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Introduction: We aimed to investigate whether sonographic peripheral cross-sectional nerve area (CSA) and progranulin (PGRN), a neuritic growth factor, are related to each other and whether they interact to predict clinical and paraclinical measures in amyotrophic lateral sclerosis (ALS). Methods: We included 55 ALS patients who had forearm median and ulnar nerve CSA, cerebrospinal fluid (CSF) PGRN and serum PGRN measures available. CSF PGRN was normalized against the CSF/serum albumin ratio (Qalb). Using age, sex, height and weight adjusted general linear models we examined CSA x CSF PGRN interaction effects on various measures. Results: There was a medium-effect size inverse relationship between CSA and CSF PGRN, but not between CSA and serum PGRN. Lower CSA values together with higher CSF PGRN levels were linked to smaller motor amplitudes. Discussion: In ALS, the constellation of peripheral nerve atrophy together with higher CSF PGRN levels indicates pronounced axonal damage. This article is protected by copyright. All rights reserved.

Juvenile myoclonic epilepsy (JME) is a common syndrome of genetic generalized epilepsies (GGEs). Linkage and association studies suggest that the gene encoding the bromodomain-containing protein 2 (BRD2) may increase risk of JME. The present methylation and association study followed up a recent report highlighting that the BRD2 promoter CpG island (CpG76) is differentially hypermethylated in lymphoblastoid cells from Caucasian patients with JME compared to patients with other GGE subtypes and unaffected relatives. In contrast, we found a uniform low average percentage of methylation (

We aimed to investigate whether sonographic peripheral cross-sectional nerve area (CSA) and progranulin (PGRN), a neuritic growth factor, are related to each other and whether they interact to predict clinical and paraclinical measures in amyotrophic lateral sclerosis (ALS).We included 55 ALS patients who had forearm median and ulnar nerve CSA, cerebrospinal fluid (CSF) PGRN, and serum PGRN measures available. CSF PGRN was normalized against the CSF / serum albumin ratio (Qalb ). Using age, sex, height, and weight adjusted general linear models, we examined CSA × CSF PGRN interaction effects on various measures.There was a medium-effect size inverse relationship between CSA and CSF PGRN, but not between CSA and serum PGRN. Lower CSA values together with higher CSF PGRN levels were linked to smaller motor amplitudes.In ALS, the constellation of peripheral nerve atrophy together with higher CSF PGRN levels indicates pronounced axonal damage. Muscle Nerve 57: 273-278, 2018.

Introduction: In this study we compare the ultrasound features in the median nerve in patients with different types of Charcot–Marie–Tooth (CMT) disease and hereditary neuropathies with liability to pressure palsies (HNPP) as a typical entrapment neuropathy. Methods: Median nerve ultrasound and conduction studies were performed in patients with CMT1A (n = 12), MFN2-associated CMT2A (n = 7), CMTX (n = 5), and HNPP (n = 5), and in controls (n = 28). Results: Median nerve cross-sectional area (CSA) was significantly increased in CMT1A, whereas, in axonal CMT2A, fascicle diameter (FD) was enlarged. CSA correlated with nerve conduction slowing in CMT1A and with axonal loss, as shown by motor and sensory nerve amplitudes in both CMT1A and CMT2A. A relatively low wrist-to-forearm-ratio (WFR 1.8) appeared to be unlikely in MFN2 and Cx32 mutations of CMT2A and CMTX, respectively. Conclusion: Differences in CSA, FD, and WFR of the median nerve can be helpful in defining subtypes of hereditary neuropathies. Muscle Nerve 47:385-395, 2013

The classical bioenergetical view of the involvement of mitochondria in neurogeneration is based on the fact that mitochondria are the central players of ATP synthesis in neurons and their failure leads to neuronal dysfunction and eventually to cell death. Mutations in at least 39 genes in inherited neurodegenerative disorders seem to alter directly or indirectly mitochondrial function. Most of these mutations do not directly affect oxidative phosphorylation, but act through disturbed mitochondrial dynamics and quality control. This, however, does not invalidate the bioenergetic hypothesis. Neurodegeneration is not necessarily associated with a gross failure of ATP production, but might rather be a consequence of local insufficiencies of ATP supply in critical compartments of neurons, like the presynaptic terminal. We hypothesize that slow disease progression, at least in a subgroup of neurodegenerative diseases, can be explained by the parallel action of subcellular ATP insufficiency and clonal expansion of somatic mitochondrial DNA mutations, and particularly deletions.

