Ethylmalonic Acid Research Articles

Ethylmalonic encephalopathy: A metabolic disorder masquerading as cerebral palsy

Many metabolic disorders may present with similar symptoms and signs as cerebral palsy. Some of these disorders which are slowly progressive are more likely to be misdiagnosed as CP. A 5-years-old boy was born at 40 weeks gestation to unrelated parents. The pregnancy was complicated by anemia and gestosis. The vaginal delivery was uncomplicated. Birth weight was 3360 g, length was 51 cm. APGAR scores were 7 and 8 at 1 and 5 min. Child developed well up to 2,5 months. At 3 months of age he manifested irritability and sleep problems. After 3 months the early developmental milestones were abnormal. Since the age of 6 months he had periodic petechial rash, acrocyanosis and diarrhea. At 1 year old the diagnosis of hypotonic cerebral palsy was made. Instead of permanent rehabilitation child had severe psychomotor dysfunction and development delay. At 5 years of life he developed his first epileptic seizure. The treatment with Levetyracetam was started. At the age of 5 years the patient had development delay and spactic diplegia. According to Gross Motor Function Classification System he has level IV. Due to atypical course of the disease and to the specific symptoms diagnosis of cerebral palsy has been questioned and the child underwent appropriate evaluation. Blood amino acids and acylcarnitines analysis revealed high C4 level. The level of ethylmalonic acid in urine was elevated. Genetic analysis revealed two heterozygous variants in the ETHE1 gene: NM_014297.3: c.487C˃G, p.R163G (CM077533) and NM_014297.3: c. 487C˃T, p.R163W (CM040420). The diagnosis of Ethylmalonic encephalopathy was confirmed. The treatment with N-acetyl-cysteine and metronidazole was started with good effect. Metabolic evaluation should be performed in all children with CP for an underlying cause, particularly in situations when there is a presence of a positive family history of “cerebral palsy”, occurrence of developmental regression, multisystemic involvement.

Short-chain acyl-CoA dehydrogenase deficiency: from gene to cell pathology and possible disease mechanisms.

Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is an inherited disorder of mitochondrial fatty acid oxidation that is characterized by the presence of increased butyrylcarnitine and ethylmalonic acid (EMA) concentrations in plasma and urine. Individuals with symptomatic SCADD may show relatively severe phenotype, while the majority of those who are diagnosed through newborn screening by tandem mass spectrometry may remain asymptomatic. As such, the associated clinical symptoms are very diverse, ranging from severe metabolic or neuromuscular disabilities to asymptomatic. Molecular analysis of affected individuals has identified rare gene variants along with two common gene variants, c.511C>T and c.625G>A. In vitro studies have demonstrated that the common variants as well as the great majority of rare variants, which are missense variants, impair folding, that may lead to toxic accumulation of the encoded protein, and/or metabolites, and initiate excessive production of ROS and chronic oxidative stress. It has been suggested that this cell toxicity in combination with yet unknown factors can trigger disease development. This association and the full implications of SCADD are not commonly appreciated. Accordingly, there is a worldwide discussion of the relationship of clinical manifestation to SCADD, and whether SCAD gene variants are disease associated at all. Therefore, SCADD is not part of the newborn screening programs in most countries, and consequently many patients with SCAD gene variants do not get a diagnosis and the possibilities to be followed up during development.

Erratum to: Novel magnetic resonance imaging findings in a patient with short chain acyl CoA dehydrogenase deficiency.

Reports on magnetic resonance imaging findings in patients with short chain acyl –Coenzyme A dehydrogenase (SCAD) deficiency, an autosomal recessive disorder caused by mutations in the acyl-Coenzyme A dehydrogenase (ACADS), are limited. Many asymptomatic carriers of ACAD variants have also been described necessitating careful evaluation of clinical and biochemical findings for an accurate diagnosis. Here we report a an infant with short chain acyl –Coenzyme A dehydrogenase (SCAD) deficiency diagnosed based on the characteristic biochemical findings and confirmed by genetic testing. He presented with refractory seizures and neuro regression at 4 months of age. His metabolic work up revealed elevated butyryl carnitine in plasma and ethyl malonic acid in urine. Magnetic resonance imaging of the brain showed cortical and basal ganglia signal changes with cortical swelling. Serial scans showed progression of the lesions resulting in cystic leukomalacia with brain atrophy. Exome sequencing revealed a novel homozygous nonsense variation, c.1146C > G (p.Y382Ter) in exon ten of ACADS which was further validated by Sanger sequencing. Both parents were heterozygous carriers. Follow up at 15 months showed gross psychomotor retardation and refractory seizures despite being on optimal doses of anti-epileptic medications, carnitine and multivitamin supplementation. This report expands the phenotypic and genotypic spectrum of SCAD deficiency.

