Fatty Acid Β-oxidation Defects Research Articles

Heptanoate Improves Compensatory Mechanism of Glucose Homeostasis in Mitochondrial Long-Chain Fatty Acid Oxidation Defect.

Defects in mitochondrial fatty acid β-oxidation (FAO) impair metabolic flexibility, which is an essential process for energy homeostasis. Very-long-chain acyl-CoA dehydrogenase (VLCADD; OMIM 609575) deficiency is the most common long-chain mitochondrial FAO disorder presenting with hypoglycemia as a common clinical manifestation. To prevent hypoglycemia, triheptanoin-a triglyceride composed of three heptanoates (C7) esterified with a glycerol backbone-can be used as a dietary treatment, since it is metabolized into precursors for gluconeogenesis. However, studies investigating the effect of triheptanoin on glucose homeostasis are limited. To understand the role of gluconeogenesis in the pathophysiology of long-chain mitochondrial FAO defects, we injected VLCAD-deficient (VLCAD-/-) mice with 13C3-glycerol in the presence and absence of heptanoate (C7). The incorporation of 13C3-glycerol into blood glucose was higher in VLCAD-/- mice than in WT mice, whereas the difference disappeared in the presence of C7. The result correlates with 13C enrichment of liver metabolites in VLCAD-/- mice. In contrast, the C7 bolus significantly decreased the 13C enrichment. These data suggest that the increased contribution of gluconeogenesis to the overall glucose production in VLCAD-/- mice increases the need for gluconeogenesis substrate, thereby avoiding hypoglycemia. Heptanoate is a suitable substrate to induce glucose production in mitochondrial FAO defect.

Free amino acid and acylcarnitine values in Ursus americanus Pallas 1780 (black bear) from Northeastern Mexico.

Ursus americanus Pallas 1780 is the largest carnivore and the only ursid in Mexico. It is considered an endangered species in the country because its distribution and population have been reduced by up to 80% because of habitat loss or furtive hunting. These problems can lead to a diet change, which could result in metabolic disorders, such as fatty acid β-oxidation defects or organic acid metabolism disorders. In our study, a free amino acid and acylcarnitine profile was characterized. Peripheral blood samples were drawn from nine free-ranging black bears in a period of five months, from June to October of 2019 in Northeastern Mexico, and 12 amino acids and 30 acylcarnitines were determined and quantified. Age differences were observed in the samples through ANOVA and post-hoc Tukey test. Only three metabolites showed a significant difference with age: alanine (Ala) [cubs vs juvenile], free-carnitine (C0) [juvenile vs cubs] and acetylcarnitine (C2) [cubs vs adults and juvenile vs cubs]. Metabolites with variability due to age were identified, making them potential biomarkers to monitor metabolic status as early diagnosis in endangered species. This is the first study of black bear amino acid and acylcarnitine profiles, and the values found could be used as reference for free amino acid and acylcarnitine concentrations in further studies of the species.

Stimulating Mitochondrial Biogenesis with Deoxyribonucleosides Increases Functional Capacity in ECHS1-Deficient Cells.

The lack of effective treatments for mitochondrial disease has seen the development of new approaches, including those that stimulate mitochondrial biogenesis to boost ATP production. Here, we examined the effects of deoxyribonucleosides (dNs) on mitochondrial biogenesis and function in Short chain enoyl-CoA hydratase 1 (ECHS1) ‘knockout’ (KO) cells, which exhibit combined defects in both oxidative phosphorylation (OXPHOS) and mitochondrial fatty acid β-oxidation (FAO). DNs treatment increased mitochondrial DNA (mtDNA) copy number and the expression of mtDNA-encoded transcripts in both CONTROL (CON) and ECHS1 KO cells. DNs treatment also altered global nuclear gene expression, with key gene sets including ‘respiratory electron transport’ and ‘formation of ATP by chemiosmotic coupling’ increased in both CON and ECHS1 KO cells. Genes involved in OXPHOS complex I biogenesis were also upregulated in both CON and ECHS1 KO cells following dNs treatment, with a corresponding increase in the steady-state levels of holocomplex I in ECHS1 KO cells. Steady-state levels of OXPHOS complex V, and the CIII2/CIV and CI/CIII2/CIV supercomplexes, were also increased by dNs treatment in ECHS1 KO cells. Importantly, treatment with dNs increased both basal and maximal mitochondrial oxygen consumption in ECHS1 KO cells when metabolizing either glucose or the fatty acid palmitoyl-L-carnitine. These findings highlight the ability of dNs to improve overall mitochondrial respiratory function, via the stimulation mitochondrial biogenesis, in the face of combined defects in OXPHOS and FAO due to ECHS1 deficiency.

