Very Long-chain acyl-CoA Dehydrogenase Deficiency Research Articles

Clinical features and mutations in seven Chinese patients with very long chain acyl-CoA dehydrogenase deficiency.

Very long chain acyl-CoA dehydrogenase deficiency (VLCADD) is an inherited metabolic disease caused by deleterious mutations in the ACADVL gene that encodes very long chain acyl-CoA dehydrogenase (VLCAD), and which can present as cardiomyopathy in neonates, as hypoketotic hypoglycemia in infancy, and as myopathy in late-onset patients. Although many ACADVL mutations have been described, no prevalent mutations in the ACADVL gene have been associated with VLCADD. Herein, we report the clinical course of the disease and explore the genetic mutation spectrum in seven Chinese patients with VLCADD. Seven Chinese patients, from newborn to 17 years old, were included in this study. Tandem mass spectrometry was performed to screen for VLCAD deficiency. All exons and flanking introns of the ACADVL gene were analyzed using polymerase chain reaction and direct sequencing. Online analysis tools were used to predict the impact of novel mutations. All cases had elevated serum levels of tetradecanoylcarnitine (C14:1) which is the characteristic biomarker for VLCADD. The phenotype of VLCADD is heterogeneous. Two patients were hospitalized for hypoactivity and hypoglycemia shortly after birth. Three patients showed hepatomegaly and hypoglycemia in infancy. The other two adolescent patients showed initial manifestations of exercise intolerance or rhabdomyolysis. Three of the patients died at the age of 6-8 months. Eleven different mutations in the ACADVL gene in the 7 patients were identified, including seven reported mutations (p.S22X, p.W427X, p.A213T, p.G222R, p.R450H, c.296-297delCA, c.1605+1G>T) and four novel mutations (p.S72F, p.Q100X, p.M437T, p.D466Y). The p.R450H and p.D466Y (14.28%, 2/14 alleles) mutations were identified in two alleles respectively. The clinical manifestations were heterog-eneous and ACADVL gene mutations were heterozygous in the seven VLCADD Chinese patients. R450H may be a relatively common mutation in Asian populations. The genotype and phenotype had a certain correlation in our patients.

Long-chain Acyl-CoA Dehydrogenase Deficiency as a Cause of Pulmonary Surfactant Dysfunction

Long-chain acyl-CoA dehydrogenase (LCAD) is a mitochondrial fatty acid oxidation enzyme whose expression in humans is low or absent in organs known to utilize fatty acids for energy such as heart, muscle, and liver. This study demonstrates localization of LCAD to human alveolar type II pneumocytes, which synthesize and secrete pulmonary surfactant. The physiological role of LCAD and the fatty acid oxidation pathway in lung was subsequently studied using LCAD knock-out mice. Lung fatty acid oxidation was reduced in LCAD(-/-) mice. LCAD(-/-) mice demonstrated reduced pulmonary compliance, but histological examination of lung tissue revealed no obvious signs of inflammation or pathology. The changes in lung mechanics were found to be due to pulmonary surfactant dysfunction. Large aggregate surfactant isolated from LCAD(-/-) mouse lavage fluid had significantly reduced phospholipid content as well as alterations in the acyl chain composition of phosphatidylcholine and phosphatidylglycerol. LCAD(-/-) surfactant demonstrated functional abnormalities when subjected to dynamic compression-expansion cycling on a constrained drop surfactometer. Serum albumin, which has been shown to degrade and inactivate pulmonary surfactant, was significantly increased in LCAD(-/-) lavage fluid, suggesting increased epithelial permeability. Finally, we identified two cases of sudden unexplained infant death where no lung LCAD antigen was detectable. Both infants were homozygous for an amino acid changing polymorphism (K333Q). These findings for the first time identify the fatty acid oxidation pathway and LCAD in particular as factors contributing to the pathophysiology of pulmonary disease.

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.

