Evaluation of mitochondrial bioenergetics, dynamics, endoplasmic reticulum-mitochondria crosstalk, and reactive oxygen species in fibroblasts from patients with complex I deficiency.

Mitochondrial complex I (CI) deficiency is the most frequent cause of oxidative phosphorylation (OXPHOS) disorders in humans. In order to benchmark the effects of CI deficiency on mitochondrial bioenergetics and dynamics, respiratory chain (RC) and endoplasmic reticulum (ER)-mitochondria communication, and superoxide production, fibroblasts from patients with mutations in the ND6, NDUFV1 or ACAD9 genes were analyzed. Fatty acid metabolism, basal and maximal respiration, mitochondrial membrane potential, and ATP levels were decreased. Changes in proteins involved in mitochondrial dynamics were detected in various combinations in each cell line, while variable changes in RC components were observed. ACAD9 deficient cells exhibited an increase in RC complex subunits and DDIT3, an ER stress marker. The level of proteins involved in ER-mitochondria communication was decreased in ND6 and ACAD9 deficient cells. |ΔΨ| and cell viability were further decreased in all cell lines. These findings suggest that disruption of mitochondrial bioenergetics and dynamics, ER-mitochondria crosstalk, and increased superoxide contribute to the pathophysiology in patients with ACAD9 deficiency. Furthermore, treatment of ACAD9 deficient cells with JP4-039, a novel mitochondria-targeted reactive oxygen species, electron and radical scavenger, decreased superoxide level and increased basal and maximal respiratory rate, identifying a potential therapeutic intervention opportunity in CI deficiency.

Inicio, diagnóstico y seguimiento de un caso con deficiencia de acil-CoA deshidrogenasa de cadena muy larga / Debut, diagnosis and outcome of a case with very long chain acyl-CoA dehydrogenase deficiency

Los ácidos grasos representan el 80% de las necesidades energéticas en periodos de estrés metabólico. La betaoxidación de los ácidos grasos es catalizada por varias enzimas, como la acil-CoA deshidrogenasa-coenzima FAD, que posee 4 formas específicas según la longitud de la cadena de acil-CoA. La acil-CoA-deshidrogenasa de cadena muy larga es una de ellas. Su déficit cursa con la acumulación intramitocondrial de ésteres de acil-CoA de cadena larga, y afecta al corazón, el músculo esquelético y el hígado. Presentamos un caso iniciado a los 22 meses de edad con un síndrome Reye-like. Confirmamos un déficit de la betaoxidación de los ácidos grasos de cadena muy larga, con las mutaciones p.A232T (c.694G>A) y p.Y201C (c.602A>G) en los alelos del gen VLCAD. Describimos su evolución durante 17 años recibiendo una dieta pobre en ácidos grasos de cadena larga y suplementos con aceite MCT (AU)

Fatty acids represent 80% of energy needs during periods of stress. Beta-oxidation of fatty acids is catalyzed by some enzymes including acyl-CoA-dehydrogenase-coenzyme FAD, wich has four different ways according to the chain length of acyl-CoA. The very-long-chain-acyl-CoA-dehydrogenase is one of them. A deficiency of this enzyme produces an accumulation of long-chain-acyl-CoA-esters in mitochondrias, affecting heart, skeletal muscle and liver. We report the case of a 22-month aged child whose first symptom was a Reye-like syndrome. We confirmed that he was affected by a deficiency in the beta-oxidation of fatty acids of very long chain. He showed some mutations in the VLCAD gene alleles: p.A232T (c.694G>A) and p.Y201C (c.602A>G). We explain the evolution in the next 17 years, following a diet with very little long chain fatty acids and MCT oil supplements (AU)

Coordinated and reversible reduction of enzymes involved in terminal oxidative metabolism in skeletal muscle mitochondria from a riboflavin-responsive, multiple acyl-CoA dehydrogenase deficiency patient.

