Cytochrome c oxidase deficiency in subacute necrotizing encephalomyelopathy

Journal oftheNeurological Sconces, 1987, 77:103-115

103

Elsevier

JNS 02785

Cytochrome c oxidase deficiency in subacute necrotizing encephalomyelopathy W. F. M. Arts l, H . R . Scholte 2, M. C.B. Loonen l, H. Przyrembel 3, J. Fernandes 4, J. M. F. Trijbels 5 and I. E . M . Luyt-Houwen 2 Departments of LNeurology, 2Biochemistryl, 3paediatrics, Erasmus University, Rotterdam; Department of 4Paediatrics, University of Groningen; and Department of 5Paediatrics, Catholic University of Nijmegen (The Netherlands) (Received 12 May, 1986) (Revised, received 30 September, 1986) (Accepted 30 September, 1986)

SUMMARY

Two new patients with Leigh's syndrome (subacute necrotizing encephalomyelopathy) due to deficiency of cytochrome c oxidase are presented and their data are compared with those of the four Leigh's syndrome patients previously reported with this deficiency. It is not possible to distinguish between the various biochemical aetiologies of Leigh's syndrome on clinical grounds. Investigation of pyruvate metabolism and of the respiratory chain will reveal the enzymatic defect in some of the patients. It has now been firmly established that a relationship exists between Leigh's syndrome and deficiency of cytochrome c oxidase. There are, however, other syndromes which are also associated with a deficiency of this enzyme. In Leigh's syndrome, the enzyme deficiency has been reported in many organ systems and in cultured fibroblasts. In the liver, however, decreased, intermediate or normal values of cytochrome c oxidase activity have been found. Selective or more widespread involvement of organ systems, due to mutations of either the nuclear or the mitochondrial DNA encoding for different subunits of the enzyme molecule (some of which may be organ- or tissue-specific), could explain the clinical and biochemical heterogeneity of syndromes associated with a cytochrome c oxidase deficiency.

Financial support was obtained from the 'Prinses Beatrix Fonds', The Hague, and the 'Willem H. Kr6ger Stichting', Rotterdam. Correspondence address: Dr. W. F. M. Arts, Department of Neurology, Westeinde Hospital, P.O. Box 432, 2501 CK The Hague, The Netherlands. 0022-510X/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

104 Key words: Cytochrome c oxidase deficiency; Encephalomyelopathy; Leigh's syndrome; Lactic acidemia

INTRODUCTION Subacute necrotizing encephalomyelopathy or Leigh's syndrome combines characteristic neuropathological abnormalities with heterogeneous clinical and biochemical findings. In the childhood-onset type, developmental retardation and failure to thrive are followed by typical respiratory abnormalities, hypotonia, ataxia, nystagmus, optic atrophy, demyelinating neuropathy, myopathy with ragged-red fibres, lactic acidosis, and in a later stage spasticity, leading to a decerebrate posture. Respiratory insufficiency is the most frequent cause of death. Until recently, demonstration of neuropathological abnormalities was the only way of proving the clinical diagnosis. However, the clinical picture in combination with a typical CT appearance (Schwartz et al. 1981; Koch et al. 1985) may now be considered to be sufficient proof of the diagnosis. Three enzyme deficiencies have hitherto been associated with Leigh's syndrome: deficiency of pyruvate carboxylase in the liver (Hommes et al. 1968); deficiency of one of the enzymes of the pyruvate dehydrogenase complex (Blass et al. 1976; Evans 1981); deficiency of cytochrome c oxidase (cytochrome aa3). The latter deficiency has been described in four patients (Willems et al. 1977; Miyabayashi et al. 1983; Hoganson et al, 1984; Miyabayashi et al. 1984). In this paper, we report two new patients with Leigh's syndrome, in whom a deficiency of cytochrome c oxidase has been demonstrated. CASE HISTORIES Patient 1 A Turkish girl, born in Holland, died at the age of 38 months. Her parents are first cousins; a younger brother is healthy. Her motor development had been slow. After a febrile illness at the age of 28 months, she could no longer walk without support. On examination, she was found to be small (length below the third percentile for age), but her weight was normal. She had severe ataxia of both trunk and limbs. Eye movements were saccadic; deep tendon reflexes were absent. The motor nerve conduction velocity in the peroneal nerve was 28 m/s (normal > 43 m/s); the sural nerve sensory conduction velocity 37 m/s (normal > 42 m/s). After an initial improvement, which lasted for two months, she suffered a relapse. New signs were bouts of severe hyperventitation, a severe tremor of the head, trunk and extremities, increasing with voluntary movements, bilateral foot drop and distal hypotonia. There was compensated metabolic acidosis (BE-8.1 mmol/1); lactate values varied from 0.74 to 8.51 retool/1 (nl < 1.8 mmol/l). Oral glucose loading produced an increase in blood lactate (from 0.74 to 3.99 mmol/l), but not of pyruvate values. Fasting did not induce hypoglycaemia. The CT scan (Fig. 1) showed bilateral round hypodensities in the region of the lentiform nucleus, typical of Leigh's syndrome (Schwartz et al. 1981; Koch et al. 1985). A needle biopsy of the liver

