Beta-oxidation Of Long-chain Fatty Acids Research Articles

Human inborn errors of long-chain fatty acid oxidation show impaired inflammatory responses to TLR4-ligand LPS.

Stimulation of mammalian cells with inflammatory inducers such as lipopolysaccharide (LPS) leads to alterations in activity of central cellular metabolic pathways. Interestingly, these metabolic changes seem to be important for subsequent release of pro-inflammatory cytokines. This has become particularly clear for enzymes of tricarboxylic acid (TCA) cycle such as succinate dehydrogenase (SDH). LPS leads to inhibition of SDH activity and accumulation of succinate to enhance the LPS-induced formation of IL-1β. If enzymes involved in beta-oxidation of fatty acids are important for sufficient responses to LPS is currently not clear. Using cells from various patients with inborn long-chain fatty acid oxidation disorders (lcFAOD), we report that disease-causing deleterious variants of Electron Transfer Flavoprotein Dehydrogenase (ETFDH) and of Very Long Chain Acyl-CoA Dehydrogenase (ACADVL), both cause insufficient inflammatory responses to stimulation with LPS. The insufficiencies included reduced TLR4 expression levels, impaired TLR4 signaling, and reduced or absent induction of pro-inflammatory cytokines such as IL-6. The insufficient responses to LPS were reproduced in cells from healthy controls by targeted loss-of-function of either ETFDH or ACADVL, supporting that the deleterious ETFDH and ACADVL variants cause the attenuated responses to LPS. ETFDH and ACADVL encode two distinct enzymes both involved in fatty acid beta-oxidation, and patients with these deficiencies cannot sufficiently metabolize long-chain fatty acids. We report that genes important for beta-oxidation of long-chain fatty acids are also important for inflammatory responses to an acute immunogen trigger like LPS, which may have important implications for understanding infection and other metabolic stress induced disease pathology in lcFAODs.

Management and Outcomes of Very Long-Chain Acyl-CoA Dehydrogenase Deficiency (VLCAD Deficiency): A Retrospective Chart Review.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is a rare genetic condition affecting the mitochondrial beta-oxidation of long-chain fatty acids. This study reports on the clinical outcomes of patients diagnosed by newborn screening with VLCAD deficiency comparing metabolic parameters, enzyme activities, molecular results, and clinical management. It is a single-center retrospective chart review of VLCAD deficiency patients who met the inclusion criteria between January 2002 and February 2020. The study included 12 patients, 7 of whom had an enzyme activity of more than 10%, and 5 patients had an enzyme activity of less than 10%. The Pearson correlation between enzyme activity and the C14:1 level at newborn screening showed a p-value of 0.0003, and the correlation between enzyme activity and the C14:1 level at diagnosis had a p-value of 0.0295. There was no clear correlation between the number of documented admissions and the enzyme activity level. Patients who had a high C14:1 value at diagnosis were started on a diet with a lower percentage of energy from long-chain triglycerides. The C14:1 result at diagnosis is the value that has been guiding our initial clinical management in asymptomatic diagnosed newborns. However, the newborn screening C14:1 value is the most sensitive predictor of low enzyme activity and may help guide dietary management.

Chapter 8 - Adrenoleukodystrophy

X-linked adrenoleukodystrophy (ALD) is a peroxisomal disorder caused by mutations in the ABCD1 gene and characterized by impaired very long-chain fatty acid beta-oxidation. Clinically, male patients develop adrenal failure and progressive myelopathy in adulthood, although the age of onset and rate of progression are highly variable. In addition, 40% of male patients develop a leukodystrophy (cerebral ALD) before the age of 18 years. Women with ALD also develop myelopathy, but generally at a later age than men and with slower progression. Adrenal failure and leukodystrophy are exceedingly rare in women. Allogeneic hematopoietic cell transplantation (HCT), or more recently autologous HCT with ex vivo lentivirally transfected bone marrow, halts the leukodystrophy. Unfortunately, there is no curative treatment for the myelopathy. In this chapter, clinical spectrum of ALD is discussed in detail.