Identification and Quantitation of Malonic Acid Biomarkers of In-Born Error Metabolism by Targeted Metabolomics.

Malonic acid (MA), methylmalonic acid (MMA), and ethylmalonic acid (EMA) metabolites are implicated in various non-cancer disorders that are associated with inborn-error metabolism. In this study, we have slightly modified the published 3-nitrophenylhydrazine (3NPH) derivatization method and applied it to derivatize MA, MMA, and EMA to their hydrazone derivatives, which were amenable for liquid chromatography- mass spectrometry (LC-MS) quantitation. 3NPH was used to derivatize MA, MMA, and EMA, and multiple reaction monitoring (MRM) transitions of the corresponding derivatives were determined by product-ion experiments. Data normalization and absolute quantitation were achieved by using 3NPH derivatized isotopic labeled compounds 13C2-MA, MMA-D3, and EMA-D3. The detection limits were found to be at nanomolar concentrations and a good linearity was achieved from nanomolar to millimolar concentrations. As a proof of concept study, we have investigated the levels of malonic acids in mouse plasma with malonyl-CoA decarboxylase deficiency (MCD-D), and we have successfully applied 3NPH method to identify and quantitate all three malonic acids in wild type (WT) and MCD-D plasma with high accuracy. The results of this method were compared with that of underivatized malonic acid standards experiments that were performed using hydrophilic interaction liquid chromatography (HILIC)-MRM. Compared with HILIC method, 3NPH derivatization strategy was found to be very efficient to identify these molecules as it greatly improved the sensitivity, quantitation accuracy, as well as peak shape and resolution. Furthermore, there was no matrix effect in LC-MS analysis and the derivatized metabolites were found to be very stable for longer time. Graphical Abstract ᅟ.

Clinical, biochemical and gene mutation characteristics of short chain acyl-coenzyme A dehydrogenase deficiency by neonatal screening

Objective: To investigate the incidence, clinical, biochemical and gene mutation characteristics of short chain acyl-coenzyme A dehydrogenase deficiency (SCADD). Method: From January, 2009 to October, 2015, a retrospective analysis of the urine organic acids and acyl-coenzyme A dehydrogenase (ACADS) gene mutation characteristics of patients diagnosed as SCADD by newborn screening using tandem mass spectrometry in Department of Genetics and Metabolism (Newborn screening Center of Zhejiang Province), Children's Hospital, Zhejiang University School of Medicine. Dietary guidance, life management and supplementation of L-carnitine were conducted, and growth and intelligence development were observed during follow-up among the SCADD patients. Result: A total of 1 430 024 neonates, seventeen cases were diagnosed with SCADD with an incidence of 1/84 117. All patients had no clinical symptoms, and intelligence and physical development were normal. Blood butylacyl-carnitine (C4) levels and the ratios increased, C4 0.713.14 μmol/L(reference value 0.03-0.48 μmol/L), C4/C2 0.07-0.23(reference value 0.01-0.04), C4/C3 0.65-2.04(reference value 0.05-0.39). Thirteen with increased urinary ethyl malonic acid (9.30-90.99 mg/g creatinine (reference value 0-6.20 mg/g creatinine )), one patient was accompanied by increased methyl succinic acid (12.33 mg/g creatinine(reference value 0-6.40 mg/g creatinine)), one subject with increased acetylglycine (3.52 mg/g creatinine(reference value 0-0.70 mg/g creatinine)). A total of 13 known mutations were detected in the ACADS gene, 1 homozygous mutation (c.1031A>G), the others are compound heterozygous mutations. One frameshift mutation (c.508_509delGC) and 12 missense mutations were detected. Common mutation were c. 1031A>G(35.3%), c. 164C>T(20.6%) and c. 991G>A(11.8%). SCADD in newborn screening program had no clinical symptoms and normal growth development after 8-42 months follow-up. Conclusion: Cases with SCADD had no clinical symptoms with an incidence of 1/84117. The c. 164C>T and c. 1031A>G may be the common mutations.