In this case report we studied alterations in mitochondrial proteins in a patient suffering from recurrent profound muscle weakness, associated with ethylmalonic-adipic aciduria, who had benefited from high dose of riboflavin treatment. Morphological and biochemical alterations included muscle lipid accumulation, low muscle carnitine content, reduction in fatty acid beta-oxidation and reduced activity of complexes I and II of the respiratory chain. Riboflavin therapy partially or totally reversed these symptoms and increased the level of muscle flavin adenine dinucleotide, suggesting that aberrant flavin cofactor metabolism accounted for the disease. Proteomic investigation of muscle mitochondria revealed decrease or absence of several flavoenzymes, enzymes related to flavin cofactor-dependent mitochondrial pathways and mitochondrial or mitochondria-associated calcium-binding proteins. All these deficiencies were completely rescued after riboflavin treatment. This study indicates for the first time a profound involvement of riboflavin/flavin cofactors in modulating the level of a number of functionally coordinated polypeptides involved in fatty acyl-CoA and amino acid metabolism, extending the number of enzymatic pathways altered in riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency.

Lipid-storage myopathy and respiratory insufficiency due to ETFQO mutations in a patient with late-onset multiple acyl-CoA dehydrogenation deficiency.

We report a patient with lipid-storage myopathy due to multiple acyl-CoA dehydrogenation deficiency (MADD). Molecular genetic analysis showed that she was compound heterozygous for mutations in the gene for electron transfer flavoprotein:ubiquinone oxidoreductase (ETFQO). Despite a good initial response to treatment, she developed respiratory insufficiency at age 14 years and has required long-term overnight ventilation. Thus, MADD is one of the few conditions that can cause a myopathy with weakness of the respiratory muscles out of proportion to the limb muscles.

Screening for medium chain acyl-CoA dehydrogenase deficiency using electrospray ionisation tandem mass spectrometry.

OBJECTIVE: To establish criteria for the diagnosis of medium chain acyl-CoA dehydrogenase (MCAD) deficiency in the UK population using a method in which carnitine species eluted from blood spots are butylated and analysed by electrospray ionisation tandem mass spectrometry (ESI-MS/MS). DESIGN: Four groups were studied: (1) 35 children, aged 4 days to 16.2 years, with proven MCAD deficiency (mostly homozygous for the A985G mutation, none receiving carnitine supplements); (2) 2168 control children; (3) 482 neonates; and (4) 15 MCAD heterozygotes. RESULTS: All patients with MCAD deficiency had an octanoylcarnitine concentration ([C8-Cn]) > 0.38 microM and no accumulation of carnitine species > C10 or < C6. Among the patients with MCAD deficiency, the [C8-Cn] was significantly lower in children > 10 weeks old and in children with carnitine depletion (free carnitine < 20 microM). Neonatal blood spots from patients with MCAD deficiency had a [C8-Cn] > 1.5 microM, whereas in heterozygotes and other normal neonates the [C8-Cn] was < 1.0 microM. In contrast, the blood spot [C8-Cn] in eight of 27 patients with MCAD deficiency > 10 weeks old fell within the same range as five of 15 MCAD heterozygotes (0.38-1.0 microM). However, the free carnitine concentrations were reduced (< 20 microM) in the patients with MCAD deficiency but normal in the heterozygotes. CONCLUSIONS: Criteria for the diagnosis of MCAD deficiency using ESI-MS/MS must take account of age and carnitine depletion. If screening is undertaken at 7-10 days, the number of false positive and negative results should be negligible. Because there have been no instances of death or neurological damage following diagnosis of MCAD deficiency in our patient group, a strong case can be made for neonatal screening for MCAD deficiency in the UK.

Evaluating newborn screening programmes based on dried blood spots: future challenges.

A UK national programme to screen all newborn infants for phenylketonuria was introduced in 1969, followed in 1981 by a similar programme for congenital hypothyroidism. Decisions to start these national programmes were informed by evidence from observational studies rather than randomised controlled trials. Subsequently, outcome for affected children has been assessed through national disease registers, from which inferences about the effectiveness of screening have been made. Both programmes are based on a single blood specimen, collected from each infant at the end of the first week of life, and stored as dried spots on a filter paper or 'Guthrie' card. This infrastructure has made it relatively easy for routine screening for other conditions to be introduced at a district or regional level, resulting in inconsistent policies and inequitable access to effective screening services. This variation in screening practices reflects uncertainty and the lack of a national framework to guide the introduction and evaluation of new screening initiatives, rather than geographical variations in disease prevalence or severity. More recently, developments in tandem mass spectrometry have made it technically possible to screen for several inborn errors of metabolism in a single analytical step. However, for each of these conditions, evidence is required that the benefits of screening outweigh the harms. How should that evidence be obtained? Ideally policy decisions about new screening initiatives should be informed by evidence from randomised controlled trials but for most of the conditions for which newborn screening is proposed, large trials would be needed. Prioritising which conditions should be formally evaluated, and developing a framework to support their evaluation, poses an important challenge to the public health, clinical and scientific community. In this chapter, issues underlying the evaluation of newborn screening programmes will be discussed in relation to medium chain acyl CoA dehydrogenase deficiency, a recessively inherited disorder of fatty acid oxidation.

