepatitis(Ruffolo and Covington, 1967), Dubin-Johnsonsyndrome (Ruffolo and Covington, 1967),Wilson’s disease (Sternlieb, 1968), mucopoly-saccharidosis (Haust, 1968),  diabetes mellitus(Bhagwat and Ross, 1971), metastatic hyper-nephroma (Djaldetti et al., 1974),  porphyria cuta-nea tarda (PCT) (Waldo and Tobias, 1973;Blekkenhorst et al., 1976), hyperthyroidism(Mandel et al., 1976), alcoholism  (Uchida et al.,1984), morbid obesity (Boon et al., 1988), and soon.  The inclusions were also found in experi-mental animals (Watrach, 1964; Simpson et al.,1974; Reid et al., 1978; Wilson-Martino et al.,1980).  In addition, they were reported even innormal human livers (Mugnaini, 1964; Wills,1965) but their pathological significance re-mains controversial.PCT, clinically characterized by cutaneousfragility, photosensitivity and hepatic dysfunc-tion, is due to reduced activity of the enzymeuroporphyrinogen decarboxylase.  We foundintramitochondrial crystalline structures in theliver biopsy from a patient with PCT.  In thispaper, we describe ultrastructures of the crystal-line inclusions and compare ultrastructural fea-tures of the inclusions thus far reported.Materials and MethodsLiver biopsy in a 57-year-old male with PCT,chronic hepatitis type C and alcoholic intake forabout 30 years, was performed.  The tissues wereimmediately fixed in a fixative solution contain-ing 1.0% glutaraldehyde, 2.0% paraformalde-hyde, 0.03% CaCl2 and 0.02% MgCl2 in 0.1mol/L cacodylate buffer (pH 7.4).  A part of thespecimen was cut into small pieces, postfixed in1% osmium tetroxide, dehydrated in a gradedseries of ethanols, and finally embedded in Epon812 through propylene oxide.  Ultrathin sections(60 nm) were cut by an ultramicrotome(Ultracut UCT, Leica, Austria), stained withuranyl acetate and lead citrate, and examinedwith a transmission electron microscope (JEM-100CXII, JEOL Ltd., Tokyo, Japan).R. Kashiwagi et al.136ResultsCharacteristic findings of the biopsy specimenwere as follows:  The smooth endoplasmic reticu-lum in some hepatocytes was hyperplastic withdilation of the cisternae.  The peroxisomes weresometimes increased in number, and their lim-iting membranes were partially continuous withthe smooth endoplasmic reticulum.  Needle-likestructures were often observed (Fig. 1).  Thecore of the structure was electron-translucentand surrounded by lysosomal substances.  Theshape of the mitochondria was not spherical asobserved in the normal liver, but was somewhatenlarged, elongated and partially narrowed.  Somematrix granules contained electron-translucentarea (Figs. 2a and 3a).Crystalline inclusions were often observedin the enlarged mitochondria (Figs. 2 and 3).They were composed of filamentous structuresoriented parallel to the longitudinal axis of theFig. 1.  Transmission electron micrograph of a needle-like structure in a hepatocyte of a patient withporphyria cutanea tarda.  The needle is electron-translucent and surrounded by lysosomal substances(arrows).  Arrowheads show crystalline inclusions in the mitochondria.mitochondria (Fig. 2).  In cross sections, thefilaments were arranged in equilateral trianglepattern, forming a hexagonal appearance as awhole (Fig. 3b).  Each filament measured ap-proximately 12 nm in diameter and was sepa-rated from an adjacent filament by a space mea-suring approximately 5 nm.  At high magnifi-cation, the filaments were composed of massiveelectron-dense substances without hollowstructures (Figs. 2b and 3b).DiscussionThe ultrastructures of liver porphyria have beendescribed by Waldo and Tobias (1973), Biempicaand colleagues (1974) and Siersema and col-leagues (1992).  The most characteristic featureof the porphyria is the presence of needle-likestructures in the hepatocytes. The structures rep-resent uroporphyrin crystals, since uropor-phyrin, crystallized in vitro, displays the sameCrystalline inclusions in hepatocyte mitochondria137Fig. 2.  Longitudinal sections of crystalline inclusions.  a:  The crystalline inclusions are composed of closelypacked filamentous structures arranged parallel to the longitudinal axis of the mitochondria.  b:  Highermagnification of the intramitochondrial inclusion.  Each filamentous structure comprises electron-densesubstances of approximately 12 nm in diameter.R. Kashiwagi et al.138Fig. 3.  Cross sections of crystalline inclusions.  a:  The inclusions are regularly spaced and closely packed.Arrows show matrix granules with electron-translucent area.  b:  Higher magnification of the intra-mitochondrial inclusion.  