P.387 - Riboflavin transporter deficiency diagnosed 30 years after onset of symptoms

Mutations in the riboflavin transporter genes SLC52A2 and SLC52A3 cause a neurodegenerative disorder formerly known as Brown–Vialetto–van Laere (BVVL) syndrome, now renamed to riboflavin transporter deficiency. We present a patient who developed hypotonia, respiratory problems and feeding problems at the age of 8 months. His respiratory problems necessitated mechanical ventilation for 8 months. During follow up global developmental delay, bilateral sensorineural deafness and profound sensory ataxia were diagnosed. Extensive additional investigations (MRI, EMG, metabolic workup, CSF analysis and muscle biopsy) in infancy did not reveal any abnormalities apart from raised ethylmalonic acid. As a mitochondrial disorder was suspected he was treated with thiamine, riboflavin, biotin and carnitine. In the ensuing years these supplements were discontinued twice. Each time parents noticed worsening of his clinical condition. A second muscle biopsy performed at the age of 15 years showed central cores. Sequencing of RYR1 gene revealed one missense mutation, which was also present in the asymptomatic mother. Whole exome sequencing performed at the age of 30 years revealed a homozygous mutation in SLC52A3 gene (c.1128C > G(p.(Tyr376*)), which led to the diagnosis of Brown–Vialetto–van Laere syndrome. Riboflavin transporter deficiencies formerly known as Brown–Vialetto–van Laere syndrome present with cranial nerve deficits including hearing loss, weakness, sensory symptoms including sensory ataxia, feeding difficulties and respiratory difficulties which are caused by a progressive axonal sensory and motor peripheral neuropathy and a cranial neuropathy. Diagnosis can only be made through molecular analysis of all genes. Treatment with oral supplementation of riboflavin is lifesaving.

Compound heterozygous mutations of ACADS gene in newborn with short chain acyl-CoA dehydrogenase deficiency: case report and literatures review.

Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is a rare autosomal recessive mitochondrial disorder of fatty acid β-oxidation, and is associated with mutations in the acyl-CoA dehydrogenase (ACADS) gene. Recent advances in spectrometric screening for inborn errors of metabolism have helped detect several metabolic disorders, including SCADD, without symptoms in the neonate period. This allows immediate initiation of treatment and monitoring, so they remain largely symptomless metabolic disease. Here, we report a 15-month-old asymptomatic male, who was diagnosed with SCADD by newborn screening. Spectrometric screening for inborn errors of metabolism 72 hours after birth revealed an elevated butyrylcarnitine (C4) concentration of 2.25 µmol/L (normal, <0.99 µmol/L). Urinary excretion of ethylmalonic acid was also elevated, as detected by urine organic acid analysis. To confirm the diagnosis of SCADD, direct sequencing analysis of 10 coding exons and the exon-intron boundaries of the ACADS gene were performed. Subsequent sequence analysis revealed compound heterozygous missense mutations c.164C>T (p.Pro55Leu) and c.1031A>G (p.Glu344Gly) on exons 2 and 9, respectively. The patient is now growing up, unretarded by symptoms such as seizure and developmental delay.

Optimization of Cu–Ag Core–Shell Solderless Interconnect Paste Technology

The use of Cu–Ag core–shell nanoparticles has been proposed as a Pb-free interconnect technology in which upon annealing, the Ag shell dewets the copper surface and forms Ag necks between Cu core particles at particle–particle contacts. Neck formation creates an electrically, thermally, and mechanically stable porous sintered network. To convert this concept into a viable manufacturing technology, optimization of particle characteristics and paste formulation is necessary with respect to sinterability, mechanical strength, and processability for typical die attach and possibly surface mount assembly reflow operations and applications. This paper describes the optimization process carried out using three core–shell powder configurations and over 50 flux formulations. The two best performing pastes consisted of 1- $\mu \text{m}$ diameter core–shell particles with a 9-nm Ag shell and fluxes containing methyl ethyl ketone, isopropanol, methyl pyrrolidone, butyl cellosolve, Terpineol, Teckros D85, Troythix XYZ, and malonic acid. These pastes exhibited the targeted shear thinning behavior necessary for screen printing and assembly processing. From a comparison of the microstructural evolution for paste and dry compacts, the presence of flux did not appear to inhibit neck formation. As the final step, printability optimization using commercial assembly equipment was performed based on squeegee speed and pressure and showed coverage and paste rheology to be comparable with those of commercial Pb-free solder pastes.