Development and pathomechanisms of cardiomyopathy in very long-chain acyl-CoA dehydrogenase deficient (VLCAD−/−) mice

Hypertrophic cardiomyopathy is a typical manifestation of very long-chain acyl-CoA dehydrogenase deficiency (VLCADD), the most common long-chain β-oxidation defects in humans; however in some patients cardiac function is fully compensated. Cardiomyopathy may also be reversed by supplementation of medium-chain triglycerides (MCT). We here characterize cardiac function of VLCAD-deficient (VLCAD−/−) mice over one year. Furthermore, we investigate the long-term effect of a continuous MCT diet on the cardiac phenotype. We assessed cardiac morphology and function in VLCAD−/− mice by in vivo MRI. Cardiac energetics were measured by 31P-MRS and myocardial glucose uptake was quantified by positron-emission-tomography (PET). Metabolic adaptations were identified by the expression of genes regulating glucose and lipid metabolism using real-time-PCR. VLCAD−/− mice showed a progressive decrease in heart function over 12months accompanied by a reduced phosphocreatine-to-ATP-ratio indicative of chronic energy deficiency. Long-term MCT supplementation aggravated the cardiac phenotype into dilated cardiomyopathy with features similar to diabetic heart disease. Cardiac energy production and function in mice with a β-oxidation defect cannot be maintained with age. Compensatory mechanisms are insufficient to preserve the cardiac energy state over time. However, energy deficiency by impaired β-oxidation and long-term MCT induce cardiomyopathy by different mechanisms. Cardiac MRI and MRS may be excellent tools to assess minor changes in cardiac function and energetics in patients with β-oxidation defects for preventive therapy.

Mitochondrial protein acetylation is driven by acetyl-CoA from fatty acid oxidation

Mitochondria integrate metabolic networks for maintaining bioenergetic requirements. Deregulation of mitochondrial metabolic networks can lead to mitochondrial dysfunction, which is a common hallmark of many diseases. Reversible post-translational protein acetylation modifications are emerging as critical regulators of mitochondrial function and form a direct link between metabolism and protein function, via the metabolic intermediate acetyl-CoA. Sirtuins catalyze protein deacetylation, but how mitochondrial acetylation is determined is unclear. We report here a mechanism that explains mitochondrial protein acetylation dynamics in vivo. Food withdrawal in mice induces a rapid increase in hepatic protein acetylation. Furthermore, using a novel LC-MS/MS method, we were able to quantify protein acetylation in human fibroblasts. We demonstrate that inducing fatty acid oxidation in fibroblasts increases protein acetylation. Furthermore, we show by using radioactively labeled palmitate that fatty acids are a direct source for mitochondrial protein acetylation. Intriguingly, in a mouse model that resembles human very-long chain acyl-CoA dehydrogenase (VLCAD) deficiency, we demonstrate that upon food-withdrawal, hepatic protein hyperacetylation is absent. This indicates that functional fatty acid oxidation is necessary for protein acetylation to occur in the liver upon food withdrawal. Furthermore, we now demonstrate that protein acetylation is abundant in human liver peroxisomes, an organelle where acetyl-CoA is solely generated by fatty acid oxidation. Our findings provide a mechanism for metabolic control of protein acetylation, which provides insight into the pathophysiogical role of protein acetylation dynamics in fatty acid oxidation disorders and other metabolic diseases associated with mitochondrial dysfunction.