Abstract 1876: Differential Expression of Mitoregulin Could Decrease Long-Chain Fatty Acid Beta-Oxidation in Pancreatic Beta Cells

Abstract 1876: Differential Expression of Mitoregulin Could Decrease Long-Chain Fatty Acid Beta-Oxidation in Pancreatic Beta Cells

Phosphoglycerate Mutase 5 Expression Modulates Hepatocellular Steatosis

Phosphoglycerate Mutase 5 (PGAM5) is a serine/threonine phosphatase that plays a role in oxidant injury sensing, mitochondrial biogenesis, mitophagy, and multiple cell death pathways.Pgam5 null mice are protected from high‐fat diet induced obesity. This metabolic phenotype was attributed to upregulation of adaptive thermogenesis in brown adipose tissue, but hepatocyte specific lipid metabolism has not been evaluated. We hypothesize that hepatic PGAM5 expression level modulates hepatic steatosis. Using primary cultures of Pgam5 knockout hepatocytes, we have shown that palmitate induced steatosis is attenuated and that treatment with the long‐chain fatty acid impairs oxygen consumption rate specifically reducing the maximal respiratory capacity. Further, over‐expression of PGAM5 in human hepatoma cells exacerbates palmitate induced steatosis. Cumulatively, these results suggest that PGAM5 regulates hepatic lipogenesis and mitochondrial long‐chain fatty acid beta‐oxidation.

Nontargeted serum metabolomics analysis and potential biomarkers for systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a multisystem chronic autoimmune disease characterized by specific clinical and serological changes. Currently, the pathogenesis of SLE remains unclear. This study aimed to identify potential diagnostic biomarkers for SLE and discover the metabolic pathways associated with the disease. We collected serum samples from 260 subjects, including patients with SLE and healthy controls (HCs). Metabolite profiles were analyzed using a nontargeted gas chromatography-mass spectrometry (GC–MS) serum metabolomics method, and differentially altered metabolites were screened and assessed using univariate and multivariate analyses. In this study, patients with SLE and HCs were distinguished by 14 significant metabolites. The levels of pipecolinic acid, glutamine, creatine, stearic acid, asparagine and uric acid were increased in serum samples from patients with SLE, whereas levels of allo-inositol, trans-4-hydroxy-L-proline, glycerol-1-phosphate, N-acetyl-L-aspartic acid, malic acid, phosphate, arabitol and ribose were decreased. Arabitol, asparagine and stearic acid were identified as potential biomarkers, and their diagnostic performance was evaluated by performing a receiver operating characteristic (ROC) curve analysis. Significantly altered pathways included aspartate metabolism, glycine and serine metabolism, cardiolipin biosynthesis, riboflavin metabolism, plasmalogen synthesis, mitochondrial beta-oxidation of long-chain saturated fatty acids, ammonia recycling, pentose phosphate pathway, arginine and proline metabolism and purine metabolism. By identifying serum biomarkers and analyzing metabolic pathways, our study contributes to a better understanding of the pathogenesis of SLE.

Strawberry fruit (Fragaria x ananassa Romina) juice attenuates oxidative imbalance with concomitant modulation of metabolic indices linked to male infertility in testicular oxidative injury.

This study investigated the protective properties of strawberry fruit on testicular oxidative injury. Oxidative injury was induced in vitro in testicular tissue homogenates by incubation with ferrous sulphate (FeSO4 ) in the presence and absence of strawberry fruit extract (SFE) for 30min at 37˚C, with gallic acid serving as the standard antioxidant drug. Induction of oxidative injury significantly reduced glutathione, cholesterol and triglyceride levels; and inhibited SOD, catalase and ENTPDase activities when compared to normal control. It also led to exacerbated nitric oxide, malondialdehyde, LDL-cholesterol levels, acetylcholinesterase, ATPase and lipase activities. These effects were, however, reversed following treatment with SFE when compared to the untreated control, except for cholesterol and triglyceride levels. Additionally, the induction of the oxidative injury led to alterations in testicular lipid metabolites that were accompanied by the activation of α-linolenic acid and linoleic acid metabolic pathways. While SFE treatment had no significant impact on the altered metabolites, it repressed pathways for mitochondrial beta-oxidation of long-chain saturated fatty acids and plasmalogen synthesis. High-performance liquid chromatography analysis of SFE revealed the presence of rutin, caffeic acid, p-coumarin and cinnamic acid. These data imply the protective potentials of strawberry fruits against testicular oxidative injury.