Determination of Ethylmalonic Acid in Human Urine by Ion Chromatography with Suppressed Conductivity

We present here a new method for the determination of EMA in urine by ion chromatography (IC) with suppressed conductivity detection. The separations on an anion exchange column have been accomplished with an optimized multi-step gradient eluent program (5 mM for 0-7 min, 5-50 mM from 7 to 30 min, 50 mM for 30-40 min, 50-5 mM from 40 to 45 min, 5mM for 45-50 min at flow-rate 0.2 mL/min) using NaOH as the eluent. The temperatures of both the column and suppressed conductivity detector were 40 °C. The suppressor current was 25 mA. The injection loop volume was 100 µL. Prior to injection, a clean-up procedure has been applied to urine samples for sulfate removal using BaCl 2 precipitation followed by elimination of cationic and some organic compounds using strongly acid cation-exchange resin and C18 solid phase extraction cartridge, respectively. Concentration range of EMA for linear external calibration curve was between 0.202 -30.278 µmol/L (r 2 =0.999). The limit of detection and the limit of quantitation were 61 nmol/L and 202 nmol/L based on the signal-to-noise ratio equal to 3 and 10, respectively. The average recoveries of EMA for normal urine samples were between 98.2-99.3 % with less than 1.1 % relative standard deviation (RSD), and for pathological sample were between 97.2-107.8% with less than 1.9% RSD in both intra-day and inter-day assays. The average concentrations of EMA normalized against creatinine for healthy human urine samples and a pathological sample were 1.14 (0.88-1.50) and 128.95 (128.04-129.33) µmol mmol -1 creatinine, respectively. DOI: http://dx.doi.org/10.17807/orbital.v7i2.574

Brain and muscle redox imbalance elicited by acute ethylmalonic acid administration.

Ethylmalonic acid (EMA) accumulates in tissues and biological fluids of patients affected by short-chain acyl-CoA dehydrogenase deficiency (SCADD) and ethylmalonic encephalopathy, illnesses characterized by neurological and muscular symptoms. Considering that the mechanisms responsible for the brain and skeletal muscle damage in these diseases are poorly known, in the present work we investigated the effects of acute EMA administration on redox status parameters in cerebral cortex and skeletal muscle from 30-day-old rats. Animals received three subcutaneous injections of EMA (6 μmol/g; 90 min interval between injections) and were killed 1 h after the last administration. Control animals received saline in the same volumes. EMA administration significantly increased thiobarbituric acid-reactive substances levels in cerebral cortex and skeletal muscle, indicating increased lipid peroxidation. In addition, carbonyl content was increased in EMA-treated animal skeletal muscle when compared to the saline group. EMA administration also significantly increased 2’,7’-dihydrodichlorofluorescein oxidation and superoxide production (reactive species markers), and decreased glutathione peroxidase activity in cerebral cortex, while glutathione levels were decreased only in skeletal muscle. On the other hand, respiratory chain complex I-III activity was altered by acute EMA administration neither in cerebral cortex nor in skeletal muscle. The present results show that acute EMA administration elicits oxidative stress in rat brain and skeletal muscle, suggesting that oxidative damage may be involved in the pathophysiology of the brain and muscle symptoms found in patients affected by SCADD and ethylmalonic encephalopathy.

SOLVENT FREE SYNTHESIS OF MALONYL CHLORIDES: A GREEN CHEMISTRY APPROACH

Methyl malonyl chloride and ethyl malonyl chloride are the acid chlorides of malonic acid diester. The present study focuses on the study of the synthesis of monomethyl malonyl chloride and monoethyl malonyl chloride. The synthesis was a three step process of selective saponification of dialkyl malonate followed by hydrolysis and then chlorination of monoalkyl malonic acid. The reaction was studied in the presence of solvent and absence of solvent (which is methylene chloride). Kinetics of the process was studied. Reaction was found to be first order by integrated rate law. The green process was evaluated with maximum conversion. The conversion of methyl malonic acid in solvent process was found to be 78.67% and in without solvent process was found to be 93.08% from the experiments carried out. The conversion of ethyl malonic acid in solvent process was found to be 84.39% and in without solvent process was found to be 98.23% from the experiments carried out.

