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Deficiency of subunits of complex I and mitochondrial encephalomyopathy.

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Deficiency of Subunits of Complex I
and Mitochonhal Encephalomyopathy
Takashi Ichiki, MD,X Masashi Tanaka, MD, PhD,? Morimitsu Nishikimi, MD, PhD,?
Hiroshi Suzuki, PhD, DMS,? Takayuki Ozawa, MD, PhD,? Masanori Kobayashi, MD,"
and Yoshiro Wada, MD+
Enzymic activities of the respiratory chain and content of immunochemically detectable subunits in NADHubiquinone oxidoreductase (Complex I) were measured in mitochondria from the skeletal muscles of 4 patients with
mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS). The rotenone-sensitive
NADH-cytochrome c reductase activity was extremely decreased, ranging from 0% to 27% of the control value. In all
patients, the content of subunits of Complex I was also reduced in parallel with the rotenone-sensitive NADHcytochrome c reductase activity. It is suggested that the variation in the degree of deficiency of Complex I subunits
could explain the clinical heterogeneity of patients with MELAS.
Ichiki T, Tanaka M, Nishikimi M, Suzuki H, Ozawa T, Kobayashi M, Wada Y.Deficiency of subunits of
Complex I and mitochondrial encephalomyopathy.Ann Neurol 1988;23:287-294
It has been reported that mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes
form a distinctive clinical syndrome (MELAS) that can
be differentiated from two other disorders: KearnsSayre syndrome and myoclonus epilepsy with raggedred fibers El]. The primary defect in MELAS is not yet
known. Pavlakis and co-workers {l} reported decreased activity of cytochrome c oxidase in 2 patients
and decreased activity of succinate-cytochrome c reductase in one patient. Kobayashi and associates [2]
found a decrease in rotenone-sensitive NADH-cytochrome c reductase activity in 2 patients. Tanaka and
associates 131 reported a decrease in the content of
subunits of NADH-ubiquinone oxidoreductase (Complex I) in heart mitochondria isolated from a patient
with mitochondrial encephalopathy and cardiomyopathy whose clinical features were consistent with
We recently examined 4 patients with clinical signs
of MELAS and analyzed biopsy samples of their skeletal muscles using biochemical, immunochemical, and
immunohistochemical methods. Here we report on 4
cases of MELAS caused by deficiency of Complex I
Case Reports
The clinical and laboratory features of 4 patients with
MELAS are summarized in Table 1.
From the *Departmentof Pediatrics, Nagoya City University Medicd School, Nagoya, and the tDepartment ofBiomedical Chemistry,
Faculty of Medicine, University of Nagoya, Nagoya, Japan.
Received May 14, 1987, and in revised form Jul 30 and Oct 5 .
Accepted for publication Oct 7, 1987.
Patient I
A 9-year-old girl had an uneventful prenatal and perinatal
history. At age 5, she had bilateral chronic otitis media. At
age 8, she experienced frequent headaches, vomiting, and
vertigo. A computed tomographic scan of the head revealed
bioccipital lucencies. One month later she had generalized
seizures with headache and vomiting and showed muscle
weakness, dementia, cortical blindness, and alternating
hemiparesis. She was hospitalized for further evaluation. She
was of short stature for her age. Blood lactate level was 58.8
mg/dl (upper limit of normal, 18 mg/dl) with a high lactate/
pyruvate ratio. Examination of a muscle biopsy sample
showed ragged-red fibers when a modified Gomori trichrome stain was used for histochemical study.
Patient 2
A 9-year-old girl had a normal gestation and birth. She was
asymptomatic and showed normal development until age 7,
when decreased exercise tolerance was noted. Also at age 7,
she developed convulsive movements abruptly. The creatine
kinase and lactic dehydrogenase levels were hgh. Cardiomyopathy was suspected, but cardiac studies were normal.
Seven months later, after a febrile illness and vomiting, she
experienced visual disorders. At age 9, she had bilateral
ptosis that was nonresponsive to edrophonium chloride. Two
months later she had a right-sided clonic convulsion. Thereafter, she also had frequent episodes of vomiting, fever, and
right hemiparesis. Five months later she was admitted to a
hospital. A computed tomographic scan of the head showed
left temporal, parietal, and occipital lucencies. Cerebral an-
Address correspondence to Dr Ozawa, Department of Biomedical
Chemistry, Faculty of Medicine, University of Nagoya, 65 Tsurumacho, Showa-ku, Nagoya 466, Japan.