SUMMARY Purpose: Levetiracetam (LEV) is a highly effective antiepileptic agent. A clinically relevant psychiatric complication of LEV treatment, however, is the provocation of irritability and aggression. Recent behavioral research indicates that personality traits may predispose to these side effects. To assess the genetic basis of the adverse psychotropic profile of LEV, a candidate gene-based two-stage association study was conducted. Methods: Polymorphisms were a priori selected according to their relevance for impulsivity and reactive-impulsive aggression. Based on data from both stages, a Bonferroni-corrected joint meta-analysis was computed. Key Findings: Stage 1 analysis included 290 patients with epilepsy and revealed a higher load of adverse psychotropic side effects of LEV in patients carrying genetic variants associated with decreased dopaminergic activity: rs1611115 (dopamine-b-hydroxylase, DBH), rs4680 (catechol-O-methyltransferase, COMT), and rs1800497 (dopamine receptor D2-associated ANKK1 TAQ-1A). Stage II analysis including 100 patients with epilepsy, and joint meta-analysis confirmed the effect of the rs1800497 polymorphism (Bonferroni corrected significance of the joint meta-analysis, p = 0.0096). Significance: Confirming the suggestion from behavioral observations that patients might be predisposed to develop irritation and aggression under treatment with LEV, the findings provide first evidence of an association of genetic variation in dopaminergic activity and the risk for psychiatric complications of LEV treatment. Replication and further work is required to prove a true causal relationship. Overall, the pharmacogenomic approach to behavioral side effects may provide a future tool to predict adverse psychotropic effects related to antiepileptic drugs.

Summary Purpose: Recent clinical studies raised concern of a cognitive teratogenicity of the major antiepileptic drug valproate. To investigate possible cerebral correlates, we established a forced self-application schedule by diluting valproate in the drinking water of pregnant Wistar rats. Methods: After application of medium (MD) and high doses (HDs) with mean daily intakes of about 470 and 720 mg/kg during the entire pregnancy, we analyzed effects on offspring performance in a series of behavioral paradigms as well as brain volumetric changes by magnetic resonance imaging (MRI). Results: While high dosages with peak serum concentrations slightly above 100 μg/ml induced early decrements in general activity and deficits in learning and memory, medium dosages led to improved watermaze performance in 30-day-old rats. MRI analyses indicated increased hippocampal volumes in the MD condition, whereas in the HD condition significantly decreased cortical and brainstem volumes were registered. Cortical volume reduction was correlated with spatial acuity in the watermaze. Conclusions: The results indicate that effects of valproate in utero on offspring cognitive capabilities might depend on total drug load differentially affecting cerebral development during adolescence in the rat.

