Mitochondrial fatty acid oxidation disorders: clinical presentation of long-chain fatty acid oxidation defects before and after newborn screening

The different long-chain fatty acid oxidation defects present with similar heterogeneous clinical phenotypes of different severity. Organs mainly affected comprise the heart, liver, and skeletal muscles. All symptoms are reversible with sufficient energy supply. In some long-chain fatty acid oxidation defects, disease-specific symptoms occur. Only in disorders of the mitochondrial trifunctional protein (TFP) complex, including long-chain 3-hydroxyacyl-coenzyme A (CoA) dehydrogenase (LCHAD) deficiency, neuropathy and retinopathy develop that are progressive and irreversible despite current treatment measures. In most long-chain fatty acid oxidation defects, no clear genotype–phenotype correlation exists due to molecular heterogeneity. However, some isolated mutations have been identified to be associated with only mild phenotypes, e.g., the V243A mutation in very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. LCHAD deficiency is due to the prevalent homozygous 1528G>C mutation and presents with heterogeneous clinical phenotypes, suggesting the importance of other environmental and genetic factors. For some disorders, it was shown that residual enzyme activity measured in fibroblasts or lymphocytes correlated with severity of clinical phenotype. Implementation of newborn screening has significantly reduced morbidity and mortality of long-chain fatty acid oxidation defects. However, the severest forms of TFP deficiency are still highly associated with neonatal death. Newborn screening also identifies a great number of mildly affected patients who may never develop clinical symptoms throughout life. However, later-onset exercise-induced myopathic symptoms remain characteristic clinical features of long-chain fatty acid oxidation defects. Disease prevalence has increased with newborn screening.     

Unique plasma metabolomic signatures of individuals with inherited disorders of long-chain fatty acid oxidation

Blood and urine acylcarnitine profiles are commonly used to diagnose long-chain fatty acid oxidation disorders (FAOD: i.e., long-chain hydroxy-acyl-CoA dehydrogenase [LCHAD] and carnitine palmitoyltransferase 2 [CPT2] deficiency), but the global metabolic impact of long-chain FAOD has not been reported. We utilized untargeted metabolomics to characterize plasma metabolites in 12 overnight-fasted individuals with FAOD (10 LCHAD, two CPT2) and 11 healthy age-, sex-, and body mass index (BMI)-matched controls, with the caveat that individuals with FAOD consume a low-fat diet supplemented with medium-chain triglycerides (MCT) while matched controls consume a typical American diet. In plasma 832 metabolites were identified, and partial least squared-discriminant analysis (PLS-DA) identified 114 non-acylcarnitine variables that discriminated FAOD subjects and controls. FAOD individuals had significantly higher triglycerides and lower specific phosphatidylethanolamines, ceramides, and sphingomyelins. Differences in phosphatidylcholines were also found but the directionality differed by metabolite species. Further, there were few differences in non-lipid metabolites, indicating the metabolic impact of FAOD specifically on lipid pathways. This analysis provides evidence that LCHAD/CPT2 deficiency significantly alters complex lipid pathway flux. This metabolic signature may provide new clinical tools capable of confirming or diagnosing FAOD, even in subjects with a mild phenotype, and may provide clues regarding the biochemical and metabolic impact of FAOD that is relevant to the etiology of FAOD symptoms.     

Disorders of mitochondrial long-chain fatty acid oxidation

Summary The oxidation of long-chain fatty acids requires a series of enzymes which are located in or on the mitochondrial membranes. These include carnitine palmitoyltransferases I and II, a carnitine-acylcarnitine translocase and, newly discovered, very long-chain acyl-CoA dehydrogenase and the mitochondrial trifunctional protein. These last two chain-shorten acyl-CoA esters to the point where they can be transferred to the more soluble medium- and short-chain-specific enzymes within the mitochondrial matrix. The disorders of long-chain fatty acid oxidation show a rather similar range of clinical and biochemical features, though with different emphasis in the different conditions. Patients with severe defects usually present early with acute attacks of hypoketotic hypoglycaemia and impaired liver function, or with cardiomyopathy or cardiac arrhythmia. In milder variants, skeletal myopathy with intermittent myoglobinuria develops later in life. 3-Hydroxyacyl-CoA dehydrogenase deficiency is unusual in producing peripheral neuropathy and retinitis pigmentosa. Treatment is based on the avoidance of fasting and replacement of normal dietary fat by medium-chain triglyceride, the medium-chain fatty acids entering the mitochondria in a carnitine-independent manner and bypassing the long-chain part of the spiral. Diagnosis must ultimately be based on direct assay of the enzyme involved, but preliminary indicators may come from determination of carnitine and intermediate metabolites in plasma, urinary organic acid profiling, and radioisotopic screening assays with lymphocytes or cultured fibroblasts.     

