Evidence for impaired gluconeogenesis in very long-chain acyl-CoA dehydrogenase-deficient mice.

Hypoketotic hypoglycaemia is a characteristic feature of fatty acid oxidation (FAO) defects. Although the underlying pathogenic mechanism is unknown, one hypothesis points to an impairment in gluconeogenesis. To study hepatic glucose production in FAO defects, we used the knockout mouse model of very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency presenting with stress-induced hypoglycaemia. We analysed metabolites of hepatic glucose production under non-stressed conditions and after stress in comparison to wildtype controls. Analysis included glycogen, glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), glycerol-3-phosphate (G3P) and dihydroxyacetone-phosphate (DHAP). We also measured the activity of the key enzyme glucose-6-phosphatase. Blood and liver glucose were found to be low after stress, and liver glycogen was depleted. In addition, hepatic G6P and F6P were significantly reduced, especially during hypoglycaemia. Importantly, the activity of the enzyme converting G6P into glucose was not impaired. These data indicate a reduced rate of gluconeogenesis. The levels of DHAP and G3P were significantly lower suggesting decreased availability of glucose precursors from glycerol. This study gives biochemical evidence of impaired gluconeogenesis as one of the causes for hypoglycaemia observed in VLCAD deficiency. Whether this is due to lack of a substrate, inhibitory effects on other gluconeogenic enzymes or impaired transcription of gluconeogenic enzymes needs to be resolved in the future.

Carnitine supplementation induces long-chain acylcarnitine production--studies in the VLCAD-deficient mouse.

Carnitine supplementation does not affect carnitine concentrations in tissues of wild-type and very long-chain acyl-CoA dehydrogenase-deficient mice, but results in an increase in long-chain acylcarnitine production.

Potential of fibrates in the treatment of fatty acid oxidation disorders: revival of classical drugs?

Exposure to fibrates leads to normalization of fatty acid oxidation (FAO) in fibroblasts from patients with myopathic forms of CPT2 deficiency or VLCAD deficiency. Correction of FAO is related to a drug-induced increase of residual enzyme activity, and this could provide a new treatment strategy for these disorders.

Bezafibrate increases very-long-chain acyl-CoA dehydrogenase protein and mRNA expression in deficient fibroblasts and is a potential therapy for fatty acid oxidation disorders.

Inherited defect in very-long-chain acyl-CoA dehydrogenase (VLCAD), a mitochondrial enzyme catalyzing the initial step of long-chain fatty acid beta-oxidation (FAO), is one of the most frequent FAO enzyme defects. VLCAD deficiency is associated with clinical manifestations varying in severity, tissue involvement and age of onset. The molecular basis of VLCAD deficiency has been elucidated but therapeutic approaches are quite limited. In this study, we tested the hypothesis that fibrates, acting as agonist of peroxisome proliferator-activated receptors (PPARs), might stimulate FAO in VLCAD-deficient cells. We demonstrate that addition of bezafibrate or fenofibric acid in the culture medium induced a dose-dependent (up to 3-fold) increase in palmitate oxidation capacities in cells from patients with the myopathic form of VLCAD deficiency, but not in cells from severely affected patients. Complete normalization of cell FAO capacities could be achieved after exposure to 500 microm bezafibrate for 48 h. Cell therapy of VLCAD deficiency was related to drug-induced increases in VLCAD mRNA (+44 to +150%; P<0.001), protein (1.5-2-fold) and residual enzyme activity (up to 7.7-fold) in patient cells. Bezafibrate also diminished the production of toxic long-chain acylcarnitines by 90% in cells harboring moderate VLCAD deficiency. Finally, real-time PCR studies indicated that bezafibrate potentially stimulated gene expression of other enzymes in the beta-oxidation pathway. These data highlight the potential of fibrates in the correction of inborn FAO defects, as most mutations associated with these defects are compatible with the synthesis of a mutant protein with variable levels of residual enzyme activity.

The early-onset phenotype of mitochondrial trifunctional protein deficiency: a lethal disorder with multiple tissue involvement.

