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Isobutyryl-CoA dehydrogenase (IBD) is an enzyme involved in the catabolism of the branched-chain amino acid valine. We report a third and a fourth child with IBD deficiency who were both detected during newborn screening with tandem mass spectrometry and so far do not receive any treatment. The diagnosis was confirmed by biochemical and molecular studies. One of the children is homozygous for the mutation M128I in the ACAD8 gene, which is predicted to affect the substrate binding cavity. The other child is compound heterozygous for a frameshift mutation F33fsins and a missense mutation V203I. It is as yet uncertain whether IBD deficiency may cause significant morbidity in affected children and whether treatment is necessary. In view of the limited experience worldwide, careful monitoring of the children is recommended.

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.

Previously undescribed medium-chain acylcarnitines were identified in a urine sample from a patient with medium-chain acyl-CoA dehydrogenase deficiency. These are the 4-methylvaleryl, 4- and 5-methylhexanoyl, 6-methyl-heptanoyl-, 6-methyloctanoyl-, 4,5-dimethylhexanoyl- and 4,7-decadienoyl-carnitines. Their chemical structures were obtained by gas chromatography–mass spectrometry analysis of their fatty acid moieties as picolinyl esters.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency (McKusick 201450) is the most commonly recognized defect of mitochondrial β-oxidation in humans. It is a serious, potentially fatal, autosomal recessive inherited defect, which may present in the first two years of life if patients experience periods of metabolic stress to the β-oxidation system (Roe and Coates 1989). A prevalent disease-causing mutation (G985), causing a lysine 304 to glutamate shift in the mature MCAD protein, is present in approximately 90% of disease alleles (Gregersen et al 1991 b; Yokota et al 1991). Only seven non-G985 mutations, which are all infrequent, have so far been reported (Tanaka et al 1992). Here we report the identification of a disease allele that results in low levels of MCAD mRNA

The effect of fasting on plasma concentrations of fatty acids has been determined in four children with medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. In addition, the in vivo rate of octanoate oxidation was measured, using [1-13C]octanoate. In the three older children (1.5-11.2 years), fasting for up to 18 h stimulated lipolysis, as reflected by the increasing concentration of free fatty acids, but with little rise in concentrations of medium-chain fatty acids, octanoate, decanoate and cis-4-decenoate. In an infant (0.5 year), lipolysis was greater and was accompanied by rising concentrations of medium-chain fatty acids. After 13.5 h there was a rapid increase in the concentration of decanoate and cis-4-decenoate. The calculated in vivo rate of octanoate oxidation was substantial in all patients studied (6.4-13.1 mumol/kg per h) despite very low MCAD activity in vitro. It is concluded that under basal conditions the in vivo oxidation rate of medium-chain fatty acids is near normal in the four children studied with MCAD deficiency.

An isotope dilution mass spectrometric assay for plasma cis-dec-4-enoic acid is described. It is quicker, more reliable and more accurate than previous methods. It confirmed previous findings that cis-dec-4-enoic acid is a reliable indicator for medium-chain acyl-CoA dehydrogenase deficiency (MCAD). The plasma cis-dec-4-enoic acid levels of both asymptomatic and symptomatic MCAD patients (3.5-71 mumol/L) are demonstrably higher than those of normal children (0.2-1.7 mumol/L), MCAD heterozygotes (0.1-1.5 mumol/L), those with other fatty acid oxidation defects (0.2-2.2 mumol/L) or those receiving high doses of valproic acid (0.2-0.4 mumol/L).

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.

The measurement of acyl-CoA dehydrogenase activity is an essential part of the investigation of patients with suspected defects of fatty acid oxidation, and recently the organometallic oxidant ferricenium hexafluorophosphate has been introduced as an electron acceptor for these assays. However, we show that when medium-chain acyl-CoA dehydrogenase activity was measured in cultured skin fibroblasts and platelets from patients with proven defects of this enzyme, there was considerable residual enzyme activity when this electron acceptor was used. The ferricenium assay is not as specific as the anaerobic ETF-linked assay in the biochemical diagnosis of medium-chain acyl-CoA dehydrogenase deficiency in fibroblasts, and therefore is of limited clinical applicability in its present form.

