Differentiation of long-chain fatty acid oxidation disorders using alternative precursors and acylcarnitine profiling in fibroblasts

The differentiation of carnitine-acylcarnitine translocase deficiency (CACT) from carnitine palmitoyltransferase type II deficiency (CPT-II) and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency from mitochondrial trifunctional protein deficiency (MTP) continues to be ambiguous using current acylcarnitine profiling techniques either from plasma or blood spots, or in the intact cell system (fibroblasts/amniocytes). Currently, enzyme assays are required to unequivocally differentiate CACT from CPT-II, and LCHAD from MTP. Over the years we have studied the responses of numerous FOD deficient cell lines to both even and odd numbered fatty acids of various chain lengths as well as branched-chain amino acids. In doing so, we discovered diagnostic elevations of unlabeled butyrylcarnitine detected only in CACT deficient cell lines when incubated with a shorter chain fatty acid, [7-2H3]heptanoate plus l-carnitine compared to the routinely used long-chain fatty acid, [16-2H3]palmitate. In monitoring the unlabeled C4/C5 acylcarnitine ratio, further differentiation from ETF/ETF-DH is also achieved. Similarly, incubating LCHAD and MTP deficient cell lines with the long-chain branched fatty acid, pristanic acid, and monitoring the C11/C9 acylcarnitine ratio has allowed differentiation between these disorders. These methods may be considered useful alternatives to specific enzyme assays for differentiation between these long-chain fatty acid oxidation disorders, as well as provide insight into new treatment strategies.     

Metabolic control during exercise with and without medium-chain triglycerides (MCT) in children with long-chain 3-hydroxy acyl-CoA dehydrogenase (LCHAD) or trifunctional protein (TFP) deficiency

Exercise induced rhabdomyolysis is a complication of long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) and mitochondrial trifunctional protein (TFP) deficiency that frequently leads to exercise avoidance. Dietary therapy for most subjects includes medium-chain triglyceride (MCT) supplementation but analysis of diet records indicates that the majority of patients consume oral MCT only with breakfast and at bedtime. We hypothesized that MCT immediately prior to exercise would provide an alternative fuel source during that bout of exercise and improve exercise tolerance in children with LCHAD deficiency. Nine subjects completed two 45 min moderate intensity (60-70% predicted maximum heart rate (HR)) treadmill exercise tests. Subjects were given 4 oz of orange juice alone or orange juice and 0.5 g MCT per kg lean body mass, 20 min prior to exercise in a randomized cross-over design. ECG and respiratory gas exchange including respiratory quotient (RQ) were monitored. Blood levels of acylcarnitines, creatine kinase, lactate, and beta-hydroxybutyrate were measured prior to and immediately after exercise, and again following 20 min rest. Creatine kinase and lactate levels did not change with exercise. There was no significant difference in RQ between the two exercise tests but there was a decrease in steady-state HR following MCT supplementation. Cumulative long-chain 3-hydroxyacylcarnitines were 30% lower and beta-hydroxybutyrate was three-fold higher after the MCT-pretreated exercise test compared to the test with orange juice alone. Coordinating MCT supplementation with periods of increased activity may improve the metabolic control of children with LCHAD and TFP deficiency following exercise.     

Effect of optimal dietary therapy upon visual function in children with long-chain 3-hydroxyacyl CoA dehydrogenase and trifunctional protein deficiency

