Sensory neuronopathy as a major clinical feature of mitochondrial trifunctional protein deficiency in adults.

INTRODUCTION: Mitochondrial trifunctional protein deficiency (MTPD) is a long-chain fatty acid oxidation disorder characterized by co-existence of rhabdomyolysis episodes and peripheral neuropathy. Two phenotypes are described: generalized mitochondrial trifunctional protein deficiency (gMTPD) and isolated long-chain-3-hydroxyacyl-CoA dehydrogenase deficiency (iLCHADD) that is always associated with the c.1528G>C mutation. Peripheral neuropathy of MTPD is commonly described in children as axonal, length-dependent and sensorimotor. OBJECTIVES: To report clinical and electrophysiological features of four independent adult MTPD patients with peripheral neuropathy. RESULTS: Onset of the disease was characterized in all patients by rhabdomyolysis episodes occurring during childhood preceded by severe hypoglycemic episodes in three patients. Peripheral nerve involvement manifesting as sensory ataxia appeared later, during adolescence or adulthood. In all cases, electroneuromyogram showed no length-dependent sensory potentials decrease characteristic of sensory neuronopathy ("ganglionopathy"). All patients harbored at least one c.1528G>C mutation. DISCUSSION: We describe MTPD as a newly hereditary etiology of sensory neuronopathy in adults, specifically in patients with c.1528G>C mutation. MTPD should be screened for by performing plasma acylcarnitines in patients with chronic sensory neuronopathy and additional suggestive features such as exercise intolerance or retinopathy.

Impact of mutations within the [Fe-S] cluster or the lipoic acid biosynthesis pathways on mitochondrial protein expression profiles in fibroblasts from patients.

Lipoic acid (LA) is the cofactor of the E2 subunit of mitochondrial ketoacid dehydrogenases and plays a major role in oxidative decarboxylation. De novo LA biosynthesis is dependent on LIAS activity together with LIPT1 and LIPT2. LIAS is an iron­sulfur (Fe-S) cluster-containing mitochondrial protein, like mitochondrial aconitase (mt-aco) and some subunits of respiratory chain (RC) complexes I, II and III. All of them harbor at least one [Fe-S] cluster and their activity is dependent on the mitochondrial [Fe-S] cluster (ISC) assembly machinery. Disorders in the ISC machinery affect numerous Fe-S proteins and lead to a heterogeneous group of diseases with a wide variety of clinical symptoms and combined enzymatic defects. Here, we present the biochemical profiles of several key mitochondrial [Fe-S]-containing proteins in fibroblasts from 13 patients carrying mutations in genes encoding proteins involved in either the lipoic acid (LIPT1 and LIPT2) or mitochondrial ISC biogenesis (FDX1L, ISCA2, IBA57, NFU1, BOLA3) pathway. Ten of them are new patients described for the first time. We confirm that the fibroblast is a good cellular model to study these deficiencies, except for patients presenting mutations in FDX1L and a muscular clinical phenotype. We find that oxidative phosphorylation can be affected by LA defects in LIPT1 and LIPT2 patients due to excessive oxidative stress or to another mechanism connecting LA and respiratory chain activity. We confirm that NFU1, BOLA3, ISCA2 and IBA57 operate in the maturation of [4Fe-4S] clusters and not in [2Fe-2S] protein maturation. Our work suggests a functional difference between IBA57 and other proteins involved in maturation of [Fe-S] proteins. IBA57 seems to require BOLA3, NFU1 and ISCA2 for its stability and NFU1 requires BOLA3. Finally, our study establishes different biochemical profiles for patients according to their mutated protein.

Molecular characterization of 82 patients with pyruvate dehydrogenase complex deficiency. Structural implications of novel amino acid substitutions in E1 protein.

