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Alpha-methylacyl-CoA racemase (AMACR) deficiency is a rare disorder of fatty acid metabolism which has recently been described in three adult cases. We have identified a further patient with clinical features of a relapsing encephalopathy, seizures and cognitive decline over a 40 year period. Biochemical studies revealed grossly elevated plasma pristanic acid levels, and a deficiency of AMACR in skin fibroblasts. Sequence analysis of AMACR cDNA identified a homozygous point mutation (c154T>C). This case adds to the phenotypic variation seen in this peroxisomal disorder and highlights the importance of screening for plasma pristanic acid levels in patients with unexplained relapsing encephalopathies. [ABSTRACT FROM AUTHOR]

Carnitine derivatives of disease-specific acyl-CoAs are the diagnostic hallmark for long-chain fatty acid β-oxidation disorders (lcFAOD), including carnitine shuttle deficiencies, very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD), long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) and mitochondrial trifunctional protein deficiency (MPTD). The exact consequence of accumulating lcFAO-intermediates and their influence on cellular lipid homeostasis is, however, still unknown. To investigate the fate and cellular effects of the accumulating lcFAO-intermediates and to explore the presence of disease-specific markers, we used tracer-based lipidomics with deuterium-labeled oleic acid (D9-C18:1) in lcFAOD patient-derived fibroblasts. In line with previous studies, we observed a trend towards neutral lipid accumulation in lcFAOD. In addition, we detected a direct connection between the chain length and patterns of (un)saturation of accumulating acylcarnitines and the various enzyme deficiencies. Our results also identified two disease-specific candidate biomarkers. Lysophosphatidylcholine(14:1) (LPC(14:1)) was specifically increased in severe VLCADD compared to mild VLCADD and control samples. This was confirmed in plasma samples showing an inverse correlation with enzyme activity, which was better than the classic diagnostic marker C14:1-carnitine. The second candidate biomarker was an unknown lipid class, which we identified as S-(3-hydroxyacyl)cysteamines. We hypothesized that these were degradation products of the CoA moiety of accumulating 3-hydroxyacyl-CoAs. S-(3-hydroxyacyl)cysteamines were significantly increased in LCHADD compared to controls and other lcFAOD, including MTPD. Our findings suggest extensive alternative lipid metabolism in lcFAOD and confirm that lcFAOD accumulate neutral lipid species. In addition, we present two disease-specific candidate biomarkers for VLCADD and LCHADD, that may have significant relevance for disease diagnosis, prognosis, and monitoring., (© 2024 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Medium chain acyl-CoA dehydrogenase (MCAD) deficiency (MCADD) is associated with ACADM gene mutations, leading to an impaired function and/or structure of MCAD. Importantly, after import into the mitochondria, MCAD must incorporate a molecule of flavin adenine dinucleotide (FAD) per subunit and assemble into tetramers. However, the effect of MCAD amino acid substitutions on FAD incorporation has not been investigated. Herein, the commonest MCAD variant (p.K304E) and 11 additional rare variants (p.Y48C, p.R55G, p.A88P, p.Y133C, p.A140T, p.D143V, p.G224R, p.L238F, p.V264I, p.Y372N, and p.G377V) were functionally and structurally characterized. Half of the studied variants presented a FAD content <65 % compared to the wild-type. Most of them were recovered as tetramers, except the p.Y372N (mainly as dimers). No correlation was found between the levels of tetramers and FAD content. However, a correlation between FAD content and the cofactor's affinity, proteolytic stability, thermostability, and thermal inactivation was established. We showed that the studied amino acid changes in MCAD may alter the substrate chain-length dependence and the interaction with electron-transferring-flavoprotein (ETF) necessary for a proper functioning electron transfer thus adding additional layers of complexity to the pathological effect of ACADM missense mutations. Although the majority of the variant MCADs presented an impaired capacity to retain FAD during their synthesis, some of them were structurally rescued by cofactor supplementation, suggesting that in the mitochondrial environment the levels and activity of those variants may be dependent of FAD's availability thus contributing for the heterogeneity of the MCADD phenotype found in patients presenting the same genotype., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

X-linked adrenoleukodystrophy (ALD) is a progressive neurodegenerative disease caused by mutations in ABCD1, the peroxisomal very long-chain fatty acid (VLCFA) transporter. ABCD1 deficiency results in accumulation of saturated VLCFAs. A drug screen using a phenotypic motor assay in a zebrafish ALD model identified chloroquine as the top hit. Chloroquine increased expression of stearoyl-CoA desaturase-1 (scd1), the enzyme mediating fatty acid saturation status, suggesting that a shift toward monounsaturated fatty acids relieved toxicity. In human ALD fibroblasts, chloroquine also increased SCD1 levels and reduced saturated VLCFAs. Conversely, pharmacological inhibition of SCD1 expression led to an increase in saturated VLCFAs, and CRISPR knockout of scd1 in zebrafish mimicked the motor phenotype of ALD zebrafish. Importantly, saturated VLCFAs caused ER stress in ALD fibroblasts, whereas monounsaturated VLCFA did not. In parallel, we used liver X receptor (LXR) agonists to increase SCD1 expression, causing a shift from saturated toward monounsaturated VLCFA and normalizing phospholipid profiles. Finally, Abcd1-/y mice receiving LXR agonist in their diet had VLCFA reductions in ALD-relevant tissues. These results suggest that metabolic rerouting of saturated to monounsaturated VLCFAs may alleviate lipid toxicity, a strategy that may be beneficial in ALD and other peroxisomal diseases in which VLCFAs play a key role.