105

Fig. 1. Patient 1, CT scan showingbilateral round hypodensitiesin the lentiformnucleus, and atrophy of the cerebellar vermis. appeared normal under light microscopy. Electron microscopy revealed a slight increase in the number of small fat droplets and in the number and size of the mitochondria, which were sometimes abnormal in shape and contained too many electron-dense granules (Fig. 2). A biopsy from the quadriceps femoris muscle, taken under local

106

Fig. 2. Patient 1, liver biopsy, showing increased fat droplets and mitochondria. Magnification x 20000. before reduction.

anesthesia, only showed a slight increase in lipid droplets in the type I fibres, while a small number of type I fibres were ragged-red, Detailed biochemical findings will be described below. She was treated unsuccessfully with thiamine 300 mg daily, and later with biotin 10 mg daily. Despite treatment with sodium bicarbonate, several severe exacerbations of acidosis occurred, leading to an increase in ataxia, tremor and hyperventilation. Alveolar hypoventilation was the immediate cause of death. Autopsy was not - performed. Patient 2

A Dutch girl, born of non-consanguineous parents, died at the age of 10.9 years. She was the second of three children; her elder brother had died at the age of 14 months with symptoms which were apparently very similar to those of his sister. Autopsy had been carried out on this boy and had revealed abnormalities around the third and fourth ventricles resembling those seen in Wernicke's encephalopathy. The younger brother is healthy. The girl could walk without support at the age of one year. After an attack of acute laryngitis, she could no longer walk and suffered episodes of mental dullness and hypotonia. Another infection at the age of two years was followed by coma with extreme

107 hypotonia, areflexia, respiratory insufficiency and short-lasting cardiac arrest. During her recovery, she had a period with opsoclonic eye movements. Later, intermittent hypotonia, tremor of the head, truncal and extremity ataxia, a horizontal gaze paresis with nystagmus, apathy and episodes of 'sighing' respiration were present. She was microcephalic and of very short stature. At three years of age, laboratory examination revealed a compensated metabolic acidosis, an increase in blood lactate (between 2 and 4mmol/1) and pyruvate (l14#mol/1; normal 45-70/~mol/1) with a corresponding increase in the lactate/pymvate ratio to 25 (normal < 15). Glucose, fructose and alanine tolerance tests were inconclusive, as was a fasting test. Therapeutic trials with high doses of thiamine and pyridoxine were unsuccessful. The girl remained severely retarded. She gradually developed a spastic tetraparesis. Finally, she developed a rapidly progressive respiratory insufficiency which was lethal. At autopsy, two hours post mortem, specimens of several organs were removed and frozen for biochemical investigations (vide infra). The neuropathological examination showed abnormalities considered to be pathognomonic for Leigh's syndrome. BIOCHEMICALINVESTIGATIONS- METHODS