Infants suspected to have very-long chain acyl-CoA dehydrogenase deficiency from newborn screening

Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is a fatty acid oxidation disorder with widely varying presentations that has presented a significant challenge to newborn screening (NBS). The Western States Regional Genetics Services Collaborative developed a workgroup to study infants with NBS positive for VLCADD. We performed retrospective analysis of newborns with elevated C14:1-acylcarnitine on NBS in California, Oregon, Washington, and Hawai'i including available confirmatory testing and clinical information.Overall, from 2,802,504 children screened, there were 242 cases screen-positive for VLCADD. There were 34 symptomatic true positive cases, 18 asymptomatic true positives, 112 false positives, 55 heterozygotes, 11 lost to follow-up, and 12 other disorders. One in 11,581 newborns had an abnormal NBS for suspected VLCADD. Comparison of analytes and analyte ratios from the NBS demonstrated statistically significant differences between true positive and false positive groups for C14:1, C14, C14:1/C2, and C14:1/C16. The positive predictive value for all true positive cases was 94%, 54%, and 23% when C14:1 was ≥2.0μM, ≥1.0μM, and ≥0.7μM, respectively. Sequential post-analytical analysis could reduce the referral rate in 25.8% of cases.This study is the largest reported follow-up of infants with NBS screen-positive results for suspected VLCADD and demonstrates the necessity of developing comprehensive and consistent long-term follow-up NBS systems. Application of clinical information revealed differences between symptomatic and asymptomatic children with VLCADD. Comparison of NBS analytes and analyte ratios may be valuable in developing more effective diagnostic algorithms.

Bezafibrate in skeletal muscle fatty acid oxidation disorders

To assess whether bezafibrate increases fatty acid oxidation (FAO) and lowers heart rate (HR) during exercise in patients with carnitine palmitoyltransferase (CPT) II and very long-chain acyl-CoA dehydrogenase (VLCAD) deficiencies. This was a 3-month, randomized, double-blind, crossover study of bezafibrate in patients with CPT II (n = 5) and VLCAD (n = 5) deficiencies. Primary outcome measures were changes in FAO, measured with stable-isotope methodology and indirect calorimetry, and changes in HR during exercise. Bezafibrate lowered low-density lipoprotein, triglyceride, and free fatty acid concentrations; however, there were no changes in palmitate oxidation, FAO, or HR during exercise. Bezafibrate does not improve clinical symptoms or FAO during exercise in patients with CPT II and VLCAD deficiencies. These findings indicate that previous in vitro studies suggesting a therapeutic potential for fibrates in disorders of FAO do not translate into clinically meaningful effects in vivo. This study provides Class I evidence that bezafibrate 200 mg 3 times daily is ineffective in improving changes in FAO and HR during exercise in adults with CPT II and VLCAD deficiencies.

Muscle MRI in patients with long‐chain fatty acid oxidation disorders

Muscle magnetic resonance imaging (MRI) is a useful tool for visualizing abnormalities in neuromuscular disorders. The value of muscle MRI has not been studied in long-chain fatty acid oxidation (lcFAO) disorders. LcFAO disorders may present with metabolic myopathy including episodic rhabdomyolysis. To investigate whether lcFAO disorders are associated with muscle MRI abnormalities. Lower body MRI was performed in 20 patients with lcFAO disorders, i.e. three carnitine palmitoyltransferase 2 deficiency (CPT2D), 12 very long-chain acyl-CoA dehydrogenase deficiency (VLCADD), three mitochondrial trifunctional protein deficiency (MTPD) and two isolated long-chain hydroxyacyl-CoA dehydrogenase deficiency (LCHADD). At the time of MRI, four patients had muscle weakness, 14 had muscle pain and 13 were exercise intolerant. Median creatine kinase (CK) level of patients at the day of MRI was 398 U/L (range 35-12,483). T1W and STIR signal intensity (SI) were markedly increased in MTPD patients from girdle to lower leg. VLCADD patients showed predominantly proximal T1W SI changes, whereas LCHADD patients mostly showed distal T1W SI changes. Prominent STIR weighted signal intensity increases of almost all muscle groups were observed in patients with VLCADD and LCHADD with very high CK (>11.000) levels. lcFAO disorders are associated with specific patterns of increased T1W and STIR signal intensity. These patterns may reflect lipid accumulation and inflammation secondary to lcFAO defects and progressive muscle damage. Future studies are needed to investigate whether muscle MRI might be a useful tool to monitor disease course and to study pathogenesis of lcFAO related myopathy.