Copyright 0 1988 by the American Neurological Association 287
Table 1 . Clinical and Laboratory Features
of 4 Patients with MELAS
Short stature
Cortical blindness
or hemianopia
hearing loss
Lactic acidosis
Positive family
Patient 1 Patient 2 Patient 3 Patient 4
MELAS = mitochondrial myopathy, encephaloparhy, lactic acidosis,
and strokelike episodes; + = present; - = absent.
giograms were normal. Blood lactate level was 49 mg/dl with
a high lactatelpyruvate ratio. Cerebrospinal fluid lactate concentration was 61 mg/dl. The following laboratory findings
were normal: complete blood cell count, erythrocyte sedimentation rate, blood electrolytes, and blood sugar. There
were increases of serum creatine kinase, lactic dehydrogenase, aldolase, and GOT. Electromyogram showed waning
phenomena. In a muscle biopsy sample of the quadriceps
femoris, numerous ragged-red fibers were seen. Electron microscopy revealed many morphologically abnormal mitochondria.
Patient 3
A 13-year-old boy had a normal prenatal and perinatal history. His detailed history has already been reported [2, 41.
Early growth and development were normal. At age 3, he
could not run swiftly. At age 7, he was admitted to a hospital
because of cardiomyopathy and cardiac failure. At age 10, he
had generalized tonic and clonic convulsions about three
times a day. He was readmitted to the hospital because of
seizures, muscle weakness, and impaired visual acuity. One
month later the blindness improved spontaneously. At age
12, he complained of blindness again, which persisted thereafter. At age 13, he was hospitalized for biochemical studies.
His height and weight were below the third percentile. H e
did not have ophthalmoplegia, myoclonus, or cardiomyopathy. There was bilateral ptosis that was nonresponsive to
edrophonium chloride. He had diffuse limb weakness, weak
tendon reflexes, and an intention tremor. A head computed
tomographic scan showed cerebral and cerebellar atrophy
with low density in the occipital region and high density in
the basal ganglia. Cerebral blood flow studies were normal.
Blood lactate levels were 26.8 to 61.1 mg/dl with a high
lactate/pyruvate ratio. Cerebrospinal fluid lactate concentra-
288 Annals of Neurology
Vol 23 N o 3 March 1988
tion was 55.2 mg/dl. Serum amino acid values were within
normal limits except alanine (6.91 mgidl; normal, less than
5.0 mg/dl). Total carnitine content in plasma was 32.2 pmoV
liter (normal, 67 2 I1 pmoVliter [mean 2 SD, n = 15]),
and free carnitine content was 15.5 pmoVliter (normal, 52 -t
10 p,mol/l [mean Ifr SD, n = 151). A muscle biopsy specimen showed ragged-red fibers. O n electron microscopy
these muscle cells were seen to contain an increased number
of glycogen granules and increased number and size of abnormal mitochondria. At age 14, he died suddenly. Posunortem examination revealed no cardiac abnormality.
Patient 4
A 15-year-old boy had a normal prenatal and perinatal history. H e developed normally until age 7, when he had generalized seizures with headache and vomiting. Thereafter he
also had muscle weakness, cortical blindness, and alternating
hemiparesis. At age 11, he was admitted to a hospital for
further evaluation. He was short in stature and demented.
Blood lactate levels were 20 to 50 mg/dl. Cerebrospinal fluid
lactate concentration was 34 mg/dl with a high lactatel
pyruvate ratio. A head computed tomographic scan revealed
diffuse atrophy with low density in the occipital region. Electromyography showed no distinctive myopathic pattern. At
age 15, a muscle biopsy sample from the left quadriceps
femoris showed an increased number of ragged-red fibers.
Materials and Methods
Muscle specimens were obtained from all patients by open
biopsy after signed permission and informed consent were
given. Control muscle specimens were obtained by diagnostic biopsy from children who ultimately were found to be
free of any muscle disease.