The objective of this prospective cohort study was to determine if dietary therapy including docosahexaenoic acid (DHA; C22:6omega-3) supplementation prevents the progression of the severe chorioretinopathy that develops in children with long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) or trifunctional protein (TFP) deficiency. Physical, biochemical, and ophthalmological evaluations, including electroretinogram (ERG) and visual acuity by evoked potential (VEP), were performed at baseline and annually following the initiation of 65-130 mg/day DHA supplementation and continued treatment with a low-fat diet. Fourteen children with LCHAD or TFP deficiency, 1-12 years of age at enrollment, were followed for 2-5 years. Three subjects with TFP beta-subunit mutations had normal appearance of the posterior pole of the ocular fundi at enrollment and no changes over the course of the study. Eleven subjects who were homozygote and heterozygote for the common mutation, c.1528G>C, had no change to severe progression of atrophy of the choroid and retina with time. Of these, four subjects had marked to severe chorioretinopathy associated with high levels of plasma hydroxyacylcarnitines and decreased color, night and/or central vision during the study. The plasma level of long-chain 3-hydroxyacylcarnitines, metabolites that accumulate as a result of LCHAD and TFP deficiency, was found to be negatively correlated with maximum ERG amplitude (Rmax) (p=0.0038, R2=0.62). In addition, subjects with sustained low plasma long-chain 3-hydroxyacylcarnitines maintained higher ERG amplitudes with time compared to subjects with chronically high 3-hydroxyacylcarnitines. Visual acuity, as determined with the VEP, appeared to increase with time on DHA supplementation (p=0.051) and there was a trend for a positive correlation with plasma DHA concentrations (p=0.075, R2=0.31). Thus, optimal dietary therapy as indicated by low plasma 3-hydroxyacylcarnitine and high plasma DHA concentrations was associated with retention of retinal function and visual acuity in children with LCHAD or TFP deficiency.     

Effects of higher dietary protein intake on energy balance and metabolic control in children with long-chain 3-hydroxy acyl-CoA dehydrogenase (LCHAD) or trifunctional protein (TFP) deficiency

The incidence of overweight and obesity is increasing among children with long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) or mitochondrial trifunctional (TFP) deficiency. Traditional treatment includes fasting avoidance and consumption of a low-fat, high-carbohydrate diet. A diet higher in protein and lower in carbohydrate may help to lower total energy intake while maintaining good metabolic control. To determine the short-term safety and efficacy of a high protein diet, subjects were admitted to the General Clinical Research Center and fed an ad-libitum high-protein diet and a high-carbohydrate diet for 6 days each using a randomized, crossover design. Nine subjects with LCHAD or TFP deficiency, age 7-14 were enrolled. Body composition was determined by DEXA. Total energy intake was evaluated daily. Resting energy expenditure and substrate utilization were determined by indirect calorimetry. Post-prandial metabolic responses of plasma glucose, insulin, leptin, ghrelin, acylcarnitines, and triglyceride were determined in response to a liquid meal. Subjects had a higher fat mass, lower lean mass and higher plasma leptin levels compared to reference values. While on the high protein diet energy consumption was an average of 50 kcals/day lower (p = 0.02) and resting energy expenditure was an average of 170 kcals/day higher (p = 0.05) compared to the high carbohydrate diet. Short-term higher protein diets were safe, well tolerated, and resulted in lowered energy intake and increased energy expenditure than the standard high-carbohydrate diet. Long-term studies are needed to determine whether higher protein diets will reduce the risk of overweight and obesity in children with LCHAD or TFP deficiency.     

Choice of oils for essential fat supplements can enhance production of abnormal metabolites in fat oxidation disorders

Patients with mitochondrial long-chain fat oxidation deficiencies are usually treated with diets containing reduced fat and increased carbohydrate, at times via gastrostomy feeding. To ensure adequate intake of essential fatty acids, supplements are provided to their diets using commercially available oils. These oils contain large quantities of non-essential fats that are preferentially oxidized and produce disease-specific metabolites (acyl-CoA intermediates) due to the genetic defect. This study describes the concentrations of these intermediates as reflected by acylcarnitines as well as the % contribution from each of four fatty acids: palmitate, oleate, linoleate, and alpha-linolenate when incubated with fibroblasts from patients with VLCAD, LCHAD, and trifunctional protein (TFP) deficiencies. Palmitate and oleate produce the majority of disease-specific acylcarnitines with these defective cell lines (79-94%) whereas linoleate and linolenate produced less (6-21%). On average, the amount of acylcarnitines decreased with increasing unsaturation (C18:1>C18:2>C18:3:34%>11%>3%, respectively. This relationship may reflect the "gatekeeper" role of carnitine palmitoyltransferase I (CPT I). A diet comparison between Canola and a combination of Flax/Walnut oils revealed that the latter, containing the least amount of non-essential fats, reduced blood acylcarnitine levels by 33-36%. The etiology of the severe peripheral neuropathy of TFP deficiency may result from the unique metabolite, 3-keto-acyl-CoA, after conversion to a methylketone via spontaneous decarboxylation. Essential fatty acid supplementation with oils should consider these findings to decrease production of disease-specific acyl-CoA intermediates.     