Clinical and biochemical monitoring of patients with fatty acid oxidation disorders

Evidence-based guidelines for monitoring patients with disorders in fatty acid oxidation (FAO) are lacking, and most protocols are based on expert statements. Here, we describe our protocol for Danish patients. Clinical monitoring is the most important measure and has the main aims of checking growth, development and diet and of bringing families to the clinic regularly to remind them of their child’s risk and review how they cope and adjust, e.g. to an acute intercurrent illness. Most of these measures are simple and can be carried out during a routine out-patient visit; we seldom do more complicated assessments by a neuropsychologist, speech therapist, or physical and occupational therapists. Paraclinical measurements are not used for short-chain and medium-chain disorders; electrocardiography (including 24 h monitoring) and echocardiography are done for most patients with long-chain and carnitine transporter deficiencies. Eye examination is done in all, and liver ultrasonography in some patients with long-chain 3-hydroxyacyl-coenzyme A dehydrogenase/tri-functional protein (LCHAD/TFP) deficiencies. Biochemical follow-up includes determination of free carnitine and acylcarnitines. Free carnitine is measured to monitor carnitine supplementation in patients with multiple acyl-coenzyme A dehydrogenase deficiency (MADD) and carnitine transporter deficiency (CTD) and to follow metabolic control and disclose deficiency states in other FAO disorders. We are evaluating long-chain acylcarnitines in patients with long-chain disorders; so far there does not seem to be any clear-cut benefit in following these levels. An erythrocyte fatty acid profile is done in patients with long-chain disorders to test for essential fatty acid and docosahexanoic acid (DHA) deficiencies. The measurement of creatine kinase is helpful in long-chain disorders. Ongoing follow-up and education of the patient is important throughout life to prevent disease morbidity or death from metabolic crises.     

Pathophysiology of fatty acid oxidation disorders

Mitochondrial fatty acid oxidation represents an important pathway for energy generation during periods of increased energy demand such as fasting, febrile illness and muscular exertion. In liver, the primary end products of the pathway are ketone bodies, which are released into the circulation and provide energy to tissues that are not able to oxidize fatty acids such as brain. Other tissues, such as cardiac and skeletal muscle are capable of direct utilization of the fatty acids as sources of energy. This article provides an overview of the pathogenesis of fatty acid oxidation disorders. It describes the different tissue involvement with the disease processes and correlates disease phenotype with the nature of the genetic defect for the known disorders of the pathway.     

Triheptanoin versus trioctanoin for long-chain fatty acid oxidation disorders: a double blinded,randomized controlled trial

Background

Observational reports suggest that supplementation that increases citric acid cycle intermediates via anaplerosis may have therapeutic advantages over traditional medium-chain triglyceride (MCT) treatment of long-chain fatty acid oxidation disorders (LC-FAODs) but controlled trials have not been reported. The goal of our study was to compare the effects of triheptanoin (C7), an anaplerotic seven-carbon fatty acid triglyceride, to trioctanoin (C8), an eight-carbon fatty acid triglyceride, in patients with LC-FAODs.

Methods

A double blinded, randomized controlled trial of 32 subjects with LC-FAODs (carnitine palmitoyltransferase-2, very long-chain acylCoA dehydrogenase, trifunctional protein or long-chain 3-hydroxy acylCoA dehydrogenase deficiencies) who were randomly assigned a diet containing 20% of their total daily energy from either C7 or C8 for 4 months was conducted. Primary outcomes included changes in total energy expenditure (TEE), cardiac function by echocardiogram, exercise tolerance, and phosphocreatine recovery following acute exercise. Secondary outcomes included body composition, blood biomarkers, and adverse events, including incidence of rhabdomyolysis.

Results

Patients in the C7 group increased left ventricular (LV) ejection fraction by 7.4% (p = 0.046) while experiencing a 20% (p = 0.041) decrease in LV wall mass on their resting echocardiogram. They also required a lower heart rate for the same amount of work during a moderate-intensity exercise stress test when compared to patients taking C8. There was no difference in TEE, phosphocreatine recovery, body composition, incidence of rhabdomyolysis, or any secondary outcome measures between the groups.

Conclusions

C7 improved LV ejection fraction and reduced LV mass at rest, as well as lowering heart rate during exercise among patients with LC-FAODs.Clinical Trial Registration: Clinicaltrials.gov NCT01379625.

