Clinical variability of isovaleric acidemia in a genetically homogeneous population.

Isovaleric acidemia (IVA) is one of the most common organic acidemias found in South Africa. Since 1983, a significant number of IVA cases have been identified in approximately 20,000 Caucasian patients screened for metabolic defects. IVA is caused by an autosomal recessive deficiency of isovaleryl-CoA dehydrogenase (IVD) resulting in the accumulation of isovaleryl-CoA and its metabolites. In total, 10 IVA patients and three carriers were available for phenotypic and genotypic investigation in this study. All patients were found to be homozygous for a single c.367 G > A (p.G123R) mutation. The amino acid substitution of a glycine to arginine resulted in a markedly reduced steady-state level of the IVD protein, which explains the nearly complete lack of IVD enzyme activity as assessed in fibroblast homogenates. Despite the genetic homogeneity of this South African IVA group, the clinical presentation varied widely, ranging from severe mental handicap and multiple episodes of metabolic derangement to an asymptomatic state. The variation may be due to poor dietary intervention, delayed diagnosis or even epigenetic and polygenetic factors of unknown origin.

Acquired multiple Acyl-CoA dehydrogenase deficiency in 10 horses with atypical myopathy.

The aim of the current study was to assess lipid metabolism in horses with atypical myopathy. Urine samples from 10 cases were subjected to analysis of organic acids, glycine conjugates, and acylcarnitines revealing increased mean excretion of lactic acid, ethylmalonic acid, 2-methylsuccinic acid, butyrylglycine, (iso)valerylglycine, hexanoylglycine, free carnitine, C2-, C3-, C4-, C5-, C6-, C8-, C8:1-, C10:1-, and C10:2-carnitine as compared with 15 control horses (12 healthy and three with acute myopathy due to other causes). Analysis of plasma revealed similar results for these predominantly short-chain acylcarnitines. Furthermore, measurement of dehydrogenase activities in lateral vastus muscle from one horse with atypical myopathy indeed showed deficiencies of short-chain acyl-CoA dehydrogenase (0.66 as compared with 2.27 and 2.48 in two controls), medium-chain acyl-CoA dehydrogenase (0.36 as compared with 4.31 and 4.82 in two controls) and isovaleryl-CoA dehydrogenase (0.74 as compared with 1.43 and 1.61 nmol min(-1) mg(-1) in two controls). A deficiency of several mitochondrial dehydrogenases that utilize flavin adenine dinucleotide as cofactor including the acyl-CoA dehydrogenases of fatty acid beta-oxidation, and enzymes that degrade the CoA-esters of glutaric acid, isovaleric acid, 2-methylbutyric acid, isobutyric acid, and sarcosine was suspected in 10 out of 10 cases as the possible etiology for a highly fatal and prevalent toxic equine muscle disease similar to the combined metabolic derangements seen in human multiple acyl-CoA dehydrogenase deficiency also known as glutaric acidemia type II.

Valproic acid metabolism and its effects on mitochondrial fatty acid oxidation: a review.

Valproic acid (VPA; 2-n-propylpentanoic acid) is widely used as a major drug in the treatment of epilepsy and in the control of several types of seizures. Being a simple fatty acid, VPA is a substrate for the fatty acid beta-oxidation (FAO) pathway, which takes place primarily in mitochondria. The toxicity of valproate has long been considered to be due primarily to its interference with mitochondrial beta-oxidation. The metabolism of the drug, its effects on enzymes of FAO and their cofactors such as CoA and/or carnitine will be reviewed. The cumulative consequences of VPA therapy in inborn errors of metabolism (IEMs) and the importance of recognizing an underlying IEM in cases of VPA-induced steatosis and acute liver toxicity are two different concepts that will be emphasized.

Genetic basis for correction of very-long-chain acyl-coenzyme A dehydrogenase deficiency by bezafibrate in patient fibroblasts: toward a genotype-based therapy.

Very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency is an inborn mitochondrial fatty-acid beta-oxidation (FAO) defect associated with a broad mutational spectrum, with phenotypes ranging from fatal cardiopathy in infancy to adolescent-onset myopathy, and for which there is no established treatment. Recent data suggest that bezafibrate could improve the FAO capacities in beta-oxidation-deficient cells, by enhancing the residual level of mutant enzyme activity via gene-expression stimulation. Since VLCAD-deficient patients frequently harbor missense mutations with unpredictable effects on enzyme activity, we investigated the response to bezafibrate as a function of genotype in 33 VLCAD-deficient fibroblasts representing 45 different mutations. Treatment with bezafibrate (400 microM for 48 h) resulted in a marked increase in FAO capacities, often leading to restoration of normal values, for 21 genotypes that mainly corresponded to patients with the myopathic phenotype. In contrast, bezafibrate induced no changes in FAO for 11 genotypes corresponding to severe neonatal or infantile phenotypes. This pattern of response was not due to differential inductions of VLCAD messenger RNA, as shown by quantitative real-time polymerase chain reaction, but reflected variable increases in measured VLCAD residual enzyme activity in response to bezafibrate. Genotype cross-analysis allowed the identification of alleles carrying missense mutations, which could account for these different pharmacological profiles and, on this basis, led to the characterization of 9 mild and 11 severe missense mutations. Altogether, the responses to bezafibrate reflected the severity of the metabolic blockage in various genotypes, which appeared to be correlated with the phenotype, thus providing a new approach for analysis of genetic heterogeneity. Finally, this study emphasizes the potential of bezafibrate, a widely prescribed hypolipidemic drug, for the correction of VLCAD deficiency and exemplifies the integration of molecular information in a therapeutic strategy.

