Background: Acylcarnitine analysis using serum samples or dried blood spots does not always provide diagnostic results to evaluate fatty acid oxidation defect. Instead, we analyzed acylcarnitines in lymphocytes loaded with stable-isotope labeled fatty acid for the diagnosis of long-chain fatty acid oxidation defects. Methods: [2H31] palmitic acid was added to glucose-free PBS solution of lymphocytes, which were collected from 3 ml of heparinized blood. The mixture was incubated for 2 hours, and washed lymphocytes were homogenized and centrifuged. Acylcarnitines in an aliquot of the supernatant, spiked with stable-isotope labeled acylcarnitines as internal standards, were analyzed by tandem mass spectrometry. Results: In lymphocytes loaded with [2H31]palmitic acid, a series of labeled acylcarnitines, from 2H31C16-to 2H1C2-acylcarnitine (d1C2AC), were detected. The ratios of d1C2AC/d31C16AC in patients with long-chain fatty acid oxidation defects, except for CPT-1 deficiency, were decreased as compared with those in controls. Decreased ratios of d27C14AC/d31C16AC were observed in patients with CPT-2 deficiency, decreased ratios of d23C12AC/ d27C14AC in patients with VLCAD deficiency, and increased ratios of d29C16OHAC/d31C16AC in patients with TFP deficiency, respectively. Conclusion: This simple test gives us diagnostic information for longchain fatty acid oxidation defects before mutation analysis, and is useful in newborn screening by tandem mass spectrometry. Background: Fatty acid oxidation defects are part of newborn screening programs worldwide. Since then disease incidences have significantly increased. New genotypes are identified with unknown clinical relevance. Methods: We performed octanoyl-CoA and palmitoyl-CoA oxidation studies in lymphocytes of newborns with acylcarnitine profiles suggestive for medium-chain acyl-CoA dehydrogenase (MCAD) or very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency, respectively. In all individuals with residual activities <50%, molecular analysis of the respective gene was performed. Results: For MCADD, two mutations were found in patients with residual activities of 0-49%. Patients with the prevalent c.985A>G mutation presented with residual activities of 0-8%, compound heterozygotes with c.199 T>C on one allele had activities of 28-49%. Heterozygotes carrying the c.199 T>C mutation presented with residual activities of 70-100 % as controls carrying no mutation. In VLCADD, we identified two mutations in patients with activities of 0-22%. The majority presented with residual activities <10%. The mild c.848 T>C mutation resulted in activities of 10-13%, the c.339 C>A mutation was associated with a residual activity >20%. Conclusion: Our functional studies identify patients with mutations that are clinically not relevant, such as the known c199T>C mutation in the ACADM gene and allow prognostic predictions in these disorders known for their molecular heterogeneity. Background: Fatty acid oxidation disorders (FAOD) are group of inborn errors of metabolism. The most common cardiac abnormality are cardiomyopathy, arrhythmias, conduction anomalies, pericardial effusion and sudden death. The aim of the study was to describe the cardiac involvement in children with FAOD from Wielkopolska Region of Poland. Material and Methods: Cardiac evaluation was done in 12 patients with FAOD (VLCAD-2, LCHAD-5, MCAD-2, SCAD-1, primary carnitine deficiency-2) compared to 30 healthy controls by ECG, Holter-ECG, standard TTE, TDI and real time 4D. Results: Standard TTE revealed abnormalities in 8 patients: hypertrophic cardiomyopathy in 5, mild TI in 1, pericardial effusion in 2. Arrhythmia was observed in 2 cases. Additionaly in 2 patients congenital heart defects were diagnosed. Compared to controls, indexes of LV systolic and diastolic function of FAOD patients differed significantly, with reduced EF, FS as well as MVand TV E/A ratio, also MAPSE and TAPSE of LVand RV were significantly reduced. FAOD patients showed a reduction in PW-TDI measurements of lateral mitral annulus. In 2 patients reverse of cardiomyopathy have been observed. Conclusions: Cardiac involved is common in children with FAOD. Asymptomatic children with FAOD have an increased prevalence of subclinical LV dysfunction Background: Most fatty acid oxidation disorders (FAOD) present in childhood with cardiac or liver involvement, but exercise intolerance, myalgia, muscle weakness, and attacks of rhabdomyolysis may characterise presentation in adulthood. Objectives: To report the clinical, biochemical and genetic findings in adults with FAOD (either diagnosed initially in adulthood (n=15), or surviving childhood (n=19)). Methods: Clinical, biochemical and genetic data for 34 unrelated adult patients, aged 16 to 60 years, followed at four UK metabolic centres, were reviewed. Patients with MCAD deficiency were excluded. Results: Patients with CPT2 (17/34), VLCAD (7/34), LCHAD (3/34), PCD (3/34), MAD (2/34) and CPT1 deficiencies (2/34) were reviewed. Attacks of rhabdomyolysis were the most frequent manifestation of CPT2 and VLCAD deficiencies. Although muscle symptoms were rare in other defects, fixed muscle weakness and multisystem involvement were seen. In this cohort, acylcarnitine profile was frequently though not always indicative of the FAOD, with cultured skin fibroblast assays and/or identification of a pathogenic mutation confirming the diagnosis. Conclusions: FAOD presenting in adulthood may result in delayed diagnosis. Diagnosis can be achieved without resorting to invasive muscle biopsy. Recognition is crucial to allow appropriate management. Longterm outcome of childhood survivors is generally good, but many remain symptomatic. Background: Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most frequent fatty acid oxidation disorder. Patients present tissue accumulation of the medium-chain fatty acids octanoic (OA) and decanoic acids. Clinically, progressive encephalopathy, drowsiness and lethargy that may develop into coma and death are found. Objective: Since the pathophysiology of the brain injury in MCADD is uncertain, in the present work we investigated the effect of intracerebroventricular OA administration on oxidative stress parameters in cerebral cortex, striatum, hippocampus and cerebral spinal fluid (CSF) of rats. Material and Methods: Animals received a single intracerebroventricular OA injection (1.66 μmol). Control animals received artificial cerebral spinal fluid in the same volume. Animals were killed 1 h after OA administration. Thiobarbituric acid-reactive species (TBA-RS) levels, carbonyl content and superoxide dismutase (SOD) and catalase activities were evaluated. Results: It was observed that OA increased TBA-RS levels and carbonyl content in all tissues tested. In addition, catalase activity was increased only in cerebral cortex and hippocampus. On the other hand, OA administration did not alter SOD activity. Conclusion: Taken together, these data suggest that OA induces oxidative stress in brain. Our results may help to explain, at least in part, the characteristic brain dysfunction observed in MCADD patients. Background: fatty acid oxidation disorders (FAOD) are rare autosomal recessive inherited metabolic conditions.This paper will highlight the clues that can be obtained from history, clinical examination, and simple bedside tests characteristic for FAOD. Method: records of 15 patients admitted between 2000 to 2003 and diagnosed as FAOD were reviewed. Results: The final diagnosis was very long chain acyl-CoA dehydrogenase deficiency in ten patients and long chain 3-hydroxyacyl-CoA dehydrogenase deficiency in five. Eighty percent had a positive family history of either a previous sudden unexplained infant death or a similar diagnosis in a first-degree relative. History of consanguinity was positive in eleven patients. Diagnosis of FAOD can be difficult because patients tend to develop symptoms only during prolonged fasting and intercurrent acute illnesses. In our series seven patients presented with acute illness and four patients presented with respiratory distress, four had heart failure due to cardiomyopathy and another two presented with convulsions due to severe metabolic acidosis. Physical findings are usually non-specific, eight had hepatomegaly and four had marked hypotonia. Conclusion: Our group of 15 cases accumulated within 3 years is inordinately large and suggest that Kuwait provides a promising venue in which to study the biochemical and molecular genetics of FAOD. Background: Selective screening for Inborn Error of Metabolism was conducted among patients of pediatric clinics. Children were observed intensive care units with acute intoxication, hypoglycemia, resistant to therapy epilepsy, coma, and liver damage Methods: Clinical observation and selective metabolic screening was performed in the Kharkiv Specialised Medical Genetic Centre: blood amino acids (HPLC, Waters), urine organic acids (GS/MS, Agilent), biochemical blood indexes, ammonia, lactate levels were measured. TMS investigation from Guthrie cards was performed in other countries. Results: An 8 month old boy was observed by the geneticist in the intensive care unit. Clinical presentation included severe gastroenteritis, diarrhea, functional liver and kidney disorder, severe lactic acidosis, respiratory violations (artificial ventilation), fatty hepatosis seen on ultrasound observation, hypertrophic cardiopathy, hypoglycemia during infectious diseases. We suspected an inborn error of metabolism in the child. TMS analyses from the DBS sample were performed. The levels of long chain hydroxylated acylcarnitines and tetradecadienoylcarnitine (C14:2) were increased. The organic acid profile showed increased levels of suberic, 3-OH-suberic and sebacic acids. The levels of lactic acid and liver enzymes in blood were increased. Long-chain 3hydroxyacyl-CoA dehydrogenase/Mitochondrial trifunctional protein deficiency was dignosed. Background: Until recently, ATP synthase deficiency was considered as a relatively rare cause of OXPHOS dysfunction, mainly caused by pathogenic mtDNA alterations. The last two years an emerging group of patients with complex V defects of nuclear origin, mostly TMEM70 mutations, have been reported. However, technical difficulties for evaluating complex Vactivity and the heterogeneous clinical picture of patients seriously hamper correct identification. Methods: A diagnostic strategy combining spectrophotometrical analysis, blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by in-gel activity staining, western blotting and diverse microscopic techniques was developed. Results: We report on a selection of complex V deficient patients harbouring pathogenic mutations in either mtDNA or nuDNA. Patients with mtDNA alterations usually have normal complex V activities measured by spectrophotometric analysis and show complex V subcomplexes in the BN-PAGE gel. Complex V deficiency due to nuDNA mutations results in a significant decrease in complex V activity using spectrophotometric techniques. The amount of detectable holocomplex V with BN-PAGE or western blotting is very low. Microscopic analysis in these patients shows aberrant mitochondrial morphology and severely decreased complex V immunoreactivities. Conclusion: Deficiencies of complex V either from mtDNA or nuDNA origin can efficiently be identified by combining several diagnostic techniques. Background: MELAS syndrome is one of the most common mitochondrial disorders. Mitochondrial proliferation may result in endothelial dysfunction and decreased nitric oxide (NO) availability leading to stroke-like episodes. This study aimed to assess NO production in adults with MELAS and the effect of oral supplementation with the NO precursors arginine and citrulline. Methods: Using stable isotope infusion techniques, we measured NO synthesis, amongst other variables, in healthy adults and subjects with MELAS before and after arginine or citrulline supplementation. Results: Adults with MELAS had lower NO synthesis rate, plasma arginine and citrulline concentrations, citrulline flux, and de novo arginine synthesis. In subjects with MELAS, arginine and, to a greater extent, citrulline supplementation increased the de novo arginine synthesis rate, the plasma concentrations and flux of arginine and citrulline, and NO production. Conclusions: The lower NO production in subjects with MELAS is primarily due to decreased de novo arginine synthesis secondary to decreased citrulline availability. De novo arginine synthesis increased markedly with citrulline supplementation, explaining the superior efficacy of citrulline in increasing NO production. The improvement in NO production with arginine or citrulline supplementation supports their use in MELAS syndrome and suggests that citrulline may have a better therapeutic efficacy. There are to date very few treatments for mitochondrial Respiratory Chain (RC) disorders and the management of patients remains largely supportive. In many cases, the RC defect is not total and some residual enzyme activity level can be measured. Thus, a therapeutic strategy aimed at stimulating residual capacities could correct partial RC deficiencies. In this context, one possible approach is to activate key factors capable to up-regulate the RC, using specific pharmacological agents. In line with this, we investigated the potential of resveratrol (RSV), a natural polyphenol compound, known to activate PGC-1alpha (Peroxisome proliferatoractivated receptor Gamma Coactivator-1 alpha), to stimulate the RC. The effects of RSV were tested in fibroblasts of controls and of five Complex I (CI) deficient patients harboring mutations in 3 nuclear genes encoding CI subunits (NDUFV1, NDUFV2 and NDUFS3). Spectrophotometric experiments indicated that exposure to RSV (75 microM, 72 h) increased the CI residual enzyme activity in controls (+30%) and in 4 out of 5 CI-deficient cells (+20 to +78%). Western-blot analysis showed that RSV significantly increased (+45 to +100%) the expression levels of mutated proteins in 4 out of 5 deficient cell lines. Finally, preliminary experiments suggest that these RSV effects might involve a mitochondrial biogenesis. Background and objectives: We recently reported on 24 statistical significant urinary organic acids from children with RCDs, disclosed through a metabolomics investigation (Reinecke, et al, Metabolomics, 2011-doi: 10 .1007/s11306-011-0309-0). The objectives here are: (1) to describe the outcome of a metabolomics investigation of urinary amino acids and acyl-carnitines from RCD patients and (2) to propose a putative biosignature for RCDs. Material and Methods: The urinary samples came largely from a previous patient group (Smuts, et al, J. Inherit. Metab. Dis., 2010-doi:10 .1007/ s10545-009-9031-8). The methods were: GC-MS analysis for the amino acids and tandem-MS for the acyl-carnitines. Statistical analysis included standard univariate analyses