Filaments are arranged in an equilateral triangle pattern, forming a hexagonal ap-pearance as a whole.  Each filament is separated from an adjacent filament by a space of approximately 5 nm.Crystalline inclusions in hepatocyte mitochondria139ultrastructural characteristics (Siersema et al.,1992).  The ultrastructure of needle-like struc-tures observed in this study is identical to that re-ported by Siersema and coworkers (1992).  Theultrastructural findings observed in the smoothendoplasmic reticulum and peroxisomes arecharacteristic features in alcoholic liver injury(Tanikawa, 1979).The dimensions of the filamentous struc-tures thus far reported are summarized in Table1.  The diameter of the individual filament rangesfrom 6 to 12 nm with the space of 5 to 20 nm.However, there are some confusions on the mea-surement of the space between filaments.  Al-though the space was reported to be 20 nm (Wills,1965) and 15 to 20 nm (Haust, 1968), the actualsize measured from the micrographs was approx-imately 5 nm.  Consequently, the ultrastructuraldimension of the filaments in this study is iden-tical to that described by Wills (1965) andHaust (1968).The inclusions have been more frequentlyreported in diseased human livers than in nor-mal human livers (Tange, 1981).  Experimen-tally, Simpson and colleagues (1974) reportedthat the inclusions were produced in the normaldog after treatment with levamisole.  The inclu-sions appear to be one of reversible pathologicalconditions:  Svoboda and Manning (1964) report-ed that the inclusions found in chronic alcoholicpatients diminished after 3 months on an adequatediet with elimination of alcohol intake.The origin of the crystalline inclusions hasbeen discussed.  Svoboda and Manning (1964)speculated they are derived from mitochondrialcristae, based on the following morphological evi-dence:  i) the continuity of the inclusions withthe cristae; and ii) the decrease of the cristae.Sternlieb and Berger (1969) also showed a closerelationship between the mitochondrial cristaeand inclusions:  the mitochondrial cristae are ar-ranged in parallel with each other at an angle ofabout 130˚ in relation to the long axis of the fila-mentous inclusions.  Furthermore, their opticaldiffraction studies indicated that they are phos-pholipid micelles or large protein molecules.  Inaddition, according to Bhagwat and Ross (1971),electron microscopic cytochemistry showed theinclusions to be rich in cytochrome oxidase andsuccinic dehydrogenase activity.Considering from some enzymatic activityon cell injury, Mugnaini (1964) speculated thatthe intramitochondrial inclusions are one typeof non-specific reaction.  Uchida and colleagues(1984) showed that the elongated mitochondriacontaining inclusions have a normal functionfrom their histochemical studies.  Recently,Daugherty and coworkers (1996) proposed anew idea on the formation of intramitochondrialinclusions based on hemodynamic alterations.They examined the ultrastructures of the liverobtained from patients with “cavernous trans-formation of the portal vein”.  Consequently,they considered that the deficiency of metabolicTable 1.  Intramitochondrial filamentous structures reported in the human liverYear Author Diameter* Space† Condition1964 Mugnaini   6   6 Normal, alcoholic dyspepsia1966 Sandborn et al.   6  –  7   5  – 15 Weil’s disease1967 Ruffolo and Covington   6  –  8 10 Dubin-Johnson syndrome, chronic viral hepatitis1970 Lundbergh and Westman   7  – Oral contraceptive use1974 Djaldetti et al.   7.5– 8.5   7.5–  8.5 Metastatic hypernephroma of the liver1968 Sternlieb   8 14.5–16‡ Wilson’s disease1965 Wills   8 – 10 20 Normal1968 Haust   8 – 10 15  – 20 Mucopolysaccharidosis1971 Bhagwat and Ross   8 – 10 20‡ Infectious hepatitis, type 1 hyperlipoproteinemia,diabetes mellitus1969 Panner and Hanss 10 10–15 Mushroom poisoning (Amanita verna)1964 Svoboda and Manning 12 20 Chronic alcoholism* Diameter of the filament (nm).† Space between the filaments (nm); ‡ center to center spacing.R. Kashiwagi et al.140substrates causes production of mitochondrialenzymes, thus producing the enlarged mito-chondria containing inclusions.  Although manyresearchers have regarded the crystalline in-clusions as the expression of degeneration, itseems likely that they are adapting in responseto the metabolic disorder as was discussed byDaugherty and colleagues (1996).References 1 Bhagwat AG, Ross RC.  