Mitochondrial potassium channels are believed to contribute to cytoprotection of injured cardiac and neuronal tissues. The following potassium channels have been described in the inner mitochondrial membrane: the ATP-regulated potassium channel, the large conductance Ca(2+)-activated potassium channel, the voltage-gated Kv1.3 potassium channel, and the twin-pore domain TASK-3 potassium channel. The putative functional roles of these channels include changes in mitochondrial matrix volume, mitochondrial respiration, and membrane potential. In addition, the activity of these channels modulates the generation of reactive oxygen species by mitochondria. In this article, we discuss recent observations on three fundamental issues concerning mitochondrial potassium channels: (i) their molecular identity, (ii) their interaction with potassium channel openers and inhibitors, and (iii) their functional properties.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive loss of motor neurons. Visualizing corresponding metabolic changes in the brain of patients with ALS with proton magnetic resonance spectroscopy ( 1 H-MRS) may provide surrogate markers for an early disease detection, for monitoring the progression and for evaluating a treatment response. The primary objective of our study was to evaluate whether modifications in MR metabolite levels occur before clinical disease onset, and whether these changes are directly linked to a distinct spatial progression pattern in the CNS. Therefore, age-dependent alterations in the cerebral and spinal metabolic profile in the mouse model of ALS overexpressing the mutated human G93A-superoxide dismutase 1 (G93A-SOD1) were determined by high-resolution MRS of tissue extracts at 14.1 Tesla. Both non-transgenic mice (control mice) and transgenic mice overexpressing the non-mutated human SOD1 (tg-SOD1) served as controls. In the spinal cord of G93A-SOD1 mice significantly decreased levels of N-acetyl aspartate were already detected 34 days postpartum, i.e. about 60 days before the average disease onset caused by motor neuron decline. In addition, glutamine and c-aminobutyric acid concentrations were significantly diminished at Day 75, which is still in the presymptomatic phase of the disease. These metabolic changes were further progressive in the course of the disease and started to involve the brainstem at Day 75. Overall, high-resolution 1 H-MRS allows a sensitive spatial and temporal metabolite profiling in the presymptomatic phase of ALS even before significant neuronal cell loss occurs.

1 Antidiabetic sulphonylureas can bind to various intracellular organelles including mitochondria. The aim of this study was to monitor the influence of antidiabetic sulphonylureas on membrane permeability in mitochondria isolated from rat skeletal muscle. 2 The effects of glibenclamide (and other sulphonylurea derivatives) on mitochondrial function were studied by measuring mitochondrial swelling, mitochondrial membrane potential, respiration rate and Ca2+ transport into mitochondria. 3 We observed that glibenclamide induced mitochondrial swelling (EC50=8.2±2.5 μM), decreased the mitochondrial membrane potential and evoked Ca2+ efflux from the mitochondrial matrix. These effects were blocked by 2 μM cyclosporin A, an inhibitor of the mitochondrial permeability transition. 4 Moreover, 30 μM glibenclamide accelerated the respiratory rate in the presence of glutamate/malate, substrates of complex I of the mitochondrial respiratory chain. 5 In conclusion, we postulate that the antidiabetic sulphonylureas activate the mitochondrial permeability transition in skeletal muscle by increasing its sensitivity to Ca2+. British Journal of Pharmacology (2005) 145, 785–791. doi:10.1038/sj.bjp.0706214

Summary: Purpose: After pilocarpine-induced epilepsy in rats, volumetric magnetic resonance imaging (MRI) reveals significant morphologic changes in functionally relevant structures of the brain. To relate structural changes to functional alteration, we studied the correlation of regional brain atrophy (e.g., of the hippocampus) with lesion-induced learning deficits in the Morris water maze. Methods: MRI experiments were performed on an MR scanner at 4.7 Tesla. For volumetric analysis, various cerebral structures were segmented in horizontal and coronal T2-weighted MR images. Before the MRI investigations, animals were trained for 10 days in a Morris water maze. Results: Volumetric MRI revealed a significant loss in hippocampal size in both the dorsal and ventral parts, correlated with an increase in ventricular size. Furthermore, significant losses were found in the relative size of thalamus, putamen, cortex, and the combined areas of perirhinal, entorhinal, and piriform cortices adjacent to the hippocampus. A significant correlation of learning performance in the Morris water maze with the relative hippocampal area and not with other areas tested was observed in pilocarpine-treated animals. Conclusions: The data provide a quantitative analysis of functionally relevant structural alterations in rats with chronic epilepsy. Water maze performance of pilocarpine-treated animals correlates with the degree of hippocampal but not with the degree of cortical damage, demonstrating the potential of this method for the investigation of cognitive impairments in relation to cerebral changes. In addition, the data point to an important role of even the residual hippocampus in memory formation. Ke yW ords: Temporal lobe epilepsy—Pilocarpine—MRI— Volumetry—Rat—Hippocampus—Learning—Water maze.