Biochemical Studies
of pyruvate, malate, and 2-ketoglutarate oxidation, we immediately isolated mitochondria from fresh skeletal muscles
essentially according to the method of Bookelman and coworkers [5]. Until determination of enzymic activities and
immunoblotting, the mitochondria were stored at - 80" C.
RATES OF PYRUVATE, MAIATE, AND 2-KETOGLUTARATE OXIDATION. The activities of pyruvate dehydrogenase com-
plex and citric acid cycle were determined in fresh muscle
mitochondria by measuring the 14C02production rate from
various labeled substrates according to the method of
Bookelman and associates [51. Concentrations of substrates
or inhibitors were 1 mM pyruvate, 1 mM malate, 1 mM 2ketoglutarate, 5 mM L-carnitine, 5 mM malonate, 5 mM
acetylcarnitine, and 1 mM arsenite. Protein in mitochondrial
preparations was measured by the method of Lowry and
associates 161 using bovine serum albumin as the standard.
Activities of NADH-c?tochrome c reductase 171, succinate-cytochrome c reductase 171, and cytochrome c oxidase 181 were measured in freeze-thawed
mitochondria. The carnitine content and carnitine palmitoyltransferase activity in the muscle homogenate were assayed
by the method of Sugiyama and co-workers [9].
Table 2. 14C02Production from Lubeled Submates i n Skeletal Muscle Mitochondria"
Substrates or Inhibitors
Patient 1
[ ~ - ' ~ ~ l p y r u v a+t ecarnitine
~ l - l ~ ~ l p y r u v a t malate
malate late + acetylcarnitine + malonate
+ arsenite
[ ~ - ' ~ ~ ) r n a ~+a acetylcarnitine
Patient 2
Patient 3
Patient 4
Control Subjectsb
38.3 -C 20.3
(22.2 -93.8)
32.8 f 11.8
(19.0 - 54.0)
4.15 & 2.39
3.09 +- 1.39
32.6 5 11.3
"Values given in nmol ''C02/midmg of mitochondrial protein.
bControl values are mean 2 SD with 10 samples.
lmmunological Studies
complex I was isolated from
beef heart mitochondria as described { 101. The flavoprotein
fraction, the iron-sulfur protein fraction, and the hydrophobic fraction were resolved from Complex I according to the
method of Galante and Hatefi 111). Antibody against Complex I was raised in rabbits by intradermal injections of 0.5 to
1 mg of enzyme protein in 1 ml of saline emulsified with 1
ml of Freund's complete adjuvant. Booster injections of the
same mixture were administered 3, 5, and 7 weeks later.
One week after the last injection, blood was withdrawn from
the carotid artery.
ELECTROPHORESIS AND IMMUNOBLOTTING. Mitochondria were electrophoresed in 9.38 to 18.75% polyacrylamide
linear gradient gels (140 x 140 X 0.75 mm) essentially as
described by Kadenbach and associates [123. The proteins on
the gels were transferred to Durapore filter (Millipore, Bedford, MA) according to the method of Towbin and coworkers [13} with the addition of 0.1% sodium dodecyl
sulfate to the electrode buffer. The binding of antibody to
the subunits of Complex I on the filter was detected by the
peroxidase-antiperoxidasemethod as reported previously [3,
141. The immunoblot strips were analyzed densitometrically
using a CS-930 chromatoscanner (Shimadzu Corporation,
Kyoto, Japan).
For immunohistochemical studies, muscle biopsy specimens were frozen in liquid nitrogencooled isopentane and cross-sectioned to 10 pm thickness in
a cryostat. Sections were incubated for 5 minutes at room
temperature with 0.1% Triton X-100, and washed twice
with cold phosphate-buffered saline. They were treated for
20 minutes with 0.03% Hz02 in methanol to block endogenous peroxidase activity. After the samples were washed
twice with cold phosphate-buffered saline, 10% normal goat
serum was layered over the sections for 20 minutes in a
humidified chamber. Then the sections were covered with
polyclonal antibody (1 :50 dilution) against Complex I for 30
minutes at 37" C. The immunohistochemical stain for Complex I was performed by the avidin-biotin complex method
of Sat0 and co-workers 1151.