Inborn Errors of Metabolism: Becoming Ready for Rare.

Inborn errors of metabolism (IEMs) are a large group of disorders that can present in any age group and must be considered in the differential diagnosis for a variety of signs and symptoms appearing in infants and children. The rarity and complexity of these conditions often make them difficult to recognize, as they may mimic more common conditions. This review article discusses some of the more commonly presenting IEMs that are important for the general pediatrician to understand when evaluating a sick patient. Many of these diseases are also on the newborn screen, which pediatricians often encounter as first-line providers. Disorders that are discussed in detail herein include disorders of amino acid metabolism, including amino acidopathies and organic acidurias; urea cycle disorders; defects in fatty acid β-oxidation; disorders of carbohydrate metabolism, including the glycogen storage diseases and galactosemia; and lysosomal storage diseases.

A mitochondrial long-chain fatty acid oxidation defect leads to transfer RNA uncharging and activation of the integrated stress response in the mouse heart.

Cardiomyopathy and arrhythmias can be severe presentations in patients with inherited defects of mitochondrial long-chain fatty acid β-oxidation (FAO). The pathophysiological mechanisms that underlie these cardiac abnormalities remain largely unknown. We investigated the molecular adaptations to a FAO deficiency in the heart using the long-chain acyl-CoA dehydrogenase (LCAD) knockout (KO) mouse model. We observed enrichment of amino acid metabolic pathways and of ATF4 target genes among the upregulated genes in the LCAD KO heart transcriptome. We also found a prominent activation of the eIF2α/ATF4 axis at the protein level that was independent of the feeding status, in addition to a reduction of cardiac protein synthesis during a short period of food withdrawal. These findings are consistent with an activation of the integrated stress response (ISR) in the LCAD KO mouse heart. Notably, charging of several transfer RNAs (tRNAs), such as tRNAGln was decreased in LCAD KO hearts, reflecting a reduced availability of cardiac amino acids, in particular, glutamine. We replicated the activation of the ISR in the hearts of mice with muscle-specific deletion of carnitine palmitoyltransferase 2. Our results show that perturbations in amino acid metabolism caused by long-chain FAO deficiency impact cardiac metabolic signalling, in particular the ISR. These results may serve as a foundation for investigating the role of the ISR in the cardiac pathology associated with long-chain FAO defects.Translational Perspective: The heart relies mainly on mitochondrial fatty acid β-oxidation (FAO) for its high energy requirements. The heart disease observed in patients with a genetic defect in this pathway highlights the importance of FAO for cardiac health. We show that the consequences of a FAO defect extend beyond cardiac energy homeostasis and include amino acid metabolism and associated signalling pathways such as the integrated stress response.

Mitochondrial Fatty Acid Oxidation Disorders Associated with Short-Chain Enoyl-CoA Hydratase (ECHS1) Deficiency.

Mitochondrial fatty acid β-oxidation (FAO) is the primary pathway for fatty acid metabolism in humans, performing a key role in liver, heart and skeletal muscle energy homeostasis. FAO is particularly important during times of fasting when glucose supply is limited, providing energy for many organs and tissues, including the heart, liver and brain. Deficiencies in FAO can cause life-threatening metabolic disorders in early childhood that present with liver dysfunction, hypoglycemia, dilated hypertrophic cardiomyopathy and Reye-like Syndrome. Alternatively, FAO defects can also cause ‘milder’ adult-onset disease with exercise-induced myopathy and rhabdomyolysis. Short-chain enoyl-CoA hydratase (ECHS1) is a key FAO enzyme involved in the metabolism of fatty acyl-CoA esters. ECHS1 deficiency (ECHS1D) also causes human disease; however, the clinical manifestation is unlike most other FAO disorders. ECHS1D patients commonly present with Leigh syndrome, a lethal form of subacute necrotizing encephalomyelopathy traditionally associated with defects in oxidative phosphorylation (OXPHOS). In this article, we review the clinical, biochemical and genetic features of the ESHS1D patients described to date, and discuss the significance of the secondary OXPHOS defects associated with ECHS1D and their contribution to overall disease pathogenesis.