Hepatic intramito-chondrial crystalloids.   Arch Pathol 1971;91:70–77. 2 Biempica L, Kosower N, Ma MH, Goldfischer S.Hepatic porphyrias. Cytochemical and ultra-structural studies of liver in acute intermittentporphyria and porphyria cutanea tarda.  ArchPathol 1974;98:336–343. 3 Blekkenhorst GH, Pimstone NR, Webber BL,Eales L.  Hepatic haem metabolism in porphyriacutanea tarda (PCT):  enzymatic studies and theirrelation to liver ultrastructure.  Ann  Clin Res1976;8:108–121. 4 Boon AP, Thompson H, Baddeley RM.  Use ofhistological examination to assess ultrastructureof liver in patients with long standing jejuno-ilealbypass for morbid obesity.  J Clin Pathol 1988;41:1281–1287. 5 Daugherty CC, Yazigi N, Bove K, Balistreri W.Mitochondrial enlargement and crystalloidmatrix arrays:  distinctive finding in childhoodportal hypertension due to cavernous trans-formation of the portal vein.  Pediatr Pathol LabMed 1996;16:263–274. 6 Djaldetti M, Mandel EM, Fishman P, Bessler H,Lewinski U.  Intramitochondrial crystalline in-clusions in metastatic hypernephroma of theliver.  Biomedicine 1974;21:158–163. 7 Haust MD.  Crystalloid structures of hepaticmitochondria in children with heparitin sulphatemucopolysaccharidosis (Sanfilippo type).  ExpMol Pathol 1968;8:123–134. 8 Lundbergh P, Westman J.  Hepatic filamentousmitochondrial inclusions associated with oralcontraceptives.  Scand J Infect Dis 1970;2:105–109. 9 Mandel EM, Fishman P, Kissin E, Har-Zaav L,Djaldetti M.  Intramitochondrial crystalline inclu-sions in the liver of a patient with hyperthyroidism.Acta Hepato-Gastroenterol 1976;23:182–186.10 Mugnaini E.  Filamentous inclusions in the ma-trix of mitochondria from human livers.  J Ultra-struct Res 1964;11:525–544.11 Panner BJ, Hanss RJ.  Hepatic injury in mush-room poisoning.  Arch Pathol 1969;87:35–45.12 Reid IM, Donaldson IA, Heitzman RJ.  Effects ofanabolic steroids on liver cell ultrastructure insheep.  Vet Pathol 1978;15:753–762.13 Ruffolo R, Covington H.  Matrix inclusion bodiesin the mitochondria of the human liver.  Am JPathol 1967;51:101–116.14 Sandborn EB, Côté MG, Viallet A.  Electronmicroscopy of a human liver in Weil’s disease(leptospirosis icterohaemorrhagica).  J PatholBacteriol 1966;92:369–374.15 Siersema PD, van Helvoirt RP, Cleton-SoetemanMI, de Bruijn WC, Wilson JHP, van Eijk HG.The role of iron in experimental porphyria andporphyria cutanea tarda.  Biol Trace Elem Res1992;35:65–72.16 Simpson CF,  Bradley RE,  Jackson RF.Crystalloid inclusions in hepatocyte mitochon-dria of dogs treated with levamisole.  Vet Pathol1974;11:129–137.17 Sternlieb I.  Mitochondrial and fatty changes inhepatocytes of patients with Wilson’s disease.Gastroenterology 1968;55:354–367.18 Sternlieb I, Berger JE.  Optical diffraction studies ofcrystalline structures in electron micrographs II.Crystalline inclusions in mitochondria of humanhepatocytes.  J Cell Biol 1969;43:448–455.19 Svoboda DJ, Manning RT.  Chronic alcoholismwith fatty metamorphosis of the liver.  Am JPathol 1964;44:645–662.20 Tange T.  Histopathological identification ofhepatic intramitochondrial crystalloid inclusions.Acta Pathol Jpn 1981;31:747–754.21 Tanikawa K.  Ultrastructural aspects of the liverand its disorders.  2nd ed.  Tokyo: Igaku-Shoin;1979.  p. 286–291.22 Uchida T, Kronborg I, Peters RL.  Giant mito-chondria in the alcoholic liver diseases—theiridentification, frequency and pathologic signifi-cance.  Liver 1984;4:29–38.23 Waldo ED, Tobias H.  Needle-like cytoplasmicinclusions in the liver in porphyria cutanea tarda.Arch Pathol 1973;96:368–371.24 Watrach AM.  Degeneration of mitochondria inlead poisoning.  J Ultrastruct Res 1964;10:177–181.25 Wills EJ.  Crystalline structures in the mito-chondria of normal human liver parenchymalcells.  J Cell Biol  1965;24:511–514.26 Wilson-Martino NA, Martino LJ, Millman-FederNG, Benitz KF.  The presence of hepatic intra-mitochondrial crystalline inclusions in polybro-minated biphenyl-treated mice.  Arch Toxicol1980;45:233–239.(Received December 7, Accepted December 21, 1998)

Crystalline Inclusions in Hepatocyte Mitochondria of a Patient with Porphyria Cutanea Tarda

http://repository.lib.tottori-u.ac.jp/files/public/0/921/20180622155752811279/42_135-140.pdf

Crystalline Inclusions in Hepatocyte Mitochondria of a Patient with Porphyria Cutanea Tarda

Abstract

Similar works

Full text

Available Versions

Tottori University Research Result Repository