Deficiency of medium-chain acyl-CoA dehydrogenase (MCAD) is an important cause of sudden death in children. The majority of surviving individuals with MCAD deficiency studied to date are homozygous for a single point mutation at bp 985 of the MCAD mRNA (A985G). We have now identified a four-base-pair deletion in exon 11 of one allele of the MCAD gene in an American child who died of MCAD deficiency. The deletion mutation results in a frameshift and premature termination codon in the mutant MCAD mRNA. The second mutant allele contained the common point mutation A985G, and thus the proband was a compound heterozygote. Protein immunoblot analysis of the child's liver proteins revealed that the mutant MCAD proteins were barely detectable. Allele-specific oligonucleotide hybridization analysis performed on amplified exon 11 of the child's MCAD gene clearly identified both mutations. MCAD RFLP analysis of the patient's DNA revealed heterozygosity at the Taq I MCAD RFLP site, thus, the two mutations are associated with different haplotypes. Therefore, we have identified a new mutation in the MCAD gene and have developed a nucleic-acid-based screening approach which allows the post mortem identification of MCAD deficiency.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, is an autosomal recessive inborn error of metabolism associated with various clinical presentations, including sudden unexplained death in young children. We have determined the Scottish frequency of the common G985 mutation found in Caucasians and in samples from Scottish patients with sudden infant death syndrome (SIDS). The heterozygote frequency of the mutation was found to be 1 in 276 (95% confidence interval: 1/76-1/2279) in 552 healthy controls and 1 in 74 (95% confidence interval: 1/27-1/377) in 233 SIDS patients: a difference that was not statistically significant (Fisher's exact test; two-sided; p = 0.316). None of the SIDS samples was found to be homozygous for the G985 mutation.

Recently, several cases of short-chain acyl-CoA dehydrogenase (SCAD) deficiency (McKusick 201470) in humans have been described. Symptoms of this disorder are variable (Turnbull et al 1984; Amendt et al 1987; Coates et al 1988) and metabolic characteristics are not well understood. A mutant mouse (BALB/cBYJ) with no SCAD activity in liver, which is the first animal model of fatty acid oxidation disorders, has been reported (Wood et al 1989). To investigate the metabolic characteristics in these mice, we performed a series of experiments using liver perfusion techniques and HPLC

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency (McKusick 20145) is the most common inherited defect of the beta-oxidation of fatty acid (Roe and Coates, 1989). It causes life-threatening attacks of hypoglycaemia and lethargy, and has led to sudden infant death in apparently asymptomatic children. It is therefore important to diagnose the enzyme deficiency in asymptomatic siblings of affected cases and in families with cases of sudden infant death syndrome and 'near miss'. The existing enzymatic methods used for the diagnosis are all either non-specific or laborious, unsuited for use in routine laboratories. The interest in defining the molecular defect(s) at the DNA level has therefore - besides the obvious interest in gaining more insight into the disease - been to develop fast and easy diagnostic tests.

Service de Biochimie Pediatrique, Hopital Debrousse, Lyon, France*Cor respondence: Ostschweizerisches Kinderspital, Claudiusstrasse 6, 9006 St. Gallen,SwitzerlandShort-chain acyl-CoA dehydrogenase (SCAD) deficiency (McKusick 201470) is a raredisorder. Up to 1996 about 10 patients in whom an enzyme defect could be confirmed infibroblasts had been described (Amendt et al 1987; Coates et al 1988; Sewell et al 1993;Bhala et al 1995; Vianey-Saban, 4 unpublished cases). So far the clinical and biochemicalpattern of the disease is heterogeneous, all patients showing at least neuromuscular signs.W e present a new patient, now 16 years old, with growth failure, muscular wasting andhypotonia since birth, severe scoliosis and restricted ventilation occurring during puberty ,but normal intellectual development.CASE REPOR TEliane K., female, was born in 1980 after a normal pregnancy as the second child ofhealthy unrelated parents. Birth weight was 2700g and length 49cm. The older sister alsohas muscular hypotrophy and ethylmalonic aciduria but to a milder degree. Eliane wasbreast fed for 6 months and then put on infant formula. During this time feeding problemsstarted and failure to thrive and psychomotor retardation became apparent. Several epi-sodes of upper airway infections were severe. Adenoidectomy was therefore performed,after which respiratory infections were less frequent, but failure to thrive and musclehypotonia remained unchanged. Routine haematological and chemical tests includingmetabolic parameters were normal. At 7 years scoliosis was diagnosed and treated byphysiotherapy. Muscles were hypotrophic and subcutaneous fat was scarce, giving theimpression of anorexia. Because the scoliosis progressed, a stabilizing operation on theJ. Inher. Metab. Dis. 20 (1997) 427– 4 3 1