Patient I Mitochondria were isolated according to Bookelman et al. (1978). Cytochrome c oxidase was measured as described by Cooperstein and Lazarow (1951), with purified reduced cytochrome c, and expressed as the first order rate constant k with respect to cytochrome c. The activities of the pyruvate and 2-oxoglutarate dehydrogenase complexes were measured in a modification of the procedure described by Taylor et al. (1973). 5~o Homogenates were made in 0.25 M sucrose, 10 mM HEPES-KOH, 1 mM EDTA, 1~o bovine serum albumin (fatty acid-poor) (pH 7.4). The incubation was carried out in plastic Eppendorfcups, with a cap covered with hyamine hydroxide (25 #1 1 M in methanol, used after evaporation of the methanol) on the inside. For the pyruvate dehydrogenase complex determinations, 20 #1 of the homogenate were pre-incubated for 6 min at 37 ° C with 5/d H20 for the blank, or with 5 #1 H20 to determine the basal activity, or with 5 #l 1 mM Mg2 ÷ -ATP to inactivate the enzyme, or with 5/A 10 mM C a 2 + C12 plus 10 mM M g 2 + C l 2 to activate the enzyme (Robinson and Sherwood 1975). A mixture containing 10 mM potassium phosphate buffer (pH 7.4), 1 mM EDTA, 2.8 mM MgC12, 1.5 mg bovine serum albumin, 1.2 mM dithiothreitol, 0.3 mM thiamine pyrophosphate, 1.6 mM NAD ÷, 0.1 mM cytochrome c (Fe3+), 0.16 mM CoASH, 10 mM L-carnitine and 75 #g carnitine acetyltransferase was added and the incubation carried out. The reactions were started by the addition of 1 mM sodium [ 1-~4C]pyruvate or [ 1-~4C]2-oxoglutaric acid (100 dpm/nmol). The radioactive substrates were diluted with the non-radioactive salts, ground in a mortar and stored at - 20 ° C. Freshly prepared solutions were used after gassing with N 2. The final volume was 0.1 ml. After 5-7 min at 37 °C the reaction was terminated with 10/~l 5 M H 2 S O 4. The tubes were opened in a vertical position, and the drop of acid put on the side wall. The cup was closed again and with a short swing the acid was made to reach the incubation mixture. Blanks were

108 run without homogenate. This was added together with the H2SO4. After standing overnight at 4 °C, the cups were opened, the caps were cut off and counted in 200/~1 1 M Tris buffer and 5 ml Instagel. Pyruvate carboxylase activity was measured for 10 min at 37 °C in a volume of 0.1 ml with 60 mM Tris-HCl (pH 7.8), 3 mM ATP, 2 mM MgC12, 0.8 #g citrate synthase, 10 mM [14C]KHCO3 (pH 9.0; 5 Ci/mol), 0.6 mM acetyl-CoA (pH 4) and 10 mM sodium pyruvate. The reaction was started with 50/~1 5 ~o liver homogenate in 0.25 M sucrose, 10 mM HEPES-KOH, 1 mM EDTA (pH 7.4), which was pretreated with 1/40 vol of 1~o Lubrol-WX in water at 0 °C for 12 min. The blanks did not contain pyruvate. The reaction was terminated with 50/~1 5 M HC1. For further details, see Scholte et al. (1986). References of other methods and assays are in Barth et al. (1983) and Mooy et al. (1984). Patient 2

Cytochrome c oxidase activity was measured according to Cooperstein and Lazarow (1951), pyruvate carboxylase activity according to Utter and Keech (1963), using a regenerating system for acetyl-CoA as proposed by Henning and Seubert (1964), pyruvate oxidation rate according to WiUems (1978), and phosphoenolpyruvate carboxykinase activity according to Harper (1962). RESULTS Patient 1