BACKGROUND: Pyruvate dehydrogenase complex (PDHc) deficiencies are an important cause of primary lactic acidosis. Most cases result from mutations in the X-linked gene for the pyruvate dehydrogenase E1α subunit (PDHA1) while a few cases result from mutations in genes for E1ß (PDHB), E2 (DLAT), E3 (DLD) and E3BP (PDHX) subunits or PDH-phosphatase (PDP1). AIM: To report molecular characterization of 82 PDHc-deficient patients and analyze structural effects of novel missense mutations in PDHA1. METHODS: PDHA1 variations were investigated first, by exon sequencing using a long range PCR product, gene dosage assay and cDNA analysis. Mutation scanning in PDHX, PDHB, DLAT and DLD cDNAs was further performed in unsolved cases. Novel missense mutations in PDHA1 were located on the tridimensional model of human E1 protein to predict their possible functional consequences. RESULTS: PDHA1 mutations were found in 30 girls and 35 boys. Three large rearrangements, including two contiguous gene deletion syndrome were identified. Novel missense, frameshift and splicing mutations were also delineated and a nonsense mutation in a mosaic male. Mutations p.Glu75Ala, p.Arg88Ser, p.Arg119Trp, p.Gly144Asp, p.Pro217Arg, p.Arg235Gly, p.Tyr243Cys, p.Tyr243Ser, p.Arg245Gly, p.Pro250Leu, p.Gly278Arg, p.Met282Val, p.Gly298Glu in PDHA1 were predicted to impair active site channel conformation or subunit interactions. Six out of the seven patients with PDHB mutations displayed the recurrent p.Met101Val mutation; 9 patients harbored PDHX mutations and one patient DLD mutations. CONCLUSION: We provide an efficient stepwise strategy for mutation screening in PDHc genes and expand the growing list of PDHA1 mutations analyzed at the structural level.

Comprehensive cDNA study and quantitative analysis of mutant HADHA and HADHB transcripts in a French cohort of 52 patients with mitochondrial trifunctional protein deficiency.

BACKGROUND: Deficiency of mitochondrial trifunctional protein (MTP) is caused by mutations in the HADHA and HADHB genes, which have been mostly delineated at the genomic DNA level and have not been always elucidated. AIM: To identify mutations in a French cohort of 52 MTP deficient patients and the susceptibility of mutations generating premature termination codons (PTCs) to the nonsense mRNA mediated decay (NMD). METHODS: Mutation screening in fibroblasts was performed at the cDNA level and real-time RT-PCR was used to compare the levels of the different PTC-bearing mRNAs before and after a treatment of fibroblasts by emetine, a translation inhibitor. RESULTS: A mutation detection rate of 100% was achieved. A total of 22 novel mutations were identified, including a large-sized genomic deletion in HADHB gene. A high proportion of all identified mutations were non-sense, frameshift and splicing mutations, generating (PTCs), distributed essentially on HADHA coding regions. We could demonstrate that the majority of mutations resulting in PTCs conform to the established rules governing the susceptibility to NMD. CONCLUSION: Our results emphasize the value of cDNA analysis in the characterization of HADHA and HADHB mutations and further strengthen the model of haploinsufficiency as a major pathomechanism in MTP defects.

Expanding the phenotype of mitochondrial disease: Novel pathogenic variant in ISCA1 leading to instability of the iron-sulfur cluster in the protein.

We report a patient carrying a novel pathogenic variant p.(Tyr101Cys) in ISCA1 leading to MMDS type 5. He initially presented a psychomotor regression with loss of gait and language skills and a tetrapyramidal spastic syndrome. Biochemical analysis of patient fibroblasts revealed impaired lipoic acid synthesis and decreased activities of complex I and II of respiratory chain. While ISCA1 is involved in the mitochondrial machinery for iron-sulfur cluster biogenesis, these dysfunctions are secondary to impaired maturation of mitochondrial proteins containing the [4Fe-4S] clusters. Expression and purification of the human ISCA1 showed a decreased stability of the [2Fe-2S] cluster in the mutated protein.

Two silent substitutions in the PDHA1 gene cause exon 5 skipping by disruption of a putative exonic splicing enhancer.

BACKGROUND: Synonymous mutations within exons may cause aberrant splicing by disrupting exonic splicing enhancer (ESE) motifs in the vicinity of non consensus splice sites. Mutational analysis of PDHA1 revealed only one silent single nucleotide substitution in exon 5 in two unrelated boys and a girl (c.483C>T and c.498C>T variants, respectively). For both patients, pyruvate dehydrogenase complex activity was low and the immunoreactive E1alpha protein was defective in cultured fibroblasts. METHODS AND RESULTS: One of the boys was a somatic mosaic for the c.483C>T variant, as shown by the variable ratio of mutant to normal alleles in fibroblast, lymphocyte and single hair root DNA. Transcript analysis in fibroblasts from the three patients revealed the presence of both normal and truncated cDNAs, with the splicing out of exon 5 predicted to result in a frame shift and premature termination (p.Arg141AlafsX11). The treatment of fibroblasts with emetine before harvesting to prevent nonsense mRNA-mediated decay increased the amount of mutant mRNA. In silico analysis revealed that each variant disrupted a putative SRp55 binding site and that the intron 5 donor splice site (5'ss) contained a weak splicing signal. Transient transfection of COS-7 or Hela cells with hybrid minigene constructs containing wild-type or mutant PDHA1 exon 5, followed by RT-PCR demonstrated that each variant resulted in the incomplete inclusion of PDHA1 exon 5, and that this defect was corrected following the restoration of a perfect consensus sequence for the 5' splice site by site-directed mutagenesis. CONCLUSION: These two synonymous mutations expand the spectrum of rare PDHA1 splicing mutations, all of which are located in non canonical splice sites.