Food withdrawal lowers energy expenditure and induces inactivity in long‐chain fatty acid oxidation–deficient mouse models

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is an inherited disorder of mitochondrial long-chain fatty acid β-oxidation (FAO). Patients with VLCAD deficiency may present with hypoglycemia, hepatomegaly, cardiomyopathy, and myopathy. Although several mouse models have been developed to aid in the study of the pathogenesis of long-chain FAO defects, the muscular phenotype is underexposed. To address the muscular phenotype, we used a newly developed mouse model on a mixed genetic background with a more severe defect in FAO (LCAD(-/-); VLCAD(+/-)) in addition to a validated mouse model (LCAD(-/-); VLCAD(+/+)) and compared them with wild-type (WT) mice. We found that both mouse models show a 20% reduction in energy expenditure (EE) and a 3-fold decrease in locomotor activity in the unfed state. In addition, we found a 1.7°C drop in body temperature in unfed LCAD(-/-); VLCAD(+/+) mice compared with WT body temperature. We conclude that food withdrawal-induced inactivity, hypothermia, and reduction in EE are novel phenotypes associated with FAO deficiency in mice. Unexpectedly, inactivity was not explained by rhabdomyolysis, but rather reflected the overall reduced capacity of these mice to generate heat. We suggest that mice are partly protected against the negative consequence of an FAO defect.-Diekman, E. F., van Weeghel, M., Wanders, R. J. A., Visser, G., Houten, S. M. Food withdrawal lowers energy expenditure and induces inactivity in long-chain fatty acid oxidation-deficient mouse models.

Child Neurology: Recurrent rhabdomyolysis due to a fatty acid oxidation disorder

Rhabdomyolysis may result from various factors, namely trauma, exercise, medications, infections, endocrine disorders, congenital myopathies, and metabolic diseases.1 Among the latter, mitochondrial fatty acid β-oxidation (FAO) defects frequently cause recurrent rhabdomyolysis. FAO disorders are recessively inherited and have a combined incidence of 1:9,300, estimated after implementation of newborn screening programs by tandem mass spectrometry (MS/MS).2 Clinical manifestations of these disorders range from sudden infant death to Reye-like syndrome, nonketotic hypoglycemia, skeletal myopathy, peripheral neuropathy, and progressive cardiomyopathy. Here, we describe an 18-month-old child presenting with episodes of recurrent rhabdomyolysis related to mitochondrial trifunctional protein deficiency (MTPD), without additional manifestations of FAO defects. We discuss the diagnosis of MTPD and review the prognosis and treatments. The authors thank Jean Ann Gilder, Scientific Communication srl, for editing the text and Vittorio Lucignano, CEINGE–Biotecnologie Avanzate, for technical assistance.

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

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

Regulation of mitochondrial fatty acid β-oxidation in human: What can we learn from inborn fatty acid β-oxidation deficiencies?

The mitochondrial fatty acid β-oxidation (FAO) pathway plays a crucial role in ATP production in many tissues with high-energy demand. This is highlighted by the diverse and possibly severe clinical manifestations of inborn fatty acid β-oxidation deficiencies. More than fifteen genetic FAO enzyme defects have been described to date, forming a large group of rare diseases. Inborn FAO disorders are characterized by a high genetic heterogeneity, with a variety of gene mutations resulting in complete or partial loss-of-function of the corresponding enzyme. The panel of observed phenotypes varies from multi-organ failure in the neonate with fatal outcome, up to milder late onset manifestations associated with significant disabilities. Diagnosis of FAO disorders has markedly improved over the last decades, but few treatments are available. The clinical, biochemical, and molecular analysis of these disorders provided new, and sometimes unexpected, data on the organization and regulation of mitochondrial FAO in humans, in various tissues, and at various stages of development. This will be illustrated by examples of FAO defects affecting enzymes of long-chain fatty acid import into the mitochondria, or Lynen helix enzymes. The involvement of the transcriptional network regulating FAO gene expression, in particular the PGC-1α/PPAR axis, as a target for pharmacological therapy of these genetic disorders, will also be discussed.