Disturbance of energy and redox homeostasis and reduction of Na+,K+-ATPase activity provoked by in vivo intracerebral administration of ethylmalonic acid to young rats

Ethylmalonic acid (EMA) accumulation occurs in various metabolic diseases with neurological manifestation, including short acyl-CoA dehydrogenase deficiency (SCADD) and ethylmalonic encephalopathy (EE). Since pathophysiological mechanisms responsible for brain damage in these disorders are still poorly understood, we investigated the ex vivo effects of acute intrastriatal administration of EMA on important parameters of energy and redox homeostasis in striatum from young rats. We evaluated CO2 production from glucose, glucose utilization and lactate production, as well as the activities of the citric acid cycle (CAC) enzymes, the electron transfer chain (ETC) complexes II–IV (oxidative phosphorylation, OXPHOS) and synaptic Na+,K+-ATPase. We also tested the effect of EMA on malondialdehyde (MDA) levels (marker of lipid oxidation) and reduced glutathione (GSH) levels. EMA significantly reduced CO2 production, increased glucose utilization and lactate production, and reduced the activities of citrate synthase and of complexes II and II–III of the ETC, suggesting an impairment of CAC and OXPHOS. EMA injection also reduced Na+,K+-ATPase activity and GSH concentrations, whereas MDA levels were increased. Furthermore, EMA-induced diminution of Na+,K+-ATPase activity and reduction of GSH levels were prevented, respectively, by the antioxidants melatonin and N-acetylcysteine, indicating that reactive species were involved in these effects. Considering the importance of CAC and ETC for energy production and Na+,K+-ATPase for the maintenance of the cell membrane potential, the present data indicate that EMA compromises mitochondrial homeostasis and neurotransmission in striatum. We presume that these pathomechanisms may be involved to a certain extent in the neurological damage found in patients affected by SCADD and EE.

Effects of a dietary sweetener on growth performance and health of stressed beef calves and on diet digestibility and plasma and urinary metabolite concentrations of healthy calves1

Two experiments were conducted to explore the effects of a sodium saccharin-based dietary sweetener (Sucram) on growth performance, health, and physiological responses of feedlot steers. In Exp. 1, 173 newly-received male calves purchased from auction barns were fed 0, 100, 200, or 300 g of Sucram/t of DM over 56 d. Overall, ADG and G:F (P > 0.10) were not different among treatments, but steers receiving 200 g Sucram/t displayed numerically greater ADG (23%). In addition, DMI was 17% greater for steers receiving 200 g of Sucram/t compared to steers fed the control diet (cubic effect, P = 0.09). The morbidity rate for respiratory disease did not differ (P > 0.50) among treatments. In Exp. 2, 15 steers (initial BW = 261 ± 28 kg) were used to evaluate the effects of Sucram on apparent total tract digestibility, plasma metabolite concentrations, and urine monoamine metabolite concentrations. Treatments consisted of ad libitum access to a 60% concentrate diet (Control), ad libitum access to Control + 200 g of Sucram/t of DM (Adlib), and Control + 200 g of Sucram/t of DM with feed intake paired to the Control (Paired). By design, steer DMI during the metabolism period did not differ (P = 0.34) between Paired and Control, but DMI tended (P = 0.14) to be 8.2% greater for Adlib than for Control. Treatments did not alter (P > 0.17) apparent total tract nutrient digestibility. Postprandial plasma citrulline concentration was lower (P = 0.03) for Adlib than for Control and tended to be lower (P = 0.13) for Paired than for Control. Plasma homocysteine concentration was reduced (P < 0.03) by feeding Sucram. Urinary concentrations of ethylmalonic acid, vanillymandelic acid, and 5-hydroxyindolacetic acid were greater (P < 0.06) for Adlib than for Control; Paired steers had a greater (P = 0.02) urine vanillymandelic acid concentration than Control steers and tended (P < 0.12) to have a greater urinary concentration of ethylmalonic and 5-hydroxyindolacetic acid than Control steers. Serum insulin was greater for Adlib than for Control steers (P = 0.04) and tended to be greater for Paired than for Control steers (P = 0.14), but serum prolactin area did not differ (P > 0.22) among treatments. Supplementation with Sucram may increase feed intake by newly-received, stressed feedlot calves. Saccharin supplementation reduced plasma homocysteine and increased urinary excretion of vanillymandelic acid, suggesting an improved activity of the dopamine reward system.