3-Methylglutaconic aciduria (3-MGA-uria) syndromes comprise a heterogeneous group of diseases associated with mitochondrial membrane defects. Whole-exome sequencing identified compound heterozygous mutations in TIMM50 (c.[341 G>A];[805 G>A]) in a boy with West syndrome, optic atrophy, neutropenia, cardiomyopathy, Leigh syndrome, and persistent 3-MGA-uria. A comprehensive analysis of the mitochondrial function was performed in fibroblasts of the patient to elucidate the molecular basis of the disease. TIMM50 protein was severely reduced in the patient fibroblasts, regardless of the normal mRNA levels, suggesting that the mutated residues might be important for TIMM50 protein stability. Severe morphological defects and ultrastructural abnormalities with aberrant mitochondrial cristae organization in muscle and fibroblasts were found. The levels of fully assembled OXPHOS complexes and supercomplexes were strongly reduced in fibroblasts from this patient. High-resolution respirometry demonstrated a significant reduction of the maximum respiratory capacity. A TIMM50-deficient HEK293T cell line that we generated using CRISPR/Cas9 mimicked the respiratory defect observed in the patient fibroblasts; notably, this defect was rescued by transfection with a plasmid encoding the TIMM50 wild-type protein. In summary, we demonstrated that TIMM50 deficiency causes a severe mitochondrial dysfunction by targeting key aspects of mitochondrial physiology, such as the maintenance of proper mitochondrial morphology, OXPHOS assembly, and mitochondrial respiratory capacity., (© 2019 Wiley Periodicals, Inc.)

Barth syndrome (BTHS) is a rare X-linked condition resulting in abnormal mitochondria, cardioskeletal myopathy, and growth delay; however, the effects of BTHS on substrate metabolism regulation and their relationships with tissue function in humans are unknown. We sought to characterize glucose and fat metabolism during rest, submaximal exercise, and postexercise rest in children, adolescents, and young adults with BTHS and unaffected controls and examine their relationships with cardioskeletal energetics and function. Children/adolescents and young adults with BTHS (n = 29) and children/adolescent and young adult control participants (n = 28, total n = 57) underwent an infusion of 6'6'H2 glucose and U- 13 C palmitate and indirect calorimetry during rest, 30-minutes of moderate exercise (50% V ˙ O 2 peak ), and recovery. Cardiac function, cardioskeletal mitochondrial energetics, and exercise capacity were examined via echocardiography, 31 P magnetic resonance spectroscopy, and peak exercise testing, respectively. The glucose turnover rate was significantly higher in individuals with BTHS during rest (33.2 ± 9.8 vs 27.2 ± 8.1 μmol/kgFFM/min, P < .01) and exercise (34.7 ± 11.2 vs 29.5 ± 8.8 μmol/kgFFM/min, P < .05) and tended to be higher postexercise (33.7 ± 10.2 vs 28.8 ± 8.0 μmol/kgFFM/min, P < .06) compared to controls. Increases in total fat (-3.9 ± 7.5 vs 10.5 ± 8.4 μmol/kgFFM/min, P < .0001) and plasma fatty acid oxidation rates (0.0 ± 1.8 vs 5.1 ± 3.9 μmol/kgFFM/min, P < .0001) from rest to exercise were severely blunted in BTHS compared to controls. Conclusion: An inability to upregulate fat metabolism during moderate intensity exercise appears to be partially compensated by elevations in glucose metabolism. Derangements in fat and glucose metabolism are characteristic of the pathophysiology of BTHS. A severely blunted ability to upregulate fat metabolism during a modest level of physical activity is a defining pathophysiologic characteristic in children, adolescents, and young adults with BTHS., (© 2019 SSIEM.)

Mutations in the ABCD1 gene that encodes peroxisomal ABCD1 protein cause X-linked adrenoleukodystrophy (X-ALD), a rare neurodegenerative disorder. More than 70% of the patient fibroblasts with this missense mutation display either a lack or reduction of the ABCD1 protein because of posttranslational degradation. In this study, we analyzed the stability of the missense mutant ABCD1 proteins (p.A616T, p.R617H, and p.R660W) in X-ALD fibroblasts and found that the mutant ABCD1 protein p.A616T has the capacity to recover its function by incubating at low temperature. In the case of such a mutation, chemical compounds that stabilize mutant ABCD1 proteins could be therapeutic candidates. Here, we prepared CHO cell lines stably expressing ABCD1 proteins with a missense mutation in fusion with green fluorescent protein (GFP) at the C-terminal. The stability of each mutant ABCD1-GFP in CHO cells was similar to the corresponding mutant ABCD1 protein in X-ALD fibroblasts. Furthermore, it is of interest that the GFP at the C-terminal was degraded together with the mutant ABCD1 protein. These findings prompted us to use CHO cells expressing mutant ABCD1-GFP for a screening of chemical compounds that can stabilize the mutant ABCD1 protein. We established a fluorescence-based assay method for the screening of chemical libraries in an effort to find compounds that stabilize mutant ABCD1 proteins. The work presented here provides a novel approach to finding therapeutic compounds for X-ALD patients with missense mutations.