as well as principal component (PCA), partial least-square discriminant (PLS-DA) and a new method of concurrent class (CONCA) analyses. Results and Conclusions: From the metabolomics analysis a putative biosignature could be formulated which comprised of 16 metabolites: lactic acid, two Krebs-cycle intermediates, four organic acids from fatty acid catabolism, seven amino acids and two acyl-carnitines. This biosignature should still be validated with more RCD cases, but is already implementable as a screening instrument to establish the need for biopsying patients for confirmative enzyme analyses on RCDs as well as for monitoring of treatment. Background: Mutations in the RRM2B gene encoding the ribonucleotide reductase (RNR) p53R2 subunit were initially reported to cause childhoodonset mitochondrial DNA (mtDNA) depletion syndrome. Objectives: To determine the frequency of RRM2B mutations in adults with multiple mtDNA deletions, and to report the association of RRM2B mutations with Kearns-Sayre syndrome (KSS). Patients and Methods: RRM2B gene sequence analysis was performed in 50 adult patients with multiple mtDNA deletions in skeletal muscle, in whom mutations in known mtDNA maintenance genes (POLG and C10orf2) had been excluded. Functional studies of RNR protein included Blue-Native gel electrophoresis (BN-PAGE) and Western blot analysis. Results: A patient with KSS had two novel missense mutations in RRM2B. BN-PAGE demonstrated reduced heterotetrameric R1/p53R2 RNR levels, despite normal p53R2 levels on Western blot, suggesting failed assembly of functional RNR as a potential disease mechanism. Another patient, who presented with late-onset progressive external ophthalmoplegia and fatigue, had a heterozygous 3 bp deletion in RRM2B. Conclusions: 4% of our cohort had RRM2B mutations. KSS has not previously been linked to nuclear gene defects; we now show that disease pathogenesis may be caused by defective RNR assembly. RRM2B mutations should be considered in the differential diagnosis of multiple mtDNA deletions presenting in adults. Background: Complex I (CI) deficiency is the most frequent cause of oxidative phosphorylation (OXPHOS) defect. X-linked AIF-deficient Harlequin (Hq) mouse is a faithful model for OXPHOS defect, with tissues-specific CI defect and phenotype closely resembling that of CI deficient-patients. No therapeutic solutions are available except "neutraceuticals" among which high-fat diet (HFD) has been proposed but never evaluated. Objectives: To evaluate the impact of HFD on disease progression in CIdefective mice. Methods: Hemizygous (Hq/Y) males were obtained by mating Hq/X females with control males and the diets given from weaning. Control diet was 8 KCal% in fat and isocaloric HFD 30 Kcal%. From one to six months, animals were monthly evaluated using an extensive battery of phenotype characterization: rotarod (indirect cerebellar ataxia evaluation), grip-test (muscular strength) and hindlimb clasping (HL, neurodegeneration marker). Results: HFD-treated Hq mice exhibited a significantly less severe loss of rotarod scores from one to six months (45 points of loss on average, n=21) when compared to Hq mice fed the control diet (100 points of loss, n=20). There was also a tendency to better HL-scores in HFD-fed mice. Conclusion: Isocaloric HFD slows down clinical neurodegeneration in CIdefective mice. This strongly favors its use in CI-defective patients. Deficiency of pyruvate dehydrogenase complex (PDHC) is the most common disorder leading to lactic acidemia. Phosphorylation of specific serine residues of the E1-alpha; subunit of the PDHC by pyruvate dehydrogenase kinase (PDK) inactivates the enzyme, whereas dephosphorylation restores PDHC activity. We recently found that phenylbutyrate prevents phosphorylation of the E1-alpha subunit of the branched-chain ketoacid dehydrogenase complex (BCKDC) and reduces plasma concentrations of neurotoxic branched chain amino acids in patients with maple syrup urine disease (MSUD), due to the deficiency of BCKDC. We hypothesized that, similarly to BCKDC, phenylbutyrate enhances PDHC enzymatic activity by increasing the portion of unphosphorylated enzyme. To test this hypothesis, we treated wild-type human fibroblasts at different concentrations of phenylbutyrate and found that it reduces the levels of phosphorylated E1-alpha; as compared to untreated cells. To investigate the effect of phenylbutyrate in vivo, we administered phenylbutyrate to C57B6 wild-type mice and we detected a significant increase in Pdhc enzyme activity and a reduction of phosphorylated E1-alpha; subunit in brains and muscles as compared to saline treated mice. Being a drug already approved for human use, phenylbutyrate has great potential for increasing the residual enzymatic activity of PDHC and to improve the clinical phenotype of PDHC deficiency. This study aimed to show feasibility of using the phosphorescence oxygen analyzer to screen for disorders with impaired cellular bioenergetics.