Tracer-based lipidomics enables the discovery of disease-specific candidate biomarkers in mitochondrial ß-oxidation disorders.

Carnitine derivatives of disease-specific acyl-CoAs are the diagnostic hallmark for long-chain fatty acid ß-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.

Studying the topology of peroxisomal acyl-CoA synthetases using self-assembling split sfGFP.

Peroxisomes are membrane-bounded organelles that contain enzymes involved in multiple lipid metabolic pathways. Several of these pathways require (re-)activation of fatty acids to coenzyme A (CoA) esters by acyl-CoA synthetases, which may take place inside the peroxisomal lumen or extraperoxisomal. The acyl-CoA synthetases SLC27A2, SLC27A4, ACSL1, and ACSL4 have different but overlapping substrate specificities and were previously reported to be localized in the peroxisomal membrane in addition to other subcellular locations. However, it has remained unclear if the catalytic acyl-CoA synthetase sites of these enzymes are facing the peroxisomal lumen or the cytosolic side of the peroxisomal membrane. To study this topology in cellulo we have developed a microscopy-based method that uses the previously developed self-assembling split superfolder (sf) green fluorescent protein (GFP) assay. We show that this self-assembling split sfGFP method can be used to study the localization as well as the topology of membrane proteins in the peroxisomal membrane, but that it is less suited to study the location of soluble peroxisomal proteins. With the method we could demonstrate that the acyl-CoA synthetase domains of the peroxisome-bound acyl-CoA synthetases SLC27A2 and SLC27A4 are oriented toward the peroxisomal lumen and the domain of ACSL1 toward the cytosol. In contrast to previous reports, ACSL4 was not found in peroxisomes.

Peroxisomal NAD(H) Homeostasis in the Yeast Debaryomyces hansenii Depends on Two Redox Shuttles and the NAD+ Carrier, Pmp47.

Debaryomyces hansenii is considered an unconventional yeast with a strong biotechnological potential, which can produce and store high amounts of lipids. However, relatively little is known about its lipid metabolism, and genetic tools for this yeast have been limited. The aim of this study was to explore the fatty acid ß-oxidation pathway in D. hansenii. To this end, we employed recently developed methods to generate multiple gene deletions and tag open reading frames with GFP in their chromosomal context in this yeast. We found that, similar as in other yeasts, the ß-oxidation of fatty acids in D. hansenii was restricted to peroxisomes. We report a series of experiments in D. hansenii and the well-studied yeast Saccharomyces cerevisiae that show that the homeostasis of NAD+ in D. hansenii peroxisomes is dependent upon the peroxisomal membrane protein Pmp47 and two peroxisomal dehydrogenases, Mdh3 and Gpd1, which both export reducing equivalents produced during ß-oxidation to the cytosol. Pmp47 is the first identified NAD+ carrier in yeast peroxisomes.

Maintenance of cellular vitamin B6 levels and mitochondrial oxidative function depend on pyridoxal 5'-phosphate homeostasis protein.

Recently, biallelic variants in PLPBP coding for pyridoxal 5'-phosphate homeostasis protein (PLPHP) were identified as a novel cause of early-onset vitamin B6-dependent epilepsy. The molecular function and precise role of PLPHP in vitamin B6 metabolism are not well understood. To address these questions, we used PLPHP-deficient patient skin fibroblasts and HEK293 cells and YBL036C (PLPHP ortholog)-deficient yeast. We showed that independent of extracellular B6 vitamer type (pyridoxine, pyridoxamine, or pyridoxal), intracellular pyridoxal 5'-phosphate (PLP) was lower in PLPHP-deficient fibroblasts and HEK293 cells than controls. Culturing cells with pyridoxine or pyridoxamine led to the concentration-dependent accumulation of pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate (PMP), respectively, suggesting insufficient pyridox(am)ine 5'-phosphate oxidase activity. Experiments utilizing 13C4-pyridoxine confirmed lower pyridox(am)ine 5'-phosphate oxidase activity and revealed increased fractional turnovers of PLP and pyridoxal, indicating increased PLP hydrolysis to pyridoxal in PLPHP-deficient cells. This effect could be partly counteracted by inactivation of pyridoxal phosphatase. PLPHP deficiency had a distinct effect on mitochondrial PLP and PMP, suggesting impaired activity of mitochondrial transaminases. Moreover, in YBL036C-deficient yeast, PLP was depleted and PMP accumulated only with carbon sources requiring mitochondrial metabolism. Lactate and pyruvate accumulation along with the decrease of tricarboxylic acid cycle intermediates downstream of α-ketoglutarate suggested impaired mitochondrial oxidative metabolism in PLPHP-deficient HEK293 cells. We hypothesize that impaired activity of mitochondrial transaminases may contribute to this depletion. Taken together, our study provides new insights into the pathomechanisms of PLPBP deficiency and reinforces the link between PLPHP function, vitamin B6 metabolism, and mitochondrial oxidative metabolism.