Ethylmalonic Acid Induces Permeability Transition in Isolated Brain Mitochondria

Predominant accumulation of ethylmalonic acid (EMA) in tissues and biological fluids is a characteristic of patients affected by short chain acyl-CoA dehydrogenase deficiency and ethylmalonic encephalopathy. Neurological abnormalities are frequently found in these disorders, but the mechanisms underlying the brain injury are still obscure. Since hyperlacticacidemia is also found in many affected patients indicating a mitochondrial dysfunction; in the present work, we evaluated the in vitro and ex vivo effects of EMA plus Ca(2+) on mitochondrial integrity and redox balance in succinate-supported brain organelles. We verified that the evaluated parameters were disturbed only when EMA was associated with exogenous micromolar Ca(2+) concentrations. Thus, we found that this short chain organic acid plus Ca(2+) dissipated the membrane potential and provoked mitochondrial swelling, as well as impaired the mitochondrial Ca(2+) retention capacity, resulting in a rapid Ca(2+) release and decreased NAD(P)H matrix content. In contrast, EMA was not able to stimulate mitochondrial hydrogen peroxide generation. We also observed that all these effects were prevented by the mitochondrial Ca(2+) uptake inhibitor ruthenium red and the mitochondrial permeability transition (MPT) inhibitors cyclosporin A (CsA) and ADP. Furthermore, mitochondria isolated from rat brains after in vivo intrastriatal administration of EMA was more susceptible to Ca(2+)-induced swelling, which was fully prevented by CsA and ADP. Finally, EMA significantly decreased striatal slice viability, which was attenuated by CsA. The data strongly indicate that EMA reduced the mitochondrial threshold for Ca(2+)-induced MPT reinforcing the role of this cation in EMA-induced disruption of mitochondrial bioenergetics. It is, therefore, presumed that EMA acting synergistically with Ca(2+) compromises mitochondrial energy homeostasis in the central nervous system that may explain at least in part the neurologic alterations presented by patients with abnormal levels of this organic acid.

Liquid chromatographic-mass spectrometric method for simultaneous determination of small organic acids potentially contributing to acidosis in severe malaria.

Acidosis is an important cause of mortality in severe falciparum malaria. Lactic acid is a major contributor to metabolic acidosis, but accounts for only one-quarter of the strong anion gap. Other unidentified organic acids have an independent strong prognostic significance for a fatal outcome. In this study, a simultaneous bio-analytical method for qualitative and quantitative assessment in plasma and urine of eight small organic acids potentially contributing to acidosis in severe malaria was developed and validated. High-throughput strong anion exchange solid-phase extraction in a 96-well plate format was used for sample preparation. Hydrophilic interaction liquid chromatography (HILIC) coupled to negative mass spectroscopy was utilized for separation and detection. Eight possible small organic acids; l-lactic acid (LA), α-hydroxybutyric acid (aHBA), β-hydroxybutyric acid (bHBA), p-hydroxyphenyllactic acid (pHPLA), malonic acid (MA), methylmalonic acid (MMA), ethylmalonic acid (EMA) and α-ketoglutaric acid (aKGA) were analyzed simultaneously using a ZIC-HILIC column with an isocratic elution containing acetonitrile and ammonium acetate buffer. This method was validated according to U.S. Food and Drug Administration guidelines with additional validation procedures for endogenous substances. Accuracy for all eight acids ranged from 93.1% to 104.0%, and the within-day and between-day precisions (i.e. relative standard deviations) were lower than 5.5% at all tested concentrations. The calibration ranges were: 2.5-2500μg/mL for LA, 0.125-125μg/mL for aHBA, 7.5-375μg/mL for bHBA, 0.1-100μg/mL for pHPLA, 1-1000μg/mL for MA, 0.25-250μg/mL for MMA, 0.25-100μg/mL for EMA, and 30-1500μg/mL for aKGA. Clinical applicability was demonstrated by analyzing plasma and urine samples from five patients with severe falciparum malaria; five acids had increased concentrations in plasma (range LA=177-1169μg/mL, aHBA=4.70-38.4μg/mL, bHBA=7.70-38.0μg/mL, pHPLA=0.900-4.30μg/mL and aKGA=30.2-32.0) and seven in urine samples (range LA=11.2-513μg/mL, aHBA=1.50-69.5μg/mL, bHBA=8.10-111μg/mL, pHPLA=4.30-27.7μg/mL, MMA=0.300-13.3μg/mL, EMA=0.300-48.1μg/mL and aKGA=30.4-107μg/mL). In conclusion, a novel bioanalytical method was developed and validated which allows for simultaneous quantification of eight small organic acids in plasma and urine. This new method may be a useful tool for the assessment of acidosis in patients with severe malaria, and other conditions complicated by acidosis.

