Carnitine palmitoyltransferase II deficiency: successful anaplerotic diet therapy.

BACKGROUND: Carnitine palmitoyltransferase II (CPT II) deficiency is an important cause of recurrent rhabdomyolysis in children and adults. Current treatment includes dietary fat restriction, with increased carbohydrate intake and exercise restriction to avoid muscle pain and rhabdomyolysis. METHODS: CPT II enzyme assay, DNA mutation analysis, quantitative analysis of acylcarnitines in blood and cultured fibroblasts, urinary organic acids, the standardized 36-item Short-Form Health Status survey (SF-36) version 2, and bioelectric impedance for body fat composition. Diet treatment with triheptanoin at 30% to 35% of total daily caloric intake was used for all patients. RESULTS: Seven patients with CPT II deficiency were studied from 7 to 61 months on the triheptanoin (anaplerotic) diet. Five had previous episodes of rhabdomyolysis requiring hospitalizations and muscle pain on exertion prior to the diet (two younger patients had not had rhabdomyolysis). While on the diet, only two patients experienced mild muscle pain with exercise. During short periods of noncompliance, two patients experienced rhabdomyolysis with exercise. None experienced rhabdomyolysis or hospitalizations while on the diet. All patients returned to normal physical activities including strenuous sports. Exercise restriction was eliminated. Previously abnormal SF-36 physical composite scores returned to normal levels that persisted for the duration of the therapy in all five symptomatic patients. CONCLUSIONS: The triheptanoin diet seems to be an effective therapy for adult-onset carnitine palmitoyltransferase II deficiency.

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.

Respiratory complex II defect in siblings associated with a symptomatic secondary block in fatty acid oxidation.

The mitochondrial oxidative phosphorylation and fatty acid oxidation pathways have traditionally been considered independent major sources of cellular energy production; however, case reports of patients with specific enzymatic defects in either pathway have suggested the potential for a complex interference between the two. This study documents a new site of interference between the two pathways, a site in respiratory complex II capable of producing clinical signs of a block in fatty acid oxidation and reduced in vitro activity of acyl-CoA dehydrogenases. The initial patient, and later her newborn sibling, had mildly dysmorphic features, lactic acidosis and a defect in mitochondrial respiratory complex II associated with many biochemical features of a block in fatty acid oxidation. Results of in vitro probing of intact fibroblasts from both patients with methyl[2H3]palmitate and L-carnitine revealed greatly increased [2H3]butyrylcarnitine; however, the ratio of dehydrogenase activity with butyryl-CoA with anti-MCAD inactivating antibody (used to reveal SCAD-specific activity) to that with octanoyl-CoA was normal, excluding a selective SCAD or MCAD deficiency. Respiratory complex II was defective in both patients, with an absent thenoyltrifluoroacetone-sensitive succinate Q reductase activity that was partially restored by supplementation with duroquinone. Although secondary, the block in fatty acid oxidation was a major management problem since attempts to provide essential fatty acids precipitated acidotic decompensations. This study reinforces the need to pursue broadly the primary genetic defect within these two pathways, making full use of increasingly available functional and molecular diagnostic tools.

Prenatal diagnosis of multiple acyl-CoA dehydrogenase deficiency: association with elevated alpha-fetoprotein and cystic renal changes.

We report the occurrence of multiple acyl-CoA dehydrogenase deficiency (MADD) in two consecutive pregnancies in a young, Caucasian, non-consanguineous couple. In the first pregnancy, the maternal serum alpha-fetoprotein was elevated. A sonogram showed growth delay, cystic renal disease, and oligohydramnios; the parents decided to terminate the pregnancy. Postmortem examination confirmed the cystic renal disease and showed hepatic steatosis, raising the suspicion of a metabolic disorder. The diagnosis of MADD was made by immunoblot studies on cultured fibroblasts. In the subsequent pregnancy, a sonogram at 15 weeks' gestation showed an early growth delay but normal kidneys. The maternal serum and amniotic fluid concentrations of alpha-fetoprotein were elevated, and the amniotic fluid acylcarnitine profile was consistent with MADD. In vitro metabolic studies on cultured amniocytes confirmed the diagnosis. A follow-up sonogram showed cystic renal changes. These cases provide additional information regarding the evolution of renal changes in affected fetuses and show a relationship with elevated alpha-fetoprotein, which may be useful in counseling the couple at risk. MADD should be considered in the differential diagnosis of elevated alpha-fetoprotein and cystic renal disease. Early growth delay may be an additional feature.

