Establishment of a practical enzymatic assay method for determination of isovaleryl-CoA dehydrogenase activity using high-performance liquid chromatography

Tanaka, Go; Sakura, Nobuo; Yofune, Hiroko; Febriani, Andi Dwi Bahagia; Nishimura, Yutaka; Sakamoto, Akiko; Ono, Hiroaki; Shigematsu, Yosuke; Kobayashi, Masao

doi:10.1016/j.cccn.2004.11.007

Cited by 16 publications

(12 citation statements)

References 9 publications

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“…The initial diagnosis of VLCAD deficiency was confirmed by an enzymatic assay for dehydrogenase activity toward palmitoyl-CoA (hereafter VLCAD activity) in lymphocytes. The method used was a modified version of the enzymatic assay method for isovaleryl-CoA dehydrogenase activity that we described previously (5). Human lymphocytes were isolated from heparinized peripheral blood using SEPARATE-L lymphocyte isolation medium (Muto Pure Chemicals, Tokyo, Japan), and 0.4% taurodeoxycholic acid (TDC) solution was added to the cell pellets to achieve a cell density of 10 6 lymphocytes/50 L. The cells were then lysed by pulsed sonic disruption (1 cycle/s with 30% duty cycle of sonic burst at 45 W) for 2 min under ice-bath conditions.…”

Section: Methodsmentioning

confidence: 99%

“…One possibility would be a test for mitochondrial acyl-CoA dehydrogenases, which produce 2-enoyl-CoA species in coordination with a reduction of electron transfer flavoprotein (ETF). Recently, we reported enzymatic diagnosis methods for isovaleric acidemia and medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, which detect the production of 3-methylcrotonyl-CoA or 2-octenoyl-CoA, respectively, using the crude lysates of lymphocytes and high performance liquid chromatography (HPLC) (5,6). The diagnosis of VLCAD deficiency can also be confirmed by a similar HPLC-based assay of 2-enoyl-CoA production.…”

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confidence: 99%

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Development of a New Enzymatic Diagnosis Method for Very-long-chain Acyl-CoA Dehydrogenase Deficiency by Detecting 2-Hexadecenoyl-CoA Production and its Application in Tandem Mass Spectrometry-based Selective Screening and Newborn Screening in Japan

et al. 2008

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ABSTRACT:The introduction of tandem mass spectrometry (MS/ MS) has made it possible to screen for very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. To confirm the diagnosis in cases with an abnormal profile of blood acylcarnitines, we developed a new enzymatic assay method for determining dehydrogenase activity toward palmitoyl-CoA (C16:0) in lymphocytes. Using this method, the production of 2-hexadecenoyl-CoA (C16:1) by crude cell lysates can be directly quantified using high performance liquid chromatography (HPLC). We applied the assay to 7 myopathic patients, 7 hypoglycemic patients, and 2 presymptomatic newborns with elevated levels of tetradecenoylcarnitine (C14:1 AC) in blood, and found impaired VLCAD activity in all of the 7 myopathic patients and both of the 2 newborns. All of the 7 hypoglycemic patients had normal level of the enzyme activity. Results of the ACADVL gene analysis were in consistent with the enzymatic diagnosis. These results suggest that MS/MS-based screening for VLCAD deficiency using blood C14:1 AC as the indicator may show a considerably high false-positive rate in selective screening of symptomatic patients. Our practical enzymatic assay can be a useful test for the accurate diagnosis of VLCAD deficiency cases screened by MS/MS. (Pediatr Res 64: 667-672, 2008)

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

Development of a New Enzymatic Diagnosis Method for Very-long-chain Acyl-CoA Dehydrogenase Deficiency by Detecting 2-Hexadecenoyl-CoA Production and its Application in Tandem Mass Spectrometry-based Selective Screening and Newborn Screening in Japan

et al. 2008

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“…Several direct and indirect methods to assay IVD activity have been published and, in addition to molecular genetic analysis, can be used to confirm a diagnosis of IVA [Hyman and Tanaka, 1986;Mohsen et al, 1998;Rhead and Tanaka, 1980;Shih et al, 1973;Tajima et al, 2005;Vockley et al, 1991;Yoshida et al, 1985]. Fibroblasts, lymphocytes, and amniocytes all have measurable amounts of IVD activity and serve as ready sources of tissue for this purpose [Ensenauer et al, 2004;Kleij et al, 1995;Mohsen et al, 1998;Vockley et al, 1991].…”