Investigation of the homogenate of a liver biopsy (Table 1) revealed an intermediate activity of cytochrome c oxidase (41 ~ of controls), while the activities of other mitochondrial enzymes were normal, The level of carnitine was in the lower control range. In a muscle homogenate (Table 1), the activity of cytochrome c oxidase was decreased (14~o of controls). Isolated muscle mitochondria (Table 2) oxidized all substrates with reduced velocities. It is interesting that the oxidation rates of the NAD+-linked substrates were decreased to 14-19~o of control rates, while the oxidation of succinate ( + rotenone) and of ascorbate ( + TMPD) were both decreased to 31-32~o of control rates. This difference may be explained by involvement of an additional (secondary?) lesion at the level of NADH-CoQ reductase, or - more likely - by a greater cytochrome c oxidase control strength (Tager et al. 1983) of the oxidation rates of the NAD ÷ -linked substrates than of those of succinate or ascorbate. Patient 2

In liver tissue, obtained by biopsy, normal levels of pyruvate carboxytase, pyruvate dehydrogenase and phosphoenolpyruvate carboxykinase activities were found. Using skeletal and heart muscle tissue, obtained post-mortem, it was not possible to isolate sufficient mitochondria to determine the cytoehrome spectra. However, cytochrome c oxidase activity was deficient, in a 10-min 1000 × g supernatant of

a

Rotenone-sensitive; b Antimycin-sensitive. n = number of controls.

Total carnitine (~mol/g) Protein (mg/g) Lactate dehydrogenase (nmol pyruvate/min/g) Creatine kinase 0zmol creatine/min/g) NADH-oxidase a 0zmol cyt. c/min/g) Succinate-cytochrome c reductase b Omol cyt. c/min/g) Cytochrome c oxidase (lst order rate constant k/nfin/mg prot.) Pyruvate dehydrogenase basal (nmol pyruvate/min/g) Pyruvate dehydrogenase after Mg2 + -ATP (nmol pyruvate/min/g) Pyruvate dehydrogenase after Ca 2 +/Mg 2 + (nmol pyruvate/min/g) 2-Oxoglutarate dehydrogenase (nmol 2-KG/min/g) Pyruvate carboxylase O~mol 14COz/rnin/g) 4.46 4. 1.11 27 4. 2 437 4. 129 144 _+ 39 524 4. 85 1056 4. 188 3.30; 3.26; 2.05

3.82 II 242 0 1068 989 6.09

(6) (5) (9) (9) (9) (9)

307 3.19 3.99 90

225 3.69 4.24 13

+ 12 4. 0.26 4. 0.43 4. 7

3.96 4. 0.09 174 4. 6

Controls

2.51 198

1.03 _+0.13 136 4. 18 84 4- 7

0.62 160 77

(7) (7) (7)

Patient 1

Controls

Patient 1

(n)

Muscle

Liver

Enzyme activities are expressed per g wet tissue weight. The average control values are given 4- SE.

TOTAL CARNITINE AND PROTEIN CONTENT AND ENZYME ACTIVITIES IN LIVER AND MUSCLE HOMOGENATE OF PATIENT 1

TABLE 1

(50) (10) (21) (16)

(59) (53)

(n)

110 TABLE 2 O X I D A T I O N RATES O F V A R I O U S S U B S T R A T E S IN I S O L A T E D M U S C L E M I T O C H O N D R I A OF PATIENT 1 The oxidation rates measured in the presence of A D P are given in nanoatoms oxygen/min/mg protein. The stimulation of the oxidation by A D P was lowered in the presence of succinate and ascorbate. There was no stimulation with the N A D ÷ -linked substrates. The P/O ratios were normal. There was some inhibition of the adenine nucleotide carrier or the Pi carrier since uncoupler stimulated ascorbate oxidation 1.5 times better than ADP, but the same stimulation factor was found in controls. Activities of basal and uncoupler stimulated Mg 2+ -ATPase, basal and detergent stimulated malonyl-CoA decarboxylase, [U-14C]palmitate oxidation to 14CO2 and ~4C-intermediates in the presence of carnitine were in the control range.

