3-Hydroxyisobutyric acid dehydrogenase deficiency: Expanding the clinical spectrum and quantitation of D- and L-3-Hydroxyisobutyric acid by an LC-MS/MS method.

A deficiency of 3-hydroxyisobutyric acid dehydrogenase (HIBADH) has been recently identified as a cause of primary 3-hydroxyisobutyric aciduria in two siblings; the only previously recognized primary cause had been a deficiency of methylmalonic semialdehyde dehydrogenase, the enzyme that is immediately downstream of HIBADH in the valine catabolic pathway and is encoded by the ALDH6A1 gene. Here we report on three additional patients from two unrelated families who present with marked and persistent elevations of urine L-3-hydroxyisobutyric acid (L-3HIBA) and a range of clinical findings. Molecular genetic analyses revealed novel, homozygous variants in the HIBADH gene that are private within each family. Evidence for pathogenicity of the identified variants is presented, including enzymatic deficiency of HIBADH in patient fibroblasts. This report describes new variants in HIBADH as an underlying cause of primary 3-hydroxyisobutyric aciduria and expands the clinical spectrum of this recently identified inborn error of valine metabolism. Additionally, we describe a quantitative method for the measurement of D- and L-3HIBA in plasma and urine and present the results of a valine restriction therapy in one of the patients.

Thermo-sensitive mitochondrial trifunctional protein deficiency presenting with episodic myopathy.

Mitochondrial trifunctional protein (MTP) is involved in long-chain fatty acid ß-oxidation (lcFAO). Deficiency of one or more of the enzyme activities as catalyzed by MTP causes generalized MTP deficiency (MTPD), long-chain hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), or long-chain ketoacyl-CoA thiolase deficiency (LCKATD). When genetic variants result in thermo-sensitive enzymes, increased body temperature (e.g. fever) can reduce enzyme activity and be a risk factor for clinical decompensation. This is the first description of five patients with a thermo-sensitive MTP deficiency. Clinical and genetic information was obtained from clinical files. Measurement of LCHAD and LCKAT activities, lcFAO-flux studies and palmitate loading tests were performed in skin fibroblasts cultured at 37°C and 40°C. In all patients (four MTPD, one LCKATD), disease manifested during childhood (manifestation age: 2-10 years) with myopathic symptoms triggered by fever or exercise. In four patients, signs of retinopathy or neuropathy were present. Plasma long-chain acylcarnitines were normal or slightly increased. HADHB variants were identified (at age: 6-18 years) by whole exome sequencing or gene panel analyses. At 37°C, LCHAD and LCKAT activities were mildly impaired and lcFAO-fluxes were normal. Remarkably, enzyme activities and lcFAO-fluxes were markedly diminished at 40°C. Preventive (dietary) measures improved symptoms for most. In conclusion, all patients with thermo-sensitive MTP deficiency had a long diagnostic trajectory and both genetic and enzymatic testing were required for diagnosis. The frequent absence of characteristic acylcarnitine abnormalities poses a risk for a diagnostic delay. Given the positive treatment effects, upfront genetic screening may be beneficial to enhance early recognition.

An autosomal dominant neurological disorder caused by de novo variants in FAR1 resulting in uncontrolled synthesis of ether lipids.

PURPOSE: In this study we investigate the disease etiology in 12 patients with de novo variants in FAR1 all resulting in an amino acid change at position 480 (p.Arg480Cys/His/Leu). METHODS: Following next-generation sequencing and clinical phenotyping, functional characterization was performed in patients' fibroblasts using FAR1 enzyme analysis, FAR1 immunoblotting/immunofluorescence, and lipidomics. RESULTS: All patients had spastic paraparesis and bilateral congenital/juvenile cataracts, in most combined with speech and gross motor developmental delay and truncal hypotonia. FAR1 deficiency caused by biallelic variants results in defective ether lipid synthesis and plasmalogen deficiency. In contrast, patients' fibroblasts with the de novo FAR1 variants showed elevated plasmalogen levels. Further functional studies in fibroblasts showed that these variants cause a disruption of the plasmalogen-dependent feedback regulation of FAR1 protein levels leading to uncontrolled ether lipid production. CONCLUSION: Heterozygous de novo variants affecting the Arg480 residue of FAR1 lead to an autosomal dominant disorder with a different disease mechanism than that of recessive FAR1 deficiency and a diametrically opposed biochemical phenotype. Our findings show that for patients with spastic paraparesis and bilateral cataracts, FAR1 should be considered as a candidate gene and added to gene panels for hereditary spastic paraplegia, cerebral palsy, and juvenile cataracts.

