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.

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.

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.

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).

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.

Valproyl-CoA inhibits the activity of ATP- and GTP-dependent succinate:CoA ligases.

BACKGROUND: Valproic acid (VPA) is an effective antiepileptic drug that may induce progressive microvesicular steatosis. The impairment of mitochondrial function may be an important metabolic effect of VPA treatment with potential adverse consequences. OBJECTIVE: To investigate the influence of VPA on the activity of GTP- and ATP-specific succinate:CoA ligases (G-SUCL and A-SUCL). METHODS: The GTP- and ATP-specific SUCL activities were measured in human fibroblasts in the reverse direction, i.e. the formation of succinyl-CoA. These were assessed at different concentrations of succinate in the presence of VPA, valproyl-CoA and zinc chloride, an established inhibitor of the enzymes. Activities were measured using an optimized HPLC procedure. RESULTS: Valproyl-CoA (1 mM) inhibited the activity of A-SUCL and G-SUCL by 45-55% and 25-50%, respectively. VPA (1 mM) had no influence on the activity of the two enzymes. DISCUSSION: Valproyl-CoA appears to affect the activity of SUCL, especially with the ATP-specific enzyme. Considering the key role of SUCL in the Krebs cycle, interference with its activity might impair the cellular energy status. Moreover, A-SUCL is bound to the nucleoside diphosphate kinase (NDPK), which is responsible for the mitochondrial (deoxy)nucleotide synthesis. An inhibition of A-SUCL might influence the activity of NDPK inducing an imbalance of nucleotides in the mitochondria and eventually mitochondrial DNA depletion. This may account for the potential liver failure associated with valproate therapy, reported in patients with deficiencies within the mitochondrial DNA replicase system such as polymerase gamma 1.

Arginine improves peroxisome functioning in cells from patients with a mild peroxisome biogenesis disorder.

BACKGROUND: Zellweger spectrum disorders (ZSDs) are multisystem genetic disorders caused by a lack of functional peroxisomes, due to mutations in one of the PEX genes, encoding proteins involved in peroxisome biogenesis. The phenotypic spectrum of ZSDs ranges from an early lethal form to much milder presentations. In cultured skin fibroblasts from mildly affected patients, peroxisome biogenesis can be partially impaired which results in a mosaic catalase immunofluorescence pattern. This peroxisomal mosaicism has been described for specific missense mutations in various PEX genes. In cell lines displaying peroxisomal mosaicism, peroxisome biogenesis can be improved when these are cultured at 30°C. This suggests that these missense mutations affect the folding and/or stability of the encoded protein. We have studied if the function of mutant PEX1, PEX6 and PEX12 can be improved by promoting protein folding using the chemical chaperone arginine. METHODS: Fibroblasts from three PEX1 patients, one PEX6 and one PEX12 patient were cultured in the presence of different concentrations of arginine. To determine the effect on peroxisome biogenesis we studied the following parameters: number of peroxisome-positive cells, levels of PEX1 protein and processed thiolase, and the capacity to ß-oxidize very long chain fatty acids and pristanic acid. RESULTS: Peroxisome biogenesis and function in fibroblasts with mild missense mutations in PEX1, 6 and 12 can be improved by arginine. CONCLUSION: Arginine may be an interesting compound to promote peroxisome function in patients with a mild peroxisome biogenesis disorder.

Differential substrate specificities of human ABCD1 and ABCD2 in peroxisomal fatty acid ß-oxidation.

