Lack of energy and tiredness are among the most serious complaints experienced by children with neuromuscular disease like those affected by a disturbance of the mitochondrial oxidative phosphorylation system. We experienced that commonly used endurance tests are difficult or impossible to execute by the latter patient group. Besides this, studies in a laboratory setting do not always reflect disabilities experienced in daily life. Moreover, many of these children are not able to follow instructions or complete questionnaires concerning their level of tiredness. Therefore, continuous activity measurement in the child's natural surrounding by accelerometers may better reflect the (lack of) energy to perform daily activities. The aim of this study was to test the feasibility, reliability and validity of the MOX accelerometer in measuring daily physical activity in patients with mitochondrial disease. By using accelerometers (MOX sensors, Maastricht Instruments), the intensity of movement and the orientation of body parts are predicted. We've used five sensors per patient (on the lower arm, upper arm, upper leg, trunk and on the wheelchair). Feasibility was defined as the reliability of data acquisition and parent reported complications with the meters. Reliability was tested using the patient's diary, and the proportion of time the accelerometer was able to provide data. Validity was tested using the correspondence with video images and the patient's diary. The first results of our study show that we are able to distinguish between ambulatory and non-ambulatory patients. Furthermore, we were able to differentiate between sitting, walking and standing episodes and the obtained data corresponded to the activities in the patients' diaries. These results will be presented in more detail at the conference.

Most eukaryotic cells depend on mitochondrial OXidative PHOSphorylation (OXPHOS) in their ATP supply. The cellular consequences of OXPHOS defects and the pathophysiological mechanisms in related disorders are incompletely understood. Using a quantitative proteomics approach we provide evidence that a genetic defect of complex‐I of the OXPHOS system may associate with transcriptional derangements of mitochondrial biogenesis through stabilization of the master transcriptional regulator PPARγ co‐activator 1α (PGC‐1α) protein. Chronic oxidative stress suppresses the gene expression of PGC‐1α but concomitant inhibition of the ubiquitin–proteasome system (UPS) can stabilize this co‐activator protein, thereby inducing its downstream metabolic gene expression programs. Thus, mitochondrial biogenesis, which lays at the heart of the homeostatic control of energy metabolism, can be deregulated by secondary impairments of the protein turnover machinery.

We studied the extent of mitochondrial involvement in chronic fatigue syndrome (CFS) and investigated whether measurement of mitochondrial respiratory chain complex (RCC) activities discriminates between CFS and mitochondrial disorders. Mitochondrial content was decreased in CFS compared to healthy controls, whereas RCC activities corrected for mitochondrial content were not. Conversely, mitochondrial content did not discriminate between CFS and two groups of mitochondrial disorders, whereas ATP production rate and complex I, III and IV activity did, all with higher activities in CFS. We conclude that the ATP production rate and RCC activities can reliably discriminate between mitochondrial disorders and CFS.

Reactive oxygen species (ROS) are known to be involved in the pathophysiology of complex regional pain syndrome type I (CRPS I). Since the mitochondrial respiratory chain is a major source of ROS, we hypothesized that mitochondria play a role in the pathophysiology of CRPS I. The hypothesis was tested by studying mitochondrial energy metabolism in muscle tissue from amputated limbs of CRPS I patients. We observed that mitochondria obtained from CRPS I muscle tissue displayed reduced mitochondrial ATP production and substrate oxidation rates in comparison to control muscle tissue. Moreover, we observed reactive oxygen species evoked damage to mitochondrial proteins and reduced MnSOD levels. It remains to be established if the mitochondrial dysfunction that is apparent at the end-stage of CRPS I is also present in earlier stages of the disease, or are secondary to CRPS I. The observation of a reduced mitochondrial energy production combined with reactive oxygen species induced damage in muscle tissue from CRPS I patients warrants further studies into the involvement of mitochondrial dysfunctioning in the pathophysiology of CRPS I.

BACKGROUND:Muscle biopsy analysis is regarded as the gold standard in diagnostic workups of patients with suspected mitochondrial disorders. Analysis of cultured fibroblasts can provide important additional diagnostic information. The measurement of individual OXPHOS complexes does not always provide sufficient information about the functional state of the complete mitochondrial energy-generating system. Thus, we optimized a high-throughput fluorescence-based methodology for oxygen consumption analysis in patient-derived cells.METHODS:We analyzed mitochondrial respiration in digitonin-permeabilized cells in the presence of a substrate mix containing pyruvate and malate, using a phosphorescent probe, 96-well plates, and a fluorescence plate reader.RESULTS:In control fibroblasts, we observed clear stimulation by ADP of the pyruvate + malate-driven respiration. Known inhibitors of the OXPHOS system and the Krebs cycle significantly reduced respiration. In patient fibroblasts with different OXPHOS deficiencies, ADP-stimulated respiratory activity was decreased in comparison to control cells. In several patients with reduced ATP production rate in muscle tissue but with normal OXPHOS enzyme activities, the fibroblasts displayed reduced respiratory activity. Finally, we observed a clear difference between control and complex I-deficient transmitochondrial cybrid cells.CONCLUSIONS:These results confirm the validity of the assay as a high-throughput screening method for mitochondrial function in digitonin-permeabilized cells. The assay allows primary and secondary mitochondrial abnormalities in muscle to be differentiated, which is of great importance with respect to counseling, and also will facilitate the search for new genetic defects that lead to mitochondrial disease.