Barth syndrome (BTHS) is a rare X-linked disorder that is characterized by cardiac and skeletal myopathy, neutropenia and growth abnormalities. The disease is caused by mutations in the tafazzin (TAZ) gene encoding an enzyme involved in the acyl chain remodeling of the mitochondrial phospholipid cardiolipin (CL). Biochemically, this leads to decreased levels of mature CL and accumulation of the intermediate monolysocardiolipin (MLCL). At a cellular level, this causes mitochondrial fragmentation and reduced stability of the respiratory chain supercomplexes. However, the exact mechanism through which tafazzin deficiency leads to disease development remains unclear. We therefore aimed to elucidate the pathways affected in BTHS cells by employing proteomic and metabolic profiling assays. Complexome profiling of patient skin fibroblasts revealed significant effects for about 200 different mitochondrial proteins. Prominently, we found a specific destabilization of higher order oxidative phosphorylation (OXPHOS) supercomplexes, as well as changes in complexes involved in cristae organization and CL trafficking. Moreover, the key metabolic complexes 2-oxoglutarate dehydrogenase (OGDH) and branched-chain ketoacid dehydrogenase (BCKD) were profoundly destabilized in BTHS patient samples. Surprisingly, metabolic flux distribution assays using stable isotope tracer-based metabolomics did not show reduced flux through the TCA cycle. Overall, insights from analyzing the impact of TAZ mutations on the mitochondrial complexome provided a better understanding of the resulting functional and structural consequences and thus the pathological mechanisms leading to Barth syndrome., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

Aims: Mitochondrial fatty acid oxidation (FAO) is an important energy provider for cardiac work and changes in cardiac substrate preference are associated with different heart diseases. Carnitine palmitoyltransferase 1B (CPT1B) is thought to perform the rate limiting enzyme step in FAO and is inhibited by malonyl-CoA. The role of CPT1B in cardiac metabolism has been addressed by inhibiting or decreasing CPT1B protein or after modulation of tissue malonyl-CoA metabolism. We assessed the role of CPT1B malonyl-CoA sensitivity in cardiac metabolism., Methods and Results: We generated and characterized a knock in mouse model expressing the CPT1BE3A mutant enzyme, which has reduced sensitivity to malonyl-CoA. In isolated perfused hearts, FAO was 1.9-fold higher in Cpt1bE3A/E3A hearts compared with Cpt1bWT/WT hearts. Metabolomic, proteomic and transcriptomic analysis showed increased levels of malonylcarnitine, decreased concentration of CPT1B protein and a small but coordinated downregulation of the mRNA expression of genes involved in FAO in Cpt1bE3A/E3A hearts, all of which aim to limit FAO. In vivo assessment of cardiac function revealed only minor changes, cardiac hypertrophy was absent and histological analysis did not reveal fibrosis., Conclusions: Malonyl-CoA-dependent inhibition of CPT1B plays a crucial role in regulating FAO rate in the heart. Chronic elevation of FAO has a relatively subtle impact on cardiac function at least under baseline conditions.

Activation of brown adipose tissue (BAT) contributes to total body energy expenditure through energy dissipation as heat. Activated BAT increases the clearance of lipids and glucose from the circulation, but how BAT accommodates large influx of multiple substrates is not well defined. The purpose of this work was to assess the metabolic fluxes in brown adipocytes during β3-adrenergic receptor (β3-AR) activation.T37i murine preadipocytes were differentiated into brown adipocytes and we used Seahorse respirometry employing a set of specific substrate inhibitors in the presence or absence of β3-AR agonist CL316,243. The main substrate used by these brown adipocytes were fatty acids, which were oxidized equally during activation as well as during resting condition. [U- 13 C]-glucose tracer-based metabolomics revealed that the flux through the TCA cycle was enhanced and regulated by pyruvate dehydrogenase (PDH) activity. Based on 13 C-tracer incorporation in lipids, it appeared that most glucose was oxidized via TCA cycle activity, while some was utilized for glycerol-3-phosphate synthesis to replenish the triglyceride pool. Collectively, we show that while fatty acids are the main substrates for oxidation, glucose is also oxidized to meet the increased energy demand during short term β3-AR activation. PDH plays an important role in directing glucose carbons towards oxidation.