In vivo stable isotope studies in three patients affected with mitochondrial fatty acid oxidation disorders: limited diagnostic use of 1-13C fatty acid breath test using bolus technique.

The in vivo oxidation of fatty acids (FA) of different chain length was investigated in three patients with documented mitochondrial FA oxidation disorders: one patient with mild multiple acyl-CoA dehydrogenase deficiency (MADM), one with medium chain acyl-CoA dehydrogenase deficiency (MCAD), and one with carnitine palmitoyltransferase I deficiency (CPT I). Breath tests were performed after oral administration of 1-13C butyric. 1-13C octanoic, and 1-13C palmitic acids. 13C/12C ratio in the expired oxidative end product CO2 was measured. The cumulative 13C elimination was calculated and expressed as a percentage of the administered dose. In the MADM patient the influence of carnitine therapy (or deprivation) on the utilization of 1-13C palmitic acid was also examined. In the MCAD and CPT I patients, the 1-13C butyric, 1-13C octanoic and 1-13C palmitic acids in vivo oxidation were similar to five healthy controls. In the MADM patient, the oxidation of 1-13C butyric and 1-13C octanoic acids were normal, whereas the metabolism of 1-13C palmitic acid ranged from 33% of 66% of controls. In this patient the serum carnitine level decreased from 60 to 27 mumol/l without carnitine supplementation. Clinically there was mild hypotonia. 1-13C palmitic acid oxidation compared to controls was 50%. After 2 further weeks of carnitine deprivation the serum carnitine was 10-15 mumol/l. Clinically he was very hypotonic and had a large liver. 1-13C Palmitic acid oxidation was 33%. After 6 weeks of readministration of carnitine (L-carnitine 100 mg/kg/day p.o.) the serum carnitine was 60 mumol/l and the patient was in good clinical condition. 1-13C palmitic acid oxidation was 66% compared to controls. Our study implies that this simple fatty acid breath test is not of diagnostic use for detection of enzymatic defects in FA oxidation disorders. The carnitine dependent 1-13C palmitic acid oxidation indicates that this test might be of some value in cases with primary or secondary carnitine deficiencies.

Acylcarnitine removal in a patient with acyl-CoA beta-oxidation deficiency disorder: effect of L-carnitine therapy and starvation.

Carnitine levels and acylcarnitine profiles in a patient with mild multiple acyl-CoA dehydrogenase deficient beta-oxidation were compared with control results. Whereas blood and urine total carnitine levels were moderately decreased, blood esterified carnitine levels in the patient were about 2-fold higher than in controls. Urinary acylcarnitine profiles presented with a larger variety of carnitine esters than in controls and included propionylcarnitine, butyrylcarnitine, 2-methylbutyrylcarnitine, hexanoylcarnitine and octanolycarnitine. Total carnitine levels in body fluids were similarly affected by chronic oral L-carnitine administration in patient and controls. By contrast, esterified carnitine level increase was 2-fold more important in controls than in patient. Whereas no qualitative changes in urinary acylcarnitine profiles were induced by L-carnitine therapy in controls, several alterations of these profiles were observed in the patient. The effect of starvation on metabolites was also studied, especially beta-oxidation rates assessed by free fatty acids to 3-hydroxybutyric acid ratios in blood from the patient in the untreated and L-carnitine treated states. In the L-carnitine-supplemented patient, the effect of starvation on the time course of carnitine levels and acylcarnitine profiles could also be documented. The ability of chronic oral L-carnitine administration to remove relatively less important amounts of acylcarnitines in the patient than in controls is further discussed, as well as qualitative alterations of acylcarnitine profiles induced by this therapy in the pathological condition.