Clinical and biological features at diagnosis in mitochondrial fatty acid beta‐oxidation defects: a French pediatric study of 187 patients

Mitochondrial fatty acid β-oxidation defects (FAODs) are a group of severe inherited metabolic diseases, most of which can be treated with favorable prognosis following diagnosis. A description of the broad range of phenotypes resulting from these defects remains incomplete, and for this study, we sought to investigate the semiology at diagnosis in a country without a newborn screening program for FAODs. Using a retrospective French multicentre study, we analyzed 187 children aged <6years at diagnosis with FAOD confirmed by enzymatic and/or molecular analyses. Clinical and biological parameters at diagnosis were assessed to screen liver, heart, neurological, and muscle symptoms. Information concerning the long-term prognosis was also collected. Predominant hepatic symptoms were observed in 89% of patients regardless of the underlying defect. The most frequent symptoms observed were hepatomegaly (92%), increased blood alanine aminotransferase (ALAT) level (82%), and steatosis (88%). Other frequent features included Reye syndrome (49%), increased gamma-glutamyltranspeptidase (GGT) (37%), and liver failure (27%). Extrahepatic features were often associated in the foreground. Hypoglycemia (75%), neurological (64%), muscle (61%), or cardiac features (55%) [as either cardiomyopathy (47%) or arrhythmias (31%)] were frequently documented. Hemodynamic events (41%) were represented by shock (31%) or sudden death (35%). Hyperammonemia (73%) and hyperlactacidemia (57%) were the two main biochemical features. Total, very-long-chain acyl-CoA dehydrogenase (VLCADD), long-chain 3-hydroxyacylCoA dehydrogenase (LCHADD), and medium-chain acyl-CoA dehydrogenase (MCADD) deficiency mortality rates were 48%, 60%, 63%, and 20% respectively. This study presents clinical features of a large cohort of patients with FAODs in a country without neonatal screening for FAODs. Our results highlight liver as the main organ involved at diagnosis regardless of age at diagnosis, classical phenotype (i.e., cardiac, hepatic, or muscular), or enzyme deficiency. Although steatosis may be observed in various inherited metabolic defects, it is a reliable indicator of FAOD and should prompt systematic screening when the diagnosis is suspected. The poor long-term prognoses reported are a strong argument for inclusion of FAODs in newborn screening programs.

Comorbidity and metabolic context are crucial factors determining neurological sequelae of hypoglycaemia

To determine risk factors for neurological sequelae following hypoglycemia. We analysed the neurological outcome in 164 patients (mean age 10y 10mo, SD 5.9) following hypoglycemia due to three diseases with various metabolic contexts, different ages at onset, and combinations with comorbidity (fever/infection, hypoxia/ischemia): glycogen storage disease type I (GSDI) (21 patients, mean age at first hypoglycemic episode 3.8mo, SD 3.5); fatty acid β-oxidation defects (FAOD) (29 patients, mean age at first hypoglycemic episode 14.8mo, SD 12.6); and hyperinsulinism (HIns) (114 patients, mean age at first hypoglycemic episode 2.3mo, SD 4.7). Risk factors of poor neurological outcome were aetiology (p<0.006), comorbidity (p<0.001), and prolonged convulsions (p<0.001). Ordinal logistic regression showed that comorbidity (p<0.001) and status epilepticus (p=0.002) were the main determinants of sequelae. Asymptomatic hypoglycemia did not lead to sequelae, whatever the aetiology. Age was not correlated to sequelae, whatever the aetiology. The highest prevalence of hypoglycemic sequelae was found in FAOD and HIns combined with comorbidity, the lowest in GSDI (p<0.001) in which hypoglycemia is often asymptomatic, associated with increased plasma lactate, and rarely combined with comorbidity. Hypoglycemia is severely deleterious for the brain in the context of fever/infection and/or hypoxia/ischemia, and status epilepticus. The metabolic context providing alternative fuels may improve neurological outcome.