Summary: Purpose: For the antiepileptic drug (AED) topiramate (TPM), neuroprotective effects have been reported in models of focal cerebral ischemia and experimental status epilepticus, but the putative mechanism of action has remained elusive. Methods: We studied the effects of TPM on mitochondrial function in the pilocarpine rat model of chronic epilepsy and in isolated mitochondria from rat brain. Results: TPM treatment in status epilepticus at doses ranging from 20 to 100 mg/kg considerably improved the survival of rats and improved CA1 and CA3 pyramidal cell survival in a dose-dependent manner. This treatment increased the activity of mitochondrial respiratory chain complex I in the CA1 and CA3 pyramidal subfields and resulted in lower seizure frequencies in chronic epileptic rats. In vitro investigations of the action of TPM on isolated rat brain mitochondria ruled out any direct effects of the drug on mitochondrial oxidative phosphorylation bu tr evealed a protective effect on hippocampal mitochondria against an external calcium challenge. This can explain its observed neuroprotective action in the concentration range tested. The in vitro effects of TPM on the calcium handling of isolated brain mitochondria was found to be comparable to the action of cyclosporin A. Conclusions: The neuroprotective action of TPM seems to be directly related to its inhibitory effect on the mitochondrial permeability transition pore. Ke yW ords: Hippocampus— Topiramate—Mitochondrial function—Neuroprotection— Epilepsy—Pilocarpine-treated rat—Mitochondrial permeability transition pore (MPT).

For neuroprotective therapy of neurodegenerative diseases creatine treatment has gained special interest because creatine has been shown to cross the blood-brain barrier, accumulate in the human brain in vivo and cause delayed neuronal cell death in a large number of animal models. Here, we used the pilocarpine model of temporal lobe epilepsy to determine whether creatine administration is able to attenuate the epilepsy-associated decrease in hippocampal N-acetyl aspartate (NAA) concentrations, impairment of mitochondrial function and neuronal cell loss. In vivo 1 H-NMR spectroscopy showed, in epileptic rats after creatine administration, higher hippocampal NAA concentrations, suggesting improved neuronal survival. However, in vitro observation of hippocampal slices from creatine-treated epileptic rats revealed a more pronounced loss of pyramidal neurons and decrease in activity of mitochondrial enzymes in hippocampal subfields. This indicates that NAA concentrations measured by in vivo 1 H-NMR spectroscopy reflect alterations of metabolism rather than neuronal cell densities. Our data indicate an adverse effect of creatine on neuronal survival under conditions of enhanced neuronal activity.

J. Reimann, W. S. Kunz, S. Vielhaber, K. Kappes-Horn and R. Schroder (2003) Neuropathology and Applied Neurobiology 29, 45–51 Mitochondrial dysfunction in myofibrillar myopathy ‘Myofibrillar myopathy’ defines a myopathic condition with focal myofibrillar destruction and accumulation of degraded myofibrillar elements. Despite the fact that a number of mutations in different genes as well as cytotoxic agents lead to the disease, abnormal accumulation of desmin is a typical, common feature. Pathological changes of mitochondrial morphology and function have been observed in animal models with intermediate filament pathology. Therefore, in the present study we tested for mitochondrial pathology in skeletal muscle of five patients with the pathohistological diagnosis of myofibrillar myopathy. Screening for large-scale mtDNA deletions and the frequent MERRF (myoclonic epilepsy; ragged red fibres) and MELAS (mitochondrial encephalomyopathy; lactic acidosis; stroke) point mutations was negative in all patients. Histologically, all muscle biopsies showed nonspecific abnormalities of the oxidative/mitochondrial enzyme stainings (histochemistry for reduced nicotinamide adenine dinucleotide, succinic dehydrogenase, cytochrome c oxidase), only one of them had ragged red fibres and a significant number of cytochrome c oxidase-negative fibres. Upon biochemical investigation, four of our patients showed pathologically low respiratory chain complex I activities. Only one of our patients had a pathologically low complex IV activity, while the measurements of the others were within low normal range. The single patient with pathological values for both complex I and IV was the one with the clear histological hallmarks (ragged red and cytochrome c oxidase-negative fibres) of mitochondrial pathology. She also was the only patient with clinical signs hinting at a mitochondrial disorder. Together with data from observations in desmin- and plectin-deficient mice, our results support the view that desmin intermediate filament pathology in these cases is closely linked to mitochondrial dysfunction in skeletal muscle.