Human peroxisomal NAD+/NADH homeostasis is regulated by two independent NAD(H) shuttle systems.

Reduced (NADH) and oxidized (NAD+) nicotinamide adenine dinucleotides are ubiquitous hydride-donating/accepting cofactors that are essential for cellular bioenergetics. Peroxisomes are single-membrane-bounded organelles that are involved in multiple lipid metabolism pathways, including beta-oxidation of fatty acids, and which contain several NAD(H)-dependent enzymes. Although maintenance of NAD(H) homeostasis in peroxisomes is considered essential for peroxisomal beta-oxidation, little is known about the regulation thereof. To resolve this issue, we have developed methods to specifically measure intraperoxisomal NADH levels in human cells using peroxisome-targeted NADH biosensors. By targeted CRISPR-Cas9-mediated genome editing of human cells, we showed with these sensors that the NAD+/NADH ratio in cytosol and peroxisomes are closely connected and that this crosstalk is mediated by intraperoxisomal lactate and malate dehydrogenases, generated via translational stop codon readthrough of the LDHB and MDH1 mRNAs. Our study provides evidence for the existence of two independent redox shuttle systems in human peroxisomes that regulate peroxisomal NAD+/NADH homeostasis. This is the first study that shows a specific metabolic function of protein isoforms generated by translational stop codon readthrough in humans.

Genetic, biochemical, and clinical spectrum of patients with mitochondrial trifunctional protein deficiency identified after the introduction of newborn screening in the Netherlands.

Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) is included in many newborn screening (NBS) programs. Acylcarnitine-based NBS for LCHADD not only identifies LCHADD, but also the other deficiencies of the mitochondrial trifunctional protein (MTP), a multi-enzyme complex involved in long-chain fatty acid ß-oxidation. Besides LCHAD, MTP harbors two additional enzyme activities: long-chain enoyl-CoA hydratase (LCEH) and long-chain ketoacyl-CoA thiolase (LCKAT). Deficiency of one or more MTP activities causes generalized MTP deficiency (MTPD), LCHADD, LCEH deficiency (not yet reported), or LCKAT deficiency (LCKATD). To gain insight in the outcomes of MTP-deficient patients diagnosed after the introduction of NBS for LCHADD in the Netherlands, a retrospective evaluation of genetic, biochemical, and clinical characteristics of MTP-deficient patients, identified since 2007, was carried out. Thirteen patients were identified: seven with LCHADD, five with MTPD, and one with LCKATD. All LCHADD patients (one missed by NBS, clinical diagnosis) and one MTPD patient (clinical diagnosis) were alive. Four MTPD patients and one LCKATD patient developed cardiomyopathy and died within 1 month and 13 months of life, respectively. Surviving patients did not develop symptomatic hypoglycemia, but experienced reversible cardiomyopathy and rhabdomyolysis. Five LCHADD patients developed subclinical neuropathy and/or retinopathy. In conclusion, patient outcomes were highly variable, stressing the need for accurate classification of and discrimination between the MTP deficiencies to improve insight in the yield of NBS for LCHADD. NBS allowed the prevention of symptomatic hypoglycemia, but current treatment options failed to treat cardiomyopathy and prevent long-term complications. Moreover, milder patients, who might benefit from NBS, were missed due to normal acylcarnitine profiles.

Peroxisomal ATP Uptake Is Provided by Two Adenine Nucleotide Transporters and the ABCD Transporters.