ACAD expression and role of mitochondrial fatty acid β‐oxidation in retinal pigment epithelium

Mitochondrial fatty acid β‐oxidation (FAO) is the main energy providing pathway in the liver, heart and skeletal muscle particularly during fasting and prolonged exercise. Among several FAO defects, only patients with 3‐hydroxy‐acyl‐CoA dehydrogenase (LCHAD) and mitochondrial trifunctional protein (TFP) are affected with progressive retinopathy, a major concern and long‐term complication of such disorders. Pigmentary retinopathy causes progressive disturbances of retinal pigment epithelium (RPE) leading to accumulation of photo‐oxidized products by inhibiting the phagolysosomal degradation of the outer segment. Accumulated undigested lipids like hydroxylated fatty acids and proteins are thought to cause retinopathy in LCHAD deficiency. To decipher the pathogenetic mechanisms of retinopathy, we did incubation studies on cultured human RPE‐cells with various concentrations of palmitate and hydroxypalmitate with/without carnitine for 48 hrs. Thin cell sections under electron microscope revealed a massive accumulation of rod shaped long tubular formations that could be due to accumulation of complex oxylipids. Further immunoblotting showed robust expression of long and medium chain ACADs and HADH with scant expression of very‐long acyl‐CoA dehydrogenase. We thus conclude that other than TFP all other major FAO enzymes in the FAO pathway are expressed in human RPE cells.

Quantitative liquid chromatography coupled with tandem mass spectrometry analysis of urinary acylglycines: Application to the diagnosis of inborn errors of metabolism

The analysis of urinary acylglycines is an important biochemical tool for the diagnosis of many organic acidemias and mitochondrial fatty acid β-oxidation defects. A new rapid analytical method has been developed for quantification of acylglycines in urine by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The method requires a simple sample preparation avoiding derivatization. It has high sensitivity, specificity, and throughput capability, and it requires minimal instrument maintenance. The use of chromatographic separation allows us to identify and quantify isomeric compounds that cannot be solved by appropriate multiple reaction monitoring (MRM) transitions. Urinary concentrations of the different acylglycines were determined using deuterated internal standards. The reference interval for the various metabolites was established using 120 healthy controls. The diagnostic usefulness of the method was demonstrated in three patients with propionic acidemia (PA), one patient with isovaleric acidemia (IVA), two patients with beta ketothiolase deficiency (BKTD), one patient with short branched chain amino acid deficiency (SBCAD), four patients with medium chain acyl-coenzyme A dehydrogenase deficiency (MCADD), one patient with isobutyryl-coenzyme A dehydrogenase deficiency (IBDHD), and one patient with multiple acyl-coenzyme A dehydrogenase deficiency (MADD).

Analysis of the Mitochondrial Complex I-V Enzyme Activities of Peripheral Leukocytes in Oxidative Phosphorylation Disorders

Mitochondrial oxidative phosphorylation defects are a common cause of mitochondrial diseases, which are characterized by multiorgan involvement and clinically heterogeneous manifestations. Diagnosis is difficult because of the lack of clinically feasible methods. In this study, mitochondrial complex I-V enzyme activity was measured in 64 patients with suspected mitochondrial disease and 200 healthy controls. Spectrophotometric assay was used for the analysis of mitochondrial complex I-V enzyme activity in peripheral leukocytes. Diagnosis was based on clinical presentation, magnetic resonance imaging (MRI), muscle pathology, and point mutation screening in mitochondrial DNA. The differential diagnosis of aminoacidopathies, organic acidurias, and fatty acid β-oxidation defects was made. Thirty-five patients (54.7%) were diagnosed with Leigh syndrome based on characteristic brain MRI. Complex enzyme activity in controls was found to be stable. A deficiency in the oxidative phosphorylation was found in 29 patients (45.3%). Twenty (31.2%) patients had isolated complex defects, complex I deficiency (n = 2, 3.1%), complex II deficiency (n = 3, 4.7%), complex III deficiency (n = 5, 7.8%), complex IV deficiency (n = 5, 7.8%), and complex V deficiency (n = 5, 7.8%). Nine patients were found to have combined deficiencies, 3 (4.7%) had combined deficiencies of complex I and IV, 2 (3.1%) had combined deficiencies of complex III and V, and 2 (3.1%) had a combined deficiency of complex I and V. In conclusion, the peripheral leukocyte oxidative phosphorylation enzyme activity assay was found to be a reliable method for the diagnosis of mitochondrial diseases.