The Galactose Index measured in fibroblasts of GALT deficient patients distinguishes variant patients detected by newborn screening from patients with classical phenotypes.

BACKGROUND: The high variability in clinical outcome of patients with Classical Galactosemia (CG) is poorly understood and underlines the importance of prognostic biomarkers, which are currently lacking. The aim of this study was to investigate if residual galactose metabolism capacity is associated with clinical and biochemical outcomes in CG patients with varying geno- and phenotypes. METHODS: Galactose Metabolite Profiling (GMP) was used to determine residual galactose metabolism in fibroblasts of CG patients. The association between the galactose index (GI) defined as the ratio of the measured metabolites [U13C]Gal-1-P/ [13C6]UDP-galactose, and both intellectual and neurological outcome and galactose-1-phosphate (Gal-1-P) levels was investigated. RESULTS: GMP was performed in fibroblasts of 28 patients and 3 control subjects. The GI of the classical phenotype patients (n = 22) was significantly higher than the GI of four variant patients detected by newborn screening (NBS) (p = .002), two homozygous p.Ser135Leu patients (p = .022) and three controls (p = .006). In the classical phenotype patients, 13/18 (72%) had a poor intellectual outcome (IQ < 85) and 6/12 (50%) had a movement disorder. All the NBS detected variant patients (n = 4) had a normal intellectual outcome (IQ ≥ 85) and none of them has a movement disorder. In the classical phenotype patients, there was no significant difference in GI between patients with a poor and normal clinical outcome. The NBS detected variant patients had significantly lower GI levels and thus higher residual galactose metabolism than patients with classical phenotypes. There was a clear correlation between Gal-1-P levels in erythrocytes and the GI (p = .001). CONCLUSIONS: The GI was able to distinguish CG patients with varying geno- and phenotypes and correlated with Gal-1-P. The data of the NBS detected variant patients demonstrated that a higher residual galactose metabolism may result in a more favourable clinical outcome. Further research is needed to enable individual prognostication and treatment in all CG patients.

Mutations in PCYT2 disrupt etherlipid biosynthesis and cause a complex hereditary spastic paraplegia.

CTP:phosphoethanolamine cytidylyltransferase (ET), encoded by PCYT2, is the rate-limiting enzyme for phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. Phosphatidylethanolamine is one of the most abundant membrane lipids and is particularly enriched in the brain. We identified five individuals with biallelic PCYT2 variants clinically characterized by global developmental delay with regression, spastic para- or tetraparesis, epilepsy and progressive cerebral and cerebellar atrophy. Using patient fibroblasts we demonstrated that these variants are hypomorphic, result in altered but residual ET protein levels and concomitant reduced enzyme activity without affecting mRNA levels. The significantly better survival of hypomorphic CRISPR-Cas9 generated pcyt2 zebrafish knockout compared to a complete knockout, in conjunction with previously described data on the Pcyt2 mouse model, indicates that complete loss of ET function may be incompatible with life in vertebrates. Lipidomic analysis revealed profound lipid abnormalities in patient fibroblasts impacting both neutral etherlipid and etherphospholipid metabolism. Plasma lipidomics studies also identified changes in etherlipids that have the potential to be used as biomarkers for ET deficiency. In conclusion, our data establish PCYT2 as a disease gene for a new complex hereditary spastic paraplegia and confirm that etherlipid homeostasis is important for the development and function of the brain.

A mutation creating an upstream translation initiation codon in SLC22A5 5'UTR is a frequent cause of primary carnitine deficiency.