Objective: The mitochondrial energy-generating system (MEGS) encompasses the mitochondrial enzymatic reactions from oxidation of pyruvate to the export of adenosine triphosphate. It is investigated in intact muscle mitochondria by measuring the pyruvate oxidation and adenosine triphosphate production rates, which we refer to as the "MEGS capacity." Currently, little is known about MEGS pathology in patients with mutations in the mitochondrial DNA. Because MEGS capacity is an indicator for the overall mitochondrial function related to energy production, we searched for a correlation between MEGS capacity and 3243A -> G mutation load in muscle of patients with the MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes) syndrome.Methods: In muscle tissue of 24 patients with the 3243A -> G mutation, we investigated the MEGS capacity, the respiratory chain enzymatic activities, and the 3243A -> G mutation load. To exclude coinciding mutations, we sequenced all 22 mitochondrial transfer RNA genes in the patients, if possible.Results: We found highly significant differences between patients and control subjects with respect to the MEGS capacity and complex I, III, and IV activities. MEGS-related measurements correlated considerably better with the mutation load than respiratory chain enzyme activities. We found no additional mutations in the mitochondrial transfer RNA genes of the patients.Interpretation: The results show that MEGS capacity has a greater sensitivity than respiratory chain enzymatic activities for detection of subtle mitochondrial dysfunction. This is important in the workup of patients with rare or new mitochondrial DNA mutations, and with low mutation loads. In these cases we suggest to determine the MEGS capacity.

We report on the clinical, molecular and biochemical findings of a patient with the rare event (<4.02 x 10(-9) per generation) of coinciding de novo mutations in the nuclear PAX6 (c.1252-1267del16) and the mitochondrial mt.RNA (Lys) (8347A-->G) genes. The boy suffers from exercise intolerance, ptosis, nystagmus, macular hypoplasia and anterior segment abnormalities evocative of Axenfeld-Rieger anomaly. The PAX6 mutation is predicted to cause haploinsufficiency. The novel mt.RNA (Lys) mutation is located close to the classic myoclonic epilepsy with ragged-red-fibers mutation, but the patient exhibits neither myoclonic epilepsy nor ragged-red-fibers. The degree of mutant mtDNA heteroplasmy, as determined by a very accurate pyrosequencing assay, varies between 31% (muscle) and 38% (fibroblasts). We discuss a potential effect of the PAX6 mutation on the mtDNA mutation rate.

Background: A reliable and sensitive complex I assay is an essential tool for the diagnosis of mitochondrial disorders, but current spectrophotometric assays suffer from low sensitivity, low specificity, or both. This deficiency is mainly due to the poor solubility of coenzyme-Q analogs and reaction mixture turbidity caused by the relatively high concentrations of tissue extract that are often required to measure complex I. Methods: We developed a new spectrophotometric assay to measure complex I in mitochondrial fractions and applied it to muscle and cultured fibroblasts. The method is based on measuring 2,6-dichloroindophenol reduction by electrons accepted from decylubiquinol, reduced after oxidation of NADH by complex I. The assay thus is designed to avoid nonspecific NADH oxidation because electrons produced in these reactions are not accepted by decylubiquinone, resulting in high rotenone sensitivity. Results: The assay was linear with time and amount of mitochondria. The Km values for NADH and 2,6-dichloroindophenol in muscle mitochondria were 0.04 and 0.017 mmol/L, respectively. The highest complex I activities were measured with 0.07 mmol/L decylubiquinone and 3.5 g/L bovine serum albumin. The latter was an essential component of the reaction mixture, increasing the solubility of decylubiquinone and rotenone. In patients with previously diagnosed complex I deficiencies, the new assay detected the complex I deficiencies in both muscle and fibroblasts. Conclusions: This spectrophotometric assay is reproducible, sensitive, and specific for complex I activity because of its high rotenone sensitivity, and it can be applied successfully to the diagnosis of complex I deficiencies.

