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Background: Mutations in the Twinkle (PEO1) gene are a recognized cause of autosomal dominant progressive external ophthalmoplegia (adPEO), resulting in the accumulation of multiple mitochondrial DNA (mtDNA) deletions and cytochrome c oxidase (COX)-deficient fibers in skeletal muscle secondary to a disorder of mtDNA maintenance. Patients typically present with isolated extraocular muscle involvement, with little apparent evidence of the clinical heterogeneity documented in other mtDNA maintenance disorders, in particular POLG-related disease., Methods: We reviewed the clinical, histochemical, and molecular genetics analysis of 33 unreported patients from 26 families together with all previous cases described in the literature to define the clinical phenotype associated with PEO1 mutations., Results: Ptosis and ophthalmoparesis were almost universal clinical features among this cohort, with 52% (17/33) reporting fatigue and 33% (11/33) having mild proximal myopathy. Features consistent with CNS involvement were rarely described; however, in 24% (8/33) of the patients, cardiac abnormalities were reported. Mitochondrial histochemical changes observed in muscle showed remarkable variability, as did the secondary mtDNA deletions, which in some patients were only detected by PCR-based assays and not Southern blotting. Moreover, we report 7 novel PEO1 variants., Conclusions: Our data suggest a shared clinical phenotype with variable mild multiorgan involvement, and that the contribution of PEO1 mutations as a cause of adPEO may well be underestimated. Direct sequencing of the PEO1 gene should be considered in adPEO patients prior to muscle biopsy.

Following the association of hereditary spastic paraparesis (HSP) with mutation in the paraplegin gene (SPG7) and mitochondrial dysfunction, we wished to investigate whether mitochondrial dysfunction might be associated with other forms of HSP. Five cases of HSP caused by mutation in the spastin gene (SPG4) and nine cases with HSP with mutation in the spastin and paraplegin genes excluded (non-SPG4/SPG7), were investigated for mitochondrial dysfunction. Muscle tissue from the HSP groups and a control group was analysed histochemically and spectrophotometrically for mitochondrial dysfunction. A significant decrease in mitochondrial respiratory chain complexes I and IV was demonstrated in the non-SPG4/SPG7 group. No abnormality was detected in the SPG4 group. We therefore conclude that there is evidence for mitochondrial dysfunction in non-SPG4/SPG7 HSP. There is no evidence for mitochondrial dysfunction in the pathogenesis of spastin-related HSP.

Mitochondrial disorders represent a multitude of clinically heterogeneous diseases in which the genetic abnormality can involve either a mitochondrial or nuclear gene. In addition to inherited defects, somatic mitochondrial DNA mutations have been implicated in the pathogenesis of neurodegenerative disease, cancer and the ageing process. The recent emergence of the first mouse models of mitochondrial disease will provide valuable insights into disease mechanisms and aid the development of realistic therapeutic strategies.

Mitochondrial DNA (mtDNA) is the only autonomously replicating source of DNA outside the nucleus. The mitochondrial genome encodes thirteen essential polypeptides of the mitochondrial respiratory chain. Defects of the mitochondrial genome can cause severe neurological and multi-systemic disorders. In many patients there is a mixture of mutated and wild-type mtDNA in the same cell (a situation termed heteroplasmy). In these patients the ratio of mutated to wild-type mtDNA is crucial and a biochemical defect only occurs with relatively high levels of mutated mtDNA within an individual cell. This threshold also seems to be critical in the development of mtDNA disease. Since the genetic defect causes a dysfunction in the terminal stage of oxidative metabolism, there is little potential for pharmacological intervention. Molecular techniques must be developed to reverse the ratio of mutated and wild-type mtDNA. In this paper we summarise our approach using both antigenomic peptide nucleic acids and cell necrosis.

The extent to which mitochondria are involved in the aging process is controversial; much of the reported decline in mitochondrial oxidations in human skeletal muscle may be due to disease and inactivity rather than age. To study true aging, mitochondrial respiratory chain function was studied in 9 young and 12 elderly athletes. No significant deterioration with age was observed. If mitochondria are involved in aging, it must be through a more subtle mechanism than a global decline in respiratory chain activity.