Peroxisomes play a crucial role in normal neurological development as exemplified by the devastating neurological consequences of a defect in the biogenesis of peroxisomes as in Zellweger syndrome. The underlying basis for the important role of peroxisomes in neurological development resides in the fact that peroxisomes catalyze a number of physiological functions, notably involving the metabolism of different lipids. Indeed, peroxisomes catalyse the beta-oxidative breakdown of certain fatty acids including: (1.) the very long-chain fatty acids C22:0, C24:0, and C26:0; (2.) pristanic acid and (3.) the bile acid intermediates di- and trihydroxycholestanoic acid which cannot be oxidized in mitochondria. Furthermore, peroxisomes catalyze the synthesis of a particular type of lipids, i.e. ether-linked phospholipids, which are highly abundant in brain, especially in myelin. The current state of knowledge with respect to the metabolic role of peroxisomes will be described in this paper with particular emphasis on the role of peroxisomes in brain., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The peroxisomal disorders (PDs) are a heterogeneous group of genetic diseases in man caused by an impairment in peroxisome biogenesis or one of the metabolic functions of peroxisomes. Thanks to the revolutionary technical developments in gene sequencing methods and their increased use in patient diagnosis, the field of genetic diseases in general and peroxisomal disorders in particular has dramatically changed in the last few years. Indeed, several novel peroxisomal disorders have been identified recently and in addition it has been realized that the phenotypic spectrum of patients affected by a PD keeps widening, which makes clinical recognition of peroxisomal patients increasingly difficult. Here, we describe these new developments and provide guidelines for the clinical and laboratory diagnosis of peroxisomal patients.

Background & Aims: Severe malnutrition in young children is associated with signs of hepatic dysfunction such as steatosis and hypoalbuminemia, but its etiology is unknown. Peroxisomes and mitochondria play key roles in various hepatic metabolic functions including lipid metabolism and energy production. To investigate the involvement of these organelles in the mechanisms underlying malnutrition-induced hepatic dysfunction we developed a rat model of malnutrition., Methods: Weanling rats were placed on a low protein or control diet (5% or 20% of calories from protein, respectively) for four weeks. Peroxisomal and mitochondrial structural features were characterized using immunofluorescence and electron microscopy. Mitochondrial function was assessed using high-resolution respirometry. A novel targeted quantitative proteomics method was applied to analyze 47 mitochondrial proteins involved in oxidative phosphorylation, tricarboxylic acid cycle and fatty acid β-oxidation pathways., Results: Low protein diet-fed rats developed hypoalbuminemia and hepatic steatosis, consistent with the human phenotype. Hepatic peroxisome content was decreased and metabolomic analysis indicated peroxisomal dysfunction. This was followed by changes in mitochondrial ultrastructure and increased mitochondrial content. Mitochondrial function was impaired due to multiple defects affecting respiratory chain complex I and IV, pyruvate uptake and several β-oxidation enzymes, leading to strongly reduced hepatic ATP levels. Fenofibrate supplementation restored hepatic peroxisome abundance and increased mitochondrial β-oxidation capacity, resulting in reduced steatosis and normalization of ATP and plasma albumin levels., Conclusions: Malnutrition leads to severe impairments in hepatic peroxisomal and mitochondrial function, and hepatic metabolic dysfunction. We discuss the potential future implications of our findings for the clinical management of malnourished children., Lay Summary: Severe malnutrition in children is associated with metabolic disturbances that are poorly understood. In order to study this further, we developed a malnutrition animal model and found that severe malnutrition leads to an impaired function of liver mitochondria which are essential for energy production and a loss of peroxisomes, which are important for normal liver metabolic function., (Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

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-d 5 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., (Copyright © 2016. Published by Elsevier Inc.)

Background: Recessive mutations in the 3-hydroxyisobutyryl-CoA hydrolase gene (HIBCH) are associated with a rare neurodegenerative disease that affects the basal ganglia. Most patients die during infancy or early childhood. Here we describe 5 adolescent and adult patients from 2 unrelated families, who presented with a movement disorder and MRI features suggestive of Leigh syndrome., Methods: Clinical and metabolic assessment was followed by autozygosity mapping and whole exome and Sanger sequencing. HIBCH enzyme activity and the bioenergetic profile were determined in patient fibroblasts., Results: The movement disorder was dominated by ataxia in one family and by dystonia in the other. All affected family members carried the identical homozygous c.913A>G (p.T305A) HIBCH mutation. Enzyme activity was reduced, and a valine challenge reduced the oxygen consumption rate., Conclusions: We report the first adult patients with HIBCH deficiency and a disease course much milder than previously reported, thereby expanding the HIBCH-associated phenotypic spectrum. © 2016 International Parkinson and Movement Disorder Society., (© 2016 International Parkinson and Movement Disorder Society.)

Introduction: Zellweger spectrum disorders (ZSDs) are characterized by a failure in peroxisome formation, caused by autosomal recessive mutations in different PEX genes. At least some of the progressive and irreversible clinical abnormalities in patients with a ZSD, particularly liver dysfunction, are likely caused by the accumulation of toxic bile acid intermediates. We investigated whether cholic acid supplementation can suppress bile acid synthesis, reduce accumulation of toxic bile acid intermediates and improve liver function in these patients., Methods: An open label, pretest-posttest design study was conducted including 19 patients with a ZSD. Participants were followed longitudinally during a period of 2.5 years prior to the start of the intervention. Subsequently, all patients received oral cholic acid and were followed during 9 months of treatment. Bile acids, peroxisomal metabolites, liver function and liver stiffness were measured at baseline and 4, 12 and 36 weeks after start of cholic acid treatment., Results: During cholic acid treatment, bile acid synthesis decreased in the majority of patients. Reduced levels of bile acid intermediates were found in plasma and excretion of bile acid intermediates in urine was diminished. In patients with advanced liver disease (n = 4), cholic acid treatment resulted in increased levels of plasma transaminases, bilirubin and cholic acid with only a minor reduction in bile acid intermediates., Conclusions: Oral cholic acid therapy can be used in the majority of patients with a ZSD, leading to at least partial suppression of bile acid synthesis. However, caution is needed in patients with advanced liver disease due to possible hepatotoxic effects., Competing Interests: Compliance with ethics guideline The study was approved by the Ethical Committee of the Academic Medical Center (AMC), Amsterdam, The Netherlands and took place between 2011 and 2014. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000. Conflict of interest Kevin Berendse, Femke C. C. Klouwer, Bart G.P. Koot, Elles M. Kemper, Sacha Ferdinandusse, Kiran V.K. Koelfat, Martin Lenicek, Frank G. Schaap, Hans R. Waterham, Frédéric Vaz, Marc Engelen, Peter L.M. Jansen, Ronald J.A. Wanders and Bwee Tien Poll-The declare that they have no conflicts of interest. Funding This work was supported by grants from Metakids, Hersenstichting, Axel Foundation and Stichting Steun Emma Kinderziekenhuis AMC, The Netherlands Informed consent Individual written informed consents were obtained from patients and/or patients’ parents.