The gene mutated in X-linked adrenoleukodystrophy (X-ALD) codes for the HsABCD1 protein, also named ALDP, which is a member of the superfamily of ATP-binding cassette (ABC) transporters and required for fatty acid transport across the peroxisomal membrane. Although a defective HsABCD1 results in the accumulation of very long-chain fatty acids in plasma of X-ALD patients, there is still no direct biochemical evidence that HsABCD1 actually transports very long-chain fatty acids. We used the yeast Saccharomyces cerevisiae to study the transport of fatty acids across the peroxisomal membrane. Our earlier work showed that in yeast the uptake of fatty acids into peroxisomes may occur via two routes, either as (1.) free fatty acid or as (2.) acyl-CoA ester. The latter route involves the two peroxisomal half-ABC transporters, Pxa1p and Pxa2p, which form a heterodimeric complex in the peroxisomal membrane. We here report that the phenotype of the pxa1/pxa2Δ yeast mutant, i.e. impaired growth on oleate containing medium and deficient oxidation of oleic acid, cannot only be partially rescued by human ABCD1, but also by human ABCD2 (ALDRP), which indicates that HsABCD1 and HsABCD2 can both function as homodimers. Fatty acid oxidation studies in the pxa1/pxa2Δ mutant transformed with either HsABCD1 or HsABCD2 revealed clear differences suggesting that HsABCD1 and HsABCD2 have distinct substrate specificities. Indeed, full rescue of beta-oxidation activity in cells expressing human ABCD2 was observed with C22:0 and different unsaturated very long-chain fatty acids including C24:6 and especially C22:6 whereas in cells expressing HsABCD1 rescue of beta-oxidation activity was best with C24:0 and C26:0 as substrates.

Characterization of L-aminocarnitine, an inhibitor of fatty acid oxidation.

The pathogenesis of hypoketotic hypoglycemia and cardiomyopathy in patients with fatty acid oxidation (FAO) disorders is still poorly understood. In vitro studies are hampered by the lack of natural mutants to asses the effect of FAO inhibition. In addition, only a few inhibitors of FAO are known. Furthermore, most inhibitors of FAO are activating ligands of peroxisome proliferator-activated receptors (PPARs). We show that l-aminocarnitine (L-AC), a carnitine analog, inhibits FAO efficiently, but does not activate PPAR. L-AC inhibits carnitine palmitoyltransferase (CPT) with different sensitivities towards CPT1 and CPT2, as well as carnitine acylcarnitine translocase (CACT). We further characterized L-AC using fibroblasts cell lines from controls and patients with different FAO defects. In these cell lines acylcarnitine profiles were determined in culture medium after loading with [U-(13)C]palmitic acid. In control fibroblasts, L-AC inhibits FAO leading to a reduction of C2-acylcarnitine and elevation of C16-acylcarnitine. In very long-chain acyl-CoA dehydrogenase (VLCAD)-deficient fibroblasts, L-AC decreased the elevated C14-acylcarnitine and increased C16-acylcarnitine. In CACT and CPT2-deficient cell lines, L-AC did not change the already elevated C16-acylcarnitine level, showing that CPT1 is not inhibited. Oxidation of pristanic acid was only partly inhibited at high L-AC concentrations, indicating minimal CACT inhibition. Therefore, we conclude that in intact cells L-AC inhibits CPT2. Combined with our observation that l-AC does not activate PPAR, we suggest that L-AC is useful to simulate a FAO defect in cells from different origin.

Demonstration of bile acid transport across the mammalian peroxisomal membrane.

It is well established that peroxisomes play a crucial role in de novo bile acid biosynthesis. The primary bile acids resulting from peroxisomal beta-oxidation are conjugated to either glycine or taurine in the peroxisomal lumen by a bile acid aminotransferase (BAT). These conjugated bile acids are subsequently secreted into the bile. In this paper we show that the export of glycine- and taurine-conjugated bile acids from mammalian peroxisomes proceeds via specific transporter. The transport activity of this protein was detected by reconstitution of peroxisomal membrane proteins in liposomes and measuring the uptake of radiolabeled substrates into these proteoliposomes. The transporter was further characterized using this assay, which led to the identification of DIDS as an inhibitor of the peroxisomal bile-acid transporter, and allowed us to establish some kinetic parameters for the transport activity.

Metabolite transport across the peroxisomal membrane.

