J. M. F. Trijbels

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.

Congenital cataract and mitochondrial myopathy of skeletal and heart muscle associated with lactic acidosis after exercise

Congenital cataract involving the nucleus, cortex, and capsule of the lens, and cardiomyopathy were found in seven of 22 children from three unrelated families. Histologic examination showed a mitochondrial myopathy of skeletal and heart muscle with storage of lipid and glycogen. When the patients performed submaximal exercise for 60 minutes they developed metabolic acidosis with lactic acidemia.

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

Biochemical Studies in the Liver and Muscle of Patients with Zellweger Syndrome

Summary: Biochemical studies have been performed in muscle, liver, leukocytes, and fibroblasts from patients suffering from the Zellweger syndrome. Oxidation rates of [1-14C]pyruvate, [U-14C]malate, and [1-14C]2-oxoglutarate were strongly reduced in skeletal muscle homogenate. Oxygen consumption in isolated skeletal muscle mitochondria could only be stimulated by ADP in the presence of ascorbate and N,N,N1,N1-tetramethyl-p-phenylenediamine. Cytochrome contents in heart muscle and liver mitochondria were normal. A very low activity of succinate-ubiquinone oxidoreductase was found in liver homogenate of two patients. From the effect of 2-thenoyltrifluoroacetone on the succinate-phenazine methosulphate oxidoreductase activity, a nearly competitive inhibition with respect to phenazine methosulphate was demonstrated in contrast with a non-competitive inhibition in controls. Normal oxidation rate of [1-14C]pyruvate and [2-14C]pyruvate was found in leucocytes and fibroblasts. Lactate and pyruvate levels were normal in serum and cerebrospinal fluid and β-hydroxybutyrate and acetoacetate levels were normal in blood. The ratios lactate/pyruvate and β-hydroxybutyrate/acetoacetate were normal as well. These findings point to a defect in the electron transport chain at the succinate-ubiquinone oxidoreductase level. This defect might be related to the absence of peroxisomes in the cells of Zellweger patients.

Infantile Refsum's disease: Biochemical findings suggesting multiple peroxisomal dysfunction

Infantile Refsums disease was diagnosed in three male patients, presenting with facial dysmorphia, retinitis pigmentosa, neurosensory hearing loss, hepatomegaly, osteopenia and delayed growth and psychomotor development.An elevated plasma phytanic acid concentration and a deficient phytanic acid oxidase activity in fibroblasts were found with an accumulation of very long chain fatty acids in plasma and fibroblasts. There were elevated pipecolic acid levels in plasma, urine and CSF, and abnormal bile acid metabolites in plasma.Deficient activity of acylCoA: dihydroxyacetone phosphate acyl transferase was found in thrombocytes and fibroblasts of these patients as well as an impairedde novo plasmalogen biosynthesis in fibroblasts. These biochemical abnormalities, previously described in the Zellweger syndrome, suggest multiple peroxisomal dysfunction in our patients.

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.

Determination of extracellular and intracellular thiopurines and methylthiopurines by high-performance liquid chromatography

The thiopurine antimetabolites 6-thioguanine and 6-mercaptopurine are important chemotherapeutic drugs in the treatment of childhood acute lymphoblastic leukaemia. Measurement of metabolites of these thiopurines is important because correlations exist between levels of these metabolites and the prognosis in childhood acute lymphoblastic leukaemia. The reversed-phase method for the determination of extracellular thiopurine nucleosides and bases was previously developed and has been modified such that methylthiopurine nucleosides, bases, thioxanthine and thiouric acid can be measured also. The anion-exchange method enables the determination of intracellular mono-, di- and triphosphate (methyl)thiopurine nucleotides in one run. Extraction on ice with perchloric acid and dipotassium hydrogenphosphate results in good recoveries for (methyl)thiopurine nucleotides in lymphoblasts and peripheral mononuclear cells and for methylthioinosine nucleotides in red blood cells. Measurement of the low concentrations of mono-, di- and triphosphate thioguanine nucleotides in red blood cells (detection limit 20 pmol/10(9) cells) is possible after extraction with methanol and methylene chloride, followed by oxidation of thioguanine nucleotides with permanganate and fluorimetric detection.

Clinical heterogeneity in respiratory chain complex III deficiency in childhood.

Six children are presented with an isolated complex III deficiency in muscle tissue. More specifically, oxidation rates and ATP+CrP production rates from both pyruvate and succinate as substrates and/or the activity of decylubiquinol:cytochrome c oxidoreductase were all markedly reduced. Complex III deficiency was also present in liver of two patients tested, but could not be demonstrated in cultured fibroblasts of four patients tested. Mitochondrial DNA, extracted from muscle, was analyzed; no deletions or common point mutations were found. Four patients presented with a multi-organ disorder. Among these patients three presented at neonatal age with neurological signs and lactate elevation in blood and CSF, of whom two had severe neonatal Fanconi syndrome. One child, aged seven years, had encephalomyopathy, ophthalmoplegia, retinopathy and Wolff-Parkinson-White syndrome. The remaining two patients exhibited myopathy only, within the first year of life. Thus, like in other respiratory chain disorders, patients with complex III deficiency may present at any age and show variable symptoms and outcome, ranging from neonatal death to failure to thrive only. Apparently there are no clinical findings which are specific for complex III deficiency.

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.