X-Linked adrenoleukodystrophy (ALD) is a peroxisomal metabolic disorder with a highly complex clinical presentation. ALD is caused by mutations in the ABCD1 gene, which leads to the accumulation of very long-chain fatty acids in plasma and tissues. Virtually all men with ALD develop adrenal insufficiency and myelopathy. Approximately 60% of men develop progressive cerebral white matter lesions (known as cerebral ALD). However, one cannot identify these individuals until the early changes are seen using brain imaging. Women with ALD also develop myelopathy, but generally at a later age than men and adrenal insufficiency or cerebral ALD are very rare. Owing to the multisystem symptomatology of the disease, patients can be assessed by the paediatrician, general practitioner, endocrinologist or a neurologist. This Review describes current knowledge on the clinical presentation, diagnosis and treatment of ALD, and highlights gaps in our knowledge of the natural history of the disease owing to an absence of large-scale prospective cohort studies. Such studies are necessary for the identification of new prognostic biomarkers to improve care for patients with ALD, which is particularly relevant now that newborn screening for ALD is being introduced.

X-linked adrenoleukodystrophy (ALD) is a severe neurodegenerative disorder caused by the accumulation of very long-chain fatty acids (VLCFA) due to mutations in the ABCD1 gene. The phenotypic spectrum ranges from a fatal cerebral demyelinating disease in childhood (cerebral ALD) to a progressive myelopathy without cerebral involvement in adulthood (adrenomyeloneuropathy). Because ABCD1 mutations have no predictive value with respect to clinical outcome a role for modifier genes was postulated. We report that the CYP4F2 polymorphism rs2108622 increases the risk of developing cerebral ALD in Caucasian patients. The rs2108622 polymorphism (c.1297G>A) results in an amino acid substitution valine for methionine at position 433 (p.V433M). Using cellular models of VLCFA accumulation, we show that p.V433M decreases the conversion of VLCFA into very long-chain dicarboxylic acids by ω-oxidation, a potential escape route for the deficient peroxisomal β-oxidation of VLCFA in ALD. Although p.V433M does not affect the catalytic activity of CYP4F2 it reduces CYP4F2 protein levels markedly. These findings open perspectives for therapeutic interventions in a disease with currently limited treatment options., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background/objectives: Nutritional deficiencies are frequently observed when treating patients with inborn errors of metabolism due to an unbalanced diet. Thus far, patients with isovaleric acidemia (IVA) who adhere to a restricted protein diet have not been investigated in this respect. We hypothesize that these patients may have a polyunsaturated fatty acid (PUFA) deficiency, leading to potential clinical complications., Subjects/methods: We examined the nutritional status by reporting on potential deficiencies in PUFAs in treated IVA patients. A general clinical chemistry work-up as well as gas chromatography flame ionization detector analysis was performed to determine PUFAs in the plasma of 10 IVA patients., Results: The general clinical chemistry tests did not indicate severe hematological abnormalities or nutritional insufficiencies. We identified a significant reduction in plasma PUFA levels, especially in omega-3 (all acids, P<0.001) and omega-6 (in particular 20:3n-6 P<0.0001 and 20:4n-6 P=0.0005) fatty acids. In addition, an elevation in omega-9 fatty acids, with the exception of 20:3n-9 and C22:1n-9, was not suggestive of complete essential fatty acid deficiency but rather indicative of isolated and/or combined omega-3 and omega-6 fatty acid depletion., Conclusions: This study emphasizes the potential nutritional insufficiencies that may occur because of therapeutic intervention in IVA.