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.

Chapter 18 - Endocrine dysfunction in adrenoleukodystrophy

X-linked adrenoleukodystrophy (ALD) is a peroxisomal disorder caused by mutations in the ABCD1 gene and characterized by impaired very long-chain fatty acid beta-oxidation. Clinically, male patients develop adrenal failure and a progressive myelopathy in adulthood, although age of onset and rate of progression are highly variable. Additionally, 40% of male patients develop a leukodystrophy (cerebral ALD) before the age of 18 years. Women with ALD also develop a myelopathy but generally at a later age than men and with slower progression. Adrenal failure and leukodystrophy are exceedingly rare in women. Allogeneic hematopoietic cell transplantation (HCT), or more recently autologous HCT with ex vivo lentivirally transfected bone marrow, halts the leukodystrophy. Unfortunately, there is no curative treatment for the myelopathy. In the following chapter, the biochemistry, pathology, and clinical spectrum of ALD are discussed in detail.

Analytical Methods for Quantitative Plasma Carnitine Determination.

Carnitine is an essential molecule for mitochondrial beta-oxidation of long-chain fatty acids and other cellular functions. Several rare, inherited disorders of carnitine metabolism occur in humans, and secondary carnitine deficiency is an important feature in a variety of clinical settings. Many of these conditions can be detected via quantitative analysis of free and esterified carnitine in plasma or urine, which thus offers an effective means for assessing the transport and initial processing of fatty acids. Here, we describe some of the methods most commonly employed for quantification of plasma carnitine and consider some of the advantages and disadvantages of these approaches. © 2019 by John Wiley & Sons, Inc.

L-Carnitine Does Not Reduce Cytotoxicity from Induction Chemotherapy for Acute Lymphoblastic Leukemia

IntroductionOff-target effects of induction therapy to treat Acute Lymphoblastic Leukemia (ALL) can lead to hepatotoxicity, cardiotoxicity, and neuropathy, particularly in patients with previous co-morbidities. Incorporation of pegylated L-asparaginase (L-ASP) into pediatric and adult ALL induction regimens has increased remission rate but results in severe hepatotoxicity. Levocarnitine (L-carnitine), a small amino-acid derivative, has been reported in small case series in children and adults with ALL to reduce asparaginase-induced hepatotoxicity. L-carnitine functions as a transport molecule and facilitates long-chain free fatty acid beta-oxidation; exogenous supplementation with L-carnitine may therefore protect hepatocytes through facilitation of fatty acid transport and maintenance of antioxidant balance in mitochondria. However, the effects of L-carnitine on ALL cell survival and possible contributions of L-carnitine to chemotherapy resistance in ALL are not known. Given the ability of L-carnitine to enhance energy production, we sought to investigate whether L-carnitine could compromise the efficacy of common induction chemotherapy agents against ALL cells.MethodsHuman ALL cells, BV173 and RS4;11 were treated with L-carnitine and either daunorubicin (DNR) or vincristine (VCR) for 72 hours (n=4-6). RS4;11 cells were additionally treated with L-ASP (n=4), and current testing to assess dexamethasone in RS4;11 is in progress. Experiments were performed in 96-well culture plates with a seeding density of 90,000-120,000 cells/well (~5 x105 cells/mL). Growth media (RPMI 1640, 1% sodium pyruvate, 1% glutamine, 10% fetal bovine serum) was supplemented to physiologic (50 μM) and supraphysiologic (100-200 μM) levels of L-carnitine (normal reference range: 25-70 μM). Chemotherapy doses were chosen to represent ~EC90 concentrations (BV173: 2 nM VCR and 25 nM DNR; RS4;11: 2 nM VCR, 20 nM DNR, and 0.1 IU/mL L-ASP). After 72 hours, cell viability was measured by trypan blue exclusion and confirmed with spectrophotometric analysis with the Alamar Blue cell viability assay. Data were analyzed using one-way analysis of variance (ANOVA) with significance defined as p<0.05.ResultsAddition of L-carnitine did not significantly affect cytotoxicity of VCR or DNR in BV173 (n=6) or cytotoxicity of VCR, DNR, or L-ASP in RS4;11 (n=4) as measured by trypan blue exclusion at 72 hours (Figure 1A). L-carnitine also did not affect cell viability assessed using Alamar Blue colorimetric assay, presented as the difference in reduction of resazurin (resorufin) compared to untreated RS4;11 or BV173 cells (Figure 1B).ConclusionOur data show L-carnitine has no significant effect on the cytotoxicity of VCR, L-ASP, or DNR in two human ALL cell lines. For providers incorporating L-carnitine into induction therapy to reduce asparaginase-induced hepatotoxicity, this data supports its use without compromising the efficacy of induction therapy. Further investigation is necessary to determine whether L-carnitine alters efficacy of glucocorticoid drugs, particularly dexamethasone, and other chemotherapies used in the treatment of ALL and across other ALL subtypes. DisclosuresNo relevant conflicts of interest to declare.