Medium chain acyl-CoA dehydrogenase deficiency in Pennsylvania: neonatal screening shows high incidence and unexpected mutation frequencies.

Medium chain acyl-CoA dehydrogenase deficiency (MCAD) is a defect in the mitochondrial oxidation of fatty acids. The disorder typically presents with episodes of vomiting and hypoglycemia, sometimes with changes in mental status and hepatic failure. These Reye's-like features may culminate in coma and death. Stress, intercurrent illness, and reaction to childhood immunization have been shown to precipitate acute metabolic episodes in MCAD patients. All cases are caused by mutations of the single MCAD gene on chromosome 1. Most clinically ascertained cases are caused by an A985G transition in exon 11. Here we report the preliminary findings of MCAD patients detected prospectively through a supplemental newborn screening program in Pennsylvania using tandem mass spectrometry. From the first 80,371 newborns screened we prospectively found nine babies with MCAD (1/8930) plus two additional newborns screened because of a previously known family history. Molecular analysis showed 56% of the detected patients to be compound heterozygotes for the A985G and a second mutation. This is in contrast to clinical retrospective studies which have found only 20% to be compound heterozygotes. We have identified two of the other mutations including a novel mutation (DG91/C92, 6-bp deletion) in one of our patients by using single-stranded conformation polymorphism (SSCP) and sequence analysis of conformers. Our results confirm that MCAD is one of the more common inborn errors of metabolism. The different mutation frequencies observed between retrospective clinical studies and our prospective newborn screening study suggest that clinical ascertainment may lead to preferential identification of the A985G mutation.

Molecular genetic characterization and urinary excretion pattern of metabolites in two families with MCAD deficiency due to compound heterozygosity with a 13 base pair insertion in one allele.

Two families with medium-chain acyl-CoA dehydrogenase (MCAD) deficiency due to compound heterozygosity are described. All patients have a 13 bp insertion in exon 11 of one allele at the MCAD gene locus. In the other allele patients in one of the families harbour the prevalent G985 mutation, and the other family possess an unidentified mutation causing reduced levels of MCAD mRNA. We demonstrate that the disease in these families is inherited as an autosomal recessive trait. Individuals heterozygous for the mutations show heterozygous/control levels of beta-oxidation activities in cultured fibroblasts (9.1-16.3 pmol/min per mg protein; control 10-17 pmol/min per mg protein), and in the excretion of the 'beta-oxidation metabolites', hexanoylglycine (< 2 mumol/mmol creatinine), suberylglycine (< 2 mumol/mmol creatinine) and phenylpropionylglycine (< 2 mumol/mmol creatinine). This shows that there is no 'negative dominance' from the mutant monomeric protein onto the normal ones, in accordance with the finding of low levels of MCAD mRNA from the allele harbouring the 13 bp insertion as well as the allele with the unidentified mutation, and the low steady-state level of enzyme protein expressed from the G985-bearing allele. In the family possessing the G985 and the 13 bp insertion mutations, two asymptomatic compound heterozygous individuals were detected. They exhibited elevated excretion of hexanoylglycine (5-15 mumol/mmol creatinine) and suberylglycine (4-13 mumol/mmol creatinine), together with beta-oxidation activity in fibroblasts in the homozygous range (2.9 pmol/min per mg protein), showing a lack of correlation between the genotype, some biochemical parameters and the clinical phenotype.

Intravenous L-carnitine and acetyl-L-carnitine in medium-chain acyl-coenzyme A dehydrogenase deficiency and isovaleric acidemia.