It has been proposed that ageing results from the accumulation of mitochondrial DNA mutations with age which interfere with respiratory chain ATP production. Insufficient ATP production impairs cell function, and tissue dysfunction ensues, leading to morbidity, decline and eventually death. Supporting this theory, mitochondrial DNA mutations accumulate with age and respiratory chain function declines dramatically in human skeletal muscle. However, the extent of decline in respiratory chain function is greater (50%) than anticipated from the low levels of mitochondrial DNA mutations (< 1%) observed in aged muscle. We hypothesized that an age-related reduction in physical activity could be an important factor in this decline and thus studied the influence of chronological age on muscle mitochondria in subjects matched for levels of physical activity. In this carefully selected group, there was little correlation between oxidative metabolism and age. However, several parameters of respiratory chain function did correlate with markers of physical activity (activity score and handgrip strength). Our results suggest that reduced physical activity is a major contribution to the decline in mitochondrial oxidations during ageing. Physical activity ameliorates and may even mask mitochondrial 'ageing' in muscle.

Objective: To test the hypothesis that low-tension glaucoma has a pathogenesis similar to Leber's hereditary optic neuropathy and results from a defect in the mitochondrial respiratory chain., Methods: Mitochondrial fractions were prepared from skeletal muscle samples collected from eight subjects with low-tension glaucoma. Their oxidative metabolism was compared with that of age- and sex-matched controls. Skeletal muscle DNA prepared from the subjects with glaucoma was also screened for the 3,460, 11,778, and 14,484 mitochondrial DNA mutations that are associated with Leber's hereditary optic neuropathy., Results: No subject with low-tension glaucoma had a defect in respiratory chain activity or one of three mitochondrial DNA mutations that are commonly associated with Leber's hereditary optic neuropathy., Conclusion: Although these results do not exclude the possibility that low-tension glaucoma is caused by an organ-specific defect of mitochondrial function, we have excluded a systemic defect of the mitochondrial respiratory chain.

Defects of the mitochondrial respiratory chain are increasingly being recognized as an important cause of neurological disease in humans. In many of these patients, the biochemical defect results from an abnormality of the mitochondrial genome. Respiratory chain defects involving complex II, which is entirely encoded by the nuclear genome, are comparatively rare. We report the clinical and biochemical findings in 2 elderly sisters who presented with late-onset neurodegenerative disease. In both patients, a partial deficiency of complex II (approximately 50% of control values) was shown to be present in mitochondria from muscle and platelets. The enzyme defect was not expressed in cultured skin fibroblasts or immortalized lymphocytes. There was an overexpression of the 70-kd flavoprotein subunit in muscle mitochondria from both patients, although we showed that this subunit is present in normal amounts in mitochondrial membranes. Our studies highlight the diversity of the clinical presentation of respiratory chain disease and that complex II deficiency should enter the differential diagnosis of certain patients with late-onset neurodegenerative disease.

We present evidence for the existence of a previously unrecognised enzyme of mitochondrial beta-oxidation in man. This enzyme, which is situated in the mitochondrial matrix and has medium- and long-chain 2-enoyl-CoA hydratase activity, was identified by studying tissues from a patient with a severe deficiency of the trifunctional protein of mitochondrial beta-oxidation. The novel enzyme is present in all tissues studied--heart, liver and cultured skin fibroblasts--but is particularly active in liver. Together with other recently identified enzymes of mitochondrial beta-oxidation, the existence of this new enzyme suggests that two mitochondrial beta-oxidation enzyme systems exist in man: one associated with the mitochondrial membrane, responsible for the beta-oxidation of long-chain fatty acids and one in the mitochondrial matrix, responsible for the oxidation of medium- and short-chain fatty acids.

Objective: To determine if diabetes insipidus, diabetes mellitus, optic atrophy, and deafness (DIDMOAD) combined with a cerebellar syndrome is associated with a systemic disorder of respiratory chain function as found in similar genetic syndromes., Case: A muscle biopsy was taken from a patient with DIDMOAD, and a mitochondrial fraction was prepared. Respiratory chain function was assessed by analysis of intermediary metabolites, histochemical analysis of muscle biopsy, measurement of the activity of individual respiratory chain complexes, oxidative flux through the respiratory chain, and cytochrome concentration and compared with a population with normal respiratory chain function. Mitochondrial DNA from skeletal muscle, brain, and pancreas was examined for major rearrangements and specific point mutations. Brain tissue was examined neuropathologically for abnormalities, particularly those previously described in association with DIDMOAD., Results: No abnormality was found in mitochondrial oxidation, individual complex activity, or cytochrome concentration. Histochemical analysis and electron microscopy showed no abnormality known to be associated with mitochondrial dysfunction. A single-base substitution at position 12308 of the mitochondrial genome was found, but no major rearrangement of mitochondrial DNA was demonstrated. Neuropathological examination revealed severe demyelination and gliosis in the optic nerves and loss of Purkinje cells associated with gliosis in the white matter in the cerebellum., Conclusions: We have found no evidence that DIDMOAD is associated with a systemic abnormality of respiratory chain function. The mitochondrial DNA single-base substitution noted is likely to be a polymorphism rather than a pathogenic point mutation. We have confirmed that DIDMOAD may be associated with a neurodegenerative disorder, but the cause of this remains undetermined.