VLCAD deficiency in a patient who recovered from ventricular fibrillation, but died suddenly of a respiratory syncytial virus infection

VLCAD deficiency is an autosomal recessive disorder caused by a defect of fatty acid oxidation. The phenotype is classified into three clinical forms on the basis of the onset of symptoms: a severe form with neonatal onset; a milder form with childhood onset; and a late-onset form. The neonatal form is the most common, and has a higher mortality rate than the others. We report the case of a newborn infant with VLCAD deficiency who developed ventricular fibrillation, which was successfully treated by intensive care, but who suddenly died after a respiratory syncytial virus infection. Early institution of i.v. glucose treatment and active immunization with vaccine, such as palivizumab (anti-RSV mAb), may be important to reduce the frequency and severity of life-threatening episodes.

Intermittent Rhabdomyolysis With Adult Onset Associated With a Mutation in the ACADVL Gene

Deficiency of very-long-chain acyl-CoA dehydrogenase (VLCAD) is an autosomal recessive disease. Most common phenotypes occur in the neonatal period or in childhood with cardiomyopathy, hepatomegaly, and hypoketogenic hypoglycemia. Juvenile/adult-onset is characterized by exercise intolerance and recurrent rhabdomyolysis triggered by prolonged exercise or fasting. This article reports a patient with the homozygous mutation c.1097G>A (p.R366H) in the ACADVL gene. In Portugal, VLCAD deficiency became part of the neonatal screening plan in 2004, and as of 2012, 8 early-onset cases have been diagnosed, giving an incidence rate of 1:97.238 per 737.902 newborns. This patient was diagnosed outside of the neonatal screening plan. Beta-oxidation defects pose a diagnostic challenge because of their transient clinical and laboratorial manifestations and the absence of morphological changes in muscle biopsy further complicate matters, especially in the late-onset forms of the disease. The adult phenotype of VLCAD deficiency is highlighted, emphasizing the need for a high suspicion index and the value of tandem mass spectrometry for the diagnosis.

Cardiac-specific VLCAD deficiency induces dilated cardiomyopathy and cold intolerance

The very long-chain acyl-CoA dehydrogenase (VLCAD) enzyme catalyzes the first step of mitochondrial β-oxidation. Patients with VLCAD deficiency present with hypoketotic hypoglycemia and cardiomyopathy, which can be exacerbated by fasting and/or cold stress. Global VLCAD knockout mice recapitulate these phenotypes: mice develop cardiomyopathy, and cold exposure leads to rapid hypothermia and death. However, the contribution of different tissues to development of these phenotypes has not been studied. We generated cardiac-specific VLCAD-deficient (cVLCAD(-/-)) mice by Cre-mediated ablation of the VLCAD in cardiomyocytes. By 6 mo of age, cVLCAD(-/-) mice demonstrated increased end-diastolic and end-systolic left ventricular dimensions and decreased fractional shortening. Surprisingly, selective VLCAD gene ablation in cardiomyocytes was sufficient to evoke severe cold intolerance in mice who rapidly developed severe hypothermia, bradycardia, and markedly depressed cardiac function in response to fasting and cold exposure (+5°C). We conclude that cardiac-specific VLCAD deficiency is sufficient to induce cold intolerance and cardiomyopathy and is associated with reduced ATP production. These results provide strong evidence that fatty acid oxidation in myocardium is essential for maintaining normal cardiac function under these stress conditions.