     

Impaired absorption of micellar long-chain fatty acid in patients with alcoholic cirrhosis

Diminished absorption of micellar long-chain fatty acid in patients with liver cirrhosis compared with controls was demonstrated during perfusion of the proximal small bowel with a micellar solution of long-chain fatty acid. Since parallel experiments showed no difference in absorption of medium-chain fatty acids, the results suggest impairment of the mucosal transport of long-chain fatty acid in cirrhosis. We suggest that this abnormality, possibly related to portal or lymphatic hypertension, contributes to fat malabsorption and malnutrition in these patients.     

Treatment recommendations in long-chain fatty acid oxidation defects: consensus from a workshop

Summary  Published data on treatment of fatty acid oxidation defects are scarce. Treatment recommendations have been developed on the basis of observations in 75 patients with long-chain fatty acid oxidation defects from 18 metabolic centres in Central Europe. Recommendations are based on expert practice and are suggested to be the basis for further multicentre prospective studies and the development of approved treatment guidelines. Considering that disease complications and prognosis differ between different disorders of long-chain fatty acid oxidation and also depend on the severity of the underlying enzyme deficiency, treatment recommendations have to be disease-specific and depend on individual disease severity. Disorders of the mitochondrial trifunctional protein are associated with the most severe clinical picture and require a strict fat-reduced and fat-modified (medium-chain triglyceride-supplemented) diet. Many patients still suffer acute life-threatening events or long-term neuropathic symptoms despite adequate treatment, and newborn screening has not significantly changed the prognosis for these severe phenotypes. Very long-chain acyl-CoA dehydrogenase deficiency recognized in neonatal screening, in contrast, frequently has a less severe disease course and dietary restrictions in many patients may be loosened. On the basis of the collected data, recommendations are given with regard to the fat and carbohydrate content of the diet, the maximal length of fasting periods and the use of l-carnitine in long-chain fatty acid oxidation defects. Competing interests: None declared     

Myocardial fatty acid oxidation in patients with impaired glucose tolerance

Abstract Aims/hypothesis. Fatty acids are an important source of energy in the myocardium. Abnormal myocardial fatty acid metabolism could contribute to the deterioration of cardiac function frequently observed in patients with Type II (non-insulin-dependent) diabetes mellitus. In our previous study, myocardial total uptake of non-esterified fatty acid (NEFA) was measured in patients with impaired glucose tolerance and found to be normal. This study aimed to investigate the subsequent metabolic steps and β-oxidation of NEFA. Methods. A total of 6 men with impaired fasting glucose (age 50 ± 2 years, BMI 29 ± 1 kg/m², means ± SEM) and 6 healthy men (50 ± 1 years, 25 ± 1 kg/m²) were studied in the fasting state. Myocardial blood flow was measured with [¹⁵O]H₂O and positron emission tomography and myocardial NEFA metabolism with [¹¹C]palmitic acid. Results. Myocardial blood flow was normal and not different between the impaired glucose tolerance and the control group (78 ± 6 vs 73 ± 13 ml/100 g/min, NS). The [¹¹C]palmitic acid uptake indices were similar between the groups (10.4 ± 0.5 vs 11.2 ± 0.8 ml/100 g/min, respectively, NS). The clearance of [¹¹C]-palmitate from the myocardium, an index of NEFA β-oxidation, was similar between the groups (half-times of activity 17.6 ± 1.6 vs 19.5 ± 2.3 min, respectively, NS) Conclusion/interpretation. The results indicate that myocardial NEFA uptake and β-oxidation are not altered in patients with IGT. Thus, it is not likely that altered NEFA metabolism contributes to the deterioration of the cardiac function in patients with IGT or Type II diabetes. [Diabetologia (2001) 44: 184–187] Received: 9 June 2000 and in revised form: 25 September 2000     

Management and outcome in 75 individuals with long-chain fatty acid oxidation defects: results from a workshop