Aim: Acylcarnitines are fatty acid oxidation (FAO) intermediates, which have been implicated in diet-induced insulin resistance. Elevated acylcarnitine levels are found in obese, insulin resistant humans and rodents, and coincide with lower free carnitine. We hypothesized that increasing free carnitine levels by administration of the carnitine precursor γ-butyrobetaine (γBB) could facilitate FAO, thereby improving insulin sensitivity., Methods: C57BL/6N mice were fed with a high fat or chow diet with or without γBB supplementation (n=10 per group). After 8weeks of diet, indirect calorimetry, glucose tolerance and insulin sensitivity tests were performed. AC profiles and carnitine biosynthesis intermediates were analyzed in plasma and tissues by tandem mass spectrometry (MS) and liquid chromatography tandem MS., Results: γBB supplementation did not facilitate FAO, was unable to curb bodyweight and did not prevent impaired glucose homeostasis in the HFD fed mice in spite of marked alterations in the acylcarnitine profiles in plasma and liver. Remarkably, γBB did not affect the acylcarnitine profile in other tissues, most notably muscle. Administration of a bolus acetylcarnitine also caused significant changes in plasma and liver, but not in muscle acylcarnitine profiles, again without effect on glucose tolerance., Conclusion: Altogether, increasing carnitine availability affects acylcarnitine profiles in plasma and liver but does not modulate glucose tolerance or insulin sensitivity. This may be due to the lack of an effect on muscle acylcarnitine profiles, as muscle tissue is an important contributor to whole body insulin sensitivity. These results warrant caution on making associations between plasma acylcarnitine levels and insulin resistance., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Peroxisomal disorders are a heterogeneous group of genetic metabolic disorders, caused by a defect in peroxisome biogenesis or a deficiency of a single peroxisomal enzyme. The peroxisomal disorders include the Zellweger spectrum disorders, the rhizomelic chondrodysplasia punctata spectrum disorders, X-linked adrenoleukodystrophy, and multiple single enzyme deficiencies. There are several core phenotypes caused by peroxisomal dysfunction that clinicians can recognize. The diagnosis is suggested by biochemical testing in blood and urine and confirmed by functional assays in cultured skin fibroblasts, followed by mutation analysis. This review describes the phenotype of the main peroxisomal disorders and possible pitfalls in (laboratory) diagnosis to aid clinicians in the recognition of this group of diseases., (Georg Thieme Verlag KG Stuttgart · New York.)

Peroxisomes are subcellular organelles involved in various metabolic processes, including fatty acid and phospholipid homeostasis. The Zellweger spectrum disorders (ZSDs) represent a group of diseases caused by a defect in the biogenesis of peroxisomes. Accordingly, cells from ZSD patients are expected to have an altered composition of fatty acids and phospholipids. Using an LC/MS-based lipidomics approach, we show that the phospholipid composition is characteristically altered in cultured primary skin fibroblasts from ZSD patients when compared with healthy controls. We observed a marked overall increase of phospholipid species containing very long-chain fatty acids, and a decrease of phospholipid species with shorter fatty acid species in ZSD patient fibroblasts. In addition, we detected a distinct phosphatidylcholine profile in ZSD patients with a severe and mild phenotype when compared with control cells. Based on our data, we present a set of specific phospholipid ratios for fibroblasts that clearly discriminate between mild and severe ZSD patients, and those from healthy controls. Our findings will aid in the diagnosis and prognosis of ZSD patients, including an increasing number of mild patients in whom hardly any abnormalities are observed in biochemical parameters commonly used for diagnosis., (Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.)

Peroxisomes are dynamic organelles that play an essential role in a variety of metabolic pathways. Peroxisomal dysfunction can lead to various biochemical abnormalities and result in abnormal metabolite levels, such as increased very long-chain fatty acid or reduced plasmalogen levels. The metabolite abnormalities in peroxisomal disorders are used in the diagnostics of these disorders. In this paper we discuss in detail the different diagnostic tests available for peroxisomal disorders and focus specifically on the important role of biochemical and functional studies in cultured skin fibroblasts in reaching the right diagnosis. Several examples are shown to underline the power of such studies.

Cofactors such as NAD, AMP, and Coenzyme A (CoA) are essential for a diverse set of reactions and pathways in the cell. Specific carrier proteins are required to distribute these cofactors to different cell compartments, including peroxisomes. We previously identified a peroxisomal transport protein in Arabidopsis (Arabidopsis thaliana) called the peroxisomal NAD carrier (PXN). When assayed in vitro, this carrier exhibits versatile transport functions, e.g. catalyzing the import of NAD or CoA, the exchange of NAD/NADH, and the export of CoA. These observations raise the question about the physiological function of PXN in plants. Here, we used Saccharomyces cerevisiae to address this question. First, we confirmed that PXN, when expressed in yeast, is active and targeted to yeast peroxisomes. Secondl, detailed uptake analyses revealed that the CoA transport function of PXN can be excluded under physiological conditions due to its low affinity for this substrate. Third, we expressed PXN in diverse mutant yeast strains and investigated the suppression of the mutant phenotypes. These studies provided strong evidences that PXN was not able to function as a CoA transporter or a redox shuttle by mediating a NAD/NADH exchange, but instead catalyzed the import of NAD into peroxisomes against AMP in intact yeast cells., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