Oxidation of unsaturated fatty acids by human fibroblasts with very-long-chain acyl-CoA dehydrogenase deficiency: aspects of substrate specificity and correlation with clinical phenotype.

The degradation of unsaturated fatty acids was examined in fibroblasts from 16 patients with very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. Analysis of acylcarnitine intermediates following incubation of intact human cells with these compounds revealed that the milder clinical phenotypes could be distinguished from the severe cardiomyopathic phenotype. These findings may reflect more effective contributions of alternate pathways in the milder forms of the disease. Incubation of VLCAD-deficient cells with cis-9 or trans-9 unsaturated fatty acids indicate that VLCAD is largely responsible for the 2,3-dehydrogenation of cis-5 or trans-5 intermediates in fibroblasts. The first two cycles of beta-oxidation with oleic and linoleic acids occur in the absence of VLCAD activity suggesting the presence of an additional acyl-CoA dehydrogenase or alternate pathway for the oxidation of these unsaturated fatty acids. These observations have clinical relevance for determining diagnosis, prognosis and strategies for dietary treatment of these patients.

Carnitine/acylcarnitine translocase deficiency (neonatal phenotype): successful prenatal and postmortem diagnosis associated with a novel mutation in a single family.

The neonatal phenotype of carnitine-acylcarnitine translocase (CACT) deficiency is one of the most severe and usually lethal mitochondrial fat oxidation disorders characterized by hypoketotic hypoglycemia, hyperammonemia, cardiac abnormalities, and early death. In this study, the proband was the daughter of consanguineous Hispanic parents. At 36 h of life, she had bradycardia and died at 4 days of age without a specific diagnosis. In a subsequent pregnancy, prenatal counseling and amniocentesis were provided. Incubation of the amniocytes from this pregnancy and fibroblasts (from the dead proband) with [16-(2)H(3)]palmitic acid and analysis by tandem mass spectrometry revealed an increasedconcentration of [16-(2)H(3)]palmitoylcarnitine, suggesting the diagnoses of either CACT or carnitine palmitoyltransferase II (CPT-II) deficiency. CACT enzyme activity was absent in both cell lines. Molecular investigation of cDNA from the dead proband and her affected sibling revealed aberrant CACT cDNA species, including exon 3 skipping, both exon 3 and 4 skipping, and a 13-bp insertion at cDNA position 388. Investigation of these cell lines for mutations affecting CACT RNA processing by analysis of CACT gene sequences, including intron and exon boundaries, revealed a single nucleotide G deletion at the donor site in intron 3 which resulted in exon skipping and a 13-bp insertion. The proband and her affected sibling were homozygous for this deletion.

2-Methylbutyryl-coenzyme A dehydrogenase deficiency: a new inborn error of L-isoleucine metabolism.

An 4-mo-old male was found to have an isolated increase in 2-methylbutyrylglycine (2-MBG) and 2-methylbutyrylcamitine (2-MBC) in physiologic fluids. In vitro oxidation studies in cultured fibroblasts using 13C- and 14C-labeled branched chain amino acids indicated an isolated block in 2-methylbutyryl-CoA dehydrogenase (2-MBCDase). Western blotting revealed absence of 2-MBCDase protein in fibroblast extracts; DNA sequencing identified a single 778 C>T substitution in the 2-MBCDase coding region (778 C>T), substituting phenylalanine for leucine at amino acid 222 (L222F) and absence of enzyme activity for the 2-MBCDase protein expressed in Escherichia coli. Prenatal diagnosis in a subsequent pregnancy suggested an affected female fetus, supporting an autosomal recessive mode of inheritance. These data confirm the first documented case of isolated 2-MBCDase deficiency in humans.

Identification of a novel mutation in patients with medium-chain acyl-CoA dehydrogenase deficiency.

A novel mutation was identified in two unrelated patients with medium-chain acyl-CoA dehydrogenase deficiency. First, a 19-year-old Caucasian female presented with a devastating illness, resulting in sudden death in adulthood which is unusual. The second patient, now a 3.5-year-old male, presented at 17 months of age with a hypoglycemic seizure and dehydration. Sequence analysis revealed a novel mutation G617T in exon 8 resulting in an arginine to leucine substitution at codon 206 (R206L). Both patients were compound heterozygous for this G617T and the common mutation A985G.

