Yonago Acta medica 1999;42:135–140

Crystalline Inclusions in Hepatocyte Mitochondria of a Patient withPorphyria Cutanea Tarda
Ryota Kashiwagi, Kinya Okamoto, Naoto Maeda, Yutaka Horie, Hironaka
Kawasaki, Hitoshi Osatake* and Takao Inoué*
Second Department of Internal Medicine and *Second Department of Anatomy, Faculty ofMedicine, Tottori University, Yonago 683-0826, Japan
Intramitochondrial crystalline inclusions were found in hepatocytes of a patient withporphyria cutanea tarda (PCT). They were composed of parallel filamentous structures
which measured approximately 12 nm in diameter. Each filament was separated froman adjacent filament by a space measuring approximately 5 nm. The mitochondria
containing such inclusions were usually elongated and enlarged. It seemed likely thatthese changes are not particular in PCT, but indicate one reversible pathological finding
in the liver.
Key words: intramitochondrial crystalline inclusions; liver; porphyria cutanea tarda; transmission electron
microscopy

Hepatic intramitochondrial inclusions, referred toas crystalline (Svoboda and Manning, 1964), crystalloid (Haust, 1968), paracrystalline (Ruffoloand Covington, 1967), or filamentous inclusion(Mugnaini, 1964), have been thus far reported.
The ultrastructure of the inclusion is characterized by the presence of closely packed filamentous 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-Johnson
syndrome (Ruffolo and Covington, 1967),
Wilson’s disease (Sternlieb, 1968), mucopolysaccharidosis (Haust, 1968), diabetes mellitus(Bhagwat and Ross, 1971), metastatic hypernephroma (Djaldetti et al., 1974), porphyria cutanea 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 experimental animals (Watrach, 1964; Simpson et al.,
1974; Reid et al., 1978; Wilson-Martino et al.,1980). In addition, they were reported even in
normal human livers (Mugnaini, 1964; Wills,1965) but their pathological significance remains controversial.

PCT, clinically characterized by cutaneousfragility, photosensitivity and hepatic dysfunction, is due to reduced activity of the enzyme
uroporphyrinogen decarboxylase. We foundintramitochondrial crystalline structures in theliver biopsy from a patient with PCT. In thispaper, we describe ultrastructures of the crystalline inclusions and compare ultrastructural features of the inclusions thus far reported.

Materials and Methods
Liver 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 containing 1.0% glutaraldehyde, 2.0% paraformaldehyde, 0.03% CaCl 2 and 0.02% MgCl 2 in 0.1mol/L cacodylate buffer (pH 7.4). A part of thespecimen was cut into small pieces, postfixed in
1% osmium tetroxide, dehydrated in a graded
series of ethanols, and finally embedded in Epon
812 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 (JEM100CXII, JEOL Ltd., Tokyo, Japan).

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Results
Characteristic findings of the biopsy specimen
were as follows: The smooth endoplasmic reticulum in some hepatocytes was hyperplastic withdilation of the cisternae. The peroxisomes weresometimes increased in number, and their limiting membranes were partially continuous withthe smooth endoplasmic reticulum. Needle-likestructures were often observed (Fig. 1). The
core of the structure was electron-translucent
and surrounded by lysosomal substances. The
shape of the mitochondria was not spherical as
observed in the normal liver, but was somewhat
enlarged, 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 structures
oriented parallel to the longitudinal axis of themitochondria (Fig. 2). In cross sections, thefilaments were arranged in equilateral triangle
pattern, forming a hexagonal appearance as awhole (Fig. 3b). Each filament measured approximately 12 nm in diameter and was separated from an adjacent filament by a space measuring approximately 5 nm. At high magnification, the filaments were composed of massive
electron-dense substances without hollow
structures (Figs. 2b and 3b).

Discussion
The ultrastructures of liver porphyria have been
described by Waldo and Tobias (1973), Biempica
and colleagues (1974) and Siersema and colleagues (1992). The most characteristic featureof the porphyria is the presence of needle-likestructures in the hepatocytes. The structures represent uroporphyrin crystals, since uroporphyrin, crystallized in vitro, displays the same

Fig. 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.

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Crystalline inclusions in hepatocyte mitochondria

Fig. 2. Longitudinal sections of crystalline inclusions. a: The crystalline inclusions are composed of closely
packed filamentous structures arranged parallel to the longitudinal axis of the mitochondria. b: Higher
magnification of the intramitochondrial inclusion. Each filamentous structure comprises electron-dense
substances of approximately 12 nm in diameter.