Metabolism of phytol to phytanic acid in the mouse, and the role of PPARalpha in its regulation.

Phytol, a branched-chain fatty alcohol, is the naturally occurring precursor of phytanic and pristanic acid, branched-chain fatty acids that are both ligands for the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARalpha). To investigate the metabolism of phytol and the role of PPARalpha in its regulation, wild-type and PPARalpha knockout (PPARalpha-/-) mice were fed a phytol-enriched diet or, for comparison, a diet enriched with Wy-14,643, a synthetic PPARalpha agonist. After the phytol-enriched diet, phytol could only be detected in small intestine, the site of uptake, and liver. Upon longer duration of the diet, the level of the (E)-isomer of phytol increased significantly in the liver of PPARalpha-/- mice compared with wild-type mice. Activity measurements of the enzymes involved in phytol metabolism showed that treatment with a PPARalpha agonist resulted in a PPARalpha-dependent induction of at least two steps of the phytol degradation pathway in liver. Furthermore, the enzymes involved showed a higher activity toward the (E)-isomer than the (Z)-isomer of their respective substrates, indicating a stereospecificity toward the metabolism of (E)-phytol. In conclusion, the results described here show that the conversion of phytol to phytanic acid is regulated via PPARalpha and is specific for the breakdown of (E)-phytol.

Acyl-CoA dehydrogenase 9 (ACAD 9) is the long-chain acyl-CoA dehydrogenase in human embryonic and fetal brain.

We recently reported the expression and activity of several fatty acid oxidation enzymes in human embryonic and fetal tissues including brain and spinal cord. Liver and heart showed expression of both very long-chain acyl-CoA dehydrogenase (VLCAD) and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) mRNA. However, while mRNA expression of LCHAD could be clearly detected in the retina and spinal cord, expression of VLCAD mRNA was low to undetectable in these tissues. Nevertheless, abundant acyl-CoA dehydrogenase (ACAD) activity was detected with palmitoyl-CoA as substrate in fetal central nervous tissue. These conflicting data suggested the presence of a different long-chain ACAD in human embryonic and fetal brain. In this study, using in situ hybridization as well as enzymatic studies, we identified acyl-CoA dehydrogenase 9 (ACAD 9) as the long-chain ACAD in human embryonic and fetal central nervous tissue. Until now, no clinical signs and symptoms of central nervous system involvement have been reported in VLCAD deficiency. A novel long-chain FAO defect, i.e., ACAD 9 deficiency with only central nervous system involvement, could, if not lethal during intra uterine development, easily escape proper diagnosis, since probably no classical signs and symptoms of FAO deficiency will be observed. Screening for ACAD 9 deficiency in patients with undefined neurological symptoms and/or impairment in neurological development of unknown origin is necessary to establish if ACAD 9 deficiency exists as a separate disease entity.

Carnitine supplementation induces long-chain acylcarnitine production--studies in the VLCAD-deficient mouse.

Carnitine supplementation does not affect carnitine concentrations in tissues of wild-type and very long-chain acyl-CoA dehydrogenase-deficient mice, but results in an increase in long-chain acylcarnitine production.

Peroxisomal trans-2-enoyl-CoA reductase is involved in phytol degradation.

Phytol is a naturally occurring precursor of phytanic acid. The last step in the conversion of phytol to phytanoyl-CoA is the reduction of phytenoyl-CoA mediated by an, as yet, unidentified enzyme. A candidate for this reaction is a previously described peroxisomal trans-2-enoyl-CoA reductase (TER). To investigate this, human TER was expressed in E. coli as an MBP-fusion protein. The purified recombinant protein was shown to have high reductase activity towards trans-phytenoyl-CoA, but not towards the peroxisomal beta-oxidation intermediates C24:1-CoA and pristenoyl-CoA. In conclusion, our results show that human TER is responsible for the reduction of phytenoyl-CoA to phytanoyl-CoA in peroxisomes.

Bezafibrate increases very-long-chain acyl-CoA dehydrogenase protein and mRNA expression in deficient fibroblasts and is a potential therapy for fatty acid oxidation disorders.