Response to therapy in carnitine/acylcarnitine translocase (CACT) deficiency due to a novel missense mutation

Deficiency of carnitine/acylcarnitine translocase (CACT) is an autosomal recessive disorder of the carnitine cycle resulting in the inability to transfer fatty acids across the inner mitochondrial membrane. Only a limited number of affected patients have been reported and the effect of therapy on this condition is still not well defined. Here, we report a new patient with this disorder and follow the response to therapy. Our patient was the product of a consanguineous marriage. He presented shortly after birth with cardiac myopathy and arrhythmia coupled with severe non-ketotic hypoglycemia. Initial metabolic studies indicated severe non-ketotic C6-C10 dicarboxylic aciduria, plasma carnitine deficiency, and a characteristic elevation of plasma C:16:0, C18:1, and C18:2 acylcarnitine species. Enzyme assay confirmed deficiency of CACT activity. Molecular studies indicated that this child was homozygous, and both parents heterozygous, for a single bp change converting glutamine 238 to arginine (Q238R). Therapy with a formula providing most of the fat via medium chain triglycerides (MCT) and carnitine supplementation reduced the concentration of long-chain acylcarnitines and reversed cardiac symptoms and the hypoglycemia. These results suggest that carnitine and MCT may be effective in treating this defect of long-chain fatty acid oxidation.     

Influence of dietary fatty acid chain-length on metabolic tolerance in mouse models of inherited defects in mitochondrial fatty acid beta-oxidation

Fasting-induced metabolic disease of all inherited deficiencies of the acyl-CoA dehydrogenases is characterized by hypoglycemia, hypoketonemia, and organic aciduria. Mice with these enzyme deficiencies are cold intolerant. To evaluate the potential role that dietary fatty acid chain-length has on a patient's ability to compensate during a metabolic challenge, we fed long-chain acyl CoA dehydrogenase (LCAD) deficient and short-chain acyl CoA dehydrogenase (SCAD) deficient mice a diet rich in medium-chain triglycerides (MCT) or long-chain triglycerides (LCT). To elucidate the importance of maintaining adequate serum glucose concentrations on compensation mechanisms during metabolic challenge, we treated LCAD-/- mice with a solution of 12.5% glucose or saline prior to fasting and a cold-challenge. We found that feeding SCAD deficient mice the LCT diet from weaning increased survival from 40 to 94% during metabolic challenge of cold tolerance. In contrast, there was no benefit to feeding the MCT diet at weaning to LCAD-/- mice; however, there was significant benefit when LCAD-/- mice were fed the MCT diet from the beginning of gestation. Survival during cold-challenge increased from 50 to 93%. In the LCAD-/- mice treated with glucose, despite maintaining serum glucose concentrations at normal or higher concentrations, the LCAD-/- mice were still unable to compensate during metabolic challenge. These results indicate the important influences dietary fatty acids may have by providing enhanced metabolic tolerance in patients with inborn errors of fatty acid oxidation. Furthermore, these studies demonstrate that there may be crucial variables involved in the treatment of these patients, including the patient's specific enzyme deficiency, the quantity and chain-length of dietary fat, which may provide positive effects, as well as the time in development when it was administered.     

Early neonatal diagnosis of long-chain 3-hydroxyacyl coenzyme a dehydrogenase and mitochondrial trifunctional protein deficiencies

Tandem mass spectrometry (MS/MS) has been introduced in several newborn screening programs for the detection of a large number of inborn errors of metabolism, including fatty acid oxidation disorders (FAOD). Early identification and treatment of FAOD have the potential to improve outcome and may be life-saving in some cases; an estimated 5% of sudden infant deaths are attributable to undiagnosed disorders of fatty acid oxidation. We report very early neonatal presentations of long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) and mitochondrial trifunctional protein (TFP) deficiencies confirmed by molecular analysis. Both patients had cardiorespiratory collapse and hypoglycemia, without a history of maternal pregnancy complications. Retrospective MS/MS analysis of the original newborn screening blood spots revealed characteristic acylcarnitine profiles. These cases are among the earliest reported presentations of LCHAD and TFP deficiencies and further illustrate the potential of MS/MS as a valuable tool for newborn screening of FAOD. However, timely analysis and reporting of results to clinicians are essential, because these disorders can manifest in the first few days of life.     