Primary carnitine deficiency is caused by a defect in the active cellular uptake of carnitine by Na+ -dependent organic cation transporter novel 2 (OCTN2). Genetic diagnostic yield for this metabolic disorder has been relatively low, suggesting that disease-causing variants are missed. We Sanger sequenced the 5' untranslated region (UTR) of SLC22A5 in individuals with possible primary carnitine deficiency in whom no or only one mutant allele had been found. We identified a novel 5'-UTR c.-149G>A variant which we characterized by expression studies with reporter constructs in HeLa cells and by carnitine-transport measurements in fibroblasts using a newly developed sensitive assay based on tandem mass spectrometry. This variant, which we identified in 57 of 236 individuals of our cohort, introduces a functional upstream out-of-frame translation initiation codon. We show that the codon suppresses translation from the wild-type ATG of SLC22A5, resulting in reduced OCTN2 protein levels and concomitantly lower transport activity. With an allele frequency of 24.2% the c.-149G>A variant is the most frequent cause of primary carnitine deficiency in our cohort and may explain other reported cases with an incomplete genetic diagnosis. Individuals carrying this variant should be clinically re-evaluated and monitored to determine if this variant has clinical consequences.

Mutated SUCLG1 causes mislocalization of SUCLG2 protein, morphological alterations of mitochondria and an early-onset severe neurometabolic disorder.

Succinate-CoA ligase (SUCL) is a heterodimer consisting of an alpha subunit encoded by SUCLG1, and a beta subunit encoded by either SUCLA2 or SUCLG2 catalyzing an ATP- or GTP-forming reaction, respectively, in the mitochondrial matrix. The deficiency of this enzyme represents an encephalomyopathic form of mtDNA depletion syndromes. We describe the fatal clinical course of a female patient with a pathogenic mutation in SUCLG1 (c.626C > A, p.Ala209Glu) heterozygous at the genomic DNA level, but homozygous at the transcriptional level. The patient exhibited early-onset neurometabolic abnormality culminating in severe brain atrophy and dystonia leading to death by the age of 3.5 years. Urine and plasma metabolite profiling was consistent with SUCL deficiency which was confirmed by enzyme analysis and lack of mitochondrial substrate-level phosphorylation (mSLP) in skin fibroblasts. Oxygen consumption- but not extracellular acidification rates were altered only when using glutamine as a substrate, and this was associated with mild mtDNA depletion and no changes in ETC activities. Immunoblot analysis revealed no detectable levels of SUCLG1, while SUCLA2 and SUCLG2 protein expressions were largely reduced. Confocal imaging of triple immunocytochemistry of skin fibroblasts showed that SUCLG2 co-localized only partially with the mitochondrial network which otherwise exhibited an increase in fragmentation compared to control cells. Our results outline the catastrophic consequences of the mutated SUCLG1 leading to strongly reduced SUCL activity, mSLP impairment, mislocalization of SUCLG2, morphological alterations in mitochondria and clinically to a severe neurometabolic disease, but in the absence of changes in mtDNA levels or respiratory complex activities.

Prediction of disease severity in multiple acyl-CoA dehydrogenase deficiency: A retrospective and laboratory cohort study.

Multiple acyl-CoA dehydrogenase deficiency (MADD) is an ultra-rare inborn error of mitochondrial fatty acid oxidation (FAO) and amino acid metabolism. Individual phenotypes and treatment response can vary markedly. We aimed to identify markers that predict MADD phenotypes. We performed a retrospective nationwide cohort study; then developed an MADD-disease severity scoring system (MADD-DS3) based on signs and symptoms with weighed expert opinions; and finally correlated phenotypes and MADD-DS3 scores to FAO flux (oleate and myristate oxidation rates) and acylcarnitine profiles after palmitate loading in fibroblasts. Eighteen patients, diagnosed between 1989 and 2014, were identified. The MADD-DS3 entails enumeration of eight domain scores, which are calculated by averaging the relevant symptom scores. Lifetime MADD-DS3 scores of patients in our cohort ranged from 0 to 29. FAO flux and [U-13 C]C2-, C5-, and [U-13 C]C16-acylcarnitines were identified as key variables that discriminated neonatal from later onset patients (all P < .05) and strongly correlated to MADD-DS3 scores (oleate: r = -.86; myristate: r = -.91; [U-13 C]C2-acylcarnitine: r = -.96; C5-acylcarnitine: r = .97; [U-13 C]C16-acylcarnitine: r = .98, all P < .01). Functional studies in fibroblasts were found to differentiate between neonatal and later onset MADD-patients and were correlated to MADD-DS3 scores. Our data may improve early prediction of disease severity in order to start (preventive) and follow-up treatment appropriately. This is especially relevant in view of the inclusion of MADD in population newborn screening programs.