Biochemical Studies
the oxidation rate of [1-'4C}pyruvate in the presence
of malate was less than normal in all 4 patients (Table
2). The oxidation rate of [l-'4C}pyruvate in the presence of carnitine was less than normal except in Patient
1. The rates of oxidation of [U-'*C}malate in the presence of acetylcarnitine plus inhibitors, and of [l-'*C}2ketoglutarate were less than normal only in Patients 2
andor 3. These results suggested a defect in the respiratory chain, which was subsequently confirmed by
more direct studies.
ENZYME ASSAYS. Rotenone-sensitive activity of
NADH-cytochrome c reductase was used as an estimate of Complex I activity. Table 3 shows that this
activity is markedly decreased in the mitochondria
isolated from the skeletal muscles of patients, ranging
from 0% to 27% of the control mean. The decreases
in the rotenone-sensitive NADH-cytochrome c reduccase activity also could be related to the patient's age:
the older patients tended to show lower enzymic activity. Succinate-cytochrome c reductase and cytochrome
c oxidase activities were in the normal range.
The carnitine content and carnitine palmitoyltransferase activity in patients' muscle tissues were within
normal limits (Table 4). We thus ruled out defects in
the carnitine acylcarnitine translocator, carnitine palmitoyltransferase deficiency, muscular carnitine deficiency, and systemic carnitine deficiency.
Immunological Studies
To determine whether the decreased enzymic activity in these patients was based on
a deficiency of some subunits in Complex I, the immunoreactive polypeptides in patients' mitochondria
were analyzed using an antibody against Complex I
Ichiki et al: Complex I Deficiency in MELAS
Table 3. Enzymic Activities of Respiratory Chain in Skeletal Muscle Mitochondria
from Patients with MELAS and from Normal Control Subjects’
Control Subjectsb
Patient 1
Patient 2
Patient 3
Patient 4
249 t 174
(82 - 637)
889 t 507
(343- 1,767)
1,554 2 753
(601 -2,873)
Values are expressed in nmoVmidmg of mitochondrial protein.
bMean f SD (n = 10).
MELAS = mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes; 1-111 = rotenone-sensitiveNADH-cytochrome c
reductase; 11-111 = succinate-cytochrome c reductase; IV = cytochrome c oxidase.
Table 4. Carnitine Content and Activity of Carnitine Palmitoyltransfrase in Muscle Tissue
Control Subjects”
Content or Activity
Patient 1
Patient 2
Patient 3
Patient 4
1.02 0.43
1.33 & 0.65
(0.39- 3.01)
2.34 ? 1.00
(0.93 -4.48)
0.25 ? 0.13
Carnitine (p,moVgmwet weight)
CPT (p,moVmin/mg protein)
CFT = carnitine palmitoyltransferase.
purified from beef heart mitochondria (Fig 1). Figure
1A shows assignment of the immunochemically detected bands to the polypeptides in the flavoprotein
fraction, the iron-sulfur protein fraction, or the hydrophobic fraction of Complex I. The amount of each
immunochemicalfy detectable subunit in Complex I
was generally decreased in the skeletal muscle mitochondria of all patients (Fig 1B). The densitometric
scan of the blots (Fig 2) demonstrates that the content
of the subunits in the mitochondria was most severely
decreased in Patient 4 and moderately decreased in
Patient 1. The total area of the peaks in the densitogram paralleled the activity of rotenone-sensitive
NADH-cytochrome c reductase (data not shown).
