J Neurol (1982) 227:201-207 Journal of
Neurology Springer-Verlag 1982
Cytochrome-C-oxidase Deficiency in Muscles of a Floppy Infant Without Mitochondrial Myopathy*
M. Rimoldi ~, E. Bottacchi ~, L. Rossi 3, F. Cornelio 2, G. Uziel 3, and S. Di Donato 3
1 Laboratory of Neurometabolic Disease and 2 Laboratory of Myopathology, Istituto Neurologico "C. Besta", Milan
~Pediatric Clinic, 1st Chair, Milan, Italy
Summary. A 4-month-old girl had hypotonia, muscle weakness and cardio- myopathy at birth and died at the age of 8 months from heart failure. Muscle biopsy showed normal muscle fibers, normal mitochondria and type II hypotrophy, in agreement with clinical and neurophysiological signs consistent with a suprasegmental lesion. Cytochrome-C-oxidase was the only respiratory chain enzyme that was profoundly decreased in the patient's muscle, but not in her brain, heart and cultured fibroblasts. We suggest that cytochrome-C- oxidase deficiency in skeletal muscle is not always associated with mito- chondrial abnormalities and may not always indicate primary muscle pa- thology.
Key words: Cytochrome-C-oxidase deficiency - Mitochondrial myopathy - Floppy infant
Zusammenfassung. Ein 4 Monate altes M~idchen litt seit Geburt an einer Muskelhypotonie verbunden mit einer Kardiomyopathie, an der es im Alter von 8 Monaten starb. Eine Muskelbiopsy zeigte normale Muskelfasern und Mitochondrien und eine Muskelhypotrophie Typ II. Dies sprach in einer Obereinstimmung mit den klinischen und elektrophysiologischen Befunden fiJr eine suprasegmentale Lasion.
Cytochrome-C-Oxidase war das einzige Enzym der Atmungskette, welches im Muskel hochgradig vermindert war. Es war hingegen normal im Gehirn- gewebe, im Herz und in der Fibroblasten-Kultur. Es wird daraus gefolgert, dab ein Mangel an Cytochrome-C-Oxidase nicht immer mit morphologisch fafSbaren Anomalien der Mitochondrien vergesellschaftet ist und dab sic nicht immer eine prim~ire Muskelerkrankung darstellt.
* This work was carried out under a grant of the Muscular Dystrophy Association, USA and research grant CT 81.00146.04 from the Consiglio Nazionale delle Ricerche, Italy
Offprint requests to." Stefano Di Donato, MD -- Laboratory of Neurometabolic Diseases, Istituto Neurologico "C. Besta" via Celoria 11 -- 20133 Milan, Italy
202 M. Rimoldi et al.
The floppy baby syndrome is frequently diagnosed in children with poor skeletal muscle control and diminished muscle tone at birth.
Many of these children suffer from central or peripheral nervous system disorders, but some have muscle diseases , including congenital myopathies, mainly defined from distinct morphological features , and metabolic myo- pathies, mainly defined from distinct biochemical features . The latter group of diseases includes many recognized disorders associated with the floppy infant syndrome, such as glycogenosis type II  and cytochrome-C-oxidase deficiency [6, 17]. In this last disease, in addi t ion to the enzyme deficiency in skeletal muscle the floppy babies have lactic acidosis, renal dysfunct ion resembling de Toni- Fancon i -Debr6 syndrome and muscle morphology consistent with the mi tochon- drial-l ipid-glycogen (MLG) myopathy described by Jerusalem et al. [ l l ] . We describe a f loppy baby with progressive lethal cardiomyopathy, moderate lactic acidosis, normal renal funct ion and central nervous system involvement, in whom a p ro found deficiency of cytochrome-C-oxidase (Cyt-C-Oxidase) was found in skeletal muscle, without any abnormali t ies of muscle mitochondria .
The propositus, a 4-month-old girl, was born in an uncomplicated delivery after 37 weeks of pregnancy. The parents were first cousins. The 3-year-old sister of the patient was normal.
The patient's weight at birth was 2150 g. At age 4 months she weighed 4000 g, but her weight declined to 3600 g at age 6 months and to 3100 g at age 8 months. Mental development seemed normal: she smiled at age 2 months and could recognize parents and objects at 4 months.
Neurological examination at age 4 months showed marked generalized hypotonia and weakness of the skeletal muscles with difficulty in grasping objects and poor head control. Deep tendon reflexes were present. Visual function and ophthalmoscopic examination of the fundi were judged normal. The liver edges was palpable two fingers beneath the right costal margin. Precordial examination revealed a protomesodiastolic thrill: the second heart sound was variably split. Electrocardiograms revealed sinus tachycardia (175/min) and signs of left ventricular hypertrophy with moderate myocardial damage. Echocardiography showed a picture consistent with asymmetric ventricular hypertrophy, with marked septal hypertrophy. Chest radiographs showed heart enlargement. The patient was digitalized at age 4 months. EMG of both proximal and distal muscles and maximal motor conduction velocities and distal latencies on the left median and peroneal nerves were normal. Repeated EEG recordings showed clear signs of diffuse brain sufferance.