Identification of enzymes involved in oxidation of phenylbutyrate.

In recent years the short-chain fatty acid, 4-phenylbutyrate (PB), has emerged as a promising drug for various clinical conditions. In fact, PB has been Food and Drug Administration-approved for urea cycle disorders since 1996. PB is more potent and less toxic than its metabolite, phenylacetate (PA), and is not just a pro-drug for PA, as was initially assumed. The metabolic pathway of PB, however, has remained unclear. Therefore, we set out to identify the enzymes involved in the ß-oxidation of PB. We used cells deficient in specific steps of fatty acid ß-oxidation and ultra-HPLC to measure which enzymes were able to convert PB or its downstream products. We show that the first step in PB oxidation is catalyzed solely by the enzyme, medium-chain acyl-CoA dehydrogenase. The second (hydration) step can be catalyzed by all three mitochondrial enoyl-CoA hydratase enzymes, i.e., short-chain enoyl-CoA hydratase, long-chain enoyl-CoA hydratase, and 3-methylglutaconyl-CoA hydratase. Enzymes involved in the third step include both short- and long-chain 3-hydroxyacyl-CoA dehydrogenase. The oxidation of PB is completed by only one enzyme, i.e., long-chain 3-ketoacyl-CoA thiolase. Taken together, the enzymatic characteristics of the PB degradative pathway may lead to better dose finding and limiting the toxicity of this drug.

Monocarboxylate transporter 1 deficiency and ketone utilization.

Ketoacidosis is a potentially lethal condition caused by the imbalance between hepatic production and extrahepatic utilization of ketone bodies. We performed exome sequencing in a patient with recurrent, severe ketoacidosis and identified a homozygous frameshift mutation in the gene encoding monocarboxylate transporter 1 (SLC16A1, also called MCT1). Genetic analysis in 96 patients suspected of having ketolytic defects yielded seven additional inactivating mutations in MCT1, both homozygous and heterozygous. Mutational status was found to be correlated with ketoacidosis severity, MCT1 protein levels, and transport capacity. Thus, MCT1 deficiency is a novel cause of profound ketoacidosis; the present work suggests that MCT1-mediated ketone-body transport is needed to maintain acid-base balance.

A novel UPLC-MS/MS based method to determine the activity of N-acetylglutamate synthase in liver tissue.

BACKGROUND: N-acetylglutamate synthase (NAGS) plays a key role in the removal of ammonia via the urea cycle by catalyzing the synthesis of N-acetylglutamate (NAG), the obligatory cofactor in the carbamyl phosphate synthetase 1 reaction. Enzymatic analysis of NAGS in liver homogenates has remained insensitive and inaccurate, which prompted the development of a novel method. METHODS: UPLC-MS/MS was used in conjunction with stable isotope (N-acetylglutamic-2,3,3,4,4-d5 acid) dilution for the quantitative detection of NAG produced by the NAGS enzyme. The assay conditions were optimized using purified human NAGS and the optimized enzyme conditions were used to measure the activity in mouse liver homogenates. RESULTS: A low signal-to-noise ratio in liver tissue samples was observed due to non-enzymatic formation of N-acetylglutamate and low specific activity, which interfered with quantitative analysis. Quenching of acetyl-CoA immediately after the incubation circumvented this analytical difficulty and allowed accurate and sensitive determination of mammalian NAGS activity. The specificity of the assay was validated by demonstrating a complete deficiency of NAGS in liver homogenates from Nags -/- mice. CONCLUSION: The novel NAGS enzyme assay reported herein can be used for the diagnosis of inherited NAGS deficiency and may also be of value in the study of secondary hyperammonemia present in various inborn errors of metabolism as well as drug treatment.

Impaired amino acid metabolism contributes to fasting-induced hypoglycemia in fatty acid oxidation defects.