Mitochondrial trifunctional protein (TFP) deficiency is a clinically heterogeneous disorder with phenotypes of different severity. Early onset, severe forms predominantly exhibit cardiomyopathy, life-threatening arrhythmias and liver dysfunction; the later-onset, milder phenotype is mainly characterized by neuromyopathic features. The mechanisms that determine these heterogeneous presentations are unknown. We performed multiple tissue immunoblots from a patient with early-onset, lethal TFP deficiency and demonstrated absent TFP antigen in all. The predominant cardiac manifestation of severe TFP deficiency reflects its essential role in myocardial energetics, not its tissue-specific expression.

Rapid, comprehensive screening of the human medium chain acyl-CoA dehydrogenase gene.

Newborn screening by tandem mass spectrometry (MS/MS) identifies patients with medium chain acyl-CoA dehydrogenase (MCAD) deficiency the most frequently observed disorder of fatty acid oxidation. Molecular genetic analysis is becoming a common tool to confirm those identified as affected by prospective screening and for carrier detection in family studies. The A985G (K304E) mutation accounts for approximately 80% of mutant alleles in MCAD deficient patients, presenting symptomatically, while greater variability of mutant alleles is observed among cases identified through prospective screening. Aside from A985G, the mutation spectrum in MCAD deficient patients is heterogeneous such that comprehensive gene analysis is required. Traditionally the MCAD gene is assayed by sequencing the entire coding region. Although effective and definitive, this approach is expensive, turn around time is slow, and is poorly amenable to a clinical service molecular genetics laboratory. Dye-binding/high-resolution thermal denaturation is a rapid and homogeneous method by which to scan a PCR product for evidence of sequence aberration. PCR is performed in capillaries in the presence of the dsDNA-binding dye LCGreen I and subsequently the DNA/dye complexes are analyzed by high-resolution thermal denaturation. DNA sequencing was limited to fragments displaying abnormal melting profiles. Of 18 specimens analyzed, 11 have a genotype consistent with MCAD deficiency and seven have a genotype consistent with carrier status. Clinical and biochemical data corroborate that the genotype results identified the affected patients and differentiates them from carriers. The entire process is homogeneous requiring no post-PCR manipulation and is completed in under 3 h.

Changes in blood carnitine and acylcarnitine profiles of very long-chain acyl-CoA dehydrogenase-deficient mice subjected to stress.

BACKGROUND: In humans with deficiency of the very long-chain acyl-CoA dehydrogenase (VLCAD), C14-C18 acylcarnitines accumulate. In this paper we have used the VLCAD knockout mouse as a model to study changes in blood carnitine and acylcarnitine profiles under stress. DESIGN: VLCAD knockout mice exhibit stress-induced hypoglycaemia and skeletal myopathy; symptoms resembling human VLCADD. To study the extent of biochemical derangement in response to different stressors, we determined blood carnitine and acylcarnitine profiles after exercise on a treadmill, fasting, or exposure to cold. RESULTS: Even in a nonstressed, well-fed state, knockout mice presented twofold higher C14-C18 acylcarnitines and a lower free carnitine of 72% as compared to wild-type littermates. After 1 h of intense exercise, the C14-C18 acylcarnitines in blood significantly increased, but free carnitine remained unchanged. After 8 h of fasting at 4 degrees C, the long-chain acylcarnitines were elevated 5-fold in knockout mice in comparison with concentrations in unstressed wild-type mice (P < 0.05), and four out of 12 knockout mice died. Free carnitine decreased to 44% as compared with unstressed wild-type mice. An increase in C14-C18 acylcarnitines and a decrease of free carnitine were also observed in fasted heterozygous and wild-type mice. CONCLUSIONS: Long-chain acylcarnitines in blood increase in knockout mice in response to different stressors and concentrations correlate with the clinical condition. A decrease in blood free carnitine in response to severe stress is observed in knockout mice but also in wild-type littermates. Monitoring blood acylcarnitine profiles in response to different stressors may allow systematic analysis of therapeutic interventions in VLCAD knockout mice.

A case of mitochondrial trifunctional protein deficiency diagnosed by acylcarnitine profile and DNA analysis in a dried blood spot of a 4-day-old boy.