HADHA, the alpha subunit of the mitochondrial trifunctional protein, is involved in long-chain fatty acid-induced autophagy in intestinal epithelial cells

Genome-wide association studies have identified autophagy-related susceptibility genes for inflammatory bowel disease (IBD); however, whether autophagy regulators can be utilized as therapeutic targets remains unclear. To identify novel microtubule-associated protein 1 light chain 3 (LC3)-interacting proteins in intestinal epithelial cells (IECs), we isolated primary IECs from green fluorescent protein (GFP)-LC3 mice. We performed immunoprecipitation with a GFP antibody and then analyzed co-immunoprecipitates by mass spectrometry. HADHA was identified as an LC3-interacting protein from primary IECs. The HADHA gene encodes the alpha subunit of the mitochondrial trifunctional protein. Given that HADHA catalyzes the last three steps of mitochondrial beta-oxidation of long-chain fatty acids, we investigated whether long-chain fatty acids induce autophagy in IECs. We found that palmitic acid induced autophagy in DLD-1, HT29, and HCT116 cells. HADHA was expressed in not only the mitochondria but also the cytosol. LC3 puncta co-localized with HADHA, which were enhanced by palmitic acid stimulation. However, LC3 puncta did not co-localize with Tom20, suggesting that HADHA was induced to associate with LC3 puncta at sites other than the mitochondria. Thus, HADHA may have extra-mitochondrial functions. Furthermore, we found that palmitic acid induced cell death in IECs, which was accelerated by bafilomycin A and chloroquine. These findings suggested that palmitic acid-induced autophagy supports the survival of IECs. Taken together, these results suggested that HADHA is involved in long-chain fatty acid-induced autophagy in IECs, thus providing new insights into the pathology of IBD and revealing novel therapeutic targets of IBD.

Renal Fanconi Syndrome Is Caused by a Mistargeting-Based Mitochondriopathy.

We recently reported an autosomal dominant form of renal Fanconi syndrome caused by a missense mutation in the third codon of the peroxisomal protein EHHADH. The mutation mistargets EHHADH to mitochondria, thereby impairing mitochondrial energy production and, consequently, reabsorption of electrolytes and low-molecular-weight nutrients in the proximal tubule. Here, we further elucidate the molecular mechanism underlying this pathology. We find that mutated EHHADH is incorporated into mitochondrial trifunctional protein (MTP), thereby disturbing β-oxidation of long-chain fatty acids. The resulting MTP deficiency leads to a characteristic accumulation of hydroxyacyl- and acylcarnitines. Mutated EHHADH also limits respiratory complex I and corresponding supercomplex formation, leading to decreases in oxidative phosphorylation capacity,mitochondrial membrane potential maintenance, and ATP generation. Activity of the Na(+)/K(+)-ATPase is thereby diminished, ultimately decreasing the transport activity of the proximal tubule cells.

The Carnitine Palmitoyl Transferase (CPT) System and Possible Relevance for Neuropsychiatric and Neurological Conditions.