X-linked adrenoleukodystrophy (ALD), a progressive neurodegenerative disease, is caused by mutations in ABCD1 and characterized by very-long-chain fatty acids (VLCFA) accumulation. In male patients, an increased plasma VLCFA levels in combination with a pathogenic mutation in ABCD1 confirms the diagnosis. Recent studies have shown that many women with ALD also develop myelopathy. Correct diagnosis is important for management including genetic counseling. Diagnosis in women can only be confirmed when VLCFA levels are elevated or when a known pathogenic ABCD1 mutation is identified. However, in 15-20% of women with ALD VLCFA plasma levels are not elevated. Demonstration that a novel sequence variant is pathogenic can be a challenge when VLCFA levels are in the normal range. Here we report two women with a clinical presentation compatible with ALD, an ABCD1 variation (p.Arg17His and p.Ser358Pro) of unknown significance, but with normal VLCFA levels. We developed a diagnostic test that is based on generating clonal cell lines that express only one of the two alleles. Subsequent biochemical studies enabled us to show that the two sequence variants were not pathogenic, thereby excluding the diagnosis ALD in these women. We conclude that the clonal approach is an important addition to the existing diagnostic array., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: Severe acute malnutrition (SAM) is a major cause of mortality in children under 5 years and is associated with hepatic steatosis. Bile acids are synthesized in the liver and participate in dietary fat digestion, regulation of energy expenditure, and immune responses. The aim of this work was to investigate whether SAM is associated with clinically relevant changes in bile acid homeostasis., Design: An initial discovery cohort with 5 healthy controls and 22 SAM-patients was used to identify altered bile acid homeostasis. A follow up cohort of 40 SAM-patients were then studied on admission and 3 days after clinical stabilization to assess recovery in bile acid metabolism. Recruited children were 6-60 months old and admitted for SAM in Malawi. Clinical characteristics, feces and blood were collected on admission and prior to discharge. Bile acids, 7α-hydroxy-4-cholesten-3-one (C4) and FGF-19 were quantified., Results: On admission, total serum bile acids were higher in children with SAM than in healthy controls and glycine-conjugates accounted for most of this accumulation with median and interquartile range (IQR) of 24.6 μmol/L [8.6-47.7] compared to 1.9 μmol/L [1.7-3.3] (p = 0.01) in controls. Total serum bile acid concentrations did not decrease prior to discharge. On admission, fecal conjugated bile acids were lower and secondary bile acids higher at admission compared to pre- discharge, suggesting increased bacterial conversion. FGF19 (Fibroblast growth factor 19), a marker of intestinal bile acid signaling, was higher on admission and was associated with decreased C4 concentrations as a marker of bile acid synthesis. Upon recovery, fecal calprotectin, a marker of intestinal inflammation, was lower., Conclusion: SAM is associated with increased serum bile acid levels despite reduced synthesis rates. In SAM, there tends to be increased deconjugation of bile acids and conversion from primary to secondary bile acids, which may contribute to the development of liver disease.

Peroxisomes are dynamic organelles that play an essential role in a variety of cellular catabolic and anabolic metabolic pathways, including fatty acid alpha- and beta-oxidation, and plasmalogen and bile acid synthesis. Defects in genes encoding peroxisomal proteins can result in a large variety of peroxisomal disorders either affecting specific metabolic pathways, i.e., the single peroxisomal enzyme deficiencies, or causing a generalized defect in function and assembly of peroxisomes, i.e., peroxisome biogenesis disorders. In this review, we discuss the clinical, biochemical, and genetic aspects of all human peroxisomal disorders currently known., (Copyright © 2015 Elsevier B.V. All rights reserved.)

X-linked adrenoleukodystrophy (ALD), a progressive neurodegenerative disease, is caused by mutations in ABCD1 and characterized by very-long-chain fatty acids (VLCFA) accumulation. Virtually all males develop progressive myelopathy (AMN). A subset of patients, however, develops a fatal cerebral demyelinating disease (cerebral ALD). Hematopoietic stem cell transplantation is curative for cerebral ALD provided the procedure is performed in an early stage of the disease. Unfortunately, this narrow therapeutic window is often missed. Therefore, an increasing number of newborn screening programs are including ALD. To identify new biomarkers for ALD, we developed an Abcd1 knockout mouse with enhanced VLCFA synthesis either ubiquitous or restricted to oligodendrocytes. Biochemical analysis revealed VLCFA accumulation in different lipid classes and acylcarnitines. Both C26:0-lysoPC and C26:0-carnitine were highly elevated in brain, spinal cord, but also in bloodspots. We extended the analysis to patients and confirmed that C26:0-carnitine is also elevated in bloodspots from ALD patients. We anticipate that validation of C26:0-carnitine for the diagnosis of ALD in newborn bloodspots may lead to a faster inclusion of ALD in newborn screening programs in countries that already screen for other inborn errors of metabolism.

Barth syndrome (BTHS) is a cardiomyopathy caused by the loss of tafazzin, a mitochondrial acyltransferase involved in the maturation of the glycerophospholipid cardiolipin. It has remained enigmatic as to why a systemic loss of cardiolipin leads to cardiomyopathy. Using a genetic ablation of tafazzin function in the BTHS mouse model, we identified severe structural changes in respiratory chain supercomplexes at a pre-onset stage of the disease. This reorganization of supercomplexes was specific to cardiac tissue and could be recapitulated in cardiomyocytes derived from BTHS patients. Moreover, our analyses demonstrate a cardiac-specific loss of succinate dehydrogenase (SDH), an enzyme linking the respiratory chain with the tricarboxylic acid cycle. As a similar defect of SDH is apparent in patient cell-derived cardiomyocytes, we conclude that these defects represent a molecular basis for the cardiac pathology in Barth syndrome., (© 2015 The Authors. Published under the terms of the CC BY 4.0 license.)