We report a Korean case, consistent with a biochemical diagnosis of trifunctional protein (TFP) deficiency, in which molecular diagnosis revealed a novel mutation in the alpha-subunit of TFP and the rare combination of two intergenic region (C/C and G/G) polymorphisms.

Deletion of the N-terminus of murine map2 by gene targeting disrupts hippocampal ca1 neuron architecture and alters contextual memory.

Microtubule-associated protein-2 (MAP2) is a brain specific A-kinase anchoring protein that targets the cyclic AMP-dependent protein kinase holoenzyme (PKA) to microtubules. Phosphorylation of MAP2 by different protein kinases is crucial for neuronal growth. The N-terminus of MAP2 contains the binding site for regulatory subunit II of cAMP-dependent protein kinase (PKA-RIIbeta). Using homologous recombination, we created a mutant line of mice (delta1-158) that express truncated MAP2 lacking the N-terminal peptide and the PKA binding site. Deletion of the PKA binding site from the MAP2 gene resulted in decreased efficiency of MAP2 phosphorylation. Biochemical and immunohistochemical studies demonstrate major changes in the morphology of hippocampal neurons in delta1-158 mice. Behavioral tests indicate that delta1-158 mice were impaired (exhibited less conditioned freezing) relative to Wild-Type (WT) controls during a test of contextual, but not during auditory cue, fear conditioning when tested at 8 weeks or 8 months of age. The delta1-158 mice displayed a heightened sensitivity to shock at 8 weeks, but not at 8 months of age. We conclude that PKA binding to MAP2 and MAP2 phosphorylation is essential for the selective development of contextual memory.

Restricted neuronal expression of ubiquitous mitochondrial creatine kinase: changing patterns in development and with increased activity.

Whereas ATP consumption increases with neural activity and is buffered by phosphocreatine (PCr), it is not known whether PCr synthesis by ubiquitous mitochondrial creatine kinase (uMtCK) supports energy metabolism in all neurons. To explore the possibility that uMtCK expression in neurons is modulated by activity and during development, we used immunocytochemistry to detect uMtCK-containing mitochondria. In the adult brain, subsets of neurons including layer Va pyramidal cells, most thalamic nuclei, cerebellar Purkinje cells, olfactory mitral cells and hippocampal interneurons strongly express uMtCK. uMtCK is transiently expressed by a larger group of neurons at birth. Neurons in all cortical layers express uMtCK at birth (P0), but uMtCK is restricted to layer Va by P12. uMtCK is detected in cerebellar Purkinje cells at birth, but localization to dendrites is only observed after P5 and is maximal on P14. Hippocampal CA1 and CA3 pyramidal neurons contain uMtCK-positive mitochondria at birth, but this pattern becomes progressively restricted to interneurons. Seizures induced uMtCK expression in cortical layers II-III and CA1 pyramidal neurons. In the cortex, but not in CA1, blockade of seizures prevented the induction of uMtCK. These findings support the concept that uMtCK expression in neurons is (1) developmentally regulated in post-natal life, (2) constitutively restricted in the adult brain, and (3) regulated by activity in the cortex and hippocampus. This implies that mitochondrial synthesis of PCr is restricted to those neurons that express uMtCK and may contribute to protect these cells during periods of increased energy demands.

Tandem mass spectrometric analysis for amino, organic, and fatty acid disorders in newborn dried blood spots: a two-year summary from the New England Newborn Screening Program.