Molecular and phenotypic heterogeneity in mitochondrial trifunctional protein deficiency due to beta-subunit mutations

The mitochondrial trifunctional protein (TFP) is a multienzyme complex of the fatty acid beta-oxidation cycle. It is composed of four alpha-subunits (HADHA) harboring long-chain enoyl-CoA hydratase and long-chain L-3-hydroxyacyl-CoA dehydrogenase (LCHAD) and four beta-subunits (HADHB) harboring long-chain 3-ketoacyl-CoA thiolase (LKAT). Mutations in either subunit can result in TFP deficiency with reduced activity of all three TFP enzymes. We characterize 15 patients from 13 families with beta-subunit mutations by clinical, biochemical, and molecular features. Three clinical phenotypes are apparent: a severe neonatal presentation with cardiomyopathy, Reye-like symptoms, and early death (n=4); a hepatic form with recurrent hypoketotic hypoglycemia (n=2); and a milder later-onset neuromyopathic phenotype with episodic myoglobinuria (n=9). Maternal HELLP syndrome occurred in two mothers independently of the fetal phenotype. Mutational analysis revealed 16 different mutations, the majority being missense mutations (n=12). The predominance of missense mutations and the milder myopathic phenotype are consistent. Based upon homology to yeast thiolase that has been characterized structurally, the mutation localization within the protein correlates with the clinical phenotype. Outer loop mutations that are expected to alter protein stability less were only present in milder forms. The degree of reduction in thiolase antigen also correlated with the severity of clinical presentation. Although TFP deficiency is highly heterogeneous, there is genotype-phenotype correlation.     

Observations regarding retinopathy in mitochondrial trifunctional protein deficiencies

Although the retina is thought to primarily rely on glucose for fuel, inherited deficiency of one or more activities of mitochondrial trifunctional protein results in a pigmentary retinopathy leading to vision loss. Many other enzymatic deficiencies in fatty acid oxidation pathways have been described, none of which results in retinal complications. The etiology of retinopathy among patients with defects in trifunctional protein is unknown. Trifunctional protein is a heteroctomer; two genes encode the alpha and beta subunits of TFP respectively, HADHA and HADHB. A common mutation in HADHA, c.1528G>C, leads to a single amino acid substitution, p. Glu474Gln, and impairs primarily long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) activity leading to LCHAD deficiency (LCHADD). Other mutations in HADHA or HADHB often lead to significant reduction in all three enzymatic activities and result in trifunctional protein deficiency (TFPD). Despite many similarities in clinical presentation and phenotype, there is growing evidence that they can result in different chronic complications. This review will outline the clinical similarities and differences between LCHADD and TFPD, describe the course of the associated retinopathy, propose a genotype/phenotype correlation with the severity of retinopathy, and discuss the current theories about the etiology of the retinopathy.     

Substrate oxidation and cardiac performance during exercise in disorders of long chain fatty acid oxidation

BackgroundThe use of long-chain fatty acids (LCFAs) for energy is inhibited in inherited disorders of long-chain fatty acid oxidation (FAO). Increased energy demands during exercise can lead to cardiomyopathy and rhabdomyolysis. Medium-chain triglycerides (MCTs) bypass the block in long-chain FAO and may provide an alternative energy substrate to exercising muscle.ObjectivesTo determine the influence of isocaloric MCT versus carbohydrate (CHO) supplementation prior to exercise on substrate oxidation and cardiac workload in participants with carnitine palmitoyltransferase 2 (CPT2), very long-chain acyl-CoA dehydrogenase (VLCAD) and long-chain 3-hydroxyacyl CoA dehydrogenase (LCHAD) deficiencies.DesignEleven subjects completed two 45-minute, moderate intensity, treadmill exercise studies in a randomized crossover design. An isocaloric oral dose of CHO or MCT-oil was administered prior to exercise; hemodynamic and metabolic indices were assessed during exertion.ResultsWhen exercise was pretreated with MCT, respiratory exchange ratio (RER), steady state heart rate and generation of glycolytic intermediates significantly decreased while circulating ketone bodies significantly increased.ConclusionsMCT supplementation prior to exercise increases the oxidation of medium chain fats, decreases the oxidation of glucose and acutely lowers cardiac workload during exercise for the same amount of work performed when compared with CHO pre-supplementation. We propose that MCT may expand the usable energy supply, particularly in the form of ketone bodies, and improve the oxidative capacity of the heart in this population.     