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Fig. 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 intramitochondrial inclusion. Filaments are arranged in an equilateral triangle pattern, forming a hexagonal appearance as a whole. Each filament is separated from an adjacent filament by a space of approximately 5 nm.

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Table 1. Intramitochondrial filamentous structures reported in the human liverYear
1964
1966
1967
1970
1974
1968
1965
1968
1971

AuthorMugnaini
Sandborn et al.
Ruffolo and Covington
Lundbergh and Westman
Djaldetti et al.
Sternlieb
Wills
Haust
Bhagwat and Ross

Diameter* Space†

Condition66
6 – 7
5 – 15
6 – 8 107–
7.5– 8.5 7.5– 8.5
8
14.5–16‡
8 – 10 20
8 – 10 15 – 20
8 – 10 20‡

Normal, alcoholic dyspepsia
Weil’s disease
Dubin-Johnson syndrome, chronic viral hepatitis
Oral contraceptive use
Metastatic hypernephroma of the liver
Wilson’s disease
NormalMucopolysaccharidosis
Infectious hepatitis, type 1 hyperlipoproteinemia,
diabetes mellitus
Mushroom poisoning (Amanita verna)
Chronic alcoholism

1969 Panner and Hanss
1010–15
1964 Svoboda and Manning1220* Diameter of the filament (nm).
† Space between the filaments (nm); ‡ center to center spacing.

ultrastructural characteristics (Siersema et al.,1992). The ultrastructure of needle-like structures observed in this study is identical to that reported by Siersema and coworkers (1992). Theultrastructural findings observed in the smooth
endoplasmic reticulum and peroxisomes arecharacteristic features in alcoholic liver injury(Tanikawa, 1979).
The dimensions of the filamentous structures thus far reported are summarized in Table
1. The diameter of the individual filament ranges
from 6 to 12 nm with the space of 5 to 20 nm.
However, there are some confusions on the measurement of the space between filaments. Although the space was reported to be 20 nm (Wills,1965) and 15 to 20 nm (Haust, 1968), the actual
size measured from the micrographs was approximately 5 nm. Consequently, the ultrastructuraldimension of the filaments in this study is identical to that described by Wills (1965) andHaust (1968).
The inclusions have been more frequently
reported in diseased human livers than in normal human livers (Tange, 1981). Experimentally, Simpson and colleagues (1974) reported
that the inclusions were produced in the normal
dog after treatment with levamisole. The inclusions appear to be one of reversible pathologicalconditions: Svoboda and Manning (1964) reported that the inclusions found in chronic alcoholicpatients diminished after 3 months on an adequate
diet with elimination of alcohol intake.

The origin of the crystalline inclusions has
been discussed. Svoboda and Manning (1964)
speculated they are derived from mitochondrial
cristae, based on the following morphological evidence: i) the continuity of the inclusions withthe cristae; and ii) the decrease of the cristae.
Sternlieb and Berger (1969) also showed a close
relationship between the mitochondrial cristae
and inclusions: the mitochondrial cristae are arranged in parallel with each other at an angle ofabout 130˚ in relation to the long axis of the filamentous inclusions. Furthermore, their optical
diffraction studies indicated that they are phospholipid micelles or large protein molecules. In
addition, according to Bhagwat and Ross (1971),
electron microscopic cytochemistry showed theinclusions to be rich in cytochrome oxidase andsuccinic dehydrogenase activity.
Considering from some enzymatic activity
on cell injury, Mugnaini (1964) speculated thatthe intramitochondrial inclusions are one typeof non-specific reaction. Uchida and colleagues(1984) showed that the elongated mitochondria
containing inclusions have a normal function
from their histochemical studies. Recently,
Daugherty and coworkers (1996) proposed a
new idea on the formation of intramitochondrial
inclusions based on hemodynamic alterations.
They examined the ultrastructures of the liverobtained from patients with “cavernous transformation of the portal vein”. Consequently,
they considered that the deficiency of metabolic139

R. Kashiwagi et al.

substrates causes production of mitochondrial
enzymes, thus producing the enlarged mitochondria containing inclusions. Although many
researchers have regarded the crystalline inclusions as the expression of degeneration, it
seems likely that they are adapting in responseto the metabolic disorder as was discussed byDaugherty and colleagues (1996).

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(Received December 7, Accepted December 21, 1998)

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