Summary: Purpose: Interictal [ 18 F]fluorodeoxyglucose (FDG) positron emission tomography (PET) demonstrates temporal hypometabolism in the epileptogenic zone of 60–90% of patients with temporal lobe epilepsy. The pathophysiology of this finding is still unknown. Several studies failed to show a correlation between hippocampal FDG-PET hypometabolism and neuronal cell loss. Because FDG is metabolized by hexokinase bound to the outer mitochondrial membrane, we correlated the glucose-oxidation capacity of hippocampal subfields obtained after surgical resection with the corresponding hippocampal presurgical FDG-PET activity. Methods: In 16 patients with electrophysiologically confirmed temporal lobe epilepsy, we used high-resolution respirometry to determine the basal and maximal glucose-oxidation rates in 400-m-thick hippocampal subfields obtained after dissection of human hippocampal slices into the CA1 and CA3 pyramidal subfields and the dentate gyrus. Results: We observed a correlation of the FDG-PET activity with the maximal glucose-oxidation rate of the CA3 pyramidal subfields (rp 0.7, p 0.003) but not for the regions CA1 and dentate gyrus. In accordance with previous studies, no correlation of the FDG-PET to the neuronal cell density of CA1, CA3, and dentate gyrus was found. Conclusions: The interictal hippocampal FDG-PET hypometabolism in patients with temporal lobe epilepsy is correlated to the glucose-oxidation capacity of the CA3 hippocampal subfield as result of impaired oxidative metabolism. Key Words: Temporal lobe epilepsy—FDG-PET—Mitochondrial oxidative phosphorylation.

Mitochondrial diseases are a heterogeneous group of disorders caused by the impairment of the mitochondrial oxidative phosphorylation system which have been associated with various mutations of the mitochondrial DNA (mtDNA) and nuclear gene mutations. The clinical phenotypes are very diverse and the spectrum is still expanding. This review gives an overview of the principal clinical phenotypes and the molecular genetic basis of mitochondrial disorders identified so far.

Mitochondrial function is a key determinant of both excitability and viability of neurons. Here, we demonstrate seizure-dependent changes in mitochondrial oxidative phosphorylation in the epileptic rat hippocampus. The intense pathological neuronal activity in pilocarpine-treated rats exhibiting spontaneous seizures resulted in a selective decline of the activities of NADH-CoQ oxidoreductase (complex I of the respiratory chain) and cytochrome c oxidase (complex IV of respiratory chain) in the CA3 and CA1 hippocampal pyramidal subfields. In line with these findings, high-resolution respirometry revealed an increased flux control of complex I on respiration in the CA1 and CA3 subfields and decreased maximal respiration rates in the more severely affected CA3 subfield. Imaging of mitochondrial membrane potential using rhodamine 123 showed a lowered mitochondrial membrane potential in both pyramidal subfields. In contrast to the CA1 and CA3 subfields, mitochondrial oxidative phosphorylation was unaltered in the dentate gyrus and the parahippocampal gyrus. The changes of oxidative phosphorylation in the epileptic rat hippocampus cannot be attributed to oxidative enzyme modifications but are very likely related to a decrease in mitochondrial DNA copy number as shown in the more severely affected CA3 subfield and in cultured PC12 cells partially depleted of mitochondrial DNA. Thus, our results demonstrate that seizure activity downregulates the expression of mitochondrial-encoded enzymes of oxidative phosphorylation. This mechanism could be invoked during diverse forms of pathological neuronal activity and could severely affect both excitability and viability of hippocampal pyramidal neurons.