The carnitine palmitoyl transferase (CPT) system is a multiprotein complex with catalytic activity localized within a core represented by CPT1 and CPT2 in the outer and inner membrane of the mitochondria, respectively. Two proteins, the acyl-CoA synthase and a translocase also form part of this system. This system is crucial for the mitochondrial beta-oxidation of long-chain fatty acids. CPT1 has two well-known isoforms, CPT1a and CPT1b. CPT1a is the hepatic isoform and CPT1b is typically muscular; both are normally utilized by the organism for metabolic processes throughout the body. There is a strong evidence for their involvement in various disease states, e.g., metabolic syndrome, cardiovascular diseases, and in diabetes mellitus type 2. Recently, a new, third isoform of CPT was described, CPT1c. This is a neuronal isoform and is prevalently localized in brain regions such as hypothalamus, amygdala, and hippocampus. These brain regions play an important role in control of food intake and neuropsychiatric and neurological diseases. CPT activity has been implicated in several neurological and social diseases mainly related to the alteration of insulin equilibrium in the brain. These pathologies include Parkinson's disease, Alzheimer's disease, and schizophrenia. Evolution of both Parkinson's disease and Alzheimer's disease is in some way linked to brain insulin and related metabolic dysfunctions with putative links also with the diabetes type 2. Studies show that in the CNS, CPT1c affects ceramide levels, endocannabionoids, and oxidative processes and may play an important role in various brain functions such as learning.

Effects of Palmitate on Mitochondrial Number and PGC1α Splicing in Primary Human Myocytes

PGC1α is an epigenetically regulated transcriptional coactivator that promotes mitochondrial biogenesis. Long chain fatty acids (LCFA), such as palmitate, decrease PGC1α expression in association with decreased mitochondrial function and increased insulin resistance in primary myocytes. Several splice variants of PGC1α have been identified and may compensate for the loss of full length PGC1α; yet, it is unclear if these variants are epigenetically regulated or altered by LCFA treatment in skeletal muscle. Here, we determined the effects of 48h of palmitate treatment on known PGC1α splice variant mRNA levels in cultured and differentiated primary human myocytes (Life Technologies). Genomic DNA and total RNA were isolated and mitochondrial number and PGC1α splice variants' expression, respectively, were determined by real time‐PCR. Palmitate treatment tended to increase mitochondrial number (P = 0.14), despite a significant decrease in the combined expression of all &lt;a name=“OLE_LINK1”&gt;PGC1α&lt;/a&gt; splice variants (P &lt; 0.05). The decrease in combined PGC1α splice variant expression appeared to be due to specific decreases in the unspliced PGC1α and A4 variants, although these were not significantly decreased on their own. In contrast, we observed a significant increase in the A2 splice variant (P &lt; 0.05) and a trend towards an increase in N‐truncated PGC1α (P = 0.11). Palmitate had no significant effect on other PGC1α splice variants. N‐truncated PGC1α has been shown to compensate for loss of unspliced PGC1α. We propose that palmitate‐induced upregulation of A2 and N‐truncated PGC1α may be sufficient to acutely increase mitochondrial number, leading to compensatory increases in the cellular capacity for beta oxidation of LCFA.

ESSENTIAL FATTY ACID DEFICIENCY IN VERY LONG-CHAIN ACYL-COA DEHYDROGENASE DEFICIENT PATIENTS

Introduction: Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD), a long-chain fatty acid (LCFA) beta-oxidation dis order, may be treated with LCFA restriction. As Essential Fatty Acids (EFAs) are LCFAs, patients may be at risk for EFA deficiency. Objectives: Investigate whether LCFA restrictions lead to EFA deficiency in VLCADD and which markers are indicative of EFA deficiency. Methods: Thirty-nine LCFA profiles of 16 VLCADD patients were determined in erythrocytes and compared to 48 healthy controls. The predictive value of EFA deficiency markers was calculated from data of a historic cohort (n=4523, 0-39yrs). Results: Linoleic acid (LA), dihomo-γ-linolenic acid (DHLA) and eicosapentaenoic acid (EPA) were significantly decreased in VLCADD patients. Patients on docosahexaenoic acid (DHA) and arachidonic acid (AA) supplementation exhibited even lower LA. Mead acid, a presumed marker for EFA-deficiency, was not increased in patients. In the historic cohort, sensitivity of MA was low for LA deficiency (24% for levels <2.5 percentile) and for DHA+AA deficiency (12% for levels <2.5 percentile). Discussion: VLCADD patients on LCFA restriction are prone to develop LA deficiency. Furthermore, MA is a specific, but not a sensitive marker for LA or EFA deficiency, neither in VLCADD patients, nor in healthy controls, nor in a large patient cohort.

