A. J. M. Janssen

Differential investigation of the capacity of succinate oxidation in human skeletal muscle.

Procedures are described for the estimation of the succinate:ubiquinone oxidoreductase and succinate:phenazine methosulfate oxidoreductase activities in post-nuclear supernatants of human skeletal muscle homogenates using 2,6-dichlorophenol indophenol as the terminal electron acceptor. The influence of ionic strength and of sucrose upon these assays and upon the succinate:cytochrome c oxidoreductase activity has been investigated. Sucrose markedly interferes with the activation of the succinate dehydrogenase complex. Succinate:cytochrome c oxidoreductase activity and succinate:phenazine methosulfate oxidoreductase activity are inhibited by increasing concentrations of ions and of sucrose. Our results lead us to propose the existence of a single acceptor site for phenazine methosulfate at the succinate dehydrogenase complex, not involved in the physiological electron flux across ubiquinone. Estimation of the enzymatic activities mentioned above allows differential investigation of the functional integrity of a large part of the respiratory chain in patients suspected of suffering from a neuromuscular disorder.

A mitochondrial encephalomyopathy: the first case with an established defect at the level of coenzyme Q

A patient is presented who had therapy-resistant epileptic seizures from the 7th day of life. Examination at the age of 17 months revealed a mentally retarded boy with epileptic seizures, generalised myoclonic contractions, and abnormal ocular movements. A cerebral CT scan showed central and cortical atrophy. Lactate levels in serum, cerebrospinal fluid and urine were elevated, the pyruvate level was raised in serum. A quadriceps muscle biopsy revealed aspecific morphologic signs of a myopathy. Biochemical analysis showed decreased substrate oxidation rates in the mitochondria associated with low rates of ATP production. Total and free carnitine levels were decreased. Investigation of the respiratory chain revealed a defect in the proximal part of respiratory chain revealed a defect in the proximal part of respiratory chain involving the region of coenzyme Q. Based on clinical and chemical data it is likely that the patient is suffering from a multi-system disorder.

Coenzyme Q- responsive Leigh's encephalopathy in two sisters

A 31‐year‐old woman had encephalopathy, growth retardation, infantilism, ataxia, deafness, lactic acidosis, and increased signals of caudate and putamen on brain magnetic resonance imaging. Muscle biochemistry showed succinate:cytochrome c oxidoreductase (complex II–III) deficiency. Both clinical and biochemical abnormalities improved remarkably with coenzyme Q10 supplementation. Clinically, when taking 300mg coenzyme Q10 per day, she resumed walking, gained weight, underwent puberty, and grew 20cm between 24 and 29 years of age. Coenzyme Q10 was markedly decreased in cerebrospinal fluid, muscle, lymphoblasts, and fibroblasts, suggesting the diagnosis of primary coenzyme Q10 deficiency. An older sister has similar clinical course and biochemical abnormalities. These findings suggest that coenzyme Q10 deficiency can present as adult Leighs syndrome. Ann Neurol 2002;52:000–000

Estimation of NADH oxidation in human skeletal muscle mitochondria

Assay procedures are described for the detection of defects in the process of NADH oxidation by the respiratory chain in human skeletal muscle biopsy specimens. The procedures allow determination of rotenone-sensitive NADH: O2 oxidoreductase and NADH: ubiquinone-1 oxidoreductase activity not only in isolated mitochondria but also in post-nuclear supernatants. The use of ferricyanide as electron acceptor for estimation of NADH dehydrogenase activity is inadequate when only applied on a disrupted mitochondrial preparation.

Pyruvate oxidation in rat and human skeletal muscle mitochondria.

Abstract Pyruvate oxidation in rat and human skeletal muscle mitochondria was studied by measuring the rate of 14 CO 2 production from [1- 14 C]pyruvate in the presence of 1 m m pyruvate, an excess of ADP, and varying amounts of citric acid cycle intermediates or carnitine. The rate of pyruvate oxidation is controlled by the availability of acetyl-CoA acceptor since addition of citric acid cycle intermediates or carnitine results in a stimulation of pyruvate oxidation. Pyruvate oxidation proceeds at its maximal rate in the presence of malate since no further stimulation is observed by addition of carnitine. It is concluded that pyruvate dehydrogenase is the rate-limiting step during pyruvate oxidation in the presence of malate. In human skeletal muscle mitochondria pyruvate oxidation proceeds maximally both in the presence of malate or carnitine. Parallel incubations of these mitochondria with [1- 14 C]pyruvate plus malate and [1- 14 C] pyruvate plus carnitine may allow one to establish disturbances in pyruvate oxidation and to discriminate between defects in the pyruvate dehydrogenase complex and in the activity of the citric acid cycle.