Short-chain acyl-CoA dehydrogenase (SCAD) is a homotetrameric mitochondrial flavoprotein that catalyses the first step in/3-oxidation of butyryl-CoA and hexanoylCoA. SCAD deficiency is associated with failure to thrive, metabolic acidosis, various degrees of neuromuscular dysfunction and abnormal urinary excretion of ethylmalonate, methylsuccinate and n-butyrylglycine (Amendt et al 1987; Coates et al 1988; Turnbull et al 1990). Recently, new variant SCAD alleles in patients with proven SCAD deficiency have been reported (Kristensen et al 1993). One of these alleles harbours adenine at position 625 (A625) of SCAD cDNA instead of guanine (G625), changing glycine at the position 209 of precursor SCAD to serine (Ser 209). Surprisingly, in 90 randomly selected controls we found that 4% were homozygous A625/A625 and 31% heterozygous A625/G625 (Kristensen et al 1994). As a result, this polymorphism is unlikely to be disease-causing itself. Significant overrepresentation of the A625 allele was found in a group of patients suspected of having a fatty oxidation defect (unpublished results). This implicates a susceptibility that may, in combination with other factors, provoke symptoms. Such a factor can, for example, be the effect of valproate and its metabolites on/3-oxidation, as presented here.

We have determined the frequency of the A985G mutation in the medium-chain acyl-CoA dehydrogenase (MCAD) gene in a cohort of 1142 healthy babies born in two Geneva hospitals. Among babies with at least one Swiss parent, heterozygotes were detected at a frequency of 1/52, with a 95% confidence range from 1/82 to 1/38. The high frequency of the carrier state for this mutation suggests that MCAD-deficient babies are born with a frequency of 1/10,000 in the Swiss population. This number is in sharp contrast with the low number of symptomatic MCAD-deficient patients diagnosed in this country. Thus, the fraction of homozygotes who remain asymptomatic is likely to be very high in the Swiss population, and possibly higher than in other countries of northern Europe.

The glycine conjugates of isocaproic, 4-methylhexanoic, 7-hydroxyoctanoic and 8-hydroxyoctanoic acids have been identified in the urine of children with medium-chain acyl-CoA dehydrogenase (MCAD) deficiency using gas chromatography-mass spectrometry of the trimethylsilyl derivatives. A quantitative study showed that the glycine conjugates of isocaproic and 4-methylhexanoics acids were excreted during acute episodes and in smaller amounts when subjects were asymptomatic. The glycine conjugates of 7-hydroxyoctanoic and 8-hydroxyoctanoic acids were detectable during acute episodes. None of the conjugates was detected in controls or controls receiving a diet containing medium-chain triglycerides. It is suggested that the glycine conjugates of isocaproic acid and 4-methylhexanoic acid are metabolites of branched-chain fatty acids and that they are specific for MCAD deficiency.

Cytochrome c oxidase (COX, EC 1.9.3.1) is part of the electron-transfer chain, where it is known as comlex IV. COX is composed of 13 different subunits: 3 are encoded by mitochondrial DNA while 10 are coded for by nuclear DNA. Given this complexity, it is not surprising that COX deficiency can cause diseases with many different clinical entities: from isolated muscle symptoms (Dimauro et al 1990) to severe progressive neurodegenerative diseases ending with death usually before the second year of life (e.g. Leigh syndrome) (Arts et al 1987). Some cases of COX deficiency have a generalized defect of this enzyme, while others have a defect restricted to one or a limited number of tissues (e.g. skeletal muscles alone)

In order to determine which are useful early diagnostic markers for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, we have analysed urine from an asymptomatic neonate. Profiling of urinary organic acids followed by peak confirmation by electron impact mass spectrometry revealed a high suberate/adipate ratio (greater than 1.0) and the presence of n-hexanoylglycine (HG). Acylcarnitine analysis by fast atom bombardment mass spectrometry (FAB-MS) was inconclusive, but FAB-MS/MS (tandem mass spectrometry) revealed diagnostic amounts of octanoylcarnitine and hexanoylcarnitine. Quantitative analysis of acylglycines by stable isotope dilution and chemical ionization mass spectrometry revealed a 30-fold increase in HG and increased suberylglycine, but no increase in 3-phenylpropionylglycine.

Two patients with hypoketotic hypoglycaemia and dicarboxylic aciduria are described. Studies of their urinary organic acids by gas chromatography-mass spectrometry (GC-MS) showed an excretion of dicarboxylic acids (adipic suberic and sebacic acids), unsaturated dicarboxylic acids (cis-octenedioic and decenedioic acids), 5-hydroxyhexanoic acid, hexanoyl-glycine and suberylglycine. Deficiency of the medium chain acyl-CoA dehydrogenase (MCAD) in fibroblasts was documented for both children. Despite a similar presentation (hypoglycaemic coma), organic acid profile (dicarboxylic aciduria and suberylglycine excretion) and enzyme deficiency (MCAD), they did not respond similarly to glucose infusion.