ACAD9, a complex I assembly factor with a moonlighting function in fatty acid oxidation deficiencies

Oxidative phosphorylation and fatty acid oxidation are two major metabolic pathways in mitochondria. Acyl-CoA dehydrogenase 9 (ACAD9), an enzyme assumed to play a role in fatty acid oxidation, was recently identified as a factor involved in complex I biogenesis. Here we further investigated the role of ACAD9's enzymatic activity in fatty acid oxidation and complex I biogenesis. We provide evidence indicating that ACAD9 displays enzyme activity in vivo. Knockdown experiments in very-long-chain acyl-CoA dehydrogenase (VLCAD)-deficient fibroblasts revealed that ACAD9 is responsible for the production of C14:1-carnitine from oleate and C12-carnitine from palmitate. These results explain the origin of these obscure acylcarnitines that are used to diagnose VLCAD deficiency in humans. Knockdown of ACAD9 in control fibroblasts did not reveal changes in the acylcarnitine profiles upon fatty acid loading. Next, we investigated whether catalytic activity of ACAD9 was necessary for complex I biogenesis. Catalytically inactive ACAD9 gave partial-to-complete rescue of complex I biogenesis in ACAD9-deficient cells and was incorporated in high-molecular-weight assembly intermediates. Our results underscore the importance of the ACAD9 protein in complex I assembly and suggest that the enzymatic activity is a rudiment of the duplication event.

The effect of valinomycin in fibroblasts from patients with fatty acid oxidation disorders

Disorders of the carnitine cycle and of the beta oxidation spiral impair the ability to obtain energy from fats at time of fasting and stress. This can result in hypoketotic hypoglycemia, cardiomyopathy, cardiac arrhythmia and other chronic medical problems. The in vitro study of fibroblasts from patients with these conditions is impaired by their limited oxidative capacity. Here we evaluate the capacity of valinomycin, a potassium ionophore that increases mitochondrial respiration, to increase the oxidation of fatty acids in cells from patients with inherited fatty acid oxidation defects. The addition of valinomycin to fibroblasts decreased the accumulation of the lipophilic cation tetraphenylphosphonium (TPP+) at low concentrations due to the dissipation of the mitochondrial membrane potential. At higher doses, valinomycin increased TPP+ accumulation due to the increased potassium permeability of the plasma membrane and subsequent cellular hyperpolarization. The incubation of normal fibroblasts with valinomycin increased [14C]-palmitate oxidation (measured as [14C]O2 release) in a dose-dependent manner. By contrast, valinomycin failed to increase palmitate oxidation in fibroblasts from patients with very long chain acyl CoA dehydrogenase (VLCAD) deficiency. This was not observed in fibroblasts from patients heterozygous for this condition. These results indicate that valinomycin can increase fatty acid oxidation in normal fibroblasts and could be useful to differentiate heterozygotes from patients affected with VLCAD deficiency.

Molecular and cellular pathology of very-long-chain acyl-CoA dehydrogenase deficiency

BackgroundVery-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD) is diagnosed in the US through newborn screening (NBS). NBS often unequivocally identifies affected individuals, but a growing number of variant patterns can represent mild disease or heterozygous carriers. AimsTo evaluate the validity of standard diagnostic procedures for VLCADD by using functional in vitro tools. MethodsWe retrospectively investigated 13 patient samples referred to our laboratory because of a suspicion of VLCADD but with some uncertainty to the diagnosis. All 13 patients were suspected of having VLCADD either because of abnormal NBS or suggestive clinical findings. ACADVL genomic DNA sequencing data were available for twelve of them. Ten of the patients had an abnormal NBS suggestive of VLCADD, with three samples showing equivocal results. Three exhibited suggestive clinical findings and blood acylcarnitine profile (two of them had a normal NBS and the third one was unscreened). Assay of VLCAD activity and immunoblotting or immunohistologic staining for VLCAD were performed on fibroblasts. Prokaryotic mutagenesis and expression studies were performed for nine uncharacterized ACADVL missense mutations. ResultsVLCAD activity was abnormal in fibroblast cells from 9 patients (8 identified through abnormal NBS, 1 through clinical symptoms). For these 9 patients, immunoblotting/staining showed the variable presence of VLCAD; all but one had two mutated alleles. Two patients with equivocal NBS results (and a heterozygous genotype) and the two patients with normal NBS exhibited normal VLCAD activity and normal VLCAD protein on immunoblotting/staining thus ruling out VLCAD deficiency. Nine pathogenic missense mutations were characterized with prokaryotic expression studies and showed a decrease in enzyme activity and variable stability of VLCAD antigen. ConclusionsThese results emphasize the importance of functional investigation of abnormal NBS or clinical testing suggestive but not diagnostic of VLCADD. A larger prospective study is necessary to better define the clinical and metabolic ramifications of the defects identified in such patients.