Death caused by perioperative fasting and sedation in a child with unrecognized very long chain acyl-coenzyme A dehydrogenase deficiency.

An adopted 3(1/2)-year-old girl with no prior medical problems died after a routine dental procedure. More than 2 years later, acylcarnitine analysis of dried blood found on her bedding revealed she had very long chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency. Perioperative oral fasting, without intravenous administration of glucose, may be detrimental to children with certain metabolic and endocrine disorders. Newborn screening by tandem mass spectrometry will detect disorders of fatty acid oxidation such as VLCAD and allow early and preventive treatment.

Evidence for a short-chain carnitine-acylcarnitine translocase in mitochondria specifically related to the metabolism of branched-chain amino acids.

Carnitine-acylcarnitine translocase (CATR) deficiency is a severe defect in fatty acid oxidation which presents early in life most frequently with hypoglycemia, hyperammonemia, and severe cardiac abnormalities. CATR exchanges acylcarnitines of various chain lengths for free carnitine across the mitochondrial membrane. In vitro studies in intact fibroblasts from patients with documented deficiency of CATR were probed with stable-isotope-labeled precursors and the resulting acylcarnitines were analyzed by tandem mass spectrometry. After a 72-h incubation with l-[(2)H(3)]carnitine the translocase-deficient cells produced acylcarnitines in which the deuterium was incorporated into short-chain acylcarnitines, C2-C5. Experiments with simultaneous incubation of l-[(2)H(3)]carnitine and l-[(13)C(6)]isoleucine produced [(13)C(5)]2-methylbutyryl-[(2)H(3)]carnitine and [(13)C(3)]propionyl-[(2)H(3)]carnitine indicating exchange of labeled acylcarnitine from inside the mitochondrial matrix with labeled free carnitine. These studies support the possible existence of a "branched-chain" carnitine-acylcarnitine translocator in mitochondria.

Familial neonatal SIDS revealing carnitine-acylcarnitine translocase deficiency.

UNLABELLED: A patient with a severe phenotype of carnitine-acylcarnitine translocase deficiency (CATR)(McKusick 212138) is reported. Prior to birth, a defect in beta-oxidation was suspected because of neonatal death of six siblings. Dietary treatment during neonatal adaptation and the subsequent six months of life and a trial of carnitine supplementation are reported. The rapidity with which long chain fatty acid metabolites can accumulate and induce secondary carnitine deficiency within a few hours after birth in an infant with CATR is noteworthy. CONCLUSION: High rates of glucose suppressed neonatal lipolysis in this infant, but did not seem sufficient to avoid secondary carnitine deficiency as in severe forms of CATR. Therefore simultaneous use of insulin and glucose may be necessary to control neonatal lipolysis. Carnitine supplementation and the possible adverse effects of MCT systematically administrated, should be further assessed in patients with CATR.

Hepatic carnitine palmitoyltransferase I deficiency presenting as maternal illness in pregnancy.

The spectrum of clinical presentation of fatty acid oxidation defects (FAOD) continues to expand. One FAOD, L-3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency has been associated with liver disease in pregnancies involving a heterozygous mother carrying an affected fetus. Hepatic carnitine palmitoyltransferase (CPT I) deficiency typically presents as a Reyelike syndrome in children between 8 and 18 mo. of age. We have investigated a family in which the mother developed liver disease consistent with acute fatty liver of pregnancy (AFLP) and hyperemesis gravidarum in her two successive pregnancies. Neither child nor their mother was found to carry the common LCHAD G1528C mutation. Both children were subsequently shown to have absent activity of CPT I. This is the first report of CPT I deficiency presenting as maternal illness in pregnancy.

Recent developments in the investigation of inherited metabolic disorders using cultured human cells.

Thepurpose of this paper is to share experience with our systems and review recent "in vitro" methods using intact cells (fibroblasts, amniocytes) in which entire metabolic pathways can be probed for inherited metabolic defects reflected by elevations of intermediates determined by tandem mass spectrometry, HPLC, or gas chromatography-mass spectrometry. Currently, one can explore the integrity of mitochondrial fat oxidation, peroxisomal degradation of methyl-branched fatty acids (e.g., pristanate), and the mitochondrial degradation of the branched chain amino acids (leucine, valine, and isoleucine). For many of the diseases, the specific defect can be recognized from the acylcarnitine profile resulting from incubation of the intact cells with stable-isotope-labeled precursors to the particular pathway. This approach has also been successful in identifying new inherited metabolic disorders, biochemical correlation with clinical phenotypes of individual defects, and sequential oxidation of fatty acids by peroxisomal-mitochondrial interaction.