At least eight inborn errors of mitochondrial fatty acid β-oxidation are currently known. In the span of the last few years, almost 200 patients suffering from one of these disorders have been diagnosed (Vianey-Liaud et al., 1987), and some of them were initially mistaken as Reye’s or sudden infant death syndrome. Since the prognosis of these patients can be greatly improved by treatment with available therapeutic measures, fast and accurate diagnosis of this group of diseases is crucial. In reality, however, the diagnosis has been difficult for many of these disorders (Editorial in Lancet, 1986).

The production of tritiated water from [9,10-3H]myristic acid can be used as a screening assay for the detection of medium-chain acyl-CoA dehydrogenase deficiency, multiple acyl-CoA dehydrogenation defects (glutaric aciduria type 2 and ethylmalonic-adipic aciduria types), and some types of hydroxydicarboxylic aciduria. Comparison with the release of tritiated water from [9,10-3H]palmitic acid may give an indication of the chain-length specificity of the metabolic defect. In a case of ethylmalonic-adipic aciduria such a prediction has been confirmed by examination of accumulated intermediates in the affected fibroblasts.

A new patient with medium-chain dicarboxylic aciduria and suberyl glycinuria during an attack of acute illness is reported. When, inadvertently he was given medium-chain triglycerides for 2 days, the excretion of abnormal metabolites of medium-chain fatty acids increased and hepatomegaly became more pronounced. During remission a low excretion of the metabolites were observed. After 16 h of fasting hypoglycaemia was accompanied by an increase of urinary dicarboxylic acids and psi-hydroxyacids similar to that found on admission. Interestingly this urinary organic acid pattern persisted 8 h after intravenous administration of glucose. In a blood sample obtained after 16 h of fasting there was hypoketonaemia and increased levels of total free fatty acids, octanoic, decanoic and cis-4-decenoic acids. These biochemical data suggest the existence of a deficiency at the level of medium-chain acyl-CoA dehydrogenase.

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.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a disorder of β- oxidation of fatty acids leading to episodes of hypoketotic hypoglycaemia. Typically, excretion of abnormal metabolites, derived from alternative pathways of fatty acid metabolism, only occurs when the patient is unwell (Gregersen et al., 1983). Two approaches have been suggested for investigation of asymptomatic patients and siblings, prolonged fasting or loading test with medium-chain triglycerides. Both tests require the analysis of urine for characteristic metabolites, preferably by gas chromatography-mass spectroscopy. Fasting is not without serious risk (Gregersen et al., 1976) and the latter test may lack specificity (Whyte et al., 1986).

Various types of dicarboxylic aciduria are known, most of them are accompanied by non-ketotic hypoglycaemia. For the differential diagnosis of these conditions several methods of investigation have been used: (1) analysis of urinary organic acids in both native and hydrolysed samples, (2) analysis of free and esterified carnitine, the latter by means of chromatographic separation and identification of acyl moieties, (3) analysis of plasma organic acids, including the so-called free fatty acids, (4) a prolonged fasting test with serial measurements of the aforementioned parameters and close monitoring of the blood glucose and (5) an oral loading test with medium chain triglycerides accompanied by the same measurements as those named in item (4). So far differentiation has been made between patients with a metabolite profile most probably characteristic of medium chain acyl-CoA dehydrogenase deficiency and other dicarboxylic acidurias, among the latter systemic carnitine deficiency. Patients belonging to the first group accumulate octanoate, decanoate and cis-4-decenoate in their plasma; they excrete hexanoylglycine, octanoylcarnitine and suberylglycine in addition to the usual C6-C10 dicarboxylic acids. There was a high prevalence of an increased plasma free fatty acid/3-hydroxybutyrate ratio.

The complex organic acidopathy known as multiple acyl-CoA dehydrogenase deficiency (MACD) appears to involve a spectrum of clinical severity with the more severe phenotypes being represented by glutaric aciduria type II (GAII; McKusick 23168) (Goodman et al., 1982) and the less severe phenotypes being represented by ethylmalonic-adipic aciduria (EMAuria McKusick 22717) (Mantagos et al., 1979). Survival beyond the newborn period in GAII cases is rare. The molecular defect appears to involve deficient electron transferring flavoprotein (ETF) or its dehydrogenase (Goodman et al., 1983). In contrast, cases of EMAuria do survive with treatment. The exact molecular defect remains unclear, but some cases appear to respond to riboflavin (Gregerson et al., 1982).