Molecular Diagnosis for a Fatal Case of Very Long-chain Acyl-CoA Dehydrogenase Deficiency in Hong Kong Chinese With a Novel Mutation

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is one of the most common fatty acid oxidation defects that cause sudden unexpected deaths in infants. The death attributed to VLCAD deficiency can be prevented by early diagnosis with expanded newborn screening using tandem mass spectrometry. A favorable outcome can be achieved with early diagnosis and prompt treatment. However, such newborn screening has not yet been available in Hong Kong. We report a 2-month-old boy who succumbed 5 hours after admission with the diagnosis of VLCAD deficiency confirmed by genetic analysis performed after death. The patient was compound heterozygous for a novel splicing mutation ACADVL NM_000018.2:c.277+2T>G; NC_000017.10:g.7123997T>G and a known disease-causing mutation ACADVL NM_000018.2:c.388_390del; NP_000009.1: p.Glu130del. Family screening was performed for at-risk siblings. The rapid downhill course of the patient clearly illustrates the need of newborn screening for early diagnosis. Our patient was asymptomatic before metabolic decompensation. However, once metabolic decompensation occurred, rapid deterioration and death followed, which obviated the opportunity to diagnose and treat. The only way to save these patients' lives and improve their outcome is early diagnosis and appropriate treatment. Therefore, we strongly urge the implementation of newborn screening using tandem mass spectrometry for VLCAD deficiency and other highly treatable inborn errors of metabolism in Hong Kong.

T.P.47 Bezafibrate does not improve fat oxidation in patients with disorders of fat metabolism; a double blind, randomized clinical trial

Bezafibrate up-regulate enzyme activities of carnitine palmitoyltransferase (CPT) II and very long-chain acyl-CoA-dehydrogenase (VLCAD) in patients with defects in these enzyme, when assessed in vitro and increased quality of life scores in an uncontrolled study in six CPT II patients. In CPT II and VLCAD deficiencies, fatty acid oxidation (FAO) is normal at rest, but residual enzyme activity is insufficient to increase FAO during exercise. We hypothesized that Bezafibrate would increase FAO and lower perceived exertion during exercise in patients with CPT II and VLCAD deficiencies. We studied the effect of Bezafibrate/placebo on total FAO by indirect calorimetry and palmitate turnover using stable isotope methodology during cycling exercise in 10 patients with recurrent episodes of rhabdomyolysis caused by CPT II (n=5) and VLCAD (n=5) deficiencies. The study design was 3-month, randomized, double-blind, cross-over (EudraCT: 2008-006704-27). Primary endpoints were FAO and heart rate during exercise. Bezafibrate lowered LDL, triglyceride and free fatty acid concentrations, but palmitate oxidation and total FAO at rest and during exercise, as well as activity levels scored by Bouchard questionnaires, were not changed by Bezafibrate. Bezafibrate does not improve clinical symptoms or FAO during exercise in patients with CPT II and VLCAD deficiencies. These findings indicate that previous in vitro studies suggesting a therapeutic potential for fibrates, do not translate into clinically meaningful effects in vivo.