Peroxisomes are essential organelles involved in various metabolic processes, including fatty acid ß-oxidation. Their metabolic functions require a controlled exchange of metabolites and co-factors, including ATP, across the peroxisomal membrane. We investigated which proteins are involved in the peroxisomal uptake of ATP in the yeast Saccharomyces cerevisiae. Using wild-type and targeted deletion strains, we measured ATP-dependent peroxisomal octanoate ß-oxidation, intra-peroxisomal ATP levels employing peroxisome-targeted ATP-sensing reporter proteins, and ATP uptake in proteoliposomes prepared from purified peroxisomes. We show that intra-peroxisomal ATP levels are maintained by different peroxisomal membrane proteins each with different modes of action: 1) the previously reported Ant1p protein, which catalyzes the exchange of ATP for AMP or ADP, 2) the ABC transporter protein complex Pxa1p/Pxa2p, which mediates both uni-directional acyl-CoA and ATP uptake, and 3) the mitochondrial Aac2p protein, which catalyzes ATP/ADP exchange and has a dual localization in both mitochondria and peroxisomes. Our results provide compelling evidence for a complementary system for the uptake of ATP in peroxisomes.

The Saccharomyces cerevisiae ABC subfamily D transporter Pxa1/Pxa2p co-imports CoASH into the peroxisome.

ATP-binding cassette (ABC) subfamily D transporters are important for the uptake of fatty acids and other beta-oxidation substrates into peroxisomes. Genetic and biochemical evidence indicates that the transporters accept fatty acyl-coenzyme A that is cleaved during the transport cycle and then re-esterified in the peroxisomal lumen. However, it is not known whether free coenzyme A (CoA) is released inside or outside the peroxisome. Here we have used Saccharomyces cerevisiae and isolated peroxisomes to demonstrate that free CoA is released in the peroxisomal lumen. Thus, ABC subfamily D transporter provide an import pathway for free CoA that controls peroxisomal CoA homeostasis and tunes metabolism according to the cell's demands.

Exploring the metabolic fate of medium-chain triglycerides in healthy individuals using a stable isotope tracer.

BACKGROUND & AIMS: Medium chain triglyceride (MCT) supplementation is often recommended as treatment for patients with long-chain fatty acid ß-oxidation (lcFAO) disorders, since they can be utilized as an energy source without the use of the defective enzyme. However, studies in mice and preterm infants suggest that not all medium-chain fatty acids (MCFA) are oxidized and may undergo elongation to long-chain fatty acids (LCFA). In this single blinded study, we explored the metabolic fates of MCT in healthy individuals using a 13C-labeled MCT tracer. METHOD: Three healthy males in rest received on two test days a primed continuous infusion of glyceryl tri[1,2,3,4-13C4]-octanoate with either an isocaloric supplementation of 1) exclusively MCT (MCT-only) or 2) a mixture of MCT, proteins and carbohydrates (MCT-mix). Gas chromatography - combustion - isotope ratio mass spectrometry (GC-C-IRMS) was used to determine 13C-enrichment of long-chain fatty acids in plasma and of 13CO2 in exhaled air. RESULTS: When provided as single energy source, an estimated 42% of administered MCT was converted to CO2. In combination with carbohydrates and proteins in the diet, oxidation of MCT was higher (62%). In both diets <1% of 13C-label was incorporated in LCFA in plasma, indicating that administered MCT underwent chain-elongation to LCT. CONCLUSIONS: Although the relative MCT oxidation rate was higher when combined with carbohydrates and protein, quantitatively more MCT was oxidized when given an isocaloric meal with solely MCT. As these results were obtained in the resting state opposed to during exercise, it is too early to give a recommendation concerning the use of MCT in lcFAO disorders. The data show that in resting healthy individuals only a very small part of the MCT is traced back as LCFA in plasma, suggesting that MCT treatment does not result in a large LCFA burden, however further research on storage of MCT in tissues is warranted. REGISTRATION: The study was registered in the Nederlands Trialregister. Protocol ID: Trial NL7417 (NTR7650).

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.

Electrophysiological Abnormalities in VLCAD Deficient hiPSC-Cardiomyocytes Can Be Improved by Lowering Accumulation of Fatty Acid Oxidation Intermediates.

Patients with very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) can present with life-threatening cardiac arrhythmias. The pathophysiological mechanism is unknown. We reprogrammed fibroblasts from one mildly and one severely affected VLCADD patient, into human induced pluripotent stem cells (hiPSCs) and differentiated these into cardiomyocytes (VLCADD-CMs). VLCADD-CMs displayed shorter action potentials (APs), more delayed afterdepolarizations (DADs) and higher systolic and diastolic intracellular Ca2+ concentration ([Ca2+]i) than control CMs. The mitochondrial booster resveratrol mitigated the biochemical, electrophysiological and [Ca2+]i changes in the mild but not in the severe VLCADD-CMs. Accumulation of potentially toxic intermediates of fatty acid oxidation was blocked by substrate reduction with etomoxir. Incubation with etomoxir led to marked prolongation of AP duration and reduced DADs and [Ca2+]i in both VLCADD-CMs. These results provide compelling evidence that reduced accumulation of fatty acid oxidation intermediates, either by enhanced fatty acid oxidation flux through increased mitochondria biogenesis (resveratrol) or by inhibition of fatty acid transport into the mitochondria (etomoxir), rescues pro-arrhythmia defects in VLCADD-CMs and open doors for new treatments.