The importance of mitochondrial fatty acid ß-oxidation (FAO) as a glucose-sparing process is illustrated by patients with inherited defects in FAO, who may present with life-threatening fasting-induced hypoketotic hypoglycemia. It is unknown why peripheral glucose demand outpaces hepatic gluconeogenesis in these patients. In this study, we have systematically addressed the fasting response in long-chain acyl-CoA dehydrogenase-deficient (LCAD KO) mice. We demonstrate that the fasting-induced hypoglycemia in LCAD KO mice was initiated by an increased glucose requirement in peripheral tissues, leading to rapid hepatic glycogen depletion. Gluconeogenesis did not compensate for the increased glucose demand, which was not due to insufficient hepatic glucogenic capacity but rather caused by a shortage in the supply of glucogenic precursors. This shortage in supply was explained by a suppressed glucose-alanine cycle, decreased branched-chain amino acid metabolism and ultimately impaired protein mobilization. We conclude that during fasting, FAO not only serves to spare glucose but is also indispensable for amino acid metabolism, which is essential for the maintenance of adequate glucose production.

Fatty acid oxidation flux predicts the clinical severity of VLCAD deficiency.

PURPOSE: Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD) is an inherited disorder of mitochondrial long-chain fatty acid ß-oxidation (LC-FAO) and is included in many newborn screening (NBS) programs worldwide. Patients may present with hypoketotic hypoglycemia, cardiomyopathy, and/or myopathy, but clinical severity varies widely and the clinical outcome is unpredictable. We investigated predictive markers that may determine clinical severity. METHODS: We developed a clinical severity score (CSS), which was determined for 13 Dutch patients with VLCADD, all of whom were diagnosed before the introduction of VLCADD in NBS to prevent bias from early diagnosis. In cultured skin fibroblasts from these patients, we measured LC-FAO flux (the rate of oleate oxidation), VLCAD activity, and acylcarnitine profiles following palmitate loading. RESULTS: The strongest correlation (r = 0.93; P < 0.0001) was observed between LC-FAO flux and the CSS. VLCAD activity measurement and the C14/C16-to-acylcarnitine ratio correlated much less. A median LC-FAO flux of 6% of control values (range 5.6-6.8%) was associated with cardiomyopathy (P < 0.01), and 32.4% (range 5.6-50.5%) was associated with myopathy (P < 0.05). CONCLUSION: Our results demonstrate a very strong correlation between LC-FAO flux in fibroblasts and the clinical severity of VLCADD. LC-FAO flux measurements may thus predict whether patients are likely to develop symptoms.

Metabolite studies in HIBCH and ECHS1 defects: Implications for screening.

3-Hydroxyisobutyryl-CoA hydrolase deficiency (HIBCHD) is a rare inborn error of the valine catabolic pathway associated with Leigh-like disease. We report a female patient who presented at the age of 5months with hypotonia, developmental delay and cerebral atrophy on MRI. Pyruvate dehydrogenase deficiency was initially suspected and decreased activity was shown in fibroblasts. Urine tandem mass spectrometry screening showed large increases in the cysteine conjugate of methacrylate previously described in HIBCHD. 3-hydroxyisobutyryl-CoA hydrolase activity in fibroblasts was below the limit of detection of the enzymatic assay and two novel HIBCH mutations were identified (c.[129dupA];[1033G>A]). Urine metabolite investigations also showed increases in 3-hydroxyisobutyryl carnitine, 2,3-dihydroxy-2-methylbutyrate and several metabolites indicating accumulation and subsequent metabolism of methacrylyl-CoA and acryloyl-CoA. The metabolites derived from acryloyl-CoA were also increased in patients with inborn errors of propionyl-CoA metabolism, indicating the involvement of a secondary propionyl-CoA pathway utilising 3-hydroxyisobutyryl-CoA hydrolase. With the exception of 3-hydroxyisobutyryl carnitine, the metabolite abnormalities were essentially the same as those observed in patients with ECHS1 mutations, a recently described disorder that also affects valine metabolism. Our findings demonstrate the benefits of urine tandem mass spectrometry screening for diagnosing HIBCH and ECHS1 defects and that propionate metabolism may play a role in their pathogenesis. These disorders should be considered during the differential diagnosis of Leigh like-diseases and hypotonia.