We have studied the control by complex III of both succinate-cytochrome c reductase and of oxidative flux measured polarographically in rat muscle and liver mitochondria using the specific inhibitor, myxothiazol, to induce partial inhibitions of complex III activity. Complex III exerted a low degree of control on electron flux through succinate-cytochrome c reductase, and a 30-50% decrease in complex III activity remained undetected by this assay. However, when overall oxidative flux was measured polarographically there was a considerable difference in the effect of lowered complex III activity on this pathway between rat muscle and liver mitochondria. Small changes in complex III activity (approximately 5% inhibition) in muscle mitochondria caused marked changes in succinate-stimulated respiration, whereas in liver mitochondria complex III had to be inhibited by about 45% before changes in maximum oxidation rates could be detected. These differences between muscle and liver mitochondria occurred despite rat liver mitochondria having at least a 4-fold lower complex III activity. This suggests that when considering the biochemical consequences of defects of the respiratory chain an important factor is the degree to which an individual complex can be lowered before major changes in overall flux occur. In addition, since many patients with respiratory chain disease present with predominantly muscle symptoms, this latter finding suggests that an understanding of the control of mitochondrial oxidations by different tissues may be important when considering the tissue-specific nature of defects of the respiratory chain.

The measurement of individual respiratory chain complexes is an important component of the investigation of diseases due to mitochondrial dysfunction. We have evaluated assays which measure complexes I to IV in human skeletal muscle mitochondria and in addition optimized these assays to provide sensitive and reliable diagnostic techniques, particularly in situations where a partial interruption at a single complex needs to identified. Using several established methods of membrane disruption we have found that optimal activities of complexes I and II are obtained by freeze-thawing the mitochondria in hypotonic potassium phosphate buffer, whereas complex III and IV activities are markedly increased by the addition of the detergent n-dodecyl-beta-D-maltoside. Complex I activity is measured in the presence of 2.5 mg.ml-1 bovine serum albumin, which increases rotenone sensitivity, and we have shown that NADH-cytochrome b5 reductase makes an important contribution to the rotenone-insensitive NADH-ubiquinone oxidoreductase activity. Complex II activity is measured after preincubation of the mitochondrial fraction with succinate to fully activate the complex. Complex I and III activities are dependent upon the length of the isoprenoid chain of the ubiquinone and ubiquinol, respectively. These assays have been used to establish a control range.

Defects of the respiratory chain are important causes of human disease and one of the most commonly used assays in the investigation of these patients is the measurement of succinate-cytochrome c reductase. However, this assay measures several components of the respiratory chain and the ability to detect a partial defect in one enzyme complex will depend on the amount of control exerted by that enzyme step on overall electron flux. We show that measurement of succinate-cytochrome c reductase activity may fail to detect partial defects of complex III and therefore is of limited diagnostic value in the identification of complex III defects. However, complex II is a major point of control of flux through succinate-cytochrome reductase and it is likely that measurement of the latter will detect defects of complex II.

1. We describe the acyl-CoA and acyl-carnitine esters which arise from the incubation of well-coupled State 3 rat skeletal-muscle mitochondrial fractions with [U-14C]hexadecanoate and [U-14C]hexadecanoyl-carnitine. 2. Acyl-CoA ester intermediates of chain length 16, 14, 12, 10 and 8 carbons were detected. 3. Although incubations were in steady state in respect of oxygen consumption, 14CO2 production and generation of acid-soluble radioactivity, quantitative analysis of acyl-CoA esters showed that steady state was not achieved in respect of all intermediates. 4. 3-Hydroxyacyl- and 2-enoyl-CoA and -carnitine esters were found under normoxic conditions. 5. Direct measurement of NAD+ and NADH shows that under identical incubation conditions our observations cannot be explained by gross perturbation of the [NAD+]/[NADH] ratio. 6. We hypothesize that there is a small pool of rapidly recycling NAD+ channelled between complex I of the respiratory chain and the newly described mitochondrial-inner-membrane-associated beta-oxidation trifunctional enzyme [Uchida, Izai, Orii and Hashimoto (1992) J. Biol. Chem. 267, 1034-1041].

