Capillary electrophoresis with capacitively coupled contactless conductivity detection for the determination of urinary ethylmalonic acid for the diagnosis of ethylmalonic aciduria.

Ethylmalonic acid is a metabolic organic acid, and its accumulation in urine is diagnostic of ethylmalonic aciduria. In this study, a simple and fast method employing capillary electrophoresis equipped with capacitively coupled contactless conductivity detection was developed for the detection of ethylmalonic acid in urine samples. The optimized electrophoretic separation was performed in 50 mmol/L 2-(N-morpholino)ethanesulfonic acid solution, buffered at a pH of 6.5, and contained 0.13 mmol/L cetyltrimethylammonium bromide as an electroosmotic modifier. Electrophoresis was run at 28 kV in reversed polarity. The linear range of ethylmalonic acid concentration was between 1 and 100 mg/L with a regression coefficient of 0.9998. This method had good intra- and interday precision with <5% relative standard deviations. The detection limit (signal-to-noise ratio = 3) and the quantification limit (signal-to-noise ratio = 10) values were 0.139 and 0.466 mg/L, respectively. Using our optimized conditions, the method was successfully employed for the detection of ethylmalonic acid in urine sample of ethylmalonic aciduria patient.

FLAD1-associated multiple acyl-CoA dehydrogenase deficiency identified by newborn screening.

BACKGROUND: Multiple acyl-CoA dehydrogenase deficiency (MADD), also known as glutaric aciduria type II, is a mitochondrial fatty acid oxidation disorder caused by variants in ETFA, ETFB, and ETFDH. Recently, riboflavin transporter genes and the mitochondrial FAD transporter gene have also been associated with MADD-like phenotype. METHODS: We present a case of MADD identified by newborn biochemical screening in a full-term infant suggestive of both medium-chain acyl-CoA dehydrogenase deficiency and MADD. Urine organic acid GC/MS analysis was also concerning for both disorders. However, panel sequencing of ETFA, ETFB, ETFDH, and ACADM was unrevealing. Ultimately, a variant in the FAD synthase gene, FLAD1 was found explaining the clinical presentation. RESULTS: Exome sequencing identified compound heterozygous variants in FLAD1: NM_025207.4: c.[442C>T];[1588C>T], p.[Arg148*];[Arg530Cys]. The protein damaging effects were confirmed by Western blot. The patient remained asymptomatic and there was no clinical decompensation during the first year of life. Plasma acylcarnitine and urinary organic acid analyses normalized without any treatment. Riboflavin supplementation was started at 15 months. CONCLUSION: Newborn screening, designed to screen for specific treatable congenital metabolic diseases, may also lead to the diagnosis of additional, very rare metabolic disorders such as FLAD1 deficiency. The case further illustrates that even milder forms of FLAD1 deficiency are detectable in the asymptomatic state by newborn screening.

Equine multiple acyl-CoA dehydrogenase deficiency (MADD) associated with seasonal pasture myopathy in the midwestern United States.

BACKGROUND: Seasonal pasture myopathy (SPM) is a highly fatal form of nonexertional rhabdomyolysis that occurs in pastured horses in the United States during autumn or spring. In Europe, a similar condition, atypical myopathy (AM), is common. Recently, a defect of lipid metabolism, multiple acyl-CoA dehydrogenase deficiency (MADD), has been identified in horses with AM. OBJECTIVE: To determine if SPM in the United States is caused by MADD. ANIMALS: Six horses diagnosed with SPM based on history, clinical signs, and serum creatine kinase activity, or postmortem findings. METHODS: Retrospective descriptive study. Submissions to the Neuromuscular Diagnostic Laboratory at the University of Minnesota were reviewed between April 2009 and January 2010 to identify cases of SPM. Inclusion criteria were pastured, presenting with acute nonexertional rhabdomyolysis, and serum, urine, or muscle samples available for analysis. Horses were evaluated for MADD by urine organic acids, serum acylcarnitines, muscle carnitine, or histopathology. RESULTS: Six horses had clinical signs and, where performed (4/6 horses), postmortem findings consistent with SPM. Affected muscle (4/4) showed degeneration with intramyofiber lipid accumulation, decreased free carnitine concentration, and increased carnitine esters. Serum acylcarnitine profiles (3/3) showed increases in short- and medium-chain acylcarnitines and urinary organic acid profiles (3/3) revealed increased ethylmalonic and methylsuccinic acid levels, and glycine conjugates, consistent with equine MADD. CONCLUSIONS AND CLINICAL IMPORTANCE: Similar to AM, the biochemical defect causing SPM is MADD, which causes defective muscular lipid metabolism and excessive myofiber lipid content. Diagnosis can be made by assessing serum acylcarnitine and urine organic acid profiles.