Pyruvate + malate Glutamate + malate Palmitoylcarnitine + malate Succinate + rotenone Ascorbate + T M P D

Patient 1

Controls _+ SE

(n)

12 16 12 34 109

84 86 71 111 344

(17) (17) (17) (17) (17)

_+ 6 + 8 + 5 + 9 _+ 16

homogenized skeletal muscle tissue, in isolated mitochondria from skeletal muscle and heart and in homogenized brain tissue (Table 3). DISCUSSION

The two patients described here bring the total number of patients with Leigh's syndrome and cytochrome c oxidase (cytochrome aa3) deficiency to 6 (Willems et al. 1977; Miyabayashi et al. 1983; Miyabayashi et al. 1984; Hoganson et al. 1984), with one possibly affected relative (the brother of our patient 2). Clinically and pathologically, it seems to be impossible to distinguish between this and other deficiencies said to be related to Leigh's syndrome. However, hypoglycemia after fasting suggests a defect in gluconeogenesis, while a rising lactate level following glucose administration indicates a defect in the pyruvate dehydrogenase complex (PDHC) or in the respiratory chain

TABLE 3 ACTIVITY O F C Y T O C H R O M E c O X I D A S E IN P A T I E N T 2 E X P R E S S E D IN nmol/min/mg PROTEIN Homogenized tissue Heart muscle Skeletal muscle Brain

11 (73-284; n = 30) 1.80 ( 1 8 - 61; n = 8)

() = range of control values; n = number of controls.

Isolated mitochondria 373 (1211-2188; n = 3) 140 (1290-3430; n = 16)

111 (Willems et al. 1977). In Leigh's syndrome associated with a deficiency of cytochrome c oxidase, only occasional elevations of lactate and pyruvate are found. These elevations may be provoked by mild exercise (Miyabayashi et al. 1983). In our patients, the lactate levels varied from normal to greatly increased, while the also increased pyruvate levels showed less variation. An increase of the lactate: pyruvate ratio was almost invariably present. Oral glucose loading showed a moderate increase of lactate, but not of pyruvate in patient 1, but no definite abnormalities in patient 2. Cytochrome c oxidase catalyzes the last step in the electron-transport chain. Cytochr0me c oxidase deficiency is associated with several heterogeneous clinical syndromes, with variable biochemical expression in different organs and probably with different modes of inheritance. A secondary deficiency has been described a.o. in Wilson's disease (Shokeir and Shreffler 1969), Menkes' kinky hair disease (French et al. 1972; Maehara et al. 1983) (in both associated with a copper deficiency), and in chronic progressive external ophthalmoplegia (Johnson et al. 1983; Maller-H0cker et al. 1983b, considered secondary by DiManro et al. 1985a). A presumably primary deficiency of cytochrome c oxidase has hitherto been described in 5 clinical syndromes: an early infantile, rapidly progressive and fatal myopathy with a renal De Toni-Fanconi-Debr6 syndrome (Van Biervliet et al. 1977; DiMauro et al. 1980; Heimann-Patterson et al. 1982; Stansbie et al. 1982; Minchom et al. 1983; Mtliler-Ht~cker et al. 1983a; Ohtani et al. 1984; Zeviani et al. 1985); an early infantile, fatal myopathy without De Toni-Fanconi-Debr6 syndrome (Rimoldi et al. 1982; Boustany et al. 1983; Trijbels et al. 1983; Sengers et al. 1984; Bresolin et al. 1985); a benign infantile myopathy in which the enzyme activity returned to normal and only slight residual weakness remained (DiMauro et al. 1983); Alpers' syndrome (Prick et al. 1983); and, finally, Leigh's syndrome. A survey of the f'mdings is Presented in Table 4. In Leigh's syndrome, the enzyme deficiency was, when measured, present in skeletal muscle, heart muscle, brain, kidney, liver and cultured fibroblasts. However, normal activities were found in liver tissue of one patient (Willems et al. 1977) and in liver tissue and cultured fibroblasts of another (DiMauro, personal communication, 1986), and an intermediate activity in liver tissue of our patient 1. The affected sibling of our second patient, the affected siblings reported by Miyabayashi et al. (1984) and the consanguinity of the parents of our first patient strongly suggest an autosomal recessive mode of inheritance for this defect. Normal amounts of immunologically cross-reacting enzymatic protein were found in the patient reported by Hoganson et al. (1984) (DiMauro et al. 1985b). Any theory on the pathophysiology and genetics of primary cytochrome c oxidase deficiency should take into account the different modes of inheritance of the associated syndromes (autosomal recessive; mitochondrial?); the presence (Leigh's syndrome) or absence (fatal myopathy with or without renal dysfunction) of immunologlcally crossreacting enzymatic protein (DiMauro et al. 1985b); and, finally, the expression of the defect in one,'severa! or many organ systems. Tissue specificity of the molecular composition of cytochrome c oxidase has been established and even overrides speciesspecificity (Jarausch and Kadenbach 1982; Kadenbach et al. 1982). Mutation of a nuclear gene controlling the synthesis of a tissue-specific subunit in muscle (and kidney),