Identification and characterization of Eci3, a murine kidney-specific Δ3,Δ2-enoyl-CoA isomerase.

Oxidation of unsaturated fatty acids requires the action of auxiliary enzymes, such as Δ(3),Δ(2)-enoyl-CoA isomerases. Here we describe a detailed biochemical, molecular, histological, and evolutionary characterization of Eci3, the fourth member of the mammalian enoyl-CoA isomerase family. Eci3 specifically evolved in rodents after gene duplication of Eci2. Eci3 is with 79% identity homologous to Eci2 and contains a peroxisomal targeting signal type 1. Subcellular fractionation of mouse kidney and immunofluorescence studies revealed a specific peroxisomal localization for Eci3. Expression studies showed that mouse Eci3 is almost exclusively expressed in kidney. By using immunohistochemistry, we found that Eci3 is not only expressed in cells of the proximal tubule, but also in a subset of cells in the tubulointerstitium and the glomerulus. In vitro, Eci3 catalyzed the isomerization of trans-3-nonenoyl-CoA to trans-2-nonenoyl-CoA equally efficient as Eci2, suggesting a role in oxidation of unsaturated fatty acids. However, in contrast to Eci2, in silico gene coexpression and enrichment analysis for Eci3 in kidney did not yield carboxylic acid metabolism, but diverse biological functions, such as ion transport (P=7.1E-3) and tissue morphogenesis (P=1.0E-3). Thus, Eci3 picked up a novel and unexpected role in kidney function during rodent evolution.

Inhibition of 3-methylcrotonyl-CoA carboxylase explains the increased excretion of 3-hydroxyisovaleric acid in valproate-treated patients.

BACKGROUND: Valproic acid (VPA) is a widely used anticonvulsant drug which affects mitochondrial metabolism including the catabolism of fatty acids and branched-chain amino acids. AIMS: To elucidate the effect of valproate on the leucine pathway through a targeted metabolomics approach and the evaluation of the effects of valproate on the activity of biotinidase and 3-methylcrotonyl-CoA carboxylase (3MCC). METHODS: Urine organic acid analysis was performed in patients under VPA therapy and healthy controls using gas-chromatography/mass spectrometry (GC-MS). Biotinidase activity was determined in plasma samples of both groups using an optimized spectrophotometric assay. After immunoprecipitation of short-chain enoyl-CoA hydratase (crotonase, ECHS1), 3MCC activity was measured in human liver homogenate using high-performance liquid chromatography (HPLC), in the absence and presence of valproyl-CoA. RESULTS: The levels of 3-hydroxyisovaleric acid (3OH-IVA), one secondary metabolite of the leucine pathway, were significantly elevated in human urine after VPA treatment. Biotinidase activity in plasma samples ranged from very low to normal levels in treated patients as compared with controls. Enzyme activity measurements revealed inhibition of 3-methylcrotonyl-CoA carboxylase by valproyl-CoA (IC(50) = 1.36 mM). Furthermore, we show that after complete immunoprecipitation of crotonase in a human liver homogenate, 3-hydroxyisovaleryl-CoA is not formed. DISCUSSION: Our results suggest the interference of VPA with the activity of 3MCC through a potential cumulative effect: direct inhibition of the enzyme activity by the drug metabolite valproyl-CoA and the inhibition of biotinidase by valproate and/or its metabolites. These interactions may be associated with the skin rash and hair loss which are side effects often reported in VPA-treated patients.

Rosuvastatin lowers coenzyme Q10 levels, but not mitochondrial adenosine triphosphate synthesis, in children with familial hypercholesterolemia.