The purpose of this study was to determine whether treatment with L-carnitine or acetyl-L-carnitine enhances the turnover of lipid or branched-chain amino acid oxidation in patients with inborn errors of metabolism. Increasing i.v. doses of L-carnitine and acetyl-L-carnitine were given to one patient with medium-chain acyl-CoA dehydrogenase deficiency and to another with isovaleric acidemia. Both patients were in stable condition and receiving oral L-carnitine supplements. The excretion of carnitine and disease-specific metabolites was measured. The incorporation of L-carnitine in the intracellular pool was demonstrated using stable isotopes and mass spectrometry. Increasing doses of either i.v. L-carnitine or acetyl-L-carnitine did not stimulate the excretion of octanoylcarnitine in the patient with medium-chain acyl-CoA dehydrogenase deficiency, nor did it raise the plasma levels of either cis-4-decenoate or octanoylcarnitine. Similarly, increasing doses of either i.v. L-carnitine or acetyl-L-carnitine did not enhance the excretion of isovalerylcarnitine in a patient with isovaleric acidemia. The excretion of isovalerylglycine actually decreased. We conclude that there was no evidence of enhanced fatty acid beta-oxidation or enhanced branched-chain amino acid oxidation in vivo by the administration of high doses of L-carnitine or acetyl-L-carnitine in these two patients. Because only one individual with each disorder was studied, the data are only indicative and may not necessarily be representative of all individuals with these disorders. Definite settlement of this issue will require further studies in additional subjects.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: diagnosis by acylcarnitine analysis in blood.

Medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency is a disorder of fatty acid catabolism, with autosomal recessive inheritance. The disease is characterized by episodic illness associated with potentially fatal hypoglycemia and has a relatively high frequency. A rapid and reliable method for the diagnosis of MCAD deficiency is highly desirable. Analysis of specific acylcarnitines was performed by isotope-dilution tandem mass spectrometry on plasma or whole blood samples from 62 patients with MCAD deficiency. Acylcarnitines were also analyzed in 42 unaffected relatives of patients with MCAD deficiency and in other groups of patients having elevated plasma C8 acylcarnitine, consisting of 32 receiving valproic acid, 9 receiving medium-chain triglyceride supplement, 4 having multiple acyl-coenzyme A dehydrogenase deficiency, and 8 others with various etiologies. Criteria for the unequivocal diagnosis of MCAD deficiency by acylcarnitine analysis are an elevated C8-acylcarnitine concentration (> 0.3 microM), a ratio of C8/C10 acylcarnitines of > 5, and lack of elevated species of chain length > C10. These criteria were not influenced by clinical state, carnitine treatment, or underlying genetic mutation, and no false-positive or false-negative results were obtained. The same criteria were also successfully applied to profiles from neonatal blood spots retrieved from the original Guthrie cards of eight patients. Diagnosis of MCAD deficiency can therefore be made reliably through the analysis of acylcarnitines in blood, including presymptomatic neonatal recognition. Tandem mass spectrometry is a convenient method for fast and accurate determination of all relevant acylcarnitine species.

Diagnosis of medium-chain acyl-CoA dehydrogenase deficiency in lymphocytes and liver by a gas chromatographic method: the effect of oral riboflavin supplementation.

The activity of medium-chain acyl-CoA dehydrogenase (MCAD) with octanoyl-CoA as a substrate was measured in human lymphocytes by a gas chromatographic technique. Phenazine methosulfate was used as the primary electron acceptor. After the addition of crotonase and subsequent hydrolysis, the reaction product 3-hydroxyoctanoic acid was quantitated by capillary gas-liquid chromatography of the trimethylsilyl derivatives. Control subjects had MCAD activities of 3.46 +/- 0.18 nmol/mg protein/min (n = 15). Five patients were investigated while receiving no therapy at all; MCAD activity ranged from 0.08 to 0.23 in four of them and was 0.65 in the fifth one. Subsequent to the long-term administration of 50-150 mg/d of riboflavin to MCAD-deficient patients (n = 11), these activities increased to an average of 0.41 in 10 patients and 2.22 in one. The activities in 15 obligate heterozygotes were 1.91 +/- 0.41 nmol/mg protein/min, thus enabling a clear distinction from controls. Neither heterozygotes nor a control responded to riboflavin. The method was also applicable to postmortem liver tissue. One patient, who had died suddenly and unexpectedly at the age of 19 mo, was correctly diagnosed as MCAD-deficient, whereas five additional children who died of the sudden infant death syndrome showed normal activities.

Riboflavin responsive multiple acyl-CoA dehydrogenase deficiency: functional evaluation of recovery after high dose vitamin supplementation.