Mitochondrial long chain fatty acid β-oxidation in man and mouse

Several mouse models for mitochondrial fatty acid β-oxidation (FAO) defects have been developed. So far, these models have contributed little to our current understanding of the pathophysiology. The objective of this study was to explore differences between murine and human FAO. Using a combination of analytical, biochemical and molecular methods, we compared fibroblasts of long chain acyl-CoA dehydrogenase knockout (LCAD −/−), very long chain acyl-CoA dehydrogenase knockout (VLCAD −/−) and wild type mice with fibroblasts of VLCAD-deficient patients and human controls. We show that in mice, LCAD and VLCAD have overlapping and distinct roles in FAO. The absence of VLCAD is apparently fully compensated, whereas LCAD deficiency is not. LCAD plays an essential role in the oxidation of unsaturated fatty acids such as oleic acid, but seems redundant in the oxidation of saturated fatty acids. In strong contrast, LCAD is neither detectable at the mRNA level nor at the protein level in men, making VLCAD indispensable in FAO. Our findings open new avenues to employ the existing mouse models to study the pathophysiology of human FAO defects.

Train, train, train! No pain, just gain

Physical training in myopathy: yes or no? This highlights a long-standing dilemma, and one that is still a matter of debate and controversy. Is it beneficial, at least to avoid muscle deconditioning, or detrimental, even harmful, because of the mechanical stress imposed to wrecked myofibres? An initial consideration is that little attention has been given to the different responses to training that the various myopathic conditions can produce. A second, consequential one, is that lack of unequivocal results and flaws in the training protocols let physicians take a somewhat empirical attitude—dictated by personal inclinations, common sense, indulgence toward patient compliance or just prudence inspired by ‘ primum non nocere - first do not harm’, an old evergreen precept (Hippocrates, 460–377 BC). In many cases, the final persuasion is that physical exercise is not recommended, through anxiety over the risk of damaging an already compromised muscle, or even triggering acute episodes of pain, rhabdomyolysis and myoglobinuria. The latter are certainly contraindications to strenuous physical exercise, for instance in several metabolic myopathies related to impaired energy metabolism, such as the lipid storage myopathies, due to defects of intra-mitochondrial transport and utilization of fatty acids (Bruno and DiMauro, 2008; e.g. carnitine-palmitoyl transferase II deficiency, electron transfer flavoprotein dehydrogenase (ETF-DH) deficiency, and very long-chain fatty acid β-oxidation defects) or glycogen (DiMauro, 2007; e.g. myophosphorylase deficiency and defects of intermediary glycolytic enzymes). Some defects of the mitochondrial respiratory chain are also associated with myoglobinuria, notably deficiency of CoQ10 (especially related to ETF-DH deficiency) and mutations in the MTCOB gene (Andreu et al. , 1999), but in most cases neither high creatine kinase nor myoglobinuric episodes are frequent symptoms of mitochondrial disorders. In addition, little, if any, attention is paid to the quantitative and qualitative features of therapeutic physical training (van der Kooi …

A novel functional assay for simultaneous determination of total fatty acid β-oxidation flux and acylcarnitine profiling in human skin fibroblasts using 2H 31-palmitate by isotope ratio mass spectrometry and electrospray tandem mass spectrometry

