[O2] was determined as function of time from the phosphorescence decay of Pd (II) meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. In sealed vials, oxygen consumption by peripheral blood mononuclear cells was linear with time, confirming its zero-order kinetics. The rate of respiration (mean ±SD, in μM O2 per min per 107 cells, set as the negative of the slope of [O2] vs. time) for adults was 2.1±0.8 (n=18), for children 2.0±0.9 (n=20), and for fetuses 0.8±0.4 (n=18), p <0.0001. For an 8-year-old patient with reduced muscle NADH dehydrogenase and pyruvate dehydrogenase activities, the rate was 0.7±0.2 (n=3) μM O2 per min per 107 cells. For a 3-month-old patient with hepatocerebral mitochondrial DNA depletion syndrome (confirmed mutation in the MPV17 gene), the rate was 0.6 μM O2 per min per 107 cells. For two siblings with reduced cytochrome oxidase on muscle neurochemistry, the rates were 5.5 and 3.0 μM O2 per min per 107 cells. This novel approach allows non-invasive (preliminary) assessment of cellular bioenergetics. Background: The process of oxidative phosphorylation (OXPHOS) establishes a mitochondrial membrane potential gradient (DY) which is utilized by ATP synthase to generate cellular energy. Methods: We developed a microscopic method to evaluate DY in cultured fibroblasts using the fluorescent compound JC-1. We tested our method on controls and OXPHOS deficient cell lines. Results: DY was significantly lower in fibroblasts from patients with isolated and combined OXPHOS complex I to IV defects, while DY values were maintained in patients with ATP synthase defects. DY reduction correlated with severity of the defect and with the percentages of heteroplasmic mtDNA mutations. Cell lines displayed differential vulnerability to rotenone. Also, this complex I inhibitor induced perinuclear mitochondrial relocalization accompanied by cytoskeletal changes. In our set-up, the complex IV inhibitor KCN had not such an effect but caused cell death when used in high concentrations. The method was found especially efficient to visualize the heterogeneity of the defect in cells from patients with heteroplasmic mtDNA mutations. Conclusions: We found that JC-1 fluorescent microscopy allows to detect and differentiate OXPHOS defects and thus may be employed to study fibroblasts from suspected patients. Background: Mitochondrial disorders are a group of complex diseases that can be caused by mutations in nuclear or mitochondrial genomes. Current molecular diagnosis requires multiple different and complementary methods, including sequencing, qPCR, Southern blot or array CGH, for the detection and quantification of mutations. These procedures are labor intensive, time consuming, and costly. Methods: We have developed and validated a "deep" coverage Next Generation Sequencing (NGS) technique in clinical settings. This approach allows simultaneous analysis of a set of nuclear genes targeted to mitochondria and the whole mitochondrial genome for point mutations and deletions. We instituted proper qualitative and quantitative controls to be analyzed along with each sample for quality assurance. Results: We demonstrated an average coverage of >500X for targeted nuclear genes and >5000X for each of the 16,569 bases of the mitochondrial genome. Nucleotide changes are correctly called with quantitative information. The limit of detection of a heteroplasmic change is calculated to be about 1.5%. Small and large insertion/deletions were correctly detected with clear breakpoints and percentage of heteroplasmy. Conclusion: Our "deep" sequencing approach provides a one-step comprehensive molecular analysis for patients with suspicion of mitochondrial diseases in a timely, accurate, and cost-effective manner suitable for clinical application. Both showed an elevated level of citrulline in serum or whole blood. The ACOMG recommends considering the next disorders, as for further investigation of elevated Citrulline levels in the NBS test: Citrullinemia Types I&II, Argininosuccinic Aciduria & Pyruvate Carboxylase Deficiency. Since introduction of Citrulline level measurement in blood, as part of the NBS tests in Israel, 5 samples were found abnormal (above 40 mmol/Ltr), of which 1 was found to be linked to E3 deficiency (thanks to Dr. Almashanu for the data). Data is being gathered from the files of thirty E3 deficienct patients. The citrulline level of each patient is then weighed in relation to time of diagnosis & metabolic crisis, if had been present. Possible Conclusions: We highly suspect that elevated Citrulline in the NBS tests or serum analysis test for amino acids may mark a defect in the Citric Acid Cycle, such as E3 deficiency. A reasonable mechanism may resemble that for elevated citrulline in Pyruvate Carboxylase Deficiency: a decline in Aspartate production, as a result of a blockage in the transformation of Pyruvate to Oxaloacetate. Objective: Fetal infection is associated with