The effect of riboflavin supplementation on muscle performance and exercise metabolism was investigated in four patients with multiple acyl-coenzyme A dehydrogenase deficiency (MAD). Maximum oxygen consumption and endurance measurements were performed to assess the patients' aerobic capacity and energy metabolism during exercise. They were tested before and after treatment with pharmacological doses of riboflavin. The initially low maximum oxygen consumption and high levels of blood lactate during submaximal exercise suggest that the oxidation of both fatty acids and carbohydrates was severely impaired. All four patients experienced a dramatic improvement in aerobic performance under riboflavin supplementation.

Molecular characterization of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: identification of a lys329 to glu mutation in the MCAD gene, and expression of inactive mutant enzyme protein in E. coli.

A series of experiments has established the molecular defect in the medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) gene in a family with MCAD deficiency. Demonstration of intra-mitochondrial mature MCAD indistinguishable in size (42.5-kDa) from control MCAD, and of mRNA with the correct size of 2.4 kb, indicated a point-mutation in the coding region of the MCAD gene to be disease-causing. Consequently, cloning and DNA sequencing of polymerase chain reaction (PCR) amplified complementary DNA (cDNA) from messenger RNA of fibroblasts from the patient and family members were performed. All clones sequenced from the patient exhibited a single base substitution from adenine (A) to guanine (G) at position 985 in the MCAD cDNA as the only consistent base-variation compared with control cDNA. In contrast, the parents contained cDNA with the normal and the mutated sequence, revealing their obligate carrier status. Allelic homozygosity in the patient and heterozygosity for the mutation in the parents were established by a modified PCR reaction, introducing a cleavage site for the restriction endonuclease NcoI into amplified genomic DNA containing G985. The same assay consistently revealed A985 in genomic DNA from 26 control individuals. The A to G mutation was introduced into an E. coli expression vector producing mutant MCAD, which was demonstrated to be inactive, probably because of the inability to form active tetrameric MCAD. All the experiments are consistent with the contention that the G985 mutation, resulting in a lysine to glutamate shift at position 329 in the MCAD polypeptide chain, is the genetic cause of MCAD deficiency in this family. We found the same mutation in homozygous form in 11 out of 12 other patients with verified MCAD deficiency.

Possible deleterious effect of L-carnitine supplementation in a patient with mild multiple acyl-CoA dehydrogenation deficiency (ethylmalonic-adipic aciduria).

A patient with riboflavin-responsive mild multiple acyl-CoA dehydrogenation deficiency of the ethylmalonic--adipic aciduria type experienced a recurrence of spontaneous hypoglycaemic episodes whilst being given supplementary L-carnitine. This phenomenon is explicable in terms of the known biochemical features of this condition and suggests caution in the carnitine supplementation of patients with defective oxidation of medium- or short-chain fatty acyl-CoA esters. This patient excreted excessive phenylpropionylglycine after an oral phenylpropionic acid load. Thus the phenylpropionic acid loading test is not completely specific for primary medium-chain acyl-CoA dehydrogenase deficiency as has been supposed.

Normalization of short-chain acylcoenzyme A dehydrogenase after riboflavin treatment in a girl with multiple acylcoenzyme A dehydrogenase-deficient myopathy.

A 12-year-old girl was shown to have carnitine-deficient lipid storage myopathy and organic aciduria compatible with multiple acylcoenzyme A (acyl-CoA) dehydrogenase deficiency. In muscle mitochondria, activities of both short-chain acyl-CoA dehydrogenase (SCAD) and medium-chain acyl-CoA dehydrogenase (MCAD) were 35% of normal. Antibodies against purified SCAD, MCAD, and electron-transfer flavoprotein were used for detection of cross-reacting material (CRM) in the patient's mitochondria. Western blot analysis showed absence of SCAD-CRM, reduced amounts of MCAD-CRM, and normal amounts of electron-transfer flavoprotein-CRM. The patient, who was unresponsive to treatment with oral carnitine, improved dramatically with daily administration of 100 mg oral riboflavin. Increase in muscle bulk and strength and resolution of the organic aciduria were associated with normalization of SCAD activity and "reappearance" of SCAD-CRM. In contrast, both MCAD activity and MCAD-CRM remained lower than normal. These results suggest that in some patients with multiple acyl-CoA dehydrogenase deficiency riboflavin supplementation may be effective in restoring the activity of SCAD, and possibly of other mitochondrial flavin-dependent enzymes.