Nutritional ketosis improves exercise metabolism in patients with very long-chain acyl-CoA dehydrogenase deficiency.

A maladaptive shift from fat to carbohydrate (CHO) oxidation during exercise is thought to underlie myopathy and exercise-induced rhabdomyolysis in patients with fatty acid oxidation (FAO) disorders. We hypothesised that ingestion of a ketone ester (KE) drink prior to exercise could serve as an alternative oxidative substrate supply to boost muscular ATP homeostasis. To establish a rational basis for therapeutic use of KE supplementation in FAO, we tested this hypothesis in patients deficient in Very Long-Chain acyl-CoA Dehydrogenase (VLCAD). Five patients (range 17-45 y; 4 M/1F) patients were included in an investigator-initiated, randomised, blinded, placebo-controlled, 2-way cross-over study. Patients drank either a KE + CHO mix or an isocaloric CHO equivalent and performed 35 minutes upright cycling followed by 10 minutes supine cycling inside a Magnetic Resonance scanner at individual maximal FAO work rate (fatmax; approximately 40% VO2 max). The protocol was repeated after a 1-week interval with the alternate drink. Primary outcome measures were quadriceps phosphocreatine (PCr), Pi and pH dynamics during exercise and recovery assayed by in vivo 31 P-MR spectroscopy. Secondary outcomes included plasma and muscle metabolites and respiratory gas exchange recordings. Ingestion of KE rapidly induced mild ketosis and increased muscle BHB content. During exercise at FATMAX, VLCADD-specific plasma acylcarnitine levels, quadriceps glycolytic intermediate levels and in vivo Pi/PCr ratio were all lower in KE + CHO than CHO. These results provide a rational basis for future clinical trials of synthetic ketone ester supplementation therapy in patients with FAO disorders. Trial registration: ClinicalTrials.gov. Protocol ID: NCT03531554; METC2014.492; ABR51222.042.14.

Electrophysiological Abnormalities in VLCAD Deficient hiPSC-Cardiomyocytes Do not Improve with Carnitine Supplementation.

Patients with a deficiency in very long-chain acyl-CoA dehydrogenase (VLCAD), an enzyme that is involved in the mitochondrial beta-oxidation of long-chain fatty acids, are at risk for developing cardiac arrhythmias. In human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs), VLCAD deficiency (VLCADD) results in a series of abnormalities, including: 1) accumulation of long-chain acylcarnitines, 2) action potential shortening, 3) higher systolic and diastolic intracellular Ca2+ concentrations, and 4) development of delayed afterdepolarizations. In the fatty acid oxidation process, carnitine is required for bidirectional transport of acyl groups across the mitochondrial membrane. Supplementation has been suggested as potential therapeutic approach in VLCADD, but its benefits are debated. Here, we studied the effects of carnitine supplementation on the long-chain acylcarnitine levels and performed electrophysiological analyses in VLCADD patient-derived hiPSC-CMs with a ACADVL gene mutation (p.Val283Ala/p.Glu381del). Under standard culture conditions, VLCADD hiPSC-CMs showed high concentrations of long-chain acylcarnitines, short action potentials, and high delayed afterdepolarizations occurrence. Incubation of the hiPSC-CMs with 400 µM L-carnitine for 48 h led to increased long-chain acylcarnitine levels both in medium and cells. In addition, carnitine supplementation neither restored abnormal action potential parameters nor the increased occurrence of delayed afterdepolarizations in VLCADD hiPSC-CMs. We conclude that long-chain acylcarnitine accumulation and electrophysiological abnormalities in VLCADD hiPSC-CMs are not normalized by carnitine supplementation, indicating that this treatment is unlikely to be beneficial against cardiac arrhythmias in VLCADD patients.

Peroxisomal Metabolite and Cofactor Transport in Humans.