We describe the use of a simple assay for beta-oxidation which depends on the release of 3H2O from [9,10-3H]hexadecanoate. This was compared with the use of [1-14C]hexadecanoate which gave comparable results when all the products of beta-oxidation were measured. The prediction that 75% of the tritium is released as 3H2O and 25% as [2-3H]acetyl units was confirmed. The assay was used successfully to demonstrate impaired beta-oxidation in tissue preparations from rats treated with etomoxir and methylenecyclopropylpyruvate which are known inhibitors of beta-oxidation. Abnormalities of beta-oxidation were also detected in skeletal muscle from patients with defects of mitochondrial oxidation.

Parkinson's disease is one of the commonest neurodegenerative disorders in Western society. Although the neuropathological changes have been well documented, the underlying biochemical defect is unknown. Toxins may play a part in the aetiology of this disorder. It has been shown that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces a Parkinson-like syndrome in both man and primates and 1-methyl-4-phenylpyridine (MPP+), a metabolite of MPTP, inhibits NADH-ubiquinone oxidoreductase (complex I) of the mitochondrial respiratory chain. We studied mitochondrial respiratory chain function in skeletal muscle from patients with Parkinson's disease because, like brain, it has a high dependence on oxidative metabolism. Our results show low activity in all complexes studied (I, II and IV). The implications of these findings are discussed in relation to the aetiology of Parkinson's disease.

Southern hybridisation demonstrates deleted mitochondrial DNAs (mtDNAs) in muscle but not in blood in a subgroup of patients with mitochondrial myopathy. The polymerase chain reaction (PCR) was used to search for low levels of rearranged mitochondrial DNAs in blood in 24 patients with mitochondrial myopathy, and 15 asymptomatic relatives, all of whom have no detectable abnormality on restriction enzyme analysis of blood mitochondrial DNA. In eight patients and two of their relatives, PCR products were obtained consistent with deletions of mitochondrial DNA. The presence or absence of a deletion was correctly predicted in 10 out of 11 patients from whom information was available from muscle DNA. No false positives were obtained in 43 controls. PCR analysis of blood may be applicable as a non-invasive screening test of affected individuals and in carrier detection.

Defects of complex I of the mitochondrial respiratory chain are important causes of neurological disease. We report studies that demonstrate a severe deficiency of complex I activity with less severe abnormalities of complexes III and IV (less than 5, 63, and 30% of control values, respectively) in a skeletal muscle mitochondrial fraction from a 22-yr-old female with weakness, lactic acidemia, and the deposition of intramuscular neutral lipid. The observation that lipid accumulates in this and other patients with complex I deficiency suggests impaired mitochondrial fatty acid oxidation. To investigate this mechanism we have shown impaired flux through beta-oxidation [( U-14C]hexadecanoate oxidation was 66% of control rate) and accumulation of specific acyl-CoA ester intermediates. The changes in fatty acid metabolism in complex I deficiency are secondary to the reduced state within the mitochondrial matrix with low NAD+/NADH ratios.

We report the cases of a 46 year old woman and of a 18 year-old boy who met the criteria for Kearns-Sayre syndrome. Additional atypic features were present in one case: family history, psychosis and acute respiratory failure. In both cases histoenzymatic analysis of the muscle biopsy and biochemical studies of mitochondria isolated from the muscle sample demonstrated mitochondrial myopathy associated with combined partial deficiency of complexes I and IV of the electron transfer chain. Although there is no correlation between clinical and biological data in the mitochondrial myopathies our cases confirm that such defects are involved in Kearns-Sayre syndrome. Improvement with coenzyme Q10 therapy in these patients is reported.