Anesthetic management for a patient with very-long-chain acyl-coenzyme A dehydrogenase deficiency

To the Editor: A 28-year-old woman with the adult form of very-longchain acyl-coenzyme A dehydrogenase (VLCAD) deficiency was scheduled for hysteroscopic myomectomy. She complained of muscle pain on fasting and hard exercise, which was alleviated by having food or resting. She was taking carnitine and could usually tolerate overnight fasting. So she fasted for 8 h before the operation, and fluid infusion was begun in the operation room with maintenance fluid containing 10 % glucose at 4.5 mg/kg/min. We used diazepam for premedication. General anesthesia was induced with thiopental, remifentanil, and rocuronium and maintained with sevoflurane and remifentanil. A diclofenac suppository was administered for postoperative analgesia. During the surgery, hyperglycemia (254 mg/dl) was treated with insulin (2 units). Lactate was slightly elevated. The operation was completed uneventfully. Neuromuscular blockade was antagonized with sugammadex; she awoke completely and made no complaint of pain, nausea, vomitting or shivering. An infusion containing 5 % glucose was continued until the next morning, with oral feeding being started the afternoon of the same day. VLCAD is one of the enzymes involved in mitochondrial long-chain fatty acid b-oxidation. A lack of VLCAD activity leads to a shortage of the energy normally derived from long-chain fatty acids [1, 2]. This means that energy is supplied only by glucose, amino acids, or lipids other than very-long-chain and long-chain fatty acids. Catabolic fasting, certain stresses, and an elevated energy demand can induce severe metabolic decompensation [2]. Additionally, the increases in acyl-coenzyme A and acylcarnitine are harmful. The symptoms of VLCAD deficiency are derived from organs/tissues such as the heart, liver, and skeletal muscle, where mitochondrial fatty acid b-oxidation is most marked. The severe form of VLCAD deficiency manifests with cardiomyopathy and has a high mortality rate [1]. Treatment of VLCAD deficiency is directed at preventing catabolic situations by the avoidance of prolonged fasting, by avoiding emotional or physical stress, and by dietary restriction of long-chain fatty acids. In the present patient, our aims were: (1) not to elevate plasma free fatty acids, (2) not to increase energy demand, and (3) to give sufficient glucose. We chose general anesthesia to avoid the physical and/or psychological stresses that could be induced by a possibly inadequate level of spinal anesthesia. Some cases have been reported in which propofol or sevoflurane was used safely in patients with VLCAD deficiency [3]. However, propofol contains long-chain fatty acids, and we therefore avoided using this agent. Some stresses (anxiety, shivering, nausea, and vomiting) can increase energy demand, and a previous report has recommended glucose, at 6 mg/kg/min, for perioperative use in infants with VLCAD deficiency; as well, it was suggested that the fasting period should not exceed 4 h in children with this condition [4]. However, it is not clear how much glucose is necessary during an operation in adult patients with VLCAD deficiency. Because glucose demand may decrease with age and because myopathic symptoms did not occur and the creatine kinase level was not elevated just after the operation in our patient, we think that the intraoperative glucose infusion she received was adequate. However, creatine kinase was elevated on postoperative day 2, and we speculate that the postoperative glucose infusion was not K. Iwata K. Tanabe (&) Y. Sugiyama M. Tanaka M. Takenaka H. Iida Department of Anesthesiology and Pain Medicine, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan e-mail: kumiko-t@m2.gyao.ne.jp

Long-term Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency in Mice Using AAV9 Gene Therapy