BACKGROUND: Tandem mass spectrometry (MS/MS) is rapidly being adopted by newborn screening programs to screen dried blood spots for >20 markers of disease in a single assay. Limited information is available for setting the marker cutoffs and for the resulting positive predictive values. METHODS: We screened >160 000 newborns by MS/MS. The markers were extracted from blood spots into a methanol solution with deuterium-labeled internal standards and then were derivatized before analysis by MS/MS. Multiple reaction monitoring of each sample for the markers of interest was accomplished in approximately 1.9 min. Cutoffs for each marker were set at 6-13 SD above the population mean. RESULTS: We identified 22 babies with amino acid disorders (7 phenylketonuria, 11 hyperphenylalaninemia, 1 maple syrup urine disease, 1 hypermethioninemia, 1 arginosuccinate lyase deficiency, and 1 argininemia) and 20 infants with fatty and organic acid disorders (10 medium-chain acyl-CoA dehydrogenase deficiencies, 5 presumptive short-chain acyl-CoA dehydrogenase deficiencies, 2 propionic acidemias, 1 carnitine palmitoyltransferase II deficiency, 1 methylcrotonyl-CoA carboxylase deficiency, and 1 presumptive very-long chain acyl-CoA dehydrogenase deficiency). Approximately 0.3% of all newborns screened were flagged for either amino acid or acylcarnitine markers; approximately one-half of all the flagged infants were from the 5% of newborns who required neonatal intensive care or had birth weights <1500 g. CONCLUSIONS: In screening for 23 metabolic disorders by MS/MS, an mean positive predictive value of 8% can be achieved when using cutoffs for individual markers determined empirically on newborns.

Diagnosis of very long chain acyl-dehydrogenase deficiency from an infant's newborn screening card.

Very long chain fatty acid dehydrogenase (VLCAD) deficiency is a rare but treatable cause of cardiomyopathy, fatty liver, skeletal myopathy, pericardial effusions, ventricular arrhythmias, and sudden death. Unrecognized, VLCAD deficiency may be rapidly progressive and fatal, secondary to its cardiac involvement. Because early diagnosis improves outcome, we present a neonate with VLCAD deficiency in whom retrospective analysis of the newborn screening card revealed that a correct diagnosis could have been made by newborn screening using tandem mass spectrometry. Our patient demonstrated a classic neonatal course with transient hypoglycemia at birth, interpreted as culture-negative sepsis, followed by a quiescent period notable only for hypotonia and poor feeding. At 3 months, he presented with cardiorespiratory failure and pericardial effusions, requiring pericardiocentesis, tracheostomy, and prolonged mechanical ventilation. Plasma free-fatty acid and acylcarnitine profiles demonstrated small but significant elevations of C14:2, C14:1, C16, and C18:1 acylcarnitine species, findings consistent with a biochemical diagnosis of VLCAD deficiency. Enteral feeds were changed to Portagen formula with marked improvement in cardiac symptoms over several weeks. To confirm the biochemical diagnosis, molecular analysis was performed by analysis of genomic DNA on a blood sample of the patient. Sequencing analysis and delineation of VLCAD mutations were performed using polymerase chain reaction and genomic sequencing. The patient was heterozygous for 2 different disease-causing mutations at the VLCAD locus. The maternal mutation was a deletion of bp 842-3 in exon 8, causing a shift in the reading frame. The paternal mutation was G+1A in the consensus donor splice site after exon 1; this splice-site mutation would likely result in decreased mRNA. The likely consequence of these mutations is essentially a null phenotype. To determine whether this case could have been picked up by tandem mass spectrometry analysis at birth when the patient was asymptomatic, acylcarnitine analysis was performed on the patient's original newborn card (after obtaining parental consent, the original specimen was provided courtesy of Dr Kenneth Pass, Director, New York State Newborn Screening Program). The blood sample had been obtained at 1 week of age and stored at room temperature for 6 months and at 70 degrees C thereafter for 18 months. Electrospray tandem mass spectrometry used a LC-MS/MS API 2000 operated in ion evaporation mode with the TurboIonSpray ionization probe source. The acylcarnitine profile obtained from the patient's original newborn card was analyzed 2 years after it was obtained. In comparison with a normal control, there was a significant accumulation of long chain acylcarnitine species, with a prominent peak of tetradecenoylcarnitine (C14:1), the most characteristic metabolic marker of VLCAD deficiency. This profile would have likely been even more significant if it had been analyzed at the time of collection, yet 2 years later is sufficient to provide strong biochemical evidence of the underlying disorder. Discussion. VLCAD was first discovered in 1992, and clinical experience with VLCAD deficiency has been accumulating rapidly. Indeed, the patients originally diagnosed with long chain acyl-CoA deficiency suffer instead from VLCAD deficiency. The phenotype of VLCAD deficiency is heterogeneous, ranging from catastrophic metabolic and cardiac failure in infancy to mild hypoketotic, hypoglycemia, and exertional rhabdomyolysis in adults. This case demonstrates that VLCAD deficiency could have been detected from the patient's own neonatal heel-stick sample. Most likely, a presymptomatic diagnosis would have avoided at least part of a lengthy and intensive prediagnosis hospitalization that had an estimated cost of $400 000. Although VLCAD is relatively rare, timely and correct diagnosis leads to dramatic recovery, so that detection by newborn screening could prevent the onset of arrhythmias, heart failure, metabolic insufficiency, and death. Fatty acid oxidation defects, including VLCAD deficiency, may account for as many as 5% of sudden infant death patients. Recent instrumentation advances have made automated tandem mass spectrometry of routine neonatal heel-stick samples technically feasible. Pilot studies have demonstrated an incidence of fatty acid oxidation defects, including short chain, medium chain, and very long chain acyl-CoA dehydrogenase deficiencies, of approximately 1/12 000. As a result, cost-benefit ratios for this approach should be systematically examined.