Neuronal Carnitine Palmitoyl Transferase1c in the Central Nervous System: Current Visions and Perspectives

The Carnitine Palmitoyl Transferase (CPT) system comprises several metabolically important enzymes of the carnitine/choline acyl transferase family. CPTs are transferases that allow the energy-neutral replacement of coenzyme A (CoA) by L-carnitine, and vice versa, when bound to acyl chains as (thio) esters. Together with acylCoA synthases and a carnitine/acyl carnitine translocase, the mitochondrial beta-oxidation of long-chain fatty acids is facilitated by distinct CPT proteins. CPT deficiencies manifest as disorders of mitochondrial fatty acid oxidation. Recently a new CPT isoform, CPT1c has been described which is found in entusiasmoc reticulum (ER) of neurons. Unlike CPT1a and CPT1b it has lower palmitoyl transferase activity. It is localised mainly in hypothalamus, amygdala and hippocampus, i.e., brain regions with roles in the control of food intake. It plays a role in energy homeostasis but its exact role in physiology of neurons is still not clear. Studies suggest a biosynthetic rather than catabolic role in long chain acyl carnitine production. The role of CPT1c may extend beyond simply the interchange of CoA and L-carnitine to acyl groups. Studies show that in the CNS the CPT1c affects ceramide levels, endocannabionoids and oxidative processes, and may play an important role in various brain functions such as learning. It is a player in insulin resistance that may occur as a result of oxidative damage and in altered redox status diseases such as metabolic cognitive syndrome, type 2 diabetes, neurodegenerative diseases and cancer. Unhealthy lifestyles, especially high sugar and fat diet in combination with other genetic and environmental risk factors, probably compromise metabolism at various levels. At the cellular level there would be increased ER stress as well as mitochondrial dysfunction leading to impaired oxidative phosphorylation and increased reliance on glucose (Warburg effect). Targeting CPT1c may provide insight on treatment of many metabolically-related diseases as well as pervasive developmental disorders.

Phenotyping a new mitochondrial fatty acid oxidation deficient mouse model

s ingediend voor het Amsterdam Kindersymposium 2013 33 Phenotyping a new mitochondrial fatty acid oxidation deficient mouse model E.F. Diekman (1,2), M.Weeghel (2), R.J. Wanders (2), G. Visser (1), S.M. Houten (2) (1) Department of Metabolic diseases, Wilhelmina Children’s Hospital, UMC Utrecht, the Netherlands (2) Laboratory Genetic Metabolic Diseases, Departments of Clinical Chemistry and Pediatrics, Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, the Netherlands INTRODUCTION Very long chain acyl-CoA dehydrogenase defi ciency (VLCADD) is an inherited disorder of mitochondrial long-chain fatty acid (FAO) beta-oxidation. Patients present with hypoglycaemia, hepatomegaly, cardiomyopathy and myopathy. Although the phenotype of the LCAD KO mouse closely resembles the phenotype in humans, this mouse model does not have myopathy. As mice have two enzymes for degradation of long-chain fatty acids (LCAD and VLCAD), we hypothesised that a model in which one VLCAD allele is deleted in the LCAD KO mouse might have myopathy. LCAD/VLCAD double KO mice are not viable [Cox 2001]. Our objective was therefore to phenotype the LCAD-/-; VLCAD+/mouse model. METHODS We cross-bred LCAD+/-; VLCAD+/mice with LCAD-/-; VLCAD+/+ and characterized the resulting 4 genotypes using metabolic cages. RESULTS Enzymatic activity was signifi cantly decreased and C14:1/C2 acylcarnitine ratio was signifi cantly increased in the LCAD-/-; VLCAD+/mice opposed to all other genotypes. Heat production was decreased in LCAD-/-; VLCAD+/+ and LCAD-/-; VLCAD+/mice, but the RER was not signifi cantly changed. Locomotor activity was reduced in the LCAD-/-; VLCAD+/opposed to all other genotypes. Creatine kinase (CK) levels –a marker for rhabdomyolysiswere measured in plasma of mice of all genotypes after fasting. CK levels were within the normal range of 0-100 U/L. CONCLUSION Our preliminary data (n=4 per genotype) suggest that although LCAD-/-; VLCAD+/mice have lower FAO fl ux compared to LCAD-/-; VLCAD+/+ mice, they do not have myopathy.