Outcome in six patients with mitochondrial trifunctional protein disorders identified by newborn screening

Before the newborn screening era, disorders of the mitochondrial trifunctional protein (TFP) complex including long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) presented with high morbidity and mortality. Data on outcome and prognosis of TFP deficiency disorders since implementation of screening are scarce.We here characterize 6 screened patients with a disorder of the TFP complex (3 of those with LCHADD) with respect to clinical presentation and molecular features.Three of 6 patients were symptomatic prior availability of screening results on days 4–5 of life. Of the three asymptomatic patients recognised by screening, one acutely died at 3 months at home during an infection. Two patients remained asymptomatic with preventive measures during follow-up until the age of 3 years. One of them had an older sibling with identical genotype born before the screening era, who became symptomatic with 15 months.We conclude that newborn screening for disorders of the TFP complex allows identification of asymptomatic cases; however, the acute presentation in 3/6 babies before screening is noteworthy and troublesome. TFP and LCHAD deficiencies remain life-threatening disorders. This is in clear contrast to other defects of long-chain fatty acid oxidation after identification by newborn screening.     

Long-chain L 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency does not appear to be the primary cause of lipid myopathy in patients with Bannayan-Riley-Ruvalcaba syndrome (BRRS)

In order to test the hypothesis that long-chain L 3-hydroxyacyl-coenzyme A dehydrogenase (LCHAD) deficiency is associated with the lipid myopathy and muscle carnitine deficiency observed in Bannayan-Riley-Ruvalcaba syndrome (BRRS), we studied the enzyme activity in cultured skin fibroblasts from three generations of a family with a clear dominant inheritance of BRRS. Enzyme activities were normal while the germline PTEN missense mutation P246L segregated with BRRS in this family. No PTEN mutations were identified in the original patient with BRRS and LCHAD deficiency. These data suggest that the previously reported case of LCHAD and BRRS either represents the coincidental concurrence of two rare genetic events or that a gene other than PTEN is related to LCHAD and BRRS.     

Skeletal muscle lipid metabolism in exercise and insulin resistance

Lipids as fuel for energy provision originate from different sources: albumin-bound long-chain fatty acids (LCFA) in the blood plasma, circulating very-low-density lipoproteins-triacylglycerols (VLDL-TG), fatty acids from triacylglycerol located in the muscle cell (IMTG), and possibly fatty acids liberated from adipose tissue adhering to the muscle cells. The regulation of utilization of the different lipid sources in skeletal muscle during exercise is reviewed, and the influence of diet, training, and gender is discussed. Major points deliberated are the methods utilized to measure uptake and oxidation of LCFA during exercise in humans. The role of the various lipid-binding proteins in transmembrane and cytosolic transport of lipids is considered as well as regulation of lipid entry into the mitochondria, focusing on the putative role of AMP-activated protein kinase (AMPK), acetyl CoA carboxylase (ACC), and carnitine during exercise. The possible contribution to fuel provision during exercise of circulating VLDL-TG as well as the role of IMTG is discussed from a methodological point of view. The contribution of IMTG for energy provision may not be large, covering approximately 10% of total energy provision during fasting exercise in male subjects, whereas in females, IMTG may cover a larger proportion of energy delivery. Molecular mechanisms involved in breakdown of IMTG during exercise are also considered focusing on hormone-sensitive lipase (HSL). Finally, the role of lipids in development of insulin resistance in skeletal muscle, including possible molecular mechanisms involved, is discussed.     