Section: Biochemical Diagonosis and Follow Upmentioning

confidence: 99%

Isovaleric acidemia: New aspects of genetic and phenotypic heterogeneity

Vockley

Ensenauer

2006

American J of Med Genetics Pt C

196

161

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Isovaleric acidemia (IVA) is an autosomal recessive inborn error of leucine metabolism caused by a deficiency of the mitochondrial enzyme isovaleryl-CoA dehydrogenase (IVD) resulting in the accumulation of derivatives of isovaleryl-CoA. It was the first organic acidemia recognized in humans and can cause significant morbidity and mortality. Early diagnosis and treatment with a protein restricted diet and supplementation with carnitine and glycine are effective in promoting normal development in severely affected individuals. Both intra-and inter-familial variability have been recognized. Initially, two phenotypes with either an acute neonatal or a chronic intermittent presentation were described. More recently, a third group of individuals with mild biochemical abnormalities who can be asymptomatic have been identified through newborn screening of blood spots by tandem mass spectrometry. IVD is a flavoenzyme that catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA and transfers electrons to the electron transfer flavoprotein. Human IVD has been purified from tissue and recombinant sources and its biochemical and physical properties have been extensively studied. Molecular analysis of the IVD gene from patients with IVA has allowed characterization of different types of mutations in this gene. One missense mutation, 932C>T (A282V), is particularly common in patients identified through newborn screening with mild metabolite elevations and who have remained asymptomatic to date. This mutation leads to a partially active enzyme with altered catalytic properties; however, its effects on clinical outcome and the necessity of therapy are still unknown. A better understanding of the heterogeneity of this disease and the relevance of genotype/phenotype correlations to clinical management of patients are among the challenges remaining in the study of this disorder in the coming years.

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“…Treatment using leucine-free formula and oral carnitine was started promptly, and she has not experienced any acute crises for 18 months. Diagnosis was confirmed by enzyme assay (Tajima G et al 2005). No cases other than IVA1 had positive results from IVG measurement.…”

Section: Resultsmentioning

confidence: 99%

Useful second‐tier tests in expanded newborn screening of isovaleric acidemia and methylmalonic aciduria

Shigematsu

Hata

Tanaka

2010

J of Inher Metab Disea

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Common use of pivalate-generating antibiotics in newborns in Japan and low cutoff value of C5-acylcarnitine (C5) to detect mild forms of isovaleric acidemia (IVA) led to 1,065 positive results from IVA screening among 146,000 newborns tested by tandem mass spectrometry over the last 3 years. Using our method to determine isovalerylglycine (IVG) levels in dried blood spots (DBS) as a second-tier test with IVG cutoff value of 0.5 nmol/ml in DBS, one patient with severe IVA was identified, and no recall of the second DBS was needed. Retrospective analysis revealed that most patients with moderate to severe forms of IVA have decreased free-carnitine levels shortly after birth and higher levels of IVG than those of C5, which suggests that this method is useful in evaluating the severity of IVA. Another second-tier test, to measure methylmalonic acid (MMA) levels in DBS by gas chromatography/mass spectrometry (GC/MS), has been developed to overcome difficulties in screening methylmalonic aciduria (MMAU) and propionic acidemia. Methanol extract from DBS was dried and derivatized using N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide. GC/MS was performed using splitless injection, electron-impact ionization, and selected ion monitoring for data recording. MMAU patients had much higher DBS concentrations of MMA (24.2-321.9 nmol/ml) than control newborns (0.34 ± 0.11 nmol/ml). MMA measurement in DBS was thought to provide useful information about the severity of MMAU, as MMAU patients with high levels of MMA had decreased levels of free carnitine and mildly increased levels of propionylcarnitine.

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Establishment of a practical enzymatic assay method for determination of isovaleryl-CoA dehydrogenase activity using high-performance liquid chromatography

Cited by 16 publications

References 9 publications

Development of a New Enzymatic Diagnosis Method for Very-long-chain Acyl-CoA Dehydrogenase Deficiency by Detecting 2-Hexadecenoyl-CoA Production and its Application in Tandem Mass Spectrometry-based Selective Screening and Newborn Screening in Japan

Development of a New Enzymatic Diagnosis Method for Very-long-chain Acyl-CoA Dehydrogenase Deficiency by Detecting 2-Hexadecenoyl-CoA Production and its Application in Tandem Mass Spectrometry-based Selective Screening and Newborn Screening in Japan

Isovaleric acidemia: New aspects of genetic and phenotypic heterogeneity

Useful second‐tier tests in expanded newborn screening of isovaleric acidemia and methylmalonic aciduria

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