Pharmacological and pathophysiological roles of carnitine/organic cation transporters (OCTNs: SLC22A4, SLC22A5 and Slc22a21)

The carnitine/organic cation transporter (OCTN) family consists of three transporter isoforms, i.e. OCTN1 (SLC22A4) and OCTN2 (SLC22A5) in humans and animals and Octn3 (Slc22a21) in mice. These transporters are physiologically essential to maintain appropriate systemic and tissue concentrations of carnitine by regulating its membrane transport during intestinal absorption, tissue distribution and renal reabsorption. Among them, OCTN2 is a sodium-dependent, high-affinity transporter of carnitine, and a functional defect of OCTN2 due to genetic mutation causes primary systemic carnitine deficiency (SCD). Since carnitine is essential for beta-oxidation of long-chain fatty acids to produce ATP, OCTN2 gene mutation causes a range of symptoms, including cardiomyopathy, skeletal muscle weakness, fatty liver and male infertility. These functional consequences of Octn2 gene mutation can be seen clearly in an animal model, jvs mouse, which exhibits the SCD phenotype. In addition, although the mechanism is not clear, single nucleotide polymorphisms of OCTN1 and OCTN2 genes are associated with increased incidences of rheumatoid arthritis, Crohn's disease and asthma. OCTN1 and OCTN2 accept cationic drugs as substrates and contribute to intestinal and pulmonary absorption, tissue distribution (including to tumour cells), and renal excretion of these drugs. Modulation of the transport activity of OCTN2 by externally administered drugs may cause drug-induced secondary carnitine deficiency. Rodent Octn3 transports carnitine specifically, particularly in male reproductive tissues. Thus, the OCTNs are physiologically, pathologically and pharmacologically important. Detailed characterization of these transporters will greatly improve our understanding of the pathology associated with common diseases caused by functional deficiency of OCTNs.

Neonatal carnitine palmitoyltransferase II deficiency associated with Dandy-Walker syndrome and sudden death

Neonatal onset of carnitine palmitoyltransferase II (CPT II) deficiency is an autosomal recessive, often lethal disorder of the mitochondrial beta-oxidation of long-chain fatty acids. It is a rare multiorgan disease which includes hypoketotic hypoglycemia, severe hepatomuscular symptoms, cardiac abnormalities, seizures and lethargy, as well as dysmorphic features. Until now, only 22 affected families have been described in the literature.An increasing number of mutations are being identified in the CPT2 gene, with a distinct genotype–phenotype correlation in most cases. Herein we report a new case of neonatal CPT II deficiency associated with Dandy-Walker syndrome and sudden death at 13days of life. CPT II deficiency was suggested by acylcarnitine analysis of dried-blood on filter paper in the expanded newborn screening. Genetic analysis of the CPT2 gene identified the presence of a previously described mutation in homozygosity (c.534_558del25bpinsT).All lethal neonatal CPT II deficiency patients previously described presented severe symptoms during the first week of life, although this was not the case in our patient, who remained stable and without apparent vital risk during the first 11days of life.The introduction of tandem mass spectrometry to newborn screening has substantially improved our ability to detect metabolic diseases in the newborn period. This case illustrates the value of expanded newborn screening in a neonate with an unusual clinical presentation, combining hydrocephalus and sudden death, that might not commonly lead to the suspicion of an inborn error of metabolism.