Ketone Bodies as a Possible Adjuvant to Ketogenic Diet in PDHc Deficiency but Not in GLUT1 Deficiency.

OBJECTIVE: Ketogenic diet is the first line therapy for neurological symptoms associated with pyruvate dehydrogenase deficiency (PDHD) and intractable seizures in a number of disorders, including GLUT1 deficiency syndrome (GLUT1-DS). Because high-fat diet raises serious compliance issues, we investigated if oral L,D-3-hydroxybutyrate administration could be as effective as ketogenic diet in PDHD and GLUT1-DS. METHODS: We designed a partial or total progressive substitution of KD with L,D-3-hydroxybutyrate in three GLUT1-DS and two PDHD patients. RESULTS: In GLUT1-DS patients, we observed clinical deterioration including increased frequency of seizures and myoclonus. In parallel, ketone bodies in CSF decreased after introducing 3-hydroxybutyrate. By contrast, two patients with PDHD showed clinical improvement as dystonic crises and fatigability decreased under basal metabolic conditions. In one of the two PDHD children, 3-hydroxybutyrate has largely replaced the ketogenic diet, with the latter that is mostly resumed only during febrile illness. Positive direct effects on energy metabolism in PDHD patients were suggested by negative correlation between ketonemia and lactatemia (r 2 = 0.59). Moreover, in cultured PDHc-deficient fibroblasts, the increase of CO2 production after 14C-labeled 3-hydroxybutyrate supplementation was consistent with improved Krebs cycle activity. However, except in one patient, ketonemia tended to be lower with 3-hydroxybutyrate administration compared to ketogenic diet. CONCLUSION: 3-hydroxybutyrate may be an adjuvant treatment to ketogenic diet in PDHD but not in GLUT1-DS under basal metabolic conditions. Nevertheless, ketogenic diet is still necessary in PDHD patients during febrile illness.

Interleukin-1 beta and interleukin-6 stimulate 2-methylaminoisobutyric acid uptake in HepG2 cells.

The metabolic response to inflammation involves an increased uptake of amino acids in the liver. It has been suggested that cytokines, such as interleukin-1 beta and interleukin-6, could be involved in this increased amino acid uptake. We investigated the role of these two inflammatory cytokines in regulating hepatic amino acid transport systems in the human hepatoma cell line, HepG2. Uptake of methylaminoisobutyric acid, the most specific known substrate of system A, and of glutamine, both transported by other sodium-dependent transport systems ASC and N, was assayed after incubation of the cells for various times with cytokines, using the cluster-tray method. Interleukin-1 beta and interleukin-6 (1000 U/ml) stimulated methylaminoisobutyric acid uptake by 36 +/- 6 and 41 +/- 4%, respectively (per cent +/- SD, n > or = 6). Under our experimental conditions, these cytokines had no effect on glutamine uptake. The stimulatory effect on methylaminoisobutyric acid uptake was not increased by combining the cytokines or by the presence of dexamethasone. The cytokine effect was abolished by cycloheximide, suggesting the involvement of de novo protein synthesis in this activation of transport system A. These data demonstrate that, in our culture conditions, interleukin-1 beta and interleukin-6 indirectly exert a stimulatory effect on methylaminoisobutyric acid transport in HepG2 cells.

[Serum activity of mitochondrial aspartate aminotransferase and extrahepatic cholestasis]. / Activité sérique de l'aspartate aminotransférase mitochondriale et cholestase extrahépatique.