We retrospectively analyzed the clinical, histological, and biochemical data of 11 children, five of which carried the maternally-inherited mitochondrial T8993C and six carrying the T8993G point mutations in the ATP synthase 6 gene. The percentage of heteroplasmy was 95% or higher in muscle and in blood. All patients had an early clinical presentation with muscle hypotonia, severe extrapyramidal dysfunction and Leigh disease demonstrated by the cranial MRI. A slower clinical progression and more frequent sensory-neuronal involvement were noted in the patients carrying the T8993C mutation in a high mutation load in muscle and blood. No histological abnormality was found. In 9 out of 11 patients a decreased ATP production was detected, and complex V activity was deficient in all children. The activities of the respiratory enzyme complexes II and IV were normal, whereas an associated combined complex I and III deficiency were present in two patients. No obvious difference was found between the biochemical parameters of the two patient groups harboring different mutations in the same gene. No correlation was found between the degree of complex V enzyme deficiency and the severity of the phenotype. We confirmed an impaired assembly/stability of complex V in our patients. This is the first report of decreased activity and impaired assembly/stability of complex V in patients with T8993C mutations measured in muscle tissue.

Cystinosis is an autosomal recessive lysosomal storage disorder caused by a defect in the lysosomal cystine carrier cystinosin. Cystinosis is the most common cause of inherited Fanconi syndrome leading to renal failure, in which the pathogenesis is still enigmatic. Based on studies of proximal tubules loaded with cystine dimethyl ester (CDME), altered mitochondrial adenosine triphosphate (ATP) production was proposed to be an underlying pathologic mechanism. Thus far, however, experimental evidence supporting this hypothesis in humans is lacking. In this study, energy metabolism was extensively investigated in primary fibroblasts derived from eight healthy subjects and eight patients with cystinosis. Patient's fibroblasts accumulated marked amounts of cystine and displayed a significant decrease in intracellular ATP content. Remarkably, overall energy-generating capacity, activity of respiratory chain complexes, ouabain-dependent rubidium uptake reflecting Na,K-ATPase activity, and bradykinin-stimulated mitochondrial ATP production were all normal in these cells. In conclusion, the data presented demonstrate that mitochondrial energy-generating capacity and Na,K-ATPase activity are intact in cultured cystinotic fibroblasts, thus questioning the idea of altered mitochondrial ATP synthesis as a keystone for the pathogenesis of cystinosis.

Mutations in SURF1, an assembly gene for cytochrome c oxidase (COX), the fourth complex of the oxidative phosphorylation system, are most frequently encountered in patients with COX deficiency. We describe a patient with Leigh syndrome harbouring a mutation in SURF1 who was reported decades ago with a tissue-specific cytochrome c oxidase deficiency.

We describe two siblings with a Sengers-like syndrome, who presented with congenital hypertrophic cardiomyopathy, infantile cataract, mitochondrial myopathy, lactic acidosis and normal mental development. A mitochondrial adenine nucleotide translocator 1 (ANT1) defect was detected since the ANT1 protein was not detectable by immmunoblotting in muscle samples of the patients. Additionally to these features of classical Sengers syndrome (OMIM 212350), we found that the mitochondrial oxidative phosphorylation, measured by biochemical analysis, was severely compromised in skeletal muscle in both children. Biochemical and morphological analysis of the fibroblasts revealed normal results. The association of significantly decreased pyruvate oxidation rates, deficient energy production and decreased multiple mitochondrial enzyme-complex activities in the muscle samples of our patients is a new finding which differs from previous results in patients with Sengers syndrome. Conclusion: we recommend a muscle biopsy and the biochemical analysis of the oxidative phosphorylation system in patients with muscle hypotonia, cardiomyopathy and congenital or infantile cataract.

In this chapter we describe about 18 years of experience with prenatal diagnosis in oxidative phosphorylation (OXPHOS) diseases in our centre. We start diagnostics of OXPHOS disorders in patients with a mitochondrial (encephalo)myopathy by preference by measuring oxidation rates of pyruvate, malate and succinate and ATP production rates from oxidation of pyruvate in a “fresh” muscle biopsy. In the same biopsy activities of the mitochondrial respiratory chain enzymes complex-I, complex-II, complex-III and complex-IV are also measured. When decreased substrate oxidation rates and ATP production rates give indication for suspicion on a complex-V or a pyruvate dehydrogenase complex (PDHC) deficiency, activities of these enzymes are also measured. In frozen muscle biopsies we only can measure the respiratory chain enzymes. In which cases now can we offer prenatal diagnosis? In about 30% of the muscle biopsies with clearly decreased substrate oxidation rates and ATP production rates, all respiratory chain enzymes, complex-V and PDHC show normal activities. In these cases it is impossible at the moment to offer prenatal diagnosis. In the remainder of the biopsies with clearly reduced substrate oxidation- and ATP production rates, decreased activities are measured of one or more of the afore mentioned enzymes. The most frequendy occurring deficiencies in fresh as well as in frozen muscle biopsies are complex-I, complex-IV or combined deficiencies of these enzymes. The next step is to search if the deficiency is also expressed in cultured fibroblasts and to exclude a mtDNA mutation as a cause of the deficiency. If the deficiency is also expressed in cultured fibroblasts and mtDNA mutations have been excluded we are willing to offer prenatal diagnosis. This chapter is aggravated on prenatal diagnosis for complex-I, complex-IV or a combined deficiency of these enzymes because the majority of the total number of requests for prenatal diagnosis that reach us concerns pregnancies in families in which the index patient was suffering from a deficiency of one of these (or both) enzymes.