Background Two separate and complementary assays, total mitochondrial fatty acid β-oxidation (FAO) flux rate and acylcarnitine profiling, have been used to establish a definitive diagnosis of FAO defects (FAOD) in cultured cells. We developed a novel functional assay for total FAO rate assay by measurement of deuterated water enrichment and to combine it with the conventional acylcarnitine profiling method into a single tracer incubation experiment. Methods Skin fibroblasts were incubated in a medium containing universal deuterium-labeled palmitate ( 2H 31-palmitate) and l-carnitine without glucose supplementation for 96 h. The culture medium was assayed for deuterated water enrichment using isotope ratio mass spectrometry (IRMS) and acylcarnitine profiling by electrospray-ionization tandem mass spectrometry (ESI/MS/MS). Results The medians of 2H 2O enrichment after 96 h of incubation of 2H 31-palmitate of the control, other inherited metabolic diseases and FAOD cell lines were 109.9, 102 and 23.1 ppm/mg protein/96 h, respectively. All fibroblasts with FAOD except carnitine uptake defective, multiple acyl-CoA dehydrogenase and short-chain 3-hydroxyacyl-CoA dehydrogenase deficient cells were well separated from the control (< 60% control median, p < 0.05) and could be identified by IRMS assay. Accumulations of disease-specific acylcarnitines due to blockage in the carnitine cycle and FAO spiral were also demonstrated by acylcarnitine profiling. Conclusions This novel functional assay is less time consuming and relatively simple by comparison to other published methods and can be used to investigate patients suspected to have FAO defects.

Medium-chain fatty acids undergo elongation before β-oxidation in fibroblasts

Although mitochondrial fatty acid β-oxidation (FAO) is considered to be well understood, further elucidation of the pathway continues through evaluation of patients with FAO defects. The FAO pathway can be examined by measuring the 3-hydroxy-fatty acid (3-OHFA) intermediates. We present a unique finding in the study of this pathway: the addition of medium-chain fatty acids to the culture media of fibroblasts results in generation of 3-OHFAs which are two carbons longer than the precursor substrate. Cultured skin fibroblasts from normal and LCHAD-deficient individuals were grown in media supplemented with various chain-length fatty acids. The cell-free medium was analyzed for 3-OHFAs by stable-isotope dilution gas-chromatography/mass-spectrometry. Our finding suggests that a novel carbon chain-length elongation process precedes the oxidation of medium-chain fatty acids. This previously undescribed metabolic step may have important implications for the metabolism of medium-chain triglycerides, components in the dietary treatment of a number of disorders.

Differential inhibitory effect of long-chain acyl-CoA esters on succinate and glutamate transport into rat liver mitochondria and its possible implications for long-chain fatty acid oxidation defects

Long-chain fatty acid β-oxidation defects are associated with a series of clinical and biochemical abnormalities, including accumulation of long-chain acyl-CoA esters which have been shown to inhibit several enzymes and transport systems that may disturb energy metabolism. Using isolated rat liver mitochondria incubated under state 3 conditions, we observed that long-chain acyl-CoA esters and their β-oxidation intermediates inhibit ATP synthesis and oxygen consumption, both with succinate (plus rotenone) and l-glutamate as respiratory substrates. When an uncoupler (2,4-dinitrophenol) was used instead of ADP, to stimulate respiration maximally, the various CoA esters showed differential effects on the oxidation of succinate and l-glutamate, respectively. With succinate as substrate, there was a strong inhibition of oxygen consumption by palmitoyl-CoA, 2,3-unsaturated, 3-hydroxy, and 3-keto-palmitoyl-CoA, in coupled as well as uncoupled mitochondria. On the other hand, with l-glutamate as substrate, inhibition was only observed under coupled conditions. The finding that acyl-CoA esters inhibit the uncoupler-induced respiration with succinate as substrate but not with glutamate, indicates that the observed inhibitory effect is most probably at the level of the transport of succinate across the mitochondrial membrane as mediated by the mitochondrial dicarboxylate carrier. This conclusion was substantiated by mitochondrial swelling studies, which showed inhibition of succinate transport by the different CoA esters whereas no effect was observed on the phosphate/hydroxyl and glutamate/hydroxyl carriers. Furthermore, long-chain acyl-CoA esters were found to potentiate the inhibitory effect of N-butylmalonate, a known inhibitor of the dicarboxylate carrier, upon oxygen consumption driven by succinate (plus rotenone). We conclude that the inhibitory effects of long-chain acyl-CoA esters on oxidative phosphorylation are dependent on the type of substrate used with the ATP/ADP carrier and the dicarboxylate carrier as targets for inhibition.