Inherited defect in very-long-chain acyl-CoA dehydrogenase (VLCAD), a mitochondrial enzyme catalyzing the initial step of long-chain fatty acid beta-oxidation (FAO), is one of the most frequent FAO enzyme defects. VLCAD deficiency is associated with clinical manifestations varying in severity, tissue involvement and age of onset. The molecular basis of VLCAD deficiency has been elucidated but therapeutic approaches are quite limited. In this study, we tested the hypothesis that fibrates, acting as agonist of peroxisome proliferator-activated receptors (PPARs), might stimulate FAO in VLCAD-deficient cells. We demonstrate that addition of bezafibrate or fenofibric acid in the culture medium induced a dose-dependent (up to 3-fold) increase in palmitate oxidation capacities in cells from patients with the myopathic form of VLCAD deficiency, but not in cells from severely affected patients. Complete normalization of cell FAO capacities could be achieved after exposure to 500 microm bezafibrate for 48 h. Cell therapy of VLCAD deficiency was related to drug-induced increases in VLCAD mRNA (+44 to +150%; P<0.001), protein (1.5-2-fold) and residual enzyme activity (up to 7.7-fold) in patient cells. Bezafibrate also diminished the production of toxic long-chain acylcarnitines by 90% in cells harboring moderate VLCAD deficiency. Finally, real-time PCR studies indicated that bezafibrate potentially stimulated gene expression of other enzymes in the beta-oxidation pathway. These data highlight the potential of fibrates in the correction of inborn FAO defects, as most mutations associated with these defects are compatible with the synthesis of a mutant protein with variable levels of residual enzyme activity.

A phytol-enriched diet induces changes in fatty acid metabolism in mice both via PPARalpha-dependent and -independent pathways.

Branched-chain fatty acids (such as phytanic and pristanic acid) are ligands for the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARalpha) in vitro. To investigate the effects of these physiological compounds in vivo, wild-type and PPARalpha-deficient (PPARalpha-/-) mice were fed a phytol-enriched diet. This resulted in increased plasma and liver levels of the phytol metabolites phytanic and pristanic acid. In wild-type mice, plasma fatty acid levels decreased after phytol feeding, whereas in PPARalpha-/- mice, the already elevated fatty acid levels increased. In addition, PPARalpha-/- mice were found to be carnitine deficient in both plasma and liver. Dietary phytol increased liver free carnitine in wild-type animals but not in PPARalpha-/- mice. Investigation of carnitine biosynthesis revealed that PPARalpha is likely involved in the regulation of carnitine homeostasis. Furthermore, phytol feeding resulted in a PPARalpha-dependent induction of various peroxisomal and mitochondrial beta-oxidation enzymes. In addition, a PPARalpha-independent induction of catalase, phytanoyl-CoA hydroxylase, carnitine octanoyltransferase, peroxisomal 3-ketoacyl-CoA thiolase, and straight-chain acyl-CoA oxidase was observed. In conclusion, branched-chain fatty acids are physiologically relevant ligands of PPARalpha in mice. These findings are especially relevant for disorders in which branched-chain fatty acids accumulate, such as Refsum disease and peroxisome biogenesis disorders.

High activity of fatty acid oxidation enzymes in human placenta: implications for fetal-maternal disease.

As the human fetus and placenta are considered to be primarily dependent on glucose oxidation for energy metabolism, the cause of the remarkable association between severe maternal pregnancy complications and the carriage of a fetus with an inborn error of mitochondrial long-chain fatty acid oxidation (FAO) has remained obscure. We analysed human term placenta and chorionic villus samples for the activities of a variety of enzymes involved in FAO, and compared the results with those obtained in human liver. All enzymes were found to be expressed, with a very high activity of two enzymes involved in the metabolism of long-chain fatty acids (CPT2 and VLCAD), whereas the activity of medium-chain acyl-CoA dehydrogenase (MCAD) was found to be low, when compared to liver. These results suggest that fatty acid oxidation may play an important role in energy generation in human placenta, and that a deficiency in the placental oxidation of long-chain FAO may result in placental dysfunction, thus causing gestational complications.

2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency in a 23-year-old man.

2-Methyl-3-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.178) deficiency is a recently described defect of isoleucine catabolism. The disorder is characterized by normal early development followed by a progressive loss of mental and motor skills. Deterioration may be rapid or may follow a slower decline with a possible stabilization of the disorder on a low-protein diet and appropriate medication. We report a 23-year-old man with 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency with a very mild clinical course. He had apparently normal early development and remained relatively well until the age of 6 years, when he contracted measles. Following this illness, his motor skills and school progress deteriorated. At 15 years he had significant dysarthria, and generalized rigidity with some dystonic and unusual posturing. He was then treated with a low-protein high-carbohydrate diet with a good response in terms of balance and gait. At 18 years he was given benzhexol (Artane), increased slowly from 2 mg to 6 mg daily, resulting in improvement in tremor and dystonia. At 23 years he can dress himself and works in sheltered employment but remains severely dysarthric.