Routine laboratory investigations were normal except for increased levels of aminotrans- ferases (SGOT 120 and SGPT 63mU/1, normal values less than 24mU/1). There was no glycosuria or proteinuria and urinary aminoacids were in the normal range. Blood lactate and pyruvate were moderately increased (lactate 2.7, 2.8, 5.3 mM; pyruvate 0.20, 0.18, and 0.26 mM; normal values for lactate less than 2 mM; normal values for pyruvate less than 0.15 raM). The lactate to pyruvate ratio was normal. Overnight fasting acetoacetate and fl-hydroxybutyrate were 76 and 136~tM, and 391 and 7271aM, respectively, in two different determinations. The fi- hydroxybutyrate to acetoacetate ratio was high (normal value less than 3), indicating poor mitochondrial oxidation of nicotinamide adenine dinucleotide (NADH). At age 7 months neurological examination revealed marked generalized muscle weakness and wasting, diffuse muscle hypotonia and no deep tendon reflexes. Her general condition was poor and echocardio- graphy revealed a worsening of the left ventricular hypertrophy. At age 8 months the patient died
Cytochrome-C-oxidase Deficiency in Muscles of a Floppy Infant 203
from heart failure. The main findings at autopsy were cardiomyopathy with asymmetric ventricular hypertrophy and diffuse fatty degeneration of the liver. Biopsies of the right biceps brachii and of skin for fibroblast culture were taken at age 4 months.
Carnitine and its esters were evaluated separately; carnitine was measured by the radiochemical method previously reported . Carnitine acetyltransferase (CAT) was determined by the method previously reported . Carnitine palmitoyltransferase (CPT) was measured by the "forward" reaction  and by the "backward" reaction, using labelled palmitoyl-1-3H-carnitine, synthesized according to the method of Stokke and Bremer . Pyruvate-dehydrogenase complex (PDHC) was measured by a radiochemical method . Succinate-cytochrome-C- reductase (Succ-Cyt-C-red) and NADH-cytochrome-C-reductase (NADH-Cyt-C-red) were measured spectrophotometrically . Cyt-C-oxidase activity was determined spectrophoto- metrically by the decrease in absorbance at 550nm reduced cytochrome-C . Oxidized cytochrome-C (Sigma), 1% in 10 mM phosphate buffer pH 7.0, was reduced by the addition of a few grains of NaBH4: the unreacted NaBH4 was eliminated by titration with 1 N HC1. Respiratory chain enzyme activities were determined in 10% skeletal muscle homogenates in TRIS-HC1- sucrose, pH 7.4 at 38C, and in homogenates of fibroblasts in TRIS-buffered saline, pH 7.4 at 30 C. Rates of oxidation of pyruvate- 1 - 14C and palmitate- 1- ~4C (A mersham) by skeletal muscle homogenates were determined as previously described . Proteins were measured by the Lowry method , non-collagen protein by the method of Lilienthal et al. . Light and electron microscopic studies of skeletal muscle were performed as previously described . The histochemical reaction for Cyt-C-oxidase was that of Seligman .
Since the rates of pyruvate and palmitate oxidat ion by muscle homogenates were shown to be slow in the pat ient ' s muscle (Table 1), we measured some of the mi tochondr ia l enzymes involved in the oxidative pathways for pyruvate and fatty
acids. Table 2 shows that the activity of Cyt-C-oxidase was p rofoundly decreased in
the pat ient ' s muscle (less than 5% of the control mean) , while the activities of the other enzyme complexes of the respiratory chain were in the no rma l range. The activities of acyltransferases (CAT and CPT) and of citrate synthase were normal , while the activity of P D H C was reduced in the pat ient ' s muscle (25% of the mean of controls). Total carni t ine content in skeletal muscle was lower than the
Table 1. Oxidation rates in skeletal muscle homogenates
Substrate Controls Patient
- L-carnitine + L-carnitine - L-carnitine + L-carnitine
Pyruvate-124C (9) 1087 +_175 2187 +570 262 292
Palmitate-l-14C (10) 17.7+_ 3.7 35.5+_ 82 7.0 6.1
Values are given as picomoles labelled CO2 produced/min/mg noncollagen protein_+SD. L-carnitine, when added, was I mM. Number of controls in parentheses
Enzyme Controls Patient
CAT (8) 2.9 _+ 0.4 2.9
CPTfbrwar d (12) 0.14_+ 0.1 0.14
CPTbackward (12) 13.3 _+ 0.9 12.8
Citrate synthase (8) 77.1 _+ 15 86.1
PDHC (4) 2.75 + 0.90 0.68
NADH-Cyt-C-red (4) 20.1 + 0.6 17.9
Succ-Cyt-C-red (4) 5.2 _+ 1.7 4.6
Cyt-C-oxidase (7) 31.8 _+ 13.3 1.4
Values are given as nanomoles subst