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Evidence for involvement of medium chain acyl-CoA dehydrogenase in the metabolism of phenylbutyrate.

ABSTRACT: Sodium phenylbutyrate is used for treating urea cycle disorders, providing an alternative for ammonia excretion. Following conversion to its CoA ester, phenylbutyryl-CoA is postulated to undergo one round of ?-oxidation to phenylacetyl-CoA, the active metabolite. Molecular modeling suggests that medium chain acyl-CoA dehydrogenase (MCAD; EC 1.3.99.3), a key enzyme in straight chain fatty acid ?-oxidation, could utilize phenylbutyryl-CoA as substrate. Moreover, phenylpropionyl-CoA has been shown to be a substrate for MCAD and its intermediates accumulate in patients with MCAD deficiency. We have examined the involvement of MCAD and other acyl-CoA dehydrogenases (ACADs) in the metabolism of phenylbutyryl-CoA. Anaerobic titration of purified recombinant human MCAD with phenylbutyryl-CoA caused changes in the MCAD spectrum that are similar to those induced by octanoyl-CoA, its bona fide substrate, and unique to the development of the charge transfer ternary complex. The calculated apparent dissociation constant (K(D app)) for these substrates was 2.16 ?M and 0.12 ?M, respectively. The MCAD reductive and oxidative half reactions were monitored using the electron transfer flavoprotein (ETF) fluorescence reduction assay. The catalytic efficiency and the K(m) for phenylbutyryl-CoA were 0.2 mM 34(-1)·sec(-1) and 5.3 ?M compared to 4.0 mM(-1)·sec(-1) and 2.8 ?M for octanoyl-CoA. Extracts of wild type and MCAD-deficient lymphoblast cells were tested for the ability to reduce ETF using phenylbutyryl-CoA as substrate. While ETF reduction activity was detected in extracts of wild type cells, it was undetectable in extracts of cells deficient in MCAD. The results are consistent with MCAD playing a key role in phenylbutyrate metabolism.

SUBMITTER: Kormanik K

PROVIDER: S-EPMC3504130 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Evidence for involvement of medium chain acyl-CoA dehydrogenase in the metabolism of phenylbutyrate.

Kormanik Kaitlyn K Kang Heejung H Cuebas Dean D Vockley Jerry J Mohsen Al-Walid AW

Molecular genetics and metabolism 20121018 4

Sodium phenylbutyrate is used for treating urea cycle disorders, providing an alternative for ammonia excretion. Following conversion to its CoA ester, phenylbutyryl-CoA is postulated to undergo one round of β-oxidation to phenylacetyl-CoA, the active metabolite. Molecular modeling suggests that medium chain acyl-CoA dehydrogenase (MCAD; EC 1.3.99.3), a key enzyme in straight chain fatty acid β-oxidation, could utilize phenylbutyryl-CoA as substrate. Moreover, phenylpropionyl-CoA has been shown ...[more]

PMID: 23141465

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Project description:BackgroundMonogenetic inborn errors of metabolism cause a wide phenotypic heterogeneity that may even differ between family members carrying the same genetic variant. Computational modelling of metabolic networks may identify putative sources of this inter-patient heterogeneity. Here, we mainly focus on medium-chain acyl-CoA dehydrogenase deficiency (MCADD), the most common inborn error of the mitochondrial fatty acid oxidation (mFAO). It is an enigma why some MCADD patients-if untreated-are at risk to develop severe metabolic decompensations, whereas others remain asymptomatic throughout life. We hypothesised that an ability to maintain an increased free mitochondrial CoA (CoASH) and pathway flux might distinguish asymptomatic from symptomatic patients.ResultsWe built and experimentally validated, for the first time, a kinetic model of the human liver mFAO. Metabolites were partitioned according to their water solubility between the bulk aqueous matrix and the inner membrane. Enzymes are also either membrane-bound or in the matrix. This metabolite partitioning is a novel model attribute and improved predictions. MCADD substantially reduced pathway flux and CoASH, the latter due to the sequestration of CoA as medium-chain acyl-CoA esters. Analysis of urine from MCADD patients obtained during a metabolic decompensation showed an accumulation of medium- and short-chain acylcarnitines, just like the acyl-CoA pool in the MCADD model. The model suggested some rescues that increased flux and CoASH, notably increasing short-chain acyl-CoA dehydrogenase (SCAD) levels. Proteome analysis of MCADD patient-derived fibroblasts indeed revealed elevated levels of SCAD in a patient with a clinically asymptomatic state. This is a rescue for MCADD that has not been explored before. Personalised models based on these proteomics data confirmed an increased pathway flux and CoASH in the model of an asymptomatic patient compared to those of symptomatic MCADD patients.ConclusionsWe present a detailed, validated kinetic model of mFAO in human liver, with solubility-dependent metabolite partitioning. Personalised modelling of individual patients provides a novel explanation for phenotypic heterogeneity among MCADD patients. Further development of personalised metabolic models is a promising direction to improve individualised risk assessment, management and monitoring for inborn errors of metabolism.

Project description:BACKGROUND: Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common inherited disorder of the mitochondrial fatty acid oxidation, caused by mutations in the ACADM gene. Since the introduction of neonatal screening for MCAD deficiency, a subgroup of newborns have been identified with variant ACADM genotypes that had never been identified before in clinically ascertained patients. In vitro residual MCAD enzyme activity has been found to facilitate risk-stratification. In this study we integrated results of in vitro (residual MCAD enzyme activities) and in vivo (clinical fasting tolerance tests, and phenylpropionic acid loading tests) tests in this subgroup of newborns, defining the consequences of variant ACADM genotypes. METHODS: Enzyme analyses were performed in leukocytes with: hexanoyl-CoA (C6-CoA) +/- butyryl-CoA (C4-CoA), and phenylpropionyl-CoA (PP-CoA). In vitro studies were performed in 9 subjects with variant ACADM genotypes, in vivo functional tests in 6 of these subjects. RESULTS: Enzyme analyses with C6-CoA, C6-CoA + C4-CoA, and PP-CoA identified significantly higher residual MCAD enzyme activities in subjects with variant ACADM genotypes when compared to patients with classical ACADM genotypes.After prolonged fasting (range 15-18.5 hours) no hypoglycaemia was observed. Increasing concentrations of free fatty acids indicated lipolysis, and ketone body concentrations were sufficient for blood glucose concentrations in 5 out of 6 subjects. Phenylpropionic acid loading clearly demonstrated in vivo residual MCAD enzyme activity in all studied subjects. CONCLUSIONS: Subjects with variant ACADM genotypes and residual MCAD enzyme activities >10% display residual MCAD enzyme activities in vitro and in vivo. Our findings support the hypothesis that the guidelines on maximal duration of fasting might be abandoned in subjects with residual MCAD enzyme activities >10% under normal conditions. An emergency regimen and parental instructions remain necessary in all subjects with MCAD deficiency, regardless of residual MCAD enzyme activity.

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Evidence for involvement of medium chain acyl-CoA dehydrogenase in the metabolism of phenylbutyrate.

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Evidence for involvement of medium chain acyl-CoA dehydrogenase in the metabolism of phenylbutyrate.

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