In this chapter the biochemical diagnosis of OXPHOS disorders is presented. The laboratory investigations in suspected patients are started with the examination of body fluids. The most important metabolite to be measured is lactate, that is frequendy found to be elevated in blood, urine and cerebrospinal fluid of patients with OXPHOS disorders. The next step in the diagnostic procedure consists of the examination of tissues. The biochemical diagnostic investigations are preferably performed in muscle tissue because in most patients the defect is expressed in muscle. Biopsy material is preferred above autopsy material. Biochemical examination of a fresh muscle sample is to be preferred because mitochondria are intact in fresh muscle thus allowing measurement of the overall oxidative capacity of the mitochondria. In a frozen muscle sample only enzyme activities of the OXPHOS complexes can be measured. In the latter case patients with a disturbance in the oxidative phosphorylation not localized in one of the OXPHOS complexes remain undiagnosed. Practical guidelines for the biochemical examinations of muscle are provided. In certain circumstances it is necessary to examine also fibroblasts. This is an absolute prerequisite in case prenatal diagnosis is requested. The interpre-tation of the biochemical investigations is discussed with special emphasis on the observed residual enzyme activities.

We describe two children carrying an inherited T899C mutation in the mitochondrial ATPase 6 gene with mild encephalopathy and normal postnatal growth followed by tall stature and obesity. No familial tall stature, endocrine anomaly or advanced skeletal age were present. Failure to thrive is a characteristic finding in most patients with a mitochondrial disease. Our observations suggest that children with encephalomyopathy, even in the presence of a significant clinical overgrowth, should be screened for a possible defect in oxidative phosphorylation.

A frequent etiology of congenital lactic acidosis is disturbed mitochondrial energy metabolism. Affected children generally present with neurologic symptoms, such as myopathy and epilepsy. Parents who have lost a child to mitochondrial disease often ask for prenatal diagnosis in subsequent pregnancies. The large number of possible mitochondrial or nuclear DNA mutations often makes the molecular defect unknown. In these cases, prenatal diagnosis rests solely on biochemical analysis. Here we report a possible pitfall in prenatal diagnosis of mitochondriopathies by biochemical methods that might occur despite all precautions. It is illustrated by a patient with isolated mitochondrial complex I deficiency and her family in the light of a new mutation (632C→T) in 1 of the 36 nuclear encoded genes of complex I ( NDUFV1 ). The girl (II.1 in Fig. 1A⇓ ) was the first child of healthy Caucasian first-degree cousins. Postnatally she showed acrocyanosis, muscular hypotonia, and a pendular nystagmus. Fundoscopy revealed bitemporal retinal depigmentation. The latencies of the visual evoked potentials were pathologically increased. Lactic acidosis (pH 7.19) was noted, with a plasma lactate concentration of 24.1 mmol/L (reference interval, 0.5–2.2 mmol/L), a lactate-to-pyruvate ratio of 57 (reference values <20), plasma alanine of 893 μmol/L (reference interval, 40–500 μmol/L), urine α-ketoglutaric acid of 1852 mmol/mol creatinine (reference interval, 159 ± 137 mmol/mol creatinine), urine lactate of 1713 mmol/mol creatinine (reference interval, 234 ± 165 mmol/mol creatinine), and cerebrospinal fluid lactate of 9.6 mmol/L (reference values <2 mmol/L). Cranial ultrasound and magnetic resonance imaging results were normal. Muscle histology revealed intracytoplasmic accumulation of glycogen. Mitochondria were ultrastructurally normal on electron microscopy. We measured the respiratory chain complex I, II+III, and IV activities in a fresh muscle biopsy specimen and in cultured fibroblasts according to standard procedures (1)(see the data supplement available with the online version of this Technical Brief, at …

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 alpha-tocopherol concentrations in his muscles and an elevated urinary leukotriene E(4) excretion indicate increased production of reactive oxygen species.