Very long-chain acyl-coA dehydrogenase (VLCAD) is the rate-limiting step in mitochondrial fatty acid oxidation. VLCAD-deficient mice and patients clinical symptoms stem from not only an energy deficiency but also long-chain metabolite accumulations. VLCAD-deficient mice were treated systemically with 1 × 1012 vector genomes of recombinant adeno-associated virus 9 (rAAV9)-VLCAD. Biochemical correction was observed in vector-treated mice beginning 2 weeks postinjection, as characterized by a significant drop in long-chain fatty acyl accumulates in whole blood after an overnight fast. Changes persisted through the termination point around 20 weeks postinjection. Magnetic resonance spectroscopy (MRS) and tandem mass spectrometry (MS/MS) revealed normalization of intramuscular lipids in treated animals. Correction was not observed in liver tissue extracts, but cardiac muscle extracts showed significant reduction of long-chain metabolites. Disease-specific phenotypes were characterized, including thermoregulation and maintenance of euglycemia after a fasting cold challenge. Internal body temperatures of untreated VLCAD−/− mice dropped below 20 °C and the mice became lethargic, requiring euthanasia. In contrast, all rAAV9-treated VLCAD−/− mice and the wild-type controls maintained body temperatures. rAAV9-treated VLCAD−/− mice maintained euglycemia, whereas untreated VLCAD−/− mice suffered hypoglycemia following a fasting cold challenge. These promising results suggest rAAV9 gene therapy as a potential treatment for VLCAD deficiency in humans.

140. Sex-Dependent Expression of Very Long Chain acyl-CoA Dehydrogenase (VLCAD) Transgene and the Role of the Liver in Biochemical and Phenotypic Correction of VLCAD Deficiency

140. Sex-Dependent Expression of Very Long Chain acyl-CoA Dehydrogenase (VLCAD) Transgene and the Role of the Liver in Biochemical and Phenotypic Correction of VLCAD Deficiency

Abstract 9376: Cardiac-Specific Very Long Chain Acyl-CoA Dehydrogenase Deficiency Induces Dilated Cardiomyopathy and Cold Intolerance in Mice

BACKGROUND: Very long chain acyl-CoA dehydrogenase (VLCAD) catalyzes the initial step of mitochondrial beta-oxidation of fatty acids with chain lengths of 14 to 20 carbons. Patients with VLCAD deficiency present with hypoketotic hypoglycemia, cardiomyopathy and sudden death that can be exacerbated by the stresses of fasting and cold. Global VLCAD knockout (KO) mice recapitulate these phenotypes. However, the contribution of cardiac VLCAD in the development of cardiomyopathy and the response to the environmental stressors is unclear. In this study, we assessed the hypothesis that cardiac-specific VLCAD deficiency is sufficient to induce cardiomyopathy and cold intolerance. METHODS AND RESULTS: A cardiac-specific homozygous VLCAD KO (csVLCAD KO) mouse was generated by crossing a VLCAD floxed-allele mouse with a transgenic mouse expressing Cre recombinase under the control of the α-myosin heavy chain promoter. Echocardiographic analysis at 6 months of age demonstrated left ventricular dilation and decreased fractional shortening (FS) in csVLCAD KO mice (for FS, 21±1% vs 29±4% in wild-type mice, p<0.05). Ultrastructural analysis of ventricular myocardium showed widening of the sarcomeric I band, increased sarcomere length and substantial lipid deposits in csVLCAD KO mice. To test if cardiac VLCAD deficiency leads to susceptibility to death caused by cold stress, csVLCAD-KO mice and controls were placed at 4°C. All csVLCAD-KO mice (n=9) became moribund within 4-10 hours, whereas only 1/9 hemizygous and 1/9 wild-type mice died. Unlike global VLCAD KO mice, csVLCAD KO mice did not develop hypoglycemia. To determine if VLCAD deficiency alters mitochondrial fatty acid oxidation in cardiomyocytes, oxygen consumption rates in response to palmitate and glutamate administration were measured. The results indicated that while mitochondrial function was largely preserved, the fatty acid oxidation rate was markedly reduced in csVLCAD-KO mice, measuring 10-15% of WT. CONCLUSIONS: Cardiac-specific VLCAD deficiency is sufficient to induce ventricular dilation and cardiac dysfunction as well as cold intolerance, which may be related to reduced fatty acid oxidation and increased lipid deposition in cardiomyocytes.