First case report of short-chain acyl-CoA dehydrogenase deficiency in China

Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is a rare autosomal recessive inborn error of mitochondrial fatty acid oxidation. It caused by rare mutations as well as polymorphic susceptibility variants. We describe the case of a 1-year-old male patient who had growth and mental retardation, seizures, fever since infancy. Urinary GC/MS showed elevated levels of ethylmalonic acid. Plasma acylcarnitines on MS/MS, elevations of C4-cartinitine are consistently present. The two polymorphic susceptibility variants of SCAD gene, c.625G>A and c.322G>A, was detected. As its highly variable clinical characteristics, there is no related report in China. This report broadens the phenotype and genotype of SCAD deficiency in China and underlines the difficulty of diagnosis.

P18 Proteome adaptations in ETHE1 deficient mice indicate a role of sulfide signaling in lipid catabolism and cytoskeleton organization via post-translational protein modifications

In patients suffering from ethylmalonic encephalopathy hydrogen sulfide accumulates in the bloodstream due to a block in mitochondrial sulfide oxidation at the level of the sulfur dioxygenase ETHE1. The chronic exposure to elevated sulfide concentrations causes fatal dysfunctions in various organ systems. In particular, the vascular endothelium is severely damaged leading to multiple necrotic and haemorrhagic brain lesions, chronic haemorrhagic diarrhea, vascular petechial purpura and orthostatic acrocyanosis. The biochemical profile, an increase in urinary and plasmatic lactate, acylcarnitine, acylglycine, and ethylmalonic acid levels, furthermore indicates disturbances in mitochondrial energy metabolism. While the effects of sulfide on cytochrome c oxidase have been thoroughly investigated, and shown to be due to an increased degradation of specific subunits, the additional systemic consequences are not completely understood. We used an Ethe1 deficient mouse model, which faithfully recapitulates the clinical and biochemical features of ethylmalonic encephalopathy, to investigate the effect of increased sulfide and persulfide concentrations on liver, kidney, muscle and brain proteomes. Our results provide evidence for a crucial role of the mitochondrial sulfide oxidation pathway in signaling. The mechanism of this regulatory function is most likely based on cysteine S-modifications. Increased sulfide concentrations promote cysteine S-sulfhydrations and thereby shift the balance between different forms of post-translational modifications. In addition, ETHE1 as a sulfur dioxygenase could directly take part in the removal of sulfhydrations by oxidizing the cysteine derived persulfide group. Notably, the main pathways affected by elevated sulfide concentrations on proteomic level are the mitochondrial oxidation of branched-chain amino acids and fatty acids as well as cytoskeleton organization. Enzymes involved in lipid catabolism are also most clearly coexpressed with Ethe1. These findings clarify the original clinical description of ethylmalonic encephalopathy that was thought to be a defect in the beta-oxidation pathway and indicate a close functional connection between sulfide oxidation and mitochondrial acyl-CoA metabolism. In addition, sulfide must be considered an important factor in the control of cytoskeleton dynamics. The increase in sulfide and persulfide concentrations elicits changes in structural proteins as well as in enzymes involved in the organization of the cytoskeleton specifically in brain and muscle of Ethe1 deficient mice. These results are consistent with the severe defects in motor activity and rapidly progressive neurological failure observed in patients and the mouse model. Thus, the present study provides a new perspective on the pathogenesis of ethylmalonic encephalopathy and also reveals new aspects about the metabolic function of mitochondrial sulfide oxidation.

Ethylmalonic acid modulates Na+, K+‐ATPase activity and mRNA levels in rat cerebral cortex

Ethylmalonic acid (EMA) accumulates in tissues of patients affected by short-chain acyl-CoA dehydrogenase deficiency and ethylmalonic encephalopathy, illnesses characterized by variable neurological symptoms. In this work, we investigated the in vitro and in vivo EMA effects on Na(+), K(+)-ATPase (NAK) activity and mRNA levels in cerebral cortex from 30-day-old rats. For in vitro studies, cerebral cortex homogenates were incubated in the presence of EMA at 0.5, 1, or 2.5 mM concentrations for 1 h. For in vivo experiments, animals received three subcutaneous EMA injections (6 μmol g(-1); 90-min interval) and were killed 60 min after the last injection. After that, NAK activity and its mRNA expression were measured. We observed that EMA did not affect this enzyme activity in vitro. In contrast, EMA administration significantly increased NAK activity and decreased mRNA NAK expression as assessed by semiquantitative reverse transcriptase polymerase chain reaction when compared with control group. Considering the high score of residues prone to phosphorylation on NAK, this profile can be associated with a possible regulation by specific phosphorylation sites of the enzyme. Altogether, the present results suggest that NAK alterations may be involved in the pathophysiology of brain damage found in patients in which EMA accumulates.