Therefore, the decrease in the enzymic activity of
Complex I in these patients is not due to the complete
absence of a single subunit, but to a deficiency of multiple subunits in Complex I. However, disproportionate and severe deficiency was observed in the 75-kD
subunit in the iron-sulfur protein fraction, the 51-kD
subunit in the flavoprotein fraction, and some of the
lower-molecular-weight subunits in the hydrophobic
230 Annals of Neurology Vol 23 No 3 March 1988
IMMUNOHISTOCHEMISTRY. Immunostaining using
an antibody against Complex I was used for light microscopic localization of Complex I in patients’ skeletal
muscles. Only a small number of ragged-red fibers
were observed in Patient 1, who was the youngest and
showed the mildest decrease in enzymic activity and
content of Complex I subunits. The ragged-red fibers
exhibited increased immunostaining (Fig 3A). In Patient 2, who showed a mild decrease in enzymic activity and in subunit content, numerous ragged-red fibers
were observed. The ragged-red fibers contained increased immunoreactive substance (Fig 3B). In Patient
3 (Fig 3C), whose deficiency of Complex I subunits
was moderate, the histological pattern was essentially
similar to that of Patient 2. In Patient 4 , who was the
oldest and showed the most severe deficiency of Complex I subunits, a large number of ragged-red fibers
was observed. The ragged-red fibers in this patient exhibited abnormal accumulation of the immunoreactive
substance (Fig 3D). The characteristic finding in the
skeletal musdes from these patients was the marked
difference in amount of immunoreactive substance between the ragged-red fibers and the non-raed-red
Fig 1, (A)Assignment of polypeptides of Complex I detected by
immunoblotting.Complex I (CI, 2 pg protein)purified from beef
heart mitochondria, theflauoprotein fraction (EP,1 pg protein),
and the iron-sugurprotein fraction (IP, 1 pg protein) were analyzed by immunoblotting. In CI, which is reported to be composed
of 25 unlike polypeptides, 16 polypeptide bands were detected. In
FP, which is composed of 3 polypeptides, the 51-kD and24-kD
polypeptides were detected, but the 9-kD polypeptide was not detected. In IP, which is composed of 6 polypeptides, the 75-kD,
49-kD, 30-kD, and 15-kD polypeptides were detected but the
18-kD and 13-kD polypeptides were not detected. The other
polypeptide bands detected in CI are presumably refirable to polypeptides in the hydrophobicfraction. The molecular mass values
indicated on the left side of the blot are according to Hat& {20}.
The actual mobilities of standtrd proteins in this electrophoretic
system are indicated on the right side of the blot with their
molecular muss in RiloaMons. (B) Immunochemical detection of
Complex I subunits in mitochondria. BHM = 25 pg betf heart
mitochondria; CI-> = 60 pg skeletal musck mitochondriafvom
normal control subjects; P I - 4 = 60 pg skeletal muscle mitochondria from Patients 1-4, respectively.
fibers. This difference was more marked than that
found between Type 1 and Type 2 fibers in normal
skeletal muscle (Fig 3E). The correspondence of the
heavily immunostained fibers with the ragged-red
fibers in the modified Gomori trichrome stain is shown
in Figure 3D,F.
MELAS is characterized by short stature, episodic
vomiting, seizures, and recurrent cerebral strokes causing hemiparesis, hemianopia, or cortical blindness.
Most patients with MELAS do not have ophthalmoplegia, retinal degeneration, heart block, and myoclonus, which are found in Kearns-Sayre syndrome or
in myoclonus epilepsy with ragged-red fibers El). As
our patients fulfilled these criteria, the diagnosis of
MELAS was made (see Table 1). The results of
biochemical (see Tables 2-4), immunochemical (see
Figs 1,2), and immunohistochemical (see Fig 3) studies
on these patients revealed that this disorder was associated with deficiency of subunits as well as activity of
Complex I.
Patients with Complex I deficiency have been reported to exhibit various symptoms. A case of fatal
infantile type Complex I deficiency was reported by
Moreadith and co-workers 1167. Their patient's conIchiki et al: Complex I Deficiency in MELAS 291
Fig 2. Densitograms of the immunoblots of Complex I subunits.
C = control human mitochondria (C3 in Fig IB). BHM =
beef heart mitochondria. P1.4 = skeletal muscle mitochondria
from Patients 1-4, respectively. The numbers are the molecular
m s s values in kilodaltons of subunits according to Hate$ {20).
Data from Figure I B .
genital lactic acidosis was found to be caused by a total
loss of the iron-sulfur clusters in Complex I that were
detectable by electron paramagnetic resonance spectroscopy. The clinical features of the infant were not
consistent with MELAS. The patients reported by
Morgan-Hughes and associates { 17, 18) had myopathy
and encephalomyopathy. Kobayashi and associates C4)
described 3 patients with Complex I deficiency, one of
whom had an early onset type; the others had MELAS.