Peroxisomes are unique subcellular organelles which play an indispensable role in several key metabolic pathways which include: (1.) etherphospholipid biosynthesis; (2.) fatty acid beta-oxidation; (3.) bile acid synthesis; (4.) docosahexaenoic acid (DHA) synthesis; (5.) fatty acid alpha-oxidation; (6.) glyoxylate metabolism; (7.) amino acid degradation, and (8.) ROS/RNS metabolism. The importance of peroxisomes for human health and development is exemplified by the existence of a large number of inborn errors of peroxisome metabolism in which there is an impairment in one or more of the metabolic functions of peroxisomes. Although the clinical signs and symptoms of affected patients differ depending upon the enzyme which is deficient and the extent of the deficiency, the disorders involved are usually (very) severe diseases with neurological dysfunction and early death in many of them. With respect to the role of peroxisomes in metabolism it is clear that peroxisomes are dependent on the functional interplay with other subcellular organelles to sustain their role in metabolism. Indeed, whereas mitochondria can oxidize fatty acids all the way to CO2 and H2O, peroxisomes are only able to chain-shorten fatty acids and the end products of peroxisomal beta-oxidation need to be shuttled to mitochondria for full oxidation to CO2 and H2O. Furthermore, NADH is generated during beta-oxidation in peroxisomes and beta-oxidation can only continue if peroxisomes are equipped with a mechanism to reoxidize NADH back to NAD(+), which is now known to be mediated by specific NAD(H)-redox shuttles. In this paper we describe the current state of knowledge about the functional interplay between peroxisomes and other subcellular compartments notably the mitochondria and endoplasmic reticulum for each of the metabolic pathways in which peroxisomes are involved.

Mitochondrial fatty acid β-oxidation (FAO) is the major pathway for the degradation of fatty acids and is essential for maintaining energy homeostasis in the human body. Fatty acids are a crucial energy source in the postabsorptive and fasted states when glucose supply is limiting. But even when glucose is abundantly available, FAO is a main energy source for the heart, skeletal muscle, and kidney. A series of enzymes, transporters, and other facilitating proteins are involved in FAO. Recessively inherited defects are known for most of the genes encoding these proteins. The clinical presentation of these disorders may include hypoketotic hypoglycemia, (cardio)myopathy, arrhythmia, and rhabdomyolysis and illustrates the importance of FAO during fasting and in hepatic and (cardio)muscular function. In this review, we present the current state of knowledge on the biochemistry and physiological functions of FAO and discuss the pathophysiological processes associated with FAO disorders.

Mitochondrial trifunctional protein (MTP) deficiency caused by HADHA or HADHB gene mutations exhibits substantial molecular, biochemical, and clinical heterogeneity and ranks among the more severe fatty acid oxidation (FAO) disorders, without pharmacological treatment. Since bezafibrate has been shown to potentially correct other FAO disorders in patient cells, we analyzed its effects in 26 MTP-deficient patient fibroblasts representing 16 genotypes. Overall, the patient cell lines exhibited variable, complex, biochemical profiles and pharmacological responses. HADHA-deficient fibroblasts showed markedly reduced alpha subunit protein levels together with decreased beta-subunit abundance, exhibited a -86 to -96% defect in LCHAD activity, and produced large amounts of C14 and C16 hydroxyacylcarnitines. In control fibroblasts, exposure to bezafibrate (400 μM for 48 h) increased the abundance of HADHA and HADHB mRNAs, immune-detectable alpha and beta subunit proteins, activities of LCHAD and LCKAT, and stimulated FAO capacities, clearly indicating that MTP is pharmacologically up-regulated by bezafibrate in human fibroblasts. In MTP-deficient patient fibroblasts, which were found markedly FAO-deficient, bezafibrate improved FAO capacities in six of 26 (23%) cases, including three cell lines heterozygous for the common c1528G > C mutation. Altogether, our results strongly suggest that, due to variable effects of HADHA and HADHB mutations on MTP abundance and residual activity, improvement of MTP deficiency in response to bezafibrate was achieved in a subset of responsive genotypes.

Introduction: We describe the natural history of patients with a Zellweger spectrum disorder (ZSD) surviving into adulthood., Methods: Retrospective cohort study in patients with a genetically confirmed ZSD., Results: All patients (n = 19; aged 16-35 years) had a follow-up period of 1-24.4 years (mean 16 years). Seven patients had a progressive disease course, while 12 remained clinically stable during follow-up. Disease progression usually manifests in adolescence as a gait disorder, caused by central and/or peripheral nervous system involvement. Nine were capable of living a partly independent life with supported employment. Systematic MRI review revealed T2 hyperintense white matter abnormalities in the hilus of the dentate nucleus and/or peridentate region in nine out of 16 patients. Biochemical analyses in blood showed abnormal peroxisomal biomarkers in all patients in infancy and childhood, whereas in adolescence/adulthood we observed normalization of some metabolites., Conclusions: The patients described here represent a distinct subgroup within the ZSDs who survive into adulthood. Most remain stable over many years. Disease progression may occur and is mainly due to cerebral and cerebellar white matter abnormalities, and peripheral neuropathy.