Arabidopsis ETHE1 Encodes a Sulfur Dioxygenase That Is Essential for Embryo and Endosperm Development

Mutations in human (Homo sapiens) ETHYLMALONIC ENCEPHALOPATHY PROTEIN1 (ETHE1) result in the complex metabolic disease ethylmalonic encephalopathy, which is characterized in part by brain lesions, lactic acidemia, excretion of ethylmalonic acid, and ultimately death. ETHE1-like genes are found in a wide range of organisms; however, the biochemical and physiological role(s) of ETHE1 have not been examined outside the context of ethylmalonic encephalopathy. In this study we characterized Arabidopsis (Arabidopsis thaliana) ETHE1 and determined the effect of an ETHE1 loss-of-function mutation to investigate the role(s) of ETHE1 in plants. Arabidopsis ETHE1 is localized in the mitochondrion and exhibits sulfur dioxygenase activity. Seeds homozygous for a DNA insertion in ETHE1 exhibit alterations in endosperm development that are accompanied by a delay in embryo development followed by embryo arrest by early heart stage. Strong ETHE1 labeling was observed in the peripheral and chalazal endosperm of wild-type seeds prior to cellularization. Therefore, ETHE1 appears to play an essential role in regulating sulfide levels in seeds.

Biochemical, molecular, and clinical characteristics of children with short chain acyl-CoA dehydrogenase deficiency detected by newborn screening in California

BackgroundShort-chain acyl-CoA dehydrogenase deficiency (SCADD) is an autosomal recessive inborn error of mitochondrial fatty acid oxidation with highly variable biochemical, genetic, and clinical characteristics. SCADD has been associated with accumulation of butyryl-CoA byproducts, including butyrylcarnitine (C4), butyrylglycine, ethylmalonic acid (EMA), and methylsuccinic acid (MS) in body fluid and tissues. Differences in genotype frequencies have been shown between patients diagnosed clinically versus those diagnosed by newborn screening. Moreover, while patients diagnosed clinically have a variable clinical presentation including developmental delay, ketotic hypoglycemia, epilepsy and behavioral disorders, studies suggest patients diagnosed by newborn screening are largely asymptomatic. Scant information is published about the biochemical, genetic and clinical outcome of SCADD patients diagnosed by newborn screening. MethodsWe collected California newborn screening, follow-up biochemical levels, and ACADS mutation data from September, 2005 through April, 2010. We retrospectively reviewed available data on SCADD cases diagnosed by newborn screening for clinical outcomes. ResultsDuring the study period, 2,632,058 newborns were screened and 76 confirmed SCADD cases were identified. No correlations between initial C4 value and follow-up biochemical markers (C4, EMA or MS levels) were found in the 76 cases studied. We found significant correlation between urine EMA versus MS, and correlation between follow-up C4 versus urine EMA. Of 22 cases where ACADS gene sequencing was performed: 7 had two or more deleterious mutations; 8 were compound heterozygotes for a deleterious mutation and common variant; 7 were homozygous for the common variant c.625G>A; and 1 was heterozygous for c.625G>A. Significant increases in mean urine EMA and MS levels were noted in patients with two or more deleterious mutations versus mutation heterozygotes or common polymorphism homozygotes. Clinical outcome data was available in 31 patients with follow-up extending from 0.5 to 60months. None developed epilepsy or behavioral disorders, and three patients had isolated speech delay. Hypoglycemia occurred in two patients, both in the neonatal period. The first patient had concomitant meconium aspiration; the other presented with central apnea, poor feeding, and hypotonia. The latter, a c.625G>A homozygote, has had persistent elevations in both short- and medium-chain acylcarnitines; diagnostic workup in this case is extensive and ongoing. ConclusionsThis study examines the largest series to date of SCADD patients identified by newborn screening. Our results suggest that confirmatory tests may be useful to differentiate patients with common variants from those with deleterious mutations. This study also provides evidence to suggest that, even when associated with deleterious mutations, SCADD diagnosed by newborn screening presents largely as a benign condition.