Identification of two novel mutations in the hypoglycemic phenotype of very long chain acyl-CoA dehydrogenase deficiency.

Very long chain acyl-CoA dehydrogenase (VLCAD) catalyzes the initial step of long chain fatty acid oxidation in the mitochondria. Patients with VLCAD deficiency have recently been observed with two clinical phenotypes. The cardiac form presents with an early onset cardiomyopathy and a high incidence of infant death, while the hypoglycemic form resembles medium chain acyl-CoA dehydrogenase (MCAD) manifesting with hypoketotic hypoglycemia. In our investigation on the molecular basis for these phenotypes, we identified two novel mutations in one VLCAD patient with the hypoglycemic form, a C953T (Pro318Leu) mutation in exon 10 resulting in a substitution of proline 318 by leucine on one allele, and a C1194A (Tyr398Stop) mutation in exon 12 which created a premature stop codon TAA on another allele. The Tyr398Stop mutation may result in a truncated protein or instable messenger RNA.

Hypoketotic hypoglycemic coma in a 21-month-old child.

We present the case of a 21-month-old child with hypoketotic hypoglycemic coma. The differential diagnosis initially included metabolic causes versus a toxicologic emergency (unripe ackee fruit poisoning). Using information obtained from the emergency department, the diagnosis was confirmed as the late-onset form of glutaric acidemia type II. This case illustrates the importance of emergency physicians in the diagnosis and management of children with inborn errors of metabolism.

Studies on the oxidation of phytanic acid and pristanic acid in human fibroblasts by acylcarnitine analysis.

The alpha-oxidation of phytanic acid and the beta-oxidation of pristanitc acid were investigated in cultured fibroblasts from controls and patients affected with different peroxisomal disorders using deuterated substrates. Formation of [omega-2H6]4,8-dimethylnonanoylcarnitine ([omega-2H6]C11-carnitine) from [omega-2H6]phytanic acid and [omega-2H6]pristanic acid was used as marker for these processes. Analysis was performed by tandem mass spectrometry. In normal cells, formation of [omega-2H6]C11-carnitine from both [omega-2H6]phytanic acid and [omega-2H6]pristanic acid was observed. When peroxisome-deficient fibroblasts were incubated with these substrates, [omega-2H6]C11-carnitine was not detectable or, in two cases, very low, which results from deficiencies in both peroxisomal alpha- and beta-oxidation. In cells with an isolated beta-oxidation defect at the level of the peroxisomal bifunctional protein, formation of [omega-2H6]C11-carnitine could also not be detected. Cells with an isolated defect in the alpha-oxidation of phytanic acid, obtained from patients affected with Refsum disease (McKusick 266500) or rhizomelic chondrodysplasia punctata (McKusick 215100), did not form [omega-2H6]C11-carnitine from [omega-2H6]phytanic acid. The observed formation of [omega-2H6]C11-carnitine from [omega-2H6]pristanic acid in these cells is in accordance with a normal peroxisomal beta-oxidation in these disorders. This study shows that separate incubation of fibroblasts with [omega-2H6]phytanic acid and [omega-2H6]pristanic acid, followed by acylcarnitine analysis in the medium by tandem mass spectrometry, can be used for screening cell lines for deficiencies in the peroxisomal alpha- and beta-oxidation pathways. Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) and pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) are branched-chain fatty acids that are constituents of the human diet. As phytanic acid possesses a beta-methyl group, it cannot be degraded by beta-oxidation. Instead, phytanic acid is first degraded by alpha-oxidation, yielding pristanic acid, which is subsequently degraded by beta-oxidation (Figure 1). Phytanic acid alpha-oxidation is thought to occur partly, and pristanic acid beta-oxidation exclusively, in peroxisomes (see Wanders et al 1995 for review). Accumulation of phytanic acid and pristanic acid is found in blood and tissues of patients affected with generalized peroxisomal disorders. In this type of disorder, no morphologically distinguishable peroxisomes are present in tissues, resulting in accumulation of metabolites that are normally metabolized in these organelles (see Wanders et al 1995 for review). The group of generalized peroxisomal disorders consists of three diseases, differing in clinical presentation. Patients suffering from the most severe disease, Zellweger syndrome (McKusick 214100), have symptoms from birth on and usually do not live beyond their first year of life. Neonatal adrenoleukodystrophy (N-ALD, McKusick 202370) has a milder presentation, whereas infantile Refsum disease (IRD, McKusick 266510) is the mildest form among the generalized peroxisomal disorders. Not only in these generalized peroxisomal disorders, but also in some isolated peroxisomal beta-oxidation defects, elevated levels of phytanic acid and pristanic acid are found (ten Brink et al 1992a). The elevated phytanic acid levels are considered to be caused by product inhibition of alpha-oxidation by accumulating pristanic acid. This is reflected in a highly elevated pristanic acid to phytanic acid ratio in plasma from patients suffering from bifunctional protein deficiency or peroxisomal thiolase deficiency (ten Brink et al 1992a). Elevated phytanic acid concentrations are also found in plasma from patients affected with classical Refsum disease and rhizomelic chondrodysplasia punctata (RCDP). As pristanic acid beta-oxidation is not disturbed in these disorders, pristanic acid levels are normal (ten Brink et al 1992