In recent years, much progress has been made with respect to the unravelling of the functions of peroxisomes in metabolism, and it is now well established that peroxisomes are indispensable organelles, especially in higher eukaryotes. Peroxisomes catalyse a number of essential metabolic functions including fatty acid beta-oxidation, ether phospholipid biosynthesis, fatty acid alpha-oxidation and glyoxylate detoxification. The involvement of peroxisomes in these metabolic pathways necessitates the transport of metabolites in and out of peroxisomes. Recently, considerable progress has been made in the characterization of metabolite transport across the peroxisomal membrane. Peroxisomes posses several specialized transport systems to transport metabolites. This is exemplified by the identification of a specific transporter for adenine nucleotides and several half-ABC (ATP-binding cassette) transporters which may be present as hetero- and homo-dimers. The nature of the substrates handled by the different ABC transporters is less clear. In this review we will describe the current state of knowledge of the permeability properties of the peroxisomal membrane.

Fatty acid oxidation in the human fetus: implications for fetal and adult disease.

Studies in the last few years have shown a remarkably high activity of fatty acid oxidation (FAO) enzymes in human placenta. We have recently shown mRNA expression as well as enzymatic activity of long-chain FAO enzymes in the human embryo and fetus. In this study we show activity of the FAO enzymes carnitine palmitoyltranferase 1, medium-chain acyl-CoA dehydrogenase and short-chain hydroxyacyl-CoA dehydrogenase in embryonic and fetal tissues. In addition, we show the presence of different acylcarnitines in fetal liver and kidney, which substantiates the notion that the mitochondrial FAO enzymes are not only present in human fetal tissues but also metabolically active. In a glucose-rich environment FAO might be necessary for additional ATP production from fatty acids, but also for the breakdown of fatty acids that are products of the turnover of membranes in the growing fetus. The importance of FAO in the human embryo and fetus is further stressed by the fact that a higher frequency of prematurity, intrauterine growth retardation, fetal morbidity and intrauterine death is noted in long-chain FAO defects. Furthermore, in animal studies, gestational loss during early embryonic development has been observed as a consequence of disturbed FAO. Finally, there are indications that regulation of activity of FAO during fetal development might not only be important for fetal life but may also have implications for health and disease in adulthood.

The peroxisomal lumen in Saccharomyces cerevisiae is alkaline.

Peroxisomes have a central function in lipid metabolism, including the beta-oxidation of various fatty acids. The products and substrates involved in the beta-oxidation have to cross the peroxisomal membrane, which previously has been demonstrated to constitute a closed barrier, implying the existence of specific transport mechanisms. Fatty acid transport across the yeast peroxisomal membrane may follow two routes: one for activated fatty acids, dependent on the peroxisomal ABC half transporter proteins Pxa1p and Pxa2p, and one for free fatty acids, which depends on the peroxisomal acyl-CoA synthetase Faa2p and the ATP transporter Ant1p. A proton gradient across the peroxisomal membrane as part of a proton motive force has been proposed to be required for proper peroxisomal function, but the nature of the peroxisomal pH has remained inconclusive and little is known about its generation. To determine the pH of Sacharomyces cerevisiae peroxisomes in vivo, we have used two different pH-sensitive yellow fluorescent proteins targeted to the peroxisome by virtue of a C-terminal SKL and found the peroxisomal matrix in wild-type cells to be alkaline (pH(per) 8.2), while the cytosolic pH was neutral (pH(cyt) 7.0). No Delta pH was present in ant1 Delta cells, indicating that the peroxisomal pH is regulated in an ATP-dependent way and suggesting that Ant1p activity is directly involved in maintenance of the peroxisomal pH. Moreover, we found a high peroxisomal pH of >8.6 in faa2 Delta cells, while the peroxisomal pH remained 8.1+/-0.2 in pxa2 Delta cells. Our combined results suggest that the proton gradient across the peroxisomal membrane is dependent on Ant1p activity and required for the beta-oxidation of medium chain fatty acids.