Vitamin B12, folic acid and mental function in the elderly

Elderly people is a vulnerable population group to specific nutrient deficiencies as vitamin B12 and folic acid, which are closely related to mental functions deterioration, especially of cognitive functions. This study was aimed to measure B12 vitamin and folic acid indicators and to establish relationships to mental function. 53 elderly, older than 60 years, living in a geriatric home were assessed. The dietary intake was evaluated by the direct weighed method, serum B12 vitamin and folic acid by radioimmunoanalysis and mental function by Foltein's mini-mental test. Dietary intake for Vit B12 was adequate and deficient for folic acid while serum levels were within normal range. Vitamin B12 levels were at marginal or deficiency values in 26,4% of the elderly and folic acid deficiency was present in 43.4%. 49% of the elderly had mental function alterations and B12 vitamin levels were significantly lower in this group. A positive association between age and mental function (elderly below 80 years had lower risk of mental impairment) and between serum B12 and mental function were found. Elderly were at risk of deficiency for both vitamins and age and mental function were associated to this risk. Further evaluation including other nutrients should be performed.     

Intrauterine cardiomyopathy and cardiac mitochondrial proliferation in mitochondrial trifunctional protein (TFP) deficiency

Because of a switch in energy-producing substrate utilization from glucose in the fetal period to fatty acids postnatally, intrauterine morbidity of fatty acid oxidation defects has widely been denied. We report the intrauterine development of severe cardiomyopathy in a child with mitochondrial trifunctional protein deficiency after 27 weeks of gestation. The child was born at 31 weeks of gestation and died on day 3 of life. Severe cardiac mitochondrial proliferation was observed. Molecular analysis of both TFP genes was performed and confirmed a homozygous mutation in the TFP α-subunit introducing a stop codon at amino acid position 256 (g.871C>T, p.R256X). Despite severe intrauterine decompensation in our patient, no HELLP-syndrome or acute fatty liver of pregnancy was observed in the mother. In the pathogenesis of maternal HELLP-syndrome, toxic effects of accumulating long-chain hydroxy-acyl-CoAs or long-chain hydroxy-acylcarnitines are suspected. In our patient, acylcarnitine analysis on day 2 of life during severest metabolic decompensation did not reveal massive accumulation of long-chain hydroxy-acylcarnitines in blood, suggesting other pathogenic factors than toxic effects. The most important pathogenic mechanism for the development of intrauterine cardiomyopathy appears to be significant cardiac energy deficiency. In conclusion, our report implicates that fatty acid oxidation does play a significant role during intrauterine development with special regard to the heart. Severe cardiac mitochondrial proliferation in TFP deficiency suggests pathophysiologically relevant energy deficiency in this condition.     

Contribution of FAT/CD36 to the regulation of skeletal muscle fatty acid oxidation: an overview

Long chain fatty acids (LCFAs) are an important substrate for ATP production within the skeletal muscle. The process of LCFA delivery from adipose tissue to muscle mitochondria involves many regulatory steps. Recently, it has been recognized that LCFA oxidation is not only dependent on LCFA delivery to the muscle, but also on regulatory steps within the muscle. Increasing selected fatty acid binding proteins/transporters on the plasma membrane facilitates a very rapid LCFA increase into the muscle, independent of any changes in LCFA delivery to the muscle. Such a mechanism of LCFA transporter translocation is activated by muscle contraction. Intramuscular triacylglycerols may also be hydrolysed to provide fatty acids for mitochondrial oxidation, particularly during exercise, when hormone-sensitive lipase and other enzymes are activated. Mitochondrial LCFA entry is also highly regulated. This however does not involve only the malonyl CoA carnitine palmitoyltransferase-I (CPTI) axis. Exercise-induced fatty acid entry into mitochondria is also regulated by at least one of the proteins (FAT/CD36) that also regulates plasma membrane fatty acid transport. Among individuals, differences in mitochondrial fatty acid oxidation appear to be correlated with the content of mitochondrial CPTI and FAT/CD36. This paper provides a brief overview of mechanisms that regulate LCFA uptake and oxidation in skeletal muscle during exercise and in obesity. We focus largely on our own work on FAT/CD36, which contributes to regulating, in a coordinated fashion, LCFA uptake across the plasma membrane and the mitochondrial membrane. Very little is known about the roles of FATP1-6 on fatty acid transport in skeletal muscle.     