OBJECTIVE: To investigate whether statin therapy affects coenzyme Q10 (CoQ10) status in children with heterozygous familial hypercholesterolemia (FH). STUDY DESIGN: Samples were obtained at baseline (treatment naïve) and after dose titration with rosuvastatin, aiming for a low-density lipoprotein cholesterol level of 110 mg/dL. Twenty-nine patients were treated with 5, 10, or 20 mg of rosuvastatin for a mean period of 29 weeks. RESULTS: We found a significant (32%) decrease in peripheral blood mononuclear cell (PBMC) CoQ10 level (P = .02), but no change in PBMC adenosine triphosphate synthesis (P = .60). Uncorrected plasma CoQ10 values were decreased significantly, by 45% (P < .01). In contrast, ratios of plasma CoQ10/total cholesterol and CoQ10/low-density lipoprotein cholesterol remained equal during treatment. CONCLUSIONS: In children with FH, rosuvastatin causes a significant decrease in cellular PBMC CoQ10 status but does not affect mitochondrial adenosine triphosphate synthesis in children with FH. Further studies should address whether (rare) side effects of statin therapy could be explained by a deterioration in CoQ10 status.

Role of isovaleryl-CoA dehydrogenase and short branched-chain acyl-CoA dehydrogenase in the metabolism of valproic acid: implications for the branched-chain amino acid oxidation pathway.

Many biological systems including the oxidative catabolic pathway for branched-chain amino acids (BCAAs) are affected in vivo by valproate therapy. In this study, we investigated the potential effect of valproic acid (VPA) and some of its metabolites on the metabolism of BCAAs. In vitro studies were performed using isovaleryl-CoA dehydrogenase (IVD), isobutyryl-CoA dehydrogenase (IBD), and short branched-chain acyl-CoA dehydrogenase (SBCAD), enzymes involved in the degradation pathway of leucine, valine, and isoleucine. The enzymatic activities of the three purified human enzymes were measured using optimized high-performance liquid chromatography procedures, and the respective kinetic parameters were determined in the absence and presence of VPA and the corresponding CoA and dephosphoCoA conjugates. Valproyl-CoA and valproyl-dephosphoCoA inhibited IVD activity significantly by a purely competitive mechanism with K(i) values of 74 ± 4 and 170 ± 12 µM, respectively. IBD activity was not affected by any of the tested VPA esters. However, valproyl-CoA did inhibit SBCAD activity by a purely competitive mechanism with a K(i) of 249 ± 29 µM. In addition, valproyl-dephosphoCoA inhibited SBCAD activity via a distinct mechanism (K(i) = 511 ± 96 µM) that appeared to be of the mixed type. Furthermore, we show that both SBCAD and IVD are active, using valproyl-CoA as a substrate. The catalytic efficiency of SBCAD turned out to be much higher than that of IVD, demonstrating that SBCAD is the most probable candidate for the first dehydrogenation step of VPA ß-oxidation. Our data explain some of the effects of valproate on the branched-chain amino acid metabolism and shed new light on the biotransformation pathway of valproate.

Toxic response caused by a misfolding variant of the mitochondrial protein short-chain acyl-CoA dehydrogenase.

BACKGROUND: Variations in the gene ACADS, encoding the mitochondrial protein short-chain acyl CoA-dehydrogenase (SCAD), have been observed in individuals with clinical symptoms. The phenotype of SCAD deficiency (SCADD) is very heterogeneous, ranging from asymptomatic to severe, without a clear genotype-phenotype correlation, which suggests a multifactorial disorder. The pathophysiological relevance of the genetic variations in the SCAD gene is therefore disputed, and has not yet been elucidated, which is an important step in the investigation of SCADD etiology. AIM: To determine whether the disease-associated misfolding variant of SCAD protein, p.Arg107Cys, disturbs mitochondrial function. METHODS: We have developed a cell model system, stably expressing either the SCAD wild-type protein or the misfolding SCAD variant protein, p.Arg107Cys (c.319 C > T). The model system was used for investigation of SCAD with respect to expression, degree of misfolding, and enzymatic SCAD activity. Furthermore, cell proliferation and expression of selected stress response genes were investigated as well as proteomic analysis of mitochondria-enriched extracts in order to study the consequences of p.Arg107Cys protein expression using a global approach. CONCLUSIONS: We found that expression of the p.Arg107Cys variant SCAD protein gives rise to inactive misfolded protein species, eliciting a mild toxic response manifested though a decreased proliferation rate and oxidative stress, as shown by an increased demand for the mitochondrial antioxidant SOD2. In addition, we found markers of apoptotic activity in the p.Arg107Cys expressing cells, which points to a possible pathophysiological role of this variant protein.