Glutamate 170 of human l-3-hydroxyacyl-CoA dehydrogenase is required for proper orientation of the catalytic histidine and structural integrity of the enzyme.

l-3-Hydroxyacyl-CoA dehydrogenase (HAD), the penultimate enzyme in the beta-oxidation spiral, reversibly catalyzes the conversion of l-3-hydroxyacyl-CoA to the corresponding 3-ketoacyl-CoA. Similar to other dehydrogenases, HAD contains a general acid/base, His(158), which is within hydrogen bond distance of a carboxylate, Glu(170). To investigate its function in this catalytic dyad, Glu(170) was replaced with glutamine (E170Q), and the mutant enzyme was characterized. Whereas substrate and cofactor binding were unaffected by the mutation, E170Q exhibited diminished catalytic activity. Protonation of the catalytic histidine did not restore wild-type activity, indicating that modulation of the pK(a) of His(158) is not the sole function of Glu(170). The pH profile of charge transfer complex formation, an independent indicator of active site integrity, was unaltered by the amino acid substitution, but the intensity of the charge transfer band was diminished. This observation, coupled with significantly reduced enzymatic stability of the E170Q mutant, implicates Glu(170) in maintenance of active site architecture. Examination of the crystal structure of E170Q in complex with NAD(+) and acetoacetyl-CoA (R = 21.9%, R(free) = 27.6%, 2.2 A) reveals that Gln(170) no longer hydrogen bonds to the side chain of His(158). Instead, the imidazole ring is nearly perpendicular to its placement in the comparable native complex and no longer positioned for efficient catalysis.

Accumulation of free 3-hydroxy fatty acids in the culture media of fibroblasts from patients deficient in long-chain l-3-hydroxyacyl-CoA dehydrogenase: a useful diagnostic aid.

BACKGROUND: The diagnosis of long-chain L-3-hydroxy-acyl-coenzyme A dehydrogenase (LCHAD) deficiency frequently requires the study of cultured fibroblasts. We developed such a test that does not require disruption and loss of the cells. METHODS: We measured free 3-hydroxy fatty acids (3-OHFAs) in media of skin fibroblasts cultures from 11 patients with a genetic deficiency of LCHAD and the associated disorder of mitochondrial trifunctional protein (MTFP). Fibroblasts were cultured for 24 h with 100 micromol/L nonisotopic palmitate added. 3-OHFAs were measured by selected-ion monitoring, stable-isotope dilution gas chromatography-mass spectrometry with [(13)C]-labeled internal standards. RESULTS: 3-OH-hexadecanoic and 3-OH-tetradecanoic FAs were increased 14- and 11-fold, respectively, in all patients with LCHAD or MTFP deficiency when compared with control fibroblast cell lines after overnight incubation with palmitate. 3-OH-dodecanoic FA demonstrated a modest, fivefold increase in LCHAD-deficient cells. The concentrations of all 3-OHFAs were similar whether or not the medium samples were hydrolyzed to release conjugated species such as acylcarnitines, suggesting that 3-OHFAs accumulate in the media as free FAs. CONCLUSIONS: Measurement of 3-OHFA excretion from LCHAD- or MTFP-deficient cell lines can be used as a diagnostic tool. Free FAs are the predominant form of these abnormal metabolic intermediates in culture media.