Decreased oxidative phosphorylation and PGAM deficiency in horses suffering from atypical myopathy associated with acquired MADD.

Earlier research on ten horses suffering from the frequently fatal disorder atypical myopathy showed that MADD (multiple acyl-CoA dehydrogenase deficiency) is the biochemical derangement behind atypical myopathy. From five horses that died as a result of this disease and seven healthy control horses, urine and plasma were collected ante mortem and muscle biopsies were obtained immediately post-mortem (2 patients and 7 control horses), to analyse creatine, purine and carbohydrate metabolism as well as oxidative phosphorylation. In patients, the mean creatine concentration in urine was increased 17-fold and the concentration of uric acid approximately 4-fold, compared to controls. The highest degree of depletion of glycogen was observed in the patient with the most severe myopathy clinically. In this patient, glycolysis was more active than in the other patients and controls, which may explain this depletion. One patient demonstrated very low phosphoglycerate mutase (PGAM) activity, less than 10% of reference values. Most respiratory chain complex activity in patients was 20-30% lower than in control horses, complex II activity was 42% lower than normal, and one patient had severely decrease ATP-synthase activity, more than 60% lower than in control horses. General markers for myopathic damage are creatine kinase (CK) and lactic acid in plasma, and creatine and uric acid in urine. To obtain more information about the cause of the myopathy analysis of carbohydrate, lipid and protein metabolism as well as oxidative phosphorylation is advised. This study expands the diagnostic possibilities of equine myopathies.

Glutaric aciduria type II [corrected] and brain tumors: a case report and review of the literature.

Heritable diseases associated with childhood tumors are sometimes defined as a probable etiologic factor or a coincidence. First of all, we must know the actual number of patients. Herein a case with medulloblastoma associated with glutaric aciduria type II [corrected] is reported for this purpose. A 5-year-old boy was admitted with nausea, vomiting, and lethargy. In medical history, consanguinity and siblings with mental-motor retardation and epilepsy are remarkable. Growth retardation, macrocephaly, lethargy, tremor, bilateral nistagmus, and papilledema were prominent features in physical examination. Noncontrast computed tomography of the brain showed a hyper dense mass in the cerebellar vermis. Gross total resection was made and the histopathology of the tumor was medulloblastoma. Besides medical history and physical findings, radiologic white matter changes in the subcortical, periventricular regions, bilateral basal ganglia, and caudate nuclei in magnetic resonance images other than tumor led us to investigate the child for glutaric aciduria type II [corrected]. The level of the 2-OH glutaric acid was determined as being 12-fold high in the urine. Chemo-radiotherapy was performed after surgery. Our case was the third patient with medulloblastoma in the literature and is still alive with no evidence of the disease 19 months after the initial diagnosis.

Outcome of organic acidurias in China.

From June 1998 to May 2007, 9566 urine samples were collected from patients with psychomotor deficits, seizures, vomiting and unconsciousness in Peking University First Hospital. Their urine organic acids profiles were analysed using gas chromatography - mass spectrometry (GCMS), GCMS solution and Inborn Errors of Metabolism Screening System software. In all patients, blood acylcarnitines were analysed using tandem mass spectrometry. One hundred and sixty-eight patients (1.76%) with organic acidurias were detected. Among them, 116 (116/ 168, 69.0%) had methylmalonic aciduria, 63 (54.3%) of these 116 patients had methylmalonic aciduria combined with homocysteinemia. Sixteen (9.5%) of those patients detected with organic acidurias had propionic aciduria, and 15 (8.9%) had multiple carboxylase deficiency. Seven (4.2%) had glutaric aciduria type 1. After dietary treatment, medicine and rehabilitation, clinical improvements were observed in more than half of the patients. Twenty-eight of the 168 patients (16.7%) recovered and led a normal life. The method of urine organic acid analysis by gas chromatography - mass spectrometry and blood acylcarnitines analysis by tandem mass spectrometry have been established and applied successfully in China, namely Beijing, Shanghai, Wuhan and Guangzhou. The prognoses of Chinese patients with organic acidurias have also improved significantly.