Serum mitochondrial aspartate aminotransferase (mAST) level and the mitochondrial aspartate aminotransferase/total aspartate aminotransferase ratio (mAST/AST) have been proposed as sensitive markers of chronic alcoholism. Their specificity, however, remains poorly defined. The purpose of this study was to compare these markers in three groups of hospitalized patients: group I, 80 patients with chronic alcoholic liver disease; group II, 51 patients with chronic liver disease without alcoholism; group III, 44 patients with extrahepatic cholestasis (due to choledocholithiasis in 21 and malignant in 23). mAST was measured after immuno-precipitation of cytoplasmic aspartate aminotransferase. The normal values of mAST (less than or equal to 2 mu/l) and mAST/AST (less than or equal to 6 p. 100) were defined in a group of 59 non alcoholic subjects without liver disease (controls). mAST was increased as compared with controls in 91 p. 100 of the patients of group I, 20 p. 100 of group II, 61 p. 100 of group III. mAST was comparable in groups I (mean +/- SD: 10 +/- 10.8) and III (10.3 +/- 12.9), and higher than in group II (1.8 +/- 2.4). m/AST was increased in 59 p. 100 of the patients of group I, 6 p. 100 of group II and 36 p. 100 of group III. It was higher in group I (8 +/- 4 p. 100) than in group III (6 +/- 4 p. 100, p less than 0.02), and particularly higher in both these groups than in group II (2 +/- 1 p. 100, p less than 0.00001). mAST was correlated to AST in each of these three groups.(ABSTRACT TRUNCATED AT 250 WORDS)

[Macroaspartate aminotransferase. Study of 5 cases and review of the literature]. / Macroaspartate aminotransférase. Etude de 5 cas et revue de la littérature.

Aspartate aminotransferase can exist as a macroenzyme, which has a higher molecular mass than the corresponding enzyme normally found in serum under physiologic or pathological conditions. This macroenzyme is often an immunoglobulin complexed-enzyme and induces persistently increased serum aspartate aminotransferase activity without any corresponding liver or muscle damage. We report 5 patients with isolated and persistent increased serum aspartate aminotransferase activity in whom a macroenzyme has been detected. Of these 5 cases, four were apparently healthy subjects and the last had chronic active hepatitis. Electrophoresis of aspartate aminotransferase isoenzymes of the subjects' serum showed an abnormal band migrating between mitochondrial and cytosolic aspartate aminotransferase. In 3 cases, the macrocomplex consisted of aspartate aminotransferase and immunoglobulin G, as shown by the immunoprecipitation method. In the patient with chronic active hepatitis, the macroenzyme disappeared after liver transplantation. As macroaspartate aminotransferase and others macroenzymes, may persist for months or even years, it is important for clinicians to be aware of their existence to avoid unnecessary invasive or costly procedures.

[Role of the serum level of mitochondrial aspartate aminotransferase as marker of alcoholic intoxication in cirrhotic patients]. / Place du taux sérique de l'aspartate aminotransférase mitochondriale comme marqueur d'intoxication chez le cirrhotique alcoolique.

The purpose of this study was to assess the diagnostic value of serum mitochondrial aspartate aminotransferase activity (mAST) and of the mitochondrial aspartate aminotransferase/total aspartate aminotransferase ratio (mAST/t AST) as markers of chronic alcoholism in cirrhotic patients. Sixty-three hospitalized cirrhotic patients (35 drinkers, 28 abstainers) were investigated. Ninety-six per cent of abstainers had normal values of mAST activity, while 89 per cent of drinkers had high levels of mAST activity. Cirrhotic patients were better divided into drinkers and abstainers by mAST activity (92 per cent) than by mean globular corpuscular volume (MCV) (81 per cent, NS) or by gamma-glutamyltransferase activity (GGT) (75 per cent, P less than 0.01). When the hospital costs of these markers were taken into account, MCV had a better "quality/price" ratio (Q/P) defined as diagnostic value/mean hospital cost (Q/P = 2.5) than MCV plus GGT (diagnostic value 61 per cent, Q/P = 1.2). The measurement of mAST activity in patients with high MCV value or with discrepancy between MCV and GGT values increased the diagnostic value of the other laboratory measurement to 89 per cent, but at a higher cost (Q/P = 0.8). Mitochondrial AST activity is a sensitive and specific marker of chronic alcoholism in cirrhotic patients. However, owing to its high cost, it should be proposed as a second marker after MCV and GGT.

Interleukin-1 beta-induced changes in the kinetic constants of L-proline uptake in human skin fibroblasts.