2

6

Alpers' syndrome

Leigh's syndrome

1

5

Muscle, returning to

9.

1

Autos. recess.

4

Fatal infantile myopathy 5 without renal dysfunction

Transient infantile myopathy

Muscle (5) Liver (2)

Autos. recess.

5

Fatal infantile 7 myopathy with De Toni-Faneoni-Debr6 syndrome

Muscle (2)

Muscle (5) Heart (2) Liver (2) Kidney (1) Brain (3) Cult. fibrobl. (1)

Mitochondrial?

Autos. recess.

nOrlTlai

Muscle (7) Kidney (1)

Decreased in (No.)

Enzyme activity

Probable mode of inheritance

No. of cases

No. of possibly affected relatives

Clinical syndrome

(2) (3) (1)

Liver

(1)

Heart (3) Liver (1) Kidney (2) Brain (3) Cult. fibroblasts (1)

Heart Liver Brain

Normal in (No.)

SYNDROMES ASSOCIATED WITH A PRESUMABLY PRIMARY CYTOCHROME c OXIDASE DEFICIENCY

TABLE 4

Slight residual weakness. One other patient with similar clinical picture described (Jerusalem et al. 1973) Relative in maternal line described by Monnens et al. 1975 See text for details

Cytochrome b in muscle may be deficient, too. Pathological findings normal in heart, liver and brain (3 patients) Cytochrome b in muscle may be deficient, too. Clinically cardiomyopathy in 2 patients

Other findings

t-O

113 necessary for the correct a s s e m b l y o f the enzyme complex, would explain both the functional deficiency o f the e n z y m e in the early-infantile fatal m y o p a t h y and the decrease in immunologically detectable enzyme protein. In Leigh's syndrome, there m a y be a defect o f a non-tissue-specific subunit, also synthesized u n d e r the control o f nuclear D N A . This unit w o u l d n o t influence the assembly o f the c y t o c h r o m e c oxidase complex, but inhibit the o p t i m a l functioning o f the c o m p l e x in m a n y tissues. H o w e v e r , even within the clinically h o m o g e n e o u s group o f patients with Leigh's syndrome, biochemical heterogeneity a p p e a r s to be present, since a n o r m a l e n z y m e activity in the liver (and cultured fibroblasts) was found in some patients, but a deficiency in others. The m o d e o f inhibition o f the c y t o c h r o m e c oxidase complex in Leigh's s y n d r o m e should be the subject o f further studies. ACKNOWLEDGEMENTS W e are grateful to Mrs. M. H. M. Vaandrager-Verduin a n d A. J. M. J a n s s e n who p e r f o r m e d some o f the assays in patient 1 a n d 2, respectively. Prof. J . F . K o s t e r gave helpful advice concerning the pyruvate d e h y d r o g e n a s e complex a s s a y in patient 1. Secretarial help was p r o v i d e d by Mrs. G. D e J o n g and Mrs. J. D o o r n b o s c h .

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