Disorders of carnitine transport and the carnitine cycle

Carnitine plays an essential role in the transfer of long-chain fatty acids across the inner mitochondrial membrane. This transfer requires enzymes and transporters that accumulate carnitine within the cell (OCTN2 carnitine transporter), conjugate it with long chain fatty acids (carnitine palmitoyl transferase 1, CPT1), transfer the acylcarnitine across the inner plasma membrane (carnitine-acylcarnitine translocase, CACT), and conjugate the fatty acid back to Coenzyme A for subsequent beta oxidation (carnitine palmitoyl transferase 2, CPT2). Deficiency of the OCTN2 carnitine transporter causes primary carnitine deficiency, characterized by increased losses of carnitine in the urine and decreased carnitine accumulation in tissues. Patients can present with hypoketotic hypoglycemia and hepatic encephalopathy, or with skeletal and cardiac myopathy. This disease responds to carnitine supplementation. Defects in the liver isoform of CPT1 present with recurrent attacks of fasting hypoketotic hypoglycemia. The heart and the muscle, which express a genetically distinct form of CPT1, are usually unaffected. These patients can have elevated levels of plasma carnitine. CACT deficiency presents in most cases in the neonatal period with hypoglycemia, hyperammonemia, and cardiomyopathy with arrhythmia leading to cardiac arrest. Plasma carnitine levels are extremely low. Deficiency of CPT2 present more frequently in adults with rhabdomyolysis triggered by prolonged exercise. More severe variants of CPT2 deficiency present in the neonatal period similarly to CACT deficiency associated or not with multiple congenital anomalies. Treatment for deficiency of CPT1, CPT2, and CACT consists in a low-fat diet supplemented with medium chain triglycerides that can be metabolized by mitochondria independently from carnitine, carnitine supplements, and avoidance of fasting and sustained exercise.     

Dietary self-selection and the regulation of protein and energy intake in chicks

Studies were conducted to determine whether chicks could regulate their protein intake independent of total energy intake in self-selection feeding trials. Day-old White Mountain cockerels were reared in electrically-heated battery brooders and given access to either a 23% protein control ration (no choice) or two diets containing 10% or 60% protein with or without supplemental amino acids. The latter were added to either improve the dietary amino acid balance or to alter plasma and brain levels of free large neutral amino acids (tryptophan, isoleucine, leucine, valine, phenylalanine, and tyrosine) which have been implicated in the neuroregulation of feed intake. Both feed and water were provided ad lib, and the location of the feed troughs within each pen were changed daily. Body weights and feed intakes were measured daily, and total calorie and protein intakes were calculated. Chicks offered 10% and 60% protein diets with no supplemental amino acids exhibited reduced weight gains and markedly higher protein intakes as compared to birds fed either the control ration or those given a choice between 10% and 60% protein diets supplemented with methionine. The higher protein consumption by chicks fed the unsupplemented diets most likely was a result of an attempt to compensate for a dietary methionine deficiency. Chicks fed the 10% and 60% protein diets supplemented with amino acids grew at a slower rate than those fed the 23% protein control diet. In general, plasma and brain data did not support a proposed relationship between certain large neutral amino acid ratios and protein or energy intake.     

Neonatal screening for defects of the mitochondrial trifunctional protein

Long-chain l-3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency has been included in the routine neonatal screening program by the German screening commission. As tandem mass spectrometry (TMS) does not discriminate between the different defects of the mitochondrial trifunctional protein (MTP) screening for isolated LCHAD deficiency includes the detection of long-chain 3-ketoacyl-CoA thiolase and complete MTP deficiencies as well. We identified 11 patients with abnormalities of the MTP out of 1.2 million newborns screened in our laboratory during the last 6 years. Treatment was started on the day the screening result was obtained (day 3 to day 9 of life). Seven of these newborns developed satisfactorily during an observation period of up to 64 months. They had isolated LCHAD deficiency, four of them caused by the typical mutation (1528 G>C), three others had no molecular genetic analysis done or were shown to have previously unknown mutations. Four children did not survive, two of them showing complete deficiency of MTP and two showing deficiency of long-chain 3-ketoacyl-CoA thiolase. We conclude that, despite the rarity of the disease, screening for MTP deficiencies is justified based on the following criteria: improved quality of life for patients with isolated LCHAD deficiency, absence of stigmatisation of babies showing mild variants without necessity of treatment, no significant increase of the total number of false positive screening results, no false negative results to our knowledge. Finally, extension of analysis to MTP deficiencies is achieved without additional costs for screening laboratories already using TMS.