Summary  At present, long-chain fatty acid oxidation (FAO) defects are diagnosed in a number of countries by newborn screening using tandem mass spectrometry. In the majority of cases, affected newborns are asymptomatic at time of diagnosis and acute clinical presentations can be avoided by early preventive measures. Because evidence-based studies on management of long-chain FAO defects are lacking, we carried out a retrospective analysis of 75 patients from 18 metabolic centres in Germany, Switzerland, Austria and the Netherlands with special regard to treatment and disease outcome. Dietary treatment is effective in many patients and can prevent acute metabolic derangements and prevent or reverse severe long-term complications such as cardiomyopathy. However, 38% of patients with very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency had intermittent muscle weakness and pain despite adhering to therapy. Seventy-six per cent of patients with disorders of the mitochondrial trifunctional protein (TFP)-complex including long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency, had long-term myopathic symptoms. Of these, 21% had irreversible peripheral neuropathy and 43% had retinopathy. The main principle of treatment was a fat-reduced and fat-modified diet. Fat restriction differed among patients with different enzyme defects and was strictest in disorders of the TFP-complex. Patients with a medium-chain fat-based diet received supplementation of essential long-chain fatty acids. l-Carnitine was supplemented in about half of the patients, but in none of the patients with VLCAD deficiency identified by newborn screening. In summary, in this cohort the treatment regimen was adapted to the severity of the underlying enzyme defect and thus differed among the group of long-chain FAO defects. Competing interests: None declared     

Effects of oral L-carnitine supplementation on in vivo long-chain fatty acid oxidation in healthy adults

Despite an abundance of literature describing the basic mechanisms of action of L-carnitine metabolism, there remains some uncertainty regarding the effects of oral L-carnitine supplementation on in vivo fatty acid oxidation in normal subjects under normal conditions. It is well known that L-carnitine normalizes the metabolism of long-chain fatty acids in cases of carnitine deficiency. However, it has not yet been shown that L-carnitine influences the metabolism of long-chain fatty acids in subjects without disturbances in fatty acid metabolism. Therefore, we investigated the effects of oral L-carnitine supplementation on in vivo long-chain fatty acid oxidation by measuring 1-[(13)C] palmitic acid oxidation in healthy subjects before and after L-carnitine supplementation (3 x 1 g/d for 10 days). We observed a significant increase in (13)CO(2) exhalation. This is the first investigation to conclusively demonstrate that oral L-carnitine supplementation results in an increase in long-chain fatty acid oxidation in vivo in subjects without L-carnitine deficiency or without prolonged fatty acid metabolism.     

Effects of long-chain monounsaturated and n-3 fatty acids on fatty acid oxidation and lipid composition in rats

Long-chain n-3 fatty acids and fat fish are reported, among multiple physiological properties, to enhance peroxisomal beta-oxidation and effect triacylglycerol status. Long-chain n-3 and monounsaturated fatty acids are the main portion of fatty acids in fat fish. The individual effect of long-chain monounsaturated fatty acids on beta-oxidation and fatty acid composition was tested and compared to the effect of n-3 polyunsaturated and saturated fatty acids in a 3-week feeding experiment of rats. To explore the contribution from long-chain monounsaturated fatty acids in these aspects, the effect of long-chain n-3 and monounsaturated fatty acids on mitochondrial and peroxisomal beta-oxidation was compared, as well as fatty acid composition of adipose tissue, liver and serum. Fatty acid oxidase, palmitoyltransferase I and II activities, the amount of serum lipids, and the fatty acid composition of lipid fractions from the organs were analysed. The peroxisomal beta-oxidation was enhanced by the n-3 fatty acids, whereas a small, significant increase with the monounsaturated fatty acids was observed. There was a stimulation of the mitochondrial oxidation with the n-3 fatty acids, but monounsaturated fatty acids gave a small, nonsignificant decrease. With n-3 fatty acids there was a considerable decrease in the levels of serum triacylglycerol, phospholipids, free fatty acids and total cholesterol, while there were only minor effects of monounsaturated fatty acids. As judged from the fatty acid composition data, there was a mobilization on n-3 fatty acids from the adipose tissue to liver and plasma with the n-3 diet. This observation was also seen with the monounsaturated fatty acid-enriched diet. In conclusion, monounsaturated fatty acids seemed to stimulate peroxisomal beta-oxidation and to increase plasma triacylglycerol, whereas the mitochondrial oxidation was slightly decreased.     