The effects of interleukin-1 beta (IL-1) on L-proline uptake in human skin fibroblasts were investigated. Exposure of the fibroblasts to IL-1 (5, 10 or 50 pg/ml) for 2 h did not change L-proline uptake. In contrast, inhibition was observed after 6 h of IL-1 treatment, and only 60% of the control uptake remained after incubation for 24 h with 10 pg of IL-1/ml. IL-1 depressed the activity of both transfer systems; the low-affinity system inhibited by alpha-(methylamino)isobutyric acid (Me-AIB), corresponding to system A, and a high-affinity transfer system which is unaffected by Me-AIB. The inhibitory effect increased as the L-proline concentration decreased. To determine whether IL-1-induced prostaglandin release influences proline uptake, indomethacin (14 microM) was added as a cyclo-oxygenase inhibitor. Indomethacin itself decreased L-proline uptake but to a lesser extent than did IL-1. When IL-1 was tested in the presence of indomethacin, the inhibition of L-proline uptake was still observed, with values between those obtained with each substance in isolation. This suggests that the inhibitory effect of IL-1 on proline uptake by skin fibroblast does not only involve the prostaglandins that accumulate in the medium, but no firm conclusion can be drawn, due to the fact that the inhibition by the two agents was not statistically independent. Kinetic analyses for 1 min combined with inhibition experiments showed that IL-1 induced a decrease in the Km and Vmax, values of the high-affinity transport system, whereas it increased the Km of system A. Therefore the two systems of proline uptake in skin fibroblasts are probably inhibited by IL-1 via different mechanisms.

Amino acid transport systems in the human hepatoma cell line Hep G2.

The human hepatoma cell line Hep G2 was used to investigate amino acid transport systems in human liver tissue. The ubiquitous transport systems responsible for the uptake of most neutral amino acids (systems A, ASC and L) were found to be present. Transport system A was predominant for proline uptake but system ASC was the major Na(+)-dependent transport system, particularly for glutamine. The specific hepatic system N was functional, but only partially mediated glutamine uptake. The study of Na(+)-independent arginine uptake demonstrated the presence of the cationic transport system Y+, reflecting the transformed nature of Hep G2 cells.

Complementation analysis of carnitine palmitoyltransferase I and II defects.

Carnitine palmitoyltransferase (CPT) consists of two activities located in the outer (CPT I) and the inner (CPT II) mitochondrial membranes. CPT II deficiency in the adult as well as in the infantile form of the disease has been shown to result from mutations in the CPT II cDNA. Nothing is known regarding the genetic defect in CPT I deficiency. We carried out complementation experiments between CPT I- and infantile CPT II-deficient cell lines. Restoration of 3H2O release from [9,10(n)-3H]-palmitate was chosen as criterion of complementation. As expected, no complementation was observed in heteropolykaryons resulting from fusions between CPT II-deficient cells. Similar results were obtained in fusions between CPT I-deficient cells, suggesting that the enzymatic defect in these cell lines results from mutations in the same gene. Conversely, complementation was observed in fusions between CPT I- and CPT II-deficient cells. These data support that CPT I and CPT II defects result from mutations in distinct genes. Palmitate oxidation by control or CPT I-deficient cell lines was decreased when cocultured with infantile CPT II-deficient cell lines. This effect, not observed in coculture including an adult CPT II-deficient cell line, was carnitine-dependent. The possible mechanism of this effect, suppressed by a high carnitine concentration, is discussed.

Mutational spectrum and DNA-based prenatal diagnosis in carnitine-acylcarnitine translocase deficiency.

Carnitine-acylcarnitine translocase (CAC) deficiency is a rare autosomal recessive disorder of long-chain fatty acid oxidation with a severe outcome. We report mutation analysis in a cohort of 12 patients. Twelve mutations were identified of which 9 have not been reported so far (G28C, D32N, R178Q, P230R, D231H, 179delG, 802delG, 69-70insTGTGC, and 609-1g>a). Altogether, including our results, 22 mutations of the CAC gene have been published to date in 23 patients demonstrating the allelic heterogeneity of CAC deficiency. DNA-based prenatal diagnosis was performed for the first time in pregnancies at risk for CAC deficiency. Two fetuses were affected and one pregnancy was terminated by family decision. Two other fetuses had normal genotype and five others were heterozygotes. All the offspring of these seven pregnancies are alive and apparently healthy.

[Production of oxygenated free radicals and the role of exogenous antioxidants in liver transplantation in rats]. / Production des radicaux libres oxygénés et rôle des antioxydants exogènes en transplantation du foie chez le rat.