considerable perinatal morbidity. As the umbilical vein regulates fetal blood flow from the placenta dysfunction of human umbilical venous endothelial cells (HUVEC) may compromise fetal blood supply. We incubated HUVEC with one of the key inflammatory mediators, LPS (Lipopolysaccharides), and measured the activity of respiratory chain enzymes. Material and Methods: Based on gestational age, HUVEC were divided into 1 mature and 2 premature groups from otherwise uncomplicated pregnancies (4 patients each) and incubated for one hour, six or twelve hours with LPS (100 ng/ml), respectively, incubation without LPS served as control. We analyzed the activity of respiratory chain enzymes and citratesynthase as a mitochondrial marker spectrophotometrically. Results: No significant differences in respiratory chain activities between the LPS-incubated cells and internal controls could be observed. However, we could demonstrate significant differences depending on gestational age: respiratory chain complexes C I+III (p<0.05) and C IV (p<0.01) showed higher activity at lower gestational age. No such differences were observed for C II+III, C V and citratesynthase. Conclusion: LPS on its own had little influence on the activities of mitochondrial respiratory chain enzymes. However, complex I+III and C IV activities are increased at lower gestational age. The most recent technological developments allows novel tools for biomarkers identification in mitochondrial respiratory chain deficiencies (RCD) through metabolome analysis. Using this approach, it has recently been shown in RCD that an extracellular creatine excess is in relation to intracellular P-creatine depletion. Furthermore, authors found that plasma creatine, appears to be the most specific and sensitive metabolite in RCD patients, exceeding lactate and alanine in magnitude of elevation and statistical significance (Shaham et al, PNAS 2010) . In order to confirm this single report, we tested plasma creatine as potential biomarker of mitochondrial dysfunction in samples obtained from 30 patients aged between 0.2-19 yrs. with pathogenic mutation (10 patients) or abnormally low RC enzyme activity in muscle (20 patients). Data showed that plasma creatine concentrations was significantly higher in mitochondrial patients (mean±SEM=81.1±3.2 vs. 57.7±2.0; p<0001) when compared with age-matched controls. In 8/30 plasma creatine clearly exceeded the upper control limit (97th percentile), and 4 of them had normal levels of plasma lactate and /or alanine on the same blood sampling. Our study confirms that plasma creatine is elevated in RCD and can be used in combination with other biomarkers for the diagnosis of mitochondrial diseases. mtDNA depletion syndrome (MDS) includes a group of autosomal disorders, generally recessive, characterized by a severe reduction in cellular mtDNA content in affected tissues. MDS is tissue-specific, affecting mostly tissues with higher energy demanding. As the cardiac muscle is one of the most energy requiring tissues, we aimed to establish control values for mtDNA copy number in myocardium in patients with cardiac involvement. We have analyzed mtDNA copy number in 14 samples of myocardium from paediatric patients (age: 4 days -11 years old) with possible mitochondrial OXPHOS disease (multiple OXPHOS deficit in 13 cases) and/or heart involvement. Total DNA was extracted by standard methods. The common mtDNA mutations were excluded and mtDNA copy number was determined by real-time PCR, using SYBR green and specific primers for target genes. The intensity of fluorescent signals was analyzed by 7500 Software v2.0.4 and relative mtDNA content was calculated by deltaCt. We performed a normal distribution and estimate 21% of cases (median 594, min-max 40-625, compared to 1293, min-max 1039-6474 in all other samples) with mtDNA depletion. The present work is an important contribution for gathering reference values in myocardium samples, given the scarcity of data in literature, namely in children. In 322 samples out of 921 muscle biopsies measured in Klinikum Schwabing between 2005 and 2009 a defect of respiration chain was found (35.0%). Isolated complex I defect was detected most frequently (118=36.6%), followed by combined complex I and IV defect (66= 20.5%). In 139 out of the 701 genetically investigated samples a positive result was found. In 35 cases (25.2%) mtDNA deletions and in 29 cases (20.9%) a depletion of mtDNA was detected. Interestingly within the 20 deletion positive cases also measured for respiratory chain activity in 13 cases no defect was detected (65 %). In contrast, within the 19 depletion positive samples measured for respiratory chain in only 3 cases no defect was found (16 %). In 253 samples genetic testing was performed initially without investigation of the respiratory chain activity due to clinical symptoms or histological findings. In 72 (28.5%) of these cases a diagnosis could be established.