Methylmalonic semialdehyde dehydrogenase deficiency: psychomotor delay and methylmalonic aciduria without metabolic decompensation.

A patient presenting with developmental delay but no episodes of metabolic acidosis was found to excrete significant amounts of methylmalonate (MMA) without any associated increased excretion of malonate, ethylmalonate, 3-hydroxypropionate, or beta-alanine. In contrast to patients with methylmalonic aciduria due to deficient mutase or impaired cobalamin metabolism, there was no increase of propionylcarnitine in blood or urine. The activity of methylmalonyl-CoA mutase and the pathway for cobalamin metabolism were also intact. The quantitative levels of the various labeled enantiomers of 3-hydroxyisobutyric (3-HIBA), 3-aminoisobutyric (3-AIBA), MMA, and propionylcarnitine were compared following separate intravenous infusions of equimolar doses of [2H8]-valine or [2H4]thymine in this patient and another with methylmalonyl-CoA mutase deficiency. Levels of labeled S- and R-3-HIBA and S- and R-3-AIBA indicated an isolated defect in methylmalonic semialdehyde dehydrogenase in this patient. This condition can be recognized by plasma MMA levels of approximately 8.5 microM (cf. 400 microM in mutase deficiency), urine MMA of 20-55 micromol/kg/24 h (cf. 1150 micromol/kg/24 h), no increase in propionylcarnitine following an oral carnitine load, and increased excretion of S-3-AIBA-nearly 10 times that observed in mutase deficiency. The ratio of R-AIBA to S-AIBA of <1 also reflects this disorder.

Pristanic acid beta-oxidation in peroxisomal disorders: studies in cultured human fibroblasts.

To investigate the individual steps of peroxisomal beta-oxidation, human fibroblasts from controls and patients affected by different peroxisomal disorders were incubated for 96 h with pristanic acid. Hereafter, 2,3-pristenic acid and 3-hydroxypristanic acid in the incubation medium were quantified by stable isotope dilution gas chromatography mass spectrometry (GC-MS). In control fibroblasts, both intermediates were formed and excreted into the medium in significant amounts. In cells from patients affected with different types of generalized peroxisomal disorders, the formation of both intermediates was absent or low, depending on the clinical severity of the disorder. In fibroblasts from patients affected with bifunctional protein deficiency, the concentrations of 2,3-pristenic acid and 3-hydroxypristanic acid in the medium were higher than in control cell lines.

A novel mutation identified in carnitine palmitoyltransferase II deficiency.

Carnitine palmitoyltransferase II (CPT II) deficiency is an autosomal recessive disorder of mitochondrial fatty-acid oxidation which presents as three distinct phenotypes (neonatal, infantile, and adult onset). CPT II exons from an adult-onset CPT II-deficient patient were amplified and directly sequenced to further investigate the molecular basis of this disorder. A novel mutation, C471T, in exon 4 of the carnitine palmitoyltransferase II gene was found which created a stop codon, TGA, at residue 124 of the protein (R124Stop). This mutation would result in severe protein truncation. This unique mutation was found on one allele while the S113L mutation, previously reported, was present on the other allele.