Revision of the current decision point for prophylactic platelet transfusion in thrombocytopenic patients requires the availability of a method that is able to provide accurate platelet counts to as low as 1 x 109/l. This study is the first to evaluate the immunoplatelet method (CD61-Imm) of the haematological analyser Cell-Dyn 4000 in direct comparison with the flow cytometric procedure. Additionally CD61-Imm results were compared with CD4000 optical (Plto) counts in the ranges 20-547 x 109/l (n = 127) and 1-35 x 109/l (n = 107). The immunoplatelet and Plto results were in good agreement between 20 x 109/l and 547 x 109/l, but for samples of < 25 x 109/l the Plto tended to overestimate the counts. We determined the limits of detection (LD) and quantification (LLQ) for all three methods using standard statistical procedures. The LD for the flow cytometric CD41a method was 0.02 x 109/l compared with 0.009 x 109/l and 1.73 x 109/l for the CD61-Imm and Plto methods respectively. The LLQCV = 15% for the CD41a method was 1.8 x 109/l compared with 1.6 x 109/l and 18.0 x 109/l for the CD61-Imm and Plto procedures. In conclusion, (i) the CD61-Imm method performance is at least equivalent to the reference flow cytometric method, and (ii) in severe thrombocytopenia the CD61-Imm count is superior to the Plto count.

Mitochondria are cellular organelles crucial for energy supply and calcium homeostasis in neuronal cells, and their dysfunction causes seizure activity in some rare human epilepsies. To directly test whether mitochondrial respiratory chain enzymes are abnormal in the most common form of chronic epilepsy, temporal lobe epilepsy (TLE), living human brain specimens from 57 epileptic patients and 2 nonepileptic controls were investigated. In TLE patients with a hippocampal epileptic focus, we demonstrated a specific deficiency of complex I of the mitochondrial respiratory chain in the hippocampal CA3 region. In contrast, TLE patients with a parahippocampal epileptic focus showed reduced complex I activity only in parahippocampal tissue. Inhibitor titrations of the maximal respiration rate of intact human brain slices revealed that the observed reduction in complex I activity is sufficient to affect the adenosine triphosphate production rate. The abnormal complex I activity in the hippocampal CA3 region was paralleled by increased succinate dehydrogenase staining of neurons and marked ultrastructural abnormalities of mitochondria. Therefore, mitochondrial dysfunction is suggested to be specific for the epileptic focus and may constitute a pathomechanism contributing to altered excitability and selective neuronal vulnerability in TLE.

Background: The therapeutic option of prophylactic platelet (PLT) transfusion in cases of severe thrombocytopenia critically depends on the availability of accurate and precise counts because clinical decisions are widely based on decision or trigger points. Although often applied in current practice at a level of 20 Gpt/L, there is increasing evidence that the trigger points could safely be reduced to10 or even 5 Gpt/L. In order to facilitate this downward revision, it is necessary to have PLT counting methods that are able to provide reliable results in the appropriate decision range. Methods: Postchemotherapy-induced pancytopenia PLT counting was performed in patients with hematological malignant disorders. This study describes a novel flow cytometric method that utilizes a PLT-specific monoclonal antibody (CD41a) in conjunction with fluorescent reference beads in order to derive absolute platelet numbers. Results: Applying a mathematical model, this flow cytometric method was shown to have a detection limit of 0.24 Gpt/L and a lower limit of quantification (coefficient of variation [CV] = 10%) of 1.1 Gpt/L. These values are a substantial improvement on previously reported results for the Technicon H1 automated instrument or manual hemocytometry. Moreover, although the flow cytometry and Technicon H3 methods were found by supplementary analyses to show a reasonably good correlation, the hematology instrument showed a distinct tendency to overestimate PLT counts at low levels. Conclusion: It is proposed that this standardized immunoplatelet method offers the best approach in evaluating, at the clinical level, the possibility of lower PLT transfusion triggers. It can be used to evaluate the performance limitations of automated hematology analyzers that are widely used at the present time. Cytometry (Comm. Clin. Cytometry) 42:284–289, 2000. © 2000 Wiley-Liss, Inc.