Medium-chain acyl-CoA dehydrogenase deficiency: metabolic effects and therapeutic efficacy of long-term L-carnitine supplementation.

Medium-chain acyl-CoA dehydrogenase deficiency is a recently described inborn error of metabolism characterized by episodes of coma and hypoketotic hypoglycaemia in response to prolonged fasting. Secondary carnitine deficiency has been documented in these patients as well as the excretion in the urine of medium-chain-length acyl carnitine esters, such as octanoylcarnitine. Based on the potential toxicity of medium-chain fatty acid metabolites and the beneficial responses of patients with other inborn errors of metabolism and secondary carnitine deficiency, oral carnitine has been proposed as treatment for children with medium-chain acyl-CoA dehydrogenase deficiency. We report the results of carefully monitored fasting challenges of an infant with this deficiency both before and after 3 months of oral carnitine therapy. Carnitine supplementation failed to prevent lethargy, vomiting, hypoglycaemia and accumulation of free fatty acids in response to fasting despite normalization of plasma carnitine levels and a marked increase in urinary excretion of acyl-carnitine esters. Potentially toxic medium-chain fatty acids accumulated in the plasma in spite of therapy. Based on this study of one patient, we stress that avoidance of fasting and prompt institution of glucose supplementation in situations when oral intake is interrupted remain the mainstays of therapy for medium-chain acyl-CoA dehydrogenase deficient patients.

Recognition of medium-chain acyl-CoA dehydrogenase deficiency in asymptomatic siblings of children dying of sudden infant death or Reye-like syndromes.

The medium-chain acyl-CoA dehydrogenase (MCAD) deficiency of mitochondrial beta oxidation has been identified in two asymptomatic siblings in a family in which two previous deaths had been recorded, one attributed to sudden infant death syndrome and the other to Reye syndrome. Recognition of this disorder in one of the deceased and in the surviving siblings was accomplished by detection of a diagnostic metabolite, octanoylcarnitine, using a new mass spectrometric technique. This resulted in early treatment with L-carnitine supplement in the survivors, which should prevent metabolic deterioration. Further studies suggest that breast-feeding may be protective for infants with MCAD deficiency. Families with children who have had Reye syndrome or in which sudden infant death has occurred are at risk for MCAD deficiency. We suggest that survivors and asymptomatic siblings should be tested for this treatable disorder.

Diagnostic and therapeutic implications of medium-chain acylcarnitines in the medium-chain acyl-coA dehydrogenase deficiency.

The medium-chain acyl-coA dehydrogenase deficiency is one of several metabolic disorders presenting clinically as Reye syndrome. Evidence is presented for a characteristic organic aciduria that distinguishes this disorder from Reye syndrome and other masqueraders characterized by dicarboxylic aciduria. The key metabolites, suberylglycine and hexanoylglycine, are excreted in high concentration only when the patients are acutely ill. More significantly, using novel techniques in mass spectrometry, the medium-chain defect is shown to be characterized by excretion of specific medium-chain acylcarnitines, mostly octanoylcarnitine, without significant excretion of a normal metabolite, acetylcarnitine, in four patients with documented enzyme deficiency. Similar studies on the urine of two patients reported with Reye-like syndromes of unidentified etiology have suggested the retrospective diagnosis of medium-chain acyl-coA dehydrogenase deficiency. Administration of L-carnitine to medium-chain acyl-coA dehydrogenase deficiency patients resulted in the enhanced excretion of medium-chain acylcarnitines. Octanoylcarnitine is prominent in the urine both prior to and following L-carnitine supplementation. The detection of this metabolite as liberated octanoic acid, following ion-exchange chromatographic purification and mild alkaline hydrolysis, provides a straightforward diagnostic procedure for recognition of this disorder without subjecting patients to the significant risk of fasting. In view of the carnitine deficiency and the demonstrated ability to excrete the toxic medium-chain acyl-coA compounds as acylcarnitines, a combined therapy of reduced dietary fat and L-carnitine supplementation (25 mg/kg/6 h) has been devised and applied with positive outcome in two new cases.