It has been suggested that, in transplantation organs, the lesions observed after conservation then reperfusion could be related to the formation of oxygenated free radicals. The aim of our work was first to verify the hypothesis that oxygenated free radical are formed after ischaemia-reperfusion of the liver, then to evaluate the effects of the allopurinol and glutathion, known antagonists of oxygenated free radicals, contained in the University of Wisconsin conservation fluid. The University of Wisconsin solution was compared with a Collins solution which does not contain oxygenated free radical antagonists. An orthotopic liver transplantation model was used in Wistar rats. Three groups were studied. In the control group, 5 rats underwent laparotomy then were closed with no surgery being performed. In the Wisconsin group (n = 6) and the Collins group (n = 6) the livers were washed and conserved in the corresponding solution at 4 degrees C before transplanting. Plasma levels of malonyldialdehyde, measured by high performance liquid chromatography, were used as a marker for the formation of oxygenated free radicals. Impaired liver function was assessed on the basis of mortality and serum transaminases, alkaline phosphatase and total bilirubin levels. Liver biopsy was performed at sacrifice. The level of malonyldialdehyde was significantly higher in the transplanted groups compared with the control group (p < 0.01). There was no difference between the Wisconsin and the Collins groups. Hepatic function was significantly reduced in the transplanted groups compared with the control group (p < 0.05). There was no significant difference between the Wisconsin and the Collins groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Impaired mitochondrial pyruvate importation in a patient and a fetus at risk.

The patient was the first child of healthy consanguineous parents. She presented at birth with hypotonia, mild facial dysmorphism, periventricular cysts, marked metabolic acidosis, hyperlactacidemia with normal lactate/pyruvate molar ratios, normoglycemia, and normal ammonia. Hyperlactacidemia was severe (5-14 mmol/l) and not corrected with bicarbonate, thiamine (10 mg/d), 2-chloropropionate (100 mg/kg/d) and a ketogenic diet. Pyruvate dehydrogenase (PDHC) activity was normal in lymphocytes and fibroblasts. Functional assays were performed in digitonin-permeabilized fibroblasts to measure oxidation rates from radiolabeled pyruvate and malate. The production of [14C]acetylcarnitine or [14C]citric cycle intermediates derived from [2-14C]pyruvate as well as the release of 14CO(2) from [1-14C]pyruvate was severely impaired, whereas decarboxylation of [U-14C]malate was normal. With increasing concentrations of [1-14C]pyruvate, the patient's fibroblasts behave like control fibroblasts incubated in the presence of alpha-cyano-4-hydroxycinnamate, a specific inhibitor of mitochondrial pyruvate uptake: a progressive increase in 14CO(2) production was observed, likely due to passive diffusion of [1-14C]pyruvate through the mitochondrial membranes. Our results are consistent with a defect of mitochondrial pyruvate transport in the patient. Mutational analysis was precluded as the cDNA sequence of the pyruvate carrier has not been identified as yet in any organism. An affected fetus was recognized in a subsequent dichorionic twin pregnancy using the coupled assay measuring [2-14C]pyruvate oxidation rates on digitonin-permeabilized trophoblasts. After selective feticide, the pregnancy was uncomplicated with delivery at 37w of a healthy female, who is currently 2-month old.

Defects in activation and transport of fatty acids.

The oxidation of long-chain fatty acids in mitochondria plays an important role in energy production, especially in skeletal muscle, heart and liver. Long-chain fatty acids, activated to their CoA esters in the cytosol, are shuttled across the barrier of the inner mitochondrial membrane by the carnitine cycle. This pathway includes four steps, mediated by a plasma membrane carnitine transporter, two carnitine palmitoyltransferases (CPT I and CPT II) and a carnitine-acylcarnitine translocase. Defects in activation and uptake of fatty acids affect these four steps: CPT II deficiency leads to either exercise-induced rhabdomyolysis in adults or hepatocardiomuscular symptoms in neonates and children. The three other disorders of the carnitine cycle have an early onset. Hepatic CPT I deficiency is characterized by recurrent episodes of Reye-like syndrome, whereas severe muscular and cardiac signs are associated with episodes of fasting hypoglycaemia in defects of carnitine transport and translocase. Convenient metabolic investigations for reaching the diagnosis of carnitine cycle disorders are determination of plasma free and total carnitine concentrations, determination of plasma acylcarnitine profile by tandem mass spectrometry and in vitro fatty acid oxidation studies, particularly in fresh lymphocytes. Application of the tools of molecular biology has greatly aided the understanding of the carnitine palmitoyltransferase enzyme system and confirmed the existence of different related genetic diseases. Mutation analysis of CPT II defects has given some clues for correlation of genotype and phenotype. The first molecular analyses of hepatic CPT I and translocase deficiencies were recently reported.