Peroxisomes are membrane-bound organelles involved in many metabolic pathways and essential for human health. They harbor a large number of enzymes involved in the different pathways, thus requiring transport of substrates, products and cofactors involved across the peroxisomal membrane. Although much progress has been made in understanding the permeability properties of peroxisomes, there are still important gaps in our knowledge about the peroxisomal transport of metabolites and cofactors. In this review, we discuss the different modes of transport of metabolites and essential cofactors, including CoA, NAD+, NADP+, FAD, FMN, ATP, heme, pyridoxal phosphate, and thiamine pyrophosphate across the peroxisomal membrane. This transport can be mediated by non-selective pore-forming proteins, selective transport proteins, membrane contact sites between organelles, and co-import of cofactors with proteins. We also discuss modes of transport mediated by shuttle systems described for NAD+/NADH and NADP+/NADPH. We mainly focus on current knowledge on human peroxisomal metabolite and cofactor transport, but also include knowledge from studies in plants, yeast, fruit fly, zebrafish, and mice, which has been exemplary in understanding peroxisomal transport mechanisms in general.

Mutagenesis separates ATPase and thioesterase activities of the peroxisomal ABC transporter, Comatose.

The peroxisomal ABC transporter, Comatose (CTS), a full length transporter from Arabidopsis has intrinsic acyl-CoA thioesterase (ACOT) activity, important for physiological function. We used molecular modelling, mutagenesis and biochemical analysis to identify amino acid residues important for ACOT activity. D863, Q864 and T867 lie within transmembrane helix 9. These residues are orientated such that they might plausibly contribute to a catalytic triad similar to type II Hotdog fold thioesterases. When expressed in Saccharomyces cerevisiae, mutation of these residues to alanine resulted in defective of ß-oxidation. All CTS mutants were expressed and targeted to peroxisomes and retained substrate-stimulated ATPase activity. When expressed in insect cell membranes, Q864A and S810N had similar ATPase activity to wild type but greatly reduced ACOT activity, whereas the Walker A mutant K487A had greatly reduced ATPase and no ATP-dependent ACOT activity. In wild type CTS, ATPase but not ACOT was stimulated by non-cleavable C14 ether-CoA. ACOT activity was stimulated by ATP but not by non-hydrolysable AMPPNP. Thus, ACOT activity depends on functional ATPase activity but not vice versa, and these two activities can be separated by mutagenesis. Whether D863, Q864 and T867 have a catalytic role or play a more indirect role in NBD-TMD communication is discussed.

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.

An UPLC-MS/MS Assay to Measure Glutathione as Marker for Oxidative Stress in Cultured Cells.

Oxidative stress plays a role in the onset and progression of a number of diseases, such as Alzheimer's disease, diabetes and cancer, as well as ageing. Oxidative stress is caused by an increased production of reactive oxygen species and reduced antioxidant activity, resulting in the oxidation of glutathione. The ratio of reduced to oxidised glutathione is often used as a marker of the redox state in the cell. Whereas a variety of methods have been developed to measure glutathione in blood samples, methods to measure glutathione in cultured cells are scarce. Here we present a protocol to measure glutathione levels in cultured human and yeast cells using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC⁻MS/MS).

Barth syndrome cells display widespread remodeling of mitochondrial complexes without affecting metabolic flux distribution.

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.

Disorders of mitochondrial long-chain fatty acid oxidation and the carnitine shuttle.

Mitochondrial fatty acid oxidation is an essential pathway for energy production, especially during prolonged fasting and sub-maximal exercise. Long-chain fatty acids are the most abundant fatty acids in the human diet and in body stores, and more than 15 enzymes are involved in long-chain fatty acid oxidation. Pathogenic mutations in genes encoding these enzymes result in a long-chain fatty acid oxidation disorder in which the energy homeostasis is compromised and long-chain acylcarnitines accumulate. Symptoms arise or exacerbate during catabolic situations, such as fasting, illness and (endurance) exercise. The clinical spectrum is very heterogeneous, ranging from hypoketotic hypoglycemia, liver dysfunction, rhabdomyolysis, cardiomyopathy and early demise. With the introduction of several of the long-chain fatty acid oxidation disorders (lcFAOD) in newborn screening panels, also asymptomatic individuals with a lcFAOD are identified. However, despite early diagnosis and dietary therapy, a significant number of patients still develop symptoms emphasizing the need for individualized treatment strategies. This review aims to function as a comprehensive reference for clinical and laboratory findings for clinicians who are confronted with pediatric and adult patients with a possible diagnosis of a lcFAOD.