Current issues regarding treatment of mitochondrial fatty acid oxidation disorders

Treatment recommendations in mitochondrial fatty acid oxidation (FAO) defects are diverse. With implementation of newborn screening and identification of asymptomatic patients, it is necessary to define whom to treat and how strictly. We here discuss critical questions that are currently under debate. For some asymptomatic long-chain defects, long-chain fat restriction plays a minor role, and a normal diet may be introduced. For patients presenting only with myopathic symptoms, e.g., during exercise, treatment may be adapted to energy demand. As a consequence, patients with exercise-induced myopathy may be able to return to normal activity when provided with medium-chain triglycerides (MCT) prior to exercise. There is no need to limit participation in sports. Progression of retinopathy in disorders of the mitochondrial trifunctional protein complex is closely associated with hydroxyacylcarnitine accumulation. A strict low-fat diet with MCT supplementation is recommended to slow or prevent progression of chorioretinopathy. Additional docosahexanoic acid does not prevent the decline in retinal function but does promote nonspecific improvement in visual acuity and is recommended. There is no evidence that L-carnitine supplementation is beneficial. Thus, supplementation with L-carnitine in a newborn identified by screening with either a medium-chain or long-chain defect is not supported. With respect to the use of the odd-chain medium-chain triglyceride triheptanoin in myopathic phenotypes, randomized trials are needed to establish whether triheptanoin is more effective than even-chain MCT. With increasing pathophysiological knowledge, new treatment options have been identified and are being clinically evaluated. These include the use of bezafibrates in myopathic long-chain defects.     

Pathophysiology of fatty acid oxidation disorders and resultant phenotypic variability

Fatty acids are a major fuel for the body and fatty acid oxidation is particularly important during fasting, sustained aerobic exercise and stress. The myocardium and resting skeletal muscle utilise long-chain fatty acids as a major source of energy. Inherited disorders affecting fatty acid oxidation seriously compromise the function of muscle and other highly energy-dependent tissues such as brain, nerve, heart, kidney and liver. Such defects encompass a wide spectrum of clinical disease, presenting in the neonatal period or infancy with recurrent hypoketotic hypoglycaemic encephalopathy, liver dysfunction, hyperammonaemia and often cardiac dysfunction. In older children, adolescence or adults there is often exercise intolerance with episodic myalgia or rhabdomyolysis in association with prolonged aerobic exercise or other exacerbating factors. Some disorders are particularly associated with toxic metabolites that may contribute to encephalopathy, polyneuropathy, axonopathy and pigmentary retinopathy. The phenotypic diversity encountered in defects of fat oxidation is partly explained by genotype/phenotype correlation and certain identifiable environmental factors but there remain many unresolved questions regarding the complex interaction of genetic, epigenetic and environmental influences that dictate phenotypic expression. It is becoming increasingly clear that the view that most inherited disorders are purely monogenic diseases is a naive concept. In the future our approach to understanding the phenotypic diversity and management of patients will be more realistically achieved from a polygenic perspective.     

Normal acylcarnitine levels during confirmation of abnormal newborn screening in long-chain fatty acid oxidation defects

Summary We report two infants identified by tandem mass spectrometry (MS/MS) of neonatal blood spot acylcarnitines and confirmed by molecular genetic analysis to have long-chain fatty acid oxidation defects. In both cases, acylcarnitine concentrations in confirmatory plasma samples were normal. None the less, molecular testing identified trifunctional protein (TFP) deficiency (McKusick 600890) and very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (McKusick 201475).     

Molecular mechanism of long-chain fatty acid uptake

Most eukaryotic cells are capable of taking up long-chain fatty acids (LCFAs) to be used for a variety of cellular processes. Low levels of cellular uptake may occur by simple diffusion of these hydrophobic molecules through the plasma membrane. However, tissues such as cardiac muscle and fat specifically and efficiently take up and release LCFAs in a regulated fashion. In cardiac myocytes and adipocytes, a recently described integral plasma membrane fatty acid transport protein (FATP) functions as an LCFA transporter. FATP may play a role in abnormalities o f LCFA uptake and metabolism in cardiac pathophysiology.     

Targeted disruption of mouse long-chain acyl-CoA dehydrogenase gene reveals crucial roles for fatty acid oxidation

Abnormalities of fatty acid metabolism are recognized to play a significant role in human disease, but the mechanisms remain poorly understood. Long-chain acyl-CoA dehydrogenase (LCAD) catalyzes the initial step in mitochondrial fatty acid oxidation (FAO). We produced a mouse model of LCAD deficiency with severely impaired FAO. Matings between LCAD +/− mice yielded an abnormally low number of LCAD +/− and −/− offspring, indicating frequent gestational loss. LCAD −/− mice that reached birth appeared normal, but had severely reduced fasting tolerance with hepatic and cardiac lipidosis, hypoglycemia, elevated serum free fatty acids, and nonketotic dicarboxylic aciduria. Approximately 10% of adult LCAD −/− males developed cardiomyopathy, and sudden death was observed in 4 of 75 LCAD −/− mice. These results demonstrate the crucial roles of mitochondrial FAO and LCAD in vivo.