Patients harboring A467T and W748S POLG1 mutations present with a broad variety of neurological phenotypes, including cerebellar ataxia, progressive external ophthalmoplegia (PEO), myoclonus, epilepsy, and peripheral neuropathy. With exception of ataxia and myoclonus, movement disorders are not typical features of POLG1 associated disorders. We report on two affected siblings compound heterozygous for A467T and W748S mutations, one suffering from choreoathetosis and apraxia of lid opening due to focal eyelid dystonia that mimicked progression of ptosis, resulting in functional blindness. So far, focal dystonia has not been reported in POLG1 mutation carriers, and should be considered when investigating patients with PEO and ptosis. Further studies on POLG1 mutations in focal dystonia are warranted.

The ratio of mtDNA and a nuclear reference gene was estimated by Southern blotting in the skeletal muscle DNA of a 3-year-old girl who suffered from congenital brain damage, focal epilepsy, hepatomegaly, malabsorption syndrome and severe myopathy. The signal ratio of mtDNA versus 18S rDNA was 22% of the mean value obtained from controls. No major deletions or insertions were found and the MERRF, MELAS and NARP mutations were ruled out. Mitochondrial DNA-encoded enzyme activities and mitochondrial respiration were reduced. The analysis of the NAD(P)H and flavoprotein redox states of intact fibres revealed the presence of mitochondrial dysfunction. In tissue sections a moderate elevation of type I and type II fibre diameter variation was detected, aberrant NADH- and succinate dehydrogenase staining and some ragged red fibres. This suggested that a mitochondrial disorder caused by a decrease in the amount of intact wild-type mtDNA was responsible for the severe myopathy.

Metabolic control analysis was applied to describe the control of mitochondrial oxidative phosphorylation in calcium (approximately 2 microM free calcium) activated saponin-skinned rat musculus soleus fibers oxidizing glutamate and malate. Under these circumstances approximately 80% of mitochondrial active-state respiration was reached due to the activation of ATP turnover by actomyosin ATPase. The flux control coefficients of H(+)-ATPase, adenine-nucleotide translocase, phosphate transporter, NADH:ubiquinone oxidoreductase and cytochrome-c oxidase were determined to be equal to 0.16 +/- 0.08 (n = 6), 0.34 +/- 0.12 (n = 5), 0.08 +/- 0.03 (n = 5), 0.01 +/- 0.006 (n = 4) and 0.09 +/- 0.03 (n = 3) using inhibitor titrations with the specific inhibitors oligomycin, carboxyatractyloside, mersalyl, rotenone and cyanide, respectively, and applying non-linear regression of the entire titration curve. The flux control coefficient of actomyosin ATPase was determined with vanadate to be equal to 0.50 +/- 0.09 (n = 6), measuring independently the vanadate-caused inhibition of fiber respiration and ATP-splitting activity. In contrast to results with isolated rat skeletal muscle mitochondria reconstituted with soluble F1-ATPase the decrease in phosphate concentration from 10 mM to 1 mM only slightly affected the distribution of flux control coefficients. This difference is caused by different kinetic properties of soluble F1-ATPase and actomyosin ATPase. Therefore, phosphate seems to be in skeletal muscle in vivo only a modest modulator of control of oxidative phosphorylation.