Lethal infantile mitochondrial disease with isolated complex I deficiency in fibroblasts but with combined complex I and IV deficiencies in muscle

A 2-month-old boy died of a lethal infantile mitochondrial disease with severe lactic acidosis and involvement of the CNS. Histochemical analysis of skeletal muscle showed that cytochrome c oxidase staining was lacking in all muscle fibers but was present in arterioles. Ragged red fibers were not seen, but some fibers showed excessive staining for succinate dehydrogenase. Biochemical analysis revealed a combined complex I and IV deficiency in skeletal muscle but only a complex I deficiency in his fibroblasts. Two-dimensional native SDS electrophoresis confirmed these enzymatic findings at the protein level. Analysis of mitochondrial translation products in fibroblasts revealed no abnormalities, and analysis of mitochondrial DNA in muscle showed no depletion, large-scale deletions, or frequently occurring point mutations. We conclude that this disease must have been the result of either a nuclear DNA mutation in a gene controlling the expression or assembly of both complex I and the muscle-specific isoform of complex IV or, alternatively, a heteroplasmic point mutation in a mitochondrial tRNA, which codon is used more often by mtDNA encoded subunits of complex I than by mtDNA encoded subunits of complex IV. A different degree of heteroplasmy in skeletal muscle and fibroblasts would then explain the curious heterogeneous tissue expression of defects in this patient. NEUROLOGY 1996;47: 243-248

Investigation of mitochondrial metabolism in small human skeletal muscle biopsy specimens. Improvement of preparation procedure

A method is presented which allows the investigation of almost the complete mitochondrial content of small human skeletal muscle biopsy specimens. Thorough mechanical disruption with a chopper apparatus results in the release of about 50% of the mitochondrial content. Subsequent treatment of the 600 x g sediment with trypsin releases another 30% of the total mitochondrial population. The biochemical characteristics of the two mitochondrial fractions obtained in these two successive steps have been compared. No obvious differences could be established. The procedure is well suited for biochemical investigation of muscle biopsy specimens from patients suspected of suffering from a mitochondrial myopathy.

Disorders of the mitochondrial respiratory chain: clinical manifestations and diagnostic approach

The clinical identification of patients with defects in the mitochondrial respiratory chain is almost impossible. We describe screening tests that should be performed in order to select those patients in whom a skeletal muscle biopsy should be carried out for more specific biochemical assays. The importance of performing in vivo function tests is stressed. The biochemical diagnosis in disorders of the respiratory chain is presented and the application of immunological methods discussed.

Deficiency of cytochromes b and aa3 in muscle from a floppy infant with cytochrome oxidase deficiency

A girl was presented suffering from generalised weakness and cardiorespiratory insufficiency. She succumbed at the age of 5 months. Lactate levels were elevated in serum, cerebrospinal fluid and urine. Histopathological examination revealed a mitochondrial myopathy. In muscle tissue the cytochrome oxydase activity was strongly reduced. The content of cytochromes b and aa3 was very low. At autopsy a cardiomyopathy was found.

Septo-optic dysplasia associated with a new mitochondrial cytochrome b mutation

We report on a 25‐year‐old patient with isolated mitochondrial complex III deficiency and a new heteroplasmic mutation (T14849C) in the cytochrome b gene. He suffered from septo‐optic dysplasia, retinitis pigmentosa, exercise intolerance, hypertrophic cardiomyopathy, and rhabdomyolysis. A HESX1 mutation was excluded as a cause of his septo‐optic dysplasia. Low α‐tocopherol concentrations in his muscles and an elevated urinary leukotriene E4 excretion indicate increased production of reactive oxygen species.