Therefore, the disorders caused by Complex I deficiency can be classified into 4 clinical types: fatal infantile type {16); myopathic type 1171; encephalomyopathic type without strokelike episodes 1181,
including the early onset type [47; and MELAS type
{2-4, 14). Similar clinical heterogeneity is reported in
patients with Complex IV deficiency C19).
Complex I contains approximately 25 unlike polypeptides, flavin mononucleotide, and 5 binuclear (2Fe2s) and 3 tetranuclear (4Fe-4s) iron-sulfur clusters.
The polypeptides of Complex I can be divided into
three groups: the flavoprotein fraction, the iron-sulfur
protein fraction, and the hydrophobic fraction 120).
Seven of the polypeptides are encoded by the mito292
Annals of Neurology Vol 23
No 3
March 1988
Fig 3. Histochemistry and imrnunohistochemistry of skeletal
muscles. (A-0) Immunostaining for Complex I in Patients 14, respectively. (I?) Immunostaining for Complex I in control
subject. (P) Mod$ed Gomori trichrome staining of the muscle
fmm Patient 4. Arrowheads indicate ragged-red fibers. (Bar =
100 p m in A-F.)
chondrial D N A 121) and constitute a major part of the
hydrophobic fraction. Since the biogenesis of Complex
I is under dual control from mitochondrial and nuclear
genomes {21, 221, deficiency of Complex I can be
caused by mutations in the structural genes in either of
the genomes or by some disorders in the expression of
the genetic information. This complexity may be responsible for the clinical heterogeneity of Complex I
deficiency. The analysis of Complex I subunits in our
patients with MELAS demonstrated disproportionate
and severe deficiency of the 75-kD, 5l-kD, and several small-molecular-weight polypeptides (Fig 1). In
contrast, Shapira and co-workers [23] reported a
marked decrease in the amounts of 75-kD and 13-kD
polypeptides in one patient with Complex I deficiency,
and 24-kD and 13-kD polypeptides in another patient.
These differences in the molecular defects might explain the clinical heterogeneity of Complex I deficiency. In addition, we previously reported 1141 that
the degree of deficiency of the Complex I subunits
varied from tissue to tissue in one patient with MELAS
(Patient 3). The distribution of enzyme deficiency
among tissues would also contribute to the variation in
the clinical features among patients with Complex I
Immunohistochemical study of skeletal muscle from
our patients with MELAS demonstrated that a large
amount of immunoreactive substance of Complex I
accumulated in the ragged-red fibers. The increased
number of mitochondria in the ragged-red fibers is
regarded as a result of stimulated proliferation of
mitochondria in response to the decreased energy supply in these cells. Since deficiency of Complex I subunits is not complete but disproportionate, the increased immunoreactivity in the ragged-red fibers is
ascribable to the residual subunits in defective mitochondria proliferated in the fibers.
Ichiki et al: Complex I Deficiency in MELAS
Several methods of treatment for Complex I deficiency have been reported. Arts and co-workers 1241
reported that a patient with myopathy due to Complex
I deficiency was successfully treated with large doses of
riboflavin. In our patients, we tried thiamine, riboflavin, biotin, carnitine, ubiquinone, and ketogenic
diet, but neither symptoms nor signs of MELAS were
improved. Recently we started oral administration of
sodium succinate to another patient with MELAS due
to Complex I deficiency, and strokelike episodes disappeared f25]. In theory, succinate increases electron
flow through Complex I1 to Complexes 111 and IV,
thus enabling these two energy coupling sites to function in spite of the disturbance in the first coupling site
(Complex I). Succinate treatment for Complex I deficiency should be further evaluated biochemically.
We thank Dr Ikuya Nonaka, National Center of Neurology and
Psychiatry, Kodaira, Tokyo, for his help in histochermstry and immunohistochemistry.
Supported in part by Grants-in-Aid for Scientific Research from the
Ministry of Education, Science, and Culture of Japan (62570128) to
M. T. and (62617002) to T. 0.;grant no. 85-04-39 from the National Center of Neurology and Psychiatry of the Ministry of Health
and Welfare of Japan to T. 0.;and grant no. 85-1106 from Yamada
Science Foundation to T.0.
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