Well-coupled mitochondrial fractions were prepared from rat skeletal muscle without the use of proteolytic enzymes. The products of [U-14C]hexadecanoate oxidation by rat skeletal muscle mitochondrial fractions were analysed by h.p.l.c. with on-line radiochemical detection. In the presence of 1 mM-carnitine, 70% of the products is acetylcarnitine. In agreement with Veerkamp et al. [Veerkamp, van Moerkerk, Glatz, Zuurveld, Jacobs & Wagenmakers (1986) Biochem. Med. Metab. Biol. 35, 248-259] 14CO2 release is shown to be an unreliable estimate of flux through beta-oxidation in skeletal muscle mitochondrial fractions. The flux through beta-oxidation is recorded unambiguously polarographically in the presence of 1 mM-carnitine and the absence of citrate cycle intermediates.

Chronic progressive external ophthalmoplegia (CPEO) associated with proximal myopathy and/or craniosomatic abnormalities is a rare syndrome in which morphological mitochondrial changes have been found in some fibres (subsarcolemmal accumulation of mitochondria or "ragged red" fibres). We report a 14-year-old boy with CPEO and a mild proximal myopathy without these characteristic "ragged red" fibres. Histochemistry of skeletal muscle showed a mosaic of fibres without detectable cytochrome oxidase activity, while other mitochondrial enzymes were normal. The total cytochrome oxidase activity and cytochrome aa3 concentration in muscle mitochondrial fractions were only 40% of normal. This case is unique in that a biochemical defect was not accompanied by morphological abnormalities and may represent an early stage of CPEO before the development of morphological changes, or alternatively, a new variant of the disease.

A case of cytochrome c oxidase deficiency primarily affecting skeletal muscle is described. The child was admitted at 4 weeks due to failure to thrive and examination at that time revealed weakness and hypotonia. His condition deteriorated until at 11 weeks respiratory arrest necessitated artificial ventilation and death occurred at 14 weeks. Biochemical investigation showed lactic acidaemia and generalised aminoaciduria. Histochemical examination of muscle obtained at biopsy showed strong reactions for some oxidative enzymes, but by contrast cytochrome c oxidase could not be detected. Cytochrome c oxidase activity was less than 5% of control values in an extract of fresh muscle. The reduced-minus oxidised absorption spectra of muscle mitochondrial fractions prepared from post-mortem tissue showed an absence of cytochrome aa3 and a partial deficiency of cytochrome b. Ultra-structural examination showed abnormal mitochondria with loss of cristae and an abnormal granular matrix. The family history suggests autosomal recessive inheritance.

Disturbances of pyruvate metabolism have been implicated in the aetiology of several neurological disorders including Leigh's disease and familial ataxia. We have re-investigated a patient whose initial description documented intermittent ataxia, a presumed disorder of pyruvate metabolism and an X-linked pattern of inheritance. Recent studies showed he had slow oxidation of pyruvate, low pyruvate dehydrogenase complex (PDC) activity and immunochemical evidence of E1 deficiency in skeletal muscle mitochondria. This is consistent with the recent finding that the gene for E1 alpha is on the X chromosome.

Quantitative cytochemical and biochemical techniques have been used in combination to study the response of mitochondrial oxidative enzymes in individual muscle fibre types to hypo- and hyperthyroidism. Hypothyroidism resulted in decreased activity of succinate dehydrogenase (SDH), L-glycerol-3-phosphate dehydrogenase (L-GPDH), and D-3-hydroxybutyrate dehydrogenase (D-HBDH) in all fibre types of both slow-twitch soleus and fast-twitch extensor digitorum longus (e.d.l.) muscles. In hyperthyroidism, only L-GPDH activity increased in e.d.l. but more marked increases were seen in soleus muscles, which also showed increased SDH activity. In addition to these alterations in the enzyme activity in individual fibre types the metabolic profile of the muscle is further modified by the hormone-induced interconversion of slow- to fast-twitch fibres and vice versa.

Deficiency of complex I is one of the most commonly reported defects of the mitochondrial respiratory chain in man. Clinical evidence of tissue specific expression of complex I deficiency has not previously been confirmed biochemically. We report here slow oxidation of NAD+-linked substrates, low activity of complex I and low amounts of immunoreactive complex I peptides in skeletal muscle mitochondria from a patient with muscle weakness and lactic acidosis. In liver mitochondria complex I activity was normal and all the immunoreactive subunits of complex I were present in normal amounts.

Fatty acids are important substrates for resting and exercising skeletal muscle. Defects of fatty acid oxidation result in muscle pain and weakness, and there is often lipid accumulation in muscle fibres. Only a few of the several possible enzyme defects have been found and some of these have responded to therapy. Some techniques for the investigation of fatty acid oxidation are outlined.