Lack of mitochondrial trifunctional protein in mice causes neonatal hypoglycemia and sudden death.

Mitochondrial trifunctional protein (MTP) is a hetero-octamer of four alpha and four beta subunits that catalyzes the final three steps of mitochondrial long chain fatty acid beta-oxidation. Human MTP deficiency causes Reye-like syndrome, cardiomyopathy, or sudden unexpected death. We used gene targeting to generate an MTP alpha subunit null allele and to produce mice that lack MTP alpha and beta subunits. The Mtpa(-/-) fetuses accumulate long chain fatty acid metabolites and have low birth weight compared with the Mtpa(+/-) and Mtpa(+/+) littermates. Mtpa(-/-) mice suffer neonatal hypoglycemia and sudden death 6-36 hours after birth. Analysis of the histopathological changes in the Mtpa(-/-) pups revealed rapid development of hepatic steatosis after birth and, later, significant necrosis and acute degeneration of the cardiac and diaphragmatic myocytes. This mouse model documents that intact mitochondrial long chain fatty acid oxidation is essential for fetal development and for survival after birth. Deficiency of MTP causes fetal growth retardation, neonatal hypoglycemia, and sudden death.

Molecular prenatal diagnosis in families with fetal mitochondrial trifunctional protein mutations.

OBJECTIVES: To evaluate the feasibility of molecular prenatal diagnosis in families with mitochondrial trifunctional protein (TFP) mutations and prospectively study the effects of fetal genotype on pregnancy outcome. TFP catalyzes the last 3 steps in mitochondrial long-chain fatty acid oxidation. STUDY DESIGN: We performed molecular prenatal diagnosis in 9 pregnancies, 8 in 6 families with isolated long-chain 3-hydroxyacyl-coenzyme A dehydrogenase (LCHAD) deficiency and one in a family with complete TFP deficiency. Analyses were performed on chorionic villous samples in 7 pregnancies and on amniocytes in 2. RESULTS: Molecular prenatal diagnosis successfully identified the fetal genotype in all 9 pregnancies. Two fetuses were affected, and both pregnancies were terminated by family decision. Two other fetuses had normal genotype and 5 others were heterozygotes. These 7 pregnancies were uncomplicated, and all the offspring are alive and apparently healthy. Genotypes of the aborted fetuses and neonates were confirmed by molecular analysis and enzymatic assays. CONCLUSIONS: Molecular prenatal diagnosis is possible and valid in guiding management of pregnancies in families with known TFP defects. Women heterozygous for TFP alpha-subunit mutations who carry fetuses with wild-type or heterozygous genotypes have uncomplicated pregnancies.

Placental floor infarction complicating the pregnancy of a fetus with long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency.

By postmortem biochemical and molecular genetic analyses, an 8-month-old infant was diagnosed with long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency, an inborn error of mitochondrial fatty acid beta-oxidation. He was born following a pregnancy complicated by a maternal floor infarction of the placenta, a disorder of unknown etiology. We speculate that the child's autosomal recessive fatty acid beta-oxidation disorder and the pregnancy complication are causally related.

Progression of hepatic damage during cold storage after procurement in a liver and kidney donor with HELLP syndrome.

BACKGROUND: HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome and acute fatty liver of pregnancy are associated with preeclampsia and fetal defects in fatty acid metabolism. This defect causes the accumulation of metabolites that are harmful to the maternal liver. CASE REPORT: We report a liver and kidney donor with HELLP syndrome and describe the progression of disease in the liver during cold storage. Before procurement, liver biopsy demonstrated minimal necrosis. However, after cold storage, repeat biopsy demonstrated more than 30% necrosis. The liver was not engrafted; the kidneys were transplanted without complication. CONCLUSION: Livers procured from patients with HELLP syndrome should be carefully evaluated for progression of hepatic damage during cold storage and transport.